Aquacultural Engineering 1 (1982) 205-214

CONTINUOUS PRODUCTION OF MICROALGAE USING A S C O U R I N G FILM R E A C T O R

T. H. ANDERSON Black & Veateh Consulting Engineers, Walnut Creek, California, USA and D. E. BRUNE Department of Agricultural Engineering, Pennsylvania State University, University Park, Pennsylvania 16802, USA

ABSTRA CT A new system for the continuous production ofmicroalgae is described and data from a series o f trial runs are presented. This system, termed the Scouring Film Reactor (SFR) is simple and inexpensive to construct and is operated by compressed air alone. This device utilizes a continuously scouring film o f glass particles to prevent wall growth and algal settling. Tests o f continuous algae production were conducted with a 40 fitre SFR and a non-scoured reactor o f similar size, using the marine alga Phaeodactylum tricornutum. Over a five-week period, production o f algae from the SFR was reliable and required no maintenance. Production from the non-scoured reactor was subject to crashes and required a high degree o f maintenance.

INTRODUCTION The uncertain reliability of algal cell production systems continues to pose a problem for many aquaculture operations in which single cell algae is the primary feed source for filter feeding aquatic organisms. Many other areas of research and development requiring continuous algal cell production also experience difficulty in meeting needs. Droop (1975) compares the use of batch and continuous systems for the production of microalgae. Continuous systems are shown to be advantageous over batch because: (1) the product is of constant composition and physiological state; (2) rates 205 Aquacultural Engineering 0144-8609[82/0001-0205/S02.75 Appfied Science Publishers Ltd, England, 1982 Printed in Great Britain

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T . H . A N D E R S O N , D. E. B R U N E

of production per volume are higher; and (3) less labour is required. Despite these advantages, however, batch culture is by far the more common method used in algae culture today. One of the main reasons for the prevalence of batch culture is that, even on a laboratory scale, non-sterile, continuous cultures have seldom been operated at a steady state for long periods of time. Often, the concentration of algae in the outflow of the reactor drops with time, a phenomenon known as a 'crash'. Algal crashes are attributed to autoflocculation and settling (Ryther, 1976), wall growth (Brune et al., 1978), and in situations in which the influent water is not filtered or sterilized, or the reactor not enclosed, predation may be a factor (Ansell et al., 1963). A variety of reactors have been used for the continuous production of algae, many of which have some provision for solving the settling or wall growth problenrs. Solution of the settling problem is usually accomplished by energy input to the media in the form of mechanical agitation (Brewer and Goldman, 1976) or compressed air (Mock and Murphy, 1970). Solution of the wall growth problem is accomplished by manual scraping (Dunstan and Menzel, 1971) or the more usual periodic draining, cleaning and refilling the reactor (Canzonier and Brunetti, 1975). Taub and Dollar (1968) used modified Erlenmeyer flasks containing glass beads in the continuous production of algae for periods of up to four months. Continuous shaking of tile reactor and subsequent scraping by the beads prevented settling and wall growth on the bottom surface. Occasional vigorous swirling prevented wall growth on the side walls. In the work presented here, settling and wall growth are prevented by using a reactor in which the walls are continuously and automatically scoured by a flowing film of glass beads. Experiments were conducted to compare the cell production characteristics of the Scouring Film Reactor (SFR) with a non-scoured continuous stirred tank reactor (NCSTR).

DESIGN METHODS AND MATERIALS

Figures 1, 2 and 3 give a detailed view of the construction of the Scouring Film Reactor unit (SFR). The SFR consists of three basic components; a tapered translucent fibre-glass cone, an air lift recircutation loop and a rotary distribution arm. The tapered cone was fabricated from 0-025 in thick 'sun-lite' fibre-glass solar glazing material obtained from the Kalwall Corporation. The cone was equipped with two ~ in marine plywood supports and a plywood flange for attachment to a moulded fibre-glass outlet. The recirculation loop consists of a }in clear plastic pipe ('Excelon') with a ~in PVC valve and drain tube. Approximately 3 in above the outlet level, the loop is equipped with an air injection port to supply the air lift action. The outlet of the loop is centred over the cone and supported by 'Plexiglas' struts. The rotary distribution arm consists of in PVC pipe, counter balanced and suspended from a

SCOURING FILM REACTOR

FOR MICROALGAE

PRODUCTION

207

(scelon

1/2" 90 e Ir Ibel eeigklse struts

~
"z : . , , ,-,.:~,

Fiberglass roiafm'cement Loyerl IOos fibdcllen Fiberglass fillet ~,/4" Morine plywood is bolted to support structure

1/2" Excelon pipe

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I/2"45 Elbow -~:-. 1 ~ / ~ I

025" t r onelucent

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, / 4 " PV C~volve
F i g . 1.

Flexible tubing ~-Hose clamp

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T . H . ANDERSON, D. E. BRUNE

1/3Z" Drill for suppocting pins .90" Elbow

I / 8 " Plastic rod 2-13/16" Stainless steel swivels

_ Plastic funnel Glued with silicone sealant I0-314' PVC pipe

PVC Counterweights f , - I / 8 " PVC Sheet 9 0 Elbow ' Excelon pipe

Fig. 2.

Side view o f Scouring Film Reactor distribution arm.

I/8"Orill

1/8"

=
13 %

6-i/2 ~

IFig. 3.

Top view of Scouring Film Reactor distribution arm.

SCOURING FILM REACTOR FOR MICROALGAE PRODUCTION

209

stainless steel swivel connected to the inlet pipe. Details of the arm are given in Figs 2 and 3.
Trial runs with S F R and N C S T R

Figure 4 shows a schematic diagram comparing the operation of the scouring film reactor and the non-scoured reactor. The operation of the SFR unit consisted of adding 125 ml of 100/~m glass beads, obtained from the Ferro Company, and injecting compressed air at a sufficient rate to maintain a water flow rate of approximately 4 litres/min to the distribution arm. A slurry of glass beads and algae flow into the external air lift pump and are transported to the distribution arm. Discharge at a 45 angle from the end of the distribution arm causes it to rotate. The beads and algae are then distributed along the wall where the beads settle rapidly, creating scouring action along all wetted surfaces. The beads reach the bottom of the reactor and recycle. In contrast, the NCSTR consisted simply of a cylindrical 'Plexiglas' column 122}in in diameter. Mixing was achieved by aeration with an air stone. Both the SFR and NCSTR were operated as continuous cultures for a period of five weeks. All pumping was done using a peristaltic pump (Cole Parmer Masterflex). In

- ~ - ~ _ - _ --,

'~Air input Oistribu arm

Light S0ufce

E x t e r n Q l oi r [ift pump

= oo --2---___

.o

I
NESTR

~Diffuser

SFR

Fig. 4.

Schematic diagram of Scouring Film Reactor (SFR) and Non-scoured Continuous Stirred Tank Reactor (NCSTR).

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T . H . A N D E R S O N , D. E. BRUNE

both reactors the inflow and outflow rates were held constant so that the volume remained constant at any given time. The dilution rate of the NCSTR was started at 0.33/day for the first few days and then adjusted to 0.5/day by changing the volume from 52 litres to 34.5 litres. Flow rate was constant at 17-28 litres/day. The initial dilution rate of the SFR was 0.67/day. After a few days, this was changed to 0.44/day by changing the flow rate from 26.3 litres/day to 17-28 litres/day. The volume was held constant at 39.5 litres.

The algal media and culture conditions

The algal culture media is presented in Table 1. The media was formulated to closely resemble the ionic composition of seawater. The salinity, however, was 28 parts per thousand. Carbon, nitrogen, and phosphorus levels were as given, except on Day 7 of the SFR run, in which they were doubled. The media was filtered to 3 - 5 / l m before use. Light levels were constant at 27-29 W/rn z (400-500 ft-c) at the liquid surface. The light source was a bank of 40 W 'daylight' fluorescent lamps. The temperature was maintained constant at 23C. The cultures were inoculated with a seed culture using the marine diatom Phaeodactylurn tricornutum (University of Texas Culture Collection #2089). It should be noted that both the SFR and NCSTR cultures were completely open to the environment. This was done in an attempt to study the behaviour of the cultures under what were, basically, field conditions. Tire NCSTR reactor was subject to frequent 'crashes'

TABLE 1 Composition of artificial seawater media

Salt
NaCI MgSO4. 7H~O MgCI2. 6t{20 CaC12 KCI NaHCO3 K~HPO4 Na2SiO3. 9H20 NaBr H3BO3 FeC13 . 6H20 NH4C1 Na2EDTA MnC12. 4H20 ZnSO4. 7H20 (NH4)6. Mo70~4 CuSO4 Co(NO3)2. 6H20

Concentration {g/litre)
20.91 5.70 4-17 0.94 0.61 0.63 0-08 0-01 0.071 0.019 0.001 5 0.03 0.05 0-001 86 0-000 20 0-000 20 0-000 05 0.000 49 28 ppt

Salinity

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so that it became necessary to maintain back-up batch cultures o f algae. When a crash occurred, the reactor was drained, cleaned, and re-started by filling it with algae from the batch cultures.

RESULTS AND DISCUSSION

The results of 40 days of continuous algae culture in both the SFR and NCSTR are given in Fig. 5. It can be seen that the production of algae in the SFR is much more reliable than that in the NCSTR. In addition, the SFR required no maintenance throughout the the testing period, while the NCSTR needed frequent cleaning, as well as maintenance of batch algae cultures to restart the reactor after a crash. The species composition in the NCSTR was mainly P. tricornutum. An occasional green flagellate was also observed. In addition, amoebae were usually present in the NCSTR, sometimes in numbers as great as 30000/ml. The amoebae were often coated with P. tricornutum. Algae which adhered to the amoebae appeared to decompose, and a single amoeba probably had as many as 50 cells attached to it at any one time. Species composition in the SFR was almost uniformly P. trieornutum, although the same green flagellate was occasionally observed. When present, in both the S F R and the NCSTR, this flagellate accounted for less than 0.5% of the biomass. No amoebae were ever observed in the SFR. In the NCSTR, wall growth and sedimentation were usually apparent within a day after each start-up. Both would become extensive within three days of start-up. In the SFR, no sedimentation ever occurred. After a period of several days a calcium carbonate scale occurred on the walls of both reactors. On the NCSTR this deposit was rinsed off with dilute acid each time the reactor was cleaned. On the SFR, however,

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O0

IO

115
Ti

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30

315

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Fig. 5.

Algal concentration from both SFR and NCSTR for 40 day period.

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ANDERSON,

D. E. BRUNE

the scale built up throughout the run. On Day 30 a filamentous blue-green bacterium began to grow on the walls of the SFR. By Day 35, the growth of the blue-green matter had become extensive, blocking much of the incoming light and resulting in the gradual decline in Phaeodactylum cell count seen at the end of the run. Irregularity of the surface of the scale is probably the reason for establishment of the blue-green bacterium, since the beads were not able to remove the scale from the reactor surface. On Day 33 of the SFR run, some of the scale was scraped off the wall and, subsequent to this, the blue-green bacterium was not able to grow in that area. The cause of the gradual decay of production from the SFR was attributed to this scale build-up. Considering the hardness of seawater and the elevated pH associated with algae culture, this may be an unavoidable problenr for marine algae applications. It is anticipated, however, that larger scale versions of the SFR will have much less slope, and that the light would come through the top of the reactor. This might eliminate the gradual decrease in production caused by the decreasing penetration of light through the reactor walls. While the SFR experienced a slow decline in cell production, the NCSTR was undergoing drastic algal cell declines every 4-5 days, requiring a complete restart of the culture of these intervals. A closer examination of data from one NCSTR run (Fig. 6) shows that the cell concentration would reach a peak and then decline exponentially as if the continuous culture were washing-out.

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Fig. 6. Declining cell production in Non-scoured Continuous Stirred Tank Reactor.

SCOURING FILM REACTOR FOR MICROALGAE

PRODUCTION

213

A better understanding of this situation may be developed by examining a mass balance on the algal cell concentration in the reactor: Rate of accumulation or symbolically: dC
--

=Ratef inflow

Rate of + Rate o f _ Rate of outflow growth decay

dt

DCo

--

DC

12aC

--

kdC

(1)

where, C = effluent algal cell concentration, Co = influent algal cell concentration = 0, D = dilution rate, ~a = algal specific growth rate, and kd = algal cell decay rate. In the case where Co = 0, eqn (1) may be written as: dC - - = [Ua--D --kd] C dt where, C = concentration at time t, and C i = initial concentration. IfIaa,D and k d are assumed constant, the solution to eqn (2) is: (2)

Ci

e [ u a - D - kat t

(3)

This equation shows that as long as the maximmn specific growth rate o f the algae is greater than the combined algal decay rate and dilution rate losses, then the culture cell density will remain stable. However, should the sum of kd and D exceed tla, then the culture will begin to wash-out. During all runs with the NCSTR, the dilution rate was well below the growth rate of the diatom Phaeodactylum. Apparently, the decay rate of the algal cells increased as a result of predation by the ever-present amoebae in the NCSTR. At the point where ka >#a - - D , cell wash-out occurs and the concentration of algae in the reactor is seen to decay exponentially with time, as illustrated in Fig. 5. The presence of the amoebae in the NCSTR and not in the SFR can probably be explained by considering a mass balance of the amoebae in both reactors. If the specific growth rate of the amoebae is less than the dilution rate of either reactor, theoretically, the amoebae should wash-out. As expected, it was not present in the SFR. However, in the NCSTR, the wall growth and sedimentation provide an ecological niche for the amoebae. The effective dilution rate of the amoebae is therefore very low and wash-out would not occur. This led to instability in the algal population in the NCSTR.
SUMMARY

The use of a scouring film of fine-diameter glass particles appears to be a very useful technique in eliminating wall growth and biofouling in the marine system described.

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1:. H. ANDERSON, D. E. BRUNE

This concept has been previously d e m o n s t r a t e d to be quite useful in maintaining system cleanliness in aquatic animal culture units (Brune, in press). By eliminating the wall growth which h a r b o u r e d algal predators, this technique has allowed for a stable and reliable means of producing algal cell biomass. The use o f scouring films is a relatively simple and e c o n o m i c a l m e t h o d of inhibiting biofihn and biofouling problems in aquatic culture systems.

REFERENCES Ansell, A. D., Raymont, J. E. G., Lander, K. F., Crowley, E. & Shackley, P. (1963). Studies on the mass culture of Phaeodaetylum. II. The growth of Phaeodactylum and other species in outdoor tanks. Limnol. Oceanogr., 8, 184-206. Brewer, P. G. & Goldman, J. C. (1976). Alkalinity changes generated by phytoplankton growth. Limnol. Oceanogr., 21,108-17. Brune, D. E. Design and development of a flowing bed reactor for brine shrimp culture. Aquacultural Engineering, 1, 63-70. Brune, D. E., Novak, J. T. & Mannebach, R. (1978). Quantifying algal carbon uptake kinetics. A paper presented to the Fiftieth Annual Water Pollution Control Federation Conference. Canzonier, W. J. & Brunetti, R. (1975). Low cost continuous algal culture system, lOth European Symposium on Marine Biology, Ostend, Belgium, 17-23 September 1975, Vol 10, 27-31. Droop, M. R. (1975). The chemostat in mariculture, lOth European Symposium on Marine Biology, Ostend, Belgium, 17-23 September 1975, Vol. 1, 71-93. Dunstan, W. M. & Menzel, D. W. (1971). Continuous cultures of natural populations of phytoplankton in dilute, treated sewage effluent. Limnol. Oceanogr., 16, 623-32. Mock, C. R. & Murphy, M. A. (1970). Techniques for raising penaeid shrimp from the egg to postlarvae. In: Proceedings o f the First Annual Workshop o f the World Mariculture SoeieO,, Baton Rouge, La., USA, 9-10 February 1970, pp. 143-56. Ryther, J. H. (1976). Marine polyculture based on natural food chains and recycled wastes. Sea Grant Technical Report. Unpublished manuscript. Taub, F. A. & Dollar, A. M. (1968). Improvement of a continuous culture apparatus for longterm use. Appl. Microbiol., 16, 232-5.