Fuel 273 (2020) 117783

Contents lists available at ScienceDirect

Fuel
journal homepage: www.elsevier.com/locate/fuel

Review article

Bio-methanol as a renewable fuel from waste biomass: Current trends and T

future perspective
⁎
Pallavi Gautama,1, Nehaa,1, S.N. Upadhyayb, S.K. Dubeya,
a
Molecular Ecology Laboratory, Centre of Advanced Study in Botany, Institute of Science, Banaras Hindu University, Varanasi 221005, India
b
Department of Chemical Engineering & Technology, Indian Institute of Technology, Banaras Hindu University, Varanasi 221005, India

A R T I C LE I N FO A B S T R A C T

Keywords: Use of abundantly available virgin and waste biomasses as feed-stock for producing gaseous (bio-gas) and liquid
Biomass fuels (bio-methanol, bio-ethanol and bio-butanol) is being considered as the sustainable and viable alternative to
Thermochemical fossil fuels (coal, natural gas and petro-fuels like gasoline and diesel). Out of these bio-methanol is being con-
Biochemical sidered as an attractive liquid fuel as well as feed-stock for the synthesis of enumerable valuable organic
Methanotrophs
compounds currently being produced from coal, natural gas, and petroleum feed stocks. This review presents an
Methanol
overview of various thermo-chemical and biochemical routes that are being explored for the sustainable pro-
Methane monooxygenase
duction of bio-methanol from waste biomass. The advantages and limitations of both the routes are discussed to
provide a brief account of their basic principles and also indicate the issues to be addressed through further
technological up-gradations for satiating the future energy demand. It focuses specially on the biochemical
conversion route which utilizes microbes as biocatalysts for methanol production under normal temperature and
pressure conditions. Available information on various process parameters affecting microbial production of bio-
methanol have been critically reviewed. To make the process cost effective certain improvements like utilization
of raw biogas instead of natural gas for methanol production and development of methane-utilizing microbes
through genetic engineering as the subject for future research are discussed. The gap existing in the current
knowledge that needs to be bridged to facilitate development of technology for large scale production of bio-
methanol at an economical rate to meet the future demands are also pointed out.

1. Introduction best option [4]. These sources of renewable energy are promising and
sustainable because of their more or less uniform global availability
Increasing human population and changing life-style of the people with each country having access to them unlike fossil fuels and nuclear
due to technological advancement have led to an exponential increase fuels. Out of the various renewable resources, the biomass consists of a
in the global demand of energy. Majority of current domestic and in- diverse group of substances of plant and animal origin which can be
dustrial operations depend primarily on fossil fuels (mainly natural gas used for gaseous, liquid and solid fuels production [5]. Waste biomass is
and petroleum fuels) for their energy requirements, which are the non- available as animal and plant residues in agricultural waste, animal
renewable sources since their reserves are limited and cannot be re- farm wastes, domestic waste, forest residue, several industrial wastes
plenished within a normal human life-span [1]. Their usage also entails and human excreta. Virgin biomass can be obtained from photo-
the issues of environmental pollution [2]. Thus, researchers are ex- synthetic microalgae and macro-algae and forests, grass-lands etc. Algal
ploring various options to come up with alternative solutions to get biomass can be grown in coastal and marginal land areas [6,7] without
over the looming energy crisis. One alternative option is nuclear energy competing with food grains.
from fissionable elements like plutonium and uranium that might help The combustion of biomass for generation of energy has been
in getting over this crisis to a large extent [3]. This however, has its own practiced since ages. However, this process causes environmental pol-
environmental and technological problems and is not a renewable so- lution and is not very efficient as the full potential of the biomass as
lution in reality. Thus, harnessing various potential renewable energy energy source is not exploited [8]. Various thermochemical and bio-
sources like geothermal, wind, solar, hydropower and biomass is the chemical processes can be used to modify the biomass into gaseous and

⁎
Corresponding author.
E-mail address: [email protected] (S.K. Dubey).
1
First two authors have equal contribution.

https://doi.org/10.1016/j.fuel.2020.117783
Received 27 December 2019; Received in revised form 6 March 2020; Accepted 2 April 2020
0016-2361/ © 2020 Elsevier Ltd. All rights reserved.
P. Gautam, et al. Fuel 273 (2020) 117783

liquid fuels having better calorific values. Presently, biomass meets getting over the issue of competition between food v/s fuels. However,
about 10% of the total global energy demand thus making it the most many researchers consider it to be an unrealistic option as algae pro-
potential candidate amongst all renewable energy sources [9]. A wide duce oils as their defense mechanism against lack of nutrients and
variety of biomass can be grown and obtained in any geographic lo- sunlight when their growth is poor [15] and are likely to give rise to a
cation depending upon the environmental conditions prevailing there major issue that in poor growth rate conditions the biofuel production
and also their storage and transport does not pose a significant problem will also be limited and unprofitable due to their direct dependence on
[10]. the amount of biomass.
Biomass derived liquid and gaseous fuels are termed as biofuels. A variety of liquid biofuels like biomethanol, bioethanol and bio-
Biofuels can be obtained from a range of cheap biomass sources and butanol can be obtained through the conversion of ligno-cellulosic
therefore can be produced practically in most places [11]. A wide biomass. Each of these liquid fuels has certain advantages as well as
variety of bio-fuels and chemicals like bio-methanol, bio-ethanol, bio- disadvantages over the other. For example bio-butanol is superior over
diesel, synthetic liquid hydrocarbons, acetic acid, formaldehyde, etc. bioethanol and biomethanol because of the reduced problem of its
can be produced from the biomass and some of these are currently demixing in biobutanol-petroleum blends, less corrosiveness, lower
available in the market [12]. Biofuels are sustainable, renewable, cause vapor pressure, higher energy content and flash point [23,24]. Despite
reduced greenhouse gas emissions, and have least or no dependence on these significant advantages of bio-butanol over other bio-alcohols, its
foreign resources, thus are superior to the conventional energy re- low productivity is the major drawback [25]. Similarly bio-methanol
sources [13]. The utility and the specific energy content of biofuels has an edge over bio-ethanol due to its higher specific energy yield
depends upon the type of feedstock used and the conversion technology [16]. Currently bio-ethanol produced through sugar and starchy feed-
employed [11]. stocks is more prevalent than biomethanol due to the compatibility of
Virgin and waste biomasses are being projected as sustainable al- bioethanol/gasoline blends with current IC engines. However, in the
ternative to conventional energy resources due to their renewable long run production of bio-methanol is likely to be more favorable due
nature and fewer environmental repercussions. A worldwide effort is to its better CO2 mitigation potential [26].
being made to replace/supplement fossil fuel based industry with bio- Further methanol is a good biofuel because it is easy to store and
fuels to reduce greenhouse gas emissions [14]. However, much ground transport being liquid at room temperature and has a high volumetric
has to be covered to achieve technical feasibility, economic viability energy density [27]. It can also be readily used as a raw material for the
and environmental sustainability of such alternatives. The major hurdle synthesis of a variety of useful organic compounds of industrial im-
in switching to bio-fuel technology is the fear of additional pressure portance [28]. It can be used as the replacement for petroleum derived
that it would put on the cultivable land for producing the required hydrocarbons and related chemicals [29]. It has a higher octane
biomass however, on the other hand a shift towards labor-intensive number compared to pure gasoline fuel [30].
agriculture would generate prospects for rural development and job Methanol can be produced through thermo-chemical as well as
opportunities for local population [15]. In an attempt to replace fossil biochemical processes. Both conversion pathways have their inherent
fuels large amount of funds are being allocated to develop renewable advantages and disadvantages. The thermo-chemical route of conver-
biofuels (or bio-refineries) that would lead to energy security and sion has been extensively studied and exploited since long. Industrial
economic upliftment of masses in developing countries [16]. However scale production of methanol through thermo-chemical processes has
the concept of bio-refinery is yet to attain technical proficiency as many been achieved but presently it is economically unattractive.
factors are yet to be optimized to achieve production efficiency. Major The biological conversion processes, on the other hand, are still
technological up gradation needs to be carried out for maximal con- being experimented at laboratory scale and lack sufficient information.
version of raw biomass into desired bio-fuels and products and to make The biochemical production of methanol requires study of the mi-
this process sustainable the byproducts obtained in each process need to crobes, their metabolic pathways and enzymes involved for proper
be put to use in other sectors for minimum waste generation [17]. understanding of the bioconversion process and knowledge of various
Biomass derived biofuels are classified as the first and the second parameters essential for scaling-up of the laboratory results to full size
generation biofuels. Bio-fuels, such as ethanol, obtained through the production units.
fermentation of polysaccharides-based biomass (sugars and starches) In this review an effort has been made to concisely assess the
[18] are the first generation bio-fuels whereas, those (e. g. bio-me- technology currently being used to convert bio-mass waste into me-
thanol) produced from the non-food biomass (virgin and waste lingo- thanol through various thermo-chemical routes directly or indirectly.
cellulosic non-edible biomasses, etc.) obtained from different sources Their merits and demerits too have been discussed to make a case in
are the second generation bio-fuels [19]. The development of second favor of bio-conversion route. The biochemical routes for methanol
generation biofuels is receiving more attention to avoid food-fuel production are discussed in relatively more detail. Various microbes are
competition in a time when the expanding population is already facing capable of producing methanol via their metabolic pathway utilizing
hunger pangs due to the reducing cultivable land area. The additional processed biomass as the feedstock. This innate tendency of microbes
need of land for growing energy crops would further complicate the can be exploited to produce methanol. The available information along
situation [18]. The requirement of vast croplands for biomass will lead with the existing gap in the knowledge is discussed thoroughly
to clearing of forests which would ultimately incur carbon debt thereby
defying its environmental sustainability, the most important argument 2. Biomass wastes as the feedstock for production of biofuel
against biofuel generation. Thus the dependence on the second gen-
eration biofuels is advantageous because there is no requirement of As pointed out earlier utilization of various types of starchy and
investment in the form of vast croplands to obtain the feedstock for sugar bearing biomass obtained from crops like corn, sugarcane, mis-
producing them. An important aspect to be considered before going canthus, etc. for the production of bio-fuels (bio-ethanol) leads to the
ahead with the production of the second generation biofuels is the ethical issue of food versus fuel. When millions of people are going to
development of new and relevant biomass conversion technologies bed daily with an empty stomach and half of the world population is
[20]. The conversion of ligno-cellulosic biomass to fuels capable of fighting starvation, in such a grave situation an additional burden on
replacing fossil fuels would require huge economic investments due to the agriculture sector to grow energy crops is not a feasible option [31].
the requirement of complex infrastructures and large amount of bio- Thus, there is a need to shift focus from energy crops to alternative
mass for generation of fuel [15]. Recently third and fourth generation substrates like biomass waste generated from various sectors [32,33].
biofuels (like bio-butanol) have been introduced which utilize algal The excessive amount of biomass waste generated from various sectors
biomass as the feedstock [1,21,22]. This class of fuel is capable of poses a problem of safe disposal. Methods like incineration and landfills

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P. Gautam, et al. Fuel 273 (2020) 117783

lead to adverse environmental consequences [34]. The utilization of for the removal of gaseous impurities [44]. Alkali compounds can
biomass waste as a feedstock for bio-fuel production has the potential corrode the downstream equipment and may cause inactivation of the
for the generation of renewable energy together with efficient and metal catalysts. These are removed by passing the syngas through
economical waste management [35]. barrier filters at high temperature [47]. These additional cleaning steps
The type of biomass (source/raw material) and the conversion route and requirement of high temperature add to the overall cost of the
play a crucial role in the economical production of bio-fuels from bio- biofuel production. Biomass conditioning prior to gasification, oxygen
mass. Based on the type and attributes of different biofuels their con- requirement and reactor maintenance are some of the major challenges
version pathways differ. Biodiesel, ethanol and biogas can be efficiently associated with biomass gasification.
produced via fermentative and microbial/enzymatic routes [36]
whereas bio-char, syngas, etc. are favorably produced through ther- 3.2. Pyrolysis
mochemical routes [16]. Pyrolysis, gasification and liquefaction are the
commonly employed thermo-chemical routes for the production of The classical pyrolysis (also known as destructive distillation of
methanol from biomass [37]. wood) was developed primarily for producing char-coal from wood and
methanol was obtained as a by-product. The modern pyrolytic process
3. Thermochemical conversion processes is the thermo-chemical route where advanced thermal treatment under
inert atmosphere converts the biomass into bio-oil, char and gases [37].
Production of methanol through thermochemical process takes It involves the high temperature (200–750 °C) heating of the biomass in
place via synthesis gas (syngas) production route. In thermochemical a non-oxidizing atmosphere. The direct thermal decomposition yields
conversion process biomass is thermally decomposed in oxygen/air various products that could act as an alternate source of energy. Pyr-
deficient conditions to produce mixture of H2 and CO, commonly olysis is the first step of all thermochemical biomass conversion pro-
known as ‘syngas’. The syngas produced is then further processed cat- cesses [48]. It is a composite process comprising reactions like iso-
alytically to produce liquid fuels like methanol [37]. Thermochemical merization, de-polymerization, dehydration, aromatization, and
conversion technologies are versatile as they have the advantage of charring [48,49]. Biomass pyrolysis begins with the loss of moisture
utilizing any kind of biomass as the feedstock unlike biochemical routes and then reactions take place in two stages where the primary processes
[38]. A brief account of various thermo-chemical processes is given are characterized by the char formation [50], fragmentation and de-
below. polymerization [51,52,53] and the secondary reactions like oil cracking
and re-polymerization [54,55]. Biomass pyrolysis can be of three types
3.1. Gasification on the basis of the heating rate, particle size, temperature and residence
time of solids- it can be slow (conventional), fast, and flash pyrolysis
The gasification process converts the biomass waste into bio-fuels of type [56,57]. However, this division is not strictly rigid because slow
industrial significance [39]. It is a complex process through which and fast heating rates are arbitrary in many cases [59]. A wide variety
carbonaceous materials of bio-mass are converted into syngas (hy- of solid (bio-char), liquid (bio-oil) and gaseous products are formed in
drogen and carbon monoxide) which is further processed to obtain this process [60]. The desired product in large amount can be obtained
methanol. Major steps involved in the process are pre-treatment of from this process by modulating the reaction conditions, for example
biomass, gasification, gas cleanup, reforming and finally utilization in liquid products can be maximized at moderate temperature, shorter
the bio-fuel production [40]. Before the biomass is subjected to the residence time and high heating rate [57]. Whereas, gaseous products
gasification process, it is pre-treated for its maximum utilization. It is or fuel gas can be obtained as a major product at a high temperature
subjected to reduction in size to increase the surface area, drying to and longer residence time but at a lower heating rate. Similarly for
maintain the required moisture conditions and densification [41]. The obtaining charcoal in substantial amount a low temperature and a
second step and most important one is the gasification. The gasification lower heating rate for longer residence time is required [39,57,58,61].
of waste biomass occurs at high temperature (800-900˚C) and the large Conventional pyrolysis, also termed as carbonization, occurs at slow
polymeric molecules are partially oxidized in the presence of gasifying heating rate and longer residence time. These conditions provide ample
agent air, oxygen or steam [5,42]. The syngas produced primarily time for repolymerization reactions to occur and thus are used for the
consists of CO and H2 and traces of CH4 along with formation of tar, production of charcoal [54,58]. The operational conditions of this
ash, alkali compounds, nitrogen and sulfur containing compounds and process lead to lesser liquid and gaseous products formation and more
water [43]. The amount and composition of syngas produced and its tar and char production due to slow devolatilization of biomass. To
impurity level depends upon the type of biomass, gasifying agent, rate obtain liquid products in higher concentration preferred temperature
of heat transfer and reactor type [44]. Sequential reactions occur during range is 350 to 450 °C [56,57,61]. The preferred technologies to obtain
gasification, the first step being the dehydration followed by the pyr- liquid products are the fast and flash pyrolysis [62]. Methanol can be
olysis and the third stage being the combustion. Finally, the fourth obtained from the pyrolytic decomposition of different types of biomass
stage of gasification is the water-gas shift reaction [45]. The optimum like pectins thereby forming methyl esters/ethers and also from uronic
temperature for each stage and the degradation kinetics depend on the acid (methoxyl groups) [57].
biomass composition and the oxidizing environment [46]. Various Fast pyrolysis (thermolysis) is rapid heating of biomass at high
parameters thus need to be optimized for designing a gasifier for the temperatures to obtain high grade bio-oil [5]. Heating rate is rapid in
large production of syngas with minimal amount of impurities as by- case of fast pyrolysis but still less than flash pyrolysis [39]. In case of
products. Various types of gasifiers namely, fixed and fluidized bed, flash pyrolysis the biomass residence time lasts for only few seconds
dual fluidized bed, and plasma gasification have been developed for the and the heating rate is extremely high [56]. Several types of reactors
production of syngas [39]. have been designed to carry out fast/flash pyrolysis of biomass. These
The side products formed in the process affects the yield of the main include fluidized bed reactors, fixed bed reactors, recirculating flui-
gaseous products and thus necessitate inclusion of an additional step of dized bed reactors, rotating cone pyrolyzer, ablative pyrolysis reactors,
cleaning up so as to sustain the downstream processing of syngas for auger or screw pyrolyzer and vacuum pyrolyzer [5,11]. The bio-oil and
methanol production [11]. Different processes are employed to obtain gaseous products obtained from fast and flash pyrolysis need to undergo
impurity free syngas. In syngas cleaning step particulates are removed further catalytic reactions for upgrading into methanol and other
by cyclone separators, wet scrubbers and electrostatic precipitators transportation fuels.
suited for large scale use. Tar is removed by catalytic reforming pro- For methanol production pyrolytic reactors are used that carry out
ducing more syngas and various sorbents and metal catalysts are used biomass gasification and produce a mixture of various gases which is

3
P. Gautam, et al. Fuel 273 (2020) 117783

purified to obtain syngas. The syngas is further converted to produce monoxide and hydrogen present in the syngas react catalytically to
methanol by catalytic reactions [63]. Although the yield is high but the form methanol along with other products like methyl formate, me-
complexity of the process, additional cleaning steps, reactor installation thane, acetone and higher alcohols (isopropyl alcohol, sec-butyl al-
and maintenance makes the process expensive. cohol, tertiary butyl alcohol, tertiary amyl ether, mixed alcohols, etc.)
[68]. The general reaction for synthesis of an alcohol from syngas is
3.3. Liquefaction given below:

Liquefaction converts wet biomass to liquid fuels in the presence of nCO + 2nH2 → Cn H(2n+ 1) OH + (n−1)H2 O (1)
catalysts and operates under low temperature (200–500 °C) and high
pressure (5–20 bar) conditions [42]. Thermal liquefaction is quite si- When n = 1, the product is methanol and when more than 1, higher
milar to the pyrolysis process and is a mosaic of various reactions like alcohols are produced. The syngas obtained through the thermo-
dehydrogenation, dehydration, decarboxylation and deoxygenation. chemical conversion of biomass has to undergo further catalytic con-
However, it varies in the requirement of temperature and pressure version to produce methanol. Prior to the methanol synthesis, syngas
conditions, type of catalyst and it mainly produces liquid products [11] has to be cleaned and ratio of CO and H2 has to be adjusted. The cat-
unlike pyrolysis. Liquefaction is of two types- direct and indirect. Direct alysts used for converting syn-gas to methanol are- high pressure cat-
liquefaction of biomass is similar to fast pyrolysis and recovers bio-oil alyst, ZnO/Cr2O3; low pressure catalyst, Cu/ZnO and Cu/ZnO/Al2O3.
from the biomass by extraction [64]. The products formed are bio-oil, When these catalysts are doped with alkali, higher alcohols are pro-
liquid tar and condensable organic vapor [37]. Indirect liquefaction duced. Use of alkali-doped sulfides such as molybdenum sulfide (MoS)
produces oil via synthesis gas (syngas) formation in two steps and thus and alkali-doped oxides (CuO/CoO/Al2O3), results in the formation of
this pathway is used for the production of methanol (partial CO hy- higher alcohols [69]. Thus, nature of the catalyst and residence time in
drogenation). The characteristics of the products formed here are in- the reactor control the type of alcohol formed.
dependent of the raw material [64]. The major plus point associated Methanol production from syngas is an exothermic reaction that
with this process is that it can convert wet biomass into biofuels [37]. In requires a catalyst together with high pressure (maintained at 300 bar)
other thermochemical processes dry biomass is required as the feed- and temperature (upto 200–400 °C) [70]. The high pressure and tem-
stock. However, major drawbacks are also associated with this process. perature are required to provide appropriate operating conditions for
The products formed in the direct liquefaction are hard to handle be- maintaining the catalytic activity [71]. However, the reaction ther-
cause of the lump formation due to the presence of excess tar [65]. The modynamically prefers low temperature and high pressure conditions
bio-oil produced through indirect liquefaction is low in yield and for the optimum methanol production due to the exothermic nature of
quality than the pyrolytic bio-oil [11]. Also, its commercial scale ap- the reaction. High temperature although leads to the increased activity
plication is further limited due to the complex operational requirements of the catalyst but the specificity of the reaction is reduced leading to
and cost ineffectiveness. The bio-oil produced has some methanol in it, enhanced formation of the side products thereby decreasing the yield of
but its separation is quite cumbersome. the main product [72]. The chemical reactions involved in the con-
The thermo-chemical processes are feasible at large scale and are version of syngas to methanol are given below:
capable of producing methanol from a variety of cheap feedstock. CO + 2H2 → CH3 OH (2)
Though the basic mechanisms of the gasification and pyrolysis pro-
cesses are more or less clear, however, several aspects still require
further investigation to generate information for design of efficient CO2 + 3H2 → CH3 OH + H2 O (3)
gasifiers and pyrolyzers. For example knowledge of effects of particle
The final yield and the selectivity of the reaction is governed by the
size of the biomass to be used, mixture of biomasses, moisture content
ratio of H2 to CO + CO2 and CO2/CO ratio, respectively [73]. Thus,
and its role, effect of minerals present in the ash/char produced during
CO2 is required to be removed to increase the selectivity as well as the
pyrolysis on syngas production, role of catalysts and the selection of
yield of methanol [41]. This gas ratio requirement makes it mandatory
most efficient and cheap catalyst, and optimal heating rate, tempera-
to modulate the syngas composition prior to the exothermic gas-phase
ture range, and residence time will be helpful in selecting the optimal
catalytic reaction for methanol synthesis. During methanol production
design of the reactor out of the various available reactor configurations.
syngas undergoes water-gas shift reaction whose utility is in main-
In case of liquefaction more investigations are required to come out
taining the H2/CO ratio > 2/1 for favoring the reaction kinetics and
with information on the effects of nature of biomass, particle size,
controlled by-product formation [74]. The water gas shift reaction is
moisture content, operating conditions like pressure and temperature,
given below:
effect of catalysts, etc. The knowledge of the involved reaction me-
chanism of product formation during liquefaction process is also not CO + H2 O↔ CO2 + H2 (4)
very clear and needs to be explored. The economic feasibility is the
major challenge with various thermochemical production pathways. Methanol is primarily achieved by the reaction between CO and H2
which is enhanced by the presence of carbon dioxide. Efforts are being
3.4. Methanol production from synthesis gas (Syngas) made to develop two-phase catalytic reactors in which un-supported
nano-sized particles or supported on high surface area supports such as
Synthesis gas (syngas) obtained through various thermochemical alumina, carbon, graphene, or silica will be used as catalysts [75]. Ef-
processes described above is made up primarily of H2 and CO with little forts are also being made to develop plasma based processes for con-
amount of CO2 and other gases [66]. Syngas has diverse role in the verting waste biomass to alcohol. The main drawback associated with
chemical industry. It is used for the production of ammonia, methanol, these processes is that they are quite expensive because of the re-
hydrogen, and in various oil refining processes [67]. Currently, almost quirement of costly metal catalysts and high temperature (200-900˚C)
all methanol used at global scale is produced from the reforming of and pressure (5–20 MPa) [76]. Also, the requirement of additional
syngas obtained from natural gas. But the advent of various thermo- cleaning steps to obtain syngas makes the process further expensive.
chemical processes like gasification, pyrolysis, etc. that use biomass as Thus a different pathway for methanol production needs to be explored
feedstock in syngas production projects a promising alternative for where cost input is efficiently reduced. The biochemical pathway pre-
methanol production. Catalytic conversion of syngas leads to the for- sents a promising alternative as it would utilize methane as a feedstock
mation of two varieties of products- hydrocarbons and alcohols de- for the methanotrophic bacteria who would convert it into methanol at
pending upon the catalytic conditions [67]. Carbon dioxide, carbon ambient pressure and temperature.

4
P. Gautam, et al. Fuel 273 (2020) 117783

4. Biochemical conversion process conditioning and purification for obtaining the feed-stock suitable for
the methanol conversion step.
The biochemical processes such as anaerobic digestion, fermenta-
tion and photo-biological conversion can be employed for the produc-
4.2. Methanol production from methanotrophic bacteria (Methanotrophs)
tion of various bio-fuels from virgin and waste biomasses [77]. The
fermentable biomass can be converted to ethanol through the alcoholic
The methanotrophs utilize methane as the sole carbon source for
fermentation, the anaerobic digestion of biomass can lead to the pro-
their metabolic needs thereby generating carbon dioxide as the final
duction of biogas (primarily methane) and bio-hydrogen can be ob-
oxidation product through a series of biochemical reactions [91–93].
tained via the dark fermentation and photo-biological routes [78–80].
The basic pathway along with the key enzymes involved in production
The anaerobic digestion of the biomass produces a gaseous mixture
of methanol from methane and its subsequent conversion to carbon
whose major constituent is methane (60–70%) and minor constituents
dioxide is depicted below:
include hydrogen sulfide, carbon dioxide, ammonia and other trace
gases [81]. The mixture of gases obtained is conventionally used for MMO MDH FDH FMDH
CH 4 → CH3 OH → HCHO → HCOOH → CO2
heating and/or generation of electricity. Through appropriate pre-
treatment this biogas can also be converted to a fuel of higher grade, The enzymes involved in this pathway sequentially are methane
e.g., methanol and other organic molecules through bio-chemical route. monooxygenase (MMO) enzyme (that converts methane into me-
In the following sections a brief account of anaerobic digestion process thanol), methanol dehydrogenase (MDH) enzyme (that converts me-
to produce biogas, its purification for obtaining the feed-stock for the thanol into formaldehyde), formaldehyde dehydrogenase (FDH) (that
methanol production through bio-route are discussed. Sufficient in- converts formaldehyde to formate) and formate dehydrogenase
formation is available in the literature on bio-ethanol production and (FMDH) (that converts formate into carbon dioxide) [94]. Above
bio-hydrogen production from biomass and hence these are not de- pathway can be modified using certain inhibitors to obtain methanol as
scribed here [82,83]. the end-product [95]. Methanotrophs convert methane into methanol
at ambient pressure and temperature in the rate limiting step of me-
4.1. Anaerobic digestion thane oxidation in the presence of the biological catalyst, methane
monooxygenase enzyme (MMO) [96]. However, to obtain methanol as
The anaerobic digestion of various biomasses produces biogas the final product, an inhibitor for the enzyme methanol dehydrogenase
consisting primarily of methane (65–70%) and other gases like carbon (MDH) is required to inhibit further oxidation [97]. Also, to support the
dioxide, hydrogen, hydrogen sulfide, etc. The actual percentage of catalytic activity of the methane monooxygenase enzyme supple-
methane depends upon the nature of the biomass used as the feed-stock mentation by the reducing equivalents is required to provide electrons
and operating conditions. The bio-gas produced through this route is for the initial oxidation step. Currently the methane, used as the feed-
more or less a carbon neutral fuel as the carbon dioxide produced stock substrate for methanol production through the thermo-chemical
during combustion is recycled back as the fresh bio-mass through route, is obtained from the natural gas fields, petroleum refineries and
photo-synthesis. Anaerobic digestion also contributes to the carbonac- shale gases [98]. Since these fossilized carbon based resources are ex-
eous domestic and industrial waste management as it converts organic haustible, the methane obtained through the anaerobic digestion of the
carbon rich wastes into useful energy by utilizing diverse groups of biomass may act as a sustainable feed-stock [88a] for methanol pro-
synergistic and anaerobic bacterial species [84,85]. The methane and duction.
ammonia present in the bio-gas obtained through anaerobic digestion There are two forms of methane monooxygenase- the more pre-
can be utilized by various methanotrophic and ammonia oxidizing valent membrane bound particulate MMO (pMMO) and the soluble
bacteria as metabolite or co-metabolite, respectively for producing cytoplasmic MMO (sMMO) [88b,99,100] On the basis of the type of
methanol [86]. An additional step however, is required for cleaning of monooxygenase enzyme present, the methanotrophs can be categorized
the biogas to get rid of the impurities and inhibitory gases like hydrogen into three groups. The Type I methanotrophs, following the ribulose
sulfide and ammonia [87]. The ratio of the concentration of con- monophosphate cycle to utilize methane, have the pMMO form, the
stituents like methane, water vapor, and carbon dioxide and carbon Type II methanotrophs have both pMMO and sMMO forms and carry
monoxide can be modulated to a desired level that could be utilized by out methane assimilation through the serine cycle. Finally, the Type X
the appropriate bacterial communities as the substrate for methanol methanotrophs has the properties similar to both Type I and Type II
production [88a]. Various bacterial communities play instrumental role species [91,98,100,101]. The metal active site of pMMO comprise of
in the production of biogas through the fermentation of organic bio- copper and thus copper deficient conditions may lead to the over-
mass. The anaerobic digestion process is cheap and economical because expression of sMMO [102,103]. In sMMO the metal active site consists
along with the utility of the main product as a renewable energy source, of divalent iron [104]. The expression of the both forms of MMO en-
the byproduct formed (the digested biomass slurry) can be utilized as zymes can be up-regulated or down-regulated by modulating the con-
an organic fertilizer. The process also helps in the waste management centration of various transition metals like gold and copper in the
and pollution control [89]. growth medium [105]. It is well established that the isolation of sMMO
The major drawbacks associated with the process is the requirement form of MMO enzyme is easier than its pMMO form. Also, sMMO has a
of an additional step for cleaning of biogas, dependence on weather wider substrate specificity than pMMO because the active site of pMMO
conditions for fulfilling optimum temperature requirement of different is more sterically hindered [106]. Thus, for optimal production of
bacterial groups and the requirement of a steady supply of feed bio- methanol a basic understanding of the structure and enzymatic me-
mass. It can only be set up in an area that can guarantee a constant chanisms involved in the oxidation with MMO is required, so that it
supply of raw materials for steady biogas production [90]. could be successfully modified or engineered to give the enhanced level
Several types of bioreactors such as- single stage, two-stage, parti- of methanol production.
tioned, submerged packed bed, fluidized bed and up-flow anaerobic This biological conversion of biomass to produce methanol is cur-
sludge bed type digesters have been developed primarily for anaerobic rently being extensively studied so that it can replace conventional
treatment of high BOD wastewater. Their suitability for the anaerobic chemical processes as it is more selective due to the specificity of the
digestion of biomass waste produced through agricultural and in- biocatalyst MMO enzyme and is also cheaper because it carries out
dustrial activities requires further investigation, particularly on pre- reactions at ambient pressure and temperature [100]. Several workers
treatment of the waste and optimization of the operating parameters have studied the potential of various methanotrophic strains for bio-
affecting biogas having maximum methane concentration, gas methanol production. Each investigator has strived for characterizing a

5
P. Gautam, et al. Fuel 273 (2020) 117783

strain of methanotroph that would produce a higher concentration of Their results showed that both CH4 and CO2 are utilized by M. sporium
methanol at a low cost. These studies have also aimed at developing a for methanol production. Their results further suggested that the raw
better understanding of the process, some of which are discussed in the biogas as such was not suitable for the methanol production, which was
next section. either because of the improper ratio of methane, carbon dioxide and
The inhibitors for methanol dehydrogenase enzyme that could be hydrogen or due to the presence of high concentration of inhibitory gas
used for producing methanol by partial oxidation of methane are H2S in the raw biogas. When a specific ratio of CH4:CO2:H2 in the
mentioned by several workers. Carver et al. [97] showed that EDTA biogas was obtained through the supplementation of deficient gases
(metal chelating agents) affected the respiratory unit of the Methylo- increased methanol productivity was obtained. H2 supplementation in
philus methylotrophus and inhibited the methanol oxidase activity the un-purified bio-gas resulted in a 3.5 fold rise in methanol produc-
completely whereas its methanol dehydrogenase activity only partially. tion. Zhang et al. [111] isolated the bacterium, Methylocaldum sp.
Frank et al. [107] showed that at high concentrations cyclopropane and SAD2, and showed its potential for methanol production using biogas
cyclopropanol act as irreversible inhibitors for MDH. Takeguchi et al. without scrubbing off H2S. They achieved a methanol production of
[108] and Furuto et al. [96] showed that cyclopropanol had the highest 276–343 mg L−1 even in the presence of 500 ppm H2S in the biogas
inhibitory effect. Lee et al. [109] investigated the effect of concentra- feed. Thus, direct utilization of biogas will further reduce the cost of
tion of Cu2+ on the ability of Methylosinus trichosporium OB3b on the methanol production. In their recent study Su et al. [112] used ther-
biosynthesis of methanol from methane. They showed that at 25 °C for motolerant methanotrophic consortia. The thermophilic MC-AD3, a
the methane: air ratio of 1:4 (v/v) and a bacterial concentration of methanotrophic consortium, was isolated from the digested slurry of
0.6 mg dry cell per mL in 12.9 mM phosphate buffer (pH 7) and 20 mM anaerobic digestion systems. It was reported that at 47 °C, 0.33 g L−1 of
sodium formate (acting as reducer) and 200 mM NaCl (acting as MDH methanol was produced by MC-AD3 that was in the range of values
inhibitor) 7.7 mM methanol could be produced in 36 h. As an inhibitor obtained using mesophilic strains. These results indicated MC AD3 as a
acting on MDH, NaCl has the advantage over cyclopropanol in that it is promising strain for methanol production. A summary of the reported
cheaper and stable. In their study using Methylosinus sporium, Yoo et al. experimental results on methanol production by methanotrophs is
[110] achieved the highest concentration of methanol presented in Table 1. The potential of different species of methano-
(6.25 μmol mg−1h−1) at 35 °C, pH 7, biogas concentration of 0.4% (v/ trophs for producing methanol under diverse optimized conditions is
v), 40 mM phosphate and 100 mM NaCl. They also concluded that clearly evident from these results.
beyond a certain concentration the MDH inhibitors negatively affect the From the foregoing discussion it is seen that some efforts have been
activity of sMMO resulting in lower methanol production. made to convert biogas to methanol. Effects of impurities like H2S, NH3,
The conversion of methane to methanol was studied in batch mode acetate, etc.; ratio of H2, CO, and CH4 in the feed gas; inhibitors; and
by Hwang et al. [100] using whole cells of Methylosinus trichosporium reducers have been reported but the results are not yet conclusive and
OB3b. They used the bacteria cultured in NMS medium and an air: adequate for utilization on large scale for producing methanol.
methane ratio of 7:3 at 30 °C. For the optimal production of methanol Similarly the information on bacterial species capable of producing
the MDH inhibitors and their concentrations were 100 mM and 0.5 mM maximum methanol concentration and the optimal temperature is also
of potassium phosphate and EDTA, respectively. The sodium formate insufficient. Further, no information is available on the suitability and
(40 mM) was taken as the alternative reducer because it leads to the use of various conventional or advanced separation techniques appro-
regeneration of the NADH cofactor which is required for a higher MMO priate for recovering methanol from the broth. However, the methods
activity. Also the highest specific growth rate and methanol production being used for recovering methanol produced through chemical route
were obtained when the culturing was done in presence of 5 µM CuSO4 may help in selecting an appropriate down processing scheme.
because the presence of copper affects the relative expression of parti-
culate and soluble MMOs as well as the cell growth. Under optimal 4.3. Large scale production of methanol from waste biomass
conditions they obtained a methanol productivity of 49.0 mg L−1h1.
Mardina et al. [93] utilized immobilized whole cells as the biocatalyst The process of bio-conversion of waste biomass into methanol
for methanol production. They used alginate-encapsulated cells of Type comprises four basic steps (a) pretreatment of biomass, (b) biogas
II methanotroph, Methylocella tundrae. These immobilized cells showed production through anaerobic route (c) purification of biogas and (d)
higher stability and reusability (i.e. upto 5 cycles) compared to the free methanol production. The pretreatment of biomass is pivotal because
cells. The optimum conditions were reported to be: incubation time without proper conditioning complex ligno-cellulosic biomass waste
(24 h), methane concentration (50%), pH (7), RPM (15), temperature cannot be acted upon by microbial enzymes [113]. The pretreatment
(30 °C), phosphate buffer concentration (100 mM), sodium formate step breaks down this resistant lignin layer and makes the waste bio-
concentration (50 mM), and inoculum load (18 mg mL−1). Due to these mass more susceptible to attack by microbes [114,115]. The pretreated
efforts the production of methanol enhanced from 0.66 to 5.18 mM. biomass is fed into the anaerobic digester for the production of biogas.
Patel et al. [88b] showed that the encapsulation of methanotrophs The biogas production takes place through four basic steps namely
improved the stability of methanol production. They immobilized Type hydrolysis, acidogenesis, acetogenesis, and methanogenesis [80,115].
II methanotroph, Methylosinus sporium by encapsulating in sodium-al- Different bacterial communities are instrumental at different stages of
ginate and silica gel. Cells encapsulated with sodium-alginate gave a biogas production. Since these bacterial communities have different
methanol productivity of 3.43 mM and maintained the methanol pro- growth rates and requirements hence carrying out anaerobic digestion
duction efficiency at 61% of initial efficiency even after 6 cycles of in two reactors operating in series provides specific conditions for the
reuse. Whereas, in case of silica gel the productivity was 3.73 mM and growth and activity of the microbes and results in an optimized output.
production efficiency after 6 cycles was 51.6% of initial efficiency. The two-stage anaerobic digester can be used for stability as well as
However, in case of free cells only 11.5% production efficiency was high rate of biogas production [116]. In the hydrolysis stage various
achieved indicating that encapsulated cells performed better than free hydrolytic bacteria play the functional role and breakdown complex
cells. polymers like carbohydrates, lipids and proteins into simpler forms
In the above studies pure methane was used as the substrate. In their [85,90,117,118,119]. These hydrolyzed products are further acted
later study Patel et al. [88a] explored the suitability of un-purified upon by various acidogenic bacteria (Clostridium, Lactobacillus, Actino-
biogas as a low cost substrate. They used immobilized whole cells of myces, Propionibacterium etc.) and get converted into various organic
Methylosinus sporium and also regulated the gas composition to get an acids, alcohols, carbon dioxide and hydrogen [85,120,121]. Thus, in
improved methanol yield. Use of un-purified biogas is advantageous as the first reactor of the anaerobic digester system, hydrolysis, acid-
it eliminates the requirement of costly pure methane as feed-stock. ification and acetogenesis reactions occur i.e. initial fermentation

6
P. Gautam, et al. Fuel 273 (2020) 117783

Table 1
Summary of selected typical studies on methanol production utilizing different methanotrophic strains.
S. No. Bacteria Constituents of reaction mixture Type of reactor Substrate used Methanol produced References

−1
1. Methylosinus trichosporium OB3b Phosphate (80 mM) Batch Methane: Air 2.7 µmol mg [144]
(1:1 v/v) h−1
2. Methylosinus Phosphate (100 mM) Na formate Continuous stirred Methane: Oxygen 267 µmol h−1 [145]
trichosporium OB3b (20 mM) membrane reactor (1:1 v/v)
3. Methylosinus trichosporium OB3b Cyclopropanol Batch Methane: Air (1:3 v/ 23 mmol g−1 (wet [146]
(0.234 µmol) v) cell wt.)
Na formate
(50 µmol)
4. Methylosinus trichosporium OB3b Cyclopropanol (67 nM) Batch Methane: Air (1:4 v/ 152 mmol [108]
Na formate v) g −1dry cell
(14.3 mM)
5. Methylosinus trichosporium OB3b Cyclopropanol (67 nM) Batch Methane: Air 0.15 mmol L−1 h−1 [96]
Na formate (14.3 mM) (1:2.6 v/v)
6. Methylosinus trichosporium OB3b NaCl Batch Methane: Air (1:5 v/ 7.7 mmol L−1 [109]
(200 mM) v)
Na formate (20 mM)
7. Methylosinus trichosporium OB3b NaCl (100 mM) Continuous Methane: Air 0.57 mmol L−1 h−1 [147]
EDTA (1 mM) (1:1 v/v)
Na formate (20 mM)
8. Methylosinus trichosporium Phosphate (400 mM) Batch Methane: Oxygen 1.12 g L−1 [132]
OB3b Na formate (20 mmol (1:1 v/v)
L−1)
9. Methylosinus trichosporium Phosphate (12.9 mM) Batch Methane: Air 290 mg L−1 [133]
OB3b NaCl (100 mM) (1:1 v/v)
EDTA (1.0 mM)
Na formate (20 mM)
10. Methylosinus trichosporium OB3b Phosphate (100 mM) Batch Air: Methane 0.393 g L−1 [100]
EDTA(0.5 mM) (7:3 v/v)
Na formate (40 mM)
11. Methylosinus sporium (encapsulated) MgCl2 (20 mM) Batch Methane: Air 3.73 mM [88b]
Phosphate (100 mM) (1:4 v/v)
Na formate (40 mM)
12. Methylosinus sporium MgCl2 (20 mM) Batch CH4:CO2:H2 7.16 mM [88a]
Phosphate(100 mM) (2:1:1)
Na formate (40 mM)
13. Methylocystis bryophila Phosphate Batch Methane: Air 4.63 mM [88c]
(100 mM) (1:1 v/v)
MgCl2 (50 mM)
Na formate (100 mM)
14. Methylosinus trichosporium OB3b Phosphate Fed-batch membrane Methane: Air 18.8 mg L−1 [148]
(12.9 mM) bioreactor (1:1 v/v)
NaCl (100 mM)
EDTA (1 mM)
Na formate
(2 mM)
15. Methylocaldum sp. Phosphate Batch Biogas: Air 0.43 g L−1 [149]
(50 mM) (1:2.5 v/v)
Na Formate (80 mM)
16. Immobilized whole-cell Methylocella Phosphate Batch Methane: Air 5.18 mM [93]
tundrae (100 mM) (1:1 v/v)
Na formate
(50 mM)
17. Methylocaldum sp. 14B Phosphate Trickle bed bioreactor Biogas: Air 0.9 g L−1 d−1 [150]
(3.6 mmol) (1:2.5 v/v)
Na Formate
(12 mmol)
18. Thermotolerant methanotrophic Na formate Batch Biogas: Air 0.33 g L−1 [112]
consortium (MC-AD3) (100 mM) (1:4 v/v)
19. Mixed methanotrophic culture NaCl Batch CH4:CO2:O2 6.35 mM [151]
(60 mM) (Biogas) (2.4:2.8:1 v/
Na formate v)
(40 mM)

through a mixed consortium of acidogenic and acetogenic bacteria. In CH3 COOH → CH 4 + CO2 (7)
the second reactor, products of previous stage are acted upon by me-
The first stage reactor (primarily acidification reactor) produces
thanogenic bacteria (Methanococcus, Methanobacterium, Methanosarcina
biogas containing high percentage of CO2 and some H2. Whereas, the
etc.) and get converted into methane and carbon dioxide [122–124].
second stage reactor (primarily methanation reactor) produces a higher
The biochemical reactions occurring in the second stage of the methane
quality biogas. The biogas mixture obtained in the second step needs to
bio-reactor can be summarized as follows:
be cleaned or upgraded to remove inhibitory gases (like hydrogen
2C2 H5 OH + CO2 → CH 4 + 2CH3 COOH (5) sulfide) obtained as side products before being fed into the methanol
producing bioreactor [125]. The removal of hydrogen sulfide is crucial
CO2 + 4H2 → CH 4 + 2H2 O (6) because it is poisonous, corrosive, and destroys the equipment and is

7
P. Gautam, et al. Fuel 273 (2020) 117783

also present in significant proportions [88a]. Biological H2S removal 4.4. Challenges associated with up-scaling for industrial production of
process is favored for this step because it eliminates the chemical and methanol by methanotrophic bacteria
disposal costs associated with existing chemical processes and also they
do not produce any harmful side products. H2S oxidation is carried out The biochemical conversion of gaseous substrates CO, hydrogen and
by phototrophic and chemotrophic bacteria using fixed-film reactors methane into methanol mediated by bacterial species requires their
(biofilter). Biofilter is packed with appropriate packing material whose availability in dissolved state in the liquid medium for interacting with
surface is covered with biofilm consisting of sulfur oxidizing bacteria the enzymatic catalysts. The gaseous substrates like hydrogen and
[126]. The biofilter medium contains sufficient micronutrients for methane are sparingly soluble in the culture media as a result these are
sulfur oxidizing bacterial communities (Thiobacillus, Acidithiobacillus, less bio-available to methanotrophs resulting in slow growth and re-
Sulfurimonas, Thiomonas, etc.). In the biological desulfurization process duced biomass production hence suboptimal activity of MMO enzyme
microbes oxidize hydrogen sulfide into elemental sulfur and/or sulfate leading to reduced methanol production [106]. Thus one of the major
in the presence of an electron acceptor [127]. The end-products thus issues associated with this process is the efficient transfer of gaseous
obtained are not toxic to the environment, the sulfate can be as such substrate to liquid culture medium [131]. A concerted effort needs to be
disposed off into the water bodies and elemental sulfur can be re- made to overcome this limitation. Another issue associated with the use
covered and used in industrial and agricultural sectors [128]. The hy- of whole cell culture is the requirement of maintaining the cell viability
drogen sulfide free bio-gas is fed into the bioreactor to be used as a and their catalytic activity. In the first step of methane oxidation, MMO
substrate for the production of methanol. The bio-reactor for methanol enzyme requires the electron donor/reducing equivalents which are
production can be either a trickle bed bioreactor or a fluidized bed again regenerated when the methanol is completely oxidized to CO2.
bioreactor. The rationale behind using fluidized bed reactors is that However, utilization of MDH inhibitors for obtaining methanol as the
they show better product generation than trickle bed bioreactors, but final product affects the metabolic pathway and as a result the reducing
due to the requirement of more pumping power and higher energy costs equivalents are not regenerated which is reflected through the loss in
a trickle bed bioreactor can be used [129]. For methanol production, the catalytic activity of MMO [132,133]. This can be overcome by the
the reaction mixture consisting of liquid nitrate mineral salt (NMS) addition of an external source of reducing agents [108]. This provides
media, methanol dehydrogenase (MDH) inhibitors like phosphate scope to explore various low-cost substances as external reducing
buffer for obtaining methanol as the end-product instead of carbon equivalents or using other approaches exhibiting maximum utility in
dioxide [97], and formate as the source of reducing power for catalytic the process. Another aspect receiving attention of researchers is the
activity of methane monooxygenase enzyme present in methanotrophic isolation of a potent bacterial strain that can carry out the maximum
bacteria [130] would be circulated in the bioreactor using a peristaltic transformation of methane to methanol. The methane monooxygenase
pump. As inoculum, potent methanotrophic cultures grown in nitrate plays the key role in this process hence a comprehensive knowledge of
mineral salts (NMS) liquid media would be added. The purified biogas the biochemistry of this enzyme is necessary. To achieve this cell-free
obtained from the hydrogen sulfide removal step would be introduced preparation of MMO enzyme is required [106]. However, purified en-
into the bioreactor at a particular flow rate as the substrate for me- zymes are unstable and also the requirement of reducing agents, en-
thanotrophic bacteria. A specific biogas: air ratio would be required to zyme inhibitors and cofactors makes it difficult to work with MMO
be maintained throughout the process. Along with sampling of gas, li- enzyme isolates [134].
quid media also needs to be sampled regularly and in another vessel
methanol produced is collected. The reactor would have to be provided 5. Ammonia oxidizing bacteria (AOB)
with sampling ports for withdrawing samples of gas and liquid. A sui-
table efficient separating process would be required to separate me- An alternative option to methane oxidizing bacteria is the utiliza-
thanol from the broth. tion of ammonia-oxidizing bacteria containing ammonia mono-
A conceptual schematic diagram showing methanol production oxygenase (AMO) enzyme, homologous to pMMO, which can convert
through waste biomass – purified biogas (CH4 as substrate by metha- methane to methanol [135]. Since, the AMO enzyme has a broad sub-
notrophs) - methanol route is shown in Fig. 1. strate specificity, it can catalyze oxidation of ammonia to act as the
primary source of energy and also co-metabolize methane into me-
thanol [136,137]. To obtain methanol utilizing ammonia mono-
oxygenase enzyme, a number of experiments have been performed with
some ammonia oxidizing bacteria such as Nitrosomonas europaea,

Fig. 1. A schematic diagram showing production of methanol by methanotrophs utilizing waste biomass.

8
P. Gautam, et al. Fuel 273 (2020) 117783

Nitrosomonas eutropha and Nitrosococcus oceani [86,138]. A positive oxidation potential in non-methanotrophic bacteria because of the
aspect with the use of ammonia oxidizing bacteria (AOB) over the limited gene tools and thus present a major ground to be covered. A
methane oxidizing bacteria (MOB) is that there is no need of inhibitors clear understanding of the physiology and biochemistry of the enzyme
that would further oxidize methanol, as AMO enzyme only partially and the reactions carried out by them is crucial for enhancing the
oxidizes methane to form methanol as the final product unlike metha- performance of the existing methanotrophic strains or to modify the
notrophs where methane is fully oxidized to carbon dioxide [138,139]. metabolic pathway through synthetic biology approaches to generate
The shake flask studies using mixed and axenic cultures of ammonia reducing equivalents required in the crucial step of methanol produc-
oxidizing bacteria fed with ammonia or hydroxylamine have been tion. The understanding of the methane oxidation machinery of me-
carried out for producing methanol from methane [86,136,138]. To thanotrophs can become clear through the genome sequences of me-
achieve the maximum methanol production through AOB, an external thanotrophic bacteria. Thus, the metabolic potential of methanotrophs
reducing power source is instrumental as they do not obtain energy can be realized by the understanding of the intricate system biological
through the oxidation of methane to methanol. Taher and Chandran approaches obtained from the sequenced genome information. The
[86] in their fed-batch reactor studies used mixed nitrifying cultures Next Generation Sequencing (NGS)-reads, assembled contigs, or scaf-
and a supply of reducing equivalents in the form of hydroxylamine and folds coupled with KEGG (Kyoto Encyclopedia of Genes and Genomes)
obtained ten times more yield of methanol than that obtained from pure pathway database mapping may result in the identification of metabolic
culture of the nitrifying bacteria [86]. They used hydroxylamine in- pathways in genomes and transcriptomes. Using these approaches,
stead of ammonia as the reducing equivalent because ammonia com- some pathways such as starch-sucrose metabolism [140,141] and sulfur
petitively inhibits the methanol production step. In their most recent metabolism [142] have been successfully identified. In a recent study,
study Su et al. [138] used mixed nitrifying enrichment cultures in a using high throughput sequencing (Miseq platform) of pmo A gene,
continuous stirred tank reactor. The product yield when hydroxylamine variety of methanotrophic bacteria have been assessed in soil [143].
was used as the electron donor was higher than when the ammonia was Based on the information generated from such type of study various
used as the reducing equivalent. Thus, it might be a promising candi- methanotrophic strains can be exploited for methanol production.
date for methanol production. However, numerous challenges such as Therefore these advanced technologies can be judiciously used for the
slow growth rates of AOB, slower reaction kinetics, etc. would have to exploratory studies of bio-methanol production pathways at genome/
be overcome before going for an industrial scale implementation of proteome level. The requirement of reducing equivalent by the methane
these results. Further the scarcity of experimental information on the monooxygenase enzyme is another barrier in the way of economic
comparison of efficacies of various methane oxidizing bacteria ne- production of bio-methanol. This can be overcome by engineering other
cessitates more work on this aspect [106]. available mono-oxygenase enzymes to utilize methane as an energy
source in heterologous hosts. Another obstacle in achieving commercial
6. Future scope and concluding remarks scale production is the gas-liquid mass transfer limitation which can be
overcome by improving the existing reactors or developing new novel
The thermo-chemical conversion of waste biomass is comparatively reactors.
advantageous because of its higher productivity, faster conversion rate, In coming decades methanol is likely to be in high demand because
established infrastructure and proven technical know-how of the ex- of its immense potential as a cleaner liquid fuel. Further, its production
isting conversion processes. Though these processes are highly suc- from methane will provide the additional advantage of reducing
cessful in producing considerable amount of methanol, these are still greenhouse effect due to accumulation of methane forming through
lacking in major information regarding proper conditioning of suitable anaerobic processes (land-fills, septic tanks, etc.) in atmosphere. The
raw materials, technical drawbacks, unattractive economics, cheaper possibility of gainful utilization of waste biomass, particularly ligno-
catalysts depending on the biomass feedstock and processes parameters cellulosic biomasses, for the production of methanol (a value added
like- temperature, particle size, residence time, and yield which need to product) through bio-chemical route makes this approach more at-
be worked out before implementing them on a commercial scale for tractive. In addition the development of an efficient bio-methanol
methanol production. The current thermo-chemical route (gasification, production process at commercial scale can also solve the problem of
pyrolysis and liquefaction) to obtain methanol through methane is quite dependence of different industries on non-renewable energy sources.
complex and thus needs to be simplified like electricity and heat gen-
eration processes to operate at commercial scale. Thus, a major thrust Declaration of Competing Interest
in the direction of research and development for resolving the techno-
economic issues related to methanol production are mandatory. The authors declare that they have no known competing financial
The production of methanol through biochemical conversion pro- interests or personal relationships that could have appeared to influ-
cess has shown promising potential at laboratory scale. But to achieve ence the work reported in this paper.
industrial scale production of methanol, amalgamation of conventional
approaches and metabolic engineering strategies are crucial. Methane Acknowledgement
oxidizing bacteria are being continuously exploited for producing me-
thanol. The first step to achieve this is the selection of appropriate Pallavi is grateful to University Grants Commission (UGC), New
strains for methanol production. For this preliminary screening, omics Delhi, India for financial assistance in the form of Junior Research
(genomics, proteomics and metabolomics) based approaches can be Fellowship. Neha is thankful to UGC-CAS (Centre of Advanced Study in
used. The major issue in using methanotrophs as biocatalyst is their Botany), for providing financial support in the form of a Junior
slow growth rates and sub-optimal product formation. To overcome this Research Fellowship. Authors express their gratitude to the
drawback, use of genetically engineered microbes, that are robust and Coordinator, CAS and FIST, Department of Botany, Institute of Science,
may utilize a range of cheap feedstock, may be a promising approach. Banaras Hindu University, Varanasi, for providing necessary facilities.
To achieve faster growth rates and increased production, expression of
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