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Applied Microbiology and Biotechnology (v.49, #5)
Degradation of dioxin-like compounds by microorganisms by R.-M. Wittich (pp. 489-499).
Polychlorinated dibenzo-p-dioxins (PCDD) and polychlorinated dibenzofurans (PCDF; PCDD/F, dioxins) have not been commercially produced in bulk amounts, as were polychlorinated biphenyls and other haloaromatic organics. Within the past two decades a lot␣of information has accumulated on the biodegradation of PCDD/F and other dioxin-like compounds because of their toxicity and because of significant environmental concern about many congeners of this class of chemicals. PCDD/F are subjected to reductive dehalogenations leading to less halogenated congeners, which can be attacked efficiently by fungal and bacterial oxidases and dioxygenases. In several cases these compounds can be utilized as carbon and energy sources. Pathways for their enzymatic degradation and the organisation of the corresponding degradative genes have been elucidated. Consequently, biotechnological applications will exploit the degradative potential of such microorganisms for bioremediation of contaminated sites.
Energy uncoupling in microbial growth under substrate-sufficient conditions by Y. Liu (pp. 500-505).
It has been observed that the correlation between ATP and biomass formation is very poor, and the observed growth yield is lowered under substrate-sufficient conditions. This indicates that the excess substrate causes uncoupling between anabolism and catabolism, which leads to the dissipation of non-growth energy. However, a quantitative description of such uncoupling remains elusive. Based on a balanced substrate reaction, a growth-yield model in relation to residual substrate concentration for substrate-sufficient continuous cultures was developed. On the basis of this yield model, a coefficient governing the uncoupling of anabolism and catabolism was defined. A model describing the effect of the residual substrate concentration on this uncoupling coefficient was further proposed. These models agree with experimental data very well. It is clearly shown that, under substrate-sufficient conditions, the variation in growth efficiency is mainly due to energy uncoupling rather than to maintenance energy expenditure.
Physiological aspects of continuous lactic acid fermentations at high dilution rates by U. Kulozik (pp. 506-510).
The effects of the substrate conditions on the volumetric productivity of Lactobacillus helveticus at different cell densities up to 60 g l−1 in a continuous stirred-tank reactor with microfiltration to retain the biomass were investigated. At low dilution rates, D, the steady-state volumetric productivity, r p, gradually increased to a maximum at D = 1.2–1.5 h−1, because of reduced product inhibition. At higher D values, r p unexpectedly decreased, although the substrate conditions further improved. The maxima of r p at different cell densities coincided with a critical specific substrate utilization rate beyond which the cell metabolism seems to be controlled through a catabolic modulator factor, and r p decreases.
Ethanol production using nuclear petite yeast mutants by A. Hutter; S. G. Oliver (pp. 511-516).
Two respiratory-deficient nuclear petites, FY23Δpet191 and FY23Δcox5a, of the yeast Saccharomyces cerevisiae were generated using polymerase-chain-reaction-mediated gene disruption, and their respective ethanol tolerance and productivity assessed and compared to those of the parental grande, FY23WT, and a mitochondrial petite, FY23ρ0. Batch culture studies demonstrated that the parental strain was the most tolerant to exogenously added ethanol with an inhibition constant. K i, of 2.3% (w/v) and a specific rate of ethanol production, q p, of 0.90 g ethanol g dry cells−1 h−1. FY23ρ0 was the most sensitive to ethanol, exhibiting a K i of 1.71% (w/v) and q p of 0.87 g ethanol g dry cells−1 h−1. Analyses of the ethanol tolerance of the nuclear petites demonstrate that functional mitochondria are essential for maintaining tolerance to the toxin with the 100% respiratory-deficient nuclear petite, FY23Δpet191, having a K i of 2.14% (w/v) and the 85% respiratory-deficient FY23Δcox5a, having a K i of 1.94% (w/v). The retention of ethanol tolerance in the nuclear petites as compared to that of FY23ρ0 is mirrored by the ethanol productivities of these nuclear mutants, being respectively 43% and 30% higher than that of the respiratory-sufficient parent strain. This demonstrates that, because of their respiratory deficiency, the nuclear petites are not subject to the Pasteur effect and so exhibit higher rates of fermentation.
High concentrations of fatty acids affect the lipase treatment of softwood thermomechanical pulps by C. Fleet; C. Breuil (pp. 517-522).
Triglycerides, a major class of wood extractives, contribute to the colloidal pitch that initiates pitch deposits. Because industrial or pilot-scale treatments with lipolytic enzymes to reduce triglyceride concentrations in pulp have not been successful in North America, we investigated such treatments at a laboratory scale. Different batches of industrial softwood chemithermomechanical pulps (CTMP) were treated with a range of concentrations of two commercial lipases: Resinase A 2X (Novo Nordisk AG) and Lipidase 10 000 (American Laboratories Inc.). A pilot-scale thermomechanical pulp (TMP) made from the same wood as the CTMP, but without the sodium hydrosulfite used in the CTMP, was also treated with the lipases. While triglycerides decreased in all the pulp treatments, the extent of their hydrolysis varied according to the ratio of triglyceride to the fatty/resin acid fraction. As this ratio can vary significantly in softwood TMP and CTMP, the success of industrial treatments of softwood mechanical pulps by commercial lipases may be related to variations in this ratio. Supporting this, adding linoleic acid to an extractives-free pulp that was spiked with olive oil reduced lipase activity by up to 55%.
Use of azo dye ligand chromatography for the partial purification of a novel extracellular peroxidase from Streptomyces viridosporus T7A by N. S. Burke; D. L. Crawford (pp. 523-530).
Crude peroxidase preparations from the lignocellulose-degrading actinomycete, Streptomyces viridosporus T7A, were shown to decolorize several azo dye isomers and showed a correlation of dye structure to degradability similar to that shown by fungal Mn-peroxidase, an enzyme not previously described in actinomycetes. Addition of the heme-peroxidase inhibitor KCN did not significantly change the ability of the T7A enzyme(s) to decompose the dyes. These results suggest that T7A may produce a Mn- or other peroxidase with similar substrate specificity to Mn-peroxidase. Affinity chromatography using immobilized azo dye isomers was used for purifying peroxidases from T7A. A significantly purified peroxidase preparation was obtained irrespective of the azo dye used. In comparison, concanavalin A lectin affinity chromatography showed very poor binding and resolution for T7A peroxidases. Azo dye affinity purification gave preparations sufficiently purified to allow amino acid microsequencing for two of the bound proteins. N-terminal amino acid sequences were found to share significant homology with a fungal Mn-peroxidase and actinomycete cellulases.
Recombinant bioprocess optimization for heterologous protein production using two-stage, cyclic fed-batch culture by C. C. Chang; D. D. Y. Ryu; C. S. Park; Jeong-Yoon Kim; D. M. Ogrydziak (pp. 531-537).
A two-stage, cyclic fed-batch bioprocess was designed, and its performance evaluated to improve rice α-amylase productivity by the yeast Yarrowia lipolytica SMY2 (MatA, ade1, ura3, xpr2), ATCC 201847, containing a replicative plasmid coding for a rice α-amlyase. Transcription of the recombinant gene is controlled by the XPR2 promoter. The first stage (or growth stage) was operated in the fed-batch mode, and the growth medium, designed to maintain a constant high cell density (i.e., 60 g/l), was fed according to a predetermined and preprogrammed optimal feed rate which, in turn, maintained the specific cell growth rate at an optimal value (i.e., 0.1 h−1). Typically, when the volume in the first stage reached a preset value, a portion of culture broth (i.e., 55%) was transferred to the second stage (or production stage). The remaining cells in the growth stage were then fed with fresh growth medium according to the bioprocess control strategy developed, while induction of α-amylase expression and its production was taking place in the second stage. The second stage was also operated in the fed-batch mode, and the production medium designed to maintain a constant high cell density and high productivity of heterologous protein was fed at a predetermined and preprogrammed rate, which maintained the specific cell growth rate at an optimal level. The volumetric α-amylase productivity achieved (1835 units l−1 h−1) from the two-stage, cyclic fed-batch culture process was twofold higher than that of the fed-batch culture process. The genetic stability of the recombinant strain and the design of optimal media for growth and production stages are also critically important to a successful implementation of the two-stage, cyclic fed-batch process for production of heterologous protein.
Fungal pretreatment by Phanerochaete chrysosporium to reduce the inhibition of methanogenesis by dehydroabietic acid by J. Hodgson; C. Laugero; R. Leduc; M. Asther; S. R. Guiot (pp. 538-544).
The white-rot basidiomycete Phanerochaete chrysosporium BKM-F-1767 was tested for its capacity to degrade dehydroabietic acid (DHA). In anaerobic treatment, this molecule is the most recalcitrant member of the resin acid group, which is known to cause operational problems to anaerobic reactors treating pulp and paper industry wastewaters. In this study the effect of DHA on different parameters, such as growth, ligninolytic enzyme activity, extracellular protein production as well as both glycerol and ammonium consumption by the fungus, was determined. Although the above parameters were affected by the addition of DHA, the results show that the fungus could still produce significant titres of ligninolytic enzymes. The fungus removed 47% of the DHA initially present in the static culture, after 10 days of incubation. Anaerobic toxicity assays showed that the treatment of DHA with P. chrysosporium reduced the methanogenesis and acetogenesis inhibition caused by DHA and allowed improved methane production by the anaerobic bacteria.
Laccase from the white-rot fungus Trametes trogii by A. M. V. Garzillo; M. C. Colao; C. Caruso; C. Caporale; D. Celletti; V. Buonocore (pp. 545-551).
The white-rot fungus Trametes trogii excretes a main laccase showing a molecular mass of 70 kDa, acidic isoelectric point and N-terminal sequence homol-ogous to that of several phenol oxidases. The purified enzyme oxidizes a number of phenolic and non-phenolic compounds; recalcitrant molecules may be converted into substrates by introducing, in the correct position, o-␣or p-orienting ring-activating groups.
Identification of tandemly repeated type VI cellulose-binding domains in an endoglucanase from the 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. 552-559).
Cellulose-binding domains (CBD) play a pivotal role during plant cell wall hydrolysis by cellulases and xylanases from aerobic soil bacteria. Recently we␣have reported the molecular characterisation of a single-domain endoglucanase from Cellvibrio mixtus, suggesting that some cellulases produced by this aerobic bacterium preferentially hydrolyse soluble cellulosic substrates. Here we describe the complete nucleotide sequence of a second cellulase gene, celB, from the soil bacterium C. mixtus. It revealed an open reading frame of 1863 bp that encoded a polypeptide, defined as cellulase B (CelB), with a predicted M r of 66 039. CelB contained a glycosyl hydrolase family 5 catalytic domain at its N terminus followed by two repeated domains, which exhibited sequence identity with type VI CBD previously found in xylanases. Full-length CelB bound to cellulose while catalytically active truncated cellulase derivatives were unable to bind the polysaccharide, confirming that CelB is a modular enzyme and that the type VI CBD homologues were functional. Analysis of the biochemical properties of CelB revealed that the enzyme hydrolyses a range of cellulosic substrates, although it was unable to depolymerise Avicel. We propose that type VI CBD, usually found in xylanases, provide an additional mechanism by which cellulases can accumulate on the surface of the plant cell wall, although they do not potentiate cellulase activity directly. These results demonstrate that C. mixtus, in common with other aerobic bacteria, is able to produce cellulases that are directed to the hydrolysis of cellulose in its natural environment, the plant cell wall.
Production of a new DNA vehicle for gene transfer using site-specific recombination by P. Kreiss; B. Cameron; A. M. Darquet; D. Scherman; J. Crouzet (pp. 560-567).
Supercoiled DNA molecules, minicircles, were produced by in vivo site-specific recombination. They contained exclusively the desired excisable fragment. Recombination was driven by bacteriophage λ integrase from a plasmid substrate containing the attP and attB recombination sites in the same orientation. Conditions for minicircle production within the lysogen Escherichia coli D1210HP were optimised. Up to 1.5 mg minicircles could be produced per litre bacterial culture, and the remaining, unrecombined plasmid comprised less than about 15% of the minicircle produced. However minicircle multimers were also produced, and comprised up to 30% of all minicircles synthesised. The parABCDE′ locus from plasmid RK2 was introduced into the minicircle fragment, resulting in minicircle dimers being reduced to less than 2% of all minicircles. The parA gene encodes a resolvase that catalyses recombination at the multimer resolution site in the parABCDE′ locus. Minicircle multimers were also resolved when parA was introduced downstream from the integrase gene of the λp L transcript in D1210HP together with a multimer resolution site carried by the minicircle fragment.
Overexpression of high-molecular-mass nitrile hydratase from Rhodococcus rhodochrous J1 in recombinant Rhodococcus cells by W. Mizunashi; M. Nishiyama; S. Horinouchi; T. Beppu (pp. 568-572).
High-molecular-mass nitrile hydratase (H-NHase, 530 kDa) is a cobalt-containing enzyme produced by Rhodococcus rhodochrous J1. For efficient production of H-NHase in R. rhodochrous ATCC12674, several plasmids were constructed. The enzyme was produced in the recombinant Rhodococcus cells only in the presence of an upstream region (approximately 4 kb) of the H-NHase gene under the control of the promoter for the amidase-NHase gene cluster from Rhodococcus sp. N-774. Although H-NHase was produced as a soluble protein in the cells, the protein did not show NHase activity. However, when the recombinant R. rhodochrous ATCC12674 cells were cultured in the presence of amide compounds, such as crotonamide and methacrylamide, markedly high NHase activity was detected. Gel-filtration chromatography revealed that the NHases produced by the cells grown in the presence and absence of the amide compounds had a molecular mass of more than 500 kDa and 50–80 kDa respectively. These results suggest that the amide compounds are essential for subunit assembly to form an enzymatically active multimer. By the use of the recombinant expression system, NHase activity 1.7 times higher than that of the original strain, R. rhodochrous J1, was achieved.
Phosphate-independent expression of the carbon-phosphorus lyase activity of Escherichia coli by G. M. Yakovleva; S.-K. Kim; B. L. Wanner (pp. 573-578).
On the basis of mutational analysis, the genes for phosphonate uptake and degradation in Escherichia coli were shown to be organized in a 10.9-kb operon of 14 genes (named phnC to phnP) and induced by phosphate (Pi) starvation [Metcalf and Wanner (1993) J Bacteriol 175: 3430–3442]. The repression of phosphonate utilization by Pi has hindered both the biochemical characterization of the carbon-phosphorus (C-P) lyase activity and the development of improved methods for phosphonate biodegradation in biotechnology. We have cloned the genes phnG to phnP (associated with C-P lyase activity) with the lac promoter to provide expression of C-P lyase in the presence of Pi. A number of strains lacking portions of the phn operon have been constructed. In vivo complementation of the strains, in which phnC to phnP (including both Pn transport and catalysis genes) or phnH to phnP (including only catalysis genes) was deleted, with plasmids carrying various fragments of the phn operon revealed that the expression of phnC-phnP gene products is essential to restore growth on minimal medium with phosphonate as the sole phosphorus source, while phnG-phnM gene products are required for C-P lyase activity as assessed by in vivo methane production from methylphosphonic acid. The minimum size of the DNA required for the whole-cell C-P lyase activity has been determined to be a 5.8-kb fragment, encompassing the phnG to phnM genes. Therefore, there is no requirement for the phnCDE-encoded phosphonate transport system, suggesting that cleavage of the C-P bond may occur on the outer surface of the inner membrane of E. coli cells, releasing the carbon moiety into the periplasm. These data are in agreement with the observation that phosphonates cannot serve as the carbon source for E.␣coli growth.
The effects of space flight on the production of monorden by Humicola fuscoatra WC5157 in solid-state fermentation by K. S. Lam; S. W. Mamber; E. J. Pack; S. Forenza; P. B. Fernandes; D. M. Klaus (pp. 579-583).
The effect of space flight on the production of the antibiotic monorden on two types of agar media, T8 and PG, by Humicola fuscoatra WC5157 was examined on board the US Space Shuttle mission STS-77 in May 1996. Paired space-flight and ground control samples were prepared using identical hardware, protocol, media, and inoculum. Inoculation occurred simultaneously for both groups 2.5 h after launch. The flight and ground samples were allowed to grow for the entire 10-day mission in a dark, thermally controlled (22 °C) environment. Post-flight HPLC analysis of the flight and ground sample extracts indicated that the production of monorden by H. fuscoatra WC5157 in the flight samples was higher than in the ground samples in both agar media. In the T8 medium, the production of monorden in the flight and ground samples was 11.6 ± 3.5 μg and 8.9 ± 1.1 μg respectively (30% increase). In the PG medium, the production of monorden in the flight and ground samples was 23.8 ± 3.3 μg and 8.2 ± 2.2 μg respectively (190% increase). The production of monorden in the flight and ground control samples was confirmed by HPLC-MS analysis.
Mineralization of synthetic humic substances by manganese peroxidase from the white-rot fungus Nematoloma frowardii by M. Hofrichter; K. Scheibner; I. Schneegaß; D. Ziegenhagen; W. Fritsche (pp. 584-588).
A manganese peroxidase preparation from the white-rot fungus Nematoloma frowardii was found to be capable of releasing up to 17% 14CO2 from 14C-labelled synthetic humic substances. The latter were prepared from [U-14C]catechol by spontaneous oxidative polymerization or laccase-catalysed polymerization. The ex-tent of humic substance mineralization was considerably enhanced in the presence of the thiol mediator glutathione (up to 50%). Besides the evolution of 14CO2, the treatment of humic substances with Mn peroxidase resulted in the formation of lower-molecular-mass products. Analysis of residual radioactivity by gel-permeation chromatography demonstrated that the predominant molecular masses of the initial humic substances ranged between 2 kDa and 6 kDa; after treatment with Mn peroxidase, they were reduced to 0.5–2 kDa. The extracellular depolymerization and mineralization of humic substances by the Mn peroxidase system may play an important role in humus turnover of habitats that are rich in basidiomycetous fungi.
Extracellular laccase production during hyphal interactions between Trichoderma sp. and Shiitake, Lentinula edodes by J.-M. Savoie; G. Mata; C. Billette (pp. 589-593).
Lentinula edodes (Berk.) Pegler was cultivated in liquid media containing malt and yeast extract. Extracellular laccase activity, measured in the culture fluids, was 5–18 times higher in cultures incubated for 29 days than in cultures incubated for 24 days. The addition of water-soluble lignin derivatives or Trichoderma sp. in cultures of L. edodes incubated for 11 days increased laccase activity 3- to 20 fold. The higher response was obtained with live mycelium of Trichoderma sp., but cell-free culture fluids of Trichoderma sp. in pure cultures were also effective. Trichoderma sp. induced changes in the laccase isoenzyme pattern as a result of the alteration of laccases secreted by L. edodes and not the induction of new isoforms.
Production of d-hydantoinase by halophilic Pseudomonas sp. NCIM 5109 by S. S. Sudge; K. B. Bastawde; D. V. Gokhale; U. R. Kalkote; T. Ravindranathan (pp. 594-599).
About 1000 bacterial colonies isolated from sea water were screened for their ability to convert dl-5-phenylhydantoin to d(−)N-carbamoylphenylglycine as a criterion for the determination of hydantoinase activity. The strain M-1, out of 11 hydantoinase-producing strains, exhibited the maximum ability to convert dl-5-phenylhydantoin to d(−)N-carbamoylphenylglycine. The strain M-1 appeared to be a halophilic Pseudomonas sp. according to morphological and physiological characteristics. Optimization of the growth parameters revealed that nutrient broth with 2% NaCl was the preferred medium for both biomass and enzyme production. d-Hydantoinase of strain M-1 was not found to be inducible by the addition of uracil, dihydrouracil, β-alanine etc. The optimum temperature for enzyme production was about 25 °C and the organism showed a broad pH optimum (pH 6.5–9.0) for both biomass and hydantoinase production. The organism seems to have a strict requirement of NaCl for both growth and enzyme production. The optimum pH and temperature of enzyme activity were 9–9.5 and 30 °C respectively. The biotransformation under the alkaline conditions allowed the conversion of 80 g l−1 dl-5-phenylhydantoin to 82 g l−1 d(−)N-carbamoylphenylglycine within 24 h with a molar yield of 93%.
The diversion of lactose carbon through the tagatose pathway reduces the intracellular fructose 1,6-bisphosphate and growth rate of Streptococcus bovis by D. R. Bond; B. M. Tsai; J. B. Russell (pp. 600-605).
Twenty strains of Streptococcus bovis grew more slowly on lactose (1.21 ± 0.12 h−1) than on glucose (1.67 ± 0.12 h−1), and repeated transfers or prolonged growth in continuous culture (more than 200 generations each) did not enhance the growth rate on lactose. Lactose transport activity was poorly correlated with growth rate, and slow growth could not be explained by the ATP production rate (catabolic rate). Batch cultures growing on lactose always had less␣intracellular fructose 1,6-bisphosphate (Fru1,6P 2) than cells growing on glucose (6.6 mM compared to 16.7 mM), and this difference could be explained by the pathway of carbon metabolism. Glucose and the glucose moiety of lactose were metabolized by the Embden-Meyerhoff-Parnas (EMP) pathway, but the galactose moiety of lactose was catabolized by the tagatose pathway, a scheme that by-passed Fru1,6P 2. A mutant capable of co-metabolizing lactose and glucose grew more rapidly when glucose was added, even though the total rate of hexose fermentation did not change. Wild-type S. bovis grew rapidly with galactose and melibiose, but these galactose-containing sugars were activated by galactokinase and catabolized via EMP. On the basis of these results, rapid glycolytic flux through the EMP pathway is needed for the rapid growth (more than 1.2 h−1) of S.␣bovis.
Isolation and characterization of two bacteriocins of Lactobacillus acidophilus LF221 by B. Bogovič-Matijašić; I. Rogelj; I. F. Nes; H. Holo (pp. 606-612).
Lactobacillus acidophilus LF221 produced bacteriocin-like activity against different bacteria including some pathogenic and food-spoilage species. Besides some lactic acid bacteria, the following species were inhibited: Bacillus cereus, Clostridium sp., Listeria innocua, Staphylococcus aureus, Streptococcus D. L. acidophilus LF221 produced at least two bacteriocins, acidocin LF221 A and acidocin LF221 B, which were purified by ammonium sulphate precipitation, ion-exchange chromatography, hydrophobic interaction and reverse-phase FPLC. The antibacterial substances were heat-stable, sensitive to proteolytic enzymes (trypsin, pepsin, pronase, proteinase K) and migrated as 3500- to 5000-Da proteins on sodium dodecyl sulphate/polyacrylamide gel electrophoresis. The sequences of 46 amino-terminal amino acid residues of peptide A and 35 of peptide B were determined. Among the residues identified, no modified amino acids were found. No significant homology was found between the amino acid sequences of acidocin LF221 A and other bacteriocins of lactic acid bacteria and 26% homology was found between acidocin LF221 B and brevicin 27. L. acidophilus LF221 may be of interest as a probiotic strain because of its human origin and inhibition of pathogenic bacteria, especially Clostridium difficile.
Pullulan content of the ethanol precipitate from fermented agro-industrial wastes by C. J. Israilides; A. Smith; J. E. Harthill; C. Barnett; G. Bambalov; B. Scanlon (pp. 613-617).
Ethanol-precipitated substances after fermentation of various agro-industrial wastes by Aureobasidium pullulans were examined for their pullulan content. Grape skin pulp extract, starch waste, olive oil waste effluents and molasses served as substrates for the fermentation. A glucose-based defined medium was used for comparison purposes. Samples were analysed by an enzyme-coupled assay method and by high-performance anion-exchange chromatography with pulsed amperometric detection after enzymic hydrolysis with pullulanase. Fermentation of grape skin pulp extract gave 22.3 g l−1 ethanol precipitate, which was relatively pure pullulan (97.4% w/w) as assessed by the coupled-enzyme assay. Hydrolysed starch gave only 12.9 g l−1 ethanol precipitate, which increased to 30.8 g l−1 when the medium was supplemented with NH4NO3 and K2HPO4; this again was relatively pure pullulan (88.6% w/w). Molasses and olive oil wastes produced heterogeneous ethanol-precipitated substances containing small amounts of pullulan, even when supplemented with nitrogen and phosphate. Overall, grape skin pulp should be considered as the best substrate for pullulan production. Starch waste requires several hydrolyis steps to provide a usable carbon source, which reduces its economic attraction as an industrial process.
Biodegradation of atrazine under denitrifying conditions by J. J. Crawford; G. K. Sims; R. L. Mulvaney; M. Radosevich (pp. 618-623).
Anaerobic biodegradation of atrazine by the bacterial isolate M91-3 was characterized with respect to mineralization, metabolite formation, and denitrification. The ability of the isolate to enhance atrazine biodegradation in anaerobic sediment slurries was also investigated. The organism utilized atrazine as its sole source of carbon and nitrogen under anoxic conditions in fixed-film (glass beads) batch column systems. Results of HPLC and TLC radiochromatography suggested that anaerobic biotransformation of atrazine by microbial isolate M91-3 involved hydroxyatrazine formation. Ring cleavage was demonstrated by 14CO2 evolution. Denitrification was confirmed by detection of 15N2 in headspace samples of K15NO3-amended anaerobic liquid cultures. In aquatic sediments, mineralization of uniformly ring-labeled [14C]atrazine occurred in both M91-3-inoculated and uninoculated sediment. Inoculation of sediments with M91-3 did not significantly enhance anaerobic mineralization of atrazine as compared to uninoculated sediment, which suggests the presence of indigenous organisms capable of anaerobic atrazine biodegradation. Results of this study suggest that the use of M91-3 in a fixed-film bioreactor may have applications in the anaerobic removal of atrazine and nitrate from aqueous media.
Rapid atrazine mineralisation in soil slurry and moist soil by inoculation of an atrazine-degrading Pseudomonas sp. strain by M. Wenk; T. Baumgartner; J. Dobovšek; T. Fuchs; J. Kucsera; J. Zopfi; G. Stucki (pp. 624-630).
The evaluation of pesticide-mineralising microorganisms to clean-up contaminated soils was studied with the widely applied and easily detectable compound atrazine, which is rapidly mineralised by several microorganisms including the Pseudomonas sp. strain Yaya 6. The rate of atrazine removal was proportional to the water content of the soil and the amount of bacteria added to the soil. In soil slurry, 6 mg atrazine kg soil−1 was eliminated within 1 day after application of 0.3 g dry weight inoculant biomass kg soil−1 and within 5 days when 0.003 g kg soil−1 was used. In partially saturated soil (60% of the maximal water-holding capacity) 15 mg atrazine kg soil−1 was eliminated within 2 days by 1 g biomass kg soil−1 and within 25 days when 0.01 g biomass kg soil−1 was used. In unsaturated soil, about 60% [U-ring-14C]atrazine was converted to 14CO2 within 14 days. Atrazine was very efficiently removed by the inoculant biomass, not only in soil that was freshly contaminated but also in soil aged with atrazine for up to 260 days. The bacteria exposed to atrazine in unsaturated sterile soil were still active after a starvation period of 240 days: 15 mg newly added atrazine kg soil−1 was eliminated within 5 days.
Microbial degradation of hydrocarbons in soil during aerobic/anaerobic changes and under purely aerobic conditions by C. Löser; H. Seidel; A. Zehnsdorf; U. Stottmeister (pp. 631-636).
Microbial hydrocarbon degradation in soil was studied during periodical aerobic/anaerobic switching and under purely aerobic conditions by using a pilot-scale plant with diesel-fuel-contaminated sand. The system worked according to the percolation principle with controlled circulation of process water and aeration. Periodical switching between 4 h of aerobic and 2 h of anaerobic conditions was achieved by repeated saturation of the soil with water. Whatever the cultivation mode, less than 50% of the diesel was degraded after 650 h because the hydrocarbons were adsorbed. Contrary to expectations, aerobic/anaerobic changes neither accelerated the rate of degradation nor reduced the residual hydrocarbon content of the soil. Obviously the pollutant degradation rate was determined mainly by transport phenomena and less by the efficiency of microbial metabolism. The total mass of oxygen consumed and carbon dioxide produced was greater under aerobic/anaerobic changing than under aerobic conditions, although the mass of hydrocarbons degraded was nearly the same. As shown by an overall balance of microbial growth and by a carbon balance, the growth yield coefficient was smaller during aerobic/anaerobic changes than under aerobic conditions.