Stable Isotope Probing and Related Technologies

The major obstacle for 15N-based DNA stable isotope probing (DNA-SIP) was insufficient separation of 15N-labeled and unlabeled DNA due to the lower nitrogen content in DNA compared with its carbon content. Extracted DNA can be loaded onto a cesium chloride (CsCl) gradient for isopycnic centrifugation and separation of labeled “heavy” DNA from unlabeled background DNA (“light” DNA). DNA-SIP experiments need to be implemented carefully in order to maximize achievable information and to avoid misinterpretation of resulting data. Using DNA-SIP, a study demonstrated the possibility that Candidate phylum TM7 is involved in toluene degradation in soils. Methanol, formaldehyde, and ammonium are the final products of the RDX degradation pathway. It is obvious that microorganisms that only use nitro-nitrogen in the ring-labeled 15N3-RDX would not be seen in this study. In a study, the authors used DNA-SIP to analyze the active methanotroph population in a peatland from the United Kingdom. By using DNA-SIP, the authors demonstrated that Methylocellaand Methylocystisare probably the most active methane utilizers in this peatland. DNA-SIP can be used in combination with metagenomics in a focused way to investigate the function of a subpopulation of environmental microorganisms. It is predicted that this approach will be adopted by more researchers in the near future. The 16S rRNA gene sequences retrieved from the SIP experiments were used to design specific probes targeting 16S rRNA gene of Acidovorax spp. and Pseudomonas spp.

This chapter focuses on RNA-based stable isotope probing (RNA-SIP) that was developed to take advantage of the features of RNA that make it an excellent biomarker for linking environmental processes—rapid turnover rates independent of cellular replication, coupled to in-depth phylogenetic information within the molecule itself. The primary aim of an RNA extraction protocol for RNA-SIP is to generate over 1µg of quantifiable RNA. Nucleic acids appear in almost all gradient fractions as revealed by high-sensitivity methods for detecting them, such as reverse transcription PCR (RT-PCR) or PCR. The chapter also talks about the RNA-SIP- directed investigation that ultimately led to the isolation of a novel Thauera strain responsible for the observed phenol degradation and to confirmation that it could be used to revive sludge that had lost phenol-degrading activity. These findings changed one's understanding of the microbes responsible for phenol degradation in aerated sludge, revealed the pitfalls of both conventional culturing and basic molecular approaches, and highlighted the importance of methods linking function with phylogeny. Manefield used RNA-SIP to compare the communities dominating the assimilation of carbon from phenol in near-identical wastewater treatment bioreactors that were operated in the same manner but differed in wastewater treatment performance. This study revealed that Acidovorax species were responsible for phenol degradation and the poor performance of one reactor was associated with two different Acidovorax populations, while the strong performance of the other was associated with a single dominant Acidovorax lineage.

This chapter reviews the methodologies used to present stable isotopically labeled substrates to microbial communities present in a wide range of environmental materials, and also discusses the wet chemical and instrumental methods used to determine compound-specific δ13C values of individual phospholipid fatty acid (PLFA). The different ways in which the δ 13C values obtained can be used to assess a variety of properties and activities of microbial communities in the environment are discussed in this chapter. The most widely used labeling techniques for PLFA- stable isotope probing (SIP) utilize 13C-enriched gases as substrates. The PLFA-SIP approach significantly extends conventional PLFA profiling methods by identifying PLFAs diagnostic of specific functional groups through their enhanced 13C signatures derived from the assimilation of applied 13C-substrate(s). In this respect, the approach has key resonances with current trends in the way that environmental microbial communities are being considered in terms of the functioning and stability of ecosystems, especially in relation to the importance of ecosystem services in the context of sustainable environments. The relative ease of preparing PLFA fatty acid methyl esters (FAMEs), combined with the high sensitivity of the gas chromatography combustion-isotope ratio mass spectrometry (GC-C-IRMS) method, makes PLFA-SIP an extremely robust methodology, which allows very large numbers of environmental samples to be studied.

The chapter on protein stable isotope probing (protein-SIP) focuses on cultivation conditions, extraction of proteins, different protein-separation techniques, methods of mass spectrometry (MS) analysis, and the calculation of stable isotope incorporation levels. The outlook includes the use of other isotopes than carbon, other molecule classes than proteins, and how protein stable isotope probing (protein-SIP) is integrated into metaproteomic approaches for the analysis of structure and function of microbial consortia. This chapter describes the method of protein-SIP by means of proof-of-principle experiments using Pseudomonas putida strain ML2 as the model organism and with fully labeled [13C]benzene or 15NH4 as a model substrate. For intact protein profiling (IPP), measurements were performed in the positive voltage polarity ion reflector mode. Mostly, recorded MS/ MS-spectra are of poor quality and mass-tags of only a few generated amino acid-ions can be defined and compared with the database. The measurements for shotgun mass mapping (SMM) were performed with external calibration using the peptide calibration standard II, which covered a mass range from m/z ~700 to 3,200. In a study, P. putida ML2, an aerobic, heterotrophic proteobacterium, was grown in mineral medium in the presence of [12C]benzene, fully labeled [13C]benzene, or [12C]benzene and labeled 15NH4Cl. In the stationary growth phase, cells were harvested, and subsequently the whole protein extracts were separated by 2-D gel electrophoresis. One of the most important challenges is the identification of peptides from samples with dynamic and variable labeling. Therefore, new algorithms for the analysis of fragmentation spectra and database search are needed.

Two promising culture-independent approaches that have been employed to assess the function and metabolic potential of uncultivated microorganisms are stable isotope probing (SIP) and metagenomics. This chapter discusses the methodology of metagenomics within the context of DNA stable isotope probing (DNA-SIP), and provides a description of the possible limitations and how these limitations can be overcome, summarizes the combined DNA-SIP and meta-genomic studies to date, and highlights future directions. The chapter also focuses on metagenomics as it relates to SIP and highlights some of the methodological considerations for cloning and characterization of labeled DNA from active and uncultivated microorganisms. A study using SIP and metagenomics with increasingly low substrate concentrations to characterize marine methylotrophs involved in C1 cycling of surface seawater was a proof-of-concept approach that utilized multiple displacement amplification (MDA) for the first time in association with DNA-SIP and metagenomics. The study also demonstrated that DNA-SIP employing near-in situ substrate concentrations may be used because the resulting low yields of DNA are still amenable to metagenomic analysis through MDA amplification. The combination of SIP, MDA, and metagenomics provides powerful access to the genomes of active-but-uncultivated microorganisms. An alternative approach for combining SIP and metagenomics is to profile the purified 13C-labeled DNA with high-throughput sequencing of cloned DNA fragments. DNA-SIP paired with metagenomics is expected to yield invaluable insight into the uncultured microbial world as the techniques become increasingly commonplace, isotopes become increasingly available and affordable, and experiments become increasingly well designed.

This chapter reviews the use of 18O-water in stable isotope probing (SIP) studies. Research groups made important contributions to understanding of adenosine triphosphate (ATP) production in mitochondria and bacteria. Richards and Boyer, employing 18O-water, showed that oxygen atoms from water can be transferred to DNA inside E. coli cells. Subsequently, it was shown that 18O-water may also be used to label DNA formed in soil. Water is a small molecule and therefore can rapidly diffuse throughout the soil environment, so that the label is relatively homogenously distributed throughout the sample and all soil organisms are exposed to similar concentrations of label. However, it is unlikely that the label will be completely homogenously distributed in soil. SIP with 18O-water may also be used to study the impact of environmental conditions such as temperature or pH on microbial population dynamics in soil. Finally, SIP with 18O-water is suitable for studies on the impact of complex nutrient sources on microbial population dynamics in soil. For instance, it is well known that plant litter quality, often gauged by measuring the lignin to nitrogen ratio, affects decomposition rates and therefore multiple nutrient cycles in soil.

This chapter examines technical considerations associated with the use of 15N in nucleic acid stable isotope probing (SIP) experiments, discusses concerns that should be considered prior to undertaking a 15N-labeling experiment, and provides an overview of different applications of 15N-SIP. The preferable approach is to use quantitative PCR (qPCR) to determine the number of 16S rRNA genes in each gradient fraction. The gene target for qPCR analysis can vary by application, and it may be desirable to use universal 16S rRNA gene-targeted primers or primers that are specific to individual domains, individual subgroups, or genera. In DNA purified by secondary gradient fractionation, nif H genes similar to Methylosinus represented 53% of those recovered while Methylocystis-like sequences represented 17% of those recovered. However, in an experiment in which 15N2-DNA-SIP was used to examine nitrogen-fixing methanotrophs in soil. There are several reasons why 15N-DNA-SIP represents an appealing method for examining nitrogen (N2)-fixing organisms. First, incubations can be carried out at realistic concentrations of substrate, as air can be evacuated from sealed containers and replaced with simulated air containing 15N2. Second, since nitrogen fixation is inhibited in the presence of mineral forms of nitrogen, problems associated with isotope dilution can largely be ignored. Experiments will need to be performed with pure cultures and environmental samples to determine whether 15N-RNA-SIP can be used effectively in microbial ecology studies.

This chapter summarizes the applications of stable isotope probing (SIP) technology in plant-soil systems and presents an overview of the progress achieved in the understanding of the plant-soil microbe interactions and their role in ecosystem functioning. The applications of phospholipid fatty acids-based SIP (PLFA-SIP) and then the applications of DNA- and RNA-based SIP in upland soils and flooded rice field soils, respectively, are described. Several studies have exploited PLFA-SIP technology to determine the plant-microbe interactions driven by rhizosphere carbon flow. In these studies, the living plants, either in the field or laboratory, are exposed to 13C-labeled CO2, and the microbial PLFAs are collected from rhizosphere soil. After pulse-labeling of rice plants with 13CO2 in a microcosm, soil samples were divided into rhizosphere and bulk soil, and the bulk soil samples were further partitioned vertically into upper layer and lower layer and horizontally into five layers with an increasing distance from roots. A study performed on grassland soil and on peatland soil, targeted mainly the root symbiont's arbuscular mycorrhizal (AM) fungi and the bacteria possibly associated with them. RNA-SIP revealed that AM fungi were labeled with 13C immediately after plant assimilation, suggesting that AM fungi preferentially used assimilates provided by plants rather than previously fixed carbon. Combining SIP with techniques such as metatranscriptomics, pyrosequencing, and community systems biology, promises a better and deeper understanding of plant-microbe interactions.

This chapter summarizes the state of the art for applications of stable isotope probing (SIP) to bio-degradation and bioremediation research. SIP is one of the many emerging tools of inquiry used by environmental microbiologists. The goals of this chapter are to catalog and analyze trends exhibited by the majority of studies published to date that are pertinent to biodegradation and bioremediation. 14C-based phospholipid fatty acid (PLFA)-SIP showed chromatographic profiles related to but distinct from known type II methanotrophs. Using PLFA-SIP analyses, a variety of comparisons were made between forest, shrubland, and pasture soils; type II methanotrophs were dominant in forest and shrubland, while type I methanotrophs dominated pasture soil. When suitable probes are available, fluorescent in situ hybridization (FISH) is an effective means of microscopic identification of microorganisms and can be combined with techniques using radioactive and stable isotopes to identify metabolically active microorganisms. The combined microscopic approaches of FISH and secondary ion mass spectrometry (SIMS) have tremendous potential to aid investigations examining the roles of bacteria in biogeochemical processes and in the biodegradation of organic pollutants. SIP is a means toward the goal of improved pollution-control technology. In general, no single technique or piece of evidence is sufficient to advance the discipline of environmental microbiology. But SIP is inherently heuristic—results have the potential to create new information and hypotheses that can be tested and confirmed with multidisciplinary approaches.

The isolation of microorganisms in pure culture has established a solid foundation of the metabolic capabilities of microorganisms, which has allowed us to understand key transformation processes in nature as well as in microbial biochemistry and molecular biology. This chapter focuses on trophic interactions involving microbes and food webs as revealed by stable isotope probing (SIP) of nucleic acids and also highlights studies using related techniques where exceptional insights have been gained in delineating trophic interactions. The concept of cooperation in anaerobic degradation of organic matter is briefly introduced to facilitate an understanding of carbon flow and trophic interactions in the anaerobic microbial food chain that is governed by both specialization of the key players and thermodynamic constraints. The thermodynamic constraints on fatty acid oxidations provide a biogeochemical framework which renders only syntrophic secondary fermenters capable of dissimilation and assimilation: only syntrophic coupling of fatty acid-oxidizing and hydrogen- and acetate-scavenging reactions makes fatty acid oxidation under methanogenic conditions exergonic. A number of studies have shown how carbon (and nitrogen) flow can be traced through microbial communities and food webs via phylogenetically identified microbes and higher-trophic-level consumers by using nucleic acid SIP and novel single-cell-based approaches of SIP.

This chapter describes the recent developments that have started to shed some light on the fermentative capacity of the colonic microbiota and its contribution to health and disease. The combined use of stable isotope probing (SIP) with analytical, gastroenterological, nanotechnological, and phylogenetic microarray techniques is highlighted in the chapter. The human gastrointestinal (GI) tract comprises a series of organs ranging from the stomach to the distal colon, with complex and dynamically changing conditions, that harbors immense microbial assemblages that are known to be critical for human health and disease. Now, many tools and techniques are available to comprehensively characterize the microbial diversity in the human gut. The study of the composition of the (intestinal) microbiota has recently gained enormous momentum through the development of DNA microarray methods to study the presence of hundreds of species at the same time. Endogenous proteins either are of secretory origin or enter the gut lumen as desquamated epithelial cells. Most microorganisms in the colon prefer to ferment carbohydrates and switch to protein fermentation when fermentable carbohydrates have been used up. The flux model currently describes the collective intestinal microbiota as a single entity. Since the bacteria that primarily used the glucose are known from the SIP results, this model could be deconvoluted into the individual microorganisms contributing to the production of the labeled metabolites. Galacto-oligosaccharides (GOS) are nondigestible carbohydrates that are resistant to GI digestive enzymes but can be fermented by specific colonic bacteria.

This chapter reviews the state of the art in isolation of new and valuable industrial enzymes from natural biodiversity, and discusses the strengths and weaknesses of each approach and shows how results with DNA stable isotope probing (DNA-SIP) indicate that this technique has substantial promise for improving the effectiveness of strategies for discovering new and valuable enzymes. The many successes of microbial enzymes from extreme environments as biocatalysts have generated an increasing demand for new, robust, and highly specific enzymes to perform all manner of transformations. Metagenomics has emerged as a powerful approach to access genes from uncultivated microbes by direct cloning of microbial DNA extracted from the environment. The study by Jeon and colleagues combined DNA-SIP and traditional microbiological methods to discover novel microorganisms involved in the degradation of the polyaromatic pollutant naphthalene. This study is an excellent example of how such techniques could be combined to discover new enzymes. The DNA-SIP gene mining approach allows the enrichment and recovery of functional genes from active and potentially uncultivated microorganisms and therefore is of great interest to the biotechnology industry. DNA-SIP is a powerful approach that can be used to enrich target genes for gene mining and thus improve and facilitate their screening in clone libraries. DNA-SIP can be combined with recent mutagenesis techniques such as gene shuffling in order to create large numbers of potentially novel biocatalysts.

As with many other molecular microbiology methods, the family of stable isotope probing (SIP) techniques based on the analysis of isotope- labeled nucleic acids, phospholipid fatty acids (PLFAs), or proteins are frequently performed at the level of the community. Single-cell studies encompass a range of techniques, including bacterial bioreporters, flow cytometry, fluorescence in situ hybridization (FISH) in combination with microautoradiography (MAR), nanoscale secondary ion mass spectrometry (nanoSIMS), and Raman spectroscopy. In general, FISH procedures are almost identical to standard epifluorescence microscopic procedures. The ability to analyze microbial communities at the level of the single cell, rather than at the level of the population or community, is one of the main advantages of Raman, and the area where it differs from conventional SIP techniques. Conventional SIP techniques tend to focus on isotope incorporation into one specific biomolecule, such as PLFAs, DNA, RNA, or proteins. Raman has a significant advantage in that it is looking at isotope incorporation by all these biomolecules simultaneously, depending of course on the exact constitution and quality of the collected Raman spectra. Raman is a sensitive technique, with isotope incorporation being detected in microbes cultured in growth media containing as low as 10% 13C. Raman spectroscopy has also been combined with optical trapping and manipulation to achieve cell sorting of isotopically labeled cells.

This chapter focuses on the application of nano-secondary ions mass spectrometry (nanoSIMS) coupled to in situ hybridization for tracking isotopically labeled cells within complex microbial communities and the importance of nanoSIMS-derived methodologies in analyzing the metabolic and phylogenetic diversity of microorganisms in the environment. The coupling of nanoSIMS and in situ hybridization allows simultaneous quantification of substrate uptake and phylogenetic identification of an individual microbial cell in a single nanoSIMS scan. Halogens such as fluorine, bromine, or iodine are used as markers for the Identification of the cells and are specifically introduced into the cells via in situ hybridization. The broad application of fluorescence in situ hybridization (FISH) on a wide variety of samples offers a big advantage for any combined method that uses FISH or in situ hybridization-related techniques for the phylogenetic identification of single cells. The chapter outlines FISH-based Identification and in situ hybridization using halogen-containing tyramides coupled to nanoSIMS, with focus on the usefulness of both approaches and the problems that can emerge. The first application of the halogen-based identification and nanoSIMS in the natural environment was performed on individual cells of the anaerobic, phototrophic bacteria inhabiting the oligotrophic, meromictic alpine Lake Cadagno. The study focused on quantification of the metabolic activities of three different bacterial species, Chlorobium clathratiforme, Chromatium okenii, andLamprocystis purpurea. Moreover, a remarkable variability in metabolic rates of individual cells of the same species was measured, showing for the first time that a microbial population is a heterogeneous group of physiologically distinct individuals.

The combination of fluorescence in situ hybridization and microautoradiography (FISH-MAR), as well as the isotope array, which are described in this chapter, are both based on the use of radioactive isotopes for revealing a specific ecophysiology and rRNA for identifying the respective organisms and provide the direct means to identify microbes that catalyze a defined process in an ecosystem. In the combination of both approaches, DNA-stable isotope probing (SIP) serves to rapidly narrow down the number of 16S rRNA genes, which might originate from substrate-consuming microbes, and FISH-MAR is then used to prove the actual functional involvement of these bacteria. Isotope arrays benefit from the fact that rRNA is labeled faster than DNA after exposure of a cell to a suitable labeled substrate, but are less sensitive than FISH-MAR because a single biomarker and not all cellular compounds contribute to the radioactive signal. If used with 14CO2 as the activity marker, isotope arrays are able to detect metabolic activity of community members that comprise not more than 1% of all cells in the investigated sample. NanoSIMS-based techniques are even more sensitive than FISH-MAR and offer a reliable quantification of the incorporated radiotracer per cell, but do not provide information about the labeled compound classes in the cell, are much more timeconsuming, and can only be performed in a few laboratories worldwide.

This chapter discusses RNA-radioisotope probing (RNA-RIP) for studying carbon metabolism in soils. Identifying the microorganisms driving bio-geochemical processes and how these are influenced by the physicochemical environment is the key to understand how microbial systems such as soils work. Thus, the use of stable isotope probing (SIP) has meant that the link between phylogeny and function for some metabolic processes can now be realized by coupling labeled biogeochemical tracers to the highly resolved, phylogenetic information held in the 16S rRNA and 18S rRNA of microbial cells. The most frequently used element in most SIP applications has been carbon in its stable isotope form of 13C. Generally, following isotopic enrichment and separation of “heavy” and “light” DNA or rRNA, the nucleic acids are further analyzed using molecular fingerprinting techniques, such as terminal restriction fragment length polymorphism (TRFLP) or denaturing gradient gel electrophoresis (DGGE), to identify and characterize the organisms that have assimilated the substrates. However, as DNA- and RNA-stable isotope probing (RNA-SIP) approaches have been applied to more ecologically relevant experiments, a new understanding is slowly emerging. These studies suggest that certain bacterial phyla can be differentiated into copiotrophic and oligotrophic categories that correspond to the r- and K-selected categories used to describe the ecological attributes of plants and animals.

In environments where growth can be fast and production of proteins and metabolites is rapid, the stable isotope probing (SIP) approach is at its best. Rapid incorporation of stable isotopes into DNA, RNA, protein, and/or metabolites will be used routinely in medical and dental research, perhaps drawing these fields far closer toward environmental microbiology than they have been in the past. Eukaryotes evolved in a sea of Bacteria and Archaea, and it would be astounding if there were not many interdependent metabolic interactions that describe the total organism as a eukaryotic/bacterial/archaeal conglomerate. This understanding will be one of the great accomplishments of the next decades and will be greatly enhanced by SIP technologies at nearly every level (DNA, RNA, protein, and metabolites). The differences between hydrogen transfer and electron transfer could be significant and offer some major challenges to the understanding of microbial ecology: challenges that may be solved in part via the application of SIP approaches and others that will require new ways of thinking and experimentation. Several papers have appeared recently on extracellular electron transport. It is clear from the work in several laboratories that microbes have mechanisms available to them to donate electrons directly to solid surfaces and to take electrons directly from solid surfaces raising the possibility that an energy realm previously not thought possible by most microbiologists could exist in sedimentary environments, utilizing various types of extracellular electron transport mechanisms to deliver energy in the form of electrons among energy sources, cells, and electron acceptors.