Methane as a resource: can the methanotrophs add value?

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Methane as a resource: can the methanotrophs add value? PJ Strong, Sihuang Xie, and William P. Clarke Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/es504242n • Publication Date (Web): 27 Feb 2015 Downloaded from http://pubs.acs.org on March 4, 2015

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Environmental Science & Technology

Methane as a resource: can the methanotrophs add value?

Authors P.J. Strong* S. Xie W.P. Clarke

Affiliation Centre for Solid Waste Bioprocessing, School of Civil Engineering, School of Chemical Engineering, The University of Queensland, St Lucia, 4072, Australia.

*Corresponding author. P.J. Strong [email protected] Tel: +61 459652099 Centre for Solid Waste Bioprocessing, School of Civil Engineering, School of Chemical Engineering, The University of Queensland, St Lucia, 4072, Australia.

ACS Paragon Plus Environment


Rationale This review considers all of the biological options for either converting methane into a product or using it directly for purposes other the production of electricity or heat. Methane is cheap and abundant, does not compete with food demand, and in the case of anaerobic digestion is renewable carbon source. It is therefore a suitable substrate by which to generate products or drive processes. To our knowledge there is currently no review that comprehensively covers the potential products or processes that could be generated using methane-consuming bacteria. Generally review papers regard methane in terms of energy capture or emissions prevention. The methanotrophs have rarely been regarded for more than their remediative abilities or for single-cell protein production. Biological reviews of the methanotrophs often cover a few individual biotechnological applications or just one in detail. In this review we explore all the avenues for biological applications, thereby providing a single source for the readers that will be readily citable. Some of the options discussed are nascent and even unproven, but represent exciting potential research avenues. It will appeal to both a popular audience that is interested in applied alternatives to mitigating anthropogenic methane emissions, as well as a scientific audience and applied researchers interested in current developments and an assessment of the available options.

The Table of Contents graphic below was created by the corresponding author.

Table of Contents graphic


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Abstract Methane is an abundant gas used in energy recovery systems, heating and transport. Methanotrophs are bacteria capable of using methane as their sole carbon source. Although intensively-researched, the myriad of potential biotechnological applications of methanotrophic bacteria has not been comprehensively discussed in a single review.

Methanotrophs can generate single-cell protein, biopolymers, components for nanotechnology applications (surface layers), soluble metabolites (methanol, formaldehyde, organic acids and ectoine), lipids (biodiesel and health supplements), growth media and vitamin B12 using methane as their carbon source. They may be genetically engineered to produce new compounds such as carotenoids or farnesene. Some enzymes (dehydrogenases, oxidase and catalase) are valuable products with high conversion efficiencies and can generate methanol or sequester CO2 as formic acid ex vivo. Live cultures can be used for bioremediation, chemical transformation (propene to propylene oxide), wastewater denitrification, as components of biosensors, or possibly for directly generating electricity.

This review demonstrates the potential for methanotrophs and their consortia to generate value while using methane as a carbon source. While there are notable challenges using a low solubility gas as a carbon source, the massive methane resource, and the potential cost savings while sequestering a greenhouse gas, keeps interest piqued in these unique bacteria.

Keywords biofuel; bioremediation; coal seam gas; natural gas; proteobacteria; methane monooxygenase



1

1. Methane

2

Methane is a colourless, odourless gas that is emitted from both natural and anthropogenic sources.

3

It provides energy or heat via combustion. However, its emission into the atmosphere has negative

4

consequences as it is a greenhouse gas with approximately twenty times the impact of carbon

5

dioxide. Anthropogenic activity accounts for the majority of global methane emissions (63 %, or

6

566 Tg CH4/year), with natural biological emissions accounting for the remainder (208 Tg CH4/year)1.

7

Anthropogenic methane emissions are generated by the use of fossil fuels, livestock farming,

8

landfilling and biomass burning. Natural sources of methane are wetlands, oceans, estuaries, rivers,

9

lakes, permafrost, gas hydrates, geological sources (terrestrial and marine), wildfires, vegetation,

10

terrestrial arthropods and wild animals 1-2,2b. The ratio of anthropogenic:natural methane production

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has increased steadily since the advent of the industrial revolution. With increased food

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requirements, greater waste generation and greater use of fossil fuels by an increasing human

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population, it will in all likelihood increase further.

14 15

The principal use of methane is as a fuel, as its combustion is highly exothermic:

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CH4(g) + 2 O2(g) →CO2(g) + 2 H2O(l) where ∆H = -891 kJ.

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Methane is primarily used for generating electricity in gas turbines or steam boilers, but is also piped

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into homes for domestic heating and cooking, or used as a vehicle fuel as compressed natural gas3.

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The reciprocating engine, gas turbine or steam turbine technologies are available over a wide range,

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from modular units of several hundred kW up to 250 MW for commercial steam turbines4. Although

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methane can be used as a transport fuel, there is currently a relative lack of infrastructure for

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fuelling vehicles compatible with natural gas. However, new approaches whereby methane is

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converted into liquid transportation fuels can take advantage of existing engines and delivery

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infrastructure5.

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From a biological perspective, methane represents a carbon and energy source for a group of

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bacteria known as methanotrophs. Methanotrophs use methane as their sole carbon source and

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directly convert methane into cellular compounds, or transform it into a substrate that drives

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processes via methanotrophs or their syntrophic interaction of other microbes. The biological

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monetisation of methane has become a topic of intense interest: in 2013 the Advanced Research

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Projects Agency (ARPA-E) within the U.S. Department of Energy granted US$34 million worth of

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funding directed towards research converting methane into liquid fuels. There are notable barriers

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to overcome, but the size of the methane resource is enormous, and in the case of anaerobic

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digestion is sustainably produced; this justifies the continued interest in these bacteria that can

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oxidise methane under ambient conditions.

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2. Methanotrophs

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Methanotrophs, a subset of the methylotrophs, can assimilate methane as their sole carbon source.

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Methanotrophs fall under proteobacteria; a major phylum of gram-negative bacteria that includes

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genera such as Escherichia, Salmonella, Vibrio, Helicobacter and Yersinia. The proteobacteria phylum

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is divided into six classes according to ribosomal RNA sequences. Two classes contain

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methanotrophs: the alpha-proteobacteria and the gamma-proteobacteria. Methanotrophs are

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traditionally classified as Type I (γ-proteobacteria) or Type II (α-proteobacteria). The Type I and II

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distinctions were primarily based on the metabolic pathway used to assimilate formaldehyde, and

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related attributes such as cell membrane composition and arrangement and cell morphology6.

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Typically, Type I methanotrophs are γ-proteobacteria that assimilate formaldehyde via the ribulose

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monophosphate pathway6b, 6d, 7, while Type II are α-proteobacteria that use the serine pathway. The

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generalised differences between Type I and Type II methanotrophs are summarised in Table 1, but

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exceptions are common and metabolic pathway flexibility may be greater than previously thought8.

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This is evident for Type I methanotrophs, which contain a sub-division denoted as Type X

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(Methylocaldum and Methylococcus species) that express enzymes associated with the serine



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pathway typical of the α-proteobacteria6b. Methane can also be oxidised by a phylum of

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thermoacidophilic bacteria known as Verrucomicrobia (which can grow at a pH below 1)9, as well as

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certain Archaea in consortia with sulfate reducing bacteria10. The ability of certain Archea to

55

anaerobically oxidise methane (where NOx11 or SOx12 replace O2) corroborates research supporting

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the concept of reverse methanogenesis13.

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Methane consumption is made possible by an enzyme known as methane mono-oxygenase (MMO),

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which occurs in a particulate form (pMMO) within an intracellular membrane or a soluble form

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(sMMO) within the cytoplasm14. Copper availability plays a defining regulatory role with regard to

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sMMO expression15, where sMMO synthesis is inhibited by higher Cu2+ concentrations16. The sMMO

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has a di-iron catalytic site, while research suggests that pMMO requires both copper and iron to be

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catalytically active7b. The sMMO can be produced by various α- or γ-proteobacteria and has a much

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broader substrate range than the pMMO. It is capable of oxidising numerous compounds, including

65

propene, butane, cyclohexane, chlorotrifluoroethylene, toluene, naphthalene, chloroform, diethyl

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ether and CO (substrates tabled in 14b). Although pMMO is expressed by most methanotrophs, some

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Methylocella spp. express sMMO exclusively17.

68 69

The metabolic activity and growth of methanotrophs are influenced factors that typically affect

70

microbial cultures, including nutrient supply, temperature, pH, macronutrients and trace metals.

71

Traditionally, the growth of γ-proteobacteria was reported to favour higher O2 concentrations and

72

low CH4 concentrations, while α-proteobacteria methanotrophs preferred low O2 and higher CH4

73

concentrations6d,

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Similarly, facultative methanotrophy was initially discounted, but has been demonstrated in certain

75

genera of α-proteobacteria7b, 21, 21b. Methanotrophs are able to assimilate CO2 and are thus affected

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by CO2 concentrations. The α-proteobacteria can assimilate up to 50 % of their biomass from CO2,

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while the γ-proteobacteria can assimilate up to 15 %22. The N source in media commonly used to

18, 19

, but γ-proteobacteria can grow efficiently under a low oxygen tension20.


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culture methanotrophs is usually nitrate (e.g. nitrate minimal salts) or ammonia. Ammonia affects

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different methanotrophs to differing extents and can be a competitive inhibitor of methane

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monooxygenase or toxic when in the intermediate forms of hydroxylamine and nitrite23. However,

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ammonium acts more as a nutrient than an inhibitor in the presence of sufficient methane.

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Nitrogenase occurs in a broad range of methanotrophs (α- and γ-proteobacteria) allowing for use of

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N2 as a nitrogen source; nitrogen-fixing may be widespread and important to nitrogen cycling in

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many environments23.

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3. Methanotrophs: prior reviews

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Many aspects of these ubiquitous bacteria have been reviewed. This includes the natural occurrence

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of methanotrophs24, their use in methane mitigation and environmental remediation6d, 14b, 25, their

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physiology, biochemical pathways for N metabolism26, C1 metabolism and assimilation22,

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enzymes involved6c,

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facultative methanotrophy7b, 21b, 30.

28

and the influence of copper on their metabolic capabilities27a,

27

, the

28c, 29

and

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Although the biotechnological applications have been reviewed in part, no single review has

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comprehensively covered all prospective products or processes. Specific topics such as biodiesel

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generation5b, epoxide production31, Polyhydroxybutyrate accumulation32 and denitrification33 have

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been covered individually, while some reviews cover many facets of methanotrophy and some

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biotechnological applications6b,

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development of various aspects of methanotrophic physiology and biochemistry, as well as views on

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the development of single cell protein production, pollutant bioremediation, whole cell catalytic

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production of propylene oxide (epoxypropane) and alludes to the possibility for electrochemically

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driving reactions that require reducing agents such as NADH. Trotsenko, et al. 34 reviewed single cell

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protein, biopolymer, ectoine, surface layers, vitamins, pollutant bioremediation and the role of

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methanotroph as plant growth-promoting bacteria. These topics, as well as extracellular

27a

. A review by Dalton

14a

discusses much of the history and



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polysaccharides, lipids for biodiesel and human health supplements, growth media, methanol,

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formaldehyde and organic acids, enzymes, enzymatic transformation of CH4 and CO2 (incorporating

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electrochemical reductant recycling), and products resulting from genetic engineering are covered in

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the current review.

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3. Methanotrophs: products and processes

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3.1. Products

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3.1.1. Single-cell protein

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Single-cell protein (SCP) production from microbes such as yeasts, fungi, algae, and bacteria surged

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in the 1950s and 1960s due to a lack of protein source and predictions of impending global

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shortages. The advent of low cost soya production in the 1970s negatively impacted microbial SCP

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research, although the production of a fungal protein was commercialised in 1985. Currently, SCP is

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the closest example of a commercially-successful biologically-generated product using methane as

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the carbon source. The commercial production of methanotrophic SCP originated as research in

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Denmark by E.B. Larsen in the 1980s and has culminated the company now known as UniBio A/S.

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The fermentation uses natural gas, technically pure oxygen, ammonia as the N source, phosphoric

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acid as the P source and supplemented with other minerals. The process is controlled at pH 6.5 and

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run at a temperature of 45°C. The concentrated biomass is sterilised by rapid heating to 140°C and

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then slowly cooled, allowing cells lysis and accessibility to the protein. The bacterial biomass consists

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predominantly of a strain of Methylococcus capsulatus (Bath) and is a promising protein source,

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based on criteria such as amino acid composition, digestibility, and animal performance and

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health35. The methanotrophic protein has been used as a protein source for several mono-gastric

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species, including pigs, broiler chickens, mink, fox, Atlantic salmon, rainbow trout, and Atlantic

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halibut35.

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Although bacteria possess a high protein content (ranging from 50 to 65 %) relative to other micro-

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organisms (30 to 60 %), they also possess the highest nucleic acid content (8 to 12 %)36, which can

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adversely affect consumers. A report regarding the safety of the UniBio protein (then known as

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Dansk BioProtein®) noted significant immune effects in rats during a toxicity study (NCSF, 2006). The

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protein was only approved for animal nutrition and was recommended for animals with a short life

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span, as the nucleic acids could cause kidney and bladder stones in longer-lived species. However,

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the risk associated with human consumption of products from animals fed on BioProtein® was

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considered negligible. Heat treatment or hydrolysis can lower RNA content. At UniBio A/S nucleic

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acids are neutralised by hydrolysis, generating a product fit for human consumption. Heat treatment

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at 60 to 65°C for 10 to 20 minutes has been used to remove nearly 90 % of the RNA from a

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Methylomonas sp. cultured for single-cell protein use37.

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Although using a C1 gas as the substrate is highly selective, large-scale continuous industrial

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fermentation is always subject to infiltration by other micro-organisms as cell lysis products or

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metabolic byproducts serve as C sources, which is problematic in a continuous process. The SCP

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strain was repeatedly contaminated by three different bacteria: a member of the Aneurinibacillus

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group, a Brevibacillus agri strain and an acetate-oxidising Ralstonia species. Fortunately, the three

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contaminant bacteria were non-toxic. Their presence was beneficial as they stabilised the culture by

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consuming metabolic byproducts that would have inhibited growth38.

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3.1.2a. Biopolymers: Internal storage polymers

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Polyhydroxy-alkanoates (PHA) such as poly(3-hydroxybutyrate), or PHB, are widespread and

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intensively-researched bacterial storage polymers seen as potential substitutes for plastics derived

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from the petroleum industry. They have beneficial properties such as biodegradability,

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biocompatibility and thermoplasticity. These biopolymers are synthesised and deposited intra-

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cellularly as granules that serve as a source of carbon, energy or reducing-power and, in exceptional



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circumstances, may comprise up to 90 % of a microbe’s dry weight39. PHB accumulation is induced

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by exposing an active culture to excess carbon while under a nutrient limitation of some sort. PHA

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yield and quality, is affected by pH, temperature and the availability of other carbon sources,

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methane, oxygen, carbon dioxide, macronutrients (nitrogen, phosphorus, sulfur, potassium,

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magnesium sodium) and trace metals (copper, iron, zinc, manganese cobalt)40. Although PHB

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production by methanotrophs and methylotrophs has been of interest for decades41, it has been re-

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evaluated in recent times5b, 40c. Cheap, or zero cost, substrates such as CH4 and CO2 are receiving

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considerable attention because of their potential to lower production costs32, 42.

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Microbe strain selection, or genetic engineering of existing strains, is critical because of the impact

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on the maximum production rate and yield. In the 1990s, heterotrophically-grown Alcaligenes

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eutrophus became the organism of choice for industrial PHB production as it produced high yields of

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high molecular weight PHBs using various economically-acceptable substrates. It was benchmarked

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against a methylotroph (Methylobacterium sp.), which only managed moderate polymers yields that

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had a low molecular weight (Mw) and were difficult to extract43. To date, many methylotrophs and

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methanotrophs have been assessed for PHA production and are tabled in a reviews by Khosravi-

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Darani, et al. 32 and Karthikeyan, et al. 40c. Polymer yields and Mw have improved among the newly-

172

isolated bacteria as well as mixed cultures. Zhang, et al.

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using methane with methanol and citric acid and obtained a high quality PHB (Mw: 1.5×106 Da) at a

174

yield 40 %. Wendlandt, et al. 45 also produced a high quality PHB with a high molecular mass of (up

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to 2.5 × 106 Da), using a Methylocystis sp. in a rapid, non-sterile process. They obtained a PHB

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content of up to 51 % using a two-stage process consisting of a continuous growth stage (dilution

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rate: 0.17 h−1) and a PHB accumulation stage under P-deficient conditions14c. Shah et al.46 enriched

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their methanotrophs for pMMO or sMMO and compared PHB accumulation under different batch

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conditions. The pMMO-rich cells displayed greater and more rapid PHB accumulation (up to 50 %

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PHB content within 120 hours) and attained a much greater biomass yield (18 g/l). The pMMO-rich

44

cultured a Methylosinus trichosporium


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cells also. Recently, researchers included silicone oil (10 % v/v) in the two-phase partitioning

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bioreactor (using 1 % methane in an air stream) with a co-culture of Methylobacterium

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organophilum and reported up to 57 % PHB under nitrogen limitation47 and improved methane

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consumption by up to 45 % in the growth stage. Methylotrophs have improved PHB accumulation

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when ammonium was the sole N source under potassium limitation48. A complex nitrogen source

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can also improve biopolymer yields when using a defined medium49. Helm, et al.

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polymer with an ultra-high average Mw of 3.1 MDa under potassium-limited conditions using

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methane-utilizing mixed culture where a Methylocystis sp. was dominant. A maximum specific PHB

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formation rate 0.08 g.g-1.h-1 and a yield coefficient of 0.45 g PHB.g-1 CH4 were obtained in further K-

190

deficiency experiments. The Mw was lower when sulfur (21 % lower) or iron (42 % lower) were

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limiting.

50

produced a

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Even after decades of considerable effort to commercialise microbial PHB production, the high

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production cost compared to traditional petrochemical-based plastics (such as polyethylene and

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polypropylene) still limits commercial application. Choi and Lee 51 performed a sensitivity analysis of

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several factors (productivity, content and yield, the cost of the carbon substrate, and the recovery

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method) that affect PHA production with a view to scale-up and found biopolymer content had

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multiple effects on the process economics. Productivity only affected equipment-related costs, but

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the yield per cell had multiple effects on process economics. Other research has also placed a firm

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emphasis on the importance of yield52. Although substrate costs can account for a major portion

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(30 %) of the production cost39b, 51-53, the costs associated with downstream processing would still

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render microbial polymer production uncompetitive with the petroleum-based polymers. An initial

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bridge to commercial production is to target higher-value polymers used in biomedical applications

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5b

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medical devices, tissue repair, artificial organ construction and nutritional/therapeutic uses55. This

, as the biocompatibility and biodegradation of PHA has shown potential for drug delivery54,



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field of research has received a promising boost: the FDA has recently approved the use of poly-4-

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hydroxybutyrate for a clinical application56.

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3.1.2b. Biopolymers: Extra-cellular polysaccharides

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The colloid and adhesive properties of extra-cellular polysaccharides (EPS) and their effects on liquid

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rheology are used in the food and non-food industries such as the pharmaceutical, textile and oil

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industries. Traditionally, industrial polysaccharides have been derived from algal and plant sources57.

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There has been an active search for producers that may be cultured using non-edible raw

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materials58. Methanotrophs satisfy this requirement as they can synthesise EPS using methane as

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their sole carbon source58-59. The genes responsible for elements of the EPS synthesis have been

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isolated from a Methylomonas sp. with the intention of genetically engineering them into other C1-

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utilising microorganisms to alter levels of EPS production for commercial production57. Malashenko

218

et al. 58 studied the dynamics of EPS production by methanotrophs in chemostats and observed EPS

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synthesis rates ranging from 0.03 to 0.43 g.g-1 dry biomass. Highest production was achieved using

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Type I isolates (Methylomonas and Methylobacter spp.). The viscosity of 0.1 % aqueous solutions of

221

EPS synthesised by the mesophilic methanotrophs varied from 2.2 to 4.0 mm2/s, which was

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comparable to the viscosity of EPS solutions synthesised by known microbial producers as well as the

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benchmark equivalent composed of 0.1 % xanthane (3.5 to 4.5 mm2/s).

224 225

However, EPS production may inhibit further synthesis as it negatively affects gas uptake.

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Chiemchaisri and Visvanathan59a studied methanotrophic EPS production in a bench-scale soil

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reactor. Methane oxidation rates (regulated by temperature and soil water content) were correlated

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to EPS production and highest EPS production occurred at 30 °C. They observed that a high oxygen

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content accelerated EPS production, but subsequently limited gas diffusion and inhibited

230

production.

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3.1.3. Internal osmo-protectants: Ectoine

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Halo-tolerant bacteria employ two primary survival strategies: they synthesise and accumulate

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intracellular organic osmo-protectants (such as ectoine, glutamate and sucrose), or they make

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structural and functional changes to their cell envelopes by altering the phospholipids composition

236

of membranes and forming glycoprotein surface layers14b, 60. Ectoine is a cyclic imino acid that is one

237

of the most widespread microbial protective measures against osmotic dehydration. It is also an

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efficient stabiliser of enzymes, nucleic acids, and DNA-protein complexes, and can be used as

239

a moisturiser in the cosmetic industry. Cosmetic formulations comprising ectoine or its derivatives

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offer excellent protection against UV-induced damage to the DNA of skin cells61. Ectoine is produced

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annually on a scale of tons in an industrial process using the halophilic γ-proteobacterium

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Halomonas elongata DSM 2581T as producer strain62.

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Because synthesis and purification is expensive, new microbial producer strains and their enzymes

245

are constantly assessed to improve the economics of microbial ectoine production. Halo-tolerant

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methanotrophs are known and isolated, with the best-known belonging to the genus

247

Methylomicrobium. Trotsenko and his colleagues demonstrated that moderately halophilic

248

methanotrophs and methylotrophs were able to accumulate up to 20 % of their dry mass as

249

ectoine34, 60. This is an area well worth further exploration, as purified ectoine has one of the highest

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retail values of the methanotroph products (approximately $1300 kg-1). Additionally, the bacteria

251

may potentially be reused multiple times: Halomonas elongata has been reused 9 times, yielding

252

ectoine at an average of 15.5% g.g-1 biomass63. However, downstream processing is complex and

253

expensive and represents a challenge to process economics.

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3.1.4. External osmo-protectants: Surface layers

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Bacterial cell surface layers are one of the most commonly observed outermost structures of

257

prokaryotes. These regular para-crystalline structures cover the entire surface of a cell and consist of



258

a single layer of identical proteins or glycoproteins. Isolated surface layer glycoproteins possess the

259

intrinsic property of self-assembly and recrystallise into isoporous lattices in suspension, onto

260

various surfaces (polymer, silicon and metal) and interfaces (air-liquid, lipid films and liposomes).

261

These characteristics and subsequent functionalising of surfaces has led to new types of

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ultrafiltration membranes, affinity structures, enzyme membranes, micro-carriers, biosensors,

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diagnostic devices, biocompatible surfaces and vaccines, as well as targeting, delivery, and

264

encapsulation systems34, 64.

265 266

As with the de novo synthesis of internal solutes such as ectoine, the osmo-adaptation of

267

methanotrophs also involves structural and functional changes to cell envelopes and changes in the

268

chemical composition of membranes60. These changes in the surface layers are potentially of

269

industrial interest. Regularly arranged glycoprotein surface layers of hexagonal and linear symmetry

270

have been observed on the outer cell walls of two halo-tolerant Methylobacter spp. Interestingly,

271

the surface layer in a Methylomicrobium sp. (consisting of tightly packed, cup-shaped sub-units) was

272

negligible when the bacteria were cultured at a neutral pH with no salt in the media 60, indicating the

273

stimulatory effect of osmotic stress. With methanotrophs, it is likely that proteins associated with

274

surface layers facilitate copper ion transport to pMMO and provide an additional mechanism to

275

maintain copper homeostasis in the cells65. Surface layers have prospective nanotechnology

276

applications in ultrafiltration because they form porous semi-permeable membranes; their

277

components undergo self-assembly and in vitro cross-linking at the surface of membranes. The

278

characteristics of isolated surface layers have allowed for various applications in biotechnology,

279

vaccine development, diagnostics, biomimetics and molecular nanotechnology34, 64b. Methanotrophs

280

may be in contention for commercial production if highly desired properties are discovered or

281

engineered for their specific proteins or glycoproteins.

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3.1.5a. Lipids: biodiesel

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Biodiesel is another possible methane-generated commodity with a potentially enormous market. It

285

may be used as a transport fuel or an energy fuel, and is advantageous compared to ethanol as it can

286

be used without any modifications to engines. Typically, animal or plant fats (triglycerides) are

287

extracted and converted via trans-esterification into biodiesel. The triglyceride consists of three long

288

chain fatty acids attached to a glycerine molecule. The triglyceride reacts with alcohol (typically

289

methanol or ethanol) in the presence of a catalyst (usually a strong base: NaOH or KOH) to yield

290

esters and glycerol (Figure 2).

291 292

Oleaginous microbes, including yeasts and various microalgae, have been researched intensively as a

293

biological means to produce diesel due to their ability to accumulate a lipid content greater than

294

20 % of their dry mass. Microbially-produced lipids are advantageous to animal or plant-based lipids

295

as they are produced in a short life cycle, may be less labour-intensive, may be less affected by

296

location, season and climate, and in certain cases may be easier to scale-up. However, there are

297

difficulties. The scale-up of dense cultures of autotrophic microalgae is difficult due to light

298

penetration66, difficulties obtaining a high lipid content with dense microbial67 and the high degree

299

of processing associated with lipid extraction and transformation5b.

300 301

Essentially, lipids from microorganisms may be divided into two types: those associated with storage

302

(non-polar lipids) and those that form structures (polar lipids). The non-polar storage lipids are

303

mainly in the form of triacylglycerides and can be trans-esterified to produce biodiesel. The

304

structural polar lipids (phospholipids) and sterols are important components of cell membranes that

305

typically have a high content of polyunsaturated fatty acids (PUFAs) and are essential nutrients for

306

humans 67. In microalgae, one of the most intensively researched oleaginous microbes, the types of

307

lipids produced range from neutral lipids to polar lipids, wax esters, sterols and hydrocarbons, as

308

well as prenyl derivatives such as tocopherols, carotenoids, terpenes, quinines and pyrrole



Page 16 of 51

309

derivatives. In methanotrophs, the two major classes of phospholipids are phosphatidyl-glycerol (PG)

310

and phosphatidyl-ethanolamine (PE), which has two derivatives: phosphatidyl methyl ethanolamine

311

(PME) and phosphatidyl dimethyl ethanolamine (PDME). Gamma-proteobacteria (Methylomonas

312

methanica, Methylomonas rubra and Methylomicrobium album BG8) contain PE and PG

313

phospholipids with predominantly C16:1 fatty acids, while alpha-proteobacteria methanotrophs

314

(Methylosinus trichosporium OB3b and CSC1) contain PG, PME and PDME with predominantly C18:1

315

fatty acids

316

production, the sugars, P and S contents are problematic to subsequent catalysts used in the

317

processing. The high hetero-atom content (specifically P and N) in the lipid fraction makes

318

downstream extraction and conversion difficult5b. Undesirable components such as sugars,

319

phosphorous and sulphur exacerbate processing problems due to gumming or catalyst inactivation.

320

This will require additional research and development of a solvent-based extraction process to

321

minimise these negative impacts5b.

68

. Although the C14 to C18, saturated or mono-unsaturated fatty acids suit diesel

322 323

Fei, et al. 5b recently reviewed the use of methanotrophs to generate lipids for biodiesel production,

324

thoroughly covering aspects from microbial lipid production to the pitfalls associated with

325

downstream processing. Lipid accumulation exhibits similarities to PHB accumulation, as both may

326

be induced by limiting the oxygen supply, nitrogen or phosphate concentrations to the microbes.

327

Similarly, a two-stage culture process is promoted as the most plausible production method; where

328

the first stage produces healthy biomass and the second stage limits a nutrient source

329

advantage of using a gas for producing a fuel is significant as it avoids the food vs. fuel debate that

330

plagues first generation biofuels. Silverman, et al.

331

methanotrophic/methylotrophic biomass into oil that is refined into a fuel, where the oil is derived

332

from the cell membrane of microorganism. Their preferred production method involves supercritical

333

CO2 extraction of the oil, which may be refined by a cracking, trans-esterification, reforming,

334

distilling, hydro-processing, and/or isomerisation. While they are focusing on using the structural

69

5b

. The

patented the conversion of lipid-containing


Page 17 of 51


335

lipids, ARPA-E funded research at the University of Washington is focussed on manipulating

336

methanotrophs to produce storage lipids. Here, methanotrophs will be genetically modified to

337

increase their lipid production and enhance the fraction of non-phosphorous-based lipids, thereby

338

aiding trans-esterification into biodiesel.

339 340

3.1.5b. Lipids: human health supplements

341

As the lipids produced by natural methanotrophic isolates are predominantly membrane-derived,

342

they are not ideal for catalytic conversion to biodiesel. However, they may have an alternative

343

higher-value application as a health supplement. There is a current patent for using methanotrophic

344

lipids in the manufacture of an oral administration for use in the treatment of animal subjects to

345

reduce plasma cholesterol levels or lower the ratio of LDL to HDL cholesterol in the plasma. The

346

composition is apparently also useful for increasing docosahexaenoic acid concentration in the

347

plasma, which acts as an immuno-protectant70. The patent is based on research the effects of three

348

different high-lipid diets on plasma lipoproteins and phospholipids in mink (Mustela vison).

349

Phospholipids from natural gas-utilising bacteria in the diet decreased plasma lipoprotein levels, the

350

LDL/HDL cholesterol ratio, and plasma phospholipid levels compared with the highly unsaturated

351

soybean oil. The decrease of plasma cholesterol was attributed to a specific mixture of phospholipids

352

containing a high level of phosphatidyl-ethanolamine, and not the dietary fatty acid composition71.

353

These phospholipids could comprise part of a formulation taken as a health supplement, or

354

potentially used as part of complimentary treatment programme.

355 356

3.1.6. Growth media and vitamin B12

357

Analogous to yeast, beef or potato dextrose extracts, soluble compounds within the methanotrophic

358

biomass may contribute to the bulk of an extract that can provide nutrients for a growth media.

359

There is a patent for a microbial growth medium derived from a microbial culture composed

360

primarily of Methylococcus capsulatus (Bath) and containing Ralstonia sp. DB3 and Brevibacillus agri



361

DB5 and optionally Aneurinibacillus sp. DB472. The nutrient medium is proposed to consist of a

362

hydrolysate, homogenate or an autolysate of the biomass, with an autolysate being preferred. The

363

addition of further compounds and nutrients such as glucose and/or nitrate and minerals (e.g. K, Ca,

364

Mg, Na, Mb, Fe, Zn, B, Co, Mn and Ni salts) further enhances the scope of this growth medium.

365

Methanotrophs and methylotrophs utilising the serine or RUMP pathways are able to produce

366

vitamin B12 (up to 800 ng/g wet mass using a Methylobacterium species), an essential vitamin to

367

many organisms73.

368 369

3.1.7. Soluble metabolic products: methanol, formaldehyde and organic acids

370

Soluble metabolic intermediates such as methanol, formaldehyde and organic acids are all potential

371

products from methanotrophs with multiple industrial uses and are required in large quantities

372

annually. Viably converting methane to methanol at ambient temperature and pressure is of great

373

interest as methanol is a more easily transportable than methane. Methanol is the first intermediate

374

formed during the conversion of methane to carbon dioxide. In the metabolic pathway (Figure 1) of

375

native bacteria, methanol is rapidly converted to formaldehyde, implying that genetic manipulation

376

or fermentation process control would be vital to ensure accumulation. Tabata and Okura 74 initially

377

failed to produce extracellular methanol from methanotrophs because it was rapidly oxidised

378

internally by methanol dehydrogenase. Extracellular methanol was detected when they selectively

379

inhibited methanol dehydrogenase using cyclopropanol. Han et al. 75 also achieved partial oxidation

380

of methane into methanol through selective inhibition of methanol dehydrogenase by using

381

relatively common media components such as phosphate, NaCl, NH4Cl or EDTA. Alternatively,

382

researchers used a thermophilic methanotroph (Methylocaludum) in a process where methanol was

383

constantly removed from the fermentation. Methanol remained in the gas phase at 50 °C and was

384

successfully condensed from the reactor headspace76. Careful selection of the production strain is

385

also vital for methanol production as production rates and yields from different isolates vary by

386

orders of magnitude. Mehta et al. 77 reported specific production of 1 mmol.hr-1.mg-1 dry mass under


Page 18 of 51

Page 19 of 51


78

387

continuous conditions, while Xin et al.

388

lower. The low productivity was attributed to the MMO specific activity of their Methylosinus

389

trichosporium strain, which was approximately 1 % of the strain used by used by Mehta et al. 77.

reported methanol production three orders of magnitude

390 391

Low methanol yields are still problematic. Although earlier research had claimed methanol

392

production up to 1 g/l79, very low yields (≤mg/l) are generally reported for methanol (and other

393

soluble metabolites). Recently, 1 g/l was reported using a high-density culture80, but the process

394

required a culture concentrated via centrifugation in 0.4 M phosphate buffer and 20 mM formic

395

acid. This process would be difficult to scale up and even then, the methanol concentration would

396

have to be improved by up to two orders of magnitude.

397 398

Formaldehyde and formic acid represent alternative products formed as intermediates during the

399

mineralisation process, while acetate, lactate, and succinate are produced under oxygen-limited

400

conditions20. Current yields according to literature are very low (mM). While a product such as

401

formaldehyde has a large global demand, it is toxic to cells as it crosslinks proteins (it is often used as

402

a fixing agent because of this quality). This quality may render a cellular and even a purely enzymatic

403

system inactive. One potential alternative is to use small molecule catalysts that mimic the methanol

404

dehydrogenase active site to convert methanol to formaldehyde. It is worth noting that there are a

405

number of companies actively pursuing these compounds. Various companies and research

406

institutions (e.g. Kiverdi, Coskata, CALYSTA Energy, NatureWorks, National Renewable Energy

407

Laboratory, Intrexon) are trying to commercialise biological production of lactate, succinate,

408

muconic acid, butanol and propanol. It will be of interest to see what yields and production

409

efficiencies can be obtained by these researchers, and how significantly production can be improved

410

by bioreactor engineering, genetic engineering and the inclusion of synthetic biology approaches.

411



412

3.1.8. Methanotroph enzymes, cell-free catalysis and electrochemistry

413

Enzymes produced by methylotrophs are themselves a valuable product. Some dehydrogenases and

414

other enzymes (including glucose-6-phosphate dehydrogenase, glutamate dehydrogenase, malate

415

dehydrogenase, alcohol oxidase and catalase) exhibit high activities34. Enzymes such as diamino-

416

butyrate acetyl-transferase from a halo-tolerant methanotroph have been cloned and expressed in

417

heterologous expression systems; this enzyme catalyses one of the key reactions of biosynthesis of

418

the bacterial osmo-protectant ectoine81.

419 420

Methane may be enzymatically converted to methanol, while the complete enzymatic oxidation of

421

methanol to CO2 has been demonstrated using the various dehydrogenases82. The conversion of

422

methane to methanol is of great industrial interest. Unfortunately, both the particulate and soluble

423

MMO require reducing equivalents, which has limited the potential for a commercial methanol

424

production from these enzymes. The sMMO requires NAD(P)H and O2 catalyses to convert methane

425

to methanol: CH4 + NAD(P)H + H+ + O2 = CH3OH + NAD(P)+ + H2O, while the pMMO requires

426

cytochromes b559/569 or c553 artificial reductants such as duroquinol and NADH83 to complete the

427

reaction. The cost of providing external reducing equivalents for the MMOs renders the economics

428

of the reaction unfeasible for producing a low-value commodity such as methanol. Normally, within

429

the cell the NAD(P)H would be regenerated in a subsequent enzymatic oxidation step. However,

430

developments in NAD(P)H regeneration may be overcome using electrochemical techniques,

431

mediators or combinations with NAD(P) reducing and NAD(P)H-oxidising enzymes.

432 433

Besides the cost associated with the enzyme co-factor /reducing equivalent, it is also difficult to

434

heterologously produce MMOs84. MMO genes have been cloned into bacterial expression systems

435

(which could enable faster and greater enzyme production), because of the relatively slow growth

436

rate of methanotrophs. However, MMOs are complex proteins (consisting of a reductase,

437

hydroxylase and regulatory protein) and a heterologous expression system has proved elusive.


Page 20 of 51

Page 21 of 51


438

Complete, active MMOs have not been produced thus far, only a partially-active sMMO with a

439

functional hydroxylase22. Active pMMO85 or sMMO86 can be obtained directly from methanotrophic

440

cultures. Although the pMMO is commonly inactive when stringently purified from native bacteria

441

(as it is removed from its lipid matrix and the closely associated proteins and co-factors), active

442

MMO can be purified from methanotrophic biomass87.

443 444

The other three key enzymes in the mineralisation of methanol to CO2 (methanol dehydrogenase,

445

formaldehyde dehydrogenase and formate dehydrogenase) may be used to generate methanol,

446

formaldehyde and formic acid. There is strong interest in enzyme-catalysed redox reactions to

447

produce electricity, fuels as well as chemical commodities from immobilised enzymes on

448

electrodes82, 88. Recently, research has not only focused on forward reaction of CH4 conversion to

449

methanol, but on the reverse process where CO2 is converted to formic acid, formaldehyde and then

450

methanol. Formate dehydrogenase alone catalyses a very useful reaction: converting CO2 into

451

formate. This was achieved by Srikanth et al.

452

demonstrated the ability to electrochemically regenerate the co-factor (NAD+). The three other key

453

enzymes that are involved in the mineralisation of methanol to CO2 have recently been

454

demonstrated capable of the complete reverse enzymatic mineralisation of CH4 displayed in

455

Figure 290. Carbon dioxide was electro-catalytically converted to formate, formaldehyde and then

456

methanol at the cathode. Reactive red was used as an electron mediator and the co-factor (NAD+)

457

was regenerated via cathodic reduction. They further enhanced the process by including carbonic

458

anhydrase, an enzyme that facilitates the hydration of CO2(g). Enzyme-electrochemical techniques

459

are of interest as the ability to transform methane into useful chemicals without the requiring a live

460

culture are highly beneficial, but there are technical, cost and process hurdles to overcome.

89

, in an electrochemical system where they also

461



Page 22 of 51

462

3.1.9 Propylene oxide production (whole cell catalysis)

463

The broad substrate range of MMOs allow these enzymes to degrade soil contaminants as well as

464

generate products, such as 1- and 2-alcohols from C1-C8 n-alkanes, 1,2-epoxides from terminal

465

alkenes and ethanol/ethanal from diethyl ether91. Alkanes are hydroxylated mostly at the terminal

466

and sub-terminal positions, while ring hydroxylation of aromatics occurs primarily at the meta

467

position. The sMMO oxygenates alkenes to epoxides with retention of stereochemistry around the

468

C=C double bond92. The range for sMMO (n-alkanes, n-alkenes, aromatic and alicyclic compounds) is

469

significantly greater than that of pMMO (n-alkanes and n-alkenes). The broad substrate range of

470

MMOs nearly enabled the commercialisation of propylene oxide production in the 1990s.

471 472

Dalton and colleagues studied the transformation of propylene (propene/methyl-ethylene) to

473

propylene oxide (epoxy-propylene/epoxy-propane) using a methanotroph31,

474

studies to establish the epoxidation rates of propylene to propylene oxide by Methylococcus

475

capsulatus (Bath) were optimised in shake-flask cultures, where adding electron donors such as

476

methanol, formaldehyde, formate or hydrogen stimulated the endogenous rate of propylene oxide

477

formation up to 50 times. Specific production rates as high as 500 mol.min-1.g-1 of cells (dry mass)

478

were obtained with methanol as the electron donor, but were only sustained for short periods of up

479

to 4 minutes94. The loss of the MMO activity and subsequent declining epoxidation rates were

480

studied further95 and subsequently demonstrated as a result of reversible product inhibition96.

481

Reactivation of the bacterial propylene oxidation mechanism could occur without growth, but the

482

process required the presence of an energy source (methane or methanol), sulfur, nitrogen and

483

oxygen. In the presence of growth substrates, cells could be reactivated after the removal of

484

propylene oxide. De novo protein synthesis was also required for reactivation of activity and cultures

485

possessing sMMO took twice as long to recover compared to cells containing pMMO. In the pilot

486

process, methanol was used as the carbon source and MMO inhibition was circumvented by

487

operating a two-stage process system that allowed the epoxide-inhibited culture to recover in a


93

. Initial laboratory

Page 23 of 51


488

separate bioreactor in the presence of methane and other nutrients. Conducting the

489

biotransformation at the optimal growth temperature for the methanotroph (45 °C) was doubly

490

advantageous, because the boiling point of propylene oxide is 34 °C: allowing simpler product

491

recovery from the gas phase. The process was run at a high cell density (30 g.l−1) and propylene

492

oxide was produced at 250 g.l−1.day−1. The authors not only demonstrated continuous pilot-scale

493

production of propylene oxide from propylene using a methanotroph, but also evaluated the

494

process for producing 1,2-epoxybutane from but-1-ene and acetaldehyde from ethane. The process

495

could unfortunately not be commercialised, as the production cost was already at parity to

496

established commercial chemical technology at the time before inclusions for transport, storage and

497

profit97. More information about propylene oxide production is available in other review articles31, 34.

498 98

499

Xin et al.

500

with methane as the electron-donating co-substrate. They circulated an optimised gas mixture

501

(methane: 35 %; propene: 20 %; oxygen: 45 %) continuously, which removed the product. In this

502

manner they were able to operate a bioreactor continuously for 25 days without any obvious loss of

503

propylene oxide productivity. More recently, Su et al.

504

production using high cell density cultures of Methylosinus trichosporim OB3b and claimed a

505

propylene oxide productivity nearly four-fold greater than the highest reported productivity by

506

optimising temperature, initial propene concentration, sodium formate and MgCl2 concentrations .

recently used a Methylomonas sp. to catalyse the epoxidation in a continuous fashion

99

optimised conditions for propylene oxide

507 508

The combination of greater productivities and the consolidated bioprocessing approach (where the

509

product is generated and removed from a single reactor maintained at a temperature above the

510

products boiling point) is worth investigating again. However, there are new challenges as the

511

traditional methods for propylene oxide production have advanced since the 1990s. The older

512

chlorohydrin process was gradually replaced by the oxidation of isobutane and its catalytic

513

conversion to propylene oxide, which in turn has been superseded by ethylbenzene oxidation, which



Page 24 of 51

514

also yields styrene as a co-product100. Until recently, commercial propene peroxidation was

515

unfeasible due to the cost of H2O2. BASF and DOW chemicals have commercialised a process that

516

converts O2 into H2O2 (continuously via the oxidation and reduction of 2-ethylanthrahydroquinone),

517

which in conjunction with propene and a catalyst (titanium silicalite-1) produces propylene oxide

518

with no byproducts other than water100-101. This is an elegant process that would be challenging to

519

improve upon. Enzymatic MMO catalysis (as opposed to live cell catalysis) could be worth pursuing,

520

but would require sufficient MMO, a stable enzyme process and cheap regeneration of the reducing

521

equivalents.

522 523

3.1.10. New products and improved efficiency of genetically engineered methanotrophs

524

There has been considerable research over the past three decades regarding the genetics of

525

methanotrophs102. Various methanotrophs have been sequenced6a,

526

genetic engineering of these methanotrophs to over-produce metabolites, or even compounds not

527

naturally synthesised by these bacteria. The proof-of-concept for metabolic engineering of

528

methanotrophs to heterologously synthesise compounds has been validated. Sharpe

529

colleagues genetically modified (GM) methanotrophs to produce high-value carotenoids, while using

530

methane or methanol as a carbon source. Carotenoids are a family of yellow to orange-red

531

terpenoid pigments that protect against oxidative damage and are desired by food, medical, and

532

cosmetic industries

533

associated enzymes to convert it to astaxanthin in a Methylomonas species106. Various C40

534

carotenoids were accumulated in the intracytoplasmic membrane system in high concentrations104.

535

Although astaxanthin is widely used as a feed supplement in poultry and aquaculture industries, it is

536

a challenge to produce in bacteria as astaxanthin generally forms a small percentage of the total

537

carotenoids. A yield of 1 to 2.4 mg.g-1 dry mass was obtained, where 90 % of the total carotenoid

538

was astaxanthin (primarily the E-isomer) by engineering a methanotroph to contain two complete

539

sets of carotenoid biosynthetic genes, proving that astaxanthin with desirable properties could be

105

103

and this has allowed the

104

and

. The research group initially expressed a canthaxanthin gene cluster and


Page 25 of 51


540

produced in methanotrophs through genetic engineering106. As astaxanthin production was strongly

541

affected by oxygen availability, bacterial haemoglobins were incorporated into the bacteria by

542

further genetic engineering. Co-expression of the haemoglobin and astaxanthin-encoding genes

543

significantly increased astaxanthin expression - as the haemoglobins likely improved the activity of

544

the oxygen-requiring enzymes. A plasmid-free production Methylomonas strain produced more

545

astaxanthin than the parent strain107. This research demonstrated the ability to engineer non-

546

traditional microbial hosts that could use methane or methanol as alternative feedstocks for

547

microbial processes, as well as improve production by metabolic engineering104, 106-108.

548 549

Recently, Intrexon Corporation announced that they had genetically modified a methanotroph to

550

produce farnesene using methane as the carbon source109. Farnesene represents an enormous

551

global commodity as it is a basic precursor for diesel, lubricants and specialty products (cosmetics,

552

rubber and plastics). While this serves as another example of successful genetic engineering, there

553

was no data substantiating any appreciable yield. Moving from initial laboratory proof-of-concept

554

(where specific production may be as below µM.g-1) to pilot and commercial scale may require

555

production to be increased by several orders of magnitude, in addition to overcoming numerous

556

biological and engineering hurdles.

557 558

Methane-oxidising bacteria have served as hosts for producing recombinant and heterologous

559

proteins, including β-glucuronidase and genetically engineered MMOs27c. There are now a number of

560

genetic tools allowing mutagenesis and expression studies with methanotrophs and these

561

techniques allow introduction of broad-host-range plasmids carrying homologous and heterologous

562

genes into methanotrophs, promoter probe fusions, transposon mutagenesis and mutagenesis by

563

marker-exchange27b. There has been a substantial advances in genetic engineering and modification

564

of the capabilities of methanotrophs since the 1980s110, and this will continue to be a challenging,



565

but rewarding field. Synthetic biology may also contribute greatly to improving the production

566

efficiencies and yields5a.

567 568

3.2 Processes

569

3.2.1. Methane mitigation

570

The remediative abilities of methanotrophs are documented and their potential for mitigating

571

methane emissions from landfills and coal mines and their ability to degrade other hazardous

572

organic compounds have been reviewed6b,

573

technologies have been demonstrated beyond the laboratory scale as adaptable field-scale systems

574

that may be engineered to meet site-specific climatic variations and ensure minimal atmospheric

575

methane emission111, where methane oxidation efficiencies as high as 100 % have been reported18,

576

112

577

passive drainage and biofiltration of landfill gas as a means of managing landfill gas emissions from

578

low to moderate gas generation landfill sites. Passively aerated biofilters operating in a temperate

579

climate achieved maximum methane oxidation efficiencies greater than 90 % and average oxidation

580

efficiencies greater than 50 % over four years of operation. Although temperature and moisture

581

within the biofilter were affected by local climatic conditions, their effect on biofilter performance

582

was overshadowed by landfill gas loading. Interestingly, microbial methane oxidation was limited by

583

outflowing biogas as it prevented diffusion of atmospheric oxygen into the biofilter. Methanotrophic

584

systems have also been combined with algae, thereby sequestering both methane and CO2 and has

585

the potential to generate additional biological products114.

. Dever et al.

113

14b, 14c, 27a, 31

. Methanotrophic methane mitigation

conducted a field scale trial at a landfill site (Sydney, Australia) investigating

586 587

3.2.2 Contaminant bioremediation

588

Methanotrophs are useful bioremediation agents because of the broad substrate range of their

589

MMO enzymes (in particular sMMO), which allows them to remove heavy metals115 and transform

590

organic pollutants116. The sMMO enzymes can transform a variety of hydrocarbons, including


Page 26 of 51

Page 27 of 51


591

alkanes, alkenes, alicyclic hydrocarbons, aromatic compounds and halogenated aliphatics31,

91, 117

592

Chlorinated compounds degradable by sMMOs include chloroform118, dichloro-ethene 119, trichloro-

593

ethylene118, 120, tetrachloro-ethene121, hydrochloro-fluorocarbons122 and even vinyl-chloride 123.

.

594 595

Methane or nutrients may be added to stimulate methanotrophs and enhance biodegradation and

596

biotransformation of contaminants. Biostimulation of methanotrophs according to the site-specific

597

needs has even been demonstrated at a field scale in situ within contaminated aquifers and soils,

598

and ex situ in bioreactors14b, 25c, 124. Their remediative capacities have also been improved by genetic

599

modification125.

600 601

Plant-microbe associations are important relationships benefiting both partners. Enhanced

602

methanotroph-plant associations may be worth pursuing to create a more stable spread of

603

methanotrophs in a soil environment in a symbiotic relationship with plant roots. Even if the

604

methanotrophs do not greatly benefit the host (as is normally the case with endophytes that provide

605

nutrients or secrete plant growth promoting factors), as long as they are actively present it is

606

environmentally beneficial. Alternatively, transgenic plants can also mobilise or degrade chlorinated

607

solvent, xenobiotic compounds, explosives and phenolic substances. A symbiotic relationship

608

between GM methanotrophs and transgenic plants could significantly enhance the bioremediation

609

of contaminated sites125d.

610 611

3.2.4 Denitrification

612

Biological wastewater denitrification systems require organic carbon to facilitate the reduction of

613

nitrate to nitrogen. This carbon requirement may be partially met by the acidogenic fermentation of

614

a portion of the organic waste entering wastewater treatment systems, which provides volatile fatty

615

acids such as acetate. However, modern wastewater treatment plants frequently need to

616

supplement their systems with a costly external carbon source such as ethanol to achieve more



617

stringent discharge limits126. Using methane as a low-cost carbon source to facilitate denitrification

618

would be highly beneficial. Incorporating methane into the denitrification process was suggested by

619

various researchers in the 1970s127, but denitrification by pure methanotrophic isolates had still not

620

been validated four decades later128.

621 622

Although methanotrophs can catalyse nitrogen cycle processes such as nitrification and nitrogen

623

fixation, they cannot perform complete denitrification

624

hypothesised that the responsible agent in the mixed methanotrophic culture was a denitrifying

625

methanol-consuming bacteria that was using a methanotrophic byproduct to perform the initial

626

reduction of nitrate to nitrite. The premise was one of syntrophy, where one organism lives off the

627

products of another organism, and this was later verified128. Methane is used by the methanotrophs

628

and they in turn provide an electron donor (such as acetate) for the denitrifying bacteria. As cheaper

629

wastewater denitrification alternatives are being investigated, there has been a recent increase in

630

research published regarding methane oxidation coupled to denitrification129. Various consortia of

631

microorganisms are capable using methane as the sole carbon source for denitrification both

632

aerobically33, 128, 130 and anaerobically33, 129, 131.

128

. Even as early as the 1970s, it was

633 634

3.2.5. Electricity generation in microbial fuel cells

635

In a microbial fuel cell (MFC) micro-organisms are attached to electrodes that harvest the electrical

636

current produced from the spontaneous oxidation of organic substrates132. Theoretically,

637

methanotrophs could be employed in a MFC using methane as the carbon source, thereby coupling

638

biological methane oxidation to electricity generation. The concept of using methane as a carbon

639

source in a microbial fuel cell was patented by Girguis and Reimers133, but, to date, no electrogenic

640

activity has been demonstrated by a methanotroph. However, it is not essential that they are

641

electrogenic as they may serve as an intermediary at the surface of a biofilm and provide organic

642

metabolites to sustain electrogenic bacteria in contact with the anode. The potential


Page 28 of 51

Page 29 of 51


643

methanotrophic metabolites that have been used as sole carbon sources in MFCs include

644

methanol134 formic acid135, or fermentation products produced under oxygen-limited conditions20

645

such as acetate136 or lactate137. However, there are significant problems associated with using

646

oxygen in the anodic chamber and the materials required to construct MFCs are expensive

647

(considering the poor power densities achieved) making industrial application unlikely.

648 649

3.2.6 Biosensors

650

Biosensors are analytical devices that use a transducer coupled to biological material that elicits a

651

signal in response to an analyte. They can be extremely sensitive as well as highly specific, but may

652

suffer from instability138 or be affected by the external environment. The ability of MMO to react

653

with methane allows the bacteria containing the enzyme, or the isolated enzyme, to be used as the

654

biological component of a biosensor. A Methylomonas culture was exploited early in the

655

development of environmental biosensors by Okada, et al. 139, where methane concentrations were

656

determined in 3 min at 30 °C at pH of 7.2. It allowed for a minimum methane measurement of

657

13 μM and was reproducible to within 5 % for more than 20 days over which more than 500 assays

658

were performed. The bacteria were also incorporated into sensors developed by Daamgard and

659

colleagues140 where oxygen consumption was measured using an internal oxygen amperometric

660

microsensor, acting as a proxy for methane presence. Wen, et al.

661

using a mixed culture of methane-oxidizing bacteria. With both biosensors the sensitivity and the

662

response time were improved by increasing the number of bacteria. Unfortunately, both biosensors

663

were restricted by environmental factors (e.g., pH and temperature) that affected the physiological

664

state of bacteria142. The use of MMO enzymes coupled to an electrode was also investigated, but this

665

was unstable and produced unusual results – presumably due to methane and oxygen retention143.

141

666


developed a similar biosensor


667

4. Overview of potential products

668

The potential products, their reported yields and production efficiencies are summarised in Table 2,

669

along with their potential global demand, potential nearness to market and relevant references.

670 671

The increasing demand for protein-rich feed by the expanding global aquaculture industry bodes

672

well for single-cell protein production. Further improvements to strains via genetic modification (to

673

generate carotenoids or improved vitamin B12 production) could enhance the nutritional value of

674

the product. Although SCP production appears feasible (as evidenced by the recent construction of a

675

new facility in Trinidad and Tobago), government initiatives/incentives may be necessary to facilitate

676

private investment and further increase this application.

677 678

Propylene oxide and ectoine production are particularly interesting as they represent extracellular

679

products. Unlike most cellular products, this enables cell reuse, which is useful for relatively slow

680

growing biomass. Once the optimal biomass concentration is attained the key constraints are the

681

rates of production and tolerance towards the product inhibition. The commercial value of ectoine

682

makes it a compound worth pursuing via improved downstream processing, new production strains,

683

and genetic engineering to improve yields and yield efficiencies. Propylene oxide has an enormous

684

market and production could be improved via improved enzyme efficiencies and product tolerance,

685

or enhanced simultaneous product removal using either new thermo-tolerant species, or improved

686

production strains engineered to exhibit thermophilic traits. With the near-commercialisation of

687

propylene oxide production by methanotrophs in the 1990s, it is worth revisiting this process,

688

although new production processes will be difficult to compete against.

689 690

Biopolymer production (polyhydroxy-alkanoates and extracellular polysaccharides) and external

691

surface layers may be limited by the production rate of the relatively slow-growing methanotrophs.

692

It can take days to weeks as opposed to hours to days when using conventional bacteria. Although


Page 30 of 51

Page 31 of 51


693

the price of petroleum precursors will have to increase before biopolymer production becomes

694

viable, a potential bridge towards economic feasibility may be focussing on producing a consistent,

695

high-quality PHB for medical applications. However, for PHAs and EPS, the low solubility of methane

696

is a great challenge, especially with high-density cultures.

697 698

Biodiesel also represents an enormous market, but polar membrane lipids with a high heteroatom

699

content from methanotrophs may not be the best microbial alternative. If attempts to genetically

700

enhance the methanotrophs to improve lipid yields and produce desired storage lipids

701

(triacyglycerols) are successful, it will be an important step towards commercialising biofuels from

702

these bacteria, as the quality of the lipid intermediates have been regarded as playing a major role in

703

the overall fuel production cost and will have a strong impact on the catalytic upgrading steps5b.

704

However, considering the lipid composition in native bacteria, use as a health supplement for

705

lowering cholesterol currently seems most appropriate.

706 707

Products that currently appear unfeasible to produce include farnesene and soluble metabolites

708

such as methanol, formaldehyde and organic acids. There is no data available that indicates that

709

farnesene is produced in any appreciable quantities in methanotrophs. Soluble metabolites such as

710

methanol, formaldehyde and organic acids are produced in low concentrations according to

711

literature. Even methanol titres of 1 g/l will not be commercially viable considering rates and

712

processing conditions; it also negatively impacts culture maintenance as reducing equivalents are

713

not regenerated. However, methanol is an important global commodity and the potential to

714

generate it under ambient conditions using cells modified via traditional genetic manipulation or

715

synthetic biology, or via enzymatic catalysis, is still a prominent topic.

716 717

Although some methanotroph enzymes possess high catalytic activities, it is unlikely that the native

718

methanotroph will be used for production. Cloning the enzymes’ genes into common expression



719

systems that use conventional carbon sources is more feasible, but this would use a traditional

720

carbon source rather than methane. It is not inconceivable that enzymes could be harvested from

721

spent methanotrophic biomass though, thereby adding value and potentially improving the

722

economic viability of another process.

723 724

Incorporating methanotrophs into biosensors and electricity generation via microbial fuel cells seem

725

unlikely options. The biosensor have a limited functional pH range and require oxygen and reducing

726

equivalents, in addition to general biosensor problems such as background noise, interference and

727

inhibition. Although patented, electricity generation in microbial fuel cells with methane as the sole

728

carbon source has not actually been demonstrated. Electricity generation seems improbable as the

729

presence of oxygen in the anodic chamber will negate electron transfer.

730 731

Regarding processes, partial denitrification aided by methanotrophs could be worth incorporating

732

into wastewater treatment plants where possible, as this could lessen the cost associated with

733

providing an external carbon source for denitrification. Other processes, such as mitigating methane

734

releases into the atmosphere and bioremediation have been demonstrated at a field scale11, 93 and

735

are technically possible, but will only be applied when there is a sufficient financial incentive to limit

736

carbon emissions or remove contaminants. GMOs with improved traits (e.g. tolerance and

737

degradation efficiency) could further enhance these applications, but the perceived of risks related

738

to genetic transfer requires extensive assessment before a GMO may be used in the environment116,

739

125a, 144

.

740 741

5. Methanotroph applications: synthetic biology, scale-up and commercialisation

742

The future of all of the bioprocesses mentioned in this review must yield a profit, or be of greater

743

value when benchmarked against conventional methane applications such as energy recovery or

744

heating, and must be achievable at scale. To ensure a consistent and optimal product or robust


Page 32 of 51

Page 33 of 51


745

process, fermentation and downstream processing must be optimised, integrated and functional at

746

scale.

747 748

The greatest technical challenge for gas fermentation at scale remains the efficient mass transfer of

749

poorly-soluble gaseous substrates into the aqueous phase145. Other major concerns are the need to

750

regenerate reducing equivalents and the cost of downstream processing. Many reactor types

751

(continuous stirred tank reactors, bubble columns, airlift reactors, trickle beds and numerous

752

variations) have been applied to gas fermentation145, and microbubble generation

753

immobilized hollow fibre membranes146 have been investigated to improve gas transfer efficiency.

754

Increasing the headspace pressure is another means of improving mass transfer. Paraffin and

755

nanoparticle addition can improve gas transfer, but at an increased cost. Cathodic reduction (directly

756

to the cells or an immobilised MMO complex) may provide the electrons required or regenerate the

757

reducing equivalents, but feasibility depends on the cost of the reduction and whether it can be

758

scaled up in an economically-viable manner.

145b

or

759 760

Another significant problem at scale with gas fermentation is foaming (especially with high density

761

cultures) and may be compounded by operating secondary reactors to the extreme of the microbe’s

762

capacity to maintain structural integrity or survive exposure to high product or metabolite

763

concentrations. This may be further aggravated by microaeration, where the small gas bubbles that

764

are required to improve gas-liquid transfer may be more easily stabilised by bacterial lysis products

765

or proteins and glycoproteins.

766 767

However, downstream processing costs may single-handedly prevent the commercialisation of these

768

products. Generally, if the product is intracellular, cells must be concentrated, lysed, and then the

769

product must be extracted, purified, concentrated and processed. There are considerable costs

770

associated with cell pretreatment147, solvent recovery, impurity removal, increases in viscosity,



771

subsequent requirements for product upgrading or modification, waste treatment and

772

environmental consequences42,

773

increases in liquid viscosity.

51

as well as technical issues dealing with particular solvents and

774 775

The capabilities and yield efficiencies of production strains may be enhanced with cloned genes, or

776

potentially even with completely synthetic additions in the future. There is hope of potentially

777

doubling the energy efficiency of MMOs by engineering a dioxygenase-like enzyme to activate

778

methane, that would allow two methane molecules to be activated for the same energy input5a.

779

Genetic engineering and synthetic biology have a large role in the future application of

780

methanotrophs and their enzymes, as indicated by the strong emphasis on these techniques in many

781

of the ARPA-E funded projects. Some projects involve completely synthetic enzymes for methane

782

activation (Arzeda Corp.), while others will re-engineer enzymes for methylation (Northwestern

783

University, Lawrence Berkeley National Laboratory), pathways (University of California Davis) or

784

methanotrophs (University of California Los Angeles), or even use phototrophic organisms (MOgene

785

Green Chemicals LLC) to produce a liquid fuels such as butanol, methanol, ethanol or dimerise

786

methane. Enzyme use is also being combined with chemical approaches for cell-free catalysis

787

(GreenLight Biosciences) and new metabolic pathways are also being engineered into methylotrophs

788

to convert methanol into butanol (University of Delaware). Synthetic biology may offer alternative

789

routes for regenerating reducing equivalents, using advanced enzymes or new metabolic pathways,

790

or improving yields and conversion efficiencies, but these are complex, time-consuming endeavours

791

and great patience and perseverance will be required.

792 793

There are still serious challenges to commercialising methanotroph applications, but the recent

794

funding allocation and intensive research committed to biologically generating transport fuels from

795

methanotrophs demonstrates the potential envisaged for these unique bacteria. Although they have

796

been known and researched for just over a century, their ability to use methane under ambient


Page 34 of 51

Page 35 of 51


797

conditions, coupled to the abilities of specialist thermophilic, halophilic and acidophilic

798

methanotrophs, as well as the potential to further improve their capabilities via genetic

799

enhancement and synthetic biology, translates to decades of intriguing research ahead.

800 801

6. Acknowledgements

802

The Centre for Solid Waste Bioprocessing gratefully acknowledges funding from Remondis. W.P.C.

803

and S.H. acknowledge funding from the Australian Research Commission (DP 140104572). The

804

authors acknowledge input from S. Freguia, T. Stewart, M. Patel, J. Bors and I. Pikaar.

805 806

7. References

807 808 809 810 811 812 813 814 815 816 817 818 819 820 821 822 823 824 825 826 827 828 829 830 831 832 833 834 835 836 837 838

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143. Chuang, J. D.; Hemond, H. F., Electrochemistry of soluble methane monooxygenase on a modified gold electrode : implications for chemical sensing in natural waters. Thesis (S.M.) Massachusetts Institute of Technology, Dept. of Civil and Environmental Engineering, 2005. http://hdl.handle.net/1721.1/31156 2005. 144. Singh, J. S., Methanotrophs: the potential biological sink to mitigate the global methane load. Current Science 2011, 100 (1), 29-30. 145. (a) Vega, J. L.; Clausen, E. C.; Gaddy, J. L., Design of bioreactors for coal synthesis gas fermentations. Resources, Conservation and Recycling 1990, 3 (2-3), 149-160; (b) Munasinghe, P. C.; Khanal, S. K., Biomass-derived syngas fermentation into biofuels: Opportunities and challenges. Bioresour Technol 2010, 101 (13), 5013-22. 146. Hickey, R. F.; Tsai, S. P.; Yoon, S. H.; Basu, R.; Tobey, R. E., Submerged membrane supported bioreactor for conversion of syngas components to liquid products. Patent US 8058058 B2: 2011. 147. Jacquel, N.; Lo, C.-W.; Wei, Y.-H.; Wu, H.-S.; Wang, S. S., Isolation and purification of bacterial poly(3-hydroxyalkanoates). Biochemical Engineering Journal 2008, 39 (1), 15-27. 148. Han, B.; Su, T.; Wu, H.; Gou, Z.; Xing, X. H.; Jiang, H.; Chen, Y.; Li, X.; Murrell, J. C., Paraffin oil as a "methane vector" for rapid and high cell density cultivation of Methylosinus trichosporium OB3b. Appl Microbiol Biotechnol 2009, 83 (4), 669-77. 149. NFCSF, Opinion on the safety of BioProtein® by the Scientific Panel on Animal Feed of the Norwegian Scientific Committee for Food Safety Revised version Adopted on the 5th of October 2006 http://www.vkm.no/dav/a0782dea9c.pdf 2006. 150. Wendlandt, K. D.; Geyer, W.; Mirschel, G.; Hemidi, F. A.-H., Possibilities for controlling a PHB accumulation process using various analytical methods. Journal of Biotechnology 2005, 117 (1), 119129. 151. Khmelenina, V. N.; Kalyuzhnaya, M. G.; Sakharovsky, V. G.; Snzina, N. E.; Trotsenko, Y. A.; Gottschalk, G., Osmoadaptation in halophilic and alkaliphilic methanotrophs. Archives of microbiology 1999, 172 (5), 321-329. 152. Taher, E.; Chandran, K., High-rate, high-yield production of methanol by ammonia-oxidizing bacteria. Environ Sci Technol 2013, 47 (7), 3167-73. 153. Eshinimaev, B. T.; Khmelenina, V. N.; Sakharovskii, V. G.; Suzina, N. E.; Trotsenko, Y. A., Physiological, biochemical, and cytological characteristics of a haloalkalitolerant methanotroph grown on methanol. Mikrobiologiya 2002, 71 (5), 596-603. 154. (a) Gretsinger, B. E.; Malashenko, Y. R.; Chernyshenko, D. V. U., patent: USSR Inventor’s Certificate no. 962594. Byull. Izobret., 1982b 1982b, 36, 36-39; (b) Gretsinger, B. E.; Malashenko, Y. R.; Karpenko, V. I.; Grinberg, T. A., Patent: USSR Inventor’s Certificate no. 973869. Byull. Izobret. 1982a, 42, 12-16. 155. Trotsenko, I. A.; Doronina, N. V.; Khmelenina, V. N., Biotechnological potential of methylotrophic bacteria: a review of current status and future prospects. Prikladnaia biokhimiia i mikrobiologiia. 2005, 41 (5), 495-503. 156. (a) Lidstrom, M. E., Metabolic Engineering of Methylotrophic Bacteria for Conversion of Methanol to Higher Value Added Products. EPA Grant Number: R826729 2001; (b) Lidstrom, M. E.; Wopat, A. E., Plasmids in methanotrophic bacteria: isolation, characterization and DNA hybridization analysis. Archives of microbiology 1984, 140 (1), 27-33. 157. Santegoeds, C. M.; Damgaard, L. R.; Hesselink, G.; Zopfi, J.; Lens, P.; Muyzer, G.; de Beer, D., Distribution of sulfate-reducing and methanogenic bacteria in anaerobic aggregates determined by microsensor and molecular analyses. Appl Environ Microbiol 1999, 65 (10), 4618-29.


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8. Figures and Tables

1394 1395

Figure 1: Methanotrophic methane mineralisation or assimilation (via the Serine or RUMP pathways).

1396 1397



1398 1399

Figure 2: Typical reaction scheme for the trans-esterification of a triglyceride into esters and glycerine.

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Table 1: Generalised differences between Type I and Type II methanotrophs 6, 27a, 40c, 110.

Genera Formaldehyde assimilation pathway Phospholipid fatty acids of intracytoplasmic membranes Membrane arrangement Preferred gas ratio Cyst formation Nitrogen fixation CO2 assimilation

Type I: -Proteobacteria Methylobacter, Methylocaldum, Methylococcus, Methylomicrobium, Methylomonas, Methylosphaera and Methylothermus genera

Type II: -Proteobacteria

Methylocella, Methylocapsa, Methylocystis and Methylosinus genera

Ribulose monophosphate pathway

Serine pathway

Bundles of 16-carbon fatty acids: 14:0, 16:0, 16:1ω7c and 16:1ω5t

Bundles of 18-carbon fatty acids: 18:1ω8c, 18:1ώ8t and 18:1ώ6c

Stacked membranes throughout cytoplasm

Peripheral membrane rings

Lower CH4:Higher O2 Yes Methylococcus, Methylomonas and Methylosphaera 5-15% of biomass

Higher CH4: Lower O2 No Common under low oxygen tension Up to 50 % of biomass

1402



1403

Table 2: Potential products and processes using methane as the primary carbon source for methanotrophs. Product Relative Global annual demand Near to market value Single cell protein. Low >25 000 tonnes Yes, under defined natural gas costs and product retail price. Commercial plant in Trinidad and Tobago. Internal storage polymers Low Equivalent to 0.6 billion Processing cost constraints. May be viable when (PHB) litres of oil used for petroleum products increase sufficiently in price. making plastics Potential for specialised polymers or via lactic acid production. Lipids: biodiesel Low 1.4 billion litres Not currently feasible due to heteroatoms. R&D needed. Potential via genetic manipulation. Lipids: dietary supplements High Kg - tonnes Possibly. Promising option. Ectoine Soluble metabolic products: Methanol

Very high

1-10 tonnes

Low

90 billion litres

Formaldehyde Organic acids

Low Low

Surface layers Extra-cellular polysaccharides (EPS)

Medium- Unknown High Low >10 tonnes medium

Growth media and Vit B12

Medium

35 billion litres 9600 tonnes (as acetic acid only)

1 tonne

Unknown. Boosts nutrition value.

>1.4 billion litres

Medium

9-10 million tonnes

Unknown. Proof of concept achieved according to press release, but no yield data. Promising, but requires improved efficiencies. Cost was parity with older technology.

None

High High

Denitrification

None low Low

Biosensors Electricity generation

Medium Low

Medium High.

Bioremediation

155 22, 34, 81-90

T: 2.4 mg astaxanthin.g-1

Low medium. Low.

GMO products: Farnesene Biotransformation of propene to propylene oxide. Process Methane mitigation

biomass No data

High

Naturally occurring. Seeded in certain instances and has worked at a field scale. Naturally occurring. Seeded in certain instances and has worked at a field scale. Achieved in laboratories. Technical requirements possibly limits this to niche WWTPs or academia. No. Technical constraints. No. Unproven. Improbable.

1404


106

6a, 27b, 27c, 104 110, 156 109

No data −1

−1

SP: 8.3 g.g .day

92

31, 34, 91-93, 98

6b, 14b, 14c, 18, 111-112 14b, 25c, 31, 91, 117, 124-125 33, 127-131

139-143, 157 133-135

Methane Oxidation and Molecular Characterization of Methanotrophs from a Former Mercury Mine Impoundment.

Acid-Tolerant Moderately Thermophilic Methanotrophs of the Class Gammaproteobacteria Isolated From Tropical Topsoil with Methane Seeps.

Two-liquid phase partitioning biotrickling filters for methane abatement: exploring the potential of hydrophobic methanotrophs.

Using Joint Interviews to Add Analytic Value.

Gammaproteobacterial methanotrophs dominate cold methane seeps in floodplains of West Siberian rivers.

Optimization of trichloroethylene oxidation by methanotrophs and the use of a colorimetric assay to detect soluble methane monooxygenase activity.

Conventional methanotrophs are responsible for atmospheric methane oxidation in paddy soils.

Can live weight be used as a proxy for enteric methane emissions from pasture-fed sheep?

A critique of the Harvard Resource-Based Relative Value Scale.

Methylocella: a gourmand among methanotrophs.

The surgical oncologist as a resource.

Indians, ethnicity as a resource and aging: You can go home again.

Value of the use of absolute lymphocyte as surrogate for CD4 count in resource poor situations.

Whole slide images add value to journal article figures.

Sense made common: how to add value to early experience.

Can we add auricular composite graft to our rhinoplasty armamentarium?

Small collaborative steps can add up to big change.

Can intermittent theta burst stimulation as add-on to psychotherapy improve nicotine abstinence? Results from a pilot study.

Moral styles: A resource for value-centered counseling.

Can a simple test of functional capacity add to the clinical assessment of diabetes?

Can focused trauma education initiatives reduce mortality or improve resource utilization in a low-resource setting?

Inclusiveness in the value base for health care resource allocation.

Results and impacts of the Resource-Based Relative Value Scale.

Incorporating practice costs into the Resource-Based Relative Value Scale.