Lactic acid fermentation by cells of Lactobacillus rhamnosus immobilized in polyacrylamide gel

Summary The process of lactic acid fermentation of lactose to lactic acid by Lactobacillus rhamnosus ATCC 7469 has been studied. The following processes have been explored: growth kinetics, as well as lactose utilization, production of lactic acid and further degradation of lactic acid. The immobilization experiments were conducted with microbial cells entrapped in polyacrylamide gels. Gels with different ratios of the monomer (acrylamide) and the cross-linking agent (N,N′-methylene-bis-acrylamide) have been tested. These were used in a repeat-batch process. The current processes inside and outside the gel particles were subjects of examination. The evolution of the activity of immobilized cells with repeated use showed that the particles served mainly as a donor of cells for the free culture. In all experiments a very high degree of conversion, 85–90% was observed. After several runs however, the particles were exhausted for microbial cells. A kinetic model of the process of lactic acid production was developed. This model allowed the evaluation of the effect of microbial growth and diffusion limitations inside the gel particles on the process rate and the separate contribution of the free and immobilized cells to the overall fermentation process upon multiple use.     

Batch fermentation with entrapped growing cells ofLactobacillus casei

Summary Growing cells ofLactobacillus casei were entrapped in-carrageenan/locust bean gum (LBG) (2:1 or 2.75%:0.25% w/w respectively) mixed gel beads (two ranges of diameter: 0.5–1.0 and 1.0–2.0 mm) to fermentLactobacillus Selection (LBS) medium and produce biomass. The results showed significant influence of initial cell loading of the beads and bead size on the fermentation rate. The highest cell release rates were obtained with 2.75%:0.25%-carrageenan/LBG small diameter gel beads. However, 17 h fermentation of LBS medium with immobilized cells resulted in substantial softening of the gel matrix, prohibiting reuse of immobilized biocatalysts as inoculum in subsequent batch fermentation. A dynamic shear rheological study showed that the gel weakness was related to chemical interactions with the medium. Results indicated that part of the matrix-stabilizing K⁺ ions diffused back to the medium. Stabilization of the gel was obtained by adding potassium ions to the LBS medium;L. casei growth was not altered by this supplementation. Fermentation of LBS medium supplemented with KCl byL. casei showed higher cell counts in the broth medium with immobilized cells than with free cells, reaching 10¹⁰ cells/ml after about 10 h with entrapped cells in 0.5–1.0 mm diameter beads and 17 h with free cells. Counts in the gel beads after fermentation were higher than 10¹¹ cells/ml and bead integrity was maintained throughout fermentation.     

The production of ethanol by immobilized yeast cells

Saccharomyces cerevisiae cells were immobilized in calcium alginate beads for use in the continuous production of ethanol. Yeasts were grown in medium supplemented with ethanol to selectively screen for a culture which showed the greatest tolerance to ethanol inhibition. Yeast beads were produced from a yeast slurry containing 1.5% alginate (w/v) which was added as drops to 0.05M CaCl₂ solution. To determine their optimum fermentation parameters, ethanol production using glucose as a substrate was monitored in batch systems at varying physiological conditions (temperature, pH, ethanol concentration), cell densities, and gel concentration. The data obtained were compared to optimum free cell ethanol fermentation parameters. The immobilized yeast cells examined in a packed-bed reactor system operated under optimized parameters derived from batch-immobilized yeast cell experiments. Ethanol production rates, as well as residual sugar concentration were monitored at different feedstock flow rates.     

Production ofLactococcus lactis biomass by immobilized cell technology

Summary Calcium alginate beads containingLactococcus lactis cells were used for three batch fermentations of milk or a commercially available growth medium (Gold Complete, Nordica) with the aim of producing concentrated cultures. Repeated fermentations did not significantly increase bead CFU counts which were between 3.3–7.8×10¹⁰ CFU/g. During the second and third fermentations, which lasted 6 h each, the bead populations decreased if the incubation was extended over 2 h. There was cell release from the beads. Fermentation media and fermentation time all had an effect on free cell counts, but none of these factors statistically interacted. Free cell counts were higher at the end of fermentations 2 and 3 than in the first fermentation and approximately 50% of the population was in the free state. Free cell counts were higher when the beads were incubated in Gold complete than in milk. Although the total bacterial population of a standard free cell fermentation was always higher than those having immobilized cells, immobilized cell technology did enable the production of dense cultures.     

Culture and nitrite uptake in immobilized Scenedesmus obliquus

Summary The green alga Scenedesmus obliquus was immobilized in Ca-alginate beads. The cell growth after immobilization was studied by cell counting. The nitrite uptake was not affected by immobilization, except that a longer lag phase was observed in immobilized cells than in free ones. That result could be due to a barrier effect of the matrix against nitrite diffusion inside the beads. The treatment of cells by glycerol prior to their immobilization in a batch reactor induced an increase of nitrite uptake by the cells. This effect disappeared after a few runs. The glycerol effect on specific rates seemed also to decrease when the number of immobilized cells increased. This decrease can be related to the decrease of light efficiency as well as substrate accessibility when a high cell concentration was used. Several alternating runs of Tris-HCl buffer containing nitrite growth medium depleted in combined nitrogen were tested. Cellular growth occurred inside the beads up to a maximum followed by a decrease of cell number in the beads.     

Influence of inorganic phosphate and immobilization on Claviceps purpurea

Summary The influence of inorganic phosphate and immobilization on cells of Claviceps purpurea strain 1029/N5 producing ergot peptides in shake culture was examined. Immobilization in Ca-alginate beads resulted in a marked reduction of some metabolic activities, i.e. the periods of alkaloid formation and cell growth were prolonged. High concentrations of inorganic phosphate (1 g/l KH₂PO₄) could reduce or stop alkaloid formation both by free and immobilized cells at any time during fermentation. The optimum phosphate concentration for alkaloid production by immobilized cells (about 0.5 mM) was a quarter of that required by free cells. This optimum shift was attributed to (i) the diminished phosphate demand of immobilized cells, due to their reduced metabolic activities, and (ii) the phosphate-dependent morphological behaviour of the biocatalyst. The observed decrease in alkaloid concentrations during later periods of the fermentation supported the idea of alkaloid-degradative enzymes, activated by high phosphate concentrations. Immobilization showed an advantageous influence on this undesirable effect. Offprint requests to: H.J. Rehm     

In situ measurement of algal growth potential in aquatic ecosystems by immobilized algae

Cells of the green algaSelenastrum capricornutum were immobilized in alginate beads. The alga was able to grow inside these beads without being grazed by zooplankton. For P-limited immobilized cells, however, a lower µ _m and initial slope of the Monod growth curve µ _m /K _s were found than for free cells.To study the feasibility of immobilized algae to estimate algal growth potentialin situ in aquatic ecosystems, a series of experiments were conducted in indoor model ecosystems (microcosms) and in a small stream. The use of immobilized algae allowed a continuous registration of algal growth potential integrated over periods with natural fluctuations in the environment. The method of encapsulation of the algae can, however, still be improved. The alginate matrix is exposed to marked degradation by microorganisms when incubated in polluted streams for a period longer than two weeks. The applicability of other types of matrices should be tested.Author for correspondence     

Production of alcohol from sugar beet molasses without heat or filter sterilization

Among three esters of p-hydroxybenzoate, n-butyl p-hydroxybenzoate was selected as the best antimicrobial substance. Molasses medium sterilized by this ester was used as a substrate for ethanol production. n-Butyl p-hydroxybenzoate (0.15% w/v) completely inhibited the growth of free yeast cell inoculum, Ca-alginate immobilized yeast inoculum and bacterial contaminants. Immobilization of the yeast cell inoculum in Ca-alginate with castor oil (6% v/v) offered a yeast cell protection against the inhibitory effect of n-butyl p-hydroxybenzoate. The presence of castor oil in this immobilization system did not affect the metabolic activity of the yeast in beads compared to the cells immobilized without castor oil. The yeast cell beads in this system completely utilized up to 25% molasses sugar with an ethanol yield of 10.58%, equal to 83% of its theoretical value. The beads were stable and could be used successfully for seven cycles of batch fermentation. The optimum fermentation temperature using this system was 35°C. Received 21 January 1997/ Accepted in revised form 05 May 1997     

Protease production by immobilized growing cells of Serratia marcescens and Myxococcus xanthus in calcium alginate gel beads

Summary Serratia marcescens and Myxococcus xanthus cells were immobilized in calcium alginate gel beads. Immobilization under various conditions had no effect on the extracellular proteolytic activity of S. marcescens cells. Protease production seemed rather to depend on the free cells in the medium. However, the stability over time of enzyme production was enhanced, as immobilization increased protease production half-life from 5 to 12 days. On the other hand, Myxococcus xanthus produced proteases inside the gel beads which could diffuse into the medium. The proteolytic activity increased as a function of the initial cell content of the beads and of the bead inoculum. Compared to free cells, immobilized cells of Myxococcus xanthus could produce 8 times more proteolytic activity, with a very low free-cell concentration in the medium.     

Studies on the respiration rate of free and immobilized cells of Cephalosporium acremonium in cephalosporin C production

Bioprocesses using filamentous fungi immobilized in inert supports present many advantages when compared to conventional free cell processes. However, assessment of the real advantages of the unconventional process demands a rigorous study of the limitations to diffusional mass transfer of the reagents, especially concerning oxygen. In this work, a comparative study was carried out on the cephalosporin C production process in defined medium containing glucose and sucrose as main carbon and energy sources, by free and immobilized cells of Cephalosporium acremonium ATCC 48272 in calcium alginate gel beads containing alumina. The effective diffusivity of oxygen through the gel beads and the effectiveness factors related to the respiration rate of the microorganism were determined experimentally. By applying Monod kinetics, the respiration kinetics parameters were experimentally determined in independent experiments in a complete production medium. The effectiveness factor experimental values presented good agreement with the theoretical values of the approximated zero‐order effectiveness factor, considering the dead core model. Furthermore, experimental results obtained with immobilized cells in a 1.7‐L tower bioreactor were compared with those obtained in 5‐L conventional fermentor with free cells. It could be concluded that it is possible to attain rather high production rates working with relatively large diameter gel beads (ca. 2.5 mm) and sucrose consumption‐based productivity was remarkably higher with immobilized cells, i.e., 0.33 gCPC/kg sucrose/h against 0.24 gCPC/kg sucrose/h in the aerated stirred tank bioreactor process. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 63: 593–600, 1999.     

Repeated fed-batch lactic acid production in a packed bed-stirred fermentor system using a pH feedback feeding method

Lactic acid production by repeated fed-batch fermentation using free and immobilized cells of Lactobacillus lactis-11 in a packed bed-stirred fermentor (PBSF) system filled with different support materials including ceramic beads, macro-activated carbon cylinders and glass fiber balls was investigated. The results showed that the optimal support materials were the ceramic beads with diameters of 1–2 mm. Compared with the free cell fermentation system, lactic acid production and volumetric productivity in the PBSF system increased by 16.6 and 12.5%, respectively. Though the concentration of free cells decreased sharply, lactic acid production remained stable in five consecutive fed-batch runs using the PBSF system. pH gradients, immobilized cell concentration and mass diffusion in the packed bed were all affected by the recirculation rate of the culture broth. Maximum lactic acid production, productivity and yield occurred at a recirculation rate of 50 mL min⁻¹.     

Production of thermostable and neutral glucoamylase using immobilized <Emphasis Type="Italic">Thermomucor indicae-seudaticae</Emphasis>

Thermomucor indicae-seudaticae was immobilized in alginate, κ-carrageenan, agarose, agar, polyacrylamide and loofah (Luffa cylindrica) sponge (as such or coated with alginate/starch/Emerson YpSs agar), and used for the production of glucoamylase in submerged fermentation. The mycelium developed from alginate-immobilized sporangiospores secreted higher glucoamylase titres (22.7 U ml⁻¹) than those immobilized in other gel matrices and the freely growing mycelial pellets (18.5 U ml⁻¹). Loofah network provided a good support for mycelial growth, but the enzyme production was lower than that attained with alginate beads. Glucoamylase production increased with inoculum density and the optimum levels were achieved when 40 calcium alginate beads (∼5 × 10⁶ immobilized spores) were used to inoculate 50 ml production medium. The alginate bead inoculum displayed high storage stability at 4°C and produced comparable enzyme titres up to 120 days. The glucoamylase production by hyphae emerged from the immobilized sporangiospores was almost stable over eight batches of repeated fermentation. Scanning electron micrographs of alginate beads, after batch fermentation, revealed extensive mycelial growth inside and around the beads.     

Continuous penicillin production by Penicillium chrysogenum immobilized in calcium alginate beads

Summary A high penicillin-producing Penicillium chrysogenum strain immobilized in calcium alginate beads was used for continuous penicillin fermentation in a bubble column and in a conical bubble fermentor. The fermentation was limited by the growth rate, dilution rates and the stability of the alginate beads. The immobilized cells lost their ability to produce penicillin in the bubble column after 48 h from beginning of the continuous fermentation. In the conical bubble fermentor the immobilized cells remained active for more than 7 days. This bioreactor ensured a good distribution of nutrients and oxygen as well as a higher mechanical stability of the alginate beads.     

Cell-recycle batch fermentation using immobilized cells of flocculent Saccharomyces cerevisiae wine strains

Five, highly flocculeng strains of Saccharomyces cerevisiae, isolated from wine, were immobilized in calcium alginate beads to optimize primary must fermentation. Three cell-recycle batch fermentations (CRBF) of grape musts were performed with the biocatalyst and the results compared with those obtained with free cells. During the CRBF process, the entrapped strains showed some variability in the formation of secondary products of fermentation, particularly acetic acid and acetaldehyde. Recycling beads of immobilized flocculent cells is a good approach in the development and application of the CRBF system in the wine industry.     

Development of a fermentation method using immobilized cells under unsterile conditions. 1. Protection of immobilized cells against anti-microbial substances

A method of protecting immobilized cells against inhibitory substances in the fermentation medium was investigated with the aim of developing a process for fermentation under unsterile conditions. It was found that yeast cells could be protected against the inhibitory effects of p-hydroxybenzoic acid esters by co-immobilizing the cells with vegetable oils. In such a system, the cells grow only in the water phase of the gel beads where most components of the fermentation medium are retained. On the other hand, the p-hydroxybenzoate that diffuses into the gel beads is retained mainly in the oil phase of the beads. Consequently, the p-hydroxybenzoate concentration in the water phase remains too low to inhibit the metabolic activities of the immobilized cells. The effectiveness of a vegetable oil in protecting the immobilized cells against an inhibitory substance depends on the partition coefficient of the substance between the oil and water, the concentration of the oil and the initial cell concentration.     

Theme 4: Immobilized Cell Technology in Wine Production

Abstract

In spite of its traditional nature, wine making is largely concerned with the progress of biotechnology. High cell density reactors have potential for enology: improved performance of alcoholic and malolactic fermentations, smaller scale fermentation facilities, adaptation to continuous processes. Among the immobilization techniques, cell entrapment in alginate beads seems to be an impressive one. Alcoholic fermentation of wine, malolactic fermentation, bottle fermentation known as “Methode champenoise” and sparkling wine are among the industrial applications. Knowledge of kinetics and physiology in microorganisms in heterogeneous media has expanded in the last few years. The use of immobilized yeast cells for the champagne method would greatly simplify “remuage”. The compared metabolism of entrapped and free cells during the bottle fermentation shows differences, but the final product does not reveal significant sensory disparity. New products can be obtained with more thoroughly controlled conditions.     

Production of acid proteinases byHumicola lutea immobilized in polyacrylamide gel

Summary Fungal spores ofHumicola lutea 120–5 were entrapped in 5% polyacrylamide gel and were cultivated for 44–48 h to form a mycelial network inside the beads. A dense mycelial growth also occurred on the surface of the beads. It was possible to reuse the immobilized mycelium for production of acid proteinases in 12 different batches without loss of mechanical stability. The inoculum size should be controlled prior to its transfer into fresh production medium. Maximal enzyme production exceeding the level of free cell fermentation was registered in the fourth to seventh cycles. According to the size of the inoculum, half of the initial production rate was reached after 7–14 batches.     

Enhancement of extracellular enzymatic activities produced by immobilized growing cells of Myxococcus xanthus

Summary The gram-negative soil bacterium Myxococcus xanthus was immobilized by entrapping into carrageenan gel beads. Unexpectedly, the growth rate was hardly increased, and the released free cell concentration remained low. However, extracellular proteolytic and bacteriolytic activities produced in the medium or inside the beads was greatly increased and (or) stabilized as compared to the control. These properties might be quite useful in view of using Myxococcus xanthus as a cloning vehicle for secretion of foreign proteins.     

Relationship between fermentation capability and fatty acid composition of free and immobilized Saccharomyces cerevisiae

Summary The fatty acid composition of Saccharomyces cerevisiae immobilized by entrapment in calcium-alginate beads or adsorption on sintered glass was compared to that of freely suspended cells under different fermentation conditions. The fermentation product ethanol was found to cause a shift towards saturation in the fatty acid composition under anaerobic conditions. Immobilized cells contained significantly higher percentages of saturated fatty acyl residues, especially of palmitic acid (16:0), and a decreased amount of oleic acid (18:1) compared with free cells. The percentage saturation of total fatty acid composition correlates positively with improved fermentation rates obtained with the immobilized cells. This enhanced saturation of fatty acid composition in immobilized cells may be due to altered osmotic conditions in the microenvironment of the cells.     

Batch fermentations with a mixed culture of lactic acid bacteria immobilized separately in κ-carrageenan locust bean gum gel beads

Summary Streptococcus salivarius subsp. thermophilus and Lactobacillus delbrueckii subsp. Bulgaricus were immobilized separately in -carrageenan-locust bean gum gel beads. The beads were prepared by a dispersion process in a two-phase system (water in oil) and two ranges of bead diameter selected by sieving (0.5–1.0 mm and 1.0–2.0 mm). Fermentations with the two strains were conducted in bench bioreactors in a supplemented whey permeate medium. Free and entrapped cells (two ranges of bead diameter and two levels of initial bead cell load) were grown in mixed culture, and carbohydrate utilization, acid production and cell growth or cell release rate measured. Fermentation rates were influenced by bead diameter and initial cell load of the beads. Beads with high initial cell density increased fermentation rates compared to low cell density beads or free cells. Smaller diameter beads (0.5–1.0 mm) showed a stable tendency (not statistically significant p _a > 0.05) towards higher cell release rates, lactose utilization, galactose accumulation and lactic acid production than did larger diameter beads (1.0–2.0 mm). Immobilization of S. salivarius subsp. thermophilus and L. delbrueckii subsp. bulgaricus in separate beads did not seem to affect protocooperation during batch fermentation, and allowed for high cell release rates into the medium.