Biodegradation of two tetrameric lignin model compounds by a mixed bacterial culture

Summary The ability of a mixed bacterial culture to decompose two tetrameric lignin model com-pounds as a sole source of carbon and energy was investigated. The mixed bacterial culture con-sisted mainly of Gram negative rods. The tetram-ers contained two types of lignin substructures, namely the most abundant β-O-4 ether structure in lignin and also the 5-5 biphenyl structure. The tetramer (MW 638) containing two phe-nolic hydroxyls was decomposed readily; after 13 days of incubation, all intermediate products formed were almost totally decomposed. The non-phenolic tetramer (MW 666) was decom-posed much more slowly; after 53 days of incuba-tion, 5% of the substrate was unchanged. When both tetramers were degraded simultaneously, the non-phenolic tetramer was decomposed similarly to the phenolic tetramer. Determination of molecular weights of cata-bolic products showed that the degradation of the non-phenolic tetramer had proceeded at least to dimer level. SKF 525A, inhibitor of cytochrome P-450, caused one catabolic product to accumulate in the culture medium. This indicates involvement of cy-tochrome P-450 in the degradation pathway of the model compounds used. We conclude that this mixed bacterial culture was able to degrade the lignin model compounds used and that free phenolic groups seem to in-crease the biodegradability significantly.     

Biodegradation of pentachlorophenol in natural soil by inoculatedRhodococcus chlorophenolicus

Rhodococcus chlorophenolicus PCP-1, a mineralizer of polychlorinated phenols, was inoculated into natural sandy loam and peaty soils with pentachlorophenol (PCP) at concentrations usually found at lightly and heavily polluted industrial sites (30 to 600 mg PCP/kg). A single inoculum of 10⁵ to 10⁸ cells per g of peat soil and as little as 500 cells/g sandy soil initiated mineralization of¹⁴C-PCP. The mineralization rates of PCP were 130 to 250 mg mineralized per kg soil in 4 months in the heavily (600 mg/kg) polluted soils and 13 to 18 mg/kg in the lightly (30 mg/kg) polluted soils. There were no detectable PCP mineralizing organisms in the soils prior to inoculation, and also there was no significant adaptation of the indigenous microbial population to degrade PCP during 4 months observation in the uninoculated soils. The inoculum-induced mineralization continued for longer than 4 months after a single inoculation. Uninoculated, lightly polluted soils (30 mg PCP/kg) also showed loss of PCP, but some of this reappeared as pentachloroanisol and other organic chlorine compounds (EOX). Such products did not accumulate in theR. chlorophenolicus-inoculated soils, where instead EOX was mineralized 90 to 98%.R. chlorophenolicus mineralized PCP unhindered by the substrate competition offered by the PCP-methylating bacteria indigenously occurring in the soils or by simultaneously inoculated O-methylatingR. rhodochrous.     

Destruction of Deinococcus geothermalis biofilm by photocatalytic ALD and sol-gel TiO2 surfaces

The aim of the present work was to explore possibilities of photocatalytic TiO₂ coating for reducing biofilms on non-living surfaces. The model organism, Deinococcus geothermalis, known to initiate growth of durable, colored biofilms on machine surfaces in the paper industry, was allowed to form biofilms on stainless steel, glass and TiO₂ film coated glass or titanium. Field emission electron microscopy revealed that the cells in the biofilm formed at 45°C under vigorous shaking were connected to the surface by means of numerous adhesion threads of 0.1--0.3 μm in length. Adjacent cells were connected to one another by threads of 0.5--1 μm in length. An ultrastructural analysis gave no indication for the involvement of amorphous extracellular materials (e.g., slime) in the biofilm. When biofilms on photocatalytic TiO₂ surfaces, submerged in water, were exposed to 20 W h m⁻² of 360 nm light, both kinds of adhesion threads were completely destroyed and the D. geothermalis cells were extensively removed (from >10⁷ down to below 10⁶ cells cm⁻²). TiO₂ films prepared by the sol-gel technique were slightly more effective than those prepared by the ALD technique. Doping of the TiO₂ with sulfur did not enhance its biofilm-destroying capacity. The results show that photocatalytic TiO₂ surfaces have potential as a self-cleaning technology for warm water using industries.     

Toxic Bacillus pumilus from indoor air, Recycled Paper Pulp,Norway Spruce, Food Poisoning Outbreaks and Clinical Samples

Forty-four B. pumilus isolates of food poisoning, clinical, environmental and industrial origins were investigated for toxin production using the boar spermatozoan motility assay, previously shown to be a sensitive method for detecting non-protein toxins from B. cereus and B. licheniformis. The three toxic isolates originated from live tree, indoor air and recycled paper pulp and were more toxic than the previously described food poisoning isolates of B. licheniformis, whereas the B. pumilus food poisoning and clinical isolates were lower in toxicity. The type strain also produced inhibitory substances. The toxic substances were insensitive to heat (100 °C, 20 min), to pH 2 or pH 10 and to digestion with pronase. The substances were readily soluble in methanol and chloroform, but less soluble in toluene. Exposure of boar spermatozoa to 1–10 μg ml^–1 (EC₅₀) of methanol soluble substance from the four strains disrupted the plasma membrane permeability barrier, induced abnormalities in the postacrosomal sheath, collapsed the mitochondrial and suppressed cytoplasmic NAD reduction. No change was observed in human peripheral blood lymphocytes exposed to concentrations of B. pumilus extract that affected spermatozoa. The toxin producing isolates were 99.4 to 99.6% similar in 16SrDNA (500 bp) to the type strain and could not be distinguished from the 41 non-toxic isolates by biochemical properties or whole cell fatty acid composition.     

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.     

Potato Crop as a Source of Emetic Bacillus cereus and Cereulide-Induced Mammalian Cell Toxicity

Bacillus cereus, aseptically isolated from potato tubers, were screened for cereulide production and for toxicity on human and other mammalian cells. The cereulide-producing isolates grew slowly, the colonies remained small (∼1 mm), tested negative for starch hydrolysis, and varied in productivity from 1 to 100 ng of cereulide mg (wet weight)⁻¹ (∼0.01 to 1 ng per 10⁵ CFU). By DNA-fingerprint analysis, the isolates matched B. cereus F5881/94, connected to human food-borne illness, but were distinct from cereulide-producing endophytes of spruce tree (Picea abies). Exposure to cell extracts (1 to 10 μg of bacterial biomass ml⁻¹) and to purified cereulide (0.4 to 7 ng ml⁻¹) from the potato isolates caused mitochondrial depolarization (loss of ΔΨm) in human peripheral blood mononuclear cells (PBMC) and keratinocytes (HaCaT), porcine spermatozoa and kidney tubular epithelial cells (PK-15), murine fibroblasts (L-929), and pancreatic insulin-producing cells (MIN-6). Cereulide (10 to 20 ng ml⁻¹) exposed pancreatic islets (MIN-6) disintegrated into small pyknotic cells, followed by necrotic death. Necrotic death in other test cells was observed only after a 2-log-higher exposure. Exposure to 30 to 60 ng of cereulide ml⁻¹ induced K⁺ translocation in intact, live PBMC, keratinocytes, and sperm cells within seconds of exposure, depleting 2 to 10% of the cellular K⁺ stores within 10 min. The ability of cereulide to transfer K⁺ ions across biological membranes may benefit the producer bacterium in K⁺-deficient environments such as extracellular spaces inside plant tissue but is a pathogenic trait when in contact with mammalian cells.     

Anaerobic fluidised bed for the purification of effluents from chemical and mechanical pulping

Anaerobic treatment has seldom been used for wastewaters from the pulp and paper industry and other branches of the chemical industry. Escape of volatile pollutants to the atmosphere, which always occurs during aerobic treatment, is avoided, and much less sludge is being produced than in an aerobic process. The greatest obstacle for using anaerobic treatment in the pulp and paper industry is the large wastewater volume, which necessitates short hydraulic detention times, because the treatment is to occur in an enclosed space. We used solid carrier particles to prevent wash-out of biomass from the reactor at high hydraulic loading, and an up-flow system in order to be able to use very small carrier particles, maximizing the surface area for biomass attachment. In this paper we describe and discuss the results obtained with this type of anaerobic reactor (fluidised bed) at bench and semitechnical scale for wastewaters from pressurized ground wood pulping and paper manufacture, sulphite pulp evaporator condensate and bleach waste. Earlier work with Kraft pulp bleaching effluent and thermomechanical pulping wastewater and evaporator condensates using anaerobic reactors is also discussed. The results obtained thus far show that there are several wastewater streams from the pulping industry, where 60 to 90% of the dissolved organic pollutants (measured as COD_Cr or TOC) was biodegraded within 4 to 24 h. The high strength waste streams (COD_Cr 2000 mg O₂ 1⁻¹) allowed organic space load of 4 to 10 kg COD_Cr m⁻³ reactor volume d⁻¹. With low strength wastes the hydraulic loading was the limiting factor.     

Transformation of chlorinated phenolic compounds in the genusRhodococcus

The ability of strains of the genusRhodococcus to transform chlorinated phenolic compounds was studied. Noninduced cells of several strains ofRhodococcus, covering at least eight species, were found to attack mono-, di-, and trichlorophenols by hydroxylation at theortho position to chlorocatechols. 3-chlorophenol and 4-chlorophenol were converted to 4-chlorocatechol, 2,3-dichlorophenol to 3,4-dichlorocatechol, and 3,4-di-chlorophenol to 4,5-dichlorocatechol. The chlorocatechols accumulated to nearly stoichiometric amounts. Other mono- and dichlorophenols were not transformed. The ability of the strains to hydroxylate chlorophenols correlated with the ability to grow on unsubstituted phenol as the sole source of carbon and energy. SeveralRhodococcus strains attacked chlorophenolic compounds by both hydroxylation and O-methylation. 2,3,4-, 2,3,5- and 3,4,5-trichlorophenol were hydroxylated to trichlorocatechol and then sequentially O-methylated to chloroguaiacol and chloroveratrole. Tetrachlo-rohydroquinone was O-methylated sequentially to tetrachloro-4-methoxy-phenol and tetrachloro-1,4-dimethoxybenzene. Several of the active strains had no known history of exposure to any chloroaromatic compound. Rhodococci are widely distributed in soil and sludge and these results suggest that this genus may play an important role in transformation of chlorinated phenolic compounds in the environment.     

A Novel Sensitive Bioassay for Detection of Bacillus cereus Emetic Toxin and Related Depsipeptide Ionophores

Of the toxins produced by Bacillus cereus, the emetic toxin is likely the most dangerous but, due to the lack of a suitable assay, the least well known. In this paper, a new, sensitive, inexpensive, and rapid bioassay for detection of the emetic toxin of B. cereus is described. The assay is based on the loss of motility of boar spermatozoa upon 24 h of exposure to extracts of emetic B. cereus strains or contaminated food. The paralyzed spermatozoa exhibited swollen mitochondria, but no depletion of cellular ATP or damage to plasma membrane integrity was observed. Analysis of the purified toxin by electrospray tandem mass spectrometry showed that it was a dodecadepsipeptide with a mass fragmentation pattern similar to that described for cereulide. The 50% effective concentration of the purified toxin to boar spermatozoa was 0.5 ng of purified toxin ml of extended boar semen⁻¹. This amount corresponds to 10⁴ to 10⁵ CFU of B. cereus cells. No toxicity was detected for 27 other B. cereus strains up to 10⁸ CFU ml⁻¹. The detection limit for food was 3 g of rice containing 10⁶ to 10⁷ CFU of emetic B. cereus per gram. Effects similar to those provoked by emetic B. cereus toxin were also induced in boar spermatozoa by valinomycin and gramicidin at 2 and 3 ng ml of extended boar semen⁻¹, respectively. The symptoms provoked by the toxin in spermatozoa indicated that B. cereus emetic toxin was acting as a membrane channel-forming ionophore, damaging mitochondria and blocking the oxidative phosphorylation required for the motility of boar spermatozoa.     

Environment-dependent mechanism of dehalogenation by Rhodococcus chlorophenolicus PCP-1

Cells and cell-free preparations of a soil-bioremediating organism, Rhodococcus chlorophenolicus PCP-1, dehalogenated polychlorophenols both under aerobic and anaerobic conditions. Under aerobic conditions molecular O₂ served as the source of oxygen for the dechlorinating para-hydroxylation reaction. Chlorophenols were dehalogenated and para-hydroxylated also under anaerobic conditions by a cyt P-450 enzyme. Water was used anaerobically as an oxygen source but the reaction required the presence of sulphite ions or iodosobenzene. When the dehalogenating enzyme was given a choice between molecular O₂ and water in the presence of sulphite ions or iodosobenzene, the oxygen was preferably derived from water.Dedicated to the honour of Prof. Dr. Norberto Palleroni for the occasion of his 70th birthday Correspondence to: J. S. Uotila