Engineering clostridia for butanol production from biorenewable resources: from cells to process integration

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ScienceDirect Engineering clostridia for butanol production from biorenewable resources: from cells to process integration Jufang Wang1,2, Xiaorui Yang2,3, Chih-Chin Chen Shang-Tian Yang2,3 Renewable feedstocks such as lignocellulosic biomass, CO2 and syngas are readily available and low cost but difficult to ferment. There are microbes that can utilize cellulose or gases to produce ethanol and organic acids as major products, but few can produce n-butanol in a significant amount. Metabolic engineering can be applied to both cellulolytic and acetogenic clostridia to produce n-butanol directly from cellulose and CO2. In addition, co-culturing these engineered microbes can utilize cellulose directly and CO2, produced via the fermentation, in an integrated process for butanol fermentation and recovery. This review focuses on the development of consolidated bioprocessing (CBP) using engineered clostridia for n-butanol production from biorenewable resources such as lignocellulosic biomass and CO2. Addresses 1 School of Bioscience & Bioengineering, South China University of Technology, Guangzhou 510006, PR China 2 William G. Lowrie Department of Chemical and Biomolecular Engineering, The Ohio State University, Columbus, OH 43210, USA 3 Department of Molecular Genetics, The Ohio State University, Columbus, OH 43210, USA Corresponding author: Yang, Shang-Tian ([email protected])

2

and

to butene and catalyzed into longer chain oligomers for aviation fuel applications. Clostridia are a versatile class of Gram-positive bacteria that can convert a wide range of substrates to organic acids (acetic, butyric, lactic, etc.) and solvents (ethanol, isopropanol, acetone, butanol, etc.) [3]. ABE production from corn (starch) and cane molasses (glucose and sucrose) with solventogenic Clostridium spp. was once the second largest fermentation process in the world [4]. However, in the conventional ABE fermentation process as practiced today in China, starch feedstocks contribute to about 70% of the overall production cost [5]. Also, approximately 34% of the carbon from the feedstocks is converted to CO2, resulting in a relatively low product yield. The high substrate cost and low product yield make biobutanol process uneconomical, and almost all biobased butanol plants had been shut down by the late 1980s. However, with the diminishing supply and rising costs of crude oils, recently there have been increasing interests in producing butanol from low-cost renewable feedstocks [6–8]. This review focuses on recent advances in metabolic engineering and process engineering for nbutanol production from biorenewable resources by anaerobic fermentation with clostridia.

Current Opinion in Chemical Engineering 2014, 6:43–54 This review comes from a themed issue on Biotechnology and Bioprocess Engineering Edited by Eleftherios Terry Papoutsakis and Nigel J TitchenerHooker

http://dx.doi.org/10.1016/j.coche.2014.09.003 2211-3398/# 2014 Elsevier Ltd. All rights reserved.

Introduction Butanol is an important industrial solvent currently produced mainly via petroleum-based processes. Biobutanol, which can be produced from renewable biomass via acetone–butanol–ethanol (ABE) fermentation, is an attractive biofuel with superior properties (higher energy density, lower volatility, water miscibility, flammability, and corrosiveness than ethanol) and excellent compatibility with existing fuel infrastructures and internal combustion engines [1,2]. Butanol can also be dehydrated www.sciencedirect.com

Metabolic engineering of solventogenic Clostridium Metabolic engineering has been widely applied for strain improvement [9,10]. Genetic tools have been developed that allow genes to be manipulated in solvent-producing clostridia, mainly the type strain Clostridium acetobutylicum ATCC 824 [11,12]. Recently, the clostridia’s n-butanol biosynthesis pathway has also been expressed for n-butanol production in various industrial microbes [13,14], including Escherichia coli [15], Saccharomyces cerevisiae [16], Pseudomonas putida [17], Bacillus subtilis [17], and Lactobacillus brevis [18]. Although these microbes possess several desirable attributes, including fast cell growth, well studied genetics, and moderate to high butanol tolerance, they cannot use cellulose directly and are often difficult to use lignocellulosic hydrolysates. Extensive research has been done on metabolic engineering of solventogenic clostridia, aiming at improving butanol production titer and yield. In general, metabolic engineering is carried out in several different aspects (see Table 1) with most work on the type strain C. acetobutylicum ATCC 824 [11,12]. Genes in acid-producing Current Opinion in Chemical Engineering 2014, 6:43–54

44 Biotechnology and Bioprocess Engineering

Table 1 Genes targeted in metabolic engineering of solventogenic clostridia and their effects on ABE fermentation Target genes

Effects Knockout Block or reduce butyrate formation Block or reduce acetate formation Increase the reassimilation of acetate and butyrate; higher and more stable butanol production Decrease acetone production Increase butanol formation Increase intracellular concentrations of ATP and NADH Increase n-butanol tolerance Prevent ‘acid crash’ caused by the accumulation of intracellular formic acid Increase aerotolerance and butanol production Enhance xylose utilization

Overexpression

References

aad, ctfAB

[20,21] [21] [22]

aad, thl adhE1, pfkA, pykA, thl, ctfAB pfkA, pykA groESL, gshAB, dnaK, hsp18, hsp90 fdh

[23,24] [25,26] [28] [29,30] [31]

gshAB talA, tal, tkl, rpe, rpi

[32,33] [46,47]

buk, ptb pta, ack

adc, ctfAB solR, spo0A

ccpA

aad: aldehyde/alcohol dehydrogenase; ack: acetate kinase; adc: acetoacetate decarboxylase; adhE1: aldehyde/alcohol dehydrogenase; buk: butyrate kinase; ccpA: catabolite control protein A; ctfAB: CoA transferase; dnaK: heat shock protein 70; fdh: formate dehydrogenase; groESL: heat shock protein, known as hsp10/60; gshAB: g-glutamylcysteine synthetase A and B; hsp18: heat shock protein 18; hsp90: heat shock protein 90; pfkA: 6-phosphofuctokinase; pta: phosphotransacetylase; ptb: phosphotransbutylase; pykA: pyruvate kinase; rpe: ribulose-5-phosphate 3-epimerase; rpi: ribose-5-phosphate isomerase; solR: a repressor of sol locus genes; spo0A: transcription factor for sporulation; tal: transaldolase; talA: transaldolase from Escherichia coli; tkl: transketolase; thl: thiolase.

(such as pta, ack, ptb, buk) and solvent-producing (adc, adhE, ctfAB) pathways have been targeted in order to increase the metabolic flux toward butanol and eliminate or downregulate the flux toward other undesired byproducts including acetone and ethanol [19,20,21,22–26]. For example, a greatly increased n-butanol titer of 16.7 g/ L was obtained by knocking out the butyrate kinase gene (buk) [20]. Knocking out both pta and buk genes with simultaneous overexpression of adhE1, which reinforced the direct butanol-forming route in C. acetobutylicum, resulted in a 60% increase in the final butanol titer (18.9 g/L) and 154% increase in the butanol yield (0.29 g/g glucose) with little acetone production compared to the wild type [21]. Overexpressing aad and thl with simultaneously downregulating CoA transferase (ctfAB) with antisense RNA decreased acetone, acetate and butyrate production with significantly increased alcohols (mainly ethanol) production [23]. In addition, it was demonstrated that overexpressing 6-phosphofructokinase ( pfkA) and pyruvate kinase ( pykA) increased intracellular concentrations of ATP and NADH in C. acetobutylicum, resulting in a 29.4% increase in the final butanol titer [27]. Some functional transcription regulators, including solR and spo0A, and chaperones groESL, grpE and htpG have also been studied for enhancing butanol production [28,30]. For example, deregulated, prolonged, and increased n-butanol production of 17.8 g/L was obtained by inactivating a putative transcriptional repressor gene (solR) [28], and n-butanol tolerance and production increased to 17 g/L when a major heat-shock protein (GroESL) was overexpressed [29]. One common problem in industrial ABE fermentation is the ‘acid crash’ that occurred when cells failed to shift from acidogenesis to solventogenesis. Overexpressing Current Opinion in Chemical Engineering 2014, 6:43–54

CoA transferase (ctfAB) and aldehyde/alcohol dehydrogenase (aad) in C. beijerinckii increased the reassimilation of acetate and butyrate and resulted in higher and more stable butanol production [22]. One recent study showed that the acid crash was caused by the intracellular accumulation of formic acid (1 mM) and could be prevented by overexpressing the formate dehydrogenase ( fdh) [31]. In addition, overexpressing the E. coli gshAB genes, encoding the enzymes for the biosynthesis of glutathione (GSH) that might be involved in scavenging the reactive oxygen species in cells, increased the robustness and aerotolerance of C. acetobutylicum and its butanol production by 37% (14.8 g/L) [32]. Since cell growth and butanol production titer are limited by butanol toxicity [34,35], extensive studies have focused on transcriptional analysis to identify genes responsible for or contributing to higher butanol tolerance and production under butanol stress [36–40]. However, butanol production and tolerance involved numerous genes in complicated metabolic and regulatory pathways associated with acidogenesis, solventogenesis, sporulation life cycle, quorum sensing, and stress responses [34], which are still poorly understood. Recently, RNAseq analysis of tolerance and stress response have revealed the presence of small, non-coding RNAs (sRNA) that play a vital role in mediating stress response and tolerance mechanism in bacteria including C. acetobutylicum [39,41–43]. The sRNAs found to regulate transcriptional and translational processes underlying stress response are potential targets for metabolic engineering, although much more work to understand their functions at the genomic scale is needed. It should be noted that butanol tolerance varies with growth conditions [35] and increasing a strain’s butanol tolerance does not guarantee a www.sciencedirect.com

Engineering clostridia for butanol production Wang et al. 45

higher butanol production [30]. Although rational metabolic engineering has made good progress in generating strains with significantly improved butanol production, so far the highest butanol-producing strains (up to 21 g/L) have been generated by mutagenesis and adaptation under butanol stress [44]. Biofuels production from lignocellulosic biomass has emerged as an important research area as current sugar and starch based biorefineries are facing great challenges economically and ecologically. Clostridia are able to metabolize pentoses present in lignocellulosic biomass, which is important to the economics of biofuels and biobased chemicals [3,6,45]. However, xylose uptake and utilization in ABE fermentation is strongly inhibited or repressed in the presence of glucose due to catabolite repression [46]. To accelerate pentose uptake and improve pentose utilization, some genes involved in carbon metabolism, including ccpA and talA, and in the pentose phosphate pathway, such as tal, tkl, rpe and rpi, were overexpressed, and glcG for the D-glucose phosphoephosphotransferase system nolpyruvate-dependent (PTS) was disrupted in C. acetobutylicum [47].

Metabolic engineering of acidogenic Clostridium Several acidogenic Clostridium such as C. tyrobutyricum can produce large quantities of butyrate as the major fermentation product from a variety of substrates [48]. Compared to native solventogenic Clostridium, a much higher product (butyrate) titer of >50 g/L can be produced by C. tyrobutyricum mutant strains with ack [49], pta [50], or ptb [51] knockout, which also showed good butanol tolerance, with >80% and 60% relative growth rate at 1.0% and 1.5% (v/v) butanol, respectively [52]. Recently, C. tyrobutyricum was engineered to produce n-butanol as the main product by overexpressing an aldehyde/alcohol dehydrogenase (adhE2) [52]. The ack knock-out mutant produced the highest butanol (up to 10 g/L) with a high yield (0.26 0.33 g/g glucose consumed) and only small amounts of ethanol and acetic acid in fermentation. Furthermore, a high butanol titer of 20 g/L with a high yield of 0.35 g/g (87% theoretical yield) using mannitol as the substrate was obtained [53]. Since butanol is the main solvent product produced, its yield can be further increased by knocking out both ack and buk genes in the acid biosynthesis pathways, to approach the theoretical value of 0.42 g/g glucose. Conventional solventogenic Clostridium usually produced only 0.2–0.25 g butanol per g glucose or starch fermented. Compared to C. acetobutylicum, whose ABE fermentation is difficult to control because of the complicated metabolic and regulatory pathways involving acidogenesis, solventogenesis and spore-forming life cycle [54], the engineered C. tyrobutyricum with a simpler butanol biosynthesis pathway is also easier to manipulate, because butanol production is growth associated and neither sporulation nor autolysis www.sciencedirect.com

would be induced by solvent (butanol) production as commonly observed with solventogenic C. acetobutylicum during the ABE fermentation [55]. It is clear that butyrate-producing clostridia with high butyrate tolerance and yield can be engineered to produce high-titer butanol with a high yield. This represents a novel metabolic engineering strategy for biobutanol production, which can also be applied to butyrate-producing cellulolytic Clostridium for butanol production from cellulose, which will be discussed in the next section.

Biofuels production from lignocellulosic biomass Lignocellulosic biomass is well-suited feedstock for biofuels production because of its low cost, large-scale availability and environmentally benign production [56]. However, lignocellulosic biomass is difficult to use as substrate in fermentation and usually has to be pretreated and hydrolyzed to simple sugars in order to be converted to biofuels by microorganisms in a sequential or separate hydrolysis and fermentation (SHF) process. Current biorefinery generally involves three processes: production of cellulases, hydrolysis of cellulose and hemicellulose, and fermentation of hexose and pentose sugars. To date, pretreatments and enzymatic hydrolysis of lignocellulosic biomass are still major obstacles in lignocellulosic biorefinery [56]. A recent National Renewable Energy Laboratory (NREL) report showed that cellulosic ethanol was produced for a product value of $3.40/gal in a SHF process, and the enzyme cost was about $0.69/gal ethanol [57]. To reduce costs, the last two processes can be combined into simultaneous saccharification and fermentation (SSF) and simultaneous saccharification and cofermentation (SSCF). Nevertheless, extensive thermochemical pretreatments and the requirement of relatively expensive cellulases for the hydrolysis of cellulose to fermentable sugars impede the development of lignocellulosic ethanol and other biofuels. Consolidated bioprocessing (CBP), which combines cellulase production, cellulose hydrolysis and fermentation, has the greatest potential in reducing the overall production cost of lignocellulosic biofuels. It has also been estimated that CBP can reduce the production cost of cellulosic ethanol by up to 70% compared to the SSF process [58]. Similar cost benefits can be expected for biobutanol production from lignocellulosic feedstock via CBP. Some progresses have been achieved on biofuels production from cellulose via CBP [58]. However, to date, CBP is mainly applied to ethanol production from cellulose using cellulolytic bacteria, including C. cellulolyticum, C. thermocellum, Thermoanaerobacterium thermosaccarolyticum and T. saccharolyticum. Metabolic engineering has been applied to enhance ethanol production and eliminate byproduct formation in cellulolytic clostridia, resulting in higher ethanol yields and productivities [59]. However, no microbe can produce butanol directly from Current Opinion in Chemical Engineering 2014, 6:43–54

46 Biotechnology and Bioprocess Engineering

cellulose to reach an economical level for industrial application. Recently, four microbes capable of producing butanol from cellulose were isolated; however, the final butanol titers were less than 0.04 g/L after 96 h fermentation [60]. Interestingly, a strain of C. acetobutylicum was capable of producing 0.6 g/L butanol on 3% (w/v) grass [61], which is still far too low to be economically feasible for industrial application. Although some strains related to C. saccharobutylicum were isolated with hemicellulosic activity and are capable of producing butanol, none of them have the true cellulase activity to utilize crystalline cellulose [62]. Thus, microbes with both fast growth on cellulosic substrates and substantial production of butanol are not available yet for CBP. As an alternative, butanol can be produced from cellulose by co-culturing cellulolytic C. thermocellum with solventogenic C. acetobutylicum or C. beijerinkii [63,64]. However, butanol production was limited to less than 2.5 g/L, probably by the low reducing sugar present in the fermentation broth produced from cellulose by C. thermocellum, resulting in low production of butyric acid in the acidogenic stage, which is required to initiate solventogenesis. Interestingly, the co-culture of C. thermocellum with C. saccharoperbutylacetonicum N1–4, which is a butanol-producing strain with a different solventogenic-initiation mechanism, produced 7.9 g/L butanol in 9 days [64]. Recently, a synergistic fungalbacterial consortium was developed for direct isobutanol production from cellulosic biomass by co-culturing cellulolytic Trichoderma reesei with engineered E. coli, achieving a titer of 1.88 g/L in 300 h [65]. However, these cocultures suffered from low productivity and required complicated operation because of different optimal growth conditions for the two very different cultures used in these processes. In order to develop efficient CBP for biobutanol production, either cellulase production must be introduced into a butanol-producing strain, or the pathway for butanol biosynthesis must be introduced into a cellulase producing strain. To date, the heterologous production of cellulases has been pursued primarily with bacterial hosts producing ethanol at high yield (engineered strains of E. coli, Klebsiella oxytoca and Zymomonas mobilis) and the yeast S. cerevisiae [56,66,67]. Cellulase expression in strains of K. oxytoca resulted in increased hydrolysis yields (but no growth without added cellulase) for microcrystalline cellulose (Avicel) and anaerobic growth on amorphous cellulose [68]. More recently, dozens of saccharolytic enzymes have been functionally expressed in S. cerevisiae [69,70]. However, to date no work on the cloning of cellulase or hemicellulase in butanol-producing C. acetobutylicum was successful to generate mutants capable of producing butanol from cellulose directly [71]. In many cases, the overexpressed cellulases appeared to be not stable or had inhibitory effect on cell growth [72]. Recently, functional mini-cellulosomes were successfully cloned, expressed, and assembled on the cell Current Opinion in Chemical Engineering 2014, 6:43–54

surface of C. acetobutylicum [73]. Although the expressed cellulases were functional, their activities were very low and none of the recombinant strains could ferment cellulose to produce ABE, suggesting the difficulty of cellulase engineering in solventogenic clostridia [74]. Engineering cellulolytic clostridia for butanol production from cellulose

An alternative to cellulase engineering is to introduce butanol synthesis pathways into cellulolytic clostridia for cellulosic butanol production. Recently, it was suggested that Thermoanaerobacterium saccharolyticum would be an idea heterologous host for butanol production because of its ability to use a variety of sugars and polysaccharides including xylane, mannan, starch and pectin [75]. A lactate deficient strain expressing the clostridia n-butanol pathway produced 1.05 g/L of n-butanol from 10 g/L xylose. However, the idea of using engineered cellulolytic Clostridium to produce butanol directly from cellulose has been hindered by the lack of genetic tools in the past [76]. With the development of new genetic tools for engineering of clostridia [77,78], it is now possible to realize the direct bioconversion from cellulose to butanol by engineering cellulolytic clostridia such as C. cellulovorans, C. cellulolyticum, and C. thermocellum, which can use cellulose and hemicellulose as carbon source directly [3]. These cellulolytic clostridia contain cellulosomes that are highly efficient in degrading and hydrolyzing crystalline cellulose and hemicellulose, and thus offer exceptional bioprocessing applications [79]. Recently, a heterologous isobutanol-producing pathway was cloned into C. cellulolyticum, which produced 0.66 g/L isobutanol from cellulose in 7–9 days [80]. Similarly, introducing and overexpressing genes in the CoA-dependent n-butanol biosynthesis pathway into cellulolytic clostridia should enable them to produce n-butanol directly from cellulose. In particular, C. cellulovorans, which can produce butyric acid as the main product from various carbon sources, including glucose, cellobiose, xylose, xylan, cellulose and hemicellulose [81], with a small amount of acetic acid and negligible amounts of lactic acid and formic acid as byproducts, is a promising host for n-butanol production from cellulosic biomass. The high butyrate/acetate ratio of 5:1–6:1 (g/g), comparable to that of C. tyrobutyricum, suggests the existence of a favorable metabolic pathway from cellulose to butyryl-CoA. Therefore, only one heterologous gene, aad or adhE2, is required to make butanol from cellulose, because a native metabolic pathway from cellulose to butyryl-CoA already exists in C. cellulovorans (see Figure 1). This not only will simplify the cloning process, but also can give higher butanol productivity. Recently, the adhE2 gene was successfully introduced into C. cellulovorans and the recombinant strain was able to produce 2 g/L n-butanol from glucose and cellobiose and 1.6 g/L from cellulose [82], which are much higher than the reported 0.4 g/L of isobutanol from cellobiose and 0.66 g/L from cellulose by engineered C. cellulolyticum www.sciencedirect.com

Engineering clostridia for butanol production Wang et al. 47

Figure 1

Figure 2

Cellulose CO2

Cellulosome

fd

Cellobiose

Formate ATP fts

2NADH + 2ATP ldh

Lactate

Acetate

Pyruvate CoA Fd

X

ack X

CO2

pta

ATP

Formyl-THF

NADH

ftc

H2

FdH2

Acetyl-CoA

2 [H]

adhE

NADH

Acetaldehyde

adhE

Ethanol

H2O

Methenyl-THF 2 [H] mtd Methylene-THF 2 [H] mtr Methyl-THF

3-Hydroxybutyryl-CoA

mtf H2O

Methyl-Co/Fe-S-Protein

Crotonyl-CoA NADH buk X

ptb

Butyryl-CoA

ATP

adhE2

NADH

CO + H2O

NADH

Acetoacetyl-CoA NADH

Butyrate

Methyl branch

Butylaldehyde

adhE2

Butanol

Butyric acid

H2 CO2

acs

cod hyda

2 [H]

CO2 + H2 2[H] Carbonyl branch

[CO]

Acetyl-CoA

Ethanol

NADH

Current Opinion in Chemical Engineering

A novel butanol fermentation pathway in cellulolytic Clostridium expressing heterologous adhE2 or aad from C. acetobutylicum with acetate, lactate and butyrate pathways knocked out to produce butanol and ethanol from cellulose. Native cellulolytic Clostridium can produce acids and ethanol, but not butanol (ack: acetate kinase; adhE2: aldehyde-alcohol dehydrogenase; buk: butyrate kinase; ldh: lactate dehydrogenase; pta: phosphotransacetylase; ptb: phosphotransbutyrylase).

using the 2-keto amino acid pathway [80]. Similar to the engineered C. tyrobutyricum, the acidogenic pathways in C. cellulovorans can be knocked down to further increase butanol yield from cellulose. With a simple butanol biosynthesis pathway, the engineered C. cellulovorans should be able to produce more butanol as the sole or main fermentation product, with a theoretical butanol yield of 0.46 g/g cellulose. Butanol as the only or main fermentation product will also simplify the downstream purification process and lower the butanol production cost significantly, compared to conventional ABE fermentation.

Biofuels production from syngas and waste gases containing CO2 Some acetogens can convert CO, CO2 and H2 into acids (mainly acetate and butyrate) and alcohols (ethanol and nbutanol) by the Wood–Ljungdahl (WL) pathway (see Figure 2), which is widely distributed among more than 100 acetogens and plays an important role in fixing or recycling CO2 in various environments on earth [83,84,85]. In this pathway, CO2 provides carbon source for methyl and carbonyl group and H2 provides www.sciencedirect.com

Acetic acid Current Opinion in Chemical Engineering

The Wood–Ljungdahl pathway in some acetogens for converting CO, CO2 and H2 to acetyl-CoA, leading to acetic acid and ethanol production. Ethanol can also be produced directly from the reduction of acetic acid by AOR. Butyric acid and butanol can also be produced from acetyl-CoA in some acetogens (acs: acetyl-CoA synthase; cod: CO dehydrogenase; fd: formate dehydrogenase; ftc: formyl-THF cyclohydrolase; fts: formyl-THF synthase; hyda: hydrogenase; mtd: methylene-THF dehydrogenase; mtf: methyltransferase; mtr: methyleneTHF reductase).

reducing power and energy for acetyl-CoA or acetic acid synthesis and cell growth. In addition, CO can also provide the reducing power necessary for acetate and biomass production. It has been suggested that acetate can be reduced to acetaldehyde by an aldehyde oxidoreductase (AOR) and then to ethanol by an alcohol dehydrogenase, which probably plays an important role in conserving ATP in ethanol-producing acetogens [85]. The WL pathway is the most efficient non-photosynthesis CO2 fixation pathways in terms of using H2 as the reducing power and energy [85]. Some acetogens, such as Clostridium aceticum and Clostridium ljungdahlii, can also use electrons derived from electrodes to reduce CO2 to organic acids (mainly acetate) in a microbial electrosynthesis process [86]. Recently, the conversion of synthesis gas (or syngas in brief) and process waste gases containing CO, CO2 and Current Opinion in Chemical Engineering 2014, 6:43–54

48 Biotechnology and Bioprocess Engineering

H2 into biofuels by microbial catalysts has gained considerable attention as a promising alternative to chemical catalysis for biofuels and chemicals production [83], although poor mass transfer properties of the gaseous substrates (mainly CO and H2) and low product yields are the biggest challenges in the commercialization of syngas fermentation technology [84]. Several acetogenic Clostridium can convert CO2 and H2 into ethanol as the main product. Among these microbes, C. ljungdahlii and C. carboxidivorans are the most popular strains for ethanol production [87]. Extensive research including medium optimization and process and reactor design has been done for ethanol production from syngas with C. ljungdahlii [88], which was able to produce ethanol at a high final concentration of 48 g/L, but the productivity was low (0.083 g/L h) [89,90]. A commercial process combining biomass gasification, fermentation and ethanol recovery has been developed by Coskata. Also, LanzaTech has developed their own proprietary microbes adapted to grow well on syngas in a high-pressure bioreactor, and has operated two demonstration facilities for ethanol production from steel mill flue gas (similar to syngas). In addition, the production of 2-hydroxyisobutyric acid (2-HIBA), a precursor used in the manufacture of PLEXIGLAS1, from syngas has also been under research and development at Evonik. In addition, production of ethanol and butanol from coalderived syngas by Butyribacterium methylotrophicum was demonstrated at conditions favoring alcohols formation [91]. A two-step fermentation process, with acetate and butyrate produced by B. methylotrophicum under conditions favoring acids production, and followed by alcohols formation by C. acetobutylicum, was also demonstrated [91]. The production of 2,3-butanediol from CO-containing industrial waste gases by three acetogenic Clostridium species (C. autoethanogenum, C. ljungdahlii, and C. ragsdalei) [92] and production of medium-chain fatty acids (acetate, butyrate, caproate and caprylate) from CO2 and H2 by a mixed culture of Clostridium spp., such as C. ljungdahlii and C. kluyveri, have also been reported [93]. More recently, the potential of carboxydotrophic bacteria, including C. ljungdahlii and C. ragsdalei, as biocatalysts for converting short-chain carboxylic acids (acetic, propionic, butyric, valeric, and caproic acids) into corresponding alcohols, using CO and H2 in the syngas as the sources of electrons and energy, has also been demonstrated [94,95]. However, low final product titer, yield, and productivity are the biggest concerns for biofuels and chemicals production from syngas and waste gases containing CO2 and H2 by these carboxydotrophic bacteria. Engineering acetogenic clostridia for butanol production from CO2/H2

Although n-butanol can be produced from CO2 and H2 by acetogens such as C. carboxidivorans [96], the product titer (usually lower than 0.5 g/L) and yield are too low to be Current Opinion in Chemical Engineering 2014, 6:43–54

commercially viable. Therefore, recent research efforts have focused on metabolic engineering of acetogenic Clostridium using similar strategies and tools described earlier [3,83]. For example, C. ljungdahlii, which naturally produced acetate and ethanol, was successfully engineered to produce n-butanol by introducing genes in the solventogenic Clostridium’s butanol biosynthesis pathway [97]. Although the engineered C. ljungdahlii produced less than 0.15 g/L n-butanol from CO, CO2 and H2, this work provided the proof of principle that a butanol producing, CO2-fixing acetogen can be created by introducing the butanol biosynthesis pathway (from acetyl-CoA to butanol) using proven metabolic engineering techniques. More recently, an acetogenic Clostridum sp. MT1962, which produced acetate (17.2 g/L) and ethanol (13.5 g/L) in a single-stage continuous fermentation fed with syngas (60% CO and 40% H2), was metabolically engineered to produce n-butanol by introducing the n-butanol biosynthesis pathway genes (thio, hbd, crt, bcd, bad, and bdh) and knocking out acetate pathway genes ( pta and aldh) and early-stage sporulation genes (spo0A and spo0J) [98]. The engineered mutant Clostridium sp. MTButOH1365 produced n-butanol (22 g/L) as the only product in the fermentation broth (no ethanol or acetate). The 22 g/L is the highest titer ever reported for n-butanol fermentation, including those from glucose in the ABE fermentation using metabolically engineered C. acetobutylicum. This process thus has a great potential for commercialization if the productivity and yield were high enough, which cannot be determined from the reported data. Similar metabolic engineering strategies can also be applied to homoacetogens including C. thermoaceticum (renamed Moorella thermoacetica) and C. aceticum, which can convert CO2 and H2 to acetate with a high yield (100%), high titer (>50 g/L) and high productivity (6 g/ L h) comparable to those from glucose [99], suggesting a high metabolic flux from CO2 to acetyl-CoA, which is the immediate precursor for both acetate and ethanol. These homoacetogens have been extensively studied for their ability to produce acetic acid [100], but not as hosts to produce alcohols. Introducing adhE2 gene into homoacetogens should enable them to produce ethanol, which has been shown by us recently (unpublished data). In theory, homoacetogens can also be engineered to produce n-butanol by also introducing genes in the pathway from acetyl-CoA to butyryl-CoA. Since both acid and butanol tolerances share similar mechanisms involving membrane H+ pump and membrane fluidity affected by membrane lipid content [101], homoacetogens with high acid tolerance should also have high butanol tolerance. Therefore, homoacetogens are attractive hosts for n-butanol production from CO2 and H2. However, the WL pathway is limited by ATP as there is no net ATP generation from CO2 to acetyl-CoA or acetate, and the conversion of acetyl-CoA to acetoacetyl-CoA requires additional ATP www.sciencedirect.com

Engineering clostridia for butanol production Wang et al. 49

that may not be sufficiently supplied when CO2 is the carbon source [85]. In other words, butanol production from CO2 fixation through the WL pathway is ATPlimited and not efficient chemoautotrophically. However, this ATP limitation can be alleviated in mixotrophic growth with organic substrates such as glucose co-fermented with CO2/H2, as predicted in a stoichiometric and energy model analysis [85].

Integrated process for butanol production from cellulose and CO2 Growing the engineered acetogens on organic substrates with CO2 fixation and H2 as the electron donor has the potential to increase product yield by 50% [85]. Furthermore, the engineered acetogens can be cocultured with cellulolytic Clostridium in a consortium to produce n-butanol as the main product. Figure 3 illustrates the co-fermentation for n-butanol production from cellulose by engineered cellulolytic C. cellulovorans and CO2-fixing acetogenic bacteria. In such a consortium, the CO2 and H2 generated in the cellulose fermentation by C. cellulovorans can be further converted to n-butanol by the CO2-fixing acetogen. Thus, the overall butanol production from lignocellulosic biomass can be enhanced to >0.45 g/g cellulose, based on 70% of the theoretical yield, with minimal CO2 released to the environment. This process concept has not been investigated in the laboratory and will need optimization in several process factors, including reactor type, media composition, pH, and temperature. Butanol production from CO2/H2 in syngas fermentation and lignocellulosic biomass in a consolidated bioprocess may be limited by low product titer and productivity due to low cell density and strong butanol inhibition in conventional fermentation processes [102]. For economical production of butanol, it is important to maintain a high density of viable and productive cells in the bioreactor in order to achieve a high volumetric productivity, which can significantly reduce the capital cost. Here we Figure 3

Butyric acid Cellulose

Mut. 1

Glucose

X

Butyryl-CoA Acetyl-CoA X

adhE2

Butanol

Mut. 2 Wood-Ljungdahl pathway

+

CO2 + H2

Acetic acid Current Opinion in Chemical Engineering

Co-fermentation for n-butanol production from cellulose by engineered cellulolytic (Mut 1) and CO2 by CO2-fixing acetogenic Clostridium (Mut 2) overexpressing adhE2. The CO2 and H2 generated in the cellulose fermentation by the cellulolytic Clostridium can be further converted to n-butanol by the CO2-fixing acetogen, thus increasing butanol yield by 50%. Also, the genes in acetate and butyrate biosynthesis pathways can be knocked out to reduce acids production, thus butanol would be the main product from cellulose. www.sciencedirect.com

discuss two process technologies, high cell density fermentation and in situ product recovery, that can contribute to economical production of biobutanol. High cell density fermentation for increased productivity

Continuous butanol production from glucose by C. acetobutylicum immobilized in a fibrous bed bioreactor (FBB) achieved a high cell density (HCD) of >50 g/L and butanol productivity of 17 g/L h, which was over 50-fold higher than conventional ABE fermentation [103]. The cell density in the FBB was 10-fold higher than that in conventional fermenters, and the higher cell density gave more than proportional increase in reactor productivity due to additional benefits such as protection from oxygen toxicity and faster culture adaptation to tolerate inhibiting fermentation products [101]. Also, the HCD fermentation can reduce cell biomass waste and increase product yield since cells can be repeatedly used in the process. Similar benefits were also observed in HCD free-cell fermentation in sequential batches and continuous butanol fermentation with cell recycle [104]. HCD fermentation with cells immobilized on the lignocellulosic biomass (particles) in a column bioreactor (airlift, upflow expanded bed or fixed bed) can be used to intensify the fermentation process, but has not been explored. Integrated fermentation with in situ butanol separation

Butanol recovery by distillation is energy-intensive and costly in conventional biobutanol production process. The limitations imposed by costly n-butanol recovery by distillation have stimulated research into alternative methods of n-butanol recovery [105]. A number of separation methods, including adsorption [106], gas stripping [107,108], liquid-liquid extraction [109], and pervaporation [110] have been studied. Extensive research has also been conducted on the development of in situ butanol recovery to realize the removal of n-butanol from the reactor simultaneously with its production. Thus, the nbutanol concentration could be prevented from exceeding the tolerance level of the culture, allowing the ABE fermentation to continue with higher substrate conversion, butanol productivity, and the final butanol concentration. Table 2 compares various in situ product recovery methods that can be used in integrated butanol fermentation. Among the separation methods, gas stripping is relatively simple to operate and shows great potential in reducing energy cost for butanol recovery and purification. Gas stripping for online butanol recovery from cultures not only can increase fermentation productivity but also facilitate butanol purification. In general, the butanol removal rate by gas stripping is proportional to the butanol concentration and is most effective for butanol recovery when the butanol concentration is higher than 10 g/L [107]. Extractive fermentation using gas stripping can increase butanol production in ABE fermentation by more than 10 fold. For example, Xue et al. used the fermentation-produced CO2/H2 as the stripping Current Opinion in Chemical Engineering 2014, 6:43–54

50 Biotechnology and Bioprocess Engineering

Table 2 Comparison of different methods in in situ product recovery Advantages

Method Gas stripping

Disadvantages

Simple and effective way; only N2 or fermentation gases (CO2 and H2) and a condenser are needed; The final ABE titer is very high (400 g/L), up to more than 500 g/L in recent research

Relatively low efficiency if butanol concentration is less than 1% More ABE residues in fermentation broth

Prevaporation

Membrane has high selectivity for butanol Low energy consumption Promising method for scale-up

Prone to fouling and clogging High cost of membrane Process stability can be an issue

Adsorption

Only sorbent is needed Complete desorption from sorbent, such as silicalite Low energy consumption

High price of adsorbents Low capacity Low selectivity Possible fouling and clogging

Liquid–liquid extraction

Only extractant is needed, such as oleyl alcohol High capacity High selectivity

High price of the extractant Difficult phase separation due to emulsion formation Extractant loss Require the regeneration of extractant

Adapted from [105] with modifications.

gas and demonstrated that by applying gas stripping intermittently in fed-batch fermentation with a butanol tolerant C. acetobutylicum strain, 173 g/L ABE were produced from 475 g/L glucose in six feeding cycles over 326 h, achieving an overall productivity of 0.53 g/L h and yield of 0.36 g/g. The intermittent gas stripping produced a highly concentrated condensate containing 196 g/L ABE (150.5 g/L butanol) which after phase separation produced a final product containing 610 g/L butanol, 40 g/L acetone, 10 g/L ethanol and no acids [102]. The fed-batch fermentation with intermittent gas stripping could also greatly reduce energy consumption and water usage by up to 90%, offering an energy efficient process for n-butanol production [102]. It is thus feasible to use the fermentation-produced CO2/H2 as the

stripping gas to continuously recover and concentrate butanol from fermentation broth to a 65% butanol solution (after condensation and phase separation), which can be readily purified to more than 99.5% by simple distillation with over 80% saving in energy consumption [102].

Conclusions and economic perspectives In summary, cellulolytic and acetogenic Clostridium can be engineered to convert cellulose, CO2 and H2, which are gaseous byproducts from clostridial fermentations, to n-butanol with high yield, titer and productivity. Converting the CO2 and H2 produced in cellulose fermentation potentially can increase the overall butanol yield by 50%, reaching a theoretical max. value of 0.63 g/g

Table 3 Comparison of economics of biobutanol production from corn using conventional ABE fermentation and from corn stover using the novel co-fermentation processes Conventional ABE fermentation a

Novel butanol fermentation b

Products Substrate cost

Acetone, butanol, ethanol (3:6:1) Corn: $170/ton 70% starch $0.24/kg corn starch

Mainly butanol (>80%) Corn stover: $70/ton 60% cellulose + hemicellulose $0.12/kg cellulose

Process

Semi-continuous process with 6 8 fermentors (CSTR) in series with a total retention time of more than 60 h Recovery by distillation; energy intensive

Sequential batch process with high cell density and online gas stripping for butanol recovery to reduce energy input

Butanol concentration Productivity Butanol yield Product cost

1.2% (w/v) 0.5 g/L h <0.25 g/g sugar $4.50/gal

1% in broth; 15% after gas stripping 0.25 1.0 g/L h 0.30 0.45 g/g cellulose $2.25/gal or less

a b

Based on studies reported in [5,110]. Based on the projected performance discussed in this paper.

Current Opinion in Chemical Engineering 2014, 6:43–54

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Engineering clostridia for butanol production Wang et al. 51

cellulose. The engineered Clostridium mutants can be used in a high-cell density fermentation process integrated with online butanol recovery for economical production of nbutanol from lignocellulosic biomass. By increasing the productivity and yield through the integrated process, the price of biobutanol can be reduced more than 50% compared to the current ABE process with corn as feedstock. A comparative economic assessment of ABE fermentation based on cellulosic and non-cellulosic feedstocks showed that butanol can be produced from cellulosic feedstocks such as corn stover, bagasse, and switch grass at a cost of $1.8–$2.3/gal if the ABE yield was 0.39 g/g (butanol yield: 0.234 g/g), whereas the butanol production cost would be much higher at $4.0/gal with corn as the feedstock [111]. For conventional ABE fermentation with corn meal or cassava, about 65% of the total production cost is from the feedstock; the other 35% is mainly from the energy used in distillation for the recovery and purification of butanol from the fermentation broth [5]. A butanol fermentation process with engineered Clostridium using cellulosic biomass such as corn stover and CO2 as low-cost substrates will be much cheaper if comparable productivity and butanol titer can be obtained (see Table 3). The much cheaper feedstock with comparable or higher butanol yield could reduce the final product cost by more than 30%. With gas stripping for butanol recovery from fermentation broth, the energy cost in distillation can be reduced by 80% and overall butanol recovery/purification cost by 50%, resulting in an additional 20% cost reduction. Therefore, biobutanol can be produced at a cost of $2.25/gal or less from cellulose using the integrated fermentation process. With the higher energy content, butanol at $2.25/gal will be cheaper than ethanol and similar to gasoline at $2.50/gal in terms of energy cost ($ per MJ). This would make biobutanol a very competitive biofuel. Large scale, economical biobutanol not only relieves the oil crisis, it also can bring environmental benefits such as emission reduction and agricultural benefits such as growing income for farmers.

Acknowledgements This work was supported by the National Science Foundation STTR program (IIP-1026648), Advanced Research Projects Agency-Energy (DEAR0000095), the Natural Science Foundation of China (21276093), and the Planned Science and Technology Project of Guangdong Province, China (2011A080403022).

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