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Preservation of estuarine sediments for lipid analysis of biomass and community structure of microbiota.

Various methods were tested for preserving estuarine sediments in the field before biochemical analysis of the microbiota. Total microbial biomass was determined as lipid phosphate (LP), and the fatty acids of the microbial lipids were used as indicators of community structure. Control samples were sieved to remove macroinvertebrates and plant materials and were extracted immediately in the field. Other samples were preserved both before and after sieving and stored for 5 days before analysis. Freezing resulted in a 50% decline in LP and significant decreases in many fatty acids. Refrigeration resulted in a 19% decrease in LP but no change in the fatty acids. Samples preserved with Formalin before sieving exhibited no significant change in LP but substantial increases in many fatty acids, which were probably derived from the macroinvertebrates. Sieved samples preserved with Formalin showed a 17 to 18% decline in LP but no change in the fatty acids. Ideally, samples should be sieved and extracted immediately in the field. However, short-term refrigeration and longer-term preservation of sieved samples with Formalin may be acceptable compromises.

Journal Article↗

Phylogenetic Analysis of Microbial Diversity in the Rhizoplane of Oilseed Rape (Brassica napus cv. Westar) Employing Cultivation-Dependent and Cultivation-Independent Approaches.

The structure of the microbial rhizoplane community of the important crop plant oilseed rape was studied by using a culture-dependent as well as a culture-independent approach based on 16S rDNA amplification. After isolation of the microbial community from the rhizoplane of oilseed rape (Brassica napus cv. Westar), the collected suspension was divided into two parts. One part was used for cultivation of bacteria onto three different growth media to establish a culture collection. From the other part of the rhizoplane suspension, genomic DNA was isolated and purified. Thereafter, 16S rDNA was amplified by PCR and cloned to obtain a library of 16S rDNA genes representative for the bacterial communities of this habitat. Phylogenetic 16S rDNA sequence analysis of 103 clones of this library revealed considerable differences from the corresponding nucleotide sequences of 111 cultured bacteria. Whereas the 16S rDNA clone library was dominated by a-Proteobacteria and bacteria of the Cytophaga-Flavobacterium-Bacteroides (CFB) phylum (51% and 30%, respectively), less than 17% of the cultured bacteria belonged to these two groups. More than 64% of the cultivated isolates were allocated to the b- and g-subclasses of the Proteobacteria, which were present in the clone library at about 14%. Most of the clones of the a-Proteobacteria of the library showed highest similarity to Bradyrhizobium sp. No such bacteria were found in the culture collection. Similarly, the second dominant group of the clone library comprising members of the CFB phylum was represented in the culture collection by a single isolate. The phylogenetic analysis of isolates of the culture collection clearly emphasized the need to use different growth media for recovery of rhizoplane bacteria. Whereas most of the a-Proteobacteria were recovered on complex medium, most of the b-Proteobacteria were isolated onto minimal media. Our results demonstrate that the combined approach pursued in this paper is necessary to explore the biodiversity of bacterial rhizoplane communities.

Journal Article↗

Trace elements in agroecosystems and impacts on the environment.

Trace elements mean elements present at low concentrations (mg kg-1 or less) in agroecosystems. Some trace elements, including copper (Cu), zinc (Zn), manganese (Mn), iron (Fe), molybdenum (Mo), and boron (B) are essential to plant growth and are called micronutrients. Except for B, these elements are also heavy metals, and are toxic to plants at high concentrations. Some trace elements, such as cobalt (Co) and selenium (Se), are not essential to plant growth but are required by animals and human beings. Other trace elements such as cadmium (Cd), lead (Pb), chromium (Cr), nickel (Ni), mercury (Hg), and arsenic (As) have toxic effects on living organisms and are often considered as contaminants. Trace elements in an agroecosystem are either inherited from soil parent materials or inputs through human activities. Soil contamination with heavy metals and toxic elements due to parent materials or point sources often occurs in a limited area and is easy to identify. Repeated use of metal-enriched chemicals, fertilizers, and organic amendments such as sewage sludge as well as wastewater may cause contamination at a large scale. A good example is the increased concentration of Cu and Zn in soils under long-term production of citrus and other fruit crops. Many chemical processes are involved in the transformation of trace elements in soils, but precipitation-dissolution, adsorption-desorption, and complexation are the most important processes controlling bioavailability and mobility of trace elements in soils. Both deficiency and toxicity of trace elements occur in agroecosystems. Application of trace elements in fertilizers is effective in correcting micronutrient deficiencies for crop production, whereas remediation of soils contaminated with metals is still costly and difficult although phytoremediation appears promising as a cost-effective approach. Soil microorganisms are the first living organisms subjected to the impacts of metal contamination. Being responsive and sensitive, changes in microbial biomass, activity, and community structure as a result of increased metal concentration in soil may be used as indicators of soil contamination or soil environmental quality. Future research needs to focus on the balance of trace elements in an agroecosystem, elaboration of soil chemical and biochemical parameters that can be used to diagnose soil contamination with or deficiency in trace elements, and quantification of trace metal transport from an agroecosystem to the environment.

Adsorption↗

Genetic diversity of bacterial strains isolated from soils, contaminated with polycyclic aromatic hydrocarbons, by 16S rRNA gene sequencing and amplified fragment length polymorphism fingerprinting.

In order to study microbial diversity in a polycyclic aromatic hydrocarbon-impacted soil, 14 bacterial strains were analyzed by 16S rRNA gene sequencing and amplified fragment length polymorphism (AFLP) analysis. Bacterial strains isolated from two different hydrocarbon-polluted sites were identified to the species level by 16S rRNA full-gene sequencing using MicroSeq 16S rRNA gene sequencing. Their genome was subsequently analyzed by high-resolution genotyping with AFLP analysis, in order to monitor species variability and to differentiate closely related strains. Cluster analysis based on AFLP fingerprinting showed intra-specific polymorphism, even among strains with 100% 16S rRNA gene sequence identity. The results show that AFLP is a powerful, highly reproducible and discriminatory tool for revealing genetic relationships in bacterial populations. The ability to differentiate and track related closely microbes is fundamental for studying structure and dynamics of microbial communities in contaminated ecosystems.

Bacteria↗

Microbial ecology to manage processes in environmental biotechnology.

Microbial ecology and environmental biotechnology are inherently tied to each other. The concepts and tools of microbial ecology are the basis for managing processes in environmental biotechnology; and these processes provide interesting ecosystems to advance the concepts and tools of microbial ecology. Revolutionary advancements in molecular tools to understand the structure and function of microbial communities are bolstering the power of microbial ecology. A push from advances in modern materials along with a pull from a societal need to become more sustainable is enabling environmental biotechnology to create novel processes. How do these two fields work together? Five principles illuminate the way: (i) aim for big benefits; (ii) develop and apply more powerful tools to understand microbial communities; (iii) follow the electrons; (iv) retain slow-growing biomass; and (v) integrate, integrate, integrate.

Biotechnology↗

Host-virus dynamics in anaerobic digesters facing abiotic inhibition.

Viruses play a major role in controlling the structure and dynamics of microbial communities in anaerobic digesters, ecosystems sensitive to disturbances that inhibit methane production. Here, we studied the interplay between abiotic disturbances, microbiome and virome composition, and process performance, to assess whether provirus induction can be triggered by abiotic stresses known to inhibit anaerobic digestion (ammonium, phenol and sodium chloride). We monitored viral dynamics in batch mesophilic anaerobic digesters fed with biowaste through shotgun metavirome sequencing. The diversity of both prokaryotes and viruses was high, with Clostridiales dominating the prokaryotic community and Caudoviricetes dominating the viromes. We identified 132 viral contigs and 19 host genera that were differentially abundant under disturbed conditions. No significant impact of the tested abiotic stresses on provirus induction was observed under the current experimental and analytical framework. The results were consistent with viruses exerting steady, background-level predation through a putative combination of kill-the-winner dynamics at the sub-genus level and piggyback-the-winner dynamics, rather than stress-triggered, synchronous lytic bursts. A few auxiliary metabolic genes were detected, potentially targeting carbon, sulfur and cofactor metabolism in anaerobic digestion. Temperate viruses were dominant, representing up to 71% of the viral genomes confirmed as complete across all conditions. Electron microscopy analysis revealed diverse virus-like particles, including head-tailed particles typical of Caudoviricetes, but also spherical, rod-shaped and spindle-shaped particles typical of archaeal viruses. Notably, we present a new virus family, Eurekaviridae, of spindle-shaped viruses associated with methanogenic archaea.

Anaerobiosis↗

In situ methods for assessment of microorganisms and their activities.

Recent technical developments in the field of molecular biology and microsensors are beginning to enable microbiologists to study the abundance, localization and activity of microorganisms in situ. The various new methods on their own bear high potential but it is the combination of studies on structure and function of microbial communities that will yield the most detailed insights in the way microorganisms operate in nature.

Bacteria↗

Terminal restriction fragment length polymorphism (T-RFLP): an emerging method for characterizing diversity among homologous populations of amplification products.

Terminal restriction fragment length polymorphism is a recent molecular approach that can assess subtle genetic differences between strains as well as provide insight into the structure and function of microbial communities. The technique has both high sensitivity and throughput making it ideal for comparative analyses.

Ecosystem↗

Cobamide-based interactions between soil bacteria can be predicted based on monoculture growth.

Interactions between microbes shape the structure and function of microbial communities. While studying interactions is key to understanding microbial communities as a whole, gaining a detailed mechanistic view is challenging due to the scale of co-occurring interactions. The model nutrient approach enables the study of a subset of interactions involving a single nutrient class and can shed light on broader interaction mechanisms involving other nutrients. Here, we focus on cobamides, the cobalamin (vitamin B12) family of enzyme cofactors, to study nutrient competition and nutrient-sharing interactions in co-cultures and tri-cultures. We examined bacteria that were previously isolated from a grassland soil and were characterized as "dependents" (require cobamides but cannot synthesize them) or "producers" (synthesize cobamides). The outcome of competition between a pair of dependents was predictable based on monoculture growth characteristics, with the dominant microbe determined by its adaptation to a specific cobamide concentration range. Moreover, cobamide producers could support the cobamide-dependent growth of dependents in co-culture and influenced the outcome of competition between dependents in tri-culture. We analyzed the metabolic capacity encoded in the genomes of producers and dependents and found that cobamides are likely the main shared nutrient in our co- and tri-cultures. These results highlight the utility of the model nutrient approach to characterize and predict interactions in bacterial consortia of increasing complexity.

Journal Article↗

daime, a novel image analysis program for microbial ecology and biofilm research.

Combinations of microscopy and molecular techniques to detect, identify and characterize microorganisms in environmental and medical samples are widely used in microbial ecology and biofilm research. The scope of these methods, which include fluorescence in situ hybridization (FISH) with rRNA-targeted probes, is extended by digital image analysis routines that extract from micrographs important quantitative data. Here we introduce daime (digital image analysis in microbial ecology), a new computer program integrating 2-D and 3-D image analysis and visualization functionality, which has previously not been available in a single open-source software package. For example, daime automatically finds 2-D and 3-D objects in images and confocal image stacks, and offers special functions for quantifying microbial populations and evaluating new FISH probes. A novel feature is the quantification of spatial localization patterns of microorganisms in complex samples like biofilms. In combination with '3D-FISH', which preserves the 3-D structure of samples, this stereological technique was applied in a proof of principle experiment on activated sludge and provided quantitative evidence that functionally linked ammonia and nitrite oxidizers cluster together in their habitat. This image analysis method complements recent molecular techniques for analysing structure-function relationships in microbial communities and will help to characterize symbiotic interactions among microorganisms.

Biofilms↗

A phylogenetic analysis of microbial communities associated with methane hydrate containing marine fluids and sediments in the Cascadia margin (ODP site 892B).

Methane hydrates represent an enormous carbon and energy source in many low temperature deep marine sediments. However, little information is available concerning the nature of the microbial communities associated with these structures. Here, we describe a phylogenetic analysis based on ribosomal DNA (rDNA) sequences obtained from sediment and fluid samples present in a region of gas hydrate formation in shallow sediments within the Cascadia margin in and around Ocean Drilling Program (ODP) Site 892B. Our studies detected diverse sulfur-utilizing microbes, methanogens, methanotrophs, and non-thermophilic members of the kingdom Crenarchaeota. This is the first culture-independent phylogenetic analysis of a gas hydrate habitat.

Archaea↗

Microbial behavior in a heterogeneous world.

Most microorganisms are motile during at least part of their life cycle, because they need to find optimal conditions in a patchy world. The sheer volume of microorganisms in the biosphere means that their motile sensory behavior also contributes to the global transformation and cycling of matter. How microorganisms move and how they orient themselves using environmental cues are integral to understanding the complex structure and function of microbial communities, but although motility in response to external stimuli was first described more than 120 years ago, understanding of the cellular and molecular mechanisms involved has only been achieved more recently.

Anaerobiosis↗

Molecular analysis of deep-subsurface bacteria.

Bacterial isolates from deep-sediment samples from three sites at the Savannah River site, near Aiken, S.C., were studied to determine their microbial community composition and DNA structure by using total DNA hybridization and moles percent G + C. Standard phenotypic identification underestimated the bacterial diversity at the three sites, since isolates with the same phenotype had different DNA structures in terms of moles percent G + C and DNA homology. The G + C content of deep-subsurface bacteria ranged from 20 to 77 mol%. More than 60% of the isolates tested had G + C values similar to those of Pseudomonas spp., and 12% had values similar to those of Acinetobacter spp. No isolates from deeper formations showed the same DNA composition as isolates from upper formations. Total-DNA hybridization and DNA base composition analysis provided a better resolution than phenotypic tests for the understanding of the diversity and structure of deep-subsurface bacterial communities. On the basis of the moles percent G + C values, deep-subsurface isolates tested seemed to belong to the families Pseudomonadaceae and Neisseriaceae, which might reflect a long period of adaptation to the environmental conditions of the deep subsurface.

Bacteria↗

Characterization of the Cricket Hindgut Microbiota with Fluorescently Labeled rRNA-Targeted Oligonucleotide Probes.

Most cricket hindgut microorganisms (60 to 80%) were detected with a universal fluorescent rRNA-targeted probe and found to be eubacteria. Group-specific probes showed that the hindguts of five different cricket species harbor similar bacterial groups, although in different proportions, and that different diets shifted the structure of the hindgut microbial community. The Bacteroides-Prevotella probe, of the eight eubacterial probes tested, stained the largest percentage of cells in all crickets.

Journal Article↗

Root exudation, phosphorus acquisition, and microbial diversity in the rhizosphere of white lupine as affected by phosphorus supply and atmospheric carbon dioxide concentration.

White lupine (Lupinus albus L.) was used as a phosphorus (P)-efficient model plant to study the effects of elevated atmospheric CO(2) concentrations on (i) P acquisition, (ii) the related alterations in root development and rhizosphere chemistry, and (iii) the functional and structural diversity of rhizosphere microbial communities, on a P-deficient calcareous subsoil with and without soluble P fertilization. In both +P (80 mg P kg(-1)) and -P treatments (no added P), elevated CO(2) (800 micromol mol(-1)) increased shoot biomass production by 20 to 35% and accelerated the development of cluster roots, which exhibit important functions in chemical mobilization of sparingly soluble soil P sources. Accordingly, cluster root formation was stimulated in plants without P application by 140 and 60% for ambient and elevated CO(2) treatments, respectively. Intense accumulation of citrate and increased activities of acid and alkaline phosphatases, but also of chitinase, in the rhizosphere were mainly confined to later stages of cluster root development in -P treatments. Regardless of atmospheric CO(2) concentrations, there was no significant effect on accumulation of citrate or on selected enzyme activities of C, N, and P cycles in the rhizosphere of individual root clusters. Discriminant analysis of selected enzyme activities revealed that mainly phosphatase and chitinase contributed to the experimental variance (81.3%) of the data. Phosphatase and chitinase activities in the rhizosphere might be dominated by the secretion from cluster roots rather than by microbial activity. Alterations in rhizosphere bacterial communities analyzed by denaturing gradient gel electrophoresis (DGGE) were related with the intense changes in root secretory activity observed during cluster root development but not with elevated CO(2) concentrations.

Atmosphere↗

Characterizing man-made and natural modifications of microbial diversity and activity in coastal ecosystems.

The impacts of growing coastal pollution and habitat alteration accompanying human encroachment are of great concern at the microbial level, where much of the ocean's primary production and biogeochemical cycling takes place. Coastal ecosystems are also under the influence of natural perturbations such as major storwns and flooding. Distinguishing the impacts of natural and human stressors is essential for understanding environmentally-induced change in microbial diversity and function. The objective of this paper is to discuss the applications and merits of recently developed molecular, ecophysiological and analytical indicators and their utility in examining anthropogenic and climatic impacts on the structure and function of coastal microbial communities. The nitrogen-limited Neuse River Estuary and Pamlico Sound, North Carolina are used as examples of ecosystems experiencing both anthropogenic (i.e., accelerating eutrophication) and climatic stress (increasing frequencies of tropical storms and hurricanes). Additional examples are derived from a coastal monitoring site (LEO) on the Atlantic coast of New Jersey and Galveston Bay, on the Gulf of Mexico. In order to assess structure, function, and trophic state of these and other coastal ecosystems, molecular (DNA and RNA-based) characterizations of the microbial taxa involved in carbon, nitrogen and other nutrient transformations can be combined with diagnostic pigment-based indicators of primary producer groups. Application of these methods can reveal process-level microbial community responses to environmental variability over a range of scales. Experimental approaches combined with strategic monitoring utilizing these methods will facilitate: (a) understanding organismal and community responses to environmental change, and (b) synthesizing these responses in the context of ecosystem models that integrate physical, chemical and biotic variability with environmental controls.

Animals↗

Adaptations in bacterial catabolic enzyme activity and community structure in membrane-coupled bioreactors fed simple synthetic wastewater.

Membrane-coupled bioreactors (MBRs) offer substantial benefits compared to conventional reactor designs for biological wastewater treatment. MBR treatment efficiency, however, has not been optimized because the effects of the MBR on process microbiology are poorly understood. In this study, the structure and function of the microbial communities growing in MBRs fed simple synthetic wastewater were investigated. In four starch-fed MBRs, the bacterial community substantially increased its alpha-glucosidase affinity (>1000-fold), while the leucine aminopeptidase and heptanoate esterase affinities increased slightly (<40-fold) or remained relatively constant. Concomitant to these physiological adaptations, shifts in the bacterial community structure in two of the starch-fed MBRs were detected by PCR-DGGE. Four of the bacterial populations detected by PCR-DGGE were isolated and exhibited specific growth rates in batch culture ranging from 0.009 to 0.22 h(-1). Our results suggest that bacterial communities growing under increasingly stringent nutrient limitation adapt their enzyme activities primarily for the nutrients provided, but that there is also a more subtle response not linked to the substrates included in the feed medium. Our research also demonstrates that MBRs can support relatively complex bacterial communities even on simple feed media.

Adaptation, Physiological↗

Bacterial function and community structure in reactors treating biopolymers and surfactants at mesophilic and thermophilic temperatures.

Microbial communities capable of degrading biopolymers and surfactants typically found in graywater were selected in continuous-flow bioreactors operated at 30, 44, 53, or 62 degrees C. The effect of temperature upon microbial activity and community composition was determined. Microbial respiration of the organic components of the medium (including linear alkylbenzene sulfonate) was detected in samples from each reactor. The microbial community in each reactor was adapted to the operating temperature. Nucleic acid-based analyses of community composition showed that distinct consortia were present at each temperature. Community complexity was inversely related to temperature. The specific maintenance rate was twofold higher at 62 degrees C than at the lower temperatures. Under starvation conditions, microbes in the 62 degrees C system lost membrane integrity 30- to 100-fold faster than microbes at lower temperatures.

Journal Article↗