A critical review of the chain elongation for biomass resource recovery: Mechanisms, advances, and challenges

BackgroundBiotechnological processes for efficient resource recovery from residual materials rely on complex conversions carried out by reactor microbiomes. Chain elongation microbiomes produce valuable medium-chain carboxylates (MCC) that can be used as biobased starting materials in the chemical, agriculture and food industry. In this study, sunflower oil is used as an application-compatible solvent to accumulate microbially produced MCC during extractive lactate-based chain elongation. The MCC-enriched solvent is harvested as a potential novel product for direct application without further MCC purification, e.g., direct use for animal nutrition. Sunflower oil biocompatibility, in situ extraction performance and effects on chain elongation were evaluated in batch and continuous experiments. Microbial community composition and dynamics of continuous experiments were analyzed based on 16S rRNA gene sequencing data. Potential applications of MCC-enriched solvents along with future research directions are discussed.ResultsSunflower oil showed high MCC extraction specificity and similar biocompatibility to oleyl alcohol in batch extractive fermentation of lactate and food waste. Continuous chain elongation microbiomes produced the MCC n-caproate (nC6) and n-caprylate (nC8) from l-lactate and acetate at pH 5.0 standing high undissociated n-caproic acid concentrations (3 g L−1). Extractive chain elongation with sunflower oil relieved apparent toxicity of MCC and production rates and selectivities reached maximum values of 5.16 ± 0.41 g nC6 L−1 d−1 (MCC: 11.5 g COD L−1 d−1) and 84 ± 5% (e− eq MCC per e− eq products), respectively. MCC were selectively enriched in sunflower oil to concentrations up to 72 g nC6 L−1 and 3 g nC8 L−1, equivalent to 8.3 wt% in MCC-enriched sunflower oil. Fermentation at pH 7.0 produced propionate and n-butyrate instead of MCC. Sunflower oil showed stable linoleic and oleic acids composition during extractive chain elongation regardless of pH conditions. Reactor microbiomes showed reduced diversity at pH 5.0 with MCC production linked to Caproiciproducens co-occurring with Clostridiumtyrobutyricum, Clostridiumluticellarii and Lactobacillus species. Abundant taxa at pH 7.0 were Anaerotignum, Lachnospiraceae and Sporoanaerobacter.ConclusionsSunflower oil is a suitable biobased solvent to selectively concentrate MCC. Extractive reactor microbiomes produced MCC with improved selectivity and production rate, while downstream processing complexity was reduced. Potential applications of MCC-enriched solvents may include feed, food and biofuels purposes.

Brewer’s spent grain (BSG) is an undervalorized organic feedstock residue composed of fermentable macromolecules, such as proteins, starch, and residual soluble carbohydrates. It also contains at least 50% (as dry weight) of lignocellulose. Methane-arrested anaerobic digestion is one of the promising microbial technologies to valorize such complex organic feedstock into value-added metabolic intermediates, such as ethanol, H2, and short-chain carboxylates (SCC). Under specific fermentation conditions, these intermediates can be microbially transformed into medium-chain carboxylates through a chain elongation pathway. Medium-chain carboxylates are of great interest as they can be used as bio-based pesticides, food additives, or components of drug formulations. They can also be easily upgraded by classical organic chemistry into bio-based fuels and chemicals. This study investigates the production potential of medium-chain carboxylates driven by a mixed microbial culture in the presence of BSG as an organic substrate. Because the conversion of complex organic feedstock to medium-chain carboxylates is limited by the electron donor content, we assessed the supplementation of H2 in the headspace to improve the chain elongation yield and increase the production of medium-chain carboxylates. The supply of CO2 as a carbon source was tested as well. The additions of H2 alone, CO2 alone, and both H2 and CO2 were compared. The exogenous supply of H2 alone allowed CO2 produced during acidogenesis to be consumed and nearly doubled the medium-chain carboxylate production yield. The exogenous supply of CO2 alone inhibited the whole fermentation. The supplementation of both H2 and CO2 allowed a second elongation phase when the organic feedstock was exhausted, which increased the medium-chain carboxylate production by 285% compared to the N2 reference condition. Carbon- and electron-equivalent balances, and the stoichiometric ratio of 3 observed for the consumed H2/CO2, suggest an H2- and CO2-driven second elongation phase, converting SCC to medium-chain carboxylates without an organic electron donor. The thermodynamic assessment confirmed the feasibility of such elongation.

Biological production of medium-chain carboxylates through chain elongation: An overview

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

BackgroundThe need for addition of external electron donors such as ethanol or lactate impairs the economic viability of chain elongation (CE) processes for the production of medium-chain carboxylates (MCC). However, using feedstocks with inherent electron donors such as silages of waste biomass can improve the economics. Moreover, the use of an appropriate inoculum is critical to the overall efficiency of the CE process, as the production of a desired MCC can significantly be influenced by the presence or absence of specific microorganisms and their metabolic interactions. Beyond, it is necessary to generate data that can be used for reactor design, simulation and optimization of a given CE process. Such data can be obtained using appropriate mathematical models to predict the dynamics of the CE process.ResultsIn batch experiments using silages of sugar beet leaves, cassava leaves, and Elodea/wheat straw as substrates, caproate was the only MCC produced with maximum yields of 1.97, 3.48, and 0.88 g/kgVS, respectively. The MCC concentrations were accurately predicted with the modified Gompertz model. In a semi-continuous fermentation with ensiled sugar beet leaves as substrate and digestate from a biogas reactor as the sole inoculum, a prolonged lag phase of 7 days was observed for the production of MCC (C6–C8). The lag phase was significantly shortened by at least 4 days when an enriched inoculum was added to the system. With the enriched inoculum, an MCC yield of 93.67 g/kgVS and a productivity of 2.05 gMCC/L/d were achieved. Without the enriched inoculum, MCC yield and productivity were 43.30 g/kgVS and 0.95 gMCC/L/d, respectively. The higher MCC production was accompanied by higher relative abundances of Lachnospiraceae and Eubacteriaceae.ConclusionsEnsiled waste biomass is a suitable substrate for MCC production using CE. For an enhanced production of MCC from ensiled sugar beet leaves, the use of an enriched inoculum is recommended for a fast process start and high production performance.

Microbial chain elongation based on methanol

Chain elongation reactor microbiomes produce valuable medium-chain carboxylates (MCC) from non-sterile residual substrates where lactate is a relevant intermediate. Gas supply has been shown to impact chain elongation performance. In the present study, the effect of nitrogen gas (N2) supply on lactate metabolism, conversion rates, biomass growth, and microbiome composition was evaluated in a lactate-fed upflow anaerobic reactor with continuous or intermittent N2 gas supply. Successful MCC production was achieved with continuous N2 gas supply at low superficial gas velocities (SGV) of 0.22 m∙h−1. Supplying N2 at high SGV (>2 m∙h−1) either continuously (2.2 m∙h−1) or intermittently (3.6 m∙h−1) disrupted chain elongation, resulting in production of short-chain carboxylates (SCC), i.e., acetate, propionate, and n-butyrate. Caproiciproducens-dominated chain-elongating microbiomes enriched at low SGV were washed out at high SGV where Clostridium tyrobutyricum-dominated microbiomes thrived, by displaying higher lactate consumption rates. Suspended growth seemed to be dominant regardless of SGV and gas supply regime applied with no measurable sludge bed formed. The highest MCC production from lactate of 10 g COD∙L−1∙d−1 with electron selectivities of 72 ± 5%was obtained without N2 gas supply at a hydraulic retention time (HRT) of 1 day. The addition of 5 g∙L−1 of propionate did not inhibit chain elongation, but rather boosted lactate conversion rates towards MCC with n-heptylate reaching 1.8 g COD∙L−1∙d−1. N2 gas supply can be used for mixing purposes and to steer lactate metabolism to MCC or SCC production.

Medium chain carboxylates (MCCs) are important precursors for biodiesel production. Using chain elongation to produce MCCs is an emerging bioenergy technology. In this study, batch tests were conducted to investigate fermentative MCC production through chain elongation from acetate, propionate, n-butyrate, and ethanol. The effect of the acid/ethanol ratio on MCC production by mixed culture was investigated. Better MCC production, especially n-caproate production, was achieved at optimal acid/ethanol ratios of 1:4, 1:3, and 1:2 with acetate, propionate, and n-butyrate as the electron acceptor, respectively. The n-caproate concentration was high, up to 41.54 mmol/L, and the highest n-caproate production efficiency was 57.96% with the n-butyrate/ethanol ratio of 1:2. The higher concentration of ethanol might stimulate the growth of chain elongation bacteria to promote chain elongation. The highest MCC production efficiency with different electron acceptors corresponded to less carbon loss and a higher chain elongation degree. In addition, with the optimal acid/ethanol ratio, the substrate was maximally utilized for chain elongation. The microbial community analysis confirmed the carbon balance analysis with the maximum relative abundance of 52.66–60.55% of the n-caproate producer Clostridium_sensu_stricto_12 enriched by the optimal acid/ethanol ratios with different volatile fatty acids (VFAs) as electron acceptors.

In order to achieve a sustainable society it is paramount to develop technologies that can aid in recycling waste streams and in reducing the environmental footprint of human activity on this planet. The introduction of this thesis underlines the necessity of transitioning towards a circular economy and presents a technology that can help with recycling and valorising organic residues. Chain elongation fermentation allows the production of medium chain carboxylates (MCC) from complex organic waste. The fermentation products can be used for a wide range of applications within agriculture and the chemical industry. In this thesis new methods for chain elongation are discovered and researched that broaden the product spectrum of the technology. Besides straight molecular chains, branched chained MCCs have been shown as dominant products in these new fermentation types. The products with a different molecular structure inherently have different physical properties that might make them better suited for certain applications within society. The research chapters elaborated on how specific selection pressures in open culture fermentations can be used to enrich microbiomes to harbour desired biocatalytic capabilities. Two different types of chain elongation fermentation are the subject of these investigations: methanol-based and ethanol-based chain elongation. These two alcohols are used by the microbiomes as electron donors within the fermentation. In order to harvest energy and grow, the organism use a metabolism where they process the energy-rich electrons from the alcohols. The electrons are subsequently used to reduce a carboxylate electron acceptor, which is simultaneously elongated in the process. Within methanol-based chain elongation microbiomes, the elongation of acetate leads to mainly butyrate formation. Depending on the pH, the microbiome could be enriched to the point that isomerization of n-butyrate to isobutyrate occurred (Chapter 2). At a pH around 6.75 no isomerization happened, but at a pH around 5.5 it did. The ratio of the n-butyrate and isobutyrate concentrations were found to be coupled to the thermodynamic equilibrium of isomerization. The responsible microorganism for isobutyrate formation was found to be closely related to an earlier described Clostridium luticellarii. When propionate was used as electron acceptor, elongation to n-valerate occurred (Chapter 3). The enriched microbiome also contained C. luticellari as dominant microorganism. The microbiome was capable to simultaneously elongate both acetate and propionate to n-butyrate, isobutyrate, n-valerate as dominant products. Also small amounts of n-caproate were formed whenever n-butyrate was present within methanol-based chain elongation microbiomes. Based on literature a metabolic pathway for methanol-based chain elongation was proposed that could describe the experimentally observed stoichiometry. Microbiomes were also enriched to perform ethanol-based chain elongation, in particular for the elongation of branched electron acceptors. When isobutyrate was fed to the microbiome together with ethanol, elongation towards isocaproate was stimulated (Chapter 4). However, due to the nature of ethanol-based chain elongation in situ acetate formation always occurs. Additionally ethanol can in some situations be directly converted towards acetate and hydrogen by other microbes that compete for substrate. This leads to a situation where the chain elongators can use an increasing amount of acetate as electron acceptor, which seemed to be preferred over isobutyrate. Limiting acetate supply led to isocaproate production up to 20% of the total products. In an attempt to control the excessive ethanol oxidation in the reactor, conditions were adjusted to limited CO2 supply (Chapter 5). Limitation of CO2 leads to a deficiency for hydrogenotrophic methanogens; they need CO2 as electron acceptor for their energy-providing, methane-producing metabolism. However, the conditions of the reactor were such that an alternative route for ethanol oxidation was stimulated. High ethanol to acetate ratios, and high (other) carboxylate to corresponding alcohol ratios created the potential for carboxylate reduction coupled to ethanol oxidation. In turn in situ acetate formation persisted, whereby straight chain elongation remained the most dominant metabolic functionality. In the general discussion hypotheses are presented that could further mechanistically explain the observed metabolic functionalities. For methanol-based chain elongation the metabolic pathway is revised, using supporting evidence from the genome of C. luticellarii. Improvements on reactor operation are suggested to increase the performances. Additionally recommendations are given on how integrated bioprocess designs could circumvent downstream processing difficulties. Finally an outlook on ethanol-based chain elongation fermentation for branched carboxylate production is presented.

Production of medium-chain carboxylic acids using sewage sludge pretreated by combined Fenton and persulfate oxidation

Electro-fermentation offers a promising strategy to upgrade organic molecules from food waste into sustainable fuels that can help decarbonize the global transportation sector. Despite its potential for decarbonization, the electro-fermentation of food waste into transportation fuel precursors such as medium chain carboxylates (MCCs) is underexplored and requires improvements in rates and selectivity.This study aims to boost the rates and selectivity of food waste electro-fermentation into MCCs using a fluidized bed reactor containing powdered activated carbon (PAC) coupled with rumen microbes enriched for microbial chain elongation. Two electrochemical H-cell reactors—R1 (control) and R2 (with PAC)—were tested under varying cathodic and anodic potential using synthetic food waste (10 g COD/L) as the substrate. R2 significantly outperformed R1, particularly at -0.8 V vs Ag/AgCl, achieving a caproic acid concentration of 6.14 ± 0.69 g COD/L—more than double that of R1 (2.54±0.57 g COD/L). Enhanced electrochemical activity and reaction kinetics in R2 were attributed to PAC’s role in expanding biofilm surface area and facilitating electron transfer. Genomic microbial community analysis revealed distinct spatial distributions across the system, especially on electrodes and PAC particles. Advanced characterization of the extra-cellular polymeric substrates (EPS) showed significant increases in EPS production and the formation of redox-active compounds in the presence of PAC.Overall, these findings highlight the ability of fluidized bed reactors with chain-elongating organisms to improve the rates and selectivity of MCC production from food waste during electro-fermentation. This technology offers a promising pathway for organic waste valorization into sustainable transportation fuels, supporting a circular carbon economy. Efforts towards testing new reactor operation modes, expanding the portfolio of products, and implementing new types of waste biomass streams are ongoing.

Metabolic interactions in chain elongation system with granular activated carbon for medium-chain carboxylates production

To convert wastes into sustainable liquid fuels and chemicals, new resource recovery technologies are required. Chain elongation is a carboxylate-platform bioprocess that converts short-chain carboxylates (SCCs) (e.g., acetate [C2] and n-butyrate [C4]) into medium-chain carboxylates (MCCs) (e.g., n-caprylate [C8] and n-caproate [C6]) with hydrogen gas as a side product. Ethanol or another electron donor (e.g., lactate, carbohydrate) is required. Competitive MCC productivities, yields (product vs. substrate fed), and specificities (product vs. all products) were only achieved previously from an organic waste material when exogenous ethanol had been added. Here, we converted a real organic waste, which inherently contains ethanol, into MCCs with n-caprylate as the target product. We used wine lees, which consisted primarily of settled yeast cells and ethanol from wine fermentation, and produced MCCs with a reactor microbiome. We operated the bioreactor at a pH of 5.2 and with continuous in-line extraction and achieved a MCC productivity of 3.9 g COD/L-d at an organic loading rate of 5.8 g COD/L-d, resulting in a promising MCC yield of 67% and specificities of 36% for each n-caprylate and n-caproate (72% for both). Compared to all other studies that used complex organic substrates, we achieved the highest n-caprylate-to-ncaproate product ratio of 1.0 (COD basis), because we used increased broth-recycle rates through the forward membrane contactor, which improved in-line extraction rates. Increased recycle rates also allowed us to achieve the highest reported MCC production flux per membrane surface area thus far (20.1 g COD/m2-d). Through microbial community analyses, we determined that an operational taxonomic unit (OTU) for Bacteroides spp. was dominant and was positively correlated with increased MCC productivities. Our data also suggested that the microbiome may have been shaped for improved MCC production by the high broth-recycle rates. Comparable abiotic studies suggest that further increases in the broth-recycle rates could improve the overall mass transfer coefficient and its corresponding MCC production flux by almost 30 times beyond the maximum that we achieved. With improved in-line extraction, the chain-elongation biotechnology production platform offers new opportunities for resource recovery and sustainable production of liquid fuels and chemicals.

Chain elongation is a relevant bioprocess in support of a circular economy as it can use a variety of organic feedstocks for production of valuable short and medium chain carboxylates, such as butyrate (C4), caproate (C6), and caprylate (C8). Alcohols, including the biofuel, butanol (C4), can also be generated in chain elongation but the bioreactor conditions that favor butanol production are mainly unknown. In this study we investigated production of butanol (and its precursor butyrate) during ethanol and acetate chain elongation. We used semi-batch bioreactors (0.16L serum bottles) fed with a range of ethanol concentrations (100-800mM C), a constant concentration of acetate (50mM C), and an initial total gas pressure of ∼112kPa. We showed that the butanol concentration was positively correlated with the ethanol concentration provided (up to 400mM C ethanol) and to chain elongation activity, which produced H2 and further increased the total gas pressure. In bioreactors fed with 400mM C ethanol and 50mM C acetate, a concentration of 114.96 ± 9.26mM C butanol (∼2.13gL-1) was achieved after five semi-batch cycles at a total pressure of ∼170kPa and H2 partial pressure of ∼67kPa. Bioreactors with 400mM C ethanol and 50mM C acetate also yielded a butanol to butyrate molar ratio of 1:1. At the beginning of cycle 8, the total gas pressure was intentionally decreased to ∼112kPa to test the dependency of butanol production on total pressure and H2 partial pressure. The reduction in total pressure decreased the molar ratio of butanol to butyrate to 1:2 and jolted H2 production out of an apparent stall. Clostridium kluyveri (previously shown to produce butyrate and butanol) and Alistipes (previously linked with butyrate production) were abundant amplicon sequence variants in the bioreactors during the experimental phases, suggesting the microbiome was resilient against changes in bioreactor conditions. The results from this study clearly demonstrate the potential of ethanol and acetate-based chain elongation to yield butanol as a major product. This study also supports the dependency of butanol production on limiting acetate and on high total gas and H2 partial pressures.

Microbial chain elongation using biomass-derived lactate can be steered to produce a variety of medium-chain carboxylates (MCC), which then need to be separated before application. In this study, we evaluated the effects of adding conductive and/or adsorbing materials to batch and continuous open-culture lactate-based chain elongation. Incubation with granular activated carbon (GAC), nickel foam (NF), and stainless steel (SS) mesh improved lactate use for chain elongation due to a ∼30% reduction in propionate formation compared to the control (no material). Isobutyrate production was stimulated in the presence of GAC and NF (up to 1.2 g·L–1, 9% electron selectivity). Adding GAC to a continuous reactor led to in situ adsorption of n-caproate. GAC showed a high affinity to n-caproate from real and artificial effluents, as well as from blends containing C2–C8 carboxylates, adsorbing 60–80% of the initial n-caproate with recoveries up to 42% after desorption. Adsorption isotherms showed that n-caproate adsorption increased with decreasing pH conditions (184–243 mg·g GAC–1). In conclusion, conductive materials changed the product spectrum and steered to isobutyrate formation in batch open-culture chain elongation. Based on the promising adsorption properties of GAC, the first design of chain elongation with in-line adsorption–recovery is proposed as well as potential direct applications of MCC-loaded porous carbons.