Selection Of Malachite Green Efficiently-degrading Bacteria And Its Application In Dyeing Wastewater Treatment

Malachite green is a kind of three phenyl methane synthetic dye.It is generally used as industrial dying.Malachite green dye has the characteristics of difficult degradation,high toxicity and high residue.During the process of using malachite green,the chemical oxygen demand(COD)in the water environment usually increases with the discharge of wastewater into environment.The wastewater with deep color obstructs the transmission of light,which seriously affects the growth of aquatic organisms.At the same time,it is easy to enrich in living organisms.The untreated wastewater may not only destroy the aquatic ecosystem,but also threaten human health.Some studies showed that the organism could remove malachite green,but some microbes only turned malachite green into leucomalachite green,or removed its color through biological adsorption,which may cause second pollution.Therefore,it is of great theoretical guiding significance and practical value for studying malachite green wastewater biological treatment.In this study,we directly isolated a strain with high efficiency to degrade malachite green from the activated sludge of a sequencing batch reactor treating high-salinity wastewater.The growth and decolorization characteristics of the isolated strain were studied systematically.And its degradation advantages were explored through degradation kinetics experiments.And the degradation performance of the strain was investigated in the biological wastewater treatment.Moreover,the bioaugmentation of the strain was verified by comparative experiment,and the difference of microbial community structure in the reaction system was studied by using molecular biological mathods.The main conclusions are as follows:(1)A strain with high efficiency to degrade malachite green was isolated and screened from a sequencing batch reactor treating high-salinity wastewater UV-visible spectroscopic analysis of the reaction product by chemical analysis showed that the strain had good degradation effect on malachite green.According to the 16S rRNA gene sequencing and NCBI database online comparison,it was confirmed that the strain was belonged to Dietzia and named it Dietzia sp.DL1.The whole genome of the pure bacteria was analyzed,and the protein sequence was annotated by the GhostKOALA online annotation platform.The protein function of the whole genome was preliminarily understood.At the same time,the possible degradate function gene tmr gene and the halogenated alkane dehalogeninic enzyme DhaA were obtained according to the functional information of the annotated gene.(2)The single factor experiment was carried out to study the growth environment of the degrading strain.The results showed that the strain DL1 could grow well with pH of 6.0?9.0,while the growth was severely inhibited under the acidic condition,and the growth state was the best at pH=8.0.Strain DL1 maintained a good growth state at ambient temperature between 25? and 35?,while the most suitable temperature was 30?.At the same time,strain DL1 was an aerobic bacterium.When the aeration volume of the cone bottle was bigger,the strain growed faster.Strain DL1 could maintain its growth state when NaCl concentration was 60 g/L,indicating that it had high salt tolerance,and almost stopped growing when the concentration reached 100 g/L.In addition,strain DL1 malachite green degrading bacteria had a very strong resistance to penicillin and could still grow well at the concentration of 256 mg/L The inhibitory concentration of tetracycline,streptomycin,oxytetracycline,vancomycin,chloramphenicol were 16 mg/L,32 mg/L,8 mg/L,16 mg/L and 64 mg/L respectively.(3)DL1 had a very high tolerance to malachite green and could degradate a large range of malachite green.When the pH of the solution was kept in the range of 7.0 to 9.0,the degradation efficiency was more than 90%.Under the salinity of 60 g/L,the strain could still maintain a good degradation efficiency,indicating that the strain not only tolerated high salinity but also degraded under high salinity.In addition,metal ions(Cu2+,Ni2+,Cd2+,Zn2+,Mg2+,Mn2+,Al3+and Fe2+)on the process of malachite green degradation in DL1 were also investigated,in which the 0.5 mM Cu2+,Cd2+,Al3+,Fe2+and other metal ions showed significant inhibition on the degradation of the dye.Mg2+facilitated in degradation.While Ni2+,Zn2+,and Mn2+had little effect on dye degradation.The best degradation conditions were pH=8,glucose 0.5 g/L and salinity 0 g/L.(4)In this study,sequence batch rectors(SBRs)were applied to invesitigate the application of strain in actual wastewater.The bioaugmentation of the strain was verified by comparative experiments,which were divided into control reactor(without strain R1)and experimental reactor(strain R2).When malachite green dye was added,the two reactor systems were affected by varying degrees.The removal of ammonia nitrogen and COD in the reactors was inhibited.When the malachite green concentration was 50 mg/L,the ammonia nitrogen removal efficiency of R1 decreased to 45.1%-52.9%;the removal efficiency of COD dropped to 50%;and the ammonia nitrogen removal efficiency of R2 remained about 80%.The COD removal efficiency fluctuated with average value at 80%.However,the R2 reactor was able to quickly adapt to the change of water quality,and the ammonia nitrogen removal efficiency was restored to 87.1%.After mixed with strain DL1,the R2 reactor could recover high ammonia nitrogen removal under high concentration of malachite green,demonstrating a stronger ability to resist the toxic impact of malachite green.The SBR system of DL1 strain was more efficient than the control system for removing malachite green.At the same time,the malachite green dye could significantly affect the microbial community structure of SBR,leading to an obvious decrease in the relative abundances of Planctomycetes,Verrucomicrobia and Nitrospirae.The abundance of genera Acinetobacter,Burkholderia,Serratia,Zoogloea,Dokdonella,and Lactococcus increased in the two reactors.The relative abundance of the functional gene tmr was analyzed by qPCR.The results showed that with the increase of the concentration of malachite green,the abundance of tmr gene generally showed an upward trend.The tmr gene abundance also showed an increasing trend in R1,but its relative abundance was always lower than that in R2.