Plasticity And Metabolic Mechanisms Of Nitrogen Heterocyclic Compounds Of Petroleum Degradation Bacteria

With the development of oil recovery technologies,the treatment of produced water in oilfields is becoming a difficult challenge.Microbial treatment technology is considered as one of the most potential solutions.However,due to lacking knowledge about the change pattern of related treatment microbial communities,and the response mechanism of bacteria to degadating toxic pollutants,the performance gap between lab studies and practical treatments has been caused,limiting the utilization of microbial remediation technology in oilfield produced water treatment.In this paper,we mainly studied the interaction mechanism between indigenous microbial community and environmental factors during the treatment of heavy oil produced water,and analyzed the global response of an oil-degrading strain Sphingobium yanoikuyae XLDN2-5 to nitrogen heterocyclic compounds.Firstly,we comprehensively investigated the plasticity of indigenous microbial community in a full-scale heavy oil-produced water(HOPW)treatment plant.Here,a newly building full-scale anoxic-oxic treatment plant with a capacity of 10,000 m~3/d was used to treat HOPW by the indigenous microbial community derived from oil reservoirs.After the treatment,the chemical oxygen demand(COD)decreased from187.5±19.5 mg/l to 97.3±9.2 mg/l,and the biological oxygen demand(BOD),total nitrogen and phosphorus decreased to 5.5±3.1 mg/l,3.8±0.6 mg/l,and 0.28±0.09mg/l,respectively.Changes to the community structure and function in all treatment stages were comprehensively analyzed using Illumina MiSeq 16S rRNA gene sequencing.In the anoxic stage,microbes such as Pseudomonas and methanogens were abundant,and the BOD increased by 248.5%.During the aerobic stage,bacteria such as Halomonas spp.,strains of Bacilli,and unclassified archaea were abundant,and the BOD decreased by 88%.This indicated that the community in the anoxic stage played a major role in enhancing the biodegradability of HOPW,whereas the community in the aerobic stage mainly mineralized BOD.These results strongly suggeste that the structure of indigenous microbial communities differed in different treatment stages to accomplish the corresponding functions.Secondly,the carbazole degradation pathway in Sphingobium yanoikuyae XLDN2-5 is systematically unraveled.Sphingomonads with higher genetic diversity are metabolically versatile to utilize various recalcitrant compounds.Sphingobium yanoikuyae XLDN2-5 is a carbazole-degrading sphingomonad that metabolizes several types of xenobiotics.This strain has been deposited in DSMZ with the accession number of DSM28039.To understand the global carbazole metabolic mechanism of this strain,proteomic data under carbazole or glucose culture conditions were gathered by using the 2D-LC-MS/MS analysis.We found that 159 proteins were up-regulated in carbazole-cultured samples,including 15 carbazole degradation enzymes.Combined with genome data,the catechol meta-cleavage cluster was characterized and transcriptionally verified by qRT-PCR.This gene cluster is organized in a special form of carLM-catS-carJK-catR-carDEFGHI,and the structure genes were distributed in three putative operons:carDEFGHI,carJK,and carLM,respectively.Surprisingly,compared to samples grown in the medium with glucose as the only carbon source,the addition of carbazole accelerated the growth of strain XLDN2-5.This may be a result of carbazole enhancing expression of the proteins involved in multiple growth-related biological processes.Additionally,because of the special organization form of the catechol meta-cleavage cluster,the expression of operon carJK in the presence of carbazole was 24.8-fold higher than that in the glucose culture conditions;the intermediates 2-oxypent-2,4-dienoic acid and 4-hydroxy-2-oxovalerate accumulated at levels approximately 55 and 1,400 times lower than those of catechol and anthranilate,respectively.Thus,this special organization form may contribute to the positive effect of carbazole on the growth of strain XLDN2-5.Finally,the regulation mechanism of the upstream gene cluster of carbazole metabolism has been preliminarily studied.Though the construction of a CRISPR system in S.yanoikuyae XLDN2-5,we found that the dCas9 protein has lethal toxicity to these kind of strains.In summary,the above studies provide a new perspective on the evolution of the genetic elements for the degradation of recalcitrant heterocycles,and shed a light on the potential of indigenous microbial community plasticity for various recalcitrant wastewater treatments.