Microbial Fermentation Research Articles

Utilization of coffee waste as a sustainable feedstock for high-yield lactic acid production through microbial fermentation

Lactic acid is an important industrial precursor; however, high substrate costs are a major challenge in microbial fermentation-based lactic acid production. Coffee waste is a sustainable feedstock alternative for lactic acid production via microbial fermentation. Herein, the feasibility of coffee waste as a feedstock was explored by employing appropriate pretreatment methods and optimizing enzyme combinations. Coffee waste pretreatment with hydrogen peroxide and acetic acid along with a combination of Viscozyme L, Celluclast 1.5 L, and Pectinex Ultra SP-L achieved the 78.9 % sugar conversion rate at a substrate concentration of 4 % (w/v). Lactiplantibacillus plantarum WiKim0126-induced fermentation with a 4 % solid loading yielded a lactic acid concentration of 22.8 g/L (99.6 % of the theoretical maximum yield) and productivity of 0.95 g/L/h within 24 h. These findings highlight the viability of coffee waste as an eco-friendly resource for sustainable lactic acid production.

Engineered biosynthesis of plant heteroyohimbine and corynantheine alkaloids in Saccharomyces cerevisiae.

An Saccharomyces cerevisiae-based microbial platform was developed for the biosynthesis of monoterpene indole alkaloids, including the universal precursor strictosidine and further modified heteroyohimbine and corynantheidine alkaloids.

The Next Food Revolution Is Here: Recombinant Microbial Production of Milk and Egg Proteins by Precision Fermentation.

Animal-based agriculture and the production of protein-rich foods from animals, particularly from ruminants, are not sustainable and have serious climate effects. A new type of alternative proteins is now on the menu, namely animal proteins produced recombinantly by microbial fermentation. This new technology, precision fermentation, is projected to completely disrupt traditional animal-based agriculture. Certain milk and egg proteins along with specific meat substitute analog components produced by precision fermentation are already entering the market. This first wave of precision fermentation products targets the use of these proteins as protein additives, and several commercial players are already active in the field. The cost-efficiency requirements involve production titers above 50 g/L which are several orders of magnitude higher than those for pharmaceutical protein manufacture, making strain engineering, process optimization, and scale-up critical success factors. This new development within alternative proteins defines a new research direction integrating biotechnology, process engineering, and sustainable food protein production.

Lactate cross-feeding between Bifidobacterium species and Megasphaera indica contributes to butyrate formation in the human colonic environment.

Butyrate, a physiologically active molecule, can be synthesized through metabolic interactions among colonic microorganisms. Previously, in a fermenting trial of human fecal microbiota, we observed that the butyrogenic effect positively correlated with the increasing Bifidobacterium population and an unidentified Megasphaera species. Therefore, we hypothesized that a cross-feeding phenomenon exists between Bifidobacterium and Megasphaera, where Megasphaera is the butyrate producer, and its growth relies on the metabolites generated by Bifidobacterium. To validate this hypothesis, three bacterial species (B. longum, B. pseudocatenulatum, and M. indica) were isolated from fecal cultures fermenting hydrolyzed xylan; pairwise cocultures were conducted between the Bifidobacterium and M. indica isolates; the microbial interactions were determined based on bacterial genome information, cell growth, substrate consumption, metabolite quantification, and metatranscriptomics. The results indicated that two Bifidobacterium isolates contained distinct gene clusters for xylan utilization and expressed varying substrate preferences. In contrast, M. indica alone scarcely grew on the xylose-based substrates. The growth of M. indica was significantly elevated by coculturing it with bifidobacteria, while the two Bifidobacterium species responded differently in the kinetics of cell growth and substrate consumption. Coculturing led to the depletion of lactate and increased the formation of butyrate. An RNA-seq analysis further revealed the upregulation of M. indica genes involved in the lactate utilization and butyrate formation pathways. We concluded that lactate generated by Bifidobacterium through catabolizing xylose fueled the growth of M. indica and triggered the synthesis of butyrate. Our findings demonstrated a novel cross-feeding mechanism to generate butyrate in the human colon.IMPORTANCEButyrate is an important short-chain fatty acid that is produced in the human colon through microbial fermentation. Although many butyrate-producing bacteria exhibit a limited capacity to degrade nondigestible food materials, butyrate can be formed through cross-feeding microbial metabolites, such as acetate or lactate. Previously, the literature has explicated the butyrate-forming links between Bifidobacterium and Faecalibacterium prausnitzii and between Bifidobacterium and Eubacterium rectale. In this study, we provided an alternative butyrate synthetic pathway through the interaction between Bifidobacterium and Megasphaera indica. M. indica is a species named in 2014 and is indigenous to the human intestinal tract. Scientific studies explaining the function of M. indica in the human colon are still limited. Our results show that M. indica proliferated based on the lactate generated by bifidobacteria and produced butyrate as its end metabolic product. The pathways identified here may contribute to understanding butyrate formation in the gut microbiota.

Recent trends in lactic acid-producing microorganisms through microbial fermentation for the synthesis of polylactic acid.

Polylactic acid (PLA) is a range of unique bioplastics that are bio-based and biodegradable. PLA is currently driving market expansion for lactic acid (LA) due to its high demand as a building block in production. One of the most practical and environmentally benign techniques for synthesising PLA is through enzymatic polymerisation of microbial LA monomers. However, microbial LA fermentation does have some limitations. Firstly, it requires the use of a nutritionally rich medium. Secondly, LA production can be disrupted by bacteriophage infection or other microorganisms. Lastly, the yield can be low due to the formation of by-products through heterofermentative pathway. Considering the potential use of PLA as a replacement for conventional petrochemical-based polymers in industrial applications, researchers are focused on exploring the diversity of LA-producing microorganisms from various niches. Their goal is to study the functional properties of these microorganisms and their ability to produce industrially valuable metabolites. This review highlights the advantages and disadvantages of lactic acid-producing microorganisms used in microbial fermentation for PLA synthesis.

Koopman modeling and optimal control for microbial fed-batch fermentation with switching operators

The modeling of microbial fed-batch fermentation with switching operators to product 1,3-propanediol (1,3-PD) still maintains a challenge because it is strongly of nonlinearity and uncertainty. Machine learning methods for learning such models have become a hot research topic, but the interpretability of existing techniques remains a challenging problem. Recently, the Koopman operator, which is a linear operator governing the eigenfunction evolution along trajectories of a nonlinear dynamical system with switching operators, has been studied for modeling complex dynamics. In this paper, we propose a Koopman modeling method based on an interpretable Koopman operator. The predominant merit of using the Koopman operator is to offer a linear infinite dimensional description of a nonlinear dynamical system with switching operators. In the proposed method, an enhanced learning-based extended dynamic mode decomposition (enhanced-EDMD) algorithm based on a novel eigenfunction construction method is proposed to obtain a finite-dimensional approximation of the Koopman operator. The convergence analysis of the enhanced-EDMD algorithm is also studied. Furthermore, to maximize the productivity of 1,3-PD and minimize the total variation in the optimal control within a time frame, an algorithm combining the model predictive control method with the enhanced learning-based EDMD (denoted by MPC-Enhanced-EDMD), based on gradient-based optimization and exact penalty function method, is proposed for devising optimal feeding rate of glycerol evolving with time. Numerical simulations are conducted by demonstrating the effectiveness of the enhanced-EDMD algorithm on the dynamics prediction and the MPC-Enhanced-EDMD method on the optimal feeding rates of glycerol.

A Physiogenomic Study of the Tolerance of Saccharomyces cerevisiae to Isoamyl Alcohol

Isoamyl alcohol is a clear, unpleasantly odorous, colorless liquid of higher alcohol that emits a fruity aroma when heavily diluted. It has received much attention in recent years as a new fuel with a high energy density. Isoamyl alcohol can be produced industrially by microbial fermentation. Still, its toxicity to host cells has limited its potential for industrial production, and the molecular mechanism of its toxic effects has not yet been elucidated. In this study, RNA-Seq technology was used to analyze the transcripts of Saccharomyces cerevisiae under normal conditions and in the presence of isoamyl alcohol (0.5 g/L and 2.5 g/L). The results showed that the expression of the cell wall (CCW12, BGL2, NCW2 and SUN4), cell membrane (ELO1, ERG2, FAA1, and OPI3), translation and other structural genes were significantly down-regulated. The expression of genes related to ATP biosynthesis, NADPH biosynthesis (ZWF1), and metal ion transport (PMC1) proteins were up-regulated. Strains with key genes knocked out were cultured without isoamyl alcohol. Combined results suggested that isoamyl alcohol may affect cell wall stability and cell membrane fluidity, and the expression of genes related to ion homeostasis and energy production may play a protective role against isoamyl alcohol stress. By maintaining cell wall stability/membrane fluidity under isoamyl alcohol pressure, improving certain ion homeostasis, and generating energy/NADPH, it is possible to overcome the toxicity of isoamyl alcohol in industrial fermentation processes to a certain extent.

Temperature-dependent microbial reactions by indigenous microbes in bentonite under Fe(III)- and sulfate-reducing conditions

Bentonite is a promising buffer material for constructing spent nuclear fuel (SNF) repositories. However, indigenous microbes in bentonite can be introduced to the repository and subsequent sealing of the repository develops anoxic conditions over time which may stimulate fermentation and anaerobic respiration, possibly affecting bentonite structure and SNF repository stability. Moreover, the microbial activity in the bentonite can be impacted by the heat generated from radionuclides decay. Therefore, to investigate the temperature effect on microbial activities in bentonite, we created microcosms with WRK bentonil (a commercial bentonite) using lactate as the electron donor, and sulfate and/or ferrihydrite (Fe(III)) as electron acceptors with incubation at 18 ℃ and 50 ℃. Indigenous WRK microbes reduced sulfate and Fe(III) at both temperatures but with different rates and extents. Lactate was metabolized to acetate at both temperatures, but only to propionate at 18 ℃ during early-stage microbial fermentation. More Fe(III)-reduction at 18 ℃ but more sulfate-reduction at 50 ℃ was observed. Thermophilic and/or metabolically flexible microbes were involved in both fermentation and Fe(III)/sulfate reduction. Our findings illustrate the necessity of considering the influence of temperature on microbial activities when employing bentonite as an engineered buffer material in construction of SNF repository barriers.

Synthesis of Poly-γ-Glutamic Acid and Its Application in Biomedical Materials.

Poly-γ-glutamic acid (γ-PGA) is a natural polymer composed of glutamic acid monomer and it has garnered substantial attention in both the fields of material science and biomedicine. Its remarkable cell compatibility, degradability, and other advantageous characteristics have made it a vital component in the medical field. In this comprehensive review, we delve into the production methods, primary application forms, and medical applications of γ-PGA, drawing from numerous prior studies. Among the four production methods for PGA, microbial fermentation currently stands as the most widely employed. This method has seen various optimization strategies, which we summarize here. From drug delivery systems to tissue engineering and wound healing, γ-PGA's versatility and unique properties have facilitated its successful integration into diverse medical applications, underlining its potential to enhance healthcare outcomes. The objective of this review is to establish a foundational knowledge base for further research in this field.

Preparation of potential organic fertilizer rich in γ-polyglutamic acid via microbial fermentation using brewer's spent grain as basic substrate

Brewer’s spent grain (BSG) is a main byproduct of the beer industry. BSG is rich in a variety of nutrients, and the search for its effective, high-value utilization is ongoing. Environmental probiotic factor γ-PGA was produced by fermenting Bacillus subtilis with BSG substrate and the fermenting grain components were analyzed. The γ-PGA yield reached 31.58 ± 0.21 g/kg of BSG. Gas chromatography-mass spectrometry and non-targeted metabolomics analyses revealed 73 new volatile substances in the fermenting grains. Furthermore, 2,376 metabolites were upregulated after fermentation and several components were beneficial for plant growth and development (such as ectoine, acetyl eugenol, L-phenylalanine, niacin, isoprene, pantothenic acid, dopamine, glycine, proline, jasmonic acid, etc). These results show that it is possible to synthesize adequate amounts of γ-PGA for use as a functional fertilizer.

Effect of Milk Origin and Seasonality of Yogurt Acid Whey on Antioxidant Activity before and after In Vitro Gastrointestinal Digestion.

Yogurt acid whey (YAW) is a by-product of Greek strained yogurt production. The disposal of YAW constitutes an environmental problem, and given the increasing demand of Greek yogurt worldwide, its handling is a challenge. However, whey-derived peptides, resulting from microbial fermentation as well as those resulting from further hydrolysis during the digestion process, have been linked to enhanced biological activities. In this study, the antioxidant capacity of 33 samples of YAW obtained from Greek dairy companies of bovine, ovine or caprine origin was investigated using both cell-free and cell-based assays. The YAW samples, their in vitro digestion products (YAW-Ds) and a fraction of the digests (less than 3 kDa; YAW-D-P3) were assessed using four biochemical assays, namely ORAC, ABTS, FRAP and P-FRAP. Our data revealed a higher antioxidant capacity for digested samples compared with undigested samples, with all four methods. ORAC values after in vitro digestion were higher for the ovine samples compared to their bovine (YAW-D and YAW-D-P3) and caprine (YAW-D-P3) counterparts. Furthermore, the YAW-D-P3 fraction derived from samples collected in the summer months exhibited higher ORAC values when compared to the respective fraction from the winter months' samples. The cellular antioxidant activity of ovine YAW-D-P3 was improved in H2O2-treated HT29 cells compared to the control H2O2-treated cells. However, YAW-D-P3 could not trigger either the pathways involving the transcription factors NF-κB or NFE2L2 or the gene expression of SOD1, CAT and HMOX1 in LPS-challenged THP-1-derived macrophages. These results suggest that YAW, and particularly YAW from ovine origin, could be used as a natural source for its antioxidant potential in human and animal nutrition.

Solid-state fermentation for gossypol detoxification and nutritive enrichment of cottonseed cake: A scale-up of batch fermentation process

Gossypol, a toxic polyphenolic compound, limits the use of cottonseed cake (CSC) in animal feed. Different approaches have been employed to detoxify gossypol and improve the nutritive properties of feed. Microbial fermentation improves the nutritive quality of CSC by increasing lysine content and reducing free and bound gossypol. In this study, microbial fermentation was scaled up under batch conditions using a prototype device at the capacity of 40 kg per day. The mixed fungal culture C. tropicalis + S. cerevisiae was used for fermentation. An industrial trial was taken to ascertain the gossypol detoxification efficiency. The fermented CSC obtained under scale-up process had 60 to 80% and 40 to 60% reduction of free and bound gossypol, respectively, compared with raw CSC. The fermented CSC demonstrated an increase in crude protein (4 to 12%) and lysine (0.3 to 0.4%) and decrease in crude fibre (3 to 11%). The fermented CSC met the standards of US Food and Drug Administration in terms of its nutritional property. Thus, the simple method described in this study could be adopted for the production of detoxified CSC for use in the animal feed industry.

Fu Brick Tea as a Staple Food Supplement Attenuates High Fat Diet Induced Obesity in Mice.

Fu brick tea (FBT), a product of microbial fermentation from primary dark tea, also known as raw material tea (RMT), has been extensively studied for its functional properties. However, its potential as a staple food supplement for weight loss remains poorly understood. This study compared the weight loss effects of orlistat, traditional plain noodles (NN), and noodles supplemented with varying amounts of RMT (RMTN) and FBT (FBTN), with the aim to elucidate their lipid-reducing effects and underlying mechanisms. Experimental trials on high fat diet fed mice revealed significant weight loss, lipid-lowering, and hypoglycemic effects upon supplementation with orlistat, RMTN, and FBTN. Moreover, supplementation with orlistat, RMTN, and FBTN effectively restored serum and liver-related index levels, mitigating high-fat diet-induced dyslipidemia. Additionally, these supplements ameliorated liver and kidney damage by inhibiting oxidative stress and inflammatory responses. Furthermore, orlistat, RMTN, and FBTN exert their anti-obesity effects primarily by modulating genes associated with lipid metabolism and inflammatory responses and through regulation of the composition and structure of the gut microbiota. Importantly, FBTN demonstrated a significantly stronger lipid-lowering effect compared to RMTN, particularly at higher tea addition ratios. In contrast, NN supplementation exhibited minimal to no weight loss effects. Based on these findings, it could be inferred that FBT holds promise as a staple food supplement to ameliorate high-fat diet-induced obesity and its associated health conditions.

An efficient and eco-friendly approach for the sustainable recovery and properties characterization of polyhydroxyalkanoates produced by methanotrophs

Synthetic biodegradable and bio-based polymers have emerged as sustainable alternatives to nonrenewable petroleum-derived polymers which cause serious environmental issues. In particular, polyhydroxyalkanoates (PHA) are promising biopolymers owing to their outstanding biodegradability and biocompatibility. The production of the homopolymer poly(3-hydroxybutyrate) (PHB) and copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) from type II methanotrophs via microbial fermentation was presented. For the efficient extraction and recovery of intracellular PHA from methanotrophs, different extraction approaches were investigated including solvent extraction using 1,3-dioxolane as a green solvent, integrated cell lysis and solvent extraction, and cell digestion without the use of organic solvents. Among various extraction approaches, the integrated method exhibited the highest extraction performance, with PHA recovery and purity exceeding 91 % and 93 %, respectively, even when the PHA content of the cells was low. Furthermore, the molecular weight, thermal stability, and mechanical properties of the recovered PHA were comprehensively analyzed to suggest its suitable practical applications. The obtained properties were comparable to that of the commercial PHA products and PHA produced from other microbial species, indicating an efficient recovery of high-quality PHA produced from methanotrophs.

Safety assessment, whole genome sequence, and metabolome analysis of Streptococcus thermophilus CICC 20372 for bone cement fermentation.

Bone is a kind of meat processing by-product with high nutritional value but low in calorie, which is a typical food in China and parts of East Asian countries. Microbial fermentation by lactic acid bacteria showed remarkable advantages to increase the absorption of nutrients from bone cement by human body. Streptococcus thermophilus CICC 20372 is proven to be a good starter for bone cement fermentation. No genes encoding virulence traits or virulence factors were found in the genome of S. thermophilus CICC 20372 by a thorough genomic analysis. Its notable absence of antibiotic resistance further solidifies the safety. Furthermore, the genomic analysis identified four types of gene clusters responsible for the synthesis of antimicrobial metabolites. A comparative metabolomic analysis was performed by cultivating the strain in bone cement at 37°C for 72h, with the culture in de Man, Rogosa, and Sharpe (MRS) medium as control. Metabolome analysis results highlighted the upregulation of pathways involved in 2-oxocarboxylic acid metabolism, ATP-binding cassette (ABC) transporters, amino acid synthesis, and nucleotide metabolism during bone cement fermentation. S. thermophilus CICC 20372 produces several metabolites with health-promoting function during bone cement fermentation, including indole-3-lactic acid, which is demonstrated ameliorative effects on intestinal inflammation, tumor growth, and gut dysbiosis. In addition, lots of nucleotide and organic acids were accumulated at higher levels, which enriched the fermented bone cement with a variety of nutrients. Collectively, these features endow S. thermophilus CICC 20372 a great potential strain for bone food processing.

Evidence of the Beneficial Impact of Three Probiotic-Based Food Supplements on the Composition and Metabolic Activity of the Intestinal Microbiota in Healthy Individuals: An Ex Vivo Study.

Scientific evidence has increasingly supported the beneficial effects of probiotic-based food supplements on human intestinal health. This ex vivo study investigated the effects on the composition and metabolic activity of the intestinal microbiota of three probiotic-based food supplements, containing, respectively, (1) Bifidobacterium longum ES1, (2) Lactobacillus acidophilus NCFM®, and (3) a combination of L. acidophilus NCFM®, Lactobacillus paracasei Lpc-37™, Bifidobacterium lactis Bi-07™, and Bifidobacterium lactis Bl-04™. This study employed fecal samples from six healthy donors, inoculated in a Colon-on-a-plate® system. After 48 h of exposure or non-exposure to the food supplements, the effects were measured on the overall microbial fermentation (pH), changes in microbial metabolic activity through the production of short-chain and branched-chain fatty acids (SCFAs and BCFAs), ammonium, lactate, and microbial composition. The strongest effect on the fermentation process was observed for the combined formulation probiotics, characterized by the significant stimulation of butyrate production, a significant reduction in BCFAs and ammonium in all donors, and a significant stimulatory effect on bifidobacteria and lactobacilli growth. Our findings suggest that the combined formulation probiotics significantly impact the intestinal microbiome of the healthy individuals, showing changes in metabolic activity and microbial abundance as the health benefit endpoint.

Microbial network and fermentation modulation of Napier grass and sugarcane top silage in southern Africa.

Feed shortage in the tropics is a major constraint to the production of livestock products such as milk and meat. In order to effectively utilize of local feed resources, the selected lactic acid bacteria (LAB) strain was used to prepare Napier grass and sugarcane top silage. The results showed that the two silages inoculated with LAB formed a co-occurrence microbial network dominated by Lactiplantibacillus during the fermentation process, regulated the microbial community structure and metabolic pathways, and improved the silage fermentation quality. This is of great significance for alleviating feed shortage and promoting sustainable production of livestock.

Investigation the nutritional properties of Auricularia auricula pulp fermented with Lactobacillus strains and their effects on gut microbiota.

Auricularia auricula is rich in bioactive components, and microbial fermentation can further dramatically increase its content and bioavailability. However, there are few studies on the relationship between fermented A. auricula pulp (FAAP) and gut microbiota. In this study, standard strains Lactobacillus plantarum 21801 and 21805 purchased from the China Center of Industrial Culture Collection were used to ferment A. auricula pulp at a ratio of 2:1, with an inoculum of 5%, a fermentation temperature of 31 °C, and a fermentation time of 22 h. The nutritional properties, aroma, and color of FAAP and their effects on the body characteristics of mice and the structure and abundance of gut microbiota are discussed. The results showed that, compared with A. auricula pulp, FAAP significantly increased the nutritional properties while maintaining favorable sensory quality and flavor profiles. Among them, the content of total polyphenols and total flavonoids reached 22.04 μg mL-1 and 20.56 μg mL-1 respectively, and the 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid free-radical scavenging rate increased to 73.21%. The consumption of FAAP had no negative effects on weight or liver and kidney function in mice and dramatically enhanced the antioxidant capacity in the liver and serum. The production of short-chain fatty acids in the gut was promoted, the relative abundance of beneficial bacteria (Lactobacillus, Bifidobacterium, norank_f__Muribaculaceae and unclassified_f__Lachnospiraceae) increased, and the growth of some pathogenic bacteria (Helicobacter, Mucispirillum, and Alloprevotella) was inhibited. These findings demonstrate that FAAP is rich in nutrients and has unique functional properties that promote host health and regulate the gut microbiota. © 2023 Society of Chemical Industry.

Production of second generation bioethanol from straw during simultaneous microbial saccharification and fermentation

Production of second generation bioethanol from straw during simultaneous microbial saccharification and fermentation

Unveiling microbial biomarkers of ruminant methane emission through machine learning.

Enteric methane from cow burps, which results from microbial fermentation of high-fiber feed in the rumen, is a significant contributor to greenhouse gas emissions. A promising strategy to address this problem is microbiome-based precision feed, which involves identifying key microorganisms for methane production. While machine learning algorithms have shown success in associating human gut microbiome with various human diseases, there have been limited efforts to employ these algorithms to establish microbial biomarkers for methane emissions in ruminants. In this study, we aim to identify potential methane biomarkers for methane emission from ruminants by employing regression algorithms commonly used in human microbiome studies, coupled with different feature selection methods. To achieve this, we analyzed the microbiome compositions and identified possible confounding metadata variables in two large public datasets of Holstein cows. Using both the microbiome features and identified metadata variables, we trained different regressors to predict methane emission. With the optimized models, permutation tests were used to determine feature importance to find informative microbial features. Among the regression algorithms tested, random forest regression outperformed others and allowed the identification of several crucial microbial taxa for methane emission as members of the native rumen microbiome, including the genera Piromyces, Succinivibrionaceae UCG-002, and Acetobacter. Additionally, our results revealed that certain herd locations and feed composition markers, such as the lipid intake and neutral-detergent fiber intake, are also predictive features for methane emissions. We demonstrated that machine learning, particularly regression algorithms, can effectively predict cow methane emissions and identify relevant rumen microorganisms. Our findings offer valuable insights for the development of microbiome-based precision feed strategies aiming at reducing methane emissions.