Batch Fermentation Research Articles

An integrated multiphase dynamic genome-scale model explains batch fermentations led by species of the Saccharomyces genus.

During batch fermentation, a variety of compounds are synthesized, as microorganisms undergo distinct growth phases: lag, exponential, growth-no-growth transition, stationary, and decay. A detailed understanding of the metabolic pathways involved in these phases is crucial for optimizing the production of target compounds. Dynamic flux balance analysis (dFBA) offers insight into the dynamics of metabolic pathways. However, explaining secondary metabolism remains a challenge. A multiphase and multi-objective dFBA scheme (MPMO model) has been proposed for this purpose. However, its formulation is discontinuous, changing from phase to phase; its accuracy in predicting intracellular fluxes is hampered by the lack of a mechanistic link between phases; and its simulation requires considerable computational effort. To address these limitations, we combine a novel model with a genome-scale model to predict the distribution of intracellular fluxes throughout batch fermentation. This integrated multiphase continuous model (IMC) has a unique formulation over time, and it incorporates empirical regulatory descriptions to automatically identify phase transitions and incorporates the hypotheses that yeasts might vary their cellular objective over time to adapt to the changing environment. We validated the predictive capacity of the IMC model by comparing its predictions with intracellular metabolomics data for Saccharomyces uvarum during batch fermentation. The model aligns well with the data, confirming its predictive capabilities. Notably, the IMC model accurately predicts trehalose accumulation, which was enforced in the MPMO model. We further demonstrate the generalizability of the IMC model, explaining the dynamics of primary and secondary metabolism of three Saccharomyces species. The model provides biological insights consistent with the literature and metabolomics data, establishing it as a valuable tool for exploring the dynamics of novel fermentation processes.IMPORTANCEThis work presents an integrated multiphase continuous dynamic genome-scale model (IMC model) for batch fermentation, a crucial process widely used in industry to produce biofuels, enzymes, pharmaceuticals, and food products or ingredients. The IMC model integrates a continuous kinetic model with a genome-scale model to address the critical limitations of existing dynamic flux balance analysis schemes, such as the difficulty of explaining secondary metabolism, the lack of mechanistic links between growth phases, or the high computational demands. The model also introduces the hypothesis that cells adapt the FBA objective over time. The IMC improves the accuracy of intracellular flux predictions and simplifies the implementation process with a unique dFBA formulation over time. Its ability to predict both primary and secondary metabolism dynamics in different Saccharomyces species underscores its versatility and robustness. Furthermore, its alignment with empirical metabolomics data validates its predictive power, offering valuable insights into metabolic processes during batch fermentation. These advances pave the way for optimizing fermentation processes, potentially leading to more efficient production of target compounds and novel biotechnological applications.

Calcium Alginate Encapsulation of Rice Starch, Instead of a Physical Mixture of Both, Regulates the Fermentation Rate and Production of Acetate

ABSTRACTCalcium alginate–encapsulated rice starch (AES) could be potentially applied as a rice analog with a significantly improved amount of resistant starch, while its effects on gut microbiota remain less clear. To this end, structural characteristics of AES and their impact on gut microbiota, fermentation rate, and short‐chain fatty acid (SCFA) production were examined using an in vitro batch fermentation method. Cooked AES showed a significantly higher amount of intermolecular interactions (∼46 times), short‐range double helices, and degree of crystallinity compared to the simple mixture of rice starch and calcium alginate (Mix), resulting in a more homogenous and densely packed network microstructure. As a result, AES, instead of Mix, showed a significantly slower gas production rate (∼17%), while relatively higher production of SCFAs, especially the ratio of acetate. Bifidobacterium pesudocatenulatum was possibly responsible for the higher production of acetate in AES. Collectively, these results show that AES has the potential to be used as a slowly fermentable carbohydrate, favoring the production of acetate in the human colon.

Response surface methodology and repeated-batch fermentation strategies for enhancing lipid production from marine oleaginous Candida parapsilosis Y19 using orange peel waste

Oleaginous yeasts are considered promising sources for lipid production due to their ability to accumulate high levels of lipids under appropriate growth conditions. The current study aimed to isolate and identify oleaginous yeasts having superior ability to accumulate high quantities of lipids; and enhancing lipid production using response surface methodology and repeated-batch fermentation. Results revealed that, twenty marine oleaginous yeasts were isolated, and the most potent lipid producer isolate was Candida parapsilosis Y19 according to qualitative screening test using Nile-red dye. Orange peels was used as substrate where C. parapsilosis Y19 produced 1.14 g/l lipids at 23.0% in batch fermentation. To enhance the lipid production, statistical optimization using Taguchi design through Response surface methodology was carried out. Total lipids were increased to 2.46 g/l and lipid content increased to 30.7% under optimal conditions of: orange peel 75 g/l, peptone 7 g/l, yeast extract 5 g/l, inoculum size 2% (v/v), pH 5 and incubation period 6 d. Furthermore, repeated-batch fermentation of C. parapsilosis Y19 enhanced lipid production where total lipids increased at 4.19 folds (4.78 g/l) compared to batch culture (before optimization). Also, the lipid content was increased at 1.7 folds (39.1%) compared to batch culture (before optimization). Fatty acid profile of the produced lipid using repeated-batch fermentation includes unsaturated fatty acids (USFAs) at 74.8% and saturated fatty acids (SFAs) at 25.1%. Additionally, in repeated-batch fermentation, the major fatty acid was oleic acid at 45.0%; followed by linoleic acid at 26.0%. In conclusion, C. parapsilosis Y19 is considered a promising strain for lipid production. Also, both statistical optimizations using RSM and repeated-batch fermentation are efficient methods for lipid production from C. parapsilosis Y19.

Batch and semi-continuous fermentation with Parageobacillus thermoglucosidasius DSM 6285 for H2 production

BackgroundParageobacillus thermoglucosidasius is a facultatively anaerobic thermophile that is able to produce hydrogen (H2) gas from the oxidation of carbon monoxide through the water–gas shift reaction when grown under anaerobic conditions. The water–gas shift (WGS) reaction is driven by a carbon monoxide dehydrogenase–hydrogenase enzyme complex. Previous experiments exploring hydrogenogenesis with P. thermoglucosidasius have relied on batch fermentations comprising defined media compositions and gas atmospheres. This study evaluated the effects of a semi-continuous feeding strategy on hydrogenogenesis.ResultsA batch and two semi-continuous fermentations, with feeding of the latter fresh media (with glucose) in either 24 h or 48 h intervals were undertaken and H2 production, carbon monoxide dehydrogenase (CODH) activity, and metabolite consumption/production were monitored throughout. Maximum H2 production rates (HPR) of 0.14 and 0.3 mmol min−1, were observed for the batch and the semi-continuous fermentations, respectively. Daily feeding attained stable H2 production for 7 days, while feeding every 48 h resulted in high variations in H2 production. CODH enzyme activity correlated with H2 production, with a maximum of 1651 U mL−1 on day 14 with the 48 h feeding strategy, while CODH activity remained relatively constant throughout the fermentation process with the 24 h feeding strategy.ConclusionsThe results emphasize the significance of a semi-continuous glucose-containing feed for attaining stable hydrogen production with P. thermoglucosidasius. The semi-continuous fermentations achieved a 46% higher HPR than the batch fermentation. The higher HPRs achieved with both semi-continuous fermentations imply that this approach could enhance the biohydrogen platform. However, optimizing the feeding interval is pivotal to ensuring stable hydrogen production.

Rational engineering of Escherichia coli strain for stable and enhanced biosynthesis of pinene.

Monoterpene α-pinene exhibits significant potential as an alternative fuel, widely recognized for its affordability and eco-friendly nature. It demonstrates multiple biological activities and has a wide range of applications. However, the limited supply of pinene extracted from plants poses a challenge in meeting the needs of the aviation industry and other sectors. Considering this, the microbial cell factory is the only viable option for achieving sustainable pinene production. This study employed a rational design model to optimize the copy number and integration site for the heterogenous pinene expression pathway in Escherichia coli (E. coli). The integrated strain with the best pinene pathway PG1 was selected. Subsequently, the resulting strain, E. coli HSY009, accumulated 49.01 mg/L of pinene after 24 h fermentation in the flask culture. To further enhance production, pinene expression cassette PG1 was sequentially integrated into three non-essential regions (44th, 58th, 23rd), resulting in an improved pinene titer. Then, the fermentation process under optimized conditions enhanced the production of pinene to 436.68 mg/L in a 5 L batch fermenter with a mean productivity of 14.55 mg/L/h. To the best of our knowledge, this work represents the maximum mean pinene productivity based on the currently available literature. The findings of this work provide valuable insights for optimizing E. coli to produce other valuable terpenoids that share the same intermediates, IPP and DMAPP. Conclusively, this research validates the model's universality and highlights its potential for application as cutting-edge biofuel precursors.

A Study of Bioactivities and Composition of a Cocktail of Supernatants Derived from Lactic Acid Bacteria for Potential Food Applications.

Growing interests in replacing conventional preservatives and antibiotics in food and pharmaceutical industries have driven the exploration of bacterial metabolites, especially those from strains with generally recognized as safe (GRAS) status, such as lactic acid bacteria (LAB). In this study, a supernatant cocktail derived from multiple LAB strains was prepared and its bioactivities-antimicrobial, antibiofilm, antioxidant, cytotoxicity, and stability-were thoroughly investigated. The cocktail's main components were identified using thermal and protease treatments, gas chromatography coupled to mass spectrometry (GC-MS), and flame ionization detection (GC-FID). The results demonstrated that the supernatant cocktail had a broad inhibition spectrum and was effective against food-related bacterial indicators with the highest activity observed on Bacillus cereus ATCC9634 (inhibition zone sizes 12.33mm) and the lowest on Enterococcus faecium DSM 13590 (3.31mm). It showed dose- and time-dependent delaying effects on the growth of tested fungi. Regarding the antibiofilm activity, it was more effective in preventing biofilm formation (40% biofilm mass reduction) than in degrading preformed biofilm (20% reduction). Additionally, the cocktail showed antioxidant capacity of 10.1 ± 0.3g Trolox equivalent (TE)/kg and dose-dependent cytotoxicity on HEK-293 and HT-29 cell lines. The main bioactive compounds in this cocktail are organic acids (particularly acetic acid), volatiles, and bacteriocin-like compounds. The antimicrobial capacity of this supernatant cocktail was highly reproducible across different fermentation batches, and it remained highly stable at 4°C. Overall, these findings provided novel insights into the functional potentials of LAB metabolites, broadening their application as customizable biopreservatives for food and pharmaceutical industry.

Alpine pasture plant species affect in vitro rumen methane production and kinetics

This study aimed to evaluate the influence of different plant species widespread in alpine pastures on in vitro rumen fermentation parameters and methane kinetic production. A total of 11 plant species were sampled at the beginning of the grazing season and used as substrates in an in vitro batch fermentation system. After 24h of fermentation, plants affected volatile fatty acids profiles, ammonia yield, and dry matter (DM) digestibilities. Carum carvi, Ranunculus. acris and Festuca rubra showed the highest total production of methane per unit of digested DM while Potentilla erecta was the species that produced less methane. In terms of methane as a percentage of the total gas, F. rubra had the highest value (28.9%) while R. acris had the lowest (24.2%). Total gas and methane production were monitored continuously and the percentage of methane in total gas was fitted with the Gompertz model. Plants differed significantly (p < .01) in methane production kinetics, including production rate decline (A), asymptotic methane concentration (B), time to maximum fermentation rate (TMFR), and maximum fermentation rate (MFR). C. carvi, Prunella grandiflora, and R. acris showed high values of MFR and the top values in the production rate decline (A > 0.9). The two grasses (F. rubra and Poa alpina) together with Hypericum maculatum showed an opposite behaviour with low values in MFR, A and a longer TMFR. The results of the methane production kinetics allow for an in-depth evaluation of plant species, adding further information to those registered at the end of fermentation.

Converting multiple hydrophobic aromatic plastic monomers into a single water-soluble substrate to increase bioavailability for the synthesis of polyhydroxyalkanoates by bacteria using batch, fed batch and continuous cultivation.

We demonstrate the proof of concept of increasing the bioavailability of carbon substrates, derived from plastic waste, for their conversion to the biodegradable polymer polyhydroxyalkanoate [PHA] by bacteria and test various approaches to PHA accumulation through batch, fed batch and continuous culture. Styrene, ethylbenzene, and toluene are produced from the pyrolysis of mixed plastic waste (Kaminsky, 2021; Miandad et al., 2017), but they are volatile and poorly soluble in water making them difficult to work with in aqueous fermentation systems. By chemically converting these aromatic compounds to benzoic acid, and subsequently to its sodium salt, we increased the solubility and reduced the volatility of the substrate supplied to Pseudomonas putida CA-3 to accumulate polyhydroxyalkanoates. 1L scale batch, fed batch, and continuous fermentations were carried out; the fed batch fermentation resulted in the maximum volumetric PHA productivity of 61.67 ± 7.34mgL-1 h-1; while batch and continuous, at a dilution rate, d = 0.2h-1, fermentations resulted in 13.30 ± 0.01 and 4.06 ± 0.01mgL-1 h-1 of PHA respectively.

Lipid production from biofilms of Marinobacter atlanticus in a fixed bed bioreactor

BackgroundBiotechnologies that utilize microorganisms as production hosts for lipid synthesis will enable an efficient and sustainable solution to produce lipids, decreasing reliance on traditional routes for production (either petrochemical or plant-derived) and supporting a circular bioeconomy. To realize this goal, continuous biomanufacturing processes must be developed to maximize productivity and minimize costs compared to traditional batch fermentation processes.ResultsHere, we utilized biofilms of the marine bacterium, Marinobacter atlanticus, to produce wax esters from succinate (i.e., a non-sugar feedstock) to determine its potential to serve as a production chassis in a continuous flow, biofilm-based biomanufacturing process. To accomplish this, we evaluated growth as a function of protein concentration and wax ester production from M. atlanticus biofilms in a continuously operated 3-D printed fixed bed bioreactor. We determined that exposing M. atlanticus biofilms to alternating nitrogen-rich (1.8 mM NH4+) and nitrogen-poor (0 mM NH4+) conditions in the bioreactor resulted in wax ester production (26 ± 5 mg/L, normalized to reactor volume) at a similar concentration to what is observed from planktonic M. atlanticus cells grown in shake flasks previously in our lab (ca. 25 mg/L cell culture). The wax ester profile was predominated by multiple compounds with 32 carbon chain length (C32; 50–60% of the total). Biomass production in the reactor was positively correlated with dilution rate, as indicated by protein concentration (maximum of 1380 ± 110 mg/L at 0.4 min−1 dilution rate) and oxygen uptake rate (maximum of 4 mmol O2/L/h at 0.4 min−1 dilution rate) measurements at different flow rates. Further, we determined the baseline succinate consumption rate for M. atlanticus biofilms to be 0.16 ± 0.03 mmol/L/h, which indicated that oxygen is the limiting reactant in the process.ConclusionThe results presented here are the first step toward demonstrating that M. atlanticus biofilms can be used as the basis for development of a continuous flow wax ester biomanufacturing process from non-sugar feedstocks, which will further enable sustainable lipid production in a future circular bioeconomy

Impact of Liquid Bio-Fertilizers on Okra (Abelmoschus esculentus L. Moench.) Plant Growth Indices using Azotobacteria and Azospirillum Consortia

This study was to make bio-fertilizers with Azotobacteria and Azospirilum consortiums and to test the influence of bio-fertilizers on Okra plant growth. This investigation was conducted using the soil from the Biological Garden of the University of Cross River State, Calabar. Pure culture and mass production was performed in batch fermentation at optimum condition by means of specific medium for Azospirilum and Azotobacter. Organisms were isolated from the soil samples and confirmed using biochemical tests. The bio-fertilizer effect on the growth parameters of Okra plants was carried out on Day 21. The post germination, phenotypic and observations showed significant differences in performance between plant length, mass, root numbers, root length, total plant length, breadth of leaves, shoot length, root mass and biological yield. The biochemical indices exibited a significant rise (P &lt; 0.05) in chlorophyll content (mg/g.fr. wt) as compared to Control (0.010±0.000), Azospirilum (0.016±0.000), Azotobacter (0.016±0.000) and consortium (0.018±0.000). Significant rise (P&lt;0.05) in carbohydrate content (%) control (51.976±0.768), Azospirilum (69.690±3), Azotobacter (69).656+2), and consortium 70).190± (5). Protein content control (0.028±0.001), Azospirilum (0.040±0.000), Azotobacter (0.060±0.001) and consortium (0.061±0.000) all significantly increased [P&lt; 0.05]. Growth is significantly affected by single injection and control when compared to plants being treated with Azotobacter, Azospirilum or both (P &lt; 0.05). According to the findings, plant mineral fixation through nitrogen fixation and phosphorous solubilization seems to be improved by bio-fertilizer inoculation as well as their mineral nutrition’s improvement.

Galacto-oligosaccharides alone and combined with lactoferrin impact the Kenyan infant gut microbiota and epithelial barrier integrity during iron supplementation in vitro

Aim: Iron supplementation to African weaning infants was associated with increased enteropathogen levels. While cohort studies demonstrated that specific prebiotics inhibit enteropathogens during iron supplementation, their mechanisms remain elusive. Here, we investigated the in vitro impact of galacto-oligosaccharides (GOS) and iron-sequestering bovine lactoferrin (bLF) alone and combined on the gut microbiota of Kenyan infants during low-dose iron supplementation. Methods: Different doses of iron, GOS, and bLF were first screened during batch fermentations (n = 3), and the effect of these factors was studied on microbiota community structure and activity in the new Kenyan infant continuous intestinal PolyFermS model. The impact of different fermentation treatments on barrier integrity, enterotoxigenic Escherichia coli (ETEC) infection, and inflammatory response was assessed using a transwell co-culture of epithelial and immune cells. Results: A dose-dependent increase in short-chain fatty acid (SCFA) production, Bifidobacterium and Lactobacillus /Leuconostoc /Pediococcus (LLP) growth was detected with GOS alone and combined with bLF during iron supplementation in batches. This was confirmed in the continuous PolyFermS model, which also showed a treatment-induced inhibition of opportunistic pathogens C. difficile and C. perfringens . In all tests, supplementation of iron alone and combined with bLF did not have a significant effect on microbiota composition and activity. We observed a strengthening of the epithelial barrier and a decrease in cell death and pro-inflammatory response during ETEC infection with microbiota fermentation supernatants from iron + GOS, iron + bLF, and iron + GOS + bLF treatments compared to iron alone. Conclusion: Overall, beneficial effects on infant gut microbiota were shown using advanced in vitro models for GOS alone and combined with bLF during low-dose iron supplementation.

Enhancement of the Degradation of Phytosterol Side Chains in Mycolicibacterium by Eliminating the Redox Sensitivity of Key Thiolase and Augmenting Cell Activity

Androstenedione (AD) is a vital intermediate in the synthesis of steroid drugs, making its efficient production critical in the steroid drug industry. Acetyl-CoA acetyltransferase (FadA5), a thiolase enzyme, plays an important role in the metabolic process of degrading phytosterol side chains in Mycolicibacterium to produce AD. This work is the first systematic analysis of the role of FadA5 in the transformation of phytosterols by Mycolicibacterium to produce AD. The relationship between redox potential and AD production was examined using resting cells, and it was confirmed that FadA5 is a key enzyme for AD production. Mutating the 87th cysteine of FadA5 to alanine reduced its redox effect, enhancing the substrate tolerance and biotransformation capacity of the strain. Co-expressing Vitreoscilla hemoglobin (VHb) and propionyl-CoA metabolized the transcription activator (PrpR), decreased intracellular reactive oxygen species levels, and improved cell viability. The AD yield of MSP-fA5C87A-VP/ΔfA5 was 2.541 g/L, an increase of 16.83% over the control strain. Using a repeated batch fermentation process, the production efficiency of the MSP-fA5C87A-VP/ΔfA5 strain was 0.658 g/L/d, which was 1.82 times higher than that of the control strain. These findings provide a theoretical basis for understanding and regulating steroid side-chain catabolism in Mycolicibacterium and offer support for the rational modification of industrial strains for steroidal drug precursor production.

A UHPLC-QE-MS-based metabolomics approach for the evaluation of fermented lipase by an engineered Escherichia coli

Using an engineered Escherichia coli to produce lipase and can easily achieve high-level expression. The investigation of biochemical processes during lipase fermentation, approached from a metabolomics perspective, will yield novel insights into the efficient secretion of recombinant proteins. In this study, the lipase batch fermentation was carried out first with enzyme activity of 36.83 U/mg cells. Then, differential metabolites and metabolic pathways were identified using an untargeted metabolomics approach through comparative analysis of various fermentation periods. In total, 574 metabolites were identified: 545 were up-regulated and 29 were down-regulated, mainly in 153 organic acids and derivatives, 160 organoheterocyclic compounds, 64 lipids and lipid-like molecules, and 58 organic oxygen compounds. Through metabolic pathways and network analysis, it could be found that tryptophan metabolism was of great significance to lipase production, which could affect the secretion and synthesis of recombinant protein. In addition, the promotion effects of cell growth by varying concentrations of indole acetic acid serve to validate the results obtained from tryptophan metabolism. This study offers valuable insights into metabolic regulation of engineered E. coli, indicating that its fermentation bioprocess can be systematically designed according to metabolomics findings to enhance recombinant protein production.

Enhancing D-lactic acid production from non-detoxified corn stover hydrolysate via innovative F127-IEA hydrogel-mediated immobilization of Lactobacillus bulgaricus T15.

The production of D-lactic acid (D-LA) from non-detoxified corn stover hydrolysate is hindered by substrate-mediated inhibition and low cell utilization times. In this study, we developed a novel temperature-sensitive hydrogel, F127-IEA, for efficient D-LA production using a cell-recycle batch fermentation process. F127-IEA exhibited a porous structure with an average pore size of approximately 1 μm, facilitating the formation of stable Lactobacillus bulgaricus clusters within the gel matrix. It also maintains excellent mechanical properties. It also maintains excellent mechanical properties. F127-IEA immobilized Lactobacillus bulgaricus T15 (F127-IEA-T15) can be used in cell-recycle fermentation for over 150 days from glucose and 50 days from corn stover hydrolysate, achieving high production rates of D-LA from glucose (2.71 ± 0.85 g/L h) and corn stover hydrolysate (1.29 ± 0.39 g/L h). F127-IEA-T15 enhanced D-LA production by adsorbing and blocking toxic substances present in corn stover hydrolysate that are detrimental to cellular activity. The newly developed hydrogels in this study provide a robust platform for large-scale extraction of D-LA from non-detoxified corn stover.

Exploration of InSitu Extraction for Enhanced Triterpenoid Production by Saccharomyces cerevisiae.

Plant-derived triterpenoids are in high demand due to their valuable applications in cosmetic, nutraceutical, and pharmaceutical industries. To meet this demand, microbial production of triterpenoids is being developed for large-scale production. However, a prominent limitation of microbial synthesis is the intracellular accumulation, requiring cell disruption during downstream processing. Destroying the whole-cell catalyst drives up production costs and limits productivity and product yield per cell. Here, insitu product extraction of triterpenoids into a second organic phase was researched to address this limitation. An organic solvent screening identified water-immiscible isopropyl myristate as a suitable insitu extractant, enabling extraction of up to 90% of total triterpenoids from engineered Saccharomyces cerevisiae. Combining isopropyl myristate and β-cyclodextrins improved extraction efficiency. In a first configuration, repeated batch fermentation with sequential product extraction and cell recycling resulted in 1.8 times higher production than a reference fermentation without insitu product extraction. In the second configuration, yeast cells were in contact with the second organic phase throughout a fed-batch fermentation to continuously extract triterpenoids. This resulted in 90% product extraction and an extended production phase. Further improvement of triterpenoid production was not achieved due to microbial host limitations uncovered through omics analyses.

Effects of aging of polyethylene microplastics and polystyrene nanoplastics on antibiotic resistance gene transfer during primary sludge fermentation

The increasing presence of nano and microplastics (NPs/MPs) in wastewater treatment plants and their inevitable accumulation in the sludge has raised serious concerns in recent years. This study investigated the effects of pristine and aged polyethylene microplastics (PEMPs), polystyrene nanoplastics (PsNPs), and their mixtures on the primary sludge fermentation process. Pristine MPs/NPs (150 μg/L and 2 g/L for PsNPs and PEMPs, respectively) underwent two weeks of weathering in the presence of humic and alginic acids. The results from a batch fermentation experiment (15 days, pH 10) revealed that the exposure to aged PEMPs/PsNPs experienced greater VFA production than pristine samples. Notably, the aged PEMPs/PsNPs mixture showed a 23.12% increase in VFA production over the pristine mixture. The relative abundance and total concentration of antibiotic resistance genes (ARGs) increased in all PEMPs/PsNPs batches compared to the control, with the most significant rise in total ARGs observed in the aged PEMPs sample. Aged PEMPs exhibited a 26.22-fold increase in tetA genes, while aged mix samples showed a 19.68-fold increase in tetM genes compared to their pristine counterparts. Both pristine and aged PEMPs/PsNPs, particularly the aged PEMPs adversely affected the microbial communities at the genus level and altered the microbial structure. Microbial richness and diversity were enhanced in samples exposed to pristine PEMPs/PsNPs and aged PsNPs but decreased in aged PEMPs and in the aged mixture group, suggesting a negative impact of aged polyethylene microplastics on microbial communities. Correlation analysis suggested that phyla Planctomycetes, Proteobacteria, and TM7 are potential hosts of ARGs. These findings manifest the substantial effects of aged nano/microplastics compared to their pristine forms, emphasizing the complex interplay between various forms of PEMPs/PsNPs and microbial dynamics in sludge fermentation processes.

Combining 2'-fucosyllactose and galactooligosaccharides exerts anti-inflammatory effects and promotes gut health

This study investigated the potential of 2'-Fucosyllactose (2'-FL) and galactooligosaccharides (GOS) combinations as a novel and cost-effective substitute for human milk oligosaccharides (HMOs) in promoting gut health and reducing inflammation. In vitro studies using Caco-2 cells showed that 2'-FL and GOS combinations (H1: GOS:2'-FL ratio of 1.8:1; H2: ratio of 3.6:1) reduced lipopolysaccharide-induced inflammation by decreasing pro-inflammatory markers, while individual treatments had no significant effects. In a mouse model of dextran sulfate sodium (DSS)-induced colitis, combined 2'-FL and GOS supplementation alleviated symptoms, improved gut permeability, and enhanced intestinal structure, with the GH1 group (H1 combo with DSS) being the most effective. 2'-FL and GOS combinations also enhanced short-chain fatty acid production in infant fecal batch fermentation and mouse fecal analysis, with GH1 showing the most promising results. GH1 supplementation altered gut microbiota in mice with DSS-induced colitis, promoting microbial diversity and a more balanced Firmicutes to Bacteroidota ratio. Infant formula products (IFPs) containing 2'-FL and GOS combinations (IFP2: 174 mg GOS and 95 mg 2'-FL per 14 g serving, 1.8:1 ratio; IFP3: 174 mg GOS and 48 mg 2'-FL per 14 g serving, 3.6:1 ratio) demonstrated gastrointestinal protective and anti-inflammatory properties in a coculture model of Caco-2 and THP-1 cells. These findings suggest that 2'-FL and GOS combinations have potential applications in advanced infant formulas and supplements to promote gut health and reduce inflammation.

Production of Succinic Acid by Metabolically Engineered Actinobacillus succinogenes from Lignocellulosic Hydrolysate Derived from Barley Straw.

Succinic acid is an industrially important component that plays a key role in food additives, dietary supplements, and precursors for biodegradable polymers. Due to environmental and economic issues, succinic acid production has become increasingly attractive. This work aimed to improve succinic acid production from lignocellulosic biomass in Actinobacillus succinogenes through genetic modifications and fermentation strategies. Firstly, the effects on succinic acid production by overexpressing genes encoding phosphoenol carboxylase, malate dehydrogenase, and fumarase were evaluated in batch fermentations of engineered A. succinogenes strains. The engineered A. succinogenes expressing PCK, MDH, and FUM (AS-PMF) showed a 1.3-fold increase in succinic acid production compared to the wild-type strain. Subsequently, the fed-batch fermentation with MgCO3 was carried out using AS-PMF, which led to producing 50 g/l of succinic acid with 0.79 g/g of yield. Finally, 22.2 g/l of succinic acid with 0.64 g/g of yield was achieved in batch fermentation from lignocellulosic hydrolysate of barley straw. These results support that sustainable succinic acid from agricultural wastes might be a promising strategy for industrial applications.

Engineering Acetobacterium wieringae for acetone production from syngas

Gas fermentation using acetogenic bacteria offers a sustainable alternative to fossil-based chemical production by converting waste gas streams, such as syngas (CO, H2, CO2), into valuable biocommodities. In this study, strain JM, a novel isolate of the acetogen Acetobacterium wieringae was engineered for autotrophic acetone production. Novel constitutive promoters native to A. wieringae JM were identified and characterized, expanding the genetic toolbox for this organism. These promoters, along with previously described promoters from Clostridium autoethanogenum, were used to control the expression of acetone production operon from C. acetobutylicum, leading to significant improvements in acetone titers. The use of multiple transcriptional units (TUs) for pathway gene expression further enhanced acetone production compared to a single operon. Additionally, the acetoacetyl-CoA acetate/butyrate CoA-transferase enzyme from C. beijerinckii was found to improve acetone production compared to its homolog from C. acetobutylicum. The plasmid-based expression system using the pMTL83151 vector (pCB102 replicon) demonstrated segregational stability and a consistent single-copy number under both selective and non-selective conditions. Fermentation conditions were optimized, with buffer capacity and gas availability identified as critical factors in batch fermentations. The physiology of autotrophic acetone production was further assessed utilizing different gas compositions which significantly impacted carbon flux to acetone. Under optimized conditions with a 20 % CO syngas blend, the engineered A. wieringae JM [pAW_PFTL-APO] strain achieved an acetone titer of 41.3 mM. This study establishes A. wieringae JM as a promising platform for sustainable acetone production from waste gases and provides a foundation for further development of gas-based biorefineries.

Synthesis of Super-High-Viscosity Poly-γ-Glutamic Acid by pgdS-Deficient Strain of Bacillus licheniformis and Its Application in Microalgae Harvesting.

Poly-γ-glutamic acid (γ-PGA) is a natural polymer whose molecular weight and viscosity are critical for its application in various fields. However, research on super-high-molecular-weight or -viscosity γ-PGA is limited. In this study, the pgdS gene in Bacillus licheniformis WX-02 was knocked out using homologous recombination, and the batch fermentation performances of the recombinant strain WX-ΔpgdS were compared to those of WX-02. Nitrate accumulation was observed in the early fermentation stages of WX-ΔpgdS, and gene transcription analysis and cell morphology observations revealed that nitrite accumulation was caused by oxygen limitation due to cell aggregation. When the aeration and agitation rates were increased to 2.5 vvm and 600 r/min, respectively, and citrate was used as a precursor, nitrite accumulation was alleviated in WX-ΔpgdS fermentation broth, while γ-PGA yield and broth viscosity reached 17.3 g/L and 4988 mPa·s. Scanning electron microscopy (SEM) showed that the γ-PGA produced by WX-ΔpgdS exhibited a three-dimensional porous network structure. At a γ-PGA concentration of 5 mg/L, the fermentation broth of WX-ΔpgdS achieved a flocculation efficiency of 95.7% after 30 min of microalgae settling. These findings demonstrate that pgdS knockout results in super-high-viscosity γ-PGA, positioning it as an eco-friendly and cost-effective biocoagulant for microalgae harvesting.