Screening Of Alkane Emulsifying Strains And Application Of Their Products In Biotransformation Production Of Tridecanedioic Acid

Tridecanedioic acid(DCA13),a widely used fine chemical,can be synthesized by microbial transformation using petroleum by-product tridecane(C13)as the substrate.The absorption of C13 is a key to achieve the biosynthesis of DCA13,but the current researches mainly focus on the catalytic oxidation process,with little attention to the absorption of C13 and its subsequent interfacial catalysis.Therefore,studying DCA13 biosynthesis from the perspective of the absorption and utilization of C13 is conducive to furthur improve its conversion efficiency.Surfactants play an important role in the absorption of C13 by microorganisms.Biosurfactants have signifacant application advantages over existing chemical surfactants(such as Tween),with not only excellent surface and interfacial activity and emulsification properties,but also structural plasticity and biodegradability.Meanwhile,the emulsifying effect is better when the biosurfacant has a similar structure to the corresponding substrate.There have been no research reports on the application of biosurfactants in the DCA13 biotransformation system.Therefore,in this paper,C13 was used as the substrate to screen microorganisms capable of synthesizing biosurfactants with specific structures,then the structural composition and physical and chemical properties of the corresponding surfactants were studied,and furthermore the biosurfactants obtained were applied to the system of biotransforming C13 to product DCA13 by Candida viswanathii(C.viswanathii).This study provides a new idea for solving the problem of the absorption and utilization of substrate C13 in the bioconversion production of DCA13,and provides a theoretical guidance for the biological manufacturing and new processes of other alkane fine chemicals.The research results obtained are as follows:(1)A biosurfactant-producing strain that can emulsify C13 efficiently was obatained.Continuous C13 selective pressure was applied to microorganisms in soil samples from long-term oil-contaminated environments.Based on the growth status of the strains and the emulsification rate of C13 in the fermentation medium,3 strains that could grew with C13 as the sole carbon source and produced biosufactant were screened out.Among them,the strain with laboratory number HG50 grew well in C13 fermentation mediumand had a relatively higher emulsification.Combining morphological observation,main physiological and biochemical indexes and 16 S r DNA molecular identification,the strain was identified as Pseudomonas aeruginosa(P.aeruginosa).(2)The surfactant produced by the strain HG50 was identified to be rhamnolipid,containing rhamnolipid homologues with a similar structure to C13,and it had better physical and chemical properties than Tween 60 and conventional rhamnolipids.The biosurfactant crude product was isolated from the fermentation broth of strain HG50 by acid precipitation,with a yield of 1.76 g/L fermentation broth.Compared with the currently reported biosurfactant production of wild P.aeruginosa(typically 1-2 g/L fermentation broth),it showed that the strain selected had good biosurfactant production ability.Combining thin layer chromatography(TLC)and Fourier infrared spectroscopy(FTIR)to analyze the components of the crude products,the biosurfactant was preliminarily confirmed to be rhamnolipid.The total rhamnolipid content in the crude product was determined to be 68.3% by phenol sulfuric acid method.Further,HG50 rhamnolipid product was prepared after purification of the crude product by organic solvent extraction combined with silica gel column chromatography.UPLC-MS was used to analyze the specific composition and structure of HG50 rhamnolipid product,taking the two components of Rha-Rha-C10-C10 and Rha-C10-C10 in the rhamnolipid standard as a reference.The result showed that the biosurfactant consisted of a series of rhamnolipid homologues,of which the most abundant homologue form was Rha-Rha-C10-C10,accounting for 12.7%.In addition,it also contained a hybrid of rhamnose with C13 and C12 lipids(Rha-Rha-C13-C12/Rha-Rha-C12-C13),which was a new type of rhamnolipid,accounting for 2.17%.It was confirmed that rhamnolipid homologues containing specific structures were successfully synthesized.By drawing the surface tension curve,the critical micelle concentration of Tween 60,rhamnolipid standard(RL)and HG50 rhamnolipid crude product(HG50 RL)were determined to be 30 mg/L,150 mg/L,and 200 mg/L respectively.The diameter of the oil discharge ring at the critical micelle concentration were 2.1 cm,5.2 cm,and 5.4 cm,respectively;the emulsification index for paraffin wax were 53%,60%,and 64%,and the emulsification index for C13 were 40%,63%,and 65%.The above indicators indicate that the rhamnolipid produced by strain HG50 has stronger surface interface and emulsifying activities than Tween 60 and ordinary rhamnolipid.In addition,compared the environmental tolerance of the above three surfactants,it was found that the stability of HG50 RL was better than Tween 60 and RL.It can maintain high surface activity in the temperature range of-20℃-100℃,p H value of 2-12,and salt concentration of 0-10 g/L.(3)The application of HG50 rhamnolipid in the system of biotransforming C13 to product DCA13 by C.viswanathii was studied and the quaternary reverse micelle enzyme catalytic system was found that constructed by HG50 RL/Exxal C13/C13/C.viswanathii crude enzyme solution has a good catalytic effect in transforming C13 to DCA13.C.viswanathii was selected for DCA13 production.The product DCA13 transformed from C13 by the strain was firstly monitored.The results showed that DCA13 accumulated mainly during the 1-3 days of transformation,and the yield reached the highest on the second day.After that,the DCA13 content dropped till below the detection limit and the corresponding C11,C9,C7 and C5 length dicarboxylic acid gradually increased.Tween 60,RL and HG50 RL successively enhanced the emulsification rate of C13 when added into C13 transformation medium respectively,whereas the C13 emulsification rate in RL and HG50 RL group were lower than Tween 60 group after inoculation with C.viswanathii,indicating that rhamnolipid had a strong inhibitory effect on the strain and its absorption process.Therefore,we selected isotridecanol(Exxal C13)as the co-surfactant,and the crude enzyme extract of C.viswanathii on the second day of fermentation as the source of the aqueous phase to construct different surfactants/Exxal C13/C13/enzyme extract quaternary reverse micellar enzyme catalytic system.The results showed that the HG50 reverse micellar enzyme catalysis system had a higher yield of DCA13,which was 12% higher than that of the traditional Tween 60 process under the same conditions.The above research initially realized the new application of biosurfactant in the DCA13 biotransformation production system.