Developing better methods for culturing rumen bacteria

Authors: Dr Peter H. Janssen, Ms Nikki Kenters and Dr Gemma Henderson

Executive summary

Goal

This project aims to develop better methods for culturing rumen bacteria, to make them available for more detailed investigation.

Context of the project

The major precursor of the methane formed in the rumen is hydrogen. This hydrogen is produced during the fermentation of feed by the rumen microbial community, and is used by methanogens to produce methane. Some of the fermentation pathways operating in the rumen produce little hydrogen and hence contribute little to methane formation. Other, parallel, fermentation pathways produce large amounts of hydrogen, and lead to significant amounts of methane. Preferentially stimulating the activity of the bacteria that use the pathways that produce little hydrogen will result in less methane formation. This approach can be termed “hydrogen management”. Much of the research into methane mitigation has focussed on inhibition of methanogens, the microorganisms that convert hydrogen to methane. Research into “hydrogen management” by manipulating the hydrogen producing bacteria is, in contrast, not being pursued, because of the lack of knowledge of the bacteria involved. This project begins a new line of research by making the first steps towards overcoming the lack of understanding of hydrogen-producing bacteria in the rumen.

Approach

The composition of microbiological media is nearly always a compromise between mimicking the environment and ease of preparation. For media used to culture rumen microbes, ease of preparation is favoured over mimicking the rumen conditions. Using previous experience in developing media for anaerobic sediment bacteria and aerobic soil bacteria, new medium formulations were trialled to produce growth media that chemically mimic the rumen. Culturing anaerobic microorganisms is generally a tedious process that limits the number of isolates, severely limiting the chances of isolating so-called uncultured species. Statistical treatment of dilution theory was applied to anaerobic culture techniques to produce the maximal number of pure cultures of rumen bacteria within any given large-scale isolation experiment. Bacteria isolated using the new media and high-throughput culturing approach were identified by sequencing their 16S rRNA genes, to confirm that the approach could isolate members of the uncultured majority of rumen bacteria

Outcomes

A new medium formulation that chemically mimics the rumen was devised, and a simple way of preparing this medium was developed. Statistical treatment of dilution theory was applied to anaerobic culture techniques to produce the maximal number of pure cultures of rumen bacteria within any given large-scale isolation experiment. The calculated expected number of pure cultures was compared with the actual number obtained, and this confirmed the applicability of the approach. Bacteria were isolated using the new media and high-throughput culturing approach, and were identified by sequencing their 16S rRNA genes. Bacteria belonging to lineages known to exist in the rumen but not previously grown in pure culture, were identified among the collection of bacteria isolated in the high-throughput culturing step. Approximately half of the 78 new isolates appeared to be members of 10 new genera. This project has generated a new approach to rapid isolation of large numbers of rumen bacteria, including representatives that form part of the so-called “uncultured majority”. This approach has provided isolates for detailed study to better understand why some rumen bacteria produce large amounts of hydrogen and so lead to large amounts of methane being formed. The methods are widely and easily applicable, and allow isolation of large number of new species at a fraction of the effort previously required.

Summary

This project has developed better methods for culturing rumen bacteria, to make them available for more detailed investigation. This research underpins the development of a good fundamental understanding of rumen bacteria, which is a critical step towards manipulation and successful “hydrogen management” as a tool to mitigate rumen methane. The advance was achieved by developing better growth media, and using a statistical approach to rapidly isolate pure cultures of fermentative bacteria. Isolates of new genera of rumen bacteria were obtained that will allow a better understanding of the rumen to be achieved.

Publications

No publications have been generated at this time. It is expected that one publication in a peer reviewed international microbial ecology journal will be a direct outcome of this project, and that isolates obtained will be characterised and named in subsequent publications. These subsequent publications will be generated from further research stemming from isolates obtained and methods developed within this project.

 

Last Updated: 13 January 2011

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