Extracellular Electron Transfer And Its Enhancement Mechanism Of Kocuria Rosea

Electroactive microorganisms?EAMs?can transfer electrons generated by metabolism from the respiratory chain to extracellular electron receptors or use extracellular electrons for respiratory metabolism,and have broad application prospects in the reduction of heavy metals.EAMs reported currently accounts for only a small part of nature,and most of them are Gram-negative bacteria,resulting in a single extracellular electron transport mechanism.Therefore,to tap the diversity of EAMs and broaden the breadth of extracellular electron transport pathways,it is necessary to focus on Gram-positive bacteria.However,the cell wall of Gram-positive bacteria is thick and dense,which resulting in unclear electrical activity characteristics.In-depth analysis of the extracellular electron transfer pathway is a major challenge for many researchers.How to improve the extracellular electron transfer efficiency of Gram-positive bacteria and tap its potential in practical applications is also a problem at present.In this research,Kocuria rosea was the research object,and electrochemical methods were used to detect its electrical activity and explore its extracellular electron transfer way.We explored ways to enhance the extracellular electron transfer efficiency of Kocuria rosea by researching the changes of the electrochemical characteristics of Kocuria rosea under the action of electronic shuttle and surfactant We revealed the effect of improved electron transfer efficiency on the conversion of uranium by Kocuria rosea through the interaction between Kocuria rosea and uranium The main results were showed as follows:1)Kocuria rosea has electrical activity characteristics proved by Electrochemical results.Cyclic voltammetry curve?CV?and differential pulse voltammetry curve?DPV?results showed two redox reactions appeared in Kocuria rosea suspension:i)Kocuria rosea transfered electrons to the electrode through Cytochrome C?c-Cyt?on the cell walls,directly.ii)The secreted riboflavin?RF?from Kocuria rosea transferred electrons to the electrode,indirectly.Amperometric?i-t?curve indicated that the response current was generated when Kocuria rosea was in the growth process,and the maximum current value could reach at about 4.4 mA.However,compared with other electroactive microorganisms?EAMs?,the current density of Kocuria rosea was too low?0.98A/m²?,which was much lower than Gram-negative bacteria such as Geobacter anodireducens?2.1 A/m²?and Geobacter sulfurreducens KN400?>2.6 A/m²?.2)The mechanisms of extracellular electron transfer in Kocuria rosea was investigated.The results showed that Kocuria rosea could attach to the cathode of the bioelectrochemical system?BES?and form a dense biofilm on carbon paper.The redox characteristics of biofilm were gradually significant with the increase of culture time.It indicated that Kocuria rosea could obtain electrons from the cathode for its own growth and metabolism in a direct contact way.The metabolites of Kocuria rosea also showed redox characteristics.The three-dimensional fluorescence spectroscopy?EEM?showed that fluorescent substances in metabolites may be NADH and RF.NADH and RF,as the members of the respiratory chain in cell,could transmit electrons in the first stage.At the same time,Coenzyme I and CoQ were extracted from the broken bacteria to further speculate the extracellular electron transfer path of Kocuria rosea's as the following sequence:NADH?FMN?Fe-S?CoQ.3)The enhancement effect of electron shuttles,surfactants on the extracellular electron transfer efficiency of Kocuria rosea was researched.The results of BES suggested that the utilization of cathode electrons of Kocuria rosea was promoted and the growth and metabolism of Kocuria rosea were accelerated with addition of RF and c-Cyt.Kocuria rosea showed stronger redox characteristics with the addition of 0.1 mmol/l RF.Compared with the control group?6.76?A and 7.12?A?,the current of reduction peak were increased by 39.1%and 22.6%,respectively.And the current of oxidation peak was 9.15?A,which was 319.7%higher than the control group?2.18?A?.However,0.1 mmol/l c-Cyt showed the opposite effect.The current of reduction peak decreased from 7.50?A to 2.34?A?a decrease of about68.8%?,and the current of oxidation peak decreased from 2.78?A to 2.04?A?a decrease of approximately 26.6%?.It indicated RF could accelerate the extracellular electron transfer of Kocuria rosea,while c-Cyt weakened the electron transfer between the electroactive substances and the electrodes.The EEM results showed the fluorescence intensity of humic acid was increased by 20%compared with the control group through improving the permeability of Kocuria rosea cell membranes by sophorolipids,while there was no significant change in morphology.The enhanced fluorescence intensity of soluble microbial by-products?SMP?were through lysozyme by destroying the cell wall of Kocuria rosea to make the content flow out.And the surface of Kocuria rosea was rough and collapsed.Compared with the control group,when Kocuria rosea was treated with permeability,the electron transfer rate of extracellular substances were increased by 7.3%and 3.58%,respectively.It indicated that the enhanced cell wall and cell membrane permeability could accelerate the electron transfer rate of Kocuria rosea.Cisplatin could effectively inhibit the split of Kocuria rosea and make it elongate.The length of common Kocuria rosea was 0.5-1.0?m,while it showed the maximum elongation efficiency in the LB containing cisplatin?1mg/L?,and the length was in the range of 8-60 um,and the number of bacteria was reduced.Kocuria rosea and their extracellular products treated with cisplatin have stronger electrochemical characteristics.It was inferred that Kocuria rosea was concentrated on the electrode in a spiral or twisted way,which could provide more adhesion space for other bacteria to achieve the maximum rate of electron transfer.4)The effects of enhanced extracellular electron transfer efficiency of Kocuria rosea on the conversion of uranium were investigated.It was found that the conversion efficiency of U?VI?could be improved by Kocuria rosea with addition of electronic shuttles?0.1 mmol/L RF and0.1 mmol/L c-Cyt?.Compared to the reduction rate?49.9%?and the removal rate?19.79%?of U?VI?in control groups,when RF was added to the system,the reduction and the removal rates of U?VI?were increased by 14.84%and 20%,respectively.And the reduction and the removal rates of U?VI?were increased by 2.41%and 10.1%with the addition of c-Cyt,respectively.It showed that RF and c-Cyt could accelerate the electron transfer between Kocuria rosea and U?VI?,resulting in the reduction and the removal rates of U?VI?were enhanced.Besides,the effect of the removal efficiency of U?VI?by Kocuria rosea was weak with addition of surfactants,while the conversion rate of U?VI?was improved,effectively.Compared to the control groups?47.2%?,the removal rate of U?VI?was enhanced by 3.2%and 0.8%when Kocuria rosea was treated with permeability at a concentration of 20 mg/L sophorolipid and 25 mg/L lysozyme,respectively.The reduction rate of U?VI?was increased by 6.45%and 1.35%with the addition of 20 mg/L sophorolipid and 25 mg/L lysozyme compared to the control group?19.9%?,respectively.However,the removal rate of U?VI?was increased by18.84%and 10.57%at 3.5 h compared to control group?13.43 mg/?L h??,respectively.It revealed that the secretion of more redox mediators could be promoted by surfactant,thus enhancing the reduction rate of U?VI?.The product of the interaction between Kocuria rosea and uranium included ammonium phosphate uranium??NH₄?₂?UO₂?₂?PO₄?₂6H₂O?,sodium uranyl phosphate hydrate?NaPUO₆?3H₂O?,sodium uranyl phosphate?Na6?UO₂?₃?PO₄?₄?and uranium-calcium oxide?CaUO₃?phases,morphology,phase structure and other characteristics of product are not affected by the electronic shuttle and surfactant,but the crystallinity was higher than the control group.In summary,this paper proves that Kocuria rosea has electrical activity,but its electricity-generating ability is weaker than Gram-negative bacteria.Kocuria rosea can use electrode electrons to promote their own growth and metabolism,and riboflavin and c-cytochrome mediate the electron transfer process between Kocuria rosea and electrodes.The electron shuttle,surfactant and cisplatin have a good promotion effect on the extracellular electron transfer efficiency of Kocuria rosea,which in turn improves the reduction ability of Kocuria rosea to uranium,but the product characteristics do not change accordingly.This paper provides a theoretical basis for the application of microorganisms in the treatment of heavy metals.