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.

Isolation, screening, and characterization of the newly isolated osmotolerant yeast Wickerhamomyces anomalus BKK11-4 for the coproduction of glycerol and arabitol.

This study explored the isolation and screening of an osmotolerant yeast, Wickerhamomyces anomalus BKK11-4, which is proficient in utilizing renewable feedstocks for sugar alcohol production. In batch fermentation with high initial glucose concentrations, W. anomalus BKK11-4 exhibited notable production of glycerol and arabitol. The results of the medium optimization experiments revealed that trace elements, such as H3BO3, CuSO4, FeCl3, MnSO4, KI, H4MoNa2O4, and ZnSO4, did not increase glucose consumption or sugar alcohol production but substantially increased cell biomass. Osmotic stress, which was manipulated by varying initial glucose concentrations, influenced metabolic outcomes. Elevated glucose levels promoted glycerol and arabitol production while decreasing citric acid production. Agitation rates significantly impacted the kinetics, enhancing glucose utilization and metabolite production rates, particularly for glycerol, arabitol, and citric acid. The operational pH dictated the distribution of the end metabolites, with glycerol production slightly reduced at pH 6, while arabitol production remained unaffected. Citric acid production was observed at pH 6 and 7, and acetic acid production was observed at pH 7. Metabolomic analysis using GC/MS identified 29 metabolites, emphasizing the abundance of sugar/sugar alcohols. Heatmaps were generated to depict the variations in metabolite levels under different osmotic stress conditions, highlighting the intricate metabolic dynamics occurring post-glucose uptake, affecting pathways such as the pentose phosphate pathway and glycerolipid metabolism. These insights contribute to the optimization of W. anomalus BKK11-4 as a whole-cell factory for desirable products, demonstrating its potential applicability in sustainable sugar alcohol production from renewable feedstocks.

Sporolactobacillus mangiferae sp. nov., a spore-forming lactic acid bacterium isolated from tree bark in Thailand.

A Gram-stain-positive, facultatively anaerobic and endospore-forming rod-shaped bacterium, designed strain CPB3-1T, was isolated from tree bark. This homofermentative strain produced dl-lactic acid from glucose. It grew at 20-45 °C, pH 4.0-9.5 and in 0-3.0 % (w/v) NaCl. It contained meso-diaminopimelic acid in cell-wall peptidoglycan and had menaquinone with seven isoprene units (MK-7) as the predominant component. The major fatty acid was anteiso-C17 : 0. The polar lipids were phosphatidylglycerol, diphosphatidylglycerol, an unknown phospholipid and an unknown lipid. Based on the results of 16S rRNA gene sequence analysis, strain CPB3-1T belonged to the genus Sporolactobacillus and was closely related to Sporolactobacillus kofuensis DSM 11701T and Sporolactobacillus spathodeae BK117-1T (both 96.7 % similarity), Sporolactobacillus inulinus NRIC 1133T and Sporolactobacillus terrae DSM 11697T (both 96.6 % similarity), and Sporolactobacillus shoreicorticis MK21-7T, Sporolactobacillus laevolacticus DSM 442T, Sporolactobacillus shoreae BK92T and Sporolactobacillus pectinivorans GD201205T (all 95.8-96.5 % similarity). The draft genome of strain CPB3-1T contained 2 930 919 bps with 3117 coding genes. The DNA G+C content was 45.1 mol%. The digital DNA-DNA hybridization values between strain CPB3-1T and closely related type strains were 19.2-24.0 %. The average nucleotide identity (84.0-87.6 %) and average amino acid identity (66.5-76.3 %) values were lower than the cut-off values for species delineation. Strain CPB3-1T was clearly distinguished from related Sporolactobacillus species based on its phenotypic and chemotaxonomic characteristics, 16S rRNA gene sequence similarity and the results of draft genome analysis. Therefore, the strain represents a novel species of the genus Sporolactobacillus, for which the name Sporolactobacillus mangiferae sp. nov. is proposed. The type strain is CPB3-1T (=JCM 35082T=TISTR 10004T).

Self-enhancement lateral flow immunoassay for COVID-19 diagnosis.

Equipment-free colorimetric-based lateral flow immunoassay (LFIA) is the most convenient and popular tool for various applications, including diagnostic tools requiring high sensitivity for the detection of pathogens. Thus, improvements and developments of LFIA are constantly being reported. Herein, we enriched the sensitivity of LFIA using the gold enhancement principle, emphasizing needlessly complicated apparatus, only one step for the strip test operation, and typical time incubation (15 min) process. Self-enhanced LFIA was then executed for subsequent flows by overlapping the additionally enhanced pad composed of gold ions and reducing agent on the conjugate pad and the sample pad. Self-enhanced LFIA was performed to detect SARS-CoV-2 antigens in saliva. The obtained result depicted that the achieved sensitivity was up to tenfold compared with that of conventional LFIA by visual measurements. The detection limits of self-enhanced LFIA detecting nucleocapsid protein antigens in the saliva sample was 0.50 and 0.10 ng/mL employed by naked eye detection and calibration curve-based calculation, respectively. When the proposed device was applied to 207 human saliva samples, the diagnostic performance presented a 96.10 % sensitivity and 99.23 % specificity. This self-enhanced LFIA could be implemented in large-scale production and demonstrates higher sensitivity with effortless use, which meets the requirements for point-of-care testing and on-field mass screening.

Enhanced biovalorization of palm biomass wastes as biodiesel feedstocks through integrated solid-state and submerged fermentations by fungal co-cultures.

Palm empty fruit bunches (EFB) were valorized into fungal lipids by oleaginous fungus Aspergillus tubingensis TSIP9 under solid-state fermentation (SSF) and submerged fermentation (SmF). An integrated SSF-SmF process increased lipid production from 116.2 ± 0.1 mg/g-EFB under SSF and 60.1 ± 0.2 under SmF up to 124.9 ± 0.5 mg/g-EFB, possibly due to the combined benefits of dispersed mycelia forming during SSF and better mass transfer during SmF. As A. tubingensis lacks sufficient ß-glucosidase, it was co-cultured with high ß-glucosidase-producing Trichoderma reesei QM 9414. The co-cultures improved overall lipid yields likely due to synergistic interaction of the two fungi. After inoculum size was optimized and the co-cultures were performed in bioreactors, the lipid yield was increased up to 205.1 ± 1.1 mg/g-EFB. The fatty acid composition of fungal lipids indicated their potential use as biodiesel feedstocks. The fungal fermentation of EFB also provided cellulose pulp residues. These strategies could be practical options for low-cost biovalorization of biomass wastes.

Weizmannia acidilactici sp. nov., a lactic acid producing bacterium isolated from soils.

The strains designed PP-18T, JC-4 and JC-7 isolated from soils, were Gram-stain-positive rods, facultative anaerobe, endospore-forming bacteria. The strains produced l-lactic acid from glucose. They showed positive for catalase but negative for oxidase, nitrate reduction and arginine hydrolysis. Strains P-18T, JC-4 and JC-7 were closely related to Weizmannia coagulans LMG 6326T (97.27-97.64%) and W. acidiproducens KCTC 13078T (96.46-96.74%) based on 16S rRNA gene sequence similarity, respectively. They contained meso-diaminopimelic acid in cell wall peptidoglycan and had seven isoprene units (MK-7) as the predominant menaquinone. The major cellular fatty acids of strain PP-18T were iso-C15:0, anteiso-C17:0, iso-C16:0 and anteiso-C15:0. The ANIb and ANIm values among the genomes of strains PP-18T, JC-4 and JC-7 are above 99.4% while their ANIb and ANIm values among them and W. coagulans LMG 6326T and W. acidiproducens KCTC 13078T were ranged from 76.61 to 79.59%. These 3 strains showed the digital DNA-DNA hybridization (dDDH) values of 20.7-23.6% when compared with W. coagulans LMG 6326T and W. acidiproducens DSM 23148T. The DNA G + C contents of strains PP-18T, JC-4 and JC-7 were 45.82%, 45.86% and 45.86%, respectively. The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol and phosphoglycolipids. The results of phenotypic and chemotaxonomic characteristics and whole-genome analysis indicated that the strains PP-18T, JC-4 and JC-7 should be represented as a novel species within the genus Weizmannia for which the name Weizmannia acidilactici sp. nov. is proposed. The type strain is PP-18T (=KCTC 33974T = NBRC 113028T = TISTR 2515T).

Metal ion removal using a low-cost coconut shell activated carbon bioadsorbent in the recovery of lactic acid from the fermentation broth.

Downstream recovery and purification of lactic acid from the fermentation broth using locally available, low-cost materials derived from agricultural residues was demonstrated herein. Surface modification of coconut shell activated carbon (CSAC) was performed by grafting with carboxymethyl cellulose (CMC) using citric acid (CA) as the crosslinking agent. A proper ratio of CMC and CA to CSAC and grafting time improved the surface functionalization of grafted nanostructured CMC-CSAC while the specific surface area and porosity remained unchanged. Lactic acid was partially purified (78%) with the recovery percentage of lactic acid at 96% in single-stage adsorption at room temperature and pH 6 with a 10:1 ratio of cell-free broth to CMC-CSAC bioadsorbent. A thermodynamic study revealed that the adsorption was exothermic and non-spontaneous while the Langmuir isotherm model explained the adsorption phenomena. The results in this study represented the potential of waste utilization as solid adsorbents in green and low-cost adsorption technology.

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.

Draft genome sequencing of Sporolactobacillus terrae SBT-1, an efficient bacterium to ferment concentrated sugar to D-lactic acid.

Recently, the industrial-scale development of microbial D-lactic acid production has been discussed. In this study, the efficiency of the new isolate Sporolactobacillus terrae SBT-1 for producing D-lactic acid under challenge conditions was investigated. The isolate SBT-1 exhibited superior activity in fermenting a very high glucose or sucrose concentration to D-lactic acid compared to the other S. terrae isolates previously reported in the literature; therefore, SBT-1 could overcome the limitations of effective lactic acid production. In batch cultivation using 360 g/L glucose, SBT-1 produced 290.30 g/L D-lactate with a sufficiently high glucose conversion yield of 86%, volumetric productivity of 3.02 g/L h, and optical purity of 96.80% enantiomer excess. SBT-1 could also effectively utilize 440 g/L sucrose as a sole carbon source to produce 276.50 g/L lactic acid with a conversion yield of 90%, a production rate of 2.88 g/L h, and an optical purity of 98%. D-Lactic acid fermentation by two other related producers, S. inulinus NRIC1133T and S. terrae NRIC0357T, was compared with fermentation by isolate SBT-1. The experimental data revealed that SBT-1 possessed the ability to ferment relatively high glucose or sucrose concentrations to D-lactic acid without obvious catabolite repression and byproduct formation compared to the two reference strains. In draft genome sequencing of S. terrae SBT-1, the results provided here can promote further study to overcome the current limitations for the industrial-scale production of D-lactic acid.

Terrilactibacillus tamarindi sp. nov., isolated from bark of Tamarindus indica.

A Gram-stain-positive, catalase-positive, facultatively anaerobic, terminal-spore-forming rod, designated strain BCM23-1T, was isolated from bark of Tamarindus indica collected from Chiang Mai Province, Thailand. This strain produced d-lactic acid from glucose. It grew at 20-45 °C (optimum, 30 °C), pH 3.5-9 (optimum, pH 7.0) and in the presence of 1-4 % (w/v) NaCl. The cell-wall peptidoglycan contained meso-diaminopimelic acid (A1γ). The major isoprenoid quinone was menaquinone 7 (MK-7). Polar lipids analysis revealed the presence of diphosphatidylglycerol, phosphatidylglycerol, an unidentified aminophospholipid, an unidentified phospholipid and an unidentified lipid. The predominant cellular fatty acids were anteiso-C17 : 0, anteiso-C15:0, and iso-C16 : 0 when cultivated on GYP agar plates. The 16S rRNA gene sequence similarity between strain BCM23-1T and Terrilactibacillus laevilacticus NK26-11T was 98.3 %. The draft genome of BCM23-1T was 3.24 Mb in size and contained 3088 coding sequences with an in silico DNA G+C content of 37.1 mol%. The values of ANIb, ANIm and digital DNA-DNA hybridization between strain BCM23-1T and T. laevilacticus NK26-11T were 89.9, 90.8 and 40.4 %, respectively. The results of phenotypic and chemotaxonomic, 16S rRNA gene sequence similarity, and whole genome analyses support strain BCM23-1T as representing a novel species of Terrilactibacillus for which the name Terrilactibacillus tamarindi sp. nov. is proposed. The type strain is BCM23-1T (=LMG 31662T=JCM 33748T=TISTR 2841T).

Free Radical Scavenging Properties and Induction of Apoptotic Effects of Fa Fraction Obtained after Proteolysis of Bioactive Peptides from Microalgae Synechococcus sp. VDW.

This study aims to determine the antioxidant activity of bioactive peptides derived from Synechococcus sp. VDW cells cultured for 21 days. Synechococcus sp. VDW protein hydrolysates were prepared with trypsin and purified by ultrafiltration with molecular mass cut-off membranes of 10, 5 and 3 kDa. The M<3 kDa (FA) fraction had the highest 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) and 2,2'-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activities, with IC50 values of (11.5±0.3) and (13.6±0.2) µg/mL, respectively. The FA fraction was separated by reversed phase HPLC to yield four subfractions (F1-4). The F4 subfraction showed the highest maximum ABTS radical scavenging activity (3.55±0.61) % and it was selected for further analysis by electrospray ionisation quadrupole time-of-flight mass spectrometry (ESI-Q-TOF-MS/MS) based on de novo peptide sequencing. Five antioxidant peptides were identified, of which AILESYSAGKTK had the highest ABTS radical scavenging activity. Furthermore, the FA fraction showed high cytotoxic activities against human cancer-derived cell lines, especially the colon cancer cell line (SW620) with an IC50 value of (106.6±21.5) µg/mL, but not the untransformed Wi38 cell line. The FA fraction activated the apoptotic pathway in SW620 cells after treatment for 24, 48 and 72 h, with the highest activities of caspases-3, -8 and -9 being observed after treatment for 72 h. These findings suggested that microalgae Synechococcus sp. VDW may be used to develop natural anticancer drugs.

Optimization of Synechococcus sp. VDW Cultivation with Artificially Prepared Shrimp Wastewater for Ammonium Removal and Its Potential for Use As a Biofuel Feedstock.

To investigate the potential of application of marine cyanobacterium for concurrent biomass production and ammonium removal, Synechococcus sp. VDW was cultured under different conditions in medium containing varying concentrations of NH4Cl. Response surface methodology (RSM) was then used to build a predictive model of the combined effects of independent variables (pH, inoculum size, ammonium concentration). At the optimum conditions of initial pH 7.4, inoculum size 0.17 (OD730) and ammonium concentration 10.5 mg L-1, the maximum ammonium removal and biomass productivity were about 95% and 34 mg L-1d-1, respectively, after seven days of cultivation. The result of fatty acid methyl ester (FAME) analysis showed that the major fatty acids were palmitic acid (C16:0), linoleic acid (C18:2 n6 cis), palmitoleic acid (C16:1) and oleic acid (C18:1 n9 cis), which accounted for more than 80% weight of total fatty acids. Further, analysis of neutral lipid accumulation using flow cytometry revealed that the mean of the fluorescence intensity increased under optimal conditions. These results indicate that Synechococcus sp. VDW has the potential for use for concurrent water treatment and production of biomass that can be applied as biofuel feedstock.

A homofermentative Bacillus sp. BC-001 and its performance as a potential L-lactate industrial strain.

Bacillus sp. BC-001 was first reported as a potent thermotolerant and homofermentative strain for an industrial-scale L-lactate production. In a flask culture, this isolate fermented both glucose and sucrose to lactate with high yield (0.96 and 0.87 g/g) and productivity (2.8 and 2.6 g/L h), respectively. The higher lactate production performance was obtained in the simultaneous saccharification and fermentation of liquefied starch (150.1 g/L final titer, 0.98 g/g yield, 3.2 g/L h productivity) and the fed-batch glucose fermentation (139.9 g/L final titer, 0.96 g/g yield, 2.9 g/L h productivity). Significant increase in lactate productivity (5.5-6.1 g/L h) was obtained from the high/heavy-inoculum seed in the stirred tank fermentor. Both calcium bases and monovalent bases were successfully employed for pH control during lactate fermentation by this isolate resulting in a versatile and simple operation. By the two-phase fermentation using the high/heavy-inoculum seed of BC-001, the fermentation reproducibility was acquired toward the pilot-scale fermentors.

Sporolactobacillus shoreicorticis sp.nov., a lactic acid-producing bacterium isolated from tree bark.

A Gram-stain-positive, lactic acid-producing bacterium designed strain MK21-7T, was isolated from tree bark collected from the north east of Thailand. This strain was a facultatively anaerobic spore-forming rod that was catalase-negative. It contained meso-diaminopimelic acid in the cell wall peptidoglycan and had seven isoprene units (MK-7) as the predominant menaquinone. Major fatty acids of MK21-7T were anteiso-C17 : 0, iso-C16 : 0, anteiso-C15 : 0 and C18 : 1ω9c. Polar lipids were phosphatidglycerol, diphosphatidylglycerol, an unknown phospholipid, three unknown glycolipids and an unknown lipid. The results of 16S rRNA gene sequence analysis indicated that it represented a member of the genus Sporolactobacillus. MK21-7T showed the highest 16S rRNA gene sequence similarity to Sporolactobacillus terrae NBRC 101527T with 98.4 % similarity and exhibited 97.6 % similarity with Sporolactobacillus kofuensis NRIC 0334T, 97.5 % with Sporolactobacillus laevolacticus NRIC 0361T, 97.3 % with Sporolactobacillus nakayamaesubsp.nakayamae NRIC 0347T and 97.1 % with Sporolactobacillus nakayamaesubsp.racemicus NBRC 101524T. Analysis of the phylogenetic relationship based on 16S rRNA and gyrB gene sequencing revealed that the position of MK21-7T was clearly separated from all related species of the genus Sporolactobacillus. It had low DNA-DNA relatedness (22.8-57.2 %) with S. terrae NBRC 101527T and related type strains. The DNA G+C content was 43.1 mol%. On the basis of the results of the phenotypic, genotypic and chemotaxonomic studies, MK21-7T should be classified as representing a novel species of the genus Sporolactobacillus for which the name Sporolactobacillus shoreicorticis sp. nov. is proposed. The type strain is MK21-7T (=NBRC 111517T=LMG 29111T=TISTR 2466T).

Terrilactibacillus laevilacticus gen. nov., sp. nov., isolated from soil.

A Gram-stain-positive, catalase-positive, facultatively anaerobic, spore-forming, rod-shaped bacterium, strain NK26-11T, was isolated from soil in Thailand. This strain produced d-lactic acid from glucose homofermentatively, and grew at 20-45 °C and pH 5-8.5. The cell-wall peptidoglycan contained meso-diaminopimelic acid. The major respiratory quinone was menaquinone 7 (MK-7), the DNA G+C content was 42.6 mol%, and the major cellular fatty acids were anteiso-C15 : 0 and anteiso-C17 : 0. On the basis of 16S rRNA gene sequences analysis, strain NK26-11T was closely related to Bacillus solimangrovi JCM 18994T (93.89 % 16S rRNA gene sequence similarity), Pullulanibacillus naganoensis LMG 12887T (93.32 %), Sporolactobacillus inulinus NRIC 1133T (92.99 %), Tuberibacillus calidus JCM 13397T (92.98 %) and Thalassobacillus devorans DSM 16966T ( < 90.93 %). Strain NK26-11T could be clearly distinguished from the closely related genera based on phenotypic characteristics and DNA G+C content, and thus represents a novel species of a new genus between the Bacillus and Sporolactobacillus cluster, for which the name Terrilactibacillus laevilacticus gen. nov., sp. nov. is proposed. The type strain of the type species is NK26-11T ( = LMG 27803T = TISTR 2241T = PCU 335T).

Regulating Pyruvate Carboxylase in the Living Culture of Aspergillus Terreus Nrrl 1960 by L-Aspartate for Enhanced Itaconic Acid Production.

Aspergillus terreus was reported as the promising fungal strain for itaconic acid; however, the commercial production suffers from the low yield. Low production yield was claimed as the result of completing the tricarboxylic acid (TCA) cycle towards biomass synthesis while under limiting phosphate and nitrogen; TCA cycle was somewhat shunted and consequently, the metabolite fluxes move towards itaconic acid production route. By regulating enzymes in TCA cycle, it is believed that itaconic acid production can be improved. One of the key responsible enzymes involved in itaconic acid production was triggered in this study. Pyruvate carboxylase was allosterically inhibited by L-aspartate. The presence of 10 mM L-aspartate in the production medium directly repressed PC expression in the living A. terreus while the limited malate flux regulated the malate/citrate antiporters resulting in the increasing cis-aconitate decarboxylase activity to simultaneously convert cis-aconitate, citrate isomer, into itaconic acid. The transport of cis-aconitate via the antiporters induced citrate synthase and 6-phosphofructo-1-kinase activities in response to balance the fluxes of TCA intermediates. Successively, itaconic acid production yield and final concentration could be improved by 8.33 and 60.32 %, respectively, compared to those obtained from the control fermentation with the shortened lag time to produce itaconic acid during the production phase.

Sporolactobacillus shoreae sp. nov. and Sporolactobacillus spathodeae sp. nov., two spore-forming lactic acid bacteria isolated from tree barks in Thailand.

Two Gram-stain-positive, endospore-forming lactic acid bacteria, designated BK92(T) and BK117-1(T), were isolated from tree barks in Thailand. Cells were catalase-negative and facultatively anaerobic rods. 16S rRNA gene sequence analysis indicated that these strains belonged to the genus Sporolactobacillus . Strains BK92(T) and BK117-1(T) showed the highest 16S rRNA gene sequence similarity to Sporolactobacillus putidus QC81-06(T) with 97.7% and 97.1% similarity, respectively. Analysis of phylogenetic relationships based on 16S rRNA and gyrB gene sequencing revealed that the positions of strains BK92(T) and BK117-1(T) were clearly separated from all related species of the genus Sporolactobacillus . Strains BK92(T) and BK117-1(T) had low DNA-DNA relatedness between each other and also with S. putidus QC81-06(T) and Sporolactobacillus vineae SL153(T). The DNA G+C content of strains BK92(T) and BK117-1(T) was 46.6 mol% and 47.4 mol%, respectively. The major fatty acids of strains BK92(T) and BK117-1(T) were anteiso-C(17 : 0) and anteiso-C(15 : 0). They contained meso-diaminopimelic acid in cell-wall peptidoglycan and had menaquinone with seven isoprene units (MK-7) as the predominant menaquinone. Based on evidence including phenotypic, genotypic and chemotaxonomic studies, strains BK92(T) and BK117-1(T) should be classified as representatives of novel species of the genus Sporolactobacillus , for which the names Sporolactobacillus shoreae sp. nov. and Sporolactobacillus spathodeae sp. nov. are proposed, respectively. The type strains are BK92(T) ( = JCM 19541(T) = LMG 28365(T) = PCU 336(T) = TISTR 2234(T)) and BK117-1(T) ( = JCM 19542(T) = LMG 28366(T) = PCU 337(T) = TISTR 2235(T)).

Manipulating pyruvate decarboxylase by addition of enzyme regulators during fermentation of Rhizopus oryzae to enhance lactic acid production.

Ethanol was found as the major by-product in lactate fermentation by Rhizopus oryzae. Several methods have been conducted in order to limit ethanol formation, thus increasing the lactate yield. The direct way to suppress ethanol production can be done by inhibition of the responsible enzymes in the related pathway. Pyruvate decarboxylase (PDC) and alcohol dehydrogenase (ADH) are responsible for ethanol production in R. oryzae. Shunting the ethanol production pathway by targeting at PDC was attempted in this study. Three compounds including 4-methylpyrazole, glyoxylic acid, and 3-hydroxypyruvate with the in vitro reversible inhibitory effect on PDC were selected from the literature and were used to regulate the living cell of R. oryzae during the fermentation. The results show that 0.1 mM 4-methylpyrazole of which the structure resembled a thiazolium ring in thiamine diphosphate, PDC cofactor, and 1.0 µm 3-hydroxypyruvate, pyruvate analog, effectively hampered ethanol production. Further observation on the enzyme expression indicated that these two regulators not only targeted PDC but also caused changes in ADH and lactate dehydrogenase (LDH) activities. This was perhaps due to the living cell of R. oryzae that responded to the presence of the regulators to balance the pyruvate flux and subsequently maintain its metabolic activities.