New Challenges For Syngas Fermentation: Towards Production of Biopolymers

Review
Received: 18 March 2015 Revised: 30 April 2015 Accepted article published: 18 May 2015 Published online in Wiley Online Library: 29 May 2015
(wileyonlinelibrary.com) DOI 10.1002/jctb.4721
New challenges for syngas fermentation:

towards production of biopolymers
Oliver Drzyzga,* Olga Revelles, Gonzalo Durante-Rodríguez, Eduardo Díaz,
José L García and Auxiliadora Prieto
Abstract
Organic wastes are a suitable feedstock for the production of value-added products that have been insufficiently exploited due
to their complexity, which challenges their transformation by conventional procedures. Gasification and pyrolysis of organic
wastes can reduce this complexity by producing syngas (CO plus H2 and other C1 gases), which can be used as a valuable
commodity by catalytic conversion into chemicals. However, the high cost and susceptibility to poisoning of chemical catalysts
have encouraged research on biocatalysts that convert C1 components of syngas into different multi-carbon compounds.
Nowadays, research on syngas fermentation is receiving much attention in order to enhance the productivity of microorganisms
by remodeling their metabolism and by optimizing the bioreactor operational conditions. This review highlights the new
technical achievements of pyrolysis as well as the new biotechnological uses of syngas for the production of bulk chemicals
and biopolymers, discussing the major bottlenecks that challenge syngas fermentation.
© 2015 Society of Chemical Industry
Keywords: syngas; gasification; pyrolysis; C1 gas fermentation; bacteria; commodity chemicals; biorefinery; polyhydroxyalkanoates
INTRODUCTION bio-based polymers (starch, PLA) and monomers (dicarboxylic

The use of oil and other fossil-derived resources such as carbu- acids) to produce biobased biodegradable polymer products
rants, plastics and commodity chemicals is deeply engrained in (composites).5 Other biodegradable polymers are completely
our current society. Although oil reserves are decreasing, the produced by biotechnological processes such as bacterial polyhy-
demand for plastic is steadily increasing. The global annual plas- droxyalkanoates (PHAs). This production is performed by several
tic production is currently about 300 million tons (in Europe companies, mainly in America (e.g. Metabolix) and in Asia (e.g.
approx. 60 MT) with an annual increase of approximately 4%.1 Tianjin GreenBio Materials Co Ltd, and TianAn Biological Mate-
More than 46 million tons of this plastics were used in the EU.2 rials). These ‘bioplastics’ or ‘green plastics’, which are produced
With a decrease of available oil, the production of plastics from as an alternative to petroleum-based plastics and which are
petrochemical resources will also become limited. On the other degraded by many microorganisms, gained in importance in
hand, synthetic plastics from petrochemical resources are harmful recent decades.6 – 9
to the environment, since they cannot be completely recycled The valorization and reuse of wastes via their bioconver-
or because recycling processes are very expensive. In addition, sion into products like bio-based plastics is one of the most
the overwhelming fraction of synthetic plastics is persistent distinctive strategies of the European Bioeconomy. Current
with regard to degradation, and since it is non-biodegradable, waste management technologies are promoting the growth of
it is therefore also not compostable. Furthermore, plastic litter a worldwide industry especially involved in the development of
makes the environment visually unpleasant, negatively impacts waste-to-biopolymers processes. Municipal solid wastes (MSW)
tourism, social sense of well-being, and acts as a hazard to animals and other waste resources, such as agricultural residues and
(especially in its form as microplastics).3 For all these reasons, the sewage sludge from water treatment plants, contain significant
chemical industry is, on one hand, continuing the production reusable carbon fractions suitable for eco-efficient revaloriza-
of traditional plastics but, on the other hand, is developing a tion processes. In this context, thermochemical conversion
broad range of biobased, biodegradable and/or compostable techniques, other than incineration/combustion, such as gasifi-
plastics. Some of these biodegradable plastics are produced cation and pyrolysis are becoming widely accepted as suitable
employing chemical processes but others are produced by a alternatives.10 – 12
combination of biotechnological and chemical processes. This
is the case of lactic acid and succinic acid, produced by fermen-
tation, and subjected to chemical polymerization processes to ∗ Correspondence to: Oliver Drzyzga, Departamento de Biología Medioambi-
produce polylactic acid (PLA) (e.g. Natureworks, Purac (lactic ental, Centro de Investigaciones Biológicas (CIB), Consejo Superior de Investi-
gaciones Científicas (CSIC), Calle Ramiro de Maeztu 9, 28040 Madrid, Spain.
acid, Lactide) or poly-butylene-succinate (PBS) (e.g. succinic acid
E-mail: [email protected]
(Biosuccinium™) by DSM and Roquette (under the joint venture
Reverdia), or by other joint ventures such as Myriant, BioAmber, Department of Environmental Biology, Center for Biological Research (CIB),
1735
and BASF, among others).4 In addition, Novamont is using various Spanish National Research Council (CSIC), Madrid, Spain
J Chem Technol Biotechnol 2015; 90: 1735–1751 www.soci.org © 2015 Society of Chemical Industry
www.soci.org O Drzyzga et al.
Figure 1. Bibliometric data of a ‘syngas/synthesis gas’ focused query in titles of publications of the databases for the last 15 years (2000 to 2014). (A) Web
of Science (Thomson Reuters, USA). (B) PubMed (National Institute of Health, USA). Light grey, ‘syngas’ focused query; dark grey, ‘synthesis gas’ focused
query; black, the sum of both queries.
Pyrolysis and gasification are thermal processes that, similarly acid, butyric acid or biofuels (e.g. ethanol and butanol) either
to incineration, use high temperatures to break down differ- by chemical catalytic conversion (e.g. Fischer-Tropsch (FT) syn-
ent kinds of waste, but consuming less oxygen than traditional thesis) or biological conversion processes. However, biological
mass-burn incineration (combustion). Whereas in the pyrolysis approaches are considered more attractive because they provide
process waste is thermally degraded in the almost absence of several advantages compared with the chemical processes (see
air (and oxygen), gasification is a process in which materials are below).
exposed to some oxygen, but not enough to allow combustion To investigate the increased worldwide interest in syngas tech-
to occur. In some industrial processes, a pyrolytic step is followed nologies we have performed a bibliometric search. Bibliometric
by a second gasification step, to facilitate that more gases can data concerning ‘syngas/synthesis gas’ in publication titles col-
be liberated from the wastes. An important product of gasifica- lected in two well-known literature databases (Pubmed and Web
tion and pyrolysis is syngas (also named synthesis gas). Syngas of Science) for the period 2000 to 2014 showed a significant
contains mainly CO, H2 and to a minor extent CO2 and other increase of published research studies (Fig. 1). Whereas the num-
gases.13 – 16 Syngas can be used as a feedstock because it can ber of technical publications on syngas is relatively high (>150
be further transformed to different chemical products by either publications per year) during this period or even before (Fig. 1(A)),
chemical or biological catalysis. Thus, syngas has been used as publications on syngas involving biological issues have increased
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a feedstock for the production of bulk chemicals such as acetic only very recently, mainly from 2006 (Fig. 1(B)). Interestingly, the
wileyonlinelibrary.com/jctb © 2015 Society of Chemical Industry J Chem Technol Biotechnol 2015; 90: 1735–1751
New challenges for syngas fermentation www.soci.org
term syngas is actually preferred over the term synthesis gas, and and biowastes). There are some biowastes that present more or
we will use it throughout this review. less homogeneous compositions (e.g. glycerol, vegetal oils, corn
Due to the increasing interest in the conversion of biomass steep liquor, molasses, milk, meat) that can be handled by different
into valuable chemicals using syngas fermentation, several recent biological processes. However, a significant portion of biowastes
review articles on this topic have been published.17 – 25 This present is poorly biodegradable and cannot be easily converted to new
review will focus on novel concepts for syngas fermentation and added value chemicals by microorganisms or other biological pro-
will address mainly the production of biopolymers and poly- cesses since it contains usually mixtures of very complex com-
mer biobricks (monomers from biological origin) as a major chal- pounds and some toxic pollutants that are highly recalcitrant
lenge of the European project SYNPOL (Biopolymers from syngas to degradation. These complex biowastes are often insufficiently
fermentation).26 exploited despite being a potential feedstock for value-added
products. Moreover, the current methods for the treatment of
these complex biowastes have major drawbacks mainly concern-
WASTE FEEDSTOCK MATERIALS FOR SYNGAS ing unwanted environmental impacts or low yield. Three major
PRODUCTION examples of these complex wastes are: (i) municipal and commer-
Innovative research and development activities should provide cial wastes; (ii) sludge derived from urban water treatments; and
solutions for sustainable processes and products as well as for (iii) agricultural wastes.
preventing and cleaning-up pollution. The valorization and the
advantageous utilization of anthropogenic derived pollutants and Municipal and commercial wastes
wastes via their bioconversion into valuable products is one of This kind of waste usually contains a mixture of biological com-
the most distinctive strategic orientations. When these new prod- pounds (lignin, carbohydrates, proteins, lipids, etc.), petrochemi-
ucts have the potential to replace existing chemical compounds cal derivatives (plastics, oils, detergents, etc.) and some inorganic
derived from fossil resources, the benefit gained by this strategy materials and, therefore, they represent a clear example of com-
is still higher. A fruitful combination of physicochemical technolo- plex wastes.32,33 As a rule of thumb, about 1 kg of household
gies with engineered biological systems can provide the means waste is generated per citizen per day. In some industrialized coun-
to reach this target, and they have been only recently recognized tries this value can even be double. By 2020, the OECD estimates
for their potential to synthesize commodities and provide services that we could be generating 45% more waste than we did in
beyond the waste treatment. Thus, over recent years, new envi- 1995.28,30 After some separation of recoverable material at source,
ronmental policies and societal concerns have triggered intensive the composition of this waste is normally as follows: one third
research efforts in the production of specialized, high-added-value that is compostable (e.g. kitchen waste); one third that is com-
chemicals and materials from biotechnological technologies that
bustible (e.g. plastics, paper, textiles), and one third that is inert
are cost-effective and benign to the environment.27
(e.g. glass, ceramics, stones, metals). Both combustion in inciner-
ators or dumping into landfill sites of these wastes generate envi-
Types of wastes and major drawbacks of the current removal ronmental damage.28 Landfilling not only takes up more and more
and recycling technologies valuable land space, but it also causes air, water and soil pollu-
On average, each of the >500 million people living in the EU tion, discharging CO2 and methane (CH4 ) into the atmosphere and
throws away around half a ton of household rubbish every year. chemicals and pesticides into the earth and groundwater. In addi-
This is on top of huge amounts of waste generated from activities tion to CH4 , the breakdown of biodegradable waste in landfill sites
such as manufacturing (360 million tons) and construction (900 may release chemicals such as heavy metals resulting in run-off
million tons), while water supply and useful energy production called leachate. Poor or incomplete burning of waste materials can
generate another 95 million tons. Altogether, the EU produces result in environmental and health damage through the release
up to 3 billion tons of waste every year.28 There are different of hazardous chemicals, including dioxins and acid gases. This, in
ways to classify the wastes depending on their origin or by their turn, is harmful to human health, as well as to plants and ani-
properties. Wastes can by classified as solid, liquid, or gaseous mals. Thus, it is important to notice that the combustible fraction
by-products of human-led biological processes, manufacturing, of municipal and commercial waste contains a large amount of
materials processing, consumption of goods, or any other human carbon that could be potentially converted to bioproducts using
activity. According to their compositions, wastes can be generally alternative processes to combustion.
classified as organic (e.g. biomass derived by-products (biowastes)
and recalcitrant products such as plastics and other petrochemical
derivatives) and inorganic (e.g. glass, ceramics, metals). Taking into The sludge derived from urban water treatments
account their origin, they have been classified as municipal or Sludge is also a complex biowaste difficult to revalorize since it has
industrial wastes. Although some wastes might be homogeneous, relatively low carbon content with a high proportion of toxic pol-
many others present very complex compositions depending on lutants. According to the figures provided to the European Com-
their origins.29 mission for the period 2003–2006, about 10 million tons DM (dry
When the complexity of the wastes increases, their removal matter) of sewage sludge were produced in the EU, but this proba-
and/or recycling becomes more difficult, requiring the integra- bly underestimates the total quantities produced.34,35 Sludge pro-
tion of different technologies to achieve more efficient processes. duction has steadily increased between 1995 and 2006 in most EU
In the particular case of biowastes, integration of the technolo- member states. This can be attributed mainly to the implementa-
gies is achieved through the concept of biorefinery.30,31 The term tion of the Urban Waste Water Treatment Directive 91/271/EEC.36
biorefinery is based on a concept analogous to a conventional According to the same sources of information, 36%, about 3.6 mil-
petrochemical refinery, and it has been widely used to describe lion tons DM, was recycled in agriculture. However, the proportion
the chemical refining of many different processes where the feed- of sludge recycled in agriculture varies widely between different
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stock is an organic material from biological origin (e.g. biomass countries. For instance in Belgium, Denmark, Spain, France, Ireland,
J Chem Technol Biotechnol 2015; 90: 1735–1751 © 2015 Society of Chemical Industry wileyonlinelibrary.com/jctb
and the UK, 50% or more of the sludge generated is applied to agri- temperatures or by using a catalyst at a much lower tempera-
cultural land. Incineration and landfilling are the main alternative ture. The first route generally requires temperatures as high as
methods to agricultural recycling for sludge management. Most 1300 ∘ C and generally involves an entrained flow gasifier. The
EU member states treat a proportion of their sludge by incinera- second route includes a fluidized bed gasifier and a downstream
tion and the residual ash is usually disposed of to landfill.36 The catalytic reformer, both operating at approximately 900 ∘ C. When
political discussion on sludge treatment and disposal is increas- gasification is oxygen-blown at temperatures above 900 ∘ C, the
ingly focusing on possible risks for soil and food due to application non-condensable vapor mixture is mostly H2 , CO and CO2 . When
of sewage sludge that may contain organic micro-pollutants. The gasification is air-blown at temperatures between 700 and 900 ∘ C,
amount of sludge that is incinerated significantly increases when the mixture also includes substantial N2 originated from the air,
recycling is discouraged or banned; however, the Landfill Directive which is known as ‘producer gas’.42
99/31/EC sets mandatory targets for the reduction of biodegrad- Therefore, the final gas composition of syngas is strongly depen-
able waste to landfill.37 Moreover, as commented above, incin- dent on the amount of oxygen and steam admitted to the gasifica-
eration has other environmental associated problems. Therefore, tion reactor as well as the time and temperature of the reaction. It is
alternative processes are needed to treat urban sludge. important to notice that when sulfur and N2 are present in syngas,
these compounds are costly to remove and can lead to the forma-
tion of health-threatening sulfur oxides (SOx) or greenhouse gases
Agricultural wastes such as nitrous oxide (N2 O).
The EU estimated that its member states produce 700 million On the other hand, pyrolysis is the thermal decomposition of the
tons of agricultural waste annually. Lignocellulosic materials volatile components of an organic substance, in the temperature
(e.g. dedicated energy crops, such as perennial grasses, forestry range 200–760 ∘ C, but in the absence of air or oxygen. This process
co-products, agricultural residuals and vegetable waste) are excel- forms syngas but also liquids and a mixture of un-reacted carbon
lent examples of feedstock biomass with the potential for flexible char (the non-volatile components) and ash, which remain as a
production of bio-based building blocks. Thus, the production of residual.
high-added-value chemicals from lignocellulosic biomass holds Conventional pyrolysis (CP) of biomass is mainly focused on the
very considerable potential, given the biomass availability (e.g. production of liquid products. In the particular case of the pyrol-
forestry residues, co-products and waste from many different ysis of algae biomass, the studies performed to date have been
sectors including, agriculture, horticulture, forestry, paper and aimed at maximizing the yields of the liquid fraction (oils).43 – 46
pulp and food processing). When dedicated energy plants and However, these oils are often acidic, unstable, and of high viscosity.
forest trees are added to the sources of lignocellulosic materials, Moreover, they contain solids, chemically dissolved water and sig-
immense opportunities for their conversion to ‘green’ chemicals nificant proportions of nitrogen compounds, making it necessary
can be readily recognized.38 However, the carbon content of to apply additional upgrading processes for the oils to be useful
lignocellulose can be only partially converted into useful chemi- for biofuel production.47,48
cals by fermentations due to the difficulties biotransforming the
lignin polymer. Conversion of these recalcitrant waste materials New technologies
to bioproducts by microorganisms through direct hydrolysis and There are other thermal waste conversion technologies that have
fermentation is now challenging. been explored recently such as plasma-arc or plasma-torch gasi-
fication processes. Among these technologies microwave plasma
gasification as well as microwave-induced pyrolysis (MIP) are
SYNGAS PRODUCTION VIA GASIFICATION emerging as the most attractive alternatives due to their higher
AND PYROLYSIS OF BIOWASTES waste conversion and product yields and, at the same time, much
Although biorefineries offer an alternative to fossil lower energy consumption for the own process.11,41,49,50 MIP not
organic-material-based petrochemical refineries, in some cases only overcomes the disadvantages of CP methods, such as slow
new and innovative processing methods to handle for instance heating and necessity of feedstock shredding, but also improves
the more complex wastes are required. Innovative processing the quality of the final pyrolysis products. Table 1 shows a compar-
methods that can be used in biorefineries include gasifica- ison between the syngas produced by MIP or CP pyrolysis of MSW,
tion or pyrolysis of organic materials followed by microbial straw and plastic fractions at two different temperatures. The data
fermentation.18,19,39 Thus, the European Commission has con- demonstrate that MIP outcompetes CP in many pyrolytic processes
sidered pyrolysis and gasification as the main viable large-scale of organic biomass because in some cases the total quantity of
option for biowaste disposal in some cases.40 syngas produced and the content of CO and H2 are higher. In addi-
tion, MIP significantly saves processing time and energy compared
with CP and often produces lower amounts of unwanted com-
Conventional technologies pounds such as CH4 or higher hydrocarbons (>C2 and aromatics) at
Gasification is generally defined as a thermochemical conversion the highest temperatures.51 However, there are several limitations
(750–850 ∘ C) of carbonaceous compounds including biomass and which prevent MIP from being widely employed in the waste man-
organic wastes into syngas mixtures consisting mainly of CO, H2 , ufacturing industry. These include the absence of sufficient data
CO2 , but also some CH4 , N2 , and smaller quantities of hydrocar- to quantify the dielectric properties of the treated waste streams,
bons > C3 (tar).41 Since there is an interaction of air or oxygen and the need for a multi-disciplinary approach to design and develop
biomass in the gasifier, the latter are classified according to the the related conversion units, and the uncertainty about the actual
way that air or oxygen is introduced into them.41 The design of costs. Nevertheless, MIP seems to be an appropriate heating
a gasifier depends upon the type of fuel used and whether the method to obtain syngas for fermentation.51 – 57 MIP reduces the
gasifier is portable or stationary. There are two thermo-chemical generation of wastes, e.g. by re-circulating part of the solid char
1738
ways to produce syngas from biomass: either by applying high to the reactor as a microwave receptor material.56 The microwave
Table 1. Fraction yields (wt%) of different wastes (A) and composition of the syngas fraction (vol%) (B) with conventional pyrolysis (CP) and
microwave induced pyrolysis (MIP) at 400 ∘ C and 800 ∘ C
at 400 ∘ C at 800 ∘ C
(A) Solid Liquid Gas Solid Liquid Gas
MSWa CP: 72.6 14.7 12.7 CP: 40.2 23.7 36.1

MIP: 60.4 12.4 27.2 MIP 43.9 7.8 48.3
Plastic CP: 79.8 9.2 11.0 CP: 11.2 36.8 53.0
MIP: 47.9 19.9 32.2 MIP: 35.2 20.3 44.5
Straw CP: 37.3 38.6 24.1 CP: 24.5 37.9 37.6
MIP: 33.4 20.0 46.6 MIP: 22.8 21.3 55.9
(B) CP MIP CP MIP
MSWa syngas total 54.2 90.5 70.6 94.6
H2 0 46.2 21.4 50.4
CO 16.3 33.3 25.6 36.2
CO2 37.9 11.0 25.5 8.0
CH4 1.1 5.1 12.7 3.3
C2 0.7 4.4 8.5 2.3
otherb 44.0 0 6.3 0
Plastic syngas total 35.5 32.7 30.1 46.2
H2 0 9.1 9.2 26.6
CO 7.3 8.8 9.6 11.5
CO2 28.2 14.8 11.3 8.1
CH4 1.2 14.1 20.3 20.0
C2 3.2 23.8 26.0 17.5
otherb 60.1 29.5 23.7 16.3
Straw syngas total 78.6 89.2 79.8 95.2
H2 0 32.4 19.8 49.1
CO 30.1 23.6 31.1 37.4
CO2 48.5 33.2 28.9 8.7
CH4 2.2 8.6 13.0 3.7
C2 0.9 2.3 2.9 1.2
otherb 18.3 0 4.2 0
a dried municipal solid waste;
b higher hydrocarbons (C , C , >C ) and aromatics; data selection taken from ref. 51
3 4 4
heating mechanism is volumetric and yields quite different prod- higher-added- value products is more expensive and highly sus-
uct distributions by favoring heterogeneous reactions between ceptible to catalyst poisoning, thus limiting the chemical yields.20
the volatiles released and the carbonaceous waste. This makes it Thus, current trends deal with the application of biocatalysts
possible to increase the concentration of valuable products, such (e.g. microorganisms) for converting the C1 syngas fraction into
as CO. Furthermore, the use of MIP will contribute to a more envi- different multi-carbon compounds, such as ethanol, butanediol or
ronmentally sustainable and energy-efficient process compared PHAs.21,26,60
with conventional heating processes. It has been demonstrated The term ‘syngas fermentation’ describes the use of microor-
in a recent study that the energy efficiency of microwave pyroly- ganisms (e.g. bacteria) as biocatalysts for the conversion of C1
sis of wheat straw was 1.5 times higher than the energy efficiency and H2 fractions of syngas to a diversity of chemicals.18,19,23,24,39
achieved by a conventional pyrolysis process.58 Table 2 summarizes the products of industrial interest that can
be obtained by chemical and biological transformations of syn-
gas. Chemicals produced from syngas fermentation include a
variety of compounds such as H2 , CH4 , carboxylic acids (e.g.
SYNGAS FERMENTATION VS CHEMICAL acetic acid, butyric acid), alcohols (e.g. ethanol, butanol), esters,
CATALYSIS or fatty acids (Table 2).61 Syngas fermentation can produce
Today syngas is an important intermediate in the chemical indus- monomers useful for biopolymer synthesis, such as C4 bifunc-
try for chemicals production by the Fischer-Tropsch process based tional compounds (e.g. butanediol, succinate, hydroxybutyrate),62
on an inorganic or metal-based catalyst. Annually, about 6 exo- or directly biopolymers (e.g. PHAs), such as polyhydroxybutyrate
joules of syngas are produced worldwide, corresponding to almost (PHB) or poly(hydroxybutyrate-co-hydroxyvalerate (P(HB-co-HV)
2% of the present total worldwide primary energy consumption.59 (Fig. 3).63,64
Currently, the world market for syngas is dominated by the ammo- Using bacteria as biocatalysts for syngas fermentation offers
nia industry. Other main applications are the production of H2 several advantages over traditional mineral-based catalysts used
for refineries (e.g. hydrogenation steps) and for the production for syngas transformation: (i) biological catalysts can operate
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of methanol.59 However, the chemical conversion of syngas to at temperatures and pressures which are closer to standard
Research related to fermentative production of chemicals from

Table 2. Products from biological fermentation and chemical catal-
ysis of syngas syngas has greatly increased in recent years when the poten-
tial for using biological means of converting CO and CO2 to
chemical catalysis biological fermentation chemicals has been recognized. With the advent of efficient
and inexpensive methods to sequence complete genomes,
methanol acetate
ethanol butyrate
the genetics of many syngas fermenting microorganisms is
dimethylether (DME) long chain fatty acids becoming better understood.39 The use of this knowledge,
diesel ethanol together with the newest tools of Systems Biology that can be
naphtha propanol applied to these microorganisms to enhance the production
ammonia (NH3 ) butanol of chemicals from gaseous C1 compounds through genetic
methane (CH4 ) 2,3-butanediol (BDO) and metabolic manipulations, has just begun.24,60 Meanwhile,
hydrogen methods such as media optimization and reactor design, are
acetone
being pursued to enhance chemicals production from existing
mevalonate
polyhydroxy-butyrate and -valerate and new CO/CO2 -fermenting microorganisms. Further genetic
enhancements combined with new fermentation media and
reactor operational conditions will continue to make fermentative
environmental conditions than traditional chemical catalysts, chemical production more competitive with other technologies
which typically use very high temperatures and at least 10 atm on a cost basis.39 Especially during the last decade, syngas fermen-
pressure; (ii) the output of biological catalysts is less sensitive to the tation has become an attractive technology for the production
ratio of CO to H2 in syngas compared with traditional/commercial of biofuels and indeed several industrial processes mainly for
catalysts that typically require a specific ratio of CO to H2 in ethanol production from syngas are already available from some
order to produce the desired chemicals; (iii) the water–gas shift companies and are produced at pilot or industrial scales (Table 3).
reaction that generates H2 , an interesting energy source and a Strategies for the production of biopolymers are currently emerg-
current biofuel, is implicit in the metabolism of autotrophic and ing as reflected in the basic idea and work flow scheme of the
unicarbonotrophic anaerobes; (iv) biological catalysts are less SYNPOL project platform illustrated in Fig. 2.
sensitive to trace amounts of contaminants in the syngas, such as
char, tar, ash, chlorine, and sulphur. Thus, most catalysts used in
the petrochemical industry are readily poisoned by sulfur-bearing BACTERIA FOR SYNGAS FERMENTATION
gases whereas syngas-consuming anaerobes are often sulphur Despite the general toxicity of CO for most living organisms, sev-
tolerant; (v) biological catalysts tend to be more product specific eral microorganisms (bacteria and archaea), both aerobic and
than inorganic catalysts; (vi) using syngas instead of dissolved anaerobic, can use CO as a source of carbon and/or energy for
sugars as feedstock for fermentations allows the uncoupling of growth. Table 4 summarizes a set of microorganisms that are able
the hydraulic retention time from the substrate supply. This offers to utilize syngas C1 compounds as substrate for growth and for
possibilities to control substrate inhibition and product formation; synthesis of different types of industrially interesting end prod-
and (vii) a simple and direct production of polymers from syngas ucts. These microorganisms can be classified as: (i) autotrophs,
using chemical catalysis does not seem to be feasible, whereas which use C1 compounds as their sole source of carbon and H2
bacterial syngas fermentation would provide such biopolymers. or light as their energy source; and (ii) unicarbonotrophs, which
Table 3. Syngas-applying companies or platforms for the production of commodity products at pilot and/or industrial scale
Feedstock for syngas production;

Enterprise, country (Website) by gasification or pyrolysis Products
Coskata Inc., USA (www.coskata.com) Natural gas, industrial gases, coal, biomass (wood, Fuels and chemicals such as ethanol
crop residues) and waste; by gasification (at lab scale also production of
propanol, butanol, butanediol,
hexanol, organic acids and fatty
acids)
INEOS Bio, Switzerland Biomass such as cellulose, hemi-cellulose and lignin, Ethanol
(www.ineos.com/businesses/ineos-bio/company) and organic waste; by gasification
OPX Biotechnologies Inc., USA (www.opxbio.com) No information available, but the use of CO2 and H2 Fatty acids
from syngas for fermentations is indicated
LanzaTech Inc., USA (www.lanzatech.com) Effluent waste gas from steel mills and coal producers; Liquid fuels (e.g. ethanol) and
woody biomass to syngas; by gasification chemicals (e.g. 2,3-butanediol)
BioMethanolChemie Nederland B.V., The Bio-feedstock like biogas, as well as other feedstock Methanol
Netherlands (www.biomcn.eu/) such as glycerine; by gasification
SyngasBiofuelsEnergy Inc., USA Air CO2 and H2 ; via water electrolysis Fuel n-butanol
(www.syngasbiofuelsenergy.com)
BRI Energy Inc., USA (www.brienergy.com) Municipal solid waste, agricultural residues, animal Ethanol and electrical energy
wastes, and coal; by gasification
SYNPOL project platform, EU (www.synpol.org) Municipal solid waste, sewage sludge and agricultural Polyhydroxyalkanoates,
residues; by microwave induced pyrolysis hydroxybutyrate, butanediol,
succinate
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Figure 2. Overview of the basic idea of the SYNPOL project platform – from different complex organic wastes to biopolymers. Abbreviations: LCA, Life
Cycle Assessment; PHA, polyhydroxyalkanoates.
use C1 compounds as their sole source of carbon and energy. balance has been an important focus of research.121 Rhodospir-
The oxidation of CO is coupled to numerous biological processes, illum rubrum is the type strain for the Rhodospirillaceae and has
such as desulfurization, methanogenesis, hydrogenogenesis, and been the subject of extensive physiological and genetic analy-
acetogenesis.119 There are three major types of CO oxidizing ses. It is capable of growth under the broad variety of conditions
bacteria: (a) phototrophic bacteria; (b) acetogenic bacteria; and (c) mentioned above, including syngas fermentation.63 The genome
aerobic carboxidotrophs. of R. rubrum has been sequenced completely and this bacterium
has been genetically manipulated for more than 20 years.122,123
Rhodospirillum rubrum appears particularly well suited for growth
Phototrophic bacteria on synthesis gas.63 Rhodospirillum rubrum can utilize CO under
Purple non-sulfur bacteria (PNSB) (𝛼-proteobacteria of the Rho- anaerobic conditions as a sole carbon and energy source in the
dospirillaceae family) are widely used as model organisms in micro- presence or absence of light. When exposed to CO, both a CODH
biology and, to an increasing extent, for systems biology. They and a CO-insensitive hydrogenase are induced. Part of the CO2
were extensively studied with respect to the molecular structure produced is assimilated into cell material and the remaining CO2 ,
of their photosynthetic apparatus and attracted further research along with H2 , are released into the environment. Although H2
activity due to their outstanding metabolic versatility and adapt- is produced as a byproduct, it is easily separated from the aque-
ability. Aerobically in the dark, they grow chemoheterotrophically ous phase due to its low solubility. The enrichment of H2 during
by respiration. When oxygen becomes limiting and light is avail- syngas fermentation has made R. rubrum a particularly attrac-
able, these facultative photosynthetic bacteria respond by syn- tive organism for the bioconversion of syngas into added value
thesizing an extensive system of light-capturing intra-cytoplasmic products. Rhodospirillum rubrum has been used for the produc-
membranes. They then switch to photoheterotrophic growth (with tion of PHA (Fig. 3) in continuous stirred tank reactors (CSTR)
an organic substrate as carbon and electron source) or to photoau- fed with syngas.63,64 One of the flux-controlling steps in syn-
totrophic growth. PNSB possess the Calvin–Benson–Bassham gas fermentation by R. rubrum is the mass transfer of CO from
cycle (Calvin cycle) which is essential for autotrophic and partially syngas to the cell culture medium in the bioreactor. Do et al.63
for photoheterotrophic growth. In darkness without O2 they can studied the growth of R. rubrum with seed corn-derived syngas
grow by fermentation or by anaerobic respiration. The ability to containing 8.8% H2 , 17.2% CO and 16.3% CO2 , observing the pro-
utilize N2 as source of organic nitrogen (via a nitrogenase) further duction of a heteropolymer of P(HB-co-HV) (𝛽-hydroxybutyrate
demonstrates the wide spectrum of metabolic abilities of these [86%] and 𝛽-hydroxyvalerate [14%]). The results of their research
bacteria. Besides, the PNSB phototrophic anaerobic bacteria can were used to develop a techno-economic model to demonstrate
use light (photon) to produce hydrogen from CO. H2 is produced the feasibility of PHA production from biomass-derived syngas
with a biocatalytic water–gas shift reaction mediated by CO dehy- fermentation.64
drogenase (CODH).120 Given this extraordinary metabolic versatil- PHB is the most widely and best characterized PHA. PHB
ity, the elucidation of key mechanisms enabling these organisms biosynthetic pathway consists of three different enzymatic
1741
to switch between different lifestyles thereby maintaining redox reactions. The first step is the condensation of two acetyl-CoA
Table 4. Overview of some bacterial products synthesized via syngas fermentation
Microorganism(s) Product(s) References
Acetobacterium woodii acetate 65,66
Acetogenium kivui acetate 67
Alkalibaculum bacchi ethanol 68
Butyribacterium methylotrophicum acetate, butyrate, ethanol 69 – 71
Carboxydothermus hydrogenoformans hydrogen, acetate, ethanol 72
Citrobacter sp. Y19 hydrogen 73
Clostridium aceticum acetate 74,75
Clostridium autoethanogenum acetate, ethanol 76
Clostridium carboxidivorans acetate, ethanol, butyrate, butanol 77 – 81
Clostridium coskatii ethanol 82
Clostridium drakei acetate 83,84
Clostridium ljungdahlii acetate, ethanol, 2,3-butanediol 62,76,85 – 90
Clostridium ragsdalei acetate, ethanol, propanol, butanol 91 – 95
Clostridium sp. MTButOH1365 ethanol, butanol 96
Clostridium sp. MAceT113 acetone 97
Clostridium sp. MTEtOH550 ethanol 98
Clostridium sp. strain P11 ethanol 99,100
Clostridium sp. MT1243 mevalonate 101
Eubacterium limosum acetate, ethanol, butyrate 102 – 104
Moorella thermoacetica acetate 105,106
Moorella sp. HUC22-1 acetate, ethanol 107
Oligotropha carboxidovorans fatty acids 133
Peptostreptococcus productus acetate 108,109
Ralstonia eutropha PHA 110
Rhodopseudomonas palustris hydrogen 111
Rhodospirillum rubrum hydrogen, PHA 63,64,112,168
Thermococcus onnurineus hydrogen 113
Anaerobic mixed culture ethanol, propanol, butanol 114,115
Anaerobic mixed culture TERI SA1 ethanol, acetic acid 116
Thermophilic anaerobic mixed culture acetate 117
(Homo)acetogenic mixed cultures acetate, ethanol, butyrate, butanol 118
molecules into acetoacetyl-CoA by 3-ketothiolase (PhaA). Fur- coded by phaC1 (Rru_A0275), phaC2 (Rru_A2413) and phaC3
ther, the reduction of acetoacetyl-CoA to 3-hydroxybutyryl-CoA (Rru_A1816) genes, respectively. The phaC1 gene is located next
is catalized by acetoacetyl-CoA reductase (PhaB). Finally, the to the phaA (Rru_A0274) and phaB (Rru_A0273) genes in the PHB
(R)-3-hydroxybutyryl-CoA monomers are polymerized into PHB by biosynthetic operon of R. rubrum.124,125
PHB synthase (PhaC). Three different PHB polymerases have been The new bioinformatics tools of Systems Biology provide
identified in the R. rubrum genome, PhaC1, PhaC2 and PhaC3 metabolic models of microorganisms at genomic scale that help
Figure 3. Photograph of a PHB-producing strain of Rhodospirillum rubrum grown with syngas plus acetate as carbon and energy sources (A) and pure
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short chain PHB isolated from a bacterial culture (B); both photographs are from CIB-CSIC, Madrid (Spain).
Figure 4. The Wood–Ljungdahl pathway generally used in acetogenic bacteria.
to approach bacterial metabolism from an integrative point of mol of CO consumed in the presence of H2 . The ratio of protons
view. Given the complexity of R. rubrum metabolism, its modeling pumped across the cell membrane versus electrons transferred
becomes a key factor to integrate the accumulated knowledge from ferredoxin to NAD+ via the Rnf complex is suggested to be
on this organism. This integration will help to gain a deeper and 1.0.126 Thus, acetogenic bacteria behave as favorable biocatalysts
holistic understanding of the metabolic processes allowing us to for the reduction of short-chain carboxylic acids into alcohols due
identify the key enzymatic and regulatory elements that can be to their high substrate and product specificity and because they
remodeled in order to increase the efficiency of PHA synthesis in are susceptible to being genetically modified to avoid side product
this organism. formation from syngas fermentation (e.g. acetate, ethanol).
The new generation sequencing techniques have facilitated the
Acetogenic bacteria complete sequencing of bacterial genomes allowing us to easily
Several anaerobic bacteria are able to ferment C1 gases such as identify the key metabolic pathways, opening possibilities for their
CO and CO2 plus H2 into chemicals, usually acetate, through the manipulation by metabolic engineering. Thus, the recent genome
acetyl-CoA pathway (Wood-Ljungdahl pathway; Fig. 4). These bac- sequencing of two acetogenic bacteria, C. ljungdahlii (4.63 Mb)60
teria are named acetogens and include (among others) species and C. carboxidivorans (5.65 Mb),127 has revealed the genes respon-
such as Acetobacterium woodii, Alkalibaculum bacchi, Butyribac- sible for the Wood–Ljungdahl pathway, including those coding
terium methylotrophicum, and many species of the genus Clostrid- for the CODH enzymes. The C. ljungdahlii genome contains five
ium such as C. aceticum, C. ljungdahlii, C. thermoaceticum, C. hydrogenase encoding genes, four of which code for Fe-only con-
autoethanogenum, C. ragsdalei, and C. carboxidivorans (Table 4). taining hydrogenases and one coding for a Ni–Fe containing
The acetyl-CoA pathway can utilize both CO and H2 as a source hydrogenase. Although the genome of C. carboxidivorans did not
of electrons and CO and CO2 as carbon sources.119 Electrons from show the presence of hydrogenase encoding genes, H2 consump-
CO are generated by an anaerobic CODH and electrons from H2 tion has been observed, suggesting the existence of a novel
are generated by a hydrogenase. The enzyme acetyl-CoA synthase hydrogenase activity.127 An electroporation procedure has been
combines a carboxyl group, which is derived directly from CO developed to transform C. ljungdahlii with plasmids bearing het-
or from the reduction of CO2 , a methyl group, which is derived erologous genes.60 In this sense, C. ljungdahlii, which does not
from a three-step reduction of CO2 , and coenzyme A (CoA) to contain genes necessary for butanol production, was successfully
form acetyl-CoA. Acetyl-CoA can then be converted to acetic acid, transformed to express butanol production genes from Clostrid-
ethanol, or other organic acids and alcohols (Fig. 4).119 For example, ium acetobutylicum, resulting in the production of 2 mmol L−1
Pérez et al.126 demonstrated the potential of acetogenic bacteria, butanol.60 Interestingly, C. carboxidivorans has been shown to pro-
such as C. ljungdahlii and C. ragsdalei, as biocatalysts for conversion duce butanol from CO because C. carboxidivorans contains genes
of short-chain carboxylic, e.g. acetic, propionic, n-butyric, isobu- for butanol production, but it cannot synthesize acetone as other
tyric, n-valeric, and n-caproic acids, into the corresponding alco- butanol producing Clostridia since it lacks CoA transferase genes
hols, using syngas as a source of electrons and energy. Biomass necessary for acetone production.61 Moreover, 2,3-butanediol pro-
yield and fermentation stoichiometry of C. ljungdahlii during syn- duction has been shown recently by three non-pathogenic ace-
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gas fermentation revealed an ATP yield of 0.4–0.5 mol of ATP per togenic Clostridium species (C. autoethanogenum, C. ljungdahlii,
and C. ragsdalei) using syngas as the sole source of carbon and (23.3 kb) encoding a membrane-bound NiFeS-hydrogenase that
energy. Through an analysis of the C. ljungdahlii genome, the com- allows the use of H2 as energy source, and the cbb gene clus-
plete pathway from CO to butanediol has been proposed giving us ter (13.3 kb) encoding most of the enzymes of the Calvin cycle
the chance to improve its production by metabolic engineering.60 for CO2 assimilation. The pHCG3 plasmid contains also a 25.01 kb
The synthesis capabilities of C. ljungdahlii from CO are not limited conjugation module (tra-trb genes) similar to that of the Ti plas-
to alcohols, but can be expanded to virtually every compound, mid from Agrobacterium tumefaciens and, therefore, it might be
for which biological pathways exist or can be artificially engi- self-transmissible to other bacteria.132,136 The cox genes are sub-
neered (synthetic biology). Thus, C. ljungdahlii and most probably ject of CO-specific coordinate transcription and their expression is
other acetogenic clostridia can be used as unique microbial pro- repressed by organic substrates.136 Although most of the genetic
duction platforms based on syngas to generate biobased chemi- determinants for the autotrophic growth of O. carboxidovorans
cal building blocks that could be useful for the synthesis of new in CO are encoded in the pHCG3 megaplasmid, a transposon
biopolymers. mutagenesis study revealed that some mutants unable to oxi-
dize CO contain disruptional insertions outside the cox-cbb-hox
Aerobic carboxydotrophs chemolithoautotrophy module, which suggests that aerobic uti-
Aerobic CO oxidizing bacteria use CO as a source of energy, which lization of CO requires additional functions that might be involved
is oxidized to CO2 with O2 as terminal electron acceptor. These in a CO-insensitive respiratory chain that channels the electrons
bacteria express an O2 -insensitive Mo-Fe-flavin-containing CODH from CODH to oxygen.129,130
encoded by the coxSLM genes. Bacteria that oxidize CO and are We have observed that O. carboxidovorans OM5 is able to
able to chemolithotrophic grow on CO levels >1% and up to produce small amounts of PHB when growing in syngas
90% (v/v) in the gas phase are named carboxydotrophs.128 – 130 (unpublished data). In this sense, a genome search revealed
Carboxydotrophic bacteria are widely distributed in nature and that O. carboxidovorans OM5 harbors genes orthologs to those
they belong to different taxonomic positions (Proteobacteria, involved in the synthesis of PHB in Ralstonia eutropha. The phaA
Firmicutes and Actinobacteria). These bacteria can grow also and phaB genes are organized in an operon, but the phaC gene
organoheterotrophically with a wide range of organic substrates. encoding the PHB synthase is located far from these genes.134,135
When growing autotrophically with CO, carboxydotrophs usu- Thus, O. carboxidovorans OM5 becomes a useful organism to
ally assimilate the CO2 by the Calvin cycle. There are also many develop efficient conversions of syngas to PHB by metabolic
newly recognized CO oxidizers, known as carboxydovores, that engineering, and as a source of chemolithoautotrophy genes that
cannot grow using elevated CO concentrations; instead, these bac- can be transferred to highly efficient PHA-producing bacteria
teria have a mixotrophic metabolism and use the oxidation of CO and hence engineer new recombinant biocatalysts for aerobic
as a supplementary energy source for maintenance or survival, biopolymers production using syngas as feedstock.
organic substrates being the major carbon and energy sources.
It is also known that CO can be oxidized under anaerobic con- Mixed cultures
ditions by some nitrate-respiring bacteria, although this anaer- Bacterial mixed culture fermentation is sometimes more suited
obic CO oxidation does not allow bacterial growth with CO as for industrial applications than pure culture fermentation. Some
sole carbon and energy source. This anaerobic oxidation of CO is of the advantages of mixed cultures are: (i) no need for highly
nitrate-dependent, suggesting that these anaerobic bacteria (car- sterile cultivation; (ii) presence of high microbial diversity, which
boxydovores) harbour a CO-insensitive respiratory chain that shut- offers increased adaptation capacity; (iii) possibility of mixed sub-
tles the electrons towards the nitrate-reductase.131 strates co-fermentation; and (iv) higher capacity for continuous
Oligotropha carboxidovorans (originally named Pseudomonas processing. The application of mixed culture presents an oppor-
carboxydovorans) is the best studied carboxidotroph and it was tunity for higher alcohols production from syngas. Indeed, Singla
isolated from soil of a waste water sewage treatment settling et al.116 enriched an anaerobic mixed bacterial culture capable of
pond. Oligotropha carboxidovorans belongs to the Bradyrhizobi- producing ethanol from syngas fermentation. The screening of
aceae family of 𝛼-proteobacteria, and strain OM5 is the type 13 anaerobic strains together with enrichment protocols helped
strain. Strain OM5 is capable of chemolithoautotrophic growth to develop an efficient mixed culture (called TERI SA1) capable
with CO (up to 90% v/v) but is also able to grow autotrophically of utilizing syngas for ethanol production. Physiological and
with CO2 plus H2 , and can utilize chemoheterotrophically organic operational parameters, i.e. initial culture medium pH, incubation
substrates (e.g. pyruvate, formate, lactate).132 Oligotropha carboxi- temperature, initial syngas pressure, and agitation speed, were
dovorans can utilize syngas for chemolithoautotrophic growth and optimized for enhanced alcohol production. Under these condi-
this type of growth leads to an increase in the production of long tions ethanol and acetic acid production by the selected mixed
chain fatty acids which are major biodiesel components. There- culture were 1.54 g L−1 and 0.8 g L−1 , respectively. Furthermore,
fore, O. carboxidovorans becomes a very interesting biological sys- up-scaling studies in a semi-continuous fermentation mode fur-
tem for bioenergy production.133 Moreover, this bacterium can ther enhanced ethanol and acetic acid production up to 2.2 g L−1
be the subject of genetic manipulation (e.g. expression of genes and 0.9 g L−1 , respectively.116
from plasmids, transposon mutagenesis studies) and its complete Mixed cultures of two or more bacterial strains can sometimes
genome is known, which facilitates its use in biotechnological offer interesting novel production characteristics due to synergis-
processes.134,135 Oligotropha carboxidovorans strain OM5 harbors tic interactions between bacterial species. Several organisms are
a circular low copy number megaplasmid pHCG3 (133.058 bp) known to convert syngas to ethanol, but very few can produce
which determines the ability of the bacterium to grow autotroph- higher alcohols alone. As a potential solution, Liu et al. reported
ically since plasmid cured mutants lose the ability to grow on on mixed culture fermentation between the syngas-fermenting
CO or CO2 . The pHCG3 plasmid contains a chemolithoautotrophy Alkalibaculum bacchi (strain CP15) and the propionic acid pro-
module (cox-cbb-hox) consisting of the cox gene cluster (14.5 kb), ducer Clostridium propionicum.114,115 The monoculture of CP15
1744
encoding the membrane-associated CODH, the hox gene cluster produced only ethanol from syngas without initial addition of
organic acids to the fermentation medium. However, the mixed enzyme inhibition. Thus, minor constituents of impure syngas can
culture produced ethanol, n-propanol and n-butanol from syn- have an adverse effect on bacterial growth and product profiles
gas. The addition of propionic, butyric and hexanoic acids to the during CO fermentation. Moreover, CO is a well-known inhibitor
mixed culture resulted in a 50% higher conversion efficiency of of hydrogenases, and therefore can inhibit the utilization of H2
these acids to their respective alcohols (propanol, butanol and from syngas by the organism. Thus, the concentration and ratio
hexanol) compared to CP15 monoculture. These results illustrate of the gases CO and H2 in the syngas is of vital importance for
one example of the great potential and advantages of mixed cul- the stability of the fermentation process and may be fine-tuned
ture syngas fermentation for the production of higher alcohols for each applied bacterial genus or species. Much literature is
in contrast to the application of pure culture fermentation. Fur- dedicated to the refinement of operational conditions for some
ther syngas-fermenting mixed cultures leading to the production bacterial strains (e.g. low cost media, nutrient substitution, nutri-
of CH4 have also been described, demonstrating the potential of ent limitation, temperature, pH, buffer, trace elements, reducing
using mixed cultures for the production of chemical compounds agents concentration, effect of nitric oxide) and future advances
instead of monocultures that often have to be kept under sterile can be expected to overcome this problematic issue of low bacte-
conditions.137,138 Therefore, it can be expected that more mixed rial growth rates.75,91 – 95,142,143
culture studies (even under non-sterile conditions) will be carried
out in the future to apply syngas fermentation for the production
of higher added value chemical compounds such as biopolymers Transferring CO and H2 from the gas phase to the liquid
as reported in other studies using other feedstock materials.139 – 141 phase of the fermentation medium
Another major challenge of syngas fermentation is overcom-
ing the gas–liquid mass transfer limitation of CO and H2 in the
TOWARDS THE EFFICIENT PRODUCTION OF aqueous phase.25,144,145 Different approaches such as high gas
and liquid flow rates, large specific gas–liquid interfacial areas,
NEW BIO-BASED PRODUCTS BY SYNGAS
increased pressure, different reactor configurations (e.g. CSTR,
FERMENTATION bubble column reactor, gas lift reactor),146 innovative impeller
Since the beginning of the 21st century, various strategies to pro- designs,147 modified fluid flow patterns,148 varying mixing times
duce fuels and chemicals from syngas have been under investiga- and speeds,149 and the use of micro-bubble dispersers,150 have
tion and development.61 Most of this exploration has been focused been examined to enhance gas solubility in the liquid phase of
on the deployment of metal-based catalytic processes. However, the fermentation medium. Many of these approaches increase
as commented above, more recently special attention has been the agitator’s power input to a volume ratio which enhances
paid to the conversion of syngas to biofuels and biochemicals bubble breakup and increases the interfacial surface area avail-
through the microbial metabolic pathways due to the preferences able for mass transfer. This strategy, however, is not economically
proposed for biocatalysts over metal-based catalysts. Currently, attractive for commercial syngas fermentation due to high energy
several syngas fermentation approaches are under evaluation in costs. In addition, higher power inputs can damage the sensitive
research laboratories and pilot plants.91 Although so far only a few microorganisms in the culture media.151 In order to achieve energy
major companies, e.g. INEOS Bio, USA/Switzerland (2008), Coskata, efficient mass transfer, alternative bioreactor configurations such
USA (2009) and LanzaTech, New Zealand/USA (2010) (Table 3), as trickling beds and airlift reactors have been investigated for
have reported the successful operation of large facilities for high syngas fermentation as well.146,152 The gas–liquid volumetric mass
ethanol production via syngas fermentation, fermentation of syn- transfer coefficient (Ka), which represents the hydrodynamic con-
gas is considered by many authors as one of the potential options ditions in a reactor, can be used as a reliable parameter to examine
for the industrial production of second-generation fuels and com- the effective mass transfer rate. In a separate study, Bredwell
modity chemicals (including bioplastics) in the near future. et al.152 reported a maximum Ka of 190 and 75 L h−1 for H2 gas in a
In spite of this increased interest, most of the existing syngas stirred tank reactor at a mixing speed of 300 rpm with and without
fermentation development plants are still at the pre-commercial micro-bubble sparging, respectively, when using a mixed culture
stage and various technical and economic challenges must be of sulfate-reducing bacteria. Munasinghe and Khanal146 reported
overcome before commercial scale plants can be established. Gen- Ka values for CO, ranging from 0.4 to 91 L h−1 for seven different
erally, slow reaction rates and the need for sterile conditions to pre- reactor configurations including a submerged composite hollow
vent media contamination are some of the disadvantages involved fiber (CHF) membrane reactor. Multi-layered CHF membranes
in biological biotech processes. However, in syngas fermentation manufactured from hydrophobic material, such as polyethylene,
the presence of CO in the gas stream ensures sterility as it is toxic to can be used to effectively enhance the gas–liquid mass transfer
most microorganisms. Mass transfer limitation is another issue that in aqueous phase. This technique is not adopted exclusively in
significantly influences fermentation efficiencies due to the slight syngas fermentation, but it has been used extensively in hydrogen
solubility of the gaseous substrates, such as CO and H2, in fermen- and oxygen mass transfer both in water and wastewater treat-
tation broth. So far, a limited range of chemicals, mainly acetate ment applications.153 – 155 Since these CHF membranes consist of
and ethanol, have been produced from syngas through fermen- a non-porous ultra-thin layer (<1 μm), it prevents the permeation
tative processes (Table 4). However, the currently used organisms of liquids on the non-pressurized side, allowing only the gases to
are not efficient producers of other desired chemicals, e.g. biopoly- pass through and increasing the damage resistance of the mem-
mers, and genetic manipulation may be required to improve their brane fibers. In these modules, higher gas saturation levels can be
efficiency.
achieved by increasing the inlet gas pressure.153 Recent studies
focused on the use of CHF membrane to enhance CO mass transfer
Low rates of bacterial cellular growth by evaluating the effects of inlet gas pressures and recirculation
The composition of the gas fed to the fermenting organisms affects flow rates on gas solubility.156 These studies confirmed that the
1745
their metabolism, both for thermodynamic reasons and due to use of CHF membranes is effective and improves the efficiency of
CO mass transfer into the aqueous phase compared with earlier producing both H2 and PHA. Their biorefinery concept considered
CHF experiments. the use of switchgrass as feedstock material and converted that
In a recent study, nanoparticles were used to enhance raw material through thermochemical methods into syngas. The
bioethanol production in syngas fermentation by C. ljungdahlii.157 syngas was then fermented by R. rubrum to produce PHB and H2
Six types of nanoparticles were tested and silica nanoparti- as by-product during the fermentation. A total daily production
cles at a concentration of 0.3 wt% were the most efficient of the biorefinery was assumed to be 12 Mg of PHA and 50 Mg of
at enhancing gas–liquid mass transfer rates. The hydrophilic H2 . Grassroots capital for the biorefinery was estimated to be $55
surfaces of silica nanoparticles were modified with hydropho- million, with annual operating costs at $6.7 million. With a market
bic functional groups such as methyl and isopropyl groups. value of $2.00 kg−1 of H2 , the cost of producing PHA was deter-
Methyl-functionalized silica nanoparticles were better than mined to be $1.65 kg−1 . The results of this analysis demonstrate
unmodified and isopropyl-functionalized silica nanoparticles at that the production of PHA from syngas fermentation using R.
enhancing mass transfer. The dissolved concentrations of CO, CO2 , rubrum is economically viable and technically feasible. The operat-
and H2 were enhanced by 273%, 200%, and 156%, respectively, ing cost of their biorefinery concept is therefore subsidized by the
by using methyl-functionalized silica nanoparticles. The use of production and sale of H2 . The final cost of producing PHA via syn-
methyl-functionalized silica nanoparticles at a silica concentration gas fermentation is less expensive than producing PHA by sugar
of 0.3 wt% during syngas fermentation by C. ljungdahlii led to fermentation, which ranged from $4–6 kg−1 . Nevertheless, the
significant increases in the levels of biomass, ethanol, and acetic major problem is that the bacterial PHA production is always inside
acid production (35%, 166%, and 29%, respectively).157 These the cells (intracellular) and high cell densities must be obtained
strategies to enhance the gas–liquid mass transfer rates of poorly to make such a biotechnological process price-competitive. High
soluble gases into the aqueous phase clearly indicate that further cell densities with C1 gaseous substrates such as syngas are not
advances can be achieved in the near future. easily obtained and they are still a challenge to microbiologists
and biotechnologists.
Low rates of bioproduction Summarizing, methods such as media optimization and reactor
It is widely observed that wild type bacteria do not produce the design are being pursued to enhance productivity from existing
desired chemical compounds in the required amounts useful for and new CO-fermenting microorganisms. With the advent of
their commercialization. To increase products yield it is necessary efficient and inexpensive methods to sequence genomes, the
to optimize both bioreactor operational conditions (see above) genetics of many CO-fermenting organisms are now becoming
and the metabolic pathways that synthesize the desired com- better understood. The use of this knowledge to enhance the
pounds. With respect to the latter, metabolic and flux models synthesis of products from CO through genetic and metabolic
derived for systems biology platforms can be of great help to ratio- manipulation has just begun.159 – 165 Further genetic enhance-
nally design new recombinant organisms that could overcome ments combined with new fermentation media and reactor
the major pathway bottlenecks. Predicted metabolic engineering set-ups will continue to make fermentative biopolymer and build-
changes can lead to delete undesired genes and/or to overexpress ing block production more competitive in comparison with other
genes that are poorly expressed or even absent in the host strain. A chemical technologies on a cost basis.
few studies on overexpressing important genes to enhance strain
productivity from syngas were published recently and thus under-
line this point.96 For example, Berzin et al.96 reported on a Clostrid- FUTURE COMERCIAL PROSPECTS
ium species (Clostridium sp. MTButOH1365) that was metabolically The vision for the sustainable production of biofuels and com-
engineered so that it lost production of ethanol and acetate while modity chemicals can be realized by the successful scale-up of
initiating the production of n-butanol (approx. 300 mmol L−1 ) from syngas fermentation. Therefore, a hybrid biotechnological pro-
syngas. This was the first report on elimination of acetate and cess that combines the newest and most efficient up-, mid- and
ethanol production genes and expression of a synthetic gene clus- down-stream technologies by the application of synthetic biol-
ter encoding n-butanol biosynthesis pathway in an acetogenic ogy, systems biology, genetic and metabolic engineering, biopro-
biocatalyst without the need for antibiotic selection to maintain cess engineering (bioreactor design including novel membrane
the introduced genes.96 technology) is expected to pave the way for the economical and
In another study, Kiriukhin and Tyurin158 eliminated genes effective utilization of organic matter, especially from renewable
unnecessary for syngas fermentation to boost an artificial inte- resources via C1 gas fermentation.166 Promotion of biofuels gen-
grated pathway performance. They engineered an acetogenic erated from syngas bioconversion can provide solutions to the
biocatalyst to selectively overproduce ethanol from syngas or energy security issues and contribute to greenhouse gases miti-
CO2 /H2 blends as the only liquid carbonaceous end product. gation strategies. The high potential of microbial catalysts to con-
An ethanol resistant mutant originally capable of producing vert C1-containing syngas and other waste gas substrates (such
only acetate from CO2 /CO was engineered to eliminate acetate as industrial effluent gases) to valuable biofuels and biochem-
production and spore formation. Bi-functional aldehyde/alcohol icals has been confirmed by various successful scientific stud-
dehydrogenase was inserted into the chromosome of the engi- ies and patent applications. A number of different catalysts for
neered mutant using a Tn7-based approach. Recombinants with mixed alcohol production from syngas were patented in the late
three or six copies of the inserted gene produced 525 mmol L−1 1970s and early 1980s, but most development efforts were aban-
and 1018 mmol L−1 of ethanol, respectively, in five independent doned after oil prices fell in the mid-1980s. However, high oil prices
single-step fermentation runs of 25 days using a syngas blend of reignited the interest. As mentioned above, several companies
60% CO and 40% H2 . Compared with the syngas blend, ethanol are developing competing technologies to commercialize prod-
production was only 64% if a CO2 plus H2 blend was used. ucts by fermentation of syngas (Table 3).22 These companies are
Choi et al.64 conducted a techno-economic analysis to demon- mainly from USA (e.g. Coskata, LanzaTech, Syngas Biofuels Energy,
1746
strate the feasibility of a gasification-based hybrid biorefinery BRI Energy, OPX Biotechnologies) but none of these operate in
Europe yet. Besides patents and patent applications, relatively little 12 Tanigaki N, Fujinaga Y, Kajiyama H and Ishida Y, Operating and envi-
published information is available concerning these private-sector ronmental performances of commercial-scale waste gasification
and melting technology. Waste Manage Res 31:1118–1124 (2013).
activities. Accordingly, we have identified a number of recent US 13 Xie Q, Kong S, Liu Y and Zeng H, Syngas production by two-stage
(e.g. 2011/0229947 2011/0138684, 2011/0104770, 2011/0236919, method of biomass catalytic pyrolysis and gasification. Bioresource
2010/7803589, 2010/0298450, 2011/0008860) and European (e.g. Technol 110:603–609 (2012).
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Research leading to these results has received funding from the 23 Bengelsdorf FR, Straub M and Dürre P, Bacterial synthesis gas (syn-
European Union’s Seventh Framework Programme for research, gas) fermentation. Environ Technol 34:1639–1651 (2013).
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duction of biofuels and commodity chemicals via syngas ferment-
agreement n∘ 311815. ing microorganisms. Curr Opin Biotechnol 27:79–87 (2014).
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New Challenges For Syngas Fermentation: Towards Production of Biopolymers

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Review

(wileyonlinelibrary.com) DOI 10.1002/jctb.4721

New challenges for syngas fermentation:

INTRODUCTION bio-based polymers (starch, PLA) and monomers (dicarboxylic

MSWa CP: 72.6 14.7 12.7 CP: 40.2 23.7 36.1

Research related to fermentative production of chemicals from

Feedstock for syngas production;

Table 4. Overview of some bacterial products synthesized via syngas fermentation

Microorganism(s) Product(s) References

Acetobacterium woodii acetate 65,66

Acetogenium kivui acetate 67

Alkalibaculum bacchi ethanol 68

Butyribacterium methylotrophicum acetate, butyrate, ethanol 69 – 71

Carboxydothermus hydrogenoformans hydrogen, acetate, ethanol 72

Citrobacter sp. Y19 hydrogen 73

Clostridium aceticum acetate 74,75

Clostridium autoethanogenum acetate, ethanol 76

Clostridium carboxidivorans acetate, ethanol, butyrate, butanol 77 – 81

Clostridium coskatii ethanol 82

Clostridium drakei acetate 83,84

Clostridium ljungdahlii acetate, ethanol, 2,3-butanediol 62,76,85 – 90

Clostridium ragsdalei acetate, ethanol, propanol, butanol 91 – 95

Clostridium sp. MTButOH1365 ethanol, butanol 96

Clostridium sp. MAceT113 acetone 97

Clostridium sp. MTEtOH550 ethanol 98

Clostridium sp. strain P11 ethanol 99,100

Clostridium sp. MT1243 mevalonate 101

Eubacterium limosum acetate, ethanol, butyrate 102 – 104

Moorella thermoacetica acetate 105,106

Moorella sp. HUC22-1 acetate, ethanol 107

Oligotropha carboxidovorans fatty acids 133

Peptostreptococcus productus acetate 108,109

Ralstonia eutropha PHA 110

Rhodopseudomonas palustris hydrogen 111

Rhodospirillum rubrum hydrogen, PHA 63,64,112,168

Thermococcus onnurineus hydrogen 113

Anaerobic mixed culture ethanol, propanol, butanol 114,115

Anaerobic mixed culture TERI SA1 ethanol, acetic acid 116

Thermophilic anaerobic mixed culture acetate 117

(Homo)acetogenic mixed cultures acetate, ethanol, butyrate, butanol 118

Figure 4. The Wood–Ljungdahl pathway generally used in acetogenic bacteria.

gasiﬁcation. A review. Waste Manage 32:625–639 (2012). 12:116–140 (2008).

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