10th. European Symposium on Marine Biology, Ostend, Belgium, September 17-23, 1975, Vol. 1:27-31.

LOW-COST CONTINUOUS ALGAL CULTURE SYSTEM.

W.J. Ganzonier and R. Brunetti.

Instituto di Biologia del Mare, Consiglio Nazionale delle Richerche, 30122 Venezia, Italy.

and

Instituto de Biologia Animale, Universita di Padova, 35100 Padova, Italy.

Abstract

An automated continuous culture system for planktonic algae, constructed from inexpensive and easily obtained components, was tested on a pilot scale as part of a laboratory unit for rearing marine invertebrates. The system has a large lighttransfer surface and is filled with a relatively small volume of a continuously circulating medium; the pilot unit occupies a minimum of floor area, 150cm x 40 cm, and can be easily placed above aquaria to economize on space. It can produce up to 10 Ltr. of diatom culture / day at a concentration equivalent to 1.200 Ug of chlorophyll a/l. No extensive maintenance is required other than a periodic refilling of the reservoir with chemically sterilized culture medium and a flushing at monthly intervals. The system, by virtue of its flexible design and limited maintenance requirements, has considerable potential for scaling up to the needs of a commercial operation.

Introduction

The continuous production of unicellular algae is an essential auxiliary procedure in the rearing and maintenance of many marine invertebrates, on both laboratory and commercial levels. A variety of batch and continuous culture systems has been used in studies on algal physiology (Meyers and Clark, 1944; Hannan and Patouillet, 1963; Mandux and Jones, 1964; Droop, 1966; Carpenter, 1968; Hare and Schmidt, 1968). Many systems of varying degree of sophistication have been developed for the production of algal cells to be used in the culture of marine invertebrates (Loosanoff, 1951; Davis and Ukeles, 1961; Taub and Dollar, 1968; Ukeles, 1973; Taub, 1974). Some of these systems have been designed for axenic culture in relatively small volumes with limited daily outputs. Others are open-vat (semi-wild) systems with mixed populations giving variable yields and in which difficulties arise from the accumulation of unpredictable quantities of bacterial and algal metabolites. Some of the basic requirements of a system which is to supply food organisms for the culture of marine invertebrates are a relative ease of construction with readily available materials and a minimum of routine maintenance. Such a system should also be relatively efficient in terms of both energy consumption and the space occupied and is flexible enough in design to be adapted to various uses and spatial configurations. It should also have the potential for scaling up to pilot plant or commercial operation. We attempted to incorporate these required features into a system that was designated for the long-term culture of experimental populations of ascidians. The system is based on a maximal utilization of the illumination by employing a large light transfer area through which the culture is pumped at relatively high velocity in order to prevent sedimentation and attachment. A partial separation of the less buoyant particulates that would otherwise settle out in the light transfer field is achieved by sedimentation in a separate chamber for which the effluent (product) is drawn-off near the bottom.

Materials and Construction

Fig. 1.

MR……… Medium Reservoir.                                                      MI……… Medium Inlet.

S   ……… Solenoid Valve.                                                          NV……..  PVC Needle Valve.

UV………  UV Sterilization Unit.                                                  WL……..  Maximum Water Level.

CF………  Cartridge Air Filter.                                                     OL……...  Outlet.

SD………  Sludge Drain.                                                              PP……...  Eheim Pump.

PGM……   Programmer.                                                               CC……..   PVC Pipe Diam. App. 12 Cm.

LTF……..   Light Transfer Field.                                                  BAL…….  Ballast UV Unit.

A scheme of the basic culture rig is presented in Fig. 1.  It is composed of a light transfer field consisting of 35 soft-glass; thin-wall tubes (1,5 Mtr. x 1 Cm. ID).                                       connected is series with Tygon-plastic tubing. The medium is forced through the field by an all-plastic magnetic drive pump (Eheim model 1048 or 1250). After passing through the light field, the culture enters the top of the gas exchange-sedimentation column (10 – 12 Cm. grey PVC thin-wall drain tube) separatory funnel is cemented. The culture then cascades over a series of cones (polypropylene powder funnels) which are attached to a central supporting rod. The outlet to the pump is slightly below the surface of the liquid in the column. The culture is harvested through an inverted siphon that collects the settled culture a few cms above the column bottom. The drain valve at the bottom of the column serves to remove the very heavy sediment of aggregated and moribund cells.

The input of the culture medium is controlled by a programmer (modified appliance programmer or inexpensive multiposition timer switch) which periodically opens the solenoid valve S (nylon appliance inlet valve) and powers the UV- unit (Canrad-Hanovia). The flow rate is controlled by the PVC needle valve NV that requires resetting at intervals of 2 – 4 days. Initially the medium was supplied from a 50 Ltr. reservoir the contents of which were circulated through a loop by a small plastic pump; a T-tube at the top of the loop led the medium at a constant head to the valve NV. However, more recently the system has been supplied from two 500 Ltr. tanks located 4 Mtr. above the inlet valve S. These tanks are alternately filled with seawater from a tube well. The water is first treated with hypochloride solution, aerated, and the hypochloride residual neutralized; the water is allowed to stand a day to settle and precipitate that forms, then dosed with nutrients (at levels specified in the formula of Ukeles, 1973) and placed on line. Illumination was supplied by a bank of three 40 W. fluorescent lamps placed 15 Cm. above the field; an equivalent bank was added 15 Cm. below the field and the lamp bank of an adjacent culture rack supplied some additional illumination. The unit has been operated at 18 degrees Celsius in a constant-temperature room and under conditions of minimum illumination, no additional cooling was required.

Maintenance

During the eight months of operation, the system required only three cleanings by flushing with a hypochlorite solution, and an occasional brush cleaning. This operation can be facilitated if the individual tubes are attached at either end to PVC manifolds using standard plastic compression fittings, a modification, which is currently being constructed. The only additional maintenance is a routine check of the flow rate from the medium supply valve and a periodic drawdown of the sludge at the bottom of the column.

Results and Discussion

The system has operated almost continuously for more than 8 months with cultures of either Phaeodactylum tricornutum Bohlin or a nano-form of Chlorella sp. Beyerinck (Andreoli personal communication). Output volumes of 2 – 10 Ltr./ day were tested. The best yields were obtained at 3 – 4 Ltr./day, giving a maintained average chlorophyll a concentration of 1.200 Ug/Ltr. Cell density average about 1 x 10-6 cells/Ml. at outlet, but occasionally reached 2 to 3 x 10-6 cells/Ml. at lower output volumes. Such yields are low when compared with those reported for other automated units (Hannan and Patouillet, 1963; Taub and Dollar, 1968; Ukeles, 1973; Taub, 1974). In most of these systems, however CO2 was added to the air supply and they all operated under considerably higher light intensities. It would not be unreasonable to expect higher yields with similar additions to the system described. One particular advantage of the large surface to volume ration of the light transfer field is the possibility of maintaining an efficient and uniform light penetration regardless of the volume of the system.  Most other systems have vat or carboy culture chambers of rectangular or cylindrical configuration, which imposes a serious limitation when scaling-up to a larger output. One exception is the plate configuration described by Hare and Schmidt (1968), but this chamber has structural and circulation limitations. The system described is easily adapted to a variety of special situations. The original unit occupies only 150 Cm. x 40 Cm. of horizontal surface and is located above the aquarium, which is served by the output. The light-transfer surface is not excessively heavy and has a modular construction; it can easily be located some distance from the other components of the system in order to utilize otherwise wasted space and to take advantage of other light sources, including natural illumination. It should be pointed out that the system is not designed to maintain axenic cultures, although the bacterial load can be held to a minimum, for example, as might be required in the rearing of some delicate bivalve larvae (Walne, 1958; Guillard, 1959). The output of the unit has been used to rear two broods of Mytilus edulis L. larvae up to setting without any indication of overt toxicity.

Acknowledgments

We would like to tank the staff of the Stazione Idrobiologica di Chioggia, of the Universita di Padova for technical assistance and its director, Prof. A. Sabbadin, for providing the facilities. The work reported is part of a research program supported by the Instituto di Biologia del Mare, Venezia, of the Consiglio Nazionale delle Ricerche.

Literature cited

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Hannan, F.J. and Patouillt, C. 1963, Gas exchange with mass cultures of algae. L. effects of light intensity and rate of carbon dioxide input on oxygen production. Appl. Microbiol. 11: 446 – 449. 

Hare, T.A. and Schmidt, R.R. 1918, Continuous dilution method for the mass culture of synchronized cells. Appl. Microbiol. 16: 496 – 499.

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Myers, J. and Clarck, L.B. 1944, Culture conditions and the development of the photosynthetic mechanism. An apparatus for the continuous culture of Chlorella. J. Gen. Physiol. 28: 103 – 112. 

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Taub, F.B. and Dollar, A.M. 1968, Improvement of a continuous culture apparatus for long-term use. Apl. Microbiol. 16: 232 – 235.

Ukeles, R. 1973, Continuous culture. A method for production of unicellular algal foods. P. 233 – 254. In Handbook of Phycological Methods. Stein, Jr. (Ed) Cambridge Univ. Press, London, New York. 348 p.

Walne, P.R. 1958, The importance of bacteria in laboratory experiments on rearing the larvae of Ostrea edulis (L). J. mar. Biol. U.K. 37: 415 – 425.

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