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Highlights • Phenol red screening plates is the best method for detecting lipolytic activity. • Substrate specificity is affected by temperature and pH. • Essential to test substrates at various pH and temperature to determine optima. • Lipolytic enzymes indigenous to Eucalyptus sp. can assist in pitch control., This study highlights the importance of determining substrate specificity at variable experimental conditions. Lipases and esterases were isolated from microorganisms cultivated from Eucalyptus wood species and then concentrated (cellulases removed) and characterized. Phenol red agar plates supplemented with 1% olive oil or tributyrin was ascertained to be the most favourable method of screening for lipolytic activity. Lipolytic activity of the various enzymes were highest at 45–61 U/ml at the optimum temperature and pH of between at 30–35 °C and pH 4–5, respectively. Change in pH influenced the substrate specificity of the enzymes tested. The majority of enzymes tested displayed a propensity for longer aliphatic acyl chains such as dodecanoate (C12), myristate (C14), palmitate (C16) and stearate (C18) indicating that they could be characterised as potential lipases. Prospective esterases were also detected with specificity towards acetate (C2), butyrate (C4) and valerate (C5). Enzymes maintained up to 95% activity at the optimal pH and temperature for 2–3 h. It is essential to test substrates at various pH and temperature when determining optimum activity of lipolytic enzymes, a method rarely employed. The stability of the enzymes at acidic pH and moderate temperatures makes them excellent candidates for application in the treatment of pitch during acid bi-sulphite pulping, which would greatly benefit the pulp and paper industry.

Lipophilic extractives naturally occurring in wood tend to coalesce during pulping to form pitch deposits, which have particularly undesirable effects on the pulping process and quality of pulp produced. A chemical characterization of different eucalypt species [Eucalyptus nitens, E. grandis, and E. dunnii (of different site qualities)] wood and generated pulp was performed. This study aimed at determining the effects of wood storage at -20 °C (for 6 months), by examining their chemical composition and indigenous microflora. Fatty acids were the main lipophilic compounds among E. dunnii (SQ3 and SQ4) and E. grandis wood extractives. The wood of E. nitens posed the least risk for pitch deposit formation, making it the most suitable Eucalyptus species for pulping. Storage of wood chips at -20 °C had a similar effect as the traditional method of seasoning (storage of wood outdoors prior to pulping) used for the reduction of lipophilic extractives. A 25 to 44% reduction of total extractives was observed in the raw material after storage. Variations in bacterial and fungal communities were observed after storage, and should be taken into consideration when conducting lab scale trials. If storage of wood chips is necessary for lab testing, it should be retained for a maximum of 3 months at -20 °C.

In the pulp and paper industry, during the manufacturing process, the agglomeration of pitch particles (composed of triglycerides, fatty acids, and esters) leads to the formation of black pitch deposits in the pulp and on machinery, which impacts on the process and pulp quality. Traditional methods of pitch prevention and treatment are no longer feasible due to environmental impact and cost. Consequently, there is a need for more efficient and environmentally friendly approaches. The application of lipolytic enzymes, such as lipases and esterases, could be the sustainable solution to this problem. Therefore, an understanding of their structure, mechanism, and sources are essential. In this report, we review the microbial sources for the different groups of lipolytic enzymes, the differences between lipases and esterases, and their potential applications in the pulping industry.

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.