Biochemical,Transcriptional and Translational Evidences of the Phenol-meta-Degradation Pathway by the Hyperthermophilic Sulfolobus solfataricus 98/2

Phenol is a widespread pollutant and a model molecule to study the biodegradation of monoaromatic compounds. After a first oxidation step leading to catechol in mesophilic and thermophilic microorganisms, two main routes have been identified depending on the cleavage of the aromatic ring: ortho involving a catechol 1,2 dioxygenase (C12D) and meta involving a catechol 2,3 dioxygenase (C23D). Our work aimed at elucidating the phenol-degradation pathway in the hyperthermophilic archaea Sulfolobus solfataricus 98/2. For this purpose, the strain was cultivated in a fermentor under different substrate and oxygenation conditions. Indeed, reducing dissolved-oxygen concentration allowed slowing down phenol catabolism (specific growth and phenol-consumption rates dropped 55% and 39%, respectively) and thus, evidencing intermediate accumulations in the broth. HPLC/Diode Array Detector and LC-MS analyses on culture samples at low dissolved-oxygen concentration (DOC  =  0.06 mg.L⁻¹) suggested, apart for catechol, the presence of 2-hydroxymuconic acid, 4-oxalocrotonate and 4-hydroxy-2-oxovalerate, three intermediates of the meta route. RT-PCR analysis on oxygenase-coding genes of S. solfataricus 98/2 showed that the gene coding for the C23D was expressed only on phenol. In 2D-DIGE/MALDI-TOF analysis, the C23D was found and identified only on phenol. This set of results allowed us concluding that S. solfataricus 98/2 degrade phenol through the meta route.     

Kinetic studies of phenol degradation by Rhodococcus sp. P1 II. Continuous cultivation

The degradation of phenol by Rhodococcus sp. P1 was studied in continuous culture systems. The organism could be adapted by slowly increasing concentration, step by step, up to 30.0 g · 1^-1 phenol in the influent. The degradation rate reached values of about 0.3 g · g dry mass^-1 ·h^-1. Large step increases in phenol concentration and addition of further substrates (e.g., catechol) were tolerated up to a certain concentration. With increasing dilution rate and increasing inlet phenol concentration the stability of the system decreased.     

Phenol degradation by Ralstonia eutropha: colorimetric determination of 2-hydroxymuconate semialdehyde accumulation to control feed strategy in fed-batch fermentations.

Phenol biodegradation by Ralstonia eutropha was modeled in different culture modes to assess phenol feeding in biotechnological depollution processes. The substrate-inhibited growth of R. eutropha was described by the Haldane equation with a Ks of 2 mg/L, a Ki of 350 mg/L and a mumax of 0.41 h(-1). Furthermore, growth in several culture modes was characterized by the appearance of a yellow color, due to production of a metabolic intermediate of the phenol catabolic pathway, 2-hydroxymuconic semialdehyde (2-hms) which was directly correlated to the growth rate and/or the phenol-degradation rate, because these two parameters are coupled (as seen by the constant growth yield of 0.68 g biomass/g phenol whatever the phenol concentration). This correlation between color appearance and metabolic activity was used to develop a control procedure for optimal phenol degradation. A mass-balance equation modeling approach combined with a filtering step using an extended Kalman filter enabled state variables of the biological system to be simulated. A PI controller, using the estimation of the phenol concentration provided by the modeling step, was then built to maintain the phenol concentration at a constant set-point of 0.1 g/L which corresponded to a constant specific growth rate of 0.3 h(-1), close to the maximal specific growth value of the strain. This monitoring strategy, validated for two fed-batch cultures, could lead, in self-cycling fermentation systems, to a productivity of more than 19 kg of phenol consumed/m(3)/d which is the highest value reported to date in the literature. This system of monitoring metabolic activity also protected the bacterial culture against toxicity problems due to the transient accumulation of phenol.     

Dynamic and steady state studies of phenol biodegradation in pure and mixed cultures.

The microbial degradation of phenol by pure and mixed cultures of Pseudomonas putida was studied in batch, phenol-stat, and continuous culture systems. In the continuous culture runs, both steady state and transient experiments were performed. From these experiments, a model for the kinetic behavior of the organisms was evolved and an analysis performed on the stability and dynamic behavior of pure and mixed cultures. The results indicate that it should be possible to achieve phenol removal from wastewaters down to levels of 1-2 ppm in a single state system. However, because of the effect of substrate inhibition on kinetic behavior of the microorganisms, long lasting transients can occur. The transient behavior of such systems cannot be solely determined from mumax or Ks parameters, but must include a consideration of the transient size and response characteristic of the organism.     

Oscillatory growth rate responses of S. Cerevisiae in continuous culture

The yeast S. cerevisiae was grown on dilute, chemically defined media in continuous culture with either glucose or ammonium sulfate as the growth-limiting ingredient. Changes in dilution rate or glucose concentration induced decaying oscillations in the numbers of yeast growing on ammonium sulfate-limited media. Spot checks indicated that Cell dry weight and Kjeldahl nitrogen followed the cell numbers during these oscillations. With glucose-limited media, there was no response to step changes in ammonium sulfate concentration, and dilution rate step changes gave non-oscillatory transient responses.     

Altered secretion of corticosteroids and prolactin in adrenal regeneration hypertensive rats.

To assess the possible role of mineralocorticoids in the onset and maintenance of hypertension in adrenal regeneration hypertensive (ARH) rats, the change in plasma mineralocorticoids, with adrenal regeneration after enucleation in ARH rats was investigated and compared with those in unilaterally nephroadrenalectomized, 1% saline-fed (UNA) rats, sham-operated, 1% saline-fed (1% NaCl) rats and water-fed (water) rats. Plasma aldosterone was determined by RIA and the other mineralocorticoids were measured by HPLC. How plasma PRL, a marker of central dopaminergic activity, affected aldosterone secretion was determined by RIA. In ARH, plasma corticosterone (B), 18-OH-DOC and aldosterone levels 2 weeks after operation were as low as 20-30% of corresponding values, but the plasma DOC level was almost 100% of the corresponding value in the other groups. Four weeks after operation plasma B increased to a level comparable with that in the other groups and the plasma aldosterone level remained low. However, plasma DOC and 18-OH-DOC levels 4 weeks after operation were as high as 120-200% of corresponding values in the other groups. Six weeks after operation, the plasma aldosterone level returned to a value comparable with that in UNA and 1% NaCl and plasma DOC and 18-OH-DOC levels returned to corresponding values in the other groups. The plasma PRL level 4 weeks after operation was significantly lower in ARH than in the other groups. These results suggest that transient DOC and 18-OH-DOC increases observed in ARH may be important in the onset of hypertension, while other factors may be involved in its maintenance and that the transient central dopaminergic hyperactivity observed in ARH may be responsible for a delayed return from aldosterone deficiency.     

Protection by natural blackwater against disturbances in ion fluxes caused by low pH exposure in freshwater stingrays endemic to the Rio Negro

Stenohaline freshwater stingrays (Potamotrygon spp.) are endemic to the very dilute (Na(+), Cl(-), Ca2(+) 300 micromol L(-1) in reference water (low DOC) to about 100 micromol L(-1) in blackwater (high DOC). In reference water, both JNain and JClin were inhibited >90%, both JNaout and JClout more than doubled, and J(Amm) did not change at pH 4.0. In blackwater, the inhibition of influxes was attenuated, the increases in outflux did not occur, and J(Amm) increased by 60% at pH 4.0. Addition of 100 micromol L(-1) Ca(2+) to reference water prevented the increases in JNaout and JClout and allowed J(Amm) to increase at pH 4.0, which demonstrates that the gills are sensitive to Ca(2+). However, addition of Ca(2+) to blackwater had no effect on the responses to pH 4.0. Addition of commercial humic acid to reference water did not duplicate the effects of natural Rio Negro blackwater at the same DOC level; instead, it greatly exacerbated the increases in JNaout and JClout at low pH and prevented any protective influence of added Ca(2+). Thus, blackwater DOC appears to be very different from commercial humic acid. Biogeochemical modeling indicated that blackwater DOC prevents Ca(2+) binding, but not H(+) binding, to the gills and that the protective effects of blackwater cannot be attributed to its higher buffer capacity or its elevated Al or Fe levels. Natural DOC may act directly at the gills at low pH to exert a protective effect and, when doing so, may override any protective action of Ca(2+) that might otherwise occur.     

Degradation of phenol by Pseudomonas putida ATCC 11172 in continuous culture at different ratios of biofilm surface to culture volume.

Pseudomonas putida ATCC 11172 was grown in continuous culture with phenol as the only carbon and energy source; a culture practically without biofilm was compared with biofilm cultures of differing surface area/volume ratios. The biofilm did not significantly affect the maximal suspended cell concentration in the effluent, but it increased the maximal phenol reduction rate from 0.23 g/liter per h (without biofilm) to 0.72 g/liter per h at the highest biofilm level (5.5 cm2 of biofilm surface per ml of reactor volume). The increase in phenol reduction rate was linear up to the surface area/volume ratio of 1.4 cm2/ml. The continuous cultures with biofilms could tolerate a higher phenol concentration of the medium (3.0 g/liter) than the nonbiofilm system (2.5 g/liter). At higher dilution rates an intermediate product, 2-hydroxymuconic semialdehyde, accumulated in the culture. When the biomass of the effluent started to decrease, the concentration of 2-hydroxymuconic semialdehyde reached a peak value. We conclude that biofilms in continuous culture have the potential to enhance the aerobic degradation of aromatic compounds.     

Degradation of phenol by Pseudomonas putida ATCC 11172 in continuous culture at different ratios of biofilm surface to culture volume

Pseudomonas putida ATCC 11172 was grown in continuous culture with phenol as the only carbon and energy source; a culture practically without biofilm was compared with biofilm cultures of differing surface area/volume ratios. The biofilm did not significantly affect the maximal suspended cell concentration in the effluent, but it increased the maximal phenol reduction rate from 0.23 g/liter per h (without biofilm) to 0.72 g/liter per h at the highest biofilm level (5.5 cm2 of biofilm surface per ml of reactor volume). The increase in phenol reduction rate was linear up to the surface area/volume ratio of 1.4 cm2/ml. The continuous cultures with biofilms could tolerate a higher phenol concentration of the medium (3.0 g/liter) than the nonbiofilm system (2.5 g/liter). At higher dilution rates an intermediate product, 2-hydroxymuconic semialdehyde, accumulated in the culture. When the biomass of the effluent started to decrease, the concentration of 2-hydroxymuconic semialdehyde reached a peak value. We conclude that biofilms in continuous culture have the potential to enhance the aerobic degradation of aromatic compounds.     

The functional quality of decomposing litter outputs from an Arctic plant community is affected by long-term exposure to enhanced UV-B

Plant exposure to enhanced UV-B radiation typically induces changes in leaf secondary metabolite profiles which will be inherited in litter, affecting litter breakdown and the carbon (C) dynamics of sensitive plant communities. A key enzyme in the decomposition process is phenol oxidase which is influenced by litter quality and, hence, a decomposition bioindicator. Here we investigated dwarf shrub litter decomposition following experimental community exposure to enhanced UV-B over two decades in the Swedish sub-Arctic. We examined the hypothesis that foliar UV-B exposure would alter litter quality to elevate phenol oxidase activity. This was tested in the field by measuring phenol oxidase activity in freshly collected mixed-community litter from under our experimental vegetation. A laboratory mesocosm was next used in a decomposition assay to investigate individual species responses over eight weeks, with an emphasis on the quality of leachate outputs from decomposing litter (from Empetrum hermaphroditum, Vaccinium vitis-idaea, Vaccinium uliginosum). In the assay bi-weekly collections of leachate were analysed for phenol oxidase activity, together with total phenolics and dissolved organic C (DOC). At the end of the assay litter mass loss and respired C were also determined. The initial assessment on field mixed-community litter found no enhanced UV-B treatment (henceforth: ‘UV-B treatment’) effect on phenol oxidase activity. However, in the controlled laboratory mesocosm assay, significant species-specific effects of the UV-B treatment were evident, with increased phenol oxidase activity in V. vitis-idaea leachate (P < 0.001) and a significant reduction (P = 0.05) in respired C. Leachate DOC release from the UV-B treatment was greater in both Vaccinium species and the effect on V. uliginosum was significant (P < 0.05). The UV-B treatment had no effect on the total phenolic concentration of litter or leachates for any species, but there were significant differences in leachate total phenolics, both over time and between species. Also the initial phenolic concentration in leachates from the decomposing litter of E. hermaphroditum was greater than both Vaccinum species. Results suggest a species specific role for UV-B in influencing enzyme function and decomposition, dependent on individual traits. This has implications for decomposition dynamics in this system and more widely. Our study highlights the value of using a laboratory assay to gain a mechanistic understanding the species level impacts of a global change factor (UV-B) on decomposition, which are otherwise obscured by community-level responses and difficult to determine under field conditions.     

Effects of progestin antagonists, glucocorticoids and estrogen on progesterone-induced protein secreted by rabbit endometrial stromal cells in culture

Progesterone enhances the synthesis of a 42 kDa protein secreted by rabbit endometrial stromal cells in primary culture. The duration of that response, the effects of estrogen and the inhibitory ability of antiprogestin steroid analogs, RU486, ZK98.299 and ZK98.734, were tested. Although there was a progressive decrease in the amount of the 42 kDa protein synthesized during a 6-day culture period, progesterone stimulated its rate of synthesis greater than 2-fold throughout that period. The addition of estrogen did not prevent the progressive decrease in the amount of the protein synthesized, nor did it enhance the progesterone effect when the culture medium contained phenol red. Estrogen alone did slightly induce 42 kDa protein synthesis by cells grown in phenol red-free medium, and the progesterone response was accentuated to the same degree. When present in a concentration that was 100-fold that of the progesterone, RU486, ZK98.299 and ZK98.734 each abolished stimulation. This antagonistic effect was overcome by addition of an equimolar concentration of progesterone. Deoxycorticosterone (DOC) also stimulated 42 kDa protein synthesis. The antiprogestins blocked this stimulatory effect, even when both steroids were in equimolar concentrations. There was no difference in the ability of ZK98.299 or ZK98.734 to block DOC stimulation, even though ZK98.734 exhibits no antiglucocorticoid activity [J. Steroid Biochem. 25 (1986) 835]. Therefore, it is likely that the DOC effect is mediated by the progesterone receptor system. These studies indicate that enhanced synthesis of the 42 kDa protein represents a progesterone receptor mediated event and that the cell culture system described can be used as a bioassay for determination of antiprogestin activity.     

Anaerobic degradation of phenol in batch and continuous cultures by a denitrifying bacterial consortium

Summary The anaerobic degradation of phenol under denitrifying conditions by a bacterial consortium was studied both in batch and continuous cultures. Anaerobic degradation was dependent on NO_f3 ^p– and concentrations up to 4 mm phenol were degraded within 2–5 days. During continuous growth in a fermenter, steady states could be maintained at eight dilution rates (D) corresponding to residence times between 12.5 and 50 h. Culture wash-out occurred at D=0.084 h^–1. The kinetic parameters obtained for anaerobic degradation of phenol under denitrifying conditions by the consortium were: maximam specific growth rate = 0.091 h^–1; saturation constant = 4.91 mg phenol/l; true growth yield = 0.57 mg dry wt/mg phenol; maintenance coefficient = 0.013 mg phenol/mg dry wt per hour. The Haldane model inhibition constant was estimated from batch culture data giving a value of 101 mg/l. The requirement of CO₂ for the anaerobic degradation of phenol with NO_f3 ^p– indicates that phenol carboxylation to 4-hydroxybenzoate was the first step of phenol degradation by this culture. 4-Hydroxybenzoate, proposed as an intermediate of phenol carboxylation under these conditions, was detected only in continuous cultures at very low growth rates (D=0.02 h^–1), but was never detected as a free intermediary metabolite either in batch or in continuous cultures. Correspondence to: N. Khoury     

The response of a nitrogen-limited chemostat culture of Escherichia coli B/r to pH and dilution rate steps

The effects of four pH steps and one dilution rate step are described for an ammonia-nitrogen-limited continuous culture of Escherichia coli B/r. Two of the pH steps, 6.06-5.49 and 5.96-5.60, led to prolonged transients in both cell density and rate-limiting nutrient concentration. The other two pH steps, 6.20-5.96 and 5.60-6.20, had almost no effect on the culture. The dilution rate step led to a sharp transition in the steady-state external pyruvate concentration. Monitoring of the external pyruvate concentration for the pH 5.96-5.60 step revealed that the transient phase continued long after the cell density and rate-limiting nutrient concentration returned to steady-state values. The implications for industrial and laboratory fermentations are discussed.     

Response of a chlorophenols degrading mixed culture to changing loads of phenol,chlorophenol and cresols

Summary A phenol and solvents degrading mixed culture from soil and sludge supplemented with Pseudomonas sp. strain B13 which harbors genes coding the sequence for chlorocatechol breakdown was acclimated to monochlorophenol degradation. Pyrocatechase activity was used as an indicator for the adaptation status of the culture.In the fully acclimated culture, strain B13 was partially replaced by hybrid strains which had acquired the chlorocatechol degrading sequence. This culture degraded changing loads of phenol, chlorophenols and cresols without accumulation of DOC (dissolved organic carbon). When high cresol concentrations were supplied simultaneously with the chlorophenols, strains were enriched which degrade cresols and 3-methylbenzoate via ortho-cleavage pathway.     

Bacterial Growth in Mixed Cultures on Dissolved Organic Carbon from Humic and Clear Waters

Interactions between bacterial assemblages and dissolved organic carbon (DOC) from different sources were investigated. Mixed batch cultures were set up with water from a humic and a clear-water lake by a 1:20 dilution of the bacterial assemblage (1.0 μm of prefiltered lake water) with natural medium (sterile filtered lake water) in all four possible combinations of the two waters and their bacterial assemblages. Bacterial numbers and biomass, DOC, thymidine incorporation, ATP, and uptake of glucose and phenol were followed in these cultures. Growth curves and exponential growth rates were similar in all cultures, regardless of inoculum or medium. However, bacterial biomass produced was double in cultures based on water from the humic lake. The fraction of DOC consumed by heterotrophic bacteria during growth was in the same range, 15 to 22% of the total DOC pool, in all cultures. Bacterial growth efficiency, calculated from bacterial biomass produced and DOC consumed, was in the order of 20%. Glucose uptake reached a peak during exponential growth in all cultures. Phenol uptake was insignificant in the cultures based on the clear-water medium, but occurred in humic medium cultures after exponential growth. The similarity in the carbon budgets of all cultures indicated that the source of the bacterial assemblage did not have a significant effect on the overall carbon flux. However, fluxes of specific organic compounds differed, as reflected by glucose and phenol uptake, depending on the nature of the DOC and the bacterial assemblage.     

Synthetic Nasonov gland pheromone enhances abundance and visitation of honeybee,Apis mellifera,in Korla fragrant pear,Pyrus sinkiangensis

1. Korla fragrant pear (Pyrus sinkiangensis Yü) depends on cross-pollination by honeybees (Apis mellifera) but may suffer from low honeybee visitation.
2. We assessed whether honeybee abundance and visitation frequency are enhanced by using synthetic Nasonov gland pheromone (NGP), which is naturally produced by worker bees to stimulate the aggregation of bees to food resources or nesting sites.
3. The response of honeybees to synthetic NGP was firstly assessed using Y-tube olfactometer tests in the laboratory, and subsequently in the field, by placing NGP lures on Korla fragrant pear trees in orchards with and without beehives. Honeybee abundance was assessed using coloured pan traps while honeybee visits were assessed by visual observations on pear flowers.
4. Y-tube olfactometer tests showed a significant preference of honeybees for NGP. In pear orchards with beehives, honeybee abundance was 2.5-fold higher on trees with NGP lures than on trees without NGP, and 2.2-fold higher in orchards in which all trees contained NGP lures than in orchards without NGP lures. Such positive effects were not observed in orchards without beehives.
5. Flower visitation by honeybees was significantly higher in trees with NGP lures than without NGP lures, irrespective of the presence (5.7-fold higher) or absence of beehives (27.6-fold higher).
6. In mixed pear-apricot orchards, honeybee abundance was higher in pear trees with NGP lures than without lures.
7. Our results show that NGP lures attract honeybees to flowering pear trees in monoculture pear and mixed pear-apricot orchards, and that this effect is greatest in orchards with beehives.

     

Temperature relationship in continous culture

The steady-state and transient behavior of a chemostat-type continuous culture of Aerobacter aerogenes was investigated with special reference to temperature. Experimentally determined kinetic parameters of the fitted steady-state model were found to be strong functions of temperature. The transient response of the culture to a step change in temperature exhibited a dynamic lag whose magnitude depended on the nature and direction of step change.     

Aerobic,phenol-induced TCE degradation in completely mixed,continuous-culture reactors

BothPseudomonas putida F1 and a mixed culture were used to study TCE degradation in continuous culture under aerobic, non-methanotrophic conditions. TCE mass balance studies were performed with continuous culture reactors to determine the total percent removed in the reactors, and to quantify the percent removed by air stripping and biodegradation. Adsorption of TCE to biomass was assumed to be negligible. This research demonstrated the feasibility of treating TCE-contaminated water under aerobic, non-methanotrophic conditions with a mixed-culture, continuous-flow system.Initially glucose and acetate were fed as primary substrates. Pnenol, which has been shown to induce TCE-degrading enzymes, was fed at a much lower concentration (20mg/L). Little degradation of TCE was observed when acetate and glucose were the primary substrates. After omitting glucose and acetate from the feed and increasing the phenol concentration to 50mg/L, TCE biotransformation was observed at a significant level (46%). When the phenol concentration in the feed was increased to 420mg/L, 85% of the incoming TCE was estimated to have been biodegraded. Under the same conditions, phenol utilization by the mixed culture was greater than that ofP. putida F1, and TCE degradation by the mixed culture (85%) exceeded that ofP. putida F1 (55%). The estimated percent-of-TCE biodegraded by the mixed culture was consistently greater than 80% when phenol was fed at 420mg/L. Biodegradation of TCE was also observed in mixed-culture, batch experiments.     

Aerobic biotreatment of acetone and methanol in a continuous flow bioreactor during unsteady state operation

The responses of a culture, enriched with acetone and methanol as dual carbon energy substrates, when growing in a continuous flow bioreactor are examined with respect to various imposed transient state operating conditions. The transients investigated include both removal from and addition to the process feed of acetone and methanol, step changes in the concentrations of acetone and methanol in the process feed, and step changes in the dilution rates employed. The capacity of the culture to achieve complete acetone oxidation, after step changes, was shown to be suspect, emphasizing the need to base performance criteria for biotreatment process operation on individual key component concentrations rather than on the lumped parameters widely used to describe pollutant loads in aqueous effluents.