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The use of mixed cultures in gas fermentations could reduce operating costs in the production of liquid chemicals such as alcohols or carboxylic acids. However, directing reducing equivalents towards the desired products presents the challenge of co-existing competing pathways. In this study, two trickle bed reactors were operated at acetogenic and chain elongating conditions to explore the fate of electron equivalents (ethanol, H2, and CO) and test pH oscillations as a strategy to target chain-elongated products. Hereby, the use of a H2-rich syngas increased gas conversion rates and the specificity towards acetic acid (86% of C-mol production, 9.0 g LEBV−1 day−1, with EBV referring to empty bed volume), while preliminary experiments with CO-rich syngas show promising results in increasing the ethanol production necessary to target chain-elongated products. On the other hand, ethanol supplementation hindered the endogenous ethanol production of the acetogenic culture but promoted butanol production (1.0 g LEBV−1 day−1) at high ethanol concentrations (9.6 g L−1) in the fresh media. Finally, pH oscillations improved chain elongation yields but negatively affected acetogenic growth, reducing production rates. [ABSTRACT FROM AUTHOR]

Clostridium ragsdalei was found to produce (S)-2-hydroxyisovalerate (2-HIV) as a novel product in addition to acetate, ethanol, and d-2,3-butanediol in heterotrophic (d-xylose), autotrophic (CO), and mixotrophic (d-xylose + CO) conditions. Mixotrophic batch processes in stirred-tank bioreactors with continuous gassing resulted in improved production of this alpha-hydroxy acid compared to batch processes solely with either d-xylose or CO. The maximal CO uptake rate was considerably reduced in mixotrophic compared to autotrophic processes, resulting in a concomitant decreased total CO2 production. Simultaneous conversion of 9.5 g L−1 d-xylose and 320 mmol CO enabled the production of 1.8 g L−1 2-HIV in addition to 1.1 g L−1 d-2,3-butanediol, 2.0 g L−1 ethanol, and 1.8 g L−1 acetate. With reduced initial d-xylose (3.1 g L−1), l-valine production started when d-xylose was depleted, reaching a maximum of 0.4 g L−1 l-valine. Using l-arabinose or d-glucose instead of d-xylose in mixotrophic batch processes reduced the 2-HIV production by C. ragsdalei. Considerable amounts of meso-2,3-butanediol (0.9–1.3 g L−1) were produced instead, which was not observed with d-xylose. The monomer 2-HIV can form polyesters that make the molecule attractive for application as bioplastic (polyhydroxyalkanoates) or new composite material. [ABSTRACT FROM AUTHOR]

Electrocatalytic CO2 reduction to CO and formate can be coupled to gas fermentation with anaerobic microorganisms. In combination with a competing hydrogen evolution reaction in the cathode in aqueous medium, the in situ, electrocatalytic produced syngas components can be converted by an acetogenic bacterium, such as Clostridium ragsdalei, into acetate, ethanol, and 2,3-butanediol. In order to study the simultaneous conversion of CO, CO2, and formate together with H2 with C. ragsdalei, fed-batch processes were conducted with continuous gassing using a fully controlled stirred tank bioreactor. Formate was added continuously, and various initial CO partial pressures (pCO0) were applied. C. ragsdalei utilized CO as the favored substrate for growth and product formation, but below a partial pressure of 30 mbar CO in the bioreactor, a simultaneous CO2/H2 conversion was observed. Formate supplementation enabled 20–50% higher growth rates independent of the partial pressure of CO and improved the acetate and 2,3-butanediol production. Finally, the reaction conditions were identified, allowing the parallel CO, CO2, formate, and H2 consumption with C. ragsdalei at a limiting CO partial pressure below 30 mbar, pH 5.5, n = 1200 min−1, and T = 32 °C. Thus, improved carbon and electron conversion is possible to establish efficient and sustainable processes with acetogenic bacteria, as shown in the example of C. ragsdalei. [ABSTRACT FROM AUTHOR]

Gas fermentation is gaining attention as a crucial technology for converting gaseous feedstocks into value-added chemicals. Despite numerous efforts over the past decade to investigate these innovative processes at a lab scale, to date, the evaluation of the technologies in relevant industrial environments is scarce. This study examines the fermentative production of acetate from biomass-derived syngas using Moorella thermoacetica. A mobile gas fermentation pilot plant was coupled to a bubbling fluidized-bed gasifier with syngas purification to convert crushed bark-derived syngas. The syngas purification steps included hot filtration, catalytic reforming, and final syngas cleaning. Different latter configurations were evaluated to enable a simplified syngas cleaning configuration for microbial syngas conversion compared to conventional catalytic synthesis. Fermentation tests using ultra-cleaned syngas showed comparable microbial growth (1.3 g/L) and acetate production (22.3 g/L) to the benchmark fermentation of synthetic gases (1.2 g/L of biomass and 25.2 g/L of acetate). Additional fermentation trials on partially purified syngas streams identified H2S and HCN as the primary inhibitory compounds. They also indicated that caustic scrubbing is an adequate and simplified final gas cleaning step to facilitate extended microbial fermentation. Overall, this study shows the potential of gas fermentation to valorize crude gaseous feedstocks, such as industrial off-gases, into platform chemicals. [ABSTRACT FROM AUTHOR]

In a circular economy approach, syngas biomethanation is a promising technology for waste to energy conversion. However, syngas can contain impurities, notably tar, that can limit the processes upgrading syngas downstream gasification. The effect of tar on syngas biomethanation is unknown. Therefore, for the first time, common tar components were tested on a consortium adapted for syngas biomethanation to assess the resistance of the microorganisms. Four light tar components (benzene, toluene, styrene and phenol) commonly found in syngas were selected and tested at different concentrations in batch tests. Adding pollutant up to inhibitory concentrations affected both the lag phase of microbial growth and the rates of the bioreactions. Hydrogenotrophic methanogens were found to be more sensitive than carboxydotrophs. Amongst the four tested pollutants, phenol appears to be the most problematic, due not only to its high inhibitory effect but also to its high solubility, allowing phenol in the syngas to reach high inhibitory concentrations. This study paves the way for further research on the resistance of syngas biomethanation to impurities contained in the syngas. [ABSTRACT FROM AUTHOR]

BACKGROUND: Isopropanol and acetone production by syngas fermentation is a promising alternative to conventional fossil carbon‐dependent production. However, this alternative technology has not yet been scaled up to an industrial level owing to the relatively low product concentrations (about 5 wt% in total). This original research aims to develop cost‐effective and energy‐efficient processes for the recovery of isopropanol and acetone from highly dilute fermentation broth (>94 wt% water) for large‐scale production (about 100 ktIPA+AC y−1). RESULTS: Vacuum distillation and pass‐through distillation enhanced with heat pumps or multi‐effect distillation were efficiently coupled with regular atmospheric distillation and extractive distillation in several innovative intensified downstream processes. Over 99.2% of isopropanol and 100% of acetone were recovered as high‐purity end‐products (>99.8 wt%). Advanced heat pumping (mechanical vapor recompression) and heat integration techniques were implemented to decrease total annual costs (0.109–0.137 USD kgIPA+AC−1), reduce energy requirements (1.348–2.043 kWth h kgIPA+AC−1) and lower CO2 emissions (0.067–0.191 kgCO2 kgIPA+AC−1), resulting in highly competitive recovery processes. CONCLUSION: The proposed three novel isopropanol and acetone recovery processes from dilute broth significantly contribute to the expansion of sustainable industrial fermentation. Furthermore, this original research is the first one to develop novel pass‐through distillation technology for the complex isopropanol–acetone–water system. All the designed processes are highly economically competitive and environmentally viable. In addition to recovering efficiently both isopropanol and acetone, the designed downstream processes offer the possibility to enhance the fermentation process by recycling all the present microorganisms and reducing fresh‐water requirements. © 2023 The Authors. Journal of Chemical Technology and Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry (SCI). [ABSTRACT FROM AUTHOR]

Clostridium ragsdalei was found to produce (S)-2-hydroxyisovalerate (2-HIV) as a novel product in addition to acetate, ethanol, and d-2,3-butanediol in heterotrophic (d-xylose), autotrophic (CO), and mixotrophic (d-xylose + CO) conditions. Mixotrophic batch processes in stirred-tank bioreactors with continuous gassing resulted in improved production of this alpha-hydroxy acid compared to batch processes solely with either d-xylose or CO. The maximal CO uptake rate was considerably reduced in mixotrophic compared to autotrophic processes, resulting in a concomitant decreased total CO2 production. Simultaneous conversion of 9.5 g L−1 d-xylose and 320 mmol CO enabled the production of 1.8 g L−1 2-HIV in addition to 1.1 g L−1 d-2,3-butanediol, 2.0 g L−1 ethanol, and 1.8 g L−1 acetate. With reduced initial d-xylose (3.1 g L−1), l-valine production started when d-xylose was depleted, reaching a maximum of 0.4 g L−1 l-valine. Using l-arabinose or d-glucose instead of d-xylose in mixotrophic batch processes reduced the 2-HIV production by C. ragsdalei. Considerable amounts of meso-2,3-butanediol (0.9–1.3 g L−1) were produced instead, which was not observed with d-xylose. The monomer 2-HIV can form polyesters that make the molecule attractive for application as bioplastic (polyhydroxyalkanoates) or new composite material.

Providing simultaneously autotrophic and heterotrophic carbon sources is a promising strategy to overcome the limits of autotrophic syngas fermentations. D-xylose and L-arabinose are particularly interesting as they can be obtained by the hydrolysis of lignocellulosic biomass. The individual conversion of varying initial concentrations of these pentoses and D-fructose as reference was studied with C. autoethanogenum in fully controlled stirred-tank reactors with a continuous syngas supply. All mixotrophic batch processes showed increased biomass and product formation compared to an autotrophic reference process. Simultaneous CO and D-xylose or L-arabinose conversion was observed in contrast to D-fructose. In the mixotrophic batch processes with L-arabinose or D-xylose, the simultaneous CO and sugar conversion resulted in high final alcohol-to-acid ratios of up to 58 g g−1. L-arabinose was superior as a mixotrophic carbon source because biomass and alcohol concentrations (ethanol and 2,3-butanediol) were highest, and significant amounts of meso-2,3-butanediol (>1 g L−1) in addition to D-2,3-butanediol (>2 g L−1) were solely produced with L-arabinose. Furthermore, C. autoethanogenum could not produce meso-2,3 butanediol under purely heterotrophic conditions. The mixotrophic production of meso-2,3-butanediol from L-arabinose and syngas, both available from residual lignocellulosic biomass, is very promising for use as a monomer for bio-based polyurethanes or as an antiseptic agent. [ABSTRACT FROM AUTHOR]

Gasifiers produce a gaseous mixture of CO/CO2/H2, also known as synthesis gas (syngas), containing varying compositions and ratios depending on the lignocellulose material types, gasifier design, and gasification conditions. Different physicochemical and thermodynamic properties of each gas type in the various syngas blends can influence syngas fermentation performance for the production of chemicals such as acetate. This study examined the effect of syngas composition (CO, CO/H2, CO/CO2/H2, and CO/H2) and its corresponding ratio on acetate production using Moorella thermoacetica, a thermophilic homoacetogen as the biocatalyst. We also investigated the effect of yeast extract addition for enhancing acetate production. A syngas fermentation study performed at a total pressure of 19 psig (2.29 atm) demonstrated that syngas fermentation in the absence of CO (30%CO2/70%H2) or at low CO proportions (21%CO/24%CO2/55%H2) resulted in the highest volumetric productivity of acetate (0.046 ± 0.001 and 0.037 ± 0.001 g/L/h, respectively). Interestingly, syngas fermentation without CO reached the highest YP/X of 22.461 ± 0.574 g-acetate/g-biomass, indicating that more acetate was produced compared to cell biomass. Higher biomass production was obtained when the CO proportion was increased up to 75% in CO/H2 fermentation. However, the cell growth and acetate production dramatically decreased with increasing CO proportion up to 99.5% CO as the sole constituent of the syngas. Even so, acetate production using 99.5% CO could be improved by adding 2 g/L yeast extract. [ABSTRACT FROM AUTHOR]

Gas–liquid mass transfer is a major issue during various bioprocesses, particularly in processes such as syngas fermentation (SNF). Since SNF involves the movement of gases into the fermentation broth, there is always a rate-limiting step that reduces process efficiency. Improving this process could lead to increased efficiency, higher production of ethanol, and reduced energy consumption. One way to improve fluid transfer between gas and liquid is by incorporating nanoparticles (NPs) into the liquid phase. This entry describes recent advances in using NPs to improve gas–liquid mass transfer during SNF. The entry also describes the basics of SNF and the impact of NPs on the process and suggests areas for future research. For example, carbon nanotubes have been found to elevate the available surface area needed for gas–liquid transfer, thus improving the process efficiency. Another area is the use of NPs as carriers for enzymes involved in syngas fermentation.

Definition: Gas–liquid mass transfer is a major issue during various bioprocesses, particularly in processes such as syngas fermentation (SNF). Since SNF involves the movement of gases into the fermentation broth, there is always a rate-limiting step that reduces process efficiency. Improving this process could lead to increased efficiency, higher production of ethanol, and reduced energy consumption. One way to improve fluid transfer between gas and liquid is by incorporating nanoparticles (NPs) into the liquid phase. This entry describes recent advances in using NPs to improve gas–liquid mass transfer during SNF. The entry also describes the basics of SNF and the impact of NPs on the process and suggests areas for future research. For example, carbon nanotubes have been found to elevate the available surface area needed for gas–liquid transfer, thus improving the process efficiency. Another area is the use of NPs as carriers for enzymes involved in syngas fermentation. [ABSTRACT FROM AUTHOR]

Gas fermentation is gaining attention as a crucial technology for converting gaseous feedstocks into value-added chemicals. Despite numerous efforts over the past decade to investigate these innovative processes at a lab scale, to date, the evaluation of the technologies in relevant industrial environments is scarce. This study examines the fermentative production of acetate from biomass-derived syngas using Moorella thermoacetica. A mobile gas fermentation pilot plant was coupled to a bubbling fluidized-bed gasifier with syngas purification to convert crushed bark-derived syngas. The syngas purification steps included hot filtration, catalytic reforming, and final syngas cleaning. Different latter configurations were evaluated to enable a simplified syngas cleaning configuration for microbial syngas conversion compared to conventional catalytic synthesis. Fermentation tests using ultra-cleaned syngas showed comparable microbial growth (1.3 g/L) and acetate production (22.3 g/L) to the benchmark fermentation of synthetic gases (1.2 g/L of biomass and 25.2 g/L of acetate). Additional fermentation trials on partially purified syngas streams identified H2S and HCN as the primary inhibitory compounds. They also indicated that caustic scrubbing is an adequate and simplified final gas cleaning step to facilitate extended microbial fermentation. Overall, this study shows the potential of gas fermentation to valorize crude gaseous feedstocks, such as industrial off-gases, into platform chemicals.

Abstract Background Ethyl acetate is a bulk chemical traditionally produced via energy intensive chemical esterification. Microbial production of this compound offers promise as a more sustainable alternative process. So far, efforts have focused on using sugar-based feedstocks for microbial ester production, but extension to one-carbon substrates, such as CO and CO2/H2, is desirable. Acetogens present a promising microbial platform for the production of ethyl esters from these one-carbon substrates. Results We engineered the acetogen C. autoethanogenum to produce ethyl acetate from CO by heterologous expression of an alcohol acetyltransferase (AAT), which catalyzes the formation of ethyl acetate from acetyl-CoA and ethanol. Two AATs, Eat1 from Kluyveromyces marxianus and Atf1 from Saccharomyces cerevisiae, were expressed in C. autoethanogenum. Strains expressing Atf1 produced up to 0.2 mM ethyl acetate. Ethyl acetate production was barely detectable (

In large-scale syngas fermentation, strong gradients in dissolved gas (CO, H2) concentrations are very likely to occur due to locally varying mass transfer and convection rates. Using Euler-Lagrangian CFD simulations, we analyzed these gradients in an industrial-scale external-loop gas-lift reactor (EL-GLR) for a wide range of biomass concentrations, considering CO inhibition for both CO and H2 uptake. Lifeline analyses showed that micro-organisms are likely to experience frequent (5 to 30 s) oscillations in dissolved gas concentrations with one order of magnitude. From the lifeline analyses, we developed a conceptual scale-down simulator (stirred-tank reactor with varying stirrer speed) to replicate industrial-scale environmental fluctuations at bench scale. The configuration of the scale-down simulator can be adjusted to match a broad range of environmental fluctuations. Our results suggest a preference for industrial operation at high biomass concentrations, as this would strongly reduce inhibitory effects, provide operational flexibility and enhance the product yield. The peaks in dissolved gas concentration were hypothesized to increase the syngas-to-ethanol yield due to the fast uptake mechanisms in C. autoethanogenum. The proposed scale-down simulator can be used to validate such results and to obtain data for parametrizing lumped kinetic metabolic models that describe such short-term responses. [ABSTRACT FROM AUTHOR]

• CO inhibition was investigated on a consortium adapted for syngas biomethanation. • The experiments were performed in continuous mode on a pressurized stirred column. • No inhibition was observed for an inlet CO partial pressure up to 5 bar. • CO is consumed faster than it is transferred, keeping a low dissolved concentration. Syngas biomethanation is a promising technology in the process chain converting wastes to methane. However, gas–liquid mass transfer is a limiting factor of the biomethanation process. To reach high methane productivity, increasing the pressure is an interesting strategy to improve mass transfer. However, the CO content in the syngas raises concerns about a potential inhibition of the microorganisms. Therefore, the aim of the research was to assess the ability to work at high CO partial pressures. In this regard, a pressurized continuous stirred column with a working volume of 10 L was implemented and a consortium adapted for syngas-biomethanation for 22 months was submitted to 100% CO and increasing pressure. No inhibition phenomenon was observed for logarithmic P CO as high as 1.8 bar (inlet pressure 5.0 bar), which was the first time that such a high CO partial pressure was tested in continuous mode. Mass transfer limitations allowed for the carboxydotrophic microorganisms to consume CO faster than it was transferred, allowing for the dissolved CO concentration to remain under inhibitory concentrations. These results question the habitual consensus that CO inhibition is a limiting factor of syngas biomethanation. [ABSTRACT FROM AUTHOR]

Synthesis gas (syngas) fermentation is a biological carbon fixation process through which carboxydotrophic acetogenic bacteria convert CO, CO2, and H2 into platform chemicals. To obtain an accurate overview of the syngas fermentation research and innovation from 2012 to 2022, a systematic search was performed on Web of Science and The Lens, focusing on academic publications and patents that were published or granted during this period. Overall, the research focus was centered on process optimization, the genetic manipulation of microorganisms, and bioreactor design, in order to increase the plethora of fermentation products and expand their possible applications. Most of the published research was initially funded and developed in the United States of America. However, over the years, European countries have become the major contributors to syngas fermentation research, followed by China. Syngas fermentation seems to be developing at "two-speeds", with a small number of companies controlling the technology that is needed for large-scale applications, while academia still focuses on low technology readiness level (TRL) research. This systematic review also showed that the fermentation of raw syngas, the effects of syngas impurities on acetogen viability and product distribution, and the process integration of gasification and fermentation are currently underdeveloped research topics, in which an investment is needed to achieve technological breakthroughs. [ABSTRACT FROM AUTHOR]

Syngas fermentation with clostridial co-cultures is promising for the conversion of CO to alcohols. A CO sensitivity study with Clostridium kluyveri monocultures in batch operated stirred-tank bioreactors revealed total growth inhibition of C. kluyveri already at 100 mbar CO, but stable biomass concentrations and ongoing chain elongation at 800 mbar CO. On/off-gassing with CO indicated a reversible inhibition of C. kluyveri. A continuous supply of sulfide led to increased autotrophic growth and ethanol formation by Clostridium carboxidivorans even at unfavorable low CO concentrations. Based on these results, a continuously operated cascade of two stirred-tank reactors was established with a synthetic co-culture of both Clostridia. An amount of 100 mbar CO and additional sulfide supply enabled growth and chain elongation in the first bioreactor, whereas 800 mbar CO resulted in an efficient reduction of organic acids and de-novo synthesis of C2-C6 alcohols in the second reactor. High alcohol/acid ratios of 4.5–9.1 (w/w) were achieved in the steady state of the cascade process, and the space-time yields of the alcohols produced were improved by factors of 1.9–5.3 compared to a batch process. Further improvement of continuous production of medium chain alcohols from CO may be possible by applying less CO-sensitive chain-elongating bacteria in co-cultures. [ABSTRACT FROM AUTHOR]

Biotransformation of lignocellulose-derived synthetic gas (syngas) into acetic acid is a promising way of creating biochemicals from lignocellulosic waste materials. Acetic acid has a growing market with applications within food, plastics and for upgrading into a wide range of biofuels and bio-products. In this paper, we will review the microbial conversion of syngas to acetic acid. This will include the presentation of acetate-producing bacterial strains and their optimal fermentation conditions, such as pH, temperature, media composition, and syngas composition, to enhance acetate production. The influence of syngas impurities generated from lignocellulose gasification will further be covered along with the means to alleviate impurity problems through gas purification. The problem with mass transfer limitation of gaseous fermentation will further be discussed as well as ways to improve gas uptake during the fermentation. [ABSTRACT FROM AUTHOR]

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Providing simultaneously autotrophic and heterotrophic carbon sources is a promising strategy to overcome the limits of autotrophic syngas fermentations. D-xylose and L-arabinose are particularly interesting as they can be obtained by the hydrolysis of lignocellulosic biomass. The individual conversion of varying initial concentrations of these pentoses and D-fructose as reference was studied with C. autoethanogenum in fully controlled stirred-tank reactors with a continuous syngas supply. All mixotrophic batch processes showed increased biomass and product formation compared to an autotrophic reference process. Simultaneous CO and D-xylose or L-arabinose conversion was observed in contrast to D-fructose. In the mixotrophic batch processes with L-arabinose or D-xylose, the simultaneous CO and sugar conversion resulted in high final alcohol-to-acid ratios of up to 58 g g−1. L-arabinose was superior as a mixotrophic carbon source because biomass and alcohol concentrations (ethanol and 2,3-butanediol) were highest, and significant amounts of meso-2,3-butanediol (>1 g L−1) in addition to D-2,3-butanediol (>2 g L−1) were solely produced with L-arabinose. Furthermore, C. autoethanogenum could not produce meso-2,3 butanediol under purely heterotrophic conditions. The mixotrophic production of meso-2,3-butanediol from L-arabinose and syngas, both available from residual lignocellulosic biomass, is very promising for use as a monomer for bio-based polyurethanes or as an antiseptic agent.

Abstract Background The replacement of fossil fuels and petrochemicals with sustainable alternatives is necessary to mitigate the effects of climate change and also to counteract diminishing fossil resources. Acetogenic microorganisms such as Clostridium spp. are promising sources of fuels and basic chemical precursors because they efficiently utilize CO and CO2 as carbon source. However the conversion into high titers of butanol and hexanol is challenging. Results Using a metabolic engineering approach we transferred a 17.9-kb gene cluster via conjugation, containing 13 genes from C. kluyveri and C. acetobutylicum for butanol and hexanol biosynthesis, into C. ljungdahlii. Plasmid-based expression resulted in 1075 mg L−1 butanol and 133 mg L−1 hexanol from fructose in complex medium, and 174 mg L−1 butanol and 15 mg L−1 hexanol from gaseous substrate (20% CO2 and 80% H2) in minimal medium. Product formation was increased by the genomic integration of the heterologous gene cluster. We confirmed the expression of all 13 enzymes by targeted proteomics and identified potential rate-limiting steps. Then, we removed the first-round selection marker using CRISPR/Cas9 and integrated an additional 7.8 kb gene cluster comprising 6 genes from C. carboxidivorans. This led to a significant increase in the hexanol titer (251 mg L−1) at the expense of butanol (158 mg L−1), when grown on CO2 and H2 in serum bottles. Fermentation of this strain at 2-L scale produced 109 mg L−1 butanol and 393 mg L−1 hexanol. Conclusions We thus confirmed the function of the butanol/hexanol biosynthesis genes and achieved hexanol biosynthesis in the syngas-fermenting species C. ljungdahlii for the first time, reaching the levels produced naturally by C. carboxidivorans. The genomic integration strain produced hexanol without selection and is therefore suitable for continuous fermentation processes.

Abstract Autotrophic syngas fermentation with clostridia enables the conversion of CO, CO2, and H2 into organic acids and alcohols. The batch process performance of Clostridium ragsdalei was studied in fully controlled and continuously gassed (600 mbar CO, 200 mbar H2, 200 mbar CO2) stirred-tank bioreactors. The final ethanol concentration varied as function of the reaction conditions. Decreasing the pH from pH 6.0–5.5 at a temperature of 37 °C increased the ethanol concentration from 2.33 g L−1 to 3.95 g L−1, whereas lowering the temperature from 37 to 32 °C at constant pH 6.0 resulted in a final ethanol concentration of 5.34 g L−1 after 5 days of batch operation. The sulphur availability was monitored by measuring the cysteine concentration in the medium and the H2S fraction in the exhaust gas. It was found that most of the initially added sulphur was stripped out within the first day of the batch process (first half of the exponential growth phase). A continuous sodium sulfide feed allowed ethanol concentrations to increase more than threefold to 7.67 g L−1 and the alcohol-to-acetate ratio to increase 43-fold to 17.71 g g−1. Graphical Abstract

In order to utilize a wider range of low-grade syngas, the syngas biomethanation was studied in this work with respect to the gas–liquid mass transfer and the reactor start-up strategy. Two reactors, a continuous stirred tank (CSTR) and a bubble column with gas recirculation (BCR-C), were used in the experiment by feeding an artificial syngas of 20% H2, 50% CO, and 30% CO2 into the reactors at 55 °C. The results showed that the CH4 productivity was slightly increased by reducing the gas retention time (GRT), but was significantly improved by increasing the stirring speed in the CSTR and the gas circulation rate in the BCR-C. The best syngas biomethanation performance of the CSTR with a CH4 productivity of 22.20 mmol·Lr−1·day−1 and a yield of 49.01% was achieved at a GRT of 0.833 h and a stirring speed of 300 rpm, while for the BCR-C, the best performance with a CH4 productivity of 61.96 mmol·Lr−1·day−1 and a yield of 87.57% was achieved at a GRT of 0.625 h and a gas circulation rate of 40 L·Lr−1·h−1. The gas–liquid mass transfer capability provided by gas circulation is far superior to mechanical stirring, leading to a much better performance of low-grade syngas biomethanation in the BCR-C. Feeding H2/CO2 during the startup stage of the reactor can effectively stimulate the growth and metabolism of microorganisms, and create a better metabolic environment for subsequent low-grade syngas biomethanation. In addition, during the thermophilic biomethanation of syngas, Methanothermobacter is the dominant genus. [ABSTRACT FROM AUTHOR]

Synthesis gas (syngas) fermentation represents a promising biological method for converting industrial waste gases, particularly carbon monoxide (CO) and carbon dioxide (CO₂) from industrial sources (e.g. steel production or municipal waste gasification), into high-value products such as biofuels, chemicals, and animal feed using acetogenic bacteria. This review identifies and addresses key challenges that hinder the large-scale adoption of this technology, including limitations in gas mass transfer, an incomplete understanding of microbial metabolic pathways, and suboptimal bioprocess conditions. Our findings emphasize the critical role of microbial strain selection and bioprocess optimization to enhance productivity and scalability, with a focus on utilizing diverse microbial consortia and efficient reactor systems. By examining recent advancements in microbial conditioning, operational parameters, and reactor design, this study provides actionable insights to improve syngas fermentation efficiency, suggesting pathways towards overcoming current technical barriers for its broader industrial application beyond the production of bulk chemicals., Competing Interests: Competing InterestsThe authors declare no competing interests., (© The Author(s) 2024.)

The aim of this study is to investigate the effect of sulfate on conversion of CO2 into organics using syngas fermentation with mixed culture. Fermentation tests were operated under H2 concentrations of 2.14 and 21.4 ​mmol/d, respectively, using substrates contained different initial concentrations of sulfate (i.e. 0, 2, 4, 6, 8, 10, and 12 ​mM). The addition of sulfate improved the acetate and formate production, and the enhancement was positively correlated with the sulfate concentrations from 2 to 8 ​mM. With 8 ​mM sulfate, the maximum acetate concentrations reached 75.4 ​± ​4.51 and 76.1 ​± ​7.77 ​mM under H2 concentrations of 2.14 and 21.4 ​mmol/d, respectively, which were 1.47 and 2.58 times higher than those of the treatment without sulfate. The biomass achieved with the sulfate addition was 52%–97% higher than that without sulfate. High-throughput pyrosequencing showed that with the presence of sulfate, Acetobacterium and Desulfovibrio were dominant in the microbial community with high relative abundance of 43% and 38%, respectively. This study suggested that the performance of syngas fermentation could be improved with co-metabolism between homoacetogen and sulfate-reducing bacteria.

Gasifiers produce a gaseous mixture of CO/CO2/H2, also known as synthesis gas (syngas), containing varying compositions and ratios depending on the lignocellulose material types, gasifier design, and gasification conditions. Different physicochemical and thermodynamic properties of each gas type in the various syngas blends can influence syngas fermentation performance for the production of chemicals such as acetate. This study examined the effect of syngas composition (CO, CO/H2, CO/CO2/H2, and CO/H2) and its corresponding ratio on acetate production using Moorella thermoacetica, a thermophilic homoacetogen as the biocatalyst. We also investigated the effect of yeast extract addition for enhancing acetate production. A syngas fermentation study performed at a total pressure of 19 psig (2.29 atm) demonstrated that syngas fermentation in the absence of CO (30%CO2/70%H2) or at low CO proportions (21%CO/24%CO2/55%H2) resulted in the highest volumetric productivity of acetate (0.046 ± 0.001 and 0.037 ± 0.001 g/L/h, respectively). Interestingly, syngas fermentation without CO reached the highest YP/X of 22.461 ± 0.574 g-acetate/g-biomass, indicating that more acetate was produced compared to cell biomass. Higher biomass production was obtained when the CO proportion was increased up to 75% in CO/H2 fermentation. However, the cell growth and acetate production dramatically decreased with increasing CO proportion up to 99.5% CO as the sole constituent of the syngas. Even so, acetate production using 99.5% CO could be improved by adding 2 g/L yeast extract.

Background: Ethyl acetate is a bulk chemical traditionally produced via energy intensive chemical esterification. Microbial production of this compound offers promise as a more sustainable alternative process. So far, efforts have focused on using sugar-based feedstocks for microbial ester production, but extension to one-carbon substrates, such as CO and CO2/H2, is desirable. Acetogens present a promising microbial platform for the production of ethyl esters from these one-carbon substrates. Results: We engineered the acetogen C. autoethanogenum to produce ethyl acetate from CO by heterologous expression of an alcohol acetyltransferase (AAT), which catalyzes the formation of ethyl acetate from acetyl-CoA and ethanol. Two AATs, Eat1 from Kluyveromyces marxianus and Atf1 from Saccharomyces cerevisiae, were expressed in C. autoethanogenum. Strains expressing Atf1 produced up to 0.2 mM ethyl acetate. Ethyl acetate production was barely detectable (< 0.01 mM) for strains expressing Eat1. Supplementation of ethanol was investigated as potential boost for ethyl acetate production but resulted only in a 1.5-fold increase (0.3 mM ethyl acetate). Besides ethyl acetate, C. autoethanogenum expressing Atf1 could produce 4.5 mM of butyl acetate when 20 mM butanol was supplemented to the growth medium. Conclusions: This work offers for the first time a proof-of-principle that autotrophic short chain ester production from C1-carbon feedstocks is possible and offers leads on how this approach can be optimized in the future. [ABSTRACT FROM AUTHOR]

Summary: Fossil fuel depletion and environmental contamination coupled with food vs fuel conflict motivated sustainable bioethanol production through syngas fermentation utilizing lignocellulosic biomass as feedstock. Herein, Enterobacter sp. RF2 strain was selected for the investigation out of the nine anaerobes that were screened for bioethanol production using the pyridinium chlorochromate assay. Under optimal conditions, Enterobacter sp. RF2 and the reference strain Clostridium ljungdahlii ATCC‐55383 produced 21.8 and 37.7 g/L of bioethanol in an anaerobic culture bottle and 25.3 and 39.6 g/L of bioethanol in a stirred tank reactor via syngas fermentation. Furthermore, infra‐red spectra and proton nuclear magnetic resonance illustrated chemical shift patterns of bioethanol. The high bioethanol producer was identified as Enterobacter hormaechei RF2 by in silico analysis. Here, it was reported for the first time that E. hormaechei RF2 is high ethanol tolerant and genetically stable bioethanol producer that can be exploited for pilot‐scale production to meet the rising demand for fossil fuels and lessen eco‐pollution. Thus, this research will pave the way for the commercialization of green chemicals which is the need of the hour in the current scenario. However, the cost of biomass conversion to bioethanol must be less than the existing price of gasoline to be competitive and get economic acceptance. Although this method is now at the pre‐commercialization stage due to lack because of techno‐economic feasibility, but usage of suitable substrates and concurrent production of different valuable products can make the entire process cost affordable. Therefore, by providing employment to the rural population through the exploitation of lignocellulosic biomass, eco‐friendly fuel to the customer, and lucrative fuel business to industries, it might thus hypothesize an intriguing future for our global economy. [ABSTRACT FROM AUTHOR]

The effect of tungsten and selenium on cell growth and production of metabolites such as acetic acid and ethanol when fermenting syngas using " Clostridium autoethanogenum" was investigated to improve the process efficiency. General concentrations of selenium and tungsten in the medium are 0.01 µM during acetogenic syngas fermentation. We conducted culture experiments at concentrations of 0, 0.001, 0.01 and 0.1 µM for each heavy metal. The effect of selenium on cell growth and total metabolite production was greater than that of tungsten as the effect of selenium on formate dehydrogenase, an important enzyme of the Wood-Ljungdahl pathway, is greater than that of tungsten. Although an increase in tungsten had a marginal effect on total metabolite production, the ethanol/acetic acid production ratio increased significantly due to a decrease in acetic acid and an increase in ethanol production. Thus, tungsten plays a key role in activating aldehyde:ferredoxin oxidoreductase, a key enzyme in the reduction of acetate to ethanol. A specific ethanol productivity of 0.462 g ethanol/g DCW∙d was obtained in a culture using 0.01 µM selenium and 0.1 µM tungsten, which was 2.18 times higher than when using 0.01 µM of both selenium and tungsten. • Effect of selenium and tungsten on syngas fermentation was investigated. • Selenium showed greater effect in cell growth and C2 compounds production. • High tungsten dosage increased ethanol/acetic acid production ratio. • High tungsten concentration in medium significantly enhanced ethanol production. [ABSTRACT FROM AUTHOR]

Synthesis gas fermentation is a microbial process, which uses anaerobic bacteria to convert CO-rich gases to organic acids and alcohols and thus presents a promising technology for the sustainable production of fuels and platform chemicals from renewable sources. Clostridium carboxidivorans and Clostridium autoethanogenum are two acetogenic bacteria, which have shown their high potential for these processes by their high tolerance toward CO and in the production of industrially relevant products such as ethanol, 1-butanol, 1-hexanol, and 2,3-butanediol. A promising approach is the coupling of gasification of biogenic residues with a syngas fermentation process. This study investigated batch processes with C. carboxidivorans and C. autoethanogenum in fully controlled stirred-tank bioreactors and continuous gassing with biogenic syngas produced by an autothermal entrained flow gasifier on a pilot scale >1200 °C. They were then compared to the results of artificial gas mixtures of pure gases. Because the biogenic syngas contained 2459 ppm O2 from the bottling process after gasification of torrefied wood and subsequent syngas cleaning for reducing CH4, NH3, H2S, NOX, and HCN concentrations, the oxygen in the syngas was reduced to 259 ppm O2 with a Pd catalyst before entering the bioreactor. The batch process performance of C. carboxidivorans in a stirred-tank bioreactor with continuous gassing of purified biogenic syngas was identical to an artificial syngas mixture of the pure gases CO, CO2, H2, and N2 within the estimation error. The alcohol production by C. autoethanogenum was even improved with the purified biogenic syngas compared to reference batch processes with the corresponding artificial syngas mixture. Both acetogens have proven their potential for successful fermentation processes with biogenic syngas, but full carbon conversion to ethanol is challenging with the investigated biogenic syngas. [ABSTRACT FROM AUTHOR]

Abstract Background Clostridium carboxidivorans P7 is capable of producing ethanol and butanol from inexpensive and non-food feedstock, such as syngas. Achieving improved ethanol and butanol production in the strain for industrial application depends on the energetics and biomass, especially ATP availability. Results This study found that exogenous addition of citrulline promoted accumulation of ATP, increased specific growth rate, and reduced the doubling time of C. carboxidivorans P7. In heterotrophic fermentation experiments, the addition of citrulline increased intracellular ATP by 3.39-fold, significantly enhancing the production of total alcohol (ethanol + butanol) by 20%. Moreover, in the syngas fermentation experiments, the addition of citrulline improved the level of intracellular ATP and the biomass by 80.5% and 31.6%, respectively, resulting in an 18.6% and 60.3% increase in ethanol and the alcohol/acid production ratio, respectively. Conclusions This is the first report that citrulline could promote the growth of C. carboxidivorans P7 and increase the level of intracellular ATP, which is of great significance for the use of C. carboxidivorans P7 to synthesize biofuels.

Energy consumption places growing demands on modern lifestyles, which have direct impacts on the world’s natural environment. To attain the levels of sustainability required to avoid further consequences of changes in the climate, alternatives for sustainable production not only of energy but also materials and chemicals must be pursued. In this respect, syngas fermentation has recently attracted much attention, particularly from industries responsible for high levels of greenhouse gas emissions. Syngas can be obtained by thermochemical conversion of biomass, animal waste, coal, municipal solid wastes and other carbonaceous materials, and its composition depends on biomass properties and gasification conditions. It is defined as a gaseous mixture of CO and H2 but, depending on those parameters, it can also contain CO2, CH4 and secondary components, such as tar, oxygen and nitrogenous compounds. Even so, raw syngas can be used by anaerobic bacteria to produce biofuels (ethanol, butanol, etc.) and biochemicals (acetic acid, butyric acid, etc.). This review updates recent work on the influence of biomass properties and gasification parameters on syngas composition and details the influence of these secondary components and CO/H2 molar ratio on microbial metabolism and product formation. Moreover, the main challenges, opportunities and current developments in syngas fermentation are highlighted in this review.

[Display omitted] • The harvesting efficiency achieved 99.2 % with the application of 60 g/L of FMT@DTAB. • PVDF integrated with extractants demonstrated enhanced butyric acid mass transfer. • Butyric acid showed improved mass transfer coefficient in PVDF with extractants. • PVDF-TDA achieved a 60 % recovery efficiency for butyric acid with 60 g/L of FMT@DTAB. Syngas fermentation utilizing acetogenic bacteria like Clostridium sp. provides a promising method for transforming CO and CO 2 -rich waste gases into valuable products such as short-chain fatty acids (SCFAs) and bio-alcohols, aiding in the reduction of greenhouse gas emissions and supporting carbon neutrality objectives. Magnetic nanoparticle-based coagulants, particularly Fe 3 O 4 @MIL-100(Fe)@TEOS@DTAB (FMT@DTAB), have recently attracted attention due to their efficient recovery and enhanced cell disruption capabilities enabled by cationic surfactant surface modifications. At a dosage of 60 g/L, FMT@DTAB has proven highly effective in achieving significant concentrations of acetic acid (7.06 g/L), butyric acid (6.27 g/L), ethanol (6.43 g/L), and butanol (5.24 g/L), along with notable harvesting efficiency (99.2 %) and intracellular ATP concentration (2.1 mM). Recent research on supported liquid membrane contactors highlights their cost-effective and environment-friendly properties, with an emphasis on minimal extractant usage. This study investigated the behavior of SCFAs using both virgin and supported liquid membrane contactors, focusing on factors such as organic extractant and membrane pore size. PVDF filled with tridodecylamine notably improved butyric acid recovery to around 60 %, with a mass flux of 14.95 ± 0.28 g/m2/h, outperforming virgin and other extractant-filled PVDF membranes. This study enhances resource efficiency and reduces industrial environmental impacts by optimizing the recovery and production of valuable chemicals from waste gases. It supports sustainable and economically viable biotechnology applications, aligning with global climate change mitigation efforts. [ABSTRACT FROM AUTHOR]

Background: The replacement of fossil fuels and petrochemicals with sustainable alternatives is necessary to mitigate the effects of climate change and also to counteract diminishing fossil resources. Acetogenic microorganisms such as Clostridium spp. are promising sources of fuels and basic chemical precursors because they efficiently utilize CO and CO2 as carbon source. However the conversion into high titers of butanol and hexanol is challenging. Results: Using a metabolic engineering approach we transferred a 17.9-kb gene cluster via conjugation, containing 13 genes from C. kluyveri and C. acetobutylicum for butanol and hexanol biosynthesis, into C. ljungdahlii. Plasmid-based expression resulted in 1075 mg L−1 butanol and 133 mg L−1 hexanol from fructose in complex medium, and 174 mg L−1 butanol and 15 mg L−1 hexanol from gaseous substrate (20% CO2 and 80% H2) in minimal medium. Product formation was increased by the genomic integration of the heterologous gene cluster. We confirmed the expression of all 13 enzymes by targeted proteomics and identified potential rate-limiting steps. Then, we removed the first-round selection marker using CRISPR/Cas9 and integrated an additional 7.8 kb gene cluster comprising 6 genes from C. carboxidivorans. This led to a significant increase in the hexanol titer (251 mg L−1) at the expense of butanol (158 mg L−1), when grown on CO2 and H2 in serum bottles. Fermentation of this strain at 2-L scale produced 109 mg L−1 butanol and 393 mg L−1 hexanol. Conclusions: We thus confirmed the function of the butanol/hexanol biosynthesis genes and achieved hexanol biosynthesis in the syngas-fermenting species C. ljungdahlii for the first time, reaching the levels produced naturally by C. carboxidivorans. The genomic integration strain produced hexanol without selection and is therefore suitable for continuous fermentation processes. [ABSTRACT FROM AUTHOR]

Waste biomass can be converted to green fuels and value-added products via thermochemical and biological conversion processes. The thermochemical processes endure limitations such as high processing costs due to high-temperature requirements. In contrast, the challenges of biological processes include low product yield and long processing time. Integrating different technologies, especially thermochemical and biological conversion processes, helps to enhance resource utilization and promote a circular economy. The combination of different technologies would help alleviate their limitations. In this respect, the integration of biological processes (e.g., syngas fermentation and anaerobic digestion) and thermochemical processes (e.g., pyrolysis, gasification, hydrothermal carbonization etc.) was the focus of this review. Integrated conversion processes often reduce the environmental impact compared to a standalone process. Hybrid pyrolysis-anaerobic digestion processes are promising from economics and ecological perspectives. However, more studies are required to understand how to effectively recycle and utilize the residue from pyrolysis and other thermochemical processes. Hydrothermal liquefaction (HTL) and pyrolysis are promising bio-oil production. However, HTL-derived oils are characterized by higher heating values and lower oxygen contents. The maturity level of most biological processes for waste biomass valorization is in the range of technology readiness level (TRL) 4-5. In contrast, thermochemical processes are expected to reach a TRL of 9 in the next two decades through detailed research and development. Moreover, the TRL of integrated processes described in the present study should also be assessed to evaluate their maturity and commercialization potential. The study will help researchers and policymakers to identify the knowledge gaps in integrating thermochemical and biological conversion processes. Motivation and novelty statement: The present review is the first of its endeavor to present the advances, progress, and prospects of combining thermochemical and biological conversion processes for biofuels and value-added chemicals production. Most of the studies available in the literature focus on the advances in thermochemical or biological processes as a standalone conversion pathway despite many possible integration scenarios available. Therein lies the motivation for the present study. The current review is helpful for new researchers and policymakers to acquire the fundamental knowledge and possible pathways to unlock full biomass conversion pathways. Therefore, the authors made a significant effort to explain each conversion pathway in the first few sections before outlining their advantages and limitation. The final section was entirely on the different possible integration scenarios followed by process integration challenges. This study aims to open up the possibility of future research and help researchers identify the knowledge gaps in integrating thermochemical and biological conversion processes. [ABSTRACT FROM AUTHOR]

Autotrophic syngas fermentation with clostridia enables the conversion of CO, CO2, and H2 into organic acids and alcohols. The batch process performance of Clostridium ragsdalei was studied in fully controlled and continuously gassed (600 mbar CO, 200 mbar H2, 200 mbar CO2) stirred-tank bioreactors. The final ethanol concentration varied as function of the reaction conditions. Decreasing the pH from pH 6.0–5.5 at a temperature of 37 °C increased the ethanol concentration from 2.33 g L−1 to 3.95 g L−1, whereas lowering the temperature from 37 to 32 °C at constant pH 6.0 resulted in a final ethanol concentration of 5.34 g L−1 after 5 days of batch operation. The sulphur availability was monitored by measuring the cysteine concentration in the medium and the H2S fraction in the exhaust gas. It was found that most of the initially added sulphur was stripped out within the first day of the batch process (first half of the exponential growth phase). A continuous sodium sulfide feed allowed ethanol concentrations to increase more than threefold to 7.67 g L−1 and the alcohol-to-acetate ratio to increase 43-fold to 17.71 g g−1. [ABSTRACT FROM AUTHOR]