Anodized graphite felt as an efficient cathode for in-situ hydrogen peroxide production and Electro-Fenton degradation of rhodamine B.

This work investigated that the graphite felt anodized by NaOH, NH4HCO3, or H2SO4 aqueous, and then as the cathode materials for in-situ hydrogen peroxide (H2O2) production and its employed for rhodamine B (RhB) degradation via Electro-Fenton (EF) process. At -0.60 V (vs. SCE), after 120 min electrolysis, the H2O2 yield by graphite felt which anodized by 0.2 M H2SO4 achieved up 110.5 mg L-1 in 0.05 M Na2SO4 electrolyte. Compared with the raw graphite felt used for cathode, the H2O2 yield increased by 15.85 times under the same conditions. The results of Raman spectroscopy demonstrated that graphite felt anodized by H2SO4 solution can be achieved the highest defect degree. For the degradation of RhB, the cathode which anodized by H2SO4 solution has the highest removal rate. For the degradation rate of RhB, the effect of applied current density, Fe2+ ions concentration, pH value were investigated. In addition, suggested that the efficient Fe3+ reduction reaction on the cathode surface was an important reason of the high efficiency of RhB degradation. 5-times continuous runs indicated that the modified cathode has remarkable stability and reusability during the EF process.

The Small RNA sr8384 Is a Crucial Regulator of Cell Growth in Solventogenic Clostridia.

Small RNAs (sRNAs) are crucial regulatory molecules in organisms and are well-known not only for their roles in the control of diverse crucial biological processes but also for their value in regulation rewiring. However, to date, in Gram-positive anaerobic solventogenic clostridia (a group of important industrial bacteria with exceptional substrate and product diversity), sRNAs remain minimally explored, and thus there is a lack of detailed understanding regarding these important molecules and their use as targets for genetic improvement. Here, we performed large-scale phenotypic screens of a transposon-mediated mutant library of Clostridium acetobutylicum, a typical solventogenic clostridial species, and discovered a novel sRNA (sr8384) that functions as a crucial regulator of cell growth. Comparative transcriptomic data combined with genetic and biochemical analyses revealed that sr8384 acts as a pleiotropic regulator and controls multiple targets that are associated with crucial biological processes through direct or indirect interactions. Notably, the in vivo expression level of sr8384 determined the cell growth rate, thereby affecting the solvent titer and productivity. These findings indicate the importance of the sr8384-mediated regulatory network in C. acetobutylicum Furthermore, a homolog of sr8384 was discovered and proven to be functional in another important Clostridium species, C. beijerinckii, suggesting the potential broad role of this sRNA in clostridia. Our work showcases a previously unknown potent and complex role of sRNAs in clostridia, providing new opportunities for understanding and engineering these anaerobes.IMPORTANCE The uses of sRNAs as new resources for functional studies and strain modifications are promising strategies in microorganisms. However, these crucial regulatory molecules have hardly been explored in industrially important solventogenic clostridia. Here, we identified sr8384 as a novel determinant sRNA controlling the cell growth of solventogenic Clostridium acetobutylicum Based on a detailed functional analysis, we further reveal the pleiotropic function of sr8384 and its multiple direct and indirect crucial targets, which represents a valuable source for understanding and optimizing this anaerobe. Of note, manipulation of this sRNA achieves improved cell growth and solvent synthesis. Our findings provide a new perspective for future studies on regulatory sRNAs in clostridia.

CRISPR-Cas12a-Mediated Gene Deletion and Regulation in Clostridium ljungdahlii and Its Application in Carbon Flux Redirection in Synthesis Gas Fermentation.

Uncovering the full potential of gas-fermenting Clostridia, attractive autotrophic bacteria capable of using synthesis gases (CO-CO2-H2) to produce a range of chemicals and fuels, for industrial applications relies on having efficient molecular tools for genetic modifications. Although the CRISPR-Cas9-mediated genome editing system has been developed in Clostridia, its use is limited owing to low GC content (approx. 30%) in these anaerobes. Therefore, the effector protein Cas12a, which recognizes T-rich instead of G-rich protospacer-adjacent motifs (PAMs), has evident advantages over Cas9 in CRISPR genome editing in Clostridia. Here, we developed the CRISPR-Cas12a system for efficient gene deletion and regulation in the gas-fermenting Clostridium ljungdahlii species. On the basis of screening for the most suitable Cas12a and significantly improved electrotransformation efficiency that bypassed poor repair efficiency of the Cas12a-caused DNA double-strand break (DSB) in C. ljungdahlii, efficient deletion (80-100%) of four genes (pyrE, pta, adhE1, and ctf) was achieved by using the CRISPR-FnCas12a system. Furthermore, a DNase-deactivated FnCas12a (ddCas12a) was adopted to construct a CRISPRi system to downregulate targeted genes, reaching over 80% repression for most of the chosen binding sites. This CRISPRi system was also used in a butyric acid-producing C. ljungdahlii strain to redirect carbon flux, leading to 20-40% reductions in ethanol titer that were accompanied by increased butyric acid titer. These results demonstrate the high efficiency of the CRISPR-FnCas12a system for genome engineering in C. ljungdahlii, which effectively expands the existing CRISPR-Cas toolbox in gas-fermenting Clostridium species and may play important roles in genetic manipulations where CRISPR-Cas9 is incompetent.

Phage serine integrase-mediated genome engineering for efficient expression of chemical biosynthetic pathway in gas-fermenting Clostridium ljungdahlii.

The real value of gas-fermenting clostridia, capable of using CO and CO2, resides in their potential of being developed into cell factories to produce various bulk chemicals and fuels. This process requires rapid chromosomal integration of heterologous chemical biosynthetic pathways, which is impeded by the absence of genetic tools competent for efficient genome engineering in these anaerobes. Here, we developed a phage serine integrase-mediated site-specific genome engineering technique in Clostridium ljungdahlii, one of the major acetogenic gas-fermenting microbes. Two heterologous phage attachment/integration (Att/Int) systems (from Clostridium difficile and Streptomyces) were introduced into C. ljungdahlii and proven to be highly active, achieving efficient chromosomal integration of a whole donor vector via single-crossover recombination. Based on this, we further realized markerless chromosomal integration of target DNA fragments through a "dual integrase cassette exchange" (DICE) strategy with the assistance of the CRISPR-Cas9 editing system. As a proof of concept, a butyric acid production pathway from Clostridium acetobutylicum was integrated into the C. ljungdahlii genome without the introduction of extra markers, enabling stable expression of the pathway genes. The resulting engineered strain produced 1.01 g/L of butyric acid within 3 days by fermenting synthesis gas (CO2/CO). More importantly, the engineered strain showed good genetic stability and maintained butyric acid production ability after continuous subculturing. The system developed in this study overcomes the deficiencies of currently available genetic tools in the chromosomal integration of large DNA fragments (rapid, markerless and stable) in C. ljungdahlii, and may be extended to other Clostridium species.

Enhanced alcohol titre and ratio in carbon monoxide-rich off-gas fermentation of Clostridium carboxidivorans through combination of trace metals optimization with variable-temperature cultivation.

Bioconversion of C1 gases to produce chemicals has good application prospects. Here, the combination of trace metals optimization using a statistical method with variable-temperature cultivation was used to enhance alcohol synthesis during CO-rich off-gas fermentation by Clostridium carboxidivorans P7. Based on ATCC medium 1754, the optimum concentration of the trace metals was found to be 5-fold Ni2+, Co2+, SeO42+, and WO42+; 3.48-fold Cu2+; 0.55-fold MoO42+; 0.5-fold Zn2+ and (NH4)2SO4·FeSO4·6H2O; and additional 44.32µM FeCl3·6H2O. The production of alcohol and organic acid changed to 4.40g/L and 0.50g/L from 2.16g/L and 2.37g/L, respectively, yielding an increase of alcohol-to-product ratio from 47.7% to 89.8%. By fermenting with the optimized medium and timed control of the incubation temperature (37°C [0-24h]-25°C [24-144h]), the alcohol titre further increased to 6.97g/L with 1.67g/L butanol and 1.33g/L hexanol, exceeding those previously reported for strain P7.

CRISPR/Cas9-Based Efficient Genome Editing in Clostridium ljungdahlii, an Autotrophic Gas-Fermenting Bacterium.

Acetogenic bacteria have the potential to convert single carbon gases (CO and CO2) into a range of bulk chemicals and fuels. Realization of their full potential is being impeded by the absence of effective genetic tools for high throughput genome modification. Here we report the development of a highly efficient CRISPR/Cas9 system for rapid genome editing of Clostridium ljungdahlii, a paradigm for the commercial production of ethanol from synthesis gas. Following the experimental selection of two promoters (Pthl and ParaE) for expression of cas9 and the requisite single guide RNA (sgRNA), the efficiency of system was tested by making precise deletions of four genes, pta, adhE1, ctf and pyrE. Deletion efficiencies were 100%, >75%, 100% and >50%, respectively. The system overcomes the deficiencies of currently available tools (more rapid, no added antibiotic resistance gene, scarless and minimal polar effects) and will find utility in other acetogens, including the pathogen Clostridium difficile.

Development of an inducible transposon system for efficient random mutagenesis in Clostridium acetobutylicum.

Clostridium acetobutylicum is an industrially important Gram-positive organism, which is capable of producing economically important chemicals in the ABE (Acetone, Butanol and Ethanol) fermentation process. Renewed interests in the ABE process necessitate the availability of additional genetics tools to facilitate the derivation of a greater understanding of the underlying metabolic and regulatory control processes in operation through forward genetic strategies. In this study, a xylose inducible, mariner-based, transposon system was developed and shown to allow high-efficient random mutagenesis in the model strain ATCC 824. Of the thiamphenicol resistant colonies obtained, 91.9% were shown to be due to successful transposition of the catP-based mini-transposon element. Phenotypic screening of 200 transposon clones revealed a sporulation-defective clone with an insertion in spo0A, thereby demonstrating that this inducible transposon system can be used for forward genetic studies in C. acetobutylicum.

Complete genome sequence of Clostridium carboxidivorans P7(T), a syngas-fermenting bacterium capable of producing long-chain alcohols.

Clostridium carboxidivorans P7(T) is an anaerobe that can ferment syngas (mainly CO or CO2 and H2) to produce acids (acetic and butyric acid), ethanol and long-chain alcohols (butanol and hexanol). Here, the first complete genome sequence for C. carboxidivorans P7(T) is presented. This anaerobic bacterium harbors a 5,732,880bp circular chromosome and a 19,902bp mega-plasmid with 4951 and 22 coding DNA sequence (CDS), respectively.