Production of L-aminoacylase by fermentation of Pseudomonas sp. BA2

The growth and enzymatic production of Pseudomonas sp. BA2 a new L -aminoacylase-producing microorganism, were studied in a bench-top fermenter. Multiple fermentations were carried out in order to determine the optical pH and temperature values. The influence of the substrate concentration on both growth and L aminoacylase activity was also investigated. The maximum growth rate and the greatest yield of enzyme were obtained when the fermentation was carried out at pH 7·5, 25°C and DOT ≥ 50%. N-Acetyl-DL -alanine, at a concentration 20 g dm^?3, was used as the sole carbon and nitrogen source. The fermentation process provided a maximum biomass concentration of 3·36 g dry weight dm^?3. The highest L -aminoacylase production (11429 U g^?1 dry weight) was obtained after 39 h of cultivation. The results were a significant improvement over those previously reported.     

Production of L‐methionine by immobilized pellets of Aspergillus oryzae in a packed bed reactor

Production of L ‐methionine by immobilized pellets of Aspergillus oryzae in a packed bed reactor was investigated. Based on the determination of relative enzymatic activity in the immobilized pellets, the optimum pH and temperature for the resolution reaction were 8.0 and 60 °C, respectively. The effects of substrate concentration on the resolution reaction were also investigated and the kinetic constants (K_m and V_m) of immobilized pellets were found to be 7.99 mmol dm^?3 and 1.38 mmol dm^?3 h^?1, respectively. The maximum substrate concentration for the resolution reaction without inhibition was 0.2 mol dm^?3. The L ‐methionine conversion rate reached 94% and 78% when substrate concentrations were 0.2 and 0.4 mol dm^?3, respectively, at a flow rate of 7.5 cm³ h^?1 using the small‐scale packed bed reactor developed. The half‐life of the L ‐aminoacylase in immobilized pellets was 70 days in continuous operation. All the results obtained in this paper exhibit a practical potential of using immobilized pellets of Aspergillus oryzae in the production of L ‐methionine. © 2002 Society of Chemical Industry     

Comparison of Two Enzyme Sequences for a Novel L-Malate Biosensor

Two novel amperometric biosensors for the determination of L -malic acid in food samples have been compared. Both sensors make use of a Clark-type O₂-electrode but differ in the enzymes used. The first sensor is composed of malate dehydrogenase (decarboxylating), also known as ‘malic enzyme’ (MDH(dec.), EC 1.1.1.40) and pyruvate oxidase (POP, EC 1.2.3.3). It covers a linear detection range from 1 μmol dm⁻³ to 0·9 mmol dm⁻³ L -malate, with a response time of 1·5 min (t₉₀) and a relative standard deviation of 3·5%. Measurements with real samples offered a good correlation with the standard enzymatic assay (difference ±7%). Stored at room temperature, the response of the sensor is constant for 8 days. The second biosensor is based on the three enzyme sequence malate dehydrogenase (MDH, EC 1.1.1.37), oxaloacetate decarboxylase (OAC, EC 4.1.1.3) and pyruvate oxidase (POP, EC 1.2.3.3). It has a non-linear calibration curve. Concentrations from 5 μmol dm⁻³ to 1 mmol dm⁻³ L -malate can be detected, within a response time of 1·5 min and with a relative standard deviation of 20%. The lower detection limit for L -malate is 2 μmol dm⁻³. The response is constant for 10 days when the sensor is stored at room temperature.     

Removal of Hydrogen Sulphide by Immobilized Thiobacillus sp. strain CH11 in a Biofilter

An autotropic Thiobacillus sp. CH11 was isolated from piggery wastewater containing hydrogen sulphide. The removal characteristics of hydrogen sulphide by Thiobacillus sp. CH11 were examined in the continuous system. The hydrogen sulphide removal capacity was elevated by the BDST (Bed Depth Service Time) method (physical adsorption) and an immobilized cell biofilter (biological conversion). The optimum pH to remove hydrogen sulphide ranged from 6 to 8. The average specific uptake rate of hydrogen sulphide was as 1·02×10⁻¹³ mol-S cell⁻¹ h⁻¹ in continuous systems. The maximum removal rate and saturation constant for hydrogen sulphide were calculated to be V_m = 30·1 mmol-S day⁻¹ (kg-dry bead)⁻¹ and K_s = 1·28 μmol dm⁻³, respectively. A criterion to design a scale-up biofilter was also studied. The maximum inlet loading in the linear region (95% removal) was 47 mmol-S day⁻¹ (kg-dry bead)⁻¹. Additionally, the biofilter exhibited high efficiency (>98·5%) in the removal of hydrogen sulphide at both low (<0·026 mg dm⁻³) and high (0·078 mg dm⁻³) concentrations. The results suggested that the Thiobacillus sp. CH11 immobilized with Ca-alginate is a potential method for the removal of hydrogen sulphide. © 1997 SCI.     

Technological and kinetic aspects of sublethal acid toxicity in microbial gum production

Chemostat culture of Xanthomonas campestris were obtained at a dilution rate of 0·05 h⁻¹ and the normal feed then supplemented with 0·58 and 1·74 mmol dm⁻³ isobutyric acid (IBA). Data revealed that the organism responded to sublethal acid stress by overproducing xanthan. The acid additions led to transient zones in the continuous cultivation profiles. By adding feed containing 1·74 mmol dm⁻³ IBA, volumetric growth rate immediately decreased from 0·059 to 0·026 g dm⁻³ h⁻¹ whereas the specific xanthan formation rate increased from 0·23 g g⁻¹ biomass h⁻¹ to a maximum 0·65 g g⁻¹ biomass h⁻¹ (with 1·0 mmol dm⁻³ IBA addition), before decreasing as the concentration of acid attained that of the feed. By monitoring the outlet CO₂ in parallel with biomass and polysaccharide levels in the IBA fermentation a 10% diversion of the total carbon flux from biomass synthesis to xanthan biosynthesis was detected. A consistent pattern of variation in activity was detected in enzymes of intermediary metabolism, suggesting an action at the regulatory level. Enhanced activities of carbon catabolism and xanthan anabolic reactions (phosphomannose isomerase) were observed in the presence of the acid. Batch experiments carried out in the pres-ence of IBA gave results which correlated with the undissociated acid form con-centration. An undissociated acid fraction of 6·5×10⁻³ mmol dm⁻³ was calculated in a set of flasks under the same conditions and a statistically vali-dated 12% increase in xanthan production was found. The maximum activation was determined to be below 1·1×10⁻² mmol dm⁻³ when a 58% specific xanthan production rate increase occurred in parallel with a 35% decrease in biomass concentration.     

Improvement of substrate-selectivity in enzyme-linked FIA-systems by differential measurement and ‘enzyme-array’

Flow injection systems with immobilized enzymes for the parallel determination of chemically similar substances such as xylose/glucose and L -leucine/L -isoleucine/L -valine are described. The determination of xylose and glucose was performed either by differential measurement or by an ‘enzyme-array’. Because of the different linear ranges of the calibration curves for xylose and glucose quantification was only possible when there was an excess of xylose (xylose 0·1–2·5 mmol dm^?3, glucose 0·001–0·1 mmol dm^?3). The parallel quantification of the branched-chain amino acids could be realized by an array arrangement using an FIA-system with several immobilized enzymes of slightly different selectivity. The total amount of branched-chain amino acids can be up to about 1·1 mmol dm^?3.     

Purification of the Penicillium citrinum Lipase Using AOT Reversed Micelles

This work describes the extraction and back-extraction of a lipase from crude extract of Penicillium citrinum using AOT reversed micelles in isooctane. The effect of pH, ionic strength, AOT concentration on the protein forward and backward transfer at 20°C was studied. The maximum protein forward extraction (32·0%) was achieved at pH 4·0 with a 50 mmol dm⁻³ acetate buffer containing 100 mmol dm⁻³ KCl and 100 mmol dm⁻³ AOT in isooctane. Proteins were back-extracted (82·7%) to a new aqueous phase containing 100 mmol dm⁻³ pH 8·0 phosphate buffer and 1000 mmol dm⁻³ KCl. No enzyme activity could be detected either in the micellar phase or in the aqueous phase after protein back-extraction. However, the lipolytic activity was recovered after hydrophobic interaction chromatography on a Phenyl Superose column. The yield obtained for the overall process was 68% for activity, 26·4% for protein recovery and the purification factor was 810-fold. A single protein band at 33000 Da was obtained for SDS–PAGE analysis for the recovered and purified enzyme. © 1997 SCI.     

Tyrosinase-glucose dehydrogenase substrate-recycling biosensor: A highly-sensitive measurement of phenolic compounds

Mushroom tyrosinase and glucose dehydrogenase from Acinetobacter calcoaceticus were immobilized in poly(vinyl)alcohol membranes and coupled with a Clark-type oxygen electrode to give a substrate (analyte) regenerating cycle for monitoring of nanomolar concentrations of phenolic compounds. In this way the response for catechol, phenol, p-cresol, p-chlorophenol and p-acetamidophenol was amplified by a factor of 450, 300, 240, 150, and 140, respectively. The resulting detection limit for catechol and phenol is 0·6 nmol dm⁻³ and 0·9 nmol dm⁻³, respectively. The measuring linear range for phenol obtained by the amplified electrode extends from 1 to 400 nmol dm⁻³. The comparison with the chemical (ascorbic acid) regeneration of the phenolic compounds demonstrates the efficiency of the enzymatic procedure. The biosensor can be used for monitoring of phenolic compounds in environmental or industrial samples.     

Biocatalysis of Chlorophyllase in Ternary Micellar Systems Using Pheophytins as Substrates

The partially purified chlorophyllase, obtained from the alga Phaeodactylum tricornutum, was assayed for its hydrolytic activity towards the pheophytin in ternary micellar systems of hexane/Tris–HCl/surfactant. A wide range of surfactants, sorbitans (Span 20, 40, 60, 80 and 85) and polysorbates (Tween 20, 40, 60, 80 and 85), was used. The use of either 50 μmol dm⁻³ of Span 85 or 1 μmol dm⁻³ of Tween 80 increased the hydrolytic activity of chlorophyllase by 110 and 23%, respectively. The optimum values of pH, enzyme content, incubation time and temperature for the hydrolytic activity of chlorophyllase were determined as 8·25, 8·00 μg protein cm⁻³, 60 min and 27·5°C, respectively. The V_max and K_m values were 6·91 nmole hydrolyzed pheophytin mg⁻¹ protein min⁻¹ and 47·2 nmole pheophytin dm⁻³, respectively, in the Span 85 medium and 10·04 and 121·00, respectively, in the Tween 80 medium. The addition of optimized amounts of individual membrane lipids, L -α-phosphatidylcholine, L -α-phosphatidyl-DL -glycerol and β-carotene increased the hydrolytic activity of chlorophyllase by 50, 36 and 10%, respectively, for Span 85 and 30, 48 and 15%, respectively, for Tween 80. Phytol showed a competitive inhibitory effect on chlorophyllase activity in both Span and Tween systems with a K_i value of 15·5 and 14·3 μmol dm⁻³, respectively. High-performance liquid chromatography and spectrophotometry analyses were used to characterize the end-products of chlorophyllase hydrolytic reaction. © 1997 SCI.     

Co-digestion of Municipal Sewage Sludges and Pre-hydrolysed Woody Agricultural Wastes

Material balance has been used to evaluate the COD behaviour and the time required for fed-batch digestion of mixtures of domestic sludges and pre-hydrolysed agricultural wastes. Pre-hydrolysis of the feed materials has been used to penetrate the strong lignocellulosic structure of these wastes as well as to increase the fraction of soluble organic substances in the mixture. The influence of the organic loading rate on the main process parameters (methane, carbon dioxide, total biogas productions and their respective conversion yields) has also been investigated. The organic load has been varied from 0·8 up to 6·1 g_COD dm⁻³ day⁻¹, corresponding to a range of volatile solids load of 0·6–4·5 g_VS dm⁻³ day⁻¹ for the material under consideration. These values are slightly higher than those usually employed in conventional digester for domestic sewage sludges. However, methane production reached a maximum rate of only 5·6 mmol dm⁻³ day⁻¹ at an organic loading rate of 4·6 g_COD dm⁻³ day⁻¹, while both CH₄ content and production of biogas rapidly fell over 2·2 g_COD dm⁻³ day⁻¹. On the whole, these results suggest that removal of lignin is necessary in order to carry out the continuous anaerobic digestion of pre-hydrolysed agricultural wastes rich in woody materials. © 1997 SCI.     

Factors affecting silver biosorption by an industrial strain of Saccharomyces cerevisiae

Factors affecting silver biosorption by Saccharomyces cerevisiae biomass, obtained as a waste product from industry, were examined. Maximum removal of silver from solution was achieved within 5 min. Increasing the concentration of biomass in experimental flasks from 1 to 8 mg cm⁻³ decreased both silver accumulation, from 224·7 to 89·5 μmol Ag g⁻¹ dry wt, and associated H⁺ ion release, from 109·4 to 31·7 μmol H⁺ g⁻¹ dry wt. The presence of 1·0 mol dm⁻³ cadmium or methionine decreased silver biosorption by 40% and 93% respectively. Boiling in 100 mmol dm⁻³ NaOH or 10 mmol dm⁻³ sodium dodecyl sulphate decreased silver biosorption by 54% and 25% respectively. A temperature increase from 4°C to 55°C decreased silver biosorption by 9%. The metabolic state of the yeast had no effect on silver biosorption. Decreasing the pH of the silver solution caused a reduction in metal removal by the biomass.     

Photocatalytic degradation of diuron [3-(3,4-dichlorophenyl)-1,1-dimethylurea] on the layer of TiO2 particles in the batch mode plate film reactor

The photocatalytic degradation of diuron in aqueous solution on TiO₂ layer of a batch mode plate reactor irradiated with ultraviolet sun-bed tubes was investigated. Dependence of the reaction rate on the diuron concentration (in the range of 0·8–8·0×10⁻⁵ mol dm⁻³) and on the light intensity (0·8–2·7×10⁻⁹ einstein cm⁻² s⁻¹) but independence on the flow rate (2·5–3·6 dm³ min⁻¹) were found. A reaction scheme was proposed following the main identified primary [3-(3,4-dichlorophenyl)-1-methyl-1-formylurea and 3-(3,4-dichlorophenyl)-1-methylurea] and secondary [3-(3,4-dichlorophenyl)-1-formylurea and 3,4-dichlorophenylurea] products. © 1998 SCI     

Nitrification of fertilizer wastewaters in a biofilm reactor

The nitrification characteristics of fertilizer wastes were investigated in a biofilm system using a submerged aerated filter. The attachment of biomass on packing media was studied. Supplement of organic carbon in the form of glucose and yeast extract enhanced biofilm formation although the nitrifiers did not require organic carbon for growth. After an attachment period, continuous operation of the reactor at different loading rates and dissolved oxygen levels was investigated. The maximum achievable nitrification rate was strongly dependent on the dissolved oxygen. In the dissolved oxygen range of 3·2–3·5 mg dm⁻³, the maximum ammonia removal rate was about 0·17 kg NH₄ N m⁻³ day⁻¹. When the dissolved oxygen was increased to 4·9 mg dm⁻³, removal rates as high as 0·41 kg NH₄ N m⁻³ day⁻¹ could be obtained. Nitrite accumulation depended on the bulk nitrogen and dissolved oxygen concentrations.     

Novel porous matrix and bioreactors for high density cultures of insulinoma cell lines: Insulin secretion and response to glucose

This paper describes the use of a novel porous matrix, Porocell, for high density, tissue-like culture of two insulinoma cell lines, CRI-D2 and CRI-D11. Both these cell lines have previously been shown not to secrete insulin in response to glucose. Porocell is a macro-porous, polymeric material manufactured in the shape of discs that are 6·2 mm in diameter and 2 mm in thickness. Insulinoma cells were cultured in two different mini-bioreactors, each containing six Porocell discs inoculated with 2·5 × 10⁶ cells per disc. In surface aerated, stirred bioreactors, the insulinoma cells grew as closely packed dense cell sheets penetrating deep into the pores of Porocell. In a second type of system, a packed-bed perfused mini-bioreactor, flat, extended monolayers of cells were observed growing throughout the Porocell matrix. In both bioreactor configurations, viable cell populations were maintained for 30 days because of the excellent oxygen and nutrient transfer properties of Porocell. CRI-D2 insulinoma cells cultured in static flasks and on Porocell did not show any insulin secretion in response to 30 min exposures in media supplemented with 5·5–16·7 mmol dm⁻³ glucose. However, in long term (14–19 day) cultures, CRI-D2 cells growing in Porocell secreted low, but measurable amounts (25–35 pmol dm⁻³) of insulin in medium supplemented with elevated (14·5 mmol dm⁻³) glucose concentrations. The glucose uptake rates of cells cultured in 4·0 mmol dm⁻³ glucose increased linearly from 1·0 to 2·3 mmol dm⁻³ day⁻¹ over a period of 19 days. At 14·5 mmol dm⁻³ glucose concentration, the uptake rate increased from 1·0 to 7·05 mmol dm⁻³ day⁻¹ over the same period of culture. Contrary to previous studies, we have demonstrated that the CRI-D2 cell line cultured at high cell density in Porocell is capable of secreting insulin when exposed to prolonged and elevated concentrations of glucose. The Porocell mini-bioreactors are easy to use, robust systems that can be used for long-term studies of primary and tumorgenic islet cell function and response to secretagogues. © 1998 SCI.     

Continuous ethanol production from carob pod extract by immobilized Saccharomyces cerevisiae in a packed-bed reactor

The continuous production of ethanol from carob pod extract by immobilized Saccharomyces cerevisiae in a packed-bed reactor has been investigated. At a substrate concentration of 150 g dm^?3, maximum ethanol productivity of 16 g dm^?3 h^?1 was obtained at D = 0·4 h^?1 with 62·3% of theoretical yield and 83·6% sugars′ utilization. At a dilution rate of 0·1 h^?1, optimal ethanol productivity was achieved in the pH range 3·5–5·5, temperature range 30–35·C and initial sugar concentration of 200 g dm^?3. Maximum ethanol productivity of 24·5 g dm^?3 h^?1 was obtained at D = 0·5 h^?1 with 58·8% of theoretical yield and 85% sugars′ utilization when non-sterilized carob pod extract containing 200 g dm^?3 total sugars was used as feed material. The bioreactor system was operated at a constant dilution rate of 0·5 h^?1 for 30 days without loss of the original immobilized yeast activity. In this case, the average ethanol productivity, ethanol yield (% of theoretical) and sugars′ utilization were 25 g dm^?3 h^?1, 58·8% and 85·5%, respectively.     

Removal of 1,1′-Dimethyl-4,4′bipyridyl Dichloride from Aqueous Solution by Natural and Activated Bentonite

The sorption of 1,1′-dimethyl-4,4′bipyridilium dichloride (paraquat) on bentonite desiccated at 110°C untreated, and acid-treated with H₂SO₄ solutions over a concentration range between 0·25 M and 1·00 M , from aqueous solution at 30°C has been studied by using batch experiments. In addition, column experiments were carried out with the bentonite sample treated with the 1·00 M H₂SO₄ solution [B-A(1·00)] by using two aqueous solutions of paraquat of different concentrations (C = 29·40 mg dm⁻³ and C = 65·38 mg dm⁻³). The experimental data points have been fitted to the Langmuir equation in order to calculate the sorption capacities (X_m) of the samples; X_m values range from 1·35×10⁵ mg kg⁻¹ for the sample acid-treated with 0·375 M H₂SO₄ [B-A(0·375)] up to 1·96×10⁵ mg kg⁻¹ for the untreated bentonite [B-N]. The removal efficiency (R) has also been calculated; R values ranging from 44·61% for the [B-A(0·375)] sample up to 67·23% for B-N. The batch experiments show that the natural bentonite is more effective than the acid-treated bentonite in relation to sorption of paraquat. The column experiments show that the B-A(1·00) sample might be reasonably used in removing paraquat, the column efficiency increasing from 37·55% for the C = 65·38 mg dm⁻³ aqueous solution of paraquat up to 66·58% for the C = 29·40 mg dm⁻³ one. © 1997 SCI.     

Lipase‐catalysed synthesis of natural ethanol esters: effect of water removal on enzyme reutilisation

Lipase‐catalysed synthesis of ethanol esters using natural substrates is presented. Initial substrate concentrations, optimised through an experimental design, were 0.8 mol dm⁻³ lauric acid and 0.58 mol dm⁻³ ethanol, with an initial esterification rate (r₀) of 17.13 mmol dm⁻³ min⁻¹. Two different water removal systems were compared: a cooling exchanger in the reactor headspace and a vacuum‐based system. The best results were obtained with the second system. After six consecutive operations with the same enzyme, no loss of activity was observed. The yields obtained in all the runs were greater than 90%. Using the same approach three different natural esters (ethyl laureate, ethyl myristate and ethyl palmitate) were synthesised at 0.9 dm³ scale. Similar results were obtained in all three cases, with chemical yields always being greater than 90%. © 2000 Society of Chemical Industry     

Improved Ethanol Production by Saccharomyces cerevisiae with EDTA,Ferrocyanide and Zeolite X Addition to Sugar Beet Molasses

The influence of ethylenediaminetetra acetic acid (EDTA), potassium ferrocyanide and zeolite X on ethanol production from sugar beet molasses by Saccharomyces cerevisiae was studied. For all of the three substances used, the effect was more pronounced when added to the fermentation medium rather than to the growth medium; 1·9 mmol dm⁻³ potassium ferrocyanide caused an increase in the final ethanol concentration of about 16·4% and 47·5% with respect to control culture on addition to growth and fermentation media respectively. The greatest stimulation in product yield was obtained with zeolite X introduced during the fermentation stage; 8·0 g dm⁻³ zeolite X increased ethanol concentration by 53·3%. © 1997 SCI.     

Reclamation of wastewater from a steel-making plant using an airlift submerged biofilm reactor

A bench-scale airlift submerged biofilm reactor was developed to test the possibility of nitrification of the final effluent discharged from a wastewater treatment process of a steel-making plant with an aim of reusing it as irrigation water. Despite the fluctuation of ammonia concentration in the wastewater (55–90 mg NH₃-N dm⁻³), the ammonia was completely converted to nitrate in the hydraulic retention time of 8 h. When decreasing the hydraulic retention time further down to 4 h, the nitrification efficiency decreased to 67·9%. However, the nitrification efficiency could be significantly enhanced by increasing the airflow rate due to an increase in both of the oxygen transfer rate and liquid circulation rate. At the aeration rate of 4 dm³ min⁻¹ and the hydraulic retention time of 4 h, the nitrification efficiency was as high as 92·6% and the nitrification rate was 34·6 mg NH₃-N dm⁻³ bed h⁻¹. © 1998 Society of Chemical Industry     

The role of sulfate as a competitive inhibitor of enzymatically‐mediated heavy metal uptake by Citrobacter sp: implications in the bioremediation of acid mine drainage water using biogenic phosphate precipitant

Heavy metals can be removed from solution via biocrystallization with enzymatically‐liberated inorganic phosphate, according to Michaelis–Menten kinetics, in free whole cells and cells immobilized within polyacrylamide gel in a flow‐through reactor. Sulfate is a competitive inhibitor of phosphate release and a predictive model was developed and shown to describe the effect of sulfate on the efficiency of phosphate release by flow‐through columns. The inhibitory effect was substantially less than anticipated in the case of metal removal by the columns. In the case of lanthanum removal metal removal efficiency was restored by increasing the substrate concentration in accordance with model predictions. In the case of uranyl ion its removal with an equivalent substrate supplement increased the activity by 20% over the initial value at a limiting flow rate. Since the initial loss in activity in the presence of 40 mmol dm⁻³ SO₄²⁻ (approximately twice the K_i value) was only approximately 20% with both metals this was considered to be a minor problem for bioprocess application. In confirmation, calculations made from a published ‘case history’ of application of the system to the bioremediation of acid mine drainage water (AMD) containing 0.22 mmol dm⁻³ of uranyl ion and 35 mmol dm⁻³ of SO showed that the benchscale model is a good representation of performance under actual load conditions. © 1999 Society of Chemical Industry