Continuous production of 4-ethylguaiacol by immobilized cells of salt-tolerant Candida versatilis in an airlift reactor

The effects of pH, temperature, aeration, and residence time on the continuous production of 4-ethyl-guaiacol (4-EG), which is one of the characteristic aroma components in soy sauce, by immobilized cells of the salt-tolerant yeast Candida versatilis were investigated using an airlift reactor. The optimum pH and temperature were about 4.0 and 30–33°C, respectively. The amount of 4-EG in the liquid was constant even during alterations of nitrogen/air ratio in the supplied gas. A large amount of 4-EG (over 20 ppm) was produced at a residence time from 5 to 28 h and 1–3 ppm of 4-EG, which was the optimum concentration in conventional soy souce, was produced at a shorter residence time of 0.5 h. The 4-EG production by immobilized C. versatilis cells using the airlift reactor was stable for 40 d. It was found that the immobilized cell method was effective for the production of 4-EG by C. versatilis cells.     

Immobilized cell microchannel bioreactor for evaluating fermentation characteristics of mixed substrate consumption and product formation

An immobilized cell microchannel bioreactor was designed to test continuous fermentation. The fermentation set-up included a bottom hydrophilic quartz channel to immobilize cells using 0.4 wt% polyethyleneimine and a top channel designed to continuously remove metabolically generated carbon dioxide using hydrophobic polypropylene. To evaluate fermentation characteristics of immobilized cells, ethanol fermentation was carried out using Saccharomyces cerevisiae and Pichia stipitis. The immobilized cell microchannel bioreactor was used to identify long-term activity of immobilized S. cerevisiae cells. The continuous flow microchannel bioreactor was operated stably over a period of 1 month. The immobilized cell microchannel bioreactor was used to examine the characteristics cells that consumed mixed substrates. The concentration ratio of glucose to xylose for simultaneous utilization of hemicellulosic sugars was evaluated using the microchannel bioreactor and the results were compared with those obtained by using conventional batch fermentation with P. stipitis.     

Effects of suboptimal environmental conditions on immobilized bacteria growing in continuous culture

Two immobilized bacterial cultures with the ability to metabolize 6-amino-2-naphthalenesulfonic acid (6A2NS) and 2-naphthalene-sulfonic acid (2NS) were investigated under suboptimal environmental growth conditions. The cultures were employed in continuously operated airlift loop-reactors. The physico-chemical growth parameters such as pH, temperature and dissolved oxygen concentration were varied. It was found that a decreasing growth rate in suboptimal conditions was compensated by increasing biomass concentration over a wide range. Operated continuously for more than 20 months, the 6A2NS-degrading system appeared to be reliable. After pH-shockloadings and long-term oxygen default, the immobilized microorganisms recovered almost immediately and stable operating conditions were achieved again within less than 48 h. These remarkable results were sustained with the 2NS degrading system.     

Pseudo-steady state oxygen-concentration profiles in an agar slab containing growing Nitrobacter agilis

A dynamic model for growth and substrate consumption of immobilized cells is evaluated by comparing experimental and calculated psendo-steady-state oxygen-concentration profiles in the support material. For this, Nitrobacter agilis cells were immobilized and cultivated in agar slabs. The system was operated as a continuous submerged-bed reactor. At different growth states oxygen-concentration profiles were determined in the slabs with a microsensor. It was shown that simulated and measured profiles agreed very well, both in steady and in pseudo-steady states.     

Effect of herbicide adjuvants on the biodegradation rate of the methylthiotriazine herbicide prometryn

A microbial community, selected by its ability to degrade triazinic herbicides was acclimatized by successive transfers in batch cultures. Initially, its ability to degrade prometryn, was evaluated using free cells or cells attached to fragments of a porous support. As carbon, nitrogen and sulfur sources, prometryn, (98.8 % purity), or Gesagard, a herbicide formulation containing 44.5 % prometryn and 65.5 % of adjuvants, were used. In batch cultures, a considerable delay in the degradation of prometryn, presumptively caused by the elevated concentration of inhibitory adjuvants, occurred. When pure prometryn was used, volumetric removal rates remarkably higher than those obtained with the herbicide formulation were estimated by fitting the raw experimental data to sigmoidal decay models, and differentiating them. When the microbial consortium was immobilized in a continuously operated biofilm reactor, the negative effect of adjuvants on the rate and removal efficiency of prometryn could not be detected. Using the herbicide formulation, the consortium showed volumetric removal rates greater than 20 g m^?3 h^?1, with prometryn removal efficiencies of 100 %. The predominant bacterial strains isolated from the microbial consortium were Microbacterium sp., Enterobacter sp., Acinetobacter sp., and Flavobacterium sp. Finally, by comparison of the prometryn removal rates with others reported in the literature, it can be concluded that the use of microbial consortia immobilized in a biofilm reactor operated in continuous regime offer better results than batch cultures of pure microbial strains.     

Continuous neomycin production by immobilized cells of Streptomyces marinensis NUV-5 in an airlift bioreactor

Neomycin production by free and calcium alginate immobilized cells was investigated in an airlift reactor. The average volumetric productivity with continuous fermentation (72.97 mg/l/h) was greater than with free cells (45.05 mg/l/h). The total neomycin produced with continuous fermentation was 62% greater than with that of free cells. Immobilized Streptomyces particles showed a half-life of 42 days during continuous fermentation under airlift conditions.     

Bifurcational and dynamical analysis of a continuous biofilm reactor

A dynamical model of a continuous biofilm reactor is presented. The reactor consists of a three-phase internal loop airlift operated continuously with respect to the liquid and gaseous phases, and batchwise with respect to the immobilized cells. The model has been applied to the conversion of phenol by means of immobilized cells of Pseudomonas sp. OX1 whose metabolic activity was previously characterized (Viggiani, A., Olivieri, G., Siani, L., Di Donato, A., Marzocchella, A., Salatino, P., Barbieri, P., Galli, E., 2006. An airlift biofilm reactor for the biodegradation of phenol by Pseudomonas stutzeri OX1. Journal of Biotechnology 123, 464-477). The model embodies the key processes relevant to the reactor performance, with a particular emphasis on the role of biofilm detachment promoted by the fluidized state. Results indicate that a finite loading of free cells establishes even under operating conditions that would promote wash out of the suspended biophase. The co-operative/competitive effects of free cells and immobilized biofilm result in rich bifurcational patterns of the steady state solutions of the governing equations, which have been investigated in the phase plane of the process parameters. Direct simulation under selected operating conditions confirms the importance of the dynamical equilibrium establishing between the immobilized and the suspended biophase and highlights the effect of the initial value of the biofilm loading on the dynamical pattern.     

Modeling of continuous L(+)-lactic acid production with immobilized R. oryzae in an airlift bioreactor

Continuous L(+)-lactic acid production was carried out in an airlift bioreactor with immobilized R. oryzae in polyurethane foam cubes. In a pseudo-steady state, the productivity of lactic acid increased with increasing dilution rate or feeding glucose concentration. A double-layer reaction-diffusion model for the pseudo-steady state process was developed to describe the bioreaction system. Using independently determined model parameters, the model prediction agreed well with the experimental results. Therefore, the model can be employed to understand the fermentation behavior, and for the process design and optimization.     

Cortisol Δ1-dehydrogenation by cells of Arthrobacter simplex immobilized by capture on the surface of unwoven cloth coated with a pyridinium-type polymer

Cells of Arthrobacter simplex having 3-ketosteroid-Δ¹-dehydromonogenase activity were immobilized by capture on the surface of unwoven cloth, which was coated with a copolymer of N-benzyl-4-vinylpyridinium chloride and styrene (soluble BVPS), a pyridinium-type polymer. The captured bacterial cells catalyzed the dehydrogenation of cortisol to prednisolone in solvents containing methanol. A fixed-bed column reactor 15 mm in diameter and 12 cm long was continuously operated. The reactor contained about 200 mg of A. simplex cells, captured on an unwoven cloth 12 cm wide, 20 cm long, and 0.5 mm thick coated with 6.5 g/m² of the soluble BVPS, and yielded approximately 90% prednisolone during 6 d when the influent cortisol concentration was 1.0 mmol/l and the space velocity was 0.57 h⁻¹ at 20°C and pH 8.2.     

Degradation of 2,4,6-TCP and a mixture of isomeric chlorophenols by immobilized Streptomyces rochei 303

We investigated the degradation of 2,4,6-trichlorophenol (2,4,6-TCP) by cells of Streptomyces rochei 303 immobilized on various carriers. Polycaproamide fibre was chosen as the optimal carrier for immobilization. The cells immobilized on this carrier degraded high-concentrations of individual chlorophenols and their mixtures: from mono- to pentachlorophenol including the most persistent meta-substituted derivatives. During continuous fermentation in a column with continuous substrate and air flow at a maximal degraded concentration of 2,4,6-TCP of 1 g/l and the specific flow rate of 0.08 h^–1, the efficiency of degradation was 720 mg 2,4,6-TCP/day (36 mg 2,4,6-TCP/day per gram of carrier). The above system of immobilized cells was operated continuously without any loss of activity for 2.5 months, the amount of degraded 2,4,6-TCP being 54 g. At a lower concentration of the reagent (150 mg/l) the system was operated without any decrease in its degradability and without any additional carbon source for 11 months. Correspondence to: L. A. Golovleva     

Integrated continuous winemaking process involving sequential alcoholic and malolactic fermentations with immobilized cells

An integrated winemaking process – including sequential alcoholic and malolactic fermentations operated continuously – was developed. For the continuous alcoholic fermentation, yeast cells (Saccharomyces cerevisiae) were immobilized either on grape stems or on grape skins, while bacterial cells (Oenococcus oeni) used for conducting continuous malolactic fermentation were immobilized on grape skins only. The produced wines were subjected to chemical analysis by HPLC (ethanol, glycerol, sugars and organic acids) and by gas chromatography (major and minor volatile compounds). The final proposed integrated continuous process permitted the production of 960 mL/d of a dry white wine, with an alcoholic strength of about 13 vol%, by using two 1.5 L tower bed reactors packed with 260 g of grape skins. The produced wines revealed a good physicochemical quality. Moreover, 67% of the malic acid concentration could be reduced in the second reactor. Both fermentative processes proved to be much more efficient than those conducted traditionally with free cells or even with immobilized cells, but in the batch mode of operation.     

Degradation of quinoline by immobilized Comamonas acidovorans in a three-phase airlift reactor

Quinolie degradation by Comamonas acidovorans was studied in a continuously operated three-phase airlift reactor. Porous glass beads were applied as support matrix for cell imobilization by colonization. Under steady-state conditions (S approximately 0), cell attachment was poor at low dilution rates but imporved considerably with increasing dilution rate. Conversion of quinoline was investigated below and above the washout for suspended culture (D(crit) = mu(max) = 0.42 h(-1)). With immobilized cells the reactor could be operated at D > mu(max), and complete conversion of quinoline was achieved as long as the specific quinoline feed rate D*S(0)/X did not exceed the maximum specific degradation rate (r(S, max)). The biofilm thickness was about 100 mum, and its efficiency was about 54% compared to suspended organisms. If quinoline overloads were supplied to the reactor, quinoline, as overloads were supplied to the reactor, quinoline, as well as its pathway intermediates, appeared in the reactor and conversion was low. Hence, the immobilized microorganisms remained viable and active. They could survive quinoline overloads. If the quinoline feed rate was reduced agains, complete conversion was reestablished. (c) 1995 John Wiley & Sons, Inc.     

Hydrogen evolution by co-immobilized chloroplasts and Clostridium butyricum

Spinach chloroplasts and Clostridium butyricum cells were immobilized in 2% agar gel. Crude ferredoxin isolated from spinach and benzyl viologen were used as electron carriers. The optimum pH for both NADP reduction by immobilized chloroplasts and for hydrogen evolution by immobilized Cl. butyricum was 8.0. The optimum temperature was between 25 and 30°C for NADP reduction by immobilized chloroplasts, and 37°C for hydrogen evolution by immobilized cells. The total amount of hydrogen evolved in 6 h was 41 μmol/mg Chl for the immobilized chloroplast-benzyl viologen-immobilized Cl. butyricum system, and 11 μmol/mg Chl for the immobilized chloroplast-ferredoxin-Cl. butyricum system. The systems evolved only a trace amount of hydrogen when dichlorophenyldimethylurea was added. The immobilized chloroplast-benzyl viologen-immobilized Cl. butyricum system evolved hydrogen continuously for 6 h, and immobilized Cl. butyricum retained the initial hydrogenase activity. However, the photoreduction activity of chloroplasts decreased to 30% of the initial activity after 6 h of reaction.     

Production of cryptotanshinone and ferruginol by immobilized cultured cells of Salvia miltiorrhiza

The production of the diterpenes cryptotanshinone and ferruginol by immobilized cultured cells of Salvia miltiorrhiza was examined. Cryptotanshinone and ferruginol were produced continuously by the immobilized cells. Much of the cryptotanshinone was released into the medium, while most of the ferruginol was retained in the cells. The production of cryptotanshinone and ferruginol by the immobilized cells was about 39% and 61% of those by cell suspensions. Re-use of the immobilized cells for the production of these compounds was also examined.     

An airlift biofilm reactor for the biodegradation of phenol by Pseudomonas stutzeri OX1

Phenol bioconversion by Pseudomonas stutzeri OX1 using either free or immobilized cells was investigated with the aim of searching for optimal operating conditions of a continuous bioconversion process. The study was developed by analyzing: (a) free-cell growth and products of phenol bioconversion by batch cultures of P. stutzeri; (b) growth of P. stutzeri cells immobilized on carrier particles; (c) bioconversion of phenol-bearing liquid streams and the establishment and growth of an active bacterial biofilm during continuous operation of an internal-loop airlift bioreactor. We have confirmed that free Pseudomonas cultures are able to transform phenol through the classical meta pathway for the degradation of aromatic molecules. Data indicate that bacterial growth is substrate-inhibited, with a limiting phenol concentration of about 600 mg/L. Immobilization tests revealed that a stable bacterial biofilm can be formed on various types of solid carriers (silica sand, tuff, and activated carbon), but not on alumina. Entrapment in alginate beads also proved to be effective for P. stutzeri immobilization. Continuous bioconversion of phenol-bearing liquid streams was successfully obtained in a biofilm reactor operated in the internal-circulation airlift mode. Phenol conversion exceeded 95%. Biofilm formation and growth during continuous operation of the airlift bioreactor were quantitatively and qualitatively assessed.     

Continuous synthesis of l-malic acid using whole-cell microreactor

Although whole-cell biocatalysis, as well as microreactor technology, are gaining importance in modern biotechnology, there are just a few literature reports on whole-cell biocatalysis in microreactors. In the present work, a continuously operated microreactor with permeabilized Saccharomyces cerevisiae cells was made out of commercially available plastic tubes and tested as a tool for the development of l-malic acid production accomplished by hydration of fumaric acid. Cells were immobilized on inner walls of microchannels by means of 3-aminopropyltriethoxysilane and glutaraldehyde and further permeabilized in order to enhance mass transfer across the membrane. The effects of different process parameters including medium pH, substrate inlet concentration and flow rate, cell permeabilization conditions, as well as catalyst stability were evaluated and the results compared to previously published data obtained within a bench-scale bioreactor. The presented microfluidic device with immobilized biocatalyst built from low cost and disposable materials could be applied for the fast development of other whole-cell biotransformations.     

Continuous production of homopolynucleotides by immobilized polynucleotide phosphorylase

A polynucleotide phosphorylase was immobilized with glutaraldehyde, via an aminopropyl spacer, on porous glass. The specific activity of the immobilized enzyme was effectively increased by the addition of an appropriate ribonucleoside diphosphate on immobilization.A homopolynucleotide could be synthesized continuously by passing a nucleoside diphosphate solution through the immobilized enzyme column. The chain length of the product depended upon the temperature and the flow rate. Polyinosinic acid, poly(I), was continuously synthesized with the immobilized enzyme for about one month without appreciable loss of activity.Polyinosinic acid-polycytidylic acid, poly(I)·poly(C), prepared from poly(I) and poly(C) synthesized with the immobilized polynucleotide phosphorylase, induced interferon-β (IFN-β) in human cultured cells as effectively as that prepared from homopolynucleotides synthesized with the free enzyme.     

Chemical oxygen demand reduction in a whey fermentation

Summary Preparations of living Pseudomonas denitrificans cells immobilized in alginate gel were used in the denitrification of water. In the presence of an exogenous carbon source the entrapped microorganisms reduced nitrate and nitrite to gaseous products and to achieve complete reduction, carbon to nitrogen ratios of over two were required. The effects on denitrification of particle size and the number of bacteria in the gel were investigated. Apparent K_m values for nitrate and nitrite reduction were calculated for free and immobilized cells. When the immobilized cells were incubated in nutrient media, an increase in reduction rate was observed and this was shown to be caused by the growth of cells within the gel particles. Immobilized P. denitrificans cells retained 75% of their initial nitrate reduction capacity after 21 days of storage at +4°C. The operational stability of the alginate-immobilized cells was studied both in batch and in a column which was operated continuously. A column (45 g of alginate-cell fibers in 80 ml) denitrified a high nitrate drinking water (100 mg NO₃/l) with a rate of 300 ml of nitrate and nitrite free water/day/g of gel. The half life for nitrate reduction was estimated to be 30 days.     

pH control of limonin debittering with entrapped Rhodococcus fascians cells

Summary Rhodococcus fascians cells were immobilized by entrapment in -carrageenan. The ability of the system to continuously degrade limonin was tested against pH. A burst of activity was observed when changing from pH 4.5 to 5.0, and a small increase could be seen above the latter value. Such behaviour was not only a response of the metabolic activity of the cells to changes in the medium pH, but to selectivity towards the chemical form of the limonin substrate, which also depends on pH. Additionally, the immobilized cells showed increased resistance against pH changes, since the system recovered almost full activity when the pH was restored to 7.0 after being operated for long periods at pH 4.0. The decrease in limonin-degrading capability of the immobilized cells at low pH values could be overcome by choosing an appropriate dilution rate.Offprint requests to: J. L. Iborra