The degradation of n-hexadecane in soil by thermophilic geobacilli

Roger Marchant, FH Sharkey, Ibrahim Banat, TJ Rahman, A Perfumo

Research output: Contribution to journalArticle

41 Citations (Scopus)

Abstract

Soil microcosms have been used to demonstrate the ability of indigenous soil thermophiles to degrade effectively a representative alkane (n-hexadecane). A fragment of the alkane mono-oxygenase gene (alkB) was amplified from thermophilic Geobacillus thermoleovorans strain T70 by PCR using degenerate primers. The amplicon demonstrated 96% sequence similarity with the alkB gene from Rhodococcus erythropolis. Critical controls ensured that the positive PCR signal detected was not a result of mesophilic soil organisms. A reverse transcription PCR (RT-PCR) assay was developed to determine if expression of the gene was inducible in the presence of an alkane or constitutively expressed in soil. In the presence of n-hexadecane, expression of the alkane mono-oxygenase gene was induced in pure cultures and soil samples and was dependent on temperature. No positive RT-PCR signal was detected at mesophilic growth temperatures either in pure cultures or in soil microcosms, whereas at 55 degrees C positive RT-PCR signals were obtained for both pure cultures of T70 and soil samples. Many different amplicons of the alkB gene fragment were obtained from the soil used in the microcosms. Thirty cloned fragments yielded 27 different sequences showing 85-96% sequence similarity with the alkB sequence of T70. To establish that the amplified alkB gene sequences from soil were derived from thermophilic geobacilli, additional strains were isolated on a selective medium containing n-hexadecane as sole carbon source. The 16S rRNA gene sequences were determined to identify the 50 isolates obtained (G. thermoleovorans, 27; G. caldoxylosilyticus, 17; G. pallidus, 2; G. toebiii, 1; Geobacillus sp., 3) representing 18 different strains and alkB gene sequences determined and deposited with the European Bioinformatics Institute.
LanguageEnglish
Pages44-54
JournalFEMS Microbiology Ecology
Volume56
Issue number1
DOIs
Publication statusPublished - Apr 2006

Fingerprint

Geobacillus
hexadecane
alkanes
degradation
Bacillus thermoleovorans
soil
reverse transcriptase polymerase chain reaction
oxygenases
nucleotide sequences
thermophilic microorganisms
genes
soil sampling
Rhodococcus erythropolis
selective media
bioinformatics
temperature
ribosomal RNA
gene expression
carbon
assays

Cite this

Marchant, Roger ; Sharkey, FH ; Banat, Ibrahim ; Rahman, TJ ; Perfumo, A. / The degradation of n-hexadecane in soil by thermophilic geobacilli. In: FEMS Microbiology Ecology. 2006 ; Vol. 56, No. 1. pp. 44-54.
@article{a955a52072fe4b9a91d9074a4b2da172,
title = "The degradation of n-hexadecane in soil by thermophilic geobacilli",
abstract = "Soil microcosms have been used to demonstrate the ability of indigenous soil thermophiles to degrade effectively a representative alkane (n-hexadecane). A fragment of the alkane mono-oxygenase gene (alkB) was amplified from thermophilic Geobacillus thermoleovorans strain T70 by PCR using degenerate primers. The amplicon demonstrated 96{\%} sequence similarity with the alkB gene from Rhodococcus erythropolis. Critical controls ensured that the positive PCR signal detected was not a result of mesophilic soil organisms. A reverse transcription PCR (RT-PCR) assay was developed to determine if expression of the gene was inducible in the presence of an alkane or constitutively expressed in soil. In the presence of n-hexadecane, expression of the alkane mono-oxygenase gene was induced in pure cultures and soil samples and was dependent on temperature. No positive RT-PCR signal was detected at mesophilic growth temperatures either in pure cultures or in soil microcosms, whereas at 55 degrees C positive RT-PCR signals were obtained for both pure cultures of T70 and soil samples. Many different amplicons of the alkB gene fragment were obtained from the soil used in the microcosms. Thirty cloned fragments yielded 27 different sequences showing 85-96{\%} sequence similarity with the alkB sequence of T70. To establish that the amplified alkB gene sequences from soil were derived from thermophilic geobacilli, additional strains were isolated on a selective medium containing n-hexadecane as sole carbon source. The 16S rRNA gene sequences were determined to identify the 50 isolates obtained (G. thermoleovorans, 27; G. caldoxylosilyticus, 17; G. pallidus, 2; G. toebiii, 1; Geobacillus sp., 3) representing 18 different strains and alkB gene sequences determined and deposited with the European Bioinformatics Institute.",
author = "Roger Marchant and FH Sharkey and Ibrahim Banat and TJ Rahman and A Perfumo",
year = "2006",
month = "4",
doi = "10.1111/j.1574-6941.2006.00061.x",
language = "English",
volume = "56",
pages = "44--54",
journal = "FEMS Microbiology Ecology",
issn = "0168-6496",
number = "1",

}

The degradation of n-hexadecane in soil by thermophilic geobacilli. / Marchant, Roger; Sharkey, FH; Banat, Ibrahim; Rahman, TJ; Perfumo, A.

In: FEMS Microbiology Ecology, Vol. 56, No. 1, 04.2006, p. 44-54.

Research output: Contribution to journalArticle

TY - JOUR

T1 - The degradation of n-hexadecane in soil by thermophilic geobacilli

AU - Marchant, Roger

AU - Sharkey, FH

AU - Banat, Ibrahim

AU - Rahman, TJ

AU - Perfumo, A

PY - 2006/4

Y1 - 2006/4

N2 - Soil microcosms have been used to demonstrate the ability of indigenous soil thermophiles to degrade effectively a representative alkane (n-hexadecane). A fragment of the alkane mono-oxygenase gene (alkB) was amplified from thermophilic Geobacillus thermoleovorans strain T70 by PCR using degenerate primers. The amplicon demonstrated 96% sequence similarity with the alkB gene from Rhodococcus erythropolis. Critical controls ensured that the positive PCR signal detected was not a result of mesophilic soil organisms. A reverse transcription PCR (RT-PCR) assay was developed to determine if expression of the gene was inducible in the presence of an alkane or constitutively expressed in soil. In the presence of n-hexadecane, expression of the alkane mono-oxygenase gene was induced in pure cultures and soil samples and was dependent on temperature. No positive RT-PCR signal was detected at mesophilic growth temperatures either in pure cultures or in soil microcosms, whereas at 55 degrees C positive RT-PCR signals were obtained for both pure cultures of T70 and soil samples. Many different amplicons of the alkB gene fragment were obtained from the soil used in the microcosms. Thirty cloned fragments yielded 27 different sequences showing 85-96% sequence similarity with the alkB sequence of T70. To establish that the amplified alkB gene sequences from soil were derived from thermophilic geobacilli, additional strains were isolated on a selective medium containing n-hexadecane as sole carbon source. The 16S rRNA gene sequences were determined to identify the 50 isolates obtained (G. thermoleovorans, 27; G. caldoxylosilyticus, 17; G. pallidus, 2; G. toebiii, 1; Geobacillus sp., 3) representing 18 different strains and alkB gene sequences determined and deposited with the European Bioinformatics Institute.

AB - Soil microcosms have been used to demonstrate the ability of indigenous soil thermophiles to degrade effectively a representative alkane (n-hexadecane). A fragment of the alkane mono-oxygenase gene (alkB) was amplified from thermophilic Geobacillus thermoleovorans strain T70 by PCR using degenerate primers. The amplicon demonstrated 96% sequence similarity with the alkB gene from Rhodococcus erythropolis. Critical controls ensured that the positive PCR signal detected was not a result of mesophilic soil organisms. A reverse transcription PCR (RT-PCR) assay was developed to determine if expression of the gene was inducible in the presence of an alkane or constitutively expressed in soil. In the presence of n-hexadecane, expression of the alkane mono-oxygenase gene was induced in pure cultures and soil samples and was dependent on temperature. No positive RT-PCR signal was detected at mesophilic growth temperatures either in pure cultures or in soil microcosms, whereas at 55 degrees C positive RT-PCR signals were obtained for both pure cultures of T70 and soil samples. Many different amplicons of the alkB gene fragment were obtained from the soil used in the microcosms. Thirty cloned fragments yielded 27 different sequences showing 85-96% sequence similarity with the alkB sequence of T70. To establish that the amplified alkB gene sequences from soil were derived from thermophilic geobacilli, additional strains were isolated on a selective medium containing n-hexadecane as sole carbon source. The 16S rRNA gene sequences were determined to identify the 50 isolates obtained (G. thermoleovorans, 27; G. caldoxylosilyticus, 17; G. pallidus, 2; G. toebiii, 1; Geobacillus sp., 3) representing 18 different strains and alkB gene sequences determined and deposited with the European Bioinformatics Institute.

U2 - 10.1111/j.1574-6941.2006.00061.x

DO - 10.1111/j.1574-6941.2006.00061.x

M3 - Article

VL - 56

SP - 44

EP - 54

JO - FEMS Microbiology Ecology

T2 - FEMS Microbiology Ecology

JF - FEMS Microbiology Ecology

SN - 0168-6496

IS - 1

ER -