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PubMed2001

DNA-based, culture-independent strategies for evaluating microbial communities in food-associated ecosystems.

Culture-independent molecular techniques are now available to study microbial ecosystems. They are opening interesting perspectives to problems related to composition and population dynamics of microbial communities in various environmental niches (e.g., soil, water) and foods. In fermented food products, estimates of true microbial diversity is often difficult chiefly on account of the inability to cultivate most of the viable bacteria. The increasing knowledge of gene sequences and the concomitant development of new culture-independent molecular techniques are providing new and effective tools to compare the diversity of microbial communities and to monitor population dynamics in minimally disturbed samples. In this review, recent advances in these techniques are reported. Possible applications to food-associated microbial ecosystems are emphasised.

Bacteria↗

PubMed2000

Rhizosphere microbial community structure in relation to root location and plant iron nutritional status.

Root exudate composition and quantity vary in relation to plant nutritional status, but the impact of the differences on rhizosphere microbial communities is not known. To examine this question, we performed an experiment with barley (Hordeum vulgare) plants under iron-limiting and iron-sufficient growth conditions. Plants were grown in an iron-limiting soil in root box microcosms. One-half of the plants were treated with foliar iron every day to inhibit phytosiderophore production and to alter root exudate composition. After 30 days, the bacterial communities associated with different root zones, including the primary root tips, nonelongating secondary root tips, sites of lateral root emergence, and older roots distal from the tip, were characterized by using 16S ribosomal DNA (rDNA) fingerprints generated by PCR-denaturing gradient gel electrophoresis (DGGE). Our results showed that the microbial communities associated with the different root locations produced many common 16S rDNA bands but that the communities could be distinguished by using correspondence analysis. Approximately 40% of the variation between communities could be attributed to plant iron nutritional status. A sequence analysis of clones generated from a single 16S rDNA band obtained at all of the root locations revealed that there were taxonomically different species in the same band, suggesting that the resolving power of DGGE for characterization of community structure at the species level is limited. Our results suggest that the bacterial communities in the rhizosphere are substantially different in different root zones and that a rhizosphere community may be altered by changes in root exudate composition caused by changes in plant iron nutritional status.

Bacteria↗

PubMed2004

Detection of genes involved in biodegradation and biotransformation in microbial communities by using 50-mer oligonucleotide microarrays.

To effectively monitor biodegrading populations, a comprehensive 50-mer-based oligonucleotide microarray was developed based on most of the 2,402 known genes and pathways involved in biodegradation and metal resistance. This array contained 1,662 unique and group-specific probes with <85% similarity to their nontarget sequences. Based on artificial probes, our results showed that under hybridization conditions of 50 degrees C and 50% formamide, the 50-mer microarray hybridization can differentiate sequences having <88% similarity. Specificity tests with representative pure cultures indicated that the designed probes on the arrays appeared to be specific to their corresponding target genes. The detection limit was approximately 5 to 10 ng of genomic DNA in the absence of background DNA and 50 to 100 ng of pure-culture genomic DNA in the presence of background DNA or 1.3 x 10(7) cells in the presence of background RNA. Strong linear relationships between the signal intensity and the target DNA and RNA were observed (r(2) = 0.95 to 0.99). Application of this type of microarray to analyze naphthalene-amended enrichment and soil microcosms demonstrated that microflora changed differently depending on the incubation conditions. While the naphthalene-degrading genes from Rhodococcus-type microorganisms were dominant in naphthalene-degrading enrichments, the genes involved in naphthalene (and polyaromatic hydrocarbon and nitrotoluene) degradation from gram-negative microorganisms, such as Ralstonia, Comamonas, and Burkholderia, were most abundant in the soil microcosms. In contrast to general conceptions, naphthalene-degrading genes from Pseudomonas were not detected, although Pseudomonas is widely known as a model microorganism for studying naphthalene degradation. The real-time PCR analysis with four representative genes showed that the microarray-based quantification was very consistent with real-time PCR (r(2) = 0.74). In addition, application of the arrays to both polyaromatic-hydrocarbon- and benzene-toluene-ethylbenzene-xylene-contaminated and uncontaminated soils indicated that the developed microarrays appeared to be useful for profiling differences in microbial community structures. Our results indicate that this technology has potential as a specific, sensitive, and quantitative tool in revealing a comprehensive picture of the compositions of biodegradation genes and the microbial community in contaminated environments, although more work is needed to improve detection sensitivity.

Bacteria↗

PubMed2005

Microbial community growth and utilization of carbon constituents during thermophilic composting at different oxygen levels.

Composting is characterized by dramatic changes in microbial community structure, to a high extent driven by changes in temperature and in the composition of the organic substrate. This study focuses on the interrelationships between decomposition of major classes in the organic material and dynamics in microbial populations during thermophilic composting of source-separated organic household waste. Experiments were performed in a 200-L laboratory reactor at 16, 2.5, and 1% O(2) in the compost atmosphere. Major classes of carbon constituents were analyzed by chemical methods, and the microbial biomass and community structure determined by fatty acid analyses with phospholipid fatty acids (PLFA) and total ester-linked fatty acids (EL) methods. At all three O(2) levels, the process was characterized by a rapid increase in microbial activity and biomass in the early thermophilic phase, although this period was delayed at the lower O(2) concentrations. Starch and fat were the main substrates utilized at all three O(2) levels during this period. The depletion of the starch fraction coincided with the beginning of a microbial biomass decrease, suggesting that starch is an important carbon substrate for the growth of thermophilic microorganisms during composting. Growth yields in the microbial community based on consumption of major carbon constituent classes in the high-activity period fell between 22 and 28%. Multivariate statistical analysis of changes in fatty acid composition revealed small, but statistically significant differences in the microbial community succession. At 16% O(2), 10 Me fatty acids from Actinomycetes and cyclopropyl fatty acids (from Gram-negative bacteria) became more important with time, whereas 18:1 omega 7t was characteristic at 2.5 and 1% O(2), indicating a more stressed bacterial community at the lower O(2) concentrations. Although adequate composting was achieved at O(2) levels as low as 2.5 and 1%, it is not recommended to compost at such low levels in large-scale systems, because the heterogeneous gas transport through the material in these systems might lead to anaerobic conditions and inefficient composting.

Actinobacteria↗

PubMed2001

Microbial community dynamics in Mediterranean nutrient-enriched seawater mesocosms: changes in abundances, activity and composition.

Quantitative and qualitative changes in bacterial communities from the Mediterranean Sea were compared in duplicate batch mesocosms with or without addition of inorganic nutrients. Methods including traditional microbial ecology techniques, molecular biology and flow cytometry were combined to determine abundances, production, cell size, activity, culturability and taxonomic diversity of bacterial cells. Addition of nutrients and confinement resulted in an increase of bacterial densities which were rapidly controlled by protozoan grazing. Changes in bacterial activity and morphology were observed during the growth phase of bacteria and under grazing pressure. The proportion of medium-size and culturable cells increased during the growth phase. These cells were preferentially consumed by grazers resulting in a strong limitation of bacterial production. As a consequence of the grazing pressure, large cells were produced and contributed to the remaining bacterial productivity after grazing. Grazing had an effect on the taxonomic composition of bacterial communities by preferentially eliminating gamma-Proteobacteria, alpha-Proteobacteria were preserved. It seems that some species from the genera Ruegeria and Cytophaga may have developed defence strategies to escape predation.

Journal Article↗

PubMed1996

Identification in situ and dynamics of bacteria on limnetic organic aggregates (lake snow).

Microbial assemblages on large organic aggregates (lake snow) of Lake Constance, Germany, were analyzed with rRNA-directed fluorescent oligonucleotide probes specific for the domain Bacteria and the alpha-, beta-, and gamma-subclasses of the class Proteobacteria. Lake snow aggregates were either collected in situ by SCUBA diving or in a sediment trap at 50 m or formed of natural lake water incubated in rolling cylinders under simulated in situ conditions. For the latter aggregates, the time course of the microbial colonization was also examined. The natural aggregates and those made in rolling cylinders were composed of the particulate organic material present in the lake and thus reflected the composition of the ambient plankton community. All types of lake snow aggregates examined were heavily colonized by microbial cells and harbored between 0.5 x 10(6) and > 2 x 10(6) cells aggregate -1. Between 55 and 100% of the microbial cells stained with 4', 6-diamidino-2-phenylindole (DAPI) could be visualized with the domain Bacteria-specific probe. In most samples, beta-subclass proteobacteria dominated the microbial community, constituting 27 to 42% of total cells as counted by DAPI staining, irrespective of the composition of the aggregates. During the time course experiments with the laboratory-made aggregates, the fraction of beta-subclass proteobacteria usually increased over time. Except for a few samples, alpha- and gamma-subclass proteobacteria were far less abundant than beta-subclass proteobacteria, constituting 11 to 25 and 9 to 33% of total cells, respectively. Therefore, we assume that a specific aggregate-adapted microbial community was established on the aggregates. Because the compositions of the microbial assemblages on natural and laboratory-made aggregates were similar, we conclude that aggregates made in rolling cylinders are good model system with which to examine the formation and microbial colonization of macroscopic organic aggregates.

Bacteria↗

PubMed2004

A simple method for differential isolation of freely dispersed and particle-associated peat microorganisms.

Differential isolation of physiologically and ecologically diverse microbial groups facilitates evaluation of their activities in the environment. Here we describe a new method for differential isolation of freely dispersed and particle-associated peat microorganisms. The method is based on sequential elution of peat. Two types of natural peat, "young" sphagnum peat and "old" reed-based peat that have significantly different molecular composition as revealed by solid-state 13C NMR, were used for the method development. Various combinations of elution parameters (pH values, surfactant concentration, and sonication) were tested. Protein yield, CFU counts, 16S rDNA gene sequence, and T-RFLP analyses were used as criteria for evaluating the effectiveness of the elution protocols. The optimal method consisted of sequential elutions with (i) pH 6.2, 0.1 M potassium phosphate buffer, (ii) the same buffer amended with 0.01% of Tween 80, and (iii) treatment (ii) amended with sonication for 2 min. This method allowed differential isolation of freely dispersed and particle-associated microorganisms with (i) highest yield, (ii) highest diversity within the each microbial fraction, and (iii) best discrimination between the freely dispersed and particle-associated microbial communities. The method was applied for characterization of peat microbial communities and revealed different taxonomic composition and similar spatial organization of sphagnum and reed-sage peat microorganisms.

Arthrobacter↗

PubMed1981

Fermentation in the rumen and human large intestine.

Fermentation of food by the microbial community of the rumen is essential for the maintenance and growth of ruminants. The microbial ecosystem and its interaction with the host are described, along with recent attempts to manipulate the composition and activity of the microbial community by adding antibiotics and other chemicals to ruminant diets. A similar microbial community and fermentation occur in the large intestine or cecum of most nonruminant animals including the large intestine of humans. The microbial ecosystems of the rumen and human large intestine are compared.

Animals↗

PubMed1997

Dynamics of a microbial community associated with manure hot spots as revealed by phospholipid fatty acid analyses.

Microbial community dynamics associated with manure hot spots were studied by using a model system consisting of a gel-stabilized mixture of soil and manure, placed between layers of soil, during a 3-week incubation period. The microbial biomass, measured as the total amount of phospholipid fatty acids (PLFA), had doubled within a 2-mm distance from the soil-manure interface after 3 days. Principal-component analyses demonstrated that this increase was accompanied by reproducible changes in the composition of PLFA, indicating changes in the microbial community structure. The effect of the manure was strongest in the 2-mm-thick soil layer closest to the interface, in which the PLFA composition was statistically significantly different (P < 0.05) from that of the unaffected soil layers throughout the incubation period. An effect was also observed in the soil layer 2 to 4 mm from the interface. The changes in microbial biomass and community structure were mainly attributed to the diffusion of dissolved organic carbon from the manure. During the initial period of microbial growth, PLFA, which were already more abundant in the manure than in the soil, increased in the manure core and in the 2-mm soil layer closest to the interface. After day 3, the PLFA composition of these layers gradually became more similar to that of the soil. The dynamics of individual PLFA suggested that both taxonomic and physiological changes occurred during growth. Examples of the latter were decreases in the ratios of 16:1 omega 7t to 16:1 omega 7c and of cyclopropyl fatty acids to their respective precursors, indicating a more active bacterial community. An inverse relationship between bacterial PLFA and the eucaryotic 20:4 PLFA (arachidonic acid) suggested that grazing was important.

Animals↗

PubMed2005

Microbial diversity in coastal subsurface sediments: a cultivation approach using various electron acceptors and substrate gradients.

Microbial communities in coastal subsurface sediments are scarcely investigated and have escaped attention so far. But since they are likely to play an important role in biogeochemical cycles, knowledge of their composition and ecological adaptations is important. Microbial communities in tidal sediments were investigated along the geochemical gradients from the surface down to a depth of 5.5 m. Most-probable-number (MPN) series were prepared with a variety of different carbon substrates, each at a low concentration, in combination with different electron acceptors such as iron and manganese oxides. These achieved remarkably high cultivation efficiencies (up to 23% of the total cell counts) along the upper 200 cm. In the deeper sediment layers, MPN counts dropped significantly. Parallel to the liquid enrichment cultures in the MPN series, gradient cultures with embedded sediment subcores were prepared as an additional enrichment approach. In total, 112 pure cultures were isolated; they could be grouped into 53 different operational taxonomic units (OTU). The isolates belonged to the Proteobacteria, "Bacteroidetes," "Fusobacteria," Actinobacteria, and "Firmicutes." Each cultivation approach yielded a specific set of isolates that in general were restricted to this single isolation procedure. Analysis of the enrichment cultures by PCR and denaturing gradient gel electrophoresis revealed an even higher diversity in the primary enrichments that was only partially reflected by the culture collection. The majority of the isolates grew well under anoxic conditions, by fermentation, or by anaerobic respiration with nitrate, sulfate, ferrihydrite, or manganese oxides as electron acceptors.

Actinobacteria↗

PubMed2005

Optical microsensors for analysis of microbial communities.

Fiber-optic microprobes connected to sensitive light meters are ideal tools to resolve the steep gradients of light intensity and spectral composition that prevail in aggregates and surface-associated microbial communities in sediments, biofilms, and microbial mats. They allow for a detailed mapping of light fields and enable insights to the complex optical properties of such highly light-scattering and -absorbing microbial systems. Used in combination with microsensors for chemical species, fiber-optic irradiance microprobes allow for detailed studies of photosynthesis regulation and of the photobiology of microbial phototrophs in intact samples under ambient microenvironmental conditions of the natural habitat. Fiber-optic microprobes connected to sensitive fluorometers enable microscale fluorescence measurements, which can be used to map (i) diffusivity and flow; (ii) distribution of photosynthetic microbes, via their photopigment autofluorescence; and (iii) activity of oxygenic photosynthesis via variable chlorophyll fluorescence measurements. Furthermore, by immobilizing optical indicator dyes on the end of optical fibers, fiber-optic microsensors for temperature, salinity, and chemical species such as oxygen, pH, and CO2 can be realized.

Biofilms↗

PubMed2026

Widespread horizontal transfer and strong selection enhance microbial adaptation in Antarctic soils.

Terrestrial Antarctica harbors compositionally diverse and functionally distinct microbial life. Yet the eco-evolutionary processes underlying adaptation to Antarctica's polyextreme conditions remain largely unknown. Here, we address how horizontal gene transfer (HGT) and de novo mutations influence microbial adaptation in 16 Antarctic soils using combined short- and long-read datasets. Phylogenetic reconciliation and mobile genetic element analysis of 676 metagenome-assembled genomes show frequent HGT across communities. While transferred genes span diverse functional categories, those involved in energy metabolism are exchanged at higher frequency. Genes for aerotrophy, i.e. the consumption of atmospheric trace gases to provide energy, carbon, and hydration, are among the most frequently disseminated. Approximately a quarter of carbon monoxide dehydrogenases and [NiFe]-hydrogenases are predicted to be horizontally acquired and are often associated with mobile genetic elements. Analysis of polymorphisms suggests widespread purifying selection, particularly for aerotrophy genes, providing further evidence that aerotrophy is critical for microbial survival in Antarctica. Genetic variation in hydrogenases is tightly associated with predicted protein structures, with intense selection acting on critical sites preserving stability and function. Together, these findings show that previously unrecognized eco-evolutionary dynamics shape the composition and function of Antarctic microbial communities, and confirm aerotrophy is a strongly selected and horizontally disseminated trait.

Antarctic Regions↗

PubMed2005

Geographic and environmental sources of variation in lake bacterial community composition.

This study used a genetic fingerprinting technique (automated ribosomal intergenic spacer analysis [ARISA]) to characterize microbial communities from a culture-independent perspective and to identify those environmental factors that influence the diversity of bacterial assemblages in Wisconsin lakes. The relationships between bacterial community composition and 11 environmental variables for a suite of 30 lakes from northern and southern Wisconsin were explored by canonical correspondence analysis (CCA). In addition, the study assessed the influences of ARISA fragment detection threshold (sensitivity) and the quantitative, semiquantitative, and binary (presence-absence) use of ARISA data. It was determined that the sensitivity of ARISA was influential only when presence-absence-transformed data were used. The outcomes of analyses depended somewhat on the data transformation applied to ARISA data, but there were some features common to all of the CCA models. These commonalities indicated that differences in bacterial communities were best explained by regional (i.e., northern versus southern Wisconsin lakes) and landscape level (i.e., seepage lakes versus drainage lakes) factors. ARISA profiles from May samples were consistently different from those collected in other months. In addition, communities varied along gradients of pH and water clarity (Secchi depth) both within and among regions. The results demonstrate that environmental, temporal, regional, and landscape level features interact to determine the makeup of bacterial assemblages in northern temperate lakes.

Bacteria↗

PubMed2006

Stability and change in estuarine biofilm bacterial community diversity.

Biofouling communities contribute significantly to aquatic ecosystem productivity and biogeochemical cycling. Our knowledge of the distribution, composition, and activities of these microbially dominated communities is limited compared to other components of estuarine ecosystems. This study investigated the temporal stability and change of the dominant phylogenetic groups of the domain Bacteria in estuarine biofilm communities. Glass slides were deployed monthly over 1 year for 7-day incubations during peak tidal periods in East Sabine Bay, Fla. Community profiling was achieved by using 16S rRNA genes and terminal restriction fragment length polymorphism (T-RFLP) of 16S rRNA genes in combination with ribotyping, cloning, and sequencing to evaluate diversity and to identify dominant microorganisms. Bacterial community profiles from biofilms grown near the benthos showed distinct periods of constancy within winter and summer sampling periods. Similar periods of stability were also seen in T-RFLP patterns from floating biofilms. Alternating dominance of phylogenetic groups between seasons appeared to be associated with seasonal changes in temperature, nutrient availability, and light. The community structure appeared to be stable during these periods despite changes in salinity and in dissolved oxygen.

Bacteria↗

PubMed2006

Community structure of Archaea and Bacteria in a profundal lake sediment Lake Kinneret (Israel).

The microbial community structure of an anoxic profundal lake sediment, i.e., subtropical Lake Kinneret, was analysed with respect to its composition by culture-independent molecular methods including terminal restriction fragment length polymorphism (T-RFLP) analysis, comparative sequence analysis, and quantitative real-time PCR. In particular we were interested in the structure, species composition, and relative abundance of the overall microbial community in the methanogenic sediment layer (0-10 cm depth). Pairwise comparison of archaeal and bacterial 16S rRNA gene T-RFLP profiles obtained from three independent samplings indicated stability of the microbial community. The numbers of Archaea and Bacteria, quantified by real-time PCR, amounted to about 10(8) and 10(10) 16S rRNA gene copies cm(-3) sediment, respectively, suggesting that Archaea may account for only a minor fraction (approximately 1%) of the total prokaryotic community. Hydrogenotrophic Methanomicrobiales and acetoclastic Methanosaeta spp. dominated T-RFLP profiles of the archaeal community. T-RFLP profiles of the bacterial community were dominated by Deltaproteobacteria, sulphate reducers and syntrophs in particular. The second most abundant group was assigned to the Bacteroidetes-Chlorobi-group. Only one bacterial group, which was affiliated with halorespiring bacteria of subphylum II of the Chloroflexi, showed variation in abundance within the sediment samples investigated. Our study gives a comprehensive insight into the structure of the bacterial and archaeal community of a profundal lake sediment, indicating that sulphate reducers, syntrophs, bacteroidetes, halorespirers and methanogens are of particular importance in Lake Kinneret sediment.

Archaea↗

PubMed2007

Microbial community dynamics during assays of harbour oil spill bioremediation: a microscale simulation study.

AIMS: Microcosm experiments simulating an oil spill event were performed to evaluate the response of the natural microbial community structure of Messina harbour seawater following the accidental load of petroleum. METHODS AND RESULTS: An experimental harbour seawater microcosm, supplemented with nutrients and crude oil, was monitored above 15 days in comparison with unpolluted ones (control microcosms). Bacterial cells were counted with a Live/Dead BacLight viability kit; leucine aminopeptidase, beta-glucosidase, alkaline phosphatase, lipase and esterase enzymes were measured using fluorogenic substrates. The microbial community dynamic was monitored by isolation of total RNA, RT-PCR amplification of 16S rRNA, cloning and sequencing. Oil addition stimulated an increase of the total bacterial abundance, leucine aminopeptidase and phosphatase activity rates, as well as a change in the community structure. This suggested a prompt response of micro-organisms to the load of petroleum hydrocarbons. CONCLUSIONS: The present study on the viability, specific composition and metabolic characteristics of the microbial community allows a more precise assessment of oil pollution. Both structural and functional parameters offer interesting perspectives as indicators to monitor changes caused by petroleum hydrocarbons. SIGNIFICANCE AND IMPACT OF THE STUDY: A better knowledge of microbial structural successions at oil-polluted sites is essential for environmental bioremediation. Data obtained in microcosm studies improve our understanding of natural processes occurring during oil spills.

Alcanivoraceae↗

PubMed2002

Changes in soil microbial community structure and function in an alpine dry meadow following spring snow melt.

Previous work in an alpine dry meadow in the Front Range of the Rocky Mountains has shown that microbial biomass is high during winter and declines rapidly as snow melts in the spring, and that this decline is associated with changes in temperature regime and substrate availability. In this study we tested the hypothesis that the summer and winter microbial communities differ in function and composition. Shifts in species composition between pre- and post-snowmelt communities were detected using reciprocal hybridization of community DNA; DNA extracted from soils sampled at different times was significantly less homologous relative to spatial replicates sampled at the same time. Fungal/bacterial ratios, as measured by direct microscopic counts and by substrate-induced respiration experiments with specific inhibitors, were higher in winter soils. Specific activity of cellulase (absolute cellulase activity per unit microbial biomass C) was higher in the winter soils than in summer soils, while specific amylase activity was not different between winter and summer. Based on most-probable number measurements, the use of the phenolic compound vanillic acid was highest in the winter, while the use of the amino acid glycine was lowest in the winter. Winter and summer soil respiration responded differently to temperature; at 0 degrees C, winter soils respired at a higher proportion of the 22 degrees C rate than did summer soils.

Biomass↗

PubMed2006

Microbial community structure and dynamics of dark fire-cured tobacco fermentation.

The Italian Toscano cigar production includes a fermentation step that starts when dark fire-cured tobacco leaves are moistened and mixed with ca. 20% prefermented tobacco to form a 500-kg bulk. The dynamics of the process, lasting ca. 18 days, has never been investigated in detail, and limited information is available on microbiota involved. Here we show that Toscano fermentation is invariably associated with the following: (i) an increase in temperature, pH, and total microbial population; (ii) a decrease in reducing sugars, citric and malic acids, and nitrate content; and (iii) an increase in oxalic acid, nitrite, and tobacco-specific nitrosamine content. The microbial community structure and dynamics were investigated by culture-based and culture-independent approaches, including denaturing gradient gel electrophoresis and single-strand conformational polymorphism. Results demonstrate that fermentation is assisted by a complex microbial community, changing in structure and composition during the process. During the early phase, the moderately acidic and mesophilic environment supports the rapid growth of a yeast population predominated by Debaryomyces hansenii. At this stage, Staphylococcaceae (Jeotgalicoccus and Staphylococcus) and Lactobacillales (Aerococcus, Lactobacillus, and Weissella) are the most commonly detected bacteria. When temperature and pH increase, endospore-forming low-G+C content gram-positive bacilli (Bacillus spp.) become evident. This leads to a further pH increase and promotes growth of moderately halotolerant and alkaliphilic Actinomycetales (Corynebacterium and Yania) during the late phase. To postulate a functional role for individual microbial species assisting the fermentation process, a preliminary physiological and biochemical characterization of representative isolates was performed.

Bacteria↗