Determination of the kinetic parameters of the phenol-degrading thermophile Bacillus themoleovorans sp. A2

Phenolic compounds are pollutants in many wastewaters, e.g. from crude oil refineries, coal gasification plants or olive oil mills. Phenol removal is a key process for the biodegradation of pollutants at high temperatures because even low concentrations of phenol can inhibit microorganisms severely. Bacillus thermoleovorans sp. A2, a recently isolated thermophilic strain (temperature optimum 65 degrees C), was investigated for its capacity to degrade phenol. The experiments revealed that growth rates were about four times higher than those of mesophilic microorganisms such as Pseudomonas putida. Very high specific growth rates of 2.8 h(-1) were measured at phenol concentrations of 15 mg/l, while at phenol concentrations of 100-500 mg/l growth rates were still in the range of 1 h(-1). The growth kinetics of the thermophilic Bacillus thermoleovorans sp. A2 on phenol as sole carbon and energy source can be described using a three-parameter model developed in enzyme kinetics. The yield coefficient Yx/s of 0.8-1 g cell dry weight/g phenol was considerably higher than cell yields of mesophilic bacteria (Yx/s 0.40-0.52 g cell dry weight/g phenol). The highest growth rate was found at pH 6. Bacillus thermoleovorans sp. A2 was found to be insensitive to hydrodynamic shear stress in stirred bioreactor experiments (despite possible membrane damage caused by phenol) and flourished at an ionic strength of the medium of 0.25(-1) mol/l (equivalent to about 15-60 g NaCl/l). These exceptional properties make Bacillus thermoleovorans sp. A2 an excellent candidate for technical applications.     

Removal of high phenol concentrations with adapted activated sludge in suspended form and entrapped in calcium alginate /cross-linked poly(N-vinyl pyrrolidone) hydrogels

The present work evaluates the aerobic removal of 0.25-2 g/L of phenol by adapted activated sludge in batch and continuous reactors, in suspended form and trapped in polymeric hydrogel beads of calcium alginate(1%) and cross-linked poly(N-vinyl pyrrolidone), x-PVP (4%). The mechanical and chemical resistance of the entrapping hydrogel was also evaluated in three different media: (I) rich in phosphate and ammonium ions; (II) using alternate P and N sources, and (III) without nutrients. The adapted consortium removed phenol concentrations up to 2 g/L more efficiently in the immobilized systems. A decrease in phenol removal rate was observed as the food/microorganisms (F/M) ratio increased. A zero-order kinetics was observed with phenol concentrations > 1 g/L and a first-order kinetics at concentrations < 1 g/L. The best response (100% removal) was in the continuous reactors using type II medium, with a hydraulic residence time (HRT) of 12.5 h, an influent pH = 5, and an F/M ratio below 0.25. The immobilizing matrix deteriorated after 170 h of use in continuous reactors, especially with media I and II, probably due to the attrition forces, to chemical weakness of the material, and to the pressure of the bacterial growth inside the bead.     

Bacteriorhodopsin production by cell recycle culture of Halobacterium halobium

When Halobacterium halobium R1 was cultured with cell recycle in a bioreactor equipped with an external hollow fiber membrane unit, the cell and bacteriorhodopsin concentrations reached in 10 days were 30.3 g cell dry weight/l and 282 mg/l, respectively. The productivity of bacteriorhodopsin (1.15 mg/l·h) was much higher than that (0.16 mg/l·h) obtained by typical batch fermentation. © Rapid Science Ltd. 1998     

Kinetics of ethanol production by immobilized Kluyveromyces marxianus cells at varying sugar concentrations of Jerusalem artichoke juice

Summary Kinetics of ethanol fermentation at varying sugar concentrations of Jerusalem artichoke tuber extract has been studied using Kluyveromyces marxianus cells immobilized in calcium alginate gel beads. A maximum ethanol concentration of 111 g/l was achieved at an initial sugar concentration of 260 g/l in 20 hours, when the immobilized cell concentration in the calcium alginate beads was 53.3 g dry wt./l bead volume. Ethanol yield remained almost unaffected by initial sugar concentration up to 250 g/l and was found to be about 88% of the theoretical. Maximum rate of ethanol production decreased from 22.5 g ethanol/l/h to 10.5 g ethanol/l/h while the maximum rate of total sugars utilization decreased from 74.9 g sugars/l/h to 28.5 g sugars/l/h as the initial substrate concentration was increased from 100 to 300 g/l. The concentration of free cells in the fermentation broth was low.     

Kinetics of biodegradation of p-xylene and naphthalene and oxygen transfer in a novel airlift immobilized bioreactor

The scope of this study included the biodegradation performance and the rate of oxygen transfer in a pilot-scale immobilized soil bioreactor system (ISBR) of 10-L working volume. The ISBR was inoculated with an acclimatized population of contaminant degrading microorganisms. Immobilization of microorganisms on a non-woven polyester textile developed the active biofilm, thereby obtaining biodegradation rates of 81 mg/L x h and 40 mg/L x h for p-xylene and naphthalene, respectively. Monod kinetic model was found to be suitable to correlate the experimental data obtained during the course of batch and continuous operations. Oxygen uptake and transfer rates were determined during the batch biodegradation process. The dynamic gassing-out method was used to determine the oxygen uptake rate (OUR) and volumetric oxygen mass transfer, K(L) a. The maximum volumetric OUR of 255 mg O(2)/L x h occurred approximately at 720-722 h after inoculation, when the dry weight of biomass concentration was 0.67 g/L.     

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.     

Isolation and selection of phenol-degrading microorganisms from industrial wastewaters and kinetics of the biodegradation

Among 22 species of microorganisms isolated from phenol-containing wastewaters, Candida parapsilopsis was found to be capable of growth on a medium with 1 g/L phenol. Kinetic parameters of phenol biodegradation in a batch reactor were determined by measuring biomass growth rates and phenol concentration as a function of fermentation time. The Haldane equation described cell growth adequately, with kinetic constants mumax = 0.174/h, KS = 11.2 mg/L and Ki = 298 mg/L.     

Repeated batch production of ethanol from Jerusalem artichoke tubers using recycled immobilized cells of Kluyveromyces fragilis

Summary Recycled immobilized cells of Kluyveromyces fragilis ATCC 28244 were used for repeated batch production of ethanol from the inulin sugars derived from Jerusalem artichoke tubers. Using 10% initial sugar concentration, a maximum ethanol concentration of 48 g/l was achieved in 7 h when the immobilized cell concentration in the Ca alginate beads was 72 g dry wt. immobilized cell/l bead volume. The maximum ethanol production rate was 13.5 g ethanol/l bioreactor volume/h. The same Ca alginate beads containing the cells were used repeatedly for 11 batch runs starting with fresh medium at the beginning of each run. The ethanol yield was found to be almost constant at 96% of the theoretical for all 11 batch runs, while the maximum ethanol production rate during the last batch run was found to be 70% of the original ethanol rate obtained in the first batch run.     

pH-controlled cell release and biomass distribution of alginate-immobilized Lactococcus lactis subsp. lactis

AIMS: To investigate the growth and release of Lactococcus lactis subsp. lactis in gel beads and to affect rates of cell release by changing the growth conditions. METHODS AND RESULTS: The rate of release and the distribution of immobilized L. lactis subsp. lactis in alginate beads were studied in continuous fermentations for 48 h. A change in operating pH from 6.5 to 9.25 initially reduced the ratio of the rates of cell release to lactate production by almost a factor of 105. Compared with fermentations at pH 6.5, growth at pH 9.25 also increased the final internal bead biomass concentration by a factor of 5 and increased the final rate of lactate production by 25%. After 48 h, the ratio of the rates of cell release to lactate production was still 10 times lower than in fermentations at pH 6.5. CONCLUSIONS: A change in the operating pH from 6.5 to 9.25 reduced rates of cell release throughout 48 h of fermentation and increased the final rates of lactate production and internal bead biomass concentration. SIGNIFICANCE AND IMPACT OF THE STUDY: These data illustrate that diffusional limitations and corresponding pH gradients can be exploited in affecting the distribution of immobilized growing cells and their concomitant release.     

Bioremediation of pentachlorophenol-contaminated soil by bioaugmentation using activated soil

The use of an indigenous microbial consortium, pollutant-acclimated and attached to soil particles (activated soil), was studied as a bioaugmentation method for the aerobic biodegradation of pentachlorophenol (PCP) in a contaminated soil. A 125-l completely mixed soil slurry (10% soil) bioreactor was used to produce the activated soil biomass. Results showed that the bioreactor was very effective in producing a PCP-acclimated biomass. Within 30 days, PCP-degrading bacteria increased from 10⁵ cfu/g to 10⁸ cfu/g soil. Mineralization of the PCP added to the reactor was demonstrated by chloride accumulation in solution. The soil-attached consortium produced in the reactor was inhibited by PCP concentrations exceeding 250 mg/l. This high level of tolerance was attributed to the beneficial effect of the soil particles. Once produced, the activated soil biomass remained active for 5 weeks at 20 °C and for up to 3 months when kept at 4 °C. The activated attached soil biomass produced in the completely mixed soil slurry bioreactor, as well as a PCP-acclimated flocculent biomass obtained from an air-lift immobilized-soil bioreactor, were used to stimulate the bioremediation of a PCP-impacted sandy soil, which had no indigenous PCP-degrading microorganisms. Bioaugmentation of this soil by the acclimated biomass resulted in a 99% reduction (from 400 mg/kg to 5 mg/kg in 130 days) in PCP concentration. The PCP degradation rates obtained with the activated soil biomass, produced either as a biomass attached to soil particles or as a flocculent biomass, were similar. Received: 31 March 1997 / Received revision: 22 July 1997 / Accepted: 25 August 1997     

High ethanol productivities using small Ca-alginate beads of immobilized cells of Zymomonas mobilis

Summary Zymomonas mobilis cells were immobilized into small 1 mm diameter beads of Ca-alginate in order to minimize mass transfer limitations and maximize immobilized cell activity. A combination of small bead size with a high cell concentration of 58 g dry wt. cell per lit. bead volume resulted in high ethanol productivities using a newly designed packed bed bioreactor system. Steady-state dilution rates ranging from 0.4 h^-1 to 3.9 h^-1 were run resulting in a maximum productivity of 102 g ethanol/l/h for an inlet substrate concentration of 100 g glu/l and 87% conversion. The bioreactor was run continuously at a fixed dilution rate for 384 h and short intermittent treatment of the beads with CaCl₂ temporarily increased ethanol productivity to a maximum of 116 g ethanol/l/h.     

A new bioreactor with a semi-fixed packing: investigation of degradation of phenol

A new bioreactor using a semi-fixed packing of frames with “sacks” made of a fabric of “Raschell” type stretched on them is proposed. The construction provides not only a large surface area of the biofilm carrier per unit volume of the apparatus, but also the possibility for an easy removal of the biomass after reaching a certain thickness of the biofilm increasing the gas velocity. Aerobic degradation of phenol in the new bioreactor, using microorganisms of the strain Pseudomonas putida, was studied. The experiments are carried out using water containing 0.7?g/l of phenol at a temperature of 27–30?°C. Different specific surface area of the packing (within 176 and 387?m²/m³) are studied. Degradation rates from 60 to 140?mg/(1?h) are attained. A retardation of the process at the end is observed, probably due to inhibition effect. This rate is 5–6 times higher than the rate observed when using free cells. At air velocity of 0.03–0.035?m/s (related to the total cross section of the bioreactor) the vibrations of the packing material lead to destruction and removal of the old biomass.     

Incorporation of phenol carbon at trace concentrations by phenol-mineralizing microorganisms in fresh water

The fate of phenol carbon at phenol concentrations ranging from 1 ng/ml to 1 microgram/ml was determined in freshwater samples. Approximately 20% of the parent phenol was incorporated into trichloroacetic acid-precipitable material by the microorganisms capable of mineralizing phenol. There was no apparent lag period before phenol incorporation commenced, and incorporation was complete within 2 h at all concentrations tested. A direct relationship was found between the initial phenol concentrations and both phenol mineralization and incorporation rates, indicating that cometabolism of phenol does not occur at concentrations that are environmentally significant. At all concentrations, approximately 80% of the initial phenol concentration was mineralized. This percentage plus the percentage of phenol incorporated at the various concentrations equaled approximately 100%. Therefore, the parent phenol does not remain in fresh water; it is either incorporated into cellular biomass or mineralized. However, the incorporated phenol carbon is subject to bioaccumulation in nature. There was no apparent lag period before thymidine was incorporated into biomass, and incorporation was complete within 4 to 8 h at all of the phenol concentrations tested. Thymidine incorporation was independent of phenol concentration at all levels tested. This is probably due to the small amount of thymidine incorporated by the phenol-mineralizing microorganisms in comparison with the amount of thymidine incorporated by the total microbial population.     

Incorporation of phenol carbon at trace concentrations by phenol-mineralizing microorganisms in fresh water.

The fate of phenol carbon at phenol concentrations ranging from 1 ng/ml to 1 microgram/ml was determined in freshwater samples. Approximately 20% of the parent phenol was incorporated into trichloroacetic acid-precipitable material by the microorganisms capable of mineralizing phenol. There was no apparent lag period before phenol incorporation commenced, and incorporation was complete within 2 h at all concentrations tested. A direct relationship was found between the initial phenol concentrations and both phenol mineralization and incorporation rates, indicating that cometabolism of phenol does not occur at concentrations that are environmentally significant. At all concentrations, approximately 80% of the initial phenol concentration was mineralized. This percentage plus the percentage of phenol incorporated at the various concentrations equaled approximately 100%. Therefore, the parent phenol does not remain in fresh water; it is either incorporated into cellular biomass or mineralized. However, the incorporated phenol carbon is subject to bioaccumulation in nature. There was no apparent lag period before thymidine was incorporated into biomass, and incorporation was complete within 4 to 8 h at all of the phenol concentrations tested. Thymidine incorporation was independent of phenol concentration at all levels tested. This is probably due to the small amount of thymidine incorporated by the phenol-mineralizing microorganisms in comparison with the amount of thymidine incorporated by the total microbial population.     

Biosorption of uranium by cross-linked and alginate immobilized residual biomass from distillery spent wash

Residual biomass from a whiskey distillery was examined for its ability to function as a biosorbent for uranium. Biomass recovered and lyophilised exhibited a maximum biosorption capacity of 165–170?mg uranium/g dry weight biomass at 15?°C. With a view towards the development of continuous or semi-continuous flow biosorption processes it was decided to immobilize the material by (1) cross-linking with formaldehyde and (2) introducing that material into alginate matrices. Cross-linking the recovered biomass resulted in the formation of a biosorbent preparation with a maximum biosorption capacity of 185–190?mg/g dry weight biomass at 15?°C. Following immobilization of biomass in alginate matrices it was found that the total amount of uranium bound to the matrix did not change with increasing amounts of biomass immobilized. It was found however, that the proportion of uranium bound to the biomass within the alginate-biomass matrix increased with increasing biomass concentration. Further analysis of these preparations demonstrated that the alginate-biomass matrix had a maximum biosorption capacity of 220?mg uranium/g dry weight of the matrix, even at low concentrations of biomass.     

Degradation of 4-aminobenzenesulfonate by alginate encapsulated cells of Agrobacterium sp. PNS-1

Studies were carried out on 4-aminobenzenesulfonate (4-ABS) degradation by free and alginate entrapped cells of Agrobacterium sp. PNS-1. Degradation rate in batch reactors with free cells was marginally higher than Ca-encapsulated cells. Comparison of Ca2+ and Ba2+ as gelling agents showed that 4-ABS removal rate was significantly less with Ba-alginate entrapped cells. Specific degradation rates, using linear regression analysis and based on the initial biomass in the beads, varied from 49.7 mg/mg biomass/h to 92.0 mg/mg biomass/h for Ca-alginate encapsulated cells for different initial 4-ABS concentrations ranging from 200 to 800 mg/L. UV spectra of the aliquots drawn at different time intervals from batch reactors did not show accumulation of any intermediate during degradation. Ca-alginate immobilized cells could be repeatedly reused upto five cycles without any loss of activity. Studies with packed bed reactors, operated in a semi-continuous mode, showed that this could be used for 4-ABS degradation.     

Removal of cadmium(II) ion from aqueous system by dry biomass, immobilized live and heat-inactivated Oscillatoria sp. H1 isolated from freshwater (Mogan Lake)

Oscillatoria sp. H1 (Cyanobacteria, microalgae) isolated from Mogan Lake was used for the removal of cadmium ions from aqueous solutions as its dry biomass, alive and heat-inactivated immobilized form on Ca-alginate. Particularly, the effect of physicochemical parameters like pH, initial concentration and contact time were investigated. The sorption of Cd(II) ions on the sorbent used was examined for the cadmium concentrations within the range of 25-250 mg/L. The biosorption of Cd(II) increased as the initial concentration of Cd(II) ions increased in the medium up to 100 mg/L. Maximum biosorption capacities for plain alginate beads, dry biomass, immobilized live Oscillatoria sp. H1 and immobilized heat-inactivated Oscillatoria sp. H1 were 21.2, 30.1, 32.2 and 27.5 mg/g, respectively. Biosorption equilibrium was established in about 1 h for the biosorption processes. The biosorption was well described by Langmuir and Freundlich adsorption isotherms. Maximum adsorption was observed at pH 6.0. The alginate-algae beads could be regenerated using 50 mL of 0.1 mol/L HCl solution with about 85% recovery.     

Comparison between entrapment methods for phenol removal and operation of bioreactor packed with co-entrapped activated carbon and Pseudomonas fluorescence KNU417

A microorganism capable of degrading phenol was isolated from crude oil contaminated soil and identified as Pseudomonas fluorescence. A porous polymer bead of polyvinyl alcohol (PVA) and Xanthan gum was found to be the best entrapment for phenol degradation in terms of bead shape (spherical form), bead strength, non-agglomeration, phenol degradation rate, and cell holding inside the bead. Activated carbon was co-immobilized with the microorganism in the bead, which readily adsorbed phenol to decrease initial phenol concentration. Due to the decreased phenol concentration, the cells needed shorter adaptation time after which the microorganism stably degraded phenol. When the bead containing microorganism with 1% of activated carbon was packed in a packed-bed bioreactor, the start-up period was shortened by 40 h and the removal efficiency of phenol during the period was increased by 28% than the case with only microorganism.     

Towards an on-line biochemical oxygen demand analyser

A simple, and effective tool to measure BOD with a 98% correlation to theoretical BOD values has been developed using a technique of continuous short term measurements of O₂ consumption rates. Measurement times are in the range of 5 to 10 h depending on the initial BOD, instead of 5 days required in the conventional BOD₅ analysis. A bioreactor containing calcium alginate entrapped activated sludge microorganisms was used with phenol and hydroxybutyric acid (HBA) as model substrates.     

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.