Nitrogen Metabolic Regulation Of Sophorolipids Synthesis, Characteristics Of Sophorolipids And Sophorolipids Production From Low-cost Substrate

Surfactants or surface active agents are widely applied in a variety of industrial sectors, such as detergent, cosmetic, petroleum, environment, and agriculture, etc. They are called "industrial monosodium glutamate", therefore, they are one of the most important classes of industrial bulk chemicals. With the growing environmental awareness, reducing pollution in origin and avioding generating secondary pollution have become consensus of the people. Biosurfactant has attracted increasing attention. Sophorolipids have attracted much more attention for their application potential in petroleum, environmental industries, cosmetics, detergents, pharmaceuticals and nanotechnology due to their high production yields, biodegradability, low ecotoxicity, biocompatibility, and production based on renewable resources. They are probably one of the most promising biosurfactants which can bring great society, economic and ecological benefits.The main research contents and results are as follows:1. Regulation of nitrogen metabolism of sophorolipids synthesisMetabolic regulation studies of sophorolipids (SLs) synthesis almost focused on effects of carbon sources on the sophorolipid-producing yeast, Candida bombicola ATCC22214. For Wickerhamiella domercqiae var. sophorolipid CGMCC1576, yeast which was screened by our laboratory, few reports on the effects of nitrogen sources on sophorolipid production and composition were available.The effects of different nitrogen sources on growth of W. domercqiae and on production and composition of SLs were studied. Organic nitrogen sources were more favorable for accumulation of biomass than inorganic ones. Presence of ammonium ion from different inorganic nitrogen sources (except NH4HCO3) greatly inhibited the production of lactonic SLs. However, when organic nitrogen sources were used, lactonic SL production was strongly increased. Increase of ammonium content in total N source led to more lactonic SL production rather than more acidic SL and the decrease of yeast biomass and pH value of the broth. Production of biomass and crystalline lactonic SLs from organic/inorganic nitrogen sources was enhanced with the increase of pH value when adjusted by sodium hydroxide or sodium citrate solution. Addition of pH controller of calcium carbonate at the beginning of the cultivation resulted in a slightly increased biomass, but in an obvious decreasing production of crystalline lactonic SL. However, adjustment of pH could not improve the production of total SLs. These results demonstrated that inorganic nitrogen source influenced SL synthesis by changing the pH value of the fermentation broth, but pH value is not the only reason.Fourier-transform infrared (FT-IR), gas chromatography mass spectrometry (GC-MS) and high performance liquid chromatography (HPLC) were employed to compare the composition of SL mixture obtained from yeast extract and ammonium sulfate as nitrogen sources. More than15acidic SL molecules and only4lactonic SL molecules were produced by using ammonium sulphate as nitrogen source, these SL molecules were separated by preparative HPLC and their structures were elucidated by MS and GC-MS. These results suggest extraordinary regulatory effects of nitrogen source on growth and SL synthesis of W. domercqiae.2. Analysis of transcription profiling of gene expression of W. domercqiae using digital gene expression tag profiling (DGE) technologyThe differences on gene expression level of W. domercqiae with two different nitrogen sources, yeast extract and ammonium sulphate were compared through gene expression profiling analyzed by Digital Gene Expression tag profiling (DGE) technology. Two libraries were built from Wickerhamiella domercqiae var. sophorolipid CGMCC1576cultured with yeast extract and ammonium sulphate as nitrogen source, which were named YE library and NS library, respectively. The YE library had6,011,524total tags including64,329distinct tags, and the NS had5,886,784sequenced total tags with72,375distinct tags.4,291differentially expressed distinct tags were detected between the two libraries, including2,020up-and2,271down-regulated distinct tags in the comparison of YE library with NS library. When FDR≤0.001and the absolute value of log2Ratio≥1was used as the threshold to judge the significance of gene expression difference,392tags were up-regulated and317tags were down-regulated significantly under ammonium sulphate condition.The sequencing tags were mapped to the W. domercqiae genome and the result showed that about96%tags could be mapped to the W. domercqiae genome. For tags of library YE and NS,70.73%and67.73%of tags could be mapped to the reference genes from W. domercqiae. After analyzing the distinctly-expressed genes involved in SL synthesis of the two libraries, many glycosyltransferase, acetyltransferase and esterase/lipase genes were up-regulated significantly when yeast extract was used as nitrogen source. These results indicated that co-regulation of these genes involved in SL synthesis. However, under ammonium sulphate condition, only2genes encoding esterase/lipase were up-regulated significantly. Nitrogen source influenced lactonic SL production significantly and it was suggested that esterification reaction of the carboxyl group of the hydroxy fatty acid with a hydroxyl group of sophorose was catalyzed by a specific lactone esterase or lipase. However, neither of both esterases has been found in the W. domercqiae genome or in other SL producing species. It was found there that27genes encoding esterase/lipase up-or down-regulated in the two libraries. These results summarized above provide useful information for our further research in constructing gene engineering strain.3. Surface and biological activity of sophorolipid molecules with different structuresSLs produced by several selected yeast strains are mixture of different SL molecules. SL molecules with different structures have been reported to show some differences in their biological and physicochemical properties and can be specifically applied in different fields.Six different SL molecules were prepared from crude SLs produced by W. domercqiae by using glucose and oleic acid as carbon sources. The staictures of the six SL molecules were elucidated by MS and GC/MS. The six SL molecules differ in the acetylation degree of sophorose, carbon chain length, unsaturation degree of fatty acid and lactonic/acidic form. The surface properties of SLs including critical micelle concentration (CMC), minimum surface tension (Min. S.T.), emulsification capacity to hydrocarbon and vegetable oils. Cytotoxicities and biodegradability were also studied and the results demonstrated that SL molecules with different structures exhibited quite different surface properties and biological activities.4. Substrates preparation from delignined corncob residue for sophorolipid synthesisLarge-scale application of sophorolipids was blocked by high production cost. One way to reduce the cost of SL production is to develop other cheap fermentative feedstock. In the dissertation, a lignocellulosic material, delignined corncob residue (DCCR) was used for providing the hydrophilic carbon source (glucose in DCCR hydrolysate), hydrophobic carbon source (single cell oil produced from an oleaginous yeast of Cryptococcus curvatus) and nitrogen source (single cell protein) for SL production.Effects of DCCR saccharification by different commercial cellulase in pH4.8H2O and different buffers including0.2M and0.05M acetate buffer of pH4.8were studied. Finally, KDN cellulase was selected for hydrolysate production.Cell growth and lipid accumulation of the oleaginous yeast of Cryptococcus curvatus from delignined corncob residue hydrolysate (DCCRH) and detoxified DCCRH were studied. In untreated hydrolysate medium, C. curvatus can growth and accumulate lipids except that0.2M acetate DCCRH medium was used. In0.05M acetate and pH4.8H2O DCCRH medium,15.32g/1and17.86g/l of biomass with40.21%and27.44%of lipid contents were obtained, respectively. Furthermore, pretreatment of DCCRH by active carbon absorption (C) and vacuum steam evaporation (Va) led to significant improvement of cell growth and lipid accumulation with highest values of20.49g/1of biomass and46.74%lipid content when cultured in0.05M acetate DCCRH medium. For0.2M acetate DCCRH medium, after detoxification, the produced biomass reached17.36g/1and lipid content increased to44.35%. The accumulated lipids were mainly constituted by neutral lipids with fatty acids as palmitic acid, stearic acid, oleic acid and linoleic acid.Orthogonal test and further optimization were applied to obtain the optimal fermentation medium for C. curvatus growth and lipid production. Under optimal yeast extract and mineral salt solution addition condition, cell growth and lipid production of C. curvatus on different DCCRH medium were compared. The result showed0.05M acetate DCCRH treated by active carbon adsorption and containing5.0g/1yeast extract and20.0ml/1mineral salt solution was the optimal DCCRH medium for SCO production. Under this condition,10.94g/1of SCO and55.41%of lipid content were obtained, respectively. After cell disruption and autoclaving, the cell homogenate containing cell debris and lipids could be served as nitrogen source and a part of lipidic substrate for SL production by W. domercqiae.5. Sophorolipid production from delignined corncob residue hydrolysateSophorolipid production by W. domercqiae from different DCCRH and detoxified DCCRH were investigated. It was found that0.2M acetate DCCRH hydrolyzed by KDN cellulase and treated by active carbon adsorption was more suitable for SL production. DCCRH detoxified by active carbon adsorption to remove the inhibitor of acetic acid increased SL production and proportion of lactonic SL in total SL. The optimizing results indicated that0.2M acetate DCCRH medium containing1.5g/1of yeast extract and60.0g/1of glucose was the optimal DCCRH medium for SL production.Effects of glucose or rapeseed oil supplement and controlling of broth pH in different1.5L bioreactor were studied. Composition of the obtained crude SLs were analyzed by HPLC. SL production from DCCRH was investigated. The results demonstrated that W. domercqiae could utilize the glucose in DCCRH and detoxified DCCRH as hydrophilic carbon source to grow and accumulate SL with relatively high yield. When W. domercqiae were cultivated on DCCRH/detoxified DCCRH and single cell oil as hydrophilic and hydrophobic carbon sources, total SL production of39.08g/l and42.06g/l was obtained. Furthermore, when cell lysate of C. curvatus was used as nitrogen source, oleic acid and detoxified DCCRH or DCCRH as complex carbon sources, production of total SL reached48.97g/l and37.19g/l, respectively. These results demonstrated that renewable DCCRH can be well utilized as low-cost substrate for the production of SL. Exploition of DCCR as substrates for SL production is a practical way to significantly reduce the cost of SL production.