This document discusses various techniques used to measure microbial biomass and metabolism in soils and aquatic environments. Key methods mentioned include measuring ATP, total adenylates, cell wall components, chlorophyll, DNA, protein, and phospholipid fatty acids. Physiological approaches include chloroform fumigation and substrate-induced respiration to estimate biomass. Techniques for assessing metabolism include measuring heterotrophic potential, productivity, decomposition, growth rates, photosynthesis, and respiration via carbon dioxide production or oxygen consumption. Specific enzyme assays can also provide insights into microbial community function and biogeochemical cycling.

1. DETERMINATION OF MICROBIAL
BIOMASS

3. • Examination of microbial biomass has been a very
common technique used in soil/aquatic
microbiology, particularly before the availability of
DNA sequencing.
• Although measurements of microbial biomass
provide information about microbial abundance,
they cannot provide information about
which microbes are present or whether they are
active.

4. Biomass an important ecological parameter.
It represents the quantity of energy stored in the
particular segment of biological community.
Biomass that is mass of the living material can be
expressed in units of weight (grams) or units of
energy (calories or joules).
Direct measurements of environmental samples is
not always possible.
The techniques used also measure mineral and
detritus particles and non-microbial biomass along
with microbes .

5. • The microbial biomass of soil is defined as the part
of the organic matter in the soil that constitutes
living microorganisms smaller than the 5-10 um3.
• It is generally expressed in the milligrams of
carbon per kilogram of soil or micrograms of
carbon per gram of dry weight of soil.
• Typical biomass carbon ranges from 1 to 5% of soil
organic matter.
• The degradation of organic compounds, such as
industrial chemicals and pesticides, can be
monitored by following changes in the soil
microbial biomass.

6. Biochemical Assay for Biomass
• Assay of specific biochemical that indicates the
presence of microorganisms.
• All microbes should have the same quantity of the
biochemical being assayed.
• There is direct correlation between the amount of
biochemical being measured and the biomass of
microorganisms.
• It is important that the biochemical to be
measured should be present only in the biomass
to be determined.

7. ATP and Total Adenylate Nucleotides:
• Present in all cells. Can be
measured with great
sensitivity.
• Measurements are for living
cells.
• Luciferin luciferase assay to
detect ATP
• HPLQ can also be used to
measure ATP.
• Method used to extract ATP
has marked effect on
sensitivity and reliability of
the assay.

10. • A factor of 250-286 is often used to convert ATP to
cellular carbon for aquatic samples.
• For soil factor of 120 is used to convert ATP
carbon to biomass.
• Some difficulties in accurately estimating biomass
based on ATP measurements.
• ATP conc may change due to nutritional or
physiological changes.
• ATP are sometimes absorbed on particles of soil
etc.
• Presence of plant and animal cells may limit
application on the method in some ecosystem.
• So total adenylate can be measured

11. • A(Total) = ATP + ADP + AMP
• The total Adenine usually remains constant and can
be used to measure both the numbers and biomass.
• Cell wall components
• Release of lactate from muramic acid and conc of
lactate is measured by enzyme or chemical assay.
• Gram+ bacteria have a ratio of 44μgMA/mg C.
• Gram –bacteria have a ratio of 12μgMA/mg C.
• It is necessary to estimate the proportions of Gram +
and Gram- bacteria in the sample
• Gram –bacteria has lipopolysacharide in cell walls that
can be quantitated by LPS method

12. • Limulus amoebocyte lysate method
• An aqueous extract from blood cells of horseshoe
crabs that reacts specifically with LPS to form a turbid
solution. Degree of turbidity is directly proportional to
lipopolysacharide conc. Can be quantitatively
measured. Mostly done for Gram-bacteria in samples.
• LPS method is very simple can detect cells as low as
10cells/ml.
• For fungal biomass chitin can be measured.

13. • Chlorophyll and other photosynthetic pigments:
• Photosynthetic algae and cyanobacteria can be
measured by photosynthetic pigments.
• Chlorophyll a extracted with solvents like acetone
or methanol and quantified by measuring
absorbance at 665m wavelength.
• Purple photosynthetic bacteria at 850nm.
• Chlorophyll content can be measured by
spectrofluorometry, which is more selective than
spectrophotometry.

14. • DNA:
• DNA conc. are relatively constant so can be used for
biomass measurements.
• In environmental samples for accurate determination
reactions wit fluorescent dyes like ethidium bromide
and spectrofluorometry are usually performed.
Purification is necessary and also removal of any
eukaryotic DNA.
• Protein
• Lowry method used for bacterial proteins but only
when background levels are negligible.
• Lipid: Polar lipids in membranes are measured for
viable cells. Ergosterol for fungi.
• PLFA phospholipid ester-linked FA analysis done.

15. Physiological approaches for
biomass

16. • Mostly based on respiratory activities.
• To select the most suitable method for determining
microbial biomass in soils both the chloroform
fumigation-incubation (FI) method and the
substrate-induced response (SIR) method have been
used Fumigating soil with chloroform and then
measuring the CO2 released from the mineralization
of microbes killed after fumigation.
• Non-fumigated soil incubated under same condition
act as control.
• Amount of Microbial C is calculated from the
difference between the CO2-C evolved from
fumigated and non fumigated samples.

17. • The soil is fumigated with chloroform to kill the
microbial population.
• After the microbes are killed by fumigation,
cytoplasm is released into the soil environment.
• The soil microbial biomass carbon is extracted
with potassium sulfate on both fumigated and
non-fumigated soil.
• The carbon content of the extract is tested and the
biomass is calculated based on the difference
between the carbon content of fumigated vs. the
non-fumigated soil.
• The carbon content is measured by dichromate.

18. • Non-physiological way is to extract organic carbon
from the fumigated soil by a 0.5M K2SO4 solution
and measured by dichromate oxidation or any
other analysis (DOC).
• Extracts from non-fumigated soil are also analyzed
and subtracted as background.
• This method is faster and better in acidic soil.
• However this is incomplete as cell walls and other
insolubles are left behind.
• Most of the times the average 33% of the biomass
organic C was extracted.
• But efficiency range is from 20 to 54%.
•

19. • Respiration rate by substrate addition for biomass
measurement.
• Peak respiration rate is assumed to be
proportional to the number of viable
microorganisms in the sample.
• Use of microbial inhibitors can help to obtain
separate estimates of bacterial and fungal
biomass.
• The results correlate with chloroform fumigation
method.

20. MEASUREMANT OF MICROBIAL METABOLISM
• Heterotrophic Potential:
• To measure uptake rates of radioactive labeled
substrates to determine the heterotrophic
potential for the utilization of that substrate.
• Rates of uptake increases with increasing
concentration of substrate to a maximal uptake
rate (Vmax).
• Vmax can be calculated by plotting a curve rates of
uptake of radiolabel C against their concentration.

21. • Turnover time of the substrate can be calculated.
• Different conc of radiolabeled substrate are
added to samples and incubating the under
conditions that stimulate the real environment.
• After incubation the cells are collected on a filter
paper and incorporated radioacitivity is counted
by liquid scintillation.
• The method was later modified to take in
account the amount of C metabolized. So
Radioactive CO2 produced during respiration is
trapped and added to the counts of incorporated
C into the cells.

22. • Measurement of heterotrophic potential also
assumes that members of the population
respond in the same way to different variation
of the solute concentration.
• The greater the percent respiration, the
greater the metabolic energy used to maintain
the microbial population.
• The lower the percent respiration the greater
the proportion of metabolic energy used for
assimilation and growth.
• The method gives an estimate for specific
heterotrophic activity for a particular
substrate not for overall heterotrophic
activity.

23. • Heterotrophs use reduced carbon compounds to
build their cell material and in most cases (an
exception are the photoheterotrophs) the carbon
compound fulfills a dual function, namely, it acts as
both a carbon and an energy source.
• In some fermenting organisms reduced carbon
compounds can act as terminal electron acceptors.
• Typically, heterotrophic cells utilize the same
carbon source for both purposes, oxidizing a part of
it to CO2 (a process called dissimilation) and using
the energy derived from this oxidation to
synthesize cell material from the other part
(assimilation).

24. • The ratio of dissimilated to assimilated carbon is
essentially dependent on the degree of reduction
of the carbon substrate used.
• The more oxidized the carbon compound, the
more of it that has to be dissimilated in order to
provide the necessary energy to drive the
synthesis processes and the less of it that can be
assimilated.
• This is reflected in the maximum growth yield
observed for different carbon sources when
plotted as a function of their energy content (i.e.,
their degree of reduction, or heat of combustion)

25. Productivity and Decomposition:
• Bacteria degrade a large number of organic substrates
and using labeled sugars amino acids, lignocellulose,
organic acids and other dissolved and particulate
substrates so bacterial uptake, respiration and turnover
of organic compounds provides data on the
decomposition and the flow of organic carbon.
• Microbial productivity has been conducted using 13C
isotope. C and N isotopes can be simultaneously
analysed by gas chromatography-mass
spectrophotometry (GC-MS).
• C13 isotope has lower sensitivity so requires larger
volumes and longer incubation

26. • Growth Rate Measurements Based on Nucleotide
Incorporation:
• DNA is proportional to biomass the rate of synthesis
reflects the growth rate of microbes.
• Incubation is done with tritiated Thymidine and
autoradiography of samples helps to determine the
rates of nucleotide incorporation.
• Radiolabeled nucleotides incorporated in RNA and
DNA have been analysed.
• The [3H]thymidine labels only bacterial DNA and
makes it very useful for studies aimed at examining
bacterial productivity.
• All growing bacteria utilize tritiated thymidine.
Labeled DNA makes up about 80%of the total labeled
macromolecules.

27. • Photosynthesis:
• Rate of primary production can be measured by
radiotracer.
• Both heterotrophic and autotrophic assimilation of
CO2 can be measured using radioactive labeled
bicarbonate by incubating the sample containing the
indigenous microbial community and determining the
amount of labelled CO2 assimilated into the cellular
organic matter.
• Cells are filtered and scintillation counting done.
• Washing filters remove unincorporated labeled
bicarbonate.
• The residual C containing organic compounds can be
oxidized with dichromate and the released CO2
trapped and quantitated.

28. • In actual field study, measurements are done in
dark bottles to differentiate the actual
photosynthetic, heterotrophic-
chemolithotrophic incorporation of 14CO2.
• Both bottles filled with water samples,
radiolabeled bicarbonate is added, bottles are
incubated for sevral hrs.
• Incorporation results from dark are subtracted
from incorporation results in the light bottles to
obtain net photosynthetic incorporation.
• Unlabeled bicarbonate in the samples is
measured to calculate the actual specific labeled
CO2 activity in the water samples.

29. Time frame for incorporation is important.
• Respiration:
• Radiolabeled CO2 released from labeled
substrates can be used to determine
decomposition rates for specific substrates.
• Mineralization is the process by which chemicals
present in organic C/matter are completely
degraded or decomposed or oxidized into easily
available forms to plants and organic carbon is
converted to CO2 by respiration. Transformation of
organic molecules in soil is mainly driven by its
microbiota such as fungi and bacteria along with
earthworms

30. • The rate of carbon dioxide production is commonly
used as a measure of microbial activity in the soil.
• The traditional method of CO2 determination involves
trapping CO2 in an alkali solution and then
determining CO2 concentration indirectly by titration
of the remaining alkali in the solution.
• This method is still commonly employed in
laboratories throughout the world due to its relative
simplicity and the fact that it does not require
expensive, specific equipment.
• However, there are several drawbacks: the method is
time-consuming, requires large amounts of chemicals
and the consistency of results depends on the
operator's skills.

31. • With this in mind, an improved method was
developed to analyze CO2 captured in alkali
traps, which is cheap and relatively simple,
with a substantially shorter sample handling
time and reproducibility equivalent to the
traditional titration method.

32. • A comparison of the concentration values
determined by gas phase flow injection analysis
(GPFIA) and titration showed no significant
difference (p > 0.05), but GPFIA has the advantage
that only a tenth of the sample volume of the
titration method is required.
• The GPFIA system does not require the purchase of
new, costly equipment.
• Furthermore, GPFIA for CO2 analysis can be equally
applied to samples obtained from either the
headspace of microcosms or from a sampling
chamber that allows CO2 to be released from alkali
trapping solutions

33. • Soil respiration is often assessed by measuring
changes in carbon dioxide (CO2) concentration
within a controlled volume over some period of
time, and rely on either spot samples or
integrated measurements.
• Methods not using radiolabeled substrates
employ rates of oxygen consumption or rates of
CO2 production.
• In aerobic condition CO2 evolution
measurement gives accurate results.
• For long term studies, rates of CO2 production
are determined using specially designed
enclosed flasks Biometer flasks.

34. • Flow through incubation system such as gas trains,
that pass a stream of CO2 free air through the flask
and trap CO2 from the effluent gas stream.
• The trapped CO2 can be quantitated by titration of
the trapping base solution with acid of known
concentration.
• Rates of oxygen consumption can also be
measured.
• Oxygen electrodes suitable for short-term
measurements.
• Microprobes used for in situ measurements.
• A new generation highly automated respirometers
has been developed

35. • It’s a computer assisted for data recorded and
plotting.
• The processor can monitor oxygen consumption
allowing for replication or variation of the
experimental conditions.
• Time saving experiments but initial cost is high.
• Specific Enzyme Assays
• A variety of enzyme assays can be used for measuring
the metabolic activities of indigenous microorganisms.
• Dehydrogenase, chitinase, nitrogenase, cellulase
denitrification enzyme activities, can assay the
metabolic function of small but important segments of
microbial community.

36. • Enzymes involved in biogeochemical cycling are
important and for maintaining community and
ecosystem.
• Different enzymes are measured by different
methods.
• For nitrogenase enzyme acetylene reduction test.
• Some general assays have been developed like
hydrolysis of fluorescein diacetate to measure
activities of lipases, proteases and esterases.
• For in situ activity studies assays should be done not
to alter the community.
• So care should be taken for temp. moisture content,
redox potential, incubation periods so as not to alter
the levels of enzyme present.