Stable Isotope Probing with 15N2 Reveals Novel Noncultivated Diazotrophs in Soil

Biological nitrogen fixation is a fundamental component of the nitrogen cycle and is the dominant natural process through which fixed nitrogen is made available to the biosphere. While the process of nitrogen fixation has been studied extensively with a limited set of cultivated isolates, examinations of nifH gene diversity in natural systems reveal the existence of a wide range of noncultivated diazotrophs. These noncultivated diazotrophs remain uncharacterized, as do their contributions to nitrogen fixation in natural systems. We have employed a novel ¹⁵N₂-DNA stable isotope probing (⁵N₂-DNA-SIP) method to identify free-living diazotrophs in soil that are responsible for nitrogen fixation in situ. Analyses of 16S rRNA genes from ¹⁵N-labeled DNA provide evidence for nitrogen fixation by three microbial groups, one of which belongs to the Rhizobiales while the other two represent deeply divergent lineages of noncultivated bacteria within the Betaproteobacteria and Actinobacteria, respectively. Analysis of nifH genes from ¹⁵N-labeled DNA also revealed three microbial groups, one of which was associated with Alphaproteobacteria while the others were associated with two noncultivated groups that are deeply divergent within nifH cluster I. These results reveal that noncultivated free-living diazotrophs can mediate nitrogen fixation in soils and that ¹⁵N₂-DNA-SIP can be used to gain access to DNA from these organisms. In addition, this research provides the first evidence for nitrogen fixation by Actinobacteria outside of the order Actinomycetales.     

Characterization of Growing Microorganisms in Soil by Stable Isotope Probing with H218O

A new approach to characterize growing microorganisms in environmental samples based on labeling microbial DNA with H₂¹⁸O is described. To test if sufficient amounts of ¹⁸O could be incorporated into DNA to use water as a labeling substrate for stable isotope probing, Escherichia coli DNA was labeled by cultivating bacteria in Luria broth with H₂¹⁸O and labeled DNA was separated from [¹⁶O]DNA on a cesium chloride gradient. Soil samples were incubated with H₂¹⁸O for 6, 14, or 21 days, and isopycnic centrifugation of the soil DNA showed the formation of two bands after 6 days and three bands after 14 or 21 days, indicating that ¹⁸O can be used in the stable isotope probing of soil samples. DNA extracted from soil incubated for 21 days with H₂¹⁸O was fractionated after isopycnic centrifugation and DNA from 17 subsamples was used in terminal restriction fragment length polymorphism (TRFLP) analysis of bacterial 16S rRNA genes. The TRFLP patterns clustered into three groups that corresponded to the three DNA bands. The fraction of total fluorescence contributed by individual terminal restriction fragments (TRF) to a TRFLP pattern varied across the 17 subsamples so that a TRF was more prominent in only one of the three bands. Labeling soil DNA with H₂¹⁸O allows the identification of newly grown cells. In addition, cells that survive but do not divide during an incubation period can also be characterized with this new technique because their DNA remains without the label.     

Stable Isotope Probing Analysis of the Diversity and Activity of Methanotrophic Bacteria in Soils from the Canadian High Arctic

The melting of permafrost and its potential impact on CH₄ emissions are major concerns in the context of global warming. Methanotrophic bacteria have the capacity to mitigate CH₄ emissions from melting permafrost. Here, we used quantitative PCR (qPCR), stable isotope probing (SIP) of DNA, denaturing gradient gel electrophoresis (DGGE) fingerprinting, and sequencing of the 16S rRNA and pmoA genes to study the activity and diversity of methanotrophic bacteria in active-layer soils from Ellesmere Island in the Canadian high Arctic. Results showed that most of the soils had the capacity to oxidize CH₄ at 4°C and at room temperature (RT), but the oxidation rates were greater at RT than at 4°C and were significantly enhanced by nutrient amendment. The DGGE banding patterns associated with active methanotrophic bacterial populations were also different depending on the temperature of incubation and the addition of nutrients. Sequencing of the 16S rRNA and pmoA genes indicated a low diversity of the active methanotrophic bacteria, with all methanotroph 16S rRNA and pmoA gene sequences being related to type I methanotrophs from Methylobacter and Methylosarcina. The dominance of type I methanotrophs over type II methanotrophs in the native soil samples was confirmed by qPCR of the 16S rRNA gene with primers specific for these two groups of bacteria. The 16S rRNA and pmoA gene sequences related to those of Methylobacter tundripaludum were found in all soils, regardless of the incubation conditions, and they might therefore play a role in CH₄ degradation in situ. This work is providing new information supporting the potential importance of Methylobacter spp. in Arctic soils found in previous studies and contributes to the limited body of knowledge on methanotrophic activity and diversity in this extreme environment.Permafrost regions occupy approximately 22% of the exposed land area of the Northern Hemisphere (63). In the past 100 years, the average temperatures in the arctic regions have increased at almost twice the average global rate (25). The melting of permafrost is one of the most important impacts of global warming on these high-latitude environments, and theoretical modeling suggests that as much as 90% of the permafrost could thaw by the end of the 21st century (29). While it has been generally reported that 15% of the total soil organic carbon is stocked in permafrost (42), a recent estimate indicates that it contains as much as 50% of the global belowground organic carbon pool (53). Carbon stocked in permafrost is now regarded as one of the most important carbon-climate feedbacks because of the size of the carbon pool and the intensity of climate change at high latitudes (46, 47). The presence of these large amounts of carbon in permafrost is raising serious concerns whether melting permafrost, and the resulting increase in microbial activity, might be a source of extensive emissions of the greenhouse gases carbon dioxide and methane (CH₄) to the atmosphere. The actual emissions of CH₄ from soils of high latitudes have been estimated to represent about 25% of the emissions from natural sources (19). Methane, which is 25 times more potent than carbon dioxide as a greenhouse gas (25), is produced by methanogenic archaea under anaerobic conditions. These microorganisms are known to inhabit permafrost environments (44, 49), and their capacity to produce methane at cold temperatures has been reported (20, 35, 44, 56). Their methanogenic activity is expected to increase as permafrost soil temperature increases (20). Moreover, large amounts of methane are stocked as methane hydrates in permafrost at an average depth of several hundred meters (33). Methane is also found in permafrost layers near the surface and could potentially be liberated to the atmosphere as permafrost melts (44).Methane can be oxidized in aerobic zones by aerobic methanotrophic bacteria or in anaerobic zones by anaerobic methanotrophic archaea (for a recent review, see reference 27). Anaerobic methane oxidizers were not covered in the context of this study, which focused exclusively on aerobic methanotrophs. These bacteria utilize methane as the sole carbon and energy source through the activity of the enzyme methane monooxygenase (MMO). Most known aerobic methanotrophs are divided into two major groups (type I and type II) based on phylogeny and carbon assimilation pathways (5). Type I methanotrophs, also known as Gammaproteobacteria methanotrophs (6) belong to the family Methylococcaceae within the Gammaproteobacteria subdivision, while type II methanotrophs (Alphaproteobacteria methanotrophs) belong to the family Methylocystaceae in the Alphaproteobacteria subdivision (5). Because of their capacity to oxidize methane, aerobic methanotrophs can significantly reduce methane emissions to the atmosphere and play an important role in the global methane cycle (12, 22). Methanotrophic activity has been observed in cold environments, and methanotrophs might contribute to the reduction of methane emissions from melting permafrost. Aerobic methanotrophic bacteria from cold environments have been reviewed in detail elsewhere (54).Most studies addressing methanotrophs from cold environments were conducted on soils from very few sites located in Northern Europe and Siberia (14, 30, 31, 40, 56-58), while methanotrophic bacterial populations in soils from the Canadian high Arctic remain mostly unexplored (41). In addition, most of these studies were conducted at low latitudes, and the pool of knowledge concerning the activity and diversity of methanotrophic bacterial populations in high Arctic soils is limited. The question being addressed in this study is whether there are active methanotrophs in the active-layer soil in the high Arctic. Therefore, the present work had two objectives: (i) to evaluate the methane oxidation capacity of three active-layer soils from the Canadian high Arctic under various incubation conditions and (ii) to identify and characterize the diversity of the active methanotrophs in these soils using stable isotope probing (SIP) of DNA (DNA-SIP) and sequencing of the 16S rRNA and pmoA genes. With this work, we identify for the first time active methanotrophs in high Arctic soils through the use of DNA-SIP.     

Linking Diversity and Stable Isotope Fractionation in Ammonia-Oxidizing Bacteria

The link between similarity in amino acid sequence for ammonia monooxygenase (AMO) and isotopic discrimination for ammonia oxidation ( l AMO ) was investigated in g -subdivision ammonia-oxidizing bacteria. The isotope effects for ammonia oxidation in pure cultures of the nitrifying strains Nitrosomonas marina , Nitrosomonas C-113a, Nitrosospira tenuis , Nitrosomonas europaea , and Nitrosomonas eutropha ranged from 14.2 to 38.2. The differences in isotope effects could not be readily explained by differential rates of ammonia oxidation, transport of NH 4 + , or accumulation of NH 2 OH or N 2 O among the strains. The major similarities and differences observed in l AMO are, however, paralleled by similarities and differences in amino acid sequences for the f -subunit of AMO (AmoA). Robust differences in l AMO among nitrifying bacteria may be expected to influence the stable isotopic signatures of nitrous oxide (N 2 O) produced in various environments.     

Methodological Considerations for the Use of Stable Isotope Probing in Microbial Ecology

Stable isotope probing (SIP) is a method used for labeling uncultivated microorganisms in environmental samples or directly in field studies using substrate enriched with stable isotope (e.g., ¹³C). After consumption of the substrate, the cells of microorganisms that consumed the substrate become enriched in the isotope. Labeled biomarkers, such as phospholipid-derived fatty acid (PLFA), ribosomal RNA, and DNA can be analyzed with a range of molecular and analytical techniques, and used to identify and characterize the organisms that incorporated the substrate. The advantages and disadvantages of PLFA-SIP, RNA-SIP, and DNA-SIP are presented. Using examples from our laboratory and from the literature, we discuss important methodological considerations for a successful SIP experiment.     

Biphenyl-Metabolizing Bacteria in the Rhizosphere of Horseradish and Bulk Soil Contaminated by Polychlorinated Biphenyls as Revealed by Stable Isotope Probing

DNA-based stable isotope probing in combination with terminal restriction fragment length polymorphism was used in order to identify members of the microbial community that metabolize biphenyl in the rhizosphere of horseradish (Armoracia rusticana) cultivated in soil contaminated with polychlorinated biphenyls (PCBs) compared to members of the microbial community in initial, uncultivated bulk soil. On the basis of early and recurrent detection of their 16S rRNA genes in clone libraries constructed from [¹³C]DNA, Hydrogenophaga spp. appeared to dominate biphenyl catabolism in the horseradish rhizosphere soil, whereas Paenibacillus spp. were the predominant biphenyl-utilizing bacteria in the initial bulk soil. Other bacteria found to derive carbon from biphenyl in this nutrient-amended microcosm-based study belonged mostly to the class Betaproteobacteria and were identified as Achromobacter spp., Variovorax spp., Methylovorus spp., or Methylophilus spp. Some bacteria that were unclassified at the genus level were also detected, and these bacteria may be members of undescribed genera. The deduced amino acid sequences of the biphenyl dioxygenase α subunits (BphA) from bacteria that incorporated [¹³C]into DNA in 3-day incubations of the soils with [¹³C]biphenyl are almost identical to that of Pseudomonas alcaligenes B-357. This suggests that the spectrum of the PCB congeners that can be degraded by these enzymes may be similar to that of strain B-357. These results demonstrate that altering the soil environment can result in the participation of different bacteria in the metabolism of biphenyl.Polychlorinated biphenyls (PCBs) are very stable chloroorganic compounds with the general formula C₁₂H_10-xCl_x. Mixtures of PCBs have been used as coolants and lubricants in transformers, capacitors, and other electrical equipment as they do not burn easily and are good insulators. It is estimated that some 1.5 million tons of PCBs were produced up to 1988 worldwide (11; http://www.atsdr.cdc.gov/cercla; http://www.epa.gov/epawaste/hazard/tsd/pcbs/pubs/about.htm). Although production of these compounds was stopped, due to their long-term persistence, many sites all over the world are still contaminated with PCBs. Moreover, not only do PCBs threaten human health in the vicinity of the contaminated area, but lower PCB congeners volatilize and migrate to places far from where they were originally released (2, 3, 16). Also, their metabolic products have environmental significance; activities of both plants and microorganisms result in formation of different intermediates and final products whose toxicity can in some cases be even higher than that of the original toxicant (24, 26; http://www.atsdr.cdc.gov/cercla).Physical-chemical methods used for the removal of PCBs often cause further natural disturbance and pollution; in contrast, biological methods of removal (i.e., bioremediation) are less expensive and more environmentally sound and thus have aroused much interest (7). These methods include the use of microorganisms and also exploitation of plants (i.e., phytoremediation) (19) and the cooperation of plants with microorganisms in the rhizosphere (i.e., rhizoremediation) (21). These bioremediation options also include the use of genetically modified bacteria (6) and/or plants (18, 23). PCBs were only recently introduced into the environment, and no completely efficient pathways for the aerobic bacterial degradation of all of these compounds have evolved (34); however, lower chlorinated PCB congeners can be degraded via the pathway that is used by aerobic bacteria to degrade biphenyl (35). Therefore, metabolism of biphenyl as a potential cometabolite of PCBs was the subject of this study.The biphenyl degradation pathway is the same in all aerobic bacteria, and enzymes of this pathway degrade biphenyl in four steps into benzoate and 2-hydroxypenta-2,4-dienoate (21). The first enzyme of the pathway, biphenyl dioxygenase, has broad substrate specificity and thus permits degradation of biphenyl-related compounds (9). Substrates for biphenyl dioxygenase comprise, in addition to biphenyl itself, other diphenyl or benzene skeletons with several substituents, including halogens and bicyclic or tricyclic fused heterocyclic aromatics (35). These substrates also include certain natural compounds, including some plant flavonoids, phenols, or terpenes (10). Bacteria capable of metabolizing biphenyl are thus pervasive members of many microbial communities in vegetated soil.As reported previously (20), there are two main problems with introduction of a new population of degrading or genetically modified microorganisms to enhance the biodegradation of PCBs in a contaminated environment: legislative barriers and the inability of strains added to the soil to survive. Therefore, the use of microorganisms for bioremediation of contaminated sites is not likely to be successful. Hence, understanding the biodegradative processes in the natural communities is necessary for planning remediation strategies. Identification of members of the community potentially responsible for the degradative process has recently been enabled by DNA-based stable isotope probing (SIP), as reviewed previously; therefore, this technique has become an efficient tool in microbial ecology (33). In this study, by tracking the transfer of ¹³C from [¹³C]biphenyl into bacterial DNA, it was possible to identify biphenyl-metabolizing bacteria in PCB-contaminated soil. To analyze how the bacterial diversity can be changed by introduction of a plant and subsequent cultivation in a greenhouse, bacteria in the rhizosphere of horseradish (Armoracia rusticana) cultivated in a contaminated soil were studied.     

Identification of Metabolically Active Bacteria in the Gut of the Generalist Spodoptera littoralis via DNA Stable Isotope Probing Using 13C-Glucose

Guts of most insects are inhabited by complex communities of symbiotic nonpathogenic bacteria. Within such microbial communities it is possible to identify commensal or mutualistic bacteria species. The latter ones, have been observed to serve multiple functions to the insect, i.e. helping in insect reproduction¹, boosting the immune response², pheromone production³, as well as nutrition, including the synthesis of essential amino acids^4, among others.    Due to the importance of these associations, many efforts have been made to characterize the communities down to the individual members. However, most of these efforts were either based on cultivation methods or relied on the generation of 16S rRNA gene fragments which were sequenced for final identification. Unfortunately, these approaches only identified the bacterial species present in the gut and provided no information on the metabolic activity of the microorganisms.To characterize the metabolically active bacterial species in the gut of an insect, we used stable isotope probing (SIP) in vivo employing ¹³C-glucose as a universal substrate. This is a promising culture-free technique that allows the linkage of microbial phylogenies to their particular metabolic activity. This is possible by tracking stable, isotope labeled atoms from substrates into microbial biomarkers, such as DNA and RNA⁵. The incorporation of ¹³C isotopes into DNA increases the density of the labeled DNA compared to the unlabeled (¹²C) one. In the end, the ¹³C-labeled DNA or RNA is separated by density-gradient ultracentrifugation from the ¹²C-unlabeled similar one⁶. Subsequent molecular analysis of the separated nucleic acid isotopomers provides the connection between metabolic activity and identity of the species.Here, we present the protocol used to characterize the metabolically active bacteria in the gut of a generalist insect (our model system), Spodoptera littoralis (Lepidoptera, Noctuidae). The phylogenetic analysis of the DNA was done using pyrosequencing, which allowed high resolution and precision in the identification of insect gut bacterial community. As main substrate, ¹³C-labeled glucose was used in the experiments. The substrate was fed to the insects using an artificial diet.     

Stable Isotope Probing with 15N Achieved by Disentangling the Effects of Genome G+C Content and Isotope Enrichment on DNA Density

Stable isotope probing (SIP) of nucleic acids is a powerful tool that can identify the functional capabilities of noncultivated microorganisms as they occur in microbial communities. While it has been suggested previously that nucleic acid SIP can be performed with ¹⁵N, nearly all applications of this technique to date have used ¹³C. Successful application of SIP using ¹⁵N-DNA (¹⁵N-DNA-SIP) has been limited, because the maximum shift in buoyant density that can be achieved in CsCl gradients is approximately 0.016 g ml⁻¹ for ¹⁵N-labeled DNA, relative to 0.036 g ml⁻¹ for ¹³C-labeled DNA. In contrast, variation in genome G+C content between microorganisms can result in DNA samples that vary in buoyant density by as much as 0.05 g ml⁻¹. Thus, natural variation in genome G+C content in complex communities prevents the effective separation of ¹⁵N-labeled DNA from unlabeled DNA. We describe a method which disentangles the effects of isotope incorporation and genome G+C content on DNA buoyant density and makes it possible to isolate ¹⁵N-labeled DNA from heterogeneous mixtures of DNA. This method relies on recovery of “heavy” DNA from primary CsCl density gradients followed by purification of ¹⁵N-labeled DNA from unlabeled high-G+C-content DNA in secondary CsCl density gradients containing bis-benzimide. This technique, by providing a means to enhance separation of isotopically labeled DNA from unlabeled DNA, makes it possible to use ¹⁵N-labeled compounds effectively in DNA-SIP experiments and also will be effective for removing unlabeled DNA from isotopically labeled DNA in ¹³C-DNA-SIP applications.     

Identification of a Novel Toluene-Degrading Bacterium from the Candidate Phylum TM7, as Determined by DNA Stable Isotope Probing

The dominant bacterium responsible for carbon uptake from toluene in an agricultural soil was identified by stable isotope probing. Samples were amended with unlabeled toluene or labeled [ring-¹³C₆]toluene, and DNA was extracted over time. Sequencing indicated that the organism involved belongs to the candidate phylum TM7. Microorganisms in this candidate phylum are of particular interest because although they have been found in a variety of habitats, no stable culture of any species exists, so their general metabolic capabilities are largely unknown.The application of PCR technology has uncovered the impressive diversity of the microbial world. It has been estimated that less than half of the recognized bacterial phyla include cultured representatives (14). Here, stable isotope probing (SIP) (a method that links function to identity in mixed microbial samples) was used to identify dominant toluene degraders in an environment previously unexposed to the contaminant but likely containing a diverse microbial community of previously undiscovered toluene degraders.Soil samples were collected from a field in Michigan previously under corn production. This field received biosolids from a wastewater treatment plant 2 to 3 years before sample collection. Following collection, soils were homogenized, sieved (4-mm screen), and stored at 4°C until use (<1 year). The microcosms consisted of phosphate-buffered mineral medium (20 ml) (10) and soil (6 g [wet weight]) in serum bottles (150 ml). The bottles were sealed with rubber stoppers and an aluminum seal. The treatment groups included no-toluene controls, autoclaved controls, and samples amended with unlabeled (1 μl, 99%; Chem Service) or labeled (1 μl, ring-¹³C₆, 99%; Cambridge Isotope Laboratories) toluene. Eight samples for each treatment (two for each time point) were incubated at room temperature (∼20°C) with reciprocal shaking. The concentrations of toluene in headspace samples (200 μl) after toluene addition (∼2 h) and at each time point were determined with a gas chromatograph (Perkin Elmer) equipped with a flame ionization detector and a capillary column (DB-624 [diameter, 0.53 mm]; J&W Scientific). The injector and detector temperatures were set at 200°C, and the column temperature was 80°C.At four time points (3, 5, 6, and 8 days after toluene addition), two samples from the labeled and unlabeled treatment groups were sacrificed for soil DNA extraction using a Powersoil DNA extraction kit (MO BIO Laboratories, Inc., Carlsbad, CA). At each time point, DNAs from two microcosms were pooled, and SIP involved terminal restriction fragment length polymorphism (TRFLP) analysis of all fractions (labeled and unlabeled). Approximately 10 μg DNA (quantified with an ND-1000 spectrometer; Nanodrop) was added to Quick-Seal polyallomer tubes (13 by 51 mm, 5.1 ml; Beckman Coulter), along with a Tris-EDTA (pH 8.0)-CsCl solution. Before the tubes were sealed (Quick-Seal tube topper; Beckman Coulter), buoyant density (BD) was determined (∼1.77 g ml⁻¹) with a model AR200 digital refractometer (Leica Microsystems, Inc.) and adjusted by adding CsCl solution or Tris-EDTA buffer. The tubes were centrifuged at 178,000 × g (20°C) for 48 h in a Stepsaver 70 V6 vertical titanium rotor (eight tubes, 5.1-ml capacity each) within a Sorvall WX 80 Ultra Series centrifuge (Thermo Scientific). Following centrifugation, a fraction recovery system (Beckman Coulter) was used for fraction (150 μl) collection. The BD of each fraction was measured, and CsCl was removed by glycogen-assisted ethanol precipitation.The fractions were PCR amplified using 27F-FAM (5′-AGAGTTTGATCMTGGCTCAG [5′ end labeled with carboxyfluorescein]) and 1492R (5′-GGTTACCTTGTTACGACTT) (Operon Biotechnologies) as previously described (4). Briefly, this involved the following conditions: 94°C (5 min); 30 cycles at 94°C (30 s), 55°C (30 s), and 72°C (1.5 min); and 72°C (5 min). The presence of PCR products was confirmed by gel electrophoresis. PCR products were purified with a QIAquick PCR purification kit (Qiagen, Inc.) (∼150 ng) and digested with HaeIII (New England Biolabs). In addition, three other enzymes (MspI, MseI, and HincII; New England Biolabs) were used for digestion of a number of heavy fractions. DNA fragments were separated by capillary electrophoresis (ABI Prism 3100 genetic analyzer; Applied Biosystems) at the Research Technology Support Facility at Michigan State University. Data were analyzed with GeneScan software (Applied Biosystems), and the percent abundance of each fragment was determined (18). Heavy-fraction ¹³C-labeled DNAs (day 8 fractions with BD values of 1.744 g ml⁻¹) were amplified, as described above, with unlabeled primers and cloned into Escherichia coli TOP10 by using a TOPO TA cloning kit (Invitrogen Corporation). E. coli clones were grown on Luria-Bertani medium solidified with 15 g agar liter⁻¹ in the presence of 50 μg ampicillin liter⁻¹ for 16 h at 37°C. Colonies with inserts were verified by PCR with primers M13 forward (5′-TGTAAAACGACGGCCAGT-3′) and M13 reverse (5′-AACAGCTATGACCATG-3′), plasmids were extracted from the positive clones with a QIAprep miniprep system (Qiagen, Inc.), and the insertions were sequenced (using primers M13 forward and M13 reverse) at the Research Technology Support Facility at Michigan State University. The partial 16S rRNA gene sequences obtained were aligned and edited with Chromas Pro (Technelysium, Pty. Ltd.). The Ribosomal Database Project (Center for Microbial Ecology, Michigan State University) analysis tool “classifier” (16) was utilized to assign taxonomic identity. In addition, the Ribosomal Database Project classifier checked the deposited sequence for chimeras.Toluene removal occurred rapidly, starting after 3 days and reaching completion after 8 days (Table ​(Table1).1). The low percentage of recoveries was likely caused by toluene sorption to the soil; however, the difference between the controls and samples clearly illustrates a biological removal mechanism. DNAs extracted over time (on days 3, 5, 6, and 8) from both labeled and unlabeled samples were subjected to ultracentrifugation, fractionation, and TRFLP on every fraction. TRFLP analyses indicated that one fragment (394 bp) was highly enriched in the heavy fractions (>1.732 g ml⁻¹) obtained from [¹³C]toluene microcosms but not in fractions with similar BD values obtained from the unlabeled controls, and further, the level of enrichment increased with time (Fig. ​(Fig.1).1). At day 3, the fragment was present in only one fraction TRFLP profile, indicating a low natural relative abundance of this organism. The peak TRFLP relative abundance values for the 394-bp fragment were 11.3%, 41.5%, and 62.1% on days 5, 6, and 8, respectively (Fig. ​(Fig.2).2). Consistent with this trend, the BD of this fragment also increased with time, with the peak relative abundances being found at BD values of 1.732 g ml⁻¹, 1.740 g ml⁻¹, and 1.744 g ml⁻¹ on days 5, 6, and 8, respectively (Fig. ​(Fig.2).2). These trends indicate that the organism represented by this fragment is directly involved in toluene transformation. A number of other TRFLP peaks were found in the heavy ¹³C fractions; however, because they were also present in the heavy ¹²C fractions, they were excluded from further analysis.Open in a separate windowFIG. 1.Dominance of the 394-bp fragment over time in TRFLP profiles from heavy fractions of soil samples amended with unlabeled (¹²C) or labeled (¹³C) toluene. The pattern is representative of other heavy-fraction samples at these time points. The BD values are given in the lower-right corners of the profiles.Open in a separate windowFIG. 2.Relative abundances of the dominant 394-bp fragment over a range of BD values from DNA extracted after 5 days (a), 6 days (b), or 8 days (c) from soil amended with either labeled (¹³C) or unlabeled (¹²C) toluene.

TABLE 1.

Ranges for percentages of toluene remaining in soil-liquid slurries over time

Using Stable Isotope Probing to Characterize Differences Between Free-Living and Sediment-Associated Microorganisms in the Subsurface

Aquifers are subterranean reservoirs of freshwater with heterotrophic bacterial communities attached to the sediments and free-living in the groundwater. In the present study, mesocosms were used to assess factors controlling the diversity and activity of the subsurface bacterial community. The assimilation of ¹³C, derived from ¹³C-acetate, was monitored to determine whether the sediment-associated and free-living bacterial community would respond similarly to the presence of protozoan grazers. We observed a dynamic response in the sediment-associated bacterial community and none in the free-living community. The disparity in these observations highlights the importance of the sediment-associated bacterial community in the subsurface carbon cycle.     

Efficacy of Organic Soil Amendments for Management of Heterodera glycines in Greenhouse Experiments

In a repeated greenhouse experiment, organic soil amendments were screened for effects on population density of soybean cyst nematode (SCN), Heterodera glycines, and soybean growth. Ten amendments at various rates were tested: fresh plant material of field pennycress, marigold, spring camelina, and Cuphea; condensed distiller’s solubles (CDS), ash of combusted CDS, ash of combusted turkey manure (TMA), marigold powder, canola meal, and pennycress seed powder. Soybeans were grown for 70 d in field soil with amendments and SCN eggs incorporated at planting. At 40 d after planting (DAP), many amendments reduced SCN egg population density, but some also reduced plant height. Cuphea plant at application rate of 2.9% (amendment:soil, w:w, same below), marigold plant at 2.9%, pennycress seed powder at 0.5%, canola meal at 1%, and CDS at 4.3% were effective against SCN with population reductions of 35.2%, 46.6%, 46.7%, 73.2%, and 73.3% compared with control, respectively. For Experiment 1 at 70 DAP, canola meal at 1% and pennycress seed powder at 0.5% reduced SCN population density 70% and 54%, respectively. CDS at 4.3%, ash of CDS at 0.2%, and TMA at 1% increased dry plant mass whereas CDS at 4.3% and pennycress seed powder at 0.1% reduced plant height. For Experiment 2 at 70 DAP, amendments did not affect SCN population nor plant growth. In summary, some amendments were effective for SCN management, but phytoxicity was a concern.     

Use of Field-Based Stable Isotope Probing To Identify Adapted Populations and Track Carbon Flow through a Phenol-Degrading Soil Microbial Community

The goal of this field study was to provide insight into three distinct populations of microorganisms involved in in situ metabolism of phenol. Our approach measured ¹³CO₂ respired from [¹³C]phenol and stable isotope probing (SIP) of soil DNA at an agricultural field site. Traditionally, SIP-based investigations have been subject to the uncertainties posed by carbon cross-feeding. By altering our field-based, substrate-dosing methodologies, experiments were designed to look beyond primary degraders to detect trophically related populations in the food chain. Using gas chromatography-mass spectrometry (GC/MS), it was shown that ¹³C-labeled biomass, derived from primary phenol degraders in soil, was a suitable growth substrate for other members of the soil microbial community. Next, three dosing regimes were designed to examine active members of the microbial community involved in phenol metabolism in situ: (i) 1 dose of [¹³C]phenol, (ii) 11 daily doses of unlabeled phenol followed by 1 dose of [¹³C]phenol, and (iii) 12 daily doses of [¹³C]phenol. GC/MS analysis demonstrated that prior exposure to phenol boosted ¹³CO₂ evolution by a factor of 10. Furthermore, imaging of ¹³C-treated soil using secondary ion mass spectrometry (SIMS) verified that individual bacteria incorporated ¹³C into their biomass. PCR amplification and 16S rRNA gene sequencing of ¹³C-labeled soil DNA from the 3 dosing regimes revealed three distinct clone libraries: (i) unenriched, primary phenol degraders were most diverse, consisting of α-, β-, and γ-proteobacteria and high-G+C-content gram-positive bacteria, (ii) enriched primary phenol degraders were dominated by members of the genera Kocuria and Staphylococcus, and (iii) trophically related (carbon cross-feeders) were dominated by members of the genus Pseudomonas. These data show that SIP has the potential to document population shifts caused by substrate preexposure and to follow the flow of carbon through terrestrial microbial food chains.     

Methanotroph Diversity in Landfill Soil: Isolation of Novel Type I and Type II Methanotrophs Whose Presence Was Suggested by Culture-Independent 16S Ribosomal DNA Analysis

The diversity of the methanotrophic community in mildly acidic landfill cover soil was assessed by three methods: two culture-independent molecular approaches and a traditional culture-based approach. For the first of the molecular studies, two primer pairs specific for the 16S rRNA gene of validly published type I (including the former type X) and type II methanotrophs were identified and tested. These primers were used to amplify directly extracted soil DNA, and the products were used to construct type I and type II clone libraries. The second molecular approach, based on denaturing gradient gel electrophoresis (DGGE), provided profiles of the methanotrophic community members as distinguished by sequence differences in variable region 3 of the 16S ribosomal DNA. For the culturing studies, an extinction-dilution technique was employed to isolate slow-growing but numerically dominant strains. The key variables of the series of enrichment conditions were initial pH (4.8 versus 6.8), air/CH₄/CO₂ headspace ratio (50:45:5 versus 90:9:1), and concentration of the medium (1× nitrate minimal salts [NMS] versus 0.2× NMS). Screening of the isolates showed that the nutrient-rich 1× NMS selected for type I methanotrophs, while the nutrient-poor 0.2× NMS tended to enrich for type II methanotrophs. Partial sequencing of the 16S rRNA gene from selected clones and isolates revealed some of the same novel sequence types. Phylogenetic analysis of the type I clone library suggested the presence of a new phylotype related to the Methylobacter-Methylomicrobium group, and this was confirmed by isolating two members of this cluster. The type II clone library also suggested the existence of a novel group of related species distinct from the validly published Methylosinus and Methylocystis genera, and two members of this cluster were also successfully cultured. Partial sequencing of the pmoA gene, which codes for the 27-kDa polypeptide of the particulate methane monooxygenase, reaffirmed the phylogenetic placement of the four isolates. Finally, not all of the bands separated by DGGE could be accounted for by the clones and isolates. This polyphasic assessment of community structure demonstrates that much diversity among the obligate methane oxidizers has yet to be formally described.     

Identification of Ciliate Grazers of Autotrophic Bacteria in Ammonia-Oxidizing Activated Sludge by RNA Stable Isotope Probing

It is well understood that protozoa play a major role in controlling bacterial biomass and regulating nutrient cycling in the environment. Little is known, however, about the movement of carbon from specific reduced substrates, through functional groups of bacteria, to particular clades of protozoa. In this study we first identified the active protozoan phylotypes present in activated sludge, via the construction of an rRNA-derived eukaryote clone library. Most of the sequences identified belonged to ciliates of the subclass Peritrichia and amoebae, confirming the dominance of surface-associated protozoa in the activated sludge environment. We then demonstrated that ¹³C-labeled protozoan RNA can be retrieved from activated sludge amended with ¹³C-labeled protozoa or ¹³C-labeled Escherichia coli cells by using an RNA stable isotope probing (RNA-SIP) approach. Finally, we used RNA-SIP to track carbon from bicarbonate and acetate into protozoa under ammonia-oxidizing and denitrifying conditions, respectively. RNA-SIP analysis revealed that the peritrich ciliate Epistylis galea dominated the acquisition of carbon from bacteria with access to CO₂ under ammonia-oxidizing conditions, while there was no evidence of specific grazing on acetate consumers under denitrifying conditions.Protozoa are the main consumers of bacteria in the environment, and as such they play a major role in controlling bacterial biomass (33) and regulating nutrient recycling (14). Therefore, being able to study the flow of carbon in food webs involving bacteria and protozoa can provide an insight into how protozoan grazing affects bacterial function in a wide range of systems.Protozoan grazing has the potential to alter the genotypic and phenotypic composition of bacterial communities (15, 17, 18, 35). Perhaps as a result of this, protozoan grazing has also been found to have an effect on the function of activated sludge systems, including nitrogen removal processes. Studies using eukaryotic inhibitors to remove grazers from activated sludge have reported a wide range of effects following reduced grazing pressure. These effects include an increase in turbidity or planktonic cell densities, and either no effect (21, 32), higher nitrification rates (16, 20), or lower (29, 30) rates of nitrification in the absence of grazers. These conflicting reports on the effect of predation on nitrification might be due to protozoa displaying feeding preferences or to the indirect ways in which protozoan grazing can affect bacterial processes. For example, it has been shown that the release of substances, such as vitamins and nucleotides, secreted by protozoa as metabolic by-products, can act as growth factors, which enhance bacterial activity, including nitrification (31). In addition, the presence of some types of grazers, particularly ciliates, has been shown to be closely related to a decrease in biochemical oxygen demand (27), and it has been reported that ciliates can alter water flux and help redistribute nutrients in flocs (8), which in turn might have an impact on nitrification rates.Although the data presented in the literature suggest that protozoan grazing is an important factor for activated sludge processes, there are still many questions as to whether its effects are directly linked to predation and possibly feeding preferences. In the present study we sought to identify protozoa assimilating carbon from autotrophic bacteria under ammonia-oxidizing conditions and acetate-consuming bacteria under denitrifying conditions using RNA-stable isotope probing (RNA-SIP).Since it was first developed, RNA-SIP has been successfully used to identify functional groups of bacteria responsible for different processes, including denitrification and benzene and phenol degradation (9, 10, 19, 25, 26). In these studies, the flow of carbon was tracked from soluble labeled substrates into the bacteria consuming it. However, recent studies have shown that it is possible to use this technique to track the flow of carbon across more than one trophic level, unraveling some of the interactions observed in microbial food webs (11, 13, 23, 24). These studies exemplify the broad range of questions that can be addressed with SIP and have begun to define the boundaries beyond which SIP has limited utility.     

Characterization of para-Nitrophenol-Degrading Bacterial Communities in River Water by Using Functional Markers and Stable Isotope Probing

Microbial degradation is a major determinant of the fate of pollutants in the environment. para-Nitrophenol (PNP) is an EPA-listed priority pollutant with a wide environmental distribution, but little is known about the microorganisms that degrade it in the environment. We studied the diversity of active PNP-degrading bacterial populations in river water using a novel functional marker approach coupled with [¹³C₆]PNP stable isotope probing (SIP). Culturing together with culture-independent terminal restriction fragment length polymorphism analysis of 16S rRNA gene amplicons identified Pseudomonas syringae to be the major driver of PNP degradation in river water microcosms. This was confirmed by SIP-pyrosequencing of amplified 16S rRNA. Similarly, functional gene analysis showed that degradation followed the Gram-negative bacterial pathway and involved pnpA from Pseudomonas spp. However, analysis of maleylacetate reductase (encoded by mar), an enzyme common to late stages of both Gram-negative and Gram-positive bacterial PNP degradation pathways, identified a diverse assemblage of bacteria associated with PNP degradation, suggesting that mar has limited use as a specific marker of PNP biodegradation. Both the pnpA and mar genes were detected in a PNP-degrading isolate, P. syringae AKHD2, which was isolated from river water. Our results suggest that PNP-degrading cultures of Pseudomonas spp. are representative of environmental PNP-degrading populations.     

Stable Isotope Ratios as a Tool for Assessing Changes in Carbon and Nutrient Sources in Antarctic Terrestrial Ecosystems

Samples of an angiosperm species, nine lichen species and a terrestrial alga, were collected from a variety of Antarctic terrestrial habitats, and were analysed for C and N stable isotope composition. Collections were made along natural gradients, the marine gradient, running from the sea coast inland and the moisture gradient, determined by melt water and precipitation runoff, and running towards the sea coast. Considerable variation in stable isotope ratios was found; δ¹³C values ranged between −16 and −32‰ and δ¹⁵N values between −23 and +23‰ The variation in stable carbon isotope ratios could be attributed in part to species specific differences, but differences in water availability also played a role, as was shown for the terrestrial alga Prasiola crispa and the lichen species Usnea antarctica. The differences in the isotope ratios of nitrogen could be retraced to the origin of nitrogen: marine or terrestrial. The nitrogen stable isotope ratios were influenced by both the marine gradient from the sea inland and the melt water and precipitation flow running in the opposite direction, towards the sea. This was shown for the lichen species Turgidosculum complicatulum and the angiosperm species Deschampsia antarctica. The variation in the C and N stable isotope ratios can be used to determine sources and pathways of N and changes in the water availability in Antarctic terrestrial ecosystems. Contrary to earlier reports the use of stable N isotope ratios is possible in this case because of the relative simplicity of the structure of the Antarctic terrestrial ecosystems.     

Comparison of Modeled and Observed Environmental Influences on the Stable Oxygen and Hydrogen Isotope Composition of Leaf Water in Phaseolus vulgaris L

In this paper we describe how a model of stable isotope fractionation processes, originally developed by H. Craig and L. I. Gordon ([1965] in E Tongiorgi, ed, Proceedings of a Conference on Stable Isotopes in Oceanographic Studies and Paleotemperature, Spoleto, Italy, pp 9-130) for evaporation of water from the ocean, can be applied to leaf transpiration. The original model was modified to account for turbulent conditions in the leaf boundary layer. Experiments were conducted to test the factors influencing the stable isotopic composition of leaf water under controlled environment conditions. At steady state, the observed leaf water isotopic composition was enriched above that of stem water with the extent of the enrichment dependent on the leaf-air vapor pressure difference (VPD) and the isotopic composition of atmospheric water vapor (AWV). The higher the VPD, the larger was the observed heavy isotope content of leaf water. At a constant VPD, leaf water was relatively depleted in heavy isotopes when exposed to AWV with a low heavy isotope composition, and leaf water was relatively enriched in heavy isotopes when exposed to AWV with a large heavy isotope composition. However, the observed heavy isotope composition of leaf water was always less than that predicted by the model. The extent of the discrepancy between the modeled and observed leaf water isotopic composition was a strong linear function of the leaf transpiration rate.     

Ubiquitous Dissolved Inorganic Carbon Assimilation by Marine Bacteria in the Pacific Northwest Coastal Ocean as Determined by Stable Isotope Probing

In order to identify bacteria that assimilate dissolved inorganic carbon (DIC) in the northeast Pacific Ocean, stable isotope probing (SIP) experiments were conducted on water collected from 3 different sites off the Oregon and Washington coasts in May 2010, and one site off the Oregon Coast in September 2008 and March 2009. Samples were incubated in the dark with 2 mM ¹³C-NaHCO₃, doubling the average concentration of DIC typically found in the ocean. Our results revealed a surprising diversity of marine bacteria actively assimilating DIC in the dark within the Pacific Northwest coastal waters, indicating that DIC fixation is relevant for the metabolism of different marine bacterial lineages, including putatively heterotrophic taxa. Furthermore, dark DIC-assimilating assemblages were widespread among diverse bacterial classes. Alphaproteobacteria, Gammaproteobacteria, and Bacteroidetes dominated the active DIC-assimilating communities across the samples. Actinobacteria, Betaproteobacteria, Deltaproteobacteria, Planctomycetes, and Verrucomicrobia were also implicated in DIC assimilation. Alteromonadales and Oceanospirillales contributed significantly to the DIC-assimilating Gammaproteobacteria within May 2010 clone libraries. 16S rRNA gene sequences related to the sulfur-oxidizing symbionts Arctic96BD-19 were observed in all active DIC assimilating clone libraries. Among the Alphaproteobacteria, clones related to the ubiquitous SAR11 clade were found actively assimilating DIC in all samples. Although not a dominant contributor to our active clone libraries, Betaproteobacteria, when identified, were predominantly comprised of Burkholderia. DIC-assimilating bacteria among Deltaproteobacteria included members of the SAR324 cluster. Our research suggests that DIC assimilation is ubiquitous among many bacterial groups in the coastal waters of the Pacific Northwest marine environment and may represent a significant metabolic process.