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 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 increasing 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 the release of methane from the rumen 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.

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

Solution

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 more easy to handle - not only for research but also for industrial purposes.

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

Results

Potential Impact

Hier Text für potential impact

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 in order 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.