Screening of cellulose-degrading bacteria and optimization of cellulase production from Bacillus cereus A49 through response surface methodology.

Cellulose-degrading microorganisms hold immense significance in utilizing cellulose resources efficiently. The screening of natural cellulase bacteria and the optimization of fermentation conditions are the hot spots of research. This study meticulously screened cellulose-degrading bacteria from mixed soil samples adopting a multi-step approach, encompassing preliminary culture medium screening, Congo red medium-based re-screening, and quantification of cellulase activity across various strains. Particularly, three robust cellulase-producing strains were identified: A24 (MT740356.1 Brevibacillus borstelensis), A49 (MT740358.1 Bacillus cereus), and A61 (MT740357.1 Paenibacillus sp.). For subsequent cultivation experiments, the growth curves of the three obtained isolates were monitored diligently. Additionally, optimal CMCase production conditions were determined, keeping CMCase activity as a key metric, through a series of single-factor experiments: agitation speed, cultivation temperature, unit medium concentration, and inoculum volume. Maximum CMCase production was observed at 150 rpm/37 °C, doubling the unit medium addition, and a 5 mL inoculation volume. Further optimization was conducted using the selected isolate A49 employing response surface methodology. The software model recommended a 2.21fold unit medium addition, 36.11 °C temperature, and 4.91 mL inoculant volume for optimal CMCase production. Consequently, three parallel experiments were conducted based on predicted conditions consistently yielding an average CMCase production activity of 15.63 U/mL, closely aligning with the predicted value of 16.41 U/mL. These findings validated the reliability of the model and demonstrated the effectiveness of optimized CMCase production conditions for isolate A49.

Transcriptomic profiling of an evolved Yarrowia lipolytica strain: tackling hexanoic acid fermentation to increase lipid production from short-chain fatty acids.

BACKGROUND: Short-chain fatty acids (SCFAs) are cost-effective carbon sources for an affordable production of lipids. Hexanoic acid, the acid with the longest carbon chain in the SCFAs pool, is produced in anaerobic fermentation of organic residues and its use is very challenging, even inhibiting oleaginous yeasts growth. RESULTS: In this investigation, an adaptive laboratory evolution (ALE) was performed to improve Yarrowia lipolytica ACA DC 50109 tolerance to high hexanoic acid concentrations. Following ALE, the transcriptomic analysis revealed several genetic adaptations that improved the assimilation of this carbon source in the evolved strain compared to the wild type (WT). Indeed, the evolved strain presented a high expression of the up-regulated gene YALI0 E16016g, which codes for FAT1 and is related to lipid droplets formation and responsible for mobilizing long-chain acids within the cell. Strikingly, acetic acid and other carbohydrate transporters were over-expressed in the WT strain. CONCLUSIONS: A more tolerant yeast strain able to attain higher lipid content under the presence of high concentrations of hexanoic acid has been obtained. Results provided novel information regarding the assimilation of hexanoic acid in yeasts.

Research advances in the identification of regulatory mechanisms of surfactin production by Bacillus: a review.

Surfactin is a cyclic hexalipopeptide compound, nonribosomal synthesized by representatives of the Bacillus subtilis species complex which includes B. subtilis group and its closely related species, such as B. subtilis subsp subtilis, B. subtilis subsp spizizenii, B. subtilis subsp inaquosorum, B. atrophaeus, B. amyloliquefaciens, B. velezensis (Steinke mSystems 6: e00057, 2021) It functions as a biosurfactant and signaling molecule and has antibacterial, antiviral, antitumor, and plant disease resistance properties. The Bacillus lipopeptides play an important role in agriculture, oil recovery, cosmetics, food processing and pharmaceuticals, but the natural yield of surfactin synthesized by Bacillus is low. This paper reviews the regulatory pathways and mechanisms that affect surfactin synthesis and release, highlighting the regulatory genes involved in the transcription of the srfAA-AD operon. The several ways to enhance surfactin production, such as governing expression of the genes involved in synthesis and regulation of surfactin synthesis and transport, removal of competitive pathways, optimization of media, and fermentation conditions were commented. This review will provide a theoretical platform for the systematic genetic modification of high-yielding strains of surfactin.

Kinetic modeling and optimization of ethanol fermentation by the marine yeast Wickerhamomyces subpelliculosus ZE75.

The study aims to enhance ethanol production by Wickerhamomyces subpelliculosus ZE75 isolated from marine sediment. In addition, analyzing the kinetic parameters of ethanol production and optimization of the fermentation conditions was performed. The marine yeast isolate ZE75 was selected as the front runner ethanol-producer, with an ethanol yield of 89.77 gL-1. ZE75 was identified relying on the phenotypic and genotypic characteristics of W. subpelliculosus. The genotypic characterization based on the Internal Transcribed Spacer (ITS) sequence was deposited in the GenBank database with the accession number OP715873. The maximum specific ethanol production rate (vmax) was 0.482 gg-1 h-1 at 175 gL-1 glucose concentration, with a high accuracy of R2 0.95. The maximum growth specific rates (µmax) were 0.141 h-1 obtained at 150 gL-1 glucose concentration with R2 0.91. Optimization of the fermentation parameters such as pH and salinity has been achieved. The highest ethanol yield 0.5637 gg-1 was achieved in a 100% natural seawater-based medium. The maximum ethanol production of 104.04 gL-1 was achieved at pH 4.5 with a specific ethanol rate of 0.1669 gg-1 h-1. The findings of the present study recommend the possibility of ethanol production from a seawater-based medium on a large scale using W. subpelliculosus ZE75.

Optimization of fermentation conditions and medium components for chrysomycin a production by Streptomyces sp. 891-B6.

BACKGROUND: Chrysomycin A (CA) is a promising antibiotic for treatment of Gram-positive bacterial infections and cancers. In order to enhance CA yield, optimization of fermentation conditions and medium components was carried out on strain Streptomyces sp. 891-B6, an UV-induced mutant with improved CA titer compared with its wide-type marine strain 891. RESULTS: Using one-way experiment, the optimal fermentation conditions for CA production in 1-L shake flask were obtained as follows: 12 days of fermentation time, 5 days of seed age, 5% of inoculum volume ratio, 200 mL of loading volume and 6.5 of initial pH. By response surface methodology, the optimal medium components determined as glucose (39.283 g/L), corn starch (20.662 g/L), soybean meal (15.480 g/L) and CaCO3 (2.000 g/L). CONCLUSION: Validation tests showed that the maximum yield of CA reached 1601.9 ± 56.7 mg/L, which was a 60% increase compared to the initial yield (952.3 ± 53.2 mg/L). These results provided an important basis for scale-up production of CA by strain 891-B6.

Elevated caproic acid production from one-stage anaerobic fermentation of organic waste and its selective recovery by electro-membrane process.

A constructed microbial consortia-based strategy to enhance caproic acid production from one-stage mixed-fermentation of glucose was developed, which incubated with acidogens (Clostridium sensu stricto 1, 11 dominated) and chain elongators (including Clostridium sensu stricto 12, Sporanaerobacter, and Caproiciproducens) acclimated from anaerobic sludge. Significant product upgrading toward caproic acid (8.31 g‧L-1) and improved substrate degradation was achieved, which can be greatly attributed to the lactic acid platform. Whereas, a small amount of caproic acid was observed in the control incubating with acidogens, with an average concentration of 2.09 g‧L-1. The strategy accelerated the shape and cooperation of the specific microbial community dominated by Clostridium sensu stricto and Caproiciproducens, which thereby contributed to caproic acid production via the fatty acid biosynthesis pathway. Moreover, the tailored electrodialysis with bipolar membrane enabled progressive up-concentration and acidification, allowing selective separation of caproic acid as an immiscible product with a purity of 82.58 % from the mixture.

An Eclectic Review on Dicarboxylic Acid Production Through Yeast Cell Factories and Its Industrial Prominence.

Dicarboxylic acid (DCA) is a multifaceted chemical intermediate, recoursed to produce many industrially important products such as adhesives, plasticizers, lubricants, polymers, etc. To bypass the shortcomings of the chemical methods of synthesis of DCA and to reduce fossil fuel footprints, bio-based synthesis is gaining attention. In pursuit of an eco-friendly sustainable alternative method of DCA production, microbial cell factories, and renewable organic resources are gaining popularity. Among the plethora of microbial communities, yeast is being favored industrially compared to bacterial fermentation due to its hyperosmotic and low pH tolerance and flexibility for gene manipulations. By application of rapidly evolving genetic manipulation techniques, the bio-based DCA production could be made more precise and economical. To bridge the gap between supply and demand of DCA, many strategies are employed to improve the fermentation. This review briefly outlines the advancements in DCA production using yeast cell factories with the exemplification of strain improvement strategies.

RT-qPCR based quantitative analysis of ARO and ADH genes in Saccharomyces cerevisiae and Metschnikowia pulcherrima strains growth white grape juice.

BACKGROUND: Yeast biosynthesizes fusel alcohols in fermentation through amino acid catabolism via the Ehrlich pathway. ARO8 and ARO9 genes are involved in the first step of the Ehrlich pathway, while ADH2 and ADH5 genes are involved in the last step. In this study, we describe RT-qPCR methods to determine the gene expression level of genes (ARO8, ARO9, ADH2, ADH5) found in Saccharomyces cerevisiae (Sc) and Metschnikowia pulcherrima (Mp) strains growth pasteurized white grape juice. METHODS AND RESULTS: We used RNA extraction and cDNA synthesis protocols. The RT-qPCR efficiency of primer pairs was evaluated by generating a standard curve through serial dilution of yeast-derived cDNA. Method performance criteria were determined for each RT-qPCR assay. Then, we evaluated the gene expression levels of the four genes in all samples. RNA extraction and cDNA synthesis from yeast samples demonstrated the method's capability to generate high-yield, high-purity nucleic acids, supporting further RT-qPCR analysis. The highest normalized gene expression levels of ARO8 and ARO9 were observed in SC1, SC4, and SC5 samples. No significant difference in ADH2 gene expression among Mp strains was observed during the examination of ADH2 and ADH5 genes (p < 0.05). We observed no expression of the ADH5 gene in Mp strains except MP6 strain. The expression of ADH2 and ADH5 genes was higher in Sc strains compared to Mp strains. CONCLUSIONS: The results suggest that the proposed RT-qPCR methods can measure gene expression of ARO8, ARO9, ADH2, and ADH5 in Sc and Mp strains growing in pasteurized white grape juice.

The branching ratio of enzymatically synthesized α-glucans impacts microbiome and metabolic outcomes of in vitro fecal fermentation.

The aim of this study was to evaluate the impacts of enzymatically synthesized α-glucans possessing α-1,4- and α-1,6-glucose linkages, and varying in branching ratio, on colonic microbiota composition and metabolic function. Four different α-glucans varying in branching ratio were synthesized by amylosucrase from Neisseria polysaccharea and glycogen branching enzyme from Rhodothermus obamensis. The branching ratios were found to range from 0 % to 2.8 % using GC/MS. In vitro fecal fermentation analyses (n = 8) revealed that the branching ratio dictates the short-chain fatty acid (SCFA) generation by fecal microbiota. Specifically, slightly branched (0.49 %) α-glucan resulted in generation of significantly (P < 0.05) higher amounts of propionate, compared to more-branched counterparts. In addition, the amount of butyrate generated from this α-glucan was statistically (P > 0.05) indistinguishable than those observed in resistant starches. 16S rRNA sequencing revealed that enzymatically synthesized α-glucans stimulated Lachnospiraceae and Ruminococcus related OTUs. Overall, the results demonstrated metabolic function of colonic microbiota can be manipulated by altering the branching ratio of enzymatically synthesized α-glucans, providing insights into specific structure-function relationships between dietary fibers and the colonic microbiome. Furthermore, the slightly branched α-glucans could be used as functional carbohydrates to stimulate the beneficial microbiota and SCFAs in the colon.

A structure-functionality insight into the bioactivity of microbial polysaccharides toward biomedical applications: A review.

Microbial polysaccharides (MPs) are biopolymers secreted by microorganisms such as bacteria and fungi during their metabolic processes. Compared to polysaccharides derived from plants and animals, MPs have advantages such as wide sources, high production efficiency, and less susceptibility to natural environmental influences. The most attractive feature of MPs lies in their diverse biological activities, such as antioxidative, anti-tumor, antibacterial, and immunomodulatory activities, which have demonstrated immense potential for applications in functional foods, cosmetics, and biomedicine. These bioactivities are precisely regulated by their sophisticated molecular structure. However, the mechanisms underlying this precise regulation are not yet fully understood and continue to evolve. This article presents a comprehensive review of the most representative species of MPs, including their fermentation and purification processes and their biomedical applications in recent years. In particular, this work presents an in-depth analysis into the structure-activity relationships of MPs across multiple molecular levels. Additionally, this review discusses the challenges and prospects of investigating the structure-activity relationships, providing valuable insights into the broad and high-value utilization of MPs.

New insights into starch, lipid, and protein interactions - Colon microbiota fermentation.

Starch, lipids, and proteins are essential biological macromolecules that play a crucial role in providing energy and nutrition to our bodies. Interactions between these macromolecules have been shown to impact starch digestibility. Understanding and controlling starch digestibility is a key area of research. Investigating the mechanisms behind the interactions of these three components and their influence on starch digestibility is of significant practical importance. Moreover, these interactions can result in the formation of resistant starch, which can be fermented by gut microbiota in the colon, leading to various health benefits. While current research has predominantly focused on the digestive properties of starch in the small intestine, there is a notable gap in understanding the colonic microbial fermentation phase of resistant starch. The benefits of fermentation of resistant starch in the colon may outweigh its glucose-lowering effect in the small intestine. Thus, it is crucial to study the fermentation behavior of resistant starch in the colon. This paper investigates the impact of interactions among starch, lipids, and proteins on starch digestion, with a specific focus on the fermentation phase of indigestible carbohydrates in the colon. Furthermore, valuable insights are offered for guiding future research endeavors.

From the grapevine to the glass: A wine metabolomics tale by FT-ICR-MS.

Wine is one of the most consumed beverages around the world. Its unique characteristics arise from numerous processes, from the selection of grapevine varieties and grapes, the effect of the terroir and geographical origin, through the biochemical process of fermentation by microorganisms, until its aging. All molecules found in wine define its chemical fingerprint and can be used to tell the story of its origin, production, authenticity and quality. Wine's chemical composition can be characterized using an untargeted metabolomics approach based on extreme resolution mass spectrometry. Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) is currently the most powerful analytical technique to analyse such complex sample, providing the most comprehensive analysis of the chemical fingerprint of wine.

Reducing transmission of high-risk antibiotic resistance genes in whole-crop corn silage through lactic acid bacteria inoculation and increasing ensiling temperature.

The microbial hosts of antibiotic resistance genes (ARGs) found epiphytically on plant materials could grow and flourish during silage fermentation. This study employed metagenomic analysis and elucidated the occurrence and transmission mechanisms of ARGs and their microbial hosts in whole-crop corn silage inoculated with homofermentative strain Lactiplantibacillus plantarum or heterofermentative strain Lentilactobacillus buchneri ensiled under different temperature (20 and 30 °C). The results revealed that the corn silage was dominated by Lactobacillus, Leuconostoc, Lentilactobacillus, and Latilactobacillus. Both the ensiling temperature and inoculation had greatly modified the silage microbiota. However, regardless of the ensiling temperature, L. buchneri had significantly higher ARGs, while it only exhibited significantly higher mobile genetic elements (MGEs) in low temperature treatments. The microbial community of the corn silage hosted highly diverse form of ARGs, which were primarily MacB, RanA, bcrA, msbA, TetA (58), and TetT and mainly corresponded to macrolides and tetracyclines drug classes. Plasmids were identified as the most abundant MGEs with significant correlation with some high-risk ARGs (tetM, TolC, mdtH, and NorA), and their abundances have been reduced by ensiling process. Furthermore, higher temperature and L. buchneri reduced abundances of high-risk ARGs by modifying their hosts and reduced their transmission in the silage. Therefore, ensiling, L. buchneri inoculation and higher storage temperature could improve the biosafety of corn silage.

Leaching Approach for ß-Glucosidase Extraction from Fermented Rice Husk in Solid State Cultivation by Aspergillus protuberus.

Environmental problems are caused by the disposal of agrowastes in developing countries. It is imperative to convert such wastes into useful products, which require enzymes such as ß-glucosidase. ß-Glucosidase has variety of applications in biotechnology including food, textile, detergents, pulp and paper, pharmaceutical and biofuel industries. ß-Glucosidase production was performed using the locally isolated Aspergillus protuberus using best growth circumstances on rice husk in solid-state fermentation (SSF). Leaching of ß-glucosidase from fermented rice husk with number of solvents to evaluate their extraction efficacy. Among the different solvents examined, acetate buffer (0.02 M, pH 5.0) proved to be the best solvent. The subsequent parameters were optimized with acetate buffer. Two washes with acetate buffer each by shaking (30 min) in a ratio of 1 g of rice husk: 5 ml of acetate buffer together attained maximum recovery of ß-glucosidase with 41.95 U/g of rice husk.

Exploring rumen fermentation and microbial populations in Dhofari goats fed a chitosan-added diet.

The use of chitosan (CHI) in ruminant diets is a promising natural modifier for rumen fermentation, capable of modulating both the rumen pattern and microbial activities. The objective of this study was to explore the rumen fermentation and microbial populations in Dhofari goats fed a diet supplemented with CHI. A total of 24 Dhofari lactating goats (body weight, 27.32 ± 1.80 kg) were assigned randomly into three experimental groups (n = 8 ewes/group). Goats were fed a basal diet with either 0 (control), 180 (low), or 360 (high) mg CHI/kg of dietary dry matter (DM) for 45 days. Feeding high CHI linearly increased (p < 0.05) the propionate level and reduced the acetate, butyrate, and total protozoa count (p < 0.05). Ruminal ammonia nitrogen (NH3-N) concentrations and the acetate:propionate ratio decreased linearly when goats were fed CHI (p < 0.05). The abundances of both Spirochetes and Fibrobacteres phyla were reduced (p < 0.05) with both CHI doses relative to the control. Both low and high CHI reduced (p < 0.05) the relative abundances of Butyrivibrio hungatei, Fibrobacter succinogenes, Ruminococcus albus, Ruminococcus flavefaciens, Selenomonas ruminantium and Neocallimastix californiae populations. Adding CHI significantly decreased (p < 0.05) the abundances of Ascomycota, Basidiomycota, and Bacillariophyta phyla compared to the control. Adding CHI to the diet reduces the abundance of fibrolytic-degrading bacteria, however, it increases the amylolytic-degrading bacteria. Application of 360 mg of CHI/kg DM modified the relative populations of ruminal microbes, which could enhance the rumen fermentation patterns in Dhofari goats.

Review: Mechanism, effectiveness, and the prospects of medicinal plants and their bioactive compounds in lowering ruminants' enteric methane emission.

Animal nutritionists continue to investigate new strategies to combat the challenge of methane emissions from ruminants. Medicinal plants (MPs) are known to be beneficial to animal health and exert functional roles in livestock due to their phytogenic compounds with antimicrobial, immunostimulatory, antioxidative, and anti-inflammatory activities. Some MP has been reported to be anti-methanogenic and can effectively lower ruminants' enteric methane emissions. This review overviews trends in MP utilization in ruminants, their bioactivity and their effectiveness in lowering enteric methane production. It highlights the MP regulatory mechanism and the gaps that must be critically addressed to improve its efficacy. MP could reduce enteric methane production by up to 8-50% by regulating the rumen fermentation pathway, directing hydrogen toward propionogenesis, and modifying rumen diversity, structure, and population of the methanogens and protozoa. Yet, factors such as palatability, extraction techniques, and economic implications must be further considered to exploit their potential fully.

Predicting ruminal degradability and chemical composition of corn silage using near-infrared spectroscopy and multivariate regression.

The aim of this study was to develop and validate regression models to predict the chemical composition and ruminal degradation parameters of corn silage by near-infrared spectroscopy (NIR). Ninety-four samples were used to develop and validate the models to predict corn silage composition. A subset of 23 samples was used to develop and validate models to predict ruminal degradation parameters of corn silage. Wet chemistry methods were used to determine the composition values and ruminal degradation parameters of the corn silage samples. The dried and ground samples had their NIR spectra scanned using a poliSPECNIR 900-1700 model NIR sprectrophotometer (ITPhotonics S.r.l, Breganze, IT.). The models were developed using regression by partial least squares (PLS), and the ordered predictor selection (OPS) method was used. In general, the regression models obtained to predict the corn silage composition (P>0.05), except the model for organic matter (OM), adequately estimated the studied properties. It was not possible to develop prediction models for the potentially degradable fraction in the rumen of OM and crude protein and the degradation rate of OM. The regression models that could be obtained to predict the ruminal degradation parameters showed correlation coefficient of calibration between 0.530 and 0.985. The regression models developed to predict CS composition accurately estimated the CS composition, except the model for OM. The NIR has potential to be used by nutritionists as a rapid prediction tool for ruminal degradation parameters in the field.

Mutation breeding of high-stress resistant strains for succinic acid production from corn straw.

The production of succinic acid from corn stover is a promising and sustainable route; however, during the pretreatment stage, byproducts such as organic acids, furan-based compounds, and phenolic compounds generated from corn stover inhibit the microbial fermentation process. Selecting strains that are resistant to stress and utilizing nondetoxified corn stover hydrolysate as a feedstock for succinic acid production could be effective. In this study, A. succinogenes CICC11014 was selected as the original strain, and the stress-resistant strain A. succinogenes M4 was obtained by atmospheric and room temperature plasma (ARTP) mutagenesis and further screening. Compared to the original strain, A. succinogenes M4 exhibited a twofold increase in stress resistance and a 113% increase in succinic acid production when hydrolysate was used as the substrate. By conducting whole-genome resequencing of A. succinogenes M4 and comparing it with the original strain, four nonsynonymous gene mutations and two upstream regions with base losses were identified. KEY POINTS: • A high-stress-resistant strain A. succinogenes M4 was obtained by ARTP mutation •  The production of succinic acid increased by 113% • The mutated genes of A. succinogenes M4 were detected and analyzed.

Adaptive laboratory evolution of Clostridium autoethanogenum to metabolize CO2 and H2 enhances growth rates in chemostat and unravels proteome and metabolome alterations.

Gas fermentation of CO2 and H2 is an attractive means to sustainably produce fuels and chemicals. Clostridium autoethanogenum is a model organism for industrial CO to ethanol and presents an opportunity for CO2-to-ethanol processes. As we have previously characterized its CO2/H2 chemostat growth, here we use adaptive laboratory evolution (ALE) with the aim of improving growth with CO2/H2. Seven ALE lineages were generated, all with improved specific growth rates. ALE conducted in the presence of 2% CO along with CO2/H2 generated Evolved lineage D, which showed the highest ethanol titres amongst all the ALE lineages during the fermentation of CO2/H2. Chemostat comparison against the parental strain shows no change in acetate or ethanol production, while Evolved D could achieve a higher maximum dilution rate. Multi-omics analyses at steady state revealed that Evolved D has widespread proteome and intracellular metabolome changes. However, the uptake and production rates and titres remain unaltered until investigating their maximum dilution rate. Yet, we provide numerous insights into CO2/H2 metabolism via these multi-omics data and link these results to mutations, suggesting novel targets for metabolic engineering in this bacterium.

Investigating the efficacy of an exopolysaccharide (EPS)-producing strain Lactiplantibacillus plantarum L75 on oat silage fermentation at different temperatures.

This study investigates the effectiveness of an exopolysaccharide (EPS)-producing strain (Lactiplantibacillus plantarum L75) alone or in combination with Saccharomyces cerevisiae on the fermentation characteristics, antioxidant capacities and microbial community successions of oat silage stored at various temperatures. A rapid decrease in pH and lactic acid accumulation was observed in silages treated with L. plantarum and S. cerevisiae (LS) as early as 3 days of ensiling (p < 0.05). Over the ensiling period of 7-60 days, L. plantarum (L)-inoculated groups showed the lowest pH, lowest ammonia nitrogen and the highest amount of lactic acid regardless of the storage temperatures. When the oat silage was stored at 15°C, LS-inoculated group exhibited a higher superoxide dismutase (SOD) activity than control and L-inoculated group. Furthermore, the proportion of Lactiplantibacillus in the combined inoculation group increased by 65.42% compared to the L-inoculated group (33.26%). Fungal community data revealed abundant Penicillium carneum in the control and L-inoculated groups stored at 15°C. Conclusively, these results showed that combined inoculation of L. plantarum L75 and S. cerevisiae improved the fermentation quality of oat silage at 15°C, thus proposing a technique for enhancing the fermentation quality of silage in regions with low temperatures during harvest season.