I have been doing biodegradation experiments in batch mode and my observation is that the OD/growth of the medium remains constant but the concentration of my target compound decreases....what should I conclude? Is the compound getting degraded by microorganisms or it has been adsorbed on the biomass?
The compound must be getting consumed by microorganisms. Have you done a biological analysis of the substance? Or a chemical analysis, whereby by - products of your target compound are in existence as a result of consumption by microorganisms? The biomass may be using your target compound as a nutrient source. Analysing the liquid content of your experiment will help to determine the source of the consumption of your target compound.
I agree with Steph. The microorganism(s) is degrading the compound. If you think the compound is adsorbed to the cell surface or absorbed into the cell, i) do a intracellular cellular content analysis (separate the cells from the culture medium and break the cells with sonication) to analyse the absorbed compounds, and ii) separate the cells from the broth and wash the cells with appropriate solvent to recover the adsorbed compounds from the cell surface then analyse the extract.
Thank you Steph and thavasi. But, i have little confusion that if there is biodegradation the microbes must be utilising it, in that case after sonication and analysing the extract we will not get the same compound. Further, the intermediates or degradation products are not reported in literature..
Using mass spectrometry to analyse the liquid component of your sample will determine the compounds that result from degradation.
Thank you Dr. Mittal. But can you suggest that which sterilisation technique will be favourable to kill the microbes, because for adsorption studies the cell surface should remain intact for dead cells also.
we are talking about two phenomena with completely different time scales, adsorption occurs in a few seconds/minutes and since you didn´t observed cell growth the equilibrium should be achieve very quickly.
So, you don´t need to kill cells, just take samples immediately after mix the substrate with the cell culture, and after that during a few minutes until you reach a constant value. I don´t know what compound and microorganisms are you working with, but for instances with phenol and a fungus mycelium in a bach reactor I did obtain more than 50% adsorption in the first seconds of contact....depending on the ratio biomass/phenol concentration. This is a very simple experiment to do.
Thank you Dr. Mittal. But , if the adsorption is not occuring on the surface as biomass growth is not observed, but the concentration of target compound is decreasing, it may happen that the microbe must be secreting some sort of extracellular enzymes which are degrading the compond. In that case, sonication of biomass and analysis cell lysate for analysing that compound may not be significant.
Exactly, Ana maria. I am agree with you, but my case is different. Because i am not getting the degradation during first few seconds or minutes, but I have anaysed the samples after attaining the complete equilibrium, i.e after hours (days) of growth and the system is kept in incubator shaker with constant shaking throughout the experiment. I am also agree with Dr. Mittal that the adsorbtion depends on several factors. Time is only one of the factors.
May be I didn]t express myself well, what I want to propose it was a simple experiment, that could give some light on the question..... As I wrote, there is no information about which compound, compound concentration , biomass concentration , at least these is very important in terms of kinetic degradation , adsorption and substrate up/take. From what I understand the only measurements were the substrate concentration in the medium and OD for biomass, and so this is a simple experiments that can me carried out easily with the same parameters.
Of course we are dealing with a very complex phenomena chemical, physical and biological which depends of several factors, where follow the concentration of the substrate in the liquid medium is very limited. But what is here is question is growth or no growth, and what happens in the first minutes of the contact with substrate and biomass is very important to study and results can give a clue....on that.
There are lots of other ways to follow the uptake and eventual assimilation of of the substrate, but are much more expensive experiments.
Thank you ana. I got what you want to convey. and Thanks to all, i will get back over it after few more experimentations.
It is also possible for the ion-exchange mechanism to occur.. If your compound is metal, it is possible for the metal ions exchange with the positive ions on the surface of biomass.
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