Biopulping process design and kinetics

Biopulping is the solid-state fermentation of wood chips as a pretreatment for mechanical pulping processes. The two organisms that are currently of the greatest interest for biopulping are the white-rot fungi, Phanerochaete chrysosporium and Ceriporiopsis subvermispora. P. chrysosporium has been shown to successfully biopulp wood (33% energy savings; 39% improvement in tear index) without the need for sterilization of the wood or nutrient supplementation. Demonstrating the practical and economical feasibility of the biopulping process requires process modeling based on accurate kinetic data. Techniques to monitor dry weight loss and growth rate as functions of time using carbon dioxide production data have been developed. Growth was shown to be linear with time on unsupplemented chips and exponential with time on supplemented chips.     

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.     

ASSESSMENT OF Cryptococcus albidus FOR BIOPULPING OF EUCALYPTUS

Cryptococcus albidus shows delignification activity in nature. It was used for the biopulping of eucalyptus wood (Eucalyptus grandis) to access its potential for industrial application in the pulp and paper industry. Enzyme analysis on days 15, 30, and 60 showed the presence of laccase and xylanase as key enzymes. The production of endo-glucanase (CMCase) and exo-glucanase (FPase) was very low. Scanning electron microscopy (SEM) showed the surface colonization of wood and loosening of wood fibers in C. albidus-treated samples. Fourier-transformation infrared spectroscopy (FT-IR) indicated the chemical modification of eucalyptus wood. Denaturing gradient gel electrophoresis (DGGE) analysis on days 15, 30, and 60 confirmed the presence of C. albidus throughout the experiments. Cryptococcu albidus was able to suppress the growth of a native population. Further, after 60 days both the control and treated eucalyptus wood chips were given kraft pulping treatment. The kappa number of pulp of control wood was 21 and for treated wood was 17. Kappa number is considered a measure of lignin content in wood; hence the treatment of eucalyptus by C. albidus (biopulping) was effective in reducing its lignin content and can be used for biopulping in the pulp and paper industry.     

Can co-culturing of two white-rot fungi increase lignin degradation and the production of lignin-degrading enzymes?

The aim of this work was to investigate the poorly understood effects of co-culturing of two white rot fungi on the production of lignin-degrading enzyme activities. Four species, Ceriporiopsis subvermispora, Physisporinus rivulosus, Phanerochaete chrysosporium and Pleurotus ostreatus were cultured in pairs to study the degradation of aspen wood and the production of lignin-degrading enzymes. Potential of co-culturing for biopulping was evaluated. Chemical analysis of decayed aspen wood blocks showed that co-culturing of C. subvermispora with P. ostreatus could significantly stimulate wood decay, when compared to monocultures. Based on the fungi tested here, however, this effect is species-specific. Other combinations of fungi were slightly stimulating or not stimulatory. The pattern of lignin degradation was altered towards the acid insoluble part of lignin especially in co-cultures where P. ostreatus was included as a partner. The use of agar plates containing the polymeric dye Poly R-478 showed elevated dye decolourization at the confrontation zone between mycelia. Laccase was significantly stimulated only in the co-culture of P. ostreatus with C. subvermispora. Manganese peroxidase activity was stimulated in co-cultures of P. ostreatus with C. subvermispora or with P. rivulosus. Immunoblotting indicated changes in lignin-degrading enzymes and/or their isoform composition in response to co-culturing. This is the first report on the effects of co-culturing of potential biopulping fungi on wood degradation, and gives basic knowledge on fungal interactions during wood decay that can be utilized in practical applications.     

Biopulping of wood chips with Phlebia brevispora BAFC 633 reduces lignin content and improves pulp quality

The white-rot fungus Phlebia brevispora BAFC 633 produces laccases in large proportions. In this work P. brevispora BAFC 633 was grown on Pinus taeda wood chips in 10-L bioreactors. To select the biopulping experimental conditions, we analyzed the variables affecting enzymatic laccase activity in the culture supernatants, indicating that the suitable incubation temperature was 30 °C in order to promote enzyme stability. Phlebia brevispora BAFC 633 secreted 744 U/g of laccase, selectively removing lignin during biotreatment of wood chips, causing a reduction in Kappa number and 10% weight loss, and creating a more open structure and better access to the pulping liquor, which would require less chemical consumption, thus diminishing the environmental impact of the chemical pulping process.These results support the biotechnological potential of P. brevispora BAFC 633 for biopulping processes and enhance the potential for bioprospecting native isolates of the microflora of our country's natural environment.     

Lignin degradation by white rot fungi on spruce wood shavings during short-time solid-state fermentations monitored by near infrared spectroscopy

Due to their outstanding capability of degrading the recalcitrant biomacromolecule lignin, white rot fungi have been attracting interest for several technological applications in mechanical pulping and wood surface modification. However, little is known about the time course of delignification in early stages of colonisation of wood by these fungi. Using a Fourier transform near infrared (FT-NIR) spectroscopic technique, lignin loss of sterilised spruce wood shavings (0.4–2.0 mm particle size) that had been degraded by various species of white rot fungi could be monitored already during the first 2 weeks. The delignification kinetics of Dichomitus squalens, three Phlebia species (Phlebia brevispora, Phlebia radiata and Phlebia tremellosa), three strains of Ceriporiopsis subvermispora as well as the white rot ascomycete Hypoxylon fragiforme and the basidiomycete Oxyporus latemarginatus were determined. Each of the fungi tested was able to reduce the lignin content of spruce wood significantly during the first week. The amount of delignification achieved by the selected white rot fungi after 2 weeks ranged from 7.2% for C. subvermispora (FPL 105.752) to 2.5% for P. radiata. Delignification was significant (P = 95%) already after 3 days treatment with C. subvermispora and P. tremellosa. Activities of extracellular ligninolytic enzymes (laccase, manganese peroxidase and/or lignin peroxidase), expressed by each of the tested fungi, were determined. Lignin was degraded when peroxidase activity was detected in the fungal cultures, but only a low level of correlation between enzyme activities and the extent of delignification was found.     

Linoleic acid peroxidation and lignin degradation by enzymes produced by Ceriporiopsis subvermispora grown on wood or in submerged liquid cultures

Ceriporiopsis subvermispora is a white-rot fungus used in biopulping processes and seems to use the fatty acid peroxidation reactions initiated by manganese-peroxidase (MnP) to start lignin degradation. The present work shows that C. subvermispora was able to peroxidize unsaturated fatty acids during wood biotreatment under biopulping conditions. In vitro assays showed that the extent of linoleic acid peroxidation was positively correlated with the level of MnP recovered from the biotreated wood chips. Milled wood was treated in vitro by partially purified MnP and linoleic acid. UV spectroscopy and size exclusion chromatography (SEC) showed that soluble compounds similar to lignin were released from the milled wood. SEC data showed a broad elution profile compatible with low molar mass lignin fractions. MnP-treated milled wood was analyzed by thioacidolysis. The yield of thioacidolysis monomers recovered from guaiacyl and syringyl units decreased by 33% and 20% in MnP-treated milled wood, respectively. This has suggested that lignin depolymerization reactions have occurred during the MnP/linoleic acid treatment.     

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.     

Improved Paper Pulp from Plants with Suppressed Cinnamoyl-CoA Reductase or Cinnamyl Alcohol Dehydrogenase

Transgenic plants severely suppressed in the activity of cinnamoyl-CoA reductase were produced by introduction of a partial sense CCR transgene into tobacco. Five transgenic lines with CCR activities ranging from 2 to 48% of wild-type values were selected for further study. Some lines showed a range of aberrant phenotypes including reduced growth, and all had changes to lignin structure making the polymer more susceptible to alkali extraction. The most severely CCR-suppressed line also had significantly decreased lignin content and an increased proportion of free phenolic groups in non-condensed lignin. These changes are likely to make the lignin easier to extract during chemical pulping. Direct Kraft pulping trials confirmed this. More lignin could be removed from the transgenic wood than from wild-type wood at the same alkali charge. A similar improvement in pulping efficiency was recently shown for poplar trees expressing an antisense cinnamyl alcohol dehydrogenase gene. Pulping experiments performed here on CAD-antisense tobacco plants produced near-identical results – the modified lignin was more easily removed during pulping without any adverse effects on the quality of the pulp or paper produced. These results suggest that pulping experiments performed in tobacco can be predictive of the results that will be obtained in trees such as poplar, extending the utility of the tobacco model. On the basis of our results on CCR manipulation in tobacco, we predict that CCR-suppressed trees may show pulping benefits. However, it is likely that CCR-suppression will not be the optimal target for genetic manipulation of pulping character due to the potential associated growth defects.     

Lignin degradation by white rot fungi on spruce wood shavings during short-time solid-state fermentations monitored by near infrared spectroscopy

Due to their outstanding capability of degrading the recalcitrant biomacromolecule lignin, white rot fungi have been attracting interest for several technological applications in mechanical pulping and wood surface modification. However, little is known about the time course of delignification in early stages of colonisation of wood by these fungi. Using a Fourier transform near infrared (FT-NIR) spectroscopic technique, lignin loss of sterilised spruce wood shavings (0.4–2.0 mm particle size) that had been degraded by various species of white rot fungi could be monitored already during the first 2 weeks. The delignification kinetics of Dichomitus squalens, three Phlebia species (Phlebia brevispora, Phlebia radiata and Phlebia tremellosa), three strains of Ceriporiopsis subvermispora as well as the white rot ascomycete Hypoxylon fragiforme and the basidiomycete Oxyporus latemarginatus were determined. Each of the fungi tested was able to reduce the lignin content of spruce wood significantly during the first week. The amount of delignification achieved by the selected white rot fungi after 2 weeks ranged from 7.2% for C. subvermispora (FPL 105.752) to 2.5% for P. radiata. Delignification was significant (P = 95%) already after 3 days treatment with C. subvermispora and P. tremellosa. Activities of extracellular ligninolytic enzymes (laccase, manganese peroxidase and/or lignin peroxidase), expressed by each of the tested fungi, were determined. Lignin was degraded when peroxidase activity was detected in the fungal cultures, but only a low level of correlation between enzyme activities and the extent of delignification was found.     

Biological treatments for contaminated soils: hydrocarbon contamination. Fungal applications in bioremediation treatment

Bioremediation is a spontaneous or controlled process in which biological, mainly microbiological, methods are used to degrade or transform contaminants to non or less toxic products, reducing the environmental pollution. The most important parameters to define a contaminated site are: biodegradability, contaminant distribution, lixiviation grade, chemical reactivity of the contaminants, soil type and properties, oxygen availability and occurrence of inhibitory substances. Biological treatments of organic contaminations are based on the degradative abilities of the microorganisms. Therefore the knowledge on the physiology and ecology of the biological species or consortia involved as well as the characteristics of the polluted sites are decisive factors to select an adequate biorremediation protocol. Basidiomycetes which cause white rot decay of wood are able to degrade lignin and a variety of environmentally persistent pollutants. Thus, white rot fungi and their enzymes are thought to be useful not only in some industrial process like biopulping and biobleaching but also in bioremediation. This paper provides a review of different aspects of bioremediation technologies and recent advances on ligninolytic metabolism research.     

Comparative studies of loblolly pine biodegradation and enzyme production by Argentinean white rot fungi focused on biopulping processes

The ability of eight white rot fungi: Coriolopsis rigida, Coriolus versicolor var. antarcticus, Peniophora sp., Phanerochaete sordida, Pycnoporus sanguineus, Steccherinum sp., Trametes elegans and Trametes villosa to selectively delignify loblolly pine (Pinus taeda) chips was studied. They were selected among 34 basidiomycetes from Argentina because of their capacity to decolorize Poly R-478 and Azure B. Fungal pretreatment caused changes in wood chemical composition as well as in physical structure. The present study allowed the identification of a new strain, potentially a candidate for use in softwoods biopulping processes. Results showed that P. sanguineus was able to reduce lignin content in 11% in 14 days of treatment, but also that P. taeda wood suffered notable structural changes of lignin and hemicelluloses during the treatment, as revealed from ¹³C CP-MAS NMR spectra. An increase of 15% in porosity of decayed wood confirmed physical changes due to fungal attack.     

Evaluation of the white-rot fungi Ganoderma australe and Ceriporiopsis subvermispora in biotechnological applications

Ganoderma australe is a white-rot fungus that causes a selective wood biodelignification in some hardwoods found in the Chilean rainforest. Ceriporiopsis subvermispora is also a lignin-degrading fungus used in several biopulping studies. The enzymatic system responsible for lignin degradation in wood can also be used to degrade recalcitrant organic pollutants in liquid effluents. In this work, two strains of G. australe and one strain of C. subvermipora were comparatively evaluated in the biodegradation of ABTS and the dye Poly R-478 in liquid medium, and in the pretreatment of Eucalyptus globulus wood chips for further kraft biopulping. Laccase was detected in liquid and wood cultures with G. australe. Ceriporiopsis subvermispora produce laccase and manganese peroxidase when grown in liquid medium and only manganese peroxidase was detected during wood decay. ABTS was totally depleted by all strains after 8 days of incubation while Poly R-478 was degraded up to 40% with G. australe strains and up to 62% by C. subvermispora after 22 days of incubation. Eucalyptus globulus wood chips decayed for 15 days presented 1–6% of lignin loss and less than 2% of glucan loss. Kraft pulps with kappa number 15 were produced from biotreated wood chips with 2% less active alkali, with up to 3% increase in pulp yield and up to 20% less hexenuronic acids than pulps from undecayed control. Results showed that G. australe strains evaluated were not as efficient as C. subvermispora for dye and wood biodegradation, but could be used as a feasible alternative in biotechnological processes such as bioremediation and biopulping.     

Studies of growth conditions in wood for three white-rot fungi and their cellulaseless mutants

White-rot fungi, which have the ability to degrade all the wood components including lignin, are of great interest in biotechnological processes based on wood and other lignocellulosic materials. It was demonstrated earlier that enough lignin can be degraded to cause a decrease in the energy demand for production of thermomechanical pulp if wood chips are pretreated by cellulaseless mutants of white-rot fungi. This paper concerns the growth conditions in wood for three white-rot fungi and their cellulaseless mutants in order to determine optimal conditions for such pretreatment processes. The pH and temperature optima have been determined as well as the growth rate in wood. The results show that the growth rate in wood. at least for Cel 44 (a cellulaseless mutant of Sporotrichum pulverulentum), is not the rate-limiting step in delignification. From different mixtures of urea and NH₄H₂PO₄ the optimal nitrogen source was determined for the mutants. The optimal C/N ratio was found to vary between 160/1 and 400/1. It is suggested that the lower the C/N ratio, the faster the growth. It was also demonstrated that both water- and acetone-extractable substances in wood supported the growth of cellulaseless mutants. When some glucose was added to the wood, the weight loss caused by Cel 44 increased. All these observations support earlier findings that lignin in wood cannot be degraded by white-rot fungi unless a more easily metabolizable carbon source is used simultaneously.     

Biodegradation of lignocellulosics: microbial, chemical, and enzymatic aspects of the fungal attack of lignin.

Wood is the main renewable material on Earth and is largely used as building material and in paper-pulp manufacturing. This review describes the composition of lignocellulosic materials, the different processes by which fungi are able to alter wood, including decay patterns caused by white, brown, and soft-rot fungi, and fungal staining of wood. The chemical, enzymatic, and molecular aspects of the fungal attack of lignin, which represents the key step in wood decay, are also discussed. Modern analytical techniques to investigate fungal degradation and modification of the lignin polymer are reviewed, as are the different oxidative enzymes (oxidoreductases) involved in lignin degradation. These include laccases, high redox potential ligninolytic peroxidases (lignin peroxidase, manganese peroxidase, and versatile peroxidase), and oxidases. Special emphasis is given to the reactions catalyzed, their synergistic action on lignin, and the structural bases for their unique catalytic properties. Broadening our knowledge of lignocellulose biodegradation processes should contribute to better control of wood-decaying fungi, as well as to the development of new biocatalysts of industrial interest based on these organisms and their enzymes.     

Assessment of selected decay Basidiomycetes for selective biodefibrillation of <Emphasis Type="Bold">Picea abies</Emphasis> wood

We studied the capacity of selected Basidiomycetes (72 species; 109 strains) to defibrillate Picea abies wood blocks, and determined the remaining cellular cohesion and the lignin content in the wood after treatment. Forty strains were sufficiently aggressive to invade non-sterile wood blocks in laboratory conditions; but only seven of them — Gloeophyllum trabeum, Gloeoporus taxicola, two strains of Phanerochaete velutina, Polyporus badius, Resinicium bicolor and Trametes versicolor produced an significant biodefibrillation. A combination of Gloeophyllum trabeum and Gloeoporus taxicola or Gloeophyllum trabeum and Resinicium bicolor created a synergetic effect and a nearly 70 % loss of the cellular cohesion. The use of selected rot fungi as pre-treatment to save wood pulping energy in several manufacturing processes is discussed.     

Selection of white-rot fungi for biopulping

Different rates of wood decay and ligninolytic activity were found in wood decayed by various white-rot fungi. Chemical and ultrastructural analyses showed wood decayed by Coriolus versicolor consisted of a nonselective attack on all cell wall components. Lignin degradation was restricted to the cell wall adjacent to hyphae or around the circumference of cell lumina. Decay by Phellinus pini, Phlebia tremellosus, Poria medullapanis and Scytinostroma galactinum was selective for lignin degradation. Secondary walls were void of lignin and middle lamellae were extensively degraded. A diffuse attack on lignin occurred throughout all cell wall layers. Variation in ligninolytic activity was found among strains of Phanerochaete chrysosporium. Differences in weight loss as well as lignin and polysaccharide degradation were also found when wood of different coniferous and deciduous tree species was decayed by various white-rot fungi.     

Comparison of methods to evaluate the potential of fungal growth on decay of spruce wood after short-time treatment

Several analytical methods were compared to evaluate characteristic wood decaying fungi for their potential to depolymerise lignin on spruce wood particles. Wood samples were treated with the white rot fungi Phlebia brevispora, Ceriporiopsis subvermispora, Merulius tremellosus, Pycnoporus sanguineus, Trametes pubescens and with the brown rot fungus Gloeophyllum trabeum. The UV absorbancies of crude ethanol extracts, total extractives content from sequential extraction, ligninolytic enzyme activities, lignin solubilisation and decrease of lignin content were compared. It was shown, that, in early decay stages, UV absorbancies of crude ethanol extracts and total extractives content correlate well with lignin degradation, increase of acid soluble lignin and increased production of ligninolytic enzymes (total peroxidase). Lignin content was determined using FT-NIR spectroscopy as well as by wet-chemical analysis, indicating a very good correlation between the two methods. According to the different analytical methods, the tested fungi can be classified into three categories based on their characteristic behaviour: brown rot, “slow” and “fast” white rot.     

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.