Influence of hydrodynamic conditions on naphthalene dissolution and subsequent biodegradation.

The influence of hydrodynamic conditions on the dissolution rate of crystalline naphthalene as a model polycyclic aromatic hydrocarbon (PAH) was studied in stirred batch reactors with varying impeller speeds. Mass transfer from naphthalene melts of different surface areas to the aqueous phase was measured and results were modeled according to the film theory. Results were generalized using dimensionless numbers (Reynolds, Schmidt, and Sherwood). In combined mass transfer and biodegradation experiments, the effect of hydrodynamic conditions on the degradation rate of naphthalene by Pseudomonas 8909N was studied. Experimental results were mathematically described using mass-transfer and microbiological models. The experiments allowed determination of mass-transfer and microbiological parameters separately in a single run. The biomass formation rate under mass transfer limited conditions, which is related to the naphthalene biodegradation rate, was correlated to the dimensionless Reynolds number, indicating increased bioavailability at increased mixing in the reactor liquid. The methodology presented in which mass transfer processes are quantified under sterile conditions followed by a biodegradation experiment can also be adapted to more complex and realistic systems, such as particulate, suspended PAH solids or soils with intrapartically sorbed contaminants when the appropriate mass-transfer equations are incorporated.

Effect of nonionic surfactants on naphthalene dissolution and biodegradation.

The effect of six nonionic surfactants, Igepal CA-720, Tergitol NPX, Triton X-100, PLE4, PLE10, and PLE23, on the dissolution rate of solid naphthalene was studied in stirred batch reactors. Results showed increased mass-transfer rates with increased surfactant concentrations up to 10 kg m-3. Dissolution experiments were adequatly described by a mechanistic mass-transfer model. Partitioning of naphthalene into the micelles and the diffusion coefficients of the micelles affected the dissolution rate most significantly. Combined dissolution and biodegradation experiments with Triton X-100 or PLE10 with naphthalene showed that the biomass-formation rate of Pseudomonas 8909N (DSM No. 11634) increased concomitantly with the mass-transfer rate under naphthalene-dissolution limited conditions up to surfactant concentrations of 6 kg m-3.

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.

Assessment of inhibition kinetics of the growth of strain P5 on pentachlorophenol under steady-state conditions in a nutristat.

A bacterium degrading pentachlorophenol (PCP) as the only source of carbon and energy was grown in a nutristat , i.e., a continuous culture with on-line measurement and control of the substrate concentration. We improved the PCP nutristat by incorporation of a personal computer with a proportional integral derivative (PID) algorithm for controlling the medium feed pump. The controlled value deviated from the average (set-point) value by 1% maximally. In the PCP nutristat (30 degrees C), the steady-state dilution rate, and hence, specific growth rate, showed a maximum value of 0.142 +/- 0.004 h-1 at set-point PCP concentrations between 37 and 168 microM. At PCP concentrations above 168 microM, the steady-state growth rate decreased because of inhibition. The growth yield coefficient was not seriously affected by the PCP concentration, suggesting that uncoupling was not the inhibitory mechanism. It was concluded that the PCP nutristat is very useful for establishing steady-state conditions that maintain growth-inhibitory PCP concentrations and high cell concentrations, conditions for which the chemostat is not suitable.

Catabolite repression of the toluene degradation pathway in Pseudomonas putida harboring pWW0 under various conditions of nutrient limitation in chemostat culture.

In earlier studies, the pathway of toluene and m- and p-xylene degradation (TOL pathway) in Pseudomonas putida (pWW0) was found to be subject to catabolite repression when the strain was grown at the maximal rate on glucose or succinate in the presence of an inducer. This report describes catabolite repression of the TOL pathway by succinate in chemostat cultures run at a low dilution rate (D = 0.05 h-1) under different conditions of inorganic-nutrient limitation. The activity of benzylalcohol dehydrogenase (BADH) in cell extracts was used as a measure of the expression of the TOL upper pathway. When cells were grown in the presence of 10 to 15 mM succinate under conditions of phosphate or sulfate limitation, the BADH activity in response to the nonmetabolizable inducer o-xylene was less than 2% of that of cells grown under conditions of succinate limitation. Less repression was found under conditions of ammonium or oxygen limitation (2 to 10% and 20 to 35%, respectively, of the BADH levels under succinate limitation). The BADH expression levels determined under the different growth conditions appeared to correlate well with the mRNA transcript levels from the upper pathway promoter (Pu), which indicates that repression was due to a blockage at the transcriptional level. The meta-cleavage pathway was found to be less susceptible to catabolite repression. The results obtained suggest that the occurrence of catabolite repression is related to a high-energy status of the cells rather than to a high growth rate or directly to the presence of growth-saturating concentrations of a primary carbon and energy source.

Influence of nonionic surfactants on bioavailability and biodegradation of polycyclic aromatic hydrocarbons.

The presence of the synthetic nonionic surfactants Triton X-100, Tergitol NPX, Brij 35, and Igepal CA-720 resulted not only in increased apparent solubilities but also in increased maximal rates of dissolution of crystalline naphthalene and phenanthrene. A model based on the assumption that surfactant micelles are formed and act as a separate phase underestimated the dissolution rates; this led to the conclusion that surfactants present at concentrations higher than the critical micelle concentration affect the dissolution process. This conclusion was confirmed by the results of batch growth experiments, which showed that the rates of biodegradation of naphthalene and phenanthrene in the dissolution-limited growth phase were increased by the addition of surfactant, indicating that the dissolution rates were higher than the rates in the absence of surfactant. In activity and growth experiments, no toxic effects of the surfactants at concentrations up to 10 g liter(sup-1) were observed. Substrate present in the micellar phase was shown to be not readily available for degradation by the microorganisms. This finding has important consequences for the application of (bio)surfactants in biological soil remediation.

Competition in chemostat culture between Pseudomonas strains that use different pathways for the degradation of toluene.

Pseudomonas putida mt-2, P. cepacia G4, P. mendocina KR1, and P. putida F1 degrade toluene through different pathways. In this study, we compared the competition behaviors of these strains in chemostat culture at a low growth rate (D = 0.05 h-1), with toluene as the sole source of carbon and energy. Either toluene or oxygen was growth limiting. Under toluene-limiting conditions, P. mendocina KR1, in which initial attack is by monooxygenation of the aromatic nucleus at the para position, outcompeted the other three strains. Under oxygen limitation, P. cepacia G4, which hydroxylates toluene in the ortho position, was the most competitive strain. P. putida mt-2, which metabolizes toluene via oxidation of the methyl group, was the least competitive strain under both growth conditions. The apparent superiority of strains carrying toluene degradation pathways that start degradation by hydroxylation of the aromatic nucleus was also found during competition experiments with pairs of strains of P. cepacia, P. fluorescence, and P. putida that were freshly isolated from contaminated soil.

Inducibility of the TOL catabolic pathway in Pseudomonas putida (pWW0) growing on succinate in continuous culture: evidence of carbon catabolite repression control.

The TOL catabolic genes in Pseudomonas putida (pWW0) are clustered in the upper operon, encoding enzymes for the conversion of toluene and xylenes to benzoate and toluates, and the meta-cleavage operon, encoding enzymes for the conversion of the benzoate and toluates to tricarboxylic acid cycle intermediates. In this study, it was shown that cells growing in a chemostat under succinate growth-limiting conditions express both the upper and meta-cleavage pathways in response to o-xylene, a nonmetabolizable effector of the XylR regulatory protein. The dilution rate maintained in the succinate-limited chemostat cultures influenced the synthesis levels of TOL pathway enzymes, their steady-state levels, and their turnover rates. Cells growing in the presence of nonlimiting concentrations of succinate in continuous culture did not express pathway enzymes in response to the addition of o-xylene, which was due to a blockage at the transcriptional level. Expression of the meta-cleavage pathway in response to 2,3-dimethylbenzoate, a nonmetabolizable effector of the XylS regulatory protein, was 93% lower in cultures exposed to succinate at nonlimiting concentrations than in the succinate-limited chemostats. The mRNA level of xylS during nonlimited growth on succinate was very low compared with that in succinate-limited cultures, suggesting that suppression of expression of the meta-cleavage pathway is regulated mainly by the level of the XylS regulator.

Growth and enrichment of pentachlorophenol-degrading microorganisms in the nutristat, a substrate concentration-controlled continuous culture.

The nutristat, a substrate concentration-controlled continuous culture, was used to grow pentachlorophenol (PCP)-degrading microorganisms. The PCP concentration control system consisted of on-line measurement of the PCP concentration in the culture vessel with a tangential filter and a flowthrough spectrophotometer. With PCP concentrations between 45 and 77 microM, a stable situation was established in the nutristat, with an average dilution rate of 0.035 +/- 0.003 h-1. Compared with those of fed-batch cultures and chemostat cultures, the growth rates of microorganisms in the PCP nutristat were significantly higher, leading to considerable time savings in the enrichment procedure. In addition, PCP accumulation to severe inhibitory levels in the culture is prevented because the set point determines the (maximum) PCP concentration in the culture. The use of the nutristat as a tool for the growth of bacteria that degrade toxic compounds is discussed.

Mathematical analysis of catabolic function loss in a population of Pseudomonas putida mt-2 during non-limited growth on benzoate.

Pseudomonas putida mt-2, harbouring the TOL plasmid PWW0, was grown continuously on benzoate in a phauxostat at a non-limited rate. The gradual decrease in the population carrying the complete TOL plasmid was caused predominantly by a growth-rate advantage of spontaneous mutants carrying a partially deleted plasmid (TOL- cells). The growth-rate difference (v) was quantified both by measuring the increase in the dilution rate (from 0.68 to 0.79 h-1; v = 0.11 h-1) and by mathematical analysis of the ingrowth of TOL- cells (v = 0.12 h-1). The latter procedure also established that the segregation rate was of the order of magnitude 10(-5) h-1. Similar values for the growth-rate advantage and the segregation rate were found when both benzoate and succinate were present in non-limiting concentrations. It is suggested that the growth-rate disadvantage of the wild-type strain is caused by inhibitory effects of an intermediate in the degradation of benzoate via the plasmid-encoded meta-pathway.

Stability of TOL plasmid pWW0 in Pseudomonas putida mt-2 under non-selective conditions in continuous culture.

Pseudomonas putida mt-2, harbouring the TOL plasmid pWW0, was grown in chemostat culture under succinate-, sulphate-, ammonium- or phosphate-limitation at different dilution rates. The fraction of mutant cells lacking the plasmid-encoded enzymes for the degradation of toluene and xylene (TOL- cells), was determined. Genetic analysis revealed that all TOL- cells isolated harboured partially deleted plasmids, lacking the TOL catabolic genes. The growth-rate advantage of the TOL- cells was quantified from the kinetics of their increase as a fraction of the total population. At a dilution rate of 0.1 h-1 no growth-rate advantage of TOL- cells was found when phosphate or ammonium were limiting. Under sulphate-limitation, ingrowth of TOL- cells was evident but did not follow a straightforward pattern. Under succinate-limitation the growth-rate advantage was the highest, particularly at low dilution rates (about 50% at D = 0.05 h-1). In phauxostat culture, at the maximal growth rate, the growth-rate advantage of TOL- cells was less than 1%. The specific activity in TOL+ cells of the plasmid-encoded enzyme catechol 2,3-dioxygenase was relatively high at a low growth rate.

Benign thymic cyst following mantle radiotherapy for Hodgkin's disease.

The case history of a patient who was treated for Stage IA Hodgkin's disease involving the left supraclavicular region is presented. Shortly after mantle field radiation therapy she developed a mass in the anterior mediastinum. Histological examination of the lesion revealed it to be a benign cyst of the thymus. We consider it important that the possibility of the existence of such a benign growth be considered when a mediastinal mass appears in a patient treated for Hodgkin's disease, before more intensive tumour therapy is given.

Survival after resection for carcinoma of the oesophagus.

During the period 1978-1984, 525 patients referred with cancer of the oesophagus or gastro-oesophageal junction were assessed for operation and cure. After investigation, 276 patients were selected and operated upon, as a rule, 4 weeks after radiotherapy (40 Gy/4 weeks). In 224 patients (81 per cent) the oesophagus and cardia were resected and reconstructed with stomach (69 per cent), colon (21 per cent), free ileal graft (7 per cent) or Roux-en-Y-oesophagojejunostomy (3 per cent). The postresectional hospital mortality was 14 per cent in all patients and decreased to 5 per cent in 1983. Mortality was higher when the colon was used for reconstruction than when the stomach was used. By postresection staging, 82 patients were found to have stages I and II tumours and 142 patients stage III tumours. Estimated 3-year survival after resection for all male patients was 28 per cent and for all female patients was 42 per cent. Estimated 3-year survival for all patients treated for adenocarcinoma was 31 per cent. Survival was better for stages I and II patients with adenocarcinoma (52 per cent) than for stage III patients (18 per cent) (P less than 0.01). Estimated 3-year survival for all patients treated for squamous cell carcinoma was 33 per cent. Estimated 3-year survival was better for stages I and II patients with squamous cell carcinoma (48 per cent) than for stage III patients (25 per cent) (P less than 0.001). It can be concluded from this study that resection of oesophagus and cardia after radiotherapy offers hope for cure in a subgroup of patients with non-advanced oesophageal cancer. The operation can be performed with acceptable mortality by experienced surgeons, especially when the stomach is used for reconstruction.

Therapy of esophageal carcinoma. Results from the Joint Group on Esophageal Carcinoma in Rotterdam.

During the period 1978-1981 172 patients were referred to the Rotterdam Joint Group on Esophageal Carcinoma. Ninety-one patients were considered for combined therapy, comprising radiation therapy and surgery, and 10 patients refused surgery. The figures given in this material are actuarial survival values corrected for intercurrent death (the actuarial overall survival in parentheses). The 4-year survival in 69 patients who completed this treatment was 39 per cent (26%) (significantly better for women compared with men; significantly better for tumors less than 2 vertebrae compared with tumors greater than or equal to 2 vertebrae). The resectability rate was 85 per cent and the operation mortality rate 20 per cent. Thirty-eight patients had curative radiation therapy with a 4-year corrected survival of 5 per cent (3%). For palliative treatment, radiation therapy and endoscopic introduction of a Celestin tube were mostly used. The results of curative as well as of palliative treatment of esophageal carcinoma have shown improvement during the past decade compared with an earlier period.

Carcinoma of the esophagus: treatment results.

Of the 172 patients with carcinoma of the esophagus or the gastro-esophageal junction seen between January 1978 and January 1981, 69 patients had combined treatment, radiotherapy and resection, and 38 had curative radiotherapy. The remaining 65 were treated palliatively. The 4-year actuarial survival of the first two treatment groups was respectively 40% and 4%. The resectability rate of the operated patients was 84% with a post-operative mortality of 20%. The tumor size and sex were two important prognostic factors. Patients with combined treatment and a tumor size of less than two corresponding underlying vertebrae, had a 4-year actuarial survival of 60%.

Carcinoma of the base of the tongue. A review of 45 cases.

The treatment results of 45 patients with carcinoma of the base of the tongue treated in the Rotterdam Radio-Therapeutic Institute in the period 1970 to 1980 are evaluated. Most patients were treated with split course radiotherapy, three patients were operated after the first course of radiotherapy. Eighteen patients with stage III and IV disease were treated with a combination of chemotherapy and radiotherapy. Overall 5-year survival was 27%, intercurrent death corrected survival was 40%. Adequate staging is a problem in carcinoma of the base of the tongue, especially in T2 and T3 lesions. A systemic combined approach of radiotherapy and surgery is recommended.

Carcinoma of the paranasal sinuses. Results of a prospective pilot study.

Sixty patients with paranasal sinus cancer were treated from January 1976 until July 1981 according to a standard protocol in which surgery is combined with a low dose of irradiation and a topically applied cytostatic drug. The local control rate was 65%. Mutilation was clearly reduced. The actuarial 2-year and 5-year survival rate was 76% and 65%, respectively. The actuarial 5-year survival rate for squamous cell carcinoma and undifferentiated carcinoma of the maxillary sinus was 52%. For adenocarcinoma of the ethmoid sinus the actuarial 5-year survival rate was 100%. The results of this prospective pilot study suggest that it may be possible to achieve better results with less aggressive treatment.

Radiotherapy of severe ophthalmic Graves' disease.

The effects of orbital irradiation on Ophthalmic Graves' Disease (OGD) were evaluated in 24 patients. All patients were irradiated after a median duration of eye symptoms of 12 months (range 3-36 months). Irradiation therapy was performed for loss of visual acuity, progressive diplopia and/or keratitis due to recent progression of eye symptoms. During a follow up period of 2-4.5 years, in 11 patients after irradiation, a mean decrease in proptosis oculi of 5.1 mm was found while 8 of these patients showed a mean increase in visual acuity of 0.26. Apparently, a decrease of activity of the inflammatory process in retrobulbar tissues can be achieved after irradiation, over a longer period of time in many patients. Furthermore concomitantly administered corticosteroids could be tapered off completely in all patients. Treatment of severe OGD of recent onset with irradiation, in combination with a short course of prednisone is very effective. This study also shows that prednisone treatment alone is not very successful because of the high recurrence rate of eye sings after decrease of the dose and the many side effects of the drug.