Bioresource Technology

Cyanobacteria have garnered interest as promising biological platforms for producing renewable biofuel, chemical feedstock, and bioactive molecules. For biotechnology applications, robust well-characterized genetic tools are required for genetically modifying cyanobacteria, but these tools are often developed for specific model strains. Here, we used broad host-range RSF1010-based plasmids to characterize a set of orthogonal constitutive promoters in diverse cyanobacterial strains. The promoters are random variants of the synthetic Escherichia coli PconII promoter. A library of PconII promoters driving a fluorescent reporter gene was first evaluated in Synechococcus elongatus and found to have a wide range of gene expression levels. A set of 25 promoter variants with graded strengths was selected after characterization in S. elongatus and three additional model cyanobacterial strains. To demonstrate the utility of these promoters, we isolated new genetically tractable cyanobacterial strains with high salt and alkalinity tolerance and transferred the subset of promoters into one of these newly isolated strains. Similar to the results with model strains, the subset of promoters had a wide range of expression levels in the non-model strain. These characterized promoters expand the genetic tools available for genetic engineering of model and non-model cyanobacterial strains. ImportanceThe use of cyanobacteria to produce renewable products will require engineered expression of many genes that affect cell growth, metabolism, and agronomic properties, leading to efficient production of biomass and desired products. Engineering the strength of gene transcription is an important element of overall gene expression levels. The set of constitutive promoters described here, with a wide range of expression strengths characterized in several diverse cyanobacterial strains, provides an important resource for genetic engineering required for biotechnology applications. Research AreasMicrobial genetics, plasmids and other genetic constructs, biotechnology Journal SecctionBiotechnology

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

In this study, we employ a two-stage dynamic metabolic control strategy to enhance the NADPH dependent biosynthesis of ethylene glycol from xylose in engineered E. coli. We evaluated the use of metabolic valves to dynamically reduce the enzymes involved in competitive pathways which compete for substrates with ethylene glycol biosynthesis, as well as regulatory pathways aimed at increasing NADPH fluxes. The performance of our initial strains with limits in pathway expression levels was improved by the addition of competitive valves, but not by increases in NADPH flux. In contrast, improving pathway expression levels, led to strains improved significantly by our regulatory valves which improved NADPH flux, but not by the competitive valves. This is consistent with a central hypothesis that faster pathways in and of themselves can compete with other metabolic fluxes by being faster and are better aided by regulatory changes capable of change rates elsewhere in metabolism. In this case in NADPH flux. Lastly, upon scale up to fed-batch bioreactors, our optimized strain, featuring dynamic control of two regulatory valves produced 140 g/L of EG in 70 hours at 92% of the theoretical yield.

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.

Crude glycerol is an underutilized waste stream. Viable routes for converting it to 1,3-propanediol (1,3-PDO) can conserve important resources and add value to its supply chain. Biological methods are appealing because they can circumvent expensive preprocessing steps while operating under mild conditions. Here, we show that the propanediol utilization pathway of Salmonella enterica serovar Typhimurium LT2 can be used to convert glycerol, including unprocessed crude glycerol, into 1,3-PDO under aerobic conditions in minimal media. Additionally, we demonstrate that high concentrations of expensive cofactors are not necessary to achieve optimal production titers. This study lays the groundwork for continual iteration on this pathway for bioprocess development. Key pointsO_LIS. enterica can produce 1,3-propanediol from crude glycerol alone C_LIO_LIGlycerol-to-1,3-propanediol conversion is dependent on expression of the propanediol utilization (Pdu) pathway C_LIO_LISub-saturating concentrations of exogenous vitamin B12 can boost cell growth and 1,3-propanediol yield C_LI

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.

Microbial fuel cell offers a promising approach to improve wastewater quality and generate bioenergy from dark fermented effluents. In this study, the use of dark-fermented palm oil mill effluent as an electron donor for bioelectricity generation was investigated using a double-chambered microbial fuel cell (MFC). The MFCs were operated at room temperature (29 {+/-} 2{degrees}C), anode electrolytes adjusted to pH 7, and a chemical catholyte as the oxidizing agent. The maximum power {+/-} 8.07 mW/m2 and 155.16 {+/-} 12.88 mA/m2, respectively, were generated from the MFCs inoculated with sludge, which was 5.9 times higher than control without inoculum. Microbial community analysis revealed the enrichment of fermentative and electrogenic representative taxa from the phyla Bacillota, Bacteroidota and Pseudomonadota on the anode electrodes. Optimizations of the running conditions were carried out, suggesting the optimum parameters of 0.5 k{Omega} external resistance, anolyte initial pH 9, and 75% DFPOME substrate concentration. Operation under the optimized conditions increased current production, wastewater treatment, and Coulombic efficiency compared to the non-optimized conditions. Multiple configurations were also evaluated, showing higher cumulative voltage, power, and current densities with the stacked MFC connections, compared to single MFC units. Parallel circuit connection produced higher power and current density than serial connection. This study demonstrated the feasibility of MFC as a promising downstream treatment for biohydrogen production processes, towards higher treatment efficiency and resource recovery.

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

Crop loss due to infection by pests and pathogens is a major barrier to the large-scale production of algal biofuels. Test systems have seen loss of green algae crops due to infection by the fungus-like Amoeboaphelidium occidentale FD01. While current antifungal compounds are effective in inhibiting the infection, their application raises the overall cost of the crop and lowers its economic viability as a biofuel source. Here we show that co-culturing environmentally harvested bacteria alongside algae crops can drastically lower the rate of infection in two different green algae species of interest for biofuel production. These bacteria-algae consortia increase the mean time to crop failure (MTTF) by up to 350% when tested under environmentally relevant conditions. While there was an increase in diversity over time, there was no statistically significant correlation between an increase in diversity and a longer MTTF. Community composition analysis reveals similarities between the bacterial genera growing alongside both green algae species even as bacterial harvest locations differed, although there was not a single dominant genus responsible for the increase in crop protection. These results show a promising new method of anti-fungal crop protection that can be applied to algal biofuels with no increase in fuel cost. HighlightsO_LIBacteria-algal cocultures protect against fungal pests without impact to productivity C_LIO_LIBacterial community composition is variable over time even as protection persists C_LIO_LIBacterial consortia can increase mean time to failure by 350% C_LI

Zymomonas mobilis is an ethanologenic Alphaproteobacterium with many interesting characteristics for fundamental research and applied microbial engineering. Although genetic engineering has been established for Z. mobilis since the 1980s, a rich set of inducible transcriptional regulators is still unavailable. In this work, seven different chemically inducible promoters have been systematically tested for their functionality in Z. mobilis. In particular, for the first time, NahR-PsalTTC, VanRAM-PvanCC, CinRAM-Pcin and LuxR-PluxB have been characterized in Z. mobilis, alongside the commonly used regulator-promoter pairs TetR-Ptet and LacI-PlacT7A1_O3O4, and the less commonly used XylS-Pm. All promoters investigated in this work are compatible with the Golden Gate modular cloning framework Zymo-Parts. Characterization was carried out with a shuttle vector backbone based on pZMO7, which has so far been rarely used for applications in Z. mobilis but seems to be completely stable without selection and generates high and uniform levels of expression. From the experimental results presented, it can be concluded that VanRAM-PvanCC and CinRAM-Pcin are particularly promising for broad use in the Z. mobilis community. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=126 SRC="FIGDIR/small/712268v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@16579e6org.highwire.dtl.DTLVardef@1262533org.highwire.dtl.DTLVardef@15456a2org.highwire.dtl.DTLVardef@3af98_HPS_FORMAT_FIGEXP M_FIG C_FIG

Current mechanical and chemical recycling strategies address less than 10% of global plastic waste, necessitating alternative valorization routes. Biological upcycling via enzymatic depolymerization combined with microbial conversion of the resulting monomers offers a promising pathway to transform mixed plastic waste into valuable alternatives. Here, we employed a single engineered Pseudomonas putida KT2440 for simultaneous co-utilization of five plastic monomers including ethylene glycol, terephthalic acid, adipic acid, 1,4-butanediol, and L-lactic acid, which can be derived from enzymatic hydrolysis of polyethylene terephthalate (PET), polybutylene adipate-co-terephthalate (PBAT), polyester-polyurethanes (PUs), and polylactic acid (PLA). Continuous fermentation over 21 days with alternating mixed-monomer feeds achieved steady state growth and complete substrate depletion, yielding adaptive mutations that informed iterative strain improvement. Further engineering enabled the biosynthesis of (R)-3-hydroxybutyrate (R-3HB), and 0.70 g L-1 R-3HB was produced directly from enzymatic hydrolysates of blended PET, PBAT, and TPU. These results establish a viable bio-based approach for upcycling realistic mixed plastics into value-added bioproducts.

Formate is emerging as an important molecule in carbon capture and utilization technologies. However, its low electron density makes formate less attractive for energy storage. Some hydrogenotrophic methanogens can reduce formate to methane, thereby upgrading it into an established energy carrier. The bottleneck in this process is that 75% of the carbon is lost as carbon dioxide, and achieving a complete formate-to-methane conversion requires co-feeding hydrogen. However, hydrogen-dependent genetic regulation of formate metabolism inhibits simultaneous formate and hydrogen utilization in hydrogenotrophic methanogens. Here, we compared the catalytic performance of the genetically modified strain Methanothermobacter thermautotrophicus {Delta}H (pFdh) with M. thermautotrophicus Z-245 by conducting continuous cultivation at different hydrogen concentrations. While M. thermautotrophicus Z-245 is a natural formatotroph, M. thermautotrophicus {Delta}H (pFdh) was engineered to enable formate utilization via episomal expression of a formate dehydrogenase-gene cassette. We found that M. thermautotrophicus {Delta}H (pFdh) can simultaneously utilize formate and hydrogen. It continuously consumed formate at {approx} 0.1 mM dissolved hydrogen, enabling a 75.6% formate-to-methane conversion efficiency. M. thermautotrophicus Z-245 showed a declining formate consumption at {approx} 0.016 mM and only reached a maximum stable efficiency of 36.3%. These results suggest that M. thermautotrophicus {Delta}H (pFdh) is largely insensitive to hydrogen-induced genetic regulation; however, it still faces redox-related metabolic limitations at dissolved hydrogen concentrations above 0.4 mM. Overall, the findings reveal a potential strategy to circumvent hydrogen-induced regulation of formate metabolism and identify M. thermautotrophicus {Delta}H (pFdh) as a promising biocatalyst for formate-to-methane conversion.

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

Lignin-derived aromatics are abundant in depolymerized lignin but remain remain untilized as carbon sources for commercial production of bulk chemicals. Among these aromatics, p-coumaric acid can be funnelled through the {beta}-ketoadipate pathway toward cis,cis-muconic acid (ccMA), a precursor of bio-based adipic and terephthalic acids. However, efficient ccMA production by Acinetobacter baylyi ADP1 is constrained by toxicity of catechol (the immediate precursor of ccMA), inefficient channelling of protocatechuate (PCA) metabolism towards ccMA production, and absence of PCA decarboxylase for converting PCA to catechol. Therefore, in this study, we engineered a modular co-culture system, combining engineered strains of A. baylyi and E. coli, for ccMA production from synthetic p-coumaric acid. Deletion of catB and catC genes and overexpression of catA in A. baylyi GJS_catA strain enabled near-stoichiometric conversion of catechol to ccMA ([~]90% carbon yield) with titres up to 56.4 mM ([~] 8 g/L) under controlled fed-batch feeding. The strain was further engineered (A. baylyi GJS2_catA) to convert p-coumaric acid to PCA. Due to the inactivity of heterologous PCA decarboxylase (aroY gene) in A. baylyi, this gene was incorporated in E. coli where it exhibited activity through PCA to catechol conversion. Upon its production by E.coli_aroY in the co-culture, catechol is instantaneously converted to ccMA by A. baylyi GJS2_catA strain. In a two-step process, 22 mM p-coumaric acid was initially converted to 20.6 mM PCA (A. baylyi GJS2_catA), which was further converted to catechol (E.coli_aroY) and finally to 18.55 mM ccMA (2.63 g L-{superscript 1}) by A. baylyi GJS2_catA. This process was validated by the valorization of lignin-derived p-coumaric acid to ccMA. While the modular strategy developed in this study substantially improves ccMA titres, it also highlights the bottlenecks in A. baylyi metabolic pathway engineering for lignin valorization. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=147 SRC="FIGDIR/small/709578v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@a83daborg.highwire.dtl.DTLVardef@168c6b6org.highwire.dtl.DTLVardef@1ce0abdorg.highwire.dtl.DTLVardef@23200b_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 engineering offers potential for improving the sustainability of agriculture by providing greater control of desired microbial functions. However, successful control of engineered functions requires greater understanding of their robustness under diverse conditions including those used for plant hydroponics. Here, we studied biomass accumulation and surfactin biosynthesis by an engineered derivative of Bacillus subtilis PY79 in common plant culture media as a model system for interrogating metabolic robustness. We report the observation that PY79 and all other B. subtilis strains that we tested, including natural isolates, exhibited difficulty growing under shaking incubation in defined media where the only nitrogen sources were inorganic. In contrast, assimilation of inorganic nitrogen sources functioned relatively robustly under static incubation in these same media. Our findings may offer some guidance for use of B. subtilis in controlled environment agriculture and could aid future efforts to identify the molecular basis for the agitation-dependent effect on nitrogen assimilation.

Hydrogenotrophic methanogens are of high environmental and biotechnological importance, converting CO2 with H2 into CH4. Despite their common metabolism, variations in the energy metabolism among these methanogens exist, likely affecting their H2 thresholds and growth yields. However, a systematic comparison of these traits for a wide range of hydrogenotrophic methanogens has been lacking. Here, we measured the H2 thresholds and growth yields of nine different hydrogenotrophic methanogens. The H2 threshold, i.e. the H2 partial pressure at which H2 consumption halts, ranged over two orders of magnitude from 1.0 {+/-} 0.5 Pa for Methanobrevibacter arboriphilus to 120 {+/-} 10 Pa for Methanosarcina mazei. Growth yields in our experimental conditions ranged from 0.51 {+/-} 0.28 gDCWx(mol CH4)-1 for Methanococcus maripaludis to 5.28 {+/-} 1.25 gDCWx(mol CH4)-1 for Methanosarcina mazei. The ATP gains, estimated from both H2 thresholds and growth yields, correlated reasonably well, confirming that these variations are due to differences in energy conservation strategies. Our results strongly differentiated the two previously proposed groups of hydrogenotrophic methanogens: methanogens with cytochromes had a high H2 threshold ([&ge;] 21 Pa) and high growth yield (> 4.0 gDCWx(mol CH4)-1), whereas methanogens without cytochromes had lower H2 threshold ([&le;] 7 Pa) and low growth yield (< 1.7 gDCWx(mol CH4)-1). Moreover, our H2 thresholds indicated that additional variations in energy metabolism exist within both groups. Overall, this study found strong variations between hydrogenotrophic methanogens, which are important to understand their environmental prevalence and biotechnological applicability.

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

Cocoa fermentation is a spontaneous microbe-driven process in which yeasts, lactic acid bacteria (LAB), and acetic acid bacteria (AAB) generate the flavor precursors that determine the sensory quality of chocolate. Although the microbial ecology of cocoa fermentation has been increasingly studied through culture-independent methods, the effect of targeted nutritional interventions on community structure within geographically defined production territories has received limited attention. Here, we employed dual-marker metabarcoding (16S rRNA V4 and ITS1) with Illumina NovaSeq 6000 sequencing to characterize bacterial and fungal communities during spontaneous fermentation of Trinitario cocoa beans subjected to amino acid and zinc supplementation in the Limon province of Costa Rica. Fifteen samples were collected at 0, 24, and 48 h from control, amino acid-supplemented, and zinc-supplemented fermentations, each in duplicate. The bacterial community comprised 292 amplicon sequence variants (ASVs) representing 88 genera across 15 phyla; the fungal community comprised 1,117 ASVs representing 248 genera across 9 phyla. Firmicutes and Proteobacteria dominated the bacterial fraction, with a pronounced shift from Tatumella-dominated fresh pulp toward Weissella- and Leuconostoc-rich assemblages during fermentation. Amino acid supplementation reduced Firmicutes at 48 h while favoring Acetobacter proliferation; zinc supplementation promoted Mucoromycota and Wickerhamomyces while sustaining Liquorilactobacillus abundance. Beta diversity analyses (Aitchison distance, weighted and unweighted UniFrac) confirmed significant compositional differences between treatments (PERMANOVA, p [&le;] 0.01), although alpha diversity indices did not differ between individual treatment pairs. Sparse Estimation of Correlations among Microbiomes (SECOM) revealed structured co-occurrence networks, including positive associations between Gluconobacter and Acetobacter and negative associations between Tatumella and several AAB genera. Predicted functional profiles (PICRUSt2) showed no significant pathway-level differences. Taken together, these results show that nutritional supplementation can reshape microbial community composition without reducing overall diversity. This provides a viable approach for steering fermentation outcomes in cocoa-producing territories that seek quality differentiation.

Industrial waste containing hydrophobic pollutants, like oils and hydrocarbons, is toxic and difficult to degrade, posing both ecological and human health risks. Biosurfactants are eco-friendly surface-active compounds produced by microorganisms, known for their ability to lower surface and interfacial tension, enhancing the solubility and bioavailability of hydrophobic compounds, facilitating their breakdown. The current study focuses on isolating biosurfactant-producing bacteria from industrial waste sources near Dhaka, Bangladesh, and characterizing their properties, determining potential usage. Using diesel-enriched nutrient agar, bacterial strains were isolated and screened for biosurfactant production by oil displacement, emulsification index (E24%), and drop collapse assay. The most promising isolates were characterized according to their biochemical activities and 16S rRNA amplicon-based sequencing. Isolation and characterization of the surfactants have been carried out using chromatographic techniques. The identified bacteria passed the drop collapse and oil displacement tests. CTAB agar assay, indicates their anionic nature, showing an emulsification index ranging 10-41%. The potential biosurfactant producers belong to Bacillus, Pseudomonas, Acinetobacter, and Enterobacterium genera. The surfactants showed antibacterial, antifungal, and plant growth promotion activity and have been characterized in terms of pH stability, salinity, adhesion, and temperature tolerance. The study successfully identified and characterized potential biosurfactant-producing bacteria from industrial waste, highlighting their efficiency in breaking down hydrophobic pollutants and hydrocarbons. These microorganisms provide a green and economical substitute for synthetic surfactants due to their biodegradability and lower toxicity. Upon further research and scaling, these bacteria can be a good source of biosurfactants for potential applications in industrial, agricultural, and biomedical fields. IMPORTANCEThe study carries high significance as it creates multi-disciplinary scopes for utilizing these environmentally adapted biosurfactant-producing bacteria in industry, agriculture, and medicine. Since the bacterial isolates have hydrocarbon degradation ability, upon optimization for higher production, industrial usage in oil refinery and other industries can be adopted. Due to their biodegradable nature, usage in wound healing bandages and as antimicrobial agents in medicine will be noteworthy. The isolates have plant growth promotion ability and utilizing them as biofertilizer will reduce the dependency on chemical fertilizers. This is the first detailed report on biosurfactant-producing bacteria from this industrial waste-polluted Turag River of Dhaka City. Moreover, it compiles detailed screening protocols and methods for analyzing such environmentally friendly microbes. Such characterization also opens the scope for optimizing the production of the surfactant compounds on a large scale and utilizing them commercially.