| Microbial involvement in iron reduction processes is the main driver of iron in the geochemical cycle,and iron-reducing bacteria are classified into respiratory and fermentative types.The iron reduction mechanisms of the two types of iron-reducing bacteria differ significantly.In this thesis,we used Clostridium sp.LQ25,a fermentative bacterium isolated from marine sediments,as experimental material to analyze the effects of electron acceptors and electron shuttles on their iron-reducing properties.The transcriptome was used to analyze the iron-reducing mechanism of fermentative iron-reducing bacteria.The results of the experiments were as follows:(1)Growth analysis of wild and mutant strains showed that wild strain LQ25 reached a maximum cell density of 1.45 ± 0.08 at the 26 th h of culture,mutant strain C reached a maximum cell density of 1.46 ± 0.03 at the 24 th h of culture,and mutant strain R reached a maximum cell density of 1.35 ± 0.21 at the 26 th h of culture.Correspondingly,wild strain LQ25 had a specific growth rate of 0.083 and a generation time of 7.8 h,mutant strain C had the highest specific growth rate and the shortest generation time of 0.104 and 6.7 h,mutant strain R had the lowest specific growth rate and the longest generation time of 0.073 and 9.5h.Stability experiments of the strains showed that the growth and iron-reducing ability of all strains remained stable without the addition of electron acceptors,with the addition of iron citrate and with the addition of iron hydroxide.(2)The iron reduction properties of the strains were influenced by different electron acceptors.With iron citrate as the electron acceptor,the iron reductase enzyme activities of wild strain LQ25 and mutant strains C and R were 4.72 ± 0.05 μmol/(h·mg),6.95 ± 0.24 μmol/(h·mg),and 3.69 ± 0.05 μmol/(h·mg),respectively.The accumulated Fe(II)concentrations were15.02±0.66 mmol/L,19.09±0.06 mmol/L,and 5.34±0.11 mmol/L,respectively.With iron hydroxide as the electron acceptor,the iron reductase enzyme activities of wild strain LQ25 and mutant strains C and R were 2.41 ± 0.03 μmol/(h·mg),2.77 ± 0.18 μmol/(h·mg),and1.73 ± 0.05 μmol/(h·mg),respectively.The cumulative Fe(II)concentrations were12.22±0.17 mmol/L,16.59±0.05 mmol/L,and 3.90±0.03 mmol/L,respectively.(3)The iron reduction properties of the strain were affected by different electron shuttles.Both exogenous riboflavin and anthraquinone-2-sulfonic acid sodium(AQS)could enhance the iron reduction ability of strain LQ25.The highest cumulative concentrations of Fe(II)were 70.73 ± 0.05 mmol/L and 435.34 ± 0.12 mmol/L when the concentrations of riboflavin and AQS were 100 mg/L and 0.8 mmol/L.The amount of riboflavin and AQS secreted by strain LQ25 showed a trend of weak enhancement and then weakening with time,and the relative fluorescence intensity could reach up to 49.50±3.92 and 51.90±6.04,respectively.the addition of the electron acceptor iron hydroxide also affected the maximum amount of riboflavin and AQS secreted by strain LQ25,and the relative fluorescence intensity could reach up to 56.23±7.93 and 61.20±3.96,respectively.7.93 and 61.20±3.96,respectively.The electron transfer respiration inhibitor experiments showed that FAD dehydrogenase and cytochrome b complex,which constitute components of the respiratory transport chain,were mainly involved in the electron transfer process of strain LQ25 under iron hydroxide conditions.(4)Transcriptome data results showed that 809 genes were significantly differentially expressed up-regulated and 807 down-regulated in pre-culture compared to mid-culture,933 genes were significantly differentially expressed up-regulated and 935 down-regulated in pre-culture compared to post-culture and 271 genes were significantly differentially expressed up-regulated and 162 down-regulated in mid-culture compared to post-culture.There were 164 genes up-regulated and 347 genes down-regulated by adding riboflavin compared with no addition of electron receptor,127 genes up-regulated and 105 genes downregulated by adding AQS compared with no addition of electron receptor and 148 genes upregulated and 63 genes down-regulated by adding AQS compared with adding riboflavin.(5)By comparing the differential genes under different incubation times and different electron shuttles,it is clear that the main functions in the process of iron reduction in strain LQ25 were riboflavin and quinones.The key genes involved in the process of riboflavin biosynthesis were rib 5,rib AB and rib H.The key genes related to quinone synthesis were Q＿GM001531 and Q＿GM001938.(6)RT-PCR experiments showed that the genes rib 5,rib AB,rib H,Q＿GM001531,and Q＿GM001938 were the key genes in the process of iron reduction in strain LQ25.RT-PCR results at different incubation times showed that riboflavin synthesis genes rib 5 and rib AB,were highly expressed in the early stage of culture,and their expression in the early stage of culture was 8.62 and 5.77 times higher than that in the middle stage of culture,respectively.The expression of quinone-related genes Q＿GM001938 and Q＿GM003268 were higher in the middle stage of culture,and their expression in the middle stage of culture were 16.8 and1.32 times higher than that in the middle stage of culture,respectively.The RT-PCR results under different electron shuttles showed that riboflavin synthesis genes rib 5,rib AB,rib H,and quinone-related gene Q＿GM001531 were higher expressed in the condition of added AQS,and the expression of the above genes in the condition of added AQS was 1.59,1.39 times higher than that in the condition of no added electron shuttles,respectively.The expression of the above genes was 1.59,1.39,1.40,and 138.36 times higher under AQS addition than without the addition of electron shuttle,respectively. |
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