Invertase reversibly immobilized onto polyethylenimine-grafted poly(GMA–MMA) beads for sucrose hydrolysis

The epoxy group containing poly(glycidyl methacrylate-co-methylmethacrylate) poly(GMA–MMA) beads were prepared by suspension polymerisation and the beads surface were grafted with polyethylenimine (PEI). The PEI-grafted beads were then used for invertase immobilization via adsorption. The immobilization of enzyme onto the poly(GMA–MMA)–PEI beads from aqueous solutions containing different amounts of invertase at different pH was investigated in a batch system. The maximum invertase immobilization capacity of the poly(GMA–MMA)–PEI beads was about 52 mg/g. It was shown that the relative activity of immobilized invertase was higher then that of the free enzyme over broader pH and temperature ranges. The Michaelis constant (K_m) and the maximum rate of reaction (V_max) were calculated from the Lineweaver–Burk plot. The K_m and V_max values of the immobilized invertase were larger than those of the free enzyme. The immobilized enzyme had a long-storage stability (only 6% activity decrease in 2 months) when the immobilized enzyme preparation was dried and stored at 4 °C while under wet condition 43% activity decrease was observed in the same period. After inactivation of enzyme, the poly(GMA–MMA)–PEI beads can be easily regenerated and reloaded with the enzyme for repeated use.     

Effect of impeller type and stirring frequency on the behavior of an AnSBBR in the treatment of low-strength wastewater

The influence of impeller type and stirring frequency on the performance of a mechanically stirred anaerobic sequencing batch reactor containing immobilized biomass on an inert support (AnSBBR - Anaerobic Sequencing Batch Biofilm Reactor) was evaluated. The biomass was immobilized on polyurethane foam cubes placed in a stainless-steel basket inside a glass cylinder. Each 8-h batch run consisted of three stages: feed (10 min), reaction (460 min) and discharge (10 min) at 30 °C. Experiments were performed with four impeller types, i.e., helical, flat-blade, inclined-blade and curved-blade turbines, at stirring frequencies ranging from 100 to 1100 rpm. Synthetic wastewater was used in all experiments with an organic-matter concentration of 530 ± 37 mg/L measured as chemical oxygen demand (COD). The reactor achieved an organic-matter removal efficiency of around 87% under all investigated conditions. Analysis of the four impeller types and the investigated stirring frequencies showed that mass transfer in the liquid phase was affected not only by the applied stirring frequency but also by the agitation mode imposed by each impeller type. The best reactor performance at all stirring frequencies was obtained when agitation was provided by the flat-blade turbine impeller.     

A study on xylitol production from sugarcane bagasse hemicellulosic hydrolysate by Ca-alginate entrapped cells in a stirred tank reactor

Candida guilliermondii FTI 20037 cells were entrapped in Ca-alginate beads and used for xylitol production from sugarcane bagasse hemicellulosic hydrolysate in a stirred tank reactor (STR). Screening design and response surface methodologies were used to determine adequate cultivation conditions for this fermentation system. Quadratic models were fitted to the experimental data by regression analysis, considering the yield (Y_P/S) and the productivity (Q_P) of the xylose-to-xylitol bioconversion as dependent variables. Using a five-fold concentrated hydrolysate, air flowrate of 1.30 l/min, agitation speed of 300 rpm, initial cell concentration of 1.4 g/l and value 6.0 for the initial pH of the fermentation medium resulted in a xylitol production of 47.5 g/l after 120 h of fermentation, corresponding to a Y_P/S of 0.81 g/g and to a Q_P of 0.40 g/l h.     

木瓜蛋白酶的固定化及其性质研究

在海藻酸钠-壳聚糖固定化木瓜蛋白酶(immobilized papin on sodium alginate-chitosan,IPSAC)的实验中,当给酶量为1 mg g1载体时,酶活性为39.2 U,酶活力回收为21.1%.在尼龙布固定化木瓜蛋白酶(knmobilized papain onnylon,IPN)的实验中,当每块尼龙布(3 cm×3 cm)给酶量为1 mg时,酶活性为35.6 U,酶活力回收为19.2%.木瓜蛋白酶(papain,PA)、IPSAC、IPN的最适pH分别为7.2、7.2和6.8.PA及IPSAC在70℃以下活性稳定;IPN在50℃以下活性稳定.IPSAC与IPN半衰期分别为59 d和66 d.     

海藻酸铝固定化酵母生产高浓度酒精的研究

以海藻酸铝凝胶代替海藻酸钙,可使固定化酵母使用寿命显著延长,其海藻酸铝凝胶耐磷酸盐能力比海藻酸钙凝胶提高六倍以上。用海藻酸铝固定化增殖酵母进行分批发酵酒精,成熟醪中酒精含量由３．５％－９．０％（Ｖ／Ｖ）提高到１１．０％（Ｖ／Ｖ）左右。在１．１Ｌ的两个多层生物反应器中,装入海藻酸铝固定化增殖酵母,采用逐步提高糖浓度方法,进行连续发酵,成熟醪接收器中酒精浓度平均为１０．３％（Ｖ／Ｖ）,总糖利用率为９２．４％。     

Water flow induced transport of Pseudomonas fluorescens cells through soil columns as affected by inoculant treatment

Abstract Water flow induced transport of Pseudomonas fluorescens cells through soil columns was measured as affected by the inoculant treatment. Bacterial cells were introduced into the topsoil of columns, either encapsulated in alginate beads of different types or mixed with bentonite clay in concentrations ranging from 0.5 to 5.0% (w/v). Survival of bacterial cells was improved with the use of alginate or bentonite. Transport, as determined by destructive sampling of the columns, was reduced with the use of alginate encapsulation. Drying of the beads had no influence on transport. The presence of bentonite in the topsoil, either pre-mixed through the soil, or applied as a slurry together with the bacteria, also reduced transport, except when 0.5% was pre-mixed through the soil. P. fluorescens cells encapsulated in alginate beads prepared with water and supplemented with skim milk powder and bentonite showed the best survival during the time of the experiment and the most reduced transport compared to the control. Therefore, cells encapsulated in this way are suitable, due to their optimal survival and reduced spread, for use in a field experiment with genetically manipulated bacteria.     

Alginate/carbon composite beads for laccase and glucose oxidase encapsulation: application in biofuel cell technology

Alginate–carbon beads were prepared in order to develop a biocompatible matrix for laccase and glucose oxidase immobilization for application in biofuel cell technology. The enzyme loading capacity was high (91%) in pure alginate beads for glucose oxidase. For laccase, the loading capacity was enhanced from 75% to 83% by introducing carbon. Desorption out of the matrix was controlled by the enzymes’ diffusion and reached a plateau after 40 h for laccase and 70 h for glucose oxidase. Two-thirds of both enzymes was irreversibly retained inside the alginate beads. This proportion increased to 80% for laccase in combined alginate/carbon beads. Half-life of the adsorbed enzyme was enhanced to 74 days for laccase in carbon/alginate beads and 45 days for glucose oxidase in pure alginate as compared to 38 days and 23 days for free enzymes, respectively.     

Development of porous collagen beads for chondrocyte culture

A method for the preparation of bioresorbable collagen beads with an open porous structure is presented. These beads were prepared from collagen-alginate composite beads by removal of the alginate component. These collagen beads were suitable for rapid proliferation of chondrocytes in a dynamic, spinner culture system. Histology and immuno-histology showed that biochemical markers of chondrocytes are present during this cell proliferation, indicating that there was control of phenotype and that cell de-differentiation had not occurred. Unlike other 3-D scaffolds that have been used, the cells were amplified from very low cell densities and were able to proliferate freely without loss of phenotype. Tracy A. Tebb, Shiao-Wen Tsai, Veronica Glattauer and Jacinta F. White have made equal contributions to this study.     

固定化细胞技术发酵玉米醪生产酒精的研究

本文报导了固定化酵母(S.cerevisiae 9064)批次发酵玉米醪生产酒精的研究。采用自制“多层分隔式生物反应器”在最适发酵条件下,成熟醪的终酒精浓度达10％(V/V)左右,发酵时间可由传统发酵的72h缩短到22h左右,酒精生产力提高了3倍。在吨级反应器连续运转试验时,固定化增殖酵母细胞活性稳定性可持续半年以上,PVA—凝胶粒的机械强度和弹性等以及醪液中酒精含量基本保持不变。     

Application of immobilized enzyme reactor in on-line high performance liquid chromatography: a review

This review summarizes all the research efforts in the last decade (1994-2003) that have been spent to the various application of immobilized enzyme reactor (IMER) in on-line high performance liquid chromatography (HPLC). All immobilization procedures including supports, kind of assembly into chromatographic system and methods are described. The effect of immobilization on enzymatic properties and stability of biocatalysts is considered. A brief survey of the main applications of IMER both as pre-column, post-column or column in the chemical, pharmaceutical, clinical and commodities fields is also reported.     

Enhanced stability of the cloned Bacillus subtilis alkaline protease gene in alginate-immobilized B. subtilis cells

Expression and stability of the cloned Bacillus subtilis alkaline protease (aprE)gene was monitored throughout the growth of free and alginate-immobilized B. subtilis cells. The time as well as the level of expression of the aprE gene in alginate-immobilized cells was found to be close to that of free cells. The multicopy plasmid that carries the aprE gene was stably maintained in alginate-immobilized cells. Plasmid stability was greatly enhanced, it reached 83% and 8% after ten growth cycles for alginate-immobilized and free cells in the absence of stress, respectively. Data presented demonstrate that immobilization of B. subtilis recombinant cells would partially solve the problem of plasmid instability in B. subtilis.     

Advances in aerobic granule formation and granule stability in the course of storage and reactor operation

Aerobic granulation is drawing increasing global interest in a quest for an efficient and innovative technology in wastewater treatment. Developed less than two decades ago, extensive research work on aerobic granulation has been reported. The instability of the granule, which is one of the main problems that hinder practical application of aerobic granulation technology, is still to be resolved. This paper presents a review of the literature in aerobic granulation focusing on factors that influence granule formation, granule development and their stability in the context of sludge granulation. The review attempts to shed light on the potential of developing granules with adequate structural stability for practical applications. The possibilities and perspective of using stored granule as inoculums for rapid startup, and as microbial supplement to enhance treatment of bioreactor systems are also discussed.     

Extracellular serine proteases from Stenotrophomonas maltophilia: Screening, isolation and heterologous expression in E. coli

A large strain collection comprising antagonistic bacteria was screened for novel detergent proteases. Several strains displayed protease activity on agar plates containing skim milk but were inactive in liquid media. Encapsulation of cells in alginate beads induced protease production. Stenotrophomonas maltophilia emerged as best performer under washing conditions. For identification of wash-active proteases, four extracellular serine proteases called StmPr1, StmPr2, StmPr3 and StmPr4 were cloned. StmPr2 and StmPr4 were sufficiently overexpressed in E. coli. Expression of StmPr1 and StmPr3 resulted in unprocessed, insoluble protein. Truncation of most of the C-terminal domain which has been identified by enzyme modeling succeeded in expression of soluble, active StmPr1 but failed in case of StmPr3.From laundry application tests StmPr2 turned out to be a highly wash-active protease at 45 °C. Specific activity of StmPr2 determined with suc-l-Ala-l-Ala-l-Pro-l-Phe-p-nitroanilide as the substrate was 17 ± 2 U/mg. In addition we determined the kinetic parameters and cleavage preferences of protease StmPr2.     

固定化地衣芽孢杆菌R08吸附Pd^2＋的研究

比较了四种固定菌体的方法。结果以聚乙烯醇一海藻酸钠包埋地衣芽孢杆菌(Bacillus licheniformis)R08菌体,制成直径约2mm的颗粒,然后用磷酸缓冲液处理,5％戊二醛溶液交联,制得的固定化R08菌体(PIRB)对Pd^2 的吸附率最高。PIRB吸附Pd^2 的最适pH值为3．5。吸附作用是一种迅速的过程。在5℃—60℃范围内,吸附作用不受温度的影响。溶液中的PIRB含量和Pd^2 起始浓度影响吸附作用,在0．5gPIRB/L、200mg Pd^2 /L、pH3．5和30℃条件下,吸附60min,吸附量达94．7mg／g干重。吸附过程符合Freundlich和Langmuir吸附等温式。Au^3 等离子抑制PIRB对Pd^2 的吸附。用1mol/L HCl洗脱PIRB所吸附的Pd^2 ,解吸率为83．6％。在填充床反应器中,在流速2mL/min、100mgPd^2 /L、2．5g PIRB(干重)、pH3．5和30℃条件下,反复吸附—解吸附,最初5批的饱和吸附量、吸附率和解吸率分别平均为44．3mgPd^2 /g干重、89．4％和82．5％。在与上述相同的条件下,PIRB对废钯催化剂处理液中的Pd^2 的吸附量为41．3mg／g,吸附率为88．6％。     

Immobilization and characterization of lipase from an indigenous Bacillus aryabhattai SE3-PB isolated from lipid-rich wastewater

Abstract

Extracellular lipase from an indigenous Bacillus aryabhattai SE3-PB was immobilized in alginate beads by entrapment method. After optimization of immobilization conditions, maximum immobilization efficiencies of 77%?±?1.53% and 75.99%?±?3.49% were recorded at optimum concentrations of 2% (w/v) sodium alginate and 0.2?M calcium chloride, respectively, for the entrapped enzyme. Biochemical properties of both free and immobilized lipase revealed no change in the optimum temperature and pH of both enzyme preparations, with maximum activity attained at 60?°C and 9.5, respectively. In comparison to free lipase, the immobilized enzyme exhibited improved stability over the studied pH range (8.5–9.5) and temperature (55–65?°C) when incubated for 3?h. Furthermore, the immobilized lipase showed enhanced enzyme-substrate affinity and higher catalytic efficiency when compared to soluble enzyme. The entrapped enzyme was also found to be more stable, retaining 61.51% and 49.44% of its original activity after being stored for 30 days at 4?°C and 25?°C, respectively. In addition, the insolubilized enzyme exhibited good reusability with 18.46% relative activity after being repeatedly used for six times. These findings suggest the efficient and sustainable use of the developed immobilized lipase for various biotechnological applications.     

Inoculant made of encapsulated Frankia: assessment of Frankia growth within alginate beads

The growth of Frankia cells within alginate beads was inhibited when the amount encapsulated exceeded 0.5 to 2.5 g protein/ml of beads. Frankia growth was observed not only in the beads incubated in nutrient media (with of without combined N), but also in those incubated in air provided they retained enough nutrients. The results allow some recommendations to be made for the preparation of Frankia inoculants.L. Frioni is with the Facultad de Agronomia, Av. Garzon 780, Montevideo, Uruguay. C. Le Roux, H.G. Diem and Y.R. Dommergues are with ORSTOM/CIRAD-Forêts BSFT Laboratory, 45 bis Av. de la Belle Gabrielle, 94736 Nogent-sur-Marne Cedex, France     

Effect of a small molecule on diffusion and swelling properties of selected polysaccharide gel beads

The effect of a small molecule (e.g., sodium fluorescein, SF) on the swelling properties of and diffusion from calcium polysaccharide (alginate or pectin) gel beads was investigated. The gel beads were prepared by ionotropic gelation, soaked in different concentrations of SF solution, and then dried. The swelling behavior and release of SF from the dried beads were investigated. After soaking in SF, the beads swelled to sizes that depended on the initial concentration of SF. However, the size of the dried beads was independent of the SF concentration. The swelling of the beads occurred quite rapidly and reached a maximum within 2 h. Although most beads swelled to a size which was less than their original size of wet beads, some of them swelled much more than their original wet size. Higher concentration of SF and lower concentration of sodium alginate provided a greater increase in weight. The release profile of SF from dried gel beads in water consists of a burst or a very rapid release phase during the first 60 min followed by a much slower release phase. The similarity of the relative weight increase and release profiles of SF, suggests that swelling might contribute to release of SF, particularly during the burst phase.     

Lipase-catalyzed selective synthesis of monolauroyl maltose using continuous stirred tank reactor

Monolauroyl maltose was synthesized by an immobilized lipase that catalyzed condensation of maltose and lauric acid in acetone using a batch reactor or a continuous stirred tank reactor. Mono- and di-lauroyl maltoses were identified by FT-IR, ¹H NMR, ¹³C NMR and MS. Monolauroyl maltose was selectively synthesized in a continuous stirred tank reactor and no diester was detected. The highest concentration of monolauroyl maltose at 28 mmol/l was obtained in 250 ml acetone when maltose was added at 4 g/d and the molar ratio of lauric acid to maltose was fixed at 4:1 at a flow rate of 0.15 ml/min for both influx and effluent without supplement of fresh molecular sieve.     

Increased neem extract content enhances drying survival of co-encapsulated Saccharomyces cerevisiae and decreases relative release of azadirachtin

Plant extracts from the neem tree can be applied as a bioinsecticidal compound in ‘attract-and-kill’ co-formulations to control soil-dwelling insect pests. Since insects are attracted to the bioinsecticide, the attract-and-kill approach benefits from a reduced dose of insecticide needed. Here, we demonstrate that a powdery neem seed kernel extract in combination with corn starch significantly enhances the drying survival of co-encapsulated CO₂-releasing Saccharomyces cerevisiae. Additionally, an increased content of the neem extract was shown to slow down the relative release of azadirachtin A. Both findings are of high relevance for the development of formulations in the field of biological pest control.