Production Of Medium-chain Fatty Acids Research Articles

Beyond Anaerobic Digestion: New Perspectives for the Development of a Biorefinery Platform for the Simultaneous Production of Medium-Chain Fatty Acids by Chain Elongation and Biogas from Food Wastes

Beyond Anaerobic Digestion: New Perspectives for the Development of a Biorefinery Platform for the Simultaneous Production of Medium-Chain Fatty Acids by Chain Elongation and Biogas from Food Wastes

Enhancing medium-chain fatty acids production via alkyl polyglucose modulation of electron acceptor generation, microbial community and metabolic traits in anaerobic fermentation of waste activated sludge

Production of medium chain fatty acids (MCFAs) through anaerobic fermentation is an essential pathway for efficient resource recovery and utilization of waste activated sludge (WAS). In this study, a novel strategy utilizing polyglucose (APG) to enhance MCFAs production from WAS by anaerobic fermentation was proposed. The results showed that the maximum MCFAs production reached 14745.0 mg COD/L with 0.3 g APG/g TSS, which was 20.2 times of that without APG treatment. The transformation of intermediates demonstrated that APG promoted the generation of electron acceptors (i.e., short-chain fatty acids) and conversion of substrate to MCFAs. Microbial community analysis indicated that APG selectively enriched microbe involved in hydrolysis, acidogenesis and chain elongation (CE). Also, the presence of APG had a negative impact on methanogens and potential excessive ethanol oxidation (EEO) microbe, which were competitors of CE bacteria in mixed-culture system. Metagenomic analysis showed an increase in relative abundance of genes encoding metabolic functions responsible for hydrolysis, acidogenesis, CE and energy generation, thus maintained high microbial activity in MCFAs biosynthesis. These enhanced metabolic functions were accompanied by a reduction in relative abundance of genes encoding methanogenesis and EEO metabolism, which together result in boosting the efficiency of substrate and energy flow to MCFAs production. This study presents a promising approach to improve the biotransformation of polysaccharides and proteins from WAS into MCFAs.

Synthesis of medium-chain fatty acids from corn stover by sequential fermentation based on substrate configuration and initial pH optimization

This work describes a novel sequential fermentation method for the production of medium-chain fatty acids (MCFAs) from corn stover, without adding exogenous electron donors/acceptors. The effects of the carbon substrate concentration, ethanol-to-acetate ratio (ED/EA), and initial pH on carbon chain elongation (CCE) by Clostridium kluyveri to produce MCFAs are investigated using ethanol and acetate as the electron donor and acceptor, respectively. A carbon substrate concentration of 300 mM C is sufficient for CCE. An initial weakly alkaline environment significantly enhances the production of caproate through buffering effects and by reducing inhibition of product formation. The maximum caproate production reached 4.41 g/L (initial pH 8.5; 300 mm C; ED/EA = 1:1). The ability of corn stover to generate a suitable ED/EA via two-step fermentation to synthesize MCFAs is also explored. Ultimately, 5.53 g/L butyrate and caproate can be obtained from corn stover, where caproate accounts for 2.79 g/L. This study offers innovative ideas for the sustainable production of bio-based chemicals.

Impact of cell voltage on synthesis of caproic acid from carbon dioxide and ethanol in direct current powered microbial electrosynthesis cell

Production of medium chain fatty acids (MCFAs) from CO2 through microbial electrosynthesis (MES) holds great potential. The present study investigated the effect of cathode voltages of − 0.8 V (MES-1), −1.0 V (MES-2) and −1.2 V vs Ag/AgCl (MES-3), on the production of MCFAs from CO2 and ethanol using an enriched culture. Direct current (DC) power supply was used to maintain constant cathode voltages. The highest amounts of caproic acid were produced in MES-2 at an average concentration of 1.51 ± 0.14 g/L with a maximum selectivity of 68 ± 7 %. Microbial diversity analysis showed abundance of the Clostridiaceae family that allowed chain elongation in all MES reactors. This study shows that potentiostatic control approach for MCFA synthesis, can be replaced by DC power supply in future MES setups. Using selective culture enrichment, MES efficiently produces MCFAs from CO2 and ethanol, with −1.0 V yielding the highest caproic acid.

Advanced Statistical Optimization for Enhanced Medium-Chain Fatty Acid Production

In recent years, the development of feed ingredients with natural additives has gained significant importance in increasing the health and quality of animal products, as well as in promoting weight gain in animals. Since Salmonella infection is a significant disease that transmits from animals to humans, the inhibition of Salmonella species can be achieved particularly through the improvement of gastrointestinal metabolism in chickens. At this point, the effectiveness of using MCFA (Medium Chain Fatty Acids) as a feed additive has been proven. MCFA are composed of a mixture of various fatty acids, including acetic acid, butyric acid, hexanoic acid, etc. Highest portion of MCFA are hexanoic acid. Besides feed additives hexanoic acid play a crucial role as primary resources in various industries, including the chemical, food, agricultural, and biofuel sectors. It is typically obtained from petrochemical-based solutions but there has been a growing focus on biotechnological production and natural sources in recent years. One of the mostly known bioprocess to produce MCFA is chain elongation (conversion of acetate and ethanol into MCFA by β oxidation reaction) by Clostridium kluyveri. However, as in most biotechnological processes, there are low yields and high costs in these reactions as well. In this study, Box-Behnken Design, a statistical experimental design method, was used to optimize the concentrations of acetate, ethanol (the two primary components of chain elongation reactions) and pH for MCFA production via chain elongation reactions with Clostridium kluyveri. Batch experiments were performed at 30°C and 37°C to also see the effect of temperature. Higher values of hexanoic acid and bacterial growth were observed at 37°C. From an economic perspective, a 14% reduction in costs has been observed with optimized components.

A Novel Sustainable and Self-Sufficient Biotechnological Strategy for Directly Transforming Sewage Sludge into High-Value Liquid Biochemicals.

Sewage sludge, as a carbon-rich byproduct of wastewater treatment, holds significant untapped potential as a renewable resource. Upcycling this troublesome waste stream represents great promise in addressing global escalating energy demands through its wide practice of biochemical recovery concurrently. Here, we propose a biotechnological concept to gain value-added liquid bioproducts from sewage sludge in a self-sufficient manner by directly transforming sludge into medium-chain fatty acids (MCFAs). Our findings suggest that yeast, a cheap and readily available commercial powder, would involve ethanol-type fermentation in chain elongation to achieve abundant MCFA production from sewage sludge using electron donors (i.e., ethanol) and acceptors (i.e., short-chain fatty acids) produced in situ. The enhanced abundance and transcriptional activity of genes related to key enzymes, such as butyryl-CoA dehydrogenase and alcohol dehydrogenase, affirm the robust capacity for the self-sustained production of MCFAs. This is indicative of an effective metabolic network established between yeast and anaerobic microorganisms within this innovative sludge fermentation framework. Furthermore, life cycle assessment and techno-economic analysis evidence the sustainability and economic competitiveness of this biotechnological strategy. Overall, this work provides insights into sewage sludge upgrading independent of additional carbon input, which can be applied in existing anaerobic sludge fermentation infrastructure as well as to develop new applications in a diverse range of industries.

Continuous chain elongation process for carbon resource recovery from excess sludge: Enhanced n-caprylate production and specific microbial functionalities

Thermal hydrolyzed sludge (THS) exhibits considerable promise in generating medium-chain fatty acids (MCFAs) through chain elongation (CE) technology. This study developed a novel continuous CE process using THS as the substrate, achieving an optimal ethanol loading rate (5.8 g COD/L/d) and stable MCFA production at 10.9 g COD/L, with a rate of 3.6 g COD/L/d. The MCFAs primarily comprised n-caproate and n-caprylate, representing 41.5 % and 54.3 % of the total MCFAs, respectively. Utilization efficiencies for ethanol and acetate were nearly complete at 100 % and 92.8 %, respectively. Key microbial taxa identified under these optimal conditions included Alcaligenes, SRB2, Sporanaerobacter, and Kurthia, which were instrumental in critical pathways such as the generation of acetyl-CoA, the initial carboxylation of acetyl-CoA, the fatty acid biosynthesis cycle, and energy metabolism. This research provides a theoretical and technical blueprint for converting waste sludge into valuable MCFAs, promoting sustainable waste-to-resource strategies.

Enhanced medium-chain fatty acid production from sewage sludge by combined electro-fermentation and anaerobic fermentation

Electro-fermentation (EF) was combined with anaerobic fermentation (AF) to promote medium-chain fatty acid (MCFA) from sewage sludge. Results showed that EF at acidification process significantly increased short-chain fatty acid (SCFA) production of by 0.5 times (82.4 mmol C/L). AF facilitated the chain elongation (CE) process by enhancing the SCFA conversion. Combined EF at acidification and AF at CE (EF-AF) achieved the highest MCFA production of 27.9 mmol C/L, which was 20 %-866 % higher than the other groups. Electrochemical analyses showed that enhanced SCFA and MCFA production was accompanied with good electrochemical performance at acidification and CE. Microbial analyses showed that EF-AF promoted MCFA production by enriching electrochemically active bacteria (EAB, Bacillus sp.). Enzyme analyses indicated that EF-AF promoted MCFA production by enriching the functional enzymes involved in Acetyl-CoA formation and the fatty acid biosynthesis (FAB) pathway. This study provided new insights into the production of MCFA from enhanced sewage sludge.

Deciphering the reduction of antibiotic resistance genes (ARGs) during medium-chain fatty acids production from waste activated sludge: Driven by inhibition of ARGs transmission and shift of microbial community

Medium-chain fatty acids (MCFAs) production from waste activated sludge (WAS) by chain extension (CE) is a promising technology. However, the effects and mechanisms of CE process on the fate of antibiotic resistance genes (ARGs) remain unclear. In this study, the results showed that the removal efficiency of ARGs was 81.15 % in CE process, suggesting its efficacy in reducing environmental risks. Further, the observed decrease in mobile genetic elements (MGEs) indicated that CE process restricted the horizontal gene transfer (HGT). Complementing this, the increase in soluble organic matters and extracellular 16 S rDNA confirmed that MCFAs production caused bacterial damage. Decreased intracellular ARGs and increased extracellular ARGs further revealed that MCFAs production impaired ARGs hosts, thereby limiting the vertical gene transfer (VGT) of ARGs. Shift of microbial community combined with co-occurrence network analysis demonstrated that functional bacteria without host potential for ARGs were enriched, but potential ARGs and MGEs hosts decreased, showing the role of functional bacterial phylogeny and selection pressure of MCFAs in reducing ARGs. Finally, partial least squares path model was used to systematic verify the mechanism of ARGs removal in CE process, which was attributed to the inhibition of ARGs transmission (HGT and VGT) and shift of microbial community.

Performance and mechanism of iron-carbon enhancing medium chain fatty acids production from waste sludge

A novel iron-carbon technique to enhance the production of medium-chain carboxylates (MCCs) from waste activated sludge (WAS) during anaerobic fermentation was proposed in this study. Results revealed that the maximal MCCs production (113.78 mmol/L) and selectivity of MCCs were significantly enhanced by 30.50% and 144.48% respectively, at 20 g/L iron-carbon. The optimal fermentation time was also shortened from 18 to 8 days. Mechanistic investigations disclosed that MCCs formation was significantly promoted by Fe2+, which was released from iron-carbon and assimilated by microorganisms. Further analysis showed that the solubilization (36.35%), hydrolysis (3.87%), and acidogenesis (33.47%) processes of WAS were promoted by iron-carbon, supplying more substrates (short-chain carboxylates (SCCs)) for the synthesis of MCCs. Iron-carbon enhanced the abundances of key microorganisms participating in the aforementioned bioprocesses, according to microbial community investigation. Additionally, the conductivity of sludge was improved by 127.84% with the addition of iron-carbon particles, thus improved the conditions for anaerobic biological activities.

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.