A photochemical fuel cell system using Anabaena N-7363

Summary A blue-green algae, Anabaena N-7363, was immobilized in 2% agar gel. The hydrogen productivity of the immobilized algae was three times higher than that of free algae. The maximum hydrogen production rate by the immobilized blue-green algae was 0.52 moles h^–1 g^–1 (of wet gel) in the medium without nitrogen sources under illumination (10,000 lux). The oxygen evolved was then removed by a reactor containing aerobic bacteria. A photo-current of 15–20 mA was continuously produced for 7 days by the photochemical fuel cell system consisting of the immobilized Anabaena reactor, the oxygen-removing reactor and the hydrogen-oxygen fuel cell. The conversion ratio of hydrogen to current was from 80% to 100%.     

l-serine production from methanol and glycine with an immobilized methylotroph

l-Serine production from methanol and glycine was attempted using immobilized resting cells of a methylotroph, Protomonas extorquens NR 1, under automatically controlled conditions. A Ca-alginate system was selected. The conditions for l-serine formation were optimized at 30°C. A concentration of glycine 100 g·l⁻¹ which was the optimum concentration for l-serine production by free resting cells was used in the reaction mixture. The optimum concentrations of methanol and dissolved oxygen were 20 g·l⁻¹ and 5 ppm, respectively. Under the optimum conditions, 11.3 g·l⁻¹ of l-serine was produced within 36 h. The selectivities (mole of l-serine/mole of substrate consumed) of l-serine from methanol and glycine were 4.5% and 95.1%, respectively. The size of gel beads affected the l-serine formation rate. The initial rate of l-serine formation decreased with an increase in the size of beads. However, the l-serine formation rate increased at elevated concentrations of dissolved oxygen, even with large sized beads. This result implies that the oxygen diffusion inside the gel beads limited the l-serine formation rate. The observed effectiveness factor of the immobilized cells could be estimated by the theoretical effectiveness factor of the zero-order reaction with respect to the dissolved oxygen.Repeated use was not feasible without reactivation of the immobilized cells. Reusability was examined by reactivation of the immobilized resting cells in appropriate media for 12 h. The reactivated immobilized resting cells were used again in the next cycle. By this procedure, several cycles of l-serine formation were made possible.     

Decolorization of azo dye by immobilized Pseudomonas luteola entrapped in alginate–silicate sol–gel beads

Pseudomonas luteola was immobilized by entrapment in alginate–silicate sol–gel beads for decolorization of the azo dye, Reactive Red 22. The influences of biomass loading and operating conditions on specific decolorization rate and dye removal efficiency were studied in details. The immobilized cells were found to be less sensitive to changes in agitation rates (dissolved oxygen levels) and pH values. Michaelis–Menten kinetics could be used to describe the decolorization kinetics with the kinetic parameters being 36.5 mg g⁻¹ h⁻¹, 300.1 mg l⁻¹ and 18.2 mg g⁻¹ h⁻¹, 449.8 mg l⁻¹ for free and immobilized cells, respectively. After five repeated batch cycles, the decolorization rate of the free cells decreased by nearly 54%, while immobilized cells still retained 82% of their original activity. The immobilized cells exhibited better thermal stability during storage and reaction when compared with free cells. From SEM observation, a dense silicate gel layer was found to surround the macroporous alginate–silicate core, which resulted in much improved mechanical stability over that of alginate beads when tested under shaking conditions. Alginate–silicate matrices appeared to be the best matrix for immobilization of P. luteola in decolorization of Reactive Red 22 when compared with previous results using synthetic or natural polymer matrices.     

Calcium alginate-Immobilized hepatic microsomes: Effect of NADPH cofactor on oxidation rates

An important function of the liver is detoxification of drugs, toxins and foreign compounds. Within the liver cell, the endoplasmic reticulum, isolated as the microsomal fraction, is especially active. Microsomal oxidation is the major oxidation pathway for many compounds, and the requirement for NADPH, an expensive cofactor, is an important consideration in bioreactor design. This paper presents design information for NADPH- and substrate-dependent oxidation rates for free and immobilized microsomes. The primary goal of this paper is determining NADPH requirements for oxidation. The effect of various initial levels of nicotinamide adenine dinucleotide phosphate (NADPH) on chlorpromazine oxidation rate has been studied for a crude hepatic microsomal fraction immobilized in calcium alginate gel. At an initial NADPH concentration of 600 nmoles/ml, immobilized microsomes accelerate to a maximal velocity of ≈ 240 nmoles min⁻¹ ml⁻¹ of oxygen consumption. In comparison, free microsomes reach a maximal velocity of approximately 150 nmoles min⁻¹ ml⁻¹ at an initial NADPH concentration of 220 nmoles/ml. By fitting the “initial” rate as a function of NADPH concentration to Michaelis-Menten kinetics, the apparent half-saturation coefficients (K_m)app are 3.5 nmole/ml for free microsomes and 134.4 nmole/ml for immobilized microsomes, however the maximum reaction velocity, V_max, for immobilized microsomes is calculated to be 322 nmoles min⁻¹ ml⁻¹ compared with 145 nmols min⁻¹ ml⁻¹ for free microsomes. Preliminary studies indicate that is is possible to obtain significant reaction rates using calcium alginate immobilized microsomes and that this system may offer advantages due to its simplicity and lower cost.     

Selective production of glutamate by an immobilized marine blue-green alga,Synechococcus sp.

Summary Among 200 strains of marine bluegreen algae isolated from the coastal areas of Japan, the marine blue-green alga Synechococcus sp. NKBG 040607 excreted glutamate at the highest rate, 82.6% of total amino acids production being glutamate. Synechococcus sp. NKBG 40607 was immobilized in calcium alginate gel. Glutamate production by immobilized cells was double that of native cells. Maximal glutamate production (25 g/cm³ gel per day) of the immobilized cells was observed under a light intensity of 144 Einstein/m² per second at a cell concentration of 7.5 mg dry cells/cm³ gel. Immobilized cells of Synechococcus sp. can use nitrate as a nitrogen source. Immobilized marine Synechococcus sp. produced 0265 mg/cm³ gel of glutamate for 7 days in the presence of chloramphenicol.     

Continuous production of acetic acid using immobilized acetobacter aceti in a three-phase fluidized bed bioreactor

Continuous operation of a three-phase fluidized bed bioreactor using immobilized cells showed that both immobilized and suspended cells contributed to the production of acetic acid. Unlike the rapid decrease in the productivity at dilution rates above 0.25 h⁻¹ in the free cell fermentation, the productivity was little affected by the dilution rate in the immobilized cell fermentation. Theoretical models were proposed for the continuous process. The models approximately agreed with the experimental results. Experimental results and/or theoretical calculations based on the kinetic models showed that suspended cells were important in the production of acetic acid if the solid holdup was small or if gel radius was large. Theoretical calculations showed that an optimal solid holdup or gel size existed at higher dilution rates because of the k_La dependence on solid holdup and particle diameter.     

Effect of culture medium on hydrogen production by sulfur-deprived marine green algae Platymonas subcordiformis

To study the effect of culture medium on hydrogen production by the marine green algae, Platymonas subcordiformis under sulfur deprivation, cell growth, hydrogen production, and starch and protein catabolism was investigated in the work. Algae cells cultured only in optimized medium required 6～8 days to reach the late logarithmic at the approximate density of (2.00 ± 0.18) × 10⁶ cells/mL, which in traditional medium needed 18～22 days to reach (1.85 ± 0.20) × 10⁶ cells/mL. Increased levels of Chlorophyll (10.74 ± 0.20 μg/mL), starch (149.50 ± 6.15 μg/mL), and protein (213.00 ± 7.36 μg/mL) were accumulated in optimized medium, which were 1.06, 1.47, and 1.87-fold of the algae cells cultured in traditional medium, respectively. The sealed culture of algae cells in sulfur-deprived optimized medium shifted to anaerobic conditions after 96 h of light illumination and produced 0.45 ± 0.12 mL H₂, but in traditional medium maintained aerobic condition and no hydrogen was produced. In addition, changes in starch and protein content during continuous light illumination indicated that more endogenous substrate was consumed in the sulfur-deprived optimized medium than that in the sulfur-deprived traditional medium.     

Algal biomass production,N uptake and N2 fixation in a synthetic medium

An experiment was conducted in the growth chamber to quantify the biomass production, N removal and N₂ fixation from a synthetic medium by Chlamydomonas reinhardtii and Anabaena flos-aquae. Nitrogen was supplied at a concentration of 100 mg liter⁻¹ of NO₃⁻−¹⁵N and NH₄⁺−¹⁵ (3·5 atom %), respectively. After 21 days Chlamydomonas reinhardtii removed an average of 83·8 and 78·7 mg N liter⁻¹ as NO₃⁻ and NH₄⁺, respectively. Averages of 0·89 and 0·71 g liter⁻¹ (first batch), 1·63 and 0·95 g liter⁻ (second batch) algal biomass were collected from NO₃⁻ and NH₄⁺ media, respectively. Uptake rates of 0·11 mg ¹⁵N g⁻¹ algae day⁻¹ from NO₃⁻ medium and 0·10 mg ¹⁵N g⁻¹ algae day⁻¹ from NH₄⁺ medium were calculated. Algal cells grown in NO₃⁻ and NH₄⁺ medium contained 71 and 65 g N kg⁻¹ (first batch), 39 and 58 g N kg⁻¹ (second batch), respectively. Anabaena flos-aquae produced averages of 0·58 and 0·46 g liter⁻¹ (first batch), 0·55 and 0·48 g liter⁻¹ (second batch) after 14 days of growth from NO₃⁻ and NH₄⁺ media, respectively. Blue-green algal biomass contained higher N (81–98 g kg⁻¹) than green algae. Isotope dilution method for determining N₂ fixation indicated that 55% and 77% of total N of blue-green algae grown in NO₃⁻ and NH₄⁺ media, respectively, was derived from the atmosphere.     

l-Glutamate production by protoplasts immobilized in carrageenan gel

Protoplastization of Brevibacterium flavum cultured in a medium containing 50 μg l⁻¹ and 5 units penicillin per ml was performed by lysozyme treatment. The protoplasts were immobilized in various polymer matrices, such as agar, polyacrylamide, calcium alginate, and κ-carrageenan and then used for l-glutamate production from glucose and urea in a batch system. The protoplasts immobilized in κ-carrageenan gels showed the highest productivity of l-glutamate being twice that of immobilized whole cells under optimum conditions. The maximum productivity reached 2.3 mg ml⁻¹ initially. The immobilized B. flavum protoplasts could be used 8 times (192 h) for l-glutamate production retaining about 22% of the initial productivity during the last reaction.     

Comparison of Immobilized and Free Amyloglucosidase Process in Glucose SyrupsProduction from White Sorghum Starch

Optimum conditions for glucose syrups production from white sorghum were studied through sequential liquefaction and saccharification processes. In the liquefaction process, a maximum dextrose equivalent (DE) of 10.98 % was achieved using 30 % (w/v) of starch and Termamyl ɑ-amylase from Bacillus licheniformis. Saccharification was performed by free and immobilized amyloglucosidase from Rhizopus mold at 1 % (w/v). DE values of 88.32 % and 79.95 % were obtained from 30 % (w/v) of starch with, respectively, free and immobilized enzyme. The immobilized Amyloglucosidase in calcium alginate beads showed reusable capacity for up to 6 cycles with 46 % of the original activity retained. The kinetic behaviour of immobilized and free enzyme gives K_m value of 22.13 and 16.55 mg mL⁻¹ and V_max of 0.69 and 1.61 mg mL⁻¹ min⁻¹, respectively. The hydrolysis yield using immobilized amyloglucosidase were lower than that of the free one. However, it is relevant to reuse enzyme without losing activity in order to trim down the overall costs of enzymatic bioprocesses as starch transformation into required products in industrial manufacturing. Hydrolysis of sorghum starch using immobilized amyloglucosidase represents a promising alternative towards the development of the glucose syrups production process and its utilization in various industries.     

Biodegradation of benzene,toluene, ethylbenzene,and o-xylene by a coculture of Pseudomonas putida and Pseudomonas fluorescens immobilized in a fibrous-bed bioreactor

A fibrous-bed bioreactor containing the coculture of Pseudomonas putida and P. fluorescens immobilized in a fibrous matrix was developed to degrade benzene (B), toluene (T), ethylbenzene (E), and o-xylene (X) in synthetic waste streams. The kinetics of BTEX biodegradation by immobilized cells adapted in the fibrous-bed bioreactor and free cells grown in serum bottles were studied. In general, the BTEX biodegradation rate increased with increasing substrate concentration and then decreased after reaching a maximum, showing substrate-inhibition kinetics. However, for immobilized cells, the degradation rate was much higher than that of free cells. Compared to free cells, immobilized cells in the bioreactor tolerated higher concentrations (>1000 mg l⁻¹) of benzene and toluene, and gave at least 16-fold higher degradation rates for benzene, ethylbenzene, and o-xylene, and a 9-fold higher degradation rate for toluene. Complete and simultaneous degradation of BTEX mixture was achieved in the bioreactor under hypoxic conditions. Cells in the bioreactor were relatively insensitive to benzene toxicity; this insensitivity was attributed to adaptation of the cells in the bioreactor. Compared to the original seeding culture, the adapted cells from the fibrous-bed bioreactor had higher specific growth rate, benzene degradation rate, and cell yield when the benzene concentration was higher than 100 mg l⁻¹. Cells in the fibrous bed had a long, slim morphology, which is different from the normal short-rod shape found for suspended cells in solution.     

Continuous production of lignin peroxidase by Phanerochaete chrysosporium

Phanerochaete chrysosporium spores were immobilized both in agarose and agar gel beads, and used for the production of lignin peroxidase in repeated batch cultures on carbon-limited medium both with 0.5 g l⁻¹ glucose and without glucose. Veratryl alcohol was used as an activator of enzyme production. The biocatalyst was more stable in agarose gel with the maximum activity of 245 U l⁻¹ obtained in a 70 h batch. The biocatalyst could be used for at least 12 batches on the glucose medium with a gradual decrease in lignin peroxidase activity after the sixth batch. Further, mycelium pellets grown on carbon-limited medium were employed both in vertical and horizontal column reactors for the continuous production of lignin peroxidase. The bioreactor produced lignin peroxidase for at least 20 days in the horizontal system at 49 h residence time, with a maximum activity of 95 U l⁻¹.     

Ethanol production by whole cell immobilization using lignocellulosic materials as solid matrix

Amongst four carriers used, rice-straw was found to be superior in terms of ethanol production. The maximum productivity (17.84 gl⁻¹ h⁻¹) corresponded to a dilution rate of 0.39 h⁻¹, the ethanol concentration being 45.80 gl⁻¹. A multistage rhomboidal bioreactor was found to partially overcome the disruption effect caused by the generation of a large volume of carbon dioxide in the column. Increases in productivity of about 12.55% and 3.6%, respectively, were achieved using rhomboidal and tapered bioreactors as compared to the cylindrical bioreactor. It was observed that the generation time of cells, in both the immobilized and free states, was around 2.5 h. The ethanol yield (Y_p/s) in the lower part of the reactor was less in comparison with other zones, where the substrate utilization efficiency was relatively higher.     

Nitrogen Fixation by Thermophilic Blue-Green Algae (Cyanobacteria): Temperature Characteristics and Potential Use in Biophotolysis

Thermophilic, nitrogen-fixing, blue-green algae (cyanobacteria) were investigated for use in biophotolysis. Three strains of Mastigocladus laminosus were tested and were found to be equally effective in biophotolysis as judged by nitrogenase activity. The alga, M. laminosus NZ-86-m, which was chosen for further study, grew well in the temperature range from 35 to 50°C, with optimum growth at 45°C, at which temperature acetylene reduction activity was also greatest. The maximum tolerable temperature was 55°C. Acetylene reduction activity was saturated at a light intensity of 1 × 10⁴ ergs cm⁻² s⁻¹. Atmospheric oxygen tension was found to be slightly inhibitory to acetylene reduction of both slowly growing and exponentially growing cultures. Nonsterile continuous cultures, which were conducted to test problems of culture maintenance, could be operated for 2 months without any significant decrease in nitrogenase activity or contamination by other algae. Nitrogen-starved cultures of M. laminosus NZ-86-m produced hydrogen at comparable rates to Anabaena cylindrica. The conversion efficiency of light to hydrogen energy at maximum rates of hydrogen production was 2.7%.     

Measurement of rates of grazing of the ostracod Cyprinotus carolinensis on blue-green algae

Measurements were made of the rates of grazing of the ostracod Cyprinotus carolinensis fed ¹⁴C-labelled filamentous blue-green algae (cyanobacteria). The grazing rate was a linear function of food concentration at densities below 1.1 µg dry weight of algae · ml^-1 and independent of concentration at densities above 11.5 µg algae · ml^-1. Starvation affected grazing rates significantly, but light vs. dark feeding, animal density, and the volume of feeding container did not. Grazing on Nostoc sp. was a linear function of ostracod size. Respiration of C. carolinensis and the blue-green algae was not a significant factor in the tests of grazing.     

Continuous solvent production from whey permeate using cells of Clostridium acetobutylicum immobilized by adsorption onto bonechar

Cells of Clostridium acetobutylicum were immobilized by adsorption onto bonechar and used in a packed bed reactor for the continuous production of solvents from whey permeate. A maximum solvent productivity of 4.1 g l⁻¹ h⁻¹, representing a yield of 0.23 g solvent/g lactose utilized, was observed at a dilution rate of 1.0 h⁻¹. The reactor was operated under stable conditions for 61 days. High concentrations of lactose in the whey permeate favored solventogenesis, while low concentrations favored acidogenesis.     

Graft copolymerization of acrylamide on chitosan-co-chitin and its application for immobilization of Aspergillus sp. RL2Ct cutinase

The synthesis of chitosan (Chs) and chitin (Chi) copolymer and grafting of acrylamide (AAm) onto the synthesized copolymer have been carried out by chemical methods. The grafted copolymer was characterized by FTIR, SEM and XRD. The extracellular cutinase of Aspergillus sp. RL2Ct (E.C. 3.1.1.3) was purified to 4.46 fold with 16.1% yield using acetone precipitation and DEAE sepharose ion exchange chromatography. It was immobilized by adsorption on the grafted copolymer. The immobilized enzyme was found to be more stable then the free enzyme and has a good binding efficiency (78.8%) with the grafted copolymer. The kinetic parameters K_M and V_max for free and immobilized cutinase were found to be 0.55 mM and 1410 μmol min⁻¹ mg⁻¹ protein, 2.99 mM and 996 μmol min⁻¹ mg⁻¹ protein, respectively. The immobilized cutinase was recycled 64 times without considerable loss of activity. The matrix (Chs-co-Chi-g-poly(AAm)) prepared and cutinase immobilized on the matrix have potential applications in enzyme immobilization and organic synthesis respectively.     

Production of molecular hydrogen with immobilized cells ofRhodospirillum rubrum

Summary Cells ofRhodospirillum rubrum have been immobilized in various gels and tested for photobiological hydrogen production. Agar proved to be the best immobilizing agent with respect to production rates as well as stability. Agar immobilized cells were also superior compared to liquid suspension cultures. Growth conditions of the cells prior to immobilization, e.g. cell age, light intensity or nutrient composition, were of primary importance for the activity in the later immobilized state. A reactor with agar immobilized cells has been operated successfully over 3000 h with a loss of the activity of about 60%. Mean rates for hydrogen production for immobilized cells in this work during the first 60 to 70 hours after immobilization were in the range of 18 to 34 μl H₂ mg⁻¹ d.w. h⁻¹ and thus by a factor of up to 2 higher than liquid cultures under the same conditions. Maximal rates of hydrogen production (57 μl H₂ ml⁻¹ immobilized cell suspension) were reached in agar gel beads with cells immobilized after 70 h growth in liquid culture in the light and a cell density of 1.0 mg ml⁻¹, 70 h after immobilization.     

Development of a monolith based immobilized lipase micro-reactor for biocatalytic reactions in a biphasic mobile system

This paper reports a simple method for producing macroporous silica-monoliths with controllable porosity that can be used for the immobilization of lipases to generate an active and stable micro-reactor for biocatalysis. A range of commercially available lipases has been examined using the hydrolysis reactions of 4-nitrophenyl butyrate in water–decane media. The kinetic studies performed have identified that a similar value for k_cat is obtained for the immobilized Candida antarctica lipase A (0.13 min⁻¹) and the free lipase in solution (0.12 min⁻¹) whilst the immobilized apparent Michaelis constant K_m (3.1 mM) is 12 times lower than the free lipase in solution (38 mM). A 96% conversion was obtained for the immobilized C. antarctica lipase A compared to only 23% conversion for the free lipase. The significant higher conversions obtained with the immobilized lipases were mainly attributed to the formation of a favourable biphasic system in the continuous flowing micro-reactor system, where a significant increase in the interfacial activation occurred. The immobilized C. antarctica lipase A on the monolith also exhibited improved stability, showing 64% conversion at 80 °C and 70% conversion after continuous running for 480 h, compared to 40 and 20% conversions under the same temperature and reaction time for the free lipase.     

Different direct effects of light intensity on the entrained activity rhythm in neotropical bats (Chiroptera,Phyllostomidae)

In the two neotropical Chiroptera species, Artibeus lituratus and Phyllostomus discolor, the influence of illumination intensity during the D-time of an LD 12:12 h on the locomotory activity rhythm was investigated by offering specific rectangular illumination schedules. In LD 12:12 (10¹:10^-4lux), A. lituratus and P. discolor exhibit a stable unimodal basic activity pattern. By increasing the light intensity during D-time to 10^-1 lux the activity pattern, especially in A. lituratus, can be modified in almost every way. This indicates a strong direct inhibitory effect of higher light intensities (> 10^-4 lux). Hereby light effects via the circadian system have practically no importance. The response of A. lituratus to changes of the D-light intensity is more pronounced than that of P. discolor. It is suggested that the different direct effects of light intensity on the activity pattern of the two bat species reflect specific ecological adaptations.