Peer-Reviewed Journal Details
Mandatory Fields
Piterina, AV. J.Barlett and J.T. Pembroke
2010
March
Diversity
Molecular Analysis of Bacterial Community DNA in Sludge Undergoing Autothermal Thermophilic Aerobic Digestion (ATAD): Pitfalls and Improved Methodology to Enhance Diversity Recovery
Published
()
Optional Fields
ATAD; thermophilic sludge; DNA extraction; PCR optimisation; PCR inhibition; microbial community analysis; DGGE; clone library
2
4
505
525
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.
http://www.mdpi.com/1424-2818/2/4/505
http://www.mdpi.com/1424-2818/2/4/505
10.3390/d2040505
Grant Details