Inactivation of guanylate kinase in Bacillus sp. TL7-3 cultivated under an optimized ratio of carbon and nitrogen sources influenced GTP regeneration capability and sporulation.

Bacillus sp. TL7-3 has potential as a dietary supplement to promote human and animal health. It produces spores that can survive in harsh environments. Thus, when supplemented with nutrients, these spores can withstand the acidic pH of the stomach and resume vegetative development in the gut when exposed to growth-promoting conditions. Spores are formed as a cellular defense mechanism when a culture experiences stress and process optimization to achieve high spore production in a typical batch process remains challenging. Existing literature on the manipulation of gene expression and enzyme activity during batch cultivation is limited. Studies on the growth patterns, morphological changes, and relevant gene expression have aided in enhancing spore production. The present study used the response surface methodology for medium optimization. The model suggested that yeast extract and NH4Cl were significant factors controlling spore production. A comparison between the high weight ratio of carbon and nitrogen (C:N) substrates (8.57:1) in the optimized and basal media (0.52:1) showed an 8.76-fold increase in the final spore concentration. The expression of major genes, including codY, spo0A, kinA, and spo0F, involved in the sporulation was compared when cultivating Bacillus sp. TL7-3 in media with varying C:N ratios. At high C:N ratios, spo0A, kinA, and spo0F were upregulated, whereas codY was downregulated. This led to decreased guanylate kinase activity, resulting in a low guanosine triphosphate concentration and inactivation of CodY, thereby reducing the repression of spo0A and CodY-repressed genes and stimulating sporulation.

Association between organic nitrogen substrates and the optical purity of D-lactic acid during the fermentation by Sporolactobacillus terrae SBT-1.

The development of biotechnological lactic acid production has attracted attention to the potential production of an optically pure isomer of lactic acid, although the relationship between fermentation and the biosynthesis of highly optically pure D-lactic acid remains poorly understood. Sporolactobacillus terrae SBT-1 is an excellent D-lactic acid producer that depends on cultivation conditions. Herein, three enzymes responsible for synthesizing optically pure D-lactic acid, including D-lactate dehydrogenase (D-LDH; encoded by ldhDs), L-lactate dehydrogenase (L-LDH; encoded by ldhLs), and lactate racemase (Lar; encoded by larA), were quantified under different organic nitrogen sources and concentration to study the relationship between fermentation conditions and synthesis pathway of optically pure lactic acid. Different organic nitrogen sources and concentrations significantly affected the quantity and quality of D-lactic acid produced by strain SBT-1 as well as the synthetic optically pure lactic acid pathway. Yeast extract is a preferred organic nitrogen source for achieving high catalytic efficiency of D-lactate dehydrogenase and increasing the transcription level of ldhA2, indicating that this enzyme plays a major role in D-lactic acid formation in S. terrae SBT-1. Furthermore, lactate racemization activity could be regulated by the presence of D-lactic acid. The results of this study suggest that specific nutrient requirements are necessary to achieve a stable and highly productive fermentation process for the D-lactic acid of an individual strain.

The outlooks and key challenges in renewable biomass feedstock utilization for value-added platform chemical via bioprocesses.

The conversion of renewable biomass feedstock into value-added products via bioprocessing platforms has become attractive because of environmental and health concerns. Process performance and cost competitiveness are major factors in the bioprocess design to produce desirable products from biomass feedstock. Proper pretreatment allows delignification and hemicellulose removal from the liquid fraction, allowing cellulose to be readily hydrolyzed to monomeric sugars. Several industrial products are produced via sugar fermentation using either naturally isolated or genetically modified microbes. Microbial platforms play an important role in the synthesis of several products, including drop-in chemicals, as-in products, and novel compounds. The key elements in developing a fermentation platform are medium formulation, sterilization, and active cells for inoculation. Downstream bioproduct recovery may seem like a straightforward chemical process, but is more complex, wherein cost competitiveness versus recovery performance becomes a challenge. This review summarizes the prospects for utilizing renewable biomass for bioprocessing.

Mathematical Modeling for Evaluating Inherent Parameters Affecting UVC Decontamination of Indicator Bacteria.

UV light is a tool associated with the denaturation of cellular components, DNA damage, and cell disruption. UV treatment is widely used in the decontamination process; however, predicting a sufficient UV dose by using traditional methods is doubtful. In this study, an in-house UVC apparatus was designed to investigate the process of the inactivation of five indicator bacteria when the initial cell concentrations and irradiation intensities varied. Both linear and nonlinear mathematical models were applied to predict the inactivation kinetics. In comparison with the Weibull and modified Chick-Watson models, the Chick-Watson model provided a good fit of the experimental data for five bacteria, Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, Streptococcus faecalis, and Bacillus subtilis. The specific death rate (kd) significantly increased when the irradiation intensity (I) increased from 1.41 W/m2 to 3.02 W/m2 and 4.83 W/m2 (P < 0.05). Statistical analysis revealed no significant difference in the kd values among the groups of tested Gram-positive bacteria, Gram-negative bacteria, and B. subtilis spores, but the kd values differed among groups (P < 0.05). The death rate coefficient (k) varied from species to species. The k values of the tested Gram-positive bacteria were higher than those of the Gram-negative bacteria. The thick peptidoglycan layer in the Gram-positive membrane was responsible for UVC resistance. The high guanine-cytosine (GC) content in bacteria also contributed to UV resistance due to the less photoreactive sites on the nucleotides. This investigation provides a good understanding of bacterial inactivation induced by UVC treatment. IMPORTANCE Prevention and control measures for microbial pathogens have attracted worldwide attention due to the recent coronavirus disease 2019 pandemic. UV treatments are used as a commercial control to prevent microbial contamination in diverse applications. Microorganisms exhibit different UV sensitivities, which are often measured by the UV doses required for decreasing the number of microbial contaminants in the logarithmic order. The maximum efficacy of UV is usually observed at 254 nm (residing in the UVC range of the light spectrum). UV technology is a nonthermal physical decontamination measure that does not require any chemicals and consumes low levels of energy while leaving insignificant amounts of chemical residues or toxic compounds. Therefore, obtaining the microbial death kinetics and their intrinsic parameters provided in this study together with the UV photoreaction rate enables advancement in the design of UV treatment systems.

A modified approach for high-quality RNA extraction of spore-forming Bacillus subtilis at varied physiological stages.

BACKGROUND: High quality RNA is required for the molecular study. Sample preparation of the spore-forming, Gram-positive bacteria like Bacillus sp., remains challenging although several methods have been proposed. Those techniques were simply developed using cell samples at certain growth stages despite some molecular studies like transcriptomic analyses require RNA samples from different physiological stages. METHODS AND RESULTS: We developed the rapid, simple yet effective cell-lysis technique with limit use of harsh reagents by modifying the kit-based protocols. Appropriate lysozyme loading (20 mg/mL), incubation time (30 min), and temperature (37 °C) enabled cell lysis and enhanced RNA extraction from both vegetative cells and endospores of Bacillus subtilis TL7-3. High RNA Integrity Numbers and ratios of A260/A280 and A260/A230 of all RNA products collected during the batch cultivation confirmed that invert mixing with absolute ethanol prevented RNA damage during protein denaturation. With the process modification of the major steps in cell lysis and RNA extraction compared with the kit-based protocols that are typically used in laboratory work, interestingly, our modified protocol, simple-yet-effective, yielded higher concentration, purity, and integrity of RNA products from all cell samples collected at different physiological stages. While the kit-based protocols either failed to provide high RNA concentration or RNA purity and integrity for all cell samples particularly during the late-log, stationary, or sporulation. CONCLUSIONS: Therefore, we can claim the significance of this modified protocol to be applicable for RNA extraction to those spore-forming Gram-positive bacteria not limited to B. subtilis growing at varied physiological stages.