Bioresource Technology

Polyhydroxyalkanoates (PHAs) are microbial polyesters that represent a sustainable alternative to fossil fuel-based plastics. These biodegradable polymers have properties similar to conventional plastics and can be produced from waste materials. The key enzyme in PHA synthesis is PHA synthase (PhaC) and despite its importance, only a few PhaCs from extremophilic microorganisms have been characterised in detail. Here we characterised PHA synthase enzyme from the thermophilic bacterium Caldimonas thermodepolymerans DSM15344, an attractive candidate for PHA production from lignocellulosic residues due to its unique properties such as utilisation of xylose-rich substrates and high PHA yields on this pentose sugar. PhaC was successfully expressed in the surrogate host E. coli BL21 (DE3) GOLD, purified to homogeneity and its enzymatic activity was confirmed in vitro. The optimal pH range and temperature were determined as 6.3-6.9 and 50 {degrees}C, respectively. The melting temperature of PhaC was detected at 57.83 {+/-} 0.04 {degrees}C and the enzyme storage stability was assessed. The tertiary structure, including the enzyme active site, was predicted using available bioinformatic tools and the predicted catalytic residues were supported by site-directed mutagenesis. This study represents the first biochemical characterisation of PHA synthase from C. thermodepolymerans, expanding the limited set of characterised thermophilic PhaC enzymes.

Microalgae are promising biological platforms for sustainable energy production because they can convert photosynthetic activity into electricity in biophotovoltaic (BPV) systems. Identifying strains that grow well at elevated temperatures and under reduced irradiance is an important step toward BPV applications in seasonally variable environments. Microalgae were isolated from Seondong Lake (Seoul, Republic of Korea) during three sampling periods and screened for growth at different temperatures. Among 45 isolates, KHUC-11 showed the highest weighted growth index based on growth rates at 25, 30, and 35 under dark conditions and was selected for further characterization. Phylogenetic reconstruction of the 45S rRNA gene cluster, internal transcribed spacer 2 (ITS2) secondary-structure comparison, and morphological characterization supported the description of KHUC-11 as Desmodesmus ecotolerans sp. nov. D. ecotolerans produced more biomass than Chlamydomonas reinhardtii CC-125 under both moderate- and low-light conditions. Under low light, its final dry biomass concentrations were 1.81 and 1.58 times those of CC-125 under mixotrophic and photoautotrophic conditions, respectively. Despite its smaller cell size, D. ecotolerans showed higher photosynthetic performance than CC-125, and screen-printed electrode (SPE)-based electrochemical assays demonstrated enhanced anodic current generation and CV-derived estimated power output. Taken together, these findings identify D. ecotolerans as a novel climate-resilient Desmodesmus species with bioelectrochemical properties that support further evaluation for BPV applications.

Anaerobic gut fungi (AGF) are an integral part of the herbivore gut microbiome, primary decomposers of lignocellulose, and play a crucial role in establishing foundational links of syntrophic associations. The syntrophy with methanogens appears beneficial for mitigating feedback inhibition and results in the augmentation of AGF activity. However, the thermodynamic advantages of the presence of methanogens to AGF remain poorly understood. This study explores the preferred pathways adopted by Cyllamyces sp. and Methanobrevibacter olleyae, using 13C-glucose as a substrate and spiked with malate and pyruvate in different experimental set-ups. The results indicated reduced biomass formation by Cyllamyces sp. in monoxenic culture with Methanobrevibacter olleyae compared to axenic AGF culture supplemented with glucose. Additionally, metabolic analyses revealed the pyruvate:ferredoxin oxidoreductase (PFO) as a key contributor to pyruvate metabolism. Malate supplementation notably enhanced the growth of Cyllamyces sp. and M. olleyae, whereas pyruvate supplementation enhanced the growth of Cyllamyces sp. only in axenic culture. This investigation provides insights into the preferred metabolic pathways operating in the hydrogenosomes of axenic anaerobic gut fungi (AGF) cultures and monoxenic AGF-methanogen co-cultures.

Acinetobacter sp. strain Tol 5 is an attractive microorganism because of its ability to utilize various aromatic compounds, including toluene. However, because Tol 5 is a non-model microorganism, the utilization of carbon sources in central carbon metabolism remains unclear. In this study, the activity of the central carbon metabolism was characterized using 13C-metabolic flux analysis (13C-MFA). Tol 5 was cultured in synthetic medium using ethanol or acetate as the sole carbon source. In addition, because toluene is degraded into pyruvate and acetyl-CoA via the toluene dioxygenase pathway, this situation was mimicked by culturing with ethanol and lactate as co-substrates. The 13C-MFA confirmed flux through the glyoxylate shunt, which bypasses part of the TCA cycle when either ethanol or acetate was used as the sole carbon source. In ethanol, the flux ratio of isocitrate dehydrogenase at the isocitrate branch point decreased markedly. Unlike acetate, the conversion of ethanol to acetyl-CoA is accompanied by NADH production. Therefore, the observed change in this flux ratio was influenced by the excess NADH generated. Under ethanol-lactate conditions, Tol 5 simultaneously utilized ethanol and lactate. The ethanol consumption rate decreased to less than half of that observed when ethanol was the sole carbon source. Most of the lactate uptake was converted to pyruvate and metabolized via the TCA cycle. The glyoxylate shunt flux was lower than that in ethanol. These metabolic characteristics provide valuable insights into the use of Tol 5 as a next-generation microbial cell factory.

Ugba, a traditional alkaline-fermented condiment produced from African oil bean seeds (Pentaclethra macrophylla Benth.), remains poorly characterised at genome resolution. This study characterised a single spontaneous ugba fermentation using genome-resolved shotgun metagenomics across Early (0-24 h), Mid (48-96 h), and Late (120-144 h) composite stages. Thirty metagenome-assembled genomes (MAGs) were recovered and grouped according to their temporal abundance patterns. The Early-stage retained MAG population was dominated by Bacteroidota-associated MAGs, which exhibited the highest carbohydrate-active enzyme density and broad glycoside hydrolase repertoires, consistent with substantial genome-encoded potential for utilisation of the plant-derived seed matrix. The Mid stage showed marked restructuring of MAG composition, while Core and Late-associated MAGs increased towards fermentation maturation. Functional differentiation was also evident among Corynebacterium MAGs: C. nuruki encoded a complete urease system, whereas C. phoceense_A encoded glutamate dehydrogenase and nitrate-reduction genes. These stage-associated changes coincided with progressive alkalinisation from pH 7.15 to 8.01. Together, the results show that temporal restructuring of reconstructed microbial populations was accompanied by differentiation in genome-encoded functional potential. This study provides a genome-resolved characterisation of microbial composition and functional potential during spontaneous ugba fermentation and expands current understanding of the microbial organisation of this traditional African fermented food.

The unprecedented rise in greenhouse gases such as carbon dioxide (CO2), and their detrimental effects on the atmosphere have intensified the current climate emergency. This challenge has inspired the development of novel carbon capture and utilisation (CCU) technologies, specifically microbial electrosynthesis (MES), for the bioelectrochemical fixation of CO2 into commodity chemical compounds (CCCs) using microbial biocatalysts. In this study, we systematically enriched and maintained robust mixed microbial communities of electroactive bacteria (EAB) from wastewater treatment sludge to serve as biocatalysts for MES. The MES performance of these biocatalysts under different operational conditions in bioelectrochemical systems (BES) reactors were investigated. BES reactors with enriched biocatalysts incubated and maintained under controlled conditions produced higher CCCs yields than those developed at ambient temperatures, while strict anaerobic operations further improved the MES efficiency compared to aerobic conditions. At an applied potential of -1000 mV vs Ag/AgCl, CO2 conversion reached up to 88.11%, significantly outperforming the lower potential (-600 mV) operation. These findings underscore the importance of systematic development of robust and stable biocatalysts, as well as maintaining strict anaerobic conditions, for achieving efficient MES performance, thereby demonstrating the potential of our approach for scalable industrial CCU applications.

Biological filtration in newly established aquaria depends on the establishment of nitrifying microorganisms that prevent accumulation of toxic ammonia and nitrite. Many aquarists use commercial nitrifying supplements to accelerate biofilter establishment and reduce ammonia and nitrite accumulation. However, the microbial composition and in situ efficacy of these products remain poorly characterized. We evaluated the ability of enrichment cultures containing comammox Nitrospira (CX) and the ammonia-oxidizing archaeon Ca. Nitrosotenuis aquarius (AQ) to serve as aquarium nitrifying supplements, tested alongside a conventional supplement (CS) containing ammonia-oxidizing bacteria. Inoculation of new aquaria containing zebrafish (Danio rerio) with nitrifying supplements resulted in distinct nitrifier profiles within biofilters of each treatment group corresponding with significant reductions in peak ammonia and nitrite concentrations, which were most evident when applied at the maximum tested supplement dose. Treating aquaria with CX, AQ, or CS maintained ammonia and nitrite at low concentrations, and CX effectively prevented ammonia and nitrite accumulation through all stages of aquarium operation and biofilter establishment. After removing all fish from the aquaria, ammonia "spike-in" experiments demonstrated biofilters treated with CX and AQ conferred long-term protection against nitrite accumulation following acute ammonia loading events. Whereas 16S rRNA gene sequences associated with CS were rarely detected within biofilter profiles, those associated with CX and AQ established early and persisted temporally. Overall, results demonstrate how nitrifiers might be differentially adapted to home aquarium conditions and the potential of AOA and comammox Nitrospira to improve biofilter establishment and maintaining safe water quality in newly established freshwater aquaria.

Microbially-mediated mineral precipitation is important in the natural world and to a number of emerging climate, energy, and water technologies. Central to many microbial mineralization processes is the preferential accumulation of precipitates on microbial surfaces. In some systems, including ureolytic microbially-induced carbonate precipitation (MICP), precipitates may entomb cells in a process termed "encapsulation." Encapsulation is known to greatly affect microbial systems, but understanding these relationships quantitatively has proven difficult, largely because nucleic acid extraction from encapsulated cells yields inconsistent results. Here, a demineralization protocol adapted from studies of natural microbialites is shown to recover genomic DNA from carbonate-encapsulated Sporosarcina pasteurii at yields indistinguishable from unmineralized cells and of sufficient quality for quantitative PCR. This method allows the degree of encapsulation to be expressed quantitatively as the amount of precipitate formed normalized to the estimated number of cells present, and allows growing populations to be tracked with increased precision. Simultaneous growth and mineralization experiments in mixed batch reactors suggest encapsulation-induced growth suppression at calcium concentrations of 250 mM, which are commonly employed in MICP under different physicochemical conditions. Variable rates of precipitate accumulation on cells, possibly stemming from local mass transport constraints, are proposed as a mechanism of calcium concentration-dependent growth suppression. Accordingly, mass transport principles are used to derive a reaction rate scaling function, allowing the observed growth and precipitation behavior to be replicated by a numerical model. These findings lay the groundwork for a more fundamental understanding of how encapsulation shapes microbial mineralization processes.

Electrochemical advanced oxidation processes (EAOPs) are increasingly investigated for tertiary wastewater treatment, yet their short-term effects on real wastewater effluent remain poorly constrained. This study investigated real wastewater treatment plant (WWTP) effluent following short-term boron-doped diamond (BDD) electrochemical treatment using an interdigitated double-diamond electrode (iDDE), combined with hydrochemical, stable-isotope and shotgun metagenomic analyses. After 10 min at 7.0 V and 1.02 A, physicochemical parameters and bulk carbon pools remained largely unchanged. In contrast, d18O-DO decreased by 31.3 +/-0.28 permille alongside a small increase in dissolved oxygen (0.16 +/-0.03 mmol/L), indicating substantial oxygen consistent with electrochemical oxygen generation. Shotgun metagenomics revealed pronounced restructuring of the recovered microbial DNA pool, including increases in Comamonadaceae (~12.3% vs. ~25.1%) and Zoogloeaceae (~3.3% vs. ~8.4%), whereas the oxidative-stress-defence gene repertoire showed no coherent response. These taxonomic changes cannot be interpreted as selective survival, as metagenomics do not resolve viability or intra-/extracellular DNA contribution. Overall, short-term iDDE treatment affected the microbial component of real WWTP effluent despite comparatively limited changes in bulk chemistry, highlighting that EAOP effects extend beyond conventional disinfection.

Eutrophication-driven hypoxia and harmful algal blooms are an expanding threat to water quality in estuaries. Engineered aeration is a technology used in freshwater and, to a lesser extent, estuarine systems to mitigate both hypoxia and algal blooms. However, the efficacy of engineered aeration has not been well studied within estuarine ecosystems and very little is known about the impact of aeration on microbial communities in either estuarine or freshwater environments. Here we present a time series investigation of microbial community response to aeration and cessation of aeration in a eutrophic estuary. Samples for 16S rRNA gene analysis were collected over about a month-long period during which time the aerators transitioned from being "on" to "off" to "on" again. Cessation of aeration selected for the dominance of a eukaryotic algae similar to Heterosigma akashiwo. After the resumption of aeration, H. akashiwo dominance decreased rapidly. Cessation of aeration also shifted the structure of the entire microbial community, where diversity decreased and the structure of microbial communities at the surface diverged from those from the bottom of the water column. These changes did not occur at non-aerated control sites. Distinct groups of taxa responded to the disruption in a successional pattern through time, with most taxa never returning to their pre-aeration-cessation abundance during the observational period. These findings demonstrate the complex community response to anthropogenic interventions and provide support for bloom suppression through aeration.

Carbohydrate inducible promoters on expression plasmids are widely used for biotechnology and basic science research. However, using carbohydrates for inducers has drawbacks including influencing bacterial metabolism, the inducer being used as a carbon source by the bacteria, high background expression, and only a subset of cells experiencing promoter activation. Furthermore, some bacteria including the pathogen Acinetobacter baumannii currently lack good carbohydrate-inducible promoters. To address this issue, a tetracycline-inducible promoter, PtetB, isolated from A. baumannii was cloned and evaluated in a number of important bacteria: Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Pseudomonas fluorescens, and Serratia marcescens. A highly sensitive luxCDABE reporter was used to evaluate promoter activity. S. marcescens strains with elevated and reduced cyclic-AMP levels were used to evaluate the impact of cAMP on PtetB function. The PtetB promoter was induced over 800-fold in A. baumannii and 10-750-fold inducible in the other tested species. Background expression was lower from PtetB in the pseudomonads than previously described carbohydrate inducible promoters. Data indicated that the PtetB promoter system may be a useful tool for future basic science research with the tested bacteria.

Antibiotic resistance genes (ARGs) travel from cities into rivers and coastal waters through wastewater treatment plants. Monitoring them normally requires metagenomic sequencing, which is sufficiently costly that most utilities can sample only occasionally. Weather and location data are freely available on a daily basis for virtually any location worldwide, making them attractive predictors for identifying where limited sequencing resources should be prioritized. However, whether such models generalize to previously unseen cities remains largely untested, as most published studies train and evaluate models within the same catchments, thereby assessing interpolation rather than geographic transferability. To address this gap, we paired 235 wastewater metagenomes collected from five European cities with 23 abiotic predictors spanning geospatial, meteorological, hydrological, radiative, and temporal domains. Model performance was evaluated using leave-one-group-out (LOGO) cross-validation, in which all samples from one city were withheld for testing while the remaining cities were used for training. CatBoost achieved a median LOGO ROC-AUC of 0.929 and a median F1-score of 0.750. Using freely available environmental reanalysis predictors (meteorological, hydrological, radiative, geospatial, and temporal) - without any metagenomic sequencing - CatBoost achieved a mean ROC-AUC of 0.722, recovering 78% of the predictive performance of the full omics-integrated model. Removing latitude and longitude reduced ROC-AUC by only 0.003, whereas replacing random cross-validation with city-wise validation reduced ROC-AUC by 0.052. Predictive performance varied across ARG classes, ranging from a ROC-AUC of 0.981 for {beta}-lactam resistance genes to 0.762 for glycopeptide resistance genes. These findings demonstrate that freely available environmental reanalysis predictors - spanning meteorological, hydrological, radiative, geospatial, and temporal domains - recover 78% of the predictive signal for antibiotic resistance gene occurrence in urban wastewater, allowing scarce sequencing capacity to be directed to the catchments where it changes a decision.

Glucoamylase is essential for industrial starch saccharification, but the limited thermostability and near-neutral pH tolerance of fungal glucoamylases necessitate cooling and acidification of liquefied starch. Here, we developed an artificial intelligence-guided strategy to simultaneously improve the thermostability, pH tolerance, and catalytic activity of glucoamylase from Penicillium oxalicum (PoGA). Two property-specific machine-learning models, CASPE-T and CASPE-A, identified substitutions associated with thermostability and pH tolerance, respectively. Experimental screening identified beneficial substitutions in 11 of 21 CASPE-T and 12 of 22 CASPE-A candidates. Folding-energy-guided recombination integrated the two traits while maintaining structural compatibility. The optimal variant, PoGA T513E/Q305N, exhibited 2.21-fold higher specific activity than the wild type, with half-life extended from 22.3 to 57.9 min at 60 degrees C and from 16.6 to 64.7 min at pH 8.0. Molecular dynamics simulations attributed these improvements to reinforcement of high-occupancy hydrogen-bonding networks, suppression of conformational fluctuations in the linker and carbohydrate-binding module, enhanced long-range dynamic coordination, and preservation of a compact catalytic architecture. At 60 degrees C and pH 6.5 without acidification, PoGA T513E/Q305N produced 219.9 g/L glucose and achieved 89.1% starch conversion, 31.4% higher than the wild type. This work provides an efficient framework for multi-objective enzyme engineering and sustainable starch biorefining.

Cellulose has long been viewed as the major load-bearing component in plant primary cell walls. Recent studies, however, suggest that pectin plays a significant role in cell wall mechanics despite a kPa-scaled modulus. Here we quantify the mechanical properties of centimeter-sized internodal cell walls of Chara corallina following treatments to remove calcium crosslinks or digest pectin. We also characterize the flow of water and gas through the cell wall as a function of pressure. We found that removal of calcium crosslinks did not affect wall strength and stress-relaxation behavior, yet it decreased the elastic modulus and increased permeability to water. In contrast, pectin removal had a dramatic impact on all measurements. Samples from which pectin had been removed exhibited lower strength and stiffness and had greater stress relaxation under constant strain. Pectin removal increased the permeability of the cell wall to water above that observed when calcium was removed and reduced the pressure at which gas "tunnels" through the cell wall ~ 2-fold. Our findings support the idea that pectin locally restricts the movement of cellulose microfibrils, with implications for understanding functional properties of primary cell walls in vascular plants and developing high-performance double-network materials.

Bacterial genomes are dynamic and harbour accessory regions that can be gained, lost, or rearranged in response to selective pressures. Members of the genus Komagataeibacter are used for bacterial cellulose production and researched as platforms for engineered living materials. However, their genomes are rich in mobile genetic elements (MGEs), including insertion sequences (ISs), genomic islands and prophage remnants, which drive genome rearrangements under agitated cultivation conditions. Whether this genome architecture contributes to genomic changes during routine strain maintenance and distribution remains poorly understood. Comparative genome sequencing of independent Komagataeibacter rhaeticus iGEM stocks revealed recurrent loss of a 210 kbp chromosomal region flanked by homologous IS5 sequences. This deletion was identified in a stock received as an agar stab and reproduced from an intact-genome population subjected to simulated agar-stab handling. Phylogenomic analysis of the affected locus revealed gene-tree/species-tree discordance, heterogeneous GC content and multiple ISs, supporting its characterization as a dynamic accessory region. Populations carrying the larger genome reduction showed improved growth and cellulose production relative to the intact genome reference, whereas populations carrying a smaller nested deletion showed reduced performance. These findings demonstrate that routine strain handling is not necessarily genomically neutral and can expose structural genomic variabilities that generate population-level genomic heterogeneity with phenotypic consequences. For bacteria with MGE-rich genomes, handling history may represent an underappreciated source of genomic and phenotypic variation between laboratories. More broadly, identifying unstable accessory regions may highlight genomic content that is dispensable under laboratory conditions, providing candidates for genome streamlining and strain improvement.

Electromicrobial production (EMP) technologies aim to combine renewable electricity, CO2, and engineered microbes to make energy-dense molecules at efficiencies exceeding photosynthesis. CO2 can be electrochemically reduced to formate, which is far easier to handle at the bench than H2 or an electrode, but formate carries only two electrons per carbon against the six in a biofuel. The remaining electrons must come from oxidizing additional formate, from H2 oxidation, or from extracellular electron uptake (EEU), and no rigorous comparison of these options coupled to the choice of carbon assimilation pathway currently exists. We calculate upper-limit efficiencies for butanol production by six carbon assimilation pathways, each paired with all three electron delivery mechanisms, using electrochemical parameters drawn from a survey of the recent literature. Electrical to butanol energy conversion efficiencies range from 35.5 to 51.7%, corresponding to solar-to-fuel efficiencies of 11.7 to 17%, so even the least efficient route exceeds the 8% theoretical ceiling of algal photosynthesis. The serine variant of the reductive glycine pathway reaches an electrical energy conversion efficiency of when using H2 oxidation, within 1.9 points of the most efficient pathway, and is the only high-efficiency option that tolerates O2. This makes an EMP system that combines electron delivery by formate coupled with the serine variant of reductive glycine pathway highly attractive, as it presents few barriers to rapid, iterative engineering in the lab, and a high theoretical ceiling. Drawing both carbon and electrons from formate costs 6.2 points against H2 at a state-of-the-art whole-cell voltage (2.2 V). However, this small penalty is amplified three-fold by any rise in the CO2-to-formate cell voltage, and reaches 13.5 points at the highest whole-cell voltages reported for scaled-up CO2-to-formate electrolyzers, where formate-only operation falls to 11.2 electrical-to-fuel and 3.7% solar-to-fuel efficiency, below the ceiling of photosynthesis, against 24.7 and 8.1% for H2 (only just above algal photosynthesis). Our choice between a formate-only system and one coupled to H2 oxidation or EEU therefore depends on our belief about the trajectory of CO2 reduction technology. If whole-cell voltages continue to fall at the rate of the past decade, formate alone is the right target, and the simplicity of its workflow is bought at low cost. However, if that improvement plateaus, the electron delivery mechanism must be swappable, and a system should be designed from the outset so that it can be. At the US Department of Energy SunShot target of 2 cents per kilowatt hour, the electricity to make a US gallon of butanol costs $1.40 for a formate-only system at the state of the art, rising to $5.45 at the highest scaled-up electrolyzer voltage reported, against $1.23 and $2.47 for formate and H2 system.

The high stability of enteric viruses in freshwater increases their risk of waterborne transmission. In aquatic environments, bacteria are known to enhance viral removal and inactivation. However, the differences in viral inactivation across freshwater ecosystems and the influence of community diversity on this process remains poorly understood. Here, we used dilution-to-extinction to generate 64 distinct freshwater communities from three lakes and three aquifers. After establishing freshwater communities in chemostat cultures, each community was challenged with two viruses, coxsackievirus B5 and human adenovirus 2. Both viruses were inactivated more rapidly in lake-derived than in groundwater-derived communities, and adenovirus was inactivated faster than coxsackievirus in both freshwater types. Community-level parameters (cell numbers, richness, evenness) were not significant predictors of viral inactivation. In contrast, differential abundance analyses identified bacterial OTUs associated with inactivation: Acidovorax sp. were associated across sources and Brevundimonas sp., Sphingopyxis sp., and Hydrogenophaga sp. showed source-specific associations. Together, these findings provide new insights into the ecological drivers of viral stability and suggest that viral inactivation may depend more strongly on specific microbial taxa and their ecological context than on overall community diversity. Identifying these associations improves our understanding of the factors governing viral persistence in freshwater environments.

In the enzymatic toolbox of depollution, hydrolytic dehalogenases are of particular interest for the bioremediation of organohalogenated compounds. To better identify and characterize these enzymes, it is important to develop appropriate sensing methods. Fluorescent probes for dehalogenase detection have been developed in the past years, however, to this date none were available for application using the common FITC parameters (i.e. excitation laser at 488 nm). To address this limitation, we herein report a new fluorescent probe, COU4-Cl, excitable in the violet (405 nm) and blue (488 nm) wavelengths adapted for the detection and characterization of haloalkane dehalogenases. Its applicability is discussed and evaluated in comparison to the previously described COU2-Cl probe only excitable in the violet (405 nm). This study shows the advantages and complementarity of the two probes, and further demonstrate their application for the discrimination of dehalogenase activity in whole cell assays.

Drought and waterlogging are prominent climate change challenges that, alone or in sequence, pronouncedly constrain agricultural crop production. A better understanding of relationships between water availability and nutrition regarding plant functioning is needed to optimise fertilisation under these challenges. We investigated impacts of drought, waterlogging and drought followed by waterlogging under two fertilisation regimes (low, normal) on various traits of wheat (Triticum aestivum). Next to biomass and photosynthesis measurements, cross-platform metabolomics was applied for in-depth metabolic analyses of flag leaves. We found detrimental effects of drought and drought-waterlogging on shoot and root biomass, especially at normal fertilisation, while photosynthetic traits were not or only slightly affected. Foliar nitrogen contents were higher in plants exposed to drought or to drought-waterlogging. Numerous primary and specialised metabolites were modulated by fertilisation and/ or watering. In particular, amino acids, benzoxazinoids and flavonoids were affected by fertilisation. Due to watering especially several amino acids changed in concentrations, with the induction pattern partly depending on fertilisation. Our study deepens the understanding of the interplay between water and nutrient availabilities as well as nutritional demands, growth and metabolism of wheat. This is highly relevant to balance sustainable agricultural nutrient management under the challenges of ongoing climate change.

Pyrogenic black carbon, such as biochar and wildfire char, possesses a reversible electron storage capacity (ESC), allowing it to serve as an electron donor or acceptor for microorganisms. However, not all the ESC is bioavailable, and the proportion available varies by culture. We hypothesized microbes capable of producing soluble redox mediators could access the ESC in biochar interior and thus utilize a higher percentage of the ESC than organisms without a mediator. To test this hypothesis, we grew wild-type Shewanella oneidensis (MR-1) and {Delta}bfe MR-1, a mutant incapable of exporting flavins, on air-oxidized wood biochar. Cell counts and redox titration show that both strains of MR-1 grew to similar cell densities and accessed comparable amounts of biochar ESC (~19%). The addition of exogenous flavin mononucleotide (FMN) yielded no measurable increase in final cell density, due to the strong adsorption of FMN to biochar. SEM images show attachment of MR-1 and {Delta}bfe to biochar surfaces and the presence of nanowires. This study demonstrates MR-1 can utilize biochar ESC for growth, through direct physical contact without the involvement of flavins. Understanding how microbes metabolize biochar provides insight into the impacts and biogeochemical roles of pyrogenic carbon.