Degradation Of Methyl Chloride By Strains L-1 And W2-1-1 Isolated From Salt Marsh In Yancheng,Northern Jiangsu Province

Soil and seawater samples were collected from the salt marshes in the coastal area of Yancheng,northern Jiangsu.The sampling environments included reed zone(L),Spartina zone(M),Suaeda zone(J),seawater(W)and mixed plant zone(H),unknown plants zone(U).DM media containing methyl chloride and methyl bromide as the sole energy source and carbon source were used for selective culture and enrichment of functional microorganisms which are capable of degrading halogenated methane(CH3Cl and CH3Br).Finally,18 strains of methyl chloride degrading bacteria were isolated,but methyl bromide-degrading bacteria were failed to find.Through 16S rDNA sequencing and homology analysis,a total of 8 methyl chloride degrading bacteria were isolated which belonged to Acinetobacter sp.(M-1 and H-3-1),Burkholderia sp.(W1-1 and L-1),Acidovorax sp.(M-2),Pseudomonas sp.(W2-1-1),Pandoraea sp.(J-2),Caulobacter sp.(H-1).The rate of degradation of CH3Cl by 8 strains of bacteria was compared by gas chromatography.It was found that the degradation rate of strain L-1 isolated from the soil of Phragmites australis was the highest,and at the same time,a strain of W2-1-1 isolated from seawater was selected.The following experiments were performed around strains W2-1-1 and L-1.The growth characteristics of strain W2-1-1 on DM medium were:round colonies with pale yellow edges and orange yellow in the middle.The growth characteristics of strain L-1 on DM medium were:round colonies,yellow,deep in the middle,moist and smooth,with neat edges.Both strains were rod-shaped,size(length × width)200nm × 100nm,no endospores,Gram-negative bacteria without fexirubin pigment.Strain W2-1-1 can utilize less carbon and nitrogen materials than strain L-1.Both can use malonate,ammonium gluconate,dextrin,lecithin,D-ribose as carbon-nitrogen source,but L-1 can also use sucrose,glucose,amino acid broth control,sorbitol,Esculin,lysine decarboxylase,bile esculin,sorbose,arginine decarboxylase,fructose broth,ornithine decarboxylase,dulcitol,arginine dihydrolase,melibiose broth,urea,inositol,mannitol,and mannose.W2-1-1 can also use acetate,arginine dihydrolase broth control,arabinose,xylose,galactose,neither can use salicin,xylitol broth,?-amygdalin,gluconate,raffinose,tartrate,N-acetylglucosamine,phenylalanine,hydrogen sulfide,sucrose,bile lysine broth,lactose,Sodium oleate,DNA,melezitose,cellobiose,rhamnose,?-galactoside(ONPG),and turanose were used as carbon and nitrogen sources.The optimal growth conditions for the two strains were the same,the optimum pH was 7.0,the optimum temperature was 30?,and the optimal salinity was 0%.However,W2-1-1 can grow at a salinity of 4%,while the growth of L-1 bacteria stops at more than 2%.The logarithmic growth phase of W2-1-1 is between 12-60 h,and the logarithmic growth phase of L-1 is between 24-60 h.According to molecular biological identification,the homology of W2-1-1 with Pseudomonas sp.Pier 14 was as high as 99%,and the homology of L-1 with Burkholderia gladioli ATCC 10248 was as high as 99%.Therefore,it was initially determined that W2-1-1 was the member of the genus Pseudomonas and L-1 belongs to the genus Burkholderia.The factors that may affect the degradation rate of CH3Cl by the strains include:1.The degradation of the bacteria on the different culture media:DM culture medium with methyl chloride as the sole energy source and carbon source and beef extract-peptone medium.2.Different electron receptors:comparison of the degradation results of NO3-and O2 as electron receptor respectively.3.whether the effect of the co-action of the two strains on the degradation of CH3Cl was stronger than that of the single strain was verified.The results showed that L-1 and W2-1-1 had lower degradation of CH3Cl when cultured on the alternative medium for a long period of time.That is,its functional properties gradually weakened.when both oxygen and NO3-were present in the reaction system as an electron acceptor,the degradation rate of methyl chloride by W2-1-1 was 60%,and the degradation rate was 43.8%in the absence of nitrate,indicating that nitrate reduction was more efficient than oxygen in the degradation of W2-1-1.The degradation rate of L-1 to methyl chloride is not much different under the above two conditions.The combined action of W2-1-1 and L-1 did not increase the degradation of CH3Cl,indicating that there was no synergy between the two strains.Previous studies have shown that the cmuA gene in the bacteria is associated with the degradation of CH3Cl,and can encode proteins with specific methyltransferase and porphyrin-like binding functions.PCR primers were designed based on the conserved regions in the cmuA gene and the unique structure of the gene.Whether the cmuA gene was contained in the W2-1-1 and L-1 bacterial genomes was identified by PCR amplification.The mechanism of the degradation of CH3Cl by these two strains was preliminary explored.No positive products were obtained by PCR amplification and sequencing.It was judged that the two strains may not contain the cmuA gene,or the currently used cmuA gene primers may not be suitable for L-1 and W2-1-1.Therefore more cmuA gene sequences need to be obtained from new strains to improve the primer sequence.Based on the results,it could not be determined whether the strains L-1 and W2-1-1 could induce the generation of a methylosterone-dependent methylation pathway when degrading CH3Cl.