Detection of Catabolic Genes in Soil Using the Polymerase Chain Reaction

Berthelet, Marc; Greer, Charles W.

doi:10.1007/978-94-017-1435-8_56

Cited by 4 publications

(2 citation statements)

References 9 publications

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“…The biofilms were grown for 8 weeks at the dates described in Table 1. Each fall, two replicate bioreactors were operated for each of the following conditions, in the absence and in the presence of hexadecane (1 ppb): river water alone (no nutrient addition, control); river water amended with glucose at 67 mol/liter of carbon; river water amended with NH 4 Cl at 80 mol/liter of nitrogen; river water amended with KH 2 PO 4 at 5 mol/liter of phosphorus; and river water amended with a combination of all three nutrients at the concentrations described above (44).…”

Section: Methodsmentioning

confidence: 99%

Influence of Nutrient Inputs, Hexadecane, and Temporal Variations on Denitrification and Community Composition of River Biofilms

Chénier

Beaumier

Fortin

et al. 2006

Appl Environ Microbiol

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Biofilms were cultivated on polycarbonate strips in rotating annular reactors using South Saskatchewan River water during the fall of 1999 and the fall of 2001, supplemented with carbon (glucose), nitrogen (NH 4 Cl), phosphorus (KH 2 PO 4 ), or combined nutrients (CNP), with or without hexadecane, a model compound representing aliphatic hydrocarbons used to simulate a pollutant. In fall 1999 and fall 2001, comparable denitrification activities and catabolic potentials were observed in the biofilms, implying that denitrifying populations showed similar activity patterns and catabolic potentials during the fall from year to year in this river ecosystem, when environmental conditions were similar. Both nirS and nirK denitrification genes were detected by PCR amplification, suggesting that both denitrifying bacterial subpopulations can potentially contribute to total denitrification. Between 91.7 and 99.8% of the consumed N was emitted in the form of N 2 , suggesting that emission of N 2 O, a major potent greenhouse gas, by South Saskatchewan River biofilms is low. Denitrification was markedly stimulated by the addition of CNP, and nirS and nirK genes were predominant only in the presence of CNP. In contrast, individual nutrients had no impact on denitrification and on the occurrence of nirS and nirK genes detected by PCR amplification. Similarly, only CNP resulted in significant increases in algal and bacterial biomass relative to control biofilms. Biomass measurements indicated a linkage between autotrophic and heterotrophic populations in the fall 1999 biofilms. Correlation analyses demonstrated a significant relationship (P < 0.05) between the denitrification rate and the biomass of algae and heterotrophic bacteria but not cyanobacteria. At the concentration assessed (1 ppb), hexadecane partially inhibited denitrification in both years, slightly more in the fall of 2001. This study suggested that the response of the anaerobic heterotrophic biofilm community may be cyclic and predictable from year to year and that there are interactive effects between nutrients and the contaminant hexadecane.

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Section: Methodsmentioning

confidence: 99%

Influence of Nutrient Inputs, Hexadecane, and Temporal Variations on Denitrification and Community Composition of River Biofilms

Chénier

Beaumier

Fortin

et al. 2006

Appl Environ Microbiol

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“…The PCR amplification procedure was modified from previously published procedures (8,28). An aliquot of extract (0.5 to 2 l of undiluted or diluted [1/10 or 1/100] DNA) was added to a final volume of 50 l of reaction mixture (Amersham Pharmacia Biotech Inc., Piscataway, N.J.) containing 10 mM TrisHCl (pH 9); 50 mM KCl; 1.5 mM MgCl 2 ; 200 M (each) dATP, dTTP, dGTP, and dCTP; and 1 M each primer (forward and reverse).…”

Section: Vol 69 2003 Impact Of Environmental Parameters On River Bimentioning

confidence: 99%

Impact of Seasonal Variations and Nutrient Inputs on Nitrogen Cycling and Degradation of Hexadecane by Replicated River Biofilms

Chénier

Beaumier

Roy

et al. 2003

Appl Environ Microbiol

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Biofilm communities cultivated in rotating annular bioreactors using water from the South Saskatchewan River were assessed for the effects of seasonal variations and nutrient (C, N, and P) additions. Confocal laser microscopy revealed that while control biofilms were consistently dominated by bacterial biomass, the addition of nutrients shifted biofilms of summer and fall water samples to phototrophic-dominated communities. In nutrient-amended biofilms, similar patterns of nitrification, denitrification, and hexadecane mineralization rates were observed for winter and spring biofilms; fall biofilms had the highest rates of nitrification and hexadecane mineralization, and summer biofilms had the highest rates of denitrification. Very low rates of all measured activities were detected in control biofilms (without nutrient addition) regardless of season. Nutrient addition caused large increases in hexadecane mineralization and denitrification rates but only modest increases, if any, in nitrification rates, depending upon the season. Generally, both alkB and nirK were more readily PCR amplified from nutrient-amended biofilms. Both genes were amplified from all samples except for nirK from the fall control biofilm. It appears that bacterial production in the South Saskatchewan River water is limited by the availability of nutrients and that biofilm activities and composition vary with nutrient availability and time of year.

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Rapid, direct extraction of DNA from soils for PCR analysis using polyvinylpolypyrrolidone spin columns

Berthelet

Whyte

Greer

1996

FEMS Microbiology Letters

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159

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Polyvinylpolypyrrolidone spin columns were used to rapidly purify crude soil DNA extracts from humic materials for polymerase chain reaction (PCR) analysis. The PCR detection limit for the tfdC gene, encoding chlorocatechol dioxygenase from the 2,4-dichlorophenoxyacetic acid degradation pathway, was 10(1)-10(2) cells/g soil in inoculated soils. The procedure could be applied to the amplification of biodegradative genes from indigenous microbial populations from a wide variety of soil types, and the entire analysis could be performed within 8 h.

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Detection of Catabolic Genes in Soil Using the Polymerase Chain Reaction

Cited by 4 publications

References 9 publications

Influence of Nutrient Inputs, Hexadecane, and Temporal Variations on Denitrification and Community Composition of River Biofilms

Influence of Nutrient Inputs, Hexadecane, and Temporal Variations on Denitrification and Community Composition of River Biofilms

Impact of Seasonal Variations and Nutrient Inputs on Nitrogen Cycling and Degradation of Hexadecane by Replicated River Biofilms

Rapid, direct extraction of DNA from soils for PCR analysis using polyvinylpolypyrrolidone spin columns

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