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A simple and inexpensive system for continuous monoxenic mass culture of marine microalgae
22 (1981) 383-387 Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
Aquaculture,
383
Brief Technical Note A SIMPLE AND INEXPENSIVE SYSTEM FOR CONTINUOUS MONOXENIC MASS CULTURE OF MARINE MICROALGAE
PASQUALE
TROTTA
Laboratory for the Biological Lesina (Italy)
Exploitation
of the Lagoons,
National
Research
Council,
(Accepted 20 May 1980)
ABSTRACT Trotta, P., 1981. A simple and inexpensive system for continuous monoxenic of marine microalgae. Aquaculture, 22: 383-387.
mass culture
A continuous monoxenic culture system for marine phytoplankters was set up utilizing low-cost and easily available materials. It consists of one or more 50-l polyethylene bags equipped with novel closing devices and tubing attachments. Supply of culture medium and air is automated, a culture-density feed-back being the controlling parameter. Illumination is obtained with a vertical series of fluorescent lamps of two types: Osram Fluora and Daylight. The intensity is about 5 klux at the bag surface. Routine operations are limited to harvesting which is achieved by periodic removal of algae suspension. The system is intended to produce adequate algae inoculum for large-volume batch cultures in hatcheries using microalgae as food for fish larvae or for invertebrates.
INTRODUCTION
The necessity for mass production of microalgae as food for filter-feeding larval stages of marine fish, or for the culture of invertebrates used as food for fish larvae, has led to the development of both discontinuous and continuous algae culture systems. These serve as a basis for inoculating larger batch cultures (from 100 1 to several cubic meters). To establish high density microalgae cultures it is necessary to keep the essential environmental factors (light, temperature, salinity, carbon-dioxide concentration, pH, mineral and organic nutrient content) at optimum levels for the algal species to be cultured. It is also important to adapt the size and configuration of the culture vessels to these specific environmental requirements as well as to the production levels to be achieved. The types of culture vessels most frequently used are spherical or cylindrical glass bottles, perspex cylinders and thin polyethylene tubes. These all provide maximum transparency, smooth walls and atoxic conditions and permit discontinuous or continuous harvesting of algae cells and the replacement of the culture medium in the most convenient manner. Complex systems of continuous algal culture have been set up in several
0044-8486/81/0000-0000/$02.50
0 1981 Elsevier Scientific Publishing Company
384
research laboratories in order to study the physiology and biochemistry of microalgae in axenic conditions (e.g., Droop, 1966). Less sophisticated but effective systems have been developed for mariculture purposes (Walne, 1966; Canzonier and Brunetti, 1975; Droop, 1975; Palmer et al., 1975). In the present paper another continuous monoxenic microalgae culture method for mariculture purposes is proposed. SYSTEM
COMPONENTS
As the basic unit a plastic bag with the following properties is used: wall thickness, 0.3 mm; width, 30 cm; length, 180 cm; volume 50 1.l Each bag is equipped with a clamp/hanger combination at the top (Fig. 1A and 2A) to fasten the bag to the ceiling of the temperature-controlled chamber. A clamp secures the closure of the bottom (Fig. 1B and C, and 2B) and there are two nylon bulkhead fittings (Fig. 1D and 2A and B), one at the bottom for the entrance and one at the top for the exit tubing. Further components of the system are: a stainless AISI 316 steel pump (flow rate: 20 l/min); a celluloseester membrane filter (Millipore CE HA 0.45 pm) with polypropylene cartridge prefilter; a solenoid valve with nylon body (washing machine valve) for the supply of fresh medium, one for each bag of the system; a solenoid gas valve to supply air to each culture unit; and a vertical series of fluorescent lamps for illumination. Each unit is prepared by heat-welding the ends of polyethylene film tubing of the desired length. Two steam-sterilized nylon bulkhead fittings (shoulder nipple and one of the gaskets, Fig. lD, 1 and 2) are introduced into the bag, prior to welding. Then the clamp/hanger combination and the bottom clamp are applied. Two circular openings, the diameter of the bulkhead fittings (Fig. lD), for the entrance and exit tubing are made with a heated iron tube. The threaded portions of the fittings are passed through the holes and the outer gaskets and nuts applied (Fig. lD, 3 and 4). The two openings should be situated on the opposite faces of the bag, each in proximity to one of the bag corners. Finally, tubing attachments (Fig. lD, 5 and 6) are inserted in the bulkhead fittings for the connection of influent and effluent tubes. The entrance fitting, near the bottom of the bag, is connected by a natural rubber tube .to the respective nylon valve through which filtered fresh medium is introduced, while theupper fitting serves as the harvest outlet. The movement of the medium into the culture system is effected by a pump of 0.5 HP that draws the culture medium (seawater fertilized according to Walne’s enrichment for Isochrysis ga2bana and other algae; Walne, 1966) from a 7,000 1 black tank. It forces it through the filter unit (Millipore 0.45 pm) and conducts it to a set of nylon valves, each of which distributes the seawater ‘Transparent polyethylene tubes have been used for algae culture purposes for more than 10 years, e.g. by Prof. Persoone and co-workers at the State University of Ghent (Persoone and Sorgeloos, 1975), BelgiCm, and at the CNEXO in Brest, France.
385
A
1
23
4
5
6
D Fig. 1. Closing devices for culture bags and fitting for tubing. A: Iron clamp/hanger combination (the plastic bag is inserted into the closing device which functions as a harness buckle). B: Clamp to secure bottom closure of culture bag (piece 2 to be inserted into piece 1 as a blocking device; piece 3 secures end with open slit, s). C: Cross section of bottom clamp with inserted culture bag. D: Nylon bulkhead fittings for entrance and exit tubing in longitudinal section and exploded view (1 shoulder nipple; 2 and 3 gaskets; 4 nut; 5 reducer; 6 tubing adapter).
to the respective bag. Air mixed with 1% of CO2 is introduced into the seawater tube, between the nylon valve and the entrance of the algae bag, through a T at a rate of 8-10 l/min. During seawater introduction, air supply is interrupted automatically by a double-throw relay controlling the solenoid seawater and air valves. Thus harvesting is a function of the introduction of fresh medium, and culture growth is continuous. Once installed, each bag is inoculated, through the exit tubing, with a monoxenic culture of the desired microalga. The set of bags is illuminated uniformly by a vertical bank of hori-
zontal fluorescent lamps (Fig. 2C). The lamps are of two types (Osram L 77/40 W and Osram Daylight lo/40 W) and arrayed alternately so as to give an intensity, at the bag surface, of about 5 klux.
Fig. 2. Details and arrangement of culture bags. A: Bulkhead fitting for exit tubing and clamp/hanger combination. B: Bulkhead fitting for entrance tubing and bottom clamp. C: Culture bags (the horizontal tube collects the overflowing algae suspension entire series of culture bags).
from the
DISCUSSION
An advantage of the system is the adaptability of the width and length of the culture bags to the desired conditions. The thickness of the plastic sheet must be increased proportionately to the volume, while maintaining maximum transparency. Polyethylene is relatively inexpensive and the bags can, therefore, easily be replaced by new ones. The inner surface of a bag is practically sterile since the tubing is manufactured at about 200°C. As an additional safeguard the bag can be steamed in a simple manner introducing the vapour from a domestic pressure cooker into the assembled unit. If properly maintained, such a system can work over several months without troubles. Its operation (pumping of culture medium, operating of air and medium valves) can be controlled by an automatic photo-switch. The latter device can automatically effect dilution of the culture when the algae concentration exceeds a preset level. The algae suspension is harvested by overflow from the bag outlet. The yield of algal biomass obtained through automation is about
387
twice the quantity obtained by harvesting only once a day, e.g. the production of a culture of Tetraselmis suecica (Butcher) using automation was about 20-30 g/day (wet weight) for each culture unit. The system has been successfully used for continuous culture of rotifers (Trotta, 1980). ACKNOWLEDGEMENTS
The author is indebted to Messrs. A. D’Amato and P. Cammarino, Lesina Laboratory, for their continuous availability and collaboration, and to Dr. M. Bilio, Director of the S.I.VAL. CO Fish Culture Research Institute, Comacchio, Italy, and also to Mr. W.J. Canzonier, Director of the Coastal Resources Applied Research Laboratory, Chioggia, Italy, for critically reviewing the manuscript.
REFERENCES Canzonier, W.J. and Brunetti, R., 1975. Low cost continuous algal culture system. 10th European Symposium on Marine Biology, Ostend, Belgium, 1: 27-31. Droop, M.R., 1966. Vitamin B,, and marine ecology. III. An experiment with chemostat. J. Mar. Biol. Assoc. U.K., 46: 659-671. Droop, M.R., 1975. The chemostat in mariculture. 10th European Symposium on Marine Biology, Ostend, Belgium, 1: 71-93. Palmer, F.E., Ballard, K.A. and Taub, F.A., 1975. A continuous culture apparatus for the mass production of algae. Aquaculture, 6: 319-331. . Persoone, G. and Sorgeloos, P., 1975. Technological improvements for the cultivation of invertebrates as food for fishes and crustaceans. I. Devices and methods. Aquaculture, 6: 275-289. Trotta, P., 1980. A simple and inexpensive system for continuous monoxenic culture of Brachionus plicatilis Miiller as a basis for mass production. In: G. Shelef, C.J. Soeder and M. Balaban (Editors), Algae Biomass. Elsevier/North Holland Biomedical Press, Amsterdam, pp. 307-314. Walne, P.R., 1966. Experiments in the large scale culture of larvae of Ostrea edulis L. Fishery Invest. Lond., Ser. 2, 25(4): l-10.
Aquaculture,
383
Brief Technical Note A SIMPLE AND INEXPENSIVE SYSTEM FOR CONTINUOUS MONOXENIC MASS CULTURE OF MARINE MICROALGAE
PASQUALE
TROTTA
Laboratory for the Biological Lesina (Italy)
Exploitation
of the Lagoons,
National
Research
Council,
(Accepted 20 May 1980)
ABSTRACT Trotta, P., 1981. A simple and inexpensive system for continuous monoxenic of marine microalgae. Aquaculture, 22: 383-387.
mass culture
A continuous monoxenic culture system for marine phytoplankters was set up utilizing low-cost and easily available materials. It consists of one or more 50-l polyethylene bags equipped with novel closing devices and tubing attachments. Supply of culture medium and air is automated, a culture-density feed-back being the controlling parameter. Illumination is obtained with a vertical series of fluorescent lamps of two types: Osram Fluora and Daylight. The intensity is about 5 klux at the bag surface. Routine operations are limited to harvesting which is achieved by periodic removal of algae suspension. The system is intended to produce adequate algae inoculum for large-volume batch cultures in hatcheries using microalgae as food for fish larvae or for invertebrates.
INTRODUCTION
The necessity for mass production of microalgae as food for filter-feeding larval stages of marine fish, or for the culture of invertebrates used as food for fish larvae, has led to the development of both discontinuous and continuous algae culture systems. These serve as a basis for inoculating larger batch cultures (from 100 1 to several cubic meters). To establish high density microalgae cultures it is necessary to keep the essential environmental factors (light, temperature, salinity, carbon-dioxide concentration, pH, mineral and organic nutrient content) at optimum levels for the algal species to be cultured. It is also important to adapt the size and configuration of the culture vessels to these specific environmental requirements as well as to the production levels to be achieved. The types of culture vessels most frequently used are spherical or cylindrical glass bottles, perspex cylinders and thin polyethylene tubes. These all provide maximum transparency, smooth walls and atoxic conditions and permit discontinuous or continuous harvesting of algae cells and the replacement of the culture medium in the most convenient manner. Complex systems of continuous algal culture have been set up in several
0044-8486/81/0000-0000/$02.50
0 1981 Elsevier Scientific Publishing Company
384
research laboratories in order to study the physiology and biochemistry of microalgae in axenic conditions (e.g., Droop, 1966). Less sophisticated but effective systems have been developed for mariculture purposes (Walne, 1966; Canzonier and Brunetti, 1975; Droop, 1975; Palmer et al., 1975). In the present paper another continuous monoxenic microalgae culture method for mariculture purposes is proposed. SYSTEM
COMPONENTS
As the basic unit a plastic bag with the following properties is used: wall thickness, 0.3 mm; width, 30 cm; length, 180 cm; volume 50 1.l Each bag is equipped with a clamp/hanger combination at the top (Fig. 1A and 2A) to fasten the bag to the ceiling of the temperature-controlled chamber. A clamp secures the closure of the bottom (Fig. 1B and C, and 2B) and there are two nylon bulkhead fittings (Fig. 1D and 2A and B), one at the bottom for the entrance and one at the top for the exit tubing. Further components of the system are: a stainless AISI 316 steel pump (flow rate: 20 l/min); a celluloseester membrane filter (Millipore CE HA 0.45 pm) with polypropylene cartridge prefilter; a solenoid valve with nylon body (washing machine valve) for the supply of fresh medium, one for each bag of the system; a solenoid gas valve to supply air to each culture unit; and a vertical series of fluorescent lamps for illumination. Each unit is prepared by heat-welding the ends of polyethylene film tubing of the desired length. Two steam-sterilized nylon bulkhead fittings (shoulder nipple and one of the gaskets, Fig. lD, 1 and 2) are introduced into the bag, prior to welding. Then the clamp/hanger combination and the bottom clamp are applied. Two circular openings, the diameter of the bulkhead fittings (Fig. lD), for the entrance and exit tubing are made with a heated iron tube. The threaded portions of the fittings are passed through the holes and the outer gaskets and nuts applied (Fig. lD, 3 and 4). The two openings should be situated on the opposite faces of the bag, each in proximity to one of the bag corners. Finally, tubing attachments (Fig. lD, 5 and 6) are inserted in the bulkhead fittings for the connection of influent and effluent tubes. The entrance fitting, near the bottom of the bag, is connected by a natural rubber tube .to the respective nylon valve through which filtered fresh medium is introduced, while theupper fitting serves as the harvest outlet. The movement of the medium into the culture system is effected by a pump of 0.5 HP that draws the culture medium (seawater fertilized according to Walne’s enrichment for Isochrysis ga2bana and other algae; Walne, 1966) from a 7,000 1 black tank. It forces it through the filter unit (Millipore 0.45 pm) and conducts it to a set of nylon valves, each of which distributes the seawater ‘Transparent polyethylene tubes have been used for algae culture purposes for more than 10 years, e.g. by Prof. Persoone and co-workers at the State University of Ghent (Persoone and Sorgeloos, 1975), BelgiCm, and at the CNEXO in Brest, France.
385
A
1
23
4
5
6
D Fig. 1. Closing devices for culture bags and fitting for tubing. A: Iron clamp/hanger combination (the plastic bag is inserted into the closing device which functions as a harness buckle). B: Clamp to secure bottom closure of culture bag (piece 2 to be inserted into piece 1 as a blocking device; piece 3 secures end with open slit, s). C: Cross section of bottom clamp with inserted culture bag. D: Nylon bulkhead fittings for entrance and exit tubing in longitudinal section and exploded view (1 shoulder nipple; 2 and 3 gaskets; 4 nut; 5 reducer; 6 tubing adapter).
to the respective bag. Air mixed with 1% of CO2 is introduced into the seawater tube, between the nylon valve and the entrance of the algae bag, through a T at a rate of 8-10 l/min. During seawater introduction, air supply is interrupted automatically by a double-throw relay controlling the solenoid seawater and air valves. Thus harvesting is a function of the introduction of fresh medium, and culture growth is continuous. Once installed, each bag is inoculated, through the exit tubing, with a monoxenic culture of the desired microalga. The set of bags is illuminated uniformly by a vertical bank of hori-
zontal fluorescent lamps (Fig. 2C). The lamps are of two types (Osram L 77/40 W and Osram Daylight lo/40 W) and arrayed alternately so as to give an intensity, at the bag surface, of about 5 klux.
Fig. 2. Details and arrangement of culture bags. A: Bulkhead fitting for exit tubing and clamp/hanger combination. B: Bulkhead fitting for entrance tubing and bottom clamp. C: Culture bags (the horizontal tube collects the overflowing algae suspension entire series of culture bags).
from the
DISCUSSION
An advantage of the system is the adaptability of the width and length of the culture bags to the desired conditions. The thickness of the plastic sheet must be increased proportionately to the volume, while maintaining maximum transparency. Polyethylene is relatively inexpensive and the bags can, therefore, easily be replaced by new ones. The inner surface of a bag is practically sterile since the tubing is manufactured at about 200°C. As an additional safeguard the bag can be steamed in a simple manner introducing the vapour from a domestic pressure cooker into the assembled unit. If properly maintained, such a system can work over several months without troubles. Its operation (pumping of culture medium, operating of air and medium valves) can be controlled by an automatic photo-switch. The latter device can automatically effect dilution of the culture when the algae concentration exceeds a preset level. The algae suspension is harvested by overflow from the bag outlet. The yield of algal biomass obtained through automation is about
387
twice the quantity obtained by harvesting only once a day, e.g. the production of a culture of Tetraselmis suecica (Butcher) using automation was about 20-30 g/day (wet weight) for each culture unit. The system has been successfully used for continuous culture of rotifers (Trotta, 1980). ACKNOWLEDGEMENTS
The author is indebted to Messrs. A. D’Amato and P. Cammarino, Lesina Laboratory, for their continuous availability and collaboration, and to Dr. M. Bilio, Director of the S.I.VAL. CO Fish Culture Research Institute, Comacchio, Italy, and also to Mr. W.J. Canzonier, Director of the Coastal Resources Applied Research Laboratory, Chioggia, Italy, for critically reviewing the manuscript.
REFERENCES Canzonier, W.J. and Brunetti, R., 1975. Low cost continuous algal culture system. 10th European Symposium on Marine Biology, Ostend, Belgium, 1: 27-31. Droop, M.R., 1966. Vitamin B,, and marine ecology. III. An experiment with chemostat. J. Mar. Biol. Assoc. U.K., 46: 659-671. Droop, M.R., 1975. The chemostat in mariculture. 10th European Symposium on Marine Biology, Ostend, Belgium, 1: 71-93. Palmer, F.E., Ballard, K.A. and Taub, F.A., 1975. A continuous culture apparatus for the mass production of algae. Aquaculture, 6: 319-331. . Persoone, G. and Sorgeloos, P., 1975. Technological improvements for the cultivation of invertebrates as food for fishes and crustaceans. I. Devices and methods. Aquaculture, 6: 275-289. Trotta, P., 1980. A simple and inexpensive system for continuous monoxenic culture of Brachionus plicatilis Miiller as a basis for mass production. In: G. Shelef, C.J. Soeder and M. Balaban (Editors), Algae Biomass. Elsevier/North Holland Biomedical Press, Amsterdam, pp. 307-314. Walne, P.R., 1966. Experiments in the large scale culture of larvae of Ostrea edulis L. Fishery Invest. Lond., Ser. 2, 25(4): l-10.