Response And Adaptation Of Farmland Soil Microorganisms To Heavy Metal Cadmium Pollution In Yellow River Delta

In the Yellow River Delta（YRD）,an important land resource and economic growth area in China,Cd pollution has the highest ecological risk index among different heavy metal pollution types.However,early monitoring and control methods of Cd pollution are still lacking in this area,raising needs to develop an effective ecological indicator of early Cd contamination risk.The potential nitrification rate（PNR）of ammoxidation is very sensitive to Cd²⁺toxicity.Thus,the gene amo A encoding the enzyme that catalyzes the ammoxidation can reflect the function of ammoxidation bacteria and the effect of Cd pollution on soil nitrogen cycle.Microbial remediation of heavy metal pollution in soil is green and economic.However,only a few microorganisms can be actually used in soil remediation.Therefore,it is necessary to screen out strains with stronger detoxification ability and fixation ability,and reveal their heavy adaptation mechanisms,which is essential for the subsequent development of efficient soil remediation microbes.Adaptive laboratory evolution（ALE）combined with whole genome sequencing（WGS）is a powerful tool to address the above requirements.In this study,we firstly analyzed the responses of the microorganisms to Cd pollution in the wheat-maize farmland soil in Dongying City.After continuous incubation with Cd²⁺for 28 d,the PNR、the abundance of amo A gene and the diversity of microbial in soil samples were detected.At the same time,Bacillus sp.BF2 was isolated from the soil incubated with exogenous Cd²⁺and then subjected to ALE.After ALE,BF2 was adapted to high concentration of Cd²⁺.In order to explore the adaptation mechanism of Cd evolved by BF2 during ALE,the fixation characteristics of the strain and its extracellular polymeric substances（EPS）on Cd²⁺were investigated.Then,we counted the types of mutations,the number of mutations and the differences in the function of the mutant genes.The results are as follows:（1）In farmland soils of YRD,AOA played a major role in the ammonia-oxidizing process,while AOB was more abundant.The PNR,the diversity and abundance of bacterial community in soil showed a Hormesis effect under low concentration（1mg/kg）of Cd²⁺.The Hormesis effect may be related to the high expression of protein disulfide isomerase;（2）The EPS produced by BF2 efficiently absorbed heavy metals.The optimal adsorption conditions were 30℃,p H 7.0,0.005 g/L,and the adsorption capacity of Cd was 178 mg/g;（3）ALE improved the minimum inhibitory concentration of BF2 to Cd,which was increased from 50 mg/L to 300 mg/L.And the proportion of protein and polysaccharide in EPS was changed.The fixation of Cd²⁺by BF2 and 250-generation strains was mainly based on cell fixation,and the fixation ability of the strain was closely related to culture growth phase;（4）When exposed to 20 mg/L Cd²⁺,BF2 preferentially fixes metal ions on the cell surface and slowly releases them into the cell.The 250-generation strains produced a large number of EPS to wrap cells,thus reducing the toxic effect on cells through extracellular isolation;（5）Whole genome sequencing revealed the mutations enriched in the 250-generation strains,including the mutation of Cd²⁺-P type ATPase and ABC transporter,which might help maintain intracellular Cd²⁺homeostasis.In this study,PNR was identified as an ecotoxicity index to effectively assess the risk of Cd pollution at the early stage of YRD.We clearly illustrated the ecological niches of AOA and AOB in the agricultural soil system studied,which will be instructive for the sustainable development of agriculture and formulation of fertilization strategy.At the same time,strains adapted to high concentration of Cd²⁺（300 mg/L）were obtained through screening and ALE domestication,and EPS with a high heavy metal adsorption capacity was also obtained.We found that synthesis and activation of Cd²⁺-P type ATPase may be the reason why the strains adapt to high Cd²⁺concentration by WGS.