| Nitrile compounds are common feedstock or intermediates that are extensively used in manufacture of solvents,plastics,rubbers and pharmaceuticals.However,nitriles are also harmful organic cyanide with long residual time and considered as environmental pollutants.The large amounts of production and persistent applications of these compounds have inevitably caused serious environmental pollution.Most nitriles,such as acetonitrile,acrylonitrile and crotononitrile etc.,are highly toxic,mutagenic and carcinogenic.The toxicity will lead to some human sicknesses.Untreated nitrile-containing wastewater discharged into a natural water area will cause the death of aquatic organisms as well as serious damage to the ecological environment.Therefore,it is crucial to exploit efficient techniques for the treatment on this class of wastewater.The elimination of nitrile compounds can be accomplished using chemical,physico-chemical and microbiological methods.Of these,the microbial method is the most accepted due to its mild,low-cost and environmentally friendly characteristics.At present,although a number of effective nitrile-degrading microorganisms have been isolated,there have been few reports in the literature demonstrating the degrading pathways and metabolic mechanism.In addition,in the application of practical wastewater treatment using Moving Bed Biofilm Reactor(MBBR)technology,effective nitrile-degrading microorganism addition and its immobilization on the carrier provided an effective way to improve the degradation of nitrile pollutants.However,most degrading bacteria are usually weak in terms of biofilm-forming capacity that lead to loss of microorganisms.Therefore,biofilm-forming bacteria addition will improve the immobilization of effective nitrile-degrading bacterium.The study indicated that biofilm-forming strains can function as a bridging organism,by which degrading bacteria can be coaggregated and adhered to develop multi-species biofilms and these beneficial biofilms are capable of holding other microorganisms in biofilms.At present,bioaugmentation technology by using degrading microorganism combined biofilm-forming bacteria on specific wastewater treatment still needs more investigations.In order to reveal the degrading mechanism of effective nitrile-degrading bacterium on aliphatic nitrile,and improve treatment effect on aliphatic nitrile wastewater,serial researches was carried out in the thesis and results are as follows:1.R.Rhodochrous BX2 was inculated in mineral salt medium supplemented with acetonitrile,acrylonitrile and crotononitrile.Bacterial growth and degradation of each aliphatic nitrile were monitored.The biodegradation process was fitted to a first-order kinetic model.The results showed that BX2 can grow using the three aliphatic nitriles as the sole sources of carbon,nitrogen and energy,respectively.The degradation efficiency was ranked according to the time to 50%substrate dissipation(t1.2)as follows:acetonitrile(2.95 h)>trans-crotononitrile(6.86 h)>acrylonitrile(11.11 h)>cis-crotononitrile(31.13 h).2.The metabolic products of acetonitrile,acrylonitrile and crotononitrile by strain BX2 were determined using GC and GC/MS.The results showed that the intermediate products were corresponding amides and carboxylic acids,which suggested that both types of nitrile-degrading enzyme systems(nitrile hydratase/amidase system and nitrilase)may be involved in aliphatic nitrile degradation.Meanwhile,acetic acid,acrylic acid or crotonic acid degradation by strain BX2 were analyzed when these carboxylic acids were used as the sole carbon source,and NH4Cl was used as nitrogen source.With the bacterial growth,all the carboxylic acids concentration decreased gradually and disappeared at the end of assay.The results confirmed that the carboxylic acids formed by aliphatic nitriles can be degraded by strain BX2 that avoid product-inhibition to nitrile degradation enzymes.3.Based on the common metabolic pathway of various aliphatic nitriles,i.e.nitrile hydratase/amidase system and nitrilase,the expression of nitrile hydratase,amidase and nitrilase were performed by qRT-PCR.Nitrile hydratase and amidase showed accompanying variations.The mRNA levels peaked at early stage and then dropped off.Whereas nitrilase-mRNA was much lower and remained relatively constant.The results suggested that both nitrile hydratase/amidase system and nitrilase may be involved in aliphatic nitrile degradation,with the nitrile hydratase/amidase system seemingly dominant in strain BX2.4.In order to verify the results of qRT-PCR,and to confirm the role of hydratase/amidase system,the temporal variations in nitrile hydratase,amidase and nitrilase activities for the degradation of the three aliphatic nitriles were analyzed by measuring the cell-free enzyme activity.nitrile hydratase and amidase still showed similar variations.The enzyme activities peaked at early stage and then decreased.Nitrilase activities were much lower and remained relatively constant in contrast to nitrile hydratase and amidase.This is consistent to the mRNA varied trend.On the basis of the consistency of mRNA level and enzyme activity,the nitrile hydratase/amidase system was identified as the dominant system responsible for the degradation of aliphatic nitriles.5.Transcriptome of strain BX2 was sequenced on the basis of RNA-seq technology.The results showed that the number of down-regulated genes(353)was much higher than that of up-regulated genes(194)under crotononitrile stress,suggesting that crotononitrile was toxic to strain BX2.The whole metaboilic level of cell decreased,while some genes expression level increased for adapting the survival environment by strengthening the metabolism on crotononitrile.Of these,expression level of three nitrile hydratase genes increased 3.91,3.31 and 1.78 times,respectively.Two amidase gene expression level increased 3.91 and 2.43 times.One nitrilase gene expression level increased 4.96 times.This was identical to the earlier results of nitrile hydratase/amidase identified as the dominant system.In addition,related metabolic genes of other substrates such as benzoate,bisphenol,chloroalkane and chloroalkene,methane and pyruvate etc.were up-regulated significantly,suggesting the wide substrate spectrum.It is important to strain BX2 for practical application in bioremediation.6.On the basis of single factors test,the cultivation conditions of crotononitrile degradation were optimized using response surface methodology based on Box-Behnken Design for the popurse of practical wastewater treatment.The maximum biodegradation rate of cis-(88.70%at 108 h)and trans-crotononitrile(88.99%at 10 h)were achieved at a substrate concentration of 1019.71 mg/L,an inoculation size of 4.44%,a temperature of 31.14 ? and a pH value of 7.17,which were higher 1.56%and 1.71%than those without optimization,respectively.Strain BX2 grew well at concentration from 1000 to 3500 mg/L,which indicated very strong tolerance to crotononitrile.The degradation effectiveness can be influenced by various additional carbon and nitrogen resources.7.Biofilm-forming strains(Bacillus mojavensis M1 and Bacillus subtilis N4)could tolerate but could not utilize 1000 mg/L crotononitrile.The biofilm biomass,BX2 CFU number,degradation rate of cis-and trans-crotononitrile from the mixed culture of BX2 with the three biofilm-forming bacteira revealed that mixed culture facilitated the biofilm formation and immobilization of nitrile-degrading strains in biofilms,therefore enhanced the biodegrading efficiency of crotononitrile.Then,the mixture of BX2 and each of biofilm-forming bacterium were inculated in crotononitrile-containing synthetic wastewater.The biofilms were developed for 72 h and the degradation rates of crotononitrile were measured.The results indicated that the mixed cultures of BX2 and biofilm-forming bacteria exerted stronger resistance against crotononitrile shock loading than BX2 alone.The multiple-species biofilm,BX2 with M1 and N4,had the strongest resistance during successive replacements of synthetic wastewater.The degradation rate of cis-and trans-crotononitrile ranged from 32.55%to 45.95%and from 67.55%to 80.95%respectively,which higher than BX2 alone(the highest values were only 17.29%and 34.79%,respectively).Therefore,the bacterial combination of BX2 with Ml and N4 was more suitable for crotononitrile-containing wastewater treatment in MBBR.The above results will be helpful to built an effective biodegradation system for nitrile-containing wastewater treatment based on the nitrile-degrading bacterium BX2.8.The MBBRs were operated for crotononitrile wastewater treatment under optimized parameters(hydraulic retention times of 24 h,aeration rates of 0.4 m3/h and packing ratio of 30%).On the 28th day after running,trans-crotononitrile in the effluent of reactor with activated sludge,BX2 and biofilm-forming bacteria were completely degraded.The removal rates of cis-crotononitrile and COD reached 93.04%and 93.91%on the 60th day,respectively.The effect was much better than other groups.Furthermore,after MBBR running for 60 days,the biofilm biomass and BX2 CFU number in reactor with activated sludge,BX2 and biofilm-forming bacteria were both higher than others.Abundance of Rhodococcus was 2.82-times higher than that of biofilm with BX2.BX2 immobilization altered biofilm microecosystem.The diversity of microbial communities and the abundance of nitrile-degrading microorganisms increased significantly.In addition,mRNA level of nitrile hydratase and nitrilase were 55.51-and 29.18-times higher than that of biofilm with activated sludge respectively,indicating reconstructed biofilm microecosystem functioned by enhancing nitrile hydratase and nitrilase gene expression.All the results showed that the multiple-species biofilm,BX2 with Ml,and N4,were inferred to have effectiveness on aliphatic nitrile-containing wastewater improvement.This approach offers a promising potential in the application for other wastewater treatment. |
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