CH4 Microbial Solutions

A pilot bioreactor has been installed on site at a coal mine undergoing remediation. Tests will be performed to evaluate the in situ performance of the reactor and the overall capabilities of these types of solutions in the field.

Thanks to Delta Brick and Climate Company for collaborating with us on this project.

Our studies into the variable kinetics of methanotrophic species has been ongoing and we soon hope to be able to publish information related to species present and conditions in high methane sampling areas vs. other natural regions. We currently have sampling data from three different mine methane leakage sites around Colorado. This data will allow us to expand operations into possible remediation methods.

As a part of ongoing studies of the soil in regions most affected by climate change samples of soil as well as measurements of temperature and pH were taken in assorted alpine regions. This was initially carried out at silver dollar lake in Arapahoe National Forest. Silver dollar lake is at 11,950 ft in elevation and is representative of alpine tundra type formations that would be found throughout the Rockies and is comparable to high latitude taiga and tundra regions of the artic. Further testing of the soil for methanotroph content and methanotroph capacity will be carried out in vitro. Soil Testing was carried out with permitting from the US Forest Service under Permit/Authorization ID: CLC1078

As a part of our efforts to network with the business community of Denver and establish connections that will help us attain our conservation goals we attended the Denver Startup Week 2023 including The Future of Energy Day which proved to be very enlightening and helpful.

One area of the methane carbon cycle that bears consideration is that of fresh water soils. There are a number of methanotrophs that inhabit the sediment in lakes around the world and serve as significant sink for the removal of methane from the environment. The main factor that determines whether lakes function as methane sinks or sources is the content of oxygen in the lake. If the lake is aerobic then it generally will function as a sink for methane consumption. If the lake is anaerobic then methanotrophs will not be able to metabolize methane and the methanogenic bacteria in the lake can instead work to produce methane. The artic region is full of lakes and ponds with more being produced all the time due to the thawing of previously frozen regions. These can serve to produce or consume methane and since many watery regions in the arctic are stagnant making them anaerobic and may to serve to produce more methane.

When crafting a strategy on how to distribute methanogenic bacteria you must take into account that they will have the needed nutrients such as oxygen. It may also be useful to distribute these species in dryer arid soils in which methanotrophs thrive that are adjacent to wetter regions that produce methane.

Methanotrophs have sensitive requirements for growth and often require specific nutrients as well as coculture species in order to thrive in vitro and in vivo. This can provide difficulties when culturing in the lab and when new colonies are seeded into the environment. Metal molecules are of special interest to methanotrophs because they are used in the active site Methane Monooxygenase (MMO) to digest methane. The most important metal to recruit for this is Copper which is incorporated into the particulate form of MMO but iron can also be incorporated into the soluble form of MMO in circumstances in which copper is not readily available. Copper as a consequence of its importance in the function of pMMO also serves as a powerful regulator of transcription and expression for a number of different protein pathways that lead to methanotroph proliferation. Methanotrophs are able to extract copper from their environment from insoluble minerals and weathering of copper rich minerals can serve as an indication of the presence of Methanotroph species.

Testing at the site of soil sampling for the presence of certain nutrients especially copper can reveal how active the growth of methanotrophs may be and as to whether they may be metabolically limited to their soluble form for carbon metabolism. When growing in the lab and eventually in the process of seeding natural environments with methanotrophs these limitations need to be accounted for as a limiting factor in growth.

Methanotrophs pose a special challenge in cultivation and expansion due to metabolic needs and limitations. It is essential to identify the strain of methanotroph that is being isolated so that proper media can be prepared. Additionally proper measuring of conditions such as salinity, pH and temperature at the sampling site can provide insights into how to properly culture these species. Some forms of methanotrophs have also been shown to increase their growth alongside other satellite bacteria that are able to clean excess contaminates produced by methanotrophs. These problems are scaled when larger bioreactors would be needed to produce larger amounts of methanotrophs.

Methane Monooxygenases (MMOs) serve the primary function for primary function of converting methane into Methanol for further digestion in Methanotrophic organisms. They come two distinct forms. The membrane bound particulate (pMMO) form and the free cytoplasmic soluble (sMMO) form. Methanotrophs can possess either of these or both fore use in metabolism.

These two different proteins have been shown to have variable forms and kinetic capabilities across the spectrum of methanotrophic microbial species. The capabilities for digestion can be directly characterized by testing the rates of digestion of CH4. This can be further determined by identifying specific species of methanotroph through 16S rRNA sequencing. The full sequence of the MMO can be then characterized through sequencing protein folding studies and X-Ray crystallography. The protein structures that lead to changes in enzyme kinetics can then be identified and utilized for making more active methanotrophic species.

A number of other groups are working towards the same goal of addressing the issues posed by methane release into the broader environment. As part of our goal to engage with the Climate Change research community we sat in with Delta Brick and Climate Company for the their public comment on the project to seal the methane escaping from Coal Basin. https://www.deltabrick.com/climate-projects

These projects provide us all a unique opportunity for collaboration with like minded people who are working towards similar goals in reducing methane release into the atmosphere. Future collaborations on such projects may involve sampling soils in high methane zones of these types of areas to test the methanotrophic capacity of microbes in these areas.

Methanotrophic organisms pose a unique challenge in culturing populations. Most methanotrophs are facultative in their reliance on methane. For them to grow without methane however they do require specialized media depending upon the lineage of the methanotroph. This can provide insights about the metabolic activity of the species and insights into what types of populations can be found by testing metabolites and other characteristics of the soil before sampling.