Bo Fu

Electron shuttling molecules (ESMs) have been proven to accelerate the electron transfer from the electrode to the electroactive microorganism in microbial electrosynthesis (MES) for higher CO₂ reduction or chemical production rate. However, the microbial electron acceptors of electroactive microorganisms and their responses to different electron shuttling molecules in MES were still unknown. In this study, three kinds of ESMs, e.g., riboflavin (B2), methyl viologen (MV) and neutral red (NR) were applied in the MES for acetate production to explore the mechanism of different ESMs on microbial interactions. The acetate concentrations were 41% and 51% higher than that of the control in B2 and NR addition. The acetogens relative abundances of control, B2, MV and NR were 0.29%, 5.68%, 22.78% and 42.89%, respectively. The microbial function profile of the microbial community on the biocathodes indicated that the performance of acetate production was more closely related to the expression of electron transport. The B2 was coupled with the NADH complex and hydrogenase, while MV and NR were coupled with the Rnf complex to support electron transfer and energy conversion via various electron transfer pathways. The study revealed that the ESMs coupled with different electron transport complexes of microorganisms to achieve electron transfer, resulting in product changes.

Little is known about the growth of formate dehydrogenase-lacking acetogen on formate as sole carbon source. Here, we analyzed formate metabolism in Clostridium bovifaecis strain BXX using different concentrations of formate. The results show that C. bovifaecis converted formate (11.5-96 mM) into acetate with molar ratio of 2.0:1∼2.6:1 by using L-cysteine in the anaerobic medium as electron source according to the stoichiometry of acetogenesis. Genome analysis of C. bovifaecis revealed genes encoding anaerobic ribonucleoside triphosphate reductase ( nrdD and nrdG ) catalyzing the oxidation of formate to CO ₂ while ATP is being reduced to the desoxy form. The existence of nrdD was verified by PCR, reverse transcription-PCR analysis and acetogenesis from formate. The process mode of acetogenesis from formate in C. bovifaecis provides insight into the unique metabolic feature of an FDH-lacking acetogen. IMPORTANCE: Wood-Ljungdahl pathway (WLP) lacking formate dehydrogenase (FDH) which catalyzes CO ₂ reduction to formate has been reported to occur acetogenesis only in the presence of formate and exogenous CO ₂ , which seems to result from the formate-rich habitats adaptation of gastrointestinal acetogens. Here, we found FDH-lacking Clostridium bovifaecis strain BXX converted formate (11.5-96 mM) into acetate with molar ratio of 2.0:1∼2.6:1 fitting the stoichiometry of acetogenesis when using formate as the sole carbon source. CO ₂ needed in the carbonyl branch of WLP was from the oxidation of formate to CO ₂ catalyzed by anaerobic ribonucleoside triphosphate reductase while ATP is being reduced to the desoxy form. L-cysteine in the anaerobic medium was the electron source of WLP. The process mode of acetogenesis from formate in C. bovifaecis provides insight into how an FDH-lacking acetogen can make a living from the simplest resources as carbon source, which has both ecological and biotechnological significance.

Acetogens are anaerobes using Wood–Ljungdahl pathway (WLP) as the terminal electron acceptor for both assimilation and dissimilation of CO ₂ and widely distributed in diverse habitats. However, their habitat adaptation is often unclear. Given that bacterial genome evolution is often the result of environmental selective pressure, hereby we analysed gene copy number, phylogeny and selective pressure of genes involved in WLP within known genomes of 43 species to study the habitat adaption of gastrointestinal acetogens. The gene copy number of formate dehydrogenase (FDH) in gastrointestinal acetogens was much lower than that of non‐gastrointestinal acetogens, and in five cases, no FDH genes were found in the genomes of five gastrointestinal acetogens, but that of the other WLP genes showed no difference. The evolutionary pattern of FDH genes was significantly different from that of the other enzymes. Additionally, seven positively selected sites were only identified in the fdhF genes, which means fdhF mutations favoured their adaptation. Collectively, reduction or loss of FDH genes and their evolutionary pattern as well as positive selection in gastrointestinal acetogens indicated their adaptation to formate‐rich habitats, implying that FDH genes catalysing CO ₂ reduction to formate as the first step of methyl branch of WLP may have evolved independently.

The acetyl-CoA pathway is an ancient pathway of CO ₂ fixation, which converts 2 mol of CO ₂ into acetyl-CoA. Autotrophic growth with H ₂ and CO ₂ via the acetyl-CoA pathway as the terminal electron accepting process is the most unique feature of acetogenic bacteria. However, the fecal acetogen Clostridium bovifaecis strain BXX displayed poor metabolic capabilities of autotrophic acetogenesis, and acetogenic utilization of glucose occurred only with the supplementation of formate. The formate-dependent acetogenic growth of Clostridium bovifaecis was associated with its lack of a gene encoding formate dehydrogenase, which may result from adaption to a formate-rich intestinal environment. This study gave insight into a unique metabolic feature of fecal acetogens. Because of the requirement of formate for the acetogenic growth of certain acetogens, the ecological impact of acetogens could be more complex and important in the formate-rich environment due to their trophic interactions with other microbes.

Self-forming dynamic membrane was used to separate high-concentration sludge fermentation liquid, and the formation mechanism and separation efficiency of dynamic membrane were investigated. The results indicated that the impact of sludge concentrations was negligible on the formation of dynamic membrane. Though membrane flux could be influenced by sludge concentration at the initial stage of membrane formation process, the influence was not obvious at the stable stage. Membrane flux was improved with increasing filter cloth pore size and stirring speeds. Moreover, the results indicated that the formation process of dynamic membrane followed the dead-end filtration model, which could be divided into four stages. Firstly, filter cloth pore was blocked by those sludge particles with the diameter similar to the pore size of filter cloth. Secondly, sludge particles formed monolayer sludge on the filter cloth. Thirdly, sludge particles formed multilayer sludge on the filter cloth. Finally, large sludge particles deposited onto the sludge layer. After formation of the dynamic membrane, the retention efficiency of sludge particles and SCOD could reach 98% and 28% , respectively, and the permeation efficiency of VFAs was over 82%. Proteins in EPS were the main component of the dynamic membrane.

Syngas fermentation into ethanol and other bioproducts by mixed cultures is considered a promising biotechnology. Effects of pH on product generation and microbial community during H₂/CO₂ utilization by acetogen enrichment cultures were investigated in this work. The maximum acetate concentration reached 95.41 mmol L⁻¹ at pH 7, which was 71.7, 21.8 and 50.9 % higher than at pH 5, 9 and 11, respectively. The maximum ethanol concentration at pH 7 was 45.7, 50, 72 % higher than that at pH 5, 9 and 11, respectively. The CO dehydrogenase (CODH) gene copy number was highest at pH 7, indicating that metabolically active acetogens reached their highest level at pH 7. The CODH gene copy number at pH 9 was lower than at pH 7, but higher than at pH 5 and 11. Correspondingly, the enrichment cultures at pH 7 had the highest species richness and diversity, while those at pH 9 had the second highest diversity, and those at pH 5 and 11 had the lowest diversity. The shift in microbial community structure and the different active acetogen contents resulting from different pHs were responsible for the differences in acetate and ethanol production.

Homoacetogens are a group of microorganisms with application potential to produce chemicals and biofuels by the bioconversion of synthesis gas. In this study, we collected waste activated sludge samples to screen homoacetogens by Hungate anaerobic technique, and studied the effect of pH on acetate and alcohol production from H2/CO2 gas. The mixed culture contained Clostridium ljungdahlii, Lysinibacillus fusiformis and Bacillus cereus. Acetate concentration achieved 31.69 mmol/L when the initial pH was 7. The mixed culture containing homoacetogen could converting H2/CO2 to acetate, which provides an efficient microbial resource for the bioconversion of synthesis gas.