Utilisation of glycerol as a carbon source for mixotrophic growth and lipid accumulation in marine microalgae

Abstract

Microalgal-based biomass is a promising feedstock for biofuel, chemicals and food production to meet the increasing demands for sustainable energy and material. Microalgae are photosynthetic microorganisms that use sunlight energy to power the assimilation of atmospheric CO2 into algal biomass, via photoautotrophy. Achieving high biomass concentrations and lipid productivities are some of the major challenges for large scale phototrophic cultivation. This thesis explored mixotrophic cultivation, in which an organic carbon source (glycerol) is used to supplement energy and carbon supplies to address the limitations of the conventional photoautotrophic cultivation.

Mixotrophy is the simultaneous assimilation of organic carbon and CO2, with the organic carbon being used as both a carbon and energy source. With a focus on reducing costs, the present study has demonstrated the possibility of reutilisation of glycerol, a biodiesel by-product, to promote algal growth and lipid accumulation. This thesis provides the first descriptive analysis of the assimilation of glycerol in microalgal metabolism under different growth parameters including the availability of light and nitrogen, and the type of nitrogen provided.

Nitrogen starvation is a well-known method to induce triacylglyceride (TAG) accumulation in microalgae, with the TAG being an ideal feedstock for biofuel or food oils. However, application of nitrogen-starvation strategies for TAG accumulation under mixotrophic conditions remains limited. In this study, the use of glycerol to enhance microalgal biomass and lipid productivities was investigated in relation to nitrogen availability.

Under nitrogen (nitrate) sufficient conditions, the biomass productivities of Nannochloropsis salina and a marine Chlorella sp. were 1.7 and 1.9 times higher in mixotrophic culture than under strictly photoautotrophic conditions, respectively. Both algae required light to assimilate glycerol. No significant algae growth was observed under heterotrophic conditions, despite apparent utilisation of both nitrate and glycerol. Under nitrate deplete conditions both species took up only minimal amounts of glycerol, thereby indicating the importance of the combined effects of carbon and nitrogen.

Mixotrophy is a well-studied growth regime for algal cultivation. However, the proliferation and potential role of heterotrophic bacteria in the presence of organic carbon has largely been neglected in the existing literature. The current study addresses these gaps by analysing the abundance and role of bacteria during mixotrophic growth. 16S rRNA sequencing confirmed the presence of two phylogenetically distinct Gram-negative bacterial isolates belonging to Alpha and Gamma subclasses of Proteobacteria (Paracoccus sp., and Marinobacter alkaliphilus and Marinobacter lipolyticus, respectively) in mixotrophic cultures of N. salina. Among these strains, M. alkaliphilus was shown to have aerobic denitrifying capabilities, which allowed it to oxidise glycerol using nitrate as a terminal electron acceptor, even in the presence of oxygen derived via algal photosynthesis. This provided an explanation for the utilisation of nitrate and glycerol that was observed in mixotrophic and heterotrophic cultures despite limited microalgal growth.

While much research has been devoted to understanding the role of nitrogen on microalgal growth, there has so far been relatively little consideration of the effect of nitrogen source on the diversity of bacteria in algal cultures. This study compared the difference between two nitrogen sources (ammonium and nitrate) on the growth of both N. salina and the associated bacteria. The performance of N. salina and the abundance, composition, and profile of bacterial groups were compared between axenic (ampicillin-containing) and non-axenic (ampicillin-free) photoautotrophic and mixotrophic cultures. The productivity of N. salina was higher with ammonium than with nitrate, with ammonium being assimilated faster than nitrate.

16S rRNA sequencing revealed the bacterial groups in the ampicillin-free cultures to include Alphaproteobacteria (predominantly Beijerinckiaceae), Gammaproteobacteria (Pseudomonadaceae and Alteromonadaceae) and Cytophagia (Cyclobacteriaceae). Pseudomonadaceae proliferated in the ammonium cultures, Alteromonadaceae in the nitrate cultures, while Beijerinckiaceae was prevalent in both ammonium and nitrate cultures. Although the presence of bacteria reduced the overall productivity of N. salina, the abundance and type of bacteria did not appear to be directly correlated with the extent of algal growth impairment. Importantly, it was revealed that both nitrate and glycerol are wasted by bacterial denitrification. As a chemically reduced form of nitrogen, ammonium did not have this problem, meaning it allows better utilisation of both nutrients for algae production rather than bacterial energy generation. These results provide a better understanding of the interactions between bacteria and algae in mixotrophic growth that may enable the development of strategies to better utilise nitrogen and carbon for mixotrophic algal cultivation regimes.

Finally, assimilation of glycerol into the acyl chains of TAG and membrane lipids in N. salina grown mixotrophically under nitrate replete and deplete conditions was investigated using metabolomic and mass-spectrometry approaches. N. salina consumed 78% and 13% of glycerol under nitrate replete and deplete conditions, respectively. Consequently, the absolute value of total 13C incorporation was 10.6 times higher in nitrate replete than deplete conditions. However, the relative abundance of 13C incorporation was found to be higher on saturated and monounsaturated acyl chains of TAG and lower on polyunsaturated acyl chains.

It was initially hypothesised in this study that glycerol would directly enhance TAG synthesis by providing a full-formed glycerol backbone on which acyl chains could be attached via the Kennedy pathway. While a disproportionate amount of fully (triple 13C) labelled lipid-associated glycerol backbones suggested that intact glycerol molecules as well as individual 13C carbon from central metabolism were used as lipid backbones, only a very low proportion of the glycerol was assimilated this way. Under nitrate replete conditions, while 43% of the carbon in the glycerol was assimilated into lipids, most of this went in membrane lipids rather than TAG. Under nitrate deplete conditions, only a small proportion (13%) of the very limited amount of glycerol that was consumed was fixed into the lipids.

The knowledge and new understanding of the importance of glycerol, nitrogen availability and source, and bacterial groups that was developed through this study will be beneficial for the development of efficient mixotrophic cultivation regimes at large scale.