I am working with Brevibacillus parabrevis, a cellulase producing bacteria and I am providing a medium containing cellulose for its growth. After the growth appearance, I want to check its cellulolytic activity by measuring cellulose concentration in the medium. So for that, can anyone suggest me the reliable, colorimetric method for estimation of cellulose?
sir,i already trid these DNS method but i cann't get the proper results. i have some dought about that because if the cellulose is hydrolysed and produces monosachharides , i.e glucose then may be these glucose is further used by an organism for its growth so....glucose may be used from the media....and i cann't able to measure glucose (reducing suger) in that.
Let me try to understand you doubt point by point:
I) I am working with Brevibacillus parabrevis, a cellulase producing bacteria and I am providing a medium containing cellulose for its growth.
II) After the growth appearance, I want to check its cellulolytic activity by measuring cellulose concentration in the medium. So for that, can anyone suggest me the reliable, colorimetric method for estimation of cellulose?
To measure cellulose i think there are not avaiable a simple colorimetric method. You must perform a full analysis like in the paper "ROCHA, G. J. M.; GONÇALVES, A. R.; OLIVEIRA, B. R.; OLIVARES, E. G.; ROSSELL, C. E. V. Steam explosion pretreatment reproduction and alkaline delignification reactions performed on a pilot scale with sugarcane bagasse for bioethanol production. Industrial Crops and Products, v. 35, n. 1, p. 274-279, 1/ 2012."
See session 2.6.
You should perfom the analysis of the media composition before and after the fermentation, separating supernatant from cells.
However you can estimate indirectly the cellulose in solid media through congo red technique:
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC241917/pdf/aem00185-0051.pdf?origin=publication_detail
http://synapse.koreamed.org/DOIx.php?id=10.4489/MYCO.2010.38.1.074&vmode=PUBREADER
You should better evaluate your goal in this case, most often a simple measure of cellulase activity will answer your question.
I performed a similar task with Clostridium thermocellum. To analyze the catalysis of cellulose, I followed the electrons. To do this, you will have to do as others suggest and monitor the fermentation products in the gas phase and the liquid phase. You can try dissolving a representative sample of cellulose in an acid and taking a TCOD measurement to figure out your initial electron quantity, then perform a TCOD after your experiment to detect the number of electron contained in products in the liquid phase. This should cover any electrons lost in fermentation products that you cannot detect with an HPLC/ UPLC, lost in SMPs, or lost in EPS (if any). Then, make sure to quantify your biomass to ensure that you are accounting for electrons stored in the cells (this will eliminate your glucose fermentation problem). From my experience, this measurement will never account for all the electrons (some always get lost somewhere in the error), but it should account for most of them.
Check out the link I added for a useful paper. I hope this helps
http://www.ncbi.nlm.nih.gov/pubmed/22055095
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