Isolation And Identification Of Three Novel Gram-positive Species Capable Of Extracellular Respiration And Preliminary Study On Extracellular Electron Transfer Mechanisms

Extracellular respiration is a novel microbial metabolism, by which microbes can oxidize organic matters and conserve energy for microbial growth coupling with transfer of electrons along the respiratory chain to extracellular electron acceptors.The well-known forms are humic acids resperation, iron respiration and electricigenic respiration. The pathway of extracellular respiration provides a new perspective for understanding the evolution and diversity of microbial respiration. The extracellular respiration is in essence the interaction between microorganisms and extracellular electron acceptors, that is, how electrons transfer along the respiratory chain to the extracellular electron acceptors. The study of mechanism of extracellular electron transfer mainly concentrated on Gram-negative bacteria and rarely on Gram-positive bacteria. Since Gram-positive bacteria are dominant microorganisms in many natural extracellular respiratory environments, it has important scientific significance and application value to search the pure cultured Gram-positive microbes capable of extracellular respiration and research their extracellular respiration capacity and extracellular electron transfer mechanisms.In this study, three Gram-positive bacteria capable of extracellular respiration were enriched, isolated and identified from compost or forest soil samples. Then,anaerobic experiments were conducted to investigate the ability of reducing extracellular substances, including humus, iron oxides and electrode. In addition,electrochemistry experiments, UV/Vis spectra and SDS-PAGE were conducted to compare the content of cytochrome c under different electron acceptor conditionsand to study the possible electron transfer chain of the new isolate. The main conclusions are as follows:1. Two strains of facultative anaerobic bacteria were isolated and designated as Sinibacillus soli GD05 T and Bacillus nitroreducens GSS08 T. A strain of strictly anaerobic bacteria was isolated, identified and designated as Desulfotomaculum ferrireducens GSS09 T.2. The above mentioned strains all had the ability to reduce AQDS by utilizing different electron donors(acetate, glucose and lactate). Strain GD05 T could be capable of Orange I decolorization, and it is not obvious that the reduction of ferrihydrite by strain GSS08 T and strain GD05 T. strain GSS09 T utilized fermentative sugars(glucose)and organic acids as effective electron donors to reduce humic acids and ferrihydrite.GSS09 T also had the ability to grow with fermentation by utilizing pyruvate. As a typical strain of Desulfotomaculum capable of Iron respiration, the ability of reducing iron oxides by GSS09 T was relatively high, therefore we select strain GSS09 T as ideal material to study the mechanism of electron transfer chain.3. The MFC of GSS09 T indicated that there were differences in the ability of harvesting electricity under different electron donors. In addition, GSS09 Tcould form electroactive biofilms by detecting with cyclic voltammetry. The assays of UV/Vis spectra and SDS-PAGE indicated that ther were differences in the content of cellular cytochrome c under different electron acceptors. The content of cellular cytochrome c under Na2SO3-reducin-condition was higher than that of HFO-reducing-condition.In conclusion, it means that electrons may be transferred to the outer surf ace of the cell to reduce extracelluar electron acceptor by the mechanism of m embrance-bouned cytochrome c, rather than conductive bacterial pili or redox mediators.