Study On Microbial Remediation Of Weak Flowing Water Polluted By Chromium-containing Black And Odorous

With the large increase of urban population and the increase of emissions,more and more rivers or lakes are seriously polluted and black and odorous water appeared and the heavy metal ions are present in sewage and waste residue.Heavy metals could not be decomposed and gradually enriched in the bottom mud,making the treatment of the original black and odorous water more difficult.Chromium,as an important industrial element,is mostly used in steelmaking,tanning,chromium plating and paint making industry.Improper treatment may easily enter the black and odorous water to form chromium-containing black and odorous water.In general,microbial remediation technology has many outstanding advantages,such as high efficiency,low cost,environmental protection and so on,but high concentration chromium in water body has the inhibition of microbial activity.In the case of combined pollution of chromiumcontaining black and odorous water,it is not clear whether the microorganism resistant to chromium in the existing studies can be effective,and whether the mechanism of resistance to chromium has changed.The applicability and effect of microbial remediation technology under flow conditions are not clear,which limits the effective application of microbial remediation technology in urban river management.In view of the phenomenon of chromium combined with black and odor pollution in urban rivers,the effect of the original water degrading bacteria becomes worse or even deactivated under the stress of high chromium concentration.In this study,a strain of water degrading bacteria resistant to high chromium concentration was screened and domesticated by microbial in-situ remediation method,and its adaptive growth environment was investigated for the chromium-containing black and odorous water in Chengdu,Sichuan Province.Physiological and biochemical analysis,characterization analysis,genome analysis and transcriptome analysis were used to explore the anti-chromium mechanism of the optimal strain.In addition,the effects of different temperature and flow conditions on the degradation effect of the optimal strains were investigated.Finally,the optimal strain was put into situ,and the effect of the optimal strain was verified by comparing the changes of water quality,microbial community structure and metabolites before and after the water body.The main research contents and achievements of this paper are as follows:(1)The typical black and odorous water in the Pi River Basin of Chengdu were selected to carry out on-site investigation and analysis of water quality and microbial community.It was found that the overall mobility of typical black and odorous water in Pi River Basin was weak,and the concentration of DO in some sections decreased significantly.The average concentration of TN in water ranged from 0.02 to 1.93 mg/L.The contents of TN and TOC in sediment were 4.30-5.30 g/kg and 9.89-25.01 g/kg,respectively.Chromium pollution was found in Baihe Island section.The chromium concentration in sediment was 22-69 mg/kg,which was a kind of class V water polluted by black odor and heavy metal chromium.According to the results of microbial community diversity,the most dominant bacteria at class level were Betaproteobacteria and Gammaproteobacteria.At the genus level,Pseudomonas was the most dominant genus in each samples.VPA(Variance Decomposition Analysis)showed that heavy metals(Cr and Pb)were the most important factors affecting microbial community(9%),followed by p H,DO,Eh(8%),and the highest degree of interpretation between the two was 15%.The results of RDA(Redundancy analysis)showed that the interpretation rate of environmental factors was 82.75%,among which p H was the highest factor,reaching 28.9%.Eh interpretation rate was 10.6% and Cr interpretation rate was 4.3%.There was a positive correlation between Cr and Pb.Anaeromyxobacter,Streptomyces and Burkholderia were highly sensitive to heavy metals,which may be caused by the inheritance of species and long-term adaptation to the environment.(2)Chrome-resistant strains SCU20 and T91 were screened from natural chromium-containing black and odorous water.The identification indicates that SCU20 is Pseudogemmo bacterbohemicus,and its storage number is CCTCC No:2020179.T91 was identified as Providencia burhodogranariea.Comparison tests on the removal rates under different initial Cr6+ concentrations found that when the initial Cr6+ concentration was 20 mg/L,and the removal rate of Cr6+ and total chromium was98.5% and 17.7% respectively in 72 hours by SCU20.The removal rate of Cr was 92.3%and the removal rate of total chromium was 26.6% in 72 hours when the initial Cr6+concentration increased to 100 mg/L and 200 mg/L.The removal efficiency of T91 strain was higher than that of SCU20 strain in the low concentration of Cr6+ solution<50 mg/L,and it was not inactivated in the high concentration of Cr6+ solution.When the initial concentration of Cr6+ was 100 mg/L,the removal rate of Cr6+ and total chromium in strain T91 was 76.8% and 28.8%,and when the initial concentration of Cr6+ was 200 mg/L,the removal rate of Cr6+ and total chromium in strain T91 was41.0% and 16.3%.The highest chromium resistance of T91 was 200mg/L.When p H was 7 and 8,the T91 could grow normally under different concentrations of Cr6+ and play the removal effect of chromium,and 1% inoculation amount was the appropriate amount.(3)Based on the above research,the characterization analysis of the chromium resistance mechanism of strain T91 showed that the cell structure collapsed and became uneven,because the functional groups on the cell wall surface were negatively charged and had a bonding reaction with chromium ions.There were a large number of COOH,C-O-C,C = O and other heavy metal adsorption functional groups on the cell wall,which interact with chromium element.Therefore,the principle of Cr removal by strain T91 is mainly to reduce Cr6+ to Cr3+ and conduct biosorption on the cell surface.The genome sequence of strain T91 showed that 4985 genes were annotated,and there were many heavy metal resistance genes,virulence related genes and antibiotic related genes on the chromosome.The expression levels of genes in T91 were increased,including multi-drug resistant protein MARA,multi-drug resistant protein MARR and cold shock protein CSPA,which indicated that these genes played an important role in the response of T91 to cadmium stress.A total of 1235 genes were expressed before and after chromium stress,among which drug-resistant protein,hydrogenase maturation factor,hydrogenase operon and outer membrane protein genes were up-regulated,while iron-regulated outer membrane virulence protein,heme binding periplasmic protein and ABC transporter solute binding protein related genes were down-regulated.The q PCR results showed that the expression levels of KDPA,KDPB,KDPC and other genes in strain T91 were down-regulated to varying degrees under chromium stress,indicating its membrane physiology and selection uniqueness.The chemotactic pathway genes of sensor CHEA and regulator CHEB and CHEY were up-regulated in the strain.The expression abundance of CHEA gene in the strain sensor increased under chromium stress,and the signal was transmitted to the reaction regulator through phosphorylation,which then feedback controlled the activity and gene expression of chemotactic regulatory factors,and protected the strain itself from heavy metal toxicity.(4)In order to investigate the removal effect of T91 strain on the black and odorous water containing chromium under different temperature and flow conditions,the black and odorous water containing chromium was taken from Baihe Island section of Pi River.The degradation performance of strain T91 at different temperatures(5℃,15℃,26℃,37℃)and different flow rates(0 m/s,0.1 m/s,0.15 m/s,0.2 m/s,0.3 m/s)was studied in laboratory experiments.The results showed that the degradation coefficient of NH3-N increased with the increase of flow rate and temperature.When the flow rate was less than 0.20 m/s or the temperature was lower than 26℃,the degradation effect of NH3-N by strain T91 was weakened when the flow rate was greater than 0.20 m/s or the temperature was higher than 26℃.The degradation effect of COD increases with the increase of temperature.The degradation coefficient ranges from 0.171 1/d to 0.319 1/d at 37℃.COD degradation coefficient increases with the increase of flow rate when the flow rate is less than 0.15 m /s,COD degradation effect gradually weakens when the flow rate is greater than 0.15 m /s.The removal rate of Cr6+ increased with the flow rate increasing,and the removal rate was the highest when the flow rate was 0.30 m/s.The removal rate of Cr6+ increased with the increase of temperature,and reached the peak value of 98.7% at 26℃.It is concluded that temperature is the most significant factor affecting the degradation rate of COD and NH3-N,and the flow rate can promote the growth and metabolism of bacteria to some extent without affecting the adsorption of substrate.Temperature,flow rate and turbulent energy are the dominant factors affecting the removal rate of chromium.(5)In order to explore the feasibility of T91 bacterial agent on the treatment of natural black and odorous water body,in situ test was carried out in the diversion channel of Pi River Basin in Chengdu to compare and analyze the changes of water quality,microbial community structure distribution and metabolites before and after the application of bacteria.The results showed that T91 could significantly reduce turbidity in water,and the degradation rate of TN was 29.8%.The degradation effect of T91 was better than 0.005 m/s at the flow rate of 0.020 m/s.After the inoculation,the water velocity changed the dominant species of bacteria,and the dominant species of bacteria in the water changed.At class level,the dominant bacteria changed from Oxyphotobacteria to Bacilli at a flow rate of 0.005 m/s.The dominant bacteria changed from Oxyphotobacteria to Acidimicrobiia at a flow rate of 0.020 m/s.At the genus level,the dominant bacteria changed from Cl500-29_marine_group to Exiguobacterium at a flow rate of 0.005 m/s.The dominant bacteria changed from Cl500-29_marine_group to Flavobacterium(3.8%)at the flow rate of 0.020 m/s.As one of the indicators of water eutrophication,the Oxyphotobacteria are the dominant bacteria before inoculation,which represents the poor water quality.After inoculation,their relative abundance decreases,which reflects the improvement of water quality.The dominant bacteria became Bacilli after the inoculation,which was a kind of good or facultative bacteria,reflecting the increase of dissolved oxygen in the water.There was a significant difference in the metabolites between the imported and exported water before and after inocication,including short-chain fatty acids,free fatty acids,lipids,amino acids,carbon dry acids,nucleotides and organic acids.There were 47 metabolites increased and 49 metabolites decreased after inoculation.In addition,there were 11 species of environmental microorganisms were significantly correlated with different metabolites.Oxyphotobacteria were significantly negatively correlated with diaminodiphenyl ether,methadone and diazepam,and proteus,bacillus and glycine.In this study,Providencia burhodogranariea,which has the ability of degrading black odor and resisting chromium,was isolated from natural polluted water,and the mechanism of resistance to chromium was revealed.We studied the degradation effect of T91 strain under different temperature and flow conditions,and carried out in situ experimental study on natural black odor water.This study provides an important basis for the microbial remediation technology of chromium-containing black and odorous water,and has important scientific value and theoretical significance for the treatment of black and odorous water and water environment protection.