Membrane-mediated extractive fermentation for lactic acid production from cellulosic biomass

Lactic acid production from cellulosic biomass by cellulase andLactobacillus delbrueckii was studied in a fermenter-extractor employing a microporous hollow fiber membrane (MHF). This bioreactor system was operated under a fed-batch mode with continuous removal of lactic acid by anin situ extraction. A tertiary amine (Alamine 336) was used as an extractant for lactic acid. The extraction capacity of Alamine 336 is greatly enhanced by addition of alcohol. Long-chain alcohols serve well for this purpose since they are less toxic to micro-organism. Addition of kerosene, a diluent, was necessary to reduce the solvent viscosity. A solvent mixture of 20% Alamine 336, 40% oleyl alcohol, and 40% kerosene was found to be most effective in the extraction of lactic acid. Progressive change of pH from an initial value of 5.0 down to 4.3 has significantly improved the overall performance of the simultaneous saccharification and extractive fermentation over that of constant pH operation. The change of pH was applied to promote cell growth in the early phase, and extraction in the latter phase.     

Gluconic acid production in bioreactor with immobilized glucose oxidase plus catalase on polymer membrane adjacent to anion-exchange membrane

Gluconic acid was obtained in the permeate side of the bioreactor with glucose oxidase (GOD) immobilized onto anion-exchange membrane (AEM) of low-density polyethylene grafted with 4-vinylpiridine. The electric resistance of the anion-exchange membranes was increased after the enzyme immobilization on the membrane. The gluconic acid productions were relatively low with the GOD immobilized by any method on the AEM. To increase the enzyme reaction efficiency, GOD was immobilized on membrane of AN copolymer (PAN) adjacent to an anion-exchange membrane in bioreactor. Uses of anion-exchange membrane led to selective removal of the gluconic acid from the glucose solution and reduce the gluconic acid inhibition. The amount of gluconic acid obtained in the permeate side of the bioreactor with the GOD immobilized on the PAN membrane adjacent to the AEM under electrodialysis was about 30 times higher than that obtained with enzyme directly bound to the AEM. The optimal substrate concentration in the feed side was found to be about 1 g/l. Further experiments were carried out with the co-immobilized GOD plus Catalase (CAT) on the PAN membrane adjacent to the AEM to improve the efficiency of the immobilize system. The yield of this process was at least 95%. The storage stability of the co-immobilized GOD and CAT was studied (lost 20% of initial activity for 90 d). The results obtained clearly showed the higher potential of the dual membrane bioreactor with GOD plus CAT bound to ultrafiltration polymer membrane adjacent to the AEM. Storage stability of GOD activity in GOD plus CAT immobilized on PAN//AEM membranes and on AEM.     

Lactic acid production from cheese whey by immobilized bacteria

The performance of immobilized Bifidobacterium longum in sodium alginate beads and on a spiral-sheet bioreactor for the production of lactic acid from cheese whey was evaluated. Lactose utilization and lactic acid yield of B. longum were compared with those of Lactobacillus helveticus. B. longum immobilized in sodium alginate beads showed better performance in lactose utilization and lactic acid yield than L. helveticus. In the spiral-sheet bioreactor, a lactose conversion ratio of 79% and lactic acid yield of 0.84 g of lactic acid/g of lactose utilized were obtained during the first run with the immobilized L. helveticus. A lactose conversion ratio of 69% and lactic acid yield of 0.51 g of lactic acid/g of lactose utilized were obtained during the first run with immobilized B. longum in the spiral-sheet bioreactor. In producing lactic acid L. helveticus performed better when using the Spiral Sheet Bioreactor and B. longum showed better performance with gel bead immobilization. Because B. longum is a very promising new bacterium for lactic acid production from cheese whey, its optimum fermentation conditions such as pH and metabolic pathway need to be studied further. The ultrafiltration tests have shown that 94% of the cell and cheese whey proteins were retained by membranes with a mol wt cutoff of 5 and 20 KDa.     

Production of cellulase and xylanase in a bubble column using immobilizedAspergillus Niger KKS

Aspergillus niger KKS, isolated from a farmland near Suwon, was immobilized on Celite and polyurethane foams. Enzyme activities produced by the immobilized cell system in a bubble column were higher than that of shake-flask culture. The enzyme productivities were twice as high. β-Glucosidase, β-xylosidase, and xylanase activities obtained in a bubble column were significant when the ground rice straw was used as a substrate.     

Solid-state fermentation of lignocellulosic plant residues from Brassica napus by Pleurotus ostreatus

Solid-state fermentation (SSF) of inedible parts of rapeseed was carried out using a white-rot fungus, Pleurotus ostreatus, to degrade lignocellulosic material for mycelial-single cell protein (SCP) production. This SSF system has the potential to be adapted to a controlled ecological life support system in space travel owing to the lack of storage space. The system for converting lignocellulosic material to SCP by P. ostreatus is simple; it can be carried out in a compact reactor. The fungal vegetative growth was better with a particle size of plant material ranging from 0.42 to 10 mm, whereas lignin degradation of the lignocellulose was the highest with particle sizes ranging from 0.42 to 0.84 mm. The addition of veratry alcohol (3,4-dimethoxybenzyl alcohol), hydrogen peroxide, and glycerol promotes lignocellulose degradation by P. ostreatus. The enhancement of bioconversion was also observed when a gas-flow bioreactor was used to supply oxygen and to maintain the constant moisture of the reactor. With this reactor, approx 85% of the material was converted to fungal and other types of biomass after 60 d of incubation.     

An electrokinetic bioreactor: using direct electric current for enhanced lactic acid fermentation and product recovery

The microbiological production of organic acids by fermentation processes is growing in commercial importance. However, the removal of product and pH control are two main issues that limit the technical and commercial viability of such processes. A laboratory scale bioreactor combining conventional electrodialysis and bipolar membrane electrodialysis has been developed for in situ product removal and pH control in lactic acid fermentation. The electrokinetic process enabled removal of the biocatalytic product (lactic acid) directly from the bioreactor system, in a concentrated form, as well as enabling good pH control without generation of troublesome salts. Moreover, end-product inhibition of glucose catabolism was reduced, resulting in a greater generation of the end-product lactic acid. An automatic pH sensor and current application system was developed and successfully implemented for lactic acid fermentation in the electrokinetic bioreactor.     

Development of a membrane-based vapor-phase bioreactor

A vapor-phase bioreactor has been developed utilizing porous metal membranes in a cylindrical design employing radial flow as opposed to traditional axial flow for the vapor stream. The system was evaluated for the biodegradation ofp-xylene (p-xylene) from a water-saturated air stream byPseudomonas putida ATCC 23973 immobilized onto sand. The biocatalyst was placed in the annular space between two cylindrical, porous stainless-steel membranes. Details of the reactor system are presented along with biological data verifying system performance. The feed flow rate andp-xylene concentration were varied between 60 and 130 cm³/min and 15–150 ppm, respectively. Continuous reactor operation was maintained for 80–200 h with removal efficiencies (based onp-xylene disappearance) between 80 and 95%. The effluent concentration histories were compared to determine the operating range of the bioreactor.     

Development of continuous fermentation using immobilized yeast cells for wine cooler process development

The continuous wine fermentation process, which employs a newly designed tapered column type bioreactor and immobilized yeast cells (Montrachet 522), was studied and its fermentation performance was compared with batch and suspended cell continuous wine fermentation systems. It was found that a stable continuous culture fermentation process could be maintained for a period of 2–3 mo when the new bioreactor system packed with immobilized yeast cells was employed. The new bioreactor containing immobilized yeast cells performed significantly better than the suspended cell culture system or batch culture. The effluent wine from the continuous fermentor system contained 7.1% (v/v) ethanol and 0.18% (w/v) residual sugar at 0.01 h^-1 dilution rate. The new continuous bioreactor system also gave 17–34 times higher maximum ethanol productivity compared to the conventional batch wine fermentation. At a low dilution rate, 0.01^-1, as high as 92% sugar to ethanol yield was achieved. Based on the results obtained from this study, the possibility of developing a continuous wine cooler fermentation process was demonstrated. A two-stage continuous wine fermentation system may be designed and operated. The grape juice can be fed into the first-stage that is operated at about 0.2 h^-1 dilution rate and the effluent from the first-stage is fed into the second-stage continuous fermentor operated at about 0.01 h^-1 dilution rate. By doing so, a wine cooler can be produced continuously and efficiently, by employing the newly designed tapered column type bioreactor charged with the immobilized yeast cells.     

Phenols removal by immobilized tyrosinase reactor in on-line high performance liquid chromatography

The development of an immobilized enzyme reactor (IMER) based on tyrosinase immobilized on aminopropyl-controlled pore glass (AP-CPG) for the removal of phenols from model aqueous solutions was reported. To elucidate the influence of the substrate nature, the apparent (V'max, K'm) and the inherent (Vmax, Km) Michaelis-Menten constants were determined by Lineweaver-Burk method and the external diffusional contributions on measured enzyme activities were removed by a graphical method. The dephenolization process was realized by recycling the phenol solutions through the bioreactor connected to a chitosan trap in order to remove the colored quinone-type products of the tyrosinase reactions. The results indicated that a complete removal of phenol derivatives in the range of 150-300 min, with the exception of 60% removal for phenol reached in 400 min, was obtained. The observed sequence: cresol > 4-methylcathecol > catechol > 4-Cl-phenol phenol was in accordance to the V'max/K'm values.     

Operability and feasibility of ethanol production by immobilizedZymomonas mobilis in a fluidized-bed bioreactor

Studies have been carried out using immobilized Z.mobilis in fluidized-bed bioreactors and have emphasized operation during high productivity and conversion. The bacteria are immobilized within small uniform beads (~1 to 1.5-mm diam) of K-carrageenan at cell loadings of 15-50 g (dry wt)/L. Conversion and productivity were measured under a variety of conditions, including feedstocks, flow rates, temperature, pH, and column sizes (up to 2.5 m tall). Volumetric productivities of 50-120 g EtOH/h-L reactor volume have been achieved. Productivities of 60 g/h-L are demonstrated from a 15% feed with residual glucose concentrations of less than 0.1% and 7.4% EtOH in the tallest fermentor. Among feeds of 10, 15, and 20% dextrose, the 15% gave the highest productivity and avoided substrate inhibition. A temperature of 30°C and pH 5 were the optimum conditions. The ethanol yield was shown to be nearly constant at 0.49 g EtOH/g glucose, or 97% of the theoretical under a variety of conditions and transients. The biocatalyst beads have been shown to remain active for two months. Nonsterile feed has been used for weeks without detrimental contamination. The advantages of this advanced bioreactor system over conventional batch technology are discussed.     

Enzymes immobilized on montmorillonite: comparison of performance in batch and packed-bed reactors

Summary The enzymes a-amylase, invertase and glucoamylase were immobilized on acid activated montmorillonite using two techniques, viz. adsorption and covalent binding, and their activities were tested in a batch and packed-bed reactor and were compared. The packed-bed reactor showed an improved performance for all immobilized enzymes, which was attributed to lowering of diffusional restrictions to mass transfer. Lower activity in case of batch reactor for immobilized invertase was due to a combined effect of loss of native conformation of enzyme on account of immobilization and mass transfer resistances due to improper diffusion of substrate to the active site of enzyme. For immobilized glucoamylase, the packed-bed reactor demonstrated exceptionally high activity that was very close to the free enzyme. Covalently bound glucoamylase showed higher activity than the free enzyme.     

Continuous attrition bioreactor with enzyme recycling for the bioconversion of cellulose

A new type of reactor, the attrition bioreactor, has been developed to increase the rate of the enzymatic hydrolysis of cellulose and also to cut pretreatment costs. It was found that the attrition bioreactor could be operated continuously or semicontinuously in conjunction with a membrane filter to produce a high cellulose conversion rate and low enzyme consumption. The membrane filter served to contain the enzyme and cellulose within the reactor while allowing sugar to permeate as a product.     

Tandem use of carboxypeptidase Y reactor and displacement chromatograph for peptide synthesis

A packed-bed enzyme reactor with immobilized carboxypeptidase Y was used in tandem with a displacement chromatograph for the preparation of N-benzoyl-L-arginyl-L-methioninamide, from N-benzoyl-L-arginine and L-methioninamide. The pumps and valves of the coupled enzyme reactor and displacement chromatograph were controlled by a microprocessor. The enzyme was immobilized on microparticulate amino-silica by glutaraldehyde and packed into a 60 X 4.6 mm I.D. column. The packed-bed reactor was used in the recirculating mode and components of the reaction mixture were subsequently separated by displacement chromatography on a 250 X 4.6 mm octadecyl-silica column using butoxyethoxyethanol as the displacer. Unreacted L-methioninamide was returned to the reaction mixture. Both the progress of the reaction and the extent of separation by displacement chromatography were monitored by high-performance liquid chromatographic analysis. The system was designed so that enzymatic peptide synthesis, separation by displacement chromatography, and column regeneration were carried out simultaneously by using two identical columns in parallel. An amount of 460 mg of N-benzoyl-L-arginyl-L-methioninamide having purity greater than 99% could be obtained in 24 h with this system. The tandem operation of the enzyme reactor and liquid chromatograph operated in the displacement mode offers a means for the synthesis and purification of peptides.     

Lactic acid production from cellulosic material by synergetic hydrolysis and fermentation

The hydrolysis process on corncob residue was catalyzed synergetically by the cellulase from Trichoderma reesei and the immobilized cellobiase. The feedback inhibition to cellulase reaction caused by the accumulation of cellobiose was eliminated efficiently. The hydrolysis yield of corncob residue was 82.5%, and the percentage of glucose in the reducing sugar reached 88.2%. The glucose in the cellulosic hydrolysate could be converted into lactic acid effectively by the immobilized cells of Lactobacillus delbrueckii. When the enzymatic hydrolysis of cellulose and the fermentation of lactic acid were coupled together, no glucose was accumulated in the reaction system, and the feedback inhibition caused by glucose was also eliminated. Under the batch process of synergetic hydrolysis and lactic acid fermentation with 100 g/L of cellulosic substrate, the conversion efficiency of lactic acid from cellulose and the productivity of lactic acid reached 92.4% and 0.938 g/(L·h), respectively. By using a fed-batch technique, the total concentration of cellulosic substrate and lactic acid in the synergetic process increased to 200 and 107.5 g/L, respectively, whereas the dosage of cellulase reduced from 20 to 15 IU/g of substrate in the batch process. The results of the bioconversion of renewable cellulosic resources were significant.     

Determination of l-phenylalanine in serum by flow-injection analysis using immobilized phenylalanine dehydrogenase and fluorimetric detection

A flow-injection system with an immobilized enzyme reactor is proposed for the determination of l-phenylalanine. Phenylalanine dehydrogenase from Rhodoccus sp. M4 was immobilized on tresylated poly (vinyl alcohol) beads (13 mum) and packed into a stainless-steel column (5 cm x 4 mm i.d.). Serum sample was deproteinized with tungstic acid and filtered through an ultrafiltration membrane. The sample solution (30 mul) was injected into the carrier stream (water). The NADH formed was detected at 465 nm (excitation at 340 nm). The calibration graph was linear for 0.9-600 mum l-phenylalanine; the detection limit was 0.3 mum. The sample throughout was 25 h(-1) without carryover. The half-life period of the immobilized enzyme was 23 days.     

Simultaneous fermentation and separation of lactic acid in a biparticle fluidized-bed bioreactor

A bioreactor configuration has been tested for simultaneous fermentation and separation of the desired inhibitory product, lactic acid. The bioreactor is a fluidized bed of immobilized Lactobacillus delbreuckii. Another solid phase of denser sorbent particles (a poly-vinyl pyridine resin) was added to this fluidized bed. These sorbent particles fell through the bed, absorbed the product, and were removed. In test fermentations, the addition of the sorbent enhanced the fermentation and moderated the fall of the pH. The biparticle fluidized-bed bioreactor utilizing immobilized microorganisms and adsorbent particles has been shown to enhance the production of lactic acid fourfold in this nonoptimized system.

     

Use of a new membrane-reactor saccharification assay to evaluate the performance of celluloses under simulated ssf conditions

A new saccharification assay has been devised, in which a continuously buffer-swept membrane reactor is used to remove the solubilized saccharification products, thus allowing high extents of substrate conversion without significant inhibitory effects from the buildup of either cellobiose or glucose. This diafiltration saccharification assay (DSA) can, therefore, be used to obtain direct measurements of the performance of combinations of cellulase and substrate under simulated SSF conditions, without the saccharification results being complicated by factors that may influence the subsequent fermentation step. This assay has been used to compare the effectiveness of commercial and special in-house-producedTrichoderma reeSci. cellulase preparations in the saccharification of a standardized microcrystalline (Sigmacell) substrate and a dilute-acid pretreated lignocellulosic substrate. Initial results strongly suggest that enzyme preparations produced in the presence of the targeted lignocellulosic substrate will saccharify that substrate more effectively. These results call into question the widespread use of the “filter paper assay” as a reliable predictor of enzyme performance in the extensive hydrolysis of substrates that are quite different from filter paper in both physical properties and chemical composition.

     

A chemiluminometric method for NADPH and NADH using a two-enzyme bioreactor and its application to the determination of magnesium in serum

Chemiluminometric methods are described for the automated flow injection analysis of NADPH and NADH using an immobilized enzyme column reactor and serum magnesium. This application is for the clinical analysis of NADPH and NADH. The reactor for NADPH and NADH contains immobilized L-glutamate dehydrogenase and L-glutamate oxidase, and that for serum magnesium immobilized hexokinase, glucose-6-phosphate dehydrogenase, L-glutamate dehydrogenase and L-glutamate oxidase. When the sample is introduced into the four-enzyme bioreactor, hydrogen peroxide is produced in proportion to the concentration of serum magnesium by the successive reactions. A co-immobilized hexokinase/glucose-6-phosphate dehydrogenase/glutamate dehydrogenase column reactor gave better efficiency compared with an enzyme column which was prepared by packing co-immobilized hexokinase/glucose-6-phosphate dehydrogenase and immobilized glutamate dehydrogenase to make two layers. Magnesium in serum was determined with 1 microL of the sample without carry-over and for an assay time of approximately 15 s. The present method is sensitive (detection limit 0.1 nmol) because Mg2+ is recycled in a column, and gives perfect linearity of the data up to 3.0 mmol/L with satisfactory precision, reproducibility, and accurate reaction recoveries.     

Ethanol production from corn starch in a fluidized-bed bioreactor

The production of ethanol from industrial dry-milled corn starch was studied in a laboratory-scale fluidized-bed bioreactor using immobilized biocatalysts. Saccharification and fermentation were carried out either simultaneously or separately. Simultaneous saccharification and fermentation (SSF) experiments were performed using small, uniform κ-carrageenan beads (1.5–2.5 mm in diameter) of co-immobilized glucoamylase and Zymomonas mobilis. Dextrin feeds obtained by the hydrolysis of 15% drymilled corn starch were pumped through the bioreactor at residence times of 1.5–4h. Single-pass conversion of dextrins ranged from 54–89%, and ethanol concentrations of 23–36 g/L were obtained at volumetric productivities of 9–15 g/L-h. Very low levels of glucose were observed in the reactor, indicating that saccharification was the rate-limiting step. In separate hydrolysis and fermentation (SHF) experiments, dextrin feed solutions of 150–160 g/L were first pumped through an immobilized-glucoamylase packed column. At 55°C and a residence time of 1 h, greater than 95% conversion was obtained, giving product streams of 162–172 g glucose/L. These streams were then pumped through the fluidized-bed bioreactor containing immobilized Z. mobilis. At a residence time of 2 h, 94% conversion and ethanol concentration of 70 g/L were achieved, resulting in an overall process productivity of 23 g/L-h. Atresidence times of 1.5 and 1 h, conversions of 75 and 76%, ethanol concentrations of 49 and 47 g/L, and overall process productivities of 19 and 25 g/L-h, respectively, were achieved.