Treatment of simulated wastewater containing toxic amides by immobilized <Emphasis Type="Italic">Rhodococcus rhodochrous</Emphasis> NHB-2 using a highly compact 5-stage plug flow reactor

Biodegradation of toxic amides by immobilized Rhodococcus rhodochrous NHB-2 has been studied to generate data for future development of reactors for the treatment of simulated wastewater containing various toxic amides. The whole resting cells were immobilized in different matrices like agar, polyacrylamide and alginate. Agar gel beads were selected for the treatment of simulated wastewater containing 100mM each acetamide, propionamide, and 10mM of acrylamide and packed in a highly compact five-stage plug flow reactor. The immobilized bacterium worked well in a broad pH range from 5 to 10, with an optimum at 8.7. The apparent K _m-value for the turnover of acetamide for the resting cells was determined to be around 40mM at pH 8.5 and 55°C, whereas the K _m-value of the purified amidase was predicted to be about 20 mM. This organism exhibited greater turnover of aliphatic amides as compared to aromatic amides. Although these cells showed maximal amide-degrading activity at 55°C, simulated wastewater treatment was carried out at 45°C, because of the greater stability of the amidase activity at that temperature. Of note, indices for overall temperature stability, based on the temperature dependence of apparent first order kinetic temperature denaturation constants, were determined to be –7.9±1.1×10^–4, and –13.7±1.3×10^–4, –14.5±0.7×10^–4, and –13.7±0.8×10^–4°Cmin, for free cells and cells immobilized in alginate, agar and polyacrylamide respectively. After 250min the reactor showed maximum degradation of acetamide, propionamide and acrylamide of about 97, 100 and 90%, respectively by using 883 enzyme activity units per reactor stage. The results of this investigation showed that R. rhodochrous NHB-2 expressing thermostable amidase could be used for the efficient treatment of wastewater containing toxic amides. Therefore, we suggest that this microbe has a very high potential for the detoxification of toxic amides from industrial effluents and other wastewaters.     

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.     

Ethanol production by immobilized whole cells ofZymomonas mobilis in a continuous flow expanded bed bioreactor and a continuous flow stirred tank bioreactor

Continuous ethanol fermentation by immobilized whole cells ofZymomonas mobilis was investigated in an expanded bed bioreactor and in a continuous stirred tank reactor at glucose concentrations of 100, 150 and 200 g L^–1. The effect of different dilution rates on ethanol production by immobilized whole cells ofZymomonas mobilis was studied in both reactors. The maximum ethanol productivity attained was 21 g L^–1 h^–1 at a dilution rate of 0.36 h^–1 with 150 g glucose L^–1 in the continuous expanded bed bioreactor. The conversion of glucose to ethanol was independent of the glucose concentration in both reactors.     

Continuous ethanol production by Zymomonas mobilis in a fluidized bed reactor. Part II: Process development for the fermentation of hydrolysed B-starch without sterilization

A continuous fluidized bed reactor operation system has been developed for ethanol production by Zymomonas mobilis using hydrolysed B-starch without sterilization. The operation system consists of two phases. In the first phase macroporous glass carriers in a totally mixed fluidized bed reactor were filled up totally with a monoculture of Z. mobilis by fast computer-controlled colonization, so that in the subsequent production phase no contaminants, especially lactic-acid bacteria, could penetrate into the carrier beads. In the production phase the high concentration of immobilized Z. mobilis cells in the fluidized bed reactor permits unsterile fermentation of hydrolysed B-starch to ethanol at short residence times. This results in wash-out conditions for contaminants from the substrate. Long-term experimental studies (more than 120 days) of unsterile fermentation of hydrolysed B-starch in the laboratory fluidized bed reactor (2.2 l) demonstrated stable operation up to residence times of 5 h. A semi-technical fluidized bed reactor plant (cascade of two fluidized bed reactors, each 55 l) was operated stably at a mean residence time of 4.25 h. Glucose conversion of 99% of the unsterile hydrolysed B-starch was achieved at 120 g glucose/l^–1 in the substrate, resulting in an ethanol concentration of 50 g·l^–1 and an ethanol space-time yield of 13 g·l^–1·h^–1. This is a factor of three compared to ethanol fermentation of hydrolysed B-starch with Z. mobilis in a continuous stirred tank reactor, which can only be operated stably under sterile conditions. Correspondence to: D. Weuster-Botz     

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.     

Continuous production of glucoamylase by immobilized growing cells ofAureobasidium pullulans

Summary Yeast-like cells ofAureobasidium pullulans were immobilized in Ca-alginate gel beads and employed for continuous production of glucoamylase in a fluidized-bed reactor (250 ml working volume). After an activation time of 48 h, to allow the in situ germination of the fungal blastospores, the reactor was operated continuously for over 150 h. A steady state enzyme concentration of 1.2–1.3 U ml^–1 of glucoamylase activity and an enzyme volumetric productivity of ca. 130 U ml^–1 h^–1 were obtained at a medium flow rate of 26 ml h^–1. Enzyme activity and volumetric productivity were influenced by fermentation conditions such as inoculum size and airflow rate.     

Transformation of arachidonic acid to 19-hydroxy- and 20-hydroxy-eicosatetraenoic acids using Candida bombicola

Candida bombicola (ATCC 22214) and C. apicola (ATCC 96134), grown on glucose (100 g l^–1) and arachidonic acid (5Z, 8Z, 11Z, 14Z-eicosatetraenoic acid; AA), 1.25 g l^–1, synthesized sophorolipid up to 0.93 g l^–1. Acid hydrolysis of sophorolipid yielded 19-hydroxy-5Z, 8Z, 11Z, 14Z-eicosatetraenoic acid (19-HETE) and 20-hydroxy-5Z, 8Z, 11Z, 14Z-eicosatetraenoic acid (20-HETE) which were identified by TLC and GC-MS; the ratio of synthesis was 73:27, respectively. Conversion of AA by immobilized Candida bombicola, suspended in beads of 2% (w/v) calcium alginate for 96 h, gave an 83% conversion of 1 g AA l^–1 to 19- and 20-HETE. There was no significant loss in the efficiency of the immobilized cells after ten uses.     

Continuous fermentation of a supplemented milk with immobilized Bifidobacterium infantis

Bifidobacterium infantis immobilized in -carrageenan - locust bean gum gel beads (1.0–2.0 mm diameter) was used to ferment. 10% reconstituted skim milk supplemented with 1% yeast extract in a continuous stirred tank reactor. Cell release rate from the gel beads into the milk and growth of free cells in the bioreactor allowed for a steady inoculation of the feed, with cell counts in the outflow varying from 1.0 to 2.2 × 10⁹ CFU/mL for dilution rates in the range 0,5 to 1,0 h^-1. High mechanical stability of the gel beads was observed in milk.     

Continuous ethanol production by Zymomonas mobilis in a fluidized bed reactor. Part I. Kinetic studies of immobilization in macroporous glass beads

A model has been developed to calculate the ethanol production in a well-mixed fluidized bed reactor. This model takes into account diffusion and the reaction inside porous glass beads and the activity of suspended cells in the fluidized bed reactor. The associated model parameters have been determined from the literature and by kinetic studies with Zymomonas mobilis in a continuous stirred tank reactor. The model permits good predictions of steady-state data in a fluidized bed reactor at residence times longer than 1–1.5 h. The immobilization of Z. mobilis in a fluidized bed reactor results in high ethanol space-time yields of more than 50 g·^–1·h^–1 at a glucose conversion of 80% (glucose in substrate: 120 gl^–1). At 99% conversion a space-time yield of 30 g·^–1·^–1 can be achieved when two fluidized bed reactors operate as cascade.     

Nitrate removal from the effluent of a fertilizer industry using a bioreactor packed with immobilized cells of <Emphasis Type="Italic">Pseudomonas stutzeri</Emphasis>and<Emphasis Type="Italic">Comamonas testosteroni</Emphasis>

A bioreactor for the removal of nitrate nitrogen (NO₃-N) from industrial effluent is described which is comprised of a glass column (60 cm × 6 cm) packed with alginate beads containing denitrifying organisms Pseudomonas stutzeri and Comamonas testosteroni. The effluent containing high concentrations of nitrate (600–950 mg l^–1) from the fertilizer industry and fusel oil (methanol as a major component) as organic carbon were used in the process. The reactor is operated in the continuous mode by injecting the pretreated nitrate-containing effluent at the top of the column. The Hydraulic retention time (HRT) was adjusted by changing the flow rates. When nitrate-containing wastewater was treated with immobilized cells, the nitrate removal rate reached a maximum 1.66 ± 0.07 Kg NO₃-N m^–3d^–1 at an influent NO₃-N concentration of 850 mg NO_3M-N l^–1within 12 h. The denitrification activity of the immobilized cells was compared with that of the free cells.     

Fermentation of xylose and rice straw hydrolysate to ethanol byCandida shehatae NCL-3501

Candida shehatae NCL-3501 utilized glucose and xylose efficiently in batch cultures. The specific rate of ethanol production was higher with mixtures of glucose and xylose (0.64–0.83 g g^–1 cells d^–1) compared to that with individual sugars (0.38–0.58 g g^–1 cells d^–1). Although the optimum temperature for growth was 30°C, this strain grew and produced appreciable levels of ethanol at 45°C. A stable ethanol yield (0.40–0.43 g g^–1 substrate utilized) was obtained between 10 g L^–1 and 80 g L^–1 of initial xylose concentration. Conversion efficiency was further improved by immobilization of the cells in calcium alginate beads. Free or immobilized cells ofC. shehatae NCL-3501 efficiently utilized sugars present in rice straw hemicellulose hydrolysate, prepared by two different methods, within 48 h. Ethanol yields of 0.45 g g^–1 and 0.5 g g^–1 from autohydrolysate, and 0.37 g g^–1 from acid hydrolysate were produced by free and immobilized cells, respectively.     

Performance of immobilized cells for dihexyl sulfosuccinate biotransformation

Possibilities of using immobilized bacterial cells for waste water treatment in a continuous process was determined. Cells ofComamonas terrigena strain N3H immobilized in calcium alginate beads were successful by used in packed bead-type reactor for continuous biotransformation of the anion-active surfactant dihexyl sulfosuccinate. Absence of calcium ions from the treated medium led to the disruption of alginate beads within 8 d of usage. When the medium was supplemented with Ca²⁺ ions the beads were stable for at least one month in the continuous process. During the whole time period the transformation effectivity was in the range of 80–100% even at the highest, flow rate of 14 mL/min. Presented at the 4th Mini-Symposium on Biosorption and Microbial Degradation, Prague, Czech Republic, November 26–29, 1996.     

Accumulation of cadmium by immobilizedZoogloea ramigera 115

Summary Zoogloea ramigera 115 was immobilized into beads of calcium-alginate and placed into batch air-bubbled column reactors. In the absence of any added nutrients the immobilized bacterium adsorbed Cd from solutions containing levels of 2 and 20 g ml^–1 per day, over a period of 21 and 20 days, respectively. Adsorption of Cd from solutions containing 20 g ml^–1 Cd was better than 90% for 16 days. Beads treated with Cd at 2 g ml^–1 never adsorbed less than 95% of the metal. Alginate adsorbed Cd as well, but inclusion of cells changed the effectiveness of adsorption. Of a 250 g ml^–1 Cd solution, alginate adsorbed 70.4% Cd in 60 min whereas alginate plus cells adsorbed 90.5% in the same time span. Temperature had no effect on adsorption by immobilized cells at levels of 2 and 10 g ml^–1 Cd. However at higher concentrations, binding was enhanced as temperature increased.Z. ramigera beads were stable during all treatments and for prolonged periods of time (21 days).     

β-Galactosidase from Talaromyces thermophilus immobilized on to Eupergit C for production of galacto-oligosaccharides during lactose hydrolysis in batch and packed-bed reactor

The β-galactosidase from Talaromyces thermophilus CBS 236.58 immobilized onto Eupergit C produced galacto-oligosaccharides (GalOS) in batchwise and continuous packed-bed mode of operation. A maximum yield of GalOS of 12, 39 and 80 g l⁻¹ was obtained for initial lactose concentrations of 50, 100 and 200 g l⁻¹, respectively, for batch conversion experiments. The immobilized enzyme could be re-used for several cycles for lactose hydrolysis and transformation. The maximum GalOS concentration of approximately 50 g l⁻¹ was obtained with the dilution rate of 0.375 h⁻¹ in a packed-bed reactor, when using an initial lactose concentration of 200 g l⁻¹. Continuous conversion of lactose in the packed-bed reactor resulted in the formation of relatively more trisaccharides than when employing the immobilized enzyme in discontinuous mode of operation.     

Batch and continuous ribonuclease production by immobilized Aspergillus clavatus cells in a bubble-collumn bioreactor

Summary Ribonuclease production using immobilized cells (IC) of Aspergillus clavatus has been studied under batch, repeated-batch and continuous fermentation conditions in a bubble-column bioreactor and compared with production by free cells, Immobilization was achieved by the method of cryostructurization in polyvinyl alcohol beads. The effect of various aeration rates in a column bioreactor has been investigated. Enzyme production by IC [42 000 units (U)·l^–1] during batch fermentations was comparable to that of a free-cell system. The specific productivity of IC was 8.5 times higher than that of free cells. In repeated batch fermentation at various aeration rates, successful reuse of IC was obtained, with comparable levels of enzyme production. Continuous ribonuclease production was achieved for 44 days at 1 vvm aeration and a dilution rate of 0.0 h^–1 with volumetric productivity (450 U·1^–1) and yield.     

Continuous solvent production by Clostridium beijerinckii BA101 immobilized by adsorption onto brick

The performance of a continuous bioreactor containing Clostridium beijerinckii BA101 adsorbed onto clay brick was examined for the fermentation of acetone, butanol, and ethanol (ABE). Dilution rates from 0.3 to 2.5 h^–1 were investigated with the highest solvent productivity of 15.8 g l^–1 h^–1 being obtained at 2.0 h^–1. The solvent yield at this dilution rate was found to be 0.38 g g^–1 and total solvent concentration was 7.9 g l^–1. The solvent yield was maximum at 0.45 at a dilution rate of 0.3 h^–1. The maximum solvent productivity obtained was found to be 2.5 times greater than most other immobilized continuous and cell recycle systems previously reported for ABE fermentation. A higher dilution rate (above 2.0 h^–1) resulted in acid production rather than solvent production. This reactor was found to be stable for over 550 h. Scanning electron micrographs (SEM) demonstrated that a large amount of C. beijerinckii cells were adsorbed onto the brick support.     

Xylitol production from barley bran hydrolysates by continuous fermentation with Debaryomyces hansenii

The continuous bioconversion of xylose-containing solutions (obtained by acid hydrolysis of barley bran) into xylitol was carried out using the yeast Debaryomyces hansenii under microaerophilic conditions with or without cell recycle. In fermentations without cell recycle, the volumetric productivities ranged from 0.11–0.6 g l^–1 h^–1 were obtained for dilution rates of 0.008–0.088 h^–1. In experiments performed with cell recycle after membrane separation, the optimum xylitol productivity (2.53 g l^–1 h^–1) was reached at a dilution rate of 0.284 h^–1.     

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.     

Optimization of lactic acid production by immobilized <Emphasis Type="Italic">Lactococcus lactis</Emphasis> IO-1

Production of lactic acid from glucose by immobilized cells of Lactococcus lactis IO-1 was investigated using cells that had been immobilized by either entrapment in beads of alginate or encapsulation in microcapsules of alginate membrane. The fermentation process was optimized in shake flasks using the Taguchi method and then further assessed in a production bioreactor. The bioreactor consisted of a packed bed of immobilized cells and its operation involved recycling of the broth through the bed. Both batch and continuous modes of operation of the reactor were investigated. Microencapsulation proved to be the better method of immobilization. For microencapsulated cells at immobilized cell concentration of 5.3 g l⁻¹, the optimal production medium had the following initial concentrations of nutrients (g l⁻¹): glucose 45, yeast extract 10, beef extract 10, peptone 7.5 and calcium chloride 10 at an initial pH of 6.85. Under these conditions, at 37 °C, the volumetric productivity of lactic acid in shake flasks was 1.8 g l⁻¹ h⁻¹. Use of a packed bed of encapsulated cells with recycle of the broth through the bed, increased the volumetric productivity to 4.5 g l⁻¹ h⁻¹. The packed bed could be used in repeated batch runs to produce lactic acid.     

Mercury removal by immobilized algae in batch culture systems

The accumulation and volatilization of mercury by non-immobilized and immobilizedChlorella emersonii have been studied in batch culture systems. Reduction in the mercury concentration in the growth medium by non-immobilized cells was highly dependent on inoculum density, whilst reduction in mercury concentration by immobilized cells was rapid at all inoculum densities. Mercury accumulation by immobilized cell biomass was significantly greater than by non-immobilized cells with 10⁶ and 10⁵ cells bead^–1 or ml^–1. Volatilization of mercury by non-immobilized cell systems was greatest at higher inoculum densities, whereas more mercury was volatilized from immobilized cell systems at lower inoculum densities, and was greatest with unstocked alginate beads. Thus, in immobilized systems, mercury removal from solution is complex and involves mercury accumulation by the cells and volatilization by the matrix and cells. Further studies of mercury accumulation and volatilization by unstocked immobilization matrices revealed that agarose volatilized much less mercury than alginate or agar. The precise mechanism of mercury volatilization by alginate remains unclear, though it is thought to be a chemical effect.