Bioresource Technology

Lignocellulosic biomass biotechnological conversion relies on enzymatic hydrolysis of recalcitrant plant cell walls, which limits conversion efficiency and economic viability and renders sugar-release dynamics difficult to predict. Mathematical modeling has therefore emerged as a key approach for elucidating the mechanisms governing enzymatic hydrolysis and predicting its dynamics. Here, a hierarchical adsorption-inhibition framework is developed, beginning with a detailed Dynamic Adsorption-Inhibition Model (DyAIM). This model is then simplified into a Reduced Adsorption-Inhibition Model (ReAIM) by pruning weak adsorption and inhibition interactions. Finally, ReAIM is further simplified into an Effective Activity Model (EAM), in which nonproductive enzyme adsorption onto lignin is represented by a reduction in effective enzymatic activity. Plant cell wall composition is expressed as four structural polymers coupled to six soluble products, with inhibition by mono- and oligosaccharides acting on five functional enzyme pools. Using glucose, xylose and mannose release at two enzyme loadings, the model hierarchy is validated in reproducing saccharification dynamics and in delivering a quantitative product-enzyme inhibition network consistent with literature trends. The model framework was further validated by prediction of saccharification dynamics at an unseen enzyme loading, demonstrating robust performance beyond the calibration conditions. Sensitivity analysis further supported the progressive simplifications adopted in ReAIM and EAM. Maintaining predictive performance across increasing levels of simplification indicates that the hierarchy effectively distinguishes essential mechanisms from dispensable complexity, enabling rational model selection.

Given the current global environmental challenges, waste biomass is an attractive renewable resource for circular economies. Gasification of biomass yields syngas (CO, CO2, and H2) that is a suitable feedstock for gas fermentation in biomanufacturing of fuels and chemicals using acetogen microbes. While it is generally known that syngas composition influences both acetogen growth and process performance, we are lacking a consistent dataset quantifying these effects under controlled fermentation conditions. Here, we mapped the metabolic response of the model-acetogen Clostridium autoethanogenum to seven synthetic syngas mixtures during exponential batch growth in bioreactor fermentations. Notably, distinct gas compositions resulted in different fermentation profiles, affecting both growth and metabolite production. Maximum specific growth rates ranged within 0.05 0.13 h-1, with slower growth for low-CO mixtures. While acetate and ethanol production yields varied between 20-133 and 76-353 mmol per gram dry cell weight, respectively, minor production of 2,3-butanediol was detected. All syngas mixtures supported co-utilization of CO and H2, though gas uptake stoichiometry only moderately correlated with syngas content. Importantly, gas uptake stoichiometry strongly influenced carbon partitioning, with higher relative H2 uptake reducing CO2 loss or even realizing CO2 fixation together with increasing carbon flow towards metabolites. Interestingly, higher syngas H2 content favored ethanol and 2,3-butanediol production, while higher H2:CO uptake ratios increased total flux through the Wood-Ljungdahl pathway rather than selectively favoring reduced by-products. Our results are valuable for a better understanding of syngas composition effects on the acetogen biocatalyst and for process engineering towards optimizing gas fermentation performance. HighlightsO_LISyngas composition affects acetogen growth, gas uptake, and carbon distribution C_LIO_LIHigher H2:CO uptake ratios increase carbon flow through the Wood-Ljungdahl pathway C_LIO_LIHigher relative H2 uptake reduces CO2 loss and increases metabolite production C_LI

BackgroundMicrobial chain-elongation by Clostridium kluyveri using the products (acetate and ethanol) derived from the electrocatalytic CO2 reduction reaction (CO2RR) represents a unique sustainable strategy for producing C4-C6 chemicals from CO2. However, direct integration of electrocatalytic effluents with anaerobic bioprocesses is often impeded by the physiological incompatibility between electrocatalytic product streams and microbial metabolism. Specifically, CO2RR effluents commonly contain formate, which cannot be utilized by C. kluyveri for chain elongation and therefore reduces the overall carbon efficiency of CO2 conversion to C4-C6 chemicals. Moreover, both formate and the elevated phosphate concentrations typical of electrochemical reaction solutions may inhibit microbial growth. ResultsWe show that formate at concentrations of up to 50 mM did not inhibit the growth of or the chain elongation by C. kluyveri. Based on this finding, we developed a modular two-step bioprocess. In the first step, the acetogen Clostridium ljungdahlii converts formate in CO2RR product mixtures into acetate, thereby generating additional substrates for second-step C. kluyveri-driven chain elongation, thus increasing the CO2RR carbon-conversion efficiency to C- C6 chemicals. To address the issue of C. ljungdahliis inhibition by high phosphate concentrations in electrocatalytic solutions, we explored the use of C. ljungdahlii biofilms for the first, i.e. the formate-conversion, step. C. ljungdahlii biofilms exhibit tolerance to concentrated electrolytes, enabling the conversion of up to 50 mM formate in CO2RR solutions. ConclusionsThe demonstrated two-step process constitutes the basis for the development of a robust and carbon-efficient biological process for the scalable upgrading of C1-C2 CO2RR products into higher-value C4-C6 chemicals.

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.

The pulp and paper industry produces large volumes of condensed kraft lignin, which is challenging to convert to single chemical products. For this purpose, tandem chemical depolymerization and bioconversion to a single atom-efficient product is a potentially promising strategy. In this study, we conducted copper-catalyzed oxidative depolymerization using pine-derived kraft lignin to generate multiple bioavailable aromatic monomers at a yield of 4.5 weight% (wt%; g monomers per g lignin) from both C--O and C--C bond cleavage, followed by counter-current extraction with a 52 wt% monomer recovery. This resulted in an oxidized lignin product containing vanillin, vanillate, 4-hydroxybenzaldehyde, 4-hydroxybenzoate, 5-formylvanillin, 5-carboxyvanillin, 5-carboxyvanillate, acetovanillone, and vanillyl glyoxylate. Based on this stream composition, we engineered the industrially relevant soil bacterium Pseudomonas putida KT2440 to catabolize the latter five compounds via overexpression of ten heterologous genes (acvABCDEFSYK-6, vceABSYK-6, ligW2SYK-6, and mdlCPP). We combined these engineered pathways with previously reported strategies for muconate production from G- and H-type monomers to generate P. putida KMM428, which utilized 93.6 {+/-} 0.2 mol% of the quantified aromatic monomers in a depolymerized kraft lignin mixture, and produced muconate at a yield of 99 {+/-} 3 mol%, on a quantified monomer basis. Together, this work increases the theoretical carbon conversion efficiency of this process by 37.6 {+/-} 0.1 mol% through incorporation of three {beta}-5 cleavage products, in addition to traditional G-type monomers.

Microalgae and cyanobacteria have emerged as promising resources for sustainable agriculture; however, integrated methodologies for the rational design of crop-specific agricultural formulations remain scarce. This study proposes an integrated framework that combines biomass production, species characterization, nutrient characterization, mixture design, nutrient profile estimation, biological validation, and statistical optimization for the rational development of agricultural formulations based on microalgae and cyanobacteria. As a proof of concept, the proposed framework was applied to formulate consortia composed of C. vulgaris, Scenedesmus sp., and A. platensis using common bean (Phaseolus vulgaris L., ecotype Sangre Toro) and rainfed rice (Oryza sativa L., cv. Fedearroz 2020) as model crops. The experimentally determined nutrient composition of the individual biomasses was integrated into a simplex-lattice mixture design coupled with response surface methodology and desirability analysis to identify crop-specific optimal formulations and estimate their nutrient profiles. The cubic model provided the best fit (P < 0.05), showing high predictive performance and a non-significant lack of fit. The optimal bean formulation consisted of 31.6% C. vulgaris and 68.4% Scenedesmus sp., whereas the optimal rice formulation comprised 62.3% A. platensis and 37.7% C. vulgaris, demonstrating distinct crop- specific responses. The optimized bean formulation exhibited higher estimated concentrations of calcium, phosphorus, iron, and zinc, whereas the rice formulation showed higher estimated potassium and Kjeldahl nitrogen contents. These findings demonstrate the feasibility of developing crop-specific microalgal formulations and highlight that different crops may require distinct formulations rather than a universal approach. The proposed approach offers a reproducible, integrated framework for the development and optimization of next-generation agricultural formulations for sustainable crop production.

Acetate is a promising carbon source for microbial biotechnology as it can be produced sustainably from lignocellulosic biomass or C1 gases. Since acetate is directly activated to acetyl-CoA, it is especially suitable for producing acetyl-CoA-derived products, showcased here with the production of 3-(3-hydroxyalkanoyloxy) alkanoic acids (HAAs). P. putida KT2440 can natively metabolize acetate, but the weak acid has also inhibitory effects on microbial growth. We present an in-depth study on the physiology of P. putida KT2440 using acetate as carbon and energy source and evaluate acetate as feedstock for the biosynthesis of HAAs. Initially, a rational engineering approach to overexpress acetyl-CoA synthetase for acetate activation resulted in an improved growth rate of 16% and reduced lag phase by six hours. To further increase the performance of P. putida KT2440 on acetate, adaptive laboratory evolution was performed. This resulted in an improvement in the growth rate from 0.4 h-1 to 0.6 h-1 and enabled growth on up to 12.5 g L-1 acetate with a shortened lag phase compared to the wild type. Whole-genome sequencing revealed mutations in proteins involved in gene expression regulation and signal transduction. This evolutionary engineering approach informed the deletions of gacS and crc, which resulted in a reduction in the lag phase from seven hours to one hour and an improvement of the growth rate by 25 %, matching the growth properties of the evolved clones. Using the evolved strains for the production of HAAs resulted in faster biomass and product formation with product titers reaching up to 94 % of that of the wild type. In conclusion, we identified mechanisms in the acetate metabolism of P. putida KT2440 and improved the growth performance of the strain by rational and evolutionary engineering, demonstrating the potential of the promising, but challenging 3rd generation feedstock acetate.

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.

Bacterial cellulose (BC) is a biopolymer that comes from natural sources. It has high purity, crystallinity, mechanical strength, and biocompatibility, which makes it suitable for various biomedical and industrial uses. Unlike plant cellulose, BC does not contain lignin or hemicellulose. This results in better material quality and simpler processing. However, producing BC on a large scale is limited by high production costs, expensive culture media, and dependence on a few bacterial strains. It is essential to find cost-effective production methods and alternative microbial sources to expand its commercial use. This study looked at the cellulose-producing ability of Acetobacter diazotrophicus, a safe and relatively unexplored bacterium, under different growth conditions. We compared bacterial growth and cellulose production using Hestrin-Schramm (HS) medium, the standard for BC production, and LB supplemented with glucose (LB+Glucose), which we explored as a more affordable option. We analyzed growth rates, inoculum age, and pH levels to find the best conditions for cellulose production. We observed faster bacterial growth in HS medium, with a doubling time of 3.906 hours, compared to 6.241 hours in LB+Glucose medium. Cellulose production was greatly affected by inoculum age, with successful synthesis from cultures that were agitated for 36 to 40 hours. The highest cellulose yield was at pH 6.0 in HS medium (4.6 mg/mL) and at pH 5.5 in LB+Glucose medium (3.6 mg/mL). FTIR analysis confirmed the presence of characteristic functional groups of bacterial cellulose. These results suggest that Acetobacter diazotrophicus is a promising and cost-effective option for producing bacterial cellulose and highlight the importance of medium composition, inoculum age, and pH for optimizing production.

Food production is a significant contributor to global greenhouse gas emissions and deforestation, exacerbated by substantial food waste. Converting food waste into yeast protein offers a sustainable solution to enhance food security and contribute to a circular economy. However, due to the diverse and variable nature of food waste substrates, numerous experimental trials are required to optimise the preprocessing steps, yeast strain selection, nutrient addition, and fermentation conditions. This study presents a hybrid modelling approach where data-driven machine learning is used to predict microbial growth kinetics from process parameters. The hybrid model was trained on a comprehensive dataset consisting of 963 fermentation experiments from 55 publications, enabling transfer learning across 46 yeast strains and 79 food waste substrates. The hybrid modelling method was integrated with Bayesian optimisation, a sequential strategy to optimise expensive-to-evaluate functions, to efficiently maximise yeast biomass growth from different food waste substrates. The utility of the hybrid model was evaluated using five test datasets selected from previous literature and was shown to facilitate an average reduction of 66% in the number of experimental trials required to identify optimal fermentation conditions compared to without using the hybrid model. This proved that the transfer of knowledge between yeast strains and food wastes improved the optimisation efficiency of real, previously published datasets compared to traditional optimisation methods. The novelty and contributions of this study include the collation of the extensive dataset, provided as supplementary material; and the demonstration that transfer learning by training the hybrid model on this heterogeneous dataset can improve the optimisation efficiency for yeast biomass growth on new strains and substrates.

This study investigates electro-fermentation candida Tropicalis SY005 to address fermentation kinetics limitation during grape must fermentation. In comparison with non-stimulated control sample, EF substantially enhanced sugar depletion, TSS drop by day 3 and generated a strongly reduced state (ORP -100 to -143mV). The oxidation-reduction shift enhanced cellular NAD+ regeneration, reducing total fermentation duration from 264 h to 72 h. GC-MS analysis showed pronounced major characteristic volatile compound confirming substantial metabolic pathway shifts in flavor of glycolytic flux. Moreover moderate electric field promoted cellular membrane electropermeabilization substantially promoting bioactive extraction.

The increasing world population necessitates new sustainable nutrient sources, making microalgae like Chlorella sorokiniana interesting due to its rich nutrient profile and sustainable cultivation methods. With genetic optimization tools like CRISPR/Cas9, microalgae as a nutrient source can be improved even further. However, degradation of the rigid cell wall of microalgae, and thereby developing protoplasts, is often necessary prior to transformation, but monitoring protoplast development in spherical, single-celled organisms like C. sorokiniana is challenging using bright-field microscopy. Carbotrace 480 and 630 were tested as fluorescent markers of the cell wall of a C. sorokiniana mutant for protoplast detection, and Carbotrace 480 was successfully used to distinguish protoplast from normal cells in a cell suspension. The enzymes Driselase, Glucanex, Snailase, and Saczyme were tested in different combinations to degrade the cell wall of the mutant, with Snailase as the most effective yielding ~60 % protoplasts. This study provides a quick and easy tool for monitoring protoplast development in the microalgae C. sorokiniana, the first step to improve C. sorokiniana as a sustainable nutrient source using genetic optimization tools like CRISPR/Cas9.

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.

While the suppression of toxic cyanobacteria by aquatic plants has long been recognized, few studies have clearly differentiated between the allelopathic effects of the plant itself and the inhibitory influence of its associated microbiome. This study aimed to clarify the primary inhibitory agent by pre-culturing Myriophyllum spicatum (Eurasian watermilfoil) under three conditions: (1) BG11 medium, (2) live Microcystis aeruginosa KW culture, and (3) a Microcystis-symbiotic microbiome (excluding Microcystis cells). After a 7-day pre-culture, Myriophyllum shoots were transferred to fresh Microcystis culture. The Myriophyllum pre-cultured in Microcystis culture exhibited rapid inhibition against Microcystis (84% within day 1), whereas the Myriophyllum pre-cultured in BG11 medium showed delayed responses (89% by day 7). In contrast, inhibition remained below 50% in the Myriophyllum pre-cultured with the Microcystis-symbiotic microbiome. Notably, plant-derived soluble compounds exhibited weak inhibitory effects, whereas the microbiome showed stronger inhibitory activity, indicating that the plant-associated microbiome plays a more dominant role than the plant itself. Exposure to Microcystis triggered significant shifts in plant-symbiotic microbial community composition, leading to rapid enhancement of inhibitory activity in the Myriophyllum microbiome. Microbial community analysis identified 28 bacterial taxa closely associated with the inhibitory response, including strains involved in organic matter degradation, adhesion, biofilm formation, and predatory behavior. Meta-transcriptomic analysis further confirmed increased expression of genes related to bacterial adhesion, biofilm formation, and carbohydrate metabolism following Microcystis exposure, highlighting functional adaptations linked to cyanobacterial suppression. These findings underline the role of microbiome-mediated cyanobactericidal mechanisms, providing new insights into a nature-based solution for mitigating Microcystis-dominated harmful algal blooms.

Inshore coastal waters are almost invariably supersaturated with respect to methane and are thereby sources of methane to the atmosphere. We investigated floating algal mats and surface waters of four contrasting inshore habitats and quantified methane concentrations, sea-to-air emissions, and microbial community composition of surface waters over a seasonal cycle to determine the potential for in-situ microbial methane production in shallow oxygen-saturated surface waters with floating algal biomass. 16S rDNA sequencing indicated that Archaea belonging to the genera Methanocorpusculum, Methanosarcina, Candidatus Methanomethylophilus, and some genera from order Methanobacteriales occurred in the floating algal mats. qPCR of the genes encoding the methyl coenzyme M reductase mcrA revealed the highest expression levels during the warmest sampling periods supporting active methane production directly in surface water. Co-occurrence of the Archaea sequences and sequences belonging to the cyanobacterium strain Nodularia PCC 9350 suggests a structural relationship. Our study underscores the significant, yet underexplored impact of methane production on the surface in aggregates of floating algal material. While Nodularia and methanogens can exist independently in surface waters, their co-occurrence in algal mats reveals where the layered mat structure creates distinct microenvironments that facilitate direct metabolic exchange and provide physical stability for both groups, thereby potentially enhancing methane production in these shallow coastal systems.

The objective of this study was to enhance the production of monensin and its derivatives in Streptomyces sp. ATCC 15413. To this end the contributions of medium composition and enzyme engineering on polyketide biosynthesis were assessed. Enzyme engineering was implemented through a single-point mutation in KS5 of the polyketide synthase (PKS). This mutation increased premonensin productivity up to 29-fold, revealing and alleviating a rate-limiting step in the multi-enzyme biosynthetic pathway. Medium optimization proved comparably effective, raising titers by at least an order of magnitude across strains. Moreover, medium optimization and ketosynthase mutagenesis acted additively in the premonensin strain, further boosting its production. Overall, our findings show that medium optimization is the dominant factor in maximizing monensin yields, while enzyme engineering can deliver targeted benefits in specific contexts.

This study aims to investigate changes in protein secondary structures (-helix, -sheet, random coils, and -turn) and macronutrient distribution in different cool-season oat varieties and steam-pressure toasting durations using synchrotron-based mid-infrared (Mid-IR) spectroscopy and Fourier Transform Infrared spectroscopy (FTIR) imaging. All oat samples, provided by the Crop Development Center at the University of Saskatchewan, were harvested over three consecutive years (2018, 2019, and 2020). The first experiment compared four oat varieties (CDC Arborg, CDC Nasser, CDC Haymaker, and Summit), while the second examined CDC Nasser oats subjected to steam-pressure toasting (SPT) at 121 for 0, 30, 60, 90, and 120 minutes. FTIR chemical imaging revealed that carbohydrates, proteins and lipids in the four oat varieties were mainly concentrated in the endosperm, aleurone layer and embryo, crease region, and remained unchanged after SPT. Peak-fitting deconvolution of the Amide I band (1700-1600 cm-1) and subsequent quantitative analysis revealed that the four oat varieties exhibited broadly similar protein secondary structure profiles, with statistically significant but subtle variety effects detected for -helix (P = 0.026), -turn (P = 0.047), and the -helix to -sheet ratio (P = 0.048), however, -sheet and random coil proportions did not differ significantly among varieties. In contrast, SPT induced pronounced structural rearrangements, with significant increase in -sheet proportion (P = 0.003) and significant decreases in random coil content (P = 0.026). Notably, 30 minutes of toasting was sufficient to significantly increase -sheet and decrease the random coil contents. These changes are consistent with heat-induced protein denaturation and intermolecular -sheet aggregation, where thermal energy breaks the hydrogen bonds that stabilize the disordered random coil conformation, causing the unfolded polypeptide chains to reassemble into highly ordered -sheet aggregates. After SPT, the peak centers of Amide I and II bands shifted to lower wavenumbers and both bands broadened while their intensities were maintained, reflecting the reorganization of the remaining protein into -sheet aggregates rather than any loss of amide-active protein. These findings suggest that, although genotype has a relatively minor effect on the protein secondary structure of oats, hydrothermal treatments fundamentally reorganize the protein matrix from a disordered to an ordered conformation, which may have implications for protein digestibility, solubility, and nutritional function.

Trace elements, such as iron, nickel, and cobalt are known to regulate methanogenic activity in anaerobic digestors used for waste valorisation, but the potential role of rare earth elements remains poorly understood. This study investigated the effects of lanthanum (La) supplementation on biogas production, methane generation, volatile fatty acid (VFA) formation, and carbohydrate utilisation in anaerobic digestion (AD). Biomethane potential (BMP) assays conducted under mesophilic conditions (37C) using methanogenic sludge granules, and glucose as substrate, were supplemented with 0.1, 1, 10, and 100 mg/L lanthanum chloride (LaCl3). Biogas production and composition was monitored over a 96-h incubation, while sacrificial, batch bioreactors were used to evaluate temporal VFA and carbohydrate profiles. La supplementation significantly enhanced biogas and methane production in a concentration-dependent manner. The highest cumulative biogas yield (478.9 mL, corresponding to 179.5 mL biogas/g COD) and methane production (285.7 mL, corresponding to 107.1 mL CH4/g COD) were observed with 100 mg/L LaCl3, corresponding to increases of 88.7% and 186%, respectively, compared with La-free controls. CO2 production also increased with La concentration, whereas hydrogen production remained comparatively low. Acetic and butyric acids represented the dominant fermentation products (80-88% of total VFAs), but profiles of accumulated VFA in the bioreactors diversified with La addition, including showing caproate production, indicating changed biodegradation dynamics in the methanogenic microbiome. These findings demonstrate that lanthanum can stimulate anaerobic digestion performance and methane generation, highlighting the potential as a novel trace element additive to enhance biogas production. Research is now required to elucidate the underlying microbial and biochemical mechanisms, and establish optimal dosing strategies for large-scale applications.

Temperature is a critical factor in microalgal production, yet its impact on microalgae and their microbiome remains poorly understood. Here, we investigated the responses of Chlorella sorokiniana and its microbial community to temperature increases in four wastewater-based cultures over two months. Two Test cultures were subjected to a thermal gradient from 20 {degrees}C to 34 {degrees}C, and two were Controls at 20 {degrees}C. Eukaryotic and bacterial communities were analyzed using 18S and 16S rRNA gene sequencing, and the microalga genome was reconstructed using PacBio HiFi sequencing and functionally annotated. Control cultures remained stable, whereas Test cultures showed a decline in C. sorokiniana relative abundance and biomass from approximately 24 {degrees}C. In contrast, photosynthetic efficiency remained stable until temperatures exceeded 33 {degrees}C. Above 22-24 {degrees}C, the bacterial community, dominated by Fontibacter, Flavobacterium, and Luteolibacter, was replaced by taxa whose relative abundance increased with rising temperature and microalgal decline, pointing to bacteria better adapted to the stress conditions. Candidate predator-prey dynamics with the protist Cercozoa also intensified as temperature increased. Microalgal genome annotation identified 28 candidate genes corresponding to seven of eight literature-informed high-temperature/heat-stress targets, primarily associated with protein homeostasis, Reactive oxygen species protection, membrane lipid remodeling, and protein degradation. Collectively, these genes likely support the microalga's response and adaptation to environmental stress. Overall, culture destabilization resulted from interacting thermal, microbial, and grazing-related processes rather than temperature alone. Early microbiome changes may therefore provide useful warning signals of culture instability and help improve the management of large-scale microalgal production systems.