Studies On The Anaerobic Sulfur Oxidation Mediated By Chemoautotrophic Sulfur Oxidation Bacteria Halothiobacillus Sp. LS2

Biological sulfur oxidation plays a key role in the natural sulfur cycle,as well as some important contributions to the environment,agriculture and metallurgy,etc.However,researches on the mechanism of sulfur oxidation of bacteria,especially the anaerobic sulfur oxidation,have some limitations.Anaerobic sulfur oxidation generally uses NO₃^-/NO₂^-,Fe³⁺,Mn(IV)as electron acceptors,which drives energy generation through the reaction coupling with denitrification,iron reduction,manganese reduction and other reactions.In addition to these electron acceptors,microorganisms in nature may also use other substances as electron acceptors for anaerobic sulfur oxidation.Halothiobacillus sp.LS2,a chemoautotrophic sulfur-oxidizing bacterium isolated from the Pearl River,contains genes encoding multiple sulfur oxidase lines and complete nitrogenase gene cluster in its genome,and can obtain nitrogen sources through nitrogen fixation under aerobic conditions.At the same time,we found that the bacteria can also use nitrogen or acetylene as the electron acceptors for sulfur oxidation under anaerobic conditions.In this study,Halothiobacillus sp.LS2 was used as the research object to investigate the anaerobic sulfur oxidation reaction of Halothiobacillus sp.LS2 with acetylene or nitrogen as electron acceptors.The sulfur oxidation kinetics and gene expression analysis of Halothiobacillus sp.LS2 were used to compare the difference of sulfur oxidation reaction of LS2.The results of anaerobic sulfur oxidation based on cell growth showed that:1)Strain LS2could grow by fixing nitrogen without adding initial nitrogen source in both aerobic and anaerobic conditions.However,the maximum number of cells obtained under nitrogen fixation condition was 1.98×10⁷cfu/mL in aerobic condition and 1.21×10⁷cfu/mL in anaerobic condition,which was only about 75%of that in the presence of nitrogen source.2)Under anaerobic conditions,the strain LS2 could perform sulfur oxidation with acetylene or nitrogen as electron acceptor.The maximum cell number was 2.05×10⁷cfu/mL when acetylene was used as electron acceptor for sulfur oxidation,and with the concentration of acetylene increasing from 7%to 20%,The maximum cell count increased from 0.96×10⁷cfu/mL to 3.31×10⁷cfu/mL.With nitrogen as the electron acceptor,the maximum cell number was 1.64×10⁷cfu/mL in the presence of nitrogen source and 1.21×10⁷cfu/mL in the absence of nitrogen source.3)In the presence of nitrogenase inhibitors,the bacteria did not grow,indicating that nitrogenase hindered the fixation of nitrogen.Sulfur oxidation kinetics analysis showed that:1)when oxygen was used as the electron acceptor,the maximum reaction rate V_maxwas 17.39?mol/L SO₄^2-h^-1(10⁷cells)^-1,K_mwas 0.05mmol/L S₂O₃^2-;When nitrogen was used as the electron acceptor,the maximum reaction rate V_maxwas 5.71?mol/L SO₄^2-h^-1(10⁷cells)^-1,K_mwas 0.11 mmol/L S₂O₃^2-,indicating that strain LS2 had a higher affinity for substrate under aerobic condition.The maximum sulfur oxidation rate is 3 times that of nitrogen as the electron acceptor.2)The K_mvalue of acetylene as the electron acceptor is the same as that of oxygen as the electron acceptor,but the maximum reaction rate is relatively smaller.3)the affinity of strain LS2 to the substrate of sodium tetrasulfate was greater than that of sodium thiosulfate.Based on gene expression analysis,1)The expression of soxB gene of sulfur oxidase in strain LS2 was increased by 10.3%when the concentration of acetylene was 5%,20%and30%,respectively,compared with that of 20%O₂.The highest expression level was found in the 30%experimental group,which was 31%higher than that in the 5%acetylene group.Therefore,the expression level of sulfur oxidase soxB gene increased with the increase of acetylene concentration,which was higher than that in the aerobic condition,indicating that acetylene may promote the sulfur oxidation reaction of strain LS2.2)Based on RNA sequencing analysis,the expression of soxB gene of sulfur oxidase changed significantly with different culture conditions.The expression level of soxB gene with nitrogen source was higher than that without nitrogen source,indicating that oxygen was more conducive to sulfur oxidation reaction when nitrogen was used as electron acceptor,and the expression level of oxygen was higher than that of anaerobic condition.It shows that nitrogen is more favorable to sulfur oxidation under aerobic condition.3)The expression of key enzyme genes related to nitrogen fixation changed significantly under different culture conditions.Some of the key genes related to energy metabolism,such as Fix complex,Rnf complex and nitrogenase,were significantly upregulated under anaerobic conditions without initial nitrogen source compared with that with initial nitrogen source.The above studies and analysis indicate that strain LS2 can mediate the sulfur oxidation reaction with acetylene or nitrogen as the electron acceptor through the action of nitrogenase under anaerobic conditions,and the energy generated can be used for the growth and metabolism of bacteria,thus proposing a new anaerobic sulfur oxidation pathway and potential electron transport or energy metabolism pathway.