Isolation And Identification Of A Humus-reducing Bacterium And Its Biochemical Mechanism For Anaerobic Conversion Of Diqua

Humus respiration is an important form of microbial anaerobic respiration,which is of great significance to the geochemical cycle of elements and the transformation of pollutants.Microbes can mediate detoxification and degradation of various organic pollutants in the environment and directly participate in the natural circulation process of important elements such as Fe and Mn during humus respiration.It is still a research hotspot in the field of environmental microbiology to explore more culturable humus-reducing bacteria resources,study the characteristics of its anaerobic transformation pollutants,and clarify the law of its degradation and transformation pollutants in the environment.Samples were taken from various environmental media in Hainan Province to enrich and separate humus-reducing bacteria with anthraquinone-2,6-disulfonate disodium（AQDS）as the sole electron acceptor.The strains were identified by morphological,physiological and biochemical characteristics,16S r RNA and whole gene sequence analysis.The reduction characteristics of strain HN05 to Fe（III）-humus and the transformation mechanism of diquat under anaerobic conditions were studied.The main conclusions are as follows:（1）The strain HN05 with the ability of reducing humus and anaerobic transformation of diquat was isolated from the sediments of Nandujiang River,Hainan Province,which was identified as Kosakonia oryzae HN05.The optimum single factor culture conditions are temperature 28-30℃,sodium chloride concentration 0.5%-1%and p H5-7.HN05 grows rapidly,and after 16 hours of cultivation,it can enter the stable growth period.（2）The electron donor spectrum showed that strain HN05 could reduce AQDS with sucrose,glucose,lactose,lactic acid,glycerol,acetic acid,ethanol and methanol as electron donors,and the order of utilization capacity was sucrose>glucose>lactose>lactic acid>glycerol>acetic acid>ethanol>methanol.（3）The results of electron acceptor spectrum showed that strain HN05 used sucrose as electron donor,and the reducing ability of the four humic analog in descending order was:AQDS>anthraquinone-2-sodium sulfonate>anthraquinone-1,5-Sodium disulfonate>anthraquinone-1-sodium sulfonate;the reducing ability to the two humic substances is in the following order:humic acid>fulvic acid;the reducing ability to the two soluble Fe（III）oxides is in the order of:ferric sodium ethylenediaminetetraacetate（Fe-EDTA）>ferric citrate;the reducing ability to four insoluble Fe（III）oxides is in the following order:ferrihydrite（HFO）>goethite>lepidocrocite>hematite.The results of intracellular electron respiration inhibition showed that when the strain HN05 used Fe-EDTA and AQDS as electron acceptors,the components of the electron transport chain of the strain only included dehydrogenase;when HFO was used as the electron acceptor,the components of the electron transport chain of HN05 included dehydrogenase,quinones.The electron transfer process of the three electron acceptors may couple proton transmembrane transport and ATP synthesis.（4）The results of anaerobic transformation of diquat by strain HN05 showed that in the humus reduction system,HN05 could directly anaerobic convert diquat with sucrose as an electron donor,and the addition of AQDS could significantly promote the transformation of diquat,and the transformation rate could reach 41.93%in 22 days.Humic analog species and concentration,sucrose concentration,temperature and p H are the key factors affecting the transformation.The optimal conditions were:AQDS0.5 mmol/L,sucrose 0.25 mmol/L,culture temperature 30℃,p H 6.5,and the transformation rate of diquat was 53.82%on 22 days under these conditions.HN05 can drive the anaerobic transformation of diquat through both biological and chemical effects,（1）The biological mechanism is:HN05 uses sucrose as an electron donor,and diquat directly acts as a microbial terminal electron acceptor for reductive degradation;（2）The chemical mechanism is:HN05 drives AQDS to form a reductive product,anthrahydroquinone-2,6-disulfonate disodium（AH₂QDS）,and AH₂QDS further undergoes redox polymerization with diquat to form single crystals.