| Microorganisms play an essential role in biodegrading petroleum hydrocarbon pollutants in the world.Biotechnology is considered to be a green technology with broader application prospects for treating oilfield-produced wastewater(OPW)containing high petroleum hydrocarbons.OPW is hard to biodegrade because of its high temperature and salinity.Current research on the use of biotechnology to treat OPW,whether using biochemical processes,microbial populations,or single strains,is always performed at room temperature.Only a few studies use biological technology to treat OPW under 45? or higher temperature,which severely limits the popularization and application of biochemical techniques in cultivating halo-tolerant,thermo-tolerant,and petrol-degrading microbes to biodegrade OPW in the oilfield.Therefore,it is essential to study how to operate biochemical processes,identify microbial distribution characteristics,and clarify biodegradation mechanisms of single strain to treat OPW under high temperature and salinity conditions.Firstly,two OPW plants were selected as research areas,one treated OPW through two-stage aerobic(O1/O2)activated sludge process in North-China Oilfield(NCO),while the other treated OPW through anaerobic/aerobic/aerobic(An/O1/O2)biofilm process in Liao-He Oilfield(LHO).These two plants were both the first to biodegrade OPW by cultivating thermo-and halo-tolerant microorganisms in China.The biochemical processes used in the two plants could biodegrade petroleum hydrocarbon well.Compared with NCO plant,LHO plant had better biodegradability,which mainly benefited from the hydrolytic acidification function of An-zone and the biofilm formation.This paper explains the different biodegradability between the two plants from the unit function,microbiota distribution,and biodegradation mechanism perspectives based on the analyses of conventional wastewater quality,GC-MS,high throughput sequencing,and microbial community diversity.The distance-based redundancy(db-RDA)analyses showed that the temperature,DO,Cl-,and organic components in two different OPW were related to the bacterial distribution.Secondly,in this paper,microbial population evolution experiments were carried out under different operating conditions and carbon sources to accurately study the effects of single environmental factors on microbial communities.The macroscopic active sludge properties and microscopic microflora distribution of each group were systematically studied.Meanwhile,the mechanism of environmental factors affecting microbial population distribution and function was studied through the quantitative change tracking of the top 5 genera,db-RDA analysis,community heatmap,Spearman correlation heatmap,phylogenetic tree and 16S function prediction in each group.Under different culture conditions and carbon sources,the results showed that all the microbial quantity(OD600,MLSS),growth character indexes(SV30,SVl,CSH,and EPS),performance indexes(the activity and distribution of enzyme,the removal of TPH,COD and petroleum hydrocarbon),microbial community distributions and homeoprotein cluster metabolic function would be affected by environmental factors.db-RDA analyses showed that the effects of environmental factors on the distribution of microbiota in each group were as follows:Cl->Temperature?Aromatic>DO>BFC>Alkane>H2O2.Spearman correlation heatmap analyses indicated that there was a relatively average positive and negative correlation between Cl-and bacteria genera,the temperature was negatively correlated with the most bacteria genera,while carbon source(alkane or aromatic),DO,H2O2 and BFC had positive correlations with most bacteria genera.Thirdly,three thermo-and halo-tolerant strains,named HPB-1(Exiguobacterium),HPB-2(Bacillus badius),HPB-3(Paenibacillus)have been isolated from NCO and LHO.Their physiological and biochemical indexes,temperature and salt tolerance,cell surface hydrophobicity(CSH),adsorption of TPH and biodegradation of petroleum hydrocarbon were analyzed in this paper.The research showed that the three gram-positive strains with different colony morphology and single cell morphology could biodegrade the petroleum hydrocarbon in OPW oilfields well.However,indigenous strains had better performances than exogenous strains for adsorbing and biodegrading petroleum hydrocarbon in OPW.There were apparent correlations among cell growth,biosurfactant(BS)yield,BS and intracellular enzymes(IE)activities,and TPH biodegradation of each strain.HPB-2 had the best performance,followed by HPB-3,and HPB-1 had the worst performance.Enzyme activity tests proved that the ortho-cleavage pathway was more dominant than the meta-cleavage pathway in the aromatic metabolism for bacterial community or single strain.It is worth noting that,microbes themselves play a leading role in TPH biodegradation compared to BS and IE.A synergistic effect occurs when mixing HPB-2 and HPB-3 compared to a single strain,and the TPH reduction was increased by 6.5%-43.8%.Finally,the draft genome analyzes method was used for non-contiguous complete genome sequencing of the three strains.All the coding genes of each strain were compared in NR,Swiss-Prot,and KEGG databases,respectively.We explained why(1)all the three strains could effectively biodegrade the actual OPW of NCO which was rich in alkanes,(2)the biodegradation performance was HPB-2>HPB-3>HPB-1 when the three strains individually biodegrading the OPW from LHO,and(3)they could adapt high temperature and salinity OPW environment from the genetic perspective.Based on the whole gene sequencing and gene retrieval related petroleum hydrocarbon metabolism of the three strains,a possible biodegradation model of petroleum hydrocarbons-key enzymes-coding genes for oil-degrading strains in high temperature and high salinity OPW was established by combining their GC-MS analyses results of biodegrading petroleum hydrocarbon,as well as their key enzymes activity and distribution analyses results.The mechanism of how strains controlled the production of key enzymes by coding genes,and transferred or released them into intracellular,periplasm,and extracellular to act on petroleum hydrocarbons or their metabolic intermediates were also clarified. |
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