This document discusses the use of stable isotopes and biomarkers to study microbial ecology. It describes how stable isotope tracers can be incorporated into microbial biomass and detected in biomarkers to identify active microbial populations. Specifically, it discusses how gas chromatography-combustion isotope ratio mass spectrometry (GC-c-IRMS) allows analysis of stable isotope ratios in specific biomarkers. It also summarizes some applications of stable isotope probing including linking populations to functions and measuring nitrification rates.

1. PRAKASH RANJAN BEHERA
ID- 20162007

2. • Isotope - Atoms with the same number of protons
but different number of neutrons are called isotopes.
• By changing the number of neutrons, isotopes still
maintain the same overall neutrality and hence the
chemical behavior remains unchange.
• Some elements have several isotopes while others may
have only one isotope.
• For example, there is only one isotope for the
element cesium while the element calcium can have six
isotopes found in nature. The simplest isotopes being
those of hydrogen

3. Application
in
agricultural
microbiology

4. Stable Isotopes and
Biomarkers in Microbial
Ecology

5. BIOMARKERS
• Biomarkers are compounds that have a
biological specificity in the sense that they are
produced only by a limited group of
organisms. such as fatty
acids and ether lipids

7. PRINCIPLE
• stable isotope tracer is incorporated into the
biomass of the metabolically active
populations, which can be detected in a variety
of biomarkers. By comparing the bio- markers
that are labelled with known biomarker
compositions of micro- organisms, active
populations can be identified

8. • Especially with the gas chromatography-
combustion isotope ratio mass
spectrometry(GC-c-IRMS), it is now possible to
analyse stable isotope ratios of specific
compounds(biomarker) with excellent
sensitivity w.r.t concentration and isotope
content .

9. The current state-of-the-art method to study the isotopic
composition of individual compounds is GC-c-IRMS.
It comprises a GC equipped with a capillary column that is
used to separate the compounds of interest at high
resolution.
The outlet of the column is attached to a miniature
oxidation reactor where the organic molecules are
combusted to CO2, N2 and H2O gas.
A reduction reactor is included for 15N analysis to convert
oxidised
nitrogen species to N2 gas.
Water is removed on-line and the purified CO2 and N2 are
led into an isotope ratio mass spectrometer (IRMS) and
hence IRMS measures the isotopic ratios between the heavy
and light isotopes (e.g. 13C/12C for carbon and 15N/14N
for nitrogen) and results are always calibrated against an

10. • Natural abundance studies use the small
differences in isotopic ratios as found in nature.
These isotope effects are caused by the
preferential use of 12C compared to 13C in many
biological and chemical processes, which is
referred to as isotopic fractionation.
• Variation in 13C/12C ratios among primary
producers occurs because of differences in
inorganic substrate, fixation pathways, or
environmental and physiological conditions
• These differences in isotopic composition can be
used to trace the origins of organic compounds in
environments.

11. • Natural abundance isotope ratios of
biomarkers can be used to study
• organic matter sources utilised by microorganisms
in complex ecosystems
• for identifying specific groups of bacteria.
• Addition of labelled substrates in combination
with
biomarker analysis enables direct identification
of microbes involved in specific processes

12. Natural abundance approach can be used to study
• The source of the carbon assimilated by
microorganisms
• To identify microbial populations involved in specific
processes e.g. methanotrophy.
• Stable isotopes have also been used extensively as
tracers for rate measurements in microbial activities
(e.g. 15N uptake and regeneration, denitrification,
nitrogen fixation,13C fixation and respiration).
• The combination of deliberately added tracers and
isotopic analysis of biomarkers provides the unique
possibility to directly link
o Microbial identity (biomarker),
o biomass (concentration of the biomarker) and
o activity (isotope assimilation).

13. TYPES OF STUDY
• Coupling between primary producers and
heterotrophic bacteria: labelling with
13
C
carbondioxide
• Linking population structure with specific
microbial
processes: labelling with specific
13
C
compounds
 stable isotope tracer is incorporated into the biomass
of the metabolically active populations, which can be
detected in a variety of biomarkers. By comparing the
bio- markers that are labelled with known biomarker
compo- sitions of micro- organisms, active

14. FUTURE PERSPECTIVES
• 15N-labelled compounds have been used to study the
competition between phytoplankton and bacteria for
various forms of nitrogen.
• The use of both organic and inorganic forms of nitrogen
and sulphur by microorganisms could be studied with
specific biomarkers containing these elements if
methods for analysing stable sulphur isotopes in
biomarkers become available.
• 13C labelling was used in combination with density
gradient centrifugation to isolate heavy, 13C-labelled
DNA from active populations in a soil sample

15. DNA-SIP
• DNA stable isotope probing (DNA-SIP) is a recently
developed
method in which the incorporation of stable
isotope from a labelled substrate is used to identify
the function of microorganisms in the
environment. The technique has now been used in
conjunction with metagenomics to establish links
between microbial identity and particular metabolic
functions. The combination of DNA-SIP and
metagenomics not only permits the detection of
rare low-abundance species from metagenomic

22. MEASUREMENT OF NITRIFICATION
RATES
• Both “tracer” and “dilution” approaches can be used to
measure nitrifica- tion rates taking advantage of the
sensitivity of stable isotope methods. In the tracer approach,
a “trace” amount (an amount low enough to avoid
perturbation of the ambient substrate concentration,
generally taken as 10% of the ambient level) of labeled
substrate (a radio or stable isotope) is added to a sample.
After incubation, the amount of label in the product is used
to compute the transformation rate.
• Unfortunately, a direct radioiso- tope tracer method is not
very useful for measuring rates of nitrification in the
environment. Capone et al. (1990) demonstrated the use of
13N to quantify nitrification rates, but the isotope is so

23. • The main approach to measuring nitrification
rates directly is to use the stable isotope, 15N, as
a tracer (Olson, 1981; Ward et al., 1984). This
approach has constraints that may limit its
application, mainly due to the facts that 15N has
a significant natural abundance and that it must
be measured using a mass spectrometer or
emission spectrometer, both more expensive and
difficult than using a scintillation counter for
radioisotopes.

24. FLUORESCENCE IN SITU
HYBRIDIZATION-
MICROAUTORADIOGRAPHY AND
ISOTOPE ARRAYS
• The combination of fluorescence in situ hybridization
(FISH) and
microautoradiography (MAR) is currently the most
widely applied tool for revealing physiological
properties of microorganisms in their natural
environment with single-cell resolution.
• For example, this technique has been used in
wastewater treatment and marine systems to describe
the functional properties of newly discovered species,
and to identify microorganisms responsible for key
physiological processes.

25. ISOTOPE ARRAY
• The principle of identifying radioactively labelled microorganisms
using rRNA-targeted oligonucleotide probes was recently also
adapted to the microarray format.
• In this so-called isotope array , community rRNA is first
extracted from an environmental sample that was incubated with
a radioactively labelled substrate, then covalently linked with a
fluorescent dye, fragmented, and hybridized with an rRNA-
targeted microarray.
• Subsequently, fluorescence and radioactivity probe signals are
quantified with a fluorescence scanner and a 𝛽 imager,
respectively.

26. • Two main advantages render the isotope array
methodologically appealing for the analysis of
complex microbial communities. The multiple probe
hybridization format offers the opportunity to identify
many microorganisms with a defined metabolic ability
in a single microarray experiment. Furthermore, the
ratio between radioactivity and fluorescence of a
probe spot provides a unique, quantitative measure
of how efficiently a probe-defined population has
incorporated the labelled substrate into its rRNA.

28. THE STIMULATION EFFECT OF LOW
DOSES 60CO GAMMA IRRADIATION
ON VIABILITY OF TRICHOGRAMMA
CHILONIS
• Wang Endong; Li Yongjun; Liu Xiaohui; Lu
Daguang (Chinese Academy of Agricultural
Sciences, Beijing (China). Inst. for Application
of Atomic Energy)

29. • There were no effect in general on viability of
Trichogramma chilonis treated with 10, 70, 150 and 300
mGy 60Co γ irradiation in eggs stage.
• 150 and 300 mGy irradiation treatment in pupae stage
induced significantly alteration of sex ratio in favor of
female adults in F1 generation and no effect on numbers of
T. cholonis adults produced per parasited H. armigera egg,
sex ratios of T. cholonis adults, longevities of T. cholonis
female adults and their parasitization rates in P
generation,however the sex ratios and numbers of T.
chilonis adults produced per parasited H. armigera egg
significantly increased in F1 and F2 generations.
• The 1000 and 2000 mGy irradiations caused certain
stimulation effects such as the sex radio altered in favor of
females and increase of numbers of T. cholonis adults
produced per parasited H. armigera egg, but also reduced
the longevities of T. cholonis female adults and their
parasitization rates, and this phenomenon only occurred in
P generation, there were no significant influence compared

30. THE RELATIVE CONTRIBUTION OF HYPHAE AND
ROOTS TO PHOSPHORUS UPTAKE BY
ARBUSCULAR
MYCORRHIZAL PLANTS, MEASURED BY DUAL
LABELLING WITH 32P AND 33P
The aim of this investigation was to measure
the relative contribution of hyphae and roots to
the total uptake of phosphorus by mycorrhizal
plants. Cucumber plants were grown in three-
compartment systems where 32P was
applied to a lateral root-free compartment
(HC) and 33P applied to an identical lateral
compartment with both roots and hyphae
(RHC). The cucumber seeds were sown into
the main root compartment (RC) which was
inoculated
with one of the following three arbuscular
mycorrhizal fungi
Scutellospora calospora
Glomus sp
Glomus caledottium

31. • The plants were harvested at17 and 27 d. The hyphal
uptake of ^"P from the HC increased as follows –
• S. calospora < Glomus sp. <G. caledonium.
• The uptake of 32P from the HC was equivalent to 7,
21 and 109% of the uptake of 33P from the RHC in
plants colonized by
S. calospora, Glomus sp. and G. caledonium,
respectively.
This indicates that the relative contribution of the roots
in total P uptake varied greatly between the three
mycorrhizal treatments .

32. CONCLUSION
• P uptake systems of roots colonized by G.
caledonium appeared to be inactive when
compared to hyphal 32P uptake. This may
have been due to feedback mechanisms being
activated because of the high hyphal P uptake.
• The roots colonized by S. calospora had higher
rates of root-P uptake
compared with the control roots, suggesting
that the root-P uptake systems have been
stimulated by the presence
of the fungus.

33. USE OF 15N STABLE ISOTOPE TO
QUANTIFY
NITROGEN TRANSFER BETWEEN
MYCORRHIZAL PLANTS

34. • Mycorrhizas (fungal roots) play vital roles in plant nutrient
acquisition, performance and productivity in terrestrial
ecosystems. Arbuscular mycorrhizas (AM) and ectomycorrhizas
(EM) are mostly important since soil nutrients, including NH+ 4,
NO3 and phosphorus, are translocated from mycorrhizal fungi to
plants.
• Individual species, genera and even families of plants could be
interconnected by mycorrhizal mycelia to form common
mycorrhizal networks (CMNs).
The function of CMNs is to provide pathways for movement or
transfer of nutrients from one plant to another.
• Both 15N external labeling or enrichment (usually expressed as
atom%) and 15N naturally occurring abundance (𝛿15N%)
techniques have been employed to trace the direction and
magnitude of N transfer between plants, with their own
advantages and limitations.
• Nitrogentransfer from the N2-fixing N donor to the non-N2-
fixing N receiver is estimated on the assumption that equal
proportions of non-labeled and externally labeled N are
transferred.

35. • The heavier stable isotope 15N is discriminated against 14N
during biochemical, biogeochemical and physiological
processes, due to a greater atomic mass. In general, non-N2-
fixing plants had greater 𝛿15N values than N2-fixing ones.
Foliar 𝛿15N often varied by 5 to 10% in the order:
• non-mycorrhizas/AMs > EMs > ericoid mycorrhizas.
• Differences in 𝛿15N % or 15N (atom%) values could thus provide
N transfer information between plants.
• A range of between 0 to 80% of one-way N transfer had been
observed from N2-fixing mycorrhizal to non-N2-fixing
mycorrhizal plants, but generally less than or around 10% in the
reverse direction. Plant-to-plant N transfer
may provide practical implications for plant performance in N-
limited habitats. Considering that N translocation or cycling is
crucial, and the potential benefits of N transfer are great in both
agricultural and natural ecosystems, more research is warranted
on either oneway or two-way N transfers mediated by CMNs
with different species and under field conditions.

36. REFERENCES AND RECOMMENDED READING
• H.T.S. Boschker, J.J. Middelburg ,Stable isotopes and biomarkers in
microbial ecology
Netherlands Institute of Ecology (NIOO-KNAW), P.O. Box 140, 4400 AC Yerseke, The Netherlands
Received 28September 2001; received in revised form 19 November 2001; accepted 15 January 2002
First published online 4 March 2002
• Stefan Radajewski*, Philip Ineson, Nisha R. Parekh
& J. Colin Murrell* ,Stable-isotope probing as a tool in microbial ecology
* Department of Biological Sciences, University of Warwick, Coventry,Warwickshire CV4 7AL, UK
† Institute of Terrestrial Ecology, Merlewood Research Station,Grange-over-Sands, Cumbria LA11 6JU, UK
• Andrew S Whiteley1, Mike Manefield1 and Tillmann Lueders2
Unlocking the ‘microbial black box’ using RNA-based stable isotope probing
technologies
• Michael Wagner1, Per H Nielsen2, Alexander Loy1,Jeppe L Nielsen2 and
Holger Daims1 ,Linking microbial community structure with
function:fluorescence in situ hybridization-microautoradiographyand isotope
arrays