Xylanases of marine fungi of potential use for biobleaching of paper pulp

Microbial xylanases that are thermostable, active at alkaline pH and cellulase-free are generally preferred for biobleaching of paper pulp. We screened obligate and facultative marine fungi for xylanase activity with these desirable traits. Several fungal isolates obtained from marine habitats showed alkaline xylanase activity. The crude enzyme from NIOCC isolate 3 (Aspergillus niger), with high xylanase activity, cellulase-free and unique properties containing 580 U l^–1 xylanase, could bring about bleaching of sugarcane bagasse pulp by a 60 min treatment at 55°C, resulting in a decrease of ten kappa numbers and a 30% reduction in consumption of chlorine during bleaching. The culture filtrate showed peaks of xylanase activity at pH 3.5 and pH 8.5. When assayed at pH 3.5, optimum activity was detected at 50°C, with a second peak of activity at 90°C. When assayed at pH 8.5, optimum activity was seen at 80°C. The crude enzyme was thermostable at 55°C for at least 4 h and retained about 60% activity. Gel filtration of the 50–80% ammonium sulphate-precipitated fraction of the crude culture filtrate separated into two peaks of xylanase with specific activities of 393 and 2,457 U (mg protein)^–1. The two peaks showing xylanase activity had molecular masses of 13 and 18 kDa. Zymogram analysis of xylanase of crude culture filtrate as well as the 50–80% ammonium sulphate-precipitated fraction showed two distinct xylanase activity bands on native PAGE. The crude culture filtrate also showed moderate activities of -xylosidase and -l-arabinofuranosidase, which could act synergistically with xylanase in attacking xylan. This is the first report showing the potential application of crude culture filtrate of a marine fungal isolate possessing thermostable, cellulase-free alkaline xylanase activity in biobleaching of paper pulp.     

Alkaline-active xylanase produced by an alkaliphilicBacillus sp isolated from kraft pulp

ABacillus sp (V1-4) was isolated from hardwood kraft pulp. It was capable of growing in diluted kraft black liquor at pH 11.5 and produced 49 IU (mol xylose min^–1 ml^–1) of xylanase when cultivated in alkaline medium at pH 9. Maximal enzyme activity was obtained by cultivation in a defined alkaline medium with 2% birchwood xylan and 1% corn steep liquor at pH 9, but high enzyme production was also obtained on wheat bran. The apparent pH optimum of the enzyme varied with the pH used for cultivation and the buffer system employed for enzyme assay. With cultivation at pH 10 and assays performed in glycine buffer, maximal activity was observed at pH 8.5; with phosphate buffer, maximal activity was between pH 6 and 7. The xylanase temperature optimum (at pH 7.0) was 55°C. In the absence of substrate, at pH 9.0, the enzyme was stable at 50°C for at least 30 min. Elecrophoretic analysis of the crude preparation showed one predominant xylanase with an alkaline pl. Biobleaching studies showed that the enzyme would brighten both hardwood and softwood kraft pulp and release chromophores at pH 7 and 9. Because kraft pulps are alkaline, this enzyme could be used for prebleaching with minimal pH adjustment.     

Purification and characterization of a cellulase-free xylanase of a moderate thermophile Bacillus licheniformis A99

Two cellulase-free xylanases were secreted by a thermophile, Bacillus licheniformis A99. Of the two, the predominant one was purified to homogeneity. The enzyme was optimally active at 60 °C, pH 6–7.5, and had a molecular weight of about 45 KDa and isoelectric point of 7.0 ± 0.2. The K _m (for birchwood xylan) and V _max were 3.33 mg/ml and 1.111 mmols mg^–1 protein min^–1 respectively. The half-life of the enzyme was 5 h at 60 °C. All cations except Hg²⁺ and Ag⁺ as well as EDTA were well tolerated and did not adversely affect xylanase activity. However, SDS inhibited the enzyme activity. The release of reducing sugars from unbleached commercial pulp sample on treatment with the enzyme indicated its potential in prebleaching of paper pulp. The enzyme caused saccharification of lignocellulosics such as wheat bran, wheat straw and sawdust. This is the first report on purification and characterization of cellulase-free xylanase from a moderate thermophile Bacillus licheniformis.     

Purification and characterization of a thermoalkalophilic xylanase from <Emphasis Type="Italic">Bacillus</Emphasis> sp.

Summary An extracellular xylanase was purified to homogeneity from the culture filtrate of a thermophilic Bacillus sp. The molecular weight of the purified xylanase was 44 kDa, as analysed by SDS/PAGE. The enzyme reaction followed Michaelis–Menten kinetics with K_m^app and V_max values of 0.025 mg/ml and 450 U/mg protein, respectively, as obtained from a Lineweaver–Burk plot. The xylanase contained no other enzyme activity except for the hydrolysis of xylan substrate. The optimal temperature of the enzyme assay was 50 °C. The optimum pH for the xylanase activity was at three peaks 6.5, 8.5 and 10.5, respectively and the enzyme was stable over a broad range of pH from pH 6 to 10.5. Metal ions tested with demetalized enzyme had no effect, with the exception of Hg²⁺ and Pb²⁺ (both strong inhibitors). Inhibition of the enzyme activity by N-bromosuccinimide (amino acid modifier) indicated the role of tryptophan residues in the catalytic function of the enzyme. Due to these outstanding properties, the xylanase of Bacillussp. finds potential applications in biopulping, biobleaching and de-inking of recycled paper and other industrial processes.     

Improved xylanase production from a haloalkalophilic Staphylococcus sp. SG-13 using inexpensive agricultural residues

The production of an alkali-stable xylanase, with dual pH optima, from haloalkalophilic Staphylococcus sp. SG-13 has been enhanced using agro-residues in submerged fermentation and a biphasic growth system. The agro-residues such as wheat bran, sugarcane bagasse, corncobs and poplar wood when used as sole carbon source, improved the xylanase yield by five-fold as compared to xylose and xylan. Staphylococcus sp. SG-13 also produced equally good amounts of xylanase when grown simply in deionized water (pH 8.0) supplemented with agro-residues as sole carbon source. In the biphasic growth system (lower layer containing agricultural residue set in agar medium with liquid medium above it), the prime substrate, wheat bran (1% w/v), resulted in maximum xylanase production of 4525 U l^–1 (pH 7.5) and 4540 U l^–1 (pH 9.2) at an agar: broth ratio of 4.0 after 48 h of incubation at 37 °C under static conditions. In general, the cost-effective agro-residues were found to be more suitable inducers for xylanase production over expensive substrates like xylan.     

Organic acids and water-soluble phenolics produced by Paxillus sp. 60/92 together show antifungal activity against Pythium vexans under acidic culture conditions

Ectomycorrhizal fungi can produce antifungal compounds in vitro as well as in symbiosis with the host plant that can reduce root diseases. The objective of this study was to isolate antifungal compounds from culture filtrate of Paxillus sp. 60/92, which can form mycorrhizas with Picea glehnii seedlings. Culture filtrate of Paxillus sp. 60/92 showed antifungal activity against Pythium vexans at pH 3–4 but not at pH 5–10, although sterile MMN-b liquid medium (pH 3–10) did not show antifungal activity. Upon separation of antifungal compounds in the culture filtrate, antifungal activity was detected in the organic acid and water-soluble phenolics fractions adjusted to pH 3. Although antifungal activity of individual fractions was lower than that of the culture filtrate, a mixture of these fractions showed antifungal activity similar to that of the culture filtrate. Furthermore, antifungal activity of oxalic acid, which is known to be produced by Paxillus involutus, was increased by mixing with the water-soluble phenolic fraction. Our findings indicate that Paxillus sp. 60/92 produces organic acids and water-soluble phenolics that together show antifungal activity at pH 3–4 against P. vexans.     

Biochemical and catalytic properties of endo-1,4-β-xylanases from Thermomyces lanuginosus (wild and mutant strains)

Endoxylanases from the thermophilic fungus, Thermomyces lanuginosus ATCC 44008 (cellulase free wild and mutant strains), were purified to homogeneity by anion-exchange and molecular-sieve chromatographic methods. The purified enzymes were monomers with molecular masses of 22 kDa (wild type) and 24 kDa (mutant), estimated by SDS-PAGE and gel filtration. As glycoproteins, the purified enzymes had 0.74% (wild type) and 11.8% (mutant) carbohydrate contents, and pI values of 5.8 and 6, respectively. The optimal pH and temperature values of wild type xylanase were determined to be pH 7 and 60 °C, whereas pH 6.7 and 70 °C, were optimal for the purified mutant enzyme (K _m and V _max values of 3.7 mg ml^–1 and 670 mol min^–1 xylose compared to the kinetic values of the purified wild type xylanase –5.1 mg ml^–1 and 385 mol min^–1 xylose). Inhibition studies suggested the possible involvement of histidine, tryptophan residues and carboxylic groups in the binding or catalysis.     

Production of alkali-tolerant cellulase-free xylanase by <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. WLUN024 with wheat bran as the main substrate

An alkali-tolerant cellulase-free xylanase producer, WLI-11, was screened from soil samples collected from a pulp and paper mill in China. It was subsequently identified as a Pseudomonas sp. A mutant, WLUN024, was selected by consecutive mutagenesis by u.v. irradiation and NTG treatment using Pseudomonas sp. WLI-11 as parent strain. Pseudomonas sp. WLUN024 produced xylanase when grown on xylosidic materials, such as hemicellulose, xylan, xylose, and wheat bran. Effects of various nutritional factors on xylanase production by Pseudomonas sp. WLUN024 with wheat bran as the main substrate were investigated. A batch culture of Pseudomonas sp. WLUN024 was conducted under suitable fermentation conditions, where the maximum activity of xylanase reached 1245 U ml⁻¹ after incubating at 37 °C for 24 h. Xylanase produced by Pseudomonas sp. WLUN024 was purified and the molecular weight was estimated as 25.4 kDa. Primary studies on the characteristics of the purified xylanase revealed that this xylanase was alkali-tolerant (optimum pH 7.2–8.0) and cellulase-free. In addition, the xylanase was also capable of producing high quality xylo-oligosaccharides, which indicated its application potential in not only pulp bio-bleaching processes but also in the nutraceutical industry.     

Extracellular ligninolytic enzyme production by Phanerochaete chrysosporium in a new solid-state bioreactor

The production of manganese-dependent peroxidase (MnP) and lignin peroxidase (LiP) by the fungus Phanerochaete chrysosporium (ATCC 24725) in a new bioreactor, the Immersion Bioreactor, which grows cells under solid-state conditions, was studied. Maximum MnP and LiP activities were 987 U l^–1 and 356 U l^–1, respectively. The polymeric dye, Poly R-478, was degraded at 2.4 mg l^–1 min^–1 using the extracellular culture filtrate.     

Production of a thermostable alkali-tolerant xylanase from Bacillus circulans AB 16 grown on wheat straw

Bacillus circulans AB 16 was able to produce 50 IU/ml of xylanase, with negligible cellulase activity when grown on untreated wheat straw. The pH optimum of the crude enzyme was 6–7 with a temperature optimum of 80 C. The enzyme showed high pH and thermal stability retaining 100% activity at 60 C, pH 8 and 9 after 2.5 h of incubation. The residual activity at 70 C after 2.5 h was 62% and 45% at pH 8 and 9, respectively. At 75 C only 22.2% activity remained at pH 8 after 1 h incubation. Since Kraft pulp is alkaline this enzyme could be used for prebleaching of pulp at temperatures up to 70 C without pH adjustment.     

Net nitrogen mineralization in soils under six indigenous tree species,an abandoned pasture and a secondary forest in the atlantic lowlands of costa rica

Nitrogen mineralization, nitrification potentials, pH, total N, C, extractable P and cations were measured in soils under 4-year-old, mono-specific stands of six fast-growing, native tree species, an abandoned pasture, and a 20-year-old secondary forest, as part of a study on the use of indigenous tree species for rehabilitation of soil fertility on degraded pastures at the La Selva Biological Station in the Atlantic humid lowlands of Costa Rica. Soil net nitrification potential rates were higher under two N-fixing, leguminous species,Stryphnodendron microstachyum Poepp. et Endl. (1.1–1.9 mg kg^–1 day^–1) andDalbergia tucurensis Donn. Smith (0.7–1.5 mg kg^–1 day^–1), than under the non-N-fixing trees in the plantation,Vochysia guatemalesis Don. Sm.,Vochysia ferruginea Mart,Dipteryx panamensis (Pittier) Record and Mell andHyeronima alchorneoides Fr. Allemao (0.2–0.8 mg kg^–1 day^–1). Values under the N-fixing trees were comparable to those found in secondary forest. There were no statistically significant differences in soil total N or in other nurtients between the species. Results of pH measurements done before and after incubation did not show any clear evidence of a pH drop attributable to nitrification.     

Production and partial characterization of xylanase by Sporotrichum thermophile under solid-state fermentation

A number of factors affecting production of xylanase, by the thermophilic fungus Sporotrichum thermophile under solid state fermentation (SSF) were investigated. Initial moisture content and type of carbon source were consecutively optimized. Solid state fermentation in a laboratory horizontal bioreactor using the optimized medium allowed the production of 320 U g^–1 of carbon source which compared favourably with those reported for other microorganisms. Optimal xylanase activity was observed at pH 5 and 70 °C. Chromogenic (fluorogenic) 4-methylumbelliferyl -glycoside of xylobiose (MUX₂) was used to characterize the xylanase multienzyme component, after separation by isoelectric focusing and native PAGE electrophoresis. The zymograms indicated one major xylanase fraction exhibiting pI and molecular mass values 4 and 90–120 kDa, respectively.     

Xylanase-induced reduction of chlorine dioxide consumption during elemental chlorine-free bleaching of different pulp types

The potential of crude xylanase from Thermomyces lanuginosus and Xylanase P (a commercial xylanase) was evaluated in bleaching of various paper pulp types. Xylanases released chromophores and reducing sugars and decreased kappa number of pulps. Chlorine-bleached, alkali-extracted bagasse and post-oxygen kraft pulps, pretreated with enzymes, gained over 5 brightness points over controls. Biobleaching of soda-aq pulp with Xylanase P produced chlorine dioxide savings of up to 30% or 4.5 kg chlorine dioxide t^–1 pulp.     

Effects of acidity on the growth of two Euglena species

Our study separates the effects of elevated protons (at pH <3) and elevated metals (Al, Cd, Cu, Fe, Ni, Zn) on the growth of E. mutabilis Schmitz, a pioneering phototroph in acid mine drainage (AMD) and E. gracilis Klebs, a closely-related species rarely found in severely AMD-impacted sites. Both species were acid tolerant, growing optimally at pH 2.5–7. At pH values typical of AMD (pH 2.5–4) in the absence of elevated metals, E. gracilis outcompeted E. mutabilis (growth rates of 1.0 and 0.8 div d^–1, respectively). Relative metal toxicities were evaluated based on the Effective Exposure causing 50% growth reduction (= EE50). With total metal additions similar to AMD levels, E. mutabilis demonstrated significantly greater tolerance to all metals, except Cu. E. gracilis showed two-fold higher tolerance to Cu²⁺ than E. mutabilis (EE50 of 91.6 vs. 45.7 pmol cell^–1). The EE50 for Zn²⁺ was similar for both species (368 pmol cell^–1 for E. gracilis and 423 pmol cell^–1 for E. mutabilis). With Cd and Ni, E. mutabilis tolerated an order of magnitude higher exposure than E. gracilis(EE50 of 1.6 vs. 0.2 pmol Cd²⁺ cell^–1; EE50 of 942 vs. 87 pmol Ni²⁺ cell^–1). Al and Fe were tolerated at high total metal concentrations (up to 100 mM) by E. mutabilis, but toxicity was evident with E. gracilisat much lower levels. E. mutabilis grew at double the Al³⁺ exposure tolerated by E. gracilis (EE50 of 398 vs. 188 pmol Al³⁺ cell^–1). There was an 18-fold difference in Fe tolerance levels between E. mutabilis and E. gracilis with EE50s of 8773 and 502 pmol Fe²⁺ cell^–1, respectively. We conclude that differential metal tolerance, particularly to Fe²⁺, accounts for the mutually exclusive distribution of E. gracilis and E. mutabilis in AMD-impacted habitats.     

Control of xylanase production without protease activity in Bacillus sp. by selection of nitrogen source

Maximum xylanase activity, of 380 IU ml^–1, with negligible protease activity, occurred when Bacillus SSP-34 was grown for 96 h with yeast extract and peptone each at 0.25%. Other concentrations of the combination gave xylanase activities less than 66% of that with the optimum nitrogen source concentration and protease activities in the range of 0.01–0.045 IU ml^–1.     

Chondrus crispus (Gigartinaceae,Rhodophyta) tank cultivation: optimizing carbon input by a fixed pH and use of a salt water well

The injection of exogenous carbon into intensively cultivated algal tanks is necessary to insure a maximum growth rate by stabilizing the dissolved inorganic carbon (DIC) pool, but represents the major part of the cultivation cost (ca. 73%). This study was conducted in paddle-wheel tanks ranging in size from 260 m² to 1000 m². Additional carbon was provided by carbon dioxide mixed into the incoming sea water through a tubular reactor. Production vs pH was analysed on 120 growth measurements covering two years of continuous cultivation. Whereas production peaked at pH 8.0–8.2, the economic optimum for pH regulation was in the range 8.4–8.5, where CO₂ injection was greatly reduced (–29%) for only a slight decrease in production (–4%). Expressed as a function of pH level, the specific carbon injection (g c gdw^–1 of Chondrus produced) showed an inverse exponential relationship, whereas gross photoconversion ratio (gdw mol photons^–1) varied according to a second degree equation with a low amplitude. The photoconversion ratio was not improved when the culture was maintained at a DIC concentration higher than the natural equilibrium (0.64 ± 0.11 gdw mol photons^–1 at 2.35 mM and 0.65 ± 0.15 gdw mol photons^–1 at 3.19 MM).A complementary source of carbon was found in underground salt water with a high and stable DIC concentration (10.15 ± 0.25 mmole Cl^–1). The mixing of the well water with natural sea water allowed another economy of CO₂ (–20% at pH 8.5) and nutrients (–12%), the total unitary cost of production being cut by about 17%.     

Optimization of pyrite bioleaching using Sulfolobus acidocaldarius

Optimization of batch pyrite bioleaching with Sulfolobus acidocaldarius was performed using statistical modelling and experimental design. First a screening design was made followed by response surface modelling. The dominating factors identified were pH, pulp density and particle size. The highest batch leaching rate after optimization was 270 mg iron·l^–1·h^–1 for 6% (w/v) pulp density, pH = 1.5 and particle size <20 m. This represents a 3.5-fold increase from the leaching rate of 80 mg iron·l^–1·h^–1 obtained under our standard laboratory conditions. Correspondence to: E. B. Lindström     

Sensitivity variations of Listeria strains to the bacteriocins, lactocin 705, enterocin CRL35 and nisin

Five strains of Listeria monocytogenes, four strains of Listeria innocua and a strain of Listeria seeligeri showed different sensitivities to lactocin 705 (17 000 AU ml^–1), enterocin CRL35 (8500 AU ml^–1) and nisin (2500 IU ml^–1) at different pHs (5, 6 and 7). The susceptibility of Listeria strains to bacteriocins at each pH was strain dependent, and it was enhanced at the low pH. L. monocytogenes had enhanced nisin tolerance while the non-nisin bacteriocins were more inhibitory with viability losses of 3–3.4 in contrast with 1.5–1.8 log cycles, respectively. Lower viability loss values were obtained with L. innocua strains with all three bacteriocins while L. seeligeri was more sensitive to nisin than to lactocin 705 or enterocin CRL35.     

Production of D(-)-lactic acid from cellulose by simultaneous saccharification and fermentation using Lactobacillus coryniformis subsp. torquens

D(–)-Lactic acid was produced from cellulose by simultaneous saccharification and fermentation (SSF) in media containing cellulolytic enzymes and Lactobacillus coryniformis subsp. torquens ATCC 25600 at 39 °C and pH 5.4, yielding 0.89 g D(–)-lactic acid g^–1 cellulose at a mean volumetric productivity of 0.5 g l^–1 h^–1. No L(+)-lactic acid was found in the medium.     

Purification and characterization of a high molecular mass serine carboxypeptidase from <Emphasis Type="Italic">Monascus pilosus</Emphasis>

Two serine carboxypeptidases, MpiCP-1 and MpiCP-2, were purified to homogeneity from Monascus pilosus IFO 4480. MpiCP-1 is a homodimer with a native molecular mass of 125 kDa composed of two identical subunits of 61 kDa, while MpiCP-2 is a high mass homooligomer with a native molecular mass of 2,263 kDa composed of about 38 identical subunits of 59 kDa. This is unique among carboxypeptidases and distinguishes MpiCP-2 as the largest known carboxypeptidase. The two purified enzymes were both acidic glycoproteins. MpiCP-1 has an isoelectric point of 3.7 and a carbohydrate content of 11%, while for MpiCP-2 these values were 4.0 and 33%, respectively. The optimum pH and temperature were around 4.0 and 50°C for MpiCP-1, and 3.5 and 50°C for MpiCP-2. MpiCP-1 was stable over a broad range of pH between 2.0 and 8.0 at 37°C for 1 h, and up to 55°C for 15 min at pH 6.0, but MpiCP-2 was stable in a narrow range of pH between 5.5 and 6.5, and up to 50°C for 15 min at pH 6.0. Phenylmethylsulfonylfluoride strongly inhibited MpiCP-1 and completely inhibited MpiCP-2, suggesting that they are both serine carboxypeptidases. Of the substrates tested, benzyloxycarbonyl-l-tyrosyl-l-glutamic acid (Z-Tyr-Glu) was the best for both enzymes. The K_m, V_max, K_cat and K_cat/K_m values of MpiCP-1 for Z-Tyr-Glu at pH 4.0 and 37°C were 1.33 mM, 1.49 mM min^–1, 723 s^–1 and 545 mM^–1 s^–1, and those of MpiCP-2 at pH 3.5 and 37°C were 1.55 mM, 1.54 mM min^–1, 2,039 s^–1 and 1,318 mM^–1 s^–1, respectively.