Available online at www.sciencedirect.
com
ScienceDirect
Pollution to products: recycling of ‘above ground’
carbon by gas fermentation
Michael Köpke and Séan D Simpson
Climate crisis and rapid population growth are posing some of
the most urgent challenges to mankind and have intensified the
need for the deployment of carbon recycling and carbon
capture and utilization (CCU) technologies. Gas fermentation
offers a solution using carbon-fixing chemolithoautotrophic
microorganisms. After a decade of scale up, the technology
has recently been commercialized with the first plant operating
successfully since 2018 and additional units under
construction. Gas fermentation offers unique feedstock and
product flexibility when compared to other available gas-to-
liquid technologies. Advancements in process technology and
synthetic biology enable a broad range of feedstocks including
emissions from industry or syngas generated from any biomass
resource to be converted into a wide range of molecules
realizing a circular economy.
Address
LanzaTech Inc., 8045 Lamon Ave, 60077 Skokie, IL, USA
Corresponding author:
Köpke, Michael (michael.koepke@lanzatech.com)
Current Opinion in Biotechnology 2020, 65:180–189
This review comes from a themed issue on Chemical Biotechnology
Edited by Christoph Wittmann and Sang Yup Lee
For a complete overview see the Issue and the Editorial
Available online 18th April 2020
https://doi.org/10.1016/j.copbio.2020.02.017
0958-1669/ã 2020 Elsevier Ltd. All rights reserved.
Introduction
The accelerating rate of extraction and combustion of
fossil resources for fuel, energy, and chemicals over the
past 100 years has resulted in carbon dioxide (CO2)
accumulation in the atmosphere to levels unprecedented
since the Pliocene Epoch (5.3–2.6 million years ago) [1].
Although the effect that elevated atmospheric CO2 will
have on the Earth’s climate has been predicted by scien-
tists for several decades, it was only in 2016 through the
Paris Agreement that nations formally laid plans to abate
atmospheric CO2 release. In each case, these plans neces-
sitate that ‘above ground’ carbon resources increasingly
displace fossil resources as feedstocks for fuel and chemi-
cal production. Numerous technology solutions have
been proposed to enable this feedstock transition
Current Opinion in Biotechnology 2020, 65:180–189
including the fermentation of farmed sugars the produc-
tion of ethanol or other bioproducts, refining of plant oils
for diesel, aviation fuel and LPG displacements, and the
recovery of methane from the anaerobic digestion of
municipal and agricultural waste to displace natural
gas. Gas fermentation is the most recent of these. This
technology uniquely offers a path to produce impactful
volumes of sustainable fuels, chemicals and food from
abundant, low value above ground carbon feedstocks
throughout the world.
In gas fermentation, a carbon-containing gaseous sub-
strate is fermented by chemolithoautotrophic microor-
ganisms (Figure 1a) [2,3]. Specialized metabolism
enables different kinds of autotrophic microorganisms
to utilize various gases. Several pathways evolved for
utilization of carbon oxides (carbon monoxide (CO),
CO2) [4,5]. About 60 years ago, Melvin Calvin elucidated
the first autotrophic CO2 fixation pathway relying on
Ribulose-1,5-bisphosphate carboxylase/oxygenase
(RuBisCo) as key enzyme found in many aerobic organ-
isms [6]. Considered to be the oldest [7] and most effi-
cient CO/CO2 fixation pathway [4,5] is the reductive
acetyl-CoA pathway found in anaerobic acetogens [2,3].
In this linear pathway, two one-carbon units are compen-
sated to form two-carbon building block acetyl-CoA. The
pathway is also known as Wood-Ljungdahl pathway in
honor of Harland G. Wood and Lars G. Ljugdahl who
elucidated the underlying biochemistry which is
described in several excellent reviews [8,9]. A variation
of the Wood-Ljungdahl pathway is also found in metha-
nogenic Archaea capable of converting CO2 to methane
(CH4) [10]. CH4 itself can be metabolized by methano-
trophs via ribulose monophosphate pathway (RuMP) or
serine pathways [11,12].
CO and CH4 can each serve as both a carbon and energy
source in the gas fermentation process, whereas CO2
requires energy to be activated. Additional energy can
for example be supplied by hydrogen (H2) present in
many feed gases or can be supplied separately on
demand. The supply of additional H2 to a process gas
stream, to increase the energy content of the inlet gas and
carbon efficiency of the overall process, is becoming
increasingly feasible via water electrolysis as the avail-
ability of renewable power increases and costs decline.
Being able to perform an internal biological Water-Gas
Shift (WGS) reaction catalyzed by carbon monoxide
dehydrogenase [7–9], gives acetogens the ability to
directly utilize a wide range of different gas mixtures
www.sciencedirect.com
 Engineered microbes in gas fermentation Köpke and Simpson 181

Figure 1

(a) Inputs Processing Gas Fermentation Outputs

Waste Gas
Industrial

Steel, Ethanol,

Fuels
Ferroalloy, Diesel
Refinery, Jet,
etc etc
Gasification
Autotrophic Water

Chemicals Nutrition
Municipal Microbe Recycle Commodities
Solid
Syngas

Fine chemicals
Waste, Materials,
Biomass etc
Residues
Gasification Clean Up
Protein,
Electrolysis Feed (Livestock,
Processing Plants, Aquaculture),
CO2

Corn Ethanol, etc

Direct Air Capture CO2
etc
Electrolysis Compression Fermentation Recovery Product Tanks

(c) (b) (d)

2.0
100%

80%

Selectivity (%)
H2:CO Ratio

60%
1.5
40%

20% Ethanol Acetate

2,3-Butanediol Biomass

1.0 0%
0 5 10 15 0 5 10 15

Time (days) Time (days)

Current Opinion in Biotechnology

Gas Fermentation Process Overview: (a) Overview of the process including input and outputs. (b) World-first commercial-scale gas fermentation
plant (Beijing Shougang LanzaTech New Energy Science & Technology Co., Ltd.). (c) Example feed gas profile from gasification of unsorted, non-
recyclable MSW (Sekisui, LanzaTech) and (d) corresponding fermentation output (Sekisui, LanzaTech).

while keeping a relatively constant energy efficiency of production with methanotrophs was scaled up by Du Pont
around 80% (Table 1). As such, a broad range of different and Norferm (10k MTA) in the late 1990’s, but subse-
gas streams including waste gases from various industrial quently shut down due to poor economics after a rela-
sources or synthesis gas (syngas), a mixture of CO, CO2, tively short period of operation. Over the past decade,
and H2 that can be generated from any biomass resource several of the open questions regarding the genetics and
through gasification can serve as input for the gas fermen- accessible product spectrum of acetogens as well as at-
tation process (Figure 1) [2,3]. scale operability have been answered through intensive
pilot and demonstration-plant operations. A first commer-
Against the odds, gas fermentation has now reached cial scale gas fermentation plant producing ethanol (46k
commercial scale. Acetogenesis was first reported in MTA) was started up in May 2018 and is successfully
1932, but for a long-time acetate was thought to be the operating since that time (Figure 1b).
sole product made by this class of organisms (hence the
name) due to perceived energetic constraints, limiting Coming of age of industrial biotechnology
their biotechnological potential. At the beginning of the Syngas has long been the feedstock for industrial chemi-
1990s, the first acetogens making other products such as cal synthesis. Using technologies such as the Fischer-
ethanol were isolated, but until a decade ago acetogens Tropsch (FT) process, supported-metal catalysts have
were still considered genetically inaccessible and con- been used since the early part of the 20th century to
cerns around energetics, mass transfer, and scale up convert syngas to a mixture hydrocarbon products
continued to be perceived as major barriers to the use [13
,14]. Commercial FT processes have predominantly
of gas fermentation as a commercial production system been based on the use of fossil resource such as coal or
[2]. In parallel, a process for single cell protein (SCP) natural gas to produce syngas. The growing demand for

www.sciencedirect.com Current Opinion in Biotechnology 2020, 65:180–189

182 Chemical biotechnology

Table 1

Feedstock flexibility of gas fermentation. Carbon and energy efficiencies for ethanol production from different feedstocks. Carbon
efficiency is a function of the reaction stoichiometry. The energy efficiency is calculated by ‘lower heating value of products’ (ethanol:
1236.5 kJ/mol) divided by ‘lower heating value of reactants’ (CO 283.0 kJ/mol; H2: 241.8 kJ/mol) — what fraction of the usable chemical
energy is converted into usable chemical energy. This can be done with the higher heating value as well. Taking one example, the
efficiency of ethanol from CO only on an LHV basis = 1236.5/(6  283.0) = 72.8%

Source Composition H2:CO Stoichiometry DG rxn Carbon Energy

ratio (kJ/ Efficiency Efficiency
rxn_mol)
Steel, Ferroalloy CO 0:1 6 CO + 3 H2O ! Ethanol + 4 CO2 216 33.3% 72.8%
Syngas (Biomass, 1:1 3 CO + 3 H2 ! Ethanol + CO2 156 66.7% 78.5%
MSW) CO + H2 2:1 2 CO + 4 H2 ! Ethanol + H2O 135 100% 80.6%
Refinery CO 5:1 1 CO + 1 CO2 + 5 H2 ! Ethanol + 2 H2O 115 100% 82.9%
+ H2 + CO2

carbon emission reduction and achieving a circular economy
has driven an interest in the use of sustainable resources such
as unsorted and non-recyclable municipal solid waste (MSW),
agricultural waste, or organic industrial waste. These feed-
stocks bring unique challenges to any conversion system; for
example they are not only inherently compositionally variable
(Figure 1c), but are also geographically highly distributed,
with lower delivered volumes available to each processing
facility [15]; moreover, syngas produced from these resources
are typically associated with elevated levels of contaminants.
The emergence of commercial gas fermentation systems
provides processors with a new option for syngas conversion
to products. To a large extent gas fermentation has more in
common with thermocatlaytic processes like FT than other
biological fermentation processes in that it operates in a truly
continuous manner (Figure 1d) and accepts simple one-
carbon inputs to produce more complex hydrocarbons. How-
ever, key features of gas fermenting microbes overcome
significant technical and economic challenges that face FT
catalysts [13
,14,15], particularly when combined with syngas
produced from these distributed, sustainable feedstocks. The
economic implication of key process requirements and the
parameters of FT catalysis and gas fermentation are compared
in Table 2.
Biological processes typically function in relatively low
pressure, low temperature environments. Gas fermentation
processes are able to maintain high product selectivity
despite variable inlet gas compositions and demonstrate
elevated tolerance to gas contaminants compared to tradi-
tional supported-metal catalysts [3,13
,14,16]. In combina-
tion these features enable reduced commercial process
capital (CAPEX) and operating (OPEX) expenditure
[13
], allowing economic operation at smaller scales, for
example, lower levels of delivered feedstock [15]. Elevated
process tolerance to gas-borne contaminants is a function of
the use of a self-replicating (microbial) catalyst that has a
finite residence time in the reaction vessel of a continuous
process. Thus, certain contaminants can be tolerated as
they simply bind to and deactivate (kill) individual
microbes, which are subsequently washed out of the reactor
as a function of the process dilution rate. While the micro-
bial ‘death rate’ remains below the growth rate the contam-
inant will not accumulate in the reactor and its presence can
be tolerated. This contrasts with a classical chemical ther-
mocatalytic process where contaminants accumulate on the
catalyst bed, deactivating the catalyst and reducing the
output of the reactor as a function of time and concentra-
tion. To prevent this, the concentration of crucial contami-
nants must be reduced to homeopathically low levels,
adding cost and complexity to the final process.
The use of microbial catalysts also offers an opportunity
to operate product flexible facilities, an exciting strategy
that represents a paradigm shift compared to the tradi-
tional chemical industry model, where plants are built to
produce a single product or a fixed product suite. Syn-
thetic biology enables a single, proven industrial chassis
to be the basis of different strains each engineered to
produce a different end-product from the same gas com-
position. In this way different products may be manu-
factured in campaigns by a single plant, enabling eco-
nomically optimal performance to be achieved as market
conditions vary. In such a scenario, the bulk of the plant
(gas production, gas clean-up, gas fermentation, water
treatment; Figure 1a) remains unaltered and fit for pur-
pose; only the downstream processing (separations) may
need to be tailored to the specific product target.
The manufacture of an array of products has been demon-
strated by gas fermentation systems with applications as
fuels, chemical intermediates, materials as well as in nutri-
tion (Figure 1a). The primary product of LanzaTech’s first
gas fermentation facility in China is ethanol, generally used
as a blending component in gasoline, but also an excellent
building block that can be upgraded to jet fuel (alcohol-to-
jet) or plastic/nylon precursors (e.g. polyethylene, butadi-
ene) through thermocatalytic dehydration and oligomeri-
zation [3]. Life-cycle assessments of the sustainable avia-
tion fuel produced from ethanol by gas fermentation shows
up to 90% greenhouse gas (GHG) emission reductions [17
]
and in October 2018, this fuel was used in a commercial

Current Opinion in Biotechnology 2020, 65:180–189 www.sciencedirect.com

 Engineered microbes in gas fermentation Köpke and Simpson 183

Table 2

Economic implication of key process requirements and parameters of FT catalysis and gas fermentation

Process Fischer-Tropsch catalysis Gas fermentation

Parameter
Gas composition Fixed: Product selectivity is directly linked to
(CO:H2 ratio) composition of delivered gas. Variable composition
syngas streams (Figure 1c) must be rectified before
gas delivery to the catalyst. Producing a fixed gas ratio
adds additional process units to the flow scheme,
increasing the CAPEX and OPEX of the final process.
Operating Generally High: Operates at pressures between 20–
pressure 300 bar [13
]. Operating at elevated pressure adds
CAPEX and OPEX to the final process.
Operating High: Operates in excess of 200 C [13
]. Operating at
temperature elevated temperature and hydrogen pressures
requires more exotic metallurgy adding significant
CAPEX and OPEX to the final process.
Process tolerance Low: Requires extensive gas clean-up to deliver high
to contaminants purity gases. Gas contaminants are catalyst poisons
that accumulate and compromise the performance of
the catalyst until regenerated or replaced. Stringent
gas clean-up facilities add CAPEX and OPEX to the
final process.
Single product Low: FT synthesis produces a distribution of products.
selectivity Not all products are high-value or even desired.
Requires that desired products must be fractionated
from a mixture.
Product High: Obtaining a desired product at high
concentration concentration requires separation and reprocessing.
Products produced in a concentrated stream.
Flexible: Product selectivity is largely independent of
feed gas ratio. Presence of an internal biological WGS
capacity within the microbial biochemistry enables the
bio-catalyst to maintain product selectivity while
consuming all available reducing equivalents
(Figure 1d). This internal WGS enables a broad array of
feed gas ratios to be used to produce the same target
molecule, eliminating the need for upstream process
units to adjust gas ratios.
Low: Operates at pressures <10 bar. Normally only a
single compressor is required.
Low: Operates at temperatures <40 C
At these lower temperatures and pressures the
material of construction can be low grade stainless.
High: Gas contaminants merely inhibit the microbe
performance. Once the inhibitor is removed, the
culture can recover. The most common microbe
inhibitors are specific molecules rather than elements.
These molecules do not necessarily need to be
removed from the gas. Inhibitory molecules can be
easily converted to non-inhibitory molecules [14].
Typical sulfur-containing gas species (H2S, COS, and
CS2) are non-inhibitory, and do not need to be
removed, often acting as a nutrient to the biocatalysts.
Other gases such as N2 are completely inert in the
process.
High: High selectivity to the target molecule
Low: Products produced in a dilute (aqueous) stream

transatlantic flight. Moreover, the microbial biomass is
nutritionally valuable, and can serve as an ingredient in
livestock and aquaculture feed formulations. Acetogen
Clostridium autoethanogenum has been shown to have a
protein composition similar to that of fishmeal [18] and
SCP from methanotroph Methylococcus capsulatus is gener-
ally recognized as safe (GRAS) [11]. However, perhaps the
biggest disruption is yet to come with advances in synthetic
biology and metabolic engineering of these microbes.
Already, reprogramming of gas fermenting organisms has
yielded production of over 50 molecules of different chain
length and chemistries (Figure 2a) [3]. Several of these
products including acetone [19
,20], isopropanol, 2,3-buta-
nediol, 3-hydroxybutyrate [21

] have already been opti-
mized for high titer, rates and selectivities and scaled up to
pilot scale (unpublished). This also included optimization
of the chassis, for example properties such as end product
tolerance or vitamin prototrophy [22
]. Through the com-
bination of feedstock and product flexibility, gas fermen-
tation enables a circular economy (Figure 2b).
Advancements in synthetic biology of gas
fermenting organisms
Since the first proof of concept for genetic engineering of
an acetogen in 2010, significant progress has been made in
both breadth and efficiency of available genetic tools as
well as the understanding of the acetogenic metabolism
and molecular mechanisms.
Today, a comprehensive set of genetic tools and parts
exist for model acetogens C. autoethanogenum and Clostrid-
ium ljungdahlii and progress has also been made in other
acetogens such as Acetobacterium woodii, Eubacterium limo-
sum or Moorella thermoacetica. This development has been
accelerated by leveraging tools developed for sugar-fer-
menting organisms and new enabling technologies such
as CRISPR-based engineering. For example, various
types of CRISPR systems (Cas9, dCas9, Cas12a, dCas12a)
have been successfully adapted to a range of acetogens for
integration, knock-out and knock-down of genes [23–27].
However, not all tools can be directly transferred to

www.sciencedirect.com Current Opinion in Biotechnology 2020, 65:180–189

184 Chemical biotechnology

Figure 2

Acids Alcohols Diols

(a) Aromatics Dienes Esters Ketones Terpenes
Carbocylic Dicarboxylic Hydroxy Dihydroxy Amino Linear Branched 1,2- 1,3- 2,3- 1,4-
C2

Acetic Ethanol MEG

[US20190185888]
[54]
C3

Lactic 3-HP Alanine n-propanol iso-propanol 1,2-PDO Acetone

[3,11] [3] [49] [US9284564B2] [3,58] [US9284564B2]
[19,20]
2-butanol MEK
[3] [3]
C4

Butyric Succinic 3-HP n-butanol iso-butanol 1,3-BDO 2,3-BDO 1,4-BDO Butylene Acetoin
[32] [3] [21,25] [21,53,57,58] [US20170335351] [21] [3,12,58] [11] [US20170283809] [3,58]
Valine
[US9297026]
C5

Isoprene
Methionine
[US20190194630] [3,12]

n-hexanol Heptadecene FAME

Caproic PHB Mevalonic Leucine [53,57] p-HBA Salicylate [63]
[US9297026] [11]
[56] [11,38,41] [3] [US10174303] Farnesene
[3,11]
C6+

Heptadecane FAEE
n-octanol
[63] [3,11]
[57] 2-amino/3,4-HBA
Caprylic Isoleucine
PHA,s [US10174303]
[56] [US9297026] Bisabolene
[61]
FABE [62]
Pentadecane [3,11]
fatty alcohols [63]
Ectoine
[59]
[11,12]

Proof of Concept Lab-scale Scale Up/Commercial

(mg/L-g/L, <5% selectivity) (>g/L &>g/L/D, >5% selectivity) (>g/L & g/L/h, >90% selectivity)

MSW Industrial Biomass CO2

(b) (c)

Re-use Re-generate Capture & Use

Gas Fermentation

Re-cycle Re-place Re-place

Chemicals Fuels

Current Opinion in Biotechnology

Strain Engineering Overview: (a) Overview of breadth of molecules produced directly from gases through gas fermentation and commercialization
status. Either references or US patent (application) numbers are provided in brackets. Abbreviations: BDO, butanediol; FAME, fatty acid methyl
ester; FAEE, fatty acid ethyl ester; FAME, fatty acid butyl ester; HB, hydroxybutyrate; HBA, hydroxybenzoate; PDO, propanediol; HP,
hydroxypropionate; PHA, polyhydroxyalkanoate; PHB, polyhydroxybutyrate. (b) Gas fermentation enables circular economy. (c)
clostridiaBiofoundry (cBioFab) for fully automated, high throughput strain engineering in context of anaerobic conditions and flammable/toxic
gases (LanzaTech).

acetogens. Key challenges are lower DNA transformation such as promoters, terminators, markers and reporters.
and recombination efficiencies compared to model organ- Over the past few years, several synthetic promoter
isms Escherichia coli or yeast, that limit certain applications libraries [23,30,31], codon usage and RBS algorithms
and necessitate an intermediate host for cloning and [32–34] have been developed that allow fine tuning of
library constructions. While incremental improvements expression with activity spanning several log units. This
in efficiency have been achieved [26] and optimized includes constitutive and inducible promoters, though a
intermediate host strains been developed [28], this often system for extreme protein overproduction for example is
adds complexity and requires alternative strategies [29] still lacking for example. With the recent report of a
unless efficiencies can be further improved. Another highly fluorescent real-time reporter system [35
] that
challenge has been the lack of validated genetic parts unlike GFP works under anaerobic conditions,

Current Opinion in Biotechnology 2020, 65:180–189 www.sciencedirect.com

 Engineered microbes in gas fermentation Köpke and Simpson 185
applications like real-time monitoring and live cell-sort-
ing now become possible in acetogens. Several excellent
reviews discussing available tools for clostridia in more
detail have recently been published [3,36,37].
Despite the advent of more sophisticated genetic tools,
most published examples of acetogenic strain engineering
remain relatively low-throughput. Performing strain selec-
tion and screening in the context of flammable, toxic and/or
explosive gases is challenging. There are no off-the-shelf
solutions, and the only system described in the literature
comprises a gastight growth chamber for well plates with
constant gas flow for aerobic methanotrophs [38]. While this
system relies on some off-the shelf components, it also
requires significant customization. Particular consideration
needs to be given to the implementation of robust safety
systems in any implementation. Further complexity is
added under anaerobic conditions but as the clostridia
Foundry for Biosystems Design (cBioFab) (Figure 2c)
established at LanzaTech shows, this is not a barrier to
the use of biofoundry-type automation. The cBioFab can
perform fully automated strain engineering and screening
of thousands of strains per cycle in anaerobic conditions
with toxic and flammable gases. For the next scale testing, a
low-cost, open-source multiple-bioreactor system was
recently described [39].
An innovative way to inform strain design in absence of
high-throughput capabilities is the use of cell-free sys-
tems for pathway prototyping and downselection. Cell-
free systems allow testing of hundreds to thousands of
different designs within days and an initial study has
shown a good correlation between observed in vitro
performance and in vivo production in gas grown C.
autoethanogenum for two example pathways [21

]. Path-
way optimization in this study was guided by a machine
learning algorithm. Enabled by the growing number of
multi-omics datasets from steady-state continuous fer-
mentations [40–42], a range of sophisticated genome-
scale models for acetogens have also been established
over the past years [41,43], including one of the first
models that includes macromolecular expression (ME-
model) [44

] as well as a kinetic ensemble model [45
]
and a spatiotemporal model [46]. These models have
been found to be effective in guiding strain engineering,
in particular as autotrophic metabolism is not always
intuitive, as it is fundamentally different from heterotro-
phic metabolism and employs redox as a main driving
force. One example includes ethanol production, for
which, in contrast to sugar fermenting organism, a
knock-out rather than overexpression of bifunctional
aldehyde/alcohol dehydrogenase (AdhE) has been found
to increase ethanol formation due to the presence of an
alternative reactions [47]. Other important findings from
multi-omics analysis and modelling includes the insight
that carbon and energy metabolism in acetogens are
highly regulated at the translational level [33] and the
www.sciencedirect.com
discovery of a new promoter motif/transcription factor
associated with crucial genes for autotrophy [48
]. It was
also found that alternative nitrogen sources can provide a
growth boost [39,49,50], which translated into improved
engineered strains [51]. One input to further inform
available models in acetogens would be data on the
amount of dissolved CO in the fermentation broth, which
so far can only be measured indirectly in absence of in-
line probes, although a new measuring platform was
recently reported [52].
Opportunities in process integration
Gas fermentation and thermocatalytic processes each
bring unique, and in many instances, complementary
advantages to fuel and chemical production from sustain-
able inputs at scale. Hybrid processes, comprising gas
fermentation with a thermocatalytic unit directly
upstream and/or downstream leverage the complemen-
tary benefits of each approach and offer opportunities to
access new renewable resources or produce new sustain-
able products. There is growing interest in leveraging
increasingly abundant, low cost, sustainable electricity
produced, for example, using photovoltaic panels or wind
turbines, as an energy resource to produce fuels and
chemicals. Approaches to achieve this are often referred
to as ‘Power-to-X’ technologies and combine electrolytic
processes supplied by renewable electricity that produce
reduced gases from either water or CO2 as a feedstock for
downstream conversion units [4]. Gas fermentation facil-
ities with a feed gas supplied entirely or in part by
electrolysis (Figure 1a) have been demonstrated to have
high faradaic efficiencies [53

] and could achieve very
high carbon efficiencies (proportion of inlet carbon cap-
tured in the fermentation products), as either the inlet gas
composition can be tailored to achieve stoichiometric
carbon fixation (Table 1) or the tail gas from the fermen-
tation process may be recycled. Gas fermentation may
offer an advantage in such process configurations, partic-
ularly where there is inconsistency in the supply of
sustainable power to the electrolysis unit causing fluctua-
tions in the gas composition delivered to the reactor. As
described above, gas fermentation processes can operate
in such circumstances with little change in the selectivity
to the target product (Table 1).
Water electrolysis to produce H2 is a relatively mature
technology and is deployed at scale in various locations
globally, while electrolytic processes to produce CO from
CO2 are still in development [53

]. For gas fermentation,
CO is energetically the preferred substrate to enable
synthesis of reduced products; acetic acid is the predomi-
nant product if only CO2 and H2 are available [3]. Acetic
acid itself can however serve as a substrate for other
organisms and continuous acetic acid production in excess
of 6 g/l/h has been demonstrated [54]. Through either co-
cultures or two-stage processes, production of, for exam-
ple, protein [55], C3–C8 acids and alcohols [3,53

,56–58]
Current Opinion in Biotechnology 2020, 65:180–189
186 Chemical biotechnology
or lipids [3,59] have been demonstrated. Co-cultures
typically combine an acetogen with one or more other
(facultative) anaerobic organisms capable of chain elon-
gation and even direct cell-to-cell exchange have been
observed [58]. In a two-stage process [55,56,59], the
fermentation broth from the gas fermentation stage is
continuously supplied to a second process that may be
aerobic, thus combining the high product selectivity
advantages of anaerobic systems with the product diver-
sity options of aerobic systems like E. coli or yeast [59],
while enabling the use of CO2 as the sole carbon source
for product synthesis. Several gas fermenting organisms
also have been shown to be capable of direct electron
transfer (electrosynthesis) [60].
Opportunities in new platforms
While commercialization of anaerobic acetogens has pro-
gressed the furthest and their form of metabolism (Wood-
Ljungdahl pathway) is considered to be the most efficient
CO2 fixation pathway [4,5], there are also other promising
platforms that offer certain advantages. Aerobic gas fer-
menting organisms such as methanotrophs (e.g. Methylo-
microbium, Methylotuvimicrobium) capable of CH4 utiliza-
tion [11,12], hydrogenotrophic ‘Knallgas’ bacteria (e.g.
Cupriavidus, formerly Ralstonia) capable of utilization of
CO2 plus H2 [60,61
] or aerobic carboxydotrophs (e.g.
Hydrogenophaga) capable of CO utilization [62], for exam-
ple, offer higher biomass concentrations and higher ATP
availability compared to anaerobes and no limitation for
oxygen-dependent reactions [63]. Some of these organ-
isms naturally produce valuable products such as PHAs
and with the emergence of genetic tools for these plat-
forms, polymer composition can be tailored and produc-
tion of several other molecules has been demonstrated
[11,60,61
,62]. Methanotrophs [11] and anaerobic metha-
nogens [64] have already been deployed at larger scale.
For the aerobic platforms, the explosive nature of oxygen
plus H2 or CH4 gas mixtures adds an additional process
engineering challenge and methanotroph fermentations
are also significantly exothermic [11], requiring careful
heat management.
Recently, autotrophy has also been successfully engi-
neered into model organisms E. coli and yeast by intro-
duction of parts of the Calvin-Benson-Bassham (CBB)
cycle and laboratory evolution [65

,66]. While this proof
of concept is an extremely exciting achievement and
offers great promise, advancing such a system to commer-
cially relevant rates will require significantly more time. A
key challenge is the low turnover numbers (1–10/s) and
high error rates (>20%) of RubisCO [6], which is also the
key enzyme in ‘Knallgas’ bacteria and aerobic carboxy-
dotrophs [60,62]. The alternative of expressing the Wood-
Ljungdahl pathway of acetogens or the methane mono-
oxygenase (MMO) of methanotrophs in E. coli and yeast
proved to be challenging, though functional expression of
the key enzyme of the Wood-Ljungdahl pathway has
Current Opinion in Biotechnology 2020, 65:180–189
been achieved in a solventogenic clostridia for example
[67]. As a work around, new synthetic pathways have been
designed [68]. Using a retrobioisynthesis approach, sev-
eral options for synthetic CO2 fixing pathways have been
predicted from the approximately 5000 metabolic
enzymes known in Nature. Of these, a malonyl-CoA-
oxaloacetate-glyoxylate (MOG) pathway showed most
promise due to the unique use of PEP carboxylase as
only carboxylating enzyme [69]. The subsequently devel-
oped crotonyl–CoA/ethylmalonyl-CoA/hydroxybutyryl-
CoA (CETCH) cycle combines not only enzymes from
nine different organisms of all three domains of life but
also an engineered enzyme. This pathway is based on
crotonyl-CoA carboxylase/reductase as carboxylating
enzyme and activity has been demonstrated in vitro
[70
]. Recently, in vitro activity of another synthetic
CO2 fixation pathway has been demonstrated in vitro
by repurposing glycolaldehyde synthase and acetyl-phos-
phate synthase into a Synthetic Acetyl-CoA (SACA)
pathway [71].
Conclusions
Gas fermentation is an attractive platform for carbon
recycling or CCU with unique feedstock and product
flexibility advantages, enabling a circular economy. The
technology has reached commercial scale, with a first 42k
MTA plant successfully operating, converting emissions
from the steel industry into ethanol. Construction of
additional units that will demonstrate other aspects of
the technology is underway. Near term opportunities for
commercialization include the use of syngas produced
from additional feedstocks including unsorted, non-recy-
clable MSW, or various agricultural residues, as well as the
use of engineered strains producing new products, cur-
rently at pilot scale.
Realizing gas fermentation at commercial scale took over
a decade of challenging technical development, a journey
though the notorious ‘valley of death’, and a significant
amount of funding. Recent advances such as high mass-
transfer bioreactors and anaerobic biofoundries [72] offer
an opportunity to accelerate this development path, but it
still requires crucial funding for scale up first-of-its-kind
technologies and a supportive legislative framework
which places a value on carbon emissions reduction.
To maximize advantages and value offered by gas fer-
mentation, strain engineering to expand the portfolio of
product targets is essential. Within a decade, synthetic
biology of acetogens has advanced from a proof of concept
manual operation to an automated, high-throughput plat-
form and has enabled the demonstration of over 50 new
products. However, it is crucial to further accelerate
development by adapting advanced transformative tools
developed in model organisms (e.g. highly multiplexed
and genome-wide engineering tools [29,73,74] or cell-free
systems using lysates from gas fermenting organisms
www.sciencedirect.com
 Engineered microbes in gas fermentation Köpke and Simpson 187
[21

]), implementing new enabling technologies (e.g.
microfluidics strain generation [75], machine learning,
artificial cells), and onboarding gas fermenting organisms
into emerging biofoundries [72]. Long term, new syn-
thetic CO2 fixing pathways [68] or even synthetic CO2
fixing organisms [76] have the potential to further
improve the process efficiencies.
On the horizon are also two-stage processes combining
gas fermentation with more traditional aerobic hosts for
conversion of CO2 to new products, and ‘Power-to-X’
processes where of renewable electricity is the dominant
source of reducing power at scale. Such approaches open
the door to possibilities beyond traditional manufacturing
applications, with the potential for gas fermentation to
provide a solution to a need for product synthesis where
resources are scarce, such as in disaster relief zones or
space missions [77].
Conflict of interest statement
The authors are employees of LanzaTech that has com-
mercial interest in gas fermentation.
CRediT authorship contribution statement
Michael Köpke: Conceptualization, Writing - original
draft, Visualization, Project administration, Funding
acquisition. Séan D Simpson: Conceptualization, Writing
- original draft, Funding acquisition.
Acknowledgements
We like to thank Andrea Schoen, Freya Burton, Laurel Harmon and Rob
Conrado from LanzaTech for input and critical review of the manuscript.
Funding: This work was supported by the U.S. Department of Energy,
Office of Biological and Environmental Research in the Office of Science
[Award Number DE-SC0018249 and DE-SC0019090]. We also thank the
following investors in LanzaTech’s technology: BASF, CICC Growth
Capital Fund I, CITIC Capital, Indian Oil Company, K1W1, Khosla
Ventures, the Malaysian Life Sciences, Capital Fund, L. P., Mitsui, the
New Zealand Superannuation Fund, Novo Holdings A/S, Petronas
Technology Ventures, Primetals, Qiming Venture Partners, Softbank
China, and Suncor.
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