| Due to their abilities for improving solubility,emulsifying properties and dispersibility,surfactants can be used in chemical products,cosmetics,detergents,foods,textiles,and pharmaceuticals,and can cover almost every aspect of industrial production and our daily life.In particular,biosurfactants produced through microbial fermentation have attracted increasing attentions in recent years because of their excellent surface activity and good environmental compatibility,biodegradability and unique biological activity.As one of the most important biosurfactants,sophorolipids has good biocompatibility and biodegradability.It is an environmentally friendly surfactant that can be used in food and daily chemicals,medical care,environmental protection,oil extraction industry and pesticides,and many other industrial and agricultural fields.At present,the higher production cost of sophorolipids limits their large-scale application.Therefore,in order to increase the production of sophorolipids and reduce their production cost,many researchers have carried out a lot of research work.The present studies tried to reduce the production of sophorolipids from two aspects which included optimization of fermentation medium and construction of recombinant strains of Starmerella bombicola through genetic engineeringThe main research contents and results are as follows:1.Optimization of fermentation medium for the production of sophorolipids from Starmerella bombicolaThe growth and reproduction of microorganisms requires continuous absorption of nutrients from the outside to obtain energy and synthesize new substances.To study the growth and reproduction of microorganisms and the synthesis of metabolites,it is necessary to understand the nutritional characteristics and culture conditions of microorganisms in order to effectively control their growth and synthesis of metabolites,and to achieve highproduction of important metabolites by fermentation of the microorganisms.Therefore,studying the nutritional characteristics of microorganisms and determining a reasonable fermentation medium is one of the key factors to realize the industrialization of microbial fermentation.The optimization of the fermentation medium is usually divided into two steps.First,the main influencing factors are determined,and then the optimal concentration of each component of the medium is further optimized by multi-factor experiments.In the present experiment,the Plackett-Burman design(PBD)and the central combination experiment(CCD)were used to optimize the fermentation medium for the production of sophorolipids of Starmerella bombicola.The yield of sophorolipid increased from 53.2 g/L of the control to 72.2 g/L,increased by about 38%.2.Functional study of Vitreoscilla hemoglobin gene in the sophorolipid producing strain Starmerella bombicolaIn deep liquid fermentation of aerobic microorganisms,it is necessary to continuously pass into sterile air,and the oxygen is dissolved by the gas phase into the liquid phase,and then absorbed by the cells,to meet the oxygen reqirement for cell growth and synthesis products.Therefore,the concentration of dissolved oxygen in the fermentation broth becomes an important limit factor for the growth of the cells and the production of the product.At present,the commonly used oxygen control methods include:(1)improving the equipment feeding capacity,such as increasing the stirring speed;(2)increase the oxygen pressure by passing into oxygen-enriching aeration;(3)controlling the rate of oxygen consumption by microbes.These methods are either limited by the stringent requirment for the fermentor or difficult to perform in industrial production.It is worth noting that there are hardly any reports on genetic improvement of the oxygen-utilizing ability of the sophorolipids-producing strains by genetic engineering techniques.In the present experiment,the Vitreoscilla hemoglobin gene vgb was integrated into the genome of Starmerella bombicola and the recombinant strain with greatly improved utilization of oxygen were obtained.In the shake flask fermentation experiment,the yield of sophorolipid increased by 18.4%,65%,and 18.2%,respectively,under different rotation conditions(100 rpm,150 rpm,200 rpm)In the fermentation experiment of 1 L fermenter,the yield of sophorolipid increased by 24%and 3 3%,respectively,at different rotational speeds(400 rpm,600 rpm).The yield of sophorolipids of the recombinant strain at 400 rpm was close to that of the wild-type strain at 600 rpm,showing excellent industrial application prospects.3.Construction of several recombinant strains expressing different cellulase gene and co-culture of the engineered strains to produce sophorolipids from lignocellulosic biomassOne of the main reasons for the large-scale industrialization of sophorolipids is the high production cost.The high production of sophorolipids can be achieved when hydrophilic and hydrophobic carbon sources are both added to the fermentation medium,and therein the hydrophilic carbon source glucose required for fermentation is about 80 g/L.Cellulose is a ?-D-glucose long chain consisting of about 14,000 glucose units and is the most abundant renewable resource in the nature.Utilization of biomass to produce soluble sugar for the production of some high-value products is believed as a substitute for the sugar from starch and can probable reduce the production cost of many important compounds include sophorolipids.In the present thesis,we constructed several recombinant strains EG1,EG2,BGL1 and CBH2 respectively expressing the major cellulase genes egl,eg2,bgll and cbh2 from Penicillium oxalicum by genetic engineering techniques.In order to make the genes work together,we adopted a strategy of co-cultivation of the strains,and the fermentation experiment was carried out with 1%RAC as a substrate.The results showed that the total amount of sophorolipid produced by the recombinant strain co-cultured with RAC was 2.6 times of the wild-type strain,which demonstrated that the recombinant strain can utilize lignocellulosic biomass to produce sophorolipids via heterogenously expressing cellulase genes from filamentous fungi. |
PDF Full Text Request