Chain Elongation with Reactor Microbiomes: Open-Culture

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Chain elongation with reactor microbiomes: openculture biotechnology to produce biochemicals Largus T. Angenent, Hanno Richter, Wolfgang Buckel, Catherine M. Spirito, Kirsten J. Steinbusch, Caroline Plugge, David P.B.T.B. Strik, Tim I.M. Grootscholten, Cees J.N. Buisman, and Hubertus V.M. Hamelers Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.5b04847 • Publication Date (Web): 08 Feb 2016 Downloaded from http://pubs.acs.org on February 11, 2016

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Environmental Science & Technology

Extensive review

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Chain elongation with reactor microbiomes: open-culture biotechnology to produce biochemicals

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Largus T. Angenent1,*, Hanno Richter1, Wolfgang Buckel2, Catherine M. Spirito1, Kirsten J. Steinbusch3,4, Caroline M. Plugge5, David P. B. T. B. Strik3, Tim I. M. Grootscholten3,6, Cees J. Buisman3,7, and Hubertus V. M. Hamelers7

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Department of Biological and Environmental Engineering, Cornell University, 226 Riley-Robb Hall, Ithaca, NY 14853, USA Laboratorium für Mikrobiologie, Fachbereich Biologie and SYNMIKRO, PhilippsUniversität, 35032 Marburg, Germany and Max-Planck-Institut für terrestrische Mikrobiologie, Karl-von-Frisch-Str. 10, 35043 Marburg, Germany Sub-Department of Environmental Technology, Wageningen University, P.O. Box 17, 6700 AA Wageningen, The Netherlands Delft Advanced Biorenewables (DAB), Julianalaan 67, 2628 BC Delft, The Netherlands

Laboratory of Microbiology, Wageningen University, Dreijenplein 10, 6703 HB Wageningen, The Netherlands Royal Cosun, P.O. Box 3411, 4800 MG Breda, The Netherlands

Wetsus - European Centre of Excellence for Sustainable Water Technology, Oostergoweg 9, 8911 MA Leeuwarden, The Netherlands

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* Corresponding author: E-mail: [email protected]; Fax: +1-607-255-4449; Tel: +1-

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Abstract:

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Chain elongation into medium-chain carboxylates, such as n-caproate and n-caprylate, with ethanol as an electron donor and with open cultures of microbial consortia (i.e., reactor microbiomes) under anaerobic conditions is being developed as a biotechnological production platform. The goal is to use the high thermodynamic efficiency of anaerobic fermentation to convert organic biomass or organic wastes into valuable biochemicals that can be extracted. Several liter-scale studies have been completed and a first pilot-plant study is underway. However, the underlying microbial pathways are not always well understood. In addition, an interdisciplinary approach with knowledge from fields ranging from microbiology and chemical separations to biochemistry and environmental engineering is required. To bring together research from different fields, we reviewed the literature starting with the microbiology and ending with the bioprocess engineering studies that already have been performed. Because understanding the microbial pathways is so important to predict and steer performance, we delved into a stoichiometric and thermodynamic model that sheds light on the effect of substrate ratios and environmental conditions on product formation. Finally, we ended with an outlook.

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1. Introduction

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A start has been made to generate renewable energy through implementation of wind energy, photovoltaics, hydropower, and anaerobic digestion among other renewable technologies. A path towards implementation is needed for renewable chemicals as well. Biochemical production from sugars or other agricultural commodities (such as corn), which is currently employed, may not be sustainable and competes with human food production. Renewable chemical production, therefore, has to come from innovative platforms utilizing other feedstock. Projects are being initiated to capture and convert carbon dioxide and carbon monoxide from industries into biochemicals. However, a vast research program to genetically modify bacteria is necessary to go beyond acetate or ethanol as biochemicals. Another platform of interest for renewable chemical production is the carboxylate platform 1, 2. Open cultures of anaerobic microbial consortia – reactor microbiomes – have the ability to efficiently convert complex organic wastes, which must be treated anyway, into biochemicals.

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Reactor microbiomes as stable biocatalysts have already been tested in thousands of fullscale anaerobic digesters all over the world to generate methane at high selectivities and rates 3, 4. Here, selectivity is based on product divided by substrate consumed. For anaerobic digesters, simple tanks are being used with a continuous feeding scheme that in principle could be operated indefinitely and that never requires sterilization. This is advantageous due to the considerable lower capital and operating costs compared to axenic systems 5, 6. The genetic spectrum of reactor microbiomes is very large due to the thousands of microbial species that are present or that are constantly introduced. In fact, to convert a potentially variable and complex biomass substrate (i.e., an organic waste stream), the concerted action of different trophic groups in a food web is required. One disadvantage of open-culture biotechnology is that only environmental conditions of the reactor microbiome can be manipulated to achieve process control. In contrast, for axenic cultures both the conditions and the genetics can be controlled. 2


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In open cultures with long cell residence times, the conversion of organic substrates will result in the production of the end product with the lowest free energy content per electron. In anaerobic sediments and digesters this is methane. Methane has an oxidation number of 8 (the number of electrons transferrable per C-atom) 7, and can be formed from a wide array of organic material ranging from carbohydrates, proteins, and fats. However, under certain environmental conditions, for example, by adding a reduced compound, such as ethanol (oxidation number of 6), and/or by inhibiting methanogens (via addition of specific antimicrobials or via reducing the pH value), the production of ncaproate (oxidation number of 5.33) or n-caprylate (oxidation number of 5.5) from acetate (oxidation number of 4) or n-butyrate (oxidation number of 5), is the one option for an open culture to conserve energy under anaerobic conditions. Anaerobic fermentation as the underlying bioprocess is important for biochemical production for several reasons: 1) the circumvention of oxygen addition reduces capital and operating costs considerably compared to aerobic bioprocessing; and 2) little energy is lost in the shift of electrons from one organic molecule to another, resulting in a very efficient conversion platform.

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n-Caproate is a medium-chain carboxylate (MCC), which is one of the most attractive products from the carboxylate platform (here, carboxylates are defined as the total of their undissociated and dissociated forms). MCCs are saturated fatty acids, which have chains with six to twelve carbons including one carboxylic group (n-caproate [C6]; nheptanoate [C7]; n-caprylate [C8]; etc.). MCCs have potential to be extracted from water due to their hydrophobic carbon-chains, leading to a relatively low maximum solubility in water when they are in their undissociated acid form. In this form and at sufficiently high concentrations, MCCs can form an oily liquid at lower pH values around or below their pKa, which spontaneous separates the MCC from water. The undissociated forms of MCCs inhibit microbial life at relatively low concentrations 8, 9. In this review, we will focus mostly on n-caproate as a platform chemical because it has already been produced at high rates and selectivities. Here, we also include n-caprylate because it: 1) is often produced simultaneously with n-caproate; 2) may become the main fermentation product in the future; and 3) is a preferred MCC as a valuable commercial product compared to ncaproate. The industrial and agricultural applications for MCCs when in the undissociated form are broad and expandable. Currently, MCCs are harvested from plant oils and animal fats and are used to manufacture products, including fragrances, pharmaceuticals, feed additives, antimicrobials, lubricants, rubbers, and dyes. Recent research has also proposed to convert MCCs into liquid biofuels as part of a C6 fuel platform 10.

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Although MCCs were known to be fermentation end products in environmental sediments and bioreactors, it was only recently that researchers envisioned a continuous MCC production process as an open-culture biotechnological production platform with a commercial future. The development of a continuous MCC process is also desirable from an environmental point of view, because it enables the production of valuable chemicals from organic waste materials. This review aims at bringing together the scientific fields needed to develop such process. First, we discuss the microbiology of the process, showing the wide variety of microbes and substrates available to produce MCCs. Second, we explain in depth the physiology and thermodynamics of pertinent microbial pathways. This is crucial as these two together determine the output of a microbiome in terms of

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product selectivity. Third, we discuss the bioprocess engineering aspects; not only the control of MCC production itself but also substrate pretreatment and product extraction and recovery (including in-situ extraction). Finally, we outline the main challenges ahead.

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2. Microbiology

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2.1 Early Work

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To our knowledge, the first publication about n-caproate production with a natural microbial inoculum (open culture) stemmed from 1868. Béchamp 11 described an experiment in which ethanol (42.5 g L-1), chalk from a river bed with environmental microbes, and precipitated albuminous matter from meat was added to water in a bottle, closed, and left for months at 20-30°C. The result was a solution that contained 8.2 g L-1 (71 mM) of n-caproate. He also reported that 6 g L-1 ethanol was left over after many months, and we assume here that the biochemical reaction stopped due to product inhibition. Likely, the chalk was not only important as an inoculum but also as a buffer to maintain a high enough pH (not measured) to prevent an immediate toxic concentration of the undissociated species. If we use an upper toxicity concentration limit of 0.87 g L-1 (7.5 mM) for undissociated n-caproic acid with an acclimated biomass (Table 1), our estimated final pH of the solution would have been 5.81. In a repeat of this experiment an n-caproate concentration of 5.0 g L-1 (43 mM) was found, which results in an estimated pH of 5.56 11. Thus, at least at the end of the fermentation period, pH values were mildly acidic.

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Barker 12 described in an elegant paper his work on n-caproate production with canal mud (open culture), which he had performed as a postdoc in the Kluyver Lab in Delft, The Netherlands. In February 1939, Barker and Taha 13 isolated a rod-shaped, spore-forming anaerobic bacterium growing on ethanol and acetate, which they named Clostridium kluyveri. Later, Barker et al. 14 found with pure cultures of C. kluyveri that the products of this fermentation were n-butyrate, n-caproate, and molecular hydrogen. A high ethanol/acetate ratio favored n-caproate and a low ratio favored n-butyrate. In addition, replacing acetate by propionate resulted in the production of n-valerate and small amounts of n-heptanoate. Finally, ethanol and n-butyrate gave rise to n-caproate, but not n-caprylate 13.

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2.2 Reverse β oxidation with ethanol as the electron donor

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Early research with C. kluyveri unraveled that chain elongation of short-chain carboxylates (SCCs), such as acetate, into n-caproate occurs via the reverse β oxidation pathway with ethanol as a source of carbon, energy, and reducing equivalents 15, 16. This seems to be the most important chain elongation pathway in the recently published biotechnology studies 17, 18. The reverse β oxidation pathway is a cyclic process (Fig. 1-2) and adds an acetyl-CoA molecule, which is derived from ethanol, to a carboxylate, elongating its carbon chain length with two carbons (C2) at a time (i.e., acetate [C2] to nbutyrate [C4], n-butyrate [C4] to n-caproate [C6], n-caproate [C6] to n-caprylate [C8], propionate [C3] to n-valerate [C5], n-valerate [C5] to n-heptanoate [C7], etc.). This cyclic process enables the oxidation of ethanol to acetyl-CoA by NAD+ and the endergonic reduction of ferredoxin by NADH. Acetyl-CoA gives rise to substrate-level

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phosphorylation, whereas reduced ferredoxin forms hydrogen and generates an electrochemical Na+/H+-gradient for electron-transport phosphorylation (Fig. 1).

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2.3 Electron donors besides ethanol

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Other molecules than ethanol have also been found to act as electron donors to elongate carboxylates (electron acceptors). For example, the chain elongation of acetate to nbutyrate with DL-lactate has been described in rumen studies 19, which was recently reviewed by Spirito et al. 20. Several different bacterial strains in pure culture were shown to convert exogenous DL-lactate into n-caproate 19, 21-24. In addition, several observations of endogenous lactate consumption and simultaneous n-caproate accumulation during batch-fed degradation of complex substrates were made with reactor microbiomes 25-28. However, proof of lactate to n-caproate conversion with a reactor microbiome was only made recently from a study during which exogenous lactate was fed to a batch bioreactor with a specific inoculum 29. Other electron donors that have been used for the production of n-caproate with pure bacterial cultures are: methanol 30; n-propanol 31, 32; amino acids in peptides 23; D-galactitol, which after an enzymatic reaction is a breakdown product from seaweed 33; and pyruvate 23, 34. Sugars, such as glucose, fructose, and sucrose, can also form Acetyl-CoA, and have been used as electron donors for unintended n-caproate production at relatively low selectivities, but high rates, in studies for which hydrogen gas production was the primary goal 35, 36.

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In addition, Zhang et al. 37 have observed n-caproate production after addition of just molecular hydrogen and carbon dioxide to a microbiome. The authors suggested that this occurred via a direct chain elongation pathway, even though: 1) previous work with pure cultures of C. kluyveri had not been able to replace ethanol by molecular hydrogen 32; and 2) a bottleneck in this direct pathway had been identified by a detailed thermodynamic modeling study 38. Until further proof is given we, therefore, assume that ethanol is still the real electron donor for n-caproate production in the Zhang et al. study 37. This because feeding molecular hydrogen and carbon dioxide to a microbiome would initiate a sequence of microbial pathways with ethanol as an intermediate (i.e., homoacetogenesis, acetate conversion into ethanol, and reverse β oxidation with ethanol). It also fits with the finding that n-caproate production with just acetate and molecular hydrogen in a microbiome was possible, but again with ethanol as an formed intermediate 17. The production rate with acetate and ethanol was considerably higher than with acetate and molecular hydrogen, likely because the pathway of acetate conversion to ethanol is observed at sluggish rates in reactor microbiomes 39.

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2.4 Electron acceptors

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A multitude of compounds have been used as electron acceptors for MCC production with pure bacterial cultures, including the mono-carboxylates acetate, propionate, nbutyrate, as well as the di-carboxylates succinate and malate 31, 32. For C. kluyveri, acetate elongation is linked with coupling a first acetyl CoA molecule to a second acetyl CoA molecule (that is derived from ethanol) to form acetoacetyl CoA in the reverse β oxidation cycle (Fig. 1). Elongating an odd-numbered electron acceptor, such as propionate, indicates that propionyl CoA could replace the first acetyl CoA in this step. Similarly, this is butyryl-CoA for n-butyrate (Fig. 2A). The highest growth rate was

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obtained with ethanol and acetate 31, indicating that the reverse β oxidation pathway of C. kluyveri may have a preference for carboxylates and electron donors with a shorter-chain carbon length. Therefore, the current record production rate of n-caproate with microbiomes may be partly explainable due to the use of ethanol and acetate as a substrate 40.

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2.5 Pure cultures of wild-type bacterial strains that can produce MCCs

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Besides two strains of C. kluyveri, we will discuss five other wild-type strains that are known to produce MCCs (Table 2). Most of the bacteria that can chain elongate are Firmicutes in the class Clostridia, but other bacteria from different locations in the phylogenetic tree can produce MCCs (Table 2), and others will almost surely be isolated. Clostridium kluyveri is by far the best-studied pure culture bacterium with respect to chain elongation. Besides the strain that was isolated from canal mud 13, C. kluyveri has also been isolated from bovine rumen 31. The rumen isolate displayed a narrow substrate range but broad temperature and pH ranges, which is unusual for ruminal bacteria. The bacterium was present at only very low levels in the rumen, while at much higher levels in silage (0.26% of bacterial 16S rRNA gene copy number). Since silage was fed to the cows, it was likely the real source of C. kluyveri. The rumen strain was able to use npropanol as an electron donor in addition to ethanol 31.

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Eubacterium pyruvativorans was isolated from sheep rumen fluid 23, 41. Its growth was supported by amino acids (i.e., alanine, glycine, serine, and threonine) or peptides with pyruvate as the central metabolite. Growth on amino acids was increased by the addition of acetate, propionate, and n-butyrate; while propionate and n-butyrate were utilized during growth to form n-valerate and n-caproate, respectively 23. Similarly to C. kluyveri with ethanol as the donor, two carbon atoms from amino acids are utilized to elongate SCCs. E. pyruvativorans seems to also conserve energy via the oxidation of pyruvate to acetate and the generation of ATP, but it cannot use ethanol. Further studies showed that besides pyruvate (from amino acids and peptides), this microbe can grow on vinyl acetate, and DL-lactate, and crotonate 23.

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Clostridium sp. BS-1 was isolated from anaerobic digester sludge from a Korean wastewater treatment plant 33. Strain BS-1 belongs phylogenetically to the genus Clostridium (Cluster IV) and is most closely related strain to Clostridium sporosphaeroides. Clostridium sp. BS-1 efficiently produces n-caproate from D-galactitol. Addition of yeast extract or SCCs, such as acetate 33 and n-butyrate 42, to the growth medium for Clostridium sp. BS-1 increased n-caproate production considerably. Eubacterium limosum was isolated from the rumen of a sheep that was fed a molassesbased diet, which contained pectin 30. E. limosum formed acetate, n-butyrate, and ncaproate during growth on methanol with carbon dioxide; plus acetate and n-butyrate, but not n-caproate, during growth on molecular hydrogen and carbon dioxide. In addition, glucose fermentation resulted in some n-caproate production. Finally, polyhydroxyl alcohols, isoleucine, valine, and L-lactate stimulated growth 30, but no information on ncaproate production was shared.

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Megasphaera elsdenii was isolated from the rumen contents of sheep 21, 43. This bacterium ferments DL-lactate, glucose, fructose, and sucrose with the formation of

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acetate, propionate, n-butyrate, n-valerate, n-caproate, molecular hydrogen, and carbon dioxide at different ratios depending on the substrate 21, 44. Energy is conserved by the oxidation of pyruvate 20. DL-lactate with glucose affects the produced MCC. With just glucose, M. elsdenii produces mainly the SCC n-butyrate 19. However, depending on substrate composition, concentration, and in-situ extraction, the main product can be ncaproate 44. Finally, Rhodospirillum rubrum was the first-ever isolated photosynthetic bacterium (from a dead mouse) and is a non-oxygenic purple photosynthetic bacterium. This strain was isolated in 1887 by Prof. von Esmarch, who invented the roll tube method 45 . Strain SI of this bacterium has been found to produce n-caproate during dark fermention of pyruvate 34.

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3. Stoichiometry and thermodynamics of reverse β oxidation with ethanol based on C. kluyveri

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3.1 Ethanol and acetate elongation to n-butyrate at high substrate conditions

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Two papers from the end of the 1960s established a scheme of energy conservation in C. kluyveri 15, 16, which was further explored in 1985 46, and recently updated in a comprehensive form by Seedorf et al. 47, to describe the complete pathway for formation of n-butyrate and n-caproate from ethanol and acetate. Here, we start with considering only n-butyrate production from acetate and ethanol at standard physiological conditions (1 M of substrates and products, a water activity of 1, and a ~101 kPa (1 atm) hydrogen partial pressure at a pH of 7.0 and a temperature of 25°C), which for reactor microbiomes is a very high concentration of ethanol and carboxylates (Eq. 1 and Fig. 1A).

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∆G0’ = −184.5 kJ reaction-1

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In the oxidative part of the reverse β oxidation pathway, 6 ethanol are oxidized by 12 NAD+ via acetaldehyde to 6 acetyl-CoA, one of which is further converted to acetate by substrate-level phosphorylation for the synthesis of 1 ATP via acetylphosphate (Fig. 1A). Therefore, the carbon of 1 ethanol is not immediately introduced in the reductive part, but rather enters the acetate pool. In the reductive part, 5 acetyl-CoA + 5 acetate (4 from Eq. 1 + 1 from ethanol oxidation) + 10 NADH give rise to 5 n-butyrate, and the remaining 2 NADH ultimately reduce 4 H+ to 2 H2 (Fig. 1A).

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Several phenomena had remained mysterious for a long time in regards to the reductive part: 1) it had been unclear how C. kluyveri is able to produce molecular hydrogen, since the physiological redox potential of NADH (E’ = −280 mV; E0’ = −320 mV) is too high to reduce protons to H2 (E0’ = −414 mV); and 2) hints existed that the metabolism allows for a larger production than 1 ATP, because the ∆G0’ of the fermentation (Eq. 1) is sufficient to produce ~ 2.5 ATP (184.5 kJ reaction-1 [= mol-1 H+]/72 kJ mol-1 ATP = 2.56) 48 , while no additional mechanisms for ATP production, such as electron-transport phosphorylation, had been identified in C. kluyveri. This was resolved by the discovery of electron bifurcation (i.e., division of electron transfer into two branches) 48 and the finding that ferredoxin-NAD reductase (Rnf) is involved 47.

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3.2 Electron bifurcation and ferredoxin-NAD reductase (Rnf)

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In the reductive part of the reverse β oxidation, the reduction of 5 acetyl-CoA to 5 butyryl-CoA involves two NADH-dependent steps ((i) and (ii) in Fig. 1A): i) the reduction of 5 acetoacetyl-CoA to 5 (S)-3-hydroxybutyryl-CoA (E0’ = −240 mV), which is catalyzed by 3-hydroxybutyryl-CoA dehydrogenase; and ii) the reduction of 5 crotonyl-CoA to 5 butyryl-CoA (E0’ = −10 mV), which is catalyzed by a complex of: A) butyryl-CoA dehydrogenase (Bcd); and B) electron transferring flavoprotein (Etf) to form BcdA-EtfBC, which is an electron bifurcating enzyme complex (Fig. 1-2). Whereas 3hydroxybutyryl-CoA dehydrogenase works close to equilibrium (∆E = +40 mV), BcdAEtfBC catalyzes a highly exergonic reaction (∆E = +270 mV), which is coupled to energy conservation by reduction of ferredoxin (E’ = −500 mV; ∆E = −220 mV) 47, 48. The two electrons from NADH are transferred to different electron acceptor molecules, resulting in two reduced products (the electrons bifurcate into two paths). For 10 NADH, 10 electrons transfer exergonically to the more positive crotonyl-CoA to produce 5 butyrylCoA (2 electrons are required to reduce 1 crotonyl-CoA in two subsequent electron transfer steps); and the other 10 electrons transfer endergonically to reduce the more negative ferredoxin (Fd-red in Fig. 1-2) 47-51.

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Next, the 10 reduced ferredoxin are re-oxidized to oxidized ferrodoxin (Fdox in Fig. 1-2) in two ways ((iii) and (iv) in Fig. 1A): iii) by reduction of 4 protons catalyzed by the hydrogenase HydA (H2-ase in Fig. 1-2) to yield 2 molecular hydrogen; and iv) by reduction of 3 NAD+ catalyzed with membrane-bound ferredoxin-NAD+ reductase (Rnf) (Fig. 1A). This Rnf is a H+/Na+ pump and regenerates NADH while forming an electrochemical H+/Na+ gradient, which drives ATP-synthesis by the F1F0-ATP synthase. Our standard reaction with ethanol and acetate generates 3 × 2 ∆µH+/Na+ (Fig. 1A). Because the synthesis of 1 ATP requires 4 ∆µH+/Na+, electron bifurcation contributes 1.5 ATP to the overall energy budget. Thus, in total 2.5 ATP are produced at an efficiency of 72 kJ mol-1 ATP 48. It is important to note that to complete this high substrate concentration pathway with Rnf and ATP synthase, the total Gibbs free energy release must reach a threshold of ≥2.5x72 (≥180) kJ reaction-1 (Req in Fig. 2B).

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3.3 Ethanol and acetate elongation to n-butyrate at low substrate conditions

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For the discussion above, we performed the calculation for standard physiological conditions, which assumes a high substrate (ethanol) concentration (1 M). At physiological conditions in our microbiomes, however, the concentrations of ethanol and the other species may be very low. Therefore, the chain elongation reaction has to be examined at these relatively low (ethanol) concentrations as well (for this section 3.3 we maintained a similarly high hydrogen partial pressure than for 3.1). For example, at a concentration of 1 mM for ethanol, acetate, n-butyrate, a hydrogen partial pressure of ~101 kPa (1 atm), a water activity of 1, a pH of 7.0, and a temperature of 25°C, the ∆G’ becomes less exergonic compared to the ∆G0’ of Eq. 1, enforcing a change in reaction stoichiometry to enable formation of only 1 ATP with a Gibbs free energy released of -77 kJ reaction-1 (Eq. 2 and Fig. 1B).

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5 Ethanol + 3 Acetate- → 4 n-Butyrate- + 1 H+ + 2 H2 + 3 H2O (+ 1 ATP)

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The low concentrations further affect the first two steps of n-butyrate synthesis ((v) and (vi) in Fig. 1B): v) in the unfavorable thiolase equilibrium (8 acetyl-CoA = 4 acetoacetylCoA + 4 CoA), the consequently low acetyl-CoA concentrations result in a very low concentration of acetoacetyl-CoA 38; and vi) in the next step, acetoacetyl-CoA is reduced to 3-hydroxybutyryl-CoA. Since this reaction is already close to equilibrium with NADH as reductant (see above), the microbe uses the more powerful NADPH to shift the equilibrium towards butyryl-CoA synthesis 48. In other words, the NADPH allows the bacteria to perform the reduction with low concentrations of acetacetyl-CoA at higher rates. Though the standard redox potentials of NAD+ and NADP+ are identical (E0’ = 320 mV), the microbes keep NADPH/NADP+ ≈ 100 (E’ = -380 mV) and NADH/NAD+ ≈ 0.05 (E’ = -280 mV) 52. Therefore, NADPH is a more powerful reducing agent. In C. kluyveri, reduction of NADP+ to NADPH is achieved by a NADH-dependent reduced ferredoxin:NADP oxidoreductase (Nfn), which functions here as an electron confurcating enzyme (Fig. 1B) 53.

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In the physiological direction, electrons from two different electron donor molecules (NADH and reduced ferredoxin [Fd-red]) transfer to only one electron acceptor (the electrons confurcate from two paths into one). For the two different electron donors ((vii) and (viii) in Fig. 1B): vii) 4 electrons transfer endergonically from 2 NADH to 2 NADP+ to form 2 NAD+ and 2 NADPH; and viii) 4 electrons transfer exergonically from 4 reduced ferrodoxin to another 2 NADP+ to from 2 NADPH and 4 oxidized ferredoxin (Fdox in Fig. 1B). Here, 4 of the 8 reduced ferredoxin are re-oxidized by this electron confurcating system and the other 4 are re-oxidized by the same process as the abovedescribed high-ethanol concentration pathway: 4 protons are reduced by H2-ase to yield 2 molecular hydrogen. Because of the electron confurcating enzyme system, however, no reduced ferredoxin is left over for electron-transport phosphorylation. Therefore, the ATP yield amounts to only 1 ATP via substrate-level phosphorylation. It is also clear that the low substrate pathway, which includes NADPH and the Nfn complex, can occur with a considerably lower total Gibbs free energy release of 77 kJ reaction-1 than for the high substrate pathway (184.5 kJ reaction-1), reaching the threshold that is tied to an efficiency of 72 kJ mol-1 ATP 48.

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3.4 Ethanol and acetate elongation to n-caproate at high substrate conditions

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The pathway in section 3.1 was over-simplified and certain aspects were omitted: 1) the stoichiometry of ethanol-acetate fermentation is in reality more flexible 13; 2) n-caproate is produced in addition to n-butyrate and molecular hydrogen 13; 3) the ratio of ethanol/acetate fermented influences the ratio of n-butyrate/n-caproate produced 13, 46; 4) the ratios of ethanol/acetate fermented and n-butryate/n-caproate produced determine to what extent molecular hydrogen is produced; 5) these ratios can theoretically determine to what extent (and in which direction) the two coupling sites of ATP conservation (substrate-level phosphorylation vs. Rnf complex/ATP synthase) have to be used, and how much ATP is consequently produced; and 6) the ethanol/acetate ratio and the dissolved hydrogen concentrations have substantial impact on thermodynamics and feasibility of the overall reactions.

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To include these six aspects, we developed a generalized stoichiometric model for the pathways described for high substrate concentrations from section 3.1, but we lowered the concentrations for some reactants and products. We still omitted the elongation to ncaprylate, which would change the stoichiometry again. By fixing the relative ratio of ethanol/acetate consumed via the parameter “a” expressed in moles ethanol (it is not a ratio per se, but rather linked to acetate) and by fixing the relative ratio of n-butyrate/ncaproate produced via “b” expressed in moles n-butyrate (linked to n-caproate), we can specify the stoichiometry (and direction) of all involved metabolic steps (Fig. 2A).

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To represent a relative ratio into moles of the above-discussed pathway, which can be compared with relevant literature 47, 48, we set the boundary for the metabolic flux to 10 moles of the two-carbon moieties consumed (i.e., between 5 ethanol vs. 5 acetate and 10 ethanol vs. 0 acetate). A theoretical minimum of 5 ethanol vs. 5 acetate is required for fermentation into n-butyrate by C. kluyveri pure cultures 54. Then, the flux through all participating reactions depends on only two parameters: “a” (between 5 and 10) defines the moles of ethanol, and consequently also the moles of acetate (10-a) consumed; and “b” (between 5 and 0) defines the moles of n-butyrate, and consequently also the moles of ncaproate ((10-2b)/3) produced. In our model, the stoichiometry of all metabolites (i.e., ethanol, acetate, n-butyrate, n-caproate, molecular hydrogen, water, intermediary metabolites, redox mediators, and the ultimate ATP) depends on parameters “a” and “b” (Fig. 2A), resulting in an overall reaction with “a” and “b” as variables (Table 3). The stoichiometry for re-oxidation of reduced ferredoxin to Fdox via H2-ase or Rnf (and ATP synthase), therefore, also varies based on “a” and “b”, which in turn determines the molecular hydrogen production and ATP production, respectively.

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As an example, we use this stoichiometric model to investigate the condition at which “a” is close to 10 (i.e., the relative ratio of ethanol/acetate is high) and “b” is close to zero (i.e., the relative ratio of n-butyrate/n-caproate is low). By filling in the model with a=10 and b=0 for biologically relevant conditions with concentrations of 10 mM for ethanol and n-caproate, a hydrogen partial pressure of ~0.1 kPa (0.001 atm), a water activity of 1, a pH of 7.0, and a temperature of 25°C, we calculated the Gibbs free energy with Eq. 3.

399

10 Ethanol → 3.33 n-Caproate- + 3.33 H+ + 6.67 H2 + 3.33 H2O (+ 3.33 ATP) (3)

400

∆G’ = −263.2 kJ reaction-1

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For these “a” and “b” values, several terms in Fig. 2A approach zero or become even negative, specifically the moles of NAD+ reduced and H+/Na+-ions pumped by the Rnf complex (and therefore the ATP produced by ATP synthase). Negative terms suggest that enzymatic reactions should operate in the reverse. A lower ATP production (or ATP consumption when the ATP synthase protein complex is operated in the reverse) is compensated for by increased production of ATP via substrate-level phosphorylation. Then, n-caproate production compared to n-butyrate production may introduce an energetic advantage due to a higher theoretical ATP yield for substrate-level phosphorylation (i.e., up to 3.33 vs. 2.5 ATP during n-caproate only vs. n-butyrate only formation, respectively) (Fig. 2A and Eq. 3).

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3.5 Scenarios to predict favorable chain elongation at biologically pertinent conditions

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Even though this stoichiometry model is theoretical, and evidence with follow-up experiments will be required, we include thermodynamic calculations to predict under which conditions chain elongation to n-caproate is favorable. Therefore, we used our overall reaction from Table 3, and calculated the Gibbs free energy ∆G’ released under the biologically relevant conditions (i.e., 10 mM concentrations for each ethanol, acetate, n-butyrate, and n-caproate; 1 for the water activity; pH=7.0; and T=298 K) to compare it to the minimum free energy required (Req in Fig. 2B) to allow the metabolic pathway for ATP production to proceed. We introduced three variables for this free energy calculation and comparison: 1) “a”; 2) “b”; and 3) the hydrogen partial pressure, resulting in a 3dimensional plot (Fig. 2B). By adding three scenarios with different hydrogen partial pressures (10 kPa; 0.1 kPa; and 0.001 kPa), we illustrated the thermodynamic effects of ethanol and/or hydrogen addition towards formation of either n-butyrate or n-caproate. This theoretical effort resulted in three observations, but it is important to note that these calculations are only based on a relatively simple model, which is different from the biological reality.

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First, the higher the relative ratio of ethanol/acetate consumed (“a” closer to 10 moles of ethanol), the more free energy is released by the fermentation (∆G’ is increasingly negative across the “a” axes in Fig. 2B), meaning that ATP production, and thereby the complete chain elongation reaction, becomes increasingly feasible. This finding had already been empirically observed because a higher ratio of ethanol/acetate consumed increased the formation of n-caproate compared to n-butyrate and promoted additional formation of molecular hydrogen by C. kluyveri 54. Thus, a high availability of ethanol compared to acetate facilitates favorable chain elongation, resulting in longer carbon chains.

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Second, the relative ratio of n-butyrate/n-caproate produced (“b”) did not influence the free energy released as much as “a” and the partial pressure of hydrogen (∆G’ does not vary considerably across the “b” axes in Fig. 2B). However, under certain specific conditions, such as a hydrogen partial pressure of 0.1 kPa (~0.001 atm) and an “a” of 7 (i.e., 7 moles of ethanol and 3 moles of acetate), a “b” of 5 is the only feasible condition (i.e., 5 moles of n-butyrate and 0 moles of n-caproate). This, because the Gibbs free energy release of 186 kJ reaction-1 is just high enough (Fig. 2B), which means that nbutyrate production is still thermodynamically feasible, but n-caproate production is not. In the specific situation of an equal relative ratio of ethanol and acetate (a=5) and only nbutyrate production (b=5), no H2 would have been produced in the stoichiometric model, but the 137 kJ Gibbs free energy release across all hydrogen partial pressures is not sufficient for a thermodynamically feasible reaction (Fig. 1A). In summary, the hydrogen partial pressure would clearly affect n-caproate production in this model.

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Third, at relatively high hydrogen partial pressures of 10 kPa (~0.1 atm), our model predicts that the entire ethanol-acetate fermentation pathway (regardless of “a” and “b”) becomes thermodynamically unfeasible because the free energy available is not enough to fulfill the total Gibbs free energy release of ~180 kJ reaction-1 with a production of 2.5 ATP (∆G’ with 10 kPa is always more positive than Req in Fig. 2B). The formation of molecular hydrogen with electrons from ferredoxin would become unfavorable (less exergonic) at high hydrogen partial pressures. This outcome, which is based on the

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assumption of using the high substrate condition pathway with Rnf and ATP synthase, however, is not the reality, because Schoberth et al. 16 have already shown that the ethanol-acetate fermentation pathway does occur at high hydrogen partial pressures.

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Here, we still shared our third observation, because it can possibly explain the empirical finding of the same authors that C. kluyveri slowed down its activity with ethanol and acetate at high hydrogen partial pressures, but did not stop it 16. One explanation for the continuing chain elongation, albeit at a slower rate, is that C. kluyveri could alter its metabolic pathway to produce less ATP via ATP synthase (i.e., 0 vs. 0.15 ATP per mol of two-carbon moieties fermented), similar as described in section 3.3 for low ethanol concentrations with the electron confurcating Nfn enzyme 48. This alternative pathway requires a lower total Gibbs free energy release of only ~77 kJ reaction-1 as discussed in section 3.3 (for 8 moles of the two-carbon moieties fermented) and would move the entire Req plane about 90 kJ reaction-1 upwards (less negative) (for 10 moles of the twocarbon moieties fermented), and therefore above the 10 kPA plane (Fig. 2B), allowing metabolism and growth to function at these high hydrogen partial pressures.

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Even with this alternative pathway, the high hydrogen partial pressures would make it thermodynamically harder to get rid of reducing equivalents (electrons) by producing more molecular hydrogen. But there is an additional route of getting rid of reducing equivalents – by continuing to elongate to a longer MCC, such as n-caprylate rather than n-caproate, because the longer the MCC, the more reduced they become. This alternative route of n-caprylate production is currently not included in our model. It may, however, explain several observations with reactor microbiomes with ethanol and acetate as substrates and relatively high levels of molecular hydrogen for which researchers have observed chain elongation towards longer carboxylates such as n-caproate and ncaprylate 17, 55.

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In summary, high partial pressures of hydrogen may not negatively impact chain elongation in systems with a long residence time, because C. kluyveri has an alternative pathway to reduce the energy demand for ATP synthesis. On the other hand, when chain elongation rates to n-caproate need to be fast in systems with short residence times, it may be imperative to maintain low hydrogen partial pressures (≤ 0.1 kPa; ≤ ~0.001 atm). Of course, with reactor microbiomes the hydrogen partial pressure should be high enough to prevent anaerobic oxidation of ethanol and MCCs.

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4. Bioprocessing

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4.1 Reverse β oxidation in open cultures

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With n-caproate production being present in anaerobic sediments as a natural function, it is not a surprise that several anaerobic fermentation studies in the past have reported reasonable titers of MCCs, including n-caproate. For example, two research papers from the McCarty Lab had shown that open-culture bioreactors, which were fed with propionate as the substrate, showed a transient increase in the concentrations of MCC species after a perturbation by ethanol 56, 57. Immediately after the perturbation, hydrogenotrophic methanogens could not keep up with hydrogen production, resulting in an elevated hydrogen partial pressure. Since anaerobic propionate oxidation cannot occur

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at elevated hydrogen partial pressures due to thermodynamically unfavorable conditions 2, the reduced product formation of MCCs was a sink for reducing equivalents. This as an alternative to propionate removal by syntrophic anaerobic oxidation to ultimate methane production 57.

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However, the elevated MCC concentrations were transient, because after reaching very low hydrogen partial pressures again, anaerobic propionate oxidation was restored and the accumulated MCC concentration declined due to degradation. This final result is pertinent because very low hydrogen gas partial pressure resulted in degradation of the SCC substrates and MCC products through β oxidation. Thus, elevated hydrogen partial pressures must be maintained for open cultures to circumvent the degradation of carboxylates. Ding et al. 36 found with hydrogen fermentation bioreactors and a low hydraulic retention time that production of n-caproate coincided with hydrogen production, which is helpful to maintain sufficiently high hydrogen partial pressures.

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4.2 MCC production as a biotechnological production platform

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To our knowledge, Levy et al. 58 performed the first study with the specific goal to produce primarily MCCs from complex biomass. They integrated open-culture fermenters, MCC extraction through ion exchange resins, and phase separation of alkanes after Kolbe electrolysis (an electrochemical decarboxylation and dimerisation of two carboxylic acids). To specifically inhibit methanogenesis, they added 2bromoethanesulfonate (BrES) to the bioreactor.

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More recently, Steinbusch et al. 17 increased production rates 10-fold and showed proofof-principle that an open culture could sustainably produce n-caproate and n-caprylate for 115 days from ethanol and acetate as a synthetic substrate. They operated a bioreactor at a pH of 7 to maintain almost all MCC product in the non-inhibiting, dissociated form without having to extract, while BrES was added to inhibit methanogenesis. Follow-up work by Grootscholten et al. 40, 59 continued a similar approach with a synthetic substrate by maintaining a close to neutral pH value, but without BrES. This resulted in a ~1000fold increase in the n-caproate production rate to ~55 g l-1 d-1 (~120 g chemical oxygen demand [COD] l-1 d-1) and an elevated n-caproate selectivity of ~80% based on COD/COD 40. The concentration of undissociated n-caproic acid remained below the upper toxicity concentration limit by maintaining a neutral pH and shortening the hydraulic residence time to several hours, because MCCs were not extracted from the bioreactor broth.

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Agler et al. 18 used a real substrate (i.e., beer from the corn-kernel-to-ethanol industry) with non-distilled, undiluted ethanol to include hydrolysis, acidogenesis, methanogenesis, and chain elongation of SCCs in the same reactor microbiome. Hydrolysis requires long residence times, and in that scenario high n-caproate production rates can only be achieved when in-line product extraction is integrated within the biotechnological production platform because a short hydraulic retention time to dilute inhibiting, undissociated MCCs is not attainable. They demonstrated that MCCs could be produced in a continuously operated bioreactor without the methanogenic inhibitor BrES. This was accomplished by maintaining a mildly acidic pH value of 5.5 with the goal to completely inhibit acetoclastic methanogenesis. Because of the much higher ratio of

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undissociated/dissociated MCCs at a pH of 5.5 compared to a pH of 7, in-line extraction was required to achieve high n-caproate production rates. Small amounts of methane (2 days) for hydrolysis of polymeric substances. Agler et al. 18 and Ge et al. 9 used beer (including the left-overs from fermentation) from the corn-kernel-to-ethanol industry at hydraulic residence times of about 15 days to achieve an n-caproate production rate of up to ~3.4 g L-1 day-1 with a reactor microbiome by including in-line product extraction 9. The electron donor ethanol is present at concentrations of 150 g L-1 (3.3 M) in the corn beer, however, this was diluted about 6 times when it was used as a substrate for these studies to prevent ethanol toxicity after periodic feeding. The electron acceptors acetate and n-butyrate were formed from the left-over corn kernel materials, the yeast cells, glycerol, and C5 sugars in the

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same chain-elongating microbiome 9 by hydrolysis and acidogenesis via primary fermentation of the carboxylate platform 2. For efficient chain elongation to MCCs it is advisable to steer the primary fermentation in the microbiome to produce n-butyrate rather than acetate as the SCC of choice, because this will decrease the need for ethanol when chain elongation starts with n-butyrate rather than with acetate 9, 61. Maintaining a sufficiently high hydrogen partial pressure can steer the primary fermentation towards nbutyrate 62.

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When ethanol is not present in the feedstock, some in-situ production of this intermediate within the microbiome can explain relatively small amounts of n-caproate formation with reactor microbiomes with complex feedstocks 36, 61, 63. Indeed, one of the intermediates of hydrolysis and acidogenesis via primary fermentation in the carboxylate platform is ethanol, which is a preferred electron donor for high-rate chain elongation. In addition, also lactate can act as a possible donor 2. Therefore, it is important to develop a microbiome technology with promising ethanol and/or lactate yields and rates for specific feedstocks 64, 65, and to couple this to chain elongation. Recently, Xiong et al. 66 reported relatively high n-caproate selectivities for a lignocellulosic carboxylate platform technology, which included lignocellulosic pre-treatment to sugars and a reactor microbiome system with in-situ MCC extraction. The paper did not specify the electron donor for chain elongation (although sugars can be electron donors), and SCCs via acidogenesis of sugars seem the likely electron acceptors.

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When an electron donor is lacking in the feedstock or when it cannot be produced by the microbiome, it must be added to achieve promising MCC production rates. Grootscholten et al. 61 added procured ethanol to food and garden waste, which they referred to as the organic fraction of municipal solid waste (OFMSW), to produce n-caproate in a singlephase reactor microbiome. Even though using a single phase may be advantageous for numerous reasons, problems with reduced hydrolysis rates were observed possibly due to undissociated medium-chain carboxylic acid inhibition at lower pH values of 5.0-5.5 since product extraction had not been integrated 61.

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Therefore, the same authors also studied procured ethanol addition to food and garden waste with a two-phase reactor microbiome and found a doubling of the MCC production rate (1.9 g MCC L-1 day-1) compared to the single-phase reactor microbiome 60. The first phase was specifically operated for hydrolysis and acidogenesis of food and garden waste to SCCs and the second phase was operated to perform chain elongation of the SCCs to MCCs by addition of procured ethanol. Because the SCCs accumulated in the first phase at a lower pH value, hydrolysis was still inhibited 60. In a recent study, the hydrolysis rates of corn beer were enhanced by operating a single-phase reactor microbiome at a mildly acidic pH level of 5.5 with efficient MCC extraction to circumvent accumulation of undissociated carboxylic acids that inhibit hydrolysis 9. The ultimate goal is to eventually achieve the same hydrolysis rates with a MCC-producing microbiome compared to a methanogenic microbiome of an anaerobic digester with low carboxylate concentrations.

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Conversion of purified cellulose with procured ethanol in a defined mixed culture including a cellulolytic bacterium and C. kluyveri showed a maximum n-caproate concentration of 4.4 g L-1 in a batch study during a 5-day period (~ 1 g L-1 day-1) 67. In a 15



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recent study, Liang and Wan 63 showed that addition of ethanol as an electron donor to brewer’s spent grain increased the n-caproate production, but that addition of lactate only increased the production of SCCs. Finally, Weimer et al. 68 added ethanol to milled switch grass and alfalfa stems to achieve an n-caproate production rate of 3.1 g L-1 day-1 during short-term batch studies with rumen fluid and an augmented C. kluyveri isolate.

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Readily biodegradable feedstock can consist of streams with SCCs or other monomeric or short-chain polymeric substances such as sucrose. Such streams require much shorter treatment times (< 2 days) than complex biodegradable feedstock, because hydrolysis is not required or proceeds more rapidly. The work by Steinbusch et al. 17 and Grootscholten et al. 40, 59 with procured ethanol and acetate as a substrate for reactor microbiomes showed promising n-caproate production rates. Notably, the ease of conversion of ethanol and acetate resulted in short residence times of ~ 4 h. The neutral pH with a controlled substrate concentration, which resulted in the short residence time, circumvented product inhibition without extraction because the concentration of undissociated n-caproic acid was calculated to be < 1.9 mM, which is lower than the estimated inhibition threshold (Table 1). The resulting n-caproate production rate is the highest ever reported with ~55 g L-1 day-1 with a MCC selectivity of ~80% 40.

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One source of ethanol and acetate substrate is effluent from a synthesis gas (syngas carbon monoxide, molecular hydrogen, and carbon dioxide) fermentation system. This effluent consists of variable ratios of ethanol and acetate in a growth medium 69. Vasudevan et al. 70 fed syngas fermentation effluent containing ethanol and acetate to a reactor microbiome and achieved n-butyrate and n-caproate production rates of 20 and 1.7 g L-1 day-1, respectively. The further shift to MCC production had not occurred due to product inhibition at the operating pH of 5.25, but should occur after integrating product extraction at these pH levels. Recently, Zhang et al. 71 introduced a gaseous substrate of carbon dioxide and molecular hydrogen to a reactor microbiome, resulting in relatively low n-caproate production rates (likely from in-situ produced ethanol and acetate as discussed in section 2.3). Traces of n-caproate were observed within a bioelectrochemical system based on the same idea - rather than feeding molecular hydrogen directly into a bioreactor, a cathode produced molecular hydrogen gas in-situ via electrolysis after applying electric power 72.

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4.5 Bioreactor configurations

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In principle, bioreactor configurations for chain elongation will have a comparable function as for anaerobic digestion. For both anaerobic bioprocesses, hydrolysis, acidogenesis, and acetogenesis will be present in the microbiome in case of a complex biodegradable feedstock. One of the main differences between the systems will be the final trophic group in the anaerobic food web – predominantly chain-elongating bacteria vs. methanogenic archaea, respectively. Therefore, for bioreactor configuration choices we can learn from anaerobic digester research 73. Both systems are characterized with relatively low microbial growth rates (compared to aerobic systems), especially for the final trophic group. Therefore, granular processes should be applicable to chain elongation systems based on the generalized granular formation model, which includes the requirement for shear and slow microbial growth rates 74-76, especially when solids are absent in the feedstock. Similar to anaerobic digestion, the choice for a bioreactor design 16


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for chain elongation with microbiomes is predominantly dependent on the feedstock used because the slowest process will limit the overall performance based on productivity, product concentrations (or titer), and product selectivity. Here, we used the same two categories as in section 4.1 - complex and readily biodegradable feedstock - to discuss bioreactor configurations.

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When a complex biodegradable feedstock is utilized, hydrolysis will be a limiting process, resulting in long residence times (> 2 days) 18, 60, 61, 66. With solids present, the same bioreactor configurations as for anaerobic digestion would be desired - a semibatch-fed bioreactor (Table 4), such as an anaerobic sequencing batch reactor (ASBR) (Fig. 3A) 9, 18 or a continuously-stirred anaerobic fermenter, or a continuously-fed, continuously-stirred tank reactor (CSTR). In addition, Grootscholten et al. 61 utilized a BIOCEL bioreactor configuration from dry anaerobic digestion research, which is a fedbatch system with percolation and recirculation of the leachate through the feedstock (Table 4). For solids residence times that are longer than ~10 days, acetoclastic methanogens must be inhibited to prevent the system from becoming an anaerobic digester. Consequently, specific process conditions (e.g., mildly acidic pH values [5 < pH < 6]) or periodic treatments (e.g., heat shocks) may be needed to inhibit the growth of acetoclastic methanogens.

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With lower solids concentrations in the feedstock and when solids residence times in the bioreactor can be maintained between 2-5 days, the chain-elongating bacteria could outcompete the acetoclastic methanogens preventing substrate competition with methanogens. This, because the growth rate for the chain-elongating bacterium C. kluyveri under ideal conditions is ~ 0.1 h-1 32 and about 5 times faster than ~ 0.02 h-1 for acetoclastic methanogens 77. However, the growth rate for C. kluyveri at higher hydrogen partial pressures may be considerably slower when an alternative pathway is utilized as described in section 3.5.

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When readily biodegradable feedstock is utilized, residence times can be considerably shortened (< 2 days). Without the presence of solids in the feedstock to potentially accumulate in or plug systems, this has led to the use of anaerobic upflow filters (Table 4 and Fig. 3B) to maintain sufficiently long solids residence times to prevent biomass washout at low hydraulic residence times of 4-18 h 40, 59, 78. By introducing a carrier material to maintain sufficient biomass concentrations as biofilms, a pH of close to 7, and low substrate concentrations with hydraulic residence times as short as 4 h, Grootscholten et al. 40 has achieved the highest reported MCC production rate to date (Table 4).

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The same authors did not report methane formation even though methanogenic-inhibiting chemicals were not utilized and the pH was maintained at ~ 7. They introduced sufficient shear forces to prevent the growth of acetoclastic methanogens in biofilms, and thus by maintaining a short solids residence time for all microbes. It is also important to note that acetoclastic methanogens were likely not present in the inoculum of the anaerobic upflow filter 40. In a different study with an anaerobic upflow filter and syngas fermentation effluent, excessive methane production needed to be prevented by reducing the pH 70, albeit in this study lower shear forces were applied. Long-term operating periods with variable conditions and feedstocks in pilot-scale bioreactors (Fig. 3C) will have to be performed to understand if a long-term shift from chain elongation to methane production 17



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is problematic with reactor microbiomes. It is clear that when a mildly acidic pH value of 5.5 is maintained with long residence times of ~ 15 days, chain elongation without the emergence of acetoclastic methanogenesis can be sustained for long-term operating periods 9.

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4.6 Bioprocess conditions

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The pH value of the fermentation broth is one of the most important bioprocess parameters for the bacterial populations that are performing chain elongation or competing pathways such as acetoclastic methanogenesis. The optimum pH for C. kluyveri to grow was determined by Kenealy et al. 32, who found an optimum of 6.4, which is close to pKa of CO2 (H2CO3) and HCO3- (6.3), making sure that CO2 is available as a growth nutrient. On the other hand, Weimer et al. 31 reported for a different strain of C. kluyveri an optimal growth pH of 7.4. However, because of the toxicity of undissociated n-caproic acid and n-caprylic acid (at mildly acidic conditions) it is difficult to find the true growth rate at the more acidic conditions due to product toxicity. On the other hand, an acidic condition by itself can reduce the growth rate, because of the energy that needs to be invested to maintain a neutral internal pH. Since studies have found relatively high chain elongation rates at a pH of ~ 5.25-5.5 9, 70, the optimum pH for C. kluyveri growth should be established in a continuously operated bioprocess with in-line extraction to exclude toxicity due to undissociated medium-chain carboxylic acids.

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Of the bioreactor studies with reactor microbiomes, the most promising MCC production rates to date have been achieved at an operating temperature of 30°C 9, 40, 60, even though the optimum temperature for several chain-elongating wild-type strains, such as C. kluyveri, E. pyruvativorans, and M. elsdenii, was found to be near 39°C 19, 23, 31, 41. It is possible that the optimum MCC production rates will be achieved at a lower temperature than the optimum growth rates for the chain-elongating bacteria due to the solvent characteristics of the MCC product, which may enhance fluidity of cell membranes, but research at different temperatures would need to be performed to confirm this. A study by Agler et al. 73 showed that promising MCC production rates could not be achieved at 55°C.

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The medium composition has been studied for the growth of different wild-type strains of chain-elongating bacteria such as C. kluyveri, Clostridium sp. BS-1, and M. elsdenii 24, 42, 54 . For the nutrient requirements of C. kluyveri, yeast extract is a necessary source of multiple microelements and B vitamins such as biotin and para-aminobenzoic acid 12. In addition, carbon dioxide is also an essential nutrient for the production of proteins in C. kluyveri 79, and would need to be added to certain easily degradable substrates. This may explain the low productivity and the long lag phase that Steinbusch et al. 17 observed in their fed-batch reactor with a reactor microbiome, because no carbon dioxide had been added to the medium with ethanol and acetate. In another study with reactor microbiomes and an easily biodegradable substrate, doubling of each of yeast extract and carbon dioxide increased MCC production rates considerably 40. For complex biodegradable substrates, however, carbon dioxide may be produced in situ during hydrolysis and acidogenesis within reactor microbiomes, and carbon dioxide was, indeed, not added to corn beer 18. In the latter study, no yeast extract was added as well, but this feedstock was obtained from a yeast fermenter with all the necessary growth nutrients present. 18


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Finally, toxicity of substrates and products should be prevented. This can be achieved by dilution of the substrate to, for example, reduce problems with ethanol toxicity, especially during periods of unusually high loading rates during perturbations. Such dilution can be implemented with water or by recirculating bioreactor effluent. Currently, we do not know the ethanol concentrations at which the acclimated microbiome or C. kluyveri becomes inhibited. Although, this concentration would likely be between 10 and 20 g L-1 (~200-400 mM) 9. Water addition to the influent would, besides ethanol, also dilute intermediates, such as SCCs or MCC products, reducing their potential for inhibition. Product extraction has been another effective method to prevent product toxicity 18.

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4.7 Extraction and separation

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Two different approaches have been developed for chain elongation with reactor microbiomes for complex biodegradable feedstocks: 1) a single-phase fermentation system at low pH with in-line product extraction 18; and 2) a two-phase fermentation system at neutral pH to achieve high MCCs concentrations without in-line extraction 60. For the latter system, the product needs to be extracted and separated via a post-process such as precipitation or extraction.

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At mildly acid pH values, extraction will considerably improve chain elongation due to the removal of inhibiting undissociated acids. In-line extraction of MCCs has been integrated with a reactor microbiome at mildly acidic pH values with membrane liquidliquid extraction (i.e., pertraction) 9, 18. The driving force was a pH gradient (5.5–9.0) to specifically extract acidic product by diffusion through membranes. This results in a low energy extraction process, requiring only energy to pump bioreactor, solvent, and extraction solutions over large surface areas of membranes (hollow-fiber membrane modules). The solvent solution was 3% tri-n-octylphosphineoxide (TOPO) in mineral oil, which preferentially extracted MCCs compared to SCCs 18. Xiong et al. 66 found increased specificities for MCCs production with a low-pH, open-culture bioreactor by integrating it with a nanofiltration membrane extraction system. Others found improved productivities with pure cultures at mildly acidic pH values with liquid-liquid extraction (10 % alamine 336 in oleyl alcohol as a solvent) 42, 44.

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But even at neutral pH values for which the extraction of undissociated carboxylic acids is less crucial, improved n-caproate yields have been reported under the presence of insitu product extraction. With ion exchange as an in-line extraction step to remove ncaproate from fermentation broth with M. elsdenii batch cultures and Amberlite IRA 400 as a resin, these yields increased with 30% at a pH value of 7 80.

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The pertraction system of Agler et al. 18 accumulated MCC product in the dissociated form into a high pH extraction solution, which was maintained at a pH of 9 by continuous chemical addition. Next, Xu et al. 81 coupled this high pH extraction solution to an in-line membrane electrolysis cell, which maintained an in-situ pH gradient between the cathode (pH of 9-10 due to hydroxyl ion production) and anode (pH of 1-2 due to proton production) chambers by utilizing electric power rather than continuous chemical addition, preventing expensive chemical costs. The electrochemical forces sustained a relatively high transfer rate of the dissociated form (anions) through the anion exchange

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membrane of the cell into the low pH anolyte. Crossing the membrane transferred the product into the undissociated form where it accumulated in concentration and selectively phase separated because of its low maximum solubility, allowing simple product separation. The combined bioreactor system, which included pertraction and membrane electrolysis, continuously produced an oily product stream containing ~90% MCCs at ambient process conditions while fed a complex substrate 81.

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5. Outlook

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Chain elongation with reactor microbiomes is poised to become an important biotechnological production platform in the coming years. Almost all of the bioprocessing studies with chain elongation, thus far, have used substrates other than purely organic waste streams, including: 1) synthetic substrates, such as procured ethanol and acetate; 2) valuable organic substrate streams, such as corn fermentation beer that includes the electron donor (i.e., ethanol); or 3) a combination of procured ethanol and real food and garden waste. During scale-up and the first industrial applications, this likely will continue. This platform technology can substantially increase its impact by developing it further to utilize purely organic wastes as substrates to produce MCC products. This can be done by either generating the required electron donor from the waste within the microbiome or in a separate (bio)process; or by using the electron donors that are already available in the organic waste stream. Besides ethanol, other electron donors may become important, which would broaden the application of this platform considerably.

823 824 825 826 827 828 829 830

Initially, MCC products may aim at a relatively small market where it has the potential to solve an industrial problem with a high-priced specialty product. For example, MCCs with uneven carbon chains, such as n-heptanoate, may be very valuable in industry. Later on, larger markets may be targeted, for example, to make a drop-in fuel. Another potential is to develop MCCs into an important platform chemical from which many other chemicals could be derived with chemical conversion technologies. The type of application would also dictate the purity of n-caproate or n-caprylate that is required and what operating units are needed to achieve this.

831 832 833 834 835 836 837 838 839 840 841 842 843 844

As a nascent field of study, a plethora of research questions exists, such as: at what ethanol concentration does the microbiome become inhibited; what is the optimum and upper temperature to maximize MCC production rates; what it the optimum pH value to maximize MCC production rates; and can the microbiome be steered to primarily produce n-caprylate rather than n-caproate? Of course, practical questions exist too, such as how to best extract and separate the MCC products from the fermentation broth or effluent to maximize the economic outlook of this platform. Most importantly is to further develop a stoichiometric and thermodynamic model that includes n-caprylate production. Not only can experiments validate and reiterate this model to gauge whether we really understand chain elongation in a microbiome, but also to answer practical questions, including the effect of changing substrate ratios and the effect of hydrogen partial pressures. We are looking forward to a very exciting research period with the ultimate objective to reach a consensus that microbiomes can produce sustainable biochemicals at an industrial scale.

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845

Acknowledgements

846 847 848 849 850 851 852 853 854

This work was supported by the U. S. Army Research Laboratory and the U. S. Army Research Office under contract/grant number W911NF-12-1-0555 and the US National Science Foundation under award number 1336186 to L.T.A.. H.R. was supported for this work by the Cornell University Agricultural Experiment Station federal formula funds, Project No. NYC-123452 received from the National Institutes for Food and Agriculture (NIFA), U.S. Department of Agriculture. C.M.S. acknowledges the STAR Fellowship Assistance Agreement no. FP-91763801-0 awarded by the U.S. Environmental Protection Agency for support. In addition, this work was supported by Wageningen University IPOP Biorefinery program and the BE-Basic partner organisation through BE-Basic.

855

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1100

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1101


TABLES AND FIGURES

1102 1103

Table 1. Characteristics of n-caproate (C6) and n-caprylate (C8). Chemical name n-Caproate n-Caprylate

n-Hexanoic acid n-Octanoic acid

Carbon chain length C6

Molecular mass

Maximum solubility 82

Density 82

Inhibition of elongation 9

pKa 82

116.16

10.82 g L-1 (~1% w/v)

0.929 kg L-1

4.88

C8

144.21

0.68 g L-1 (~0.1% w/v)

0.910 kg L-1

7.5 mM (0.87 g L-1) NA

1104

27


4.89


1105

1106 1107 1108 1109

Page 28 of 34

Table 2. Wild-type bacterial strains that produce n-caproate.

Phylum and Class*

Clostridium kluyveri Firmicutes Clostridia

Clostridium kluyveri Firmicutes Clostridia

Eubacterium pyruvativorans Firmicutes Clostridia

Clostridium sp. BS-1 Firmicutes Clostridia

Eubacterium limosum Firmicutes Clostridia

Megasphaera elsdeni Firmicutes Negativicutes&

Origin

Canal mud

Silage

Sheep rumen

Sheep rumen

Sheep rumen

Temperature optimum (°C) Pertinent tested electron donor

34

39

39

Anaerobic digester sludge 37

39

38

37

Ethanol

Ethanol and n-propanol

D-Galactitol

Methanol

DL-Lactate and sugars

Pyruvate

MCC formed

n-Caproate

n-Caproate

Amino acids, peptides, and pyruvate n-Caproate

n-Caproate

n-Caproate

n-Caproate

Yes Positive RF

Yes Negative LC-3

n-Caproate during dark fermentation NA Negative NA

No No No Yes Saccharolytic Positive Positive Positive Positive Gram reaction K1 3231B I-6T BS-1 Strain designation DSM 555 NA ATCC-BAA574 NA Strain collection 13 31 23, 41 33 References * http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Root & Recently re-classified as Clostridia 83 NA: not applicable

28


Rhodospirillum rubrum Proteobacteria Alphaproteobacteria Dead mouse or tap water

DSM 20543

DSM 20460

DSM 467

30

21

45

Page 29 of 34

1110 1111 1112 1113 1114


Table 3. Stoichiometry for fermentation of ethanol and acetate into n-butyrate, ncaproate, and molecular hydrogen, dependent on parameters “a” (moles of ethanol consumed when linked to acetate consumption) and “b” (moles of n-butyrate produced when linked to n-caproate production). For parameters “a” and “b” the following statements are valid: 5≤a≤10; if a=5 then b=5; if a=6 then 2≤b≤5; if a ≥7 then 0≤b≤5. Substrates Compound Ethanol (Acetate- + H+)

1115 1116 1117

Products Compound Quantity (mole) (n-Butyrate- + H+) b (n-Caproate- + H+) (10-2b)/3 H2 (6a+2b-40)/3 H2O* (40/3-a-2b/3) (ATP) (20-b)/6 *1 mole water, which is produced during condensation of 1 mol Pi and 1 mol ADP to form 1 mol ATP, is consumed during ATP hydrolysis. Therefore, 1 net mole water is consumed per mol acetate produced from acetyl-CoA during substrate-level phosphorylation, and this was considered when balancing the overall equation. Quantity (mole) a 10-a

1118

29



1119

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Table 4. Feedstocks and other parameters for MCC production. Feedstock

Biocatalysts

Experimental conditions

Maximum volumetric production rate (g L-1 day-1) (Concentration [g L-1]) Yield n-Caproate n-Heptanoate n-Caprylate

Reference

Semi-batch pH 5.5 Extraction Semi-batch pH 5.5 Extraction Batch Uncontrolled pH Extraction Fed-batch pH 5.5, 6, and uncontrolled pH No extraction Two phase fed-batch and anaerobic filter pH 6.7-7.3 No extraction Batch pH 6.7-7.0 No extraction Batch Uncontrolled pH No extraction Batch Uncontrolled pH No extraction

2.1 (-) 0.79 mol C 3.4 (-) 0.70 COD (30) 0.6 (2.7) -

small

small

18

small

small

9

NA

NA

66

0.27 (1.5) -

0.1 (0.5) -

61

26.0 (12.6) 0.72 mol e-

1.09 (0.9) 0.04 mol e-

0.87 (0.4) 0.03 mol e-

60

0.1 (4.4) (0.8) 3.1 (6.1) -

NA

NA

67

NA

NA

63

NA

NA

68

Fed-batch pH 7 No extraction Upflow filter pH 6.5-7.0 No extraction Upflow filter pH 6.5-7.2 No extraction Upflow filter pH 6.5-7.0 No extraction Upflow filter pH 5.25 No extraction

0.5 (8.2) 0.62 mol C 16.6 (11.1) 0.85 mol e57.4 (12.0) 0.81 mol e6.9 (4.9) 0.33 mol e1.7 (1.0) 0.1 COD

-

0.1 (0.3) 0.02 mol C 0.9 (0.6) 0.06 mol e1.8 (0.9) small (small) 0.02 mol e-

17

Complex biodegradable feedstock Corn beer

Reactor microbiome

Corn beer

Reactor microbiome

Acid pretreated willow Ethanol and food/garden waste

Reactor microbiome

Ethanol and food/garden waste

Reactor microbiome

Ethanol and cellulose

C. kluyveri ATCC 8527 and a cellulolytic bacterium Reactor microbiome

Reactor microbiome

Ethanol and brewers’ spent grain Ethanol and C. kluyveri ATCC milled 3231B and rumen microbiome switch grass or alfalfa stems Readily biodegradable feedstock

1120

Ethanol and acetate

Reactor microbiome

Ethanol and acetate

Reactor microbiome

Ethanol and acetate

Reactor microbiome

Ethanol and propionate

Reactor microbiome

Syngas effluent

Reactor microbiome

-

-

4.5 (3.2) 0.23 mol e-

NA – not available

1121

30


59

40

78

70

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1122

FIGURE HEADINGS

1123 1124 1125 1126 1127 1128 1129 1130 1131 1132 1133

Figure 1. Chain elongation pathway with ethanol and acetate to n-butyrate for C. kluyveri: A. Ethanol-acetate fermentation by C. kluyveri at biological conditions with relative high substrate concentrations (1 M for substrates and products); and B. Ethanolacetate fermentation by C. kluyveri at biological conditions with relative low substrate concentrations (1 mM for substrates and products). Redox-factors are highlighted in blue; Classical energy conservation in red; and more recent described mechanisms of energy conservation in yellow; roman numerals explained in main text. Abbreviations: BcdAEtfBC = butyryl-CoA dehydrogenase-electron transferring flavoprotein complex; CoA=Coenzyme A; F0F1 = H+/NA+-pumping ATP synthase complex; Fdox = oxidized ferredoxin; Fd-red = reduced ferredoxin; H2-ase = hydrogenase; Nfn= NADH-dependent reduced ferredoxin:NADP oxidoreductase; Rnf = ferredoxin-NAD reductase complex.

1134 1135 1136 1137 1138 1139 1140 1141 1142 1143 1144 1145 1146 1147 1148 1149 1150 1151 1152 1153 1154 1155 1156

Figure 2. Chain elongation pathway with ethanol and acetate to n-butyrate and ncaproate for C. kluyveri: A. Generalized stoichiometric model for fermentation of ethanol and acetate into n-butyrate, n-caproate, and molecular hydrogen by C. kluyveri. The variable “a” represents the moles of ethanol when linked to moles of acetate (consumed), which must be 5≤a≤10; while “b” represents the moles of n-butyrate when linked to the moles of n-caproate (produced), which must be 5≥b≥0; boundaries and exact meanings of these variable are explained in the text. Redox-factors are highlighted in blue; Classical energy conservation in red; and more recent described mechanisms of energy conservation in yellow; and Abbreviations: AA=acetacetyl; AC=acetyl; BcdAEtfBC=butyryl-CoA dehydrogenase-electron transferring flavoprotein complex; BU=butyryl; CoA=Coenzyme A; CR=crotonyl; F0F1=H+/Na+-pumping ATP synthase complex; Fdox = oxidized ferredoxin; Fd-red = reduced ferredoxin; H2-ase = hydrogenase; HA=hexanoyl; HB=hydroxybutyryl; HE=Hex-2-enoyl; HH=hydroxyhexanoyl; KH=2ketohexanoyl; Nfn= NADH-dependent reduced ferredoxin:NADP oxidoreductase; Rnf = ferredoxin-NAD reductase complex; and B. Three-dimensional plots of the Gibbs free energy (∆G’) released from fermentation of ethanol and acetate into n-butyrate, ncaproate, and molecular hydrogen, dependent on “a” and “b” for three different hydrogen partial pressures (10 kPa; 0.1 kPa; and 0.001 kPa). This is compared to the Gibbs free energy required for synthesis of ATP (Req) according to the generalized stoichiometric model when -72 kJ free energy are required per mol ATP formed 48. The standard Gibbs free energy are taken from Kleerebezem and van Loosdrecht 84, with 10 mM concentrations for ethanol, acetate, n-butyrate, and n-caproate, a water activity of 1, a pH of 7.0, and a temperature of 25°C.

1157 1158 1159 1160

Fig. 3. Views of bioreactor setups: A. Anaerobic sequencing batch reactor (ASBR) with extraction modules for corn beer into n-caproate conversion 9, 18; B. Upflow filter for ethanol and SCC to MCC conversion 40, 59, 60; and C. Pilot-scale system from ChainCraft BV (www.chaincraft.nl).

31



Figure 1. Chain elongation pathway with ethanol and acetate to n-butyrate for C. kluyveri: A. Ethanolacetate fermentation by C. kluyveri at biological conditions with relative high substrate concentrations (1 M for substrates and products); and B. Ethanol-acetate fermentation by C. kluyveri at biological conditions with relative low substrate concentrations (1 mM for substrates and products). Redox-factors are highlighted in blue; Classical energy conservation in red; and more recent described mechanisms of energy conservation in yellow; roman numerals explained in main text. Abbreviations: BcdA-EtfBC = butyryl-CoA dehydrogenase-electron transferring flavoprotein complex; CoA=Coenzyme A; F0F1 = H+/NA+-pumping ATP synthase complex; Fdox = oxidized ferredoxin; Fd-red = reduced ferredoxin; H2-ase = hydrogenase; Nfn= NADH-dependent reduced ferredoxin:NADP oxidoreductase; Rnf = ferredoxin-NAD reductase complex. 117x233mm (300 x 300 DPI)


Page 32 of 34

Page 33 of 34


Figure 2. Chain elongation pathway with ethanol and acetate to n-butyrate and n-caproate for C. kluyveri: A. Generalized stoichiometric model for fermentation of ethanol and acetate into n-butyrate, n-caproate, and molecular hydrogen by C. kluyveri. The variable “a” represents the moles of ethanol when linked to moles of acetate (consumed), which must be 5≤a≤10; while “b” represents the moles of n-butyrate when linked to the moles of n-caproate (produced), which must be 5≥b≥0; boundaries and exact meanings of these variable are explained in the text. Redox-factors are highlighted in blue; Classical energy conservation in red; and more recent described mechanisms of energy conservation in yellow; and Abbreviations: AA=acetacetyl; AC=acetyl; BcdA-EtfBC=butyryl-CoA dehydrogenase-electron transferring flavoprotein complex; BU=butyryl; CoA=Coenzyme A; CR=crotonyl; F0F1=H+/Na+-pumping ATP synthase complex; Fdox = oxidized ferredoxin; Fd-red = reduced ferredoxin; H2-ase = hydrogenase; HA=hexanoyl; HB=hydroxybutyryl; HE=Hex-2-enoyl; HH=hydroxyhexanoyl; KH=2-ketohexanoyl; Nfn= NADH-dependent reduced ferredoxin:NADP oxidoreductase; Rnf = ferredoxin-NAD reductase complex; and B. Threedimensional plots of the Gibbs free energy (∆G’) released from fermentation of ethanol and acetate into n-



butyrate, n-caproate, and molecular hydrogen, dependent on “a” and “b” for three different hydrogen partial pressures (10 kPa; 0.1 kPa; and 0.001 kPa). This is compared to the Gibbs free energy required for synthesis of ATP (Req) according to the generalized stoichiometric model when -72 kJ free energy are required per mol ATP formed 48. The standard Gibbs free energy are taken from Kleerebezem and van Loosdrecht 84, with 10 mM concentrations for ethanol, acetate, n-butyrate, and n-caproate, a water activity of 1, a pH of 7.0, and a temperature of 25°C. 176x236mm (300 x 300 DPI)


Page 34 of 34

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Figure 3. Views of bioreactor setups: A. Anaerobic sequencing batch reactor (ASBR) with extraction modules for corn beer into n-caproate conversion 9, 18; B. Upflow filter for ethanol and SCC to MCC conversion 40, 59, 60; and C. Pilot-scale system from ChainCraft BV (www.chaincraft.nl). 155x57mm (300 x 300 DPI)


Chain Elongation with Reactor Microbiomes: Open-Culture

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