Production of alkaline protease with immobilized cells of bacillus subtilis PE-11 in various matrices by entrapment technique

The purpose of this investigation was to study the effect ofBacillus subtilis PE-11 cells immobilized in various matrices, such as calcium alginate, k-Carrageenan, ployacrylamide, agar-agar, and gelatin, for the production of alkaline protease. Calcium alginate was found to be an effective and suitable matrix for higher alkaline protease productivity compared to the other matrices studied. All the matrices were selected for repeated batch fermentation. The average specific volumetric productivity with calcium alginate was 15.11 U/mL/hour, which was 79.03% higher production over the conventional free-cell fermentation. Similarly, the specific volumetric productivity by repeated batch fermentation was 13.68 U/mL/hour with k-Carrageenan, 12.44 U/mL/hour with agar-agar, 11.71 U/mL/hour with polyacrylamide, and 10.32 U/mL/hour with gelatin. In the repeated batch fermentations of the shake flasks, an optimum level of enzyme was maintained for 9 days using calcium alginate immobilized cells. From the results, it is concluded that the immobilized cells ofB subtilis PE-11 in calcium alginate are more efficient for the production of alkaline protease with repeated batch fermentation. The alginate immobilized cells ofB subtilis PE-11 can be proposed as an effective biocatalyst for repeated usage for maximum production of alkaline protease. Published: October 21, 2005     

Enhanced production of a thermostable mannanase by immobilized cells of <Emphasis Type="Italic">Bacillus</Emphasis><Emphasis Type="Italic">subtilis</Emphasis> on various membranes

Cells of the thermophilic Bacillus subtilis WY34 were immobilized on various formaldehyde-activated polymer membranes and the immobilized cells were used for the production of thermostable mannanase in flasks. The results showed that polyethersulfone membranes (PES) and nylon-6 membranes were the most suitable supports for cell immobilization to produce the mannanase. Moreover, PES and nylon-6 membranes immobilized cells provided 1.78- and 1.74-fold higher mannanase activity compared to the control after 4 days of cultivation, respectively. The immobilized cells on PES and nylon-6 membranes had good stability and retained 131.5 and 114.3% of ability of enzyme production even after six cycles of repeated batch fermentation, respectively. Active cell growth was observed by scanning electron microscopy (SEM) after 16 days (four cycles) repeated batch cultivation. Therefore, the membrane-immobilized cells of B. subtilis WY34 can be proposed as an effective biocatalyst for repeated usage for production of the thermostable mannanase.     

Improvement of acid phosphatase production by immobilization of Humicola lutea mycelium in polyurethane sponge

Acid phosphatase production by the fungus Humicola lutea 120-5, immobilized in polyurethane sponge, was studied under semicontinuous shake flask fermentation and compared to the enzyme secretion by free cells. The effect of parameters such as the carrier content and the duration of the batch in repeated batch experiments on the phosphatase production half-life was investigated. The best results were obtained with 1.0 g of sponge cubes (about 1.0 cm per side) per culture flask using 72 h runs. In these conditions the half-life of enzyme production by immobilized biocatalyst was 15 sequential cycles (45 days) compared to three cycles (9 days) for the free mycelium. The maximal phosphatase titre registered in free cell fermentation was 2500 U/l (i.e. 100%), while the relative enzyme activity of the optimal immobilized system was over 100% during the whole half-life time of 45 days. Significant improvement (200–215%) in the yield was observed in one-third of this period or 15 days. The supernatant medium obtained at any stage of the repeated batch cultures did not contain free cells and, due to the low pH (3.0–3.5), the whole process was carried out without any bacterial contamination. In comparison with free cell fermentation, the significant improvement of the acid phosphatase production by polyurethane sponge-immobilized H. lutea mycelium as well as its operation stability was confirmed by scanning electron microscopy.     

Production of l (+) lactic acid by Lactobacillus delbrueckii immobilized in functionalized alginate matrices

The role of functionalized alginate gels as immobilized matrices in production of l (+) lactic acid by Lactobacillus delbrueckii was studied. L. delbrueckii cells immobilized in functionalized alginate beads showed enhanced bead stability and selectivity towards production of optically pure l (+) lactic acid in higher yields (1.74Yp/s) compared to natural alginate. Palmitoylated alginate beads revealed 99% enantiomeric selectivity (ee) in production of l (+) lactic acid. Metabolite analysis during fermentation indicated low by-product (acetic acid, propionic acid and ethanol) formation on repeated batch fermentation with functionalized immobilized microbial cells. The scanning electron microscopic studies showed dense entrapped microbial cell biomass in modified immobilized beads compared to native alginate. Thus the methodology has great importance in large-scale production of optically pure lactic acid.     

Ethanol production from concentrated food waste hydrolysates with yeast cells immobilized on corn stalk

The aim of the present study was to examine ethanol production from concentrated food waste hydrolysates using whole cells of S. cerevisiae immobilized on corn stalks. In order to improve cell immobilization efficiency, biological modification of the carrier was carried out by cellulase hydrolysis. The results show that proper modification of the carrier with cellulase hydrolysis was suitable for cell immobilization. The mechanism proposed, cellulase hydrolysis, not only increased the immobilized cell concentration, but also disrupted the sleek surface to become rough and porous, which enhanced ethanol production. In batch fermentation with an initial reducing sugar concentration of 202.64 ± 1.86 g/l, an optimal ethanol concentration of 87.91 ± 1.98 g/l was obtained using a modified corn stalk-immobilized cell system. The ethanol concentration produced by the immobilized cells was 6.9% higher than that produced by the free cells. Ethanol production in the 14th cycle repeated batch fermentation demonstrated the enhanced stability of the immobilized yeast cells. Under continuous fermentation in an immobilized cell reactor, the maximum ethanol concentration of 84.85 g/l, and the highest ethanol yield of 0.43 g/g (of reducing sugar) were achieved at hydraulic retention time (HRT) of 3.10 h, whereas the maximum volumetric ethanol productivity of 43.54 g/l/h was observed at a HRT of 1.55 h.     

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.

     

Itaconic acid production by immobilizedAspergillus terreus from xylose and glucose

Summary Aspergillus terreus NRRC 1960 spores were entrapped in calcium alginate gel beads or alternotely the fungal mycelium was immobilized either on Celite R-626 or in agar gel cubes, and the biocatalyst was employed both in repeated batch and in continuous column reactors to produce itaconic acid from D-xylose or D-glucose. The highest itaconic acid yield obtained in a submerged culture batch fermentation was 54.5% based on total initial glucose (55 g/l) with a volumetric productivity of 0.32 g/l h, and 44.8% from xylose (67 g/l) with a productivity of 0.20 g/l h. In a repeated batch fermentation mycelium immobilized in agar gel had a productivity of 0.112 g/l h, and mycelium grown from spores immobilized in calcium alginate gel 0.06 g/l h, both from xylose (60 g/l). With the best immobilized biocatalyst system used employing Celite R-626 as a carrier, volumetric productivities of 1.2 g/l h from glucose and 0.56 g/l h from xylose (both at 60 g/l) were obtained in continuous column operation for more than 2 weeks.     

Production of pullulanase by free and immobilized cells of <Emphasis Type="Italic">Bacillus licheniformis</Emphasis> NRC22 in batch and continuous cultures

The production of extracellular pullulanase by Bacillus licheniformis NRC22 was investigated using different fermentation modes. In batch culture maximal enzyme activity of 18 U/ml was obtained after 24 h of growth. In continuous fermentation by the free cells, maximal reactor productivity (4.15 KU/l/h) with enzyme concentration of 14.8 U/ml and specific productivity of 334.9 U/g wet cells/h was attained at a dilution rate of 0.28/h, over a period of 25 days. B. licheniformis NRC22 cells were immobilized on Ca-alginate. The immobilization conditions with respect to matrix concentration and cell load was optimized for maximal enzyme production. In repeated batch operation, the activity of the immobilized cells was stable during the 10 cycles and the activity remained between 9.8 and 7.7 U/ml. Continuous production of pullulanase by the immobilized cells was investigated in a packed–bed reactor. Maximal reactor productivity (7.0 KU/h) with enzyme concentration of 16.8 U/ml and specific productivity of 131.64 U/g wet cells/h was attained at dilution rate of 0.42/h. The enzyme activity in the effluent started to decline gradually to the level of 8.7 U/ml after 25 days of the operation.     

Room and low temperature brewing with yeast immobilized on gluten pellets

A biocatalyst prepared by the immobilization of a cryotolerant strain of Saccharomyces cerevisiae on gluten pellets was used for batch and continuous fermentation at low temperatures. The immobilized yeast showed important operational stability in repeated batch fermentations without a decrease of activity even at 0 and 5°C. Repeated batch fermentations using the biocatalyst resulted in improvement of ethanol productivity in comparison with bottom brewing fermentation and free cells using the same yeast strain. At 0 and 10°C, the fermentation rate was four and seven times higher than that of free cells, respectively. For immobilized yeast, diacetyl and polyphenol contents were lower and the alcohol concentration higher at low temperatures (0–7°C) when compared to free cells. Fine clarity was also observed in the beers. Continuous brewing using gluten-supported biocatalyst had an operational stability of 3 months with relatively high productivity and without contamination. Polyphenol and bitterness contents were lower in the continuous process than those of batch fermentations, but at low temperature (5°C) they were higher. The diacetyl content was higher than in batch fermentations and beers had a fine aroma and taste.     

Batch and continuous ribonuclease production by immobilized Aspergillus clavatus cells in a bubble-collumn bioreactor

Summary Ribonuclease production using immobilized cells (IC) of Aspergillus clavatus has been studied under batch, repeated-batch and continuous fermentation conditions in a bubble-column bioreactor and compared with production by free cells, Immobilization was achieved by the method of cryostructurization in polyvinyl alcohol beads. The effect of various aeration rates in a column bioreactor has been investigated. Enzyme production by IC [42 000 units (U)·l^–1] during batch fermentations was comparable to that of a free-cell system. The specific productivity of IC was 8.5 times higher than that of free cells. In repeated batch fermentation at various aeration rates, successful reuse of IC was obtained, with comparable levels of enzyme production. Continuous ribonuclease production was achieved for 44 days at 1 vvm aeration and a dilution rate of 0.0 h^–1 with volumetric productivity (450 U·1^–1) and yield.     

Successive cultivation of Fusarium oxysporum on rice chaff for economic production of fibrinolytic enzyme

Production of fibrinolytic enzyme by successive solid state cultivation of Fusarium oxysporum on rice chaff (RC) was studied. From recycling 20?g dry RC six times by pure batch culture, an overall of 23200IU fibrinolytic enzyme was obtained. A total of 27400IU was obtained when the same amount of RC was used for six cycles with repeated batch fermentation employing immobilized whole cells, which also resulted in 30%?(w/w) loss by weight of the substrate, while only 8%?(w/w) of RC was degraded in the initial pure batch fermentation.     

High alcohol production by repeated batch fermentation using an immobilized osmotolerant Saccharomyces cerevisiae

A repeated batch fermentation system was used to produce ethanol using an osmotolerant Saccharomyces cerevisiae (VS₃) immobilized in calcium alginate beads. For comparison free cells were also used to produce ethanol by repeated batch fermentation. Fermentation was carried for six cycles with 125, 250 or 500 beads using 150, 200 or 250 g glucose L⁻¹ at 30°C. The maximum amount of ethanol produced by immobilized VS₃ using 150 g L⁻¹ glucose was only 44 g L⁻¹ after 48 h, while the amount of ethanol produced by free cells in the first cycle was 72 g L⁻¹. However in subsequent fed batch cultures more ethanol was produced by immobilized cells compared to free cells. The amount of ethanol produced by free cells decreased from 72 g L⁻¹ to 25 g L⁻¹ after the fourth cycle, while that of immobilized cells increased from 44 to 72 g L⁻¹. The maximum amount of ethanol produced by immobilized VS₃ cells using 150, 200 and 250 g glucose L⁻¹ was 72.5, 93 and 87 g ethanol L⁻¹ at 30°C. Journal of Industrial Microbiology & Biotechnology (2000) 24, 222–226. Received 16 September 1999/ Accepted in revised form 22 December 1999     

Investigations on neomycin production with immobilized cells of<Emphasis Type="Italic">Streptomyces marinensis</Emphasis> Nuv-5 in calcium alginate matrix

The purpose of this investigation was to study the effect ofStreptomyces marinensis NUV-5 cells immobilized in calcium alginate for the production of neomycin. The effect of various parameters, such as the effect of alginate concentration (1%, 2%, 3%, 4%, and 5% wt/vol), the effect of cation (caCl₂, BaCl₂, and SrCl₂), the concentration of cation (0.01M, 0.125M, 0.25M, 0.375M, and 0.5M), the curing times (1, 6, 11, 16, and 21 hours), and the diameter of the bead (1.48, 2.16, 3.24, 4.46, and 5.44 mm), on neomycin production and bead stability were studied. The effect of maltose (4%, 3%, 2%, and 1% wt/vol) and sodium glutamate (0.6%, 0.3%, 0.15%, and 0.075%) wt/vol) concentration on neomycin production was also studied. Better neomycin production was achieved with optimized parameters, such as alginate at 2% wt/vol, 0.25M CaCl₂, 1-hour curing time, and 3.24 mm bead diameter. Effective neomycin production was achieved with 3% wt/vol maltose and 0.6% wt/vol sodium glutamate concentration. The repeated batch fermentations were conducted (every 96 hours) using the optimized alginate beads, employing the production medium with 3% wt/vol maltose and 0.6% wt/vol sodium glutamate along with minerals salts solution. The increase in antibiotic production was observed up to the 5th cycle, and later gradual decrease in antibiotic production was observed. Comparison of the total antibiotic production with free cells and immobilized cells was also done. An enhanced antibiotic productivity of 32% was achieved with immobilized cells over the conventional free-cell fermentation, while 108% more productivity was achieved over the washed free-cell fermentation. From these results it is concluded that the immobilized cells ofS marinensis NUV-5 in calcium alginate are more efficient for the production of neomycin with repeated batch fermentation.     

Production of tylosin and nikkomycin by immobilized Streptomyces cells

Summary Mycelia of Streptomyces sp. T 59-235 and Streptomyces tendae Tü 901 (producing the antibiotics tylosin and nikkomycin, resp.) were immobilized in different carriers. With both organisms best antibiotic production was observed in calcium alginate gel.Influence of aeration, cell density and flow rate on antibiotic production was investigated in batch fermentation and in a continuous system (air-bubbled reactor).In batch fermentation, immobilization prolongued the production phase from 72 to 120 h (Streptomyces T 59-235) and from 72 to 96 h (S. tendae). The relative productivity of immobilized cells was 40 to 50% compared to that of free mycelia in both cases.In continuous tylosin fermentation highest production rate was observed in a medium nearly saturated with oxygen.Nikkomycin production by immobilized S. tendae could be maintained for longer than 350 h in a continuous system. The production rate depended on cell density and flow rate of the medium. The maximum specific productivity was 100% compared to that of free mycelium in batch culture.     

固定化根霉发酵生产脂肪酶

以聚氨酯为少根根霉固定化载体,对固定化后的细胞连续重复批次发酵进行了研究。优化了重复批次发酵培养基组成。在取代发酵液40mL,取代培养基组成为全脂豆粉3%,花生油0.5％条件下,固定化菌体摇瓶实验可连续使用140h,重复9批次。酶的时空产率提高6倍。5L发酵罐小试固定化菌体可连续发酵6批次。固定化细胞连续发酵,大大缩短了发酵的时间,酶的时空产率获得大幅提高。     

Comparative study of spent grains and delignified spent grains as yeast supports for alcohol production from molasses

Fresh, defrosted and delignified brewer's spent grains (BSG) were used as yeast supports for alcoholic fermentation of molasses. Glucose solution (12%) with and without nutrients was used for cell immobilization on fresh BSG, without nutrients for cell immobilization on defrosted and with nutrients for cell immobilization on delignified BSG. Repeated fermentation batches were performed by the immobilized biocatalysts in molasses of 7, 10 and 12 initial Baume density without additional nutrients at 30 and 20 degrees C. Defrosted BSG immobilized biocatalyst was used only for repeated fermentation batches of 7 initial Baume density of molasses without nutrients at 30 and 20 degrees C. After immobilization, the immobilized microorganism population was at 10(9) cells/g support for all immobilized biocatalysts. Fresh BSG immobilized biocatalyst without additional nutrients for yeast immobilization resulted in higher fermentation rates, lower final Baume densities and higher ethanol productivities in molasses fermentation at 7, 10 and 12 initial degrees Be densities than the other above biocatalysts. Adaptation of defrosted BSG immobilized biocatalyst in the molasses fermentation system was observed from batch to batch approaching kinetic parameters reported in fresh BSG immobilized biocatalyst. The results of this study concerning the use of fresh or defrosted BSG as yeast supports could be promising for scale-up operation.     

Process and technoeconomics of ethanol production by immobilized cells

Summary A two-stage fermentation process has been developed for continuous ethanol production by immobilized cells of Zymomonas mobilis. About 90–92 kg/m³ ethanol was produced after 4 h of residence time. Entrapped cells of Zymomonas mobilis have a capability to convert glucose to ethanol at 93% of the theoretical yield. The immobilized cell system has functioned for several weeks, and experience indicates that the carrageenan gel apparently facilitates easy diffusion of glucose and ethanol.The simplicity and the high productivity of the plug-flow reactor employing immobilized cells makes it economically attrative. An evaluation of process economics of an immobilized cell system indicates that at least 4 c/l of ethanol can be saved using the immobilized cell system rather than the conventional batch system. The high productivity achieved in the immobilized cell reactor results in the requirement for only small reactor vessels indicating low capital cost. Consequently, by switching from batch to immobilized processing, the fixed capital investment is substantially reduced, thus increasing the profitability of ethanol production by fermentation.     

Ethanol production from nonsterilized carob pod extract by free and immobilized Saccharomyces cerevisiae cells using fed-batch culture

The production of ethanol from carob pod extract by free and immobilized Saccharomyces cerevisiae cells in batch and fed-batch culture was investigated. Fed-batch culture proved to be a better fermentation system for the production of ethanol than batch culture. In fed-batch culture, both free and immobilized S. cerevisiae cells gave the same maximum concentration (62 g/L) of final ethanol at an initial sugar concentration of 300 g/L and F = 167 mL/h. The maximum ethanol productivity (4.4 g/L h) was obtained with both free and immobilized cells at a substrate concentration of 300 g/L and F = 334 mL/h. In repeated fed-batch culture, immobilized S. cerevisiae cells gave a higher overall ethanol concentration compared with the free cells. The immobilized S. cerevisiae cells in Ca-alginate beads retained their ability to produce ethanol for 10 days. (c) 1994 John Wiley & Sons, Inc.     

Improvement of cell lysis activity of immobilized lysozyme with reversibly soluble-insoluble polymer as carrier

Summary Hen egg white lysozyme was immobilized by covalent binding to a polymer showing reversibly soluble-insoluble characteristics with pH change. The retention of the specific activity of the immobilized enzyme can be as high as 41% of that of the free enzyme. The immobilized enzyme could be used in repeated batch lysis of M. lysodeikticus cells and to enhance the release of intracellular proteins 1.4 folds when compared with batch operation.     

Ethanol production from starch by immobilized Aspergillus awamori and Saccharomyces pastorianus using cellulose carriers

A simultaneous saccharification and fermentation (SSF) process was investigated to produce ethanol using two kinds of cellulose carriers that were respectively suitable for immobilization of Aspergillus awamori and Saccharomyces pastorianus. The maximum ethanol concentration attained by the batch operation was 25.5 g l⁻¹. Under suitable conditions, both cellulose carriers with immobilized cells could be reused efficiently for three cycles. The total amount of ethanol production was 66.0 g (per 1 l working volume) after the repeated operation. Ethanol productivity mainly depends on a saccharification process. There is a limit in durability in the repeated batch operation, and it is important to maintain high activity of the fungus in order to produce ethanol efficiently. Journal of Industrial Microbiology & Biotechnology (2001) 27, 52–57. Received 11 December 2000/ Accepted in revised form 02 June 2001