THREAD: Estimation and Activity of the Soil Microbial Biomass

EWEN E.F.Mcpherson at uel.ac.uk
Wed Sep 7 04:27:34 EST 1994


   Well, I promised that my next post would be scientific..... 

     
   Following my reply to Dorothea regarding the estimation of
activity, Douglas at Aberystwyth has suggested that we try to
start a thread on the subject. I mentioned one of my papers in
the post, so what I'm  going to do is hack a few para's out of
it RE: Biomass/Activity for the general edification.  
     
   A fundamental measure of the soil microbial community is that
of Biomass. Here are some of the techniques popularly usedfor the
estimation and  ennumeration of organisms in the Soil Microbial
Biomass (SMB) and the measurement of their activity;- I'm not
going to go into too great detail here, and I'm not going to make
criticism or observation of the techniques until the thread gets
going.   
     
     
   1/ DNA/RNA reassociation; By extraction of DNA/RNA from a soil 
sample, magnification and purification of the substances, and  
measuring the homology (and therefore inversely) the
dissimilarity of the sequences by reassociation. The work of
Torsvik has suggested firstly that less than 10% of the soil
microbial biomass is capable of being cultured by recognised
methods, and that the dissimilarity of sequences as shown by high
reassociation times suggests a) a far greater diversity of
species in the SMB than has previously been suspected and b)far
greater numbers of organisms than have been suggested by
"classical" microbiologiocal techniques
     
   2/ Substrate-induced Respiration;-By measuring the conversion
of a known amount of a readily-assimilable carbon source, i.e.
glucose, into Carbon dioxide by respiring micro-organisms, an
estimation can  be obtained both of the carbon extant in the
biomass and of the relative activity of the SMB.  

   3/ Chloroform Fumigation/extraction or Fumigation/incubation
(often in association with Substrate-induced respiration). By
killing off the SMB by fumigation with a biocide (and I include
the various other fumigation techniques here) it is possible to
either directly estimate the amount of soluble Carbon (and
therefore by conversion the SMB Carbon)by chemical methods or
again by respiration (of the C contained in the "dead" biomass
by a survivor/introduced population) to carbon dioxide. It is
also possible to use the soil from this technique to estimate
levels of other elements, such as Biomass Nitrogen, and the
resultant C:N ratio can sometimes be informative of the community
structure.

   4/ Microbial Lipids, particularly Phospholipids. The chemical
extraction,purification and identification of lipids from a soil
sample has been shown to have great potential for not only
characterising the various subsets of the SMB but also measuring
the total biomass, activity and metabolic status of the
community. 
     Phospholipids are a component of all cell membranes. They
are not found as storage lipids, remain in reasonably constant
proportion to the bacterial biomass and rapidly disappear after
cell death. However, there are persistent doubts regarding the
utility of PL as a measure of microbial biomass due to its lower
concentration in fungi than bacteria . If so, any change in
proportions of these two groups would also produce an apparent,
though artificial change in total biomass.

   5/ Ergosterol; Ergosterol is a sterol specific to fungi, and
can be readily extracted and purified from a soil sample, and by
HPLC, a measure of the fungal biomass can be obtained.

   6/ "Classical" Microbiology techniques. Yes, your friends and
mine, The various techniques derived from medical microbiology
using differential agars, inhibitors and so on that we all did
in Microbiology 101. These techniques come in for a lot of
criticism, especially in the light of "sexier" techniques like
DNA homologies, but for a good basic look at what;s in there, you
can't beat a Plate Count on NA, PDA or SXA. Although it has been
suggested that less than 10% of the SMB is cultivatable by these
techniques, the use of more complex agars and inhibitors or
promoters has extended the utility.

Methods for the estimation of activity;-

  1/ Adenosine Tri-phosphate. By shattering the cells in a sample
and liberating the ATP present, a luminometric technique can
estimate the ATP present,yielding both a measure of the activity
of the sample and of the relative size of the soil microbial
biomass.

     (As I've said, I'm not going to indulge in criticism of
techniques Yet. Personally, I do not like ATP as a measure, but
I'ld like to see what the rest of you have to say first..)

  2/ Estimation of Dehydrogenase Activity.
This technique measures the activity of dehydrogenase (DHA)
enzymes.  These enzymes are produced by the soil microbial
community to enable them to breakdown organic matter. 
Measurement of the activity of these enzymes over a given time
will reflect the level of microbial metabolic activity in the
soil.

Dehydrogenase enzymes catalyse the dehydrogenation (the removal
of hydrogen) from organic compounds, a fundamental step in their
breakdown.  There are many dehydrogenase enzymes.  They operate
endogenously (within the cell), and each one is highly specific
for a particular reaction.  The general formula for the process
is:
     XH2 + A  =  X + AH2

where XH2 is an organic compound and A is a hydrogen acceptor. 
The reaction releases electrons, which are normally captured by
oxygen.

However, as we've seen from Dorothea's post, there are problems
associated with using this method in ceratin environments, and
I also have further criticisms about its application.

This test utilises this release of electrons to indirectly
measure the amount of dehydrogenation that has resulted from the
catalytic action of the enzymes.  It does so by providing an
alternative to oxygen as the normal electron acceptor.  The
chemical used is 2,3,5-Triphenyltetrazolium chloride (TTC), a
colourless and water-soluble quaternary ammonium salt which is
readily reduced (captures electrons) to form Triphenyl formazan
(TPF), an intensely red and methanol soluble product (Fig. 1). 
Competition between TTC and oxygen is minimised by immersing the
soil in water in which ample TTC has been dissolved.


Right, that's a short and admittedly incomplete list of some of
the techniques used in Soil Microbial Ecology for the estimation
of the Size and activity of the SMB.

What I'ld like to do now is start the thread like this;-
1/ What techniques do you use for estimation of the SMB ?
2/ Can you justify chosing them or do you, like me, have problems
with some of the assumptions that these STANDARD techniques 
make ?
3/ Are you working on any Alternative techniques ? Please don't
be afraid to put them up for examination/discussion/assassination.

4/ Lets consider theory and concepts. Douglas at Aberystwyth said
this to me;-
     " I get very nervous when people talk about viable-but-not-
culturable (since viable MEANS culturable) when what they mean
is 'showing metabolic activity under some condition but not
capable of dividing under any condition that WE tried'".

     Well, me too. How about the rest of you ? Why ?

5/ What do you take the phrases "Biomass" and "Activity of the
Biomass" to encompass ? Is there such a thing as Viable and Non-
Viable biomass ? Does Biomass-Carbon encompass intra- and extra-
cellular metabolites ?


Right, that's enough from me for just now. Lets hear your
thoughts and get this discussion Moving.

High Thoughts, Colleagues,

Regards,

Ewen
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