Medium chain carboxylic acids production from waste biomass: Current advances and perspectives

Biotechnology Advances 37 (2019) 599–615

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Research review paper

Medium chain carboxylic acids production from waste biomass: Current advances and perspectives

T

Qinglian Wu1, Xian Bao1, Wanqian Guo , Bing Wang, Yunxi Li, Haichao Luo, Huazhe Wang, Nanqi Ren ⁎

State Key Laboratory of Urban Water Resource and Environment, Harbin Institute of Technology, Harbin 150090, PR China

ARTICLE INFO

ABSTRACT

Keywords: Anaerobic fermentation Chain elongation Medium chain carboxylic acid Caproate Reverse β oxidation Waste biomass Renewable energy

Alternative chemicals to diverse fossil-fuel-based products is urgently needed to mitigate the adverse impacts of fossil fuel depletion on human development. To this end, researchers have focused on the production of biochemical from readily available and affordable waste biomass. This is consistent with current guidelines for sustainable development and provides great advantages related to economy and environment. The search for suitable biochemical products is in progress worldwide. Therefore, this review recommends a biochemical (i.e., medium chain carboxylic acids (MCCAs)) utilizing an emerging biotechnological production platform called the chain elongation (CE) process. This work covers comprehensive introduction of the CE mechanism, functional microbes, available feedstock types and corresponding utilization strategies, major methods to enhance the performance of MCCAs production, and the challenges that need to be addressed for practical application. This work is expected to provide a thorough understanding of the CE technology, to guide and inspire researchers to solve existing problems in depth, and motivate large-scale MCCAs production.

1. Introduction According to the latest BP Statistical Review of World Energy (BP, 2018), global average fossil fuel consumption increased at an average annual rate of 1.7% in the past decade, and reached 2.2% in 2017 (the highest since 2013). The continuously increasing use of fossil fuel will lead to the rapid depletion of these resources in the foreseeable future (Bao et al., 2019a). To decelerate this process, renewable energy, such as solar energy, wind energy, and hydropower, have been implemented (Angenent et al., 2016). Furthermore, renewable chemicals also need to be developed to replace a diverse range of fossil-fuel-based products to mitigate the adverse impacts of fossil fuel depletion on human development. Considering economic and environmental sustainability, researchers have put more emphasis on biochemical production from waste biomass instead of human food crops (e.g., grain, sugar, oilseed crops) (Lee et al., 2014). Anaerobic biotechnology is an important biochemical production platform since little energy is lost in the anaerobic process. It also requires much lower capital and operating costs than aerobic processes (Angenent et al., 2016). Ethanol, butanol, lactate (the carboxylates in this context including dissociated

carboxylates and corresponding undissociated carboxylic acids), and short chain carboxylic acids (SCCAs) are the main anaerobic products (Van Eerten-Jansen et al., 2013; Wu et al., 2017; Wu et al., 2016). These biochemicals are high-value and broad-spectrum; however, their characteristic of complete miscibility with the fermentation broth makes the subsequent products separation/extraction process energetically costly and thereby largely limit their further exploitability. For example, SCCAs derived from waste biomass are mostly used in the situations when purity is unimportant, such as providing a carbon source for sewage treatment plant to facilitate nitrogen and phosphorus removal (Bao et al., 2019b; Lee et al., 2014; Liu et al., 2016). Ethanol extraction with energy-intensive distillation generally accounts for 8–15% of its total production cost (Baeyens et al., 2015). Lactate extraction using crystallization separation, esterification separation, and molecular distillation are also very costly (Lopez-Garzon and Straathof, 2014). The high-cost of separation/extraction processes is a challenging technical bottleneck for biochemical applications. Recently, chain elongation (CE) technology, an anaerobic carboxylate platform based on a reverse β oxidation pathway, has provided an effective option overcoming this barrier. This is achieved by upgrading miscible organics like SCCAs, ethanol, and lactate to form less hydrophilic medium chain carboxylic

Corresponding author. E-mail address: [email protected] (W. Guo). 1 These authors contributed equally to this work and should be considered as co-first authors. ⁎

https://doi.org/10.1016/j.biotechadv.2019.03.003 Received 3 November 2018; Received in revised form 1 March 2019; Accepted 3 March 2019 Available online 05 March 2019 0734-9750/ © 2019 Elsevier Inc. All rights reserved.

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acids (MCCAs) using reactor microbiomes (open culture of microbial consortia). (Agler et al., 2012a; Ge et al., 2015; Kucek et al., 2016a; Steinbusch et al., 2011; Zhu et al., 2015). The open culture CE process is also envisioned as a commercially viable biochemical production platform (Angenent et al., 2016). MCCAs, the straight chain mono-carboxylates with 6–12 carbon atoms including caproate, heptylate, caprylate, etc., are considered to be more profitable than traditional anaerobic products (Agler et al., 2012a; Steinbusch et al., 2011; Zhu et al., 2015). Moreover, MCCAs feature higher energy density as well as stronger hydrophobicity than its precursors. To be specific, the energy contained in 1 mol caproate (3452 kJ) is higher than its reactants of 2 mol ethanol (2638 kJ) (Steinbusch et al., 2011). The solubility of caproate, heptylate, and caprylate in water in their undissociated forms are as low as 10.82, 2.42, and 0.68 g/L, respectively, while ethanol, lactate, and SCCAs are entirely miscible with water except for the five‑carbon valerate (with high solubility of 49.7 g/L) (Xu et al., 2018). Such low solubility of MCCAs means that they can be separated as an oily fluid from the fermentation broth once the required concentrations are reached and phase separation appeared without external energy or chemicals input. MCCAs can be widely applied in industry and agriculture (Angenent et al., 2016). Currently, MCCAs can be directly utilized as antimicrobial agents (Desbois, 2012) and food additives (Xu et al., 2015). Besides, as a versatile platform chemical, MCCAs are also used to manufacture products including pharmaceuticals, fragrances, lubricants, rubbers, and dyes (Angenent et al., 2016; Choi et al., 2013). They can also be further processed into liquid biofuels including diesel (Levy et al., 1984) and aviation fuel (Harvey and Meylemans, 2014). Despite these versatile applications, the high cost of current MCCAs production limits its large-scale utilization. MCCAs are usually derived from plant or animal oils, or petroleum (Ge et al., 2015; Jeon et al., 2013). Besides, caproate can also be produced through carbonylation of ethylene with CO and water, oxidation of propionaldehyde, and oxidation of hydrocarbon compounds (Wasewar and Shende, 2011); heptylate can be produced by oxidizing heptaldehyde derived from the pyrolysis of castor oil (Das et al., 1989); and caprylate can be produced though the oxidation of octanal, and oxidation and dehydrogenation of octanol. Obviously, these chemical MCCAs production methods require expensive investments for raw material and facilities. In contrast, wastebiomass-based MCCAs production is a more economically and environmentally attractive approach. To take advantage of MCCAs to replace fossil-fuel-based products, scaling up MCCAs production from current lab scale to industrial level while ensuring the high MCCAs yield and its economic benefit are essential. To provide guidance for future research, this work provides a comprehensive state-of-the-art review to introduce the CE mechanism, functional strains, reactor microbiomes, and MCCAs production strategies (based on three feedstock types), with special emphasis on wastebiomass-based MCCAs production, strategies to enhance MCCAs production performance, and major challenges ahead to scale up MCCAs production and corresponding recommendations.

pathways in the second step of cyclic reverse β oxidation are similar. Caproate production by the CE process with both ethanol and lactate as EDs is thermodynamically feasible under standard scondition (1 atm, 25 °C, and 1 M) (Eqs. (1)–(2)) (Coma et al., 2016; Zhu et al., 2017). When ethanol is used as the ED, CE starts with ethanol oxidation to acetaldehyde and then into acetyl-CoA catalyzed by ethanol dehydrogenase and acetaldehyde dehydrogenase, respectively. About 1/6 of the acetyl-CoA is converted into acetate by substrate-level phosphorylation along with energy (ATP) harvest (Eq. (3)), and another 5/6 of the acetyl-CoA enters the cyclic reverse β oxidation pathway (Angenent et al., 2016). In each cycle, acetyl-CoA is first coupled to another CoA derivative to form a CoA derivative with a two‑carbon increase (Lynen and Ochoa, 1953). In the first cycle of acetate elongation into butyrate (Eq. (4)), acetyl-CoA is coupled to another acetyl-CoA catalyzed by acetoacetyl CoA thiolase (the original name is acetyl-CoA C-acetyltransferase) to generate acetoacetyl-CoA. Next, a series of enzymatic reactions (involving the enzymes NAD- and NADP-dependent 3-hydroxybutyryl-CoA dehydrogenase, 3-hydroxybutyryl-CoA dehydratase, and NAD-dependent butyryl-CoA dehydrogenase) are carried out until generation of the critical intermediate butyryl-CoA, which is then transferred one CoA to acetate (electron acceptor (EA)) along with butyrate generation and acetyl-CoA release under catalysis by acetate CoA transferase. The released acetyl-CoA then reacts with other acetylCoA provided by ethanol oxidation to start acetate elongation once again. Meanwhile, acetyl-CoA is coupled to butytyl-CoA to start the second cycle of butyrate elongation into caproate (Eq. (5)) with 3-ketohexanoyl-CoA, 3-hydroxyhexanoyl-CoA, hex-2-enoyl-CoA, and hexanoyl-CoA as intermediates. During the reverse β oxidation process, energy (ATP) is harvested only via proton translocation from the oxidation of reduced ferredoxin (Fdred), which occurs in the strongly exergonic process of crotonyl-CoA reduction to butyryl-CoA (E0′ = −10 mV) with NADH (E0′ = −320 mV) (Eq. (6)) and hex-2enoyl-CoA reduction to hexanoyl-CoA (E0′ = −10 mV) with NADH (E0′ = −320 mV) (Eq. (7)) (Cavalcante et al., 2017; González-Cabaleiro et al., 2013; Seedorf et al., 2008; Spirito et al., 2014).

C2 H6 O + 2C2 H3 O2 + H+ + 2H2

C6 H11 O2 + 3H2 O,

= 2C3 H5 O3 + C2 H3 O2 + 2H+

=

2

CoA + 2FdOX + 2NADH + 2H+

enoyl

Hexanoyl

G0

G0 (5)

183.5 kJ/mol

NAD+ ,

(3) (4)

182.5 kJ/mol

+2

Hex

G0 = 49.6 kJ/mol

5C6 H11O2 + C2 H3 O2 + 4H2 O + 2H2 + H+,

6C2 H6 O + 5C4 H7 O2

CE involves electron donor (ED) oxidation and the following cyclic reverse β oxidation pathway (Ge et al., 2015; Roghair et al., 2018). ED is the energy-rich reduced compound that is the prerequisite substance for the CE process (Agler et al., 2012a; Spirito et al., 2014). The first step of ED oxidation provides the required energy, reducing equivalents (NADH), and the essential intermediate acetyl-CoA. So far, ethanol and lactate have been identified as the most ideal EDs to induce CE process (Cavalcante et al., 2017; Zhu et al., 2015). Therefore, the mechanism of the CE process is introduced based on EDs of ethanol and lactate and CE products of caproate, heptylate, and caprylate. As shown in Fig. 1, for different ED-guided CE process, the differences lie mainly in the first step of ED oxidation, and the metabolic

(2)

5C4 H7 O2 + C2 H3 O2 + 4H2 O + 2H2 + H+, =

2. Mechanism of CE

G0

195.6 kJ/mol

C2 H3 O2 + 2H2 + H+,

6C2 H6 O + 4C2 H3 O2

Crotonyl

(1)

126.7 kJ/mol

C6 H11O2 + 2H2 O + 2CO2 , =

C2 H6 O + H2 O

G0

G0

Butyryl =

CoA + 2Fd red2

115.7 kJ/mol

(6)

CoA + 2FdOX + 2NADH + 2H+ CoA + 2Fd red2 + 2 NAD+ ,

G0 =

118.4 kJ/mol

(7)

When lactate is used as ED, it is firstly oxidized to pyruvate with lactate dehydrogenase; then pyruvate is further oxidized to acetyl-CoA in conjunction with an equimolar CO2 release and ATP synthase catalyzed by pyruvate dehydrogenase. As with the use of ethanol as ED, part of the acetyl-CoA is also converted into acetate by substrate-level phosphorylation (Eq. (8)). It is noteworthy that propionate can be generated through the acrylate pathway with lactyl-CoA, acrylyl-CoA, and propionyl-CoA as intermediates (Kucek et al., 2016a; Prabhu et al., 2012). The acrylate pathway competes for lactate‑carbon flow with the CE pathway (Kucek et al., 2016a), and it is hard to extend propionate 600

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Fig. 1. CE pathway with ethanol and lactate as EDs. Adapted from Seedorf et al. (2008a), Prabhu et al. (2012), and Spirito et al. (2014). Substrates and products are highlighted in red and blue, respectively. The key enzymes in the metabolic pathway are labeled with numbers of 1–20 that are explained at the lower right of figure. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

further to form heptylate (Grootscholten et al., 2013b). This results in reduced MCCA selectivity. Therefore, the acrylate pathway flow should be restricted in order to achieve higher MCCAs yield. The acetyl-CoA produced by lactate oxidation enters cyclic reverse β oxidation by reacting with another acetyl-CoA, as described above, to elongate acetate into butyrate (Eq. (9)) and then to caproate (Eq. (10)). Since the second step of cyclic reverse β oxidation is similar to the use of ethanol as ED, the details are omitted here. In addition, it should be noted that when even‑carbon acetate or butyrate as EAs are replaced by odd‑carbon propionate or valerate, odd‑carbon heptylate can be generated as the major CE product. Therefore, through a whole CE process directed by

suitable EDs, acetate can be elongated via an orderly process to form butyrate, caproate, and caprylate; and propionate can be elongated into valerate, heptylate, and nonanoate, step-by-step.

C3 H5 O3 + H2 O

C2 H3 O2 + 2H2 + CO2 ,

C3 H5 O3 + C2 H3 O2 + H+

9.5 kJ/mol

C4 H7 O2 + H2 O + CO2 , =

C3 H5 O3 + C4 H7 O2 + H+

97.9 kJ/mol

(8)

G0 (9)

97.7 kJ/mol

C6 H11O2 + H2 O + CO2 , =

601

G0 =

G0 (10)

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39 °C 7.0 Pyruvate, amino acids, peptides Caproate 1.16 g/L

–

(Wallace et al., 2004)

39 °C 7.4 Methanol

–

(Genthner et al., 1981)

Caproate 0.54 g/L

Firmicutes Clostridia Sheep rumen Firmicutes Clostridia Sheep rumen

(Jeon et al., 2016; Jeon et al., 2017)

(Jeon et al., 2010; Jeon et al., 2013)

(Zhu et al., 2017)

So far, several wild-type strains that can carry out the CE process to produce MCCAs have been isolated and characterized. The origin, living conditions, substrates, and MCCAs production performances of these strains have some differences, which are compared in Table 1 and introduced in detail as follows to make better use of these strains. Clostridium kluyveri is the best-studied pure culture strain among the identified CE bacteria. C. kluyveri was first isolated and enriched from the mud of a canal in 1937, and then became a model organism for studying fatty acid generation and oxidation (Barker, 1937; Lynen, 1953; Stadtman, 1953). C. kluyveri was also easily isolated from sediment sources (Barker and Taha, 1942) and the rumens of large herbivores (Weimer and Stevenson, 2012). Generally, members of C. kluyveri grow anaerobically using ethanol and acetate as energy/carbon sources, and synthesize butyrate, caproate, and H2 as their main products. Moreover, C. kluyveri can also metabolize other alcohols and organic acids and correspondingly produce different products. For example, when C. kluyveri was fed with propanol and acetate, the products were propionate, butyrate, valerate, caproate, and a trace of heptylate. When ethanol and succinate were fed, the main products were acetate, butyrate, and caproate (Kenealy and Waselefsky, 1985). However, when aiming at caproate production, ethanol and acetate were still the optimal substrates for C. kluyveri (Seedorf et al., 2008). In addition to these pure substrates, C. kluyveri also achieved a high rate of MCCAs production from the actual waste biomass of syngas fermentation effluent (mainly consisting of ethanol and acetate) (Gildemyn et al., 2017). Moreover, C. kluyveri can also play a key role in the open culture CE process. C. kluyveri was co-cultured with mixed rumen microorganisms, as a result, the co-culture microbiome largely shortened the CE time (48–72 h) and enhanced caproate production (6.11 g/L) from raw materials of cellulosic biomass and ethanol (Weimer et al., 2015). Steinbusch et al. (2011) reported that C. kluyveri (57.8%) dominated the microbial populations in the MCCAs production reactor with ethanol and acetate as substrates. In addition, C. kluyveri was also greatly enriched in the CE reactors fed with beer (Agler et al., 2012a), H2 and CO2 (Zhang et al., 2013), and glycerol (with ethanol as the intermediate) (Leng et al., 2019). Megasphaera elsdenii was originally isolated from sheep rumen samples (Elsden et al., 1956) and named Peptostreptococcus elsdenii by Gutierrez et al. (1959). It was later reclassified by Rogosa (1971). M. elsdenii is a multifunctional strain and can utilize different carbon sources including lactate, glucose, fructose, and sucrose to produce SCCAs (C2–C5) and caproate (C6), H2, and CO2 (Choi et al., 2013; Elsden et al., 1956; Marounek et al., 1989; Weimer and Moen, 2013). M. elsdenii can even utilize corn stover hydrolysates as substrate and achieve efficiently simultaneous production of butyrate and caproate using batch extractive fermentation (Nelson et al., 2017). When glucose is used as the sole substrate of M. elsdenii, butyrate is the predominant product (Marounek et al., 1989). With sucrose as the substrate, acetate/ butyrate supplementation nearly doubles the caproate production. In

(Elsden et al., 1956; Jeon et al., 2013) (Barker and Taha, 1942; Yin et al., 2017)

(Lanjekar et al., 2014)

5.29 g/(L·d) 0.34 g/(L·h) 0.41 g/(L·h) – –

–

Caproate 16.6 g/L Caproate 32 g/L Caproate 9.7 g/L Caproate 28.42 g/L Caproate 8.42 g/L

Main MCCAs Highest caproate production Highest caproate production rate References

Caproate –

30–40 °C 5.0–6.5 Lactate 30–40 °C 5.5–7.5 Fructose 38 °C 7.4 Lactate, sucrose 34 °C 6.8 Ethanol Optimum temperature Optimum pH Optimum ED

37 °C – Glucose

Firmicutes Negativicutes Cow rumen Firmicutes Negativicutes Feces Firmicutes Clostridia Canal mud Affiliated phylum Affiliated class Origin

Firmicutes Negativicutes Sheep rumen

Earlier CE studies, including the investigation of CE mechanism and CE behavior of specific strains, were conducted with pure culture fermentation since it is convenient to control the optimal process parameters for specific bacterial strains. However, because pure culture fermentation requires strict sterilization and high-purity substrates, it limits available feedstock types, complicates operation, and goes against large-scale production (Agler et al., 2012a; Oleskowicz-Popiel, 2018). In recent years, MCCAs production is more inclined to be carried out with mixed culture fermentation (reactor microbiome) which can adapt to nonsterile conditions and utilize complex substrates. To guide the selection of microorganism sources, both pure CE strains and reactor microbiomes are introduced for carrying out MCCAs production. 3.1. Pure culture strains

Firmicutes Clostridia Anaerobic digestion sludge 37 °C 6.8 D-galactitol

Firmicutes Clostridia CE reactor microbiome

Eubacterium pyruvativorans Eubacterium limosum Ruminococcaceae bacterium CPB6 Clostridium sp. BS-1 Megasphaera hexanoica Megasphaera indica Megasphaera elsdenii Clostridium kluyveri

Table 1 Characteristics and performances of main pure CE strains.

3. Microorganisms for CE

602

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contrast, acetate/butyrate addition has little effect on caproate production when lactate is used as the substrate (Choi et al., 2013). Weimer and Moen (2013) reported that M. elsdenii preferred to utilize lactate rather than glucose when both substrates were existed, and acetate and propionate were main products but hardly any caproate was detected from lactate. However, caproate and butyrate became the main products when M. elsdenii used glucose, but both the selectivity and production of caproate were low (Weimer and Moen, 2013). In addition, seven strains of M. elsdenii were recently isolated from pig feces on a Japanese farm (Yoshikawa et al., 2018), and found to be able to utilize lactate to produce valerate as the predominant product. However, M. elsdenii was scarce in open culture fermentation. The absence of M. elsdenii was reported by Kucek et al. (2016c) and Nzeteu et al. (2018), where caproate productions were carried out from lactate. Genus Megasphaera was also reportedly present at the beginning of a CE process and then diminished quickly to < 1% (Duber et al., 2018). Other CE reactor microbiome also did not have detectable enrichment of M. elsdenii, indicating that this strain could not adapt to the open culture fermentation and was less competitive than the other microorganisms present. Megasphaera indica (NMBI-10T), another strain of genus Megasphaera, was isolated from human feces in 2014 (Lanjekar et al., 2014). M. indica can utilize a variety of carbohydrates including glucose, lactose, cellobiose, rhamnose, galactose, and sucrose; and caproate and succinate were the two main products from glucose fermentation. Megasphaera hexanoica (MHT), the closest taxonomic relative of M. indica (Jeon et al., 2017), was isolated from a cow rumen (Jeon et al., 2016). M. hexanoica is a caproate-producing bacterial strain. It can metabolize fructose to produce C2–C8 carbon-chain carboxylates, and supplementation with acetate, propionate and butyrate as EAs enhanced MCCAs production (Jeon et al., 2016). Recently, M. hexanoica was co-cultured with Clostridium tyrobutyricum (an excellent strain to produce acetate and butyrate) to produce caproate using a submerged hollow-fiber membrane bioreactor, and a caproate concentration of 10.08 g/L and productivity of 0.69 g/(L·h) were obtained (Kim et al., 2018). Clostridium sp. BS-1, affiliated to the genus Clostridium IV, was isolated in recent years from anaerobic sludge in a wastewater treatment plant (Jeon et al., 2010). Based on its phenotypic and phylogenetic characteristics, Clostridium sp. BS-1 represents a novel genus within Clostridium IV, and was thus renamed Caproiciproducens galactitolivorans (Kim et al., 2015). The optimized medium composition for C. galactitolivorans is: 15.5 g/L yeast extract, 10.13 g/L tryptone, 0.04 g/L FeSO4·7H2O, 0.85 g/L sodium acetate, and 6.47 g/L sodium butyrate (Jeon et al., 2013). C. galactitolivorans can utilize various carbohydrates including glycerol, L-arabinose, L-ribose, D-xylose, L-galactose, D-glucose, D-fructose, D-mannose, dulcitol, glucosamine, D-cellobiose, starch, glycogen, D-tagatose, and L-fucose. The main metabolic end products are H2, CO2, ethanol, acetate, butyrate, and caproate (Kim et al., 2015). With galactitol as substrate, C. galactitolivorans can produce caproate as the primary product. Addition of acetate increases caproate production (2.9 g/L), which was largely increased to 32 g/L with a production rate of 0.34 g/(L·h) using in situ extractive fermentation to remove the caproate generated (Jeon et al., 2013). C. galactitolivorans can also utilize glucose to produce caproate but is less efficient than with galactitol (Jeon et al., 2010). Moreover, it was found that a co-culture of C. galactitolivorans and other strains isolated from sludge, such as Clostridium sp. BS-6, BS-7 and BS-8, achieved higher caproate production than did the pure culture of C. galactitolivorans (Jeon et al., 2010). In an open culture CE reactor with thin stillage as substrate, the reactor microbiome was dominated by a Clostridium IV species with 95% similarity to C. galactitolivorans, and its domination led to the production of caprylate and caprate (caproate was still the primary product with a concentration of 8.1 g/L) (Andersen et al., 2017). Moreover, there was a significant linear correlation between caprylate production and the ratio of C. galactitolivorans to Clostridium sp. BS-6 (p = 0.03). The more the relative abundance of C.

galactitolivorans with respect to Clostridium sp. BS-6, the higher the caprylate production (Andersen et al., 2017). Recently, other new strain that was also affiliated to Clostridium IV, called Ruminococcaceae bacterium CPB6 was isolated by Zhu et al. (2017) from a microbiome. It was found responsible for caproate production from lactate. This strain prefers pH of 5.0–6.5, temperature of 30–40 °C, and lactate as ED for caproate production. In addition, other substrates like starch, maltose, glucose, arbutin, salicin, pyruvate, lactate, formate, acetate, butyrate, malate, fumarate, citrate, oxaloacetate, and 2-oxoglutarate can also be utilized by Ruminococcaceae bacterium CPB6 (Tao et al., 2017b). In a caproate production system from food waste (lactate as intermediate), Clostridium sp. with 99% similarity to Ruminococcaceae bacterium CPB6 and Clostridium sp. MT1 played a key role in caproate production (Nzeteu et al., 2018), indicating that Ruminococcaceae bacterium CPB6 could also be enriched and work in the open culture CE process. In one of the first papers on the microbiome composition of CE bioreactors from Agler et al. (2012a), a positive correlation between Ruminococcaceae (16S) and caproate production was also found. Eubacterium limosum, initially isolated from the rumen fluid of sheep, can also carry out the CE process to produce butyrate and caproate from methanol and acetate. However, in the CE process conducted by E. limosum, caproate (0.78 mM C) was only a by-product, while butyrate (35.68 mM C) was the major product (Genthner et al., 1981). Afterwards, it was reported that caproate could become the main product by culturing E. limosum with methanol, butyrate, and CO2 as feedstocks (Lindley et al., 1987; Tarasov et al., 2011). However, because of its low caproate selectivity, E. limosum is more often studied for butyrate production from syngas fermentation nowadays (Park et al., 2017; Song et al., 2017). During the process of metabolizing syngas (CO2, H2, and CO), E. limosum is was used to produce acetyl-CoA for subsequent utilization (Song et al., 2018). In an open culture fermentation reactor for the co-production of 1,3-propanediol and caproate from glycerol, E. limosum was also greatly enriched as a glycerol degrader and worked synergistically with C. kluyveri, which was responsible for upgrading ethanol and acetate (Leng et al., 2019). Eubacterium pyruvativorans, also isolated from sheep rumen (Wallace et al., 2003), is a non-saccharolytic, non-alcohol, and incomplete lactate-utilization strain (Wallace et al., 2004). However, it is able to utilize pyruvate derived from amino acids or peptides rapidly, to growth and harvest energy (ATP) (Wallace et al., 2003). E. pyruvativorans can produce valerate and caproate (the predominant product) by coupling two‑carbon from amino acids to propionate and butyrate, respectively, through the reverse β oxidation pathway (Wallace et al., 2004). Thus, this strain can be supplemented to facilitate MCCAs production when a protein-rich substance is used as feedstock. 3.2. Mixed culture fermentation Mixed culture fermentation, also called a reactor microbiome, consists of a group of microorganisms living in an artificial (bioreactor) environment and does not rely on specific strains (Oleskowicz-Popiel, 2018; Zheng et al., 2018). Steinbusch et al. (2011) provided the first hint that CE under mixed culture could be turned into a biotechnology production platform technology. Compared with pure culture, open culture fermentation has the capacity to sustain stable function over a long operating period even with nonsterile feedstock (Agler et al., 2012a; Ge et al., 2015). In addition, by cooperating and interacting with other microbes, the microorganisms in mixed culture fermentation show a higher resilience level in functionality and organization (Oleskowicz-Popiel, 2018). It is therefore more effective to conduct waste biomass based MCCAs production using a well-functioning reactor microbiome rather than using any pure strain. Microorganism resources such as sewage sludge, natural wastes, or another reactor microbiome, can be used as the inoculum for MCCAs production (Agler et al., 2012a; Duber et al., 2018; Kucek et al., 2016c; Scarborough et al., 2018). Before reaching a satisfying MCCAs 603

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production performance, a purposeful operating period is needed to adapt the microorganism to specific substrates, and to enrich CE functional microbiota. This process was defined as microbiome shaping by Agler et al. (2012b) who demonstrated that the main microbiome shaping methods were to purposefully (1) change the feedstock composition; (2) adjust operating conditions; and (3) change the bioreactor history by provisionally imposing perturbations including oxygen addition, heat shock, etc. By using a natural microbiome with a long-term microbiome shaping process (pH 5, temperature of 30 °C, ethanol as the main feedstock, and in-line extraction of product), the caproate production rate and specificity from yeast fermentation beer (mainly consisting of dilute ethanol, leftover biomass, and yeast cells) reached 1.5 g/(L·d) and 79%, respectively (Agler et al., 2012a). More than 50% of the assigned reads in the shaped microbiome belonged to Clostridium spp., and other detected species, like the members of the genera Ethanoligenens, Bifidobacterium, and Desulfitobacterium, were associated with hydrolysis and ethanol oxidation (Agler et al., 2012a). To shape a wellfunctioning CE microbiome, timely product extraction is significant because the toxicity of undissociated MCCAs could hinder the enrichment of functional bacteria (Spirito et al., 2014). Weimer et al. (2015) reported that an undissociated caproate concentration of 6.9 mM exhibited toxicity in a batch test with pH 5.7, and Ge et al. (2015) found the threshold concentration at which undissociated caproate showed toxicity was about 7.5 mM in a long-term continuous reactor with pH 5.5. In addition, microbiome structures vary greatly depending on the feedstock. C. kluyveri, Clostridium butyricum, and Escherichia coli that rarely exist in anaerobic digestion sludge (the inoculum) were co-enriched substantially and constituted a synergistic relationship to utilize glycerol to produce 1, 3-propanediol and caproate (Leng et al., 2017). Zhu et al. (2015) used Chinese yellow water (mainly containing lactate, ethanol, polysaccharide, and SCCAs) as feedstock and pit mud (a special fermentation mud devoted to providing the distinct aroma of Chinese strong-aroma liquor by its unique microflora) as inoculum to produce caproate (12.93 g/L, 2.46 g/(L·d)). A unique microbiome dominated by Clostridium IV (79.07%) was shaped after 90 days of cultivation, while the raw pit mud only contained 12.71% of Clostridium IV. It is noteworthy that pit mud could be a superior microorganism resource for MCCAs production since it is rich in CE functional bacteria (Hu et al., 2015; Hu et al., 2014; Zhu et al., 2017). Specifically, the active populations in pit mud that were mainly involved in caproate production were Clostridium IV and other Clostridium spp. (Tao et al., 2017a; Zhu et al., 2015). In addition, with the same microbiome (Clostridium spp. > 50%) from Agler et al. (2012a) as initial microorganism resources, the microbial composition changed greatly in the follow-up studies with different substrates and operating conditions (Kucek et al., 2016a; Kucek et al., 2016b; Kucek et al., 2016c; Xu et al., 2018). After changing the feedstock to lactate and butyrate, the main species became Rhodocyclaceae K82 spp. (63.3%) after 30 days, and then Acinetobacter spp. (62.9%) after 140 days, respectively. The maximum caproate production rate reached 6.9 g COD/(L·d) at a lactate loading rate of 9.1 g COD/(L·d) (Kucek et al., 2016a). When the ethanol/acetate ratio in the synthetic substrate was increased to 15, the microbiome structure varied from predominantly Acinetobacter spp. (55.5%) during days 130–145 to the overwhelming abundance of Rhodocyclaceae K82 spp. (70.8%) during days 160–170. Caprylate became the primary product with a high production rate of 19.4 g COD/(L·d) and caprylate/ caproate ratio of 11 g COD/g COD (Kucek et al., 2016b). When wine lees (consisting of settled yeast cells and ethanol) was used as a feedstock and the broth-recycle rate for the in-line product extraction was largely increased, Bacteroides spp. became the dominant species. The total MCCAs production rate, yield, and specificity was 3.9 g COD/(L·d), 67%, and 72%, respectively, and a relatively high caprylate/caproate ratio of 1 was obtained (Kucek et al., 2016c). When the lactate-rich effluent generated from acid whey was used as feedstock, Bacteroidales spp. (21.7%) and Clostridiales spp. (12.6%) were most abundant in the microbiome, and a caproate production rate of 1.68 g/(L·d) was

obtained (Xu et al., 2018). It was noted that the best-known CE strains (like C. kluyveri and M. eladenii) mentioned above were not greatly enriched in the microbiome and also did not play an important role in MCCAs production. This is likely because these pure strains were less competitive than the other microorganisms in the mixed culture fermentation. In summary, the microbial composition varies greatly during the microbiome shaping process in response to different substrates and operating conditions, but they all contribute to more stable and effective MCCAs production performance. In future studies, the relationships between bioreactor performance, operating conditions, feedstock types, and microbial community succession should be more deeply understood to enhance microbiome shaping efficiency. 4. MCCAs production from waste biomass Waste biomass containing large amounts of biodegradable organics and abundant nutrients, is a promising feedstock for biofuel/chemical production (Agler et al., 2012b). Since the precursors for MCCAs production by the CE pathway are available in many waste biomass or can be anaerobically produced from organic wastes or lignocellulosic materials under suitable conditions, CE-based MCCAs production shows higher competitiveness than the previous, more costly chemical synthesis methods. Table 2 summarizes the published studies for MCCAs production from waste biomass. Based on their component characteristics and corresponding MCCAs production strategies, waste biomass can be divided into three types: (I) contains both ED and EA, and thus needs no exogenous ED and EA; (II) can be used to produce EA in situ and exogenous ED must be supplied; and (III) can be used to produce both ED and EA in situ and needs no exogenous substance. This classification clearly show the available waste biomass and corresponding MCCAs production strategies (summarized in Fig. 2). It also includes relevant research. 4.1. Feedstock type I The first type of waste biomass, containing both ED and EA, is the best feedstock for MCCAs production. After proper pretreatment, such as impurity removal, disintegration, dilution, concentration, etc., the waste biomass along with suitable microbiome can be fed into a onestage bioreactor (batch reactor, or continuously fed reactor like continuous stirred tank reactor (CSTR), upflow anaerobic filter (UAF), etc.) to carry out the CE process. The substrate concentration, feed rate, and corresponding CE influencing factors need to be optimized to achieve high-efficiency MCCAs production. The factors include the organic loading rate (OLR), hydraulic retention time (HRT), solid retention time (SRT), pH, temperature, etc. Byproducts from the wine industry are the representative of this feedstock type, which generally contain plenty of ethanol or lactate (superior EDs for CE) and slight SCCAs (EAs). In addition, other organics existed in the byproducts, such as residual corn kernels, polysaccharides, yeast cells, etc., can also be converted into SCCAs through anaerobic hydrolysis and acidification, and the SCCAs generated in situ, along with preexisting SCCAs can be incorporated into MCCAs (Agler et al., 2012a). Ge et al. (2015) converted complex yeast-fermentation beer to caproate by operating a continuous anaerobic reactor equipped with an in-line membrane liquid-liquid products extraction unit, and obtained an average caproate production rate of 3.38 g/(L·d) (7.52 g COD/(L·d)) and caproate yield of 70.3%, over 55 days of continuous operation. Yellow water, comes from the fermentation mash used in Chinese strong-flavour liquor production (occupying the largest market share (70%) of all Chinese liquor production) (Zhao et al., 2012) and mainly consists of lactate, ethanol, glucose, and SCCAs., is also a suitable substrate for MCCAs production. Zhu et al. (2015) obtained a 2.46 g/(L·d) caproate production rate with diluted yellow water as the sole substrate without product extraction. Recently, Wu et al. (2018) obtained a high MCCAs yield (about 80.34%) from actual Chinese 604

Pit mud CE reactor microbiome

Anaerobic digestion sludge

Sewage sludge + pit mud

Lignocellulosic stillage

Chinese liquor-making wastewater

605 Batch reactor

Mixed ruminal microbes + C. kluyveri

CE reactor microbiome

C. kluyveri + mixed culture

Granular sludge

Food waste

Food waste

a

Leach-bed reactor + membrane electrolysis Batch reactor

Anaerobic digester sludge

Vegetable and salad waste Grass

The production/production rate/yield refers to caproate if no special designation.

Leach-bed reactor

Batch reactor

Anaerobic digester sludge

Continuous stirred tank reactor (CSTR) + UAF + in-line product extraction Upflow anaerobic sludge blanket

Acid Whey

Type III Acid Whey

Lactate production microbiome + CE microbiome

Batch reactor

Activated sludge

Modified rotting box

Brewer's spent grain + ethanol Switchgrass + ethanol

OFMSW + ethanol

No inoculum (Acidification) + CE reactor microbiome No inoculum

Batch UAF + continuous UAF

Batch reactor

Batch reactor

OFMSW + ethanol

No inoculum

Batch reactor UAF + in-line product extraction

CE reactor microbiome

Type II OFMSW + ethanol

UAF + in-line product extraction

C. kluyveri

Batch reactor

UAF + in-line product extraction

CE reactor microbiome CE reactor microbiome

Yeast fermentation beer Syngas fermentation effluent Syngas fermentation effluent Syngas fermentation effluent Chinese yellow water Wine Lees (40% ethanol)

Natural microbiome

Type I Yeast fermentation beer

Reactors

Upflow anaerobic filter (UAF) + in-line product extraction UAF + in-line product extraction UAF

Inoculum

Waste biomass

Table 2 Summary of MCCAs production performance from actual waste biomassa.

Clostridia (50%), Actinobacteria (14.2%), Sphingobacteriales (8.3%), Deltaproteobacteria (8.3%), Bacilli (8.3%) Clostridium spp. (~42%)

Clostridium IV (28%), Lactobacillus (26%)

Coriobacteriaceae (37.4 ± 7.2%), Ruminococcaceae (20.5 ± 8.4%), Prevotellaceae (17.9 ± 8.5%) –

Bacteroidales OUT (21.7 ± 9.1%), Clostridiales OUT (12.6 ± 4.9%)

C. kluyveri

–

–

–

–

Clostridium IV, Veillonella, Bacteroides

Lactobacillus, Roseburia, Atopobium, Olsenella, Pseudoramibacter (> 95%)

Clostridium IV (79.07%) Bacteroides spp., Ruminococcus spp.

Rhodocyclaceae K82 spp. (70.8%)

C. kluyveri

– –

Clostridium spp. (> 50%)

Dominated microbes

10 g/L

8.1 g/L

4.09 g/L

3 g/L

10.45 g/L

–

6.1 g/L

8–31.5 g/L (SCCAs+MCCAs) 0.9 g/L

2.7 g/L (caproate); 1.5 g/ L (heptylate); 0.5 g/L (caprylate) 12.6 g/L

7.51 g/L

–

12.93 g/L –

–

–

– 1 g/L

–

MCCAs production

3 g/(L·d)

–

0.99 g/(L·d)

–

3.2 g/(L·d)

1.68 g/(L·d)

2.03 g/(L·d)

–

–

1.9 g/(L·d)

–

19.4 g COD/(L·d) (caprylate) 2.46 g/(L·d) 1.57 g/(L·d) (caproate); 1.43 g/ (L·d) (caprylate) 2.6 g/(L·d) (caproate); 0.27 g/ (L·d) (caprylate)

4.64 g/(L·d)

3.6 g/(L·d) 1.7 g/(L·d)

2.1 g/(L·d)

MCCAs production rate

–

–

> 70%

7–24%

58–83%

53.5%

–

–

–

78%

–

80.34%

18%

73% 67%

61%

90%

70.3% 9.6%

–

MCCAs yield

(Nzeteu et al., 2018)

(Reddy et al., 2018a)

(Bolaji and Dionisi, 2017) (Khor et al., 2017)

(Duber et al., 2018)

(Xu et al., 2018)

(Kannengiesser et al., 2016) (Liang and Wan, 2015) (Weimer et al., 2015)

(Grootscholten et al., 2014)

(Grootscholten et al., 2013d)

(Wu et al., 2018)

(Scarborough et al., 2018)

(Zhu et al., 2015) (Kucek et al., 2016c)

(Ge et al., 2015) (Vasudevan et al., 2014) (Gildemyn et al., 2017) (Kucek et al., 2016b)

(Agler et al., 2012a)

References

Q. Wu, et al.

Biotechnology Advances 37 (2019) 599–615

Biotechnology Advances 37 (2019) 599–615

Q. Wu, et al.

Fig. 2. Strategies for MCCAs production from the three feedstock types.

liquor-making wastewater (CLMW) without additional introduction of EDs and EAs. They found that the coexistence of ethanol and lactate could form a cooperative relationship to facilitate substrate utilization, and hence enhance MCCAs production. In addition to the liquid byproducts mentioned above, wine lees, the main solid waste from the wine industry consisting primarily of settled yeast cells, and ethanol (86.5 g/L, 1.88 M), was also used as feedstock. A high MCCAs production rate (7 g COD/(L·d)) and MCCAs yield (67%) was obtained (Kucek et al., 2016c). Additionally, the lignocellulosic stillage was also verified to be potentially useful for producing MCCAs, and a caproate production rate of 2.6 g/(L·d) and caprylate production rate of 0.27 g/(L·d) was achieved. However, the conversion ratio of stillage to MCCAs was still low (18 ± 2.1%) due to the low biodegradability of lignocellulose, which remains to be further enhanced to improve the economy of lignocellulosic biorefinery (Scarborough et al., 2018). In general, upgrading the by-products from distillery to MCCAs is more commercially viable for recycling than the current, more expensive recycling methods like distilling ethanol, extracting lactate, and manufacturing esterification liquid. Therefore, the CE biotechnology is expected to be adopted by the wine industry to provide a more sustainable wine producing path. In addition to the wine industry, the effluent of waste-derived syngas fermentation also provides a superior source of diluted ethanol and acetate. Vasudevan et al. (2014) conducted a proof-of-concept study on the feasibility of MCCAs production from actual syngas fermentation effluent using a reactor microbiome. The final production

rate of caproate and butyrate was ~1.7 and 20 g/(L·d), respectively. The low caproate selectivity was ascribed to the limited elongation of butyrate into caproate because of product toxicity. Afterwards, benefiting from an in-line product extraction unit, Gildemyn et al. (2017) obtained a higher caproate production rate of 40 mM/L (4.64 g/(L·d)), caprylate production rate of ~2.19 mM/L, and carbon conversion ratio of > 90% from actual syngas fermentation effluent using a pure culture of C. kluyveri. It is worth mentioning that, the highest caprylate production rate (19.4 g COD/(L·d)) and specificity (96%) reported so far, were achieved using a synthetic syngas-fermentation effluent. This was mainly attributed to the high ethanol/acetate ratio of 15, in-line product extraction, and a specially shaped microbiome (Kucek et al., 2016b). Therefore, syngas fermentation effluent is also a superior feedstock for MCCAs production. Moreover, since both CE and syngas fermentation are anaerobic technologies and require similar temperature, pH, growth nutrients, and other anaerobic conditions (Kucek et al., 2016b), these two waste-derived technologies have the potential to be coupled in the future to share nutrients and recover high-value MCCAs, with the prerequisite of timely MCCAs extraction (Agler et al., 2012a). 4.2. Feedstock type II The second type of waste biomass is the organics that can be used to produce EA (SCCAs) in situ through hydrolysis and acidification but lacks available ED. Therefore, external ED needs to be supplemented to 606

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enable the CE process. Since this feedstock type requires two sequential fermentation steps (SCCAs generation and subsequent CE) to finally produce MCCAs, the available reactors can be divided into one-stage and two-stage types. The one-stage reactors integrate feedstock hydrolysis/acidification and CE processes in one reactor, which might result in reduced MCCAs yield because the hydrolysis rate of the feedstock could be limited by the toxicity of the added EDs and undissociated MCCAs. In the research by Grootscholten et al. (2013d), the feasibility of MCCAs production from the organic fraction of municipal solid waste (OFMSW) was verified, while the MCCAs production was seriously limited due to the severely suppressed OFMSW hydrolysis in the one-stage reactor. Afterwards, Grootscholten et al. (2014) separated the OFMSW primary fermentation and CE into two sequential reactors (two-stage reactor), and almost 5-fold higher caproate production was obtained compared with the previous one-stage system. Recently, Reddy et al. (2018a) also obtained high butyrate (8.9 g/L) and caproate (8.1 g/L) production from food waste by operating a two-stage reactor. Compared with a one-stage reactor, the two-stage reactor allows the separation of hydrolysis/acidification and CE into two different reactors. This has the advantages of (1) eliminating the toxicity effects of exogenous ED and generated MCCAs on hydrolysis process; (2) allowing separate control of the optimum operating conditions for the different processes; and (3) avoiding the adsorption of the generated MCCAs to the granular feedstock (Grootscholten et al., 2013d; Grootscholten et al., 2014). The main drawbacks of the two-stage reactor are the more complicated operation process and increased facility cost. Thus, the reactor type should be properly chosen by systematically considering the feedstock characteristics and economic calculations. To produce MCCAs efficiently from the second feedstock type, in addition to the substrate concentration and related operating conditions mentioned above (a two-stage reactor needs to have the process parameters in each of the different reactors optimized), the ED addition dosage and rate should also be optimized to ensure successful CE and meanwhile avoid waste from excessive ED. In addition, appropriate product extraction strategies should be applied to prevent the toxicity of undissociated MCCAs to related bacteria. A life cycle assessment conducted by Chen et al. (2017) quantified the factors associated with caproate production from OFMSW and supermarket food waste in both lab-scale and pilot-scale systems. The results showed that ethanol addition and extraction solvent recovery have substantial influences on the life-cycle impacts. Thus future study should focus on reducing the ED dosage and improving the recovery efficiency of the extraction solvent.

arrangement achieved a final conversion efficiency of > 50% without adding any exogenous substances. In contrast, Duber et al. (2018) operated a one-stage upflow anaerobic sludge blanket (UASB) reactor (pH 5.5 and 30 °C) to produce caproate from acid whey with the preexisting and in situ generated lactate as ED. A higher MCCAs yield of 58–83% was achieved with an average caproate production rate of 0.11 g/(L·h) and a caproate/carboxylate ratio of 83%. The differences in MCCAs production performance between the two studies utilizing acid whey as substrate mainly depends on the amount of lactate that was already present in their acid whey. The acid whey used in Duber et al. (2018) was partly fermented in the factory and laboratory before utilization, thus most lactose was converted to lactate (average lactose/ lactate molar ratio was 0.78 (mM C/mM C)). However, the acid whey used by Xu et al. (2018) possessed a much higher lactose concentration (1030–1430 mM C) than lactate concentration (272–438 mM C), thus a lactate acid-producing step was needed to supply lactate for the subsequent CE process. This is also the main reason that Duber et al. (2018) could achieve a higher MCCAs yield from acid whey in a one-stage reactor. Recently, Nzeteu et al. (2018) verified the feasibility of caproate production only from food waste without external ED supplementation. The high caproate production of 23 g COD/L (10 g/L) and caproate production rate of 3 g/(L·d) were obtained under high H2 partial pressure and neutral pH, but caproate specificity was only about 40%. In addition, it is worth noting that maintaining neutral pH by adding alkaline chemicals is too costly for real FW fermentation since the pH of food waste acidification is normally as low as 4–5.5. Moreover, as an energy substance, H2 input also greatly improved the MCCAs production cost. Before this, food waste was also used to produce caproate under the premise of external ethanol addition (Reddy et al., 2018a), resulting in increased MCCAs production cost. In fact, the current waste biomass based MCCAs production relies mainly on the addition of exogenous ED. However, not only the EA (SCCAs) but also the ED (ethanol or lactate) can be produced by anaerobically treating the complicated organics (containing starches, sugars, protein, cellulose, etc.) under corresponding conditions. Therefore, considering the high cost to supply additional ethanol or lactate produced off site, in situ ethanol/lactate production before or during the CE process under the favorable conditions and microbiome is strongly recommended. In addition, in view of the different process parameters and microbiomes involved in the formation of ED and EA formations, a possible MCCAs production strategy is also recommended: carrying out ED and EA productions separately and then combining the generated ED and EA to produce MCCAs (Fig. 2d). The feedstock for this MCCAs production strategy could be two different kinds of waste biomass that are suited to producing ED and EA, respectively, or one kind of waste biomass to produce ED and EA in separate reactors under respective optimal conditions.

4.3. Feedstock type III The third type of feedstock is capable of producing both ED and EA as intermediates for subsequent MCCAs production. Currently, the representative waste biomass mainly includes glycerol, acid whey, food waste, and sludge. Similar to the feedstock type II, both one-stage and two-stage reactors are appropriate reactor types. The related process parameters for the two reactor types should be optimized, and corresponding measures should be taken to avoid their respective defects, as mentioned above. Leng et al. (2017) operated a one-stage reactor and achieved the co-production of 1,3-propanediol and caproate from crude glycerol (a by-product of biodiesel production). In this case, 1,3-propanediol was first obtained from glycerol fermentation with ethanol, acetate, and butyrate as byproducts, and then caproate was formed after a few days of lag phase. During the CE process, ethanol was used as ED to elongate acetate to caproate. Thus, caproate is indirect from glycerol, and caproate production from glycerol was not a direct bioconversion. To harvest MCCAs (mainly caproate) from acid whey (a Greek-yogurt waste stream), Xu et al. (2018) operated a sequential twostage reactor to utilize acid whey by first performing thermophilic lactate production (pH 5 and 50 °C) and then conducting a mesophilic CE process (pH 5 and 30 °C) with different microbiomes. This

5. Strategies for enhancing MCCAs production performance To achieve development of a superior and sustainable form of bioenergy, high bioenergy production rate and yield are essential to ensure its economic benefit. However, similar to other anaerobic technologies, there are also some adverse factors limiting MCCAs production performance. Therefore, more research is needed to drive advances in the CE technology to overcome these barriers. Herein, the main limiting factors for MCCAs production and the potential solution strategies (Fig. 3) are introduced based on existing studies. 5.1. Avoiding product inhibition In the CE process, once the generated MCCAs reaches a certain concentration (toxicity limit), the CE functional bacteria cannot grow and metabolize normally because of the toxicity effect from undissociated MCCAs. As a result, both ED oxidation and reverse β oxidation processes would stop. Therefore, preventing the feedback 607

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Fig. 3. Outline of the strategies for enhancing MCCAs production.

hollow fiber membranes, showed better performance to selectively extract MCCAs from fermentation broth (pH 5–5.5) into the alkaline extraction solution (pH 9) with 3% tri-n-octylphosphine oxide as extractant (Agler et al., 2012a; Ge et al., 2015; Kucek et al., 2016a; Kucek et al., 2016b). The advantage of pertraction extraction over the traditional liquid-liquid extraction mentioned above is that it avoids contact of the extraction solvent with the microbiome and fermentation broth. A high MCCAs production rate of ~3.38 g/(L·d) (7.52 g COD/(L·d)) with the caproate yield of ~70.3% and caproate/ethanol ratio of ~1.2 were obtained in a continuous CE reactor (operational pH 5.5) equipped with a pertraction extraction unit (Ge et al., 2015). The mildly acidic pH of 5.5 was used to form a pH gradient with the extraction buffer (pH 9) to accomplish the extraction of undissociated MCCAs. Meanwhile, acetoclastic methanogenesis could be suppressed by the acidic pH (Angenent et al., 2016). This extraction technology consumes little energy, while the collecting dissociated carboxylate (R-COO−) in the alkaline extraction solution needs to be further converted to the undissociated form (R-COOH) to achieve phase separation and subsequent application. This process requires addition of chemicals like HCl. To reduce the cost of this step, a membrane electrolysis cell was integrated with a pertraction extraction unit to continuously separate undissociated MCCAs using the pH gradient between the two electrolysis chambers (Xu et al., 2015). The membrane electrolysis cell could continuously separate MCCAs by phase separation in the anode chamber, and an oily liquid containing about 90% caproic acid and caprylic acid was obtained (Xu et al., 2015). In the future, to simplify production, more research should focus on establishing a stably integrated system to accomplish MCCAs production, extraction, and separation simultaneously. Another way to avoid product inhibition of the microorganisms is to

inhibition of MCCAs on microorganisms is a prerequisite for sustainable MCCAs production. Extracting MCCAs is the most direct approach to avoid product inhibition, which also makes it a fundamental step for the industrial production of MCCAs (Cavalcante et al., 2017). The traditional methods for extracting carboxylate from fermentation broths include precipitation, liquid-liquid extraction, distillation, membrane dialysis, electrodialysis, and ion exchange (Wasewar and Shende, 2011). Among these available methods, liquid-liquid extraction, based on reversible chemical complexation, has been used to transfer MCCAs from the biotic medium to an extraction medium with a suitable extractant. The extractant is generally a viscous liquid that dissolves in diluents, and the diluents provide a higher solubility for extractants through specific reactions (Wasewar and Shende, 2011). Trialkylphosphine oxide dissolved in kerosene was initially used to extract caproate (Wang et al., 2001). The extractant tri-n-butyl phosphate (TBP) with diluents of benzene and toluene was also proven to be effective for caproate extraction (Wasewar and Shende, 2010). Afterwards, to achieve simultaneous MCCAs production and extraction, Choi et al. (2013) operated an in situ biphasic extractive fermentation system (a single vessel reactor), which could timely extract > 90% of the caproate from the fermentation broth into the extractant (10% (v/v) alamine 336 in oleyl alcohol). As a result, this in situ biphasic extractive fermentation system presented about four times higher caproate production capacity (caproate yield of 0.5 g/g-sucrose and production rate of 0.2 g/(L·h)) compared with that without an in situ extraction unit. Using this biphasic extractive fermentation system, Jeon et al. (2013) also obtained the relatively high caproate concentration of 32 g/L and caproate production rate of 0.34 g/(L·h) from galactitol. Recently, pertraction extraction, which is a liquid-liquid extraction process carried out using 608

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dilute the substrates and products since the inhibition effect occurs only when undissociated MCCAs reach the toxic-limit concentration. Dilution can be implemented by adding water to the influent or by recirculating bioreactor effluent (Angenent et al., 2016). In addition to avoid the toxicity of MCCAs, dilution can also reduce the toxicity effect of the substrate (like ethanol) on microorganisms (Angenent et al., 2016). The ethanol concentration likely to inhibit CE bacteria is between 10 and 20 g/L (~200–400 mM) (Ge et al., 2015). However, dilution could diminish the MCCAs production rate and improve the subsequent extraction cost (Liu et al., 2017). Thus, timely product extraction may be the best approach to prevent product inhibition.

acidic pH itself not only can reduce methanogenic activity, but can also make part of the MCCAs exist in the undissociated forms (the pKa of the carboxylic acids was 4.8–4.9) (Agler et al., 2012a). The methanogens are susceptible to the toxicity of undissociated MCCAs (Cavalcante et al., 2017; Chen et al., 2008; Reddy et al., 2018b) and undissociated SCCAs at concentrations above 5 mM are sufficient to inhibit methanogens (Anderson, 1982). In contrast, CE functional bacteria resist the toxic effects of undissociated MCCAs within a certain concentration range (Steinbusch et al., 2011). The CE bacteria are inhibited at undissociated caproic acid concentrations of 6.9 mM (Weimer et al., 2015), 7.5 mM (Ge et al., 2015), and 17.2 mM (Duber et al., 2018), respectively. The undissociated carboxylic acids concentrations mainly depended on the pH. It is noteworthy that too high undissociated carboxylic acid concentrations could poison all anaerobes including CE bacteria. Therefore, there might be a specific undissociated carboxylic acids concentration and corresponding pH that can exactly inhibit methanation and simultaneously ensure normal MCCAs production, and the pH might vary with different substrates and reactors. These areas merit further investigations. Another kind of substrate consumption involves sulfate-reducing bacteria, which utilize the same kinds of substrates (H2, acetate, propionate, and butyrate) as CE bacteria and methanogens (Lovley and Klug, 1983; Lovley and Phillips, 1987; Stams et al., 2005). When the feedstock contains very little or no sulfur, the competition for substrates mainly occurs among methanogens and CE bacteria; however, when the feedstock contains sufficient sulfur, sulfate-reducing bacteria are predominant. This is because the sulfidogenic process is thermodynamically more favorable than the other two processes are (Cavalcante et al., 2017). Therefore, appropriate measures need to be taken to suppress sulfate-reducing bacteria when sulfur-rich substances are used. In addition to inhibiting methanogenic archaea, CHCl3 also inhibits acetate-consuming sulfate-reducing bacteria (Chidthaisong and Conrad, 2000), and thus could be used if needed.

5.2. Preventing competition reactions Although open culture fermentation has the advantage of remaining stable function even when facing nonsterile feedstock and unexpected disturbance, the open culture environment and nonsterile substrates also introduce other bacteria with diverse functions. In addition, these bacteria generally possess multiple metabolic pathways; thus some other thermodynamically feasible bioprocess could also exist. As a result, several biological competition reactions occur during the CE process to consume substrates or intermediates, directly resulting in a lower substrate utilization ratio and MCCAs yield. These competition reactions mainly involve substrate consumption from methanogens and sulfate-reducing bacteria, excessive ethanol oxidation to acetate, carboxylic acids oxidation, and the acrylate pathway (in lactate-guided CE). To ensure effective MCCAs production, these competitive processes should be limited. 5.2.1. Preventing supererogatory substrate consumption Substrate consumption by methanogens includes acetoclastic methanogens consuming acetate and hydrogenotrophic methanogens consuming H2 and CO2. Acetate is the main EA for CE, and thus the acetate consumption by acetoclastic methanogens rather than CE bacteria directly reduces MCCAs yield, which should be strictly controlled. For hydrogenotrophic methanogenesis, Agler et al. (2012a) and Grootscholten et al. (2014) reported that methane generation only from H2 and CO2 did not involve competition for the substrate used for MCCAs production. This was based on the premise of preventing excessive ethanol oxidation by (1) suppressing other ethanol-oxidizing microorganisms except for the MCCAs producers, and (2) raising H2 partial pressure to make this process thermodynamically unfavorable. To guarantee satisfactory MCCAs yield, acetoclastic methanogens need to be inhibited completely. The currently main methods are to add methanogen inhibitors such as 2-bromoethanesulfonic acid (2-BES) and CHCl3, or to decrease pH to weakly acidic value. 2-BES generally contributes to inhibition of acetoclastic methanogens, while CHCl3 can suppress both acetoclastic and hydrogenotrophic methanogens (Chidthaisong and Conrad, 2000). Steinbusch et al. (2011) achieved sustainable caproate (8.17 g/L) and caprylate (0.32 g/L) production for 115 d by adding 10 g/L 2-BES to suppress methanogenesis and controlling pH at 7 to make the generated MCCAs occur in dissociated (nontoxic) forms. However, the high cost and dosage for chemical addition also significantly increased the MCCAs production cost. Thermal pretreatment of the inoculum could exterminate most of the initial methanogens and retain the CE-related bacteria since most of the CE functional bacteria possess heat-resistant spores and can survive extreme temperature conditions (Sauer et al., 1995; Steinbusch et al., 2009). In addition, based on the considerable differences in growth rates between methanogens and CE bacteria, Grootscholten et al. (2013c) reported that the suspended methanogens could be washed-out by shortening the HRT to 4 h (improving the upflow velocity) in a UAF reactor, and achieved the highest MCCAs production rate (57.4 g/(L·d)) so far, at pH 6.5–7. However, this method might only be applicable in an upflow reactor. Controlling an appropriate weakly acidic pH could be a more general method for inhibiting methanogensis. The weakly

5.2.2. Avoiding excessive oxidation of ethanol to acetate In the ethanol-guided CE process, for every six molecules of ethanol, one molecule of ethanol needs to be anaerobically oxidized into acetate to harvest one ATP via substrate level phosphorylation, and this process is performed by CE functional bacteria (Roghair et al., 2018). However, increased oxidation of ethanol to acetate (higher than 1/6) could occur along with the CE process, which is called excessive ethanol oxidation and is mainly performed by ethanol-oxidizing microorganisms rather than by the CE bacteria (Roghair et al., 2018). The ethanol-oxidizing microorganisms could come from an open culture environment, and the exact species responsible have not been identified yet (Roghair et al., 2018). The direct result of excessive ethanol oxidation is the reduction of essential intermediates of acetyl-CoA, and consequent decrease in the MCCAs production. Considering the possible positive effects of ethanol-oxidizing microorganisms, the most economical and environmentally friendly method of control is to elevate H2 partial pressure to make the excessive ethanol oxidation process thermodynamically infeasible. Moreover, since CE bacteria can oxidize ethanol at a higher H2 partial pressure than other ethanol-oxidizing microorganisms can (Li et al., 2008), a proper H2 partial pressure range might exist that would guarantee a normal CE process. As reported by Grootscholten et al. (2014), H2 partial pressure above 0.03 atm can avoid excessive ethanol oxidation, and meanwhile, the H2 partial pressure must be lower than 0.1 atm to ensure ethanol oxidation into acetyl-CoA and acetate. The H2 partial pressure in the CE process can be adjusted artificially by increasing or decreasing H2 flow. It is worth noting that, CO2 content would also affect H2 partial pressure by reacting with hydrogenotrophic methanogen and thus affect excessive ethanol oxidation (Roghair et al., 2018). When the CO2 content is low enough, hydrogenotrophic methanogenesis is limited (Ge et al., 2015), and as a result, the high H2 partial pressure might limit the excessive ethanol oxidation. 609

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Conversely, the high CO2 content could lead to low H2 partial pressure and thus facilitate excessive ethanol oxidation. Therefore, H2 and CO2 need to be adjusted simultaneously to avoid excessive ethanol oxidation.

accumulation was observed in their batch CE process with lactate-enriched wastewater as feedstock and pit-mud as microbiome. However, their process was operated as a batch reactor, and therefore not an open-culture. Then, if the pit-mud inoculum did not include a microbe with the acrylate pathway due to an lengthy selection process in pit mud systems, diversion of carbon into this pathway would not occur. In a scaled-up version with a real biomass waste, such microbes with the acrylate pathway would surely be constantly introduced. In the following study, Zhu et al. (2017) utilized their newly isolated CE strain of Ruminococcaceae bacterium CPB6 to produce caproate from lactate. High caproate production of 16.6 g/L, with a maximum production rate of 5.29 g/(L·d), was obtained from 45.1 g/L lactate, but still no propionate generation was observed. This may be because the genes about acrylate pathway are not present in this bacterium. This indicated that in addition to being related to the residual lactate concentration, the acrylate pathway might also be related to the microorganisms present (Cavalcante et al., 2017).

5.2.3. Preventing carboxylic acids oxidation In the anaerobic environment, both MCCAs and SCCAs might be oxidized to form corresponding shorter‑carbon-chain carboxylic acids along with CO2 and H2O generation. To make the carboxylic acids oxidation thermodynamically feasible, the H2 partial pressure should be lower than 10−4 atm (Stams, 1994). Under the premise of limiting hydrogenotrophic methanogenesis, the H2 partial pressure in the CE reactor is unlikely to be below 10−4 atm since H2 would be released continuously in the reverse β oxidation process (Seedorf et al., 2008). Besides, as mentioned above, to avoid excessive ethanol oxidation, H2 partial pressure should be at least above 0.03 atm (Grootscholten et al., 2014). This means that carboxylic acids oxidation could not occur as long as excessive ethanol oxidation does not occur in the CE process, or else the H2 partial pressure needs to be elevated properly.

5.3. Adjusting the gas composition Appropriate gas composition in the reactor is essential for efficient MCCAs production since the CE process requires the participation of certain gases and the excess or shortage of some gases may make the process infeasible. Moreover, the requirement for gas composition is not fixed but varies with substrate components, which makes it hard to control. H2 and CO2 are the major and functional gas components. Just as mentioned above, oxidizing ED to acetyl-CoA, avoiding excessive ethanol oxidation, and preventing carboxylic acids oxidation requires specific H2 partial pressure. Moreover, H2 could also be used as a raw material to synthesize MCCAs. Steinbusch et al. (2011) reported both caproate and caprylate production were improved when 200 mL/h H2 was continuously flushed into the medium (containing ethanol and acetate), and consequently they considered H2 to be an available ED for CE. However, a thermodynamic and kinetic analysis conducted by González-Cabaleiro et al. (2013) verified that elongating acetate directly with H2 into butyrate or caproate is biochemically and thermodynamically infeasible. The generation of butyrate and caproate from acetate and H2 involves two successive steps (i.e., acetate reduction to ethanol and acetate elongation with ethanol as ED) (González-Cabaleiro et al., 2013). In addition, excess H2 could react with CO2 to generate acetate and ethanol by homoacetogenesis and subsequent acetate reduction to ethanol, respectively. Then, the in situ generated acetate and ethanol could be further transformed to MCCAs through the reverse β oxidation pathway (Angenent et al., 2016; Grootscholten et al., 2013c). However, it is noteworthy that carboxylate reduction into its alcohols may never become a great biotechnological production platform. The reduction ratio of carboxylate into corresponding alcohols is very low (about 55% for acetate, 50% for propionate, and 46% for butyrate based on the electron balance) due to the very low reduction rates (Steinbusch et al., 2008). This is the reason why Zhang et al. (2013) obtained a low production rate of caproate (31.4 mM C/(L·d)) and caprylate (19.1 mM C/(L·d)) when they only pumped H2 and CO2 as substrates into a mixed culture. CO2 is an essential substance for ethanol-guided CE because the corresponding CE functional bacteria (such as C. kluyveri) require CO2 as carbon source to synthesize protein (Seedorf et al., 2008). CO2 provides about 30% of the carbon for cellular synthesis, and the remaining 70% comes from organic carbon (Jungermann et al., 2010). Previously, many researchers supplied exogenetic CO2 to enhance MCCAs production in the ethanol-guided CE process, and obtained predictable effects (Grootscholten et al., 2013c; Grootscholten et al., 2014). CO2 supplementation was also needed to stimulate caproate formation from other EDs such as methanol (Chen et al., 2016; Tarasov et al., 2011). However, when real waste biomass is used as substrate for MCCAs production, exogenous CO2 might not be needed because it can be

5.2.4. Suppressing the acrylate pathway In the lactate-guided CE process, in addition to the CE pathway, there could also be the competing acrylate pathway in which lactate is transformed into propionate with lactyl-CoA, acrylyl-CoA, and propionyl-CoA as intermediates (Hetzel et al., 2003; Kucek et al., 2016a; Prabhu et al., 2012; Whanger and Matrone, 1967). This pathway is mediated by propionyl-CoA transferase, lactyl-CoA dehydratase, and acrylyl-CoA reductase (Brockman and Wood, 1975; Hetzel et al., 2003; Schulman and Valentino, 1976). The propionate formation from the acrylate pathway branches the lactate‑carbon flow and thereby lowers MCCAs selectivity (Kucek et al., 2016a). Moreover, the acrylate pathway is irreversible, that is, once the lactate enters the acrylate pathway, it cannot return to the reverse β oxidation pathway (Kucek et al., 2016a). Therefore, the acrylate pathway should be inhibited to concentrate lactate‑carbon flow into MCCAs production. It was reported that the acrylate pathway generally occurs in cases where lactate concentration is high, which induces the formation of the key substance of lactyl-CoA that further directs lactate‑carbon flux toward propionate (Prabhu et al., 2012; Strauber et al., 2016). However, under lactatelimited conditions, propionate could not be formed due to the absence of lactate to induce the formation of lactyl-CoA with propionyl-CoA transferase (Prabhu et al., 2012). Kucek et al. (2016a) found that, once the residual lactate concentration increased, the propionate productivity increased and caproate productivity declined. In view of this, Kucek et al. (2016a) proposed that propionate formation from lactateguided CE could be restrained by maintaining a residual lactate concentration near zero. In addition, their studies suggested that an operation pH near 5.0 and in-line product extraction would also contribute to suppression of propionate formation. However, it should be noted that the low pH of 5 might be not suitable for reactors without product extraction units since acidic condition at pH 5.0 makes some of the MCCAs exist in the undissociated states, which does not support CE bacterial metabolism (Agler et al., 2012a). Therefore, other strategies to avoid the acrylate pathway for the reactors without MCCAs extraction need to be proposed in the future. In addition, when the acrylate pathway cannot be effectively suppressed, converting the product (propionate) of the acrylate pathway into MCCAs might be another way to reutilize the dispersive lactate‑carbon flow and reduce the loss of lactate‑carbon. Recently, Wu et al. (2018) reported that utilizing lactate and ethanol as the co-EDs of the CE process could facilitate reutilization of propionate to produce heptylate and thus enhance total MCCAs yield. The specific transformation pathways remain to be revealed. In addition, it is also noteworthy that the acrylate pathway might not necessarily exist in the lactate-guided CE process. It was reported by Zhu et al. (2015) that a high caproate concentration of 23.4 g/L and production rate of 1.08 g/(L·d) were obtained but no propionate 610

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produced in situ from the hydrolysis and acidification of complex organics. Moreover, when a lactate-rich substance is used as feedstock, in addition to the CO2 produced from hydrolysis and acidification, an equimolar CO2 would be released in the lactate-guided CE process when a three‑carbon pyruvate is oxidized to a two‑carbon acetyl-CoA by pyruvate decarboxylase (Wu et al., 2018). Thereby, H2 might be appropriately supplied in the lactate-guided CE process to fix the excess CO2 into the CE substrates (acetate and ethanol) to further form MCCAs. In summary, appropriately adjusting CO2 and H2 partial pressures are significant for MCCAs production, and the adjusting strategies vary with different feedstock. For example, CO2 and H2 need to be supplemented into the ethanol-guided and lactate-guided CE processes, respectively, and the optimal CO2 and H2 contents for the specific feedstock remain to be determined.

utilizing ethanol and propionate as substrates, and the heptylate concentration and production rate reached 3.2 g/L and 4.5 g/(L·d), respectively, which showed the potential to replace the traditional heptylate production methods. However, the low heptylate selectivity of 23% still remains to be improved to meet the requirements of industrialized production (Grootscholten et al., 2013b). The low heptylate selectivity is mainly ascribed to the formation of by-products, including propanol, valerate, and caproate. The main synthetic pathways for these by-products are summarized in Table 3. Propanol is likely formed from the reactions of propionate and ethanol (Pathway #1) (Smith and Mccarty, 1989) and propionate reduction (Pathway #2) (Wu and Hickey, 1996). Valerate, as the intermediate of heptylate production, is mainly generated from propionate elongation (Pathway #3) (Coma et al., 2016). Caproate can be derived from the associative reactions of ethanol oxidation into acetate and subsequent acetate elongation with ethanol (Pathway #5) (Grootscholten et al., 2013b). Moreover, since propanol is also an available ED for CE (Coma et al., 2016; Weimer and Stevenson, 2012), valerate and caproate might also be formed by elongating acetate (Pathway #4) (Waselefsky and K., 1985) and propionate (Pathway #6), respectively, with propanol as ED, (Kenealy and Waselefsky, 1985). In order to avoid the needless formation of the by-products mentioned above, appropriate H2 partial pressure in the reactor should be applied. This is because excessively low H2 partial pressure would induce ethanol oxidation into acetate that further leads to even‑carbon carboxylate formation; while excessively high H2 partial pressure makes propionate reduce to propanol, not only resulting in decrease of available propionate, but also promotion of valerate and caproate generation. Therefore, high heptylate selectivity requires a specific H2 partial pressure that remains to be investigated in detail. In addition, the incomplete elongation of intermediate valerate also occupies plenty of carbon-flow, resulting in low heptylate selectivity. It was reported that the valerate selectivity reached 28% in the propionate elongation process (Grootscholten et al., 2013b), in contrast, the butyrate selectivity was only 9% in the acetate elongation process (Grootscholten et al., 2013a). The lower efficiency for further elongation of valerate than of butyrate could be caused by the higher toxicity of heptylate than caproate to microorganisms. Therefore, the longer the carbon chain, the more necessary is timely product extraction.

6. Production of longer-chain MCCAs The product toxicity, energy harvest, intermediate concentration, and microbiological properties limit the further elongation of carboxylate (González-Cabaleiro et al., 2013), as a result, the MCCAs attainable from the open culture CE process are mainly caproate (C6), heptylate (C7), and caprylate (C8). Among these, caproate and caprylate could be produced at high productivities and specificities (Agler et al., 2012a; Ge et al., 2015; Grootscholten et al., 2013a; Kucek et al., 2016b), while heptyrate is generally produced with low selectivity (Grootscholten et al., 2013b). Regardless of the currently low production efficiency, heptylate is still of great research value because of its strong hydrophobicity and high energy density (Van Eerten-Jansen et al., 2013). In view of that the caproate production has been specifically introduced in a previous review (Cavalcante et al., 2017), this section mainly focuses on the research status and production characteristics of longer-chain hyptylate and caprylate. 6.1. Heptylate production Heptylate can be generated using the CE technology with odd‑carbon propionate or valerate as EA. As early as 1948, Bornstein and Barker (1948) detected heptylate (0.3 g/L, 2 mM) generation from the substrates of ethanol and propionate with a CE type strain of C. kluyveri. Afterwards, in an open culture fermentation reactor fed with propionate and ethanol, Smith and Mccarty (1989) observed an improved heptylate concentration (~1.5 g/L, 12 mM) once ethanol and propionate were increasedly supplemented. The heptylate observed in the two early studies were attributed to the elongation of propionate. In recent years, Grootscholten et al. (2013d) produced heptylate from actual waste biomass of OFMSW, and a fourfold higher heptylate production (1.6 g/L) was obtained when ethanol was supplemented after the OFMSW acidification (compared with a control test without ethanol addition). However, caproate (2.7 g/L) was still the main product since even‑carbon SCCAs (acetate and butyrate) were the major products of OFMSW acidification. To further improve the heptylate production, Grootscholten et al. (2013b) performed an open culture CE process

6.2. Caprylate production Caprylate is the direct product of further caproate elongation, which is generally produced with a much lower yield than caproate when even‑carbon acetate/butyrate is used as EA. However, in contrast with caproate, caprylate almost has a double value and is also a preferred precursor for some manufactures like feed industry (Kucek et al., 2016a). In addition, the low solubility of caprylate in water (0.68 g/L, 4.72 mM) can be easily reached through CE technology, and thus the generated caprylate is more easily separated than caproate (the solubility is 10.82 g/L, 93.27 mM) as oily state by phase separation. Steinbusch et al. (2011) first observed negligible caprylate

Table 3 Generation pathways of main by-products in the heptylate production process. By-products

Pathway

Equation

∆Go (kJ/mol)

References

Propanol

#1 #2 #3 #4

C3H5O2− + C2H6O → C3H8O + C2H3O2− C3H5O2− + H+ + 2H2 → C3H8O + H2O C3H5O2− + C2H6O → C5H9O2− + H2O C2H3O2− + C3H8O → C5H9O2− + H2O

−2.3 −51.8 −30.2 −36.3

C2H6O + H2O → C2H3O2− + H+ + 2H2 2C2H3O2− + H+ + C2H6O + 2H2 → C6H11O2− + 3H2 C3H5O2− + C3H8O → C6H11O2− + H2O

49.5 −126.7 −36.3

Smith and Mccarty (1989b) Wu and Hickey (1996) Coma et al. (2016) Kenealy and Waselefsky (1985) Grootscholten et al. (2013b)

Valerate Caproate

From ethanol and propionate Propionate reduction Elongating propionate with ethanol Elongating acetate with propanol

#5 Ethanol oxidation into acetate; elongating acetate with ethanol #6 Propionate elongation with propanol

611

Kenealy and Waselefsky (1985)

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production (0.3 g/L) from a batch open culture CE reactor with ethanol, acetate, and H2 as substrates. Afterwards, using an UAF reactor, Grootscholten et al. (2013a) improved caprylate production to 0.6 g/L (4 mM, near its solubility in water) with a production rate of 0.9 g/(L·d) and a selectivity of 6%, while caproate was still the major product, with much higher concentration of 11.1 g/L and selectivity of 85%. Furthermore, by shortening HRT to prevent methanation, Grootscholten et al. (2013c) improved the caprylate concentration by 50%, and the caprylate concentration (0.9 g/L) was higher than its solubility in water for the first time. For the purpose of resource reutilization, OFMSW was used as the substrate in one-stage and two-stage CE systems, and similar caprylate concentrations of 0.6 g/L (4 mM) and 0.4 g/L (3 mM), respectively, were obtained (Grootscholten et al., 2013d; Grootscholten et al., 2014). Recently, from actual waste biomass of wine lees, which inherently contain ethanol (ethanol ratio on a COD basis of 40%, ethanol concentration of 180.5 g COD/L), the relatively high caprylate specificity of 36% and caprylate/caproate ratio of 1 (total MCCAs production rate of 7 g COD/(L·d)) were obtained (Kucek et al., 2016c). The better caprylate production performance from Kucek et al. (2016c) compared with the above mentioned studies was mainly attributed to superior substrates with larger amounts of available ethanol, and the well-working in-line product extraction equipment. So far, the highest caprylate production rate of 19.4 g COD/(L·d), caprylate specificity of 96%, and caprylate/caproate ratio of 11 were obtained from the synthetic syngas-fermentation effluent (Kucek et al., 2016b). They attributed such a high caprylate selectivity and production rate to a high ethanol/acetate of 11 in the feedstock and to the in-line product extraction. It was verified that a high ethanol/acetate ratio benefits caprylate production; conversely, a high acetate/ethanol ratio results in butyrate production (Angenent et al., 2016; Kucek et al., 2016b; Spirito et al., 2018). High proportions of ethanol contribute to more available acetylCoA, which is able to guide continuous CE cycles until the generation of caprylate (C8) and even caprate (C10). Therefore, organics with a high ethanol/acetate ratio could be the optimal substrates, and two different organics that inherently contain ethanol and acetate, respectively, could also be combined in proper proportion to produce caprylate. In addition, timely product extraction is also vital for enhancing caprylate production due to the high toxicity of undissociated caprylic acid seriously damages microbial cells. Moreover, the toxicity increases with the chain length of MCCAs, which might be one of the reasons that nonanoate (C9) and caprate (C10) were hardly observed experimentally.

After each CE cycle with two carbon atoms coupling to carboxylic acids, the price of the product increases considerably due to its solubility in water decreasing by > 10-fold along with greatly increased energy density (Xu et al., 2018). Therefore, in addition to caproate, future CE studies should be more focused on the production of longerchain MCCAs. 7. Conclusions and future perspectives MCCAs production from waste biomass, and based on the emerging CE technology, exhibits numerous advantages related to energy, economy, and environment, and accords with the current guidelines toward resource recovery and environmental protection (waste treatment and greenhouse gas emission reduction). At the same time, many existing challenges remain to be overcome to achieve these advantages. Table 4 highlights the main advantages and challenges for MCCAs production and utilization. The key advantages are as follows: (1) the production of MCCAs provides a superior alternative bioenergy source with high energy density, high security, and versatility, and thus reduces dependence on fossil energy (coal, crude, crude gas, etc.); (2) the low cost for MCCAs production, separation, and extraction facilitates the large-scale utilization of MCCAs-based products; and (3) the reutilization of widespread waste biomass (including agricultural residuals) as feedstock avoids the long period of conflicts between food and biofuel, and eases the environmental burden. To achieve these potential advantages, scaling up MCCAs production from current lab scale to industrial level while maintaining superior MCCAs production performance, system stability, and economic benefit, is necessary. The major challenges that need to be overcome through future studies and corresponding recommendations are as follows: (1) There is a need to explore a large number of available and affordable waste biomass sources as feedstock, and to establish a complete collection and storage network to satisfy the high demand for raw materials. ➢ Recommendation: Except for the existing reports, the waste biomass used for other anaerobic technologies, such as diversified agricultural by-products (corn fiber, wheat straw, manure, corn stover, etc.), protein-rich sewage sludge, and carbohydrate-rich microalgae, could also be suitable candidates for MCCAs production. Moreover, the use of multiple substances with complementary properties (for instance, two feedstocks, one containing ED and on with EA) are also suggested for better MCCAs production. To determine suitable feedstocks for large-scale MCCAs production, comprehensive evaluations covering MCCAs production performance and total utilization cost of each potential feedstock are needed. Equally important, a strong policy enforced by governments is required to sort, collect, store, and transport the available waste biomass.

Table 4 Potential advantages and challenges for MCCAs production and utilization. Advantages ➢ Energy benefit High energy density High security Versatility Wide distribution for production and supply ➢ Economy efficiency Low production/separation/extraction costs Enlarge the utilization of MCCAs-based products Stabilize the prices of fossil fuel Sustainable development of chemical industry ➢ Environmental friendliness Waste biomass treatment and reutilization Alleviate environmental pollution Reduce greenhouse gas emission Slow the exploitation of fossil energy

• • • • • • • • • • • •

Challenges ➢ Available waste biomass Appropriate feedstock sources Waste biomass collection Storage method and facility ➢ Technological improvement

• • •

(2) There is a need for technology improvements that focus on substrate pretreatment, shaping of high-efficiency microbiomes, and keeping the microbiomes resilient in functionality. These, along with the use of strategies to enhance MCCA yield and purity, are vital for achieving economical and efficient MCCAs production.

pretreatment • Proper • Microbe source and shaping • Keep microbiome resistant CE efficiency • Enhance • Timely product extraction the product purity • Improve • Produce long-chain MCCAs

➢ Recommendation: (I) Specific pretreatment methods (such as physical: pyrolysis, ultrasound, mechanical disintegration; chemical: acid, alkaline, ozone; biological: enzymatic hydrolysis, saccharification; or combinations of them) and corresponding process parameters for each feedstock (especially the cellulose and hemicellulose-rich substances) need to be tailored based on their characteristics, to overcome the rate612

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(II)

(III)

(IV)

(V)

limiting step of slow or low hydrolysis. The selected pretreatment strategy should be easy to implement, cost-effective, and without undesirable hazardous by-product formation. To build a high-efficiency CE reactor microbiome, the key issue is to understand deeply the relationships between the microbial community structure and CE performance, and how the external/ internal factors (operating parameters, perturbations, new microbes inflow, products toxicity, etc.) influence microbiome structure and bioreactor performance. This can be achieved by using omics approaches including metagenomics, metatranscriptomics, metaproteomics, and metabolomics. In addition, a training CE reactor microbiome with enough resilience to outcompete new incoming microbes is also significant for keeping MCCAs production stable. Thus, the relationships between CE bacteria and other microorganisms need to be evaluated by characterizing the phylogeny and population dynamics under changed operating conditions. In addition, diversified microbes potentially carrying out the CE pathway, especially the strains with high tolerance to toxicity and inhibitors, should be exploited. Their corresponding habitats and CE behaviors also need to be investigated. The advanced genetic modification techniques and metabolic engineering strategies by which strains can be equipped with industrially relevant levels can also be applied to obtain higher MCCAs production performance. Avoiding competitive reactions that disperse carbon flow is the prerequisite to ensuring a high MCCAs yield while the available solutions are still immature or scarce, especially for the suppression of the acrylate pathway and methanation process. More and better-targeted methods with the characteristics of efficiency, affordability, ease of implementation, and compatibility with the CE process should be developed in future studies. Systems for timely product extraction are crucial for large-scale MCCA production. Combined MCCAs production and product extraction systems require reasonable coordination of design and operation conditions in both sections to simplify production step and reduce cost. For this, mathematical modeling and precise automatic control equipment could be applied. Furthermore, more researchers should focus on developing integrated systems to accomplish simultaneous MCCAs production, extraction, and separation. To maximize MCCAs production profits, the corresponding construction and maintenance costs for integrated production systems need to be optimized. Also recommended is more research on producing high-purified longer chain MCCAs like nonanoate, caprate, etc. Shaping the reactor microbiome to primarily produce these types of MCCAs by combining it with specific substrate composition (possibly with a high ED/EA ratio), operating conditions, and product extraction unit might be feasible. Isolating and exploiting the pure strains that are good at producing longer-chain MCCAs is also significant. In addition, the types of MCCAs-based chemicals should also be broadened and popularized under either prompting or compulsion by governments to promote high profits and sustainable development of the chemical industry.

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