Using straw hydrolysate to cultivate Chlorella pyrenoidosa for high-value biomass production and the nitrogen regulation for biomass composition
Introduction
Microalgae are well known for their abilities to rapidly accumulate valuable biocomponents, including proteins, lipids/fatty acids, polysaccharides and pigments (Chisti, 2006). The microalgal protein is favorable in composition (Becker, 2004) and has been recommended by World Health Organization (WHO) as a well-balanced protein source for animal and human since 1973 (FAO/WHO, 1973). The microalgal lipid is rich in polyunsaturated fatty acids (PUFA) (Behrens and Kyle, 1996), which are the valuable fatty acids for animals and related to many critical metabolic pathways (Salem et al., 1999).
Based on the high biomass quality and rapid growth rate, microalgae has been long applied as one of the most important feed additive source (Brown et al., 1997) and basic biochemical material for several other applications (Borowitzka, 1986). Currently, the global animal husbandry and aquaculture is highly dependent upon marine capture fisheries for sourcing key nutrients dietary (Tidwell et al., 2006). Because of the climate change and overfishing, the marine protein and fatty acid sources are in long term decline (Albertgj and Marc, 2008), thus the alternative is urgently demanded (Carter and Hauler, 2000, Kongwah et al., 2014). Microalgal biomass is an advantageous alternative for feed additive production (Patnaik et al., 2006), thus is potentially needed for large-scale production (Becker, 2007).
High productivity of biomass is the foundation of large-scale cultivation of microalgae. Compared with autotrophic cultivation, heterotrophic cultivation is effective to overcome light limitation and significantly increase the microalgal biomass productivities (Miao and Wu, 2006, Perez-Garcia et al., 2011), yet the cost is expensive and sensitive on the carbon source.
Straw hydrolysate is considered as a cost-effective substrate for heterotrophic cultivation of microorganism (Zeng et al., 2013). In recent years, a novel treatment process based on the ammonium sulfite delignification method has been largely applied in China to produce lignin-based fertilizer and lignocellulose from million tons of straw (Qu, 2016), which can provide a low-cost lignocellulose source at industrial scale. The hydrolysate produced from straw lignocellulose was a potential and promising substrate for heterotrophic cultivation of microalgae, yet has not been well researched.
The feasibility of using straw hydrolysate to heterotrophically cultivate microalgae for high-value biomass production should be carefully examined before application. Besides of the productivity, the quality of biomass produced from straw substrate should also be evaluated. Different applications of microalgae require different compositions of microalgal biomass. It is necessary to produce microalgal biomass according to the specific requirement of the target application.
The current research on heterotrophic cultivation of microalgae is mainly limited in the lipid accumulation for energy use (Cho et al., 2011, Singhasuwan et al., 2015), yet failed to pay attention on other valuable components (such as protein). Some researchers have reported the low protein content of microalgae at dark condition (Fan et al., 2014, Fan et al., 2012), which made the heterotrophic production of microalgal biomass with high protein content seems infeasible. If the heterotrophic biomass is only limited for lipid-related application, the utilization would be restrained.
Due to the molecular components, the lipid and protein synthesis have different demands on nitrogen source (Duchars and Attwood, 1989). Increasing nitrogen concentration in the medium was an effective approach to improve protein content in biomass for many heterotrophic microorganism species, such as Hansenula polymorpha (Egli et al., 1986) and Lachancea thermotolerans (Schnierda et al., 2014). The effect of nitrogen concentration on the microalgae at heterotrophic condition is still unknown. The effect of nitrogen concentration in the medium for the growth and biomass composition of microalgae should be clarified to improve and optimize the cultivation of microalgae in straw hydrolysate medium.
In this research, heterotrophic cultivation of microalga based on straw substrate and the nitrogen regulation strategy on biomass composition were investigated. Chlorella pyrenoidosa, a widely used valuable microalgal species, was used as the experimental species in this research. Gradient amounts of the sodium nitrate were added into the low-nitrogen hydrolysate solution to prepare the medium with 10 g L−1 of glucose and different initial nitrogen concentrations. Both the contents and compositions of protein and fatty acid in the microalgal biomass were measured to analyze the biomass quality of C. pyrenoidosa produced from straw hydrolysate medium.
Section snippets
Hydrolysate preparation
The wheat straw material used in this study was the industrial wheat straw pulp provided by Tranlin Papermaking Co. Ltd., Shandong, China. The wheat straw was collected from the local farm field and then cut into pieces. The pieces were boiled in ammonium sulfite solution (130–160 °C, 2–2.5 h) for lignin extraction and then sent into vacuum washers to purify the straw pulp which was mainly composed by cellulose. The pulp was used to make hydrolysate. More information about the ammonium sulfite
Growth and nutrients assimilation of C. pyrenoidosa in hydrolysate medium
Different from the artificial medium (such as BG11-based medium), the composition of straw hydrolysate was very complex. Glucose and xylose were the main organic matters in the hydrolysate. The glucose and xylose concentration in raw hydrolysate was 49.1 ± 0.8 and 12.5 ± 0.4 g L−1, respectively. Various growth stimulating substances (Vitamin B1, B3, B5, B6, B7, B9, Vitamin A, 20 common free fatty acids, heteroauxin, etc.) were detected from the hydrolysate, implying that the hydrolysate has plentiful
Conclusions
C. pyrenoidosa well adapted to straw hydrolysate substrate. The intrinsic growth rate was around 0.09 h−1 when TN was 45–360 mg L−1. Higher amount of TN showed inhibition. Increasing nitrogen concentration significantly increased protein content and decreased lipid content in biomass. At the cultivation condition, 90 mg L−1 and 1080 mg L−1 of nitrogen concentration in medium was recommended for lipid and protein accumulation, respectively. Microalgal biomass varied in proportions of protein (10–62%),
Acknowledgements
This study was supported by the National Natural Science Foundation of China (No. 21521064) and the Collaborative Innovation Center for Regional Environmental Quality, China. The straw pulp used in this research was provided by Tranlin Papermaking Co. Ltd., Shandong, China. The Cellic Ctec 2 enzyme was provided by Novozymes (China) Investment Co. Ltd.
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