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BackgroundCorn stover (CS) is evaluated as the most favorable candidate feedstock for butanol production via microbial acetone–butanol–ethanol (ABE) fermentation by Clostridium acetobutylicum. By independent acid pretreatment and enzymatic hydrolysis, fermentable sugars (mainly glucose and xylose) were released, of which glucose was naturally utilized as the most preferred carbon source by C. acetobutylicum. However, the ABE fermentation using corn stover hydrolysate (CSH) without detoxification is typically limited to poor sugars utilization, butanol production and productivity. In the presence of pretreatment-derived inhibitors, the intracellular ATP and NADH, as important factors involved in cell growth, solventogenesis initiation and stress response, are exceedingly challenged owing to disrupted glucose phosphotransferase system (PTS). Therefore, there is a necessity to develop effective engineering approaches to overcome these limitations for high-efficient butanol production from CSH without detoxification.ResultsPTS-engineered C. acetobutylicum strains were constructed via overexpression and knockout of gene glcG encoding glucose-specific PTS IICBA, which pleiotropically regulated glucose utilization, cell growth, solventogenesis and inhibitors tolerance. The PTSGlcG-overexpressing strain exhibited high fermentation efficiency, wherein butanol production and productivity was 11.1 g/L and 0.31 g/L/h, compared to those of 11.0 g/L and 0.15 g/L/h with the PTSGlcG-deficient strain. During CSH culture without detoxification, the PTSGlcG-overexpressing strain exhibited desirable inhibitors tolerance and solventogenesis with butanol production of 10.0 g/L, increased by 300% and 400% compared to those of 2.5 and 2.0 g/L with the control and PTSGlcG-deficient strains, respectively. As a result of extra glucose and 10 g/L CaCO3 addition into CSH, butanol production and productivity were further maximized to 12.5 g/L and 0.39 g/L/h. These validated improvements on the PTSGlcG-overexpressing strain were ascribed to not only efficient glucose transport but also its cascading effects on intracellular ATP and NADH generation, solventogenesis initiation and inhibitors tolerance at the exponential growth phase.ConclusionThe PTSGluG regulation could be an effective engineering approach for high-efficient ABE fermentation from lignocellulosic hydrolysates without detoxification or wastewater generation, providing fundamental information for economically sustainable butanol production with high productivity.

Serine/threonine protein kinases (STKs) are important for signal transduction and involved in multiple physiological processes, including cell growth, central metabolism, and sporulation in bacteria. However, the role of STKs in solventogenic clostridia remains unclear. Here, we identified and comprehensively investigated six STK candidates in Clostridium beijerinckii . These STKs were classified into four groups with distinct characteristics via analysis of genetic organizations, prediction of protein domains, and multiple sequence alignment. Cbei0566 is a member of the PrkA family with 41% identity to PrkA from Bacillus subtilis , and both Cbei0666 and Cbei0813 are two-component-like STKs. Cbei1151 and Cbei1929 belong to the Hanks family STKs and consist of a cytoplasmic catalytic domain, a transmembrane region, and extracellular sensor domains. In-frame deletion mutants of cbei0566 , cbei0666 , cbei1929 , and cbei2661 displayed similar cell growth with wild type. Both Δ cbei0666 and Δ cbei2661 improved acetone-butanol-ethanol (ABE) production by 14.3% (19.2 g/L vs. 16.8 g/L), and the sporulation frequencies of Δ cbei0566 , Δ cbei1929 , and Δ cbei2661 significantly decreased to 35.5%, 55.1% and 44.8%, respectively. The restored phenotypes after genetic complementation demonstrated their direct link to STKs deletion. Remarkably, overexpressing cbei0566 contributed to 41.5% more spore formation and cbei1929 overexpression enhanced ABE production from 19.3 to 24.2 g/L, along with 25% less acids. These results revealed that Cbei0566 and Cbei1929 had prominent regulatory functions. This study expands the current knowledge of the existence and functions of STKs in prokaryotes and highlights the importance of STK-mediated signaling networks in developing superior strains. Key points • First reported serine/threonine protein kinases in solventogenic clostridia • Six STKs with distinct properties possessed diverse functions in C. beijerinckii • Cbei1929 and Cbei0566 remarkably regulated solventogenesis and sporulation

Biobutanol outperforms bioethanol as an advanced biofuel, but is not economically competitive in terms of its titer, yield and productivity associated with feedstocks and energy cost. In this work, the synergistic effect of calcium and zinc was investigated in the acetone-butanol-ethanol (ABE) fermentation by Clostridium acetobutylicum using glucose, xylose and glucose/xylose mixtures as carbon source(s). Significant improvements associated with enhanced glucose/xylose utilization, cell growth, acids re-assimilation and butanol biosynthesis were achieved. Especially, the maximum butanol and ABE production of 16.1 and 25.9 g L−1 were achieved from 69.3 g L−1 glucose with butanol/ABE productivities of 0.40 and 0.65 g L−1 h−1 compared to those of 11.7 and 19.4 g/L with 0.18 and 0.30 g L−1 h−1 obtained in the control respectively without any supplement. More importantly, zinc was significantly involved in the butanol tolerance based on the improved xylose utilization under various butanol-shock conditions (2, 4, 6, 8 and 10 g L−1 butanol). Under the same conditions, calcium and zinc co-supplementation led to the best xylose utilization and butanol production. These results suggested that calcium and zinc could play synergistic roles improving ABE fermentation by C. acetobutylicum. Calcium and zinc play synergistic roles in glucose/xylose utilization and butanol tolerance of Clostridium acetobutylicum.

Cellulase serves as a pivotal enzymatic catalyst in the bioconversion of lignocellulosic biomass into fermentable sugars and subsequent bioproducts. However, its high cost poses a significant challenge, making the overall lignocellulosic biorefinery process economically burdensome. To mitigate this issue and enhance cost-effectiveness, it becomes imperative to employ in-house built and highly potent cellulases. This study focused on effective cellulase production by Trichoderma reesei RUT C30 and PB3 strains using solid and liquid inducers mixture with varying ratios. By optimizing the SLI mixture at a ratio of 1:5 (w/w), we successfully attained maximum cellulase production and productivity, reaching 29.5 FPU/mL and 204.8 FPU/L/h, respectively. Furthermore, this strategy yielded comparatively elevated levels of cellobiase and xylanase production, achieving 69.6 CBU/mL and 250.1 IU/mL, respectively, in contrast to the control process utilizing either solid or liquid inducers. Ultimately, hydrolysis of 10% (w/v) H 2 SO 4 -pretreated corn stover (HPCS) utilizing a crude enzyme dosage of 15 FPU/g-HPCS liberated 45.5 g/L of glucose and 18.2 g/L of xylose, underscoring the immense potential of this methodology in alleviating the cost burden associated with lignocellulosic biorefinery.

The results demonstrated that among the total 365 investigated samples, the proportion of samples detecting three target herbicides simultaneously, two herbicides, one herbicide, and none were 0.3%, 6.4%, 65.2%, and 23.2%, respectively. For samples with only one detected herbicide, the detection rates in Jiyuan, Luohe, Puyang, Luoyang, Xuchang, and Hebi were relatively high (ranging from 44% to 100%), whereas those in Sanmenxia, Nanyang, Xinxiang, and Kaifeng were relatively low (ranging from 6% to 20%). Regarding individual herbicides, the detection rates of mesosulfuron-methyl, quinclorac, halosulfuron-methyl, diflufenican, imazethapyr, pyroxasulfone, imazapic, fomesafen, and atrazine were 1.2%, 3.8%, 5.5%, 6.1%, 8.7%, 8.7%, 10.4%, 11.0%, and 33.9%, respectively. Based on these findings, the current reliance on long-acting effect herbicides for weed management in China’s agricultural management practices was systematically analyzed. Within the framework of agricultural sustainability, it is proposed that there is an urgent need to promote the concept of scientific herbicide use among farmers and that pesticide scientists must recognize the extreme importance of continuous innovation and the development of alternative herbicides with new mechanisms of action as a long-term strategic goal.