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.     

Biodegradation of dichloroacetic acid by entrapped and adsorptive immobilized Xanthobacter autotrophicus GJ10

The degradation of dichloroacetic acid (DCA) by free, Ca-alginate entrapped and adsorptive immobilized cells of Xanthobacter autotrophicus GJ10 has been studied in various experimental systems. Entrapped cells tolerated increasing concentrations of DCA better than free cells. Free and adsorptive immobilized cells degraded DCA most effectively at maximum O₂ supply, 34°C and an initial pH value of 8.0. The degradation of high DCA concentrations led to a decrease in the pH value and to a stagnation of mineralization, particularly with free or entrapped cells. Due to the stabilization of pH, the supplementation of acetate or succinate resulted in a complete degradation of higher DCA concentrations. Higher degradation rates than in shake cultures were achieved in air-bubble and packed-bed fermentors. DCA was mineralized faster by free or entrapped X. autotrophicus GJ10 than by adsorptive immobilized cells, which, however, were able to remove higher DCA concentrations. The results of the recent investigations with immobilized X. autotrophicus GJ10 are an important prerequisite for the application of this bacterium in waste treatment systems. Correspondence to: U. Heinze     

Degradation of 2-chloroethanol by wild type and mutants of Pseudomonas putida US2

A strain of Pseudomonas putida was isolated that was able to degrade 2-chloroethanol. The degradation proceeded via 2-chloroacetaldehyde and chloroacetate to glycolate. In crude extracts the enzymes for this degradation pathway could be detected. All enzymes proved to be inducible. The dehalogenase that catalyzed the dehalogenation of chloroacetate to glycolate was further characterized. It consisted of a single polypeptide chain with a molecular mass of 28 kDa. After induction the dehalogenase was expressed at a high level. In a mutant resistant to high concentrations of 2-chloroethanol the dehalogenase was no longer expressed. The mechanism of resistance seemed to be due to the inability to convert chloroacetate and export of this compound out of the cell.Non-standard abbreviations CEO 2-chloroethanol - DCPIP 2,6-dichlorophenolindophenol - FPLC fast protein liquid chromatography - PAGE polyacrylamide gelelectrophoresis - PES phenazine ethosulfate - PMS phenazine methosulfate - PQQ pyrroloquinoline quinone     

The Influence of Glucose and Fructose on the Degradation of 2-Chlorophenol by Pseudomonas putida CP1

Summary Pseudomonas putida CP1 grew on 2-chlorophenol when supplied as the sole source of carbon. Chlorophenol degradation was stimulated in the presence of low concentrations of glucose (0.05–1%, w/v). Substrate removal was inhibited and there was a significant fall in pH with concentrations of glucose greater than 1.0% (w/v). When the pH was controlled at pH 7.0 inhibition of substrate removal was alleviated. The rate of removal of 2-chlorophenol was greater in the presence of fructose than in the presence of glucose. P. putida CP1 formed clumps of cells when grown on 2-chlorophenol and fructose but not on glucose. When the organism was grown on a combination of 2-chlorophenol and an additional carbon source clumping was present but to a lesser degree.     

Screening of encapsulated microbial cells for the degradation of inorganic cyanides

Summary Different encapsulation matrices were screened to encapsulate cells ofPseudomonas putida for degradation of inorganic cyanides. Degradation of NaCN by free cells and cells immobilized in agar, alginate or carrageenan matrices was studied. The rate of NaCN degradation was monitored for 120 h by measuring pH, bacterial growth, dissolved and gaseous NH₃ and gaseous CO₂. Alginate-immobilized cells degraded NaCN more efficiently than free cells or agar- or carrageenan-immobilized cells.     

Substrate-dependent autoaggregation of <Emphasis Type="Italic">Pseudomonas putida</Emphasis> CP1 during the degradation of mono-chlorophenols and phenol

A bacterium, CP1, identified as Pseudomonas putida strain, was investigated for its ability to grow on and degrade mono-chlorophenols and phenols as sole carbon sources in aerobic shaking batch culture. The organism degraded up to 1.56 mM 2- and 3-chlorophenol, 2.34 mM 4-chlorophenol and 8.5 mM phenol using an ortho-cleavage pathway. P. putida CP1, acclimated to degrade 2-chlorophenol, was capable of 3-chlorocatechol degradation, while P. putida, acclimated to 4-chlorophenol degradation, degraded 4-chlorocatechol. Growth of P. putida CP1 on higher concentrations of the mono-chlorophenols, ≥1.56 mM 4-chlorophenol and ≥0.78 mM 2- and 3-chlorophenol, resulted in decreases in cell biomass despite metabolism of the substrates, and the formation of large aggregates of cells in the culture medium. Increases in cell biomass with no clumping of the cells resulted from growth of P. putida CP1 on phenol or on lower concentrations of mono-chlorophenol. Bacterial adherence to hydrocarbons (BATH) assays showed cells grown on the higher concentrations of mono-chlorophenol to be more hydrophobic than those grown on phenol and lower concentrations of mono-chlorophenol. The results suggested that increased hydrophobicity and autoaggregation of P. putida CP1 were a response to toxicity of the added substrates. Journal of Industrial Microbiology & Biotechnology (2002) 28, 316–324 DOI: 10.1038/sj/jim/7000249 Received 27 June 2001/ Accepted in revised form 09 February 2002     

Aerobic batch degradation of phenol using immobilized Pseudomonas putida

Alginate concentrations between 2 and 4% had little effect on the degradation rate of phenol by alginate-immobilized Pseudomonas putida. Ten-degree shifts from 25°C resulted in approximately 30% slower degradation. Maximal degradation rates were favored at pH 5.5–6.0. The response of degradation rate to increased air flow in the bubble column used was almost linear and an optimal higher than 16 vol vol⁻¹ was indicated, although free cells appeared in the reaction medium above 12 vol vol⁻¹. When the initial phenol concentration was raised, degradation rate was not significantly affected until levels higher than 1200 mg ml⁻¹ where performance was markedly reduced. Increasing the ratio of total bead volume to medium volume gave progressively smaller increases in degradation rate. At a medium volume to total bead volume ratio of 5:1, the maximum degradation rate was 250 mg L⁻¹ h⁻¹. Received 24 November 1998/ Accepted in revised form 27 January 1999     

Degradation of 4-chlorophenol in municipal wastewater by adsorptiv immobilized Alcaligenes sp. A 7-2

Summary Alcaligenes sp. A 7-2 has been applied in a packed-bed fermenter to degrade 4-chlorophenol in municipal wastewater continuously. With sterile wastewater degradation rates up to 300 mol/l/h were reached when precultivated Alcaligenes sp. A 7-2 had been adsorbed onto the Lecaton-packed-bed-material.The natural microbial population of the wastewater was not able to degrade 4-chlorophenol. Beside an accumulation of the haloaromatic compound a yellow-greenish substance exhibiting the spectral characteristics of 5-chloro-2-hydroxymuconic acid semialdehyde was found.This compound caused a rapid decrease in metabolic activity of the microbial culture.With non-sterile wastewater Alcaligenes sp. A 7-2 could not be established as member of the natural mixed population. Due to the poor retainment of the specialized strain in the packed-bed the degradation capacity of the fermentation system decreased and 4-chlorophenol was accumulated.     

Ethanol production from whey with immobilized living yeast

Summary Living Kluyveromyces fragilis yeast cells were succesfully entrapped in calcium alginate gel beads at cell loadings of 4 to 16 g yeast (0.8 to 3.2 g d.m.) per 1 g of sodium alginate. In batch systems, about 90 % conversion in 48 h was obtained both with free and immobilized yeast using demineralized whey of 5 to 10 % lactose content as substrate. In continuous packed-bed column operation nearly a constant 2 % product ethanol concentration could be maintained at 5 % substrate lactose level for at least one month.     

Biodegrading of pyrene by a newly isolated Pseudomonas putida PL2

Polycyclic aromatic hydrocarbons (PAHs) are a class of persistent organic compounds derived from natural sources and anthropogenic processes, which have been recommended as priority pollutants. Degradation of PAHs in the environment is becoming more necessary and urgent. In the current study, strain PL2, which is capable of growing aerobically on pyrene (PYR) as the sole carbon source, was isolated from hydrocarbons-contaminated soil and then identified as Pseudomonas putida by morphological and physiological characteristics as well as 16S rDNA sequence. The strain PL2 was able to degrade 50.0% of the pyrene at 28°C within 6 days in the presence of 50 mg/L pyrene, while the strain PL2 degraded 50.0% of the pyrene within 2 days when a solution of 50 mg/L pyrene and 50 mg/L phenanthrene was used. In addition, phenanthrene was shown to increase the biodegradation efficiency of pyrene by the strain PL2. The order of degradation by the strain PL2 was pH 6.0 > pH 7.0 > pH 5.0 > pH 8.0. The degradation rate of PYR in the soil by the strain PL2 reached 70.0% at the 10^th day. The dynamics of PYR degradation in soil by PL2 was fit to the first order model and the strain PL2 was shown to efficiently degrade PYR in soil. The current study showed that P. putida PL2 was a novel bacterium that could degrade pyrene and holds great promise for use in PAHs bioremediation in soil.     

Degradation of phenol by a mixed culture of Pseudomonas putida and Cryptococcus elinovii adsorbed on activated carbon

Summary Phenol degradation by a defined mixed culture of Pseudomonas putida P8 and Cryptococcus elinovii H 1, which were immobilized by adsorption on activated carbon, was studied.The immobilized mixed culture was able to degrade phenol up to 17 g/l and degraded it faster than the pure cultures, depending on a complementary metabolism of the two microorganisms.Storage experiments revealed an excellent longterm storage capability of the biocatalyst: activated carbon with adsorbed cells of Pseudomonas putida P8 and Cryptococcus elinovii H1 could be stored up to 12 months without decrease on degradation capacity.Scanning electron micrographs showed that Pseudomonas putida P8 had grown through the pore system of the activated carbon into the inside of the carbon particles.     

Immobilization of glucose isomerase to ion-exchange materials

Glucose isomerase (D -xylose ketol-isomerase EC 5.3.1.5) from Bacillus Coagulans was partially purified and immobilized by adsorption to anion exchangers. The highest activities were obtained when the enzyme was adsorbed to DEAE-cellulose. On immobilization to DEAE-cellulose the measured optimum pH value for enzyme activity shifted from 7.2 to 6.8. There was no appreciable difference between the heat stabilities of soluble and immobilized enzyme. The K_{m app} values for the immobilized enzyme were found to be 0.25M in the presence of 0.01M Mg²⁺ and 0.19M with 0.005M Mg²⁺, while those enzyme were 0.11 and 0.17M, re spectively. Under conditions of contimuous of D -glucose, a decrease of activity with time was observed, but this decrease was less at a low Mg²⁺ concentration and was affected by column geometry. There were no appreciable diffusional limitation effects in packed-bed columns.     

Manganese‐oxidizing bacteria mediate the degradation of 17α‐ethinylestradiol

Manganese (II) and manganese‐oxidizing bacteria were used as an efficient biological system for the degradation of the xenoestrogen 17α‐ethinylestradiol (EE2) at trace concentrations. Mn²⁺‐derived higher oxidation states of Mn (Mn³⁺, Mn⁴⁺) by Mn²⁺‐oxidizing bacteria mediate the oxidative cleavage of the polycyclic target compound EE2. The presence of manganese (II) was found to be essential for the degradation of EE2 by Leptothrix discophora, Pseudomonas putida MB1, P. putida MB6 and P. putida MB29. Mn²⁺‐dependent degradation of EE2 was found to be a slow process, which requires multi‐fold excess of Mn²⁺ and occurs in the late stationary phase of growth, implying a chemical process taking place. EE2‐derived degradation products were shown to no longer exhibit undesirable estrogenic activity.     

Biodegradation of 4-chlorophenol by entrappedAlcaligenes sp. A 7-2

Summary The degradation of 4-chlorophenol by free and by Ca-alginate-immobilized cells ofAlcaligenes sp. A 7-2 has been studied. Increasing concentrations of 4-chlorophenol (0.4–0.55 mM) were better tolerated and more quickly degraded by the immobilized organisms than by free cells. The capability for haloarene-degradation is inducible. In semicontinuous fermentation at pH 7 a minimal degradation time of 5 h for degrading 0.2 mM 4-chlorophenol was reached. Fermentation temperature was shown to be important for inducing the degradation capability, but to be less important for the degradation rate by induced organisms. High-frequency feeding of small amounts of 4-chlorophenol (0.05 mM) was more favourable than low-frequency feeding of larger amounts (0.15 mM).Continuous fermentation with unbuffered medium allowed a degradation rate of about 2 mmol·l^-1·d^-1; with buffered medium a higher degradation rate of nearly 4 mmol·l^-1·d^-1 was reached, but the Ca-alginate beads dissolved.     

Immobilization of the methanogenic bacterium Methanosarcina barkeri

Whole cells of the methanogen Methanosarcina barkeri were immobilized in an alginate network which was crosslinked with Ca²⁺ ions. The rates of methanol conversion to methane of entrapped cells were found to be in the same range as the corresponding rates of free cells. Furthermore, immobilized cells were active for a longer period than free cells. The particle size of the spherical alginate beads (1.2 mm-3.7 mm ?) and thus diffusion had no obvious influence on the turnover of methanol. The half-value period for methanol conversion activity determined in a buffer medium was approximately 4 days at 37°C for entrapped cells. The apparent K_m value K for such cells was nearly 140mM and the V_max value was about 1.2 μmol methanol/min/mg entrapped protein. Therefore the high rates of methanol degradation measured, e.g., 0.5 μmol methanol/min/mg entrapped protein, indicated that the immobilization technique preserved the cellular functions of this methanogenic bacterium.     

Phenol degradation performance by isolated Bacillus cereus immobilized in alginate

Phenol degradation by Bacillus cereus AKG1 MTCC9817 and AKG2 MTCC 9818 was investigated and degradation kinetics are reported for the free and Ca-alginate gel-immobilized systems. The optimal pH for maximum phenol degradation by immobilized AKG1 and AKG2 was found to be 6.7 and 6.9, respectively, while 3% alginate was optimum for both the strains. The degradation of phenol by free as well as immobilized cells was comparable at lower concentrations of phenol (100–1000 mg l⁻¹). However, the degradation efficiency of the immobilized strains was higher than that of the free strains at higher phenol concentrations (1500–2000 mg l⁻¹), indicating the improved tolerance of the immobilized cells toward phenol toxicity. More than 50% of 2000 mg l⁻¹ phenol was degraded by immobilized AKG1 and AKG2 within 26 and 36 days, respectively. Degradation kinetics of phenol by free and immobilized cells are well represented by the Haldane and Yano model.     

Degradation of mixtures of monochlorophenols and phenol as substrates for free and immobilized cells of Alcaligenes sp. A7-2

Summary The biodegradation of the three isomeric monochlorophenols 2-(2CP), 3- (3CP) and 4-chlorophenol (4CP) and phenol by the constructed strain Alcaligenes sp. A7-2 was investigated. Mineralization took place in the order: phenol >4CP >2CP >3CP, whereas 3CP was mineralized only co-metabolically. In substrate mixtures with phenol, degradation of 4CP was decelerated but degradation of 2CP was accelerated. Free cells in batch culture showed biphasic growth with an equimolar mixture of 2CP and 4CP as substrates, perhaps due to diauxie. Degradation patterns obtained with free cells in batch culture were confirmed with immobilized cells in continuous culture. Immobilized cells of Alcaligenes sp. A7-2 built up a biofilm on the lava that was used as filling material in the packed-bed reactors. The continuous cultures remained stable despite increasing input rates of chlorophenol and phenol mixtures up to 1.16 mMo1.1^–1.h^–1 for several weeks. Correspondence to: H.-J. Rehm     

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     

The adsorption of Fusarium flocciferum spores on celite particles and their use in the degradation of phenol

Summary Spores of Fusarium flocciferum were inserted in porous celite beads. The effects of bead size, adsorption time course, washing cycle and spore concentration on spore loading were investigated. Cell loadings up to 50% (dry weight/beads) were obtained. The degradation of phenol using adsorbed cells was studied in batch experiments. The immobilized cell system was shown to efficiently degrade high concentrations of the substrate (up to 2.0 g/l) and to remain active for more than 2 motths. The oxygen uptake rate of free and immobilized cells was determined at various concentrations of phenol. The kinetic constants K _s=85 mg/l, K _i=345 mg/l and SMI=170 mg/l were estimated from the experimental data by linearization of the Haldane function for the free cells. The uptake rates exhibited by the confined cells were lower (30%) than those obtained for free cells and no significant differences were found for phenol concentrations between 150 and 1200 mg/l.     

Degradation of cationic surfactants using Pseudomonas putida A ATCC 12633 immobilized in calcium alginate beads

In this study, the degradation of tetradecyltrimethylammonium bromide (TTAB) by freely suspended and alginate-entrapped cells from the bacteria Pseudomonas putida (P. putida) A ATCC 12633 was investigated in batch cultures. The optimal conditions to prepare beads for achieving a higher TTAB degradation rate were investigated by changing the concentration of sodium alginate, pH, temperature, agitation rate and initial concentration of TTAB. The results show that the optimal embedding conditions of calcium alginate beads are 4 % w/v of sodium alginate content and 2 × 10⁸ cfu ml^?1 of P. putida A ATCC 12633 cells that had been previously grown in rich medium. The optimal degradation process was carried out in pH 7.4 buffered medium at 30 °C on a rotary shaker at 100 rpm. After 48 h of incubation, the free cells degraded 26 mg l^?1 of TTAB from an initial concentration of 50 mg l^?1 TTAB. When the initial TTAB concentration was increased to 100 mg l^?1, the free cells lost their degrading activity and were no longer viable. In contrast, when the cells were immobilized on alginate, they degraded 75 % of the TTAB after 24 h of incubation from an initial concentration of 330 mg l^?1 of TTAB. The immobilized cells can be stored at 4 °C for 25 days without loss of viability and can be reused without losing degrading capacity for three cycles.