Succinic acid has been considered an important molecule in the transition of chemical manufacturing from fossil-based to sustainable and future-proof processes. While there has been extensive research on biotechnological succinic acid production from biomass, attempts to roll out bio-succinic acid are impeded by its high price and remaining sustainability issues. Both drawbacks are interconnected and can be traced back to the used feedstocks and a wasteful expenditure of acid and base, among others. In this opinion, we discuss biochemical principles and metabolic pathways of next-generation carbon assimilation and low-pH fermentations to address these drawbacks. For this reason, we chart the potential for producing succinic acid from sustainable next-generation feedstocks based on electron, carbon and ATP balances as well as relevant thermodynamic considerations. Furthermore, we summarize key advances in low-pH succinic acid synthesis using acid-tolerant yeasts and assess the suitability of selected acid tolerance mechanisms for industrial application. Eventually, we aim to inspire researchers to synthesize innovative approaches to realize competitive and sustainable biotechnological succinic acid production.

Cytochrome P450 monooxygenases (P450s) not only play many physiological roles in oxidative metabolism but are also promising biocatalysts for the synthesis of organic molecules. Bacillus subtilis strain 168, which possesses eight P450 genes, has been extensively studied as a model bacterium, but the catalytic function of CYP107J1 remains to be fully elucidated. Here, we investigated the catalytic function of CYP107J1. Because the genes encoding the reductase components of CYP107J1 could not be identified from the genome sequence, putidaredoxin (Pdx) and its reductase (PdR) were first used to reconstitute the enzymatic activity of CYP107J1. The enzyme showed oxidation activity toward 4-alkylbenzoic acids with a carbon chain length of 3-8, although the activity was low. To enhance the activity of CYP107J1, over that observed when using heterologous redox partners, we next engineered CYP107J1 into a hydrogen peroxide-driven form (i.e., peroxygenase). Only two amino acid substitutions in the active site of CYP107J1 were required for a peroxygenase variant exhibiting 28 times higher catalytic activity toward 4-hexylbenzoic acid than the wild-type enzyme supported by Pdx and PdR. This highly active enzyme enabled detailed characterization of CYP107J1. Interestingly, the engineered enzyme not only oxidized 4-alkylbenzoic acids but also efficiently produced the valuable dye indigo by simply mixing the enzyme with indole and hydrogen peroxide. Enzymes of the CYP107J subfamily are widely distributed among bacteria of genus Bacillus. The findings from this study will facilitate further exploitation of the catalytic potential of CYP107J subfamily enzymes.

Polyhydroxyalkanoates (PHAs) are intracellular microbial polyesters whose commercial deployment is strongly influenced by downstream processing costs and environmental burden. Here, we demonstrate a nearly solvent-free, mild aqueous strategy for the recovery of PHAs directly from wet bacterial biomass using crude enzyme systems derived from Tenebrio molitor. Under optimized conditions (0.6 wt% crude protein, pH 7.6, 40°C), near-quantitative (≥ 95%) recovery of PHB and PHB/HV and up to 60% recovery of mcl-PHA were achieved without prior biomass drying. Proteomic analysis identified abundant digestive hydrolases, including α-amylase and cathepsin and recombinant validation confirmed their contribution to polymer release, demonstrating that targeted enzyme combinations can substantially enhance mcl-PHA recovery (up to 94%-98%). Gel permeation chromatography, NMR and thermal analysis demonstrated preservation of polymer molecular integrity and crystallinity comparable to chloroform extraction. Life cycle assessment revealed a three- to seven-fold reduction in carbon footprint relative to conventional solvent and classical enzymatic methods, primarily due to elimination of drying and solvent use. These findings establish biologically driven biomass hydrolysis as a scalable downstream strategy and highlight insect-derived enzyme systems as promising tools for integrated microbial biopolymer processing.

High-quality silage enhances palatability and feed intake; however, the effects of co-fermentation with flavouring agents and lactic acid bacteria (LAB) on its flavour quality, core microbiota, and taste-active amino acids remain unclear. This study investigated the effects of fermentation using Lactiplantibacillus plantarum (LP) alone or in combination with phenethyl acetate (LPP) on the flavour profile of alfalfa silage and its subsequent influence on feed intake. Both LP and LPP significantly improved fermentation quality versus control (CK), with markedly higher feed intake-LP > CK and LPP > LP. Key flavour compounds, including dimethyl trisulfide, 4-ethylphenol and β-damascenone, were significantly increased in the LP alone group. Contrarily, essential taste-related amino acids including aspartic acid, alanine, proline, histidine, isoleucine, and valine were decreased, except for arginine. These metabolic shifts collectively contributed to enhanced feed intake. The addition of LPP increased phenylethyl alcohol, benzyl alcohol and hexanal, and decreased arginine, contributing to enhanced palatability. Aryl alcohol dehydrogenase, proline aminopeptidase, histidine dehydrogenase, and branched-chain amino acid transaminase from LP played a crucial role in the formation of phenylethyl alcohol, proline, histidine and isoleucine during fermentation. These results provide insights into how LAB and flavouring agents jointly regulate flavour development in high-quality alfalfa silage.

ABSTRACTFish aquaculture faces significant economic losses from disease outbreaks. Vaccination is the most effective prevention strategy, and bacterial extracellular vesicles (EVs) show promise as vaccine platforms due to their strong immuno‐stimulating properties. However, the application of EVs derived from pathogenic bacteria is limited by toxicity risks and production challenges. Alternatively, genetic engineering of non‐pathogenic microorganisms is being explored to produce tailored EVs to deliver antigens and serve as carriers of therapeutic proteins. Recently, we have engineered the model cyanobacterium Synechocystis sp. PCC 6803 for the expression of the reporter green fluorescent protein (sfGFP) and its targeting to EVs. Here, taking advantage of the Synechocystis sfGFP‐loaded EVs, the stability of vesicles and their cargo was evaluated in the long term when stored under different temperature conditions and after freeze‐drying. The possibility of using Synechocystis EVs as a tool for eliciting specific/adaptive immune responses was assessed in European seabass, a high commercial value fish, by following the amount of total and sfGFP‐specific immunoglobulins produced after immunisation through injection. Synechocystis EVs were shown to be resilient nanostructures that can induce specific immune responses in fish with additional adjuvant features. This represents a biotechnological breakthrough towards a novel antigen‐carrier platform for sustainable fish‐pathogen control.

ABSTRACTSelenium (Se) is an essential trace element whose toxicity depends on its oxidation state. Microorganisms detoxify Se(VI) and Se(IV) by reducing them to elemental selenium [Se(0)], forming selenium nanoparticles (SeNPs) with antimicrobial activity. Stenotrophomonas bentonitica BII‐R7 exhibits remarkable tolerance and reduction capacity toward toxic Se oxyanions, making it a promising candidate for bioremediation and green nanotechnology. In this study, cells exposed to Se(IV) were fractionated into cytoplasmic and membrane components and analysed at 24, 168 and 720 h. Spherical SeNPs were observed in the cytoplasm, while irregular aggregates formed in the membrane fraction, suggesting compartment‐specific reduction pathways. The delayed formation of SeNPs in membranes supports a time‐dependent, multimodal mechanism. Homogeneous biogenic SeNPs (160–180 nm) produced by intact S. bentonitica cells exhibited antimicrobial activity against Escherichia coli CET101 and Staphylococcus aureus ATCC 25923. Flow cytometry revealed strong, time‐dependent cytotoxicity. In E. coli, SeNPs induced 21.1% membrane depolarization, 62.8% ROS accumulation and DNA damage at 48 h, indicating a ROS‐mediated mechanism. In contrast, S. aureus showed early membrane depolarization at 12 h, with only 4.37% active cells and minimal ROS levels, and a significant drop in viability at 24 h (31.3%), suggesting a ROS‐independent mechanism driven by membrane disruption. These findings highlight the strain‐specific toxicity of SeNPs and their potential as eco‐friendly, broad‐spectrum antimicrobials.

ABSTRACTWe live in a time of global crises: a deteriorating environment that is struggling to provide all the resources and services we demand of it, changing climate and its consequences for the biosphere, its habitats, inhabitants and biodiversity, conflicts‐divisive ideologies‐competition for resources, increasing societal inequalities and human deprivations, and a youth mental health pandemic, to name but just a few. Most of these crises are self‐made, the result of human decisions, and their acceptance/toleration by society. Policies and practices at all levels of society that created, exacerbate and launch new crises are, at worst, self‐serving and, at best, faulted through a lack of understanding. In democracies, citizens can hold decision‐makers to account but, to do this, they must understand the issues and be able to imagine better policies. We also live in a digital world in which a flood of mostly inconsequential information and misinformation pollutes our brains, enhancing pre‐existing biases and creating new ones, and numbing our mental ability to think clearly and reach sensible decisions. But sensible decisions are urgently needed at all levels to fix problems and reduce future self‐harm. Sensible decisions require sourcing the best available relevant information, and a process to convert information into understanding, understanding into clear decision options, and the choice of a decision option that leads to an action that represents best practice. Critical thinking is the enabling cognitive process of this decision pathway, because it selects the best available information through demanding evidence‐basing, seeks critical discourse between experts and stakeholders that agnostically explores solution space to find plausible options, and whittles down options inter alia through plausibility, due diligence, bottleneck analysis, cost‐benefit analysis, and benchmarking filtering. Crucially, it rejects biases, influencing factors, and other constraints on options, and is an effective barrier to the information flood. The problem is that critical thinking capacity is not widely available among either decision makers or stakeholders. There is an urgent need to rapidly roll out effective education programmes in which critical thinking teaching is solidly embedded. Since biases accumulate with age, the teaching of critical thinking must begin with the very young. However, the very young are not able to comprehend the complex abstract issues underpinning critical thinking. Embedding the teaching of critical thinking in a suitable educational context, and integrating it into curricula, is another challenge. To address these two challenges, the International Microbiology Literacy Initiative is developing a storytelling programme for children, called the Critical Thinking MicroChats Gallery, within the curriculum of societally relevant microbiology it is creating. MicroChats illustrate the principal practical elements of critical thinking, like bottlenecks, cost: benefit, benchmarking, the need for discussions and other points of view, employing readily relatable, relevant microbially centric scenarios. MicroChats suggest class discussion topics to encourage children to imagine the application of each element in other contexts to reinforce principles and hone critical thinking skills. Critical thinking, and especially the cultivation of the habit of asking ‘why’ and requiring plausible justification for policies/actions, is a shield against bias, prejudice, propaganda, misinformation and the incessant pressures of social media. It promotes a healthy mind and the attainment of the developmental potential of individuals. Increasing critical thinking in society will raise the quality of decision making at all levels and thereby improve sustainability/reduce the human footprint on our planet, and promote the individual sense of responsibility and global citizenship necessary for the improvement of the condition of humanity and its relationship with Planet Earth.