Industrial Crops and Products 106 (2017) 21–30

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Industrial Crops and Products

journal homepage: www.elsevier.com/locate/indcrop

Exploiting the potential of gas fermentation

Stephanie Redl a,1 , Martijn Diender b,1 , Torbjørn Ølshøj Jensen a , Diana Z. Sousa b ,
Alex Toftgaard Nielsen a,∗
a
Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Hørsholm, Denmark
b
Laboratory of Microbiology, Wageningen University, Wageningen, The Netherlands

a r t i c l e i n f o a b s t r a c t

Article history: The use of gas fermentation for production of chemicals and fuels with lower environmental impact is a
Received 15 July 2016 technology that is gaining increasing attention. Over 38 Gt of CO2 is annually being emitted from industrial
Received in revised form 20 October 2016 processes, thereby contributing significantly to the concentration of greenhouse gases in the atmosphere.
Accepted 8 November 2016
Together with the gasification of biomass and different waste streams, these gases have the potential for
Available online 19 November 2016
being utilized for production of chemicals through fermentation processes. Acetogens are among the most
studied organisms capable of utilizing waste gases. Although engineering of heterologous production of
Keywords:
higher value compounds has been successful for a number of acetogens, the processes are challenging due
Syngas fermentation
Biomass gasification
to the redox balance and the lack of efficient engineering tools. In this review, we address the availability
Co-cultures of different gaseous feedstock and gasification processes, and we focus on the advantages of alternative
Mixotrophy fermentation scenarios, including thermophilic production strains, multi-stage fermentations, mixed
Acetogens cultures, as well as mixotrophy. Such processes have the potential to significantly broaden the product
Thermophiles portfolio, increase the product concentrations and yields, while enabling the exploitation of alternative
and mixed feedstocks. The reviewed processes also have the potential to address challenges associated
with product inhibition and may contribute to reducing the costs of downstream processing. Given the
widespread availability of gases, such processes will likely significantly impact the transition towards a
more sustainable society.
© 2016 Elsevier B.V. All rights reserved.

1. Introduction
There is an increasing demand for processes that reduce carbon-
emissions and ensure carbon neutral and sustainable production
of energy and commodities for the steadily growing population
(Pachauri et al., 2014). Previous advances in the production of first
generation biofuels have raised the feed vs. fuel debate. A promising
technology that has gained increasing attention within recent years
is gas fermentation, a process in which microorganisms anaerobi-
cally convert a gaseous substrate into biofuels and biochemicals.
Several microorganisms have the ability to utilize CO2 and CO as
Abbreviations: 3HP, 3-hydroxypropionic acid; 4HB, 4-hydroxybutyrate; ATP,
adenosine triphosphate; BDO, butanediol; GHG, greenhouse gas; MEK, methyl ethyl
ketone; MSW, municipal solid waste; NADH/NAD+, nicotinamide adenine dinucleo-
tide; NADPH/NADP+, nicotinamide adenine dinucleotide phosphate; NETL, National
Energy Technology Laboratory; PFOR, pyruvate:ferredoxin oxidoreductase; PHA,
polyhydroxyalkanoates; VFA, volatile fatty acids; VSS, volatile suspended solids;
WLP, Wood-Ljungdahl pathway.
∗ Corresponding author.
E-mail address: atn@biosustain.dtu.dk (A.T. Nielsen).
1
These authors contributed equally.
energy and carbon source. Acetogenic bacteria are the most stud-
ied and have the greatest industrial potential, making use of the
Wood-Ljungdahl pathway (WLP) to convert CO2 (and CO). Com-
pared to the Calvin-Benson-Bassham cycle, which is used by plants,
algae, cyanobacteria, purple bacteria, and some proteobacteria, the
WLP is a highly energy efficient CO2 fixation pathway (Hawkins
et al., 2013). Microorganisms employing the WLP are therefore
relevant as biotechnological platforms for the production of biofu-
els and biochemicals from one-carbon compounds, and potentially
decrease our dependence on fossil resources.
Metabolic traits of gas-fermenting bacteria have been reviewed
recently (Daniell et al., 2016; Dürre and Eikmanns, 2015; Latif et al.,
2014). The scope of this article is to assess the potential of gas fer-
mentation and ways to exploit this potential. We would like to draw
attention to alternative production scenarios, including multi-stage
processes, co-cultures, mixotrophy, and thermophilic production
strains. To date, pure cultures of mesophilic strains are deployed,
with a focus mainly on ethanol, and on 2,3-BDO production. The
aforementioned alternative production scenarios on the contrary
would broaden the spectrum of products that can be produced from
CO and CO2 . Additionally, it would be possible to explore combina-

http://dx.doi.org/10.1016/j.indcrop.2016.11.015
0926-6690/© 2016 Elsevier B.V. All rights reserved.
22 S. Redl et al. / Industrial Crops and Products 106 (2017) 21–30
tions of different industrial feedstock streams (gas and sugar) and
to take advantage of variable process conditions.
2. Feedstock availability
The present review focuses on the conversion of CO and CO2
as carbon source by gas fermenting microorganisms. Hereby, CO
serves as carbon source and electron donor. When CO2 serves as
sole carbon source, an additional electron donor is required. Below,
we describe some of the sources of CO, CO2 , and electron donors,
as well as their industrial availability.
2.1. Off-gases from industry, heat and energy generation
CO- and CO2 -rich waste gases are an attractive substrate for gas
fermentation. Many industrial processes produce large amounts of
carbon-rich gases that are often left unused, thereby contribut-
ing to elevated concentrations of CO2 and CO in the atmosphere.
In 2011, about 23% of the total CO2 emissions were derived from
industrial processes (van der Hoeven, 2013), the second largest
sector contributing to CO2 emissions, after electricity- and heat-
generation installations. More than 40% of the CO2 emissions in
2011 were derived from generation of electricity and heat (van der
Hoeven, 2013), which worldwide relies heavily upon coal combus-
tion (Gutmann, 2014). Overall, the anthropogenic CO2 emissions
account to 38 Gt/year (Edenhofer et al., 2014).
Industrial processes produce CO2 emissions through chemical
reactions that do not involve combustion, of which the following
three sub-sectors are the main-contributors: iron and steel (27%),
non-metallic minerals (27%), and chemicals and petrochemicals
(16%) (International Energy Agency, 2007). For example, 60% of
the CO2 emissions from cement production come from inevitable
chemical reactions in the process (Cement Sustainability Initiative,
2014). Those emissions cannot be prevented by heat and energy
generation with renewables, thus alternative strategies for reduc-
ing GHG emission are required. According to the world steel
association, 1.7 × 109 t of crude steel were produced worldwide in
2014 (World Steel Association, 2015a) and 1.9 tons CO2 are emit-
ted per ton crude steel produced (World Steel Association, 2015b),
which accounts to an annual CO2 emission of 3.2 × 109 t. In con-
clusion, large amounts of carbon rich off-gases are available and
their conversion into chemicals and fuels has the potential to sig-
nificantly decrease GHG emissions (Handler et al., 2015; Ou et al.,
2013).
Off-gases from electricity and heat generation, as well as indus-
trial waste gases are supposedly a substrate that comes free of
charge. Currently, gas fermentation processes closest to commer-
cialization are based on industrial waste gases (LanzaTech, 2016).
However, not all waste gases are equally suitable for microbial gas
fermentation, since there are demands with regards to the conti-
nuity of the gas stream, the carbon content, as well as the purity of
the gas.
The CO and CO2 content of the off-gas is dependent on the sec-
tor, but is also heavily dependent on process parameters and can
therefore vary between production sites. For example, the off-gas
from power plants contains only 3–4% (gas-fired) to 13–14% (coal-
fired) CO2 , but process improvements such as chemical looping
combustion and oxyfuel-technology (O2 -fired instead of air-fired)
can increase the power-efficiency and the CO2 -content of the off-
gas (International Energy Agency, 2014). The off-gases of other
industrial processes contain high percentage of CO2 , for exam-
ple the production of ethylene oxide emitting nearly 100% CO2
(International Energy Agency, 2014). As it is the case for electricity
and heat generation, the off-gas composition can be greatly influ-
enced by the process parameters: for example, the CO2 content of
cement kiln waste gas increases from <50% CO2 (air-fired) to up to
100% when being O2 -fired (International Energy Agency, 2014).
2.2. Gasification of low-value carbonaceous materials to syngas
The availability of substrate for gas fermentation is broadened
immensely when considering the amount of feedstock that can be
converted into carbon- and energy-rich gas streams via gasifica-
tion. Gasification is defined as the “thermo-chemical conversion
of carbonaceous feedstock to gaseous products through a partial
oxidation process at elevated temperatures” (Mohammadi et al.,
2011). Besides fossil fuels, there is a broad range of more sustainable
options: lignocellulosic energy crops such as willow, switchgrass,
etc. can serve as feedstock for syngas production. The use of lig-
nocellulosic energy crops has the advantage that their prices are
“more stable as they only participate in the energy market” (Daniell
et al., 2016). Also algae is an abundant biomass that is suitable as
feedstock for gasification (Azadi et al., 2015).
Another option is lignocellulosic biomass waste, which is not
suitable for food production or starch- and sugar-based produc-
tion of chemicals and biofuels. The impact of this option is even
more significant when taking into account that the lignin-fraction
is not utilized in sugar-based production (both 1st and 2nd gen-
eration production). In wheat straw the lignin content is around
20% (Sheldon, 2014), and can be as high as 44.5% in some woody
biomass (Vassilev et al., 2012). This kind of lignin-rich feedstock,
suitable for gasification, accumulates as agricultural residues or as
forestry by-products. Crop production (for food, feed, or produc-
tion of 1st generation biofuels), generates large amounts of residue,
with a residue/crop ratio of 1:1.4 for conventional crops (Kim and
Dale, 2004). Another interesting source for lignocellulosic biofuels
include residues from 2nd generation biofuels production. In the
process of ethanol production from corn stover, for example, 5.9-
times (by mass) more lignocellulosic residues are generated than
ethanol (McAloon et al., 2000).
Municipal solid waste (MSW) or industrial waste could also
serve as feedstock for gasification. A total of 251 × 106 t of MSW was
generated in the US in 2012 after annual increases during the last
decades (U. S. Environmental Protection Agency, 2014). Especially
those parts of the complex waste that are of organic origin possess
great potential as starting material for gasification. Although their
carbon content is high, with 13% by mass (Staley and Barlaz, 2009),
they are currently left unrecycled. In particular, the biodegrada-
tion of the aforementioned waste fraction has been identified as
one of the main challenges for direct exploitation (Drzyzga et al.,
2015). Additionally, sludge from waste water treatment could act
as potential feedstock for gasification, but is of lesser relevance,
since its carbon-content is relatively low (Drzyzga et al., 2015).
The feedstock requirements for gasification technologies are
generally considered flexible (Daniell et al., 2016). However, there
are certain requirements, for example with regards to the moisture
content of the feedstock (Piccolo and Bezzo, 2009). Additionally, the
technology of large-scale gasifiers restricts the feed rate to around
2000 t per day (Griffin and Schultz, 2012). There are different gasifi-
cation technologies available, and fluidized bed gasification is most
suitable for large scale gas production, when taking throughput,
costs, complexity, and efficiency into account (Alauddin et al., 2010;
Mohammadi et al., 2011). The efficiency of conventional biomass
gasification, when comparing the lower heating value of the pro-
duced syngas with that of the gasification feedstock, is around
85% for biomass and coal (Ptasinski, 2008). Currently, advances
in biomass gasification technologies are made (Heidenreich and
Foscolo, 2015), thus more efficient gasification technologies are
likely to emerge.
The syngas composition depends on the feedstock (Tiquia-
Arashiro, 2014), but can be greatly influenced by the gasification
 S. Redl et al. / Industrial Crops and Products 106 (2017) 21–30
Table 1
Theoretical yields for the production of some compounds of interest from CO, CO2 /H2 , and CO/H2 respectively by C. ljungdahlii.
CO CO2 /H2
mol/mol CO g/g CO mol ATP/mol producta mol/mol CO2
acetate 0.250 0.527 1.13 0.500
acetone 0.125 0.259 1.25 0.333
ethanol 0.167 0.274 1.13 0.500
i-propanol 0.111 0.238 1.75 0.333
butanol 0.083 0.221 2.75 0.250
a
The estimated energy yield is displayed taking into account the minimal energy yield of the C. ljungdahlii metabolism (Diender et al., 2015; Schuchmann and Müller,
2014).
technology and gasification parameters used (Drzyzga et al., 2015;
McKendry, 2002). Syngas typically contains 40–50% N2 , 15–20%
H2 , 10–15% CO, 10–15% CO2 and 3–5% CH4 (McKendry, 2002). The
syngas composition subsequently can be shifted towards a cer-
tain composition by gas cleaning and reforming, such as (reverse)
water-gas-shift or gas drying.
In mid-2016, 95 active gasification plants are operating world-
wide (23 in the USA and 72 in the rest of the world) according
to the NETL gasification plant database of the U.S. Department of
Energy. However, only 16 plants utilize waste or biomass to date,
the rest are still petro-fueled. The produced syngas is not yet uti-
lized in gas fermentation processes, but is mainly channeled to the
chemical industry for production of fuels, fertilizer, etc. (National
Energy Technology Laboratory, 2016). The technology for biomass
gasification for syngas production exists, but economic incentives,
as well as the necessary infrastructure are required (Roddy, 2013).
Until now, only few economic analyses have been published in
the scientific literature with respect to fermentation of biomass-
derived syngas (Choi et al., 2010; Piccolo and Bezzo, 2009; Spath
and Dayton, 2003).
2.3. CO and H2 as alternative carbon and electron sources in
microbial processes
Beyond the utilization of off-gases from industry or syngas gen-
erated by gasification of certain feedstock, there are alternative
ways to supply the microorganisms with carbon and electrons. Due
to its high energy content, CO is in general preferred by gas ferment-
ing microorganisms over CO2 and H2 . Besides reverse water-gas
shift reaction, some other methods make it possible to generate
CO from CO2 . For example, light-driven processes can be used to
perform splitting of CO2 into CO (Service, 2009; Woolerton et al.,
2010).
In microbial fermentation of C5 and C6 sugars, the substrate
is reduced to CO2 . Therefore microbial processes such as pro-
duction of 1st and 2nd generation biofuels provide an additional
source of CO2 . The fermentation off-gases contain nearly 100% CO2
(International Energy Agency, 2014). For fermentation of C5/C6
sugars, the theoretical mass yield of the conversion is 0.51 g ethanol
and 0.49 g CO2 per g sugar.
As mentioned before, fixation of CO2 by microorganisms
requires an additional electron source. The concept of photosyn-
thesis, where electrons are made available by induction with light,
can also be exploited for gas fermentation as cyanobacteria are
naturally fixing CO2 through photosynthesis. Recently, Sakimoto
et al. described a method for artificially photosensitizing the ace-
togen Moorella thermoacetica. In this hybrid approach, the cells
were loaded with cadmium sulfide nanoparticles, which enabled
acetate production via photosynthesis (Sakimoto et al., 2016).
Some microorganisms can also directly accept electrons from an
electrode for the reduction of fixed CO2 , in a process called elec-
trosynthesis. The process is described to be far more efficient than
23
CO/H2
g/g CO2 mol ATP/mol producta mol/mol CO g/g CO mol ATP/mol producta
0.671 0.125 0.5 1.054 0.625
0.440 −0.750 0.25 0.518 0.250
0.523 −0.375 0.5 0.822 0.125
0.455 0.500 0.25 0.536 0.500
0.421 −0.250 0.25 0.662 0.750
photosynthesis (Lovley and Nevin, 2013). However, electrosynthe-
sis technology is still awaiting large-scale demonstration.
Hydrogen can serve as an electron donor to enable fixation
of CO2 for many microorganisms. In addition to the natural
hydrogen content of syngas, there are several processes that can
lead to the production of H2 . For instance, H2 can be produced
by hydrogenogenic microorganisms (Das and Veziroǧlu, 2001).
Another option is production of H2 by electrolysis of water, and
technologies of several companies have reached commercial levels
(Bertuccioli et al., 2014). Already about 4% of the global hydrogen
production is derived from water electrolysis (Gandía et al., 2013).
The required electricity could be generated by renewables, espe-
cially wind power and photovoltaics, to allow energy storage in
form of hydrogen during times of excess energy production.
In conclusion, there is a substantial amount of gaseous sub-
strates, electron sources, and feedstock for gasification available.
Making use of the available resources holds a promise for chem-
ical and fuel production with reduced carbon emission and full
exploitation of available resources such as biomass and renewable
energy. However, the potential of gas fermentation and its role in
the transition towards a bio-based economy can only be exploited if
the processes are also economically attractive. Hence, the gaseous
substrates have to be converted by the microbial production organ-
isms to a product of sufficiently high value.
3. Status of current gas fermentation processes
Bacteria fermenting CO2 - and CO-rich gases have a range of
natural products which are mainly of interest as biocommodities
or biofuels, and some are already on their way to being commer-
cialized. Acetate is the typical product of acetogenic bacteria and
advances have been made to improve the production of natural
acetate producers by genetic modification (Straub et al., 2014).
Butyrate and 2,3-BDO are other natural products of gas ferment-
ing bacteria that are of interest as biocommodity (Bengelsdorf et al.,
2013; Cho et al., 2014; Köpke et al., 2011). However, syngas fermen-
tation processes closer to commercialization generally aim at the
production of biofuels. Multiple acetogenic bacteria can naturally
produce ethanol (Abubackar et al., 2011) or butanol (Dürre, 2016).
Some other natural products of potential industrial interest have
been described, such as 2-oxobutyrate (Daniell et al., 2016), hex-
anol (Daniell et al., 2016), polymers and polymer building blocks
(Drzyzga et al., 2015), PHA (Brown, 2007), 3HP/4HB (Hawkins et al.,
2013), 5-aminovulinic acid, and vitamin B12 (Wiegel, 1994). Table 1
shows the theoretical production yields for a range of relevant
compounds, and significant production of certain biochemicals
have already been achieved by gas fermentation as summarized
in Table 2.
Generally, the natural products that can be produced with
higher yields have relatively low commercial value. This, combined
with relatively high costs of fermentation and gasification reactors,
makes such processes less attractive when compared to conven-
tional production based on fossil resources or on 1st generation
24 S. Redl et al. / Industrial Crops and Products 106 (2017) 21–30

Table 2
Examples of volumetric productivity, titer, and yield of some important gas fermentation target products.

Volumetric Titer Yield References Comments

productivity

Acetate 479 mmol/l/day 833 mM n.a. (Straub et al., 2014) A. woodii (ME), controlled batch
7.92 mmol/l/day 10 mM n.a. (Kim et al., 2016) Multistage process of
carboxydotrophic, hydrogenogenic
culture converting CO into H2 /CO2 ,
off-gas in second reactor converted
mainly to acetate by the acetogen T.
kivui
11.92 mmol/l/day 97.7 mM n.a. (Xu et al., 2015) Acetogenic enrichment culture grown
on H2 /CO2

Ethanol 193 mmol/l/day 450 mM 0.14 mol/mol (Richter et al., 2013) C. ljungdahlii ERI-2 Yield calculated
CO from 28% of introduced CO present in
carbon of ethanol
0.25 mmol/l/day 5.5 mM n.a. (Xu et al., 2015) Acetogen enrichment culture grown on
H2 /CO2

Butanol n.a. 25.6 mM n.a. (Köpke and Liew, 2012) C. autoethanogenum (GM)
3.5 mmol/l/day 6 mM 0.074 mol/mol (Diender et al., 2016) Co-culture of C. kluyveri and C.
CO autoethanogenum
2.7 mmol/l/daya 8 mM 0.0080 mol/mol (Ramió-Pujol et al., C. carboxydivorans P7 at 25 ◦ C
CO 2015)

Hexanol 2 mmol/l/day 4 mM 0.05 mol/mol (Diender et al., 2016) Co-culture of C. kluyveri and C.
CO autoethanogenum
2.1 mmol/l/daya 5 mM 0.0038 mol/mol (Ramió-Pujol et al., C. carboxydivorans P7 at 25 ◦ C
CO 2015)

Acetone 10.9 mmol/l/day 52 mM n.a. (Hoffmeister et al., A.woodii (GM)

2016)

Butyrate 227 mmol/l/day 220 mM n.a. (Vasudevan et al., Chain-elongating open mixed culture
2014) fed with effluent of reactor syngas
fermenting C. ljungdahlii ERI-2
8.5 mmol/l/day 11 mM 0.136 mol/mol (Diender et al., 2016) Co-culture of C. kluyveri and C.
CO autoethanogenum
6.3 mmol/l/daya 6 mM 0.0135 mol/mol (Ramió-Pujol et al., C. carboxydivorans P7 at 25 ◦ C
CO 2015)

Caproate 14.7 mmol/l/day 8.6 mM n.a. (Vasudevan et al., Chain-elongating open mixed culture
2014) fed with effluent from reactor with
syngas fermenting C. ljungdahlii ERI-2
2.5 mmol/l/day 6 mM 0.074 mol/mol (Diender et al., 2016) Co-culture of C. kluyveri and C.
CO autoethanogenum
4.6 mmol/l/daya 6 mM 0.0073 mol/mol (Ramió-Pujol et al., C. carboxydivorans P7 at 25 ◦ C
CO 2015)
a
Converted from mmol/g protein/h to volumetric production rates using protein concentrations and maximal production rates reported.

bio-production (Piccolo and Bezzo, 2009; Wei et al., 2009). There-
fore, attempts of producing higher value compounds from CO2 and
CO gases with genetically engineered strains are being pursued.
Two main approaches can be taken: engineering of an autotrophic
strain to produce a heterologous compound, or engineering of a
heterotrophic producer to utilize gaseous substrates. Initial suc-
cess to the latter approach, conferring autotrophy on heterotrophic
host organisms, has been achieved within the recent years. Engi-
neering of CO2 fixation in a heterotroph was reported for the first
time when the 3HP/4HB pathway was engineered into a hyperther-
mophilic host, Pyrococcus furiosus, for the production of n-butanol
from H2 /CO2 (Keller et al., 2013). Efforts have also been made to
engineer the WLP for CO into E. coli (Burk et al., 2009), however, only
low rates have been reported (Daniell et al., 2016). Antonovsky et al.
(2016) recently published a hybrid approach of rational metabolic
engineering and laboratory evolution. The evolved E. coli strain
employs a fully functional Calvin-Benson-Bassham cycle to fix CO2 ,
while energy and reduction equivalents are generated by another
supplied compound. The developed strain has been the first report
of a fully functional carbon fixation cycle in a heterologous host
(Antonovsky et al., 2016).
More research is focused on the engineering of gas-fermenting
microorganisms. Successful heterologous production in Clostridium
autoethanogenum, C. ljungdahlii, and Acetobacterium woodii has
been reported for acetone (Becker et al., 2009; Hoffmeister et al.,
2016), n-Butanol (Dürre, 2016; Köpke et al., 2010), butyrate (Ueki
et al., 2014), isoprene (Beck et al., 2013; Chen et al., 2013), iso-
propanol (Köpke et al., 2012), and MEK (Mueller et al., 2013). The
recently published review by Liew et al. gives an excellent overview
of the advances in genetic engineering of syngas-fermenting bac-
teria (Liew et al., 2016). Cueto-Rojas et al. gives an overview of the
potential products which can be formed from gaseous (and non-
gaseous) substrates when taking the Gibbs free energy content per
electron into account (Cueto-Rojas et al., 2015).
4. Alternative gas fermentation scenarios
Progress is being made in the field of genetic modification
of gas fermenting microorganisms. However, pathways (natural
or heterologous) have to make it possible to harvest the energy
that can be generated by product formation. The metabolism of
syngas-fermenting bacteria operates at a thermodynamic limit
(Schuchmann and Müller, 2014) and the ATP yield for a given
product is dependent on the production strain (Bengelsdorf et al.,
2013; Bertsch and Müller, 2015). The expression of heterolo-
gous pathways therefore often disturbs the energy balance of the
cell. To fully exploit the potential of gas fermentation, alternative
approaches such as multi-stage fermentation, mixed fermentation,
 S. Redl et al. / Industrial Crops and Products 106 (2017) 21–30
co-cultivation, mixotrophy, and thermophilic production strains
therefore need to be considered.
4.1. Multi-stage fermentation
As the formation of products from gas fermentation is influenced
by environmental factors and interesting products are mainly
produced under conditions non-optimal for growth, high concen-
trations of desired products can often not be obtained in a single
processing step. Therefore, applying multiple processing stages
could broaden the portfolio of products obtained from syngas fer-
mentation together with higher end-product concentrations. One
example is the combination of fermentation of easily accessible
C5- and C6-compounds of the lignocellulosic biomass with syn-
gas fermentation. The residual lignin-rich biomass, not converted
via sugar fermentation, can be gasified, making previously inac-
cessible carbon available for syngas fermenters. Additionally, as
mentioned above, CO2 off-gas from the sugar fermentation might
be introduced to the gas fermentation, reducing waste emission
and increasing the overall yield of the process. This process can be
seen as an upgraded version of the sugar platform accessing the full
range of carbon present in the initial material.
Syngas fermentation by acetogens generally results in a mix-
ture of both alcohols and acids under optimal growth conditions
(Bertsch and Müller, 2015). Lowering pH and decreasing the con-
centration of yeast extract has shown to move the spectrum
towards alcohol production (Abubackar et al., 2012). In addition,
many microorganisms cannot tolerate the presence of CO at high
concentrations, requiring partly removal of the toxic gas in order
to operate efficiently. To produce interesting amounts of one type
of product, and to do this efficiently, the fermentation process can
be performed in multiple stages.
In a study by Kim et al. (2016), acetate was produced as
an end-product in a multistage fermentation process using steel
mill off-gas as feedstock. In the first part of the process the
hydrogenogenic Thermococcus onnurineus converted a large part of
the CO fraction in the gas to H2 and CO2 . The resulting H2 /CO/CO2
mixture was subsequently fed into a reactor containing the homo-
acetogenic Thermoanaerobacter kivui, allowing for generation of
acetate as the main end-product at a maximal volumetric pro-
duction rate of 0.33 mmol/l/h. Absence of the initial biological
gas-treatment step caused T. kivui not to grow in the mentioned
study due to CO inhibition (Kim et al., 2016). Recently, T. kivui
was shown to be capable of adapting to growth on CO up to
100% headspace composition, but this took several transfers with
increasing CO pressures (Weghoff and Müller, 2016).
A multistage approach can also be used for the production of
ethanol, generating acetate in the first step, followed by solven-
togenesis in the second system (Grethlein and Jain, 1992). Richter
et al. (2013) designed a two stage system for producing ethanol
from syngas using C. ljungdahlii. The first reactor was employed
using acetogenic conditions, producing acetate as the main end-
product. The broth was subsequently fed to a second fermenter,
which employed a lower pH causing acetate to be converted to
ethanol. An ethanol production rate of 0.37 g/l/h was achieved with
this system with maximal concentrations up to 450 mM (Richter
et al., 2013). Carbon and hydrogen recovery in the ethanol end-
product were 28% and 74%, respectively. Performance of different
clostridial strains were tested in this system showing C. ljungdahlii
PETC as the most optimal producer in this case (Martin et al., 2016).
In addition to production of relatively simple products such as
acetate and ethanol, longer chain VFAs could be produced via a sim-
ilar multistage system. These VFAs are produced via the process
of chain elongation, in which shorter chain VFAs are converted to
longer chain VFAs via reverse ␤-oxidation (Angenent et al., 2016).
Strains performing this metabolism often grow optimally around
25
pH 7 and lowering the pH significantly decreases their efficiency
(Ganigué et al., 2016). Vasudevan and co-workers have shown that
effluent from a syngas reactor, containing acetate and ethanol,
could be used to feed a reactor performing chain elongation, indi-
rectly obtaining elongated acids from syngas (Vasudevan et al.,
2014). A pure culture of C. ljungdahlii strain ERI-2 was grown in
the first stage in order to ferment the syngas, while a mixed cul-
ture obtained from a chain-elongating reactor was used in the
second part of the system. The separation of these two processes
allows both systems to function at optimal conditions, obtaining
production rates for butyrate and caproate of 20 and 1.7 g/l/day,
respectively.
For the production of longer chain lipids (C16-C18) from syn-
gas, Hu and coworkers constructed a two-stage system (Hu et al.,
2016). This was done by growing M. thermoacetica in the first stage
on syngas, mainly producing acetate as the final product. The out-
going broth was fed into an aerobic reactor containing a genetically
engineered Yarrowia lipolytica, a widely used oleaginous yeast.
The system accumulated up to 18 g/l C16-C18 lipids at a rate of
0.19 g/l/h. This system nicely shows how carbon from syngas can be
fixated into more complex products by using a multi-stage system.
In theory, such a two-stage system would allow for production of
other complex products as the second reactor can contain different
genetically altered strains.
Not only biological processes can be coupled for production
of bio-based chemicals. The integration of bio-based processes
with chemical processes enables the production of chemicals
rarely obtained from biological systems. A collaboration of Lan-
zaTech with Invista and SK Innovation proposes production of
1,3-butadiene via a two-step process involving a biological and
chemical step (Green Chemicals Blog, 2014; Invista, 2015). Butadi-
ene precursors, 2,3-BDO and 1,3-BDO are generated biologically via
syngas fermentation. Subsequently these products can be thermo-
catalytically dehydrated to form butadiene. Additionally, there are
attempts to make production of butadiene a one-step process,
involving an engineered biological catalyst. Both the one- and two-
step process are still in the development stage, but these initiatives
show the possibilities for combining different processes in order to
expand the scope of syngas derived products.
4.2. Mixed cultures and defined co-cultures
The utilization of open or defined mixed cultures has some
advantages over pure culture approaches (Drzyzga et al., 2015):
(i) microbial interactions can result in the production of different
final products, (ii) some strains may detoxify the environment from
a toxic compound or reduce the concentration of a certain prod-
uct allowing for a more efficient conversion of the gas or increased
product yield, (iii) cultures are more robust and resilient to changes
in environmental conditions, (iv) less prone to contamination by
foreign bacteria or collapse due to phage infection. Trials for syngas
conversion by open mixed cultures resulted mainly in the produc-
tion of methane, hydrogen, or acetate.
In general, methanogens are poor CO utilizers (Daniels et al.,
1977; Diender et al., 2015), and grow slowly on the substrate.
In mixed cultures it was observed that methane could be pro-
duced indirectly from CO. Under thermophilic conditions, CO was
first converted to hydrogen, subsequently acting as substrate for
methanogenesis. Under mesophilic conditions it was observed that
CO was mainly converted to acetate, which subsequently acted as
substrate for methanogenesis. (Sipma et al., 2003, 2004). Grow-
ing methanogens in a mixed culture containing carboxydotrophs,
such as acetogens and hydrogenogens, does not only enable pro-
duction of intermediate substrates, but also for the removal of CO
as a toxic compound. Open mixed cultures not adapted to growth
on CO were shown to be able to convert syngas to biogas (Guiot
26 S. Redl et al. / Industrial Crops and Products 106 (2017) 21–30
et al., 2011). After enrichment and adaptation of anaerobic sludge,
a CO conversion efficiency of 75% was obtained. The overall sys-
tem was mass transfer-limited, but a specific activity of 20 mmol
CH4 /gVSS/day is expected to be achieved under thermophilic con-
ditions. Complementation of traditional anaerobic digestion with
gasification of the non-degraded fraction and subsequent fermen-
tation of syngas could result in a significant increase in biogas yield,
which for municipal waste can be up to six times higher than in
traditional anaerobic digestion (Guiot et al., 2011). However, the
additional energy investment for the gasification may reduce the
energy efficiency of the overall process.
In the work of Alves et al. (2013), a thermophilic enrich-
ment produced mainly H2 and subsequently acetate. Predominant
microorganisms in this culture included Desulfotomaculum, Ther-
mincola, and Thermoanaerobacter (Alves et al., 2013). Another study
on CO conversion to H2 by mixed cultures was performed by
Liu et al. (2016). Here methanogens were inhibited with chloro-
form in order to obtain production of mainly hydrogen (Liu et al.,
2016). CO conversion to hydrogen was 91% in a continuous reac-
tor operated with gas recycling, with an average production rate of
6.1 mmol/gVSS/day.
Acetate production from syngas by mixed cultures has also been
pursued, obtaining concentrations of up to 23 g/l at a production
rate of 0.72 g/l/day, and a maximal yield of 0.663 gacetate /gCO (Nam
et al., 2016). Also mixtures of acetate and ethanol could be obtained
by mixed cultures (Xu et al., 2015); however, their final concentra-
tions being lower compared to other approaches. In both studies,
pH was found to be of major influence on the production capacity of
the mixed cultures, being optimal at pH 7. Recently, Ganigué et al.
(2016) studied formation of higher alcohols by open mixed cultures
fed with syngas. Chain elongated products were generated in these
mixed cultures at higher pH while higher alcohols were formed at
lower pH, showing that pH is an essential parameter for produc-
tion of acids or alcohols in a mixed culture (Ganigué et al., 2016).
Higher alcohols were also produced via mixed cultures by feeding
syngas and longer chain VFAs (Liu et al., 2014). Carboxydotrophic
bacteria, such as C. ljungdahlii, are able to convert carboxylic acids
into their corresponding alcohols, using syngas as electron donor.
Propionic acid, n-butyric acid, isobutyric acid, n-valeric acid, and n-
caproic acid are converted into n-propanol, n-butanol, isobutanol,
n-pentanol, and n-hexanol (Perez et al., 2013).
Mixed cultures are more robust than pure cultures, but their
complexity and potential competition within the system often
results in a poor understanding of these systems and in a low yield
of desired products. An alternative is to use defined synthetic co-
cultures, which are less complex while still having the benefits from
microbial interactions. Defined co-cultures are currently largely
applied in food industrial fermentation processes, such as produc-
tion of dairy products and alcoholic beverages (Bader et al., 2010).
In the case of syngas fermentation, Diender et al. (2016) described
a co-culture of C. autoethanogenum and C. kluyveri, capable of pro-
ducing butyrate, caproate, and their respective alcohols from CO as
substrate. Butyrate, caproate, butanol, and hexanol were produced
at rates of 8.5, 2.5, 3.5, and 2.0 mmol/l/day, respectively. Other
configurations of defined co-cultures and bio-augmentation of
open-culture systems might stimulate production of different bio-
chemicals from syngas. Defined co-cultures have advantages over
open cultures, which have to deal with competition for substrate
and potential removal of interesting products. Such competition
was observed in a sulfate reducing system where methanogens,
sulfate reducers, and acetogens competed for utilization of H2 ,
resulting in lower efficiency of the overall system (Sipma et al.,
2006). Next to applications in food industry, patents have been
filed on the use of co-cultures in applications used for butanol pro-
duction from syngas (Datta and Reeves, 2013) and production of
biofuels using photosynthetic co-cultures (Contag, 2015). Table 2
gives an overview of achieved key production data of some relevant
compounds by gas fermentation processes.
The use of mixed and co-cultures shows that microbial inter-
actions allow for the production of different end-products. Due
to competition for substrate and complexity of mixed communi-
ties, co-cultures are in theory more efficient and easier to control.
However, it remains to be assessed whether co-cultures are robust
enough to maintain over long periods in continuous systems. An
alternative use for co-cultures would be to act as bio-augmentation
in open non-sterile systems. However, further study is required to
assess feasibility of such an approach.
4.3. Mixotrophy
Some organisms are able to use two or more carbon/electron
sources simultaneously, as observed in some phototrophs using
both CO2 and organic substrates at the same time for growth. Dur-
ing mixotrophic growth with sugars, electrons, and CO2 released
from the fermentation can be fixed via the WLP. Fast et al. (2015)
described an anaerobic, non-photosynthetic, mixotrophic fermen-
tation in which 2 CO2 and 8 electrons from glycolysis are used in
the WLP (pathway coupling), generating 3 acetyl-CoA instead of
the 2 acetyl-CoA normally generated. This causes the fermentation
to have a net CO2 exhaust of zero. Despite that the pathway from
glycolysis to acetyl-CoA yields 8 electrons (the amount required in
the WLP), an additional electron donor can be required as the gly-
colysis mainly yields low redox potential electrons (mainly in the
form of NADH, −340 mV). Only when a pyruvate ferredoxin oxi-
doreductase (PFOR), generating reduced ferredoxin, is present, all
8 electrons can be stoichiometrically used in the WLP (Table 3). In
other cases the more reduced electrons have to be derived from H2
(−414 mV) via bifurcation processes or directly from CO (−520 mV)
in order to complete the WLP (Table 3). Mixotrophic growth with
CO as additional substrate could also result in higher yields of
more reduced products. Therefore, depending on the degree of
reduction of the final end-product, the respective electron donor
is either generated or has to be supplied (Fast et al., 2015). One
of the challenges in mixotrophic cultivation is to prevent catabo-
lite repression, causing one of the substrates not to be used in
the presence of the other (Görke and Stülke, 2008). For the use of
sugars and H2 /CO2 or CO, both the WLP and the glycolysis need
to be active to have the desired effect. Toxic properties of CO
might stimulate mixotrophy as its oxidation by the microorgan-
isms can prevent inhibition, and might be necessary for preserving
other catabolic routes active. An indirect application of the con-
cept of mixotrophy is the co-utilization of lower value products
from syngas-fermenting bacteria with sugars. Chotani et al. (2012)
described for example a method to enhance isoprene production in
E. coli by co-utilization of acetate and glucose (Chotani et al., 2012).
4.4. Overview of products from alternative gas fermentation
scenarios
Fig. 1 gives an overview of the product range that can
be achieved with multistage fermentation, mixed cultures, and
defined co-cultures in comparison to the products of pure cul-
tures of natural and engineered hosts. Considering the approaches
involving non-engineered syngas fermenters, multistage fermenta-
tion is currently the most suitable option for generating a relatively
broad scope of products from gas waste streams. Especially options
combining the conversion of syngas by native organisms with the
generation of products by genetically engineered strains using the
products of the syngas fermenters are interesting. These systems
do however lack microbial interaction, which may be necessary
for the production of different products like higher alcohols. An
avenue to explore is the utilization of mixed and co-cultures for
 S. Redl et al. / Industrial Crops and Products 106 (2017) 21–30 27

Table 3
Stoichiometric conversion of different combinations of electron donors is displayed for the formation of acetyl-CoA or acetate.

Pathway Electron donor CO2 generated Acetyl-CoA formation Acetate formation

Number of molecules ATP yielda Number of molecules ATP yielda

WLP 4 CO 2 1 0.125 1 1.125

4 H2 −2 1 −0.875c 1 0.125c
2 H2 + 2 COb 0 1 −0.75c 1 0.625c
Glycolysisd C6 sugar 2 2 2 2 4
Glycolysis + WLP (in presence of PFOR) C6 sugar 0 3 1.125 3 4.125
Glycolysis + WLPe (H2 ) C6 sugar + 28 H2 −14 10 −6 10 4
Glycolysis + WLPe (CO) C6 sugar + 4 CO 2 4 0.25 4 4.25
a
For the WLP, energy yields can vary strongly per organism, depending on the type of enzymes present and final end-product. Here the estimated energy yield is displayed
taking into account the minimal energy yield of the C. ljungdahlii metabolism (Diender et al., 2015; Schuchmann and Müller, 2014).
b
Data is displayed under the assumption that CO and H2 are co-utilized.
c
A negative ATP yield is obtained for the WLP up to formation of acetyl-CoA from H2 /CO2 or a syngas mixture rich in hydrogen, this yield becomes positive when acetate
or acetate derived products (e.g. ethanol) are formed.
d
This reaction yields 8 electrons that need to be re-oxidized via other pathways (not included).
e
It is assumed that the reaction from pyruvate to acetyl-CoA is not catalysed by a PFOR enzyme and that the electrons derived from glycolysis and acetyl-CoA formation
are all in the form of NADH and are reinvested in the WLP.

Fig. 1. Product portfolio of native and engineered acetogens utilizing CO, CO2 and H2 (grey arrows). Multi-stage fermentation (orange arrows) and mixed co-cultures (green
arrows) broaden the range of potential products. TAGs: triacylglycerides. MEK: methyl ethyl ketone.

syngas conversion. An added value of mixed and co-cultures is the
potential to utilize non-CO tolerant microbes, like methanogens,
to grow on syngas by-products. Finally, mixotrophy is interesting
for increasing the yield of already existing platforms or decreasing
CO2 exhaust from these systems. However, suitable strains need to
be selected or engineered to have multiple pathways active at the
same time.
4.5. Thermophilic gas fermenting microbes
Thermophilic production strains are known to have certain
advantages over mesophiles and have attracted significant atten-
tion also for gas fermentation. The risk of contamination is reduced,
since most contaminants are mesophiles (Bosma et al., 2013), and
the oxygen solubility (although also the CO and H2 solubility) is
reduced at elevated temperatures (Tiquia-Arashiro, 2014). Fur-
thermore, the use of a thermophilic production host offers the
opportunity of low cost downstream processing via condensation
and distillation, since the product enters the gas phase and can
be condensed from the gas stream leaving the reactor. Since the
product does not accumulate in the fermentation broth, it solves
problems with product toxicity and inhibition of production path-
ways.
Several anaerobic, carboxydotrophic thermophiles have been
characterized to date (Henstra et al., 2007; Tiquia-Arashiro, 2014).
The product spectrum of those thermophilic carboxydotrophs com-
prises acetate, formate, H2 S, H2 , and CH4 (Henstra et al., 2007;
Tiquia-Arashiro, 2014). The first thermophilic acetogen to naturally
produce ethanol was the strain Moorella sp. HUC22-1, published in
2004 (Sakai et al., 2005, 2004). Efforts are being made to develop the
genetic toolbox of acetogenic thermophiles in order to broaden the
range of products. Although production of industrially relevant het-
erologous products have not been reported so far, most progress is
made with M. thermoacetica (Iwasaki et al., 2013; Kita et al., 2013a,
2013b). Recently, we analyzed a hypothetical process in which ace-
tone is produced in a bubble column fermentation at 60 ◦ C from
corn stover-derived syngas with a M. thermoacetica strain with
regard to thermodynamic and economic aspects (manuscript in
preparation). We found that the process has the potential to be
economically interesting, if the high costs for biomass gasification
are reduced.
Fig. 2 shows compounds with relatively low boiling point, which
may be interesting product candidates for thermophilic production
strains. In M. thermoacetica, the generation of 1 acetyl-CoA requires
0.5 ATP (Eq. (1)); net ATP is only generated in the subsequent step
of acetate formation. Acetyl-CoA formation from CO leads to a net
28 S. Redl et al. / Industrial Crops and Products 106 (2017) 21–30
Fig. 2. Compounds with relatively low boiling point are interesting products for gas fermentation processes using thermophiles. The temperature range and optimum of the
thermophilic acetogen M. thermoacetica (Drake and Daniel, 2004) is highlighted in orange.
Fig. 3. The ATP yield and the mol of CO2 consumed/produced based on the energy generation mechanism in M. thermoacetica proposed by Schuchmann and Müller, 2014.
MEK: methyl ethyl ketone.
ATP balance of +0.5 ATP (Eq. (2)).
Acetyl-CoA from H2 /CO2 : 4 H2 + 2CO2 − > 1acetyl-CoA
+ 2 H2 O − 0.5ATP
Acetyl-CoA from CO : 4CO + 2 H2 O − > 1acetyl-CoA
+ 0.5ATP + 2CO2
For the production of 1 mol acetone, 2 acetyl-CoA are required.
Fig. 3 shows the ATP yield per 1 mol of product. The ATP yield calcu-
lations are based on the energy conservation mechanism proposed
by Schuchmann and Müller, 2014. For the production of ethanol, 1
acetyl-CoA is reduced to acetaldehyde and further to ethanol, with
NADH as electron donor in each step. The 2 NAD+ can generate an
additional 0.5 ATP. Acetone can be further reduced with NADH to
isopropanol, which generates an additional 0.25 ATP. The produc-
tion of isoprene via the mevalonate pathway requires 3 acetyl-CoA,
2 NADH, and 3 ATP. The energy requirement for isoprene produc-
tion is relatively high, and 4 ATP are required per mol product when
H2 /CO2 serves as substrate. For the production of methyl ethyl
ketone (MEK), 2 acetyl-CoA are converted via pyruvate into ace-
tolactate, which is decarboxylated into acetoin. Acetoin is further
reduced with NADH into (R,S)-2,3-BDO, which undergoes conver-
sion to MEK (Mueller et al., 2013). In comparison, the production
of the native product of M. thermoacetica yields 1.5 ATP for the
growth on CO, and 0.5 ATP for the growth on H2 /CO2 . Assuming that
0.5 ATP is the lower limit to maintain and gain the biomass, only
the production of the more reduced compounds acetone, ethanol,
isopropanol, and MEK from CO generates enough energy. For the
other production scenarios, by-product formation would be neces-
sary.
5. Conclusion
Vast amounts of industrial waste gases are currently emit-
(1) ted to the atmosphere. Together with gasification of other waste
streams and underutilized fractions of biomass, these gases have
the potential to serve as substrates for gas fermentation processes
for the production of biochemicals. Such processes are attract-
ing increasing attention since they may help to significantly curb
the emission of greenhouse gases, while at the same time having
(2)
the potential of decreasing the production cost of certain chem-
icals. A significant amount of research is currently focused on
developing tools for metabolic engineering of novel acetogenic
production organisms, and novel products are constantly being
pursued. Heterologous production of higher value compounds has
been successful for some strains, but the reactions are often chal-
lenging due to the redox balance, and mainly ethanol and BDO
production is reaching commercial levels. Currently, there are only
few examples of biomass-based gasification plants operating at a
commercial scale, and none of these are reported to utilize syn-
gas for gas fermentation processes. To investigate the economic
aspect of these processes, it is therefore important to also conduct
techno-economic studies. Alternative fermentation processes have
the potential to address many of the challenges associated with pro-
duction of biochemicals through gas fermentation. In this review
we have focused on the recent development of processes utiliz-
ing for example multi-stage fermentation, mixed communities,
defined co-cultures, mixotrophic processes, as well as thermophilic
production organisms. Such production scenarios have the poten-
tial for significantly broadening the product portfolio, increasing
production titers and yields, exploiting different and also mixed
feedstock streams, removing inhibitors and decreasing the cost of
downstream processing. The future will undoubtedly show further
development of industrial processes from this rapidly emerging
field of research.
 S. Redl et al. / Industrial Crops and Products 106 (2017) 21–30
Acknowledgements
This work was supported by The Novo Nordisk Foundation and
a Ph.D. grant from the People Programme (Marie Curie Actions)
of the European Union Seventh Framework Programme FP7-
People-2012-ITN, under grant agreement No. 317058, “BACTORY”.
Research of M. Diender and D.Z. Sousa is supported by a ERC grant
(project 323009) of the European Union Seventh Framework Pro-
gramme FP7 and a Gravitation grant (project 024.002.002) of the
Netherlands Ministry of Education, Culture and Science and the
Netherlands Science Foundation(NWO).
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