Next Article in Journal
Next Article in Special Issue
Previous Article in Journal
Previous Article in Special Issue
Diversity 2010, 2(4), 505-526; doi:10.3390/d2040505
Article

Molecular Analysis of Bacterial Community DNA in Sludge Undergoing Autothermal Thermophilic Aerobic Digestion (ATAD): Pitfalls and Improved Methodology to Enhance Diversity Recovery

1
, 2
 and 1,*
Received: 13 February 2010; in revised form: 21 March 2010 / Accepted: 24 March 2010 / Published: 31 March 2010
(This article belongs to the Special Issue Biological Diversity Assessed by Molecular Methods)
Download PDF [478 KB, uploaded 31 March 2010]
Abstract: Molecular analysis of the bacterial community structure associated with sludge processed by autothermal thermophilic aerobic digestion (ATAD), was performed using a number of extraction and amplification procedures which differed in yield, integrity, ability to amplify extracted templates and specificity in recovering species present. Interference to PCR and qPCR amplification was observed due to chelation, nuclease activity and the presence of thermolabile components derived from the ATAD sludge. Addition of selected adjuvant restored the ability to amplify community DNA, derived from the thermophilic sludge, via a number of primer sets of ecological importance and various DNA polymerases. Resolution of community profiles by molecular techniques was also influenced by the ATAD sludge extraction procedure as demonstrated by PCR-DGGE profiling and comparison of taxonomic affiliations of the most predominant members within 16S rRNA gene libraries constructed from ATAD DNA extracted by different methods. Several modifications have been shown to be necessary to optimize the molecular analysis of the ATAD thermal niche which may have general applicability to diversity recovery from similar environments.
Keywords: ATAD; thermophilic sludge; DNA extraction; PCR optimisation; PCR inhibition; microbial community analysis; DGGE; clone library ATAD; thermophilic sludge; DNA extraction; PCR optimisation; PCR inhibition; microbial community analysis; DGGE; clone library
This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Export to BibTeX |
EndNote


MDPI and ACS Style

Piterina, A.V.; Bartlett, J.; Pembroke, J.T. Molecular Analysis of Bacterial Community DNA in Sludge Undergoing Autothermal Thermophilic Aerobic Digestion (ATAD): Pitfalls and Improved Methodology to Enhance Diversity Recovery. Diversity 2010, 2, 505-526.

AMA Style

Piterina AV, Bartlett J, Pembroke JT. Molecular Analysis of Bacterial Community DNA in Sludge Undergoing Autothermal Thermophilic Aerobic Digestion (ATAD): Pitfalls and Improved Methodology to Enhance Diversity Recovery. Diversity. 2010; 2(4):505-526.

Chicago/Turabian Style

Piterina, Anna V.; Bartlett, John; Pembroke, J. Tony. 2010. "Molecular Analysis of Bacterial Community DNA in Sludge Undergoing Autothermal Thermophilic Aerobic Digestion (ATAD): Pitfalls and Improved Methodology to Enhance Diversity Recovery." Diversity 2, no. 4: 505-526.

Diversity EISSN 1424-2818 Published by MDPI AG, Basel, Switzerland RSS E-Mail Table of Contents Alert