Fungal pitch control in the draft pulping and bleaching
of Eucalyptus spp.

Gerhardus C Scheepers, Tim Rypstra, Theodorus H de Koker and Bernard J H Janse

Abstract

Wood and pulp samples as well as pitch deposits were collected at a kraft pulp mill and the composition of their dichloromethane extracts was determined. Pitch inducing triglycerides comprised 15.5% of volatile E. grandis extract, 0% of pulp and 10.1% of volatile pitch. To identify South African fungal strains that could reduce the triglyceride content of E. grandis, the lipase activities of 381 South African isolates were screened. Four of these strains reduced the triglyceride content of E. grandis significantly when incubated for a period of four weeks.

Introduction

Extractives are released during the pulping of wood and bleaching of pulp. In pulping, wood extractives increase consumption of pulping chemicals, impair the colour and brightness of unbleached pulp (Nelson et al., 1970), and cause a sticky deposit on pulping equipment. This deposit is referred to as pitch and perceived to be organic in nature. Wood triglycerides induce pitch deposition (Allen, 1977; Fischer and Messner, 1992; Fujita et al., 1992; Suckling and Ede, 1990). Several successful fungal pretreatment trials have been done to decrease the triglyceride content of softwoods and aspen (Blanchette et al., 1992; Brush et al., 1994; Farrell et al., 1997; Fischer et al., 1994; Fischer et al., 1995; Rocheleau et al., 1998).

Eucalyptus species are extensively used in the Southern Hemisphere and pitch problems are encountered despite the relatively low extractive content (ca. 0.4% of oven dry wood mass) of the species. In this investigation the composition of extracts of pulp and wood from a South African kraft pulp mill was determined. The effect of fungal pretreatment of E. grandis was also investigated. Firstly, the lipase activities of 381 fungal strains isolated in South Africa were evaluated. Eight of these strains were selected and their lipase activities were quantified. Thereafter, the abilities of four of the eight strains to decrease E. grandis wood and pulp triglyceride content were evaluated.

Materials and methods

Wood, pulp and pitch sampling
Six year old Eucalyptus grandis logs were obtained from the logyard of a kraft pulp mill in the KwaZulu/Natal region of South Africa. The logs were left in the plantations for six weeks after harvesting prior to delivery to the pulp mill logyard. The logs were debarked and chipped. The pulp mill feedstock comprised several Eucalyptus species and hybrids with E. grandis comprising more than 50% of the total feedstock. The mill has a C/DEoDED bleaching regime. Pulp samples were taken from the brown stock decker mat, CD mat, D1 inlet, E2 mat and bleach stock decker mat. Pitch was sampled from the bleach stock decker outlet. All samples were stored at -20C until analysed.

Fungal strains and screening for lipase activity
Two Thermomyces lanuginoses strains (Haarhoff et al., 1998) and Phanerochaete pseudomagnoliae nom. prov. were amongst the 381 South African fungal strains (de Koker et al., 1998) screened for lipase activity using the Tween 80 plate and tributyrin deep agar diffusion methods of Paterson and Bridge (1994). The media were inoculated with an agar plug and activity zones measured every 24 hours for 7 days.

Ophiostoma piliferum Cartapip 58 (CAR 58) was used as control strain in the Tween 80 and tributyrin assays repeated on the eight selected strains. Both O. piliferum Cartapip 58 and Phanerochaete chrysosporium BKM-F-1767 were used as control strains in the p-nitrophenyl palmitate (pNPP) assay also performed on these eight strains. The liquid olive oil medium (OOM) used to grow the fungal strains on for the pNPP assay contained 1% olive oil and 1.5% malt extract. The final inoculum consisted of 10 day old macerated (Waring blender) fungal biomass from OOM medium.

Only P. chrysosporium BKM-F-1767 was used as control strain in the wood chip treatment. All strains were incubated at 23C except for the strains of T. lanuginoses, which were incubated at 50C.

pNPP lipase activity assay
Lipase activity was determined with the p-nitrophenyl palmitate (pNPP) method. Solution A contained 30 mg pNPP (Sigma) dissolved in 10 ml propan-2-ol. Solution B contained 0.207 g sodium deoxycholate (Sigma) and 0.1 g gum arabic (Sigma) dissolved in 90 ml 0.05 M phosphate buffer (pH 8). Solution A was added in small quantities to Solution B while stirring continuously until all was dissolved. This substrate solution was prepared daily, prewarmed to 37C and dispensed in 2.5 ml quantities into test tubes. Culture supernatant (100 ml) of OOM grown cultures was added to the substrate solutions and incubated at 37C for 15 minutes. Enzyme activity was stopped by boiling for 3 minutes and the tubes were then placed on ice. Absorbance was measured at 410 nm against an appropriate blank. Lipase from Rhizopus arrhizus (Sigma) was used for production of standard curves. One unit of lipase will hydrolyse 1.0 microequivalent of fatty acid from a triglyceride in 1 hr at pH 7.7 at 37C.

Wood chip treatment trial
Six year old Eucalyptus grandis trees were felled, debarked and chipped after ca. 6 weeks. Inocula were grown in OOM medium containing 0.5% yeast extract. Autoclaved (121C for 15 min) E. grandis wood chips (500 g oven dry equivalent) at 70% moisture content (based on oven dry mass) were inoculated in triplicate with macerated (Waring blender) mycelia and incubated for four weeks in cotton wool stoppered containers at 30C except for strain MED 4B1, which was incubated at 50C. Two hundred grams of the wood chips were ground in a Wiley mill, with a 0.40 mm (40 mesh) sieve and extracted with dichloromethane.

Dichloromethane extractions
Extractive contents of wood and pulp samples were determined according to Tappi method T 204 om-88. Two thimbles per wood sample, each containing approximately two grams of air dry ground wood (1 g heartwood and 1 g sapwood) and two thimbles per pulp sample, each containing approximately 10 g of air dry pulp, were extracted with dichloromethane for 5 hours. The dichloromethane was evaporated from the extracts in a fume hood. The extractives were weighed and stored at -20C for analysis by gas chromatography.

Gel permeation chromatography (GPC)
Samples were dissolved in tetrahydrofuran. Analyses were done using a Spectra Physics 8875 autosampler, HP1100 isocratic pump and Spectra Physics GPC software. The molecular mass distribution of wood and pulp extracts were determined by passing extracts (flow rate: 1.25 ml.min-1) through four in series 300 7.8 micron Phenogel 10 columns (Phenomenex) with packing pore sizes of 10 nm, 50 nm, 100 nm and 1000 nm respectively. Molecular masses were determined using polystyrene standards and an Erma 7510 refractive index (RI) detector.

Gas chromatography
Extracts were methylated with diazomethane (Christie, 1992). Methylated extracts were analysed with a Hewlett Packard 6890 gas chromatograph equipped with a flame ionisation detector. Column: Chrompack SimDist Ulti Metal, 10 m 0.53 mm i.d., 0.17 m coating. Carrier gas: nitrogen. Injection method: 10 mg of each sample was dissolved in 200 l chloroform/methanol (2:1) and 2 l was injected with a 20:1 split ratio. Temperature program: Injector 250C, initial column temperature 130C, ramp rate 5C/min, final column temperature 390C.

Results and discussion

The composition of E. grandis wood extract
The <900 Da range comprised 71.4% and the >900 Da range 28.6% of the total wood extract. Ohtani et al. (1986) and Ohtani and Shigemoto (1991) reported that the high molecular mass components of several hardwoods used in Japanese kraft pulping mills as well as pitch from these mills were mostly polymerised aliphatic hydrocarbons. Triglycerides, which have molecular masses in the range of 800-900 Da, were the compounds with the highest molecular mass that could be detected by GC analysis.

Table 1 shows that there were some differences in the composition of E. grandis heartwood and sapwood dichloromethane extractives though the total extractive contents were similar. -sitosterol constituted a large portion of the total volatile E. grandis heartwood and sapwood extracts. b-sitosterol is a major fraction of the sterol content of many hardwoods (Fengel and Wegener 1989). There was little difference in the triglyceride content of the sapwood and heartwood. Triglyceride content was generally within the range of 15-30% of volatile dichloromethane wood extractives.

Table 1. Dichloromethane extract composition of E. grandis heart- and sapwood. Total extractive content is given as a percentage of oven dry wood mass. All other values are given as a percentage of total volatile extract.

The composition of Eucalyptus spp. pulp extract and pitch
The dichloromethane extractable levels of pulp remained much the same despite washing and bleaching stages (Table 2). The same compounds found in the E. grandis wood extract were present in the pulp extracts. However, in the latter stages of bleaching a significant proportion of the pulp extractives was fatty acid amides coming from additives. Due to the digestion process the contribution of the >900 Da range changed from 28.6% to 30.1%. The >900 Da range comprised 51.6% of bleach stock decker mat pulp extract, indicating that the high molecular mass compounds were less readily removed.

A lot of pulp fibres were attached to the sticky pitch deposits. Free fatty acids, sterols, triglycerides and fatty acid amides were the major constituents of the volatile fraction of pitch (Table 2). Most of the fatty acid amides were N, N -dimethylpalmitamide. The presence of additives in the small amounts of pitch that were deposited does not imply that it induced pitch deposition. This pulp mill experienced minimal pitch deposition since the use of additives was instigated.

Table 2. The chemical composition of Eucalyptus spp. dichloromethane pulp extracts and pitch. Total extractives are given as a percentage of oven dry pulp mass. All other values are given as a percentage of total volatile extract.

Table 2

Triglycerides constituted 10.1% of the volatile pitch deposits though pulp extracts contained no triglycerides. This indicates either that triglycerides were in suspension in the white water or that very small amounts carried by the pulp, deposited over an extended period of time. It is also possible that the triglycerides were deposited in periods when feedstock with a high triglyceride content was pulped.

Screening for lipase activity
Only 42 strains (11% of the 381 screened strains) gave a high degree of discolouration on Tween 80 plates whilst 50 strains (13%) created a high degree of clarity of the tributyrin media. The eight strains selected from the screening were assayed again for lipase activity and the results are shown in Table 3. On Tween 80 all eight strains had a faster growth rate and all gave larger lipase active zones than the control O. piliferum CAR 58. On tributyrin media the two thermophilic T. lanuginoses strains, MED 2D and MED 4B1, performed better than Cartapip 58 (O. piliferum CAR 58 ) in terms of lipase production (depth of lipase active zone, data not shown) and was equal in lipase activity (clarity of lipase active zone, Table 3). Strain MTZ 95 was comparable in terms of the depth of the lipase active zone, but did not match the lipase activity of the control and the two thermophilic strains.

Table 3. Lipase activity of eight selected strains and the controls, O. piliferum CAR 58 and P. chrysosporium BKM -F-1767, on tributyrin and Tween 80 as well as units of enzyme activity measured with the pNPP assay. The discolouration effect of the strains on the solid media are graded from + (weak effect) to +++ (striking effect). ND = not determined

Table 3

pNPP lipase activity of selected strains
Due to maceration of the biomass prior to inoculation, cell-associated lipases (intracellular and/or mucilage-bound lipases) were released into the culture supernatant giving cell-associated lipase activity on day 1 (Table 3). The lipase activity on day 4 is attributed solely to free lipases released into the supernatant without any agitation of the biomass. Strains T. lanuginoses MED 2D, T. lanuginoses MED 4B1 and P. pseudomagnoliae nom. prov., showed a higher lipase activity on day 4 than day 1, indicating a high free lipase activity. The control strain, O. piliferum CAR 58, had both high cell-associated and free lipase activities. However, P. chrysosporium BKM-F-1767, which is known to reduce the total dichloromethane soluble wood extractives (Farrell et al., 1997), showed high cell-associated but low free lipase activity. Since both control strains reduced the total dichloromethane soluble wood extractives, the inability to produce free lipases does not seem to have a negative effect on the ability of a fungus to hydrolyse wood extractives when growing on wood.

Wood chip treatment trial
The strains BKM-F-1767, BLK 10A, MTZ 95 and P. pseudomagnoliae nom. prov. all whitened the wood during growth, indicating ligninolysis. T. lanuginoses MED 4B1 did not discolour the wood. The variation in extractive composition of the sterile controls made it difficult to compare the ability of different fungal strains to reduce the triglyceride content (Table 4). However, all the strains decreased the wood triglyceride content significantly, although some increased the total extractive content. This indicates that a decrease in total extractive content can not be used as an indication of the ability of a fungus to combat pitch. The final triglyceride content was lowest for P. pseudomagnoliae nom. prov. treated samples while MTZ 95 caused the highest percentage reduction (45%) in triglyceride content. Because of the higher triglyceride content of sterile controls, more triglycerides would be released into pulping process waters by the sterile controls than the treated samples.

Table 4. Extractive composition and content of sterile E. grandis treated with five different fungal strains. Total extractive content is given as a percentage of oven dry wood mass. All other values are given as a percentage of total volatile extract.

Table 4

Conclusions

Involatile compounds constituted a large portion of wood and pulp extracts. b-sitosterol was a major constituent of volatile E. grandis wood and pulp extractives. The triglyceride content of volatile E. grandis wood extractives was ca. 15%.

The dichloromethane extractable levels of pulp remained much the same despite washing and bleaching stages. Though no triglycerides were found in the pulp extractives, it constituted 10% of the volatile fraction of pitch deposits. Wallis and Wearne (1999) also found that kraft cooking eliminated triglycerides from E. globulus pulp extract. The fact that triglycerides were found in the pitch deposits indicate either that triglycerides were in suspension in the white water or that trace amounts carried by the pulp deposited over an extended period of time. It is also possible that the triglycerides were deposited in periods when feedstock with a high triglyceride content was pulped.

It was clear from the results of the Tween 80, tributyrin and pNPP assays that most of the eight selected strains had the potential for effective triglyceride breakdown. The presence of free rather than cell-associated lipases did not seem to determine the ability of the fungus to reduce the triglyceride content of E. grandis wood chips.

The apparent natural variation in extractive composition of the sterile controls made it difficult to compare the ability of different fungal strains to reduce extractive and triglyceride content. The four selected fungi compared well to P. chrysosporium BKM-F-1767 in reducing the triglyceride content of E. grandis wood chips. Although all of the selected strains decreased the wood triglyceride content, the total extractive content of the wood chips was increased in some cases. This result indicates that the reduction of the total extractive content can not be used as a measure of the ability of a fungal strain to combat pitch. The effect on the triglyceride content should rather be employed as measure.

Acknowledgements

The authors are grateful to Saralene Thomas for her technical assistance in screening the fungal strains as well as Mondi Kraft Ltd., The Foundation for Research and Development, and The Technology and Human Resources for Industry Programme for supporting this project.

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