Family-10 and Family-11 xylanases differ in their capacity to enhance the bleachability of hardwood and softwood paper pulps

Enzyme-aided bleaching of softwood and hardwood kraft pulps by glycosyl hydrolase family-10 and -11 xylanases and a family-26 mannanase was investigated. The ability to release reducing sugar from pulp xylan and to enhance bleachability is not a characteristic shared by all xylanases. Of the six enzymes tested, two xylanases belonging to family 11 were most effective at increasing bleachability and improving final paper brightness. None of the enzymes had a deleterious effect on pulp fibre integrity. The efficiency of individual xylanases as bleach enhancers was not dependent on the source microorganism, and could not be predicted solely on the basis of the quantity or nature of products released from pulp xylan. Cooperative interactions between xylanase/xylanase and xylanase/mannanase combinations, during the pretreatment of softwood and hardwood pulps, were investigated. Synergistic effects on reducing-sugar release and kappa number reduction were elicited by a combination of two family-10 xylanases. Pretreatment of kraft pulp with mannanase A from Pseudomonas fluorescens subsp. cellulosa and any one of a number of xylanases resulted in increased release of reducing sugar and a larger reduction in kappa number than obtained with the xylanases alone, confirming the beneficial effects of family-26 mannanases on enzyme-aided bleaching of paper pulp. Received: 6 January 1997 / Received revision: 10 April 1997 / Accepted: 19 April 1997     

Lignin-hemicellulose complexes restrict enzymatic solubilization of mannan and xylan from dissolving pulp

Mannan and xylan present in bleached softwood dissolving pulp were found to be partially resistant to hemicellulases even after repeated enzyme treatment. Despite the additional effect of an endoglucanase from Gloeophyllum sepiarium, which increased the␣accessibility of mannan and xylan to a mannanase from Sclerotium rolfsii and to a xylanase from Thermomyces lanuginosus, the enzyme mixture solubilized only half of the hemicellulose present in the pulp. Half of the remaining hemicellulose present in the pulp appeared to be entrapped within the cellulose matrix while the other half was associated with lignin-carbohydrate complexes. The latter hemicellulose portion was isolated and characterized. Chromatography and spectroscopic techniques revealed the presence of two types of lignin-carbohydrate complex, a galactoglucomannan-lignin complex (degree of polymerization DP 50–60) and a xylan-lignin complex (DP >200). Received: 8 December 1997 / Received revision: 30 April 1998 / Accepted: 8 May 1998     

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.     

Investigation of lignin-carbohydrate complexes in kraft pulps by selective enzymatic treatments

The occurrence of covalent bonds between residual lignin and polysaccharides in birch and pine kraft pulps was investigated by specific enzymatic treatments. Pure enzymes degrading cellulose, xylan and mannan were used both separately and in combination. Comparison of the molar masses of polysaccharides and lignin in the orginal pulps and in the residual pulps after enzymatic treatments showed that residual lignin in birch kraft pulp is linked at least to xylan. A minor portion may also be linked to cellulose. In pine kraft pulp some of the residual lignin appears to be linked to cellulose, glucomannan and xylan. The linkages between lignin and cellulose and hemicelluloses may be either native or formed during pulp processing. The results also provided new information on the synergistic action of cellulose- and hemicellulose-degrading enzymes on pulp fibres. The synergism appears to be mainly due to the structure of the pulp fibres, with different layers of cellulose sheets, hemicelluloses and lignin. On the other hand the results also provided information about fibre structure. The degradation of xylan clearly enhanced the action of enzymes on cellulose, suggesting that xylan partially covers the cellulose. A similar phenomenon was not observed in the simultaneous hydrolysis of glucomannan and cellulose. However, the results suggest that glucomannan does interact with cellulose, possibly by non-covalent linkages. Received: 8 July 1998 / Received revision: 7 October 1998 / Accepted: 11 October 1998     

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.     

On-line enzyme activity determination using the stopped-flow technique: application to laccase activity in pulp mill waste-water treatment

An automated system for on-line measurement of enzyme activity is proposed. The system uses a flow injection manifold in the stopped-flow mode to measure initial reaction rates. The time during which the flow is halted is selected in such a way as to optimise the enzyme/substrate ratio for the correct determination of activity values. The proposed system was used to determine the activity of laccase produced by the fungus Trametes versicolor immobilised on nylon in a fixed-bed reactor used for treating pulp mill waste water. Received: 17 February 1997 / Received revision: 23 April 1997 / Accepted: 27 April 1997     

A bioassay for methylated galacturonan on pulp-fiber surfaces

A colorimetric bioassay has been developed enabling separation of wood pulp qualities based on the quantity of methyl-esterified galacturonan exposed on fiber surfaces. Thermomechanical pulp (TMP) and chemi-TMP (CTMP) had, respectively, 3.1 and 0.7 g galacturonan on the surface per mg wood pulp. The presence of galacturonan on the surface of TMP and CTMP was further confirmed by immunogold localization.     

A comparison of enzyme-aided bleaching of softwood paper pulp using combinations of xylanase, mannanase and α-galactosidase

Enzymatic pretreatment of softwood kraft pulp was investigated using xylanase A (XylA) from Neocallimastix patriciarum in combination with mannanase and α-galactosidase. Mannanase A (ManA) from Pseudomonas fluorescens subsp. cellulosa and ManA from Clostridium thermocellum, both family 26 glycosyl hydrolases, are structurally diverse and exhibit different pH and temperature optima. Although neither mannanase was effective in pretreating softwood pulp alone, both enzymes were able to enhance the production of reducing sugar and the reduction of single-stage bleached κ number when used with the xylanase. Sequential incubations with XylA and P. fluorescens ManA produced the largest final κ number reduction in comparison to control pretreated pulp. The release of galactose from softwood pulp by α-galactosidase A (AgaA) from P. fluorescens was enhanced by the presence of ManA from the same microorganism, and a single pretreatment with these enzymes, in combination with XylA, gave the most effective κ number reduction using a single incubation. Results indicated that mixtures of hemicellulase activities can be chosen to enhance pulp bleachability. Received: 16 August 1999 / Received revision: 13 December 1999 / Accepted: 15 December 1999     

Removal of organic pollutants and of nitrate from wastewater from the dairy industry by denitrification

The aim of this work was to remove nitrate-N and organic pollutants from wastewater of the dairy industry by denitrification. An artificially prepared wastewater, containing 250 mg/l nitrate-N and 1.5 g/l whey powder, was completely denitrified with removal of 90%–93% of the chemical oxygen demand (COD) of the whey powder by suspended or immobilized mixed cultures and by a suspended or immobilized pure culture that was isolated from the mixed culture inoculum. For the above COD/nitrate-N ratio of 6:1, the results indicated that the organic compounds of the wastewater served as electron donors for complete denitrification and that there was no need to add an external carbon source. In batch denitrification assays the suspended or immobilized mixed cultures proved to be more active and reacted faster than the isolated pure cultures. In continuous denitrification processes with immobilized pure or mixed cultures, the alginate beads, used for immobilization, were not stable for more than 12 days of incubation. The mixed free cultures removed the nitrate-N and COD continuously with no change of their activity for at least 15 days at an optimum hydraulic retention time of 0.27 days with a loading rate of 900 mg nitrate-N l⁻¹ day⁻¹. Received: 13 October 1997 /  Received revision: 16 December 1997 / Accepted: 19 December 1997     

Effects of directly soluble and fibrous rapidly acidifying chemical oxygen demand and reactor liquid surface tension on granulation and sludge-bed stability in upflow anaerobic sludge-blanket reactors

 In recent years, it has become clear that the rapidly acidifying chemical oxygen demand (RACOD) content of the waste water and the surface tension of the reactor liquid contribute to the phenomenon of granular growth in upflow anaerobic sludge-blanket reactors (UASB). By adding 20% of directly soluble RACOD, in the form of a sucrose/starch mixture, on top of the original COD load and by adjusting the reactor liquid surface tension below 50 mN m⁻¹ with linear alkylbenzenesulphonate, granular growth and sludge-bed stability could be enhanced significantly within 40 days. Carrot pulp, a waste product having a high short-chain fatty acid precursor potential, was applied as an alternative fibrous RACOD source. Best results were obtained when adding the carrot pulp freshly to the laboratory-scale UASB reactor in an in-recycle liquefying chamber. This concept of adding carrot pulp waste product as a granular growth supplement by means of an in-recycle liquefying chamber therefore merits testing in practice. Received: 30 October 1996 / Received version: 3 February 1997 / Accepted: 10 February 1997     

Nitrification, denitrification, and dechlorination in bleached kraft pulp mill wastewater

This study deals with combining the biologi cal removal of organic halogens with the removal of nitrogen from bleached kraft pulp mill wastewater in fluidized-bed reactors under nitrifying and denitrifying conditions. Untreated and biotreated bleached kraft pulp mill wastewaters had no detrimental effect on nitrification or denitrification. The nitrifying biofilm reactor, pregrown on synthetic inorganic feed with ammonia, removed without a lag phase adsorbable organic halogens [7.2 mg Cl (g biomass volatile solids)⁻¹day⁻¹] from bleached kraft pulp mill wastewater and selected chlorophenols from synthetic wastewater. Electron microscopical examination of the biofilm showed that bacteria, morphologically similar to the nitrifying species Nitrosomonas or Nitrobacter, and Nitrosospira were dominant. The denitrifying fluidized-bed reactor, pregrown on nitrate and methanol, denitrified without a lag phase bleached kraft pulp mill wastewater. Under denitrifying conditions, 35% of the total organic carbon content of untreated bleached kraft pulp mill waste water was removed. The reducing power delivered by untreated bleached kraft pulp mill wastewater for denitrification was 2 mmol electrons/mmol carbon mineralized. Dechlorination under denitrifying conditions was negligible. Received: 21 November 1996 / Received revision: 27 January 1997 / Accepted: 1 February 1997     

Production of 1,3-propanediol by Clostridium butyricum in continuous culture with cell recycling

The continuous fermentation of 1,3-propanediol from glycerol by Clostridium butyricum was subjected to cell recycling by filtration using hollow-fibre modules made from polysulphone. The performance of the culture system was checked at a retention ratio (dilution rate/bleed rate) of 5, dilution rates between 0.2 h⁻¹ and 1.0 h⁻¹ and glycerol input concentrations of 32 g l⁻¹ and 56 g l⁻¹. The near-to-optimum propanediol concentration of 26.5 g l⁻¹ (for 56 g l⁻¹ glycerol) was maintained up to a dilution rate of 0.5 h⁻¹ and then decreased while the propanediol productivity was highest at 0.7 h⁻¹. The productivity could be increased by a factor of four in comparison to the continuous culture without cell recycling. By application of the model of Zeng and Deckwer [(1995) Biotechnol Prog 11: 71–79] for cultures under substrate excess, it was shown that the limitations resulted exclusively from product inhibition and detrimental influences from the cell recycling system, such as shear stress, were not involved. Received: 20 October 1997 / Received revision: 12 December 1997 / Accepted: 14 December 1997     

Pullulan content of the ethanol precipitate from fermented agro-industrial wastes

Ethanol-precipitated substances after fermentation of various agro-industrial wastes by Aureobasidium pullulans were examined for their pullulan content. Grape skin pulp extract, starch waste, olive oil waste effluents and molasses served as substrates for the fermentation. A glucose-based defined medium was used for comparison purposes. Samples were analysed by an enzyme-coupled assay method and by high-performance anion-exchange chromatography with pulsed amperometric detection after enzymic hydrolysis with pullulanase. Fermentation of grape skin pulp extract gave 22.3 g l⁻¹ ethanol precipitate, which was relatively pure pullulan (97.4% w/w) as assessed by the coupled-enzyme assay. Hydrolysed starch gave only 12.9 g l⁻¹ ethanol precipitate, which increased to 30.8 g l⁻¹ when the medium was supplemented with NH₄NO₃ and K₂HPO₄; this again was relatively pure pullulan (88.6% w/w). Molasses and olive oil wastes produced heterogeneous ethanol-precipitated substances containing small amounts of pullulan, even when supplemented with nitrogen and phosphate. Overall, grape skin pulp should be considered as the best substrate for pullulan production. Starch waste requires several hydrolyis steps to provide a usable carbon source, which reduces its economic attraction as an industrial process. Received: 24 October 1997 / Received revision: 10 February 1998 / Accepted: 15 February 1998     

Identification and quantification of radical reaction intermediates by electron spin resonance spectrometry of laccase-catalyzed oxidation of wood fibers from beech (Fagus sylvatica)

During laccase-catalyzed oxidation of beech wood fibers in an aqueous suspension, phenoxy radicals were detected in steady-state concentrations by electron-spin resonance (ESR) spectrometry of the suspension liquid, suggesting that colloidal lignin functions as a mediator between laccase and the fiber lignin matrix. Phenoxy radicals were observed directly, whereas ESR spin-trapping techniques gave no evidence for reduced oxygen species, such as the superoxide or hydroxyl radical. A reaction mechanism involving parallel direct oxidation of the lignin on fiber surfaces and a phenol/phenoxy cyclic mediator process in the suspension liquid could accordingly describe laccase-catalyzed oxidation of beech wood fibers. Cytochrome c assays for detection of superoxide in systems involving lignin oxidized by oxidoreductases should be used with caution, as cytochrome c may be reduced by species other than superoxide. Received: 24 March 1997 / Received revision: 27 May 1997 / Accepted: 1 June 1997     

Mode of depolymerisation of hemicellulose by various mannanases and xylanases in relation to their ability to bleach softwood pulp

Endo-mannanases and endo-xylanases cleave different heteromannans and xylans yielding mainly dimers and trimers of the corresponding sugars as end-products. However, in the early stages of hydrolysis, four purified mannanases and four xylanases from fungal and bacterial origin, examined in this study, showed a different pattern of released oligomers (determined up to the pentamers). Furthermore, some of these enzymes showed a preference for cleaving the polysaccharides in the middle of the chain while others acted more at the end. When the increase in the specific fluidity of mannan and xylan solutions per reducing sugar released (K _v) was measured against the bleaching effect of the enzymes on softwood kraft pulp, a correlation was found. A xylanase from Penicillium simplicissimum (K _v = 0.15 l mPa⁻¹s⁻¹g⁻¹) and a mannanase from Sclerotium rolfsii (K _v = 0.12 l mPa⁻¹s⁻¹g⁻¹) applied in a O(QX)P bleaching sequence (O = oxygen delignification, X = treatment with hemicellulolytic enzymes, Q = chelation of metals, P = treatment with hydrogen peroxide in alkaline solution) gave a high brightness increase of 3.0% and 1.9% ISO respectively. A less significant brightness increase was obtained with enzymes showing lower K _v values, such as a xylanase from Schizophyllum commune (K_v = 0.051  l mPa⁻¹s⁻¹g⁻¹, 0.2% ISO) and a bacterial mannanase (K _v = 0.061 l mPa⁻¹s⁻¹g⁻¹,0.5% ISO). Received: 19 December 1996 / Received revision: 20 February 1997 / Accepted: 22 February 1997     

Microbial oxidative metabolism of diclofenac: production of 4′-hydroxydiclofenac using Epiccocum nigrum IMI354292

The 4′-hydroxylated metabolite of diclofenac was produced by biocatalysis for probing specific human drug-metabolising enzymes (CYP2C9). An initial screen of 11 microorganisms was carried out (50 ml scale) to identify the organism best suited to the regioselective conversion of diclofenac to its 4′-hydroxylated metabolite. From this screen, the fungus Epicoccum nigrum IMI354292 was selected as the most suitable microorganism. Scale-up was carried out in a 30-l fermenter to which 2 g diclofenac was added. After 48 h, 50% of the diclofenac had been converted to it 4′-hydroxylated metabolite. The broth was then extracted with ethyl acetate and purified by chromatography and crystallisation. This yielded 0.3 g 4′-hydroxydiclofenac with a purity of at least 99%. The 4′-hydroxydiclofenac produced by E. nigrum was characterised by HPLC, mass spectrometry and NMR. Received: 28 July 1997 / Received revision: 8 December 1997 / Accepted: 14 December 1997     

Substrate selectivity of various lipases in the esterification of cis- and trans-9-octadecenoic acid

The substrate selectivity of numerous commercially available lipases from microorganisms, plants and animal tissue towards 9-octadecenoic acids with respect to the cis/trans configuration of the CC double bond was examined by the esterification of cis- and trans-9-octadecanoic acid (oleic and elaidic acid respectively) with n-butanol in n-hexane. A great number of lipases studied, e.g. those from Pseudomonas sp., porcine pancreas or Carica papaya, were unable to discriminate between the isomeric 9-octadecenoic acids. However, lipases from Candida cylindracea and Mucor miehei catalysed the esterification of oleic acid 3–4 times faster than the corresponding reaction of elaidic acid and therefore have a high preference for the cis isomer. Of all biocatalysts examined, only recombinant lipases from Candidaantarctica favoured elaidic acid as substrate. While the preference of Candida antarctica lipase B for the trans isomer was quite low, Candida antarctica lipase A had an extraordinary substrate selectivity and its immobilized enzyme preparation [Chirazyme L-5 (3) from Boehringer] esterified elaidic acid about 15 times faster than oleic acid. Received: 29 October 1998 / Received revision: 18 December 1998 / Accepted: 21 December 1998     

Potato pulp: microbiological characterization, physical modification, and application of this agricultural waste product

Potato pulp, one of the agricultural waste products obtained in high quantities during starch production, contains starch, cellulose, hemicelluloses, pectin, proteins, free amino acids and salts. It exhibits physical and physicochemical properties of a typical colloid. It is mainly used, in a dried and pelleted form, as cattle feed. Its autochthonic microbial flora (bacteria, fungi) was identified and studied with a view towards the degradative potential of the microorganisms and ways of conserving the pulp for subsequent technical applications; 33 isolates (28 bacteria, 4 fungi, 1 yeast), belonging to 15 genera were characterized. Biological conservation was possible at very low oxygen pressure, brought about by the autochthonic anaerobic microorganisms causing acidification. Chemical conservation was achieved with sorbic aicd. By treatment with hot water vapour under pressure (autoclaving), followed by a pressure release procedure, intact cells in the pulp (both potato cells and microorganisms, not spores) were destroyed, and their contents and wall fragments were set free. This process resulted in low drying costs and was a prerequisite for the production of a powder that can be used as glue or as animal feed. Received: 18 April 1997 / Received revision: 12 June 1997 / Accepted: 14 June 1997     

Isolation and characterization of a marine bacterium capable of utilizing 2-methylphenanthrene

A marine bacterium isolated from a coastal hydrocarbon-polluted sediment has been described and attributed on the basis of its phenotypic and genotypic characteristics to the genus Sphingomonas sp. This strain was capable of using an alkylated phenanthrene 2-methylphenanthrene, as sole source of carbon and energy. In experiments, 2-methylphenanthrene (0.2 g/l) was added as crystals to the culture medium. After 5 days of aerobic growth at 30 °C, 70% was degraded and the complete dissipation occurred after 20 days. Furthermore, the strain could degrade various kinds of polyaromatic compounds, but failed to grow on aliphatic hydrocarbons. Received: 27 December 1996 / Received last revision: 10 June 1997 / Accepted: 14 June 1997     

Factors affecting the activation of pulps with laccase

Activation of fibres by radical formation is the first step when aiming at oxidative coupling of new functional groups on the fibre bound lignin. In this work, factors affecting the amount of phenoxy radicals created to unbleached TMP, CTMP, softwood kraft and hardwood kraft pulp fibres in the laccase catalysed oxidation were determined by EPR. Laccase was able to catalyse the oxidation of all the pulps studied. The reactivity of the pulp was found to be affected by both the physical accessibility of lignin in the fibres and the chemistry of the surface lignin accessible to laccase. Laccase dosage, use of extra oxygen in the laccase catalysed radicalization reaction, treatment time and also the amount and type of low-molecular weight compounds (LMWC) present in the pulp were all found to contribute to the radical content of pulp fibres measured after the enzymatic reaction. It could not been excluded that two types of reactions take place during the radical formation in fibres. Within the fibre matrix there may be both fibre material bound and soluble lignin fragments differing with respect to accessibility, molecular weight or chemical structure which can be radicalized at various rates, and the formed radicals may also undergo cross-coupling reactions reducing the amount of the total radicals.