Biotechnology for Biofuels

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.

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

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

Probiotic-based encapsulation offers unique advantages over purified enzymes, such as increased protection from thermal-, pH-, and protease-mediated degradation, for oral therapeutic delivery applications. However, one of the major disadvantages of whole-cell systems is lower reaction rate due to substrate-product transport limitations imposed by the cell membrane and/or wall. In this work, we explore the potential of different lactic acid bacteria (LAB) - Lacticaseibacillus rhamnosus GG (LGG), Lactococcus lactis (Ll), and Lactiplantibacillus plantarum (Lp) - as expression hosts for recombinant Anabaena variabilis phenylalanine ammonia-lyase (AvPAL*). AvPAL* is used as a therapeutic to treat Phenylketonuria (PKU), a rare autosomal recessive metabolic disorder. Among the three species tested, LGG showed the highest PAL activity followed by L. lactis. Next, we attempted to overcome mass transfer limitation in whole-cell biocatalysts in two ways - expression of heterologous transporters and treatment with different chemical surfactants. Engineered strains expressing heterologous transporters exhibited approximately 3-4-fold increased PAL activity, while chemical treatment did not improve reaction rates. This work highlights the challenges and advances in realizing the potential of LAB as biotherapeutics. Impact StatementOral delivery of phenylalanine ammonia-lyase (PAL) using engineered probiotics is a promising therapeutic strategy to treat Phenylketonuria (PKU). Although PAL expression has been reported in probiotic strains of Limosilactobacillus reuteri, Lactococcus lactis, and E. coli, a systematic comparison of lactic acid bacteria (LAB) is underexplored. This study explores the potential of multiple LAB as hosts for PAL expression and investigates strategies to improve whole cell enzymatic activity. The findings from this study provide a foundation for implementing LAB-based delivery of PAL and indicate an important step towards development of probiotic platform for PKU management.

Yarrowia lipolytica is a yeast of industrial interest exhibiting remarkable lipolytic and proteolytic capacities, with a high potential for white biotechnology applications. This yeast can be isolated from a wide range of natural, polluted or anthropogenic environments, including various food products. The present study aims to increase the data on Y. lipolytica phenotypic diversity by evaluating the growth parameters and secreted enzymatic activities of 28 wild-type Y lipolytica (and Yarrowia sp.) strains isolated from various environments across 10 countries. These data could facilitate the selection of appropriate strains for specific research purposes, particularly when wild-type strains are prioritized over genetically engineered ones, like for food-related applications. Notably, strain SWJ-1b exhibited an outstanding combination of favourable characteristics, with optimum (or near) performances for both growth and enzymatic parameters.

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.

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

Sourdough starters contain simple microbial communities typically consisting of a few bacterial species and one or two yeast species. The yeast Maudiozyma humilis and the lactic acid bacterium Fructilactobacillus sanfranciscensis often co-occur in sourdough starters, and have been presumed to exist in a trophic relationship supported by glucose cross-feeding. However, previous research has highlighted a lack of evidence showing that yeast strains consume the glucose that F. sanfranciscensis produces. We have investigated the interaction between sourdough isolates of M. humilis and F. sanfranciscensis in a synthetic wheat sourdough medium, allowing us to control substrate composition and use flow cytometry to enumerate living and dead cells. M. humilis fitness was found to be lower in co-culture with F. sanfranciscensis than when grown alone. Analysis of spent medium composition highlighted the reliance of M. humilis on glucose rather than maltose for growth. Comparisons of predicted and measured co-culture metabolite content also revealed that F. sanfranciscensis consumed less maltose in co-culture than when grown alone. For the first time, we examined potential amino acid cross-feeding between M. humilis and F. sanfranciscensis, and found that within the pairing, F. sanfranciscensis was the main producer of amino acids. Our findings suggest that the M. humilis-F. sanfranciscensis interaction is likely to be neutral, or even competitive, with the strain identity of F. sanfranciscensis playing a defining role in the observed dominance of the bacteria and spent medium metabolite composition. ImportanceThe association of the yeast Maudiozyma humilis and the bacterium Fructilactobacillus sanfranciscensis in sourdough starters is well-documented, and together this pairing makes key functional and organoleptic contributions to the final bread product. Their relationship has historically been thought to be stabilised by cross-feeding of glucose to M. humilis. However, this theory has been drawn into question by recent research which found no evidence that M. humilis consumes the glucose produced by F. sanfranciscensis. Our understanding of cooperation, coexistence, and competition in microbial consortia affects approaches to ecosystem management in a broad variety of applied fields. The significance of our research is in demonstrating that this pairing does not interact mutualistically within a specified setting, providing support for neutral or competitive interactions as drivers of ecological stability. Research areas:

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.

The growth of cultures and formation of mucilage blooms in reaction to salt stress of cyanobacterial cultures are investigated with a focus on the influence of pH. In non-buffered medium, cultures show their pH increasing from 6.5 just after inoculation, up to 11 during the exponential phase. We record the time-evolution of concentration and pH, with different initial OD0. In a second set of experiments, we extract the doubling time of the unbuffered cultures in comparison with those inoculated in pH-buffered BG11 media at four different pH from 6.3 to 10.5 : in the most acid media, all cultures die or grow very slowly. At pH = 10.5, we obtain the fastest growth for all four strains, allowing to qualify these cyanobacteria as being alkaliphiles, though for all strains with comparable initial OD0, the doubling time is shorter for unbuffered cultures. Following a previous study [31]), we finally investigate the influence of pH on mucilage formation and biomass uplift induced by salt stress, involving EPS floculation by cations. Our results show that operating in buffered media significantly influences the mucilage formation, though the observed regimes cannot be simply correlated to the pH value.

Source-separated organics (SSO) are widely processed via anaerobic digestion to produce biogas, yet alternative conversion pathways could generate higher-value products. Here, we demonstrate long-term continuous production and recovery of medium-chain carboxylic acids (MCCAs) from SSO via microbial chain elongation using a bench-scale anaerobic bioreactor operated for 911 days. The reactor was fed with SSO samples collected from two full-scale municipal organics processing facilities in Toronto, Canada, capturing facility-specific and seasonal variability in SSO composition. MCCA production depended strongly on the availability of lactate as an electron donor, which varied with SSO preprocessing operations and outdoor collection temperatures. To mitigate product inhibition, an in-line extraction system using hollow-fiber polydimethylsiloxane (PDMS, also known as silicone) membranes was integrated with the anaerobic membrane bioreactor, providing a robust and solvent-free alternative to solvent-based extraction methods. Maximum MCCA yields reached 0.31 g MCCA/ g VSfeed, with notable octanoic acid production (up to 20% of total MCCA), and production rates up to 0.84 g L-1 d-1. Acidification of the alkaline extract produced a phase-separated MCCA-rich oil ([~]95% purity) without addition of downstream separation steps. Microbial community analysis of the reactor revealed enrichment of putative chain-elongating bacteria, including Eubacterium and Pseudoramibacter species, while shifts in SSO feedstock microbiomes influenced substrate availability and product spectra. These results demonstrate the feasibility of sustained MCCA production from municipal organic waste streams and highlight opportunities to integrate chain elongation with existing anaerobic digestion infrastructure.

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.

Todays canola quality rapeseed press cake (RPC) is a protein-rich co-product with potential as human food, but its application is limited due to antinutritional compounds and bitter taste. It remains, however, unknown how introduction of raw RPC to a food matrix affects sensory perception and which metabolites drive the sensation. Here, raw RPC from whole or dehulled seeds was introduced into snack bars at 0%, 7%, 14%, and 21%, and sensory responses were correlated to selected known RPC-derived bitter compounds. A trained panel evaluated 13 RPC-characteristic sensory attributes, and the bitter compounds sinapic acid, kaempferol 3-O-(2'''-O-sinapoyl-{beta}-sophoroside) (KSS), KSS-hexose, selected bitter glucosinolates, and goitrin were quantified using targeted LC-MS/MS. Most dose-dependent sensory responses increased up to 14% RPC and then plateaued, whereas astringent mouthfeel increased almost linearly across the full dose range. Dehulling intensified several odor- and flavor-related attributes but did not increase bitterness or protein content in the final product. Principal component analysis linked bitterness and astringency positively with KSS, KSS-hexose, and goitrin. Dose-over-threshold analysis further showed that goitrin, but not progoitrin, reached concentrations relevant for bitterness perception. Together, the results demonstrate that raw RPC contributes distinct dose-dependent sensory attributes and that metabolite transformations in the food matrix shape final sensory profiles. These findings provide a basis for developing RPC-containing foods and for breeding rapeseed lines with improved sensory characteristics. HIGHLIGHTSO_LIThis study presents the first sensory panel assessment of rapeseed press cake (RPC)-containing in food products (snack bars) made from whole and dehulled seeds. C_LIO_LI13 RPC-characteristic sensory attributes are identified. C_LIO_LISensory profiles of the tasted snack bars differed significantly, influenced by the dosage of RPC and by the dehulling treatment. Bitterness and astringency are positively correlated with the RPC dosage. C_LIO_LIGoitrin, kaempferol 3-O-(2'''-O-sinapoyl-{beta}-sophoroside) (KSS) and sinapic acid are RPC-derived bitter compounds that correlate with bitter taste of RPC-containing snack bars. C_LIO_LIApproximately 90% of glucosinolates introduced with the RPC are not detected in the snack bars, and goitrin levels in snack bars accounts for only [~]10% of introduced progoitrin. C_LIO_LIGoitrin is - for the first time - reported to contribute to the perceived bitterness of an RPC-containing food product. C_LI

Reported avocado dietary fiber (DF) content and composition are inconsistently reported, particularly during ripening. Thus, this study aimed to characterize the amount and type of DF in Hass avocados and evaluate DF changes during ripening. Unripe (day 0), ripe (day 5), and overripe (day 12) Hass avocados were freeze-dried and defatted. DF was analyzed using non-starch polysaccharide (NSP) enzymatic-chemical methods. Per 100g of as-is avocado, unripe contained 3.96g total DF, ripe 3.68g, and overripe 3.26g. In ripe avocados, DF comprised 43% soluble (SDF) and 57% insoluble dietary fiber (IDF). SDF consisted primarily of rhamnogalacturonan-1 and arabinan pectins, while IDF was predominantly cellulose (32%), hemicelluloses (23%), and lignin (2%). Total DF decreased with ripening, with pectin undergoing solubilization and depolymerization, while cellulose and hemicelluloses remained stable. These findings are important as dietary fibers differentially influence intestinal microbial fermentation and health benefits.

This article presents the development of an advanced modeling and simulation platform for carbon capture systems, with a focus on integrated process analysis from upstream CO2 capture through to bioethanol production. The platform supports the evaluation of CO2 mitigation technology by coupling mathematical bioprocess models with an interactive desktop application. The biological system employs Chlorella vulgaris microalgae to fix CO2 through photosynthesis and generate carbohydrate substrates, which are subsequently converted to bioethanol by Saccharomyces cerevisiae yeast via fermentation. The simulation integrates three established kinetic models--the Monod, Logistic, and Luedeking-Piret models--to predict biomass growth, substrate consumption, and ethanol yield under varying operational conditions. A closed-loop CO2 recycling subsystem captures fermentation off-gases and reintroduces them into the bioreactor, enhancing overall carbon utilization efficiency. Three representative simulation scenarios demonstrated process efficiencies ranging from 1.09% to 93.78% of the theoretical maximum CO2-to-ethanol conversion efficiency, confirming the platforms capacity to evaluate a wide operational envelope. The Electron/React-based desktop application provides real-time visualization, interactive 3D bioreactor models, and a simulation history module, making it accessible to researchers, engineers, and students. The platform serves as a digital twin that bridges rigorous bioprocess mathematics with intuitive user interaction, providing a cost-effective tool for designing and optimizing sustainable carbon capture and biofuel production systems.

Small signaling peptides (SSPs) are critical regulators of plant growth, development, and responses to biotic and abiotic stress, yet their role in the C4 grass Sorghum bicolor is largely uncharacterized. To help fill this knowledge gap, 219 S. bicolor genes that encode SSPs were identified based on SSP sequences previously identified in Arabidopsis thaliana, Oryza sativa, Zea mays, Triticum aestivum, and Brachypodium distachyon. The 219 sorghum genes were assigned to 19 gene families, analyzed for the presence of motifs, and aligned with genes that encode SSPs in other plants using phylogenetic analysis. Expression of the 219 SSP encoding genes in sorghum organs, during stem development, and in stem tissues and cell types revealed distinct spatial, temporal and developmental patterns of expression. Genes associated with the SbCEP and SbRGF families were preferentially expressed in roots, whereas SbEPF genes were expressed in stems and panicles. The expression of genes during bioenergy sorghum stem growth and development was investigated because stems account for [~]80% of harvested biomass and serve as conduits for water and nutrient transport between leaves and roots. During stem development, 28 SSP encoding sorghum genes in several families (CLE, EPF, CEP, GASS, PSY, ES, PSK, CAPE, POE) were expressed at higher levels in zones of cell proliferation. For example, the TDIF homologs SbCLE41 and SbCLE42 were expressed at high levels in nascent stem nodes where they may regulate cambial activity and vascular bundle cell differentiation. A different set of 15 genes in the CIF, POE, CAPE, PSY, CEP, RALF, and CLE families were expressed at higher levels in zones of stem tissue differentiation highlighted by elevated expression of 5 SbRALFs in the stem nodal plexus. Cell type specific expression of many SSP encoding sorghum genes was also observed in fully elongated internodes indicating gene expression is regulated with high spatial resolution. Overall, the results provide a foundation of information for analysis of SSP functions in sorghum that can be integrated with knowledge of sorghum gene regulatory networks to modulate traits important for production of sorghum crops.

Microbial fuel cells (MFCs) represent a promising technology for the simultaneous treatment of wastewater and bioelectricity generation. In this study, the MFCs are conceived as functional modules to be integrated into hydroponic cultivation systems, acting as a prosthetic rhizosphere capable of coupling wastewater treatment and bioelectrochemical activity with plant nutrition improvement. We compared the electrochemical performance of different microbial consortia comprising the electroactive bacterium Shewanella oneidensis, the plant growth promoting rhizobacterium (PGPR) Pseudomonas putida, and the plant biomass-degrading fungus Ophiostoma piceae, along with the supplementation with the quorum sensing (QS) analogue molecule 1{square} dodecanol. These microbial consortia are tested in MFCs fed with wastewater and root exudates to analyze enhanced feedstock assimilation, electricity production, and the generation of plant growth-promoting substances (PGPS). From an electrochemical perspective, we evaluated planktonic growth, anode adhesion, substrate consumption, and the production of redox-active molecules and PGPS such as flavins and siderophores respectively alongside key electrical production parameters, including current output and power. Among the different microbial configurations tested, the consortium combining S. oneidensis, P. putida, and O. piceae exhibited the highest electrical production potential. Moreover, within this framework, we detected the extracellular production of siderophores in MFCs containing P. putida, suggesting a potential role supporting hydroponic crop growth. Furthermore, the addition of 1-dodecanol led to an improvement of the bioelectrochemical parameters. These results highlight the potential of synthetic microbial consortia in MFC-based systems not only to enhance electricity generation from wastewater but also to provide added value in integrated hydroponic applications through rhizosphere-like functions.

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.

During drought, numerous compounds accumulate in plant tissues, but their physiological roles remain unclear - they may function as osmolytes, osmoprotectants, or merely arise as by-products of stress-induced metabolic shifts. We developed an experimental approach to link accumulation patterns with specific functions, using Scots pine (Pinus sylvestris L.) saplings subjected to water deprivation and subsequent rewatering as a model system. We monitored changes in relative water content (RWC) and osmotic adjustment dynamics, employed untargeted primary metabolite profiling for preliminary screening of compounds correlated with water status, and performed quantitative GC-MS and LC-MS analyses of selected metabolites. Major inorganic cations (K, Ca{superscript 2}, Mg{superscript 2}) were also quantified to assess their potential roles. Our results revealed that tryptophan, valine, and lysine - though generally present in low abundance - exhibited selective accumulation under severely reduced RWC ([&le;] 70%), suggesting their involvement as osmoprotectants. Major organic acids, particularly shikimic acid, showed trends consistent with osmotic adjustment. Notably, neither sucrose nor inorganic cations appeared to function as primary osmolytes in this context. The proposed approach offers a viable strategy for identifying compounds involved in plant adaptation to water deficit, with potential applications in breeding programs aimed at improving drought tolerance. HighlightsAn approach to identify osmolytes and osmoprotectants was implemented Accumulation of Trp, Val and Lys was consistent with their role in osmoprotection Osmotic adjustment relied predominantly on organic acids than on inorganic ions Monosaccharides but not sucrose correlates with changes in needle water status

BackgroundGlobally, seagrass ecosystems are threatened by anthropogenic activities that are leading to increased levels of eutrophication, coastal pollution and thermal conditions. Consequently, there is a growing need to develop new approaches that work to mitigate these stressors and enhance restoration efforts in seagrass meadows. One promising strategy is to identify, isolate and characterize microbial consortia that are likely to support seagrass productivity. However, our current understanding of key microbial functions that support plant growth in marine systems is limited. Based on evidence from terrestrial plant-microbe systems, seagrass-associated bacteria are expected to provide the plant with nitrogen and phosphorus resources while detoxifying sulfur and producing phytohormones. Here, we sequenced 61 bacterial cultures isolated from the rhizosphere, rhizoplane, and endosphere of the seagrass, Zostera marina to identify a consortium of six putative plant growth promoting (PGP) candidates. ResultsOur cultivation approach using plant-based media allowed us to isolate 201 bacteria from Z. marina, which reflected 18% of the total microbial diversity of the starting inoculum. Genomic and phenotypic analyses of the 61-sequenced pure-cultures revealed that most of the sequenced taxa were able to mobilize nitrogen primarily through catabolic pathways, including denitrification (51%), dissimilatory nitrate reduction to ammonia (71%), and C-N bond cleavage (83%). Six of the isolates, which represent new lineages of Agarivorans, coded for the nitrogenase gene cassette. Additionally, 52% of the genomes had genes for sulfur and/or thiosulfate oxidation, 88.5% for phosphorus solubilization, and 60.5% for IAA production. Genomic analysis also revealed that some pathways, including denitrification and dissimilatory nitrite to ammonia DNRA, required cross-species cooperation as no one taxa contained all the genes needed to complete these metabolic pathways. Based on draft genome models and results from phenotypic assays, isolates Streptomyces sp. (Iso23 and Iso384), Mesobacillus sp (Iso127), Roseibuim sp. (Iso195), Peribacillus sp. (Iso49), and Agarivorans sp. (Iso311) represent a minimal microbial community that is likely to promote seagrass growth and enhance restoration efforts. ConclusionOur work provides a detailed genomic and phenotypic analysis of bacteria isolated from Z. marina and identifies a minimal microbial community with complementary PGP traits. Isolating, identifying and characterizing bacteria that promote seagrass growth is critical towards enhancing restoration efforts of seagrass meadows.