Microbial and enzymatic control of pitch in the pulp and paper industry

Pitch control is an important aspect in pulp and paper manufacture, and the first example where microbial biotechnology provided successful solutions in this industrial sector. Triglycerides cause deposits in softwood mechanical pulping, and both microbial and enzymatic products have been commercialized to be applied on wood and pulp, respectively. The former are based on colorless strains of sapstain fungi. The latter are improved lipases, including thermostable variants from directed evolution. These enzymes are among the additives of choice in pulping of high-resin-content softwoods. However, lipases are not useful when pitch originates from other lipids, such as steroids and terpenes, and the sapstain inocula are also only partially effective. In the search for stronger biocatalysts to degrade recalcitrant lipids, the potential of white-rot fungi and their enzymes has been demonstrated. When inocula of these fungi are used, wood treatment must be controlled to avoid cellulose degradation. However, the efficiency and selectivity of the laccase-mediator system permits its integration as an additional bleaching stage. A double benefit can be obtained from these treatments since pitch is controlled at the same time that residual lignin is removed facilitating the implementation of totally chlorine free pulp bleaching.     

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.     

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.     

Elimination and detoxification of softwood extractives by white-rot fungi

The ability of several white-rot fungal strains to remove and detoxify acetone extractives (pitch or resin) in Scots pine sapwood was investigated in stationary laboratory batch assays. Fungal pretreatment provided up to 62% total pitch reduction and significant decreases in pitch toxicity. The best strains were Bjerkandera sp. strain BOS55, Stereum hirsutum and Trametes versicolor that eliminated over 93% of the problematic triglyceride fraction and 58–87% of other lipophilic extractive classes in only 2 weeks. Fungal removal of the wood extractives was accompanied by a 7.4–16.9-fold decrease in their inhibitory effect, as determined in the Microtox bioassay. Wood pretreatment by Bjerkandera sp. and T. versicolor caused limited losses of woody mass (less than 4% in 4 weeks); whereas S. hirsutum led to somewhat higher mass losses (7% in 4 weeks). These results indicate the potential of white rot fungi to control pitch deposition problems in pulping and to reduce the aquatic toxicity caused by naturally-occurring lipophilic extractives in forest industry effluents.     

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.     

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.     

Colour and AOX removal from pulping effluents by algae

A mixed culture of algae was used to treat pulping mill effluent in terms of removing both colour and adsorbably organic halides (AOX). The removal of AOX from pulping effluent increased with increasing initial colour value of the effluent. However, for the total mill effluent (composed of both pulping and bleaching effluents), AOX removal was found to be independent of initial colour value, and was around 70%. Up to 80% removal of colour from pulping effluent was achieved within 30 days under continuous lighting conditions. It was found that algae reduced the colour of pulping effluent of relatively low initial colour more efficiently than that of high initial colour. Under simulated field lighting conditions, up to 60% colour removal from pulping effluent was observed after 60 days of exposure, whereas for the total mill effluent it was up to 64% after 45 days. Total organic carbon and lignin (UVA₂₈₀) were also removed to a significant extent, suggesting that the mechanism of colour removal might not be transformation of the coloured lignin molecules to non-coloured ones. Analysis of alkaline extraction of the algal biomass and material balance findings indicated that the main colour removal mechanism was metabolism rather than adsorption. The experimental results were also analysed using multiple regression techniques and a mathematical model was developed to express the removal of colour from pulping effluents in terms of initial colour value, exposure time and lighting periods as well as interactions between these variables. Received: 12 January 1999 / Revision received: 25 March 1999 / Accepted: 26 March 1999     

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.     

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%.     

Using agar extraction waste of Gracilaria lemaneiformis in the papermaking industry

In China, fiber supply lags behind the growing demand for paper and paperboard products. The increasing consumption of paper products necessitated the need for new fiber sources. The red alga Gracilariopsis lemaneiformis is economically cultivated on a large scale in China for industrial agar extraction. During the extraction processes, considerable amounts of solid residues are produced as extraction wastes. In this study, we explored the potential of using the agar extraction residues as raw materials for pulping and papermaking. The results show that the extraction wastes of G. lemaneiformis could indeed be utilized for papermaking. Evaluation of the paper handsheets showed that a higher content of algal material resulted in paper that had lower strength and permeability but higher waterproof and greaseproof characteristics, as well as better antimicrobial effects. The results indicated that alga extraction residues could be employed as functional fillers to produce paper products that are potentially useful in the food-packaging industry.     

Kinetics of polysaccharide degradation during ethanol–alkali delignification of giant reed—Part 1. Cellulose and xylan

The degradation kinetics of the principal polysaccharides (cellulose and xylan) of the agro-fibre crop Arundo donax L. (giant reed) during ethanol–alkali delignification is reported. Based on the properties of a multi-component reaction system, the degradation kinetics of both polysaccharides was accurately described in terms of two simultaneous irreversible first-order reactions corresponding to removal of two kinetically homogeneous fractions. The moderate cellulose losses during pulping (about 4.5%) result mainly from the removal of the more reactive cellulose fraction, that accounted for 4% of initial cellulose. The bulk of the cellulose (96%) degrades slowly with three orders lower rate with pulping progress. The apparent activation energy of cellulose fractions degradation was estimated as 105.2 and 106.5 kJ mol⁻¹, respectively. Substantial loss of xylan during pulping (about 55%, as a homoxylan) is caused by fast removal of the first very reactive fraction, covering about 48% of total xylan. The degradation rate of the second xylan fraction is only one order higher of the bulk cellulose degradation. The activation energy of xylan fractions degradation was found as 74.4 and 140.9 kJ mol⁻¹, respectively.     

碱性木聚糖酶研究进展*

木聚糖是一种在自然界中含量仅次于纤维素的丰富的可再生资源,木聚糖酶是一类可以将木聚糖水解成单糖和寡糖的酶,利用木聚糖酶将木聚糖分解后的产物被广泛应用于食品、造纸以及纺织等行业。木聚糖酶按其对酸碱环境的耐受能力分为碱性木聚糖酶、中性木聚糖酶和酸性木聚糖酶,其中碱性木聚糖酶适合应用于造纸工业中,尤其在造纸的制浆、促进漂白及废纸脱墨等多种工艺中,可以显著提高纸张质量,有效降低氯气排放量,从而减少对环境的污染。随着生物技术的进步,利用基因工程技术可以对碱性木聚糖酶进行分子改造,以提高其耐碱、耐热能力,扩大其在工业应用中的条件范围。介绍碱性木聚糖酶在分子改造方面的研究进展以及其在造纸漂白和制浆、废纸脱墨中的应用。     

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.     

Farmland treatment and utilization of straw pulp mill effluents

Straw pulp production accounts for 74% of the total raw pulp production in China. The pulping waste, containing high contents of silicate and high pH, is difficult to treat. Instead of the conventional alkaline process, ammonia sulfite pulping discharges neutral effluent with nitrogen, phosphorus, potassium, sulfur and organic matter which are potential fertilizers and water resources for agriculture. The effect of effluent from ammonia sulfite pulping on rice growth and yield, was studied in Baigezhuang farm, Hebei province. Diluted rice straw digester waste or composite waste were irrigated on a paddy field. In plot tests, rice output increased by 16.2–25.3% with 0.7–6% pulp digester waste and by 22.9–37.9% with 2.7–5.4% composite waste. In field tests, rice output increased by 8.6–15.9% with 2.7–4.1% composite waste. Due to clean water dilution and paddy purification, the removal efficiency of biochemical oxygen demand (BOD), chemical oxygen demand (COD), suspended solid (SS), sulfide and phenol can reach over 97%. Rice quality also tested had higher protein content than the control. The study suggests that the effluents from ammonia sulfite pulping can be widely used in the farming industry. The ammonia sulfite process is a good approach to overcome pulp pollution in rural areas.     

Fractionation of wheat straw by atmospheric acetic acid process

Fractionation of wheat straw was investigated using an atmospheric acetic acid process. Under the typical conditions of 90% (v/v) aqueous AcOH, 4% H(2)SO(4) (w/w, on straw), ratio of liquor to straw (L/S) 10 (v/w), pulping temperature 105 degrees C, and pulping time 3h, wheat straw was fractionated to pulp (cellulose), lignin and monosaccharides mainly from hemicellulose with yields of approximately 50%, 15% and 35%, respectively. Acetic acid pulp from the straw had an acceptable strength for paper and could be bleached to a high brightness over 85% with a short bleaching sequence. Acetic acid pulp was also a potential feedstock for fuels and chemicals. The acetic acid process separated pentose and hexose in wheat straw to a large extent. Most of the pentose (xylan) was dissolved, whereas the hexose (glucan) remained in the pulp. Approximately 30% of carbohydrates in wheat straw were hydrolyzed to monosaccharides during acetic acid pulping, of which xylose accounted for 70% and glucose for 12%. The acetic acid lignin from wheat straw showed relatively lower molecular weight and fusibility, which made the lignin a promising raw material for many products, such as adhesive and molded products.     

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.     

Effect of bio-treatment on the lipophilic and hydrophilic extractives of wheat straw

Wheat straw, an important papermaking raw material in China, was treated with a white-rot fungus of Phanerochaete chrysosporium ME446, and the lipophilic and hydrophilic extractives from the control and bio-treated samples were analyzed by GC and GC–MS. Bio-treatment of wheat straw could alter the chemical composition of both the lipophylic and hydrophilic extractives. Sugars and phenolic substances such as coniferyl alcohol, 4-hydroxycinnamic acid, 1-guaiacylglycerol and ferulic acid were substantially degraded or consumed by the fungus. More lipophilic substances such as wax, glycerides and steryl esters were degraded into the corresponding components, resulting in much higher concentrations of fatty acids and sterols in the bio-treated samples. Obviously, the bio-treatment of wheat straw was of benefit to pitch control in pulping and papermaking processes, in the view of degradation of the more lipophilic substances. In addition, the bio-treatment could increase the lignin concentration in hot-water extractives of wheat straw.     

Lignin from rice straw Kraft pulping: effects on soil aggregation and chemical properties

Lignin contained in pulping liquor that is generated during the pulping process for papermaking is a disposal problem for the pulp and paper industry. Separating lignin and other organic components from pulping liquor with inorganic acids may improve its applicability to fields as a beneficial soil amendment while offering a potential disposal alternative. Sulfuric acid-precipitated lignin from rice straw pulping liquor applied at rates of 1.67 and 3.34 g C kg(-1) soil was incubated to evaluate its effects on soil properties over 8 weeks of incubation. Addition of this acid-precipitated lignin at these rates decreased soil pH by 0.24-0.53 units over 8 weeks of incubation, suggesting that this sulfuric acid-precipitated lignin from pulping liquor may have potential as a soil acidifying agent. Soil electrical conductivity (EC) only increased by up to 0.36 d Sm(-1), but highest EC levels were less than 4 d Sm(-1), indicating that lignin applied at both rates would not cause salinity problems. Application of this lignin increased soil organic C by 1.46 and 3.13 g C kg(-1), and total soil N by 0.07 and 0.17 g N kg(-1) over the incubation period. Lignin improved the macroaggregation of >2mm size fraction, and increased wet microaggregate stability of >2mm and 0.5-0.25 mm aggregates compared to a nonamended control. The results of this study suggest that this acid-precipitated lignin from pulping liquor may have potential as a beneficial soil amendment.     

Predicting the most appropriate wood biomass for selected industrial applications: comparison of wood,pulping, and enzymatic treatments using fluorescent-tagged carbohydrate-binding modules

Background

Lignocellulosic biomass will progressively become the main source of carbon for a number of products as the Earth’s oil reservoirs disappear. Technology for conversion of wood fiber into bioproducts (wood biorefining) continues to flourish, and access to reliable methods for monitoring modification of such fibers is becoming an important issue. Recently, we developed a simple, rapid approach for detecting four different types of polymer on the surface of wood fibers. Named fluorescent-tagged carbohydrate-binding module (FTCM), this method is based on the fluorescence signal from carbohydrate-binding modules-based probes designed to recognize specific polymers such as crystalline cellulose, amorphous cellulose, xylan, and mannan.

Results

Here we used FTCM to characterize pulps made from softwood and hardwood that were prepared using Kraft or chemical-thermo-mechanical pulping. Comparison of chemical analysis (NREL protocol) and FTCM revealed that FTCM results were consistent with chemical analysis of the hemicellulose composition of both hardwood and softwood samples. Kraft pulping increased the difference between softwood and hardwood surface mannans, and increased xylan exposure. This suggests that Kraft pulping leads to exposure of xylan after removal of both lignin and mannan. Impact of enzyme cocktails from Trichoderma reesei (Celluclast 1.5L) and from Aspergillus sp. (Carezyme 1000L) was investigated by analysis of hydrolyzed sugars and by FTCM. Both enzymes preparations released cellobiose and glucose from pulps, with the cocktail from Trichoderma being the most efficient. Enzymatic treatments were not as effective at converting chemical-thermomechanical pulps to simple sugars, regardless of wood type. FTCM revealed that amorphous cellulose was the primary target of either enzyme preparation, which resulted in a higher proportion of crystalline cellulose on the surface after enzymatic treatment. FTCM confirmed that enzymes from Aspergillus had little impact on exposed hemicelluloses, but that enzymes from the more aggressive Trichoderma cocktail reduced hemicelluloses at the surface.

Conclusions

Overall, this study indicates that treatment with enzymes from Trichoderma is appropriate for generating crystalline cellulose at fiber surface. Applications such as nanocellulose or composites requiring chemical resistance would benefit from this enzymatic treatment. The milder enzyme mixture from Aspergillus allowed for removal of amorphous cellulose while preserving hemicelluloses at fiber surface, which makes this treatment appropriate for new paper products where surface chemical responsiveness is required.

     

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.