ABSTRACT. Selected adventures, lessons therefrom, and thoughts on future directions will be presented drawing from a career of activity in the field.

ABSTRACT. Burning fossil fuels is highly contributing to the emissions of greenhouse gases which accelerate climate change. The Iran war impacted the global trade of fossils and triggered an energy crisis in Europe. This shows how important it is to become independent from these raw materials. In the MiMiWin project we teamed up with two partners and together we are dedicated to recycle mixed plastics via gasification into valuable products. We intend to convert CO2 along with CO or H2 to produce acetate, butyrate, isobutyrate and isobutanol.

Anaerobic acetogens such as Clostridium luticellarii and Eubacterium callanderi can use those substrates to gain acetyl-CoA, a central metabolic intermediate, via the Wood-Ljungdahl pathway. Acetyl-CoA can then be converted to acetate, via acetyl-phosphate, thereby obtaining ATP. These two acetogens can also elongate acetyl-CoA to butyrate and hexanoate via reverse β-oxidation. Isobutyrate is an important intermediate to produce isobutanol and is in C. luticellarii presumably produced by the action of an enzyme, called isobutyryl-CoA mutase (ICM). This enzyme consists of two subunits, a large subunit (IcmA) which catalyzes the reaction and a small subunit (IcmB) which is responsible for binding of the cofactor vitamin B12. Both genes were cloned into the plasmid pJIR751 under the control of a constitutive promoter. During heterotrophic growth experiments of E. callanderi [pJIR751_Pfd_icm], isobutyrate production was absent. It turned out that the ICM likely requires two additional enzymes for proper functionality. An adenosyltransferase (ATR) which binds and activates the cofactor cob(II)alamin and a G-protein metallochaperone (Mea) which loads the active cofactor to the ICM. These genes were subcloned and introduced into E. callanderi yielding the strains E. callanderi [pJIR751_Pfd_ATR_icm] and E. callanderi [pJIR751_Pfd_ATR_icm_Mea]. Unfortunately, isobutyrate production remained absent in respective heterotrophic and autotrophic growth experiments. Altered concentrations of the inactive form of the vitamin B12 cofactor, cyanocobalamin and the activated form adenosylcobalamin also did not have an effect in our experimental setups for isobutyrate production. Therefore, RT-(q)PCR assays were executed, in order to analyze whether the recombinant genes were expressed and hopefully these results provide insights to unravel the unknown bottleneck.

ABSTRACT. Necrotic enteritis caused by Clostridium perfringens is one of the most economically damaging diseases in commercial broiler production across South Asia. In Punjab, Pakistan, daily mortality of 10 to 15 birds in flocks of 50,000 is not unusual and it keeps happening even when antibiotics are being used. Overcrowding, poor biosecurity, and near total dependence on antimicrobials have created conditions where the disease keeps cycling back regardless of treatment.

Colistin sulfate is the antibiotic most commonly reached for when NE strikes in Pakistani poultry operations. It's a last resort drug critically important in human medicine and it's not reliably working anymore. Sub-therapeutic dosing and overuse in food animals have been linked globally to the spread of mobilized colistin resistance through mcr genes. Whether that's happening in Pakistani flocks is simply unknown. No one has looked.

This study will fill that gap. We'll collect intestinal samples from NE-affected birds across commercial broiler farms in Okara District, isolate and identify C. perfringens under anaerobic conditions, and run antimicrobial susceptibility testing with a focus on colistin. PCR will screen for mcr-1 through mcr-9, with sequencing used to confirm any resistance determinants detected.

What we find won't stay contained to a poultry context. Colistin-resistant C. perfringens moving through the food chain is a public health problem - one that sits squarely at the animal-human interface. Pakistan's livestock sector has a serious AMR data deficit, and this study is designed to produce the kind of evidence that antibiotic stewardship policy actually needs to move forward.

ABSTRACT. The transition toward sustainable biofuels has intensified research efforts in biobutanol production due to its high energy density and compatibility with existing fuel infrastructure. However, its efficient production from lignocellulosic biomass remains challenging due to incomplete carbon utilization, particularly the poor co-metabolism of glucose and xylose and the formation of lignocellulose-derived inhibitory compounds (LDMICs) during pretreatment and enzymatic hydrolysis. While Saccharomyces cerevisiae has limited capacity to metabolize pentose sugars (e.g., xylose), solventogenic Clostridium species can metabolize both hexoses (e.g., glucose) and pentoses but are constrained by low product yields and product inhibition. Coculture systems that integrate the complementary metabolic capabilities of these microorganisms provide a promising strategy to overcome these limitations. This study investigated coculture interactions between Clostridium saccharoperbutylacetonicum and Saccharomyces cerevisiae during fermentation of wheat straw hydrolysate (WSH) for acetone-butanol-ethanol (ABE) production. Biomass was pretreated with 1% (w/v) dilute sulfuric acid at 15% (w/v) solids and autoclaved (121 °C; 1 h), followed by enzymatic hydrolysis. The resulting hydrolysate contained 100 g/L total reducing sugars (TRS), comprising 44.7% glucose, 51.1% xylose, and 4.3% arabinose. Batch fermentations were performed anaerobically at 30 °C with 60 g/L TRS in WSH-based P2 medium, using mono- and cocultures. In the control P2 medium, with the same carbon source ratio as WSH, the maximum growth of the coculture, as measured by the optical density (OD600nm), was ∼30% greater than that of the monoculture. ABE and butanol production were ∼9.1% and ∼25.1%, respectively, greater than in the monoculture. There was no growth observed in 100% WSH Clostridium monoculture, while the coculture exhibited over 75% reduction in growth, with no butanol detected during fermentation. However, growth was restored in 50% WSH (hydrolysate + control medium), in which the coculture achieved ∼6% greater growth than the Clostridium monoculture. Despite this, butanol production in 50% WSH was ∼15.5% and ∼41.7% lesser than that of mono- and coculture, respectively, when compared to the growth of C. saccharoperbutylacetonicum in the control medium. These findings demonstrate that while coculture systems improve microbial growth and solvent production under defined conditions, the presence of LDMICs in the WSH adversely affects butanol production.

ABSTRACT. With the recent awareness about the consequences of global warming, research for alternatives to fossil fuel resources and petrochemistry has received interest in the last decades. Thanks to their ability to convert lignocellulose-derived sugars into industrially relevant compounds like C2-C4 alcohols, solventogenic microorganisms such as Clostridium acetobutylicum are promising candidates for the bioproduction of fuels and platform molecules. While genetic engineering of those bacteria was still complex and time-consuming a few years ago, the development of efficient genetic tools now makes it possible to modify their metabolism. Another strategy to improve fermentation capacities of those microorganisms relies on the use of cocultures, which can contribute to the improvement of carbon yield, the expansion of substrate and product repertoire or the maintenance of a synergistic environment. In this work, an ethanol and acetate hyperproducing mutant of Clostridium acetobutylicum was constructed using a CRISPR-Cas9 based tool. A bottom-up approach was then implemented to set-up a semi-synthetic consortium comprising the mutant strain and an anaerobic microbial partner.

ABSTRACT. Climate change is driving the urgent need for innovative technologies that convert carbon emissions into valuable products. Gas fermentation has emerged as an attractive route to produce fuels and chemicals by recycling waste gases and gasified organic solid waste using microbes. Acetogens that use the Wood-Ljungdahl pathway (WLP) to fix carbon oxides (CO and CO2) into metabolic products are being used as biocatalysts in industrial gas fermentation. However, metabolic engineering of novel acetogen cell factories is hindered by our limited understanding of the functions of genes within and adjacent to the C1-fixing gene cluster. Here, we applied CRISPR/nCas9-mediated single-gene deletions, bottle and bioreactor fermentations, proteomics, and bioinformatics in the model gas-fermenting acetogen Clostridium autoethanogenum to investigate the roles of genes with unclear functions associated with the C1-fixing gene cluster. Deletion of two genes adjacent to this cluster – a hypothetical protein (hp) and a CooT nickel binding protein (nbp) resulted in significant growth defects and altered by-product profiles, alongside impacts on complex nutrient utilisation and on the expression of C1-fixation and ethanol synthesis pathway proteins. Deletions within the cluster revealed that acsV, cooC, and cooC2 were essential for autotrophy, likely due to their requirement for proper function of the key enzyme complex CODH/ACS. Notably, their deletions also affected heterotrophic growth and distribution of carbon and redox fluxes. In contrast, genes presumably linked to the glycine cleavage system showed more specialised functions, with lpdA essential for autotrophy while gcvH contributing to WLP activity. Altogether, these findings expand our understanding of genotype-phenotype relationships linked to C1-fixation in acetogens and identify potential targets for engineering improved industrial strains for gas fermentation.

ABSTRACT. Nowadays, one of the planet's main concerns is to minimize the emission of CO2 pollutants and to find viable alternatives to petroleum. One possible approach is to produce second generation bioethanol through bacteria fermentation via consolidated bioprocessing (CBP). In this bioprocess, thermophilic bacterias are used instead of yeasts. They are anaerobic, have the ability of fermenting directly from biomass and can be genetically modified to produce ethanol at higher titers. By taking advantage of these residues, it would be possible both to reduce waste and to increase bioethanol production. While Clostridium thermocellum is the main focus of CBP due to its cellulolytic capacity, Thermoanaerobacterium saccharolyticum stands out for its ability to produce ethanol in high yield and its fermentation pathway has been extensively studied in order to be recapitulated in C. thermocellum. Although T. saccharolyticum's optimal growth temperature is 55 °C, several growth and metabolic parameters are temperature-dependent, lowering the temperature may improve certain conditions. Our hypothesis is that reducing the temperature could allow a higher ethanol tolerance, since both factors (i.e. temperature and ethanol) have chaotropic effects, thereby improving ethanol yield. Based on this, we intend to investigate how the yield of ethanol is affected by changing the temperature to 35, 45, 55 and 60 °C, evaluating whether lower temperatures can improve ethanol production. Fermentations were conducted in 125mL batch serum bottles with 20 mL of media and 0.5mL of inoculum. The media used were TSC6 (rich) and MTC-6 (defined), both containing high concentrations of cellobiose (approximately 140g/L) to ensure achieving the maximum limits of ethanol production. The fermentations were carried out for 14 days and samples were collected at time points 0, 3, 7 and 14 days, to track fermentation products through time by HPLC analysis. The preliminary results showed that strain LL1049 achieved the highest ethanol yield compared to LL1145, likely due to its use of cofactors (i.e. NADPH). In contrast, the wild-type strain LL1025 showed the lowest ethanol production, as expected. The preliminary results were also consistent with the observed increase in ethanol production at lower temperatures.

ABSTRACT. Thermophilic bacteria not only provide fundamental understanding of life at high temperatures but also have potential as industrial catalysts. In processes, elevated temperatures can result in higher reaction rates, improved substrate solubility and mass transfer, reduced contamination risks, and lower cooling requirements. Thermophiles are also a valuable source of thermostable enzymes and serve as model systems for studying protein thermostability. Despite these advantages, the metabolic capabilities of thermophiles remain incompletely characterized, and genome annotations often lack experimental validation. It is therefore crucial to provide high-quality reference data for data-driven research, such as metabolic modelling and machine learning-guided protein engineering, and to improve functional annotation of thermophilic enzymes. This study will therefore highlight the importance of functional annotations of pyrophosphate- and ATP-dependent phosphofructokinase in Acetivibrio thermocellus, as understanding the central parts of glycolysis is key to utilizing A. thermocellus for industrial biofuel production. This study further experimentally validated a total of four putative glycerol kinases (Gk) and glycerol-3-phosphate dehydrogenases (G3PDH) genes from three thermophilic bacteria: A. thermocellus, Thermoanaerobacter saccharolyticum, and Thermoanaerobacter wiegelli. The glycerol genes were heterologously expressed in E. coli BL21 from the pTrc99a plasmid, and enzyme activities were assayed at 55 °C in heat-treated cell-free extracts. The A. thermocellus gene clo1313_0073 encoded a functional GTP-dependent glycerol kinase, showing no detectable activity with ATP. Two T. saccharolyticum genes (tsac_0494 and tsac_1204) and one T. wiegelli gene (thewi_2009) also encoded for active glycerol kinases. Among the G3PDH candidate genes, only clo1313_1192 from A. thermocellus displayed NADH-dependent G3PDH activity, while the putative FAD-dependent homologs from T. saccharolyticum (tsac_0493 and tsac_1205) and T. wiegelli (thewi_2008) could not be stably expressed in E. coli. These findings provide experimentally confirmed annotations for key thermophilic metabolism genes, supply reliable enzymatic data for metabolic and machine learning-based models, and improve the functional annotation of thermophilic genomes.

ABSTRACT. Increased carbon emissions are a growing global sustainability concern. Efficient valorization of waste streams into value-added products such as biofuels and biochemicals can help reduce our environmental carbon footprint. For this, gasification of organic waste into syngas (CO+H₂+CO₂) can serve as an attractive substrate for microbial gas fermentation. While acetogen gas fermentation with CO-rich waste gases has been commercialized, utilization of low-CO syngas mixes presents a particular challenge due to reduced carbon availability. In this regard, adaptive laboratory evolution (ALE) represents a promising strategy to adapt and generate superior acetogen strains for low-CO syngas growth. In another work, we detected slower growth (µmax ~ 0.08 h⁻¹) of the model-acetogen Clostridium autoethanogenum on synthetic low-CO syngas (7%) compared to CO-rich mixtures (µmax ~ 0.10–0.13 h⁻¹) in bioreactor batch growth. Here, we implemented ALE on the same low-CO syngas to obtain strains with improved growth under CO-limited conditions. After 20 serial bottle transfers, no change in µmax could be seen potentially because of high variability in µmax determination at low culture optical densities (OD). We thus explored bottle headspace flushing and re-pressurization to enhance CO availability that increased final ODs. This approach will now be used to restart ALE. This work will establish a feasible framework for further improving acetogenic performance utilizing ALE with additional strategies.

ABSTRACT. As manufacturing industries are making a shift toward more sustainable practices, acetogens such as Clostridium autoethanogenum have emerged as attractive biocatalysts for biomanufacturing of fuels and chemicals from one-carbon waste gases and gasified solid waste. However, genetic engineering of new acetogen cell factories is hindered by slow growth, laborious protocols, and the necessity of stringent anaerobic conditions, which limits the capacity for genetic design testing in vivo. Cell-free gene expression (CFE) systems have recently gained popularity for prototyping genetic constructs and exploring metabolic pathways in vitro. While CFE has been established for C. autoethanogenum lysates used aerobically, an alternative approach is needed that would maintain the activities of oxygen-sensitive reactions. This work aims to integrate a previously established three-enzyme oxygen-scavenging cascade into a C. autoethanogenum CFE system to allow testing of oxygen-sensitive proteins and pathways under ambient conditions. We have heterologously expressed, purified, and quantified the proteins making up the cascade and mediating the oxygen-sensitive 4Fe-4S cluster assembly. The scavenging capability is tested using a resazurin fluorescence assay and the system’s ability to maintain anaerobic conditions is confirmed by observing in vitro assembly and degradation of 4Fe-4S clusters. MS-identification of key metabolites, including central metabolite acetyl-CoA and its precursors, is also performed to verify metabolic activity. Our work would enable the use of established CFE systems, with minimal amendments, to more extensively explore C. autoethanogenum metabolism under atmospheric conditions.

ABSTRACT. Accumulation of greenhouse gases from combustion of fossil fuels drives climate change and threatens biosustainability on Earth. Microbial gas fermentation provides an attractive route for the capture of CO2 toward biomanufacturing of value-added products. Acetogens are attractive biocatalysts for this process as they can use CO2 as their sole carbon source in the presence of H2. However, metabolic engineering of novel cell factories is hindered by slow and complex genetic engineering workflows, including inefficient plasmid curing following genome editing. Here, we developed different approaches to optimize plasmid curing from genetically engineered strains of the model acetogen Clostridium autoethanogenum. Interestingly, a CRISPR/Cas9-based curing plasmid (C-plasmid) targeting the origin of replication both in the target editing plasmid and in the C-plasmid did not improve curing over a non-targeting control plasmid. Strikingly, plasmid curing by making cells electrocompetent (ECCs) and by non-transformative electroporation of ECCs or buffer-washed glycerol stocks showed 14–100% curing efficiencies across the approaches for five different genetically engineered C. autoethanogenum strains. The most time-efficient approach with non-transformative electroporation of buffer-washed glycerol stocks also cured an editing plasmid from Escherichia coli, with ∼97% efficiency. This work both improves genetic engineering workflows for C. autoethanogenum by significantly accelerating plasmid curing and offers methods to potentially ease plasmid curing in other microbes.

ABSTRACT. The global push toward sustainable biofuels has placed Clostridium-based acetone-butanol-ethanol fermentation at the center of industrial biotechnology. Yet a fundamental bottleneck remains underappreciated the feedstock itself. Most fermentation research optimizes what happens inside the bioreactor while the plant material entering it receives far less attention. This disconnect between upstream crop biology and downstream microbial performance represents a missed opportunity. Energy crops like sorghum, sugarcane bagasse, and dedicated lignocellulosic species vary enormously in their cell wall architecture, lignin content, hemicellulose-to-cellulose ratios, and fermentable sugar profiles. All traits directly shaped by genetics and breeding selection. High lignin biomass increases pretreatment costs and generates inhibitory compounds that suppress Clostridium growth and solvent yields. Conversely, genotypes with lower syringyl to guaiacyl lignin ratios, reduced recalcitrance, and elevated pentose content show measurably better compatibility with ABE fermentation systems. This review synthesizes current evidence on how targeted plant breeding including quantitative trait loci mapping, marker-assisted selection, and emerging genomic approaches can systematically improve biomass composition for Clostridium fermentation. We examine traits that are prioritized in recent energy crop improvement programs and evaluate how selection for cell wall digestibility, moisture retention, and low-inhibitor profiles translates into fermentation gains. We also highlight where plant breeding programs and fermentation research remain poorly integrated, and propose a framework for coordinated feedstock to fermentation optimization. Bridging plant genetics with microbial biotechnology is not a peripheral concern. It is a prerequisite for economically viable biobutanol production. This perspective aims to open that conversation across disciplines.

ABSTRACT. There is a growing need for sustainable alternatives to fossil-based fuel and chemical production. Gas fermentation using acetogens offers a promising route for converting waste gases and gasified solid waste into value-added products. However, development of novel acetogen cell factories is hindered by slow and laborious genetic engineering workflows. Recently, cell-free gene expression (CFE) systems have emerged as powerful platforms for rapid prototyping of genetic elements that have also potential to accelerate metabolic engineering of acetogens. In this work, we optimised a previously developed CFE platform for the obligate anaerobic, gas-fermenting model-acetogen Clostridium autoethanogenum. Comparison of cell lysates from autotrophic and heterotrophic cultures with and without pH adjustment in CFE yielded no measurable GFP expression but significant luciferase expression, with pH-adjusted lysates exhibiting higher expression than non-adjusted. We detected maximum luciferase expression with 1.5 U/µL T7 RNA polymerase and 32 mM magnesium glutamate after 1 h, followed by declining luminescence. Notably, luminometer-based quantification showed higher signal-to-noise ratios and lower variability than plate readers. Importantly, significantly reduced luciferase expression was detected for freeze-thawed lysates. Luciferase expression driven by the Pthl promoter achieved ~10% of protein expression quantified for the T7 promoter. Next, we aim to use the optimised CFE platform for testing various genetic parts on luciferase expression. This work established an optimised CFE platform for C. autoethanogenum towards accelerating metabolic engineering of acetogen cell factories.

ABSTRACT. Chain elongation by Clostridium kluyveri converts ethanol and acetate into medium-chain fatty acids (MCFA) via the reverse β-oxidation (r-βOx) pathway, with caproate (C6) as the primary target for biorefinery applications. Although the ethanol to acetate (E:A) ratio is established as a key metabolic determinant, the isolated effect of controlled pH on caproate synthesis, carbon selectivity, and productivity in pure culture remains insufficiently characterised. Five batch fermentations were performed (3L bioreactors, 37°C, anaerobic) spanning uncontrolled pH (6.4 - ~5.9) and controlled conditions at 6.2, 6.5, and 6.8, with initial E:A ratios of 1.2–4.9. Caproate, butyrate, ethanol, acetate, and biomass were monitored over 3–20 d. Controlled pH 6.5 with E:A ≈ 4.8 maximised caproate production (182.6 mM), carbon selectivity (84.4%), and volumetric productivity (Qp = 0.93 mM/h). pH 6.2 at equivalent E:A yielded the highest caproate to ethanol stoichiometric efficiency (Yca/Et = 0.59 mol/mol), exceeding the theoretical limit of 0.33 mol/mol when calculated over the active production period, consistent with acetate co-contribution to the elongation process. Uncontrolled fermentations grew faster (µmax = 0.094 h⁻¹) but produced less caproate (80–118 mM, S_Ca = 58–67%), as the lack of pH control caused progressive acidification that limited productive metabolism. When E:A was reduced to 1.2 at pH 6.2, carbon flux shifted toward butyrate (Bu_max = 197 mM, S_Ca = 37%), demonstrating that ethanol supply and not pH could determines the second elongation cycle proceeds. At pH 6.8, caproate production decreases to 41–113 mM despite thermodynamic favourability (ΔGr = −74 kJ/mol), a pattern consistent with reduced r-βOx enzymatic efficiency above pH 6.5, as suggested by the observed growth and product profiles. These results establish pH 6.5 and E:A > 4 as co-determining conditions for selective caproate production in C. kluyveri pure culture, and provide a quantitative basis for pH optimisation in ethanol based MCFA bioprocess design.