Culture-dependent and -independent approaches establish the complexity of a PAH-degrading microbial consortium

2005 ◽  
Vol 51 (11) ◽  
pp. 897-909 ◽  
Author(s):  
Marc Viñas ◽  
Jordi Sabaté ◽  
Caterina Guasp ◽  
Jorge Lalucat ◽  
Anna M Solanas
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Microbial Consortium ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Sole Source ◽  
Rrna Gene ◽  
Dominant Group ◽  
Clone Libraries ◽  
Variable Regions ◽  
Culture Dependent

A microbial consortium (AM) obtained by sequential enrichment in liquid culture with a polycyclic aromatic hydrocarbon (PAH) mixture of three- and four-ringed PAHs as a sole source of carbon and energy was examined using a triple-approach method based on various cultivation strategies, denaturing gradient gel electrophoresis (DGGE), and the screening of 16S and 18S rRNA gene clone libraries. Eleven different sequences by culture-dependent techniques and seven by both DGGE and clone libraries were obtained. The comparison of three variable regions (V3–V5) of the 16S rRNA gene between the sequences obtained yielded 19 different microbial components. Proteobacteria were the dominant group, representing 83% of the total, while the Cytophaga–Flexibacter–Bacteroides group (CFB) was 11% and the Ascomycota fungi 6%. β-Proteobacteria were predominant in the DGGE and clone library methods, whereas they were a minority in culturable strains. The highest diversity and number of noncoincident sequences were achieved by the cultivation method that showed members of the α-, β-, and γ-Proteobacteria; CFB bacterial group; and Asco mycota fungi. Only six of the 11 strains isolated showed PAH-degrading capability. The bacterial strain (AMS7) and the fungal strain (AMF1), which were similar to Sphingomonas sp. and Fusarium sp., respectively, achieved the greatest PAH depletion. The results indicate that polyphasic assessment is necessary for a proper understanding of the composition of a microbial consortium.Key words: microbial consortium, microbial diversity, PAHs, DGGE, 16S rRNA gene.

scholarly journals The Existence of Endophytic Actinobacteria from Rhododendron zoelerri Revealed by Culture-Dependent and Culture-Independent Approaches

2018 ◽  
Vol 25 (2) ◽  
pp. 54
Author(s):  
Yulin Lestari ◽  
Lia Aseptin Murdini ◽  
Dedy Duryadi Solihin
Keyword(s):  
Community Structure ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Morphological Characteristics ◽  
Rrna Gene ◽  
Culture Independent ◽  
Reference Strains ◽  
Culture Dependent ◽  
Endophytic Actinobacteria

Endophytic actinobacteria from medicinal plant may play a significant role in producing bioactive compounds. The information regarding their diversity is an important.  Rhododendron are traditionally used for treating human disorders. One of the selected Rhododendron used in this study was R.  zoelleri from Papua origin, which has been conserved and grown in Cibodas Botanical Garden, West Java, Indonesia. The aim of this study was to assess the existence of endophytic actinobacteria from R. zoelleri based on a culture-dependent and their community structure based on a culture-independent approach. Culturable actinobacteria were isolated and cultured on HV medium. Polymerase Chain Reaction-Denaturing Gradient Gel Electrophoresis (PCR-DGGE) targeting the metagenomic 16S rRNA was used to analyse the structure of the actinobacterial community. Six culturable endophytic actinobacteria (200 cfu/g fresh weight) from R. zoelleri were successfully isolated, three isolates from leaf, and the other isolates were obtained from stem. The six culturable isolates were RZP 1.3, RZP 1.1, RZP 2.2, RZPB 1.1, RZPB 7.1, RZPB 4.1. Based on their morphological characteristics, the endophytes have Streptomyces characters. The existence of Streptomyces spp. were also confirmed with molecular analysis based on 16S rRNA gene. The phylogenetic analysis based on 16S rRNA gene to the reference strains available in EzTaxon-e database showed that six isolates were closely related to S. djakartensis strains of NBRC 15409ᵀ(99.19%), S. tritolerans strains of DAS 165T(99.90%), S. coelicoflavus strains of NBRC 15399T(99.59). However, they showed differences in morphological characteristics as compared with the reference strains. The metagenomic analysis of the DGGE profile based on 16S rRNA gene showed the community structure of endophytic actinobacteria from R. zoelleri which was represented by 13 DGGE bands. The bands were closely related to Agromyces, Gordonia, Microbacterium, Micromonospora, Propionibacterium, Saccharomonospora, Streptomyces which have 93.18%-100% similarity. Based on the data, it showed diversity of endophytic actinobacteria from R. zoelleri which may be further assess for their novelty and bioprospecting.

scholarly journals Molecular Diversity Assessment of Plant Growth Promoting Rhizobacteria Using Denaturing Gradient Gel Electrophoresis (DGGE) of 16s rRNA Gene

2017 ◽  
Vol 5 (1) ◽  
pp. 72-80
Author(s):  
Umesh Prasad Shrivastava
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
16S Rdna ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Uttar Pradesh ◽  
Plant Growth Promoting Rhizobacteria ◽  
Melting Behavior ◽  
Rrna Gene ◽  
Dgge Profile ◽  
Diversity Assessment

The rhizobacteria were isolated from rhizosphere of rice plant of different fields of 4 districts of Nepal and 5 districts of Bihar and Uttar Pradesh, adjoining states of India with Nepal. The DGGE analysis was performed for diversity analysis. For the construction of dendrogram, 16S rRNA gene was amplified by two different sets of primers. The DGGE ladder consisting of PCR amplified products of nine pure bacterial cultures were obtained. The first DGGE ladder was prepared by 400 bp fragment of 16S rDNA with GC clamp and the second DGGE ladder was prepared with 200 bp fragment of 16S rDNA with GC clamp. The perpendicular DGGE of these amplicons based on their melting behavior clearly demonstrated separation of different isolates. The 16S rDNA fragment amplified with primer set of V2-V3 regions with GC clamp showed separation between 40-60% of denaturant. The DGGE profile based on primer set F352T and 519r for various bacteria present in soil samples of 5 districts of India and 4 districts of Nepal revealed that the number of bands which might be specific for diazotrophic isolates varied from 2 to 11. The dendrogram constructed based on DGGE profile of various samples of 5 districts of India and 4 districts of Nepal showed that all the samples could be clustered in nine groups with 58-96% similarity to each other. Among all these 37 samples, only Var-4 and Var-5 showed 100% similarity, no other samples from any site showed 100% similarity. Int. J. Appl. Sci. Biotechnol. Vol 5(1): 72-80

scholarly journals Mucosa-Associated Faecalibacterium prausnitzii Phylotype Richness Is Reduced in Patients with Inflammatory Bowel Disease

2015 ◽  
Vol 81 (21) ◽  
pp. 7582-7592 ◽  
Author(s):  
Mireia Lopez-Siles ◽  
Margarita Martinez-Medina ◽  
Carles Abellà ◽  
David Busquets ◽  
Miriam Sabat-Mir ◽  
...  
Keyword(s):  
Inflammatory Bowel Disease ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Bowel Disease ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Gastrointestinal Disease ◽  
Rrna Gene ◽  
Content Type ◽  
Faecalibacterium Prausnitzii ◽  
Inflammatory Bowel

ABSTRACTFaecalibacterium prausnitziidepletion in intestinal diseases has been extensively reported, but little is known about intraspecies variability. This work aims to determine if subjects with gastrointestinal disease host mucosa-associatedF. prausnitziipopulations different from those hosted by healthy individuals. A new species-specific PCR-denaturing gradient gel electrophoresis (PCR-DGGE) method targeting the 16S rRNA gene was developed to fingerprintF. prausnitziipopulations in biopsy specimens from 31 healthy control (H) subjects and 36 Crohn's disease (CD), 23 ulcerative colitis (UC), 6 irritable bowel syndrome (IBS), and 22 colorectal cancer (CRC) patients. The richness ofF. prausnitziisubtypes was lower in inflammatory bowel disease (IBD) patients than in H subjects. The most prevalent operational taxonomic units (OTUs) consisted of four phylotypes (OTUs with a 99% 16S rRNA gene sequence similarity [OTU99]), which were shared by all groups of patients. Their distribution and the presence of some disease-specificF. prausnitziiphylotypes allowed us to differentiate the populations in IBD and CRC patients from that in H subjects. At the level of a minimum similarity of 97% (OTU97), two phylogroups accounted for 98% of the sequences. Phylogroup I was found in 87% of H subjects but in under 50% of IBD patients (P= 0.003). In contrast, phylogroup II was detected in >75% of IBD patients and in only 52% of H subjects (P= 0.005). This study reveals that even though the main members of theF. prausnitziipopulation are present in both H subjects and individuals with gut diseases, richness is reduced in the latter and an altered phylotype distribution exists between diseases. This approach may serve as a basis for addressing the suitability ofF. prausnitziiphylotypes to be quantified as a putative biomarker of disease and depicting the importance of the loss of these subtypes in disease pathogenesis.

Identification and occurrence of tetrad-forming Alphaproteobacteria in anaerobic–aerobic activated sludge processes

Microbiology ◽  
2004 ◽  
Vol 150 (11) ◽  
pp. 3741-3748 ◽  
Author(s):  
Man-Tak Wong ◽  
Fea Mein Tan ◽  
Wun Jern Ng ◽  
Wen-Tso Liu
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Organic Substrate ◽  
Rrna Gene ◽  
Soluble Phosphate ◽  
Biological Phosphorus Removal ◽  
Dominant Group ◽  
Chemical Profiles ◽  
Group A ◽  
Activated Sludge Processes

In an acetate-fed anaerobic–aerobic membrane bioreactor, a deteriorated enhanced biological phosphorus removal (EBPR) community was developed (as determined based on the chemical profiles of organic substrate, soluble phosphate, and intracellular carbohydrate and polyhydroxyalkanote (PHA) concentrations). Microscopic observations revealed the dominance of tetrad-forming organisms (TFOs), of which the majority stained positively for PHA under anaerobic conditions. Fluorescence in situ hybridization (FISH) confirmed that the Alphaproteobacteria (85·0±7·0 % of total cells) were the most dominant group. A 16S rRNA gene clone library specific for the Alphaproteobacteria indicated that most 16S rRNA gene clones (61 % of total clones) were closely affiliated with ‘Defluvicoccus vanus’, forming a cluster within subgroup 1 of the Alphaproteobacteria. Combined PHA staining and FISH with specific probes designed for the members of the ‘Defluvicoccus’ cluster suggested diversity within this TFO cluster, and that these TFOs were newly identified glycogen-accumulating organisms in EBPR systems. However, these ‘Defluvicoccus’-related TFOs were only seen in low abundance in 12 different EBPR and non-EBPR systems, suggesting that they were not the key populations responsible for the deterioration of full-scale EBPR processes.

scholarly journals Change in Bacterial Community Structure during In Situ Biostimulation of Subsurface Sediment Cocontaminated with Uranium and Nitrate

2004 ◽  
Vol 70 (8) ◽  
pp. 4911-4920 ◽  
Author(s):  
Nadia N. North ◽  
Sherry L. Dollhopf ◽  
Lainie Petrie ◽  
Jonathan D. Istok ◽  
David L. Balkwill ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Microbial Communities ◽  
Quantitative Pcr ◽  
Rrna Gene ◽  
Gene Sequences ◽  
16S Rrna Gene Sequences ◽  
Clone Libraries ◽  
Subsurface Sediments

ABSTRACT Previous studies have demonstrated that metal-reducing microorganisms can effectively promote the precipitation and removal of uranium from contaminated groundwater. Microbial communities were stimulated in the acidic subsurface by pH neutralization and addition of an electron donor to wells. In single-well push-pull tests at a number of treated sites, nitrate, Fe(III), and uranium were extensively reduced and electron donors (glucose, ethanol) were consumed. Examination of sediment chemistry in cores sampled immediately adjacent to treated wells 3.5 months after treatment revealed that sediment pH increased substantially (by 1 to 2 pH units) while nitrate was largely depleted. A large diversity of 16S rRNA gene sequences were retrieved from subsurface sediments, including species from the α, β, δ, and γ subdivisions of the class Proteobacteria, as well as low- and high-G+C gram-positive species. Following in situ biostimulation of microbial communities within contaminated sediments, sequences related to previously cultured metal-reducing δ-Proteobacteria increased from 5% to nearly 40% of the clone libraries. Quantitative PCR revealed that Geobacter-type 16S rRNA gene sequences increased in biostimulated sediments by 1 to 2 orders of magnitude at two of the four sites tested. Evidence from the quantitative PCR analysis corroborated information obtained from 16S rRNA gene clone libraries, indicating that members of the δ-Proteobacteria subdivision, including Anaeromyxobacter dehalogenans-related and Geobacter-related sequences, are important metal-reducing organisms in acidic subsurface sediments. This study provides the first cultivation-independent analysis of the change in metal-reducing microbial communities in subsurface sediments during an in situ bioremediation experiment.

scholarly journals Effects of Antibiotics on the Bacterial Community, Metabolic Functions and Antibiotic Resistance Genes in Mariculture Sediments during Enrichment Culturing

2020 ◽  
Vol 8 (8) ◽  
pp. 604
Author(s):  
Meng-Qi Ye ◽  
Guan-Jun Chen ◽  
Zong-Jun Du
Keyword(s):  
Antibiotic Resistance ◽  
Bacterial Community ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
High Throughput ◽  
Antibiotic Resistance Genes ◽  
Control Group ◽  
Rrna Gene ◽  
Metabolic Functions ◽  
Culture Dependent

The effect of antibiotics on the diversity and functioning of indigenous microorganisms in the environment has attracted much attention. In this study, effects of exposure to six different antibiotics on the bacterial community, metabolic functions and antibiotic resistance genes (ARGs) in marine sediments during enrichment culturing were investigated. Classical culture-dependent method and high-throughput 16S rRNA gene sequencing method were both applied. In the culture-dependent analysis, the obtained 1549 isolates belonged to four phyla (Proteobacteria, Firmicutes, Bacteroidetes and Actinobacteria) and 155 genera. Proteobacteria and Firmicutes were the dominant phyla. The diversity and abundance of obtained bacteria after antibiotic processing exhibited different degrees of decrease. Enrichment culturing for different time could also affect the bacterial community composition. Some genera of bacteria were not isolated in the control group, but they could be isolated in the antibiotic-treated groups. In high-throughput 16S rRNA gene amplicon sequencing analyses, all the effective reads were clustered into 2822 OTUs at 97% similarity cutoff; they were annotated to 49 phyla, 103 class, 220 orders, 347 families, 624 genera and 1122 species. An alpha diversity analysis indicated that the community diversity and richness decreased under antibiotic exposure. The changes at the genus level were much more obvious. Only 48 genera of 129 genera were shared by all the samples. A total of 29 genera which were not detected in the initial control sample could be detected in at least one antibiotic-treated group. SIMPER analysis showed that OTU2543 and OTU1450 were the most common taxa to the dissimilarity of bacterial community between antibiotic-treated groups and the control group. OTU2034 and OUT2543 were the most contributive taxa to dissimilarity of groups incubating for different time. Metabolism was the predominant bacterial function. A total of 30 ARGs were detected in the samples. This study mainly focused on the changes of microbiota under the selective pressure of antibiotics for different time and the results demonstrated that the antibiotic could affect the bacterial diversity and richness in the marine ecosystem.

scholarly journals Intrinsic challenges in ancient microbiome reconstruction using 16S rRNA gene amplification

2015 ◽  
Vol 5 (1) ◽  
Author(s):  
Kirsten A. Ziesemer ◽  
Allison E. Mann ◽  
Krithivasan Sankaranarayanan ◽  
Hannes Schroeder ◽  
Andrew T. Ozga ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Dna Amplification ◽  
Amplicon Sequencing ◽  
Universal Primers ◽  
Rrna Gene ◽  
Dental Calculus ◽  
Shotgun Metagenomics ◽  
Variable Regions ◽  
V3 Region

Abstract To date, characterization of ancient oral (dental calculus) and gut (coprolite) microbiota has been primarily accomplished through a metataxonomic approach involving targeted amplification of one or more variable regions in the 16S rRNA gene. Specifically, the V3 region (E. coli 341–534) of this gene has been suggested as an excellent candidate for ancient DNA amplification and microbial community reconstruction. However, in practice this metataxonomic approach often produces highly skewed taxonomic frequency data. In this study, we use non-targeted (shotgun metagenomics) sequencing methods to better understand skewed microbial profiles observed in four ancient dental calculus specimens previously analyzed by amplicon sequencing. Through comparisons of microbial taxonomic counts from paired amplicon (V3 U341F/534R) and shotgun sequencing datasets, we demonstrate that extensive length polymorphisms in the V3 region are a consistent and major cause of differential amplification leading to taxonomic bias in ancient microbiome reconstructions based on amplicon sequencing. We conclude that systematic amplification bias confounds attempts to accurately reconstruct microbiome taxonomic profiles from 16S rRNA V3 amplicon data generated using universal primers. Because in silico analysis indicates that alternative 16S rRNA hypervariable regions will present similar challenges, we advocate for the use of a shotgun metagenomics approach in ancient microbiome reconstructions.

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