Vol. 13(3), pp. 356-365, 15 January, 2014 DOI: 10.5897/AJB2013.12899 ISSN 1684-5315 ©2014 Academic Journals http://www.academicjournals.org/AJB African Journal of Biotechnology Full Length Research Paper Method optimization for denaturing gradient gel electrophoresis (DGGE) analysis of microflora from Eucalyptus sp. wood chips intended for pulping Lucretia Ramnath 1 , Tamara Bush 2 and Roshini Govinden 1 * 1 Discipline of Microbiology, School of Life Sciences, College of Agriculture, Engineering and Science, University of KwaZulu-Natal, Westville Campus, P/Bag X54001, Durban, 4000, South Africa. 2 Discipline of Chemistry, School of Chemistry and Physics, College of Agriculture, Engineering and Science, University of KwaZulu-Natal, Westville Campus, P/Bag X54001, Durban, 4000, South Africa. Accepted 6 December, 2013 Eucalyptus is the predominant exotic wood species used in South African pulp and paper industry. Once chipped and stored in piles, the wood becomes vulnerable to microbial degradation and spontaneous combustion. The denaturing gradient gel electrophoresis (DGGE) technique was optimized for the detection of microbial diversity in the wood. Wood chips were collected and milled to different specifications. The 16S and 18S rRNA genes were amplified using 338F-GC/518R and 933F- GC/1387R for bacteria and NS26/518R-GC and EF4F/518R for fungi. Several gel gradients were examined to determine optimal separation. A comparison of DGGE profiles revealed greater diversity in the milled wood chips amplified using primer sets of 338F-GC/518R (16S) and NS26/518R-GC (18S) with gradients of 30/60% (16S) and 25/50% (18S), respectively. Once optimized, this protocol was tested against five samples to assess its applicability to wood chip samples. Profiles were generated and amplicons excised from gels, re-amplified and sequenced to determine origin of DNA. Using this technique, 18 bacterial and 12 fungal species were identified, compared to ten bacterial and nine fungal isolates which were identified using the culturing technique and standard rRNA gene sequence analysis. The optimised DGGE is an appropriate tool for microbial community studies of Eucalyptus wood chips. Key words: Wood chips, Eucalyptus, community analysis using denaturing gradient gel electrophoresis (DGGE), microfloral variations. INTRODUCTION Eucalyptus sp. is the predominant hardwood in com- mercial plantations for the production of pulp and paper (FAO, 2005). Within a few days following harvesting, the wood is debarked, chipped at the mill and stored in piles, and thus remains wet and may hold over 50% of their weight as water (Brown et al., 1994). The availability of water initiates the growth of bacteria and promotes fermentation (Li et al., 2006). Storage of wood chips in piles leads to redistribution of the moisture resulting in a wet outer surface and drier inner part (Bedane et al., 2011; Noll and Jirjis, 2012) and may influence microbial succession within the chip piles based *Corresponding author. E-mail: govindenr@ukzn.ac.za. Tel: +2731 260 8281. Fax: +2731 260 7809. Abbreviations: PCR-DGGE, Polymerase chain reaction-denaturing gradient gel electrophoresis; ARISA, automated ribosomal intergenic spacer analysis; T-RFLP, terminal restriction fragment length polymorphism; PDA, potato dextrose agar.