Biodegradation of dichloroacetic acid by freely suspended and adsorptive immobilized Xanthobacter autotrophicus GJ10 in soil

Xanthobacter autotrophicus GJ10 was applied in a packed-bed fermentor to degrade dichloroacetic acid (DCA) in batch-, semicontinuous and continuous culture. Degradation has been studied with freely suspended and adsorptive immobilized cells. To imitate natural soil systems, the fermentor was filled with sand. Concentrations of up to 20 mm DCA were degraded completely. If higher initial concentrations were used, the decrease in pH value inhibited further growth and degradation. In continuous culture the fermentor was inoculated additionally with activated sludge. Over a period of 2 weeks the specialized strain could be retained and no decrease in metabolic activity was observed. A decrease in degradation of DCA was observed when succinate was added as a second substrate. The haloacid dehalogenase was found to be induced by DCA. Non-induced cells showed typical repression of catabolites and diauxic growth with succinate as co-substrate. The results demonstrate that X. autotrophicus GJ10 might be suitable for applications in biological waste treatment systems. Correspondence to: H.-J. Rehm     

Influence of a supplementary carbon source on biodegradation of pyridine by freely suspended and immobilizedPimelobacter sp.

The effect of the presence of supplementary glucose or acetate on the growth and pyridine-degrading activity of freely suspended and calcium-alginate-immobilizedPimelobacter sp. was investigated. Although the supplementary carbon sources could be degraded simultaneously with pyridine,Pimelobacter sp. exhibited a preference for pyridine over supplementary carbon sources. Thus, the pyridine-degrading activity of the freely suspended cells was not decreased significantly by the addition of either glucose (1.5–6 mM) or acetate (6–24 mM) to the pyridine (6–24 mM). In the semi-continuous immobilized cell culture, immobilized cells also exhibited a preference for pyridine over supplementary carbon sources and did not switch their substrate preference throughout the culture. Owing to a high cell concentration, the volumetric pyridine degradation rate at 24 mM pyridine in the immobilized cell culture was approximately six times higher than that in the freely suspended cell culture. Furthermore, the immobilized cells could be reused 16 times without losing their pyridine-degrading activity during the culture period tested. Taken together, the use of immobilizedPimelobacter sp. for the degradation of pyridine is quite feasible because of the preference for pyridine over supplementary carbon sources, the high volumetric pyridine degradation rate, and the reusability of immobilized cells.     

Biodegradation of 2-chloroethanol by freely suspended and adsorbed immobilized Pseudomonas putida US2 in soil

The degradation of 2-chloroethanol by Pseudomonas putida US2 was investigated in batch, repeated batch and continuous cultures especially in a packed-bed fermenter with sand. The degradation of 2-chloroethanol was connected with a release of protons, which led to a decrease of the pH in the medium. Higher initial concentration than 25 mM 2-chloroethanol were not degraded completely because they entailed a decrease of the pH to 5.0, which inhibited further growth and degradation. P. putida US2 showed a typical repression of catabolites and diauxic growth with succinate as cosubstrate. The addition of succinate as a second substrate caused a decrease in degradation of 2-chloroethanol. Activated sludge added to adsorbed cultures in a continuous fermentation did not lead to a decrease in metabolic activity. After 2 weeks of continuous cultivation the specialized strain could be retained.     

Influence of phenol on biodegradation of p-nitrophenol by freely suspended and immobilized Nocardioides sp. NSP41

The effect of the presence of an alternate toxiccompound (phenol) on the p-nitrophenol(PNP)-degrading activity of freely suspended andcalcium alginate immobilized Nocardioides sp.NSP41 was investigated. In the single substrateexperiments, when the concentration of phenol and PNPwas increased to 1400 mg l^-1 and 400 mg l^-1,respectively, the initial cell concentrations in thefreely suspended cell culture should be higher than1.5 g dry cell weight l^-1 for completedegradation. In the simultaneous degradationexperiment, when the initial concentration of phenolwas increased from 100 to 400 mg l^-1, thespecific PNP degradation rate at the concentration of200 mg l^-1 was decreased from 0.028 to 0.021h^-1. A freely suspended cell culture with a highinitial cell concentration resulted in a highvolumetric degradation rate, suggesting the potentialuse of immobilized cells for simultaneous degradation.In the immobilized cell cultures, althoughsimultaneous degradation of PNP and phenol wasmaintained, the specific PNP and phenol degradationrate decreased. However, a high volumetric PNP andphenol degradation rate could be achieved byimmobilization because of the high cell concentration.Furthermore, when the immobilized cells were reused inthe simultaneous degradation of PNP and phenol, theydid not lose their PNP- and phenol-degrading activityfor 12 times in semi-continuous cultures. Takentogether, the use of immobilized Nocardioidessp. NSP41 for the simultaneous degradation of PNP andphenol at high concentrations is quite feasiblebecause of the high volumetric PNP and phenoldegradation rate and the reusability of immobilizedcells.     

Influence of phenol on the biodegradation of pyridine by freely suspended and immobilized Pseudomonas putida MK1

AIMS: To study the effect of co-contaminants (phenol) on the biodegradation of pyridine by freely suspended and calcium alginate immobilized bacteria. METHODS AND RESULTS: Varying concentrations of phenol were added to free and calcium alginate immobilized Pseudomonas putida MK1 (KCTC 12283) to examine the effect of this pollutant on pyridine degradation. When the concentration of phenol reached 0.38 g l(-1), pyridine degradation by freely suspended bacteria was inhibited. The increased inhibition with the higher phenol levels was apparent in increased lag times. Pyridine degradation was essentially completely inhibited at 0.5 g l(-1) phenol. However, immobilized cells showed tolerance against 0.5 g l(-1) phenol and pyridine degradation by immobilized cell could be achieved. CONCLUSIONS: This works shows that calcium alginate immobilization of microbial cells can effectively increase the tolerance of P. putida MK1 to phenol and results in increased degradation of pyridine. SIGNIFICANCE AND IMPACT OF THE STUDY: Treatment of wastewater stream can be negatively affected by the presence of co-pollutants. This work demonstrates the potential of calcium alginate immobilization of microbes to protect cells against compound toxicity resulting in an increase in pollutant degradation.     

Effect of pyridine on the fatty acid composition of Pimelobacter sp.

Abstract The fatty acid composition of Pimelobacter sp. is of the complex type. When pyridine was used as a sole inhibitory substrate, the fatty acid composition of Pimelobacter sp. was quite different from that within other readily available substrates. When compared with the fatty acid composition in a complex medium, the proportion of isopalmitic acid ( iso -C_16:0) drastically decreased from 68.4% to 7.7%, while the proportion of anteisoheptadecanoic acid ( anteiso -C_17:0) remained almost constant at ca. 7%. Concomitantly, this decrease of the branched-chain fatty acid was accompanied by the increase of the straight-chain, especially long-chain, fatty acid such as heptadecanoic (C_17:0), octadecenoic (Cig:i), 10-methylheptadecanoic (10-me-18) and 10-methyloctadecanoic (10-me-19) acids. Consequently, in response to membrane active organic solvents, Pimelobacter sp. was found to regulate its membrane fatty acid composition in a fashion different from Gram-negative bacteria.     

Production of cellulase by freely suspended and immobilized cells of Trichoderma reesei

Trichoderma reesei (QM 9123) was immobilized within the open porous network of reticulated polyurethane foam matrices, and the growth pattern, glucose consumption and cellulase production were compared with those of freely suspended cells. It was found that the method of immobilization was simple and had no detrimental effect on cell activity. Various production media, to be used after the cultivation of T. reesei were tried. It was found that a nitrogen source-free production medium gave the highest enzyme titers of 1.5 × 10³ FPA U l⁻¹. Similar results were obtained with both freely suspended and immobilized cells.     

Biodegradation of p-cresol by immobilized cells of Bacillus sp. strain PHN 1

The Bacillus sp. strain PHN 1 capable of degrading p-cresol was immobilized in various matrices namely, polyurethane foam (PUF), polyacrylamide, alginate and agar. The degradation rates of 20 and 40 mM p-cresol by the freely suspended cells and immobilized cells in batches and semi-continuous with shaken cultures were compared. The PUF-immobilized cells achieved higher degradation of 20 and 40 mM p-cresol than freely suspended cells and the cells immobilized in polyacrylamide, alginate and agar. The PUF- immobilized cells could be reused for more than 35 cycles, without losing any degradation capacity and showed more tolerance to pH and temperature changes than free cells. These results revealed that the immobilized cell systems are more efficient than freely suspended cells for degradation of p-cresol.     

Biodegradation of phenol by enzymes from Pseudomonas sp. immobilized onto ultrafiltration membranes

A method of immobilizing enzymes from Pseudomonas sp. that decompose phenol on polymeric ultrafiltration membranes is described. Transport-separation properties of neutral and enzymic membranes have been compared and the optimal ultrafiltration process parameters of a model phenol solution have been determined. The immobilized enzyme system was applied to the biodegradation of phenol in coke wastewaters.     

Real-time in situ monitoring of freely suspended and immobilized cell cultures based on mid-infrared spectroscopic measurements.

Glucose and lactate profiles in Chinese hamster ovary cell cultures were accurately monitored in real time and in situ during three bioreactor batch cultures lasting 11,15, and 15 days performed within a 60-day period. Monitoring was accomplished using in situ-collected mid-infrared spectra analyzed with a priori one-time established partial least-squares regression models. The robustness of the technique was demonstrated by application of these models without modification after 2.3 years. Neither recalibration nor instrument maintenance was required during the 2.3-year period, except for the daily filling of liquid nitrogen for detector cooling during operation. The lactate calibration model yielded accurate absolute concentration estimations during each of the batch cultures with standard errors of estimate from 1 to 3 mM. The a priori-established glucose calibration model yielded concentration estimations with an off-set, which was constant throughout a culture. Adjustment of the off-set before inoculation resulted in accurate concentration estimations with Standard errors of estimate of approximately 1 mM for each of the bioreactor cultures. Sensitivity in detecting differences of 0.5 mM and selectivity against variation of one metabolite while the other was kept constant was demonstrated during standard additions of either glucose or lactate. The sensor system proved to be reliable, simple, accurate, sterile, and capable of long-term automatic operation and is considered to be mature enough to be routinely applied for in situ (on-line) cell culture monitoring.     

Use of ATP to characterize biomass viability in freely suspended and immobilized cell bioreactors

This work describes investigations into the viability of cells growing on 3,4-dichloroaniline (34DCA). Two bioreactors are employed for microbial growth, a continuous stirred tank (CST) bioreactor with a 2-L working volume, and a three-phase air lift (TPAL) bioreactor with a 3-L working volume. Experiments have been performed at several dilution rates between 0.027 and 0.115 h(-1) in the CST bioreactor and between 0.111 and 0.500 h(-1) in the TPAL bioreactor. The specific ATP concentration was calculated at each dilution rate in the suspended biomass in both bioreactors as well as in the immobilized biomass in the TPAL bioreactor. The ATP was extracted from the cells using boiling tris-EDTA buffer (pH 7.75), and the quantity determined using a firefly (bioluminescence) technique. The cultures were inspected under an electron microscope to monitor compositional changes. Results from the CST bioreactor showed that the biomass-specific ATP concentration increases from 0.44 to 1.86 mg ATP g(-1) dry weight (dw) as dilution rate increases from 0.027 to 0.115 h(-1). At this upper dilution rate the cells were washed out. The specific ATP concentration reached a limiting average value of 1.73 mg ATP g(-1) dw, which is assumed to be the quantity of ATP in 100% viable biomass. In the TPAL bioreactor, the ATP level increased with dilution rate in both the immobilized and suspended biomass. The specific ATP concentration in the immobilized biomass increased from approximately 0.051 mg ATP g(-1) dw at dilution rates between 0.111 and 0.200 h(-1) to approximately 0.119 mg ATP g(-1) dw at dilution rates between 0.300 and 0.500 h(-1). This indicates that the immobilized biomass contained a viable cell fraction of around 5%. Based on these results, kinetic data for freely suspended cells should not be applied to the modeling of immobilized cell systems on the assumption that immobilized biomass is 100% viable. (c) 1993 John Wiley & Sons, Inc.     

Biodegradation of 4-chlorophenol by adsorptive immobilized Alcaligenes sp. A 7-2 in soil

Summary Alcaligenes sp. A 7-2 immobilized on granular clay has been applied in a percolator to degrade 4-chlorophenol in sandy soil. Good adsorption rates on granular clay were achieved using cell suspensions with high titres and media at pH 8.0. The influence of various parameters such as aeration rate, pH, temperature, concentration of 4-chlorophenol and size of inoculum on the degradation rate were investigated. During fedbatch fermentations under optimal culture conditions, concentrations of 4-chlorophenol up to 160 mg·1^–1 could be degraded. Semicontinuous culture experiments demonstrated that the degradation potential in soil could be well established and enhanced by the addition of immobilized bacteria. Continuous fermentation was performed with varying 4-chlorophenol concentrations in the feed and different input levels. The maximum degradation rate was 1.64 g·1^–1·day^–1. Offprint requests to: H.-J. Rehm     

Biotransformation of codeine to morphine in freely suspended cells and immobilized cultures of Spirulina platensis

Both freely suspended cells and immobilized cultures of Spirulina platensis, a blue-green alga, biotransformed exogenously fed codeine, an opium alkaloid, to morphine. The external addition of codeine to the culture medium did not affect the growth of S. platensis. Immobilization of Spirulina in a calcium alginate gel matrix was optimized by using 2% (w/v) sodium alginate and reducing the concentration of nutrients of Zarrouk's medium, which caused destabilization of the calcium alginate gel. The accumulation of morphine increased gradually and reached maxima of 330g 100ml^–1 culture at 105h in freely suspended and 351g 100ml^–1 at 96h in immobilized Spirulina cultures. Accumulation of morphine was detected only in the medium, whereas cells did not show accumulation. The immobilized Spirulina cultures showed marginally higher conversion of codeine to morphine over freely suspended cultures.     

Biodegradation of malachite green by Ochrobactrum sp.

This study presents the biodegradation of malachite green (MG), a triphenylmethane dye, using a novel microorganism isolated from textile effluent contaminated environment. The organism responsible for degradation was identified as Ochrobactrum sp JN214485 by 16S rRNA analysis. The effect of operating parameters such as temperature, pH, immobilized bead loading, and initial dye concentration on % degradation was studied, and their optimal values were found to be 30 °C, 6, 20 g/L and 100 mg/L, respectively. The analysis showed that the extracellular enzymes were responsible for the degradation. The biodegradation of MG was confirmed by UV–visible spectroscopic and FTIR analysis. The phytotoxicity test concluded that the degradation products were less toxic compared to MG. The kinetics of biodegradation was studied and the activation energy was found to be 10.65 kcal/mol.     

Biodegradation of p-toluenesulphonamide by a Pseudomonas sp.

A bacterium capable of utilising p-toluenesulphonamide was isolated from activated sludge. The isolated strain designated PTSA was identified as a Pseudomonas sp. using chemotaxonomic and genetic studies. Pseudomonas PTSA grew on p-toluenesulphonamide in a chemostat with approximately 90% release of sulphate and 80% release of ammonium. The isolate was also able to grow on 4-carboxybenzenesulphonamide and 3,4-dihydroxybenzoate but did not grow on p-toluenesulphonate. The transient appearance of 4-hydroxymethylbenzenesulphonamide and 4-carboxybenzenesulphonamide during p-toluenesulphonamide degradation proves oxidation of the methyl group is the initial attack in the biodegradation pathway. Both metabolites of p-toluenesulphonamide degradation were identified by high-performance liquid chromatography-mass spectrometry. 4-Carboxybenzenesulphonamide is probably converted into 3,4-dihydroxybenzoate and amidosulphurous acid. The latter is a chemically unstable compound in aqueous solutions and immediately converted into sulphite and ammonium. Both sulphite and ammonium were formed during degradation of 4-carboxybenzenesulphonamide.     

Lipolytic activity of suspended and membrane immobilized lipase originating from indigenous Burkholderia sp. C20

In this work, a simple, inexpensive, and efficient method of preparing immobilized lipase is presented. The lipase originating from a newly isolated indigenous strain Burkholderia sp. C20 was immobilized onto cellulose nitrate (CN) membrane via filtration. The CN-immobilized lipase was able to retain 60% of its original activity after repeated uses for nine times. The thermal stability of the lipase was also slightly improved after immobilization. The optimal reaction conditions of CN-lipase were pH 9.0 and 55 degrees C, which are similar to those for the suspended lipase. Both suspended and immobilized lipase could hydrolyze the six oil substrates examined, while immobilized lipase displayed less specificity over the oil substrates. Kinetic analysis shows that the dependence of lipolytic activity of both suspended and immobilized lipase on oil substrate concentration can be described by Michaelis-Menten model with good agreement. The estimated kinetic constants for suspended lipase (v(max)=243.9 U/mg, K(m)=0.024 mM) and immobilized lipase (v(max)=32.8 U/mg, K(m)=5.61 mM) were quite different. Employment of immobilization seemed to result in a decrease in v(max) and an increase in K(m), most likely due to the mass transfer resistance arising from formation of micelles during the lipase immobilization process.     

两株假单胞菌对吡啶和喹啉的生物降解

【目的】探究2株假单胞菌(Pseudomonas)对吡啶和喹啉的降解。【方法】基于16S rRNA序列同源性和基因间区分析,对分离菌株进行分类鉴定。通过分光光度法和电喷雾电离质谱法(Electrospray Ionisation/Mass Spectrometry,ESI/MS)确定分离菌株对吡啶和喹啉的降解性能。通过质粒消除验证降解质粒的存在,同时克隆了可能的降解基因。【结果】鉴定结果表明,两株分离细菌隶属于Pseudomonas,并将其命名为XJUHX-1和XJUHX-12。降解数据表明,2株菌株分别耐受吡啶和喹啉,同时分别检测到4种和2种吡啶和喹啉的可能降解产物。结果还表明,消除质粒后的菌株对吡啶和喹啉的降解能力降低。扩增的编码NADH还原酶部分的降解喹啉oxoR基因和编码硝酸还原酶的降解吡啶的nifH基因,同时在E.coli中表达了43kDa和16kDa的蛋白。【结论】2株Pseudomonas具有降解吡啶和喹啉的能力。     

Degradation of 3,4-dichloroaniline in synthetic and industrially produced wastewaters by mixed cultures freely suspended and immobilized in a packed-bed reactor

Summary Degradation of 3,4-dichloroaniline (34DCA) in aqueous by undefined cultures of free and immobilized cells was examined. Batch cultures of freely suspended cells and continuous degradation in a packed-bed reactor were studied using both synthetically concocted and industrially produced waste-waters. 34DCA was found to be degraded with a concomitant evolution of chloride ions into the bulk medium. The [acked bed reactor with biomass immobilized on celite diatomaceous earth was found to be capable of degrading over 98% of the 34DCA present in a synthetically concocted inlet stream at a concentration of 250 mg l^–1. Residence times of less than 4 h were employed, giving an overall volumetric degradation rate for the packed bed of 90 mg l^–1 h^–1. The industrially produced wastewater contained, in addition to 34DCA, aniline, 4-chloroaniline, 2,3-dichloroaniline (23DCA) and 3,4-dichloronitrobenzene. The biomass enriched on the synthetic 34DCA waste-water was found to be capable of degrading these compounds in addition to 34DCA with the exception of 23DCA. 34DCA degradation efficiencies of over 95% were obtained for the industrial waste-water at reactor residence times of 4.6 h, giving volumetric degradation rates of 24 mg l^–1 h^–1. Offprint requests to: A. G. Livingston     

Biodegradation of 2,4-dinitrotoluene by a Pseudomonas sp.

Previous studies of the biodegradation of nonpolar nitroaromatic compounds have suggested that microorganisms can reduce the nitro groups but cannot cleave the aromatic ring. We report here the initial steps in a pathway for complete biodegradation of 2,4-dinitrotoluene (DNT) by a Pseudomonas sp. isolated from a four-member consortium enriched with DNT. The Pseudomonas sp. degraded DNT as the sole source of carbon and energy under aerobic conditions with stoichiometric release of nitrite. During induction of the enzymes required for growth on DNT, 4-methyl-5-nitrocatechol (MNC) accumulated transiently in the culture fluid when cells grown on acetate were transferred to medium containing DNT as the sole carbon and energy source. Conversion of DNT to MNC in the presence of 18O2 revealed the simultaneous incorporation of two atoms of molecular oxygen, which demonstrated that the reaction was catalyzed by a dioxygenase. Fully induced cells degraded MNC rapidly with stoichiometric release of nitrite. The results indicate an initial dioxygenase attack at the 4,5 position of DNT with the concomitant release of nitrite. Subsequent reactions lead to complete biodegradation and removal of the second nitro group as nitrite.