scholarly journals Application of Denaturing High-Performance Liquid Chromatography in Microbial Ecology: Fermentor Sludge, Compost, and Soil Community Profiling

2008 ◽  
Vol 75 (4) ◽  
pp. 956-964 ◽  
Author(s):  
Andreas Otto Wagner ◽  
Cornelia Malin ◽  
Paul Illmer
Keyword(s):  
High Performance Liquid Chromatography ◽  
Liquid Chromatography ◽  
Microbial Ecology ◽  
High Performance ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Community Analysis ◽  
Microbial Community Analysis ◽  
Rrna Gene ◽  
Sludge Compost ◽  
Fingerprinting Methods

ABSTRACT Genetic fingerprinting methods, such as denaturing gradient gel electrophoresis (DGGE), are used in microbial ecology for the analysis of mixed microbial communities but are associated with various problems. In the present study we used a new alternative method: denaturing high-performance liquid chromatography (dHPLC). This method was previously shown to work with samples from water and gut flora but had not yet been applied to complex environmental samples. In contrast to other publications dealing with dHPLC, we used a commonly available HPLC system. Samples from different origins (fermentor sludge, compost, and soil), all ecologically significant, were tested, and the 16S rRNA gene was amplified via PCR. After optimization of the HPLC elution conditions, amplicons of pure cultures and mixed microbial populations could be separated successfully. Systematic differentiation was carried out by a cloning approach, since fraction collection of the peaks did not result in satisfactory fragment separation. dHPLC was evaluated as a tool for microbial community analysis on a genetic level and demonstrated major improvements compared to gel-based fingerprinting methods, such as DGGE, that are commonly used in microbial ecology.

scholarly journals Development of a Denaturing High-Performance Liquid Chromatography Method for Detection of Protist Parasites of Metazoans

2008 ◽  
Vol 74 (14) ◽  
pp. 4336-4345 ◽  
Author(s):  
Christofer Troedsson ◽  
Richard F. Lee ◽  
Vivica Stokes ◽  
Tina L. Walters ◽  
Paolo Simonelli ◽  
...  
Keyword(s):  
High Performance Liquid Chromatography ◽  
Liquid Chromatography ◽  
High Performance ◽  
18S Rrna ◽  
18S Rrna Gene ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Chromatography Method ◽  
Factors Affecting ◽  
Triethylammonium Acetate

ABSTRACT Increasingly, diseases of marine organisms are recognized as significant biotic factors affecting ecosystem health. However, the responsible disease agents are often unknown and the discovery and description of novel parasites most often rely on morphological descriptions made by highly trained specialists. Here, we describe a new approach for parasite discovery, utilizing denaturing high-performance liquid chromatography (DHPLC) reverse-phase ion-paring technology. Systematic investigations of major DHPLC variables, including temperature, gradient conditions, and target amplicon characteristics were conducted to develop a mechanistic understanding of DNA fragment separation by DHPLC. As a model system, 18S rRNA genes from the blue crab (Callinectes sapidus) and a parasitic dinoflagellate Hematodinium sp. were used. Binding of 18S rRNA gene PCR amplicons to the DNA separation column in the presence of triethylammonium acetate (TEAA) was inversely correlated with temperature and could be predicted based on the estimated DNA helicity of the PCR amplicon. Amplicons of up to 498 bp were resolved as single chromatographic peaks if they had high (>95%) DNA helicity. Amplicons that differed by as few as 2 bp could be resolved. Separation of 18S rRNA gene PCR amplicons was optimized by simultaneous manipulation of both temperature and solvent gradients. The optimal conditions included targeting regions of high DNA helicity (>95%), temperatures in the range of 57 to 63°C, and a linear acetonitrile gradient from 13.75 to 17.5% acetonitrile in 0.1 M TEAA (55 to 70% buffer B) over a 9-min period. Under these conditions, amplicons from a variety of parasites and their hosts can be separated and detected by DHPLC.

scholarly journals Biodegradation of an Alicyclic Hydrocarbon by a Sulfate-Reducing Enrichment from a Gas Condensate-Contaminated Aquifer

2003 ◽  
Vol 69 (1) ◽  
pp. 434-443 ◽  
Author(s):  
Luis A. Rios-Hernandez ◽  
Lisa M. Gieg ◽  
Joseph M. Suflita
Keyword(s):  
Mass Spectrometry ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Community Analysis ◽  
Gas Condensate ◽  
Microbial Community Analysis ◽  
Gas Chromatography Mass Spectrometry ◽  
Rrna Gene ◽  
Contaminated Aquifer ◽  
Sulfate Reducing ◽  
Gradient Gel Electrophoresis

ABSTRACT We used ethylcyclopentane (ECP) as a model alicyclic hydrocarbon and investigated its metabolism by a sulfate-reducing bacterial enrichment obtained from a gas condensate-contaminated aquifer. The enrichment coupled the consumption of ECP with the stoichiometrically expected amount of sulfate reduced. During ECP biodegradation, we observed the transient accumulation of metabolite peaks by gas chromatography-mass spectrometry, three of which had identical mass spectrometry profiles. Mass-spectral similarities to analogous authentic standards allowed us to identify these metabolites as ethylcyclopentylsuccinic acids, ethylcyclopentylpropionic acid, ethylcyclopentylcarboxylic acid, and ethylsuccinic acid. Based on these findings, we propose a pathway for the degradation of this alicyclic hydrocarbon. Furthermore, a putative metabolite similar to ethylcyclopentylsuccinic acid was also found in samples of contaminated groundwater from the aquifer. However, no such finding was evident for samples collected from wells located upgradient of the gas condensate spill. Microbial community analysis of the ECP-degrading enrichment by denaturing gradient gel electrophoresis revealed the presence of at least three different organisms using universal eubacterial primers targeting 550 bp of the 16S rRNA gene. Based on sequence analysis, these organisms are phylogenetically related to the genera Syntrophobacter and Desulfotomaculum as well as a member of the Cytophaga-Flexibacter-Bacteroides group. The evidence suggests that alicyclic hydrocarbons such as ECP can be anaerobically activated by the addition to the double bond of fumarate to form alkylsuccinate derivatives under sulfate-reducing conditions and that the reaction occurs in the laboratory and in hydrocarbon-impacted environments.

scholarly journals Detection and Discovery of Crustacean Parasites in Blue Crabs (Callinectes sapidus) by Using 18S rRNA Gene-Targeted Denaturing High-Performance Liquid Chromatography

2008 ◽  
Vol 74 (14) ◽  
pp. 4346-4353 ◽  
Author(s):  
Christofer Troedsson ◽  
Richard F. Lee ◽  
Tina Walters ◽  
Vivica Stokes ◽  
Karrie Brinkley ◽  
...  
Keyword(s):  
High Performance Liquid Chromatography ◽  
Liquid Chromatography ◽  
High Performance ◽  
18S Rrna ◽  
Callinectes Sapidus ◽  
18S Rrna Gene ◽  
Rrna Gene ◽  
Sequence Information ◽  
Blue Crabs ◽  
Crustacean Parasites

ABSTRACT Recently, we described a novel denaturing high-performance liquid chromatography (DHPLC) approach useful for initial detection and identification of crustacean parasites. Because this approach utilizes general primers targeted to conserved regions of the 18S rRNA gene, a priori genetic sequence information on eukaryotic parasites is not required. This distinction provides a significant advantage over specifically targeted PCR assays that do not allow for the detection of unknown or unsuspected parasites. However, initial field evaluations of the DHPLC assay suggested that because of PCR-biased amplification of dominant host genes it was not possible to detect relatively rare parasite genes in infected crab tissue. Here, we describe the use of a peptide nucleic acid (PNA) PCR hybridization blocking probe in association with DHPLC (PNA-PCR DHPLC) to overcome inherent PCR bias associated with amplification of rare target genes by use of generic primers. This approach was utilized to detect infection of blue crabs (Callinectes sapidus) by the parasitic dinoflagellate Hematodinium sp. Evaluation of 76 crabs caught in Wassaw Sound, GA, indicated a 97% correspondence between detection of the parasite by use of a specific PCR diagnostic assay and that by use of PNA-PCR DHPLC. During these studies, we discovered one crab with an association with a previously undescribed protist symbiont. Phylogenetic analysis of the amplified symbiont 18S rRNA gene indicated that it is most closely related to the free-living kinetoplastid parasite Procryptobia sorokini. To our knowledge, this is the first report of this parasite group in a decapod crab and of this organism exhibiting a presumably parasitic life history.

scholarly journals Photodegradation of carbon dots cause cytotoxicity

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Yue-Yue Liu ◽  
Nan-Yang Yu ◽  
Wen-Di Fang ◽  
Qiao-Guo Tan ◽  
Rong Ji ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
High Performance Liquid Chromatography ◽  
Liquid Chromatography ◽  
Carbon Dots ◽  
High Performance ◽  
Community Analysis ◽  
Cytotoxic Effects ◽  
Hek 293 ◽  
Alkyl Radicals ◽  
Flight Mass Spectrometry

AbstractCarbon dots (CDs) are photoluminescent nanomaterials with wide-ranging applications. Despite their photoactivity, it remains unknown whether CDs degrade under illumination and whether such photodegradation poses any cytotoxic effects. Here, we show laboratory-synthesized CDs irradiated with light degrade into molecules that are toxic to both normal (HEK-293) and cancerous (HeLa and HepG2) human cells. Eight days of irradiation photolyzes 28.6-59.8% of the CDs to <3 kilo Dalton molecules, 1431 of which are detected by high-throughput, non-target high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry. Molecular network and community analysis further reveal 499 cytotoxicity-related molecules, 212 of which contain polyethylene glycol, glucose, or benzene-related structures. Photo-induced production of hydroxyl and alkyl radicals play important roles in CD degradation as affected by temperature, pH, light intensity and wavelength. Commercial CDs show similar photodegraded products and cytotoxicity profiles, demonstrating that photodegradation-induced cytotoxicity is likely common to CDs regardless of their chemical composition. Our results highlight the importance of light in cytocompatibility studies of CDs.

scholarly journals Optimization of denaturing high performance liquid chromatography technique for rapid detection and identification of acetic acid bacteria of interest in vinegar production

2013 ◽  
Vol 2 (1s) ◽  
pp. 5 ◽  
Author(s):  
Noelia Isabel Sagarzazu ◽  
Maribel Martínez ◽  
Cristina Algarra ◽  
Javier Butrón ◽  
Carlos J. González-Navarro ◽  
...  
Keyword(s):  
High Performance Liquid Chromatography ◽  
Acetic Acid ◽  
Liquid Chromatography ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
High Performance ◽  
Acetic Acid Bacteria ◽  
Gene Region ◽  
Rrna Gene ◽  
Wild Strains

This paper evaluates the use of denaturing high performance liquid chromatography (DHPLC) technology for the discrimination of genetic differences in the 16S rRNA and alcohol dehydrogenase (AdhA) genes among bacterial species based on its efficiency and sensitivity to enable the detection and discrimination of different genetic sequences. In order to optimize DHPLC protocols for the analysis of 16S rRNA gene fragments amplified from bacteria, DNA isolated from 22 different strains representing main bacterial groups of interest in food microbiology was analyzed. While the use of 16S rRNA gene did not allow to difference two wild strains of <em>Acetobacter malorum</em>, this region revealed as useful to differentiate them from some pathogenic bacteria as <em>Escherichia coli</em>, <em>Salmonella typhimurium</em>, <em>Listeria monocytogenes</em>, <em>Listeria innocua</em>, <em>Clostridium perfringens </em>or <em>Sthapylococcus aureus</em>, from spoilage microorganisms as <em>Xantomonas vesicatoria</em> and <em>Alicyclobacillus</em> spp., and also from lactic acid bacteria as <em>Lactobacillus plantarum</em>, <em>Lactobacillus casei</em>, <em>Lactobacillus sakei</em>, <em>Lactobacillus acidophilus</em>, <em>Streptococcus thermophilus </em>and <em>Lactococcus lactis</em> that may suppose technological risk during vinegar production. The results demonstrate that 16S rRNA gene region is not adequate for the discrimination of the acetic acid bacteria (AAB) strains, so AdhA gene was selected to identify the two wild strains of <em>Acetobacter malorum</em>. Also 6 different reference strains of AAB were separated based on differences in AdhA gene region. DHPLC technology is able to discriminate between these two wild strains of <em>A. malorum</em> based on differences existing in the AdhA gene region. The data obtained indicate that the technique is capable of identifying most bacteria at species level and even at strain level with optimization of the protocols. This is of particular relevance in the case of AAB due to their poor recovery on culture media and difficulties in detection of viable but non cultivable cells.

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