Glycerol from novel strains of halophytic microalgae

Glycerol is a new biofuel which underpins a commercial CHP technology as a result of the novel McNeil combustion cycle patented by Aquafuel, UK. This research explored glycerol production in new strains of halotolerant microalgae Dunaliella (T35, T36 and T37) and Asteromonas (T33a, T33b and T33c) obtained from Namibia. The aim was to: (a) Determine the optimum conditions of pH value, temperature and salinity on the specific growth rate and doubling time and parameters of glycerol production, (b) Investigate metabolites associated with glycerol accumulation and dissimilation in Dunaliella cells exposed to salinity stress under continuous light or dark regimes. (c) Maximize glycerol recovery from microalgal cultures using novel supercritical carbon dioxide (SC-CO2) extraction technology. Dunaliella and Asteromonas strains were shown to withstand large variations in the salinity of the growth medium 0.5 - 4.0 M NaCl. The optimum conditions for their growth were 1.0 M NaCl, 30 oC, pH 7.5 to 9.0 and 45 μmol m-2 s-1. Cultivation at 1.0 M NaCl produced 46.1 - 65.2 pg/cell of glycerol in Dunaliella and 53.8 to 67.1 pg/cell of glycerol in Asteromonas, but transfer of cells from 1.0 to 4.0 M NaCl for 240 min maximized glycerol production to 179.5 - 237.6 pg/cell in Dunaliella and 128.7 - 184.2 pg/cell in Asteromonas. The amount of glycerol produced by Dunaliella isolated from Namibia was between 3.3 and 5.2 fold greater than other species investigated (D. salina, D. quartolecta, D. parva and D. polymorpha). Transfer of cells to high salinities did not disrupt cell integrity, although cells shrank by 35 to 62 % of their original volume due to water efflux from the cell. The starch and glycerol pools of Dunaliella T35 were investigated after 24 h exposure to 1.0 and 4.0 M NaCl in dark and light regimes in order to investigate how starch degradation contributed to glycerol production. In 1.0 M NaCl, the amount of starch was 45 and 222 μg/mg of dry biomass under dark and light regimes respectively, and glycerol, 163 and 174 μg/mg of dry biomass. Under parallel conditions at 4.0 M NaCl, the amount of starch decreased to 27 and 146 μg/mg of dry biomass and glycerol increased to 592 and 706 μg/mg of dry biomass in the dark and light, respectively. This suggested that the contribution of starch breakdown to glycerol synthesis increased with increasing salinity stress in the Namibian strain of Dunaliella. Hyperosmotic shock decreased the pool size of proline and increased the pools size of pyruvate and glycerol. The degree of cell rupture after SC-CO2 extraction of glycerol from Dunaliella was dependent on the applied pressure. Cells were ruptured and glycerol was released at 100 bar and the degree of cell rupture increased with pressure to 200 bar in the presence of chloroform. At 350 bar in the absence of chloroform SC-CO2 extraction of glycerol produced ~6 % higher glycerol levels than sonication. Namibian strains of Dunaliella and Asteromonas T33c investigated are best suited for glycerol production compared to Asteromonas T33a, T33b and other strains of Dunaliella (D. salina, D. quartolecta, D. parva and D. polymorpha). Cells cultivated in 1.0 M NaCl to generate biomass followed by transfer to hyperosmotic stress for glycerol accumulation was shown to be optimum for glycerol production. SC-CO2 is shown to be an effective, ‘green’ technology for glycerol extraction from Dunaliella