Search Results (44)

This study investigates the valorization of carrot discard, a carbohydrate-rich agricultural residue, for the production of 2,3-butanediol (2,3-BDO). The fermentation process was evaluated using two strains, Bacillus licheniformis DSM 8785 and Bacillus amyloliquefaciens DSM 7. Two process configurations were compared: separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF). Additionally, to determine substrate and product inhibition thresholds, fermentation assays were conducted in semi-defined media with glucose concentrations ranging from 20 to 120 g/L. The SHF strategy proved more effective than the SSF configuration. Under the SHF configuration, B. amyloliquefaciens demonstrated superior performance, yielding 16.7 g/L of 2,3-BDO. In contrast, B. licheniformis was notable for its high capacity for acetoin synthesis, producing 24.2 g/L of acetoin in addition to 10.9 g/L of 2,3-BDO. Therefore, these findings demonstrate that carrot discard is a viable feedstock for the co-production of 2,3-BDO and acetoin. Full article

2,3,5-Trimethylpyrazine (TMP) is an alkyl pyrazine with broad application prospects in the fields of food additives and medicine. L-threonine-3-dehydrogenase (TDH) is a key enzyme in the biosynthesis pathway of TMP. To explore the efficient and environmentally friendly production method of TMP, we constructed recombinant strains overexpressing the BlTDH gene and its mutant BlTDH (N157A) using Bacillus licheniformis YC7. The TMP yield of recombinant strains with pHT01-BlTDH (N157A) reached 15.35 ± 1.51 mg/L, which was significantly higher than that of strains with pHT01-BlTDH (9.86 ± 1.24 mg/L) and strains with vector pHT01 (2.35 ± 0.58 mg/L). To further increase the TMP yield of strain YC7/pHT01-BlTDH (N157A), the fermentation process was optimized by single-factor experiments, and the response surface test was conducted using the Box–Behnken design. The results revealed that the substrate ratio, IPTG concentration and fermentation time had significant effects on the yield of TMP, and the degree of influence was substrate ratio > fermentation time > IPTG concentration. The optimization results of response surface indicated that the optimal fermentation conditions were as follows: substrate ratio of 1:2, IPTG concentration of 1.0 mM, and fermentation time of 4 d. Under these conditions, the TMP yield reached 44.52 ± 0.21 mg/L, which was around 0.005 mg/L different from the predicted value (45.515 mg/L), and increased by 29.17 mg/L compared with the initial condition. The optimization of fermentation conditions significantly increased the yield of TMP produced by recombinant strains, which provided a theoretical basis and strain resources for industrial production of TMP. Full article

The increasing demand for industrial enzymes calls for cost-effective and sustainable production strategies. This study investigates the potential of industrial wastewater as an alternative fermentation medium for enzyme synthesis, aligning with the principles of the circular bioeconomy. Four wastewater types from Québec, Canada—beverage wastewater (BW), pulp and paper mill activated sludge (PPMS), food industry wastewater (FIW), and starch industry wastewater (SIW)—were evaluated for their potential to support protease, amylase, and lipase production using Bacillus licheniformis, Bacillus amyloliquefaciens, and Bacillus megaterium. Initial screening identified SIW as optimal for amylase production with B. amyloliquefaciens, and PPMS for protease production with B. megaterium. Optimization using the Box–Behnken design was then performed, followed by scale-up experiments in 5 L bioreactors. B. amyloliquefaciens achieved 5.73 ± 0.01 U/mL of amylase at 48 h under 40 g/L total solids, 30 °C, and a 2% inoculum size, while B. megaterium produced the highest protease of 55.41 ± 3.54 U/mL at 24 h. Lipase production remained negligible across all media and strains. These findings demonstrate the feasibility of the potential of wastewater-based enzyme production, reducing reliance on expensive synthetic substrates, mitigating environmental burdens, and contributing to the transition to a circular bioeconomy. Full article

This study isolated five acid-producing strains (XQ₁ and YZ₁–YZ₄) from naturally fermented pomace of Rosa roxburghii Tratt (RRT) in Guizhou’s karst region. Genetic and phenotypic analyses identified XQ₁, YZ₂, and YZ₄ as Lactobacillus plantarum (L. plantarum), YZ₃ as Weissella cibaria, and YZ₁ as Bacillus licheniformis. A comparative evaluation with commercial strain AC revealed that XQ₁, YZ₂, and YZ₄ exhibited superior acidification (reaching the stationary phase at 40 h) and tolerance to acidic conditions (pH 3.0), ethanol (6% v/v), bile salts (0.3%), and osmotic stress (6.5% NaCl), along with broad-spectrum antimicrobial activity against Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Shigella dysenteriae, and Pseudomonas aeruginosa. Their cell-free supernatants (CFSs) showed comparable superoxide dismutase activity and total antioxidant capacity (2.54–2.66 FeSO₄·7H₂O eq mmol/L) to AC (2.68), with DPPH radical scavenging exceeding 50%. YZ₃ displayed weaker acid production, tolerance, and limited antimicrobial effects. Safety assessments confirmed non-hemolytic activity and antibiotic susceptibility. In conclusion, the L. plantarum strains XQ₁, YZ₂, and YZ₄ demonstrated strong ensiling potential and remarkable probiotic properties, establishing them as promising indigenous microbial resources for the preservation of RRT pomace and other food products. Full article

Direct-fed microbials (DFMs) have shown the potential to improve livestock performance and overall health. Extensive research has been conducted to identify new DFMs and understand their mechanisms of action in the gut. Bacillus species are multifunctional spore-forming bacteria that exhibit resilience to harsh conditions, making them ideal candidates for applications in the feed industry and livestock production. This study investigates the mode of action of B. licheniformis and B. subtilis in the rumen using diverse in vitro techniques. Our results revealed that both strains germinated and grew in sterile rumen and intestinal contents from dairy cows and bulls. Gas composition analysis of in vitro cultures in a medium containing 40% rumen fluid demonstrated that germination of B. licheniformis and B. subtilis strains reduced oxygen levels, promoting an anaerobic environment favorable to rumen microbes. Enzymatic activity assays showed that B. licheniformis released sugars from complex substrates and purified polysaccharides in filtered rumen content. Additionally, the combination of B. licheniformis and B. subtilis survived and grew in the presence of a commercial monensin dose in rumen fluid media. The effects of B. licheniformis and B. subtilis on rumen fermentation activity and microbiota were studied using an in vitro batch fermentation assay. In fermenters that received a combination of B. licheniformis and B. subtilis, less CO₂ was produced while dry matter degradation and CH₄ production was comparable to the control condition, indicating better efficiency of dry matter utilization by the microbiota. The investigation of microbiota composition between supplemented and control fermenters showed no significant effect on alpha and beta diversity. However, the differential analysis highlighted changes in several taxa between the two conditions. Altogether, our data suggests that the administration of these strains of Bacillus could have a beneficial impact on rumen function, and consequently, on health and performance of ruminants. Full article

Purpose: This study aimed to utilize genetically engineered Bacillus licheniformis for the production of ergothioneine (EGT). Given the value of EGT and the application of Bacillus licheniformis in enzyme preparation production, we cloned the key enzymes (EanA and EanB) from Chlorbium limicola. Through gene alignment, new ergothioneine synthase genes (EanAN and EanBN) were identified and then expressed in Bacillus licheniformis to construct strains. Additionally, we investigated the factors influencing the yield of EGT and made a comparison with Escherichia coli. Methods: The relevant genes were cloned and transferred into Bacillus licheniformis. Fermentation experiments were conducted under different conditions for yield analysis, and the stability of this bacterium was also evaluated simultaneously. Results: The constructed strains were capable of producing EGT. Specifically, the yield of the EanANBN strain reached (643.8 ± 135) mg/L, and its stability was suitable for continuous production. Conclusions: Genetically engineered Bacillus licheniformis demonstrates potential in the industrial-scale production of EGT. Compared with Escherichia coli, it has advantages, thus opening up new possibilities for the application and market supply of EGT. Full article

Bacillus licheniformis 24 (BL24) is an efficient, non-pathogenic producer of 2,3-butanediol (2,3-BD). However, during inulin fermentation, the strain produces large amounts of exopolysaccharides (EPS), which interfere with the process’ performance. The present study aims to investigate the effect that inactivation of the sacB gene, encoding levansucrase in BL24, has on 2,3-BD production efficiency. Knockout of the sacB gene was accomplished via insertional inactivation. The sacB-knockout variant formed 0.57 g/L EPS from sucrose and 0.7–0.8 g/L EPS from glucose and fructose, a 15- and 2.5-fold reduction relative to the wild type, respectively. Likewise, during batch fermentation with soluble inulin Frutafit^® CLR, the mutant BLΔsacB produced significantly less EPS than the wild type, allowing the maintenance of pH at values favoring 2,3-BD synthesis. At pH 6.50, BLΔsacB reached a record titer of 128.7 g/L 2,3-BD, with productivity of 1.65 g/L/h, and a yield of 85.8% of the theoretical maximum. The obtained concentration of 2,3-BD is two-fold higher compared to that of the wild type. Subsequent RT-qPCR assays confirmed a successful sacB knockout. Three of the genes involved in inulin hydrolysis (sacA, sacC, and fruA) maintained their expression levels compared to the wild type, while that of levB increased. Although total EPS accumulation could not be completely eliminated via sacB gene knockout alone, the overall reduction in EPS content has enabled the highest yield of 2,3-BD from inulin to date, a promising result for the industrial production from inulin-rich substrates. Full article

Diosgenin (DSG) is a phytosterol saponin mainly found in Dioscorea zingiberensis C.H. Wright. It has shown promising results in treating various diseases such as cancer, diabetes, arthritis, asthma, and cardiovascular diseases. Diosgenin is also an important medicinal chemical for synthesizing various steroid medicines. The production of diosgenin by acid hydrolysis generates a large amount of wastewater, leading to severe environmental pollution. However, producing diosgenin through microbial fermentation can effectively reduce environmental pollution. Numerous studies have demonstrated that various microorganisms can produce diosgenin via solid-state fermentation. Nevertheless, due to the complexity, high maintenance costs, uneven heat production, and other characteristics of solid-state fermentation, it is not commonly used in the industrial production of diosgenin. In contrast, liquid fermentation offers advantages such as simple operation, easy maintenance, and stable fermentation, making it more suitable for the industrial production of diosgenin. However, few studies have focused on producing diosgenin using liquid fermentation. In this study, endophytic Bacillus licheniformis SYt1 was used to produce diosgenin via liquid fermentation, with Dioscorea tuber powder as a substrate. Soxhlet extraction and silica gel column chromatography were employed to identify the diosgenin from the liquid fermentation products. Suitable fermentation conditions were screened and identified. The environmental variables that significantly affect the diosgenin yield were determined by the Plackett–Burman design (P-BD) with eight factors. The three factors (peptone, yeast extract powder and inorganic salt) with the greatest influence on the diosgenin yield were selected and further optimized using a response surface methodology (RSM). The final culture conditions were determined to be 35.79 g/L of peptone, 14.56 g/L of yeast extract powder, and 1.44 g/L of inorganic salt. The yield of diosgenin under these conditions was 132.57 mg/L, which was 1.8 times greater than the yield under pre-optimization conditions. This effective, clean, and promising liquid fermentation method possesses the potential to replace the traditional acid hydrolysis method for the industrial production of diosgenin. Full article

The exploration of microbial genetic resources for the production of fFen-flavor Baijiu has not only enriched the microbial library for baijiu production but has also laid the foundation for process improvement and strain optimization in baijiu brewing. In this study, a total of 177 fungal isolates were screened, including Saccharomyces cerevisiae, non-Saccharomyces cerevisiae, molds, and some pathogenic bacteria. Among them, Saccharomyces cerevisiae was the most abundant with 119 isolates, playing a major role in the fermentation of baijiu production. A total of 148 bacterial isolates were obtained from the fermentation mash samples, showing greater diversity compared to fungi. Bacillus species were the most abundant, with 94 isolates. Bacillus licheniformis, in particular, can produce a rich enzymatic system and flavor precursors, making it an important contributor to the sensory quality of baijiu. Lactic acid bacteria were the second most abundant, with 16 isolates. Additionally, five pathogenic fungal species were identified, including Candida pelliculosa, Candida lusitaniae, Fusarium oxysporum, Fusarium solani and Talaromyces marneffei. Six pathogenic bacterial species were also isolated, namely Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus xylosus, Moraxella osloensis, Actinomyces meyeri and Stenotrophomonas maltophilia. Finally, two strains of high acetate ethyl ester-producing yeast and lactate-degrading bacteria with good tolerance to temperature, pH, and ethanol concentration were identified as Saccharomyces cerevisiae and Bacillus licheniformis, respectively. Full article

The rise of agro-industrial activities over recent decades has exponentially increased lignocellulose biomasses (LCB) production. LCB serves as a cost-effective source for fermentable sugars and other renewable chemicals. This study explores the use of microbial consortia, particularly thermophilic consortia, for LCB deconstruction. Thermophiles produce stable enzymes that retain activity under industrial conditions, presenting a promising approach for LCB conversion. This research focused on two microbial consortia (i.e., microbiomes) that were analyzed for enzyme production using a cheap medium, i.e., a mixture of spent mushroom substrate (SMS) and digestate. The secreted xylanolytic enzymes were characterized in terms of temperature and pH optima, thermal stability, and hydrolysis products from LCB-derived polysaccharides. These enzymes showed optimal activity aligning with common biorefinery conditions and outperformed a formulated enzyme mixture in thermostability tests in the digestate. Phylogenetic and genomic analyses highlighted the genetic diversity and metabolic potential of these microbiomes. Bacillus licheniformis was identified as a key species, with two distinct strains contributing to enzyme production. The presence of specific glycoside hydrolases involved in the cellulose and hemicellulose degradation underscores these consortia’s capacity for efficient LCB conversion. These findings highlight the potential of thermophilic microbiomes, isolated from an industrial environment, as a robust source of robust enzymes, paving the way for more sustainable and cost-effective bioconversion processes in biofuel and biochemical production and other biotechnological applications. Full article

Soy molasses, a by-product from the processing of soy protein concentrate, is a low-cost feedstock for fermentation processes due to its high content of fermentable sugars. This work investigates the use of soy molasses for growing Bacillus species, aiming at their potential application as plant growth promoters. Firstly, six Bacillus strains were screened for their ability to grow in increasing concentrations of soy molasses in a microplate assay. Following this, shaken-flask assays for growth and γ-polyglutamic acid (γ-PGA) production by three Bacillus strains in medium E and soy molasses media with 28 and 56 g L⁻¹ of total reducing sugars (TRS) were carried out. An in vivo experiment evaluated the effect of the bacterial fermented broths on the germination and initial development of maize. Soy molasses supported the growth of Bacillus amyloliquefaciens, Bacillus subtilis, and Bacillus licheniformis in concentrations of 28 and 56 g L⁻¹ TRS, but it was inhibitory at 112 and 224 g L⁻¹ TRS. In soy molasses media, growth was not always associated with γ-PGA production, which was a maximum of 56 g L⁻¹ TRS for B. amyloliquefaciens and B. licheniformis. Fermented broths with B. subtilis and B. licheniformis in soy molasses media (56 and 28 g L⁻¹ TRS, respectively) applied to maize seeds resulted in the highest Vigor Indexes of the seedlings, which correlated negatively with the broth pH and were not impacted by the γ-PGA and indole acetic acid produced by the bacteria. The low-cost and easily available feedstock soy molasses constitutes a potential culture medium for the growth of plant growth-promoting bacteria. Full article

The solid-state fermentation (SSF) efficiency of Bacillus licheniformis ATCC 21424 (BL) on various agro-industrial by-products such as oilseed cakes [hemp (HSC), pumpkin (PSC), and flaxseed (FSC)] was evaluated by examining the nutritional composition, reducing sugars, and in vitro protein digestibility (IVPD) for use in animal nutrition. SSF significantly decreased crude protein, along with changes in the total carbohydrates (p < 0.05) for all substrates fermented. An increase in crude fat for HSC (1.04%) and FSC (1.73%) was noted, vs. PSC, where the crude fat level was reduced (−3.53%). Crude fiber does not differ significantly between fermented and nonfermented oilseed cakes (p > 0.05). After fermentation, neutral detergent fiber (NDF) and acid detergent fiber (ADF) significantly increased for HSC and FSC (p < 0.05), as well as for PSC despite the small increase in ADF (4.46%), with a notable decrease in NDF (−10.25%). During fermentation, pH shifted toward alkalinity, and after drying, returned to its initial levels for all oilseed cakes with the exception of PSC, which maintained a slight elevation. Further, SSF with BL under optimized conditions (72 h) increases the reducing sugar content for FSC (to 1.46%) and PSC (to 0.89%), compared with HSC, where a reduction in sugar consumption was noted (from 1.09% to 0.55%). The viable cell number reached maximum in the first 24 h, followed by a slowly declining phase until the end of fermentation (72 h), accompanied by an increase in sporulation and spore production. After 72 h, a significant improvement in water protein solubility for HSC and FSC was observed (p < 0.05). The peptide content (mg/g) for oilseed cakes fermented was improved (p < 0.05). Through gastro-intestinal simulation, the bacterial survivability rate accounted for 90.2%, 101.5%, and 85.72% for HSC, PSC, and FSC. Additionally, IVPD showed significant improvements compared to untreated samples, reaching levels of up to 65.67%, 58.94%, and 80.16% for HSC, PSC, and FSC, respectively. This research demonstrates the advantages of oilseed cake bioprocessing by SSF as an effective approach in yielding valuable products with probiotic and nutritional properties suitable for incorporation into animal feed. Full article

The purpose of this study was to assess the impact of various concentrations of Bacillus licheniformis-fermented products (BLFP) on the growth and productivity of laying ducks (Anas platyrhynchos) subjected to heat stress during eight weeks of a feeding trial. A total of 150 one-day-old Brown Tsaiya ducks of both sexes were divided into five groups, with each group having three replicates and 10 ducks each for evaluation of growth performance. The treatment groups received dietary supplements of BLFP at levels of 0.1%, 0.2%, and 0.3%, along with a group receiving flavomycin (F) at 5 ppm, all over a 24-week period. The fermentation process in this study utilized a B. licheniformis strain (ATCC 12713) for the production of the spores through solid-state fermentation. The control group was given a basal diet consisting of yellow corn and soybean meal. The results showed that as compared to the flavomycin group, ducks in the 0.3% BLFP group had significantly higher body weights and better feed conversion rates. In addition, during the three weeks, the BLFP group showed higher feed consumption as compared to the control group. The jejunum villi length was significantly increased in the 0.2% BLPF group as compared to the control and flavomycin groups. This study also found that the flavomycin group had a significantly higher egg conversion rate, while the 0.1–0.3% BLFP groups had improved feed intake and the 0.3% group had significantly enhanced egg yolk color. Additionally, the 0.2% BLFP group showed substantial decreases in IL-1$\mathsf{\beta }$, TNF-$\mathsf{\alpha }$, IL-6, and IL-10 levels in the liver as well as an uptick in the tight junction protein Occludin gene expression in the colon when compared to the control group. Furthermore, the expression of the heat shock protein 70 in the gut upregulated in the 0.1% and 0.2% BLFP groups. In conclusion, these observations demonstrate that dietary supplementation of 0.2% BLFP is an ideal concentration to increase gut morphology, alleviate inflammatory response, and promote gut integrity in heat-stressed laying ducks. Full article

Food waste is a major issue that is increasingly affecting our environment. More than one-third of food is wasted, resulting in over $400 billion in losses to the U.S. economy. While composting and other small recycling practices are encouraged from person-to-person, it is not enough to balance the net loss of 80 million tons per year. Currently, one of the most promising routes for reducing food waste is through microbial fermentation, which can convert the waste into valuable bioproducts. Among the compounds produced from fermentation, 2,3-butanediol (2,3-BDO) has gained interest recently due to its molecular structure as a building block for many other derivatives used in perfumes, synthetic rubber, fumigants, antifreeze agents, fuel additives, and pharmaceuticals. Waste feedstocks, such as food waste, are a potential source of renewable energy due to their lack of cost and availability. Food waste also possesses microbial requirements for growth such as carbohydrates, proteins, fats, and more. However, food waste is highly inconsistent and the variability in composition may hinder its ability to be a stable source for bioproducts such as 2,3-BDO. This current study focuses specifically on post-consumer food waste and how 2,3-BDO can be produced through a non-model organism, Bacillus licheniformis YNP5-TSU during non-sterile fermentation. From the dining hall at Tennessee State University, 13 food waste samples were collected over a 6-month period and the compositional analysis was performed. On average, these samples consisted of fat (19.7%), protein (18.7%), ash (4.8%), fiber (3.4%), starch (27.1%), and soluble sugars (20.9%) on a dry basis with an average moisture content of 34.7%. Food waste samples were also assessed for their potential production of 2,3-BDO during non-sterile thermophilic fermentation, resulting in a max titer of 12.12 g/L and a 33% g/g yield of 2,3-BDO/carbohydrates. These findings are promising and can lead to the better understanding of food waste as a defined feedstock for 2,3-BDO and other fermentation end-products. Full article

During the production of nattokinase (NK) by Bacillus species, certain by-products such as poly-γ-glutamic acid (γ-PGA) are simultaneously synthesized. The impact of γ-PGA synthesis on NK production remains unclear. In this study, we knocked out the pgsC gene, a component of the γ-PGA synthetase cluster (pgsBCA), and constructed a novel recombinant strain, Bacillus licheniformis BL11. Next, we compared the fed-batch fermentation profiles of BL11 and its parental strain BL10, conducted transcriptional analysis, and measured intracellular ATP content. We also optimized glucose-feeding strategies under varying oxygen supply conditions. Our results indicated that the utilization rates of glucose and soybean meal were both improved in the pgsC-deficient strain BL11, and NK activity was enhanced. Furthermore, the transcriptional levels of genes involved in glycolysis and the TCA cycle were relatively upregulated in BL11. The maximal NK activity reached 2522.2 FU/mL at 54 h of fermentation using a constant glucose-feeding rate of 5.0 g/(L·h) under high oxygen supply conditions. The newly developed recombinant strain B. licheniformis BL11, along with the optimized feeding strategy, shows promise for large-scale NK production. Full article