scholarly journals Stable-Isotope Probing-Enabled Cultivation of the Indigenous BacteriumRalstoniasp. Strain M1, Capable of Degrading Phenanthrene and Biphenyl in Industrial Wastewater

2019 ◽  
Vol 85 (14) ◽  
Author(s):  
Jibing Li ◽  
Chunling Luo ◽  
Dayi Zhang ◽  
Xixi Cai ◽  
Longfei Jiang ◽  
...  
Keyword(s):  
Stable Isotope ◽  
Industrial Wastewater ◽  
Culture Conditions ◽  
Stable Isotope Probing ◽  
Comprehensive Treatment ◽  
Rrna Gene ◽  
Content Type ◽  
Ralstonia Pickettii ◽  
Industrial Parks ◽  
Polycyclic Aromatic

ABSTRACTTo identify and obtain the indigenous degraders metabolizing phenanthrene (PHE) and biphenyl (BP) from the complex microbial community within industrial wastewater, DNA-based stable-isotope probing (DNA-SIP) and cultivation-based methods were applied in the present study. DNA-SIP results showed that two bacterial taxa (VogesellaandAlicyclobacillus) were considered the key biodegraders responsible for PHE biodegradation only, whereasBacillusandCupriaviduswere involved in BP degradation.VogesellaandAlicyclobacillushave not been linked with PHE degradation previously. Additionally, DNA-SIP helped reveal the taxonomic identity ofRalstonia-like degraders involved in both PHE and BP degradation. To target the separation of functionalRalstonia-like degraders from the wastewater, we modified the traditional cultivation medium and culture conditions. Finally, an indigenous PHE- and BP-degrading strain,Ralstonia pickettiiM1, was isolated via a cultivation-dependent method, and its role in PHE and BP degradation was confirmed by enrichment of the 16S rRNA gene and distinctive dioxygenase genes in the DNA-SIP experiment. Our study has successfully established a program for the application of DNA-SIP in the isolation of the active functional degraders from an environment. It also deepens our insight into the diversity of indigenous PHE- and BP-degrading communities.IMPORTANCEThe comprehensive treatment of wastewater in industrial parks suffers from the presence of multiple persistent organic pollutants (POPs), such as polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs), which reduce the activity of activated sludge and are difficult to eliminate. Characterizing and applying active bacterial degraders metabolizing multiple POPs therefore helps to reveal the mechanisms of synergistic metabolism and to improve wastewater treatment efficiency in industrial parks. To date, SIP studies have successfully investigated the biodegradation of PAHs or PCBs in real-world habitats. DNA-SIP facilitates the isolation of target microorganisms that pose environmental concerns. Here, an indigenous phenanthrene (PHE)- and biphenyl (BP)-degrading strain in wastewater,Ralstonia pickettiiM1, was isolated via a cultivation-dependent method, and its role in PHE and BP degradation was confirmed by DNA-SIP. Our study provides a routine protocol for the application of DNA-SIP in the isolation of the active functional degraders from an environment.

scholarly journals Stable Isotope Probing of an Algal Bloom To Identify Uncultivated Members of the Rhodobacteraceae Associated with Low-Molecular-Weight Polycyclic Aromatic Hydrocarbon Degradation

2011 ◽  
Vol 77 (21) ◽  
pp. 7856-7860 ◽  
Author(s):  
Tony Gutierrez ◽  
David R. Singleton ◽  
Michael D. Aitken ◽  
Kirk T. Semple
Keyword(s):  
Stable Isotope ◽  
Polycyclic Aromatic Hydrocarbon ◽  
Aromatic Hydrocarbon ◽  
Algal Bloom ◽  
Stable Isotope Probing ◽  
Gene Copy ◽  
Rrna Gene ◽  
Content Type ◽  
Degrading Bacteria ◽  
Polycyclic Aromatic

ABSTRACTPolycyclic aromatic hydrocarbon (PAH)-degrading bacteria associated with an algal bloom in Tampa Bay, FL, were investigated by stable isotope probing (SIP) with uniformly labeled [13C]naphthalene. The dominant sequences in clone libraries constructed from13C-enriched bacterial DNA (from naphthalene enrichments) were identified as uncharacterized members of the familyRhodobacteraceae. Quantitative PCR primers targeting the 16S rRNA gene of these uncultivated organisms were used to determine their abundance in incubations amended with unlabeled naphthalene and phenanthrene, both of which showed substantial increases in gene copy numbers during the experiments. As demonstrated by this work, the application of uniformly13C-labeled PAHs in SIP experiments can successfully be used to identify novel PAH-degrading bacteria in marine waters.

scholarly journals Identification of Benzo[a]pyrene-Metabolizing Bacteria in Forest Soils by Using DNA-Based Stable-Isotope Probing

2015 ◽  
Vol 81 (21) ◽  
pp. 7368-7376 ◽  
Author(s):  
Mengke Song ◽  
Chunling Luo ◽  
Longfei Jiang ◽  
Dayi Zhang ◽  
Yujie Wang ◽  
...  
Keyword(s):  
Stable Isotope ◽  
Forest Soils ◽  
Contaminated Soils ◽  
Stable Isotope Probing ◽  
Soil Microcosms ◽  
Content Type ◽  
The Family ◽  
Polycyclic Aromatic ◽  
Uncultivated Bacteria ◽  
Different Soils

ABSTRACTDNA-based stable-isotope probing (DNA-SIP) was used in this study to investigate the uncultivated bacteria with benzo[a]pyrene (BaP) metabolism capacities in two Chinese forest soils (Mt. Maoer in Heilongjiang Province and Mt. Baicaowa in Hubei Province). We characterized three different phylotypes with responsibility for BaP degradation, none of which were previously reported as BaP-degrading microorganisms by SIP. In Mt. Maoer soil microcosms, the putative BaP degraders were classified as belonging to the genusTerrimonas(familyChitinophagaceae, orderSphingobacteriales), whereasBurkholderiaspp. were the key BaP degraders in Mt. Baicaowa soils. The addition of metabolic salicylate significantly increased BaP degradation efficiency in Mt. Maoer soils, and the BaP-metabolizing bacteria shifted to the microorganisms in the familyOxalobacteraceae(genus unclassified). Meanwhile, salicylate addition did not change either BaP degradation or putative BaP degraders in Mt. Baicaowa. Polycyclic aromatic hydrocarbon ring-hydroxylating dioxygenase (PAH-RHD) genes were amplified, sequenced, and quantified in the DNA-SIP13C heavy fraction to further confirm the BaP metabolism. By illuminating the microbial diversity and salicylate additive effects on BaP degradation across different soils, the results increased our understanding of BaP natural attenuation and provided a possible approach to enhance the bioremediation of BaP-contaminated soils.

scholarly journals Metagenomics and stable isotope probing offer insights into metabolism of polycyclic aromatic hydrocarbons degraders in chronically polluted seawater

2019 ◽  
Author(s):  
Ella T. Sieradzki ◽  
Michael Morando ◽  
Jed A. Fuhrman
Keyword(s):  
Polycyclic Aromatic Hydrocarbons ◽  
Los Angeles ◽  
Stable Isotope ◽  
Aromatic Hydrocarbons ◽  
Degradation Pathway ◽  
Stable Isotope Probing ◽  
Rrna Gene ◽  
Port Of Los Angeles ◽  
Polycyclic Aromatic ◽  
Bacterial Biodegradation

AbstractBacterial biodegradation is a significant contributor to remineralization of polycyclic aromatic hydrocarbons (PAHs): toxic and recalcitrant components of crude oil as well as byproducts of partial combustion chronically introduced into seawater via atmospheric deposition. The Deepwater Horizon oil spill demonstrated the speed at which a seed PAH-degrading community maintained by low chronic inputs can respond to an acute pollution. We investigated the diversity and functional potential of a similar seed community in the Port of Los Angeles, a chronically polluted site, using stable isotope probing with naphthalene, deep-sequenced metagenomes and carbon incorporation rate measurements at the port and in two sites further into the San Pedro Channel. We show a switch in the composition of the PAH degrading community from diverse early-responding generalists to late-blooming specialized degraders. This switch demonstrates the ability of the local seed community of degraders at the Port of LA to incorporate carbon from PAHs independently of a labile-hydrocarbon degrading succession. We were able to directly show that assembled genomes belonged to naphthalene degraders by matching their 16S-rRNA gene with experimental stable isotope probing data. Surprisingly, we did not find a full PAH degradation pathway in any of those genomes and even when combining genes from the entire microbial community. We use metabolic pathways identified in those genomes to generate metagenomic-based recommendations for future optimization of PAHs bioremediation.

scholarly journals Stable-Isotope Probing Identifies Uncultured Planctomycetes as Primary Degraders of a Complex Heteropolysaccharide in Soil

2015 ◽  
Vol 81 (14) ◽  
pp. 4607-4615 ◽  
Author(s):  
Xiaoqing Wang ◽  
Christine E. Sharp ◽  
Gareth M. Jones ◽  
Stephen E. Grasby ◽  
Allyson L. Brady ◽  
...  
Keyword(s):  
Stable Isotope ◽  
16S Rrna ◽  
Soil Bacteria ◽  
Stable Isotope Probing ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Content Type ◽  
Degrading Bacteria ◽  
Aerobic Soil

ABSTRACTThe exopolysaccharides (EPSs) produced by some bacteria are potential growth substrates for other bacteria in soil. We used stable-isotope probing (SIP) to identify aerobic soil bacteria that assimilated the cellulose produced byGluconacetobacter xylinusor the EPS produced byBeijerinckia indica. The latter is a heteropolysaccharide comprised primarily ofl-guluronic acid,d-glucose, andd-glycero-d-mannoheptose.13C-labeled EPS and13C-labeled cellulose were purified from bacterial cultures grown on [13C]glucose. Two soils were incubated with these substrates, and bacteria actively assimilating them were identified via pyrosequencing of 16S rRNA genes recovered from13C-labeled DNA. Cellulose C was assimilated primarily by soil bacteria closely related (93 to 100% 16S rRNA gene sequence identities) to known cellulose-degrading bacteria. However,B. indicaEPS was assimilated primarily by bacteria with low identities (80 to 95%) to known species, particularly by different members of the phylumPlanctomycetes. In one incubation, members of thePlanctomycetesmade up >60% of all reads in the labeled DNA and were only distantly related (<85% identity) to any described species. Although it is impossible with SIP to completely distinguish primary polysaccharide hydrolyzers from bacteria growing on produced oligo- or monosaccharides, the predominance ofPlanctomycetessuggested that they were primary degraders of EPS. Other bacteria assimilatingB. indicaEPS included members of theVerrucomicrobia, candidate division OD1, and theArmatimonadetes. The results indicate that some uncultured bacteria in soils may be adapted to using complex heteropolysaccharides for growth and suggest that the use of these substrates may provide a means for culturing new species.

scholarly journals Identification of a Toluene-Degrading Bacterium from a Soil Sample through H218O DNA Stable Isotope Probing

2011 ◽  
Vol 77 (17) ◽  
pp. 5995-5999 ◽  
Author(s):  
Angela Woods ◽  
Maribeth Watwood ◽  
Egbert Schwartz
Keyword(s):  
Stable Isotope ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Stable Isotope Probing ◽  
Rrna Gene ◽  
Soil Dna ◽  
Content Type ◽  
Degrading Bacterium ◽  
Rhodococcus Jostii ◽  
Oxygen Atoms

ABSTRACTDNA stable isotope probing (DNA-SIP) with H218O was used to identify a toluene-degrading bacterium in soil amended with 48 ppm toluene. After quantification of toluene degradation rates in soil, DNA was extracted from soil incubated with H218O, H216O, H216O and 48 ppm toluene, or H218O and 48 ppm toluene. A single DNA band formed along a cesium chloride gradient after isopycnic centrifugation of extracts from soils incubated with H216O. With extracts from soils to which only H218O was added, two distinct DNA bands formed, while three bands formed when DNA extracted from soil incubated with both H218O and toluene was analyzed. We suggest that this third band formed because toluene does not contain any oxygen atoms and toluene-degrading organisms had to transfer oxygen atoms from H218O into metabolic intermediates to form nucleic acidsde novo. We extracted the third DNA band and amplified a large fraction of the bacterial 16S rRNA gene. Direct sequencing of the PCR product obtained from the labeled DNA, as well as cloned 16S rRNA amplicons, identified a known toluene degrader,Rhodococcus jostiiRHA1. A toluene-degrading bacterial strain was subsequently isolated from soil and shown to beRhodococcus jostiiRHA1. Finally, quantitative real-time PCR analysis showed that the abundance of the 16S rRNA gene ofRhodococcus jostiiRHA1 increased in soil after toluene exposure but not in soils from which toluene was withheld. This study indicates that H218O DNA-SIP can be a useful method for identifying pollutant-degrading bacteria in soil.

scholarly journals Anaerobic Methyltert-Butyl Ether-Degrading Microorganisms Identified in Wastewater Treatment Plant Samples by Stable Isotope Probing

2012 ◽  
Vol 78 (8) ◽  
pp. 2973-2980 ◽  
Author(s):  
Weimin Sun ◽  
Xiaoxu Sun ◽  
Alison M. Cupples
Keyword(s):  
Wastewater Treatment ◽  
Stable Isotope ◽  
Wastewater Treatment Plant ◽  
Treatment Plant ◽  
Stable Isotope Probing ◽  
Rrna Gene ◽  
Butyl Ether ◽  
Content Type ◽  
Plant Samples ◽  
Mtbe Degradation

ABSTRACTAnaerobic methyltert-butyl ether (MTBE) degradation potential was investigated in samples from a range of sources. From these 22 experimental variations, only one source (from wastewater treatment plant samples) exhibited MTBE degradation. These microcosms were methanogenic and were subjected to DNA-based stable isotope probing (SIP) targeted to both bacteria and archaea to identify the putative MTBE degraders. For this purpose, DNA was extracted at two time points, subjected to ultracentrifugation, fractioning, and terminal restriction fragment length polymorphism (TRFLP). In addition, bacterial and archaeal 16S rRNA gene clone libraries were constructed. The SIP experiments indicated bacteria in the phylaFirmicutes(familyRuminococcaceae) andAlphaproteobacteria(genusSphingopyxis) were the dominant MTBE degraders. Previous studies have suggested a role forFirmicutesin anaerobic MTBE degradation; however, the putative MTBE-degrading microorganism in the current study is a novel MTBE-degrading phylotype within this phylum. Two archaeal phylotypes (generaMethanosarcinaandMethanocorpusculum) were also enriched in the heavy fractions, and these organisms may be responsible for minor amounts of MTBE degradation or for the uptake of metabolites released from the primary MTBE degraders. Currently, limited information exists on the microorganisms able to degrade MTBE under anaerobic conditions. This work represents the first application of DNA-based SIP to identify anaerobic MTBE-degrading microorganisms in laboratory microcosms and therefore provides a valuable set of data to definitively link identity with anaerobic MTBE degradation.

scholarly journals DNA stable isotope probing reveals contrasted activity and phenanthrene-degrading bacteria identity in a gradient of anthropized soils

2019 ◽  
Vol 95 (12) ◽  
Author(s):  
Florian Lemmel ◽  
Florence Maunoury-Danger ◽  
Corinne Leyval ◽  
Aurélie Cébron
Keyword(s):  
Stable Isotope ◽  
Contaminated Soils ◽  
Amplicon Sequencing ◽  
Shannon Index ◽  
Stable Isotope Probing ◽  
Rrna Gene ◽  
Degrading Bacteria ◽  
Polycyclic Aromatic ◽  
Contamination Levels ◽  
Almost All

ABSTRACT Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous soil organic pollutants. Although PAH-degrading bacteria are present in almost all soils, their selection and enrichment have been shown in historically high PAH contaminated soils. We can wonder if the effectiveness of PAH biodegradation and the PAH-degrading bacterial diversity differ among soils. The stable isotope probing (SIP) technique with 13C-phenanthrene (PHE) as a model PAH was used to: (i) compare for the first time a range of 10 soils with various PAH contamination levels, (ii) determine their PHE-degradation efficiency and (iii) identify the active PHE-degraders using 16S rRNA gene amplicon sequencing from 13C-labeled DNA. Surprisingly, the PHE degradation rate was not directly correlated to the initial level of total PAHs and phenanthrene in the soils, but was mostly explained by the initial abundance and richness of soil bacterial communities. A large diversity of PAH-degrading bacteria was identified for seven of the soils, with differences among soils. In the soils where the PHE degradation activities were the higher, Mycobacterium species were always the dominant active PHE degraders. A positive correlation between PHE-degradation level and the diversity of active PHE-degraders (Shannon index) supported the hypothesis that cooperation between strains led to a more efficient PAH degradation.

scholarly journals Phyllobacterium loti sp. nov. isolated from nodules of Lotus corniculatus

2014 ◽  
Vol 64 (Pt_3) ◽  
pp. 781-786 ◽  
Author(s):  
Maximo Sánchez ◽  
Martha-Helena Ramírez-Bahena ◽  
Alvaro Peix ◽  
María J. Lorite ◽  
Juan Sanjuán ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Sequence Similarity ◽  
Carbon Sources ◽  
Lotus Corniculatus ◽  
Culture Conditions ◽  
Rrna Gene ◽  
Content Type ◽  
Link Type ◽  
The 16S Rrna Gene

Strain S658T was isolated from a Lotus corniculatus nodule in a soil sample obtained in Uruguay. Phylogenetic analysis of the 16S rRNA gene and atpD gene showed that this strain clustered within the genus Phyllobacterium . The closest related species was, in both cases, Phyllobacterium trifolii PETP02T with 99.8 % sequence similarity in the 16S rRNA gene and 96.1 % in the atpD gene. The 16S rRNA gene contains an insert at the beginning of the sequence that has no similarities with other inserts present in the same gene in described rhizobial species. Ubiquinone Q-10 was the only quinone detected. Strain S658T differed from its closest relatives through its growth in diverse culture conditions and in the assimilation of several carbon sources. It was not able to reproduce nodules in Lotus corniculatus. The results of DNA–DNA hybridization, phenotypic tests and fatty acid analyses confirmed that this strain should be classified as a representative of a novel species of the genus Phyllobacterium , for which the name Phyllobacterium loti sp. nov. is proposed. The type strain is S658T( = LMG 27289T = CECT 8230T).

scholarly journals Heterologous Expression of Polycyclic Aromatic Hydrocarbon Ring-Hydroxylating Dioxygenase Genes from a Novel Pyrene-Degrading Betaproteobacterium

2012 ◽  
Vol 78 (10) ◽  
pp. 3552-3559 ◽  
Author(s):  
David R. Singleton ◽  
Jing Hu ◽  
Michael D. Aitken
Keyword(s):  
Polycyclic Aromatic Hydrocarbon ◽  
Aromatic Hydrocarbon ◽  
Enrichment Culture ◽  
Metagenomic Library ◽  
Stable Isotope Probing ◽  
Content Type ◽  
Close Proximity ◽  
Polycyclic Aromatic ◽  
Dominant Member ◽  
Pah Metabolism

ABSTRACTA betaproteobacterium within the familyRhodocyclaceaepreviously identified as a pyrene degrader via stable-isotope probing (SIP) of contaminated soil (designated pyrene group 1 or PG1) was cultivated as the dominant member of a mixed bacterial culture. A metagenomic library was constructed, and the largest contigs were analyzed for genes associated with polycyclic aromatic hydrocarbon (PAH) metabolism. Eight pairs of genes with similarity to the α- and β-subunits of ring-hydroxylating dioxygenases (RHDs) associated with aerobic bacterial PAH degradation were identified and linked to PG1 through PCR analyses of a simplified enrichment culture. In tandem with a ferredoxin and reductase found in close proximity to one pair of RHD genes, six of the RHDs were cloned and expressed inEscherichia coli. Each cloned RHD was tested for activity against nine PAHs ranging in size from two to five rings. Despite differences in their predicted protein sequences, each of the six RHDs was capable of transforming phenanthrene and pyrene. Three RHDs could additionally transform naphthalene and fluorene, and these genotypes were also associated with the ability of theE. coliconstructs to convert indole to indigo. Only one of the six cloned RHDs was capable of transforming anthracene and benz[a]anthracene. None of the tested RHDs were capable of significantly transforming fluoranthene, chrysene, or benzo[a]pyrene.

scholarly journals Identifying the Active Microbiome Associated with Roots and Rhizosphere Soil of Oilseed Rape

2017 ◽  
Vol 83 (22) ◽  
Author(s):  
Konstantia Gkarmiri ◽  
Shahid Mahmood ◽  
Alf Ekblad ◽  
Sadhna Alström ◽  
Nils Högberg ◽  
...  
Keyword(s):  
Brassica Napus ◽  
Stable Isotope ◽  
Oilseed Rape ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Rhizosphere Soil ◽  
Stable Isotope Probing ◽  
Biofuel Production ◽  
Content Type ◽  
Bacteria And Fungi

ABSTRACT RNA stable isotope probing and high-throughput sequencing were used to characterize the active microbiomes of bacteria and fungi colonizing the roots and rhizosphere soil of oilseed rape to identify taxa assimilating plant-derived carbon following 13CO2 labeling. Root- and rhizosphere soil-associated communities of both bacteria and fungi differed from each other, and there were highly significant differences between their DNA- and RNA-based community profiles. Verrucomicrobia, Proteobacteria, Planctomycetes, Acidobacteria, Gemmatimonadetes, Actinobacteria, and Chloroflexi were the most active bacterial phyla in the rhizosphere soil. Bacteroidetes were more active in roots. The most abundant bacterial genera were well represented in both the 13C- and 12C-RNA fractions, while the fungal taxa were more differentiated. Streptomyces, Rhizobium, and Flavobacterium were dominant in roots, whereas Rhodoplanes and Sphingomonas (Kaistobacter) were dominant in rhizosphere soil. “Candidatus Nitrososphaera” was enriched in 13C in rhizosphere soil. Olpidium and Dendryphion were abundant in the 12C-RNA fraction of roots; Clonostachys was abundant in both roots and rhizosphere soil and heavily 13C enriched. Cryptococcus was dominant in rhizosphere soil and less abundant, but was 13C enriched in roots. The patterns of colonization and C acquisition revealed in this study assist in identifying microbial taxa that may be superior competitors for plant-derived carbon in the rhizosphere of Brassica napus. IMPORTANCE This microbiome study characterizes the active bacteria and fungi colonizing the roots and rhizosphere soil of Brassica napus using high-throughput sequencing and RNA-stable isotope probing. It identifies taxa assimilating plant-derived carbon following 13CO2 labeling and compares these with other less active groups not incorporating a plant assimilate. Brassica napus is an economically and globally important oilseed crop, cultivated for edible oil, biofuel production, and phytoextraction of heavy metals; however, it is susceptible to several diseases. The identification of the fungal and bacterial species successfully competing for plant-derived carbon, enabling them to colonize the roots and rhizosphere soil of this plant, should enable the identification of microorganisms that can be evaluated in more detailed functional studies and ultimately be used to improve plant health and productivity in sustainable agriculture.

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