Medium-chain Carboxylic 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.

Influence of pH and temperature on the performance and microbial community during the production of medium-chain carboxylic acids using winery effluents as substrate.

Winery effluents containing high ethanol concentrations and diverse organic matter are ideal substrates for producing medium-chain carboxylic acids via fermentation and chain elongation. However, the process needs to be better understood. This study presents novel insights into the bioconversion mechanisms of medium-chain carboxylic acids by correlating fermentation and chain elongation kinetic profiles with the study of microbial communities at different pH (5 to 7) conditions and temperatures (30 to 40°C). It was found that high productivities of MCCA were obtained using a native culture and winery effluents as a natural substrate. Minor pH variations significantly affected the metabolic pathway of the microorganisms for MCCA production. The maximal productivities of hexanoic (715mg/L/d) and octanoic (350mg/L/d) acids were found at pH 6 and 35°C. Results evidence that the presence of Clostridium, Bacteroides, and Negativicutespromotes the high productions of MCCA. The formation of heptanoic acid was favor when Mogibacterium and Burkholderia were present.

Combining chain elongation and oleogel extraction for the production and recovery of caproate from beef cattle manure wastewater

Producing and recovering bioproducts from organic wastes is vital for building a circular economy. Livestock waste can serve as a feedstock for bioproduct production, such as medium chain carboxylic acids (MCCAs). This study aims to (1) optimize the conversion of organics in beef cattle manure to caproate and (2) develop an innovative oleogel-based extraction method for caproate recovery. With intrinsic chain elongation bacteria in cattle manure, we achieved a maximum caproate production rate of 8.59 g/L/d and the maximum concentration of 11.09 g/L with ethanol as the electron donor. Chain elongators such as Clostridium, Eubacterium, Caproiciproducens were detected. Oleogel beads were fabricated and enabled high mass transfer rates, achieving a cumulative caproate extraction efficiency of 87.39±0.96 % in 180 min. In subsequent desorption process, caproate accounted for 86.18±0.32 % of all carboxylic acids and alcohols desorbed from oleogel beads. This study demonstrates the technical feasibility of producing bioproducts from an abundant agricultural waste.

Bio-based production of medium-chain carboxylic acids from food waste and sludge without chemical addition: The pivotal role of mix ratio and pretreatment

Medium-chain carboxylates such as caproic acid, have a plethora of applications, ranging from food additives to bioplastics, and can be produced via microbial chain elongation (CE), a process that can be more sustainable than conventional production routes. During CE short fatty acids accept electrons from donors as lactate or ethanol elongating into medium-chain carboxylates with higher economic value and easier recoverability. Nowadays, waste activated sludge (WAS) and food waste (FW) are the most abundant waste steams generated in European cities impacting dramatically on environment, society, and economic sectors. A novel sustainable biotechnology is here proposed to produce marketable caproic acid from mixed WAS and FW with in-situ self-formed lactate without buffering agents addition. Different mix ratios between WAS and FW, and thermal pretreatment strategies were compared by fermentation batch tests. The production of lactate as electron donor (ED), together with acetate and butyrate, and hydrogen assured high production of caproate only in presence of thermal pretreated WAS, able to reduce the potential risk of lactate accumulation and drop in pH. The findings of this study demonstrate that the initial soluble carboydrate/protein ratio significantly affected the pH of the fermentation broth in the first hours of fermentation, and that only using FW and pretreated WAS mixed using a ratio of 60/40 in terms of volatile solids, CE pathway and lactate utilization as ED were promoted obtaining the highest percentage of caproate (about 21% of the total produced carboxylic acids; 2 gCOD/L).

Simultaneous medium chain carboxylic acids and 1,3-propanediol production in a bioaugmented lactate-based chain elongation induced with glycerol

The objective was to investigate the impact of the bioaugmentation on chain elongation process using glycerol, lactate and lactose as substrates in an open culture fermentation. In the batch trials the highest selectivity for chain elongation product, i.e. caproate, was observed in trials inoculated with co-culture of Megasphaera elsdenii and Eubacterium limosum grown on glycerol (28.6%), and in non-bioaugmented open culture run on lactose + lactate (14.8%). The results showed that E. limosum, out of two bioaugmented strains, was able to survive in the open culture. A continuous open culture fermentation of glycerol led to caproate and 1,3-propanediol (1,3-PDO) formation, while lactate addition led to 1,3-PDO and short chain carboxylates production. Moving the process into batch mode triggered even-carbon chain elongation. Presence of E. limosum promoted odd-carbon chain elongation and valerate production. Imaging flow cytometry combined with machine learning enabled the discrimination of Eubacterium cells from other microbial strains during the process.

Acidogenic fermentation of food waste to generate electron acceptors and donors towards medium-chain carboxylic acids production

This study investigated the optimum pH, temperature, and food-to-microorganisms (F/M) ratio for regulating the formation of electron acceptors and donors during acidogenic fermentation to facilitate medium-chain carboxylic acids (MCCAs) production from food waste. Mesophilic fermentation at pH 6 was optimal for producing mixed volatile fatty acids (719 ± 94 mg COD/g VS) as electron acceptors. Under mesophilic conditions, the F/M ratio (g VS/g VS) could be increased to 6 to generate 22 ± 2 g COD/L of electron acceptors alongside 2 ± 0 g COD/L of caproic acid. Thermophilic fermentation at pH 6 was the best condition for producing lactic acid as an electron donor. However, operating at F/M ratios above 3 g VS/g VS under thermophilic settings significantly reduced lactic acid yield. A preliminary techno-economic evaluation revealed that converting lactic acid and butyric acid generated during acidogenic fermentation to caproic acid was the most profitable food waste valorization scenario and could generate 442–468 €/t VS/y. The results presented in this study provide insights into how to tailor acidogenic fermentation reactions to desired intermediates and will help maximize MCCAs synthesis.

Caproate production from Enset fiber in one-pot two-step fermentation using anaerobic fungi (Neocallimastix cameroonii strain G341) and Clostridium kluyveri DSM 555

BackgroundLignocellulosic biomass plays a crucial role in creating a circular bioeconomy and minimizing environmental impact. Enset biomass is a byproduct of traditional Ethiopian Enset food processing that is thrown away in huge quantities. This study aimed to produce caproate from Enset fiber using Neocallimastix cameroonii strain G341 and Clostridium kluyveri DSM 555 in one-pot two-step fermentation.ResultsThe process started by growing N. cameroonii on Enset fiber as a carbon source for 7 days. Subsequently, the fungal culture was inoculated with active C. kluyveri preculture and further incubated. The results showed that N. cameroonii grew on 0.25 g untreated Enset fiber as the sole carbon source and produced 1.16 mmol acetate, 0.51 mmol hydrogen, and 1.34 mmol formate. In addition, lactate, succinate, and ethanol were detected in small amounts, 0.17 mmol, 0.08 mmol, and 0.7 mmol, respectively. After inoculating with C. kluyveri, 0.3 mmol of caproate and 0.48 mmol of butyrate were produced, and hydrogen production also increased to 0.95 mmol compared to sole N. cameroonii fermentation. Moreover, after the culture was supplemented with 2.18 mmol of ethanol during C. kluyveri inoculation, caproate, and hydrogen production was further increased to 1.2 and 1.36 mmol, respectively, and the consumption of acetate also increased.ConclusionA novel microbial cell factory was developed to convert untreated lignocellulosic Enset fiber into the medium chain carboxylic acid caproate and H2 by a co-culture of the anaerobic fungi N. cameroonii and C. kluyveri. This opens a new value chain for Enset farmers, as the process requires only locally available raw materials and low-price fermenters. As the caproate production was mainly limited by the available ethanol, the addition of locally produced ethanol-containing fermentation broth (“beer”) would further increase the titer.Graphical

Physiological and stoichiometric characterization of ethanol-based chain elongation in the absence of short-chain carboxylic acids

Hexanoate is a valuable chemical that can be produced by microorganisms that convert short-chain- to medium-chain carboxylic acids through a process called chain elongation. These microorganisms usually produce mixtures of butyrate and hexanoate from ethanol and acetate, but direct conversion of ethanol to hexanoate is theoretically possible. Steering microbial communities to ethanol-only elongation to hexanoate circumvents the need for acetate addition and simplifies product separation. The biological feasibility of ethanol elongation to hexanoate was validated in batch bioreactor experiments with a Clostridium kluyveri-dominated enrichment culture incubated with ethanol, acetate and butyrate in different ratios. Frequent liquid sampling combined with high-resolution off-gas measurements allowed to monitor metabolic behavior. In experiments with an initial ethanol-to-acetate ratio of 6:1, acetate depletion occurred after ± 35 h of fermentation, which triggered a metabolic shift to direct conversion of ethanol to hexanoate despite the availability of butyrate (± 40 mCmol L−1). When only ethanol and no external electron acceptor was supplied, stable ethanol to hexanoate conversion could be maintained until 60–90 mCmol L−1 of hexanoate was produced. After this, transient production of either acetate and butyrate or butyrate and hexanoate was observed, requiring a putative reversal of the Rnf complex. This was not observed before acetate depletion or in presence of low concentrations (40–60 mCmol L−1) of butyrate, suggesting a stabilizing or regulatory role of butyrate or butyrate-related catabolic intermediates. This study sheds light on previously unknown versatility of chain elongating microbes and provides new avenues for optimizing (waste) bioconversion for hexanoate production.

Trophic interactions shape the spatial organization of medium-chain carboxylic acid producing granular biofilm communities.

Granular biofilms producing medium-chain carboxylic acids (MCCA) from carbohydrate-rich industrial feedstocks harbor highly streamlined communities converting sugars to MCCA either directly or via lactic acid as intermediate. We investigated the spatial organization and growth activity patterns of MCCA producing granular biofilms grown on an industrial side stream to test (i) whether key functional guilds (lactic acid producing Olsenella and MCCA producing Oscillospiraceae) stratified in the biofilm based on substrate usage, and (ii) whether spatial patterns of growth activity shaped the unique, lenticular morphology of these biofilms. First, three novel isolates (one Olsenella and two Oscillospiraceae species) representing over half of the granular biofilm community were obtained and used to develop FISH probes, revealing that key functional guilds were not stratified. Instead, the outer 150-500 µm of the granular biofilm consisted of a well-mixed community of Olsenella and Oscillospiraceae, while deeper layers were made up of other bacteria with lower activities. Second, nanoSIMS analysis of 15N incorporation in biofilms grown in normal and lactic acid amended conditions suggested Oscillospiraceae switched from sugars to lactic acid as substrate. This suggests competitive-cooperative interactions may govern the spatial organization of these biofilms, and suggests that optimizing biofilm size may be a suitable process engineering strategy. Third, growth activities were similar in the polar and equatorial biofilm peripheries, leaving the mechanism behind the lenticular biofilm morphology unexplained. Physical processes (e.g., shear hydrodynamics, biofilm life cycles) may have contributed to lenticular biofilm development. Together, this study develops an ecological framework of MCCA-producing granular biofilms that informs bioprocess development.

Efficient conversion of wine lees into medium-chain carboxylic acids by mixed culture using an in-line extraction system

This study evaluates a process to generate medium-chain carboxylic acids (MCCA) from wasted wine lees. The effect of pH, feeding mode, organic loading rate, and the in-line extraction of the acids on the MCCA production rate was evaluated. Both the electron donor (ethanol) and acceptor (acetate) were generated in situ from the waste. First, the reactor was operated as a sequencing batch reactor (SBR), and pH 5.5 and 5.2 were evaluated. The caproate productivity was ten times higher at pH 5.5 than at 5.2. An in-line extraction system was next implemented, and the operation as SBR and the continuous mode was studied. The continuous mode allowed an increase of the residual ethanol; however, the feeding with concentrated wine lees caused an acetate over-accumulation. Caprylate was produced and extracted over caproate (caprylate/caproate ratio of 1.2) by avoiding the bioreactor overload and keeping a low ethanol broth concentration. In conclusion, the maximal MCCA production rate (143.4 ± 14.8 mmol C/L-d) was obtained when the organic loading rate was gradually increased in the continuous operation; under such conditions, Eubacterium pyruvativorans predominated. The in-line extraction system allowed the production and recovery of MCCA.

Predicting the performance of chain elongating microbiomes through flow cytometric fingerprinting.

As part of the circular bio-economy paradigm shift, waste management and valorisation practices have moved away from sanitation and towards the production of added-value compounds. Recently, the development of mixed culture bioprocess for the conversion of waste(water) to platform chemicals, such as medium chain carboxylic acids, has attracted significant interest. Often, the microbiology of these novel bioprocesses is less diverse and more prone to disturbances, which can lead to process failure. This issue can be tackled by implementing an advanced monitoring strategy based on the microbiology of the process. In this study, flow cytometry was used to monitor the microbiology of lactic acid chain elongation for the production of caproic acid, and assess its performance both qualitatively and quantitatively. Two continuous stirred tank reactors for chain elongation were monitored flow cytometrically for over 336 days. Through community typing, four specific community types could be identified and correlated to both a specific functionality and genotypic diversity. Additionally, the machine-learning algorithms trained in this study demonstrated the ability to predict production rates of, amongst others, caproic acid with high accuracy in the present (R² > 0.87) and intermediate accuracy in the near future (R² > 0.63). The identification of specific community types and the development of predictive algorithms form the basis of advanced bioprocess monitoring based on flow cytometry, and have the potential to improve bioprocess control and optimization, leading to better product quality and yields.

Electrolysis of medium chain carboxylic acids to aviation fuel at technical scale

For mobility, especially in aviation, cargo weight and volume of the energy vector are substantially more limited than in any other form of transportation. Therefore, energy vectors with high mass and volume specific energy density are needed, for which carbon based liquid fuels are unmatched. Concurrently, there is an imperative of deriving these fuels from non-fossil sources. Here, we show the scale-up of the production of aviation drop-in fuel-like mixtures (mainly n-decane) that are obtained by Kolbe electrolysis of medium chain carboxylic acids (n-hexanoic acid), which can be derived from biomass. We increased the scale to 1 L volume and 100 cm2 electrode surface area within a single stack electrolysis cell allowing straightforward numbering-up. The obtained electrolysis performance is unprecedented at technical scale with a maximum n-decane production rate of 0.73 g L−1h−1 cm−2, a selectivity for n-decane between 76.3 % and 97.8 % and Coulombic efficiencies up to 68 %.

Advances in understanding entire process of medium chain carboxylic acid production from organic wastes via chain elongation

Chain elongation is an environmentally friendly biological technology capable of converting organic wastes into medium chain carboxylic acids (MCCAs). This review aims to offer a comprehensive analysis of MCCA production from organic wastes via chain elongation. Seven kinds of organic wastes are introduced and classified as easily degradable and hardly degradable. Among them, food waste, fruit and vegetable waste are the most potential organic wastes for MCCA production. Combined pretreatment technologies should be encouraged for the pretreatment of hardly degradable organic wastes. Furthermore, the mechanisms during MCCA production are analyzed, and the key influencing factors are evaluated, which affect the MCCA production and chain elongation efficiency indirectly. Extracting MCCA simultaneously is the most important way to improve MCCA production efficiency, and technologies for sequentially extracting different kinds of MCCAs are recommended. Finally, some perspectives for future chain elongation researches are proposed to promote the large-scale application of chain elongation.

Analysis of secondary waste from a plastics recycling plant for the production of carboxylic acids

The production of bio-based platform chemicals through the chain elongation of short-chain carboxylic acids to medium-chain carboxylic acids by ethanol-acetat fermentation can be a contribution to the circular economy. To avoid further waste, secondary waste that already contains short-chain carboxylic acids can be used. The potential for the production of bio-based carboxylic acids from a secondary waste of a recycling plant for plastic waste is examined in this paper. Therefore, practical experiments with the process water of a recycling plant for plastic waste were conducted in order to assess the potential for carboxylic acids production. At the end of the experiment, the concentrations achieved by chain elongation in the secondary waste result in 496 mg/L butyric acid and 87 mg/L caproic acid and the concentration in the extraction solvent is 933 mg/L caproic acid. To conclude, chain elongation of carboxylic acids in secondary waste, in this case the process water from a treatment plant for plastic waste, is generally possible. In order to estimate the total potential for the production, the fluctuations of the quality of the process water have to be considered.

Preparation and Application of Carbon-Based Materials in the Production of Medium-Chain Carboxylic Acids by Anaerobic Digestion: A Review

The main purpose of this article is to explore the mechanism of action of carbon-based materials in the anaerobic digestion (AD) production of medium-chain carboxylic acids (MCCA). Currently, there are various methods to increase production, but there is no review on how carbon-based materials improve MCCA. This paper first introduced the chain elongation (CE) technology, focusing on the factors affecting the production of MCCA by AD, such as pH, temperature, the ratio of electron donor (ED) to an electron acceptor (EA), substrate type, and other related factors. This article introduces the preparation and characteristics of carbon-based materials, as well as the effect and mechanism of adding carbon-based materials to AD acid production. Finally, the shortcomings of the current research were pointed out, and future research directions were prospected, aiming to provide a reference for improving the efficiency of AD of MCCA using carbon-based materials.

Experimental evaluation of temperature, nutrients, and initial concentration on medium-chain carboxylic acids production from winery wastes.

In the wine industry, grape processing is accompanied by waste generation, such as grape stalks, winery wastewater, and grape pomace (GP). GP can be used to produce value-added compounds such as medium-chain carboxylic acids (MCCA). This work aimed to determine the operational conditions (temperature, addition of nutrients, and initial waste concentration) to improve MCCA production using waste GP from the winery industry as a substrate. The electron donor (ethanol) and electron acceptor (acetate) were directly generated from the GP and consecutively used to produce MCCA. The treatment with high concentration, temperature, and nutrient addition promotes caproic acid's maximal yield and concentration (0.11 ± 0.02 g MCCA/g TS). Nutrients' presence and temperature significantly affected electron acceptor production. The addition of nutrients and 30 °C leads to elevated acetate production. However, at 37 °C, butyrate and MCCA were mainly produced without adding nutrients, and high ethanol consumption was observed. A higher metabolic diversification was observed at 37 °C than at 30 °C. Temperature and nutrient availability significantly affected the metabolic pathway and the type of carboxylic acid produced.

Advances in Electricity-Steering Organic Waste Bio-Valorization for Medium Chain Carboxylic Acids Production

Medium chain carboxylic acids (MCCAs, e.g., caproic acid, caprylic acid, etc.) with 6–12 carbon atoms are valuable platform chemicals produced from organic waste via microbial chain elongation metabolism named as reversed β-oxidation and fatty acid-biosynthesis cyclical pathway. Recently, many articles reported that electricity could not only serve as the external electron donor and provide the reduction equivalent required for chain elongation but also regulate the microbiome structure and metabolic behaviors to promote MCCAs formation. Electricity-steering MCCAs bioproduction has become an appealing technique to valorize low-value organic waste, paving an alternative pathway for net-zero carbon emission energy systems and sustainable socio-economic development. However, the MCCAs’ bioproduction from organic waste steered by electric field has not been comprehensively reviewed. From a systematical analysis of publicly available literature, we first covered the basic working principle, fermentation architecture, functional microflora, and metabolic pathway of MCCAs production driven by electricity. The strategies of substrate modulation, applied voltage/current regulation, electrode optimization, and microbial cooperation and stimulation for boosting electricity-driven MCCAs bioproduction are then scrutinized and extensively discussed. Ultimately, the pressing knowledge gaps and the potential path forward are proposed to provide pointers for consistently higher MCCAs yield and the transition from laboratory to market.

Competitive Reactions during Ethanol Chain Elongation Were Temporarily Suppressed by Increasing Hydrogen Partial Pressure through Methanogenesis Inhibition.

Organic waste streams can be converted into high-value platform chemicals such as medium-chain carboxylic acids (MCCAs) using mixed microbial communities via chain elongation. However, the heterogeneity of waste streams and the use of complex microbial communities can lead to undesirable reactions, thus decreasing process efficiency. We explored suppressing excessive ethanol oxidation to acetate (EEO) by increasing the hydrogen partial pressure (PH2) through hydrogenotrophic methanogenesis inhibition by periodically adding 2-bromoethanesulfonate (2-BES) to an MCCA-producing bioreactor to reach 10 mM of 2-BES upon addition. The bioreactor was fed with pretreated food waste and brewery waste containing high concentrations of short-chain carboxylic acids and ethanol, respectively. While 2-BES addition initially reduced EEO, some methanogens (Methanobrevibacter spp.) persisted and resistant populations were selected over time. Besides changing the methanogenic community structure, adding 2-BES also changed the bacterial community structure due to its impact on PH2. While we demonstrated that PH2 could be manipulated using 2-BES to control EEO, methods that do not require the addition of a chemical inhibitor should be explored to maintain optimum PH2 for long-term suppression of EEO.

Polyhydroxyalkanoates and 5-aminolevulinic acid production by a mixed phototrophic culture using medium-chain carboxylic acids from winery effluents

This work aimed to obtain polyhydroxyalkanoates (PHA) and 5-aminolevulinic acid (5-ALA) from medium-chain carboxylic acids (MCCA) using a mixed culture enriched in Rhodopseudomnas palustris. MCCA, obtained from residual wine lees, were tested in batch photofermentation experiments. First, the influence of individual MCCA (hexanoic, heptanoic, and octanoic acids) was evaluated; then, the MCCA coming directly from a fermentation reactor (LC-effluent) or after acids extraction (HC-effluent) were studied. Nutrient supplementation, bicarbonate, and acetic acid addition were also tested. Results showed that PHA production was higher in hexanoic (328 mg PHA/L) compared to heptanoic (152 mg PHA/L) and octanoic (164 mg PHA/L) acids. Bicarbonate addition and acetic acid as co-substrate improved the MCCA consumption, the PHA content and production rate. The HC-effluent, without nutrient supplementation, was allowed to increase 2.5 times the PHA content (reaching 40 % w/w and 584 mg/L) and to double 5-ALA production (7.6 µM) compared to the LC-effluent condition.

Ferric oxide stimulates medium-chain carboxylic acids synthesis from waste activated sludge via ethanol-driven chain elongation: Mechanisms and implications

Nowadays, conductive iron-containing materials (i.e., Fe3O4 and zerovalent iron) have attracted greatly attention in improving medium-chain carboxylic acids (MCCA) from waste activated sludge (WAS). However, the feasibility and mechanism of semi-conductive iron oxide, i.e., ferric oxide (Fe2O3), in stimulating MCCA synthesis from WAS via ethanol-driven chain elongation (CE) has been unclear. Therefore, this work is aimed to fill up the knowledge gap. Results showed that the MCCA yield in the Fe2O3-supplemented fermenter attained at 9162 mg COD/L (i.e., 6268 mg COD/L caproate and 2895 mg COD/L caprylate), which was 2.4 times that of the control system. Kinetic analysis proved that Fe2O3 enhanced both the potential and rate of MCCA synthesis. Mechanism analysis indicated that Fe2O3 facilitated the individual steps for MCCA production, i.e., hydrolysis, acidification and CE. Further investigation disclosed that the dissimilatory iron reduction (DIR) induced by Fe2O3 corrosion and released ferrous ions improved enzymes activities of hydrolysis and acidification, while the promotion of CE was mainly ascribed to increased electron transfer efficiency by crystalline Fe2O3. Further, Fe2O3 promotes electron transfer through several mechanisms, including stimulating extracellular polymeric substance (EPS) excretion, increasing EPS electroactivity, and enhancing electron transport system activity. Microbial analysis revealed that Fe2O3 induced microflora structure shifting towards substrates transformation, iron reduction and MCCA generation, and up-regulated the key enzymes in CE pathways. This work provides an efficient strategy for MCCA generation and WAS management.