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Biotechnological conversion of methane to methanol: evaluation of progress and potential

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Sources of methane are numerous, and vary greatly in their use and sustainable credentials. A Jekyll and Hyde character, it is a valuable energy source present as geological deposits of natural gas, however it is also potent greenhouse gas, released during many waste management processes. Gas-to-liquid technologies are being investigated as a means to exploit and monetise non-traditional and unutilised methane sources. The product identified as having the greatest potential is methanol due to it being a robust, commercially mature conversion process from methane and its beneficial fuel characteristics. Commercial methane to methanol conversion requires high temperatures and pressures, in an energy intensive and costly process. In contrast methanotrophic bacteria perform the desired transformation under ambient conditions, using methane monooxygenase (MMO) enzymes. Despite the great potential of these bacteria a number of biotechnical difficulties are hindering progress towards an industrially suitable process. We have identified five major challenges that exist as barriers to a viable conversion process that, to our knowledge, have not previously been examined as distinct process challenges. Although biotechnological applications of methanotrophic bacteria have been reviewed in part, no review has comprehensively covered progress and challenges for a methane to methanol process from an industrial perspective. All published examples to date of methanotroph catalysed conversion of methane to methanol are collated, and standardised to allow direct comparison. The focus will be on conversion of methane to methanol by whole-cell, wild type, methanotroph cultures, and the potential for their application in an industrially relevant process. A recent shift in the research community focus from a mainly biological angle to an overall engineering approach, offers potential to exploit methanotrophs in an industrially relevant biotechnological gas-to-liquid process. Current innovations and future opportunities are discussed.
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AIMS Bioengineering, 5(1): 138.
DOI: 10.3934/bioeng.2018.1.1
Received: 14 November 2017
Accepted: 14 January 2018
Published: 19 January 2018
http://www.aimspress.com/journal/Bioengineering
Review
Biotechnological conversion of methane to methanol: evaluation of
progress and potential
Charlotte E. Bjorck1, Paul D. Dobson2 and Jagroop Pandhal1,*
1 Department of Chemical and Biological Engineering, University of Sheffield, Mappin Street,
Sheffield S1 3JD, UK
2 The National Centre for Text Mining, Manchester Institute of Biotechnology, Princess Street,
Manchester M1 7DN, UK
* Correspondence: Email: j.pandhal@sheffield.ac.uk; Tel: +441142224914.
Abstract: Sources of methane are numerous, and vary greatly in their use and sustainable credentials.
A Jekyll and Hyde character, it is a valuable energy source present as geological deposits of natural
gas, however it is also potent greenhouse gas, released during many waste management processes.
Gas-to-liquid technologies are being investigated as a means to exploit and monetise non-traditional
and unutilised methane sources. The product identified as having the greatest potential is methanol
due to it being a robust, commercially mature conversion process from methane and its beneficial
fuel characteristics. Commercial methane to methanol conversion requires high temperatures and
pressures, in an energy intensive and costly process. In contrast methanotrophic bacteria perform the
desired transformation under ambient conditions, using methane monooxygenase (MMO) enzymes.
Despite the great potential of these bacteria a number of biotechnical difficulties are hindering
progress towards an industrially suitable process. We have identified five major challenges that exist
as barriers to a viable conversion process that, to our knowledge, have not previously been examined
as distinct process challenges. Although biotechnological applications of methanotrophic bacteria
have been reviewed in part, no review has comprehensively covered progress and challenges for a
methane to methanol process from an industrial perspective. All published examples to date of
methanotroph catalysed conversion of methane to methanol are collated, and standardised to allow
direct comparison. The focus will be on conversion of methane to methanol by whole-cell, wild type,
methanotroph cultures, and the potential for their application in an industrially relevant process. A
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recent shift in the research community focus from a mainly biological angle to an overall engineering
approach, offers potential to exploit methanotrophs in an industrially relevant biotechnological gas-
to-liquid process. Current innovations and future opportunities are discussed.
Keywords: methanotrophs; methane monooxygenase; gas-to-liquid; methane partial oxidation;
biocatalysis; methanol synthesis
Abbreviations: MMO: Methane monooxygenase; GTL: Gas-to-liquid; CNG: Compressed natural
gas; LNG: Liquified natural gas; MTBE: Methyl tert-butyl ether; DME: Dimethyl ether; MTO:
Methanol-to-olefin; GHG: Greenhouse gas; DNA: Deoxyribose nucleic acid; RNA: Ribonucleic acid;
MMO: Methane monooxygenase; sMMO: Soluble methane monooxygenase; pMMO: Particulate
methane monooxygenase; MDH: Methanol dehydrogenase; FADH: Formaldehyde dehydrogenase;
FDH: Formate dehydrogenase; RuMP: Ribulose monophosphate; NADH: Nicotinamide adenine
dinucleotide; PQQ: Pyrroloquinoline quinine; GMO: Genetically modified organism; AMO:
Ammonia monooxygenase; EDTA: Ethylenediaminetetraacetic acid; GC-MS: Gas chromatography-
mass spectrometry; ISPR: In situ product removal; CytC: Cytochrome c; PHB: Polyhydroxybutyrate;
TCE: Trichloroethylene; rRNA: Ribosomal ribonucleic acid; OD: Optical density; DEAE:
Diethylaminoethanol; MBR: Membrane bioreactor
1. Introduction
Methane is simultaneously a valuable energy resource, significant global waste product and a
potent green-house gas (GHG). The volume of waste methane released from anthropogenic sources
is increasing, in addition to natural gas sources becoming increasingly remote and diffuse [1,2]. Gas-
to-liquid (GTL) technologies are being developed to exploit and monetise a range of underutilised
methane resources through chemical conversion to liquid hydrocarbon products that are more readily
stored and transported. Methane to methanol conversion is receiving increased research interest due
to the drive towards sustainable technologies and renewable fuels.
Compared to methane, methanol can be easily used as a feedstock for further chemical
conversion, is suitable for use in the current transportation fuel infrastructure, has a greater energy
density, and burns with fewer toxic by-products [3]. Methanol is produced commercially from
methane via syngas, however the two-step process requires high temperatures (about 900 °C) and
pressures (3 MPa) and as such is energy intensive [4]. Despite the costs associated with the process,
chemical conversion has been successfully commercialised, however the high volumes of methane
necessary to make large-scale processes economically viable are not applicable for marginal fields
and waste methane sources.
In contrast to chemical routes, the oxidation of methane to methanol is performed biologically
by methane monooxygenase (MMO) enzymes in a single step at ambient temperature and pressure.
Unique to methanotrophic bacteria, MMO enzymes catalyse the initial oxidation of methane to
methanol, ultimately allowing the use of methane as a sole carbon and energy source [5]. The
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biological conversion offers methane to methanol conversion in an energy efficient and
environmentally benign manner. In addition, biotechnological processes are well suited to small-
scale operations, appropriate for remote and diffuse methane sources, and require low capital
investment. The potential to exploit methanotrophs for the partial oxidation of methane to methanol
has been explored, however progress towards an industrially relevant biocatalytical process to date
has been minimal due to a range of issues.
Herein, we have identified five major challenges that exist as barriers to a viable conversion
process that, to our knowledge, have not previously been examined as distinct process challenges.
This review will comprehensively analyse recent progress in these areas from an industrial
perspective, in addition to providing tabulated and standardised data for all published examples to
date of the whole cell bioconversion of methane to methanol using methanotrophic bacteria. In
summary, the potential to exploit methanotrophs in a biotechnological GTL process is vast, however
implementation is hindered by the factors identified. Progress in this will be facilitated by the recent
shift in the research community from biologically focused research to take a holistic, engineering
approach, and further work is required at the interface of these disciplines. Current innovations and
future opportunities are discussed.
2. Setting the scene: the energy and environmental context
It is now widely accepted that a significant deviation from the unsustainable global energy
situation is necessary. The scientific consensus is that the Earth’s climate is being affected by human
activities [6], attributed to the release of GHGs, with atmospheric concentrations at unprecedented
levels [6]. Combustion of fossil fuels for energy production is responsible for the majority of GHG
emissions, whilst also being available as finite resources, and so alternative fuels that are both
sustainably sourced and produce lower emissions are of interest.
As such, methane has received increased attention from the scientific community. A Jekyll and
Hyde character, sources of methane can be divided into anthropogenic and natural, while there is also
a distinction between those that are traditionally utilised commercially and those that result in
atmospheric and biogenic accumulation. The resource most frequently exploited is geological
deposits of fossil formed natural gas, used predominantly for energy generation. As a product of the
anaerobic decay of biomass, however, it can be considered either a renewable carbon source or a
potent GHG, depending on its final use or treatment.
2.1. Methane sources and uses
The main component of natural and shale gases, methane is considered a next-generation carbon
feedstock due to the vast global reserves [7], with geological deposits of the fossil formed gas
frequently exploited for energy generation. In addition, it is the main constituent of biogas, produced
by the microbial digestion of biomass under anaerobic conditions, and as such is a product of many
waste management processes and agricultural activity including enteric fermentation and rice
cultivation [1]. However methane is an abundant and potent GHG. The greatest source to the
atmosphere is as a result of anaerobic decay of biomass, with anthropogenic contribution through
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industrial waste production on the increase [8]. Also the energy sector is responsible for significant
methane emissions released during fossil fuel exploration, extraction and transportation.
Anthropogenic methane emissions in 2010 were estimated to be 481 billion m3 methane,
equivalent in global warming potential to 6867 Mt carbon dioxide [1], with atmospheric methane
concentrations at unprecedented levels, having increased from 715 ppb to 1774 ppb over the past 300
years [6]. This, combined with a global warming potential 25 times greater than carbon dioxide, have
resulted in methane being the second most significant greenhouse gas after carbon dioxide,
contributing more than one-third of current anthropogenic warming [1]. Methane has a much shorter
global atmospheric lifetime (12 years) compared with carbon dioxide (5200 years) [6], so it would
be possible to rapidly reduce atmospheric concentrations through a reduction in emissions. Methane
mitigation strategies offer both the potential to curb atmospheric accumulation and the associated
climate impact in addition to providing a valuable industrial fuel source and chemical feedstock.
Global natural gas production in 2013 was estimated to be 3369.9 billion m3, with proven
reserves of 185.7 trillion m3 [9]. Of this it is estimated that between 30% and 80% is “stranded
gas” [10], defined as natural gas that is wasted or unused because the gas field may be too small or
remote for production to be economically feasible [2]. An additional environmental concern is the
considerable amount of associated gas that is flared or vented during oil production encouraging the
implementation of restrictions on such processes. In 2008, 139 billion m3 of natural gas was flared
globally; equal to 4% of global natural gas production, and resulted in the release of more than
278 Mt of carbon dioxide [11].
Combining the volume of anthropogenic waste methane emissions, stranded and associated gas
demonstrates the huge amount of unutilised global methane sources and the waste of a valuable
resource.
2.2. Gas-to-liquid technologies
One difficulty in the use of methane is that it occurs as a gas under ambient conditions (boiling
point 164 °C) and so storage and transportation are costly, further compounded for diffuse and
remote non-traditional sources. Conversion to compressed natural gas (CNG) or liquefied natural
gas (LNG) are energy intensive and require large capital investment, in addition to being hazardous
due to their high pressure (2125 MPa) and low temperature (164 °C) [2]. This, and the potential to
monetise unutilised methane sources has initiated interest in GTL technologies to chemically convert
methane to liquid hydrocarbon products that are more readily transported. The most widely deployed,
commercially demonstrated GTL technologies utilise the Fischer-Tropsch conversion process to
produce diesel, naphtha and waxes [10], although other conversion technologies are being
investigated to generate products including methanol, dimethyl ether (DME) and olefins. GTL
processes offer market diversification and an opportunity to harness remote natural gas resources,
although high costs, price risks, reliability and technical difficulties have hindered implementation [4].
A number of factors impact the suitability and success of such technologies including scale, capital
cost and potential markets for products [4]. Large-scale projects offer economies of scale but
typically require high capital investment and a constant high volume input of methane, not often
available at remote sources. Ultimately, the commercial viability of a plant is determined by natural
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gas and product prices. Volatility and uncertainty in these markets make justifying the large capital
investment problematic. Interest in small-scale, modular GTL units has increased for reasons
contrasting large-scale projects, such as the suitability for use at low volume gas sources and reduced
capital investment. It is anticipated that unit cost and reliability will have the greatest impact on the
uptake and success of such technologies [4].
2.3. Methanol as a sustainable liquid fuel
Conversion to methanol is an attractive option due to the range of applications and growing
market; global methanol demand reached 70 Mt in 2015 [12].
Traditionally used as a solvent and feedstock, methanol is utilised in the synthesis of industrially
relevant compounds including acetic acid, formaldehyde, methyl tert-butyl ether (MTBE), and
dimethyl ether (DME), whilst the methanol-to-olefin (MTO) process can be used to produce ethylene
or propylene which can be further processed into a range of hydrocarbon and organic polymeric
materials (Figure 1).
Figure 1. Possible industrial transformations from methanol, including production
processes and products [13].
Methanol also offers great potential as an energy carrier or fuel, with an energy density greater
than that of methane (15.6 MJ L1 and 36.6 × 103 MJ L1 respectively). There are five main fuel
applications for methanol: directly as a transportation fuel; blended with petrol; converted to DME to
be used as a diesel replacement; in the production of biodiesel via trans-esterification; in fuel cells to
generate electricity. The current energy situation has increased interest in methanol in transportation
fuel applications due to the potential for sustainable, carbon neutral sources, low cost compared to
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other fuels, and clean burning [14], in addition to offering a range of advantages over both methane,
and current petrochemical fuels. Methanol exhibits favourable combustion properties, increased
engine performance and greater efficiencies over those achieved with gasoline [3]. Another
advantage of methanol over petroleum based fuels is that it is considered both safer, with a lower or
comparable toxicity, and more environmentally benign in case of uncontrolled release as it is highly
biodegradable [15].
Perhaps the greatest advantage of methanol as a fuel is related to the GHG emission reduction
potential. Comparison of life cycle carbon intensity analysis shows that emissions from methanol as
a fuel are heavily dependent on the feedstock source. Methanol from natural gas has slightly lower
carbon emissions compared with those from conventional gasoline fuels. However, sustainably
produced bio-methanol is the lowest of results calculated [3]. Combustion of methanol produces
fewer toxic by-productsabout half as much carbon monoxide and an eighth as much nitrogen
oxides (NOx)compared with gasoline [16]. In terms of point-of-use emissions for transportation
fuels, methanol generates lower carbon dioxide emissions per unit energy than conventional
petrochemical fuels [17]. It is possible to blend methanol with petrol to increase the octane value and
reduce the cost, without the need for any engine modifications [18], however at high levels it is
corrosive requiring specific compatible engines. In an interdisciplinary report prepared by MIT [19]
on the future role of natural gas as an energy source, conversion to methanol is identified as having
the lowest cost and GHG emissions in comparison with alternative liquid fuel products as well as
being the only potential transformation that has been produced for a long period at an industrial scale.
2.4. Conversion of methane to methanol
In theory, conversion of methane gas to liquid products offers many advantages, however the
current reality is that processes are energetically inefficient and costly. The highly inert nature of
saturated hydrocarbons makes chemical transformation challenging, and although high temperatures
and pressures can be employed to promote reaction, this often results in loss of selectivity and low
yields. As the dissociation energy of the CH bond in methane (440 kJ mol1) is greater than
methanol (393 kJ mol1), under oxidising conditions, the product methanol reacts preferentially to
methane forming a mixture of products including carbon monoxide, carbon dioxide, formaldehyde,
and formic acid [20].
Current commercial methanol production overcomes selectivity issues by utilising a two-step
process in which fossil methane is first converted to syngas via steam reforming, followed by the
metal-catalysed methanol synthesis step in an overall endothermic process = +116 kJ mol1) (Figure 2).
Initial conversion of methane and water to carbon monoxide and hydrogen is an energy intensive
process, operating at temperatures around 900 °C and pressures of 3 MPa [4]. Harsh reaction
conditions necessitate costly equipment that account for approximately 60% of the process capital
costs [21,22]. In addition, the overall process has a conversion rate of ~25% and selectivity ~70% [21].
Interest in a direct, single step oxidation of methane to methanol is vast, driven by many
potential advantages over the conventional two-step process. In contrast to the indirect commercial
process, the single step oxidation of methane to methanol is an exothermic
reaction (ΔH° = 128 kJ mol1) (Figure 3), avoiding the energy intensive, inefficient and expensive
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syngas formation step. Mild reaction conditions also negate the need for specialist, costly equipment.
Despite significant interest and effort in the direct partial oxidation of methane to methanol, many
difficulties remain unsolved and as such the selective CH bond activation required often is
described as the “Holy Grail” of Chemistry [23].
Figure 2. Energies of indirect methanol formation from methane via syngas [24].
Figure 3. Energies of direct methanol formation from methane [24].
In summary, methane is a major contributor to the climate change problem that could instead be
exploited as a chemical industry feedstock and as a fuel. Conversion to methanol is an attractive
proposition but existing processes are difficult to apply to less accessible methane sources.
Essentially this is due to difficult chemistry that biology has already evolved mechanisms to exploit.
Due to it being an efficient energy store, convenient fuel suitable for use in the existing transport fuel
infrastructure and raw-material for synthetic hydrocarbons, arguments exist for the implementation
of a “methanol economy” as an alternative to the current fossil-fuel based situation [25].
3. Methanotrophic bacteria
In nature, methanotrophic bacteria are able to perform the controlled partial oxidation of
methane to methanol at high conversion and selectivity, allowing the use of methane as a sole carbon
and energy source. This unique ability makes methanotrophs a valuable candidate for the
bioconversion of methane to methanol. Driven by the prospect of commercial exploitation for
biocatalysis and bioremediation, interest in these microorganisms has increased over the last 30 years [5].
3.1. Methanotrophs: a brief introduction
The first methanotroph was isolated by Söhngen in 1906 which he named Bacillus methanicus [26].
Since then the most significant contribution was made by Whittenbury and his colleagues in 1970, in
which over 100 methane-utilising bacteria were isolated, characterised and compared [27]. Playing
an important role in the global methane cycle, methane-oxidising bacteria are found across a broad
range of natural environments, present in nearly all samples taken from soil, swamps, rivers, oceans,
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ponds and sewage sludge, reportedly representing up to 8% of the total “heterotrophic”
population [28]. As the majority of methane is naturally produced through the anaerobic decay of
organic matter, they are found primarily at oxic-anoxic interfaces. The majority of known
methanotrophs are aerobic, however, methane oxidation is known to occur in anaerobic
environments by coupling oxidation to sulphate [29] and nitrite reduction [30]. As expected from
their prevalence within the environment, methanotrophs are found in both mesophilic and extreme
environments. Strains have been isolated from temperatures as low as 4 °C [31] and as high as
72 °C [32] and it has been demonstrated that populations of methanotrophs in nature adapt to
different temperatures [5].
Two populations of methanotrophs have been identified that exist depending on environmental
methane availability [33]. Low affinity methanotrophs are able to utilise methane at high
concentrations (>40 ppm), and are observed in soils with high methane exposure, accounting for all
isolated methanotroph cultures known to date. High affinity methanotrophs are able to oxidise
ambient methane concentrations (~ 2 ppm) and although their existence within soil samples has been
verified using molecular techniques, isolation of such bacteria has not yet been possible. Analysis of
nucleic acids (DNA and RNA), phospholipids, methane oxidation rates and stable isotope
probing (SIP) using 13C labelled methane has provided characterisation information, and confirmed
relatively low abundance of these high affinity methanotrophs in soils [34]. In contrast to the
relatively high abundance of methanotroph populations present in the environment, these low affinity
methanotrophs account for <0.01% of total bacteria biomass in soils, attributed to low atmospheric
methane concentrations [34].
Methane monooxygenase (MMO) enzymes catalyse the initial oxidation of methane to
methanol, followed by sequential oxidation to formaldehyde by methanol dehydrogenase (MDH),
oxidation of formaldehyde to formate by formaldehyde dehydrogenase (FADH), and finally formate
to carbon dioxide by formate dehydrogenase (FDH). Formaldehyde is assimilated into biomass by
either the ribulose monophosphate (RuMP) pathway, or the serine pathway. Figure 4 illustrates the
metabolism of methane by methanotrophs.
Figure 4. Pathways for the oxidation of methane and assimilation of formaldehyde in
methanotrophic bacteria [5,35].
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Traditionally, all aerobic methane-oxidising bacteria were of the phylum Proteobacteria, and
classified into two major groups: Type I and Type II, based on differences in physiological and
morphological traits, with Type X methanotrophs further differentiated from Type I [36,37]. Recent
characterisation of several new genera and species, and the subsequent increase in the diversity of
known methanotrophs, meant this system was no longer useful to characterise all known species,
resulting in an update in the taxonomical description used to classify these organisms.
Methanotroph species are now known in Proteobacteria, Verrucomicrobia and candidate
phylum NC10. Proteobacteria methanotrophs are divided into two classes; Alphaproteobacteria and
Gammaproteobacteria. Current classification further divides Gammaproteobacteria, of the order
Methylococcales, into three families: Methylococcaceae, which is further separated into Type Ia,
including a total of 13 genera, and Type Ib including four genera (Methylococcus, Methylocaldum,
Methylogaea and Methyloparacoccus); Methylothermaceae, Type Ic, (genera: Methylothermus,
Methylohalobius, Methylomarinovum); and Crenotrichaceae which includes a single
genus (Crenothrix polyspora) that to date has not been isolated as a pure culture. Methanotrophs of
the family Methylococcaceae, of which the majority are Type Ia, utilise the RuMP cycle for carbon
assimilation and have intracytoplasmic membranes arranged as a uniform array of bundles of
vesicular disks distributed evenly across the cell. Differing from Type Ia methanotrophs in the
expression of low levels of the ribulose-1,5-bisphosphate carboxylase enzyme, in addition to the
RuMP pathway, genera formally identified as Type X were renamed Type Ib. The methanotrophic
Alphaproteobacteria have been divided into two families; Methylocystaceae, Type IIa (genera:
Methylocystis, Methylosinus) and Beijerinckiaceae, Type IIb (genera: Methylocella, Methylocapsa,
Methyloferula) methanotrophs. Type II methanotrophs of the family Methylocystacea, utilise the
serine pathway for formaldehyde assimilation, with the intracytoplasmic membrane arranged as
stacks of vesicles in parallel to the cell membrane. Species of the family Beijerinckiaceae, identified
as Type IIb, differ from Type IIa methanotrophs in that cells of Methylocella and Methylocapsa do
not contain an intracytoplasmic membrane, while in Methyloferula they are found only on one side
of the cell. Extremophilic methanotrophs belonging to the phylum Verrucomicrobia, of the genus
Methylacidiphilium are sometimes described as Type III. Able to grow across a wide range of
temperatures, they are unique in comparison with all other known methanotrophs due to their
extremely acidophilic phenotype [38,39]. As with the majority of methanotrophs they possess
pMMO but lack the familiar formaldehyde assimilation pathways, instead utilising the Calvin-
Benson cycle for carbon fixation [40,41]. Anaerobic methane oxidation, coupled with nitrite
reduction, has been observed in bacteria of the candidate phylum NC10, and named Ca.
Methylomirabilis oxyfera [30]. See reviews by Knief [42], and Semrau [43] for comprehensive
reviews of the current taxonomy of aerobic methanotrophs.
Methylosinus trichosporium OB3b (for “oddball” strain 3b) [44] and Methylococcus
capsulatus (Bath) (originally isolated from the hot water baths in Bath, UK) [45], have proven
themselves to be the experimental workhorses. The majority of investigation into the effect of
environmental growth conditions, metabolic characterisation work, and consideration into
commercial applications has been performed using these two strains leading to for full genomic
characterisation [44,46], further enhancing molecular and system level research in the species.
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3.2. Biochemistry of methane oxidation using methane monooxygenase (MMO)
Unique to methanotrophs is possession of MMO enzymes that catalyse the oxidation of methane
to methanol. The initial reaction in the sequential oxidation to carbon dioxide, it is oxygen dependent
and the most chemically difficult step. The monooxygenase splits the OO bond of dioxygen using
two reducing equivalents, with one of the oxygen atoms incorporated into methane to form methanol
and the other reduced to water [47].
Two types of MMO have been found in methanotrophic bacteria; a soluble cytoplasmic
form (sMMO) and a particulate membrane-bound form (pMMO). All methanotrophs, with the
exception of members of the genera Methylocella [48] and Methyloferula [49], have the ability to
produce pMMO, however Type II methanotrophs are also able to produce sMMO. For those strains
able to produce both forms, the environmental growth conditions are responsible for dictating the
type of enzyme expressed within the cell, with dependence primarily on the availability of copper.
Under conditions of copper excess (>0.85 µmol g1 dry weight of cells) pMMO is produced
preferentially, while under conditions of limited copper availability, sMMO is generated, although
the two are not mutually exclusive [50]. The dependence on copper availability is attributed to its
presence in the active site of the pMMO enzyme [51].
The relative ease of isolation of sMMO has resulted in it being thoroughly studied and fully
characterised [47,52]. sMMO is known to be made up of three protein components: a
hydroxylase (MMOH), a regulatory protein (MMOB), and a reductase (MMOR). The hydroxylase
protein is made up of three polypeptide subunits, arranged as a α2β2γ2 dimer, and contains a di-iron
active site where oxygen and methane react using electrons supplied from NADH oxidation at the
reductase, facilitated by the regulatory protein [47]. In contrast, significantly less is known about the
biocatalysis and structure of pMMO, despite it being more prevalent in nature, due to difficulties in
isolation and stability of the membrane bound protein. pMMO is known to be comprised of three
subunits: PmoA, PmoB and PmoC, arranged in a trimeric α3β3γ3 complex, with the di-copper active
site on the soluble part of the PmoB subunit [53,54]. A number of recent reviews give full structural
and mechanistic details of sMMO and pMMO [47,5557]. Despite their similar function within the
cell, sMMO and pMMO are not related structurally or genetically.
Both forms of MMO are able to co-oxidise a range of organic substrates in the presence of
methane although they do not support in vivo growth [58,59]. sMMO exhibits broader substrate
specificity and is able to catalyse a larger number of biotransformations than pMMO. Preferential
oxidation of smaller substrates by the pMMO system led to understanding that access to the active
site of pMMO is sterically more restricted than sMMO [60]. The differences between the two forms
of MMO have been predicted to lead to specific phenotypic differences. Cells that contain pMMO
have greater growth yields, attributed to a reduction in the energetic requirement, and exhibit a
higher affinity for methane than those containing sMMO [61,62]. This is related to differences in
reducing power utilisation between pMMO and sMMO. Electrons required for the initial oxidation
step are provided by NADH in the sMMO catalysed reaction, whereas pMMO utilises reducing
equivalents provided by the MDH co-factor pyrroloquinoline-quinone (PQQ) [61,62].
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3.3. Utilising methanotrophic bacteria as biocatalysts for the oxidation of methane to methanol
The majority of biological methods to exploit this single step transformation are based on
utilising or mimicking powerful MMO enzymes that activate the CH bond in methane in a highly
selective process at ambient temperature and pressure. MMO-catalysed partial oxidation of methane
to methanol has a number advantages over thermochemical oxidation routes, including higher
selectivity, improved process efficiency and safety, milder reaction conditions and energy savings,
all leading to associated economic benefits.
A number of approaches have been investigated to exploit the powerful oxidising ability of
methanotrophic bacteria, with varying potential for use in industrial processes. Below is a brief
overview of these, including consideration of their suitability for use in an industrially relevant
process. As the method with greatest potential, exploiting whole cell methanotroph cultures will be
the focus of this review.
3.3.1. Whole cell methanotroph cultures
Whole cell methanotroph cultures have the potential to be a relatively cheap route for the
bioconversion of methane to methanol. The generation of biomass is reasonably simple and cost
effective, whilst the more involved molecular operations, such as the synthesis of key MMO
enzymes and necessary reducing equivalents, are controlled entirely by the bacteria. Whole cells also
have the capacity for self-maintenance and replication. Moreover, there are downstream processing
benefits because although the biochemical reactions occur within the intracellular space, the
methanol accumulates extracellularly, which facilitates product isolation.
Although currently the preferred option, whole cell biocatalysts do pose a number of challenges.
Being closely specialised to a particular niche constrains their deployment in dissimilar
biotechnological process operating conditions. High cell density culture also has proven difficult,
which has been attributed to gas-liquid transfer limitations [63,64]. The complex nature of cellular
metabolism in the case of methanotrophic bacteria presents the risk of over oxidation to
formaldehyde, in addition to complications associated with interrupting the natural biochemical
pathways of the cell. An added level of process difficulty exists as it is necessary to design a biphasic
growth process for both cell growth (enzyme manufacturing phase) and bioconversion (methanol
production phase).
3.3.2. MMO enzyme isolates
Using methanotrophic bacteria for the desired transformation is ultimately a way to exploit the
powerful MMO enzyme. Substantial research efforts mean MMO enzymes are fully characterised
with a high level of understanding of their biochemistry. An alternative strategy uses enzyme isolates
from cell cultures. By avoiding various complex cellular interactions, and therefore performing only
the desired reaction, the particular benefit of the isolate strategy is that it avoids over oxidation of
methanol through normal cellular metabolism. The bacteria still perform the difficult MMO
production and, in the absence of other cellular components, process interactions are simplified and
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cellular toxicity is not an issue. Using cell-free preparations of the MMO enzyme however poses
difficulties in isolation and purification attributed to instability of the purified enzyme [57].
Complications associated with working with an integral membrane bound protein hinder the use of
pMMO, although cytoplasmic sMMO is more readily isolated. Typically stabilisation is achieved by
enzyme immobilisation on or in artificial matrices. Even so, Evolution did not optimise Nature’s
catalysts for technical process conditions and so stability, activity and lifetime become process
issues [65]. Additionally, cofactor dependency and the necessary supply of exogenous reducing
equivalents favours the use of whole cells. The energy requirements for the system for both biomass
production and bioconversion are equivalent to using a whole-cell culture, without the advantage of
cell maintenance.
3.3.3. Genetically modified organisms
It is possible to combine the advantages of whole-cell systems with optimised reaction
processes. By using recombinant microorganisms containing artificial synthetic pathways,
methanotrophs offer the potential for specific biotransformations (beyond just methane oxidation to
methanol) and improved product yields. Progress here is currently hindered by the inability to
express functional MMO proteins in Escherichia coli [6668]. Away from scientific ability and
innovation, the production and use of GMOs (genetically modified organisms) poses significant
ethical consideration [69,70]. In the case of methanotrophs, Calysta have patented a process for the
biological oxidation of hydrocarbons using a genetically engineered form of Methylosinus
trichosporium OB3b, although a lack of published data to verify the system exists [71].
3.3.4. Synthetic MMO analogues
The thorough characterisation and understanding of the biochemistry of MMO enzymes
suggests the design of synthetic biomimetic catalysts with the potential to offer the advantages of
using enzyme isolates combined with the stability of a thermochemical process. Biologically-
inspired organometallic compounds might be designed so as to maximise selectivity, yield, reaction
rate and conversion efficiency, whilst increasing tolerance to process conditions compared with
purified enzymes, avoiding issues relating to instability of isolated membrane bound proteins, and
being less susceptible to product inhibition [72]. However, taken together these objectives constitute
a challenging optimisation problem that likely necessitates a similarly complicated molecular
machine to MMO enzymes, which will challenge chemical synthesis. This must be contrasted with
the ease and efficiency with which methanotrophs produce powerful MMO enzymes, especially
given that our rapidly increasing ability to design proteins [73] with improved properties should
overcome many of the limitations of MMO enzymes. Despite efforts, synthesis of a chemically
active MMO analogue has not yet been achieved [72].
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3.3.5. Ammonia-oxidising bacteria
An alternative option utilises ammonia-oxidising bacteria containing the ammonia
monooxygenase (AMO) enzyme, a pMMO homologue. Similar in both structure and function to
pMMO, under typical cellular conditions, AMO catalyses the oxidation of ammonia (NH3) to
hydroxylamine (NH2OH), followed by the hydroxylamine oxidoreductase catalysed oxidation to
nitrate (NO2). The metabolism of ammonia generates reducing equivalents for the cell, whilst
carbon dioxide is used as a carbon source [74]. Being similar in structure to pMMO, the low
substrate specificity of AMO also allows it to oxidise methane to methanol [75,76].
Despite the potential of ammonia oxidising bacteria, a number of challenges exist before
commercial implementation will be possible, including slow reaction rates, high costs and technical
immaturity.
4. Challenges and potential strategies associated with the methanotroph catalysed conversion
of methane to methanol
The major challenges faced in developing an industrially relevant biological partial methane
oxidation process are described below, in addition to approaches investigated to overcome these and
optimise reaction conditions. The success of these can be measured in terms of greater biomass
concentration, improved methanol yields and enhanced enzyme activity.
4.1. Challenge I: gas-liquid mass transfer limitations
As in the majority of fermentation processes, biomass and cellular product generation will be
limited by the availability of metabolic gases, especially at high cell densities. This problem is
intensified by the sparingly soluble nature of gases such as oxygen and methane.
It has been demonstrated that the low rates of gas-liquid mass transfer of methane in aqueous
culture is a growth limiting factor [63,64] which is responsible, in part, for slow growth and
difficulty in high biomass production. As MMO is a growth associated enzyme, high cell density
cultivation can result in an increase in MMO containing biomass [77], which is desirable for the
proposed biotransformation.
A number of process factors can be addressed to optimise the solubility of gaseous substrates in
methanotroph culture including reactor design, gas delivery method and temperature. As with all
gases, the solubility of methane decreases with increasing temperature [78], and so to maximise
dissolved methane and availability for the methanotroph culture, a low temperature is desired. This
parameter is restricted, however, by the optimum temperature for culture growth and maintenance, as
well as for MMO reaction and stability. The impact of temperature is further detailed in Section 4.6.2.
4.1.1. Optimised reactor design to maximise mass transfer
Current investigation has focused on employing membrane reactors for the methanotroph
catalysed methane oxidation. The delivery of gaseous substrates through two porous membranes
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allows the separate feed of methane and air to the reactor, reducing risks involved in using
potentially explosive mixtures of gases (methane in air is explosive between 5% v/v and 15% v/v).
The gases are delivered to the methanotroph culture through membrane contactors that offer a large
surface area and avoid bubble formation, both of which optimise gas-liquid mass transfer.
Duan et al. first demonstrated methane bioconversion in a dense silicon tube stirred membrane
reactor. Through improved methane delivery to the liquid phase, methanotroph culture was possible
at high cell densities up to 17.3 dry cell g/L, producing 0.95 g/L methanol after 40 h [79]. Pen et al.
since designed and demonstrated methanotroph biocatalysis in a novel recirculating macroporous
membrane bioreactor (MBR). The mass transfer achieved was twice that observed in a batch reactor
in similar conditions, producing 120 mg/L methanol after 24 h [80]. Calysta’s commercial
FeedKind® protein process is performed in a patented loop reactor optimised for gas-liquid mass
transfer. Rapid liquid flow is used to drive substrate gases downwards against gravity, faster than
they rise, leading to in situ pressurisation of the gases and consequently increased gas dissolution [81].
4.1.2. Paraffin oil as a Methane Vector
In a novel approach to increase the mass transfer of methane from the gas phase to the liquid
medium, Han et al. found that adding water-immiscible organic compounds in which methane has a
higher solubility, showed significant improvement on cell density. With the addition of 5% (v/v)
paraffin oil in the NMS medium, cell density of M. trichosporium OB3b reached 14 g/L (dry weight),
around seven times higher than the control after 240 hours culture [63]. Higher concentrations of
paraffin did not improve cell growth, suggesting that methane transfer is not the only limiting factor,
and also attributed to the fact that cell growth was observed in the oil phase which could act as a
barrier to metal ions and nutrient substrates. Although paraffin in the liquid medium has been shown
to enhance methanotroph growth, the effect on methanol synthesis has not been investigated.
4.2. Challenge II: over oxidation of methane beyond methanol
As part of the natural biochemical pathway in methanotrophs, methanol is further metabolised
through formaldehyde and formate to carbon dioxide, so it is necessary to stop the reaction at the
methanol oxidation level. One approach to enhance the production of methanol is to suppress over
oxidation by inhibition of the MDH enzyme as shown in Figure 5. A number of compounds have
been identified as MDH inhibitors with varying degrees of success in enhancing methanol production
as some have been observed to also reduce MMO activity. The efficiency of methanol conversion is
known to be impacted by the nature and concentration of such inhibitors.
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Figure 5. The MDH inhibited pathway of methane oxidation to methanol in
methanotrophic bacteria. Sections in red do not occur if 100% of produced methanol is
extracted from the cell.
4.2.1. Cyclopropane-derived inhibitors
Cyclopropane-derived compounds have been found to act as irreversible inhibitors of
MDH [82]. The ring opening reaction of the cyclopropane functionality with pyrroloquinoline
quinone (PQQ), the coenzyme of MDH, results in deactivation of the MDH [82,83].
Treatment of cell suspensions of M. trichosporium OB3b with cyclopropanol show extracellular
methanol accumulation under a methane atmosphere [8385]. At a cyclopropanol concentration of
6.18 µM, MDH activity in M. trichosporium OB3b has been shown to decrease by 79%, although at
this level, simultaneous reduction in pMMO activity of 12% was observed [85]. After 100 hours,
M. trichosporium OB3b produced 152 mmol/g (dry cell) methanol which is 51 times higher than
produced under conventional conditions although direct comparisons of this data is not possible
due to the lack of a control, and the distinct differences between conventional and optimum
conditions employed, including temperature and cell density.
4.2.2. High salt concentrations
A range of inorganic compounds have been investigated offering MDH inhibition with varying
effects on MMO activity. These are considered advantageous over inhibitors such as cyclopropanol
as they are cheaper, more chemically stable and exhibit reversible nature. It is believed that
electrostatic interactions between MDH and cytochrome cL, the primary electron acceptor, can be
disrupted by high salt concentrations in the culture medium, thus deactivating the enzyme [86,87].
Initially observed in Methylosinus trichosporium OB3b cell-free extracts [88], and
Methanomonas methanooxidans microbial culture [89], the addition of phosphate resulted in
methanol accumulation attributed to MDH inhibition. Complete inhibition of MDH has been
observed in cell-free extracts of M. trichosporium at 150 mM phosphate [88], with 120 mM
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phosphate concentration offering maximum inhibition in whole-cell suspensions of
M. trichosporium [90]. At a concentration of 80 mM phosphate ions, 80% of the MDH activity was
inhibited, however this was found to simultaneously reduce MMO activity by 16%, and FDH by
20% [90]. Extracellular accumulation of methanol has been maximised using a phosphate
concentration of 400 mM, generating 0.96 g/L methanol using a relatively high cell density of
17.3 g dry cell L1, after 43 hours [79]. Using a mixed microbial culture, Han et al. observed
maximum methanol production, and highest methane-to-methanol conversion ratio, at a phosphate
concentration of 40 mM [91]. Inhibition with phosphate was found to be fully reversible, with
enzyme activity completely restored after washing of the cells with low concentration phosphate
buffer [90]. The inhibition mode of phosphate on MDH was found to be uncompetitive, suggesting
phosphate binds to a site on the enzyme or enzyme-substrate complex other than the active site [90].
A sodium chloride concentration of 300 mM has been shown to inhibit 100% of MHD activity
in M. trichosporium OB3b, while also reducing pMMO activity by 50% [92]. Under an optimal
concentration of 200 mM sodium chloride, 7 mM methanol accumulated after 36 hours, compared
with no methanol accumulation under the same conditions in an absence of salt [92]. In a
methanotroph based consortia, maximum methanol accumulation of 0.5 mmol was observed with an
optimum 100 mM sodium chloride concentration, in addition to a conversion ratio of almost 80% [91].
Electron microscopy has shown that at concentrations above 100 mM, sodium chloride disrupts
cell structure and, significantly, the intracytoplasmic membrane where pMMO is found [93].
Ethylenediaminetetraacetic acid (EDTA) also inhibits MDH activity, through chelation of metals
present in the enzyme, and is not known to impact cell morphology [87]. Kim et al. used a
combination of sodium chloride and EDTA, thus allowing reduced sodium chloride concentrations
but maintaining MDH inhibition. The study demonstrated that the combination of 1 mM EDTA and
100 mM sodium chloride was optimal for methanol production [93]. At higher concentrations of
EDTA, methanol production was reduced, attributed to inhibition of MMO. The efficiency of lone
EDTA as an MHD inhibitor was much lower compared with other inhibitors. An optimal
concentration of 50 µM in a mixed methanotroph consortia produced methanol at a conversion rate
of just 43% [91].
Ammonium chloride is also known as an MDH inhibitor, inducing maximum methane-to-
methanol conversion of 80%, and optimal methanol accumulation at a concentration of 40 mM in a
mixed microbial culture [91].
4.2.3. Carbon dioxide
Xin et al. investigated the effect of various concentrations of carbon dioxide on the production
of methanol using M. trichosporium IMV 3011 [35,94]. They demonstrated that the addition of
carbon dioxide to methanotroph cultures under a methane/oxygen atmosphere resulted in the
extracellular accumulation of methanol. It was found that 40% v/v carbon dioxide resulted in an
optimum accumulation of 14 µmol/L methanol in a sealed flask after 24 h compared with a control
where no methanol was detected [35]. At concentrations above 40% v/v methanol synthesis was
lower, attributed to greater inhibition of methanol oxidation causing NADH limitation within the
cells. Using an appropriate carbon dioxide concentration in the gas feedstock is believed to offer
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partial inactivation of MDH, allowing simultaneous accumulation of methanol and NADH recycling
through complete oxidation of methane to carbon dioxide. Although reducing the maximum
theoretical efficiency to 50%, the advantage of this method is that an external source of reducing
equivalents is not needed and instead the natural NADH regenerating cycle can be exploited.
4.3. Challenge III: product inhibition
As with ethanol fermentation, the oxidation of methane to methanol by methanotrophic bacteria
is hindered by product inhibition [35,83]. Methanol was first shown to be toxic to most methanotroph
strains at concentrations as low as 0.01% v/v [27], supported by the work of Adegbola where
methanol was found to completely inhibit growth at 40 g/L [95]. It has since been demonstrated that
the pMMO enzyme in M. trichosporium OB3b is directly inhibited at levels as low as 10 mM
methanol, confirmed by the complete inhibition of propene epoxidation [83].
It has been hypothesised that under stress conditions, methanotrophs may excrete various other
products in addition to methanol, that could have negative effects on the bacterial oxidation ability.
An inability to identify unknown compounds by GC-MS and the ultimate loss of oxidation ability
after successive media renewals, suggest this is not the mechanism by which biocatalyst activity is
lost [96].
4.3.1. In situ product removal (ISPR)
One method to overcome this issue is immediate removal of the methanol product using in situ
product removal (ISPR). Maintaining the methanol concentration below inhibitory levels encourages
methane oxidation, while also maximising product recovery by preventing over oxidation. ISPR
necessitates consideration of reactor design and operation but has the added benefit of reducing
downstream processing and associated costs [97].
A number of published examples of continuous and semi-continuous methanol biosynthesis
utilise membrane reactors in which methanol is removed in the reaction media as a means to
maximise product yield. In a semi-continuous process utilising an ultrafiltration cell, a suspension of
M. trichosporium OB3b was investigated for methanol production. After incubation for 90 minutes
the reaction mixture was filtered, separating product methanol from the cell suspension. This
procedure was repeated five times producing a total of 36.1 µmol methanol compared to 19.6 µmol
after 6 h in a batch reactor under the same conditions [83]. Xin et al. utilised a membrane reactor
with a reaction volume of 40 mL and a continuous buffer feed to remove produced methanol from
the cell suspension. The reactor was run for 198 h without loss of productivity and generated a
total ~23 µmol methanol at a rate of 0.13 µmol/h [35] . This was in comparison with a batch reaction
under the same conditions in which a calculated total ~3.7 µmol (18.8 µmol/L quoted) methanol was
produced, as a consequence of product inhibition. Continued productivity can be attributed to
methanol removal, in addition to allowing a portion of the methanol produced to oxidise to carbon
dioxide, generating NADH and maintaining MMO activity.
Pen et al. performed successive reaction medium renewals over a 22 h methanol production
process, demonstrating increased methane oxidation activity compared to a process without media
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renewal. A plateau in methanol concentration was observed at 22 h for both the reaction with and
without media change, and the total methanol quantity produced was also comparable: 16.5 mg for
the consistent reaction media and 18.0 mg after 3 medium renewals. The apparent lack of
improvement on methanol production was attributed to a limit to bacterial oxidation capacity [96].
4.4. Challenge IV: maintaining catalytic activity and methanotroph viability
One issue in using whole cell cultures for catalysis is the need to maintain the physiological
activity, catalytic activity and viability of the microbes.
During the MMO catalysed oxidation of methane, two electrons are used to split the OO bond
in molecular oxygen, supplied by the cell in the form of NADH or cytochrome c (CytC) depending
on whether sMMO or pMMO are utilised. Under standard cell conditions, reducing equivalents are
regenerated from NAD+ and CytCox during oxidation of methanol via formaldehyde and formate to
carbon dioxide. However, interruption of metabolic pathways by MDH inhibition and extraction of
methanol results in the sequential oxidation of methanol to carbon dioxide not being possible, thus
preventing regeneration of reducing equivalents. Eventually exhaustion of the energy source results
in loss of MMO activity and cell viability (Figure 5).
An important point in considering the suitability of an electron source for an industrial process,
it that it is low cost and sustainable.
4.4.1. Formate addition
The addition of external metabolic electron donors to the reaction media overcomes this issue,
allowing continued production of methanol. Formate is a preferred choice as a downstream
metabolite of the process of interest, employed in the majority of studies [79,80,83,85,92,93,96,98,99].
Formate added to the reaction mixture is oxidised by FDH in the cell, generating an electron and
carbon dioxide. Mehta et al. [98] were first to demonstrate the restoration of methanol synthesis by
formate addition through regeneration of NADH2. The rate of methanol synthesis in an MDH
inhibited methanotroph culture was observed to fall off after 6 h, attributed to depletion of reducing
equivalents, and the addition of 40 mM sodium formate to the reaction mixture restored biocatalytic
activity to the previous level. Takeguchi et al. reported methanol accumulation in MHD inhibited
M. trichosporium OB3b increased with increasing sodium formate concentration in the reaction
media up to a maximum 14.3 mmol/L [85].
In a study to establish the optimal reaction conditions for methanol synthesis, varying sodium
formate concentration was investigated [92]. In agreement with Takeguchi et al., methanol synthesis
increased with sodium formate addition, although an optimum concentration of 20 mM formate was
determined above which there was no increase in methanol accumulation. In conflict however,
Duan et al. found that under the reaction conditions employed, increased sodium formate
concentrations between 10 and 80 mM resulted in almost equivalent maximum methanol production,
and the accumulation rate decreased with increasing formate concentration [79]. This lead to the
suggestion that to maximise rate of methanol synthesis, formate should be added to the media at low
concentrations throughout reaction.
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The effect on methanol synthesis of supplementing the microbial culture with formate
throughout reaction was investigated by Pen et al.. In a culture where maximum methanol synthesis
had been achieved, and oxidation activity dropped off, the addition of 20 mM sodium formate did
not restart bacterial activity. This suggests that once lost, methane oxidation activity is irreversible.
When formate was added whilst the bacteria were still active, an adverse effect was observed. The
methanol production rate immediately dropped off compared with a culture without formate addition,
and a lower total methanol concentration was achieved (50 mg/L compared with 120 mg/L) [96].
It is believed that sodium formate can be used by the cell in the serine pathway for carbon
fixation. This process would compete with NADH regeneration, with the two processes in
equilibrium, and could explain the noted trend in increased methanol production with formate
addition [96].
4.4.2. Use of cellular regeneration pathways
An alternative method to ensure the sustained activity of MMO is to use the cells’ natural
regeneration mechanism through the complete oxidation of methane to carbon dioxide [35,94]. By
supplying methane to the cell and not extracting methanol, reducing equivalents are generated that
can be utilised during the MMO catalysed oxidation of methane. Through alternating between
methanol production and regeneration cycles, semi continuous methanol biosynthesis can be
maintained. Although use of the methane feedstock in this way reduces the overall process yield, it is
necessary to maintain the viability of the cell and provides a relatively cheap and simple solution.
This principle has been demonstrated successfully by Xin et al. for the biosynthesis of methanol
from carbon dioxide with M. trichosporium IMV 3011 [94,100]. The hydrogenase enzymes
responsible for the oxidation of methanol to carbon dioxide, via formaldehyde and formate, are able
to catalyse the reverse reactions, although progressing against the natural biochemical pathway is an
energy intensive process requiring an electron for each sequential reduction. In batch experiments
after continuous reaction for 48 hours, almost 100% of the methanol synthesis ability of the cells was
lost, however by alternate reaction for 24 hours and regeneration for 12 hours with methane and
air (1:10, v/v) there was no notable loss in methanol synthesis after 9 cycles [94]. It was proposed,
due to the rapid resumption of cellular viability and reduction ability, that regeneration of reducing
equivalents was responsible rather than growth of additional cells that would be considerably slower.
4.4.3. Using poly-β-hydroxybutyrate cellular energy store
In addition to NADH as a direct source of reducing equivalents, the ability of the cell to store
energy in the form of poly-β-hydroxybutyrate (PHB) has been considered. PHB is a lipid, produced
and accumulated as an intracellular carbon and energy storage molecule by a variety of
microorganisms in response to stress conditions, and undergoes metabolism releasing reducing
equivalents when standard energy sources are not available [100]. PHB accumulation has been
observed in methanotrophic bacteria [27,28,101], and is known to be synthesised by the RuMP and
serine pathways in response to nitrogen, phosphate and oxygen limitation [100]. PHB production is
believed to be mainly non-growth associated, with maximum accumulation occurring during late
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growth and early stationary phases [102]. The main factor that determines the extent of PHB
production is availability of the synthetic precursor, acetyl-CoA. Type II methanotrophs employing
the serine pathway are the most effective producers able to accumulate up to 80% PHB by dry
weight [103,104].
Thomson et al. noted the metabolism of PHB in the presence of C2 compounds, attributed in
part to the satisfaction of energy requirements in utilising non-growth associated substrates [105].
Additionally, the capacity for M. trichosporium OB3B to degrade trichloroethylene (TCE) in an
sMMO catalysed reaction was enhanced 160% in cells containing 10% PHB compared with 2%
PHB [106]. Similarly a positive correlation has been noted between PHB content and TCE oxidation
ability in a mixed methanotroph culture, supported by the observed increase oxidation rate on
addition of the PHB monomer, β-hydroxybutyrate [107]. It is believed that the finite supply of
reducing equivalents within the cell is supplemented by the metabolism of stored PHB.
4.4.4. Microbial electrosynthesis
Microbial electrosynthesis is the process by which electrons can be transferred from an
electrode to living cells to provide energy for biocatalytic synthetic processes. A relatively new
concept, the direct supply of electrons from an electrode to microbes was initially investigated by
Gregory et al. for the anaerobic respiration of Geobacteraceae [108]. The idea has been further
developed to utilise an applied current in an electrochemical cell to drive microbial metabolism for
the production of a range of fuels and chemicals [109,110]. In a novel approach, it is proposed that
microbial electrosynthesis could be used to supply reducing equivalents directly to the methanotroph
culture in place of formate.
To date only a small number of bacterial species have been found to accept electrons directly
from an electrode, with no electrotrophic methanotrophs identified. Should such species be identified
and isolated, and using renewably sourced electrical power, it may be possible to provide a cheap
and sustainable source of electrons for methane oxidation.
4.4.5. Cell integrity
It has been demonstrated, that the addition of high concentrations of sodium chloride as an
MDH inhibitor has a negative impaction on cell integrity and methane oxidation ability by disrupting
the structure of intracytoplasmic membranes [93]. This discovery prompted investigation into
alternative MDH inhibitors that do not negatively impact cell morphology.
Pen et al. employed flow cytometry to investigate the integrity of the bacterial culture before
and after a methanol production experiment. At the beginning of the experiment, 81% of cells were
viable, with damaged cells constituting 5% of the bacterial population, which dropped to 41% viable
and 38% dead after 48 h methanol production [96]. Loss in methane oxidation ability of the cells is
believed to be related to loss in cell membrane integrity. Despite the presence of 41% viable cells,
the oxidation activity loss was measured as 97%, which indicated that the loss of biocatalytic activity
of the cells was due to both cell death and an alternative mechanism [96].
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Pen et al. noted that the proportion of viable cells halved and the bacterial concentration was
reduced by 23% after 48 h of methanol synthesis. The reduction in optical density (OD)
measurements was attributed to cell lysis and absorption of bacteria onto the bioreactor [96]. Cell
lysis would result in the release of MMO enzymes from the cell into the reaction media. However,
the low stability and activity of MMO isolates suggest the catalytic contribution from these to be
minimal.
The effect of fresh biocatalyst addition during the methane oxidation process has also been
investigated and is surprisingly shown to have a negative impact on methanol production. After 22 h,
at the point of loss in methanol oxidation activity, addition of fresh methanotroph culture to double
the total biocatalyst concentration, showed a sudden and strong decrease in the methanol
concentration, with 70% of the produced methanol lost after a further 24 h [96]. Biocatalyst addition
at a stage whilst the bacteria were still viable and activity high, also resulted in methanol
consumption by the bacteria, even in the presence of MDH inhibitors. The explanation for this
previously unreported phenomenon is that the accumulated methanol gives rise to configuration
changes on the PQQ group, restoring the ability for electron transfer from PQQ to cytochrome CL,
ultimately overriding the sodium chloride MDH inhibition and restoring methanol oxidation [96].
4.5. Challenge V: toxicity of source methane impurities
The oxidation of methane by methanotrophs is well known to be sensitive to impurities in the
methane feedstock, attributed to the low substrate specificity of the oxidation enzymes in
methanotrophs including MMO, MDH and FDH [27] (the names of which do not describe the
specificity for such substrates but their metabolic function within the cell). Non-growth hydrocarbons
are co-oxidised by the bacteria, producing toxic metabolites that have the potential to disrupt
metabolic pathways through both competitive inhibition of catalysts and accumulation of toxic
products, which ultimately results in cell death [84,105]. Ethane is particularly problematic as it is
generally the most abundant organic compound in natural gas after methane, and is oxidised through
ethanol and acetaldehyde to acetate followed by build-up in the cells.
Although free from higher hydrocarbons, methane biogas - the product of industrial anaerobic
digestion-contains a mixture of gases depending on the feedstock composition. Primarily
methane (30%70%) and carbon dioxide (25%50%), it also contains trace impurities including
hydrogen sulphide (<2000 ppm), ammonia (<100 ppm) and organic chlorine and silicon
compounds [111]. These impurities pose process difficulties if biogas sources of methane are utilised.
The removal of impurities in the methane feedstock can be achieved by the implementation of
gas purification processes. Options are vast depending on the methane source and problematic
contaminants. Generally however a number of stages are required resulting in a complex, energy
intensive and expensive process [112].
4.5.1. Microbial consortium
Interestingly, methanotroph cultures are abundant in natural gas environments such as around
petroleum seeps and vents where longer chain gaseous alkanes including ethane, propane and butane
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are present at inhibitory concentrations [113115]. It is well known that natural methanotrophic
populations exist in symbiotic communities with a range of organisms, including a variety of alkane
oxidising organisms able to utilise both C1 and C2 substrates [116]. It is believed that within such
situations methanotrophs are able to selectively utilise methane whilst potentially toxic C2 alkanes
are removed by other bacterial species. Of the methanotrophic strains identified, only those of the
genus Methylocella are able to grow on substrates containing CC bonds [48,117,118], in contrast to
numerous bacteria that are capable of growth on linear alkanes C2C9 but not methane [113].
Han et al. were the first to investigate methanol production using a mixed culture consortium
from a natural environment [91]. Isolated from a land fill site, 16S rRNA gene analysis identified the
key species present as Methylosinus sporium NCIMB 11126, M. trichosporium OB3b and
M. capsulatus (Bath). Maximum methanol accumulation was demonstrated, with sodium chloride
MDH inhibition, at a production rate of 9 µmol/mg h. No change to microbial community structure
was observed over the 24 h time course experiment, suggesting stability of the methanotroph
community and methane oxidation process.
This has been demonstrated commercially by Norferm AS who have developed an industrial
process utilising a synthetic bacterial community including M. capsulatus (Bath) to convert natural
gas, to a bacterial biomass product known as BioProtein® [116].
4.6. Challenge VI: optimised biotechnological conditions and consideration of requirements of the
biphasic process
The first stage in the biocatalytic process is growth of biomass, during which the bacteria
multiply and manufacture the critical MMO enzymes. This is followed by the bioconversion of
methane to methanol which can be considered the production phase and the specific reaction of
interest. Unsurprisingly the optimum conditions for these two processes differ which presents the
option for using a single vessel with compromised conditions, or a two-step process allowing
conditions tailored to culture and reaction separately.
4.6.1. Copper concentration
It is well documented that the concentration of copper in the reaction medium is responsible for
the expression of sMMO and pMMO, and consequently the bacterial growth rate. For strains able to
produce both MMO forms, under conditions of copper excess pMMO is made preferentially, while
under conditions of limited copper availability sMMO is present [50]. Excess copper beyond that
required to switch from sMMO to pMMO expression increases the activity of pMMO [119], and it
has been demonstrated that cells producing pMMO have a faster growth rate and higher catalytic
activity with methane [120]. Cells producing pMMO have greater growth yields than those
expressing sMMO attributed in part to the reduced energy requirements on the cell [61,62].
In terms of methanol synthesis, it is proposed that the copper concentration has an indirect
effect in the form of MMO synthesised and the relative enzymatic activity. Markowska &
Michalkiewicz showed a positive correlation between copper content in the media and methanol
synthesis by M. trichosporium OB3b up to 1.0 µmol/L, above which productivity decreased [121].
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4.6.2. Temperature
It has been observed that the optimum temperature is different for MMO activity and cell
growth in methanotrophic bacteria. Mehta et al. found the optimum temperature for methanol
accumulation in M. trichosporium OB3b to be 35 °C [90], confirmed by Lee et al. who noted that
maximum sMMO activity is observed at 35 °C whereas the rate of cell growth is maximised at
37 °C [122]. Above 40 °C led to a decrease in methanol accumulation [90], attributed to instability in
the MMO system. Takeguchi et al., however, observed maximum methanol accumulation in
M. trichosporium OB3b by pMMO at 25 °C [85]. Increasing temperature resulted in reduced
methanol accumulation attributed to instability of the pMMO enzyme at elevated temperatures.
4.6.3. pH
The effect of pH on methanotroph growth rate and MMO activity is less sensitive compared to
the temperature and copper concentration effect. In M. trichosporium OB3b the optimal pH for
sMMO activity was shown to be 6.26.4 compared with pH 7.0 for cell growth [122]. In
M. trichosporium OB3b expressing pMMO optimal methanol synthesis was observed at pH 6.5 [90].
4.6.4. Cell concentration
The cell concentration in the reaction medium influences extracellular methanol accumulation
as MMO is a growth associated enzyme. Mehta et al. found maximum methanol production in
M. trichosporium OB3b at a cell concentration of 4 mg ml1 [90], in close agreement with the
findings of Xin et al. at 3 mg/ml [35]. In contradiction Lee et al. noted an optimum cell concentration
of 0.6 mg/ml for methanol synthesis [92]. It is believed that the maximum cell concentration able to
be supported is limited by the availability of methane in the reaction mixture, related to gas-liquid
mass transfer limitations and as such delivery methods and culture conditions.
The methanol production kinetics, for reaction in a membrane bioreactor, exhibited the same
profile at biocatalytic concentrations between 11 u/ml and 150 u/ml, with little correlation between
total methanol production and biocatalyst concentration [80]. This behaviour was shown to be related
to oxygen limitation due to absorption of bacteria and fouling of bioreactor membranes.
4.6.5. Bacterial strain
To date Methylosinus trichosporium OB3b has been the preferred methanotroph species for
investigation into the potential for a biocatalytic partial methane oxidation process, aided by the
wealth of characterisation data available. The application of alternative species, in particular
extremophilic and extremotolerant methanotrophs, able to either thrive or tolerate living in extreme
environments, offers huge potential in terms of maximising methanol yield through process
efficiencies.
The use of psychrophilic methanotrophs, capable of growth at low temperatures, would be well
suited to tackling gas-liquid mass transfer limitations. The solubility of methane increases with
24
AIMS Bioengineering Volume 5, Issue 1, 138.
decreasing temperature, however this is currently constrained in methanol biosynthesis by the
optimum temperature of M. trichosporium OB3b which is 30 °C. A number of psychrophilic
methanotrophs have been identified and isolated from a range of low temperature ecosystems
including Siberian tundra bogs, Antarctic lakes, and bottom sediments and water from Pacific and
Atlantic Oceans [123]. The potential of such species in an industrial process is limited as the rates of
in vivo methane oxidation and in vitro metabolic activity have been demonstrated to decrease
significantly as the temperature is lowered from 30 °C to 5 °C [124].
MMO isolated from thermophilic methanotrophs has the potential to be better suited to
industrial process conditions, offering increased stability under high temperature reaction conditions
which would promote increased reaction rates. Tolerance and active growth of such species in
environments of elevated temperatures suggests development of metabolic systems to survive under
these extreme conditions. Isolated and characterised by Bodrossy et al., strain HB was isolated from
Japanese and Hungarian hot springs, capable of growth at temperatures up to 70 °C [32].
Representing a new genus, the name Methylothermus was proposed, however this organism was
not extensively characterised and is no longer extant. With investigation prompted by loss of the HB
strain, the only truly thermophilic methanotroph currently known is strain MYHT, described as
Methylothermus thermalis. Isolated from a Japanese hot spring, and capable of growth at
temperatures 3767 °C (optimum 5759 °C), it is closely related to the HB strain [125].
The biochemical and molecular mechanisms by which methanotrophs are able to survive in
such harsh conditions is still uncertain, although de novo synthesis of ectoine as a stress protectant is
known [123]. Further understanding of responses to stress conditions is needed, in addition to
bioenergetic and genetic aspects of extremophile adaptation. Advances have been made, and as such
we are increasingly able to isolate and culture extremophilic methanotrophs, which furthers the
potential for industrial biotechnological applications.
MMO activity is not only dependent on the expression of either soluble or particulate forms,
regulated by copper availability, but also the methanotroph species in question. An example of this is
the low methanol productivity recorded by Xin et al. attributed to low specific MMO activity of
M. trichosporium 3011, which is about one percent compared with M. trichosporium OB3b [35]. It
would therefore be reasonable to expect methanotroph species to exist with MMO activities greater
than that in OB3b, which would offer the potential for higher methanol production rates.
4.6.6. Citric acid as a Krebs cycle substrate
The effect of various organic chemicals on the growth of M. trichosporium OB3b was studied
as a means to improve the cell density by Xing et al. [126]. The addition of vitamins, amino acids
and organic acids involved in the Krebs cycle and serine pathway of Type II methanotrophs were
anticipated to enhance assimilation of formaldehyde to biomass. Addition of citrate had the most
pronounced positive effect, at an optimal concentration of 0.015 mmol/L the cell density was
0.66 g/L (dry weight), more than 3.5 times that of the control, after 4 days cultivation [126]. It is
believed the addition of such organic acids alters the metabolic flow of formaldehyde from oxidation
to formate, and ultimately carbon dioxide, into the Krebs cycle for cell growth. Although the addition
25
AIMS Bioengineering Volume 5, Issue 1, 138.
of citric acid was shown to greatly increase the cell density of the bacterial culture, the effect on
methanol synthesis was not investigated.
5. Literature examples to date of the methanotroph facilitated methane oxidation to
methanol
Table 1 reviews work published on whole cell bioconversion of methane to methanol utilising
wild type methanotrophic bacteria. Sorted by year of publication, the utilised bacterial strains are
listed along with reaction conditions and the process yield. Where multiple data for different reaction
conditions are published in the same article, the conditions with the highest productivity are listed. If
different reaction modes are investigated, these too are listed independently. The volume of methanol
produced is recorded as published, in addition to the calculated volume of methanol in mmol/L/h dry
cell mass for comparison.
26
AIMS Bioengineering Volume 5, Issue 1, 138.
Table 1. Summary of experimental biological conversion on methane to methanol.
Ref.
Bacteria strain
Process mode and
reaction vessel
Reaction
volume
(mL)
Gas
Feedstock
Cell density
(dry weight
basis) (g/L)
pH
Total
production
period
(hours)
MDH inhibition
method
Exogenous reducing
agent
Quoted amount
of methanol
produced
Calculated
methanol
produced
(mmol/L/h)
[90]
Methylosinus
trichosporium OB3b
(NCIB 11131)
Batch
5
CH4:air
(1:1 v/v)
3.00 × 103
6.5
3
80 mM
phosphate
-
2.7 µmol/mg/ h
8.1 × 103
[127]
Unidentified isolate
1 (from digester
sludge)
Batch
250
100% CH4
0.3
a
24
-
-
0.5 g/L
0.65
[127]
Unidentified isolate
2 (from digester
sludge)
Batch
250
100% CH4
0.3
a
24
-
-
1.0 g/L
1.30
[98]
Methylosinus
trichosporium NCIB
11131 (OB3b)
Batch
Cells immobilised on
DEAE-cellulose
5
CH4:O2
(1:1 v/v)
3.6
6.4
12
100 mM
phosphate
40 mM sodium
formate at 6 h
50 µmol/mg
15
[98]
Methylosinus
trichosporium NCIB
11131 (OB3b)
Continuous
Stirred membrane
reactor with ISPR
Cells immobilised on
DEAE-cellulose
50
CH4:O2
(1:1 v/v)
2
6.4
70
100 mM
phosphate
Sodium formate
multiple pulsed
addition throughout
reaction
267 µmol/h
5.34
[99]
Methylosinus
trichosporium OB3b
Batch
3.5
CH4:air
(1:3 v/v)
Positive
pressure
0.36 ×103 (wet
cell wt.)
7.0
120
0.234 µmol
Cyclopropanol
50 µmol
sodium formate
23 mmol/g (wet
cell wt.)
6.90 × 105
Continued on next page
27
AIMS Bioengineering Volume 5, Issue 1, 138.
Ref.
Bacteria strain
Process mode and
reaction vessel
Reaction
volume
(mL)
Gas
Feedstock
Cell density
(dry weight
basis) (g/L)
pH
Total
production
period
(hours)
MDH inhibition
method
Exogenous reducing
agent
Quoted amount
of methanol
produced
Calculated
methanol
produced
(mmol/L/h)
[85]
Methylosinus
trichosporium OB3
Batch
3.5
CH4:air (1:4
v/v) Positive
pressure
1.39
7.0
~3
251 µM
cyclopropanol
14.3 mM sodium
formate
3 mmol/g dry
cell
1.39
[85]
Methylosinus
trichosporium OB3b
Batch
3.5
CH4:air (1:4
v/v) Positive
pressure
3.46 × 102
7.0
100
67 nM
cyclopropanol
14.3 mM sodium
formate
152 mmol/g dry
cell
5.26 × 102
[83]
Methylosinus
trichosporium OB3b
Batch
17.5
CH4:air
(1:2.6 v/v)
3.46 × 102
7.0
7.5
67 nM
cyclopropanol
14.3 mM sodium
formate
19.6 µmol
0.15
[83]
Methylosinus
trichosporium OB3b
Repeated batch
5 cycles of 1.5 h
17.5
CH4:air
(1:2.6 v/v)
3.46 × 102
7.0
7.5
67 nM
cyclopropanol
14.3 mM sodium
formate
36.1 µmol
0.28
[35]
Methylosinus
trichosporium IMV
3011
Batch
25
CO2:CH4:O2:
N2
(40:20:20:20
v/v)
3
7.0
24
CO2
Oxidation of a
portion of metabolic
methanol for NADH
regeneration
14 µmol/L
5.83 × 104
[35]
Methylosinus
trichosporium IMV
3011
Batch
25
CO2:CH4:O2:
N2
(40:20:20:20
v/v)
3
7.0
30
CO2
Oxidation of a
portion of metabolic
methanol for NADH
regeneration
18.8 µmol/L
6.26 × 104
[35]
Methylosinus
trichosporium IMV
3011
Continuous
Stirred membrane
reactor with ISPR
40
CO2:CH4:O2:
N2
(40:20:20:20
v/v)
3
7.0
198
CO2
Oxidation of a
portion of metabolic
methanol for NADH
regeneration
23 µmol
0.13 µmol/h
2.90 × 103
Continued on next page
28
AIMS Bioengineering Volume 5, Issue 1, 138.
Ref.
Bacteria strain
Process mode and
reaction vessel
Reaction
volume
(mL)
Gas
Feedstock
Cell density
(dry weight
basis) (g/L)
pH
Total
production
period
(hours)
MDH inhibition
method
Exogenous reducing
agent
Quoted amount
of methanol
produced
Calculated
methanol
produced
(mmol/L/h)
[92]
Methylosinus
trichosporium OB3b
Batch
5
CH4:air
(1:5 v/v)
0.6
7.0
36
200 mM NaCl
20 mM sodium
formate
7.7 mmol/L
0.21
[121]
Methylosinus
trichosporium OB3b
Batch
500
CH4:O2
(1:2 v/v)
~1.5 × 1012 dm3
7
72
-
-
47.6 µmol/L
6.61 × 104
[93]
Methylosinus
trichosporium OB3b
Batch
100
CH4:air
(1:3 v/v)
0.6
7.0
28
100 mM NaCl
1 mM EDTA
20 mM sodium
formate
13.2 mM
0.47
[93]
Methylosinus
trichosporium OB3b
Repeated batch
3 cycles of 8 h
100
CH4:air
(1:3 v/v)
0.6
7.0
24
100 mM NaCl
1 mM EDTA
20 mM sodium
formate
2.17 µmol/h/mg
dry cell wt.
1.30 × 106
[93]
Methylosinus
trichosporium OB3b
Continuous
1,000
CH4:air
(1:1 v/v)
0.6
7.0
24
100 mM NaCl
1 mM EDTA
20 mM sodium
formate
13.7 mM
0.57
[79]
Methylosinus
trichosporium OB3b
Batch
Sealed flask
10
CH4:O2
(1:1 v/v)
17.3
6.3
40
400 mM
phosphate
20 mmol/L sodium
formate
1.12 g/L
0.87
[79]
Methylosinus
trichosporium OB3b
Continuous
Bubble free membrane
reactor
300
CH4:O2
(1:1 v/v)
17.3
6.3
40
400 mM
phosphate
20 mmol/L sodium
formate
0.95 g/L
0.74
[91]
Mixed methanotroph
consortium from
landfill soil
Batch
100
Artificial
biogas:air
(4:6 v/v)
4 × 103
a
a
100 mM NaCl
-
1.49 g/g
(g CH3OH per
g CH4)
Continued on next page
29
AIMS Bioengineering Volume 5, Issue 1, 138.
a Complete reaction conditions not given; b Catalytic concentration defined as “the mass of bacteria required to form 1 µg of methanol in the reaction media within 1h in a 50 mL-batch reactor incubated at 30 °C under stirring at
160 rpm and with a volume ratio Vgas/Vliq of 9”.
Ref.
Bacteria strain
Process mode and
reaction vessel
Reaction
volume
(mL)
Gas
Feedstock
Cell density
(dry weight
basis) (g/L)
pH
Total
production
period
(hours)
MDH inhibition
method
Exogenous reducing
agent
Quoted amount
of methanol
produced
Calculated
methanol
produced
(mmol/L/h)
[91]
Mixed methanotroph
consortium from
landfill soil
Batch
5,000
reactor,
reaction
volume
not stated
CH4:air
(1:9 v/v)
a
a
24
100 mM NaCl
-
9.0 µmol/h mg
cell
[80]
Methylosinus
trichosporium OB3b
Batch
50
CH4:air
(1:1 v/v)
Vgas/Vliq = 9
70 u/mL b
7.0
24
12.9 mM
phosphate
100 mM NaCl
1.0 mM EDTA
20 mM sodium
formate
290 mg/L
0.38
[96]
Methylosinus
trichosporium OB3b
Fed-batch
Membrane bio-reactor
(MBR)
150
CH4:air
(1:1 v/v)
11 u/mL b
7.0
48
12.9 mM
phosphate
100 mM NaCl
1.0 mM EDTA
20 mM sodium
formate
18.8 mg
8.15 × 102
30
AIMS Bioengineering Volume 5, Issue 1, 138.
6. Conclusion
Methane is an abundant natural resource used in the generation of heat and electrical power.
Most notably, methane is extracted from geological fossil deposits. A significant proportion of these
are identified as stranded methane deposits. GTL technologies are being developed as a means to
exploit these remote, often diffuse sources. An additional and often undervalued resource is methane
produced during anaerobic digestion that results in large volumes released as a waste product from
numerous industrial processes. Consequently atmospheric methane concentrations are at
unprecedented levels that, as a potent greenhouse gas, are a major cause for concern. This has led to
the instigation of various methane abatement schemes.
GTL technologies offer the opportunity to convert methane into a liquid hydrocarbon fuel that is
more readily handled and transported than the gas precursor. Of the range of possible products,
methanol is considered an attractive option with the potential for a “methanol economy” to fulfil
both the energy and hydrocarbon feedstock demands currently satisfied by fossil fuels. The
commercial production of methanol from methane is an energy intensive two-step process.
A direct, single-step oxidation would be an attractive option with the potential for energy and
cost savings. Methanotrophic bacteria utilise powerful MMO enzymes to perform the desired
reaction with a high level of selectivity under mild conditions. It is proposed that the methanotroph
catalysed oxidation of methane to methanol is a potential GTL technology. The advantages of such a
process are the low energy and cost requirements; suitability for small scale, modular processes thus
allowing use of diffuse and remote gas sources; and the contribution towards methane abatement.
Figure 6 summarises the five main challenges faced in developing an industrially relevant
biocatalytic methane oxidation process, and the potential strategies to overcome these.
Figure 6. Current challenges and potential strategies for the methanotroph biocatalysed
conversion of methane to methanol.
Relatively slow growth and the inability to obtain high cell density cultures is attributed to low
methane solubility being a growth limiting factor. This can be addressed through process and reactor
31
AIMS Bioengineering Volume 5, Issue 1, 138.
design to allow optimised gas-liquid mass transfer. In terms of methanol synthesis, the first problem
arises as methanol is not the final product in the oxidation of methane, but a precursor used by the
cell to generate electrons and synthesise various essential metabolites. For this reason, it is necessary
to inhibit the MDH enzyme, which results in the accumulation of methanol but also causes depletion
of cellular reducing equivalents. It is possible to provide exogenous electrons to the biocatalyst from
a number of sources, although partial suppression of MDH, rather than complete inhibition, allows a
portion of methanol to be fully oxidised and so exploits the cell’s natural regeneration pathways.
Additionally, methanol is toxic to most methanotroph strains and so must be removed from the
reaction medium before growth and methanol synthesis is affected. Implementation of ISPR methods
in the initial process design avoids biocatalyst poisoning whilst also maximising methanol
production yields. Although the low specificity of the MMO enzymes is one of the factors that give
methanotrophs such potential for applications in biotechnological processes, it presents a
complication in biocatalysis of methane. The co-oxidation of contaminants in the feedstock methane
gas are further metabolised generating toxic by-products that accumulate within the cell, causing
death and loss of biocatalytic activity. It is proposed that use of either natural or synthetic microbial
consortia will overcome this, mimicking natural conditions where methanotrophs are able to
selectively utilise methane whilst potentially toxic compounds are removed by other bacterial species
that utilise higher hydrocarbons. As new strains of methanotrophs are constantly being discovered,
isolated and characterised, this also offers potential to identify extremophilic species that may
address some of the identified challenges. It is believed that continued progress in these areas will
ultimately allow the development of a technically feasible and economically viable methanotroph
bioconversion of methane to methanol.
Acknowledgements
CEB and JP thanks go to the E-Futures Doctoral Training Centre for the funding that supported
this work. PDD thanks BBSRC award BB/M006891/1.
Conflict of interest
All authors declare that there are no conflicts of interest.
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... Including MDH inhibitors, several other factors like headspace gases, cell mass, and formate concentration also influence methanol production. Several MDH inhibitors of various compositions, such as NaCl (100-200 mM), ethylenediaminetetraacetic acid (EDTA) (0.05-1 mM), phosphate (12.9-400 mM), cyclopropanol (67 nM-251 µM), MgCl 2 (5-10 mM), and NH 4 Cl (40 mM), have been used individually or in combinations to increase methanol production [6,9,10]. In addition, several approaches demonstrate the cell immobilization of methanotrophs to improve methanol yield and operational stability [4,[11][12][13]. ...
... Selection of MDH inhibitors is crucial to achieve a high yield of methanol by MOB. Many studies have presented several chemical compounds, such as sodium chloride, phosphate, chelating agents, magnesium chloride, ammonium chloride, and cyclopropanol, individually or in combinations, as MDH activity inhibitors [6,9,10,21]. In this study, to investigate new chemical compounds as MDH inhibitors for methanol production, different chelating agents were tested using cells of the isolate, M. trichosporium M19-4. ...
... Cell concentration influences the methanol accumulation in the reaction mixture as methane monooxygenase is a growthassociated enzyme that oxidizes methane to methanol [10]. To assess the effect of cell concentration of M. trichosporium M19-4 on extracellular methanol accumulation, dry cell weight (DCW)/ml in the range of 0.5-2.5 mg were treated in the reaction mixture. ...
... CH 3 OH is a useful feedstock for further chemical synthesis, a fuel with high energy density, and an excellent hydrogen carrier that provides safe and clean energy. However, the chemical process of producing CH 3 OH through direct functionalization of CH 4 is energy intensive (the reaction temperature is approximately 900°C and the operation pressure is 0.5-4 MPa) due to the high energy to cleave the C-H bond of CH 4 (104 kcal mol -1 ) [4]. ...
... Although CH 4 bioconversion shows great potential as an economical GTL technology, it still faces some di culties in achieving e cient conversion for commercialization. One of the di culties occurs due to the low solubility of CH 4 and oxygen (O 2 ) in water. Since the conventional methanotrophic process is generally conducted in the aqueous phase [8][9][10][11][12][13], the limitation of the amount of dissolved CH 4 results in a low productivity of CH 3 OH. ...
... The microbial cells are suspended in the aqueous phase or immobilized on the membrane as a bio lm and catalyze the oxidation of organic chemicals in the aqueous phase. These MBRs have been employed for aerobic wastewater treatment [29,30], synthesis of ne chemicals [31,32], and CH 3 OH production [33,34], in which CH 4 and O 2 were supplied through two independent membrane modules; premixing was not performed to keep the risk of explosion low. However, the disadvantage of membrane aerated reactors is the requirement of gas pressurizing to assist gas diffusion through the membrane into the aqueous phase, which is energy-consuming. ...
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Full-text available
Background Methane (CH4), as one of the major energy sources, easily escapes from the supply chain into the atmosphere because it exists in a gaseous state under ambient conditions. Compared to carbon dioxide (CO2), CH4 is 25 times more potent at trapping radiation; thus, the emission of CH4 to the atmosphere causes severe global warming and climate change. To mitigate CH4 emissions and utilize them effectively, the direct biological conversion of CH4 into liquid fuels, such as methanol (CH3OH), using methanotrophs is a promising strategy. However, supplying biocatalysts in an aqueous medium with CH4 involves high energy consumption due to vigorous agitation and/or bubbling, which is a serious concern in methanotrophic processes, because the aqueous phase causes a very large barrier to the delivery of slightly soluble gases. Results An inverse membrane bioreactor (IMBR), which combines the advantages of gas-phase bioreactors and membrane bioreactors, was designed and constructed for the bioconversion of CH4 into CH3OH in this study. In contrast to the conventional membrane bioreactor with biofilms that are immersed in an aqueous phase, the biofilm in the IMBR was placed to face a gas phase to supply CH4 directly from the gas phase to bacterial cells. Methylococcus capsulatus (Bath), a representative methanotroph, was used to demonstrate the bioconversion of CH4 to CH3OH in the IMBR. Cyclopropanol and sodium formate were supplied from the aqueous phase as a selective inhibitor of methanol dehydrogenase, preventing further CH3OH oxidation, and as an electron donor, respectively. After optimizing the inlet concentration of CH4, the mass of immobilized cells, the cyclopropanol concentration, and the gas flow rate, continuous CH3OH production can be achieved over 72 h with productivity at 0.88 mmol L⁻¹ h⁻¹ in the IMBR, achieving a longer operation period and higher productivity than those using other types of membrane bioreactors reported in the literature. Conclusions The IMBR can facilitate the development of gas-to-liquid (GTL) technologies via microbial processes, allowing highly efficient mass transfer of substrates from the gas phase to microbial cells in the gas phase and having the supplement of soluble chemicals convenient.
... The selective expression of these MMOs is controlled by Cu availability. In addition to the CH 4 oxidation, MMOs also oxidize various carbon substrates, including alkanes, alkenes, aromatics, and heterocyclics [4,[27][28][29]. The Cu:biomass ratio regulates the expression of different MMOs; this unique biological phenomenon is termed the copper-switch [30][31][32][33]. ...
... CH 3 OH is biosynthesized as a metabolic intermediate during growth, via the oxidation of CH 4 . While CH 3 OH is used as a solvent, fuel, and antifreeze, it is also a critical platform chemical to produce several commodity chemical compounds, including acetic acid, formaldehyde, methyl tert-butyl ether, and dimethyl ether [28]. Several studies present CH 3 OH production through MMO-catalyzing CH 4 oxidation using OB3b cell mass (Table 3). ...
... Different concentrations of cyclopropanol (67 nM-251 µM), ethylenediaminetetraacetic acid (EDTA) (0.05-1 mM), phosphate (12.9-400 mM), NaCl (100-200 mM), MgCl 2 (5-10 mM), and NH 4 Cl (40 mM) were used individually or in combination to achieve the effective suppression of MDH to increase CH 3 OH production. Of all inhibitory compounds, cyclopropanol was the most effective in terms of inhibiting MDH activity; however, increased CH 3 OH accumulation was not possible due to the unstable nature of cyclopropanol in aerobic conditions [17,28,70]. Higher concentrations of methanol are achieved through pMMO activity of OB3b as opposed to sMMO. ...
Article
Rising greenhouse gas concentrations in the atmosphere are having deleterious effects on biotic and abiotic systems, causing serious global concerns. Bioprocess technology is able to utilize these gases to manufacture high-value products and provide an excellent alternative to currently expensive conventional carbon sources in use. Methanotrophs are ubiquitous and utilize reduced carbon substrates without C−C bonds, such as methane, methanol, methylamine, and formaldehyde. Notably, methanotrophs produce single-cell proteins, polyhydroxyalkanoates, methanol, and exopolysaccharides, using a unique metabolic pathway during their growth process. Many industries have developed copyright-protected bioprocesses to utilize methane through methanotrophic pathways and manufacture high-value products. Among methanotrophs, Methylosinus trichosporium OB3b is one of the most important methane oxidizing bacteria; it has been studied extensively to identify potential applications including methanol and biopolymer production, and the bioremediation of environmental contaminants. This paper summarizes the characteristics and versatile role of methanotrophs and Methylosinus trichosporium OB3b. In addition, commercially established biological conversions of methane were constituted.
... sMMO is the only monooxygenase that can activate methane, but there are a range of other monooxygenases 58 that can utilize a wide range of hydrocarbon substrates and the use of these enzymes in chemical transformations could be of great interest in the future. Recently, the prospects of using sMMO to make methanol as part of a gas to liquids process has been reviewed, 65 and a number of challenges were identified that present obstacles should this route to exploit natural gas be pursued. These include gas−liquid mass transfer limitation and the potential poisoning of the enzyme by impurities in the natural gas. ...
Article
The direct transformation of methane to methanol remains a significant challenge for operation at a larger scale. Central to this challenge is the low reactivity of methane at conditions that can facilitate product recovery. This review discusses the issue through examination of several promising routes to methanol and an evaluation of performance targets that are required to develop the process at scale. We explore the methods currently used, the emergence of active heterogeneous catalysts and their design and reaction mechanisms and provide a critical perspective on future operation. Initial experiments are discussed where identification of gas phase radical chemistry limited further development by this approach. Subsequently, a new class of catalytic materials based on natural systems such as iron or copper containing zeolites were explored at milder conditions. The key issues of these technologies are low methane conversion and often significant overoxidation of products. Despite this, interest remains high in this reaction and the wider appeal of an effective route to key products from C-H activation, particularly with the need to transition to net carbon zero with new routes from renewable methane sources is exciting.
... Voies d'oxydation du CH4 en CO2 avec assimilation du formaldéhyde. (Bjorck et al., 2018) modifiée d'après (Hanson & Hanson, 1996). (Pimenov et al., 2000) ont permis de mettre en évidence la réalisation de ce processus dans ces écosystèmes. ...
Thesis
Les sources hydrothermales océaniques sont caractérisées par la formation de dépôts de sulfure massif d’associations minéralogiques complexes autour de la zone d’émission du fluide hydrothermal. La base de la chaîne trophique de ces écosystèmes est assurée par la production primaire chimio-synthétique des micro-organismes spécifiques à ces écosystèmes. L’objectif de ce travail de thèse était d’identifier et de caractériser le fonctionnellement de ces communautés microbiennes en interaction à l'interface entre la biosphère et la géosphère. Afin de répondre à cet objectif, des approches innovantes de colonisation ex situ de substrats naturels de sulfures massifs ont été réalisées grâce à des incubations en mésocosmes. Cinq incubations en bioréacteur Gas-lift ont été réalisées à partir de cheminées et de fluides prélevés sur trois champs hydrothermaux de l’Atlantique (Lucky Strike, Snake Pit et TAG). L’utilisation de fluide non filtré comme base minérale a permis l’enrichissement de micro-organismes originaux jusqu’alors non détectés dans les inventaires moléculaires. Dans l’ensemble, les minéraux associés à ces incubations ont été colonisés par des communautés d’hyperthermophiles dont la structure de population est similaire à celle des fractions liquides. De plus, un nouveau mécanisme de tolérance à l’H2 considéré jusqu’alors comme inhibiteur de la croissance chez les Thermococcales, a été décrit. Ce trait métabolique original constitue un élément pour comprendre leur distribution ubiquiste dans les écosystèmes hydrothermaux. Ces travaux permettent d’étendre nos connaissances sur la diversité hyperthermophile de ces écosystèmes.
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Chapter
Synthetic biology is a new interdisciplinary area that involves the application of engineering principles to biology. One of the common goals of the synthetic biology project is adopting an integrative omics approach. The word omics can be used as an umbrella term to describe the various fields of biological sciences that end with the suffix ‐omics, like genomics, transcriptomics, proteomics, metabolomics, etc. It is a rapidly evolving, multidisciplinary, and emerging field that aims at the collective characterization and quantification of pools of biological molecules that translate into the structure, function, and dynamics of an organism. As the world looks for sources of clean, efficient, and affordable energy, the research and development of biofuels grabs its attention. With the help of microbial technology, biorefinery‐related research is actively pursued in many countries to manufacture microbial strains and to produce a wide range of value‐added biochemicals like alternative fuels. Biofuels of the first and second generation are made of food and non‐food sources, which might not be affordable to developing countries. So, other approaches like metabolic engineering and rDNA technology using algae and other sources are being developed to meet the ever‐growing energy demand. This review discusses the prospects of how information that can be extracted from multi‐omics data can be applied in finding new horizons in biorefineries using microbial technologies for the production of various biofuels and bioenergy resources.
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The technology of methanol production from the fermentation of methanol‐producing bacteria (methanotroph) is still in the research phase. Maximizing the methanol production through bacteria fermentation in this process is a challenge to researchers. By optimizing the biological process, this technology can potentially fill the demand gap in methanol industries. Methylosinus trichosporium is a methanotroph that is used for the production of methanol in this analysis. Using OD600 analysis, the rate and growth profile for M. trichosporium is determined. The one factor at a time (OFAT) method was chosen to optimize the methanol production (percentage of methane, pH, and phosphate ion), and the optimum condition for this bacterium was determined. M. trichosporium showed a specific growth rate at 0.02 h–1 with the exponential phase reaching the maximum at 90 h of the cultivation period. Optimization analysis showed that the maximum methanol production by this bacterium was at a 1:1 methane‐air ratio, a pH of 6.3–6.5, and 40 mM of phosphate ions. The ability of M. trichosporium to produce methanol was proved in this analysis with the maximum rate of production at 0.072 mg L–1 h–1. © 2022 Society of Chemical Industry and John Wiley & Sons, Ltd. This article is protected by copyright. All rights reserved
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We describe the phase-transfer biocatalytic process for methane-to-methanol conversion based on the spontaneous phase-separable membrane micro-CSTR (μCSTR). The developed system has several critical benefits for operating the conversion reaction, including (a) preventing the whole-cell biocatalyst loss while operating the reactor continuously, (b) introducing a powerful mixing effect to significantly improve the mass transfer efficacy of methane to the biocatalyst, (c) facilitating the product separation process by employing the spontaneous phase separation system, and (d) providing an excellent platform for more complex, high-value-added conversion reactions.
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Air pollution is a topic of important global concern because it has contributed significantly to an increase in the earth's global warming potential and contributed to severe health and environmental impacts. In this review, the different bioreactor configurations commonly used for waste gas treatment, namely the biofilters, the biotrickling filters and the bioscrubbers, and their industrial applications were compared in terms of the type of inoculum, the packing material/media, removal efficiency and elimination capacity. Typically, biofilters are operated under the following range of operating conditions: gas residence time = 15–60 s; gas flow rate = 50–300,000 m³ h⁻¹; temperature = 15–30 °C; pH = 6.0–7.5; filter area = 100–3000 m²; relative humidity >95.0%; and removal efficiencies >75.0% depending on the waste gas composition and concentration. The biotechnological approaches for resource recovery, i.e., the conversion of C1 gaseous compounds (CO, CO2 and CH4) to liquified value-added products or biofuels have been discussed. From this review, it was evident that the performances of different aerobic, anoxic and/or anaerobic lab, pilot and full-scale bioreactors for waste gas treatment and resource recovery depend on the composition, the individual concentration of pollutants present in the waste gas and the gas flow rate. Although most of the research on product recovery from waste gas is rather limited to lab/pilot-scale studies, there are some key commercialized technologies that have proven to be economical at the full-scale. Thus, this review, comprehensively presents a complete overview of the current trends and limitations of conventional waste gas treatment systems, the benefits of novel bioreactor configurations and their potential to be applied for resource recovery from waste gases.
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Methane-oxidizing bacteria are characterized by their capability to grow on methane as sole source of carbon and energy. Cultivation-dependent and -independent methods have revealed that this functional guild of bacteria comprises a substantial diversity of organisms. In particular the use of cultivation-independent methods targeting a subunit of the particulate methane monooxygenase (pmoA) as functional marker for the detection of aerobic methanotrophs has resulted in thousands of sequences representing “unknown methanotrophic bacteria.” This limits data interpretation due to restricted information about these uncultured methanotrophs. A few groups of uncultivated methanotrophs are assumed to play important roles in methane oxidation in specific habitats, while the biology behind other sequence clusters remains still largely unknown. The discovery of evolutionary related monooxygenases in non-methanotrophic bacteria and of pmoA paralogs in methanotrophs requires that sequence clusters of uncultivated organisms have to be interpreted with care. This review article describes the present diversity of cultivated and uncultivated aerobic methanotrophic bacteria based on pmoA gene sequence diversity. It summarizes current knowledge about cultivated and major clusters of uncultivated methanotrophic bacteria and evaluates habitat specificity of these bacteria at different levels of taxonomic resolution. Habitat specificity exists for diverse lineages and at different taxonomic levels. Methanotrophic genera such as Methylocystis and Methylocaldum are identified as generalists, but they harbor habitat specific methanotrophs at species level. This finding implies that future studies should consider these diverging preferences at different taxonomic levels when analyzing methanotrophic communities.
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Gene synthesis is a fundamental technology underpinning much research in the life sciences. In particular, synthetic biology and biotechnology utilize gene synthesis to assemble any desired DNA sequence, which can then be incorporated into novel parts and pathways. Here, we describe SpeedyGenes, a gene synthesis method that can assemble DNA sequences with greater fidelity (fewer errors) than existing methods, but that can also be used to encode extensive, statistically designed sequence variation at any position in the sequence to create diverse (but accurate) variant libraries. We summarize the integrated use of GeneGenie to design DNA and oligonucleotide sequences, followed by the procedure for assembling these accurately and efficiently using SpeedyGenes.
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The kinetic parameters of CH4 oxidation (Km, Vmax, apparent threshold = Tha) were measured using different oxic soils (cultivated cambisol, forest luvisol, meadow cambisol, paddy soil) both in a fresh state and after 3 weeks preincubation under high CH4 mixing ratios (20%). The preincubation resulted in an increase of the most probable number of methanotrophic bacteria. In fresh soils, CH4 oxidation followed Michaelis‐Menten kinetics with a low Km (30–51 nM CH4), low Vmax (0.7–3.6 nmol CH4 h⁻¹g⁻¹dw soil), and low Tha (0.2–2.7 ppmv CH4). In preincubated soils, CH4 oxidation exhibited biphasic kinetics in which two different CH4 saturation curves were apparently superimposed on each other. Eadie‐Hofstee plots of the data showed two activities with different kinetic parameters: a high‐affinity activity with low Km (13–470 nM CH4), low Vmax (2.1–150.0 nmol CH4 h⁻¹g⁻¹dw) and low Tha (0.3–4.1 ppmv CH4) being similar to the kinetic parameters in fresh soils; and a low‐affinity activity with high Km (1740–27 900 nM CH4), high Vmax (270–3 690 nmol CH4 h⁻¹g⁻¹dw) and high Tha (11–45 ppmv CH4) being similar to the kinetic parameters known from methanotrophic bacteria. The low‐affinity activity was also observed in a soil over a deep natural gas source which was permanently exposed to high CH4 mizing ratios (>5% CH4). Bacteria culturable as methanotrophs are probably responsible for the low‐affinity activity which is typical for the soils exposed to high CH4 mixing ratios. However, the bacteria responsible for the high‐affinity activity are still unknown. This activity is typical for the soils exposed to only ambient CH4 mixing ratios. Both high‐ and low‐affinity activities were inhibited by autoclaving and by acetylene.
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A quasi-total loss of the bacterial hydroxylating activity was identified to be responsible for methanol production stop. Different strategies acting on the reaction mixture were implemented to apprehend the biocatalyst behavior in view to extend methanol production. Activity monitoring showed first that sodium formate addition did not maintain the biocatalyst activity and even disrupted bacterial equilibrium when added into the reaction mixture with still active biocatalysts. Reaction medium renewals had no influence on methanol production and highlighted a limited hydroxylating potential of the biocatalyst while addition of fresh biocatalysts in the reaction mixture resulted in methanol consumption. Finally, performing hydroxylation directly in the native bacterial culture appeared as a way to enhance methanol production by both release of intracellular methanol accumulated in the cells during cultivation and effective production by methane hydroxylation.
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Before delving into the taxonomy of the methylotrophic bacteria, it is worth taking a light-hearted look at the subject of taxonomy itself. What is taxonomy and why is it necessary?
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The paper critiques proposals for de-carbonizing transport and offers a potential solution which may be attained by the gradual evolution of the current fleet of predominantly low-cost vehicles via the development of carbon-neutral liquid fuels. The closed-carbon cycles which are possible using such fuels offer the prospect of maintaining current levels of mobility with affordable transport whilst neutralizing the threat posed by the high predicted growth of greenhouse gas emissions from this sector. Approaches to de-carbonizing transport include electrification and the adoption of molecular hydrogen as an energy carrier. These two solutions result in very expensive vehicles for personal transport which mostly lie idle for 95% of their life time and are purchased with high-cost capital. The total cost of ownership of such vehicles is high and the impact of such vehicles in reducing greenhouse gas emissions from transport is therefore likely to be low due to their unaffordability for a large number of customers. Conversely, powertrains and fuel systems capable of using renewable alcohols in high concentrations have minimal additional cost over existing models as they are made from abundant materials with low embedded energy levels. The use of ethanol and methanol in internal combustion engines is reviewed and it is found that the efficiency and performance of engines using these fuels exceeds that of their fossil fuel counterparts. Low-carbon-number alcohols and, where necessary, more energy-dense hydrocarbons can be supplied using feed stocks from the biosphere up to the biomass limit from biofuels and, beyond the biomass limit, from the atmosphere and oceans using captured CO 2 and hydrogen electrolysed from water. Using the hydrogen in a synthesized fuel rather than as an independent energy carrier can be thought of as a pragmatic implementation of the hydrogen economy. This avoids the extremely high infrastructure and distribution costs which accompany the use of molecular hydrogen. The production of liquid fuels from CO2 and water are reviewed in which fully-closed carbon cycles are theoretically possible with the development of large-scale renewable energy generation and CO2 capture from the atmosphere. An approach to the latter problem where CO 2 concentration and release based on bipolar membrane electrodialysis, developed by the co-authors from PARC, is described in detail and initial results from a laboratory scale device are reported. The development of a Tri-Flex-Fuel vehicle, capable of operating on any combination of gasoline, ethanol, and methanol, using a single fuel system is also described. The low additional technology and materials costs of such vehicles demonstrates that compatible, affordable transport can be developed which provides a feasible means of vehicle evolution towards decarbonized transport without the consequences of huge stranded assets which would be imposed on the automotive industry by the revolution which would be required to mass-produce hydrogen fuel cell vehicles and battery-electric vehicles. Copyright © 2009 Lotus Cars Ltd. Published by SAE International with permission.
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Methanol can be made from gas, coal, or wood. It is stored and used in existing equipment.