Production Of Medium-chain Fatty Acids Research Articles

Fermentation of Organic Residues to Beneficial Chemicals: A Review of Medium-Chain Fatty Acid Production

Medium-chain fatty acids (MCFAs) have a variety of uses in the production of industrial chemicals, food, and personal care products. These compounds are often produced through palm refining, but recent work has demonstrated that MCFAs can also be produced through the fermentation of complex organic substrates, including organic waste streams. While “chain elongation” offers a renewable platform for producing MCFAs, there are several limitations that need to be addressed before full-scale implementation becomes widespread. Here, we review the history of work on MCFA production by both pure and mixed cultures of fermenting organisms, and the unique metabolic features that lead to MCFA production. We also offer approaches to address the remaining challenges and increase MCFA production from renewable feedstocks.

Circular bioeconomy solutions: driving anaerobic digestion of waste streams towards production of high value medium chain fatty acids

This review outlines the road map for the production of medium chain fatty acids (MCFAs) from organic wastes by adapting the conventionally methanogenic process of anaerobic digestion (AD) for the production of volatile acids and the subsequent chain elongation, as well as for the production of hydrogen. The article analyses the state-of-the-art of these processes and discusses the modifications involved, the bottlenecks, challenges and opportunities. In particular, the various methods of inhibition of methanogenesis are described, their applicability is discussed and investigation of the use of naturally occurring inhibitors like bromoform is suggested. The review concludes that the using AD for MCFA production has a strong potential as a circular economy solution, avoiding many issues with the use of AD for biogas production while creating more economically viable products. The main bottleneck identified here is the downstream separation and extraction of the products. Standardization across studies and implementation of the various methods at pilot scale levels are critical to develop a clearer idea of the viability of the MCFA production process. More in-depth studies on the inhibition process and detailed cost–benefit and life cycle analysis are also recommended to for the use of this process and for its acceptance as a bioeconomy solution.

Potential of medium chain fatty acids production from municipal solid waste leachate: Effect of age and external electron donors

A large quantity of leachate is generated during municipal solid waste collection operation and in landfills due to the large amount of organic waste and high humidity. The content of medium chain fatty acids (MCFAs) in the leachate is a low cost feedstock for bio-based chemical and fuel production processes. The aim of this study is to investigate the MCFA production potential of three leachate ages through chain elongation process under uncontrolled pH batch test. Moreover, the effect of using different external electron donors (ethanol, methanol and a mixture of both) is studied. The experiment consists of characterizing the samples then adding external electron donors with a specific ratio to leachate samples under mesophilic temperature. For this investigation, also a statistical analysis is done, which shows the production of MCFAs is highly influenced by leachate age. The results indicate that the production of even-numbered acids increase from 600 to 1,000 mg/L by the end of the ethanol chain elongation experiment for young leachate. However, a higher MCFA production of more than 1,000 mg/L is achieved by using the mixture of methanol and ethanol as electron donor. Furthermore, all methanol chain elongation experiments lead to an odd-numbered production of MCFAs, such as pentanoic and heptanoic acids. These results confirm the potential improvement of MCFA production from leachate through choosing the optimal leachate age and electron donor. Overall, producing MCFAs from leachate is a good example of circular bio-economy because waste is used to produce biochemicals, which closes the material cycle.

Diagnosing and Predicting Mixed-Culture Fermentations with Unicellular and Guild-Based Metabolic Models.

Multispecies microbial communities determine the fate of materials in the environment and can be harnessed to produce beneficial products from renewable resources. In a recent example, fermentations by microbial communities have produced medium-chain fatty acids (MCFAs). Tools to predict, assess, and improve the performance of these communities, however, are limited. To provide such tools, we constructed two metabolic models of MCFA-producing microbial communities based on available genomic, transcriptomic, and metabolomic data. The first model is a unicellular model (iFermCell215), while the second model (iFermGuilds789) separates fermentation activities into functional guilds. Ethanol and lactate are fermentation products known to serve as substrates for MCFA production, while acetate is another common cometabolite during MCFA production. Simulations with iFermCell215 predict that low molar ratios of acetate to ethanol favor MCFA production, whereas the products of lactate and acetate coutilization are less dependent on the acetate-to-lactate ratio. In simulations of an MCFA-producing community fed a complex organic mixture derived from lignocellulose, iFermGuilds789 predicted that lactate was an extracellular cometabolite that served as a substrate for butyrate (C4) production. Extracellular hexanoic (C6) and octanoic (C8) acids were predicted by iFermGuilds789 to be from community members that directly metabolize sugars. Modeling results provide several hypotheses that can improve our understanding of microbial roles in an MCFA-producing microbiome and inform strategies to increase MCFA production. Further, these models represent novel tools for exploring the role of mixed microbial communities in carbon recycling in the environment, as well as in beneficial reuse of organic residues.IMPORTANCE Microbiomes are vital to human health, agriculture, and protecting the environment. Predicting behavior of self-assembled or synthetic microbiomes, however, remains a challenge. In this work, we used unicellular and guild-based metabolic models to investigate production of medium-chain fatty acids by a mixed microbial community that is fed multiple organic substrates. Modeling results provided insights into metabolic pathways of three medium-chain fatty acid-producing guilds and identified potential strategies to increase production of medium-chain fatty acids. This work demonstrates the role of metabolic models in augmenting multi-omic studies to gain greater insights into microbiome behavior.

Medium-Chain fatty acids and long-chain alcohols production from waste activated sludge via two-stage anaerobic fermentation

Traditional bioenergy recovery in the form of short chain fatty acids (SCFAs) from waste activated sludge (WAS) is generally limited by economic unattractiveness and complexity of products separation. Herein, a novel biotechnology process of two-stage anaerobic fermentation for converting the WAS into high energy density, easy-separated medium chain fatty acids (MCFAs) and long-chain alcohols (LCAs) was evaluated. In this process, the WAS was first converted to WAS alkaline fermentation liquid (WASAFL), serving as electron acceptors (EAs) and inoculum, then adding ethanol as electron donor (ED) for chain elongation (CE). The co-production of MCFAs and LCAs during CE were studied under three different ED to EA ratios, i.e., 3:1, 4:1 and 5:1. Experimental results demonstrated that when the ratio of ED to EA increased from 3:1 to 5:1, the production of MCFA and LCAs respectively increased from 5.57 ± 0.17 and 2.58 ± 0.18 to7.67 ± 0.48 and 4.21 ± 0.19 g COD/L. A similar observation was made in the total product electron efficiency, increasing from 59.9% to 72.1%. However, the highest total product selectivity (i.e., 68.0%) and highest products production yield (i.e., 59.77%) were not achieved at the ED to EA ratio of 5:1 due to toxicity caused by higher accumulation of n-caproate. The kinetic analysis further confirmed that high ratio of ED to EA induced improvement in product maximum yield, production rate for both MCFAs and LCAs. Microbial community analysis indicated that Clostridium, Caproiciproducens, Acinetobacter, Exilispira, and Oscillibacter were clearly enriched in the CE reactor and had positive correlation with MCFAs and LCAs production.

Medium chain fatty acids production from anaerobic fermentation of waste activated sludge

The utilization of waste activated sludge (WAS) to recover energy in the form of methane or short-chain fatty acids (SCFAs) is generally restricted by the low energy density of products and poor degradability of WAS. Herein, this study reported a novel alternative WAS fermentation technology to produce high-energy medium-chain fatty acids (MCFAs) from WAS in a one-stage anaerobic fermentation system using ethanol as electron donor. The MCFAs production and WAS degradation at different ethanol levels were investigated. The increased ethanol levels resulted in increasing MCFAs production (from 1875 to 6115 mg chemical oxygen demand (COD)/L) and selectivity (from 30.3 to 56.2%). The main MCFAs products were n-caproate and n-caprylate at lower level of ethanol, while n-caproate was the sole MCFA product at higher level of ethanol with longer chain alcohol (i.e., n-hexanol) produced as well. The ethanol markedly increased WAS degradation, with the greatest degradation (0.72 g COD/g volatile solids (VS)) being 1.9 times of that without ethanol (0.38 mg COD/mg VS, at 0 mmol/L), which was ascribed to the advancement of sludge solubilization, hydrolysis and acidification. Microbial community revealed that the ethanol participation induced the community shift to the favorable direction for hydrolysis-acidification and chain elongation in anaerobic WAS fermentation.

Anaerobic chain elongation of cornstalk for MCFAs production via mixed culture: pH regulation

Anaerobic fermentation of waste biomass via microbial chain elongation for producing medium-chain fatty acids (MCFAs) is becoming an emerging and economically viable technology. However, due to the large abundant of microbial species and great diversity of microbial metabolic pathways in open cultures, how to achieve the high yields of MCFAs is an important topic. In this study, the effect of pH, as an important parameter for process control, on the production and composition of volatile fatty acids (VFAs) in two cycles of anaerobic chain elongation process with cornstalk as feedstock was investigated, and the 16S rRNA high throughput sequencing was employed to elucidate the mechanism of pH affecting chain elongation process. The results showed that pH significantly influenced the VFAs production and their spectra. At pH 5.6, the VFAs production was 10.0 g/L, which was increased to 15.9 g/L at pH 7.0, and was further increased to 18.8 g/L at pH 9.0. In addition, the metabolic pathways and the VFAs composition in anaerobic fermentation were greatly changed with pH regulation. Under acidic and neutral conditions (pHs of 5.6 and 7.0), the chain elongation was enhanced, and butyrate and caproate were the major products accounting for more than 80%. Specially, under the neutral condition, the caproate concentration reached (3.49±0.17) g/L. However, the pH of 9.0 seriously inhibited the chain elongation, and the major fermentation products were acetate (93.2%) and propionate (5.5%). The second-cycle fermentation with supplementing additional ethanol as an electron donor indicated that pH played the more important role in the chain elongation process for a high level MCFAs production. In the second cycle, the chain elongation reaction did not occur under the acidic conditions. However, when the pH was adjusted from 5.6 to 7.0, the chain elongation reaction was significantly stimulated, and the caproate concentration quickly increased from 1.88 to 11.8 g/L. The results of microbial community analysis showed that the fermentative bacteria of Firmicutes were the dominate population under the neutral pH condition accounting for 49.9%, which was far higher than those under the acidic and alkaline conditions. The strains relating to the chain elongation, such as Clostridium IV , Clostridium XI , Clostridium XIVa , Clostridium XIVb , Clostridium XVIII and Clostridium sensu stricto , were enriched under the neutral and acidic conditions, but not under the alkaline condition. It should be noted that the well-known pure culture bacteria relating to chain elongation, such as Clostridium Kluyveri , were not found in this study. In the second cycle, no significant difference on the relative abundance of strains relating to chain elongation was found between neutral and acidic conditions, suggesting that these strains could survive under the acidic conditions but their activity was inhibited. When the pH was adjusted from 5.6 to 7.0, their activities could quickly recover to produce caproate. The above findings provided an effective regulating strategy for selective production of MCFAs from waste biomass with open cultures of microbial consortia.

Zerovalent Iron Effectively Enhances Medium-Chain Fatty Acids Production from Waste Activated Sludge through Improving Sludge Biodegradability and Electron Transfer Efficiency.

A novel zerovalent iron (ZVI) technique to simultaneously improve the production of medium-chain fatty acids (MCFAs) from waste activated sludge (WAS) and enhance WAS degradation during anaerobic WAS fermentation was proposed in this study. Experimental results showed that the production and selectivity of MCFAs were effectively promoted when ZVI was added at 1-20 g/L. The maximum MCFAs production of 15.4 g COD (Chemical Oxygen Demand)/L and MCFAs selectivity of 71.7% were both achieved at 20 g/L ZVI, being 5.3 and 4.8 times that without ZVI (2.9 g COD/L and 14.9%). Additionally, ZVI also promoted WAS degradation, which increased from 0.61 to 0.96 g COD/g VS when ZVI increased from 0 to 20 g/L. The microbial community analysis revealed that the ZVI increased the populations of key anaerobes related to hydrolysis, acidification, and chain elongation. Correspondingly, the solubilization, hydrolysis, and acidification processes of WAS were revealed to be improved by ZVI, thereby providing more substrates (short-chain fatty acids (SCFAs)) for producing MCFAs. The mechanism studies showed that ZVI declined the oxidation-reduction potential (ORP), creating a more favorable environment for the anaerobic biological processes. More importantly, ZVI with strong conductivity could act as an electron shuttle, contributing to increasing electron transfer efficiency from electron donor to acceptor. This strategy provides a new paradigm of transforming waste sludge into assets by a low-cost waste to bring significant economic benefits to sludge disposal and wastewater treatment.

Influence of tetraconazole on the proteome profile of Saccharomyces cerevisiae Lalvin T73™ strain

This work aimed to evaluate the modifications on the proteome profile of Saccharomyces cerevisiae T73™ strain as a consequence of its adaptive response to the presence of tetraconazole molecules in the fermentation medium. Pasteurised grape juices were separately supplemented with tetraconazole or a commercial formulation containing 12.5% w/v of tetraconazole at two concentration levels. In addition, experiments without fungicides were developed for comparative purposes.Proteome profiles of yeasts cultured in the presence or absence of fungicide molecules were different. Independently of the fungicide treatment applied, the highest variations concerning the control sample were observed for those proteins involved in metabolic processes, especially in the metabolism of nitrogen compounds. Tetraconazole molecules altered the abundance of several enzymes involved in the biosynthesis of amino acids, purines, and ergosterol. Moreover, differences in the abundance of several enzymes of the TCA cycle were found. Changes observed were different between the active substance and the commercial formulation. SignificanceThe presence of fungicide residues in grape juice has direct implications on the development of the aromatic profile of the wine. These alterations could be related to changes in the secondary metabolism of yeasts. However, the molecular mechanisms involved in the response of yeasts to fungicide residues remains quite unexplored. Through this exhaustive proteomic study, alterations in the amino acids biosynthesis pathways due to the presence of the tetraconazole molecules were observed. Amino acids are precursors of some important higher alcohols and ethyl acetates (such as methionol, 2-phenylethanol, isoamyl alcohol or 2-phenylacetate). Besides, the effect of tetraconazole on the ergosterol biosynthesis pathway could be related to a higher production of medium-chain fatty acids and their corresponding ethyl acetates.

Effects of medium-chain fatty acids as alternatives to ZnO or antibiotics in nursery pig diets.

The objective of this experiment was to evaluate the effects of medium-chain fatty acids (MCFA) on nursery pig performance in place of ZnO and carbadox. In this trial, 360 weanling pigs (DNA 200 × 400; 5.4 ± 0.07 kg BW) were fed for 35 d, with 6 pigs/pen and 10 replicate pens/treatment. Upon weaning, pigs were weighed and allotted to pens based on BW in a completely randomized design to one of six treatment diets: 1) Negative control (no added ZnO or carbadox); 2) Control + 3,000 ppm ZnO in phase 1 and 2,000 ppm ZnO in phase 2; 3) Control + 50 g/ton carbadox; 4) Control + C6:C8:C10 MCFA blend; 5) Control + Proprietary Oil Blend (Feed Energy Corp.); and 6) Control + monolaurate blend (FORMI GML from ADDCON). Treatment diets were fed through two dietary phases and a common diet fed through phase three. Pigs and feeders were individually weighed on a weekly basis to determine average daily gain (ADG) and average daily feed intake (ADFI). From days 0 to 19, pigs being fed the ZnO or Carbadox diets had the greatest ADG. These pigs had significantly higher (P < 0.05) ADG than pigs fed the control or Feed Energy Proprietary Oil Blend, whereas pigs fed the C6:C8:C10 blend or FORMI GML diets had similar (P > 0.05) ADG compared with those fed carbadox. These effects were primarily driven by feed intake, which was greatest (P < 0.05) in pigs fed ZnO and carbadox. Treatment diet had a marginally significant effect (P = 0.078) on G:F. Increased day 19 BW (P < 0.05) was observed for pigs fed ZnO and carbadox compared with the negative control, whereas other treatments were intermediate. Additionally, blood data and fecal scores were collected throughout the trial. On day 21, pigs fed ZnO or carbadox had higher (P < 0.0001) glucose values than those fed the Feed Energy Proprietary Oil Blend, with other diets being intermediate, showing potential health benefits of carbadox. Although ZnO resulted in higher glucose values, it may also contribute to hepatic issues. Although replacing ZnO and carbadox with MCFA did not result in significant changes in gut microflora, it did affect fecal consistency by softening the feces during the treatment period. Overall, these results show that ZnO and carbadox are valuable additives to help maximize growth performance in early stages of the nursery. Some MCFA products, like FORMI GML, may result in similar performance, whereas others restrict it. Thus, additional research is needed to study the effectiveness of MCFA to replace ZnO or feed-based antibiotics.

Review on the production of medium and small chain fatty acids through waste valorization and CO2 fixation

The developing approaches in the recovery of resources from biowastes for the production of renewable value-added products and fuels, using microbial cultures as bio-catalyst have now became promising aspect. In the path of anaerobic digestion, the microorganisms are assisting transformation of a complex organic feedstock/waste to biomass and biogas. This potentiality consequently leads to the production of intermediate precursors of renewable value-added products. Particularly, a set of anaerobic pathways in the fermentation process, yields small-chain fatty acids (SCFA), and medium-chain fatty acids (MCFA) via chain elongation pathways from waste valorization and CO2 fixation. This review focuses on the production of SCFA and MCFA from CO2, synthetic substrates and waste materials. Moreover, the review introduces the metabolic engineering of Escherichia coli and Saccharomyces cerevisiae for SCFAs/MCFAs production. Furtherly, it concludes that future critical research might target progress of this promising approach as a valorization of complex organic wastes.

A mass spectrometry-based high-throughput screening method for engineering fatty acid synthases with improved production of medium-chain fatty acids.

Microbial cell factories have been extensively engineered to produce free fatty acids (FFAs) as key components of crucial nutrients, soaps, industrial chemicals, and fuels. However, our ability to control the composition of microbially synthesized FFAs is still limited, particularly, for producing medium-chain fatty acids (MCFAs). This is mainly due to the lack of high-throughput approaches for FFA analysis to engineer enzymes with desirable product specificity. Here we report a mass spectrometry (MS)-based method for rapid profiling of MCFAs in Saccharomyces cerevisiae by using membrane lipids as a proxy. In particular, matrix-assisted laser desorption/ionization time-of-flight (MALDI-ToF) MS was used to detect shorter acyl chain phosphatidylcholines from membrane lipids and a higher m/z peak ratio at 730 and 758 was used as an indication for improved MCFA production. This colony-based method can be performed at a rate of ~2 s per sample, representing a substantial improvement over gas chromatography-MS (typically >30 min per sample) as the gold standard method for FFA detection. To demonstrate the power of this method, we performed site-saturation mutagenesis of the yeast fatty acid synthase and identified nine missense mutations that resulted in improved MCFA production relative to the wild-type strain. Colony-based MALDI-ToF MS screening provides an effective approach for engineering microbial fatty acid compositions in a high-throughput manner.

Multidimensional Metabolic Engineering for Constructing Efficient Cell Factories

Microbial production of medium-chain fatty acids (MCFAs) is challenging, as they are rare metabolites and toxic to cells. Recently, Zhu et al. performed multidimensional engineering in Saccharomyces cerevisiae, including engineering fatty acid synthases and improving cellular tolerance to MCFAs, to build an efficient MCFA cell factory.

Medium-Chain Fatty Acid Synthesis by "Candidatus Weimeria bifida" gen. nov., sp. nov., and "Candidatus Pseudoramibacter fermentans" sp. nov.

Chain elongation is emerging as a bioprocess to produce valuable medium-chain fatty acids (MCFA; 6 to 8 carbons in length) from organic waste streams by harnessing the metabolism of anaerobic microbiomes. Although our understanding of chain elongation physiology is still evolving, the reverse β-oxidation pathway has been identified as a key metabolic function to elongate the intermediate products of fermentation to MCFA. Here, we describe two uncultured chain-elongating microorganisms that were enriched in an anaerobic microbiome transforming the residues from a lignocellulosic biorefining process. Based on a multi-omic analysis, we describe "Candidatus Weimeria bifida" gen. nov., sp. nov., and "Candidatus Pseudoramibacter fermentans" sp. nov., both predicted to produce MCFA but using different substrates. The analysis of a time series metatranscriptomic data set suggests that "Ca Weimeria bifida" is an effective xylose utilizer since both the pentose phosphate pathway and the bifid shunt are active. Furthermore, the metatranscriptomic data suggest that energy conservation during MCFA production in this organism is essential and occurs via the creation of an ion motive force using both the RNF complex and an energy-conserving hydrogenase. For "Ca Pseudoramibacter fermentans," predicted to produce MCFA from lactate, the metatranscriptomic analysis reveals the activity of an electron-confurcating lactate dehydrogenase, energy conservation via the RNF complex, H2 production for redox balance, and glycerol utilization. A thermodynamic analysis also suggests the possibility of glycerol being a substrate for MCFA production by "Ca Pseudoramibacter fermentans." In total, this work reveals unknown characteristics of MCFA production in two novel organisms.IMPORTANCE Chain elongation by medium-chain fatty acid (MCFA)-producing microbiomes offers an opportunity to produce valuable chemicals from organic streams that would otherwise be considered waste. However, the physiology and energetics of chain elongation are only beginning to be studied, and many of these organisms remain uncultured. We analyzed MCFA production by two uncultured organisms that were identified as the main MCFA producers in a microbial community enriched from an anaerobic digester; this characterization, which is based on meta-multi-omic analysis, complements the knowledge that has been acquired from pure-culture studies. The analysis revealed previously unreported features of the metabolism of MCFA-producing organisms.

Modeling a co-culture of Clostridium autoethanogenum and Clostridium kluyveri to increase syngas conversion to medium-chain fatty-acids

Microbial fermentation of synthesis gas (syngas) is becoming more attractive for sustainable production of commodity chemicals. To date, syngas fermentation focuses mainly on the use of Clostridium species for the production of small organic molecules such as ethanol and acetate. The co-cultivation of syngas-fermenting microorganisms with chain-elongating bacteria can expand the range of possible products, allowing, for instance, the production of medium-chain fatty acids (MCFA) and alcohols from syngas. To explore these possibilities, we report herein a genome-scale, constraint-based metabolic model to describe growth of a co-culture of Clostridium autoethanogenum and Clostridium kluyveri on syngas for the production of valuable compounds. Community flux balance analysis was used to gain insight into the metabolism of the two strains and their interactions, and to reveal potential strategies enabling production of butyrate and hexanoate.The model suggests that one strategy to optimize the production of medium-chain fatty-acids from syngas would be the addition of succinate. According to the prediction, addition of succinate would increase the pool of crotonyl-CoA and the ethanol/acetate uptake ratio in C. kluyveri, resulting in a flux of up to 60% of electrons into hexanoate. Another potential way to further optimize butyrate and hexanoate production would be an increase of C. autoethanogenum ethanol production. Blocking either acetaldehyde dehydrogenase or formate dehydrogenase (ferredoxin) activity or formate transport, in the C. autoethanogenum metabolic model could potentially lead to an up to 150% increase in ethanol production.

Acidogenic fermentation of cassava wastewater for volatile fatty acids production

Acidogenic fermentation (AF) of cassava wastewater was investigated with adapted and unadapted biomass with and without methanogenic inhibition techniques to enhance accumulation of volatile fatty acids (VFA) in batch tests. Subsequently, the possibility of VFA chain elongation for the production of medium-chain fatty acids (MCFA) by means of ethanol addition to the fermentation broth was evaluated. With unadapted sludge, 84.1% and 66.1% acidification could be obtained for thermally inhibited and non-inhibited conditions respectively, results 10% and 45% higher than the degrees of acidification obtained under the same conditions with adapted sludge. Results show that neither sludge adaptation nor heat treatment but rather the pH of the fermentation broth was most relevant for enhancing VFA production, the optimum pH range being 5.5 to 6. The main product was butyric acid (87% of total acid produced), and addition of ethanol to the fermentation broth promoted MCFA formation.

Multidimensional engineering of Saccharomyces cerevisiae for efficient synthesis of medium-chain fatty acids

Medium-chain fatty acids (MCFAs; C6–C12) are valuable molecules used for biofuel and oleochemical production; however, it is challenging to synthesize these fatty acids efficiently using microbial biocatalysts due to the cellular toxicity of MCFAs. In this study, both the endogenous fatty acid synthase (FAS) and an orthogonal bacterial type I FAS were engineered for MCFA production in the yeast Saccharomyces cerevisiae. To improve cellular tolerance to toxic MCFAs, we performed directed evolution of the membrane transporter Tpo1 and strain adaptive laboratory evolution, which elevated the MCFA production by 1.3 ± 0.3- and 1.7 ± 0.2-fold, respectively. We therefore further engineered the highly resistant strain to augment the metabolic flux towards MCFAs. This multidimensional engineering of the yeast at the single protein/enzyme level, the pathway level and the cellular level, combined with an optimized cultivation process, resulted in the production of >1 g l−1 extracellular MCFAs—a more than 250-fold improvement over the original strain. Efficient microbial production of medium-chain fatty acids (MCFAs; C6–C12)—valuable molecules in the oleochemical and biofuel industry—is challenging due to their cellular toxicity. Now, this work improves the production of extracellular MCFAs to over 1 g l−1 by systematically engineering yeast at multiple levels.

Unveiling the mechanisms of medium-chain fatty acid production from waste activated sludge alkaline fermentation liquor through physiological, thermodynamic and metagenomic investigations

Effective sludge treatment with bioenergy production is attracting increasing interests as large quantities of waste activated sludge (WAS) are produced during the wastewater treatment. In this study, a new biotechnical process for converting the WAS alkaline fermentation liquor (WASAFL) into valuable, easy-separated medium chain fatty acids (MCFAs) through chain elongation (CE) was investigated, which may provide a new insight into sludge treatment. In the process, ethanol was served as the electron donor (EDs) and WASAFL were main electron acceptors (EAs). The MCFAs productions were investigated under three different ED to EA ratios (i.e., 1:2, 1:1 and 2:1). The result showed that MCFAs production was increased from 2.88 ± 0.01 to 5.28 ± 0.18 g COD/L with the increase of ED to EA ratio. However, the highest MCFA selectivity was achieved at 72.9% when the ED to EA ratio was 1:1. The decrease in the selectivity at high ED:EA ratio is mainly due to the production of higher alcohol (i.e., n-butanol and n-hexanol). The thermodynamic analysis confirmed all CE processes for MCFAs production from WASAFL were exothermic reactions, with the spontaneity and energy release of the reactions increased with the ethanol level. The microbial community analysis showed that the relative abundances of Clostridium, Oscillibacter, Leptolinea and Exilispira were positively correlated with the MCFAs production. The metagenomic analysis suggested that both the reverse β-oxidization pathway and fatty acid biosynthesis pathway contributed to the CE process in the studied system. The functional enzymes were mainly associated within Clostridium, with Clostridium Kluyveri, Clostridium botulinum and Clostridium magnum being likely the key species responsible for the CE process.

Optimizing the production of short and medium chain fatty acids (SCFAs and MCFAs) from waste activated sludge using different alkyl polyglucose surfactants, through bacterial metabolic analysis

Alkyl polyglucose is an environmentally-friendly biosurfactant, which is able to enhance short-chain fatty acids production with different carbon chain lengths and concentrations, during sludge anaerobic fermentation. This presents a promising strategy for sludge re-utilization by effectively converting hazardous sludge into value-added compounds. The maximum yield of short-chain fatty acids produced from sludge was 479.3 and 462.2 mg COD/g VSS, following pretreatment with APG06 and APG1214, respectively. To the best of our knowledge, the short-chain fatty acid production performance by sludge fermentation reported here, achieved a higher level than reported in previous studies. Additionally, these findings indicate that the production of medium-chain fatty acids from sludge can be induced by alkyl polyglucoses. Finally, the microbial community and enzyme activity were also assessed to reveal the mechanism of short-/medium-chain fatty acids biosynthesis under alkyl polyglucose pretreatment. This study demonstrates that alkyl polyglucose provides an environmentally-friendly and effective strategy for enhancing the production of short-/medium-chain fatty acids from waste activated sludge. These findings are useful for the assessment of alkyl polyglucose-assisted production of short-/medium-chain fatty acids, as well as for understanding the interactions between short-/medium-chain fatty acids and microbial communities with key enzymes, to establish short-/medium-chain fatty acids metabolic pathways during sludge fermentation.

Unravelling a microbial synergy to boost caproate production via carboxylates chain elongation with ethanol

Chain elongation of volatile fatty acids for medium chain fatty acids production (e.g. caproate) is an attractive approach to treat wastewater anaerobically and recover resource simultaneously. Undefined microbial consortia can be tailored to achieve chain elongation process with selective enrichment from anaerobic digestion sludge, which has advantages over pure culture approach for cost-efficient application. Whilst the metabolic pathway of the dominant caproate producer, Clostridium kluyveri, has been annotated, the role of other coexisting abundant microbiomes remained unclear. To this end, an ethanol-acetate fermentation inoculated with fresh digestion sludge at optimal conditions was conducted. Also, physiological study, thermodynamics and 16 S rRNA gene sequencing to elucidate the biological process by linking the system performance and dominant microbiomes were integrated. Results revealed a possible synergistic network in which C. kluyveri and three co-dominant species, Desulfovibrio vulgaris, Fusobacterium varium and Acetoanaerobium sticklandii coexisted. D. vulgaris and A. sticklandii (F. varium) were likely to boost the carboxylates chain elongation by stimulating ethanol oxidation and butyrate production through a syntrophic partnership with hydrogen (H2) serving as an electron messenger. This study unveils a synergistic microbial network to boost caproate production in mixed culture carboxylates chain elongation.