Comprehensive comparative study on n-caproate production by Clostridium kluyveri: batch vs. continuous operation modes.

Recently, there has been notable interest in researching and industrially producing medium-chain carboxylic acids (MCCAs) like n-caproate and n-caprylate via chain elongation process. This study presents a comprehensive assessment of the behavior and MCCA production profiles of Clostridium kluyveri in batch and continuous modes, at different ethanol:acetate molar ratios (1.5:1, 3.5:1 and 5.5:1). The highest n-caproate concentration, 12.9 ± 0.67 g/L (92.9 ± 1.39 % MCCA selectivity), was achieved in batch mode at a 3.5:1 ratio. Interestingly, higher ratios favored batch mode selectivity over continuous mode when this was equal or higher to 3.5:1. Steady state operation yielded the highest n-caproate (9.5 ± 0.13 g/L) and n-caprylate (0.35 ± 0.020 g/L) concentrations at the 3.5:1 ratio. Increased ethanol:acetate ratios led to a higher excessive ethanol oxidation (EEO) in both operational modes, potentially limiting n-caproate production and selectivity, especially at the 5.5:1 ratio. Overall, this study reports the efficient MCCA production of both batch and continuous modes by C. kluyveri.

Enhancing bacterial biodegradation of n-hexane by utilizing the adsorption capacity of non-degrading fungi.

Biodegradation of hydrophobic volatile organic compounds (VOCs) such as n-hexane is limited by their poor accessibility. Constructing fungal-bacterial degradation alliances is an effective approach, but the role of those fungi without the capability to degrade VOCs may have been overlooked. In this study, a non-n-hexane-degrading fungus, Fusarium keratoplasticum FK, was utilized to enhance n-hexane degradation by the bacterium Mycobacterium neworleansense WCJ. It was shown that strain WCJ removed 64.84% of n-hexane (at a concentration of 648.20 mg L-1) over 3 d, and 84.04% after introducing strain FK. Microbial growth kinetic studies revealed that the growth of strain WCJ was also promoted. Through a stepwise adsorption-degradation experiment combined with qPCR technology, it was found that the strain WCJ could utilize the n-hexane pre-adsorbed by strain FK, with an increase in copy number from 108.2662 to 108.7731. Therefore, the non-degrading fungi can improved the accessibility of n-hexane by providing n-hexane adsorbed by the mycelium to the degrading bacteria. In addition, the adsorption tests and characterization of the fungal samples before and after Soxhlet extraction indicated that the adsorption of n-hexane on strain FK conformed to Lagergren's pseudo-second-order kinetics and Freundlich adsorption isotherms, and was correlated with the presence of lipids and nonpolar groups. This study emphasizes the potential role of non-degrading fungi in bioremediation and proposes a viable strategy to enhance the bacterial degradation of hydrophobic VOCs.

Optimization of methane production from solid tuna waste: Thermal pretreatment and co-digestion.

Fish canning industries generate large amounts of solid waste during their processing operations, creating a significant environmental challenge. Nonetheless, this waste can be efficiently and sustainably treated through anaerobic digestion. In this study, the potential of biogas production from anaerobic digestion of thermally pretreated and co-digested solid tuna waste was investigated. The thermal pretreatment of raw fish viscera resulted in a 50 % increase in methane yield, with a production of 0.27 g COD-CH4/g COD added. However, this pretreatment did not lead to a significant increase in biogas production for cooked tuna viscera. When non-thermally pretreated raw viscera was tested, a large accumulation of volatile fatty acids and long chain fatty acids was observed, with levels reaching 21 and 6 g COD/L, respectively. On the other hand, anaerobic co-digestion of cooked tuna viscera with fat waste significantly enhanced methane production, achieving 0.87 g COD-CH4/g COD added. In contrast, co-digestion of cooked tuna viscera with dairy waste and sewage sludge resulted in notably lower yields of 0.36 and 0.46 g COD-CH4/g COD added, respectively. These results may be related to the C/N ratio, which was found to be within the optimal range for anaerobic digestion only in the tuna and fat waste co-digestion assay.

Unlocking the potential of one-carbon gases (CO2, CO) for concomitant bioproduction of ß-carotene and lipids.

This study investigates the use of a Yarrowia lipolytica strain for the bioconversion of syngas-derived acetic acid into ß-carotene and lipids. A two-stage process was employed, starting with the acetogenic fermentation of syngas by Clostridium aceticum, metabolising CO, CO2, H2, to produce acetic acid, which is then utilized by Y. lipolytica for simultaneous lipid and ß-carotene synthesis. The research demonstrates that acetic acid concentration plays a pivotal role in modulating lipid profiles and enhancing ß-carotene production, with increased acetic acid consumption leading to higher yields of these compounds. This approach showcases the potential of using one-carbon gases as substrates in bioprocesses for generating valuable bioproducts, providing a sustainable and cost-effective alternative to more conventional feedstocks and substrates, such as sugars.

Carbon dioxide as key player in chain elongation and growth of Clostridium kluyveri: Insights from batch and bioreactor studies.

Chain elongation technology allows medium-chain fatty acids (MCFAs) production as an alternative to fossil resources. Clostridium kluyveri generates n-caproate primarily from ethanol and acetate, presumably requiring CO2 for growth. Here, the impact of CO2 on C. kluyveri was explored. Bottle studies revealed the bacterium's adaptability to low CO2 levels, even in conditions with minimal dissolved NaHCO3 (0.0003 M) and unfavorable pH (below 6) under 1 bar CO2. Bioreactor investigations demonstrated a direct correlation between CO2 availability and bacterial growth. The highest n-caproate production (11.0 g/L) with 90.1 % selectivity was achieved in a bioreactor with continuous CO2 supply at 3 mL/min. Additional bottle experiments pressurized with 1 bar CO2 and varying ethanol:acetate ratios (1:1, 2:1, 4:1) also confirmed CO2 consumption by C. kluyveri. However, increasing the ethanol:acetate ratio did not enhance n-caproate selectivity, likely due to overly acidic pH conditions. These findings provide insights into chain-elongators responses under diverse conditions.

Effect of electron acceptors on product selectivity and carbon flux in carbon chain elongation with Megasphaera hexanoica.

Megasphaera hexanoica is a bacterial strain following the reverse ß-oxidation pathway to synthesize caproate (CA) using lactate (LA) as an electron donor (ED) and acetate (AA) or butyrate (BA) as electron acceptors (EA). Differences in the type and concentration of EA lead to distinctions in product distribution and energy bifurcation of carbon fluxes in ED pathways, thereby affecting CA production. In this study, the effect of various ratios of AA, BA, and AA+BA as EA on carbon flux and CA specific titer during the carbon chain elongation in M. hexanoica was explored. The results indicated that the maximum levels of CA were 18.81 mM and 31.48 mM when the molar ratios of LA/AA and LA/BA were 10:1 and 3:1, respectively. Meanwhile, when AA and BA were used as combined EA (LA, AA, and BA molar amounts of 100, 23, and 77 mM), a maximum CA production of 39.45 mM was obtained. Further analysis revealed that the combined EA exhibited a CA production carbon flux of 49 % (4.3 % and 19.5 % higher compared to AA or BA, respectively) and a CA production specific titer of 45.24 mol (80.89 % and 58.51 % higher compared to AA or BA, respectively), indicating that the effective carbon utilization rate and CA production efficiency were greatly improved. Finally, a scaled-up experiment was conducted in a 1.2 L (working volume) automated bioreactor, implying high biomass (optical density at 600 nm or OD600 = 1.809) and a slight decrease in CA production (28.45 mM). A decrease in H2 production (4.11 g/m3) and an increase in CO2 production (0.632 g/m3) demonstrated the appropriate metabolic adaptation of M. hexanoica to environmental changes such as stirring shear.

Effect of pH and medium composition on chain elongation with Megasphaera hexanoica producing C4-C8 fatty acids.

Introduction: Chain elongation technology, which involves fermentation with anaerobic bacteria, has gained attention for converting short and medium chain substrates into valuable and longer-chain products like medium chain fatty acids (MCFAs). In the recent past, the focus of studies with pure chain elongating cultures was on species of other genera, mainly Clostridium kluyveri. Recently, other chain elongators have been isolated that deserve further research, such as Megasphaera hexanoica. Methods: In this study, batch studies were performed in bottles with two different media to establish the optimal conditions for growth of M. hexanoica: (a) a medium rich in different sources of nitrogen and (b) a medium whose only source of nitrogen is yeast extract. Also, batch bioreactor studies at pH values of 5.8, 6.5 and 7.2 were set up to study the fermentation of lactate (i.e., electron donor) and acetate (i.e., electron acceptor) by M. hexanoica. Results and discussion: Batch bottle studies revealed the yeast extract (YE) containing medium as the most promising in terms of production/cost ratio, producing n-caproate rapidly up to 2.62 ± 0.24 g/L. Subsequent bioreactor experiments at pH 5.8, 6.5, and 7.2 confirmed consistent production profiles, yielding C4-C8 fatty acids. A fourth bioreactor experiment at pH 6.5 and doubling both lactate and acetate concentrations enhanced MCFA production, resulting in 3.7 g/L n-caproate and 1.5 g/L n-caprylate. H2 and CO2 production was observed in all fermentations, being especially high under the increased substrate conditions. Overall, this study provides insights into M. hexanoica's behavior in lactate-based chain elongation and highlights optimization potential for improved productivity.

Integrated fermentative process for lipid and ß-carotene production from acetogenic syngas fermentation using an engineered oleaginous Yarrowia lipolytica yeast.

An engineered Yarrowia lipolytica strain was successfully employed to produce ß-carotene and lipids from acetic acid, a product of syngas fermentation by Clostridium aceticum. The strain showed acetic acid tolerance up to concentrations of 20 g/L. Flask experiments yielded a peak lipid content of 33.7 % and ß-carotene concentration of 13.6 mg/g under specific nutrient conditions. The study also investigated pH effects on production in bioreactors, revealing optimal lipid and ß-carotene contents at pH 6.0, reaching 22.9 % and 44 mg/g, respectively. Lipid profiles were consistent across experiments, with C18:1 being the dominant compound at approximately 50 %. This research underscores a green revolution in bioprocessing, showing how biocatalysts can convert syngas, a potentially polluting byproduct, into valuable ß-carotene and lipids with a Y. lipolytica strain.

Autotrophic production of polyhydroxyalkanoates using acidogenic-derived H2 and CO2 from fruit waste.

The environmental concerns regarding fossil plastics call for alternative biopolymers such as polyhydroxyalkanoates (PHAs) whose manufacturing costs are however still too elevated. Autotrophic microbes like Cupriavidus necator, able to convert CO2 and H2 into PHAs, offer an additional strategy. Typically, the preferred source for CO2 and H2 are expensive pure gases or syngas, which has toxic compounds for most PHAs-accumulating strains. In this work, for the first time, H2 and CO2 originating from an acidogenic reactor were converted autotrophically into poly(3-hydroxybutyrate) P(3HB). During the first stage, a mixed microbial community continuously catabolized melon waste into H2 (26.7 %) and CO2 (49.2 %) that were then used in a second bioreactor by C. necator DSM 545 to accumulate 1.7 g/L P(3HB). Additionally, the VFAs (13 gCOD/L) produced during acidogenesis were processed into 2.7 g/L of P(3HB-co-3HV). This is the first proof-of-concept of using acidogenic-derived H2 and CO2 from fruit waste to produce PHAs.

Sequential bioconversion of C1-gases (CO, CO2, syngas) into lipids, through the carboxylic acid platform, with Clostridium aceticum and Rhodosporidium toruloides.

Syngas (CO, CO2, H2) was effectively bioconverted into lipids in a two-stage process. In the first stage, C1-gases were bioconverted into acetic acid by the acetogenic species Clostridium aceticum through the Wood-Ljungdahl metabolic pathway in a stirred tank bioreactor, reaching a maximum acetic acid concentration of 11.5 g/L, with a production rate of 0.05 g/L·h. Throughout this experiment, samples were extracted at different periods, i.e., different concentrations, to be used in the second stage, aiming at the production of lipids from acetic acid. The yeast Rhodosporidium toruloides, inoculated in the acetogenic medium, was able to efficiently accumulate lipids from acetic acid generated in the first stage. The best results, in terms of lipid content, dry biomass, biomass yield (Y(X/S)) and lipid yield (Y(L/S)) were 39.5% g/g dry cell weight, 3 g/L, 0.35 and 0.107, respectively. In terms of abundance, the lipid profile followed the order: C18:1 > C16:0 > C18:2 > C18:0 > Others. Experiments were also performed to determine the toxicity exerted by high concentrations of acetic acid on R. toruloides, resulting in inhibition at initial acid concentrations around 18 g/L leading to a higher lag phase and being lethal to the yeast at initial acetic acid concentrations around 22 g/L and above. This research paves the way for a novel method of growing oleaginous yeasts to produce sustainable biofuels from syngas or C1-pollutant gases.

Comparative assessment of the performance of one- and two-liquid phase biotrickling filters for the simultaneous abatement of gaseous mixture of methanol, α-pinene, and hydrogen sulfide.

A gaseous mixture of methanol (M), α-pinene (P), and hydrogen sulfide (H) was treated in one/two-liquid phase biotrickling filters (OLP/TLP-BTFs) at varying inlet concentrations and at an empty bed residence time (EBRT) of 57 s. The performance of TLP-BTF [BTF (A)] improved significantly in terms of M and P removal due to the presence of silicone oil at 5% v/v. The maximum elimination capacities (ECs) of M, P, and H in BTF (A) were obtained as 309, 73, and 56 g m-3 h-1, respectively. While, the maximum ECs achieved in the BTF operated without silicone oil [BTF (B)] were 172, 28, and 21 g m-3 h-1 for M, P, and H removal, respectively. Increasing the inlet concentration of H from 32 to 337 ppm inhibited P removal in both the BTFs. The presence of silicone oil enhanced gas-liquid mass transfer, prevented the BTF from experiencing substrate inhibition effects and allowed reaching high ECs for M and P. The experiments showed promising results for the long-term operation of removal of M, P, and H mixture in a one-stage TLP-BTF with the decreasing negative effects of M and H on P degradation.

Neural network-based performance assessment of one- and two-liquid phase biotrickling filters for the removal of a waste-gas mixture containing methanol, α-pinene, and hydrogen sulfide.

The performance of one- and two-liquid phase biotrickling filters (OLP/TLP-BTFs) treating a mixture of gas-phase methanol (M), α-pinene (P), and hydrogen sulfide (H) was assessed using artificial neural network (ANN) modeling. The best ANN models with the topologies 3-9-3 and 3-10-3 demonstrated an exceptional capacity for predicting the performance of O/TLP-BTFs, with R2 > 99%. The analysis of causal index (CI) values for the model of OLP-BTF revealed a negative impact of M on P removal (CI = -2.367), a positive influence of P and H on M removal (CI = +7.536 and CI = +3.931) and a negative effect of H on P removal (CI = -1.640). The addition of silicone oil in TLP-BTF reduced the negative impact of M and H on P degradation (CI = -1.261 and CI = -1.310, respectively) compared to the OLP-BTF. These findings suggested that silicone oil had the potential to improve P availability to the biofilm by increasing the concentration gradient of P between the air/gas and aqueous phases. Multi-objective particle swarm optimization (MOPSO) suggested an optimum operational condition, i.e. inlet M, P, and H concentrations of 1.0, 1.1, and 0.3 g m-3, respectively, with elimination capacities (ECs) of 172.1, 26.5, and 0.025 g m-3 h-1 for OLP-BTF. Likewise, one of the optimum operational conditions for TLP-BTF is achievable at inlet concentrations of 4.9, 1.7, and 0.8 g m-3, leading to the optimum ECs of 299.7, 52.9, and 0.072 g m-3 h-1 for M, P, and H, respectively. These results provide important insights into the treatment of complex waste gas mixtures, addressing the interactions between the pollutant removal characteristics in OLP/TLP-BTFs and providing novel approaches in the field of biological waste gas treatment.

Production of short-chain fatty acids (SCFAs) as chemicals or substrates for microbes to obtain biochemicals.

Carboxylic acids have become interesting platform molecules in the last years due to their versatility to act as carbon sources for different microorganisms or as precursors for the chemical industry. Among carboxylic acids, short-chain fatty acids (SCFAs) such as acetic, propionic, butyric, valeric, and caproic acids can be biotechnologically produced in an anaerobic fermentation process from lignocellulose or other organic wastes of agricultural, industrial, or municipal origin. The biosynthesis of SCFAs is advantageous compared to chemical synthesis, since the latter relies on fossil-derived raw materials, expensive and toxic catalysts and harsh process conditions. This review article gives an overview on biosynthesis of SCFAs from complex waste products. Different applications of SCFAs are explored and how these acids can be considered as a source of bioproducts, aiming at the development of a circular economy. The use of SCFAs as platform molecules requires adequate concentration and separation processes that are also addressed in this review. Various microorganisms such as bacteria or oleaginous yeasts can efficiently use SCFA mixtures derived from anaerobic fermentation, an attribute that can be exploited in microbial electrolytic cells or to produce biopolymers such as microbial oils or polyhydroxyalkanoates. Promising technologies for the microbial conversion of SCFAs into bioproducts are outlined with recent examples, highlighting SCFAs as interesting platform molecules for the development of future bioeconomy.

Production of biofuels from C1 -gases with Clostridium and related bacteria-Recent advances.

Clostridium spp. are suitable for the bioconversion of C1 -gases (e.g., CO2 , CO and syngas) into different bioproducts. These products can be used as biofuels and are reviewed here, focusing on ethanol, butanol and hexanol, mainly. The production of higher alcohols (e.g., butanol and hexanol) has hardly been reviewed. Parameters affecting the optimization of the bioconversion process and bioreactor performance are addressed as well as the pathways involved in these bioconversions. New aspects, such as mixotrophy and sugar versus gas fermentation, are also reviewed. In addition, Clostridia can also produce higher alcohols from the integration of the Wood-Ljungdahl pathway and the reverse ß-oxidation pathway, which has also not yet been comprehensively reviewed. In the latter process, the acetogen uses the reducing power of CO/syngas to reduce C4 or C6 fatty acids, previously produced by a chain elongating microorganism (commonly Clostridium kluyveri), into the corresponding bioalcohol.

Accumulation of lipids by the oleaginous yeast Yarrowia lipolytica grown on carboxylic acids simulating syngas and carbon dioxide fermentation.

Volatile fatty acids (VFAs) can be considered as low-cost carbon substrates for lipid accumulation by oleaginous yeasts. This study demonstrates that a common mixture of VFAs, typically obtained from the anaerobic fermentation of C1-gases by some acetogenic bacteria, can be used in a second aerobic fermentation with the yeast Yarrowia lipolytica to obtain lipids as precursors of biodiesel. In the batch experiments, the preference of Yarrowia lipolytica W29 for acetic acid over butyric and caproic acids was demonstrated, with the highest consumption rate reaching 0.664 g/L·h. In the bioreactor experiments, the amount initial biomass inoculated, as well as the initial acid concentration, were found to have a significant influence on the process. Though the lipid content was relatively low, it can be optimized and further improved. Oleic, linoleic and palmitic acids accounted for about 80 % of the fatty acids in the lipids, which makes them suitable for biodiesel.

Enhanced solventogenesis in syngas bioconversion: Role of process parameters and thermodynamics.

Biofuels, such as ethanol and butanol, obtained from carbon monoxide-rich gas or syngas bioconversion (solventogenesis) are an attractive alternative to traditional fermentation processes with merits of no competition with food production and sustainability. However, there is a lack of comprehensive understanding of some key process parameters and mechanisms enhancing solventogenesis during the fermentation process. This review provides an overview of the current state of the art of the main influencing factors during the syngas fermentation process catalyzed by acetogenic species as well as undefined mixed cultures. The role of syngas pressure, syngas components, fermentation pH, temperature, trace metals, organic compounds and additional materials is overviewed. As a so far hardly considered approach, thermodynamic calculations of the Gibbs free energy of CO conversion to acetic acid, ethanol, butyric acid and butanol under different CO pressures and pH at 25, 33 and 55 °C are also addressed and reviewed. Strategies for enhancing mass transfer and longer carbon chain solvent production are considered as well.

Effect of Endogenous and Exogenous Butyric Acid on Butanol Production From CO by Enriched Clostridia.

Butanol is a potential renewable fuel. To increase the selectivity for butanol during CO fermentation, exogenous acetic acid and ethanol, exogenous butyric acid or endogenous butyric acid from glucose fermentation have been investigated using CO as reducing power, with a highly enriched Clostridium sludge. Addition of 3.2 g/L exogenous butyric acid led to the highest 1.9 g/L butanol concentration with a conversion efficiency of 67%. With exogenous acetate and ethanol supply, the butanol concentration reached 1.6 g/L at the end of the incubation. However, the presence of acetic acid and ethanol favoured butanol production to 2.6 g/L from exogenous butyric acid by the enriched sludge. Finally, exogenous 14 g/L butyric acid yielded the highest butanol production of 3.4 g/L, which was also among the highest butanol concentration from CO/syngas fermentation reported so far. CO addition triggered butanol production from endogenous butyric acid (produced from glucose, Glucose + N2) with as high as 58.6% conversion efficiency and 62.1% butanol yield. However, no efficient butanol production was found from glucose and CO co-fermentation (Glucose + CO), although a similar amount of endogenous butyric acid was produced compared to Glucose + N2. The Clostridium genus occupied a relative abundance as high as 82% from the initial inoculum, while the Clostridia and Bacilli classes were both enriched and dominated in Glucose + N2 and Glucose + CO incubations. This study shows that the supply of butyric acid is a possible strategy for enhancing butanol production by CO fed anaerobic sludge, either via exogenous butyric acid, or via endogenous production by sugar fermentation.

Engineering Acetobacterium woodii for the production of isopropanol and acetone from carbon dioxide and hydrogen.

The capability of four genetically modified Acetobacterium woodii strains for improved production of acetone from CO2 and hydrogen was tested. The acetone biosynthesis pathway was constructed by combining genes from Clostridium acetobutylicum and Clostridium aceticum. Expression of acetone production genes was demonstrated in all strains. In bioreactors with continuous gas supply, all produced acetic acid, acetone, and, surprisingly, isopropanol. The production of isopropanol was caused by an endogenous secondary alcohol dehydrogenase (SADH) activity at low gas-feeding rate. Although high amounts of the natural end product acetic acid of A. woodii were formed,14.5 mM isopropanol and 7.6 mM acetone were also detected, showing that this is a promising approach for the production of new solvents from C1 gases. The highest acetic acid, acetone, and isopropanol production was detected in the recombinant A. woodii [pJIR750_ac1t1] strain, with final concentrations of 438 mM acetic acid, 7.6 mM acetone, and 14.5 mM isopropanol. The engineered strain A. woodii [pJIR750_ac1t1] was found to be the most promising strain for acetone production from a gas mixture of CO2 and H2 and the formation of isopropanol in A. woodii was shown for the first time.

Influence of feedstock mix ratio on microbial dynamics during acidogenic fermentation for polyhydroxyalkanoates production.

The nature of microbial populations plays an essential role in the production of volatile fatty acids (VFA) during acidogenesis, the first stage in polyhydroxyalkanoates (PHA) production using mixed cultures. However, the composition of microbial communities is generally affected by substrate alterations. This work aimed to unravel the microbial dynamics in response to a gradual change in the feedstock composition in an acidogenic reactor, with subsequent PHA production. To achieve this, co-digestion of cheese whey and brewery wastewater (BW) was carried out for the production of VFA, in which the ratio of these feedstocks was varied by gradually increasing the proportion of BW from 0 up to 50% of the organic content. Bacteria such as Megasphaera, Bifidobacterium or Caproiciproducens were the most abundant in the first stages of the co-digestion. However, when BW reached 25% of the organic load, new taxa emerged and displaced the former ones; like Selenomonas, Ethanoligenens or an undefined member of the Bacteroidales order. Accordingly, the production of butyric acid dropped from 52 down to 27%, while the production of acetic acid increased from 36 up to 52%. Furthermore, the gradual increase of the BW ratio led to a progressive drop in the degree of acidification, from 72 down to 57%. In a subsequent approach, the VFA-rich streams, obtained from the co-digestion, were used as substrates in PHA accumulation tests. All the tests yielded similar PHA contents, but with slightly different monomeric composition. The overall results confirmed that the microbiome was altered by a gradual change in the feedstock composition and, consequently, the VFA profile and the monomeric composition of the biopolymer also did.