Production Of Medium-chain Fatty Acids Research Articles

Lactate-mediated medium-chain fatty acid production from expired dairy and beverage waste

Fruits, vegetables, and dairy products are typically the primary sources of household food waste. Currently, anaerobic digestion is the most used bioprocess for the treatment of food waste with concomitant generation of biogas. However, to achieve a circular carbon economy, the organics in food waste should be converted to new chemicals with higher value than energy. Here we demonstrate the feasibility of medium-chain carboxylic acid (MCCA) production from expired dairy and beverage waste via a chain elongation platform mediated by lactate. In a two-stage fermentation process, the first stage with optimized operational conditions, including varying temperatures and organic loading rates, transformed expired dairy and beverage waste into lactate at a concentration higher than 900 mM C at 43 °C. This lactate was then used to produce >500 mM C caproate and >300 mM C butyrate via microbial chain elongation. Predominantly, lactate-producing microbes such as Lactobacillus and Lacticaseibacillus were regulated by temperature and could be highly enriched under mesophilic conditions in the first-stage reactor. In the second-stage chain elongation reactor, the dominating microbes were primarily from the genera Megasphaera and Caproiciproducens, shaped by varying feed and inoculum sources. Co-occurrence network analysis revealed positive correlations among species from the genera Caproiciproducens, Ruminococcus, and CAG-352, as well as Megasphaera, Bacteroides, and Solobacterium, indicating strong microbial interactions that enhance caproate production. These findings suggest that producing MCCAs from expired dairy and beverage waste via lactate-mediated chain elongation is a viable method for sustainable waste management and could serve as a chemical production platform in the context of building a circular bioeconomy.

Tandem Acidic CO2 Electrolysis Coupled with Syngas Fermentation: A Two-Stage Process for Producing Medium-Chain Fatty Acids.

The tandem application of CO2 electrolysis with syngas fermentation holds promise for achieving heightened production rates and improved product quality. However, the significant impact of syngas composition on mixed culture-based microbial chain elongation remains unclear. Additionally, effective methods for generating syngas with an adjustable composition from acidic CO2 electrolysis are currently lacking. This study successfully demonstrated the production of medium-chain fatty acids from CO2 through tandem acidic electrolysis with syngas fermentation. CO could serve as the sole energy source or as the electron donor (when cofed with acetate) for caproate generation. Furthermore, the results of gas diffusion electrode structure engineering highlighted that the use of carbon black, either alone or in combination with graphite, enabled consistent syngas generation with an adjustable composition from acidic CO2 electrolysis (pH 1). The carbon black layer significantly improved the CO selectivity, increasing from 0% to 43.5% (0.05 M K+) and further to 92.4% (0.5 M K+). This enhancement in performance was attributed to the promotion of K+ accumulation, stabilizing catalytically active sites, rather than creating a localized alkaline environment for CO2-to-CO conversion. This research contributes to the advancement of hybrid technology for sustainable CO2 reduction and chemical production.

Improved production of medium-chain fatty acids by biochar: Effect of biochar dosage and mechanism

Chain elongation (CE) is a promising technology that can produce medium-chain fatty acids (MCFAs) from sewage sludge, reaching the dual benefits of waste management and clean energy production. The feasibility of using biochar to enhance CE efficiency was examined in this study, where the underlying promoting mechanisms and optimal biochar dosage were also simultaneously explored. Biochar improved the carbon conversion and electron transfer efficiency of CE. The highest caproate concentration (3347.95 mg/L) occurred in the group with 10 g/L biochar addition, and caprylate was most accumulated (612.36 mg/L) when biochar dosage was 20 g/L. The electroactive genera positively related to MCFAs (e.g., norank_f__JG30-KF-CM45 and Acetobacterium) were noticeably enriched by low dosage, but the acidification genera proliferated better under excessive dosage. The different MCFAs production induced by varied biochar dosages was closely related to the genera Dysgonomonadaceae. The strengthened dominant CE pathways, FAB, might account for the promoted MCFAs production.

Research on the Resource Recovery of Medium-Chain Fatty Acids from Municipal Sludge: Current State and Future Prospects.

The production of municipal sludge is steadily increasing in line with the production of sewage. A wealth of organic contaminants, including nutrients and energy, are present in municipal sludge. Anaerobic fermentation can be used to extract useful resources from sludge, producing hydrogen, methane, short-chain fatty acids, and, via further chain elongation, medium-chain fatty acids. By comparing the economic and use values of these retrieved resources, it is concluded that a high-value resource transformation of municipal sludge can be achieved via the production of medium-chain fatty acids using anaerobic fermentation, which is a hotspot for future research. In this study, the selection of the pretreatment method, the method of producing medium-chain fatty acids, the influence of the electron donor, and the technique used to enhance product synthesis in the anaerobic fermentation process are introduced in detail. The study outlines potential future research directions for medium-chain fatty acid production using municipal sludge. These acids could serve as a starting point for investigating other uses for municipal sludge.

Influence of adding strategy of biochar on medium-chain fatty acids production from sewage sludge

Production of medium-chain fatty acids (MCFAs) from sewage sludge has dual effects on valuable sludge disposal and renewable energy generation, while low efficiency limits its application. Biochar addition is considered an effective method to improve MCFAs production. In this study, the influence of biochar adding strategies (i.e., adding biochar in acidification or chain elongation (CE) processes) on MCFAs production was explored. Results showed that by adding biochar in the acidification process, MCFAs accumulation increased by over 114%, accompanied by the highest carbon conversion efficiency (134.66%) and electron transfer efficiency of MCFAs (94.22%) by the terminal CE. Adding biochar before the acidification process better enriched CE bacteria (e.g., Paraclostridium) and strengthened the dominant metabolic pathway. In contrast, the biochar added before the CE process priorly enriched the bacteria capable of degrading organics, like unclassified_f__Dysgonomonadaceae, norank_f__norank_o__OPB41, and Acetobacterium. The differences in excessive ethanol oxidation and short-chain fatty acids accumulation induced by varied adding strategies might be responsible for this.

Enhancement of medium-chain fatty acids production from sludge anaerobic fermentation liquid under moderate sulfate reduction

In recent years, there has been a marked increase in the production of excess sludge. Chain-elongation (CE) fermentation presents a promising approach for carbon resource recovery from sludge, enabling the transformation of carbon into medium-chain fatty acids (MCFAs). However, the impact of sulfate, commonly presents in sludge, on the CE process remains largely unexplored. In this study, batch tests for CE process of sludge anaerobic fermentation liquid (SAFL) under different SCOD/SO42− ratios were performed. The moderate sulfate reduction under the optimum SCOD/SO42− of 20:1 enhanced the n-caproate production, giving the maximum n-caproate concentration, selectivity and production rate of 5.49 g COD/L, 21.4% and 4.87 g COD/L/d, respectively. The excessive sulfate reduction under SCOD/SO42− ≤ 5 completely inhibited the CE process, resulting in almost no n-caproate generation. The variations in n-caproate production under different conditions of SCOD/SO42− were all well fitted with the modified Gompertz kinetic model. Alcaligenes and Ruminococcaceae_UCG-014 were the dominant genus-level biomarkers under moderate sulfate reduction (SCOD/SO42− = 20), which enhanced the n-caproate production by increasing the generation of acetyl-CoA and the hydrolysis of difficult biodegradable substances in SAFL. The findings presented in this work elucidate a strategy and provide a theoretical framework for the further enhancement of MCFAs production from excess sludge.

Metabolic engineering of Saccharomyces cerevisiae for de novo production of odd-numbered medium-chain fatty acids

Odd-numbered fatty acids (FAs) have been widely used in nutrition, agriculture, and chemical industries. Recently, some studies showed that they could be produced from bacteria or yeast, but the products are almost exclusively odd-numbered long-chain FAs. Here we report the design and construction of two biosynthetic pathways in Saccharomyces cerevisiae for de novo production of odd-numbered medium-chain fatty acids (OMFAs) via ricinoleic acid and 10-hydroxystearic acid, respectively. The production of OMFAs was enabled by introducing a hydroxy fatty acid cleavage pathway, including an alcohol dehydrogenase from Micrococcus luteus, a Baeyer-Villiger monooxygenase from Pseudomonas putida, and a lipase from Pseudomonas fluorescens. These OMFA biosynthetic pathways were optimized by eliminating the rate-limiting step, generating heptanoic acid, 11-hydroxyundec-9-enoic acid, nonanoic acid, and 9-hydroxynonanoic acid at 7.83 mg/L, 9.68 mg/L, 9.43 mg/L and 13.48 mg/L, respectively. This work demonstrates the biological production of OMFAs in a sustainable manner in S. cerevisiae.

Metagenomic analysis towards understanding the effects of ammonia on chain elongation process for medium chain fatty acids production

The production of medium chain fatty acids (MCFAs) through chain elongation (CE) from organic wastes/wastewater has attracted much attention, while the effects of a common inhibitor-ammonia has not been elucidated. The mechanism of ammonia affecting CE was studied by metagenomic. The lag phase duration of caproate production was increased, and the maximum caproate production rate was decreased by 43.4 % at 4 g-N/L, as compared to 0 g-N/L. And hydrochar (HC) alleviated the inhibition of ammonia at 4 g-N/L. Metagenomic analysis indicated that ammonia induced UBA4085 sp.FDU78 as the dominant microorganism, and metabolic reconstruction revealed its potential CE ability. Furthermore, ammonia inhibited the reverse β oxidation pathway and Acetyl-CoA production pathway. The tolerance of UBA4085 sp.FDU78 to ammonia was associated with the uptake of inorganic ions, energy conservation, and synthesis of osmoprotectants. The present study provided a deep-insight on the ammonia tolerance mechanism on the CE process.

Novel anaerobic fermentation paradigm of producing medium-chain fatty acids from food wastes with self-produced ethanol as electron donor

As food waste (FW) is a largely available and carbon-rich feedstock, bulk production of medium-chain fatty acids (MCFAs) via chain elongation (CE) from FW is of great interest to biotechnology. However, the development of this emerging biotechnology is limited by the high cost from external electron donor (ED) input. To solve this bottleneck, replacing external ED input with self-produced ED in the system is a key. Therefore, this study provided a novel anaerobic fermentation paradigm of inoculating yeast to internally generate ethanol from FW as ED for subsequent MCFAs production. Batch experimental results demonstrated that cumulative 1540 mg COD/L of MCFAs was produced from FW with endogenous ethanol when inoculating 4.50 × 107 cells/ml-FW Saccharomyces cerevisiae in the fermenter over 20 days. In contrast, only a very small amount of MCFAs were detected in the control (58.98 mg COD/L). In continuous operation over 61 days, around 1300 mg COD/L of MCFAs was steadily obtained in the long-term fermenter with yeast assisted, which was almost 1.49 times higher than that from the control. The enriching S. cerevisiae responsible for on-site ethanol generation and chain-elongating bacteria including Caproiciproducens and Oscillibacter jointly promoted FW-derived MCFAs productions. Higher abundance of genes encoding substrate degradation, ethanol biosynthesis and CE further ensured the higher MCFAs production in the yeast-assisted system. This biotechnology does not require major changes to the design and operation of existing anaerobic fermentation infrastructure. Instead, the utilization of FW was enlarged through producing high-value MCFAs while waiving the cost of external ED by using inexpensive cheap yeast.

Urine pretreatment enhances energy recovery by boosting medium-chain fatty acids production from waste activate sludge through anaerobic fermentation

Medium-chain fatty acids (MCFAs) represent a promising form of energy recovered from waste activated sludge (WAS) through anaerobic fermentation. However, the low WAS degradability hindered the MCFAs generation from WAS. In this study, urine pretreatment of 5 – 25 wt% was applied to enhance the MCFAs production in anerobic WAS fermentation with ethanol as an electron donor. Urine pretreatment (5 – 25 wt%) dose-dependently improved the MCFAs production by 0.8 – 3.3 times (from 2.4 to 4.4 – 12.8 g chemical oxygen demand (COD)/L) and enhanced the MCFAs selectivity by 0.8 – 3.5 times (from 12.2 % to 21.4 % – 54.4 %). Urine pretreatment (5 – 25 wt%) improved WAS degradation by up to 54.8 % and enhanced electron transfer efficiency from substrates (i.e. WAS and ethanol) to MCFAs by up to 18.6 %, which contributed to the improvement in MCFAs production. According to model-based analysis, the MCFAs production potential (Pm) rose from 2.71 ± 0.09 g COD/L with 0 % urine pretreatment to 4.51 – 13.11 g COD/L with 5 – 25 wt% urine pretreatment, while the lag phase (λ) decreased from 5.30 d to 4.67 – 3.08 d, which may be the reason for the improved MCFAs production. Additionally, urine pretreatment significantly facilitated each step involved in the MCFAs generation, i.e. solubilization, hydrolysis, acidification, and chain elongation, by up to 600 %, 51 %, 17 %, and 42 %, respectively. This study for the first time reported an innovative method, urine pretreatment, enhances energy recovery by MCFAs from WAS through anaerobic fermentation, which potentially brings wastewater treatment plants economic and environmental profits.

Optimizing continuous medium-chain fatty acid production from biohydrogenic palm oil mill effluent: Operational parameters and microbial dynamics

Palm oil mill effluent (POME) was dark fermented into biohydrogen for clean energy production with volatile fatty acids (VFAs) as a byproduct. The VFAs remaining 70% in hydrogenic POME offer potential substrate for conversion into valuable medium-chain fatty acids (MCFA), presenting an opportunity for resource recovery and environmental mitigation. This study investigated the influence of varying operating parameters on continuous MCFA production from biohydrogenic POME through a stirred tank reactor. Optimizing MCFA production necessitates a 5.4 g/L ethanol load supplemented with 2.6 g/L NaHCO3 and 1.5 L-CO2/L/d while maintaining a pH of 6.5 and a temperature of 35 °C. Under optimized conditions, a maximum MCFA production of 2.2 g-COD/L was achieved, consisting primarily of 1.8 g-COD/L caproic acid, 0.2 g-COD/L heptanoic acid, and 0.2 g-COD/L caprylic acid, translating to a total MCFA yield of 0.5 g-COD/g-COD and MCFA selectivity of 51% with Caproiciproducens sp. (45%), Lactobacillus sp. (21%), and Clostridium sp. (14%) as predominant bacteria. Notably, CO2 loading significantly enhances MCFA production, particularly caproic and heptanoic acids. Remarkably, the system maintained stable MCFA production for an impressive 220 days. The microbial community in the reactor effectively removed total solids, volatile solids, chemical oxygen demand (COD), suspended solids of 42%, 61%, 31%, and 95%, respectively. Key bacteria like Caproiciproducens sp., C. kluyveri, Clostridium sp., and Sporanaerobacter sp. possess most of the enzymes required for the reverse β-oxidation pathway, an energy-efficient pathway for MCFA generation. This study makes a significant contribution to the field of biowaste valorization, specifically focusing on anaerobic fermentation for bio-based chemical production. Despite challenges related to butyric acid accumulation and inhibition, these findings offer promising potential for scaling up the fermentation process.

Integrated biogas upgrading and medium-chain fatty acids production for more efficient resource recovery

The conversion of carbon dioxide (CO2) from biogas into medium-chain fatty acids (MCFAs) represents an eco-friendly resource recovery approach to reduce dependence on fossil fuels and combat global climate change. This study presented the novel concept of integrated resource recovery by coupling biogas upgrading and MCFAs production for the first time. Initially, the impact of different initial ethanol concentrations on chain elongation was examined, determining that an ethanol concentration of 160 mmol/L maximized MCFAs yield at 45.7 mmol/L. Subsequently, using this optimal ethanol supply, the integrated strategy was implemented by connecting two bioreactors in series and maintaining continuous operation for 160-day. The results were noteworthy: upgraded bio-methane purity reached 97.6 %, MCFAs production rate and carbon-flow distribution reached 24.5 mmol/L d−1 and 76.1 %, respectively. In summary, these promising outcomes pioneer a resource recovery approach, enabling the high-value utilization of biogas and the conversion of CO2 into valuable bio-chemicals.

Promotion of medium-chain fatty acids and long-chain alcohols production by zero-valent iron: Effect of particle sizes

The effect of zero-valent iron (ZVI) particle size on biological chain elongation (CE) was explored. The addition of ZVI particles with diameters of 50 nm-1000 µm stimulated both medium-chain fatty acids (MCFAs) and long-chain alcohols (LCAs) production, and increased carbon conversion rate of CE process by 2.8–12.2 times. ZVI particle size of 75 µm exhibited the best performance in promoting MCFAs production, achieved the highest MCFAs production of 2164.2 mg/L, which was 11.9 times of control group. ZVI particle size of 1000 µm showed the best performance in stimulating LCAs production, obtained the highest LCAs production of 434.0 mg/L, which was 33.4 times of the control group. Microbial community analysis suggested that different ZVI particle sizes significantly affected genus Clostridium_sensu_stricto_12 and Clostridium_sensu_stricto_7. Functional enzyme analysis revealed that 75 µm ZVI particles induced the strengthened reverse β oxidation pathway, while 1000 µm ZVI particles enhanced the fatty acid biosynthesis pathway.

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.

Peracetic acid (PAA)-based pretreatment effectively improves medium-chain fatty acids (MCFAs) production from sewage sludge

Peracetic acid (PAA), known for its environmentally friendly properties as a oxidant and bactericide, is gaining prominence in decontamination and disinfection applications. The primary product of PAA oxidation is acetate that can serve as an electron acceptor (EA) for the biosynthesis of medium-chain fatty acids (MCFAs) via chain elongation (CE) reactions. Hence, PAA-based pretreatment is supposed to be beneficial for MCFAs production from anaerobic sludge fermentation, as it could enhance organic matter availability, suppress competing microorganisms and furnish EA by providing acetate. However, such a hypothesis has rarely been proved. Here we reveal that PAA-based pretreatment leads to significant exfoliation of extracellular polymeric substances (EPS) from sludge flocs and disruption of proteinic secondary structures, through inducing highly active free radicals and singlet oxygen. The production of MCFAs increases substantially to 11,265.6 mg COD L−1, while the undesired byproducts, specifically long-chain alcohols (LCAs), decrease to 723.5 mg COD L−1. Microbial activity tests further demonstrate that PAA pretreatment stimulates the CE process, attributed to the up-regulation of functional genes involved in fatty acid biosynthesis pathway. These comprehensive findings provide insights into the effectiveness and mechanisms behind enhanced MCFAs production through PAA-based technology, advancing our understanding of sustainable resource recovery from sewage sludge.

Different Electron Donors Drive the Variation in the Performance of Medium-Chain Fatty Acid Production from Waste-Activated Sludge

Different Electron Donors Drive the Variation in the Performance of Medium-Chain Fatty Acid Production from Waste-Activated Sludge

Engineering Escherichia coli for cost-effective production of medium-chain fatty acids from soy whey using an optimized galactose-based autoinduction system

Medium-chain fatty acids (MCFAs) are essential chemical feedstocks. Microbial production of MCFAs offers an attractive alternative to conventional methods, but the costly media and external inducers limit its practical application. To address this issue and make MCFA production more cost-effective, an E.coli platform was developed using soy whey as a medium and galactose as an autoinducer. We first designed an efficient, stringent, homogeneous, and robust galactose-based autoinduction system for the expression of pathway enzymes by rationally engineering the promoter of the galactose-proton symporter (GalP). Subsequently, the intracellular acetyl-CoA availability and NADH regeneration were enhanced to improve the reversal of the β-oxidation cycle. The resulting strain yielded 8.20 g/L and 16.42 g/L MCFA in pH-controlled batch fermentation and fed-batch fermentation with glucose added using soy whey as medium, respectively. This study provided a cost-effective and promising platform for MCFA production, as well as future strain development for other value-added chemicals production.

Sodium percarbonate effectively enhances medium-chain fatty acids production from waste activated sludge via facilitating electron acceptor formation, providing inorganic carbon and enriching functional microorganisms

Medium-chain fatty acids (MCFA) production from waste activated sludge (WAS) has been a promising approach for resource and energy recovery. However, the poor sludge biodegradability and existence of competitive microorganisms result in inefficient electron donor utilization and low MCFA output. Therefore, a percarbonate (SPC)-based strategy is proposed to effectively enhance MCFA production. SPC pretreatment largely boosts substrate accessibility and organics transformation through the dual function of chemical oxidation and alkaline hydrolysis in the acidogenic fermentation stage, providing abundant electron acceptors directly available for functional microorganisms in the chain elongation (CE) stage. Consequently, MCFA production is significantly enhanced from 3522 mg COD/L of the control to 10,437 mg COD/L of 0.15 g SPC/g TSS, while decreased to 1898 mg COD/L at 0.25 g SPC/g TSS. Meanwhile, long-chain alcohols (LCA) production is apparently reduced by 68.3%, implying a decrease in the flow of electrons to byproducts. Importantly, SPC pretreatment with an appropriate level can provide inorganic carbon source for CE microbes to further increase the MCFA yield. Mechanistic studies suggest that SPC pretreatment with appropriate levels improves microbial metabolisms contributing to MCFA production and functional gene expression involved in CE pathways. Additionally, SPC enriches functional microorganisms from 19.25% to 59.75%, such as MCFA (i.e., Proteiniclasticum sp.) and butyrate (i.e., Romboutsia sp. and Alkaliphilus sp.) producers.

Permanganate (PM) pretreatment improves medium-chain fatty acids production from sewage sludge: The role of PM oxidation and in-situ formed manganese dioxide

Medium-chain fatty acids (MCFAs) production from sewage sludge is mainly restricted by the complex substrate structure, competitive metabolism and low electron transfer rate. This study proposes a novel permanganate (PM)-based strategy to promote sludge degradation and MCFAs production. Results show that PM pretreatment significantly increases MCFAs production, i.e., attaining 12,036 mg COD/L, and decreases the carbon fluxes of electron acceptor (EA)/electron donor (ED) to byproducts. Further analysis reveals that PM oxidation enhances the release and biochemical conversion of organic components via disrupting extracellular polymers (EPS) structure and reducing viable cells ratio, providing directly available EA for chain elongation (CE). The microbial activity positively correlated with MCFAs generation are apparently heightened, while the competitive metabolism of CE (i.e., methanogensis) can be completely inhibited. Accordingly, the functional bacteria related to critical bio-steps and dissimilatory manganese reduction are largely enriched. Further mechanism exploration indicates that the main contributors for sludge solubilization are 1O2 (61.6 %) and reactive manganese species (RMnS), i.e., Mn(V)/Mn(VI) (22.3 %) and Mn(III) (∼16.1 %). As the main reducing product of PM reaction, manganese dioxide (MnO2) can enable the formation of microbial aggregates, and serve as electron shuttles to facilitate the carbon fluxes to MCFAs during CE process. Overall, this strategy can achieve simultaneous hydrogen recovery, weaken competitive metabolisms and provide electron transfer accelerator for CE reactions.

Simultaneous biogas upgrading and medium-chain fatty acids production using a dual membrane biofilm reactor

Utilizing H2-assisted ex-situ biogas upgrading and acetate recovery holds great promise for achieving high value utilization of biogas. However, it faces a significant challenge due to acetate's high solubility and limited economic value. To address this challenge, we propose an innovative strategy for simultaneous upgrading of biogas and the production of medium-chain fatty acids (MCFAs). A series of batch tests evaluated the strategy's efficiency under varying initial gas ratios (v/v) of H2, CH4, CO2, along with varying ethanol concentrations. The results identified the optimal conditions as initial gas ratios of 3H2:3CH4:2CO2 and an ethanol concentration of 241.2 mmol L−1, leading to maximum CH4 purity (97.2 %), MCFAs yield (54.2 ± 2.1 mmol L−1), and MCFAs carbon-flow distribution (62.3 %). Additionally, an analysis of the microbial community's response to varying conditions highlighted the crucial roles played by microorganisms such as Clostridium, Proteiniphilum, Sporanaerobacter, and Bacteroides in synergistically assimilating H2 and CO2 for MCFAs production. Furthermore, a 160-day continuous operation using a dual-membrane aerated biofilm reactor (dMBfR) was conducted. Remarkable achievements were made at a hydraulic retention time of 2 days, including an upgraded CH4 content of 96.4 ± 0.3 %, ethanol utilization ratio (URethanol) of 95.7 %, MCFAs production rate of 28.8 ± 0.3 mmol L−1 d−1, and MCFAs carbon-flow distribution of 70 ± 0.8 %. This enhancement is proved to be an efficient in biogas upgrading and MCFAs production. These results lay the foundation for maximizing the value of biogas, reducing CO2 emissions, and providing valuable insights into resource recovery.