The ever-increasing human demand for energy is leading to depletion of the planet’s fossil fuel reserves. Past records have shown that the price of fossil fuel has always been increasing and it is expected to rise continuously with demand in future. Use of fossil fuel generates significant amounts of greenhouse gases, which are contributing to climate change. These factors clearly highlight the need of a cheaper, renewable, and an eco-friendly fuel source. Past research has suggested that biodiesel from algae could be a possible alternative, as microalgae have been shown to produce the highest lipid yield in the lowest area when compared to other biological sources. However, despite having a high lipid yield per area, biodiesel produced by using the lipids from growing algae using current cultivation methods will not be able to meet the increasing energy demands. Furthermore, the cost of algal biomass production is very high for economic implementation on an industrial scale. Therefore the research reported here focuses on optimizing microalgal performance for biodiesel production, both phototrophically and heterotrophically. Under phototrophic conditions we analysed three different approaches to optimise lipid production; use of chemical inhibitors, phosphorus starvation and combined nitrogen and phosphorus starvation. Methionine sulfoximine (MSX) was used as a chemical inhibitor for nitrogen assimilation for instant nitrogen starvation in cultures, as nitrogen starvation or limitation has been shown to increase the lipid content of microalgae. Use of MSX induced rapid lipid accumulation in cells and prolonged higher lipid content (2-fold higher) in cells as
compared to the controls. Phosphorus starvation of cells led to lower biomass but showed up to three-fold higher cellular lipid content without affecting the photosynthetic efficiency. Combined nitrogen and phosphorus starvation also resulted in lower biomass but higher cellular lipid content; however combined nitrogen and phosphorus starvation had no synergistic effect on the lipid synthesis over individually starved nitrogen or phosphorus conditions. Also, comparison of the parameters measuring the photosynthetic physiology implied that nitrogen starvation had a more dominant effect on the photosynthetic performance of the cells than phosphorus starvation in the combined nutrient starved group. As a result, the combined nitrogen and phosphorus-starved cells behaved similarly to the nitrogen starved rather than the phosphorusstarved cells in terms of photosynthetic performance. Overall all the tested condition resulted in higher cellular neutral lipid as compared to their own control group, however the neutral lipid measured per ml was still similar to the control groups. This was mainly due to the lower biomass generated under nutrient limited/starved conditions. Therefore, investigation of other modes of cultivation such as heterotrophy and mixotrophy was done to overcome this problem.
Scenedesmus sp. was used for heterotrophic studies using molasses as the carbon source in growth media. Under heterotrophic conditions, Scenedesemus sp. performed much better than in phototrophic conditions in terms of both biomass (2.5-fold higher) and much higher lipid content (neutral lipid per ml). Surprisingly, the heterotrophically grown Scenedesmus sp. still retained their chlorophyll in the dark, indicating the possibility of retention of active photosynthetic machinery (5 ng chlorophyll per cell was measured in heterotrophic cells). Exposure of stationary phase heterotrophically grown cells to light caused stimulation of growth that in turn increased the biomass and total neutral lipid content even further. Low rates of oxygen evolution (0.2
femtomol O2.min-1.μm-3) suggested the presence of an active photosynthetic apparatus in heterotrophically grown cells, which might have played a significant role in the observed growth stimulation in stationary phase cells. Further investigations revealed an interesting set of changes to the photosynthetic apparatus in heterotrophically grown cells, including more than two-fold lower absorption cross-sectional area and NPQ values, higher electron transport flux, and two-fold higher connectivity between reaction centers as compared to the photosynthetically grown cultures.
Eventually, when all the tested methods were compared with respect to the total neutral lipid per ml, neutral lipid productivity, and final cell number normalised to the values obtained under the control conditions of the respective experiments, we found a combination of heterotrophy followed by mixotrophy is likely to be a better way of producing algal biomass for the production of biodiesel and other co-products
