Studies on chromium(VI) adsorption-desorption using immobilized fungal biomass

The aim of this study was to investigate the Cr(VI) biosorption potential of immobilized Rhizopus nigricans and to screen a variety of non-toxic desorbing agents, in order to find out possible application in multiple sorption-desorption cycles. The biomass was immobilized by various mechanisms and evaluated for removal of Cr(VI) from aqueous solution, mechanical stability to desorbents, and reuse in successive cycles. The finely powdered biomass, entrapped in five different polymeric matrices viz. calcium alginate, polyvinyl alcohol (PVA), polyacrylamide, polyisoprene, and polysulfone was compared for biosorption efficiency and stability to desorbents. Physical immobilization to polyurethane foam and coir fiber was less efficient than polymer entrapment methods. Of the different combinations (%, w/v) of biomass dose compared for each matrix, 8% (calcium alginate), 6% (polyacrylamide and PVA), 12% (polyisoprene), and 10% (polysulfone) were found to be the optimum. The Cr sorption capacity (mg Cr/g sorbent) of all immobilized biomass was lesser than the native, powdered biomass. The Cr sorption capacity decreased in the order of free biomass (119.2) > polysulfone entrapped (101.5) > polyisoprene immobilized (98.76) > PVA immobilized (96.69) > calcium alginate entrapped (84.29) > polyacrylamide (45.56), at 500 mg/l concentration of Cr(VI). The degree of mechanical stability and chemical resistance of the immobilized systems were in the order of polysulfone > polyisoprene > PVA > polyacrylamide > calcium alginate. The bound Cr(VI) could be eluted successfully using 0.01 N NaOH, NaHCO3, and Na2CO3. The adsorption data for the native and the immobilized biomass was evaluated by the Freundlich isotherm model. The successive sorption-desorption studies employing polysulfone entrapped biomass indicated that the biomass beads could be regenerated and reused in more than 25 cycles and the regeneration efficiency was 75-78%.     

Biosorption of gold by immobilized fungal biomass

The characteristics of polyvinyl alcohol (PVA) and calcium alginate as immobilization matrices were examined and compared for the uptake of gold by a fungal biomass. PVA-immobilized biomass showed superior mechanical strength and chemical stability. In addition, PVA beads were also stable under a wider range of pH (1-13). The lower mass transfer resistance in PVA beads was evident from kinetic studies which showed a significantly shorter period of time for the immobilized PVA beads to achieve 80% gold removal as compared with immobilized alginate beads. Calculated rate constants and maximum rates for the uptake of gold by both immobilized PVA and immobilized alginate biosorbent revealed a much more rapid uptake phenomenon by the former. BET analyses also indicated a larger surface area and larger pore size distribution in PVA beads, further indicating a lower resistance to mass transfer. Gold biosorption in the immobilized PVA bead could be modeled by both the Langmuir and Freundlich adsorption isotherms.     

Imaging and thermal studies of wheat gluten/poly(vinyl alcohol) and wheat gluten/thiolated poly(vinyl alcohol) blends

The morphology of wheat protein (WG) blends with polyvinyl alcohol (PVA) and respectively with thiolated polyvinyl alcohol (TPVA) was investigated by atomic force (AFM) and transmission electron microscopy (TEM) as well as by modulated dynamic scanning calorimetry (MDSC). Thiolated additives based on PVA and other substrates were previously presented as effective means of improving the strength and toughness of compression molded native WG bars via disulfide-sulfhydryl exchange reactions. Consistent with our earlier results, AFM and TEM imaging clearly indicate that the addition of just a few mole percent of thiol to PVA was sufficient to dramatically change its compatibility with wheat protein. Thus, TPVA is much more compatible with WG and phase separates into much smaller domains than in the case of PVA, although there are still two phases in the blend: one WG-rich phase and another TPVA-rich phase. The WG/TPVA blend has phase domains ranging in size from 0.01 to 0.1 microm, which are roughly 10 times smaller than those of the WG/PVA blend. MDSC further illustrates the compatibilization of the protein with TPVA via the dependence of the transition temperatures on composition.     

Immobilization of maltogenase onto polyurethane microparticles from poly(vinyl alcohol) and hexamethylene diisocyanate

A series of porous polyurethane (PU) microparticles from poly(vinyl alcohol) (PVA) and hexamethylene diisocyanate (HMDI) using different ratios of components were obtained by one step method. Molar compositions of PU microparticles were estimated by determination of nitrogen, isocyanate and hydroxyl groups. PU carriers which were synthesized using optimal initial molar ratios of PVA and HMDI were applied for immobilization of maltogenase (MG) from Bacillus stearothermophilus. Immobilized enzyme exhibited higher catalytic activity and enhanced temperature stability in comparison with the native MG. Maximal loading 7.78 mg/g wet carrier was reached when PU microparticles with initial molar ratio of PVA and HMDI = 1:3 was used as a carrier for immobilization. The high efficiency of immobilization (EI) was obtained using PU microparticles when initial molar ratio of HMDI and PVA was 1:1–1:10. High stability of MG immobilized onto PU microparticles during storage was demonstrated. Immobilized starch hydrolyzing enzyme was successfully tested in batch and column type reactors for hydrolysis of potato starch. MG immobilized onto PU enables easy separation from the reaction medium and reuse of the immobilized preparation over seven reaction cycles in bath operation and at least three cycles in column type reactor.     

Adsorptive immobilization of a Pseudomonas strain on solid carriers for augmented decolourization in a chemostat bioreactor

Pseudomonas GM3, a highly efficient strain in cleavage of azo bonds of synthetic dyes under anoxic conditions, was immobilized via adsorption on two types of carriers, porous glass beads and solid PVA particles. The cells were cultivated in a nutrient medium, adsorbed on sterile carriers, stabilized as biofilms in repeated batch cultures, and introduced into a chemostat activated sludge reactor for augmented decolourization. The microbial cells were quickly adsorbed and fixed on the PVA surface, compared to a slow and linear immobilization on the glass surface. The porous structure of glass beads provided shelter for the embedded cells, giving a high biomass loading or thick biofilm (13.3 mg VS ml-1 carrier) in comparison with PVA particles (4.8 mg VS ml-1 carrier), but the mass transfer of substrate in the biofilm became a significant limiting factorin the thicker biofilms (effectiveness factor eta = 0.31). The microbial decolourization rate per volume of carriers was 0.15 and 0.17 mg dye ml-1 of glass beads and PVA particles, respectively. In augmented decomposition of a recalcitrant azo dye (60 mg l-1), the immobilized Pseudomonas cells in porous glass beads gave a stable decolourization efficiency (80-81%), but cells fixed on solid PVA particles showed an initial high colour removal of 90% which then declined to a stable removal efficiency of 81%. In both cases, the colour removal efficiency of the chemostat bioreactor was increased from < 10% by an activated sludge to approximately 80% by the augmented system.     

Wheat gluten-thiolated poly(vinyl alcohol) blends with improved mechanical properties

A multifunctional macromolecular thiol (TPVA) obtained by esterification of poly(vinyl alcohol) (PVA) with 3-mercaptopropionic acid was characterized by a combination of NMR, IR, transmission electron microscopy (TEM), and differential scanning calorimetry (DSC), and was used as a wheat gluten (WG) reactive modifier. The effect of TPVA molecular weight (M(w) = 2000, 9500, 50 000, and 205 000) and blend composition (5, 20, and 40% w/w TPVA/WG) on the mechanical properties of compression-molded bars indicates that TPVA/WG blends increase the fracture strength by up to 76%, the elongation by 80%, and the modulus by 25% above WG. In contrast, typical WG additives such as glycerol and sorbitol improve flexibility but decrease modulus and strength. Preliminary investigations of suspension rheology, water uptake, molecular weight distribution and electron microscopy of TPVA/WG and PVA/WG blends illustrate the different protein interactions with PVA and TPVA. Further work is underway to determine whether TPVA and WG form protein conjugates or microphase-separated morphologies.     

Ligninolytic enzyme production by Phanerochaete chrysosporium immobilized on different carriers

In this study, several different carriers were employed in a Phanerochaete chrysosporium BVH-F-1767 cell immobilization study. Polystyrene foam was shown to be the optimum carrier material from organism biomass measurements and maximum MnP production (915.62 U L(-1)). The maximum MnP activity of polystyrene foam system was achieved 2-5 days sooner than with the other carrier systems studied. It was thus clear that the polystyrene foam approach shortened the culture cycle. Analysis of the carrier mechanisms employed in this study revealed that polystyrene foam had larger internal spaces and a greater surface area, and thus the potential to enhance the transfer efficiency of oxygen and nutrients to the fungus and accelerate its growth. The mycelia of the fungus were able to associate closely with the unique internal pore structure of the polystyrene foam, providing a more quiescent microenvironment and helping to maintain the stability of the cultivation system.     

Immobilization of porcine pancreatic lipase on glycidyl methacrylate grafted poly vinyl alcohol

Graft copolymerization of glycidyl methacrylate (GMA) on to polyvinyl alcohol (PVA) using benzophenone (BP) as initiator was carried out. Grafted PVA was used as carrier for pancreatic lipase immobilization. The effects of GMA and BP concentrations as well as grafting reaction times on grafting yields and activities of the immobilized lipase were determined. The influence of enzyme concentrations was also studied. The optimal conditions for the grafting reaction were: 1 h at 15 mM BP and 2.3 M GMA, the optimum enzyme concentration for immobilization was 1 mg/ml. After optimization of the immobilization process a physical and chemical characterization of the immobilized enzyme was performed. Furthermore, the thermal, pH, storage and operational stability of the immobilized enzyme in comparison to the free form was tested.     

Improvement of Process Stability of Microbiological Quinoline Degradation in a Three‐Phase Fluidized Bed Reactor

The biological degradation of quinoline by suspended and immobilized Comamonas acidovorans was studied under continuous and discontinuous operating conditions in a three‐phase fluidized bed reactor. C. acidovorans degrades quinoline into biomass and carbon dioxide. Quinoline and the intermediates of its metabolic pathway are found only by quinoline shockloads. The continuous degradation of quinoline by suspended biomass was only possible, if the dilution rate was less than the growth rate (μ_max =0.42 h^–1) and the concentration of a shockload was less than 1 kg/m³. A concentration greater than 1 kg/m³ led to an irreversible damage of the cells. Hence, two different carrier materials were used for immobilization by attachment, to increase the stability of the process. Using immobilization of biomass on carriers decouples the hydrodynamic retention time and the growth rate of the microorganisms. A comparison of the carrier material showed no differences with respect of activity and stability of the biofilm. The process stability of a three‐phase fluidized bed reactor was increased by immobilized biomass. The degradation of toxic shockloads was only possible with immobilized biomass. A dynamic model has been developed to describe the concentration profile of quinoline, 2‐hydroxyquinoline as metabolite and the suspended biomass. A comparison of the measured and calculated values showed good agreement.     

Ethanol production by Kluyveromyces lactis immobilized cells in copolymer carriers produced by radiation polymerization

The conditions for batch and continuous production of ethanol, using immobilized growing yeast cells of Kluyveromyces lactis, have been optimized. Yeast cells have been immobilized in hydrogel copolymer carriers composed of polyvinyl alcohol (PVA) with various hydrophilic monomers, using radiation copolymerization technique. Yeast cells were immobilized through adhesion and multiplication of yeast cells themselves. The ethanol production of immobilized growing yeast cells with these hydrogel carriers was related to the monomer composition of the copolymers and the optimum monomer composition was hydroxyethyl methacrylate (HEMA). In this case by using batch fermentation, the superior ethanol production was 32.9 g L(-1) which was about 4 times higher than that of cells in free system. The relation between the activity of immobilized yeast cells and the water content of the copolymer carriers was also discussed. Immobilized growing yeast cells in PVA: HEMA (7%: 10%, w/w) hydrogel copolymer carrier, were used in a packed-bed column reactor for the continuous production of ethanol from lactose at different levels of concentrations (50, 100 and 150) g L(-1). For all lactose feed concentrations, an increase in dilution rates from 0.1 h(-1) to 0.3 h(-1) lowered ethanol concentration in fermented broth, but the volumetric ethanol productivity and volumetric lactose uptake rate were improved. The fermentation efficiency was lowered with the increase in dilution rate and also at higher lactose concentration in feed medium and a maximum of 70.2% was obtained at the lowest lactose concentration 50 g L(-1).     

Immobilization of glucoamylase on porous silicas

The influence of the pore structure of silica carriers (macroporous silica gels, silochromes and porous glasses) on the catalytic activity of immobilized glucoamylase (exo 1,4-α-d-glucosidase, 1,4-α-d-glucan glucohydrolase EC 3.2.1.3) has been studied. The dependence of the immobilized glucoamylase activity, in units g^?1, on the carrier pore diameter was found to pass through a maximum within a range 70–100 nm. Macroporous silica gels can be used with success as carriers for glucoamylase immobilization instead of porous glasses and silochromes.     

Performance of three phase fluidized bed reactor for quinoline degradation on various supports at steady state and dynamic conditions

Quinoline degradation by Comamonas acidovorans was investigated in a three phase fluidized bed reactor at dilution rates below and above the critical value (mu(max) = 0.42 h(-1)). Quinoline was used as the sole source of carbon, nitrogen, and energy. Two attachment carriers, polyurethane foam (Bayvitec(R)) and modified cellulose (Aquacel(R)), and a gel entrapment carrier (polyvinyl alcohol) were studied and compared with regard to their effectiveness to immobilize cells. Attachment and biofilm formation was best at higher dilution rates, regardless of carrier type used. Except for the maximum biomass concentration on the carrier, Y(V) (biomass per volume of solid particles), there was no significant difference in reactor performance between the investigated carriers under stationary conditions. The highest value for Y(V) was found for the gel entrapment carrier (Y(V) = 35 g L(-1)). In a long-term run (66 days), the gel entrapment carrier established a permanent biofilm on the surface of the gel beads after 900 h of cultivation time. Complete quinoline mineralization was achieved at a dilution rate of 2.0 h(-1), which is 4.7 times higher than the critical dilution rate. Identical substrate overloads were applied to the gel entrapment and the cellulose carrier by a step increase of the quinoline feed concentration at a dilution rate of 0.8 h(-1) (D approximately 2mu(max)). The cells survived the overload, but the accumulation of quinoline and quinoline degradation products and the degradation efficiency were different for the two systems during the overload, showing the influence of the carrier type on the dynamic performance and stability of the process. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 295-303, 1997.     

聚乙烯醇-明胶复合膜固定化重组大肠杆菌NAD激酶的研究

为提高烟酰胺腺嘌呤二核苷酸(NAD)激酶的稳定性,采用复合膜对NAD激酶进行固定化研究。选用聚乙烯醇(PVA)、聚乳酸(PLA)、海藻酸钠(SA)和明胶(GEL)膜材料固定化NAD激酶。通过单因素实验确定最佳固定化条件为:PVA∶GEL为4∶1,加酶量为0.6 mL,固定化时间为6h,固定化温度为35℃,此时酶活力回收率达到最高值84%。固定化酶酶学性质分析结果表明,与游离酶进行比较,固定化后NAD激酶的最适温度由50℃提高至55℃,最适pH由8.0降至7.0,NAD激酶的热稳定性和pH稳定性均得到显著提高,但固定化酶的亲和力降低。固定化NAD激酶重复利用6次后,酶活性依然可维持初始酶活性的75%以上,表明聚乙烯醇-明胶复合膜固定化酶具有良好的操作稳定性。     

Adsorptive Immobilization of a Pseudomonas Strain on Solid Carriers for Augmented Decolourization in a Chemostat Bioreactor

Pseudomonas GM3, a highly efficient strain in cleavage of azo bonds of synthetic dyes under anoxic conditions, was immobilized via adsorption on two types of carriers, porous glass beads and solid PVA particles. The cells were cultivated in a nutrient medium, adsorbed on sterile carriers, stabilized as biofilms in repeated batch cultures, and introduced into a chemostat activated sludge reactor for augmented decolourization. The microbial cells were quickly adsorbed and fixed on the PVA surface, compared to a slow and linear immobilization on the glass surface. The porous structure of glass beads provided shelter for the embedded cells, giving a high biomass loading or thick biofilm (13.3 mg VS ml^?1 carrier) in comparison with PVA particles (4.8 mg VS ml^?1 carrier), but the mass transfer of substrate in the biofilm became a significant limiting factor in the thicker biofilms (effectiveness factor η = 0.31). The microbial decolourization rate per volume of carriers was 0.15 and 0.17 mg dye ml^?1 of glass beads and PVA particles, respectively. In augmented decomposition of a recalcitrant azo dye (60 mg l^?1), the immobilized Pseudomonas cells in porous glass beads gave a stable decolourization efficiency (80 - 81%), but cells fixed on solid PVA particles showed an initial high colour removal of 90% which then declined to a stable removal efficiency of 81%. In both cases, the colour removal efficiency of the chemostat bioreactor was increased from < 10% by an activated sludge to ～80% by the augmented system.     

Optimization of immobilization conditions for vinegar production. Siran,wood chips and polyurethane foam as carriers for Acetobacter aceti

A complete experimental design has been developed to study the properties of three different solid carriers (Siran, wood chips and polyurethane foam) in the immobilization of acetic acid bacteria. Temperature-controlled reactors of 450-ml volume were employed to compare a standard immobilization procedure consisting of consecutive discontinuous acetic acid fermentations in the presence of the carrier. On reaching the final saturation condition, the different immobilized carriers were removed and introduced into identical sterilized reactors. These were then submitted to several semi-continuous fermentation cycles with the aim of characterising and comparing their acetification properties. Immobilization and acetification data obtained in this study have been evaluated in order to determine the best carrier material on the basis of several technical criteria. Polyurethane foam was the most successful because it allows a huge number of immobilized cells in the shortest time and leads to the highest acetification rate of the three assayed carriers.     

聚氨酯固定化热带假丝酵母发酵木糖醇

固定在多孔聚氨酯载体中的热带假丝酵母(Candida tropicalis), 可有效地利用玉米芯半纤维素水解液生产木糖醇。在摇瓶条件下, 采用分批发酵方式, 确立了适宜的发酵工艺参数为: 接种量7%, 聚氨酯加入量1.0 g/100 mL, 温度30°C, 初始pH值6.0, 分段改变摇床转速进行溶氧调节, 其中0~24 h 为200 r/min; 24 h~46 h为140 r/min。聚氨酯固定化提高了菌体对发酵抑制物的耐受力, 固定化细胞密度高, 发酵性能稳定, 发酵产率和体积生产速率都有所提高。水解液未经脱色与离子交换便可转化成木糖醇, 大幅降低了成本, 显示了良好的应用前景。固定化细胞连续重复进行12批次21 d的发酵, 木糖醇得率平均为67.6%, 体积生产速率平均为1.92 g/(L·h)。     

谷氨酰胺合成酶产生菌的固定化研究

谷氨酰胺合成酶产生菌的固定化在酶法合成谷氨酰胺中的应用具有重要意义。实验首先从味精废水中筛选出谷氨酰胺合成酶高产菌株LNU018,然后分别用海藻酸钠、聚乙烯醇(PVA)为载体对谷氨酰胺合成酶高产菌棒杆菌进行固定化。探讨了固定化条件对固定化小球结构、机械强度、弹性、稳定性和培养后菌体的谷氨酰胺合成酶的活性情况的影响,分析确定最佳的固定化条件。研究结果表明,5%的海藻酸钠、11%的聚乙烯醇形成的固定化菌球大小合适,有弹性,但5%的海藻酸钠能更好的保持酶活性,比11%聚乙烯醇高16%,其为最佳的固定化条件。     

Immobilization of Paenibacillus macerans NRRL B-3186 cyclodextrin glucosyltransferase and properties of the immobilized enzyme

Cyclodextrin glucosyltransferase from Paenibacillus macerans NRRL B-3186 was immobilized on aminated polyvinylchloride (PVC) by covalent binding with a bifunctional agent (glutaraldehyde). The immobilized activity was affected by the length of the hydrocarbon chain attached to the PVC matrix, the amount of the protein loaded on the PVC carrier, and glutaraldehyde concentration. The activity of the immobilized enzyme was 121 units/gram carrier, the specific activity calculated on bound protein basis was 48% of the soluble enzyme. Compared to the free enzyme, the immobilized form exhibited: a higher optimal reaction temperature and energy of activation, a higher K_m (Michaelis constant) and lower V_max (maximal reaction rate), improved thermal stability and resistance to chemical denaturation. The operational stability was evaluated in repeated batch process and the immobilized enzyme retained about 85% of the initial catalytic activity after being used for 14 cycles.     

环氧交联剂和氨基载体固定化海洋假丝酵母脂肪酶*

游离酶经过固定化后,稳定性和环境耐受性得到提高,在食品、医药、化工、环境和皮革等领域可以很好的提高酶的利用率并降低生产成本,具有极大的应用潜力。新型交联剂在固定化酶工艺的应用极大推进了固定化酶研究的深入。借助新型交联剂聚乙二醇二缩水甘油醚(PEGDGE),利用氨基载体LX-1000HA固定化海洋假丝酵母脂肪酶,结合单因素和正交试验优化得到交联及固定化条件为:交联温度30℃,交联2h,交联剂浓度0.75%,pH7.0,加酶量800U,载体量0.5g,固定化2h,固定化温度45℃。根据上述最佳固定化工艺,制备得到固定化酶LX-1000HA-PEGDGE-CRL在最适条件下测得酶活达到160.81U/g,约为此前制备的固定化酶LX-1000HA-GA-CRL(由LX-1000HA和戊二醛交联脂肪酶得到)和LX-1000EA-PEGDGE-CRL(由短链氨基载体LX-1000EA和PEGDGE交联脂肪酶得到)酶活的2倍,发现固定化酶LX-1000HA-PEGDGE-CRL的最适反应温度相比于游离酶提高15℃;在70℃的环境中3h后酶活仍存留70%;循环使用6次后残留65%左右的酶活;酸碱耐受性和储存稳定性也表现良好,4℃保存30天后剩余约70%的初始酶活。同时,将制备的固定化酶LX-1000HA-PEGDGE-CRL与游离酶、固定化酶LX-1000HA-GA-CRL、固定化酶LX-1000EA-PEGDGE-CRL进行了比较,发现固定化酶LX-1000HA-PEGDGE-CRL在温度耐受性和重复使用性等方面具有更好的使用效果。     

An approach for the improved immobilization of penicillin G acylase onto macroporous poly(glycidyl methacrylate‐co‐ethylene glycol dimethacrylate) as a potential industrial biocatalyst

The use of penicillin G acylase (PGA) covalently linked to insoluble carrier is expected to produce major advances in pharmaceutical processing industry and the enzyme stability enhancement is still a significant challenge. The objective of this study was to improve catalytic performance of the covalently immobilized PGA on a potential industrial carrier, macroporous poly(glycidyl methacrylate‐co‐ethylene glycol dimethacrylate) [poly(GMA‐co‐EGDMA)], by optimizing the copolymerization process and the enzyme attachment procedure. This synthetic copolymer could be a very promising alternative for the development of low‐cost, easy‐to‐prepare, and stable biocatalyst compared to expensive commercially available epoxy carriers such as Eupergit or Sepabeads. The PGA immobilized on poly(GMA‐co‐EGDMA) in the shape of microbeads obtained by suspension copolymerization appeared to have higher activity yield compared to copolymerization in a cast. Optimal conditions for the immobilization of PGA on poly(GMA‐co‐EGDMA) microbeads were 1 mg/mL of PGA in 0.75 mol/L phosphate buffer pH 6.0 at 25°C for 24 h, leading to the active biocatalyst with the specific activity of 252.7 U/g dry beads. Chemical amination of the immobilized PGA could contribute to the enhanced stability of the biocatalyst by inducing secondary interactions between the enzyme and the carrier, ensuring multipoint attachment. The best balance between the activity yield (51.5%), enzyme loading (25.6 mg/g), and stability (stabilization factor 22.2) was achieved for the partially modified PGA. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 32:43–53, 2016