Dear colleagues!
I am trying to figure out how to do an extraction for soil microorganisms for further metabolomic analyses. I am only interested in the microbiota part, so I would like to discard any organic matter present in the samples.
Do you know any useful method for that? Any suggestions that could help me?
Thanks for your help!
Good question and wonder how people do meta-transcriptomics !
Just possible guesse (As you said, any suggestions would help!):
1. Dilute, filter (specific micrometer to separate particles, protozoans from bacteria!), wash, repeat several times and harvest several times to enrich enough to be able to extract metabolites ! Seems like a difficult preposition.
2. Easier would be to grow as many as possible on say bacterial and fungal media (different!) separately, grow under different conditions (media, pH, temperatures, durations)- then pool to extract them or extract them separately and pool to analyze ! But this does not capture the meta-metabolome (bacteria that would not grow at all!).
Best wishes.
This is certainly a difficult task as the complexity of a soil environment is hard to deconvolute. I suspect that analysis of crude soil samples would be complicated further by the lack of abundance of metabolites of interest. You can beat the low abundance hurdle by extracting even more soil, but perhaps the method below will be appealing to you.
I currently run the Bacterial Pipeline for a natural products group. We sample soil from time to time, but usually isolate bacteria from the orifices of mammals. For our pipeline we make dilutions of the sample swabs (1:100, 1:1000, 1:5000) and plate ~150 uL onto three or four different media agars. We let them grow for 1-2 weeks and then transfer isolated colonies to a 96-well deep well plate with 50% rich media supplemented with 15% glycerol (one colony per 96-well). We grow this master plate for 3-7 days, transfer each well to a respective location in a 24-deep well plate with three different media types (ie Minimal, Rich, and mammalian derived medias - so 1 96 well plate equals 4 24 well plates X 3 media types). We let those small-scale fermentation shake for 7 days and perform an ethyl acetate extractions (3x). The extracts are subsequently dried and resuspended in 100 uL of DMSO and submitted to bioassay. After we find active wells we reconfirm the activity in triplicate, submit for 16s sequencing, and analyze with LC-PDA-ESIMS. Finally we start the dereplication process. With each swab that we take for isolation we also collect one for meta-genomic analysis to assess our cultivation effectiveness.
Good luck.
Any process of soil extraction will also contribute metabolites from plant root exudates, insects, apart from soil organic matter. Thus, it will be wise to fabricate a model soil, augment it with a microbial consortium and then proceed for metabolomics. Otherwise, you can cultivate microorganism from soil on different media, get isolates, pool them and then proceed.
Still it will be a difficult job.
Best luck
Thanks for your help.
Yes, I understand the difficulty of such process. Just I asked that in case there is any methodology to estimate the metabolism of the microbes.
I though in have two soil samples, one normal and one fumigated. The problem that I see is that anyway fumigating, the microbial metabolome will be there and I will detect it.
Thanks again!
There are many methodologies to estimate the metabolism of the organisms. Herein are articles and useful l linkages of webpages:
(1) Oumarou Abdou-Arbi et al (2014) Exploring metabolism flexibility in complex organisms through quantitative study of precursor sets for system outputs. BMC Systems Biology 2014, 8:8 (attached). The electronic version of this article is the complete one and can be found online at: http://www.biomedcentral.com/1752-0509/8/8
(2) Jens Nielsen (2003) It Is All about Metabolic Fluxes, Journal of Bacteriology, 185(24) 7031–7035 (attached).
(3) Mlawule R. Mashego et al (2007) Microbial metabolomics: Past, Present and Future Methodologies. Biotechnol Lett. 29(1):1-16(attached).
(4) Methodology on monitoring and modelling of microbial metabolism. http://www.tnw.tudelft.nl/nl/over-faculteit/afdelingen/biotechnology/research-groups/cell-systems-engineering/research-projects-cse/methodology-on-monitoring-and-modelling-of-microbial-metabolism/
Regards
http://www.tnw.tudelft.nl/nl/over-faculteit/afdelingen/biotechnology/research-groups/cell-systems-engineering/research-projects-cse/methodology-on-monitoring-and-modelling-of-microbial-metabolism/
- Badges
- Science topic
- Similar topics
- Agricultural Science
- Soil Science
- Soil Analysis