Isolation, Identification And Electricity Production Capacity Of A Strain Exoelectricigen CL-1 From Soil

For electrochemical active biofilm(EAB)formation,the soil was sampled from a rice paddy field and was inoculated into a half-cell which was carried out on a 16-channel potentiostat using a three-electrode configuration.An exoelectrogenics CL-1 was successfully isolated from enriched EAB by repeated electrochemical selection,enrichment,acclimation and isolation.Comparison with sequences in GenBank suggested that the strain CL-1 was most closely related to Geobacter sulfurreducens subsp.ethanolicus OSK2A,with 98.5%sequence similarity.CL-1 was Gram-negative bacterium with white,small and smooth colonies on agar plate while pink in liquid medium.Scanning electron microscopy(SEM)and confocal laser scanning microscopy(CLSM)were used to image the biofilm attached on electrode and the results showed that CL-1 was baculiform and the thickness of the biofilm was around 30μm.To optimize the growth condition and electricity generation of CL-1,growth curves,the optimum growth temperature and pH,salinity tolerance range,the utilization of different carbon source and other relevant physiological and biochemical properties were measured and tested.The results showed that strain CL-1 was a strictly anaerobic bacterium with 37°C as the optimum growth temperature,8.0 as the optimum growth pH,sodium acetate and ethanol as its carbon sources,and could survive in 1%NaCl solution.Moreover,the electricity generation capacity of strain CL-1from sodium acetate or ethanol was assessed by half-cells and microbial fuel cells(MFCs).In half-cells,the maximum current densityachieved from sodium acetate and ethanol were 900μA/cm~2 and750μA/cm~2,respectively.The biomass of the biofilm(at the maximum current density)was also measured and the result showed that the biomass of the biofilmusing ethanolas substrate was larger than that using sodium acetate,which was contrary to the electricity production.Furthermore,the output voltages of MFCs were continuously monitored using Keithley instrument and polarization data were collected by varying the external resistance using a resistance decade box during the stable power production stage of each batch experiment.The maximum power output,maximum current density and internal resistance of two-chamber MFC using CL-1 from acetate acid and ethanol were 800 mW/m~2,2 A/m~2,500Ωand 350mW/m~2,0.78 A/m~2,700Ω,respectively.Therefore,the optimum substrate of CL-1,whose current density and power output were higher than that of the reported model bacteria,G.sulfurreducens,was acetate acid.In order to investigate the electron transfer mechanism of strain CL-1 between biofilm and electrode surface,the three-electrode system with ITO electrode was adopted to expose the specific redox cytochrome c changing in cell out membrane.The spectrum curves were collected with the applied potentials decreasing from 0.3 V to-0.6 V when it showed the maximum current density.For the spectral absorption peaks at different potential positions,the cytochrome c usually detected at the potential range from-0.3 V to-0.6 V.This research put forward a feasible method on seeking pure microbes with high electrochemical activity andeffective enrichment of electrochemically active microbes.It might provide more prospects on power density output of MFC and exploration of electron transfer mechanism for exoelectrogenics.