Laccase treatment of recycled blue dyed paper: physical properties and fiber charge

Recycled blue colored paper was treated with laccase under various combinations of physical and chemical parameters including enzyme concentration, temperature, oxygen, and reaction time. Laccase treatment of recycled dyed pulp increased acid group content, tear index, tensile index, and color removal in a dose-dependent manner. Lengthening the treatment time from 2 to 4 h was beneficial to acid group content (12% increase), dye removal, and tensile index but had a detrimental 8% decrease on the tear index. A higher reaction temperature (65 vs. 45 degrees C) had a beneficial effect on acid group content (+31%), and tensile index (+26%) and a slightly negative effect on tear index (-5%), but significantly reduced the ability of laccase to remove color. Comparison of reactions subjected to different levels of oxygen supplementation showed the greatest beneficial effect for laccase treatment with slow oxygen bubbling. The experimental results indicate that laccase treatment increases fiber carboxylic acid content and tensile strength, in addition to reducing the color of the enzyme treated paper.     

Response of ectomycorrhizal fungi to benomyl and nocodazole: growth inhibition and microtubule depolymerization

The growth of seven ectomycorrhizal fungi was tested in the presence of the antimicrotubule drugs benomyl and nocodazole. The polymerization stage of the cytoplasmic microtubules in the hyphal cells was visualized by indirect immunofluorescence microscopy after a 3-h drug treatment. Nocodazole reduced the growth of all the fungi tested at concentrations of 2 and 4 g ml^-1 and caused strong depolymerization of microtubules in all other species except Hebeloma cylindrosporum. Benomyl inhibited the growth and depolymerized the microtubules in the ascomycete Cenococcum geophilum, while in the basidiomycetes it reduced the growth and depolymerized the microtubules only in H. cylindrosporum. The role of the microtubule cytoskeleton and the target of the benzimidazole-derived drugs in fungal cells are discussed.     

Choline: Its role in the growth of filamentous fungi and the regulation of mycelial morphology

Abstract Choline is an essential metabolite for the growth of filamentous fungi. It occurs most notably as a component of the major membrane phospholipid, phosphatidyl choline (lecithin), and fulfills a major role in sulphate metabolism in the form of choline- o -sulphate in many species. Choline is usually synthesised endogenously, but exogenous choline can also be taken up, either to compensate for metabolic deficiencies in choline-requiring mutants such as those of Aspergillus nidulans and Neurospora crassa , or as a normal function by species such as Fusarium graminearum which do not require added choline for growth. F. graminearum has a highly specific constitutive uptake system for this purpose. Recent studies have begun to indicate that choline also plays an important role in hyphal and mycelial morphology. Over a wide range of concentrations, choline influences mycelial morphology, apparently influences mycelial morphology, apparently by controlling branch initiation. At high concentrations of added choline, branching is inhibited but specific growth rate is unaffected, leading to the production of rapidly extending, sparsely branched mycelia. Reduction of choline concentration allows a progressive increase in branching. Additionally, in choline-requiring mutants which have a very reduced content of choline, multiple tip-formation and apical branching occurs. Just prior to cessation of growth in choline-starved cultures of A. nidulans choline-requiring mutants, hyphal morphology changes due to a brief phase of unpolarised growth to produce spherical swellings called ballons, at or near hyphal apices. The precise mechanism by which choline affects fungal morphology is not yet known, although in A. nidulans it appears to be at least partially due to the influence of membrane composition on the synthesis of the hyphal wall polymer chitin. Several hypotheses for the possible mode of action of choline in affecting fungal morphology are discussed here.     

A microculture chamber and improved method for combined light and electron microscopy of filamentous fungi

A microculture chamber has been developed which enables specific fungal hyphae to be followed in their living state before being processed and longitudinally sectioned for electron microscopy. Photomicrographs and their corresponding electron micrographs are presented which illustrate the effectiveness of this approach. Examples of the application of the method are presented.     

Effects of different growth forms of Mucor indicus on cultivation on dilute-acid lignocellulosic hydrolyzate, inhibitor tolerance, and cell wall composition

The dimorphic fungus Mucor indicus was grown in different forms classified as purely filamentous, mostly filamentous, mostly yeast-like and purely yeast-like, and the relationship between morphology and metabolite production, inhibitor tolerance and the cell wall composition was investigated. Low concentrations of spores in the inoculum with subsequent aeration promoted filamentous growth, whereas higher spore concentrations and anaerobic conditions promoted yeast-like growth. Ethanol was the main metabolite with glycerol next under all conditions tested. The yields of ethanol from glucose were between 0.39 and 0.42 g g⁻¹ with productivities of 3.2–5.0 g l⁻¹ h⁻¹. The ethanol productivity of mostly filamentous cells was increased from 3.9 to 5.0 g l⁻¹ h⁻¹ by the presence of oxygen, whereas aeration of purely yeast-like cells showed no such effect. All growth forms were able to tolerate 4.6 g l⁻¹ furfural and 10 g l⁻¹ acetic acid and assimilate the sugars, although with different consumption rates. The cell wall content of the fungus measured as alkali insoluble materials (AIM) of the purely yeast-like cells was 26% of the biomass, compared to 8% of the pure filaments. However, the chitosan concentration of the filaments was 29% of the AIM, compared to 6% of the yeast-like cells.     

Effects of nocodazole and brefeldin A on microtubule cytoskeleton and membrane organization in the homobasidiomyceteSchizophyllum commune

Summary The effects of nocodazole and brefeldin A (BFA) on the growth of dikaryotic hyphae inSchizophyllum commune corresponded with the development of abnormal structures in the apical region of treated hyphae. Microtubules (MTs) were totally depolymerized after 1 h nocodazole treatment, which correlated with strong branch formation in the apical cells. One reason for branching could be the shift in the position of apical vesicles from the center to the side of the tip, observed in some nocodazole-treated hyphae. After 2 h growth in the presence of nocodazole the apical cells had malformed or swollen tips, or tips of normal shape but containing only a few apical vesicles. After 0.5 h treatment with BFA, almost all the leading hyphae had swollen apical parts in which the endoplasmic reticulum (ER) formed an interconnected network and perturbed Golgi particles were found. The orientation of MTs in the BFA-treated hyphae often followed that of the interconnected ER network, which suggested an association between MTs and ER. The results of the experiments with nocodazole suggest that, in filamentous homobasidiomycetes the subtle organization of cytoplasm necessary for the polar growth at the apex is maintained only in the presence of an intact MT cytoskeleton. The BFA experiments indicated that the secretion pathway inS. commune is sensitive to BFA. In addition rapid change in apical morphology in the BFA-treated hyphae emphasizes the importance of correct orientation of components of the secretory pathway for normal apical growth to continue.Abbreviations BFA brefeldin A - EM electron microscopy - ER endoplasmic reticulum - IIF indirect immunofluorescence - MBC methylbenzimidazole-2-ylcarbamate - MT microtubule - MVB multivesicular body - RER rough endoplasmic reticulum     

Improved methods for the preparation of high molecular weight DNA from large and small scale cultures of filamentous fungi

Improved methods are described for the isolation of pure, high molecular weight DNA from small and large scale cultures of filamentous fungi. The methods depend on the extraction of DNA under conditions which prevent nuclease activity and contamination by carbohydrate. The small scale method depends on enzymatic digestion of the wall whereas the large scale method uses partial damage followed by autolysis. High yields of DNA are obtained by both methods and the DNA is suitable for restriction analysis. Southern Blotting, RFLP analysis, dot blotting and the production of gene libraries. The small scale method can be used for the simultaneous analysis of multiple cultures.     

Homology of the root adhesin of Pseudomonas fluorescens OE 28.3 with porin F of P. neruginosa and P. syringae

Summary A transformation system for the thermophilic cellulolytic fungus Talaromyces sp. CL240 has been developed, using the phleomycin resistance gene from Streptoalloteichus hindustanus (Sh ble) as a dominant selectable marker. The plasmids (pAN8-1 and pUT720) carrying the Sh ble gene under the control of the Aspergillus nidulans glyceraldehyde-3-phosphate dehydrogenase (gpd) promoter, allowed selection of phleomycin-resistant transformants. A new promoter sequence cloned from chromosomal DNA of Trichoderma reesei (pUT737) was also able to drive efficient expression of the Sh ble gene in Talaromyces sp. CL240, resulting in the selection of transformants that were highly resistant to phleomycin.     

Description of heterotrophic bacteria occurring in paper mills and paper products

AIMS: To isolate aerobic mesophilic bacilli and thermophilic bacteria from different paper mill samples and to evaluate their potential harmfulness. METHODS AND RESULTS: A total of 109 mesophilic and 68 thermophilic isolates were purified and characterized by automated ribotyping and partial 16S rDNA sequencing. The mesophilic isolates belonged to the genera Bacillus (13 taxa), Brevibacillus (three taxa) and Paenibacillus (five taxa). The thermophilic bacteria represented seven taxa of Bacillus, Geobacillus or Paenibacillus, four of proteobacteria and one of actinobacteria. The most frequently occurring bacteria were Bacillus cereus, B. licheniformis, Pseudoxanthomonas taiwanensis and bacteria closely related to Paenibacillus stellifer, P. turicensis or Leptothrix sp. One mill was contaminated throughout with bacteria of a novel mesophilic genus most closely related to Brevibacillus centrosporus and another with bacteria of a novel thermophilic genus most closely related to Hydrogenophilus thermoluteolus. One B. cereus isolate producing haemolytic diarrhoeal enterotoxin was detected and all the tested B. licheniformis isolates produced a metabolite toxic to boar sperm cells. CONCLUSIONS: The bacilli and thermophilic bacteria isolated represent species which should not present occupational hazards in paper mill environments. The most harmful bacterium detected was B. licheniformis and potentially also B. cereus. SIGNIFICANCE AND IMPACT OF THE STUDY: Knowledge of the microbial diversity in a paper mill provides a rational basis for development of an effective controlling programme. A database constructed from the fingerprints generated using automated ribotyping helps to identify and trace the contamination routes of bacteria occurring in paper mills.     

Use of pH auxostats to grow filamentous fungi in continuous flow culture at maximum specific growth rate

Abstract The pH auxostat employs a pH-derived feedback control of biomass concentration and allows continuous flow cultures of organisms to be grown at their maximum specific growth rate. We examine three modifications of the pH auxostat system for the growth of filamentous fungi and discuss the suitability of each method according to the biomass concentration desired, the medium used and the equipment available. Fusarium graminearum and Geotrichum candidum were used for this work and it was possible to maintain steady state pH auxostats of these filamentous fungi for up to 20 days. pH auxostats have not previously been described for filamentous fungi.     

Physicochemical characteristics of microbial content in a modified anaerobic inclining-baffled reactor (MAI-BR) treating recycled paper mill effluent (RPME)

Abstract

Microbial content formed in bioreactors plays a significant role in the anaerobic process. Therefore, the physicochemical characteristics of microbial content in a modified anaerobic inclining-baffled reactor (MAI-BR) treating recycled paper mill effluent (RPME) were investigated using Fourier transform infrared (FTIR), X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), thermogravimetric (TG), and derivative thermogravimetric (DTG) analyses, scanning electron microscopy-energy dispersive X-ray spectroscopy (SEM-EDS), Brunauer-Emmett-Teller (BET), and surface area analyzer. FTIR spectra revealed that the microbial content had stronger characteristic peaks corresponding to alcohols, water, lipids carbohydrates, proteins, and mineral compounds. Calcite, muscovite, and lepidolite were the prevalent mineral phases found by XRD analysis. The elemental of these minerals like C, Ca, N, O, and Si was confirmed by XPS results. The microbial content samples from each compartment showed similar thermal behavior. SEM images showed that straight rod-shaped and Methanosaeta-like microorganisms were predominant, whereas C, O, and Ca were noticed by EDS on the surface of granules. The BET surface areas and pores of granules are found to decline throughout the reactor’s compartment, where Compartment 1 had the largest values. Thus, the findings of this study establish further understanding of the physicochemical properties of microbial content formed in MAI-BR during the RPME treatment.     

Isolation and analysis of functional homologues of the secretion-related SAR1 gene of Saccharomyces cerevisiae from Aspergillus niger and Trichoderma reesei

The Aspergillusniger and Trichodermareesei genes encoding the functional homologues of the small GTP-binding protein SAR1p, which is involved in the secretion pathway in Saccharomyces cerevisiae, have been cloned and characterised. The A. niger gene (sarA) contains five introns, whereas the T. reesei gene (sar1) has only four. In both cases the first intron is at the same position as the single S. cerevisiae SAR1 intron. The encoded proteins show 70–80% identity to the SAR1 protein. Complementation of S. cerevisiaesar1 and sec12 mutants by expression vectors carrying the A. nigersarA and T. reesei sar1 cDNA clones confirmed that the cloned genes are functional homologues of the S. cerevisiae SAR1 gene. Three mutant alleles of the A. nigersarA gene (D29G, E109K, D29G/E109K), generated by site-directed mutagenesis, revealed a thermosensitive dominant-negative phenotype in the presence of the wild-type sarA allele. This result contrasts with the situation in S. cerevisiae, where similar mutations have a thermosensitive phenotype. Taken together, our results indicate that the sarA gene is involved in an essential function in A. niger. Received: 21 January 1997 / Accepted: 21 June 1997     

Environmental assessment of enzyme assisted processing in pulp and paper industry

Background Aims and Scope  The pulp and paper (P&P) industry is traditionally known to be a large contributor to environmental pollution due its large consumptions of energy and chemicals. Enzymatic processing, however, offers potential opportunities for changing the industry towards more environmentally friendly and efficient operations compared to the conventional methods. The aims of the present study has been to investigate whether the enzyme technology is a more environmentally sound alternative than the conventional ways of producing paper. The study addresses five enzyme applications by quantitative means and discusses the environmental potential of a range of other enzyme applications by qualitative means. Methods  LCA is used as analytical tool and modelling is facilitated in SimaPro software. Foreground LCA data are production/company specific and collected from P&P technology service providers, specific P&P companies and P&P researchers. The background data on energy systems, auxiliary chemicals, etc. are primarily taken from the ecoinvent database. Results  The study shows that fossil energy consumption and potential environmental impacts (global warming, acidification, nutrient enrichment, photochemical smog formation) induced by enzyme production are low compared with the impacts that they save when applied in bleach boosting, refining, pitch control, deinking, and stickies control. Discussion  The general explanation is that small amounts of enzyme provide the same function as large amounts of chemicals and that enzymatic processes generally require less fossil energy inputs than conventional processes. Data quality assessments and sensitivity analyses indicate that this observation is robust for all processes except deinking, although the results are subject to uncertainty and much variation. Conclusions and Recommendations  The environmental improvements that can be achieved by application of enzymatic solutions in the P&P industry are promising. To get a greater penetration of enzymatic solutions in the market and to harvest the environmental advantages of biotechnological inventions, it is recommended that enzymatic solutions should be given more attention in, for instance, ‘Best Available Technology’ notes within the framework of the European Directive on Integrated Pollution Prevention and Control (IPPC). ESS-Submission Editor: Roland Hischier (roland.hischier@empa.ch)     

Influence of commercial fungicides on the germination,growth and virulence of four species of entomopathogenic fungi

The influence of 15 commercially available fungicides on the germination, growth and virulence of Metarhizium anisopliae, Beauveria bassiana, Isaria fumosorosea, and Lecanillium longisporum was evaluated. The influence of the fungicides on conidial germination was dependant on the fungicide type and dose. Most fungicides retarded conidial germination of all the fungi tested at 10× and at the recommended rate of application, however, their toxicity declined at lower concentrations. Most of the fungicides inhibited mycelial growth of B. bassiana, whereas L. longisporum growth was unaffected. Only two and eight fungicides influenced mycelial growth of I. fumosorosea and M. anisopliae, respectively. None of the fungicides influenced the virulence of B. bassiana and L. longisporum, however, tolylfluanid and azoxystrobin reduced the virulence of M. anisopliae and I. fumosorosea, respectively. These studies clearly show that certain fungicides have the potential to inhibit germination of entomopathogenic fungi in vitro but appear to have little or no effect on their virulence against target insects.     

Identification of aerobic mesophilic bacilli isolated from board and paper products containing recycled fibres

AIMS: To identify aerobic mesophilic bacteria isolated from coreboard, kitchen roll paper and food packaging boards containing recycled fibres and to create a rapid fingerprint-based database for their identification. METHODS AND RESULTS: A total of 197 isolates and 20 relevant type strains were characterized by automated ribotyping and as far as possible identified by the similarities of their riboprints to the relevant type strains. One strain from each unidentified ribotype, a total of 87 strains, was subjected to partial 16S rDNA sequencing and in most cases also to fatty acid analysis and physiological tests. From the isolates 113 and seven different ribotypes were generated belonging to the genera Bacillus and Paenibacillus, respectively. The dominating species, or closest related to them, were B. simplex (22.8% of isolates), B. licheniformis (18.3%) and B. amyloliquefaciens (12.7%); 5.1% of the isolates were identified as B. cereus, a potential food-borne pathogen. In particular, this species was present in one food packaging board (26.3% of isolates). Based on these results, 40.1% of the isolates and 45.0% of ribotypes were so different from the relevant type strains that they may represent novel species. CONCLUSIONS: All isolates were aerobic spore-formers, indicating that all non-spore-formers were eliminated during the drying stage of the processes. Although many isolates could be affiliated to described species of Bacillus or Paenibacillus, a significant proportion of the isolates could not be identified unambiguously as members of a described species. SIGNIFICANCE AND IMPACT OF THE STUDY: A RiboPrint identification database, composed of 120 composite patters, was established for bacteria originating from the pulp and paper industry. Considering the discrimination power of ribotyping, this database will be extremely useful in future for the reliable and rapid identification of bacteria isolated from pulp and paper industrial sources.     

Microbial Ecological Model of Filamentous Bulking and Mechanisms

The paper proposes a prototype mathematical model for the interactions among filamentous bacteria, floc-forming bacteria and protozoa within completely mixed activated sludge systems, which is capable of describing the practical coexistence of three microorganisms in a generic way. The systems with substrate fluctuation and without substrate fluctuation are investigated, respectively. The results show that filamentous bulking will occur within an interested range of substrate concentrations under unsuitable dilution rates, particularly at low substrate concentrations. When substrate concentration fluctuates, the same-frequency synchronous control parameters are beneficial to prevent filamentous bulking. Thus, the present investigations not only reveal the cause of the problem of filamentous bulking theoretically, but also may provide a crucial theoretical foundation for overcoming it.     

Enzymatic degradation of mould stains on paper analysed by colorimetry and DRIFT-IR spectroscopy

Melanin is chemically and by physical characteristics very similar to lignin, a major constituent of wood, and therefore ligninolytic enzymes of white-rot fungi were tested for their ability to selectively degrade melanin. Melanin degradation was studied both in liquid suspensions of melanin and on melaninised paper samples. Liquid suspension samples were tested for changes in their chemical composition (appearance and relative representation of functional groups and chemical bonds) with FTIR spectrometry. Changes in colour of melaninised paper samples were investigated with a colorimeter. Effectiveness of the treatment (bleaching) was determined as a change in lightness (ΔL). Melanin was oxidised in the liquid suspensions, and the intensity of modification varied depending on the procedure employed. The most pronounced changes in melanin were observed in laccase-1-hydroxybenzotriazole (HBT) treatment at heightened air pressure. The most prominent discoloration of the melaninised paper samples (and no visually detectable damage to the integrity of the paper) was, like in the case of the liquid suspensions, observed after laccase-HBT treatment.     

Evaluation of filter paper collection of urine samples for detection and measurement of organic acidurias by capillary electrophoresis

There is little doubt that mental retardation has been prevented in most babies diagnosed by newborn screening programs for inborn errors and the cost-benefit ratios of these programs have been reported as highly positive. In a previous work we optimised a CE method for quick profiling of organic acidurias, which characterize a large number of inborn errors, so that it permits the separation, detection and even identification in less than 15 min of 22 organic acids in urine samples related to a wide range of metabolic disorders. In the present work we have studied the adequacy of filter paper collection of urine samples to simplify this step, always difficult in babies, when it is not performed by training personnel. The studied parameters were: media and conditions for re-extraction to give the best sensitivity and a more simple procedure when the samples are measured by CE, interferences coming from the diaper, recoveries obtained, possible correction of recoveries with creatinine and stability of the compounds. The whole method we report has the advantages of easy sample collection, easy shipping or delivery, and rapid analysis. Moreover, this method of collection and analysis allows the identification and quantitation of fumaric, methylmalonic, N-acetylaspartic, pyroglutamic and homogentisic acids, as well as glutaric acid for which screening is considered especially advisable.     

Effects of a pulp and paper mill on the ecology of the La Trobe River,Victoria, Australia

Four riverine animal communities were measured to assess the impact of extensively treated wastewater from a pulp and paper mill on the lower La Trobe River in Victoria, Australia. Benthic macroinvertebrates in channel and bankside habitats were sampled using a new air-lift corer. Population density was expressed in relation to substrate volume. Other communities measured at five sites upstream and downstream of the mill's wastewater outfall were the zooplankton, and the animals associated with submerged littoral vegetation. Ten environmental variables were also measured during the two sampling periods.A total of 50 benthic macro-invertebrate taxa were dominated by Oligochaeta, Chironomidae and Bivalvia. Benthic communities upstream and downstream of the outfall were very similar. Benthic samples showed large unexplained variation between stations and seasons, despite the similarity of stations and the stratified sampling design, but within-sample variation was small. There was some evidence that benthic faunal patchiness was associated with patterns of stream-bed scouring and deposition in periods of high flow. Littoral samples collected 28 taxa, dominated by Decapoda and Hemiptera. The benthic and littoral communities were quite distinct, with only three species common to both.Only two of the biological and environmental variables responded to wastewater from the mill: total dissolved solids rose by 20–25% over upstream levels; and zooplankton density increased by 2–3 orders of magnitude. It was concluded that wastewater treatment had successfully avoided the major environmental problems often associated with pulp and paper mills.