Degradation of 4-aminobenzenesulfonate by a two-species bacterial coculture

The mutualistic interactions in a 4-aminobenzenesulfonate (sulfanilate) degrading mixed bacterial culture were studied. This coculture consisted of Hydrogenophaga palleronii strain S1 and Agrobacterium radiobacter strain S2. In this coculture only strain S1 desaminated sulfanilate to catechol-4-sulfonate, which did not accumulate in the medium but served as growth substrate for strain S2. During growth in batch culture with sulfanilate as sole source of carbon, energy, nitrogen and sulfur, the relative cell numbers (colony forming units) of both strains were almost constant. None of the strains reached a cell number which was more than threefold higher than the cell number of the second strain. A mineral medium with sulfanilate was inoculated with different relative cell numbers of both strains (relative number of colony forming units S1:S2 2200:1 to 1:500). In all cases, growth was found and the proportion of both strains moved towards an about equal value of about 3:1 (strain S1:strain S2). In contrast to the coculture, strain S1 did not grow in a mineral medium in axenic culture with 4-aminobenzenesulfonate or any other simple organic compound tested. A sterile culture supernatant from strain S2 enabled strain S1 to grow with 4-aminobenzenesulfonate. The same growth promoting effect was found after the addition of a combination of 4-aminobenzoate, biotin and vitamin B₁₂. Strain S1 grew with 4-aminobenzenesulfonate plus the three vitamins with about the same growth rate as the mixed culture in a mineral medium. When (resting) cells of strain S1 were incubated in a pure mineral medium with sulfanilate, up to 30% of the oxidized sulfanilate accumulated as catechol-4-sulfonate in the culture medium. In contrast, only minor amounts of catechol-4-sulfonate accumulated when strain S1 was grown with 4ABS in the presence of the vitamins.Abbreviations 4ABS 4-aminobenzenesulfonate - CFU colony forming units - 4CS catechol-4-sulfonate - 4HB 4-hydroxybenzoate     

Bacterial catabolism of sulfanilic acid via catechol-4-sulfonic acid

Abstract A sulfanilic acid (4-aminobenzenesulfonic acid) degrading culture consisting of two strains (strain S1 and S2), was studied. Only strain S1 was able to attack sulfanilic acid. When strain S1 was cultavated in a mineral medium with sulfanilic acid an intensive violet colour was observed. The accumulating metabolite was isolated from the culture supernatant. By comparison with an authentic compound the metabolite was identified as catechol-4-sulfonic acid by thin layer and high performance liquid chromatography and by UV- and H-NMR spectroscopy. The occurrence of catechol-4-sulfonic acid indicates that there is no release of the sulfonic group before ring cleavage.     

Isolation of a Bacterial Strain with the Ability To Utilize the Sulfonated Azo Compound 4-Carboxy-4′-Sulfoazobenzene as the Sole Source of Carbon and Energy

A bacterial strain (strain S5) which grows aerobically with the sulfonated azo compound 4-carboxy-4′-sulfoazobenzene as the sole source of carbon and energy was isolated. This strain was obtained by continuous adaptation of “Hydrogenophaga palleronii” S1, which has the ability to grow aerobically with 4-aminobenzenesulfonate. Strain S5 probably cleaves 4-carboxy-4′-sulfoazobenzene reductively under aerobic conditions to 4-aminobenzoate and 4-aminobenzene-sulfonate, which are mineralized by previously established degradation pathways.It is generally assumed that sulfonated azo dyes are not degraded under aerobic conditions (14). Nevertheless, there have been some reports which suggest a conversion of certain sulfonated azo dyes under aerobic conditions (3, 7, 8, 13, 15). Furthermore, certain carboxylated analogs of sulfonated azo compounds are utilized aerobically as the sole source of carbon and energy by specifically adapted bacteria (11, 12, 16, 17). However, unequivocal evidence for the productive mineralization of a sulfonated azo compound by bacteria is lacking. In the present article the first observation of the utilization of a sulfonated azo compound as the sole source of carbon and energy by a bacterial strain is reported.Previously, a mixed bacterial culture which mineralizes sulfanilate (4-aminobenzenesulfonate) was isolated. This coculture consisted of the strains “Hydrogenophaga palleronii” S1 and Agrobacterium radiobacter S2 (4, 5). Because sulfanilate occurs as an azoaryl structural element in many azo dyes, it was of interest whether this mixed culture could adopt the ability to reduce azo bonds and release sulfanilate as growth substrate. Therefore, the model sulfonated azo compound 4-carboxy-4′-sulfoazobenzene (CSAB) was synthesized by nitro-amine condensation starting with sulfanilic acid and 4-nitrobenzoic acid (1). The precipitated CSAB was separated from the reaction mixture by filtration and purified by repeated dissolution in alkali and precipitation with acid. The identity and purity of the bright orange product were analyzed by UV-visible light spectroscopy, elementary analysis, and high-pressure liquid chromatography (HPLC). For the solid material obtained, molar extinction coefficients of 23.74 and 1.13 mM⁻¹ cm⁻¹ in water were determined at the wavelengths of 326 and 434 nm, respectively. The elementary analytic results were consistent with the structure of CSAB. The purity of the preparation was tested by HPLC with a reversed-phase column and a solvent gradient from 1 to 90% (vol/vol) methanol and 0.3% (vol/vol) H₃PO₄. A single band which showed absorbance at a wavelength of 326 nm was eluted. At 210 nm a minor contaminant (about 15% of the signal intensity of CSAB) was detected. This compound was clearly different from either 4-nitrobenzoate or sulfanilate.The mixed culture was grown in repeated batch cultures in a mineral medium with sulfanilate (5 mM). About every 2 weeks the culture was transferred (1:10 [vol/vol]) to fresh medium, in which the sulfanilate concentration was subsequently reduced and the CSAB concentration increased (±0.5 mM each). The color of the azo dye disappeared after 2 months. The culture was transferred to a solid mineral medium with CSAB as the sole source of carbon. From this culture was obtained strain S5, which grew aerobically with the sulfonated azo compound CSAB as its sole source of carbon and energy and with a doubling time of 9.5 h (Fig. ​(Fig.1).1). The complete disappearance of the dye was demonstrated by the loss of the orange color from the medium and by HPLC analysis, whereas CSAB was not degraded in a sterile control flask. Based on its colony morphology and the results obtained with the commercial identification system Biolog GN, this strain strongly resembled “H. palleronii” S1. Recently, it was demonstrated that, in the presence of low concentrations of biotin, cyanocobalamin, and 4-aminobenzoate, strain S1 also grows in axenic culture with sulfanilate (2). Therefore the adaptation experiment was repeated in the presence of these three substances with a pure culture of strain S1. This experiment also resulted in the isolation of a strain which grew in axenic culture with CSAB as the sole source of carbon and energy. Open in a separate windowFIG. 1Aerobic growth of strain S5 with CSAB as the sole source of carbon and energy. The growth was determined photometrically (OD₅₄₆), and the turnover of CSAB was measured by HPLC with a reversed-phase column and a solvent gradient consisting of H₂O, methanol, and 0.3% H₃PO₄ with increasing concentrations of methanol (1 to 90%). An OD₅₄₆ of 1 corresponded to 0.33 mg of protein ml⁻¹.To ensure that the genetic backgrounds of strains S5 and S1 were identical, the genes for the 16S rRNAs were amplified by PCR with different universal primers (6) and sequenced in comparison to the corresponding gene from the type strain, H. palleronii DSM 63. It was found that the sequences from strains S1 and S5 were > 99.8% identical (there were only two discrepancies between the two sequences), but they showed only 97.7 to 97.9% identity with the 16S rRNA gene from H. palleronii DSM 63. It was therefore concluded that strain S5 was derived from strain S1 and that the strains do not belong to the species H. palleronii.A reductive cleavage of the azo bond of CSAB would result in the formation of 4-aminobenzoate and sulfanilate. Like the parent strain, S1, strain S5 grew in the presence of sulfanilate, 4-aminobenzoate, and 4-sulfocatechol. The doubling times with these compounds were 6.2 to 6.4 h. We therefore investigated whether reductive cleavage of CSAB by strain S5 occurs. Strain S5 was grown aerobically with 5 mM CSAB, and cell extracts were prepared (10) in different buffers. These cell extracts were incubated aerobically in cuvettes containing 50 mM Tris-HCl buffer (pH 8.0), 0.5 mM CSAB, 1 mM NADH, or 1 mM NADPH and with various mixtures of possible cofactors. The enzyme activity was measured spectrophotometrically at the absorption maximum for CSAB (at a wavelength of 434 nm), but no significant decrease in absorbance was observed. Neither addition of a membrane fraction nor performing the enzyme assays under anaerobic conditions (9) improved the turnover of CSAB in the cell-free system. Furthermore, there was no significant increase in azo reductase activity when harvested cells were resuspended in the culture supernatant instead of Tris-HCl buffer.The maximal enzyme activities observed for cell extracts were only about 30% of the activities found for intact cells. This suggested that during the disruption of the cells some important components of the azo reductase system were destroyed or some cofactors were present in only limiting quantities.Because it was difficult to obtain reproducible enzyme activities with cell extracts, the turnover of CSAB by resting cells was investigated. Cells of strain S5 were grown with CSAB (5 mM), harvested by centrifugation, resuspended in Tris-HCl at an optical density at 546 nm (OD₅₄₆) of 5.3, and incubated in a water bath shaker (140 rpm; 30°C) with 0.5 mM CSAB (Fig. ​(Fig.2).2). Thus, the transient accumulation of two metabolites in the supernatants was observed by reversed-phase HPLC (column size, 250 by 4.6 mm) (SIL 100; Grom, Herrenberg, Germany). The solvent system consisted of a solvent gradient with increasing concentrations of methanol, starting with 1% (vol/vol) methanol, 98.9% (vol/vol) water, and 0.1% H₃PO₄. The flow rate was 0.7 ml min⁻¹. The metabolites formed were identified as sulfanilate and 4-sulfocatechol by comparison of their retention times and in situ UV-visible light-spectra with authentic standards. Surprisingly, the concentration of 4-sulfocatechol in the medium increased (and decreased) during the experiment more rapidly than the concentration of sulfanilate (Fig. ​(Fig.2).2). 4-Sulfocatechol also temporarily accumulated when resting cells of strain S1 were incubated with sulfanilate (4, 5). This suggested that in the resting-cell assay the initial activity of the sulfanilate-converting enzyme was higher than the activity of the 4-sulfocatechol-oxidizing enzyme protocatechuate-3,4-dioxygenase type II. Presumably, the activity of the sulfanilate-converting enzyme decreased during the experiment more rapidly than the activity of protocatechuate-3,4-dioxygenase type II. No accumulation of 4-aminobenzoate or protocatechuate was found by HPLC analysis during the experiment. In a control experiment with cells of strain S1 grown with 4-aminobenzenesulfonate, no turnover of CSAB was observed by HPLC analysis. Open in a separate windowFIG. 2Conversion of CSAB (•) to sulfanilate (▪) and 4-sulfocatechol (□) by resting cells of strain S5. Strain S5 was grown in a mineral medium with CSAB as the sole source of carbon and energy, and resting cells were prepared as described in the text.The detection of sulfanilate derived from CSAB suggested a reductive cleavage of CSAB, yielding sulfanilate as one of the reduction products. This reaction should also proceed in the absence of oxygen. Therefore, resting cells were incubated under anaerobic conditions with CSAB. Surprisingly, the rate of CSAB turnover under anaerobic conditions was <2% of the turnover rate under aerobic conditions.A further indication of a reductive cleavage of CSAB into sulfanilate and 4-aminobenzoate was obtained by growing strain S5 with CSAB or a complex medium (HPG medium) (4). When the cells were grown in a mineral medium with CSAB and the turnover of the substrates was analyzed by HPLC, it was found that resting cells converted CSAB, 4-aminobenzoate, or 4-aminobenzenesulfonate with specific activities of 0.012, 0.026, and 0.011 μmol min⁻¹ mg of protein⁻¹, respectively. In contrast, after growth of the cells in HPG medium, these activities were only 0.007, 0.010, and 0.003 μmol min⁻¹ mg of protein⁻¹, respectively. Incubation of resting cells with CSAB and different potential inhibitors of ring cleavage dioxygenases showed that the turnover of CSAB was almost completely inhibited by the addition of 8-hydroxyquinoline or 2,2′-bipyridyl (1 mM each). The presence of 4-nitrocatechol (0.25 mM) also resulted in a pronounced reduction of the rate of CSAB turnover (6% of the rate in the absence of the inhibitor). In this system as well the formation of 4-sulfocatechol was observed.The degradation of sulfanilate and 4-aminobenzoate by strain S1 has been previously studied (5). The proposed degradation pathway for CSAB and its reduction products is shown in Fig. ​Fig.3.3. Open in a separate windowFIG. 3Proposed pathway for the degradation of CSAB by strain S5. 4AB, 4-aminobenzoate; 4ABS, 4-aminobenzenesulfonate (sulfanilate); 3,4DHB, 3,4-dihydroxybenzoate (protocatechuate); 4SC, 4-sulfocatechol; 2H4CMSA, 2-hydroxy-4-carboxymuconic semialdehyde; 3SM, 3-sulfomuconate; 4SL, 4-carboxymethyl-4-sulfobut-2-en-4-olide (4-sulfolactone); MA, maleylacetate; 3OA, 3-oxoadipate; TCC, tricarboxylic acid cycle.To obtain some information about the substrate specificity, resting cells were incubated with CSAB, 4,4′-dicarboxyazobenzene (DCAB), 4-hydroxy-4′-sulfoazobenzene, methyl orange [4-(N,N-dimethyl)-4′-sulfoazobenzene; color index (C.I.) 13025], orange II {4-[(2-hydroxy-1-naphthalenyl)azo]-benzenesulfonic acid; C.I. 15510}, or sunset yellow FCF {6-hydroxy-5-[(4-sulfophenyl)azo]-2-naphthol-6-sulfonic acid; FD&C no. 6; C.I. 15985}. Of these compounds, only CSAB and DCAB were converted by resting cells. DCAB was also utilized by strain S5 as the sole source of carbon and energy. Furthermore, no growth of strain S5 was found with acid black 24 and 52, acid blue 113, acid red 1, amaranth, direct red 81, direct yellow 4 and 50, mordant yellow 3, and naphthol blue black.The results presented in this study suggest that bacterial cultures with the ability to aerobically degrade simple sulfonated azo dyes may be obtained after preadaptation to sulfonated aminoaromatics and/or when reductive cleavage of the azo bond gives rise to an aerobically assimilable aminoaromatic structure, like 4-aminobenzoate. This selection scheme circumvents the problems observed during attempts to adapt bacteria with the ability to degrade carboxylated azo compounds for the degradation of sulfonated azo compounds (12). The ability of strain S5 to mineralize CSAB suggests that it is possible to degrade sulfonated azo dyes under aerobic conditions if biological systems which can grow and can mineralize the reduction products are available.

Nucleotide sequence accession number.

The nucleotide sequences for the 16S rRNAs from strains S5 and S1 have been deposited in the GenBank data library under accession no. {"type":"entrez-nucleotide","attrs":{"text":"AF019037","term_id":"3004497","term_text":"AF019037"}}AF019037 and {"type":"entrez-nucleotide","attrs":{"text":"AF019073","term_id":"3004495","term_text":"AF019073"}}AF019073, respectively.     

Horizontal transfer of catabolic plasmids in the process of naphthalene biodegradation in model soil systems

The process of naphthalene degradation by indigenous, introduced, and transconjugant strains was studied in laboratory soil microcosms. Conjugation transfer of catabolic plasmids was demonstrated in naphthalene-contaminated soil. Both indigenous microorganisms and an introduced laboratory strain BS394 (pNF142::TnMod-OTc) served as donors of these plasmids. The indigenous bacterial degraders of naphthalene isolated from soil were identified as Pseudomonas putida and Pseudomonas fluorescens. The frequency of plasmid transfer in soil was 10^?5–10^?4 per donor cell. The activity of the key enzymes of naphthalene biodegradation in indigenous and transconjugant strains was studied. Transconjugant strains harboring indigenous catabolic plasmids possessed high salicylate hydroxylase and low catechol-2,3-dioxygenase activities, in contrast to indigenous degraders, which had a high level of catechol-2,3-dioxygenase activity and a low level of salicylate hydroxylase. Naphthalene degradation in batch culture in liquid mineral medium was shown to accelerate due to cooperation of the indigenous naphthalene degrader P. fluorescens AP1 and the transconjugant strain P. putida KT2442 harboring the indigenous catabolic plasmid pAP35. The role of conjugative transfer of naphthalene biodegradation plasmids in acceleration of naphthalene degradation was demonstrated in laboratory soil microcosms.     

Production of B-group vitamins by two Rhizobium strains in chemically defined media

Twenty-eight strains of Rhizobium spp. were tested for their ability to grow in chemically-defined medium lacking growth factors. Two strains, R. meliloti GR4B and Rhizobium spp. ( Acacia ) GRH28, were selected, on the basis of their good growth under the conditions imposed, for further quantification of the production of water-soluble vitamins (thiamine, niacin, riboflavin, pantothenic acid and biotin) in chemically defined media amended with different compounds (mannitol, glucose or sodium succinate) as sole carbon sources. Qualitative and quantitative production of vitamins in chemically-defined media was significantly affected by the use of C sources of a different nature and the age of the cultures. Strain GRH28 produced all the vitamins analysed, and high biological levels of biotin (14 ng ml^–1 culture) were detected after 6 d of culture in mineral medium amended with mannitol. Pantothenic acid was the vitamin detected in the highest amounts (up to 1 μg ml^–1 of culture) in culture supernatant fluids of strain GR4B grown for 6 d with succinate as sole carbon source.     

Syntrophic interactions during degradation of 4-aminobenzenesulfonic acid by a two species bacterial culture

During synthrophic growth of Hydrogenophaga palleronii (strain S1) and Agrobacterium radiobacter (strain S2) with 4-aminobenzene sulfonate (4ABS) only strain S1 desaminates 4ABS by regioselective 3,4-dioxygenation. The major part of the metabolite catechol-4-sulfonate (4CS) is excreted and further metabolized by strain S2. Although both organisms harbour activities of protocatechuate pathways assimilation of the structural analog 4CS requires first of all enzyme activities with broader substrate specificity: protocatechuate 3,4-dioxygenase and carboxymuconate cycloisomerase activities were identified which in addition to the natural substrates also convert 4CS requires first of all enzyme activities with Carboxymethyl-4-sulfobut-2-en-4-olide (4SL) was identifed as a metabolite. Its further metabolism requires a desulfonating enzyme which eliminates sulfite from (4SL) and generates maleylacetate. Convergence with the 3-oxoadipate pathway is catalyzed by a maleyl acetate reductase, which was identified in cell-free extracts of both organisms S1 and S2. Characteristically, only strain S1 can oxidize sulfite and thus contributes to the interdependence of the two bacteria during growth with 4ABS.     

Characterization of a Symbiotic Coculture of Clostridium thermohydrosulfuricum YM3 and Clostridium thermocellum YM4

Clostridium thermohydrosulfuricum YM3 and C. thermocellum YM4 were isolated from a coculture which was obtained from an enrichment culture inoculated with volcanic soil in Izu Peninsula, Japan. Strain YM3 had advantages over reported C. thermohydrosulfuricum strains in that it fermented inulin and could accumulate ethanol up to 1.3% (wt/vol). The highest ethanol yield obtained was 1.96 mol/mol of anhydroglucose unit in cellobiose. Strain YM4 had features different from those reported in C. thermocellum strains: it formed spores rarely (at a frequency of <10^-5), it required CO₂ and Na₂CO₃ for growth, and it fermented sucrose. Strain YM4 completely decomposed 1% Avicel within 25 h when the inoculum constituted 2% of the culture medium volume, and it produced 0.22 U of Avicelase and 2.21 U of carboxymethylcellulase per ml of the medium. The doubling times on Avicel, cellobiose, and glucose were 2.7, 1.1, and 1.6 h, respectively. Reconstructed cocultures of strains YM3 and YM4 were very stable and degraded Avicel more rapidly than did strain YM4 monoculture. Without yeast extract, neither microorganism was able to grow. However, the coculture grew on cellulose without yeast extract and produced ethanol in high yield. Moreover, cell-free spent culture broth of strain YM3 could replace yeast extract in supporting the growth of strain YM4. The symbiotic relationship of the two bacteria in cellulose fermentation is probably a case of mutualism.     

Horizontal transfer of catabolic plasmids in the process of naphthalene biodegradation in model soil systems

The process of naphthalene degradation by indigenous, introduced, and transconjugant strains was studied in laboratory soil microcosms. Conjugation transfer of catabolic plasmids was demonstrated in naphthalene-contaminated soil. Both indigenous microorganisms and an introduced laboratory strain BS394 (pNF142::TnMod-OTc) served as donors of these plasmids. The indigenous bacterial degraders of naphthalene isolated from soil were identified as Pseudomonas putida and Pseudomonas fluorescens. The frequency of plasmid transfer in soil was 10(-5)-10(-4) per donor cell. The activity of the key enzymes of naphthalene biodegradation in indigenous and transconjugant strains was studied. Transconjugant strains harboring indigenous catabolic plasmids possessed high salicylate hydroxylase and low catechol-2,3-dioxygenase activities, in contrast to indigenous degraders, which had a high level of catechol-2,3-dioxygenase activity and a low level of salicylate hydroxylase. Naphthalene degradation in batch culture in liquid mineral medium was shown to accelerate due to cooperation of the indigenous naphthalene degrader P. fluorescens AP1 and the transconjugant strain P. putida KT2442 harboring the indigenous catabolic plasmid pAP35. The role of conjugative transfer of naphthalene biodegradation plasmids in acceleration of naphthalene degradation was demonstrated in laboratory soil microcosms.     

Growth in Coculture Stimulates Metabolism of the Phenylurea Herbicide Isoproturon by Sphingomonas sp. Strain SRS2

Metabolism of the phenylurea herbicide isoproturon by Sphingomonas sp. strain SRS2 was significantly enhanced when the strain was grown in coculture with a soil bacterium (designated strain SRS1). Both members of this consortium were isolated from a highly enriched isoproturon-degrading culture derived from an agricultural soil previously treated regularly with the herbicide. Based on analysis of the 16S rRNA gene, strain SRS1 was assigned to the β-subdivision of the proteobacteria and probably represents a new genus. Strain SRS1 was unable to degrade either isoproturon or its known metabolites 3-(4-isopropylphenyl)-1-methylurea, 3-(4-isopropylphenyl)-urea, or 4-isopropyl-aniline. Pure culture studies indicate that Sphingomonas sp. SRS2 is auxotrophic and requires components supplied by association with other soil bacteria. A specific mixture of amino acids appeared to meet these requirements, and it was shown that methionine was essential for Sphingomonas sp. SRS2. This suggests that strain SRS1 supplies amino acids to Sphingomonas sp. SRS2, thereby leading to rapid metabolism of ¹⁴C-labeled isoproturon to ¹⁴CO₂ and corresponding growth of strain SRS2. Proliferation of strain SRS1 suggests that isoproturon metabolism by Sphingomonas sp. SRS2 provides unknown metabolites or cell debris that supports growth of strain SRS1. The role of strain SRS1 in the consortium was not ubiquitous among soil bacteria; however, the indigenous soil microflora and some strains from culture collections also stimulate isoproturon metabolism by Sphingomonas sp. strain SRS2 to a similar extent.     

Genome Sequence of Ralstonia sp. Strain PBA, a Bacterium Involved in the Biodegradation of 4-Aminobenzenesulfonate

Ralstonia sp. strain PBA was isolated from textile wastewater in a coculture with Hydrogenophaga sp. strain PBC. Here we present the assembly and annotation of its genome, which may provide further insights into the mechanism of its interaction with strain PBC during 4-aminobenzenesulfonate degradation.     

Adaptation of Saccharomyces cerevisiae cells to high ethanol concentration and changes in fatty acid composition of membrane and cell size

Background

Microorganisms can adapt to perturbations of the surrounding environment to grow. To analyze the adaptation process of the yeast Saccharomyces cerevisiae to a high ethanol concentration, repetitive cultivation was performed with a stepwise increase in the ethanol concentration in the culture medium.

Methodology/Principal Findings

First, a laboratory strain of S. cerevisiae was cultivated in medium containing a low ethanol concentration, followed by repetitive cultivations. Then, the strain repeatedly cultivated in the low ethanol concentration was transferred to medium containing a high ethanol concentration and cultivated repeatedly in the same high-ethanol-concentration medium. When subjected to a stepwise increase in ethanol concentration with the repetitive cultivations, the yeast cells adapted to the high ethanol concentration; the specific growth rate of the adapted yeast strain did not decrease during repetitive cultivation in the medium containing the same ethanol concentration, while that of the non-adapted strain decreased during repetitive cultivation. A comparison of the fatty acid composition of the cell membrane showed that the contents in oleic acid (C_18:1) in ethanol-adapted and non-adapted strains were similar, but the content of palmitic acid (C_16:0) in the ethanol-adapted strains was lower than that in the non-adapted strain in media containing ethanol. Moreover, microscopic observation showed that the mother cells of the adapted yeast were significantly larger than those of the non-adapted strain.

Conclusions

Our results suggest that activity of cell growth defined by specific growth rate of the yeast cells adapted to stepwise increase in ethanol concentration did not decrease during repetitive cultivation in high-ethanol-concentration medium. Moreover, fatty acid content of cell membrane and the size of ethanol-adapted yeast cells were changed during adaptation process. Those might be the typical phenotypes of yeast cells adapted to high ethanol concentration. In addition, the difference in sizes of the mother cell between the non-adapted and ethanol strains suggests that the cell size, cell cycle and adaptation to ethanol are thought to be closely correlated.     

Isolation of a Bacterial Strain with the Ability To Utilize the Sulfonated Azo Compound 4-Carboxy-4′-Sulfoazobenzene as the Sole Source of Carbon and Energy

A bacterial strain (strain S5) which grows aerobically with the sulfonated azo compound 4-carboxy-4′-sulfoazobenzene as the sole source of carbon and energy was isolated. This strain was obtained by continuous adaptation of “Hydrogenophaga palleronii” S1, which has the ability to grow aerobically with 4-aminobenzenesulfonate. Strain S5 probably cleaves 4-carboxy-4′-sulfoazobenzene reductively under aerobic conditions to 4-aminobenzoate and 4-aminobenzene-sulfonate, which are mineralized by previously established degradation pathways.     

Kinetic study and mathematical modelling of killer and sensitive S. Cerevisiae strains growing in mixed culture

This paper presents a kinetic study of two yeasts growing in pure and mixed batch cultures. Two winemaking strains were used: S. cerevisiae K1 possessing the K2 killer character and S. cerevisiae 522D sensitive to the K2 killer toxin. Initially the kinetics of growth of the two strains were analysed in pure culture. In this case, the kinetic profiles of biomass production have shown that the growth rate of the K1 strain is slightly superior to the 522D strain. During the fermentation, the viability for both populations was higher than 90%. Fermentations in mixed culture with an initial percentage in killer strain of 5 and 10% with respect to the total population were carried out. The results showed a more important decrease in the percentage of total viable yeasts when the initial concentration of killer yeast increased. However, the kinetic profiles of total biomass (killer plus sensitive yeasts) were very similar for both fermentations. A mathematical model was proposed to simulate the microbial growth of the killer and sensitive strain developing in pure and mixed cultures. This mathematical model consists in three main reactions: the evolution of the killer toxin in the culture medium, the duplication and the mortality rates for each microbial population. The results of the simulation appeared in agreement with the experimental data.     

Behavior of sulfate reducing bacteria under oligotrophic conditions and oxygen stress in particle-free systems related to drinking water

The response of sulfate reducing bacteria (SRB) to oxygen stress under oligotrophic conditions in particle-free systems was studied in (i) sterile Berlin drinking water; (ii) mineral medium; and (iii) in coculture experiments with aerobic bacteria. Using a polyphasic approach including anaerobic cultivation, fluorescent in situ hybridization (FISH) and digital image analysis, the behavior of the strains zt3l and zt10e, isolated from Berlin groundwater and affiliated to the family Desulfovibrionaceae, was compared to the type strains Desulfomicrobium baculatum and Desulfovibrio desulfuricans. Anaerobic deep agar dilution series were performed for the determination of cell culturability. FISH and subsequent digital image analysis of probe-conferred fluorescence intensities were used for the assessment of metabolic activity. For the in situ identification of both isolates in coculture tests, two strain-specific oligonucleotides were developed and evaluated. The total cell counts of stressed SRB in drinking water decreased during the course of the assay dependent on the strain. Both environmental isolates could be cultured for a longer period than cells of D. baculatum and D. desulfuricans, respectively. The FISH intensities showed a strain-specific behavior. When exposed to simultaneous oxygen stress and carbon limitation in mineral medium, total cell counts of all four strains remained constant throughout a period of 72 days. The rate of culturability differed between the investigated strains. The decrease of metabolic activity as assessed by FISH was a strain-specific property. Exposure of SRB to oxygen stress and carbon starvation in coculture experiments with Aquabacterium commune resulted in strain dependent prolonged culturability and a delayed decrease of the metabolic activity compared to pure culture tests for all strains tested. Total cell counts of SRB were constant throughout the whole experiment.     

Isolation and partial characterization of bacteria in an anaerobic consortium that mineralizes 3-chlorobenzoic Acid

A methanogenic consortium able to use 3-chlorobenzoic acid as its sole energy and carbon source was enriched from anaerobic sewage sludge. Seven bacteria were isolated from the consortium in mono- or coculture. They included: one dechlorinating bacterium (strain DCB-1), one benzoate-oxidizing bacterium (strain BZ-2), two butyrate-oxidizing bacteria (strains SF-1 and NSF-2), two H(2)-consuming methanogens (Methanospirillum hungatei PM-1 and Methanobacterium sp. strain PM-2), and a sulfate-reducing bacterium (Desulfovibrio sp. strain PS-1). The dechlorinating bacterium (DCB-1) was a gram-negative, obligate anaerobe with a unique "collar" surrounding the cell. A medium containing rumen fluid supported minimal growth; pyruvate was the only substrate found to increase growth. The bacterium had a generation time of 4 to 5 days. 3-Chlorobenzoate was dechlorinated stoichiometrically to benzoate, which accumulated in the medium; the rate of dechlorination was ca. 0.1 pmol bacterium day. The benzoate-oxidizing bacterium (BZ-2) was a gram-negative, obligate anaerobe and could only be grown as a syntroph. Benzoate was the only substrate observed to support growth, and, when grown in coculture with M. hungatei, it was fermented to acetate and CH(4). One butyrate-oxidizing bacterium (NSF-2) was a gram-negative, non-sporeforming, obligate anaerobe; the other (SF-1) was a gram-positive, sporeforming, obligate anaerobe. Both could only be grown as syntrophs. The substrates observed to support growth of both bacteria were butyrate, 2-dl-methylbutyrate, valerate, and caproate; isobutyrate supported growth of only the sporeforming bacterium (SF-1). Fermentation products were acetate and CH(4) (from butyrate, isobutyrate, or caproate) or acetate, propionate, and CH(4) (from 2-dl-methylbutyrate or valerate) when grown in coculture with M. hungatei. A mutualism among at least the dechlorinating, benzoate-oxidizing, and methane-forming members was apparently required for utilization of the 3-chlorobenzoate substrate.     

Growth of the syntrophic anaerobic acetogen, strain PA-1, with glucose or succinate as energy source

Strain PA-1 (S. Barik, W.J. Brulla, and M.P. Bryant, Appl. Environ. Microbiol. 50:304-310, 1985) is an anaerobic, gram-negative rod that in pure culture decarboxylates succinate to propionate and that grows syntrophically as an acetogen with the H2 utilizer Methanospirillum hungatei if glucose, pyruvate, aspartate, or fumarate is provided. In pure culture, strain PA-1 grows optimally in a medium containing 5% ruminal fluid, 0.1% yeast extract, a 4:1 N2-CO2 gas phase, and 20 mM succinate. With the PA-1 plus M. hungatei coculture, good growth was obtained with 7.5 mM glucose and tryptophan could replace the yeast extract. Strain PA-1 in pure culture grew quite well in glucose medium if the large headspace was flushed intermittently with N2. Flushing with H2 inhibited this growth.     

The genusCandida berkhout

Basic nutritional conditions were studied in 30 strains ofCandida albicans comprising slightly, moderately and highly pathogenic cultures. It was found that the given strains had no special requirements as far as growth substances were concerned and that even after tenfold transfer they continued to reproduce on minimum medium. A mixture of the most important amino acids only slightly stimulated growth, but tended to stimulate alcohol and total acid synthesis. Its stimulating effect was strongest in the group of highly pathogenic strains. The only treatment which markedly stimulated and accelerated reproduction, especially in the initial phase of growth of the culture, was a combination of four vitamins—biotin, calcium pantothenate, nicotinic acid and thiamine. Differences were also found in the dry weight increase; cultures with a rapid, slow and moderate rate of growth were differentiated, according to the size of their economic coefficient. The group of slightly pathogenic strains also had different economic coefficients from moderately and highly pathogenic strains. There was no significant difference between moderately and highly pathogenic strains. One strain differed from all the rest by reproducing well in minimum medium without amino acids or vitamins; its rate of reproduction in minimum medium was the same as in medium enriched with amino acids.     

Effect of immobilization of a bacterial consortium on diuron dissipation and community dynamics

This work intended to study the relationship between diuron herbicide dissipation and the population dynamics of co-cultivated Delftia acidovorans WDL34 (WDL34) and Arthrobacter sp. N4 (N4) for different cell formulations: free cells or immobilization in Ca-alginate beads of one or both strains. GFP-tagged WDL34 and N4 Gram staining allowed analyzing the cell growth and distribution of each strain in both beads and culture medium in the course of the time. Compared to the free cell co-culture of WDL34 and N4, immobilization of WDL34 in Ca-alginate beads co-cultivated with free N4 increased the dissipation rate of diuron by 53% (0.141 mg ml⁻¹ h⁻¹). In that case, immobilization strongly modified the final equilibrium among both strains (highest total N4 to WDL34 ratio). Our results demonstrated that the inoculant formulation played a major role in the cell growth of each cultivated strain possibly increasing diuron dissipation. This optimized cell formulation may allow improving water and soil treatment.