Production of glycerol by immobilizedPichia farinosa

Summary Cells of the osmophilic yeastPichia farinosa were immobilized in sintered glass Raschig rings for the production of glycerol. The kinetics of production were observed under different conditions in batch, fed-batch and semicontinuous fermentations in fixed-bed column reactors and compared with those of free cells. 2.6 × 10⁹ cells/g sintered glass were adsorbed. The glycerol productivity amounted to 8.1 g/l per day. The highest concentration reached in batch culture was 86 g/l with immobilized cells. Fermentations using immobilized cells were accelerated compared to fermentations using free cells and maximum yield and productivity were reached at lower initial sugar concentrations. Using scanning electron microscopy it was observed that the shape of the cells was related to the sugar concentration in the medium. The experiments show thatP. farinosa produces glycerol with a high and constant productivity over long periods of time.     

Relationship between fermentation capability and fatty acid composition of free and immobilized Saccharomyces cerevisiae

Summary The fatty acid composition of Saccharomyces cerevisiae immobilized by entrapment in calcium-alginate beads or adsorption on sintered glass was compared to that of freely suspended cells under different fermentation conditions. The fermentation product ethanol was found to cause a shift towards saturation in the fatty acid composition under anaerobic conditions. Immobilized cells contained significantly higher percentages of saturated fatty acyl residues, especially of palmitic acid (16:0), and a decreased amount of oleic acid (18:1) compared with free cells. The percentage saturation of total fatty acid composition correlates positively with improved fermentation rates obtained with the immobilized cells. This enhanced saturation of fatty acid composition in immobilized cells may be due to altered osmotic conditions in the microenvironment of the cells.     

Glycerol production by cells of Saccharomyces cerevisiae immobilized in sintered glass

Summary Cells of Saccharomyces cerevisiae were immobilized in sintered glass Raschig rings for the production of glycerol. It can be shown that sintered glass with a porosity of 60% and pore dimensions of 60 to 100 m has a good adsorption capacity for cells of S. cerevisiae. This sintered glass was used in a fixed-bed loop reactor with a working volume of 81. Eight glycerol fermentations were carried out semicontinuously and led to glycerol yields of 26.2 to 29.5 g/l.     

Biofilm of Pseudomonas C12B on glass support as catalytic agent for continuous SDS removal

The purpose of this work was to investigate the biodegradation of Sodium dodecylsulphate, a common surfactant used in commercial detergent formulations, by immobilized cells of the surfactant-degrading bacterium Pseudomonas C12B. Cells were immobilized by adsorption on porous glass beads with either unmodified or silanized surface. Data showed a direct relation between the SDS concentration in the medium and formation of the biofilm on glass beads. Bioreactors with Pseudomonas C12B cells immobilized on both types of porous glass beads were prepared. Both types showed equivalent efficiency to remove SDS. This biocatalyst was also effective to remove anionic surfactants from commercial dishwashing liquid (Jar) and shampoo (Clear) under continuous operation.     

Growth and removal of nitrogen and phosphorus by free-living and chitosan-immobilized cells of the marine cyanobacterium Synechococcus elongatus

This study investigated the growth rate of chitosan-immobilized cells of the marine cyanobacterium Synechococcus elongatus and its potential application in the removal of nitrogen and phosphorus for wastewater treatment. Immobilized cell cultures had a lag phase of growth due to the immobilization method, and their growth rate was similar to that of free-living cell cultures. Ammonia removal was higher in free cells (54%) than in immobilized cells (29%), but nitrate removal was similar in immobilized (38%) and free cells (44%); phosphorus removal was more efficient in free cells (88%) than in immobilized cells (77%). Chlorophyll a and protein content were higher in immobilized cells. Our study demonstrates that S. elongatus immobilized into chitosan capsules can remove nutrients and is able to maintain a growth rate comparable to that of free cells in culture.     

Inert supports for lactic acid fermentation —a technological assessment

Production of lactic acid using Lactobacillus delbrueckii NRRL B445 recently renamed L. rhamnosus was studied in continuously recycled packed reactors at pH 6.3 and 42° C. Four inert adsorbent supports were used for immobilization: Raschig rings of sintered glass (Schott, FRG), beads of sintered glass (Schott), beads of porous glass (Poraver; Dennert, FRG) and irregular ceramic particles (Otto Feuerfest, FRG). The best support was found to be the beads of sintered glass, yielding the highest volumetric lactic acid productivity. Zeta potentials of L. rhamnosus showed the cells to be negatively charged at all pHs studied, the charge becoming less negative with increasing ionic strength. The surface charge did not control adhesion. A comparison between the immobilization carried out in batch and continuous tests with the different supports demonstrated that extrapolation from batch adsorption curves to continuous operation can introduce large errors. The effect of dilution rate was also stidied: a saturation concentration of adsorbed cells was achieved at all dilution rates, i.e., the immobilized cell component was almost invariant. Different diameters of Poraver beads were tested; clear evidence for mass transfer limitation was shown. Finally, the effects of pH and substrate concentration under immobilization were evaluated. The results indicate that pHs above or below the optimum for suspended cell systems can be used in the immobilized reactor while maintaining lactic acid productivity. To simplify downstream processing by keeping the glucose concentration close to zero in the effluent, the glucose concentration in the feed has to be chosen in conjunction with the dilution rate. Correspondence to: M. J. T. Carrondo     

Adsorptive Immobilization of a Pseudomonas Strain on Solid Carriers for Augmented Decolourization in a Chemostat Bioreactor

Pseudomonas GM3, a highly efficient strain in cleavage of azo bonds of synthetic dyes under anoxic conditions, was immobilized via adsorption on two types of carriers, porous glass beads and solid PVA particles. The cells were cultivated in a nutrient medium, adsorbed on sterile carriers, stabilized as biofilms in repeated batch cultures, and introduced into a chemostat activated sludge reactor for augmented decolourization. The microbial cells were quickly adsorbed and fixed on the PVA surface, compared to a slow and linear immobilization on the glass surface. The porous structure of glass beads provided shelter for the embedded cells, giving a high biomass loading or thick biofilm (13.3 mg VS ml^?1 carrier) in comparison with PVA particles (4.8 mg VS ml^?1 carrier), but the mass transfer of substrate in the biofilm became a significant limiting factor in the thicker biofilms (effectiveness factor η = 0.31). The microbial decolourization rate per volume of carriers was 0.15 and 0.17 mg dye ml^?1 of glass beads and PVA particles, respectively. In augmented decomposition of a recalcitrant azo dye (60 mg l^?1), the immobilized Pseudomonas cells in porous glass beads gave a stable decolourization efficiency (80 - 81%), but cells fixed on solid PVA particles showed an initial high colour removal of 90% which then declined to a stable removal efficiency of 81%. In both cases, the colour removal efficiency of the chemostat bioreactor was increased from < 10% by an activated sludge to ～80% by the augmented system.     

Physiological tests for yeast brewery cells immobilized on modified chamotte carrier

In this study yeast cell physiological activity was assessed on the basis of the in situ activity of two important enzymes, succinate dehydrogenase and pyruvate decarboxylase. FUN1 dye bioconversion and cellular ATP content were also taken as important indicators of yeast cell activity. The study was conducted on six brewing yeast strains, which were either free cells or immobilized on a chamotte carrier. The experimental data obtained indicate clearly that, in most cases, the immobilized cells showed lower enzyme activity than free cells from analogous cultures. Pyruvate decarboxylase activity in immobilized cells was higher than in planktonic cell populations only in the case of the Saccharomyces pastorianus 680 strain. However, in a comparative assessment of the fermentation process, conducted with the use of free and immobilized cells, much more favorable dynamics and carbon dioxide productivity were observed in immobilized cells, especially in the case of brewing lager yeast strains. This may explain the higher total cell density per volume unit of the fermented medium and the improved resistance of immobilized cells to environmental changes.     

Glycerol production by semicontinuous fed-batch fermentation with immobilized cells of Saccharomyces cerevisiae

Summary Semicontinuous fed-batch glycerol fermentations with Saccharomyces cerevisiae cells immobilized in sintered glass Raschig rings were carried out in fixed-bed loop reactors with a working volume of either 0.8 l or 8 l. The influence of biomass, temperature and CO₂ gassing on the glycerol yield was examined. The highest glycerol yield of 85 g l^–1 was achieved at 30° C and average CO₂ gassing rate of 0.4 v/v m with a theoretical glycerol yield of 67%. Fed-batch fermentations with free cells indicated an inhibition mechanism of the glycerol produced, affecting the fermentation capacity of the yeast strain used.Offprint requests to: H.-J. Rehm     

Ribonuclease production by free and immobilizedAspergillus clavatus cells

Several fungal strains ofAspergillus andPenicillium were immobilized by cryopolymerization in polyvinyl alcohol cryogel beads.Aspergillus clavatus was the best producer of extracellular ribonuclease. Enzyme productivity and growth of free and immobilized cells in shake flasks and agitated bioreactor were studied. Ribonuclease production and growth behaviour depended on concentrations of glucose, peptone and soybean in the culture medium. Enzyme production was influenced by agitation and aeration intensity. In repeated batch, shake-flask cultures, the immobilized cells showed 2 to 3.5 times higher enzyme activity than free cells. The optimal conditions in a bioreactor were at 150 rev/min agitation speed and 0.5 vol/vol.min aeration. Enzyme productivity of immobilized cells (237 units/g dry mycelium.h) was 2.1 times higher than the productivity of free cells in a bioreactor, and 2.3 times higher than that of a shake-flask culture.R.J. Manolov is with the Institute of Microbiology, Department of Enzymes, Bulgarian Academy of Sciences, Georgy Bonchev Street 26, 1113 Sofia, Bulgaria.     

Degradation of oily sludge from a flotation unit by free and immobilized microorganisms

Summary The degradation rate of hydrocarbons in oily sludge obtained from a flotation unit by free and immobilized cells in shaking flasks and in a stirred tank reactor was investigated. For the biodegration of 3.3% hydrocarbons free cells and cells immobilized on granular clay were used. Free cells needed 7–8 weeks to use 30% of the 3.3% hydrocarbons, whereas with immobilized cells the same result was obtained after 3–4 weeks only. In shaken flasks with high hydrocarbon concentrations (8%), immobilized Candida parapsilosis degraded 90% of the hydrocarbons in the oily sludge within 3 weeks, while free cells degraded only 27.5% in the same period. In degradation experiments with a bioreactor, free and immobilized cells of the isolate ISO-OS BÜ 20 showed better results compared to cultures in shaken flasks due to better aeration and mixing. Free cells degraded 50% of the 5% hydrocarbon-containing oily sludge in 7 weeks, whereas immobilized cells gave the same result after only 4 weeks.Offprint requests to: H.-J. Rehm     

Tannase enzyme production by entrapped cells of Aspergillus niger FETL FT3 in submerged culture system

The ability of immobilized cell cultures of Aspergillus niger FETL FT3 to produce extracellular tannase was investigated. The production of enzyme was increased by entrapping the fungus in scouring mesh cubes compared to free cells. Using optimized parameters of six scouring mesh cubes and inoculum size of 1 × 10⁶ spores/mL, the tannase production of 3.98 U/mL was obtained from the immobilized cells compared to free cells (2.81 U/mL). It was about 41.64% increment. The immobilized cultures exhibited significant tannase production stability of two repeated runs.     

Immobilization ofPenicillium raistrickii spores and mycelium growth for 15α-hydroxylation of 13-ethylgon-4-en-3,17-dione

Spores ofPenicillium raistrickii immobilized by gel entrapment, microencapsulation and surface adsorption were allowed to grow into mycelial form, in order to accomplish hydroxylation of 13-ethylgon-4-en-3,17-dione at the 15α-position. The hydroxylating activity of both free and immobilized cells at different pH and substrate concentration as well as under starvation conditions was studied. The productivity of cells immobilized by several techniques is compared.     

Contributions of immobilized and free cells of salt-tolerantZygosaccharomyces rouxii andCandida versatilis to the production of ethanol and 4-ethylguaiacol

Summary The contribution of immobilized cells and free cells released from gel beads to ethanol production by the salt-tolerant yeastsZygosaccharomyces rouxii andCandida versatilis, and 4-ethylguaiacol (4-EG) production byC. versatilis were investigated using an airlift reactor. The amounts of ethanol produced by free cells were about 65% and about 90% of total ethanol in the reactor forZ. rouxii andC. versatilis, respectively. It was found that immobilized cells gave a much lower specific productivity of ethanol (ethanol production per hour per cell) than free cells of both yeasts, especially ofC. versatilis. 4-EG was produced mainly by immobilized cells ofC. versatilis; the amount of 4-EG produced by free cells was about 20% of the total 4-EG, in contrast to the results of ethanol production. However, the specific productivity of 4-EG (4-EG production per hour per cell) by immobilized and free cells was fairly similar.     

Influence of the oxygenation level on d-xylose fermentation by free and agar-entrapped cultures of Candida shehatae

This paper investigates the effects of the oxygenation level on the performance of d-xylose alcoholic fermentation by free- and immobilized-cell batch cultures of Candida shehatae (ATCC 22984). Yeast cells were immobilized in composite agar layer/microporous membrane structures. Fermentations were performed under varying oxygenation levels corresponding to different O₂ flow rates (OFRs). Low OFRs enhanced the fermentation performance of free and immobilized yeasts. The best ethanol yield coefficient, obtained at an OFR of 5 mmol O₂ h^–1 dm^–3 for both culture modes, was slightly higher (0.425 g g^–1) for immobilized cultures than for their free counterparts (0.39 g g^–1). More sustained aeration inhibited ethanol production by free and immobilized organisms. However, this inhibition was more pronounced for agar-entrapped cultures. Xylitol production of free cultures normally decreased as the OFR increased. At high OFR, however, immobilized organisms surprisingly produced more xylitol than at lower OFR or in anaerobiosis. This effect is discussed by referring to the mass transfer limitations that occur inside the immobilized-cell structures. Gel-entrapped cultures displayed higher specific and volumetric production rates of ethanol and xylitol than free-cell cultures.     

Physiology of polyol formation by free and immobilized cells of the osmotolerant yeast Pichia farinosa

Summary Some physiological data of cells of Pichia farinosa immobilized on sintered glass Raschig rings were compared with data from free cells. Glucose consumption and productivity of total polyols (arabitol, glycerol and erythritol) showed a simultaneous inter-lag phase. Enzymes that catalyse steps of the pentosephosphate pathway (glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, transaldolase and polyol dehydrogenase) showed a distinct increase after transfer of the cells into production medium. The activity of glycerol-3-phosphate dehydrogenase was generally low. Only alcohol dehydrogenase presented the inter-lag phase mentioned above.Offprint requests to: H.-J. Rehm     

Hydrolysis of rice bran oil using immobilized lipase in a stirred batch reactor

Candida cylindracea lipase was immobilized by adsorption on acid washed glass beads. It was observed that protein loading of the support depends on the size of the particle, with smaller particle containing higher amount of protein per unit weight. Initial reaction rate linearly varied up to enzyme concentration of 17.25 U/mL. Amount of free fatty acids produced was linearly proportional up to the enzyme loading of 1650 μg/g of bead. Achievement of chemical equilibrium took longer time in the case of less protein loading. Degree of hydrolysis was found to decrease in second and third consecutive batch operations on repeated use of immobilized lipase.     

Physiological aspects of free and immobilized Aspergillus niger cultures producing citric acid under various glucose concentrations

Similarities and differences between cultures of free and immobilized Aspergillus niger were identified under various glucose concentrations. Growth and citric acid production rates were compared, and the macro-morphology and fine structure of the mycelia examined to determine which parameters were significant in the production of citric acid. With free cultures the diameter of pellets was inversely related to glucose concentration, while biomass levels were lower for immobilized cultures than the equivalent free cultures. Rates of citric acid production were higher with immobilized mycelium, especially at higher glucose levels. The morphology that characterized high citric acid productivity was that of swollen hyphal tips which were seen at concentrations over 100 kg/m³ glucose in both free and immobilized mycelium. Although there is a characteristic morphology associated with high productivity it does not account for the difference observed between free and immobilized mycelia. The increased glucose uptake and productivity was not due to an increased surface area either, since the immobilized system was slightly lower in total surface area than the equivalent free cultures. The major difference was in the mean diffusion path in the two systems.     

Immobilization of cultured Catharanthus roseus cells using a fibreglass substratum

Summary Cultured Catharanthus roseus cells were immobilized using geometrically identical needled fibreglass mats prepared with a range of surface coatings. The phenyl (PS), polyglycol (PG), aldehyde (CHO), alkyl (CTMS), and silanol (AW) coatings, along with the untreated glass (HC) surface, produced surfaces with a range of surface tensions. The immobilization efficiency of the substratum, measured as the percentage of cells immobilized, increased with increasing substratum surface tension in the order PS < PG < CHO < CTMS < AW < HC. The dependence of immobilization efficiency on substratum surface tension can be described using a thermodynamic model of adhesion that considers the extent of plant cell adhesion to be a function of the surface tensions of the substratum, the suspending liquid, and the plant cells. In addition, this dependence also demonstrates the fundamental role of adhesion in the immobilization process involving a glass fibre matrix. However, cell entrapment processes are also implicated. The untreated glass fibre substratum (HC), which demonstrated the greatest immobilization efficiency, was used for further characterization of the immobilization strategy. Maximum inoculum biomass was determined to be approximately 1.9 g cells (fresh weight)/g substratum (dry weight) to achieve greater than 90% immobilization efficiency. The growth rate of immobilized cultures was slower than suspension cultures, probably due to mass transfer limitations. Production of the indole alkaloids, tryptamine, catharanthine, and ajmalicine, was also suppressed relative to suspension-cultured cells. These results are considered in relation to other immobilization strategies and their apparent effects on cellular processes. Offprint requests to: F. Dicosmo     

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