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Research protocol on stable isotope probing to elucidate the role of soil microorganisms in nutrient cycling and soil quality A A B non-labeled DNA newly grown labeled DNA Figure 4. Isopycnic centrifugation of DNA extracted from mixed conifer soil incubated with H218O (tubes A) or H216O (tube B) for 10 days. Dr. Egbert Schwartz Northern Arizona University Requisition 12750 (IAEA staff: Joseph Adu-Gyamfi) 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 nonlabeled DNA. i. ii. iii. 13Carbon 15Nitrogen 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 2 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 3 d. Characterization of microorganisms in the plants rhizosphere V. List of Published SIP studies 4 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 15N or 18O 13C, 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 nonlabeled 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. 5 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. 6 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 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. 7 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 while the other had incorporated 15N 14N atoms 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 appeared in 15N strand formed and DNA in which both strands contained subsequent generations thereby establishing the 15N 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 8 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 9 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 10 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 11 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. 12 15Nitrogen As previously mentioned replication in E.Coli. 15N 15N was used by Messelson and Stahl to study DNA 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 13 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 neutron relative 12C or 14N while 18O used in SIP contain one extra 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 labeled organic matter while in H218O. 18O- 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. 14 A A B B C C D D E E 18 Timecourse of O labeling of DNA in Ponderosa Pine soil. A = DNA 18 extracted from soil incubated with H2 O for 0 days, B = DNA 18 extracted from soil incubated with H2 O for 6 days, C= DNA 18 extracted from soil incubated with H2 O for 21 days, D = DNA 16 extracted from soil incubated with H2 O for 6 days, E = DNA 16 extracted from soil incubated with H2 O for 21 days. 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 nonlabeled 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. 15 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 16 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 17 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 18 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 19 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 Change in position of DNA bands as the samples are spun in an ultracentrifuge 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. 20 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 21 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 22 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 DNASIP. 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 23 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 nonlabeled 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. 24 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 25 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 26 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 27 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 28 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. 29 This 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. 30 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. 31 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 Harvesting of fractions from an ultracentrifuge tube 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 realtime PCR analysis. 24 Quantify the DNA in each fraction using a QUBIT from Invitrogen inc. 32 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 nonlabeled treatments contain the genomes of microorganisms that assimilated the added substrate. 0.3 not labeled DNA labeled DNA Fraction of total DNA 0.25 0.2 sample 2 sample 4 sample 6 sample 4W 0.15 0.1 0.05 0 1.6 1.7 1.8 density (g/ml) 1.9 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) 33 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 34 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. 35 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 36 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 37 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 38 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. 39 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-1,3,5trinitro-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 40 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 H218O. 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 H218O 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 41 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.14622920.2006.01182.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.14622920.2007.01414.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 42 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 43 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.15746941.2012.01345.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 44 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.0597311 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:10.1111/14622920.12078 Andreoni, V., & Gianfreda, L. (2007). Bioremediation and monitoring of aromaticpolluted 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 45 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 46 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/s00248-0119826-7 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.15746941.2009.00809.x Borodina, E., Cox, M. J., McDonald, I. R., & Murrell, J. C. (2005). Use of DNA-stable isotope probing and functional gene probes to investigate the diversity of methyl chloride-utilizing bacteria in soil. Environmental microbiology, 7(9), 1318–28. doi:10.1111/j.1462-5822.2005.00819.x Bressan, M., Roncato, M.-A., Bellvert, F., Comte, G., Haichar, F. Z., Achouak, W., & Berge, O. (2009). Exogenous glucosinolate produced by Arabidopsis thaliana has 47 an impact on microbes in the rhizosphere and plant roots. The ISME journal, 3(11), 1243–57. doi:10.1038/ismej.2009.68 Brinkmann, N., Martens, R., & Tebbe, C. C. (2008). Origin and diversity of metabolically active gut bacteria from laboratory-bred larvae of Manduca sexta (Sphingidae, Lepidoptera, Insecta). Applied and environmental microbiology, 74(23), 7189–96. doi:10.1128/AEM.01464-08 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 Burkhardt, E.-M., Akob, D. M., Bischoff, S., Sitte, J., Kostka, J. E., Banerjee, D., Scheinost, A. C., et al. (2010). Impact of biostimulated redox processes on metal dynamics in an iron-rich creek soil of a former uranium mining area. Environmental science & technology, 44(1), 177–83. doi:10.1021/es902038e 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 Chauhan, A., Cherrier, J., & Williams, H. N. (2009). Impact of sideways and bottom-up control factors on bacterial community succession over a tidal cycle. Proceedings of the National Academy of Sciences of the United States of America, 106(11), 4301– 6. doi:10.1073/pnas.0809671106 Chauhan, A., & Ogram, A. (2006a). Fatty acid-oxidizing consortia along a nutrient gradient in the Florida Everglades. Applied and environmental microbiology, 72(4), 2400–6. doi:10.1128/AEM.72.4.2400-2406.2006 Chauhan, A., & Ogram, A. (2006b). Phylogeny of acetate-utilizing microorganisms in soils along a nutrient gradient in the Florida Everglades. Applied and environmental microbiology, 72(10), 6837–40. doi:10.1128/AEM.01030-06 Chauhan, A., Pathak, A., & Ogram, A. (2012). Composition of methane-oxidizing bacterial communities as a function of nutrient loading in the Florida everglades. Microbial ecology, 64(3), 750–9. doi:10.1007/s00248-012-0058-2 Chen, Y., Dumont, M. G., Neufeld, J. D., Bodrossy, L., Stralis-Pavese, N., McNamara, N. P., Ostle, N., et al. (2008). Revealing the uncultivated majority: combining DNA stable-isotope probing, multiple displacement amplification and metagenomic 48 analyses of uncultivated Methylocystis in acidic peatlands. Environmental microbiology, 10(10), 2609–22. doi:10.1111/j.1462-2920.2008.01683.x Chen, Y., & Murrell, J. C. (2010). When metagenomics meets stable-isotope probing: progress and perspectives. Trends in microbiology, 18(4), 157–63. doi:10.1016/j.tim.2010.02.002 Chen, Y., Neufeld, J. D., Dumont, M. G., Friedrich, M. W., & Murrell, J. C. (2010). Metagenomic analysis of isotopically enriched DNA. Methods in molecular biology (Clifton, N.J.), 668, 67–75. doi:10.1007/978-1-60761-823-2_4 Chen, Y., Vohra, J., & Murrell, J. C. (2010). Applications of DNA-stable isotope probing in bioremediation studies. Methods in molecular biology (Clifton, N.J.), 599, 129– 39. doi:10.1007/978-1-60761-439-5_9 Chen, Y., Wu, L., Boden, R., Hillebrand, A., Kumaresan, D., Moussard, H., Baciu, M., et al. (2009). Life without light: microbial diversity and evidence of sulfur- and ammonium-based chemolithotrophy in Movile Cave. The ISME journal, 3(9), 1093–104. doi:10.1038/ismej.2009.57 Churchland, C., Weatherall, A., Briones, M. J. I., & Grayston, S. J. (2012). Stable-isotope labeling and probing of recent photosynthates into respired CO2, soil microbes and soil mesofauna using a xylem and phloem stem-injection technique on Sitka spruce (Picea sitchensis). Rapid communications in mass spectrometry : RCM, 26(21), 2493–501. doi:10.1002/rcm.6368 Cupples, A. M. (2011). The use of nucleic acid based stable isotope probing to identify the microorganisms responsible for anaerobic benzene and toluene biodegradation. Journal of microbiological methods, 85(2), 83–91. doi:10.1016/j.mimet.2011.02.011 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 Cébron, A., Bodrossy, L., Chen, Y., Singer, A. C., Thompson, I. P., Prosser, J. I., & Murrell, J. C. (2007). Identity of active methanotrophs in landfill cover soil as revealed by DNA-stable isotope probing. FEMS microbiology ecology, 62(1), 12– 23. doi:10.1111/j.1574-6941.2007.00368.x Cébron, A., Bodrossy, L., Stralis-Pavese, N., Singer, A. C., Thompson, I. P., Prosser, J. I., & Murrell, J. C. (2007). Nutrient amendments in soil DNA stable isotope probing experiments reduce the observed methanotroph diversity. Applied and environmental microbiology, 73(3), 798–807. doi:10.1128/AEM.01491-06 Cébron, A., Louvel, B., Faure, P., France-Lanord, C., Chen, Y., Murrell, J. C., & Leyval, C. (2011). Root exudates modify bacterial diversity of phenanthrene degraders in 49 PAH-polluted soil but not phenanthrene degradation rates. Environmental microbiology, 13(3), 722–36. doi:10.1111/j.1462-2920.2010.02376.x Date, Y., Nakanishi, Y., Fukuda, S., Kato, T., Tsuneda, S., Ohno, H., & Kikuchi, J. (2010). New monitoring approach for metabolic dynamics in microbial ecosystems using stable-isotope-labeling technologies. Journal of bioscience and bioengineering, 110(1), 87–93. doi:10.1016/j.jbiosc.2010.01.004 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 DeLorenzo, S., Bräuer, S. L., Edgmont, C. A., Herfort, L., Tebo, B. M., & Zuber, P. (2012). Ubiquitous dissolved inorganic carbon assimilation by marine bacteria in the Pacific Northwest coastal ocean as determined by stable isotope probing. PloS one, 7(10), e46695. doi:10.1371/journal.pone.0046695 DeRito, C. M., & Madsen, E. L. (2009). Stable isotope probing reveals Trichosporon yeast to be active in situ in soil phenol metabolism. The ISME journal, 3(4), 477– 85. doi:10.1038/ismej.2008.122 DeRito, C. M., Pumphrey, G. M., & Madsen, E. L. (2005). Use of field-based stable isotope probing to identify adapted populations and track carbon flow through a phenol-degrading soil microbial community. Applied and environmental microbiology, 71(12), 7858–65. doi:10.1128/AEM.71.12.7858-7865.2005 Dolinsek, J., Lagkouvardos, I., Wanek, W., Wagner, M., & Daims, H. (2013). Interactions of Nitrifying Bacteria and Heterotrophs: Identification of a Micavibriolike, Putative Predator of Nitrospira. Applied and environmental microbiology. doi:10.1128/AEM.03408-12 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 Dumont, M. G., & Murrell, J. C. (2005). Stable isotope probing - linking microbial identity to function. Nature reviews. Microbiology, 3(6), 499–504. doi:10.1038/nrmicro1162 Dumont, M. G., Pommerenke, B., Casper, P., & Conrad, R. (2011). DNA-, rRNA- and mRNA-based stable isotope probing of aerobic methanotrophs in lake sediment. Environmental microbiology, 13(5), 1153–67. doi:10.1111/j.14622920.2010.02415.x 50 Dumont, M. G., Radajewski, S. M., Miguez, C. B., McDonald, I. R., & Murrell, J. C. (2006). Identification of a complete methane monooxygenase operon from soil by combining stable isotope probing and metagenomic analysis. Environmental microbiology, 8(7), 1240–50. doi:10.1111/j.1462-2920.2006.01018.x Dunford, E. A., & Neufeld, J. D. (2010). DNA stable-isotope probing (DNA-SIP). Journal of visualized experiments : JoVE, (42). doi:10.3791/2027 Egert, M., De Graaf, A. A., Maathuis, A., De Waard, P., Plugge, C. M., Smidt, H., Deutz, N. E. P., et al. (2007). Identification of glucose-fermenting bacteria present in an in vitro model of the human intestine by RNA-stable isotope probing. FEMS microbiology ecology, 60(1), 126–35. doi:10.1111/j.1574-6941.2007.00281.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 Freitag, T. E., Chang, L., & Prosser, J. I. (2006). Changes in the community structure and activity of betaproteobacterial ammonia-oxidizing sediment bacteria along a freshwater-marine gradient. Environmental microbiology, 8(4), 684–96. doi:10.1111/j.1462-2920.2005.00947.x Frias-Lopez, J., Thompson, A., Waldbauer, J., & Chisholm, S. W. (2009). Use of stable isotope-labelled cells to identify active grazers of picocyanobacteria in ocean surface waters. Environmental microbiology, 11(2), 512–25. doi:10.1111/j.14622920.2008.01793.x Friedrich, M. W. (2006). Stable-isotope probing of DNA: insights into the function of uncultivated microorganisms from isotopically labeled metagenomes. Current opinion in biotechnology, 17(1), 59–66. doi:10.1016/j.copbio.2005.12.003 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.0227409 Gallagher, E., McGuinness, L., Phelps, C., Young, L. Y., & Kerkhof, L. J. (2005). 13Ccarrier DNA shortens the incubation time needed to detect benzoate-utilizing denitrifying bacteria by stable-isotope probing. Applied and environmental microbiology, 71(9), 5192–6. doi:10.1128/AEM.71.9.5192-5196.2005 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 51 Ginige, M. P., Bowyer, J. C., Foley, L., Keller, J., & Yuan, Z. (2009). A comparative study of methanol as a supplementary carbon source for enhancing denitrification in primary and secondary anoxic zones. Biodegradation, 20(2), 221–34. doi:10.1007/s10532-008-9215-1 Ginige, M. P., Keller, J., & Blackall, L. L. (2005). Investigation of an acetate-fed denitrifying microbial community by stable isotope probing, full-cycle rRNA analysis, and fluorescent in situ hybridization-microautoradiography. Applied and environmental microbiology, 71(12), 8683–91. doi:10.1128/AEM.71.12.86838691.2005 Glaubitz, S., Labrenz, M., Jost, G., & Jürgens, K. (2010). Diversity of active chemolithoautotrophic prokaryotes in the sulfidic zone of a Black Sea pelagic redoxcline as determined by rRNA-based stable isotope probing. FEMS microbiology ecology, 74(1), 32–41. doi:10.1111/j.1574-6941.2010.00944.x Glaubitz, S., Lueders, T., Abraham, W.-R., Jost, G., Jürgens, K., & Labrenz, M. (2009). 13C-isotope analyses reveal that chemolithoautotrophic Gamma- and Epsilonproteobacteria feed a microbial food web in a pelagic redoxcline of the central Baltic Sea. Environmental microbiology, 11(2), 326–37. doi:10.1111/j.14622920.2008.01770.x Graue, J., Engelen, B., & Cypionka, H. (2012). Degradation of cyanobacterial biomass in anoxic tidal-flat sediments: a microcosm study of metabolic processes and community changes. The ISME journal, 6(3), 660–9. doi:10.1038/ismej.2011.120 Graue, J., Kleindienst, S., Lueders, T., Cypionka, H., & Engelen, B. (2012). Identifying fermenting bacteria in anoxic tidal-flat sediments by a combination of microcalorimetry and ribosome-based stable-isotope probing. FEMS microbiology ecology, 81(1), 78–87. doi:10.1111/j.1574-6941.2011.01282.x Gupta, V., Smemo, K. A., Yavitt, J. B., & Basiliko, N. (2012). Active methanotrophs in two contrasting North American peatland ecosystems revealed using DNA-SIP. Microbial ecology, 63(2), 438–45. doi:10.1007/s00248-011-9902-z Gutierrez, T., Singleton, D. R., Aitken, M. D., & Semple, K. T. (2011). Stable isotope probing of an algal bloom to identify uncultivated members of the Rhodobacteraceae associated with low-molecular-weight polycyclic aromatic hydrocarbon degradation. Applied and environmental microbiology, 77(21), 7856– 60. doi:10.1128/AEM.06200-11 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.14622920.2006.01182.x 52 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 Hamberger, A., Horn, M. A., Dumont, M. G., Murrell, J. C., & Drake, H. L. (2008). Anaerobic consumers of monosaccharides in a moderately acidic fen. Applied and environmental microbiology, 74(10), 3112–20. doi:10.1128/AEM.00193-08 Han, B., Chen, Y., Abell, G., Jiang, H., Bodrossy, L., Zhao, J., Murrell, J. C., et al. (2009). Diversity and activity of methanotrophs in alkaline soil from a Chinese coal mine. FEMS microbiology ecology, 70(2), 40–51. doi:10.1111/j.1574-6941.2009.00707.x Hatamoto, M., Imachi, H., Yashiro, Y., Ohashi, A., & Harada, H. (2007). Diversity of anaerobic microorganisms involved in long-chain fatty acid degradation in methanogenic sludges as revealed by RNA-based stable isotope probing. Applied and environmental microbiology, 73(13), 4119–27. doi:10.1128/AEM.00362-07 He, R., Wooller, M. J., Pohlman, J. W., Catranis, C., Quensen, J., Tiedje, J. M., & Leigh, M. B. (2012). Identification of functionally active aerobic methanotrophs in sediments from an arctic lake using stable isotope probing. Environmental microbiology, 14(6), 1403–19. doi:10.1111/j.1462-2920.2012.02725.x He, R., Wooller, M. J., Pohlman, J. W., Quensen, J., Tiedje, J. M., & Leigh, M. B. (2012). Diversity of active aerobic methanotrophs along depth profiles of arctic and subarctic lake water column and sediments. The ISME journal, 6(10), 1937–48. doi:10.1038/ismej.2012.34 Henneberger, R., Chiri, E., Blees, J., Niemann, H., Lehmann, M. F., & Schroth, M. H. (2013). Field-scale labelling and activity quantification of methane-oxidizing bacteria in a landfill-cover soil. FEMS microbiology ecology, 83(2), 392–401. doi:10.1111/j.1574-6941.2012.01477.x Herrmann, S., Kleinsteuber, S., Chatzinotas, A., Kuppardt, S., Lueders, T., Richnow, H.H., & Vogt, C. (2010). Functional characterization of an anaerobic benzenedegrading enrichment culture by DNA stable isotope probing. Environmental microbiology, 12(2), 401–11. doi:10.1111/j.1462-2920.2009.02077.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 53 Hori, T., Noll, M., Igarashi, Y., Friedrich, M. W., & Conrad, R. (2007). Identification of acetate-assimilating microorganisms under methanogenic conditions in anoxic rice field soil by comparative stable isotope probing of RNA. Applied and environmental microbiology, 73(1), 101–9. doi:10.1128/AEM.01676-06 Huang, W. E., Ferguson, A., Singer, A. C., Lawson, K., Thompson, I. P., Kalin, R. M., Larkin, M. J., et al. (2009). Resolving genetic functions within microbial populations: in situ analyses using rRNA and mRNA stable isotope probing coupled with single-cell raman-fluorescence in situ hybridization. Applied and environmental microbiology, 75(1), 234–41. doi:10.1128/AEM.01861-08 Hunger, S., Schmidt, O., Hilgarth, M., Horn, M. A., Kolb, S., Conrad, R., & Drake, H. L. (2011). Competing formate- and carbon dioxide-utilizing prokaryotes in an anoxic methane-emitting fen soil. Applied and environmental microbiology, 77(11), 3773– 85. doi:10.1128/AEM.00282-11 Héry, M., Singer, A. C., Kumaresan, D., Bodrossy, L., Stralis-Pavese, N., Prosser, J. I., Thompson, I. P., et al. (2008). Effect of earthworms on the community structure of active methanotrophic bacteria in a landfill cover soil. The ISME journal, 2(1), 92– 104. doi:10.1038/ismej.2007.66 Ishii, S., Ohno, H., Tsuboi, M., Otsuka, S., & Senoo, K. (2011). Identification and isolation of active N2O reducers in rice paddy soil. The ISME journal, 5(12), 1936– 45. doi:10.1038/ismej.2011.69 Ito, T., Yoshiguchi, K., Ariesyady, H. D., & Okabe, S. (2011). Identification of a novel acetate-utilizing bacterium belonging to Synergistes group 4 in anaerobic digester sludge. The ISME journal, 5(12), 1844–56. doi:10.1038/ismej.2011.59 Ito, T., Yoshiguchi, K., Ariesyady, H. D., & Okabe, S. (2012). Identification and quantification of key microbial trophic groups of methanogenic glucose degradation in an anaerobic digester sludge. Bioresource technology, 123, 599–607. doi:10.1016/j.biortech.2012.07.108 Jakobs-Schönwandt, D., Mathies, H., Abraham, W.-R., Pritzkow, W., Stephan, I., & Noll, M. (2010). Biodegradation of a biocide (Cu-N-cyclohexyldiazenium dioxide) component of a wood preservative by a defined soil bacterial community. Applied and environmental microbiology, 76(24), 8076–83. doi:10.1128/AEM.01092-10 Jechalke, S., Franchini, A. G., Bastida, F., Bombach, P., Rosell, M., Seifert, J., Von Bergen, M., et al. (2013). Analysis of structure, function and activity of a benzene degrading microbial community. FEMS Microbiology Ecology, n/a–n/a. doi:10.1111/1574-6941.12090 Jensen, S., Neufeld, J. D., Birkeland, N.-K., Hovland, M., & Murrell, J. C. (2008). Methane assimilation and trophic interactions with marine Methylomicrobium in 54 deep-water coral reef sediment off the coast of Norway. FEMS microbiology ecology, 66(2), 320–30. doi:10.1111/j.1574-6941.2008.00575.x Jia, Z. (2011). [Principle and application of DNA-based stable isotope probing---a review]. Wei sheng wu xue bao = Acta microbiologica Sinica, 51(12), 1585–94. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/22379799 Jia, Z., & Conrad, R. (2009). Bacteria rather than Archaea dominate microbial ammonia oxidation in an agricultural soil. Environmental microbiology, 11(7), 1658–71. doi:10.1111/j.1462-2920.2009.01891.x Jones, M. D., Crandell, D. W., Singleton, D. R., & Aitken, M. D. (2011). Stable-isotope probing of the polycyclic aromatic hydrocarbon-degrading bacterial guild in a contaminated soil. Environmental microbiology, 13(10), 2623–32. doi:10.1111/j.1462-2920.2011.02501.x Jones, M. D., Singleton, D. R., Carstensen, D. P., Powell, S. N., Swanson, J. S., Pfaender, F. K., & Aitken, M. D. (2008). Effect of incubation conditions on the enrichment of pyrene-degrading bacteria identified by stable-isotope probing in an aged, PAHcontaminated soil. Microbial ecology, 56(2), 341–9. doi:10.1007/s00248-0079352-9 Jones, M. D., Singleton, D. R., Sun, W., & Aitken, M. D. (2011). Multiple DNA extractions coupled with stable-isotope probing of anthracene-degrading bacteria in contaminated soil. Applied and environmental microbiology, 77(9), 2984–91. doi:10.1128/AEM.01942-10 Kalyuzhnaya, M. G., Lapidus, A., Ivanova, N., Copeland, A. C., McHardy, A. C., Szeto, E., Salamov, A., et al. (2008). High-resolution metagenomics targets specific functional types in complex microbial communities. Nature biotechnology, 26(9), 1029–34. doi:10.1038/nbt.1488 Kasai, Y., Takahata, Y., Manefield, M., & Watanabe, K. (2006). RNA-based stable isotope probing and isolation of anaerobic benzene-degrading bacteria from gasoline-contaminated groundwater. Applied and environmental microbiology, 72(5), 3586–92. doi:10.1128/AEM.72.5.3586-3592.2006 Kellermann, M. Y., Wegener, G., Elvert, M., Yoshinaga, M. Y., Lin, Y.-S., Holler, T., Mollar, X. P., et al. (2012). Autotrophy as a predominant mode of carbon fixation in anaerobic methane-oxidizing microbial communities. Proceedings of the National Academy of Sciences of the United States of America, 109(47), 19321–6. doi:10.1073/pnas.1208795109 Kerkhof, L. J., Williams, K. H., Long, P. E., & McGuinness, L. R. (2011). Phase Preference by Active, Acetate-Utilizing Bacteria at the Rifle, CO Integrated Field Research Challenge Site. Environmental science & technology. doi:10.1021/es102893r 55 Kittelmann, S., & Friedrich, M. W. (2008a). Novel uncultured Chloroflexi dechlorinate perchloroethene to trans-dichloroethene in tidal flat sediments. Environmental microbiology, 10(6), 1557–70. doi:10.1111/j.1462-2920.2008.01571.x Kittelmann, S., & Friedrich, M. W. (2008b). Identification of novel perchloroethenerespiring microorganisms in anoxic river sediment by RNA-based stable isotope probing. Environmental microbiology, 10(1), 31–46. doi:10.1111/j.14622920.2007.01427.x Kovatcheva-Datchary, P., Egert, M., Maathuis, A., Rajilić-Stojanović, M., De Graaf, A. A., Smidt, H., De Vos, W. M., et al. (2009). Linking phylogenetic identities of bacteria to starch fermentation in an in vitro model of the large intestine by RNAbased stable isotope probing. Environmental microbiology, 11(4), 914–26. doi:10.1111/j.1462-2920.2008.01815.x Kristiansen, A., Lindholst, S., Feilberg, A., Nielsen, P. H., Neufeld, J. D., & Nielsen, J. L. (2011). Butyric acid- and dimethyl disulfide-assimilating microorganisms in a biofilter treating air emissions from a livestock facility. Applied and environmental microbiology, 77(24), 8595–604. doi:10.1128/AEM.06175-11 Kunapuli, U., Lueders, T., & Meckenstock, R. U. (2007). The use of stable isotope probing to identify key iron-reducing microorganisms involved in anaerobic benzene degradation. The ISME journal, 1(7), 643–53. doi:10.1038/ismej.2007.73 Kuppardt, S., Chatzinotas, A., & Kästner, M. (2010). Development of a fatty acid and RNA stable isotope probing-based method for tracking protist grazing on bacteria in wastewater. Applied and environmental microbiology, 76(24), 8222–30. doi:10.1128/AEM.01632-10 Langenheder, S., & Prosser, J. I. (2008). Resource availability influences the diversity of a functional group of heterotrophic soil bacteria. Environmental microbiology, 10(9), 2245–56. doi:10.1111/j.1462-2920.2008.01647.x Lear, G., Song, B., Gault, A. G., Polya, D. A., & Lloyd, J. R. (2007). Molecular analysis of arsenate-reducing bacteria within Cambodian sediments following amendment with acetate. Applied and environmental microbiology, 73(4), 1041–8. doi:10.1128/AEM.01654-06 Lee, T. K., Lee, J., Sul, W. J., Iwai, S., Chai, B., Tiedje, J. M., & Park, J. (2011). Novel biphenyl-oxidizing bacteria and dioxygenase genes from a korean tidal mudflat. Applied and environmental microbiology, 77(11), 3888–91. doi:10.1128/AEM.00023-11 Leigh, M. B., Pellizari, V. H., Uhlík, O., Sutka, R., Rodrigues, J., Ostrom, N. E., Zhou, J., et al. (2007). Biphenyl-utilizing bacteria and their functional genes in a pine root zone contaminated with polychlorinated biphenyls (PCBs). The ISME journal, 1(2), 134–48. doi:10.1038/ismej.2007.26 56 Li, T., Mazéas, L., Sghir, A., Leblon, G., & Bouchez, T. (2009). Insights into networks of functional microbes catalysing methanization of cellulose under mesophilic conditions. Environmental microbiology, 11(4), 889–904. doi:10.1111/j.14622920.2008.01810.x Lin, Y.-S., Lipp, J. S., Elvert, M., Holler, T., & Hinrichs, K.-U. (2012). Assessing production of the ubiquitous archaeal diglycosyl tetraether lipids in marine subsurface sediment using intramolecular stable isotope probing. Environmental microbiology. doi:10.1111/j.1462-2920.2012.02888.x Liou, J. S.-C., Derito, C. M., & Madsen, E. L. (2008). Field-based and laboratory stable isotope probing surveys of the identities of both aerobic and anaerobic benzenemetabolizing microorganisms in freshwater sediment. Environmental microbiology, 10(8), 1964–77. doi:10.1111/j.1462-2920.2008.01612.x Liu, F., & Conrad, R. (2010). Thermoanaerobacteriaceae oxidize acetate in methanogenic rice field soil at 50°C. Environmental microbiology, 12(8), 2341–54. doi:10.1111/j.1462-2920.2010.02289.x Liu, F., & Conrad, R. (2011). Chemolithotrophic acetogenic H2/CO2 utilization in Italian rice field soil. The ISME journal, 5(9), 1526–39. doi:10.1038/ismej.2011.17 Liu, P., Qiu, Q., & Lu, Y. (2011). Syntrophomonadaceae-affiliated species as active butyrate-utilizing syntrophs in paddy field soil. Applied and environmental microbiology, 77(11), 3884–7. doi:10.1128/AEM.00190-11 Liu, W., Wei, X., Yuan, J., & Huang, L. (2011). [Applications and perspectives of DNA stable-isotope probing in metagenomics: a review]. Sheng wu gong cheng xue bao = Chinese journal of biotechnology, 27(4), 539–45. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/21847987 Liu, Y.-J., Liu, S.-J., Drake, H. L., & Horn, M. A. (2011). Alphaproteobacteria dominate active 2-methyl-4-chlorophenoxyacetic acid herbicide degraders in agricultural soil and drilosphere. Environmental microbiology, 13(4), 991–1009. doi:10.1111/j.1462-2920.2010.02405.x Lolas, I. B., Chen, X., Bester, K., & Nielsen, J. L. (2012). Identification of triclosandegrading bacteria using stable isotope probing, fluorescence in situ hybridization and microautoradiography. Microbiology (Reading, England), 158(Pt 11), 2796– 804. doi:10.1099/mic.0.061077-0 Longnecker, K., Da Costa, A., Bhatia, M., & Kujawinski, E. B. (2009). Effect of carbon addition and predation on acetate-assimilating bacterial cells in groundwater. FEMS microbiology ecology, 70(3), 456–70. doi:10.1111/j.1574-6941.2009.00767.x Lu, H., & Chandran, K. (2010). Diagnosis and quantification of glycerol assimilating denitrifying bacteria in an integrated fixed-film activated sludge reactor via 13C 57 DNA stable-isotope probing. Environmental science & technology, 44(23), 8943–9. doi:10.1021/es102124f Lu, L., & Jia, Z. (2012). Urease gene-containing Archaea dominate autotrophic ammonia oxidation in two acid soils. Environmental microbiology. doi:10.1111/14622920.12071 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 Lueders, T., Kindler, R., Miltner, A., Friedrich, M. W., & Kaestner, M. (2006). Identification of bacterial micropredators distinctively active in a soil microbial food web. Applied and environmental microbiology, 72(8), 5342–8. doi:10.1128/AEM.00400-06 Luo, C., Xie, S., Sun, W., Li, X., & Cupples, A. M. (2009). Identification of a novel toluene-degrading bacterium from the candidate phylum TM7, as determined by DNA stable isotope probing. Applied and environmental microbiology, 75(13), 4644–7. doi:10.1128/AEM.00283-09 Madsen, E. L. (2006). The use of stable isotope probing techniques in bioreactor and field studies on bioremediation. Current Opinion in Biotechnology, 17(1), 92–97. doi:10.1016/j.copbio.2005.12.004 Mahmood, S., Paton, G. I., & Prosser, J. I. (2005). Cultivation-independent in situ molecular analysis of bacteria involved in degradation of pentachlorophenol in soil. Environmental microbiology, 7(9), 1349–60. doi:10.1111/j.14622920.2005.00822.x Manefield, M., Griffiths, R., McNamara, N. P., Sleep, D., Ostle, N., & Whiteley, A. (2007). Insights into the fate of a 13C labelled phenol pulse for stable isotope probing (SIP) experiments. Journal of microbiological methods, 69(2), 340–4. doi:10.1016/j.mimet.2007.01.019 Manefield, M., Whiteley, A. S., Griffiths, R. I., & Bailey, M. J. (2002). RNA stable isotope probing, a novel means of linking microbial community function to phylogeny. Applied and environmental microbiology, 68(11), 5367–73. Retrieved from http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=129944&tool=pmcent rez&rendertype=abstract Manefield, M., Whiteley, A. S., Ostle, N., Ineson, P., & Bailey, M. J. (2002). Technical considerations for RNA-based stable isotope probing: an approach to associating 58 microbial diversity with microbial community function. Rapid communications in mass spectrometry : RCM, 16(23), 2179–83. doi:10.1002/rcm.782 Martin, F., Malagnoux, L., Violet, F., Jakoncic, J., & Jouanneau, Y. (2012). Diversity and catalytic potential of PAH-specific ring-hydroxylating dioxygenases from a hydrocarbon-contaminated soil. Applied microbiology and biotechnology. doi:10.1007/s00253-012-4335-2 Martin, F., Torelli, S., Le Paslier, D., Barbance, A., Martin-Laurent, F., Bru, D., Geremia, R., et al. (2012). Betaproteobacteria dominance and diversity shifts in the bacterial community of a PAH-contaminated soil exposed to phenanthrene. Environmental pollution (Barking, Essex : 1987), 162, 345–53. doi:10.1016/j.envpol.2011.11.032 Martineau, C., Whyte, L. G., & Greer, C. W. (2008). Development of a SYBR safe technique for the sensitive detection of DNA in cesium chloride density gradients for stable isotope probing assays. Journal of microbiological methods, 73(2), 199– 202. doi:10.1016/j.mimet.2008.01.016 Martineau, C., Whyte, L. G., & Greer, C. W. (2010). Stable isotope probing analysis of the diversity and activity of methanotrophic bacteria in soils from the Canadian high Arctic. Applied and environmental microbiology, 76(17), 5773–84. doi:10.1128/AEM.03094-09 Martínez-Lavanchy, P. M., Dohrmann, A. B., Imfeld, G., Trescher, K., Tebbe, C. C., Richnow, H.-H., & Nijenhuis, I. (2011). Detection of monochlorobenzene metabolizing bacteria under anoxic conditions by DNA-stable isotope probing. Biodegradation, 22(5), 973–82. doi:10.1007/s10532-011-9456-2 Maxfield, P. J., Dildar, N., Hornibrook, E. R. C., Stott, A. W., & Evershed, R. P. (2012). Stable isotope switching (SIS): a new stable isotope probing (SIP) approach to determine carbon flow in the soil food web and dynamics in organic matter pools. Rapid communications in mass spectrometry : RCM, 26(8), 997–1004. doi:10.1002/rcm.6172 Maxfield, P. J., Hornibrook, E. R. C., & Evershed, R. P. (2008). Acute impact of agriculture on high-affinity methanotrophic bacterial populations. Environmental microbiology, 10(7), 1917–24. doi:10.1111/j.1462-2920.2008.01587.x Mayali, X., Weber, P. K., Brodie, E. L., Mabery, S., Hoeprich, P. D., & Pett-Ridge, J. (2012). High-throughput isotopic analysis of RNA microarrays to quantify microbial resource use. The ISME journal, 6(6), 1210–21. doi:10.1038/ismej.2011.175 Mayumi, D., Yoshimoto, T., Uchiyama, H., Nomura, N., & Nakajima-Kambe, T. (2010). Seasonal change in methanotrophic diversity and populations in a rice field soil assessed by DNA-stable isotope probing and quantitative real-time PCR. Microbes 59 and environments / JSME, 25(3), 156–63. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/21576868 McLean, J. S., Fansler, S. J., Majors, P. D., McAteer, K., Allen, L. Z., Shirtliff, M. E., Lux, R., et al. (2012). Identifying low pH active and lactate-utilizing taxa within oral microbiome communities from healthy children using stable isotope probing techniques. PloS one, 7(3), e32219. doi:10.1371/journal.pone.0032219 Meyer, R. L., Saunders, A. M., & Blackall, L. L. (2006). Putative glycogen-accumulating organisms belonging to the Alphaproteobacteria identified through rRNA-based stable isotope probing. Microbiology (Reading, England), 152(Pt 2), 419–29. doi:10.1099/mic.0.28445-0 Michinaka, A., & Fujii, T. (2012). Efficient and direct identification of fructose fermenting and non-fermenting bacteria from calf gut microbiota using stable isotope probing and modified T-RFLP. The Journal of general and applied microbiology, 58(4), 297–307. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/22990490 Miyatake, T., MacGregor, B. J., & Boschker, H. T. S. (2009). Linking microbial community function to phylogeny of sulfate-reducing Deltaproteobacteria in marine sediments by combining stable isotope probing with magnetic-bead capture hybridization of 16S rRNA. Applied and environmental microbiology, 75(15), 4927–35. doi:10.1128/AEM.00652-09 Miyatake, T., Macgregor, B. J., & Boschker, H. T. S. (2013). Depth-related differences in organic substrate utilization by major microbial groups in intertidal marine sediment. Applied and environmental microbiology, 79(1), 389–92. doi:10.1128/AEM.02027-12 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 Monard, Cécile, Vandenkoornhuyse, P., Le Bot, B., & Binet, F. (2011). Relationship between bacterial diversity and function under biotic control: the soil pesticide degraders as a case study. The ISME journal, 5(6), 1048–56. doi:10.1038/ismej.2010.194 Moreno, A. M., Matz, C., Kjelleberg, S., & Manefield, M. (2010). Identification of ciliate grazers of autotrophic bacteria in ammonia-oxidizing activated sludge by RNA stable isotope probing. Applied and environmental microbiology, 76(7), 2203–11. doi:10.1128/AEM.02777-09 Morris, S. A., Radajewski, S., Willison, T. W., & Murrell, J. C. (2002). Identification of the functionally active methanotroph population in a peat soil microcosm by stableisotope probing. Applied and environmental microbiology, 68(3), 1446–53. 60 Retrieved from http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=123758&tool=pmcent rez&rendertype=abstract 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.14622920.2007.01414.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 Nelson, C. E., & Carlson, C. A. (2012). Tracking differential incorporation of dissolved organic carbon types among diverse lineages of Sargasso Sea bacterioplankton. Environmental microbiology, 14(6), 1500–16. doi:10.1111/j.14622920.2012.02738.x Nercessian, O., Noyes, E., Kalyuzhnaya, M. G., Lidstrom, M. E., & Chistoserdova, L. (2005). Bacterial populations active in metabolism of C1 compounds in the sediment of Lake Washington, a freshwater lake. Applied and environmental microbiology, 71(11), 6885–99. doi:10.1128/AEM.71.11.6885-6899.2005 Neufeld, J. D., Boden, R., Moussard, H., Schäfer, H., & Murrell, J. C. (2008). Substratespecific clades of active marine methylotrophs associated with a phytoplankton bloom in a temperate coastal environment. Applied and environmental microbiology, 74(23), 7321–8. doi:10.1128/AEM.01266-08 Neufeld, J. D., Chen, Y., Dumont, M. G., & Murrell, J. C. (2008). Marine methylotrophs revealed by stable-isotope probing, multiple displacement amplification and metagenomics. Environmental microbiology, 10(6), 1526–35. doi:10.1111/j.14622920.2008.01568.x Neufeld, J. D., Dumont, M. G., Vohra, J., & Murrell, J. C. (2007). Methodological considerations for the use of stable isotope probing in microbial ecology. Microbial ecology, 53(3), 435–42. doi:10.1007/s00248-006-9125-x Neufeld, J. D., Schäfer, H., Cox, M. J., Boden, R., McDonald, I. R., & Murrell, J. C. (2007). Stable-isotope probing implicates Methylophaga spp and novel Gammaproteobacteria in marine methanol and methylamine metabolism. The ISME journal, 1(6), 480–91. doi:10.1038/ismej.2007.65 Neufeld, J. D., Vohra, J., Dumont, M. G., Lueders, T., Manefield, M., Friedrich, M. W., & Murrell, J. C. (2007). DNA stable-isotope probing. Nature protocols, 2(4), 860–6. doi:10.1038/nprot.2007.109 61 Neufeld, J. D., Wagner, M., & Murrell, J. C. (2007). Who eats what, where and when? Isotope-labelling experiments are coming of age. The ISME journal, 1(2), 103–10. doi:10.1038/ismej.2007.30 Nicholson, W. L., Fedenko, J., & Schuerger, A. C. (2009). Carbon-13 (13C) labeling of Bacillus subtilis vegetative cells and spores: suitability for DNA stable isotope probing (DNA-SIP) of spores in soils. Current microbiology, 59(1), 9–14. doi:10.1007/s00284-009-9387-x Nielsen, J. L., Nguyen, H., Meyer, R. L., & Nielsen, P. H. (2012). Identification of glucose-fermenting bacteria in a full-scale enhanced biological phosphorus removal plant by stable isotope probing. Microbiology (Reading, England), 158(Pt 7), 1818– 25. doi:10.1099/mic.0.058818-0 Noll, M., Frenzel, P., & Conrad, R. (2008). Selective stimulation of type I methanotrophs in a rice paddy soil by urea fertilization revealed by RNA-based stable isotope probing. FEMS microbiology ecology, 65(1), 125–32. doi:10.1111/j.15746941.2008.00497.x Oka, A. R., Phelps, C. D., McGuinness, L. M., Mumford, A., Young, L. Y., & Kerkhof, L. J. (2008). Identification of critical members in a sulfidogenic benzene-degrading consortium by DNA stable isotope probing. Applied and environmental microbiology, 74(20), 6476–80. doi:10.1128/AEM.01082-08 Osaka, T., Ebie, Y., Tsuneda, S., & Inamori, Y. (2008). Identification of the bacterial community involved in methane-dependent denitrification in activated sludge using DNA stable-isotope probing. FEMS microbiology ecology, 64(3), 494–506. doi:10.1111/j.1574-6941.2008.00473.x Osaka, T., Yoshie, S., Tsuneda, S., Hirata, A., Iwami, N., & Inamori, Y. (2006). Identification of acetate- or methanol-assimilating bacteria under nitrate-reducing conditions by stable-isotope probing. Microbial ecology, 52(2), 253–66. doi:10.1007/s00248-006-9071-7 Pawelczyk, S., Bumann, D., & Abraham, W.-R. (2011). Kinetics of carbon sharing in a bacterial consortium revealed by combining stable isotope probing with fluorescence-activated cell sorting. Journal of applied microbiology. doi:10.1111/j.1365-2672.2011.04964.x Pester, M., Bittner, N., Deevong, P., Wagner, M., & Loy, A. (2010). A “rare biosphere” microorganism contributes to sulfate reduction in a peatland. The ISME journal, 4(12), 1591–602. doi:10.1038/ismej.2010.75 Pilloni, G., Von Netzer, F., Engel, M., & Lueders, T. (2011). Electron acceptor-dependent identification of key anaerobic toluene degraders at a tar-oil-contaminated aquifer by Pyro-SIP. FEMS microbiology ecology, 78(1), 165–75. doi:10.1111/j.15746941.2011.01083.x 62 Powell, S. N., Singleton, D. R., & Aitken, M. D. (2008). Effects of enrichment with salicylate on bacterial selection and PAH mineralization in a microbial community from a bioreactor treating contaminated soil. Environmental science & technology, 42(11), 4099–105. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/18589972 Pratscher, J., Dumont, M. G., & Conrad, R. (2011a). Assimilation of acetate by the putative atmospheric methane oxidizers belonging to the USCα clade. Environmental microbiology, 13(10), 2692–701. doi:10.1111/j.14622920.2011.02537.x Pratscher, J., Dumont, M. G., & Conrad, R. (2011b). 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 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 Pumphrey, G. M., & Madsen, E. L. (2008). Field-based stable isotope probing reveals the identities of benzoic acid-metabolizing microorganisms and their in situ growth in agricultural soil. Applied and environmental microbiology, 74(13), 4111–8. doi:10.1128/AEM.00464-08 Pumphrey, G. M., Ranchou-Peyruse, A., & Spain, J. C. (2011). Cultivation-independent detection of autotrophic hydrogen-oxidizing bacteria by DNA stable-isotope probing. Applied and environmental microbiology, 77(14), 4931–8. doi:10.1128/AEM.00285-11 Qiu, Q., Noll, M., Abraham, W.-R., Lu, Y., & Conrad, R. (2008). Applying stable isotope probing of phospholipid fatty acids and rRNA in a Chinese rice field to study activity and composition of the methanotrophic bacterial communities in situ. The ISME journal, 2(6), 602–14. doi:10.1038/ismej.2008.34 Radajewski, S, Ineson, P., Parekh, N. R., & Murrell, J. C. (2000). Stable-isotope probing as a tool in microbial ecology. Nature, 403(6770), 646–9. doi:10.1038/35001054 Radajewski, S, & Murrell, J. C. (2002). Stable isotope probing for detection of methanotrophs after enrichment with 13CH4. Methods in molecular biology (Clifton, N.J.), 179, 149–57. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/11692859 Radajewski, Stefan, Webster, G., Reay, D. S., Morris, S. A., Ineson, P., Nedwell, D. B., Prosser, J. I., et al. (2002). Identification of active methylotroph populations in an acidic forest soil by stable-isotope probing. Microbiology (Reading, England), 63 148(Pt 8), 2331–42. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/12177327 Rahman, M. T., Crombie, A., Moussard, H., Chen, Y., & Murrell, J. C. (2011). Acetate repression of methane oxidation by supplemental Methylocella silvestris in a peat soil microcosm. Applied and environmental microbiology, 77(12), 4234–6. doi:10.1128/AEM.02902-10 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 Rasche, F., Lueders, T., Schloter, M., Schaefer, S., Buegger, F., Gattinger, A., HoodNowotny, R. C., et al. (2009). DNA-based stable isotope probing enables the identification of active bacterial endophytes in potatoes. The New phytologist, 181(4), 802–7. doi:10.1111/j.1469-8137.2008.02744.x Redmond, M. C., & Valentine, D. L. (2012). Natural gas and temperature structured a microbial community response to the Deepwater Horizon oil spill. Proceedings of the National Academy of Sciences of the United States of America, 109(50), 20292–7. doi:10.1073/pnas.1108756108 Redmond, M. C., Valentine, D. L., & Sessions, A. L. (2010). Identification of novel methane-, ethane-, and propane-oxidizing bacteria at marine hydrocarbon seeps by stable isotope probing. Applied and environmental microbiology, 76(19), 6412–22. doi:10.1128/AEM.00271-10 Reichardt, N., Barclay, A. R., Weaver, L. T., & Morrison, D. J. (2011). Use of stable isotopes to measure the metabolic activity of the human intestinal microbiota. Applied and environmental microbiology, 77(22), 8009–14. doi:10.1128/AEM.05573-11 Roh, H., Yu, C.-P., Fuller, M. E., & Chu, K.-H. (2009). Identification of hexahydro-1,3,5trinitro-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 Rui, J., Qiu, Q., & Lu, Y. (2011). Syntrophic acetate oxidation under thermophilic methanogenic condition in Chinese paddy field soil. FEMS microbiology ecology, 77(2), 264–73. doi:10.1111/j.1574-6941.2011.01104.x Saidi-Mehrabad, A., He, Z., Tamas, I., Sharp, C. E., Brady, A. L., Rochman, F. F., Bodrossy, L., et al. (2012). Methanotrophic bacteria in oilsands tailings ponds of northern Alberta. The ISME journal. doi:10.1038/ismej.2012.163 64 Saito, T., Ishii, S., Otsuka, S., Nishiyama, M., & Senoo, K. (2008). Identification of novel betaproteobacteria in a succinate-assimilating population in denitrifying rice paddy soil by using stable isotope probing. Microbes and environments / JSME, 23(3), 192–200. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/21558708 Sakai, N., Kurisu, F., Yagi, O., Nakajima, F., & Yamamoto, K. (2009). Identification of putative benzene-degrading bacteria in methanogenic enrichment cultures. Journal of bioscience and bioengineering, 108(6), 501–7. doi:10.1016/j.jbiosc.2009.06.005 Saleh-Lakha, S., Miller, M., Campbell, R. G., Schneider, K., Elahimanesh, P., Hart, M. M., & Trevors, J. T. (2005). Microbial gene expression in soil: methods, applications and challenges. Journal of microbiological methods, 63(1), 1–19. doi:10.1016/j.mimet.2005.03.007 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 Schwartz, E. (2007). Characterization of growing microorganisms in soil by stable isotope probing with H218O. 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 Schwarz, J. I. K., Lueders, T., Eckert, W., & Conrad, R. (2007). Identification of acetateutilizing Bacteria and Archaea in methanogenic profundal sediments of Lake Kinneret (Israel) by stable isotope probing of rRNA. Environmental microbiology, 9(1), 223–37. doi:10.1111/j.1462-2920.2006.01133.x Sharp, C. E., Stott, M. B., & Dunfield, P. F. (2012). Detection of autotrophic verrucomicrobial methanotrophs in a geothermal environment using stable isotope probing. Frontiers in microbiology, 3, 303. doi:10.3389/fmicb.2012.00303 Shen, J.-P., Zhang, L.-M., Di, H. J., & He, J.-Z. (2012). A review of ammonia-oxidizing bacteria and archaea in Chinese soils. Frontiers in microbiology, 3, 296. doi:10.3389/fmicb.2012.00296 Shrestha, M., Abraham, W.-R., Shrestha, P. M., Noll, M., & Conrad, R. (2008). Activity and composition of methanotrophic bacterial communities in planted rice soil studied by flux measurements, analyses of pmoA gene and stable isotope probing of phospholipid fatty acids. Environmental microbiology, 10(2), 400–12. doi:10.1111/j.1462-2920.2007.01462.x 65 Singh, B. K., & Tate, K. (2007). Biochemical and molecular characterization of methanotrophs in soil from a pristine New Zealand beech forest. FEMS microbiology letters, 275(1), 89–97. doi:10.1111/j.1574-6968.2007.00885.x Singh, B. K., Tate, K. R., Kolipaka, G., Hedley, C. B., Macdonald, C. A., Millard, P., & Murrell, J. C. (2007). Effect of afforestation and reforestation of pastures on the activity and population dynamics of methanotrophic bacteria. Applied and environmental microbiology, 73(16), 5153–61. doi:10.1128/AEM.00620-07 Singleton, D. R., Hunt, M., Powell, S. N., Frontera-Suau, R., & Aitken, M. D. (2007). Stable-isotope probing with multiple growth substrates to determine substrate specificity of uncultivated bacteria. Journal of microbiological methods, 69(1), 180–7. doi:10.1016/j.mimet.2006.12.019 Singleton, D. R., Jones, M. D., Richardson, S. D., & Aitken, M. D. (2012). Pyrosequence analyses of bacterial communities during simulated in situ bioremediation of polycyclic aromatic hydrocarbon-contaminated soil. Applied microbiology and biotechnology. doi:10.1007/s00253-012-4531-0 Singleton, D. R., Ramirez, L. G., & Aitken, M. D. (2009). Characterization of a polycyclic aromatic hydrocarbon degradation gene cluster in a phenanthrene-degrading Acidovorax strain. Applied and environmental microbiology, 75(9), 2613–20. doi:10.1128/AEM.01955-08 Singleton, D. R., Richardson, S. D., & Aitken, M. D. (2011). Pyrosequence analysis of bacterial communities in aerobic bioreactors treating polycyclic aromatic hydrocarbon-contaminated soil. Biodegradation, 22(6), 1061–73. doi:10.1007/s10532-011-9463-3 Singleton, D. R., Sangaiah, R., Gold, A., Ball, L. M., & Aitken, M. D. (2006). Identification and quantification of uncultivated Proteobacteria associated with pyrene degradation in a bioreactor treating PAH-contaminated soil. Environmental microbiology, 8(10), 1736–45. doi:10.1111/j.1462-2920.2006.01112.x Sueoka, K., Satoh, H., Onuki, M., & Mino, T. (2009). Microorganisms involved in anaerobic phenol degradation in the treatment of synthetic coke-oven wastewater detected by RNA stable-isotope probing. FEMS microbiology letters, 291(2), 169– 74. doi:10.1111/j.1574-6968.2008.01448.x Sul, W. J., Park, J., Quensen, J. F., Rodrigues, J. L. M., Seliger, L., Tsoi, T. V, Zylstra, G. J., et al. (2009). DNA-stable isotope probing integrated with metagenomics for retrieval of biphenyl dioxygenase genes from polychlorinated biphenylcontaminated river sediment. Applied and environmental microbiology, 75(17), 5501–6. doi:10.1128/AEM.00121-09 66 Sun, W., & Cupples, A. M. (2012). Diversity of five anaerobic toluene-degrading microbial communities investigated using stable isotope probing. Applied and environmental microbiology, 78(4), 972–80. doi:10.1128/AEM.06770-11 Sun, W., Sun, X., & Cupples, A. M. (2012). Anaerobic methyl tert-butyl ether-degrading microorganisms identified in wastewater treatment plant samples by stable isotope probing. Applied and environmental microbiology, 78(8), 2973–80. doi:10.1128/AEM.07253-11 Sun, W., Xie, S., Luo, C., & Cupples, A. M. (2010). Direct link between toluene degradation in contaminated-site microcosms and a Polaromonas strain. Applied and environmental microbiology, 76(3), 956–9. doi:10.1128/AEM.01364-09 Tillmann, S., Strömpl, C., Timmis, K. N., & Abraham, W.-R. (2005). Stable isotope probing reveals the dominant role of Burkholderia species in aerobic degradation of PCBs. FEMS microbiology ecology, 52(2), 207–17. doi:10.1016/j.femsec.2004.11.014 Tourna, M., Freitag, T. E., & Prosser, J. I. (2010). Stable isotope probing analysis of interactions between ammonia oxidizers. Applied and environmental microbiology, 76(8), 2468–77. doi:10.1128/AEM.01964-09 Uhlik, O., Jecna, K., Mackova, M., Vlcek, C., Hroudova, M., Demnerova, K., Paces, V., et al. (2009). Biphenyl-metabolizing bacteria in the rhizosphere of horseradish and bulk soil contaminated by polychlorinated biphenyls as revealed by stable isotope probing. Applied and environmental microbiology, 75(20), 6471–7. doi:10.1128/AEM.00466-09 Uhlik, O., Leewis, M.-C., Strejcek, M., Musilova, L., Mackova, M., Leigh, M. B., & Macek, T. (2012). Stable isotope probing in the metagenomics era: A bridge towards improved bioremediation. Biotechnology advances, 31(2), 154–65. doi:10.1016/j.biotechadv.2012.09.003 Uhlik, O., Musilova, L., Ridl, J., Hroudova, M., Vlcek, C., Koubek, J., Holeckova, M., et al. (2012). Plant secondary metabolite-induced shifts in bacterial community structure and degradative ability in contaminated soil. Applied microbiology and biotechnology. doi:10.1007/s00253-012-4627-6 Uhlik, O., Wald, J., Strejcek, M., Musilova, L., Ridl, J., Hroudova, M., Vlcek, C., et al. (2012). Identification of bacteria utilizing biphenyl, benzoate, and naphthalene in long-term contaminated soil. PloS one, 7(7), e40653. doi:10.1371/journal.pone.0040653 Uhlík, O., Jecná, K., Leigh, M. B., Macková, M., & Macek, T. (2009). DNA-based stable isotope probing: a link between community structure and function. The Science of the total environment, 407(12), 3611–9. doi:10.1016/j.scitotenv.2008.05.012 67 Van der Zaan, B. M., Saia, F. T., Stams, A. J. M., Plugge, C. M., De Vos, W. M., Smidt, H., Langenhoff, A. A. M., et al. (2012). Anaerobic benzene degradation under denitrifying conditions: Peptococcaceae as dominant benzene degraders and evidence for a syntrophic process. Environmental microbiology, 14(5), 1171–81. doi:10.1111/j.1462-2920.2012.02697.x Vandieken, V., Pester, M., Finke, N., Hyun, J.-H., Friedrich, M. W., Loy, A., & Thamdrup, B. (2012). Three manganese oxide-rich marine sediments harbor similar communities of acetate-oxidizing manganese-reducing bacteria. The ISME journal, 6(11), 2078–90. doi:10.1038/ismej.2012.41 Vandieken, V., & Thamdrup, B. (2013). Identification of acetate-oxidizing bacteria in a coastal marine surface sediment by RNA-stable isotope probing in anoxic slurries and intact cores. FEMS microbiology ecology. doi:10.1111/1574-6941.12069 Wang, Y., Chen, Y., Zhou, Q., Huang, S., Ning, K., Xu, J., Kalin, R. M., et al. (2012). A culture-independent approach to unravel uncultured bacteria and functional genes in a complex microbial community. PloS one, 7(10), e47530. doi:10.1371/journal.pone.0047530 Wawrik, B., Boling, W. B., Van Nostrand, J. D., Xie, J., Zhou, J., & Bronk, D. A. (2012). Assimilatory nitrate utilization by bacteria on the West Florida Shelf as determined by stable isotope probing and functional microarray analysis. FEMS microbiology ecology, 79(2), 400–11. doi:10.1111/j.1574-6941.2011.01226.x Wawrik, B., Callaghan, A. V, & Bronk, D. A. (2009). Use of inorganic and organic nitrogen by Synechococcus spp. and diatoms on the west Florida shelf as measured using stable isotope probing. Applied and environmental microbiology, 75(21), 6662–70. doi:10.1128/AEM.01002-09 Webster, G., Rinna, J., Roussel, E. G., Fry, J. C., Weightman, A. J., & Parkes, R. J. (2010). Prokaryotic functional diversity in different biogeochemical depth zones in tidal sediments of the Severn Estuary, UK, revealed by stable-isotope probing. FEMS microbiology ecology, 72(2), 179–97. doi:10.1111/j.1574-6941.2010.00848.x Webster, G., Watt, L. C., Rinna, J., Fry, J. C., Evershed, R. P., Parkes, R. J., & Weightman, A. J. (2006). A comparison of stable-isotope probing of DNA and phospholipid fatty acids to study prokaryotic functional diversity in sulfatereducing marine sediment enrichment slurries. Environmental microbiology, 8(9), 1575–89. doi:10.1111/j.1462-2920.2006.01048.x Wegener, G., Bausch, M., Holler, T., Thang, N. M., Prieto Mollar, X., Kellermann, M. Y., Hinrichs, K.-U., et al. (2012). Assessing sub-seafloor microbial activity by combined stable isotope probing with deuterated water and 13C-bicarbonate. Environmental microbiology, 14(6), 1517–27. doi:10.1111/j.1462-2920.2012.02739.x 68 Wegener, G., Niemann, H., Elvert, M., Hinrichs, K.-U., & Boetius, A. (2008). Assimilation of methane and inorganic carbon by microbial communities mediating the anaerobic oxidation of methane. Environmental microbiology, 10(9), 2287–98. doi:10.1111/j.1462-2920.2008.01653.x Whiteley, A. S., Manefield, M., & Lueders, T. (2006). Unlocking the “microbial black box” using RNA-based stable isotope probing technologies. Current opinion in biotechnology, 17(1), 67–71. doi:10.1016/j.copbio.2005.11.002 Whiteley, A. S., Thomson, B., Lueders, T., & Manefield, M. (2007). RNA stable-isotope probing. Nature protocols, 2(4), 838–44. doi:10.1038/nprot.2007.115 Winderl, C., Penning, H., Netzer, F. von, Meckenstock, R. U., & Lueders, T. (2010). DNA-SIP identifies sulfate-reducing Clostridia as important toluene degraders in tar-oil-contaminated aquifer sediment. The ISME journal, 4(10), 1314–25. doi:10.1038/ismej.2010.54 Woods, A., Watwood, M., & Schwartz, E. (2011). Identification of a toluene-degrading bacterium from a soil sample through H(2)(18)O DNA stable isotope probing. Applied and environmental microbiology, 77(17), 5995–9. doi:10.1128/AEM.05689-11 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 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 Yamasaki, S., Nomura, N., Nakajima, T., & Uchiyama, H. (2012). Cultivationindependent identification of candidate dehalorespiring bacteria in tetrachloroethylene degradation. Environmental science & technology, 46(14), 7709–16. doi:10.1021/es301288y Yu, C.-P., & Chu, K.-H. (2005). A quantitative assay for linking microbial community function and structure of a naphthalene-degrading microbial consortium. Environmental science & technology, 39(24), 9611–9. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/16475342 Zemb, O., Lee, M., Gutierrez-Zamora, M. L., Hamelin, J., Coupland, K., Hazrin-Chong, N. H., Taleb, I., et al. (2012). Improvement of RNA-SIP by pyrosequencing to identify putative 4-n-nonylphenol degraders in activated sludge. Water research, 46(3), 601–10. doi:10.1016/j.watres.2011.10.047 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 69 of strongly acidic soils. The ISME journal, 6(5), 1032–45. doi:10.1038/ismej.2011.168 Zhang, L.-M., Offre, P. R., He, J.-Z., Verhamme, D. T., Nicol, G. W., & Prosser, J. I. (2010). Autotrophic ammonia oxidation by soil thaumarchaea. Proceedings of the National Academy of Sciences of the United States of America, 107(40), 17240–5. doi:10.1073/pnas.1004947107 Zhang, S., Wang, Q., & Xie, S. (2012). Stable isotope probing identifies anthracene degraders under methanogenic conditions. Biodegradation, 23(2), 221–30. doi:10.1007/s10532-011-9501-1 Zhu, H., Singleton, D. R., & Aitken, M. D. (2010). Effects of nonionic surfactant addition on populations of polycyclic aromatic hydrocarbon-degrading bacteria in a bioreactor treating contaminated soil. Environmental science & technology, 44(19), 7266–71. doi:10.1021/es100114g 70