Chapter 11
Physiological Methods
Todd R. Sandrin, David C. Herman and Raina M. Maier
Information Box 11.1 Measurement of Microbial Biomass in Soil—The Chloroform Fumigation Method
Step 1. Collect a representative soil sample (see Chapter 8).
Step 2. Perform a fumigation experiment as outlined:
To determine Fc:
1. Fumigate a portion of the soil sample with chloroform.
2. Inoculate.
3. Incubate for 10 days.
4. Determine CO2 as an estimate of biomass mineralization 5 Fc.
To determine Ufc:
1. Incubate a second portion of the soil sample for 10 days.
2. Determine CO2 as an estimate of basal mineralization levels 5 Ufc.
Step 3. Estimate Kc by performing a mineralization experiment using
Section 11.2.4).
14
C-labeled glucose and a soil inoculum (see
TABLE 11.1 Several Common Methods Used for the Measurement of Microbial Activity in Environmental Samples a
Test
Basis of test
Measurement
of respiration
gases
Measurement of oxygen
Basal respiration
utilization or CO2
measurements reflect
production in an environmentalmicrobial metabolism of
sample.
organic substrates present
The flux of respiration
in the environment.
gases provides an
However, respiration by
indication of overall metabolic other components, such
activity, which
as plant roots in soil or
can reflect the level of
algae in water, will
microbial activity.
also be included,
depending on the
environment.
Respiration of
radiolabeled
substrates
Metabolism of a
radiolabeled substrate
is monitored by
measuring the evolution
of labeled CO2.
This method is used to
The use of radiolabeling imparts a The concentration of
determine the potential
high sensitivity
substrate added is often
for metabolism of a
to the measurement, thus
greater than the
foreign substrate, such as an short incubation periods
concentration present in the
organic pollutant. Also, overall can be used. The use of
environment; thus the rate
heterotrophic potential can be specific radiolabeled compounds of metabolism may be
estimated by determining the shows the
overestimated unless
turnover of organic
potential for degradation of that corrective procedures are
substrates that occur naturally specific substrate.
used.
in the
environment.
Microelectrodes
Probes with tips ,20 mm
Microelectrodes have
Can monitor real-time
The instrumentation is
been designed to
activity at critical interfaces
delicate and relatively
measure specific
in biological systems. Especially expensive.
respiratory activities, including useful to monitor
oxygen
the interdependence of
in diameter can be
inserted into environmental
samples to provide a
Application
Advantages
Disadvantages
Field chambers can be
Incubation of samples in
built and installed in situ,
closed chambers
to monitor flux of respiration
(microcosms) in order to
gases in relatively
monitor the flux of
undisturbed samples.
respiration gases can create
The addition of an organic
an artificial environment.
substrate to the sample
CO2 production can be
can be used to indicate the level of underestimated due to pHpotential microbial activity.
dependent retention of
inorganic carbon as
bicarbonate.
Incorporation of
radiolabeled
thymidine into
cellular DNA
Adenylate
energy
charge (AEC)
continuous monitoring of
activity.
utilization and nitrate
respiration.
aerobic and anaerobic processes in
the
environment.
Microorganisms will scavenge The rate of DNA
When using a short
Not all bacteria will
DNA precursors, such as
synthesis provides a
incubation time, thymidine
incorporate exogenously
thymidine, from their
reasonable estimate of
incorporation is thought to
supplied thymidine into
environment. By radiolabeling the rate of cell division,
measure bacterial DNA
DNA. Also, estimating
thymidine,
thus providing an
production because the rate of
microbial activity requires
the rate of incorporation
estimate of microbial biomass bacterial incorporation of
the development of a
into DNA can be
production.
thymidine is thought to be much conversion factor relating
measured.
faster than for other organisms
thymidine incorporation to
which may be present in
biomass production.
environmental samples.
AEC is a weighted ratio
of ATP to total adenylates.
ATP is quantified using a
luciferin–luciferase substrate–
enzyme
system.
AEC values reflect a
continuum between an
active microbial
community (AEC . 0.8)
and a community with a
high proportion of dead
or moribund cells (AEC , 0.4).
AEC can establish the
AEC is not necessarily a
presence of a metabolically active direct measure of microbial
community without
activity because adenylates
the need to incubate the sample or are present in all living
add a surrogate substrate.
organisms and can also be
released by decaying cells.
Dehydrogenase
assay
This assay measures
The rate of reduction of
Actively respiring
The amount of tetrazolium
the rate of oxidation–reduction tetrazolium salts reflects the microorganisms can be visualized salts reduced depends on
reactions (electron transport overall activity by all respiring microscopically
many factors including
chain activity) by monitoring microorganisms.
by the deposition of
sample incubation
the reduction of a tetrazolium
pigmented tetrazolium salt
conditions. Direct
salt by
reduction products within the cell. comparison of activity
actively respiring
The assay has a high sensitivity, between samples can be
microorganisms.
and can be used to measure
made only when the assay
activity in low productivity
is performed under
environments.
identical conditions.
Hydrolysis of
fluorescein
diacetate
Hydrolysis of fluorescein
diacetate is performed
by a variety of enzymes
including esterases, proteases,
and lipases.
The assay measures enzymatic Fluorescein diacetate hydrolysis
activity, thus providing an
produces a highly fluorescent
estimate
product, fluorescein, which is
of total microbial activity
easily detected.
in an environmental
sample.
Enzymes involved in
hydrolysis can be
intracellular or
extracellular.
Stable isotope
probing (SIP)
An isotopically labeled
(e.g., 13C) substrate is
added to a sample.
Analysis of 13C
distribution into DNA
(DNA-SIP) or cell lipids
(PFLA-SIP) provides
information about the active
populations in the community.
This technique identifies the
populations within
a community that are actively
metabolizing
the labeled substrate added.
Links phylogeny with
function even if the
functional populations
cannot be cultured. Can
be used to measure
carbon flow through
trophic levels in the environment.
PFLA-SIP is more sensitive
in terms of numbers but
gives only very general
taxonomic information.
DNA-SIP is less sensitive
in terms of number but
gives precise phylogenetic
information. SIP is
technically difficult and not
yet standardized.
Microarray
A matrix of immobilized
Allows high throughput
nucleic acid probes is
screening of gene expression
used to query the nucleic acid in a sample.
makeup in a sample.
Allows direct and rapid
inquiry of how
microorganisms respond
to and interact with their
environment.
Cost prohibitive in many
cases although
technological advances are
decreasing costs associated
with microarray fabrication.
Data interpretation is also
problematic.
Proteomics
Analysis of the proteins
expressed by a
microorganism under a
prescribed set of
environmental conditions.
a
Allows identification of
Provides the best
Analysis is expensive,
proteins that are differentially information available describing a difficult, and timeexpressed and, thus, likely
microbial response to a stimulus. consuming. The technique
important in microbial
has not yet been made userresponses to environmental
friendly.
conditions.
This table is meant to highlight advantages and disadvantages of these techniques, and should not be considered as comprehensive.
TABLE 11.2 General and Specific Enzyme Assays That Can Be Used to Measure Microbial Activity
Enzyme
Substrate
Description of assay
Dehydrogenase
Triphenyltetrazolium
Dehydrogenases convert triphenyltetrazolium chloride to triphenylformazan; the
triphenylformazan is extracted with methanol and quantitated spectrophotometrically.
Phosphatase
p-Nitrophenol
phosphate
Phosphatases convert the p-nitrophenol phosphate to p-nitrophenol, which is extracted
in aqueous solution and quantitated spectrophotometrically.
Protease
Gelatin
Gelatin hydrolysis, as an example of proteolytic activity, can be measured by the
determination of residual protein.
Amylase
Starch
The amount of residual starch is quantitated spectrophotometrically by the intensity of
the blue color resulting from its reaction with iodine.
Chitinase
Chitin
Production of reducing sugars is measured using anthrone reagent.
Cellulase
Cellulose,
carboxymethylcellulose
Production of reducing sugars is measured using anthrone reagent. Cellulases alter the
viscosity of carboxymethylcellulose, a quantity that can be measured.
Nitrogenase
Acetylene
Nitrogenase, besides reducing dinitrogen gas (N2) to ammonia (NH3), is also capable of
reducing acetylene (C2H2) to ethylene (C2H4); the rate of formation of ethylene can be
monitored using a gas chromatograph, and the rate of nitrogen fixation can be
calculated using an appropriate conversion factor.
Nitrate reductase
Nitrate
Dissimilatory nitrate reductase can be assayed by the disappearance of nitrate or by
measuring with a gas chromatograph the evolution of denitrification products, such as
nitrogen gas and nitrous oxide, from samples; denitrification can be blocked at the
nitrous oxide level by the addition of acetylene, permitting a simpler assay procedure.
From Atlas and Bartha, 1993.