Production Of Medium-chain Fatty Acids Research Articles

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.

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

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. 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. 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.

Effect of inoculum source, electron donors, and substrates on medium-chain fatty acid production from palm oil mill effluent

Selecting an appropriate inoculum source and electron donor for chain elongation is crucial to optimizing medium-chain fatty acid (MCFA) production from organic waste. The investigation examined the impact of various factors, including inoculum source, electron donors, and substrate composition, on MCFA production from palm oil mill effluent (POME). The substrate containing a mixture of ethanol, lactic acid, and acetic acid exhibited the highest MCFA production, reaching 0.4–0.5 g-COD/L. Among the cultures tested, the compost (CP)-enriched culture demonstrated the highest MCFA production (0.5 g-COD/L) and was characterized by the dominance of Sporanaerobacter sp., Clostridium sp., and Oscillibacter sp. The mesophilic condition (35 °C) has higher MCFA production than the thermophilic condition (55 °C), with the mesophilic system favoring caproic acid, heptanoic acid, and caprylic acid, while the thermophilic was more suitable for propionic acid production. The highest MCFA yield obtained from POME was 0.2 g-COD/g-COD, with a selectivity of 13.6 % for MCFA production. Lactobacillus sp., Caproiciproducens sp., and Clostridium sp. were dominant microbial species and played important roles in specific MCFA production. These findings provide valuable insights for optimizing MCFA production processes from POME and highlight the importance of selecting appropriate conditions and microbial communities to enhance MCFA yields.

Redox mediators stimulated chain elongation process in fluidized cathode electro-fermentation systems for caproate production

Medium chain fatty acids (MCFAs), the secondary products of traditional anaerobic fermentation, can be produced via chain elongation (CE), a process often retarded due to the difficulty during interspecies electron transfer (IET). This study employed redox mediators, neutral red (NR), methyl viologen (MV), and methylene blue (MB) as electron shuttles to expedite the electro-fermentation for caproate production by improving IET. Results showed that MV increased the MCFAs production by promoting acetate to ethanol conversion, leading to the highest MCFAs selectivity of 68.73%. While NR was indicated to improve CE by encouraging H2 production, and the biocathode had the highest electrical activity due to the smallest internal resistance and largest capacitance increase of 96% than the control. A higher proportion of Sutterella, Prevotella, and Hydrogenophaga, linked with the H2 mediated interspecies electron transfer (MIET) during CE process, was observed across redox mediators supplied groups compared to the control. The presence of mediators led to an elevated abundance of key enzymes for enhanced CE process and electron transfer. This study provided the perspective of the stimulated electron transfer for improved MCFAs production in electro-fermentation systems.

Production of medium chain fatty acids from ensiled potato peels; effect of inoculum type and kinetic study

Medium chain fatty acids (MCFAs) are fatty acids containing 6 to 12 carbon atoms with a wide range of industrial application. They can be produced by the fermentation of waste biomass through a process called chain elongation (CE). During CE, the type of inoculum used plays a key role in determining the optimal yield of MCFAs. In this study, we showed, for the first time, the use of three different inocula including leachate, rumen fluid and digestate from a biogas reactor for the batch fermentation of ensiled potato peels for MCFAs production. Results showed that the highest chain elongation was obtained when leachate was used as inoculum with a maximum yield of 57, 4 and 26 g/kgVS for caproic acid, heptanoic acid and caprylic acid respectively. A kinetic study shows that the production of MCFAs from ensiled potato peels was better described by the first-order model than by the modified Gompertz model.

Amino acids as in-situ electron donors drive medium chain fatty acids production from sludge acidogenic fermentation liquid by electro-fermentation enhancement

The production of medium chain fatty acids (MCFAs) from waste biomass by chain elongation often requires the supplementation of exogenous chemical electron donor (ED), to support the chain elongator. This study explored the feasibility of producing MCFAs using amino acids in acidogenic fermentation liquid from protein-rich sludge hydrolysate, and proposed a two-stage electro-fermentation process to realize MCFAs production without exogenous chemical ED. Stage I aimed to convert organic substrates into short chain fatty acids (SCFAs) through anodic alkaline acidogenic fermentation. Stage II utilized residual amino acids as in-situ ED in acidogenic fermentation liquid to generate MCFAs in cathodic chamber under open circuit (OC) and 0.9 V conditions. Results indicated that acetate (6.9 g/L) and butyrate (4.5 g/L) were accumulated as the main products in stage I, and 2.7 g/L of amino acids remained in acidogenic fermentation liquid. In stage II, the remained amino acids were consumed as in-situ ED to drive the conversion of acetate and butyrate into caproate, and 1.1 g/L and 3.01 g/L caproate were respectively determined at OC and 0.9 V conditions. Microbial community and function prediction mechanisms revealed the presence of abundant chain elongator (Clostridium_sensu_stricto_12) and electroactive bacteria (Desulfovibrio) in cathodic biofilm, as well as the organic degrader (Pseudomonas) in plankton, these microbes synergistically converted SCFAs and amino acids into MCFAs. This study for the first time explored the feasibility of using amino acids as alternative chemical ED to drive chain elongation, and provides a novel technical solution foralleviating dependence on exogenous chemical ED in the production of MCFAs from waste biomass.

Co-production of lactate and volatile fatty acids through repeated-batch fermentation of fruit and vegetable waste: Effect of cycle time and replacement ratio

In this study, repeated-batch fermentation was used to convert fruit and vegetable waste to lactate and volatile fatty acids (VFAs), which are essential carbon sources for medium-chain fatty acids (MCFAs) production. The effect of cycle time and replacement ratio on acidification in long-term fermentation was investigated. The results showed that they had a significant impact on product yield, productivity, and type of products. Considering the yield, productivity, and lactate/VFAs ratio, a replacement ratio of 30% and a cycle time of 2 d may be more suitable for further production of MCFAs. Its productivity and lactate/VFAs ratio were 4.07 ± 0.24 g/(L·d) and 5 ± 0.6, respectively. The lactic acid bacteria, such as Enterococcus (63%) and Lactobacillus (33%), stabilized in the reactor, resulting in the generation of both lactate and VFAs by heterolactic fermentation. The present study demonstrated a new strategy with the potential to recover high-value products from organic waste streams.

Medium chain fatty acids production from anaerobic fermentation of food wastes: The role of fermentation pH in metabolic pathways

Using food waste (FW) as a sustainable feedstock for the production of valuable medium-chain fatty acids (MCFAs) would alleviate greenhouse gas emissions while allowing the efficient recovery of carbon contained in this biowaste. The fermentation pH is a crucial factor controlling the type and yields of the MCFAs derived from FW-fed fermentation by steering the biological reactions involved. However, the mechanism shifts that occur at varying pH values are not well understood. Therefore, this study assessed the potential role of fermentation pH (i.e., 5, 7, and 10) on the carbon recovery from FW as MCFAs and the associated mechanisms. Predominantly MCFAs (5.52 g COD/L) were synthesized at pH 5 with caproate formed as the main high-value product. Abundant but a bit fewer MCFAs (1.08 g COD/L) were yielded at pH 7, in which caproate also was the prevalent MCFAs. However, no obvious MCFAs were synthesized under pH 10. The pH also drove the change of key microbial communities that served as chain elongators, which were maximally enriched at pH 5 but were scarce at pH 10. More importantly, mildly acidic pH (i.e., 5) drove less ethanol oxidization, ensuring the effective usage of electron donor for higher MCFAs yield. This study revealed how pH steers the metabolic pathways of MCFAs production from FW in open-culture anaerobic fermentation from a metagenomic viewpoint.

Impact of phytosterol addition on fermentation progress and volatile compounds synthesis during alcoholic fermentation in synthetic and natural grape musts

Lipid nutrition is an important factor for yeast during alcoholic fermentation. Although recent research reports have revisited the role of sterols during alcoholic fermentation, our knowledge of lipids assimilation and volatile compound biogenesis remains partial. This study aimed to find out more about the impact of grape must phytosterol content on fermentative kinetics, nitrogen assimilation by yeast and fermentative aroma synthesis. To that end, experimental fermentations were performed in synthetic and Chardonnay musts supplemented with different phytosterol concentrations (0, 1, 3 and 5 mg/L). Sterols addition significantly increased the maximum CO2 production rate while reducing fermentation duration. This can be explained by higher nitrogen assimilation by yeast due to sterols, which leads to higher yeast growth and better viability at the end of the fermentation process. Regarding the aromatic profile, sterol addition also significantly increased acetate esters, ethyl esters, fusel alcohols and medium-chain fatty acids production. These new advances highlight the major role of phytosterols in fermentation control and wine aroma profile.

Co-electron donors driven medium-chain fatty acids production: Roles of electron donors, reaction kinetics and metabolic pathways

Energy conversion of waste activated sludge alkaline fermentation liquor (WASAFL) to medium-chain fatty acids (MCFAs) is promising for sludge treatment and carbon recovery. However, the single electron donor (ED) fermentation for MCFAs production has irreparable defects. To resolve the respective shortcomings of single electron donor (ED) and improve the MCFAs production efficiency from WASAFL, a novel biotechnical process utilizing ethanol and lactate as co-EDs within different combination ratios were investigated. The results verified that MCFAs production was highest with ethanol to lactate ratio of 1:3 (6988.54 ± 208.18 mg COD/L), being 1.46 and 1.87 times of that with ethanol and lactate as single ED. The kinetic analysis results confirmed that ethanol to lactate ratio of 1:3 resulted in the highest MCFAs yield and formation rate. The microbial taxa results uncovered that the relative abundance of Sphaerochaeta and Haloimpatiens showed positive correlation with MCFAs production. The metabolic pathway analysis indicated that the ethanol oxidization, lactate oxidization, acrylate pathway, reverse β oxidization and fatty acid biosynthesis pathway might take place in the WASAFL fermentation system, contributing to the WASAFL-to-MCFAs conversion.