Biotechnology Advances 37 (2019) 508–518

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Biotechnology Advances
journal homepage: www.elsevier.com/locate/biotechadv

Research review paper

New bioproduction systems for chemicals and fuels: Needs and new T
development
An-Ping Zenga,b
a
Beijing Advanced Innovation Center for Soft Matter Science and Engineering, Beijing University of Chemical Technology, North Third Ring Road 15, Chaoyang District,
100029 Beijing, China
b
Institute of Bioprocess and Biosystems Engineering, Hamburg University of Technology, Denickestrasse 15, D-21073 Hamburg, Germany

ARTICLE INFO ABSTRACT

Keywords: Currently used bioproduction systems for chemicals and fuels are primarily based on sugar-containing sub-
Biorefinery strates. They have inherent limitations regarding substrate sustainability and affordability, product spectrum
C1 bioeconomy and yield, and costs for up- and downstream processing. To overcome some of these major burdens new bio-
Feedstock production strategies and systems are being developed, including biorefinery, electro-biotechnology (use of
Bioproduction
electricity for biosynthesis) and C1 bioeconomy using synthetic biological systems based on C1 carbon feed-
Synthetic biology
stocks such as CO, CO2, methane, methanol and formic acid. In this article, the promises, development trends
Electro-biotechnology
CO2 and challenges of these new bioproduction systems and concepts are briefly summarized and discussed.
Formic acid
1,3-propanediol

1. Introduction produced bio-based.

One of the primary goals of bioeconomy is the development of new
bioproduction systems to produce fuels, chemicals and other materials
at large scale from renewable resources or various feedstocks including
industrial waste streams using bio-based conversion technologies.
Bioeconomy is viewed as a consequent response to the global challenges
facing humankind and our living environment: limited fossil resources
and its increasing shortage for secured energy supply, global climate
change and environmental pollution. It has been formulated as strategic
research and development goals of many nations and regions world-
wide. In Europe, EU has once made the ambitious goal of producing
30% of all industrial chemicals and materials based on renewable ma-
terials and/or bioprocesses by the year 2030. To this end, there have
been great efforts in the last decades to develop sustainable and eco-
nomically competitive bioproduction processes of chemicals and fuels.
Fig. 1 summaries the potentials of bioproduction of chemicals and fuels
and the development status of selected products (graph generated by
end 2014). Classic fermentations products such as amino acids, anti-
biotics, food and feed additives are not included here. It is clear that
bioproductions of chemicals and fuels have huge impacts in many
sectors of agriculture, industry, healthcare and our daily life. With the
fast development in enabling technologies like functional genomics and
synthetic biology it is believed that probably all the major known bulk
organic chemicals and many hereto unthinkable new products could be
On the other side, only a few of the bioproducts and bioprocesses
have passed through the levels of laboratory and pilot plant trials and
become economically competitive to existing chemical routes. Some of
the products such as lactic acid, succinic acid and n-butanol had
reached industrial commercialization in some regions of the world (e.g.
China, USA and Europe) a few years ago, but currently have a hard time
to compete with chemical production processes because of the falling
oil price or unsure environmental policy. Overall, the chemical market
is worth more than €3.6 trillion per year globally, but only about 5% of
this market is addressed biotechnologically so far, although micro-
organisms have the potential to produce a vast range of chemicals
covering all the major sectors of the chemical market (Fig. 1). Present
bioproduction processes are primarily based on sugars or sugar-con-
taining materials including organic wastes or cellulosic materials. From
technological points of view they suffer at least from two major pro-
blems: 1. limited supply of sugar-based substrates for large-scale bio-
production and 2. high costs of available substrates (accounting for up
to 50% of the total costs in many cases) and up- and downstream
processing (nearly the other half of the total cost).
The limited penetration of bio-based products into the chemical
market is to a large extend also due to the fact that today there is an
over dependency on a small number of model microorganisms such as
E. coli, yeast or acetogens which lack many of the robust traits required
for industrial fermentation, resulting in thereby low product titers, low

E-mail address: aze@tuhh.de.

https://doi.org/10.1016/j.biotechadv.2019.01.003
Received 15 December 2017; Received in revised form 4 January 2019; Accepted 5 January 2019
Available online 11 January 2019
0734-9750/ © 2019 Elsevier Inc. All rights reserved.
A.-P. Zeng
Fig. 1. Potential of bioproduction of chemicals and fuels and development status of selected products.
productivity or poor fermentation performance, especially when using
cellulosic and waste materials as feedstocks. Indeed, a roadmap report
(National Research Council, 2015) from the US National Academy of
Sciences underlines the need for new microbes by concluding that
“expanding the palette of domesticated microbial platforms for bio-
manufacturing is seen as critical to expanding the repertoire of feed-
stocks and chemical products”.
2. New bioproduction systems: development needs and trends
To realize the promises of bioeconomy new bioproduction systems
which are more economical and sustainable are desperately needed.
This calls for fundamental innovations and new concepts in bioprocess
engineering for the use of renewable resources at an efficiency sig-
nificantly improved in comparison to today's production systems. Fig. 2
illustrates some of the key concepts and recognizable development
trends of bioproduction systems. Other important developments such as
in the areas of photo-biotechnology and marine biotechnology are not
included here and the readers interested in those areas may refer to
corresponding reviews.
2.1. Biorefinery
Biorefinery is one of the prominent concepts for new bioproduction
systems which has received great attention in the past years. The
principle idea is to make use of the substrate(s) and energy flows in the
production system completely and synergistically. A biorefinery pro-
duces normally several product streams and energy in one and the same
facility or from a certain starting feedstock, e.g. biomass. The different
concepts and achievements of biorefinery have been extensively re-
viewed in the past (Kamm et al., 2006; Aresta et al., 2015; de Jong and
Jungmeier, 2015; Chandel et al., 2018). Despite of the successful es-
tablishment of biorefinery plants for the processing of sugarcanes and
energy crops (e.g. for bioethanol production), plant oils, and organic
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Biotechnology Advances 37 (2019) 508–518
wastes (e.g. biogas biorefinery), the biorefinery of lignocellulosic ma-
terials, microalgae and marine resources still remains challenging for
industrial applications.
Even for “established” biorefineries there are still big rooms for
future development, for example, biorefinery plant for the production
of bioethanol. The presently used bioethanol refinery concepts for the
processing of corn and other grains (Ferreira et al., 2014; Reis and Hu,
2017; Reis et al., 2017) or sugarcanes (Nogueira and Capaz, 2015) can
be further expanded regarding the use of “co-product”, namely stillage
(from grain based processes) or vinasse (from sugarcane based process)
as depicted in Fig. 3. Stillage or vinasse is obtained after ethanol eva-
poration and distillation. The volume of thin stillage or vinasse can be
several times greater than that of ethanol. Stillage and vinasse contains
many organic compounds including about 1–2% glycerol. The glycerol
content could be even higher if the microbial strain or fermentation
process conditions are slightly adjusted. Reis et al. (2017) reviewed the
technologies for different uses or value-upgrading of thin stillage in
corn-to-ethanol process. It is stated that the recovery of co-products in
stillage is one of the key drivers for the economic sustainability of
bioethanol process, which can account for nearly 25% of the total
revenue for some ethanol plants. Here we will focus on glycerol, which
is a very interesting substrate for the generation of several important
bulk chemicals such as 1,3-propanediol and n-butanol (Zeng and Biebl,
2002; Westbrook et al., 2019) and, in general, a very attractive sub-
strate for producing a number of useful chemicals in other fermentation
processes. Presently, stillage/vinasse containing residual glycerol is
either further processed into distillers dried grains with solubles
(DDGS) by energy-intensive drying and pelleting or directly used for
fertilization/irrigation or cultivation of fungi for the production of
biomass. DDGS is sold as animal feed or used in some cases for biogas
production.
If the residual glycerol in stillage and vinasse can be recoved or used
for bioconversion, glycerol can become a desired “byproduct” from
bioethanol production in huge amount. The world production volume
A.-P. Zeng Biotechnology Advances 37 (2019) 508–518

Fig. 2. Concepts and development trends of new and sustainable bioproduction systems for chemicals and fuels.

of bioethnol was more than 26 billions of gallons (98.4 billions of litre)
in 2017 (data from International Energy Agency: https://www.iea.org/
tcep/transport/biofuels/), this could give more than 1000 Mio. t/a of
stillage and vinasse worldwide and thus 10–20 Mio. t of glycerol.
Assuming that only 10% of this glycerol potential would be used, there
would be more than 1 Mio. t of glycerol available for bioconversion. In
fact, the use of glycerol in stillage and vinasse can enhance the economy
and ecology of the bioethanol biorefinery in several aspects:
▪ First, glycerol can be converted into value-added products, thus
increasing the overall economy of bioethanol production. For ex-
ample, Oehmke and Zeng (2015) developed a very simple and ef-
ficient bioprocess to convert glycerol in thin stillage directly into 3-
hydroxypropionaldelyde (3-HPA) at a conversion yield of 95% using
the probiotic bacterium Lactobacillus reuteri. 3-HPA can be dehy-
drated into acrolein by shifting the pH value, the latter can be re-
moved from the culture broth via simple destillation. 3-HPA and
acrolein have a wide range of applications in healthcare, food and
chemical industries, e.g. for the production of maloic acid, acrylic
acid and acryamide. Acrolein has a market volume larger than
10 Mio. t/a worldwide and is presently produced from the oxidation
of propene.
▪ Second, the further processing (like drying into animal feed) of
stillage can be improved because of reduced viscosity (thus reduced
energy costs for drying) due to the depletion of glycerol.
▪ Third, glycerol in stillage could lead to “accidental” formation of 3-
HPA or acrolein if the stillage is not properly stored or processes,
leading to safety concern if the stillage is further processed into
DDGS for feed application, or causing toxicity to microorgamisms if
the stillage is used for biogas production or cultivation of biomass.
The deliberate bioconversion of glycerol and the removal of 3-HPA
as acrolein can avoid these undesiarble situsations.
▪ Finally, the bacterium L. reuteri used for the bioconversion of gly-
cerol is known to have probiotic effects for human and could be left
in stillage/vinasse. DDGS with L. reuterie enrichment may be also
benificial as feed for animals.
The above example illustrates the importance of using co- or by-
products of biorefinery processes. One of the co- or by-product of most
biorefinery processes is CO2. In the bioethanol production one third of

Fig. 3. An extented biorefinery concept for the production of bioethanol, biobased acrolein, and DDGS (distillers dried grains with solubles) enhanced with probiotic
bacteria. What remains to be explored is the reuse of the large amount of carbon dioxide released from ethanol fermentation.

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the carbon source is converted into CO2 (Fig. 3). CO2 reuse is, therefore,
gaining great attention nowadays and will be further described in a
section below.
Two other new developments of biorefiney are worth mentioning.
One of the trends is to move from fuel-focued biorefinery to more
chemically focued biorefinery as demonstrated in the European multi-
nation collaboratory project EuroBioRef (http://www.eurobioref.org/).
EuroBioRef has intended to bridge the gap between agriculture and
chemical industry by integrating the whole biomass chain into a multi-
feedstock (non-edible), multi-process (chemical, biochemical, thermo-
chemical), multi-products (aviation fuels and chemicals), and com-
mercially viable and adaptable approach for a sustainable bio-economy
in Europe. Another very interesting development is the integration of
the present biorefinery concepts with developments in electro-
biotechnology and one-carbon (C1) biosynthesis.

Fig. 5. Best case scenario for the e-driven biosynthesis of 1,3-propanediol.

2.2. Electro-biotechnology
In the past years, the use of electricity for chemical and biochemical
conversions has received great interest due to the increasingly avail-
ability of regenerative electricity from wind and solar energy at rea-
sonable prices and the need for “Power to Products” (Lovley and Nevin,
2013; Harnisch and Holtmann, 2017; Zeng and Kaltschmitt, 2017). To
this end, bioelectrochemical systems (BES) and more recently microbial
electrosynthesis (MES) have been studied as fundamentally new bio-
production systems of biofuels and chemicals based on electricity as
energy source (Rabaey and Rozendal, 2010; Lovley, 2012; Angenent
and Rosenbaum, 2014; Rosenbaum and Franks, 2014; Rosenbaum and
Henrich, 2014) (Fig. 4).
In BES two types of bioelectrochemical processes involving mi-
crobes or enzymes can take place: (1) delivering electrons from organic
compounds to the anode and generating thereby electricity and an
oxidized product such as CO2, and (2) using electrons from the cathode
to reduce a substance or to regenerate cofactors, generating thereby
more reduced products. Purely electrochemical reactions (termed as
abiotic in Fig. 5), such as the electrochemical formation of H2 or organic
acids like formic or acetic acid, can be carried out in BES as well which
can be coupled with biological processes, leading to secondary BES.
Fig. 4. Principles of bioelectrochemical systems (BES) with the underlying
redox (bio)reactions on the anode and cathode and their broad applications.
Previous studies have been concentrated on the anode reactions, e.g. for gen-
eration of electricity from wastewater in form of microbial fuel cell and in
biofilms or enzymatic fuel cell. The use of cathode reactions for microbial and
enzymatic electrosynthesis of value-added products is gaining more and more
importance. The key scientific questions to be addressed and methods and tools
to be developed are also outlined. (The central part of the graph is adapted from
Harnisch et al., 2015).
Electro-biotechnology provides a fundamentally new possibility to
engineer redox processes of bioproduction systems through extra-
cellular uptake or supply of reducing equivalents in the form of elec-
trons. The electrons can be used as universal energy and reducing
equivalents for biochemical reactions. Furthermore, the redox potential
of bioreaction systems can be adjusted or controlled by varying the
strength of the electric current or potential, leading to hereto un-
thinkable new bioreaction windows and bioprocesses. Compared to
reducing equivalents derived from carbohydrates electrons from re-
generative energy are not only more sustainable but also cost-compe-
titive (Rabaey et al., 2011).
Electro-biotechnology has a wide range of attractive applications in
bioelectrochemical synthesis, electrochemical energy conversion, and
treatment or reuse of waste materials, and in environmental remedia-
tion (Fig. 4.). BES such as microbial fuel cell (MFC), enzymatic fuel cell
(EFC) and microbial electrolysis cell (MEC) have been studied for
decades. These studies have led to the discoveries of several electro-
active enzymes and microorganisms, such as species of Geobacter and
Shewannella, and three general mechanisms of electron transfer be-
tween electrodes and biocatalysts (Sydow et al., 2014). However, the
molecular details of the electron transfer mechanisms and their ex-
tendibility into other relevant microorganisms such as clostridia and
methanogens are still poorly understood (Shi et al., 2016). While MFC,
EFC and MEC have been intensively studied and some of the technol-
ogies have reached pilot plant demonstration or even industrial uses
(Haas et al., 2018), MES is still in its very early stage of laboratory
development. Although the first laboratory study of microbial electro-
chemical fermentation can be dated back to as early as the 1980's
(Hongo and Iwahara, 1979), MES has first received great attention as a
potentially disruptive bioproduction process in biotechnology only after
the seminal studies of Nevin et al. (2011) and Li et al. (2012) which
described successful microbial electrosynthesis of multi-carbon organic
compounds such as acetate, 2-oxobutyrate and isobutanol from CO2 in
BES. The sequential integration of electrochemical and microbial pro-
cesses (abiotic + biotic or in the opposite order), also called secondary
MES, opens additional technological possibilities for electro-bio-
technology (Suastegui et al., 2016). Secondary MES is presently carried
out in two separate steps or reactors; it is of great scientific and tech-
nological interest to carry out such integrated processes within a single
step or in a single bioreactor.
In the following, the promise and challenges of electrochemically
supported biosynthesis of value-added products are illustrated with the
microbial production of 1,3-propanediol (PDO), an appealing bio-based
compounds with a wide range of applications, e.g. as monomer for a
new type of polyester or as building block and intermediate in cos-
metics and personal care products. The natural microbial pathway for
PDO production is based on the anaerobic conversion of glycerol by
different organisms such as Klebsiella and Clostridia (Zeng and Sabra,

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2011). A synthetic bioproduction route based on combining microbial

pathways of glycerol formation from glucose (e.g. in yeast) and sub-
sequent conversion of glycerol to PDO (e.g. in K. peumoniae) was de-
veloped (Nakamura and Whited, 2003) and commercialized by Dupont
Tate & Lyle. Chen et al. (2015) developed a glycerol-independent
pathway for producing PDO directly from glucose. In all the PDO
pathways, the product yield is limited by the supply of reducing power,
i.e. a part of the substrate is oxidized to deliver the reducing power
needed for the PDO biosynthesis. If the reducing power can be gener-
ated from electricity the product yield of these processes should be able
to be greatly increased. For example, the maximum yield of PDO from
anaerobic glycerol fermentation is 0.65–0.72 mol/mol. With a re-
generation of the reducing power from electricity the theoretical yield
could be increased to 1.0 mol/mol. Furthermore, the formation of oxi-
dative byproducts could be simultaneously reduced, leading to simpli-
fied downstream processing, the latter represents a major cost factor for
the PDO bioproduction (Xiu and Zeng, 2008). The feasibility of using
BES for enhanced PDO formation from glycerol fermentation has been
demonstrated by several groups (Zhou et al., 2013; Xafenias et al.,
2015; Utesch et al., 2018). A theoretical best-case calculation indicates
that e-driven biosynthesis of PDO is both technologically possible and
economically attractive (Fig. 5). Fig. 6. Clostridia as promising cell factories for the production of fuels and
chemicals from a variety of substrates. The native spectrums of products and
Of particular interest is the second generation of the e-driven bio-
substrates can be expanded using synthetic biology. Reducing power [H] is
synthesis of PDO based on CO2 and electricity. Many challenges and
needed for many of the pathways. [H] is ideally supplied using regenerative
technical barriers should be overcome to realize the promises. First, an electricity in bioelectrochemical systems. Hybrid systems combining electro-
electron-active microorganism should be identified or engineered for chemical generation of formate from CO2 or H2 from water with biosynthesis
efficient cofactor regeneration through the direct or indirect use of are also of great interest.
electricity. While several microorganisms (e.g. species of Geobacter and
Shewanella) have been shown to generate electricity on the anode, there
electrode of a conventional two-chamber bioelectrochemical system
are nearly no microorganisms which have been unambigously shown to
into a rod shaped ensemble. This “All in One” electrode can be easily
directly take electrons from the cathode. Electron transfer from cathode
put into common bioreactors to perform electrochemically supported
to cells is normally observed in biofilm or in microbiome (Rabaey and
bioprocesses. It can be used as hydrogen or oxygen donor, as an elec-
Rozendal, 2010; Lovely and Nevin 2013; Lovley, 2012; Angenent and
trodialysis unit and/or electrocatalytic surface for biological or che-
Rosenbaum, 2014). Recently, Choi et al. (2014) reported that one of the
mical conversion processes. The efficiencies of this electrode has been
important PDO producers Clostridium pasteurianum is electron-active.
characterized and its advantages and usefulness compared to the classic
This result is interesting although it has not yet been confirmed by other
H-Cell BES are demonstrated by glycerol fermentations with C. pas-
research groups and is thus still of debate. C. pasteurianum belongs to
teurianum DSM 525 (Utesch et al., 2018).
the genus of Clostridium, which has a long history of commercial uses,
The effects of electrochemistry on fermentation processes and con-
with C. acetobutylicum and C. ljungdahlii as the most prominent ex-
sequently the reaction rates reported for electro-biosynthesis are gen-
amples. C. pasteurianum has several favorable physiological properties
erally still very low for technical applications. One of the key issues is
and metabolic capability. It was already reported in the 1970's that this
the lack of mechanistic and quantitative understanding of electron
microbe is able to reduce CO2 to formate with the enzyme reduced
transfer mechanisms in microorganisms other than those related to the
ferredoxin: CO2 oxidoreductase (Scherer and Thauer, 1978). Moreover,
generation of electricity as in MFC. It is still not known how industrially
C. pasteurianum belongs to the very few of clostridia capable of N2
appealing microorganisms such as species of clostridia exchange elec-
fixation and has been extensively studied as a model organism in this
trons with electrodes for the purpose of biosynthesis. Although mole-
respect. For a sustainable bioproduction the supply of nitrogen source is
cular biological tools are generally available for studying or even en-
also an important aspect to be considered. Since the end of 1990's this
gineering these microorganisms, they have not been used to specifically
microbe has been intensively studied for the production of PDO and n-
explore questions related to electro-biosynthesis. Another serious
butanol (Biebl, 2001; Groeger et al., 2016). In recent years, genetic
burden is the fact that very different cultivation systems (e.g. the classic
tools and omics-based analyses have also been developed for the sys-
H-cell BES) have been used in the basic researches which are hardly
tems and synthetic biology studies of C. pasteurianum (Pyne et al., 2014;
applicable in process engineering (e.g. for an exact control of the pro-
Bruder et al., 2016; Groeger et al., 2017; Schmitz et al., 2018). In a long
cess parameters) and often not usable for a truly quantitative and sys-
term, it is to expect that C. pasteurianum could become a microbial cell
tematic understanding of the underlying mechanisms and interactions
factory superior in sustainable supply of reducing power and nitrogen
(e.g. artifacts due to ion transfer and other abiotic effects) in the in-
source (Fig. 6).
herently complex BES with multiple parameters (Krieg et al., 2018).
The work of Choi et al. (2014) was carried out in a classic H-Cell
The scale-up of BES presents additional challenges and have been
bioelectrochemical system (BES). It has several drawbacks and limita-
seldom studied for biosynthesis purpose.
tions associated with the use of membrane to separate the two elec-
From an engineering point of view the major challenge is due to the
trolytic chambers, making pH control and scale-up difficult. Recently,
fact that electron transfer from an electrode is surface-dependent. In
Utesch and Zeng (2018) showed that iron ion shift between the two
contrast, bioproduction processes are typically volume-dependent.
chamber presents a problem for glycerol fermentation, because the
Thus, process and scale-up strategies different from the ones in the
product selectivity of this fermentation process is strongly dependent
classic biochemical engineering seem to be needed. Enzmann et al.
on the availability of iron. In fact, new bioreactor design is needed to
(2018) discussed the scale-up issues of BES and made an interesting
make e-driven bioproduction at large scale feasible (Krieg et al., 2018;
comparison with bioreactors and scale-up strategies applied in photo-
Rosa et al., 2017). Utesch and Zeng (2017) designed therefore an “All in
biotechnology. Since light transfer is also primarily surface dependent,
One” electrode to combine the working electrode and the counter

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A.-P. Zeng
electro-biotechnology and photo-biotechnology share many common
aspects regarding scale-up. Owing to intensive study of photo-bio-
technology in the last years it is more advanced and has resulted in first
industrial applications. Some of the process and scale-up strategies in
photo-biotechnology such as numbering-up of bioreactors may be
adapted to the field of electro-biotechnology. Recent advances in ma-
terial sciences for novel electrodes, in bioprocess engineering for more
suitable bioreactors and process strategies, and in genome editing,
metabolic engineering, systems and synthetic biology for more pow-
erful quantitative analysis and targeted biomolecular engineering pro-
vide great opportunities for more quantitative and systematic funda-
mental studies of microbial and enzymatic electrosynthesis processes,
with the final goal to establish electro-biotechnology as a transforma-
tive new technology.
2.3. C1-based bioproduction systems
Microbial production systems have achieved great success in the
production of large-volume fuels, chemicals and materials from sugar-
type substrates or biomass (including cellulose), but the sustainable
supply and the costs of sugars and sugar-containing biomass are two
major constraints for large-scale bioproduction of chemicals and fuels,
calling for the use of more abundant and cheap substrates. One-carbon
(C1) feedstocks such as methane, methanol, formaldehyde, formate, CO
and CO2 have recently received great attention for bioproduction
(Haynes and Gonzalez, 2014; Bar-Even et al., 2013; Olah, 2013; Dürre
and Eikmanns, 2015; Yishai et al., 2017). They can be generated from
biomass including organic wastes, but are also available from fossil
sources or industrial wastes. Among them the use of CO and CO2 has
probably attracted the greatest interest because of the urgent need to
reduce their emission to the environment and to realize decarboniza-
tion of the industry. CO as a component of syngas (comprising H2 and
CO, and often also CO2) is more accessible for biosyntesis than CO2
because of its high energy content. For biological use of CO2 external
energy has to be applied, because CO2 has the mostly oxidized form of
carbon and cannot take part in bioreaction without an energy donor.
In plants, CO2 is fixed using light energy during photosynthesis and
channeled into carbon metabolism via the well-known Calvin-Benson
cycle (Calvin and Benson, 1948). From a technical point of view,
photosynthesis has a relatively low energy efficiency (less than 3%).
Technologically, CO2 fixation pathways in microorganisms, in parti-
cular those using energy in molecular hydrogen to drive highly energy-
demanding reactions such as those in the Wood-Ljungdahl pathway
(WL pathway, also known as reductive acetyl-CoA pathway)
(Schuhman et al., 1972), the dicarboxylate/4-hydroxybutyrate cycle
(Di-4HB cycle) (Huber et al., 2008), and the 3-hydroxypropionate-4-
hydroxybutyrate cycle (3HP- 4HB cycle) (Berg et al., 2008) are of
particular interest. Microorganisms utilize hydrogen via hydrogenases,
and H2 can be generated technologically in several ways, among others
the electrolysis of water. H2 is also a major component of syngas and
certain industrial waste gas streams.
Although there are still several technical and economic limitations
in the use of CO2 and H2 for large-scale bioproduction, recent pro-
gresses in this direction are promising (Dürre and Eikmanns, 2015).
Prominent examples of microbial CO2 bioconversion (in combination
with H2 or H2 + CO as syngas) are the production of ethanol, acetate
and 2,3-butanediol using acetogens such as Clostridium auto-
ethanogenum, Clostridium ljungdahlii and Acetobacterium woodii (Köpke
et al., 2010; Hoffmeister et al., 2016). These anaerobic bacteria can
grow autotrophically on CO2 + H2 or syngas but at very low growth
rates and biomass formation, leading to relatively low product con-
centrations (typically in the range of less than 10–50 gL−1) and low
volumetric productivities (typically less than 0.1–0.3 gL−1 h−1 in batch
culture). Optimization of cultivation conditions, e.g. by employing
mixed autotrophic and chemotrophic growth of cells, can significantly
improve the biomass formation, product concentration and
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Biotechnology Advances 37 (2019) 508–518
productivity. For example, Kantzow et al. (2015) improved the biomass
concentration (up to 3.2 gL−1) of A. woodii grown in a medium con-
taining 2 g/L yeast extractwithin a process time of 7.5 h in a batch-
culture for acetate formation from CO2 + H2. The higher biomass
concentration is mainly attributed to cell growth from yeast extract
consumed. As the result, an impressive acetate concentration of 59.2 g/l
was achieved with a volumetric productivity of 0.77 gL−1 h−1. The
volumetric productivity was further improved to above 6.1 gL−1 h−1 in
a continuous culture with total cell retention and at a high dilution rate
(0.35 h−1). However, the acetate concentration was decreased to 17.6
gL−1 under the given conditions. It should be noted that the internal
cell retention through a submerged membrane module as used in the
study of Kantzow et al. (2015) is hardly to be carried out over a long
operation time to reach a steady-state and is not suitable for scale-up.
As pointed out by the authors, acetogens like A. woodii seem to be
sensitive to shear force which hinters the use of external cross-flow
membrane filtration for cell recycle. The cross-flow membrane filtration
is more effective and can be used for long-term operation.
Another challenge of current gas fermentation systems is the limited
mass transfer of gases, especially CO and H2, into the bioreactor.
Furthermore, acetogens are sensitive to oxygen and some components
of waste gases. Thus, new bioreactor design and process engineering
strategies are needed to establish acetogen fermentation as a new bio-
production system. A further drawback of acetogens is the limited
spectrum of products and product selectivity. Metabolic engineering or
synthetic biology of acetogens has been carried out to produce non-
native products like acetone and butanol (Dürre and Eikmanns, 2015).
However, the product concentration and productivity of these en-
gineered acetogens and other hosts such as E. coli for CO2 utilization are
very low.
A very promising integrated bioproduction system is the conversion
of syngas to metabolites like acetate and/or ethanol by an acetogen
microbe and the subsequent conversion of these metabolites to higher
value-added products by another microorganism in cascade or in mixed
culture (Charubin et al., 2018). The group of Stephanopoulos (Hu et al.,
2016) used this approach to produce microbial lipids in a two-stage
process. In the first stage, an anaerobic culture of the acetogen Moorella
thermoacetica converts syngas to acetic acid. The culture supenatant of
the first stage is then fed to a second bioreactor to use acetic acid as a
substrate for aerobic production of lipids in an engineered oleaginous
yeast Yarrowia lipolytica. The integrated continuous bench-scale reactor
system could produce 18 gL−1 microbial lipids directly from syngas,
with an overall productivity of 0.19 g·L−1·h−1 and a lipid content ac-
counting for 36% of the biomass. In an optimized second-stage bior-
eactor system with Y. lipolytica grown on semicontinuously fed acetic
acid (3%) and with cell recyle using cross-flow filtration, they achieved
a lipid titer, yield, and productivity of 115 g L−1, 0.16 g g−1, and
0.8 g·L−1·h−1, respectively (Xu et al., 2017). Gildemyn et al. (2017)
used Clostridium kluyveri in continuous bioreactor to convert a mixture
of ethanol and acetic acid contained in an effluent from syngas fer-
mentation into medium-chain carboxylic acids such as n-caproic acid. A
productivity of 0.19 g·L−1·h−1 of n-caproic acid and a carbon conver-
sion efficiency higher than 90% were achieved. In the study of Hu et al.
(2016) cascade cultures were employed. It would be interesting to ex-
amine if co-cultures (mixed cultures) can be used to achieve an in-
tegrated conversion of syngas. Co-cultures and mixed cultures have a
great potential in industrial biotechnology, but have been not well
explored except for applications in food biotechnology and in waste
treatment (Sabra et al., 2013; Sabra and Zeng, 2014). More fundrea-
mental and bioprocess engineering researches in this area are needed,
especially with respect to the bioproduction of a target compound.
Methane is another attractive gaseous C1 feedstock for bioproduc-
tion. It is a major component of natural gas and biogas and has 3 times
higher green-house effect than CO2. In the recent years there has been
great interest to biotechnologically convert methane gas to liquid fuels
(Bio-GTL). Haynes and Gonzalez (2014) and Fei et al. (2015) discussed
A.-P. Zeng
the impact and major challenges of Bio-GTL using methane as a carbon
source and pointed out future research needs. In nature, methane is
known to be used by methanotrophic microorganisms (methanotrophs)
such as Methylococcus or Methylomonas species (Trotsenko and Murrell,
2008). These microorganisms can grow on methane as the sole carbon
and energy sources. They oxidize methane to methanol by using the
enzyme methane monooxygenase (MMO) under aerobic conditions
(Hakemian and Rosenzweig, 2007) (Fig. 7). MMO requires the cofactor
NADH or NADPH to activate O2 and to insert one oxygen atom into the
highly stable CeH bond of methane, forming thereby a C-OH group.
The cofactor required is regenerated by the subsequent oxidation of
methanol into formaldehyde using the enzyme methanol dehy-
drogenase (MDH). Anaerobic methane oxidation (AMO) has been
speculated to occur in anoxic marine and freshwater sediments, where
methanotrophic archaea and microbial consortia may oxidize methane
using different terminal electron acceptors such as sulfate, nitrate, ni-
trite and metals (Haroon et al., 2013; Knittel and Boetius, 2009).
However, neither pure cultures nor microbial consortia for AMO have
been isolated and cultivated in laboratory so far. From a biotechnolo-
gical point of view, the biological conversion of methane is closely re-
lated to the biological utilization of methanol and formaldehyde.
Methanol itself is an attractive feedstock for new bioproduction
system(s). In addition to biological conversion of methane to methanol
mentioned above methanol can be chemically effectively produced
from methane or natural gas. Presently, it is industrially produced
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Biotechnology Advances 37 (2019) 508–518
Fig. 7. From waste and regenerative electricity to bulk che-
micals and liquid fuels via biogas, electrobiosynthesis and
synthetic biology (modified from Zeng and Kaltschmitt 2017).
The central part of the electrochemical system symbolizes
potential simultaneous uses of the anode and cathode pro-
cesses for the oxidation of methane and reduction of CO2 (e.g.
via generation of O2 and H2 from water electrolysis) respec-
tively. From CH4 and CO2 a common intermediate for-
maldehyde can be obtained which can be channeled into
cellular metabolism for the production of chemicals and li-
quid fuels through native or synthetic metabolic pathways.
The strategy could be termed as “electro-biorefinery of waste
and C1-feedstocks twined with synthetic biology”.
mainly from syngas by hydrogenation of carbon monoxide. As a sub-
strate for bioproduction methanol is more reduced than most sugars
(50% more electrons per carbon atom compared to glucose) and thus
principally well suitable for the biosynthesis of many metabolites such
as alcohols, carboxylic acids, and fatty acids (Witaker et al. 2015).
Methanol and formaldehyde can be utilized by methylotrophic micro-
organisms such as Baccilus methanolicus (Zhang et al., 2017; Zhang
et al., 2018). Several natural metabolic pathways have been identified
for methanol assimilation such as the ribulose-monophosphate (RuMP)
pathway, the xylulose monophosphate (XuMP) pathway, and the serine
pathway (Zhang et al., 2017). In the RuMP and the serine pathways
methanol is first oxidized to become formaldehyde (HCHO) which is
then channeled into the cellular metabolism. The RuMP pathway is
bioenergetically more favorable than the serine pathway and is the
mostly studied one so far (Whitaker et al., 2015). In the RuMP pathway
HCHO is fixed into the pentose phosphate pathway (PPP) intermediate
ribulose-5-phosphate. Thus, the RuMP pathway shares many inter-
mediates with PPP and can have different configurations with different
output for the regeneration of ribulose-5-phosphate.
The technical use of native methylotrophs for bioconversion of
methane or methanol is very challenging and even not feasible in a
short term (Whitaker et al., 2015). This is because autotrophic growth
of these microbes on methane or methanol is relatively slow and gen-
erates very low biomass and little metabolites. Genetic tools for meta-
bolic engineering of typical methylotrophs are still not well developed.
A.-P. Zeng
Fig. 8. (A) Reductive glycine (rGly) pathway as a novel synthetic route for the
fixation of carbon dioxide and assimilation of formate (from Cotton et al.,
2018). (B) The reversed glycine cleavage system with the key reactions for
glycine synthesis from CO2 and NH3. The flux of the rGly pathway is presently
too low to sustain formatotrophic growth of microorganisms. To significantly
increase the flux many questions should be answered regarding the mechan-
isms, kinetics and regulation of the rGly pathway and the glycine cleavage
system.
Among others, the transfer efficiency for foreign DNA is very inefficient
or restricted. Thus, efforts have been put on creating synthetic methy-
lotrophic microorganisms using industrial microorganisms such as E.
coli (Müller et al., 2015; Witaker et al., 2017; Chen et al., 2018;
Gonzalez et al., 2018; Meyer et al., 2018) and Corynebacterium gluta-
micum (Leßmeier et al., 2015; Witthoff et al., 2015; Tuyishime et al.,
2018) for the use of methanol in the last years.
Müller et al. (2015) first demonstrated the incorporation of me-
thanol into the central metabolism of E. coli by heterologous expression
of the RuMP pathway genes coding for NAD-dependent methanol de-
hydrogenase (MDH2), hexulose-6-phosphate synthase (Hps) and 6-
phospho-3-hexuloisomerase (Phi) from B. methanolicus. However,
growth of E. coli on methanol or incorporation of methanol into bio-
mass was not confirmed. Witaker et al. (2017) later showed the in-
corporation of methanol into biomass component and a metabolite,
flavonoid naringenin, at the presence of yeast extract by proper en-
gineering of the RuMP pathway in E. coli. Very recently, several groups
(Chen et al. 2018; Gonzalez et al., 2018; Meyer et al., 2018; He et al.,
2018) improved methanol assimilation in E. coli grown on co-sub-
strates. Of particular interest is the development of E. coli strains with
methanol-dependent growth and product formation, the so-called syn-
thetic methanol auxotrophy (Chen et al., 2018; Meyer et al., 2018).
These methanol-dependent strains can achieve a much higher con-
sumption rate of methanol in a certain stoichiometry or ratio to the
other substrate such as xylose (Chen et al., 2018) or gluconate (Meyer
et al., 2018). Mutations of several native genes/pathways in the host
strain are necessary for these purposes, such as the genes encoding
adenylate cyclase (cyaA) and the formaldehyde detoxification operon
(frmRAB), NAD(H) homeostasis/biosynthesis (nadR) and phospho-
pentomutase (deoB). Chen et al. (2018) further engineered their me-
thanol auxotrophic strain to produce ethanol or 1-butanol to final titers
of 4.6 gL−1 and 2.0 gL−1 from a mixture of methanol and xylose, with
43% and 71% of the carbon in ethanol and 1-butanol produced being
derived from methanol, respectively. Nevertheless, the contribution of
carbon and energy from methanol or the RuMP pathway to the cell
growth is still limited in this strain. This is because the heterologous
establishment of the RuMP pathway for active cell growth requires
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Biotechnology Advances 37 (2019) 508–518
addressing at least three major challenges: 1. sufficient formaldehyde
supply, 2. efficient formaldehyde assimilation, and 3. active regenera-
tion of ribulose-5-phosphate as formaldehyde acceptor. He et al. (2018)
proposed an interesting modular approach to deal with these chal-
lenges. They demonstrated that two of the three requirements can be
met by using external sarcosine to supply formaldehyde and xylose to
supply ribulose-5-phosphate. Further optimization was then focused on
formaldehyde assimilation, resulting in a strain in which the RuMP
pathway provides nearly all the biomass and energy needed for cell
growth.
A parallel development of C. glutamicum for the utilization of me-
thanol has taken place in the last years. Witthoff et al. (2015) and
Leßmeier et al. (2015) first introduced genes coding for the key en-
zymes (e.g. MDH, Hps, Phi) of the RuMP pathway into C. glutamicum
and demonstrated the utilization of methanol as an auxiliary substrate
in a glucose-based medium and its positive effect on cell growth upon
deletion of several endogenous genes to prevent methanol oxidation to
CO2 (via aldehyde dehydrogenases) and formaldehyde detoxification
pathway (via frmRAB operon). The incorporation of methanol into the
secreted product cadaverine was also confirmed by C13-labeling
(Leßmeier et al., 2015). Recently, Tuyishime et al. (2018) engineered C.
glutamicum into a methanol-dependent synthetic methylotroph for
growth on a mixture of methanol and xylose. Adaptive laboratory
evolution was then applied to improve methanol utilization. The
evolved mutant showed a significantly increased cell growth in a
minimal medium containing methanol and xylose. In this study, a
methanol-dependent production of glutamate (up to about 90 mgL−1)
was also shown.
The development of synthetic methylotrophic microorganisms
briefly outlined above suggests their potential application in bio-
conversion of methanol into useful chemicals although the product title
and productivity achieved are still quite low.
As mentioned in the previous section formate is another promising
C1 feedstock for new bioproduction processes. It can be produced from
different sources including CO2 by hydrogenation or electrochemical
reduction (Fig. 8). Electrochemical conversion of CO2 into formate is
considered the most efficient route in the (electro)chemical conversion
of CO2 (Del Castillo et al. 2015, 2017). Yishai et al. (2016) gave an
excellent overview of formate production from different sources and the
biological routes for formate assimilation. They discussed the concept
of “Formate Bioeconomy” and concluded that natural formatotrophes
(e.g. some species of acetogens and methanogens) are either inefficient
in terms of energetic efficiency or difficult to cultivate or engineer. An
alternative approach to adapt industrial microorganisms for growth on
formate and/or using it for biosynthesis in synthetic pathways, i.e. by
transferring natural formate assimilation pathways or designing de
novo non-natural pathways (Bar-Even et al., 2013). One of the im-
portant issues in selecting the host organism is its tolerance to formate,
since the acid form of formate, formic acid, is known to be quite toxic to
some microorganisms (Nicholls, 1975; Warnecke and Gill, 2005). It is
also important to design efficient formate assimilation routes that could
be easily implemented in the selected host organisms and support the
production of a wide variety of products. A systematic study con-
sidering issues related to resource-usage efficiency, thermodynamic
profile, kinetic capacity, and connectivity to the endogenous metabolic
network of model microbes (E. coli and S. cerevisiae) resulted in the
identification of the reductive glycine (rGly) pathway as the most fa-
vorable route for aerobic formate assimilation (Bar-Even et al., 2013).
The rGly pathway starts from formate and yields glycine which is
then converted into serine and further to pyruvate, a key intermediate
of biosynthesis (Fig. 8). The whole pathway can be divided into three
steps, the reduction step, the CeC bond forming step and the deami-
nation step:
1. The reduction step belongs to the folate-dependent one-carbon
metabolism, in which formate is ligated to tetrahydrofolate (THF)
A.-P. Zeng
catalyzed by formate tetrahydrofolate ligase and then reduced to
5,10-methylenetetrahydrofolate (5,10-CH2-THF) through methe-
nyltetrahydrofolate cyclohydrolase and methylenetetrahydrofolate
dehydrogenase. In this step one molecular ATP and NADPH is
needed, respectively.
2. The CeC bond forming step is the most important step of the re-
ductive glycine pathway, which is the “real” synthesis step. Glycine
is synthesized from 5,10-CH2-THF by the glycine cleavage/synthase
system (GCS) with the incorporation of NADH, NH3 and CO2. The
GCS system is also known as the glycine decarboxylase complex
which consists of foure proteins, i.e. H-protein, T-protein, P-protein
and L-protein. The H-protein is a lipoyl-bearing transferase, which
acts as a shuttle for intermediate products and is responsible for
interacting with other three proteins in the system.
3. The deamination step converts glycine to serine using another mo-
lecule of 5,10-CH2-THF yielded from the reduction step, and the
deamination of serine catalyzed by serine deaminase yields pyruvate
finally.
The group of Bar-Even (Yishai et al., 2016) first showed the op-
eration of the reductive step of formate assimilation and the deamina-
tion step of glycine in recombinant E. coli. The same group later de-
monstrated that the rGly pathway for C-C-bond formation can be
realized in E. coli through rational integration of native and foreign
enzymes of the tetrahydrofolate and glycine cleavage systems and to
satisfy all of the cellular glycine and serine requirements from the as-
similation of formate and CO2 (Yishai et al., 2016; Yishai et al., 2018).
To achieve this, it was necessary to overexpress four endogenous genes
encoding the four proteins of the GCS system and three foreign genes
from Methylobacterium extorquens encoding formate-THF ligase (Me-
FTL, activity not native to E. coli), 5,10-methenyl-THF cyclohydrolase
(Me-Fch) and 5,10-methylene-THF dehydrogenase (Me-MtdA) respec-
tively. The overexpression of the endogenous gene encoding the bi-
functional 5,10-methenyl-THF cyclohydrolase/5,10-methylene-THF
dehydrogenase (Ec-FolD) was not enough to support growth on glycine
derived from formate, presuably because of the inhibition of Ec-FolD by
10-formyl-THF. The recombinant E. coli was shown to obtain about
10% of its cellular carbon from formate and CO2 and the rest from
glucose with a relativley fast growth (a doubling time of 1.7 h) in shake
flask culture. It is also shown that the biosynthesis of serine from for-
mate and CO2 did not lower the growth rate compared to culture with
externally added serine.
Tashiro et al. (2018) also engineered an E. coli strain to use formate
via the reductive glycine pathway. In addition to overexpression of the
four endogeneous GCS genes, similarly as done by Yishai et al. (2016),
three foreign genes coding for formate-THF ligase (FTL), 5,10-me-
thenyl-THF cyclohydrolase (Fch) and 5,10-methylene-THF dehy-
drogenase (MtdA) have to be expressed. Tashiro et al. (2018) tested
FTL, Fch and MtdA from C. ljungdahlii and A. woodii, respectively. It
turned out that the genes from C. ljungdahlii are more suitable. An E. coli
strain further optimized regarding the formation of serine and pyruvate
was than used in an integrated electrochemical systems to demonstrate
the use of formate generated from CO2 by electrochemical synthesis.
The compatablility of the culture (M9 minimal medium) with the
electrochemical system was confirmed. However, the formate genera-
tion rate and cell growth in this systems were extremly low, with for-
mate being formed at about 10 mM/day and biomass reaching an
OD600nm of 0.47 after 8 days. The cell growth in the working electro-
chemical system had a unusually long lag phase (5 days) compared to
growth (lag phase 1 day) in the same system without electric current.
The authors attributed this to a possible growth inhibition by the for-
mation of reactive oxygen species such as H2O2. There are might be
other unkonwn reasons such as reactions of some of the medium
components on the elctrodes, making them unavailable for cell growth.
To achieve a fully formatotrophic growth on formate and even CO2
as the sole carbon source the reductive flux via the THF enzymes and
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Biotechnology Advances 37 (2019) 508–518
the GCS system should be increased by at least an order of magnitude
(providing thus 100% of biomass carbon instead of only 10%) (Yishai
et al., 2016). In this context, the reaction rate (flux) of the reversed GCS
may represent a major limiting factor. GCS is a quite complicated multi-
enzyme system with sophisticated regulation mechanisms (Kikuchi,
1973). Its reversibility has been principally demonstrated (Motokawa
and Kikuchi, 1974; Yishai et al., 2016). However, little quantitative
information is avaibale about how to increase its reaction rate. In fact, it
is not know which one is the limiting reaction step for glycine synthesis
in the reversed GCS. Mathematic modeling of the in vitro and in vivo
activities of reversed GCS may be very helpful for a guided design and
optimization of the rGly pathway. To this end, many questions should
be answered, for example regarding the mechanisms of NH3 uptake in
the T protein and CO2 fixation by the P protein, the kinetics of the GCS
complex and the regulation of the rGly pathway. The supply of reducing
power and energy should be also properly engineered to increase the
overall reductive flux from formate and CO2. Directed evolution of
microorganisms may be a powerful approach for further optimization of
microbes using C1 feedstocks. For example, Doering et al. (2018) ap-
plied an adaptive evolution strategy to optimize the enzymatic steps of
formatotrophic growth of E. coli.
3. Concluding remarks
Great efforts have been made in the last decade on delevloping
biotechnological production systems/processes for producing chemicals
and fuels. Unlike for fine chemicals and high-value intermediates used
as ingredients in pharma, food and healthcare industries, and except for
bioethnol, the biological production of bulk chemicals and commodities
has had rather limited success in view of its great potential. One of the
major limitations is the sustainable availabililty of affordable feed-
stocks. New bioproduction systems and strategies are being developed
to address this bottleneck. In this article, progresses in the areas of
biorefinery, electro-biotechnology and C1-based bioproduction have
been briefly discussed. Although the biorefinery concept has been
commercially in use since long time, e.g. for the processing of crops,
biorefinaries of lignocellulosic feedstocks, syngas, algaes, organic
wastes and marine resources are still under active development. Even
for the well established biorefineries such as those for biofuels (e.g.
bioethanol) there are still rooms for innovation, e.g. the full use of side
streams and the effluenet gas CO2. The biotechnological utilization of
CO2 is one of the exciting and challenging topics for new bioproduction
systems in the coming years. It is closely related to the other two topics
briefly addressed in this article: electro-biotechnology and C1-based
bioproduction. Because CO2 is the mostly oxidized and thus “energy-
poor” compound, energy has to be supplied in converting it into any
useful product. Electricity from renewable energy is probably the most
sustainable and affordable energy form. However, the transfer of elec-
tric energy into cells is still poorly understood and the electrochemical
systems developed so far for electrobiosynthesis are far from efficient
for practical applications. A number of fundamental and technological
issues as briefly outlined in Fig. 4 have to be addressed to push eletro-
biotechnology forward. Great fundamental research progresses have
been made in the use of methane, methanol, formate, and CO and CO2
for the biosynthesis in the last years. New and promising pathways have
been steadily proposed and experimentally apporved. While the auto-
trophic productions of acetate and ethanol from syngas or CO2 + H2
using acetogens have achieved commercial or precommercial stage of
technological readiness, the titer, yield and productivity of allmost all
products based on non-natural pathways are still too low. This also
applies to many “new” routes of electricity-added biosynthesis. Al-
though synthetic biology is powerful in designing de novo or non-nat-
ural metabolic pathways aimed at the use of electricity and C1 feed-
stocks, it is still a challenging task to push it beyond the “proof-of-
concept” trap.
In addition to using systems metabolic engineering for strain
A.-P. Zeng
optimization, new strategies in bioreactor design and operation and in
integrated bioprocess engineering are equally important to realize the
promise of C1– and electricity-based bioproduction. Bubble columns
and airlift reactors may find their renosaunce, since these bioreactors
are especially suitable for C1-based and electricity-aided biosynthesis
because of their higher surface/volume ratio and higher mass transfer
compared to standard stirred tank bioreactors. Integrated electro-
chemical conversion of CO2 to formate or chemical conversion of me-
thane and methanol to formate and the subsequent bioconversion of
formate into pyruvate and then targeted final products could unify the
electro-biotechnology and C1-based bioproduction. It is worth men-
tioning that another very promising integrated bioproduction system is
the conversion of C1 feedstocks to ethanol, acetate, and/or lactate by
one microorganism and their conversion to higher value-added pro-
ducts by another microorganism in cascade or in mixed culture.
Acknowledgements
This work has been financially supported by the Beijing Advanced
Innovation Center for Soft Matter Science and Engineering (China) and
by the Bundesministeriums für Bildung und Forschung (BMBF,
Germany) through the project BIOCHEM, Projektzeichen 031B0270.
References
Angenent, L.T., Rosenbaum, M.A., 2014. Microbial electrocatalysis to guide biofuel and
biochemical bioprocessing. Biofuels 4, 131–134.
Aresta, M., Dibendetto, A., Dumeignil, F., 2015. Biorefineries – An Introduction. De
Gruyter.
Bar-Even, A., Noor, E., Flamholz, A., Milo, R., 2013. Design and analysis of metabolic
pathways supporting formatotrophic growth for electricity-dependent cultivation of
microbes. Biochim. Biophys. Acta Bioenerg. 1827, 1039–1047.
Berg, L.A., Kockelkorn, D., Buckel, W., et al., 2007. A 3-hydroxypropionate/4-hydro-xy-
butyrate autotrophic carbon dioxide assimilation pathway in archaea. Science 318,
1782–1786.
Biebl, H., 2001. Fermentation of glycerol by Clostridium pasteurianum — batch and con-
tinuous culture studies. J. Ind. Microbiol. Biotechnol. 27, 18–26.
Bruder, M.R., Pyne, M.E., Moo-Young, M., Chung, D.A., et al., 2016. Extending CRISPR-
Cas9 technology from genome editing to transcriptional engineering in the genus
Clostridium. Appl. Environ. Microbiol. 82, 6109–6119.
Calvin, M., Benson, A.A., 1948. The path of carbon in photosynthesis. Science 107,
476–480.
Chandel, A.K., Garlapati, V.K., Singh, A.K., Antunes, F.A.F., da Silva, S.S., 2018. The path
forward for lignocellulose biorefineries: Bottlenecks, solutions, and perspective on
commercialization. Bioresour. Technol. 264, 370–381.
Charubin, K., Bennett, R.K., Fast, A.G., Papoutsakis, E.T., 2018. Engineering Clostridium
organisms as microbial cell-factories: challenges and opportunities. Met. Eng (In
print).
Chen, Chang-Ting, Chen, Frederic Y.-H., Bogorad, Igor W., Wu, Tung-Yun, Zhang, Ruoxi,
Lee, Abrax S., Liao, James C., 2018. Synthetic methanol auxotrophy of Escherichia
coli for methanol-dependent growth and production. Metab. Eng. 49, 257–266.
Chen, Z., Geng, F., Zeng, A.P., 2015. Protein design and engineering of a de novo pathway
for microbial production of 1,3-propanediol from glucose. Biotechnol. J. 10,
284–289.
Choi, O., Kim, T., Woo, H.M., Um, Y., 2014. Electricity-driven metabolic shift through
direct electron uptake by electroactive heterotroph Clostridium pasteurianum. Sci. Rep.
4, 6961–6971.
Cotton, C.A., Edlich-Muth, C., Bar-Even, A., 2018. Reinforcing carbon fixation: CO2 re-
duction replacing and supporting carboxylation. Curr. Opin. Biotechnol. 49, 49–56.
Del Castillo, A., Alvarez-Guerra, M., Solla-Gullón, J., Sáez, A., Montiel, V., Irabien, A.,
2015. Electrocatalytic reduction of CO2 to formate using particulate Sn electrodes:
Effect of metal loading and particle size. Appl. Energy 157, 165–173.
Del Castillo, A., Alvarez-Guerra, M., Solla-Gullón, J., Sáez, A., Montiel, V., Irabien, A.,
2017. Sn nanoparticles on gas diffusion electrodes: Synthesis, characterization and
use for continuous CO2 electroreduction to formate. J. CO2 Util. 18, 222–228.
Dürre, P., Eikmanns, B.J., 2015. C1-carbon sources for chemical and fuel production by
microbial gas fermentation. Curr. Opin. Biotechnol. 35, 63–72.
Enzmann, F., Stöckl, M., Zeng, A.P., Holtmann, D., 2018. Same but different - Scaling up
and Numbering up in electrobiotechnology and photobiotechnology. Eng. Life Sci (In
print).
Ferreira, J.A., Lennartsson, P.R., Taherzadeh, M.J., 2014. Production of ethanol and
biomass from thin stillage using food-grade Zygomycetes and Ascomycetes fila-
mentous fungi. Energies 7, 3872–3885.
Gildemyn, S., Molitor, S.B., Usack, J.G., Nguyen, M., Rabaey, K., Angenent, L.T., 2017.
Upgrading syngas fermentation effluent using Clostridium kluyveri in a continuous
fermentation. Biotechnol. Biofuels 10, 83.
Gonzalez, Jacqueline E., Bennett, R. Kyle, Papoutsakis, E. Terry, Antoniewicz, Maciek R.,
2018. Methanol assimilation in Escherichia coli is improved by co-utilization of
517
Biotechnology Advances 37 (2019) 508–518
threonine and deletion of leucine-responsive regulatory protein. Metab. Eng. 45,
67–74.
Groeger, C., Sabra, W., Zeng, A.P., 2016. Simultaneous production of 1,3-propanediol and
n-butanol by Clostridium pasteurianum: in situ gas stripping and cellular metabolism.
Eng. Life Sci. 16, 664–674.
Groeger, C., Wang, W., Sabra, W., Utesch, T., Zeng, A.P., 2017. Metabolic and proteomic
analyses of product selectivity and redox regulation in Clostridium pasteurianum
grown on glycerol under varied iron availability. Microb. Cell Factories 16 (1), 64.
Haas, T., Krause, R., Weber, R., Demler, M., Schmid, G., 2018. Technical photosynthesis
involving CO2 electrolysis and fermentation. Nat. Catalysis 1, 32–39.
Hakemian, A.S., Rosenzweig, A.C., 2007. The biochemistry of methane oxidation. Annu.
Rev. Biochem. 76, 223–241.
Harnisch, F., Holtmann, D., 2017. Electrification of Biotechnology: Status quo. Adv.
Biochem. Eng. Biotechnol. 98, 1–14.
Harnisch, F., Rosa, F.M., Kracke, F., Virdis, B., Krömer, J., 2015. Electrifying white bio-
technology: engineering and economic potential of electricity-driven bio-production.
ChemSusChem 8, 758–766.
Haroon. M, F., Hu, S., Shi, Y., Imelfort, M., Keller, J., Hugenholtz, P., Yuan, Z., Tyson, G.
W., 2013. Anaerobic oxidation of methane coupled to nitrate reduction in a novel
archaeal lineage. Nature 500: 567–570.
Haynes, C.A., Gonzalez, R., 2014. Rethinking biological activation of methane and con-
version to liquid fuels. Nat. Chem. Biol. 10, 331–339.
He, H., Edlich-Muth, C., Lindner, S.N., Bar-Even, A., 2018. Ribulose Monophosphate
Shunt Provides Nearly All Biomass and Energy Required for Growth of E. coli. ACS
Synth Biol (In print).
Hoffmeister, S., Gerdom, M., Bengelsdorf, F.R., Linder, S., Flüchter, S., Öztürk, H.,
Blümke, W., May, A., Fischer, R.-J., Bahl, H., Dürre, P., 2016. Acetone production
with metabolically engineered strains of Acetobacterium woodii. Metab. Eng. 36,
37–47.
Hongo, M., Iwahara, M., 1979. Application of electro-energizing method to L-glutamate
fermentation. Agric. Biol. Chem. 43, 2075–2081.
Hu, P., Chakraborty, S., Kumar, A., Woolston, B., Liu, H., Emerson, D., Stephanopoulosa,
G., 2016. Integrated bioprocess for conversion of gaseous substrates to liquids. Proc.
Natl. Acad. Sci. U. S. A. 113, 3773–3778.
Huber, H., Gallenberger, M., Jahn, U., et al., 2008. A dicarboxylate/4-hydroxybutyrate
autotrophic carbon assimilation cycle in the hyperthermophilic Archaeum Ignicoccus
hospitalis. Proc. Natl. Acad. Sci. U. S. A. 105, 7851–7856.
de Jong, E., Jungmeier, G., 2015. Biorefinery concepts in comparison to petrochemical
refineries. In: Pandey, A., Höfer, R., Larroche, C. (Eds.), Industrial Biorefineries and
White Biotechnology. Elsevier B.V, pp. 3–33.
Kamm, B., Gruber, P.G., Kamm, M., 2006. Biorefieries - Industrial Processes and Products.
Wiley-VCH, Weinheim.
Kantzow, C., Mayer, A., Weuster-Botz, D., 2015. Continuous gas fermentation by
Acetobacterium woodii in a submerged membrane reactor with full cell retention. J.
Biotechnol. 212, 11–18.
Kikuchi, G., 1973. The glycine cleavage system: composition, reaction mechanism, and
physiological significance. Mol. Cell. Biochem. 1, 169–187.
Knittel, K., Boetius, A., 2009. Anaerobic oxidation of methane: progress with an unknown
process. Annu. Rev. Microbiol. 63, 311–334.
Köpke, M., Held, C., Hujer, S., Liesegang, H., Wiezer, A., Wollherr, A., Ehrenreich, A.,
Liebl, W., Gottschalk, G., Dürre, P., 2010. Clostridium ljungdahlii represents a micro-
bial production platform based on syngas. Proc. Natl. Acad. Sci. U. S. A. 107,
13087–13092.
Krieg, T., Madjarov, J., Rosa, L.F.M., Enzmann, F., Harnisch, F., Holtmann, D., Rabaey, K.,
2018. Adv. Biochem. Eng. Biotechnol. 43, 2075.
Leßmeier, L., Pfeifenschneider, J., Carnicer, M., Heux, S., Portais, J.C., Wendisch, V.F.,
2015. Production of carbon-13-labeled cadaverine by engineered Corynebacterium
glutamicum using carbon-13-labeled methanol as co-substrate. Appl. Microbiol.
Biotechnol. 99, 10163–10176.
Li, H., Opgenorth, P.H., Wernick, D.G., Rogers, S., Wu, T., Higashide, W., Malati, P., Huo,
Y., Cho, K.M., Liao, J.C., 2012. Integrated electromicrobial conversion of CO2 to
higher alcohols. Science 335, 1596.
Lovley, D.R., 2012. Electromicrobiology. Annu. Rev. Microbiol. 66, 391–409.
Lovley, D.R., K.P. Nevin, K.P., 2013. Electrobiocommodities: powering microbial pro-
duction of fuels and commodity chemicals from carbon dioxide with electricity. Curr.
Opin. Biotechnol. 24, 385–390.
Meyer, Fabian, Keller, Philipp, Hartl, Johannes, Gröninger, Olivier G., Kiefer, Patrick,
Vorholt, Julia A., 2018. Methanol-essential growth of Escherichia coli. Nat. Commun.
9, 1508.
Motokawa, Y., Kikuchi, G., 1974. Glycine metabolism by rat liver mitochondria.
Reconstruction of the reversible glycine cleavage system with partially purified
protein components. Arch. Biochem. Biophys. 164, 624–633.
Müller, J.E.N., Meyer, F., Litsanov, B., Kiefer, P., Potthoff, E., et al., 2015. Engineering
Escherichia coli for methanol conversion. Met. Eng. 28, 190–201.
Nakamura, C.E., Whited, G.M., 2003. Metabolic engineering for the microbial production
of 1,3-propanediol. Curr. Opin. Biotechnol. 14, 454–459.
National Research Council, 2015. Industrialization of Biology: A Roadmap to Accelerate
the Advanced Manufacturing of Chemicals. The National Academies Press,
Washington, DC. https://doi.org/10.17226/19001.
Nevin, K.P., Hensley, S.A., Franks, A.E., Summers, Z.M., Ou, J., Woodard, T.L.,
Snoeyenbos-West, O.L., Lovley, D.R., 2011. Electrosynthesis of organic compounds
from carbon dioxide is catalyzed by a diversity of acetogenic microorganisms. Appl.
Environ. Microbiol. 77, 2882–2886.
Nicholls, P., 1975. Formate as an inhibitor of cytochrome c oxidase. Biochem. Biophys.
Res. Commun. 67, 610–616.
Nogueira, L.A.H., Capaz, R.S., 2015. Ethanol from sugarcane in Brazil: economic
A.-P. Zeng
perspectives. In: Pandey, Ashok, Höfer, Rainer, Larroche, Christian (Eds.), Industrial
Biorefineries and White Biotechnology. Elsevier B.V, pp. 237–246.
Oehmke, S., Zeng, A.P., 2015. Recovery of biologically produced 3-hydro-
xypropionaldehyde and its dehydrated product acrolein. Eng. Life Sci. 15, 133–139.
Olah, G.A., 2013. Towards oil independence through renewable methanol chemistry.
Angew. Chem. Int. Ed. 52, 104–107.
Pyne, M.E., Moo-Young, M., Chung, D.A., Chou, C.P., 2014. Expansion of the genetic
toolkit for metabolic engineering of Clostridium pasteurianum: chromosomal gene
disruption of the endogenous CpaAI restriction enzyme. Biotechnol. Biofuels 7, 163.
Rabaey, K., Rozendal, R.A., 2010. Microbial electrosynthesis - revisiting the electrical
route for microbial production. Nat. Rev. Microbiol. 8, 706–716.
Rabaey, K., Girguis, P., Nielsen, L.K., 2011. Metabolic and practical considerations on
microbial electrosynthesis. Curr. Opin. Biotechnol. 22, 371–377.
Reis, C.E.R., Hu, B., 2017. Vinasse from sugarcane ethanol production: better treatment or
better utilization? Front. Energy Res. 5, 7.
Reis, C.E.R., Rajendran, A., Hu, B., 2017. New technologies in value addition to the thin
stillage from corn-to-ethanol process. Rev. Environ. Sci. Biotechnol. 16, 175–206.
Rosa, L.F.M., Hunger, S., Gimkiewicz, C., Zehnsdorf, A., Harnisch, F., 2017. Paving the
way for bioelectrotechnology: Integrating electrochemistry into bioreactors. Eng. Life
Sci. 17, 77–85.
Rosenbaum, M.A., Franks, A.E., 2014. Microbial catalysis in bioelectrochemical tech-
nologies: status quo, challenges and perspectives. Appl. Microbiol. Biotechnol. 98,
509–518.
Rosenbaum, M.A., Henrich, A.W., 2014. Engineering microbial electrocatalysis for che-
mical and fuel production. Curr. Opin. Biotechnol. 29, 93–98.
Sabra, W., Zeng, A.-P., 2014. Mixed microbial cultures for industrial biotechnology:
Success, chance, and challenges. In: Grunwald, P. (Ed.), Pan Stanford series on
Biocatalysis: Vol. 1. Industrial Biocatalysis. Pan Stanford Publication, Singapore, pp.
205–238.
Sabra, W., Dietz, D., Zeng, A.P., 2013. Substrate-limited co-culture for efficient produc-
tion of propionic acid from flour hydrolysate. Appl. Microb. Biotechnol. 97,
5771–5777.
Scherer, P.A., Thauer, R.K., 1978. Purification and properties of reduced ferredoxin: CO2
oxidoreductase from Clostridium pasteurianum, a molybdenum iron-sulfur-protein.
Eur. J. Biochem. 85, 125–135.
Schmitz, R., Sabra, W., Hong, Y., Utesch, T., Zeng, A.P., 2018. Improved electro-
competence and metabolic engineering of Clostridium pasteurianum revealed a new
regulation pattern of glycerol mono- and cosubstrate fermentation. Eng. Life Sci (In
print).
Schulman, M., Wood, H.G., Ljungdahl, L.G., et al., 1972. Total synthesis of acetate from
CO2 V. determination by mass analysis of the different types of acetate formed from
13
CO2 by heterotrophic bacteria. J. Bacteriol. 109, 633–644.
Shi, L., Dong, H.L., Reguera, G., et al., 2016. Extracellular electron transfer mechanisms
between microorganisms and minerals. Nat. Rev. Microbiol. 14, 651–662.
Suastegui, M., Matthiesen, J.E., Carraher, J.M., Hernandez, N., Quiroz, N.R., Okerlund,
A., Cochran, E.W., Shao, Z., Tessonnier, J.P., 2016. Combining metabolic engineering
and electrocatalysis: Application to the production of polyamides from sugar. Angew.
Chem. Int. Ed. 55, 2368–2373.
Sydow, A., Krieg, T., Mayer, F., Schrader, J., Holtmann, D., 2014. Appl. Microbiol.
Biotechnol. 98, 8481–8495.
Tashiro, Y., Hirano, S., Matson, M.M., Atsumi, S., Kondo, A., 2018. Electrical-biological
hybrid system or CO2 reduction. Metab. Eng. 47, 211–218.
Trotsenko, Y.A., Murrell, J.C., 2008. Metabolic aspects of aerobic obligate methano-
trophy. Adv. Appl. Microbiol. 63, 183–229.
Tuyishime, P., Wang, Y., Fan, L., Zhang, Q., Li, Q., Zheng, P., Sun, J, Ma, Y., 2018.
Engineering Corynebacterium glutamicum for methanol-dependent growth and glu-
tamate production. Metab. Eng. 49, 220–231.
Utesch, Tyll, Zeng, An-Ping, 2018. A Novel “All in One” Electrolysis Electrode and
518
Biotechnology Advances 37 (2019) 508–518
Bioreactor Enable Better Study of Electrochemical Effects and Electricity-Aided
Bioprocesses. Eng. Life Sci. 18, 600–610.
Utesch, T., Sabra, W., Prescher, C., Baur, J., Arbter, P., Zeng, A.P., 2018. Enhanced
electron transfer of different mediators for strictly opposite shifting of metabolism in
Clostridium pasteurianum grown on glycerol in a new electrochemical bioreactor.
Biotechnol. Bioeng (Under revision).
Warnecke, T, Gill, R.T., 2005. Organic acid toxicity, tolerance, and production in
Escherichia coli biorefining applications. Microb. Cell Fact. 4, 25.
Westbrook, A.W., Miscevic, D., Kilpatrick, S., Bruder, M.R., Moo-Young, M., Chou, C.P.,
2019. Strain engineering for microbial production of value-added chemicals and fuels
from glycerol. Biotechnol. Adv (In print).
Whitaker, W.B., Joned, J.A., Bennett, R.K., Gonzalez, J.E., Vernacchio, V.R., Collins, S.M.,
Palmer, M.A., Schmidt, S., Antoniewicz, M.R., Koffas, M.A., Papoutsakis, E.T., 2017.
Engineering the biological conversion of methanol to specialty chemicals in Escheri-
chia coli. Metab. Eng. 39, 49–59.
Whitaker, W.B., Sandoval, N.R., Bennett, R.K., Fast, A.G., Papoutsakis, E.T., 2015.
Synthetic methylotrophy: engineering the production of biofuels and chemicals based
on the biology of aerobic methanol utilization. Curr. Opin. Biotechnol. 33, 165–175.
Witthoff, S., Schmitz, K., Niedenfuhr, S., Noh, K., Noack, S., Bott, M., Marienhagen, J.,
2015. Metabolic engineering of Corynebacterium glutamicum for methanol meta-
bolism. Appl. Environ. Microbiol. 81, 2215–2225.
Xafenias, N., Anunobi, M.O., Mapelli, V., 2015. Electrochemical startup increases 1,3-
propanediol titers in mixed-culture glycerol fermentations. Process Biochem. 50,
1499–1508.
Xiu, Z.L., Zeng, A.P., 2008. Present state and perspective of downstream processing of
biologically produced 1,3-propanediol and 2,3-butanediol. Appl. Microbiol.
Biotechnol. 78, 917–926.
Xu, J., Liu, N., Qiao, K., Vogg, S., Stephanopoulos, G., 2017. Application of metabolic
controls for the maximization of lipid production in semicontinuous fermentation.
Proc. Natl. Acad. Sci. U. S. A. 114, 5308–5316.
Yishai, O., Goldbach, L., Tenenboim, H., Lindner, S.N., Bar-Even, A., 2017. Engineered
Assimilation of Exogenous and Endogenous Formate in Escherichia coli. ACS Synth.
Biol. 6, 1722–1731.
Yishai, O., Bouzon, M., Döring, V., Bar-Even, A., 2018. In vivo assimilation of one-carbon
via a synthetic reductive glycine pathway in Escherichia coli. ACS Synth. Biol.
https://doi.org/10.1021/acssynbio.8b00131. (In print).
Yishai, O., Lindner, S.N., de la Cruz, J., Tenenboim, H., Bar-Even, A., 2016. The formate
bio-economy. Curr. Opin. Chem. Biol. 35, 1–9.
Zeng, A.P., Biebl, H., 2002. Bulk-chemicals from biotechnology: the case of microbial
production of 1,3-propanediol and the new trends. In: Schügerl, K., Zeng, A.P. (Eds.),
Tools and Applications of Biochemical Engineering Science. Adv. Biochem. Eng.
Biotechnol. 74. pp. 237–257.
Zeng, An-Ping, Kaltschmitt, Martin, 2016. Green electricity and bio-wastes via biogas to
bulk-chemicals and fuels. Eng. Life Sci. 16, 211–221. https://doi.org/10.1002/elsc.
201400262.
Zeng, A.P., Sabra, W., 2011. Microbial production of diols as platform chemicals: recent
progresses. Curr. Opin. Biotech. 22, 749–757.
Zhang, Wenming, Zhang, Ting, Wu, Sihua, Wu, Mingke, Xin, Fengxue, Dong, Weiliang,
Ma, Jiangfeng, Zhang, Min, Jiang, Min, 2017. Guidance for engineering of synthetic
methylotrophy based on methanol metabolism in methylotrophy. RSC Adv. 7,
4083–4091.
Zhang, Wenming, Song, Meng, Yang, Qiao, Dai, Zhongxue, Zhang, Shangjie, Xin,
Fengxue, Dong, Weiliang, Ma, Jiangfeng, Jiang, Min, 2018. Current advance in
bioconversion of methanol to chemicals. Biotechnol. Biofuels 11, 260.
Zhou, M., Chen, J., Freguia, S., Rabaey, K., Keller, J., 2013. Carbon and electron fluxes
during the electricity driven 1,3-propanediol biosynthesis from glycerol. Environ. Sci.
Technol. 47, 11199–11205.