mar-fish

REVIEWS

An appraisal of methods for linking environmentalprocesses 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 revolu-

tion in the development of methods and technology

available to investigate the ecological roles of

microorganisms in the environment. As a conse-

quence, microbial ecologists have gained a better

understanding of the functional aspects of microor-

ganisms 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 meth-

ods linking microbial function with phylogeny,

including a description and advantages and limita-

tions of each method. Examples are also provided to

illustrate their application. The ongoing improve-

ments 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

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 con-

fronted 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 func-

tions 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

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: [email protected]

123

Rev Environ Sci Biotechnol (2010) 9:153185

DOI 10.1007/s11157-010-9205-8

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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 sub-

strates and their high cost, particularly of radioactive

substrates. In addition, ex situ incubation of commu-

nity samples with isotopically labelled substrates

(stable or radioactive) does not necessarily reflect in

situ conditions. Care must be taken to achieve condi-

tions 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 func-

tion 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 impli-

cating 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-

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

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)

Fig. 1 Number of publications between 2007 and 2010 for allfunction-identity methods reviewed

154 Rev Environ Sci Biotechnol (2010) 9:153185

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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 com-

monly 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.

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 spec-

trometry (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 group-

specific 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, incuba-

tions 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 mole-

cules in the cell and label incorporation is indepen-

dent of cell replication. In PLFA-SIP, there is no need

to purify unlabelled from labelled biomarkers, unlike

DNA and RNA-SIP (see below).

Radioactive isotope probing

13C-lipids

13C-DNA

13C-RNA

13C-Proteins

14C-RNA

Biomolecule Basic method Assignation of

taxonomic identity Name of method

GC-c-IRMS profile

Density gradient centrifugation

Density gradient centrifugation

MALDI-MS, MS/MS

14C-RNA:12C-DNA hybridisation on

profiles or library clones

Comparison with profiles of known species

DGGE, T-RFLP, clone libraries

DGGE, T-RFLP, clone libraries

Comparison with PMF profiles in databases

Direct band or clone sequencing

PLFA-SIP

DNA-SIP

RNA-SIP

Protein-SIP

Pulse Extraction of biomolecule

Fig. 2 Schematicrepresenting the basic steps

in the different isotope

probing techniques

reviewed. (See text for

definition of abbreviations)

1 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.

Rev Environ Sci Biotechnol (2010) 9:153185 155

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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 micro-

organisms 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 pre-

sented in Table 2. Overall, these studies provide a

picture of microbial biogeochemical processes and

microbial trophic interactions in different environ-

ments. These examples indicate that changes in land

use practices, rising temperatures and the widespread

use of inorganic fertilisers can affect microbe-medi-

ated methane cycling processes, with potential nega-

tive implications in the global warming phenomenon.

PLFA-SIP has been combined with DNA/RNA-

SIP 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 concen-

trations of 13C-labelled glucose and acetate. The

authors found that when using glucose the identity of

the glucose consumers was unclear when both PLFA-

and 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 PLFA-

SIP. Chen et al. (2008a) compared unlabelled mRNA

(gene transcripts) with PLFA-SIP profiles of

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 polymor-

phism (T-RFLP) and sequence analysis to assess

active methanotroph populations with enhanced tax-

onomic 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 con-

tinue 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 chlo-

ride (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. Alterna-

tively, 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 (Cup-

ples 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


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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)


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.

(2007)


in the environment

Grassland soil Fungi readily assimilated plant-derived carbon before

bacterial communities did

Deines et al.

(2007)

Trophic interactions 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

soil versus pasture

land soil

Type II methanotrophs were more active in forest and

shrub land soils and Type I methanotrophs were more

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 Beijerinckiaceaespecies to Methylococcaceae and Methylocystaceaespecies. 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/Methlocapsaspp. and Sphagnum/Eriophorum-covered land favoured aputative novel methanogen


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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 confi-

dence. This method has the potential to investigate a

wide range of uncultured species and, unlike PLFA-

SIP 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 concen-

trations 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 concen-

tration 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

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 (Radajew-

ski 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 met-

hanotrophy, methylotrophy, denitrification and nitro-

gen fixation, as well as carbon flow in the ecosystems

(trophic interactions), and to the study of biodegra-

dation 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 methodolog-

ical 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-DNA-

SIP 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 centrifuga-

tion to improve the separation of 15N-labelled DNA

from high G ? C unlabelled DNA. This strategy

opened up the possibility of deciphering the identity

Table 2 continued


Lerch et al.

(2009)

Degradation of xenobiotics: 2,4-

Dichlorophenoxiacetic acid

Agricultural soil A succession of microbial communities degraded 2,4-

Dichlorophenoxiacetic 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

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

fungal-to-bacterial PLFA-carbon increased under

elevated CO2 compared with ambient conditions


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Table 3 DNA-SIP examples from 2008 to 2010


Jensen et al.

(2008)


Methanotrophy

Deep water coral

reef sediments

Methylomicrobium and Gammaproteobacteria were activemicrobes involved in methane oxidation and members of

Gammaproteobacteria, Alphaproteobacteria, Deferribacterand Bacteroidetes were putative cross-feeders

Neufeld et al.

(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. andGammaproteobacteria (methanol, methylamines,dimethylsulfide) and Alphaproteobacteria and theCytophaga-Flexibacter-Bacteroidetes guild(methylbromide)

Neufeld et al.

(2008b)


Methylotrophy

Surface seawater Methylophaga-like phylotypes were identified as the mostactive 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 IImethanotrophs 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

Type I, Type II methanotrophs, and methylotrophs

(Methylopila spp. and Hyphomicrobium spp.) were the mostactive methanotrophs

Hery et al.

(2008)


methanotrophy

Landfill cover soil 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 classwere 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 ofHyphomicrobium spp.

Saito et al.

(2008)


denitrification

Rice paddy soils Bukholderia spp. and Rhodocyclales spp. dominated (readilyavailable) succinate consumption under denitrifying

conditions in water logged soils

Buckley et al.

(2008)


Nitrogen fixation in

methanotrophs

Grassland soil Using 15N2 it was found that Methylocystis-like species fixedN2 in the soil in response to methane addition. Thus, N2fixation by methanotrophs in the soil was demonstrated for

the first time

Jia and

Conrad

(2009)


Autotrophic ammonia

oxidation

Agricultural soils Despite a higher abundance of archaeal ammonia-oxidizing

gene amoA, members of the domain Bacteria activelyconsumed 13CO2, showing a predominant role in

autotrophic ammonia oxidation in these soils

Wawrik et al.

(2009)


N cycle

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

Synechococcus and diatoms have a high plasticity innitrogen assimilation processes

Chen et al.

(2009)


Sulphur oxidation and

denitrification

Cave freshwater

(plus biofilm)

The most active microbes assimilating 13CO2 were

Thiobacillus spp., Nitrospira spp. and CandidatusNitrotoga spp. suggesting an important role of sulphur and

ammonia/nitrite oxidisers in this environment


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Table 3 continued


Webster et al.

(2010)

Distribution of active

microbes in the environment

Estuarine sediments Uncultivated microorganisms play important metabolic roles

in different zones in tidal sediments. Gammaproteobacteriaand 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 sulphatereducing conditions was dominated by Crenarchaeota

Pumphrey

and Madsen

(2008)

Trophic interactions: Plant

microbe

Agricultural soil Burkholderia spp. dominated the carbon acquisition frombenzoic acid, present in plant root exudates and a product of

plant decomposition in soil

Haichar et al.

(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 andspecific to monocots were Sphingomonadales

Bressan et al.

(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 carbonfrom exudates of Merkur cultivar and Acidovorax spp. fromDesiree cultivar

Qiu et al.

(2009)


microbe

Rice rhizosphere Sphingomonadales and Methylocystacea were more active incarbon 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 Acetovibriospp., of 13C-glucose Clostridium spp. andPorphyromonadaceae members, and of 13C-acetate thearchaeal Methanoculleus. All of these were implicated inthe methanisation of cellulose

Gihring et al.

(2009)


flow

Marine sediments Degradation of detrital organic matter derived from 13C-

enriched Spirullina cells implicates alphaproteobacterialdenitrifiers 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. Gammaproteobacteriawere more active in high intensity light (no vegetation

cover), Rhodococcus and Enterobacter were more activeduring ambient light, and Betaproteobacteria were mostactive during darkness

Jones et al.

(2008)

Degradation of xenobiotics:

Pyrene

Industrial site soil Caulobacter spp. and members of the uncultured Pyrenegroup 2 from Gammaproteobacteria were the dominantpyrene degraders in polyaromatic hydrocarbon-

contaminated 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 twoequivalent field and laboratory conditions

Oka et al.

(2008)


Benzene

Sulfidogenic

enrichment

cultures

A Desulfobacteraceae species was the first one to assimilatecarbon 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 dominantfungal degrader of phenol in soil. Isolation efforts were

successful and demonstrated that this fungus degraded

phenol in situ


123

of microbes involved in the nitrogen cycle (Buckley

et al. 2008). Another interesting approach to DNA-

SIP has been the use of isotopic oxygen (18O)

incorporated as H218O into the system. This alternative

allowed Schwartz (2007) to obtain a substrate inde-

pendent analysis of microbial communities. The

author suggested that by using H218O 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 comprehen-

sive and meaningful result. Another significant

improvement to DNA-SIP was achieved by Neufeld

et al. (2008b) by using multiple displacement ampli-

fication 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 possi-

bility 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 trifluoro-

acetate (CsTFA)/formamide solution by equilibrium

density gradient centrifugation followed by gradient

fractionation and precipitation of RNA with isopropa-

nol. 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).

Table 3 continued


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 Achromobacterand Pseudomonas, and functional genes were recoveredfrom a cosmid library constructed from labelled DNA

Uhlik et al.

(2009)


Biphenyl

Agricultural soil and

horseradish

rhizosphere

Paenibacillus spp. dominated assimilation of carbon frombiphenyl in pentachlorobenzene contaminated bulk soil,

whereas Hydrogenophaga spp. dominated in horseradishrhizosphere, indicating a plant-associated effect in

community structure and biodegradation capacity. Bipheyl

dioxigenase genes were similar to those of Pseudomonasalcaligenes B-357

Nicholson

et al. (2009)


flow and spore-forming cells

Pure laboratory

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

Methylomicrobium and Gammaproteobacteria were activemicrobes involved in methane oxidation and members of

Gammaproteobacteria, Alphaproteobacteria, Deferribacterand Bacteroidetes were putative cross-feeders

Tourna et al.

(2010)


Nitrification

Cultures A comparison between DNA-denaturing gradient gel

electrophoresis (DGGE), RNA-DGGE and stable-isotope-

probing (SIP)-DGGE profiling of Nitrospira culturesdemonstrated that SIP is more sensitive to changes in

activity of organisms with relatively low yields and activity


123

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 confirm13C 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 con-

sumption 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 compar-

ison 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 DNA-

SIP 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.

(2005) determined that the lower limit of detection of

RNA-SIP is 105106 cells/g soil. Additionally, RNA-

SIP 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 biodeg-

radation and rhizosphere interactions. Table 4 summa-

rises 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 hydro-

carbon-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 incorpora-

tion 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 sed-

iment 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 13C-

DNA 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 perchloroeth-

ene (PCE) in river sediments, Kittelmann and Fried-

rich (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


123

Table 4 RNA-SIP examples from 2008 to 2010


Brinkmann et al.

(2008)

Trophic interactions:

Animal-microbe

Gut of Manducasexta larvae

Enterococcus spp. were most dominant in the gut after feeding thelarvae 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

First study to demonstrate that diatom-associated bacteria actively

consume carbon products derived from their algal host

Kovatcheva-

Datchary et al.

(2009)


Human-microbe

Human colon in

vitro model

Ruminococcus bromii was found to be the primary starch degrader,with Bifidobacterium adolescentis, Prevotella spp., andEubacterium rectale involved further down the carbonassimilation chain

Frias-Lopez et al.

(2009)


Food webs

Seawater surface Labelled cyanobacteria Prochlorococcus and Synechococcus wereconsumed by protozoa from the Haptophyta, Stramenopiles andAlveolata groups. The method was successfully applied toidentify cyanobacterial predators

Glaubitz et al.

(2009)


Food webs

Marine pelagic

water

Marine dark CO2 fixation in pelagic environments was attributed

to Gammaproteobacteria and the Sulfiromonas cluster ofEpsilonproteobacteria. Euplotes spp. ciliates were potentialgrazers of these autotrophic bacteria

Hamberger et al.

(2008)

Biogeochemical

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, Chloroflexiand Planctomycetes dominated carbon assimilation fromcellulose, and Intrasporangiaceae and Micrococcaceae fromcellobiose and glucose. Under anaerobic conditions, members of

Kineosporiaceae, cluster II Clostridiaceae and Bacteroidetesdominated cellulose carbon assimilation and cluster I

Clostridiaceae from cellobiose and glucose

Hatamoto et al.

(2008)


Carbon flow

Methanogenic

sludge

Members of Syntrophaceae, Tepidanaerobacter spp., andClostridium spp. were the most active degraders of butyrate insludge under methanogenic conditions

Moreno et al.

(2010)


Protozoa-bacteria

Activated sludge The ciliate Epistylis galea was the dominant grazer from bacteriaassimilating CO2 under ammonia oxidising conditions. No

grazing on acetate consuming bacteria was detected

Langenheder and

Prosser (2008)

Ecosystem

functioning:

Resource availability

Soil 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

Monard et al.

(2008)

Ecosystem

functioning:


Soil and earthworm

casts

Differences in active degraders of glucose and acetate were

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

functioning:


Rice field soil 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

Degelmann et al.

(2009)

Ecosystem

functioning:


Forest soil Facultative aerobes Rahnella and Ewingella spp. dominated andoutcompeted anaerobes in the rapid fermentation of glucose in

forests soils in microcosms that simulated anoxic

microenvironments after rainfall


123

the community profiles generated from heavy frac-

tions of acetate-amended PCE cultures versus acetate

only cultures, unique sequences were found for PCE-

dechlorinating 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 dechlo-

rinating bacteria were successfully identified with

RNA-SIP. Collectively, these studies have demon-

strated that by using the correct controls and exper-

imental 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

a 15N pulse. Addison et al. (2010) observed that 15N-

labelled 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 centrifuga-

tion time did not solve the problem. The authors

highlighted the difficulty in separation of 15N-

labelled RNA in CsTFA and argued that co-

mingling 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 15N-

DNA-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

Table 4 continued


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 intransforming tetrachloroethene to cis-dichloroethene indehalorespiringmicrocosms, whereas Dehalococcoides spp.were the predominant in ethene-producing microcosms.

Geobacteraceae, Desulfobacteraceae and Desulbobulbaceaewere 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 grouphere 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 theacquisition of carbon from benzene under aerobic conditions

Huang et al.

(2009)

Xenobiotic

degradation:

Naphthalene

Groundwater Acidovorax spp. were the key organisms in naphthalenedegradation in situ displaying high substrate affinity, while

Pseudomonas putida and P. fluorescens were low affinitynaphthalene degraders

Sueoka et al.

(2009)

Xenobiotic

degradation: Phenol

Sludge (denitrifying

conditions)

Azoarcus spp. was the primary degrader of phenol. Microbulbifer,Pelagiobacter, Pseudomonas, and Thauera spp. also assimilatedcarbon from phenol either as primary consumers, intermediate

consumers or cross-feeders


123

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 electro-

phoresis and selected proteins are digested with

trypsin, purified and analysed by matrix-assisted laser

desorption ionisation/mass spectrometry (MALDI-

MS) 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 non-

labelled 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 non-

labelled 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 enrich-

ment of the proteins, which potentially could mean

obtaining differences in levels of isotope (substrate)

incorporation per species in a complex system. This

method can provide direct information on the meta-

bolic pathways being used in the assimilation of a

carbon or nitrogen source.

Despite the increased sensitivity and good taxo-

nomic 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, overlap-

ping 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 aromat-

icum 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 calcu-

lated 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 enrich-

ment 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 13C-

benzene or 15N-ammonium (and 12C-benezene) as

sources of carbon and nitrogen. Two dimensional gel

electrophoresis and MALDI-MS analysis and


123

quantification of ten proteins allowed them to deter-

mine 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 demon-

strated the applicability of this method when using13C 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 and15N-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 avera-

gine model which assumes an average molecular

composition and mass of amino acids to calculate the

number of C and N atoms per peptide. After MALDI-

MS, 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 bacte-

rial 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 electro-

blotting and hybridised with denatured labelled RNA.

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 microorgan-

isms 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 environ-

ments. 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 quanti-

tative 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. SeS-

PERA is reliant on the limited size separation capacity

of agarose gels, which may impede clear separation of

hybrid molecules from complex environmental sam-

ples. Nonetheless, SeSPERA constitutes a promising

variation of radioactive isotope probing.


123

3 Probe-based methods

The majority of probe-based methods have in com-

mon 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 probe-

defined 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 oligonu-

cleotide 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 40,6-diamidino-2-phenylindole (DAPI) allows quantifica-

tion 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

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 microorgan-

isms 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

Type of probe Basic method Assignation of function Name of method

Pulse Hybridisation of sample with probe

Microautoradiography Fluorescence

microscopy and TEM microscopy

MAR-FISH MAR-positive

cells

Fluorescence microscopy

Isotope imaging by

Secondary ion mass spectrometry

FISH-SIMSIncrease in

isotope ratio per cell

Raman microspectroscopy

FISH-RAMAN Red shifts in biomolecules

per cell

Fluorescence microscopy

RNA extraction

14C-RNA +

fluorescent dye Isotope Arrays

Fragmentation +

Hybridisation with taxonomic probe array

Fluorescence imaging and -imaging Fluorescent

spots = identity Radioactive

spots = labelled taxa

13C-RNA Isotope ratio mass spectrometry

SSU-IRMS Labelled taxa as per rRNA Biotin probe hybridisation

Magnetic bead capture

F-probe

EL-FISH Fluorescence microscopy

Isotope imaging by NanoSims

Increase in isotope ratio

per cell HRP-probe

+ H-F-tyramide

Isotope imaging by NanoSims SIMSISH Increase in isotope ratio per cell

H-probe

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

cell is not achieved prior to the hybridisation step, the

probe may not reach its target, resulting in inadequate

quantification. Secondly, interaction between ribo-

somes 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 accessi-

bility. To artificially increase the amount of target

rRNA, cells have been incubated with chloramphen-

icol 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 thatincrease signal intensity and target accessibility

(Stoecker et al. 2010). These and the concomitant

technological improvements in the imaging equip-

ment and software have substantially improved the

ability of FISH to detect targeted cells.

3.1 Fluorescence in situ hybridisation-

microautoradiography (FISH-MAR)

FISH-MAR (also known as substrate-tracking auto-

radiography (STAR-FISH) and microautoradiogra-

phy-FISH (MICRO-FISH)) allows the taxonomic

identification of microbes that incorporated a radio-

active 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,

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 probe-

targeted 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 theactivity 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, back-

ground 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 sub-

strates 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, FISH-

MAR has undergone a series of makeovers that have

improved the method. These have resulted in Q-


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MAR-FISH, Het-CO2-FISH-MAR and MAR-CARD-

FISH, 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, lipopolysacha-

rides and peptidoglycans derived from dead cells

(Kragelund et al. 2008). Through FISH-MAR analysis,

Chloroflexi spp. have been implicated in the consump-

tion of bacterial detritus carbon in sludge (Miura and

Okabe 2008). The role that Beta- and Gammaproteo-

bacteria 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 phos-

phate 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 Actinobac-

teria species were found to constitute important

consumers of glucose, mannose and galactose impli-

cating them as potential carbon and energy providers

for PAOs (Kong et al. 2008). In relation to the

nitrogen cycle in activated sludge, addition of meth-

anol 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 environ-

ments. 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 prefer-

entially over D-asparagine. This was in contrast to a

large proportion of prokaryotic D-asparagine consum-

ers 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 concen-

trations, Nitrosomonas spp. Nm-143 lineage members

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 Q-

FISH-MAR. Their procedure included the prepara-

tion of a standard curve with pure cultures of a

filamentous bacterium incubated with different con-

centrations 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 Q-

FISH-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 14CO2assimilation into bacterial cells as a means of


123

userHighlight

identifying active heterotrophs (Het-CO2-FISH-

MAR; Hesselsoe et al. 2005). The rationale is based

on the observation that all heterotrophic organisms

assimilate CO2 during biosynthesis in various carbox-

ylation reactions. Therefore, it is possible to isotopi-

cally 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 bioreme-

diation 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 micro-

organisms 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-FISH-

MAR, Candidatus Microthrix parvicellaa filamen-

tous bacteria from sludgewas incubated with 14CO2and 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 conven-

tional approach. Conversely, only those that grew

aerobically were positive with Het-CO2-FISH-MAR.

By switching from anaerobic incubation with 14CO2and oleic acid to aerobic conditions, the cells became

MAR-positive. Cells had assimilated 14CO2 in com-

parable 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 exten-

sively in immunoassays, is a methodological improve-

ment 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. oligo-

trophic environments). In this technique, the fluoro-

phore of the FISH probe is replaced by the horseradish

peroxidise enzyme (HRP), the catalyser. The HRP-

probe 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, includ-

ing 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 fluo-

rescent 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


123

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 envi-

ronments by way of tracking assimilation of labelled

amino acids, glucose, ATP or dimethylsulfoniopro-

pionate. 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, fresh-

water 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 incorpora-

tion by specific cells identi

mar-fish

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microbial function

suite of methods

development of methods

microbial ecologists

microbial ecology hasm

reviewsan appraisal

incorporation of isotopes

enriched isotopes