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Chemical Engineering Problems in large Scale Culture of Algae
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PAUL M. COOK S T A N F O R D RESEARCH INSTITUTE, STANFORD, CALIF.
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RINCIPLES of chemical engineering apply to biological slant culture furnished by the Plant Biology Division, Carnegie as well as chemical processes. This paper is concerned with Institution of Washington, Stanford, Calif. I. Chlorella is a unicellular green alga. The individual cells are solitary and the use of these principles in studying the possibility of growing spherical in shape, normally 2 to 10 microns in diameter. Realgae on a large scale in a controlled process. The abundance and rapid growth of algae as observed in nature and in fundaproduction takes place through the formation within the adult cell of autospores, usually 2, 4, 8, or 16 in number. These automental studies on photosynthesis led to the suggestion of possible large scale algae culture to supplement the supply of protein and fat. spores become complete cells and are then liberated by rupture A survey [Stanford Research Institute Project, Preliminary Study of the adult cell wall. Requirements for photosynthesis in liquid medium include of Large Scale Chlorella Culture (1949), sponsored by Research Corp., New York] investigating the technical and economic carbon dioxide, water, light, mineral, and micro nutrients. feasibility of an algae culture process showed that although mass Recent studies (6) have shown that the chemical composition of growth appeared techniChlorella can be altered by cally possible, data were changing the environmental I n the development of a process to culture algae on a large conditions under which it is insufficient for an economic scale for the possible production of feed, many chemical grown, Normal, r a p i d l y evaluation. Several novel methods were considered engineering problems must be solved. Research a t the growing cells have a protein for obtaining m a x i m u m Stanford Research Institute using the organism Chlorella content of over 50%. By reducing the concentration pyrenoidosa has resulted in the development on a laboragrowth. of fixed nitrogen in the cultory scale of a continuous process for growing algae. The An experimental program ture medium, cells with a system maintains constant known optimum conditions [Stanford Research Instilipide content of over 85% for maximum growth. A tentative design for a large tute Project, Development have been grown. Other scale process is based on limited experimental data and of Chlorella Culture Procstudies ( I ) with several difseveral assumptions. ess (1950), sponsored by ferent organisms determined Laboratory experiments using glass columns have been Research Corp., New York] growth and reproduction carried out with both artificial light and sunlight, Temwas undertaken with the rates and found Chlorella perature, carbon dioxide aeration, total culture volume, objectives of determining cell population density, nutrient concentration, and agitapyrenoidosa to give the highthe optimum conditions for tion conditions can be held constant. The variables have est yield. maximum growth and designing a tentative large been studied to determine optimum growth conditions. Growth of algae in the The influences affecting the growth of Chlorella have past has been accomplished scale process for engineerbeen considered in detail and a tentative design has been largely on a batch basis. A ing and economic evaluabatch culture is one which drawn up for a process to grow algae on a large scale. The tion. is started with a small indesign divides the process into two primary parts: the oculum and is allowed to culture farm and theprocessingplant. In theculture farm, GROWTH OF ALGAE grow for extended time where the growth takes place, a continuous system conperiods without adding or stantly maintains optimum conditions by supplying fresh The science and engineerremoving anything from the medium and continually harvesting the algae. In the ing of natural growth procc u l t u r e . Thus conditions process plant the harvest is separated from the spent esses have developed slowly within the culture are conbecause of the very large medium and dried. stantly changing as the alga number of uncontrolled or grows, removing its requireeven unknown v a r i a b l e s ments from the medium. The many data on growth and influencing growth. Because of the simplicity of the system yield of Chlorella were difficult to correlate, because no continin which algae grow, scientists have used them for years in uous control on the conditions within the culture was provided. fundamental studies of photosynthesis. However, even with Analysis of growth in batch cultures, however, did indicate that algae many more variables must be considered than in the during a small period of the life of the culture, the alga grows most complex chemical engineering process. The organism selected for use in these studies, and about which . very rapidly. By maintaining the conditions in the culture constant a t this point of maximum growth, it was possible that a considerable fund of information has been accumulated, is considerably greater yields could be obtained. Chlorella pyrenoidosa [(Emerson’s strain) obtained from an agar
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INDUSTRIAL AND ENGINEERING CHEMISTRY CONTINUOUS CULTURE SYSTEM
An experimental apparatus was designed that would maintain constant conditions within an algae culture. The basis for this system !vas a cont,inuous supply of nutrient and a continuous removal or harvest of cells. Thus a constant number of cells would remain in the system, the average age of the cells would be const,ant, and the nutrieiit,s in the medium would he held at a fixed concentration.
Vol. 43, No. 10
nesium sulfate, and 0.018 M potassium dihydrogeri phosphate. Iron was not added, because numerous tests showed that the tap water used contained iron in concentrations sufficient for mRsimum growth. A column constructed for operation out of doors is similni, in construction and operatiou to the art'ificial light colunii~. I t is mounted on a pivot so that the column may be operated noriiial to sunlight,, in a vertical position, or at any angle desired, Population density is maintained constant by recycling culture through a cell enclosed in a black cylinder cont,aiiiing a constant-ixitr:i~sity light source and a photoelectric cell. This circuit activatos a solenoid which controls the entrance of fresh medium as s h o w in Figure 2. A photograph of the sunlight column in opei,atioii is shown in Figure 3. GROWTH UNDER CONSTANT CONDITIONS
In a continuous system n-here the population density is held constant, the amount of growth is measured by the oveiflocror harvest obtained per unit time, from a conftant weight of reproducing and groning cells. Thus rate of hi2rwht. Kr1, is defined as:
where V Ois the volume of overflow, V , is volume of cuituie, and t d is time in days. The yield in grams per liter per day, Yd, is thP product of the rate of harvest, Kd,and the population den-
PHOTO TUBE
CULTURE OVERFLOW Figure 1. Continuous Culture Apparatus Artificial light 4-inch column
A borosilicate glass column 4 inches in diameter and 6 ieet in height was constructed. This column, shown schematically in Figure 1, is illuminated by three 100-watt fluorescent lights, placed about 0.75 inch from the column wall, and spaced 90' apart around the inside of a sheet-metal reflector. A cooling tube is sealed into the top part of the unit and extends almost to the bottom. Temperature is maintained by a temperature controller which regulates the flow of cooling water. Aeration is provided by means of an inlet tube which enters at the bottom of the column. Two other tubes are sealed into the column near the bottom: one for the introduction of new medium, and the other for sampling. Volume of the culture is maintained constant by an overflow outlet at a level of 10 liters. The population density of Chlorellu in the culture is held constant by means of a photoelectric cell circuit which activates a solenoid valve allo~ring the entry of ne\v medium. This medium is supplied from glass bottles as indicated in Figurc 1. A continuous supply of sterile medium is available from the supply bottles. The aeration mixture is controlled by pressure reducers and needle valves on sources of compressed air and carbon dioxide. Each gas is metered through manometer flowmeters. Cotton gas filters are used, all connections are sterile, and the usual aseptic techniques are employed. This culture apparatus assures a closed, sterile system. The composition of the medium used in all experiments reported here was 0.025 2c;r potassium carbonate, 0.020 M mag-
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Population Density Control in Contiriiioiis Culture Process
sity, D,, in grams per liter, and is expressed as grams of material grown in a given period of time per unit volume of cwlture. Yield is defined here as:
The rate of harvest of Chlorella when maintained under constant culture conditions is a function of the population density and light intensity. ,4s the light conditions in t h e artificial
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light column are constant, experimental results are reported in terms of population density. The result of one typical experiment is shown in Figure 4, where volume of overflow is plotted against time. The increase in volume of overflow is constant with time and the slope of the line is the rate of harvest on an hourly basis. In several experiments, particularly a t high population densities, a considerable period of time was required to reach equilibrium conditions and to obtain increase in overflow volume per unit time. Figure 5 shows the rate of harvest versus population density for several experiments conducted in the artificial light column with optimum culture conditions as determined to date. Yield is plotted against population density in Figure 6 for these same results. This curve shows a maximum yield at a density of 0.36 gram dry weight per liter. The maximum production obtained from such a system is 0.48 gram dry weight per liter each day. Similar data have been obtained with the sunlight column. In this case the light condition is not constant. The results of one experiment are shown in Figure 7 , where overflow volume has again been plotted against time. The daily weather conditions are shown as a qualitative indication of light conditions. This experiment was carried out a t a population density of 0.27 gram per liter with the column in a vertical position. The daily average yield for this experiment was 0.279 gram per liter and the yield for clear days was 0.351 gram per liter. OPTIMUM CONDITIONS O F CULTURE
The optimum nutrient and culture conditions for maximum growth as found in this study do not vary greatly from present common practices of Chlorella culture. Five per cent carbon dioxide in air appears to be the best aeration mixture. There was no detectable change in rate of growth when the carbon dioxide was varied Z!Z 1 % in the author's apparatus. An optimum temperature of 25' C. confirms some results given in the litera-
Figure 3.
Continuous Culture Column Operating in Sunlight on Roof
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PRO C E S S CHLORELLA PYRENOIDOSA 40 ARTIFICIAL LIGHT lGURE 4 4"CULUMN V,-lO LITERS 0,s .362 GRAMS /LITER Run started- 4PM, 10-17-49
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INDUSTRIAL AND ENGINEERING CHEMISTRY
ture. An aeration rate of 3 cubic feet per hour or more in the 4-inch column is optimum. The mineral nutrients are optimum over a wide range. By using water from different sources, different rates of growth were obtained even after supplementing with Arnon’s A-4 and B-7 micro nutrient solutions. It is apparent that the effect of micro nutrients on Chlorella growth is not fully understood and that increased growth rates might result from improved media.
CONTINUOUS CULTURE PROCESS C H L O R E L L A P Y R E N O IDO S A ARTIFICIAL LIGHT
intensity of the light. It also has been shown ( 2 ) that, at and above a certain critical intensity of light, a cell is light-saturated and has reached its maximum rate of photosynthesis. At a lower critical intensity, the maximum growth rate is reached (2, 3 ) . Therefore, we can expect to gain no further benefit from increased light intensity, with the exception of having more volume growing a t maximum rate oming t o greater penetration of light through the culture. I t has been demonstrated that a t various wave lengths and intensities of light a Chlorella culture closely follows BeerLambert’s law on the absorption of light. This means that a t high light intensities Chlorella absorbs considerably more light than can be used, owing to the light-saturation point of each cell. In a practical system, then, under high light intensities, a considerable portion of the light will be wasted. KO practical system has been visualized t o overcome this inherent difficulty. More data still must be obtained on the light-saturation point of
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CONTINUOUS CULTURE PROCESS C H L O R E L L A PYRENOIDOSA 0 SUNLIGHT ”FIGURE 7 4” C O L U M N Vc=12 LITERS Dc=0.27 GRAMS/LITER Daily overflow and weather conditions shown.
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COYSIDERATIONS I S LARGE SC4LE PROCESS DESIGS
In developing a process for the large scale culture of Chlorella,
it is necessary first to consider the limitations as determined by nature. Because solar radiation provides the energy, sunlight as received on the earth a t the particular geographical location in question is the fird limitation. The mechanism of photssynthesis and the physiology of the organism to be considered are the second. Within these parameters a process must be developed which will most efficiently produce organic matter using the sun’s energy. In other words, how can we most efficiently use sunlight to synthesize organic matter? From a study of solar radiation and a study of the mechanism of photosynthesis as it occurs in the organism Chlorella, a tentative large scale process has been developed. Only the more significant factors contributing to this design are cited here. Light. Light from the sun supplies the energy for the conversion of carbon, oxygen, hydrogen, and nitrogen to Chlorella. The intensity of sunlight varies from time to time over a wide range, so that there is no one light condition to be studied in reference to maximum conversion. I t has been demonstrated ( 4 ) that Chlorella exposed to light to high intensity will grow rapidly a t first, then at a diminishing rate until injury takes place and death of the cell occurs. For a given cuIture the completeness of this cycle is a function of the length of exposure and the
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Chlorella under various conditions, but it is now clear that as much as 85% of the light is wasted under certain conditions of population density and culture, even though the culture is deep enough to permit light extinction. This seriously limits the possible amount of conversion of light in the synthesis of organic matter. I n spite of this, experiments show a conversion of approximately 2.5% of the incident solar radiation in the production of organic matter. Some improvement map be expected from maintaining a greater depth of culture, inasmuch as the culture in the column is not absorbing all the light at tinies of maximum intensity. POPULATION DENSITY.For maximum production under the optimum culture conditions as determined to date, population density should be maintained at 0.36 gram dry weight per liter. This can vary 3~10% without an important reduction in yield. If conditions can be found which will cause but little loss of yield
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INDUSTRIAL AND ENGINEERING CHEMISTRY
while operating a t much higher population densities, certain obvious savings in equipment and processing will result. It should be one of the objectives of future work to determine conditions that will permit high production rates under more dense culture conditions.
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TURBULENCE. A necessary condition for optimum growth is turbulence or agitation. This serves three primary purposes: to keep cells in suspension and, therefore, a t uniform density; to maintain equilibrium conditions between the necessary nutrients and each cell; and to remove cells under high light intensity conditions to lower light intensities to prevent their injury and thus maintain them a t maximum growth rates. This can be obtained by mechanical or gaseous agitation or by turbulent flow conditions of the culture medium. ARCHITECTURE. It is important to know how much of the available sunlight actually reaches the Chlorella culture, as this must be the source of energy for a practical system. Because this value is a function of the architecture and the transparency of the material used, calculations were made for various shapes of both glass and plastics. It was desirable to compare (with a minimum of direct experimentation) the efficacy of various shapes of culture vessels as admitters of sunlight. The basic shapes investigated analytically were: 1. 2. 3. 4.
A flat horizontal surface A vertical cylinder An inclined cylinder always normal to sunlight A horizontal cylinder
Figure 8 shows graphically the amount of nonreflected energy transmitted into each of the four architectures considered. April 12 has been used because it represents the weighted yearly average of insolation a t Palo Alto. Light absorbed by the transparent material must be subtracted to calculate the light transmitted to the culture. Although the inclined cylinder is slightly more efficient than a horizontal cylinder, economic considerations show clearly that a horizontal cylinder or some modification thereof is the more practical architecture. OTHERCONDITIONS.Temperature must be maintained at 20" to 25' C. for maximum production. During periods at night with no illumination, no adverse effects have been observed after allowing the temperature to fall as low as 7 " C. A closed and sterile system is necessary to maintain an uncontaminated culture. There are strong indications that certain molds and bacteria will cause a considerable decrease in growth rates. A system not requiring sterile conditions would result in appreciable savings and should be the basis of more experimental work.
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TENTATIVE LARGE SCALE PROCESS
The major conditions have been considered and must now be incorporated into a simple, cheap, efficient system. This 12quires not only inexpensive construction and ease of control and operation, but also maximum efficiency. A continuous process is the most practical method of meeting such requirements. Conditions must be constantly maintained at the optimum point for maximum yield. Each cell must receive the required nutrients, proper population density must be maintained, and temperature must be correct. The process for large scale algae culture described below is only tentative, as it is based on data from laboratory experiments. The design was prepared so that preliminary engineering and economic calculations could be made. The results of these pixliminary evaluations were used to show what areas of research and development held the most promise for process improvement. In Figure 9 is shown a simple block flow diagram illustrating the tentative large scale continuous process. The design divides the process into two primary parts: the culture farm and the processing plant. Growth takes place in long horizontal tanks of the culture farm. Conditions are maintained at optimum by introducing fresh medium a t frequent intervals along the tank. Because growth is constant in all portions of the tank, it is necessary to provide an equal flow of fresh media into each inlet of the tank, thus maintaining constant ppulation density. With equal dilution throughout the tanks ut removal only at the end, the flow rate of the culture in the tank increases as the end is approached. To obtain proper agitation, the culture is recycled in sufficient quantity to obtain turbulent flow conditions. The necessary nutrients, including car-
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Figure 9.
Flow Diagram of Continuous Culture Process
bon dioxide as carbonate and bicarbonate, are introduced with the fresh medium. Additional carbon is supplied from an atmosphere of carbon dioxide and air maintained over the culture. The harvested culture flows to the processing plant, where the cells are separated from the medium by one of several possible systems. Waste combustion gases from the sterilization and dehydration processes supply enough carbon dioxide for growth requirements and one variation of the process calls for a carbonation of the spent medium. Make-up water, to which the necessary nutrients have been added, is sterilized and incorporated with the spent medium for return to the culture tanks. LITERATURE CITED
(1) Ketchum, B. H., Liliick, L., and Redfield, A. C., J. Cellular Comp. Physiol., 24, 120 (1949). (2) Myers, J., J. Gen. Physiol., 29, 419 (1946). (3) Ibid., p. 429. (4) Myers, J., and Burr, G. O., Ibid., 24, 46 (1940). (5) Spoehr, H. A., and Milner, H. W., Plant Physiol., 24, 120 (1949). R E C E I V ~April D 4, 1951. Presented before t h e Divisions of Industrial and Engineering Chemistry a n d Agricultural a n d Food Chemistry, Symposium on Chemical Engineering Aspects of Food Technology, a t the 119th Meeting of t h e AMERICANCHEMICALSOCIETY,Boston, Mass.
