Chapter-4 Isolation and screening of microalgae for carbon sequestration and its lipid content Abstract: Samples collected from Gujarat coast and from polluted habitat, were enriched for isolation and screening of potential microalgae for carbon sequestration and its lipid content. Most of the unicellular eukaryotic microalgae have higher lipid and biomass productivity. Out of the studied strains, Monoraphidium minutum and Chlorella variabilis were selected because of their higher lipid, biomass productivity and CO 2 tolerance. 1. Introduction: Isolation and screening of microalgae for higher lipid productivity is important criterion for biodiesel production and CO 2 sequestration (Dunahay, 1998). The tolerance of CO 2 is one of the important characteristics which should be evaluated while selecting the microalgal strain in different media as well as in varied ph (Griffiths et al., 2009; Wang et al., 2008). The present chapter deals with the selection of promising microalgal strains from the coastal environment. 2. Materials and methods: 2.1. Isolation of microalgal samples: The collected microalgal samples from described assessed sites in Chapter- 3 (Table-1) were inoculated for enrichment. Each individual sample distributed and 116
poured in different autoclaved media (BG11, modified ZM, ASN-3, BBM, ACB and F- 2) under aseptic conditions and allowed for 7 10 days for enrichment. After enrichment, all the samples were taken for serial dilution (10-1, 10-2, 10-3 ) and, streaking were done on solid CSMCRI-agarose/ agar containing media plates under aseptic conditions. Isolated colonies which were observed after 5-20 days were picked and transferred in autoclaved fresh respective medium (10 ml.) as well as plates in sterile laminar flow chamber (Rippka, 1988; Anderson, 2005). Some of the microalgae like Monoraphidium and Navicula isolated by single cell isolation technique (Anderson, 2005). Purity of the culture was examined under optical microscope with different magnifications (10X, 40X and 100X). The media composition for BG 11 (Blue green 11; Stainer et al., 1971), modified ZM (Zarrouk s media; Zarrouk, 1966), ACB (Algae culture broth, Himedia), F/2 media (Guillard, 1975) and BBM (Bold basal media; Kantz and Bold, 1969) were used. All media were prepared in distilled water and autoclaved. One liter of Zarrouk s medium consists of (part A) NaHCO 3 (16.80 g/l) and K 2 HPO 4 (0.50 g/l); (part B) NaNO 3 (2.50 g/l), K 2 SO 4 (1.00 g/l), NaCl (1.00 g/l), MgSO 4 7H 2 O (0.20 g/l), EDTA-Na 2 2H 2 O (0.08 g/l), CaCl 2 2H 2 O (0.04 g/l), and FeSO 4 2H 2 O (0.01 g/l); trace elements mixture A 5 (part C 10 ml/l): 1.00 ml, trace elements mixture B 6 (part D 1.0 ml/l): 1.00 ml; part C (mg/l): H 3 BO 3 2.86, MnCl 2 4H 2 O 1.810 g, ZnSO 4 7H 2 O 0.222 MoO 3 0.015, and CuSO 4 5H 2 O 0.074 (the used amount is 10 ml/l); part D mg/l: NH 4 VO 3 22.9, NiSO 4 7H 2 O 47.8, NaWO 2 17.9, Ti 2 (SO 4 ) 3 6H 2 O, and 117
Co(NO 3 ) 2 6H 2 O 4.4 (the amount used was 1.0 ml/l). The ph of the media was maintained at 10.0 0.02 (Zarrouk, 1966). BG-11 media composition was having NaNO 3 (1.5 g/l), K 2 HPO 4 (0.04 g/l), MgSO 4.7H 2 O (0.075 g/l), CaCl 2.2H 2 O (0.036 g/l), FeC 6 H 5 O 7 NH 4 OH (0.006 g/l), C 6 H 8 O 7 (0.006 g/l), C 10 H 14 N 2 Na 2 O 8 (0.001 g/l), Na 2 CO 3 (0.02 g/l). In addition to this trace metal mix (A5) was used 1ml/l. The trace metal mix solution was prepared separately with composition of H 3 BO 3 (2.86 g/l), MnCl 2.4H 2 O (1.81 g/l), ZnSO 4.7H 2 O (0.222 g/l), NaMoO 4.2H 2 O (0.39 g/l), CuSO 4.5H 2 O (0.079 g/l), Co(NO 3 ) 2.6H 2 O (49.4 g/l) in distilled water. The ph of the final medium was 7.1 ± 0.2 (Stainer et al., 1971). The detailed composition of the of autoclaved F/2 media in distilled water was as follows: NaCl (29.23 g/l), KCl (1.105 g/l), MgSO 4 7H 2 O (11.09 g/l), Tris-base (1.21 g/l), CaCl 2 2H 2 O (1.83 g/l), NaHCO 3 (0.25 g/l), and a 3.0 ml trace metal solution that was composed of 281.3 mg NaNO 3, 21.2 mg NaH 2 PO 4 H 2 O, 16.35 mg Na 2 EDTA, 11.8 mg FeCl 3 6H 2 O, 675 MnCl 2 4H 2 O, 37.5 CoCl 2 6H2O, 37.5 ZnSO 4 7H 2 O, 22.5 Na 2 MoO 4, 0.375 mg vitamin B1, and 0.188 of biotin (Guillard, 1975). The algae culture broth was prepared in sterile distilled water and, final composition of the media consist of NH 4 Cl (0.05 g/l), CaCl 2 (0.058 g/l), K 2 HPO 4 (0.25 g/l), FeCl 3 (0.003g/L),, MgSO 4 (0.513 g/l), NaNO 3 (1g/L). The final ph of the medium was adjusted up to 7.0 0.02. 2.2. Mass propagation and culture maintenance: 118
Culture volume of each isolated monoalgal culture successively increased by adding fresh autoclaved medium under laminar flow conditions. Isolated colonies of microalgae identified morphologically by using standard monographs (Desikachary, 1959) under 40X and 100X magnification of Carl Zeiss Microscope. The isolated cultures were also maintained as mother culture as well as on solid agar containing media plates and slant test tubes. 2.3. Screening of microalgal strains: Isolated microalgal strains were checked for their growth in different media (BG11, ZM, ACB, BBM) with some selective range of ph 3.0-10) to know their optimum growth. Growth was monitored at OD 540, dry weight (oven dry weight at 80ºC) and ph. All the cultures were harvested in stationary phase by centrifugation at 8000 rpm. The promising microalgae of higher biomass productivity were grown at 1000 ml volumetric scale. After harvesting and drying of microalgal biomass, total lipid extracted, separated and weighed gravimetrically. Microalgae having higher biomass productivity and lipid content (lipid productivity) were selected for further experiments. The total lipid was extracted from freeze-dried biomass using Chloroform: Methanol in 2:1 ratio (Bligh and Dyer, 1959). The extracted total lipid for each sample was weighed gravimetrically after removal of solvent and, %lipid calculated (Bligh and Dyer, 1959). All sample analyzed in triplicates. The resistance of microalgal strains towards CO 2 was checked by inhibition of growth in sterile CO 2 incubator under different CO 2 %. The 100 ml of each selected microalgal culture having OD 540 of 0.25 was inoculated in 250 ml flask containing favorable media. All culture flasks transferred 119
in CO 2 incubator at 50µM photons/m 2 /sec. light and 30ºC temp. for incubation wherein mouth of the flasks kept open for direct exposure of CO 2. The light source was provided externally and light transmitted through glass door of CO 2 incubator. The OD 540 and ph were checked daily. 3. Results and discussions: The Oscillatoria spp., Phormidium spp., Lyngbya Chlorella spp., Monoraphidium, Navicula, Chroococcus, Spirulina, Synechococcus, Synechocystis and Scenedesmus were isolated and identified as shown in Table 1. Out of total 17 strains, unicellular microalgae were chosen for the study. The most of the strains were alkalophilic but some of them were able to tolerate acidic conditions eg. Chlorcoccum, Synechoccoccus, and Synechocystis showed growth even when ph was acidic (6.80, 4.0 and 3.0) in BBM and BG11, which showed the resistance of these strains. The highest OD 540 was observed in Monoraphidium minutm and Chlorella variabilis in ZM as shown in Table 2. The Monoraphidum minutum, Synechococcus, Chlorococcum and Chlorella had higher biomass productivity (Table 3). Monoaraphidium minutum showed maximum biomass productivity (24.75 mg/l/day) at ph 8.0 in ZM. Monoraphidium minutum also showed comparable biomass productivity (23.83 mg/l/day) at ph 10.0 in the same ZM. The second most promising strain was Chlorella variabilis with 23.57 mg/l/day and 24.75 mg/l/day at ph 10.0 and ph 8.0 in ZM, respectively. Both M. minutum and C. variabilis were also having good growth in ACB after ZM as shown in Table 2 & Table 3. These both strains had CO 2 tolerance of 2%. The total lipid% of the 120
four promising microalgae was estimated. The lipid% of Monoraphidum minutum, Synechococcus sp, Chlorococcum and Chlorella were 20.93±1.12, 14.60±0.43, 14.07±0.58 and 15.3±0.55, respectively. 4. Conclusion: Out of the studied strains, Monoraphidium minutum and Chlorella variabilis were selected because of their higher lipid, biomass productivity and CO 2 tolerance. Chlorella variabilis was also selected because of its ongoing successful trial of mass cultivation in open ponds by CSIR-CSMCRI team. 121
Table 1. Isolated microalgal strains from different sites. Major region Sub-site Code Isolated strains Vasad Ankleshwar MAHI A-DP A-CP2 A-AML2 A-AML Chlorella, Lyngbya Chlorella Scenedesmus Chlorella Oscillatoria, Phormidium Surat- Hazira Somanath Su- IE Su-CS Som-CS Madhavpur M-CS Phormidium Bet-Dwarka D-CS Chroococcus, Scenedesmus, Monoraphidium Navicula Synechococcus, Spirulina Synechocystis, Chlorococcum, Phormidium 122
Table 2. OD 540 of the selected microalgal strains on 18 th day in different media and ph. Name of the strain/genus Initial ph BG11 ZM ACB BBM 7.1 6.0 3.0 10.0 8.0 6.0 3.0 8.0 6.0 3.0 6.8 4.0 Chlorococcum Synechococcus Synechocysits Chlorella Variabilis Monoraphidium minutum Chroococccus Scenedesmus Mean 0.82 0.51 0.40 0.46 0.55 0.65 0.29 0.71 0.99 NG 1.52 0.75 SD 0.00 0.02 0.01 0.06 0.00 0.04 0.00 0.10 0.05 0.00 0.01 0.43 Mean 0.95 0.84 0.43 0.68 1.71 0.42 0.41 0.65 0.44 NG 0.40 NG SD 0.03 0.03 0.00 0.06 0.03 0.01 0.01 0.00 0.00 0.00 0.00 0.00 Mean 0.88 0.79 0.68 0.45 0.30 0.50 0.00 0.48 0.57 NG 0.30 NG SD 0.03 0.02 0.00 0.01 0.00 0.01 0.00 0.03 0.01 0.00 0.00 0.00 Mean 0.44 0.30 NG 1.79 1.88 0.63 0.00 0.92 1.21 NG 1.10 NG SD 0.02 0.00 0.00 0.05 0.08 0.01 0.00 0.03 0.01 0.00 0.01 0.01 Mean 0.92 0.43 NG 1.81 1.89 0.70 0.00 1.74 1.22 NG 0.72 NG SD 0.01 0.04 0.00 0.02 0.05 0.00 0.00 0.01 0.03 0.00 0.00 0.00 Mean 0.42 0.44 NG 1.08 1.37 0.95 0.31 1.22 0.42 NG 0.45 NG SD 0.01 0.04 0.00 0.02 0.05 0.00 0.02 0.01 0.01 0.00 0.00 0.00 Mean 0.99 0.64 0.29 0.71 0.69 0.53 NG 0.89 0.96 NG 0.73 NG SD 0.01 0.00 0.00 0.02 0.01 0.01 0.00 0.01 0.02 0.01 0.03 0.00 Italic value in the table shows optimum growth. NG shows that no growth was observed. 123
Table 3. Biomass productivity (mg/l/day) of some of the selected microalgal strains. Name of the strain/genus Chlorococcum Synechococcus Synechocystis Chlorella variabilis Monoraphidium minutum Chroococccus Scenedesmus BG11 ZM ACB BBM Growth inhibition by minimum CO 2 (%) Initial ph 7.1 6 3 10 8 6 3 8 6 3 6.8 4 Mean 10.80 6.72 5.27 6.06 7.24 8.56 3.82 9.35 13.04 NG 20.01 9.88 2 SD 0.01 0.26 0.13 0.79 0.01 0.53 0.09 1.32 0.66-0.13 0.66 Mean 12.51 11.06 5.66 8.95 22.52 5.53 5.40 8.56 5.79 NG 5.27 NG 1 SD 0.40 0.40 0.04 0.79 0.40 0.13 0.13 0.04 0.03-0.03 - Mean 11.59 10.40 8.95 5.93 3.95 6.58 0.01 6.32 7.51 NG 3.95 NG 1 SD 0.40 0.26 0.01 0.13 0.05 0.13 NG 0.40 0.13-0.01 - Mean 5.79 3.95 NG 23.57 24.75 8.30 NG 12.11 15.93 NG 14.48 NG 2 SD 0.26 0.01 0.01 0.66 1.05 0.13 0.01 0.40 0.13-0.20 - Mean 12.11 5.66 NG 23.83 24.89 9.22 NG 22.91 16.06 NG 9.48 NG 2 SD 0.13 0.53 0.00 0.26 0.66 0.00 NG 0.13 0.40-0.00 - Mean 5.53 5.79 NG 14.22 18.04 12.51 4.08 16.06 5.53 NG 5.93 NG 0.5 SD 0.13 0.53 0.00 0.26 0.66 0.00 0.26 0.13 0.04-0.10 - Mean 13.04 8.43 3.82 9.37 9.16 7.03 NG 11.31 12.64 NG 9.66 NG SD 0.13 0.00 0.00 0.26 0.13 0.13 0.00 0.13 0.26-0.40-0.5 Italic value in the table shows optimum biomass productivity. NG shows that no growth was observed. 124
Lipid (%) Fig. 1. Total lipid content of four promising microalgae at 18 th day. 25.00 20.00 15.00 10.00 5.00 0.00 Monoraphidium minutumchlorella variabilis Synechococcus Chlorococcum 125
References: Andersen, R. A. (Ed.). (2005). Algal culturing techniques. Academic press. Desikachary, T. V. (1959). Cyanophyta. 686 pp. Indian Council of Agricultural Research, New Delhi. Dunahay, T., Benemann, J., & Roessler, P. (1998). A look back at the US Department of Energy's aquatic species program: biodiesel from algae (Vol. 328). Golden: National Renewable Energy Laboratory Griffiths, M. J., & Harrison, S. T. (2009). Lipid productivity as a key characteristic for choosing algal species for biodiesel production. Journal of Applied Phycology, 21(5), 493-507. Guillard, R. R. (1975). Culture of phytoplankton for feeding marine invertebrates. In Culture of marine invertebrate animals (pp. 29-60). Springer US. Kantz, T., & Bold, H. C. (1969). Morphological and taxonomic investigations of Nostoc and Anabaena in culture. University of Texas. Rippka, R. (1988). Isolation and purification of cyanobacteria. Methods in enzymology, 167, 3. Stanier, R. Y., Kunisawa, R., Mandel, M., & Cohen-Bazire, G. (1971). Purification and properties of unicellular blue-green algae (order Chroococcales). Bacteriological reviews, 35(2), 171. 126
Wang, B., Li, Y., Wu, N., & Lan, C. Q. (2008). CO 2 bio-mitigation using microalgae. Applied Microbiology and Biotechnology, 79(5), 707-718. 127