| Developing renewable and clean energy source is of top priority for environmental scientists due to the decreasing storage of the unrenewable fossil fu el and the increasing pollution toward the environment. Biodiesel is widely considered as a promising substitute to fossil fuel. Raw materials such as vegetable oils and discarded dinning oils, from which biodiesel is produced, could hardly satisfy nowaday s expending market demand. Microbial oil, benefits from its unique advantage such as short production cycle with no affected by the geographic environment and seasons, is gradually introduced to energy field and leading the trend of biodiesel development. However, seeking for cheap carbon source to reduce costs of culture media becomes one of the essential factors for large scale microbial oil production and application. On the other hand, wastes containing abundant carbon sources are generated from lignoce llulose during the biorefinary with chemical treatment. Considering the wide-range carbon source adaptation of many oleaginous microorganisms, we adopted Rhodococcus opacus PD630 and DSM 1069 in testing the following lignocellulose wastes as carbon sources for oil production, including, pyrolysis light oil, effluent from pretreatment process for bio-ethanol production, and Kraft lignin from pulp and paper industries. Furthermore, the characterization methods such as gel permeation chromatography(GPC), nucl ear magnetic resonance(NMR), and scanning electron microscope(SEM) were used to preliminarily discuss the mechanism. The goal of this study is in the purpose of providing new carbon source for microbial oil production as biodiesel precursors, as well as supporting the utilization of wastes from lignocellulose treatments. The work in this paper covers the following 3 parts.First, the growth and oil accumulation of the two types of bacteria grown in pyrolysis light oil was studied. The experiments were conducted under two different p H conditions, acid(p H=4) and neutral(p H=7). Both results showed proliferation and the biomass growth is positively related to the substrate concentration within certain range. Further experiment presented a favorable growth when fully adapted cells were further transferred into light oil medium where nitrogen source was scarce. Results showed that bacteria grew faster in acid environment and PD630 and DSM 1069 reached their peak biomass of 0.82 g/L and 0.90 g/L, respectively, 10.81% and 9.76% higher than the values when being cultured under p H=7 condition. Moreover, the peak value of lipid content(p H=4) in the PD630 and DSM 1069 cells occurred at16 h, counting 25.8% and 22.0% of the cell dry weight(CDW). In the case of p H=4, the main composition of fatty acid was saturated fatty acid in DSM 1069, while the proportion decreased and more unsaturated fatty acid was synthesized with p H=7. On the other hand, PD630 always accumulated saturated fatty acid far more than unsaturated f atty acid in either conditions, especially when p H=7. Analysis result from 31 P NMR and 13 C NMR proved that after short adaption periods both strains can attack aliphatic compounds, as well as some of the aromatic oligomers and monomers in light oil.Different lignocellulosic pretreatment effluent also contains various organic compounds such as sugars and lignin derivatives. In this study, effluent from autohydrolysis and organosolv pretreatment were used as the research objects. Preliminary experiment was firstly conducted to investigate the growth of both the bacteria with the single sugar, including glucose, xylose, galactose, arabinose, mannose, as a sole carbon source under different concentrations. It was found that cells mainly metabolized the hexose, and the growth rates increased with the increase of sugar concentrations. R. opacus PD630 can use the glucose, galactose and mannose, while DSM 1069 can only digest the glucose and mannose. When the autohydrolysis effluent was directly used as substrate, cells died rapidly due to the inhibitory effect of by-products, such as HMF and furfural that usually generated during the pretreatment process. The detoxification treatment combining overliming with adsorption can effectively remove these by-products. After detoxification, the maximum bacterial biomass of R. opacus PD630 and DSM 1069 in pine autohydrolysate(DPAH) occred at 72 h, which were 0.96 g/L and 1.03 g/L respectively. In the case of detoxified sweetgum autohydrolysate(DSAH), after longer lag phas e, the maximum of the two bacteria were 1.18 g/L(PD630) and 1.06 g/L(DSM 1069). Moreover, R. opacus PD630 accumulated as much as 28.6% of its cell dry weight(CDW) in lipids while growing on DSAH that translates to 0.25 g/l lipid yield. The accumulation of SCOs reached the level of oleagenicity in DSM 1069 cells(28.3% of CDW) as well, while cultured on DPAH with the maximum lipid yield of 0.31 g/ L. The composition of the obtained microbial oils varied depending on the substrates provided. In case of organosolv pretreatment, the effluent is predominantly enriched with degraded hemicellulose as well as lignin monomers and oligomers. Results showed that R. opacus DSM 1069 can s imultaneously use the glucose and aromatic compounds for cell proliferation and lipid accumulation. The maximum lipid content was up to 26.88% at 48 h. Upon transesterification, the composition of FAMEs were determined to be largely composed of even numbered monounsaturated and saturated fatty acids that included oleic, palmitic, and st earic acid.The degradation of Kraft lignin extracted from black liquid of pulping and paper industry(BL-Kraft lignin) and commercial Kraft lignin were investigated. It can be seen that the maximum lipid accumulation in DSM 1069 growth on BL-Kraft lignin was only 6%. GPC, 13 C NMR and HSQC-NMR analysis showed that bacteria mainly utilize the high content extractives as the carbon source. Commercial Kraft lignin was chosen in the further study due to its high lignin content(88.11%). Results showed poor bacterial growth when KL was used directly as a substrate. Consequently, oxygen-pretreatment(O2-pretreatment) was introduced to lower Kraft lignin molecular weight. The bioconversion of O2-Kraft lignin to lipids proved unsuccessful under pretreatment A. However, when the O2-pretreatment severity was increased to largely decrease molecular weight of Kraft lignin, DSM 1069 exhibited viable growth patterns and cell proliferation was pronounced an increase of ~460 times compared to initial inoculum size. The maximum lipid accumulation(0.067 mg/ml) was measured after 36 h at 14.21% based on CDW and was mainly composed of palmitic(C16:0) and stearic(C18:0) acids. Together with GPC, 31 P NMR, HSQC-NMR results, it can be concluded that R.opacus DSM 1069 possesses the set of enzymes that can degrade lignin fragments to monomers and oligomers and convert them to smaller aromatics(such as protocatechuic acid). Subsequently, these components can be imported into cells for aromatic catabolism that in R.opacus proceeds via the β-ketoadipate pathway. By the late stages of fermentation the more complicated and polymerized lignin was left behind, however, the lower Mw O2-Kraft lignin fraction was converted to lipids.Overall, this study investigated the possibility of using lignocellulose waste during the biorefinary process as microbial oils production feedstocks, thereby providing a new route to achieve economy of microbial oil production. The data obtained in this study is of importance for further deceasing of the baiodiesel cost and improving its widely application in furture. |
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