Methane as a Resource: Can the Methanotrophs Add Value

Critical Review pubs.acs.org/est

Methane as a Resource: Can the Methanotrophs Add Value? P. J. Strong,* S. Xie, and W. P. Clarke

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

1. INTRODUCTION

2. METHANE

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 a 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. This review is intended to 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.

Methane is a colorless, odorless gas that is emitted from both natural and anthropogenic sources. It provides energy or heat via combustion. However, its emission into the atmosphere has negative consequences as it is a greenhouse gas with approximately 20 times the impact of carbon dioxide. Anthropogenic activity accounts for the majority of global methane emissions (63%, or 566 Tg CH4/year), with natural biological emissions accounting for the remainder (208 Tg CH4/year).1 Anthropogenic methane emissions are generated by the use of fossil fuels, livestock farming, landfilling, and biomass burning. Natural sources of methane are wetlands, oceans, estuaries, rivers, lakes, permafrost, gas hydrates, geological sources (terrestrial and marine), wildfires, vegetation, terrestrial arthropods, and wild animals.1,2,2b The ratio of anthropogenic:natural methane production has increased steadily since the advent of the industrial revolution. With increased food requirements, greater waste generation, and greater use of fossil fuels by an increasing human population, it will in all likelihood increase further. The principal use of methane is as a fuel, as its combustion is highly exothermic:

© 2015 American Chemical Society

Received: Revised: Accepted: Published: 4001

September 1, 2014 February 19, 2015 February 27, 2015 February 27, 2015 DOI: 10.1021/es504242n Environ. Sci. Technol. 2015, 49, 4001−4018

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Environmental Science & Technology Table 1. Generalized Differences between Type I and Type II Methanotrophs6,27a,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: γ-Proteobacteria

Type II: α-Proteobacteria

Methylobacter, Methylocaldum, Methylococcus, Methylomicrobium, Methylomonas, Methylosphaera and Methylothermus genera ribulose monophosphate pathway bundles of 16-carbon fatty acids: 14:0, 16:0, 16:1ω7c, and 16:1ω5t

Methylocella, Methylocapsa, Methylocystis and Methylosinus genera serine pathway bundles of 18-carbon fatty acids: 18:1ω8c, 18:1ώ 8t, and 18:1ώ 6c peripheral membrane rings higher CH4:lower O2 no common under low oxygen tension up to 50% of biomass

stacked membranes throughout cytoplasm lower CH4:higher O2 yes Methylococcus, Methylomonas and Methylosphaera 5−15% of biomass

between Type I and Type II methanotrophs are summarized in Table 1, but exceptions are common and metabolic pathway flexibility may be greater than previously thought.8 This is evident for Type I methanotrophs, which contain a subdivision denoted as Type X (Methylocaldum and Methylococcus species) that express enzymes associated with the serine pathway typical of the α-proteobacteria.6b Methane can also be oxidized by a phylum of thermoacidophilic bacteria known as Verrucomicrobia (which can grow at a pH below 1),9 as well as certain Archaea in consortia with sulfate reducing bacteria.10 The ability of certain Archea to anaerobically oxidize methane (where NOx11 or SOx12 replace O2) corroborates research supporting the concept of reverse methanogenesis.13 Methane consumption is made possible by an enzyme known as methane mono-oxygenase (MMO), which occurs in a particulate form (pMMO) within an intracellular membrane or a soluble form (sMMO) within the cytoplasm.14 Copper availability plays a defining regulatory role with regard to sMMO expression,15 where sMMO synthesis is inhibited by higher Cu2+ concentrations.16 The sMMO has a di-iron catalytic site, while research suggests that pMMO requires both copper and iron to be catalytically active.7b The sMMO can be produced by various α- or γ-proteobacteria and has a much broader substrate range than the pMMO. It is capable of oxidizing numerous compounds, including propene, butane, cyclohexane, chlorotrifluoroethylene, toluene, naphthalene, chloroform, diethyl ether, and CO (substrates tabled in Jiang et al14b). Although pMMO is expressed by most methanotrophs, some Methylocella spp. express sMMO exclusively.17 The metabolic activity and growth of methanotrophs are influenced factors that typically affect microbial cultures, including nutrient supply, temperature, pH, macronutrients, and trace metals. Traditionally, the growth of γ-proteobacteria was reported to favor higher O2 concentrations and low CH4 concentrations, while α-proteobacteria methanotrophs preferred low O2 and higher CH4 concentrations,6d,18,19 but γproteobacteria can grow efficiently under a low oxygen tension.20 Similarly, facultative methanotrophy was initially discounted but has been demonstrated in certain genera of αproteobacteria.7b,21,21b Methanotrophs are able to assimilate CO2 and are thus affected by CO2 concentrations. The αproteobacteria can assimilate up to 50% of their biomass from CO2, while the γ-proteobacteria can assimilate up to 15%.22 The N source in media commonly used to culture methanotrophs is usually nitrate (e.g., nitrate minimal salts) or ammonia. Ammonia affects different methanotrophs to differing extents and can be a competitive inhibitor of methane monooxygenase or toxic when in the intermediate forms of hydroxylamine and nitrite.23 However, ammonium acts more as a nutrient than an inhibitor in the presence of sufficient

CH4(g) + 2O2(g) → CO2(g) + 2H 2O(l) where ΔH = −891 kJ

Methane is primarily used for generating electricity in gas turbines or steam boilers but is also piped into homes for domestic heating and cooking or used as a vehicle fuel as compressed natural gas.3 The reciprocating engine, gas turbine, or steam turbine technologies are available over a wide range, from modular units of several hundred kW up to 250 MW for commercial steam turbines.4 Although methane can be used as a transport fuel, there is currently a relative lack of infrastructure for fueling vehicles compatible with natural gas. However, new approaches whereby methane is converted into liquid transportation fuels can take advantage of existing engines and delivery infrastructure.5 From a biological perspective, methane represents a carbon and energy source for a group of bacteria known as methanotrophs. Methanotrophs use methane as their sole carbon source and directly convert methane into cellular compounds or transform it into a substrate that drives processes via methanotrophs or their syntrophic interaction of other microbes. The biological monetization of methane has become a topic of intense interest: in 2013 the Advanced Research Projects Agency (ARPA-E) within the U.S. Department of Energy granted US $34 million worth of funding directed toward research converting methane into liquid fuels. There are notable barriers to overcome, but the size of the methane resource is enormous and in the case of anaerobic digestion is sustainably produced; this justifies the continued interest in these bacteria that can oxidize methane under ambient conditions.

3. METHANOTROPHS Methanotrophs, a subset of the methylotrophs, can assimilate methane as their sole carbon source. Methanotrophs fall under proteobacteria; a major phylum of Gram-negative bacteria that includes genera such as Escherichia, Salmonella, Vibrio, Helicobacter, and Yersinia. The proteobacteria phylum is divided into six classes according to rRNA sequences. Two classes contain methanotrophs: the alpha-proteobacteria and the gamma-proteobacteria. Methanotrophs are traditionally classified as Type I (γ-proteobacteria) or Type II (α-proteobacteria). The Type I and II distinctions were primarily based on the metabolic pathway used to assimilate formaldehyde and related attributes such as cell membrane composition and arrangement and cell morphology.6 Typically, Type I methanotrophs are γproteobacteria that assimilate formaldehyde via the ribulose monophosphate pathway,6b,d,7 while Type II are α-proteobacteria that use the serine pathway. The generalized differences 4002

DOI: 10.1021/es504242n Environ. Sci. Technol. 2015, 49, 4001−4018

Critical Review

Environmental Science & Technology

protein source for several monogastric species, including pigs, broiler chickens, mink, fox, Atlantic salmon, rainbow trout, and Atlantic halibut.35 Although bacteria possess a high protein content (ranging from 50 to 65%) relative to other micro-organisms (30 to 60%), they also possess the highest nucleic acid content (8 to 12%),36 which can adversely affect consumers. A report regarding the safety of the UniBio protein (then known as Dansk BioProtein) noted significant immune effects in rats during a toxicity study (NCSF, 2006). The protein was only approved for animal nutrition and was recommended for animals with a short life span, as the nucleic acids could cause kidney and bladder stones in longer-lived species. However, the risk associated with human consumption of products from animals fed on BioProtein was considered negligible. Heat treatment or hydrolysis can lower RNA content. At UniBio A/S nucleic acids are neutralized by hydrolysis, generating a product fit for human consumption. Heat treatment at 60 to 65 °C for 10 to 20 min has been used to remove nearly 90% of the RNA from a Methylomonas sp. cultured for single-cell protein use.37 Although using a C1 gas as the substrate is highly selective, large-scale continuous industrial fermentation is always subject to infiltration by other micro-organisms as cell lysis products or metabolic byproducts serve as C sources, which is problematic in a continuous process. The SCP strain was repeatedly contaminated by three different bacteria: a member of the Aneurinibacillus group, a Brevibacillus agri strain, and an acetateoxidizing Ralstonia species. Fortunately, the three contaminant bacteria were nontoxic. Their presence was beneficial as they stabilized the culture by consuming metabolic byproducts that would have inhibited growth.38 5.1.2a. Biopolymers: Internal Storage Polymers. Polyhydroxy-alkanoates (PHA) such as poly(3-hydroxybutyrate), or PHB, are widespread and intensively researched bacterial storage polymers seen as potential substitutes for plastics derived from the petroleum industry. They have beneficial properties such as biodegradability, biocompatibility. and thermoplasticity. These biopolymers are synthesized and deposited intracellularly as granules that serve as a source of carbon, energy, or reducing-power and, in exceptional circumstances, may comprise up to 90% of a microbe’s dry weight.39 PHB accumulation is induced by exposing an active culture to excess carbon while under a nutrient limitation of some sort. PHA yield and quality is affected by pH, temperature, and the availability of other carbon sources, methane, oxygen, carbon dioxide, macronutrients (nitrogen, phosphorus, sulfur, potassium, magnesium sodium), and trace metals (copper, iron, zinc, manganese cobalt).40 Although PHB production by methanotrophs and methylotrophs has been of interest for decades,41 it has been re-evaluated in recent times.5b,40c Cheap, or zero cost, substrates such as CH4 and CO2 are receiving considerable attention because of their potential to lower production costs.32,42 Microbe strain selection, or genetic engineering of existing strains, is critical because of the impact on the maximum production rate and yield. In the 1990s, heterotrophically grown Alcaligenes eutrophus became the organism of choice for industrial PHB production as it produced high yields of high molecular weight PHBs using various economically acceptable substrates. It was benchmarked against a methylotroph (Methylobacterium sp.), which only managed moderate polymers yields that had a low molecular weight (Mw) and were difficult to extract.43 To date, many methylotrophs and

methane. Nitrogenase occurs in a broad range of methanotrophs (α- and γ-proteobacteria) allowing for use of N2 as a nitrogen source; nitrogen-fixing may be widespread and important to nitrogen cycling in many environments.23

4. METHANOTROPHS: PRIOR REVIEWS Many aspects of these ubiquitous bacteria have been reviewed. This includes the natural occurrence of methanotrophs,24 their use in methane mitigation and environmental remediation,6d,14b,25 their physiology, biochemical pathways for N metabolism,26 C1 metabolism, and assimilation,22,27 the enzymes involved,6c,28 and the influence of copper on their metabolic capabilities27a,28c,29 and facultative methanotrophy.7b,21b,30 Although the biotechnological applications have been reviewed in part, no single review has comprehensively covered all prospective products or processes. Specific topics such as biodiesel generation,5b epoxide production,31 polyhydroxybutyrate accumulation,32 and denitrification33 have been covered individually, while some reviews cover many facets of methanotrophy and some biotechnological applications.6b,27a A review by Dalton14a discusses much of the history and development of various aspects of methanotrophic physiology and biochemistry, as well as views on the development of single cell protein production, pollutant bioremediation, and whole cell catalytic production of propylene oxide (epoxypropane) and alludes to the possibility for electrochemically driving reactions that require reducing agents such as NADH. Trotsenko et al.34 reviewed single cell protein, biopolymer, ectoine, surface layers, vitamins, pollutant bioremediation, and the role of methanotroph as plant growth-promoting bacteria. These topics, as well as extracellular polysaccharides, lipids for biodiesel and human health supplements, growth media, methanol, formaldehyde and organic acids, enzymes, enzymatic transformation of CH4 and CO2 (incorporating electrochemical reductant recycling), and products resulting from genetic engineering are covered in the current review. 5. METHANOTROPHS: PRODUCTS AND PROCESSES 5.1. Products. 5.1.1. Single-Cell Protein. Single-cell protein (SCP) production from microbes such as yeasts, fungi, algae, and bacteria surged in the 1950s and 1960s due to a lack of protein source and predictions of impending global shortages. The advent of low cost soya production in the 1970s negatively impacted microbial SCP research, although the production of a fungal protein was commercialized in 1985. Currently, SCP is the closest example of a commercially successful biologically generated product using methane as the carbon source. The commercial production of methanotrophic SCP originated as research in Denmark by E. B. Larsen in the 1980s and has culminated the company now known as UniBio A/S. The fermentation uses natural gas, technically pure oxygen, ammonia as the N source, phosphoric acid as the P source, and supplemented with other minerals. The process is controlled at pH 6.5 and run at a temperature of 45 °C. The concentrated biomass is sterilized by rapid heating to 140 °C and then slowly cooled, allowing cells lysis and accessibility to the protein. The bacterial biomass consists predominantly of a strain of Methylococcus capsulatus (Bath) and is a promising protein source, based on criteria such as amino acid composition, digestibility, and animal performance and health.35 The methanotrophic protein has been used as a 4003


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been derived from algal and plant sources.57 There has been an active search for producers that may be cultured using nonedible raw materials.58 Methanotrophs satisfy this requirement as they can synthesize EPS using methane as their sole carbon source.58,59 The genes responsible for elements of the EPS synthesis have been isolated from a Methylomonas sp. with the intention of genetically engineering them into other C1utilizing microorganisms to alter levels of EPS production for commercial production.57 Malashenko et al.58 studied the dynamics of EPS production by methanotrophs in chemostats and observed EPS synthesis rates ranging from 0.03 to 0.43 g· g−1 dry biomass. Highest production was achieved using Type I isolates (Methylomonas and Methylobacter spp.). The viscosity of 0.1% aqueous solutions of EPS synthesized by the mesophilic methanotrophs varied from 2.2 to 4.0 mm2/s, which was comparable to the viscosity of EPS solutions synthesized by known microbial producers as well as the benchmark equivalent composed of 0.1% xanthane (3.5 to 4.5 mm2/s). However, EPS production may inhibit further synthesis as it negatively affects gas uptake. Chiemchaisri and Visvanathan59a studied methanotrophic EPS production in a bench-scale soil reactor. Methane oxidation rates (regulated by temperature and soil water content) were correlated to EPS production and the highest EPS production occurred at 30 °C. They observed that a high oxygen content accelerated EPS production but subsequently limited gas diffusion and inhibited production. 5.1.3. Internal Osmo-Protectants: Ectoine. Halo-tolerant bacteria employ two primary survival strategies: they synthesize and accumulate intracellular organic osmo-protectants (such as ectoine, glutamate, and sucrose), or they make structural and functional changes to their cell envelopes by altering the phospholipids composition of membranes and forming glycoprotein surface layers.14b,60 Ectoine is a cyclic imino acid that is one of the most widespread microbial protective measures against osmotic dehydration. It is also an efficient stabilizer of enzymes, nucleic acids, and DNA−protein complexes and can be used as a moisturizer in the cosmetic industry. Cosmetic formulations comprising ectoine or its derivatives offer excellent protection against UV-induced damage to the DNA of skin cells.61 Ectoine is produced annually on a scale of tons in an industrial process using the halophilic γ-proteobacterium Halomonas elongata DSM 2581T as producer strain.62 Because synthesis and purification is expensive, new microbial producer strains and their enzymes are constantly assessed to improve the economics of microbial ectoine production. Halo-tolerant methanotrophs are known and isolated, with the best-known belonging to the genus Methylomicrobium. Trotsenko and his colleagues demonstrated that moderately halophilic methanotrophs and methylotrophs were able to accumulate up to 20% of their dry mass as ectoine.34,60 This is an area well worth further exploration, as purified ectoine has one of the highest retail values of the methanotroph products (approximately $1300 kg−1). Additionally, the bacteria may potentially be reused multiple times: Halomonas elongata has been reused 9 times, yielding ectoine at an average of 15.5% g·g−1 biomass.63 However, downstream processing is complex and expensive and represents a challenge to process economics. 5.1.4. External Osmo-Protectants: Surface Layers. Bacterial cell surface layers are one of the most commonly observed outermost structures of prokaryotes. These regular paracrystalline structures cover the entire surface of a cell and

methanotrophs have been assessed for PHA production and are tabled in a reviews by Khosravi-Darani et al.32 and Karthikeyan et al.40c Polymer yields and Mw have improved among the newly isolated bacteria as well as mixed cultures. Zhang et al.44 cultured a Methylosinus trichosporium using methane with methanol and citric acid and obtained a high quality PHB (Mw: 1.5 × 106 Da) at a yield 40%. Wendlandt et al.45 also produced a high quality PHB with a high molecular mass of (up to 2.5 × 106 Da), using a Methylocystis sp. in a rapid, nonsterile process. They obtained a PHB content of up to 51% using a two-stage process consisting of a continuous growth stage (dilution rate: 0.17 h−1) and a PHB accumulation stage under P-deficient conditions. 14c Shah et al. 46 enriched their methanotrophs for pMMO or sMMO and compared PHB accumulation under different batch conditions. The pMMOrich cells displayed greater and more rapid PHB accumulation (up to 50% PHB content within 120 h) and attained a much greater biomass yield (18 g/L). The pMMO-rich cells also. Recently, researchers included silicone oil (10% v/v) in the two-phase partitioning bioreactor (using 1% methane in an air stream) with a coculture of Methylobacterium organophilum and reported up to 57% PHB under nitrogen limitation47 and improved methane consumption by up to 45% in the growth stage. Methylotrophs have improved PHB accumulation when ammonium was the sole N source under potassium limitation.48 A complex nitrogen source can also improve biopolymer yields when using a defined medium.49 Helm et al.50 produced a polymer with an ultrahigh average Mw of 3.1 MDa under potassium-limited conditions using methane-utilizing mixed culture where a Methylocystis sp. was dominant. A maximum specific PHB 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-deficiency experiments. The Mw was lower when sulfur (21% lower) or iron (42% lower) were limiting. Even after decades of considerable effort to commercialize microbial PHB production, the high production cost compared to traditional petrochemical-based plastics (such as polyethylene and polypropylene) still limits commercial application. Choi and Lee51 performed a sensitivity analysis of several factors (productivity, content, and yield, the cost of the carbon substrate, and the recovery method) that affect PHA production with a view to scale-up and found biopolymer content had multiple effects on the process economics. Productivity only affected equipment-related costs, but the yield per cell had multiple effects on process economics. Other research has also placed a firm emphasis on the importance of yield.52 Although substrate costs can account for a major portion (30%) of the production cost,39b,51−53 the costs associated with downstream processing would still render microbial polymer production uncompetitive with the petroleum-based polymers. An initial bridge to commercial production is to target higher-value polymers used in biomedical applications,5b as the biocompatibility and biodegradation of PHA has shown potential for drug delivery,54 medical devices, tissue repair, artificial organ construction, and nutritional/therapeutic uses.55 This field of research has received a promising boost: the FDA has recently approved the use of poly-4-hydroxybutyrate for a clinical application.56 5.1.2b. Biopolymers: Extracellular Polysaccharides. The colloid and adhesive properties of extracellular polysaccharides (EPS) and their effects on liquid rheology are used in the food and nonfood industries such as the pharmaceutical, textile, and oil industries. Traditionally, industrial polysaccharides have 4004


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lipids are mainly in the form of triacylglycerides and can be transesterified to produce biodiesel. The structural polar lipids (phospholipids) and sterols are important components of cell membranes that typically have a high content of polyunsaturated fatty acids (PUFAs) and are essential nutrients for humans.67 In microalgae, one of the most intensively researched oleaginous microbes, the types of lipids produced range from neutral lipids to polar lipids, wax esters, sterols, and hydrocarbons, as well as prenyl derivatives such as tocopherols, carotenoids, terpenes, quinines, and pyrrole derivatives. In methanotrophs, the two major classes of phospholipids are phosphatidyl-glycerol (PG) and phosphatidyl-ethanolamine (PE), which has two derivatives: phosphatidyl methyl ethanolamine (PME) and phosphatidyl dimethyl ethanolamine (PDME). Gamma-proteobacteria (Methylomonas methanica, Methylomonas rubra, and Methylomicrobium album BG8) contain PE and PG phospholipids with predominantly C16:1 fatty acids, while alpha-proteobacteria methanotrophs (Methylosinus trichosporium OB3b and CSC1) contain PG, PME, and PDME with predominantly C18:1 fatty acids.68 Although the C14 to C18, saturated or monounsaturated fatty acids suit diesel production, the sugars, P and S contents are problematic to subsequent catalysts used in the processing. The high heteroatom content (specifically P and N) in the lipid fraction makes downstream extraction and conversion difficult.5b Undesirable components such as sugars, phosphorus, and sulfur exacerbate processing problems due to gumming or catalyst inactivation. This will require additional research and development of a solvent-based extraction process to minimize these negative impacts.5b Fei et al.5b recently reviewed the use of methanotrophs to generate lipids for biodiesel production, thoroughly covering aspects from microbial lipid production to the pitfalls associated with downstream processing. Lipid accumulation exhibits similarities to PHB accumulation, as both may be induced by limiting the oxygen supply, nitrogen or phosphate concentrations to the microbes. Similarly, a two-stage culture process is promoted as the most plausible production method; where the first stage produces healthy biomass and the second stage limits a nutrient source.5b The advantage of using a gas for producing a fuel is significant as it avoids the food vs fuel debate that plagues first generation biofuels. Silverman et al.69 patented the conversion of lipid-containing methanotrophic/methylotrophic biomass into oil that is refined into a fuel, where the oil is derived from the cell membrane of microorganism. Their preferred production method involves supercritical CO2 extraction of the oil, which may be refined by a cracking, transesterification, reforming, distilling, hydroprocessing, and/ or isomerization. While they are focusing on using the structural lipids, ARPA-E funded research at the University of Washington is focused on manipulating methanotrophs to produce storage lipids. Here, methanotrophs will be genetically modified to increase their lipid production and enhance the fraction of nonphosphorus-based lipids, thereby aiding transesterification into biodiesel. 5.1.5b. Lipids: Human Health Supplements. As the lipids produced by natural methanotrophic isolates are predominantly membrane-derived, they are not ideal for catalytic conversion to biodiesel. However, they may have an alternative higher-value application as a health supplement. There is a current patent for using methanotrophic lipids to manufacture an oral administration for use in the treatment of animal subjects to reduce plasma cholesterol levels or lower the ratio of LDL to HDL

consist of a single layer of identical proteins or glycoproteins. Isolated surface layer glycoproteins possess the intrinsic property of self-assembly and recrystallize into isoporous lattices in suspension, onto various surfaces (polymer, silicon, and metal) and interfaces (air−liquid, lipid films, and liposomes). These characteristics and subsequent functionalizing of surfaces have led to new types of ultrafiltration membranes, affinity structures, enzyme membranes, microcarriers, biosensors, diagnostic devices, biocompatible surfaces, and vaccines, as well as targeting, delivery, and encapsulation systems.34,64 As with the de novo synthesis of internal solutes such as ectoine, the osmo-adaptation of methanotrophs also involves structural and functional changes to cell envelopes and changes in the chemical composition of membranes.60 These changes in the surface layers are potentially of industrial interest. Regularly arranged glycoprotein surface layers of hexagonal and linear symmetry have been observed on the outer cell walls of two halo-tolerant Methylobacter spp. Interestingly, the surface layer in a Methylomicrobium sp. (consisting of tightly packed, cupshaped subunits) was negligible when the bacteria were cultured at a neutral pH with no salt in the media,60 indicating the stimulatory effect of osmotic stress. With methanotrophs, it is likely that proteins associated with surface layers facilitate copper ion transport to pMMO and provide an additional mechanism to maintain copper homeostasis in the cells.65 Surface layers have prospective nanotechnology applications in ultrafiltration because they form porous semipermeable membranes; their components undergo self-assembly and in vitro cross-linking at the surface of membranes. The characteristics of isolated surface layers have allowed for various applications in biotechnology, vaccine development, diagnostics, biomimetics, and molecular nanotechnology.34,64b Methanotrophs may be in contention for commercial production if highly desired properties are discovered or engineered for their specific proteins or glycoproteins. 5.1.5a. Lipids: Biodiesel. Biodiesel is another possible methane-generated commodity with a potentially enormous market. It may be used as a transport fuel or an energy fuel and is advantageous compared to ethanol as it can be used without any modifications to engines. Typically, animal or plant fats (triglycerides) are extracted and converted via transesterification into biodiesel. The triglyceride consists of three long chain fatty acids attached to a glycerine molecule. The triglyceride reacts with alcohol (typically methanol or ethanol) in the presence of a catalyst (usually a strong base: NaOH or KOH) to yield esters and glycerol (Figure 2). Oleaginous microbes, including yeasts and various microalgae, have been researched intensively as a biological means to produce diesel due to their ability to accumulate a lipid content greater than 20% of their dry mass. Microbially produced lipids are advantageous to animal- or plant-based lipids as they are produced in a short life cycle, may be less labor-intensive, may be less affected by location, season, and climate, and in certain cases may be easier to scale-up. However, there are difficulties. The scale-up of dense cultures of autotrophic microalgae is difficult due to light penetration,66 difficulties obtaining a high lipid content with dense microbial,67 and the high degree of processing associated with lipid extraction and transformation.5b Essentially, lipids from microorganisms may be divided into two types: those associated with storage (nonpolar lipids) and those that form structures (polar lipids). The nonpolar storage 4005


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Figure 1. Methanotrophic methane mineralization or assimilation (via the Serine or RUMP pathways). Adapted from Hanson and Hanson.6b

through selective inhibition of methanol dehydrogenase by using relatively common media components such as phosphate, NaCl, NH4Cl, or EDTA. Alternatively, researchers used a thermophilic methanotroph (Methylocaludum) in a process where methanol was constantly removed from the fermentation. Methanol remained in the gas phase at 50 °C and was successfully condensed from the reactor headspace.76 Careful selection of the production strain is also vital for methanol production as production rates and yields from different isolates vary by orders of magnitude. Mehta et al.77 reported specific production of 1 mmol·h−1·mg−1 dry mass under continuous conditions, while Xin et al.78 reported methanol production 3 orders of magnitude lower. The low productivity was attributed to the MMO specific activity of their Methylosinus trichosporium strain, which was approximately 1% of the strain used by used by Mehta et al.77 Low methanol yields are still problematic. Although earlier research had claimed methanol production up to 1 g/L,79 very low yields (≤mg/L) are generally reported for methanol (and other soluble metabolites). Recently, 1 g/L was reported using a high-density culture,80 but the process required a culture concentrated via centrifugation in 0.4 M phosphate buffer and 20 mM formic acid. This process would be difficult to scale up, and, even then, the methanol concentration would have to be improved by up to 2 orders of magnitude. Formaldehyde and formic acid represent alternative products formed as intermediates during the mineralization process, while acetate, lactate, and succinate are produced under oxygenlimited conditions.20 Current yields according to the literature are very low (mM). While a product such as formaldehyde has a large global demand, it is toxic to cells as it cross-links proteins (it is often used as a fixing agent because of this quality). This quality may render a cellular and even a purely enzymatic system inactive. One potential alternative is to use small molecule catalysts that mimic the methanol dehydrogenase active site to convert methanol to formaldehyde. It is worth noting that there are a number of companies actively pursuing these compounds. Various companies and research institutions (e.g., Kiverdi, Coskata, CALYSTA Energy, NatureWorks, National Renewable Energy Laboratory, Intrexon) are trying to commercialize biological production of lactate, succinate, muconic acid, butanol, and propanol. It will be of interest to see what yields and production efficiencies can be obtained by these researchers, and how significantly production can be improved by bioreactor engineering, genetic engineering, and the inclusion of synthetic biology approaches. 5.1.8. Methanotroph Enzymes, Cell-Free Catalysis, and Electrochemistry. Enzymes produced by methylotrophs are themselves a valuable product. Some dehydrogenases and other enzymes (including glucose-6-phosphate dehydrogenase, gluta-

cholesterol in the plasma. The composition is apparently also useful for increasing docosahexaenoic acid concentration in the plasma, which acts as an immunoprotectant.70 The patent is based on research of the effects of three different high-lipid diets on plasma lipoproteins and phospholipids in mink (Mustela vison). Phospholipids from natural gas-utilizing bacteria in the diet decreased plasma lipoprotein levels, the LDL/HDL cholesterol ratio, and plasma phospholipid levels compared with the highly unsaturated soybean oil. The decrease of plasma cholesterol was attributed to a specific mixture of phospholipids containing a high level of phosphatidyl-ethanolamine and not the dietary fatty acid composition.71 These phospholipids could comprise part of a formulation taken as a health supplement or potentially used as part of complementary treatment program. 5.1.6. Growth Media and Vitamin B12. Analogous to yeast, beef, or potato dextrose extracts, soluble compounds within the methanotrophic biomass may contribute to the bulk of an extract that can provide nutrients for a growth media. There is a patent for a microbial growth medium derived from a microbial culture composed primarily of Methylococcus capsulatus (Bath) and containing Ralstonia sp. DB3 and Brevibacillus agri DB5 and optionally Aneurinibacillus sp. DB4.72 The nutrient medium is proposed to consist of a hydrolysate, homogenate, or an autolysate of the biomass, with an autolysate being preferred. The addition of further compounds and nutrients such as glucose and/or nitrate and minerals (e.g., K, Ca, Mg, Na, Mb, Fe, Zn, B, Co, Mn, and Ni salts) further enhances the scope of this growth medium. Methanotrophs and methylotrophs utilizing the serine or RUMP pathways are able to produce vitamin B12 (up to 800 ng/g wet mass using a Methylobacterium species), an essential vitamin to many organisms.73 5.1.7. Soluble Metabolic Products: Methanol, Formaldehyde, and Organic Acids. Soluble metabolic intermediates such as methanol, formaldehyde, and organic acids are all potential products from methanotrophs with multiple industrial uses and are required in large quantities annually. Viably converting methane to methanol at ambient temperature and pressure is of great interest as methanol is a more easily transportable than methane. Methanol is the first intermediate formed during the conversion of methane to carbon dioxide. In the metabolic pathway (Figure 1) of native bacteria, methanol is rapidly converted to formaldehyde, implying that genetic manipulation or fermentation process control would be vital to ensure accumulation. Tabata and Okura74 initially failed to produce extracellular methanol from methanotrophs because it was rapidly oxidized internally by methanol dehydrogenase. Extracellular methanol was detected when they selectively inhibited methanol dehydrogenase using cyclopropanol. Han et al.75 also achieved partial oxidation of methane into methanol 4006


Critical Review

Environmental Science & Technology mate dehydrogenase, malate dehydrogenase, alcohol oxidase, and catalase) exhibit high activities.34 Enzymes such as diamino-butyrate acetyl-transferase from a halo-tolerant methanotroph have been cloned and expressed in heterologous expression systems; this enzyme catalyzes one of the key reactions of biosynthesis of the bacterial osmo-protectant ectoine.81 Methane may be enzymatically converted to methanol, while the complete enzymatic oxidation of methanol to CO2 has been demonstrated using the various dehydrogenases.82 The conversion of methane to methanol is of great industrial interest. Unfortunately, both the particulate and soluble MMO require reducing equivalents, which has limited the potential for a commercial methanol production from these enzymes. The sMMO requires NAD(P)H and O2 catalyzes to convert methane to methanol: CH4 + NAD(P)H + H+ + O2 = CH3OH + NAD(P)+ + H2O, while the pMMO requires cytochromes b559/569 or c553 artificial reductants such as duroquinol and NADH83 to complete the reaction. The cost of providing external reducing equivalents for the MMOs renders the economics of the reaction unfeasible for producing a lowvalue commodity such as methanol. Normally, within the cell the NAD(P)H would be regenerated in a subsequent enzymatic oxidation step. However, developments in NAD(P)H regeneration may be overcome using electrochemical techniques, mediators, or combinations with NAD(P) reducing and NAD(P)H-oxidizing enzymes. Besides the cost associated with the enzyme cofactor/ reducing equivalent, it is also difficult to heterologously produce MMOs.84 MMO genes have been cloned into bacterial expression systems (which could enable faster and greater enzyme production), because of the relatively slow growth rate of methanotrophs. However, MMOs are complex proteins (consisting of a reductase, hydroxylase, and regulatory protein), and a heterologous expression system has proved elusive. Complete, active MMOs have not been produced thus far, only a partially active sMMO with a functional hydroxylase.22 Active pMMO 85 or sMMO 86 can be obtained directly from methanotrophic cultures. Although the pMMO is commonly inactive when stringently purified from native bacteria (as it is removed from its lipid matrix and the closely associated proteins and cofactors), active MMO can be purified from methanotrophic biomass.87 The other three key enzymes in the mineralization of methanol to CO2 (methanol dehydrogenase, formaldehyde dehydrogenase, and formate dehydrogenase) may be used to generate methanol, formaldehyde, and formic acid. There is strong interest in enzyme-catalyzed redox reactions to produce electricity, fuels as well as chemical commodities from immobilized enzymes on electrodes.82,88 Recently, research has not only focused on forward reaction of CH4 conversion to methanol but also on the reverse process where CO2 is converted to formic acid, formaldehyde, and then methanol. Formate dehydrogenase alone catalyzes a very useful reaction: converting CO2 into formate. This was achieved by Srikanth et al.,89 in an electrochemical system where they also demonstrated the ability to electrochemically regenerate the cofactor (NAD+). The three other key enzymes that are involved in the mineralization of methanol to CO2 have recently been demonstrated capable of the complete reverse enzymatic mineralization of CH4 displayed in Figure 2.90 Carbon dioxide was electrocatalytically converted to formate, formaldehyde, and then methanol at the cathode. Reactive red was used as an

Figure 2. Typical reaction scheme for the transesterification of a triglyceride into esters and glycerine.

electron mediator, and the cofactor (NAD+) was regenerated via cathodic reduction. They further enhanced the process by including carbonic anhydrase, an enzyme that facilitates the hydration of CO2(g). Enzyme-electrochemical techniques are of interest as the ability to transform methane into useful chemicals without requiring a live culture are highly beneficial, but there are technical, cost, and process hurdles to overcome. 5.1.9. Propylene Oxide Production (Whole Cell Catalysis). The broad substrate range of MMOs allows these enzymes to degrade soil contaminants as well as generate products, such as 1- and 2-alcohols from C1−C8 n-alkanes, 1,2-epoxides from terminal alkenes and ethanol/ethanal from diethyl ether.91 Alkanes are hydroxylated mostly at the terminal and subterminal positions, while ring hydroxylation of aromatics occurs primarily at the meta position. The sMMO oxygenates alkenes to epoxides with retention of stereochemistry around the CC double bond.92 The range for sMMO (n-alkanes, nalkenes, aromatic, and alicyclic compounds) is significantly greater than that of pMMO (n-alkanes and n-alkenes). The broad substrate range of MMOs nearly enabled the commercialization of propylene oxide production in the 1990s. Dalton and colleagues studied the transformation of propylene (propene/methyl-ethylene) to propylene oxide (epoxy-propylene/epoxy-propane) using a methanotroph.31,93 Initial laboratory studies to establish the epoxidation rates of propylene to propylene oxide by Methylococcus capsulatus (Bath) were optimized in shake-flask cultures, where adding electron donors such as methanol, formaldehyde, formate, or hydrogen stimulated the endogenous rate of propylene oxide formation up to 50 times. Specific production rates as high as 500 mol·min−1·g−1 of cells (dry mass) were obtained with methanol as the electron donor but were only sustained for short periods of up to 4 min.94 The loss of the MMO activity and subsequent declining epoxidation rates were studied further95 and subsequently demonstrated as a result of reversible product inhibition.96 Reactivation of the bacterial propylene oxidation mechanism could occur without growth, but the process required the presence of an energy source (methane or methanol), sulfur, nitrogen, and oxygen. In the presence of growth substrates, cells could be reactivated after the removal of propylene oxide. De novo protein synthesis was also required for reactivation of activity, and cultures possessing sMMO took twice as long to recover compared to cells containing pMMO. In the pilot process, methanol was used as the carbon source, and MMO inhibition was circumvented by operating a two-stage process system that allowed the epoxideinhibited culture to recover in a separate bioreactor in the presence of methane and other nutrients. Conducting the biotransformation at the optimal growth temperature for the methanotroph (45 °C) was doubly advantageous, because the 4007


Critical Review

Environmental Science & Technology boiling point of propylene oxide is 34 °C: allowing simpler product recovery from the gas phase. The process was run at a high cell density (30 g·L−1), and propylene oxide was produced at 250 g·L−1·day−1. The authors not only demonstrated continuous pilot-scale production of propylene oxide from propylene using a methanotroph but also evaluated the process for producing 1,2-epoxybutane from but-1-ene and acetaldehyde from ethane. The process could unfortunately not be commercialized, as the production cost was already at parity to established commercial chemical technology at the time before inclusions for transport, storage, and profit.97 More information about propylene oxide production is available in other review articles.31,34 Xin et al.98 recently used a Methylomonas sp. to catalyze the epoxidation in a continuous fashion with methane as the electron-donating cosubstrate. They circulated an optimized gas mixture (methane: 35%; propene: 20%; oxygen: 45%) continuously, which removed the product. In this manner they were able to operate a bioreactor continuously for 25 days without any obvious loss of propylene oxide productivity. More recently, Su et al.99 optimized conditions for propylene oxide production using high cell density cultures of Methylosinus trichosporim OB3b and claimed a propylene oxide productivity nearly 4-fold greater than the highest reported productivity by optimizing temperature, initial propene concentration, sodium formate, and MgCl2 concentrations. The combination of greater productivities and the consolidated bioprocessing approach (where the product is generated and removed from a single reactor maintained at a temperature above the products boiling point) is worth investigating again. However, there are new challenges as the traditional methods for propylene oxide production have advanced since the 1990s. The older chlorohydrin process was gradually replaced by the oxidation of isobutane and its catalytic conversion to propylene oxide, which in turn has been superseded by ethylbenzene oxidation, which also yields styrene as a coproduct.100 Until recently, commercial propene peroxidation was unfeasible due to the cost of H2O2. BASF and DOW chemicals have commercialized a process that converts O2 into H2O2 (continuously via the oxidation and reduction of 2-ethylanthrahydroquinone), which in conjunction with propene and a catalyst (titanium silicalite-1) produces propylene oxide with no byproducts other than water.100,101 This is an elegant process that would be challenging to improve upon. Enzymatic MMO catalysis (as opposed to live cell catalysis) could be worth pursuing but would require sufficient MMO, a stable enzyme process, and cheap regeneration of the reducing equivalents. 5.1.10. New Products and Improved Efficiency of Genetically Engineered Methanotrophs. There has been considerable research over the past three decades regarding the genetics of methanotrophs.102 Various methanotrophs have been sequenced,6a,103 and this has allowed the genetic engineering of these methanotrophs to overproduce metabolites or even compounds not naturally synthesized by these bacteria. The proof-of-concept for metabolic engineering of methanotrophs to heterologously synthesize compounds has been validated. Sharpe104 and colleagues genetically modified (GM) methanotrophs to produce high-value carotenoids, while using methane or methanol as a carbon source. Carotenoids are a family of yellow to orange-red terpenoid pigments that protect against oxidative damage and are desired by food, medical, and cosmetic industries.105 The research group initially

expressed a canthaxanthin gene cluster and associated enzymes to convert it to astaxanthin in a Methylomonas species.106 Various C40 carotenoids were accumulated in the intracytoplasmic membrane system in high concentrations.104 Although astaxanthin is widely used as a feed supplement in poultry and aquaculture industries, it is a challenge to produce in bacteria as astaxanthin generally forms a small percentage of the total carotenoids. A yield of 1 to 2.4 mg·g−1 dry mass was obtained, where 90% of the total carotenoid was astaxanthin (primarily the E-isomer) by engineering a methanotroph to contain two complete sets of carotenoid biosynthetic genes, proving that astaxanthin with desirable properties could be produced in methanotrophs through genetic engineering.106 As astaxanthin production was strongly affected by oxygen availability, bacterial hemoglobins were incorporated into the bacteria by further genetic engineering. Coexpression of the hemoglobin and astaxanthin-encoding genes significantly increased astaxanthin expression - as the hemoglobins likely improved the activity of the oxygen-requiring enzymes. A plasmid-free production Methylomonas strain produced more astaxanthin than the parent strain.107 This research demonstrated the ability to engineer nontraditional microbial hosts that could use methane or methanol as alternative feedstocks for microbial processes, as well as improve production by metabolic engineering.104,106−108 Recently, Intrexon Corporation announced that they had genetically modified a methanotroph to produce farnesene using methane as the carbon source.109 Farnesene represents an enormous global commodity as it is a basic precursor for diesel, lubricants, and specialty products (cosmetics, rubber, and plastics). While this serves as another example of successful genetic engineering, there was no data substantiating any appreciable yield. Moving from initial laboratory proof-ofconcept (where specific production may be as below μM·g−1) to pilot and commercial scale may require production to be increased by several orders of magnitude, in addition to overcoming numerous biological and engineering hurdles. Methane-oxidizing bacteria have served as hosts for producing recombinant and heterologous proteins, including β-glucuronidase and genetically engineered MMOs.27c There are now a number of genetic tools allowing mutagenesis and expression studies with methanotrophs, and these techniques allow introduction of broad-host-range plasmids carrying homologous and heterologous genes into methanotrophs, promoter probe fusions, transposon mutagenesis, and mutagenesis by marker-exchange.27b There have been substantial advances in genetic engineering and modification of the capabilities of methanotrophs since the 1980s,110 and this will continue to be a challenging but rewarding field. Synthetic biology may also contribute greatly to improving the production efficiencies and yields.5a 5.2. Processes. 5.2.1. Methane Mitigation. The remediative abilities of methanotrophs are documented, and their potential for mitigating methane emissions from landfills and coal mines and their ability to degrade other hazardous organic compounds have been reviewed.6b,14b,c,27a,31 Methanotrophic methane mitigation technologies have been demonstrated beyond the laboratory scale as adaptable field-scale systems that may be engineered to meet site-specific climatic variations and ensure minimal atmospheric methane emission,111 where methane oxidation efficiencies as high as 100% have been reported.18,112 Dever et al.113 conducted a field scale trial at a landfill site (Sydney, Australia) investigating passive drainage 4008


Critical Review


1970s, it was hypothesized that the responsible agent in the mixed methanotrophic culture was a denitrifying methanolconsuming bacteria that was using a methanotrophic byproduct to perform the initial reduction of nitrate to nitrite. The premise was one of syntrophy, where one organism lives off the products of another organism, and this was later verified.128 Methane is used by the methanotrophs, and they in turn provide an electron donor (such as acetate) for the denitrifying bacteria. As cheaper wastewater denitrification alternatives are being investigated, there has been a recent increase in research published regarding methane oxidation coupled to denitrification.129 Various consortia of microorganisms are capable of using methane as the sole carbon source for denitrification both aerobically33,128,130 and anaerobically.33,129,131 5.2.4. Electricity Generation in Microbial Fuel Cells. In a microbial fuel cell (MFC) micro-organisms are attached to electrodes that harvest the electrical current produced from the spontaneous oxidation of organic substrates.132 Theoretically, methanotrophs could be employed in a MFC using methane as the carbon source, thereby coupling biological methane oxidation to electricity generation. The concept of using methane as a carbon source in a microbial fuel cell was patented by Girguis and Reimers,133 but, to date, no electrogenic activity has been demonstrated by a methanotroph. However, it is not essential that they are electrogenic as they may serve as an intermediary at the surface of a biofilm and provide organic metabolites to sustain electrogenic bacteria in contact with the anode. The potential methanotrophic metabolites that have been used as sole carbon sources in MFCs include methanol,134 formic acid,135 or fermentation products produced under oxygen-limited conditions20 such as acetate136 or lactate.137 However, there are significant problems associated with using oxygen in the anodic chamber and the materials required to construct MFCs are expensive (considering the poor power densities achieved) making industrial application unlikely. 5.2.5. Biosensors. Biosensors are analytical devices that use a transducer coupled to biological material that elicits a signal in response to an analyte. They can be extremely sensitive as well as highly specific but may suffer from instability138 or be affected by the external environment. The ability of MMO to react with methane allows the bacteria containing the enzyme, or the isolated enzyme, to be used as the biological component of a biosensor. A Methylomonas culture was exploited early in the development of environmental biosensors by Okada et al.,139 where methane concentrations were determined in 3 min at 30 °C at pH of 7.2. It allowed for a minimum methane measurement of 13 μM and was reproducible to within 5% for more than 20 days over which more than 500 assays were performed. The bacteria were also incorporated into sensors developed by Daamgard and colleagues140 where oxygen consumption was measured using an internal oxygen amperometric microsensor, acting as a proxy for methane presence. Wen et al.141 developed a similar biosensor using a mixed culture of methane-oxidizing bacteria. With both biosensors the sensitivity and the response time were improved by increasing the number of bacteria. Unfortunately, both biosensors were restricted by environmental factors (e.g., pH and temperature) that affected the physiological state of bacteria.142 The use of MMO enzymes coupled to an electrode was also investigated, but this was unstable and produced unusual results−presumably due to methane and oxygen retention.143

and biofiltration of landfill gas as a means of managing landfill gas emissions from low to moderate gas generation landfill sites. Passively aerated biofilters operating in a temperate climate achieved maximum methane oxidation efficiencies greater than 90% and average oxidation efficiencies greater than 50% over four years of operation. Although temperature and moisture within the biofilter were affected by local climatic conditions, their effect on biofilter performance was overshadowed by landfill gas loading. Interestingly, microbial methane oxidation was limited by outflowing biogas as it prevented diffusion of atmospheric oxygen into the biofilter. Methanotrophic systems have also been combined with algae, thereby sequestering both methane and CO2 and has the potential to generate additional biological products.114 5.2.2. Contaminant Bioremediation. Methanotrophs are useful bioremediation agents because of the broad substrate range of their MMO enzymes (in particular sMMO), which allows them to remove heavy metals115 and transform organic pollutants.116 The sMMO enzymes can transform a variety of hydrocarbons, including alkanes, alkenes, alicyclic hydrocarbons, aromatic compounds, and halogenated aliphatics. 31,91,117 Chlorinated compounds degradable by sMMOs include chloroform,118 dichloroethene,119 trichloroethylene,118,120 tetrachloroethene,121 hydrochlorofluorocarbons,122 and even vinyl chloride.123 Methane or nutrients may be added to stimulate methanotrophs and enhance biodegradation and biotransformation of contaminants. Biostimulation of methanotrophs according to the site-specific needs has even been demonstrated at a field scale in situ within contaminated aquifers and soils and ex situ in bioreactors.14b,25c,124 Their remediative capacities have also been improved by genetic modification.125 Plant-microbe associations are important relationships benefiting both partners. Enhanced methanotroph-plant associations may be worth pursuing to create a more stable spread of methanotrophs in a soil environment in a symbiotic relationship with plant roots. Even if the methanotrophs do not greatly benefit the host (as is normally the case with endophytes that provide nutrients or secrete plant growth promoting factors), as long as they are actively present it is environmentally beneficial. Alternatively, transgenic plants can also mobilize or degrade chlorinated solvent, xenobiotic compounds, explosives, and phenolic substances. A symbiotic relationship between GM methanotrophs and transgenic plants could significantly enhance the bioremediation of contaminated sites.125d 5.2.3. Denitrification. Biological wastewater denitrification systems require organic carbon to facilitate the reduction of nitrate to nitrogen. This carbon requirement may be partially met by the acidogenic fermentation of a portion of the organic waste entering wastewater treatment systems, which provides volatile fatty acids such as acetate. However, modern wastewater treatment plants frequently need to supplement their systems with a costly external carbon source such as ethanol to achieve more stringent discharge limits.126 Using methane as a low-cost carbon source to facilitate denitrification would be highly beneficial. Incorporating methane into the denitrification process was suggested by various researchers in the 1970s,127 but denitrification by pure methanotrophic isolates had still not been validated four decades later.128 Although methanotrophs can catalyze nitrogen cycle processes such as nitrification and nitrogen fixation, they cannot perform complete denitrification.128 Even as early as the 4009


4010

none - low

low

medium low

denitrification

biosensors electricity generation

medium high

high

high

high

none

bioremediation

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

>1.4 billion L 9−10 million tonnes

Achieved in laboratories. Technical requirements possibly limits this to niche WWTPs or academia. No. Technical constraints. No. Unproven. Improbable.

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.

Potentially already on the market as heterologously produced enzymes. Unknown. Boosts nutrition value.

Unknown.

No. Product inhibition as negates gas diffusion. Rates and yields too low compared to other producers.

No. Production rates may be limiting.

Unknown. Possible to generate as 20% of the biomass. Production rates may be limiting. Yields low according to literature. Improbable using natural isolates. Potentially possible via engineered organisms.

Not currently feasible due to heteroatoms. R&D needed. Potential via genetic manipulation. Possibly. Promising option.

kg - tonnes >1 tonne

enzyme transformations GMO products: carotenoids GMO products: farnesene biotransformation of propene to propylene oxide Process methane mitigation

10 tonnes

unknown

kg - tonnes

1.4 billion L

Processing cost constraints. May be viable when petroleum products increase sufficiently in price. Potential for specialized polymers or via lactic acid production.

Yes, under defined natural gas costs and product retail price. Commercial plant in Trinidad and Tobago.

near to market

low - high low medium low medium

medium

growth media and Vit B12

low low

extracellular polysaccharides (EPS)

very high low

ectoine soluble metabolic products: methanol formaldehyde organic acids

medium high low medium

35 billion L 9600 tonnes (as acetic acid only)

high

lipids: dietary supplements

surface layers

1−10 tonnes 90 billion L

low

lipids: biodiesel

equivalent to 0.6 billion L of oil used for making plastics

low

internal storage polymers (PHB)

>25 000 tonnes

global annual demand

low

relative value

single cell protein

product

Table 2. Potential Products and Processes Using Methane as the Primary Carbon Source for Methanotrophs

no data SP: 8.3 g·g−1·day−192

T: 4.96 g·L−1153 SP: 0.850 g·L−1·day−1153 T: 800 ng Vit B12·g−1 wet biomass73 T: 150 μg cobalamin·g−1 dry biomass155 no data T: 2.4 mg astaxanthin·g−1106

no data T: formate 90 mg·g−1 20 T: acetate 30 mg·g−120 T: succinate 0.8 mg·g−120 T: lactate 0.9 mg·g−120 no data

Y: 0.02 SP: 0.7 g·L ·day T: 0.7 g·g−1 cell41a Y: 0.54 g·g−1 CH414c,150 SP: 21 g·L−1·day−114c,45 Y: 0.259 g·g−1 CH45b (theoretical) Y: 0.259 g·g−1 CH45b (theoretical) T: 0.21 g·g−1 cell151 T: 1 g·L−179,80

g·g CH4148 −1 −1148

−1

highest titer (T), yield (Y), or specific productivity (SP)

139−143, 157 133−135

6b, 14b, c, 18, 27a, 31, 111, 112 14b, 25c, 31, 91, 115−117, 124, 125 33, 127−131

22, 34, 81−90 6a, 27b, c, 104−108, 110, 156 109 31, 34, 91−93, 98, 99

72,73

57−59, 154

34, 60

34, 20, 74−79,

70, 71

5b, 66, 67

5b, 30, 32, 39−46, 48, 148

35, 37, 38, 149, 36

relevant literature

Environmental Science & Technology Critical Review


Critical Review


6. OVERVIEW OF POTENTIAL PRODUCTS The potential products, their reported yields, and production efficiencies are summarized in Table 2, along with their potential global demand, potential nearness to market, and relevant references. The increasing demand for protein-rich feed by the expanding global aquaculture industry bodes well for singlecell protein production. Further improvements to strains via genetic modification (to generate carotenoids or improved vitamin B12 production) could enhance the nutritional value of the product. Although SCP production appears feasible (as evidenced by the recent construction of a new facility in Trinidad and Tobago), government initiatives/incentives may be necessary to facilitate private investment and further increase this application. Propylene oxide and ectoine production are particularly interesting as they represent extracellular products. Unlike most cellular products, this enables cell reuse, which is useful for relatively slow growing biomass. Once the optimal biomass concentration is attained, the key constraints are the rates of production and tolerance toward the product inhibition. The commercial value of ectoine makes it a compound worth pursuing via improved downstream processing, new production strains, and genetic engineering to improve yields and yield efficiencies. Propylene oxide has an enormous market, and production could be improved via improved enzyme efficiencies and product tolerance or enhanced simultaneous product removal using either new thermotolerant species or improved production strains engineered to exhibit thermophilic traits. With the near-commercialization of propylene oxide production by methanotrophs in the 1990s, it is worth revisiting this process, although new production processes will be difficult to compete against. Biopolymer production (polyhydroxy-alkanoates and extracellular polysaccharides) and external surface layers may be limited by the production rate of the relatively slow-growing methanotrophs. It can take days to weeks as opposed to hours to days when using conventional bacteria. Although the price of petroleum precursors will have to increase before biopolymer production becomes viable, a potential bridge toward economic feasibility may be focusing on producing a consistent, highquality PHB for medical applications. However, for PHAs and EPS, the low solubility of methane is a great challenge, especially with high-density cultures. Biodiesel also represents an enormous market, but polar membrane lipids with a high heteroatom content from methanotrophs may not be the best microbial alternative. If attempts to genetically enhance the methanotrophs to improve lipid yields and produce desired storage lipids (triacyglycerols) are successful, it will be an important step toward commercializing biofuels from these bacteria, as the quality of the lipid intermediates has been regarded as playing a major role in the overall fuel production cost and will have a strong impact on the catalytic upgrading steps.5b However, considering the lipid composition in native bacteria, use as a health supplement for lowering cholesterol currently seems most appropriate. Products that currently appear unfeasible to produce include farnesene and soluble metabolites such as methanol, formaldehyde, and organic acids. There is no data available that indicates that farnesene is produced in any appreciable quantities in methanotrophs. Soluble metabolites such as

methanol, formaldehyde, and organic acids are produced in low concentrations according to the literature. Even methanol titers of 1 g/L will not be commercially viable considering rates and processing conditions; it also negatively impacts culture maintenance as reducing equivalents are not regenerated. However, methanol is an important global commodity, and the potential to generate it under ambient conditions using cells modified via traditional genetic manipulation or synthetic biology, or via enzymatic catalysis, is still a prominent topic. Although some methanotroph enzymes possess high catalytic activities, it is unlikely that the native methanotroph will be used for production. Cloning the enzymes’ genes into common expression systems that use conventional carbon sources is more feasible, but this would use a traditional carbon source rather than methane. It is not inconceivable that enzymes could be harvested from spent methanotrophic biomass though, thereby adding value and potentially improving the economic viability of another process. Incorporating methanotrophs into biosensors and electricity generation via microbial fuel cells seem unlikely options. The biosensors have a limited functional pH range and require oxygen and reducing equivalents, in addition to general biosensor problems such as background noise, interference, and inhibition. Although patented, electricity generation in microbial fuel cells with methane as the sole carbon source has not actually been demonstrated. Electricity generation seems improbable as the presence of oxygen in the anodic chamber will negate electron transfer. Regarding processes, partial denitrification aided by methanotrophs could be worth incorporating into wastewater treatment plants where possible, as this could lessen the cost associated with providing an external carbon source for denitrification. Other processes, such as mitigating methane releases into the atmosphere and bioremediation, have been demonstrated at a field scale11,93 and are technically possible but will only be applied when there is a sufficient financial incentive to limit carbon emissions or remove contaminants. GMOs with improved traits (e.g., tolerance and degradation efficiency) could further enhance these applications, but the perceived risks related to genetic transfer requires extensive assessment before a GMO may be used in the environment.116,125a,144

7. METHANOTROPH APPLICATIONS: SYNTHETIC BIOLOGY, SCALE-UP, AND COMMERCIALIZATION The future of all of the bioprocesses mentioned in this review must yield a profit, or be of greater value when benchmarked against conventional methane applications such as energy recovery or heating, and must be achievable at scale. To ensure a consistent and optimal product or robust process, fermentation and downstream processing must be optimized, integrated, and functional at scale. The greatest technical challenge for gas fermentation at scale remains the efficient mass transfer of poorly soluble gaseous substrates into the aqueous phase.145 Other major concerns are the need to regenerate reducing equivalents and the cost of downstream processing. Many reactor types (continuous stirred tank reactors, bubble columns, airlift reactors, trickle beds, and numerous variations) have been applied to gas fermentation,145 and microbubble generation145b or immobilized hollow fiber membranes146 have been investigated to improve gas transfer efficiency. Increasing the headspace pressure is another means of improving mass transfer. Paraffin and nanoparticle addition 4011



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can improve gas transfer but at an increased cost. Cathodic reduction (directly to the cells or an immobilized MMO complex) may provide the electrons required or regenerate the reducing equivalents, but feasibility depends on the cost of the reduction and whether it can be scaled up in an economically viable manner. Another significant problem at scale with gas fermentation is foaming (especially with high density cultures) and may be compounded by operating secondary reactors to the extreme of the microbe’s capacity to maintain structural integrity or survive exposure to high product or metabolite concentrations. This may be further aggravated by microaeration, where the small gas bubbles that are required to improve gas−liquid transfer may be more easily stabilized by bacterial lysis products or proteins and glycoproteins. However, downstream processing costs may single-handedly prevent the commercialization of these products. Generally, if the product is intracellular, cells must be concentrated and lysed, and then the product must be extracted, purified, concentrated, and processed. There are considerable costs associated with cell pretreatment,147 solvent recovery, impurity removal, increases in viscosity, subsequent requirements for product upgrading or modification, waste treatment, and environmental consequences42,51 as well as technical issues dealing with particular solvents and increases in liquid viscosity. The capabilities and yield efficiencies of production strains may be enhanced with cloned genes or potentially even with completely synthetic additions in the future. There is hope of potentially doubling the energy efficiency of MMOs by engineering a dioxygenase-like enzyme to activate methane, that would allow two methane molecules to be activated for the same energy input.5a Genetic engineering and synthetic biology have a large role in the future application of methanotrophs and their enzymes, as indicated by the strong emphasis on these techniques in many of the ARPA-E funded projects. Some projects involve completely synthetic enzymes for methane activation (Arzeda Corp.), while others will re-engineer enzymes for methylation (Northwestern University, Lawrence Berkeley National Laboratory), pathways (University of California Davis), or methanotrophs (University of California Los Angeles) or even use phototrophic organisms (MOgene Green Chemicals LLC) to produce a liquid fuel such as butanol, methanol, ethanol, or dimerize methane. Enzyme use is also being combined with chemical approaches for cell-free catalysis (GreenLight Biosciences), and new metabolic pathways are also being engineered into methylotrophs to convert methanol into butanol (University of Delaware). Synthetic biology may offer alternative routes for regenerating reducing equivalents, using advanced enzymes or new metabolic pathways, or improving yields and conversion efficiencies; but these are complex, time-consuming endeavors, and great patience and perseverance will be required. There are still serious challenges to commercializing methanotroph applications, but the recent funding allocation and intensive research committed to biologically generating transport fuels from methanotrophs demonstrates the potential envisaged for these unique bacteria. Although they have been known and researched for just over a century, their ability to use methane under ambient conditions, coupled with the abilities of specialist thermophilic, halophilic, and acidophilic methanotrophs, as well as the potential to further improve their capabilities via genetic enhancement and synthetic biology, translates to decades of intriguing research ahead.

Critical Review

AUTHOR INFORMATION

Corresponding Author

*Phone: 61 459652099. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The Centre for Solid Waste Bioprocessing gratefully acknowledges funding from Remondis. W.P.C. and S.X. acknowledge funding from the Australian Research Commission (DP 140104572). The authors acknowledge input from S. Freguia, T. Stewart, M. Patel, J. Bors and I. Pikaar.

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