M9 Media Broth turning green - (Feb/19/2014 )
There are quite of few options to evaluate the possibility of using the bugs you isolate for bioremediation. Microbes interact with geochemical cycles in so many different ways that it is hard to say what would be the best option for your project.
I cited the redox changes because some elements are quite prone to suffer redox transformations by organisms even if they are not involved in respiration. But looking to some Eh-pH diagrams for W, it is unlikely under neutral oxic conditions, but it will be different in anoxic sediments. This will help http://link.springer.com/article/10.1007%2FBF02650012
With a pH around 7 the question is if the W is really a problem. Is it movable? Can affect to xxx? Many remediation strategies are based on the transformation of some compounds or elements in less movable/soluble forms.
LB culturing as primary isolation method will select for some heterotrophic microbes disregarding if they are capable of withstand W or not. (by the way, culturing temp?) So, it is likely that you will have many colonies that won't be useful for the following tests.... unless you want to use the biomass as a biosorbent system to bind W and you don't care about the viability of the bugs in the soil. Although this approach is more common for treatment of effluents and liquid wastes. e.g. Omar, N. B., Merroun, M. L., González-Muñoz, M. T. & Arias, J. M. (1996). Brewery yeast as a biosorbent for uranium. Journal of Applied Microbiology 81, 283–287.
The measurement of the pH of a soil it is not something universally standardised, just be sure that all the measurements are performed in the same way.
plants too? O_o
@Phil Geis I am primarily addressing tungsten uptake by bacteria in soil. In the same soil I decided to plant some species (of Brassica napus) and see how the control plants, those planted in soil with tungsten but without the bacteria, and the test plants, those with tungsten + bactera, differ in their morphological characters. Apart from that I also plan to assay the activity of the peroxidase enzyme, measure the free proline and total phenol in the test and control. This should give me a basic idea about the bioremediation capabilities of my bugs, or do you think it will Sir? Sir, the mine from where I sampled my soil had the scheelite ore, which is calcium tungstate. But I am using sodium tungstate...will that affect considerably?
@El Crazy Xabi Sir, please elaborate on what is meant by a movable form of tungsten. I have actually never come across this terminology before. Sir, the culturing temperature has been around 35-37 degrees C. I do intend to use my bacteria as a biosorbent system. Sir, please let me know if figuring out the content of tungsten by atomic absorption spectroscopy of a broth culture inoculated with bacteria is an ok way to screen out the tungsten accumulating bugs or not. I intend to detect the levels of tungsten (through AAS) after 24 hours of inoculation and then finally at 74 hours of inoculation. The broths that show a decrease in W at 72 hours as compared to the original W conc would be selected for the bioremediation experiment. Sir, does it sound okay? And Sir unfortunately, I am unable to access the full papers of the references that you sent to me.
I am figuring out a way to attach the picture of the green M9 Broth!
Sodium tungstate is a soluble salt while scheelite is practically not soluble (ppm levels) unless you use acids. That affects to the mobility and bioavailability of the element. The total concentration of an element in a soil may be important but in remediation other fractions as extractables by different methods are also analysed. Any book on soil pollution and/or remediation or soil characterisation handbook should have all this information.
If you culture the bugs at 35-37° C it is OK if you are gonna use them as a biomass sorbent as they are not that likely to proliferate in soil, that temp is really hot for a soil, but also depends on the climate regime.... Releasing broth into the soil I don't think is a good idea. It is high in nutrients and may reach aquifers and cause eutrophication.
For the accumulation, do you use a control with W uninoculated? Measure that as W concentration reference. Sometimes you may get adsorption to the flask or precipitation, and evaporation is a common cause that also changes the concentration, not only the bacteria. But in this case it would be dependent on the amount of biomass (cell density) in your culture, not on their efficiency on absorption/uptake!! For that, you should expose a known amount of biomass (usually given as dry weight basis) under a given W concentration in a osmotically balanced and maybe buffered solution (it can be a mineral base solution used for mineral media preparation; e.g. M9 without C-source nor trace elements)... but avoiding any combination that might cause chemical precipitation, e.g. I cannot use phosphate containing solutions when I test U because it precipitates, which happens with most non-alkaline cations.
By the way... do you plan on remove the bugs from the soil after treatment? Usually biosorbents are use for treating running waters, aquifers or subsuperficial plumes where they are packaged and removed after treatment. Keep in mind that the behaviour of solutions in the lab may be relevant to aquifer decontamination but soils are completely different.
If you need a paper send me a pm
Sir, I kept the culturing temperatures at around 37 degrees as it gets really hot at the place where the mines are situated. The temp touch 48 degress in peak summers. Sir, since scheelite is not soluble, then do you think I would have isolated any relevant bugs? Because I used sodium tungstate to screen them out.
As for the addition of broth directly into the soil, could I dilute it and then release it? Also Sir, I intend to autoclave the soil before adding in the broth, do you think it will reduce the chances of eutrophication?
Sir for bioremediation, I am going to use an uninoculated W concentration as reference. Sir, before I proceed for the bioremediation experiment, I will inoculate a dried biomass of my bugs in the M9 broth, (without a C source) as you said, with a pre-decided conc of tungsten at which my bugs would remain viable and grow (will do MIC to determine the right W concentration). Then with the help of atomic absorption spectroscopy I will determine which inoculated broths show a decrease in W conc after 24 and 72 hours.
Sir, here's a bit of a problem that I would like you to help me out with. How should I separate out my bug's biomass from the inoculated M9 broth cultures without bursting them? I wish to collect only the broth without the cells after 24 and 72 hours of inoculation to check the W conc in it. Should I simple centrifuge them at low rpms, like at around 2000 rpm? So that my cells do not burst and release whatever W they might have accumulated back into the broth that will be collected for AAS analysis?
I will make sure I don't use a broth that may cause precipitation of W.
No Sir, I do not plan on removing the bugs from the soil after treatment. This is actually only a preliminary analysis that I am doing.
Sir could you please provide me with the brewery yeast as biosorbent paper? Also the following paper.
Enterococcus faecalis strain LZ-11 isolated from Lanzhou reach of the Yellow River is able to resist and absorb Cadmium. by Wu G1, Sun M, Liu P, Zhang X, Yu Z, Zheng Z, Chen Y, Li X.
P.S. By the way Sir, I am really very grateful to you for all the help that you are providing me with. I would have hit a roadblock, had it not been for this guidance. :)
here is the paper: http://www.jeb.co.in/journal_issues/201309_sep13/paper_03.pdf
(its free)
p.phadtare on Fri Feb 28 16:51:11 2014 said:
Arsenic accumulating bacteria isolated from soil for possible application in bioremediation.Sir, if you could provide me with this paper too, then it would be great!
Thanks for the paper!!
Can you help me understand the peroxidase, proline and phenol analyses? What purpose do these serve?
Assume you'll use soil - both inoculated and uninoculated. I understand the temperature but wonder if the pH (e.g.) of your experiment is relevant to the mine environment.
If tungsten in the mine is present as the largely insoluble scheelite (or insoluble wolframite), what application do you see for the precipitation of tungsten from the soluble sodium salt solution? Are you addressing some stage of tungsten processing?
Not sure why you'd deny the bugs a carbon source in the M9 medium. Suggest you take supernatant samples at, and various times after, exposure. Run the control on centrifugation - cfu/ml, protein in medium, etc.
@Phil Geis. Sir, I am doing the peroxidase assay to check the stress levels in the plants grown in soil with a high level of tungsten. The peroxidase activity is known as an indicator of heavy metal toxicity in plants. Hence I'd like to compare the peroxidase levels in a negative control with test (with my bacteria) to draw conclusions about bioremediation.
Higher conc of tungsten in the soil results in higher proline contents, hence i'd like to use proline levels as an indicator as well. Similarly for total phenol, it is known to follow the same trend.
Sir, I am providing the bugs a carbon source in the M9 medium in the form of sodium acetate. I intend to take the supernatant samples at 24 and 48 hours of exposure. Sir, I didn't get the last thing that you said, about running the control on centrifugation -cfu/ml, prt... I will centrifuge my samples at about 8000 rpm for separation of the biomass with the supernantant.
Thank you for interest in my queries! :)