Increased salinity improves the thermotolerance of mesophilic nitrification

Nitrification is a well-studied and established process to treat ammonia in wastewater. Although thermophilic nitrification could avoid cooling costs for the treatment of warm wastewaters, applications above 40 °C remain a significant challenge. This study tested the effect of salinity on the thermotolerance of mesophilic nitrifying sludge (34 °C). In batch tests, 5 g NaCl L^?1 increased the activity of aerobic ammonia-oxidizing bacteria (AerAOB) by 20–21 % at 40 and 45 °C. For nitrite-oxidizing bacteria (NOB), the activity remained unaltered at 40 °C, yet decreased by 83 % at 45 °C. In a subsequent long-term continuous reactor test, temperature was increased from 34 to 40, 42.5, 45, 47.5 and 50 °C. The AerAOB activity showed 65 and 37 % higher immediate resilience in the salt reactor (7.5 g NaCl L^?1) for the first two temperature transitions and lost activity from 45 °C onwards. NOB activity, in contrast to the batch tests, was 37 and 21 % more resilient in the salt reactor for the first two transitions, while no difference was observed for the third temperature transition. The control reactor lost NOB activity at 47.5 °C, while the salt reactor only lost activity at 50 °C. Overall, this study demonstrates salt amendment as a tool for a more efficient temperature transition for mesophilic sludge (34 °C) and eventually higher nitrification temperatures.     

Immobilization of chitosan-modified invertase on alginate-coated chitin support via polyelectrolyte complex formation

Saccharomyces cerevisiae invertase was chemically modified with chitosan and further immobilized on sodium alginate-coated chitin support. The yield of immobilized protein was determined as 85% and the enzyme retained 97% of the initial chitosan-invertase activity. The optimum temperature for invertase was increased by 10 °C and its thermostability was enhanced by about 9 °C after immobilization. The immobilized enzyme was stable against incubation in high ionic strength solutions and was four-fold more resistant to thermal treatment at 65 °C than the native counterpart. The biocatalyst prepared retained 80% of the original catalytic activity after 50 h under continuous operational regime in a packed bed reactor.     

Hydrolysis of inulin from Jerusalem artichoke by inulinase immobilized on aminoethylcellulose

Purified inulinase (inulase, 2,1-β-d-fructan fructanohydrolase, EC 3.2.1.7) of Kluyveromyces fragilis has been immobilized on 2-aminoethyl-cellulose by treatment with 2% glutaraldehyde in 0.05 m phosphate buffer, pH 7.0, for 2 h at room temperature. The immobilized enzyme preparation had 39.3 units inulinase activity per gram dried matrix, with 53.4% recovery yield of activity, and showed good operational stability in the presence of substrate, inulin or the tuber extract of Jerusalem artichoke. Optimum pH and temperature were 5.5 and 45°C, respectively. In a batch reactor, the conversion was 90% ($\text{d}\text{-fructose/}\text{d}\text{-glucose}\text{= 76/24}$) and 34 mg d-fructose per ml was produced from the artichoke tuber extract by the immobilized inulinase in 20 h. In column reactor packed with 28 ml immobilized enzyme, the following conditions were found to be optimal: height/diameter ratio of column, 10.3; space time, 3.8 h; temperature, 40°C. Operation under these conditions gave 90% conversion of a 7% inulin solution and the productivity was 102 mmol l^?1 h^?1.     

Immobilization of catalase on cotton fabric oxidized by sodium periodate

Cotton fabric was first oxidized with sodium periodate, and then employed to immobilize catalase. Optimization studies for oxidation of the fabric and immobilization of the enzyme were performed. The properties of the immobilized catalase were examined and compared with those of the free enzyme. A high activity of the immobilized enzyme was obtained when the fabric was oxidized at 40°C and pH 6.0 for 8h in a bath containing 0.20 mol L^?1 sodium periodate and the enzyme was immobilized at 4°C for 24h with a catalase dosage of 120.0 U mL^?1. The immobilized enzyme exhibited optimum activity at 40°C, while the free enzyme had optimal temperature of 30°C, suggesting that the immobilized catalase could be used in a broader temperature range. Both the immobilized and free enzyme had pH optima of 7.0. The staining test and reusability showed that the catalase was fixed covalently on the oxidized cotton fabric.     

Genetic and physiological alterations occurring in a yeast population continuously propagated at increasing temperatures with cell recycling

This work investigated the effects of increasing temperature from 30°C to 47°C on the physiological and genetic characteristics of Saccharomyces cerevisiae strain 63M after continuous fermentation with cell recycling in a system of five reactors in series. Steady state was attained at 30°C, and then the temperature of the system was raised so it ranged from 35°C in the last reactor to 43°C in the first reactor or feeding reactor with a 2°C difference between reactors. After 15 days at steady state, the temperature was raised from 37°C to 45°C for 25 days at steady state, then from 39°C to 47°C for 20 days at steady state. Starter strain 63M was a hybrid strain constructed to have a MAT a/α, LYS/lys, URA/ura genotype. This hybrid yeast showed vigorous growth on plates at 40°C, weak growth at 41°C, positive assimilation of melibiose, positive fermentation of galactose, raffinose and sucrose. Of 156 isolates obtained from this system at the end of the fermentation process, only 17.3% showed the same characteristics as starter strain 63M. Alterations in mating type reaction and in utilization of raffinose, melibiose, and sucrose were identified. Only 1.9% of the isolates lost the ability to grow at 40°C. Isolates showing requirements for lysine and uracil were also obtained. In addition, cell survival was observed at 39–47°C, but no isolates showing growth above 41°C were obtained.     

Immobilization of antibodies through the surface regions having the highest density in lysine groups on finally inert support surfaces

Immobilization of anti-horseradish peroxidase on glyoxyl-agarose proceeds rapidly, and after the immobilization, it was found that the antibody captured almost the same amount of peroxidase than the free antibody. After boiling the antibodies in the presence of SDS and mercaptoethanol, more than 95% of the immobilized antibodies presented the four subunits attached to the support.The reduction of the preparation converts the glyoxyl groups into very hydrophilic and inert hydroxyl-groups. That way, the final support was fully unable to adsorb any protein under any condition, and the only adsorbed proteins on the immobilized antibody are these recognized by the antibody. The immobilized antibody maintained intact their capacity to capture peroxidase after 20 weeks of storage at 4 °C.The high functionality of the immobilized antibody and the fully inert surface suggest that this technique may be a very suitable one to immobilize antibodies for biosensor design or immuno-chromatographic matrices.     

Characteristic of immobilized cephalosporin C acylase and its application in one-step enzymatic conversion of cephalosporin C to 7-aminocephalosporanic acid

Cephalosporin C (CPC) acylase is an enzyme which hydrolyzes CPC to 7-aminocephalosporanic acid (7-ACA) directly, and therefore has great potential in industrial application. In this study, the CPC acylase from a recombinant Escherichia coli was purified to high purity by immobilized metal affinity chromatography, and the CPC acylase was covalently attached to three kinds of epoxy supports, BB-2, ES-V-1 and LX-1000EP. The immobilized CPC acylase with LX-1000EP as the support shows the highest activity (81 U g⁻¹) suggesting its potential in industrial 7-ACA production. The activity of immobilized enzyme was found to be optimal at pH between 8.5 and 9.5 and to increase with temperature elevation until 55 °C. Immobilized CPC acylase showed good stability at pH between 8.0 and 9.5 and at temperature up to 40 °C. To avoid product degradation, the production of 7-ACA utilizing immobilized enzyme was carried out at 25 °C, pH 8.5 in a designed reactor. Under optimal reaction conditions, a very high 7-ACA yield of 96.7% was obtained within 60 min. In the results of repeated batch production of 7-ACA, 50% activity of the initial cycle was maintained after being recycled 24 times and the average conversion rate of CPC reached 98%.     

Efficient production of methionine from 2-amino-4-methylthiobutanenitrile by recombinant Escherichia coli harboring nitrilase

Methionine as an essential amino acid has been attracting more attention for its important applications in food and feed additives. In this study, for efficient production of methionine from 2-amino-4-methylthiobutanenitrile, a codon-optimized nitrilase gene was newly synthesized and expressed, and the catalytic conditions for methionine production were studied. The optimal temperature and pH for methionine synthesis were 40 °C and 7.5, respectively. The recombinant nitrilase was thermo-stable with half-life of 5.52 h at 40 °C. The substrate loading was optimized in given amount of catalyst and fixed substrate/catalyst ratio mode to achieve higher productivity. Methionine was produced in 100 % conversion within 120 min with a substrate loading of 300 mM. The production of methionine with the immobilized resting cells in packed-bed reactor was investigated. The immobilized nitrilase exhibited good operation stability and retained over 80 % of the initial activity after operating for 100 h. After separation, the purity and the total yield of methionine reached 99.1 and 97 %, respectively. This recombinant nitrilase could be a potential candidate for application in production of methionine.     

Continuous production of fructose-syrups from inulin by immobilized inulinase from recombinantSaccharomyces cerevisiae

Recombinant exoinulinase was partially purified from the culture supernatant ofS. cerevisiae by (NH₄)₂SO₄ precipitation and PEG treatment. The purified inulinase was immobilized onto Amino-cellulofine with glutaraldehyde as a cross-linking agent. Immobilization yield based on the enzyme activity was about 15%. Optimal pH and temperature of immobilized enzyme were found to be 5.0 and 60°C, respectively. The enzyme activity was stably maintained in the pH ranges of 4.5 to 6.0 at 60°C. 100% of enzyme activity was observed even after incubation for 24 hr at 60°C. In the operation of a packed-bed reactor containing 412 U inulinase, dahalia inulin of 7.5%(w/v) concentration was completely hydrolyzed at flow rate of 2.0 mL/min at 60°C, resulting in a volumetric productivity of 693 g-reducing sugars/L/h. Under the reaction conditions of 1.0 mL/min flow rate with 2.5% inulin at 60°C, the reactor was successfully operated over 30 days without loss of inulinase activity.     

Temperature increases from 55 to 75 °C in a two-phase biogas reactor result in fundamental alterations within the bacterial and archaeal community structure

Agricultural biogas plants were operated in most cases below their optimal performance. An increase in the fermentation temperature and a spatial separation of hydrolysis/acetogenesis and methanogenesis are known strategies in improving and stabilizing biogas production. In this study, the dynamic variability of the bacterial and archaeal community was monitored within a two-phase leach bed biogas reactor supplied with rye silage and straw during a stepwise temperature increase from 55 to 75 °C within the leach bed reactor (LBR), using TRFLP analyses. To identify the terminal restriction fragments that were obtained, bacterial and archaeal 16S rRNA gene libraries were constructed. Above 65 °C, the bacterial community structure changed from being Clostridiales-dominated toward being dominated by members of the Bacteroidales, Clostridiales, and Thermotogales orders. Simultaneously, several changes occurred, including a decrease in the total cell count, degradation rate, and biogas yield along with alterations in the intermediate production. A bioaugmentation with compost at 70 °C led to slight improvements in the reactor performance; these did not persist at 75 °C. However, the archaeal community within the downstream anaerobic filter reactor (AF), operated constantly at 55 °C, altered by the temperature increase in the LBR. At an LBR temperature of 55 °C, members of the Methanobacteriales order were prevalent in the AF, whereas at higher LBR temperatures Methanosarcinales prevailed. Altogether, the best performance of this two-phase reactor was achieved at an LBR temperature of below 65 °C, which indicates that this temperature range has a favorable effect on the microbial community responsible for the production of biogas.     

Trichloroethylene removal and bacterial variations in the up-flow anaerobic sludge blanket reactor in response to temperature shifts

Trichloroethylene (TCE) degradation and the variations of bacteria composition and structure in the up-flow anaerobic sludge blanket (UASB) reactor were investigated by increasing the operating temperature from 20 to 40 °C. The influent was supplemented with 36.5 mg/L of TCE. There was a rise in the chemical oxygen demand (COD) removal efficiency from 20 to 35 °C and a decline when temperature enhanced to 40 °C. It reached maximum at 35 °C. In addition, TCE removal efficiency increased with temperature varying from 20 to 35 °C, and it dropped dramatically to 78.38 % at 40 °C, which presumably because the genus of Dehalobacter, a kind of bacteria with the ability to dechlorinate TCE to the corresponding chlorinated products, was not detected at 40 °C according to sequencing results. The Illumina MiSeq platform was adopted to explore the bacteria composition and structure in response to temperature shifts. The results indicated that temperature impacted greatly on the dominance and presence of specific populations at different taxonomic levels. Importantly, the class Dehalococcoidia was detected from 25 to 40 °C, in which there were many well-known Dehalococcoides sp. strains that were capable of complete dechlorination of TCE to ethene. It also suggested the potential function of the dominant genera (non-dechlorinating bacteria and dechlorinating bacteria) in the reactor.     

A comparative study of immobilized amyloglucosidase in a packed bed reactor and a continuous feed stirred tank reactor

The catalytic activity of amyloglucosidase covalently attached to DEAE-cellulose was studied in a packed bed reactor and a continuous feed stirred tank reactor (CSTR) for the reaction maltose → glucose. At low flow rates mass-transfer limitations in the bed reactor lead to lower conversions for this reactor compared to the CSTR. Simple theoretical expressions for these reactors were compared with the experimental results. There are significant differences between the kinetic parameters and pH profile of the immobilized and free enzyme. The immobilized enzyme also showed greater stability at 50°C than did free amyloglucosidase. The temperature dependence of the reaction rate was the same for immobilized and free enzyme.     

Monitoring and control of enzymic sucrose hydrolysis using on-line biosensors

Summary Previously reported flow microcalorimeter devices for enzymic reaction heat measurement, enzyme thermistors, have here been extended with systems for on-line sample treatment. Glucose analysis was performed by intermittent flow injections of 50 l samples through such an enzyme thermistor device containing immobilized glucose oxidase and catalase. Sucroce analysis was performed by allowing diluted samples to continuously pass through an additional enzyme thermistor containing immobilized invertase. The reaction heats were recorded as temperature changes in the order of 10–50 m°C for concentrations of 0.05–0.30 M glucose or sucrose present in the original non-diluted samples.The performance of this system was investigated by its ability to follow concentration changes obtained from a gradient mixer. The system was applied to monitoring and controlling the hydrolysis of sucrose to glucose and fructose in a plug-flow reactor with immobilized invertase. The reactor was continuously fed by a flow of scurose of up to 0.3 M (100 g/l). Glucose and remaining sucrose were monitored in the effluent of the column. By using flow rate controlled feed pumps for sucrose and diluent the influent concentration of sucrose was varied while the overall flow rate remained constant.On-line control of the effluent concentration of lucose and sucrose was achieved by a proportional and integral regulator implemented on a microcomuter. Preset concentration of glucose in the effluent could be maintained over an extended period of time espite changes in the overall capacity of the invertase reactor. Long delay times in the sensor system and the enzyme column made it necessary to carefully tune the control parameters. Changes of set-point value and temperature disturbances were used to verify accuracy of controlling performance.     

Photochemical system for regenerating NADPH from NADP with use of immobilized chloroplasts

Spinach chloroplasts were immobilized in 2% agar gel. Crude ferredoxin and NADP–ferredoxin oxidoreductase isolated from spinach were used as electron carriers. The activity of the NADP reduction by immobilized chloroplasts increased with increasing ferredoxin concentration and the maximum activity was obtained at 8μM ferredoxin. The saturation of NADP reduction was observed at a light intensity of over 1000 lx. The optimum pH and temperature of NADP reduction were 8 and 25°C, respectively. The reduced NADP in a reaction medium increased linearly with increasing reaction time under illumination. NADP was continuously reduced for 2 hr with a hollow-fiber reactor containing immobilized chloroplasts. NADPH and NADP were separated with a hollow-fiber dialyzer from ferredoxin and NADP–ferredoxin oxidoreductase, which were reused. The conversion ratio of NADP to NADPH was from 40 to 80%.     

Chemo-enzymatic epoxidation of oleic acid and methyl oleate in solvent-free medium

Chemo-enzymatic epoxidation of oleic acid (OA) and its methyl ester has been performed using hydrogen peroxide and immobilized lipase from Candida antarctica (Novozym® 435). The purpose of the study was to characterize the reaction under solvent-free conditions. The reaction temperature had a significant impact on epoxidation of OA. At lower temperatures, the substrate conversion was hindered by the formation of solid epoxystearic acid product. Nearly 90% conversion of OA to the epoxide product was obtained after 6?h at 50°C. Longer reaction times at 40°C and above resulted in by-product formation and eventually lowered the product yield. In contrast, the reaction with methyl oleate (MO) was less influenced by temperature. Almost complete epoxidation was achieved at 40–60°C; the higher the temperature the shorter was the reaction time. The main epoxidation product obtained was epoxystearic acid methyl ester (EME), and the remaining was epoxystearic acid (EA) formed by the hydrolytic action of the lipase. Recycling of the lipase for epoxidation of MO at 50°C indicated that the immobilized enzyme was prone to activity loss.     

Hydrolysis of Galacto-oligosaccharides in Soymilk by κ-carrageenan-entrapped α-galactosidase from Aspergillus Oryzae

Summary α-Galactosidase was immobilized in κ-carrageenan. The optimum pH of the soluble enzyme and immobilized enzyme was 4.8. The optimum temperature of the soluble enzyme was 50 °C and that of the immobilized enzyme was increased to 53 °C. The immobilized enzyme was used in batch, repeated batch, and in the continuous mode to degrade the raffinose family sugars present in soymilk. Two hours incubation with free and immobilized α-galactosidase resulted in 88 and 75% reduction in raffinose family oligosaccharides in soymilk respectively. In the repeated batch, 61% reduction was obtained in the fourth cycle. A fluidized bed reactor was designed to treat soymilk continuously. The performance of immobilized α-galactosidase was also tested in a fluidized bed reactor at different flow rates and 92% reduction of raffinose family oligosaccharides in soymilk was obtained at 25 ml h⁻¹ flow rate. The study revealed that immobilized α-galactosidase in continuous mode is efficient in reducing the oligosaccharides present in the soymilk.     

Decreased D2-40 immunoreactivity in stored paraffin sections and methods for preserving it

D2-40, a monoclonal antibody against podoplanin, is a selective marker of lymphatic endothelium and is widely used for research on and diagnosis of pathology of lymphatic vessels. We examined the relation between the duration of tissue section storage and changes in immunostaining by D2-40 antibody; we evaluated also the effects of preservation methods on changes in immunostaining during storage. Staining by D2-40 was attenuated by long-term preservation of scalp skin and lymph node sections at room temperature. The attenuation of D2-40 staining in stored sections was improved by preservation at low temperature, i.e., 4° or ? 30° C. We investigated also the immunostaining of preserved tissue sections using NZ-1 and Lyve-1, which are antibodies against lymphatic endothelium markers. Staining by NZ-1 or Lyve-1 antibody was detected clearly in sections that had been stored for 16 weeks. Our study suggests that either long-term storage of D2-40 immunostained tissue sections should be avoided or the section should be preserved at low temperature.     

Preparative-scale kinetic resolution of racemic styrene oxide by immobilized epoxide hydrolase

Epoxide hydrolase from Aspergillus niger was immobilized onto the modified Eupergit C 250 L through a Schiff base formation. Eupergit C 250 L was treated with ethylenediamine to introduce primary amine groups which were subsequently activated with glutaraldehyde. The amount of introduced primary amine groups was 220 μmol/g of the support after ethylenediamine treatment, and 90% of these groups were activated with glutaraldehyde. Maximum immobilization of 80% was obtained with modified Eupergit C 250 L under the optimized conditions. The optimum pH was 7.0 for the free epoxide hydrolase and 6.5 for the immobilized epoxide hydrolase. The optimum temperature for both free and immobilized epoxide hydrolase was 40 °C. The free epoxide hydrolase retained 52 and 33% of its maximum activity at 40 and 60 °C, respectively after 24 h preincubation time whereas the retained activities of immobilized epoxide hydrolase at the same conditions were 90 and 75%, respectively. Immobilized epoxide hydrolase showed about 2.5-fold higher enantioselectivity than that of free epoxide hydrolase. A preparative-scale (120 g/L) kinetic resolution of racemic styrene oxide using immobilized preparation was performed in a batch reactor and (S)-styrene oxide and (R)-1-phenyl-1,2-ethanediol were both obtained with about 50% yield and 99% enantiomeric excess. The immobilized epoxide hydrolase was retained 90% of its initial activity after 5 reuses.     

Immobilization of cyclodextrin glucanotransferase on amberlite IRA-900 for biosynthesis of transglycosylated xylitol

Cyclodextrin glucanotransferase (CGTase) fromThermoanaerobacter sp. was adsorbed on the ion exchange resin Amberlite IRA-900. The optimum conditions for the immobilization of the CGTase were pH 6.0 and 600 U CGTase/g resin, and the maximum yield of immobilization was around 63% on the basis of the amount ratio of the adsorbed enzyme to the initial amount in the solution. Immobilization of CGTase shifted the optimum temperature for the enzyme to produce transglycosylated xylitol from 70°C to 90°C and improved the thermal stability of immobilized CGTase, especially after the addition of soluble starch and calcium ions. Transglycosylated xylitol was continuously produced using immobilized CGTase in the column type packed bed reactor, and the operating conditions for maximum yield were 10% (w/v) dextrin (13 of the dextrose equivalent) as the glycosyl donor, 10% (w/v) xylitol as the glycosyl acceptor, 20 mL/h of medium flow rate, and 60°C. The maximum yield of transglycosylated xylitol and productivity were 25% and 7.82 g·L⁻¹·h⁻¹, respectively. The half-life of the immobilized CGTase in a column type packed bed reactor was longer than 30 days.     

Galactose oxidase: applications of the covalently immobilized enzyme in a packed bed configuration.

Galactose oxidase (E.C. 1.1.3.9) was covalently immobilized to chemically modified porous silica particles by reaction of the native enzyme with pendant benzoyl azide groups on the carrier. The enzyme loading on the carrier was 100-150 units per milliliter. The immobilized enzyme was incorporated into a hardware assembly suitable for the determination of galactose or lactose concentrations in complex biological fluids. The prototype instrument as described is suitable for continuous, on-line monitoring or discrete sample analysis. Reaction conditions can be readily provided which maintain global first order kinetics within the reactor and strict linearity of the procedure over a wide range of sample concentrations. Auto-inactivation of the immobilized enzyme can be prevented by K3Fe(CN)6 and long-term reactor stability can be achieved by the periodic application of the reagent to the enzyme reactor in situ.