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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
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
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.
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
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.
While Brown and Escombe were doing their work, a new theory burst upon the physical world when Max Planck announced his Quantum Theory. This stated that the heat radiation from a black body is emitted in discrete quanta of energy. Pronounced in this way, the theory would fail to switch any biologist on-until one realises that heat radiation from a black body refers merely to a selected range of the electro-magnetic spectrum of radiation which includes visible light, the energy source of photosynthesis. The theory therefore implies that light is composed of minute particles called quanta or photons: and it further states that when one electron is displaced by
where E = the energy released; v = the frequency of the light emitted; and h = Planck's Constant-the factor relating energy and frequency. A few years after the Quantum Theory was announced, Einstein showed that Planck's Equation could be applied to photochemical reactions. ‘The principle of photochemical equivalence is that, in molecular or atomic terms, the absorption of a single quantum of energy (the photon, hv ergs) is required to initiate a chemical process.’ (69)
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
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
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
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)
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.
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
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)
To some marine research workers, however, the connection between phytoplankton and copepod did not appear as direct as numerous people implied. G. (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
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)
Perhaps it would be as well at this point to investigate population growth a little more closely. Let us take a hypothetical case first, as set out in the figures in Table 1. We start with a culture inoculum of 20 individuals of a type which practises vegetative multiplication in which a mother unicell divides into two daughter cells. If we plot the logarithm of the total cell number against the time interval, we get curve A as seen in Fig. 1. This curve can be divided into six sections as marked:
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
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.
One other point is worth noting. Counts are given for 18 intervals of 12 hours. The average yield per 12-hour interval over these 18 counts is 1,046, which is approximately a quarter of the highest yield
Ketchum and Redfield set out to test the feasibility of obtaining the greatest possible production in the shortest time by determining the maximum daily yield of a diatom and maintaining the culture at this peak. They chose Nitzschia closterium. The maximum daily
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
‘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.
In that year, Butlerow discovered that formaldehyde under alkaline conditions would condense to form an optically inactive syrup showing some of the properties of hexose sugars. At that time it was known that the generalised formula for a hexose sugar was C6H12O6 or 6(CH2O) or 6(H.CHO). Baeyer in 1870 expressed the opinion that the syrup of Butlerow might originate through the condensation of six molecules of formaldehyde:
and suggested in fact that this might be the way in which grape sugar (glucose) was formed in the plant. Maybe because of the facileness of such an idea, Baeyer dreamed up a theory of photosynthesis which went as follows. Sunlight might split carbon dioxide into carbon monoxide and oxygen; the latter could escape and the former be bound to chlorophyll by some unexplained but fortuitous reaction. The carbon monoxide might then be reduced to formaldehyde by the hydrogens of water, and six molecules of formaldehyde could then condense to form a hexose sugar. Thus —
2O —→ 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
(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
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
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
(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
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.
Its ubiquity in nature would indicate that Chlorella is not too fussy about its nutrients. It can be grown on a completely inorganic medium without the addition of any of the vitamin B complex or other vitamin-like factors. It is truly 100% autotrophic; and the usual inorganic elements required in higher plant nutrition are sufficient sustenance. It is very catholic in its requirements for nitrogen — using either ammonium (which must be buffered) or nitrate ions; but it can utilise acetamide, urea, uric acid, peptone and several amino acids such as alanine or asparagine. Carbon dioxide is the usual carbon source but it will use bicarbonate. All the normal inorganic elements are required: hence chemicals containing phosphorus, potassium, magnesium, calcium, sulphur, iron, copper, zinc, manganese and several other trace elements are constituents of a suitable culture medium. The alga does not seem too particular about pH and the culture fluid can vary from about 6(89) to about 7.5.(86) It is expedient to use a chelating agent to hold the iron in solution and therefore metabolically available. To do this, citric acid or, better still, ethylene diamine tetraacetic acid is added in some form to
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.
Now we must diverge for a little to take in some theory which will enable us to follow the next stage in the Chlorella story. Photosynthesis is a reduction process; so the ultimate products of this process and their derivatives contain carbon which displays some degree of reduction. A scale for expressing the degree of reduction of carbon can be devised with carbon dioxide at the bottom with a reduction value of 0, since carbon dioxide is the most highly oxidised form of carbon in the photosynthetic process; and methane at the top of the scale with 100, since methane is the most highly reduced form of carbon. We can refer to a compound in terms of its R-value, meaning the degree to which it is reduced; and every carbon compound must fit somewhere along this scale. All organic compounds in the biological world will fall between these extremes, and their R-values can be worked out. A high R-value infers a high degree of reduction and a low value, a low degree of reduction. Here are some actual R-values, starting with the least reduced and proceeding to the highly reduced.
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
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
Chlorella could be varied at will.
For example:
‘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.
‘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
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
Let us look at two chains which man depends on for protein — those which produce fish and meat.
There are two points to focus on here. Firstly, as one goes along either of these chains (or any other) the energy value of a potential foodstuff in calories per gram hardly increases at all: an oil produced by a phytoplankton member will not be greatly increased in energy value (and hence food value) from an oil found in the last member of the chain. So there is no increase in food value with an increase in the number of steps in the chain. Secondly — and of much greater significance, in the change-over from one trophic level to another the total amount of energy passed on must diminish: one cannot have a greater mass of zooplankton than the mass of phytoplankton which can support it. Entropy sees to this! It is generally accepted that there is about a 90% loss in amount of potential food-stuff (i.e. potential energy for heterotrophs) at Each change-over in the chain. So:
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
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
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