Growth of phototrophic biofilms from limestone monuments under laboratory conditions

In the current study, five phototrophic biofilms from different Southern Europe limestone monuments were characterised by molecular techniques and cultivated under laboratory conditions. Phototrophic biofilms were collected from Orologio Tower in Martano (Italy), Santa Clara-a-Velha Monastery and Ajuda National Palace, both in Portugal, and Seville and Granada Cathedrals from Spain. The biofilms were grown under laboratory conditions and periodically sampled in order to monitor their evolution over a three-month period. Prokaryotic communities from natural samples and cultivated biofilms were monitored using denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rRNA gene fragments in conjunction with clone sequencing and phylogenetic analysis. DNA-based molecular analysis of 16S rRNA gene fragments from the natural green biofilms revealed complex and different communities composition with respect to phototrophic microorganisms. The biofilms from Orologio Tower (Martano, Italy) and Santa Clara-a-Velha Monastery (Coimbra, Portugal) were dominated by the microalga Chlorella. The cyanobacterium Chroococcidiopsis was the dominating genus from Ajuda National Palace biofilm (Lisbon, Portugal). The biofilms from Seville and Granada Cathedrals (Spain) were both dominated by the cyanobacterium Pleurocapsa. The DGGE analysis of the cultivated biofilms showed that the communities developed differently in terms of species establishment and community composition during the three-month incubation period. The biofilm culture from Coimbra (Portugal) showed a remarkable stability of the microbial components of the natural community in laboratory conditions. With this work, a multiple-species community assemblage was obtained for further stone colonisation experiments.     

Comparison of Two Fingerprinting Techniques, Terminal Restriction Fragment Length Polymorphism and Automated Ribosomal Intergenic Spacer Analysis, for Determination of Bacterial Diversity in Aquatic Environments

We investigated bacterial diversity in different aquatic environments (including marine and lagoon sediments, coastal seawater, and groundwater), and we compared two fingerprinting techniques (terminal restriction fragment length polymorphism [T-RFLP] and automated ribosomal intergenic spacer analysis [ARISA]) which are currently utilized for estimating richness and community composition. Bacterial diversity ranged from 27 to 99 phylotypes (on average, 56) using the T-RFLP approach and from 62 to 101 genotypes (on average, 81) when the same samples were analyzed using ARISA. The total diversity encountered in all matrices analyzed was 144 phylotypes for T-RFLP and 200 genotypes for ARISA. Although the two techniques provided similar results in the analysis of community structure, bacterial richness and diversity estimates were significantly higher using ARISA. These findings suggest that ARISA is more effective than T-RFLP in detecting the presence of bacterial taxa accounting for <5% of total amplified product. ARISA enabled also distinction among aquatic bacterial isolates of Pseudomonas spp. which were indistinguishable using T-RFLP analysis. Overall, the results of this study show that ARISA is more accurate than T-RFLP analysis on the 16S rRNA gene for estimating the biodiversity of aquatic bacterial assemblages.     

Comparison of two fingerprinting techniques, terminal restriction fragment length polymorphism and automated ribosomal intergenic spacer analysis, for determination of bacterial diversity in aquatic environments

We investigated bacterial diversity in different aquatic environments (including marine and lagoon sediments, coastal seawater, and groundwater), and we compared two fingerprinting techniques (terminal restriction fragment length polymorphism [T-RFLP] and automated ribosomal intergenic spacer analysis [ARISA]) which are currently utilized for estimating richness and community composition. Bacterial diversity ranged from 27 to 99 phylotypes (on average, 56) using the T-RFLP approach and from 62 to 101 genotypes (on average, 81) when the same samples were analyzed using ARISA. The total diversity encountered in all matrices analyzed was 144 phylotypes for T-RFLP and 200 genotypes for ARISA. Although the two techniques provided similar results in the analysis of community structure, bacterial richness and diversity estimates were significantly higher using ARISA. These findings suggest that ARISA is more effective than T-RFLP in detecting the presence of bacterial taxa accounting for <5% of total amplified product. ARISA enabled also distinction among aquatic bacterial isolates of Pseudomonas spp. which were indistinguishable using T-RFLP analysis. Overall, the results of this study show that ARISA is more accurate than T-RFLP analysis on the 16S rRNA gene for estimating the biodiversity of aquatic bacterial assemblages.     

NESTED AUTOMATED RIBOSOMAL INTERGENIC SPACER ANALYSIS: A RAPID AND ACCURATE METHOD FOR COMPARISON OF BACTERIAL COMMUNITY COMPOSITION

Nested automated ribosomal intergenic spacer analysis (ARISA) was used to examine the community structure of epilithic biofilms in freshwater streams experiencing different levels of human impact. This molecular fingerprinting technique generated reproducible profiles of bacterial community structure that varied significantly between stream sites. Nested ARISA was determined to be a cost-effective, high-throughput approach to assess bacterial community composition from very small sample volumes, requiring little sampling effort and without the need for taxonomic identification of individual organisms. In combination with multidimensional scaling, nested ARISA provides a rapid and sensitive method to carry out complex analyses of bacterial community structure.

PRACTICAL APPLICATIONS

Nested automated ribosomal intergenic spacer analysis (ARISA) provides a high-throughput molecular method with which to screen large numbers of environmental samples for differences in microbial community structure. This sensitive approach benefits assessments from small sample volumes or environments exhibiting reduced microbial biomass (both aquatic and terrestrial). Differences in bacterial community structure (obtained from ARISA profiles) could be used to characterize the impact of anthropogenic disturbance on freshwater systems, analogous to the current use of macroinvertebrate indicators of freshwater ecological health.     

A dynamic programming algorithm for binning microbial community profiles

MOTIVATION: A number of community profiling approaches have been widely used to study the microbial community composition and its variations in environmental ecology. Automated Ribosomal Intergenic Spacer Analysis (ARISA) is one such technique. ARISA has been used to study microbial communities using 16S-23S rRNA intergenic spacer length heterogeneity at different times and places. Owing to errors in sampling, random mutations in PCR amplification, and probably mostly variations in readings from the equipment used to analyze fragment sizes, the data read directly from the fragment analyzer should not be used for down stream statistical analysis. No optimal data preprocessing methods are available. A commonly used approach is to bin the reading lengths of the 16S-23S intergenic spacer. We have developed a dynamic programming algorithm based binning method for ARISA data analysis which minimizes the overall differences between replicates from the same sampling location and time. RESULTS: In a test example from an ocean time series sampling program, data preprocessing identified several outliers which upon re-examination were found to be because of systematic errors. Clustering analysis of the ARISA from different times based on the dynamic programming algorithm binned data revealed important features of the biodiversity of the microbial communities.     

Coupling 16S-ITS rDNA clone libraries and automated ribosomal intergenic spacer analysis to show marine microbial diversity: development and application to a time series

We outline an approach to simultaneously assess multilevel microbial diversity patterns utilizing 16S-ITS rDNA clone libraries coupled with automated ribosomal intergenic spacer analysis (ARISA). Sequence data from 512 clones allowed estimation of ARISA fragment lengths associated with bacteria in a coastal marine environment. We matched 92% of ARISA peaks (each comprising >1% total amplified product) with corresponding lengths from clone libraries. These peaks with putative identification accounted for an average of 83% of total amplified community DNA. At 16S rDNA similarities <98%, most taxa displayed differences in ARISA fragment lengths >10 bp, readily detectable and suggesting ARISA resolution is near the 'species' level. Prochlorococcus abundance profiles from ARISA were strongly correlated (r2=0.86) to Prochlorococcus cell counts, indicating ARISA data are roughly proportional to actual cell abundance within a defined taxon. Analysis of ARISA profiles for 42 months elucidated patterns of microbial presence and abundance providing insights into community shifts and ecological niches for specific organisms, including a coupling of ecological patterns for taxa within the Prochlorococcus, the Gamma Proteobacteria and Actinobacteria. Clade-specific ARISA protocols were developed for the SAR11 and marine cyanobacteria to resolve ambiguous identifications and to perform focused studies. 16S-ITS data allowed high-resolution identification of organisms by ITS sequence analysis, and examination of microdiversity.     

Molecular diversity studies of bacterial communities of oil polluted microbial mats from the Etang de Berre (France)

The biodiversity of microbial mats inhabiting the oil-contaminated lagoon Etang de Berre was determined by molecular approaches. The fingerprint of denaturing gradient gel electrophoresis (DGGE) and automatic ribosomal intergenic spacer analysis (ARISA) of mats exposed to different pollution levels showed specific microbial communities for each site but similar diversity richness. Species composition of the mats were compared by constructing 16S rRNA libraries. Amplified rDNA restriction analysis (ARDRA) of clone libraries confirmed their similar level of diversity richness. Phylogenetic analysis of the 16S rRNA sequences showed that the classes gamma and alpha of Proteobacteria were abundantly present in both sites whereas phylotypes related to the delta-Proteobacteria and to the uncultured WS3 group were mainly found in the site with the highest pollution. Identification of the species involved in oil degradation by combining culture-based approaches and DGGE, showed that enrichment cultures were constituted by members of the Rhodobacterales and species related to Rhodococcus, Sphingomonas, Xanthomonas and Microbacterium, all of them known for their ability to degrade hydrocarbons. Our findings suggest that oil pollution has not affected the biodiversity richness of the mats. However, the populations involved in hydrocarbon degradation represent a minor fraction of the mat communities in the Etang de Berre.     

Fungal endophytic communities in grapevines (Vitis vinifera L.) respond to crop management

We studied the distribution of fungal endophytes of grapevine (Vitis vinifera L.) plants in a subalpine area of northern Italy, where viticulture is of high economic relevance. We adopted both cultivation-based and cultivation-independent approaches to address how various anthropic and nonanthropic factors shape microbial communities. Grapevine stems were harvested from several locations considering organic and integrated pest management (IPM) and from the cultivars Merlot and Chardonnay. Cultivable fungi were isolated and identified by internal-transcribed-spacer sequence analysis, using a novel colony-PCR method, to amplify DNA from fungal specimens. The composition of fungal communities was assessed using a cultivation-independent approach, automated ribosomal intergenic spacer analysis (ARISA). Multivariate statistical analysis of both culture-dependent and culture-independent data sets was convergent and indicated that fungal endophytic communities in grapevines from organically managed farms were different from those from farms utilizing IPM. Fungal communities in plants of cv. Merlot and cv. Chardonnay overlapped when analyzed using culture-dependent approaches but could be partially resolved using ARISA fingerprinting.     

Diversity of phototrophic components in biofilms from Piperno historical stoneworks

We investigated phototrophic microorganisms dwelling on stone walls made of Piperno, a volcanic rock frequently used as construction material in historical buildings in Naples, Italy. Biofilms from three historical buildings in the center of the city and from a natural Piperno quarry located in a suburban area were examined. Light and electron microscopy, and molecular biology techniques allowed the identification of 17 species belonging to Cyanobacteria, Rhodophyta, Bacillariophyta, and Chlorophyta. Cyanobacteria were the dominant components in all the biofilms. No significant differences in microbial composition were observed for biofilms collected in autumn and spring, with minor exceptions for the quarry samples, where environmental conditions were relatively more stable than in the city. Results are discussed in comparison with information on microbial communities dwelling on other kinds of substrata commonly used in historical buildings in the Neapolitan area.     

Salt marsh plant-microorganism interaction in the presence of mixed contamination

In this study we investigated under laboratory conditions, the combined effect of the presence of two contaminants (Cu and PAHs) and plants (Halimione portulacoides) on a salt marsh microbial community, in terms of genetic structure, abundance and capacity to remove those contaminants. Plants changed the microbial community structure (evaluated by ARISA), but only in the treatments without PAHs. Also, in the presence of plants, Cu displayed lower toxicity (ToxScreen test) than in its absence. Nevertheless, the presence of plants interfered with the degradation (decreasing it up to 45%) of higher molecular weight PAHs by sediment microorganisms. This was in agreement with the lower microbial numbers (quantified by DAPI) observed in the presence of the plants, pointing for a competition between plants and microorganisms for nutrients. These results are new for salt marshes, and highlight the need for fertilization in order to obtain optimal effects of rhizoremediation for this type of compounds.     

Analyzing salt-marsh fungal diversity: comparing ARISA fingerprinting with clone sequencing and pyrosequencing

In a previous study from our laboratory we used automated ribosomal intergenic spacer analysis (ARISA) to assess salt-marsh fungal diversity (Torzilli et al. 2006). The results demonstrated that different salt-marsh plants harbor distinct fungal communities, thereby supporting the hypothesis that substratum type is an important factor in determining fungal community composition. However, ARISA of several pure cultures of salt-marsh fungi indicated that an operational taxonomic unit (OUT) in an ARISA community profile may represent more than one taxon. To assess the extent to which such ambiguity might have affected the interpretation of our ARISA fingerprinting, we have now fingerprinted and sequenced clones derived from the same fungal DNA used for our ARISA community profiles. Results from this confirmed that an ARISA OTU may represent multiple taxa and that a given taxon may be represented by more than one OTU. Nonetheless, sequencing still confirmed the importance of substratum in determining community composition, and indicated that despite ambiguities associated with OTU's, ARISA may be used to provide a quick snapshot of diversity which can be further refined using sequencing methods. In addition, we compared the fungal diversity from short-form Spartina alterniflora as revealed by clone sequencing with that obtained from pyrosequencing, which avoids the cloning biases of traditional sequencing, and provide greatly expanded depth of coverage. Pyrosequencing significantly enhanced the characterization of fungal diversity compared to traditional clone sequencing.     

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.     

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.     

Automated approach for ribosomal intergenic spacer analysis of microbial diversity and its application to freshwater bacterial communities.

An automated method of ribosomal intergenic spacer analysis (ARISA) was developed for the rapid estimation of microbial diversity and community composition in freshwater environments. Following isolation of total community DNA, PCR amplification of the 16S-23S intergenic spacer region in the rRNA operon was performed with a fluorescence-labeled forward primer. ARISA-PCR fragments ranging in size from 400 to 1,200 bp were next discriminated and measured by using an automated electrophoresis system. Database information on the 16S-23S intergenic spacer was also examined, to understand the potential biases in diversity estimates provided by ARISA. In the analysis of three natural freshwater bacterial communities, ARISA was rapid and sensitive and provided highly reproducible community-specific profiles at all levels of replication tested. The ARISA profiles of the freshwater communities were quantitatively compared in terms of both their relative diversity and similarity level. The three communities had distinctly different profiles but were similar in their total number of fragments (range, 34 to 41). In addition, the pattern of major amplification products in representative profiles was not significantly altered when the PCR cycle number was reduced from 30 to 15, but the number of minor products (near the limit of detection) was sensitive to changes in cycling parameters. Overall, the results suggest that ARISA is a rapid and effective community analysis technique that can be used in conjunction with more accurate but labor-intensive methods (e.g., 16S rRNA gene cloning and sequencing) when fine-scale spatial and temporal resolution is needed.     

Quantifying Microbial Diversity: Morphotypes, 16S rRNA Genes, and Carotenoids of Oxygenic Phototrophs in Microbial Mats

We quantified the diversity of oxygenic phototrophic microorganisms present in eight hypersaline microbial mats on the basis of three cultivation-independent approaches. Morphological diversity was studied by microscopy. The diversity of carotenoids was examined by extraction from mat samples and high-pressure liquid chromatography analysis. The diversity of 16S rRNA genes from oxygenic phototrophic microorganisms was investigated by extraction of total DNA from mat samples, amplification of 16S rRNA gene segments from cyanobacteria and plastids of eukaryotic algae by phylum-specific PCR, and sequence-dependent separation of amplification products by denaturing-gradient gel electrophoresis. A numerical approach was introduced to correct for crowding the results of chromatographic and electrophoretic analyses. Diversity estimates typically varied up to twofold among mats. The congruence of richness estimates and Shannon-Weaver indices based on numbers and proportional abundances of unique morphotypes, 16S rRNA genes, and carotenoids unveiled the underlying diversity of oxygenic phototrophic microorganisms in the eight mat communities studied.     

Biodiversity of a Burkholderia cepacia population isolated from the maize rhizosphere at different plant growth stages.

A Burkholderia cepacia population naturally occurring in the rhizosphere of Zea mays was investigated in order to assess the degree of root association and microbial biodiversity at five stages of plant growth. The bacterial strains isolated on semiselective PCAT medium were mostly assigned to the species B. cepacia by an analysis of the restriction patterns produced by amplified DNA coding for 16S rRNA (16S rDNA) (ARDRA) with the enzyme AluI. Partial 16S rDNA nucleotide sequences of some randomly chosen isolates confirmed the ARDRA results. Throughout the study, B. cepacia was strictly associated with maize roots, ranging from 0.6 to 3.6% of the total cultivable microflora. Biodiversity among 83 B. cepacia isolates was analyzed by the random amplified polymorphic DNA (RAPD) technique with two 10-mer primers. An analysis of RAPD patterns by the analysis of molecular variance method revealed a high level of intraspecific genetic diversity in this B. cepacia population. Moreover, the genetic diversity was related to divergences among maize root samplings, with microbial genetic variability markedly higher in the first stages of plant growth; in other words, the biodiversity of this rhizosphere bacterial population decreased over time.     

Automated Approach for Ribosomal Intergenic Spacer Analysis of Microbial Diversity and Its Application to Freshwater Bacterial Communities

An automated method of ribosomal intergenic spacer analysis (ARISA) was developed for the rapid estimation of microbial diversity and community composition in freshwater environments. Following isolation of total community DNA, PCR amplification of the 16S-23S intergenic spacer region in the rRNA operon was performed with a fluorescence-labeled forward primer. ARISA-PCR fragments ranging in size from 400 to 1,200 bp were next discriminated and measured by using an automated electrophoresis system. Database information on the 16S-23S intergenic spacer was also examined, to understand the potential biases in diversity estimates provided by ARISA. In the analysis of three natural freshwater bacterial communities, ARISA was rapid and sensitive and provided highly reproducible community-specific profiles at all levels of replication tested. The ARISA profiles of the freshwater communities were quantitatively compared in terms of both their relative diversity and similarity level. The three communities had distinctly different profiles but were similar in their total number of fragments (range, 34 to 41). In addition, the pattern of major amplification products in representative profiles was not significantly altered when the PCR cycle number was reduced from 30 to 15, but the number of minor products (near the limit of detection) was sensitive to changes in cycling parameters. Overall, the results suggest that ARISA is a rapid and effective community analysis technique that can be used in conjunction with more accurate but labor-intensive methods (e.g., 16S rRNA gene cloning and sequencing) when fine-scale spatial and temporal resolution is needed.     

PCR-DGGE analysis of lactic acid bacteria and yeast dynamics during the production processes of three varieties of Panettone

Aims:  To study lactic acid bacteria (LAB) and yeast dynamics during the production processes of sweet-leavened goods manufactured with type I sourdoughs.
Methods and Results:  Fourteen sourdough and dough samples were taken from a baking company in central Italy during the production lines of three varieties of Panettone. The samples underwent pH measurements and plating analysis on three solid media. The microbial DNA was extracted from both the (sour)doughs and the viable LAB and yeast cells collected in bulk, and subjected to PCR-denaturing gradient gel electrophoresis (DGGE) analysis. The molecular fingerprinting of the cultivable plus noncultivable microbial populations provide evidence of the dominance of Lactobacillus sanfranciscensis , Lactobacillus brevis and Candida humilis in the three fermentation processes. The DGGE profiles of the cultivable communities reveal a bacterial shift in the final stages of two of the production processes, suggesting an effect of technological parameters on the selection of the dough microflora.
Conclusions:  Our findings confirm the importance of using a combined analytical approach to explore microbial communities that develop during the leavening process of sweet-leavened goods.
Significance and Impact of the Study:  In-depth studies of sourdough biodiversity and population dynamics occurring during sourdough fermentation are fundamental for the control of the leavening process and the manufacture of standardized, high-quality products.