Isolation And Identification Of Cd-resistant Plant Growth-promoting Rhizobacteria And Their Adsorption Effect And Mechanism Studies On Cadmium

Due to rapid growth of industrialization,civilization and population,the demand for food supply is sharply increasing.In contrast,the arable field is not increasing,but is being contaminated by heavy metals,especially for cadmium(Cd).Excessive Cd accumulation in soil can adversely affect plants,animals,microbes,and humans.Therefore,it is imperative to remediate Cd contaminated arable land.Bioremediation is a novel,cost-effective,environment friendly and sustainable remediation technology in situ,which consists of phytoremediation,animal remediation,microbiology remediation and combination remediation.Among them,plant growth-promoting rhizobacteria(PGPR)are extensively studied and applied,which not only can assist hyperaccumulators to remediate Cd contaminated soil,but also can adsorb Cd(II)being biosorbents.However,their adsorption mechanism is affected by Cd(II)concentrations,microbial cell wall compositions,and cellular physiological functions,which is unclear.This article mainly works on the above issues and the results are as follows:1.Isolation and identification of Cd-resistant plant growth-promoting bacteriaThirteen high Cd-resistant(at least 5 mM)rhizobacteria were screened from rhizosphere soil of dominant plants(Eragrostis ferruginea,Eremochloa ophiuroides,Bidens pilosa,Colocasia esculenta,and Solanum nigrum)in 5 sample areas,near a 60-year lead-zinc mine in Guangxi,China.16S rRNA gene sequencing and phylogenetic tree analysis were carried out and they were finally clustered into four genera(Curtobacterium,Chryseobacterium,Cupriavidus,and Sphingomonas)and three phyla(Actinobacteria,Bacteroidetes,and Proteobacteria).Soil metagenomics analysis showed that Sphingomonas had the highest proportion and were common strain types,while Cupriavidus,Chryseobacterium,and Curtobacterium were rare ones.Cupriavidus necator GX-5(CP002878),Sphingomonas sp.GX-15(MF959440),and Curtobacterium sp.GX-31(MF959445)were selected as the most promising plant growth-promoting rhizobacteria(PGPR)based on the analysis of PGP traits.Pot experiments indicated that inoculating GX-5,-15,and GX-31 significantly increased plant growth and Cd(II)uptake by accumulator Brassica napus.There existed significant difference in promotion effect among strains,which were GX-31>GX-5>GX-15.PGPR GX-5,-15 and GX-31 were stored in the China General Microbiological Culture Collection Center(CGMCC),and the preservation numbers were:CGMCC1.13645,GCMCC1.13644 and CGMCC1.13643,respectively.2.Adsorption isotherms and adsorption kinetic models of Cd(II)by dead and live biomass of Cd-resistant PGPRAccording to the analysis of the influence factors of adsorption(pH value,dosage of bacterial biosorbent,Cd(II)concentration and adsorption time),the optimum adsorption conditions were as follows:pH adjusted to6,and at a dose of 1 g/L,as dead and live bacteria of PGPR being the biosorbents.The adsorption process can be roughly divided into a rapid process at the beginning stage and a slow process at the second stage.During the first 30 minutes,the adsorption amount of live and dead cells of GX-5,-15 and GX-31 accounted for at least 86%of the total amount,and that of GX-31 could reach as high as 98%.Correlation coefficient R~2showed that the adsorption process of Cd(II)by live and dead biomass of GX-5 fitted Freundlich isotherm well,while that of GX-31 obeyed Langmuir isotherm.Interestingly,Langmuir and Freundlich isotherms could better describe dead and live cells of GX-15 for Cd(II)adsorption,respectively.Meanwhile,the dead cells are more favorable for Cd(II)adsorption than the live cells,and adsorption capacity,affintiy and intensity can be ordered as GX-31>GX-15>GX-5 among strains based on the analysis of constants of Langmuir and Freundlich isotherms.The pseudo-seconde-order model was more suitable to simulate the whole kinetic adsorption behavior of all the biomass,and the chemisorption process might be involved.3.Adsorption effects and mechanisms of intracellular accumulation and extracellular adsorption of Cd(II)by live biomass of Cd-resistant PGPRThree Cd-resistant PGPR,Cupriavidus necator GX-5,Sphingomonas sp.GX-15,and Curtobacterium sp.GX-31,were used to study bioaccumulation and biosorption mechanisms under different initial Cd(II)concentrations(20,50 and 100 mg/L).Removal efficiency and adsorption capacity of the assessed PGPR could be ordered as GX-31>GX-15>GX-5.At low concentrations,the removal efficiency was the highest and the adsorption capacity was the weakest.On the contrary,at high concentrations,the removal efficiency was the lowest and the adsorption capacity was the strongest.Physical entrapment,ion exchange,and complexation were involved in biosorption processes and performance were inconsistent among strains.The dominant adsorption mechanism for GX-5 was bioaccumulation,while the dominant mechanisms for GX-15 and GX-31 were both biosorptions under the experimental conditions.4.Adsorption effects and mechanisms of Cd(II)by dead and live biomass of Cd-resistant PGPRThe removal efficiency and adsorption capacity of live and dead biosorbents of C.necator GX-5,Sphingomonas sp.GX-15,and Curtobacterium sp.GX-31 were quite different and strongly affected by initial Cd(II)concentrations.Whether the cells were living or dead,the adsorption capacity among strains could be listed as GX-31>GX-15>GX-5.SEM-EDX,FTIR analysis and adsorption studies indicated that both live and dead cells had the ability to adsorb Cd(II),but dead biomass was superior to live biomass within the experimental conditions tested,especially when Cd(II)concentration was low.