Nov 7, 2017 - and 2,3-butanediol but is rapidly expanding to heterologous products. Syngas fermentation can achieve high carbon- efficiencies; however, the ...
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ScienceDirect Overcoming the energetic limitations of syngas fermentation Bastian Molitor1, Esteban Marcellin2 and Largus T Angenent3 The fermentation of synthesis gas (including carbon monoxide, carbon dioxide, and hydrogen) with anaerobic acetogens is an established biotechnological process that has recently been transferred to a commercial scale. The natural product spectrum of acetogens is natively restricted to acetate, ethanol, and 2,3-butanediol but is rapidly expanding to heterologous products. Syngas fermentation can achieve high carbonefficiencies; however, the underlying metabolism is operating at a thermodynamic limit. This necessitates special enzymatic properties for energy conservation by acetogens. Therefore, the availability of cellular energy is considered to restrain the efficient production of energy-intense products with complex production pathways. The optimization of the feed-gas composition and other process parameters, genetic engineering, and integration with other biotechnologies is required to overcome this limitation.
Addresses 1 Department of Biological and Environmental Engineering, Cornell University, Riley-Robb Hall, Ithaca, NY 14853, United States 2 Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Queensland 4072, Australia 3 University of Tu¨bingen, Center for Applied GeoSciences, Ho¨lderlinstr. 12, 72074 Tu¨bingen, Germany Corresponding author: Angenent, Largus T (l.angenent@uni-tuebingen. de)
Current Opinion in Chemical Biology 2017, 41:84–92 This review comes from a themed issue on Energy Edited by Matthew W. Kanan For a complete overview see the Issue and the Editorial Available online 7th November 2017 http://dx.doi.org/10.1016/j.cbpa.2017.10.003 1367-5931/ã 2017 Elsevier Ltd. All rights reserved.
utilization of syngas fermentation can off-set the use of fossil sources by replacing traditional production routes for fuels and chemicals [4]. In the long run, the process can contribute to a bio-economy that is based on direct recycling of CO2, and might become intrinsically carbonneutral. This is also because the source of the gases is flexible. Syngas can be generated from the gasification of biomass or municipal waste, and from renewable natural gas (mainly methane [CH4]) by steam-reforming [2]. The renewable natural gas has to come from a source other than fossil natural gas to contribute to a sustainable bioeconomy. For reviews on the production of renewable natural gas with Power-to-Gas technologies, the reader is referred to recent reviews on this topic [5,6]. Also, CO is the main component of certain gas streams from heavy industries [7]. The utilized microbial catalysts, which microbiologists refer to as acetogens, mainly produce acetate as their natural fermentation product [8]. The conditions can be steered toward the production of alternative natural products such as ethanol, and 2,3-butanediol [9,10]. Furthermore, progress on the implementation of molecular biology tools and genetic engineering strategies now allows to broaden the product spectrum to non-natural fermentation products or to optimize selective production of natural products [11,12,13]. This review briefly summarizes the knowledge on the variety of acetogens capable of syngas fermentation, the achieved product spectrum, and new insight into the underlying physiology. The focus, however, is on the most recent achievements for overcoming the energetic limitations of syngas fermentation by metabolic modeling, genetic engineering, and integration with other bioprocessing technologies.
Open culture-based versus pure culturebased production platforms Introduction Besides the need for alternative production routes for fuels and platform chemicals from renewable resources, technologies that enable mitigation of green-house gases are pertinent for a sustainable future [1]. The fermentation of reduced gases (i.e., carbon monoxide [CO] and hydrogen [H2]), together with carbon dioxide (CO2) is known as syn(thesis)gas fermentation. This platform technology has already proven to have enormous potential to contribute to a future bio-economy [2,3]. The Current Opinion in Chemical Biology 2017, 41:84–92
For the implementation of a syngas fermentation process either open cultures or pure cultures can be considered [14]. For open cultures, process parameters can be optimized to establish a microbiome that potentially generates a product of interest. The spectrum of possible products is limited to naturally occurring products, since genetically engineered strains cannot be specifically maintained in an open culture. However, the products may result from a combination of metabolic pathways available in different microbes. Thus far, the achieved volumetric production rates and titers have been low in www.sciencedirect.com
Overcoming the energetic limitations of syngas fermentation Molitor, Marcellin and Angenent 85
open cultures [15–17], and it is not known whether they can be improved to industrially relevant rates and titers. Higher production rates and titers have been reported with pure cultures [14,18–21]. Also the product spectrum can be extended by genetic engineering [2]. Important bacteria capable of syngas fermentation are discussed in the next section.
The spectrum of acetogens capable of syngas fermentation Most commonly used acetogens for syngas fermentation are bacteria in the genus Clostridium, however, a few other species have also interesting properties (Table 1). Other recent reviews present more complete lists [2,10,22]. The products that naturally occur with syngas fermentation are mainly acetate, ethanol, and to a lesser extent 2,3-butanediol [23]. Other acetogens have a broader product spectrum and Clostridium carboxidivorans, for example, also naturally produces n-butanol and n-hexanol (Table 1) [18]. The increasing availability of genome sequences may result in the identification of more efficient production hosts, the capabilities of producing other interesting products, and strategies for genetic engineering [24–27].
The Wood-Ljungdahl pathway and the physiology of acetogens Acetogens are capable to fix CO2 via the Wood-Ljungdahl pathway (WLP) [8,28,29]. This linear process is considered the oldest carbon-fixation pathway [30]. It results in acetyl-CoA, which is further utilized for biomass growth and acetate production. While the production of one mole of acetate results in the formation of one mole of ATP by substrate-level phosphorylation, the pathway does not generate net ATP because it also utilizes one mole of ATP for the activation of formate to formyl-tetrahydrofolate (Figure 1) [31,32]. For further cellular energyconservation in the form of ATP for biomass growth and cellular maintenance processes, some acetogens, such as Moorella thermoacetica, contain an energy-converting hydrogenase (Ech), which generates a membrane potential [33,34]. M. thermoacetica also contains cytochromes and menaquinone while the exact mechanism of energy conservation (i.e., the generation of a membrane potential to drive ATP synthase) in this acetogen has not been elucidated (Figure 1) [32]. These acetogens typically are not efficient alcohol producers from syngas [35]. Researchers only discovered a few years ago how acetogens without cytochromes can generate additional cellular energy for
Table 1 Important microbes capable of syngas fermentation Organism
Rnf/ATP synthetase (H+/Na+)
Acetobacterium woodii Butyribacterium
Na+
n-Butanol production Clostridium
[26,79]
AOR No
methylotrophicum
Gaseous Substrates H2/CO2 coupling ion not determined
Products
Interesting features
References
Acetate; ethanol only when fermenting sugars nda
Highest reported acetate [19,37,76–78] production, genetic tools H2/CO2/CO Acetate, nbutyrate, ethanol, n-butanol
H+
Yes
H2/CO2/CO
Acetate, ethanol, 2,3-butanediol, lactate
Industrially relevant, genetic tools, CRISPR/ Cas9 reported, GEMb
H+
Yes
H2/CO2/CO
Hexanol-ButanolEthanol fermentation
[82–84]
Clostridium ljungdahlii
H+
Yes
H2/CO2/CO
Acetate, n-butyrate, ethanol, n-butanol, ncaproate, n-hexanol Acetate, ethanol, 2,3-butanediol, lactate
[13,24,55,85,86]
Eubacterium limosum Moorella thermoacetica
Na+
nd
H2/CO2/CO
Acetate, n-butyrate
Genetic tools, CRISPR/ Cas9 reported, widely used model organism, GEMb n-Butyrate production
H+ (Ech and cytochromes instead of Rnf) H+ (Ech and cytochromes instead of Rnf)
No
H2/CO2
Acetate
[34,89,90]
Yes
H2/CO2
Acetate, Ethanol
Thermophilic, contains cytochromes and menaquinone, GEMb Used to study microbial electrosynthesis, contains cytochromes and ubiquinone
[12,56,80,81] Clostridium carboxidivorans
Sporomusa ovata
a b
autoethanogenum
[87,88]
[91–93]
nd, not determined. GEM, genome-scale metabolic model.
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Current Opinion in Chemical Biology 2017, 41:84–92
86 Energy
Figure 1
cathode H+ H2 CO2
H2
Na+
CO Fdred
NADPH/ CO2 Fdred/H2 formate -ATP NADPH
H2ase (Hyt) Fdred
CO
formyl-THF
H+
H2ase (Ech)
H2
2,3BD/ lactate
Fdred
NAD(P)H
methyl-FeS-P
CO biomass
Fdred acetyl-CoA
biomass
Cyt ? MQH2
n-butyrate/ n-butanol/ n-caproate/ n-hexanol
MQ
Na+/ H+
+ATP
Fdred
acetate*
2
pyruvate 1 NADH
acetaldehyde NAD(P)H ethanol
Fdred Rnf
NADH
Fdred
NADH
Nfn NADPH
H2
+ATP
ATPase
Na+/ H+
H2ase (Hyd) Fdred NADH Current Opinion in Chemical Biology
Schematic overview of energy-conserving mechanisms in acetogens without providing exact stoichiometries. The Wood-Ljungdahl pathway (WLP) is used for the generation of acetyl-CoA from CO2 and/or CO. CO2 is stepwise reduced to a methyl-group and combined with a carbonyl-group and CoA to form acetyl-CoA. Diverse hydrogenases oxidize H2 to provide reducing cofactors. Electron-bifurcation plays a pivotal role for that. Electron-bifurcating complexes are the hydrogenases Hyt and Hyd, the transhydrogenase Nfn, formate dehydrogenase, and likely methylenetetrahydrofolate reductase (not shown). Reduced ferredoxin can also be generated in the CO dehydrogenase reaction when CO is oxidized to CO2. Acetyl-CoA is the central intermediate from which biomass and fermentation products are derived. For the generation of ethanol, acetyl-CoA can be directly reduced to acetaldehyde and further to ethanol (route 1, orange). A second route involves the generation of acetate first. The asterisk highlights that only undissociated acetic acid is reduced to acetaldehyde by an aldehyde oxidoreductase and further to ethanol by an alcohol dehydrogenase (route 2, purple). The activation of formate to formyl-THF consumes ATP and the production of acetate yields ATP by substrate-level phosphorylation. Further cellular energy is derived from membrane-associated complexes. The Rnf-complex pumps protons (C. ljungdahlii-type) or sodium ions (A. woodii-type) across the membrane while oxidizing reduced ferredoxin (blue shaded box). In M. thermoacetica an energy-converting hydrogenase (Ech) is involved in generating a membrane potential. M. thermoacetica also contains cytochromes (Cyt) and menaquinones (MQ), although these are likely not involved in an electron-transport chain. The exact mechanism which couples the Ech to energyconserving reactions is not known (grey shaded box). The membrane potential is consumed by an FoF1 ATP synthetase (ATPase) to generate ATP. Steps directly involved in energy-conservation are shown in red. Reducing power can be derived from a cathode (box in top right corner). Very likely this microbial electrosynthesis involves the generation of H2 as an intermediate for acetogens. Recently, it was described that biofilm formation in C. ljungdahlii is increased with high sodium-salt concentration, which can have an impact on microbial electrosynthesis but also on syngas fermentation systems. 2,3BD, 2,3-butanediol; Fd, ferredoxin; FeS-P, corrinoid iron-sulfur protein; THF, tetrahydrofolate.
biomass growth [36]. This is performed by utilization of the membrane-associated ferredoxin-dependent Rnf complex [37–39]. In addition, electron bifurcation plays an important role in all known acetogens [32,40]. Electron bifurcation allows for the coupling of an unfavorable Current Opinion in Chemical Biology 2017, 41:84–92
reduction reaction (i.e., the generation of reduced lowpotential ferredoxins [E00 = 500 mV]) and a favorable reduction reaction (i.e., the generation of reduced NAD(P)H [E00 = 320 mV]) from the oxidation of H2 (E00 = 414 mV) [41]. The Rnf complex utilizes the www.sciencedirect.com
Overcoming the energetic limitations of syngas fermentation Molitor, Marcellin and Angenent 87
reduced ferredoxin partly for the generation of a membrane potential that, in turn, drives an ATP synthetase [32]. Meanwhile, CO (E00 = 520 mV) can directly reduce ferredoxin. However, stoichiometrically 2/3 of the carbon is lost as CO2 when only CO is used as a substrate, and the co-feeding of H2 is necessary for optimized carbon efficiencies [7]. The Rnf complex can be proton-dependent or sodium-dependent [37–39], and the number of c-ring subunits in the ATP synthetase determines the overall energy efficiency [31,42].
regenerates redox cofactors without flow through acetyl-CoA [45]. In a one-stage system, a high flux through acetyl-CoA is necessary to keep up ATP requirements, which at high acetate concentrations leads to a metabolism crash (see section ‘Overcoming energetic limitations of acetogenic metabolism’ for more details) [46]. Importantly, not every acetogen encodes for the AOR enzyme that is required to perform the second route. This explains why some acetogens produce ethanol readily when growing on syngas and others do not [27].
When acetogens ferment sugars, the metabolism is often referred to as homoacetogenic, because the intermediary produced CO2 (from decarboxylation reactions in glycolysis) is further converted into additional acetyl-CoA via the WLP. This is also called acetogenic mixotrophy and can be used to increase carbon efficiency from sugars, and enable more energy-intense production routes [43]. This strategy can be interesting when sugars are supplied to the fermentation together with optimized gas mixes. In this case, the gas is the main source of carbon and energy, while the breakdown of sugars via glycolysis generates additional ATP through substrate-level phosphorylation, which can be used for energy-intense production pathways. The CO2 generated during glycolysis is not wasted but fixated into more acetyl-CoA, when surplus reducing equivalents are available, for example, by supplying H2 gas [44].
Currently, the new field of microbial electrosynthesis is generating interesting results on specific physiological traits of acetogens. In this process, microbes take up electrons from a cathode most likely indirectly via the generation of H2 first as an intermediate [47–50]. Recently, biofilm formation of Clostridium ljungdahlii was reported for the first time in this context [51].
For the production of ethanol there are two possible routes known (Figure 1). In the first route, acetyl-CoA is directly reduced into ethanol with acetaldehyde as an intermediate with NAD(P)H as the source of reducing equivalents [24]. In this route, no ATP is produced by substrate-level phosphorylation, but also no reduced ferredoxin is required, which can be used to build-up a membrane potential with the Rnf complex. In the second route, acetate is produced first, which supplies ATP by substrate-level phosphorylation. Then, undissociated acetic acid is reduced to acetaldehyde with reduced ferredoxin by an aldehyde oxidoreductase (AOR), and acetaldehyde is concomitantly reduced to ethanol with NAD(P)H [31]. During homoacetogenesis the first route is more efficient because less reduced ferredoxin is available to the cell. During gas fermentation, especially with CO-rich gas mixtures, the second route is more energy efficient, allowing for ethanol production as an overflow mechanism [45]. The bioprocessing strategy is very important for the optimization toward ethanol production in a fermentation with wild-type microbes, because the overflow mechanism is believed to depend on high undissociated acetic acid levels [45]. In a two-stage system, the overflow mechanism leads to high ethanolto-acetate ratios in the second stage, because it is supplied with high amounts of acetate from the first stage [21]. This triggers ethanol production and undissociated acetic acid is reduced by AOR and reduced ferredoxin, which www.sciencedirect.com
The different physiological properties can have an important influence on the growth rate and the available energy for bioproduction in different acetogens and for different targeted products, and have to be considered for the choice of a host as a production platform.
Overcoming energetic limitations of acetogenic metabolism To become an economically competitive biotechnology, syngas fermentation facilities need to be versatile and rely on the production of multiple products. Expanding the product spectrum of gas-fermenting microbes is, therefore, crucial (Figure 2). One way to accelerate progress on this end is through rational design and systems biology using in silico designs [52,53]. As discussed before, producing energy-intense molecules with complex biochemical pathways from syngas is considered to be limited by the availability of cellular energy in the form of ATP [29,32,54]. Modern strain engineering uses genome-scale metabolic models to first design and test efficient pathways that maximize energy efficiency while avoiding undesired by-product formation before genetic engineering of the microbe is performed in the lab. The first genome-scale metabolic model (GEM) of an acetogen was published for C. ljungdahlii [55] followed by GEMs for M. thermoactica [34] and Clostridium autoethanogenum [56]. These GEMs have been used to predict phenotypes [57] and to estimate intracellular flux patterns [58]. In conjunction with shadow-price analysis, the C. autoethanogenum GEM was used to find a pathway utilizing arginine to supply additional ATP, which increases cellular energy availability for the generation of energyintense products [59]. A refined version of this model was used to show that coupling carbon and redox metabolism with transcriptomics data is necessary to accurately predict growth phenotypes, and to further substantiate Current Opinion in Chemical Biology 2017, 41:84–92
88 Energy
Figure 2
GEM reconstruction
Metabolic Modelling
Systems Biology
Strain design
Data Integration
Metabolic Engineering X X
X
X
1600 400 RNAseq (40hrs)
RNAseq 41hrs
S. ν = 0 ν
0 80 RNAseq (40hrs)
0 novel ORF νi
Mathematical representation of metabolism
peptide tag 0 novel ORF peptide tag
Multi-omics analysis
Genetic manipulation of acetogens using for example Clos-Tron or CRISPR/CAS9
Superior gas fermentation strains with expanded product spectrum Current Opinion in Chemical Biology
Genome-scale metabolic models (GEMs) provide a comprehensive understanding of the physiology and metabolism of acetogens. The new genomic era allows to gain understanding of microbial physiology at the systems level. Genome-scale metabolic models represent a valuable framework for integrative analysis which can be used for strain design. Eventually this leads to superior gas fermentation strains and processes, overcoming the energetic limitations of syngas fermentation.
the current hypothesis that the methylene-tetrahydrofolate reductase reaction is ferredoxin-reducing by means of electron bifurcation (Figure 1), which is one of the remaining open questions about energy conservation in acetogens [46]. Insight into the regulation of acetogenic metabolism was achieved using continuous bioreactors at different steady-state biomass levels that showed a shift in byproduct formation (acetate/ethanol ratio). Past a certain biomass concentration, steady state was unachievable. First, the H2 consumption of the culture dropped, accompanied with higher CO to CO2 reduction activity. This was hypothesized to be due to the impacted ability of ATP generation and higher ATP requirements. Higher CO2 production rates divert carbon away from acetyl-CoA production. This results in the depletion of the acetylCoA pool and leads to an imbalance in metabolism with an ocillatory behaviour that follows. Therefore, one layer of regulation of the distribution of carbon toward different products in the metabolism, is by the ATP homeostasis requirement. A second layer of regulation, specifically for ethanol production, is hypothesized to be the intracellular undissociated acetic acid concentration [45]. The bioreactor design has an important influence. In a one-stage Current Opinion in Chemical Biology 2017, 41:84–92
bioreactor as used by Valgepea, et al. [46], the generation of biomass and fermentation products (except CO2) involves a flux through acetyl-CoA, and the acetyl-CoA pool plays a pivotal role in metabolism. It has been reported before that bypassing the flux through acetylCoA can circumvent this crash by using a two-stage fermentation system in which acetate is generated in the first stage, and fed to a second stage [45]. Thus, reduced ferredoxin can be regenerated by reducing undissociated acetic acid to ethanol in the second stage, while allowing high CO consumption. The same effect should be achieved by feeding acetate to a one-stage system when the metabolism is about to collapse. This would allow for cofactor regeneration without the need for flux through acetyl-CoA, and therefore, would allow for ongoing product formation. Together with molecular biology tools for acetogens, which become readily available, GEMs are a powerful tool for strain optimization [56]. The first example of genetic manipulation of an acetogen was described by Ko¨pke, et al. [24]. Tools for genetic engineering, such as Clos-Tron, which is a group II intron-based retrohoming www.sciencedirect.com
Overcoming the energetic limitations of syngas fermentation Molitor, Marcellin and Angenent 89
gene-disruption tool [60], are now commonly used [11,31,56]. Further methods and tools for genetic engineering of acetogens are constantly being developed [38,61–64], and now also include CRISPR/Cas9 technology [12,13]. These developments are facilitating genetic investigations of acetogen physiology, the potential to optimize autotrophic production of naturally occurring products, and the avenues to increase the product spectrum of acetogens. The potential is demonstrated exemplarily by two recent studies. In the first example, isogenes (i.e., each of two or more genes encoding for enzymes with identical function), which encode for carbon monoxide dehydrogenase (CODH), were studied. The most important CODH isogene for carbon fixation was identified, and it was demonstrated that genetic inactivation of an unimportant CODH could improve autotrophic growth [65]. In the second example, the missing genetic experiments were provided to confirm that AOR is critical for ethanol formation in acetogens, and inactivation of the bifunctional alcohol/aldehyde dehydrogenase AdhE led to consistently enhanced autotrophic ethanol production [11].
Alternative strategies to overcome limitations of syngas fermentation While genetic engineering strategies to expand the product spectrum and to optimize the energy availability in syngas-fermenting microbes are getting increasingly feasible, another interesting opportunity is the combination with other (bioprocessing) technologies. The main products of syngas fermentation, which are the C2 compounds acetate and ethanol, are the substrates for chain elongation via the reverse b-oxidation pathway [66,67]. By placing syngas fermentation and chain elongation in series, C1 (CO, CO2) has now been elongated to C8 (n-caprylic acid, which is also referred to as n-octanoic acid) with an open-culture process and a pure-culture process [68,69]. In addition, two defined co-culture studies with C. ljungdahlii or C. autoethanogenum and Clostridium kluyveri, and one open culture study, have combined syngas fermentation, chain elongation, and biological reduction to alcohols in one bioreactor. However, the low product specificities and the pH incompatibilities may be difficult to overcome [70,71,72]. Two-stage bioprocessing is a potential route to overcome energetic limitations and to allow for the production of much more energy-dense products such as lipids [73], malic acid [74], or polyhydroxyalkanoates (PHAs) [75]. For example, the production of acetate by syngas fermentation can be optimized in a first stage under anaerobic conditions, while this acetate is then converted in a second stage under aerobic conditions, which allows for the generation of high cellular energy levels through oxidative phosphorylation. Then the CO2, which is produced as an unwanted byproduct in the aerobic process, can be re-entered into the syngas fermentation to optimize the carbon-footprint of the combined processes. www.sciencedirect.com
Conclusion Syngas fermentation with acetogens offers great potential for a future bio-economy. Molecular biology tools are being developed to a level that allows extended metabolic engineering. With this, the product spectrum can be increased and pathways can be implemented to provide further cellular energy for complex pathways. Combined with high quality metabolic models, the field is poised to make considerable leaps in the near future. These models help to increase the knowledge on acetogenic metabolism which can be also harnessed for optimization of process parameters. Understanding the role of the AOR enzyme, and the ATP homeostasis allow to optimize bioreactor design (two-stage system) or feeding strategies (one-stage system) to increase alcohol production. The combination of syngas fermentation with downstream (bio)processes can further help to make the overall process economically feasible. The combination with chain elongation for the production of medium-chain carboxylic acids and alcohols up to C8 shows promising results. Finally, the combination with aerobic processes offers the potential to generate energy-dense products, including lipids and PHAs, without the need for genetic engineering.
Conflict of interest The authors declare no conflicts of interest.
Acknowledgements BM was funded through a postdoctoral research fellowship from the German Research Foundation (DFG, MO2933/1-1) at the time of preparing the manuscript. EM is grateful to the Australian Research Council and to Lanzatech (ARC LP140100213) and the Queensland Government for his Accelerate fellowship. LA acknowledges support from the Alexander von Humboldt Foundation in the framework of the Alexander von Humboldt Professorship endowed by the Federal Ministry of Education and Research in Germany.
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