Applied Microbiology and Biotechnology

Fermentation of C1 gases is an emerging technology where waste gases are bio converted into value-added products. This study navigates the gas fermentation potential of Gordonia rubripertincta to produce carotenoids. The crucial carbon monoxide dehydrogenase (CODH) enzyme, necessary for gas uptake by the microbe, was found to be present in G. rubripertincta through blastp on NCBI website. The organism was then used for gas fermentation experiments in a continuous stirred tank reactor (CSTR) in different modes of reactor operation resulting in the production of about 500 mg pigment/g WCW (wet cell weight). Two important reactor parameters, molybdenum content and pH, were optimized for enhanced carotenoid production. Overall, G. rubripertincta was observed to be an efficient candidate organism for C1 gas fermentation. KEY HIGHLIGHTSO_LIGordonia rubripertincta synthesises aerobic carbon monoxide dehydrogenase enzyme. C_LIO_LIIt is a potential gas fermenting microbe that gives carotenoids as product. C_LIO_LIThe gas uptake efficiency of the microbe is more in fed-batch discontinued mode. C_LIO_LIIn FB-D, the resultant carotenoids are 500+9 mg/g wet cell weight (WCW). C_LIO_LIMo/pH of 20 mg/7.0 resulted in highest carotenoids, i.e., 134+41 mg/g WCW. C_LI GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=87 SRC="FIGDIR/small/722808v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@8b1185org.highwire.dtl.DTLVardef@2b6f90org.highwire.dtl.DTLVardef@1a9697dorg.highwire.dtl.DTLVardef@14c9dc8_HPS_FORMAT_FIGEXP M_FIG C_FIG

Biological sequences are known to be not random. Thus, the comparison of in silico restriction fragment distributions of random and biological sequences may be an indicator of this non-randomness. Our analyses show that for most of the tested combinations of restriction enzyme and genome sequence the fragments per Megabase of the biological sequence deviate at least more then 10% from the corresponding random sequence. This deviation goes into both directions, i.e. clearly increased values are as common as clearly decreased values. Although there is no species- or restriction-enzyme-specific effect, a clear impact of the GC content both of the restriction site and of the genome sequence can be seen. In contrast to the random sequences, the genome sequences show distinct peaks in their fragment length distributions, hinting to repetitive elements such as transposons.

Chitosan is an oligosaccharide derived from chitin that is protonated at acidic pH to form a polycation. Its positive charge promotes the interaction with negatively charged components of the yeast cell surface, which has been associated with increased cell permeability and growth inhibition. In this study, we investigated the interaction of chitosan with the cell surface and its permeabilizing capacity in three yeast species displaying distinct susceptibility profiles, Saccharomyces cerevisiae, Candida albicans and Debaryomyces hansenii. We evaluated the correlation between differential susceptibility and chitosan association at the cell surface, as well as cell permeabilization, by integrating growth analyses with surface-binding assays, including FITC-conjugated chitosan to monitor surface association and cellular integration over time, and ultrastructural examination by transmission electron microscopy (TEM). Our results showed that chitosan exhibited varying effects on the growth and permeability of each yeast strain, with D. hansenii being the most susceptible. Furthermore, we observed the incorporation of chitosan onto the cell surface and confirmed its role as a permeabilizing agent. Finally, we used chitosan-induced permeabilization as a method to measure the activity of selected enzymes in situ, demonstrating its potential for studying metabolic functions in permeabilized yeast cells. Overall, our findings establish chitosan as a strain-dependent antifungal agent and a useful tool for functional biochemical analyses in yeast.

The search for antimicrobials with a low propensity to select resistance has intensified in response to the global antimicrobial resistance crisis. Norway spruce resin (Picea abies) has long been used in Northern European wound care traditions and has shown broad antimicrobial activity in earlier microbiological studies. In the present study, we evaluated whether prolonged exposure to medical-grade spruce resin promotes reduced susceptibility in clinically relevant bacterial species. A 20-day serial-passage experiment was performed with Staphylococcus aureus, Pseudomonas aeruginosa, and Enterococcus faecalis using sub-inhibitory resin concentrations and broth microdilution readouts at baseline, day 10, and day 20. Resistance development was predefined as a [≥]4-fold increase in inhibitory concentration. Baseline inhibitory concentrations were 1.25% for S. aureus, 5.0% for P. aeruginosa, and 2.5% for E. faecalis. After 20 days, inhibitory concentrations were 2.5%, 10.0%, and 2.5%, respectively, corresponding to at most 2-fold changes and remaining below the predefined threshold for resistance development. Validation and vehicle-control arms indicated that these shifts were not attributable to medium transfer or solvent-related bias. These findings suggest that medical-grade Norway spruce resin has a low short-term tendency to select for reduced susceptibility under serial-passage conditions. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=134 SRC="FIGDIR/small/723837v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@160479forg.highwire.dtl.DTLVardef@1fe1e95org.highwire.dtl.DTLVardef@89dec3org.highwire.dtl.DTLVardef@17ff134_HPS_FORMAT_FIGEXP M_FIG C_FIG

Fungal contamination of food with yeast and moulds is associated with major economic losses due to spoilage and also poses health risks in the form of mycotoxin production. The strain Pantoea agglomerans APC 4211 isolated from leaves of Ilex aquifolium (holly tree) has broad spectrum antifungal activity against a variety of food spoilage fungi. Genomic analysis of the strain confirmed the presence of biosynthetic gene clusters potentially encoding for the enzymatic machinery required for the production of the antifungal lipopeptide herbicolin A. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis of the cell-free supernatant (CFS) confirmed the presence of molecular masses corresponding to herbicolin A (1300.8 Da), and herbicolin B (1138 Da). Purified herbicolin A has desirable properties for biotechnological applications, including potent antifungal activity against a range of spoilage fungi, thermal stability and resistance to proteases. Herbicolin A has low cytotoxicity against epithelial cell lines and has minimum inhibitory concentrations (MICs) lower than those of some commercial antifungal drugs (0.2 - 2.5 {micro}g/ml). In a model dairy system (10% skim milk), herbicolin A demonstrated excellent solubility and stability, effectively eliminating Aspergillus niger and Penicillium notatum at a concentration of 5 {micro}g/mL. In conclusion, herbicolin A is a potent, naturally occurring antifungal agent with the potential to be applied as a biopreservative in food systems, providing a safe, clean-label, and efficient compound for synthetic preservatives replacement. HighlightsO_LIHerbicolin A has a strong potential as a natural preservative for food protection C_LIO_LIHerbicolin A shows lower MICs than several antifungal agents C_LIO_LIHerbicolin A is stable under heat and resistant to proteolytic degradation C_LIO_LIHerbicolin A has strong solubility and stability in a model dairy system C_LIO_LIHerbicolin A indicates low cytotoxicity against epithelial cell lines C_LI Data summaryThe authors confirm all supporting data, code and protocols have been provided within the article or through supplementary data files.

Sewer biofilms represent dynamic interfaces for exchange of bacteria and antibiotic resistance genes between biofilms and the overlying wastewater. Using inline, biofilm reactors, the movement of bacteria and 16S rRNA and carbapenemase genes (blaKPC, blaVIM, blaNDM, blaOXA-48-like, and blaIMP) between wastewater and sewer biofilms was investigated. Established, complex biofilms without these {beta}-lactamase (bla) genes, absorbed resistant bacteria within two minutes of exposure to high concentrations of resistant cultures in lab settings. Carbapenem-resistant organisms from these high-concentration source biofilms transferred to downstream biofilms over 60 minutes of representative sewer shear flows. Mass balances of bacteria and genes in biofilms versus wastewater under representative shear flow showed that biofilms exposed to resistant cultures contributed more to the wastewater than to the downstream biofilms. In field studies, established, complex biofilms without target carbapenem-resistant bacteria and genes from wastewater within hours and then stabilized between 2 to 15 days, not varying by more than 0.5 MPN/cm2 or 0.5 log gene copies (GC)/cm2. In contrast, metagenomic profiles of the bacterial community species continued to change up to 21 days. Established biofilms with resistant bacteria and genes exposed to tertiary-treated wastewater without target carbapenemase genes or meropenem antibiotics did not lose resistant genes or bacteria over nine days of exposure (i.e., < 1 log GC/cm2 reduction). Results show that sewer biofilms contribute to the resistance-gene signal found in sewer wastewater by absorbing and releasing bacteria and genes. Consideration of sewer biofilm dynamics is essential for more accurately interpreting wastewater bacterial concentrations in wastewater-based epidemiology studies. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=77 SRC="FIGDIR/small/726639v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@19f6ce0org.highwire.dtl.DTLVardef@1a507c8org.highwire.dtl.DTLVardef@1a2013dorg.highwire.dtl.DTLVardef@ff8613_HPS_FORMAT_FIGEXP M_FIG C_FIG

Colletotrichum siamense is a predominant causal agent of anthracnose in rubber tree and numerous economically important crops, causing severe yield losses worldwide. Conidial germination represents a critical early step for successful infection, while the high-osmolarity glycerol (HOG) MAPK pathway and ergosterol biosynthesis individually govern fungal development, stress adaptation and fungicide responses. However, the molecular crosstalk between these two modules remains largely elusive in phytopathogenic fungi. Here, we identified CsErg5B, a sterol C-22 desaturase homolog, as a direct target of the HOG- regulated transcription factor CsAtf1 in C. siamense. CsErg5B was indispensable for ergosterol biosynthesis, conidial germination, appressorium formation, and full virulence. The {Delta}CsErg5B mutant showed increased conidiation but severely impaired germination, and exhibited elevated resistance to fludioxonil while hypersensitivity to azole fungicides. Epistasis analysis using the {Delta}CsErg5B/{Delta}CsCyp51G1 double mutant - where CsCyp51G1 serves as another downstream target of CsAtf1 - revealed that CsErg5B functions as the predominant downstream effector of CsAtf1 in modulating conidial development and fludioxonil sensitivity. Furthermore, overexpression of CsErg5B significantly rescued the defects in conidial germination and fludioxonil sensitivity in both {Delta}CsAtf1 and {Delta}CsPbs2 mutants. Taken together, our findings uncover a HOG MAPK - CsAtf1 - CsErg5B regulatory axis that connects HOG MAPK signaling to ergosterol homeostasis, thereby governing conidial germination and fungicide sensitivity in C. siamense. This study provides novel insights into the regulatory network underlying fungal development and fungicide response, and offers promising molecular targets for the integrated management of plant anthracnose.

Numerous studies have shown that the abundance of antibiotic-resistant bacteria (ARBs) or antibiotic-resistance genes (ARGs) in soil increases after irrigation with wastewater. However, it is unclear whether this increase is due to the selection effects of pharmaceutical residues in the irrigation water or the continuous introduction of ARBs and ARGs with the wastewater. Further, it is unclear how the binding of antibiotics to natural colloids (1-1000 nm) affects their biological effects compared to truly dissolved substances (< 1 nm). We conducted competition experiments with resistant and susceptible Acinetobacter baylyi BD413 strains in wastewater, as well as in colloidal and truly dissolved extracts of soils irrigated with wastewater. Although the concentrations of our six target antibiotics were far below the measured minimum selective concentrations of the tested strains, we demonstrate that the resistant strain was favored in the wastewater and the colloidal extracts. In contrast, the truly dissolved fractions exhibited weaker and more variable selective effects. A non-targeted analysis revealed the presence of 82 additional substances in our extracts, including further antibiotics, pesticides, and different non-antibiotic drugs that may influence the selection of our resistant A. baylyi BD413 strain. Our findings suggest that antibiotic resistance is selected for in wastewater and wastewater-irrigated soils. This cannot be explained by antibiotic concentrations alone, but may also arise from the effects of complex mixtures of co-occurring contaminants, particularly those associated with colloidal particles.

Edible seaweed has the potential to become a valuable marine resource for food applications due to its potential health benefits and ecological sustainability. The brown seaweed Alaria esculenta is rich in essential minerals, vitamins, and dietary fibers, making it a nutritious food source. Fermentation, as a traditional preservation method, can enhance seaweed shelf-life and be useful for the development of new foods/ beverages. In this study, the effects of fermentation of A. esculenta, by the lactic acid bacterium (LAB) Lactiplantibacillus plantarum, on the nutritional profile, and the content of potentially toxic elements, was investigated. L. plantarum was successfully cultivated on A. esculenta using two modes of operation, submerged (SmF) and solid-state fermentation (SSF), resulting in production of cells and lactic acid, and reduction of the pH to below 4.3 within 3 days, which was not achieved in parallel spontaneous fermentations using indigenous seaweed microbiota. A. esculenta s macro-nutritional profile was altered, reducing mannitol but increasing fucose and glucose content (after acid hydrolysis) while also concentrating the protein content. LAB fermentation significantly increased the concentration of antioxidant phenolic compounds, such as phloroglucinol, syringic acid, and epicatechin, compared to untreated samples. However, lipophilic compounds like carotenoids decreased after both spontaneous and LAB-fermentation. A reduction in total mineral content was observed after LAB fermentation and water soaking, and SmF with L. plantarum effectively reduced arsenic and iodine levels. Overall, fermentation using L. plantarum showed potential as a bio-preservation method for the edible brown seaweed, A. esculenta, improving its nutritional profile and enhancing food safety.

Quaternary ammonium compounds (QACs) are high production volume biocidal compounds increasingly scrutinized for their potential to promote antimicrobial resistance spread. This study compared the release of QACs, QAC resistance indicator genes (qacE/qacE{Delta}1), and QAC tolerant bacteria from livestock and human waste streams into the environment. Five livestock farms with on-farm biogas plants (BGPs), a rural and an urban municipal wastewater treatment plant (WWTP) were studied in parallel. In WWTPs, <1% of incoming QACs were discharged with treated wastewater but 10-20% were transferred to sewage sludge. QAC concentrations in sewage sludge far exceeded those in raw and digested manure. The qacE/qacE{Delta}1 genes were detected in all samples with a higher relative abundance in solid than liquid samples. Relative abundances of QAC tolerant fast growing heterotrophic bacteria cultivated under high nutrient conditions at 37{degrees}C were higher in human than livestock waste streams. Providencia and Pseudomonas dominated the cultivated QAC tolerant bacteria in both systems but showed higher QAC tolerance when originating from human waste streams. Additionally, Enterobacteriaceae with higher QAC tolerance were cultivated from human waste streams. Most QAC tolerant strains carried antibiotic resistances without strong system differences. Only few strains carried the qacE/qacE{Delta}1 gene indicating that other mechanisms must be responsible for the increased QAC tolerance. In conclusion, QACs, qacE/qacE{Delta}1, and viable QAC tolerant bacteria including potential pathogenic bacteria were released from livestock and human waste streams into the environment with highest abundances in a post-pandemic sewage sludge sample. Highlights- QACs most abundant in human waste streams, especially biosolids - Higher relative abundance of QAC tolerant bacteria in human waste streams - Pseudomonas and Providencia dominated QAC tolerant bacteria in both waste streams - Enterobacteriaceae with higher QAC tolerance abundant in human waste streams - Most QAC tolerant strains carried additional antibiotic resistances Environmental implicationMunicipal wastewater treatment plants (WWTPs) and livestock farms are hotspots for antimicrobial resistance (AMR) propagation. We compared the simultaneous occurrence of quaternary ammonium compounds (QACs), resistance genes (RGs), QAC-tolerant bacteria, and their multidrug-resistance status in livestock and human waste streams. QACs, indicators of QAC tolerance and AMR occurred in both systems but were higher in WWTPs, especially sewage sludge. Our findings highlight the need for prudent disinfectant use and enhanced waste treatments to reduce the risks of spreading micropollutants, pathogens, and AMR via organic fertilizers or treated wastewater recycled in circular agricultural practice.

Hydrogenophilus thermoluteolus TH-1 is a thermophilic hydrogen-oxidizing bacterium capable of producing poly(3-hydroxybutyrate) (PHB) from CO2. To redirect carbon flux for producing other useful biomaterials, we disrupted the acetoacetyl-CoA reductase genes (phaB1 and phaB2), which are central to the primary PHB synthesis pathway. Unexpectedly, the resulting {Delta}phaB1B2 mutant still accumulated PHB under autotrophic conditions, reaching approximately 25-35 % of the wild-type level. Furthermore, PHB accumulation in the mutant was significantly restored when fatty acids (butyrate and oleate) were used as carbon sources, whereas acetate and malate resulted in reduced accumulation. These results suggest the existence of a PhaB-independent PHB synthesis pathway. We propose that intermediates from the {beta}-oxidation of fatty acids are converted to (R)-3-hydroxybutyryl-CoA, bypassing the disrupted PhaB enzymes. Additionally, the basal PHB production from non-fatty acid sources implies the involvement of a reverse {beta}-oxidation pathway. This study highlights the metabolic versatility of strain TH-1 for future metabolic engineering.

Cyclic diguanosine monophosphate (c-di-GMP) is a ubiquitous bacterial second messenger that regulates diverse cellular processes, including colony morphology, motility, biofilm formation, and virulence. It is synthesized by diguanylate cyclases (DGCs) containing the GGDEF domain and degraded by phosphodiesterases (PDEs) containing the EAL domain. However, studies on the genetic and physiological characteristics of c-di-GMP metabolism in Pantoea ananatis are lacking. In this study, we identified 26 predicted c-di-GMP metabolism-related genes in the P. ananatis PA13 genome: 9 encode GGDEF-only domain proteins, 5 encode dual GGDEF/EAL domain proteins, and 12 encode EAL-only domain proteins. We constructed overexpression strains and mutants of 26 DGC- and PDE-encoding genes, and then assessed their Congo Red binding, mucoid and rugose phenotypes, pellicle formation, and swimming motility. We identified 14 of 26 DGC and PDE proteins that affect phenotype changes. Among the 26 DGC- and PDE-overexpressing strains, 13 exhibited the phenotypic changes described above, with some showing alterations in multiple phenotypes simultaneously. Notably, overexpression of dgcM induced changes across all phenotypes. Among the 26 DGC and PDE mutants, the pdeC mutant increased pellicle formation and Congo red binding, the pdeM mutant reduced the mucoid phenotype, and the pdeS mutant, which shows high similarity to ydiV, an anti-FlhD factor, increased swimming motility. Overexpression strains and mutants of 14 DGC and PDE proteins that exhibited phenotypic changes had higher intracellular c-di-GMP levels than the wild type. This study provides important insight into the role of the c-di-GMP network in the plant pathogen P. ananatis. IMPORTANCEPantoea ananatis is a versatile bacterium that causes significant diseases in various economically important plants. To survive and infect hosts, bacteria use a key signaling molecule called c-di-GMP to switch between swimming freely and forming protective communities known as biofilms. Despite its importance, the specific genes governing this signaling network in P. ananatis remained unknown. In this study, we systematically identified and characterized 26 genes responsible for regulating c-di-GMP levels in P. ananatis PA13. By analyzing mutants and overexpressing these genes, we pinpointed 14 critical factors that control essential behaviors such as motility, pellicle formation, and colony appearance. Notably, we discovered specific genes, such as dgcM and pdeS, that act as master regulators of these traits. This comprehensive functional map of the c-di-GMP network provides essential insights into how this pathogen adapts to its environment, offering potential targets to control plant infections.

Antibiotic residues exceeding selective concentrations for antibiotic-resistant bacteria have been detected in various environments, including manure, wastewater, and effluents from wastewater treatment plants. When these residues come into contact with soils, for instance, due to wastewater irrigation or fertilization with manure, they interact with soil constituents. Soil colloids (1-1000 nm), such as montmorillonite, have been observed to adsorb pharmaceuticals, including antibiotics. We investigated the effect of colloids on the bioavailability of ciprofloxacin and found, that added to bacterial growth medium, montmorillonite reduces, but does not completely prevent, the growth-inhibitory effect of the antibiotic. The bacteria were able to grow at up to roughly double the concentration of ciprofloxacin in the presence of montmorillonite. We show that the incomplete deactivation of ciprofloxacin was most probably caused by medium components that decreased the adsorption of ciprofloxacin to montmorillonite. We conclude that a selective potential of this highly active antibiotic in contaminated soils, which also contain nutrients enabling bacterial growth, cannot be ruled out. Environmental implicationAntibiotics such as ciprofloxacin are frequently detected in water bodies and soils due to wastewater irrigation or manure application. These residues raise concerns about environmental toxicity and antibiotic resistance. This study demonstrates that montmorillonite, a common clay mineral in soils, significantly reduces the antimicrobial efficacy of environmental ciprofloxacin concentrations by sorption. The findings reveal a natural attenuation mechanism that may influence the environmental fate and bioavailability of antibiotics. Understanding such interactions is critical for predicting antibiotic behavior in terrestrial systems and for designing more accurate environmental risk assessments.

Pathogen cross-contamination during food production is primarily controlled through environmental sanitation. However, sanitizer efficacy is often studied in bench-scale experiments that poorly approximate the fluid dynamics of sanitization and limits our understanding of commercial sanitization efficacy. This study paired computational fluid dynamics (CFD) estimates of shear stress with experimental measurements of Listeria innocua reduction on stainless steel following treatment with 100 ppm hypochlorite sanitizer. At the pilot-scale, sanitizer spray manually applied by researchers achieved a 2.6 {+/-} 0.4 log CFU/surface reduction; however, microbial reduction from manual operation of sanitizer spray equipment differed significantly between researchers (p < 0.05). Microbial reduction varied by location following stationary, bench-scale spray application of sanitizer for 3 s. The greatest reduction was at the point of sanitizer spray impingement (7.5 {+/-} 0.5 log CFU/surface) and directly adjacent to the impingement point (6.4 {+/-} 0.7 log CFU/surface) where shear stress was the highest. Significantly less microbial reduction (0.4 {+/-} 0.1 log CFU/surface) occurred where shear stress was lowest in the fluid-film of sanitizer running down from the impingement point (p < 0.05). Static submersion of inoculated coupons in sanitizer for 3 s resulted in a log reduction of 2.3 {+/-} 0.1 log CFU/surface. Discrepancies between bench-scale spraying, pilot-scale spraying, and submerged coupons demonstrate the need for sanitizer efficacy testing under realistic conditions to better estimate the risk reduction achieved through sanitation programs. IMPORTANCESanitation is critical for controlling pathogen cross-contamination during food production. These findings highlight the limitations of traditional approaches to sanitizer efficacy testing, not because they are invalid, but because they do not reflect the level of microbial reduction typically achieved in application. We demonstrate that these differences in outcomes are attributable to fluid dynamics and exposure, which are not well approximated in submerged coupon experiments. Accurate estimation of microbial reduction from sanitizer application is needed to guide food safety policy decisions. For example, overestimation of the risk reduction conferred by sanitizer treatment may result in food safety policies that neglect other sources of microbial reduction within sanitation programs.

In vegetarian diets, phytate is known to disrupt the adsorption of minerals. Fortifying foods with phytase, a therapeutic enzyme known to mitigate phytate, might increase the uptake of important nutrients. Phytase is susceptible to environmental stress such as heat and acidic conditions encountered during food processing. Therefore, we developed and optimized a core-shell microparticle composed of a phytase-chitosan core and a shell consisting of cross-linked alginate-{kappa}-carrageenan. Ethanol was used to precipitate the microparticles, and the ethanol concentration was optimized along with the chitosan and phytase ratio and the alginate-carrageenan concentration, to form stable core-shell microparticles. The optimized core-shell microparticles have a loading capacity of 32.7% with a high encapsulation efficiency of 80.3% and uniform micro-size with a diameter of 3.2 {micro}m and a poly-dispersity index of 0.178. Loaded phytase retained 62.7% enzymatic activity after heat treatment and digestion conditions. These results indicate that core-shell microparticles are suitable for retaining enzyme activity within the food matrix under typical food processing conditions. HighlightsO_LIDevelopment of size-controlled core-shell microparticles to protect phytase C_LIO_LIPhytase-chitosan microparticles are surrounded by an alginate-{kappa}-carrageenan shell C_LIO_LIOptimization achieved 32.7% loading capacity with a uniform size of 3.2 {micro}m C_LIO_LICore-shell microparticles retained 62.7% enzyme activity after heat and digestion C_LIO_LIPhytase powder (2 mg) is required for a single maize meal C_LI

Black Soldier Fly larvae (BSFL, Hermetia illucens) are highly effective for the bioconversion of food waste. However, their rearing process often produces substantial ammonia emissions, which are malodorous and environmentally concerning. We investigated the co-cultivation of BSFL with the sulfur-oxidizing bacterium Thiobacillus thioparus as a strategy to mitigate ammonia release. Importantly, under conditions where ammonia emissions were significantly reduced, neither larval growth nor bacterial viability was negatively affected. Furthermore, even when the initial bacterial inoculum was reduced to 3.3*105 CFU/g-food wastes, the bacterium rapidly recovered to functional levels and effectively controlled ammonia emissions. This indicates the absence of harmful interaction or nutrient competition between BSFL and T. thioparus. These findings suggest an efficient method for controlling ammonia in large-scale BSFL waste treatment. By reducing the required bacterial inoculum, this approach enables scalable microbial co-culturing with environmental and production benefits.

A challenge in using plant biomass is its highly recalcitrant nature, which makes it economically infeasible to utilize. In natural environments, various microbes, including bacteria and fungi, are reported to decompose plant cell wall materials such as cellulose and hemicellulose, and there may be undescribed microbes that contribute to the degradation of plant biomass. We focused on isolating novel plant biomass-degrading bacteria and screened more than 100 isolates from the Tomakomai experimental forest in Hokkaido, Japan. Among them, one novel Bacillus species was chosen for whole-genome sequencing. Comparative genomics and a carbon source utilization assay indicated that the isolate belongs to a subspecies of Bacillus subtilis, which we named B. sp. TTS1. Glucose, cellobiose, xylose, xylan, mannose, or mannan was used as the sole carbon source in the minimum medium, and the growth of this bacterium was determined. Furthermore, a proteomic analysis of B. sp. TTS1 was performed using culture supernatants from various polysaccharide-containing media. In the present study, several key enzymes involved in plant biomass degradation were identified, namely {beta}-1,4-mannanase and xylanase, and they were highly enriched in all tested polysaccharides.

Recent evidence suggests that the gut microbiome plays a role in the development and treatment response of pancreatic ductal adenocarcinoma (PDAC). However, the functional impact of tumor location and preoperative biliary stenting (PBS) on microbial composition and metabolism remains poorly understood. In this prospective study, preoperative stool specimens were collected from patients undergoing surgery for PDAC at Heidelberg University Hospital, Germany, between March 2020 and July 2021. Whole-genome shotgun metagenomic sequencing was performed to characterize microbial composition and functional pathways. A total of 63 preoperative stool samples were analyzed, including 40 patients with pancreatic head tumors (63.5%) and 23 with body/tail tumors (36.5%). Microbial community composition differed significantly according to tumor location (Bray-Curtis, p=0.005), with enrichment of Ruminococcus bromii in body/tail tumors. Among patients with pancreatic head tumors, PBS was associated with reduced alpha diversity (Shannon index, p=0.04), depletion of taxa including members of the Eubacteriales and Clostridiales orders as well as the genera Raoultella and Prevotella, and reduced abundance of selected genes involved in secondary bile acid metabolism. PBS was also associated with a higher rate of major postoperative complications according to Clavien-Dindo >3a (28.6% vs 3.8%; p=0.04). These findings suggest that biliary intervention may induce functional dysbiosis characterized by reduced microbial diversity and impaired bile acid metabolism, potentially disrupting host- microbiome crosstalk and contributing to adverse postoperative outcomes in pancreatic cancer.

BackgroundThe use of autologous or allogeneic cell therapies has now entered to the clinical practice in several fields of medicine, especially in oncology and hematology. From this regard, 2D-cell manufacturing is complex and costly and bioreactors have attracted major interest for efficient and cost-effective mass production of cells. Bioreactors have several advantages such as homogeneous repartition of nutrients and gas, control of all culture parameters and increased yield. However, the important shear stress generated by those bioreactors is an important disadvantage as it can affect cell survival or cell quality. This important shear stress is the result of the mixing method using either blades (used in stirred-tanked bioreactors) or gas bubbles (used in airlift bioreactors). Another downside of the use of bioreactors is the difficulty to scale-up. As the volume increases, the shear stress generated by blades radically increases leading to cell death and a decrease of cell quality. DescriptionIn this study, we describe a bioreactor developed using a different mixing method effectively reducing the shear stress and facilitating scale-up. This bladeless method uses an inclination of the bioreactor as well as rotation to mix fluids in a container. Here we described different steps that led to the adaptation of this bioreactor, initially developed for fragile microalgae culture, for mammalian cell culture amplification. The bioreactor was tested to amplify a natural killer (NK) cell line NK92 which is an IL-2 dependent cell line used in clinical trials for cancer therapy. We have tested the influence of 1-The number of cells seeded; 2-The influence of the rotation speed on cell growth and viability; 3-The influence of the bioreactor angle on the above parameters; 4-The duration of the culture. ResultsCells were initially seeded at 2.5.105 / ml in a volume of 380 ml. According to the rotation speed of 15, 30, 45 and 60 rpm, we have observed an increase of cell numbers at day 3 (3-fold), day 5 (7-fold) and day 7 (10-fold) compared to seeding, the best expansion being obtained at day 7 with a rotation speed of 45 rpm. The optimal angle of rotation was found to be 3 degree, with an optimal amplification at day 7 versus day 3 (p < 0.01). The viability was also found to be optimal in the latter condition. ConclusionsThese preliminary results demonstrate that NK92 cells could be amplified using this bioreactor. In the best tested condition, neither cell viability nor cell growth was impacted. These results strongly suggest the potential use of this device in future clinically applicable conditions.

Cyanobacteria are diverse photosynthetic microorganisms of great interest for fundamental science and sustainable biotechnological applications. However, their polyploidy makes genetic manipulation challenging and time-consuming. The development of CRISPR/Cas tools has greatly accelerated genome editing and metabolic engineering of few cyanobacterial model species. In this work, we extend the CRISPR/Cas12a system for targeted gene deletion in the non-model cyanobacterium Cyanothece PCC 7425, interesting for its ability to perform intracellular calcium carbonate (CaCO3) biomineralization, nitrogen fixation, etc. We demonstrate for the first time its tractability to gene knockout by generating deletion mutants of four genes (cax3-cax4, gor, and sodB) acting in metabolism and/or response to stresses, using Cas12a mediated homologous recombination. Importantly, full chromosome segregation was rapidly achieved after a single round of selection in all cases. All mutants were genotypically and phenotypically characterised. Moreover, biochemical analysis in the case of{Delta} sodB mutant further confirmed its targeted deletion. Overall, CRISRPR/Cas12a provides a rapid and efficient system for genome editing in Cyanothece PCC 7425, establishing this organism as a versatile model for studying oxidative stress pathways, metal toxicity and moreover, the still poorly known mechanism(s) of intracellular CaCO3 biomineralization. Key PointsO_LIRapid and efficient CRISPR/Cas12a editing established in Cyanothece PCC 7425. C_LIO_LIFully segregated knockout mutants obtained after single selection round. C_LIO_LIPlatform for nuclear waste bioremediation and other biotechnological applications. C_LI