Fermentation

The ideal yeast for the production of alcohol-free beer does not form ethanol, produces a distinct and pleasant taste and has probiotic properties. This study characterized the potential of a wine yeast, Saccharomyces cerevisiae CCM 9181, to be an ideal alcohol free beer strain. It was found to be maltose-negative, and the ethanol content in fermented all-malt wort has never exceeded the legal limit of 0.5% v/v. Its specific growth rate (µ) was the highest at 25 °C (μ = 0.41 ± 0.01 h⁻¹) and it was not affected by iso-α-bitter acids (15–50 IBU, international bitterness units). A response surface methodology was used to optimize the temperature and pitching rate affecting the formation of total higher alcohols and esters. A statistical analysis of the experimental data revealed that temperature affected esters most significantly, while both temperature and pitching rate had the most significant effects on higher alcohols. The sensory properties of beers were evaluated by trained panelists and they were described as malty, clove-like, having a very mild bitterness and a bouquet of white wine. The survival rate of S. cerevisiae CCM 9181 after simulated passage through the gastrointestinal tract was investigated as a first step to evaluate its probiotic properties. Our analyses show that Saccharomyces cerevisiae CCM9181 is a suitable candidate for the large-scale commercial production of alcohol-free beer and has probiotic potential that needs to be studied further. Full article

The production of secondary metabolites can be improved with the supply of precursors both in submerged and solid-state fermentation (SSF). Microorganisms assimilate the precursors and biotransform them to excrete compounds of commercial interest. The raw materials used in SSF, frequently agro-industrial residues, may contain molecules that serve as precursors for secondary metabolites. However, supplying a precursor can dramatically improve crop production. Commonly, precursors are added as part of the liquid with which the solid material to be fermented is moistened. However, recently it has been proposed to take advantage of the oxygen supply for the gradual supply of volatile precursors. It can help to avoid toxicity problems with the precursors. The present work reviews the strategies to supply precursors to improve the production of secondary metabolites in solid-state fermentation. Full article

Lignocellulosic biomasses have a very important role as raw materials to produce biobased chemicals. However, a sustainable, efficient, and economically competitive way to convert lignocellulosic biomass into these chemicals has still not been achieved. This study is related to the selective separation and conversion of birch wood C5 carbohydrates into furfural during the H₃PO₄–NaH₂PO₄-catalyzed hydrothermal pretreatment simultaneously preserving cellulose in the lignocellulosic leftover for glucose production by the enzymatic hydrolysis. The ratio of H₃PO₄–NaH₂PO₄ in the catalyst solution was changed (3:0, 2:1, 1:1, and 1:2). Results show that around 64.1 to 75.9% of available C5 carbohydrates were converted into furfural. The results of birch wood lignocellulosic leftover chemical composition analysis show that cellulose losses during the pretreatment stage did not reach more than 10% of the initial amount. Based on the enzymatic hydrolysis screening experiments, a suitable catalyst for pretreatment was selected and an in-depth study was carried out. Enzymatic hydrolysis experiments were organized based on the three-factor central composite face-centered design. The variable parameters were treatment time (24–72 h), enzyme load (10–20 U/g cellulose), and substrate amount in reaction media (10–20%). At optimal conditions, 49.9 ± 0.5% of available cellulose in lignocellulosic leftover was converted into glucose. Full article

L-Cysteine is a widely used unique sulfur-containing amino acid with wide application in the food, pharmaceutical, and agricultural industries. This paper concludes the advantages and disadvantages of chemical hydrolysis, enzymatic biotransformation, and fermentation for the synthesis of L-cysteine. Meanwhile, a detailed introduction is given to the biosynthesis of L-cysteine, metabolic engineering strategies, and the latest progress in reported L-cysteine fermentation bacteria. Finally, insights are provided on the development direction of increasing the production of biosynthetic L-cysteine in the future. This review provides ideas for the future development of more efficient L-cysteine biosynthetic pathways. Full article

The role of volatile fatty acids (VFAs) in ruminal fermentation is well studied, but their effects on the ileal microbiome and epithelial health remain less explored. In this study, we investigated the impact of the exogenous oral infusion of three VFAs, sodium acetate (NaAc), propionate (NaPr), and butyrate (NaBu), on the VFA concentrations in ileal contents, as well as ileal microbiome profiling and epithelial health parameters (inflammatory cytokine and tight junctions) in goats. The data demonstrated that the oral infusion of three VFAs can enhance VFA production by increasing the proportions of each individual VFA and the total VFAs. Then, the microbiome revealed distinct microbial succession patterns and altered microbial diversities in response to the oral infusion of VFA treatments. Moreover, the oral infusion of each VFA had unique effects on the ileal bacterial community, with potential implications for epithelial health. Notably, the oral infusion of VFAs demonstrated potential anti-inflammatory effects, as evidenced by reduced IL-6 levels in the NaPr group and increased IL-10 levels in the NaAc group. Notably, the oral infusion of VFAs did not directly impact the tight junction concentrations, such as Claudin1, Occludin, and ZO-1. Lastly, the correlation analyses identified specific relationships between the ileal bacteria and epithelial health parameters, and Prevotella was positively correlated with IL-6 and IL-1β, while Bifidobacterium was positively correlated with IL-10. These results highlighted the crosstalk between VFAs, the ileal microbiome, and the health of the small intestine. Our findings had significant implications for optimizing ruminant nutrition, enhancing epithelial health, and promoting sustainable livestock production practices. Full article

This study demonstrates that the fermentation of Tetragonia tetragonioides (T. tetragonioides) by Bacillus subtilis (B. subtilis) subsp. spizizenii enhances its antioxidant, anti-inflammatory, and skin-moisturizing activities. Fermented T. tetragonioides extracts (FTEs) showed a significant increase (p < 0.05) of approximately 1.3 to 3.07 times in their total polyphenol content (TPC), total flavonoid content (TFC), and vanillic acid content compared to pre-fermentation T. tetragonioides extracts (TEs). Additionally, the 2,2-diphenyl-1-picrylhydrazyl (DPPH)- and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical-scavenging activities were significantly higher (p < 0.05) in FTEs than in TEs, showing an increase of about 1.25 to 1.64 times. The anti-inflammatory effects, evaluated through the inhibition of nitric oxide (NO) in RAW 264.7 cells stimulated by lipopolysaccharide (LPS), revealed that FTEs exhibited significant (p < 0.05) NO inhibition activity at less than half the concentration of TEs. It is particularly noteworthy that the FTE at 200 μg/mL significantly suppressed the expression of the cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), and tumor necrosis factor-alpha (TNF-α) proteins. In HaCaT cells, FTEs substantially (p < 0.001) increased the mRNA expression of filaggrin (FLG), hyaluronan synthase (HAS)-1, and HAS-3, indicating improved skin protection and moisturization. In conclusion, this study confirms that T. tetragonioides’ antioxidant, anti-inflammatory, and skin-moisturizing activities are enhanced by B. subtilis fermentation, suggesting the potential of FTEs as a cosmeceutical ingredient. Full article

Increasing greenhouse gas emissions and decreasing fossil fuel supplies necessitate the development of alternative methods for producing petroleum-based commodities. Plastics are also primarily petroleum-based goods with rising demand, thus there is growing interest in plastic substitutes. Polyhydroxyalkanoates (PHAs) are naturally produced biopolymers that are utilized by microorganisms as a source of energy and carbon storage. Poly-3-hydroxybutyrate (PHB) is a member of the PHA family and is considered the most promising candidate to replace polyethylene (PE). PHB is naturally produced by Cupriavidus necator, but recombinant production has also been recently established. This study is the first to investigate the heterologous production of PHB with recombinant Acetobacterium woodii using CO₂ + H₂ as a carbon and energy source. The introduction of a synthetic PHB production pathway resulted in the production of 1.23 g/L CDW and 1.9% PHB/cell dry weight (CDW), which corresponds to a production of 23.5 mg/L PHB. PHB quantification was simplified using LipidGreen2 fluorescence measurements. Full article

Digesters at water resource recovery facilities (WRRFs) operating at different temperatures within the mesophilic and thermophilic temperature range is a flexibilization concept to contribute to heat management. Four 25 L digesters were fed with sewage sludge from a municipal WRRF and were operated at 37, 43, 47 and 53 °C, respectively, to describe changes in the overall process performance and the microbiota. Specific methane yield and COD degradation rates were the highest at 47 °C, only being up to 7% higher compared with at 37 °C. The increase in pH and concentrations of NH₄-N and PO₄-P above 43 °C were statistically significant. The effect on the microbial community was strong, indicating both a constant specialization towards thermophilic organisms as well as a change from acetoclastic to hydrogenotrophic/methylotrophic methanogenesis. The influence of temperature on process-engineering and physicochemical aspects was rather small compared with the changes in the microbiota. Full article

Wastewater with a low carbon/nitrogen (C/N) ratio is widespread and difficult to treat. The addition of an external carbon source is an effective method for treating such wastewater. Therefore, we aimed to prepare a self-releasing carbon biofilm carrier using agricultural waste (corncobs), polyvinyl alcohol, and sponge iron in various ratios to provide a carbon source that would facilitate denitrification, providing an optimal environment for microorganisms. We found that the carbon release of the MAC biofilm carrier that accumulated over 60 d was 116.139 mg of chemical oxygen demand (COD)·g⁻¹, whereas the accumulated total nitrogen release was approximately 0 mg·(g·d)⁻¹. The NO₃⁻-N removal rate after 24 h reached 98.1%, whereas the theoretical use rate of the carbon source (in terms of COD) was stable at 90.34%. In addition, the sum of the abundances of the denitrifying and cellulose-degrading bacteria was 49.89%. Furthermore, biofilm carriers are used as functional carriers that contribute to cellulose degradation, a process in which sponge iron produces Fe²⁺ to provide electron donors and shuttles for denitrifying bacteria and forms the iron cycle, thereby inducing an increase in microbial abundance; this increase then facilitates the microbial degradation of cellulose and synergistic denitrification through interspecific bacterial cooperation. This study provides a new and effective method for enhancing the denitrification of wastewater with low C/N ratios. Full article

AA10 family lytic polysaccharide monooxygenases (AA10 LPMOs) are mainly distributed in bacteria. Because of their characteristics of oxidative degradation of crystalline polysaccharides, such as cellulose and chitin, they have great application potential in industrial biomass conversion and have attracted wide attention. Efficient heterologous expression of LPMOs by recombinant engineering bacteria has become the main strategy for the industrial production of enzymes. The research progress of AA10 LPMOs’ heterologous expression systems was reviewed in this paper. The construction strategies of its diversified heterologous expression system were introduced based on the design and processing of the expression host, vector, and LPMOs gene. The effects of different expression systems on the soluble expression of LPMOs and the development direction of the construction of LPMOs’ heterologous expression systems were discussed. The broad application prospect of LPMOs in the biomass conversion and biofuel industry has been prospected. Full article

Dosidicus gigas is a kind of low-fat food with poor gel properties. Fermentation has been proved to be an effective food processing method that could improve the gel properties of meat. Here, we inoculated D. gigas with two strains, Lacticaseibacillus casei and Staphylococcus carnosus, that have been approved for use in meat processing, and studied their impact on the quality of the product. Compared with the uninoculated samples, inoculation with L. casei and mixed inoculation with L. casei and S. carnosus were able to significantly reduce pH during fermentation. The plate counting results showed that L. casei may have adapted well to the environment in the inoculated groups, while the growth of Staphylococcus may have been inhibited in the mixed inoculated group. 16s rRNA sequencing confirmed that inoculation significantly altered the bacterial composition of squid surimi sausages. Both inoculation with L. casei and mixed inoculation with L. casei and S. carnosus were able to inhibit the accumulation of the main biogenic amines, and in the mixed inoculated group, the main biogenic amines were lower. Compared with unfermented squid surimi sausages, mixed inoculation changed the texture, gel properties, color, and appearance of squid surimi sausages. These results showed that mixed inoculation can not only ensure safety, but also improve the quality of squid surimi sausages. Full article

Yarrowia lipolytica is a widely used microorganism in biotechnology since it is capable of producing a wide range of products (lipase, citric acid, polyols). A less-studied related strain is Y. divulgata, which is also capable of erythritol production in even higher concentration than most Y. lipolytica wild strains from glycerol as renewable feedstock. Thus, the aim of this work was to investigate Y. divulgata’s complex utilisation based on erythritol fermentation from glycerol to establish a Yarrowia-based biorefinery in which both the fermentation broth and separated cells are converted into high added-value products (erythritol, bioemulsifier, cosmetic ingredient, i.e., skin moisturizer). An important parameter of erythritol fermentation is an adequate oxygen level, so both the constant oxygen level and oxygen absorption rate were investigated regarding the three target products. DO (dissolved oxygen) = 10, 20, 30, 40% was examined in the bioreactor, and a KLa range of 18–655 h⁻¹ was investigated in both the bioreactor and in different types of shaking flasks, applying two different glycerol levels (100–150 g/L). The results showed that the Yarrowia divulagata NCAIM 1485 strain could produce one of the highest amounts of erythritol (44.14 ± 1 g/L) among wild-type yeasts from 150 g/L glycerol beside a K_La value of 655 h⁻¹. Cell-lysates skin hydrating activity was the highest (12%) when DO = 20% (K_La 26.4 h⁻¹) was applied. In all cases, the collected samples had an emulsification index above 69% which did not decrease below 54% after 24 h, showing good stability. Since Y. divulgata fermentations resulted in three high added-value products at the same time from a renewable raw material (glycerol), we concluded that it is suitable for complex utilisation in a microbial biorefinery, since the fermentation broth can be used for the isolation of a sweetener and bioemulsifier; meanwhile, the separated cells can be processed for cosmetic application as a skin moisturizer. Full article

The present study investigates the effect of 50 mL AMCB taken daily as a dietary supplement on the rumen fermentation, microbiota, and production performance of 40 Holstein dairy cows in the transition period with 2.76 ± 0.48 parity and 650 ± 25 kg body weight. AMCB supplementation stabilized rumen pH, improved rumen microbiota richness and partial probiotic colonization, and considerably increased dry matter intake, milk production, protein content, and yield. Moreover, after calving, AMCB supplementation considerably reduced the serum blood urea nitrogen, malondialdehyde, hydrogen peroxide, alanine aminotransferase, and aspartate transaminase levels and increased the serum immunoglobulin G and A levels. The results indicated that AMCB dietary supplementation improved postpartum dry matter intake, production performance, partial immune function, antioxidant capacity, and rumen microbiota richness in Holstein dairy cows in the transition period. AMC is an excellent candidate for use as a rumen buffer. Full article

In this work, microbial lipid production from non-detoxified Eucalyptus bark hydrolysate (EBH) with oleaginous xylose-utilizing Ashbya gossypii strains was explored. The best producing strain from a set of engineered strains was identified in synthetic media mimicking the composition of the non-detoxified EBH (SM), the lipid profile was characterized, and yeast extract and corn steep liquor (CSL) were pinpointed as supplements enabling a good balance between lipid accumulation, biomass production, and autolysis by A. gossypii. The potential of the engineered A. gossypii A877 strain to produce lipids was further validated and optimized with minimally processed inhibitor-containing hydrolysate and high sugar concentration, and scaled up in a 2 L bioreactor. Lipid production from non-detoxified EBH supplemented with CSL reached a lipid titer of 1.42 g/L, paving the way for sustainable single-cell oil production within the concept of circular economy and placing lipids as an alternative by-product within microbial biorefineries. Full article

This work aimed at producing lactic acid (LA) from sugarcane bagasse after steam explosion at 195 °C for 7.5 and 15 min. Enzymatic hydrolysis was carried out with Cellic CTec3 and Cellic HTec3 (Novozymes), whereas fermentation was performed with Bacillus coagulans DSM2314. Water washing of pretreated solids before enzymatic hydrolysis improved both hydrolysis and fermentation yields. The presence of xylo-oligosaccharides (XOS) in substrate hydrolysates reduced hydrolysis efficiency, but their effect on fermentation was negligible. The presence of fermentation inhibitors in C5 streams was circumvented by adsorption on activated carbon powder with no detectable sugar losses. High carbohydrates-to-LA conversions (Y_p/s) of 0.88 g·g⁻¹ were obtained from enzymatic hydrolysates of water-washed steam-exploded materials that were produced at 195 °C, in 7.5 min, and the use of centrifuged-but-never-washed pretreated solids decreased Y_p/s by 16%. However, when the detoxified C5 stream was added at a 10% ratio, Y_p/s was raised to 0.93 g·g⁻¹ for an LA productivity of 2.55 g·L⁻¹·h⁻¹. Doubling the pretreatment time caused a decrease in Y_p/s to 0.78 g·g⁻¹, but LA productivity was the highest (3.20 g·L⁻¹·h⁻¹). For pretreatment at 195 °C for 7.5 min, the elimination of water washing seemed feasible, but the use of longer pretreatment times made it mandatory to eliminate fermentation inhibitors. Full article

The use of chemical fertilizers is essential for agricultural development when soils do not have the nutritional balance required for plants. The continuous use of chemical fertilizers has changed the soil physicochemical and biological properties. Biofertilizer production has been considered as an alternative to reduce chemical fertilizers dependence and the environmental impact. The aim of this study is the evaluation of three technologies for the production of biofertilizers and bioenergy at technical, economic, environmental, and social levels. Ammonium sulfate and digestate-based biofertilizers were obtained via anaerobic digestion; biochar was produced via gasification; and amino acids as plants biostimulants were obtained via protein hydrolysis. Different indicators were calculated for elucidating the sustainability of the processes. Technical, economic, environmental, and social analysis is performed for each of the biorefineries processing stage (complex and stand-alone) without considering the agronomic stage or other value chain links. Scenario 1 presented a positive impact on the economic, environmental, and social aspects since this process has a payback period of 10 years, a carbon footprint of 0.67 kg CO_2-eq/kg product, and a potential to generate nine jobs in the Colombian context due to the products portfolio, in contrast with scenario 2 and 3. As conclusion, the integration of biofertilizers and bioenergy in biorefineries have the potential to expand the range of bioproducts and to increase the process sustainability. Full article

This study aimed at investigating the feasibility of using a highly specialized bacterial inoculum harboring lactic acid bacteria (LAB) and lactate-oxidizing, hydrogen-producing bacteria (LO-HPB) to produce either lactate or biohydrogen and butyrate from several agro-industrial resources via dual-phase dark fermentation. The feedstocks were fruit–vegetable waste, cheese whey, coffee wastewater, tequila vinasse, and maize processing wastewater, and were tested in both mono- and co-fermentation. The results obtained indicated that the biocatalyst used was able to perform a dual-phase lactate fermentation, producing high lactate (13.1–36.4 g/L), biohydrogen (0.2–7.5 NL H₂/L_feedstock, equivalent to 0.3–1.7 mol H₂/mol hexose), and butyrate (3.3–13.9 g/L) with all the tested feedstocks. A series of self-fermentation tests were also performed with crude cheese whey and fruit–vegetable waste for comparison purposes. Compared to inoculum-aided fermentations, the self-fermentation exhibited a reduced bioconversion efficiency. Short-length 16S rRNA gene sequencing analysis showed that LO-HPB was the dominant microbial group (86.0%) in the biocatalyst, followed by acetic acid bacteria (5.8%) and LAB (5.7%). As expected, the molecular analysis also showed significant differences in the microbial community structure of the biocatalyst and those that evolved from self-fermentation. Besides lactate fermentation and oxidation, the biocatalyst also assisted the bi-phasic lactate fermentation via oxygen consumption, and apparently, via substrate hydrolysis. Overall, this study can lay the foundation for robust inoculum development, which is of special significance in the field of dark fermentation, and proposes an innovative bioprocess for agro-industrial valorization through a trade-off approach, tailoring the metabolic pathway to the target product(s). Full article