Biomass Enhancement in Chlamydomonas reinhardtii using Steady C/N Ratio Microfluidic Perfusion Bioreactor, Vibrational Stress Priming, and Malate Synthase Expression

Abstract

Microalgae, a photosynthetic microorganism, has recently attracted attention as an effective resource for both renewable energy and food security. This is because sequestration of more CO2 and composition of more nutrition than terrestrial plants help microalgae mitigate climate change and secure food shortage in the future. Therefore, efficient process operation on microalgae cultivation is of importance. In this dissertation, three strategies were developed and applied to enhance microalgal biomass productivity with less time and cost at large-scale.<br/> The first one is cultivation with steady unbalanced C/N ratio. Microalgae-derived secondary metabolites include antioxidants (lutein, astaxanthin, etc) and neutral lipids (TAG), most of which are useful substances. These substances are produced in cells cultivated in a nutrient-depleted environment. In order to improve total productivity, increase in microalgal biomass should be accompanied well with increase in intracellular content. Thus, traditional nutrient depletion method can no longer be viable drastically constraining cellular growth. Recently, cultivation with unbalanced C/N ratio has been reported to increase biomass at a certain level simultaneously with TAG content increase. However, because batch process also has depleted nutrient over time, this method has the same limitations for enhancement of total products yield as traditional methods. In this study, continued supply of low concentrations of nutrients with the maintenance of unbalanced C/N ratio was proposed to enhance secondary metabolites production A microfluidic continuous perfusion system was designed and tested to culture microalgae, Chlamydomonas reinhardtii, under constant nutrient concentration slightly lower than normal condition with high C/N ratio. When cultured in 7.5%/7.5% of NH4+ /PO42-, C. reinhardtii showed a 2.4-fold increase in TAG production with a 3.5-fold increase in biomass compared to level obtained under an only NH4+ depleted condition. This is because nutrients continue to be supplied in small quantities without depletion of nutrients. Maintaining the unbalanced C/N ratio can improve the total TAG productivity by increasing the biomass. <br/> The second is cellular stress response preparedness against turbulence in bioreactors. Turbulence agitates all the nutritional components for cellular proliferation. However, severe hydrodynamic shear fields by the turbulence decreases cell viability that detrimentally influence maximum yieldable biomass. Vibrational wave treatment has been used to increase proliferation of microalgae. When directly applied at large scale, however, it costs much setting up massive vibration generating system, and turbulence can offset positive effects of vibration on microalgae proliferation. Stress priming is the phenomenon that primed cells with activated stress response by milder stress can prepare themselves for harsh stress and exhibit greater survival rate. In this study, vibration pretreatment (between 10–30 Hz and 0.15–0.45 G) was used to prime the cells for enhanced biomass. When exposed to 10 Hz at 0.15 G for 72 h and inoculated in baffled flasks of large shear fields (0.292 Pa for the average wall shear force (aveWSF) and 184 s−1 for the average shear strain rate (aveSSR)), microalgae showed 27% increase in biomass as well as 39% increase in corresponding amount of heterologous protein (i.e. GFP-3HA). The level of TRP11 transcript was increased both at vibration treatment and at shaking cultivation. Ca2+-signaling pathway closely relates with stress response. In plants, calmodulin (CaM)-binding transcription factor (CAMTA) is involved in expression of CBF/CREB1 master transcriptional regulator under stressful conditions. Although Ca2+-signaling pathway in Chlamydomonas has not yet been studied in detail, our results show that stress primed microalgae with vibrations can lead to improved proliferation that results in increased biomass production at industrial scale bioprocesses.<br/> Last but not at least, cellular metabolism itself could be additionally activated by malate. Malate has a close relation to biomass increase. High uptake of malate leads to biomass increase, reducing non-growth associated coefficient (maintenance energy). In addition, in plants, efficient distribution of intracellular malate to organelles was also elucidated to relate to biomass. Redox balance, especially in chloroplast, is carried out with reducing equivalents such as NADPH. Because its pool should be tightly regulated to maintain its capacity, intermediate metabolite, malate, play a role as an high potential energy carrier without disturbing reducing equivalents pool, and the malate is exported to other organelles. In this study, transgenic Chlamydomonas expressing malate synthase in chloroplast was developed. Stable expression of malate synthase enables malate production in chloroplast and distribution of more malate could be carried out in the transgenic cells. Transgenic Chlamydomonas under glyoxylate treatment showed 19% more increase in microalgal biomass than wild-type. By RNA analysis, the levels of malate dehydrogenase (MDH4) in TCA cycle, acetyl-CoA synthetase (ACS3), and isocitrate lyase (ICL1) and malate synthase (MAS1) of glyoxylate shunt, were significantly more expressed, which was consistent with reported metabolic flux analysis of heterotrophic cultivated cells. More meticulous analysis are necessary, but, in the transgenic microalgae with malate synthase overexpression, the metabolism is likely to more rely on energy production via TCA cycle and glyoxylate cycle than on photosynthesis, resulting in increase in microalgal biomass.