Isolation and characterization of a new spore-forming sulfate-reducing bacterium growing by complete oxidation of catechol

A new mesophilic sulfate-reducing bacterium, strain Groll, was isolated from a benzoate enrichment culture inoculated with black mud from a freshwater ditch. The isolate was a spore-forming, rod-shaped, motile, gram-positive bacterium. This isolate was able of complete oxidation of several aromatic compounds including phenol, catechol, benzoate, p-and m-cresol, benzyl alcohol and vanillate. With hydrogen and carbon dioxide, formate or O-methylated aromatic compounds, autotrophic growth during sulfate reduction or homoacetogenesis was demonstrated. Lactate was not used as a substrate. SO _inf4 ^sup2- , SO _inf3 ^sup2- , and S₂O _inf3 ^sup2- were utilized as electron acceptors. Although strain Groll originated from a freshwater habitat, salt concentrations of up to 30 g·l^-1 were tolerated. The optimum temperature for growth was 35–37°C. The G+C content of DNA was 42.1 mol%. This isolate is described as a new species of the genus Desulfotomaculum.     

Isolation and characterization of a thermophilic benzoate-degrading,sulfate-reducing bacterium,Desulfotomaculum thermobenzoicum sp. nov.

A new thermophilic sulfate-reducing bacterium, strain TSB, that was spore-forming, rod-shaped, slightly motile and gram-positive, was isolated from a butyrate-containing enrichment culture inoculated with sludge of a thermophilic methane fermentation reactor. This isolate could oxidize benzoate completely. Strain TSB also oxidized some fatty acids and alcohols. SO _inf4 ^sup2- , SO _inf3 ^sup2- , S₂O _inf3 ^sup2- and NO _inf3 ^sup- were utilized as electron acceptors. With pyruvate or lactate the isolate grew without an external electron acceptor and produced acetate. The optimum temperature for growth was 62°C. The G+C content of DNA was 52.8 mol%. This isolate is described as a new species, Desulfotomaculum thermobenzoicum.     

Isolation of a Bacterial Strain Able To Degrade Branched Nonylphenol

Conventional enrichment of microorganisms on branched nonylphenol (NP) as only carbon and energy source yielded mixed cultures able to grow on the organic compound. However, plating yielded no single colonies capable, alone or in combination with other isolates, of degrading the NP in liquid culture. Therefore, a special approach was used, referred to as “serial dilution-plate resuspension,” to reduce culture complexity. In this way, one isolate, TTNP3, tentatively identified as a Sphingomonas sp., was found to be able to grow on NP in liquid culture. Remarkably, this isolate was able to be filtered through a 0.45-μm-pore-diameter filter. Moreover, isolate TTNP3 did not form visible colonies on mineral medium with NP, and it formed visible colonies on R₂A agar only after a prolonged incubation of 1 week. High-performance liquid chromatography and gas chromatography-mass spectroscopy analysis of the culture media indicated that the strain starts the degradation of NP with a fission of the phenol ring and preferably uses the para isomer of NP and not the ortho isomer. No distinct accumulation of an intermediary product could be observed.     

Replica filter assay of human beta-adrenergic receptors expressed in E.coli

We have developed a replica filter assay that permits the direct identification of bacterial colonies expressing membrane receptors. E.coli transformed with appropriate phage or plasmid vectors containing human beta-adrenergic receptor cDNAs were grown on LB/agar plates. Bacterial colonies transferred onto nitrocellulose filters showed specific [125I]-iodocyanopindolol binding. beta-adrenergic receptors expressed in bacteria retained their pharmacological properties when transferred onto filters. This strategy, which is considerably simplified and more rapid compared to similar methods based upon expression of receptor genes in eukaryotic cells, may be a useful tool for cloning membrane receptors.     

Benzene Oxidation Coupled to Sulfate Reduction

Highly reduced sediments from San Diego Bay, Calif., that were incubated under strictly anaerobic conditions metabolized benzene within 55 days when they were exposed initially to 1 (mu)M benzene. The rate of benzene metabolism increased as benzene was added back to the benzene-adapted sediments. When a [(sup14)C]benzene tracer was included with the benzene added to benzene-adapted sediments, 92% of the added radioactivity was recovered as (sup14)CO(inf2). Molybdate, an inhibitor of sulfate reduction, inhibited benzene uptake and production of (sup14)CO(inf2) from [(sup14)C]benzene. Benzene metabolism stopped when the sediments became sulfate depleted, and benzene uptake resumed when sulfate was added again. The stoichiometry of benzene uptake and sulfate reduction was consistent with the hypothesis that sulfate was the principal electron acceptor for benzene oxidation. Isotope trapping experiments performed with [(sup14)C]benzene revealed that there was no production of such potential extracellular intermediates of benzene oxidation as phenol, benzoate, p-hydroxybenzoate, cyclohexane, catechol, and acetate. The results demonstrate that benzene can be oxidized in the absence of O(inf2), with sulfate serving as the electron acceptor, and suggest that some sulfate reducers are capable of completely oxidizing benzene to carbon dioxide without the production of extracellular intermediates. Although anaerobic benzene oxidation coupled to chelated Fe(III) has been documented previously, the study reported here provides the first example of a natural sediment compound that can serve as an electron acceptor for anaerobic benzene oxidation.     

Plasmid transformation and replica filter plating of Acholeplasma laidlawii

The restriction deficient mutant 8195 of Acholeplasma laidlawii strain JA1 was transformed by the promiscuous streptococcal plasmid vector pNZ18 at a frequency of 4 x 10(-4)/cfu. The plasmid was maintained without structural rearrangements but was lost in the absence of a selection pressure, i.e. kanamycin or neomycin. Transformed primary colonies were easily recognized due to a different colony morphology. Replica filter plating, previously not obtained with mycoplasmas, was achieved using pNZ18 as a marker by incubating the replica filters with the cell side down on the new agar plates. These findings should greatly facilitate the genetic and functional analysis of A. laidlawii.     

Evidence That Ceriporiopsis subvermispora Degrades Nonphenolic Lignin Structures by a One-Electron-Oxidation Mechanism

The white-rot fungus Ceriporiopsis subvermispora is able to degrade nonphenolic lignin structures but appears to lack lignin peroxidase (LiP), which is generally thought to be responsible for these reactions. It is well established that LiP-producing fungi such as Phanerochaete chrysosporium degrade nonphenolic lignin via one-electron oxidation of its aromatic moieties, but little is known about ligninolytic mechanisms in apparent nonproducers of LiP such as C. subvermispora. To address this question, C. subvermispora and P. chrysosporium were grown on cellulose blocks and given two high-molecular-weight, polyethylene glycol-linked model compounds that represent the major nonphenolic arylglycerol-(beta)-aryl ether structure of lignin. The model compounds were designed so that their cleavage via one-electron oxidation would leave diagnostic fragments attached to the polyethylene glycol. One model compound was labeled with (sup13)C at C(inf(alpha)) of its propyl side chain and carried ring alkoxyl substituents that favor C(inf(alpha))-C(inf(beta)) cleavage after one-electron oxidation. The other model compound was labeled with (sup13)C at C(inf(beta)) of its propyl side chain and carried ring alkoxyl substituents that favor C(inf(beta))-O-aryl cleavage after one-electron oxidation. To assess fungal degradation of the models, the high-molecular-weight metabolites derived from them were recovered from the cultures and analyzed by (sup13)C nuclear magnetic resonance spectrometry. The results showed that both C. subvermispora and P. chrysosporium degraded the models by routes indicative of one-electron oxidation. Therefore, the ligninolytic mechanisms of these two fungi are similar. C. subvermispora might use a cryptic LiP to catalyze these C(inf(alpha))-C(inf(beta)) and C(inf(beta))-O-aryl cleavage reactions, but the data are also consistent with the involvement of some other one-electron oxidant.     

Regulation of the Synthesis and Activity of Ammonia Monooxygenase in Nitrosomonas europaea by Altering pH To Affect NH(inf3) Availability

The obligately ammonia-oxidizing bacterium Nitrosomonas europaea was incubated in medium containing 50 mM ammonium. Changes in the concentration of nitrite, the pH, and the NH(inf4)(sup+)- and NH(inf2)OH-dependent O(inf2) uptake activities of the cell suspension were monitored. The NH(inf4)(sup+)-dependent O(inf2) uptake activity doubled over the first 3 h of incubation and then slowly returned to its original level over the following 5 h. The extent of stimulation of NH(inf4)(sup+)-dependent O(inf2) uptake activity was decreased by lowering the initial pH of the medium. Radiolabeling studies demonstrated that the stimulation of NH(inf4)(sup+)-dependent O(inf2) uptake activity involved de novo synthesis of several polypeptides. Under O(inf2)-limited conditions, the stimulated NH(inf4)(sup+)-dependent O(inf2) uptake activity was stabilized. Rapid, controlled, and predictable changes in this activity could be caused by acidification of the medium in the absence of ammonia oxidation. These results indicate that the NH(inf4)(sup+)-dependent O(inf2) uptake activity in N. europaea is strongly regulated in response to NH(inf3) concentration.     

Anaerobic degradation of halogenated benzoic acids by photoheterotrophic bacteria

Abstract From light-exposed enrichment cultures containing benzoate and a mixture of chlorobenzoates, a pure culture was obtained able to grow with 3-chlorobenzoate (3-CBA) or 3-bromobenzoate (3-BrBA) as the sole growth substrate anaerobically in the light. The thus isolated organism is a photoheterotroph, designated isolate DCP3. It is preliminarily identified as a Rhodopseudomonas palustris strain. It differs from Rhodopseudomonas palustris WS17, the only other known photoheterotroph capable of using 3-CBA for growth, in its independence of benzoate for growth with 3-CBA and in its wider substrate range: if grown on 3-CBA, it can also use 2-CBA, 4-CBA or 3,5-CBA.     

Degradation of Monochlorinated and Nonchlorinated Aromatic Compounds under Iron-Reducing Conditions

The capacity for Fe(sup3+) to serve as an electron acceptor in the microbial degradation of monochlorinated and nonchlorinated aromatic compounds was investigated in anoxic sediment enrichments. The substrates tested included phenol, benzoate, aniline, their respective monochlorinated isomers, o-, m-, and p-cresol, and all six dimethylphenol isomers. Phenol and 2-, 3-, and 4-chlorophenol were utilized by anaerobic microorganisms, with the concomitant reduction of Fe(sup3+) to Fe(sup2+). The amount of Fe(sup2+) produced in the enrichments was 89 to 138% of that expected for the stoichiometric degradation of these substrates to CO(inf2), suggesting complete mineralization at the expense of Fe reduction. Under Fe-reducing conditions, there was initial loss of benzoate and 3-chlorobenzoate but not of 2- or 4-chlorobenzoate. In addition, there was initial microbial utilization of aniline but not of the chloroaniline isomers. There was also initial loss of o-, m-, and p-cresol in our enrichments. None of the dimethylphenol isomers, however, was degraded within 300 days. Furthermore, we tested the capacity of an Fe-reducing, benzoate-grown culture of Geobacter metallireducens GS-15 to utilize monochlorinated benzoates and phenols. G. metallireducens was able to degrade benzoate and phenol but none of their chlorinated isomers, suggesting that the degradation of chlorophenols in our sediment enrichments may be due to novel Fe-reducing organisms that have yet to be isolated.     

Comparison of the Efficacies of Chloromethane, Methionine, and S-Adenosylmethionine as Methyl Precursors in the Biosynthesis of Veratryl Alcohol and Related Compounds in Phanerochaete chrysosporium

The effect on veratryl alcohol production of supplementing cultures of the lignin-degrading fungus Phanerochaete chrysosporium with different methyl-(sup2)H(inf3)-labelled methyl precursors has been investigated. Both chloromethane (CH(inf3)Cl) and l-methionine caused earlier initiation of veratryl alcohol biosynthesis, but S-adenosyl-l-methionine (SAM) retarded the formation of the compound. A high level of C(sup2)H(inf3) incorporation into both the 3- and 4-O-methyl groups of veratryl alcohol occurred when either l-[methyl-(sup2)H(inf3)]methionine or C(sup2)H(inf3)Cl was present, but no significant labelling was detected when S-adenosyl-l-[methyl-(sup2)H(inf3)]methionine was added. Incorporation of C(sup2)H(inf3) from C(sup2)H(inf3)Cl was strongly antagonized by the presence of unlabelled l-methionine; conversely, incorporation of C(sup2)H(inf3) from l-[methyl-(sup2)H(inf3)]methionine was reduced by CH(inf3)Cl. These results suggest that l-methionine is converted either directly or via an intermediate to CH(inf3)Cl, which is utilized as a methyl donor in veratryl alcohol biosynthesis. SAM is not an intermediate in the conversion of l-methionine to CH(inf3)Cl. In an attempt to identify the substrates for O methylation in the metabolic transformation of benzoic acid to veratryl alcohol, the relative activities of the SAM- and CH(inf3)Cl-dependent methylating systems on several possible intermediates were compared in whole mycelia by using isotopic techniques. 4-Hydroxybenzoic acid was a much better substrate for the CH(inf3)Cl-dependent methylation system than for the SAM-dependent system. The CH(inf3)Cl-dependent system also had significantly increased activities toward both isovanillic acid and vanillyl alcohol compared with the SAM-dependent system. On the basis of these results, it is proposed that the conversion of benzoic acid to veratryl alcohol involves para hydroxylation, methylation of 4-hydroxybenzoic acid, meta hydroxylation of 4-methoxybenzoic acid to form isovanillic acid, and methylation of isovanillic acid to yield veratric acid.     

From hybridization image to numerical values: a practical,high throughput quantification system for high density filter hybridizations

Hybridization to sets of bacterial colonies or PCR products arrayed on high density filters is used in a number of experimental schemes. In many cases it is desirable to collect quantitative information (‘hybridization signatures’) rather than indications on ‘positive’ and ‘negative’ colonies. We present a practical system, based on an imaging plate analyser and a customized version of commercial software, that makes such quantification feasible, and define its performance in terms of reproducibility and linearity. The system is far superior to methods based on autoradiography and should be useful in many projects that involve the increasingly popular high density filter format.     

Kinetics of Inhibition of Methane Oxidation by Nitrate, Nitrite, and Ammonium in a Humisol

The kinetics of inhibition of CH(inf4) oxidation by NH(inf4)(sup+), NO(inf2)(sup-), and NO(inf3)(sup-) in a humisol was investigated. Soil slurries exhibited nearly standard Michaelis-Menten kinetics, with half-saturation constant [K(infm(app))] values for CH(inf4) of 50 to 200 parts per million of volume (ppmv) and V(infmax) values of 1.1 to 2.5 nmol of CH(inf4) g of dry soil(sup-1) h(sup-1). With one soil sample, NH(inf4)(sup+) acted as a simple competitive inhibitor, with an estimated K(infi) of 8 (mu)M NH(inf4)(sup+) (18 nM NH(inf3)). With another soil sample, the response to NH(inf4)(sup+) addition was more complex and the inhibitory effect of NH(inf4)(sup+) was greater than predicted by a simple competitive model at low CH(inf4) concentrations (<50 ppmv). This was probably due to NO(inf2)(sup-) produced through NH(inf4)(sup+) oxidation. Added NO(inf2)(sup-) was inherently more inhibitory of CH(inf4) oxidation at low CH(inf4) concentrations, and more NO(inf2)(sup-) was produced as the CH(inf4)-to-NH(inf4)(sup+) ratio decreased and the competitive balance shifted. NaNO(inf3) was a noncompetitive inhibitor of CH(inf4) oxidation, but inhibition was evident only at >10 mM concentrations, which also altered soil pHs. Similar concentrations of NaCl were also inhibitory of CH(inf4) oxidation, so there may be no special inhibitory mechanism of nitrate per se.     

Metabolism of Melamine by Klebsiella terragena

Experiments were conducted to determine the pathway of melamine metabolism by Klebsiella terragena (strain DRS-1) and the effect of added NH(inf4)(sup+) on the rates and extent of melamine metabolism. In the absence of added NH(inf4)(sup+), 1 mM melamine was metabolized concomitantly with growth. Ammeline, ammelide, cyanuric acid, and NH(inf4)(sup+) accumulated transiently in the culture medium to maximal concentrations of 0.012 mM, 0.39 mM, trace levels, and 0.61 mM, respectively. In separate incubations, in which cells were grown on either ammeline or ammelide (in the absence of NH(inf4)(sup+)), ammeline was metabolized without a lag while ammelide metabolism was observed only after 3 h. In the presence of 6 mM added NH(inf4)(sup+) (enriched with 5% (sup15)N), ammeline, ammelide, and cyanuric acid accumulated transiently to maximal concentrations of 0.002 mM, 0.47 mM, and trace levels, respectively, indicating that the added NH(inf4)(sup+) had little effect on the relative rates of triazine metabolism. These data suggest that the primary mode of melamine metabolism by K. terragena is hydrolytic, resulting in successive deaminations of the triazine ring. Use of (sup15)N-enriched NH(inf4)(sup+) allowed estimates of rates of triazine-N mineralization and assimilation of NH(inf4)(sup+)-N versus triazine-N into biomass. A decrease in the percent (sup15)N in the external NH(inf4)(sup+) pool, in conjunction with the accumulation of ammelide and/or triazine-derived NH(inf4)(sup+) in the culture medium, suggests that the initial reactions in the melamine metabolic pathway may occur outside the cytoplasmic membrane.     

Dissimilatory Nitrate Reduction in Anaerobic Sediments Leading to River Nitrite Accumulation

Recent studies on Northern Ireland rivers have shown that summer nitrite (NO(inf2)(sup-)) concentrations greatly exceed the European Union guideline of 3 (mu)g of N liter(sup-1) for rivers supporting salmonid fisheries. In fast-flowing aerobic small streams, NO(inf2)(sup-) is thought to originate from nitrification, due to the retardation of Nitrobacter strains by the presence of free ammonia. Multiple regression analyses of NO(inf2)(sup-) concentrations against water quality variables of the six major rivers of the Lough Neagh catchment in Northern Ireland, however, suggested that the high NO(inf2)(sup-) concentrations found in the summer under warm, slow-flow conditions may result from the reduction of NO(inf3)(sup-). This hypothesis was supported by field observations of weekly changes in N species. Here, reduction of NO(inf3)(sup-) was observed to occur simultaneously with elevation of NO(inf2)(sup-) levels and subsequently NH(inf4)(sup+) levels, indicating that dissimilatory NO(inf3)(sup-) reduction to NH(inf4)(sup+) (DNRA) performed by fermentative bacteria (e.g., Aeromonas and Vibrio spp.) is responsible for NO(inf2)(sup-) accumulation in these large rivers. Mechanistic studies in which (sup15)N-labelled NO(inf3)(sup-) in sediment extracts was used provided further support for this hypothesis. Maximal concentrations of NO(inf2)(sup-) accumulation (up to 1.4 mg of N liter(sup-1)) were found in sediments deeper than 6 cm associated with a high concentration of metabolizable carbon and anaerobic conditions. The (sup15)N enrichment of the NO(inf2)(sup-) was comparable to that of the NO(inf3)(sup-) pool, indicating that the NO(inf2)(sup-) was predominantly NO(inf3)(sup-) derived. There is evidence which suggests that the high NO(inf2)(sup-) concentrations observed arose from the inhibition of the DNRA NO(inf2)(sup-) reductase system by NO(inf3)(sup-).     

A Simple, High-Precision, High-Sensitivity Tracer Assay for N(inf2) Fixation

We describe a simple, precise, and sensitive experimental protocol for direct measurement of N(inf2) fixation using the conversion of (sup15)N(inf2) to organic N. Our protocol greatly reduces the limit of detection for N(inf2) fixation by taking advantage of the high sensitivity of a modern, multiple-collector isotope ratio mass spectrometer. This instrument allowed measurement of N(inf2) fixation by natural assemblages of plankton in incubations lasting several hours in the presence of relatively low-level (ca. 10 atom%) tracer additions of (sup15)N(inf2) to the ambient pool of N(inf2). The sensitivity and precision of this tracer method are comparable to or better than those associated with the C(inf2)H(inf2) reduction assay. Data obtained in a series of experiments in the Gotland Basin of the Baltic Sea showed excellent agreement between (sup15)N(inf2) tracer and C(inf2)H(inf2) reduction measurements, with the largest discrepancies between the methods occurring at very low fixation rates. The ratio of C(inf2)H(inf2) reduced to N(inf2) fixed was 4.68 (plusmn) 0.11 (mean (plusmn) standard error, n = 39). In these experiments, the rate of C(inf2)H(inf2) reduction was relatively insensitive to assay volume. Our results, the first for planktonic diazotroph populations of the Baltic, confirm the validity of the C(inf2)H(inf2) reduction method as a quantitative measure of N(inf2) fixation in this system. Our (sup15)N(inf2) protocols are comparable to standard C(inf2)H(inf2) reduction procedures, which should promote use of direct (sup15)N(inf2) fixation measurements in other systems.     

Isolation of Escherichia coli O157:H7 from retail fresh meats and poultry.

A total of 896 samples of retail fresh meats and poultry was assayed for Escherichia coli serogroup O157:H7 by a hydrophobic grid membrane filter-immunoblot procedure developed specifically to isolate the organism from foods. The procedure involves several steps, including selective enrichment, filtration of enrichment culture through hydrophobic grid membrane filters, incubation of each filter on nitrocellulose paper on selective agar, preparation of an immunoblot (by using antiserum to E. coli O157:H7 culture filtrate) of each nitrocellulose paper, selection from the filters of colonies which corresponded to immunopositive sites on blots, screening of isolates by a Biken test for precipitin lines from metabolites and antiserum to E. coli O157:H7 culture filtrate, and confirmation of isolates as Vero cell cytotoxic E. coli O157:H7 by biochemical, serological, and Vero cell cytotoxicity tests. E. coli O157:H7 was isolated from 6 (3.7%) of 164 beef, 4 (1.5%) of 264 pork, 4 (1.5%) of 263 poultry, and 4 (2.0%) of 205 lamb samples. One of 14 pork samples and 5 of 17 beef samples contaminated with the organism were from Calgary, Alberta, Canada, grocery stores, whereas all other contaminated samples were from Madison, Wis., retail outlets. This is the first report of the isolation of E. coli O157:H7 from food other than ground beef, and results indicate that the organism is not a rare contaminant of fresh meats and poultry.     

Denitrifying Bacteria in the Earthworm Gastrointestinal Tract and In Vivo Emission of Nitrous Oxide (N(inf2)O) by Earthworms

Earthworms (Lumbricus rubellus and Octolasium lacteum) and gut homogenates did not produce CH(inf4), and methanogens were not readily culturable from gut material. In contrast, the numbers of culturable denitrifiers averaged 7 x 10(sup7) and 9 x 10(sup6) per g (dry weight) of gut material for L. rubellus and O. lacteum, respectively; these values were 256- and 35-fold larger than the numbers of culturable denitrifiers in the soil from which the earthworms were obtained. Anaerobically incubated earthworm gut homogenates supplemented with nitrate produced N(inf2)O at rates exceeding that of soil homogenates. Furthermore, living earthworms emitted N(inf2)O under aerobic conditions, and N(inf2)O emission was stimulated by acetylene. For earthworms collected from a mildly acidic (pH 6) beech forest soil, the rates of N(inf2)O emission for earthworms and soil averaged 884 and 2 pmol per h per g (fresh weight), respectively. In contrast, for earthworms collected from a more acidic (pH 4.6) oak-beech forest soil, N(inf2)O emission by earthworms and soil averaged 145 and 45 pmol per h per g (fresh weight), respectively. Based on the extrapolation of this data, earthworms accounted for an estimated 16 and 0.25% of the total N(inf2)O produced at the stand level of these beech and oak-beech forest soils, respectively.     

Properties of Desulfovibrio carbinolicus sp. nov. and Other Sulfate-Reducing Bacteria Isolated from an Anaerobic-Purification Plant

Several sulfate-reducing microorganisms were isolated from an anaerobic-purification plant. Four strains were classified as Desulfovibrio desulfuricans, Desulfovibrio sapovorans, Desulfobulbus propionicus, and Desulfovibrio sp. The D. sapovorans strain contained poly-beta-hydroxybutyrate granules and seemed to form extracellular vesicles. A fifth isolate, Desulfovibrio sp. strain EDK82, was a gram-negative, non-spore-forming, nonmotile, curved organism. It was able to oxidize several substrates, including methanol. Sulfate, sulfite, thiosulfate, and sulfur were utilized as electron acceptors. Pyruvate, fumarate, malate, and glycerol could be fermented. Because strain EDK82 could not be ascribed to any of the existing species, a new species, Desulfovibrio carbinolicus, is proposed. The doubling times of the isolates were determined on several substrates. Molecular hydrogen, lactate, propionate, and ethanol yielded the shortest doubling times (3.0 to 6.3 h). Due to the presence of support material in an anaerobic filter system, these species were able to convert sulfate to sulfide very effectively at a hydraulic retention time as short as 0.5 h.