Evaluation of some gelling agents for immobilization of aerobic microbial cells in alginate and carrageenan gel beads

Summary Different gelling agents were used to immobilized viable cells in either alginate or -carrageenan gel beads. Based on cell leakage from the gel beads, oxygen and glucose diffusion coefficients and toxicity of the gelling agents, SrCl₂ was found to be the best for immobilization of aerobic microbial cells in, not only alginate but also carrageenan gel beads.     

Effect of gelation temperature and gel-dissolving solution on cell viability and recovery of two Pseudomonas putida strains co-immobilized within calcium alginate or κ-carrageenan gel beads

Summary Since a lethal effect of an increased temperature (42°C) on Pseudomonas putida strains PaW8 and PaW130 was demonstrated, strictly ionotropic gels such as calcium alginate or -carrageenan type X 0909 were used for cell co-immobilization, rather than a thermoionotropic -carrageenan gel. Among the variety of gel-dissolving solutions tested, only a 0.05M Na₂CO₃/0.02M citric acid solution was able to preserve around 100 % of the cell viability. A complete cell recovery was obtained from calcium alginate gel beads, while only 6 % of viable cells was recovered from the ionotropic -carrageenan gel.     

Production of citric acid with immobilized Yarrowia lipolytica

Summary Citric acid was produced with immobilized Yarrowia lipolytica yeast in repeated batch-shake-flask and air-lift fermentations. In active and passive immobilization methods calcium alginate, -carrageenan, polyurethane gel, nylon web and polyurethane foams were tested as carriers in repeated-batch fermentations. The highest citric acid productivity of 155 mg l^–1 h^–1 was reached with alginate-bead-immobilized cells in the first batch. A decrease in bead diameter from 5–6 mm to 2–3 mm increased the volumetric citric acid productivity threefold. In an air-lift bioreactor the highest citric acid productivity of 120 mg l^–1 h^–1 with a product concentration of 16.4 g l^–1 was obtained with cells immobilized in -carrageenan beads. Offprint requests to: H. Kautola     

Enhanced hydrogen production from aromatic acids by immobilized cells of Rhodopseudomonas palustris

Cells of the purple non-sulphur bacterium Rhodopseudomonas palustris DSM 131 were immobilized in agar, agarose, -carrageenan or sodium alginate gel. With alginate beads, prepared by an emulsion technique, and an optimal cell load of 10 mg dry weight/ml gel, the hydrogen production from aromatic acids was doubled as compared to that resulting from liquid cultures. Hydrogen yields of 60%, 57%, 86% or 88% of the maximal theoretical value were obtained from mandelate, benzoylformate, cinnamate or benzoate respectively. Benzoate concentrations above 16.5 mM were inhibitory. During a period of 55 days the process of hydrogen evolution with immobilized cells was repeated in five cycles with slowly decreasing efficiency.     

Continuous fermentation of a supplemented milk with immobilized Bifidobacterium infantis

Bifidobacterium infantis immobilized in -carrageenan - locust bean gum gel beads (1.0–2.0 mm diameter) was used to ferment. 10% reconstituted skim milk supplemented with 1% yeast extract in a continuous stirred tank reactor. Cell release rate from the gel beads into the milk and growth of free cells in the bioreactor allowed for a steady inoculation of the feed, with cell counts in the outflow varying from 1.0 to 2.2 × 10⁹ CFU/mL for dilution rates in the range 0,5 to 1,0 h^-1. High mechanical stability of the gel beads was observed in milk.     

Studies on immobilization of the thermophilic bacterium Thermus aquaticus YT-1 by entrapment in various matrices

Summary Immobilization of the thermophilic bacterium Thermus aquaticus YT-1 has been studied using various entrapment techniques. Alginate, -carrageenan, agar, agarose and polyacrylamide were tested as supports during operation at 65°C at which the cells are known to produce protease when grown free in solution. Alginate showed toxic effects and no viability was observed after entrapment in Ca alginate or even after exposure of free living cells to sodium alginate. Polyacrylamide was observed to be the best support. Protease activity was closely related to the appearance of free cells in the medium.     

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.     

Analysis of immobilized cell bioreactors for desulfurization of flue gases and sulfite/sulfate-laden wastewater

Sulfur dioxide (SO₂) is one of the major pollutantsin the atmosphere that cause acid rain. Microbialprocesses for reducing SO₂ to hydrogen sulfide(H₂S) have previously been demonstrated byutilizing mixed cultures of sulfate-reducing bacteria(SRB) with municipal sewage digest as the carbon andenergy source. To maximize the productivity of theSO₂-reducing bioreactor in this study, variousimmobilized cell bioreactors were investigated: a stirredtank with SRB flocs and columnar reactors with cellsimmobilized in either -carrageenan gel matrix orpolymeric porous BIO-SEP^TM beads. Themaximum volumetric productivity for SO₂reduction in the continuous stirred-tank reactor (CSTR)with SRB flocs was 2.1 mmol SO₂/h·l. The-carrageenan gel matrix used for cellimmobilization was not durable at feed sulfiteconcentrations greater than 2000 mg/l or at sulfite feedrate of 1.7 mmol/h·l. A columnar reactor withmixed SRB cells that had been allowed to grow intohighly stable BIO-SEP polymeric beads exhibited thehighest sulfite conversion rates, in the range of16.5 mmol/h·l (with 100% conversion) to20 mmol/h·l (with 95% conversion). In addition toflue gas desulfurization, potential applications of thismicrobial process include the treatment ofsulfate/sulfite-laden wastewater from the pulp and paper,petroleum, mining, and chemical industries.     

Comparison of different methods of immobilization for lignin peroxidase production by Phanerochaete chrysosporium

Summary Phanerochaete chrysosporium was immobilized in agar, agarose and -carrageenan gel beads, nylon web, and polyurethane foam, and used for the production of lignin peroxidase in shake cultures on a carbon-limited medium. Nylon was found to be the best carrier, with the maximum lignin peroxidase activity (340 U/l) reached on the 7th day. The enzyme production rate was significantly lower with freely suspended mycelial pellets. Both nylon and polyurethane based biocatalysts were active for at least 38 days after the addition of veratryl alcohol. Best results were obtained when a spore inoculum was used instead of day-old pellets. -Carrageenan was found unsuitable as a carrier for lignin peroxidase production.     

New solution method for equations of reaction and mass transfer in biocatalyst particles

Summary For numerical solution of the reaction-mass transfer equations for immobilised biocatalysts it may be better to start integration at the particle surface and proceed inwards: calculations are targetted on the region to which practically interesting changes are often confined (because concentrations are effectively zero in the interior); and during iterative solution wrong initial estimates may be rejected after detecting anomalies early in the integration.Symbols C_b substrate concentration in bulk (mol m^–3) - c dimensionless substrate concentration (C/C_b) (-) - D_e effective diffusion coefficient (m²s^–1) - Da Damkohler number (V.r_o ²/D_e.K_s) (-) - K_s substrate concentration kinetic coefficient (mol m^–3) - k_e external mass transfer coefficient (ms^–1) - r_o bead radius (m) - Sh Sherwood number (k_e.r_o/D_e) (-) - V maximum rate per unit volume in beads (mol m^–3s^–1) - x dimensionless distance from bead centre (r/r_o) (-) - dimensionless kinetic coefficient (K_s/C_b) (-) - _o effectiveness factor (-)     

Immobilized growing lactic acid bacteria with κ-carrageenan — locust bean gum gel

Summary A cell entrapment process using -carrageenan — locust bean gum gel is presented. Streptococcus thermophilus, Lactobacillus bulgaricus and S. lactis were immobilized in small gel beads (0.5–1.0 mm and 1.0–2.0 mm diameter) and fermentations in bench bioreactors were conducted. Viability of entrapped cells, lactose utilization, lactic acid production and cell release rates were measured during fermentation. The procedure was effective for S. thermophilus, L. bulgaricus and S. lactis, and the viability of these bacteria remained very high throughout entrapment steps and subsequent storage. Bead diameter influenced the fermentation rate: smaller beads (0.5–1.0 mm) permitted an increase in release rates, lactose utilization and acid production by entrapped cells, approximating values attained with free cells.     

Production of vitamin B12 by immobilized cells of a propionic acid bacterium

Summary The ability of immobilized cells of propionic acid bacteria to form vitamin B₁₂ has been investigated. Propionibacterium arl AKU 1251 having a considerable activity to produce the vitamin was selected as a test organism among six strains of propionic acid bacteria tested. The whole cells were entrapped with urethane prepolymers, photo-crosslinkable resin prepolymers or several other materials such as -carrageenan, agar or sodium alginate, and their vitamin B₁₂ productivity was compared. Based on the criteria of the convenience of preparation and the stability of the cell-entrapping gels, a hydrophilic urethane prepolymer, PU-9, was employed as gel material. Satisfactory vitamin B₁₂ production was obtained when 5–10 g of wet cells precultured to the late exponential growth phase were entrapped with 1 g of the prepolymer. Addition of a suitable amount of cobaltous ion and of 5,6-dimethyl benzimidazole to the culture medium was effective for the production of the vitamin by the immobilized cells. The repeated use of the immobilized cells was successfully achieved when a suitable amount of cells were entrapped and allowed the proliferation of cells inside gel matrices.     

High Rate of N2 Fixation by East Siberian Cryophilic Soil Bacteria as Determined by Measuring Acetylene Reduction in Nitrogen-Poor Medium Solidified with Gellan Gum

For evaluating N₂ fixation of diazotrophic bacteria, nitrogen-poor liquid media supplemented with at least 0.5% sugar and 0.2% agar are widely used for acetylene reduction assays. In such a soft gel medium, however, many N₂-fixing soil bacteria generally show only trace acetylene reduction activity. Here, we report that use of a N₂ fixation medium solidified with gellan gum instead of agar promoted growth of some gellan-preferring soil bacteria. In a soft gel medium solidified with 0.3% gellan gum under appropriate culture conditions, bacterial microbiota from boreal forest bed soils and some free-living N₂-fixing soil bacteria isolated from the microbiota exhibited 10- to 200-fold-higher acetylene reduction than those cultured in 0.2% agar medium. To determine the N₂ fixation-activating mechanism of gellan gum medium, qualitative differences in the colony-forming bacterial components from tested soil microbiota were investigated in plate cultures solidified with either agar or gellan gum for use with modified Winogradsky''s medium. On 1.5% agar plates, apparently cryophilic bacterial microbiota showed strictly distinguishable microbiota according to the depth of soil in samples from an eastern Siberian Taiga forest bed. Some pure cultures of proteobacteria, such as Pseudomonas fluorescens and Burkholderia xenovorans, showed remarkable acetylene reduction. On plates solidified with 1.0% gellan gum, some soil bacteria, including Luteibacter sp., Janthinobacterium sp., Paenibacillus sp., and Arthrobacter sp., uniquely grew that had not grown in the presence of the same inoculants on agar plates. In contrast, Pseudomonas spp. and Burkholderia spp. were apparent only as minor colonies on the gellan gum plates. Moreover, only gellan gum plates allowed some bacteria, particularly those isolated from the shallow organic soil layer, to actively swarm. In consequence, gellan gum is a useful gel matrix to bring out growth potential capabilities of many soil diazotrophs and their consortia in communities of soil bacteria.In 1967, Schöllhorn and Burris discovered that nitrogenase from an N₂-fixing rhizobium of soybean can reduce acetylene to produce ethylene (C₂H₄) (32), a reaction analogous to the conversion of the natural substrate N₂ into ammonia. Shortly afterwards, it was shown that this acetylene reduction activity parallels N₂ reduction by nitrogenase (13), and since then, acetylene reduction assays have been widely used in the evaluation of biological N₂ fixation. An acetylene reduction assay is generally performed under the following conditions: precultured bacterial cells are suspended into N-free or -deficient liquid medium containing a carbon source, usually d-glucose or d-mannitol (35) at 0.5 to 2.0%, and exposed for 24 h or less at a representative room temperature, e.g., 25°C (2). However, this method is not applicable to free-living, microaerobic N₂-fixing bacteria, which have been regarded as notoriously difficult to culture. To solve this problem, Döbereiner and her group developed a soft gel method (7), which used 0.2% agar as a gel matrix for the medium. Due to a vertical gradient of dissolved oxygen concentrations, these microaerobes formed a thin layer at the particular depth of the medium that contained an ideal level of dissolved oxygen (10). Also, significant activities in acetylene reduction assays were observed for N₂-fixing microaerobes, particularly those from the rhizoplane of monocotyledonous crop plants (e.g., Azospirillum and Herbaspirillum spp.) (1, 9, 40). To date, these soft gel media solidified with 0.2% agar have been widely used as the most basic method for the screening of free-living or difficult-to-culture N₂-fixing bacteria (2, 16).In an agar composed of soft gel, however, the layer formation of highly transparent colony-forming bacteria is often obscured and is more difficult to observe than comparable layer formation in water due to the higher turbidity of the agar gel, and some members of the soil bacterial community do not show any positive response in acetylene reduction assays under these conditions. These drawbacks to the usage of agar as a soft gel matrix delayed the recognition that free-living N₂ fixers make a potent contribution to the support of ecosystems under adverse soil conditions. Hashidoko et al. developed an improved soft gel medium for growth of N₂-fixing bacteria in 2002 (15). In their study, 0.2% agar was replaced with 0.3% gellan gum, a bacterial extracellular polysaccharide (EPS) produced by Sphingomonas elodea (a synonym of Sphingomonas paucimobilis) ATCC 31461 (12, 17, 18). Initially, gellan gum was used for the purpose of preparing a highly transparent soft gel medium that was better for culturing microaerobic N₂-fixing rhizobacteria. It had other favorable physical properties: when 0.3% gellan gum containing Winogradsky''s mineral mixture was autoclaved, the medium remained in a liquid form over a period of several hours while cooling to room temperature. Even after the gellan gum had been solidified, the soft gel was easily liquefied upon mechanical agitation. The liquefied medium was able to resolidify after a short period of time, so it was easy to uniformly disperse inoculants into the soft gel medium. The outstanding transparency (14) and other properties of this gel matrix enable easy visualization of transparent colony-forming N₂-fixing bacteria and also allow observation of their responses to various concentrations of dissolved oxygen and cell motilities (15).In many preliminary experiments, nitrogen-poor gellan gum media allowed high growth of diazotrophs, but this study was needed to compare gellan gum with agar as a gel matrix for N₂ fixation. Because Siberian boreal forest soils have been noted for their low N₂-fixing capability (3), we first cultured bacterial microbiota from the eastern Siberian Taiga forest bed in gellan gum medium. A quantitative comparison of N₂ fixation behaviors of free-living soil bacteria was attempted to investigate gellan gum as a potential N₂ fixation-promoting soft gel matrix. We here first report on the efficacy of gellan gum as a soft gel matrix for monitoring acetylene reduction by the use of free-living N₂-fixing soil bacteria.     

Quantitative recovery of fungal biomass grown on solid κ- carrageenan media

The use of a dissolvable solid matrix, -carrageenan, to quantify biomass grown on solid media was studied. A firm gel was obtained with 2% (w/v) -carrageenan and 20 mM K+ which could be easily dissolved in demineralized water. Direct quantification of Coniothyrium minitans biomass grown on this medium was feasible. No effects of the dissolution on the amount of biomass recovered were detected.     

Gellan gum gel as entrapment matrix for high temperature fermentation processes: A rheological study

High temperature rheological measurements (60–80°C) were carried out on gellan gum gel. The experiments were conducted in order to optimize the behavior of the gel as an entrapment matrix for fermentation processes of dairy fluids using highly thermophilic microorganisms. At 60°C, no significant decrease of rheological parameters of the gel was observed for 15 days, as compared to 23°C. At 80°C, the gel weakened abruptly. The gel was extremely sensitive to calcium loss (optimum concentration 0.1% CaCl₂) due to chelating agents. Gellan gum gel could be an efficient entrapment matrix for fermentation processes at temperature up to 60°C in dairy fluids.     

Second-derivative Fourier transform infrared spectra of seaweed galactans

The Fourier transform infrared spectra of agar, agarose, -, -, and -carrageenan, and ofChondrus canaliculatus, Iridaea ciliata, I. membranacea, I. laminarioides andGracilaria chilensis polysaccharides were recorded in the 4000–400 cm^-1 region. The bands in the second derivative mode are sharper and more bands are resolved than in the normal spectra.Agar, agarose andG. chilensis phycocolloids exhibit diagnostic bands at 790 and 713 cm^-1. -, - and -carrageenans, and native carrageenan-type polysaccharides fromC. canaliculatus andIridaea species exhibit bands at around 1160, 1140, 1100, 1070, 1040, 1008, 610, and 580 cm^-1. Therefore, FT-IR spectroscopy in the second-derivative mode may be applied to differentiate between agar- and carrageenan-types seaweed galactans.     

Hybridoma cells in a protein-free medium within a composite gel perfusion bioreactor

A composite gel system has been developed combining the chemical and physical properties of calcium alginate and agarose gels. The results of growing composite gel immobilized hybridoma SPO1 cells in a protein-free medium within a fluidized-bed perfusion bioreactor are presented in this paper. During the continuous operation of this system, the total cell density reached 3.9×10⁷ cells per ml of beads (viability 79.6%). The specific productivity of monoclonal antibody of the immobilized hybridoma cells reached more than 1.5 g per 10⁶ viable cells per hour, compared with 0.5 for non-immobilized viable cells grown in a one liter agitated bioreactor with the same medium. Significant increases in cell metabolic activities, including substrate utilization and byproduct formation, were also observed. Leaching of materials from the beads was evident and the major fraction of released materials was alginate.     

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.     

Degradation of bromoxynil by resting and immobilized cells of Agrobacterium radiobacter 8/4 strain

The Agrobacterium radiobacter 8/4 strain capable of degradation of bromoxynil, ioxynil and dichlobenil, arylnitrile herbicides was isolated from soil and cell entrapment was investigated. Three immobilization techniques was used: Caalginate, Ca-pectate and -carrageenan technique, and resting cells. The highest degradation rates were obtained with Ca-alginate and -carrageenan entrapped cells.     

Immobilization of Proteus vulgaris for the reduction of 2-oxo acids with hydrogen gas or formate to d-2-hydroxy acids

For the stereoselective reduction of 2-oxo acids by hydrogen gas or formate to d-2-hydroxy acids, anaerobically grown Proteus vulgaris cells were immobilized in alginate, -carrageenan, chitosan, polyurethane and polyacrylamide acylhydrazide. With the exception of the last matrix, immobilization led to a decrease in the apparent activity, probably caused by diffusional limitations. Chitosan or polyurethane-entrapped cells kept their initial catalytic activity for over more than 600 h in a continuous working period. In both matrices the cells could be partially reactivated by incubation of the immobilisates in growth medium. Polyurethane-immobilized cells (and also cell membranes) were repeatedly usable. After 30 batch operations, 30–40% of the initial reduction rate was still detectable. Chitosan-immobilized cells did not lose any activity during 17 months of storage at 4° C under exclusion of oxygen. Correspondence to: H. Simon