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Microbial Carbon and Nitrogen


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#1 TNM

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Posted 05 July 2009 - 03:51 AM

I'm trying to measure microbial C and N from soil samples using the chloroform technique. I've tried both the CHCl3-fumigation and direct addition with CHCl3 technique but unable to detect any differences between the control (unfumigated samples) and fumigated samples. I've tried incubating the samples for 1d, 5d, and 14d but unable to detect any differences. I'm analysing the samples using the titration method for microbial C and spectrophotometer for microbial N as i don't have acces to a TOC analyser. Running out of ideas here.
Most protocols mentioned that EtOH-free CHCl3 has to be used. How important is it and why? Please advice. Also, what is the simplest method for removing EtOH from CHCl3? Clearly i must be doing something wrong. would really appreciate it if anyone can provide me with a working methodology that yields results....desperately need help as time is of the essence here.

#2 GeorgeWolff

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Posted 05 July 2009 - 04:42 AM

Can you provide a reference for this methiod? Are you trying to quantitate lipid C and N?
I'm not familiar with a chloroform extraction procedure that would extract all C and N molecules. to the latter, a micro Kjedahl application would prob be the best.
How did you (try to) validate your methods?

#3 TNM

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Posted 07 July 2009 - 02:46 AM

Hi.
Thanks for your reply and query.

I'm trying to extract microbial carbon and nitrogen after chlorofom fumigation with 2M KCl (for microbial N) and 0.5M K2SO4 (for microbail C) respectively. Similarly this is done with the controls or unfumigated samples.

For microbial N, samples are diluted in 2M KCl (1:4 ratio) and measured spectrophotometrically at 570 nm.

For microbial C, samples are analysed colorimetrically via titration. For the lattter, samples are digested in potassium dichromate and concentrated H2SO4 followed by the addition of ferroin indicator (1,10-phenanthroline ferrous sulfate) and titrated using 3.3 mM ferrous ammonium sulfate.

The difference between fumigated and unfumigated samples are meant to indicate microbial N and C respectively.

Problem is, no differences are noted between either fumigated and unfumigated samples. Ive repeated the experiment with incubation with chloroform for 1, 5 and 14 days. Ive even tried introducing bacterial culture into the soil thinking that the inability to detect differences may be attributed to the lower sensitivity of the assay. Hence i thought by increasing bacterial counts in soil may result in a noticeable difference between fumigated and unfumigated samples. But unfortunately, no such luck in detecting any positive differences.

Reference for Microbial C:
Nelson DW, Sommers LE (1982) Total carbon, organic carbon and organic matter. In Page AL, Miller RH, Keeny DR (Eds). Methods of soil Analysis. part 2. Chemical and microbiological properties.

Li Q, Lee Allen H, Wollum AG (2004) Microbial biomass and bacterial functional diversity in forest soils: effects of organic matter removal, compaction, and vegetation control. Soil Biology & Biochemistry; 36: 571-579.

Reference for Microbial N:
Amato M aand Ladd JN (1988) Assay for microbial biomass based on ninhydrin reactive nitrogen in extracts of fumigated soils. Soil Biol Biochem. 20: 107-114.

In response to validating the data, I'm first trying to establish the working methodology based on the protocol which i've summarised above which i must say has been published in numerous other publications. Hence clearly it is meant to generate positive results. Anyhow, if it works i then intend to send the samples to another laboratory for validation using a TOC/TIC analyser (for microbial C) and Lachat flow injection autoanalyser (for Microbial N). Of course it would be easier for me to send the samples over but the fees they charge costs a bomb.

Appreciate any input and suggestions!

#4 GeorgeWolff

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Posted 07 July 2009 - 10:52 AM

I understand these methods have been published (haven't access to enire paper) but I fail to see the technical basis for quantitative and apparently exclusive extraction of total microbial N or C using salt solutuions.
Would you please explain the technical basis?

Edited by GeorgeWolff, 07 July 2009 - 10:53 AM.


#5 303microbialist

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Posted 07 July 2009 - 12:31 PM

Hi.
Thanks for your reply and query.

I'm trying to extract microbial carbon and nitrogen after chlorofom fumigation with 2M KCl (for microbial N) and 0.5M K2SO4 (for microbail C) respectively. Similarly this is done with the controls or unfumigated samples.

For microbial N, samples are diluted in 2M KCl (1:4 ratio) and measured spectrophotometrically at 570 nm.

For microbial C, samples are analysed colorimetrically via titration. For the lattter, samples are digested in potassium dichromate and concentrated H2SO4 followed by the addition of ferroin indicator (1,10-phenanthroline ferrous sulfate) and titrated using 3.3 mM ferrous ammonium sulfate.

The difference between fumigated and unfumigated samples are meant to indicate microbial N and C respectively.

Problem is, no differences are noted between either fumigated and unfumigated samples. Ive repeated the experiment with incubation with chloroform for 1, 5 and 14 days. Ive even tried introducing bacterial culture into the soil thinking that the inability to detect differences may be attributed to the lower sensitivity of the assay. Hence i thought by increasing bacterial counts in soil may result in a noticeable difference between fumigated and unfumigated samples. But unfortunately, no such luck in detecting any positive differences.

Reference for Microbial C:
Nelson DW, Sommers LE (1982) Total carbon, organic carbon and organic matter. In Page AL, Miller RH, Keeny DR (Eds). Methods of soil Analysis. part 2. Chemical and microbiological properties.

Li Q, Lee Allen H, Wollum AG (2004) Microbial biomass and bacterial functional diversity in forest soils: effects of organic matter removal, compaction, and vegetation control. Soil Biology & Biochemistry; 36: 571-579.

Reference for Microbial N:
Amato M aand Ladd JN (1988) Assay for microbial biomass based on ninhydrin reactive nitrogen in extracts of fumigated soils. Soil Biol Biochem. 20: 107-114.

In response to validating the data, I'm first trying to establish the working methodology based on the protocol which i've summarised above which i must say has been published in numerous other publications. Hence clearly it is meant to generate positive results. Anyhow, if it works i then intend to send the samples to another laboratory for validation using a TOC/TIC analyser (for microbial C) and Lachat flow injection autoanalyser (for Microbial N). Of course it would be easier for me to send the samples over but the fees they charge costs a bomb.

Appreciate any input and suggestions!



I'd switch to an ATP assay if you can. We use the chloroform fumigation method too, but it I'm not inclined to believe in it based on comparisons with DNA extraction concentrations and community membership/structure data.

Edited by 303microbialist, 07 July 2009 - 12:43 PM.


#6 303microbialist

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Posted 07 July 2009 - 12:42 PM

I understand these methods have been published (haven't access to enire paper) but I fail to see the technical basis for quantitative and apparently exclusive extraction of total microbial N or C using salt solutuions.
Would you please explain the technical basis?



I don't do the bench work for this analysis in my lab, but the general idea is that the first unfumigated run measures all the free (non-intercellular) C or N in the soil sample. Then the second run where you fumigate with chloroform releases "all" cellular (not just lipid) C and N into the solution. By taking the difference between the two samples you are supposedly left with a measure of C and N that was initially inside intact living cells. Of course as you mention, C containing molecules that don't dissolve in the salt solution will be left out.

One advantage over the ATP assay is that you could potentially infer relative abundances of fungi/arthropods vs bacteria/archaea (chitin=higher N ratio)... DNA assays are of course confounded by DNA from dead cells...

Edited by 303microbialist, 07 July 2009 - 12:46 PM.


#7 GeorgeWolff

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Posted 08 July 2009 - 01:34 AM

If the work carries your name in publication, you should be prepared to defend it. On its face, the method does not appear to be appropriate to the conclusions you propose to draw. Of all ther diverse proteins, carbohydrates, lipids, extracellular material, etc. in soil - to presume a quantitative and even differential extraction by a salt solution is pretty hard to accept. To your example, no way is chitin solubilized by the protocol.

Curious - with what are you "fumigating" the soil and how is that to be done?

#8 303microbialist

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Posted 08 July 2009 - 01:31 PM

If the work carries your name in publication, you should be prepared to defend it. On its face, the method does not appear to be appropriate to the conclusions you propose to draw. Of all ther diverse proteins, carbohydrates, lipids, extracellular material, etc. in soil - to presume a quantitative and even differential extraction by a salt solution is pretty hard to accept. To your example, no way is chitin solubilized by the protocol.

Curious - with what are you "fumigating" the soil and how is that to be done?


Absolutely no way chitin or peptidoglycan is going to be solubilized. To clarify, we report dissolved microbial carbon and nitrogen as an indicator of microbial biomass. It might not be an exact quantification, but I do think you can see changes across samples that reflect overall microbial abundance. I want to change how we do this, but I'm not exactly in charge around here. We simply mix the liquid chlorofom into the soil sample to disrupt the cell membranes, then vacuum out. We don't report the C:N ratio as measure who makes up the biomass, but we do get a good bit of variance between samples...

#9 GeorgeWolff

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Posted 08 July 2009 - 03:48 PM

I see "fumigation" means adding and subsequently evaporating chlrooform.

But why do you think this? What is the technical basis? Has your lab validated this with artificial mixtures in soil?

#10 303microbialist

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Posted 08 July 2009 - 04:14 PM

I see "fumigation" means adding and subsequently evaporating chlrooform.

But why do you think this? What is the technical basis? Has your lab validated this with artificial mixtures in soil?


By validation you mean like trying this on some soil mixture that we know has no microbes in it and seeing if we are getting a difference in fumigated vs non-fumigated treatments? One reason to think it works, is because unlike the starter of this topic, we always get a difference between fumigated and non-fumigated treatments of the same sample indicating the addition of cellular contents to solution after chloroform mediated cell lysis. I will go through and read the lit on this methodology, since I'm getting ready to write my first paper as a primary author, and will include some of these data. We certainly did not invent this technique though.

Edited by 303microbialist, 08 July 2009 - 04:15 PM.


#11 GeorgeWolff

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Posted 09 July 2009 - 01:16 AM

I understrand you to report C/N ratios in salt extracts of "fumigated" soils correlate to relative viable biological populations - bacteria, fungi, etc. I'd certainly establish different populations in soils to extract and validate that the method works - it's accuracy, precision, range as well as it's applicability to different species and forms (e.g. fungal spores).




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