Production of sorbitol and gluconic acid by permeabilized cells of Zymomonas mobilis

Summary Zymomonas mobilis is able to convert glucose and fructose to gluconic acid and sorbitol. The enzyme, glucose-fructose oxidoreductase, catalysing the intermolecular oxidation-reduction of glucose and fructose to gluconolactone and sorbitol, was formed in high amounts [1.4 units (U)·mg^-1] when Z. mobilis was grown in chemostats with glucose as the only carbon source under non-carbon-limiting conditions. The activity of a gluconolactone-hydrolysing lactonase was constant at 0.2 U·mg^-1. Using glucose-grown cells for the conversion of equimolar fructose and glucose mixtures up to 60% (w/v), a maximum product concentration of only 240 g·1^-1 of sorbitol was found. The gluconic acid accumulated was further metabolized to ethanol. After permeabilizing the cells using cationic detergents, maximum sorbitol and gluconic acid concentrations of 295 g·1^-1 each were reached; no ethanol production occurred. In a continuous process with -carrageenan-immobilized and polyethylenimin-hardened, permeabilized cells no significant decrease in the conversion yield was observed after 75 days. The specific production rates for a high yield conversion ( > 98%) in a continuous two-stage process were 0.19 g·g^-1·h^-1 for sorbitol and 0.21 g·g^-1·h^-1 for gluconic acid, respectively. For the sugar conversion of cetyltrimethylammonium bromide-treated -carrageenan-immobilized cells a V _max of 1.7 g·g^-1·h^-1 for sorbitol production and a K _m of 77.2 g·1^-1 were determinedOffprint requests to: B. Rehr     

Fructose production by immobilizedArthrobacter cells

Summary ImmobilizedArthrobacter cells (NRRL-B-3728) were used for continuous isomerization of glucose to fructose in a bioreactor system. The system utilized stationary phase (55h) cells (2.2×10⁹ CFU/ml saline) immobilized onto K-carrageenan (3% w/v) beads [cells were heated at 65°C for 10 min to inactivate endogenous proteolytic enzymes]. Immobilized-cell preparations were hardened using three different glutaraldehyde systems. Glutaraldehyde (0.2 M) treated-immobilized cells (pH 7.0, 5°C for 30 min) exhibited good gel strength and high glucose isomerase activities. Maximal bioreactor isomerization of 44% was achieved when a buffered feedstock containing 40% glucose was fed into the column (60°C) at a flow rate of 0.2 ml/min. The biological half-life of glucose isomerase activities in this system was 400 h. Scanning electron microscopy revealed large numbers of cells distributed within the beads. A thin layer surrounding the beads following glutaraldehyde treatment was mainly due to cross-linking reactions between cell proteins and glutaraldehyde. This layer prevented leaking of cells during continuous isomerization reaction.     

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.     

Single-pot conversion of cephalosporin C to 7-aminocephalosporanic acid in the absence of hydrogen peroxide

In this study, d-amino acid oxidase (DAAO) and catalase (CAT) in the permeabilized recombinant Pichia pastori cells were well investigated. It appeared that their thermal stability was negatively correlated with the apparent enzymatic activities. The frozen-melted cells presented the best stability and the lowest apparent activities of DAAO and CAT, whereas the cetyltrimethylammonium bromide (CTAB) permeabilized cells displayed the weakest stability and the highest apparent activities of the two enzymes. Simultaneous action of DAAO and CAT in the CTAB-permeabilized cells and glutaryl-7-aminocephalosporanic acid acylase (GA) immobilized on carrier contributed to the conversion of cephalosporin C (CPC) to 7-aminocephalosporanic acid (7-ACA) with a yield of 76.2%. During such a reaction cycle, no visible activity loss occurred at the immobilized GA, whereas the loss rates of DAAO and CAT activities were about 0.029 and 1.13 U min⁻¹, respectively. Nevertheless, this problem could be easily solved by continuous feeding of the new permeabilized cell suspension at the rate of 6 ml h⁻¹ to the reactor. Following such a fed-batch strategy, these permeabilized cells and the immobilized GA could be efficiently reused for 6 and 15 reaction cycles, respectively, yielding around 76% 7-ACA at each reaction cycle.     

Evaluation of succinic acid continuous and repeat-batch biofilm fermentation by <Emphasis Type="Italic">Actinobacillus succinogenes</Emphasis> using plastic composite support bioreactors

Continuous and repeat-batch biofilm fermentations using Actinobacillus succinogenes were performed with immobilized and suspended-cell systems. For the immobilized continuous system, plastic composite supports (PCS) containing 50% (w/w) polypropylene (PP), 35% (w/w) ground soybean hulls, 5% (w/w) dried bovine albumin, 2.5% (w/w) soybean flour, 2.5% (w/w) yeast extract, 2.5% (w/w) dried red blood cells, and 2.5% (w/w) peptone, or PP tubes (8.5 cm in length) were arranged around the agitator shaft in a grid formation. Agitation was controlled at 125 rpm and 150 rpm. Samples were taken at dilution rates of 0.2, 0.4, 0.6, 0.8, 1.0, and 1.2 h^–1 and analyzed for succinic acid production and glucose consumption (g l^–1). For PCS bioreactors, the highest final succinic acid concentrations (10.1 g ^–1, 10.4 g l^–1) and percentage yields (62.6%, 71.6%) occurred at the dilution rate of 0.2 h^–1. PCS disks were evaluated in a repeat-batch biofilm reactor. Suspended-cell batch fermentations were performed in flasks and a repeat-batch bioreactor. The maximum concentration of succinic acid produced was 40 g l^–1. Peak succinic acid percentage yields in continuous and repeat-batch fermentations of A. succinogenes were observed in suspended-cell continuous fermentations at a dilution rate of 1.0 h^–1 (76.2%) and in PCS repeat-batch fermentations with an initial glucose concentration of 40 g l^–1 (86.7%).     

Continuous production of gibberellic acid in a fixed-bed reactor by immobilized mycelia of Gibberella fujikuroi in calcium alginate beads

Summary The continuous production of gibberellic acid with immobilized mycelia of Gibberella fujikuroi was maintained over a hundred days in a tubular fixed-bed reactor. Free mycelium at the beginning of the storage phase was harvested from G. fujikuroi shake-flask culture and was immobilized by ionotropic gelation in calcium alginate beads.The continuous recycle production system consisted of a fixed-bed reactor, a container in which the culture medium was heated, stirred and aerated, and valves for sample withdrawal or reactant addition during the first 1320 h (55 days). A two-phase continuous extractor was then added for the last 960 hours (40 days). Free and immobilized mycelium shake-flask cultures with the same strain used in the continuous culture system were also realized to compare growth, maintenance and production parameters. The results show about the same gibberellic acid productivity in both free and immobilized mycelium shakeflask cultures: 0.384 and 0.408 mgGA₃·gBiomass^-1 ·day^-1, respectively, whereas in the continuous system the gibberellic acid production is about twice as large for a similar biomass: 0.768 mgGA₃·gBiomass^-1·day^-1. Several factors affecting the overall productivity of the immobilized systems were found to be: the quality and the quantity of mycelia in the biocatalyst beads and the immobilization conditions.     

Continuous production of ethanol using yeast cells immobilized in preformed cellulose beads

 Saccharomyces cerevisiae cells were immobilized on preformed cellulose beads by adsorption. The fermentation capacity of the immobilized yeast cells was found to be practically independent of the hydrogen ion concentration between pH 3.1 and 6.25. The fermentation capacity was maximal at 30 °C. The immobilized yeast cells were used for continuous production of ethanol in a fluidized-bead reactor. The average values characteristic for the process were an ethanol concentration of 41.9±0.1 g l^-1, a fermentation efficiency of 82.9±2.1% and a volumetric productivity of 3.94±0.52 g l^-1 h^-1. Received: 9 October 1995/Accepted: 22 April 1996     

The simultaneous production of sorbitol from fructose and gluconic acid from glucose using an oxidoreductase of Zymomonas mobilis

Summary The production of sorbitol and gluconic acid by toluene-treated, permeabilized cells of Zymomonas mobilis has been evaluated. From a 60% total sugar solution (300 g/l glucose and 300 g/l fructose), a sorbitol concentration of 290 g/l and a gluconic acid concentration of 283 g/l were achieved after 15 h in a batch process using free toluene-treated cells. A continuous process with immobilized cells was developed and only a small loss of enzyme activity (less than 5%) was evident after 120 h. With a strongly basic anion exchange resin and an eluent of 0.11 M Na₂B₄O₇/0.11 M H₃BO₃, good separation of sorbitol and gluconic acid was achieved.     

Performance of immobilized cells for dihexyl sulfosuccinate biotransformation

Possibilities of using immobilized bacterial cells for waste water treatment in a continuous process was determined. Cells ofComamonas terrigena strain N3H immobilized in calcium alginate beads were successful by used in packed bead-type reactor for continuous biotransformation of the anion-active surfactant dihexyl sulfosuccinate. Absence of calcium ions from the treated medium led to the disruption of alginate beads within 8 d of usage. When the medium was supplemented with Ca²⁺ ions the beads were stable for at least one month in the continuous process. During the whole time period the transformation effectivity was in the range of 80–100% even at the highest, flow rate of 14 mL/min. Presented at the 4th Mini-Symposium on Biosorption and Microbial Degradation, Prague, Czech Republic, November 26–29, 1996.     

Enhanced continuous production of lovastatin using pellets and siran supported growth of Aspergillus terreus in an airlift reactor

Lovastatin, a hypocholesterolemic agent, is a secondary metabolite produced by filamentous microorganism Aspergillus terreus in submerged batch cultivation. Lovastatin production by pellets and immobilized siran cells was investigated in an airlift reactor. The process was carried out by submerged cultivation in continuous mode with the objective of increasing productivity using pellet and siran supported growth of A terreus. The continuous mode of fermentation improves the rate of lovastatin production. The effect of dilution rate and aeration rate were studied in continuous culture. The optimum dilution rate for pellet was 0.02 h⁻¹ and for siran carrier was 0.025 h⁻¹. Lovastatin productivity using immobilized siran carrier (0.0255 g/L/h) was found to be greater than pellets (0.022 g/L/h). The productivity by both modes of fermentation was found higher than that of batch process which suggests that continuous cultivation is a promising strategy for lovastatin production.     

Biological treatment of phenolic wastes: Comparison between free and immobilized cell systems

Summary Phenol degradation by free and immobilized cells ofFusarium flocciferum was studied in a chemostat at steady-state conditions. For the free cell system the dilution rates varied from 0.02 to 0.13h^–1, with a total phenol removal up to 0.08h^–1. Wash-out seemed to set in at 0.11h^–1. The immobilized cells showed virtually complete phenol utilization at 1g/l, over a period of four months. At D=0.2h^–1 and above 1g/l phenol, the complete phenol removal is not achieved: a progressive increase in the outlet concentration was observed attaining a value of 284mg/l at 1.5g/l.     

An automated flow injection analysis system for on-line monitoring of glucose and L-lactate during lactic acid fermentation in a recycle bioreactor

The study concerns on-line sequential analysis of glucose and L-lactate during lactic acid fermentation using a flow injection analysis (FIA) system. Enzyme electrodes containing immobilized glucose oxidase and L-lactate oxidase were used with an amperometric detection system. A 12-bit data acquisition card with 16 analog input channels and 8 digital output channels was used. The software for data acquisition was developed using Visual C++, and was devised for sampling every hour for sequential analyses of lactate and glucose. The detection range was found to be 2–100 g l^–1 for glucose and 1–60 g l^–1 for L-lactate using the biosensors. This FIA system was used for monitoring glucose utilization and L-lactate production by immobilized cells of Lactobacillus casei subsp. rhamnosus during a lactic acid fermentation process in a recycle batch reactor. After 13 h of fermentation, complete sugar utilization and maximal L-lactate production was observed. A good agreement was observed between analysis data obtained using the biosensors and data from standard analyses of reducing sugar and L-lactate. The biosensors exhibited excellent stability during continuous operation for at least 45 days.     

Degradation of 2-methylnaphthalene by free and immobilized cells of Pseudomonas sp. strain NGK1

A Pseudomonas sp. strain NGK1 (NCIM 5120) capable of utilizing 2-methylnaphthalene (2-MN) was immobilized in various matrices namely, polyurethane foam (PUF), alginate, agar and polyvinyl alcohol (PVA) (1.5 × 10¹² c.f.u. g^–1 beads). The degradation rates of 25 and 50 mM 2-MN by freely suspended cells (2 × 10¹¹ c.f.u. ml^–1) and immobilized cells in batches, semi-continuous with shaken culture and continuous degradation in a packed-bed reactor were compared. The PUF-immobilized cells achieved higher degradation of 25 and 50 mM of 2-MN than freely suspended cells and the cells immobilized in alginate, agar or PVA. The PVA- and PUF-immobilized cells could be reused for more than 30 and 20 cycles respectively, without losing any degradation capacity. The effect of dilution rates on the rate of degradation of 25 and 50 mM 2-MN with freely suspended and immobilized cells were compared in the continuous system. Increase in dilution rate increased the degradation rate only up to 1 h^–1 in free cells with 25 mM 2-MN and no significant increase was observed with 50 mM 2-MN. With immobilized cells, the degradation rate increased with increase in dilution rate up to 1.5 h^–1 for 25 mM and 1 h^–1 for 50 mM 2-MN. These results revealed that the immobilized cell systems are more efficient than freely suspended cells for biodegradation of 2-MN.     

Immobilization of the slime mould Dictyostelium discoideum for the continuous production of recombinant human antithrombin III

Recombinant vegetative Dictyostelium discoideum cells were immobilized inside a porous matirx by an inorganic membrane that was permeable to nutrients but not to cells, in order to produce recombinant human antithrombin III. Cells so entrapped could reach up to 15 times higher biomass densities compared with organisms growing freely in suspension. The high cell concentration maintained in the immobilized cell bioreactor caused an increase in specific and volumetric productivity. In continuous operation a maximum volumetric antithrombin productivity of 56 ng h ^–1 ml ^–1 catalyst bulk volume was attained at a dilution rate of 0.016 h ^–1. In addition, the good retention of metabolic activity for several weeks as well as the convenient form of storage and regeneration of the catalytic system were shown. Correspondence to: H. Tiltscher     

Degradation kinetics of phenol by immobilized cells of Candida tropicalis in a fluidized bed reactor

Degradation kinetics of phenol by free and agar-entrapped cells of Candida tropicalis was studied in batch cultures. The initial phenol degradation rate achieved with free cells was higher than that obtained with immobilized cells, when phenol concentrations up to 1000 mg l^–1 were used. However, at higher phenol concentrations, the behaviour was quite different. The initial degradation rate of the immobilized yeast cells was about 10 times higher than that of the free cells, at a phenol concentration of 3500 mg l^–1. The semicontinuous and continuous degradation of phenol by immobilized yeast cells was also investigated in a multi-stage fluidized bed reactor. The highest phenol removal efficiencies and degradation rates as well as the lowest values of residual phenol and chemical oxygen demand were obtained in the semicontinuous culture when phenol concentrations up to 1560 mg l^–1 were used.     

Optimization of lactic acid production by immobilized <Emphasis Type="Italic">Lactococcus lactis</Emphasis> IO-1

Production of lactic acid from glucose by immobilized cells of Lactococcus lactis IO-1 was investigated using cells that had been immobilized by either entrapment in beads of alginate or encapsulation in microcapsules of alginate membrane. The fermentation process was optimized in shake flasks using the Taguchi method and then further assessed in a production bioreactor. The bioreactor consisted of a packed bed of immobilized cells and its operation involved recycling of the broth through the bed. Both batch and continuous modes of operation of the reactor were investigated. Microencapsulation proved to be the better method of immobilization. For microencapsulated cells at immobilized cell concentration of 5.3 g l⁻¹, the optimal production medium had the following initial concentrations of nutrients (g l⁻¹): glucose 45, yeast extract 10, beef extract 10, peptone 7.5 and calcium chloride 10 at an initial pH of 6.85. Under these conditions, at 37 °C, the volumetric productivity of lactic acid in shake flasks was 1.8 g l⁻¹ h⁻¹. Use of a packed bed of encapsulated cells with recycle of the broth through the bed, increased the volumetric productivity to 4.5 g l⁻¹ h⁻¹. The packed bed could be used in repeated batch runs to produce lactic acid.     

Biodegradation of phenol by immobilized Aspergillus awamori NRRL 3112 on modified polyacrylonitrile membrane

Covalent immobilization of Aspergillus awamori NRRL 3112 was conducted onto modified polyacrylonitrile membrane with glutaraldehyde as a coupling agent. The polymer carrier was preliminarily modified in an aqueous solution of NaOH and 1,2-diaminoethane. The content of amino groups was determined to be 0.58 mgeq g⁻¹. Two ways of immobilization were used—in the presence of 0.2 g l⁻¹ phenol and without phenol. The capability of two immobilized system to degrade phenol (concentration—0.5 g l⁻¹) as a sole carbon and energy source was investigated in batch experiments. Seven cycles of phenol biodegradation were conducted. Better results were obtained with the immobilized system prepared in the presence of phenol, regarding degradation time and phenol biodegradation rate. Scanning electron micrographs of the polyacrylonitrile membrane/immobilized Aspergillus awamori NRRL at the beginning of repeated batch cultivation and after the 7th cycle were compared. After the 7th cycle of cultivation the observations showed large groups of cells. The results from the batch experiments with immobilized system were compared to the results produced by the free strain. Phenol biodegradation experiments were carried out also in a bioreactor with spirally wound membrane with bound Aspergillus awamori NRRL 3112 in a regime of recirculation. 10 cycles of 0.5 g l⁻¹ phenol biodegradation were run consecutively to determine the degradation time and rate for each cycle. The design of the bioreactor appeared to be quite effective, providing large membrane surface to bind the strain.     

Glutathione production coupled with an ATP regeneration system

Summary Escherichia coli cells possessing glutathione synthetase and acetate kinase activities were immobilized with carrageenan gel. To enhance the operational stability, immobilized cells were treated with hardening agent, glutaraldehyde in the presence of hexamethylenediamine. The continuous production of glutathione was investigated using the column packed with immobilized Escherichia coli cell preparations. Glutathione was continuously produced by this column in the presence of acetyl phosphate and the half-life of this column was calculated to be 8 days at the flow rate of S.V.=0.1 h^–1 at 37°C.     

Ethanol production by immobilized whole cells ofZymomonas mobilis in a continuous flow expanded bed bioreactor and a continuous flow stirred tank bioreactor

Continuous ethanol fermentation by immobilized whole cells ofZymomonas mobilis was investigated in an expanded bed bioreactor and in a continuous stirred tank reactor at glucose concentrations of 100, 150 and 200 g L^–1. The effect of different dilution rates on ethanol production by immobilized whole cells ofZymomonas mobilis was studied in both reactors. The maximum ethanol productivity attained was 21 g L^–1 h^–1 at a dilution rate of 0.36 h^–1 with 150 g glucose L^–1 in the continuous expanded bed bioreactor. The conversion of glucose to ethanol was independent of the glucose concentration in both reactors.     

Effect of medium and temperature of storage on viability of lactic acid bacteria immobilized in κ-carrageenan-locust bean gum gel beads

Summary The influence of various storage solutions and temperature (4°C and 25°C) on viability ofStreptococcus salivarius subsp.thermophilus andLactobacillusdelbrueckii subsp.bulgaricus entrapped in κ-carrageenan-locust bean gum mixed gel beads was studied. The immobilized strains could be stored at 4°C in all storage solutions studied for at least 14 and 11 days respectively before counts decreased to 10⁵c.f.u./mL, which was considered to be the practical limit for their use as inoculum in a fermentation process. The most effective storage solutions for preserving cell viability at 4°C were NaCl, glycerol and sorbitol solutions forS. thermophilus, and PO₄ buffer and sorbitol solutions forL. bulgaricus. At 25°C,S. thermophilus could be stored for over 14 days in all solutions except glycerol, andL. bulgaricus for 4 days in 10% sorbitol.