Tuatara: Volume 22, Issue 2, June 1976
The Biological and Economic Importance of Algae. Part 4: the Industrial Culturing of Algae
The Biological and Economic Importance of Algae. Part 4: the Industrial Culturing of Algae
Because of a constant effervescence of new facts, new theories and new things to see, research must seem a most satisfying occupation to the onlooker. To the participant, however, certain aspects of it can be viewed as anything but satisfying. Research is something of a paradox. Admittedly it leads to the acquisition of fresh knowledge and pegs out with a small measure of certainty our progress towards some understanding of the things around us. But in achieving this we expose fresh surfaces of the unknown, for solving one problem automatically uncovers a galaxy of new ones and in a way our condition after the experiment is worse than before it. In essence, therefore, research is one step nearer uncertainty. What an appalling prospect - to spend a lifetime chipping away at the fringe of what appears to be an infinity of the unknown, knowing that you will never be able to write the last word on anything!
Although a research project must have a beginning, it is not always easy to discern when or where its conception occurred. Often the starting point is quite arbitrary from an historical point of view. Nor is there an end to a research project once started, because the ultimate goal assumes the character of an intellectual mirage that continuously recedes into the aether - into an ‘Expanding Universe’ of perpetuating enquiry. Most present-day research workers are like runners in the middle of a relay race; they are just part of a continuum, having no direct and personal relationship with the beginning and of course never with the end. They get interested in a topic and do not always have - or take - the opportunity to view their research in relation to its real beginnings and development up to the point where they begin. This is lamentable in many ways.
Another of the tantalising things about research is that nobody knows what side-issues are going to emerge along the way - or if page 2 one of these could eclipse the original line of work. It might be disastrous if one were to succumb to the temptation of diverting from the main stream to follow one of these fringe enticements. But then a side-issue might turn out to be a real winner. Who can tell?
A fourth feature is that as in forms of creative art, some research is spawned ahead of its time - its value not appreciated nor utility realised: it may even have to hibernate while awaiting, for its further development, a ‘triggering mechanism’ to emerge from research in unrelated fields. Whereas other work appears and is used immediately because its application is obvious or its results are just what somebody has been waiting for to answer their particular problem. So a research worker never knows when, where or by whom his results will be used. There is both impersonality and unpredictability about the follow-on.
But despite these philosophical impediments, research work is still very alluring and seductive and just like a mirage lures one on remorselessly despite there being really no end to the quest. It is a kind of habit-forming addiction that chelates many very securely, an opiate for the enquiring mind and a curse which derives from the sapiens part of Homo.
While the cosmologists cannot say at this moment if the Universe is expanding, the demographers know only too well that the earth's population is not merely expanding, but exploding. Unfortunately, these newcomers are neither photosynthetic nor nitrogen-fixing; they, like us, are all too heterotrophic and have to buy their sugars and proteins. But where will they get a cheap and at the same time nutritious food? ‘Having seen how easily planktonic algae yield to laboratory culture, Science has tried to cultivate algae industrially for food-hoping through advanced technology to be able to improve on Nature's productivity.’ (75) So the theme for this article explores the feasibility on a large industrial scale of growing and harvesting microscopic algae as food for humans.
We could begin by discussing the more recent important and critical papers starting with the pilot-scale growing of Chlorella. But to begin this way might be to lose much of the interest of the discussion because we would miss completely the point that research projects like living organisms - undergo an evolution, a fact all too infrequently realised. Instead, it is intended to retrace history a little and follow this work from one particular point in time in order to review its ontogeny. And as we proceed we will be able to discern very clearly the expression of those idiosyncrasies of research already mentioned, all of which when working together help to mould the evolution of a project: the dependence of new work on a background of earlier and often unrelated research, the ever-receding horizon, the side issues which arise, the inhumation of results until new avenues or new techniques for use have appeared, the new applications of results never conceived of earlier. All are here to be seen. So let us page 3 start a traverse through time and piece together the evolution of one particularly fascinating line of work.
By the year 1800 it was realised that most inorganic elements found in ashed plant material were involved in the metabolism of that plant. In 1804 de Saussure published work which reported the first use of water cultures to investigate the mineral requirements of plants. He grew Polygonum persicaria, a fairly common weed, and Bidens cannabina in dilute solutions of various salts. Among other things, he found that of the various minerals required, nitrates were indispensable for the growth of some plants. Further work centering on the essentiality of inorganic elements was conducted by growing plants in solution-culture using sand, quartz, pumice, acid-washed charcoal and even fragments of platinum as a supporting medium. Except for platinum, these supporting media suffered one drawback: they introduced a source of impurity in the form of extraneous chemicals which could upset the results of an experiment. To overcome this problem, de Cassincourt, John and Boussingault grew plants in media which had been boiled in acid; but their results were inconclusive. Salm-Horstmar (1856) developed this idea of acid-washing the supporting media and showed the necessity in plant nutrition for nitrogen, phosphate, sulphur, calcium, potassium, magnesium, silicon, iron and manganese; and even described the deficiency symptoms shown in plants as a result of a lack of an individual element. For instance he identified ‘grey speck’ in oats as a manganese deficiency.
But acid-washing sand and other types of inert media is a timeconsuming job; and human nature being mainly homozygous and dominant for laziness is always on the look-out for easier ways of doing things. Conceivably this is what motivated Sachs to experiment with the growing of plants in solutions of chemicals-what we now know as water-culture, or ‘hydroponics’. This he did very successfully at the School of Forestry in Tharandt near Dresden. During his research he evolved the use of solutions of constant composition containing all the elements thought necessary in plant nutrition. He ‘succeeded in growing healthy plants by alternatively transferring them from one solution, containing a portion of the ash constituents, to another which contained the remainder.’ (116)
So Sachs set the pattern for water culture. But once more improvement in the system was effected by Knop, who yet again was able to produce a simpler way of doing things - by altering the kinds of chemicals used so that only one solution was necessary for growing the plants in. Knop's solution was also defined in terms of molar ratios. Because of its ease of use, this solution was the one that became widely known and therefore adopted.page 4
The Russian Famintzin (1871) has been recognised as probably the first to appreciate and preach the necessity for learning the nutritive needs of algae through the use of culture solutions. He used Knop's solution. This idea was taken up by Molisch and Beneke; and the over-all similarity of the nutritional requirements of higher plants and algae became apparent. ‘The following 20 years appear to have been relatively barren in the cultivation of algae for it was not until 1890 that the Dutch bacteriologist Beyerinck became interested in the problem. At first, he followed the simplest and most obvious procedure, that of attempting to grow algae in water from their natural habitat, and in his classical paper describing the isolation of Chlorella and Scenedesmus in bacteria-free cultures, he used ditch water solidified with gelatine as the culture medium. The career of Chlorella as a botanical and physiological “guinea pig” was launched in this work. Beyerinck soon found it helpful to enrich water from the natural habitat with various inorganic and organic substances, and from this standpoint his work marked a return to the type of investigations of Famintzin, Knop and other students of the nutrition of flowering plants.’ (7)
From the turn of the 20th century, phycologists concentrated more on procuring pure bacterium-free cultures of algae. Such names as Chodat, Grintzesco and students, Moore, Chick and Pringsheim stand out for their contributions. Chick's paper is interesting because she managed to isolate Chlorella pyrenoidosa from polluted water, grow it in bacterium-free culture and study many aspects of its nutrition including its apparent preference for reduced rather than oxidised forms of nitrogen.(25)
Pringsheim should be remembered particularly because he was the first person to set up a collection of pure cultures of algae, both septic and aseptic. He acted as the first focal point for amassing such cultures as well as a dissemination centre for those requiring pure cultures.
In 1883 a German botanist, Reinke, reported that the rate of photosynthesis increased proportionally with the increase in light intensity until light saturation was reached, when the response curve flattened out. Over the years 1898-1901 Brown and Escombe had also been conducting experiments on photosynthesis at the Jodrell Laboratory at Kew Gardens. Brown and Escombe's ‘main object of the research was, in the first place, to obtain a direct measure of the rate of photosynthesis in a leaf, when it is surrounded by an atmosphere containing an amount of carbon dioxide not far removed from the normal amount of 0.03 per cent; and secondly to obtain more definite information on the “energetics” of the leaf, especially as regards its power of absorbing and transforming the solar radiation incident upon it ’.(9) In the course of this work they became the first to discern that intermittent illumination could permit a greater amount page 5 of photosynthesis than continuous light. They also used air enriched with carbon dioxide. Their estimation of carbon dioxide fixation depended on titration methods applied to air before and after contact with a leaf.
Blackman, a British plant physiologist, published a paper in 1905 that will always be regarded as a milestone in the history of photosynthesis.(6) It embodied his Law of Limiting Factors, which implied that the rate of any process affected by several factors is controlled mainly by the factor in shortest supply-or in laymen's terms, ‘the speed of a convoy is mainly determined by the speed of the slowest ship.’ Blackman's experiments showed that in strong light an increase in temperature led to an increase in photosynthesis, but that in weak light there was hardly any increase in photosynthesis despite an increase in temperature. He realised that photosynthesis must involve a photochemical reaction somewhere, and therefore that the rates of such reactions would not be affected by temperature unless other limiting factors were operating. This reasoning was based on the fact that photochemical reactions, not being controlled by enzymes, are not affected by temperature; whereas the rates of biological reactions mediated by enzymes usually double or treble when subjected to a 10° C. rise in temperature. From his results, Blackman inferred that photosynthesis must consist of two kinds of reactions:
a photochemical or ‘light’ reaction-a physico-chemical reaction not mediated by enzymes and involving one reaction only;
a ‘dark’ reaction or set of purely chemical reactions mediated by enzymes and processing the products of the photochemical reaction, involving a whole series of reactions and therefore taking time.
This ‘dark’ reaction came to be called the ‘Blackman Reaction’. His interpretation of the experiments was that at low light levels the photochemical reaction was rate-limiting; whereas at high light levels, the flattening-out occurred because the dark reactions could not process quickly enough the products formed by the photochemical step, i.e. the dark reactions became rate-limiting. So was borne the concept that photosynthesis consists of two main classes of reaction.
- E = hv
J. S. Haldane graduated in Medicine at Edinburgh in 1881 and became a demonstrator to Professor Carnelley at Dundee. With Carnelley, he investigated the chemical composition and bacterial content of air in such places as dwellings, schools and sewers; and from these prosaic beginnings there began a lifetime of very fundamental human physiological research. Not very long after this, he moved to Oxford where he developed an accurate gravimetric method for determining carbon dioxide and moisture in air. The earlier work in Dundee aroused his interest in the composition of air, especially in situations where men were exposed to the dangers of ‘foul air’; in this way he became involved in problems peculiar to mines-such as ‘black damp’ and ‘after damp’, carbon monoxide poisoning and related problems. Around the turn of the century he developed methods and apparatus for analysing air, and for investigating blood gases and the derivatives of haemoglobin.
In 1898 he discovered that when potassium ferricyanide was added to solutions of oxyhaemoglobin or the carbon monoxide-haemoglobin complex, the gas combined with the haemoglobin was set free. Because of this reaction and his background of gas analysis, he thought that the volume of oxygen or other gas combined with haemoglobin should be capable of estimation much more easily and accurately than by using the mercury air-pump. Around this time Dupré had developed an apparatus for estimating urea in urine. It was known that urea when treated with sodium hypobromite released its nitrogen in gaseous form. So when urine was mixed with hypobromite. nitrogen was produced in proportion to the amount of urea present. The nitrogen was collected in a burette inverted over water: the volume of nitrogen gas could be easily read off from the burette graduations. Haldane adapted Dupré's ureometer to the estimation of oxygen in blood. But in 1902, he and Barcroft redesigned the apparatus and produced a new constant volume apparatus capable of giving accurate results for estimating oxygen and carbon dioxide on page 7 as little as 1 ml of blood. And so was born the first manometer or manometric microrespirometer. Barcroft later produced the differential manometer.
We move now to Berlin - the Kaiser Wilhelm Institute, where a scion of the famous German banking family of Warburg had begun a lifetime of biochemical research - predominantly in respiration, with several major excursions into photosynthesis. Otto Warburg published a paper in 1919 which was very important because of the experimental innovations it embodied. This is really where our prologue ends and our story on Chlorella begins.
It is as well at this point to write down the photosynthetic equation to grasp several points of extreme significance.
6CO2 + 12H2O - LIGHT -→ C6H12O6 + 6O2 + 6H2O
There are four different chemical compounds involved in this reaction of which two are gases. These gases could be estimated chemically, as Brown and Escombe did for carbon dioxide and as Haldane did for oxygen as well as carbon dioxide. By the year 1919 Barcroft and Haldane's manometers were widely used for blood gas analysis and generally employed in the measurement of respiration manometrically. So here now was a marvellous piece of apparatus for investigating chemical reactions involving gases. It possessed features which appealed immediately to those involved in quantitative work. Without dismantling the apparatus it was possible to collect and estimate within the system one or more of the gaseous end-products. This eliminated errors inherent in volumetric analytical procedure. Using this apparatus analysis could be carried out with a high degree of accuracy. Systems could be investigated using semimicro- and microamounts of reactants. Because the apparatus was small, its physical environment was easy to control while the experiment was in progress.
Warburg seemed most interested in the kinetics of photosynthesis. Obviously Barcroft's manometer provided an ideal system for measuring photosynthesis because of the involvement of gases. But what kind of plant material could be used in such a small container? This must have presented a dilemma, compounded no doubt by another difficult-to-satisfy requirement at that time-the material would have to be bacteriologically sterile. Bacteria and other nonphotosynthetic micro-organisms usually found as contaminants are heterotrophic, and in their metabolism take in oxygen and give out carbon dioxide-the complete reverse of photosynthesis. Obviously one could not investigate photosynthesis with non-sterile plant material. But what could be used? Sterile plant-tissue culture was a long way off: there were no antibiotics to help in the sterilisation of small page 8 aquatic plants such as Lemna, Wolfia or Spirodela. Warburg consulted a botanist uncle, who suggested using a green alga Chlorella. Beyerinck had isolated this organism in axenic culture quite a few years earlier, and it would more than likely have been available from Pringsheim's collection of algal cultures. This was truly a brilliant suggestion! The alga is microscopically small and eminently suited to this kind of micro-experimentation. It could also be maintained indefinitely in a sterile condition and grown on a completely inorganic and chemically-reproducible medium whose components could be got in pure form off the laboratory shelf. What better could be used? Little could Warburg have realised the fashion he was establishing! He also used an enhanced carbon dioxide concentration. It must be emphasised that Chlorella was chosen possibly for no other reason than that it would have been one of very few algae available at that time in a bacteriologically-free condition.
In the course of his experiments he used intermittent illumination, and found an increase in the amount of carbon dioxide reduced compared with the amount reduced under continuous illumination. This was also what Brown and Escombe had found. Warburg tried to explain this phenomenon, and of two possible explanations postulated by him chose the one in which he thought photosynthesis proceeded twice as fast during a brief flash plus dark period as during the same length of time under continuous light.(143)
Warburg went on to do further work on photosynthesis. Having now such a sensitive apparatus as the manometer for measuring volumes of gases so accurately, and being aware of Einstein's application of Planck's Equation to photochemical reactions, Warburg no doubt could see the possibility of determining the efficiency of the photosynthetic process - the number of quanta required to make one molecule of carbon dioxide combine with one molecule of water.
Let us investigate this a little more closely. If we burn glucose (C6H12O6) in air and measure the amount of heat given out, we obtain the figure of approximately 672 kilocalories per gram mole of glucose. Because there are six atoms of carbon in glucose, 672 kilocals per gram mole of glucose is equivalent to 112 kilocals per gram atom of carbon. If we re-write the photosynthetic equation slightly differently, this point can be appreciated.
CO2 + 2H2O -→ CH2O + O2 + H2O
Here the amounts of reactants and products have been divided by six to reduce the compounds involving carbon to terms of a gram-atom of carbon. Visible light extends from the shorter wavelength violet to the longer wavelength red; and since the energy of a quantum (i.e. photon) of light depends on the wavelength of that light, photons of violet light contain more energy than those of red. However, in the photochemical reaction of photosynthesis, quanta of the weakest page 9 wavelength (red) are all that are normally required: so we can carry out the forthcoming calculations on the basis of red light.
The gram molecular weight of any substance always contains the same number of molecules - 6.02 X 1023, a figure known as Avogadro's Number. Recalling Einstein's Law of Photochemistry that one quantum of light effects a direct photochemical change in one molecule, we can see that it will require 6.02 X 1023 photons to produce a like effect in every molecule of a gram molecular weight of chlorophyll. 6.02 X 1023 photons of red light have an energy content of 40 kilocalories; and one gram molecular weight of carbon dioxide gives rise to a sugar monomer (CH2O) whose energy equivalent is 112 kilocalories. Obviously, at least three photons are going to be required; if three only were used, the efficiency of energy utilisation would be 112/3X40X100 = 92%, which would represent a fabulously high degree of efficiency; if 5 were used, the efficiency would be 56%; and 35% if 8 were used. The question was -how many are used?
Having now an ideal type of plant and an elegant apparatus for measuring photosynthesis, what more could one want for investigating the quantum efficiency of this, the most important reaction in the biological kingdom? So Warburg and Negelein set about to measure this efficiency, which they found to be 70% -i.e. that 4 photons were required for every molecule of carbon dioxide absorbed or oxygen evolved. But this represents an efficiency which makes no allowance for energy of activation. The results of Warburg and Negelein were published in 1923.(144)
These two papers of Warburg's had far-reaching implications. He had developed manometry to a very high level while using the Barcroft-Warburg manometer. He also set a fashion in his choice of experimental plant - Chlorella - which was ideal as a physiological laboratory plant in botany.
Not long after Warburg and Negelein published their paper an American, Emerson, joined their laboratory and also worked on Chlorella - adapting the methods of Warburg to investigate the effect of respiratory inhibitors on this alga. Emerson returned to America and began working at the Californian Institute of Technology, bringing with him (one suspects) a culture of Chlorella.
The reasons put forward by Warburg to explain the increase in photosynthesis brought on by intermittent illumination were accepted for about twelve years. But in the early 1930's Emerson and Arnold at the Californian Institute of Technology began to re-investigate the phenomenon. Following in the footsteps of Warburg, they used a Barcroft-Warburg manometer with slight modifications and Chlorella as experimental material. In some of their work they also used 5% carbon dioxide in air.(45)page 10
Their results showed that Warburg's explanation was incorrect. They demonstrated that photosynthesis involves a light reaction not affected by temperature yet effected at great speed, and a dark reaction whose rate was governed by temperature and whose duration was much greater than the light reaction. The higher fixation of carbon dioxide under intermittent illumination was due to the fact that the dark period provided the occasion to process the reactants of the light reaction and thus relieve the pressure on the dark-reaction enzyme systems of a constant choking by the products of the light reaction. Their experiments were done mainly with Chlorella pyrenoidosa, but they also experimented with Chlorella vulgaris.
Warburg and Negelein's paper was also accepted for many years; but in the end the high efficiency they purported to show became too much of a straitjacket. People could not reconcile the then current chemical hypothesis about photosynthesis with this high quantum efficiency; and further-an aspect which was even more serious and of even greater importance-most workers were unable in the main to duplicate this result. Therefore the value of the original results in establishing the efficiency of the light reaction of photosynthesis came under a pall of doubt. Consequently various groups of investigators in America began a re-examination of Warburg and Negelein's experimental technique and results - but nearly always using Chlorella as their experimental plant.
Again Emerson featured in the refutation of Warburg's work. Emerson and Lewis at the Carnegie Institution at Stanford University investigated exhaustively the quantum efficiency of photosynthesis and always got a figure of double that found by Warburg and Negelein.(46) The discrepancy between the two sets of results sparked off much research, and techniques other than manometric were used to try to resolve this dilemma. These included micro-gas analysis of oxygen and carbon dioxide; measurement of oxygen by chemical titration or by polarographic methods; measuring unused heat of radiation with micro-photocalorimeters; measuring calorimetrically the difference in the heating of a leaf when photosynthesising and when not doing so. The methods of culture, the influence of nutrients and traces of various elements, and the age of the algae were varied over wide ranges; but the empirical maximum efficiency was nearly always between 8 to 10 photons per molecule of carbon dioxide reacting.(36)
So, as a result of Warburg's experiments in photosynthesis and particularly because of the controversy they kindled (especially the one on quantum efficiency), a lot of people got to know a lot of information about Chlorella. And this was not the only way in which chemists and physiologists became aware of this alga.
It had been believed for many years that the oxygen evolved in photosynthesis came from the carbon dioxide-that one substituted the two atoms of oxygen in carbon dioxide for one molecule of water.page 11
But in the early 1930's the bacteriologist, van Niel, hypothesised that in photosynthesis the water molecule was split and that the oxygen evolved came from the water. Credence for this was suggested by van Niel from considering the parallel case in certain photosynthetic sulphur bacteria which were able to acquire hydrogen from hydrogen sulphide while depositing sulphur.
CO2 + 2H2S -→ CH2O + H2O + 2S
CO2 + 2H2A -→ CH2O + H2O + 2A
How could this hypothesis be tested?
By this time, elements were known to exist in isotopic variation. Oxygen has one of these isotopes with an atomic weight of 18 instead of 16. Chemically, these isotopes cannot be distinguished; but physically this can be done by using a mass spectrometer, an instrument able to separate isotopes because of the differing behaviour of molecules of differing mass in a powerful magnetic field. Ruben and his team carried out an experiment with O18.(125) They prepared both water and carbon dioxide with O18 instead of O16. Chlorella was allowed to photosynthesise with CO216 and H2O18 and also with CO218 and H2O16 and the O2 evolved was assessed for its isotope content. They found O218 was formed only when using H2O18 and not H2O16 So, van Niel's hypothesis about the origin of the O2 in photosynthesis was vindicated; and it became obvious that a lot of new thinking had to be done about this process. In 1937, Ruben in association with Hassid and Kamen had begun investigating photosynthesis with the short-lived carbon isotope C11. This form of carbon was a bit too short-lived for easy working; and aware of the existence of a longer-lived isotope, Ruben and Kamen in 1940 discovered a way of obtaining quantities of C14 which they used in their further studies on photosynthesis. (124) This discovery and its application opened up new vistas in biochemistry. Calvin and his team carried on research into photosynthesis and after extensive experimental work were able to unravel the chemistry of this process. Throughout their investigations Chlorella was the organism used. So was heralded in the Golden Era of radioactive isotope technique and discovery during which many older theories and hypotheses were buried beyond recall.
But in all this work, Chlorella was grown in small quantities only. Nobody had evolved techniques for large-scale culture. These were pre-antibiotic days; and people had not begun to expand the volumes of their cultures even to gallons of sterile medium. Later, when antibiotics arrived and micro-organisms were cultured industrially on a very large scale, things changed dramatically: but just before the era of antibiotics, one or two folk were beginning to mass-produce in sterile culture because investigation of certain problems demanded page 12 access to large amounts of micro-algae obtained from pure and sterile culture.
One of the problems faced by the marine invertebrate zoologist working at the turn of this century was the unravelling of the lifecycles of many marine animals. In their life-cycles, coelenterates, molluscans, annelids and crustacea have planktonic larval forms; and at that stage in our knowledge it became necessary to culture these larvae through metamorphoses to find out which larval form belonged to what life-cycle. Seeing these larval forms were planktonic, it was thought that they would in all likelihood be dependent on phytoplankton primarily if not entirely as a source of sustenance. Grave was among the first who reported being able to rear larvae on diatoms.(59) These he obtained by putting sea-bottom sand in aquaria and using whatever diatoms grew under his conditions. While successful, this method suffered from the drawback of providing a culture of somewhat capricious and uncertain composition.
Allen in 1905, assisted by Nelson from 1907, began experiments to attempt the growing of larval forms in sterile sea-water enriched with diatom cultures.(1) They achieved considerable success with their culturing of diatoms and managed to get about eighteen species into persistent culture, although many were not entirely free of adulterant organisms. One diatom favoured for feeding trials was Nitzschia closterium forma minutissima. They found this particularly useful. It was small enough for larvae to draw into their mouths by ciliary currents and it remained suspended throughout the culture liquid without setting to the bottom. They were unable to detect a diminution in size of the individual frustules despite the fact that the organism had been held in continuous culture for more than two years. All culturing was done in 125 ml flasks using 60 mls of medium.
Copepods provide an important link in the first conversion step from marine phytoplankton to marine zooplankton: in other words they are the main grazers, the ruminants of the marine pasture. ‘Of the common species that are frequently to be seen in the plankton, probably the most important is Calanus finmarchicus. This is wide spread in all oceans except the Antarctic and is very common in the northern hemisphere, where it may be found as deep as 4,000 m, although it is much more frequent near the surface. In these northern seas its role is quite outstanding as a link in the chain of production, making available the protein of the phytoplankton to pelagic fish, whales, and other creatures of importance to man. An instance of this is the staple part it plays in the food of the herring, which is the most massive population of food fish available to the peoples of north-west Europe.’ (149)page 13
To some marine research workers, however, the connection between phytoplankton and copepod did not appear as direct as numerous people implied. G. L. Clarke had this to say: ‘The traditional impression of copepod production is that it follows and is dependent upon the growth of diatoms. But in attempting to determine more precisely the relations between copepods and diatoms, recent investigators have proposed certain widely differing theories, all of which need confirmation. A comparison of these theories and of the plankton investigations from which they emanate, will be valuable and, it is hoped, helpful in penetrating further into this important problem.’(27) One of the disturbing things in the phytoplankton-copepod complex was that several investigators found no apparent direct relationship between the spring flush of diatoms and Calanus — that the diatoms could be present but they may not be accompanied at the same time by grazing Calanus. Steeman Nielsen produced a hypothesis to explain this but its applicability depended on knowing something about the feeding habits of this copepod — what it ate and how much food it needed.(133)
To try to provide information on this topic, experiments were conducted by Fuller in 1937 at the Woods Hole Oceanographic Institution on some of the feeding habits of Calanus, using a diatom Nitzschia closterium.(54) ‘In most of these laboratory tests the concentration of diatoms used was considerably higher than that ordinarily found in Nature.’(26) Either the diatom would have been concentrated by filtering sea-water or else it must have been cultured. Clarke said, in 1937, that exceedingly few species had been cultured successfully by anyone. The alternative was to collect diatoms by filtering sea-water whenever the diatom counts were high. However, this latter method was also beset with a few disadvantages, such as collecting protozoons or particles of detritus of the same size as the diatoms being harvested; this, of course, would lead to contamination. Copepods and other crustacea would also be inevitably collected. One would also get a mixture of diatoms, not all of which might be fodder for Calanus. Allen also pointed out that not all representatives of a particular diatom species gathered from the sea would be uniformly representative in chemical composition. At the Scripps Institution of Oceanography it has been found that some larger population near the surface of the sea show nearly 50 per cent in decadent condition.’(3)
Fuller does not mention anywhere in his paper that the diatoms were artificially cultured; but for several reasons this must have been so. He specifically mentions using Nitzschia closterium Plymouth strain — presumably obtained in culture from Plymouth Marine Biological Laboratory in England. Secondly, for reasons just given, it would have been preferable to work with a pure culture; and we have already seen Clarke's remark that the concentration of diatoms page 14 used in most of the tests was higher than found in nature. And, thirdly, a paper by Ketchum and Redfield in 1938 and published as a contribution from Woods Hole specifically dealt with a method for maintaining a continuous supply of a marine diatom Nitzschia closterium by culturing it in the then large volume of 26 litres.(80)
‘Culture techniques by means of which large supplies of unicellular organisms can be continually available are greatly in demand. This is especially true of unicellular plants, since they are convenient organisms for the study of photosynthetic and other metabolic processes.’
‘The problem is essentially one of the maintenance of a growing population. So long as no factors develop which limit the rate of multiplication, increase in a culture or population is directly proportional to the number of organisms present. The growth of the population is logarithmic during the initial period. Some factor or factors in the environment, however, sooner or later lower the division rate. These factors may be limiting nutrient concentrations, formation of inhibitory excretory products, production of non-viable cells in the process of division, or, in the case of photosynthetic plants, limiting light intensity.’
‘It was pointed out by Hjort, Jahn and Ottestad (1933), in a study of whaling in the Antarctic, that the most advantageous way to exploit a population is to keep it at a level at which the greatest number of new organisms are produced in unit time. This paper presents an application of this principle to a culture method. After procuring the cell concentration at which the greatest daily yield is obtained the culture is maintained at this concentration.’(80)
|1||= lag phase|
|2||= acceleration phase|
|3||= exponential phase (also referred to as logarithmic phase)|
|4||= retardation phase|
|5||= stationary phase|
|6||= decline phase (also referred to as death phase)|
Table 1 also sets out in column three the cell number increase per time interval. From 48 hours to 120 hours it will be seen that the increase in cell number doubles every 12 hours. For an organism page 15 undergoing vegetative multiplication in which mitosis in one cell can give rise to two daughter cells only, this rate of division represents peak efficiency in cell proliferation.
|Time Interval in hours||Total Cell Number||Cell Number Increase per Time Interval|
If we now plot the logarithm of the increase in cell number per time interval, we get Curve B in which there is an inflection at 156 hours. This represents the point of maximum cell-number increase (i.e. yield) for this organism under the prevailing conditions of growth and in a limited volume. Now, something is beginning to affect cell division — maybe the depletion of a nutrient, the build-up of some excretory product, or the density of cells affecting the penetration of light into the culture where the cultured organism is an alga. But if at this point one removes a volume of the culture containing 4,224 cells and makes good the deficit of medium by adding an equal volume of new nutrient solution, it should be possible by doing this repeatedly to harvest the organism at its maximum production.
|Initial Count cells/litre||Final Count cells/litre||Duration of Growth Period hours||Daily Yield cells/litre|
|8.84 X 109||11.36 X 109||25||2.42 X 109|
|7.70 X 109||11.15 X 109||41||2.02 X 109|
|8.10 X 109||12.92 X 109||45||2.57 X 109|
The total volume of their culture was 26 litres — a different story from the 60 mls of culture used by Allen and Nelson. Although they stated that Nitzschia Could be obtained in bacterium-free culture, they made no specific mention of having carried out their experiments under completely sterile conditions. Not that this would have mattered much, seeing they were working with sea-water and a marine form: airborne contaminants would not find it easy to grow in this environment. Air with 5% carbon dioxide was used.
‘The culture method described above has the advantage, not obtainable with natural collections, of producing a continuous supply of cells in a pure state. The culture can, moreover, be grown under various controlled environmental conditions and an ample quantity of material is produced daily for a variety of chemical and physiological tests. It is believed that a similar method can be applied advantageously to many other unicellular organisms required for physiological research.’
In a later paper(79) they applied their technique to the sterile culture of several Chlorophyceae: Stichococcus bacillaris, Chlorella pyrenoidosa, C. vulgaris, Scenedesmus obliquus, S. basilensis. The volume of culture was 8 litres. Of these five, Chlorella pyrenoidosa was found to be superior to the others both in the number and weight of cells produced.
Another reason for wanting large quantities of an alga was that many people were interested in the chemical composition of unicellular algae. Such work could only be done if pure and reasonably large samples of material were available. Up till this point in time sufficient material for this kind of research could be obtained only when a water-bloom occurred; but such phenomena were completely unpredictable and the choice of alga more than somewhat vicarious. Retovsky attacked this problem of large-scale culture and evolved a technique whereby he grew Scenedesmus obliquus and a species of Navicula in 70 litres of culture solution.(122) He was able to harvest 25 g dry weight of Scenedesmus in several weeks and about 20 g of Navicula. From the latter he managed for the first time to extract and crystallise the carotenoid pigment fucoxanthin. The technique and apparatus he used were adapted from the brewing and fermentation industry.
Now we must turn to consider the background and then the contribution to our story of one particular organisation, a famous page 18 privately-endowed American research organisation founded by its benefactor with the original donation of 10,000,000 one-dollar shares in United Steel Corporation — the Carnegie Institution of Washington. This was inaugurated in 1902 by Arthur Carnegie, who in 1907 added another 2,000,000 dollars and in 1911 another 10,000,000. A reserve fund of about 3,000,000 dollars was set aside by Carnegie and the income from this was to be added to the endowment fund of the institution. As a result of this latter investment, a further 5,000,000 dollars was paid over by the Carnegie Corporation of New York in 1931. The articles of incorporation declare in general ‘that the objects of the Corporation shall be to encourage, in the broadest and most liberal manner, investigation, research and discovery, and the application of knowledge to the improvement of mankind.
‘The Institution is essentially an operation Organisation. It attempts to advance fundamental research in fields not normally covered by the activities of other agencies, and to concentrate its attention upon specific problems, with the idea of shifting attack from time to time to meet the more pressing needs of research as they develop with increase of knowledge. Some of these problems require the collaboration of several investigators, special equipment, and continuous effort. Many close relations exist among activities of the Institution, and a type of organisation representing investigations in astronomy, in terrestrial sciences, in biological sciences, and in historical research has been effected. Conference groups on various subjects have played a part in bringing new vision and new methods to bear upon many problems.’
In the original organisation of the institution there was among the biological sciences a section devoted to botany and this was referred to as the Division of Plant Biology. A plan for botanical research was drawn up which had as its central theme an investigation of the relationship of vegetation to environment in the United States. This involved the establishment and maintenance of a laboratory at Tucson, Arizona — opened in 1903. The main purpose of this laboratory was to look at the methods by which plants perform their functions under the extraordinary conditions peculiar to deserts. In 1906 the botanical research work was organised into a Department of Botanical Research. In that year the distribution of chlorophyll in desert plants was looked into and it was found that chlorophyll in those plants lacking or having only rudimentary leaves was to be found particularly in branch and stem tissue. In 1910 plans were announced for dealing with some of the botanical problems by investigating plant physiology and chemistry; and to this end Dr H. A. Spoehr was appointed to the staff from the University of Chicago.(12) To understand the tenor of the research work he embarked upon, it is necessary once more to dip into the past — to 1861 to be exact.page 19
- 6 X CH2O —→ 6(CH2O) or C6H12O6
When Spoehr was first appointed, he quickly became involved in research on photosynthesis and the chemical effects of radiant energy in plant processes. His first work centred around the formation of formaldehyde and the conditions under which it could be synthesised in the laboratory.(13) He also tried to find if formaldehyde could be formed from acids such as malic, tartaric and citric through decomposition by light, and much effort was devoted to the effect of blue-violet light on malic acid.
During the year 1914 a phytochemical laboratory had been completed and facilities were now available for a study of light and its relation to organisms. With these new facilities Spoehr resumed his investigations on the formation of formaldehyde and its possible occurrence as an intermediate in photosynthesis. But after exposing solutions of pure formaldehyde and weak alkalies to sunlight in glass flasks for five months, he could not find a trace of sugar formation in the presence of calcium carbonate, magnesium carbonate, potassium page 20 bicarbonate and potassium hydroxide either in the light or the dark.(14)
In 1921 a new laboratory for the Department of Botanical Research was established at Carmel in California which offered more modern facilities for investigating photosynthesis and the compounds formed under the influence of light. In 1922 Spoehr referred to the fact that the primary photolysis of carbon dioxide had not received experimental support, and he came to the conclusion that photosynthesis is not a simple splitting of carbonic acid initiated by light.(15)
Yearbook 26 for the years 1926-27 reports the appointment to the staff of Dr Harold H. Strain. It also contains the first report of the extraction in this laboratory of plant pigments from leaves — the first isolated being carotene. With this carotene, experiments were conducted to see if formaldehyde could be formed by the photo-oxidation of this pigment in a stream of pure oxygen — the basic idea being to test the theory of Ewart. No positive reactions were found to indicate the presence of formaldehyde. ‘In the course of the investigations on photosynthesis in plants it has become clearly evident that more precise knowledge of the cell constituents is a prerequisite to an understanding of the processes concerned and this applies to the pigments of the chloroplast, for although these pigments have been subject to many investigations, little is known of their fundamental physical and chemical properties.’(16) They therefore went ahead and extracted carotene but found they had then to determine such physical constants as molecular weight, melting points, etc.
Another reorganisation was under way for the botanical section, and Stanford University campus was chosen as the site for a new physiological laboratory. But meantime, during 1928, pigment extraction was still proceeding and xanthophyll was isolated from spinach. Chlorophyll was also extracted from spinach and sunflower.(17) Planning for the new laboratory went ahead and this was opened in 1929. Dr Spoehr was appointed chairman of a reorganised group known as the Division of Plant Physiology; and mention was first made of the appointment to the staff of Mr H. W. Milner — who with Dr H. H. Strain will feature prominently in our story later on. The first work to emanate from their new laboratory dealt with further investigation of the physical constants of carotene and xanthophyll.(18) This work was begun because of a need for more information on the role of the yellow pigments. Maybe this was an overhang from Spoehr's earlier work on the effect of blue-violet rays on the supposed photolysis of organic acids — carotene and xanthophyll having absorption spectral peaks in the blue-violet region.
Pigment work dominated the research of this group for quite a number of years, during which time the isomers of carotene were separated and characterised; their molecular weights, absorption spectra, degree of unsaturation had also been accurately determined. Xanthophylls were also investigated, and the wide range of their page 21 variability and distribution gradually became known. The earlier separations were made with column chromatography, for which technique this laboratory became world famous along with the person responsible for the major part of its development — H. H. Strain. The essentials of column chromatography were not new — the technique had been discovered many years previously by a Russian, Tswett, who was the first person to separate some of the leaf pigments including the two chlorophylls. But for reasons unknown — maybe due to a language barrier or maybe because Tswett was a name unknown as a chemist in the international field — the technique he demonstrated never took on until Strain and his associates began to apply it. During this period the photosynthetic pigments of a purple sulphur bacterium were investigated; otherwise all the pigment work up to this point had been done on angiospermic plants.
In 1938 Emerson and Lewis were appointed as research associates. Their task was a critical reinvestigation of the quantum efficiency of photosynthesis,(19) to which reference has already been made. But it is well to point out again that the organism they used was Chlorella, more than likely brought by Emerson from Warburg's laboratory.
In the year 1940-41 Strain and company began the extraction of pigments from blue-green algae, starting with Chroococcus and Aphanizomenon.(20) This latter piece of work must have drawn back the curtains on a whole new horizon of research because it initiated a large investigation of the pigments of algae from all the groups of this diverse section of the plant kingdom. They seemed surprised at the diversity of photosynthetic pigments found in the algae compared with the higher plants. Undoubtedly the realisation was forced upon them that if they wanted to isolate pigments from algae, they just had to work with pure cultures of algae. Consequently we find in the annual report covering 1941-42 that work had begun on the diatom Nitzschia closterium which was grown in pure culture in 10-litre vessels.(21) They also reported the effect of different kinds of light on the variation in the amount of one of the xanthophylls of Nitzschia — diadinoxanthin. At that point in time, little could they have imagined what that last observation would open to them.
The variation in the quantity of diadinoxanthin in Nitzschia in response to the use of ‘neon’ light compared with the ordinary white light led these workers to postulate that products of probable functional importance may be varied a great deal in response to changes in external or environmental conditions. ‘By careful control of external conditions it may become possible to vary at will the chemical products of nature's greatest factory, the green part of plants.’(21)
In the Year Book covering 1942-43 the annual report of the Division of Plant Biology includes a section entitled ‘Biochemistry of Algae’. In it the following statement was made:(22) ‘For page 22 experimental purposes algae offer some striking advantages over land plants. Although the isolation of most algae in pure culture is often associated with many difficulties — there can be made available an almost limitless supply of material which is very favourable for biochemical and physiological investigation… . Some of these organisms can also be subjected to a wide range of experimental conditions, such as temperature, light intensity, and salinity of the culture solutions, without injury.’ They further went on to say that other workers had reported that various environmental factors may influence the pigment concentration in various plants, especially that of chlorophyll; and in the previous Year Book, they had shown how the concentration of diadinoxanthin in Nitzschia varied with the quantity of light. This stimulated Dr Spoehr and Mr Milner to have a look at another alga whose responses to variation in artificial environments in the laboratory were now fairly well known. They found that pigment concentration in Chlorella pyrenoidosa was influenced by light, along with other factors. For instance, both chlorophyll and carotene concentrations per unit dry weight could be varied by a factor of about 25. The extent of these variations had obvious practical implications to them; and they reckoned that more general use might be made of controlled environments for efficient production of other specific substances. They thought that ‘capacity for variation with change in environment may not be confined to pigments’. What a prophetic statement this later turned out to be!
One of the features about photosynthetic pigment analysis is that not much pigment solution is need for characteristation because chromatography and visible light absorption spectroscopy are so sensitive and definitive in the identification of pigments. In algae these pigments would be the most highly coloured compounds whereas fats, carbohydrates and other chemicals of metabolic significance present in amounts much greater than pigments would be colourless for the most part. Thin-layer and gas-liquid chromatographic techniques had not even been thought of in those days; and consequently one had to rely more on ‘bucket chemistry’ and process large amounts of raw material if colourless compounds were to be isolated and characterised. Hence the statement, ‘For the investigations on other components of these plants, larger quantities of material were required, especially because it was desired to determine the influence of certain environmental factors on the production of particular compounds… . Although a number of micro-organisms, including some diatoms, were cultured in large amounts, special attention was given to Chlorella pyrenoidosa, because we had had more experience with this organism than with any of the others and the effects in changes of environmental conditions could be more rapidly worked out with this one organism. Also, the methods of page 23 chemical analysis which were to be applied to these algal investigations could be more satisfactorily tested on this material than on any other which had thus far been cultured.’(22)
And so was born large-scale culture of Chlorella at the Carnegie Institution. The choice of this alga may have resulted from Emerson's earlier association with the Institution while re-examining the quantum efficiency of photosynthesis. The group working on Chlorella grew the alga in 15-litre containers outdoors and in 2-litre containers in the laboratory under artificial light. Both systems used carbon dioxide either in air or in nitrogen. Yields were usually about 2 g/litre fresh weight, although the dry weight could vary from 11 to 39%.
For those not familiar with Chlorella it may be as well to describe this organism.
Chlorella is a microscopic green alga belonging to the large group of algae called Chlorophyceae, and within that group has been assigned to the order Chlorococcales, the family Oocysteceae and sub-family Chlorelloideae.(53)
It has a real grassy-green colour and contains cholorphylls ‘a’ and ‘b’, just like our clovers, grasses and other land plants. Chlorella is an exceedingly common genus. It is found in fresh and salt waters — in fact it was first isolated by Beijerinck from green-coloured fresh water. It has been isolated from waters which vary from very nutrient poor (oligotrophic) water to very rich polluted water (eutrophic); from mineral springs, mucilage of other coccoid algae, from aerial habitats, rocks, tree trunks and soil. It is a common air-borne alga. It can be endozoic in animals (this is why Hydra has a green colour): in fact the first description of Chlorella was given by Brandt in 1881 when he described Zoochlorella. Beijerinck recognised that endozoic Zoochlorellas could live independently of sponges and Hydra and referred these algae to a new genus — Chlorella. ‘A peculiar niche of Chlorella is the sap of trees, often around wounds. From this habitat the only auxotroph, Ch. protothecoides, was isolated, but the sap of trees is not the only environment where this species can grow.(53)
The individual cells are microscopic and solitary — not colonial like many other algae, nor coenobial like other genera among the Chlorococcaceae such as Scenedesmus. In size these cells range from about 5-10 microns in diameter — that is about 0.005-0.01 mm or 0.0002-0.0004 inches. Chlorella has no flagella and therefore is non-motile, although it is reported very occasionally to produce flagellated cells;(51) and whereas other green algae may produce flagellated reproductive cells, Chlorella never does. It does not practise sexual reproduction — only vegetative multiplication by normal cell division to produce 2, 4, 8, 16, 32 (and under very favourable growing conditions, even 64) daughter cells held within the mother cell wall. These daughter cells are kept for a time within the parent cell wall and are referred to as autospores, although they are not page 24 encapsulated in a thick spore wall as are many other algal spore bodies. When the parent wall ruptures and releases the daughter cells, these are able to begin growth immediately. One of the interesting things about Chlorella is that as a result of autospore formation the Whole of the cytoplasm of the mother cell is divided amongst the autospores — only the parent wall remains undistributed. Thus there is no loss of cytoplasm between one generation and another. No asexual reproduction by means of zoospores is known.
The Chlorella cell appears to have a simple structure. The thin cell wall seems to possess an inner layer of cellulose — although there may be some doubt about this. Among the cell contents is according to species a chloroplast of varying shape which houses the photosynthetic pigments and apparatus. Pyrenoids can be readily seen in some species and starch grains can be found. There is a single nucleus, but no flagella nor eyespot. The cell does not have an exterior coating of mucilage around the cell wall. Vacuoles may or may not be present.
As already mentioned, it never practises sexual reproduction, an interesting fact having quite important consequences. When gametes unite a zygote is formed; and in many (if not most) fresh-water algae, this zygote envelopes itself in a hard wall and forms a zygospore. This is a spore body capable of withstanding adverse conditions, and in this state the organism can hibernate for considerable periods of time. The interruption in the growth pattern of an alga induced by sexual reproduction and subsequent zygospore formation can be very considerable. But in the case of Chlorella there is no interruption in the continuity of its growth brought about by zygospore formation. It just grows and divides, grows and divides — for ever and a day.
|Sodium dihydrogen phosphate||621.0||"|
|Disodium hydrogen phosphate||89.0||"|
|Ferrous sulphate as EDTA complex||6.95||"|
Laboratory studies have shown that the optimum temperatures are 25°C. during the day and 15°C, at night. (One strain has been isolated which grows at the phenomenal temperature of 39°C. Apparently this strain is the most efficient photosynthesiser yet found.) For maximum yield, Chlorella requires at least 400 foot candles of unilateral illumination.
|Triolein (a fat)||72.48|
Notice particularly the figures for cellulose (a carbohydrate; and starch would have the same value) and triolein (a fat). Because Spoehr and Milner had so many cultures running at once, they were unable to analyse the harvested alga from each culture for individual components. So to start with, they determined the energy level of page 26 the total organic content of one treatment rather than the amounts of particular constituents. Seeing these constituents were formed as a result of photosynthesis, the R-value was a very appropriate index of the storage material formed — i.e. an overall measure of how far the carbon dioxide had been reduced. High R-values indicated that more highly reduced compounds had been formed — such as fats and hydrocarbons, which would also be high in terms of metabolic energy potential for heterotrophs (such as humans); whereas low R-values indicated that carbohydrates and other less highly reduced forms of carbon low in metabolic energy potential had been formed as a result of photosynthesis.
They first made a survey of leaves of a number of higher plants and found the R-value to fall within a fairly narrow range-from about 30 to 40. When algae such as Chlorella were analysed, they found a greater degree of flexibility in the effect of environment in changing the composition of the alga — so much so that the R-value could vary from 38 to 58, depending on conditions of growth. Among some of the things found were that the highest yields and highest R-values were obtained with 5% carbon dioxide and high light intensities: 10% carbon dioxide under high light conditions gave lower yields and lower R-values. Nitrogen and potassium levels in the nutrient solution also seemed to have a marked effect. No nitrogen at all produced low yields but the highest R-values of 57 to 58; and addition of small quantities of ammonium ion gave maximum yield but a lower R-value of 53. Some of the highest yields accompanied by the highest R-values were got with cultures high in potassium.
As a result of this large-scale culturing it was found by Spoehr, Milner and Hardin — and possibly to their surprise — that ‘the lower plants, for example Chlorella, appear in some respects to be more flexible than the higher plants. The alga can grow under a wide variety of environmental conditions, and thereby undergoes considerable change in composition… . This change is not only quantitative but results in products of different chemical composition.’(22)
During these investigations the group discovered that an antibiotic material could be obtained from Chlorella pyrenoidosa. Fatty acids extracted from the alga initially showed no antibiotic behaviour, but this kind of property developed on exposure to light and air, due — it was thought — to photo-oxidation of some component of the fatty acids. They also found that Chlorella fatty acids contained some highly unsaturatued components. The photo-oxidation of these was reckoned to produce the antibacterial substance.(23) This fatty acid discovery, along with their experiments on the effects of environmental change on over-all chemical composition, more than likely led to the following statement:(23) ‘Considerable theoretical interest attaches to the production of cells with a high R-value, that is, cells containing a relatively large proportion of fats or hydrocarbons. These page 27 investigations were resumed during the present year by Spoehr and Milner, primarily with a view to discovering conditions favourable for the growth of cells having a high R-value.’ Some of these conditions were found, and it was discovered that the composition of Chlorella could be varied at will.
|Protein %||Carbohydrate %||Lipid %|
|Experimental regime A could produce cells containing||…..||58.0||37.5||4.5|
‘It is a rather remarkable phenomenon that the same species of organism should show a variation in composition ranging from 4.5 to 85.6 per cent lipoid, depending upon the conditions under which it is grown.’ … ‘Perhaps the most striking feature of this property is the large percentage of highly reduced carbon compounds, probably in the form of fat, which these organisms are capable of synthesizing.’(23) The figures just quoted were calculated on an ash-free basis. If quoted in terms of total composition of Chlorella they would be slightly lower; but this would have no effect on the over-all picture nor on the conclusions reached.
The Year Book for 1947-48 is most interesting because under the report of the Director of the Division of Plant Biology is a section entitled ‘Chlorella as a Source of Food’ by H. A. Spoehr and Harold W. Milner. It is worth quoting extensively from this section. ‘The growing of food for the earth's population is still in the hands of millions of independent-minded farmers; the plants they raise as crops were brought into cultivation by primitive man thousands of years ago. The increased production necessary to feed the constantly increasing population has been accomplished thus far largely through the introduction of machinery. From many sides serious question is raised whether these methods of food production are adequate to meet the demands of population growth. Food production, being based fundamentally upon the process of photosynthesis, is essentially a biological industry. It would seem to be therefore, a compelling function of biological science to explore all possible means of contributing to the solution of this problem. It would appear more over, that biological science, in order to bridge the gap between discovery and application, may have to make a slightly greater effort toward application until the industries dependent upon it develop to the same point of awareness and acceptance of scientific research the case in the relations between technology and the physical sciences.page 28
‘In so complex a problem as increasing the world's food production, involved as it is in innumerable climatic, nutritional, economic, and social complications, it would be temerarious to advocate a revolutionary change in methods of food production. Nevertheless, every effort must be made to improve existing methods and at the same time to explore all possible means which might supplement older, well established practices. In the course of such exploration it would be almost providential if success were to come rapidly; it is rather to be expected that it can be attained only on the basis of a great deal of patient and painstaking research. Nor will it be advisable to place confidence in any single approach, or method, but instead numerous roads must be followed and any lead that seems promising should be pursued industriously and critically.
‘As a result of investigations conducted in this laboratory on the influence of environment on the chemical composition of plants, it was found that the percentages of fat, protein, and carbohydrate produced by the alga Chlorella can be modified within wide limits. Carbohydrates are relatively plentiful in the World supply; fats and proteins, on the other hand, are in deficit. Through the proper selection of culture conditions Chlorella can be made to produce about 50% of its dry weight as protein, and under other conditions the same organism will produce as high as 75 per cent of its dry weight as, fat. In fairly large scale laboratory experiments such yields have been found to have a high degree of reproducibility and some features of these investigations have been presented in previous reports.’(24)
This appears to be the first announcement in the English-speaking world about the possibility of industrially culturing an alga for food — a most novel method of food production but one necessitated by the spectre of increasing millions of mouths. At this point it might not be out of place to see why such a spectre existed despite the involvement of about three-quarters of the world in a war, and the ravaging of life and property on a scale never seen before in the history of man. Two discoveries just prior to the war and one incident during the war opened a great vista on international health problems because they pointed the way to the control and in some cases the elimination of man's oldest scourges — insect-borne protozoan parasites. Disease and epidemic have always been the greatest controllers of population.
The Swiss firm Geigy had been looking for a chemical which would insect-proof woollen fabrics and carpets against clothes moth and carpet beetle. While conducting research in this field, Dr Herman Mueller in the summer of 1939 found that one chemical under test had a most startling effect on the laboratory flies he used for assessing page 29 the insecticidal power of his chemical candidates. After spraying, these flies became very agitated, flew about in an unco-ordinated and drunken fashion for a time, then fell to the floor where they kicked feebly before dying.
World War II had just started in Europe and once again the possibility loomed large of widespread pestilence and epidemic which up till then had always followed hard on the heels of moving armies and derelict refugees. Having found his chemical to work so well on flies and mosquitoes, Mueller tried it on lice collected from refugees fleeing across the Swiss border from the Nazis. These lice were of the kind known to carry typhus fever. Again the chemical worked; and as a result the Swiss Red Cross sprayed all refugees entering the country. Not long after this the Colorado potato beetle appeared in Switzerland and began attacking the potato crops. Once again, Mueller's miracle chemical came to the rescue and the Colorado beetle disappeared from Switzerland, and Europe was thus freed of a devastating pest.
But these incidents, while revelationary and important, did not produce the same impact as one which occurred later. The big test came in late summer - early autumn of 1943 when the Allied armies on entering Naples found a full-blown typhus epidemic raging. They set up delousing stations and dusted every one of 1,300,000 Neapolitan civilians at the rate of 70,000 a day. In three weeks the epidemic had been stopped and Naples was freed of typhus — the first time in the history of mankind that an epidemic of this kind had ever been arrested — particularly in times of war when conditions were propitious for its upsurge and spread.(97)
Mueller's miracle chemical was DDT. Also about this time Imperial Chemical Industries in England had discovered a new and different insecticidal chemical — the gamma isomer of benzene hexa-chloride, which while not lasting as long as DDT was swifter in its action. In the early post-war years both these insecticides were used extensively on an international scale to control some of the oldest and greatest health scourges — malaria, tsetse fly and other insect-borne protozoan parasites. For the first time in man's history, the migratory locust was having the extent of its depredation severely curtailed. But also in these early post-war years, the standards of earning and therefore living and over-all nutrition had increased greatly along with the newly-found freedom from insect-borne diseases. Populations began to increase; and the world was brought face to face with the prospect of crisis of alarming international implication — population pressure.
Unfortunately man is neither photosynthetic nor nitrogen-fixing; he is utterly and for ever heterotrophic. Since he is not predated upon by any other heterotroph, he occupies the last position in all food-chains. As population numbers increase this position becomes page 30 more perilous in terms of enough to eat since the amount available to him on a per capita basis gets less; and the efficiency of many of the food chains cannot be manipulated at all or can be improved only to a limited extent in order that more food is available at the end for man. The base of all food chains is plant life because only plants through their capacity to photosynthesize can convert physical energy into chemical energy — the currency of the biological world. From the plant level upwards, all levels in any food chain are heterotrophic.
Let us look at two chains which man depends on for protein — those which produce fish and meat.
- Fish: phytoplankton —→ zooplankton —→ small fish —→ large fish —→ man.
- Meat: pasture plants —→ herbivorous animal (ruminant usually) —→ man.
- 10,000 lb of phytoplankton
- —→ 1,000 lb of zooplankton
- —→ 100 lb of small fish
- —→ 10 lb of large fish
- —→ 1 lb of man.
Thus in this chain man acquires 1/10,000 of the usable food value of the original phytoplankton — and the rest is lost in feeding and sustaining the intermediary stages in the chain.(114) On land the picture appears slightly better.
10,000 lb of pasture
- —→ 1,000 lb of ruminant
- —→ 100 lb of man.
However, not every acre of the earth's land surface can support ruminants — in fact very little of the earth's land surface is suitable for this purpose. So on a global-acre basis, the longer fish chain may yield in total more energy than the shorter ruminant chain. But why not go direct from the plant trophic level to man? This was the page 31 theoretical idea behind the industrial culturing of algae. Could theory be converted into practice? Was it possible to manipulate plants using modern factory facilities and technology?
Sparked by Spoehr and Milner's ideas, people began research along similar lines in several other countries. England became interested. Some of her colonies had perennial problems of famine and malnutrition which might be alleviated to a small extent if there was an easy way to provide a cheap form of protein. Work was undertaken by Imperial Chemical Industries at their research station at Jealott's Hill. Soil has never been a plentful commodity in Holland and any method of producing more food is of great interest to the Dutch because of their shortage of farmland. Like England, they were conducting intensive research on ways of improving agricultural production and the use of photosynthetic organisms — no matter how novel — was fair grist to their mill. Much of the Dutch work was carried out by the Solar Energy Research Group located at the Agricultural University, Wageningen. Israel was another interested country. They, unlike Holland, had a lot of soil but little water: consequently, any method which resulted in a greater protein production per unit of water used was of immense interest to them. Japan, too, saw good prospect in this method of food production, With minimal amounts of farmland for raising meat and millions of mouths to feed, she began to investigate the use of Chlorella — especially at the Tokugawa Institute of Biological Research in Tokyo.
It must be pointed out, however, that Spoehr and Milner were not the first to conceive the idea of industrial cultivation of an alga and follow up with experimental work to assess feasibility. We must go back to Germany once again — for Harder and von Witsch had been experimenting at Gottingen since 1939 on the laboratory culture of the diatom Nitzschia palea. A diatom was more than likely chosen because an organism of this kind can produce oil instead of starch as a photosynthetic end-product. It may have become necessary in Germany during the Second World War — as in the First — to exploit the possibility of increasing fat production. Later, due to a study of the literature and because of further investigations, Gummert, Meffert and Stratmann chose Chlorella as a more suitable organism to work with, hoping to capitalise on the ‘biological utilisation of huge quantities of carbon dioxide from waste gases available in the industrial district of the Ruhr’.(61)
At this particular point maybe we should put the questions — why all the stir about growing algae industrially? What are the inherent problems associated with growing our crops in soil? Will the algae be easier to grow? Perhaps it is pertinent therefore to review the page 32 advantages and disadvantages of growing an alga industrially as a source of food compared with growing a conventional crop in soil. Let's take wheat or lucerne (alfalfa) as examples of conventional crops. Before sowing, a suitable seed-bed must be prepared to guarantee a good start for the germinating seed. The land usually has to be ploughed, disced and harrowed to reduce the soil particles to a suitable tilth so that the drilling of seed can be achieved. An over-all dressing of lime may also be necessary to offset soil acidity. The seed is usually drilled at a time when the anticipated weather after seed-sowing will be conducive to good germination. It may also be necessary to dress the seed with a fungicide; or if the crop is leguminous as with lucerne, to be inoculated with a good strain of symbiotic nitrogen-fixing bacteria, or even pelletted. Assuming that climatic conditions have been favourable, germination occurs after seven days or so, the seedlings emerge from the soil and after several weeks begin to branch out across the vacant space between the rows. In the meantime the sun has been radiating down something of the order of 3.5 X 106 kilocalories of energy per acre per day suitable for photosynthesis, but the amount of plant surface to absorb this energy has been minimal — non-existent for a while.
The seed-bed is also an incubator for weed seeds, and in many cases the weeds are away before the crop. If this is the case, a selective weedkiller must be used. The crop in its seedling stage covers only part of the ground: and even in its mature stage covers the ground for only part of the year. This is a waste of sunlight. Weeds can never be eliminated completely and these subsequently compete for soil nutrients as well as light and moisture. With the best of machinery and perfection in application, it is not possible to get the fullest return from fertilisers or nutrients. These may be leached past the root zone by rain or immobilised in the soil by chemical fixation as is particularly the case with phosphate.
Now the insects, the birds, the fungi, bacteria, viruses and other pests begin to take their toll. The climate can also become a problem — even to wiping out the crop. Harvesting, too, has its share of problems. So, all in all, the life of a crop is fraught with considerable hazard from the seedling to the store.
Most civilisations and countries in the world use a cereal or graminaceous seed-bearing plant as the basis of their diet; but only the seed is used — much of the rest is not metabolically available even if it were capable of being eaten. Think of the wastage of photosynthetic and metabolic effort in an acre of wheat in the form of leaves, awns, stems and root-systems! Humans can metabolise only a-linked polysaccharides such as starch; but in a wheat plant the amount of starch may not equal the amount of non-assimilable B-linked polysaccharides like cellulose and other hemi-celluloses. According to Walter,(142) a mere 30% of the dry matter produced by page 33 spring wheat goes into the kernels and serves for nutrition: the remaining 70% goes into roots and leaves which are not available to us nutritionally. One cannot think of any autotrophic crop plant which is eaten in its entirety — roots, leaves and stems, at least not among the Gramineae; and this group contains the greatest starch producers in the world.
In the case of Chlorella there is no need to prepare a seed-bed — no ploughing, discing or harrowing. Once the nutrient solution is prepared and inoculation made, there will be no failures due to climate, pests or other natural phenomena; and grown in closed system, Chlorella remains absolutely pure. Even if grown in open culture, it seems to remain comparatively free of contamination. When a harvest is made, we harvest everything that is grown; and in this regard the return on capital is very high, not only financially but also metabolically because Chlorella has hardly any cellulose and there is no wastage because of useless parts of the plant body. Theoretically, it is almost entirely edible. Being single-celled, Chlorella has no need for structural materials since it floats in its medium. Most of its nutrients are converted into chemicals which in theory can be looked upon as food. As the team at the Carnegie Institution found out, one merely alters the growing medium to get different amounts of protein, carbohydrate and fat. It would be difficult to name any higher plant whose composition when viewed as potential food could be altered so easily or tailored so accurately. Harvesting can be done all the year round since the algae are always at the height of their growing season because they are being harvested at the point of optimum catch.
It would appear that there are fewer problems in the growing of algae than land plants. But can the algae produce more than land plants? Let us examine some figures on land-based production and see whether they favour conventional crops or Chlorella. The following data were assembled by Farrington Daniells.(36)
1 square foot of land in the U.S.A. receives on the average about 1 kilocalorie of solar radiation per minute.
This occurs over about 500 minutes per day.
There are 42,000 square feet per acre.
Therefore he calculates that the amount of heat originating from the sun and falling on one acre per day = 21 X 106 kilocals.
About half the solar radiation reaching the soil is infra-red and therefore of no use in photosynthesis.
Assuming an efficiency in photosynthesis of about 33% (8 photons — see above, but it could be a lower efficiency), approximately 1/2 X 1/3 X (21 X 106) kilocals could be stored by plants, i.e. about 3.5 X 106 kilocals/acre/day under Optimum conditions for growth.
In 1950-51 in the U.S.A. the average corn crop was 33 bushels/acre which is equivalent to less than 2 tons of organic matter/ page 34 acre/year (about 1 ton of kernels and 1 ton of roots, leaves, stalk and cobs).
Under really good conditions with hybrid corn plus fertilisers and good pest control, one could get about 100 bushels/acre: this is equivalent to 5-6 tons/acre/year.
Silage (dry-weight) would give about 2 1/2 tons/acre/year.
Wheat and hay about 1 ton/acre/year.
Aspen forest after 10 years growth, about 2 tons of wood, leaves and twigs/acre/year.
Algae in some lakes, about 2 tons of organic matter/acre/year.
Organic matter such as sugar or wood when burnt in air produces 4 kilocals/gram; this is about 3.5 X 106 kilocals/ton.
3.5 X 106 kilocals/acre/day is a reasonable value for storage of an acre of sunlight as calculated on the basis of experiments with algae under optimum laboratory conditions.
Therefore theoretically 1 ton of organic matter dry weight of algae could be grown/acre/day and this would approximate 3.5 X 106 kilocals/ton.
This high efficiency cannot be even approached, let alone achieved, in agriculture. On an average in the temperate zone, 2 tons per acre is produced in one summer of about 100 days. A yield of this size is about 2% of the theoretical yield under optimum conditions such as can be achieved with laboratory culture of algae. In the field, low carbon dioxide and high light intensity are two of the limitations.
A similar estimate of 2% use of solar radiation was made by Rabinowitch(121) Wassinck also calculated the optimal use of sunshine by agricultural crops in the Netherlands and obtained figures of between 1 and 2%, estimating on a dry weight basis while assuming the average composition of the crop to be represented by the formula CH2O.(145) Beets and mangels were highest, followed by maize — with onions giving a figure of 0.45%. Small culture short-term experiments with Chlorella, assuming quantum yield of 0.10 - 0.12 per mol of oxygen evolved and quanta of 50 kilocals/mol hv had demonstrated a calculated efficiency of about 25%.(145) Wassink and associates conducted experiments for about four days using Chlorella, and in about 30 determinations of efficiency the majority gave values between 12-21%.(146) This group also studied the efficiency of Chlorella growth in mass culture of 300 litres volume under normal day and night regime. In full light the efficiency was 2.6 - 2.7%; in reduced light (22% of incident light), the figure became 6.3%. Mass cultures similarly organised inside but at slightly higher constant temperatures and under continuous light, gave:—
13.3% at 24° C. and a lower light level (with a maximum of 19.7%)page 35
10.9% at 32° C. and a lower light level
4.6% at 32° C. and a higher light level
From their work, these Dutch investigators concluded:
|1.||Semiaseptic Chlorella cultures in large tanks are capable of producing high efficiency (about 15% or more of incident light).|
|2.||Under conditions of natural illumination in summer, the efficiency was found to be 2 to 3%.|
|3.||In both indoor and outdoor mass cultures, lower light intensities give rise to higher efficiency.|
|4.||In proportion to the amount of energy received in 24 hours, the efficiency of the indoor cultures is higher than that of the outdoor ones. This may be due to the more favourable indoor temperatures and to the regular spreading of the energy over the whole 24-hour period. Especially, excessive illumination (far above the limiting range of photosynthesis) around noon may be a factor in the low outdoor efficiency.’|
In parallel with their outdoor Chlorella tests, a plot of grass was also cultivated. This was regularly watered and fertilised with nutrient solution. Three successive harvests over the summer and autumn gave an average efficiency of 2.6% of the incident light (without the infra-red). Beet seedlings were also grown in the laboratory under fluorescent light at 20° C. and successive harvests were made. Efficiency figures of 11-15% were recorded on a dry weight basis and a chemical composition of CH2O. Earlier experiments of colleagues in the same laboratory had given values of 12-19%.
So, the picture for higher plants seems much the same as for algae. Indoor culture under conditions of low illumination results in a much higher efficiency of light energy conversion than outdoors. Excessive illumination seems to reduce efficiency under natural outdoor conditions. Burlew(11) stated that extrapolation of pilot-plant figures indicated that an annual yield of 17.5 tons/acre dry weight did not seem ‘an unreasonable expectation with the present technological knowledge’. Production figures of this kind were one of the carrots that dangled before these folk and spurred them on to fervid investigation.
As we have seen, Spoehr and Milner had by 1948 shown it was possible by altering the composition of the culture medium of Chlorella to grow a crop of this alga to a food-value specification. They envisaged the possibility that Chlorella could be grown as a supplementary source of food. But was this idea feasible from an engineering point of view? Let us call to mind once again the Theory of Optimum Catch and what this must imply. If we want to grow an alga industrially and keep it growing under conditions of optimum harvest, we are committed to do certain things which will greatly affect operational requirements and equipment design. It will have to be a continuous process, and this means that the concentration page 36 of nutrients must be constant. Thus, chemical analysis of the medium must be done continuously so that depleted nutrients can be replaced. All nutrients and environmental factors must be held at optimum to keep the generation time as short as possible. Agitation will be necessary not only to hold the medium and temperature uniform but also to ensure that cells are not allowed to settle, so that they are kept as long as possible under a suitable intensity of light. A carbon dioxide-enriched air mixture must be continuously pumped in and contaminants excluded. Light will have to be available; and harvesting continuous. And while satisfying all the above criteria, construction and particularly maintenance must be as cheap as possible.
Such an investigation was outside the scope of the Carnegie Institution's Department of Plant Biology and needed therefore scrutiny of a different kind. The Carnegie Institution thus enlisted the services of the Research Corporation of New York to organise research on the bio-engineering aspects of the problem. As a result the Research Corporation let a contract to the Stanford Research Institute late in 1948 to culture Chlorella on a pilot plant scale. ‘The basic problem was to discover whether it might be feasible as well as practical to grow large quantities of algae in a controlled system. This problem required detailed answers to several specific questions:
|1.||Is it technically possible to grow algae in large quantities in any controlled system?|
|2.||If so, what type of system should be used?|
|3.||What are the optimum conditions for maximum growth?|
|4.||How valuable is the material produced?|
|5.||What is the cost of producing large amounts of Chlorella?’|
These were the questions set down by Cook who was responsible for the Stanford Research Institute survey.(33)
Continuous culture raised many uncertainties. For this reason, a small pilot plant was set up in a laboratory using artificial light and a similar one was set up in the open for study under sunlight. In these, light, temperature, aeration, total volume and population density were controlled. A sterile medium was quite an achievement because up till now people had mainly used the batch system of growth. Such a method of culturing is tolerably easy compared with the organisation of a sterile continuous system. One could not extrapolate directly the parameters of batch-growing to continuous cultures and many variables had to be explored, such as optimum temperatures, composition of culture solution, rate and composition of aeration systems, optimum population density, artificial light versus sunlight.
Data from this first apparatus gave sufficient information to enable a more practical pilot plant to be set up — keeping in focus the dual considerations of maximum production and minimum cost. The organism used was Chlorella pyrenoidosa. One interesting point was page 37 known before pilot-plant investigations began — that high-protein Chlorella grew more rapidly than high-fat Chlorella. Consequently one aim was to grow Chlorella in a medium capable of producing high-protein as quickly as possible. It was found that under constant culture conditions the growth rate of high-protein Chlorella depended on population density and light intensity.
From the information gained during these studies, Cook set out a tentative design for a large-scale Chlorella continuous culture system. But the results were not conclusive enough to permit the Research Corporation to finance further investigations and the contract between the Research Corporation and Stanford Research Institute was terminated in early 1950. However, certain features of the whole concept of Chlorella mass culture still stoked the fires of imagination in many people and towards the end of 1950 the American Research and Development Corporation of Boston took the Stanford Research Institute project reports for a closer economic assessment. Its review revealed that considerable doubt prevailed about the favourable economics of large-scale culture. There was still a wide knowledge gap with a host of attendant ‘ifs and buts’ between the pilot-scale plant of the Stanford Research Institute and a full-scale plant of industrial proportions. So the Carnegie Corporation of New York joined forces with the Carnegie Institution to provide funds for a firm engaged in consulting and research development to enable them to pursue this project further. This firm was Arthur D. Little Inc. of Cambridge, Massachusetts.
The system chosen by them was completely different from the one used by the Stanford Research Institute, and consisted of growing the alga in a broad, flat, thin-walled tube of polythene four feet wide and a few inches deep. A 160-foot length of tubing held 1200 gallons of culture solution in a layer 2-3 inches deep. An air mixture containing 5% carbon dioxide was circulated over the solution throughout the tube. The culture solution was circulated continuously and part of it was bypassed through a heat exchanger to effect temperature control. A centrifuge was used to harvest the Chlorella. The unit was operated over a period of three months, during which time the yield averaged about a third of an ounce per square yard per day — equivalent to about 16 tons per acre per year. Nearly 100 lb. dry weight of Chlorella were produced using this pilot plant.(106)
The best large-scale yields were about half the estimated potential of 40 tons per acre per year. But even the achieved rate of production of protein and fat was well above that of a conventional crop. The estimated cost of production was 25-30 cents per pound. Milner wrote that ‘if the yield could be doubled and the cost cut in half, algae would immediately become highly competitive with farm produce in a free-economy market’.(106)page 38
The material harvested from this plant was used mainly in investigations of its nutritive properties and its possible applications as an industrial raw material. But what was needed was ‘a demonstration plant having an area of about one acre. Such a plant would be large enough to give operating experience on which the design of a commercial unit could safely be based; and it would provide enough algae for experiments on processing them as food.’ But such a plant never materialised — in fact no further pilot plant experiments seem to have been undertaken on behalf of the Carnegie Instiution. The only other experimental investigation of this kind to attain the record of the printed page was done by Thacker and Babcock of Charles Pfizer and Co. Inc. of Brooklyn, New York.(138) This firm had had considerable experience in the industrial growing of microorganisms for the extraction of chemicals and antibiotics.
|1.||Either the population of the earth must increase to a point such that a new source of foods or feeds is mandatory regardless of cost, or|
|2.||A major research break-through must occur either in equipment design or the discovery of some algal species as yet unknown which exhibits greatly enhanced growth under less stringent conditions, or|
|3.||The algae must be shown to be a source of some extremely valuable materials.’|
This paper of Thacker and Babcock's sounded loud and clear the death knell of the industrial cultivation of Chlorella. But not too long before this work appeared, criticism had come from another quarter and of a different kind. Once again let's have a look at the photosynthetic equation and present it in a slightly different form in order to highlight further aspects of this wondrous reaction which are relevant to an appreciation of the criticism to be discussed.page 39
In its modern version, the summarising equation for photosynthesis is
But it is frequently forgotten that this is a summarising equation of a very complex set of chemical reactions, and as such it cannot help but be a gross understatement. The equation should be represented as:
The absence of any One of these factors arrests the Whole process. If we grow the alga industrially we arrange things so that none of these will be absent; but for commercial and feasibility studies we have to range these nutrients in order of decrease in ready availability and increase in costs. When we do this some limitations become apparent.
Light would be no problem. The ordinary diurnal pattern of day and night does not permit as good utilisation of light energy as does flashing light, but there is still plenty of daylight — too much in a way for highly efficient photosynthesis in the field. A suitable temperature normally creates no problem — rather the converse is the case since it is usually necessary to get rid of excess heat. Water does not create difficulties in most places. But with carbon dioxide we encounter our first problem. The ordinary atmosphere contains 0.03% by volume of carbon dioxide; and it is found that high yields of Chlorella can be obtained only if the carbon dioxide concentration is stepped up to about 5%. Now this represents a 150-fold increase above atmospheric level. In certain areas this presents no problem since gaseous effluent from certain industries is rich in carbon dioxide. This is why the Germans experimented with Chlorella culture in the Ruhr Valley, to capitalise on large quantities of this gas. But where would culture factories in non-industrial areas or countries get their increased carbon dioxide from? It could be fractionated out of ordinary air fairly easily; but it takes money to put carbon dioxide into cylinders! Oil-fired power stations might be one available source. The world is certainly not short of inorganic carbonates. Just consider all the marble and limestone deposits, not to mention the incredible quantities of coral that exist! But again, money is required to release all this ‘unavailable’ carbon dioxide. So here is our first problem — a difficulty in supplying an enriched source of the key compound which is the main one elaborated into complex materials such as fats, page 40 carbohydrates and proteins as a result of photosynthesis. If we have to hitch our industrial production of algae to the heavy industry areas of the world, the freight charges from these locations to the underdeveloped areas where the product is required most will certainly detract from cheap and easy purchase by those in need. We defeat our purpose by having to site the culture factories in highly developed areas — not where the hunger is!
Now let us consider the case of nitrogen. Here we must refer to Hugh Nicol who in 1956 spoke out against the feasibility of the whole scheme as envisaged by the more ardent extrapolators of the laboratory results.(110) Chlorella has to be provided with nitrogen already fixed in some compound or other: it cannot by any known fixation process tap any of the 75% by volume content of nitrogen in the atmosphere. It is necessary to go back to Publication No. 300 of the Carnegie Institution entitled ‘Algal Culture from Laboratory to Pilot Plant’, edited by John S. Burlew.(11) On page 7 of this work Burlew wrote: ‘Looking into the future we may forecast some of the long term effects of the culture of algae. Assuming that half the per capita requirement of 65 grams of protein a day was to be obtained from algae, the total area required for algal culture would be less than a million acres for the present world population. This is such a relatively small area — just a little bigger than the state of Rhode Island — that we may conclude that the introduction of algal culture need not displace existing agricultural crops. Rather, we may consider it a means of utilising land not suited for agriculture. The use of increasing areas of such land for algal culture would be a means of keeping the food supply in balance with a steadily growing population.’
Nicol(110) took this proposition and did some interesting calculations, as follows: ‘Take the world population as 3,000,000,000 (3 X 109): If each person required 65 gms of protein daily, half that figure (to get the amount contributed by algae) = 32.5 gms.
So one man requires 32.5 gms/day of Chlorella protein.
For one year, one man therefore requires (32.5 X 365) gms.
Generally 6.25 gms of protein is considered equivalent to 1 gm of nitrogen.
So 1 man requires 32.5 X 365/6.25 gms of nitrogen per year if supplied as Chlorella.
And 3 X 109 people require 32.5 X 365 X (3 X 109)/6.25 gms of nitrogen per year.page 41
This is equivalent to 32.5 X 365 X (3 X 109)/6.25 X 2204 X 453 metric tons of nitrogen per year = 5.7 X 106 metric tons of nitrogen per year.’
At the time Nicol made these calculations, coal was an essential ingredient in the synthetic production of sulphate of ammonia by the Haber Bosch process. The coal was coked, heated to red heat and steam was then passed over it. The chemical action was that the coke (i.e. carbon) reduced the steam to hydrogen gas, and carbon monoxide was produced by the oxygen combining with the carbon. The hydrogen gas was then combined in the presence of catalysts with nitrogen extracted from the air.
‘In this process it had been determined that 1 ton of nitrogen required 5 tons of coal for ultimate conversion to ammonia.
‘So the amount of nitrogen required by 3 X 109 people is equivalent to (5.7 X 106) X 5 tons of coal per year, = 2.9 X 107 metric tons of coal a year approximately, i.e. 29,000,000 metric tons per year.’
Chlorella is not nitrogen-fixing and must be supplied with its nitrogen all the time. So, we are faced with the following problem. If we want to grow Chlorella for making supplementary protein sufficient to give each person half his daily protein intake, we need to ‘fix’ 5,700,000 metric tons of nitrogen gas Each Year to add to the culture fluid for growing the Chlorella. This would require approximately 29,000,000 metric tons of coal Each Year — a colossal amount of coal to mine and prepare annually just to make the required amount of nitrogenous fertiliser for cultivating Chlorella on the scale envisaged, with no allowances being made for its use in agriculture or other spheres. The assumption is made that the alga uses all the nitrogen provided.
We can extrapolate these figures a little further to accentuate the fatuity of this laboratory test-tube dream. When nitrogen is ‘fixed’ synthetically it comes out of the factory as ammonia or ammonium sulphate — the latter preferably because it is solid and much more convenient to handle.
28 tons of nitrogen are equivalent to 132 tons of ammonium sulphate, So 5.7 X 106 metric tons of nitrogen are equivalent to (5.7 X 106) X 132/28 metric tons of ammonium sulphate = 26.8 X 106 metric tons of ammonium sulphate.
From a production point of view at the time Nicol was writing, the situation was more than a bit farcical since the largest factory in page 42 England could make only about half a million tons of sulphate of ammonia a year; and this was a large factory by world standards. According to the United Nations Statistical Year-book 1954 the world production of all forms of nitrogenous fertiliser for the year 1950/51 amounted to 3,572,000 metric tons of nitrogen, and the world consumption to 3,482,000 metric tons. This left a world surplus of about 90,000 metric tons of nitrogen which would be equivalent to 424,200 metric tons of sulphate of ammonia. Not very much when compared with the required 26.8 X 106 tons! And, of course, the total world production of nitrogenous fertiliser for that year expressed as sulphate of ammonia (16,840,000 metric tons) is also far short of the target! Even if we look at the production figures for 1968/69, there was a world surplus of nitrogen equivalent to only 8,840,000 metric tons of sulphate of ammonia — and the world population had increased considerably since Nicol originally made his calculations: and this surplus would have been due in no small measure to the invention by Imperial Chemical Industries of the steam-reforming naphtha process for making hydrogen.
So the industrial cultivation of Chlorella on the scale envisaged as a source of additional protein seemed to be doomed before it got beyond the pilot-plant scale, due to limitations imposed by the required provision of carbon dioxide and some form of fixed nitrogen. Had Chlorella been able to fix nitrogen itself as some of the blue-green algae can do, the story might have been very different. But there were other rocks also which brought about the shipwreck of this project — the most important of these latter being of a physical nature and not chemical.
The cells of Chlorella range from about 5-10 microns in diameter, i.e. about 0.0002 - 0.0004 inches in diameter. How does one go about harvesting such an organism? In the pilot plants mentioned earlier, one of the features stressed was the necessity for constant agitation and circulation of the liquid in order to get high turbulence and therefore exposure to light — thus achieving as high a photosynthetic rate as possible. This constant movement would militate against agglutination of cells into masses, possibly an unwanted feature from a light-penetration point of view. The cell wall carbohydrates do not seem to have an exterior mucilaginous layer, and this would also tend to keep the cells discrete. So how does one harvest possibly thousands of gallons of Chlorella culture each day? It has been stated that in a large-scale culture unit the algal suspension will be quite dilute — less than 1% of algal cells dry weight.(11) So the harvesting system has to be one capable of handling large volumes of liquid and small amounts of incredibly fine particles of solid matter. Further, the page 43 liquid has to be recovered since it still contains much usable nutrient; what has been used by the growing alga is merely added to this spent solution to make fresh nutrient.
Cells of this size would go through the pores of a coarse filter — indeed it would have to be a very fine filter to cope with this particular size. If a suitable filter could be found, how would one pass such large volumes through it? Pressure filter-presses are expensive to operate and require a lot of man-hours in manipulation. Filter-beds of sand would work as they do in waterworks; but how could the alga be separated from the sand? Maybe the strategy of adsorption could be used. Well, there's charcoal; but this is so expensive, and again the separation of the alga becomes a problem. Aluminium sulphate added to water causes a flocculation of suspended material due to the formation of colloidal aluminium hydroxide acting much like sodium silicate in the refining of wines: and this idea might be used for the adsorption of the algal cells. But how would one get rid of the aluminium after settling?
Even if it were possible to harvest the algal mass successfully and cheaply, the problems do not end there since the alga degrades very quickly if not processed further immediately. This is to be expected really because the harvested mass is wet — unlike the harvest of many terrestrial crops, where the moisture content is reduced naturally to a level at which microbial degradation and endogenous anaerobic respiration do not occur. In such a wet condition, microbial degradation could begin very quickly; or large compacted amounts of wet cells might begin to autolyse because the cells are still respiring whether harvested or not. Conditions in such compactions of harvested cells might quickly become anaerobic and spoilage of this nature could occur. Burlew says that when algal cells are removed from their growing medium by centrifugation they form a thick paste which contains about 75% of water.(11) In this condition the material spoils quickly — in less than an hour in a hot room. In operating experimental plants, investigators froze the material immediately after centrifuging; but this could hardly be envisaged as a factory process in the large-scale culture of Chlorella just for protein, alone. After the cells have been frozen, the harvested material can be kept without spoilage for a long time.
Obviously the material cannot be stored wet except in a frozen state. It could, however, be dried, and Burlew lists three ways whereby this might be accomplished: by freeze-drying, by spray-drying, and by simultaneously defatting and dehydrating with a solvent. He said the material harvested experimentally was freeze-dried; and this process was least likely to degrade more labile constituents such as vitamins. But, again, freeze-drying is an expensive process. It is easy to freeze-dry discrete pellets such as the garden pea (Pisum sativum): but not so easy to do the same with a wet mass of page 44 material. The process of spray-drying offers a much better possibility: and this is a process well understood and practised in industry. Again, the harvested material has to be processed very quickly to prevent spoilage. After spray-drying the material keeps indefinitely. The third way uses a solvent mixture which simultaneously defats and dehydrates the harvested material.(11)
Tamiya mentions centrifugation as the method of separation used in Tokyo.(136) The algal cake is washed with water, then either dried in a vacuum dryer or extracted with alcohol to remove chlorophyll and other soluble substances. The dehydrated algal mass was then ground in a mill to a fine powder.
Centrifugation has been the harvesting method used in all the preliminary experimental work and pilot-plant investigations, and it is an easy matter to centrifuge out the algal cells. This is a satisfactory method on a laboratory and pilot-plant scale but not on an industrial scale. ‘The cost of a very high speed centrifuge increases rapidly with the increased size that is necessary to handle large volumes of liquid. Growing the culture at as high a density as possible will decrease the cost of centrifuging. Even for a dense suspension, it may be economical to use two centrifuges in series. The first one, large enough to handle the whole flow, could operate at a moderate speed to give partial separation. The concentrated material from this centrifuge could then be separated completely by a smaller machine operated at a higher speed.’(11)
Another method suggested was to employ a first stage of gravity sedimentation to achieve partial concentration, followed by centrifugation of this concentrated cell suspension. It was found possible to concentrate the cell suspension about 70 times in one laboratory set-up before drawing off and centrifuging.(37)
By 1957, when Thacker and Babcock published their critical examination of the mass culture of algae, no cheap and satisfactory means of harvesting had been devised. Even these authors wrote: ‘Very little work has yet been done on the recovery of the algal cells from the culture medium. However, the process should be relatively simple, thought not necessarily inexpensive.’
In 1962 an interesting paper appeared where harvesting of algae on a laboratory scale had been achieved by the process of froth flotation. While culturing algae in their laboratory, Levin, Clendenning, Gibor and Bogar had noticed that cultures of algae frothed under continuous aeration and on occasions were seen to produce rings of algae in the culture tubes just above the level of the medium.(93) This observation gave them the idea that it might be possible to harvest algae by froth flotation. They noticed that the algae investigated — which included a high temperature-tolerant strain of Chlorella — produced a natural frothing agent whose optimum action seemed dependent on a fairly low pH, a suitable aeration rate page 45 and several other factors. They found that 88% of the cells in 1200 ml of culture fluid could be harvested in 18 minutes. The major drawback of this method was that the PH of the culture (about 7.5-8.0) had to be adjusted to about 3.0 in preparation for harvesting; and if the cell-free culture liquor was to be used again, the pH had to be readjusted to 6.8. This pH regulation needed sizable quantities of acid and alkali. But again, the introduction of chemicals of this kind set up further complications. The harvested material undergoes degradation if stored at low pH's or if not frozen or dried very soon after harvesting. These authors say, ‘It will, therefore, be necessary to readjust the pH of the harvest. This should be done before final drying and in a manner that will not dilute the harvest with water. A harvest with a packed cell volume of 0.120 and a pH of 3.0 resulting from feed adjustment with HCl would contain 0.073% NaCl after neutralisation with NaOH… . This meant that upon drying, the sodium chloride concentration would be somewhat over 1% — a distinct disadvantage.’ They were hoping to find ways of eliminating this salt level; but failing that they thought the dried harvest could be diluted with some other form of food and so reduce this concentration of salt.
But what about the concentration of salt in the spent medium after readjustment to pH 6.8? How would one get around this? They suggested it might be possible to use nitric acid for acidification and ammonia for readjustment, and in doing so recharge the spent liquor with a nitrogen compound ready for the alga to use. Nitric acid and ammonia are more expensive than hydrochloric acid and caustic soda; but the difference in cost might be justified under these circumstances. This they were intending to investigate.
|1.||the initial concentration or removal of algal cells from culture medium to produce a slurry of 1-2% solids content wet weight — the primary concentration;|
|2.||further removal of water from this slurry to raise the solids content to 10-15% — the secondary concentration (a step they deemed necessary because of the economics involved);|
|3.||final drying to produce a product capable of being stored indefinitely, having a moisture content of about 15%.|
Golueke and Oswald wrote: ‘Technical and economic problems in the initial concentration or removal processes have been a major obstacle to the large-scale production of planktonic algae as a feed-stuff. The difficulties lie largely in the nature of the product, namely, the size, specific gravity, and morphology of the algal cells, their limited concentration, and relatively low market value.page 46
‘A combination of small size (5 to 10μ) and low specific gravity results in a settling rate that is too slow to permit the use of settling as a routine procedure for harvesting algae. The small size of the algal cell also necessitates the use of screens or filters having a pore size within the micropore range. The limited concentrations — 200 mg/l in ponds designed to produce algae for livestock feeding to 2,000 mg/l in ponds operated for the production of algae for human consumption — involve the handling of large volumes of liquid in order to recover a comparatively small amount of product. Because of the probable low market value of the algae, only a limited amount of money can be spent on harvesting if the over-all process is to be economically feasible.
‘A variety of methods that are suitable for the removal of solids in low concentrations from large volumes of water may be applied to the harvesting of algae. Among them are filtration, flotation, centrifugation, precipitation, ion exchange, passage through a charged zone, and ultrasonic vibration. In our studies we tried all of the above methods, but found only two that met the criteria of low cost and reliability.’
These two were chemical precipitation and centrifugation; but they thought the latter would be a borderline operation because of power requirements and capitalisation costs. Chemical precipitation was tried using lime, alum or synthetic organic polyvalent cationic polymers. The idea behind this approach is that some chemicals (e.g. alum) when added to water produce a gelatinous insoluble hydroxide which as it settles entraps the algal cells; but the aluminium hydroxide precipitate has to be removed from the algal cell mass at the end of the operation. The best removing agent found was sulphuric acid; however, its use required washing the harvested algae and centrifuging — all added labour and materials cost. By raising the pH of the algal suspension with lime to about 11, it was possible to produce a gelatinous floc which would settle. Combining the lime treatment with ferrous sulphate brought about a distinct improvement in precipitation; but the advantages were offset by ending up with an algal slurry and a supernatant in which the iron content was objectionable — its concentration too high for a food or feedstuff or for discharge of the supernatant into receiving waters. Another problem was that the addition of iron and alum would precipitate most of the nutrients left in the supernatant which could therefore not be reconstituted as new medium for culture.
It has been found that Chlorella cells carry a negative charge. For this reason cationic organic polyelectrolyte flocculants are the only ones suitable. Several of these were tried and found to be effective in low dosage rates. Again, one presumes the major problem is how to separate the algae from the polymer for if this cannot be done easily, tests would have to be carried out on the residual toxicity page 47 of these polymers in the dried algal mass. In any case, a low rate of recoverability of the polymer might turn out to be expensive since these compounds are synthetic and possibly rather costly.
Secondary concentration as a single operation could be achieved best by using a centrifuge with a solid rotating bowl. To preserve the food quality as much as possible, the algal mass should be exposed to high temperature for the least possible time. The best method found was to spread the slurry as a thin film on the exterior of a drum heated from the inside by steam.
The cheapest way to achieve secondary concentration along with final drying involved using a sandbed and allowing drainage and evaporation to effect dewatering and dehydration. In pilot studies, slurry from stage 1 was applied to a sandbed to a depth of 5 inches and left for 24-48 hours. The particle-size of this sand was specially selected so that the weight of sand adhering to the algal mass after drying would be as small as possible. After 48 hours the dewatered material had a solids content of 7-10% total wet weight with a ‘consistency of soft cream cheese’. It took a further 5-7 days drying to reduce the moisture content to 15-20%. When dried, the algal mass formed chips; and these had to be passed at least twice over screens to remove adhering sand. Such a system worked well in fine weather; but the sandbeds would have to be covered in times of rain.
However, there were some serious drawbacks to this method too. Long exposure to air and sunlight (particularly ultra-violet light) brings about destruction of photolabile compounds such as vitamins as well as possible oxidation of other substances. Maybe there is some destruction of the algal protein. And there is still the problem of some residual sand.
Matthern, in his article ‘The Potential of Algae as Food’, reports on a very interesting possibility relating to the harvesting of algae in solution.(99) He refers to this as ‘biological coagulation’. ‘At one time in our laboratory a mold contaminant grew up overnight in the culture. The mold mycelium were completely covered with algal cells, massed into half inch diameter clusters, and settled to the bottom of the culture vessel leaving a clear liquid. If this or any similar acting micro-organisms can be proven a safe, desirable food, biological coagulation would conserve considerable energy used in centrifuging large volumes of liquid and simplify the entire harvesting process.’
Well, there seems no end to the problems encountered! And every theoretical advancement appears to be nullified by some disadvantage which follows in its wake. So, despite all the feverish research work, the wonderful horizons of applicability, the humanitarian goads page 48 driving many of these research workers, and the fantastic prospects based on what seemed to be simple premises, the industrial culturing of Chlorella never became a viable proposition — never got beyond a couple of pilot plants and a library of literature cloistering published dreams, designs and derelict results. To date, Science has not been successful in bettering Nature in this sphere; and even with advanced technology is unable industrially to improve on Nature's productivity. One does not see nor hear of massive algal factories churning out protein or carbohydrate for the undernourished millions. Once again we witness the phenomenon of the promise of plenty for the impoverished remaining unfulfilled because what looks so easy theoretically becomes surprisingly difficult practically. In any case, the technological environment from which fringe benefits (such as the industrial culturing of algae) can develop is lacking in the areas where dietary protein is so hard to acquire. But this is not the end to industrial algal cultivation. The saga of Chlorella might well have been closed at this stage were it not for a completely new and unrelated field of endeavour which subcutaneously had been festering away but had only now come to the surface.
Many people regard evolution as a biological phenomenon to be taught to or recognised as such by students of botany, zoology and geology only. But this is utterly fallacious. There is evolution in everything that man has done and is still doing. Call it progress, development, change, advancement — call it what you like; but it is still evolution, History is evolution, on a shorter time-base than the biological variety. But what difference does the time-base make? So long as a sequence of activity or interest embodies change — be it for better or for worse — this is evolution! So by briefly reviewing the history of a seemingly-unrelated technical development, let us take some welcome respite from Chlorella for a while before being brought back to it in a completely new context.
The accurate date of the Chinese discovery of gunpowder is a detail of history that is more than likely lost to posterity for ever: it just appeared during the dynasty described as China's most creative age — the T'ang Dynasty, which spawned her most renowned poets and painters along with gunpowder. This was an invention whose impact on society would possibly be felt in all countries for all time. In tune with the prevailing philosophy of the dynasty, the Chinese first allowed it to be used for fireworks only. Not for another 400 years was it realised to have advantages as a weapon of war. Records show that its first military use was in 1161 A.D. when ‘it was formed into hand grenades and employed in war’. But the appearance of gunpowder marks the beginning of a very interesting evolution — that of rockets and rocketry.page 49
From illustrations it is known that the Chinese used rocket-assisted arrows; and when the capital of the Honan Province — Kai-feng fu — was attacked in 1232 by the Mongols, the Chinese defenders used weapons described as arrows of flying fire. They also must have dropped from the city walls something similar to a bomb or grenade. The noise of these was likened to ‘Heaven-shaking thunder’. Apparently the Chinese can also take the credit for being the inventors of rockets in the true sense of the word; and it did not take long after their appearance in the Far East to reach the Far West, for rockets were used by the Tartars in the Battle of Legnica in 1241, by the Arabs in 1249 on the Iberian Peninsula and in 1288 when Valencia was attacked.
Something approximating the first firearm appeared about 1325, consisting of a closed tube partially filled with gunpowder which was used to propel a ball charge. The principle and practicality were amply demonstrated even if the performance left a lot to be desired. Military engineers became interested and began improving and developing thse rudimentary rockets and firearms. The French historian Jean Froissant suggested in 1400 that better aim could be got by firing rockets from tubes. It did not take long for new designs in rockets to appear since many ordnance masters took an active interest in experimentation. Consequently such things as the staged-or step-rocket appeared, as well as the clustered and winged rocket. These experiments and designs were obviously the outcome of considering rockets as weapons of war; but great promise seemed not to be fulfilled and ultimately they were more or less abandoned—mainly because of their unpredictable and disconcerting habit of exploding while being manufactured. By 1500 they were no longer considered useful in land battles. One writer claimed that ‘war rockets persisted longest in industrially backward countries; to make rockets one needed only a workshop, whereas to make cannon and shot required a foundry’.(47) Nevertheless, people were still interested in them; and as guns and cannon were improved, there was some technical spin-off applied to the development of rockets. For instance, a German army officer, Colonel Kristoff Friedrich von Geissler, designed a wooden rocket weighing 132 lb with a charge of 16 lb of gunpowder. But their use in war was very sporadic.
However, interest persisted like a dormant plague. They reached the limelight once again after a series of battles in India towards the end of the eighteenth century when Haider Ali, Prince of Mysore, used them very successfully. His rockets had one great improvement. They were made from metal tubes which could tolerate much higher internal pressures. They also weighed from 6 lb to 12lb and used a 10 ft bamboo stick as a flight stabiliser. Because of these features they had a range of about 1-1 1/2 miles, and although not very accurate in flight were very effective against cavalry. His son, Tipu Sahib, page 50 developed and expanded the use of this form of weaponry. Just before 1800, the British suffered some surprising defeats in India — particularly in battles at Seringpatam in 1792 and 1799. These were viewed with consternation by the British who very quickly investigated this type of weapon. This disturbing news incited a Royal Artillery major — William Congreve — to do a little rocket research for himself. Resulting from this work, he produced a greatly improved design which in no time made its presence felt in quite a number of battles. The greatest improvement he introduced was a new and very safe way to compact the powder charge in the case. In earlier times compaction was achieved by ramming the dry powder into the case, and to do this without entrapping air with the powder would have been impossible. If during compaction the powder was rammed very hard, the air would have been compressed with a resulting rise in temperature — to such an extent that the powder could (and obviously in many cases, did) explode. Congreve got around this problem in a very neat way by simply wetting the powder with alcohol, where-upon the entrapped and compressible air was replaced by an incompressible liquid. From this point, the use of rockets in war took a dramatic turn. They could now be made with a much greater margin of safety and therefore in greater quantities, particularly as Congreve also invented a mechanical method of tamping the powder charge.
Congreve rockets weighed anything from 25 lb to 60 lb and had a firing range of about three miles. At the time they were equivalent in performance and weighed about the same as the charge of a ponderous 10 in mortar; but, of course, they were much more mobile. They looked like giant sky-rockets with a guide stick 12-16 ft long. They had many advantages. Since they were recoilless, their launching was simple—either from a thin copper tube to give initial directional guidance, from a collapsible A-frame if a massed bombardment was wanted, or even from a small boat.
About the middle of the nineteenth century, a second Englishman effected another significant advance in rocket design by putting on the outside of the case curved vanes so placed that the exhaust gases would impinge on them. The rocket now acquired a spin which resulted in a much better flight stabilisation, with great improvement in performance and handling. But from this point on, rockets were fighting a losing battle with artillery which by now was much enhanced in performance and accuracy; the only superiority rockets had was their ease of use in difficult terrain. They were exploited for several non-military uses, such as line-casting in time of shipwreck, and more than likely were the forerunner of whaling harpoons with explosive charges.
Such has been the evolution of the rocket. Now for a few words about the evolution of the propellant. Roger Bacon wrote guardedly about the various formulae of gunpowder in his book Epistola which page 51 appeared about 1248; and from what he said, he must have done a little experimentation on the various amounts of saltpetre, sulphur and charcoal that were necessary to achieve a good gunpowder. About the same time, Albertus Magnus of Germany wrote about the formulae for the powder charge of rockets in his book De mirabilis mundi. Because of the presence of charcoal, the gunpowder was referred to as ‘black powder’. It appears that an early formula required about 75% saltpetre, 15% charcoal and 10% sulphur. In those days no doubt the ingredients were pretty impure, especially the saltpetre. Apparently as time went by the black powder mixture became a little too explosive, possibly because the purity of the saltpetre was getting better due to improved methods of preparation. A later formula required 60% saltpetre, 25% charcoal and 15% sulphur. And this formula was used for centuries—till after the end of the First World War. But there was still one fault with black powder rockets which resulted from Congreve's improvements. The use of alcohol to moisten the black powder before tamping the charge allowed a rocket to be made with a good charge; but alcohol being volatile evaporated from the rocket on storage. As a result, cracks appeared in the powder due to accidental dropping, shaking during transport or changes in temperature. Explosions would occur when such rockets were fired, and they were described as ‘brittle’ because of this feature.
After World War I research was directed to try and overcome this ‘brittleness’ of black powder, and at the same time every endeavour was made to devise a smokeless powder. The latter was successfully formulated and found not to be ‘brittle’ and much less subject to changes in temperature. This new smokeless powder was possible because of two earlier discoveries. The German chemist Schonbein accidentally discovered nitrocellulose (or as it is more commonly called, gun-cotton) in 1845. In 1860 Nobel was producing nitroglycerine commercially. These materials readily found use in firearms, explosives and charges for heavy armament; but 50 more years were to pass before rocketry would be enhanced by their use. Nobel later went on to devise a new formulation called ‘double-base powder’ — a mixture of nitrocellulose and nitroglycerine, hence the name ‘double-base’. This powder was much preferred for the reason that it could produce an exhaust velocity at least twice that of black powder.
Dreaming and scheming are integral parts of man's make-up. One of these dreams has been to travel through space to the moon and further. This more than likely was borne out of and nurtured by an ever-increasing interest in the science of astronomy. A great knowledge of the movements of planetary bodies was known long before the Christian area. But such things as distances between earth and the nearest planet were not known, nor was there any understanding of a lack of atmosphere away from the earth. However, once man had page 52 learnt that these heavenly bodies were real physical bodies, an urge welled up in him to visit them for no other good reason than he was born inquisitive and they were there. In Elizabethan times fantasies existed wherein man was drawn by wild swans or carried aloft on the dew to visit the moon. With the development of the telescope and the realisation of the physical implications of Newton's Laws, the problems of journeying into space became apparent. In Paris in the early nineteenth century, Ruggieri, an Italian rocket maker, sent rats and mice into space by rockets and provided parachutes for their return. One of his intended experiments involved sending a small boy aloft by using a rocket cluster, but the police intervened and the experiment never got off the ground.
That famous book, From the Earth to the Moon, by Jules Verne, was published in 1865. In this he wrote about sending passengers in a space-ship on a flight around the moon, interpreting correctly several aspects of such a journey—for example, weightlessness in free fall. By the late nineteenth century inventors had begun to consider reaction propulsion; and we find all sorts of designs for flying machines powered by steam, or aeroplanes and airships driven by rockets. ‘Many of the concepts were ingenious and prophetic in nature considering that at the time the only mechanical prime movers and sources of controllable kinetic energy were the steam engine and clockwork. Technology had not advanced sufficiently for any of these reaction-powered devices to be practical despite the fact that the principles involved in many cases were sound.’ (47) The only thing lacking was a suitable source of power — but this was soon to come.
In 1881 there was an attempted assassination of the Czar Alexander II of Russia; and for his part in the plot an explosives maker, Nicolai Ivanovitch Kobalchich, was arrested and put into prison. While there he conceived, wrote about and drew plans of a rocket aeroplane powered by a succession of explosions of compressed powder candles. After his execution, these writings and drawings lay in the prison archives till the overthrow of the government in 1917. Another inventor, the German Ganswindt, also thought of the idea of a rocket propulsion system not unlike that of Kobalchich, using steel cartridges to hold the explosive charge. But Ganswindt's mental peregrinations went further: his idea was to provide sufficient speed to attain escape velocity — namely, to leave the earth's gravitational field. It appears that Ganswindt was the first to recognise the rocket as a source of power for space flight.
But a young Russian mathematics teacher from Barovsk published an article in 1895 which set the tone and direction of much subsequent research. K. E. Tsiolkovski was among the first to understand the importance of exhaust velocity and the reason why black powder of any formula would be useless as a power source for rockets. He conceived the use of liquid propellants such as liquid hydrogen and page 53 oxygen, because using these would allow greater efficiencies to be achieved. He also contributed ideas on the design of the space vehicle. Maybe because of a language barrier and a retiring nature, his ideas were not appreciated at the time and he was little known for many years. In any case, he was much too far ahead of his time — a penalty for innovation and advanced thinking. The concept failed to catch on, although the recognition was beginning to spread that rocket propulsion was the key to space flight.
World War I saw very little use of rocket weapons. In the Smithsonian Report for 1919 an American, Goddard, mentioned the idea of using liquid fuels for propulsion but laid no great emphasis on the idea. In 1923 an obscure German mathematics teacher of Roumanian origin, Herman Oberth, published a small pamphlet, ‘The Rocket into Interplanetary Space’, in which he stressed the potentiality of rockets to achieve great velocity. In his opinion, technology had reached the stage where man might see his idea of a space vehicle become a reality within several decades. In a later work, ‘Way to Space Travel’, in 1929, he had not only produced designs for high interplanetary space vehicles using cluster liquid-propellant motors but also hypothesised on electrical propulsion and the ion rocket, thereby anticipating work on electrostatic propulsion by thirty years.
The ideas of rocket-powered flight and even rocket space-flight journeys were now making a transition from the impossible fantasy state to the highly probable, thanks to the publications of Tsiolkovski, Goddard and Oberth. In the period 1927-33 rocket and space flight societies were formed in the U.S.S.R., Germany, Austria, United States and Great Britain. The setting up of these societies helped to focus the interest and attention of the public on experiments that were undertaken. Dreams were great, financial support limited, successes few; and every society had its share of cranks and crackpots. But by and large the aim of these societies was centred more on interplanetary flight than war rockets.
The Germans had been interested in rocketry for some time and had formed the German Rocket Society who, as a group, carried out numerous experiments. This society performed its last experiment on a lake near Berlin in 1933 and soon after disbanded. The German Government carried on with the work begun by this group, and out of this background evolved the German military rocketry which culminated in the V2 rockets used against England in World War II. The Russians were also very early on the scene of rocketry with the formation of the Gas Dynamics Laboratory in Leningrad in 1929. They, too, were conducting research on liquid rocket engines and their early prototypes were fired with toluene and nitrogen tetroxide.
World War II finished and once more man had sufficient undistracted leisure to resume his day-dreaming; but now he could envisage page 54 the realisation of some of his earlier dreams because of a vastly more advanced technology at his disposal—the result of having to fight a war. Now the prospect of flying to the moon was well within the bounds of possibility. Very quickly the wartime rockets were modified to explore the upper atmosphere, and with this newly-acquired knowledge man was casting his sights on extra-terrestrial flight. Of course there were many problems to overcome in an adventure of this kind; and these would have fallen into two main classes—engineering and biological. It is the latter group in which we are interested.
It is well known in mountaineering that the higher you climb the lower the temperature at which water boils; and along with this the colder the atmosphere, the lower the oxygen content of the air, the harder to breathe, the stronger the insolation, and the more unavailable free water becomes because it now freezes. Above about 25,000 feet, climbers have to carry their own oxygen supplies for breathing because the atmosphere is so rarified; not only is the air pressure low but this is also the case with the oxygen concentration. Protective clothing has to be worn and water melted for drinking. If man's environment is so hostile at 25,000 feet, what will it be like at 25,000 miles out in space and at 250,000 miles on the moon's surface? It's not until we try to escape from our environment that we realise how restrictive it is. We get a small appreciation of this insularity when we travel by air at high altitudes. This must be done in a cabin in which the air pressure is artificially maintained at ordinary atmospheric levels. We could not fly at high altitudes if this were not done; and who amongst us does not have some appreciation of what might happen if this pressurised cabin is ruptured by an explosion? A suitable temperature also has to be maintained artificially in these pressurised cabins. Why is there an oxygen mask above the head of every passenger in these planes?
So if man wishes to go to the moon, he has to take his own environment with him. If he intends to spend merely a few days out in space or on the moon, the biological problems — although big — are not so bad; but if he wants to spend weeks or months, then troubles really begin as one would expect when an organism is required to exist in a completely enclosed eco-system. He has to take oxygen — there's none to speak of in space: he has to take water — there's none of that either in space. And then there's food — nothing grows in space. So much for the input requirements of this biological machine called man. Now for the output because, like inanimate machines, man also takes in a high-energy fuel and produces an exhaust of low-energy waste. Unfortunately, he is not a perfect combustion engine like a furnace which burns organic matter to carbon dioxide and an ash of inorganic materials: when he excretes, the excreted matter is not fully broken down. There is water in the form of perspiration and urine, water-soluble chemicals in urine, and generally page 55 insoluble material in the form of faeces. Carbon dioxide is also exhaled. So what does one do with this excreted material — convert space into another toilet for man?
If he hopes to spend long periods in space, man must devise recycling systems in his space ecosystem in the same way as recycling systems occur on earth, for what is Earth but a giant space-ship which, because of its mass and gravitational force resulting from revolution on its own axis has been able to hold to itself an atmosphere of oxygen, water, carbon dioxide and nitrogen, and through biological evolution is now replete with unmanned recycling systems which were going concerns long before man first walked in an upright fashion.
The primary recycling system on earth is the carbon cycle because this is the conveyor belt for energy. The passage of energy is a one-way system but the regeneration of the conveyor is cyclic. Through the agency of the chlorophyll of green plants, seventeen other chemical elements, a suitable temperature, water, and the sun as a source of energy, carbon dioxide is transformed into sugars as a result of photosynthesis. In the process, energy is encapsulated so to speak mainly in the carbohydrate molecule. This is the energy that drives the biological world. Another recycling system is the nitrogen cycle where nitrogenous compounds are converted back to nitrogen gas. By courtesy of the nitrogen-fixing organisms, this nitrogen is re-elaborated into the amino acid glutamic acid which, by an ingenious set of chemical reactions, shuttles its nitrogen into other amino acids and hence into protein. In time, the protein is broken down and its nitrogen released to go back into circulation. Another cycle is the sulphur cycle—and in fact there is a recycling system for every element used in biological chemistry. Now, all this is driven by the sun, the ultimate provider of energy for the whole of our solar system and planets.
If a recycling system could be set up in space along similar lines, then Man could live in space for extended periods. The sun still shines out there; so here is the required source of energy for that inventive and therefore adventurous heterotroph called Man. It's up to his ingenuity to supply the rest which, in this context, refers to the other indispensible ingredients of photosynthesis. There is a source of carbon dioxide present—man's breath, his pulmonary excretory product. There is, of course, water—perspiration and urine, for what is urine but an aqueous solution of various metabolic end-products soluble in water (e.g. urea). There is a source of nitrogen present in the form of urinary urea and faecal material. And the photosynthetic organism to do all the conversion—none other than Chlorella! The use of a recycling system embodying Chlorella can solve some very awkward problems in space such as the elimination of waste products, the conservation of water and the page 56 refurnishing of oxygen as well as the recycling of inorganic elements necessary in human nutrition.
A recycling system such as this seems more than somewhat repugnant to our sensitivities because of the direct use of human metabolic waste products. But we should not lose sight of the fact that man has used human excrement as a fertiliser on the soil for centuries. In the soil the excrement is broken down by heterotrophic organisms before being re-utilised by autotrophs such as soil algae and other green plants. But from the excreta, when freshly applied plants can assimilate whatever available chemicals are present—such as urea. In reality there is little difference between what is proposed in a space ship and what occurs commonly in agriculture. It would be a funny type of agriculture that prevented an animal from casting manure on the pasture! The recycling principle is the same although the practice is greatly condensed in time in the autotrophic reconstitution — as envisaged in a space vehicle.
It could be said that Spoehr and Milner were primarily concerned with the practical demonstration of the concept that Chlorella culture had wonderful potential as a supplementary source of protein and fat, since these were the essentials deficient in so much of the world's diet. This concept immediately focussed attention on several important questions, such as — what would be the nutritional status of Chlorella cultivated now in the glass and stainless steel womb of those horrid hydroponic systems of industry compared with other food nurtured in humus-blessed soil culture? Grown in this fashion, would Chlorella be lacking any of the essential vitamins and other ‘goodness’ —things which people imagine to be lacking in land-based crops dosed with ‘artificial’ out-of-the-bag fertilisers so antigenic to composters the world over? If this Chlorella appeared nutritious, would it be palatable? Was there any possibility of ‘recessive’ toxicities which might appear only when a diet contained large amounts of Chlorella? So it devolved on others with different interests and skills to investigate these and other finer aspects of the importance of Chlorella in nutrition. Man was now very interested in space and his imagination ran riot to the extent that he could envisage orbiting space-stations as well as long-term excursions to the outer planets. Research into the use of Chlorella in space was gaining momentum since not only did this organism offer a means of regenerating a spaceman's environment but at the same time and in the same process if managed properly it could help to meet some of the nutritional needs of man. These would be a little more demanding in space than on earth. Investigations into the many-sided project of putting and keeping a few men in space became more intense while the prospect of using algae to feed the starving and under-nourished page 57 millions on earth was receding in the face of numerous unforeseen difficulties.
- inorganic elements
|Protein (N X 6.25)||55.5%|
|Vitamins micrograms/g Ascorbic acid||14.6|
All living organisms make up their protein molecules from 24 amino acids; but not all living organisms can synthesise threonine, valine, methionine, leucine, isoleucine, lysine, phenylalanine, tryptophane, histidine. Therefore food protein must contain these. What amounts of these are found in Chlorella? Some comparative figures can be found in Table 3.
From this we can see that Chlorella protein is fit for human consumption and in fact is about on a par for essential amino acid levels with soyabean protein. The sulphur-containing methionine is the only one describable as being low; and this is regrettable.
|Amino Acid||Beef Cuts||Soyabean||Chlorella||Egg||Essential Amino Acids|
|Cystine Total Sulphur-containing||.079||.111||trace||.146|
|% element in alga — dry weight|
Of the elements listed, the one most deficient is calcium; but as a human usually gets this so easily from other foods, a low content of calcium is not of great concern. Unfortunately there are no figures given for zinc or manganese—two necessary trace elements.
Because carbohydrates are the most readily acquired form of food, one would not be concerned about the kinds of carbohydrate available from Chlorella. But it has been said that about one-third of the page 59 Chlorella carbohydrate content is available.(89) Among the vitamins and associated compounds are found vitamins A, all members of the B complex, C, E and K, along with choline, biotin, lipoic acid, inositol and p-amino-benzoic acid.
So the over-all picture of Chlorella as a foodstuff appears to be reasonably good except for a deficiency of sodium and iodine. and a low value for calcium and methionine. It seems that the level of vitamin C is low and would have to be augmented. In terms of comparative nutritive value, Chlorella appears higher than brewer's yeast and groundnut meal in protein level, but is less than skimmed milk protein;(55), and the algal protein is thought to compare favourably with gluten as a protein source.(102) The availability of its fat to a higher animal is not known. In an early investigation(76) a Chlorella ‘soup’ was given to patients in a leprosarium in Venezuela over a period of four years. It was found to be both palatable and nutritious and although there was no control group, the patients seemed to have shown signs of improvement.
The next important questions needing investigation were freedom from toxicity, acceptability and palatability. From animal and human feeding trials it appears that there is no problem from toxicity. According to U.S. Army experiments, up to 100gms per day dry weight of alga can be taken without problems. ‘Some concern has however been expressed by medical men on the effect on humans of the continual consumption of high concentrations of chlorophyll.’(98) As mentioned above, patients in a leprosarium in Venezuela were fed amounts of algae up to 35 g per day per man for periods varying between one and three years. No toxic effects were described.(76) In another study it was found that intakes of up to 2380g of a mixture of Chlorella and Scenedesmus consumed over a period of about a month resulted in no toxicity symptoms becoming visible.(118)
But the picture on acceptability and palatability is not nearly as clear cut. It has been said that freshly growing young algae have a mild grass-like flavour.(104) The texture of this material is similar to that of soup but seems unpalatable to most people. Dried Chlorella is described in one instance as having a vegetable-like flavour akin to raw lima beans or pumpkin. Tamiya(136) reported that dried Chlorella ‘has an appearance, flavour and taste similar to those of powdered green tea and powdered seaweed “Aonori” (Enteromorpha compressa) which are relished as drinks and food ingredient, respectively, by Japanese people. This characteristic, however, has both favourable and unfavourable aspects in applying algal powder to various foods. Because it is reminiscent of these pre-existing foods, it can be mixed with them or substituted for them. However, it is not as immediately acceptable when mixed with foods to which tea page 60 or seaweeds are not usually added. Furthermore, since tea and seaweeds ordinarily are taken in rather small quantities at a time, there is a limit in quantity of application of algal powder if used in place of these conventional foods. The intense green color of algal powder is also a factor limiting the quantity of its application as an ingredient of foods. For instance, if noodles are colored almost black by the addition of a large quantity of algae, they will not be easily accepted even if their taste is itself not unagreeable.’
When he visited America, Tamiya conducted palatability trials using two different groups of people—American and Japanese.(136) The dried algal material was added in varying concentrations to bread, noodles, soups and ice cream. From these tests he concluded that foods containing Chlorella appealed more to Japanese than Americans, presumably because the Japanese are ‘more accustomed to foods including seaweeds and other vegetables which are similar to Chlorella not only in appearance, but also in odour and taste. It was strongly realised how difficult an undertaking is the introduction of a new item into human foods. Closely associated with multifarious psychological factors, the gustatory sense is affected not simply by the palatability of substances, but also by the traditional prepossessions of people or of individuals.’ Attempts were made to get around the problem of the intense green colour by treating the algal cells with alcohol. When partially decolourised in this way, the extracted material was found to be milder in odour and taste, and as a result could be added to foodstuffs in higher concentrations than could be used with the non-decolourised material. The Japanese National Institute of Nutrition conducted some experiments on digestibility; and Hayami and Shino had this to say in the institute's annual report for 1958:(63) ‘The majority of the unpalatable smell and taste were removed by the processing, but the remaining smell and taste of the decolorized Chlorella were intolerable and impaired the apetite, when we took considerable amounts of this substance.’ In these experiments 30 gms of decolourised Chlorella were fed each day for nine days to five subjects.
Powell, Nevels and McDowell reported an interesting experiment carried out under the auspices of the United States Army Medical Research and Nutrition Laboratory.(118) Five volunteers embarked on a feeding trial lasting almost two months in which the intake of algal material added to a basal diet started at 10 g per man per day, and was subsequently increased at regular intervals to 20, 50, 100, 200 and finally 500 g. The algal material in this case was a homogeneous mixture of Chlorella and Scenedesmus strains provided by the Japan Nutrition Association. The authors reported that ‘All subjects tolerated diets supplemented with algae in amounts up to 100 gms per man per day. Even at the 10- and 20 gm feeding levels, however, certain symptoms were noted. The algae taste was strong and somewhat disagreeable. It was dominant in all foods and was page 61 compared to bitter spinach or strong green tea. This taste tended to linger several hours. After several days, however, the subjects grew more accustomed to the taste of algae and soon tolerated it quite well. Algae discolored food to which it was added. Chocolate cake, for example, turned olive drab in color… .’ They go on to say that at the 200g feeding level nausea was noted. In their conclusions they thought that daily intakes of 100g can be well tolerated at least for a short time, and that although the taste and odour were moderately unpleasant at first, all volunteers soon grew accustomed to them. In the authors' opinions, the greenish discolouration of foods was not a problem, and the most acceptance preparations containing algal additives were cookies, chocolate cake, gingerbread and cold milk. An intake of more than 100 g a day resulted in gastro-intestinal problems such as nausea, vomiting, abdominal distention, flatulence, lower abdominal cramping pains and bulky hard stools. Finally, they reckoned that heat-treated and dried algae can be tolerated as a food supplement but further processing will be necessary if algae are to be useful as a major food source.
Another interesting experiment concerning the feeding of algae to humans was reported by workers from the University of Nebraska.(34) The main purpose of their investigation was to see if these micro-algae could be used as a principal source of protein in a diet for humans. To do this they analysed the food intake and the excreted waste for nitrogen and were able to produce a nitrogen balance sheet. Their paper reports the results of two experiments. The first dealt with the feeding of Scenedesmus obliquus in biscuit form made from green lyophilised material, and then from ethanol-extracted material; the second concerned feeding Chlorella pyrenoidosa in lyophilised and extracted forms to provide a nitrogen level of 6 and 10 g per day for 10 days. In the first experiment the subjects were able to digest the algal diet of 92.5 g of lyophilised material which provided 7.4 g of nitrogen per day; and they complained about the green colour of the biscuits and the bitter taste of the whole algae. Another of their concerns was algal halitosis ‘which made the subjects some-what less acceptable to their compatriots’. The reaction to the ethanol-extracted material was surprising and contrary to the experience of others in that it was less well tolerated than the unextracted, despite the almost complete removal of the green colour and the bitter taste. All that remained was a faint forage-like flavour. One clear-cut result was that acceptance was better when the algal material was incorporated into one food rather than into numerous items which made up one meal. In the second experiment which involved different subjects from those used in the first, acceptability of the algal additive was much better than in the first experiment, and there were no complaints about a bitter taste.
It was found from this work that a positive nitrogen balance could be achieved and maintained in humans using micro-algae as page 62 the source of protein. A daily intake of 8-10g of algal nitrogen was sufficient to maintain this—meaning that more nitrogen was being retained than excreted. When the nitrogen balance was worked out for each of these experiments it was found that in the first experiment more than 30% of the dietary nitrogen was not absorbed on the way through the intestinal tract; and likewise in the second experiment over 40% was excreted — despite a positive balance for nitrogen having been established. Thus the digestibility of algal nitrogen in experiment 1 was 68% and 59% in experiment 2 when the daily intake was 10g of algal nitrogen per day. These figures were much lower than those quoted by earlier workers; but there could be little doubting such digestibility figures since they had been determined by direct analysis of input and output. Such losses in digestibility required explanation since they represented extreme inefficiency.
Two reasons were considered to account for such losses: inability of the digestive process to disrupt the algal cell wall, and the presence of nitrogen in some form which could not be made available for absorption from the digestive tract. Let us examine the second possibility first. Nitrogen can exist in the cells in many forms: it is found in chlorophyll, DNA and RNA bases, amino sugars and amides—the non-protein nitrogen fraction which can amount to 15-20% of the total nitrogen. The rest is ‘true’ protein nitrogen — the polymerisation product of amino acids. Chlorella pyrenoidosa may contain chlorophyll up to 6.6% of the total weight, depending on how it is grown. DNA and RNA nitrogen would not amount to much; and amino sugars and amides would be expected to be metabolised readily by humans. About 3.7% of the total protein dry weight of this alga is thought to occur in the cell wall as bound protein and probably resistant to proteolytic attack.(34) If allowance is made for this cell wall protein plus some chlorophyll and some unmetabolised purine and pyrimidine bases, there would be an improvement in the digestibility figures; but such adjustments could not account for 30 and 40% of nitrogen not being absorbed. In any case a positive nitrogen balance was obtained; so this eliminates the possibility of there being large amounts of unavailable forms of nitrogen.
These two papers and other reports in the literature refer to a bitterness in harvested and dried Chlorella. This presented quite a problem and considerable attention was devoted to chemical means of removing it. Matthern(98) says that the acceptability of algae as food depends on several factors including removal both of chlorophyll and this bitterness. Chlorophyll can be got rid of by extracting with methanol or ethanol or by ‘solarization of the algae’ (illumination at very high light intensities destroys the chlorophyll) but he states that ‘the undesirable bitter after taste is most easily destroyed by preparing a roux of the algae in butter or oil’. page 63 A chemical removal can be effected by carrying out a Soxhlett extraction with ethanol for one hour. This bitterness was thought to be due to some long chain unsaturated fatty acids associated with the fats found in the alga. A completely white product could be got by extracting the algal mass with methanol first and then treating the residue with 3% hydrogen peroxide. Another way around this bitter after-taste would be to mix the harvested alga with other foods, using the palatability of the latter to mask the alga's peculiar flavour. Whether this has been done is not quite clear from the literature. But various algal food preparations have been made including green noodles, algal date and fruit bars, algal oatmeal cookies and an oatmeal cereal, three different algal soups, and mashed potato with algae. This latter was said to be most acceptable and contained 50% algae (dry weight). An algal-onion soup was found to be very delectable.(99)
In the early literature on Chlorella, one does not find reference to this bitterness, yet Matthern devotes much attention to it. This variance may be a reflection of the use of different strains of Chlorella. Should the industrial culture of algae become a viable proposition, careful attention would have to be paid to their growth characteristics and differences in palatability. This is nothing new: we meet this phenomenon in assessing varietal differences in all manner of our normal edible crops.
If we feed Chlorella to humans, will their digestive processes be able to extract most of the protein held within the cells? These cells are from 5 to 10 microns in diameter. Will they pass straight through the intestinal tract? Will we be excreting much and intercepting little of this protein? If so, the whole exercise is pointless! These questions were partly answered by the research carried out by the University of Nebraska group mentioned earlier, for in trying to explain the high loss through excretion of algal nitrogen they put forward the idea that the digestive process was unable to effect a disruption of the algal cell wall. One can infer from this that algal cells can go through the intestinal tract retaining much of the protein intact within the cells. Hence the high loss. That this can occur was shown by Japanese work with Scenedesmus where electron micrographs showed intact cells in the faeces of humans after feeding trials.(137) The following experiment was performed and demonstrates this point well. Fresh baker's yeast was fed to humans and it was determined that only 52% of the nitrogen in this yeast protein was biologically available. But when the yeast was drum-dried, up to 90% became available.(88) It appeared therefore that the drying was somehow altering the yeast cell wall and making it more permeable to digestive enzymes. This hypothesis was reinforced by further experiments which demonstrated that yeast protein isolated free from cells also showed a good availability.(107)page 64
The same phenomenon has been found to apply in the case of Chlorella. The protein in fresh algal cells is not available to animals. If the cells are oven-dried first, then the protein content could be got at and its nutritional value realised.(103) It had also been found that mechanical disruption of the cells allows a greater utilisation of the protein; and even treatment with methanol leads to increased protein availability because of a partial disruption of the cell wall.(87)
Much subsequent work has been done on disintegration of intact cells and here is a quote on the situation from a Swedish source: (64) ‘Before the protein content of microbial cells can be utilised by the digestive system in higher non-ruminant animals and in man, the cell walls must accordingly be degraded or made more penetrable. This can be achieved by special heat treatment or by enzymatic, chemical or mechanical cell wall degradation. A properly designed heat treatment when drying yeast and microalgae is probably the only processing needed when the cells are to be used for feeding purposes. However, when the cells are to be used in foods the heat treatment might be less suitable. The high temperature could affect the functional properties of the protein and the solubility of cell components in an unfavourable manner. Cell wall degradation by means of enzymes or by mechanical methods could be expected to release a more intact cell material. Chemicals might also be used if the cell content is not exposed to strong chemicals or extreme temperatures. So far, however, enzymatic, mechanical and chemical treatments have been used only on a small scale.’
To investigate in a meaningful way the disintegration or disruption of cells to get at the protein inside, it becomes necessary to look at the cell wall structure. In Chlorella, this consists of a two-phase system in which microfibrils of cellulose are embedded in a continuous matrix of hemicellulosic material. These microfibrils are irregularly interwoven into a continuous network over the cell wall and throughout its thickness. In this network the microfibrils tend to be oriented at right angles to each other. So each cell has quite an envelope around it which must be ruptured before protein can be made available for attack by digestive enzymes.
There seem to be two possibilities of approach if one wants to get at the interior of the Chlorella cell: attack either the cellulose or the hemicellulose. Cellulose is a polymer of glucose with a B1-4 linkage between the units. This is a very difficult linkage to break: not many organisms possess the enzyme cellulase which can attack it. It takes prolonged boiling with strong acid to duplicate chemically the action of this enzyme cellulase.
The hemicellulose fraction of the cell wall on hydrolysis gives rise mainly to galactose, with mannose, arabinose, xylose and rhamnose present in small amounts; but uronic acids cannot be detected in any quantity. So this hemicellulose is of the pentosan-hexosan type and page 65 not the polyuronide type. Hemicelluloses are easily attacked by pectolytic enzymes, and many can dissolve in fairly weak caustic soda.
With this knowledge at hand, let us see how Chlorella cells have been fractured chemically, taking the attack on cellulose first. For several reasons one would not contemplate the use of acid hydrolysis of cellulose by strong acid at 100° C. for several hours. But experiments have been done using the gastric juice of the common snail (Helix pomatia) as a source of cellulase.(111) This works in a laboratory; but can you imagine scaling this up to an industrial level? One could grow on the algal mass an organism which actively excretes a cellulolytic enzyme. This does not appear to have been done. But a cellulolytic enzyme has been extracted from the fungus Trichoderma and used to see what could be achieved.(66) While having some effect on the release of intracellular protein, it was not as effective as rupturing the cell wall. To date, the approach of attacking the cellulose does not seem very feasible.
Now for the attack on hemicellulose! It is well known that urea disrupts those hydrogen bonds of proteins which give them their spatial configuration, and thereby denatures the protein. Urea also disperses the protein in a low molecular weight complex. Mitsuda used this idea of extraction with urea after a preliminary soaking in a solvent mixture of hexane and methanol to remove pigments. It is interesting to see that when he pretreated the algal mass with N/NaOH for five minutes at 100° C. before urea extraction he obtained a protein recovery of about two and a half times the amount achieved with 8M urea on its own. Conceivably, this difference reflects the effects of caustic soda on helping to remove the hemicellulose matrix. Again, one could grow on the algal mass a fungus which excretes hemicellulolytic enzymes. Indeed, this has been done using the common fungus Rhizopus nigricans. By this means, an increased digestibility of the algal cells was obtained. But one would want to know a great deal about Rhizopus before embarking on a treatment like this. The cure might be worse than the disease.
Other physical methods such as freezing and thawing, mechanical disruption by ball-mill grinding or sonication have been tried. Boiling followed by roller drying has also been used. Hedenskog and workers found the best disintegration to be had by passing a suspension of algae through a continuous disintegrator based on the idea of grinding with Ballotini beads and rotating discs.(66) In this specially-designed machine they could process 100 litres of suspension per hour with a cylinder volume of 5 litres and achieve 70-90% disintegration. Using this system and subsequent extraction with urea or phosphate buffers to extract the protein, they could obtain twice as much protein as by extracting undisintegrated material. They seem to think this machine could be used in large-scale production. But the whole process of bursting the cell wall of Chlorella to allow access to its protein seems difficult and—one imagines—expensive when page 66 elevated to an industrial-scale level. What a pity man was not a ruminant! Food would be ever so much cheaper and the range of foods wider!
Having disrupted the cell wall and extracted the protein, one now has to recover the protein. Where caustic soda alone has been used, the method is simple; one merely adjusts the pH to the iso-electric point and all the protein precipitates. Where urea is used, acid precipitation of the protein with trichloroacetic or metaphosphoric acid is enough to bring the protein out of solution and allow recovery of the urea. The Swedish workers looked at the idea of precipitating the protein straight from the distintegrated cell suspension, thus eliminating an extraction step. They achieved a yield of about 80% of amino acid nitrogen using acids, salts and heating as precipitation agents. The end-product is referred to as protein concentrate.(64)
Work has been done on the production of protein concentrates with a low content of nucleic acid. It has been found that the content of RNA in the protein precipitate from disintegrated cells decreases as the precipitation pH increases. For baker's yeast, the RNA content in the protein precipitate is reduced from 10% to 2% when the precipitation pH is increased from 4 to 9. An even better removal of RNA is obtained if sodium chloride is present during precipitation. ‘The RNA content in the protein concentrates from Scenedesmus obliquus is less than 1% compared with 2-3% in the whole cells. No further processing is therefore necessary for the use of such algal concentrates as a food protein.’(65) This interest in RNA removal will be referred to later.
Apparently, methanol breaks the cell wall but does not destroy it; and Matthern referred to the successful use of a Morehouse colloidal mill for fracturing this wall.(98) The Japanese reported a different approach to this problem. By a combination of autolysis and butanol treatment, they were able to extract a cell-free protein which proved as digestible as casein when treated with trypsin. Its biological value was inferior to that of casein because of low content of sulphur-containing amino acids; but its high lysine content could make it an ideal supplement for improving the nutritional value of cereals.(4)
Before leaving this subject of cell wall disintegration and associated problems, we must once more refer to the question of colour. The green colour of Chlorella would not be noticed when the alga is prepared for eating in certain ways. But people react differently to colours in food; and to be universally acceptable it is better to decolourise the algal harvest. Mechanical methods of cell disruption would be of no use in decolourisation: so ultimately chemical treatment such as extraction with an easily recoverable solvent would still have to be used.
One possible technique for simultaneous pigment extraction and drying which does not appear to have been tried extensively is the use of a non-polar solvent or solvent mixture capable of forming an page 67 azeotrope with water. Such a process could be found to have several virtues. One would expect the fat extraction to be more complete than with ethanol: the process should be completed in a much shorter period of time than with a solvent like ethanol; there should be no loss of protein from the algal cell mass using non-polar solvents; ridding the extracted algal mass of solvent would be much quicker with a non-polar mixture or single solvent than with a partially polar one like ethanol. The Viobin solvent rendering process(92) using ethylene dichloride at atmospheric pressure (71.4° C.) or under partial vacua (38° C.) has been in practice for some considerable time. With this process very good simultaneous extraction of fats and water has been achieved from the rendering of inedible meat packing-house materials.
Much of the information just quoted was not available to Spoehr and Milner in 1948. Their enthusiasm for the whole idea was fired and sustained solely (presumably) because of their analytical figures on total protein and total fat content and also by the fact that the culturing of the organism could alter the amounts of end products. Now, we can appreciate how well-founded from a food-value point of view their enthusiasm and optimism was.
So far we have dealt only with Chlorella; and the impression may have been created that this was the only organism capable of being grown in this way, or that no others had been tried. Before discussing this aspect, there are several points which should be brought to the forefront because they are most important and in fact are crucial to the whole project.
Firstly, we must go back to Warburg's uncle who suggested that Chlorella be used for the early experiments on photosynthesis. This has already been referred to; but now we should examine the hidden importance of this incredibly serendipitous choice. As pointed out earlier, Chlorella could well have been chosen solely for the reason that it was available in axenic culture even in those far-off days. Admittedly, this was an eminent reason for its choice. But was Warburg's uncle aware of the fact that Chlorella belongs to a group of algae, the Chlorococcales, which are renowned for their lack of sexual reproduction? Was he aware of the implications of this fact as they applied to this alga when suggested as a tool for physiological experiments? If Chlorella is grown in culture, it just grows and divides vegetatively without any interruption in its growth cycle for sexual reproduction. As pointed out earlier, many freshwater green algae form zygospores after zygote formation; these are resting bodies which can remain dormant for long periods of time. If an alga reproduced sexually and then formed a perennating spore, it would certainly not make a suitable choice for industrial culturing. Obviously, an alga chosen for this purpose must be capable of page 68 growing all the time. And this is a characteristic of Chlorella. So when we muse on Warburg's choice of an experimental organism and how this was ultimately used for investigating industrialisation of algal growth, how unwittingly fortuitous were the Carnegie Institution workers and those that came after them! Had there been no necessity to doubt Warburg's results, and had Emerson not worked at Warburg's laboratory and subsequently at the Carnegie Institution Laboratories at Stanford—thereby coming into contact with Spoehr, Milner and Strain, would Chlorella ever have been used in this investigation? The ‘if's’ of evolution.
|Freshwater Forms Ankistrodesmus sp. (5)||Group Chlorophyta||Order Chlorococcales|
|"||falcatus v. spirilliformis (41)||"||"|
|Chlorella sp. (5)|
|Dactylococcus infusorium (120)||"||"|
|Dictyosphaerium pulchellum (41)||"||"|
|Oocystis naegelii (120)||"||"|
|Tetraedon sp.(5)||"||"page 69|
|ALGA Chlamydomonas sp.(5)||GROUP Chlorophyta||ORDER Volvocales|
|Chlorogonium elongatum (120)||"||"|
|Haematococcus pluvialis (120)||"||"|
|Sphaerella lacustris (120)||"||"|
|Pandorina morum (41)||"||"|
|Volvox tertius (41)||"||"|
|Coccomyxa elongata (120)||"||"|
|Gloeocystis sp. (5)||"||"|
|Uronema gigas (70)||"||"|
|Pleurococcus commutata (120)||"||"|
|Stigeoclonium sp. (70)||"||"|
|Cylindrocystis brebissonii (127)||"||Conjugales|
|Mesotaenium caldariorum (120)||"||"|
|Staurastrum cristatum (127)||"||"|
|Oedogonium sp. (5)||"||Oedogoniales|
|Euglena gracilis (41)||Euglenophyta|
|Mischococcus sphaerocephalus (120)||"|
|Nostoc punctiforme (49)||Cyanophyta|
|Anabaena cylindrica (49)||"|
|Navicula sp.(122)||Bacillariophytapage 70|
|Platymonas subcordiformis (41)||"||"|
|Dunaliella bioculata (41)||"||"|
|Euglena viridis var. maritima (41)||Euglenophyta|
|Porphyridium cruentum (41)||Rhodophyta|
|Phaeodactylum tricornutum (80)||Bacillariophyta (formerly known as Nitzschia closterium f. minutissima)|
|Nitszchia palea (151)||Bacillariophyta|
The brackish water Chaetomorpha linum has also been suggested.(150)
Scenedesmus has also been the subject of a great deal of research; and this organism, like Chlorella, does not practise sexual reproduction. Scenedesmus also belongs to the Chlorococcales. Others from this same group are listed in Table 4.
Other green algae have been experimented with which do not belong to this order of the Chlorophyta. Stichococcus was the subject of investigation by Ketchum and Redfield. This belongs to the order Ulotrichales in which both asexual and sexual reproduction are common methods of cell-number increase. Unlike Chlorella—which is spherical and unicellular, Stichococcus is described as having cylindrical cells in very short filaments which tend to dissociate in moist conditions. Interestingly enough, however, cell division and fragmentation are the only known methods of cell multiplication according to Prescott. Ulothrix and Hormidium, also from this family, were suggested for investigation by Tamiya.(135) Both of these are truly filamentous algae. There were several reasons which prompted this approach. Firstly, they grow on the surface of the culture solution and tend to be buoyed up because of the entrapping of oxygen gas in the mass of filaments. Thus they can be grown in relatively deep ponds, and can also be harvested very easily. Secondly they would be easy to dehydrate by merely squeezing or pressing the algal mass. In laboratory trials the growth rate of Ulothrix was found comparable with that of a fast-growing Chlorella so long as the Ulothrix was maintained in its ‘vegetative’ form; and this could be done almost indefinitely when the culture was illuminated continuously at a constant temperature (25° C.). When subjected to outdoor culture, this alga alternated its growth phase between vegetative and zoospore production; and once the zoosporic condition page 71 was assumed, growth ceased almost immediately and the culture was eventually eliminated and replaced by some other rapidly-growing unicellular green alga. Tamiya wrote: ‘Several attempts were made to keep the alga in its vegetative form under outdoor conditions, but they proved utterly unsuccessful; thus, the idea of using filamentous algae as material for mass culture was tentatively abandoned.’
Interestingly enough it was found by Hindak and Pribil that the biomass production of some filamentous algae is higher than that of Scenedesmus quadricauda.(70) The filamentous forms investigated were Hormidium, Ulothrix, Uronema gigas and Stigeoclonium. They, also found that these algae contained amounts of proteins, lipids, cellulose and ash similar to those found in production strains of Chlorella and Scenedesmus but that the digestibility of proteins in vitro was about a third higher in the filamentous forms compared with Scenedesmus quadricauda. ‘This fact is probably associated with the structure of the cell wall and with the type of reproduction. The filamentous algae reproduce vegetatively by cell division into two daughter cells, in consequence of which their cell wall is composed of lamellae of different ages, some of them originating even in the previous division. This lamellar structure of the cell wall probably makes it possible for digestive enzymes to enter the cell interior.’ In this study the algal mass was harvested, washed with distilled water and dried at 105° C., after which the various chemical analyses were done without further mechanical treatment to break up the cells.
So some of the filamentous algae are superior to Chlorella and Scenedesmus in terms of mass culture in that the former are easier to harvest and the proteins of those investigated are more readily accessible without additional treatment. But whether the advantageous features can be exploited to the full under outdoor conditions of cultivation is a moot point — because undoubtedly the experience of Tamiya already quoted will also be shared by others. If the conditions which induce zoospore formation were known (whether caused by change in temperature, light or some other factor) it might still be possible to culture Ulothrix in certain locations —- e.g. in the tropics or subtropics, where some environmental factor responsible for this change may not fluctuate to the extent seen in temperate areas.
Another member of this same group — Stichococcus — might be worth further investigation. Milner(105) showed that the protein content of Stichococcus bacillaris under certain cultural conditions had reached 59.1%. This was not as high a figure as he had obtained for Chlorella but was still high enough to command attention as a candidate for culturing. Pruess and his team also found under their cultural conditions the protein content of Stichococcus to equal that of one strain of Chlorella vulgaris; and further, that under comparable conditions in tank fermentation culture, Stichococcus produced almost as much biomass as C. vulgaris. As pointed out earlier, increase in cell number in Stichococcus seems to be by fragmentation and cell page 72 division only; and one would expect it to exhibit a constant growth rate under outdoor conditions uninterrupted by excursions into asexual reproduction by zoospores or sexual reproduction. But if one uses Stichococcus the problem of harvesting increases since it is at most quasi-filamentous — unlike other Ulotrichalean genera, and one is faced with the problem of separating what is virtually a unicell although not nearly so small as Chlorella. Maybe, therefore, the various members of the Ulotricales although possessing distinct advantages have for one reason or another to be deleted from the list of possible candidates.
Euglena has also been suggested for industrial culturing; and here again is an unusual organism. Although the subject of continuing argument about whether it is vegetable or animal, Euglena does possess photosynthetic pigment and can use light and carbon dioxide to make its own sugars—just like any respectable plant (but highly aberrant animal). Yet it is not known to practise sexual or asexual reproduction — only binary fission. So there would be no interruption of the life-cycle by the formation of spore bodies — sexual or asexual. It has one drawback — it is not completely autotrophic, but requires the presence of vitamin B12. It is therefore auxotrophic. To offset this disadvantage, however, it is motile — unlike most in the list. It might be possible to capitalise on this property for harvesting, using its phototactic response to light. Apparently this tactic has already been used successfully.(52)
Another peculiarity of Euglena is that it does not have a normal rigid cell wall like Chlorella, and as a result it can alter its shape. This lack of rigidity could mean that its cells are easier to rupture than those of Chlorella for purposes of getting at the cellular protein.
Extensive reference has already been made to Ketchum and Redfield's paper on the culturing of a marine diatom Nitzschia closterium, and also to von Denffer's and later von Witsch and Harder's work with Nitzschia palea. Allen and Nelson in 1907 mentioned their successful growing of Nitzschia closterium forma minutissima. They also stated that over a period of two years' continuous culture it was not possible to detect a diminution in size of the frustule. This is an interesting remark. One of the characteristics of diatom division is that the size of the frustule usually gets smaller and smaller because the hypotheca of one mother cell becomes the epitheca of one of its daughter cells. If in Nitzschia there appears no change in frustule size, it seems that somehow the smaller daughter frustule of this diatom swells to normal size after division (as is known to occur in some) so that a critically small size which can induce sexual reproduction in some is never reached. Maybe Nitzschia does not sexually reproduce easily if at all. Were this the case, the diatom makes a good organism for mass culturing because of the page 73 possible rarity of sexual reproduction and thus the lack of a non-growing phase in a culture cycle. Once again, serendipity may have smiled on a scientist or two!
From what has been written one can see that not all the nails in the coffin of the Chlorella project were chemical: the question of harvesting the alga was a physical one. Looking back over the years and reviewing the evolution of the Chlorella project, one can see all too easily how Chlorella came to be used for the pilot experiments: its choice was a foregone conclusion—a victim of the circumstance that it had been used so much experimentally. But Chlorella proved difficult to harvest; and this was a very bad feature. When failure of the whole project was seen to be due to this factor, it is difficult to see why nobody was prepared to say ‘Let's sacrifice some efficiency or quality and use an organism which is (or should be) much easier to harvest!’ Although as Table 4 shows, many genera and species had been investigated including numerous Chlorococcaleans, it is a great wonder that nobody bothered to look up another Chlorococcalean as a possible candidate—namely Eremosphaera. This organism is reported to be fairly widely distributed and not difficult to isolate: yet it just seems to have been missed by those who have gone out into the biological highways and byways and made isolates from all sorts of places. Here is an organism that shares with Chlorella several of the necessary features for industrial culture and, one imagines, would be very worth while investigating. It grows on a completely inorganic medium, is spherical in shape, and is described ‘as one of the largest green algal cells’.(119) Increase in cell number is by the same method as in Chlorella—by formation of autospores, there being no sexual or asexual reproduction. Under laboratory conditions this is a very easy organism to grow. Cells can attain a diameter of 500 microns or more—about 50 to 100 times the diameter of Chlorella. Of course, there are a lot of ‘unknowns’ about Eremosphaera which would want to be looked at as a prelude to industrial culture. But with the hindsight now available, one feels that people's thought were too heavily infected with Chlorella and Scenedesmus and such things as their high rates of biomass production and crude protein content to allow the idea to seep through that it might be better to accept lower levels of these features and try to find an organism that on a size-basis should be much easier to harvest. There appears little sense in growing an organism with a cost advantage of high production that will ultimately be offset by the cost disadvantage of difficult harvesting. But, as in many situations, it's so easy to be wise after the event!
From a present point of view the first attempts to grow Chlorella would have been under microbiologically sterile conditions, since the pilot and large-scale experiments were really an extrapolation of page 74 normal laboratory sterile methods. Despite difficulties, sterile culture — if feasible economically — has numerous advantages: but it also has disadvantages. If non-sterile open air culture was possible, the whole operation could be cheaper and easier to run on an industrial scale. With this end in sight, several groups of research workers experimented with non-sterile culture of algae.
The first group appears to have been Gummert, Meffert and Stratmann. They experimented with Chlorella both in tanks in a glasshouse and in trenches in open-air systems, and in each case used no artificial illumination or constant temperature. The only concession made was to cool the greenhouse culture tanks during the summer months. The tanks were 82-122 cm long, 82-66 cm wide and 15-21 cm deep; the trenches were 9 m long, 70 cm wide and 20-24 cm deep and were filled to a depth of 9-15 cm. Aeration was such that the concentration was about 1% carbon dioxide by volume. A higher concentration would have been better but an ‘open’ system does not permit such concentrations to be used—a disadvantage when compared with a ‘closed’ system. The strain of Chlorella pyrenoidosa used was one which excelled in its ability to remain in suspension as well as its rate of growth.
Harvesting was conducted according to the ‘Theory of Optimum Catch’. A comparison of growth curves between open-air trenches and greenhouse tanks showed the former to be more productive under the prevailing conditions where the cultures were exposed to sunlight for 10 to 12 hours day. The depth of the culture accounted for the difference in the two systems—the open-air trenches, being shallow, gave the higher yield. The greatest yield of organic matter occurred during the middle to late summer.
One of the big problems, of course, is contamination and nobody could expect a culture to remain pure under open-air conditions. Contaminants included algae and protozoa. In spring they found Chroococcus minutus (a blue-green) and Pseudanabaena catenata in the summer; occasionally Chlamydomonas and Scenedesmus appeared. But the cell count of these was always less than 5% of the Chlorella. Protozoa caused problems, although they appeared troublesome mainly when conditions were unsatisfactory for algal growth—such as prolonged cloudiness. It was found, however, that Scenedesmus showed a greater ‘resistance’ to protozoa than Chlorella.
The Japanese also experimented with outdoor culture in an ‘open circulation system’.(78) Here the basic system was an open shallow pond in which the algal suspension was circulated by means of movable pipes equipped with jets for ejection of fresh culture fluid. The pipes were submerged in the suspension and either rotated or moved back and forth slowly in a horizontal plane. In this way the whole suspension was moved slowly and agitated. This was the earlier version. Scenedesmus was used in warmer seasons and Chlorella in cooler.page 75
The whole project looked promising — so much so that the Japan Nutrition Association became interested, and a pilot plant was assembled to investigate the industrial cultivation of algae further. The culture ponds were 20 metres in diameter with a small tower at the centre. This ‘tower’ had two sprinkling arms extending from the centre to the periphery sited along the diameter of the pond; and these arms were moved in a circular sweep by a motor in the central tower. Solution and carbon dioxide were injected into the pond along the sprinkler arms. In a year's run under Tokyo conditions a yield of 8.6g D.W./sq.metre/day was obtained — which worked out at approximately 13 metric tons/acre/year.
In 1963 the Czechs set up a completely new type of open-air system at Trebon in South Bohemia.(128) Basically, the principle was to allow an algal suspension to flow over an inclined surface having a slope of about 10% and facing south. The first unit explored ways of organising the surface to get aeration as well as illumination. One system had troughs running at right angle to the length of the inclined plain, where the suspension in one trough spilt over into the next trough just below it. In the second, the whole length of the inclined plane consisted of a corrugated surface where the corrugations ran at right angles to the long side of the inclined plane. The corrugations were 12cm from crest to crest. The third system was comprised of a flat inclined plane with 4cm high transverse strip-line baffles 12 cm apart. The organism they mainly used was Scenedesmus quadricauda which, because it has larger and heavier cells than Chlorella, will settle out and can be filtered and centrifuged much more easily. It also was a high temperature strain giving maximal production at 34-35° C.(83)
To overcome alterations of the solution concentration by rainfall or evaporation, adjustment of the total volume of nutrient to a fixed volume was made each morning. This was done either by replacing the evaporated water or by centrifuging the surplus. Extra nutrients were added when adjusting for rain.
Autotrophic contaminants occurred occasionally but never amounted to more than 1% of the total cell number. Chlorococcum and Euglena were found over summer: and a diatom Nitzschia appeared at times. Certain infusoria and colourless flagellates were found throughout the whole of the cultivating season.
A later report describes a larger unit also at Trebon where 900m2 of inclined surface was used. The plane consisted of glass plates mounted on a steel frame with an inclination of 1:30 and again facing south. Now, the surface was arranged so that the algal suspension ran in parallel streams separated by dividing walls. Baffles, mounted at right angles to the direction of flow, caused sufficient aeration and turbulence to keep the algae suspended uniformly. The total volume of liquid flowing was 54,000 litres.page 76
At night the whole of the suspension was held in a tank and aerated with stirring—mainly to conserve the heat content of the suspension as well as to save power. On rainy days, the suspension was also allowed to run to this tank and so eliminate dilution with rainwater.
The algal concentration achieved was 1.5-2-0g dry weight of organic matter/litre: and the average production during a growing season of 150 days was about 10g organic matter D.W./sq.metre/ day. The plant was thought capable of producing 17.5g D.W./ sq.metre/day. This would mean a production of about 2.36 metric tons in 150 days. Apparently a unit with a surface area of 200 hectares (2 X 106 sq. metres or 494 acres) was projected for use in Bulgaria: and over a cultivation period of six months, the harvest was anticipated to be about 6,300 tons of algal mass which is equivalent to about 13.5 metric tons/acre/year. A yield of this level corresponds to the production of a large fodder-yeast factory.
Russian scientists also became interested in open-air cultivation, and began experimental work in 1957 when a large-scale system was set up at the University of Leningrad's Biological Institute. In the Leningrad climate, optimal harvests of about 10-20g D.W./sq.metre/ day were realised. Resulting from this, further open-air systems were set up at the Viliams Institute near Moscow (1961), in Middle Asia near Tashkent (in 1960), and also in Tadjikistan (in 1965). The algae used were not only Chlorella and Scenedesmus but also Ankistrodesmus.(60)
A small plant for commercial production has also been set up in Bulgaria. The organism used is Scenedesmus obliquus with which yields up to 45g/sq.metre have been obtained.
Among the species of Chlorella investigated for industrial culture was Chlorella pyrenoidosa. In fact, Ketchum and Redfield found this species to give the best yield of harvested cells of all the species they grew. But if we read the work of Fott and Novakova dealing with the classification of Chlorella, we find this species is no longer taxonomically valid.(53) So what organism were the earlier workers growing? It is most interesting, therefore, to take a quick look at what was a taxonomic mess and see how and why this had to be resolved. What is the point of growing an organism whose exact identity is not known? How is it possible to duplicate someone else's research if the same organism cannot be obtained?
Around the turn of the century the first investigators of Chlorella were mainly microbiologists such as Beijerinck, Artari, Chodat, Chick and others. In those days microscopes were not as good optically as they are today and observations on morphology and cellular detail of an organism with a diameter of 4-5 microns would not yield a mass of physical characteristics of suitable calibre for critical page 77 differentiation of species on sound taxonomic grounds. In any case, microalgae at that time constituted a kind of biologists' no man's land — people not being too sure whether they were animal or vegetable. Beijerinck and his contemporaries were microbiologists and physiologists and because of their background would not have been over-anxious to define morphologically the organisms they worked with according to accepted taxonomic procedures. Rather, they tended to define their organisms physiologically according to cultural criteria, since this was standard practice in bacterial classification. Consequently they gave very little morphological detail. Species names were assigned to various isolates, but the taxonomy of Chlorella seems to have been a field which most people left well alone. To most — even today — Chlorella looks somewhat nondescript and barren of diagnostic features which could be used as easy but faithful indicators of species.
Yet because of Warburg's experiments and the ensuing controversies, of the widespread use of Chlorella in early work on photo-synthesis and its adoption as a plant physiological tool, of Spoehr and Milner's suggestions about its use as a food and source of protein, and of its suggested role in space travel, its importance was such that its taxonomy clearly needed investigation. Meanwhile people continued to use the alga and possibly thought the amount of microscopic detail insufficient to distinguish between species. The practice arose of using cultures which had been given ‘strain’ numbers or names depending on the person or place associated with their isolation — such as ‘Emerson's strain’ referred to earlier. Over the last forty years since Chlorella has become an indispensible laboratory organism, it has been used primarily by physiologists; and these people would not be interested in (nor more than likely capable of) undertaking a rigorous taxonomic investigation of the genus. But it is still imperative to have trustworthy taxonomy which will tell us as accurately as possible what we are working with. No physiological work can be of great value unless the identity of the experimental organism is known to the best of our ability — as well as where it fits into the scheme of things. Despite what many physiologists and others think, taxonomy is not like the dodo. Man was born a classifying animal and as long as he remains this way, taxonomy will never become a botanical redundancy heading unwittingly along the path to extinction: it is still and will always remain an indispensible skill and discipline. Its ramifications are legion.
The problems associated with Chlorella taxonomy have been discussed by Fott and Novakova as follows:—
‘The names of species, used to designate the various strains of Chlorella in culture collections, were made up not on the basis of a careful study of the species as described by classical authors, but quite randomly by the isolator, who was unconcerned with the taxonomy of the isolated alga. These names are designations of strains rather page 78 than correct names arrived at after thorough morphological study. A good example of this is Chlorella pyrenoidosa Chick sensu auctores, which is an assemblage of various Chlorella species. Sometimes Chlorellas with large oil droplets simulating the pyrenoid are placed in this species!
‘Unfortunately, the morphological and structural details of Chlorella cells are not easily discernible as they are at the limit of resolution of the light microscope. Moreover, physiologists are not trained to notice small morphological features, and are usually occupied with experiments and with the maintenance of cultures. Up to now about 50 taxa of Chlorella have been described but only a few of them satisfy the principal rules of the Botanical Code. Before the issue of the monograph of Shihira et Krauss (1965), only four species could be recognised with certainty; they were the only species to be provided with a good description, iconotype and type culture. These are: Chlorella vulgaris Beijerinck, Ch. protothecoides Kruger, Ch. saccharophila (Kruger) Migula and Ch. zofingiensis Donz. Among the other species which are maintained in culture collections, some could be identified by existing descriptions. By means of these cultures, we have compiled new diagnoses and fixed the lectotypes, either from the original description or from our drawings. Those species of Chlorella that have been described solely on the basis of their behaviour in various media (sometimes unnatural) without reference to morphological descriptions and drawings, are nomine confusa and, if no description exists, it is not possible to recognise and identify them.
‘The physiological and biochemical responses to different growth conditions can yield, of course, results that are available for taxonomy. But, experiments by Kruger, Artari, Chodat and his students to study the different growth of Chlorella species on various sugars and to classify the ability to use different nitrogen sources, showed that these criteria are unreliable, quite apart from the difficulty of repeating the experiments. The results are quantitatively uncertain, as the distinction between good and bad growth is rather vague.
‘Shihira et Krauss (1965) attempted a comprehensive treatment of the genus, based primarily on physiological characters, and distinguished species by the responses, to various sugars, nitrogen sources and other compounds. They demonstrated a great diversity of nutritional requirements, recognising 20 species and 8 varieties from 41 isolates. From these 28 taxa, three-quarters (22) were defined by the observation of a single strain. In principle, growth responses could provide good taxonomical criteria if they are quantitatively clearly different. For example, a species may be distinguished on the basis that it requires thiamin and does not grow in its absence. But the majority of characteristics used for species identification in Shihira et Krauss's monograph are expressed by degree and not by presence or absence. Thus, growth of Chlorella vulgaris Beijerinck page 79 is “always promoted by glucose”. Growth of Chlorella mutabilis Shihira et Krauss on the other hand, is sometimes promoted by glucose, but sometimes inhibited. It seems clear that, under these circumstances, growth responses with glucose cannot be used as decisive criteria to distinguish the species (Shihira et Krauss, 1.c.p.57).
‘Growth characteristics can only be used in taxonomy if there is a qualitatively clear response to the experiment: the culture either grows or does not grow. The fact that glucose supports good growth or supports growth only very slightly cannot be used for distinguishing taxa in the range of species (1.c.p.58, the difference between Ch. infusionum and Ch. simplex). Shihira and Krauss' observations on Chlorella physiology and growth requirements provide important evidence of the physiological variability within the genus Chlorella. These properties are mostly (in three-quarters of the strains examined) related to a single strain. If these physiological abilities could be studied in more strains, they could define taxonomic units in the range of form.
‘Some of the physiological characteristics observed by Shihira et Krauss (and by Kessler et al.) are combined with morphological and structural features; they can, therefore, be used to distinguish the higher taxa, species and varieties. Thus Ch. fusca Shihira et Krauss is characterised not only by its morphology, structure of the protoplast, reproduction, etc. Ch. vulgaris var. autotrophica (Shihira et Krauss) comb. nova is distinguished by its obligate autotrophy as well as by the morphology and structure of the cell.
‘Kessler and his co-workers separated Chlorella strains according to their biochemistry. They examined the hydrogenase activity in single strains, the production of secondary carotenoids and the resistance to low pH (Kessler 1965, Kessler et al. 1962, 1963), while also observing the habit of cells (Soeder 1963). On the basis of biochemical and physiological properties, 9 groups of Chlorella strains can be discerned (Kessler in a letter 1966, Kessler 1967), of which 7 coincide exactly with our taxonomic units; of the remaining 2 species, 1 is the heterotrophic Ch. protothecoides Kruger and the other is Chlorella IV, which in our opinion does not belong to the genus Chlorella. Biochemical and physiological investigations of Chlorella indicate that, in the majority of cases, one quality does not characterise a morphologically-defined species but may occur in a number of species on the other hand, various morphologically-defined species can include infraspecific units (varieties, forms) exhibiting the same physiological and biochemical properties.
‘In consideration of all these facts and peculiarities of Chlorella, we were obliged to use a mode of investigation slightly different from that commonly used for higher plants or algae. The main taxonomical rule used is expressed in our motto and is cited from the physiological work of Artari (1906, p. 189) on Chlorella: “The systematics of plants is based on their morphological characteristics.” During the page 80 study of Chlorella taxonomy we became convinced that morphological attributes, e.g. shape and size of the cell, structure of the protoplast, reproduction and release of autospores, yield enough characteristics for description and differentiation of species.'
For most work, three species of Chlorella have been used — C. pyrenoidosa, C. vulgaris and C. ellipsoidea; although many people have referred to their Chlorella as a culture number from some collection. From the taxonomic study by Fott and Novakova it appears that the name C. pyrenoidosa is no longer applicable. Depending on the collection from which the isolate was taken, this species is synonymous with C. vulgaris; C. vulgaris var. vulgaris; C. Kessleri; C. fusca var. fusca or var. vacuolata; C. homosphaera, or C. minutissima. C. vulgaris and C. ellipsoidea still stand as species, although various isolates with these names in culture collections have had to be assigned to other species.
After all these years of physiological and biochemical research on Chlorella, it is more than somewhat shattering to read Fott and Novakova's paper and be made to realise that much of the earlier work has been done with cultures of unknown or uncertain taxonomic identity and is therefore of doubtful validity. How much of this work can be compared with any degree of certainty? Was a single species cited by two or more investigators (e.g. Chlorella pyrenoidosa) in fact the same species? How much earlier controversy or apparent contradiction of results was without foundation because the taxonomy of the day was not definitive enough? Trying to unify the results of this research is like tilting at windmills because the earlier classification of Chlorella was not accurate to the extent that species could be easily separated and recognised. Where isolates have been kept identification — although late — can still be applied, and the research using such cultures is still valuable. But if cultures have not been kept, the published research has lost a lot of its value.
Throughout this article so far one has mentioned solely the autotrophic ability of Chlorella and other microalgal contestants for industrialisation as if no alternatives existed for growing these organisms. Much work has been done on the heterotrophic growth of algae, and many will happily proliferate in the absence of light providing there is a suitable source of reduced carbon present. In fact, a number of green algae will grow heterotrophically; but of those that do, most seem to belong to the Chlorococcales. Included amongst these versatile élite are Chlorella and its colourless counterpart Prototheca, Scenedesmus, Ankistrodesmus, Chlorococcum, Dictyosphaerium and Pediastrum.(152) It must not be imagined that the apparent physiological contradiction of heterotrophy in a thorough-going autotroph is some newly-discovered piece of information.page 81
Even in 1898 Beijerinck knew that Chlorella could be grown in the dark provided glucose was used as its source of reduced carbon. Scenedesmus quadricauda can also readily metabolise glucose.
If cheap sources of available sugars can be found, why not use these as a source of energy rather than rely on the ability of the algae to fix carbon dioxide? This is what the Japanese have done: they grow Chlorella heterotrophically on a medium containing glucose. Obviously this has advantages: no problems about light penetration in a medium, no problems about getting rid of extra heat from sunlight, a much simpler industrial set-up is then required. But there seems little point in growing an alga heterotrophically. Why not use a yeast instead? There would have to be some special chemical component in the alga to justify growing it heterotrophically.
Vincent has this to say on this aspect.(141) ‘Advocates of algae as a source of cheap protein have held up photosynthesis as a prime advantage of algae. In fact, although the carbon is provided “free”, a high price will have to be paid to perform the cultivation in such a way as to let in light and carbon dioxide. A high price may have to be paid for supplementary carbon dioxide and for agitation machinery if the highest yields are thought to be necessary. To some extent, a vicious circle may be created in which the high growth rates are necessary in order to justify the expensive plant which makes them possible. In terms of the cost of carbon used by the culture, it may be cheaper to grow yeast with its higher specific growth rate and to pay for the substrate used than to invest the money in special equipment for algal cultivation. This is certainly indicated as a harsh commercial reality in Japan where the major producer of algae (as a source of vitamins, growth factors for lactobacilli, and green colouring matter for fermented drink) uses heterotrophic cultivation on a glucose-containing medium rather than photosynthetic growth (Enebo, 1969). From my personal experience it is relatively simple to grow cultures containing 30-40g dry weight/1 in heterotrophic growth of Chlorella pyrenoidosa strain 7-11-05, and the problems are the normal ones such as maintenance of sterility and gas transfer. To provide protein, however, yeast would be a more attractive proposition under such conditions because of its higher growth rate. Thus in general it may be concluded that the ability to fix carbon dioxide may not qualify algae as the thriftiest source of single-cell protein. To claim carbon dioxide fixation as an advantage only makes economic sense if there is in fact a shortage of fixed carbon. On a World scale we have molasses, starch, cellulose and hemicellulose, sulphite liquor, whey and various vegetable wastes which are not even completely used at the moment, not to mention fossil fuels.
‘Is there then a need for more fixed carbon? The answer lies in the distribution, evenness of supply and ready fermentability of these sources of carbon, coupled again with the technical sophistication and investment necessary to exploit them. The geographical distribution page 82 of fresh and fossil fermentable materials is not ideal. The World is divided by political barriers and, in any case, transport is prohibitively expensive. It may be that to use local mineral resources to the full we have to construct new food-yielding ecosystems with the most efficient carbon fixer available plus a nitrogen-fixation step integrated into the system (although the nitrogen-fixation step might be chemical). Locally, the use of fossil fuels plus inorganic nitrogen to produce yeasts or bacteria might be the best solution. Paradoxically this seems to be happening where the food supplies are already good, simply because of the relative abundance of investment capital and the advanced technology available. Elsewhere, with a surplus of fermentable carbohydrate material, a microbial upgrading such as suggested for cassava might be ideal.
‘Evidently local conditions define the optimum solution. Where there is a deficiency of both fossil and fermentable materials—and many areas of the World suffer from a calorie as well as from a protein deficiency — the carbon-fixation step is the limiting one in local food supplies. Given the fixed carbon, many solutions are possible. We must thus look to the most efficient carbon fixer to give a better all-round food supply. This naturally leads us to consider not only algae but higher plants, the basis of conventional farming, which in fact have a huge development potential in terms of possible increases in yield.'
More recent Continental work on growing of micro-algae has centred on the use of Scenedesmus quadricauda. The European plants are out in the open and therefore subject to aerial contamination. Yet Simmer states that ‘autotrophic contaminants occurred only occasionally and never exceeded 1% of all cells in the suspension’. But the development of bacteria seemed to remain at such levels as not to influence unfavourably the productivity of the algal biomass. ‘Provided the cultures were in a good physiological state and no unfavourable cultivation factors appeared (e.g. persistent rainfall), the suspension grew without a remarkable bacterial pollution and no conspicuous development of autotrophic or heterotrophic contaminants was observed.’
This lack of contamination is interesting. Gummert and his co-workers(61) reported that Scenedesmus cultures showed a greater resistance to contamination by protozoa than did Chlorella. Maybe an antibiotic like chlorellin reported on earlier by Spoehr and Milner could be responsible? But it has been shown that the production of chlorellin in species other than C. vulgaris is absent or occurs only under very high density population conditions. Chlorellin showed antibiotic activity against Staphylococcus aureus but was not stated to be effective against protozoa and other bacteria.
Protozoa and bacteria are heterotrophic in nutrition and require already-elaborated carbon compounds. Such have been found in the growing medium of at least one species of Chlorella. Maksimova(96) page 83 showed that in the case of C. pyrenoidosa not only were such compounds released during growth but also during the release of autospores from the parent cell. Among the compounds found were free amino and keto-acids as well as various polysaccharides. Smith and his team(129) found that C. pyrenoidosa TX7-11-05 in axenic culture excreted numerous organic acids (including gluconic and galacturonic acids), eighteen amino acids, polysaccharides and short-chain peptides. Is it any wonder then that contaminant bacteria although heterotrophic can proliferate in the growing medium of Chlorella—assuming other species to behave similarly to C. pyrenoidosa. If much organic matter were excreted this could support a lot of contamination by heterotrophs: but if little were excreted, then the level of contamination could be very low and so prevent the build-up of a large heterotrophic contamination level. In this way a low organic-matter excretion rate could produce a better result than the excretion of an antibiotic, since the latter would undoubtedly be selective and the former broad-spectrum — which is what is wanted. Maybe Scenedesmus excretes less organic matter into its medium than Chlorella! Maybe this is why Scenedesmus can be grown more easily outside! An interesting point emerges where this the answer, since anything excreted into the medium is photosynthate lost, i.e. is lost production. The more that is retained within the cell, the more efficient is the organism as a food-synthesiser and the greater the crop yield. One would like to see excretion loss' figures for the algae used industrially. There seems no mention of the production of an antibiotic substance by Scenedesmus.
Earlier it was mentioned that Chlorella does not practise sexual reproduction or asexual reproduction by zoospores — only vegetative multiplication — and how this was an important feature in any candidate for industrial cultivation since there are no spore-forming stages to interrupt the growth. One other consequence of its asexual proclivity is that there is a remarkable uniformity in all cultures of any one clone even after thousands of divisions. Because there is no sexual reproduction, there is no meiosis and therefore no recombination of genes — only the odd mutation brought about by chromosome deletion, inversion, translocation or maybe point mutation during mitosis. Thus there is no multiplicity of genotypes within any one species of Chlorella. Each species is really a single genotype — a feature uncommon in the world of living organisms but characteristic of the order Chlorococcales to which Chlorella belongs. So one imagines the rate of evolution to be very much slower in Chlorella than in Chlamydomonas. Once established, a culture of Chlorella just goes on growing uninterrupted by the formation of either sexual or asexual resting spores. It thus maintains a uniformity of reaction page 84 to its environment — a feature found in few freshwater algae. This genetic stability has its drawbacks too, because it is not an easy matter to sort our useful variants which might possess features of greater value in intensive culturing than those of the parent. But seeing we are always trying to improve on Nature's productivity, it is only natural that either we select strains for different growing or other characteristics or try deliberately to induce mutation rather than rely on selection from wild populations or atypical habitats. Both of these approaches have been looked at.
Concerning strain-selection, Tamiya had this to say:(135) ‘The selection of suitable algal strains is as important a matter as the design of the culture unit. Thus far, the search has been mostly for fast-growing, hardy, algae, although other features, such as the chemical composition and nutritive value, are also important. The choice of algal strains may influence the choice of culture conditions as much as the culture conditions influence the choice of the algae. Other conditions being equal, the desiderata for an algal strain are (a) that it be resistant to contamination by other organisms, (b) that the tendency toward frothing or excretion of organic matter from the cells during growth be as little as possible, and (c) that the cells be neither so large as to precipitate easily during culture nor so small as to cause difficulties in the process of separation from the culture medium. In most of the outdoor cultures strains of Chlorella and Scenedesmus have been used because many of them fulfil, although not always satsifactorily, the above-mentioned conditions.’
We mentioned previously that the optimum growing temperature for Chlorella is 25°C. and that one of the problems in industrial culture was the necessity to cool the growing chamber to keep somewhere near this optimum. Obviously, if strains could be found with higher temperature optima this would help to make things a little easier. The first to be successful in this direction were Sorokin and Myers, who obtained inocula from ‘warm local surface waters’.(131) From these they managed to isolate ultimately a strain (TX 7-11-05) with a growth optimum temperature of 39° C., whereas C. pyrenoidosa Emerson's Strain had its optimum at 25-26° C. They said in their paper that from manometric studies of TX 7-11-05 this strain seemed to have the ‘highest rate of photosynthesis per unit quantity of cell material of any organism so far observed’. Other studies along similar lines were done in which temperature-tolerant strains were isolated from various soils and waters around Tokyo.(126) It was found that the thermophilic strains grew well in summer but not in winter, while mesophilic strains of ordinary temperature tolerance grew in all months, but less in summer.
Kok(82) managed to get some strains of Chlorella to adapt to higher temperatures but he could not get these to adapt to high light intensity. He also managed to isolate a strain of Scenedesmus which grew well at 20° C., and also at 45° C.page 85
Komarek and Ruzicka(83) used a strain of Scenedesmus quadricauda which was able to produce its maximum dry weight production at 34-35° C.
As we have seen, the most troublesome problem encountered in this whole project was the inability to separate easily and in large quantities the cells from the nutrient solution — the problem of harvesting; the cells of Chlorella are so small. Kihlberg wrote: ‘Micro-organisms can be easily modified genetically. Mutations, induced by chemical or physical means and followed by large-scale screening programs, will give variants with required qualities, e.g. faster growth rate or the ability to grow at high temperatures or a deficient cell wall integrity.’(81) One of the ‘required qualities’ that could be selected for would be an increase in cell size. Genetic principles and practice have been applied to try and get around this problem of the size of Chlorella cells; and the more interesting of these will now be briefly reviewed.
In his paper given at the 1959 UNESCO Symposium on Algology in New Delhi, Mayer suggested that it might be possible to find a species or variety of Chlorella with a slightly increased size.(101) He and an associate had found, for instance, that ‘coumarin at certain concentrations increases the average diameter of Chlorella cells, but it is uncertain whether this would be practicable under large scale mass culture conditions’. Rather than rely on adding coumarin, von Witsch commented after Mayer's paper that it might be better to try and rear polyploid Chlorella. So let us investigate these two aspects and see what has been found.
Pearsall mentioned that he had been able by treatment with camphor to obtain a mutant of Chlorella vulgaris.(113) This mutant had double the cell size of its parent strain but the same generation time, which meant that the mutant could produce twice the dry weight in the same period of time. This observation does not seem to have been pursued further and maybe it was found to be impracticable. Exploration of a phenomenon discovered by a Japanese might have held promise. Higashiyama found that growing Chlorella on glucose, fructose, galactose, mannose, xylose and arabinose produced ‘gigantism’ in the cells;(68) and further, that cell division was stimulated by glucose and galactose. However the amounts of protein nitrogen and dry matter per unit volume of giant Chlorella were much less than those of control cells. So this idea would lead nowhere.
Rather than rely on chemicals in the medium to produce an increase in cell size, one could use mutagenic agents in the hope that a mutant might appear which had all the known desirable cultural features along with an increased cell size. Many people have induced mutations of various kinds in Chlorella. Some have used radiation, X-rays and ultra-violet; and there have been pigment mutants produced by treatment with streptomycin. But it seems that none of page 86 the mutants investigated had enhanced capabilities of growth; many in fact could not be investigated further since the pigment systems seemed to be the most easily affected metabolic areas. Tugarinov said in relation to streptomycin-produced mutants that they were all without exception characterised by the entire absence or by a very low content of chlorophylls ‘a’ and ‘b’.(139) Scenedesmus obliquus has been treated with N-nitrosomethylurea; and this has produced lethal mutants or induced auxotrophic mutants of various kinds which would be useless for industrial purposes because of the appearance of this new requirement for growth factors. Chlorella pyrenoidosa (whatever species this was) can claim special mention since it has been subjected to a radiation dose of two megarads; yet a small percentage of cells survived which after three weeks grew at the original rate.(57) However, they were bigger although it is not known if the larger cell size was due to polyploidy or just merely polysomy.
Let us refer to von Witsch's statement concerning the possibility of producing polyploids. When one looks through the literature on the genetics of those algae considered as high-ranking candidates for industrial culturing, there is a surprising lack of reference to the induction of polyploidy in Chlorella and Scenedesmus. This is very interesting. Von Witsch undoubtedly had it in mind that the production of polyploids with colchicine could be achieved in algae as easily as in higher plants. But this appears not to be the case. In her book ‘The Chromosomes of the Algae’ Godward reviews numerous papers and from their results extracted the following information. ‘There is a high threshold for the effects of colchicine on green algae as compared with that in the higher plants. Four hours in 0.005 to 0.001% colchicine is enough to produce c-mitosis in Allium cepa: but the algae are seldom affected by less than 0.5%.’ Some algae seem immune to colchicine—e.g. Chaetophora, Spirogyra and Euglena; others will become diploid only after a week's treatment —such as Hydrodictyon. Ready reference to polyploid induction in Chlorella and Scenedesmus is most difficult to find and Godward makes no reference to this in her book. It is known that colchicine and other spindle-inhibitors are ineffective on Euglena which has no evident spindle. Maybe the situation in Euglena prevails also in Chlorella and Scendesmus, since Pickett-Heaps has pointed out that cytokinesis and spindle-formation in some algae are atypical, and that we are not entitled to extrapolate to all the algae what is known about the mitotic apparatus in higher plants.(117) He describes cytological features in algae concerning spindle-formation and other organelles which are markedly different from higher plants; and it may be that some peculiarity relating to spindle formation precludes the induction by colchicine treatment of polyploids in Chlorella and Scenedesmus. There is reference to polyplody in the unicellular alga, page 87 Stichococcus;(84) this organism, however, belongs to the Ulotrichales — an order of the green algae considered to be more advanced than the Chlorococcales.
In view of the lack of purposely-produced polyploids and the ready production of usable mutants, people directly concerned with exploring the industrial possibilities of growing algae still seem to favour the use of locally-discovered strains — just as Tamiya did. For instance, Palamar-Nordvynsteva looked at introduced strains of Chlorella as well as local strains from around the neighbourhood of Kiev.(112) It was shown that the local strains had advantages in growth and accumulation of dry weight over introduced ones. Dilov and Yordanova also came to the same opinion when they looked at thermophilic strains of Chlorella for growing on a semi-production basis, choosing finally an isolate from the Petrich-Sandanski region of Bulgaria.(39) And Sorokin, after isolating the first high temperature strain TX7-11-05, isolated another form (called 1-9-30) which was superior in terms of its synthesis of organic matter;(130) this one likewise came from the Woller Creek on the campus of the University of Texas.
So, although Leonard remarked that ‘The quest for new species might well go beyond the sampling of ponds and puddles’, and that ‘Chemically- and radiation-induced mutations could produce the optimum combination of physical and physiological characteristics’,(91) it seems that to date our efforts at producing suitable genetical variants have not met with success.
Previously it was stated that ‘the industrial culturing of Chlorella never became a viable proposition—never got beyond a couple of pilot-plants and a library of literature cloistering published dreams, designs and derelict results’. As a means of providing protein for the proletariat on the scale envisaged, this statement is true. But one minor commercial outlet can claim to sustain the industrial culturing of algae on a small scale. And this we find in Japan; algal harvest is not being used as a source of protein but as a source of a growth factor for Lactobacillus which the Japanese use to make a drink derived from milk —after the style of a liquid yoghurt. The growing of the required algae is done heterotrophically.
From what has been said, it is obvious there are numerous constraints which make the industrial culture of Chlorella a much more difficult operation than what it appeared from an armchair or on paper. Among the chemical constraints is the incredible quantity of fixed nitrogen required to culture Chlorella or a similar alga on page 88 the scale envisaged. To an algologist, there seems a theoretical answer to this impassé, since some algae are known which can fix nitrogen gas from the air just as the nodule-bacteria on the roots of legumes do. These biochemical élite are found amongst the blue-green algae: so why not culture industrially a blue-green alga capable of performing this remarkable reaction?
It should be obvious by now that the investigation of an alga for industrial cultivation requires that a large amount of research be done to see if that alga possesses characteristics which fit it for this purpose. Such research needs the use of pure and bacteriologically-free cultures. Keeping this point in mind, let us investigate the blue-green algae.
Blue-green algae differ from other algae in numerous ways. They have no distinct cell organelles; they tolerate much higher temperatures, pH's and salt concentrations in their growing media; they show a greater resistance to ultra-violet light than other micro-organisms. Many are enclosed in thick mucilaginous sheaths which engender them with great powers of survival, but unfortunately harbour bacteria. This latter property makes blue-green algae very, difficult to obtain in axenic culture and probably has accounted for our early lack of knowledge of their physiology. Due to these cultural difficulties — and maybe because of their slower growth — they were not chosen as common tools in the types of physiological work recounted earlier and thus did not get caught up in controversies as did Chlorella — because controversy forced people to get to know Chlorella whether they wanted to or not.
Concerning the first isolations of blue-green algae, Allen reports on the early history as follows:(2) ‘The first claims for pure cultures of Myxophyceae were made by Tischutkin in 1897, Bouilhac in 1898, and Beijerinck in 1902. Since none of these authors reported tests of their cultures for contaminants in media favourable for the growth of bacteria, and since later experience has shown that cultures which are apparently pure when grown on mineral media may give rise to an abundant bacterial flora on organic media, it seems possible that these investigators obtained only unialgal cultures free from gross contamination. Bouilhac's cultures can certainly not have been pure since he isolated them only by successive transfers in liquid mineral media.’
The first undeniably pure axenic culture of a blue-green alga was achieved by Pringsheim in 1913. Harder in 1917 modified Pringsheim's technique and managed to get several more bacterium-free blue-green algal cultures. Frank was possibly the first to propose that certain blue-green algae might be able to fix nitrogen; but because his cultures were impure and because other algae subsequently obtained in pure culture were found incapable of growth in the absence of combined nitrogen, the general belief prevailed that all algae were incapable of nitrogen fixation. As was mentioned earlier, Chlorella was obtained page 89 in axenic culture back in the days of Beijerinck. But the obtaining of a bacterium-free culture of Chlorella is a much easier job than freeing a culture of a blue-green alga from bacteria: Chlorella has no enveloping mucilaginous sheath from which contaminating bacteria would be very difficult to eradicate. Drewes (1928) is credited with the first indisputable demonstration that nitrogen-fixation occurred in an axenic culture of a blue-green alga. By a laborious method of sub-culturing, he obtained axenic cultures of a Nostoc and two Anabaenas and showed nitrogen-fixation to occur in Nostoc punctiforme and Anabaena variabilis. Knowing how mucilaginous Nostoc is and what a wonderful haven this mucilage must be for bacteria, one can but applaud his achievement and admire the fortitude with which repeated sub-culturing must have been done to obtain such a result. A new approach to ridding uni-algal blue-green cultures of contaminating bacteria was devised by Allison and Morris in 1930, who successfully used ultra-violet radiation to produce an axenic culture of Nostoc muscorum.
The culture of rice is a centuries-old practice confined, like the major population density of the world, to the warm parts of the globe — the tropics. But where did all the nitrogen come from throughout the ages to sustain the prolonged cropping of untold millions of tons of this cereal of the Orient? Human and animal excrement would have provided but a tiny fraction of the required amount; so what was the major source of this indispensable element — for nothing grows without nitrogen in some form or another? Howard focussed attention on this intriguing question when writing about rice production in India, in which country rice had been cropped on the same land for long periods of time without adding any fertiliser to the soil.(72)
In India, the growing season of rice is divided into three periods:(38)
1. The waterlogged period (from transplantation time to harvest time) in which there are a few inches to several feet of water above the soil and which is distinguished by the growth of abundant algae:
2. the dry period, which follows in winter after harvest and during which conditions remain very suitable for microbiological activity:
3. the desiccation period, commencing after winter when the soil temperature frequently exceeds 50° C.
The continuous growing of rice without fertiliser application implies the possibility of nitrogen-fixation in the soil or growing medium or the symbiosis of a nitrogen-fixing organism with the rice plant.
In 1914, Harrison and Aiyer showed that the abundant growth of algae in the first period — the waterlogged phase — was beneficial in aerating the upper layers of the submerged soils.(62) Some other experiments were designed to see if nitrogen-fixation occurred during page 90 this phase; arising from these, algae were implicated as possible agents of such fixation. These ideas led De to investigate more rigorously if algae were active in this sphere. Many soil samples from different rice-growing parts of India were collected, covered with distilled water and kept in light. Algae grew in these soil cultures — especially those of high pH in which blue-greens proliferated readily. Analyses done after three months showed a considerable increase in total nitrogen over the range of 4-35%. De found that considerable fixation occurred only in those cultures producing abundant growth of blue-greens. He then went ahead and isolated blue-green algae from rice soils, grew them in a nitrogen-free medium and tested their ability to fix nitrogen. Three species of Anabaena showed this ability but Phormidium foveolarum failed. He also showed that a considerable part of the nitrogen fixed by the alga could be found in the external medium in an organic form.
During World War II a Japanese, Watanabe, organised the collection of more than 600 samples of blue-green algae from rice fields in various regions of Asia.(148) He investigated these for their nitrogen-fixing capabilities, but found only 13 with this faculty. One, Tolypothrix tenuis from Borneo, was a very strong nitrogen-fixer, and when inoculated into experimental paddy fields produced an increase in the total nitrogen of the rice plants.
The paddy-field nitrogen-fixing blue-green algae found in tropical areas were rare in Japan, North China, Manchuria, Korea and Sakhalin — i.e. in the colder rice-growing areas. Maybe the paddy fields of these areas could be inoculated with Tolypothrix—leading to increased yields. To do this would require culturing the alga—but how could this be done since no previous large-scale culturing of blue-green algae had been achieved? Watanabe tried out three systems.(147) The first was a tank-culture system with illumination through glass windows in the tank. The second was a closed-circulation system — the same as described earlier for Chlorella culture by A. D. Little and Company of Cambridge, Massachusetts. The third consisted of growing the alga on the surface of sieved pumice granules wet with nutrient medium and kept in plastic tubes using aeration with carbon dioxide-enriched air. The last method was found successful; and the granules impregnated with algae were used for inoculating paddy fields. Prior to inoculation the paddy fields were top-dressed with lime to raise the pH to a favourable level — to about 7.5, a pH much more conducive to the successful growing of blue-greens.
Using this form of inoculum it was possible to introduce Tolypothrix into many paddy fields and thereby increase ultimately the nitrogen levels of the rice. The results were good enough to consider the practice of inoculating rice fields as a matter of course. It was also proposed to look at Anabaena cylindrica with the same idea in mind.page 91
Blue-green algae are also important in water-supplies but from a nuisance point of view, and people had also been trying to get some of these offending species into culture. Gerloff had this to say:(56) ‘Although species of blue-green algae produce serious nuisance blooms and growths in lakes and streams, surprisingly little work had been done on their nutrition and physiology under controlled conditions in the laboratory. This is undoubtedly due to the difficulties that have been encountered in growing many species of Myxophyceae in pure culture, a point stressed in a recent publication by Rodhe (1948). He was unable in spite of numerous attempts and modifications of the nutrient medium, to grow such common bloom-producers as Anabaena flos-aquae. Aphanizomenon flos-aquae, Coelosphaerium naegelianum or Microcystis aeruginosa in a nutrient solution which was suitable for continuous growth of forty species belonging to Chlorococcales, Volvocales, Conjugatae, Heterokontae, and diatoms.’ Until basic physiological studies can be done — for which axenic cultures are imperative. it is impossible to investigate the parameters of growing conditions which apply in natural surroundings, such as those referred to above. Ultimately Gerloff and his team were able to get 10 ‘bloom’ blue-green algae into pure culture.
In 1951 Allen published a review on the cultivation of the blue-greens and gave details of many aspects of nutrition which she had been able to discover from working with 30 axenic cultures.(2)
This brings us up to the period when much experimentation was being done on Chlorella in many countries; and against this now familiar background, the following comments by Meier are rather interesting.(104) In an article entitled ‘Possibilities of Photosynthesis in Lower Plants’ Meier compared photosynthetic capabilities of higher and lower plants, using algae in comparison with more conventional crop plants. He pointed out that by growing algae under really optimum conditions, it was conceivable to organise a 20% conversion of solar energy to chemical energy in the form of dry matter. But he also pointed out that ‘a whole series of constraints arise which profoundly affect the choice of feasible processes for culturing, handling and consuming the algae that might be so grown’. The most fundamental of these constraints was to be found in the scarcity of natural resources — particularly carbon dioxide, in his opinion. He was also mindful of the problem of providing nitrogen compounds because he suggested that ‘some of the blue-green species, especially among the Nostocaceae and Rivulariaceae are able to fix their own nitrogen — a highly advantageous property. The culture of these organisms, however, is still less flexible, and the composition is less variable than for the alternatives which are scattered broadly over several phyla.’ Later on he also writes ‘in areas of temperate climate, where little or no synthetic ammonia is available, the prospects of growing nitrogen-fixing blue-green algae appear to be most page 92 favourable’ … ‘A nutrient medium for blue-greens is somewhat different than for Chlorella, and the optimum pH is around 10 instead of 5-6. Partial amino-acid analyses of proteins in actively-growing blue-greens indicate that they should be suitable food materials.
‘Too little is known about the physiology of these organisms to put forward any more explicit description of a large-scale process, but, if laboratory data are any criterion, it appears that a feasible economic target would involve the fixing of two tons of nitrogen per acre per year. The process designs employing blue-green algae would need to be less elaborate and expensive than that described for Chlorella. This is aided by the high pH which might permit economic use of carbon dioxide from the atmosphere, and also by the observation that bacteria do not seem to inhibit growth.’ So here is an early appreciation of the peculiarities of the blue-greens and how these peculiarities could possibly be capitalised upon in industrial cultivation.
Apart from the capacity of some to fix nitrogen, the blue-green algae have certain features which make them very suitable candidates as a substitute for Chlorella. Some of these features include the following; many are larger in size. and some have gas vacuoles which can therefore make them easier to harvest; they do not have membrane-bound organelles and this makes extraction of their cells easier; they can tolerate higher temperatures and higher pH's which combination of factors could keep their cultures purer than those of other algae. Having no liquid vacuoles, they are tolerant of much higher salt concentrations in their growth media. But while they grow easily in nature — albeit in funny and physiologically peculiar habitats such as hot springs and highly natreous situations, they cannot be said to yield as readily to large-scale artificial culture as Chlorella.
We find one, Synechococcus lividus, being actively investigated in relation to space flight ecosystems.(40) This was isolated from a hot spring in Yellowstone Park and was found to thrive at temperatures of 125° F. It could survive and function at temperatures as high as 187° F. Normal varieties of Chlorella would have to be grown at temperatures of 90° F. and this would require the use of cooling apparatus. Even a high temperature strain of Chlorella which is able to grow at 103° F. would need cooling compared with this Synechococcus. In a space vehicle it would be more economical to heat the algal system to 125° F. than to cool it to 90° F. Bacterial contamination may not be a problem at high temperature. However, this blue-green alga was not being grown for the purpose of nitrogen-fixation — only because of its thermophilic properties.
As Kratz and Myers point out,(85) up till about the mid 1950's people had been interested in the blue-green algae mainly because some of them possess this unusual but highly desirable capacity of being able to fix nitrogen. Not much had been discovered about their general physiology in the same way as had been done for the page 93 green algae such as Chlorella. ‘No study has been carried out to the point of developing a blue-green alga as a reliable tool organism for physiological studies.
‘There have been two obstacles to such development. One may be attributed to the difficulties of isolation and the lack of species in pure culture which suspend readily and submit in simplicity of manipulation possible with green algae such as Chlorella and Scenedesmus. A second difficulty arises from inadequate provision of carbon dioxide inherent in most of the culture methods used. Nutritional requirements have been defined in terms of the maximum growth yield obtained in cultures in cotton-stoppered flasks. Methods have not been developed for routine study of growth rate, a characteristic far more useful and interpretable for physiological work.’
Some of the problems encountered in the growing of nitrogen-fixing blue-greens were gradually being resolved because of the recognition from agricultural fields of study that cobalt and molybdenum might be necessary for growing these algae since they, like legume-nodule bacteria. were able to fix nitrogen. Also as was pointed out earlier, they were not easy to get into axenic culture. So no studies on growth or growth rates could be undertaken until it had been discovered how to grow them, as well as how to get them into pure bacterium-free culture.
Florenzano, Balloni and Materassi undertook successfully the large-scale non-sterile culture of isolates of Nostoc punctiforme and Anabaena cylindrica.(49) They were able to convert this into a semi-continuous system without too much trouble or loss in productivity. From their research, they felt that these blue-green algae were more interesting from the point of view of large-scale culture than the green algae. Under favourable conditions they could achieve 7-8 g dry weight/sq. metre/day.
We arrive at the point where blue-green algae do not appear as a contender of any kind for the industrial culture of algae, despite the pristine capability of some for fixing nitrogen, their ease of culture under peculiar conditions as shown by Watanabe, and the demonstration by Florenzano, Balloni and Materassi, that they could be grown in non-sterile culture to produce yields of 7-8 g dry weight/sq. metre/day.
We move now to the southern limits of the Saharan Desert, to the vicinity of Lake Tchad, and watch a spectacle which has been enacted countless times over the last few centuries.(8) The location is Kanem in the region just north of Tchad. Whilst tending their flocks the Kanembou men pass the time of day in what must be sparse shade of a thorn-bush, playing a doleful air on a one-stringed guitar-like page 94 instrument hollowed out from a calabash. Meantime the Kanembou women seek out those depressions in the desert which hold water (called wadis) and on whose surface grows a greenish scum. They then proceed to wade through the water, dragging after them a large closely-woven basket in which this thick green sludge collects. When full, the baskets are dragged to the side of the wadi and left a short time for water to seep out and so concentrate the sludge inside. More liquid is decanted; and when the sludge is sufficiently thick it is poured into earthernware jars which the women then carry on their heads up the side of the wadi. Where they find a flattish surface of sand they scoop out shallow flat depressions into which they pour the contents of the jars. Because of the dryness and porosity of the sand and the heat of the sun, the water soon dissipates and leaves a large flat cake of dark green material. Using a stick or finger, one woman scores the cake into segments to simplify breaking it in smaller pieces when thoroughly dry. This material is eaten in several ways — such as making it into a spicy sauce which is then poured over little balls or cakes of boiled millet.
In this sequence of activities the Kanembou women have been harvesting and drying a blue-green alga, now known as Spirulina platensis. What an incredibly easy procedure to follow to gain access to a source of food containing 54% crude protein dry weight! No problems in the growing of it — it is just there; no problems in the harvesting — it is merely filtered through a closely-woven basket. There would not be many sources of such naturally-occurring high protein food so readily available in the world! But of course it is highly localised (fortunately for the people of Kanem) and therefore not much good for the hungry peoples around the globe. They call this material ‘Dié’, ‘Dihé’, or ‘Douhé’.
Although this practice had been carried on for a very long time. it was not investigated closely until about 1939 or 1940 when a French pharmacist stationed at Fort Lamy near Lake Tchad sent a sample back to France for identification. This was made and its botanical description written up by Dangéard.(35) There were other things to think about in France at this time and presumably nothing further was done. But the war finished; and the new spectre of world-wide food shortage began to become apparent. This motivated Spoehr and Milner to carry out their research work at the Carnegie Institution on the possibility of growing Chlorella as a means of contributing to the world's food supply. As we have seen, U.S.A., England, Germany, Israel, Japan and Sweden were very active in this field — but not so France: a few more years were to pass before she would become interested in this area of research.
In the late 1950's a Frenchman, Brandily, was getting to know the Kanem area of Tchad very well by being involved in filming an episode of a sequence called ‘Lands without Tracks’. He had seen how the Kanembou women extract Spirulina from the water and page 95 make ‘dié’. This must have impressed him considerably because he wrote a popular article for Sciences et Avenir entitled, ‘For a long time now a primitive tribe of Tchad have been using the food of the year 2000’ (from which the above description of its collection is taken).(8) The Belgian Trans-Saharan Expedition in 1964/5 also came across ‘dié’ at Fort Lamy and located another source of it at the oasis of Ounianga Kebir — 750 miles to the north-east of Fort Lamy.(90)
Meanwhile, around 1962 the Institute Français de Pétrole had been conducting a survey of research and information on the industrial culturing of Chlorella and Scenedesmus. Such an undertaking — if feasible — would be worth while from their point of view because of the large amounts of carbon dioxide gas which were available and going to waste in this industry. About this time a public conference was held, having as its theme ‘The Republic of Tchad’, and it appears that Brandily gave a lecture during which reference was made to the fact that the people of Kanem collected and ate an alga. This point came to the notice of the I.F.P., who commissioned Brandily to collect more samples from Tchad. These arrived in France about March 1963 and study of them was begun immediately by the I.F.P. who discovered that the alga was not the green alga Chlorella as Brandily had suspected in his article but a blue-green alga called Spirulina.(28) Unlike Chlorella and Scenedesmus which had never been eaten traditionally by anyone, Spirulina has been used as food for a long time and is still today a staple for the Kanembou. This point plus the obvious ease of harvesting and the fact that it was incredibly rich in protein whetted the research appetite of the I.F.P. who began investigating immediately the possibility of its industrial culture.
The seventh World Petroleum Congress was held in Mexico City in 1967 at which the I.F.P. reported their work on Spirulina, its nutritional value and the method of culture at low cost which could allow it to be grown for food supplementation.(31)
Mexico City is situated in the Valley of Mexico — a natural land-locked basin, ‘entirely surrounded by mountains of volcanic origin’.(32) In times past there was a cluster of five lakes in this valley. In winter they were discrete; but after the summer rains they often coalesced to form one large lake called the Lake of the Moon. This summer flooding was frequently disastrous because the fresh water of the two southernmost lakes became contaminated with water from Lake Texcoco to the north. Lake Texcoco was highly saline and charged with sodium salts; and this water, pervading the fresh water of the southern lakes, wrought immense havoc in the highly productive cropping areas (called Chinampas) of the southern area.
The appearance of these natreous waters in the Valley of Mexico is somewhat enigmatic since the volcanoes surrounding this area including the well-known Popocatapetl are andesitic and thus of acid page 96 nature. So where did all the sodium salts come from? It is thought that at some time in the geological past there must have been rifting in this valley although there is no apparent evidence of it today. If this were the case, these sodium salts would have come from basic igneous rocks normally associated with rifting. Subsequent leaching of this kind of parent rock material would have removed the easily soluble constituents such as sodium and associated anions (or anions formed as a result of weathering — such as carbonate), and these could have collected subterraneanly only in such a land-locked basin.
In Aztec times, the city of Tenochtitlan was built on an island in Lake Texcoco. Some time about the fourteenth century the climate became much wetter and flooding brought the Aztec economy to the brink of disaster, because of the contamination of the highly productive cropping areas by this natreous water. As a result, a huge dyke ten miles long was built across the lake to the north of the Aztec capital. The dyke ensured that the capital and the crop-producing areas in the south were always in fresh water and free from contamination by water from the rest of Lake Texcoco. The city of Tenochtitlan grew and expanded over land reclaimed from the lake and has ultimately become what we know today as Mexico City.(32)
Caustic soda is a very important basic chemical in industry, usually obtained by the electrolysis of sodium chloride. Large subterranean quantities of sodium chloride and carbonate were discovered in the waters under Mexico City, and a private company — Sosa Texcoco — was formed to extract this material and engage in the manufacture of products and derivatives for industry. They put bores down and pumped from underground this highly natreous solution into solar evaporators, using Mexico City's sun and arid atmosphere to get rid of the water and crystallise the dissolved salts. They had not been in operation very long when they noticed to their surprise an emerald green colouration appearing around the periphery of their evaporators which, on closer examination, turned out to be a blue-green alga called Spirulina platensis.(28) Around this time Farrer had written his article called ‘Tecuitlatl; a glimpse of Aztec food technology’; this appeared in Nature 1966.(48) It was then realised that the contaminant appearing in the crystallising pans was in all likelihood an Aztec foodstuff of ancient vintage; and further that it appeared to be the same species as that found in Tchad. Information of this kind caused the contaminant in the solar evaporators to be viewed in a completely different light rather than just of nuisance value.
Now for some information about Spirulina! It is a blue-green alga and is classified in the Oscillatoriaceae, which in turn belong in the order Nostocales. It is a multi-cellular organism having the shape of a spiral filament about 0.2 - 0.3 mm in length. It has one page 97 chlorophyll only, namely ‘a’, and like other prokaryotic cells has no membrane-bound cell organelles.
|Disodium hydrogen phosphate||0.94||"|
|+ EDTA and trace elements|
Spirulina when grown in natural conditions in Africa contains 40-45% crude protein: grown artificially, it contains up to 70% crude protein dry weight. To achieve such a high nitrogen figure one might have expected Spirulina to be a nitrogen-fixing blue-green like some of its taxonomic bed-mates in the order Nostocales. Yet the main Nostocalean nitrogen-fixers are characterised by having heterocysts: : Spirulina has none of these. It has been reported that Oscillatoria sub-brevis(108) and Lyngbya aestuarii,(140) both from the same family as Spirulina, fix nitrogen; but the literature does not report Spirulina as having this capability. However, it is odd that the French medium requires 195 mg/litre of potassium nitrate which one would not expect to be necessary at all were it a nitrogen-fixer. This amount of potassium nitrate is about five times less than is used in Kuhl's formula for the Chlorella medium; and yet the crude protein figure for Spirulina is 70% — much higher than that for Chlorella TX7-11-05 which is 55%. So there is something unusual going on.
What about Spirulina as a food source — is it nutritious? Is it better than Chlorella? What does chemical analysis reveal? Table 5 sets out the gross analysis of artificially grown Spirulina.
|Analysis % D.W.||I.F.P.||Sosa-Texcoco|
|Protein (N X 6.25)||75.3||67.03|
|Ash — at 550° C.||4.87||6.89|
|Index of digestibility||80||83|
|Ash insoluble in HCl||0.05||0.19|
|FAO Provisional Pattern(30)||Cow's Milk(30)||Eggs(30)||Beef(30)||Soya-meal(30)||Spirulina—I.F.P.(29)||Spirulina—Sosa-Texcoco(29)||Chlorella(99)||Yeast. Candida utilis(132)||Bacterial Protein—Esso-Nestle(132)||Whole Wheat F.A.O.(81)|
|Sulphur amino acids Total||4.2||3.3||5.4||3.7||3.2||3.6||3.4||1.2||2.0||2.6||4.0|
|Note: The figure for total sulphur amino-acids is the sum of the methionine and cystine figures.|
Returning to the question of protein! Although it is high, it is necessary to know what the spectrum of this protein is like for those amino-acids which man is unable to synthesise: for no matter how high in protein a foodstuff is, it is useless on its own if one of these amino-acids is missing. Such a foodstuff if deficient in this respect could be mixed with another containing the missing amino-acid and in this way we would overcome the problem. These figures are given in Table 6, in comparison with levels for these same acids which F.A.O. reckon should be present in a diet. Figures for other foods old and new are also given.
Although earlier work on the digestibility of Spirulina showed that its digestibility was about 76%(30) and rats when fed this alga showed little increase in weight compared with those fed on casein, more recent results report a digestibility of 84% with rats.(73) This latest work quotes a protein efficiency ratio of 2.3 compared with 2.5 for casein.
One last problem remains to be mentioned; and this applies to protein made by Chlorella, Spirulina, or any form of single-cell protein manufactured by heterotrophs such as food yeasts and bacteria. All living organisms contain poly-nucleotide molecules — DNA and RNA. DNA is the conveyor belt for genetic information and RNA is the agent through which the DNA message is transcribed into biochemical and physiological actuality. Both of these molecules contain purine and pyrimidine bases: the purines are adenine and guanine, while cytosine, uracil, and thymine are the pyrimidines. We can forget about the pyrimidine bases since the discussion to follow concerns only the purine compounds, adenine and guanine.
The two compounds are similar in overall structure to other naturally-occurring purines such as hypoxanthine, xanthine and uric acid. Like many complex organic molecules in living organisms, adenine and guanine undergo constant synthesis and breakdown. The normal breakdown route in mammals for these two purines is: adenine/guanine —→ inosine —→ hypoxanthine —→ xanthine —→ uric acid —→ allantoin —→ allantoic acid —→ urea —→ glyoxylic acid. Each of these reactions is mediated by an enzyme whose presence is genetically determined. Among the many things that man shares with the higher apes is the lack of uricase; this enzyme effects the reaction uric acid —→ allantoin. Under normal circumstances uric acid in man is got rid of by excretion in urine as sodium urate. But in those individuals with a genetic tendency to primary over-production of uric acid, it is possible for the acid to be precipitated in crystalline form in joints — a painful condition called gout; or the acid can accrete and form stones in the urinary tract — the familiar kidney stones, whose removal is also a painful business. Clinical research has revealed that a level of about 7 mg uric acid/100 ml of serum is a level which separates the majority of patients with primary gout from most normal people.page 100
It has been known for many years that feeding yeast can lead to an increase in uric acid excretion in urine, due to yeast's high content of nucleic acid; but up till now nobody has worried too much about this since yeast was usually eaten in small quantities merely as a source of the Vitamin B Complex. Now that yeast and other micro-organisms may be eaten in much larger quantities as a source of protein, the level of intake must be known in terms of uric acid production. Eating 2g of single-cell protein nucleic acid per day seems probably a safe daily intake, and this is roughly equivalent to 30g of yeast protein since dried yeast contains about 8-15% nucleic acid. Whereas 3g of nucleic acid ingested per day brings the serum level up to more than 7mg of uric acid per 100 ml of serum. It is considered in the light of present knowledge that this level might put some normal people at risk. Three grams of nucleic acid is equivalent to 45g of yeast protein. This latter dosage elevates the urinary acid level from approximately 600 mg per day on a normal diet to the vicinity of 1300 or more — and this is considered to be a highly undesirable level.(42) It is a cruel twist of evolutionary fate that one of the basic molecules that constitutes our gene bank should also constitute a risk to health.
How does Spirulina fare from the point of view of nucleic acid content? A figure quoted by the I.F.P. is 4.1% on a dry-weight basis.(29) This is much lower than those figures given above for yeasts (8-15%) and for bacteria which range from 20-25%. A high nucleic acid content is a characteristic of rapidly growing cells; and the higher RNA content of yeasts and bacteria is due to their faster growth rate compared with algae. So maybe the lesson to be learnt from this fact is — do not strive to find an alga with an ever-decreasing generation time. A shortened generation time can lead to an increased production rate; but it looks as though other disadvantages might follow in the wake.
The use of Spirulina by humans is well and truly tried of course but only in very isolated places. Hunger is now global. So let us mention a few avenues that have been suggested or experimented with to see how this material could be used. Because of its good protein quality, Spirulina can be used as a supplement for human feeding in the same way as a very good quality soya flour. One could dispense it in milk or use it as a substitute for milk where such a necessity arises.
The Infant Food Programme of FAO/UNICEF/WHO has become interested in this source of protein. As a result recipes for compound foods have been designed: for instance there is a powder formulated specifically for weaning babies and there is also a preparation in the form of pasta designed for four to twelve-year-olds.(73)
There are people in the vicinity of Tchad who were apparently unaware of this kind of high proteinaceous food. Under the auspices page 101 of FAO more than 6,000 meals based on Spirulina were distributed to these folk. The results were excellent.
When reading about Spirulina, one comes across references to certain water-birds using this alga as food. For instance, in his book ‘Conquest of Mexico’, Cortez through his ghost-author Gomara writes of Lake Texcoco as being covered in winter by birds feeding on the tecuitlatl (Spirulina) growing on its surface. Spirulina constitutes the bulk of the diet of the lesser flamingo found literally by the millions in the lakes of the Rift Valley of Kenya; and ducks feed on the same food in Lakes Yoan and Katam near the oasis of Ounianga Kebir.
- Phoenicopterus — the greater flamingo, is found in Europe, the Caspian Sea, the Persian Gulf, North-west India, the Atlantic seaboard of North America and the islands of subtropical and tropical America:
- Phoeniconaias — the lesser flamingo, is found in East and South Africa, Madagascar and North-west India:
- Phoenicoparrus — includes three different species which are confined to the Andes in South America.
One wonders if the Aztecs got the idea of eating Spirulina from observing the fact that water-fowl fed so voraciously on it; and having grasped the significance of this observation assumed that what was good for the birds was also good for them. Maybe this is how the Kanembou recognised the value of dié. It is surprising therefore that others such as the people around Ounianga Kebir and those near the Rift Valley Lakes did not take a cue from the ducks and flamingos, page 102 because had they observed and appreciated this avian habit they would have had access to a source of very good protein.
In following this review of the industrial culturing of algae, we have covered much ground and pursued many lines of thought and experiment — some productive, some abortive. Not all bright ideas work; not all laboratory results can survive escalation even to pilot-plant scale. What looks so deceptively simple in nature becomes so devilish difficult in industry; what in the mind seems so easy to do is often so difficult to translate to reality. We have seen how this research required answers not only from numerous sections of botany but from many other sections of science. Like hunger, disease and poverty, research recognises no boundaries. There is no telling where it will meander to collect its facts.
Along the way we have seen numerous examples of some of the idionsyncrasies of research mentioned in the introduction; the ever-receding horizon, the dependence of new work on unrelated research, new applications undreamed of earlier, and others. Research on a macro-scale such as we have just seen is not a smooth progression; rather, it proceeds in stages — is even peristaltic, which for many reasons must be the case as one can now appreciate.
However, it can be said that following the story of Chlorella as an evolution is more interesting and rewarding than merely reading a review of the main features of efforts to exploit microalgae. When presented in this way, the story is so much richer and somehow seems to come to life more readily.
Following an evolutionary development allows one to distil time and recover the essentials, leaving a residue of material inconsequential to the evolutionary sequence. Such distillation leads to a view of this sequence as a smooth progression, which possibly permits a better and easier comprehension of the overall development.
So much for the philosophy — how about the practicality of producing protein for the people? Single cell protein — whether of algal, fungal or bacterial origin — is here to stay: it must be. That this is so is apparent when one considers a statement made by Bunker:(10). A bullock weighing 10 hundredweight can synthesize rather less than a pound of protein a day, whereas 10 hundredweight of yeast could in the same time produce over 50 tons of protein.' There can be no doubt where the efficiency lies! Those 50 tons of yeast protein may be synthesised in an area much less than that required by the bullock too, although the technical know-how required to tend the bullock cannot be compared with the sophisticated background, knowledge and equipment required to grow the yeast. But the bullock along with other ruminants can do something neither the yeast nor any alga can do — namely, break cellulose down as a page 103 carbon source while elaborating protein: and there is an incredible amount of cellulose being continuously produced by land-based plants which is not used profitably by organisms other than ruminants. While using this cellulose the ruminant produces protein which is still for many a much more acceptable and tasty form than that from yeast; and those with access to this kind of animal protein are among the world's privileged. However, so many millions of people are just not getting enough protein of any kind, and for them the origin and palatability of the protein would be of little or no consequence. If algal protein could be produced readily and tolerably cheaply, it might help to fill an ever-increasing lack on this globe of ours.
An incredible amount of research has been done and many people have tried hard to get these algal projects to work: but success on the scale required is not yet within our reach. At this point in time, it is anybody's guess whether they will work sufficiently to be able to help out with this protein problem. Vincent brought the whole concept into sharp focus when he wrote:(141) ‘Direct use of algae as human food is at first sight attractive because it offers the shortest food chain and therefore the highest yield of food protein per unit of light energy or unit area of growing space. The psychological, technical and nutritional problems involved in direct use are immense and would involve very sophisticated technology to produce what would probably never be a very satisfactory product.’ The implications of the first sentence constitute the carrot which has dangled in front of the many research workers from Spoehr and Milner onwards. But, as can now be seen, the problems are still very real and still unfortunately in the main unsolved.
Hunger and malnutrition occur mainly in the tropical and sub-tropical areas. These areas are also at present characterised by an over-all lack of technological skills and background. The industrial cultivation of microalgae needs to be located where advanced technology and skill already exist; and these are to be found predominantly in the temperate regions. So the distance between the sites of hunger and the algal factories must introduce another set of difficulties. If algal protein can be produced in the temperate regions while the hunger exists in the tropics, a transport gap immediately appears between the producer and the user; and apart from the physical effort involved in transport, the spectre of increased cost automatically raises its ugly head — the costs of packaging for lengthy transport, loading, actual transport, unloading, and final dissemination to the point of use. Even if industrial algal culture could be set up in tropical regions, there would be difficulties to surmount to locate such production units where the hunger was most intense and the protein least expensive for the ultimate user. Here is one difficulty. Kihlberg points out that ‘The temperature range used in the production of yeast, bacteria, and algae often lies near 28-32, 28-38 and 30-40° C. respectively. The aerobic growth of microorganisms is an exothermic page 104 process and cooling is required to keep the temperature at the optimum level. This implies high costs, particularly in tropical regions where refrigeration would be necessary since the temperature gradient between cooling water and fermenter will be too small.’ The majority of chemicals (excluding carbon dioxide) would have to be transported to the site. A source of carbon dioxide would need to be at hand. A good and constant supply of water would be a primary requirement — and this necessity eliminates the construction of such a plant in arid areas and maybe in areas subject to extreme monsoon desiccation. This limitation on factory location immediately removes the possibility of growing algae for protein in areas of great hunger, since hunger on a macro-scale is possibly associated more with drought and lack of water than any other single environmental factor.
From a productivity point of view it almost tops the list, as can be seen from the following figures assembled by Vincent:(141)
it has already been used as food for centuries without apparent toxicity of any kind:
it has a high protein content with good digestilibity properties:
the essential amino acid spectrum is fairly good:
the availability of its protein is good because mere drying ruptures the cell wall:
the nucleic acid content presents no problem:
it is easy to harvest:
culture does not have to be sterile:
it grows at a higher temperature than most other algae and cooling is not such a problem:
apart from decolouration there is minimal processing.
|Protein Source||Yield (dry weight of protein kg./ha./yr.)|
|Chlorella pyrenoidosa TX 7-11-05||44,700|
|Filamentous algae (Ulothrix, Uronema and others))||20,000|
|Chlorella pyrenoidosa (Emerson)||15,700|
From a practical point of view, it is at the top because it is at present the only alga capable of being grown and harvested as food for humans in large quantities. It can be grown industrially page 105 using an artificially-designed medium. But it also has the great advantage that it can be grown under completely natural conditions as in the Tchad area or under semi-natural conditions as is done by Sosa-Texcoco in Mexico City. This feature, combined with its somewhat exotic growing environment which permits a virtually pure culture to be achieved, would seem capable of further exploitation — especially since it might be possible for this to be done in tropical areas without the prerequisite of advanced technology.
Under truly natural conditions, Spirulina has been found growing in Lake Tchad, at Ounianga Kebir Oasis, in lakes in the Kenya Rift Valley, in Lake Mugunga in the Congo (south of the laval plain) and in some lakes in Ethiopia. Under semi-natural conditions, it grows in the solar evaporators of Sosa-Texcoco. The feature common to these areas is the alkalinity of the water. But why should these waters be alkaline? The answer seems to be that these areas are what a geologist refers to as rift areas — where basic igneous rock has come to the surface and been subjected to normal weathering processes. If the water leaching this kind of rock has been able to collect on the surface, we find alkaline lakes — and subsequently the growth of Spirulina. It is interesting to recall that in the geological past Lake Tchad was about a thousand miles or more in diameter. The oasis of Ounianga Kebir, where the Belgian Trans-Saharan Expedition also found Spirulina, is about 750 miles north-east of the present Lake Tchad and in times past would more than likely have been a part of Lake Tchad. This could explain the highly natreous nature of both sorts of water which leads to the presence of Spirulina. Reference has already been made to the possibility of rifting in the Valley of Mexico to give the alkaline water there. Geologists can tell us where other rift areas exist and they might be able to provide information on probable areas of subterranean water containing sodium carbonate. Such data could give us clues to locations where deliberate outdoor culture might be established without a great deal of technological requirement. If only we could find masses of water similar to those of Tchad and Texcoco which naturally and without further additions of chemicals would form a growth medium free of charge!
One also wonders if the Dead Sea waters might on dilution yield a natural medium wherein Spirulina or a more suitable isolate would grow. The Dead Sea has also been formed in a rift area. The temperature is high, as is the insolation; and maybe dilution with water would bring the salt concentration down to a level more conducive to a living organism. Not that the Dead Sea even at its present salinity of 32% is entirely devoid of life, because Elazari-Volcani has reported finding a blue-green alga (Aphanocapsa) in its sediments.(43) If the nutrients are present to sustain this organism, they might with a little dilution and a bit of luck support the growth of Spirulina. Analytical figures of waters from the Dead Sea System page 106 indicate that the bicarbonate figure may be low,(94) but it may be sufficient to act as a collector of carbon dioxide and thus allow a culture system to function.
By investigating the distribution of flamingos it should also be possible to locate natreous waters and lakes. These birds seem to be associated with this unusual kind of habitat since this is also the habitat of Spirulina on which the flamingos depend for food — either directly in the case of the lesser or indirectly in the case of the greater. This is the picture in Africa. The South American species are recorded from mountainous areas of Chile near Antofagasta and in the neighbouring areas of Bolivia; and these species are reputedly filter-feeders. It is known that salt deserts occur in Chile; and one assumes because of the presence of flamingos that the same must apply in Bolivia. Thus these flamingo-inhabiting areas should be looked at to see if Spirulina occurs there. Similar areas known to be frequented by flamingos occur also in India.
It is thus possible that flamingos could be used as a good biological indicator of water containing Spirulina naturally or as sites for potential culture. It has been calculated that a million lesser flamingos eat about 200 tons of alga a day. Since a million or more of this species will congregate for months on relatively small bodies of water such as Lake Nakuru which is about six miles by four miles, one's mind boggles at the amount of algal biomass eaten and more particularly at the productivity of such an area.(77) Kahl predicts that when the productivities of such lakes are measured ‘they will be found to be one of the most — if not the most — productive ecosystems in the world’.(77)
We might well ask the question — what makes such lakes so productive? One of the first answers would be lack of competition, since very few algae could tolerate such high temperatures and insolation in combination with a very high alkalinity and dissolved salt content. The osmotic potentialities of the waters of such areas must be incredibly high. Also, their high sodium content more than likely imposes a physiological barrier of a non-osmotic kind to most plants except Spirulina and some of its blue-green algal cohorts, since sodium in such concentrations would be expected to induce symptoms of metabolic disturbance akin to toxicity. For reasons which may be associated with their origin in the days of the primaeval soup, some blue-green algae can grow in such incredibly harsh physiological conditions. But, on the other hand, the alkaline pH renders certain inorganic nutrients insoluble and therefore unavailable (such as iron); and one wonders in what form phosphate is present since most inorganic phosphates are insoluble — especially under alkaline conditions. However, there must be available phosphate, iron and other nutrients present to allow such prodigious productivity. There is a question mark associated with nitrogen since we do not know for sure whether Spirulina is nitrogen-fixing or requires some nitrogen page 107 to be present — not that the latter would be a problem with so many birds around. So what accounts for this productivity? More than likely it is due to the presence of sodium carbonate, since an alga growing in a medium so rich in carbonate has access right outside the cell membrane to quantities of potential carbon dioxide the like of which could not be exceeded by industry in the Ruhr Valley or petroleum refineries anywhere. As we have seen with Chlorella, high yields can be got only by using fairly high concentrations of carbon dioxide — other things not being limiting. So the world's natreous lakes if rich in sodium carbonate are Nature's equivalent of industry's bounteous quantities of free and unused carbon dioxide. Unlike the situation in industry where the carbon dioxide goes to waste, the presence of sodium in these lakes automatically concentrates the gas as carbonate which, as it is depleted by algal photosynthesis, is constantly renewed by absorption from the atmosphere. What could be simpler?
Having such a concentrated source of carbon dioxide right outside the cell membrane is an interesting situation since the gradient along the diffusion path for carbon dioxide (or its equivalent, carbonate or bicarbonate) must be very steep. Once inside the cell membrane, the carbon dioxide does not have to pass through another membrane before photosynthesis occurs since Spirulina, being a blue-green alga and therefore prokaryotic, has no membrane-bound chloroplasts. Rather, the carbon dioxide must enter into combination with its acceptor molecule which must be exposed in the cytoplasm unbounded by a membrane. Maybe in this unusual habitat of high sodic lakes and for this reason, the prokaryotic state allows a photosynthetic rate superior to that of eukaryotes like Chlorella, since impedance to diffusion of carbon dioxide may well be less in prokaryotes. This may also help to explain the enormous productivity seen in these lakes.
The exploitation of alkaline lakes in this way would not alleviate the world's protein position because the number of such lakes seems limited. But they do have the advantage of having water ‘on tap’, the nutrients necessary for growth, and a built-in method for concentrating carbon dioxide: furthermore and of great importance, the whole system is free and renewable without charge. No industry can match this! In their locations, therefore, they could contribute something. The local people of Lake Tchad use woven baskets as their only piece of equipment: such could hardly be described as sophisticated. Yet it gives them access to one of the most concentrated forms of protein in the world. Admittedly the Kanembou have also got used to the taste and texture of Spirulina. But one wonders if other people would accept this food of alkaline waters.
Science can effect many improvements for mankind. These, however, are sometimes rejected because in the final analysis the most immutable feature of human nature is the psyche of man; and page 108 this rather than the intelligence of man still controls so much — even such things as his eating habits, which occasionally will not alter even in the face of hunger.
Since this article went to press, a reference has been found to experiments conducted in Mauritius over the period 1942-45 on the cultivation of an alga for human food. The organism, Pleurococcus, was grown by slowly irrigating sloping slabs of concrete to which the alga became attached. After harvest and drying, the green powder contained 40-50% moisture, 8-10% crude protein and 18mg ascorbic acid (presumably 18mg%). The dried powder mixed badly with water but could be incorporated with flour and yeast into biscuits. The taste of the powder was slightly bitter but easily masked. It was thought that such a food if produced in quantity could be used together with yeast to meet the then immediate needs of malnutrition.
This information was contained in the final report of the Director of the Medical and Health Department of Mauritius 1942-45 under the sub-title of ‘Nutritional Investigations’, p. 78.
I wish to thank Professor J. Bradley (Geology Department), Dr S. T. H. Scoones (Romance Languages Department) and Mr D. R. Winchester (Geography Department) for their assistance in several matters and also the Publications Committee of Victoria University of Wellington for a grant which offsets the increased cost of publishing this article as a single number of Tuatara.
I am most grateful to Mr P. J. C. Dart, representative of the British Council in New Zealand, for obtaining the information on the algal experiments in Mauritius.
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