Differential Responses of Nitrate Reducer Community Size,Structure, and Activity to Tillage Systems

The main objective of this study was to determine how the size, structure, and activity of the nitrate reducer community were affected by adoption of a conservative tillage system as an alternative to conventional tillage. The experimental field, established in Madagascar in 1991, consists of plots subjected to conventional tillage or direct-seeding mulch-based cropping systems (DM), both amended with three different fertilization regimes. Comparisons of size, structure, and activity of the nitrate reducer community in samples collected from the top layer in 2005 and 2006 revealed that all characteristics of this functional community were affected by the tillage system, with increased nitrate reduction activity and numbers of nitrate reducers under DM. Nitrate reduction activity was also stimulated by combined organic and mineral fertilization but not by organic fertilization alone. In contrast, both negative and positive effects of combined organic and mineral fertilization on the size of the nitrate reducer community were observed. The size of the nitrate reducer community was a significant predictor of the nitrate reduction rates except in one treatment, which highlighted the inherent complexities in understanding the relationships the between size, diversity, and structure of functional microbial communities along environmental gradients.The transition from intensive tillage to various forms of conservation tillage began more than 50 years ago with the development of herbicides which have replaced mechanical cultivation. Since then, the principles of no-till cropping have been extensively adopted by farmers worldwide. This cropping system, also known as direct seeding, mimics natural systems by leaving the soil mostly undisturbed and permanently covered with crop residues or living plants. The benefits of reducing tillage in sustainable agriculture are now well recognized for various environmental and economic reasons (14). Leaving all residues of the previous crop on the soil surface protects against evaporative water loss, wind erosion, and surface water runoff. Concomitant with reduced erosion, no-till cropping can also result in enhanced soil carbon storage in the topsoil layer, with estimated carbon sequestration rates of 30 to 60 g C m² year⁻¹ (27, 50). In turn, these changes in soil organic matter and soil structure under a no-till cropping system can affect microbial communities (20). Thus, the microbial biomass is most often higher in no-till systems than in conventional tillage systems (11, 26). Analysis of the structure or activity of soil microbial communities has also revealed significant differences between conventional tillage and minimal tillage or no-tillage systems (25, 29). However, although the effect of tillage practices on the total soil microbial community in relation to soil organic matter management has frequently been investigated, knowledge of the changes in N-cycling microbial communities induced by no-till management is limited and is mainly focused on N process rates (3, 11, 32).The aim of this work was to determine how conversion from conventional tillage to no-till affects microorganisms involved in the N cycle. For this purpose, we used the nitrate reducing community as a model functional guild (40). Prokaryote nitrate reducers constitute a wide taxonomic group with a shared ability to produce energy from the dissimilatory reduction of nitrate to nitrite, the first step of denitrification and of the dissimilatory processes of reduction of nitrate to ammonium (39). Nitrate reduction by denitrification is of great importance, since the resulting nitrite is then reduced to N₂O or N₂ gases, which can lead to considerable nitrogen losses in agriculture and emissions of the N₂O greenhouse gas (4, 13). We hypothesized that higher C and N contents in the no-till system will result in increased nitrate reduction rates and nitrate reducer abundance combined with shifts in the community composition. Relationships between the size, activity, and structure of the nitrate reducer community in the studied cropping systems were also investigated. The structure and size of the nitrate reducer community were assessed by fingerprinting and real-time PCR using the narG and napA genes, encoding the membrane-bound and periplasmic nitrate reductases, respectively, as molecular markers (40, 41). The potential activity of the nitrate reducing community was determined by colorimetric measurement of the nitrite produced during nitrate reduction.     

Role of Plant Residues in Determining Temporal Patterns of the Activity,Size, and Structure of Nitrate Reducer Communities in Soil

The incorporation of plant residues into soil not only represents an opportunity to limit soil organic matter depletion resulting from cultivation but also provides a valuable source of nutrients such as nitrogen. However, the consequences of plant residue addition on soil microbial communities involved in biochemical cycles other than the carbon cycle are poorly understood. In this study, we investigated the responses of one N-cycling microbial community, the nitrate reducers, to wheat, rape, and alfalfa residues for 11 months after incorporation into soil in a field experiment. A 20- to 27-fold increase in potential nitrate reduction activity was observed for residue-amended plots compared to the nonamended plots during the first week. This stimulating effect of residues on the activity of the nitrate-reducing community rapidly decreased but remained significant over 11 months. During this period, our results suggest that the potential nitrate reduction activity was regulated by both carbon availability and temperature. The presence of residues also had a significant effect on the abundance of nitrate reducers estimated by quantitative PCR of the narG and napA genes, encoding the membrane-bound and periplasmic nitrate reductases, respectively. In contrast, the incorporation of the plant residues into soil had little impact on the structure of the narG and napA nitrate-reducing community determined by PCR-restriction fragment length polymorphism (RFLP) fingerprinting. Overall, our results revealed that the addition of plant residues can lead to important long-term changes in the activity and size of a microbial community involved in N cycling but with limited effects of the type of plant residue itself.Modern agricultural practices include a return of plant residues to soil, as this is considered sustainable to the environment. It is now recognized that the conversion of native land into cultivated systems leads to carbon losses, which can be up to 20 to 40% (17). Postharvest plant residues therefore represent an important source of carbon, helping to replenish soil organic matter that decomposes as a result of cultivation. Decomposing plant residues are also a source of nutrients, such as nitrogen, with reduced nitrate leaching compared to mineral fertilizers, which is beneficial for water quality (3). In addition, leaving the plant residue on the soil surface limits water losses by evaporation and prevents soil erosion by wind or water (15).The biochemical composition of plant residues is one of the most important factors influencing their decomposition in soil (14, 28, 29, 51). Indeed, Manzoni et al. (28), using a data set of 2,800 observations, showed previously that the patterns of decomposition were regulated by the initial residue stoichiometry. Several other factors such as climatic conditions, soil type, or localization of the residue in the soil (incorporated or on the soil surface) were also reported previously to influence decomposition (2, 24, 29, 44). Microorganisms are the major decomposers of organic matter in soil, and therefore, the diversity and activity of the microbial community during plant residue decomposition has received much attention (6, 23, 26, 27, 35). It was shown previously that the biochemical composition of plant residues influences microbial respiration (8) and microbial community structure (7, 37). The recent development of carbon-labeling approaches has furthered our knowledge of the microorganisms that actively assimilate the carbon derived from various plant residues (10, 31). However, most of those studies focused on microorganisms involved in C mineralization, and in contrast, very little is known about the effect of plant residue decomposition on the microbial communities involved in biochemical cycles other than the carbon cycle. Thus, despite the influence of plant residues on nitrogen cycling (1, 4, 5, 16, 20), studies assessing the effect of the presence and composition of plant residues on the ecology of microbial communities involved in nitrogen cycling are rare (21, 32, 36).The dissimilatory reduction of nitrate into nitrite is the first step in the processes of denitrification and the dissimilatory reduction of nitrate to ammonium (33, 41). The reduction of nitrate by denitrification leads to losses of nitrogen, which is often a limiting nutrient for plant growth in agriculture. Two types of dissimilatory nitrate reductases, differing in location, have been characterized: a membrane-bound nitrate reductase (Nar) and a periplasmic nitrate reductase (Nap) (9, 53). Nitrate reducers can harbor either Nar, Nap, or both (40, 47). Nitrate reducers are probably the most taxonomically diverse functional community within the nitrogen cycle, with members in most bacterial phyla and also archaea (42). Because of this high level of diversity of heterotrophs sharing the ability to produce energy from nitrate reduction, nitrate reducers are an excellent model system to investigate the response of the N-cycling community to plant residue addition.The aim of this work was to determine how the incorporation of plant residues with contrasting biochemical compositions into soil affects the nitrate-reducing community. For this purpose, we monitored the dynamics of the potential activity, size, and structure of the nitrate-reducing community after the addition of wheat, rape, or alfalfa residues to soil in a field experiment. As the nature and availability of the substrate change during residue decomposition (38, 39, 48), the influence of the incorporation of different plant residues on the nitrate-reducing community was investigated at several sampling times for 11 months.     

Succession of Bacterial Populations during Plant Residue Decomposition in Rice Field Soil

The incorporation of rice residues into paddy fields strongly enhances methane production and emissions. Although the decomposition processes of plant residues in rice field soil has been documented, the structure and dynamics of the microbial communities involved are poorly understood. The purpose of the present study was to determine the dynamics of short-chain fatty acids and the structure of bacterial communities during residue decomposition in a rice field soil. The soil was anaerobically incubated with the incorporation of rice root or straw residues for 90 days at three temperatures (15, 30, and 45°C). The dynamics of fatty acid intermediates showed an initial cumulative phase followed by a rapid consumption phase and a low-concentration quasi-steady state. Correspondingly, the bacterial populations displayed distinct successions during residue decomposition. Temperature showed a strong effect on the dynamics of bacterial populations. Members of Clostridium (clusters I and III) were most dominant in the incubations, particularly in the early successions. Bacteroidetes and Chlorobi were abundant in the later successions at 15 and 30°C, while Acidobacteria were selected at 45°C. We suggest that the early successional groups are responsible for the decomposition of the easily degradable fraction of residues, while the late successional groups become more important in decomposing the less-degradable or resistant fraction of plant residues. The bacterial succession probably is related to resource availability during residue decomposition. The fast-growing organisms are favored at the beginning, while the slow-growing bacteria are better adapted in the later stages, when substrate availability is limiting.Rice residues, including root and straw residues, serve as the major carbon source in paddy fields. It has been estimated that the amounts of organic matter supplied annually to paddy fields range from 1,700 to 3,470 kg ha⁻¹, and more than 65% of them were derived from plant residues (19, 23). The incorporation of rice residues into paddy fields helps sustain soil organic matter, improve physical and chemical properties, and increase nutrient availability (15, 39). However, it also strongly enhances methane production and emissions (6, 45, 46).Numerous studies have been carried out on residue decomposition and CH₄ production in rice field soils (11-13, 22). The rate of decomposition usually is separated into a fast phase and a slowdown phase (24). According to the dynamics of the intermediates H₂ and fatty acids and the activities of polysaccharolytic enzymes, Glissmann and Conrad (13) proposed five stages for residue decomposition, including the production and consumption of reducing sugars, the production and consumption of H₂ and fatty acids, and the production of CH₄. The rate of decomposition was affected by the composition of residues (11, 18). Root residues generally are decomposed slower than straw residues (24). The fermentation pathway also could differ depending on the residue material, resulting in different fatty acid intermediates (13).A complex microbial assemblage consisting of hydrolytic, fermenting, homoacetogenic, syntrophic, and methanogenic microorganisms are involved in the anaerobic decomposition of organic residues (5, 41, 48). Plant residues are composed of complex components. With the decomposition process, the proportion of labile component decreases while the resistant components relatively accumulate. Changes in the activity and structure of the microbial community thus are anticipated during the processes of residue decomposition. However, little has been known about the dynamics of microbial populations during residue decomposition in anoxic rice soil. Using culture-independent methods, Weber et al. (47) showed that the structure of the bacterial community shifted between early and late stages. Microscopic observation revealed the differential colonizations of bacterial populations on different parts of straw residue, indicating the effects of residue quality and niche condition (18). The microbial community appears to form a spatially well organized architecture, with the fermenting bacteria colonizing the residue particles, while the syntrophic bacteria and methanogens mainly inhabit the adjacent soil during the decomposition process (12).Air temperature exhibits a large seasonal variation in southeastern Asia, where rice is widely cultivated. The lowest and highest records in our research site, for instance, were −5 and 40°C, respectively, in 2006, when we collected the soil samples. It has been demonstrated that temperature has a strong effect on residue decomposition and CH₄ production (8, 22, 33). However, it is uncertain whether the effect also is reflected in the structure and function of degrading microbial communities in the soil. Therefore, the purpose of our project was to determine the effect of temperature on microbial communities during the processes of plant residue decomposition in a Chinese rice field soil. The dynamics of methanogenesis and methanogenic archaea have been reported previously (33). Here, we show the results on fatty acid intermediates and the responsible bacterial communities.     

Pyrosequencing-Based Assessment of Soil pH as a Predictor of Soil Bacterial Community Structure at the Continental Scale

Soils harbor enormously diverse bacterial populations, and soil bacterial communities can vary greatly in composition across space. However, our understanding of the specific changes in soil bacterial community structure that occur across larger spatial scales is limited because most previous work has focused on either surveying a relatively small number of soils in detail or analyzing a larger number of soils with techniques that provide little detail about the phylogenetic structure of the bacterial communities. Here we used a bar-coded pyrosequencing technique to characterize bacterial communities in 88 soils from across North and South America, obtaining an average of 1,501 sequences per soil. We found that overall bacterial community composition, as measured by pairwise UniFrac distances, was significantly correlated with differences in soil pH (r = 0.79), largely driven by changes in the relative abundances of Acidobacteria, Actinobacteria, and Bacteroidetes across the range of soil pHs. In addition, soil pH explains a significant portion of the variability associated with observed changes in the phylogenetic structure within each dominant lineage. The overall phylogenetic diversity of the bacterial communities was also correlated with soil pH (R² = 0.50), with peak diversity in soils with near-neutral pHs. Together, these results suggest that the structure of soil bacterial communities is predictable, to some degree, across larger spatial scales, and the effect of soil pH on bacterial community composition is evident at even relatively coarse levels of taxonomic resolution.The biogeographical patterns exhibited by microbial communities have been examined in a wide range of environments, and studies focusing on microbial biogeography continue to be published at a rapid pace. We know that microbial community diversity and composition can vary considerably across space, and this variation is theorized to be linked to changes in a number of biotic or abiotic factors (22, 36, 41). There are numerous overarching reasons for this interest in understanding microbial biogeography. For example, comparing microbial patterns to those commonly observed in plant and animal taxa is of intense theoretical interest (22, 25). From a more practical standpoint, studies of microbial biogeography can often provide key insights into the physiologies, environmental tolerances, and ecological strategies of microbial taxa, particularly those difficult-to-culture taxa that often dominate in natural environments. However, perhaps the most important rationale for studying microbial biogeography is the most basic one: microbes are diverse, ubiquitous, and abundant, yet their biogeographical patterns and the factors driving these spatial patterns often remain poorly understood.No single biogeographical pattern is shared by all microorganisms, just as there is no single biogeographical pattern followed by all “macrobial” (i.e., plant and animal) communities (31). The specific biogeographical patterns exhibited by microorganisms are variable and highly dependent on a number of factors, including the taxonomic group in question (29), the degree of phylogenetic resolution at which the communities are examined (e.g., Pseudomonas) (7), and the spatial scale of the study (40). However, some common patterns emerge if we specifically examine the biogeography of soil microorganisms. In particular, the structure and diversity of soil bacterial communities have been found to be closely related to soil environmental characteristics (5, 37, 47), and soil pH is often correlated with the observed biogeographical patterns (19, 24). However, due to the paucity of detailed and comprehensive studies of soil bacterial biogeography, particularly across larger spatial scales, our understanding of soil microbial biogeography remains incomplete.Previous studies of soil bacterial biogeography have focused on either surveying a few soils in detail or surveying a larger number of soils by techniques that offer less detailed phylogenetic information. For example, a few recent studies used pyrosequencing or Sanger sequencing-based techniques to deeply survey the diversity and composition of the bacterial communities within a single soil or a few soils (1, 14, 20, 39, 42). Such studies are valuable in that they provide our best assessments of overall bacterial diversity and community structure and the relative abundances of specific bacterial taxa within soils. However, because such studies often examine only a limited number of soils, they do not allow for robust assessment of biogeographical patterns and the factors that may drive these patterns. Other studies have examined bacterial communities across a larger number of soils, using more limited techniques, such as fingerprinting methods that offer little specific phylogenetic information on bacterial community structure or techniques that describe communities at very coarse levels of taxonomic resolution (18, 19). A comprehensive assessment of the biogeographical patterns exhibited by soil bacterial communities requires both depth (individual communities surveyed at a reasonable level of phylogenetic detail) and breadth (examining a sufficiently large number of samples to assess spatial patterns). With the recent development of the bar-coded pyrosequencing technique (23), we need not sacrifice depth for breadth, or vice versa. This was demonstrated in several recent studies (2, 12, 17, 28) that used bar-coded pyrosequencing to simultaneously analyze relatively large numbers of individual samples, surveying the bacterial community in each sample to an extent that would be difficult (or prohibitively expensive) using standard cloning and Sanger sequencing techniques.Here we apply the bar-coded pyrosequencing technique to examine the structure and diversity of bacterial communities in 88 soils collected from across North and South America. This work expands on a previous fingerprinting-based survey of bacterial communities across a similar set of soils (19), using the pyrosequencing technique to extend the analyses and to answer the following questions. Which taxa are most abundant in soil? How does the phylogenetic structure of bacterial communities vary across the continental scale? Which environmental factors best predict bacterial community structure and diversity? Are some soil bacterial phyla more diverse than others?     

Soil Microbial Community Responses to Multiple Experimental Climate Change Drivers

Researchers agree that climate change factors such as rising atmospheric [CO₂] and warming will likely interact to modify ecosystem properties and processes. However, the response of the microbial communities that regulate ecosystem processes is less predictable. We measured the direct and interactive effects of climatic change on soil fungal and bacterial communities (abundance and composition) in a multifactor climate change experiment that exposed a constructed old-field ecosystem to different atmospheric CO₂ concentration (ambient, +300 ppm), temperature (ambient, +3°C), and precipitation (wet and dry) might interact to alter soil bacterial and fungal abundance and community structure in an old-field ecosystem. We found that (i) fungal abundance increased in warmed treatments; (ii) bacterial abundance increased in warmed plots with elevated atmospheric [CO₂] but decreased in warmed plots under ambient atmospheric [CO₂]; (iii) the phylogenetic distribution of bacterial and fungal clones and their relative abundance varied among treatments, as indicated by changes in 16S rRNA and 28S rRNA genes; (iv) changes in precipitation altered the relative abundance of Proteobacteria and Acidobacteria, where Acidobacteria decreased with a concomitant increase in the Proteobacteria in wet relative to dry treatments; and (v) changes in precipitation altered fungal community composition, primarily through lineage specific changes within a recently discovered group known as soil clone group I. Taken together, our results indicate that climate change drivers and their interactions may cause changes in bacterial and fungal overall abundance; however, changes in precipitation tended to have a much greater effect on the community composition. These results illustrate the potential for complex community changes in terrestrial ecosystems under climate change scenarios that alter multiple factors simultaneously.Soil microbial communities are responsible for the cycling of carbon (C) and nutrients in ecosystems, and their activities are regulated by biotic and abiotic factors such as the quantity and quality of litter inputs, temperature, and moisture. Atmospheric and climatic changes will impact both abiotic and biotic drivers in ecosystems and the response of ecosystems to these changes. Feedbacks from ecosystem to the atmosphere may also be regulated by soil microbial communities (3). Although microbial communities regulate important ecosystem processes, it is often unclear how the abundance and composition of microbial communities correlate with climatic perturbations and interact to effect ecosystem processes. As such, much of the ecosystem climate change research conducted to date has focused on macroscale responses to climatic change such as changes in plant growth (43, 44), plant community composition (2, 37), and coarse scale soil processes (14, 18, 21, 26), many of which may also indirectly interact to effect microbial processes. Studies that have addressed the role of microbial communities and processes have most often targeted gross parameters, such as microbial biomass, enzymatic activity, or basic microbial community profiles in response to single climate change factors (22, 28, 29, 33, 61, 63).Climate change factors such as atmospheric CO₂ concentrations, warming, and altered precipitation regimes can potentially have both direct and indirect impacts on soil microbial communities. However, the direction and magnitude of these responses is uncertain. For example, the response of soil microbial communities to changes in atmospheric CO₂ concentrations can be positive or negative, and consistent overall trends between sites and studies have not been observed (1, 28, 34-36). Further, depending on what limits ecosystem productivity, precipitation and soil moisture changes may increase or decrease the ratio of bacteria and fungi, as well as shift their community composition (8, 50, 58). Increasing temperatures can increase in microbial activity, processing, and turnover, causing the microbial community to shift in favor of representatives adapted to higher temperatures and faster growth rates (7, 46, 60, 64, 65). Atmospheric and climatic changes are happening in concert with one another so that ecosystems are experiencing higher levels of atmospheric CO₂, warming, and changes in precipitation regimes simultaneously. Although the many single factor climate change studies described above have enabled a better understanding of how microbial communities may respond to any one factor, understanding how multiple climate change factors interact with each other to influence microbial community responses is poorly understood. For example, elevated atmospheric [CO₂] and precipitation changes might increase soil moisture in an ecosystem, but this increase may be counteracted by warming (10). Similarly, warming may increase microbial activity in an ecosystem, but this increase may be eliminated if changes in precipitation lead to a drier soil condition or reduced litter quantity, quality, and turnover. Such interactive effects of climate factors in a multifactorial context have been less commonly studied even in plant communities (45), and detailed studies are rarer still in soil microbial communities (25). Clearly, understanding how microbial communities will respond to these atmospheric and climate change drivers is important to make accurate predications of how ecosystems may respond to future climate scenarios.To address how multiple climate change drivers will interact to shape soil microbial communities, we took advantage of a multifactor climatic change experiment that manipulated atmospheric CO₂ (+300 ppm, ambient), warming (+3°C, ambient) and precipitation (wet and dry) in a constructed old-field ecosystem that had been ongoing for 3.5 years at the time of sampling. Previous work on this project has demonstrated direct and interactive effects of the treatments on plant community composition and biomass (15, 30), soil respiration (56), microbial activity (30), nitrogen fixation (21), and soil carbon stocks (20). These results led us to investigations of how the soil bacterial and fungal communities, important regulators of some of these processes, were responding using culture-independent molecular approaches. Our research addresses two overarching questions. (i) Do climatic change factors and their interactions alter bacterial and fungal abundance and diversity? (ii) Do climatic change factors and their interactions alter bacterial or fungal community composition?     

Cultivation of Fastidious Bacteria by Viability Staining and Micromanipulation in a Soil Substrate Membrane System

Soil substrate membrane systems allow for microcultivation of fastidious soil bacteria as mixed microbial communities. We isolated established microcolonies from these membranes by using fluorescence viability staining and micromanipulation. This approach facilitated the recovery of diverse, novel isolates, including the recalcitrant bacterium Leifsonia xyli, a plant pathogen that has never been isolated outside the host.The majority of bacterial species have never been recovered in the laboratory (1, 14, 19, 24). In the last decade, novel cultivation approaches have successfully been used to recover “unculturables” from a diverse range of divisions (23, 25, 29). Most strategies have targeted marine environments (4, 23, 25, 32), but soil offers the potential for the investigation of vast numbers of undescribed species (20, 29). Rapid advances have been made toward culturing soil bacteria by reformulating and diluting traditional media, extending incubation times, and using alternative gelling agents (8, 21, 29).The soil substrate membrane system (SSMS) is a diffusion chamber approach that uses extracts from the soil of interest as the growth substrate, thereby mimicking the environment under investigation (12). The SSMS enriches for slow-growing oligophiles, a proportion of which are subsequently capable of growing on complex media (23, 25, 27, 30, 32). However, the SSMS results in mixed microbial communities, with the consequent difficulty in isolation of individual microcolonies for further characterization (10).Micromanipulation has been widely used for the isolation of specific cell morphotypes for downstream applications in molecular diagnostics or proteomics (5, 15). This simple technology offers the opportunity to select established microcolonies of a specific morphotype from the SSMS when combined with fluorescence visualization (3, 11). Here, we have combined the SSMS, fluorescence viability staining, and advanced micromanipulation for targeted isolation of viable, microcolony-forming soil bacteria.     

Comparison of Effects of Compost Amendment and of Single-Strain Inoculation on Root Bacterial Communities of Young Cucumber Seedlings

Compost amendment and inoculations with specific microorganisms are fundamentally different soil treatment methods, commonly used in agriculture for the improvement of plant growth and health. Although distinct, both methods affect the rhizosphere and the plant roots. In the present study we used a 16S rRNA gene approach to achieve an overview of early consequences of these treatments on the assemblage of plant root bacterial communities. For this purpose, cucumber seedlings were grown, under controlled conditions, in perlite potting mix amended with biosolid compost or straw compost, or inoculated with Streptomyces spp. A uniform trend of response of root bacterial communities for all treatments was observed. Root bacterial density, measured as bacterial targets per plant tef gene by real-time PCR, was reduced in 31 to 67%. In addition, increased taxonomic diversity accompanied shifts in composition (α-diversity). The magnitude of change in these parameters relative to the perlite control varied between the different treatments but not in relation to the treatment method (compost amendments versus inoculations). Similarity between the compositions of root and of potting mix bacterial communities (β-diversity) was relatively unchanged. The abundance of Oxalobacteraceae was >50% of the total root bacterial community in the untreated perlite. Root domination by this group subsided >10-fold (straw compost) to >600-fold (Streptomyces sp. strain S1) after treatment. Thus, loss of dominance appears to be the major phenomenon underlining the response trend of the root bacterial communities.Environmental concern over conventional agricultural fertilization and disease control measures has led to increased interest in finding environmentally friendly alternatives. The most explored ones include compost amendments (18, 36) and the application of different microbial preparations (11, 19, 37). These are widely distinct applications. The first approach adds to the amended medium not only a rich and diverse consortium of biological agents but also organic matter and nutrients. It was confirmed that the efficacy of such treatments involves the response of the soil, the plant, and the rhizosphere microbial communities (19, 56). The activities of rhizosphere microorganisms alter the rhizosphere and thus affect plant health and root growth and development (24). Therefore, one of the main objectives of compost amendment or of inoculation with specific microbial strains is manipulation of the plant rhizosphere conditions, particularly via manipulation of the microbial community composition (32).The response of rhizosphere bacterial communities to different anthropogenic and other disturbances has been discussed in terms of resilience (3, 32). Generally, the introduction of new microorganisms produces only restricted spatial and temporal effects on the soil, rhizosphere, and root microbial communities (4, 29, 35). Thus, the plant growth-promoting effect of such treatments may be related to microbial events occurring during the early stages of plant development. Such early effects were pointed out for inoculants of different bacterial species (14, 42) and for compost amendment (15, 21, 50).Consequences of compost amendment or of single species inoculation often include shifts in the plant roots hormonal balance or a plant systemic response, namely, induced systemic resistance (8, 38, 52). Thus, direct or indirect activities of the introduced microorganisms may result in similar modifications of the root habitat. If so, bacterial assemblages of treated roots may share qualitative and quantitative characteristics different from those exhibited by untreated roots.The objective of the present study was therefore to describe and compare responses of bacterial communities of young plant roots to the application of compost or bacterial inoculants. This was performed in a simple model comprised of cucumber seedlings grown in potting mixes amended with compost or inoculated with Streptomyces spp. isolated from the two different composts.     

Characterization of Airborne Microbial Communities at a High-Elevation Site and Their Potential To Act as Atmospheric Ice Nuclei

Bacteria and fungi are ubiquitous in the atmosphere. The diversity and abundance of airborne microbes may be strongly influenced by atmospheric conditions or even influence atmospheric conditions themselves by acting as ice nucleators. However, few comprehensive studies have described the diversity and dynamics of airborne bacteria and fungi based on culture-independent techniques. We document atmospheric microbial abundance, community composition, and ice nucleation at a high-elevation site in northwestern Colorado. We used a standard small-subunit rRNA gene Sanger sequencing approach for total microbial community analysis and a bacteria-specific 16S rRNA bar-coded pyrosequencing approach (4,864 sequences total). During the 2-week collection period, total microbial abundances were relatively constant, ranging from 9.6 × 10⁵ to 6.6 × 10⁶ cells m⁻³ of air, and the diversity and composition of the airborne microbial communities were also relatively static. Bacteria and fungi were nearly equivalent, and members of the proteobacterial groups Burkholderiales and Moraxellaceae (particularly the genus Psychrobacter) were dominant. These taxa were not always the most abundant in freshly fallen snow samples collected at this site. Although there was minimal variability in microbial abundances and composition within the atmosphere, the number of biological ice nuclei increased significantly during periods of high relative humidity. However, these changes in ice nuclei numbers were not associated with changes in the relative abundances of the most commonly studied ice-nucleating bacteria.Microbes are abundant in the atmosphere, with both cultivation-dependent and molecular approaches showing that the atmosphere harbors a diverse assemblage of bacteria and fungi, including taxa also commonly found on leaf surfaces (5, 49) and in soil habitats (30). The abundance and composition of airborne microbial communities are variable across time and space (14, 24, 27, 33, 47, 48, 69). However, the atmospheric conditions responsible for driving the observed changes in microbial abundances are unknown. The diversity of airborne microorganisms, and the factors influencing diversity levels, also remains poorly characterized. One reason for these limitations in knowledge is that until recently, culture-based microbiological methods have been the standard, and it is well-recognized that such methods capture only a small portion of the total microbial diversity (59). As demonstrated in a number of recent studies (6, 13, 22, 23, 33, 52, 59, 63, 73), advances in culture-independent techniques allow far more of the microbial diversity present in the atmosphere to be surveyed and the spatiotemporal variability in microbial communities to be examined.Microbes are often considered passive inhabitants of the atmosphere, dispersing via airborne dust particles. However, recent studies suggest that many atmospheric microbes may be metabolically active (3, 4, 64), even up to altitudes of 20,000 m (34). Some airborne microbes may alter atmospheric conditions directly by acting as cloud condensation nuclei (7, 25, 56) and/or ice nuclei (IN) (19, 41, 56, 57, 61); this hypothesis is supported by the observation that most ice nuclei in snow samples are inactivated by a 95°C heat treatment (16, 17). However, the overall contribution of airborne microbes to atmospheric processes such as ice nucleation remains unclear.The best-studied ice-nucleating microbes are gram-negative bacteria that have also been isolated from leaf surfaces, including Pseudomonas syringae, Pseudomonas fluorescens, Erwinia herbicola, Xanthomonas campestri, and Sphingomonas spp. (45). These bacteria have been cultured extensively, and their ice-nucleating activity has been traced to a membrane-bound glycoprotein (40, 42, 70). However, their specific influence on atmospheric processes remains, at this point, largely anecdotal. Less is known about the ice-nucleating activities of fungi, but a few studies have shown that fungi can be effective ice nucleators, capable of initiating ice nucleation at temperatures as high as −2°C (41, 61). At this point, all known ice-nucleating microorganisms are amenable to culture-based studies, but given that the vast majority of microorganisms have yet to be cultured, it is likely that other ice-nucleating microbes remain undiscovered.The work presented here addresses three overarching questions. (i) Are microbial abundances altered by changes in atmospheric conditions? (ii) How is the diversity and composition of airborne microbial communities influenced by changes in atmospheric conditions? (iii) Can we identify known and novel ice-nucleating microbes in the atmosphere by testing for correlations between taxa abundances and the concentrations of biological ice nuclei? To address these questions, we combined epifluorescence microscopy, tagged pyrosequencing, Sanger sequencing, and an ice nucleation assay with atmospheric measurements to characterize the microbial communities at a high-elevation research site.     

Strategy for Extracting DNA from Clay Soil and Detecting a Specific Target Sequence via Selective Enrichment and Real-Time (Quantitative) PCR Amplification

We present a simple strategy for isolating and accurately enumerating target DNA from high-clay-content soils: desorption with buffers, an optional magnetic capture hybridization step, and quantitation via real-time PCR. With the developed technique, μg quantities of DNA were extracted from mg samples of pure kaolinite and a field clay soil.Isolating and characterizing DNA sequences for use in molecular methods are integral to evaluating microbial community diversity in soil (6, 21, 22, 24, 37). Any isolation protocol should maximize nucleic acid isolation while minimizing copurification of enzymatic inhibitors. Although several methods that focus on extraction of total community DNA from environmental soil and water samples have been published (7, 21, 26, 34), the lack of a standard nucleic acid isolation protocol (32) reflects the difficulty in accomplishing these goals, most likely due to the complex nature of the soil environment.DNA extraction is especially difficult for soils containing clay (3, 5), given the tight binding of DNA strands to clay soil particles (7, 10, 20). Additionally, extracellular DNA binds to and is copurified with soil humic substances (10), which inhibit the activity of enzymes such as restriction endonucleases and DNA polymerase (6, 13, 23). Although clay-bound DNA can be PCR amplified in the absence of inhibitors (1), it is often the case that inhibitors are present in the soil environment, among them bilirubin, bile salts, urobilinogens, and polysaccharides (40). Of these inhibitors, humic substances have been found to be the most recalcitrant (36).A promising technique for isolating specific target sequences from soil particles and enzymatic inhibitors is the magnetic capture hybridization-PCR technique (MCH-PCR) presented by Jacobsen (19) and used to obtain high detection sensitivities (11, 38).We have found no evidence in the published literature of the use of MCH-PCR on soils that have high clay contents and here present a three-step strategy for isolating specific DNA sequences from the most difficult soil environment—clay that contains humic substances—and enumerating a specific target sequence from the crude extract.     

Vegetation and Soil Environment Influence the Spatial Distribution of Root-Associated Fungi in a Mature Beech-Maple Forest

Although the level of diversity of root-associated fungi can be quite high, the effect of plant distribution and soil environment on root-associated fungal communities at fine spatial scales has received little attention. Here, we examine how soil environment and plant distribution affect the occurrence, diversity, and community structure of root-associated fungi at local patch scales within a mature forest. We used terminal restriction fragment length polymorphism and sequence analysis to detect 63 fungal species representing 28 different genera colonizing tree root tips. At least 32 species matched previously identified mycorrhizal fungi, with the remaining fungi including both saprotrophic and parasitic species. Root fungal communities were significantly different between June and September, suggesting a rapid temporal change in root fungal communities. Plant distribution affected root fungal communities, with some root fungi positively correlated with tree diameter and herbaceous-plant coverage. Some aspects of the soil environment were correlated with root fungal community structure, with the abundance of some root fungi positively correlated with soil pH and moisture content in June and with soil phosphorous (P) in September. Fungal distribution and community structure may be governed by plant-soil interactions at fine spatial scales within a mature forest. Soil P may play a role in structuring root fungal communities at certain times of the year.In temperate forests, most trees form relationships with ectomycorrhizal (ECM) fungi, and the diversity of this fungal group alone can approach 100 species within a forest stand (17, 20, 60). The ECM mutualism may be necessary for the success of some native plant species, as approximately 90% of roots of some tree species are colonized by ECM fungi (65). Nevertheless, we still know surprisingly little about what controls the community structure and distribution of root-associated fungi in forest systems (44, 46). The occurrence of root-associated fungi may broadly reflect soil environmental conditions and the presence of preferred plant hosts (28, 61), but how these factors interact to influence the diversity, distribution, and community structure of these fungi within forest habitat patches at a local scale is uncertain.The distribution of root-associated fungi may be primarily a species response to local soil environmental conditions. For example, both the quality (i.e., nutrient content) and the quantity of soil organic matter are known to influence the diversity of ECM communities (18, 20, 32). ECM fungi also vary in drought tolerance (14, 36), resistance to fire (61, 65), and tolerance to soil acidity (19) and temperature (56). Changes in soil chemistry, especially as they relate to pH and the availability of nitrogen (N) and phosphorous (P), might favor selection of fungi most capable of tolerating environmental extremes (2, 28, 29).Plant distribution and identity may, however, play the strongest role in structuring the below-ground diversity of root-associated fungi. Many ECM fungi can colonize a wide range of plant species, and plant species can be host to a large number of ECM fungi (63), especially those in the families Russulaceae and Thelephoraceae (34, 35, 62). Moreover, some ECM fungi are also specific to certain tree species (e.g., Suillus and Rhizopogon species are specific to species in the family Pinaceae [38, 39]). At the local scale, fungal distribution and richness might be influenced by differences in root growth and architecture (30, 42), by the distance to the bole of the tree (11, 42, 49), or by the presence of neighboring trees (29, 64). Temporal changes in ECM communities could be associated with seasonal changes in plant physiology and phenology (3, 8, 17).An often overlooked factor influencing root-associated fungi of tree roots is the occurrence of herbaceous plant species within forest stands. Many species of parasitic, achlorophyllous angiosperms obtain carbon (C) from ECM fungi that colonize tree roots (43), and some autotrophic plants could also obtain C from ECM fungi during certain times of the year (58). Herbaceous plants also influence the cycling of nutrients, including N, P, and K (potassium) (31, 50), within forests, which could affect the distribution of root-associated fungi. Herbaceous plants can also produce secondary compounds that inhibit colonization of tree roots (68).In this study, we examine the effect of soil environment and plant distribution on root-associated fungi of tree roots in a mature beech-maple forest at two points in the growing season. We predict that plant distribution, both the distribution of host trees and that of herbaceous plants, influences fungi associated with tree roots in terms of both community structure and diversity. Molecular typing protocols, including a site-specific database of fungal sequences and fingerprints, were used to identify fungi on tree roots (i.e., beech or maple trees) to the species level.     

Microbial Community Structure and Denitrification in a Wetland Mitigation Bank

Wetland mitigation is implemented to replace ecosystem functions provided by wetlands; however, restoration efforts frequently fail to establish equivalent levels of ecosystem services. Delivery of microbially mediated ecosystem functions, such as denitrification, is influenced by both the structure and activity of the microbial community. The objective of this study was to compare the relationship between soil and vegetation factors and microbial community structure and function in restored and reference wetlands within a mitigation bank. Microbial community composition was assessed using terminal restriction fragment length polymorphism targeting the 16S rRNA gene (total bacteria) and the nosZ gene (denitrifiers). Comparisons of microbial function were based on potential denitrification rates. Bacterial community structures differed significantly between restored and reference wetlands; denitrifier community assemblages were similar among reference sites but highly variable among restored sites throughout the mitigation bank. Potential denitrification was highest in the reference wetland sites. These data demonstrate that wetland restoration efforts in this mitigation bank have not successfully restored denitrification and that differences in potential denitrification rates may be due to distinct microbial assemblages observed in restored and reference (natural) wetlands. Further, we have identified gradients in soil moisture and soil fertility that were associated with differences in microbial community structure. Microbial function was influenced by bacterial community composition and soil fertility. Identifying soil factors that are primary ecological drivers of soil bacterial communities, especially denitrifying populations, can potentially aid the development of predictive models for restoration of biogeochemical transformations and enhance the success of wetland restoration efforts.Wetlands provide more ecosystem services (e.g., flood control, water purification, nutrient cycling, and habitat for wildlife) per hectare than any other ecosystem (16). Riparian wetlands, in particular, are sites of intense biogeochemical activity and play an important role in improving water quality, recycling nutrients, and detoxifying chemicals (41). Changing patterns of land use over the last century have resulted in the loss of over half of the wetlands in the contiguous United States (17) and about 60% of wetlands in the Midwestern United States (82). The loss of ecosystem services through conversion of wetlands to alternative (primarily agricultural) land uses exacerbates nutrient pollution and eutrophication of downstream ecosystems (57). Declines in wetland acreage have continued despite a federal policy goal of no-net-loss of wetland acreage and function adopted in 1990 (7, 55). Wetland mitigation projects provide compensation for impacted wetlands and aim to replace the critical functions provided by wetlands. Despite decades of wetland mitigation, however, restoration efforts frequently fail to reestablish desired levels of ecosystem services. Restoration outcomes remain uncertain, and more information is necessary in order to improve monitoring and assessment of wetland development (13, 18, 50, 80).One approach to wetland compensation is through mitigation banks. These sites are areas that are restored, established, enhanced, or preserved for replacement of wetlands that will be affected by future land use change. Mitigation banks are considered “third-party” compensatory mitigation, where the permittee (e.g., developer planning to destroy a wetland) is responsible for purchasing wetland credits in acreage, but the wetland bank is established and managed by another party (24). Wetland mitigation banks have unique characteristics that distinguish them from smaller individual restoration projects (7, 69, 81). Due to their size, wetland mitigation banks are especially heterogeneous and may have a great deal of within-site variability in hydrology and nutrient status, making it challenging to implement a single restoration design. Thus, wetland mitigation banks require intense management and monitoring for improved success (7, 69, 81).Restoration efforts such as mitigation banks aim to replace chemical, physical, and biological ecosystem functions of wetlands that have been lost through anthropogenic disturbance (24). Monitoring of wetland mitigation sites has largely focused on measures of macro-scale community structure (e.g., vegetation surveys) (52) along with measures of hydrology and soil type (24). Measurement of vegetation is a common proxy for wetland performance but does not provide an accurate assessment of wetland function (6, 52). Quantitative assessment is achievable, however, for ecosystem services such as water quality improvement through nitrate removal, where well-characterized microbial mechanisms underlie denitrification processes.The link between microbial community structure and function in a restoration context is a topic of current interest (33). Relating microbial community composition and dynamics to chemical, physical, and biological variables can help to reveal important ecological drivers of microbial communities and their activities (26, 35, 42). Conserved bacterial functional genes related to specific biogeochemical transformations allow evaluation of the community structure of microbial populations directly involved in these processes (49, 60, 63, 77, 79). Assessing the diversity of microorganisms that are specifically involved in denitrification is possible through amplification of the nosZ gene, which encodes the catalytic subunit of nitrous oxide reductase, the enzyme responsible for the final step of denitrification (60, 63, 66). Phylogenetically diverse microorganisms can carry out denitrification though the majority of previously described denitrifiers belong to subphyla within the Proteobacteria (53, 56, 60, 61). Denitrification is a facultative process that occurs only under anaerobic conditions (53, 75). Complete denitrification to N₂ is more prevalent in anaerobic, saturated wetland ecosystems (14, 76), and incomplete denitrification to N₂O is the less desirable, more common endpoint of denitrification under more aerobic, drier conditions (14, 62). While the environmental factors (e.g., oxygen, carbon, nitrate, and pH) that influence bulk denitrification rates have been well characterized (31, 72), the influence of these factors on the composition of denitrifier communities, particularly in a restoration context, is unclear. Understanding the relationship between the microbial populations responsible for nitrogen transformations and easily measured environmental parameters (e.g., soil chemical and physical measures) could lead to assessment metrics that are linked directly to ecosystem functions such as denitrification and bridge the current gap in functional assessment methods (36, 60, 70).The objectives of this study were (i) to compare the microbial and plant community composition in restored wetlands to the composition in adjacent reference floodplain forest wetlands; (ii) to assess the relationship between microbial community composition (based on terminal restriction fragment length polymorphism [T-RFLP]) and potential denitrification activity throughout the mitigation bank; and (iii) to examine soil factors correlated with microbial community composition using both phylogenetic and functional gene markers. As soil environmental conditions affect microbial community structure and activity, we expected that sites where wetland hydrology and soil chemistry have been successfully restored would harbor microbial assemblages that are similar in composition and denitrification function to those observed in reference wetlands within this mitigation bank.     

Assessment of Bias Associated with Incomplete Extraction of Microbial DNA from Soil

DNA extraction bias is a frequently cited but poorly understood limitation of molecular characterizations of environmental microbial communities. To assess the bias of a commonly used soil DNA extraction kit, we varied the cell lysis protocol and conducted multiple extractions on subsamples of clay, sand, and organic soils. DNA, as well as bacterial and fungal ribosomal gene copies as measured by quantitative PCR, continued to be isolated in successive extractions. When terminal restriction fragment length polymorphism was used, a significant shift in community composition due to extraction bias was detected for bacteria but not for fungi. Pyrosequencing indicated that the relative abundances of sequences from rarely cultivated groups such as Acidobacteria, Gemmatimonades, and Verrucomicrobia were higher in the first extraction than in the sixth but that the reverse was true for Proteobacteria and Actinobacteria. This suggests that the well-known phylum-level bacterial cultivation bias may be partially exaggerated by DNA extraction bias. We conclude that bias can be adequately reduced in many situations by pooling three successive extractions, and additional measures should be considered when divergent soil types are compared or when comprehensive community analysis is necessary.The vast majority of soil bacteria (1, 7, 27) and fungi (13, 29) cannot be cultured via traditional laboratory techniques and must be identified using molecular methods. Successful characterization of microbial communities is therefore often dependent on DNA that is extracted from the environment. However, extraction of high-quality DNA from soil can be problematic (8, 11, 22, 26). Commercial DNA extraction kits are now commonly used in the assessment of taxonomic and functional diversity, community composition, and population abundance (e.g., references 19, 21, 23, 25, and 31). Studies comparing various kits (18, 32) or comparing commercial kits to other methods (2, 10, 24) have shown that DNA yield and purity vary depending on methodology and soil type. While these comparative studies are valuable, it is still unclear to what extent these protocols yield genomic DNA representative of the microbial community found within soil.Our objective in this study was to optimize and assess the bias of a widely used commercial soil DNA extraction kit. We hypothesized that cell lysis would be enhanced and DNA would be removed from adsorption sites by conducting multiple extractions on a single sample, thereby increasing genomic DNA yield and obtaining a more complete survey of microbial taxa. This hypothesis was tested by (i) varying the extraction protocol and measuring DNA yield for three soils with differing characteristics and (ii) examining extraction bias in the genomic DNA obtained from successive extractions by using an improved method. Analytical replicates rather than biological replicates were used in order to focus strictly on variation and bias introduced through methodology, although multiple soil types were analyzed to determine whether biases detected were consistent.     

In Situ Phylogenetic Structure and Diversity of Wild Bradyrhizobium Communities

Bacteria often infect their hosts from environmental sources, but little is known about how environmental and host-infecting populations are related. Here, phylogenetic clustering and diversity were investigated in a natural community of rhizobial bacteria from the genus Bradyrhizobium. These bacteria live in the soil and also form beneficial root nodule symbioses with legumes, including those in the genus Lotus. Two hundred eighty pure cultures of Bradyrhizobium bacteria were isolated and genotyped from wild hosts, including Lotus angustissimus, Lotus heermannii, Lotus micranthus, and Lotus strigosus. Bacteria were cultured directly from symbiotic nodules and from two microenvironments on the soil-root interface: root tips and mature (old) root surfaces. Bayesian phylogenies of Bradyrhizobium isolates were reconstructed using the internal transcribed spacer (ITS), and the structure of phylogenetic relatedness among bacteria was examined by host species and microenvironment. Inoculation assays were performed to confirm the nodulation status of a subset of isolates. Most recovered rhizobial genotypes were unique and found only in root surface communities, where little bacterial population genetic structure was detected among hosts. Conversely, most nodule isolates could be classified into several related, hyper-abundant genotypes that were phylogenetically clustered within host species. This pattern suggests that host infection provides ample rewards to symbiotic bacteria but that host specificity can strongly structure only a small subset of the rhizobial community.Symbiotic bacteria often encounter hosts from environmental sources (32, 48, 60), which leads to multipartite life histories including host-inhabiting and environmental stages. Research on host-associated bacteria, including pathogens and beneficial symbionts, has focused primarily on infection and proliferation in hosts, and key questions about the ecology and evolution of the free-living stages have remained unanswered. For instance, is host association ubiquitous within a bacterial lineage, or if not, do host-infecting genotypes represent a phylogenetically nonrandom subset? Assuming that host infection and free-living existence exert different selective pressures, do bacterial lineages diverge into specialists for these different lifestyles? Another set of questions addresses the degree to which bacteria associate with specific host partners. Do bacterial genotypes invariably associate with specific host lineages, and is such specificity controlled by one or both partners? Alternatively, is specificity simply a by-product of ecological cooccurrence among bacteria and hosts?Rhizobial bacteria comprise several distantly related proteobacterial lineages, most notably the genera Azorhizobium, Bradyrhizobium, Mesorhizobium, Rhizobium, and Sinorhizobium (52), that have acquired the ability to form nodules on legumes and symbiotically fix nitrogen. Acquisition of nodulation and nitrogen fixation loci has likely occurred through repeated lateral transfer of symbiotic loci (13, 74). Thus, the term “rhizobia” identifies a suite of symbiotic traits in multiple genomic backgrounds rather than a taxonomic classification. When rhizobia infect legume hosts, they differentiate into specialized endosymbiotic cells called bacteroids, which reduce atmospheric nitrogen in exchange for photosynthates from the plant (35, 60). Rhizobial transmission among legume hosts is infectious. Rhizobia can spread among hosts through the soil (60), and maternal inheritance (through seeds) is unknown (11, 43, 55). Nodule formation on hosts is guided by reciprocal molecular signaling between bacteria and plant (5, 46, 58), and successful infection requires a compatible pairing of legume and rhizobial genotypes. While both host and symbiont genotypes can alter the outcome of rhizobial competition for adsorption (34) and nodulation (33, 39, 65) of legume roots, little is known about how this competition plays out in nature.Rhizobia can achieve reproductive success via multiple lifestyles (12), including living free in the soil (14, 44, 53, 62), on or near root surfaces (12, 18, 19, 51), or in legume nodules (60). Least is known about rhizobia in bulk soil (not penetrated by plant roots). While rhizobia can persist for years in soil without host legumes (12, 30, 61), it appears that growth is often negligible in bulk soil (4, 10, 14, 22, 25). Rhizobia can also proliferate in the rhizosphere (soil near the root zone) of legumes (4, 10, 18, 19, 22, 25, 51). Some rhizobia might specialize in rhizosphere growth and infect hosts only rarely (12, 14, 51), whereas other genotypes are clearly nonsymbiotic because they lack key genes (62) and must therefore persist in the soil. The best-understood rhizobial lifestyle is the root nodule symbiosis with legumes, which is thought to offer fitness rewards that are superior to life in the soil (12). After the initial infection, nodules grow and harbor increasing populations of bacteria until the nodules senesce and the rhizobia are released into the soil (11, 12, 38, 40, 55). However, rhizobial fitness in nodules is not guaranteed. Host species differ in the type of nodules they form, and this can determine the degree to which differentiated bacteroids can repopulate the soil (11, 12, 38, 59). Furthermore, some legumes can hinder the growth of nodules with ineffective rhizobia, thus punishing uncooperative symbionts (11, 27, 28, 56, 71).Here, we investigated the relationships between environmental and host-infecting populations of rhizobia. A main objective was to test the hypothesis that rhizobia exhibit specificity among host species as well as among host microenvironments, specifically symbiotic nodules, root surfaces, and root tips. We predicted that host infection and environmental existence exert different selective pressures on rhizobia, leading to divergent patterns of clustering, diversity, and abundance of rhizobial genotypes.     

Functional diversity and electron donor dependence of microbial populations capable of U(VI) reduction in radionuclide-contaminated subsurface sediments

In order to elucidate the potential mechanisms of U(VI) reduction for the optimization of bioremediation strategies, the structure-function relationships of microbial communities were investigated in microcosms of subsurface materials cocontaminated with radionuclides and nitrate. A polyphasic approach was used to assess the functional diversity of microbial populations likely to catalyze electron flow under conditions proposed for in situ uranium bioremediation. The addition of ethanol and glucose as supplemental electron donors stimulated microbial nitrate and Fe(III) reduction as the predominant terminal electron-accepting processes (TEAPs). U(VI), Fe(III), and sulfate reduction overlapped in the glucose treatment, whereas U(VI) reduction was concurrent with sulfate reduction but preceded Fe(III) reduction in the ethanol treatments. Phyllosilicate clays were shown to be the major source of Fe(III) for microbial respiration by using variable-temperature Mössbauer spectroscopy. Nitrate- and Fe(III)-reducing bacteria (FeRB) were abundant throughout the shifts in TEAPs observed in biostimulated microcosms and were affiliated with the genera Geobacter, Tolumonas, Clostridium, Arthrobacter, Dechloromonas, and Pseudomonas. Up to two orders of magnitude higher counts of FeRB and enhanced U(VI) removal were observed in ethanol-amended treatments compared to the results in glucose-amended treatments. Quantification of citrate synthase (gltA) levels demonstrated a stimulation of Geobacteraceae activity during metal reduction in carbon-amended microcosms, with the highest expression observed in the glucose treatment. Phylogenetic analysis indicated that the active FeRB share high sequence identity with Geobacteraceae members cultivated from contaminated subsurface environments. Our results show that the functional diversity of populations capable of U(VI) reduction is dependent upon the choice of electron donor.Uranium contamination in subsurface environments is a widespread problem at mining and milling sites across North America, South America, and Eastern Europe (1). Uranium in the oxidized state, U(VI), is highly soluble and toxic and thus is a potential contaminant to local drinking-water supplies (46). Nitrate is often a cocontaminant with U(VI) as a result of the use of nitric acid in the processing of uranium and uranium-bearing waste (6, 45). Oxidized uranium can be immobilized in contaminated groundwater through the reduction of U(VI) to insoluble U(IV) by indirect (abiotic) and direct (enzymatic) processes catalyzed by microorganisms. Current remediation practices favor the stimulation of reductive uranium immobilization catalyzed by indigenous microbial communities along with natural attenuation and monitoring (5, 24, 40, 44, 65, 68, 69). Microbial uranium reduction activity in contaminated subsurface environments is often limited by carbon or electron donor availability (13, 24, 44, 69). Previous studies have indicated that U(VI) reduction does not proceed until nitrate is depleted (13, 16, 24, 44, 68, 69), as high nitrate concentrations inhibit the reduction of U(VI) by serving as a competing and more energetically favorable terminal electron acceptor for microorganisms (11, 16). The fate and transport of uranium in groundwater are also strongly linked through sorption and precipitation processes to the bioreduction of Fe minerals, including oxides, layer-silicate clay minerals, and sulfides (7, 23, 53).In order to appropriately design U(VI) bioremediation strategies, the potential function and phylogenetic structure of indigenous subsurface microbial communities must be further understood (24, 34, 46). Conflicting evidence has been presented on which microbial groups, Fe(III)- or sulfate-reducing bacteria (FeRB or SRB), effectively catalyze the reductive immobilization of U(VI) in the presence of amended electron donors (5, 44, 69). The addition of acetate to the subsurface at a uranium-contaminated site in Rifle, Colorado, initially stimulated FeRB within the family Geobacteraceae to reduce U(VI) (5, 65). However, with long-term acetate addition, SRB within the family Desulfobacteraceae, which are not capable of U(VI) reduction, increased in abundance and a concomitant reoxidation of U(IV) was observed (5, 65). At a uranium-contaminated site in Oak Ridge, Tennessee, in situ and laboratory-based experiments successfully employed ethanol amendments to stimulate denitrification followed by the reduction of U(VI) by indigenous microbial communities (13, 24, 44, 48, 50, 57, 68). In these studies, ethanol amendments stimulated both SRB and FeRB, with SRB likely catalyzing the reduction of U(VI). This suggests that the potential for bioremediation will be affected by the choice of electron donor amendment through effects on the functional diversity of U(VI)-reducing microbial populations. As uranium reduction is dependent on the depletion of nitrate, the microbial populations mediating nitrate reduction are also critical to the design of bioremediation strategies. Although nitrate-reducing bacteria (NRB) have been studied extensively in subsurface environments (2, 15, 19, 24, 56, 58, 70), the mechanisms controlling the in situ metabolism of NRB remain poorly understood.The dynamics of microbial populations capable of U(VI) reduction in subsurface sediments are poorly understood, and the differences in the microbial community dynamics during bioremediation have not been explored. Based on the results of previous studies (13, 44, 49, 57, 68, 69), we hypothesized that the activity of nitrate- and Fe(III)-reducing microbial populations, catalyzing the reductive immobilization of U(VI) in subsurface radionuclide-contaminated sediments, would be dependent on the choice of electron donor. The objectives of the present study were (i) to characterize structure-function relationships for microbial groups likely to catalyze or limit U(VI) reduction in radionuclide-contaminated sediments and (ii) to further develop a proxy for the metabolic activity of FeRB. Microbial activity was assessed by monitoring terminal electron-accepting processes (TEAPs), electron donor utilization, and Fe(III) mineral transformations in microcosms conducted with subsurface materials cocontaminated with high levels of U(VI) and nitrate. In parallel, microbial functional groups (i.e., NRB and FeRB) were enumerated and characterized using a combination of cultivation-dependent and -independent methods.     

Impacts of Inter- and Intralaboratory Variations on the Reproducibility of Microbial Community Analyses

With the advent of molecular biological techniques, especially next-generation sequencing and metagenomics, the number of microbial biogeography studies is rapidly increasing. However, these studies involve the synthesis of data generated by different laboratories using different protocols, chemicals, etc., all with inherent biases. The aim of this study was to assess inter- as well as intralaboratory variations in microbial community composition when standardized protocols are applied to a single soil sample. Aliquots from a homogenized soil sample from a rice field in Italy were sent to five participating laboratories. DNA was extracted by two investigators per laboratory using an identical protocol. All DNA samples were sent to one laboratory to perform DNA quantification, quantitative PCR (QPCR), and microarray and denaturing gradient gel electrophoresis (DGGE) analyses of methanotrophic communities. Yields, as well as purity of DNA, were significantly different between laboratories but in some cases also between investigators within the same laboratory. The differences in yield and quality of the extracted DNA were reflected in QPCR, microarray, and DGGE analysis results. Diversity indices (Shannon-Wiener, evenness, and richness) differed significantly between laboratories. The observed differences have implications for every project in which microbial communities are compared in different habitats, even if assessed within the same laboratory. To be able to make sensible comparisons leading to valid conclusions, intralaboratory variation should be assessed. Standardization of DNA extraction protocols and possible use of internal standards in interlaboratory comparisons may help in rendering a “quantifiable” bias.Microorganisms comprise a major part of total biomass and biodiversity (21, 41-43, 49). They play a critical role in biogeochemical processes and ecosystem functioning (16). However, knowledge of ecology and functioning of environmental microbial communities is still far from complete, mainly due to our inability to grow the majority of environmental microbes under laboratory conditions. The introduction of culture-independent DNA- and RNA-based techniques has led to a revolution in environmental microbiology, yielding a wealth of information on community compositions in an ever-growing range of habitats. Phylogenetic as well as functional microarrays (51) and metagenomic techniques (41, 47) enable in-depth analyses and comparison of whole microbial communities in a high-throughput manner.The collective goal of all environmental microbial ecology studies is 2-fold: (i) to obtain an overall understanding of microbial community composition, dynamics, and functioning and (ii) to identify regulating mechanisms. Reaching these goals will necessitate the integrated analyses of data generated in different laboratories and from different habitats. The first step in most if not all environmental microbial community studies is the extraction of total DNA from environmental samples in a way that reflects the in situ community composition as closely as possible. Numerous methods, protocols, and commercial kits have been developed to improve and optimize quantity and quality of extracted community DNA from a wide range of natural environments (4, 8, 28, 37, 39). However, up-to-date bias-free extraction methods are not available, especially not for complex and highly variable matrices, like soil. Beside the challenge of lysing all cells, the incomplete removal of compounds interfering with downstream processing render the development of a bias-free protocol a “mission impossible.” Assessments of the bias introduced by DNA extraction with different methods and kits on microbial community profiling revealed that a perfect protocol fitting all types of environments is not feasible (10, 17, 20, 45). However, in light of the global biodiversity debate, assessment of local and global patterns of microbial diversity and their controlling factors (19, 26) necessitates the comparison of data collected in multiple habitats and processed in different laboratories.In contrast to other scientific disciplines, intercalibration of protocols is not common practice in environmental microbiology. Interlaboratory comparisons (ring analyses) have been applied commonly in food control, veterinary, forensic, and soil studies to evaluate, for example, Salmonella diagnostic accuracy (25), virus isolation (18), enzyme-linked immmunosorbent assay methods (2), mitochondrial DNA sequencing (30), soil microbial biomass C (3), and quantitative PCR (QPCR) (11). Ring analyses assessing the reproducibility of DNA extraction and subsequent community analyses between different laboratories have not been carried out so far in environmental microbial ecology.A microbial functional guild that has been investigated intensively using molecular techniques is represented by the methanotrophs (aerobic methane-oxidizing bacteria [MOB]), which can be found in a wide variety of environments (27). The unique contribution of these bacteria to the global methane cycle has rendered the diversity and ecology of MOB hot topics for decades (9, 14, 34, 46, 48). By using methane as single source of carbon and energy, these microbes represent the only biological sink of the greenhouse gas methane under aerobic conditions (13). Aerobic MOB belong to the Gamma- and Alphaproteobacteria and the Verrucomicrobia (13, 34) and have the following features that enable linking function and identity. Assimilating methane facilitates the application of stable isotope probing of diagnostic lipids and of RNA/DNA (6, 29, 33). Besides this, the key gene in methane oxidation (for methanemonooxygenase subunit A, pmoA) reflects the phylogeny of these bacteria, facilitating a direct link between methane consumption and taxonomy. These features have made this group of microbes a model group for studies in environmental microbial ecology. Combined with the broad distribution and high environmental relevance, this group is highly suited to perform a ring analysis on reproducibility of DNA extraction and subsequent community profiling.In the present study, five independent laboratories from Norway, Finland, Netherlands, Germany, and Austria extracted DNA from the same rice field soil sample, using identical protocols and performed by two different investigators per laboratory. Subsequently, the extracted DNA was sent to one laboratory, where DNA quantification, QPCR, microarray, and denaturing gradient gel electrophoresis (DGGE) analyses were performed by one and the same person. The impacts of inter- as well as intralaboratory variations of DNA extraction are discussed, and recommendations for comparative studies are presented.     

Coral-Associated Bacteria and Their Role in the Biogeochemical Cycling of Sulfur

Marine bacteria play a central role in the degradation of dimethylsulfoniopropionate (DMSP) to dimethyl sulfide (DMS) and acrylic acid, DMS being critical to cloud formation and thereby cooling effects on the climate. High concentrations of DMSP and DMS have been reported in scleractinian coral tissues although, to date, there have been no investigations into the influence of these organic sulfur compounds on coral-associated bacteria. Two coral species, Montipora aequituberculata and Acropora millepora, were sampled and their bacterial communities were characterized by both culture-dependent and molecular techniques. Four genera, Roseobacter, Spongiobacter, Vibrio, and Alteromonas, which were isolated on media with either DMSP or DMS as the sole carbon source, comprised the majority of clones retrieved from coral mucus and tissue 16S rRNA gene clone libraries. Clones affiliated with Roseobacter sp. constituted 28% of the M. aequituberculata tissue libraries, while 59% of the clones from the A. millepora libraries were affiliated with sequences related to the Spongiobacter genus. Vibrio spp. were commonly isolated from DMS and acrylic acid enrichments and were also present in 16S rRNA gene libraries from coral mucus, suggesting that under “normal” environmental conditions, they are a natural component of coral-associated communities. Genes homologous to dddD, and dddL, previously implicated in DMSP degradation, were also characterized from isolated strains, confirming that bacteria associated with corals have the potential to metabolize this sulfur compound when present in coral tissues. Our results demonstrate that DMSP, DMS, and acrylic acid potentially act as nutrient sources for coral-associated bacteria and that these sulfur compounds are likely to play a role in structuring bacterial communities in corals, with important consequences for the health of both corals and coral reef ecosystems.Dimethylsulfoniopropionate (DMSP) is an organic sulfur compound implicated in the formation of clouds via its cleavage product dimethyl sulfide (DMS) and therefore has the potential to exert major cooling effects on climate (9, 38). The production of DMSP is mainly restricted to a few classes of marine macro- and microalgae (27, 68), with the main producers being phytoplankton species belonging to prymnesiophyte and dinoflagellate taxa (28, 62, 67). Recently, significant concentrations of DMSP and DMS have been recorded in association with animals that harbor symbiotic algae such as scleractinian corals and giant clams (7, 8, 68), raising questions about the role of coral reefs in sulfur cycling. The densities of symbiotic dinoflagellates (genus Symbiodinium, commonly known as zooxanthellae) in coral tissues are similar to those recorded for dinoflagellates in phytoplankton blooms (11, 68). Since dinoflagellates are among the most significant producers of DMSP and high intracellular concentrations of DMSP have been found in both cultured zooxanthellae (26) and scleractinian corals (6-8, 25), these observations suggest that endosymbiotic zooxanthellae have an integral role in sulfur cycling in oligotrophic reef waters.Most of the DMSP produced by planktonic dinoflagellates is exuded into the surrounding water, where it is degraded by bacteria via two possible pathways: the first one converts a large fraction (ca. 75%) of dissolved DMSP to methylmercaptopropionate, which is subsequently incorporated into the biomass of microbial cells (22, 27, 66). The second pathway transforms the remaining part of the dissolved DMSP to equimolar concentrations of DMS and acrylic acid (43, 66, 72). This metabolic pathway for DMSP degradation has been identified in the alphaproteobacterial species Sulfitobacter sp. and the enzyme involved (DMSP-dependent DMS lyase [DddL]) characterized (10). Another pathway for DMS formation (without production of acrylate) has been described for Marinomonas sp. and the gene responsible, dddD, identified. In addition, the protein DddR has been directly implicated in the regulation of the gene encoding DddD (66). The DMS produced by these enzymes are then released into the surrounding water (27). Prior to the 1980s, diffusion of supersaturated DMS from the oceans to the atmosphere was thought to be the major removal pathway of this compound from the oceans (35, 72). More recently, however, it has been estimated that between 50 and 80% of the DMS produced by DMSP-degrading bacteria is degraded directly by other types of bacteria (58, 59), although the populations and metabolic pathways involved in the degradation of DMS are still poorly understood.Coral-associated bacterial communities are known to be diverse and highly abundant (12, 30, 48, 49, 52). These dynamic communities exploit a number of habitats associated with corals, including mucus on coral surfaces (48), intracellular niches within coral tissues (3, 16, 45, 47, 52), spaces within coral skeletons (15, 51), and seawater surrounding corals (16, 61). Each of these habitats is believed to harbor different bacterial populations (4, 52). Despite high bacterial diversity, corals have been reported to harbor species-specific microbial communities for beneficial effects; however, their role in coral health is poorly understood (47-50). In coral reef environments, bacteria are dependent upon organic compounds produced by photoautotrophic organisms such as endosymbiotic zooxanthellae (48); therefore, photosynthates translocated to coral tissues and mucus may determine microbial communities closely associated with corals (48, 52). The high levels of DMSP and DMS produced by corals, coupled with the dependence of DMSP and DMS conversion on processes typically involving bacteria, suggest that corals are likely to harbor bacterial species involved in the cycling of these compounds. To investigate the potential of the organosulfur compound DMSP and its breakdown products, DMS and acrylic acid, to drive coral-associated microbial communities, we used these compounds as sole carbon sources to isolate bacteria from two coral species (Montipora aequituberculata and Acropora millepora) and then directly compared these microbial communities with coral-associated microbiota identified using culture-independent analyses. Genes implicated in the metabolism of DMSP were also characterized from isolated strains, confirming that bacteria associated with corals have the potential to metabolize organic sulfur compounds present in coral tissues.     

The Carnivorous Pale Pitcher Plant Harbors Diverse,Distinct, and Time-Dependent Bacterial Communities

The ability of American carnivorous pitcher plants (Sarracenia) to digest insect prey is facilitated by microbial associations. Knowledge of the details surrounding this interaction has been limited by our capability to characterize bacterial diversity in this system. To describe microbial diversity within and between pitchers of one species, Sarracenia alata, and to explore how these communities change over time as pitchers accumulate and digest insect prey, we collected and analyzed environmental sequence tag (454 pyrosequencing) and genomic fingerprint (automated ribosomal intergenic spacer analysis and terminal restriction fragment length polymorphism) data. Microbial richness associated with pitcher plant fluid is high; more than 1,000 unique phylogroups were identified across at least seven phyla and 50 families. We documented an increase in bacterial diversity and abundance with time and observed repeated changes in bacterial community composition. Pitchers from different plants harbored significantly more similar bacterial communities at a given time point than communities coming from the same genetic host over time. The microbial communities in pitcher plant fluid also differ significantly from those present in the surrounding soil. These findings indicate that the bacteria associated with pitcher plant leaves are far from random assemblages and represent an important step toward understanding this unique plant-microbe interaction.Characterization of the phyllosphere is fundamental to our understanding of the ecology and evolution of plant populations and plant diversity and their interactions with other organisms (46, 64, 66). The carnivorous pitcher plant genus Sarracenia is an obvious system to address basic questions in plant-microbe interaction because each pitcher (a modified leaf) of the plant contains a microcosm composed of larval insects, fungi, algae, rotifers, nematodes, and bacteria that, together, ultimately break down nutrients from insect prey for the plant (1, 10, 20, 28, 37). Each pitcher represents a naturally defined and discrete community with a finite volume and a discrete life span (each leaf lasts only one season). Several investigations have explored species interactions within Sarracenia pitchers (13, 20, 34, 54), and competition, predation and dispersal frequency appear to be important drivers of community composition in the system (1, 20, 43, 44). Studies involving community patterns on a larger scale within pitchers, however, are few, and the processes that produce these patterns remain unknown (33).     

Impact of an 8-Year-Old Transgenic Poplar Plantation on the Ectomycorrhizal Fungal Community

The long-term impact of field-deployed genetically modified trees on soil mutualistic organisms is not well known. This study aimed at evaluating the impact of poplars transformed with a binary vector containing the selectable nptII marker and β-glucuronidase reporter genes on ectomycorrhizal (EM) fungi 8 years after field deployment. We generated 2,229 fungal internal transcribed spacer (ITS) PCR products from 1,150 EM root tips and 1,079 fungal soil clones obtained from the organic and mineral soil horizons within the rhizosphere of three control and three transformed poplars. Fifty EM fungal operational taxonomic units were identified from the 1,706 EM fungal ITS amplicons retrieved. Rarefaction curves from both the root tips and soil clones were close to saturation, indicating that most of the EM species present were recovered. Based on qualitative and/or quantitative α- and β-diversity measurements, statistical analyses did not reveal significant differences between EM fungal communities associated with transformed poplars and the untransformed controls. However, EM communities recovered from the root tips and soil cloning analyses differed significantly from each other. We found no evidence of difference in the EM fungal community structure linked to the long-term presence of the transgenic poplars studied, and we showed that coupling root tip analysis with a soil DNA cloning strategy is a complementary approach to better document EM fungal diversity.The poplar has become a model tree species in genetic engineering as it can easily be transformed and clonally propagated and has a small genome size (7, 77, 80). Tree growth, agronomic traits, and timber quality can be improved through genetic engineering (61), thereby avoiding the long reproductive cycles of conventional breeding (47, 59, 83). However, concerns have arisen about the potential impact of genetically modified (GM) trees on the environment (10). The potential environmental hazards linked to GM trees differ from those associated with transgenic crop plants at both spatial and temporal scales (84) because trees are long-lived perennials, unlike annual crop plants. They display several biotic interactions with soil microbial communities such as bacteria and fungi. Interactions between GM trees and these communities could result in exposure to the expression of new traits over several decades, a period longer than those for GM crop plants.Impact studies of GM plants on nontarget organisms usually focus on the potential risk linked to transgene expression (expected effects) that confers a genetic advantage to the transformed plant rather than on unforeseen (pleiotropic) effects from transgene insertion or the expression of other transgene components such as selection markers or reporter genes. The nptII gene, encoding neomycin phosphotransferase II (EC 2.7.1.95), and the GUS gene, encoding β-glucuronidase (GUS; EC 3.2.1.31), are frequently used for genetic selection of transformed cells and for monitoring transgene presence and expression during transgenic plant lifetime (76). The products of the nptII and GUS genes have been subjected to safety assessment studies and were shown to be nondeleterious to human and animal health (21, 23, 27, 51). Nevertheless, pleiotropic effects in crop plants transformed with the nptII and GUS genes have been observed (2, 15, 17, 43). Pleiotropic effects from GM trees coexpressing such selectable markers have also been recorded. For example, Pasonen et al. (56) showed a significant decrease in the number of root tips colonized by Paxillus involutus associated with a line of chitinase-transformed silver birch in vitro. Similar results have been observed in vivo with P. involutus associated with a line of lignin-modified silver birches (72).Many trees in temperate, boreal, tropical, and subtropical forests establish mutualistic interactions with ectomycorrhizal (EM) fungi (42, 66, 67, 68). EM fungi are a polyphyletic group comprising over 5,000 species (49) that play key roles in biogeochemical soil processes and plant health. They represent one-third of the total microbial biomass in the soil of boreal forests (32). Fine roots colonized by EM fungi, also called EM root tips or ectomycorrhizae, display a fungal mantle from which extends the extraradical mycelium to prospect the soil for nutrient uptake. These two anatomical parts can be sampled for EM fungus molecular identification, but some studies have highlighted dissimilarities between the EM fungal diversity recorded in root tip sampling and that recorded in extraradical mycelium sampling (26, 37, 39).Given the potential cumulative effects caused by the presence and stable constitutive expression of transgenes over years on GM tree fitness and on the environment, impact studies of GM trees require long-term field trials (5, 72, 84). In this study, we investigated the potential long-term impact on the EM fungal community of hybrid poplars transformed with the binary vector containing the selectable nptII marker and GUS reporter genes, field deployed for 8 years. This plantation was part of the first confined field trial of transgenic trees in Canada. Hybrid poplars constitutively expressed the nptII gene for kanamycin resistance driven by the NOS promoter (30). The activity of the NOS promoter has been shown to increase in the lower part of transgenic tobacco plants (4). Such a vertical gradient has also been observed in transgenic hybrid poplars, where the NOS promoter activity was 2.4-fold higher in roots than in leaves (87).As no direct negative impact of nptII or GUS gene expression on fungal organisms has been reported in the literature, we first tested the null hypothesis (H₀) that the EM fungal community recorded from transgenic poplars was similar to that from untransformed poplars. Second, since the EM fungal diversity picture can be influenced by the sampling method, we contrasted the EM fungal community recovered from root tips with that recorded in soil cloning analyses. Internal transcribed spacer (ITS) sequences from the nuclear rRNA were produced from both EM root tips and extraradical mycelia to compare the EM fungal communities associated with three control and three transgenic poplars. EM fungal communities were characterized by measuring the usual qualitative and quantitative EM species diversity within each community (α-diversity) and then estimating the nucleotide diversity between EM communities in relation to EM phylotype relative abundances (quantitative β-diversity).     

Diversity of Glycosyl Hydrolases from Cellulose-Depleting Communities Enriched from Casts of Two Earthworm Species

The guts and casts of earthworms contain microbial assemblages that process large amounts of organic polymeric substrates from plant litter and soil; however, the enzymatic potential of these microbial communities remains largely unexplored. In the present work, we retrieved carbohydrate-modifying enzymes through the activity screening of metagenomic fosmid libraries from cellulose-depleting microbial communities established with the fresh casts of two earthworm species, Aporrectodea caliginosa and Lumbricus terrestris, as inocula. Eight glycosyl hydrolases (GHs) from the A. caliginosa-derived community were multidomain endo-β-glucanases, β-glucosidases, β-cellobiohydrolases, β-galactosidase, and β-xylosidases of known GH families. In contrast, two GHs derived from the L. terrestris microbiome had no similarity to any known GHs and represented two novel families of β-galactosidases/α-arabinopyranosidases. Members of these families were annotated in public databases as conserved hypothetical proteins, with one being structurally related to isomerases/dehydratases. This study provides insight into their biochemistry, domain structures, and active-site architecture. The two communities were similar in bacterial composition but significantly different with regard to their eukaryotic inhabitants. Further sequence analysis of fosmids and plasmids bearing the GH-encoding genes, along with oligonucleotide usage pattern analysis, suggested that those apparently originated from Gammaproteobacteria (pseudomonads and Cellvibrio-like organisms), Betaproteobacteria (Comamonadaceae), and Alphaproteobacteria (Rhizobiales).Microorganisms producing diverse glycosyl hydrolases (GHs) are widespread and typically thrive in environments where plant materials tend to accumulate and deteriorate (42, 73). The habitats of microorganisms with great GH diversity are the ruminant animal rumen, mouse bowel, and rabbit cecum (10, 24, 26, 28, 49, 74). Microorganisms associated with soil invertebrates in general and with soil earthworms in particular carry out metabolic processes that contribute to element cycling and are essential in sustaining processes which their hosts are unable to perform (20, 52, 72, 76). Although some species of earthworms produce cellulases (15, 55), they generally rely on microbes inhabiting their gastrointestinal (GI) tracts to perform cellulose utilization processes (31, 47, 77). Casts are of special interest in this respect. Considering that the overall numbers of cellulolytic microbes in earthworm casts are greater than those in soil (57), earthworm casts seem to play an important role in the decomposition of plant litter, serving as an inoculum for cellulosic substrates (9). It is important to note that microorganisms from preingested substratum (soil or plant litter) are predominant in the gut lumen (20); however, microbial populations in earthworm casts differ from those in soil in terms of diversity and the relative abundance of different taxa (29, 57, 63). It is anticipated that the enzymatic repertoire of such microbial communities must be especially broad toward diverse sugar-based polymeric, oligomeric, and monomeric substrates; however, among approximately 115 families of GHs with thousands of members known to date (12), none of the GHs have been derived from microorganisms of earthworm-associated microbial communities.The aim of the present work was therefore to examine the diversity of GHs in metagenome libraries derived from fresh casts of Aporrectodea caliginosa and Lumbricus terrestris earthworms via functional screening. Other important tasks of this work were to characterize individual enzymes and to gain insight into their structural-functional features. Finally, we performed sequence analysis of large contiguous DNA fragments of fosmids harboring the genes for GHs to associate them with the organism(s) that may produce them, which was complemented by conventional small-subunit (SSU) rRNA clone library sequencing analysis.