Continuous alcohol production from whey permeate using immobilised cell reactor systems

The present paper reports the performance of a bioreactor packed with alginate-entrapped Kluyveromyces marxianus NCYC179 for continuous fermentation of whey permeate to ethanol. A maximum ethanol productivity as 28.21 gl^?1 h^?1 was attained at D=0.42h^?1 and 75% lactose consumption (substrate feed rate in the inflowing medium was 200 g lactose I^?1). However, the higher dilution rates (0.6-1.Oh^?1) resulted in poor productivities and higher substrate washout in the effluent samples. The maximum specific ethanol production (qpi) and maximum specific lactose uptake (qsi) of the immobilised Kluyveromyces marxianus NCYC179 was found to be 3.88g ethanol/g immobilised cell/hx10^?2 and 8.75g lactose consumed/g immobilised cell/hx10^?2 respectively. A bead size of 2.5 mm in diameter and activation period of 24h of alginate beads in lactose solution (10%) prior to their packing in column reactor were found to support the efficient working of the bioreactor. The immobilised cell bioreactor system was operated continuously at a constant dilution rate of 0.15h^?1 and 10% lactose for 562 h without any significant change in the efficiency (varied from 84 to 88% of theoretical) and viability of the entrapped yeast cells (dropped from 84 to 81%).     

Effect of Immobilisation on the Production of α-Amylase by an Industrial Strain of Bacillus amyloliquefaciens

The effect of immobilisation of an industrial strain of Bacillus amyloliquefaciens in calcium alginate beads on production of α-amylase was investigated using lactose-based media in shake flasks and in a 0.3 dm³ glass fermenter. Although the microorganism was a good α-amylase producer in batch cultures of free cells, it was unable to produce the enzyme for extended periods either in repeated batch cultures, or in continuous cultivation. In each case, parallel tests with cells immobilised in calcium alginate beads gave still further reduced enzyme yields, and the free cells released into the broth from these beads probably contributed substantially to any amylase produced during these extended fermentations. After prolonged use, the core of the alginate beads accumulated hard insoluble material, with viable immobilised cells confined to a surface layer.     

Production of lactic acid from beet molasses by calcium alginate immobilized Lactobacillus delbrueckii IFO 3202

Lactic acid was produced from pretreated beet molasses by the homofermentative organism Lactobacillus delbrueckii subsp delbrueckii IFO 3202 entrapped in calcium alginate gel using batch, repeated batch and continuous fermentation systems. In batch fermentation studies successful results were obtained with 2.0–2.4 mm diameter beads prepared from 2% sodium alginate solution. The highest effective yield (82.0%) and conversion yield (90.0%) were obtained from substrate concentrations of 52.1 and 78.2 g dm⁻³ respectively. The gel beads produced lactic acid for 14 consecutive batch fermentations without marked activity loss and deformation. In the continuous fermentation, the highest lactic acid (4.22%) was obtained at a dilution rate of 0.1 h⁻¹ while the highest productivity (13.92 g dm⁻³ h⁻¹) was obtained at a dilution rate of 0.4 h⁻¹. © 1999 Society of Chemical Industry     

Immobilization of α‐chymotrypsin for use in batch and continuous reactors

α‐Chymotrypsin from bovine pancrease (EC 3.4.21.1) was entrapped in Ca‐alginate gel particles to carry out hydrolysis of N‐acetyl‐L ‐phenylalanine methyl ester (APME) in batch as well as continuous fixed bed reactor. The enzyme was covalently modified with homo‐bifunctional polyethylene glycol derivatives in order to reduce its leakage from the beads; 85% modification of the ∈‐NH₂ groups of lysine residues caused reduction in the enzyme activity by 50%. However, this modification was helpful in a long run because it reduced both enzyme leakage and deactivation. Effective diffusivities and the distribution coefficients of the substrate and the product were determined experimentally, and later used in simulation of a batch experiment employing the beads. A continuous fixed bed reactor with the gel beads was operated to study the deactivation of the enzyme. During a 15‐day period, the enzyme showed about 15% loss in the conversion which occurred only during the first 5 days. After that the enzyme did not deactivate further which demonstrates that this method can be applied for continuous reactions. © 2000 Society of Chemical Industry     

Optimization of lactic acid production from whey by L casei NRRL B‐441 immobilized in chitosan stabilized Ca‐alginate beads

The production of lactic acid from whey by Lactobacillus casei NRRL B‐441 immobilized in chitosan‐stabilized Ca‐alginate beads was investigated. Higher lactic acid production and lower cell leakage were observed with alginate–chitosan beads compared with Ca‐alginate beads. The highest lactic acid concentration (131.2 g dm^?3) was obtained with cells entrapped in 1.3–1.7 mm alginate–chitosan beads prepared from 2% (w/v) Na‐alginate. The gel beads produced lactic acid for five consecutive batch fermentations without marked activity loss and deformation. Response surface methodology was used to investigate the effects of three fermentation parameters (initial sugar, yeast extract and calcium carbonate concentrations) on the concentration of lactic acid. Results of the statistical analysis showed that the fit of the model was good in all cases. Initial sugar, yeast extract and calcium carbonate concentrations had a strong linear effect on lactic acid production. The maximum lactic acid concentration of 136.3 g dm^?3 was obtained at the optimum concentrations of process variables (initial sugar 147.35 g dm^?3, yeast extract 28.81 g dm^?3, CaCO₃ 97.55 g dm^?3). These values were obtained by fitting of the experimental data to the model equation. The response surface methodology was found to be useful in optimizing and determining the interactions among process variables in lactic acid production using alginate–chitosan‐immobilized cells. Copyright © 2005 Society of Chemical Industry     

Performance of different immobilized‐cell systems to efficiently produce ethanol from whey: fluidized batch,packed‐bed and fluidized continuous bioreactors

BACKGROUND: The bioconversion of whey into ethanol by immobilized Kluyveromyces marxianus in packed‐bed and fluidized bioreactors is described. Both batch and continuous cultures were analyzed using three different strains of K. marxianus and the effect of the operating mode, temperature, and dilution rates (D) were investigated. RESULTS: All immobilized strains of K. marxianus (CBS 6556, CCT 4086, and CCT 2653) produced similar high yields of ethanol (0.44 ± 0.01 g EtOH g^?1 sugar). Significant variations of conversion efficiencies (66.1 to 83.3%) and ethanol productivities (0.78 to 0.96 g L^?1 h^?1) were observed in the experiments with strain K. marxianus CBS 6556 at different temperatures. High yields of ethanol were obtained in fluidized and packed‐bed bioreactors continuous cultures at different D (0.1 to 0.3 h^?1), with the highest productivity (3.5 g L^?1 h^?1) observed for D = 0.3 h^?1 in the fluidized bioreactor (87% of the maximal theoretical conversion), whereas the highest ethanol concentration in the streaming effluent (28 g L^?1) was obtained for D = 0.1 h^?1. Electronic micrographs of the gel beads showed efficient cell immobilization. CONCLUSION: Batch and continuous cultivations of immobilized K. marxianus in fluidized and packed‐bed bioreactors enable high yields and productivities of ethanol from whey. Copyright © 2012 Society of Chemical Industry     

Biotransformation of rifamycin B to rifamycin S using immobilised whole cells of Humicola spp. in a fluidised bed reactor

The biotransformation of rifamycin B to rifamycin S using immobilised whole cells of a Humicola sp. in a fluidised bed reactor exhibited a linear relationship between loading of the immobilised whole cells and conversion, in both batch and continuous operations; the conversion of rifamycin B was higher in batch than continuous operation for a given residence time. The immobilised whole cells were obtained by copolymerisation of the acetone-dried cells with acrylamide. They showed maximum activity at pH 7.8 and 50°C. It was found that aeration effects on the reaction in continuous operation were different from those in batch operation for the same residence time. In batch operation, the conversion of rifamycin B to rifamycin S increased with increased rate of aeration and reached a constant maximum value at aeration rates above 20.8 vvm. In continuous operation, the steady state conversion reached a maximum value in the range of aeration rate between 5.8 and 12.5 vvm; beyond this range the steady state conversion decreased steadily owing to cell leakage. The half life for the operational stability at 40°C was about 13 h.     

Processing efficiency of immobilized non-growing bacteria: Biocatalytic modeling and experimental analysis

Modeling methods used to optimize the biocatalytic efficiency of freely suspended cells have been applied to non-growing microbial cells entrapped within a macro-porous carrier. The catalytic rate, which is dependent on cell concentration inside the biocatalyst beads, coincided with catalytic parameters for freely suspended cells. Immobilized non-growing cell systems could be optimized utilizing the characteristics of freely suspended cells without requiring extensive experimentation to define catalytic behaviour inside the biocatalyst. A dynamic diffusion–reaction model was developed and validated using experimental data for thiodiglycol degradation by Alcaligenes xylosoxidans subsp. xylosoxidans immobilized within macro-porous poly(vinyl alcohol) cryogel in a completely mixed batch bioreactor.     

Ethanol production by alginate immobilised yeast in a fluidised bed bioreactor

Yeast, immobilised in alginate beads of known standard size and mechanical strength, has been utilised in a novel design of fluidised bed bioreactor which avoids problems of particle flotation and gas logging. Circulating substrate simultaneously entered the top and bottom of the bed. The bioreactor operated reliably for periods of up to 20 days. Increasing alginate concentration in the range 1–5% (w/w) had little effect on the performance of the immobilised yeast in converting ethanol to glucose but reduced the tendency of beads to split. Increasing bead diameter in the range 1–5 mm increased the tendency to split and reduced overall conversion of glucose. A model was developed to describe the consumption of glucose within beads based on Michaelis–Menten kinetics and the diffusion of glucose into beads. Application of the model to experimental results showed maximum reaction velocity to be independent of bead diameter and alginate concentration. The model confirmed that the observed reduction in ethanol yield compared with free yeast cells was caused by the lower substrate concentration towards the centre of the bead as opposed to any change in the metabolic rate of the immobilised cells.     

Optimization of pullulan production using Ca‐alginate‐immobilized Aureobasidium pullulans by response surface methodology

BACKGROUND: The production of pullulan from synthetic medium by Aureobasidium pullulans P56 immobilized in Ca‐alginate beads was investigated using batch and repeated batch fermentation systems. RESULTS: The highest pullulan concentration (19.52 ± 0.37 g dm^?3) was obtained with 2.0‐2.4 mm beads prepared from 2% sodium alginate solution. Pullulan production was mainly accomplished by immobilized fungal cells since leaked cells in the fermentation medium comprised 17.4% of the total fungal population at the end of fermentation. The pullulan proportion was 84.5% of the total polysaccharide in the fermentation medium. Response surface methodology was used to investigate the effects of three fermentation parameters (initial pH, agitation speed and incubation time) on the concentration of pullulan. Results of the statistical analysis showed that the fit of the model was good in all cases. The maximum pullulan concentration of 21.07 ± 0.48 g dm^?3 was obtained at the optimum concentrations of process variables (pH 7.31, agitation speed 191.5 rpm, incubation time 101.2 h). The gel beads produced pullulan under the optimized conditions for six consecutive batch fermentations without marked activity loss and deformation. CONCLUSION: The results of this study suggest that the immobilization of A. pullulans cells in Ca‐alginate gel beads is suitable for batch and repeated batch production of pullulan. Copyright © 2007 Society of Chemical Industry     

Production of pharmaceuticals: Amines from alcohols in a continuous flow fixed bed catalytic reactor

This paper reports the use of an immobilised ruthenium complex in a continuous flow process for the N-alkylation of morpholine with benzyl alcohol. The ruthenium-based catalyst was supported on a phosphine bound polymer. Screening experiments were first performed in a batch reactor, with a 16 vol% mixture of morpholine and benzyl alcohol (stoichiometric molar ratio of 1:1) in toluene as the solvent. Operating at 110 °C for 24 h, it was shown that high conversions (>99%) into the desired tertiary amine could be achieved. This reaction was then shown to be viable in a continuous flow reactor, where the catalytic polymer beads were retained in the bed. Operating at 150 °C and using p-xylene as a solvent, the conversion into the desired tertiary amine was shown to be as high as 98%. This approach is clearly very promising, as it provides a greener and more atom efficient route for the production of secondary and tertiary amines in the pharmaceutical industry.     

Modelling of phenol biodegradation by Candida tropicalis immobilised in alginate gel beads

Phenol‐degrading yeast Candida tropicalis were immobilised in alginate gel beads and photographed by scanning electron microscopy. Batch phenol biodegradation experiments were done in shaking flasks under varying conditions such as initial phenol concentrations and bead loadings. A mathematical model was proposed to simulate the batch phenol biodegradation process in the immobilised system, which took into account the internal and external mass transfer resistances of phenol and oxygen and the double‐substrate phenol–oxygen intrinsic kinetics. The validation of this model was done by the comparison between the model simulations and the experimental measurements of phenol concentration profiles in the main liquid phase. Moreover, the time and radius courses of phenol, oxygen, and cell concentration profiles within the alginate gel beads were reasonably predicted by the proposed model.     

Production of Hexanoic Acid by Free and Immobilised Cells of Megasphaera elsdenii: Influence of in-situ Product Removal Using Ion Exchange Resin

The objective of this work was to improve the production of hexanoic acid by the anaerobic rumen bacterium, Megasphaera elsdenii, using product removal and immobilised cell approaches. Hexanoic acid, the major product of glucose metabolism by M. elsdenii strain ATCC25940, was produced at concentrations of 2–3 g dm⁻³ in stirred batch cultures. With pH controlled manually at 7, maximum concentrations of hexanoic acid increased to 6–8 g dm⁻³ with yields (g product per g glucose used) of approximately 30%. When an anion exchange resin, Amberlite IRA 400, was added during early stages of culture to minimise product inhibition, growth was not impaired and cell lysis, which was commonly seen during the stationary phase in control fermentations, was prevented. The presence of resin in pH-controlled, stirred batch fermentations increased the rate of glucose consumption and doubled hexanoic acid productivity: the equivalent of 11 g dm⁻³ of hexanoic acid was made with an estimated yield of up to 39%. Cells were immobilised successfully in κ-carrageenan and, when cell densities in inocula were sufficiently high, rates of glucose consumption and product formation were similar to free cells. Including resin in cultures of immobilised cells had effects similar to those above. Using a fed-batch mode with immobilised cells cultured in the presence of resin further increased final concentrations of hexanoic acid (up to 19 g dm⁻³) but yields were lower (20–30%) and productivity did not increase. These results show that production of volatile fatty acids can be improved significantly by product stripping onto an anion exchange resin. © 1997 SCI.     

In situ reactive extraction of lactic acid from fermentation media

Extractive lactic acid fermentation was investigated in the presence of sunflower oil and Alamine‐336 (with oleyl alcohol as the diluent solvent). Lactic acid was produced in various media at 37 °C using Lactobacillus delbrueckii (NRRL‐B 445). First, the effects of oleyl alcohol (33.3%, v/v), immobilisation, and immobilisation in the presence of sunflower oil (5, 10, 15%, v/v) on lactic acid production were investigated. It was found that oleyl alcohol did not affect production while addition of sunflower oil increased lactic acid production from 10.22 to 16.46 gdm^?3. On the other hand, a toxic effect was observed for oleyl alcohol solutions containing 15–50% (v/v) Alamine‐336. A maximum total lactic acid concentration of 25.59 gdm^?3 was obtained when an oleyl alcohol solution containing 15% (v/v) Alamine together with immobilised cells with 15% (v/v) sunflower oil was used. This value was about 2.5 times that obtained from fermentation without organic solutions. © 2001 Society of Chemical Industry     

L(+)‐Lactic acid production using poly(vinyl alcohol)‐cryogel‐entrapped Rhizopus oryzae fungal cells

A new immobilized biocatalyst based on Rhizopus oryzae fungal cells entrapped in poly(vinyl alcohol)‐cryogel was evaluated in both the batch and semi‐batch processes of L (+)‐lactic acid (LA) production, when glucose, acid hydrolysates of starch or gelatinized potato starch were used as the main substrates. Under the batch conditions, the immobilized biocatalyst developed produced LA with yields of 94% and 78% from glucose and acid starch hydrolysates, respectively. Semi‐batch conditions enabled product yields of 52% and 45% to be obtained with the corresponding substrates. The highest process productivity (up to 173 g L^?1) was reached under semi‐batch conditions. Potato starch (5–70 g L^?1) was also transformed into lactic acid by immobilized R. oryzae. It was shown that long‐term operation of the immobilized biocatalyst (for at least 480 h) produced a low decrease in metabolic activity. Copyright © 2006 Society of Chemical Industry     

Biosorption of heavy metals from aqueous solution using modified activated carbon: comparison of linear and nonlinear methods

BACKGROUND: Biosorption of heavy metals from aqueous solution by modified activated carbon with Phanerochaete chrysosporium immobilised in Ca‐alginate beads was investigated using a batch system and comparison of linear and nonlinear methods. RESULTS: The amount of Cu(II), Zn(II) and Pb(II) ion sorption by the beads was as follows: activated carbon with P. chrysosporium immobilised in Ca‐alginate beads (ACFCA) (193.4, 181.8, 136.6 mg g^?1) > activated carbon immobilised in Ca‐alginate beads (ACCA) (174.8, 162.0, 130.7 mg g^?1) > P. chrysosporium (F) (148.8, 125.6, 120.4 mg g^?1) > activated carbon (AC) (138.8, 112.3, 109.3 mg g^?1) > plain Ca‐alginate beads (PCA) (125.4, 105.2, 98.2 mg g^?1). The widely used Langmuir and Freundlich isotherm models were utilised to describe the biosorption equilibrium process. CONCLUSION: The results of this study suggest that the immobilisation of modified activated carbon with P. chrysosporium in Ca‐alginate beads is suitable for a batch system. The isotherm parameters were estimated using linear and nonlinear regression analyses. The surface charge density of the biosorbents varied with the pH of the medium; the maximum biosorption of heavy metal ions on the biosorbents was obtained when the pH was between 5.6 and 7.4. Copyright © 2008 Society of Chemical Industry     

A continuous alcohol fermentation byKluyveromyces fragilis using jerusalem artichoke

In this study, a continuous fermentation of inulin in the tubers of Jerusalem artichoke (JA) for alcohol production was investigated. Experiments were also conducted on the fermentation of mashed JA tubers, without extracting the juice. In a continuous fermentation of the juice of JA tubers, alcohol productivity was increased by 3.8 times as compared with that obtained in a batch fermentation. The liquefaction of mashed JA tubers by enzymes, pectinase and cellulase followed by fermentation of liquefied solution byK. fragilis was found as effective as direct fermentation of the juice. The results of this study are expected to provide valuable information in the utilization of Jarusalem artichoke for ethanol production.     

Studies of L-phenylalanine production by immobilised aminoacylase in stabilized calcium alginate beads

Aminoacylase I (EC 3.5. 1.14) was immobilised by entrapment in uncoated calcium alginate beads and calcium alginate beads coated with chitosan, polyethyleneimine and polyethyleneimine-glutaraldehyde for the production of L-phenylalanine by the hydrolysis of a racemic mixture of N-acetyl-DL-phenylalanine. The operational stability, thermal stability, effects of pH and temperature and kinetic constants, K_m and V_max values of free and immobilised enzymes were studied. Scanning electron micrographs revealed differences in the structures of the surfaces of coated and uncoated beads. Chitosan-coated calcium alginate beads was found to be the best among the immobilised systems studied. The activity and the optimum temperature of immobilised aminoacylase were higher than those of the free enzyme. In addition, the beads showed stable activity under operational conditions. The immobilised aminoacylase lost about 20% of its initial activity after ten cycles of reuse. Polyethyleneimine and polyethyleneimine-glutaraldehyde treatments were also found to enhance the operational stability of the enzyme but its activity was greatly reduced.     

Pressure Cycling to Enhance an Immobilized Enzymic Reaction by Enzyme Entrapment in a Pressure-Sensitive Gel

The enzyme β-galactosidase was entrapped in poly(N-isopropylacrylamide) gel. The gel swelled or collapsed sharply upon the rise or fall of environmental pressure. The gel beads, prepared by reverse suspension polymerization, were placed in a stirred batch reactor filled with a solution of the substrateo-nitrophenol β-D -galactopyranoside. Operations under constant or cycling pressure were performed. A considerable increase in conversion rate was observed under pressure cycling operation, compared with isobaric operations at either atmospheric or high pressure. This result could be explained by the enhancement of mass transfer inside the gel beads during the pressure cycling operation.     

Hydrodynamic considerations on optimal design of a three‐phase airlift bioreactor with high solids loading

The hydrodynamic study of a three‐phase airlift (TPAL) bioreactor with an enlarged gas–liquid dual separator was carried out. Different lengths and diameters of the draft tube were tested to show how the design of the separator zone affects the hydrodynamic performance of the TPAL reactor. Ca‐alginate beads with entrapped yeast biomass at different loadings (0, 7, 14 and 21% v/v) were used in order to mimic the solid phase of conventional high cell density systems, such as those with cells immobilized on carriers or flocculating cells. Important information on multiphase flow and distribution of gas and solid phases in the internal‐loop airlift reactor (ALR) with high solids loading was obtained, which can be used for suggesting optimal hydrodynamic conditions in a TPAL bioreactor with high solids loading. It is finally suggested that the ALR with a dual separator and a downcomer to riser cross‐sectional area ratio (A_D/A_R) ranging from 1.2 to 2.0 can be successfully applied to batch/continuous high cell density systems, where the uniform distribution of solid phase, its efficient separation of particles from the liquid phase, and an improved residence time of air bubbles inside the reactor are desirable. Copyright © 2003 Society of Chemical Industry