Influence of agitation and aeration on growth and antibiotic production by <Emphasis Type="Italic">Xenorhabdus nematophila</Emphasis>

The effect of agitation and aeration on the growth and antibiotic production by Xenorhabdus nematophila YL001 grown in batch cultures were investigated. Efficiency of aeration and agitation was evaluated through the oxygen mass transfer coefficient (K _L a). With increase in K _L a, the biomass and antibiotic activity increased. Activity units of antibiotic and dry cell weight were increased to 232 U ml⁻¹ and 19.58 g l⁻¹, respectively, productivity in cell and antibiotic was up more than 30% when K _L a increased from 115.9 h⁻¹ to 185.7 h⁻¹. During the exponential growth phase, DO concentration was zero, the oxygen supply was not sufficient. So, based on process analysis, a three-stage oxygen supply control strategy was used to improved the DO concentration above 30% by controlling the agitation speed and aeration rate. The dry cell weight and activity units of antibiotic were further increased to 24.22 g l⁻¹ and 249 U ml⁻¹, and were improved by 24.0% and 7.0%, compared with fermentation at a constant agitation speed and a constant aeration rate (300 rev min⁻¹, 2.5 l min⁻¹).     

Photoautotrophic Production of Lipids by Some <Emphasis Type="Italic">Chlorella</Emphasis> Strains

The microalgae Chlorella protothecoides UTEX 25, Chlorella sp. TISTR 8991, and Chlorella sp. TISTR 8990 were compared for use in the production of biomass and lipids under photoautotrophic conditions. Chlorella sp. TISTR 8990 was shown to be potentially suitable for lipid production at 30°C in a culture medium that contained only inorganic salts. For Chlorella sp. TISTR 8990 in optimal conditions in a stirred tank photobioreactor, the lipid productivity was 2.3 mg L⁻¹ h⁻¹ and after 14 days the biomass contained more than 30% lipids by dry weight. To attain this, the nitrogen was provided as KNO₃ at an initial concentration of 2.05 g L⁻¹ and chelated ferric iron was added at a concentration of 1.2 × 10⁻⁵ mol L⁻¹ on the ninth day. Under the same conditions in culture tubes (36 mm outer diameter), the biomass productivity was 2.8-fold greater than in the photobioreactor (0.125 m in diameter), but the lipid productivity was only 1.2-fold higher. Thus, the average low-light level in the photobioreactor actually increased the biomass specific lipid production compared to the culture tubes. A light-limited growth model closely agreed with the experimental profiles of biomass production, nitrogen consumption, and lipid production in the photobioreactor.     

Enhanced fructooligosaccharides and inulinase production by a <Emphasis Type="Italic">Xanthomonas campestris</Emphasis> pv. <Emphasis Type="Italic">phaseoli</Emphasis> KM 24 mutant

Xanthomonas campestris pv phaseoli produced an extracellular endoinulinase (9.24 ± 0.03 U mL⁻¹) in an optimized medium comprising of 3% sucrose and 2.5% tryptone. X. campestris pv. phaseoli was further subjected to ethylmethanesulfonate mutagenesis and the resulting mutant, X. campestris pv. phaseoli KM 24 demonstrated inulinase production of 22.09 ± 0.03 U mL⁻¹ after 18 h, which was 2.4-fold higher than that of the wild type. Inulinase production by this mutant was scaled up using sucrose as a carbon source in a 5-L fermenter yielding maximum volumetric (21,865 U L⁻¹ h⁻¹) and specific (119,025 U g⁻¹ h⁻¹) productivities of inulinase after 18 h with an inulinase/invertase ratio of 2.6. A maximum FOS production of 11.9 g L⁻¹ h⁻¹ and specific productivity of 72 g g⁻¹ h⁻¹ FOS from inulin were observed in a fermenter, when the mutant was grown on medium containing 3% inulin and 2.5% tryptone. The detection of mono- and oligosaccharides in inulin hydrolysates by TLC analysis indicated the presence of an endoinulinase. This mutant has potential for large-scale production of inulinase and fructooligosaccharides.     

Effect of aeration and agitation on the protease production by S<Emphasis Type="Italic">taphylococcus aureus</Emphasis> mutant RC128 in a stirred tank bioreactor

The modified rotating simplex method has been successfully used to determine the best combination of agitation rate and aeration rate for maximum production of extracellular proteases by Staphylococcus aureus mutant RC128, in a stirred tank bioreactor operated in a discontinuous way. This mutant has shown altered exoprotein production, specially enhanced protease production. Maximum production of proteases (15.28 UP/ml), measured using azocasein as a substrate, was obtained at exponential growth phase when the bioreactor was operated at 300 rpm and at 2 vvm with a volumetric oxygen transfer coefficient (K _L a) of 175.75 h⁻¹. These conditions were found to be more suitable for protease production.     

Alcoholic fermentation of xylose and mixed sugars using recombinant <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> engineered for xylose utilization

Previously, a Saccharomyces cerevisiae strain was engineered for xylose assimilation by the constitutive overexpression of the Orpinomyces xylose isomerase, the S. cerevisiae xylulokinase, and the Pichia stipitis SUT1 sugar transporter genes. The recombinant strain exhibited growth on xylose, under aerobic conditions, with a specific growth rate of 0.025 h⁻¹, while ethanol production from xylose was achieved anaerobically. In the present study, the developed recombinant yeast was adapted for enhanced growth on xylose by serial transfer in xylose-containing minimal medium under aerobic conditions. After repeated batch cultivations, a strain was isolated which grew with a specific growth rate of 0.133 h⁻¹. The adapted strain could ferment 20 g l⁻¹ of xylose to ethanol with a yield of 0.37 g g⁻¹ and production rate of 0.026 g l⁻¹ h⁻¹. Raising the fermentation temperature from 30°C to 35°C resulted in a substantial increase in the ethanol yield (0.43 g g⁻¹) and production rate (0.07 g l⁻¹ h⁻¹) as well as a significant reduction in the xylitol yield. By the addition of a sugar complexing agent, such as sodium tetraborate, significant improvement in ethanol production and reduction in xylitol accumulation was achieved. Furthermore, ethanol production from xylose and a mixture of glucose and xylose was also demonstrated in complex medium containing yeast extract, peptone, and borate with a considerably high yield of 0.48 g g⁻¹.     

Effects of Corn Steep Liquor on Growth Rate and Pyrene Degradation by Pseudomonas strains

The growth rates and pyrene degradation rates of Pseudomonas sp. LP1 and Pseudomonas aeruginosa LP5 were increased in corn steep liquor (CSL) supplemented. On pyrene alone the highest specific growth rate of LP1 was 0.018 h⁻¹, while on CSL-supplemented pyrene MSM, the value was 0.026 h⁻1. For LP5 the highest growth rate on CSL-supplemented pyrene-MSM was 0.034 h⁻¹. Conversely, on pyrene alone the highest rate was 0.024 h⁻¹. CSL led to marked reduction in residual pyrene. In the case of Pseudomonas sp. LP1 values of residual pyrene were 58.54 and 45.47%, respectively, for the unsupplemented and supplemented broth cultures, showing a difference of 13.09%. For LP5 the corresponding values were 64.01 and 26.96%, respectively, showing a difference of 37.05%. The rate of pyrene utilization by LP1 were 0.08 and 0.11 mg l⁻¹ h⁻¹ on unsupplemented and supplemented media, respectively. The corresponding values for LP5 were 0.07 and 0.015 mg l⁻¹ h⁻¹, respectively. These results suggest that CSL, a cheap and readily available waste product, could be very useful in the bioremediation of environments contaminated with pyrene.     

Production of nisin Z using <Emphasis Type="Italic">Lactococcus lactis</Emphasis> IO-1 from hydrolyzed sago starch

A membrane bioreactor for production of nisin Z was constructed using Lactococcus lactis IO-1 in continuous culture using hydrolyzed sago starch as carbon source. A strategy used to enhance the productivity of nisin Z was to maintain the cells in a continuous growth at high cell concentration. This resulted in a volumetric productivity of nisin Z, as 50,000 IU l⁻¹ h⁻¹ using a cell concentration of 15 g l⁻¹, 30^°C, pH 5.5 and a dilution rate of 1.24 h⁻¹. Adding 10 g l⁻¹ YE and 2 g l⁻¹ polypeptone, other inducers were unnecessary to maintain production of nisin. The operating conditions of the reactor removed nisin and lactate, thus minimizing their effects which allowed the maintenance of cells in continuous exponential growth phase mode with high metabolic activity.     

Continuous nisin production with bioengineered <Emphasis Type="Italic">Lactococcus lactis</Emphasis> strains

Nisin production in continuous cultures of bioengineered Lactococcus lactis strains that incorporate additional immunity and regulation genes was studied. Highest nisin activities were observed at 0.2 h^–1 dilution rate and 12.5 g l^–1 fructose concentration for all strains. Recombinant strains were able to produce greater amounts of nisin at dilution rates below 0.3 h⁻¹ compared to the control strain. However, this significant difference disappeared at dilution rates of 0.4 and 0.5 h^–1. For the strains LL27, LAC338, LAC339, and LAC340, optimum conditions for nisin production were determined to be at 0.29, 0.26, 0.27, and 0.27 h^–1 dilution rates and 11.95, 12.01, 11.63, and 12.50 g l^–1 fructose concentrations, respectively. The highest nisin productivity, 496 IU ml^–1 h^–1, was achieved with LAC339. The results of this study suggest that low dilution rates stabilize the high specific nisin productivity of the bioengineered strains in continuous fermentation. Moreover, response surface methodology analysis showed that regulation genes yielded high nisin productivity at wide ranges of dilution rates and fructose concentrations.     

Utilization of <Emphasis Type="Italic">Anabaena</Emphasis> sp. in CO<Subscript>2</Subscript> removal processes

This paper focuses on modelling the growth rate and exopolysaccharides production of Anabaena sp. ATCC 33047, to be used in carbon dioxide removal and biofuels production. For this, the influence of dilution rate, irradiance and aeration rate on the biomass and exopolysaccharides productivity, as well as on the CO₂ fixation rate, have been studied. The productivity of the cultures was maximum at the highest irradiance and dilution rate assayed, resulting to 0.5 g_bio l⁻¹ day⁻¹ and 0.2 g_eps l⁻¹ day⁻¹, and the CO₂ fixation rate measured was 1.0 gCO₂ l⁻¹ day⁻¹. The results showed that although Anabaena sp. was partially photo-inhibited at irradiances higher than 1,300 μE m⁻² s⁻¹, its growth rate increases hyperbolically with the average irradiance inside the culture, and so does the specific exopolysaccharides production rate. The latter, on the other hand, decreases under high external irradiances, indicating that the exopolysaccharides metabolism hindered by photo-damage. Mathematical models that consider these phenomena have been proposed. Regarding aeration, the yield of the cultures decreased at rates over 0.5 v/v/min or when shear rates were higher than 60 s⁻¹, demonstrating the existence of thus existence of stress damage by aeration. The behaviour of the cultures has been verified outdoors in a pilot-scale airlift tubular photobioreactor. From this study it is concluded that Anabaena sp. is highly recommended to transform CO₂ into valuable products as has been proved capable of metabolizing carbon dioxide at rates of 1.2 gCO₂ l⁻¹ day⁻¹ outdoors. The adequacy of the proposed equations is demonstrated, resulting to a useful tool in the design and operation of photobioreactors using this strain.     

Effect of growth rate on the production of<Emphasis Type="SmallCaps">l</Emphasis>-proline in the fed-batch culture of<Emphasis Type="Italic">Corynebacterium acetoacidophilum</Emphasis>

Corynebacterium acetoacidophilum RYU3161 was cultivated in al-histidine-limited fed-batch culture. To investigate the effect of cell growth on thel-proline production, 5l fed-batch culture was performed using an exponential feeding rate to obtain the specific growth rates (μ) of 0.04, 0.06, 0.08, and 0.1 h⁻¹. The results show that the highest production ofl-proline was obtained at μ=0.04 h⁻¹. The specificl-proline production rate (Q_p) increased proportionally as a function of the specific growth rate, but decreased after it revealed the maximum value at μ=0.08 h⁻¹. Thus, the highest productivity ofl-proline was 1.66 g L⁻¹ h⁻¹ at μ=0.08 h⁻¹. The results show that the production of L-proline inC. acetoacidophilum RYU3161 has mixed growth-associated characteristics.     

Alginate molecular mass produced by <Emphasis Type="Italic">Azotobacter vinelandii</Emphasis> in response to changes of the O<Subscript>2</Subscript> transfer rate in chemostat cultures

Alginate production by Azotobacter vinelandii growing in chemostat cultures was evaluated under different O₂ transfer rates (OTR). As a result of modifying the culture’s agitation rate from 300 to 500 rpm, the OTR increased from 9 to 15.1 mmol l⁻¹ h⁻¹ and a slight variation in the alginate production (1.7–2.2 g l⁻¹) was observed. At a constant growth rate (0.1 h⁻¹), the mean molecular mass of the alginate was strongly influenced by changes in the OTR, varying from 860 to 1,690 kDa. These results support a possible relationship between alginate polymerization-depolymerization process and the O₂ uptake rate.     

Enhanced degradation of phenol by <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. CP4 entrapped in agar and calcium alginate beads in batch and continuous processes

Phenol is one of the major toxic pollutants in the wastes generated by a number of industries and needs to be eliminated before their discharge. Although microbial degradation is a preferred method of waste treatment for phenol removal, the general inability of the degrading strains to tolerate higher substrate concentrations has been a bottleneck. Immobilization of the microorganism in suitable matrices has been shown to circumvent this problem to some extent. In this study, cells of Pseudomonas sp. CP4, a laboratory isolate that degrades phenol, cresols, and other aromatics, were immobilized by entrapment in Ca-alginate and agar gel beads, separately and their performance in a fluidized bed bioreactor was compared. In batch runs, with an aeration rate of 1 vol⁻¹ vol⁻¹ min⁻¹, at 30°C and pH 7.0 ± 0.2, agar-encapsulated cells degraded up to 3000 mg l⁻¹ of phenol as compared to 1500 mg l⁻¹ by Ca-alginate-entrapped cells whereas free cells could tolerate only 1000 mg l⁻¹. In a continuous process with Ca-alginate entrapped cells a degradation rate of 200 mg phenol l⁻¹ h⁻¹ was obtained while agar-entrapped cells were far superior and could withstand and degrade up to 4000 mg phenol l⁻¹ in the feed with a maximum degradation rate of 400 mg phenol l⁻¹ h⁻¹. The results indicate a clear possibility of development of an efficient treatment technology for phenol containing waste waters with the agar-entrapped bacterial strain, Pseudomonas sp. CP4.     

Optimization of culture conditions and continuous production of chitosan by the fungi,Absidia coerulea

The production of chitosan from the mycelia ofAbsidia coerulea was studied to improve cell growth and chitosan productivity. Culture conditions were optimized in batch cultivation (pH 4.5 agitator speed of 250 rpm, and aeration rate of, 2 vvm) and the maximum chitosan concentration achieved was 2.3 g/L under optimized conditions. Continuous culture was carried out successfully by the formation of new growth spots under optimized conditions, with a chitosan productivity of 0.052 gL⁻¹ h⁻¹, which is the highest value to date, and was obtained at a dilution rate of 0.05 h⁻¹. Cell chitosan concentrations reached about 14% in the steady state, which is similar to that achieved in batch culture. This study shows that for the continuous culture ofAbsidia coerulea it is vital to control the medium composition.     

Continuous production of citric acid from dairy wastewater using immobilized<Emphasis Type="Italic">Aspergillus niger</Emphasis> ATCC 9142

The continuous production of citric acid from dairy wastewater was investigated using calcium-alginate immobilizedAspergillus niger ATCC 9142. The citric acid productivity and yield were strongly affected by the culture conditions. The optimal pH, temperature, and dilution rate were 3.0, 30°C, and 0.025 h⁻¹, respectively. Under optimal culture conditions, the maximum productivity, concentration, and yield of citric acid produced by the calcium-alginate immobilizedAspergillus niger were 160 mg L⁻¹ h⁻¹, 4.5 g/L, and 70.3% respectively. The culture was continuously perfored for 20 days without any apparent loss in citric acid productivity. Conversely, under the same conditions with a batch shake-flask culture, the maximum productivity, citric acid concentration, and yield were only 63.3 mg L⁻¹ h⁻¹, 4.7 g/L and 51.4%, respectively. Therefore, the results suggest that the bioreactor used in this study could be potentially used for continuous citric acid production from dairy wastewater by applying calcium-alginate immobilizedAspergillus niger.     

Improving intracellular production of recombinant protein in <Emphasis Type="Italic">Pichia pastoris</Emphasis> using an optimized preinduction glycerol-feeding scheme

High-cell-density production of recombinant growth hormone of Lateolabrax japonicus (rljGH) expressed intracellularly in Pichia pastoris was investigated. In the regular strategy of induction at a cell density of 160 g l⁻¹, short duration of intracellular rljGH accumulation (17 h) resulted in a low final cell density of 226 g l⁻¹. Thus, a novel strategy of induction at a cell density of 320 g l⁻¹ was investigated. In this strategy, the preinduction glycerol-feeding scheme had a significant effect on the post-induction production. Constant glycerol feeding led to a decrease of the specific rljGH production and specific production rate because of low preinduction specific growth rate. This decrease was avoided by exponential glycerol feeding to maintain a preinduction specific growth rate of 0.16 h⁻¹. The results from exponential glycerol feeding indicated that the rljGH production depended on the preinduction specific growth rate. Moreover, mixed feeding of methanol and glycerol during induction improved the specific production rate to 0.07 mg g⁻¹ h⁻¹ from 0.043 mg g⁻¹ h⁻¹. Consequently, both high cell density (428 g l⁻¹) and high rljGH production could be achieved by the novel strategy: growing the cells at the specific growth rate of 0.16 h⁻¹ to the cell density of 320 g l⁻¹ and inducing the expression by mixed feeding.     

Effects of temperature,photosynthetic photon flux density,photoperiod and O<Subscript>2</Subscript> and CO<Subscript>2</Subscript> concentrations on growth rates of the symbiotic dinoflagellate, <Emphasis Type="Italic">Amphidinium</Emphasis> sp.

Symbiotic dinoflagellates of the species Amphidinium are expected to be pharmaceutically useful microalgae because they produce antitumor macrolides. A microalgae production system with a large number of cells at a high density has been developed for the efficient production of macrolide compounds. In the present study, the effects of culture conditions on the cellular growth rate of dinoflagellates were investigated to determine the optimum culture conditions for obtaining high yields of microalgae. Amphidinium species was cultured under conditions with six temperature levels (21–35°C), six levels of photosynthetic photon flux density (15–70 μmol photons m⁻² s⁻¹), three levels of CO₂ concentration (0.02–0.1%), and three levels of O₂ concentration (0.2–21%). The number of cells cultured in a certain volume of solution was monitored microscopically and the cellular growth rate was expressed as the specific growth rate. The maximum specific growth rate was 0.022 h⁻¹ at a temperature of 26°C and O₂ concentration of 5%, and the specific growth rate was saturated at a CO₂ concentration of 0.05%, a photosynthetic photon flux density of 35 μmol photons m⁻² s⁻¹ and a photoperiod of 12 h day⁻¹ upon increasing each environmental parameter. The results demonstrate that Amphidinium species can multiply efficiently under conditions of relatively low light intensity and low O₂ concentration.     

Long-term production of soluble human Fas ligand through immobilization of <Emphasis Type="Italic">Dictyostelium discoideum</Emphasis> in a fibrous bed bioreactor

The production of recombinant glycoproteins in Dictyostelium discoideum by conventional cell culture methods was limited by low cell density as well as low growth rate. In this work, cotton towel with a good adsorption capability for D. discoideum cells was used as the immobilization matrix in an external fibrous bed bioreactor (FBB) system. With batch cultures in the FBB, the concentration of immobilized cells in the cotton fiber carrier increased to 1.37 × 10⁸ cells per milliliter after 110-h cultivation, which was about tenfold higher than the maximal cell density in the conventional free-cell culture. Correspondingly, a high concentration of soluble human Fas ligand (hFasL; 173.7 μg l⁻¹) was achieved with a high productivity (23 μg l⁻¹ h⁻¹). The FBB system also maintained a high density of viable cells for hFasL production during repeated-batch cultures, achieving a productivity of 9∼10 μg l⁻¹ h⁻¹ in all three batches studied during 15 days. The repeated-batch culture using immobilized cells of D. discoideum in the FBB system thus provides a good method for long-term and high-level production of hFasL.     

Production of <Emphasis Type="SmallCaps">l</Emphasis>-phenylalanine from glycerol by a recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis>

The production of l-phenylalanine is conventionally carried out by fermentations that use glucose or sucrose as the carbon source. This work reports on the use of glycerol as an inexpensive and abundant sole carbon source for producing l-phenylalanine using the genetically modified bacterium Escherichia coli BL21(DE3). Fermentations were carried out at 37°C, pH 7.4, using a defined medium in a stirred tank bioreactor at various intensities of impeller agitation speeds (300–500 rpm corresponding to 0.97–1.62 m s⁻¹ impeller tip speed) and aeration rates (2–8 L min⁻¹, or 1–4 vvm). This highly aerobic fermentation required a good supply of oxygen, but intense agitation (impeller tip speed ~1.62 m s⁻¹) reduced the biomass and l-phenylalanine productivity, possibly because of shear sensitivity of the recombinant bacterium. Production of l-phenylalanine was apparently strongly associated with growth. Under the best operating conditions (1.30 m s⁻¹ impeller tip speed, 4 vvm aeration rate), the yield of l-phenylalanine on glycerol was 0.58 g g⁻¹, or more than twice the best yield attainable on sucrose (0.25 g g⁻¹). In the best case, the peak concentration of l-phenylalanine was 5.6 g L⁻¹, or comparable to values attained in batch fermentations that use glucose or sucrose. The use of glycerol for the commercial production of l-phenylalanine with E. coli BL21(DE3) has the potential to substantially reduce the cost of production compared to sucrose- and glucose-based fermentations.     

Bioethanol production in batch mode by a native strain of <Emphasis Type="Italic">Zymomonas mobilis</Emphasis>

Two wild strains of Zymomonas mobilis were isolated (named as ML1 and ML2) from sugar cane molasses obtained from different farms of Santander, Colombia. Initially, selection of the best ethanol-producer strains was carried out using ethanol production parameters obtained with a commercial strain Z. mobilis DSM 3580. Three isolated strains were cultivated in a culture medium containing yeast extract, peptone, glucose and salts, at pH 6 and 32°C with stirring rate of 65 rpm during 62 h. The best results of ethanol production were obtained with the native strain ML1, reaching a maximum ethanol concentration of 79.78 g l⁻¹. ML1 and ML2 strains were identified as Z. mobilis, according to the morphology, biochemical tests and molecular characterization by PCR of specific DNA sequences from Z. mobilis. Subsequently, the effect of different nitrogen sources on production of ethanol was evaluated. The best results were obtained using urea at a 0.73 g/l. In this case, maximum concentration of ethanol was 83.81 g l⁻¹, with kinetic parameters of yield of ethanol on biomass (Y_P/X) = 69.01(g g⁻¹), maximum volumetric productivity of ethanol (Qp_max) = 2.28 (g l⁻¹ h⁻¹), specific productivity of ethanol (q_P) = 3.54 (h⁻¹) and specific growth rate (μ) = 0.12 h⁻¹. Finally, we studied the effect of different culture conditions (pH, temperature, stirring, C/N ratio) with a Placket-Burman′s experimental design. This optimization indicated that the most significant variables were temperature and stirring. In the best culture conditions a significant increase in all variables of response was achieved, reaching a maximum ethanol concentration of 93.55 g l⁻¹.     

Kinetics of growth and caffeine demethylase production of Pseudomonas sp. in bioreactor

The effect of various initial caffeine concentrations on growth and caffeine demethylase production by Pseudomonas sp. was studied in bioreactor. At initial concentration of 6.5 g l^?1 caffeine, Pseudomonas sp. showed a maximum specific growth rate of 0.2 h^?1, maximum degradation rate of 1.1 g h^?1, and caffeine demethylase activity of 18,762 U g CDW^?1 (CDW: cell dry weight). Caffeine degradation rate was 25 times higher in bioreactor than in shake flask. For the first time, we show highest degradation of 75 g caffeine (initial concentration 20 g l^?1) in 120 h, suggesting that the tested strain has potential for successful bioprocess for caffeine degradation. Growth kinetics showed substrate inhibition phenomenon. Various substrate inhibition models were fitted to the kinetic data, amongst which the double-exponential (R ² = 0.94), Luong (R ² = 0.92), and Yano and Koga 2 (R ² = 0.94) models were found to be the best. The Luedeking–Piret model showed that caffeine demethylase production kinetics was growth related. This is the first report on production of high levels of caffeine demethylase in batch bioreactor with faster degradation rate and high tolerance to caffeine, hence clearly suggesting that Pseudomonas sp. used in this study is a potential biocatalyst for industrial decaffeination.