Research protocol on stable isotope probing to elucidate the role of soil microorganisms in nutrient cycling and soil quality

Dr. Egbert Schwartz

Northern Arizona University

Requisition 12750

(IAEA staff: Joseph Adu-Gyamfi)

non-labeled

DNA

newly grown

labeled DNA

Figure 4. Isopycnic centrifugation of DNA extracted from mixed conifer soil incubated with H2

18O (tubes A) or H216O (tube B) for 10 days.

A A B

non-labeled

DNA

newly grown

labeled DNA

Figure 4. Isopycnic centrifugation of DNA extracted from mixed conifer soil incubated with H2

18O (tubes A) or H216O (tube B) for 10 days.

A A B

2

Table of Contents

I. Introduction to centrifugation and stable isotope probing.

II. Stable Isotope Probing experimental design

a. Cost of isotopes limits size of experiments

b. Most SIP experiments to date involve a single labeled substrate

c. Type of stable isotope impacts separation of labeled DNA from non-

labeled DNA.

i. 13Carbon

ii. 15Nitrogen

iii. 18Oxygen

d. In SIP experiments a majority of atoms in “labeled” DNA must be the

heavy isotope

e. Adding high concentrations of a labeled substrate may introduce a

fertilizer artifact into the experiment

f. The labeled substrates, once added to soil, will be incorporated into a

variety of compounds, making interpretation of experimental results more

difficult with extended incubations.

III. Stable Isotope Probing Experimental Protocol

a. Incubation of soil with labeled substrate

b. DNA extraction

c. DNA quantification

d. Centrifugation

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i. Rotor Choice

1. Vertical Rotor

ii. Fixed Angle Rotor

iii. The compromise between speed and length of centrifuge spin.

iv. Guanine/cytosine content affects density of DNA

v. DNA binding dyes may be used to exaggerate the impact of GC

content

e. Preparing the ultracentrifuge tubes for isopycnic centrifugation

f. Photographing DNA Bands Within the Centrifuge Tubes

g. Fractionation of centrifuge tube contents.

h. Purifying DNA from CsCl Solution

i. The SIP experiment is successful if significantly more heavy DNA is

present in the labeled substrate treatment than the non-labeled substrate

treatment.

j. Analysis of DNA fractions

i. Quantitative PCR

ii. Sequencing of fractions

IV. Use of SIP in elucidating the role of microorganisms in nutrient

cycling and soil quality

a. Carbon Cycle

b. Nitrogen Cycle

i. Nitrogen Fixation

ii. Ammonia oxidation

c. Food web analysis

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d. Characterization of microorganisms in the plants rhizosphere

V. List of Published SIP studies

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Introduction

Stable isotope probing is a technique that is used to identify the microorganisms

in environmental samples that use a particular growth substrate. The method relies on

the incorporation of a substrate that is highly enriched in a stable isotope, such as 13C,

15N or 18O and allows identification of active microorganisms by the selective recovery

and analysis of isotope-enriched cellular components. DNA and rRNA are the most

informative taxonomic biomarkers and 13C-labelled molecules can be purified from non-

labeled nucleic acid by density-gradient centrifugation. The future holds great promise

for SIP, particularly when combined with other emerging technologies such as

metagenomics.

The report is divided into five separate sections: 1. An introduction to

centrifugation and stable isotope probing, 2. Experimental design of stable isotope

probing experiments, 3. A detailed experimental protocol for stable isotope probing

experiments 4. A discussion of possible applications of stable isotope probing to

nutrient cycling and soil quality is given, and 5. A list of published SIP studies is given in

the Reference section. Stable Isotope Probing sometimes refers to the study of

incorporation of stable isotopes into any biomolecule including RNA, DNA,

phospholipid fatty acids, and proteins. This report is limited to the application of stable

isotope probing to the study of DNA and RNA.

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VI. Introduction to centrifugation and stable isotope probing.

Centrifugation has played an extremely important role in biological research. It

has been used to separate cell types, organelles within a cell and a variety of biological

macromolecules. Among the most famous researchers was the Swedish chemist

Theodore Svedberg who received the Nobel Prize in 1926. The Svedberg unit, for

sedimentation rate is named after him, and he was the first to show that pure proteins

could be separated from each other through ultra-centrifugation.

There are several types of centrifugation including differential centrifugation,

rate zonal centrifugation and isopycnic centrifugation. In differential centrifugation

particles of various densities are sedimented at different rates thereby separating them.

In rate zonal centrifugation the sample is not homogenously distributed throughout the

centrifuge tube but rather is layered on top of the density gradient. Differences in

sedimentation rates are still exploited to separate particles from each other. It is less

likely that particles will cross contaminate in Rate zonal centrifugation than in

differential centrifugation. In isopycnic centrifugation the particles do not sediment.

Instead the density gradient is designed in such a way that the particles will migrate

within the density gradient to the location where the particles density is equal to the

density of the gradient. Isopycnic centrifugation is used in SIP so that both labeled and

non-labeled DNA hang in the middle of the tube.

In isopycnic centrifugation a variety of gradient media are used to separate

biomolecules. In SIP cesium chloride (CsCl) is most commonly used to separate labeled

DNA from non-labeled DNA while cesium trifluoroacetate (CsTFA) is used to separate

labeled RNA from non-labeled RNA. However in the 1960’s and 1970’s other media,

such as sodium iodide, were also employed to separate labeled DNA molecules.

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In SIP a highly labeled substrate, as much as 99 atom% but rarely less than 50

atom%, is added to an environmental sample. Microorganisms in the sample will

assimilate the substrate thereby incorporating heavy isotopes from the substrate into

their nucleic acids. These nucleic acids will have higher buoyant densities than those of

organisms that did not assimilate the labeled substrate, either because they were not

active or because they assimilated other non-labeled substrates present in the

environmental sample. The difference in buoyant density is subsequently exploited to

separate the labeled nucleic acids from non-labeled nucleic acids along a density

gradient of CsCl or CsTFA, generated in an ultracentrifuge. The labeled nucleic acids

are subsequently recovered from the tube and can be analyzed in a variety of ways

including quantitative PCR or next generation sequencing such as pyrosequencing or

sequencing on the illumina platform.

Representation of isopycnic centrifugation. At time = 0 (A) all compounds, such as labeled and non-labeled nucleic acids are homogenously mixed. After centrifugation (B) the compounds hang in the density gradient according to their buoyant density. Labeled nucleic acids are more dense and would position lower in the centrifuge tube. None of the compounds pellet in isopycnic centrifugation

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Stable Isotope Probing has it origins in the 1950’s and 60’s when a large number

of researchers focused on how DNA in cells replicated. The classic studies of Messelson

and Stahl (1958) showed a new strand DNA formed by using an old strand as a template

also known as semi-conservative replication. In these experiments DNA was labeled

with 15N, the heavier stable isotope of nitrogen. The researchers were able to analyze the

DNA in their experiments after one generation through isopycnic centrifugation; 2

distinct DNA bands formed in their ultra-centrifuge tubes, one containing 14N atoms

while the other had incorporated 15N atoms. When labeled DNA from subsequent

generations where included in the comparisons a third DNA band appeared. The only

reasonable interpretation of these results was that, after one generation, DNA with one

14N strand and one 15N strand formed and DNA in which both strands contained 15N

appeared in subsequent generations thereby establishing the principle of

semiconservative DNA replication. During this important period of innovation in the

field of centrifugation, and biology in general, studies were limited to pure cultures and

labeled substrates were not administered to environmental samples. These were

experiments in the fields if cell and molecular biology and not microbial ecology.

The first report of a Stable Isotope Probing study was published in the journal

Nature in 2000 and was authored by Radajewski, Ineson, Parkeh and J.C. Murrell. The

report was entitled “stable-isotope probing as a tool in microbial ecology” and involved

the use of 13C-methane to label DNA of methane oxidizers in environmental samples.

Subsequently numerous SIP studies appeared not only using 13C labeled substrates but

also compounds labeled with 15N or 18O. Theoretically any atom present in nucleic

acids including, Carbon, Nitrogen, Oxygen, Hydrogen and Phosphorus, can be used in

SIP to label nucleic acids. Phosphorus has only radioactive isotopes and to date no SIP

9

Overview of Stable Isotope Probing. (Taken from Marc G. Dumont & J. Colin Murrell Nature Reviews Microbiology 3, 499-504 (June 2005) doi:10.1038/nrmicro1162

10

studies have employed this element. Furthermore the element is relatively heavy

compared to Carbon, Nitrogen and Oxygen and a one or two neutron difference between

the isotopes would only increase buoyant density by a small fraction. Deuterium can be

used in SIP. DNA from E.coli grown in D2O based media can be separated along a CsCl

gradient from DNA extracted from E.coli grown in media with H2O, but to date no

environmental studies employing deuterium have been published. Hydrogen atoms on

molecules may exchange more readily with water molecules, which may complicate

interpretation of SIP experiments that use deuterium. Most of the SIP studies to date

are in the field of bioremediation and focus on the degradation of pollutants in

environmental samples.

II Stable Isotope Probing experimental design

Before designing a SIP experiment, it is important that the researcher is aware of

several limitations imposed on SIP experiments and she must adjust her experimental

design to accommodate these limitations.

Cost of isotopes limits size of experiments

SIP experiments require that nucleic acids are heavily labeled, in excess of 50

atom%, and therefore the substrate added to an environmental sample must also be

heavily labeled. Highly labeled substrates are expensive. 95 atom % H218O for instance

can cost upwards of $200 a mL. Also it is important that the label of the added

substrate is not diluted with non-labeled substrates already present in the soil. If for

instance, the object is to study degradation of labeled litter in soil the carbon already

present in soil may dilute the added labeled litter. A small amount of labeled litter in a

large amount of non-labeled soil will not produce nucleic acids sufficiently labeled for

11

SIP experiments. Consequently SIP experiments are often conducted on small

quantities of soil (1-5 grams) and often relatively large quantities of substrate are added

to produce enough labeled nucleic acids. As discussed below these experimental

limitations may lead to fertilizer artifacts.

Most SIP experiments to date involve a single labeled substrate

To perform SIP a highly labeled substrate must be added to an environmental

sample. Usually this substrate is purchased from a chemical company and is delivered

to the environmental sample in pure form. For some substrates, including 13CO2, 13CH4

and H218O, that is representative of environmental conditions. But in other studies,

especially those focusing on pollution degradation or carbon metabolisms in soil, the

addition of a single substrate does not conform to realistic circumstances. For instance,

it is highly unlikely that pure benzene or glucose is ever added to an environmental

sample. More likely, benzene pollutes soil in combination with other compounds

including toluene, ethylbenzene and xylene. Thus SIP experiments in which multiple

labeled substrates are added to a soil remain rare. Some scientists would argue that

adding multiple substrates at once defeats the main purpose of SIP experiments which

is to identify microbial populations that assimilate specific compounds. However, in

studies of soil quality or nutrient cycling we are especially keen to understand the role of

microorganisms in degrading complex cocktails of substrates such as litter or organic

nitrogen.

Type of stable isotope impacts separation of labeled DNA from non-labeled

DNA

Stable isotopes are more dense because the nuclei of the atom incorporates extra

neutrons. An extra neutron can shift the density of a small element substantially but

12

may have less impact, on a relative basis, on larger elements. Furthermore the

concentrations of different elements in nucleic acids are not equivalent. Thus while 15N

is on a percentage basis, relative to 14N, only slightly less heavy than 13C is to 12C, there is

substantially less N in nucleic acids than C, so that it is more difficult to label nucleic

acids sufficiently for SIP with 15N than 13C.

13Carbon

There is a long and rich history of the use of carbon isotopes in biology dating

back to the classical experiments on photosynthesis of Kammen, Benson and Calvin

employing 14C. The first experiments that demonstrated stable isotope probing was

feasible in environmental samples used 13C labeled methane. The methane was the sole

carbon source for methane oxidizers and as the communities were incubated with 13C

methane substantial quantities of 13C (DNA) formed. Methane was a particularly good

choice for the first SIP experiments because it is relatively easy to remove methane from

soil samples so that in an incubation with newly added 13C-methane almost all the added

methane is labeled with 13C. In studies of litter decomposition this is not feasible

because soil organic matter cannot be removed from soil without severely disturbing the

sample. Therefore the added labeled litter will be diluted by the 12C in the soil organic

matter. Microorganisms in soil are often limited by the availability of labile carbon.

Therefore SIP studies that employ labile carbon are particularly susceptible to

fertilization artifacts. Most SIP experiments to date involved 13C substrates, partly

because there is great interest in metabolism of organic compounds in our environment,

but also because 13C labeled substrates are readily available. Specifically, most studies to

date have focused on degradation of pollutants in the environment and assimilation of

methane or simple compounds such as acetate.

13

15Nitrogen

As previously mentioned 15N was used by Messelson and Stahl to study DNA

replication in E.Coli. 15N has also been used in SIP to study nitrogen fixation and to

identify pollutant degraders in environmental samples. The table below lists the limited

number of studies that have used 15N SIP. It is the most difficult of the elements to use

in SIP because 15N labeled nucleic acids are only slightly more dense than 14N labeled

nucleic acids. The maximum shift in buoyant density that can be achieved in CsCl

gradients for 15N labeled nucleic acids is approximately 0.016 g ml-1 DNA, relative to

0.036 g ml- for 13C labeled isotopes. GC content of microbial genomes can result in

DNA samples that vary in buoyant density by as much as 0.05 g ml-1. Some researchers

employ a DNA binding dye, bis-benzimide and further discussed later in the report, to

help separate 15N labeled DNA from non-labeled DNA.

18Oxygen

Paul Boyer and coworkers studied DNA replication in E. coli using H218O to label

DNA showing that branch oxygen atoms of E. coli DNA are almost entirely derived from

water and that 18O labeling of DNA is not due to formation of hydration shells around

DNA. When analyzing environmental samples of microorganisms by stable isotope

probing (SIP), labeling the DNA with H218O, instead of organic or nitrogenous

compounds, offers important advantages because water cannot be used as an energy,

carbon, or nitrogen source. As a result, addition of the label is unlikely to influence

microbial growth rates in soil directly and microbial communities can be exposed to the

label for long periods of time because they are not exposed to abnormally high substrate

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concentrations. Because all organisms incorporate water into their DNA, performing

SIP with H218O is a method for identifying microorganisms that have grown during

incubation with H218O, as well as, microorganisms that have not grown (i.e., did not

incorporate the label) but survived the incubation. Though there are fewer oxygen atoms

in DNA than carbon (Radajewski et al., 2003), more neutrons can be added to DNA

through 18O than any other isotope. There are, on average, 11 oxygen atoms per

nucleotide unit in DNA whereas on average 3.75 nitrogen and 14.75 carbon atoms are

present per unit DNA. The carbon and nitrogen isotopes used in SIP contain one extra

neutron relative 12C or 14N while 18O has 2 more neutrons than the more prevalent

oxygen isotope 16O. Thus by labeling DNA with 18O it is possible to introduce 22 extra

neutrons per unit DNA while only 3.75 or 14.75 extra neutrons can be added with 15N

and 13C respectively.

To test if soil DNA could be labeled sufficiently to use H218O in SIP of soil

microbial communities, 0.2 ml of 95 atom% H218O was added to one gram of Ponderosa

Pine soil, with a moisture content of 8%. DNA was extracted from soil and isopycnic

centrifugation produced 2 or 3 DNA bands after 7 or 21 days of incubation, respectively

(Schwartz, 2007). The third band appears to form after microorganisms feed on 18O-

labeled organic matter while in H218O. DNA extracted from Ponderosa pine soil

incubated with H216O for 6 or 21 days did not produce multiple bands after isopycnic

centrifugation indicating that changes in % GC of bacterial genomes in soil did not cause

the formation of multiple DNA bands.

15

There are important advantages in using H218O over other types of labels, such as NH4

+,

in order to measure growth in soil. These include:

1. More neutrons can be added to DNA when H218O is used than when 15N is added

as a label in SIP experiments. As a result labeled DNA separates from non-

labeled DNA along a CsCl gradient whereas in experiments with 15N a continuous

smear of DNA forms. Migration of DNA within this smear is controlled by both

DNA label incorporation and GC content of the DNA whereas in 18O-SIP changes

in % GC are not sufficient to cause DNA to migrate to the lower labeled band. In

18O-SIP, but not 15N-SIP, only two DNA fractions are retrieved (labeled and

unlabeled DNA) making subsequent analysis much more simple and cost

effective.

A A B B C C D D E E

Timecourse of 18

O labeling of DNA in Ponderosa Pine soil. A = DNA

extracted from soil incubated with H2

18O for 0 days, B = DNA

extracted from soil incubated with H2

18O for 6 days, C= DNA

extracted from soil incubated with H2

18O for 21 days, D = DNA

extracted from soil incubated with H2

16O for 6 days, E = DNA

extracted from soil incubated with H2

16O for 21 days.

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2. In contrast to an organic or nitrogenous molecule, water by itself does not induce

growth because it is not used as an energy, carbon or nitrogen source. As a result

fertilization artifacts by adding large quantities of labeled substrate to soil are

avoided.

3. Water can easily be distributed homogenously throughout soil because it is a

small molecule that does not interact with the cation exchange capacity of soil. As

a result microorganisms in soil are uniformly exposed to the label. In SIP with

15NH4+, microorganisms will incorporate unlabeled ammonium, formed through

N mineralization, as well as labeled ammonium into their DNA. Because some

organisms may grow in N-mineralization hot spots not all microorganisms are

exposed to the same amount of 15NH4+.

4. During precipitation events water is normally added to soil while semiarid soils

in northern Arizona never receive pure ammonium solutions.

5. H218O SIP allows study of environmental manipulations that do not involve

substrate assimilation including the impact of temperature, moisture, soil bulk

density and pH on ammonia oxidizing microorganisms. These environmental

parameters could be very important in determining which genotype grows fastest

in soils.

In SIP experiments a majority of atoms in “labeled” DNA must be the

heavy isotope

As previously mentioned the majority of atoms of an element in nucleic acids

must be the heavy isotope in order to have sufficient difference in buoyant density to

separate labeled and non-labeled nucleic acids along a CsCl or CsTFA density gradient.

Consequently SIP experiments should only be designed for labeled substrates that may

17

be obtained with 50 atom% heavy isotope or higher. Furthermore the researcher needs

to consider to what extent the added labeled substrate will be diluted by non-labeled

substrates already present in the environmental sample. It may, for instance be difficult

to do SIP with 13C-methane in an environmental sample that produces large quantities

of 12C-methane. Similarly when using 15N2, it may be prudent to replace the atmosphere

of the sample thereby removing the 14N2 from the incubation.

Adding high concentrations of a labeled substrate may introduce a

fertilizer artifact into the experiment

Because high concentrations of substrate are required in small environmental

samples, SIP experiments are susceptible to fertilizer artifacts. When studying glucose

assimilators in soil, for instance, it is important to recognize that addition of glucose will

alter the active microbial community in soil. Therefore results may not reflect the

community that would assimilate low concentrations of substrate. In soils

microorganisms are often limited by labile carbon and adding a large amount of labile

carbon to a soil may not reflect average conditions in the environment. Often in

bioremediation experiments microorganisms are exposed to high concentrations of

carbon substrates but SIP studies of nutrient cycling or soil quality will need to be

designed in such a way that the fertilization artifact is avoided.

The labeled substrates, once added to soil, will be incorporated into a

variety of compounds, making interpretation of experimental results more

difficult with extended incubations.

Once microorganisms assimilate the added labeled substrate and produce new

biomolecules, a wide range of labeled organic compounds are formed in soil including

proteins, lipids, nucleic acids, carbohydrates, cell wall components and smaller

18

metabolites. These compounds also serve as excellent substrates for microbial growth

so that the longer the incubation proceeds the more types of microorganisms will

become labeled. While it is reasonable to presume that the first labeled nucleic acids are

derived from organisms that assimilated the original added labeled substrate, once the

incubation proceeds further it will become increasingly difficult to ascribe labeled

nucleic acids to specific substrate assimilators. By conducting a SIP time series analysis

it is feasible to track an element such as carbon through different microbial populations

as the incubation proceeds. This approach may be highly suitable for studies of nutrient

cycling and soil quality.

III Stable Isotope Probing Experimental Protocol

Incubation of soil with labeled substrate

1. Combine 1 g of soil with 200 μL of H218O (95 atom%) or other labeled substrate and

place in a 15-mL Falcon tube. Stir the soil with a small spatula so that it becomes

homogenously moist. The moisture content of the soil matters because unlabeled H2O

present in the soil, prior to addition of labeled H2O, will dilute the label present during

the incubation. If the soil is too wet, so that addition of 200 μL of H218O results in a soil

completely saturated with H2O, it may be necessary to air dry the soil before adding the

H218O. If 13C or 15N labeled substrates are used it is important to add a sufficient quantity

of substrate to cause growth of the microbial population. It is likely that at least 50µg

substrate/g soil is required. The more substrate is added the more likely there will be a

fertilization artifact.

2. Incubate the soil with the labeled H2O or labeled substrate for ~1 week in a Falcon

tube at room temperature (approximately 20 °C). Keep the tube closed to avoid

19

evaporation of H2O and drying of the soil sample. A preliminary experiment to

determine the optimum incubation time to allow formation of labeled DNA which can

be detected along the CsCl gradient is highly recommended. More than one week may be

required to produce sufficient labeled DNA. If the sample is incubated too long the label

will turnover and DNA of organisms that did not originally assimilate the substrate will

become labeled. For many 13C labeled substrates incubation times are shorter than one

week; 24 to 72 hours may be sufficient to label a large fraction of the microbial

population.

3. After incubation, the soil may be frozen at -20°C or -80°C until time is available for

DNA extraction. If the soil is frozen at -20°C, DNA should be extracted within a month,

whereas soil may be stored at -80°C for at least a year.

4. A non-labeled substrate control must be included in the experiment to ascertain that

nucleic acids in the labeled treatment did become labeled during the incubation. There

must be significantly greater quantities of heavy nucleic acids (for DNA usually greater

than 1.72 g/mL) in the labeled treatment than in the non-labeled substrate control.

DNA extraction

Extract the DNA from soil using a commercially available soil DNA extraction kit

according to the manufacturer’s instructions. The frozen soil should not be thawed prior

to extraction. Extract only half of the incubated sample at one time, leaving the other

half frozen, in case the first attempt at SIP analysis is unsuccessful. During the

extraction procedure it is important to maximize yield, not purity, of the DNA because

subsequent centrifugation of the DNA on a CsCl gradient will further purify the DNA.

The yield can be improved by eluting the DNA, in the final step of purification, with

larger amounts of elution buffer, because the DNA does not need to be concentrated for

20

centrifugation. Some researchers include a phenol/chloroform step in their protocol to

improve DNA yield. One microgram of DNA is sufficient to perform SIP.

DNA quantification

Nucleic acids should be quantified before they are loaded onto the cesium

chloride gradient. Quantification can be done via fluorescent methods such as the use of

picogreen a fluorescent DNA binding dye and the QUBIT system. Alternatively a

spectrophotometer can be used to quantify the DNA. If using a spectrophotometer it is

important to measure the absorbance at 230, 260 and 280nm. The absorbance at 260

nm will be used to calculate the concentration of DNA and the ratio of absorbance at

260 nm over 230 nm or 260 nm over 280 nm are indicative of the cleanliness of the

DNA. If the A260/A280 or the A260/A230 are not over 1.5 the DNA is not very clean

and the A260 is not a reliable measurement of DNA concentration.

Change in position of DNA bands as the samples are spun in an ultracentrifuge

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Centrifugation

Rotor Choice

Both fixed angle and nearly vertical rotors may be used in SIP experiments. Vertical

rotors appear to set up the density gradient faster and therefore require shorter spin

times. However, it appears that greater separation is feasible with a fixed angle rotor.

Furthermore contaminants in DNA such as humic acids may be pelleted in a fixed angle

rotor.

Vertical Rotor

An example of a vertical rotor used in SIP analysis is the TLN-100 from Beckman. It

positions the tubes at a 9 degree angle, so it is referred to as a nearly vertical rotor.

In a nearly vertical rotor, such as the Beckman TLN-100 the tubes are nearly upright in the rotor

22

If the samples are spun too fast CsCl will precipitate in the centrifuge tubes. This graph shows how fast the TLN-100 rotor can be spun without having CsCl precipitate

23

In isopycnic centrifugation the gradient media needs to be of a density similar to the

molecules the researcher is attempting to hang in the middle of the tube. DNA has a

density of approximately 1.7 g/mL. The number is not exact since it varies due to

differences in GC content. As a result Cesium Chloride is a good salt to use for DNA-

SIP. RNA has a higher buoyant density and will pellet when centrifuged in a cesium

chloride media. For RNA SIP Cesium Tri Fluoro Acetate is routinely used as a media to

set up a density gradient. Cesium chloride will precipitate from the media if the samples

are spun too fast. The graph above shows the relationships, at different temperatures

between the density of the cesium chloride solution and how fast the samples are spun.

Cesium chloride will not precipitate at any point below these curves. Precipitation of

cesium chloride during isopycnic centrifugation should be avoided as it will impact

separation of labeled and non-labeled DNA.

The speed at which a rotor is spun generates varying levels of RCF in different rotor. The graph shows the relationship between RCF and rpm for the TLN-100 rotor

24

The speed at which samples are spun, in rotations per minute, is not very

informative in SIP studies. The density gradient that forms is dependent on the amount

of g-force, often expressed in rotational centrifugal force (RCF). While RCF is

dependent on how fast the samples are spun in rpm the relationship between rpm and

rcf varies between different rotors. Therefore, SIP publications should always report

isopycnic procedures in rcf and not rpm.

One of the challenges in SIP is to separate labeled DNA from non-labeled DNA

even though there may be only small differences in buoyant density. This is especially

the case when the nucleic acids are not fully labeled with the isotopes because non-

labeled substrate was present in the incubation. The speed at which the centrifuge is

spun will determine the difference in densities from the top of the centrifuge tube to the

bottom. If the samples are spun very fast, there will be a large difference in densities

along the gradient, resulting in relatively tight nucleic acid bands that are positioned

close together in the tube. If the samples are spun more slowly the bands will be more

diffuse but also positioned further apart. It may require more time to set up a fully

formed gradient when samples are spun more slowly.

25

The faster the samples are spun the steeper the density gradient becomes. This graph shows the relationship between centrifugation speed and the steepness of the density gradient for the TLN-100 rotor

26

Fixed Angle Rotor

In the fixed angle rotor the tubes are positioned at more of an angle than in the

near vertical rotor. In the case of the TLA-110 rotor, a rotor commonly used in SIP, the

tubes are placed at a 28 degree angle. This allows contaminating compounds to pellet

more readily from the samples.

Position of tubes in a fixed angle rotor. The Beckman TLA-110 is shown

27

If the samples are spun too fast CsCl will precipitate in the centrifuge tubes. This graph shows how fast the TLA-100 rotor can be spun without having CsCl precipitate

28

The faster the samples are spun the steeper the density gradient becomes. This graph shows the relationship between centrifugation speed and the steepness of the density gradient for the TLA-110 rotor

29

The compromise between speed and length of centrifuge spin

The faster the samples are spun the greater the difference in buoyant density

between the top and bottom of the centrifuge tubes. As a result compounds that differ

only slightly in buoyant density such as partially labeled nucleic acids and non-labeled

nucleic acids will position very close together along the density gradient if the centrifuge

tube is spun fast. However, if the tubes are spun very slowly it may take a relatively long

time to form the density gradient, so that the researcher must make a compromise

between length of spin and degree of separation between labeled and non-labeled

nucleic acids.

Guanine/cytosine content affects density of DNA

Incorporation of heavy isotopes is not the only determinant of buoyant density of

nucleic acids. Nucleotide composition, specifically the ratio of guanidine cytosine (GC)

over adenosine thymidine (AT) nucleotides, will also affect the buoyant density of

nucleic acids by as much as 0.05 g ml-1. Nucleic acids with high GC content are more

dense that nucleic acids with high AT content. The GC content can vary substantially

between different microbial genomes. Consequently non-labeled DNA extracted from

soil will spread over a range of densities. The impact of GC content on SIP results

requires the inclusion of a non-labeled but identically treated control. The scientist

must ascertain that the high concentrations of heavy DNA formed during a SIP

incubation is due to assimilation of the isotopically labeled substrate and not because a

microbial population grew that contained a genome with a high GC content.

DNA binding dyes may be used to exaggerate the impact of GC content

Several studies have used DNA binding dyes, such as bis-benzimide, to

preferentially bind AT rich regions to manipulate buoyant density of DNA. This

30

approach can be exploited in SIP where GC rich DNA can contaminate labeled DNA. By

including a second spin in which the DNA binding dye is included GC rich DNA can be

separated from labeled DNA. This experimental approach is especially useful when 15N

is the isotope used in SIP experiments.

Preparing the ultracentrifuge tubes for isopycnic centrifugation

5. Weigh 1 mL of CsCl solution to determine that it has the correct density. A saturated

CsCl solution will have a density of 1.9 g/mL. Be certain that no crystals remain in the

solution. By holding the solution up to the light it is possible to see any remaining

transparent CsCl crystals.

6. Add a saturated solution of CsCl solution to each centrifuge tube placed on a scale to

confirm that each tube receives an identical amount of CsCl. The amount added to the

tube depends on the volume of the tube used. In the case of 4.7 ml TLA-110 rotor tubes

we add 3.6 ml of saturated CsCl solution. The final density of the tube contents, after

the DNA and gradient buffer are added should be approximately 1.72 g/mL.

7. If the researcher wants to visualize the DNA after isopycnic centrifugation, add 0.5 μL

of fresh SYBR Green I DNA stain to the DNA extracted from soil. Older SYBR I stain can

increase the time required to get good DNA separation along the cesium chloride

gradient. The DNA should still be in the microcentrifuge tube used to elute the DNA

from the column in the final step of DNA extraction (Step 4). Alternatively, the

researcher may elect to not include a DNA binding dye, in which case the DNA cannot

be visualized and no photograph of the tube will be taken after centrifugation.

8. Add the DNA/SYBR-Green mixture to the CsCl solution in the centrifuge tubes. Add

300 μL of gradient buffer (200mM Tris pH 8.0, 200mM KCl, 2mM EDTA) to each tube.

31

Fill each centrifuge tube to the top with H2O and invert the tubes several times to

thoroughly mix the contents.

9. Weigh each tube to ensure that they all weigh within 0.01 g of each other. If

necessary, add sterile H2O to balance the tubes.

10. Load the tubes in the TLA-110 rotor and centrifuge in an ultracentrifuge at

approximately 176,000 X g for approximately 72 h.

11. Decelerate the centrifuge as slowly as possible. When the rotor is removed from the

centrifuge and the tubes are extracted from the rotor, extreme care needs to be taken to

avoid bumps, so that the gradients established in the tubes are not disturbed.

Photographing DNA Bands Within the Centrifuge Tubes

12. Carefully transfer the tubes from the rotor into a rack that has been placed on top of

a UV transilluminator in a dark room. The two bands formed during centrifugation

should be readily visible.

13. Photograph the tubes at this time using an exposure greater than 1 sec. Because of

the long exposure time, immobilize the camera on a test tube rack or camera stand.

Fractionation of centrifuge tube contents

Once the isopycnic centrifugation is complete and labeled DNA has separated

from non-labeled DNA the fractions will still need to be collected. A needle is used to

puncture the bottom of the tube while a second needle is used to pierce the tube on top.

The gradient media will drip from the bottom of the tube and 16 to 50 fractions of 100 to

250 microliter each may be collected. The density of each fraction is measured with a

digital refractometer before the DNA is precipitated from each fraction.

32

Purifying DNA from CsCl

Solution

16. Add ~500 μL of H2O and ten μL

of glycogen solution (10 µg/µL) to

each DNA fraction and shake the

tubes by hand.

17. Add 1 mL of isopropanol to each

tube and vigorously mix the contents.

18. Centrifuge the tubes in a

microcentrifuge at full speed for 30

min.

19. Discard the supernatant from the

tube, taking care not to dislodge the

pellet.

20. Add 500 μL of 70% ethanol to the tube and shake gently. Centrifuge at full speed for

5 min.

21. Discard the supernatant from the tube into a waste beaker.

22. Remove the last bit of liquid from the bottom of the tube with a pipette. Air dry the

pellet for ~30 min.

23. Dissolve the pellet in sterile H2O or TE buffer. The DNA is now ready for subsequent

analysis including production of clone libraries, generation of T-RFLP patterns, or real-

time PCR analysis.

24 Quantify the DNA in each fraction using a QUBIT from Invitrogen inc.

Harvesting of fractions from an ultracentrifuge tube

33

The SIP experiment is successful if significantly more heavy DNA is

present in the labeled substrate treatment than the non-labeled substrate

treatment.

Most of the DNA isolated from the centrifuge tubes will have a lower buoyant

density (1.68 to 1.72 g/mL) but in the labeled treatment there should also be DNA with a

higher buoyant density. The fractions with a density greater than 1.72 g/mL and that

contain significantly more DNA in the labeled treatments than in the control non-

labeled treatments contain the genomes of microorganisms that assimilated the added

substrate.

0

0.05

0.1

0.15

0.2

0.25

0.3

1.6 1.7 1.8 1.9

Fra

ctio

n o

f to

tal

DN

A

density (g/ml)

sample 2sample 4sample 6sample 4W

labeled DNA

not labeled DNA

This graph shows the DNA concentrations in different fractions taken from the tube. In a the heavy isotope treatments (samples 2, 4 and 6, represented by filled symbols) there is DNA in heavy fractions that is absent from the non-labeled control treatments (sample 4W represented by open circles)

34

Analysis of DNA fractions

The labeled fractions may be combined into one labeled DNA pool or, alternatively each

fraction can be analyzed separately. Any type of DNA analysis is feasible but

quantitative PCR and next generation sequencing are the two most common types of

analyses.

Quantitative PCR

In quantitative PCR the fluorescence generated by dyes binding to PCR product is

measured after each cycle. The resulting sigmoidal curve can be related to a set of

standard curves to determine the original abundance of gene copies in a DNA fraction.

This approach quantifies the growth or the extent to which a pre-determined microbial

population has assimilated the added substrate. The benefit from this approach is that

it is highly quantitative. The downside is that it can only be used for microbial

populations or functional genes for which PCR primers have been developed.

Sequencing of fractions

The DNA in the fractions may also be used in next generation sequencing, including

pyrosequencing and illumina based sequencing. Usually an amplicon, such as a

fragment of the bacterial 16S rRNA gene, is analyzed. However, increasingly scientist

are shot gun sequencing all the DNA in SIP samples in order to identify booth functional

genes and genes indicative of taxonomic structure. However, the quantity of DNA

obtained through SIP analysis is not sufficient to analyze directly in shotgun sequencing.

Therefore the DNA has to first be used in whole genome amplification. Commonly

multiple displacement amplification, a non-PCR based amplification technique, is used

to amplify small amounts of DNA to obtain sufficient quantities for genomic analyses.

PCR based amplification approaches are likely to introduce biases into the analysis

35

Use of SIP in elucidating the role of microorganisms in nutrient cycling

and soil quality

The majority of SIP studies have focused on degradation of pollutants in

environmental samples or assimilation of simple carbon compounds. Studies of

nutrient cycling or soil quality, however, require understanding of how complex

structures with multiple compounds, such as plant biomass, are assimilated by

microorganisms in soil. There are two possible approaches to identifying

microorganisms that assimilate complex organic structures. The first is to label the

plant with 13CO2. The challenge is that for SIP to work the majority of C atoms in the

plant need to be of the 13C variety, thus the plant should be grown from a seed in an

atmosphere consisting predominately of 13CO2. This is expensive, though not

impossible. In the second approach newly growing organisms are labeled with 18O

during 18O-water incubations. In this approach the growing microorganisms in soil with

only 18O-water are compared to the growing microorganisms in soil with 18O-water and

plant litter. This approach is likely cheaper and allows comparison of many different

kinds of naturally grown litter but does not provide a direct connection between litter

and labeled organisms. Rather the organisms that are labeled in the litter treatment but

absent from labeled DNA in the treatment without litter are identified as the litter

assimilators.

36

Assimilation of complex organic structures such as plant litter can be studies with 18O-water SIP. In this approach the labeled DNA of an environmental sample without plant litter is compared to the labeled DNA of a sample with plant litter. The microorganisms present in the labeled DNA from the litter treatment but absent in the control represent the organisms that grew due to the presence of plant litter

37

There have been several SIP studies that relate to soil quality and nutrient

cycling and these are described in the sections below:

Carbon Cycle

The few SIP studies that focused on the carbon cycle have employed either simple

sugars such as glucose or more complex polymers such as cellulose in order to identify

the organisms that assimilate and therefore decompose these carbon compounds in soil.

To date there have been no shotgun sequencing studies of SIP fractions in order to

identify genes involved in carbon catabolism.

Nitrogen Cycle

Nitrogen Fixation

SIP with 15N is more difficult than SIP with 13C or 18O because of the small

changes in buoyant density caused by incorporation of 15N into nucleic acids. The list of

publications that employed 15N are shown earlier in the report and several of these

studies investigated nitrogen fixers in environmental samples via SIP with 15N.

However, considering the large interest in nitrogen fixation among the soil microbial

ecology community, it is surprising not more reports have followed these initial

publications. It is likely that nitrogen fixation studies with 15N-SIP are extremely

challenging.

Ammonia oxidation

Ammonia oxidizers catalyze the first rate limiting step in nitrification, converting

ammonia to nitrite. These organisms may be bacterial or archaeal and are autotrophic

38

so that their nucleic acids can be labeled with 13CO2. Alternatively, it is feasible to study

the impact of environmental parameters such as ammonia availability on ammonia

oxidizers with 18O-water SIP. Here the abundance of ammonia oxidizing genes such as

bacterial amoA or archaeal amoA is compared in the labeled DNA between

environmental treatments of elevated ammonia and ambient ammonia.

Food web analysis

If environmental samples are exposed to labeled substrates for extended periods of time

the isotope will turn over, first being incorporated into biomolecules of the organism

that assimilated the substrate but subsequently becoming part of organisms that feed on

the original assimilating microorganisms. This can be an artifact if the objective is to

identify the organism that assimilated the labeled substrate. But it can also be exploited

in studies of the soil food web. Here samples are taken over time and the isotope is

followed into nucleic acids of different groups of organisms. There are very few SIP

studies of soil food webs but it is likely that SIP will provide new insights into the soil

food web.

Characterization of microorganisms in the plants rhizosphere

Plants interact with a large number of microorganisms in the rhizosphere,

releasing root exudates which may consist of simple sugars and amino acids and

forming symbiotic relationships with mycorrhizal fungi to obtain nutrient and/or water

from soil. Several studies have tried to follow organic carbon from the plant into nucleic

acids of microorganisms in the rhizosphere. Plants are exposed to high concentrations

of 13CO2 before soil attached to roots is used for nucleic acid extraction. One challenge

39

with this experimental approach is that often the plant contains large amounts of 12C-

organic compounds including sugars that will be released as exudate. Consequently it is

difficult to label the nucleic acids of microorganisms in the rhizopshere sufficiently for

SIP. There have been successful RNA-SIP studies of the rhizosphere but DNA-SIP

studies have required extensive periods of exposing the plant to labeled 13CO2, often in

excess of a month. As a result it is not possible to determine if the organisms

represented in the labeled DNA are rhizosphere organisms or of they feed on

rhizosphere organisms. To date there have been no metagenomic studies of SIP –DNA

obtained from rhizosphere organisms.

40

List of SIP studies that employ 15N labeled molecules

Bell, T. H., Yergeau, E., Martineau, C., Juck, D., Whyte, L. G., & Greer, C. W. (2011). Identification of nitrogen-incorporating bacteria in petroleum-contaminated arctic soils by using [15N]DNA-based stable isotope probing and pyrosequencing. Applied and environmental microbiology, 77(12), 4163–71. doi:10.1128/AEM.00172-11 Buckley, D. H., Huangyutitham, V., Hsu, S.-F., & Nelson, T. A. (2007a). Stable isotope probing with 15N2 reveals novel noncultivated diazotrophs in soil. Applied and environmental microbiology, 73(10), 3196–204. doi:10.1128/AEM.02610-06 Buckley, D. H., Huangyutitham, V., Hsu, S.-F., & Nelson, T. A. (2007b). Stable isotope probing with 15N achieved by disentangling the effects of genome G+C content and isotope enrichment on DNA density. Applied and environmental microbiology, 73(10), 3189–95. doi:10.1128/AEM.02609-06 Cadisch, G., Espana, M., Causey, R., Richter, M., Shaw, E., Morgan, J. A. W., Rahn, C., et al. (2005). Technical considerations for the use of 15N-DNA stable-isotope probing for functional microbial activity in soils. Rapid communications in mass spectrometry : RCM, 19(11), 1424–8. doi:10.1002/rcm.1908 Cupples, A. M., Shaffer, E. A., Chee-Sanford, J. C., & Sims, G. K. (2007). DNA buoyant density shifts during 15N-DNA stable isotope probing. Microbiological research, 162(4), 328–34. doi:10.1016/j.micres.2006.01.016 Gallagher, E. M., Young, L. Y., McGuinness, L. M., & Kerkhof, L. J. (2010). Detection of 2,4,6-trinitrotoluene-utilizing anaerobic bacteria by 15N and 13C incorporation. Applied and environmental microbiology, 76(5), 1695–8. doi:10.1128/AEM.02274-09 Roh, H., Yu, C.-P., Fuller, M. E., & Chu, K.-H. (2009). Identification of hexahydro-1,3,5-trinitro-1,3,5-triazine-degrading microorganisms via 15N-stable isotope probing. Environmental science & technology, 43(7), 2505–11. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/19452908

A list of SIP studies that employed 18O isotopes

41

Aanderud, Z. T., & Lennon, J. T. (2011). Validation of heavy-water stable isotope probing for the characterization of rapidly responding soil bacteria. Applied and environmental microbiology, 77(13), 4589–96. doi:10.1128/AEM.02735-10 Adair, K., & Schwartz, E. (2011). Stable isotope probing with 18O-water to investigate growth and mortality of ammonia oxidizing bacteria and archaea in soil. Methods in enzymology, 486, 155–69. doi:10.1016/B978-0-12-381294-0.00007-9 Schwartz, E. (2007). Characterization of growing microorganisms in soil by stable isotope probing with H2

18O. Applied and environmental microbiology, 73(8), 2541–6. doi:10.1128/AEM.02021-06 Schwartz, E. (2009). Analyzing microorganisms in environmental samples using stable isotope probing with H2

(18)O. Cold Spring Harbor protocols, 2009(12), pdb.prot5341. doi:10.1101/pdb.prot5341 Woods, A., Watwood, M., & Schwartz, E. (2011). Identification of a toluene-degrading bacterium from a soil sample through H2

18O DNA stable isotope probing. Applied and environmental microbiology, 77(17), 5995–9. doi:10.1128/AEM.05689-11

A list of SIP publications related to the soil carbon cycle:

Degelmann, D. M., Kolb, S., Dumont, M., Murrell, J. C., & Drake, H. L. (2009). Enterobacteriaceae facilitate the anaerobic degradation of glucose by a forest soil. FEMS microbiology ecology, 68(3), 312–9. doi:10.1111/j.1574-6941.2009.00681.x Eichorst, S. A., & Kuske, C. R. (2012). Identification of cellulose-responsive bacterial and fungal communities in geographically and edaphically different soils by using stable isotope probing. Applied and environmental microbiology, 78(7), 2316–27. doi:10.1128/AEM.07313-11 Gan, Y., Qiu, Q., Liu, P., Rui, J., & Lu, Y. (2012). Syntrophic oxidation of propionate in rice field soil at 15 and 30°C under methanogenic conditions. Applied and environmental microbiology, 78(14), 4923–32. doi:10.1128/AEM.00688-12

42

Haichar, F. E. Z., Achouak, W., Christen, R., Heulin, T., Marol, C., Marais, M.-F., Mougel, C., et al. (2007). Identification of cellulolytic bacteria in soil by stable isotope probing. Environmental microbiology, 9(3), 625–34. doi:10.1111/j.1462-2920.2006.01182.x Hori, T., Müller, A., Igarashi, Y., Conrad, R., & Friedrich, M. W. (2010). Identification of iron-reducing microorganisms in anoxic rice paddy soil by 13C-acetate probing. The ISME journal, 4(2), 267–78. doi:10.1038/ismej.2009.100 Monard, C., Binet, F., & Vandenkoornhuyse, P. (2008). Short-term response of soil bacteria to carbon enrichment in different soil microsites. Applied and environmental microbiology, 74(17), 5589–92. doi:10.1128/AEM.00333-08 Murase, J., & Frenzel, P. (2007). A methane-driven microbial food web in a wetland rice soil. Environmental microbiology, 9(12), 3025–34. doi:10.1111/j.1462-2920.2007.01414.x Murase, J., Shibata, M., Lee, C. G., Watanabe, T., Asakawa, S., & Kimura, M. (2012). Incorporation of plant residue-derived carbon into the microeukaryotic community in a rice field soil revealed by DNA stable-isotope probing. FEMS microbiology ecology, 79(2), 371–9. doi:10.1111/j.1574-6941.2011.01224.x Schellenberger, S., Kolb, S., & Drake, H. L. (2010). Metabolic responses of novel cellulolytic and saccharolytic agricultural soil Bacteria to oxygen. Environmental microbiology, 12(4), 845–61. doi:10.1111/j.1462-2920.2009.02128.x

A list of SIP studies that investigate ammonia oxidizers in environmental

samples:

Adair, K., & Schwartz, E. (2011). Stable isotope probing with 18O-water to investigate growth and mortality of ammonia oxidizing bacteria and archaea in soil. Methods in enzymology, 486, 155–69. doi:10.1016/B978-0-12-381294-0.00007-9

43

Avrahami, S., Jia, Z., Neufeld, J. D., Murrell, J. C., Conrad, R., & Küsel, K. (2011). Active autotrophic ammonia-oxidizing bacteria in biofilm enrichments from simulated creek ecosystems at two ammonium concentrations respond to temperature manipulation. Applied and environmental microbiology, 77(20), 7329–38. doi:10.1128/AEM.05864-11 Pratscher, J., Dumont, M. G., & Conrad, R. (2011). Ammonia oxidation coupled to CO2 fixation by archaea and bacteria in an agricultural soil. Proceedings of the National Academy of Sciences of the United States of America, 108(10), 4170–5. doi:10.1073/pnas.1010981108 Xia, W., Zhang, C., Zeng, X., Feng, Y., Weng, J., Lin, X., Zhu, J., et al. (2011). Autotrophic growth of nitrifying community in an agricultural soil. The ISME journal, 5(7), 1226–36. doi:10.1038/ismej.2011.5 Zhang, L.-M., Hu, H.-W., Shen, J.-P., & He, J.-Z. (2012). Ammonia-oxidizing archaea have more important role than ammonia-oxidizing bacteria in ammonia oxidation of strongly acidic soils. The ISME journal, 6(5), 1032–45. doi:10.1038/ismej.2011.168

A list of SIP studies related to food webs:

Bernard, L., Chapuis-Lardy, L., Razafimbelo, T., Razafindrakoto, M., Pablo, A.-L., Legname, E., Poulain, J., et al. (2012). Endogeic earthworms shape bacterial functional communities and affect organic matter mineralization in a tropical soil. The ISME journal, 6(1), 213–22. doi:10.1038/ismej.2011.87 Wüst, P. K., Horn, M. A., & Drake, H. L. (2011). Clostridiaceae and Enterobacteriaceae as active fermenters in earthworm gut content. The ISME journal, 5(1), 92–106. doi:10.1038/ismej.2010.99

A list of SIP studies focused on the rhizosphere

44

Bressan, M., Roncato, M.-A., Bellvert, F., Comte, G., Haichar, F. Z., Achouak, W., & Berge, O. (2009). Exogenous glucosinolate produced by Arabidopsis thaliana has an impact on microbes in the rhizosphere and plant roots. The ISME journal, 3(11), 1243–57. doi:10.1038/ismej.2009.68 Drigo, B., Pijl, A. S., Duyts, H., Kielak, A. M., Gamper, H. A., Houtekamer, M. J., Boschker, H. T. S., et al. (2010). Shifting carbon flow from roots into associated microbial communities in response to elevated atmospheric CO2. Proceedings of the National Academy of Sciences of the United States of America, 107(24), 10938–42. doi:10.1073/pnas.0912421107 Haichar, F. el Z., Marol, C., Berge, O., Rangel-Castro, J. I., Prosser, J. I., Balesdent, J., Heulin, T., et al. (2008). Plant host habitat and root exudates shape soil bacterial community structure. The ISME journal, 2(12), 1221–30. doi:10.1038/ismej.2008.80 Haichar, F. el Z., Roncato, M.-A., & Achouak, W. (2012). Stable isotope probing of bacterial community structure and gene expression in the rhizosphere of Arabidopsis thaliana. FEMS microbiology ecology, 81(2), 291–302. doi:10.1111/j.1574-6941.2012.01345.x Lu, Y., & Conrad, R. (2005). In situ stable isotope probing of methanogenic archaea in the rice rhizosphere. Science (New York, N.Y.), 309(5737), 1088–90. doi:10.1126/science.1113435 Lu, Y., Rosencrantz, D., Liesack, W., & Conrad, R. (2006). Structure and activity of bacterial community inhabiting rice roots and the rhizosphere. Environmental microbiology, 8(8), 1351–60. doi:10.1111/j.1462-2920.2006.01028.x Prosser, J. I., Rangel-Castro, J. I., & Killham, K. (2006). Studying plant-microbe interactions using stable isotope technologies. Current opinion in biotechnology, 17(1), 98–102. doi:10.1016/j.copbio.2006.01.001 Rangel-Castro, J. I., Killham, K., Ostle, N., Nicol, G. W., Anderson, I. C., Scrimgeour, C. M., Ineson, P., et al. (2005). Stable isotope probing analysis of the influence of liming on root exudate utilization by soil microorganisms. Environmental microbiology, 7(6), 828–38. doi:10.1111/j.1462-2920.2005.00756.x

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Alphabetical List of Published SIP Studies

Aanderud, Z. T., & Lennon, J. T. (2011). Validation of heavy-water stable isotope probing for the characterization of rapidly responding soil bacteria. Applied and environmental microbiology, 77(13), 4589–96. doi:10.1128/AEM.02735-10

Adair, K., & Schwartz, E. (2011). Stable isotope probing with 18O-water to investigate growth and mortality of ammonia oxidizing bacteria and archaea in soil. Methods in enzymology, 486, 155–69. doi:10.1016/B978-0-12-381294-0.00007-9

Addison, S. L., McDonald, I. R., & Lloyd-Jones, G. (2010). Stable isotope probing: technical considerations when resolving (15)N-labeled RNA in gradients. Journal of microbiological methods, 80(1), 70–5. doi:10.1016/j.mimet.2009.11.002

Akob, D. M., Kerkhof, L., Küsel, K., Watson, D. B., Palumbo, A. V, & Kostka, J. E. (2011). Linking specific heterotrophic bacterial populations to bioreduction of uranium and nitrate in contaminated subsurface sediments by using stable isotope probing. Applied and environmental microbiology, 77(22), 8197–200. doi:10.1128/AEM.05247-11

Andeer, P., Strand, S. E., & Stahl, D. A. (2012). High-sensitivity stable-isotope probing by a quantitative terminal restriction fragment length polymorphism protocol. Applied and environmental microbiology, 78(1), 163–9. doi:10.1128/AEM.05973-11

Anderson, R., Wylezich, C., Glaubitz, S., Labrenz, M., & Jürgens, K. (2013). Impact of protist grazing on a key bacterial group for biogeochemical cycling in Baltic Sea pelagic oxic/anoxic interfaces. Environmental microbiology. doi:10.1111/1462-2920.12078

Andreoni, V., & Gianfreda, L. (2007). Bioremediation and monitoring of aromatic-polluted habitats. Applied microbiology and biotechnology, 76(2), 287–308. doi:10.1007/s00253-007-1018-5

Antony, C. P., Kumaresan, D., Ferrando, L., Boden, R., Moussard, H., Scavino, A. F., Shouche, Y. S., et al. (2010). Active methylotrophs in the sediments of Lonar Lake, a saline and alkaline ecosystem formed by meteor impact. The ISME journal, 4(11), 1470–80. doi:10.1038/ismej.2010.70

Aslett, D., Haas, J., & Hyman, M. (2011). Identification of tertiary butyl alcohol (TBA)-utilizing organisms in BioGAC reactors using 13C-DNA stable isotope probing. Biodegradation, 22(5), 961–72. doi:10.1007/s10532-011-9455-3

46

Auclair, J., Lépine, F., Parent, S., & Villemur, R. (2010). Dissimilatory reduction of nitrate in seawater by a Methylophaga strain containing two highly divergent narG sequences. The ISME journal, 4(10), 1302–13. doi:10.1038/ismej.2010.47

Avrahami, S., Jia, Z., Neufeld, J. D., Murrell, J. C., Conrad, R., & Küsel, K. (2011). Active autotrophic ammonia-oxidizing bacteria in biofilm enrichments from simulated creek ecosystems at two ammonium concentrations respond to temperature manipulation. Applied and environmental microbiology, 77(20), 7329–38. doi:10.1128/AEM.05864-11

Barclay, A. R., Morrison, D. J., & Weaver, L. T. (2008). What is the role of the metabolic activity of the gut microbiota in inflammatory bowel disease? Probing for answers with stable isotopes. Journal of pediatric gastroenterology and nutrition, 46(5), 486–95. doi:10.1097/MPG.0b013e3181615b3a

Barret, M., Gagnon, N., Kalmokoff, M. L., Topp, E., Verastegui, Y., Brooks, S. P. J., Matias, F., et al. (2013). Identification of Methanoculleus spp. as Active Methanogens during Anoxic Incubations of Swine Manure Storage Tank Samples. Applied and environmental microbiology, 79(2), 424–33. doi:10.1128/AEM.02268-12

Bastida, F., Jechalke, S., Bombach, P., Franchini, A. G., Seifert, J., Von Bergen, M., Vogt, C., et al. (2011). Assimilation of benzene carbon through multiple trophic levels traced by different stable isotope probing methodologies. FEMS microbiology ecology, 77(2), 357–69. doi:10.1111/j.1574-6941.2011.01118.x

Bastida, F., Rosell, M., Franchini, A. G., Seifert, J., Finsterbusch, S., Jehmlich, N., Jechalke, S., et al. (2010). Elucidating MTBE degradation in a mixed consortium using a multidisciplinary approach. FEMS microbiology ecology, 73(2), 370–84. doi:10.1111/j.1574-6941.2010.00889.x

Baytshtok, V., Kim, S., Yu, R., Park, H., & Chandran, K. (2008). Molecular and biokinetic characterization of methylotrophic denitrification using nitrate and nitrite as terminal electron acceptors. Water science and technology : a journal of the International Association on Water Pollution Research, 58(2), 359–65. doi:10.2166/wst.2008.391

Baytshtok, V., Lu, H., Park, H., Kim, S., Yu, R., & Chandran, K. (2009). Impact of varying electron donors on the molecular microbial ecology and biokinetics of methylotrophic denitrifying bacteria. Biotechnology and bioengineering, 102(6), 1527–36. doi:10.1002/bit.22213

Beckmann, S., Lueders, T., Krüger, M., Von Netzer, F., Engelen, B., & Cypionka, H. (2011). Acetogens and acetoclastic methanosarcinales govern methane formation in abandoned coal mines. Applied and environmental microbiology, 77(11), 3749–56. doi:10.1128/AEM.02818-10

47

Bell, T. H., Yergeau, E., Martineau, C., Juck, D., Whyte, L. G., & Greer, C. W. (2011). Identification of nitrogen-incorporating bacteria in petroleum-contaminated arctic soils by using [15N]DNA-based stable isotope probing and pyrosequencing. Applied and environmental microbiology, 77(12), 4163–71. doi:10.1128/AEM.00172-11

Bengtson, P., Basiliko, N., Dumont, M. G., Hills, M., Murrell, J. C., Roy, R., & Grayston, S. J. (2009). Links between methanotroph community composition and CH oxidation in a pine forest soil. FEMS microbiology ecology, 70(3), 356–66. doi:10.1111/j.1574-6941.2009.00751.x

Bernard, L, Maron, P. A., Mougel, C., Nowak, V., Lévêque, J., Marol, C., Balesdent, J., et al. (2009). Contamination of soil by copper affects the dynamics, diversity, and activity of soil bacterial communities involved in wheat decomposition and carbon storage. Applied and environmental microbiology, 75(23), 7565–9. doi:10.1128/AEM.00616-09

Bernard, Laetitia, Chapuis-Lardy, L., Razafimbelo, T., Razafindrakoto, M., Pablo, A.-L., Legname, E., Poulain, J., et al. (2012). Endogeic earthworms shape bacterial functional communities and affect organic matter mineralization in a tropical soil. The ISME journal, 6(1), 213–22. doi:10.1038/ismej.2011.87

Binga, E. K., Lasken, R. S., & Neufeld, J. D. (2008). Something from (almost) nothing: the impact of multiple displacement amplification on microbial ecology. The ISME journal, 2(3), 233–41. doi:10.1038/ismej.2008.10

Blazewicz, S. J., & Schwartz, E. (2011). Dynamics of 18O incorporation from H₂ 18O into soil microbial DNA. Microbial ecology, 61(4), 911–6. doi:10.1007/s00248-011-9826-7

Bodelier, P. L. E., Bär-Gilissen, M.-J., Meima-Franke, M., & Hordijk, K. (2012). Structural and functional response of methane-consuming microbial communities to different flooding regimes in riparian soils. Ecology and evolution, 2(1), 106–27. doi:10.1002/ece3.34

Bombach, P., Chatzinotas, A., Neu, T. R., Kästner, M., Lueders, T., & Vogt, C. (2010). Enrichment and characterization of a sulfate-reducing toluene-degrading microbial consortium by combining in situ microcosms and stable isotope probing techniques. FEMS microbiology ecology, 71(2), 237–46. doi:10.1111/j.1574-6941.2009.00809.x

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