Fermentation

Recently, the extensive spread of some algae along coastlines has surged into unmanageable thick decomposition layers. This study aimed to demonstrate the use of Sargassum horneri as a biomass resource for ethanol production through the continuous hydrolysis, enzymatic saccharification, and fermentation process. Sugars from S. horneri were obtained using a combination of thermal acid hydrolysis and enzymatic saccharification. The optimal conditions for thermal acid hydrolysis involved a 10% (w/v) S. horneri slurry treated with 100 mM H₂SO₄ at 121 °C for 60 min; enzymatic saccharification using 16 U/mL Cellic CTec2 further boosted the monosaccharide concentration to 23.53 g/L. Fermentation experiments were conducted with mannitol-adapted Saccharomyces cerevisiae BY4741 using S. horneri hydrolysate. Enhanced ethanol production was observed in the hydrolysate, particularly with mannitol-adapted S. cerevisiae BY4741, which yielded 10.06 g/L ethanol. Non-adapted S. cerevisiae produced 8.12 g/L ethanol, as it primarily utilized glucose and not mannitol. Regarding ethanol fermentation using 5 L- and 500 L-scale fermenters, the ethanol concentrations reached 10.56 g/L and 7.88 g/L with yields of 0.51 and 0.45, respectively, at 48 h. This study confirmed the economic viability of ethanol production using waste seaweed with optimized pretreatment conditions and the adaptive evolution of S. cerevisiae to mannitol. Full article

High production cost is one of the major factors that limit the market growth of polyhydroxyalkanoates (PHAs) as a biopolymer. Improving PHA synthesis performance and utilizing low-grade feedstocks are two logical strategies for reducing costs. As an oleaginous yeast, Y. lipolytica has a high carbon flux through acetyl-CoA (the main PHB precursor), which makes it a desired cell factory for PHB biosynthesis. In the current study, two different metabolic pathways (NBC and ABC) were introduced into Y. lipolytica PO1f for synthesizing PHB. Compared to the ABC pathway, the NBC pathway, which includes NphT7 to redirect the lipogenesis pathway and catalyze acetoacetyl-CoA synthesis in a more energy-favored reaction, led to PHB accumulation of up to 11% of cell dry weight (CDW), whereas the ABC pathway resulted in non-detectable accumulations of PHB. Further modifications of the strain with the NBC pathway through peroxisomal compartmentalization and gene dose overexpression reached 41% PHB of CDW and a growth rate of 0.227 h⁻¹. A low growth rate was observed with acetate as the sole source of carbon and energy or glucose as the sole substrate at high concentrations. Using a co-substrate strategy helped overcoming the inhibitory and toxic effects of both substrates. Cultivating the engineered strain in the optimal co-substrate condition predicted by response surface methodology (RSM) led to 83.4 g/L of biomass concentration and 31.7 g/L of PHB. These results offer insight into a more cost-effective production of PHB with engineered Y. lipolytica. Full article

In the evolving landscape of sustainable biopharmaceutical process development, the utilization of bacteria in the production of various compounds via fermentation has attracted extensive attention from scientists. A successful fermentation process and the release of its associated products hinge on the synergy between an efficient bacterial strain and the formulation of a suitable growth medium. Balancing all nutrient levels of a growth medium to maximize microbial growth and the product quality is quite an intricate task. In this context, significant advancements have been achieved via the strategic implementation of design-of-experiment (DOE) methodologies and the utilization of parallel microbioreactor systems. This work presents a case study of the fermentation growth medium optimization of a Gram-negative bacterium of the Neisseriaceae family that releases outer membrane vesicles (OMVs), which represent a potential vaccine platform. To achieve this, the ability of Sartorius MODDE^®13 DOE software to explore multiple variables and their interactions was combined with the functionality of a Sartorius Ambr^® 15F parallel microbioreactor system. The findings reported in this study have led to the design of a well-suited fermentation medium for a Gram-negative bacterium and an improvement in the quality of the OMVs produced from it. Full article

Trans-2-decenoic acid has a wide range of applications, including those in medicine, food, and health care. Therefore, the industrial production of trans-2-decenoic acid is particularly important. However, few studies have focused on medium-chain unsaturated fatty acids. Therefore, we aimed to optimize the fermentation process of decanoic acid biocatalysis to synthesize trans-2-decenoic acid using an engineered Escherichia coli constructed in the laboratory. Early-stage culture and the effect of the seed liquid culture time, culture temperature, inoculum amount, induction temperature, dissolution effects of the substrate solvent, metal ions, and substrate loading on the titer of trans-2-decenoic acid were evaluated. Based on a single-factor experimental optimization, a Box–Behnken design (BBD) was used for response surface testing using the substrate feeding concentration, inducer concentration, and MnCl₂ concentration as response variables and trans-2-decenoic acid production as the response value. The optimal fermentation process was as follows: Seed culture time of 20 h, culture temperature of 37 °C, inoculation amount of 1%, induction temperature of 30 °C, substrate flow of 0.15 g/L, inducer concentration of 5.60 g/L, and MnCl₂ concentration of 0.10 mM. Under these conditions, the average production of trans-2-decenoic acid was 1.982 ± 0.110 g/L, which was 1.042 g/L higher than that obtained in the basic LB medium. Compared with that of the previous period, the titer of the trans-2-decenoic acid studied increased by 1.501 ± 0.110 g/L, providing a basis for further research on the fermentation process of the biocatalytic decanoic acid synthesis of trans-2-decenoic acid. Full article

Vitamin C, a water-soluble vitamin with strong reducing power, cannot be synthesized by the human body and participates in a variety of important biochemical reactions. Vitamin C is widely used in the pharmaceutical, food, health care, beverage, cosmetics, and feed industries, with a huge market demand. The classical two-step fermentation method is the mainstream technology for vitamin C production. D-sorbitol is transformed into L-sorbose by Gluconobacter oxydans in the first step of fermentation; then, L-sorbose is transformed into 2-keto-L-gulonic acid (2-KGA) by a coculture system composed of Ketogulonicigenium vulgare and associated bacteria; and finally, 2-KGA is transformed into vitamin C through chemical transformation. The conversion of L-sorbose into 2-KGA in the second fermentation step is performed by K. vulgare. However, considering the slow growth and low 2-KGA production of K. vulgare when cultured alone, it is necessary to add an associated bacteria to stimulate K. vulgare growth and 2-KGA production. Although the mechanism by which the associated bacteria promote K. vulgare growth and 2-KGA production has extensively been studied, this remains a hot topic in related fields. Based on the latest achievements and research, this review summarizes the metabolic characteristics of K. vulgare and associated bacteria and elucidates the mechanism by which the associated bacteria promote the growth and 2-KGA production of K. vulgare. Full article

Several emerging mosquito control technologies require mass releases of adult male mosquitoes. Previous studies resulted in the generation of a laboratory female-specific larvicidal yeast strain targeting the GGT gene, which facilitated the laboratory sex separation of male Culex quinquefasciatus mosquitoes. Global deployment of this yeast-based sex-separation technology requires engineering second generation yeast strains which can be used in industrial-scale fermentations to support global mosquito control programs. In this study, the RNA-guided Cas-CLOVER system was used in combination with piggyBac transposase to generate robust Saccharomyces cerevisiae strains with multiple integrated copies of the insecticidal GGT shRNA expression cassette. Top expressing Cas-CLOVER strains killed Culex quinquefasciatus female larvae which consumed the yeast, facilitating male sex separation. Scaled fermentation resulted in kilogram-scale production of the yeast, which can be heat-killed and dried for global deployment to mosquito mass-rearing facilities. Full article

Testosterone (TS) is an important androgen drug and a precursor of steroid drug synthesis. Ketoreductase 2 (KR-2) (GenBank accession no. ABP64403.1) is observed to stereo-selectively catalyze the bioreduction of 4-androstene-3,17-dione (4-AD) to testosterone and contribute to the regeneration of NADH using isopropanol as a co-substrate. The Km value of KR-2 was 2.22 mmol/L with 4-AD, and the optimal pH was 6.5–7.0. The enzyme is stable and demonstrates relatively high-level enzyme activity at 40 °C. Acetone significantly inhibits this activity. This inhibition was overcome using an intermittent vacuum during the reaction process. Finally, the amount of TS reached 65.42 g/L after a 52 h reaction with 65.8 g/L 4-AD, 10% isopropanol, and 2 g/L β–NAD⁺ at 40 °C, with a conversion rate of 98.73%. A total of 6.15 g of TS was obtained from 6.58 g of 4-AD after the reaction and purification; the HPLC purity was 99.82%, and the overall yield was 92.81%. This enzyme provides a promising route for the green biosynthesis of testosterone for industrial applications. Full article

The clinical antibiotic gentamicin is a mixture of several difficult-to-separate components, the minor group of which is gentamicin C1a, a precursor for the synthesis of the high-efficacy and low-toxicity antibiotic etimicin. This study aimed to achieve the high production of gentamicin as well as gentamicin C1a. In this study, the influence of organic and inorganic salts on the gentamicin production was screened and label-free proteomics was used to determine the mechanisms responsible for the effects. In 25 L fermentation experiments, the addition of 0.1% CaCl₂ and 0.3% sodium citrate increased gentamicin titers by 11.5% (2398 μg/mL vs. 2150 μg/mL), while the C1a ratio increased from 38% to 42%. The results showed that CaCl₂ downregulated the synthesis and metabolism of the tetrapyrrole pathway and the GenK protein (0.08-fold) in the gentamicin synthesis pathway, whereas sodium citrate downregulated key proteins in the glycosylation pathway and tricarboxylic acid pathway. Thus, CaCl₂ caused changes in methylation during the synthesis of gentamicin, increasing the proportion of gentamicin C1a. In contrast, sodium citrate inhibited primary metabolism to promote the production of secondary metabolites of gentamicin. This study provided a basis for the co-production of gentamicin C1a mono-component and gentamicin multicomponent. Full article

A potentially novel probiotic strain, YY-112, was previously isolated and identified as Lactiplantibacillus pentosus using 16S rDNA sequencing. The whole genome analysis showed that strain YY-112 has the potential to metabolize carbohydrates in the gastrointestinal environment and to regulate immunity. Further, comparative genomics analysis revealed that strain YY-112 was Lactiplantibacillus plantarum (L. plantarum) with more unique genes. The results of in vivo tests showed that L. plantarum YY-112 had no adverse effects and restored the damaged spleen and intestinal barrier of immunocompromised mice. L. plantarum YY-112 recovered the normal levels of lymphocytes, serum cytokines (Interferon-γ, tumor necrosis factor α, and interleukin-6), immunoglobulins (Ig) (IgA, IgM, and IgG) and lipids (albumin, cholesterol, triglyceride). Additionally, L. plantarum YY-112 might indirectly enhance the immune system by improving the intestinal microbiota structure. These results supported the potential of L. plantarum YY-112 as a probiotic to regulate the immune system of hosts. Full article

The commercial potential of Trametes versicolor laccases in the degradation of various persistent contaminants is significant. Despite numerous attempts through rational metabolic engineering to enhance the properties of laccases, the outcomes have proven unsatisfactory for practical implementation. The present study successfully generated two novel mutants, namely, TA-04 and TA-15, derived from Trametes versicolor ATCC20869, utilizing atmospheric and room temperature plasma (ARTP). The laccase activities of TA-04 and TA-15 showed a significant increase to 136.507 ± 4.827 U/mg DCW and 153.804 ± 6.884 U/mg DCW, respectively, which were 1.201 and 1.354 times than that of the original strain. The laccase activities of the mutant strains TA-04 and TA-15 surpassed that of the original strain by 10.372% and 18.914%, respectively, at a higher pH level of five. Sequencing analysis of TA-04 and TA-15 revealed that several alternative amino acids within their active regions may enhance their catalytic characteristics under a higher temperature and pH condition. This study employed ARTP mutagenesis to propose two highly efficient microbial mutants derived from Trametes versicolor ATCC20869, exhibiting enhanced laccase activities. These mutants hold promising potential for the degradation of diverse environmental pollutants. Full article

The increasing popularity of Greek yogurt generates large amounts of acid whey worldwide. The use of yogurt acid whey in animal nutrition is limited. The aim of this study was to determine the effect of a yogurt acid whey powder (YAWP) addition to maize forage prior to ensiling on the nutritional, microbial and fermentation quality of maize silage. Depending on the addition level of the YAWP to maize forage, there were the following four experimental treatments: YAWP 0, 2.5, 5 and 10% w/w. An increasing YAWP inclusion level linearly increased the maize silage dry matter, crude protein and ash concentrations, whereas it reduced the crude fiber, neutral-detergent fiber and acid-detergent fiber concentrations. The silage pH decreased quadratically with the increasing YAWP level, with the lower plateau noted for the YAWP 5% addition. Concentrations of total bacteria in the silage and Lactobacillus spp. decreased linearly with the YAWP increase. The silage acetic acid content decreased linearly, whereas propionic acid, lactic acid and the ratio of lactic to acetic acid increased linearly with the increasing YAWP level. The ammonia-N content decreased linearly with the increasing YAWP level. In conclusion, the incorporation of the 5 and 10% YAWP addition in silage preparation improved the nutritional and fermentative quality of the produced silage. Full article

Anaerobic co-digestion (AcoD) of animal waste and agro-industrial by-products has been widely studied and employed to increase biogas production potential and enhance process stability. This study evaluated the AcoD of cattle manure (CM) and brewer’s residual yeast (RY) in semi-continuous biodigesters, focusing on energy potential (biogas and methane yields) and process stability. Four treatments were assessed, each with different proportions (% of volatile solids) of CM and RY: 100:0, 88:12, 78:22, and 68:32. Trials were conducted in 30-L tubular reactors at room temperature with a hydraulic retention time of 30 days. The inclusion of RY led to a gradual rise in biogas and methane production, with more significant reductions in solid content than mono-digestion of CM. The addition of RY resulted in daily CH₄ production increases of 18.5, 32.3, and 51.9% for treatments with 12, 22, and 32% of RY, respectively, compared to the control treatment. Therefore, AcoD demonstrated a higher potential for energy recovery. However, RY introduced elevated H₂S levels in the biogas. Caution is advised when adding this co-substrate to AcoD due to potential process influence and biogas application restrictions. Full article

Enzymatic biodiesel production is a potential eco-friendly alternative to the conventional chemical route which requires extensive study to reduce the costs associated with the application of commercial enzymes. Thus, this study aimed to develop a bioprocess using residues from macauba (Acrocomia aculeata) as raw material for lipase production in solid-state fermentation (SSF) by Yarrowia lipolytica. Then, the product obtained was used as a biocatalyst for the conversion (hydrolysis/esterification) of macauba acidic oil to biodiesel esters. Firstly, different SSF parameters (inoculum concentration, initial moisture content, and carbon and nitrogen levels) were investigated in a factorial design approach, using the cake from macauba fruit. Afterwards, moisture and urea concentration were shown to be statistically significant variables for lipase production. Lipase productnivities were 12.6 ± 0.6 U g⁻¹ h⁻¹ (at 24 h) for macauba fruit cake and 11.6 ± 1 U g⁻¹ h⁻¹ (at 20 h) for macauba pulp and peel cake. The solid enzymatic preparation (biocatalyst) showed optimized values at pH 6–7 at 37 °C, remaining stable (>70% retention) for 90 days at room temperature. Finally, enzymatic hydrolysis of the acidic oil from macauba reached 96% conversion (72 h) to fatty acids, and esterification of fatty acids reached 72% (biodiesel yield of 67%). The bioprocess described is a promising alternative for an integral and self-sufficient valorization of the macauba products. Full article

Cellular adhesion plays an important role in numerous fundamental physiological and pathological processes. Its measurement is relatively complex, requires sophisticated equipment, and, in most cases, cannot be carried out without breaking the links between the studied cell and its target. In this contribution, we propose a novel, nanomotion-based, technique that overcomes these drawbacks. The applied force is generated by the studied cell itself (nanomotion), whereas cellular movements are detected by traditional optical microscopy and dedicated software. The measurement is non-destructive, single-cell sensitive, and permits following the evolution of the adhesion as a function of time. We applied the technique on different strains of the fungal pathogen Candida albicans on a fibronectin-coated surface. We demonstrated that this novel approach can significantly simplify, accelerate, and make more affordable living cells–substrate adhesion measurements. Full article

Lignocellulose consists of cellulose, hemicellulose, and lignin and is a sustainable feedstock for a biorefinery to generate marketable biomaterials like biofuels and platform chemicals. Enormous tons of lignocellulose are obtained from agricultural waste, but a few tons are utilized due to a lack of awareness of the biotechnological importance of lignocellulose. Underutilizing lignocellulose could also be linked to the incomplete use of cellulose and hemicellulose in biotransformation into new products. Utilizing lignocellulose in producing value-added products alleviates agricultural waste disposal management challenges. It also reduces the emission of toxic substances into the environment, which promotes a sustainable development goal and contributes to circular economy development and economic growth. This review broadly focused on lignocellulose in the production of high-value products. The aspects that were discussed included: (i) sources of lignocellulosic biomass; (ii) conversion of lignocellulosic biomass into value-added products; and (iii) various bio-based products obtained from lignocellulose. Additionally, several challenges in upcycling lignocellulose and alleviation strategies were discussed. This review also suggested prospects using lignocellulose to replace polystyrene packaging with lignin-based packaging products, the production of crafts and interior decorations using lignin, nanolignin in producing environmental biosensors and biomimetic sensors, and processing cellulose and hemicellulose with the addition of nutritional supplements to meet dietary requirements in animal feeding. Full article

Lipases are enzymes that catalyze the hydrolysis of ester bonds of triacylglycerols at the oil–water interface, generating free fatty acids, glycerol, diacylglycerol, and monoacylglycerol, which can be produced from the fermentation of agro-industrial by-products rich in fatty acids, such as cupuaçu fat cake. In this study, Yarrowia lipolytica IMUFRJ50682 was used for lipase production from cupuaçu fat cake in solid-state fermentation (SSF) associated with soybean meal. The 2:1 ratio of cupuaçu fat cake/soybean meal increased the lipase activity of Y. lipolytica via SSF by approximately 30.3-fold compared to that in cupuaçu without supplementation. The optimal conditions for Y. lipolytica to produce lipase were obtained by supplementation with peptone, urea, and soybean oil (all at 1.5% w/v), reaching values of up to 70.6 U g⁻¹. These results demonstrate that cupuaçu fat cake associated with soybean meal can be used for lipase production and adds value to cupuaçu by-products. Furthermore, the proper processing of by-products can contribute to improving the economic viability of the biotechnological processing industry and help prevent the accumulation of waste and environmental pollution. Full article

Direct interspecies electron transfer (DIET) between electroactive microorganisms (EAMs) offers significant potential to enhance methane production, necessitating research for its practical implementation. This study investigated enhanced methane production through DIET in an anaerobic digester bio-augmented with EAMs. A horizontal anaerobic digester (HAD) operated for 430 days as a testbed to validate the benefits of bioaugmentation with EAMs. Anaerobic digestate slurry, discharged from the HAD, was enriched with EAMs in a bioelectrochemical auxiliary reactor (BEAR) under an electric field. This slurry enriched with EAMs was then recirculated into the HAD. Results showed bio-augmentation with EAMs led to an increase in volatile solids removal from 56.2% to 77.5%, methane production rate from 0.59 to 1.00 L/L.d, methane yield from 0.26 to 0.34 L/g CODr, and biogas methane content from 59.9% to 71.6%. It suggests that bio-augmentation enhances DIET, promoting the conversion of volatile fatty acids to methane and enhancing resilience against kinetic imbalances. The enrichment of EAMs reached optimal efficacy under an electric field intensity of 2.07 V/cm with a mean exposure time of 2.53 days to the electric field in the BEAR. Bio-augmentation with externally enriched EAMs is a feasible and effective strategy to optimize anaerobic digestion processes. Full article

The sensory quality and health benefits of Pu-erh tea are mainly determined by microbial fermentation processing. The directed exogenous inoculation of specific microorganisms is an effective method to improve the quality and flavor of Pu-erh tea. In this study, Lactobacillus plantarum and Saccharomyces cerevisiae were introduced into the fermentation processes of Pu-erh tea, as they are the main contributors to enzyme secretion, to change the tea’s functional components. The raw tea materials, spontaneous fermentation tea and microbiological fermentation tea were analyzed by microbiomics and metabolomics. A total of 248 metabolites were characterized, 71 of which were identified as essential metabolites involved in the metabolic changes. These essential metabolites were produced by specific dominant microbial species with multivariate analysis methods. Metabolites essential to the sensory quality and health benefits of Pu-erh tea, such as flavonoids and free amino acids, were increased in tea samples inoculated with Lactobacillus plantarum and Saccharomyces cerevisiae following fermentation. Fungal diversity decreased after fermentation, and both the diversity and richness of bacteria were significantly decreased. In conclusion, our results demonstrate the advantages of Lactobacillus plantarum and Saccharomyces cerevisiae in forming the unique sensory characteristics of Pu-erh tea, and they indicate that the microbial composition is a key factor in altering the tea’s metabolic profile. Our work establishes a theoretical foundation for the promotion of the safety and quality of Pu-erh tea through exogenous inoculation with Lactobacillus plantarum and Saccharomyces cerevisiae. Full article

The tung tree (Vernicia fordii Hemsl.), as a woody oilseed crop, has been cultivated in China for thousands of years, and its leaves are rich in cellulose and proteins. The tung leaf is an alternative raw material for the traditional ethanol fermentation of food crops. In this work, the effects of the simultaneous saccharification fermentation of tung leaves at different substrate concentrations on gas production characteristics, reducing sugars, pH, oxidation–reduction potential (ORP), and ethanol yield were investigated during bioethanol production. In addition, the effect of the initial fermentation pH on the ethanol fermentation of tung leaves was explored. The results showed that during bioethanol production from tung leaves, the pH of the fermentation broth showed a continuous decreasing trend. Moreover, the ORP showed a decreasing trend and then rebounded, and the concentration of reducing sugars initially increased and then decreased. The optimal ethanol yield of 4.99 g/L was obtained when the substrate concentration was 100 g/L. Changes in the initial pH have little effect on yeast activity, but such changes can affect the yeast cell wall structure and substance transport, leading to differences in the ethanol yield. When the initial pH is 7, the maximum ethanol yield is 5.22 g/L. The experimental results indicate that the utilization of tung leaves for bioethanol production has a good potential for development. Full article