Lipid Production Of An Walnut Endophyte By Fermentation Of Cotton Stalk Hydrolysate And Analysis For Its Metabolic Base

The third generation biodiesel processing from microbial lipids using low-cost lignocellulosic feedstocks has attracted many attentions. Endophytes isolated from oleaginous plants possibly have the capacity to accumulate lipids similar to the hosts. However, little work has been reported in terms of endophytic bacteria isolation from oleaginous plants and their lipid production using lignocellulosic hydrolysate as substrate.A new oleaginous endophyte HB1310 has been isolated from the thin-shelled walnut with high oil content, and identified as Bacillus subtilis on the basis of both 16S rDNA gene sequencing and examination of its physiological and biochemical properties. This strain effectively accumulates cellular lipids using cotton stalk hydrolysate as a substrate. The optimum C/N ratio, culture temperature, and pH value in batch fermentation were determined to be 50/1,30 ℃, and 6.5 respectively, and under such conditions, the lipid content could reach higher than 30%. On the basis, the dynamic and fitting analysis was developed for the batch fermentation of lipid production and a good fitting dynamic mode was built. In order to understand the cytological basis for lipid inclusion formation of walnut endophyte HB1310, the variations in the fluorescent intensity and lipid inclusion formation of cells collected at different sampling times were examined using nile red fluorescent staining and transmission electron microscopy, which illustrated the potential of this bacterium to be useful for cellular lipid production. The fatty acid profile of the produced bacterial lipids showed that the major constituents are myristic, palmitic, stearic, oleic, and linoleic acids with an estimated cetane number of about 61.8, indicating that the lipid produced by this strain may be suitable for biodiesel production.Alternative temperature and pH regulation and fed-batch fermentation were developed for the batch fermentation of lipid production from fermentation of cotton stalk hydrolysate by walnut endophyte HB1310. The results showed that, the biomass of walnut endophyte, glucose and xylose utilization rate were significantly improved, the bacterial lipid content lower than the control in earlier fermentation stage while higher than the control in the later fermentation stage under alternative temperature regulation mode, and the increasing range could reach 8%～13%. The alternative pH regulation improved little for the lipid accumulation in walnut endophyte HB1310, only the treatment of "pH7.0 for 24 h + pH6.5" improved the bacterial lipid accumulation in the later fermentation stage (after 48 h). Fed-batch fermentation effectively promote the lipid accumulation of walnut endophyte, all the three fed mode developed in this research realized the slight increase of strain biomass and great improvement of lipid content, and the increasing rang of lipid content reached 18%-24%.The genome sequencing of walnut endophyte HB1310 was developed, and the results showed that, this bacterial genome contained a 4.1 Mb chromsome with GC content of 43.5%. 4314 CDSs were predicted in this genome, of which 3351 CDSs coded products possessed specific function. The glycosis pathway, pentose phosphate pathway and fatty acid synthesis way were detailed analyzed based on the pathway compared with KEGG database, the pathway of xylose metabolism of walnut endophyte entering the pentose phosphate pathway was preliminarily illustrated. Moreover, the fatty acid synthetase FAS genes probably correlated with the saturated and unsaturated fatty acids synthesis.Based on the genome annoted KEGG metabolism pathway and the property for lipid production and substrate utilization, the flux calculation and metabolic flux analysis were developed for key note metabolites in potential pathway. The reaction conversing xylose to xylulose was introduced in the pentose phosphate pathway, which provided a rational analysis for the bacterial utilization of glucose and xylose in cotton stalk hydrolysate. Meanwhile, the correlation between the activities of key enzymes in fatty acid synthesis and lipid production were analyzed, the results showed that the fatty acid synthetase activity showed high positive correlation with the bacterial lipid yield, indicating the fatty acid synthetase play a positive regulation role in lipid production of walnut endophyte HB1310. Our research also reconstructed the metabolic network based on metabolic pathways in the stationary phase (48 h), the global metabolic flux analysis indicated that the carbon skeleton mainly converted to the fatty acid synthesis way. The NADPH synthesis at the note of 6-phosphogluconate mainly provided reducing power NADPH for fatty acid synthesis at this stage.This research isolated a walnut endophyte HB1310 capable of producing lipid using cotton stalk hydrolysate as substrate, the optimal production of bacterial lipid was obtained through regulation of the fermentation process, the cytological basis for bacterial lipid production and complete genome data were analyzed, and the metabolic network was also reconstructed, providing theoretic basis for further research of the fatty acid biosynthesis mechanism and effective regulation for lipid production of walnut endophyte HB1310.