Ex-situ single-culture biomethanation operated in trickle-bed configuration: Microbial H2 kinetics and stoichiometry for biogas conversion into renewable natural gas.

Biomethanation converts carbon dioxide (CO2) emissions into renewable natural gas (RNG) using mixed microbial cultures enriched with hydrogenotrophic archaea. This study examines the performance of a single methanogenic archaeon converting biogas with added hydrogen (H2) into methane (CH4) using a trickle-bed bioreactor with enhanced gas-liquid mass transport. The process in continuous operation followed the theoretical reaction of hydrogenotrophic methanogenesis (CO2 + 4 H2 → CH4 + 2 H2O), producing RNG with over 99 % CH4 and more than 0.9 H2 conversion efficiency. The Monod constants of H2 uptake were experimentally determined using kinetic modelling. Also, a dimensionless parameter was used to quantify the ratio between the H2 mass transfer rate and the maximum attainable H2 consumption rate. Single-culture biomethanation averts the formation of secondary metabolites and bicarbonate buffer interferences, resulting in lower demands for H2 than mixed-culture biomethanation.

Microbial electrosynthesis from CO2 reaches productivity of syngas and chain elongation fermentations.

Carbon-based products are essential to society, yet producing them from fossil fuels is unsustainable. Microorganisms have the ability to take up electrons from solid electrodes and convert carbon dioxide (CO2) to valuable carbon-based chemicals. However, higher productivities and energy efficiencies are needed to reach a viability that can make the technology transformative. Here, we show how a biofilm-based microbial porous cathode in a directed flow-through electrochemical system can continuously reduce CO2 to even-chain C2-C6 carboxylic acids over 248 days. We demonstrate a threefold higher biofilm concentration, volumetric current density, and productivity compared with the state of the art. Most notably, the volumetric productivity (VP) resembles those achieved in laboratory-scale and industrial syngas (CO-H2-CO2) fermentation and chain elongation fermentation. This work highlights key design parameters for efficient electricity-driven microbial CO2 reduction. There is need and room to improve the rates of electrode colonization and microbe-specific kinetics to scale up the technology.

Functional plasticity of HCO3- uptake and CO2 fixation in Cupriavidus necator H16.

Despite its prominence, the ability to engineer Cupriavidus necator H16 for inorganic carbon uptake and fixation is underexplored. We tested the roles of endogenous and heterologous genes on C. necator inorganic carbon metabolism. Deletion of ß-carbonic anhydrase can had the most deleterious effect on C. necator autotrophic growth. Replacement of this native uptake system with several classes of dissolved inorganic carbon (DIC) transporters from Cyanobacteria and chemolithoautotrophic bacteria recovered autotrophic growth and supported higher cell densities compared to wild-type (WT) C. necator in batch culture. Strains expressing Halothiobacillus neopolitanus DAB2 (hnDAB2) and diverse rubisco homologs grew in CO2 similarly to the wild-type strain. Our experiments suggest that the primary role of carbonic anhydrase during autotrophic growth is to support anaplerotic metabolism, and an array of DIC transporters can complement this function. This work demonstrates flexibility in HCO3- uptake and CO2 fixation in C. necator, providing new pathways for CO2-based biomanufacturing.

Engineered acetogenic bacteria as microbial cell factory for diversified biochemicals.

Acetogenic bacteria (acetogens) are a class of microorganisms with conserved Wood-Ljungdahl pathway that can utilize CO and CO2/H2 as carbon source for autotrophic growth and convert these substrates to acetate and ethanol. Acetogens have great potential for the sustainable production of biofuels and bulk biochemicals using C1 gases (CO and CO2) from industrial syngas and waste gases, which play an important role in achieving carbon neutrality. In recent years, with the development and improvement of gene editing methods, the metabolic engineering of acetogens is making rapid progress. With introduction of heterogeneous metabolic pathways, acetogens can improve the production capacity of native products or obtain the ability to synthesize non-native products. This paper reviews the recent application of metabolic engineering in acetogens. In addition, the challenges of metabolic engineering in acetogens are indicated, and strategies to address these challenges are also discussed.

An efficient cre-based workflow for genomic integration and expression of large biosynthetic pathways in Eubacterium limosum.

Acetogenic Clostridia are obligate anaerobes that have emerged as promising microbes for the renewable production of biochemicals owing to their ability to efficiently metabolize sustainable single-carbon feedstocks. Additionally, Clostridia are increasingly recognized for their biosynthetic potential, with recent discoveries of diverse secondary metabolites ranging from antibiotics to pigments to modulators of the human gut microbiota. Lack of efficient methods for genomic integration and expression of large heterologous DNA constructs remains a major challenge in studying biosynthesis in Clostridia and using them for metabolic engineering applications. To overcome this problem, we harnessed chassis-independent recombinase-assisted genome engineering (CRAGE) to develop a workflow for facile integration of large gene clusters (>10 kb) into the human gut acetogen Eubacterium limosum. We then integrated a non-ribosomal peptide synthetase gene cluster from the gut anaerobe Clostridium leptum, which previously produced no detectable product in traditional heterologous hosts. Chromosomal expression in E. limosum without further optimization led to production of phevalin at 2.4 mg/L. These results further expand the molecular toolkit for a highly tractable member of the Clostridia, paving the way for sophisticated pathway engineering efforts, and highlighting the potential of E. limosum as a Clostridial chassis for exploration of anaerobic natural product biosynthesis.

Pressure fermentation to boost CO2-based poly(3-hydroxybutyrate) production using Cupriavidus necator.

CO2-based poly(3-hydroxybutyrate) (PHB) can be produced by the versatile bacterium Cupriavidus necator through chemolithoautotrophic fermentation, using a gas mixture consisting of CO2, H2, and O2. Despite offering a propitious route for carbon-neutral bioplastic manufacturing, its adoption is currently hampered by the wide explosive range of the required gas mixture, as well as the limited gas-to-liquid mass transfer rates. To address these challenges, pressure fermentation was applied as a robust and effective strategy, while ensuring safe operation by adhering to the limiting O2 concentration, utilizing state-of-the-art bioreactors. Consequently, exponential growth could be prolonged, boosting CO2-based PHB production from 10.8 g/L at 1.5 bar up to 29.6 g/L at 3 bar. The production gain closely aligns with the theoretical calculations, except for when the pressure was increased up to 4 bar. Overall, the demonstrated increase in PHB production underscores the potential of pressure fermentation to enhance aerobic gas fermentation.

Industrial biotechnology goes thermophilic: Thermoanaerobes as promising hosts in the circular carbon economy.

Transitioning away from fossil feedstocks is imperative to mitigate climate change, and necessitates the utilization of renewable, alternative carbon and energy sources to foster a circular carbon economy. In this context, lignocellulosic biomass and one-carbon compounds emerge as promising feedstocks that could be renewably upgraded by thermophilic anaerobes (thermoanaerobes) via gas fermentation or consolidated bioprocessing to value-added products. In this review, the potential of thermoanaerobes for cost-efficient, effective and sustainable bioproduction is discussed. Metabolic and bioprocess engineering approaches are reviewed to draw a comprehensive picture of current developments and future perspectives for the conversion of renewable feedstocks to chemicals and fuels of interest. Selected bioprocessing scenarios are outlined, offering practical insights into the applicability of thermoanaerobes at a large scale. Collectively, the potential advantages of thermoanaerobes regarding process economics could facilitate an easier transition towards sustainable bioprocesses with renewable feedstocks.

Development of a Recombineering System for the Acetogen Eubacterium limosum with Cas9 Counterselection for Markerless Genome Engineering.

Eubacterium limosum is a Clostridial acetogen that efficiently utilizes a wide range of single-carbon substrates and contributes to metabolism of health-associated compounds in the human gut microbiota. These traits have led to interest in developing it as a platform for sustainable CO2-based biofuel production to combat carbon emissions, and for exploring the importance of the microbiota in human health. However, synthetic biology and metabolic engineering in E. limosum have been hindered by the inability to rapidly make precise genomic modifications. Here, we screened a diverse library of recombinase proteins to develop a highly efficient oligonucleotide-based recombineering system based on the viral recombinase RecT. Following optimization, the system is capable of catalyzing ssDNA recombination at an efficiency of up to 2%. Addition of a Cas9 counterselection system eliminated unrecombined cells, with up to 100% of viable cells encoding the desired mutation, enabling creation of genomic point mutations in a scarless and markerless manner. We deployed this system to create a clean knockout of the extracellular polymeric substance (EPS) gene cluster, generating a strain incapable of biofilm formation. This approach is rapid and simple, not requiring laborious homology arm cloning, and can readily be retargeted to almost any genomic locus. This work overcomes a major bottleneck in E. limosum genetic engineering by enabling precise genomic modifications, and provides both a roadmap and associated recombinase plasmid library for developing similar systems in other Clostridia of interest.

Autotrophic adaptive laboratory evolution of the acetogen Clostridium autoethanogenum delivers the gas-fermenting strain LAbrini with superior growth, products, and robustness.

Microbes able to convert gaseous one-carbon (C1) waste feedstocks are increasingly important to transition to the sustainable production of renewable chemicals and fuels. Acetogens are interesting biocatalysts since gas fermentation using Clostridium autoethanogenum has been commercialised. However, most acetogen strains need complex nutrients, display slow growth, and are not robust for bioreactor fermentations. In this work, we used three different and independent adaptive laboratory evolution (ALE) strategies to evolve the wild-type C. autoethanogenum to grow faster, without yeast extract and to be robust in operating continuous bioreactor cultures. Multiple evolved strains with improved phenotypes were isolated on minimal media with one strain, named "LAbrini", exhibiting superior performance regarding the maximum specific growth rate, product profile, and robustness in continuous cultures. Whole-genome sequencing of the evolved strains identified 25 mutations. Of particular interest are two genes that acquired seven different mutations across the three ALE strategies, potentially as a result of convergent evolution. Reverse genetic engineering of mutations in potentially sporulation-related genes CLAU_3129 (spo0A) and CLAU_1957 recovered all three superior features of our ALE strains through triggering significant proteomic rearrangements. This work provides a robust C. autoethanogenum strain "LAbrini" to accelerate phenotyping and genetic engineering and to better understand acetogen metabolism.

Two-stage conversion of syngas and pyrolysis aqueous condensate into L-malate.

Hybrid thermochemical-biological processes have the potential to enhance the carbon and energy recovery from organic waste. This work aimed to assess the carbon and energy recovery potential of multifunctional processes to simultaneously sequestrate syngas and detoxify pyrolysis aqueous condensate (PAC) for short-chain carboxylates production. To evaluate relevant process parameters for mixed culture co-fermentation of syngas and PAC, two identical reactors were run under mesophilic (37 °C) and thermophilic (55 °C) conditions at increasing PAC loading rates. Both the mesophilic and the thermophilic process recovered at least 50% of the energy in syngas and PAC into short-chain carboxylates. During the mesophilic syngas and PAC co-fermentation, methanogenesis was completely inhibited while acetate, ethanol and butyrate were the primary metabolites. Over 90% of the amplicon sequencing variants based on 16S rRNA were assigned to Clostridium sensu stricto 12. During the thermophilic process, on the other hand, Symbiobacteriales, Syntrophaceticus, Thermoanaerobacterium, Methanothermobacter and Methanosarcina likely played crucial roles in aromatics degradation and methanogenesis, respectively, while Moorella thermoacetica and Methanothermobacter marburgensis were the predominant carboxydotrophs in the thermophilic process. High biomass concentrations were necessary to maintain stable process operations at high PAC loads. In a second-stage reactor, Aspergillus oryzae converted acetate, propionate and butyrate from the first stage into L-malate, confirming the successful detoxification of PAC below inhibitory levels. The highest L-malate yield was 0.26 ± 2.2 molL-malate/molcarboxylates recorded for effluent from the mesophilic process at a PAC load of 4% v/v. The results highlight the potential of multifunctional reactors where anaerobic mixed cultures perform simultaneously diverse process roles, such as carbon fixation, wastewater detoxification and carboxylates intermediate production. The recovered energy in the form of intermediate carboxylates allows for their use as substrates in subsequent fermentative stages.

Erratum: A TetR-family protein (CAETHG_0459) activates transcription from a new promoter motif associated with essential genes for autotrophic growth in acetogens.

[This corrects the article DOI: 10.3389/fmicb.2019.02549.].

Methanotrophs mediated biogas valorization: Sustainable route to polyhydroxybutyrate production.

This study explores the ability of methanotrophs to convert biogas into biopolymers, addressing H2S as a limitation in the utilization of biogas as a carbon source for bioconversion. Transcriptomic analysis was conducted to understand the growth and changes in the expression patterns of Type I and II methanotrophs under varying H2S concentrations. Results suggested that Type II methanotrophs can possess a native H2S utilization pathway. Both Type I and II methanotrophs were evaluated for their growth and polyhydroxybutyrate (PHB) production from biogas. Methylocystis sp. MJC1 and Methylocystis sp. OK1 exhibited a maximum biomass production of 4.0 and 4.5 gDCW/L, respectively, in fed-batch culture, aligning with the transcriptome data. Furthermore, Methylocystis sp. MJC1 produced 2.9 g PHB/L from biogas through gas fermentation. These findings underscore biogas-based biotechnology as an innovative solution for environmental and industrial challenges with further optimization and productivity enhancement research expected to broaden the potential in this field.

The enhancement of energy supply in syngas-fermenting microorganisms.

After the second industrial revolution, social productivity developed rapidly, and the use of fossil fuels such as coal, oil, and natural gas increased greatly in industrial production. The burning of these fossil fuels releases large amounts of greenhouse gases such as CO2, which has caused greenhouse effects and global warming. This has endangered the planet's ecological balance and brought many species, including animals and plants, to the brink of extinction. Thus, it is crucial to address this problem urgently. One potential solution is the use of syngas fermentation with microbial cell factories. This process can produce chemicals beneficial to humans, such as ethanol as a fuel while consuming large quantities of harmful gases, CO and CO2. However, syngas-fermenting microorganisms often face a metabolic energy deficit, resulting in slow cell growth, metabolic disorders, and low product yields. This problem limits the large-scale industrial application of engineered microorganisms. Therefore, it is imperative to address the energy barriers of these microorganisms. This paper provides an overview of the current research progress in addressing energy barriers in bacteria, including the efficient capture of external energy and the regulation of internal energy metabolic flow. Capturing external energy involves summarizing studies on overexpressing natural photosystems and constructing semiartificial photosynthesis systems using photocatalysts. The regulation of internal energy metabolic flows involves two parts: regulating enzymes and metabolic pathways. Finally, the article discusses current challenges and future perspectives, with a focus on achieving both sustainability and profitability in an economical and energy-efficient manner. These advancements can provide a necessary force for the large-scale industrial application of syngas fermentation microbial cell factories.

Sustainable media feedstocks for cellular agriculture.

The global food system is shifting towards cellular agriculture, a second domestication marked by cultivating microorganisms and tissues for sustainable food production. This involves tissue engineering, precision fermentation, and microbial biomass fermentation to establish food value chains independent of traditional agriculture. However, these techniques rely on growth media sourced from agricultural, chemical (fossil fuels), and mining supply chains, raising concerns about land use competition, emissions, and resource depletion. Fermentable sugars, nitrogen, and phosphates are key ingredients derived from starch crops, energy-intensive fossil fuel based processes, and finite phosphorus resources, respectively. This review explores sustainable alternatives to reduce land use and emissions associated with cellular agriculture media ingredients. Sustainable alternatives to first generation sugars (lignocellulosic substrates, sidestreams, and gaseous feedstocks), sustainable nitrogen sources (sidestreams, green ammonia, biological nitrogen fixation), and efficient use of phosphates are reviewed. Especially cellulosic sugars, gaseous chemoautotrophic feedstocks, green ammonia, and phosphate recycling are the most promising technologies but economic constraints hinder large-scale adoption, necessitating more efficient processes and cost reduction. Collaborative efforts are vital for a biotechnological future grounded in sustainable feedstocks, mitigating competition with agricultural land and emissions.

Phenotypic and genomic characterization of Methanothermobacter wolfeii strain BSEL, a CO2-capturing archaeon with minimal nutrient requirements.

A new variant of Methanothermobacter wolfeii was isolated from an anaerobic digester using enrichment cultivation in anaerobic conditions. The new isolate was taxonomically identified via 16S rRNA gene sequencing and tagged as M. wolfeii BSEL. The whole genome of the new variant was sequenced and de novo assembled. Genomic variations between the BSEL strain and the type strain were discovered, suggesting evolutionary adaptations of the BSEL strain that conferred advantages while growing under a low concentration of nutrients. M. wolfeii BSEL displayed the highest specific growth rate ever reported for the wolfeii species (0.27 ± 0.03 h-1) using carbon dioxide (CO2) as unique carbon source and hydrogen (H2) as electron donor. M. wolfeii BSEL grew at this rate in an environment with ammonium (NH4+) as sole nitrogen source. The minerals content required to cultivate the BSEL strain was relatively low and resembled the ionic background of tap water without mineral supplements. Optimum growth rate for the new isolate was observed at 64°C and pH 8.3. In this work, it was shown that wastewater from a wastewater treatment facility can be used as a low-cost alternative medium to cultivate M. wolfeii BSEL. Continuous gas fermentation fed with a synthetic biogas mimic along with H2 in a bubble column bioreactor using M. wolfeii BSEL as biocatalyst resulted in a CO2 conversion efficiency of 97% and a final methane (CH4) titer of 98.5%v, demonstrating the ability of the new strain for upgrading biogas to renewable natural gas.IMPORTANCEAs a methanogenic archaeon, Methanothermobacter wolfeii uses CO2 as electron acceptor, producing CH4 as final product. The metabolism of M. wolfeii can be harnessed to capture CO2 from industrial emissions, besides producing a drop-in renewable biofuel to substitute fossil natural gas. If used as biocatalyst in new-generation CO2 sequestration processes, M. wolfeii has the potential to accelerate the decarbonization of the energy generation sector, which is the biggest contributor of CO2 emissions worldwide. Nonetheless, the development of CO2 sequestration archaeal-based biotechnology is still limited by an uncertainty in the requirements to cultivate methanogenic archaea and the unknown longevity of archaeal cultures. In this study, we report the adaptation, isolation, and phenotypic characterization of a novel variant of M. wolfeii, which is capable of maximum growth with minimal nutrients input. Our findings demonstrate the potential of this variant for the production of renewable natural gas, paving the way for the development of more efficient and sustainable CO2 sequestration processes.

Recombinant Production of Pseudomonas aeruginosa Rhamnolipids in P. putida KT2440 on Acetobacterium woodii Cultures Grown Chemo-Autotrophically with Carbon Dioxide and Hydrogen.

The establishment of sustainable processes for the production of commodity chemicals is one of today's central challenges for biotechnological industries. The chemo-autotrophic fixation of CO2 and the subsequent production of acetate by acetogenic bacteria via anaerobic gas fermentation represents a promising platform for the ecologically sustainable production of high-value biocommodities via sequential fermentation processes. In this study, the applicability of acetate-containing cell-free spent medium of the gas-fermenting acetogenic bacterium A. woodii WP1 as the feeder strain for growth and the recombinant production of P. aeruginosa PAO1 mono-rhamnolipids in the well-established nonpathogenic producer strain P. putida KT2440 were investigated. Additionally, the potential possibility of a simplified production process without the necessary separation of feeder strain cells was elucidated via the cultivation of P. putida in cell-containing A. woodii culture broth. For these cultures, the content of both strains was investigated by examining the relative quantification of strain-exclusive genes via qPCR. The recombinant production of mono-rhamnolipids was successfully achieved with maximum titers of approximately 360-400 mg/L for both cell-free and cell-containing A. woodii spent medium. The reported processes therefore represent a successful proof of principle for gas fermentation-derived acetate as a potential sustainable carbon source for future recombinant rhamnolipid production processes by P. putida KT2440.

In vitro protein digestion and carbohydrate colon fermentation of microbial biomass samples from bacterial, filamentous fungus and yeast sources.

This study evaluated the nutritional quality of different microbial biomass samples by assessing their protein digestibility and carbohydrate fermentability in the colon using in vitro methods. Four microbial samples were produced: one hydrogen-oxidizing bacterial strain (Nocardioides nitrophenolicus KGS-27), two strains of filamentous fungi (Rhizopus oligosporus and Paecilomyces variotii), and one yeast strain (Rhodotorula babjevae). The microorganisms were grown in bioreactors, harvested and dried before analysis. The commercial fungal product Quorn was used as a reference. The protein digestibility of the microbial samples was analysed using the INFOGEST in vitro model, followed by quantification of N-terminal amine groups. An in vitro faecal fermentation experiment was also performed to evaluate the degradation of carbohydrates in microbial biomass samples and formation of short-chain fatty acids (SCFA). The fungal biomass samples had higher protein hydrolysis (60-75 %) than the bacterial sample (12 %) and Quorn (45 %), while the yeast biomass had the highest protein digestibility (85 %). Heat-treatment of the biomass significantly reduced its protein digestibility. Total dietary fibre (DF) content of fungal biomass was 31 - 43 %(DW), mostly insoluble, whereas the bacterial biomass contained mainly soluble DF (total DF: 25.7 %, of which 23.5 % were soluble and 2.2 % insoluble). After 24 h of colonic in vitro fermentation, SCFA production from the biomass of Paecilomyces, Quorn and Rhodotorula was similar to that of wheat bran, while 17 % and 32 % less SCFA were produced from the biomass of Rhizopus and the bacterial strain, respectively. Further studies are needed to clarify the reasons for the observed differences in protein digestibility and DF fermentability, especially regarding the cell wall structures and role of post-processing.

Recent progress in engineering Clostridium autoethanogenum to synthesize the biochemicals and biocommodities.

Excessive mining and utilization fossil fuels has led to drastic environmental consequences, which will contribute to global warming and cause further climate change with severe consequences for the human population. The magnitude of these challenges requires several approaches to develop sustainable alternatives for chemicals and fuels production. In this context, biological processes, mainly microbial fermentation, have gained particular interest. For example, autotrophic gas-fermenting acetogenic bacteria are capable of converting CO, CO2 and H2 into biomass and multiple metabolites through Wood-Ljungdahl pathway, which can be exploited for large-scale fermentation processes to sustainably produce bulk biochemicals and biofuels (e.g. acetate and ethanol) from syngas. Clostridium autoethanogenum is one representative of these chemoautotrophic bacteria and considered as the model for the gas fermentation. Recently, the development of synthetic biology toolbox for this strain has enabled us to study and genetically improve their metabolic capability in gas fermentation. In this review, we will summarize the recent progress involved in the understanding of physiological mechanism and strain engineering for C. autoethanogenum, and provide our perspectives on the future development about the basic biology and engineering biology of this strain.

CO2-based production of phytase from highly stable expression plasmids in Cupriavidus necator H16.

BACKGROUND: Existing plasmid systems offer a fundamental foundation for gene expression in Cupriavidus necator; however, their applicability is constrained by the limitations of conjugation. Low segregational stabilities and plasmid copy numbers, particularly in the absence of selection pressure, pose challenges. Phytases, recognized for their widespread application as supplements in animal feed to enhance phosphate availability, present an intriguing prospect for heterologous production in C. necator. The establishment of stable, high-copy number plasmid that can be electroporated would support the utilization of C. necator for the production of single-cell protein from CO2. RESULTS: In this study, we introduce a novel class of expression plasmids specifically designed for electroporation. These plasmids contain partitioning systems to boost segregation stability, eliminating the need for selection pressure. As a proof of concept, we successfully produced Escherichia coli derived AppA phytase in C. necator H16 PHB- 4 using these improved plasmids. Expression was directed by seven distinct promoters, encompassing the constitutive j5 promoter, hydrogenase promoters, and those governing the Calvin-Benson-Bassham cycle. The phytase activities observed in recombinant C. necator H16 strains ranged from 2 to 50 U/mg of total protein, contingent upon the choice of promoter and the mode of cell cultivation - heterotrophic or autotrophic. Further, an upscaling experiment conducted in a 1 l fed-batch gas fermentation system resulted in the attainment of the theoretical biomass. Phytase activity reached levels of up to 22 U/ml. CONCLUSION: The new expression system presented in this study offers a highly efficient platform for protein production and a wide array of synthetic biology applications. It incorporates robust promoters that exhibit either constitutive activity or can be selectively activated when cells transition from heterotrophic to autotrophic growth. This versatility makes it a powerful tool for tailored gene expression. Moreover, the potential to generate active phytases within C. necator H16 holds promising implications for the valorization of CO2 in the feed industry.