The biotechnological control of pitch in paper pulp manufacturing

At present, microbial and enzymatic preparations for the control of triglyceride-containing pitch deposits during the manufacture of mechanical and sulfite paper is commercially available. However, biotechnological products for pitch control in other pulping processes, such as alkaline pulping, are under development. These products include new fungi for the removal of steroids involved in pitch deposit formation in chlorine-free pulps, to be used as a biological pretreatment of wood before pulping. Simultaneously, tailor-made enzymes are being produced using protein-engineering techniques, enabling the specific removal of pitch contaminant compounds from paper pulp.     

Degradation and detoxification of softwood extractives by sapstain fungi

Wood extractives (resin) cause pitch deposition problems and effluent toxicity in pulp and papermaking. The ability of six sapstaining fungi to degrade and detoxify extractive constituents in Scots pine sapwood was examined, and the results were compared with those obtained with the commercial depitching fungus Cartapip (Ophiostoma piliferum). Pestalotiopsis crassiuscula and O. piliferum were the best strains and they provided high reductions of total resin (50–60% in 6 weeks). Both strains were highly effective in the degradation of individual extractive components including triglycerides, diglycerides and free fatty acids. Although all strains displayed moderate to high pitch degradation, their detoxifying capacity was limited. Two important exceptions were Ceratocystis deltoideospora and O. piliferum that caused a 11–14-fold decrease in toxicity (Microtox bioassay). These results indicate the potential of wood pretreatment with the selected sapstain fungi for minimizing pitch problems and decreasing effluent toxicity in pulping.     

Fungal degradation of lipophilic extractives in eucalyptus globulus wood

Solid-state fermentation of eucalypt wood with several fungal strains was investigated as a possible biological pretreatment for decreasing the content of compounds responsible for pitch deposition during Cl2-free manufacture of paper pulp. First, different pitch deposits were characterized by gas chromatography (GC) and GC-mass spectrometry (MS). The chemical species identified arose from lipophilic wood extractives that survived the pulping and bleaching processes. Second, a detailed GC-MS analysis of the lipophilic fraction after fungal treatment of wood was carried out, and different degradation patterns were observed. The results showed that some basidiomycetes that decreased the lipophilic fraction also released significant amounts of polar extractives, which were identified by thermochemolysis as originating from lignin depolymerization. Therefore, the abilities of fungi to control pitch should be evaluated after analysis of compounds involved in deposit formation and not simply by estimating the decrease in the total extractive content. In this way, Phlebia radiata, Funalia trogii, Bjerkandera adusta, and Poria subvermispora strains were identified as the most promising organisms for pitch biocontrol, since they degraded 75 to 100% of both free and esterified sterols, as well as other lipophilic components of the eucalypt wood extractives. Ophiostoma piliferum, a fungus used commercially for pitch control, hydrolyzed the sterol esters and triglycerides, but it did not appear to be suitable for eucalypt wood treatment because it increased the content of free sitosterol, a major compound in pitch deposits.     

Biological Processing of Pine Logs for Pulp and Paper Production with Phlebiopsis gigantea

Phlebiopsis gigantea (=Phanerochaete gigantea) is a white rot fungus that rapidly colonizes cut stumps, stems, and branches of pine. Two laboratory and several field studies showed that inoculation of red pine logs, Pinus resinosa, with P. gigantea reduced the pitch content of wood, facilitated bark removal, modified wood cells, and controlled detrimental sapstain. Isolations from inoculated logs revealed up to 100 and 80% colonization of the sapwood by P. gigantea after 8 weeks in the field and 32 days in the laboratory, respectively. Logs colonized by P. gigantea in both the laboratory and field showed a 9 to 71% reduction in pitch content, as well as a significant enhancement of bark removal. Examination with Simons' stain of refined wood fibers from inoculated logs revealed an increase in cell wall porosity. Blue stain fungi that cause dark discoloration of the sapwood were inhibited by inoculation with P. gigantea. These studies demonstrate that biological processing of logs with P. gigantea can result in substantial benefits to the pulp and papermaking process.     

Fungal Degradation of Lipophilic Extractives in Eucalyptus globulus Wood

Solid-state fermentation of eucalypt wood with several fungal strains was investigated as a possible biological pretreatment for decreasing the content of compounds responsible for pitch deposition during Cl₂-free manufacture of paper pulp. First, different pitch deposits were characterized by gas chromatography (GC) and GC-mass spectrometry (MS). The chemical species identified arose from lipophilic wood extractives that survived the pulping and bleaching processes. Second, a detailed GC-MS analysis of the lipophilic fraction after fungal treatment of wood was carried out, and different degradation patterns were observed. The results showed that some basidiomycetes that decreased the lipophilic fraction also released significant amounts of polar extractives, which were identified by thermochemolysis as originating from lignin depolymerization. Therefore, the abilities of fungi to control pitch should be evaluated after analysis of compounds involved in deposit formation and not simply by estimating the decrease in the total extractive content. In this way, Phlebia radiata, Funalia trogii, Bjerkandera adusta, and Poria subvermispora strains were identified as the most promising organisms for pitch biocontrol, since they degraded 75 to 100% of both free and esterified sterols, as well as other lipophilic components of the eucalypt wood extractives. Ophiostoma piliferum, a fungus used commercially for pitch control, hydrolyzed the sterol esters and triglycerides, but it did not appear to be suitable for eucalypt wood treatment because it increased the content of free sitosterol, a major compound in pitch deposits.     

High concentrations of fatty acids affect the lipase treatment of softwood thermomechanical pulps

Triglycerides, a major class of wood extractives, contribute to the colloidal pitch that initiates pitch deposits. Because industrial or pilot-scale treatments with lipolytic enzymes to reduce triglyceride concentrations in pulp have not been successful in North America, we investigated such treatments at a laboratory scale. Different batches of industrial softwood chemithermomechanical pulps (CTMP) were treated with a range of concentrations of two commercial lipases: Resinase A 2X (Novo Nordisk AG) and Lipidase 10 000 (American Laboratories Inc.). A pilot-scale thermomechanical pulp (TMP) made from the same wood as the CTMP, but without the sodium hydrosulfite used in the CTMP, was also treated with the lipases. While triglycerides decreased in all the pulp treatments, the extent of their hydrolysis varied according to the ratio of triglyceride to the fatty/resin acid fraction. As this ratio can vary significantly in softwood TMP and CTMP, the success of industrial treatments of softwood mechanical pulps by commercial lipases may be related to variations in this ratio. Supporting this, adding linoleic acid to an extractives-free pulp that was spiked with olive oil reduced lipase activity by up to 55%. Received: 7 August 1997 / Received last revision: 8 December 1997 / Accepted: 14 December 1997     

The role of microorganisms in the formation of pitch deposits in pulp and paper mills

The cause of pitch deposit formation seems still not fully understood. The work reported here demonstrates that microorganisms effect the agglomeration of emulgated resin droplets and the formation of sticky precipitates.

Pitch deposits from mills consist mainly of ethanol-soluble resins. It is also the ethanol-soluble fraction of wood resins which forms stable emulsions and which is easily agglomerated by microorganisms.

Pitch deposits, collected from various pulp and paper mills, were all found to contain large amounts of microorganisms. Sterile resin emulsions prepared from pitch deposits remained stable over long periods. After inoculation with microorganisms the emulsions were destabilized and the resins completely precipitated as sticky lumps.

Various bacteria and fungi are capable of agglomerating the resins, but species isolated from water, pulp and slime in paper mills were usually most effective. Resins from fresh wood were precipitated at a faster rate than aged resins.

Problems of pitch formation can be considerably reduced when microbial growth is kept under control in the production system. To be effective, the control measures, e.g. slimicides, must be applied at the right place and time, and in the correct concentrations. This presupposes a thorough knowledge of the plant's microbiological condition which can only be obtained by microbiological examination. Practical cases of the appropriate application of biocides in pulp and paper mill systems are discussed.     

Biopulping: An overview of developments in an environmentally safe paper-making technology

Abstract: Treatment of wood chips with lignin-degrading fungi prior to pulping has been shown to have great potential for mechanical as well as chemical pulping on a laboratory scale. Ceriporiopsis subvermispora , when grown on aspen or loblolly pine for 4 weeks, was found to be superior to other fungi. On aspen there was an energy savings of 47%, and an increase in burst and tear indices of 22% and 119%, respectively. With loblolly pine, energy savings amounted to 37%, while burst and tear indices increased by 41% and 54%, respectively. The weight loss was only 6%, but a decrease in optical properties had to be accepted. After sulfite cooking of wood chips pretreated for 2 weeks, the Kappa number decreased by 30% with hard- and softwood. Tensile and tear indices decreased by only 10%, while the brightness of unbleached pulp increased by 4% with birch. Information obtained by immunoelectron microscopy and differential staining led to the conclusion that the biopulping effect obtained after 2 weeks of incubation cannot be explained by the direct action of enzymes on lignin or polysaccharides. Instead, a low molecular mass agent is considered to be responsible for the biopulping effect. These results have changed the aims of biopulping from an emphasis on removing the bulk of lignin to an emphasis on a short-term process, lasting 2 weeks and yielding a low mass loss. Data on these kinetics of fungal development and the degree of asepsis will help to scale-up the process. An advanced chip pile is assumed to be the most feasible process design, rather than a controlled enclosed reactor.     

Use of laccase in pulp and paper industry

Laccase, through its versatile mode of action, has the potential to revolutionize the pulping and paper making industry. It not only plays a role in the delignification and brightening of the pulp but has also been described for the removal of the lipophilic extractives responsible for pitch deposition from both wood and nonwood paper pulps. Laccases are capable of improving physical, chemical, as well as mechanical properties of pulp either by forming reactive radicals with lignin or by functionalizing lignocellulosic fibers. Laccases can also target the colored and toxic compounds released as effluents from various industries and render them nontoxic through its polymerization and depolymerization reactions. This article reviews the use of both fungal and bacterial laccases in improving pulp properties and bioremediation of pulp and paper mill effluents.     

Effect of volatiles from bacteria and yeast on the growth and pigmentation of sapstain fungi

Sapstain fungi affect the appearance of wood due to colonisation by pigmented hyphae but without producing significant strength losses. This is due to the production of melanin in the fungal cell walls of the staining fungi. Any biological control strategy targeted against this type of deterioration would therefore be considered successful if it inhibited either fungal growth or pigment production. Previous work has established that specific bacterial and yeast isolates selected on the basis of agar screening studies could significantly reduce levels of staining in wood block tests. This paper presents the results of a study to examine the role of volatile organic compounds (VOCs) produced by four bacterial and three yeast isolates on the growth and pigment production by a range of five sapstain fungi on three media types. VOCs from three of the four bacterial strains tested completely inhibited growth of the five target sapstain fungi but only when the antagonists were grown on tryptone soya media. When antagonists were grown on either malt agar or a low nutrient medium levels of inhibition were either significantly reduced or non-existent. Yeast antagonists generally produced lower levels of growth inhibition than the bacteria but a Williopsis mrakii isolate gave 100% inhibition of three of the five sapstain fungi. Production of inhibitory VOCs was highly dependent on the specific antagonist as well as its growth substrate and all five sapstain fungi showed varying sensitivities to the VOCs produced. Not all fungi were inhibited, growth of O. piliferum and A. pullulans being stimulated by the VOCs from antagonists but only when grown under low nutrient conditions. In some instances, where growth was only slightly reduced, the level of pigmentation of the sapstain colony was significantly reduced compared with corresponding controls. The implications of this work for the biological control of sapstain fungi are discussed.     

Time course of fungal removal of lipophilic extractives from Eucalyptus globulus wood

Free and esterified sitosterol, the main lipophilic constituents of eucalypt wood extractives, have been associated with the formation of pitch deposits during manufacturing of environmentally-sound paper pulp from Eucalyptus globulus wood. These, and other lipophilic compounds, were analyzed by gas chromatography-mass spectrometry in the course of wood treatments (up to 7 weeks) with four extractive-degrading fungi in order to optimize biotechnological control of pitch deposition in eucalypt pulp (with moderate loss of wood weight). In contrast to commercialized fungi used in pitch control, which are not able to degrade sitosterol, the fungi investigated in this paper produced a rapid decline of both free and esterified sterols in wood. The degradation rate of steroid hydrocarbons and squalene was moderate, and the amount of steroid ketones (probably formed during oxidative degradation of steroids) and triglycerides increased at different stages of wood treatment. Up to 95% removal of total steroids (including free and esterified sterols, steroid ketones and steroid hydrocarbons) by fungi was obtained at the end of wood treatment under the solid-state fermentation conditions used. The most promising results from the point of view of industrial applicability, however, were obtained after 1-2 weeks of treatment with either Phlebia radiata or Poria subvermispora, which enabled 70% steroid removal with a moderate wood weight loss of 1-4%.     

Biodegradation of aspen steryl esters and waxes by two filamentous fungi with or without other carbon sources

Aspergillus luchuensis and Cunninghamella elegans were evaluated for their growth on steryl esters and waxes, which are a major cause of pitch deposition in aspen pulping. These fungi hydrolysed aspen steryl esters and waxes into their constituent sterol and fatty acid moieties. A. luchuensis and C. elegans were also grown on steryl esters and waxes supplemented with glucose and triglycerides in order to provide a more accurate assessment of how these fungi behave on aspen wood. Both fungi consumed glucose before steryl esters and waxes while they degraded the triglycerides and steryl esters and waxes simultaneously.     

Microbial lipases as virulence factors

Up to now, lipases of microbial origin are known to be very useful in a palette of industrial applications. But it becomes more and more obvious that extracellular lipases also play a role during microbial infections. This review will focus on the virulent traits of these secreted enzymes from bacteria and fungi. Special emphasis will be laid on Candida albicans research. This human pathogenic fungus possesses a lipase gene family, which is expressed and differentially regulated under a variety of in vitro conditions. First results show that this isoenzyme family is also expressed during an experimental infection.

In addition, putative functions of extracellular lipases of pathogenic micro-organisms will be discussed.     

Aspergilli and lignocellulosics: Enzymology and biotechnological applications

Abstract: Aspergilli are versatile ascomycetes that are able to transform at a rapid rate a wide spectrum of lignin-related aromatic compounds. While it is clear that these fungi can degrade phenolic and polysaccharide components from lignocellulosic material, the status regarding degradation of high-molecular mass lignins is controversial. This review compiles data from the literature as well as that from the authors' laboratory with the aim of clarifying this point. The main body of evidence points towards the inability of aspergilli alone to degrade lignin free of low-molecular mass contaminants. Nevertheless, the ability of this genus to efficiently degrade hemicelluloses makes it an essential participant in the complex microbial system necessary for wood decay under natural conditions. Aspergilli are known to overproduce high levels of hemicellulolytic enzymes. Out of the large array of these enzymes that act in concert to degrade lignocellulosic material, only endoxylanases of aspergilli are described in so far as these are the main activities required for enzyme-aided bleaching. The biochemical features of the endoxylanases from Aspergillus niger are briefly described as these serve to illustrate how a complex family of isozymes is necessary to deal with the structural and chemical heterogeneity of xylans. Emphasis is placed on the biotechnological applications of lignocellulosic materials transformed by aspergilli. The key application areas are biopulping and biobleaching where a reduction in the use of environmentally harmful chemicals traditionally used in the pulp and paper industry is envisaged. Waste water treatment represents another vast application area where aspergilli have been shown to be effective not only in colour removal but also in the bioconversion of potentially noxious substances into useful bioproducts.     

Application of enzymes in the pulp and paper industry

The pulp and paper industry processes huge quantities of lignocellulosic biomass every year. The technology for pulp manufacture is highly diverse, and numerous opportunities exist for the application of microbial enzymes. Historically, enzymes have found some uses in the paper industry, but these have been mainly confined to areas such as modifications of raw starch. However, a wide range of applications in the pulp and paper industry have now been identified. The use of enzymes in the pulp and paper industry has grown rapidly since the mid 1980s. While many applications of enzymes in the pulp and paper industry are still in the research and development stage, several applications have found their way into the mills in an unprecedented short period of time. Currently the most important application of enzymes is in the prebleaching of kraft pulp. Xylanase enzymes have been found to be most effective for that purpose. Xylanase prebleaching technology is now in use at several mills worldwide. This technology has been successfully transferred to full industrial scale in just a few years. The enzymatic pitch control method using lipase was put into practice in a large-scale paper-making process as a routine operation in the early 1990s and was the first case in the world in which an enzyme was successfully applied in the actual paper-making process. Improvement of pulp drainage with enzymes is practiced routinely at mill scale. Enzymatic deinking has also been successfully applied during mill trials and can be expected to expand in application as increasing amounts of newsprint must be deinked and recycled. The University of Georgia has recently opened a pilot plant for deinking of recycled paper. Pulp bleaching with a laccase mediator system has reached pilot plant stage and is expected to be commercialized soon. Enzymatic debarking, enzymatic beating, and reduction of vessel picking with enzymes are still in the R&D stage but hold great promise for reducing energy. Other enzymatic applications, i.e., removal of shives and slime, retting of flax fibers, and selective removal of xylan, are also expected to have a profound impact on the future technology of the pulp and paper-making process.     

Methods for Facilitating Microbial Growth on Pulp Mill Waste Streams and Characterization of the Biodegradation Potential of Cultured Microbes

The kraft process is applied to wood chips for separation of lignin from the polysaccharides within lignocellulose for pulp that will produce a high quality paper. Black liquor is a pulping waste generated by the kraft process that has potential for downstream bioconversion. However, the recalcitrant nature of the lignocellulose resources, its chemical derivatives that constitute the majority of available organic carbon within black liquor, and its basic pH present challenges to microbial biodegradation of this waste material. Methods for the collection and modification of black liquor for microbial growth are aimed at utilization of this pulp waste to convert the lignin, organic acids, and polysaccharide degradation byproducts into valuable chemicals. The lignocellulose extraction techniques presented provide a reproducible method for preparation of lignocellulose growth substrates for understanding metabolic capacities of cultured microorganisms. Use of gas chromatography-mass spectrometry enables the identification and quantification of the fermentation products resulting from the growth of microorganisms on pulping waste. These methods when used together can facilitate the determination of the metabolic activity of microorganisms with potential to produce fermentation products that would provide greater value to the pulping system and reduce effluent waste, thereby increasing potential paper milling profits and offering additional uses for black liquor.     

Main lipophilic extractives in different paper pulp types can be removed using the laccase–mediator system

Lipophilic extractives in wood and other lignocellulosic materials exert a negative impact in pulp and paper manufacturing causing the so-called pitch problems. In this work, the appropriateness of an enzymatic treatment using the laccase–mediator system for pitch biocontrol is evaluated. With this purpose, three pulp types representative for different raw materials and pulping processes—eucalypt kraft pulping, spruce thermomechanical pulping, and flax soda-anthraquinone pulping—were treated with a high-redox-potential laccase from the basidiomycete Pycnoporus cinnabarinus in the presence of 1-hydroxybenzotriazole as a redox mediator. The gas chromatography and gas chromatography/mass spectrometry analyses of the lipophilic extractives from the enzymatically treated pulps revealed that the laccase–mediator treatment completely or greatly removed most of the pitch-causing lipophilic compounds present in the different pulps including: (1) free and conjugated sitosterol in eucalypt paper pulp; (2) resin acids, sterol esters, and triglycerides in spruce pulp; and (3) sterols and fatty alcohols in the flax pulp. Different amounts of free and conjugated 7-oxosterols were found as intermediate products in the oxidation of pulp sterols. Therefore, the laccase–mediator treatment is reported as an efficient method for removing pitch-causing lipophilic compounds from paper pulps obtained from hardwood, softwood, and nonwoody plants.     

Chemical characterization of pitch deposits produced in the manufacturing of high-quality paper pulps from hemp fibers

The composition of pitch deposits occurring in pulp sheets and mill circuits during soda/anthraquinone pulping and elemental chlorine-free pulp bleaching of bast fibers of industrial hemp (Cannabis sativa) was studied. Pitch deposits were extracted with acetone, and the extracts analyzed by gas chromatography (GC) and gas chromatography/mass spectrometry (GC/MS). Acetone extracts (15-25% of pitch deposits) were constituted by the defoamers used at the mill and by lipophilic extractives from hemp fibers. Acetone-insoluble residues (75-85% of pitch deposits) were analyzed by pyrolysis-GC/MS in the presence and absence of tetramethylammonium hydroxide. These residues were constituted by salts of fatty acids (arising from hemp fibers) with calcium, magnesium, aluminum and other cations that were identified in the deposits. It was concluded that inappropriate use of defoamer together with the presence of multivalent ions seemed to be among the causes of hemp extractives deposition in the pitch problems reported here.     

Assessment of wood-inhabiting Basidiomycetes for biokraft pulping of softwood chips

Wood-inhabiting Basidiomycetes have been screened for various applications in the pulp and paper industry and it is evident that different fungi need to be used to suit the specific requirements of each application. This study assessed the suitability of 278 strains of South African wood-decay fungi for the pre-treatment of softwood chips for kraft pulping. The influence of these fungi on kappa number, yield and strength properties of pulp was evaluated. A number of these strains were more efficient in reducing kappa number than the frequently used strains of Phanerochaete chrysosporium and Ceriporiopsis subvermispora. Six strains of Stereum hirsutum and a strain of an unidentified species were able to reduce the kappa number significantly without a significant influence on the pulp yield. Treatment of wood with two strains of S. hirsutum, one strain of Peniophora sp. and a strain of an unidentified species resulted in paper with improved strength properties.     

制浆造纸生物技术研究进展

制浆造纸工业是国民经济的主要支柱之一,但也是资源消耗和环境污染的大户。近年来,国外用于制浆造纸工业的生物技术研究异常活跃,除废水生物处理外,木聚糖酶助漂、脂肪酶控制树脂、木片真菌预处理和酶法废纸脱墨等工艺已经在生产中得到实际应用,生物制浆、漆酶漂白工艺也已进入中试阶段。结合以草浆为主的特点,我国的制浆造纸生物技术研究也已日趋活跃起来。