Enrichment, Isolation, and Phylogenetic Identification of Polycyclic Aromatic Hydrocarbon-Degrading Bacteria from Elizabeth River Sediments†

ABSTRACT The diversity of indigenous bacteria in sediments from several sites in the Elizabeth River (Virginia) able to degrade multiple polycyclic aromatic hydrocarbons (PAHs) was investigated by the use of classical selective enrichment and molecular analyses. Enrichment cultures containing naphthalene, phenanthrene, fluoranthene, or pyrene as a sole carbon and energy source were monitored by denaturing gradient gel electrophoresis (DGGE) to detect changes in the bacterial-community profile during enrichment and to determine whether the representative strains present were successfully cultured. The DGGE profiles of the final enrichments grown solely on naphthalene and pyrene showed no clear relationship with the site from which the inoculum was obtained. The enrichments grown solely on pyrene for two sample sites had >80% similarity, which suggests that common pyrene-degrading strains may be present in these sediments. The final enrichments grown on fluoranthene and phenanthrene remained diverse by site, suggesting that these strains may be influenced by environmental conditions. One hundred and one isolates were obtained, comprising representatives of the actinomycetes and alpha-, beta-, and gammaproteobacteria, including seven novel isolates with 16S rRNA gene sequences less than 98% similar to known strains. The ability to degrade multiple PAHs was demonstrated by mineralization of 14C-labeled substrate and growth in pure culture. This supports our hypothesis that a high diversity of bacterial strains with the ability to degrade multiple PAHs can be confirmed by the combined use of classical selective enrichment and molecular analyses. This large collection of diverse PAH-degrading strains provides a valuable resource for studies on mechanisms of PAH degradation and bioremediation.

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Comparing Metabolic Functionalities, Community Structures, and Dynamics of Herbicide-Degrading Communities Cultivated with Different Substrate Concentrations

Applied and Environmental Microbiology ◽

10.1128/aem.02536-12 ◽

2012 ◽

Vol 79 (1) ◽

pp. 367-375 ◽

Cited By ~ 23

Author(s):

Erkin Gözdereliler ◽

Nico Boon ◽

Jens Aamand ◽

Karen De Roy ◽

Michael S. Granitsiotis ◽

...

Keyword(s):

Denaturing Gradient Gel Electrophoresis ◽

Nucleic Acid Content ◽

Cell Physiology ◽

Rrna Gene ◽

Low Concentrations ◽

Degrading Bacteria ◽

Gradient Gel Electrophoresis ◽

Herbicide Concentration ◽

Single Cell Physiology ◽

High Concentrations

ABSTRACTTwo 4-chloro-2-methylphenoxyacetic acid (MCPA)-degrading enrichment cultures selected from an aquifer on low (0.1 mg liter−1) or high (25 mg liter−1) MCPA concentrations were compared in terms of metabolic activity, community composition, population growth, and single cell physiology. Different community compositions and major shifts in community structure following exposure to different MCPA concentrations were observed using both 16S rRNA gene denaturing gradient gel electrophoresis fingerprinting and pyrosequencing. The communities also differed in their MCPA-mineralizing activities. The enrichments selected on low concentrations mineralized MCPA with shorter lag phases than those selected on high concentrations. Flow cytometry measurements revealed that mineralization led to cell growth. The presence of low-nucleic acid-content bacteria (LNA bacteria) was correlated with mineralization activity in cultures selected on low herbicide concentrations. This suggests that LNA bacteria may play a role in degradation of low herbicide concentrations in aquifers impacted by agriculture. This study shows that subpopulations of herbicide-degrading bacteria that are adapted to different pesticide concentrations can coexist in the same environment and that using a low herbicide concentration enables enrichment of apparently oligotrophic subpopulations.

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Screening and Identification of Glufosinate-Degrading Bacteria from Glufosinate-Treated Soils

Weed Science ◽

10.1614/ws-06-202.1 ◽

2007 ◽

Vol 55 (6) ◽

pp. 631-637 ◽

Cited By ~ 3

Author(s):

Chau-Ling Hsiao ◽

Chiu-Chung Young ◽

Ching-Yuh Wang

Keyword(s):

Denaturing Gradient Gel Electrophoresis ◽

Bacterial Strains ◽

High Concentration ◽

Degrading Bacteria ◽

Gradient Gel Electrophoresis ◽

Screening And Identification ◽

Polymerase Chain ◽

Competition Pressure ◽

Rdna Analysis

In order to select efficient and competitive glufosinate-degrading bacteria, two soils which had been treated with glufosinate annually for more than 5 yr were screened. Three strains tolerant to this herbicide were identified by 16S rDNA analysis asBurkholderia sacchari,Serratia marcescens, andPseudomonas psychrotolerans. In addition, a moderately tolerant strain,P. citronellolis, was isolated from a soil which had received glufosinate treatment for only 6 mo. In culture medium containing high concentration of glufosinate, the former three strains showed significant ability to degrade this glutamine synthetase inhibitor, suggesting that glufosinate-degrading bacteria would be readily found in soils after a long-term induction or selection. A subsequent biodegradation experiment showed that 30 and 50% of glufosinate was degraded 7 and 21 d after treatment (DAT), respectively, in sterilized soils inoculated with the above-mentioned three tolerant strains. While more than 30% of the glufosinate in nonsterilized soils was degraded 7 DAT by the indigenous edaphic microbes, inoculation with the three selected strains enhanced glufosinate degradation to nearly 50%. A study on the competition from edaphic microorganisms in soils by polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE) analysis revealed that within 21 d after inoculation (DAI), the propagation ofB. sacchariandP. psychrotoleranswas not affected, whereas that of the less tolerantP. citronelloliswas inhibited. This observation suggests that a long-term herbicide exposure is a promotive factor in generating bacterial strains having high degradation efficiency and showing vigorous propagation under the competition pressure arising from indigenous microbes.

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Isolation of Adherent Polycyclic Aromatic Hydrocarbon (PAH)-Degrading Bacteria Using PAH-Sorbing Carriers

Applied and Environmental Microbiology ◽

10.1128/aem.66.5.1834-1843.2000 ◽

2000 ◽

Vol 66 (5) ◽

pp. 1834-1843 ◽

Cited By ~ 264

Author(s):

Leen Bastiaens ◽

Dirk Springael ◽

Pierre Wattiau ◽

Hauke Harms ◽

Rupert deWachter ◽

...

Keyword(s):

Polycyclic Aromatic Hydrocarbon ◽

Aromatic Hydrocarbon ◽

Bacterial Species ◽

Sole Source ◽

Rrna Gene ◽

Bacterial Strains ◽

High Adhesion ◽

Degrading Bacteria ◽

Polycyclic Aromatic ◽

Adhesion Efficiency

ABSTRACT Two different procedures were compared to isolate polycyclic aromatic hydrocarbon (PAH)-utilizing bacteria from PAH-contaminated soil and sludge samples, i.e., (i) shaken enrichment cultures in liquid mineral medium in which PAHs were supplied as crystals and (ii) a new method in which PAH degraders were enriched on and recovered from hydrophobic membranes containing sorbed PAHs. Both techniques were successful, but selected from the same source different bacterial strains able to grow on PAHs as the sole source of carbon and energy. The liquid enrichment mainly selected for Sphingomonasspp., whereas the membrane method exclusively led to the selection ofMycobacterium spp. Furthermore, in separate membrane enrichment set-ups with different membrane types, three repetitive extragenic palindromic PCR-related Mycobacterium strains were recovered. The new Mycobacterium isolates were strongly hydrophobic and displayed the capacity to adhere strongly to different surfaces. One strain, Mycobacterium sp. LB501T, displayed an unusual combination of high adhesion efficiency and an extremely high negative charge. This strain may represent a new bacterial species as suggested by 16S rRNA gene sequence analysis. These results indicate that the provision of hydrophobic sorbents containing sorbed PAHs in the enrichment procedure discriminated in favor of certain bacterial characteristics. The new isolation method is appropriate to select for adherent PAH-degrading bacteria, which might be useful to biodegrade sorbed PAHs in soils and sludge.

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16S rRNA PCR-Denaturing Gradient Gel Electrophoresis of Oral Lactobacillus casei Group and Their Phenotypic Appearances

ISRN Microbiology ◽

10.1155/2013/342082 ◽

2013 ◽

Vol 2013 ◽

pp. 1-6 ◽

Cited By ~ 7

Author(s):

S. Piwat ◽

R. Teanpaisan

Keyword(s):

16S Rrna ◽

Gel Electrophoresis ◽

Acid Production ◽

Lactobacillus Casei ◽

Denaturing Gradient Gel Electrophoresis ◽

Inhibitory Effect ◽

Rrna Gene ◽

Bacterial Strains ◽

Closely Related Species ◽

Gradient Gel Electrophoresis

This study aimed to develop a 16S rRNA PCR-denaturing gradient gel electrophoresis (DGGE) to identify the species level of Lactobacillus casei group and to investigate their characteristics of acid production and inhibitory effect. PCR-DGGE has been developed based on the 16S rRNA gene, and a set of HDA-1-GC and HDA-2, designed at V2-V3 region, and another set of CARP-1-GC and CARP-2, designed at V1 region, have been used. The bacterial strains included L. casei ATCC 393, L. paracasei CCUG 32212, L. rhamnosus ATCC 7469, L. zeae CCUG 35515, and 46 clinical strains of L. casei/paracasei/rhamnosus. Inhibitory effect against Streptococcus mutans and acid production were examined. Results revealed that each type species strain and identified clinical isolate showed its own unique DGGE pattern using CARP1-GC and CARP2 primers. HDA1-GC and HDA2 primers could distinguish the strains of L. paracasei from L. casei. It was found that inhibitory effect of L. paracasei was stronger than L. casei and L. rhamnosus. The acid production of L. paracasei was lower than L. casei and L. rhamnosus. In conclusion, the technique has been proven to be able to differentiate between closely related species in L. casei group and thus provide reliable information of their phenotypic appearances.

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MICROBIAL COMMUNITY DURING BIOREMEDIATION EXPERIMENTAL ON OIL SPILL IN COASTAL OF PARI ISLAND

Jurnal Ilmu dan Teknologi Kelautan Tropis ◽

10.29244/jitkt.v3i1.7838 ◽

2011 ◽

Vol 3 (1) ◽

Author(s):

Lies Indah Sutiknowati

Keyword(s):

Oil Spill ◽

Deoxyribonucleic Acid ◽

Denaturing Gradient Gel Electrophoresis ◽

Gas Chromatography Mass Spectrometry ◽

Oil Degradation ◽

Sequence Determination ◽

Blast Search ◽

Degrading Bacteria ◽

Gradient Gel Electrophoresis

There is an information how to identify hydrocarbon degrading bacteria for bioremediation of marine oil spill. We have Bioremediation treatment for degradation of oil spill on Pari island and need two kind of experiment there are tanks experiment (sampling 0 to 90 days) and semi enclosed system (sampling 0 to 150 days). Biostimulation with nutrients (N and P) was done to analyze biodegradation of hydrocarbon compounds. Experiment design using fertilizer Super IB and Linstar will stimulate bacteria can degrade oil, n-alkane, and alkane as poly aromatic hydrocarbon. The bacteria communities were monitored and analyzed by Denaturing Gradient Gel Electrophoresis (DGGE) and Clone Library; oil chemistry was analyzed by Gas Chromatography Mass Spectrometry (GCMS). DNA (deoxyribonucleic acid) was extracted from colonies of bacteria and sequence determination of the 16S rDNA was amplified by primers U515f and U1492r. Strains had been sequence and had similarity about 90-99% to their closest taxa by homology Blast search and few of them suspected as new species. The results showed that fertilizers gave a significant effect on alkane, PAH and oil degradation in tanks experiment but not in the field test. Dominant of the specific bacteria on this experiment were Alcanivorax, Marinobacter and Prosthecochloris. Keywords: Bioremediation, Biostimulation, DGGE, PAH, Pari Island

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Development and Validation of a Nested-PCR-Denaturing Gradient Gel Electrophoresis Method for Taxonomic Characterization of Bifidobacterial Communities

Applied and Environmental Microbiology ◽

10.1128/aem.69.11.6380-6385.2003 ◽

2003 ◽

Vol 69 (11) ◽

pp. 6380-6385 ◽

Cited By ~ 52

Author(s):

R. Temmerman ◽

L. Masco ◽

T. Vanhoutte ◽

G. Huys ◽

J. Swings

Keyword(s):

Nested Pcr ◽

Gel Electrophoresis ◽

Denaturing Gradient Gel Electrophoresis ◽

Temporal Changes ◽

Rrna Gene ◽

Specific Pcr ◽

Gradient Gel Electrophoresis ◽

Species Specific ◽

Taxonomic Characterization

ABSTRACT The taxonomic characterization of a bacterial community is difficult to combine with the monitoring of its temporal changes. None of the currently available identification techniques are able to visualize a “complete” community, whereas techniques designed for analyzing bacterial ecosystems generally display limited or labor-intensive identification potential. This paper describes the optimization and validation of a nested-PCR-denaturing gradient gel electrophoresis (DGGE) approach for the species-specific analysis of bifidobacterial communities from any ecosystem. The method comprises a Bifidobacterium-specific PCR step, followed by purification of the amplicons that serve as template DNA in a second PCR step that amplifies the V3 and V6-V8 regions of the 16S rRNA gene. A mix of both amplicons is analyzed on a DGGE gel, after which the band positions are compared with a previously constructed database of reference strains. The method was validated through the analysis of four artificial mixtures, mimicking the possible bifidobacterial microbiota of the human and chicken intestine, a rumen, and the environment, and of two fecal samples. Except for the species Bifidobacterium coryneforme and B. indicum, all currently known bifidobacteria originating from various ecosystems can be identified in a highly reproducible manner. Because no further cloning and sequencing of the DGGE bands is necessary, this nested-PCR-DGGE technique can be completed within a 24-h span, allowing the species-specific monitoring of temporal changes in the bifidobacterial community.

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Differences in Microbial Activity and Microbial Populations of Peat Associated with Suppression of Damping-Off Disease Caused by Pythium sylvaticum

Applied and Environmental Microbiology ◽

10.1128/aem.00313-06 ◽

2006 ◽

Vol 72 (10) ◽

pp. 6452-6460 ◽

Cited By ~ 43

Author(s):

Paul J. Hunter ◽

Geoff M. Petch ◽

Leo A. Calvo-Bado ◽

Tim R. Pettitt ◽

Nick R. Parsons ◽

...

Keyword(s):

Microbial Activity ◽

Denaturing Gradient Gel Electrophoresis ◽

Small Subunit ◽

Disease Suppression ◽

Rrna Gene ◽

Damping Off ◽

Small Subunit Rrna ◽

Pythium Sylvaticum ◽

Gradient Gel Electrophoresis ◽

Microbial Groups

ABSTRACT The microbiological characteristics associated with disease-suppressive peats are unclear. We used a bioassay for Pythium sylvaticum-induced damping-off of cress seedlings to identify conducive and suppressive peats. Microbial activity in unconditioned peats was negatively correlated with the counts of P. sylvaticum at the end of the bioassay. Denaturing gradient gel electrophoresis (DGGE) profiling and clone library analyses of small-subunit rRNA gene sequences from two suppressive and two conducive peats differed in the bacterial profiles generated and the diversity of sequence populations. There were also significant differences between bacterial sequence populations from suppressive and conducive peats. The frequencies of a number of microbial groups, including the Rhizobium-Agrobacterium group (specifically sequences similar to those for the genera Ochrobactrum and Zoogloea) and the Acidobacteria, increased specifically in the suppressive peats, although no single bacterial group was associated with disease suppression. Fungal DGGE profiles varied little over the course of the bioassay; however, two bands associated specifically with suppressive samples were detected. Sequences from these bands corresponded to Basidiomycete yeast genera. Although the DGGE profiles were similar, fungal sequence diversity also increased during the bioassay. Sequences highly similar to those of Cryptococcus increased in relative abundance during the bioassay, particularly in the suppressive samples. This study highlights the importance of using complementary approaches to molecular profiling of complex populations and provides the first report that basidiomycetous yeasts may be associated with the suppression of Pythium-induced diseases in peats.

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MICROBIAL COMMUNITY DURING BIOREMEDIATION EXPERIMENTAL ON OIL SPILL IN COASTAL OF PARI ISLAND

Jurnal Ilmu dan Teknologi Kelautan Tropis ◽

10.28930/jitkt.v3i1.7838 ◽

2011 ◽

Vol 3 (1) ◽

Author(s):

Lies Indah Sutiknowati

Keyword(s):

Oil Spill ◽

Deoxyribonucleic Acid ◽

Denaturing Gradient Gel Electrophoresis ◽

Gas Chromatography Mass Spectrometry ◽

Oil Degradation ◽

Sequence Determination ◽

Blast Search ◽

Degrading Bacteria ◽

Gradient Gel Electrophoresis

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MOLECULAR IDENTIFICATION OF YEAST OF THE PULQUE BY PCR-DGGE, A TRADITIONAL MEXICANBEVERAGE

International Journal of Research -GRANTHAALAYAH ◽

10.29121/granthaalayah.v3.i3.2015.3026 ◽

2015 ◽

Vol 3 (3) ◽

pp. 1-15

Author(s):

Marcial-Quino J. ◽

Garcia-Ocón B. ◽

Mendoza-Espinoza J.A. ◽

Gómez-Manzo S. ◽

Sierra-Palacios E

Keyword(s):

Gel Electrophoresis ◽

Fermentation Process ◽

Denaturing Gradient Gel Electrophoresis ◽

Rapid Identification ◽

Rrna Gene ◽

Beverage Samples ◽

D2 Domain ◽

Gradient Gel Electrophoresis ◽

26S Rrna

Currently it is well known that yeasts play an essential role in the production of different beverages. In this paper, were identified some of the yeasts involved in the fermentation process of the pulque, a Mexican traditional beverage. Samples were collected from different regions of Mexico and yeasts were detected directly from samples without cultivation. Identifying the yeasts was obtained using amplification the D1/D2 domain of the 26S rRNA gene and Denaturing Gradient Gel Electrophoresis (DGGE). The results of DGGE showed different profiles of bands in each of the analyzed samples, indicating the presence of several species of yeast, which was also confirmed by sequencing of the bands corresponding to the domain D1/D2, succeeded in identifying five species of yeasts. The results obtained in this work demonstrated that the technique used for identification of yeasts of pulque was efficient. Besides, the optimization of this method could also allow rapid identification of yeasts and help understand the role of these in the fermentation process of this beverage, as well as the isolation of strains of interest for biotechnological purposes such as production of ethanol or metabolites with nutraceutical activity.

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Denaturing Gradient Gel Electrophoresis Analysis of the 16S rRNA Gene V1 Region To Monitor Dynamic Changes in the Bacterial Population during Fermentation of Italian Sausages

Applied and Environmental Microbiology ◽

10.1128/aem.67.11.5113-5121.2001 ◽

2001 ◽

Vol 67 (11) ◽

pp. 5113-5121 ◽

Cited By ~ 240

Author(s):

Luca Cocolin ◽

Marisa Manzano ◽

Carlo Cantoni ◽

Giuseppe Comi

Keyword(s):

Gel Electrophoresis ◽

Denaturing Gradient Gel Electrophoresis ◽

Rrna Gene ◽

Dynamic Changes ◽

Good Tool ◽

Reverse Transcription Pcr ◽

Dna And Rna ◽

Fermented Sausages ◽

Brochothrix Thermosphacta ◽

Gradient Gel Electrophoresis

ABSTRACT In this study, a PCR-denaturing gradient gel electrophoresis (DGGE) protocol was used to monitor the dynamic changes in the microbial population during ripening of natural fermented sausages. The method was first optimized by using control strains from international collections, and a natural sausage fermentation was studied by PCR-DGGE and traditional methods. Total microbial DNA and RNA were extracted directly from the sausages and subjected to PCR and reverse transcription-PCR, and the amplicons obtained were analyzed by DGGE. Lactic acid bacteria (LAB) were present together with other organisms, mainly members of the family Micrococcaceae and meat contaminants, such as Brochothrix thermosphacta andEnterococcus sp., during the first 3 days of fermentation. After 3 days, LAB represented the main population, which was responsible for the acidification and proteolysis that determined the characteristic organoleptic profile of the Friuli Venezia Giulia fermented sausages. The PCR-DGGE protocol for studying sausage fermentation proved to be a good tool for monitoring the process in real time, and it makes technological adjustments possible when they are required.

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