Screening,Identification And Optimization Of Fermentation Conditions Of Rhamnolipid-producing Bacteria

Rhamnolipid,a biosurfactant produced by microorganisms,is widely used to deal with environmental problems such as oil pollution,organic pollution,and heavy metal pollution.In our study,three strains of rhamnolipid-producing bacteria were screened from the food waste oil aging soil and morphological,physiological and DNA molecular marker identification were conducted.Single factor experiment,PB experiment and Response Surface experiments were carried out to optimize the fermentation conditions of the strains.At the same time,the application of the strains in three aspects of crude oil degradation,corn oil emulsification and the control of agricultural pathogens was preliminarily explored.The results were as follows:(1)Three strains of rhamnolipid-producing bacteria,named CT4,CT5 and CT7,were screened from environmental samples using CTAB-methylene blue plates,and their yields of rhamnolipid detected by anthrone sulfate method were 7.43 g/L,8.02 g/L and 7.46 g/L,respectively.(2)Combined morphological,physiological,16S r DNA and gyr B gene identification,CT4was identified as Klebsiella quasipneumoniae,a G^-bacteria,with rod-shaped cells,no spores,and round colonies,off-white,opaque,the colony surface is viscous,and the edges are neat;CT5 and CT7 were identified as Pseudomonas aeruginosa,G^-bacteria,rod-shaped,no spore-producing,round colonies,gray-green,opaque,the colony surface is wet,the edges are uneven,and with pungent odor.Functional gene identification found that CT4 contained two rhamnolipid synthesis-related genes:rhl R,and rhl C;CT5 and CT7 contained five rhamnolipid synthesis-related genes:rhl A,rhl B,rhl AB,rhl R,and rhl C.(3)The optimal fermentation condition(L)of CT4 was optimized by experiments as:corn oil 9.899%,Na NO₃ 10.532 g,Na₂HPO₄ 1.2 g,KH₂PO₄ 0.4 g,Mg SO₄ 0.9 g,Ca Cl₂ 0.1 g,trace element solution 1 m L,inoculum volume 3%,and the initial p H value 7.916.The theoretical yield was estimated to be about 24.323 g/L after 72h fermentation,and the measured yields(24.014 g/L)was 3.23 times higher than before.The optimal fermentation condition(L)of CT5was:soybean oil 6%,Na NO₃ 8.249 g,Na₂HPO₄1.165 g,KH₂PO₄ 0.389 g,Mg SO₄0.3 g,Ca Cl₂0.1 g,trace element solution 1 m L,inoculum volume 2%,initial p H The value 7.095.The theoretical yield was 25.551 g/L after 72h fermentation,and the measured yield(25.146 g/L)was 3.14 times higher than before.The optimal fermentation condition(L)of CT7 was:corn oil 8%,Na NO₃ 9.841 g,Na₂HPO₄ 0.4 g,KH₂PO₄ 1.2 g,Mg SO₄ 0.3 g,Ca Cl₂ 0.1 g,trace element solution 1 m L,inoculum volume 2.174%,the initial p H value 6.436.The theoretical yield was 22.025 g/L after 72h fermentation,the measured yield(22.253 g/L)was 2.98 times higher than before.(4)Application experiments found that the petroleum degradation rates of strains CT4,CT5 and CT7 were 41.8%,43.5%and 31.3%,after inoculated into petroleum-containing medium for 72 h.For emulsification of corn oil,the metabolites of strains CT4,CT5 and CT7assessed by emulsification coefficients which were 60%,53%and 66%.In control study of agricultural pathogenic bacteria,we found that CT5 could inhibit Fusarium oxysporum,Rhizoctonia solani,Bipolaris sorokiniana,Botryosphaeria dothidea,Sclerotium rolfsi.Sclerotium rolfsii,while CT4 and CT7 had no inhibitory effect on the growth of selected agricultural pathogens.