Accepted Article
Article Type: Original Article
Seasonal Isolation of Microalgae from Municipal Wastewater for Remediation and Biofuel Applications
Kyoung C. Park, Crystal E.G. Whitney, Catherine Kozera, Stephen J.B. O’Leary, Patrick J. McGinn*
Aquatic and Crop Resources Development, National Research Council of Canada, 1411 Oxford St., Halifax, N.S., Canada B3H 3Z1
*Corresponding author: Patrick J. McGinn Aquatic and Crop Resources Development, National Research Council of Canada, 1411 Oxford St., Halifax, N.S., Canada B3H 3Z1 Tel: 1 902 293 2746 Email: [email protected]
Abstract Aims: The objective of the study was to isolate microalgae strains from treated municipal wastewater in both summer and winter seasons in order to identify strains better suited for nutrient remediation and biofuel production under either cooler or warmer temperatures.
Methods and Results: Fifty-six strains in total were isolated and identified by DNA sequencing from effluent samples collected from a local wastewater treatment plant during the summer and winter of 2011. Screening of 41 isolates based on fatty acid productivity at either 22 or 10 °C resulted in the selection of
12 strains organized into two groups of 6 - the M (mild) and C (cool) groups, respectively. Four of the Cgroup strains were isolated from the winter sample, while four of the M-group isolates were isolated from
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an 'Accepted Article', doi: 10.1111/jam.12818 This article is protected by copyright. All rights reserved.
Accepted Article
the summer sample. Fatty acid pools in M-group strains were heavily regulated in response to growth temperature while C-group strains were more insensitive. In three of the six C-group strains, the rates of biomass and fatty acid productivity at 10 °C exceeded the corresponding rates at 22 °C. Conversely, M-
group were always more productive at 22 compared to 10 °C. Mixotrophic strategies to enhance productivity were generally unsuccessful in M-group strains at 22°C but proved more effective in C-
group cultures at 10 °C.
Conclusions: In general, C group strains appeared better suited for growth in municipal wastewater at 10 °C, while M group strains were better suited at 22 °C. On balance, C-group isolates were more likely to
come from winter wastewater samples while M-group strains were more likely to come from the summer sample.
Significance and Impact: Our results demonstrate that the effects of temperature on microalgal growth for wastewater remediation can be mitigated somewhat by isolation and careful selection of strains adapted to seasonal wastewater conditions.
Keywords: Wastewater, bioremediation, biomass productivity, microalgae, CO2 capture, biofuel, fatty acids, temperature
Introduction In recent years, tremendous efforts, both public and private, have been undertaken to produce
microalgae-based biofuels. However, comparatively little effort has being conducted in Northern regions due to strong seasonal effects on climate and solar insolation levels, which are likely to negatively affect feasibility and production costs. However, these disadvantages can be potentially mitigated by careful selection of strains that can be grown in free and abundant wastewater and at lower temperatures. It has been shown that some freshwater strains grow and accumulate lipid in municipal wastewater media as efficiently as in traditional laboratory growth media (Li et al. 2011; Zhou et al. 2011; McGinn et al. 2012; Park et al. 2012).
This article is protected by copyright. All rights reserved.
Accepted Article
microalgal biofuel production using municipal wastewater, provided suitable strains with acceptable growth rates at the lower temperature ranges can be isolated and successfully cultivated.
Even though there are some reports of the effects of low temperatures on fatty acid profiles in
microalgae (Joh et al. 1993; Thompson 1996; McLarnon-Riches et al. 1998; Jiang and Gao 2004), information related to the potential for microalgae to remediate wastewater under such conditions is still rather limited. However, it has been generally established that membrane lipids are strongly influenced by temperature changes. When temperature is decreased, more unsaturated fatty acids are produced in microalgae membranes; while more saturated fatty acids are produced when temperature is increased (Guschina and Harwood 2006).
To overcome cold-weather disadvantages in Northern regions, we isolated 56 strains of
microalgae that were tolerant to cold-temperatures (5°C) from a secondary municipal wastewater, from which we further down-selected to a group of 19 isolates. We compared their biomass and lipid production at two temperatures, 10 and 22°C, which represented the approximate range of temperatures
expected in wastewater effluent in Northern latitudes over an annual cycle. We also explored the bioremediation efficiency of these strains and their potential to grow mixotrophically as a strategy to augment growth and biomass production.
Materials and Methods Microalgae strain isolation Municipal wastewater was obtained from the Mill Cove Waste Water treatment plant (Bedford,
Nova Scotia) in June, 2010 (summer sample) and January, 2011 (winter sample). Secondary wastewater effluent was collected in carboys (15 l) and maintained in an incubator for one month at 5°C under continuous illumination of 50 µmol photons m-2 s-1. Carboys were passively aerated using 0.2 µm PTFE membrane filter (Pall Life Sciences, Port Washington). After a month of incubation in these conditions, a small volume (100 μl) of wastewater was transferred onto ten solidified agar plates containing modified Bold’s basal medium (Bold 1949), as described elsewhere (Park et al.2012). A total of 56 microalgae isolates (36 and 20 from the summer and winter samples, respectively) were eventually obtained after serial plating on agar containing ampicillin/chloramphenicol/streptomycin (100 μg ml-1 for each). Strains
sensitive to antibiotics were transferred without them until no bacterial colonies developed.
This article is protected by copyright. All rights reserved.
and phosphate, respectively. Centrate was diluted to 1% (v/v) in MCWW to give 32.8 mg l-1, 4.46 mg l-1
Accepted Article
and 300 mg l-1 for ammonium, phosphate, and total organic carbon, respectively.
Specific growth rate, biomass and lipid-FAMEs production measurement Specific growth rate (μ, units of d-1) was measured by cell counts from day 1 to day 4, using the
equation: μ (d-1) =
(1)
where N2 and N1 are the cell numbers sampled at times T2 (day 4) and T1 (day 1), respectively.
The biomass productivity (BP) and fatty acid productivity (FAP) were calculated using the equations, as described by Griffiths and Harrison (2009) BP (mg l-1d-1) =
(2)
FAP (mg l-1d-1) =
(3)
where B2 and B1 are the biomass at times T2 and T1, respectively and FA2 and FA1 are the fatty acid contents of the biomass (in % dry biomass wt.) at times T2 and T1, respectively.
Cells were harvested by filtration using 25 mm Whatman GF/F filters and were freeze dried using
a Freezone 4.5 l Benchtop Freeze Dryer ( Labconco, USA). Mean biomass (n=2) were determined by gravimetric analysis (Zhu and Lee 1997) and the filtered biomass was used for the analysis of fatty acids. Fatty acid content in the biomass was determined by direct transesterification to methyl esters (FAMEs) as previously described (McGinn et al. 2012). The filtered biomass was placed directly into tubes using 1ml of anhydrous toluene (Sigma-aldrich) to extract lipid along with 2 ml of 5% acetyl chloride (Fluka)/anhydrous methanol (Sigma-Aldrich), heated for 1h at 100°C. Derivatized FAME was washed with 5 ml of milli-Q water with 5% NaCl and 4 ml of milli-Q water with 2% NaHCO3. The samples were then made anhydrous with sodium sulfate (Caledon) and evaporated at 37°C under a stream of nitrogen. Total dried derivatized FAME was diluted to 5 mg ml-1 with hexane and analyzed (Ehimen et al. 2010)
using an Agilent 7890 gas chromatograph with an Omegawax 250 column.
This article is protected by copyright. All rights reserved.
Accepted Article
improved the manuscript. This work was supported by NRC Canada’s flagship program in Algal Carbon Conversion.
Conflict of Interest The authors declare that there are no conflicts of interest.
References Benemann, J., Woertz, I. and Lundquist, T. (2012) Life cycle assessment for microalgae oil production. Disruptive Science and Technology 1,68-78. Benson, D.A., Karsch-Mizrachi, I., Lipman, D.J., Ostell, J., Rapp, B.A. and Wheeler, D.L. (2000) GenBank. Nucleic Acids Res 28,15-18.
Bold, H.C. (1949) The morphology of Chlamydomonas chlamydogama, sp. nov. Bull. Torrey Bot. Club 76,101-108.
Cerón-García, M.C., García Camacho, F., Sánchez Mirón, A., Fernández, J.M., Chisti, Y. and Molina Grima, E. (2006) Mixotrophic production of marine microalga Phaeodactylum tricornutum on various carbon sources. J Microbiol Biotechnol 16,689-694. Danielewicz, M.A., Anderson, L.A. and Franz, A.K. (2011) Triglycerol profiling of marine microalgae by mass spectrometry. J Lipid Res 52,2101-2108. Dalsgaard, J., St John, M., Kattner, G., Muller-Navarra, D. and Hage, W. (2003) Fatty acid trophic markers in the pelagic marine environment. Adv Mar Biol 46,225-340.
Ehimen, E.A., Sun, Z.F. and Carrington, C.G. (2010) Variables affecting the in situ transesterification of microalgae lipids. Fuel 89,677-684. Guschina, I.A. and Harwood, J.L. (2006) Lipids and lipid metabolism in eukaryotic algae. Prog Lipid Res 45,160-186. Griffiths, M.J. and Harrison, S.T.L. (2009) Lipid productivity as a key characteristic for choosing algal species for biodiesel production. J Appl Phycol 21,493-507. Ingram, L.O., Van Baalen, C. and Calder, J.A. (1973) Role of reduced exogenous organic compounds in the physiology of the blue-greeen bacteria (algae): photoheterotrophic growth of an “autotrophic” bluegreen bacterium. J Bacteriol 114,701-705. Jiang, H. and Gao, K. (2004) Effects of lowering temperature during culture on the production of polyunsaturated fatty acids in the marine diatom Phaeodactylum tricornutum (bacillariophyceae). J Phycol 40,651-654. Joh, T., Yoshida, T., Yoshimoto, M., Miyamoto, T. and Hatano, S. (1993) Composition and positional distribution of fatty acids in polar lipids from Chlorella ellipsoidea differing in chilling susceptibility and frost hardiness. Physiol Plant 89,285-290. Kaplan, D., Richmond, A.E., Dubinsky, Z. and Aaronson, S. (1986) In Handbook for Microalgal Mass Culture ed. Richmond, A. pp. 147-198. CRC Press, Boch Raton, FL., USA.
This article is protected by copyright. All rights reserved.
Accepted Article
Down-Selection to 19 Isolates and Cultivation at 10 °C Isolates which demonstrated superior fatty acid production with a minimum 2.2 mg l-1d-1in the
initial screen of 41 strains were selected for further study at 10 °C. In addition, at least one strain from each species was also selected for evaluation of growth at low temperature. The combination of these two criteria resulted in the down-selection to 19 isolates from 41 (Table 2). With the exception of the lower cultivation temperature, all other conditions were the same as those used for the initial screen at 22 °C. For the growth experiments at 10 °C, the selected strains were sub-cultured at the lower temperature for two weeks before inoculation (Table 2).
Mixotrophic cultivation of top 6 selected strains with MCWW at 10 °C or 22 °C Based on the results of the screen of 19 isolates grown at 10 and 22 °C (cool and mild
temperature, respectively), 12 strains which showed better growth characteristics under either cool (6 isolates) or mild (6 isolates) temperatures were selected for further study under mixotrophic cultivation conditions. Two replicate batch cultures of each isolate were grown in 250-ml Erlenmeyer flasks containing 150 ml of MCWW media for either 10 days (10°C) or 8 days (22°C). The initial light intensity was approximately 75 µmol photons m-2 s-1 but was increased to 125 µmol photons m-2 s-1 at day 3. During growth, CO2 was gradually supplemented to the air-stream to a final concentration of 1.5% (v/v) by day 4. The pH of the culture at the final day was around 8. For mixotrophic cultivation, MCWW was supplemented with 3 different sources of carbon: glycerol (50 mmol); acetate (14 mmol) or centrate (1%; centrate was obtained from the Annacis Island Wastewater Treatment Plant, Vancouver, B.C.).
MCWW-nutrient level and biochemical information Wastewater samples were taken at day 0 and the final day of cultivation to assess the extent of
microalgal mediated N and P removal. Ten mL of culture were filtered using 0.2 μm filter and stored until analysis. Residual nutrients in the sample were measured using a DR 2800 spectrophotometer (Hach, Loveland, CO, U.S.A.) with the following commercial assay kits supplied by Hach Co. (CO, USA) : ammonia-N by the salicylate method with a detection range of 2-47 mg N /L (supplier # TNT832), phosphorus-P by the ascorbic acid method with a detection range of 0.15-4.5 mg P/L (supplier # TNT843), total nitrogen by the Kjeldahl method (supplier # TNT880) and low range total organic carbon (TOC) by direct digestion and oxidation to CO2 with a detection range of 0.3-20 mg/L (supplier #
2760345)The initial nutrient concentrations in MCWW were 20.8 mg l-1 and 2.15 mg l-1 for ammonium
This article is protected by copyright. All rights reserved.
and phosphate, respectively. Centrate was diluted to 1% (v/v) in MCWW to give 32.8 mg l-1, 4.46 mg l-1
Accepted Article
and 300 mg l-1 for ammonium, phosphate, and total organic carbon, respectively.
Specific growth rate, biomass and lipid-FAMEs production measurement Specific growth rate (μ, units of d-1) was measured by cell counts from day 1 to day 4, using the
equation: μ (d-1) =
(1)
where N2 and N1 are the cell numbers sampled at times T2 (day 4) and T1 (day 1), respectively.
The biomass productivity (BP) and fatty acid productivity (FAP) were calculated using the equations, as described by Griffiths and Harrison (2009) BP (mg l-1d-1) =
(2)
FAP (mg l-1d-1) =
(3)
where B2 and B1 are the biomass at times T2 and T1, respectively and FA2 and FA1 are the fatty acid contents of the biomass (in % dry biomass wt.) at times T2 and T1, respectively.
Cells were harvested by filtration using 25 mm Whatman GF/F filters and were freeze dried using
a Freezone 4.5 l Benchtop Freeze Dryer ( Labconco, USA). Mean biomass (n=2) were determined by gravimetric analysis (Zhu and Lee 1997) and the filtered biomass was used for the analysis of fatty acids. Fatty acid content in the biomass was determined by direct transesterification to methyl esters (FAMEs) as previously described (McGinn et al. 2012). The filtered biomass was placed directly into tubes using 1ml of anhydrous toluene (Sigma-aldrich) to extract lipid along with 2 ml of 5% acetyl chloride (Fluka)/anhydrous methanol (Sigma-Aldrich), heated for 1h at 100°C. Derivatized FAME was washed with 5 ml of milli-Q water with 5% NaCl and 4 ml of milli-Q water with 2% NaHCO3. The samples were then made anhydrous with sodium sulfate (Caledon) and evaporated at 37°C under a stream of nitrogen. Total dried derivatized FAME was diluted to 5 mg ml-1 with hexane and analyzed (Ehimen et al. 2010)
using an Agilent 7890 gas chromatograph with an Omegawax 250 column.
This article is protected by copyright. All rights reserved.
Accepted Article
Statistical analysis For the statistical analysis of each results, we performed a t-test using the GraphPad (GraphPad
Software, Inc. San Diego, CA). Results at the P
Article Type: Original Article
Seasonal Isolation of Microalgae from Municipal Wastewater for Remediation and Biofuel Applications
Kyoung C. Park, Crystal E.G. Whitney, Catherine Kozera, Stephen J.B. O’Leary, Patrick J. McGinn*
Aquatic and Crop Resources Development, National Research Council of Canada, 1411 Oxford St., Halifax, N.S., Canada B3H 3Z1
*Corresponding author: Patrick J. McGinn Aquatic and Crop Resources Development, National Research Council of Canada, 1411 Oxford St., Halifax, N.S., Canada B3H 3Z1 Tel: 1 902 293 2746 Email: [email protected]
Abstract Aims: The objective of the study was to isolate microalgae strains from treated municipal wastewater in both summer and winter seasons in order to identify strains better suited for nutrient remediation and biofuel production under either cooler or warmer temperatures.
Methods and Results: Fifty-six strains in total were isolated and identified by DNA sequencing from effluent samples collected from a local wastewater treatment plant during the summer and winter of 2011. Screening of 41 isolates based on fatty acid productivity at either 22 or 10 °C resulted in the selection of
12 strains organized into two groups of 6 - the M (mild) and C (cool) groups, respectively. Four of the Cgroup strains were isolated from the winter sample, while four of the M-group isolates were isolated from
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an 'Accepted Article', doi: 10.1111/jam.12818 This article is protected by copyright. All rights reserved.
Accepted Article
the summer sample. Fatty acid pools in M-group strains were heavily regulated in response to growth temperature while C-group strains were more insensitive. In three of the six C-group strains, the rates of biomass and fatty acid productivity at 10 °C exceeded the corresponding rates at 22 °C. Conversely, M-
group were always more productive at 22 compared to 10 °C. Mixotrophic strategies to enhance productivity were generally unsuccessful in M-group strains at 22°C but proved more effective in C-
group cultures at 10 °C.
Conclusions: In general, C group strains appeared better suited for growth in municipal wastewater at 10 °C, while M group strains were better suited at 22 °C. On balance, C-group isolates were more likely to
come from winter wastewater samples while M-group strains were more likely to come from the summer sample.
Significance and Impact: Our results demonstrate that the effects of temperature on microalgal growth for wastewater remediation can be mitigated somewhat by isolation and careful selection of strains adapted to seasonal wastewater conditions.
Keywords: Wastewater, bioremediation, biomass productivity, microalgae, CO2 capture, biofuel, fatty acids, temperature
Introduction In recent years, tremendous efforts, both public and private, have been undertaken to produce
microalgae-based biofuels. However, comparatively little effort has being conducted in Northern regions due to strong seasonal effects on climate and solar insolation levels, which are likely to negatively affect feasibility and production costs. However, these disadvantages can be potentially mitigated by careful selection of strains that can be grown in free and abundant wastewater and at lower temperatures. It has been shown that some freshwater strains grow and accumulate lipid in municipal wastewater media as efficiently as in traditional laboratory growth media (Li et al. 2011; Zhou et al. 2011; McGinn et al. 2012; Park et al. 2012).
This article is protected by copyright. All rights reserved.
Accepted Article
microalgal biofuel production using municipal wastewater, provided suitable strains with acceptable growth rates at the lower temperature ranges can be isolated and successfully cultivated.
Even though there are some reports of the effects of low temperatures on fatty acid profiles in
microalgae (Joh et al. 1993; Thompson 1996; McLarnon-Riches et al. 1998; Jiang and Gao 2004), information related to the potential for microalgae to remediate wastewater under such conditions is still rather limited. However, it has been generally established that membrane lipids are strongly influenced by temperature changes. When temperature is decreased, more unsaturated fatty acids are produced in microalgae membranes; while more saturated fatty acids are produced when temperature is increased (Guschina and Harwood 2006).
To overcome cold-weather disadvantages in Northern regions, we isolated 56 strains of
microalgae that were tolerant to cold-temperatures (5°C) from a secondary municipal wastewater, from which we further down-selected to a group of 19 isolates. We compared their biomass and lipid production at two temperatures, 10 and 22°C, which represented the approximate range of temperatures
expected in wastewater effluent in Northern latitudes over an annual cycle. We also explored the bioremediation efficiency of these strains and their potential to grow mixotrophically as a strategy to augment growth and biomass production.
Materials and Methods Microalgae strain isolation Municipal wastewater was obtained from the Mill Cove Waste Water treatment plant (Bedford,
Nova Scotia) in June, 2010 (summer sample) and January, 2011 (winter sample). Secondary wastewater effluent was collected in carboys (15 l) and maintained in an incubator for one month at 5°C under continuous illumination of 50 µmol photons m-2 s-1. Carboys were passively aerated using 0.2 µm PTFE membrane filter (Pall Life Sciences, Port Washington). After a month of incubation in these conditions, a small volume (100 μl) of wastewater was transferred onto ten solidified agar plates containing modified Bold’s basal medium (Bold 1949), as described elsewhere (Park et al.2012). A total of 56 microalgae isolates (36 and 20 from the summer and winter samples, respectively) were eventually obtained after serial plating on agar containing ampicillin/chloramphenicol/streptomycin (100 μg ml-1 for each). Strains
sensitive to antibiotics were transferred without them until no bacterial colonies developed.
This article is protected by copyright. All rights reserved.
and phosphate, respectively. Centrate was diluted to 1% (v/v) in MCWW to give 32.8 mg l-1, 4.46 mg l-1
Accepted Article
and 300 mg l-1 for ammonium, phosphate, and total organic carbon, respectively.
Specific growth rate, biomass and lipid-FAMEs production measurement Specific growth rate (μ, units of d-1) was measured by cell counts from day 1 to day 4, using the
equation: μ (d-1) =
(1)
where N2 and N1 are the cell numbers sampled at times T2 (day 4) and T1 (day 1), respectively.
The biomass productivity (BP) and fatty acid productivity (FAP) were calculated using the equations, as described by Griffiths and Harrison (2009) BP (mg l-1d-1) =
(2)
FAP (mg l-1d-1) =
(3)
where B2 and B1 are the biomass at times T2 and T1, respectively and FA2 and FA1 are the fatty acid contents of the biomass (in % dry biomass wt.) at times T2 and T1, respectively.
Cells were harvested by filtration using 25 mm Whatman GF/F filters and were freeze dried using
a Freezone 4.5 l Benchtop Freeze Dryer ( Labconco, USA). Mean biomass (n=2) were determined by gravimetric analysis (Zhu and Lee 1997) and the filtered biomass was used for the analysis of fatty acids. Fatty acid content in the biomass was determined by direct transesterification to methyl esters (FAMEs) as previously described (McGinn et al. 2012). The filtered biomass was placed directly into tubes using 1ml of anhydrous toluene (Sigma-aldrich) to extract lipid along with 2 ml of 5% acetyl chloride (Fluka)/anhydrous methanol (Sigma-Aldrich), heated for 1h at 100°C. Derivatized FAME was washed with 5 ml of milli-Q water with 5% NaCl and 4 ml of milli-Q water with 2% NaHCO3. The samples were then made anhydrous with sodium sulfate (Caledon) and evaporated at 37°C under a stream of nitrogen. Total dried derivatized FAME was diluted to 5 mg ml-1 with hexane and analyzed (Ehimen et al. 2010)
using an Agilent 7890 gas chromatograph with an Omegawax 250 column.
This article is protected by copyright. All rights reserved.
Accepted Article
improved the manuscript. This work was supported by NRC Canada’s flagship program in Algal Carbon Conversion.
Conflict of Interest The authors declare that there are no conflicts of interest.
References Benemann, J., Woertz, I. and Lundquist, T. (2012) Life cycle assessment for microalgae oil production. Disruptive Science and Technology 1,68-78. Benson, D.A., Karsch-Mizrachi, I., Lipman, D.J., Ostell, J., Rapp, B.A. and Wheeler, D.L. (2000) GenBank. Nucleic Acids Res 28,15-18.
Bold, H.C. (1949) The morphology of Chlamydomonas chlamydogama, sp. nov. Bull. Torrey Bot. Club 76,101-108.
Cerón-García, M.C., García Camacho, F., Sánchez Mirón, A., Fernández, J.M., Chisti, Y. and Molina Grima, E. (2006) Mixotrophic production of marine microalga Phaeodactylum tricornutum on various carbon sources. J Microbiol Biotechnol 16,689-694. Danielewicz, M.A., Anderson, L.A. and Franz, A.K. (2011) Triglycerol profiling of marine microalgae by mass spectrometry. J Lipid Res 52,2101-2108. Dalsgaard, J., St John, M., Kattner, G., Muller-Navarra, D. and Hage, W. (2003) Fatty acid trophic markers in the pelagic marine environment. Adv Mar Biol 46,225-340.
Ehimen, E.A., Sun, Z.F. and Carrington, C.G. (2010) Variables affecting the in situ transesterification of microalgae lipids. Fuel 89,677-684. Guschina, I.A. and Harwood, J.L. (2006) Lipids and lipid metabolism in eukaryotic algae. Prog Lipid Res 45,160-186. Griffiths, M.J. and Harrison, S.T.L. (2009) Lipid productivity as a key characteristic for choosing algal species for biodiesel production. J Appl Phycol 21,493-507. Ingram, L.O., Van Baalen, C. and Calder, J.A. (1973) Role of reduced exogenous organic compounds in the physiology of the blue-greeen bacteria (algae): photoheterotrophic growth of an “autotrophic” bluegreen bacterium. J Bacteriol 114,701-705. Jiang, H. and Gao, K. (2004) Effects of lowering temperature during culture on the production of polyunsaturated fatty acids in the marine diatom Phaeodactylum tricornutum (bacillariophyceae). J Phycol 40,651-654. Joh, T., Yoshida, T., Yoshimoto, M., Miyamoto, T. and Hatano, S. (1993) Composition and positional distribution of fatty acids in polar lipids from Chlorella ellipsoidea differing in chilling susceptibility and frost hardiness. Physiol Plant 89,285-290. Kaplan, D., Richmond, A.E., Dubinsky, Z. and Aaronson, S. (1986) In Handbook for Microalgal Mass Culture ed. Richmond, A. pp. 147-198. CRC Press, Boch Raton, FL., USA.
This article is protected by copyright. All rights reserved.
Accepted Article
Down-Selection to 19 Isolates and Cultivation at 10 °C Isolates which demonstrated superior fatty acid production with a minimum 2.2 mg l-1d-1in the
initial screen of 41 strains were selected for further study at 10 °C. In addition, at least one strain from each species was also selected for evaluation of growth at low temperature. The combination of these two criteria resulted in the down-selection to 19 isolates from 41 (Table 2). With the exception of the lower cultivation temperature, all other conditions were the same as those used for the initial screen at 22 °C. For the growth experiments at 10 °C, the selected strains were sub-cultured at the lower temperature for two weeks before inoculation (Table 2).
Mixotrophic cultivation of top 6 selected strains with MCWW at 10 °C or 22 °C Based on the results of the screen of 19 isolates grown at 10 and 22 °C (cool and mild
temperature, respectively), 12 strains which showed better growth characteristics under either cool (6 isolates) or mild (6 isolates) temperatures were selected for further study under mixotrophic cultivation conditions. Two replicate batch cultures of each isolate were grown in 250-ml Erlenmeyer flasks containing 150 ml of MCWW media for either 10 days (10°C) or 8 days (22°C). The initial light intensity was approximately 75 µmol photons m-2 s-1 but was increased to 125 µmol photons m-2 s-1 at day 3. During growth, CO2 was gradually supplemented to the air-stream to a final concentration of 1.5% (v/v) by day 4. The pH of the culture at the final day was around 8. For mixotrophic cultivation, MCWW was supplemented with 3 different sources of carbon: glycerol (50 mmol); acetate (14 mmol) or centrate (1%; centrate was obtained from the Annacis Island Wastewater Treatment Plant, Vancouver, B.C.).
MCWW-nutrient level and biochemical information Wastewater samples were taken at day 0 and the final day of cultivation to assess the extent of
microalgal mediated N and P removal. Ten mL of culture were filtered using 0.2 μm filter and stored until analysis. Residual nutrients in the sample were measured using a DR 2800 spectrophotometer (Hach, Loveland, CO, U.S.A.) with the following commercial assay kits supplied by Hach Co. (CO, USA) : ammonia-N by the salicylate method with a detection range of 2-47 mg N /L (supplier # TNT832), phosphorus-P by the ascorbic acid method with a detection range of 0.15-4.5 mg P/L (supplier # TNT843), total nitrogen by the Kjeldahl method (supplier # TNT880) and low range total organic carbon (TOC) by direct digestion and oxidation to CO2 with a detection range of 0.3-20 mg/L (supplier #
2760345)The initial nutrient concentrations in MCWW were 20.8 mg l-1 and 2.15 mg l-1 for ammonium
This article is protected by copyright. All rights reserved.
and phosphate, respectively. Centrate was diluted to 1% (v/v) in MCWW to give 32.8 mg l-1, 4.46 mg l-1
Accepted Article
and 300 mg l-1 for ammonium, phosphate, and total organic carbon, respectively.
Specific growth rate, biomass and lipid-FAMEs production measurement Specific growth rate (μ, units of d-1) was measured by cell counts from day 1 to day 4, using the
equation: μ (d-1) =
(1)
where N2 and N1 are the cell numbers sampled at times T2 (day 4) and T1 (day 1), respectively.
The biomass productivity (BP) and fatty acid productivity (FAP) were calculated using the equations, as described by Griffiths and Harrison (2009) BP (mg l-1d-1) =
(2)
FAP (mg l-1d-1) =
(3)
where B2 and B1 are the biomass at times T2 and T1, respectively and FA2 and FA1 are the fatty acid contents of the biomass (in % dry biomass wt.) at times T2 and T1, respectively.
Cells were harvested by filtration using 25 mm Whatman GF/F filters and were freeze dried using
a Freezone 4.5 l Benchtop Freeze Dryer ( Labconco, USA). Mean biomass (n=2) were determined by gravimetric analysis (Zhu and Lee 1997) and the filtered biomass was used for the analysis of fatty acids. Fatty acid content in the biomass was determined by direct transesterification to methyl esters (FAMEs) as previously described (McGinn et al. 2012). The filtered biomass was placed directly into tubes using 1ml of anhydrous toluene (Sigma-aldrich) to extract lipid along with 2 ml of 5% acetyl chloride (Fluka)/anhydrous methanol (Sigma-Aldrich), heated for 1h at 100°C. Derivatized FAME was washed with 5 ml of milli-Q water with 5% NaCl and 4 ml of milli-Q water with 2% NaHCO3. The samples were then made anhydrous with sodium sulfate (Caledon) and evaporated at 37°C under a stream of nitrogen. Total dried derivatized FAME was diluted to 5 mg ml-1 with hexane and analyzed (Ehimen et al. 2010)
using an Agilent 7890 gas chromatograph with an Omegawax 250 column.
This article is protected by copyright. All rights reserved.
Accepted Article
Statistical analysis For the statistical analysis of each results, we performed a t-test using the GraphPad (GraphPad
Software, Inc. San Diego, CA). Results at the P
