Problem

Greenhouse Effect

Solar radiation powers the Earth’s climate system. Considering that the Earth’s temperature has been nearly constant for many centuries, the incoming solar energy must be nearly in balance with the outgoing radiation.
Nearly half of the incoming short-wave solar radiation is absorbed by the Earth’s surface. The amount of short-wave radiation reflected back to space by gases and aerosols, clouds and the Earth’s surface is around 30 %, the rest of it is absorbed in the atmosphere. The long-wave radiation emitted from the Earth’s surface is to a great extent absorbed by certain atmospheric constituents such as water vapour, carbon dioxide (CO₂), methane (CH₄), nitrous oxide (N₂O) and other greenhouse gases. The downward directed part of this longwave radiation adds heat to the lower layers of the atmosphere and to the Earth’s surface, which is known as the greenhouse effect.

As a natural process, the greenhouse effect makes for an average temperature of 15°C on Earth. Without the gases in the atmosphere, the average temperature would be about -18°C. Humans increase the greenhouse effect directly by emitting greenhouse gases such as CO₂, CH₄, N₂O and chlorofluorocarbons. Furthermore, pollutants such as carbon monoxide (CO), volatile organic compounds (VOC), nitrogen oxides (NO_x) and sulphur dioxide (SO₂), which by themselves are insignificant greenhouse gases, have an indirect effect on the greenhouse effect: They alter the abundance of important greenhouse gases such as CH₄ and ozone (O₃) through chemical reactions or by acting as precursors of secondary aerosols.

The enhancement of the natural greenhouse effect results in significant warming of the Earth’s surface and therefore leads to a change of climate. There are many indicators of climate change. These include physical responses such as changes in, for example, the surface temperature, atmospheric water vapour, precipitation, glaciers, ocean and land ice, and sea level.

References

1. Cubasch U, Wuebbles D, Chen D, Facchini MC, Frame D, Mahowald N, Winther JG (2013) Introduction. In: Stocker TF, Qin D, Plattner GK, Tignor M, Allen SK, Boschung J, Nauels A, Xia Y, Bex V, Midgley PM, eds. (2013) Climate Change 2013: The Physical Science Basis. Contribution of Working Group I to the Fifth Assessment Report of the Intergovernmental Panel on Climate Change. Cambridge, New York: Cambridge University Press

That's Why We Target Methane

Gases absorbing and emitting radiation within the thermal infrared range in the atmosphere are called greenhouse gases. They are responsible for the greenhouse effect. The main greenhouse gases are carbon dioxide, methane, nitrous oxides and fluorinated gases or, in short, F-gases.
The relative contribution of each of these greenhouse gases to climate change depends roughly on three main factors:

1. The amount of a respective gas in the atmosphere.
2. The duration of stay in the atmosphere.
3. The intensity of their impact on the global temperature.

For each of these gases a Global Warming Potential (GWP) has been calculated which is a measure of the total energy that a gas absorbs over a particular period of time compared to carbon dioxide. Besides, it is also based on the degradation rate of the respective gas. Thus, the GWP describes the ability of a gas to trap heat in the atmosphere. The larger the GWP the bigger the effect of the particular gas on global warming.
As shown in the figure on the right, the GWPs (100 years) of the primary greenhouse gases differ significantly. Since the GWP is calculated in relation to carbon dioxide, the GWP of carbon dioxide is defined as 1. The 100 year GWP for methane is 25 implying that methane will trap 25-times more heat than carbon dioxide over the next 100 years.

Carbon dioxide is the main greenhouse gas emitted by human activities. As part of the Earth’s carbon cycle, carbon dioxide is also naturally present in the atmosphere: It is constantly exchanged between the atmosphere, ocean and land surface as it is produced and absorbed by many microorganisms, plants and animals. But human activities affect the carbon cycle by adding more carbon dioxide to the atmosphere and by damaging natural sinks, for example forests which would otherwise remove carbon dioxide from the atmosphere.
Nitrous Oxide, like carbon dioxide, is also naturally present in the atmosphere as it is part of the global nitrogen cycle: Nitrogen circulates between the atmosphere, plants, animals and bacteria breaking it down in the oceans and soils. Even though it has a variety of natural sources, human activities such as wastewater management or nitrogen fertilization increase the amount of nitrous oxide in the atmosphere. These molecules stay in the atmosphere for about 120 years but can be removed by certain types of bacteria or even destroyed by chemical reactions or ultraviolet radiation.

In contrast to other greenhouse gases, fluorinated gases do not have natural sources and only arise from human activities. F-gases are emitted through different industrial processes and many of them have a very high global warming potential in comparison to other greenhouse gases. Therefore, even very small atmospheric concentrations can have great effects on global temperatures. Additionally they have very long atmospheric lifetimes, lasting up to thousands of years. F-gases are removed from the atmosphere only when they get destroyed by sunlight in the far upper atmosphere. In general, fluorinated gases are the most potent and longest lasting sort of greenhouse gases emitted by humans.

Methane is the second most important greenhouse gas. It is emitted by several natural sources such as wetlands but also by different human activities like leakage from natural gas and livestock farming. Methane can be removed from the atmosphere by natural processes in soil and chemical reactions in the atmosphere. As its lifetime in the atmosphere is, compared to other greenhouse gases, quite short (~12 years), targeting methane emissions as a means of reducing the overall greenhouse gas emissions in a relatively short period of time is a reasonable approach to reduce global warming. Globally, around 60 % of all methane emissions originate from human activities like industry, agriculture and waste management.

Even though the total methane emissions decreased by around 10 % between 1990 and 2012 the emissions associated with agricultural activities did not. This is mostly due to the intensification of livestock farming. Methane is set free in the course of digestion in ruminant animals like cattle and emitted into the atmosphere. Therefore, by increasing livestock production methane emissions are increased as well.

Methane is formed in a process called methanogenesis which, for example, takes place in the rumen of cattle. However, the methane is not further utilized by any of the microorganisms living in the animal’s digestive tract nor by the animal itself, so it gets exhaled to the atmosphere and thus contributes to global warming. Read here what has been done in the past against these emissions, or see below to find out how exactly methanogenesis takes place.

References

1. U.S. Department of State (2007). Projected Greenhouse Gas Emissions. In: Fourth Climate Action Report to the UN Framework Convention on Climate Change. U.S. Department of State, Washington, DC, USA.
2. IPCC (2007). Climate Change 2007: Mitigation of Climate Change. Contribution of Working Group III to the Fourth Assessment Report of the Intergovernmental Panel on Climate Change B. Metz, O.R. Davidson, P.R. Bosch, R. Dave, L.A. Meyer (eds). Cambridge University Press, Cambridge, United Kingdom and New York, NY, USA.
3. http://epa.gov/climatechange/ghgemissions/

The Origin of Methane

Methane from biological sources can originate in various different environments like peat bogs, deep-sea hydrothermal vents and the digestive tracts of animals. Despite this variation there are certain common aspects: All biologically formed methane originates from methanogenic archaea and all these methanogens are strictly anaerobic organisms [1]. However, there is not only one mode of methanogenesis. Some methanogens perform hydrogenotrophic methanogenesis which mostly relies on the reduction of carbon dioxide while hydrogen usually acts as the electron donor. The methylotrophic methanogens, in contrast, are additionally able to use a variety of other compounds for methane generation, for example certain methyl compounds. Lastly, in acetoclastic methanogenesis acetate is used as a substrate for methane formation [2,3].

Hydrogenotrophic methanogens comprise, for example, the order Methanobacteriales and species like Methanobrevibacter ruminantium. Methanosarcinales is the most important order of the methylotrophic methanogens with species like Methanosarcina barkeri. Representatives of both orders and both modes of methanogenesis can be found in the rumen of cattle, one of the most important biological methane sources [4].

In the hydrogenotrophic pathway, carbon dioxide is initially reduced with electrons taken from the oxidation of hydrogen or alternative electron donors. The resulting formyl group is then subsequently reduced. Involved in this process are several unique cofactors to which the groups of the different oxidation states - some of them cyclic - are being bound until methane is released. The reduction reactions are coupled to the export of sodium ions across the cell membrane.
The methylotrophic pathway, in which C1 compounds like methanol or methylamines can be utilized, joins the hydrogenotrophic pathway at a late stage. A total of four C1 molecules is needed here, one of which is oxidized to provide electrons for the reduction of the other three. In the acetoclastic pathway, the electrons required for reduction are taken from the oxidation of the carbonyl carbon of acetate [2,3].

References

1. Bapteste E, Brochier C, Boucher Y (2005) Higher-level classification of the Archaea: evolution of methanogenesis and methanogens. Archaea 1:353-63
2. Costa KC, Leigh JA (2014) Metabolic versatility in methanogens. Curr Opin Biotechnol 29:70-5
3. Galagan JE, Nusbaum C, Roy A et al. (2002) The Genome of M. acetivorans Reveals Extensive Metabolic and Physiological Diversity. Genome Res 12:532-42
4. Hook SE, Wright ADG, McBride BW (2010) Methanogens: Methane Producers of the Rumen and Mitigation Strategies. Archaea 2010:ID 945785

Digestion in Ruminants

Like all ruminants, cattle are herbivores, which means that they mainly feed on plants – the most demonstrative example for this might be a cow grazing on a meadow. For the vegetable components to be properly utilized, however, the cow needs a stomach completely different from the kind you find in other mammals like, for example, humans. Indeed, the stomach of ruminants consists of four components: rumen, reticulum and omasum are succeeded by the abomasum, and those four compartments work closely together in the digesting process. Thanks to this, the grazing cow is able to utilize cellulose to gain energy, which is not possible for most other mammals.

When a cow ingests food, it is swallowed without being chewed at first, passes through the reticulum and enters the rumen. Here, fermentation is carried out by microorganisms, which can take 20 to 50 hours. Next, small balls are formed from the processed food in the reticulum by muscle contractions, and these balls are regurgitated, chewed and mixed with saliva. The resulting food mush is then swallowed once again; this time, though, it does not enter the rumen after passing the reticulum, but is channelled into the omasum. Thus, the reticulum works as a kind of filter separating the unprocessed food of the first swallowing from the processed mush of the second.

The importance of the rumen in digestion arises from the numerous microorganisms living in it. In fact, each gram of rumen contents contains about 10¹⁰ to 10¹¹ bacteria belonging to 300-400 different species, resulting in a great variety of metabolic profiles. Most of these organisms are obligate anaerobes, meaning that they can only survive and grow in the absence of oxygen. However, a certain amount of oxygen entering the rumen with every swallow can hardly be prevented; thus, there are also a few facultative anaerobes present capable of consuming oxygen.

Among the billions of microorganisms in the rumen there are different groups specialized on degrading compounds like cellulose, starch, or lactate. The sugars set free by breaking down these substances are subsequently fermented. The size and structure of the rumen ensure that the food is properly mixed and that the microorganisms can work on it for a sufficiently long time. During this process different kinds of gases originate. The so-called volatile fatty acids (VFAs) like acetic, propionic, and butyric acid are particularly important for the animal, as they pass through the rumen wall into the bloodstream, serving as a main source of energy. Unfortunately, alongside the VFAs greenhouse gases like carbon dioxide and methane are produced and released into the atmosphere by eructation.

All rumen microbes are well adapted to the conditions prevailing in this special ecosystem. Its temperature averages at about 39°C, but is also dependent on external influences. The pH value is relatively constant, ranging between about 6.5 and 7.0, although acids are permanently produced – this constancy is due to bicarbonate contained in and swallowed with the saliva and to ammonium set free in the course of the deamination of amino acids. Under certain nutritional conditions, though, stronger deviations of the pH value may occur due to the rapid proliferation of bacteria like Streptococcus bovis which produce lactic acid.

In newborn ruminants, the microflora and -fauna of the rumen is not yet properly developed. However, through contact with the mother animal or aerosols a rapid colonization occurs and a microbial population emerges, ensuring the functionality of the rumen. In the view of this, it is imaginable to influence the fermentation processes in the rumen by dispensing microbial probiotics to the calf directing the composition of the population in a certain way. Furthermore, there are different approaches to particularly influence methane production inside the rumen by dispensing so-called direct-fed microbial (DFM); these are, however, as yet restricted to making use of alternative metabolic pathways competing with methanogenesis.

References

1. Hristov AN, Oh J, Firkins JL, Dijkstra J, Kebreab E, Waghorn G, Makkar HP, Adesogan AT, Yang W, Lee C, Gerber PJ, Henderson B, Tricarico JM (2013) Special topics – Mitigation of methane and nitrous oxide emissions from animal operations: I. A review of enteric methane mitigation options. J. Anim. Sci. 91(11):5045-69
2. Jeyanathan J, Martin C, Morgavi DP (2014) The use of direct-fed microbials for mitigation of ruminant methane emissions: a review. Animal 8(2):250-61
3. Madigan MT, Martinko JM, Stahl DA, Clark DP (2012) Brock Biology of Microorganisms. 13th ed. San Francisco: Pearson Education. pp. 762-6
4. Weimer PJ (1992) Cellulose Degradation by Ruminal Microorganisms. Crit Rev Biotechnol 12(3):189-223

Idea

That's What We Are Going to Do

The greenhouse effect is a well-known phenomenon describing the synergy of several gases in the atmosphere of the Earth, which leads to positive as well as negative consequences. Without greenhouse gases the Earth would be a very cold place and life would not be possible the way we know it. Nevertheless, due to increasing emission of gases like methane and carbon dioxide the effect is further enhanced and thus causes global warming.
Among other things, the greenhouse gas methane is a product of naturally occuring enteric fermenation of cows. Because the consumption of dairy products and meat is increasing, mass husbandry is widely spread. Hence, the emission of methane attributed to cows rises to unnatural dimensions.

Since most people are not willing to change their daily nutrition and reduce their consumption of several products significantly (see our survey) an elimination of methane emissions caused by cows would be useful. However, former approaches merely regard either the methanotrophs responsible for methane production or the substrates they need.
Our project idea is to reduce global methane emission right at one of the sources, the rumen of cows, without affecting resident organisms. Our idea is to provide the digestive tract with a specially modified microorganism, named E. cowli, which is planned to metabolize the methane before its emission and, therefore, prevent the enhanced greenhouse effect and global warming.

Approach

To reduce the methane emission caused by cows, we needed a microorganism which is able to metabolize this greenhouse gas right after its production in the rumen. Therefore, our project is based on the enzyme complex methane monooxygenase (sMMO) from the bacterium Methylococcus capsulatus, the model organism for obligate methanotrophic bacteria, native to multiple habitats such as soils and the oceans.
The sMMO is a multi-enzyme complex consisting of three subunits: a hydroxylase (MMOH), a reductase (MMOR), and a regulator (MMOB). The hydroxylase itself again consists of the three subunits α, β and γ. Hence, the complete complex encompasses an overall mass of 330 kDa and enables M. capsulatus to turn methane into a natural intermediate of the cellular metabolism. Thus, our aim was to isolate the DNA segments encoding the six MMO subunits (MMOX, MMOY, MMOB, MMOZ, MMOD, MMOC) from M. capsulatus and equip the well-characterised intestinal bacterium Escherichia coli with them. Thus, the transformed E. cowli would become capable of metabolizing methane.

After isolating the genes encoding each MMO subunit the biggest challenge would be to stably produce the functional enzyme complex in E. coli. To facilitate protein folding our plan, therefore, was to introduce a special combination of chaperone proteins. Having determined the right combination for proper producibility of all subunits in E. coli the enzyme complex would be assembled in one final construct, thus creating our methane degrading E. cowli. Establishing a stable expression of the MMO complex in E. coli and enabling the degradation of methane would be the first step towards a genetically modified bacterium administered to cows.

Nevertheless the question of its feasibility as well as the efficacy in a non-laboratory environment has to be addressed. Although E. coli is a natural inhabitant, the designed E. cowli is possibly not able prevail inside the rumen due to the presence of inhibiting substances. Furthermore, the safety of introduction of a genetically modified organism has to be considered. To protect the cow from E. cowli and vice versa we also evaluted the beneficial effects of entrapment inside an alginate matrix. Therefore, we contacted Dr. Ulf Prüße, an expert for immobilization of enzymes and bacteria. Read the whole interview here.

Past Approaches

Past strategies for reducing the methane emissions from ruminants like cattle can be subdivided into two main approaches: Firstly, approaches aimed directly at the methanogens, and secondly, approaches aimed at other microorganisms of the rumen which provide the methanogens with substrates for methanogenesis [1]. The methanogens engaged in such symbioses are also called protozoa-associated methanogens (PAMs) and account for about 37 % of the methane produced in the rumen [2].
The substrates for methanogenesis vary greatly among different methanogenic organisms because they use different modes of methanogenesis. For example, the archaeon Methanobrevibacter ruminantium (order Methanobacteriales) utilizes carbon dioxide, hydrogen and formate for methane production, whereas Methanosarcina barkeri (order Methanosarcinales) can grow on a wider range of substrates like acetate or methylamines, in addition to hydrogen and carbon dioxide [1].
One obvious approach for the reduction of methane emissions is changing the diet fed to the animal. For example, the amount of methane emitted by cattle is lower when the diet consists mostly of concentrate, which is rich in nutrients and low in fibre content, rather than a mixture of concentrate and forage [3]. Another possibility is grinding the food before feeding so that it resides in the rumen for a shorter period. This way, there would be less time for the microorganisms, including methanogens, to work on the food [1].
Apart from the type of food, the latter can also be supplemented by, for example, fatty acids which would then inhibit the protozoa associated with the methanogens. To this aim, it was found that medium-chain saturated fatty acids with chain lengths of 8-10 are most effective [4]. However, the use of unsaturated fatty acids which can act as hydrogen sinks and thus inhibit methanogenesis is also discussed [1]. Another approach by which the association between protozoans and methanogens may be split is defaunation, i.e. the removal of the protozoans from the rumen. As a large amount of methane emissions is accounted for by PAMs, this treatment would act on an equally large source of methane emissions [1,2].

There have also been vaccines developed in order to stimulate the ruminant’s immune system so that it acts on the methanogens [1]. However, such vaccines are usually directed against certain groups of methanogens only. In some cases, this might give those methanogens responsible for most of the emissions an advantage and thus increase methane emissions instead of decreasing them [5].
Aside from the above-mentioned ones there are several further attempts to stop methane emissions in the rumen, but it also has to be considered that although by this process a substantial amount of energy is lost, methanogenesis still has a point in ruminal ecology. The removal of hydrogen prevents inhibition of the fermentative processes which are essential for the cattle’s digestion [6].

Therefore, decreasing the rate of methanogenesis might be positive for the climate, but probably not so for the cattle’s digestion. In contrast, no such disadvantages come along with our idea of degrading methane because methanogenesis itself would not be prevented. Therefore, the methanogens could still fulfill their important role in the cattle’s digestion. As the methane is not further utilized anyway, degrading it should not cause any damage to the animal.

References

1. Hook SE, Wright ADG, McBride BW (2010) Methanogens: Methane Producers of the Rumen and Mitigation Strategies. Archaea 2010:ID 945785
2. Belanche A, de la Fuente G, Newbold CJ (2014) Study of methanogen communities associated with different rumen protozoal populations. FEMS Microbiol Ecol [published ahead of print] doi: 10.1111/1574-6941.12423
3. Rooke JA, Wallace JJ, Duthie CA, McKain N, Motta de Souza S, Hyslop JJ, Ross DW, Waterhouse T, Roehe R (2014) Hydrogen and methane emissions from beef cattle and their rumen microbial community vary with diet, time after feeding and genotype. Br J Nutr 112:398-407
4. Dohme F, Machmüller A, Wasserfallen A, Kreuzer M (2001) Ruminal methanogenesis as influenced by individual fatty acids supplemented to complete ruminant diets. Lett Appl Microbiol 32:47-51
5. Williams YJ, Popovski S, Rea SM, Skillman LC, Toovey AF, Northwood KS, Wright AD (2009) A Vaccine against Rumen Methanogens Can Alter the Composition of Archaeal Populations. Appl Environ Microbiol 75:1860-6
6. Sharp R, Ziemer CJ, Stern MD (1998) Taxon-specific associations between protozoal and methanogen populations in the rumen and a model rumen system. FEMS Microbiol Ecol 26:71-8

Methanotrophs

Methanotrophic bacteria accomplish the remarkable task of utilizing the inert compound methane as their sole source of carbon and energy. They are usually found where anaerobic and aerobic environments come together, the former providing methane and the latter oxygen. They use methane by oxidizing it to methanol, which is then further converted to formaldehyde and can be assimilated into the cellular biomass [1,2].
Methanotrophs can be subdivided into two classes which are classified mainly by phylogenetic analyses, but also by physiological and morphological differences. Type I methanotrophs belong to the γ-Proteobacteria, while type II methanotrophs are members of the α-Proteobacteria. They differ in the way the assimilation of formaldehyde, subsequent to methane oxidation, takes place [3].

Methylococcus capsulatus, a type I member, is considered a model methanotroph and has been extensively studied in recent years [4]. However, due to several characteristics it is much harder to handle than other model organisms like, for example, Escherichia coli. M. capsulatus is thermophilic with a temperature optimum of 45°C and has a rather slow growth rate. Therefore, it would be useful if enzymes like those catalyzing the degradation of methane could be expressed in an organism easier to handle - not only for research but also for industrial purposes.

References

1. Dalton H (2005) The Leeuwenhoek Lecture 2000: The natural and unnatural history of methane-oxidizing bacteria. Phil Trans R Soc 360:1207-22
2. Merkx M, Koop DA, Sazinsky MH, Blazyk JL, Müller J, Lippard SJ (2001) Dioxygen Activation and Methane Hydroxylation by Soluble Methane Monooxygenase: A Tale of Two Irons and Three Proteins. Angew Chem Int Ed 40:2782-807
3. Ward N, Larsen Ø, Sakwa J et al. (2004) Genomic Insights into Methanotrophy: The Complete Genome Sequence of Methylococcus capsulatus (Bath). PLoS Biol 2:e303
4. Hakemian AS, Rosenzweig AC (2007) The Biochemistry of Methane Oxidation. Annu Rev Biochem 76:223-41

Methane Monooxygenase

Methane monooxygenases are enzyme complexes which provide organisms called methanotrophs with the ability to use methane as their sole source of carbon and energy. There are two basic forms of the enzyme: A membrane-bound particulate one (pMMO) and a cytoplasmic soluble one (sMMO). While the genes for the pMMO complex are present in almost all methanogens, sMMO is only present in a smaller group of organisms. The expression of the respective forms is dependent on the copper concentration, mediated by a so-called “copper switch”. If the copper concentration is high, pMMO is expressed and the expression of sMMO is inhibited. If, in contrast, the copper concentration is low, sMMO is expressed. The two MMO forms also differ in their substrate specificity in that sMMO has a much broader substrate range which includes, for example, long-chain alkanes or aromatic molecules [1]. pMMO, despite being much more common than sMMO, is far less characterized due to its membrane-bound state and the analytical difficulties coming along with it [2]. Therefore, we chose sMMO as our subject matter. The enzyme complex catalyzes the oxidation of methane to methanol, additionally involving an oxygen molecule and NAD(P)H:

The sMMO complex consists of three main components. The first component, the hydroxylase MMOH, is made up of the three different subunits α, β and γ which are arranged as a heterodimer (α₂β₂γ₂). The α-subunit houses the active site of the enzyme where the oxidation of methane takes place. The reductase component MMOR oxidizes NAD(P)H and shuttles the electrons to the hydroxylase. The electron transfer is facilitated by a [2Fe-2S]-cluster and an FAD cofactor bound to the reductase. The regulatory protein MMOB seems to have a function in modulating electron transfer because it binds to the same site of MMOH as MMOR and thus there is a competitive relationship between the reductase and the regulatory protein [3]. The active site of the enzyme contains a non-heme diiron center. Under resting conditions the iron ions are both in the ferric state [Fe^III-Fe^III] and can be reduced by uptake of electrons from NAD(P)H to the mixed-value [Fe^III-Fe^II] and diferrous [Fe^II-Fe^II] states. These transitions are important for the activation of dioxygen and thus methane hydroxylation [4,5]. The genes for the subunits are arranged in a 5,5-kb operon which additionally contains another open reading frame formerly designated orfY and later mmoD. MMOD binds to the hydroxylase in a similar fashion as MMOB and MMOR, yet the function has long stayed unclear [6]. It has been suggested, however, to be part of the above-mentioned 'copper switch' regulating the expressions of sMMO and pMMO, respectively [7].

References

1. Murrell JC, McDonald IR, Gilbert B (2000) Regulation of expression of methane monooxygenases by copper ions. Trends Microbiol 8:221-5
2. Hakemian AS, Rosenzweig AC (2007) The Biochemistry of Methane Oxidation. Annu Rev Biochem 76:223-41
3. Wang W, Iacob RE, Luoh RP, Engen JR, Lippard SJ (2014) Electron Transfer Control in Soluble Methane Monooxygenase. J Am Chem Soc 136:9754-62
4. Basch H, Mogi K, Musaev DG, Morokuma K (1999) Mechanisms of the Methane → Methanol Conversion Reaction Catalyzed by Methane Monooxygenase: A Density Functional Study. J Am Chem Soc 121:7249-56
5. Tinberg CE, Lippard SJ (2010) Dioxygen Activation in Soluble Methane Monooxygenase. Acc Chem Res 44:280-8
6. Merkx M, Lippard SJ (2001) Why OrfY? Characterization of MMOD, a long overlooked component of the soluble methane monooxygenase from Methylococcus capsulatus (Bath). J Biol Chem 277:5858-65
7. Semran JD, Jagadevan S, DiSpirito AA, Khalifa A, Scanlan J, Bergman BH, Freemeier BC, Baral BS, Bandow NL, Vorobev A, Haft DH, Vuilleumier S, Murrel JC (2013) Methanobactin and MmoD work in concert to act as the “copper-switch” in methanotrophs. Environ Microbiol 15:3077-86

Potential Impact

“With great power comes great responsibility.” - Uncle Ben (and Voltaire)

On the basis of this quote we thought about other possible applications of E. cowli. Not only could the isolation and successful production of the sMMO have a great impact on the greenhouse effect, but it could also facilitate addressing problems of safety, pollution and energy.

If the sMMO is immobilized and introduced into the cow’s rumen the worldwide methane emissions could be reduced by 164 million tons, based on a total number of 1.5 billion cows on Earth. In effect, this equals 5 billion tons of carbon dioxide! More pictorially, our application can compensate the impact 2.8 million cars, assuming that one car releases approximately 1,800 t of carbon dioxide annually. See our example calculation.

In addition to the effect on the world's climate, the application in cows has another positive impact: safety. Earlier this year, for instance, high amounts of methane accumulated in a cow barn in Germany and a single spark caused the building to blow up [1]. By reducing the methane emissions of cows incidents like this could be prevented.

As the sMMO is able to degrade a broad variety of substrates such as one of the major water pollutants, the industrial solvent trichloroethylene (TCE), our project can serve as a foundation for a new approach of biological water treatment or environmental care in general [2].

One could also exploit the ability of the sMMO to convert methane to methanol: Methane can serve as a source of energy, but due to its gaseous state it is very difficult to transport compared to liquid methanol. So far, methane is chemically converted to methanol for transportation and later on further to components of diesel fuel and gasoline or to propylene and ethylene, important precursors of important chemical substances. Using the sMMO to oxidize methane is a much more economical and eco-friendly approach for utilizing methane as a resource for energy production [3]. Hence, our project has a huge significance not only for climate but also for a wide-ranging spectrum of other problems and opportunities. Therefore, the isolation and successful expression of the sMMO has a huge impact on various areas and provides multiple possibilities for application.

References

1. http://www.bbc.com/news/world-europe-25922514 (10.10.2014)
2. Brussea GA, Tsien HC, Hanson RS, Wackett LP (1990) Optimization of trichloroethylene oxidation by methanotrophs and the use of a colorimetric assay to detect soluble methane monooxygenase activity. Biodegradation 1:19-29
3. Tinberg CE, Lippard SJ (2011) Dioxygen Activation in Soluble Methane Monooxygenase. Acc Chem Res 44:280-8

Engineering Principles

While planning our project and putting it into practice, we always kept in mind some fundamental principles of engineering. Firstly, our project was well planned and all available information was collected and evaluated beforehand, allowing us to adjust our construct before actually starting in the wet lab. Also, we kept the whole construct as simple and made use of as many standard biobricks from the iGEM registry as possible. Those parts not available in the registry (see our new parts) were built into the assembly standard making them available to all users of the Registry of Standard Biological Parts.
Additionally, we ensured the functionality of each subunit by building His­-constructs of all mmo genes to test them separately. This allowed us to evaluate their functionality and, if necessary, to adjust them for a better performance. Furthermore, as it is the duty of an engineer to not only take responsibility for the positive but also the negative consequences of a construction, we never left safety and ethics out of our equations.

Expert Interview

After successful and functional expression of the sMMO-complex, we encountered questions addressing the practicability of our project. Can E. cowli survive inside the inhospitable environment of the cow's rumen or is the activity affected by it, are just two of the many questions we had to face. To address these question we talked to Dr. Ulf Prüße of the Thünen-Institute Braunschweig, an expert in immobilization of enzymes and bacteria.

1. Our findings suggest that E. coli is inhibited by conjugated fatty acids in the rumen. Is there a possibility to avoid this inhibition using, for example, immobilization?
No, that’s usually not possible. The immobilization matrix allows the transport of low molecular weight substrates, products, nutrients etc. through the matrix by diffusion. Otherwise the cell would die. There is a molecular cut-off of the matrix which depends on the matrix material itself and partially also on the immobilization procedure, but this cut-off is much higher than the molecular weight of fatty acids.
However, the extent of inhibition may be reduced by two facts:
- If a concentration gradient of the fatty acids is built up inside the immobilization matrix. The prerequisite for that is that the inhibitory fatty acids must be removed or digested inside the beads. If, for example, the cells were able to digest the fatty acids faster than they diffuse through the matrix, a high, inhibitory concentration would be avoided.
- If the diffusion coefficient of the inhibitory compounds in the matrix is reduced. This might be achieved by using a hydrophilic matrix material in which more hydrophobic compounds like fatty acids cannot diffuse so easily.

2. Which kind of immobilization would you recommend for our project considering the rumen as milieu?
The rumen is a very complex milieu so that it is not easy to predict how immobilisates will behave. A synthetic matrix material like polyvinylalcohol cannot be digested and would be stable in the rumen but will end up in the manure and later on somewhere in the environment. This wouldn’t be a good idea. Therefore, a natural biodegradable material like alginate or carrageenan should be preferred. As these materials consist of natural polysaccharides, they might be degraded by the enzymes in the rumen. How fast this will occur needs to be checked.

3. Is the production of alginate, beads expensive or difficult?
Of course, there are additional costs for the immobilization procedure, but those are in general not too high. It is always a question whether the benefit of immobilization is higher than its costs. This cannot generally be answered but has to be checked from case to case. Immobilization in alginate isn’t difficult at all. You just need an alginate solution with the cells and drop it e.g. with a syringe into another solution containing calcium ions, e.g. CaCl₂ or Ca-lactate, leading immediately to Ca-alginate beads. (Calcium acts as a crosslinker for the alginate chains.) It’s so easy that anyone can do it, even in their own kitchen. In fact, similar procedures using alginate are part of the modern molecular cuisine offered in restaurants.

4. How much alginate is contained in the alginate beads?
This depends on the alginate type which is used, i.e. its molecular weight and thus its viscosity in a solution. Typically, a 1 – 4 wt.% alginate solution is used to prepare beads. This is more or less also the alginate content of the resulting beads.

5. What is the ordinary concentration of cells included in the alginate beads?
Usually, an initial cell concentration of 10⁷ - 10⁹ is used when the beads are prepared.

6. Is E. coli able to grow inside the alginate beads?
Yes, they are. Typically, the beads with the initial cell loading are cultivated for some time (depends on how fast the cells are growing) in order to let them grow inside the beads. At the end a large part the whole bead volume can consist of cells provided that they are still provided with the necessary nutrients.

7. Are the ingredients of alignate beads in any way toxic for E. coli or the cow?
No, not at all. Alginate is a natural polysaccharide produced from algae. It is also used as thickener for food applications, e.g. for yoghurt or ice cream.

8. Under what circumstances do the alginate beads disintegrate? (shearing, pH, phosphate concentration? any components of the rumen fluid?)
The pH value is usually not critical as long as it is within a physiological range. A disintegration may occur if considerable phosphate concentrations are present as in that case calcium phosphate will precipitate. The removal of calcium as crosslinker will lead to a dissolution of the beads. A mechanical disintegration needs considerable shear rates.

9. Are the alginate beads able to protect E. coli against other bacteria occuring inside the rumen. For example ciliates are known to ingest many bacteria such as E. coli?
Yes. No other organism can penetrate the alginate bead.

10. Are the alginate beads able to protect E. coli when ruminated?
I’m not sure but I’m afraid that the beads will be destroyed by chewing.