Immobilization of Lactobacillus rhamnosus in polyvinyl alcohol/calcium alginate matrix for production of lactic acid

Immobilization of Lactobacillus rhamnosus ATCC7469 in poly(vinyl alcohol)/calcium alginate (PVA/Ca-alginate) matrix using “freezing–thawing” technique for application in lactic acid (LA) fermentation was studied in this paper. PVA/Ca-alginate beads were made from sterile and non-sterile PVA and sodium alginate solutions. According to mechanical properties, the PVA/Ca-alginate beads expressed a strong elastic character. Obtained PVA/Ca-alginate beads were further applied in batch and repeated batch LA fermentations. Regarding cell viability, L. rhamnosus cells survived well rather sharp immobilization procedure and significant cell proliferation was observed in further fermentation studies achieving high cell viability (up to 10.7 log CFU g⁻¹) in sterile beads. In batch LA fermentation, the immobilized biocatalyst was superior to free cell fermentation system (by 37.1%), while the highest LA yield and volumetric productivity of 97.6% and 0.8 g L⁻¹ h⁻¹, respectively, were attained in repeated batch fermentation. During seven consecutive batch fermentations, the biocatalyst showed high mechanical and operational stability reaching an overall productivity of 0.78 g L⁻¹ h⁻¹. This study suggested that the “freezing–thawing” technique can be successfully used for immobilization of L. rhamnosus in PVA/Ca-alginate matrix without loss of either viability or LA fermentation capability.

     

Immobilization of microbial cells using polyvinyl alcohol (PVA) — polyacrylamide gels

Summary A novel cell immobilization technique is developed in which polyvinyl alcohol is crosslinked with boric acid with addition of an acrylamide as a polymerizing agent. The presence of the polymerizing agent overcomes the problem of swelling of PVA gels in aqueous solution. The new immobilization method was used to entrap a phenol-degrading microorganism, a species of Pseudomonas. Phenol was successfully degraded in a shake-flask test of the beads demonstrating that cell viability was maintained following the immobilization procedure.     

PVA-Ca(NO3)2法包埋固定氧化亚铁硫杆菌研究

首次报道了把聚乙烯醇(PVA)、海藻酸钠混合水溶胶和氧化亚铁硫杆菌混合后滴入1%~5%(W/V)的Ca(NO3)2溶液中凝固成型,并把成型后的颗粒置-20℃条件下冷冻1d,从而形成固定化颗粒,把该颗粒在摇瓶中进行分批培养,对Fe2 最大氧化速率可达2.45g/(L.h)。而且整个固定化操作简单,颗粒不粘连、强度高、稳定性好,可以同时消除PVA-H3BO3法中PVA颗粒的粘连膨胀和H3BO3对微生物的毒性,具有很好的应用价值。     

Hydrodynamic characteristics of immobilized cell beads in a liquid-solid fluidized-bed bioreactor

This study examined the hydrodynamic characteristics of a liquid-solid fluidized-bed bioreactor using elastic particles (PVA gel beads) of various diameters as carriers. The drag coefficient-Reynolds number, velocity-voidage, and expansion index-Reynolds number relationships observed during fluidization of PVA gel beads in a fluidized bed in our experiments were compared with the published results. Predictions made from previous correlations were examined with our new experimental findings, revealing the inadequacy of most of these correlations. Thus, new correlations describing the above-mentioned relationships are suggested. The drag coefficient of immobilized cell beads is larger than that of free cell ones at the same Reynolds number because the surface of the immobilized cell beads is rougher. For multiparticle systems, the correction factor, f(epsilon), is a function of the falling gel bead properties (Reynolds number) as well as the fluidized gel bead properties (Archimedes number), and depend strongly on the bed voidage (epsilon). A new simple relation was developed to predict easily the epsilon value from 0.5-0.9 at 4,986 < A(r) < 40,745 or 34 < Re(t) < 186. For all the immobilized cell beads used in this study, the prediction error of the bed voidage was less than 5% at epsilon > 0.5. The prediction equations in this study can be further applied to analyzing the hydrodynamic characteristics of a fluidized-bed reactor using similar entrapped elastic particles as carriers.     

Biodesulfurization using Pseudomonas delafieldii in magnetic polyvinyl alcohol beads

AIMS: To immobilize Pseudomonas delafieldii R-8 cells in magnetic polyvinyl alcohol (PVA) beads for biodesulfurization. METHODS AND RESULTS: Magnetic PVA beads were prepared by a freezing-thawing technique under liquid nitrogen. The beads have distinct super-paramagnetic properties and their saturation magnetization is 8.02 emu g(-1). The desulfurization rate of the immobilized cells could reach 40.2 mmol kg(-1) h(-1). Desulfurization patterns of dibenzothiophene in model oil with the immobilized and free cells were represented by the Michaelis-Menten equation. The Michaelis constant for both immobilized and free cells was 1.3 mmol l(-1). CONCLUSIONS: The cells immobilized in magnetic PVA beads could be stably stored and be repeatedly used over 12 times for biodesulfurization. The immobilized cells could be easily separated by magnetic field. SIGNIFICANCE AND IMPACT OF THE STUDY: Magnetic PVA beads are easy to prepare. The immobilization process in the paper is to increase the efficiency of cells and to decrease the cost of operations.     

High-rate ferrous iron oxidation by immobilized Acidithiobacillus ferrooxidans with complex of PVA and sodium alginate

By four different methods, Acidithiobacillus ferrooxidans cells were immobilized by the complex of PVA and sodium alginate. The beads formed by these different methods were evaluated in terms of relative mechanical strength, biological activity, dilatability, and so on. The results indicate that the technique utilizing the complex of PVA and sodium alginate crosslinked with Ca(NO(3))(2) is more appropriate for the immobilization of A. ferrooxidans than any others. So the PVA-calcium nitrate beads were used in batch and continuous culture. A maximum ferrous iron oxidation rate of 4.6 g/l/h was achieved in batch culture. Long-time performance of packed-bed bioreactor was evaluated systematically over 40 days, depending on the conversion ratio of ferrous iron and the residence time. At a residence time of 2.5 h, 96% of the initial ferrous iron was oxidized. This study shows this new immobilization technique will be a feasible and economical method for A. ferrooxidans.     

Enhanced efficacy of nitrifying biomass by modified PVA_SB entrapment technique

In this study, we developed a novel technique for preparing polyvinyl alcohol (PVA) hydrogel as an immobilizing matrix by the addition of sodium bicarbonate. This resulted in an increase in the specific surface area of PVA_sodium bicarbonate (PVA_SB) hydrogel beads to 65.23 m² g^?1 hydrogel beads, which was approximately 85 and 14 % higher than those of normal PVA and PVA_sodium alginate (PVA_SA) hydrogel beads, respectively. The D _e value of PVA_SB hydrogel beads was calculated as 7.49 × 10^?4 cm² s^?1, which was similar to the D _e of PVA_SA hydrogel beads but nearly 38 % higher than that of the normal PVA hydrogel beads. After immobilization with nitrifying biomass, the oxygen uptake rate and the ammonium oxidation rate of nitrifying biomass entrapped in PVA_SB hydrogel beads were determined to be 19.53 mg O₂ g MLVSS^?1 h^?1 and 10.59 mg N g MLVSS^?1 h^?1, which were 49 and 43 % higher than those of normal PVA hydrogel beads, respectively. Scanning electron microscopy observation of the PVA_SB hydrogel beads demonstrated relatively higher specific surface area and revealed loose microstructure that was considered to provide large spaces for microbial growth. This kind of structure was also considered beneficial for reducing mass transfer resistance and increasing pollutant uptake.     

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.     

Immobilization of Rhizopus oryzae in a modified polyvinyl alcohol gel for L(+)-lactic acid production

The production of L(+)-lactic acid (LA) by Rhizopus oryzae immobilized in polyvinyl alcohol (PVA) was investigated. To decrease diffusional resistance, we modified the PVA gel through the addition of sodium alginate and phosphate esterification. The production of L(+)-LA improved notably in the immobilized Rhizopus oryzae. Maximum L(+)-LA production (106.27 g/L), with a yield of 73.1 % and rate of 2.95 g/L·h, was obtained at a temperature of 38 °C, 6 % PVA, and 0.8 % sodium alginate. The immobilized R. oryzae was stable in 14 serial-batch cultures using non-growth medium. The immobilized beads also displayed good tolerance to low temperature and long-term storage at 4 °C with the preservation of biochemical properties.     

Immobilization of Pseudomonas delafieldii with magnetic polyvinyl alcohol beads and its application in biodesulfurization

Pseudomonas delafieldii was immobilized in magnetic polyvinyl alcohol (PVA) beads using a hydrophilic magnetic fluid, which was prepared by a co-precipitation method. The beads had distinct super-paramagnetic properties and were compared with immobilized cells in non-magnetic PVA beads. Their desulfurizing activity was increased slightly from 8.7 to 9 mmol sulfur kg(-1) (dry cell) h(-1). The main advantages was that the magnetic immobilized cells maintain a high desulfurization activity and remain in good shape after 7 times of repeated use, while the non-magnetic immobilized cells could only be used for 5 times. Furthermore, the magnetic immobilized cells could be easily collected or separated magnetically from the biodesulfurization reactor.     

Equilibrium and kinetics studies of adsorption of copper (II) on chitosan and chitosan/PVA beads

The adsorption of Cu(II) ions from aqueous solution by chitosan and chitosan/PVA beads was studied in a batch adsorption system. Chitosan solution was blended with poly(vinyl alcohol) (PVA) in order to obtain sorbents that are insoluble in aqueous acidic and basic solution. The adsorption capacities and rates of Cu(II) ions onto chitosan and chitosan/PVA beads were evaluated. The Langmuir, Freundlich and BET adsorption models were applied to describe the isotherms and isotherm constants. Adsorption isothermal data could be well interpreted by the Langmuir model. The kinetic experimental data properly correlated with the second-order kinetic model, which indicates that the chemical sorption is the rate-limiting step. The Cu(II) ions can be removed from the chitosan and chitosan/PVA beads rapidly by treatment with an aqueous EDTA solution. Results also showed that chitosan and chitosan/PVA beads are favourable adsorbers.     

Biotransformation of penicillin V to 6-aminopenicillanic acid using immobilized whole cells of E. coli expressing a highly active penicillin V acylase

The production of 6-aminopenicillanic acid (6-APA) is a key step in the manufacture of semisynthetic antibiotics in the pharmaceutical industry. The penicillin G acylase from Escherichia coli has long been utilized for this purpose. However, the use of penicillin V acylases (PVA) presents some advantages including better stability and higher conversion rates. The industrial application of PVAs has so far been limited due to the nonavailability of suitable bacterial strains and cost issues. In this study, whole-cell immobilization of a recombinant PVA enzyme from Pectobacterium atrosepticum expressed in E. coli was performed. Membrane permeabilization with detergent was used to enhance the cell-bound PVA activity, and the cells were encapsulated in calcium alginate beads and cross-linked with glutaraldehyde. Optimization of parameters for the biotransformation by immobilized cells showed that full conversion of pen V to 6-APA could be achieved within 1?hr at pH 5.0 and 35°C, till 4% (w/v) concentration of the substrate. The beads could be stored for 28 days at 4°C with minimal loss in activity and were reusable up to 10?cycles with 1-hr hardening in CaCl₂ between each cycle. The high enzyme productivity of the PVA enzyme system makes a promising case for its application for 6-APA production in the industry.     

The use of polysiloxane/polyvinyl alcohol beads as solid phase in IgG anti-Toxocara canis detection using a recombinant antigen

Immunodetection of human IgG anti-Toxocara canis was developed based on ELISA and on the use of polysiloxane/polyvinyl alcohol (POS/PVA) beads. A recombinant antigen was covalently immobilized, via glutaraldehyde, onto this hybrid inorganic-organic composite, which was prepared by the sol-gel technique. Using only 31.2 ng antigen per bead, a peroxidase conjugate dilution of 1:10,000 and a serum dilution of 1:200 were adequate for the establishment of the procedure. This procedure is comparable to that which utilizes the adsorption of the antigen to conventional PVC plates. However, the difference between positive and negative sera mean absorbances was larger for this new glass based assay. In addition to the performance of the POS/PVA bead as a matrix for immunodetection, its easy synthesis and low cost are additional advantages for commercial application.     

聚乙烯醇-海藻酸盐共固定化冰核活性细菌——黄单胞菌TS206的研究

冰核活性细菌固定化在食品冷冻浓缩中的应用具有重要意义,冰核活性和抗渗漏能力是衡量其性能的两个重要技术指标。研究采用PVA和海藻酸盐作为固定化载体,通过两者优良性能的互补而建立对冰核活性细菌Xanthomonas ampelina TS206的共固定化技术。结果表明,细胞投入量对冰核活性有较大影响,基础固定化条件对固定化技术指标的综合评分影响程度大小的顺序依次为：海藻酸钠浓度＞硼酸浓度＞PVA浓度＞CaCl2浓度,各因素的较优水平是：海藻酸钠浓度1%,硼酸浓度5%,PVA浓度8%,CaCl2浓度1.1%;研究还发现冰核活性与固定化凝胶珠的添加量正相关,与固化时间相关性较小,渗漏量受固定化凝胶珠的添加量和固化时间影响不显著。     

Fabrication of hyaluronic acid hydrogel beads for cell encapsulation

Hyaluronic acid (HA) hydrogel beads were prepared by photopolymerization of methacrylated HA and N-vinylpyrrolidone using alginate as a temporal spherical mold. Various fabrication conditions for preparing the hydrogel beads, such as the concentration of methacrylated HA and UV irradiation time, were optimized to control swelling properties and enzymatic degradability. A new concept for cell encapsulation is proposed in this paper. Viable cells were directly injected into the hydrogel beads using a microinjection technique. When bovine articular chondrocytes were injected into HA hydrogel beads and cultivated for 1 week, the cells could proliferate well within the HA beads. HA hydrogel beads could be potentially used as injectable cell delivery vehicles for regenerating tissue defects.     

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.     

Purification of aquarium water by PVA gel-immobilized photosynthetic bacteria during goldfish rearing

This study was conducted to evaluate the ability of a PVA-gel beads filtration (PVA) system using photosynthetic bacteria to purify water. To accomplish this, duplicate long-term goldfish rearing experiments were conducted using four different types of aquarium systems (COF, PSB, EMC, and PVA). The results revealed that the concentrations of NH₄ ⁺-N on the day of a goldfish’s death were significantly higher than the concentrations on other days for all the aquarium systems. In addition, the mean concentration of NH₄ ⁺-N during goldfish rearing occurred in the following order: COF system > EMC system > PSB system > PVA system. Furthermore, the mean values of all other ion concentrations (NO₃ ⁻-N, NO₂ ⁻-N, and PO₄ ²⁻-P) were found to be lowest in the PVA system. As a result, there was more prominent decomposition of organic matter in the aquarium tank containing the PVA system, as well as less turbid aquarium water and more active goldfish. Additionally, the PVA-gel beads resulted in almost complete denitrification, even after six-months of goldfish rearing. To our knowledge, this is the first study to report that PVA gel-immobilized photosynthetic bacteria have the ability to purify water. Overall, the results of this study indicate that this immobilized photosynthetic bacteria system has the potential for use as a component in circulating filtration systems.     

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.     

Cell immobilization using PVA crosslinked with boric acid

A new cell immobilization technique is described in which polyvinyl alcohol is crosslinked with boric acid, with the addition of a small amount of calcium alginate. The presence of the calcium alginate improves the surface properties of the beads, preventing agglomeration. A pure culture of phenol-degrading Pseudomonas was immobilized in the PVA-alginate beads. Phenol was successfully degraded in a fluidized bed of the beads, indicating that cell viability was maintained following the immobilization procedure. The PVA-alginate beads proved to be very strong and durable, with no noticeable degradation of the beads after 2 weeks of continuous operation of the fluidized bed.