Bioresource Technology

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

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.

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.

1.Abiotic stresses like nitrogen deficiency and soil salinity are major factors contributing to low crop yields. The use of selective biofertilizers alleviates both types of stress. In this study, we investigated the biofertilizer activity and plant growth-promoting properties (PGP) of Rhodococcus jialingiae RS1 through cytosolic proteome remodelling. We cultured RS1 under two conditions, i) without and ii) with 6% NaCl, in nitrogen-deficient defined Burks medium. Under dual stress of nitrogen limitation and salt stress, Orbitrap LC-MS/MS proteomics revealed one-quarter of the proteome remodelling, particularly the upregulation of ribosomal synthesis and protein repair systems. As expected, we found high expression of EctC, an ectoine synthase, a key enzyme in osmolyte biosynthesis. Additionally, ribosomal and translational-associated factors, including RpsL, RpsS, RpsT, RpsR1, RplV, RplL, RplA, and elongation factor Tuf, were highly expressed, suggesting enhanced translational fidelity under dual stress. High levels of DNA protection protein, Dps suggest dual stress may lead to DNA damage. Upregulation of chaperones, environmental sensors (KinE), and redox transcriptional factors like WhiB3, Hsp18, AhpC, and MetE suggests protein misfolding and oxidative stress. Metabolic modulations were evident through high expression of IlvA, NAD-dependent glutamate dehydrogenase, lipid/envelope-remodelling enzymes, cutinase/esterases, lipases, endopeptidases like NlpC/P60 and transport systems. In contrast, proteins involved in urease structural components (urea-G), nitrogen regulators and ammonium transporters (GlnK and Amt) were downregulated. Dual stress may lead to an energy crisis, prompting strategic shifts away from high-ATP-dependent ureolytic nitrogen-scavenging pathways towards lower-energy nitrogen-assimilating routes, such as IlvA-mediated deamination and NAD-dependent glutamate dehydrogenation. Genetic manipulations of the above-mentioned genes or their homologues across the genera of microbes, plants, and crops may enhance resilience to abiotic stresses. Our studies reveal stress-responsive genes and biochemical pathways that could be used to improve transgenic efficacy in nitrogen-limited, saline soil and other (a)biotic stresses. Global Proteome Profiling of Rhodococcus jialingiae RS1 to Develop Transgenics O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC="FIGDIR/small/724437v1_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@1719d80org.highwire.dtl.DTLVardef@1b6b59org.highwire.dtl.DTLVardef@24d367org.highwire.dtl.DTLVardef@1b33224_HPS_FORMAT_FIGEXP M_FIG C_FIG

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

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.

Microbial ammonia oxidation, the first and rate-limiting step of nitrification, plays a central role in soil nitrogen cycling. It is most relevant in agricultural soils as nitrifiers compete with crops for ammonia-based fertilizers. Therefore, synthetic nitrification inhibitors are widely used alongside fertilizers to reduce the activities of dominant drivers of this process, i.e. ammonia-oxidizing archaea (AOA) and bacteria (AOB). However, the physiological responses of ammonia oxidizers remain poorly resolved. Here the response of the AOA Nitrososphaera viennensis to the nitrification inhibitors 3,4-dimethylpyrazole phosphate (DMPP) and allylthiourea (ATU) were investigated using a combination of functional genomics, physiological assays, and relief experiments. The results overturn earlier assumptions that DMPP and ATU act by chelating free copper. Both compounds affected ammonia oxidation and triggered broader shifts in energy metabolism and stress-response pathways, which diverged markedly between the two inhibitors. We propose a competitive inhibition of the ammonia monooxygenase complex with DMPP as it can be alleviated by additional ammonia and elicits activation of urea acquisition, while ATU acted as a non-competitive inhibitor generally inducing quiescence. Both modes of inhibition were associated with clear transcriptomic and proteomic signals that will be advantageous for the identification of mechanisms of other nitrification inhibitors in the future. Key word: Ammonia-oxidizing archaea, nitrification, nitrification inhibitors, archaea, nitrogen cycle

Brown rot wood-degrading fungi release carbon (C) from deadwood but leave behind a large fraction of C sequestered in lignin residues or as fungal metabolites. The strength of sequestration in these C residuals remains unclear, but proteobacteria-dominated bacterial communities have been implicated in metabolizing C from decay residues, possibly erasing the C sequestration potential assumed for brown rot. Here, we paired a model brown rot fungus (Rhodonia placenta) with a model Alphaproteobacterium (Rhodopseudomonas palustris) to track fungal release and bacterial utilization of C derived from decaying wood. We found that fungal decay products generated by R. placenta could be used by R. palustris for growth, and later decay stages contained more usable substrates than early stages. High performance liquid chromatography with mass spectrometry identified a range of aromatic and non-aromatic compounds in the fungal-decayed wood, but after 95 days of bacterial growth, R. palustris preferentially consumed non-aromatic acids over aromatic lignin monomers. Genes involved with aromatic compound degradation were unimportant for bacterial growth, and RNA sequencing revealed that aromatic compound degradation genes were repressed on decayed wood extract. Randomly barcoded transposon sequencing failed to identify a solitary catabolic pathway used by R. palustris, suggestive of substrate co-utilization, and surprisingly, showed that genes involved with copper toxicity were essential. Finally, we found that genes involved with biosynthesis of certain cofactors and amino acids were no longer essential on decayed wood extract, suggesting these nutrients were readily accessible. This study helps lay the foundation to understand potential bacterial-fungal interactions in decayed wood. Graphical abstractTo explore how brown rot fungi support and compete with bacterial partners in the wood decay environment, the model brown rot fungus Rhodonia placenta was used to degrade aspen wafers which were then infused into bacterial growth medium. By leveraging the range of molecular biology tools available for the model Alphaproteobacterium Rhodopseudomonas palustris, we discovered that R. palustris preferentially consumes short organic acids instead of aromatic lignin monomers which it would otherwise consume if provided in isolation. Additionally, R. palustris scavenged certain amino acids (AAs) and enzyme cofactors including methionine, biotin, and PLP from the decayed wood extract, highlighting these as key shared resources for bacterial-fungal partnerships. We found that R. placenta increased the concentration of certain metals (Cu and Al) inducing a metal stress response in R. palustris, indicating that metal toxicity could be an important mode of competition between fungi and bacteria in the wood decay environment. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=93 SRC="FIGDIR/small/723453v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@16f31fcorg.highwire.dtl.DTLVardef@13a9b34org.highwire.dtl.DTLVardef@a37dcforg.highwire.dtl.DTLVardef@198bf1c_HPS_FORMAT_FIGEXP M_FIG C_FIG

Plant growth promoting microbes enhance developmental progression of the host by influencing its nutrient availability or by deploying secondary metabolites responsible for manipulating the hormonal crosstalk. Microbacterium bengalense sp. nov. GB16_1_BI (Accession number: SRX9280401), a newly identified ammonium releasing Actinomycetota, could enhance plant growth by manipulating rhizosphere bacteria. Amplicon sequencing of the 16S rRNA V3-V4 region from the rhizosphere of the black rice (Chakhao Poireiton) showed that GB16_1_BI could inhibit most bacteria. However, GB16_1_BI inoculation encouraged the growth of rare bacteria specific to waterlogged rice rhizosphere. Analysis of the OTUs using PICRUSt2 (Phylogenetic investigation of communities by reconstruction of unobserved states) showed increased abundance in the marker genes for nitrogen cycling (nifH, nrfA and nrt) but not for nifD or nifK which was also reflected in the ANOSIM analysis in the OTUs of the N-fixing bacteria. Marker genes for methane metabolism (comA, comB, cofG and cofH) were also more abundant in the inoculated plants than the control; however, ANOSIM studies did not support this observation in the OTUs of methane cycling bacteria. Both Methylosinus and Methylocystis, the two most abundant methanotrophic OTUs, are also known to be nitrogen fixers. Hence, GB16_1_BI could influence plant growth predominantly by manipulating nitrogen cycling microbes. The genome sequence as well as untargeted metabolome analyses of GB16_1_BI showed abundance of secondary metabolites with probable antimicrobial activity. GB16_1_BI could utilize varied carbohydrates and amino acid as energy source and form persister-like cells may help it to survive in the soil in absence of the host plant.

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.

Odd-chain carboxylates such as valerate and heptanoate are ecologically relevant metabolites and promising platform chemicals, yet the factors leading to their formation during secondary lactate fermentations remain poorly understood. Here, a continuous anaerobic bioreactor was operated for 297 days under mildly acidic conditions to evaluate how lactate:propionate molar ratios shape product spectrum in lactate fermentations. Valerate was the predominant odd-chain product under all conditions, reaching concentrations up to 110 mM, while heptanoate accumulated only at low levels (<10 mM). At low lactate concentrations (10-20 g/L), product selectivity strongly depended on the lactate:propionate ratio. When lactate:propionate ratios were around 1.2 mol/mol, odd-chain products were favored, whereas higher ratios (up to 4.8 mol/mol) shifted metabolism toward caproate and butyrate formation. However, this trend was not maintained at higher lactate concentrations (30-40 g/L; lactate not fully consumed), where odd-chain selectivities remained high even at lactate:propionate ratios of 4.8 mol/mol. Pathway analysis indicated that under high-lactate conditions up to 30% of lactate was redirected toward propionate and acetate formation, likely via the acrylate pathway. Microbial community analysis revealed a stable dominance of Caproiciproducens spp., that could be correlated to valerate production. Overall, this work provides mechanistic insights into the ecology of lactate fermentations and offers a framework for steering product selectivity in engineered anaerobic systems. HighlightsValerate was the dominant product, reaching up to 110 mM. Lactate:propionate ratios drive product selectivities. High lactate concentrations activated in situ propionate formation pathways. Caproiciproducens dominance was associated with sustained valerate production.

Microbial communities are central to the biogeochemical cycling of nutrients, critically shaping ecosystem functioning and influencing climate change mitigation. Mangrove ecosystems are among the most important global carbon sinks that enable large amounts of carbon to be sequestered and stored. However, gaps persist in understanding the fundamental aspects of microbial-driven carbon cycling in these environments. This research explores the microbial taxonomic and functional diversity related to carbon cycling in selected tropical mangrove sediments across various locations and depths. Sequencing data analyses based on the 16S rRNA gene revealed distinct microbial community composition but conserved predicted functions across the different mangrove locations. Depth was a strong influence on the functional composition, with carbon-related pathways and metabolic strategies differing between top and bottom sediments. Putative functional gene abundance analyses revealed that carbon fixation processes were among the top carbon-related pathways, suggesting the key role of mangrove microbial communities in sustaining long-term carbon storage. Within these communities, Desulfobacterota appeared as a primary contributor to carbon fixation, while Chloroflexota played a significant role in carbon metabolism and methane cycling. Co-occurrence network analyses also revealed that these microbial groups were among the keystone taxa in mangrove sediments. Our study adds on to the body of knowledge on the mangrove microbiome and their carbon metabolic processes, which helps to improve strategies for managing and leveraging these vital carbon sinks.

Methane is a powerful greenhouse gas. Typically, a large fraction of the methane formed in coastal sediments is removed via anaerobic methane oxidation (AOM). Here, we demonstrate the potential for a range of AOM pathways in brackish coastal sediments by ANME-2a archaea. At our study site, geochemical profiles indicate that AOM is primarily restricted to a shallow, metal-oxide-rich sulfate-methane transition zone (SMTZ). ANME-2a were the sole methanotrophs detected, and metatranscriptomics showed the highest expression levels of the ANME-2a genes in the SMTZ. AOM activity was observed in sediment incubations with various electron acceptors, including sulfate, metal oxides, and the organic matter analogue graphene oxide. Highest potential rates were observed in sediments from below the SMTZ, pointing towards fast stimulation of the deeper methanotrophic community when alleviating the electron acceptor limitation. The variety of AOM pathways and persistence of methanotrophs below the SMTZ likely contribute to the resilience of the microbial methane filter in brackish coastal sediments.

Riverine ecosystems particularly in industrialized environment are subjected to chronic press disturbances, resulting from the decadal release of synthetic organic compounds and other xenobiotics. While indigenous microbial communities are highly sensitive to such stressors, the resulting metabolic restructuring and functional reshaping of the microbiome, driven by these long-term anthropogenic pressures remains poorly characterized. In this study, a microbial ecology of Bhadar River flowing across the Jetpur Industrial Estate, (Jetpur) were studied. Using a cross-sectional comparative approach, soil/sediment samples were collected from the diverse polluted and non-polluted sites from the estate. The taxonomic profiling using 16S rRNA gene amplicon sequencing, taxo-phenomic shifts (through metaphenomics) was studied, while the functional potential of metabolic pathways was validated using high-resolution shot-gun metagenomic study. Due to prolong pollution, the samples were rich in sulphur (9809 to 12391 mg/L), where polluted samples were having elevated COD (2432 to 4150 mg/L) as well as BOD (1000 to 1420 mg/L) values, along with the presence of heavy metals (e.g., Fe, Mg). Results revealed a distinct taxonomic shift at both the bacterial and archaeal levels. In non-polluted sites Proteobacteria (33 to 57%) dominated along with Acidobacteria and Actinobacteria, with diverse genera like Alcaligenes and Serratia. Whereas, polluted sites exhibited marked increase in Bacteroidetes (13 to 29%), Firmicutes, and Synergistetes and genera like Alkalitalea, Mesotoga and Desulfomicrobium, reflecting anaerobic, fermentative, and sulfate-reducing phenotypes. The archaeal communities at polluted sites were dominated by Euryarchaeota (78 to 99%), specifically methanogenic genera of Methanosaeta and Methanocalculus, contrasting with the Methanomassiliicoccus dominance in non-polluted areas. The alpha-diversity was marginally higher in polluted sites (Shannon: 4.11 to 4.81 vs. 3.81 to 5.39 (non-polluted)), but beta-diversity underscored clear separation (94% variance explained by pollution). The shot-gun metagenomic analysis indicated a substantial enhancement in anaerobic metabolic capacities within the polluted microbiome, primarily in sulphur respiration (dissimilatory sulfate reduction), methanogenesis (elucidating biogenic pathways), along with nitrogen cycling (identifying key denitrification and ammonification genes). The polluted microbiome have developed the potential to metabolise/degrade complex aromatic compounds (pcaK for benzoate/protocatechuate transport) and heavy metal resistance. The strong positive co-occurrences among anaerobic phyla (Thermotogae, Synergistetes, Bacteroidetes) in polluted sites was established, indicating syntrophic interactions for xenobiotic metabolism. These findings provide a theoretical ecological model for perturbed industrial ecosystems, emphasizing the role of habitat selection in shaping microbial functional diversity and demonstrate the remarkable adaptation of autochthonous communities to persistent press disturbances.

This study investigates the potential of the garden rhizosphere as a source of electrochemically active bacteria (EAB) for operating microbial fuel cells (MFCs). We evaluated a diverse array of garden flora, including vegetables (lettuce, Chinese cabbage), flowering plants (August lily, peppermint), and woody species (pine, oak, ginkgo, and bush clover). Among the tested groups, MFCs inoculated with peppermint and ginkgo rhizosphere microbiotas exhibited the highest current densities within their respective categories, significantly outperforming control groups without plant components. 16S rRNA gene microbial community analysis revealed that the initial rhizosphere environment acts as a decisive selective pressure, shaping distinct anode biofilms based on plant types (herbaceous vs. woody). Despite these structural differences in microbial assembly, high current generation was achieved in both peppermint and ginkgo systems, suggesting a high degree of functional redundancy within the rhizosphere-derived consortia. These findings demonstrate that various garden ecosystems can serve as robust biological reservoirs for MFC operation, where diverse microbial configurations are capable of sustaining efficient bio-electrochemical energy conversion.

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.

Malic acid is a C4 dicarboxylic acid traditionally produced from petroleum and widely used in the food industry. As a sustainable alternative, it can also be produced as a value-added platform chemical from biomass. Previously, the Escherichia coli strain XZ658 was engineered to produce L-malate via the carbon-fixation reductive branch of the TCA cycle. In this study, we further improved this system by relieving allosteric regulation of citrate synthase, addressing redox imbalance, and enhancing malate export. These modifications approximately doubled the L-malate titer in the final strain MO128 compared to XZ658 under simple batch fermentation conditions. The process achieved a high mass yield of 1.2 g malate g-{superscript 1} glucose, highlighting the carbon-fixation capacity of the reductive TCA pathway for fermentative malate production.

Cyanobacterial natural products are a rich source of bioactive compounds, yet their heterologous production remains challenging. This study investigates the feasibility of expressing the lyngbyatoxin A (LTXA) biosynthetic gene cluster in a fungal host. The lyngbyatoxin biosynthetic genes (ltxA, ltxB, ltxC) were individually cloned and expressed in Aspergillus oryzae NSAR1 under the control of an inducible promoter. Metabolite production was assessed using LC- MS, and transcriptional analysis was performed by RT-PCR. Codon-optimized constructs and precursor feeding experiments were employed to evaluate pathway functionality. No production of LTXA or pathway intermediates was detected upon co-expression of ltxA-C despite confirmed transcription of ltxB and ltxC. RT-PCR analysis revealed truncation of the ltxA transcript, suggesting incompatibility with fungal transcriptional or splicing machinery. In contrast, expression of a codon-optimized ltxC enabled biotransformation of indolactam V to LTXA in A. oryzae, confirming functional expression of the prenyltransferase. These results highlight transcriptional limitations as a key barrier to heterologous expression of cyanobacterial NRPS pathways in fungal hosts, while demonstrating that downstream tailoring enzymes can remain functional. This work provides insights for future engineering of fungal platforms for cyanobacterial natural product biosynthesis.

Many environmental bacteria do not readily grow under laboratory conditions and population establishment often occurs stochastically. Although the scout hypothesis has been proposed to explain stochastic population establishment in environmental bacteria, how stochastic population establishment is shaped by individual cell growth behaviors in environmental isolates remains unclear. In the present study, we focused on the ammonia-oxidizing bacterium Nitrosomonas sp. PY1 and showed that environmentally responsive individual cell growth behavior, incorporating time-dependent stochastic growth initiation, shapes both deterministic and stochastic population establishment dynamics. Using single-cell observation, we revealed that PY1 altered cell growth behavior in response to surrounding biomass production ({Delta}Vt). These {Delta}Vt-dependent changes in growth behavior were suppressed by the addition of its own cell-free supernatant (CFS), indicating the presence of a growth regulation mechanism via cell-cell communication. Replicate cultures under the same conditions showed that the population establishment of PY1 was stochastic, whereas the model strain Nitrosomonas europaea exhibited synchronized population establishment, consistent with previous reports. This stochasticity in PY1 was also eliminated by the addition of CFS. Finally, a simulation model based on {Delta}Vt-dependent cell growth behavior of PY1 successfully reproduced synchronized population establishment in the presence of CFS. By contrast, the stochastic population establishment observed in the absence of CFS was successfully reproduced by a model incorporating {Delta}Vt-independent growth initiation following a Weibull distribution. Such environmentally responsive changes in population establishment dynamics may contribute to the low isolation success of environmental bacteria and sudden blooms of the rare biosphere.

Northern peatlands store large carbon stocks but are sensitive to disturbance. Hydrology, vegetation, herbivory and snow conditions may affect the soil microorganisms driving methane (CH) and nitrous oxide (N2O) cycling. We investigated how reindeer exclusion and snow depth (increased and reduced relative to ambient) manipulations (ongoing for three seasons) influenced archaeal and bacterial communities in a boreal rich fen. Metagenomic (MG) and metatranscriptomic (MT) sequencing were combined with pore-water chemistry and CH flux measurements to link the microbiome to ecosystem processes. Microbial communities differed between outside and inside the exclosure. However, these patterns primarily reflected underlying hydrological variation. Slightly wetter inside plots showed higher expression of denitrification genes (norB, nosZ) and lower (nirS+nirK)/nosZ ratios, indicating greater potential for complete denitrification to N2 instead of N2O. Methane dynamics were mainly associated with vegetation: plots associated with Carex rostrata exhibited lower pmoA/mcrA ratios and elevated CH fluxes. Snow manipulations had subtle effects: reduced snow depth decreased the expression of taxa dependent on microbial interactions, while the effect to the investigated metabolic marker genes was small. Overall hydrology, leading to variations in redox conditions and nutrient availability, together with vegetation appeared as the primary drivers on microbial greenhouse gas processes in this peatland.