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Applied Microbiology and Biotechnology (v.48, #4)
Potato pulp: microbiological characterization, physical modification, and application of this agricultural waste product by F. Mayer; Jan-Otto Hillebrandt (pp. 435-440).
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.
Biodegradation of the pesticide 4,6-dinitro-ortho-cresol by microorganisms in batch cultures and in fixed-bed column reactors by D. Gisi; G. Stucki; K. W. Hanselmann (pp. 441-448).
A mixed culture of microorganisms able to utilize 4,6-dinitro-ortho-cresol (DNOC) as the sole source of carbon, nitrogen and energy was isolated from soil contaminated with pesticides and from activated sludge. DNOC was decomposed aerobically in batch cultures as well as in fixed-bed column reactors. Between 65% and 84% of the substrate nitrogen was released as nitrate into the medium, and 61% of the carbon from uniformly 14C-labelled DNOC was recovered as 14CO2. The mixed microbial culture also decomposed 4-nitrophenol and 2,4-dinitrophenol but not 2,3-dinitrophenol, 2,6-dinitrophenol, 2,4-dinitrotoluene, 2,4-dinitrobenzoic acid or 2-sec-butyl-4,6-dinitrophenol (Dinoseb). Maximal degradation rates for DNOC by the bacterial biofilm immobilized on glass beads in fixed-bed column reactors were 30 mmol day−1 (l reactor volume)−1, leaving an effluent concentration of less than 5 μg l−1 DNOC in the outflowing medium. The apparent K s value of the immobilized mixed culture for DNOC was 17 μM. Degradation was inhibited at DNOC concentrations above 30 μM and it ceased at 340 μM, possibly because of the uncoupling action of the nitroaromatic compound on the cellular energy-transducing mechanism.
The effect of nisin concentration and nutrient depletion on nisin production of Lactococcuslactis by W. S. Kim; R. J. Hall; N. W. Dunn (pp. 449-453).
The kinetics of nisin production was studied in batch cultures using a construct of Lactococcuslactis subsp. lactis C2SmPrt−, containing a transposon (TnNip) that encodes nisin production. The introduction of TnNip into C2SmPrt−significantly lowered the specific growth rate and the maximum A 620 reached was reduced from 15.2 to 11.0. The effect of nisin concentration and nutrient depletion on nisin production of the construct, C2SmPrt−(TnNip), was examined. Nisin production was found to be inhibited by high concentrations of nisin, when grown in excess nutrient, even though growth of the culture continued because nutrient limitation was not operating. However, in low nutrient concentrations nisin production was limited by nutrient depletion. The specific growth rate of C2SmPrt−(TnNip) was altered, by using different nutrient concentrations and different sugars, in order to examine the relationship between nisin production and growth. Nisin production was shown to be growth-associated for most of growth, but near the end of growth, when the specific growth rate was 0.05 h−1 or less, the production ceased.
Cytochrome c peroxidase from a methylotrophic yeast: physiological role and isolation by M. V. Gonchar; L. B. Kostryk; A. A. Sibirny (pp. 454-458).
Mutant strains of the methylotrophic yeast Hansenula polymorpha defective in catalase (cat) and in glucose repression of alcohol oxidase synthesis (gcr1) have been isolated following multiple UV mutagenesis steps. One representative gcr1 cat mutant C-105 grows during batch cultivation in a glucose/methanol medium. However, growth is preceded by a prolonged lag period. C-105 and other gcr1 cat mutants do not grow on methanol medium without an alternative carbon source. A large collection of second-site suppressor catalase-defective (scd) revertants were isolated with restored ability for methylotrophic growth (Mth+) in the absence of catalase activity. These Mth+ gcr1 cat scd strains utilize methanol as a sole source of carbon and energy, although biomass yields are reduced relative to the wild-type strain. In contrast to the parental C-105 strain, H2O2 does not accumulate in the methanol medium of the revertants. We show that restoration of methylotrophic growth in the suppressor strains is strongly correlated with increased levels of the alternative H2O2-destroying enzyme, cytochrome c peroxidase. Cytochrome c peroxidase from cell-free extracts of one of the scd revertants has been purified to homogeneity and crystallized.
Identification and quantification of radical reaction intermediates by electron spin resonance spectrometry of laccase-catalyzed oxidation of wood fibers from beech (Fagus sylvatica) by C. Felby; B. R. Nielsen; P. O. Olesen; L. H. Skibsted (pp. 459-464).
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.
Characterization of Leuconostoc mesenteroides NRRL B-512F dextransucrase (DSRS) and identification of amino-acid residues playing a key role in enzyme activity by V. Monchois; M. Remaud-Simeon; R. R. B. Russell; P. Monsan; R.-M. Willemot (pp. 465-472).
Dextransucrase (DSRS) from Leuconostoc mesenteroides NRRL B-512F is a glucosyltransferase that catalyzes the synthesis of soluble dextran from sucrose or oligosaccharides when acceptor molecules, like maltose, are present. The L. mesenteroides NRRL B-512F dextransucrase-encoding gene (dsrS) was amplified by the polymerase chain reaction and cloned in an overexpression plasmid. The characteristics of DSRS were found to be similar to the characteristics of the extracellular dextransucrase produced by L. mesenteroides NRRL B-512F. The enzyme also exhibited a high homology with other glucosyltransferases. In order to identify critical amino acid residues, the DSRS sequence was aligned with glucosyltransferase sequences and four amino acid residues were selected for site- directed mutagenesis experiments: aspartic acid 511, aspartic acid 513, aspartic acid 551 and histidine 661. Asp-511, Asp-513 and Asp-551 were independently replaced with asparagine and His-661 with arginine. Mutation at Asp-511 and Asp-551 completely suppressed dextran and oligosaccharide synthesis activities, showing that at least two carboxyl groups (Asp-511 and Asp-551) are essential for the catalysis process. However, glucan-binding properties were retained, showing that DSRS has a two-domain structure like other glucosyltransferases. Mutations at Asp-513 and His-661 resulted in greatly reduced dextransucrase activity. According to amino acid sequence alignments of glucosyltransferases, α-amylases or cyclodextrin glucanotransferases, His-661 may have a hydrogen-bonding function.
Possible roles for a non-modular, thermostable and proteinase-resistant cellulase from the mesophilic aerobic soil bacterium Cellvibrio mixtus by C. M. G. A. Fontes; J. H. Clarke; G. P. Hazlewood; T. H. Fernandes; H. J. Gilbert; L. M. A. Ferreira (pp. 473-479).
The widespread presence of cellulose-binding domains in cellulases from aerobic bacteria and fungi suggests the existence of a strong selective pressure for the retention of these non-catalytic modules. The complete nucleotide sequence of the cellulase gene, celA, from the aerobic soil bacterium Cellvibrio mixtus, was determined. It revealed an open reading frame of 1089 bp that encoded a polypeptide, defined as cellulase A (CelA), of M r 41 548. CelA displayed features characteristic of an endo-β-1,4-glucanase, rapidly decreasing the viscosity of the substrate while releasing only moderate amounts of reducing sugar. Deletion studies in celA revealed that removal of 78 nucleotides from the 5′ end or 75 from the 3′ end of the gene led to the complete loss of cellulase activity of the encoded polypeptides. The deduced primary structure of CelA revealed an N-terminal signal peptide followed by a region that exhibited significant identity with the catalytic domains of cellulases belonging to glycosyl hydrolase family 5. These data suggest that CelA is a single-domain endoglucanase with no distinct non-catalytic cellulose-binding domain. Analysis of the biochemical properties of CelA revealed that the enzyme hydrolyses a range of soluble cellulosic substrates, but was inactive against Avicel, xylan or any other hemicellulose. CelA was resistant to proteolytic inactivation by pancreatic proteinases and surprisingly, in view of its mesophylic origin, was shown to be thermostable. The significance of these findings in relation to the role of single-domain cellulases in plant cell wall hydrolysis by aerobic microorganisms is discussed.
Biosynthetic production of type II fish antifreeze protein: fermentation by Pichia pastoris by M. C. Loewen; X. Liu; P. L. Davies; A. J. Daugulis (pp. 480-486).
Sea raven type II antifreeze protein (SRAFP) is one of three different fish antifreeze proteins isolated to date. These proteins are known to bind to the surface of ice and inhibit its growth. To solve the three-dimensional structure of SRAFP, study its ice-binding mechanism, and as a basis for engineering these molecules, an efficient system for its biosynthetic production was developed. Several different expression systems have been tested including baculovirus, Escherichia coli and yeast. The latter, using the methylotrophic organism Pichia pastoris as the host, was the most productive. In shake-flask cultures the levels of SRAFP secreted from Pichia were up to 5 mg/l. The recombinant protein has an identical activity to SRAFP from sea raven serum. In order to increase yields further, four different strategies were tested in 10-l fermentation vessels, including: (1) optimization of pH and dissolved oxygen, (2) mixed feeding of methanol and glycerol with Muts clones, (3) supplementation of amino acid building blocks, and (4) methanol feeding with Mut+ clones. The mixed-feeding/Muts strategy proved to be the most efficient with SRAFP yields reaching 30 mg/l.
Efficient production of a functional mouse/human chimeric Fab′ against human urokinase-type plasminogen activator by Bacillus brevis by Y. Inoue; T. Ohta; H. Tada; S. Iwasa; S. Udaka; H. Yamagata (pp. 487-492).
Expression/secretion vectors for the production of Fab′ and single-chain (sc) Fab′ by Bacillus brevis have been constructed. For the production of Fab′, the cDNAs encoding the L chain and Fd′ fragment (Fd with the hinge region) of a mouse-human chimeric Fab′ against human urokinase-type plasminogen activator were fused directly with the translation-start and signal-peptide-encoding regions of the mwp gene, the gene for one of the major cell-wall proteins of Bacillus brevis. The two fused genes were placed tandemly downstream from the promoter of the cell-wall protein gene operon (cwp) of B. brevis. For the production of scFab′, the two cDNAs were linked with a synthetic oligonucleotide encoding a flexible peptide linker of 17 or 24 amino acids, and fused with the translation start and signal-peptide-encoding regions of the mwp gene. Fab′ was efficiently produced by B. brevis, being accumulated at a level of 100 mg/l in the culture medium in a simple shake-flask culture, which is the highest level obtained so far for a gram-positive bacterium. On the other hand, the scFab′ remained at a level of a few milligrams per liter in the culture medium. The Fab′ produced by B. brevis showed comparable antigen-binding activity to that of the parental antibody. The L chain and Fd′ fragment, constituting the Fab′, had the correct N-terminal amino acid sequences. These results indicate that B. brevis is a very promising host for the production of native Ig fragments.
Competition of plasmid-bearing Pseudomonas putida strains catabolizing naphthalene via various pathways in chemostat culture by A. E. Filonov; W. A. Duetz; A. V. Karpov; R. R. Gaiazov; I. A. Kosheleva; A. M. Breure; I. F. Filonova; J. G. van Andel; A. M. Boronin (pp. 493-498).
Plasmid-carrying Pseudomonas putida strains degrade naphthalene through different biochemical pathways. The influence of various combinations of host bacteria and plasmids on growth characteristics and competitiveness of P. putida strains was studied in chemostat culture at a low dilution rate (D=0.05 h−1) with naphthalene as the sole source of carbon and energy. Under naphthalene limitation, the plasmid-bearing strains degrading naphthalene that use catechol 1,2-dioxygenase for catechol oxidation (ortho pathway), were the most competitive. The strains bearing plasmids that control naphthalene catabolism via catechol 2,3-dioxygenase (meta pathway), were less competitive. Under these conditions the strain carrying plasmid pBS4, which encodes for naphthalene catabolism via gentisic acid, was the least competitive.
Genetic immobilization of cellulase on the cell surface of Saccharomyces cerevisiae by T. Murai; M. Ueda; H. Atomi; Y. Shibasaki; N. Kamasawa; M. Osumi; T. Kawaguchi; M. Arai; A. Tanaka (pp. 499-503).
We tried genetically to immobilize cellulase protein on the cell surface of the yeast Saccharomyces cerevisiae in its active form. A cDNA encoding FI-carboxymethylcellulase (CMCase) of the fungus Aspergillus aculeatus, with its secretion signal peptide, was fused with the gene encoding the C-terminal half (320 amino acid residues from the C terminus) of yeast α-agglutinin, a protein involved in mating and covalently anchored to the cell wall. The plasmid constructed containing this fusion gene was introduced into S. cerevisiae and expressed under the control of the glyceraldehyde-3-phosphate dehydrogenase promoter from S. cerevisiae. The CMCase activity was detected in the cell pellet fraction. The CMCase protein was solubilized from the cell wall fraction by glucanase treatment but not by sodium dodecyl sulphate treatment, indicating the covalent binding of the fusion protein to the cell wall. The appearance of the fused protein on the cell surface was further confirmed by immunofluorescence microscopy and immunoelectron microscopy. These results proved that the CMCase was anchored on the cell wall in its active form.
Effect of growth rate on α-amylase production by Streptomyces sp. IMD 2679 by H. E. M. Mc Mahon; C. T. Kelly; W. M. Fogarty (pp. 504-509).
The α-amylase of Streptomyces sp. IMD 2679 was subject to catabolite repression. Four different growth rates were achieved when the organism was grown at 40 °C and 55 °C in the presence and absence of cobalt, with an inverse relationship between α-amylase production and growth rate. Highest α-amylase yields (520 units/ml) were obtained at the lowest growth rate (0.062 h−1), at 40 °C in the absence of cobalt, while at the highest growth rate (0.35 h−1), at 55 °C in the presence of cobalt, α-amylase production was decreased to 150 units/ml. As growth rate increased, the rate of specific utilisation of the carbon source maltose also increased, from 46 to 123 μg maltose (mg biomass)−1 h−1. The pattern and levels of α-glucosidase (the enzyme degrading maltose) detected intracellularly in each case, indicate that growth rate effectively controls the rate of feeding of glucose to the cell, and thus catabolite repression.
Changes in alginate molecular mass distributions, broth viscosity and morphology of Azotobacter vinelandii cultured in shake flasks by C. Peña; N. Campos; E. Galindo (pp. 510-515).
The effect of different aeration conditions during the culture of Azotobacter vinelandii on the production and molecular mass of alginate was evaluated in shake flasks. In baffled flasks, the bacteria grew faster and produced less alginate (1.5 g/l) than in conventional (unbaffled) flasks (4.5 g/l). The viscosity of the culture broth was also influenced by the type of flask. Higher final viscosities were attained in unbaffled flasks [520 cP (520 mPa s)] as compared to baffled flasks (30 cP). This latter phenomenon was closely related to the changes in the molecular mass distribution. In either cases, the mean molecular mass increased with culture age; however, at the end of the fermentation, the mean molecular mass of the alginate obtained in unbaffled flasks was fivefold higher than that obtained in baffled flasks. As the culture proceeded, the cells of Azotobacter grown in unbaffled flasks increased in diameter, whereas those cultured in baffled flasks decreased in size.
Analysis of the exopolysaccharides produced by Lactobacillus delbrueckii subsp. bulgaricus NCFB 2772 grown in continuous culture on glucose and fructose by G. J. Grobben; W. H. M. van Casteren; H. A. Schols; A. Oosterveld; G. Sala; M. R. Smith; J. Sikkema; J. A. M. de Bont (pp. 516-521).
The exopolysaccharides produced by Lactobacillus delbrueckii subsp. bulgaricus NCFB 2772 grown in defined medium were investigated. At equal cell densities, the strain produced 95 mg l−1 exopolysaccharides with glucose and 30 mg l−1 with fructose as the carbohydrate source. High-performance size-exclusion chromatography of the exopolysaccharides produced on glucose showed the presence of two fractions with relative molecular masses (M r) of 1.7 × 106 and 4 × 104 in almost equal amounts. The exopolysaccharides produced on fructose contained mainly a fraction of low M r of 4 × 104. The high-M r fraction of the purified exopolysaccharides produced on glucose appeared to have a sugar composition of galactose, glucose and rhamnose in the molar ratio of 5:1:1, whereas the low-M r weight fraction contained galactose, glucose and rhamnose in the molar ratio of approximately 11:1:0.4. The purified exopolysaccharide fractions produced on fructose showed comparable ratios. The high-molecular-mass fractions contained terminally linked galactose, 1,2,3-linked galactose, 1,3,4-linked galactose, 1,3-linked glucose and terminally linked rhamnose. The low-molecular-mass fractions contained mainly 1,3-linked galactose and 1,6-linked galactose and lower amounts of other sugar linkages. The production of the high-M r fractions appeared to be dependent on the carbohydrate source, whereas the low-M r fractions were produced more continuously.
Influence of phosphate on rhamnose-containing exopolysaccharide rheology and production by Klebsiella I-714 by J. Farrés; G. Caminal; J. López-Santín (pp. 522-527).
Physiological conditions enhancing rhamnose-containing polysaccharide synthesis by Klebsiella I-714 were studied in batch culture (0.3-l and 2-l bioreactors). The four carbon sources tested, sucrose, sorbitol, Neosorb and Cerelose, allowed exopolysaccharide production. Larger amounts of polymer were produced when high carbon/nitrogen ratios and complex nitrogen sources were used. Exopolysaccharide synthesis was greatest at 30 °C, which was a suboptimal growth temperature. A reduction in the phosphate content of the medium enhanced rhamnose-containing polysaccharide production. When the initial carbon source concentration was augmented, byproducts other than exopolysaccharide were formed. Rhamnose-containing polysaccharide rheology can be modulated by changing the phosphate content of the medium.
Isolation and characterization of a marine bacterium capable of utilizing 2-methylphenanthrene by M. Gilewicz; Ni'matuzahroh; T. Nadalig; H. Budzinski; P. Doumenq; V. Michotey; J. C. Bertrand (pp. 528-533).
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.
Microbial colonization of polyurethane foam matrices in horizontal-flow anaerobic immobilized-sludge reactor by M. B. Varesche; M. Zaiat; L. G. T. Vieira; R. F. Vazoller; E. Foresti (pp. 534-538).
This paper presents the anaerobic biomass characterization and the bacterial framework inside polyurethane foam matrices taken from a horizontal-flow anaerobic immobilized-sludge (HAIS) reactor treating a glucose-based substrate. Ultrastructure polyurethane foam analyses carried out using scanning electron microscopy (SEM) in samples treated with hexamethyldisylazane showed three different patterns of biomass retention inside the polyurethane foam matrices: micro-granules ranging from 270 μm to 470 μm were entrapped in the porous medium thin multi-cellular films were attached to the inner surface, and individual cells adhered to the support. The use of SEM and epifluorescence microscopy permitted inferences to be made on the bacteriological composition of the immobilized sludge formed by different morphotypes (rods, cocci and filaments) and on the ecological significance of their framework inside the matrices. Polyurethane matrices were found to offer excellent conditions for anaerobic growth and retention, favoring the flux of substrate and products. Such outstanding characteristics were confirmed by the short start-up period observed during the operation of the HAIS reactor.
Relationship between cadmium sensitivity and degree of plasma membrane fatty acid unsaturation in Saccharomyces cerevisiae by N. G. Howlett; S. V. Avery (pp. 539-545).
The sensitivity of Saccharomyces cerevisiae to the redox-active metal copper has recently been found to be influenced by cellular fatty acid composition. This study sought to investigate whether fatty acid composition affected plasma membrane permeabilisation and whole-cell toxicity induced by the redox-inactive metal cadmium. S. cerevisiae NCYC 1383 was enriched with the polyunsaturated fatty acids linoleate (18:2) and linolenate (18:3) by growth in 18:2- or 18:3-supplemented medium. Incorporation of the exogenous fatty acids resulted in them comprising more than 65% of the total fatty acids in plasma membrane lipids. Inhibition of cell division in the presence of Cd(NO3)2 was accentuated by growth in the presence of a polyunsaturated fatty acid. Furthermore, susceptibility to Cd2+-induced plasma membrane permeabilisation increased with the degree of fatty acid unsaturation. Thus, during exposure to Cd2+, K+ efflux from 18:2- and 18:3-enriched cells was up to 2.5-fold or 3-fold greater, respectively than that from unsupplemented cells. In addition, reductions in cell viability during exposure to Cd2+ were most marked in polyunsaturated-fatty-acid-supplemented cells. At certain times, unsupplemented Cd2+-exposed cells displayed up to 7-fold greater viability than supplemented Cd2+-exposed cells. The study demonstrates that the toxicity of the redox-inactive metal Cd2+ towards S. cerevisiae becomes markedly amplified with increased cellular and plasma membrane fatty acid unsaturation.
Novel l-specific cleavage of the urethane bond of t -butoxycarbonylamino acids by whole cells of Corynebacterium aquaticum by T. Ohshiro; M. Shinji; Y. Morita; Y. Takayama; Y. Izumi (pp. 546-548).
Microorganisms capable of cleaving the urethane bond of t-butoxycarbonyl (Boc) amino acids in a whole-cell reaction were screened among stock cultures, and Corynebacterium aquaticum IFO12154 was the most promising. The conversion of Boc-Ala to Ala was stimulated by CoSO4 in the medium and reaction mixture. The optimum whole-cell concentration was 25 mg lyophilized cells/ml. Boc-l-Met was the best substrate for this reaction, and other Boc-L-amino acids, as well as benzyloxycarbonyl-l-amino acids with hydrophobic residues, were also good substrates. Boc-d- and Z-d-amino acids were inert. When the reactions had proceeded for 24 h with each substrate at 10 mM, the molar conversion rates from Boc-l-, dl- and d-Met were 100%, 50%, and 0% respectively. From 150 mM Boc-l-Met, 143 mM l-Met was formed at a molar yield of 95.3%.
The ability of soil-borne fungi to degrade organophosphonate carbon-to-phosphorus bonds by T. Krzyśko-Lupicka; W. Strof; K. Kubś; M. Skorupa; P. Wieczorek; B. Lejczak; P. Kafarski (pp. 549-552).
The ability of a wide variety of soil-borne fungal strains to degrade four structurally different com pounds containing PC bonds, namely the naturally occurring amino acid ciliatine, the popular herbicide glyphosate, phosphonoacetic acid and 2-amino-3-phosphonopropionic acid, was studied in order to show that soil fungi may play an important role in the biodegradation of organophosphonates. Most of the strains appeared to utilize ciliatine as the sole source of phosphorus for growth. Only a limited number of strains were able to grow on the other phosphonates used in this work. The strains of Trichoderma harzianum, Scopulariopsis sp. and Aspergillus niger chosen for more detailed study show the ability to degrade ciliatine, glyphosate and also amino(3-methoxyphenyl)mehtylphosphonic acid effectively.
Complete degradation of tetrachloroethene in coupled anoxic and oxic chemostats by J. Gerritse; G. Kloetstra; A. Borger; G. Dalstra; A. Alphenaar; J. C. Gottschal (pp. 553-562).
Anaerobic tetrachloroethene(C2Cl4)-dechlorinating bacteria were enriched in slurries from chloroethene-contaminated soil. With methanol as electron donor, C2Cl4 and trichloroethene (C2HCl3) were reductively dechlorinated to cis-1,2-dichloroethene (cis-C2H2Cl2), whereas, with l-lactate or formate, complete dechlorination of C2Cl4 via C2HCl3, cis-C2H2Cl2 and chloroethene (C2H3Cl) to ethene was obtained. In oxic soil slurries with methane as a substrate, complete co-metabolic degradation of cis-C2H2Cl2 was obtained, whereas C2HCl3 was partially degraded. With toluene or phenol both of the above were readily co-metabolized. Complete degradation of C2Cl4 was obtained in sequentially coupled anoxic and oxic chemostats, which were inoculated with the slurry enrichments. Apparent steady states were obtained at various dilution rates (0.02–0.4 h−1) and influent C2Cl4-concentrations (100–1000 μM). In anoxic chemostats with a mixture␣of␣formate and glucose as the carbon and electron source, C2Cl4 was transformed at high rates (above␣140 μmol l−1 h−1, corresponding to 145 nmol Cl− min−1 mg protein−1) into cis-C2H2Cl2 and C2H3Cl. Reductive dechlorination was not affected by addition of 5 mM sulphate, but strongly inhibited after addition of 5 mM nitrate. Our results (high specific dechlorination rates and loss of dechlorination capacity in the absence of C2Cl4) suggest that C2Cl4-dechlorination in the anoxic chemostat was catalysed by specialized dechlorinating bacteria. The partially dechlorinated intermediates, cis-C2H2Cl2 and C2H3Cl, were further degraded by aerobic phenol-metabolizing bacteria. The maximum capacity for chloroethene (the sum of tri-, di- and monochloro derivatives removed) degradation in the oxic chemostat was 95 μmol l−1 h−1 (20 nmol min−1 mg protein−1), and that of the combined anoxic → oxic reactor system was 43.4 μmol l−1 h−1. This is significantly higher than reported thus far.
Comparative biotransformation of pentachlorophenol in soils by solid substrate cultures of Lentinula edodes by B. C. Okeke; A. Paterson; J. E. Smith; I. A. Watson-Craik (pp. 563-569).
Sterilised and non-sterilised soils contaminated with pentachlorophenol (PCP) were inoculated with solid substrate cultures of Lentinula edodes LE2 (“shiitake” mushroom) to simulate monoculture bioremediation treatments and treatments in which the fungus competes with natural microflora. With monocultures of L. edodes, rates of PCP depletion were rapid for the initial 4 weeks and, although thereafter the rate decreased, 99% biotransformation was obtained in 10 weeks. In mixed culture, PCP biotransformation by L.␣edodes was markedly slower and only 42% of the PCP was depleted after 10 weeks. Maximal rates of PCP transformation, biomass (ergosterol) accumulation and oxidative enzymes (phenol oxidase and manganese-peroxidase) production were observed after 2 weeks of incubation. In monocultures, phenol oxidase activity was 195.5 U g−1 and Mn-peroxidase 138.4 U g−1. In mixed cultures, fungal enzyme activities were markedly lower: 70.33 U g−1 for phenol oxidase and 85.0 g−1 for Mn-peroxidase. Analyses of soil metabolites after 10 weeks revealed that monocultures of L.edodes had eliminated both PCP and pentachloroanisole. Pentachloroanisole, however, was detected in soils with the mixed microflora. Both dechlorination and mineralisation of the xenobiotic compound were effected by L. edodes LE2.
The effect of low temperature (5–29 °C) and adaptation on the methanogenic activity of biomass by R. H. Kettunen; J. A. Rintala (pp. 570-576).
The influence of low temperature (5–29 °C) on the methanogenic activity of non-adapted digested sewage sludge and on temperature/leachate-adapted biomass was assayed by using municipal landfill leachate, intermediates of anaerobic degradation (propionate) and methane precursors (acetate, H2/CO2) as substrates. The temperature dependence of methanogenic activity could be described by Arrhenius-derived models. However, both substrate and adaptation affected the temperature dependence. The adaptation of biomass in a leachate-fed upflow anaerobic sludge-blanket reactor at approximately 20 °C for 4 months resulted in a sevenfold and fivefold increase of methanogenic activity at 11 °C and 22 °C respectively. Both acetate and H2/CO2 were methanized even at 5 °C. At 22 °C, methanogenic activities (acetate 4.8–84 mM) were 1.6–5.2 times higher than those at 11 °C. The half-velocity constant (K s) of acetate utilization at 11 °C was one-third of that at 22 °C while a similar K i was obtained at both temperatures. With propionate (1.1–5.5 mM) as substrate, meth‐anogenic activities at 11 °C were half those at 22 °C. Furthermore, the residual concentration of the substrates was not dependent on temperature. The results suggest that the adaptation of biomass enables the achievement of a high treatment capacity in the anaerobic process even under psychrophilic conditions.