Choonawala, B.B. and F.M. Swalaha - eWISA
SPIRULINA SP. PRODUCTION IN BRINE EFFLUENT FROM COOLING TOWERS Choonawala, B.B*. and Swalaha, F.M*. Department of Biotechnology, Durban Institute of Technology, P.O. Box 1334, Durban, 4000 *
Durban Institute of Technology, P.O. Box 1334, Steve Biko Campus, Durban , 4000 Tel: 031 2042350 Fax: 031 2042793 E-mail: [email protected]
ABSTRACT Spirulina is a blue-green, multicellular, filamentous cyanobacterium that can grow to sizes of 0.5 millimetres in length. It is an obligate photoautotroph and has a pH growth range from 8.3 to 11.0. Brine effluent from cooling towers of electricity generating plants may provide an ideal growth medium for Spirulina sp. based on its growth requirements, i.e. high alkalinity and salinity. The aim of this research was to optimise the growth of Spirulina sp in cooling tower brine effluent in order to produce Spirulina sp as a viable feed supplement. Supplementation experiments were based on two statistical fractional factorial experiments, the first consisting of eight flasks and the second, 19. Salts from Zarrouks medium (2), NaNO3, NaCl, K2SO4 and NaHCO3 (Fractional Factorial 1 [FF1]) and K2HPO4, MgSO4.7H2O, CaCl2, FeSO4.7H2O, NaEDTA and a trace metals solution (Fractional Factorial 2 [FF2]) were used to supplement brine effluent. These fractional factorial experiments screened for the effects of the 10 factors above with either the supplement being present or absent in a specific flask. Flasks containing 250 ml brine effluent were inoculated with 5.8 x 107 cells of Spirulina sp and incubated for ten days on a shaker at 250C. A 5 ml sample from each flask, was removed every 24 hours and the turbidity read at 550 nm to determine growth. Growth rate and amount of Spirulina produced in each culture flask was calculated by converting absorbance to concentration using a standard curve. Concentrations of Spirulina sp. produced were calculated every 24 hours and the effects of each combination was assessed at 172 hours when the maximum concentrations were reached. After 172 hours a maximum of 193 mg.l-1 of Spirulina was found in brine effluent which was supplemented with NaCl and NaNO3. The specific growth rate was calculated to be 0.03 h-1 and the doubling time of the culture was 23 hours. A maximum concentration of 193 mg.l-1 was obtained in brine effluent supplemented with CaCl2 and FESO4.7H20. The specific growth rate was 0.02 h-1 and the doubling time was 34.65 hrs. A Pareto chart displayed the magnitude of the effects of each factor from the data set. The chart for the first set of experiments indicated that the NaCl had the largest positive effect on the growth of Spirulina in brine effluent. The order of the effects from the Pareto chart were NaCl > NaHCO3 ~ NaNO3 > K2SO4. For the second experiment, FeS04.7H2O and CaCl2 had the largest positive effect on the growth of Spirulina. The order of the effects from the Pareto chart were> FeS04.7H2O>CaCl2>MgSO4.7H2O > K2HPO4 > NaEDTA > Trace Metals. Overall, supplementation increased Spirulina sp. yield by 86%. These experiments show that effluent medium needs to be supplemented in order to obtain higher yields and that NaCl, FeSO47H2O and CaCl2 have the largest effect on growth.
INTRODUCTION Spirulina is a blue-green, multi-cellular (1), filamentous cyanobacterium (2). Its name is derived from the latin word for ‘helix’ or ‘spiral’, denoting the physical configuration of the organism as it forms swirling microscopic strands. Even though it is single-celled, Spirulina is relatively large, attaining sizes of 0.5 millimetres in length. The diameter of the cells ranges from 1 to 3 µm in the smaller species and from 3 to 12 µm in the larger. The prolific reproductive capacity of the cells and their proclivity to adhere in colonies makes Spirulina sp. a large and easily gathered plant mass (2). A basic issue in the production of photoautotrophic organisms in general and Spirulina sp. in particular is to maintain a continuous culture with an optimal population density. The basic demand in this respect is to provide growth conditions that will not be too much different from the optimal for Spirulina sp. The pH should be maintained as high as possible to create exclusive surroundings for Spirulina sp. with out limiting growth (2). Spirulina thrives in warm alkaline fresh water bodies and can readily tolerate progressive changes in pH. The culture, however, could quickly deteriorate when the pH is changed abruptly, as may happen in a growth medium which is not well buffered (2). Spirulina sp. thrives in very warm waters of 32 to 450C and has even survived in temperatures of 600C (1). The culture of Spirulina sp. depends on a suitable growth medium such as saline water. The large scale cultivation of microalgae and the use of its biomass as a source of certain food constituents was considered as early as the 1950's. It is being developed as the ‘food for the future’ because of its remarkable ability to synthesise high-quality concentrated food more efficiently than any other algae. Most notably Spirulina sp. is 65 to 71% percent complete protein, with all essential amino acids in perfect balance (3). It also provides a high concentration of many other nutrients, chelated minerals, pigmentations, rhamose sugars (complex natural plant sugars) trace elements, enzymes -that are in an easily assimilable form (2). The primary aim in developing a culture media is ensuring that the required nutrients are present in an appropriate form and at non-inhibitory concentrations. This objective can be challenging when one considers the diversity of microorganisms in nature as well as their diverse nutritional requirements (5). Oilfield brine, a wastewater from the petroleum industry contains high levels of dissolved salts and residual hydrocarbons (4). Treatment of the brine with hydrocarbon degrading bacteria and dilution to desired salinity produces a diluent that is suitable for preparation of a growth medium for Spirulina sp. The brine effluent to be used in this study will be obtained from Eskom and is the result of the cooling tower blow-down at the power station. After initial cultivation is achieved, the media can then be subjected to optimisation studies in order to find the conditions, appropriate nutrients, nutrient concentration, temperature, aeration etc, that will best support the growth of the organism. An optimisation strategy that is commonly used in the laboratory is the one-variable-at-a-time method in which an independent variable is tested and optimised while all other variables are kept constant (5). When a large number of variables are tested, valuable time and resources are wasted due to large experiments. An alternative strategy for optimisation is to use statistically designed experiments that allow an investigation of more that one independent variable at
a time in smaller experiments (5). A screening experiment determines which few process variables out of many candidates have an important effect on process performance. A tractional factorial design is a fraction of a full-factorial design which decreases the number of experiments and allows for optimisation (7). The growth of Spirulina sp. on wastewater is an attractive possibility. It will not compete with conventional agriculture for land, it is less dependant on favourable weather conditions and its yield is much higher than that of agriculture. Despite having to sterilise the biomass before it can be used for animal or human consumption, in the long run, two goals will be accomplished: recycling of industrial wastes and the production of protein biomass (4).
MATERIALS AND METHODS Inoculum preparation: 20 ml of Spirulina sp. (CSIR, Upington) grown in Zarrouks (6) medium at 200C on a shaker, was centrifuged at 7700 x g for 10 minutes. The remaining pellet was washed twice in distilled water, resuspended, counted in a Nebauer Counting chamber to a cell density of 5.8 x107 cells per ml and used as an inoculum. Growth Estimation: 250 ml of brine effluent supplemented with salts (Table 1 and Table 2) was inoculated with Spirulina sp. The flasks were incubated on a shaker at 160 rpm at 250C for 10 days and exposed to al diurnal light cycle. Two five ml samples from each flask, were removed every 24 hours and the turbidity was read at 550 nm to determine growth. Standard curves were used to convert absorbances to concentrations of biomass. Specific growth rates and doubling times were calculated. Fractional-Factorial experiments: The components of Zarrouks medium were used to supplement the brine effluent in two fractional factorial experiments, the first of which consisted of 8 flasks (Table 1) and the second 19 flasks (Table 2). A Pareto ranks effects in descending order according to the largest effect a supplement has on an experiment. Cube plots were used to pinpoint maximum biomass produced for 3-factor interactions. Contour graphs were used to show maximum biomass produced from 2-factor interactions. Design Expert 7 (Industat Pro, Cape Town) was used to design and interpret all fractional-factorial experiments.
Table 1 Fractional factorial 1 FF0408 - four factors and eight experiments. The table indicates the presence and absence of the four supplements used in the first experiment.
Run
NaNO3
NaCl
K2SO4
NaHCO3
A1
-
-
-
-
A2
-
-
+
+
A3
-
+
-
+
A4
-
+
+
-
A5
+
-
-
+
A6
+
-
+
-
A7
+
+
-
-
A8
+
+
+
+
Table 2 Fractional Factorial 2 - FF0616 with three centre points - six factors and 16 experiments. The table indicates the presence and absence of the six supplements used in the second experiment. Run
FeSO4.7H2O
CaCl2
MgSO4.7H2 O
K2HPO4
Na.EDTA
Trace metals
B1
-
-
-
-
-
-
B2
+
-
-
-
+
+
B3
-
+
-
-
+
+
B4
+
+
-
-
-
-
B5
-
-
+
-
+
-
B6
+
-
+
-
-
+
B7
-
+
+
-
-
+
B8
+
+
+
+
-
-
B9
-
-
-
+
-
+
B 10
+
-
-
+
+
-
B 11
-
+
-
+
+
-
B 12
+
+
-
+
-
+
B 13
-
-
+
+
+
+
B 14
+
-
+
+
-
+
B 15
-
+
+
+
-
-
B 16
+
+
+
+
+
+
B 17
0
0
0
0
0
0
B 18
0
0
0
0
0
0
B 19
0
0
0
0
0
0
RESULTS Growth curve results for FF1
220 200
-1
Spirulina (mg.l )
180 160 140 120 Run A2 (K2SO4)
100
Run A4 (NaCl and K2SO4) Run A5 (NaNO3, NaHCO3)
80
Run A7 (NaNO3 and NaCl)
60 0
50
100
150
200
250
300
Time (hrs)
Figure 1Growth of Spirulina sp. in brine effluent supplemented with NaCl, NaNO3, K2SO4 and NaHCO3 Table 3 Analysis of variance (Partial sum of Squares - Type III) )of the model calculated to fit FF1 Source
Sum of Squares
df
Mean Square
F Value
p-value Prob>
Model
26021.64
7
3717.38
6939.43
Durban Institute of Technology, P.O. Box 1334, Steve Biko Campus, Durban , 4000 Tel: 031 2042350 Fax: 031 2042793 E-mail: [email protected]
ABSTRACT Spirulina is a blue-green, multicellular, filamentous cyanobacterium that can grow to sizes of 0.5 millimetres in length. It is an obligate photoautotroph and has a pH growth range from 8.3 to 11.0. Brine effluent from cooling towers of electricity generating plants may provide an ideal growth medium for Spirulina sp. based on its growth requirements, i.e. high alkalinity and salinity. The aim of this research was to optimise the growth of Spirulina sp in cooling tower brine effluent in order to produce Spirulina sp as a viable feed supplement. Supplementation experiments were based on two statistical fractional factorial experiments, the first consisting of eight flasks and the second, 19. Salts from Zarrouks medium (2), NaNO3, NaCl, K2SO4 and NaHCO3 (Fractional Factorial 1 [FF1]) and K2HPO4, MgSO4.7H2O, CaCl2, FeSO4.7H2O, NaEDTA and a trace metals solution (Fractional Factorial 2 [FF2]) were used to supplement brine effluent. These fractional factorial experiments screened for the effects of the 10 factors above with either the supplement being present or absent in a specific flask. Flasks containing 250 ml brine effluent were inoculated with 5.8 x 107 cells of Spirulina sp and incubated for ten days on a shaker at 250C. A 5 ml sample from each flask, was removed every 24 hours and the turbidity read at 550 nm to determine growth. Growth rate and amount of Spirulina produced in each culture flask was calculated by converting absorbance to concentration using a standard curve. Concentrations of Spirulina sp. produced were calculated every 24 hours and the effects of each combination was assessed at 172 hours when the maximum concentrations were reached. After 172 hours a maximum of 193 mg.l-1 of Spirulina was found in brine effluent which was supplemented with NaCl and NaNO3. The specific growth rate was calculated to be 0.03 h-1 and the doubling time of the culture was 23 hours. A maximum concentration of 193 mg.l-1 was obtained in brine effluent supplemented with CaCl2 and FESO4.7H20. The specific growth rate was 0.02 h-1 and the doubling time was 34.65 hrs. A Pareto chart displayed the magnitude of the effects of each factor from the data set. The chart for the first set of experiments indicated that the NaCl had the largest positive effect on the growth of Spirulina in brine effluent. The order of the effects from the Pareto chart were NaCl > NaHCO3 ~ NaNO3 > K2SO4. For the second experiment, FeS04.7H2O and CaCl2 had the largest positive effect on the growth of Spirulina. The order of the effects from the Pareto chart were> FeS04.7H2O>CaCl2>MgSO4.7H2O > K2HPO4 > NaEDTA > Trace Metals. Overall, supplementation increased Spirulina sp. yield by 86%. These experiments show that effluent medium needs to be supplemented in order to obtain higher yields and that NaCl, FeSO47H2O and CaCl2 have the largest effect on growth.
INTRODUCTION Spirulina is a blue-green, multi-cellular (1), filamentous cyanobacterium (2). Its name is derived from the latin word for ‘helix’ or ‘spiral’, denoting the physical configuration of the organism as it forms swirling microscopic strands. Even though it is single-celled, Spirulina is relatively large, attaining sizes of 0.5 millimetres in length. The diameter of the cells ranges from 1 to 3 µm in the smaller species and from 3 to 12 µm in the larger. The prolific reproductive capacity of the cells and their proclivity to adhere in colonies makes Spirulina sp. a large and easily gathered plant mass (2). A basic issue in the production of photoautotrophic organisms in general and Spirulina sp. in particular is to maintain a continuous culture with an optimal population density. The basic demand in this respect is to provide growth conditions that will not be too much different from the optimal for Spirulina sp. The pH should be maintained as high as possible to create exclusive surroundings for Spirulina sp. with out limiting growth (2). Spirulina thrives in warm alkaline fresh water bodies and can readily tolerate progressive changes in pH. The culture, however, could quickly deteriorate when the pH is changed abruptly, as may happen in a growth medium which is not well buffered (2). Spirulina sp. thrives in very warm waters of 32 to 450C and has even survived in temperatures of 600C (1). The culture of Spirulina sp. depends on a suitable growth medium such as saline water. The large scale cultivation of microalgae and the use of its biomass as a source of certain food constituents was considered as early as the 1950's. It is being developed as the ‘food for the future’ because of its remarkable ability to synthesise high-quality concentrated food more efficiently than any other algae. Most notably Spirulina sp. is 65 to 71% percent complete protein, with all essential amino acids in perfect balance (3). It also provides a high concentration of many other nutrients, chelated minerals, pigmentations, rhamose sugars (complex natural plant sugars) trace elements, enzymes -that are in an easily assimilable form (2). The primary aim in developing a culture media is ensuring that the required nutrients are present in an appropriate form and at non-inhibitory concentrations. This objective can be challenging when one considers the diversity of microorganisms in nature as well as their diverse nutritional requirements (5). Oilfield brine, a wastewater from the petroleum industry contains high levels of dissolved salts and residual hydrocarbons (4). Treatment of the brine with hydrocarbon degrading bacteria and dilution to desired salinity produces a diluent that is suitable for preparation of a growth medium for Spirulina sp. The brine effluent to be used in this study will be obtained from Eskom and is the result of the cooling tower blow-down at the power station. After initial cultivation is achieved, the media can then be subjected to optimisation studies in order to find the conditions, appropriate nutrients, nutrient concentration, temperature, aeration etc, that will best support the growth of the organism. An optimisation strategy that is commonly used in the laboratory is the one-variable-at-a-time method in which an independent variable is tested and optimised while all other variables are kept constant (5). When a large number of variables are tested, valuable time and resources are wasted due to large experiments. An alternative strategy for optimisation is to use statistically designed experiments that allow an investigation of more that one independent variable at
a time in smaller experiments (5). A screening experiment determines which few process variables out of many candidates have an important effect on process performance. A tractional factorial design is a fraction of a full-factorial design which decreases the number of experiments and allows for optimisation (7). The growth of Spirulina sp. on wastewater is an attractive possibility. It will not compete with conventional agriculture for land, it is less dependant on favourable weather conditions and its yield is much higher than that of agriculture. Despite having to sterilise the biomass before it can be used for animal or human consumption, in the long run, two goals will be accomplished: recycling of industrial wastes and the production of protein biomass (4).
MATERIALS AND METHODS Inoculum preparation: 20 ml of Spirulina sp. (CSIR, Upington) grown in Zarrouks (6) medium at 200C on a shaker, was centrifuged at 7700 x g for 10 minutes. The remaining pellet was washed twice in distilled water, resuspended, counted in a Nebauer Counting chamber to a cell density of 5.8 x107 cells per ml and used as an inoculum. Growth Estimation: 250 ml of brine effluent supplemented with salts (Table 1 and Table 2) was inoculated with Spirulina sp. The flasks were incubated on a shaker at 160 rpm at 250C for 10 days and exposed to al diurnal light cycle. Two five ml samples from each flask, were removed every 24 hours and the turbidity was read at 550 nm to determine growth. Standard curves were used to convert absorbances to concentrations of biomass. Specific growth rates and doubling times were calculated. Fractional-Factorial experiments: The components of Zarrouks medium were used to supplement the brine effluent in two fractional factorial experiments, the first of which consisted of 8 flasks (Table 1) and the second 19 flasks (Table 2). A Pareto ranks effects in descending order according to the largest effect a supplement has on an experiment. Cube plots were used to pinpoint maximum biomass produced for 3-factor interactions. Contour graphs were used to show maximum biomass produced from 2-factor interactions. Design Expert 7 (Industat Pro, Cape Town) was used to design and interpret all fractional-factorial experiments.
Table 1 Fractional factorial 1 FF0408 - four factors and eight experiments. The table indicates the presence and absence of the four supplements used in the first experiment.
Run
NaNO3
NaCl
K2SO4
NaHCO3
A1
-
-
-
-
A2
-
-
+
+
A3
-
+
-
+
A4
-
+
+
-
A5
+
-
-
+
A6
+
-
+
-
A7
+
+
-
-
A8
+
+
+
+
Table 2 Fractional Factorial 2 - FF0616 with three centre points - six factors and 16 experiments. The table indicates the presence and absence of the six supplements used in the second experiment. Run
FeSO4.7H2O
CaCl2
MgSO4.7H2 O
K2HPO4
Na.EDTA
Trace metals
B1
-
-
-
-
-
-
B2
+
-
-
-
+
+
B3
-
+
-
-
+
+
B4
+
+
-
-
-
-
B5
-
-
+
-
+
-
B6
+
-
+
-
-
+
B7
-
+
+
-
-
+
B8
+
+
+
+
-
-
B9
-
-
-
+
-
+
B 10
+
-
-
+
+
-
B 11
-
+
-
+
+
-
B 12
+
+
-
+
-
+
B 13
-
-
+
+
+
+
B 14
+
-
+
+
-
+
B 15
-
+
+
+
-
-
B 16
+
+
+
+
+
+
B 17
0
0
0
0
0
0
B 18
0
0
0
0
0
0
B 19
0
0
0
0
0
0
RESULTS Growth curve results for FF1
220 200
-1
Spirulina (mg.l )
180 160 140 120 Run A2 (K2SO4)
100
Run A4 (NaCl and K2SO4) Run A5 (NaNO3, NaHCO3)
80
Run A7 (NaNO3 and NaCl)
60 0
50
100
150
200
250
300
Time (hrs)
Figure 1Growth of Spirulina sp. in brine effluent supplemented with NaCl, NaNO3, K2SO4 and NaHCO3 Table 3 Analysis of variance (Partial sum of Squares - Type III) )of the model calculated to fit FF1 Source
Sum of Squares
df
Mean Square
F Value
p-value Prob>
Model
26021.64
7
3717.38
6939.43
