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

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.

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

The dynamics and impact of microbial contaminants in industrial sugarcane bioethanol production in Brazil were investigated through a two-year metagenomic study across two biorefineries. Shotgun metagenomic sequencing revealed that temporal shifts in the contaminant microbiome dynamics within production seasons were more pronounced than inter-annual or inter-mill variations. While Saccharomyces spp. dominated, bacterial communities, primarily within the Firmicutes phylum and dominated by the genera Lactobacillus, Limosilactobacillus, and Bacillus, exhibited dynamic changes. Correlation analyses with industrial process parameters revealed a complex interplay: lower Lactobacillus levels in one mill were associated with increased ethanol yield, whereas higher levels in another mill correlated with reduced yeast viability and increased flocculation. The presence of Limosilactobacillus was linked to decreased yeast viability, whereas Bacillus showed potential for inhibiting both Lactobacillus and Limosilactobacillus. These findings highlight the nuanced and species-specific impacts of bacterial contaminants on bioethanol production, underscoring the need for strain-level functional studies and targeted interventions to optimize fermentation efficiency and stability in industrial settings.

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.

This paper evaluated and compared the relative microalgal biomass accumulation of rocking, floating, and stationary bag photobioreactors. Microalga Neochloris oleoabundans was grown in these photobioreactors in batch mode for 24 days under illumination. The 50 L plastic bags (cell suspension volume 25 L) were placed on the surface of a rocking platform, an artificial pond or a stationary platform. In the pond, waves were generated by electrical fans which shake and mix microalgal cells within the plastic bags. The bags were supplied with 5% CO2 in air under elevated pressure inside of the bags. The rocking bag method significantly increased biomass yields to approximately 3-4 g * L-1, as compared to 0.16 g * L-1 in the floating photobioreactor and only 0.03 g * L-1 in the stationary type photobioreactor.

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.

Exopolysaccharides (EPS) produced by lactic acid bacteria (LAB) and other microorganisms have attracted considerable interest due to their structural diversity and physicochemical properties, which makes them valuable across various industrial applications. To achieve high cell densities and maximize EPS yields, microorganisms are typically cultivated in nutrient-rich media containing yeast extract. However, yeast extract may contain high molecular weight polysaccharides that are not metabolized by the bacteria. This can lead to an overestimation of EPS yields and contamination of the bacterial EPS, potentially resulting in misinterpretation of their structure and biological activity. In this study, we demonstrate the presence of high molecular weight -mannan and {beta}-glucan in yeast extract in EPS isolates using both ultrafiltration and the commonly used trichloroacetic acid/ethanol (TCA/EtOH) precipitation method. These polysaccharides were characterized by size-exclusion chromatography, high-performance anion-exchange chromatography, and nuclear magnetic resonance spectroscopy. Their abundances were estimated to range from 10 to 50 mg/L in MRS medium, depending on the supplier of the yeast extract. The main contaminant identified was yeast -mannan. By cultivating L. rhamnosus GG (ATCC 53103) and L. pentosus KW1 and isolating their respective EPS, we illustrate how these yeast extract contaminants affect the structural interpretation of the EPS and that the contaminants can be completely removed by ultrafiltration of the growth medium prior to bacterial cultivation. In conclusion, we emphasize the necessity of stringent controls in the production and purification of microbial EPS, with particular attention to the chemical purity of medium constituents.

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

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.

A critical process in traditional Japanese indigo dyeing is the microbial reduction of indigo within the dye suspension, which consists solely of sukumo (fermented indigo leaves), wood ash lye, and microbial nutrients such as wheat bran. Although sukumo has long been recognized as a potential microbial source, the microbial community dynamics during its production is still largely unexplored, and its contribution to dyeing performance via microbial supply remains poorly characterized. In this study, we investigated the significance of sukumo as a microbial source, in addition to its established role as a pigment source. We conducted a time-series analysis of microbial communities throughout the four-month fermentation process of sukumo, using weekly samples collected from two geographically distinct indigo dyeing studios. Microbial profiling revealed similar successional patterns between the two sites. Notably, in the later stages of fermentation, known indigo-reducing bacteria, such as the genus Oceanobacillus and the family Tissierellaceae, emerged at both locations. Laboratory-scale dyeing experiments using immature sukumo demonstrated that supplementation with a small amount of mature sukumo restored dyeing activity and increased the abundance of Oceanobacillus and Tissierellaceae. Furthermore, the addition of the indigo-reducing isolate Tissierellaceae strain TU-1 to the immature sukumo-based dye suspension led to a marked enhancement in dyeing performance. These findings highlight the critical role of sukumo as a microbial source in traditional indigo dyeing and suggest that prolonged fermentation is essential for nurturing functional indigo-reducing bacteria. This insight provides a foundation for improving dye suspension performance through targeted microbial community management.

The urgent need for sustainable solutions to mitigate climate change has intensified research into carbon capture, utilization, and storage (CCUS) strategies. Biological approaches, particularly involving extremophilic microorganisms, offer promising alternatives to conventional methods due to their adaptability and potential for bioproduct synthesis. In this study, we report the complete genome sequencing and functional characterization of isolate BS253, derived from a hypersaline alkaline lake in Brazils Pantanal region. Using a hybrid sequencing strategy combining Oxford Nanopore long reads and Illumina short reads, we assembled a circular chromosome of 3.76 Mb and identified two plasmids. Phylogenetic and comparative genomic analyses identified the isolate as Vreelandella zhaodongensis. Digital DNA-DNA hybridization (dDDH % = 71.4%) and ANI (96,83%) values supported the designation of BS253 as a distinct subspecies of V. zhaodongensis. The genome reveals genes associated with salt and alkali tolerance, hydrocarbon and plastic degradation, and the biosynthesis of secondary metabolites. Phenotypically, BS253 is a moderately halophilic, facultatively anaerobic, Gram-negative rod exhibiting biosurfactant activity, with an emulsification index of 51.7% under defined culture conditions. These findings highlight BS253 as a metabolically versatile extremophile with potential applications in different types of industries and biotechnological CCUS systems. ImportanceMicroorganisms adapted to extreme environments represent an untapped source of biotechnologically valuable traits. Vreelandella zhaodongensis BS253, isolated from a hypersaline alkaline lake in the Brazilian Pantanal, expands the known diversity of extremophiles and offers metabolic features with relevance to sustainable bioprocesses. Its complete genome reveals genes involved in salt and alkali tolerance, plastic and hydrocarbon degradation, and the biosynthesis of biosurfactant-like compounds, positioning this strain as a promising chassis for applications in emerging carbon capture, utilization, and storage (CCUS) strategies. The ability of BS253 to produce bioemulsifying molecules under defined nutritional conditions, combined with pathways for degrading recalcitrant pollutants, reinforces its potential for environmentally friendly industrial processes. By characterizing BS253 at the genomic and physiological levels, this work provides foundational information for future exploitation of extremophiles in biotechnological innovations aimed at reducing carbon emissions and supporting circular bioeconomy initiatives.

Postharvest losses and rapid nutrient degradation due to fruit spoilage necessitate alternative preservation methods. Wine production presents a viable approach to minimizing fruit waste while retaining essential nutrients. In this study, mixed fruit wines (watermelon, banana, and pineapple) were produced using Saccharomyces cerevisiae isolated from palm wine as a starter culture. After secondary fermentation, the wines maintained an acidic pH range (2.29{+/-}0.1 to 3.25{+/-}0.2), a stable fermentation temperature (26.50{+/-}1.1{degrees}C to 27.00{+/-}1.1{degrees}C), specific gravity values of 1.021{+/-}0.02 kg/L and 1.027{+/-}0.03 kg/L, and total acidity levels of 1.57{+/-}0.2% and 2.11{+/-}0.1% for Wines A and B, respectively. The final alcohol content was 8.40{+/-}2.9% in Wine A and 9.84{+/-}3.6% in Wine B. Proximate analysis demonstrated the retention of key nutrients post-clarification and maturation, and sensory evaluation indicated a higher consumer preference for Wine B (P>0.05). These findings highlight the potential of indigenous S. cerevisiae strains from palm win for efficient wine fermentation and support the utilization of mixed fruits as a sustainable raw material for value-added wine production. This approach not only mitigates fruit wastage but also provides an economic avenue for enhancing fruit utilization.

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.

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.

Methanogenesis is a crucial component of Earths carbon cycle and a source of methane for biofuel production. The presence of higher energy electron acceptors, such as iron(III) oxides and quinones, is believed to significantly impact methanogenesis. This study investigated the physiological and proteomic responses of the type I Methanosarcina, M. barkeri, to the artificial quinone 9,10-anthraquinone-2,7-disulfonate disodium (2,7-AQDS), using H2/CO2 as substrates. Our findings revealed that during 2,7-AQDS reduction, cellular growth ceased. The lack of energy conservation was associated with direct inhibition of both methanogenesis and CO2 utilization, corroborated by a significant downregulation of the enzymes involved in this metabolic pathway. Furthermore, the significant upregulation of specific subunits of the reversible Ech hydrogenase suggests that this enzyme redirects electrons from H2 towards the most energetically favorable reaction (2,7-AQDS reduction), rather than the reduction of ferredoxin, which is a highly energy-demanding process, essential for initiating the CO2 reduction pathway. Additionally, it is conceivable that Ech homologues in other hydrogenotrophic methanogens also participate in the reduction of higher energy-yielding electron acceptors. These findings provide novel insights into how quinones, particularly in their oxidized state, directly impact methanogenesis, thereby influencing both artificial and natural methanogenic environments.

We report the isolation and characterization of Streptomyces albidoflavus MD102, a strain that can be used as a microbial chassis for the heterologous production of secondary metabolites. This strain, closely related to the widely used S. albidoflavus J1074, exhibits a compact genome, exceptional genetic tractability, rapid growth, and susceptibility to antibiotics. Whole-genome sequencing revealed the metabolic capabilities of S. albidoflavus MD102, highlighting its versatility in supporting the production of diverse secondary metabolites. Employing CRISPR/Cas9-assisted genome editing tools, we created mutant strains with reduced genome and cleaner chromatographic background. In addition to the deletion of several biosynthetic gene clusters (BGC), we inserted the global regulator bldA gene and geranyl diphosphate synthase (gpps) genes and an additional {Phi}BT1-attB attachment site into the chromosome to enhance the strains capability in producing secondary metabolites. S. albidoflavus MD102 will be a new addition to the repertoire of existing Streptomyces chassis, contributing to the advancement of secondary metabolite discovery and synthetic microbiology. IMPORTANCEThe pursuit of a universal Streptomyces microbial chassis for the heterologous production of secondary metabolites has proven elusive, prompting a more pragmatic approach to develop a suite of Streptomyces chassis. The current study introduces Streptomyces albidoflavus MD102 as a promising heterologous chassis with rapid growth, susceptibility to common antibiotics, and genetic tractability. Its close phylogenetic relation with the widely used versatile S. albidoflavus J1074 chassis and the traits gained from strain improvement place the engineered S. albidoflavus MD102 strains as useful chassis for the heterologous production of microbial secondary metabolites. A notable feature of S. albidoflavus MD102 that distinguishes it from J1074 and other Streptomyces chassis is the presence of metabolic genes in its genome putatively responsible for the degradation of aromatic compounds. This characteristic may endow the strain with the capability to convert petrogenic polycyclic aromatic hydrocarbons (PAHs) and substituted aromatics into valuable secondary metabolites.