Activity-dependent fluorescent labeling of bacteria that degrade toluene via toluene 2,3-dioxygenase

Alternative substrates for the toluene 2,3-dioxygenase pathway of several pseudomonads served as enzyme-activity-dependent fluorescent probes for the bacteria. Phenylacetylene and cinnamonitrile were transformed to fluorescent and brightly colored products by Pseudomonas putida F1, Pseudomonas fluorescens CFS215, and Burkholderia (Pseudomonas) strain JS150. Active bacteria transformed phenylacetylene, producing bright yellow solutions containing the putative product 2-hydroxy-6-oxo-7-octyn-2,4-dienoate. Transformation of cinnamonitrile resulted in bright orange solutions due to accumulation of the putative product 2-hydroxy-6-oxo-8-cyanoocta-2,4,7-trienoate. Chemical and physical properties of the products supported their identification, which indicated that the first three enzymes of the pathway catalyzed product formation. Phenylacetylene labeled bacteria with green fluorescence emission; bacteria were concentrated on black 0.2-μm-pore-size polycarbonate filters containing polyvinylpyrrolidone (PVP) as a wetting agent. Bacteria labeled with cinnamonitrile were fluorescent orange; labeling was effective with bacteria trapped on PVP-free polycarbonate filters. Production of the enzymes involved in labeling of P. putida F1 and P. fluorescens CFS215 was induced by growth (on arginine) in the presence of toluene; cells grown on arginine without toluene were not labeled. Labeling of P. putida F1 by phenylacetylene was inhibited by toluene, indicating that the same enzymatic pathway was required for transformations of both substrates. Bacteria expressing other toluene-degrading enzymatic pathways were not fluorescently labeled with phenylacetylene. Received: 30 July 1997 / Received revision: 1 November 1997 / Accepted: 21 November 1997     

A genetically modified solvent-tolerant bacterium for optimized production of a toxic fine chemical

The aim of the study was to investigate whether toxic fine chemical production can be improved using the solvent-tolerant Pseudomonas putida S12 in a two-liquid-phase system consisting of aqueous media and a water-immiscible octanol phase with production of 3-methylcatechol from toluene as the model conversion. For this purpose the genes involved in this conversion, todC1C2BAD from P. putida F1, were introduced into P. putida S12 with high stable expression. Production of 3-methylcatechol was monitored in batch incubations with different media using a single medium and a two-liquid medium–octanol system. The maximum concentration of 3-methylcatechol increased two-fold using the two-liquid medium–octanol system, irrespective of the selected medium. Received: 29 December 1999 / Received revision: 29 February 2000 / Accepted: 6 March 2000     

Biotransformation of isoquinoline, phenanthridine, phthalazine, quinazoline, and quinoxaline by Streptomyces viridosporus

Streptomyces viridosporus T7A (ATCC␣39115), during growth in tryptone/yeast extract broth, cometabolized five heterocyclic nitrogen-containing compounds. The metabolites produced from the azaarenes were identified by high-performance liquid chromatography, UV/visible absorption spectroscopy, and mass spectrometry. Isoquinoline was transformed to 1(2H)-isoquinolinone (14%), phenanthridine to 6(5H)-phenanthridinone (25%), phthalazine to 1(2H)-phthalazinone (46%), quinazoline to 2,4(1H,3H)-quinazolinedione (4%), and quinoxaline to 2(1H)-quinoxalinone (8%) and 1-methyl-2(1H)-quinoxalinone (12%). Received: 29 July 1997 / Received revision: 19 November 1997 / Accepted: 29 November 1997     

Lignin-hemicellulose complexes restrict enzymatic solubilization of mannan and xylan from dissolving pulp

Mannan and xylan present in bleached softwood dissolving pulp were found to be partially resistant to hemicellulases even after repeated enzyme treatment. Despite the additional effect of an endoglucanase from Gloeophyllum sepiarium, which increased the␣accessibility of mannan and xylan to a mannanase from Sclerotium rolfsii and to a xylanase from Thermomyces lanuginosus, the enzyme mixture solubilized only half of the hemicellulose present in the pulp. Half of the remaining hemicellulose present in the pulp appeared to be entrapped within the cellulose matrix while the other half was associated with lignin-carbohydrate complexes. The latter hemicellulose portion was isolated and characterized. Chromatography and spectroscopic techniques revealed the presence of two types of lignin-carbohydrate complex, a galactoglucomannan-lignin complex (degree of polymerization DP 50–60) and a xylan-lignin complex (DP >200). Received: 8 December 1997 / Received revision: 30 April 1998 / Accepted: 8 May 1998     

Facilitative effect of Lotustenuis on Paspalumdilatatum in a lowland grassland of Argentina

We studied the responses in growth and N content of the perennial grass Paspalum dilatatum to the substitution of Lotus tenuis for a whole group of species, the dicotyledons of a natural grassland community, in the Salado lowland Pampas of Argentina. Two kinds of manipulations were performed in the field: removal of alien dicots with herbicide application, and introduction of L. tenuis, resulting in a combination of four treatments, arranged in a 2 × 2 factorial randomized block design. Leaf area per tiller of P. dilatatum was higher when it was growing near L. tenuis; this increase was the result of a greater leaf elongation rate and slower leaf senescence. In the vicinity of L. tenuis, P. dilatatum exhibited an increase in tiller production and a decrease in tiller death. More tillers were functional at the end of the growing season and their aboveground biomass was 5␣times higher than for plants growing in plots where the community dicots were removed. This increment was accompanied by a higher N content. Growth enhancement of P. dilatatum plants when L. tenuis was the␣immediate neighbour is interpreted as the result of facilitation mediated by higher N availability, and not as a consequence of a release from competition exerted by the community dicots. Competition and facilitation did not interact to produce an increase in the vegetative output of Paspalum dilatatum plants growing under these field conditions. It is on these grounds that Lotus tenuis might be considered as a keystone species in the managed grassland. Received: 5 July 1997 / Accepted: 12 December 1997     

Physiological stress in batch cultures of Pseudomonas putida 54G during toluene degradation

Physiological stress associated with toluene exposure in batch cultures of Pseudomonas putida 54G was investigated. P. putida 54G cells were grown using a continuous vapor phase feed stream containing 150 ppmv or 750 ppmv toluene as the sole carbon and energy source. Cells were enumerated on non-selective (R2A agar plates) and a selective minimal medium incubated in the presence of vapor phase toluene (HCMM2). Differential recovery on the two media was used to evaluate bacterial stress, culturability and loss of toluene-degrading capability. A majority of the bacteria were reversibly stressed and could resume active colony formation on selective medium after passage on non-selective medium. A small fraction of the bacterial cells suffered an irreversible loss of toluene degradation capability and were designated as Tol⁻ variants. Numbers of stressed organisms increased with duration of toluene exposure and toluene concentration and coincided with accumulation of metabolic intermediates from incomplete toluene degradation. Respiring cell numbers in the batch cultures decreased as injury increased, indicating a possible relationship between respiring and injured cells. Rate expressions for injury, for formation of Tol⁻ variants and for growth of Tol⁻ variants were determined by calibrating a theoretical model to the results obtained. These rate expressions can be used to calibrate bioreactor models, and provide a basis for better design and control of bioremediation systems. Received 01 July 1996/ Accepted in revised form 25 March 1997     

Non-toluene-associated respiration in a Pseudomonas putida 54G biofilm grown on toluene in a flat-plate vapor-phase bioreactor

Non-toluene-associated respiration (NTAR) within a Pseudomonas putida 54G biofilm growing on toluene as sole external carbon source was evaluated using oxygen microelectrodes in a flat-plate vapor-phase biological reactor. Two fluorescent probes, 2,4-diamidino-2-phenylindole and 5-cyano-2,3-ditolyltetrazolium chloride, were used to evaluate the number of total and respiring cells respectively within the biofilm. Biofilm samples were also analyzed for viable and toluene-culturable cells by spread-plating on non-selective and selective media respectively. Fractions of viable stressed, respiring and non-respiring cells within the biofilm were evaluated. The NTAR rate was positively correlated with the fraction of viable stressed and non-respiring cells within the biofilm, which suggested the capability of some cells to grow at the expense of leakage and lysis products coming from injured and dead cells. This effect was more pronounced at higher toluene concentration. Results suggest that NTAR should be incorporated into mathematical models of biofilm reactors degrading volatile organic carbon compounds. Received: 4 January 1997 / Received revision: 20 March 1997 / Accepted: 27 March 1997     

High levels of antioxidant enzymes correlate with delayed senescence in nonnetted muskmelon fruits

We investigated the senescence process in two nonnetted muskmelon (Cucumis melo L.)␣varieties␣Clipper and Jerac differing in their storage life. Our results indicate that senescence in Jerac (the short-storage-life variety, less than 7 d) is the result of lipid peroxidation by free radicals, membrane phospholipid breakdown, and a drop in the level of antioxidants, resulting in increased membrane leakiness. By contrast, evidence is presented that the high levels of two enzymes implicated in antioxidative defence, superoxide dismutase (SOD) and catalase, combined with changes in the three different classes of SOD during the storage stage, are involved in delaying the senescence process in Clipper and this could explain, at least, to some extent, the long storage life of Clipper (longer than 14 d). Received: 15 April 1997 / Accepted: 17 July 1997     

A counter-selectable marker for genetic transformation of the yeast Schwanniomyces alluvius

We report here a counter-selectable marker system for genetic transformation of the yeast Schwanniomyces alluvius, based on the complementation of uracil auxotrophs defective in either orotidine-5′-phosphate decarboxylase (URA3) or orotidine-5′-pyrophosphatase (URA5). Uracil auxotrophs of S. alluvius were obtained by ethyl methanesulphonate mutagenesis and complemented using the ura3 gene from S. cerevisiae. A␣transformation frequency of approximately 10⁴/μg DNA was obtained, which is tenfold higher than results described in earlier reports. Transformants were analysed by Southern blot hybridisation and were found to be mitotically stable. The extrachromosomal nature of the transforming DNA was confirmed by Southern hybridisation and plasmid rescue. The rescued plasmid DNA had a restriction pattern identical to that of the parent plasmid. Received: 19 August 1996 / Received last revision: 30 April 1997 / Accepted: 4 May 1997     

Competition of plasmid-bearing Pseudomonas putida strains catabolizing naphthalene via various pathways in chemostat culture

Plasmid-carrying Pseudomonas putida strains degrade naphthalene through different biochemical pathways. The influence of various combinations of host bacteria and plasmids on growth characteristics and competitiveness of P. putida strains was studied in chemostat culture at a low dilution rate (D=0.05 h⁻¹) with naphthalene as the sole source of carbon and energy. Under naphthalene limitation, the plasmid-bearing strains degrading naphthalene that use catechol 1,2-dioxygenase for catechol oxidation (ortho pathway), were the most competitive. The strains bearing plasmids that control naphthalene catabolism via catechol 2,3-dioxygenase (meta pathway), were less competitive. Under these conditions the strain carrying plasmid pBS4, which encodes for naphthalene catabolism via gentisic acid, was the least competitive. Received: 24 February 1997 / Received revision: 22 May 1997 / Accepted: 25 May 1997     

The diversion of lactose carbon through the tagatose pathway reduces the intracellular fructose 1,6-bisphosphate and growth rate of Streptococcus bovis

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. Received: 3 June 1997 / Received revision: 10 September 1997 / Accepted: 6 January 1998     

Nitrate and phosphate ion removal from water by Phormidium laminosum immobilized on hollow fibres in a photobioreactor

Removal of nitrate and phosphate ions from water, by using the thermophilic cyanobacterium Phormidium laminosum, immobilized on cellulose hollow fibres in the tubular photobioreactor at 43 °C, was studied by continuously supplying dilute growth medium for 7 days and then secondarily treated sewage (STS) for 12 days. The concentrations of NO⁻ ₃ and PO³⁻ ₄ in the effluent from the dilute growth medium decreased from 5.0 mg N/l to 3.1 mg N/l, and from 0.75 mg P/l to 0.05 mg P/l respectively, after a residence time of 12 h. The concentrations of NO⁻ ₃ and PO³⁻ ₄ in the effluent from STS decreased from 11.7 mg N/l to 2.0 mg N/l, and from 6.62 mg P/l to 0.02 mg P/l respectively, after a residence time of 48 h. The removal rates of nitrogenous␣and phosphate ions from STS were 0.24 and 0.11 mmol day⁻¹ l reactor⁻¹ respectively, under the same conditions. Although, among nitrogenous ions, nitrate and ammonium ions were efficiently removed by P.␣laminosum, the nitrite ion was released into the effluent when STS was used as influent. Treatment of water with thermophilic P. laminosum immobilized on hollow fibres thus appears to be an appropriate means for the removal of inorganic nitrogen and phosphorus from treated wastewater. Received: 15 August 1997 / Received last revision: 18 November 1997 / Accepted: 29 November 1997     

The solvent efflux system of Pseudomonas putida S12 is not involved in antibiotic resistance

The active efflux system contributing to the solvent tolerance of Pseudomonas putida S12 was characterized physiologically. The mutant P. putida JK1, which lacks the active efflux system, was compared with the wild-type organism. None of 20 known substrates of common multi-drug-resistant pumps had a stronger growth-inhibiting effect on the mutant than on the wild type. The amount of [¹⁴C]toluene accumulating in P. putida S12 increased in the presence of the solvent xylene and in the presence of uncouplers. The effect of uncouplers confirms the proton dependency of the efflux system in P. putida S12. Other compounds, potential substrates for the solvent pump, did not affect the accumulation of [¹⁴C]toluene. These results show that the efflux system in P. putida S12 is specific for organic solvents and does not export antibiotics or other known substrates of multi-drug-resistant pumps. Received: 15 February 2000 / Received revision: 16 June 2000 / Accepted: 18 June 2000     

High rates of microbial sulphate reduction in a mesophilic ethanol-fed expanded-granular-sludge-blanket reactor

In a mesophilic (30–35 °C), sulphidogenic, ethanol-fed expanded-granular-sludge-blanket reactor, sulphate, at loading rates of up to 10.0–12.0 g Sl⁻¹␣day⁻¹, was removed with an average efficiency of more than 80%. The pH was between 7.7 and 8.3 and the maximal total dissolved sulphide concentration was up to 20 mM S (650 mg S/l). The alkaline pH was maintained by either a pH-control unit with sodium hydroxide or by stripping part of the sulphide and CO₂ from the recycle with nitrogen gas. The superficial upstream liquid velocity (v _up) was 3.0–4.5 m/h. The ratio of ethanol to sulphur was near stoichiometry. At alkaline pH, the activity of the acetotrophic sulphate-reducing bacteria, growing on acetate, was strongly enhanced, whereas at pH below 7.7 the acetotrophic sulphate-reducing bacteria were inhibited by aqueous H₂S. With regard to the removal efficiency and operational stability, external stripping with N₂ and pH control were equally successful. Received: 2 December 1996 / Received revision: 13 March 1997 / Accepted: 15 March 1997     

Effects of phenolic compounds on the growth and the fatty acid composition of Lactobacillus plantarum

The effects of different phenolic compound concentrations on the fatty acid composition of Lactobacillus plantarum isolated from traditional home-made olive brines were determined. Increasing amounts of caffeic and ferulic acids induced a gradual increase in the amounts of myristic, palmitoleic, stearic and 9,10-methylenehexadecanoic (C17Δ, where Δ represents the cyclopropane group) acid with a concomitant decrease of lactobacillic acid (C₁₉Δ). On the other hand, the addition of tannins induced an increase in the C₁₉Δ level at the expense of vaccenic acid content. The presence of acidic phenols and tannins also affected bacterial growth, inducing the most obvious effect with tannin at 1 g l⁻¹. Received: 1 July 1997 / Received revision: 9 September 1997 / Accepted: 15 September 1997     

Enantioselective reduction of 3,4-methylene-dioxyphenylacetone using Candida famata and Zygosaccharomyces rouxii

In an effort to prepare 3,4-methylene-dioxyphenyl-(S)-isopropanol from 3,4-methylene-dioxyphenylacetone, an initial screen of microbes indicated that Candida famata could catalyze this reaction efficiently at low substrate concentration. A dilute, large-scale process was developed to provide experimental material for the chemical synthesis to be explored. However, the productivity number of this process [0.134 g product (g␣wet␣weight cells)⁻¹ day⁻¹ was too low to be practical. C.␣famata was also extremely sensitive to concentrations of both the ketone and the alcohol greater than 2 g/l. A more extensive screen of yeast and fungi revealed that Zygosaccharomyces rouxii was more tolerant to higher substrate concentrations and had a higher productivity number [0.8 g (g wet weight cells)⁻¹ day⁻¹]. These characteristics suggested that Z. rouxii could be used in a large-scale process at high substrate concentrations. Received: 8 July 1996 / Received revision: 9 September 1996 / Accepted: 18 September 1996     

Continuous cultures of <Emphasis Type="Italic">Pseudomonas putida</Emphasis> mt-2 overcome catabolic function loss under real case operating conditions

The long-term performance and stability of Pseudomonas putida mt-2 cultures, a toluene-sensitive strain harboring the genes responsible for toluene biodegradation in the archetypal plasmid pWW0, was investigated in a chemostat bioreactor functioning under real case operating conditions. The process was operated at a dilution rate of 0.1 h⁻¹ under toluene loading rates of 259 ± 23 and 801 ± 78 g m⁻³ h⁻¹ (inlet toluene concentrations of 3.5 and 10.9 g m⁻³, respectively). Despite the deleterious effects of toluene and its degradation intermediates, the phenotype of this sensitive P. putida culture rapidly recovered from a 95% Tol⁻ population at day 4 to approx. 100% Tol⁺ cells from day 13 onward, sustaining elimination capacities of 232 ± 10 g m⁻³ h⁻¹ at 3.5 g Tol m⁻³ and 377 ± 13 g m⁻³ h⁻¹ at 10.9 g Tol m⁻³, which were comparable to those achieved by highly tolerant strains such as P. putida DOT T1E and P. putida F1 under identical experimental conditions. Only one type of Tol⁻ variant, harboring a TOL-like plasmid with a 38.5 kb deletion (containing the upper and meta operons for toluene biodegradation), was identified.     

Modelling the subgenual organ of the honeybee, Apis mellifera

In a recent study on the honeybee (Apis mellifera), the subgenual organ was observed moving inside the leg during sinusoidal vibrations of the leg (Kilpinen and Storm 1997). The subgenual organ of the honeybee is suspended in a haemolymph channel in the tibia of each leg. When the leg accelerates, the inertia causes the haemolymph and the subgenual organ to lag behind the movement of the rest of the leg. To elucidate the biophysics of the subgenual organ system of the honeybee, two mathematical models to simulate the experimentally observed mechanical response are considered. The models are a classical mass-spring model and a newly developed tube model consisting of an open-ended, fluid-filled tube occluded by an elastic structure midway. Both models suggest that the subgenual organ included in the haemolymph channel resembles that of an overdamped system. In resembling the biophysics of the subgenual organ system in the honeybee, we consider the tube model to be the better of the two because it simulates a mechanical response which complies best with the experimental data, and the physical parameters in the model can be related to the␣constituent parts of the subgenual organ included in the haemolymph channel. Received: 25 July 1997 / Accepted in revised form: 8 December 1997     

Thermophilic biodegradation of BTEX by two consortia of anaerobic bacteria

Two thermophilic anaerobic bacterial consortia (ALK-1 and LLNL-1), capable of degrading the aromatic fuel hydrocarbons, benzene, toluene, ethylbenzene, and the xylenes (BTEX compounds), were developed at 60 °C from the produced water of ARCO'S Kuparuk oil field at Alaska and the subsurface water at the Lawrence Livermore National Laboratory gasoline-spill site, respectively. Both consortia were found to grow at 45–75 °C on BTEX compounds as their sole carbon and energy sources with 50 °C being the optimal temperature. With 3.5 mg total BTEX added to sealed 50-ml serum bottles, which contained 30 ml mineral salts medium and the consortium, benzene, toluene, ethylbenze, m-xylene, and an unresolved mixture of o- and p-xylenes were biodegraded by 22%, 38%, 42%, 40%, and 38%, respectively, by ALK-1 after 14 days of incubation at 50 °C. Somewhat lower, but significant, percentages of the BTEX compounds also were biodegraded at 60 °C and 70 °C. The extent of biodegradation of these BTEX compounds by LLNL-1 at each of these three temperatures was slightly less than that achieved by ALK-1. Use of [ring-¹⁴C]toluene in the BTEX mixture incubated at 50 °C verified that 41% and 31% of the biodegraded toluene was metabolized within 14 days to water-soluble products by ALK-1 and LLNL-1, respectively. A small fraction of it was mineralized to ¹⁴CO₂. The use of [U-¹⁴C]benzene revealed that 2.6%–4.3% of the biodegraded benzene was metabolized at 50 °C to water-soluble products by the two consortia; however, no mineralization of the degraded [U-¹⁴C]benzene to ¹⁴CO₂ was observed. The biodegradation of BTEX at all three temperatures by both consortia was tightly coupled to sulfate reduction as well as H₂S generation. None was observed when sulfate was omitted from the serum bottles. This suggests that sulfate-reducing bacteria are most likely responsible for the observed thermophilic biodegradation of BTEX in both consortial cultures. Received: 12 July 1996 / Received revision: 31 December 1996 / Accepted: 31 January 1997