Isolation,Identification And Extracellular Electron Transfer Characterization Of Electrochemical Ly Active Microorganisms

Extracellular electron transfer(EET)of electrochemically active microorganisms(EAM)represents a communicative bridge between the interior and exterior of the cells.It promotes the reduction/oxidation of extracellular soluble and solid phase electron acceptor/donor,and plays vital role in biogeochemical cycling of metals and carbon and in biosynthesis of bioenergy.Presently,most of the isolated EAM belong to Proteobacteria and are Gram-negative.Besides,EET of EAM are mainly confined in Gram-negative Shewanella oneidensis MR-1 and Geobacter sulfurreducens,whereas,widely spread Gram-positive bacteria and fungi,that contain dense cellular walls,have rarely been reported.This study aims to elucidate EET mechanism in Gram-positive bacteria and fungi.We firstly isolate and identify EAM from different environmental systems.Then we study the EET of Gram-positive bacteria and fungi that identified to be electrochemically active.Furthermore,we characterize the effect of EET in Gram-positive bacteria and fungi on extracellular reduction of soluble and insoluble electron accepor that can not freely diffuse to cytoplasma space.Main contents and results are summarized as follows:1.Compared to anaerobic anode,micro-aerobic anode captures more energy from microbial fuel cell.However,most of the previous researches were focus on functioning bacteria in anaerobic anode,functioning bacteria in micro-aerobic anode was rarely studied.Herein,we used the traditional aerobic screening technology to isolate functioning bacteria from a micro-aerobic anode.Four pure cultures Bacillus sp.WS-XY1,Aeromonas sp.WS-XY2,Citrobacter sp.WS-XY3 and Bacterium strain WS-XY4 were obtained.WS-XY1 was belonged to Firmicutes,WS-XY2 and WS-XY3 were belonged to Proteobacteria,whereas WS-XY4 was possibly a new species.Cyclic voltammetry and chronoamperometry analysis demonstrated all of them showed electrochemical activity by direct extracellular electron transfer,and micro-aerobic anode could select bacteria that have similar electrochemical activity to proliferate on the anode.We further conclude that functioning bacteria in micro-aerobic anode are more efficiency than that of anaerobic anode may be the reason that micro-aerobic anode has better performance than anaerobic anode.Therefore,a thorough study of functioning bacteria in micro-aerobic anode will significantly promote the energy recovery from microbial fuel cell.2.Cyclic voltammetry and chronoamperometry were employed to characterize electrochemical activity of three yeast Pichia stipitis,Pachysolen tannophilus and Saccharomyces cerevisiae.Results indicated that all of them have electrochemical activity that similar to each other.Besides,cyclic voltammetry was applied to identify the electrochemical activity of three xylan bacteria Geobacillus sp.XB4,Geobacillus sp.XB8 and Bacteria strain XM1 and two cellulose bacteria Bacillus sp.CB1 and Geobacillus sp.CA.Results demonstrated that all of them have redox active substance on their cell surface,whereas no redox active compounds in the medium supernatant.The isolation and identification of these electrochemically active microorganisms provide a wide range of microorganisms for further study of extracellular electron transfer.3.Three model dense cell wall microorganisms(Bacillus sp.WS-XY1 and the yeast Pichia stipitis,Pachysolen tannophilus)were identified to be electrochemically active.Further analysis indicated that the three microorganisms were able to secrete flavins to mediate their EET.These results demonstrated that flavins are ubiquitous mediators for microorganism EET and will play an important role in the application of microorganisms in geochemical mineral cycling and bioelectrochemical systems.4.As fungal biomass increased with the decrease of pH,we studied effect of pH on riboflavin secretion in Pachysolen tannophilus.Results indicated Pachysolen tannophilus secretes riboflavin efficiently at pH 3.In addition,riboflavin enhanced Pachysolen tannophilus reduced extracellular soluble and solid phase electron acceptor,which will help to reduce heavy metal toxity in acid emvironment.5.With the increase of culture time,electrochemical activity of Bacillus sp.WS-XY1 changed drastically.Electrochemical study indicated spore formed by Bacillus sp.WS-XY1 vegetative cell can transfer electron direct to electrode.Furthermore,the spore could catalyze oxygen reduction and still have electrochemical activity after treated for 5 hours at 100?.Spore is a promising cathode catalyst that can regeneration by germination and sporulation subsequently.