A continuous culture apparatus for the mass production of algae

Aquaculture, 6 (1975) 319-331 o Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

A CONTINUOUS CULTURE APPARATUS FOR THE MASS PRODUCTION OF ALGAE

FRED E. PALMER,

KATHLEEN

Institute for Food Science Seattle, Wash. (U.S.A.)

A. BALLARD*

and Technology,

and FRIEDA

College of Fisheries,

B. TAUB University

of Washington,

*Present address: Fishery Biologist, U.S. Fish and Wildlife Service, Patchogue, (Received November 26th, 1974)

N.Y.

(U.S.A.)

ABSTRACT Palmer, F.E., Ballard, K.A. and Taub, F.B., 1975. A continuous culture apparatus for the mass production of algae. Aquaculture, 6: 319-331. A multi-stage, continuous culture apparatus has been designed and tested for the production of algae for larval molluscs and crustacea. A single-line system produced a maximum of 2.4 x 10” cells/day, or 5 g ash-free dry weight of Monochrysis lutheri. Multipleline systems are recommended for hatcheries. The flow rate affected algal cell density, yield, biomass, protein level, and residual nitrate. Maximal cell yield occurred at 10 1 flow per day, a dilution rate of 63% of the volume of the first growth carboy, or 30% of the volume of the total system. The system is also adaptable to growth of larger planktonic algae or mixed cultures of algae and protozoa and/or rotifers.

INTRODUCTION

The increasing commercial interest in the hatchery propagation of molluscs and crustacea for human consumption has made it urgent that more modern methods be developed for providing feeds for these invertebrates. Considerable success has been attained in finding suitable diets of algae for clam and oyster larvae and in providing methods for producing large amounts of feed. However, as these methods are commonly practiced, they require extensive labor and space and are frequently unreliable. Batch cultures are currently used in most hatcheries. Algal cells are inoculated into large tanks of enriched seawater, incubated with illumination for several days, and then harvested (Loosanoff and Davis, 1963). Charact@ristically, production in batch cultures is initially limited by low cell density and subsequently by inadequate remaining nutrient and self-shading; Continuous culture is better suited to hatchery production because (1) growth rate and production can be maintained near maximum in a few growth vessels, thus reducing the space requirement; (2) cells of more uniform and

Air ond Cop flow meters

-

L

1111

3 Fig.1. A. Configuration of the nutrient and seawater supply. One volume of nutrient concentrate was added with two volumes of 50% artificial sea water by using a narrower and a wider tube through the pump. Each carboy has a filtered air inlet. The position of the yield collection bottle is shown behind the gas tank. The CO, is mixed with compressed air from the pump shown on the left. B. A top view of the unit with a double line of carboys., The second set of fluorescent lights is not shown, so as to minimize confusion with the gas lines. (Engineering drawings and detailed specifications of components are available at the cost of copying from the Washington Sea Grant Communications Program, Division of Marine Resources, University of Washington, Seattle, Wash. 98195, U.S.A.)

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controlled quality are produced; and (3) the culture unit lends itself to automation and thus reduces labor. Study of a single-line unit was adequate to explore the influence of culture conditions, but it is anticipated that multiple-line systems would be used in hatcheries to increase the total output and to assure cell production during routine maintenance of the units. METHOD AND APPARATUS

The alga used was Monochrysis lu theri. The continuous culture algal production line consists of three growth chambers (carboys) connected in series and illuminated with fluorescent lights. These chambers are supplied continuously with a mixture of COz in air; fresh nutrient is supplied to the first growth chamber (Fig.1). Culture is forced by gas pressure from each growth vessel through an overflow tube. Nutrients are metered from the reservoir to the first growth vessel by a peristaltic-type tubing pump. In the following description it should be understood that the frame, lights, and shaker will accommodate two parallel units (FigA), although a single-line unit was used in these studies (Fig.2). Conventional 20-I (5-gallon) borosilicate glass (Pyrex brand) carboys were chosen for the first growth vessel and medium reservoirs, and 10-l (2.5-gallon)

Fig.2. Photograph of the recommended

algal production

unit with a single line of carboys.

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carboys were used for the second and third growth vessels, where denser algal cultures would greatly reduce light penetration. The vessels are closed with No. 12 green SteriI Kaps (Baltimore Biological Laboratories), bored to accept glass tubing for sampling, medium additions, culture output, and other functions. Surgical grade silicone rubber tubing is used where flexible tubing is desired and is connected to glass tubing by Beckmann unions (Beckman Instruments, Fullerton, Calif.) of teflon and aluminum. This coupling system reduces the risk of plastic tubing slipping from the glass or permanently binding to it, and the system is autoclavable, well protected from contamination, and non-toxic. All glass tubing is 6 mm OD except for the overflow tubes used to transfer culture out of the culture vessels; these tubes are 8 mm OD to minimize back pressure. Illumination is provided continuously by 8 cool white 40-W, high output fluorescent tubes (F48 T12 CW HO, General Electric Company). The intensity is about 2500 f&candles (1 ft-candle = 10.76 lux) from each side, measured at the center of the unit, using a cosine corrected G.E. 213 meter with the blue filter removed. A mixture of 2% CO? in air is obtained by mixing compressed COz with pumped air. Rates of flow are controlled with pinch clamps and monitored with flow meters, and the COz is passed through a valve, which closes if the power to the air pump is interrupted. Nutrient medium is provided via silicone tubing by a Model 375 Sage Instruments roller pump, if use of a single-line unit is intended. Otherwise, a Model 1201 Harvard apparatus finger pump is used in a multiple-line system. The latter pump is recommended for a double system, as shown in Fig.1, because of its more powerful motor. The growth vessels are mounted on an Eberbach Corp. shaker, Model 5900. The structural components are perforated angle steel and plywood. Culture medium The growth medium stock concentrate is prepared in two portions. Solution I consists of KH2P04, 20 g/l; thiamine HCl, 0.2 g/l; biotin, 1.0 mg/l; and vitamin Blz, 2 mg/l. Solution II consists of Na,EDTA (the disodium salt of ethylenediamine tetra-acetic acid), 7.9 g/l; FeCl, 6Hz0, 6.4 g/l; NaN03, 150 g/l; and trace metal mix, 5.0 ml/l. The trace metal mix has the following composition: CuClz 5Hz0, 3.90 g/l; ZnSO,, * 7Hz0, 8.8 g/l; CoClz - 6Hz0, 4.4 g/l; MnClz* 4Hz0, 72.0 g/l; and (NH4)Mo04, 2.5 g/l. Stock solution III, composed of NH4Cl, 25.0 g/l, is included in the original instructions (Davis, 1960) but was omitted in these experiments. All stock solutions are prepared with distilled water, sterilized by autoclaving, and stored under refrigeration. The continuous cultures received a mixture of 1 part concentrated nutrient solution and 2 parts dilute artificial sea water (50% Instant Ocean, Aquarium Systems, Inc., Eastlake, Ohio). The nutrient solution contained 54 ml each of solutions I and II in 18 1 of autoclaved distilled water. The artificial sea water is prepared by dissolving the dry salt mix in hot tap water. By using narrower silicone rubber pump tubing, 0.635 cm OD (0.318 cm ID) for the nutrient l

l

323

solution and wider tubing for the artificial sea water, 0.794 cm OD (0.476 cm ID), one part of the nutrient solution was added to two parts of the sea water. This procedure results in the appropriate nutrient concentration (0.1% of the concentrations of the stock solutions) and a final salinity of about 1.3°/00, which gives good results with M. Zutheri. Other species may require higher salinities. Since only the nutrient concentrate is handled aseptically, and twothirds of the flow may be considered “pasteurized” at best, the time and labor involved in sterilization is lessened. Reduction in the volume of aseptic nutrient solution would further reduce labor. The culture is started by inoculating about 11 of an axenic culture of algae into 16 I of sterile medium in a carboy. This carboy culture may be preincubated or immediately placed in the unit and all tubing attached. A flow of 2% COZ (0.2 to 5.0 I/min) to each vessel will give good results. The speed and stroke of the shaker can be adjusted so that good mixing occurs in both the 20-l and 10-I carboys. The culture volume contained in the carboys are maintained at about 16 1 in the first stage and about 7 1 in the second and third stages. As long as nutrients are flowing into the first stage culture, the overflow tube will maintain approximately the same volume on the shaker as it would if the culture were static. The output from the third stage may be delivered directly to the animals to be fed, or it may be collected and stored, or a feedback system may be used to increase production before delivery of the yield. The feedback of algal cells from the final growth flask back into the initial growth flask was accomplished by the addition of another outlet tube inserted into the culture in the final flask and connected by plastic tubing back through the metering pump. This volume is not counted in the yield. Feedback techniques are often used in sewage plants to assure the maintenance of an adequate density of cells at high flow rates. The theoretical relationships between dilution rate, population density, and cell yield in chemostat continuous culture have been described by Herbert (1964) and Kubitschek (1970). Properly defined, a chemostat is a culture whose growth rate is controlled by the concentration of a single limiting nutrient; the cell density declines only slightly as the system flow is increased, until at a point approaching the maximal growth rate, the cell density declines sharply with increasing flow. The growth rate of the cultures described here is limited by light as well as nutrient at operational cell densities. Growth rate may be better described by an equation such as P’Pmax

L-S ( L(K, + S) + SK1 )

where = instantaneous growth rate (cc = (1nN - ln.No)/t expressed as specific /J growth per day, equalling dilution rate at steady state conditions). = maximal growth rate of algae. Pmax

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= light intensity at the mid-depth of the culture (as can be calculated by measuring the light absorbance and applying Beers laws). s = substrate (nitrate) concentration. = concentration of nutrient that will yield l/2 maximal growth if light KS is not limiting. = light intensity that will yield l/2 maximal growth if nutrient is not Ll limiting. This equation was developed by D. McKenzie, who found it adequate to describe growth of Chlumydomonas reinhardtii growing in 0.5-l continuous cultures (Taub, 1973). L

RESULTS

AND DISCUSSION

Results show that algal cell density increases in successive carboys (Fig.3). These densities were maintained as approximate steady states. The greatest growth is produced in the first carboy (16 1 of culture), where nutrient enrichment is introduced; additional growth occurs in the cells that have been washed over into the second and third carboys (7 1 each), as the cells have additional time to use residual nutrient and absorb light. Cell density decreased in all carboys as the flow rate was increased beyond 6 1. The decrease in density was slight between flow rates of 6 and 8 1, but dropped more rapidly as the flow rate was further increased, especially beyond 16 l/day. However, even at 30 l/day the cultures maintained a steady state; p = 30/16 =

LT g G.o_ I z D

ALGAL

POPULATION

DENSITY

VS. FLOW

_

VESSEL

3

-

VESSEL

2

-

VESSEL

I

FLOW

RATE

RATE

(liter/day)

Fig.3. Density of Monochrysis cells over a range of flow rates. The density is consistently higher in the downstream carboys.

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1.9/day. This gradual decline of population density with increasing flow, over a broad range of flow rates, supports the hypothesis that light, as well as nitrate, was also a limiting factor in these cultures. As cell density decreased, more light penetration occurred, permitting a higher growth rate and maintenance of steady state conditions. The maximum cell production of 2.1 x lo1 ’ cells/day(density X volume produced) occurred at 10 l/day (Fig.4). At lower flows, cell production was less because a smaller volume of only slightly denser suspension was produced. The daily production of 2 X 10” cells/day from this 30-l continuous culture equals the harvest of a 1 000-l batch culture of 2 X 10’ cells/l. The equivalent yield by batch culture would require 10 days of handling through transfers from flask to carboy to tank. Therefore, the replacement of lower density batch cultures with high density continuous cultures should reduce the volumes of culture medium needed, the space required, and the time for prolonged grow out. Further reduction in labor should be possible, since the continuous culture method is more amenable to automation. Pumping a portion of the dense, nutrient-starved cells of the last carboy into the first carboy (Fig.4) did increase daily production to 2.4 X 1011 cells/ day at’a flow rate of 18 l/day and a backfeeding rate of 1.35 l/day, or 7.5%. Since the 1.35 1 is diverted from the last flask, its cells are not collected in that day’s .yield. However, this is only a. 20% daily increase over the produc-

24

o 7.5 % FEEDBACK

1 o 9.4%

2

4

6

9

10

12

FEEDBACK

14

16

FLOW

I6

(I/day

20

22

24

26

26

30

32

I

Fig.4. Daily production of Monochrysis cells (cells/day = cells/ml x ml/day) over a range of flow rates. The solid circles correspond to the data in Fig.3. Note that maximal daily production occurs at a faster flow rate than that providing maximal density (Fig.3). The open circles refer to production levels at given flow rates where percentage feedback was varied (percent = volume of feedback from third carboy to first, divided by daily flow, e.g., 9.4% - 0.94 l/10 l/day).

14.5 12.0 7.5 3.1

15.5 16.0 12.0 5.1

12.0 14.0 20.5 17.0 7.5

10.4

From Unit I Protype (Ballard, 1972).

10.7

5.5 10.2 14.0 23.5 2 2 4 6

560 000 600 400 100

670 1900 3 200 5 800 6700

2 680 2 800 3 700 6400 9000

3

1

3

1

2

Daily production ash-free dry w (mg)

Cell density (X lo9 cells/l)

3.0

Flow (I/day)

31.8 33.1 14.9 23.2

29.9

1

Protein (%)

24.7 42.7 20.5 24.0

24.5

2

27.8 45.0 27.0 26.7

23.6

3

13.2 1 800 2 300 3 172

1

0.4 527 2 175 2 935

2

NO; in culture medium (erg atm/l)

1.2 2.5 1 500 1363

3

Cell density, daily production of biomass, percent protein, and residual nitrate in stages 1, 2 and 3, at different flow rates

TABLE I

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tion at 10 l/day without feedback. The increased cell yield is obtained at the cost of slightly greater complexity and increased risk of tubing failure in the pump. For hatchery use, production units should be managed for maximum daily production of dry weight and protein, not for maximal cell density or maximal growth rate. The relationship between flow rate and cell density, daily production of biomass (ash-free dry weight), percent protein, and residual nitrate (Table I) was studied on an earlier prototype apparatus designated Unit I (Ballard, 1972). Although cell production was maximal at 10 l/day, there was a lack of agreement between cell production and biomass production, caused by variations in weight per cell. Cell weights varied widely, from 1.7 to possibly as high as 8 or even 14 X lo-* mg/cell; the heavier cells tended to occur at the faster dilution rates. Some of the variation may have been an artifact of the weighing method; possible sublimation of unrinsed salts, such as nitrates, may have occurred during the ashing process. A different estimate of biomass should be used in future methods, e.g., organic carbon should be measured by an infrared COz analyzer. To the extent that optical density is a good estimate of biomass, hatcheries could use it to estimate yield and feeding levels. In repeated experiments on earlier prototypes (Ballard, 1972), maximal cell density always occurred at approximately 10 l/day, but not all densities were as high as in Unit I. The percent protein in Unit I (Table I) varied from 15 to 45% of dry weight. This variation agrees with the range of 21 to 53% reported earlier in smaller cultures (Taub, 19’71), but it did not occur in a high-to-low sequence as it had in the smaller cultures. In l-l cultures, the highest proportions of protein occurred in the first flask and at the highest rates of flow. In the larger cultures, highest protein production tended to occur in the second or third carboys at the higher flow rates. The more intense self-shading may be responsible for the altered relationship between protein and biomass. The protein level tended to remain constant from day to day while the flow rate was constant. Kern (1973) studied the effects of varying protein levels of M. Zutheri and starch mixtures on juvenile oysters (Crussostrea gigus); oyster growth was not significantly reduced unless the mixtures were less than 14% protein. It is therefore important that the algal protein level is known, before it is mixed with a protein-free material such as starch. The residual nitrate in Unit I (Table I) was higher in the first carboy and increased with faster flow rates. Although the medium was designed to have 2 000 pg atm/l of nitrate, as high as 3 172 ,ug atm/l was recovered. This increase was probably due to evaporation of water during autoclaving and during culturing with bubbled dry air. The residual nitrate suggests that cultures could have produced more protein and biomass, given more time in the light. The residual nitrate might create a problem in the feeding waters, although the algal suspension would probably be highly diluted prior to the addition to oyster or clam tanks. Alternatively, lower concentration of nitrate might be used, since the concentrations are much above the rate-limiting con-

328

centration. More detailed information on these and other experiments can be found in Ballard (1972). Thus, using continuous cultures, it is possible to provide a more uniform product day by day by selecting growth conditions to obtain the desired composition of the product. For example, by selecting the flow rate, a product of high or low protein cells could be produced consistently. Light intensity or initial nutrient substrate could also be used as controlling variables. It is a characteristic of batch cultures, on the other hand, that their composition will vary continuously as they get older (Taub and Dollar, 1965). In practice, this may mean that if batch cultures are used, the entire culture must be used each day if it is considered desirable to provide a uniform feed. Hatcheries may find it desirable to decrease the number of culture stages to two growth vessels per line and to increase the number of parallel lines. The highest growth rate does occur in the first carboy; however, its output is more subject to oscillations. In part, the selection of a 20-l bottle, followed by two 10-l bottles, was made to increase the exposure of the cells to light. The light intensity of all eight tubes is inhibitory to M. Zutheri cells unless the cultures are sufficiently dense to provide significant selfshading. However, if the cultures are dense, nearly all of the light is absorbed before reaching the interior of the culture. In the first stage culture, if the cell density is 7.5 X lo9cells/l and light reaching the most exposed cells is 2 000 ft-candles, the light reaching the center of the culture is 60 ft-candles (Ballard, 1972). It was found that below 10 X lo9 cells/l, better growth was obtained using six fluorescent tubes and that above 20 X lo9 cells/l, the use of all eight tubes could be recommended. Operation of the unit on a shaker had no direct effect on production except that algae had less tendency to attach to the vessel walls, which resulted in longer periods of operation between cleaning the vessels. The two shakers we tested required frequent repairs after they had been in service about 1 year. It was found that algal growth on the vessel walls could be effectively scrubbed free by use of fiber scouring pads wrapped around teflon-coated magnetic stirring bars inside the vessels and manipulated by a strong, handheld permanent magnet outside the vessel (Oswald et al., 1962). An ambient temperature of 25°C was as high as could be tolerated by M. Zutheri, using this unit. Culture failure due to higher temperature occurred during the summer. Slightly warmer ambient temperatures would be satisfactory if the fluorescent tube ballasts were placed on the outside of the growth unit, since they are a major source of heat. However, our results agree with those of Ukeles (1961) that 27°C is about the maximum temperature at which M. lutheri will grow. The major labor required to operate the production unit is to supply sea water and nutrients. If the sea water used in the other hatchery areas has been treated by filtration so that the water is essentially free of live algae and protozoa, no further treatment is required. Highly concentrated pasteurized _or sterile nutrients can be mixed with the sea water by metering pumps, thus

329

minimizing the frequency of replenishing the nutrients. If such sea water is not available, it is necessary to remove or kill the algae and protozoa. A pasteurization temperature of 66°C (150°F) for 30 min is satisfactory, or a higher temperature for a shorter time may be used. This heat treatment could be done continuously as part of an in-line system. The remaining maintenance consists mainly of changing the 50 lb COZ tanks (9 X 55-inch cylinders of 2% CO* usually last several weeks), monitoring flow rates, and inspecting tubing and connections for leaks. It will occasionally be necessary to replace operating vessels with clean ones. It is good practice to always have on hand a carboy containing a culture from 1-3 weeks old. If it is necessary to replace the vessels, the standby culture and clean second and third stage vessels with clean tubing can be connected into the system with little loss of production. Over a period of about 1 year, the average duration of uninterrupted production was about 6 weeks. In hatchery use, where the testing of equipment is not the objective, the unit should operate for longer periods without interruption. It is recommended that the cell density in the output be monitored in some way such as counting in a hemocytometer or by measuring turbidity. If visual observations are relied upon, a subsample of standard depth should be compared to colored standards. Visual observations of a deep tank with the unaided eye are often misleading and may contribute to operation of the unit at a poor level of production. If cell numbers per unit volume are lower than expected, the operator can investigate potential sources of trouble such as poor quality water, poorly prepared nutrients, or contamination. Extracellular algal products of M. Zutheri are not expected to be a problem; Heller (1970) demonstrated that only about 5% of the fixed carbon in rapidly growing cultures was excreted as organic material. Monochrysis from these units has also been used to cultivate the protozoan Parauronema virginianurn Strain 211, the rotifer Brachionus plicatilis, and the copepod Tigriopus sp. Combined continuous cultures of a freshwater alga and protozoan (Taub, 1973) suggests that stable mixtures of an alga and herbivore can be grown at certain flow rates. Since mixtures of organisms may provide a more balanced diet, mixed cultures may be useful. Our experience indicates that increased output of practical significance would be difficult to obtain from a unit of this basic design, and then only at the cost of increased complexity. The benefits of using larger growth vessels are decreased by the light loss in the culture. In general, the use of additional production units would be more effective than attempting to use a largerscale unit of similar design. It is recognized that this design is simple and unsophisticated. However, a unit of this type is easily constructed and need not be expensive, while the design and testing of more advanced designs may be some time in the future.

330

ADDENDUM

Over $30 x lo6 has been invested in the design of mass algal culture units; almost all of the technical information has been published in the open literature. Some modifications of the earlier units would be necessary, since most were designed for a high temperature, fast growing strain of Chlorella. The production rates often were expressed as O2 produced; the dry weight of algae can be estimated by multiplying by 0.94 = (180 g of algae)/(192 g of 0,). Miller and Ward (1966) provide a review of more sophisticated mass algal culture units, most of which were designed for space vehicle life-support systems. Hatcheries wishing information beyond that review may request a literature search on algae by National Aeronautics and Space Administration, Scientific and Technical Information Office, Washington, D.C. 20546, or by the National Technical Information Service, U.S. Department of Commerce, Springfield, Va. 22151. ACKNOWLEDGMENTS

Special thanks are due William Doke, who made many engineering improvements over earlier prototypes, built the unit pictured, and drew Fig.1. Mr Dale Kalamasz assisted in the preparation of media and other support activities. The work was supported by the Washington Sea Grant Program under the National Oceanic and Atmospheric Administration, U.S. Department of Commerce. College of Fisheries Contribution No.427.

REFERENCES

ProductionCharacteristics of ThreeContinuousCulturesof lMonochrysislutheri. Thesis, Univ.Wash.,Seattle,Wash.,43 pp.* Davis,H.C., 1960. SeaWaterEnrichmentMedium.U.S. Bur.Comm. Fish.Biol. Lab., Milford, Conn., mimeographed. Heller,J., 1970. The extracellularproductsof Monochtyis lutheri and theirutilizationby bacteria.Thesis,Univ.Wash.,Seattle,Wash.,83 pp.* Herbert,D., 1964. Multi-stagecontinuousculture.In: I. Malek, K. Beran and J. Hospodka Ballard, K.A., 197 2. The

(Editor), Continuous Cultivation of Microorganisms. Proc. 2nd Symp. Czechoslovak Acad. Sci., Prague. pp.22-44. Kern, R.B., 1973. A survey of some potential artifical foods for juvenile oysters. Thesis, Univ. Wash., Seattle, Wash., 67 PP.* Kubitachek, H.E., 1970. Introduction to Research with Continuous Cultures. Prentice-Hall, Englewood Cliffs, N.J., 195 pp. Loosanoff, V.L. and Davis, H.C., 1963. Rearing of bivalve molluscs. In: F.S. Russell (Editor), Advances in Marine Biology. Academic Press, New York, N.Y., Vol. I, pp. l-136.

*Available at cost of copying from the Washington Sea Grant Communications Program, Division of Marine Resources, University of Washington, Seattle Wash. 98195, U.S.A.

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Miller, R.L. and Ward, C.H., 1966. Algal bioregenerative systems. In: K. Kammermeyer (Editor), Atmosphere in Space Cabins and Closed Environments. Appleton-CenturyCrofts, New York, N.Y., pp. 186-222. Oswald, W.J., Golueke, C.G., Brewer, J.W. and Gee, H.K., 1962. Microbiological waste conversion in control of isolated environments. USAFCambridge Res. Lab., Off. Aerosp. Res., Bedford, Mass., (ASTA 278079, 46 pp. Taub, F.B., 1971. Algal culture as a source of food. Proc. First Annu. Workshop, World Mariculture Sot., 1970, pp. 101-117. Taub, F.B., 1973. Biological Models of Freshwater Communities. Ecological Research Series, EPA-660/3-73-008, 00 pp. (Available from the Superintendent of Documents, U.S. Government Printing Office, Washington, D.C. 20402.) Taub, F.B. and Dollar, A.M., 1965. Control of protein level of algae, Chlorella. J. Food Sci., 30: 359-364. Ukeles, R., 1961. The effect of temperature on the growth and survival of several marine algal species. Biol. Bull., 120: 255-264.