Effects of cell entrapment on nucleic acid content and microbial diversity of mixed cultures in biological wastewater treatment

The effects of entrapment on nucleic acid content and microbial diversity of mixed cultures in biological municipal wastewater treatment were investigated. Deoxyribonucleic acid content increased 1.6-5.5 times more in alginate entrapped cells than in free and polyvinyl alcohol (PVA) entrapped cells. PVA entrapment resulted in 1.1- to 5.9-fold more increases in ribonucleic acid content compared to that experienced by free and alginate entrapped cells. Entrapment in carrageenan changed the bacterial community structure more than the alginate and PVA entrapments (35-80% versus 0-35%) as determined by single-strand conformation polymorphism analyses. The change in the bacterial community structure of alginate entrapped cells was less time dependent than that of PVA entrapped cells. This study enhances understandings on the physiology of entrapped cells and their community evolution in wastewater treatment environments.     

Effects of cell entrapment on growth rate and metabolic activity of pure cultures commonly found in biological wastewater treatment

The effects of cell entrapment on the growth rate and metabolic activity of major groups of bacteria (betaproteobacteria and gammaproteobacteria) in biological municipal wastewater treatment were investigated. Three different cell entrapment media (alginate, carrageenan and polyvinyl alcohol) and three cell-to-matrix ratios (0.1%, 0.2% and 0.6%, w v⁻¹) were examined. Representative species of betaproteobacteria were Alcaligenes faecalis and Comamonas testosteroni whereas Pseudomonas putida was a gammaproteobacteria species studied. Free (non-entrapped) cells were included in the study for comparative purpose. Results indicated that the entrapment, type of entrapment media, and cell-to-matrix ratio had significant effects on the growth and metabolic activity of major groups of bacteria in wastewater treatment. Polyvinyl alcohol entrapped cells had the highest specific growth and specific substrate utilization rates. Increase of cell-to-matrix ratio (from 0.1% to 0.2% or 0.6%) did not improve the specific growth and specific substrate utilization rates. The relative performances provided by different entrapment media of the three species studied were quite consistent. This study showed that the suitable choices of entrapment media and cell-to-matrix ratio are important but similar for major groups of bacteria in wastewater treatment.     

Reject water treatment by improvement of whole cell anammox entrapment using polyvinyl alcohol/alginate gel

Reject water treatment performance was investigated by whole cell anammox sludge entrapped polyvinyl alcohol/sodium alginate gel in the stirred tank reactor (STR). The whole experiment was conducted through Phase 1 and Phase 2 in which synthetic wastewater and modified reject water were used as feeding medium, respectively. The anammox reactor demonstrated quick start-up after 22 days as well as stable and relatively high nitrogen removal rate of more than 8.0 kg-N m⁻³ day⁻¹ during the two both phases even under moderately low temperature of 25 ± 0.5°C during the last 2 months of Phase 2. The matured brownish red PVA beads had good characteristics with buoyant density of 1.10 g cm⁻³, settling velocity of 141 m h⁻¹ and diameter of 4 mm. The bacterial community was identified by 16S rDNA analysis revealing the concurrent existence of KSU-1 and new kind anammox bacterium Kumadai-I after changing influent from synthetic wastewater to reject water. It was speculated that Kumadai-I might play a role as “promotion” factor together with KSU-1 on high nitrogen removal rate. These results demonstrate the potential application of whole cell anammox entrapment by PVA/alginate gel for achieving stable and high-rate nitrogen removal from high ammonium with low C/N ratio contained wastewaters, such as reject water, digester liquor or landfill leachate.     

Production of daunorubicin by immobilized growing Streptomyces peucetius cells

Summary Streptomyces peucetius cells were immobilized by entrapment in calcium alginate and a photosensitive synthetic polymer, and used for the production of daunorubicin (daunomycin), which is known to be an antitumour reagent. The use of cultivation media removed insoluble components in a natural medium prevented rapid decrease in daunorubicin titer after maximum production. These entrapped cells could be used at least five times for repeated daunorubicin production; the total cultivation period was 45 days.     

Entrapment of a Trigonopsis variabilis D‐amino acid oxidase variant F54Y for oxidative deamination of cephalosporin C

Trigonopsis variabilis D ‐amino acid oxidase (TvDAAO) is an enzyme used in the industrial bioconversion of cephalosporin C (CPC) into 7‐aminocephalosporanic acid, a crucial biosynthetic nucleus for a wide spectrum of semi‐synthetic cephem antibiotics. Using homology modeling and site‐directed mutagenesis, we have previously shown that the TvDAAO variant F54Y possesses improved catalytic activity and thermostability. To further explore its industrial application, the conditions for immobilization of the enzyme were examined in the present investigation. The results showed that entrapment in a calcium alginate (Ca‐alginate) matrix using 2% alginate, 500 mM CaCl₂, and 15 min stabilization appeared to be optimal for the immobilization of F54Y. The entrapped enzyme allowed complete CPC conversion. The entrapped enzyme also showed good operational stability and retained at least 90% of its original activity after 20 reaction cycles. To conclude, the entrapment of F54Y in Ca‐alginate appeared to be a simple and efficient biocatalysis system with potential application in the antibiotics industry.     

Relationship between respirometric activity and community of entrapped nitrifying bacteria: Implications for partial nitrification

Activated sludge obtained from two municipal wastewater treatment facilities (WWTF) was used as seed sludge for enriched nitrifiers, which were later entrapped in polyvinyl alcohol. Seed sludge from one WWTF was acclimated to high ammonia level (1813 mg NH₃-N l^?1) through the return of sludge digester supernatant back to primary clarifier while seed sludge from the other WWTF was un-acclimated. To elucidate on how to control partial nitrification by entrapped cells, which could be different from suspended cells, kinetics of entrapped enriched nitrifiers were studied using a respirometric assay. The community of nitrifiers within the entrapment matrix, which was observed by fluorescence in situ hybridization (FISH) technique, was related to the nitritation and nitratation kinetics based on oxygen uptake rate. Maximum oxygen uptake rate, and substrate and oxygen affinities of both ammonia oxidizing bacteria (AOB) for nitritation and nitrite oxidizing bacteria (NOB) for nitratation in entrapped cells were lower than those of corresponding suspended cells. Under dissolved oxygen (DO) limiting conditions, nitratation was more suppressed than nitritation for suspended cells, while for the entrapped cells, the results were the contrary. A free ammonia (FA) inhibition affected only the un-acclimated sludge. Either FA inhibition or DO limitation might not be a sole effective control parameter to achieve partial nitrification by entrapped cells. FISH results revealed that Nitrosomonas europaea was the dominant AOB while Nitrobacter species was the dominant NOB in all cases. Heterotrophs were also present in the entrapment at 22.8 ± 18.6% and 41.5 ± 4.3% of total bacteria for acclimated and un-acclimated originated sludge. The availability of substrate and oxygen governed the distributions of AOB, NOB and heterotrophs within the entrapment and nitritation kinetics of entrapped nitrifiers.     

Mixed photo-cross-linked polyvinyl alcohol and calcium-alginate gels for cell entrapment

Living cells may be immobilized by gel entrapment under very mild conditions. The ionotropic gelation of alginate with bivalent cations such as Ca²⁺, as well as photo-induced gelation of polyvinyl alcohol (PVA) bearing photosensitive stilbazolium (SbQ) groups, are procedures that are compatible with most bioactive materials. In the search for more stable and stronger alginate gel beads, experiments have been carried out to investigate mixed gels from alginate and PVA-SbQ. The swelling capacities, diffusion properties, and potential toxic effect of the binary gel beads have been evaluated. The gel beads of selected PVA-SbQ/alginate mixtures were applied successfully as carriers in a denitrification process with continuous feeding of unsterilized water medium. Under such conditions, the purely synthetic PVA-SbQ network is expected to have a longer lifespan than a natural biopolymer such as alginate.     

Continuous production of l-serine by immobilized growing Corynebacterium glycinophilum cells

Summary Auxotrophic mutant cells of Corynebacterium glycinophilum with high l-serine production activity were immobilized by entrapment with various gel materials, such as synthetic prepolymers and natural polysaccharides. The entrapped cells were used for estimation of l-serine productivity in a medium supplemented with glycine as a precursor. Based on the above criteria, including cell growth in gels and cell leakage from gels, calcium alginate was the most suitable gel material. Continuous l-serine fermentation with calcium alginate-entrapped growing cells was successfully achieved in an air-bubbled reactor for at least 13 days.     

Diethyl phthalate degradation by the freeze-dried,entrapped Bacillus subtilis strain 3C3

Microbial degradation is the key treatment for diethyl phthalate (DEP) of which the efficacy is subdued by substrate toxicity. DEP-degrading Bacillus subtilis strain 3C3 adopted cell size alteration as one of the adaptive mechanisms in response to DEP stress at high concentrations. Nevertheless, to enhance cell tolerance in the protected environment and to facilitate practical treatment operation, cell entrapment was optimized with the entrapment yield at 89 ± 1% in a modified minimal salt medium-containing alginate matrix and the freeze-dried, entrapped cells were then formulated. Among several compounds tested, incorporation of sucrose proved to be beneficial as a cryoprotectant sustaining cell biodegradation efficiency (97%) and viability (≥90%) during freeze drying, storage under a vacuum condition at low temperatures, rehydration and as an additional matrix filler to reinforce the bead structure. The effective DEP treatment of the formulated, entrapped cells was demonstrated in a packed bed continuous system in which 70% DEP removal at hydraulic retention time (HRT) of 30 min was occurred and was enhanced up to 90% when HRT was increased to 60 min. The work demonstrates an effective preparation and a potential application of the formulated entrapped DEP-degrading cells for DEP treatment.     

Immobilized catalase-containing yeast cells: Preparation and enzymatic properties

The cell of Saccharomyces cerevisiae previously induced for catalase (EC 1.11.1.6) activity were immobilized by entrapment of intact cells in acrylamide polymerized by γ irradiation (100 kR). Yeast cells showed an enhancement in catalase activity on entrapment, an effect similar to that observed on treatment with organic solvents like toluene. The cells pretreated with toluene, however, showed complete loss of catalase activity on entrapment. The entrapped enzyme exhibited a narrow pH optimum, reduced K_m for H₂O₂, and a decrease in thermostability. The temperature optimum of catalase was also decreased from 60 to 40°C on immobilization. A tenfold decrease in the activation energy was also observed. The enzyme in the entrapped cells was, however, stable toward inactivation by γ irradiation. Unlike the intact cells, the entrapped yeast cells did not have the ability to induce catalase.     

Survival of Bifidobacterium longum Immobilized in Calcium Alginate Beads in Simulated Gastric Juices and Bile Salt Solution

Bifidobacterium longum KCTC 3128 and HLC 3742 were independently immobilized (entrapped) in calcium alginate beads containing 2, 3, and 4% sodium alginate. When the bifidobacteria entrapped in calcium alginate beads were exposed to simulated gastric juices and a bile salt solution, the death rate of the cells in the beads decreased proportionally with an increase in both the alginate gel concentration and bead size. The initial cell numbers in the beads affected the numbers of survivors after exposure to these solutions; however, the death rates of the viable cells were not affected. Accordingly, a mathematical model was formulated which expressed the influences of several parameters (gel concentration, bead size, and initial cell numbers) on the survival of entrapped bifidobacteria after sequential exposure to simulated gastric juices followed by a bile salt solution. The model proposed in this paper may be useful for estimating the survival of bifidobacteria in beads and establishing optimal entrapment conditions.     

Production of l (+) lactic acid by Lactobacillus delbrueckii immobilized in functionalized alginate matrices

The role of functionalized alginate gels as immobilized matrices in production of l (+) lactic acid by Lactobacillus delbrueckii was studied. L. delbrueckii cells immobilized in functionalized alginate beads showed enhanced bead stability and selectivity towards production of optically pure l (+) lactic acid in higher yields (1.74Yp/s) compared to natural alginate. Palmitoylated alginate beads revealed 99% enantiomeric selectivity (ee) in production of l (+) lactic acid. Metabolite analysis during fermentation indicated low by-product (acetic acid, propionic acid and ethanol) formation on repeated batch fermentation with functionalized immobilized microbial cells. The scanning electron microscopic studies showed dense entrapped microbial cell biomass in modified immobilized beads compared to native alginate. Thus the methodology has great importance in large-scale production of optically pure lactic acid.     

Redirecting cellular metabolism by immobilization of cultured plant cells: A model study with Coffea arabica

Suspension-cultured cells of Coffea arabica have been immobilized by entrapment in calcium alginate gels to mimic natural aggregation. The production of methylxanthine alkaloid was increased up to 13-fold by the immobilization. This increased production has been ascribed to organization of the entrapped cells through physicochemical interactions between the polymer (alginate) and the plant cell wall. It has been shown that the metabolic changes induced by the immobilization are reversible.     

Effects of immobilization and environmental stress on growth and production of non-polar metabolites of Tagetes minuta cells

Tagetes minuta (marigold) cells were entrapped under sterile conditions in agarose, κ-carrageenan, agar and alginate. The effects of different supports on the growth rate of the entrapped cells during incubation for one week under standard conditions [I] were studied. In the second part (weeks 2 and 3) of the experiment the effects of low temperature (10°C) [II], intermittent N₂ gassing [III], and omission of carbohydrates from the medium [IV]—superimposed on that of entrapment—on growth rate and the production of non-polar secondary metabolites were investigated. Compared to free cells, the impact of agarose on growth during the first week was nil, while the inhibition of growth increased in the order κ-carrageenan, agar and alginate, probably as a result of increasing rigidity of the support. In the second period the plant cells clearly had reached the stationary phase of the growth cycle in all cases. Again the pattern of growth on agarose closely followed that of free cells, i.e. a small increase in cases I and III, and a small decrease under the other two conditions. Low temperature [II] had the greatest effect on cell growth and cell release, probably as a result of gel structure at this temperature. Similarly to the effects on growth, the impact on secondary metabolite production was most pronounced in the case of alginate combined with low temperature. Both the omission of carbohydrates, and Nin2 gassing resulted in low concentrations of non-polar compounds in the media. The major trend observed was a shift away from mainly intracellular compounds in the case of free cells to mainly extracellular compounds in the case of entrapped cells at 10°C.     

Enhanced specific antibody productivity of calcium alginate-entrapped hybridoma is cell line-specific

In order to determine whether the enhanced specific antibody productivity (q _MAb ) of calcium alginate-entrapped hybridoma is cell line-specific, calcium alginate-entrapped hybridomas (4A2 and DB9G8) were cultivated under the condition where we had previously observed significantly enhancedq _MAb of calcium alginate-entrapped S3H5/2bA2 hybridoma. Unlike S3H5/2bA2 hybridoma, neither 4A2 nor DB9G8 hybridomas showed persistently enhancedq _MAb when they were entrapped in calcium alginate beads. The enhancedq _MAb of entrapped 4A2 and DB9G8 hybridomas, which was 2–3 times higher than theq _MAb of free-suspended cells in a control experiment, was observed only during the early stage of the culture. During the early stage of the culture, the viable cell concentration decreased probably due to cell damage during the entrapment process. As cell growth resumed, theq _MAb decreased to the similar level ofq _MAb of free-suspended cells within 5–7 days. Thus, we conclude that the enhancedq _MAb of calcium alginate-entrapped hybridomas is cell line-specific.     

Immobilization of alcohol dehydrogenase by gel entrapment of cells of Saccharomyces cerevisiae

A stable immobilized preparation of alcohol dehydrogenase (ADH) (EC 1.1.1.1) was obtained by entrapment of ADH-containing Saccharomyces cerevisiae cells in polyacrylamide, polymerized by gamma-rays (100 kR). The permeability barrier for the substrate through the cell membrane was found to be eliminated on entrapment. The stability characteristics, pH-activity profile and other properties of the entrapped ADH are presented. A four-fold enhancement in K_m for NAD⁺ was observed on entrapment, whereas K_m for ethanol was not altered.     

Immobilization of Saccharomyces uvarum cells in porous beads of polyacrylamide gel for ethanolic fermentation

Summary A procedure which does not involve the use of an immiscible organic solvent phase is described for the entrapment of yeast cells in porous beads of polyacrylamide gel. The cells are rapidly dispersed at 4° C in an aqueous solution containing sodium alginate and acrylamide-N,Nmethylene-bis-acrylamide monomer, and the suspension is immediately dropped into a solution of calcium formate to give calcium alginate coated beads. Polyacrylamide gel forms within the bead. The calcium alginate is subsequently leached out of the composite bead with either sodium citrate or potassium phosphate buffer solution. Cells of Saccharomyces uvarum ATCC 26 602 entrapped in such polyacrylamide beads ferment cane molasses in batch mode at higher specific ethanol productivity than a free cell suspension. Their volumetric productivity in continuous fermentation is higher than that of Ca²⁺-alginate immobilized cells.NCL Communication No. 4383     

Fatty acid impurities in alginate influence the phenol tolerance of immobilized Escherichia coli

Summary A short time after the immobilization of Escherichia coli in calcium alginate substantial modifications of the fatty acid patterns of the cells were observed. This effect could be related to lipid impurities in the commercial alginate product used, which could be taken up, at least in part by the microorganisms. The impurities were mainly free fatty acids but sterols were also detected. Immobilization of the cells in alginate material extracted by chloroform or ethanol decreased the tolerance of the cells to phenol as compared with cells immobilized in raw alginate. This effect was diminished if the immobilized cells were exogenously supplied with palmitic acid, which is the main constituent of the fatty acids extracted from alginate. These results indicate that not only fatty acids but also other ingredients of commercial alginate have physiological effects on cells entrapped in this gel material. Offprint requests to: H.-J. Rehm     

Culture of chondrocytes in alginate gel: Variations in conditions of Gelation influence the structure of the alginate gel, and the arrangement and morphology of proliferating chondrocytes

Summary Sodium alginate, which gels in the presence of calcium ions, is commonly used for culture of anchorage-independent cells, such as chondrocytes. Normally, the gel appears microscopically homogeneous but, depending on the conditions of gelation, it may contain a varying number of small channels that extend inward from the surface. We have examined the influence of these channels on the morphology of cultured chondrocytes entrapped in alginate beads. Growth-plate or articular chondrocytes cultured in alginate normally proliferate and form rounded cell clusters but, in alginate beads containing numerous channels, many chondrocytes become aligned and form columns similar to those in the growth plate in vivo. As the pattern of cellular growth and morphology in alginate is profoundly influenced by the presence of channels in the gel, further studies were conducted to determine what specific conditions of gelation affect their formation. The channels are especially numerous when both the alginate and the gelling solutions lack sodium ions or other monovalent cations. The channels are cavities in the gel formed by particulate blocking of the rapid diffusion of calcium ions from the gelling solution into the boundary of the calcium alginate solution, and hence they extend inward from cells at the surface of the alginate gel. An understanding of the conditions under which these channels develop makes it possible either to avoid their formation or, alternatively, to enhance the number of channels in order to encourage proliferating cells to grow in radial columns, rather than in a less organized pattern characteristic of most culture systems.     

Studies on the accumulation of heavy metal elements in biological systems

Summary Cells of a Daucus carota suspension culture were entrapped in a matrix of calcium alginate. The immobilised cells, incubated in a buffer mixture of sucrose, nitrate, KCl, CaCl₂, 2-(N-morpholino)-ethane sulphonic acid at pH 5.5, hydroxylated digitoxigenin. When compared under the same incubation conditions, freely suspended cells biotransformed digitoxigenin at a faster rate. Periplogenin formation was maximal at pH 5.3 and temperatures of 26°–34°C. The hydroxylase activity of the entrapped cells adapted to the presence of 20 mM CaCl₂ over a 12 day incubation. The diffusion barrier established on entrapment of the cells could not be overcome by addition of detergents or methanol. Controlled addition of chloroform (at 1/4 and 1/2 saturation) did stimulate hydroxylation of digitoxigenin without adversely affecting cell viability. The rate of hydroxylation of digitoxigenin was linear over an immobilised cell concentration of 0–7 mg dry weight and a digitoxigenin concentration of 0–20 mg/L. Five consecutive batch bioconversions at a rate greater than 60% could be achieved before the biocatalyst was inactivated. The results are discussed in relation to improving the hydroxylation reaction by immobilised D. carota and other reactions performed by immobilised plant cells.