- Email: [email protected]
A turbidostat vessel for the continuous culture of marine microalgae
Aquacultural Engineering 7 (1988) 89-96
A Turbidostat Vessel for the Continuous Culture of Marine Mieroalgae* I. Laing and E. Jones Ministry of Agriculture, Fisheries and Food, Directorate of Fisheries Research, Fisheries Experiment Station, Benarth Road, Conwy, Gwynedd LL32 8UB, Great Britain (Received 21 August 1986; accepted 15 September 1987)
ABSTRACT A 40 litre turbidostat vessel in which the alga culture is contained in polyethylene tubing supported around a core of six 80 W fluorescent lamps is described. The marine unicellular flagellates Tetraselmis suecica (Kylin) Butch. and Isochrysis aft. galbana Green were produced by continuous culture operation in the vessel. Average production of 0"56 x 1011 T. suecica cells day - i for 33 days and 5"18 x 1011 I. galbana cells day - 1for 21 days was obtained.
INTRODUCTION A successful method for the efficient production of marine microalgae in an 80 litre internally illuminated turbidostat culture vessel was described in a previous paper (Laing and Jones, 1983). It was shown that production of the marine unicellular flagellates Tetraselmis suecica (Kylin) Butch and Isochrysis aft. galbana Green (CLONE T.-Iso) by this method was similar to that from the 200 litre culture vessels operated semi-continuously. Disadvantages of the system were the high capital cost of the vessels and the time lost between culture trials for cleaning and sterilising them. This paper describes a 40 litre turbidostat culture vessel designed to overcome these disadvantages by using polyethylene tubing, heat-sealed *The reference to proprietary products in this paper should not be construed as an official endorsement of these products, nor is any criticism implied of similar products which have not been mentioned. 89 © Crown copyright, 1988
90
1. Laing, E. Jones
at the bottom to form a bag, to contain the algal culture. The yields of the above algal species over a number of culture trials are evaluated.
DESIGN A N D C O N S T R U C T I O N Figure 1 shows the general arrangement of the vessel. For durability and ease of construction the framework for supporting the polyethylene algal tube was made from galvanised 'Kee Klamp' fittings, but other forms of scaffold products would be equally suitable. The framework consisted of three 2-5 m lengths of 33.7 mm diameter, No. 6 'Kee Klamp' pipe, with feet (fittings No. 61) located on the end of each upright. Six collars (No. 75 fittings) were also attached to support the top and bottom plates. The
collar . t ./Algal take 1 ~__~,. off point Weldmesh jacket
Galvanised
/ Sensor
!
Fluorescent lamp
/ Clamp to fasten jacket ~" - ]'
I
Frame constructed from Kee Klamps
No 6 pipe
Plate sup collars
Fig. 1.
/
Outer corrugated PVC sheet
"/ ~l~ y t h e n e
--
algal tube
PVC core tube
General arrangement of the vessel.
Turbidostat vessel for marine microalgae culture
~ Media input 25ram Dia
91
Strengthening bars No 45
Cylinder support collar Air input 15ram Dia
Top and bottom plates (PVC) ,
I
-- Acrylic cylinder
No 61
G Fig. 2.
Support frame arrangement.
assembled frame was strengthened with six 90 ° connectors (No. 45 fittings) constructed in the manner shown in Fig. 2. The top and bottom plates were 46 cm diameter and made from 12 m m thick white 'DARVIC' rigid polyvinyl chloride sheet. Both plates had the centres removed to accommodate the 15 cm diameter, 165 cm long, transparent acrylic cylinder, which in turn was suspended through the top and bottom plates and supported by a 12 m m thick polyvinyl chloride collar attached to the acrylic cylinder by means of three equally-spaced O B A screws. Three equally-spaced holes (35 m m diameter) were drilled on a 37 cm pitch circle diameter to allow the plates to slide freely on the upright pipes. This allowed for adjusting the final position of the top and bottom plates with the six No. 75 fittings, three locked under each plate, to support the weight of the assembled vessel. The top plate was also provided with a 25 m m diameter medium input pipe and a 15 m m diameter air inlet pipe. The polyethylene algal bag was made from a 160 cm length of 71 cm wide, heavy or extra heavy gauge clear layflat tubing, heat-sealed across the bottom. It was placed around the acrylic cylinder as shown in Fig. 3.
92
I. Laing, E. Jones
Inner acrylic cylinder White corrugated PVC j a c k e t
Polyethylene algal tube
Details of __
j a c k e t fasteners
Galvanised mesh jacke v
Fig. 3.
Algal culture tube arrangement.
An outer support jacket 150 cm × 90 cm made from galvanised weld mesh was placed around the algal bag, and fastened by five clamps along its length. The light sensor housing and a 150 cm × 90 cm sheet of reflective material (white corrugated pvc) were held in position against the mesh by 12.7 mm power belting. Illumination was provided by six 80 W, 150 cm length, daylight fluorescent tubes mounted in the transparent extruded acrylic cylinder, which projected beyond both the top and bottom plates to prevent water splashing the lamp holders. The lamps were mounted around a hollow core, 19 mm diameter bore polyvinyl chloride pipe through which electrical cables were fed to the bottom lamp holders. The core tube was fitted with three plates, the bottom plate made from 6 mm thick polyvinyl chloride sheet with six
Turbidostat vesselfor marine microalgae culture
93
lamp holders equally spaced on a 90 m m pitch circle diameter. This plate slid freely on the core tube and could be locked in position by an O B A screw. The centre plate was made from 6 m m thick aluminium plate drilled to support and position the fluorescent tubes; this plate was positioned two-thirds of the way up the core tube and secured by an O B A screw. The complete light assembly was suspended from the top plate which was made from a 12 nun thick, 19 cm diameter, polyvinyl chloride disc. The disc had three sections removed to allow the heat to dissipate from the light tubes and to allow access for the electrical cables connected to the top lamp holders. A central boss, 40 mm diameter by 40 m m long, was attached to the disc, and a 3 m m deep circular groove, 15 cm diameter, was machined for it to locate on the end of the acrylic cylinder. The top plate slid freely on the core tube and could be clamped by an OBA screw to suspend the light unit at the correct position within the acrylic cylinder.
OPERATION A 160 cm length was cut from a roll of 28 in (71 cm) wide polyethylene layflat tubing. The tubing was free of potential contaminants due to the heat used in the manufacturing process and no further sterilisation was necessary. The cut length was heat-sealed across the width of one end and positioned around the acrylic cylinder containing the lamps. The six nuts and bolts securing the outer supporting mesh jacket were fastened and the outer reflective sheet of white corrugated plastic held in place by 12.7 m m nylon power belting, which also supported the sensor housing unit against the outer surface of the culture. The polyethylene tubing was filled with 38 litres of nutrient-enriched sea water medium that had been filtered through a sterile 0.45 /~m particle retention cartridge filter. Nutrient salts were added at 2.5 ml litre- 1 of sea water from the standard nutrient stock solution described by Walne (1966). This was 2"5 times the usual amount, and was to ensure that nutrient levels did not become limiting at the high cell densities at which the cultures were maintained. A 2.5 cm diameter circle was cut from the tubing, with its centre about 7 cm above the water level. Into this was fitted a ] in (1.9 cm) rigid PVC tank connector. A 150 cm length of 1.5 cm bore flexible PVC tubing was run from this into a 125 litre collecting vessel. This overflow allowed for automatic harvesting of the culture into the reservoir.
L Laing, E. Jones
94
A supply of filtered air, enriched with sufficient carbon dioxide to maintain culture pH at 7.6-7.8 (about 0.25% CO2 by volume) was introduced through a 0.4 cm bore 150 cm long acrylic tube inserted into the top of the culture. A flow rate of about 15 litres m i n - 1 ensured efficient mixing of the culture. Cooling water at a flow rate of about 0-35 litre min- ~ was allowed to run down over the outer culture surface in order to maintain culture temperature at 2 I°C +_ 1 °C. Inoculae were 2 litre cultures of T. suecica or I. galbana grown for 4-5 days in medium prepared from autoclaved sea water. The method for automatic harvesting of the culture, which began 3-4 days from inoculation, was as described for 80 litre vessels by Laing and Jones (1983). The 40 litre polyethylene bag was discarded at the end of each culture trial. A new clean bag was then fitted to the vessel and the above operating procedures repeated. ALGAL PRODUCTION Continuous (turbidostat) culture was maintained for 18-24 days with/. galbana and 24-69 days with T. suecica. Yields obtained when these species were maintained at a range of pre-determined cell densities are shown in Fig. 4. Maximum daily yields of 6.5-9.0 × 10 ~1 I. galbana cells 12 1
I. g a l b a n a
~
T
8
4-
0 x
2--
9 o '~-
11 13 15 17 19 21 23 2 5 27
Cell density ( c e l l s ~ u l - l x 10 - 3 ) 1.0 F
T. s u e c i c a
T
-~ 0.6 c~
0.4 O. 7
9 11 13 15 17 19 21 23 2 5 27 Cell density ( c e l l s / u l - l x l O - 2 )
Fig. 4.
Daily yield (numbers of cells x 10 -~) for a range of cell densities of I. galbana and T. suecica in 40 iitre, internally-illuminated turbidostat cultures.
95
Turbidostat vessel for marine microalgae culture
and 0.70-0.87 × 101~ T. suecica cells were obtained at culture densities of 1 9 0 0 0 - 2 1 0 0 0 cells /A -~ and 2 1 0 0 - 2 3 0 0 cells ~1-1, respectively. These 'optimum' cell densities were higher than those described for 80 litre vessels (15 000-17 000 and 1900-2100 cells /~1-1 respectively). Average yields, achieved over a number of culture trials, are shown in Table 1. The mean yield for T. suecica of 0.56 × 10 ~1 cells day -~ for 33 days over the eight trials compares with an average of 0-60 × 101~ cells day- 1 for 40 days in nine trials with an 80 litre turbidostat culture vessel.
TABLE 1
Production of T. suecica and L galbana from a Number of Culture Trials Trial no. T. suecica Average yield (cells × 10-Jl day-l) Harvesting period (days) L galbana Average yield (cells × 10 -II day -j ) Harvesting period (days)
1
2
3
4
5
6
7
8
9
0"66
0.55
0.46
0"59
0"58
0'48
0"44
0"55
0"55
69
24
24
25
40
24
35
24
32
4"40
5.78
5.44
21
18
24
Towards the end of the harvesting period there was a tendency for the cells to stick to the polyethylene around the lamps. This could be due either to the high cell densities at which cultures were maintained or to the nature of the material. It caused a reduction in illumination intensity and probably contributed to the shorter harvesting period from 40 litre as compared with 80 litre vessels. However, the culture trials in the polyethylene bag vessels were run successively, whereas the 80 litre vessels, which were made from glass and acrylic, required 2-3 days between trials for cleaning and sterilising.
CONCLUSION The 40 litre vessel described has been shown to give a similar yield to 80 hire vessels, the estimated total cost of constructing one of these vessels is only about one-third of that for a vessel of similar design but made entirely from re-usable materials.
96
L Laing, E. Jones
REFERENCES Laing, I. & Jones, E. (1983). Large-scale turbidostat culture of marine microalgae. Aquacultural Engineering, 2,203-12. Walne, P. R. (1966). Experiments in the large-scale culture of the larvae of Ostrea edulis L. Fishery lnvest., Lond., Ser. 2, 25 (4), 53.
A Turbidostat Vessel for the Continuous Culture of Marine Mieroalgae* I. Laing and E. Jones Ministry of Agriculture, Fisheries and Food, Directorate of Fisheries Research, Fisheries Experiment Station, Benarth Road, Conwy, Gwynedd LL32 8UB, Great Britain (Received 21 August 1986; accepted 15 September 1987)
ABSTRACT A 40 litre turbidostat vessel in which the alga culture is contained in polyethylene tubing supported around a core of six 80 W fluorescent lamps is described. The marine unicellular flagellates Tetraselmis suecica (Kylin) Butch. and Isochrysis aft. galbana Green were produced by continuous culture operation in the vessel. Average production of 0"56 x 1011 T. suecica cells day - i for 33 days and 5"18 x 1011 I. galbana cells day - 1for 21 days was obtained.
INTRODUCTION A successful method for the efficient production of marine microalgae in an 80 litre internally illuminated turbidostat culture vessel was described in a previous paper (Laing and Jones, 1983). It was shown that production of the marine unicellular flagellates Tetraselmis suecica (Kylin) Butch and Isochrysis aft. galbana Green (CLONE T.-Iso) by this method was similar to that from the 200 litre culture vessels operated semi-continuously. Disadvantages of the system were the high capital cost of the vessels and the time lost between culture trials for cleaning and sterilising them. This paper describes a 40 litre turbidostat culture vessel designed to overcome these disadvantages by using polyethylene tubing, heat-sealed *The reference to proprietary products in this paper should not be construed as an official endorsement of these products, nor is any criticism implied of similar products which have not been mentioned. 89 © Crown copyright, 1988
90
1. Laing, E. Jones
at the bottom to form a bag, to contain the algal culture. The yields of the above algal species over a number of culture trials are evaluated.
DESIGN A N D C O N S T R U C T I O N Figure 1 shows the general arrangement of the vessel. For durability and ease of construction the framework for supporting the polyethylene algal tube was made from galvanised 'Kee Klamp' fittings, but other forms of scaffold products would be equally suitable. The framework consisted of three 2-5 m lengths of 33.7 mm diameter, No. 6 'Kee Klamp' pipe, with feet (fittings No. 61) located on the end of each upright. Six collars (No. 75 fittings) were also attached to support the top and bottom plates. The
collar . t ./Algal take 1 ~__~,. off point Weldmesh jacket
Galvanised
/ Sensor
!
Fluorescent lamp
/ Clamp to fasten jacket ~" - ]'
I
Frame constructed from Kee Klamps
No 6 pipe
Plate sup collars
Fig. 1.
/
Outer corrugated PVC sheet
"/ ~l~ y t h e n e
--
algal tube
PVC core tube
General arrangement of the vessel.
Turbidostat vessel for marine microalgae culture
~ Media input 25ram Dia
91
Strengthening bars No 45
Cylinder support collar Air input 15ram Dia
Top and bottom plates (PVC) ,
I
-- Acrylic cylinder
No 61
G Fig. 2.
Support frame arrangement.
assembled frame was strengthened with six 90 ° connectors (No. 45 fittings) constructed in the manner shown in Fig. 2. The top and bottom plates were 46 cm diameter and made from 12 m m thick white 'DARVIC' rigid polyvinyl chloride sheet. Both plates had the centres removed to accommodate the 15 cm diameter, 165 cm long, transparent acrylic cylinder, which in turn was suspended through the top and bottom plates and supported by a 12 m m thick polyvinyl chloride collar attached to the acrylic cylinder by means of three equally-spaced O B A screws. Three equally-spaced holes (35 m m diameter) were drilled on a 37 cm pitch circle diameter to allow the plates to slide freely on the upright pipes. This allowed for adjusting the final position of the top and bottom plates with the six No. 75 fittings, three locked under each plate, to support the weight of the assembled vessel. The top plate was also provided with a 25 m m diameter medium input pipe and a 15 m m diameter air inlet pipe. The polyethylene algal bag was made from a 160 cm length of 71 cm wide, heavy or extra heavy gauge clear layflat tubing, heat-sealed across the bottom. It was placed around the acrylic cylinder as shown in Fig. 3.
92
I. Laing, E. Jones
Inner acrylic cylinder White corrugated PVC j a c k e t
Polyethylene algal tube
Details of __
j a c k e t fasteners
Galvanised mesh jacke v
Fig. 3.
Algal culture tube arrangement.
An outer support jacket 150 cm × 90 cm made from galvanised weld mesh was placed around the algal bag, and fastened by five clamps along its length. The light sensor housing and a 150 cm × 90 cm sheet of reflective material (white corrugated pvc) were held in position against the mesh by 12.7 mm power belting. Illumination was provided by six 80 W, 150 cm length, daylight fluorescent tubes mounted in the transparent extruded acrylic cylinder, which projected beyond both the top and bottom plates to prevent water splashing the lamp holders. The lamps were mounted around a hollow core, 19 mm diameter bore polyvinyl chloride pipe through which electrical cables were fed to the bottom lamp holders. The core tube was fitted with three plates, the bottom plate made from 6 mm thick polyvinyl chloride sheet with six
Turbidostat vesselfor marine microalgae culture
93
lamp holders equally spaced on a 90 m m pitch circle diameter. This plate slid freely on the core tube and could be locked in position by an O B A screw. The centre plate was made from 6 m m thick aluminium plate drilled to support and position the fluorescent tubes; this plate was positioned two-thirds of the way up the core tube and secured by an O B A screw. The complete light assembly was suspended from the top plate which was made from a 12 nun thick, 19 cm diameter, polyvinyl chloride disc. The disc had three sections removed to allow the heat to dissipate from the light tubes and to allow access for the electrical cables connected to the top lamp holders. A central boss, 40 mm diameter by 40 m m long, was attached to the disc, and a 3 m m deep circular groove, 15 cm diameter, was machined for it to locate on the end of the acrylic cylinder. The top plate slid freely on the core tube and could be clamped by an OBA screw to suspend the light unit at the correct position within the acrylic cylinder.
OPERATION A 160 cm length was cut from a roll of 28 in (71 cm) wide polyethylene layflat tubing. The tubing was free of potential contaminants due to the heat used in the manufacturing process and no further sterilisation was necessary. The cut length was heat-sealed across the width of one end and positioned around the acrylic cylinder containing the lamps. The six nuts and bolts securing the outer supporting mesh jacket were fastened and the outer reflective sheet of white corrugated plastic held in place by 12.7 m m nylon power belting, which also supported the sensor housing unit against the outer surface of the culture. The polyethylene tubing was filled with 38 litres of nutrient-enriched sea water medium that had been filtered through a sterile 0.45 /~m particle retention cartridge filter. Nutrient salts were added at 2.5 ml litre- 1 of sea water from the standard nutrient stock solution described by Walne (1966). This was 2"5 times the usual amount, and was to ensure that nutrient levels did not become limiting at the high cell densities at which the cultures were maintained. A 2.5 cm diameter circle was cut from the tubing, with its centre about 7 cm above the water level. Into this was fitted a ] in (1.9 cm) rigid PVC tank connector. A 150 cm length of 1.5 cm bore flexible PVC tubing was run from this into a 125 litre collecting vessel. This overflow allowed for automatic harvesting of the culture into the reservoir.
L Laing, E. Jones
94
A supply of filtered air, enriched with sufficient carbon dioxide to maintain culture pH at 7.6-7.8 (about 0.25% CO2 by volume) was introduced through a 0.4 cm bore 150 cm long acrylic tube inserted into the top of the culture. A flow rate of about 15 litres m i n - 1 ensured efficient mixing of the culture. Cooling water at a flow rate of about 0-35 litre min- ~ was allowed to run down over the outer culture surface in order to maintain culture temperature at 2 I°C +_ 1 °C. Inoculae were 2 litre cultures of T. suecica or I. galbana grown for 4-5 days in medium prepared from autoclaved sea water. The method for automatic harvesting of the culture, which began 3-4 days from inoculation, was as described for 80 litre vessels by Laing and Jones (1983). The 40 litre polyethylene bag was discarded at the end of each culture trial. A new clean bag was then fitted to the vessel and the above operating procedures repeated. ALGAL PRODUCTION Continuous (turbidostat) culture was maintained for 18-24 days with/. galbana and 24-69 days with T. suecica. Yields obtained when these species were maintained at a range of pre-determined cell densities are shown in Fig. 4. Maximum daily yields of 6.5-9.0 × 10 ~1 I. galbana cells 12 1
I. g a l b a n a
~
T
8
4-
0 x
2--
9 o '~-
11 13 15 17 19 21 23 2 5 27
Cell density ( c e l l s ~ u l - l x 10 - 3 ) 1.0 F
T. s u e c i c a
T
-~ 0.6 c~
0.4 O. 7
9 11 13 15 17 19 21 23 2 5 27 Cell density ( c e l l s / u l - l x l O - 2 )
Fig. 4.
Daily yield (numbers of cells x 10 -~) for a range of cell densities of I. galbana and T. suecica in 40 iitre, internally-illuminated turbidostat cultures.
95
Turbidostat vessel for marine microalgae culture
and 0.70-0.87 × 101~ T. suecica cells were obtained at culture densities of 1 9 0 0 0 - 2 1 0 0 0 cells /A -~ and 2 1 0 0 - 2 3 0 0 cells ~1-1, respectively. These 'optimum' cell densities were higher than those described for 80 litre vessels (15 000-17 000 and 1900-2100 cells /~1-1 respectively). Average yields, achieved over a number of culture trials, are shown in Table 1. The mean yield for T. suecica of 0.56 × 10 ~1 cells day -~ for 33 days over the eight trials compares with an average of 0-60 × 101~ cells day- 1 for 40 days in nine trials with an 80 litre turbidostat culture vessel.
TABLE 1
Production of T. suecica and L galbana from a Number of Culture Trials Trial no. T. suecica Average yield (cells × 10-Jl day-l) Harvesting period (days) L galbana Average yield (cells × 10 -II day -j ) Harvesting period (days)
1
2
3
4
5
6
7
8
9
0"66
0.55
0.46
0"59
0"58
0'48
0"44
0"55
0"55
69
24
24
25
40
24
35
24
32
4"40
5.78
5.44
21
18
24
Towards the end of the harvesting period there was a tendency for the cells to stick to the polyethylene around the lamps. This could be due either to the high cell densities at which cultures were maintained or to the nature of the material. It caused a reduction in illumination intensity and probably contributed to the shorter harvesting period from 40 litre as compared with 80 litre vessels. However, the culture trials in the polyethylene bag vessels were run successively, whereas the 80 litre vessels, which were made from glass and acrylic, required 2-3 days between trials for cleaning and sterilising.
CONCLUSION The 40 litre vessel described has been shown to give a similar yield to 80 hire vessels, the estimated total cost of constructing one of these vessels is only about one-third of that for a vessel of similar design but made entirely from re-usable materials.
96
L Laing, E. Jones
REFERENCES Laing, I. & Jones, E. (1983). Large-scale turbidostat culture of marine microalgae. Aquacultural Engineering, 2,203-12. Walne, P. R. (1966). Experiments in the large-scale culture of the larvae of Ostrea edulis L. Fishery lnvest., Lond., Ser. 2, 25 (4), 53.