Mar Fish

Rev Environ Sci Biotechnol (2010) 9:153185
DOI 10.1007/s11157-010-9205-8
REVIEWS
An appraisal of methods for linking environmental

processes to specific microbial taxa
Maria-Luisa Gutierrez-Zamora Mike Manefield
Published online: 14 May 2010

Springer Science+Business Media B.V. 2010
Abstract The last decade has witnessed a revolution in the development of methods and technology
available to investigate the ecological roles of
microorganisms in the environment. As a consequence, microbial ecologists have gained a better
understanding of the functional aspects of microorganisms in marine, groundwater and freshwater
systems, soils, sediments, hot springs, wastewater
treatment plants, landfills, the rhizosphere and the
animal gut. This review provides a compilation and
critical comparison of the currently available methods linking microbial function with phylogeny,
including a description and advantages and limitations of each method. Examples are also provided to
illustrate their application. The ongoing improvements of these function-identity methods points to a
bright future in our understanding of complex
ecological processes and to improved management
of microbe dependent ecosystem services.
Keywords Function-identity methods
Isotope probing FISH NanoSIMS
Isotope array Raman SSU-IRMS
M.-L. Gutierrez-Zamora M. Manefield (&)

Centre for Marine BioInnovation, School of
Biotechnology and Biomolecular Sciences,
University of New South Wales, Sydney, NSW, Australia
e-mail: manefield@unsw.edu.au
1 Introduction
For many decades, it has been the aim of microbial
ecologists to identify the diversity of microorganisms
present in the environment and to understand their
roles in biotic and abiotic interactions. Unlike other
disciplines in ecology, microbial ecology is confronted with the major difficulty that its subject of
study is microscopic and that the majority of the
individuals are not culturable in the laboratory (Head
et al. 1998). These difficulties have, however, fuelled
a myriad of technological developments, from early
optical microscopy to micro-manipulation with lasers
and DNA amplification of single cells (Binga et al.
2008). In particular, the last decade has witnessed the
development of a suite of methods for deciphering
which microorganisms are performing selected functions in the environment, with the ultimate aim to
understand their ecological roles. These methods are
commonly referred to as function-identity methods
because they aim at linking ecological processes to
specific microbial taxa.
Function-identity methods have typically made
use of stable or radioactive isotopes of atoms present
in biological molecules. These methods rely on the
incorporation of artificially enriched isotopes into the
biomolecules of microorganisms that have consumed
an isotopically labelled substrate after incubation.
The incorporation of isotopes into the microbial
biomass indicates substrate specific metabolic
activity. Using isotopes in microbial ecology has
123
154
Table 1 List of methods available in functional molecular microbial ecology

Isotope probing methods
Probe-based methods
Phospholipid-derived fatty acid-stable isotope probing

(PLFA-SIP)
Fluorescence in situ hybridisation-microautoradiography (FISH-MAR)
DNA-stable isotope probing (DNA-SIP)
Fluorescence in situ hybridisation-secondary ion mass spectrometry

(FISH-SIMS)
RNA-stable isotope probing (RNA-SIP)
FISH-Raman spectroscopy (FISH-Raman)
Protein-stable isotope probing (Protein-STP)
Secondary-ion mass spectrometry-in situ hybridisation (SIMSISH)
Radioactive isotope probing (RIP)
Element labelling-FISH (EL-FISH)

Isotope arrays
Small subunit-isotope ratio mass spectrometry (SSU-IRMS)
the enormous advantage of allowing researchers to

trace the flow of elements within communities and to
draw conclusions on the metabolic activities of
microorganisms. Some drawbacks of working with
isotopes include limited availability of labelled substrates and their high cost, particularly of radioactive
substrates. In addition, ex situ incubation of community samples with isotopically labelled substrates
(stable or radioactive) does not necessarily reflect in
situ conditions. Care must be taken to achieve conditions as close as possible to those in situ. Further, health
and environmental hazards related to the use of
radioactive materials, as well as the processing of
licences and compliance with regulations limits to
some extent the use of radioactive based techniques in
comparison with stable isotope methods. Despite this,
the use of isotopes in this discipline has proved to be a
powerful tool to determine which microbial cells are
responsible for an observed process and to what extent
are they involved. The degree of discovery allowed by
the use of isotopic substrates has certainly outweighed
these shortcomings.
The purpose of this review is to describe the
methods developed to date that link microbial function with taxonomic identity and, to compare their
advantages and limitations. This review also presents
a compilation of recent significant examples where
such methods have been applied. Overall, it provides a
general view of the direction that methodological
development is taking in microbial ecology.
The selection of an appropriate method for implicating microbes in an environmental process of
interest depends on the type of questions being
asked. The current methodological toolbox offers
techniques that answer two distinct types of function-
123
identity questions: Which microbes are performing

this task? (open question) Or is a specific microbe or
group of microbes performing this task? (closed
question). Based on this distinction, the available
methods can be grouped into two broad categories: (a)
Isotope probing methods (for open questions) and (b)
Probe-based methods (closed questions; Table 1).
Each one of these methods has had different uptake
in the scientific community over the years. In general,
stable isotope probing (SIP) methods have been
applied more broadly than probe-based methods
according to the scientific literature for the years
between 2007 and 2010 (Fig. 1). This may simply be a
reflection of the fact that SIP methods were developed
earlier than some FISH-based methods or that the
latter represent a more expensive alternative and have
not been tested thoroughly yet. Despite this trend, we
have compiled here the characteristics of each one of
Fig. 1 Number of publications between 2007 and 2010 for all

function-identity methods reviewed

Fig. 2 Schematic
representing the basic steps
in the different isotope
probing techniques
reviewed. (See text for
definition of abbreviations)
155
Biomolecule
13
13
Name of method
Comparison with profiles

of known species
GC-c-IRMS
profile
C-DNA
Density gradient
centrifugation
DGGE, T-RFLP,
clone libraries
DNA-SIP
C-RNA
Density gradient
centrifugation
DGGE, T-RFLP,
clone libraries
RNA-SIP
MALDI-MS,
MS/MS
Comparison with PMF

profiles in databases
Protein-SIP
13
Extraction of
biomolecule
Assignation of
taxonomic identity
C-lipids
13
Pulse
Basic method
C-Proteins
14
14
C-RNA:12C-DNA
hybridisation on
profiles or library
clones
C-RNA
these to provide a comprehensive view of the

available alternatives for function-identity methods.
2 Isotope probing methods

All isotope probing studies start with the incubation
of the experimental sample with a 13C or 15N labelled
substrate or 14C-substrates for radioactive isotope
probing for a period that varies depending on the
method and environment studied. The addition of the
labelled substrate followed by incubation is commonly referred to as the pulse. After pulsing, the
biomolecules are extracted, processed according to
each methods protocols and the taxonomic identity
of the consumers of the label is determined. A flow
chart of the basic steps in the different isotope
probing techniques is shown in Fig. 2. Stable isotope
probing (SIP) can be applied in field trials because, as
opposed to radioactive isotopes, stable isotopes are
innocuous to the environment. In general, however,
these methods do not allow quantitative estimations
of the relative abundance of specific taxa consuming
a substrate. This is particularly so for the methods
based on PCR because abundance information is
substantially altered after amplification. A second
consequence of PCR is that isotopic content of
biomolecules is diluted. For this reason, the level of
isotopic incorporation per taxonomic group can only
be obtained through PCR-free strategies such as
Protein-SIP or a radioactive approach (see below).
Overall, a shared advantage of isotope probing
methods is the fact that active members of a
community can be identified without any prior
knowledge of their identity.
Direct band or clone

sequencing
PLFA-SIP
Radioactive
isotope probing
2.1 Phospholipid-derived fatty acid-stable

isotope probing (PLFA-SIP)
This method, developed in the late 1990s, is based on
the extraction of signature lipid biomarkers from the
membranes of microorganisms in an environmental
sample. After a 13C labelled substrate pulse, all the
lipids in the sample are extracted and analysed by gas
chromatography-combustion-isotopic ratio mass spectrometry (GC-c-IRMS). The resulting spectra indicate
the content of phospholipid-derived fatty acids in the
sample and their corresponding 13C enrichment, if
present. The lipid profile is then compared to groupspecific profiles of previously cultured organisms. In
this way, the signature lipids and their isotopic
enrichment enable the identification of taxa involved in
the consumption of the labelled substrate.
The sensitivity1 of this method is high, as only
small amounts of label incorporation are needed to
detect labelled biomarkers. For this reason, incubations with labelled substrates can be carried out at
near in situ concentrations, resulting in a realistic
incorporation of the label into cell membranes. This
method is useful for analysing microbial communities
with low cell numbers and low carbon incorporation
rates, as phospholipids are naturally abundant molecules in the cell and label incorporation is independent of cell replication. In PLFA-SIP, there is no need
to purify unlabelled from labelled biomarkers, unlike
DNA and RNA-SIP (see below).
A number of methods are compared here in terms of their

sensitivity. In this context, sensitivity refers to the degree of
labelling required for a particular method to generate a result.
123
156
This method, however, has a low phylogenetic

resolution, because it relies on pre-determined profiles
from cultured microorganisms (Murase et al. 2006).
Therefore, the taxonomic identity of associated microorganisms is difficult to confirm and impossible to
determine if there are no cultivated close relatives.
Since its development in 1998 (Boschker et al. 1998),
this method has been widely used and applied to
diverse environments (Evershed et al. 2006). In most
recent years, PLFA-SIP has been useful in identifying
active microbial populations in soils and sediments,
disentangling animal/plantmicrobe interactions and
understanding the ecology of methane oxidation. A
summary of the most recent achievements (2007
2010) in microbial ecology using PLFA-SIP is presented in Table 2. Overall, these studies provide a
picture of microbial biogeochemical processes and
microbial trophic interactions in different environments. These examples indicate that changes in land
use practices, rising temperatures and the widespread
use of inorganic fertilisers can affect microbe-mediated methane cycling processes, with potential negative implications in the global warming phenomenon.
PLFA-SIP has been combined with DNA/RNASIP analyses as a means of overcoming the limitation
of low phylogenetic resolution. Webster et al. (2006)
supplemented marine sediment enrichment cultures
under sulphate reducing conditions with low concentrations of 13C-labelled glucose and acetate. The
authors found that when using glucose the identity of
the glucose consumers was unclear when both PLFAand DNA-SIP were applied. However, PLFA- and
DNA-SIP could resolve the specific identity of active
acetate consumers. Qiu et al. (2008), successfully
combined PLFA-SIP with RNA-SIP to identify
methanotrophic bacteria in rice rhizosphere under in
situ conditions. Conversely, Bengtson et al. (2009)
were unsuccessful in a similar combined approach
obtaining only PLFA profiles of methanotrophs in
forest soil horizons, but no information from DNA/
RNA SIP assays. These authors were unable to obtain
sufficient isotopic enrichment of nucleic acids for
efficient density separation (see below). Obtaining
successful results with this approach may be a matter
of fine-tuning the right substrate concentrations.
A few studies have also included additional
molecular analysis to expand the reach of PLFASIP. Chen et al. (2008a) compared unlabelled mRNA
(gene transcripts) with PLFA-SIP profiles of
123
methanogenic communities of peatland soils to assess

which genes were expressed in these systems. Singh
and Tate (2007) combined PLFA-SIP with pmoA
specific terminal restriction fragment length polymorphism (T-RFLP) and sequence analysis to assess
active methanotroph populations with enhanced taxonomic resolution. A further methodological
improvement of this technique was achieved by
increasing the diagnostic phospholipid profiles in the
databases (Bodelier et al. 2009), which should continue to improve the resolution of this method. In
conclusion, despite having low taxonomic resolution,
PLFA-SIP is still found useful in a number of
environments. This method is mainly used as a
primary screen of active microbes at broad taxonomic
levels.
2.2 DNA-stable isotope probing (DNA-SIP)

This method is based on the extraction and use of
DNA as a biomarker molecule from a microbial
community that has been fed with a 13C or 15N
labelled growth substrate. The extracted DNA is a
mixture of heavy and light molecules, which are
separated by their buoyant density in caesium chloride (CsCl) solutions by equilibrium density gradient
centrifugation. Fractions containing the labelled
DNA can be visualised by UV light if the gradient
solution contains ethidium bromide enabling direct
extraction of labelled DNA with a syringe. Alternatively, the gradient solution is fractionated, then DNA
of every fraction is precipitated out of the CsCl
solution with polyethylene glycol and visualised by
agarose gel electrophoresis. DNA is then used as a
template for PCR to amplify 16S rRNA genes. These
PCR products have been analysed by denaturing
gradient gel electrophoresis (DGGE; from where
specific bands can be sequenced; Haichar et al. 2007;
Bressan et al. 2009), or used to generate clone
libraries for taxonomic identification of those
microbes that incorporated the label (Baytshtok
et al. 2009; Jensen et al. 2008). T-RFLP analysis of
DNA from heavy fractions has also been used in
combination with clone library T-RFLP patterns to
assign taxonomic identity to specific microbes (Cupples and Sims 2007; Gihring et al. 2009). From heavy
DNA, it is also possible to look for functional genes
of interest or to amplify the whole genome of the
157
Table 2 PLFA-SIP examples from 2007 to 2010

Reference
Type of study
Environment
Main finding
Lu et al.
(2007)
Distribution of active microbes

in the environment
Rice rhizosphere soil
Gram negative bacteria were most actively incorporating

plant-derived carbon in the vicinity of the rhizosphere as
opposed to bulk soil
Balasooriya
et al.
(2008)
Distribution of active microbes

in the environment
Wetland soil
Gram negative bacteria were most actively incorporating

plant-derived carbon in superficial, dryer soils. Gram
positive were more active in deeper, wetter layers and
assimilated root-derived carbon much slower than gram
negatives
Denef et al. Distribution of active microbes

(2007)
in the environment
Grassland soil
Fungi readily assimilated plant-derived carbon before

bacterial communities did
Deines et al. Trophic interactions

(2007)
Freshwater
Methane oxidising bacteria were used as a carbon and

energy source by macro-invertebrate chironomid larvae
Wegener
et al.
(2008)
Biogeochemical processes:
sulphate-driven anaerobic
methane oxidation
Marine cold seep

sediments
Archaea associated sulphate reducing bacteria displayed

autotrophic growth only when methane was present,
suggesting the presence of an electron shuttle mechanism
between the members of this consortium
Shrestha
et al.
(2008)
methanotrophy
Rice plants
rhizosphere
Type I methanotrophs were of particular importance in the

rhizosphere of rice plants, as evidenced by their enhanced
activity and population size in comparison with other
methanotrophs
Qiu et al.
(2008)
methanotrophy
In situ rice plants

rhizosphere
Type I methanotrophs had a predominant role in the active

assimilation of methane in rice fields
Singh and
Tate
(2007)
methanotrophy
Forest soil
Type II methanotrophs were the predominant

methanotrophs in these pristine soils
Singh et al.
(2007)
methanotrophy
Forest and shrub land Type II methanotrophs were more active in forest and
soil versus pasture
shrub land soils and Type I methanotrophs were more
land soil
active in pasture land soils
Tate et al.
(2007)
methanotrophy
Forest and pasture soil Active Type II methanotrophs predominated in forest soil
and Type I methanotrophs in neighbouring pasture land
soil
Singh et al.
(2009)
methanotrophy
Forest and pasture soil Shifts in land use practices (from pastures to forested land)
have resulted in shifts from Type I to Type II
methanotrophs in the soil
Dorr et al.
(2010)
methanotrophy
Forest and farmland

soil
Changes in land use practices, from forest to farmland,

have induced a community shift from Beijerinckiaceae
species to Methylococcaceae and Methylocystaceae
species. Afforested land was a greater CH4 sink than
farmland
Menyailo
et al.
(2010)
methanotrophy
Grassland soil
Different Siberian tree species did not affect community

composition of soil methanotrophs, but strongly altered
CH4 oxidation rates
Knoblauch
et al.
(2008).
methanotrophy
Permafrost soils
There was a shift from Type I to Type II methanotrophs

when methane oxidation activity was compared at in situ
temperatures (0C) and at higher temperatures (22C)
Maxfield
et al.
(2008)
methanotrophy
Agricultural soils
A decrease of more than 70% of methanotrophs was

observed in fertilised soils as compared to non-fertilised
controls. The shift in active microbial communities was
attributed to the fertiliser salt induced effect
Chen et al.
(2008a)
methanotrophy
Peatland soils
Different plant covers induced changes in methanotrophs.

Calluna-covered soil favoured Methylocella/Methlocapsa
spp. and Sphagnum/Eriophorum-covered land favoured a
putative novel methanogen
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158
Table 2 continued
Reference
Type of study
Environment
Lerch et al.
(2009)
Degradation of xenobiotics: 2,4- Agricultural soil

Dichlorophenoxiacetic acid
A succession of microbial communities degraded 2,4Dichlorophenoxiacetic acid, with proteobacteria being

the principal degraders. Labelled fatty acids were present
in biomass long after peak degradation
Jin and
Evans
(2010)
consumption of plant derived
carbon
Ratios of bacterial-to-total PLFA-carbon decreased and

fungal-to-bacterial PLFA-carbon increased under
elevated CO2 compared with ambient conditions
Desert soil
community by multiple displacement amplification

(MDA) for metagenomic analysis (Chen et al. 2008b;
Neufeld et al. 2008b; Sul et al. 2009).
This method provides a much higher phylogenetic
resolution than PLFA-SIP as it exploits taxa DNA
sequence differences and the large existing databases
that compile this information. Thus, DNA sequences
can be assigned to a taxonomic group with confidence. This method has the potential to investigate a
wide range of uncultured species and, unlike PLFASIP it does not rely on information obtained only
from cultured species. Since genomic DNA is
obtained, it also provides direct access to functional
genes.
DNA-SIP has a number of disadvantages. Firstly,
in order to efficiently label DNA, the labelled
substrate concentration often has to be higher than
observed in situ and, therefore, higher than that used
in PLFA-SIP. Consequently, DNA-SIP has lower
sensitivity. This also means that the higher concentrations of substrate may artificially favour certain
microorganisms, resulting in culture bias. Secondly,
the duration of the pulse has to be long enough (up to
40 days) to ensure that DNA becomes sufficiently
labelled, as labelling depends on cell replication.
Consequences of long incubation times are a
potential culture bias effect and cross feeding. That
is, the incorporation of the label into non-primary
consumers that feed from metabolites excreted by
labelled cells or dead labelled cells. Therefore, to
obtain reliable results with DNA-SIP it is important
to determine the right balance between the concentration of labelled substrate that reflects in situ
conditions and the concentration that will be enough
to achieve sufficient labelling. Thirdly, the label may
become diluted in the DNA of its consumers if there
is simultaneous growth on an unlabelled carbon
source. The resulting DNA may have the same
123
Main finding
buoyant density as DNA from non-consumers with

high G ? C content, which will again bias the results.
For this reason, DNA used in DNA-SIP experiments
needs a 13C content of at least 50 atom% to avoid
overlapping in the same gradient fraction (Radajewski et al. 2003).
Radajewski et al. described this groundbreaking
method for the first time in 2000 (Radajewski et al.
2000). Numerous reviews have been published that
summarise the discoveries made with DNA-SIP since
then (Friedrich 2006; Kreuzer-Martin 2007; Madsen
2006; Neufeld et al. 2007a). Most recently, DNA-SIP
has been applied to study the involvement of
microbes in biogeochemical processes such as methanotrophy, methylotrophy, denitrification and nitrogen fixation, as well as carbon flow in the ecosystems
(trophic interactions), and to the study of biodegradation of xenobiotic compounds. A summary of these
studies published between 2008 and 2010 has been
compiled in Table 3. For the purpose of this review
only investigations that accomplished a methodological improvement were considered here.
In recent years, DNA-SIP has undergone a number
of technological makeovers. For example, Gallagher
et al. (2005) and Neufeld et al. (2007b) added
archaeal DNA or glycogen, respectively, as carriers
of DNA from the gradient fractions to improve DNA
recovery from CsCl gradients. Applying 15N-DNASIP was particularly problematic because the shift in
buoyant density achieved with heavy nitrogen in DNA
was less than half of that occurring naturally from
high G ? C content DNA. To eliminate this problem,
Buckley et al. (2007) included two centrifugation
steps instead of one, and a DNA intercalating
compound (bis-benzimide) on the second centrifugation to improve the separation of 15N-labelled DNA
from high G ? C unlabelled DNA. This strategy
opened up the possibility of deciphering the identity
159
Table 3 DNA-SIP examples from 2008 to 2010

Reference
Type of study
Environment
Main finding
Jensen et al.
(2008)
Methanotrophy
Deep water coral

reef sediments
Methylomicrobium and Gammaproteobacteria were active

microbes involved in methane oxidation and members of
Gammaproteobacteria, Alphaproteobacteria, Deferribacter
and Bacteroidetes were putative cross-feeders
Neufeld et al. Biogeochemical processes:

(2008a)
methylotrophy and trophic
interactions: Food webs
Surface seawater
from algal bloom
Algal blooms provided C1 compound precursors that were

actively incorporated by: Methylophaga spp. and
Gammaproteobacteria (methanol, methylamines,
dimethylsulfide) and Alphaproteobacteria and the
Cytophaga-Flexibacter-Bacteroidetes guild
(methylbromide)
Neufeld et al. Biogeochemical processes:

(2008b)
Methylotrophy
Surface seawater
Methylophaga-like phylotypes were identified as the most

active methanol consumers. Using low concentrations of
labelled substrate it was possible to achieve DNA-SIP and
multiple displacement amplification for metagenomic
analysis
Moussard
et al.
(2009).
Methylotrophy
Estuarine sediments
Type I (Methylophaga spp.) rather than Type II

methanotrophs were the most active consumers of methane,
methanol and methylamine
Chen et al.
(2008b)
Methanotrophy
Peatland soils
DNA-SIP combined with multiple displacement amplification

and fosmid library analysis showed that Methylocystis spp.
were the dominant methanotrophs in these soils
Han et al.
(2009)
Methanotrophy
Coal mine alkaline

soils
Hery et al.
(2008)
methanotrophy
Landfill cover soil
Type I, Type II methanotrophs, and methylotrophs

(Methylopila spp. and Hyphomicrobium spp.) were the most
active methanotrophs
Bacterial methane oxidation was significantly increased by
the presence of earthworms likely due to a bacterial growth
stimulation phenomenon
Osaka et al.
(2008)
Methanotrophy
Activated sludge
Type-X methanotrophs of the Gammaproteobacteria class

were the dominant key players in methane-dependent
denitrification
Baytshtok
et al. (2009) Methylotrophy under
denitrifying conditions
Batch reactors
By alternating between C1 carbon sources, a switch in active

methylotrophs was observed, evidencing the facultative
nature of Methyloversatilis spp. and the obligate nature of
Hyphomicrobium spp.
Saito et al.
(2008)
Rice paddy soils
Bukholderia spp. and Rhodocyclales spp. dominated (readily

available) succinate consumption under denitrifying
conditions in water logged soils
Buckley et al. Biogeochemical processes:

(2008)
Nitrogen fixation in
methanotrophs
Grassland soil
Using 15N2 it was found that Methylocystis-like species fixed

N2 in the soil in response to methane addition. Thus, N2
fixation by methanotrophs in the soil was demonstrated for
the first time
Jia and
Conrad
(2009)
Agricultural soils
Despite a higher abundance of archaeal ammonia-oxidizing

gene amoA, members of the domain Bacteria actively
consumed 13CO2, showing a predominant role in
autotrophic ammonia oxidation in these soils
Wawrik et al. Biogeochemical processes:

(2009)
N cycle
Marine microcosms
Using a variety of 15N-labelled substrates, it was shown that

Synechococcus and diatoms have a high plasticity in
nitrogen assimilation processes
Chen et al.
(2009)
Cave freshwater
(plus biofilm)
The most active microbes assimilating 13CO2 were

Thiobacillus spp., Nitrospira spp. and Candidatus
Nitrotoga spp. suggesting an important role of sulphur and
ammonia/nitrite oxidisers in this environment
denitrification
Autotrophic ammonia
oxidation
Sulphur oxidation and
denitrification
123
160
Table 3 continued
Reference
Type of study
Environment
Main finding
Webster et al. Distribution of active

Estuarine sediments
(2010)
microbes in the environment
Uncultivated microorganisms play important metabolic roles

in different zones in tidal sediments. Gammaproteobacteria
and Marine Group I archaea dominated aerobic carbon
assimilation from glucose in the aerobic zone. Anaerobic
acetate assimilation was mainly dominated by
Epsilonproteobacteria. Acetate assimilation under sulphate
reducing conditions was dominated by Crenarchaeota
Pumphrey
Trophic interactions: Plant
and Madsen microbe
(2008)
Agricultural soil
Burkholderia spp. dominated the carbon acquisition from

benzoic acid, present in plant root exudates and a product of
plant decomposition in soil
Haichar et al. Trophic interactions: Plant

(2008)
microbe
Plant rhizosphere
Bacteria assimilating labelled root exudates from 4 different

plants were identified. Bacteria consuming exudates from
all plants were Sphingobacteriales and Myxococcus and
specific to monocots were Sphingomonadales
Bressan et al. Trophic interactions: Plant

(2009)
microbe
Plant rhizosphere
Rhizobiacea and fungal communities were the most active

microbes assimilating carbon from glucosinolates, a
biocidal exudate produced by Brassica plants
Rasche et al.
(2009)

microbe
Potato rhizosphere
A cultivar-dependent microbial differentiation was observed:

Acinetobacter spp. were more actively incorporating carbon
from exudates of Merkur cultivar and Acidovorax spp. from
Desiree cultivar
Qiu et al.
(2009)

microbe
Rice rhizosphere
Sphingomonadales and Methylocystacea were more active in

carbon assimilation from methane in young roots whereas
Methylophilales were more active in older roots.
Li et al.
(2009)
Trophic interactions: Carbon

flow
Municipal solid
waste
The most active degraders of 13C-celluose were Acetovibrio

spp., of 13C-glucose Clostridium spp. and
Porphyromonadaceae members, and of 13C-acetate the
archaeal Methanoculleus. All of these were implicated in
the methanisation of cellulose
Gihring et al. Trophic interactions: Carbon

(2009)
flow
Marine sediments
Degradation of detrital organic matter derived from 13Cenriched Spirullina cells implicates alphaproteobacterial
denitrifiers as important members of this process
Lear et al.
(2009)
Ecosystem functioning:
Human impact
River sediments
The composition of acetate assimilating bacteria was affected

by different amounts of light, e.g. Gammaproteobacteria
were more active in high intensity light (no vegetation
cover), Rhodococcus and Enterobacter were more active
during ambient light, and Betaproteobacteria were most
active during darkness
Jones et al.
(2008)
Degradation of xenobiotics:
Pyrene
Industrial site soil
Caulobacter spp. and members of the uncultured Pyrene

group 2 from Gammaproteobacteria were the dominant
pyrene degraders in polyaromatic hydrocarboncontaminated soils irrespective of biostimulation treatments
Liou et al.
(2008)
Benzene
Coal tar waste

contaminated
sediments
Different incubation parameters in situ and in laboratory

assays revealed a broad range of bacteria involved in
benzene consumption, with Pelomonas dominating in two
equivalent field and laboratory conditions
Oka et al.
(2008)
Benzene
Sulfidogenic
enrichment
cultures
A Desulfobacteraceae species was the first one to assimilate

carbon from benzene and was crucial to the degradation of
benzene in sulfidogenic enrichment cultures
De Rito and
Madsen
(2009)
Phenol
Agricultural soil
The fungus Trichosporum multisporum was the dominant

fungal degrader of phenol in soil. Isolation efforts were
successful and demonstrated that this fungus degraded
phenol in situ
123
161
Table 3 continued
Reference
Type of study
Environment
Main finding
Luo et al.
(2009)
Toluene
Agricultural soil
A member of the Candidate T7 phylum was the dominant

toluene degrader in soils previously free of toluene
contamination. This is the first study to report toluene
degradation capacity of this phylum which lacks culturable
representatives
Sul et al.
(2009)
Biphenyl
River sediments
Dominant biphenyl degraders belonged to Achromobacter

and Pseudomonas, and functional genes were recovered
from a cosmid library constructed from labelled DNA
Uhlik et al.
(2009)
Biphenyl
Agricultural soil and Paenibacillus spp. dominated assimilation of carbon from

horseradish
biphenyl in pentachlorobenzene contaminated bulk soil,
rhizosphere
whereas Hydrogenophaga spp. dominated in horseradish
rhizosphere, indicating a plant-associated effect in
community structure and biodegradation capacity. Bipheyl
dioxigenase genes were similar to those of Pseudomonas
alcaligenes B-357
Nicholson
Trophic interactions: Carbon Pure laboratory
et al. (2009) flow and spore-forming cells cultures
Tested the suitability of DNA-SIP to study typically dormant

cells, e.g. spores in the environment. Spores displayed a
normal physiology under labelling experiments which made
them suitable for this analysis
Jensen et al.
(2008)
Methanotrophy
Deep water coral

reef sediments
Tourna et al.
(2010)
Nitrification
Cultures
Methylomicrobium and Gammaproteobacteria were active

microbes involved in methane oxidation and members of
Gammaproteobacteria, Alphaproteobacteria, Deferribacter
and Bacteroidetes were putative cross-feeders
A comparison between DNA-denaturing gradient gel
electrophoresis (DGGE), RNA-DGGE and stable-isotopeprobing (SIP)-DGGE profiling of Nitrospira cultures
demonstrated that SIP is more sensitive to changes in
activity of organisms with relatively low yields and activity
of microbes involved in the nitrogen cycle (Buckley

et al. 2008). Another interesting approach to DNASIP has been the use of isotopic oxygen (18O)
incorporated as H18
2 O into the system. This alternative
allowed Schwartz (2007) to obtain a substrate independent analysis of microbial communities. The
author suggested that by using H18
2 O it may be
possible to study the impact of water and moisture
fluctuation in soil or sediment on microorganisms.
DeRito et al. (2005) used DNA-SIP in combination
with secondary ion mass spectrometry to obtain images
of the cells that had incorporated isotopic carbon. This
approach demonstrated that when several methods are
combined, it is possible to achieve a more comprehensive and meaningful result. Another significant
improvement to DNA-SIP was achieved by Neufeld
et al. (2008b) by using multiple displacement amplification of picogram amounts of DNA from gradient
fractions. In doing so, they overcame the need of long
incubations to obtain enough labelled DNA for
metagenomic library construction. They were able to
incubate with in situ concentrations of the label,

obtaining a more realistic view of the carbon dynamics
in the system. This strategy also expanded the possibility of retrieving information from functional genes.
2.3 RNA-stable isotope probing (RNA-SIP)
This method is based on the assimilation of the labelled
growth substrate into the RNA of its consumers. After
an incubation period (hours to days), total RNA is
extracted and the heavy molecules are separated from
lighter unlabelled molecules in a caesium trifluoroacetate (CsTFA)/formamide solution by equilibrium
density gradient centrifugation followed by gradient
fractionation and precipitation of RNA with isopropanol. The density of each fraction is determined by
weight or by refractometry to ensure the correct
formation of a density gradient. The distribution of
domain specific rRNA across the gradient fractions can
be quantified by fluorometry and by real time reverse
transcription (RT)-PCR (Lueders et al. 2004).
123
162
Following reverse transcription and amplification, the

microbial communities are analysed by fingerprinting
techniques such as DGGE, T-RFLP, single stranded
conformational polymorphism (SSCP) or clone library
construction and sequencing. Bands from a DGGE gel
that increase in intensity in the heavy fractions in a time
course experiment and that do not appear in the heavy
fractions of an unlabelled control represent those
microbes that assimilated carbon from the substrate.
These are then excised from the gels, purified and
sequenced to obtain their identity. The isotopic ratio of
the original RNA can be obtained by IRMS to confirm
13
C enrichment.
Both in RNA and DNA-SIP different strategies of
labelling the target cells have been used. These
include: (1) addition of soluble labelled substrates
directly to cultures or microcosms containing target
microbes, (2) exposing plants to 13CO2 to study
rhizospheric plantmicrobe interactions or the consumption of plant debris in soils, and (3) labelling of
cells in culture which will serve as a food source for
organisms in a higher trophic levels.
RNA molecules are more abundant and have a
higher turnover than DNA in active cells, therefore
the incubation times and substrate amount required to
achieve efficient labelling can be reduced in comparison with DNA-SIP. Additionally, labelling occurs
independently of cell replication, so it happens faster
than in DNA. This has the advantage that active non
replicating cells are also labelled (Manefield et al.
2007). Since less incubation time and substrate are
required, this method can obtain labelled RNA with
concentrations closer to in situ conditions, although it
still requires a higher degree of label incorporation
compared to PLFA-SIP. The minimum 13C content of
RNA for separation by density was determined
empirically to be 20 atom% (Manefield et al. 2002).
Consequently its sensitivity is higher than DNA-SIP
but lower than PLFA-SIP. Since rRNA contains
phylogenetic information, this method allows a
higher taxonomic resolution than PLFA-SIP but
equivalent to that of DNA-SIP.
Despite needing less incubation time than DNASIP experiments, cross feeding effects can occur if
incubation is prolonged or large amounts of substrate
are used. Given that higher amounts of label are
required than for PLFA-SIP, its ability to detect
consumers of a substrate that are low in abundance is
more limited than the latter. Rangel-Castro et al.
123
(2005) determined that the lower limit of detection of

RNA-SIP is 105106 cells/g soil. Additionally, RNASIP is only applicable to environments from which
good quality RNA can be extracted.
In the past 2 years, RNA-SIP has found several
applications across microbial ecology, providing
insights into the functional dynamics of niches such
as methanotrophy, methanogenesis, xenobiotic biodegradation and rhizosphere interactions. Table 4 summarises the most recent advances (20082010) achieved
with RNA-SIP. Here we comment on those studies that
have assessed directly or indirectly the limitations of
this technique and contributed to its improvement.
As with DNA-SIP, functional gene sequences can be
accessed owing to the fact that mRNA is co-extracted
with rRNA during sample preparation. Labelled
mRNA can be used to investigate the expression of
specific genes during a pulse. This idea was explored by
Huang et al. (2009) who used a combination of mRNA/
rRNA-SIP and Raman microspectroscopy-FISH to
identify naphthalene degraders in polyaromatic hydrocarbon-contaminated groundwater and the genes
involved in the process (see below).
Intrinsic to SIP is the fact that it is restricted to
assimilatory processes since it follows the incorporation of the (carbon) isotope into biomass. However,
Lear et al. (2007) challenged this aspect of the method
and applied RNA-SIP in combination with DNA-SIP
to study bacterial populations involved in arsenate
reduction in Cambodian aquifers. Using aquifer sediment microcosms amended with 13C-acetate, they
found a direct link between inputs of carbon and the
increased prevalence of arsenic-reducing microbial
populations. This was confirmed when genes for
arsenate reductase arrA were amplified from the 13CDNA fractions. Arsenate V reduced to arsenate III
becomes a more mobile and hazardous form of
arsenate. Thus, this study implied that exogenous
organic matter dislodged into aquifers could directly
stimulate the growth and activity of these bacteria,
which, in turn, make this compound more hazardous.
Reductive dehalogenation is another dissimilatory
process that has recently been studied with RNA-SIP.
In investigating the biodegradation of perchloroethene (PCE) in river sediments, Kittelmann and Friedrich (2008a) identified novel PCE-dehalorespiring
populations. Labelled acetate was used as an electron
donor and carbon source soon after dechlorination
products appeared in the microcosms. By comparing
163
Table 4 RNA-SIP examples from 2008 to 2010

Reference
Environment
Main finding
Brinkmann et al. Trophic interactions:

(2008)
Animal-microbe
Gut of Manduca
sexta larvae
Enterococcus spp. were most dominant in the gut after feeding the
larvae with labelled tobacco leaves. This environment/system
supported low diversity of active bacteria
Sapp et al. (2008) Trophic interactions:

Algae-microbe
Seawater
microcosms and
pure cultures
Human colon in
vitro model
First study to demonstrate that diatom-associated bacteria actively

consume carbon products derived from their algal host
KovatchevaDatchary et al.
(2009)
Type of study
Trophic interactions:
Human-microbe
Ruminococcus bromii was found to be the primary starch degrader,

with Bifidobacterium adolescentis, Prevotella spp., and
Eubacterium rectale involved further down the carbon
assimilation chain
Frias-Lopez et al. Trophic interactions:

(2009)
Food webs
Seawater surface
Labelled cyanobacteria Prochlorococcus and Synechococcus were

consumed by protozoa from the Haptophyta, Stramenopiles and
Alveolata groups. The method was successfully applied to
identify cyanobacterial predators
Glaubitz et al.
(2009)
Marine pelagic
water
Marine dark CO2 fixation in pelagic environments was attributed

to Gammaproteobacteria and the Sulfiromonas cluster of
Epsilonproteobacteria. Euplotes spp. ciliates were potential
grazers of these autotrophic bacteria
Food webs
Hamberger et al. Biogeochemical

(2008)
processes: Carbon
flow
Soil from acidic fen A diverse group of facultative aerobes and obligate anaerobes
fermented xylose and glucose under acidic conditions. These
were linked to active acid-tolerant methanogens and
Crenarchaeota through carbon flow
Schellenberger
et al. (2009)
Biogeochemical
processes: Carbon
flow
Agricultural soil
Under aerobic conditions, members of Bacteroidetes, Chloroflexi

and Planctomycetes dominated carbon assimilation from
cellulose, and Intrasporangiaceae and Micrococcaceae from
cellobiose and glucose. Under anaerobic conditions, members of
Kineosporiaceae, cluster II Clostridiaceae and Bacteroidetes
dominated cellulose carbon assimilation and cluster I
Clostridiaceae from cellobiose and glucose
Hatamoto et al.
(2008)
Carbon flow
Methanogenic
sludge
Members of Syntrophaceae, Tepidanaerobacter spp., and

Clostridium spp. were the most active degraders of butyrate in
sludge under methanogenic conditions
Moreno et al.
(2010)
Protozoa-bacteria
Activated sludge
The ciliate Epistylis galea was the dominant grazer from bacteria
assimilating CO2 under ammonia oxidising conditions. No
grazing on acetate consuming bacteria was detected
Langenheder and Ecosystem

Soil
Prosser (2008)
functioning:
Resource availability
Monard et al.
(2008)
Pulsing with the common metabolite benzoate at different

concentrations into the same soil samples resulted in marked
shifts in community structure. Evidence was provided that
resource limitation has an effect on diversity of active microbes
Ecosystem
Soil and earthworm Differences in active degraders of glucose and acetate were
functioning:
casts
detected between bulk soil and soil pre-treated with
earthworms as soil bioturbation agents that typically allow better
nutrient distribution in soils
Noll et al. (2008) Ecosystem

Rice field soil
functioning:
Degelmann et al. Ecosystem
Forest soil
(2009)
functioning:
Fertilisation of soil with urea strongly increased the activity of

Type I methanotrophs Methylomicrobium and Methylocaldum,
despite the presence of both Type I and Type II methanotrophs in
the unamended soil
Facultative aerobes Rahnella and Ewingella spp. dominated and
outcompeted anaerobes in the rapid fermentation of glucose in
forests soils in microcosms that simulated anoxic
microenvironments after rainfall
123
164
Table 4 continued
Reference
Type of study
Environment
Main finding
Bastias et al.
(2009)
Ecosystem
functioning: Human
impact
Soil subjected to
prescribed
burning
The prescribed burning of forest soils reduced the diversity of

cellulose-degrading fungi
Kittelmann and
Friedrich
(2008a)
Xenobiotic
degradation:
Tetrachloroethene
River sediments
Chloroflexi (Lahn cluster) spp. were the most active in

transforming tetrachloroethene to cis-dichloroethene in
dehalorespiringmicrocosms, whereas Dehalococcoides spp.
were the predominant in ethene-producing microcosms.
Geobacteraceae, Desulfobacteraceae and Desulbobulbaceae
were also involved in tetrachloroethene degradation
Kittelmann and
Friedrich
(2008b)
Xenobiotic
degradation:
Tetrachloroethene
Tidal flat sediments The most dominant bacteria involved in tetrachloroethene

dechlorination were Dehalococcoidetes spp., and a novel group
here designated as Tidal flat Chloroflexi Cluster related to
Dehalococcoides spp.
Aburto and Ball

(2009)
Xenobiotic
degradation:
Benzene
Groundwater
Members of Acidovorax spp. and Milika spp. dominated the

acquisition of carbon from benzene under aerobic conditions
Huang et al.
(2009)
Xenobiotic
degradation:
Naphthalene
Groundwater
Acidovorax spp. were the key organisms in naphthalene

degradation in situ displaying high substrate affinity, while
Pseudomonas putida and P. fluorescens were low affinity
naphthalene degraders
Sueoka et al.
(2009)
Xenobiotic
degradation: Phenol
Sludge (denitrifying Azoarcus spp. was the primary degrader of phenol. Microbulbifer,
conditions)
Pelagiobacter, Pseudomonas, and Thauera spp. also assimilated
carbon from phenol either as primary consumers, intermediate
consumers or cross-feeders
the community profiles generated from heavy fractions of acetate-amended PCE cultures versus acetate
only cultures, unique sequences were found for PCEdechlorinating microbes, which corresponded to
Chloroflexi spp. distantly related to Dehalococcoides
spp. The same strategy was applied soon after to
identify the PCE-degraders in tidal flat sediments
(Kittelmann and Friedrich 2008b). A novel group of
PCE-dechlorinating bacteria, which were designated
the Tidal Flat Chloroflexi Cluster, and a population
closely related to Dehalobium chlorocoercia DF-1
were identified in the heavy fractions of 13C-acetate
amended PCE microcosms. Thus, dominant dechlorinating bacteria were successfully identified with
RNA-SIP. Collectively, these studies have demonstrated that by using the correct controls and experimental set up, RNA-SIP can be applied to identify
microorganisms involved in dissimilatory processes.
The use of 15N as a tracer in RNA-SIP has only
recently been tested to study microbes involved in the
nitrogen cycle. Since the content of nitrogen in RNA
is approximately 2.5 times less than carbon, it
presents the difficulty of a lower density gain after
123
a 15N pulse. Addison et al. (2010) observed that 15Nlabelled RNA increased in buoyant density compared
to unlabelled RNA when centrifuged individually,
but not when centrifuged together. Similarly, labelled
RNA extracted from a paper mill effluent microcosm
after a 15N2 pulse, showed a limited separation by
density centrifugation in CsTFA. Longer centrifugation time did not solve the problem. The authors
highlighted the difficulty in separation of 15Nlabelled RNA in CsTFA and argued that comingling interactions of 14N- and 15N-RNA may
have occurred during gradient centrifugation that
reduced the resolution of the gradient. A second
ultracentrifugation step with bis-benzimide could
potentially solve this problem (as observed in 15NDNA-SIP), however, this remains to be tested.
2.4 Protein-stable isotope probing (Protein-SIP)

This method was developed based on the fact that
proteins are the biomolecules that can provide the
most direct link to a metabolic process, since they
catalyse reactions directly (Jehmlich et al. 2008a).

The aim of the method is to obtain a protein profile
that describes the identity of a microbial species and
to measure concomitantly the incorporation of stable
isotopes into its atoms. To achieve this, the proteins
are extracted from cells grown with a labelled
substrate used as a carbon or nitrogen growth source.
These are separated by two dimensional gel electrophoresis and selected proteins are digested with
trypsin, purified and analysed by matrix-assisted laser
desorption ionisation/mass spectrometry (MALDIMS) to obtain a peptide mass fingerprint (PMF) for
each protein. The PMF of proteins derived from the
unlabelled substrate are then compared to public
databases to obtain the identity of the microbes that
possess the proteins. A tandem mass spectrometry
analysis (MS/MS) is also used to validate the identity
of the peptides by generating a peptide tag that can
be used to identify a protein in a database.
By comparing the mass of the peaks in the PMF
spectra of the non-labelled with the labelled samples,
shifts in mass (increase) can be detected per peptide
and the amount of label incorporation can be
calculated per protein. The atom% of 13C and 15N
incorporation (incorporation efficiency) is calculated
taking into account theoretical natural abundance
values. Thus, heavy proteins can be identified from
a mixture of labelled and unlabelled ones. To assign a
taxonomic identity to the heavy proteins, the nonlabelled counterpart (derived from two dimensional
gel from non-labelled control sample) is investigated
by PMF database analysis as described above. The
labelled PMF cannot be used directly since database
search algorithms are based on PMF from nonlabelled sources. Finally, those proteins that become
labelled can be used to directly link function with
microbial identity.
This technique requires only 12 atom% isotopic
enrichment to detect labelling; therefore, it has a 10
fold higher sensitivity compared with RNA/DNA-SIP.
In consequence, pulses of labelled substrate can be
administered at lower concentration and less duration.
The taxonomic resolution that can be obtained is
comparable to that from DNA/RNA-SIP if MS/MS
peptide tag identification is employed, because
related databases are used. This technique allows a
direct quantification of the level of isotopic enrichment of the proteins, which potentially could mean
obtaining differences in levels of isotope (substrate)
165
incorporation per species in a complex system. This

method can provide direct information on the metabolic pathways being used in the assimilation of a
carbon or nitrogen source.
Despite the increased sensitivity and good taxonomic resolution, a disadvantage of Protein-SIP is
that proteins from different organisms can have
identical amino acid composition, with consequent
ambiguous taxa identification. According to the
authors of this method (Jehmlich et al. 2009) this
can be overcome by analysing at least three peptides
per protein in each case. However, this can result in a
very time consuming analysis. Additionally, overlapping spots in two dimensional gels may complicate
the selection of relevant proteins from complex
samples and potentially cause cross-contamination.
One component of the taxonomic assignment of
peptides is based on PMF profiles in existing
databases, which means that the resolution will be
limited to the profiles contained in these databases if
an MS/MS analysis is not done in parallel. Using
parallel MS/MS for every sample implies that a very
large amount of data will need to be handled and
processed, making this procedure potentially tedious.
This method was first published in 2008 (Jehmlich
et al. 2008a) using the strain Aromatoleum aromaticum EbN1, which is able to grow on toluene as a sole
carbon source under denitrifying conditions. The
authors demonstrated that it was possible to detect
labelled proteins exclusively from this strain when
grown within an artificial mixed culture unable to
utilise toluene. The identity of a number of proteins
from this test strain was first confirmed. Then, by
comparing the mass of the peaks in the PMF spectra
from non-labelled and labelled samples, they calculated an average 13C incorporation level of 82.6% in
this culture. The fact that this value was lower than
expected was attributed to cross feeding effects in
this mixed culture. All proteins with isotopic enrichment corresponded exclusively to strain EbN1. In this
work, the authors demonstrated for the first time that
it is possible to link functional information from a
specific microbe to its identity using proteins.
A second study (Jehmlich et al. 2008b) tested the
method using both a 15N and 13C labelled substrate.
Pseudomonas putida ML2 was grown with 13Cbenzene or 15N-ammonium (and 12C-benezene) as
sources of carbon and nitrogen. Two dimensional gel
electrophoresis and MALDI-MS analysis and
123
166
quantification of ten proteins allowed them to determine that 13C and 15N isotopes were incorporated into
the proteins with an efficiency of 90 atom% for 15N
and 98 atom% for 13C.This was in good agreement
with the expected levels. Thus, this study demonstrated the applicability of this method when using
13
C and 15N isotopes simultaneously.
To improve the method, Jehmlich et al. (2009)
compared two alternative fingerprint strategies to
eliminate the two dimensional gel electrophoresis
step. Proteins were extracted from P. putida ML2
cells labelled through assimilation of 13C-benzene and
15
N-NH4Cl as growth substrates. Protein isotopic
incorporation was compared using intact protein
profiling (IPP) and shotgun mass mapping (SMM).
IPP is based on the profiling of low molecular weight
proteins using MALDI-MS followed by a comparison
of the profile with a fingerprint from a reference strain.
SMM is based on profiling by MALDI-MS of protein
fragments generated by trypsin digestion of the total
protein content of the cells. Calculation of the isotope
content of the samples was done as before (labelled
vs. unlabelled spectra), but incorporating the averagine model which assumes an average molecular
composition and mass of amino acids to calculate the
number of C and N atoms per peptide. After MALDIMS, an additional MS/MS analysis was performed to
obtain a spectra profile for a database comparison to
assign identity. The authors concluded that the SMM
approach was better than the IPP approach because it
provided better accuracy, less uncertainty and is
independent of reference strains.
To date, this method has only been tested in
artificial communities supplemented with one bacterial species able to degrade the tested substrate. It still
awaits validation with environmental samples or
complex mixtures.
2.5 Radioactive isotope probing (RIP)
This method constitutes the radioactive counterpart
of SIP. It was first proposed and described by
Nikolausz et al. (2007), and is based on the labelling
of nucleic acids after a radioactive pulse. RNA
extracted from cells grown on labelled substrates is
subjected to reverse transcription and clone library
construction or DGGE profiling. This DNA profile is
then transferred onto a nylon membrane via electroblotting and hybridised with denatured labelled RNA.
123
This step is necessary because the radioactive label is

lost during the PCR amplification process which
consequently impedes the direct analysis of the gel
for radioactivity. The membrane is processed with
phosphor imaging technology for the detection of
hybridised bands, which correspond to microorganisms that assimilated the carbon source. By excision
and sequencing of the bands the identity of the active
microbes is resolved. Alternatively, single stranded
DNA generated from the clones in the library can be
dotted onto a nylon membrane and processed in the
same way to find clones that incorporated the
radioactive label.
This method is relatively simple and requires less
sophisticated equipment as compared to isotope
arrays (see below). A radioactive label allows the
use of less labelled substrate for pulse experiments in
comparison with stable isotopes, reducing potential
cross-feeding effects. Disadvantages to this method
include those related to manipulation of radioactive
substances as discussed before.
After the first report from Nikolausz et al. (2007),
this method has not been applied in other environments. However, recently, Franchini et al. (2009),
built on the development of this technique and
proposed the use of DNA:14C-RNA hybrid molecules
to generate a community fingerprint of active
microbes in a method termed Sequence Specific
Primer Extension RNA Analysis (SeSPERA). In their
approach, they hybridised a taxon-specific probe to
labelled RNA for cDNA construction using a reverse
transcriptase lacking RNase H activity, which leaves
the RNA template intact. After elongation, the hybrid
is treated with RNase T1 to eliminate non-hybridised
RNA and to create a duplex molecule that varies in
length from species to species. These hybrids are
separated by agarose gel electrophoresis to create a
profile, which is then blotted onto a membrane for
further phosphor imaging analysis of radioactive
label. This method has the potential to yield quantitative information of active consumers of a labelled
substrate to some extent. It utilises a taxonomic probe
to define identity of consumers, which limits the
taxonomic resolution to the probe being used. SeSPERA is reliant on the limited size separation capacity
of agarose gels, which may impede clear separation of
hybrid molecules from complex environmental samples. Nonetheless, SeSPERA constitutes a promising
variation of radioactive isotope probing.
167
3 Probe-based methods
within a sample and allows resolution to the cellular

level in situ. FISH methods also provide information
on spatial arrangement of organisms, which SIP
methods cannot achieve. Any FISH protocol has the
disadvantage of relying on the use of a probe to target
a specific group within a community. This means that
it is necessary to have prior knowledge of the
microbial community under investigation or at least
an understanding of the diversity of microbes likely
to be present in a sample. Although it is possible to
use a universal probe to target high level taxa, the use
of a probe sets the limit of discovery of this technique
in comparison with SIP methods. A combination of
fluorescently labelled probes can be used to identify
bacteria from different taxonomic groups, however,
there is only a limited number of fluorophores that
can be applied simultaneously (Wagner et al. 2006).
This imposes a limit in the diversity of microorganisms that can be detected. This shortcoming can be
overcome by an initial investigation of substrate
consumers with SIP methods followed by design of
relevant probes to apply with FISH based analysis.
This strategy has been termed the full cycle rRNA
approach (Amann et al. 1995; Ginige et al. 2004).
A series of problems come into play when using
taxonomic probes. Firstly, if total permeability of the
The majority of probe-based methods have in common the use of fluorescence in situ hybridisation
(FISH). A flow chart that depicts the basic steps in the
different probe-based methods is shown in Fig. 3. In
FISH based methods, the identification of probedefined microbial taxa precedes the identification of
the microbes involved in assimilation of a substrate.
In brief, the microbial community of interest is
probed with a fluorescently labelled DNA oligonucleotide that binds to a specific sequence in target
rRNA. The probe can be selected to bind organisms
at different taxonomic levels, e.g. species-specific to
domain-specific, and/or multiple probes can be used
to locate multiple taxonomic groups. When the probe
binds to its target, it becomes fluorescent, labelling
the cells that belong to the chosen taxa. Fluorescence
of single cells, aggregates or biofilms can then be
detected with epifluorescence microscopy or confocal
laser scanning microscopy. A counterstain with 40 ,6diamidino-2-phenylindole (DAPI) allows quantification of all cells.
The main advantage of FISH analysis above SIP
methods is that it permits quantification of the
relative abundance of specific microbial groups
Type of probe
Microautoradiography
F-probe
Pulse
Hybridisation of
sample with probe
H-probe
HRP-probe
+ H-F-tyramide
14
C-RNA
+
fluorescent dye
RNA
extraction
13
C-RNA
Fragmentation
+
Hybridisation with
taxonomic
probe array
Biotin probe
hybridisation
Assignation of function
Basic method
Fluorescence
microscopy and
TEM microscopy
Fluorescence
microscopy
Isotope imaging
by
Secondary ion
mass spectrometry
Fluorescence
microscopy
Raman
microspectroscopy
Isotope imaging by NanoSims
Fluorescence
microscopy
Isotope imaging
by NanoSims
Fluorescence imaging and -imaging
Magnetic bead
capture
Isotope ratio mass

spectrometry
Name of method
MAR-positive
cells
MAR-FISH
Increase in
isotope ratio
per cell
FISH-SIMS
Red shifts in
biomolecules
per cell
FISH-RAMAN
Increase in
isotope ratio
per cell
SIMSISH
Increase in
isotope ratio
per cell
EL-FISH
Fluorescent
spots = identity
Radioactive
spots = labelled
taxa
Labelled taxa as
per rRNA
Isotope Arrays
SSU-IRMS
Fig. 3 Schematic representing the basic steps in the different probe-based methods reviewed. See text for definition of abbreviations.
F-probe = Fluorophore-labelled probe; H-probe = Halogen-labelled probe; H-F = Halogen and fluorophore-label
123
168
cell is not achieved prior to the hybridisation step, the

probe may not reach its target, resulting in inadequate
quantification. Secondly, interaction between ribosomes and proteins or secondary and tertiary structure
of rRNA may render the target inaccessible to the
probe. Thirdly, the intensity of the signal depends on
the number of target ribosomes, therefore if the cells
are inactive, the signal intensity may be low.
Fourthly, target specificity can be a problem, as the
probes can potentially bind to unwanted targets,
especially when discerning between two closely
related species. Additionally, FISH is difficult to
perform in soil systems given that soil has high
background fluorescence. Thus, not all environmental
samples are suitable for FISH (Wagner et al. 2006).
A number of developments have been made to
overcome inherent limitations in FISH studies.
Multiple probes have been used to target independent
sites of the same rRNA molecule to increase its
fluorescent signal (Lee et al. 1993). Alternatively,
unlabelled helper probes (Fuchs et al. 2000) or
peptide nucleic acid probes (Worden et al. 2000)
have been employed to increase target site accessibility. To artificially increase the amount of target
rRNA, cells have been incubated with chloramphenicol prior to fixation (Ouverney and Fuhrman 1997).
In addition, the amplification of the fluorescent signal
has been achieved by applying the catalysed reporter
deposition technique (Schonhuber et al. 1997;
CARD-FISH). Recently, doubly labelled probes (50 and 30 - end dye labelling) have been developed that
increase signal intensity and target accessibility
(Stoecker et al. 2010). These and the concomitant
technological improvements in the imaging equipment and software have substantially improved the
ability of FISH to detect targeted cells.
3.1 Fluorescence in situ hybridisationmicroautoradiography (FISH-MAR)
FISH-MAR (also known as substrate-tracking autoradiography (STAR-FISH) and microautoradiography-FISH (MICRO-FISH)) allows the taxonomic
identification of microbes that incorporated a radioactive carbon label after a pulse experiment. Pulsed
samples are first prepared for FISH analysis. Then, to
determine the microbial involvement in consumption
of the substrate, the FISH-processed sample is
subjected to an autoradiographic procedure. In brief,
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the microscopic slide containing the sample is

overlayed with a silver emulsion, exposed for a few
days, developed and analysed by confocal laser
scanning microscopy. The emulsion layer absorbs
the beta particles emitted by the radioactive carbon
and causes the deposition in the emulsion of silver
grains on sites adjacent to the cells emitting the
radiation. The silver grain deposition is observed with
the transmission mode of the microscope. The image
is then compared and overlayed with that obtained
with the fluorescence mode to correlate probetargeted cells with grain accumulation.
FISH-MAR has a high lateral resolution of 0.5
2 lm, which means that it can be used to detect the
activity of single cells. It is a highly sensitive method,
as incorporation into all biomolecules is detected, as
opposed to SIP methods where only one biomarker
molecule is detected. As a consequence of this,
incubation times with the label are shorter than in SIP
experiments, reducing cross-feeding effects.
One of the main disadvantages of FISH-MAR is
that it is labour intensive and time consuming,
resulting in low throughput (Nielsen and Nielsen
2005). In addition, fundamental experimental factors
such as biomass/radioactive substrate ratio, background levels of unlabelled substrate, presence of
other electron donors and acceptors, length and
conditions of incubation, length of exposure and
development time, need to be determined a priori by
trial and error. Furthermore, development of the
emulsion has to be determined empirically to avoid
biases in grain saturation and signal-to-noise ratio.
The sensitivity of the method is limited by the
sensitivity of the radiographic emulsion and the
thickness of the sample section analysed. It is also
necessary to run duplicate or triplicate samples to
quantify adsorption of the radiolabel onto cell
surfaces. Also, as described above, radioactive substrates are expensive and hazardous.
The FISH-MAR approach was described by Lee
et al. (1999) and Ouverney and Fuhrman (1999). Ten
years later, over 30 papers have been published that
have reported the use of this method. These have been
reviewed elsewhere (Okabe et al. 2004; Rogers et al.
2007; Wagner et al. 2006), so in this review only those
published between 2008 and 2010 are reported. It is
important to mention that since its conception, FISHMAR has undergone a series of makeovers that have
improved the method. These have resulted in Q-
MAR-FISH, Het-CO2-FISH-MAR and MAR-CARDFISH, which are discussed below.

FISH-MAR has been employed most extensively in
the study of microbial communities in wastewater
treatment plants. It has led to the discovery in activated
sludge of a novel group of epiphytic bacteria involved
in the consumption of amino acids and proteins (Xia
et al. 2008). It has allowed the elucidation of the role of
Bacteroidetes in degradation of sugars, lipopolysacharides and peptidoglycans derived from dead cells
(Kragelund et al. 2008). Through FISH-MAR analysis,
Chloroflexi spp. have been implicated in the consumption of bacterial detritus carbon in sludge (Miura and
Okabe 2008). The role that Beta- and Gammaproteobacteria play in the degradation of estrogen has also
been identified through FISH-MAR in waste water
treatment plants (Zang et al. 2008).
FISH-MAR has been applied to understand phosphate accumulating organisms (PAOs) in activated
sludge. A model for anaerobic carbon metabolism of
polyhydroxyalkanoates in Accumulibacter spp., an
important PAO member, has been determined (Burow
et al. 2008). Additionally, Firmicutes and Actinobacteria species were found to constitute important
consumers of glucose, mannose and galactose implicating them as potential carbon and energy providers
for PAOs (Kong et al. 2008). In relation to the
nitrogen cycle in activated sludge, addition of methanol and acetate was found to enhance nitrate uptake
in denitrifying sludge with Azoarcus spp. being the
most active bacteria assimilating carbon from acetate
and methanol (Hagman et al. 2008). Deciphering this
functional information in activated sludge is essential
for the rational design and operation of wastewater
treatment plants.
FISH-MAR has also been recently applied to link
function with identity in seawater and lagoon environments. The functional roles of microbes in deep ocean
biogeochemistry have been investigated by Varela
et al. (2008a, b).These authors observed that members
of the cluster SAR202 consume L-asparagine preferentially over D-asparagine. This was in contrast to a
large proportion of prokaryotic D-asparagine consumers in deep ocean waters of the North Atlantic. Other
studies have investigated communities of nitrifiers
from zero-discharge marine aquaculture biofilters by
using FISH-MAR analysis (Foesel et al. 2008). In this
saline environment with fluctuating ammonia concentrations, Nitrosomonas spp. Nm-143 lineage members
169
were found to be the most active ammonia oxidisers,

whereas Nitrospira marina species were the most
active nitrite oxidising bacteria. In lagoon planktonic
environments differences in the metabolic behaviour
of Betaproteobacteria highlighted the functional roles
of Polynucleobacter clades C and D under different
environmental conditions, pointing to their potential
ecological roles in situ (Alonso et al. 2009).
3.2 Variations on the FISH-MAR strategy:
Q-FISH-MAR, Het-CO2-FISH-MAR
and MAR-CARD-FISH
FISH-MAR is considered a semi-quantitative tool, as
quantification of silver grains can vary between slides
and between different radioactive substrates used for
the same sample. At best, quantitative data can be
obtained by measuring the percentage of the total
silver grain area corresponding to probe-defined
bacterial groups in comparison with total DAPI
counts, as was shown by Cottrell and Kirchman
(2003) and Malmstrom et al. (2004). However,
attempts to improve the quantitative capacity of
FISH-MAR were described by Nielsen et al. (2003)
in what they termed Quantitative- FISH-MAR or QFISH-MAR. Their procedure included the preparation of a standard curve with pure cultures of a
filamentous bacterium incubated with different concentrations of labelled substrate. After MAR was
performed on these, the number of silver grains per
cell was plotted against counts per minute per cell to
obtain the standard curve. The environmental sample
was incubated with the same substrate and was spiked
with the standard cells containing defined specific
radioactivity and pre-stained with DAPI. The counts
per minute of the target cells were then inferred from
the standard curve. This way the authors calculated a
specific activity per target cell in situ and the
substrate affinity for the uptake of acetate of two
different filamentous bacteria in sludge. To date, no
other similar study has been performed, owing
perhaps to the labour intensive process and large
amounts of radioactive substrate needed. Since QFISH-MAR needs an internal standard of bacteria
with known radioactive isotopic composition, the
comparison of quantitative data from different studies
may be problematic.
FISH-MAR has also been used to monitor 14CO2
assimilation into bacterial cells as a means of
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170
identifying active heterotrophs (Het-CO2-FISHMAR; Hesselsoe et al. 2005). The rationale is based
on the observation that all heterotrophic organisms
assimilate CO2 during biosynthesis in various carboxylation reactions. Therefore, it is possible to isotopically label active heterotrophic cells by incubation
with 14CO2 in parallel with an unlabelled substrate of
interest. This enables assessment of the consumption
of multiple unlabelled compounds with a single
labelled compound and the investigation of complex
organic substrates whose radioactive counterparts may
not be available. For example, this method was used to
assess the substrate preference (diesel or glucose) of
two bioaugmentation mixtures proposed for bioremediation of diesel-contaminated soils (Hesselsoe et al.
2008). It was concluded that a rapid assessment of the
substrate preferences of bioaugmentation mixtures
could be attained to circumvent costs and time
involved in microcosm evaluation studies.
With this approach, incubation times can be shorter
than MAR, as the cell specific isotope labelling is more
than one order of magnitude faster than that obtained
with a reduced carbon substrate (Hesselsoe et al. 2005).
The assimilation of 14CO2 is, however, not a direct
quantitative measure of substrate incorporation, as it
may depend on the background concentration of the
inorganic carbon pool during metabolism of available
substrates. Similarly, labelling of autotrophic microorganisms will occur independently from substrate
assimilation. In addition, differences in growth rates,
CO2 assimilation and isotope dilution (presence of
unlabelled CO2 in samples) may introduce variations
in cell specific label incorporation. Also, conditions
need to be optimised for a specific system to ensure
appropriate labelling and to reduce background noise.
In the original application of Het-CO2-FISHMAR, Candidatus Microthrix parvicellaa filamentous bacteria from sludgewas incubated with 14CO2
and oleic acid (its main carbon and energy source)
under aerobic and anaerobic conditions, as well as
with 14C-oleic acid in parallel to compare the
approach with the conventional use of a reduced
carbon substrate (Hesselsoe et al. 2005). It was
observed that cells were MAR-positive in both
aerobic and anaerobic conditions using the conventional approach. Conversely, only those that grew
aerobically were positive with Het-CO2-FISH-MAR.
By switching from anaerobic incubation with 14CO2
and oleic acid to aerobic conditions, the cells became
123
MAR-positive. Cells had assimilated 14CO2 in comparable rates to filaments incubated aerobically
without transitions. The authors concluded that these
bacteria can store oleic acids incorporated during
anaerobiosis and subsequently use them for growth
under aerobic conditions. This may explain why this
bacterium grows extremely well in nutrient removal
plants under alternating aerobic/anaerobic conditions.
This physiological phenomenon would not have been
observed using 14C-oleic acid alone.
Catalysed reporter deposition (CARD), used extensively in immunoassays, is a methodological improvement to FISH that has enhanced several FISH-MAR
studies. MAR-CARD-FISH overcomes the problem in
FISH-MAR of low signal intensity of fluorescently
labelled microbes with very low cell numbers. It
increases the signal-to-noise ratio in environments
with high background fluorescence and detects cells
that may have very low rRNA contents (e.g. oligotrophic environments). In this technique, the fluorophore of the FISH probe is replaced by the horseradish
peroxidise enzyme (HRP), the catalyser. The HRPprobe is hybridised with its target rRNA and the cells
are incubated with the fluorescently labelled tyramide
reporter. Tyramides are phenolic compounds that can
penetrate through the cell membrane. In contact with
HRP, tyramides form highly reactive intermediates
that bind to electron rich moieties of proteins, including HRP itself. Thus, the HRP catalyses the deposition
of fluorescent tyramide molecules in its vicinity. In this
way, numerous fluorescent signals can be introduced at
the hybridisation point in situ. This is also known as the
tyramide signal amplification method. The result is a
greatly enhanced FISH sensitivity compared with
probes with a single fluorophore. The introduction by
diffusion of a large molecule (HRP is 40 kDa) with the
probe into the cell needs a carefully controlled
permeabilisation step to achieve the correct targeting
of cells (Pernthaler et al. 2002). This method has the
additional step of conjunction of tyramide with fluorescent dyes and HRP with the phylogenetic probe,
which results in more experimental costs.
The protocol for CARD-FISH was first described
by Pernthaler et al. (2002). In this work the authors
demonstrated enhanced sensitivity of this method
above conventional FISH when applying the method
to detect the activity of marine bacterioplankton,
which typically has low cell numbers. Two years
later, Teira et al. (2004), published an improved
protocol and combined it for the first time with

microautoradiography (MAR-CARD-FISH). The
authors obtained a two-fold increased detection rate
of archaea in deep sea waters in comparison with
previous studies. Interestingly, they also found that in
deep sea waters of the North Atlantic, archaea were
more abundant than bacteria, and that archaea took up
a larger proportion of L-aspartic acid than bacteria.
Since then, MAR-CARD-FISH has been used to
identify active microorganisms in a variety of environments by way of tracking assimilation of labelled
amino acids, glucose, ATP or dimethylsulfoniopropionate. Over 20 papers have been published since it
was first described. Studies from the last 3 years are
compiled in Table 5. Recent work has focused on
active microbes in the marine water column, freshwater environments and hot springs. In these, the
general aim was to use MAR-CARD-FISH to unveil
spatial and seasonal distribution patterns of active
microbes or the effects of environmental disturbances
on their activity rather than to determine the role of
microbes in the degradation of a substrate.
3.3 FISH-Secondary ion mass spectrometry
(FISH-SIMS)
In FISH-SIMS, after the microbial cells of the probed
taxa are identified by FISH, the cell/aggregates of
interest are mapped by software-assisted imaging and
the glass slide containing the sample is directly
marked with a diamond knife. This glass slide is then
placed in a secondary ion mass spectrometer, which
determines the isotopic composition of the targeted
cells by capturing the secondary ions emitted from
the sample after sputtering with a caesium ion beam
across its surface.
An advantage of FISH-SIMS is that it allows the
direct determination of labelled substrate incorporation by specific cells identified by FISH. As opposed
to MAR-FISH, it precludes the use of radioactive
isotopes, as the SIMS instrument is able to detect the
mass changes that result from stable isotope incorporation. Additionally, it allows analysis of incorporation of a range of stable isotopes (3H, 18O, 15N, etc.)
SIMS instruments have ion microprobes with
lateral beam resolution of 115 lm, which is enough
to scan an average bacterial cell but is limited in
comparison with the resolution offered by similar
technologies (NanoSIMS see below). Another
171
drawback of this technique is the fact that two

expensive instruments are required (confocal laser
scanning microscope and SIMS instrument). Additionally, in order to achieve an accurate match
between a cell and its isotopic content, an exact
mapping of the samples between instruments is
needed. Thus, it can result in false positives and the
accuracy is difficult to evaluate.
Orphan et al. (2001) described the use of FISH-SIMS
for the first time in studying the natural abundance of
13
C of anaerobic methane oxidising archaea in marine
environments. The authors were able to determine the
d13C profile from the periphery to the interior of
separate cell-aggregates containing ANME-2 archaea
in the core and Desulfosarcina/Desulfococcus bacteria
on the periphery. They found a large depletion of d13C
in both groups which could only be explained by
consumption of the naturally depleted methane. Subsequent studies using FISH-SIMS (Orphan et al. 2002)
revealed that ANME-1 archaea are active methane
oxidisers either as single cells or as aggregates. The
authors also observed a greater diversity of microbes
and associations in anaerobic oxidation of methane
(AOM) than had previously been reported. AOM was
found to involve both bacteria and archaea and to occur
as pure or mixed species consortia.
Further studies with 15N-labelling of active cells
through assimilation of labelled amino acids and
ammonia revealed a de-coupling of metabolic activity
in the members of the ANME-2/sulphate reducing
symbiosis (Orphan 2009). Members of this symbiosis
had a tendency for higher 15N uptake when coaggregated than as individual cells. Metagenomic
analysis of the consortium indicated the presence of
nitrogen fixing genes in ANME-2 (Pernthaler et al.
2008). Therefore, further FISH-SIMS analyses were
carried out which specifically identified the ANME-2
members as the primary nitrogen fixers of this consortium by localising and quantifying 15N assimilation in
individual cells of ANME-2/sulphate reducing bacteria aggregates (Dekas et al. 2009). These studies have
contributed to the understanding of the carbonnitrogensulphur cycle in deep seep environments.
Treude et al. (2007) used SIMS in combination
with other methods (CARD-FISH and beta-micro
imaging) to detect the distribution of stable carbon
isotopes in the bulk biomass of a methanotrophic
microbial mat from submarine gas seeps. In combination with radiotracer analysis the authors identified
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172
Table 5 MAR-CARD-FISH examples from 2007 to 2010

Reference
Type of study
Environment
Main finding
Varela et al.
(2008b)
Spatial distribution of active

Marine: North
Atlantic
There was a decrease of picoplankton activity of bacteria and

archaea with depth, as per D- and L-aspartic acid uptake. The
relative abundance of Crenarchaeota I decreased in deep
waters
Zhang et al.
(2009)

Marine: South
China Ocean
The activity of Crenarchaeota I increased with depth, as per

aspartic acid uptake, whereas activity from bacteria
decreased. Prokaryotic activity was higher inside rather than
outside cyclonic eddies, dominated by Crenarchaeota in the
mesopelagic zone
Grote et al.
(2008)

Marine: Baltic
From total chemoautotrophic prokaryotes, members of the
and Black Sea
Epsilonproteobacteria constituted the major active group in
the dark deep redoxclines of these seas (70100%)
Alonso-Saez
and Gasol
(2007)
Seasonal distribution of active

Marine:
Mediterranean
Sea
Roseobacter sp. was the most active bacterial group all year
round in comparison with Gammaproteobacteria,
Bacteriodetes and SAR11, which showed a variable uptake
activity throughout the year
Vila-Costa
et al. (2007)
Marine:
Mediterranean
Sea
A steady increase in sulphur assimilation from winter to

summer was detected in all bacteria, with Roseobacter sp.,
SAR11 and Gammaproteobacteria, being the most active
groups. This may be a consequence of
dimethylsulfoniopropionate being used as both a carbon and
sulphur source in summer
Alonso-Saez
et al. (2008)
Marine: Arctic
Ocean
Archaea were more active in winter than in summer, when

they became almost undetectable. In summer, bacteria were
more active than in winter. Betaproteobacteria, Roseobacter
and Gammaproteobacteria were less abundant but more
active than SAR11 and Bacteroidetes, indicating that cell
number was not directly proportional to activity
Obernosterer Spatial distribution of active

et al. (2008)
Marine: South
Pacific Ocean
Across a transect from Marquises Island to Chile,

Bacteriodetes members were active across surface and
deeper layers, but that Gammaproteobacteria were more
active in the top micro layer (250400 lm), whereas
Alphaproteobacteria dominated in the subsurface layers
Perez and
Sommaruga
(2007)
Active community structure in

relation to environmental
parameters
Freshwater:
Alpine lake
Increased UV radiation had a negative effect on bacterial

activity via increased photoalteration of organic matter.
Changes in behaviour of Actinobacteria and
Betaproteobacterial assemblages in time evidenced changes
in the community structure
Salcher et al.
(2008)
Seasonal and spatial distribution

of active microbes in the
environment
Freshwater:
Mesotrophic
lake
A moderate seasonal variation in abundance of active

bacteria was observed at the top layers, but marked increase
of PnecC and Beta IV clades of Proteobacteria in deeper
anoxic layers. Distinct vertical ecophysiological niches in
the lake were found
Wilhartitz
et al. (2009)
Freshwater:
Alpine karstic
aquifer
A significantly lower percentage of active bacteria in the

planktonic phase was found in comparison with the
sediment, suggesting a potential surface-associated benefit
for active bacteria
Hatzenpichler Biogeochemical processes:

et al. (2008)
Ammonia oxidation
Hot spring
The first thermophilic ammonia oxidiser was described. A

novel thermophilic archaea growing in an enrichment
culture showed ammonia oxidising activity. The microbe
belongs to the widely distributed group I.1b of the
Crenarchaeota
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hot spots of methanotrophy close to the surface of the

mats. Further applications of FISH-SIMS include the
recent report from Dattagupta et al. (2009) where
they observed that the exoskeleton of the macroinvertebrate amphipod Niphargus ictus, endemic to the
Frasassi cave waters in Italy, was abundant with
sulphur oxidising bacteria Thiotrix spp. living in
symbiosis with this animal. It was found that these
bacteria were chemoautotrophs and did not derive
carbon from their host, but instead benefited from the
mobility of the animal between oxic and anoxic
sulphur rich environments. The animal obtained a
benefit from the reduced sulphide toxicity. This was
the first example found of a non-marine symbiosis
between an animal and chemoautotrophic bacteria.
3.4 FISH-RAMAN
This method allows for the detection of stable isotope
labelled cells in a FISH sample by detecting the
covalent bond vibrational shifts (red shifts) of the
molecules contained in those cells. Molecules that
contain 13C label produce longer wavelengths within
the Raman spectra. These are detected by confocal
Raman microspectroscopy and displayed as peaks for
different biomolecules within a cell. The spectral
shifts of the relevant molecules (nucleic acids,
proteins, the essential amino acid phenylalanine)
correlate with the label content of the cells, hence this
method offers a quantitative approach, e.g. amounts
of label incorporated per cell.
This method has a lateral resolution of 1 lm.
Therefore, in addition to giving single cell information, it also provides information into which cellular
molecules incorporated the label. Analysis time is
typically 1 min per cell, which reduces considerably
the time employed as compared to the FISH-MAR
technique. The Raman laser analysis is performed
directly on the same microscopic slide used for FISH,
thus, as with SIMSISH, it precludes the use of two
different machines for the full identity-function
analysis, and permits a direct screening of label
incorporation. FISH-RAMAN has similar advantages
to FISH-MAR, however, as opposed to the latter,
FISH-RAMAN uses stable rather than radioactive
isotopes. Consequently, FISH-RAMAN can be coupled to SIP analysis with a single pulse, which reduces
the cost and time of experimentation as compared to a
SIP-FISH-MAR approach. Importantly, it could also
173
provide a quantitative analysis of labelled carbon

incorporation by different cells in a sample.
This method requires calibration curves using
cultured microorganisms to obtain quantitative information, which may be limiting for some environments
where no cultured representatives exist. Despite its
single machine advantage, its composite equipment
(fluorescent equipped-Raman microscope) may be
expensive to acquire (Neufeld and Murrell 2007).
In the seminal FISH-RAMAN paper, Huang et al.
(2007) demonstrated Pseudomonas spp. in situ
involvement in naphthalene degradation in groundwater, and discovered a large variation of 13C incorporation within different pseudomonad cells. The
authors showed that there were either activity differences within a clonal population or activity differences
within the different targeted pseudomonads. Furthermore, the authors applied a combination of RNA-SIP
and FISH-RAMAN (Huang et al. 2009) to explore
naphthalene degradation in the entire microbial community of this polluted groundwater, finding that
Acidovorax spp. played a dominant role in naphthalene degradation. The high affinity of Acidovorax spp.
for naphthalene versus pseudomonads lower affinity
implied the existence of a niche specialisation that has
allowed the diversification (and co-existence) of the
microbial communities in situ.
3.5 The NanoSIMS approach: Secondary Ion
Mass Spectrometry In Situ Hybridisation
(SIMSISH) and Element Labelling-FISH
(EL-FISH)
3.5.1 SIMSISH
In this technique, the fluorescent dye of the taxonomic probe used in FISH is replaced by the stable
isotope of a halogen gas (e.g. 127Iodine), elements
rarely present in biomass. The probe is hybridised
with its target and the isotopic signal is detected by a
NanoSIMS instrument, which when coupled to
sophisticated imaging software creates a quantitative
mass image. A quantitative mass image contains at
each pixel a number of counts that are directly
proportional to the selected atomic mass abundance
in the sample. Counts from several atomic masses can
be obtained in parallel from the same pixel location
enabling the calculation of isotope ratios. Also, cells
that contain isotopically enriched atoms of C, N, O,
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174
P, and S can be detected simultaneously. This enables

the determination of the involvement of a cell in the
assimilation of substrates labelled with stable isotopes of any of these atoms.
The NanoSIMS instrument is the latest development in secondary ion mass spectrometry technology.
It uses the same principle of SIMS (bombardment of
a Cs beam into a solid sample and detection of the
secondary ions emitted by the sample separated
according to mass), but has a greatly enhanced
sensitivity to isotopes and an increased lateral
resolution of 50 nm, hence the prefix Nano. Consequently, this method provides an improved spatial
resolution compared to that of conventional SIMS (1
15 lm) and FISH-RAMAN (1 lm). The NanoSIMS
instrument has a sensitivity to isotopes that exceeds
autoradiography by about 1000 times (Williams
2006), and it can measure multiple isotopes simultaneously. It provides a means to analyse microbial
identity and function with one highly sensitive and
precise instrument. In addition, a NanoSIMS instrument can determine quantitative information on
isotopic enrichment per cell, and it is compatible
with stable and radioactive isotopes.
The main disadvantage of this method is that
instrument availability is limited owing to cost
(Neufeld et al. 2007a), thus restricting its widespread
use. The method also experiences all the limitations
of in situ hybridisation discussed above. High background levels may be found if the environment where
the sample comes from contains a high concentration
of the halogen used in the taxonomic probe. Additionally, sample fixation and probe hybridisation
could modify the cell isotopic composition by
introducing exogenous C and N.
The use of NanoSIMS in microbial ecology was first
described by Lechene et al. in 2006 (Lechene et al.
2006). The authors used pure cultures of the N2-fixing
marine bacterium Teredinibacter turnerae and Escherichia feacalis grown in a 15N atmosphere. Mass images
of 12C,13C 14N and 15N were obtained in parallel after
120 h of incubation. They demonstrated that T. turnerae fixed nitrogen and incorporated it into its biomolecules, while E. feacalis did not. Furthermore, they
demonstrated that there were large differences in
15 14
N/ N ratios within cells of the same population,
which indicated a high variability in uptake rates.
The following year, the same authors (Lechene
et al. 2007), published direct measurements of N2
123
fixation by individual T. turnerae bacteria inside the

gills of its host shipworm Lyrodus pedicellatus. They
demonstrated, for the first time, that the fixed
nitrogen was supplied directly to the host, which
normally survives on a N-poor diet, as its basic
source of food is wood. Although these were
revolutionary studies, they did not analyse complex
microbial systems composed of uncultured organisms
and did not use taxonomic probes to identify the
microbes involved.
It was not until SIMSISH was developed by Li et al.
(2008) that complex microbial mixtures and probes
were studied using NanoSIMS. The authors designed
16S rRNA probes containing iodised cytidine (IEUB338) that were targeted to an artificially mixed
community of Escherichia coli fed with 13C-glucose
and Bacillus subtilis fed with unlabelled glucose. They
observed that the hybridisation of the iodised probe
co-localised with the 13C isotope signal with E. coli
cells only. The isotopic composition of E. coli cells
cultured on different 13C or 15N substrate mixtures
obtained with NanoSIMS was in agreement with data
from isotope ratio mass spectrometry data. Furthermore, these authors (Li et al. 2008) analysed uncultured archaeal and bacterial communities from an
anaerobic municipal solid waste bioreactor leachate
consuming 13C-methanol. They observed that some Iprobe targeted cells had an enriched carbon composition, but that some cells with an enriched carbon
composition displayed no detectable iodine signal,
suggesting that bacteria not targeted by probe EUB338
were involved in methanol metabolism. When the
archaeal probe ARC915 was used, the iodine signal
was also co-localised with clusters of cells that showed
carbon enrichment. Thus, this study demonstrated that
in a single scan it was possible to observe isotope
assimilation by 16S rRNA probe-directed microorganisms from complex environments.
Insights into the metabolic behaviour of cyanobacterium Trichodesmium were provided by FinziHart et al. (2009). These authors combined transmission electron microscopy (TEM) with NanoSIMS
analysis and found that this non-heterocyst forming
cyanobacterium segregates CO2 fixation from N2
fixation by the formation of transient sub-cellular
granules (cyanophycin granules) which are nitrogen
hot-spots. Maximum photosynthesis rates occurred
during the morning, whereas N2-fixation rates peaked
in the afternoon. These results provided evidence of
the temporal-spatial differentiation of metabolic

pathways in single bacterial species.
3.5.2 Element labelling-FISH (EL-FISH)

This method applies the same principle as CARD
FISH (enzyme mediated tyramide deposition), but it
differs from the latter in that the amplification
substrate (tyramide) has a halogen-containing fluorescent dye. The reason for this composite label is to
allow signal identification with both a FISH protocol
as well as a SIMSISH protocol. After hybridisation,
the probed cells show increased fluorescence resulting from the tyramide amplification signal and an
elevated deposition of the halogen element carried by
the tyramide molecules, hence the name elementlabelling FISH. This way, the halogen acts as the
phylogenetic marker when a NanoSIMS instrument is
used, while it is compared simultaneously with the
isotopic enrichment in C or N in the cell that results
from the catabolic incorporation of a labelled
substrate. This method allows the simultaneous
identification of cells in mixed communities by both
NanoSIMS and fluorescence microscopy.
The main advantage of this method over SIMSISH
is that it increases the sensitivity of cell detection by
amplifying the signal of low abundance rRNA cells.
This protocol increases the signal-to-noise ratio to
10100 fold above FISH probes (Behrens et al.
2008), enabling phylogenetic identification and cell
detection independent of metabolic state. It also
extends the range of elements used for cell labelling
in comparison with the SIMSISH protocol described
by Li et al. (2008) since those authors limited the
method protocol to an iodine label.
This method shares the same disadvantages as
SIMSISH, but requires a fluorescence microscope in
addition to the NanoSIMS instrument, which further
increases experimental costs. In addition, it shares the
disadvantages described above for FISH. Also, oligonucleotide probes covalently modified with halogen
atoms may have an altered melting behaviour which
may result in lower detection sensitivity. Although
this method overcomes some of the disadvantages of
MAR-CARD-FISH, the incorporation of large probes
into cells and the construction of elaborate composite
probes can potentially be problematic.
175
This method was recently described by Behrens

et al. (2008). They tested the methodological principle and the possible metabolite transfer between an
epibiont bacteria and its partner. A two species
consortium of the filamentous cyanobacteria Anabaena and Rhizobium sp. was incubated with
NaH13CO3 and 15N. Rhizobium did not show any
13
C or 15N enrichment when grown in pure culture,
but only when it was grown in co-culture with
Anabaena. They also observed that cells not attached
to a heterocyst did not show 13C or 15N enrichments,
but were detected by NanoSIMS since they had
increased fluorine abundance. This suggested that
both C and N fixed by Anabaena were transferred to
Rhizobium, the epibiont, when they were in close
proximity. It also demonstrated that EL-FISH can
identify cells independent of their metabolic state.
When analysing a multispecies oral biofilm
(Behrens et al. 2008), the probe-targeted Cytophaga-Flavobacterium group was identified by elevated intracellular fluorine levels. Contrary to
expectations, not all the cells belonging to this
group metabolised the 13C-labelled amino acids,
whereas cells that were not a target of the probe did
incorporate the label. Using quantitative mass imaging, the authors calculated the relative label incorporation of individual cells in the samples. The
observed heterogeneity of label incorporation indicated differential metabolic capacity of individual
cells within the population.
In a similar study, Musat et al. (2008) investigated
ammonium and organic carbon assimilation in three
anaerobic phototrophic bacterial species from an
alpine lake. The authors observed metabolic variation
between cells of the same species, concluding that
one species contained physiologically different individuals with enhanced rates of C and N assimilation.
They also showed that these individuals were the
least abundant species. Consequently, organisms that
may be low in cell numbers can have increased
metabolic activity, potentially playing an important
role in ecosystem dynamics.
SIMSISH and EL-FISH offer an unprecedented
capacity to establish the link between identity and
function in complex microbial communities. Importantly, NanoSIMS-based approaches allow investigations on the physiological role of individual cells
within populations as well. This technique has a very
123
176
promising future provided it becomes widely accessible to researchers.

3.6 Isotope arrays
This method allows a simultaneous investigation of
community composition and specific substrate consumption by its members via direct detection of 16S
rRNA on a DNA probe microarray (Adamczyk et al.
2003). Community RNA extracted from an environmental sample previously incubated with radioactively labelled substrate is linked with a fluorescent
dye, then fragmented and hybridised with the rRNA
targeted phylogenetic probes fixed in the microarray.
Fluorescence and radioactivity signals are measured
and quantified with a fluorescence scanner and betaimager, respectively, to identify community composition (by fluorescence) and substrate consumers (via
the radioactive signal).
An isotope array allows the application of many
taxonomic probes in parallel, providing a higher
throughput than any one of the other probe-dependent methods. It has the capacity to simultaneously
link a large number of microbes from different taxa
in a sample with a specific activity (substrate
uptake), and it can detect low abundance microbes.
Importantly, in contrast to SIP-based methods, it
allows a PCR-independent approach to assigning
taxonomic identity to active microbes which,
hybridisation biases aside, may allow a quantitative
estimation of the active community members.
Additionally, it is less tedious than methods such
as FISH-MAR.
A drawback of this method is the fact that it only
identifies organisms for which probes are readily
available, limiting its discovery capacity. Also,
studies on the incorporation of a labelled substrate
depend on the availability of suitable microarrays. As
this method relies on the use of probes, one may
encounter target accessibility issues and hybridisation
problems. Since hybridisation capacity varies
between probes, an increased stringency may cause
false negatives for some sequences, or lower stringency may result in false positives. This method also
requires efficient signal-to-noise ratio adjustments.
The limit of detection of a beta-imager used to
analyse the radioactive signal is around 125 lm,
which limits the size of the probe spot that can be
applied on an array, consequently limiting the
123
number of probes that can be loaded on one chip.

This method relies on an efficient extraction of RNA
and does not allow genetic or genomic investigations.
The isotope array was developed and described by
Adamczyk et al. in 2003 (Adamczyk et al. 2003). The
initial application was carried out on the community
composition and CO2 fixation of ammonia oxidising
bacteria (AOB) in nitrifying activated sludge. The
community structure of the active AOB obtained with
the microarray was compared and confirmed with
FISH, and the 14C incorporation into AOB was
confirmed by FISH-MAR. This first report utilised
only a small number of manually spotted probes in
the microarray.
In further trials, Hesselsoe et al. (2009) tested a
multi probe chip targeting the Rhodocyclales group
(RHC-PhyloChip) in activated sludge. The aim of
this work was to investigate Rhodocyclales metabolic
behaviour in activated sludge under oxic and anoxic
denitrifying conditions and supplied with different
carbon sources (propionate, butyrate and toluene).
This study combined the previously described strategy of 14CO2-assimilation based labelling of RNA
(Hesselsoe et al. 2005). This isotope array study
revealed that the majority of the members of Rhodocyclales displayed some involvement with nitrogen
metabolism. The 14CO2 incorporation activity varied
with the different carbon sources, showing some
specialisation in its use. The authors concluded that
within Rhodocyclales there exists a functional niche
overlap in activated sludge that contributes to the
metabolic versatility of this group.
3.7 Small subunit-isotope ratio mass

spectrometry (SSU-IRMS)
In this method, the RNA extracted from a microbial
community after a pulse is captured by biotinlabelled probes. These are then retrieved using
streptavidin coated magnetic beads, which bind to
the biotin probe. The isotopic content of RNA is
determined by IRMS and the identity of the active
microbes is determined by the specific probes used to
retrieve the SSU rRNA.
This method has a high taxonomic resolution,
similar to RNA-SIP. However, unlike this one the
incorporation of a label into rRNA at low levels can
be detected by IRMS. Therefore, labelled substrates
can be added at realistic concentrations, limiting

cross feeding effects. It is a method that is free of
PCR biases since it analyses RNA molecules directly.
Consequently, it avoids PCR artifacts and provides
realistic estimations of RNA abundance. It is less
time consuming than the majority of other methods
and can be used in conjunction with natural abundance studies.
In order to determine isotope content, it is
necessary to obtain large amounts of RNA (10
100 lg) from a sample, which is not possible from
environments with low cell numbers or activity. In
comparison to other methods that use probes to
acquire taxonomic information, it is not possible to
obtain cell specific information, and it does not allow
a spatial determination of bacterial cell arrangements.
Because it uses probes to retrieve specific taxonomic
groups from a mixture, it may still suffer from some
hybridisation biases, such as target accessibility and/
or target specificity. Thus, its taxonomic resolution
relies on the efficiency of existing probes.
The SSU-IRMS method was first described by
MacGregor et al. (2002).They demonstrated that the
small subunit of rRNA of E. coli grown in culture
could be isolated from total RNA by magnetic bead
capture. The natural abundance isotopic content of
these molecules was measured by IRMS and was
shown to reflect that of the growth substrate in the
media. In subsequent work (MacGregor and Amann
2006), the authors isolated the SSU rRNA by bead
capture from total RNA extracted from estuarine
sediments and microbial mats. RNA was separated by
SSCP in high strength polyacrylamide gels to obtain
an RNA profile. Selected bands of interest (those
bands that disappeared from the profile after RNase
H-probe targeted cleavage) were excised and
sequenced for taxonomic identification. In further
studies, coastal sediments were labelled with several
13
C-substrates and the incorporation of the label into
bacterial and eukaryotic rRNA and PLFA was
analysed (MacGregor et al. 2006). The authors
compared the isotopic content of the lipid biomarkers
with the rRNA biomarkers of both eukaryotic and
bacterial populations. The bacterial members of the
community had much greater 13C content than
eukaryotic cells, which implied that bacteria were
the main consumers of the 13C-labelled compounds.
The authors, however, did not attempt to investigate
further the identity of those bacteria.
177
A recent study by Miyatake et al. (2009) further

improved the method by employing a micro-elemental analyser-IRMS that enhanced the sensitivity of the
analysis tenfold and substantially reduced the background level contamination of the blank. This
approach included the use of a nested series of
probes from Deltaproteobacteria to Desulfoproteobacteriacea to increase the taxonomic depth of the
technique. The rationale was that an increase in
labelling with decreasing target range would implicate the specific group in carbon assimilation from
the substrate. By testing the independent consumption of 13C-glucose, 13C-propionate and 13C-acetate
in anoxic marine sediments, they observed decreasing
levels of labelling with decreasing levels of target
range with regards to glucose. When propionate and
acetate were used as carbon sources, increased
labelling with decreasing target was obtained. This
implied that, in this environment, members of the
Desulfobacteriacea family were major consumers of
propionate and acetate, to a lesser degree, which are
important carbon sources of sulphate-reducing bacteria in marine sediments.
Recently, Pearson et al. (2008) employed the
magnetic bead capture technology to retrieve SSU
rRNA from sediments from an oil spill site in a coastal
environment. The authors were able to measure
isotopic carbon content from the isolated RNA by
the recently described Spooling-Wire Micro-combustion (SWiM) Isotope Ratio Mass Spectrometry.
By comparing the d13C of the RNA with the highly
depleted d13C of petroleum in the oil spill, the authors
demonstrated bacterial involvement in oil degradation. Phylogenetic assignation of the putative degraders was achieved by PCR amplification of the 16S
rRNA genes and not directly from the highly depleted
RNA. Additionally, this method was not able to
determine a taxa-specific isotopic depletion/enrichment. They were only able to quantify the microbial
assimilation of petroleum hydrocarbons based on the
13
C depletion from the total community RNA. Taxaspecific quantitative estimation of the degradation of
petroleum hydrocarbons still remains to be elucidated.
A modification of the SSU-capture method was
recently described by Van Mooy and Devol (2008).
The authors were interested in defining the nutrient
limitations of Prochlorococcus bacteria in waters of
the North Pacific subtropical gyre. Radiolabelled
phosphate uptake was used as an indicator of RNA
123
178
synthesis and cellular activity in the presence and

absence of a nitrogen source. Labelling of heterotrophic phosphate consuming bacteria was achieved, but
Prochlorococcus spp. were specifically separated
from the total community RNA by magnetic bead
capture, as described previously by MacGregor et al.
(2002). Radioactivity of RNA rather than its stable
isotope content was determined to selectively quantify the activity of these members of the microbial
community. It was found that nitrogen rather than
phosphate was limiting the activity of these populations. This method was termed Ribotrace or
radioisotope-based tracking of RNA synthesis by
hybridisation and capture.
4 Concluding remarks
This overview of the existing methods designed to
link the identity of microorganisms with processes in
the environment shows that a revolution in microbial
ecology has taken place in the past decade, with a
large number of sophisticated methods being developed in a very short time. As a consequence,
knowledge of microbial activity in complex systems
such as soils, sediments, sludge, landfill, the rhizosphere, the animal gut, contaminated sites and
freshwater and marine systems has increased substantially. Microbial ecologists have an extensive
toolbox from which to select an appropriate method.
Inherent to this diversity of techniques, there is
variation on the capacity and availability of each
method and as a consequence uptake has varied
(Fig. 1). Table 6 provides a summary of characteristics for each technique reviewed here to aid in the
assessment of the methods. Ultimately, the selection
of the most appropriate technique will depend on the
type of question being asked, the ease of use of the
method and accessibility of instrumentation and
isotopic substrates required.
Development of this suite of methods has shown
that the relationship between environmental processes and microbial phylotypes is more complex
than initially anticipated. Stable isotope probing (SIP)
studies have sometimes implicated unexpected bacteria in specific environmental processes, and have
helped in determining specialist versus generalist
microbial guilds involved in the consumption of
123
substrates. SIP studies have also revealed the influence of human activity on the activity of functional
clades of microbes (Singh et al. 2007, 2009; Maxfield
et al. 2008; Lear et al. 2009). Probe-based studies
have been revolutionary in that functional information is now being accessed at the single cell level.
NanoSIMS and FISH-Raman based studies have
uncovered that metabolic differentiation within
clonal lineages appears to be common in the microbial world.
The methods reviewed here have enabled identification of key microbes in several important environmental processes. The number of examples of
defined relationships between ecosystem functions
and microbial phylogeny is set to increase rapidly in
the following years. Certainly, there is still room for
improvement in this area of method development.
However, a great challenge now lies in integrating
this new knowledge into conceptual frameworks that
enable us to predict the consequences of manipulating environments in specific ways for ecosystem
function. Integrating the knowledge acquired through
the methods reviewed here should guide us in
resolving issues such as how to manipulate activated
sludge, contaminated soil or decentralised composting systems to improve ecological services and
minimise the negative impacts of human activity.
Other issues to resolve include how to increase the
activity of phosphate accumulating bacteria, reduce
bulking by filamentous bacteria, achieve more efficient denitrification and fatty acid and protein
removal in wastewater treatment plants. Likewise,
important questions to explore are for example: can
we enhance in situ biodegradation of pollutants by
facilitating or inhibiting specific microbial taxa? Can
we favour specific methanogens over sulphate or
nitrate reducing clades in anaerobic digestors? The
vast amount of information gained from applying
these sophisticated methods will most certainly result
in addressing these issues and questions in an
effective way.
The development of methods to link function with
phylogeny in the microbial world has greatly
improved our understanding of microbial ecology.
The near future will witness the further application and
improvement of the newly developed methods. These
promise to unveil functional aspects of microbial
ecosystems that have been complicated to obtain in the
past and perhaps unpredicted. From this perspective,
Species
Species
Species
Species
Species
Species
Family
FISH-MAR
FISH-SIMS
SIMSISH
EL-FISH
FISH-Raman
Isotope arrays
SSU-IRMS
No
No
Yes
Yes
Yes
Yes
Yes
No
No
No
No
No
Hours
Hours
Hours
Hours
Hours to (25) days
Hours to days
(45) Minutes to
(48) hours
Hours
Hours
Hours to (14) days
Hours to (40) days
Hours to (30) days
Spatial
Range of
resolution incubation times
ND
No
170 lM2 mM
No
Yes
4300 lM
50200 mM
Yes
500 lM500 mM
Yes
Yes
500 lm3 mM
15 mm150 mM
No
ND
1 nM6.6 mM (most in
the lM range)
66 mM
0.5 mM2 mM
No
No
No
300 lM40 mM
C-acetate
CH4
13
Differences in
metabolic rates
10 lM2 mM
10100 lM
13
1010,000 ppm
Range of incubation
concentrations
No
No
No
No
No
No
No
No
ND
Yes
ND
No
IRMS
b-imager
Fluorescence imager
Raman equipped
fluoresecence
microscope
NanoSIMS ? Fluorescence
microscope
NanoSIMS
SIMS
Fluorescence microscope
Phosphorimager
MALDI/MS
No
No
No
Applicable to Need of costly

dissimilatory specialised
processes*
equipment
Stable
Radioactive
Stable
Stable or radioactive
Radioactive
Radioactive
Stable
Stable
Stable
Stable
Type of
isotope
* In this context, dissimilatory processes can be studied through identification of relevant genes or enzymes or through detection of active taxa in synchrony with appearance of
dissimilation products, as demonstrated by Kittelmann and Friedrich (2008a,b). ND not demonstrated; MALDI/MS matrix-assisted laser desorption ionisation/mass spectrometer;
SIMS secondary ion mass spectrometer; IRMS Isotope ratio mass spectrometer
Species
Species
RNA-SIP
Species
Species
DNA-SIP
RIP
Class
PLFA-SIP
Protein-SIP
Phylogentic
resolution
Method
Table 6 Summary of characteristics for each technique relevant to applicability

179
123
180
the future vision of environmental biotechnology is

increasingly vivid.
Acknowledgments Maria-Luisa
Gutierrez-Zamora
is
supported by a University International Postgraduate Award
from the University of New South Wales. Mike Manefield is
supported by the Environmental Biotechnology Cooperative
Research Centre.
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Rev Environ Sci Biotechnol (2010) 9:153185

An appraisal of methods for linking environmental

Published online: 14 May 2010

M.-L. Gutierrez-Zamora M. Manefield (&)

Rev Environ Sci Biotechnol (2010) 9:153185

Table 1 List of methods available in functional molecular microbial ecology

Phospholipid-derived fatty acid-stable isotope probing

Fluorescence in situ hybridisation-microautoradiography (FISH-MAR)

DNA-stable isotope probing (DNA-SIP)

Fluorescence in situ hybridisation-secondary ion mass spectrometry

RNA-stable isotope probing (RNA-SIP)

FISH-Raman spectroscopy (FISH-Raman)

Protein-stable isotope probing (Protein-STP)

Secondary-ion mass spectrometry-in situ hybridisation (SIMSISH)

Radioactive isotope probing (RIP)

Element labelling-FISH (EL-FISH)

the enormous advantage of allowing researchers to

identity questions: Which microbes are performing

Fig. 1 Number of publications between 2007 and 2010 for all

Rev Environ Sci Biotechnol (2010) 9:153185

Comparison with profiles

Comparison with PMF

these to provide a comprehensive view of the

2 Isotope probing methods

Direct band or clone

2.1 Phospholipid-derived fatty acid-stable

A number of methods are compared here in terms of their

This method, however, has a low phylogenetic

Rev Environ Sci Biotechnol (2010) 9:153185

methanogenic communities of peatland soils to assess

2.2 DNA-stable isotope probing (DNA-SIP)

Rev Environ Sci Biotechnol (2010) 9:153185

Table 2 PLFA-SIP examples from 2007 to 2010

Distribution of active microbes

Rice rhizosphere soil

Gram negative bacteria were most actively incorporating

Distribution of active microbes

Gram negative bacteria were most actively incorporating

Denef et al. Distribution of active microbes

Fungi readily assimilated plant-derived carbon before

Deines et al. Trophic interactions

Methane oxidising bacteria were used as a carbon and

Marine cold seep

Archaea associated sulphate reducing bacteria displayed

Type I methanotrophs were of particular importance in the

In situ rice plants

Type I methanotrophs had a predominant role in the active

Type II methanotrophs were the predominant

Forest and farmland

Changes in land use practices, from forest to farmland,

Different Siberian tree species did not affect community

There was a shift from Type I to Type II methanotrophs

A decrease of more than 70% of methanotrophs was

Different plant covers induced changes in methanotrophs.

Rev Environ Sci Biotechnol (2010) 9:153185

Degradation of xenobiotics: 2,4- Agricultural soil

A succession of microbial communities degraded 2,4Dichlorophenoxiacetic acid, with proteobacteria being

Ratios of bacterial-to-total PLFA-carbon decreased and

community by multiple displacement amplification

buoyant density as DNA from non-consumers with

Rev Environ Sci Biotechnol (2010) 9:153185

Table 3 DNA-SIP examples from 2008 to 2010

Deep water coral