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In the pulp and paper industry, during the manufacturing process, the agglomeration of pitch particles (composed of triglycerides, fatty acids, and esters) leads to the formation of black pitch deposits in the pulp and on machinery, which impacts on the process and pulp quality. Traditional methods of pitch prevention and treatment are no longer feasible due to environmental impact and cost. Consequently, there is a need for more efficient and environmentally friendly approaches. The application of lipolytic enzymes, such as lipases and esterases, could be the sustainable solution to this problem. Therefore, an understanding of their structure, mechanism, and sources are essential. In this report, we review the microbial sources for the different groups of lipolytic enzymes, the differences between lipases and esterases, and their potential applications in the pulping industry.

Alkalophilic Cellulosimicrobium cellulans CKMX1 isolated from mushroom compost is an actinomycete that produces industrially important and environmentally safer thermostable cellulase-free xylanase, which is used in the pulp and paper industry as an alternative to the use of toxic chlorinated compounds. Strain CKMX1 was previously characterized by metabolic fingerprinting, whole-cell fatty acids methyl ester analysis, and 16S rDNA and was found to be C. cellulans CKMX1. Crude enzyme (1027.65 U/g DBP) produced by C. cellulans CKMX1, having pH and temperature optima of 8.0 and 60 °C, respectively, in solid state fermentation of apple pomace, was used in the production of bleached wheat straw pulp. Pretreatment with xylanase at a dose of 5 U/g after pulping decreased pulp kappa points by 1.4 as compared with the control. Prebleaching with a xylanase dose of 5 U/g pulp reduced the chlorine charge by 12.5%, increased the final brightness points by approximately 1.42% ISO, and improved the pulp strength properties. Xylanase could be substituted for alkali extraction in C–Ep–D sequence and used for treating chemically bleached pulp, resulting in bleached pulp with higher strength properties. Modification of bleached pulp with 5 U of enzyme/g increased pulp whiteness and breaking length by 1.03% and 60 m, respectively; decreased tear factor of pulp by 7.29%; increased bulk weight by 3.99%, as compared with the original pulp. Reducing sugars and UV-absorbing lignin-derived compound values were considerably higher in xylanase-treated samples. Cellulosimicrobium cellulans CKMX1 has a potential application in the pulp and paper industries.

This study investigated the effects of cyanobacteria from pulp-and-paper waste-treatment systems on biological toxicity removal and biodegradation of certain wastewater contaminants. In field and batch studies, using the Microtox®assay, cyanobacterial biomass and final wastewater toxicity were significantly correlated. In softwood-based wastewater, a decrease in toxicity was negatively correlated with cyanobacterial biomass, but the correlation was positive in hardwood-based wastewater. In the softwood-based wastewater, toxicity remained higher in the light than it was in the dark, whereas in hardwood-based wastewater, toxicity was lower in the light than it was in the dark. All of these results were light-dependent, suggesting that the photosynthetic growth of cyanobacteria is required to induce significant effects. When grown in mixed cultures with bacterial degraders, cyanobacteria from pulp-and-paper waste-treatment systems generally impeded the biodegradation of the wastewater contaminants phenol and dichloroacetate (DCA). However, there was one case where the cyanobacterium Phormidium insigne improved the bacterial degradation of DCA. Doubling inorganic nutrient concentrations did not improve phenol or DCA biodegradation in the majority of cases, indicating that nutrient competition is not a major factor. These data suggest that cyanobacteria play an important role during the biological treatment of contaminants, and, hence, toxicity removal in pulp-and-paper waste-treatment systems.Key words: cyanobacteria, heterotrophic bacteria, biodegradation, pulp and paper waste-treatment, wastewater toxicity.

The ability to differentiate functional and structural diversity of bacterial communities present in activated sludges adapted to elementally (ECF) and totally (TCF) chlorine-free bleaching effluents was evaluated. Community function was evaluated through substrate utilization patterns in BiologGN®microplates, and taxonomic structure was evaluated by fluorescent in situ hybridization using probes targeting the Eubacteria; the alpha, beta, and gamma subclasses of the Proteobacteria; and gram-positive bacteria with high GC content. Over 6-week sampling periods, ECF- and TCF-adapted sludge bacterial communities presented reproducible substrate utilization patterns that through principal components (PCs) analysis, separated the ECF samples from the TCF samples. Application of the fluorescent in situ hybridization technique was complicated by the intense autofluorescence of the bleaching effluent sludge samples that interfered with detection of specific hybridization signals. The most notable difference in community structure detected using the chosen set of probes was the relatively greater proportion of cells of the alpha subclass in TCF sludge (27%) than in ECF sludge (6%). Nonspecific hybridization with beta and gamma probes was relatively high, but both sludges appeared to have similar proportions of cells of the beta (20–22%) and gamma (11–12%) subclasses. The two sludges presented relatively few gram-positive cells with high GC content (

The relationship between the abundance of three functional genes and their corresponding biochemical reaction rates was investigated in several activated sludge and mill effluent microbial communities. Gene probes were prepared for two key denitrification genes (nirS and nirK) and for one nitrogen-fixation gene (nifH) and were validated using a variety of strains of known nir and nif genotype. ATP-based measures of viable cell numbers were used to provide total population sizes. In certain microbial communities (activated sludge enrichment cultures and multiple samples taken from the same mill primary clarifier), a strong correlation was observed between gene abundance and biochemical activity rates. However, when comparing several different nonenriched activated sludge bioreactors and separate primary clarifier microbial communities, the ratio of specific gene abundance to biochemical activity rates varied widely. These results suggest that in cases where a microbial community is not fully induced for a given biochemical activity or when very different communities are compared, quantitative gene probing can give a better measure of a community's potential to carry out the encoded function than can the relevant biochemical assay. However, the gene quantitation method employed here probably underestimated the true number of probed genes present in the microbial communities due to nirS and nifH genes in the communities having reduced DNA sequence similarity with the probes used.Key words: denitrification, nitrogen fixation, quantitative hybridization, activated sludge, primary clarifier.

Bacterial community compositions from 10 pulp- and paper-mill treatment systems were compared using both traditional and molecular techniques. 16S-RFLP (Random Fragment Length Polymorphisms) analysis was used to examine the genotypic profiles of the whole bacterial community of each treatment system. Although all the communities shared approximately 60% of their DNA band pattern, as determined by computer-assisted cluster analysis, each community displayed a unique profile that was stable over time under normal operating parameters. Reverse Sample Genome Probing (RSGP) and 16S-RFLP were used to compare the culturable bacterial communities of several geographically separated pulp-mill biotreatment system communities. There was little overlap in the composition of the culturable community between mills at the genus level. Furthermore, RSGP variation was almost as high within a mill as between mills. Partial sequences of the 16S rRNA genes from culturable isolates identified Bacillus spp., Pseudomonas spp., and Xanthobacter as some of the dominant species. Finally, several 16S rRNA genes from two whole community 16S RNA gene libraries were partially sequenced and identified as similar to unknown α-, β-, and γ-Proteobacteria, Ralstonia, Alcaligenes, Nitrospira, Firmicutes, and clones representing the new Holophaga/Acidobacterium phylum. These findings suggest that although these pulp- and paper-mill biotreatment communities perform similar functions, they are populated by unique mixtures of species.Key words: biodiversity, 16S-RFLP, secondary treatment, pulp mills.

Pulp and paper secondary waste-treatment systems in Brazil, Canada, New Zealand, and the U.S.A. contained dynamic cyanobacterial communities, some of which exceeded heterotrophic bacterial biomass. No other viable photoautotrophic populations were detected in the ponds. Regardless of geographical location, Oscillatoriales including Phormidium, Geitlerinema, and Pseudanabaena were the dominant taxa. As well, Chroococcus (Chroococcales) was an important genus in Brazil and New Zealand. The possible impact of cyanobacteria on waste-treatment efficiency deserves further study given their large biomass and diverse metabolic characteristics.Key words: cyanobacteria, blue-green algae, heterotrophic bacteria, community structure, pulp and paper secondary waste treatment.

Three of four isolates, representing phylogenetically distinct groupings of low-temperature basidiomycetes (LTB), were capable of utilizing wheat straw, and to a lesser extent conifer wood at 15°C. A cottony snow mould LTB (LRS 013) and a fruit rot LTB (LRS 241) grown on straw significantly degraded filter paper, carboxymethylcellulose (CMC), p-nitrophenyl β-glucopyranoside (i.e., β-glucosidases), and xylan. Enzymes produced by Coprinus psychromorbidus (LRS 067) were limited to xylanases from straw and wood and β-glucosidases from wood. A sclerotia-forming LTB (LRS 131) exhibited poor growth on both substrates, and did not produce detectable quantities of extracellular enzymes. None of the LTB isolates tested degraded avicel. The temperature optima of CMCases and xylanases in the filtrates from the straw medium ranged from 25°C to 55°C, and with the exception of LRS 067, significant activity was observed at 5°C. Two cellulases (25 and 31 kDa) and two xylanases (24 and 34 kDa) were observed on zymograms for LRS 013 and 241. Reduction of enzymes with 2-mercaptoethanol adversely affected their activity on zymograms, and an additional cellulase band was observed for non-reduced samples. This study indicates that LTB produce an array of cellulolytic and xylanolytic enzymes, and that some of these enzymes possess low-temperature optima which may facilitate degradation of plant fibre under low-temperature conditions.Key words: psychrophile, snow mould, cellulase, xylanase, enzymes.

Resin acids, a group of diterpenoid carboxylic acids present mainly in softwood species, are present in many pulp mill effluents and toxic to fish in recipient waters. They are considered to be readily biodegradable. However, their removal across biological treatment systems has been shown to vary. Recent studies indicate that natural resin acids and transformation products may accumulate in sediments and pose acute and chronic toxicity to fish. Several resin acid biotransformation compounds have also been shown to bioaccumulate and to be more resistant to biodegradation than the original material. Until recently, the microbiology of resin-acid degradation has received only scant attention. Although wood-inhabiting fungi have been shown to decrease the level of resin present in wood, there is no conclusive evidence that fungi can completely degrade these compounds. In contrast, a number of bacterial isolates have recently been described which are able to utilize dehydroabietic or isopimaric acids as their sole carbon source. There appears to be an unusually high degree of substrate specificity with respect to the utilization of abietane congeners and the presence of substituents. Pimaranes do not appear to be attacked to the same extent as the abietanes. This paper reviews the occurrence, chemistry, toxicity, and biodegradation of resin acids in relation to the biological treatment of pulp and paper mill effluents.Key words: resin acids, biodegradation, pulp mill effluents.

Xylanase production from a Bacillus subtilis gene cloned into a strain of Escherichia coli was monitored. Although this gene was expressed in E. coli at several temperatures, efficient xylanase secretion did not occur; the observed protein release apparently depended on cell leakage or lysis. Screening for a better microbial protein secretor free of cellulase selected Bacillus cereus No. 518. A bidirectional vector plasmid (pMK3) was employed to carry the cloned gene into this B. cereus strain. Transformation was carried out by electroporation. Total xylanase production by the new pMK3-borne gene in B. cereus was similar to that from E. coli but the xylanase was shown to be normally secreted. The xylanase gene products from the E. coli and B. cereus hosts were shown to function identically. Both xylanases improved the delignification of unbleached softwood and hardwood kraft pulps, thus reducing the Cl2 required to achieve a given degree of bleaching, without altering the physical properties of the fibers. Using a target kappa number (lignin content) of 5, xylanase pretreatment of aspen kraft (chemical) pulp led to a 22% savings of chlorine. Adsorbable organic halogens in the bleachery effluent were also lowered by more than 50%.Key words: Bacillus subtilis, endoxylanase gene, bleaching, kraft pulp properties.

Water samples from the wastewater treatment system of a bleach kraft mill and from the river that supplies the mill were plated on six different media and culturable isolates were screened for substrate utilization patterns, taxonomic characters, plasmid content, and resistance to ampicillin, streptomycin, kanamycin, tetracycline, naldixic acid, mercury, nickel, copper, cobalt, cadmium, and zinc. A cluster analysis of the substrate utilization profiles and taxonomic characters revealed that Pseudomonas, Acinetobacter, and Acidovorax spp. were common among the culturable isolates from the river, while Ancylobacter aquaticus, Klebsiella spp., and an unknown group of pleomorphic Gram-negative methylotrophs were common among the culturable isolates from the mill treatment system. Of isolates from the settling pond and aerated lagoon, 78 and 64% carried plasmids, while only 56% of isolates from the river carried plasmids. Plasmids were significantly associated with resistance to cadmium but not with any other resistance characters. Large numbers of plasmid-carrying A. aquaticus strains and pleomorphic methylotrophs accounted for high plasmid incidence levels in the mill treatment system, and the ability to dechlorinate simple aliphatic substrates was found in these two groups as well as in one Pseudomonas strain.Key words: pulp and paper, wastewater treatment, chlorinated organics, culturable heterotrophs, methylotrophs, plasmids.

A mixed culture in which cellulose is capable of being converted to methane and carbon dioxide was obtained from an inoculum procured from a sewage-treatment plant and maintained in a synthetic medium containing tissue paper and an inorganic salt and vitamin mixture. The culture was tested for its ability to degrade 12 different paper and cotton products under batch conditions in 3-ℓ anaerobic fermenters. This culture degraded 6–8 mmol/ℓ per week of cellulose, expressed as glucose equivalents, with total gas yields of 0.3 m3/kg of cellulose degraded. The gas produced contained between 56 and 59% of methane. Maximum cellulose degradation occurred at chemical oxygen demand: nitrogen: phosphorus level of 80:5:1 and was adversely affected by high stirring rate. Also the presence of higher proportions of lignin in cellulose products adversely affected the ability of this culture to degrade cellulose.

The coliform flora of a pulp and cardboard mill that uses birch as the raw material and ammonium sulphate as the process chemical was studied. Escherichia coli was observed to multiply in the mill. It persisted as the dominant thermotolerant coliform in the effluent. Klebsiellae were encountered among total coliforms only. The E. coli strains isolated had the biochemical characteristics and maximum growth temperatures typical to the species. However, serotyping and hemolysin test differentiated these strains from pathogenic and fecal E. coli.

Alternaria solani produced two homogeneous exocellular polysaccharide fractions when grown in shake culture with fructose as the carbon source and the NH4NO3 concentration of the medium 1.7 g/l. Chromatographic techniques indicated that fraction I contained glucose, galactose, and glucosamine in a 2.4:1:1 ratio whereas fraction II contained glucose, galactose, and mannose in a 6.4:1:1.2 ratio. Fraction I was found at levels seven times greater than II. Total polysaccharide produced was 1.8% of the dry weight of the mycelium and 1.2% of the available carbohydrate.Growth and polysaccharide production were greatest with fructose as the carbon source. Decreasing levels of growth occurred with mannitol, glucose, and sucrose, respectively. Exocellular polysaccharides were not detected with mannitol as carbon source. At 10 days, growth on fructose is maximum at NH4NO3 concentrations of 1.5 to 2.5 g/l, but exocellular polysaccharide production is maximum at 0.8 g/l. When produced on a medium of 2.0 g/l NH4NO3 the nitrogen content of I is double that when the medium is 0.8 g/l NH4NO3. In addition the ratio of I to II is about four times greater.

Multiple electrophoretic species of arylsulfatase were demonstrated in sonicates of Proteus rettgeri by paper electrophoresis. Subsequent treatment of the sonicates with streptomycin sulfate and DEAE-cellulose chromatography resolved enzyme activity into four fractions. Electrophoretic examination of each chromatographic fraction revealed sulfatase heterogeneity comparable to that of the initial extract. These electrophoretic components differed in their general response to a number of arylsulfatase inhibitors. The results are discussed in relation to the finding of multiple species of arylsulfatase in other biological systems.

A substance present in culture filtrates of enterotoxigenic staphylococci that is distinct from alpha-, beta-, or delta-lysins causes vomiting; in cats when injected by the intraperitoneal route. An emetic fraction containing only vestigial traces of lysins can be resolved into three protein components and one polysaccharide by use of the Durrum ionophoresis method. The only component of this group that gave indications of enterotoxicity was a protein with specific mobility, relative to the others. Hemolytic proteins were separated by paper ionophoresis using the method of McKinley, Maw, Oliver, and Common.

An adaptation of a moist-chamber method is described whereby Verticillium albo-atrum, V. dahliae, and V. nigrescens can readily be isolated from herbaceous and woody hosts and easily identified. Segments of infected stems, petioles, and roots cut transversely can be observed microscopically at daily or frequent intervals to determine the origin of outgrowing fungal hyphae in the absence of troublesome contaminants.

The reaction of Paramecium caudatum to solutions containing the ash of cigarette paper was consistent with the response of Paramecium aurelia previously reported. After this confirmation, both these species of Paramecium were used as test organisms during a search for the causative agents for the "Paramecium reaction". A qualitative spectrochemical analysis of cigarette paper ash showed that its chief constituent is calcium (97%) with 12 other detected elements present at levels too low to elicit any response from paramecia. Hydroxyl ions in concentrations equivalent to 0.001 M Ca(OH)2, 0.0008 M KOH, or 0.002 M NaOH simulated but did not duplicate the effect of standard cigarette paper ash solution (as of one cigarette paper in 20 ml water). While neutralization caused the three hydroxide solutions to lose their toxicity, the toxicity of cigarette paper ash solution was not appreciably affected by neutralization. Equivalent solutions of facial tissue, filter paper, and a paper towel, burned the same way as cigarette paper, were used as controls. Each of them affected paramecia differently and none simulated the Paramecium reaction. Further, a comparison between ashes from a paperless cigarette, a whole cigarette, and one with two extra roll-your-own wrappers demonstrated a dependence of the Paramecium reaction on cigarette paper ash as well as a direct correlation between the reaction intensity and the amount of cigarette paper ash present.