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.     

Biodegradation of pyridine by freely suspended and immobilized Pimelobacter sp.

Freely suspended and Ca-alginate-immobilized cells of Pimelobacter sp. were used for degradation of pyridine. When the pyridine concentration was up to 2 g l^–1, freely suspended cells completely degraded pyridine regardless of the initial cell concentrations used. However, when the pyridine concentration increased to 4 g l^–1, the initial cell concentration in freely suspended cell culture should be higher than 1.5 g dry cell weight l^–1 for complete degradation of pyridine. In addition, a freely suspended cell culture with a high initial cell concentration resulted in a high volumetric pyridine-degradation rate, suggesting the potential use of immobilized cells for pyridine-degradation. When the immobilized cells were used for pyridine-degradation, neither specific pyridine-degradation rate nor tolerance against pyridine was improved. However, a high volumetric pyridine-degradation rate in the range 0.082–0.129 g l^–1 hr^–1 could be achieved by the immobilized cells because of the high cell concentration. Furthermore, when the immobilized cells were reused in degrading pyridine at a concentration of 2–4 g l^–1 they did not lose their pyridine-degrading activity for 2 weeks. Taken together, the data obtained here showed the feasibility of using immobilized cells for pyridine-degradation.     

Continuous degradation of phenol(s) by Pseudomonas putida P8 entrapped in polyacrylamidehydrazide

Summary The continuous degradation of phenol by immobilized cells of Pseudomonas putida P8 under different conditions was investigated. The bacterial cells were entrapped in polyacrylamidehydrazide (PAAH) and cultivated in a columnar fluidized-bed bioreactor. Working with a dilution rate of 0.067 h^-1 the phenol content in the feed was varied to find the capacity of an one-stage system with complete phenol degradation.Under sterile conditions and with phenol as the sole carbon source a degradation rate of 7.2 g·l^-1·d^-1 was reached whereas in non-sterile waste water only 3.12 g·l^-1·d^-1 were degraded. In any case the immobilized cells showed a stable phenol degradation activity and even simultaneously fed cresols or 4-chlorophenol were utilized completely.     

Degradation of 4-Chlorophenol at Low Temperature and during Extreme Temperature Fluctuations by Arthrobacter chlorophenolicus A6

Low average temperatures and temperature fluctuations in temperate soils challenge the efficacy of microbial strains used for clean up of pollutants. In this study, we investigated the cold tolerance of Arthrobacter chlorophenolicus A6, a microorganism previously shown to degrade high concentrations of 4-chlorophenol at 28°C. Luciferase activity from a luc-tagged derivative of the strain (A6L) was used to monitor the metabolic status of the population during 4-chlorophenol degradation. The A6L strain could degrade 200–300 g mL^–1 4-chlorophenol in pure cultures incubated at 5°C, although rates of degradation, growth and the metabolic status of the cells were lower at 5°C compared to 28°C. When subjected to temperature fluctuations between 5 and 28°C, A6L continued to degrade 4-chlorophenol and remained active. In soil microcosm experiments, the degradation rates were significantly faster the first week at 28°C, compared to 5°C. However, this difference was no longer seen after 7 days, and equally low 4-chlorophenol concentrations were reached after 17 days at both temperatures. During 4-chlorophenol degradation in soil, CFU and luciferase activity values remained constant at both 5 and 28°C. However, once most of the 4-chlorophenol was degraded, both values decreased by 1–1.5 logarithmic values at 28°C, whereas they remained constant at 5°C, indicating a high survival of the cells at low temperatures. Because of the ability of A. chlorophenolicus A6 to degrade high concentrations of 4-chlorophenol at 5°C, together with its tolerance to temperature fluctuations and stress conditions found in soil, this strain is a promising candidate for bioaugmentation of chlorophenol-contaminated soil in temperate climates.This revised version was published online in November 2004 with corrections to Volume 48.     

Direct alcoholic fermentation of starchy biomass using amylolytic yeast strains in batch and immobilized cell systems

Summary Direct alcoholic fermentation of dextrin or soluble starch with selected amylolytic yeasts was studied in both batch and immobilized cell systems. In batch fermentations, Saccharomyces diastaticus was capable of fermenting high dextrin concentrations much more efficiently than Schwanniomyces castellii. From 200 g·l^–1 of dextrin S. diastaticus produced 77 g·l^–1 of ethanol (75% conversion efficiency). The conversion efficiency decreased to 59% but a higher final ethanol concentration of 120 g·l^–1 was obtained with a medium containing 400 g·l^–1 of dextrin. With a mixed culture of S. diastaticus and Schw. castellii 136 g·l^–1 of ethanol was produced from 400 g·l^–1 of dextrin (67% conversion efficiency). S. diastaticus cells attached well to polyurethane foam cubes and a S. diastaticus immobilized cell reactor produced 69 g·l^–1 of ethanol from 200 g·l^–1 of dextrin, corresponding to an ethanol productivity of 7.6g·l^–1·h^–1. The effluent from a two-stage immobilized cell reactor with S. diastaticus and Endomycopsis fibuligera contained 70 g·l^–1 and 80 g·l^–1 of ethanol using initial dextrin concentrations of 200 and 250 g·l^–1 respectively. The corresponding values for ethanol productivity were 12.7 and 9.6 g·l^–1·h^–1. The productivity of the immobilized cell systems was higher than for the batch systems, but much lower than for glucose fermentation.     

Degradation kinetics of phenol by immobilized cells of Candida tropicalis in a fluidized bed reactor

Degradation kinetics of phenol by free and agar-entrapped cells of Candida tropicalis was studied in batch cultures. The initial phenol degradation rate achieved with free cells was higher than that obtained with immobilized cells, when phenol concentrations up to 1000 mg l^–1 were used. However, at higher phenol concentrations, the behaviour was quite different. The initial degradation rate of the immobilized yeast cells was about 10 times higher than that of the free cells, at a phenol concentration of 3500 mg l^–1. The semicontinuous and continuous degradation of phenol by immobilized yeast cells was also investigated in a multi-stage fluidized bed reactor. The highest phenol removal efficiencies and degradation rates as well as the lowest values of residual phenol and chemical oxygen demand were obtained in the semicontinuous culture when phenol concentrations up to 1560 mg l^–1 were used.     

Kinetics of chlorophenol degradation by benzoate-induced culture of Rhodococcus erythropolis M1

A pure culture of Rhodococcus erythropolis was isolated with the ability to degrade 2-chlorophenol, 4-chlorophenol and 2,4-dichlorophenol. Degradation of 2-chlorophenol by the uninduced culture of Rhodococcus erythropolis began after a prolonged lag period and complete mineralization of the substrates took 45 days. With the aim of reducing the lag period and subsequently improving the rate of degradation, the cells of the isolate were induced with benzoate, phenol, toluene and catechol individually. Benzoate-induced cells showed the highest rate of degradation and were thus used for the study of the degradation kinetics of 2-chlorophenol, 4-chlorophenol and 2,4-dichlorophenol. Complete mineralization of these substrates was achieved up to a concentration of 300, 100 and 50 mg l^–1 respectively. Degradation of the chlorophenols was initiated without any significant lag and took the remarkably short time periods of 84, 64 and 144 h for the highest concentrations of the substrate. Evaluation of kinetic parameters showed chlorophenol degradation to follow substrate inhibition kinetics. This is evident from the decrease in specific growth rate, growth yield and substrate uptake rate with increase in the initial substrate concentrations. Toxicity of the chlorophenols was observed to depend on the position of chlorine on the benzene ring and the degree of chlorination.     

Degradation of caffeine by immobilized cells of Pseudomonas putida strain C 3024

Summary A caffeine-resistant strain of Pseudomonas putida was isolated from soil and was grown with caffeine as the sole source of carbon, energy and nitrogen. Cells were immobilized in agar gel particles which were continuously supplied with a caffeine solution (0.52 g · l^–1, D=1.0 h^–1) in a homogeneously mixed aerated reaction vessel. In the presence of the ATPase inhibitor arsenate the caffeine was removed by the immobilized cells at an average rate of 0.25 mg caffeine · h^–1 · (mg cell carbon)^–1 during 6 days. Thereafter a rapid decline of activity was observed. From a similar system without arsenate supplied with a growth medium containing a limiting amount of caffeine (0.13 g · l^–1) the caffeine was almost completely oxidized by the immobilized cells. The concentration of the remaining caffeine was 1.4 mg · l^–1, which is much lower than the substrate constant for caffeine (9.7 mg · l^–1) observed with freshly harvested suspended resting cells.     

Production of mead by immobilized cells of Hansenula anomala

Summary Mead was produced by immobilized cells of Hansenula anomala in calcium alginate gels. The immobilized cell beads of 3 mm diameter packed in column reactors of dimensions 2.2x60, 4x40 and 8x80 cm, produced mead containing maximum concentrations of ethanol and ethyl acetate of 70 g/l and 730 mg/l, respectively at a dilution rate of 0.1 h^–1. The maximum alcohol productivity achieved was 23.1 g/l·h at a dilution rate of 0.33 h^–1. With intermittent regenerations of the cells the reactor operated continuously for 110 days. This process enables the quick production of matured mead by a single culture and the elimination of the traditionally used long aging periods.     

Aerobic nitrate and nitrite reduction in continuous cultures of Escherichia coli E4

Nitrate and nitrite was reduced by Escherichia coli E4 in a l-lactate (5 mM) limited culture in a chemostat operated at dissolved oxygen concentrations corresponding to 90–100% air saturation. Nitrate reductase and nitrite reductase activity was regulated by the growth rate, and oxygen and nitrate concentrations. At a low growth rate (0.11 h^–1) nitrate and nitrite reductase activities of 200 nmol · mg^–1 protein · min^–1 and 250 nmol · mg^–1 protein · min^–1 were measured, respectively. At a high growth rate (0.55 h^–1) both enzyme activities were considerably lower (25 and 12 nmol mg^–1 · protein · min^–1). The steady state nitrite concentration in the chemostat was controlled by the combined action of the nitrate and nitrite reductase. Both nitrate and nitrite reductase activity were inversely proportional to the growth rate. The nitrite reductase activity decreased faster with growth rate than the nitrate reductase. The chemostat biomass concentration of E. coli E4, with ammonium either solely or combined with nitrate as a source of nitrogen, remained constant throughout all growth rates and was not affected by nitrite concentrations. Contrary to batch, E. coli E4 was able to grow in continuous cultures on nitrate as the sole source of nitrogen. When cultivated with nitrate as the sole source of nitrogen the chemostat biomass concentration is related to the activity of nitrate and nitrite reductase and hence, inversely proportional to growth rate.     

Degradation of carbondisulphide by a Thiobacillus isolate

During experiments investigating the purification of waste gas a bacterium capable of using carbon disulphide (CS₂) als sole energy source was isolated. It could be identified as a Thiobacillus sp.; however, the species remains unclear. Both the properties of T. thioparus and T. thiooxidans have been observed. Since the organism could be used for removing CS₂ in the environment, the degradation kinetics have been investigated by different methods. Substrate concentrations of up to 100 mg CS₂·l^–1 were oxidized at maximum rates of 2.5 mg CS₂·g^–1 protein·min^–1 at pH 7.0 and at 30°C. CS₂ levels above 150 mg CS₂·l^–1 caused termination of degradative activity. Correspondence to: Ch. Plas     

Degradation of 2-methylnaphthalene by free and immobilized cells of Pseudomonas sp. strain NGK1

A Pseudomonas sp. strain NGK1 (NCIM 5120) capable of utilizing 2-methylnaphthalene (2-MN) was immobilized in various matrices namely, polyurethane foam (PUF), alginate, agar and polyvinyl alcohol (PVA) (1.5 × 10¹² c.f.u. g^–1 beads). The degradation rates of 25 and 50 mM 2-MN by freely suspended cells (2 × 10¹¹ c.f.u. ml^–1) and immobilized cells in batches, semi-continuous with shaken culture and continuous degradation in a packed-bed reactor were compared. The PUF-immobilized cells achieved higher degradation of 25 and 50 mM of 2-MN than freely suspended cells and the cells immobilized in alginate, agar or PVA. The PVA- and PUF-immobilized cells could be reused for more than 30 and 20 cycles respectively, without losing any degradation capacity. The effect of dilution rates on the rate of degradation of 25 and 50 mM 2-MN with freely suspended and immobilized cells were compared in the continuous system. Increase in dilution rate increased the degradation rate only up to 1 h^–1 in free cells with 25 mM 2-MN and no significant increase was observed with 50 mM 2-MN. With immobilized cells, the degradation rate increased with increase in dilution rate up to 1.5 h^–1 for 25 mM and 1 h^–1 for 50 mM 2-MN. These results revealed that the immobilized cell systems are more efficient than freely suspended cells for biodegradation of 2-MN.     

Effect of soil moisture on bioremediation of chlorophenol-contaminated soil

A chlorophenol-contaminated soil was tested for the biodegradability in a semi-pilot scale microcosm using indigenous microorganisms. More than 90% of 4-chlorophenol and 2,4,6-trichlorophenol, initially at 30 mg kg^–1, were removed within 60 days and 30 mg pentachlorophenol kg^–1 was completely degraded within 140 days. The chlorophenols were degraded more effectively under aerobic condition than under anaerobic condition. Soil moisture had a significant effect with the slowest degradation rate of chlorophenols at 25% in the range of 10–40% moisture content. At 25–40%, the rate of chlorophenol degradation was directly related to the soil moisture content, whereas at 10–25%, it was inversely related. Limited oxygen availability through soil agglomeration at 25% moisture content might decrease the degradation rate of chlorophenols.     

Fermentative production of 1,3-propanediol from glycerol by Clostridium butyricum up to a scale of 2m3

Summary The conversion of glycerol to 1,3-propanediol (PD) by Clostridium butyricum DSM 5431 was studied in anaerobic culture. Growth and product formation were optimal at pH = 7.0 and T = 35° C, while aeration rate and stirrer speed were found to have no significant influence. As increasing amounts of initial glycerol led to inhibition of growth, cultivations were done in fed-batch operation. Comparative cultivations were carried out in an air-lift (ALR) and a stirred-tank reactor (STR) having equal working volumes (V _L = 30 l) and no difference in product formation was found. The process was scaled up to reactor sizes of 1.2 m³ (ALR) and 2.0 m³ (STR). The same results were obtained irrespective of reactor volume as well as reactor type (STR/ALR). PD concentrations of approximately 50–58 g·l^–1 and overall productivities of 2.3–2.9 g·l^–1 ·h^–1 could be reached. Offprint requests to: W.-D. Deckwer     

Malate degradation by Schizosaccharomyces yeasts included in alginate beads

Schizosaccharomyces yeasts can be used for deacidification of grape musts. To this aim, we studied malic acid degradation by yeasts included in double layer alginate beads in a bubble column reactor. Use of immobilized micro-organisms allowed a continuous process with high dilution rates giving a deacidification capacity of 0.032 g of malate/hour/dm³/g of beads. The pneumatic agitation was very convenient in this case.List of Symbols D h^–1 Dilution rate for continuous culture - h Residence time for continuous culture - dM/dt kg/(m³ · h) Rate of degradation of malic acid - dS/dt kg/(m³ · h) Rate of consumption of glucose - _max h^–1 Maximal specific rate of growth     

Degradation of phenol by a defined mixed culture immobilized by adsorption on activated carbon and sintered glass

Summary A defined mixed culture of the yeast Cryptococcus elinovii H1 and the bacterium Pseudomonas putida P8 was immobilized by adsorption on activated carbon and sintered glass, respectively. Depending on its adsorption capacity for phenol the activated carbon system could completely degrade 17 g/l in batch culture, whereas the sintered glass system was able to degrade phenol up to 4 g/l. During semicontinuous degradation of phenol (1 g/l) both systems reached constant degradation times with the fourth batch that lasted 8 h when using the activated carbon system and 10 h in the sintered glass system. In the course of continuous degradation of phenol the activated carbon system reached a maximum degradation rate of 9.2 g l^–1 day^–1 compared to 6.4 g l^–1 day^–1degraded by the sintered glass system. 2-Hydroxymuconic acid semialdehyde could be identified and quantitatively determined as a metabolite of phenol degradation by P. putida P8. Increased membrane permeability under the influence of phenol was demonstrated by the examination of K⁺ efflux from P. putida P8. Offprint requests to: H.-J. Rehm     

Degradation of n-alkanes by Candida parapsilosis and Penicillium frequentans immobilized on granular clay and aquifer sand

Summary The immobilization intensity of cells of Penicillium frequentans and Candida parapsilosis on materials such as granular clay, granular clay + aquifer sand and aquifer sand alone, was followed by scanning electron microscopy (SEM). The results demonstrate that the granular clay was the best adsorbent for both organisms, followed by the mixture of both granular clay and aquifer sand. Poor adhesion of cells was detected on using aquifer sand alone with C. parapsilosis.The highest degree of degradation of the alkane mixture (C₁₂–C₁₈) used was achieved by cells immobilized on granular clay, followed by those cells adsorbed on clay and sand. The weakest degradation was observed with cells immobilized on the sand alone.     

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     

Studies on the variables and kinetics of SCP production from nopal fruit juice

Summary Submerged batch cultivation under controlled environmental conditions of pH 3.8, temperature 30°C, and K_La200 h^–1 (above 180 mMO₂ l ^–1 h^–1 oxygen supply rate) produced a maximum (12.0 g·l ^–1) SCP (Candida utilis) yield on the deseeded nopal fruit juice medium containing C/N ratio of 7.0 (initial sugar concentration 25 g·l ^–1) with a yield coefficient of 0.52 g cells/g sugar. In continuous cultivation, 19.9 g·l ^–1 cell mass could be obtained at a dilution rate (D) of 0.36 h^–1 under identical environmental conditions, showing a productivity of 7.2 g·l ^–1·h^–1. This corresponded to a gain of 9.0 in productivity in continuous culture over batch culture. Starting with steady state values of state variables, cell mass (C_X–19.9 g·l ^–1), limiting nutrient concentration (C_ln–2.5 g·l ^–1) and sugar concentration (C_S–1.5 g·l ^–1) at control variable conditions of pH 3.8, 30°C, and K_La 200 h^–1 keeping D=0.36 h^–1 as reference, transient response studies by step changes of these control variables also showed that this pH, temperature and K_La conditions are most suitable for SCP cultivation on nopal fruit juice. Kinetic equations obtained from experimental data were analysed and kinetic parameters determined graphically. Results of SCP production from nopal fruit juice are described.Nomenclature C_ln concentration of ammonium sulfate (g·l ^–1) - C_S concentration of total sugar (g·l ^–1) - C_X cell concentration (g·l ^–1) - D dilution rate (h^–1) - K_ln Monod's constant (g·l ^–1) - m maintenance coefficient (g ammonium sulfate cell^–1 h^–1) - m_(S) maintenance coefficient (g sugar g cell^–1 h^–1) - t time, h - Y yield coefficient (g cells/g ammonium sulfate) - Y_m maximum of Y - Y_S yield coefficient based on sugar consumed (g cells · g sugar^–1) - Y_S(m) maximum value of Y_S - ^µm maximum specific growth rate constant (h^–1)     

Dynamics of production oftrans-zeatin andtrans-zeatin riboside by immobilized cytokinin-autonomous and cytokinin-dependent tobacco cells

Two strains of cultured tobacco cells (Nicotiana tabacum L. cv. Wisconsin 38) differing in their requirement for exogenous cytokinins (cytokinin-dependent and cytokinin-autonomous) were immobilized on polyphenylenoxide (Sorfix) activated with glutaraldehyde. Columns packed with immobilized cells were continually eluted with diluted Murashige and Skoog's medium lacking or supplemented with synthetic cytokinin (6-benzylaminopurine; BA). Purified samples of column eluates were fractionated by HPLC, andtrans-zeatin (t-Z) andtrans-zeatin riboside (t-ZR) content was estimated by enzyme immunoassay. Both cytokinin-autonomous and cytokinin-dependent tobacco cells produced and excretedt-Z and its riboside, and there were significant quantitative differences between the strains. The steady-state excretion rate oft-Z was 19.8 ng · g^–1 dw · h^–1 and 4 ng · g^–1 dw · h^–1, respectively, and that oft-ZR 4 ng · g^–1 dw · h^–1 and 1 ng · g^–1 dw · h^–1, respectively. Exposure of cytokinin-dependent cells to BA after 72 h of starving for this synthetic cytokinin caused temporary increase in excretion of both zeatin and its riboside. After the application of 5 M BA for 24 h, the excretion rate oft-ZR reached 5 ng · g^–1 dw · h^–1 (5-fold increase), and that oft-Z achieved 12 ng · g^–1 dw · h^–1 (3-fold increase). The elevation oft-Z excretion was delayed about 13 h compared witht-ZR excretion, which started increasing almost immediately after BA application. A pulse of BA in lower concentration (1.5 M for 30 h) provoked lower response.