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

Diversity

Anna V. Piterina²  John Bartlett¹ 

[1] Centre for Sustainability, Institute of Technology Sligo, Sligo, Ireland; E-Mail:;Department of Chemical and Environmental Sciences, Material and Surface Science Institute, University of Limerick, Limerick, Ireland; E-Mail:

关键词: ATAD;    thermophilic sludge;    DNA extraction;    PCR optimisation;    PCR inhibition;    microbial community analysis;    DGGE;    clone library;   

DOI  :  10.3390/d2040505

来源: mdpi

【 摘 要 】

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.

【 授权许可】

CC BY   
© 2010 by the authors; licensee Molecular Diversity Preservation International, Basel, Switzerland.