The effect of long-term preservation of bacterial cells immobilized in poly(vinyl alcohol) cryogel on their viability and biosynthesis of target metabolites

The effect of cell storage at ?18°C for 18–24 months on reproductive capacity was investigated for various microorganisms (gram-positive and gram-negative bacteria, yeasts, and filamentous fungi) immobilized in poly(vinyl alcohol) cryogel. To examine the viability of immobilized cells after defrosting, the bioluminescent method of intracellular ATP determination was used. A high level of metabolic activity of immobilized cells after various periods of storage was recorded for Streptomyces anulatus, Rhizopus oryzae, and Escherichia coli, which are producers of the antibiotic aurantin, L(+)-lactic acid, and the recombinant enzyme organophosphate hydrolase, respectively. It was shown that the initial concentration of immobilized cells in cryogel granules plays an important role in the survival of Str. anulatus and Pseudomonas putida after 1.5 years of storage. It was found that, after slow defrosting in the storage medium at 5°C for 18 h of immobilized cells of the yeast Saccharomyces cerevisiae that had been stored for nine months, the number of reproductive cells increased due to the formation of ascospores.     

Immobilization of hydrocarbon-oxidizing bacteria in poly(vinyl alcohol) cryogels hydrophobized using a biosurfactant

A simple biosurfactant-based hydrophobization procedure for poly(vinyl alcohol) (PVA) cryogels was developed allowing effective immobilization of hydrocarbon-oxidizing bacteria. The resulting partially hydrophobized PVA cryogel granules (granule volume 5 microl) contained sufficient number (6.5 x 10(3)) of viable bacterial cells per granule, possessed high mechanical strength and spontaneously located at the interface in water-hydrocarbon system. Such interfacial location of PVA granules allowed high contact of immobilized biocatalyst with hydrophobic substrate and water phase, thus providing bacterial cells with mineral and organic nutrients. As a result, n-hexadecane oxidation efficiency of 51% after 10-day incubation was achieved using immobilized biocatalyst. PVA cryogels with increased hydrophobicity can be used for immobilization of bacterial cultures performing oxidative transformations of water-immiscible organic compounds. Immobilization of in situ biosurfactant producing Rhodococcus bacteria into PVA cryogel is discussed. PVA cryogel granules with entrapped alkanotrophic rhodococcal cells were stable after 10-month storage at room temperature.     

Adsorptive immobilization of rhodococcal cells in hydrophobized derivatives of wide-pore polyacrylamide cryogel

Adsorption of Rhodococcus ruber cells on columns with polyacrylamide cryogel (CryoPAAG) partially hydrophobized by different quantities (0.2, 1, and 5 mol %) of chemically grafted n-dodecane residues has been studied. The adsorption capacity (1.1 x 10(9) cells/g) of gel carrier for rhodococcal cells and the optimal content (1 mol %) of hydrophobizing groups were determined. The respirometric method showed the high catalytic activity and functional stability of immobilized bacterial cells. Respiratory activity of immobilized rhodococci in the presence of a model mixture of oil hydrocarbons exceeded the respective parameter for free cells by 12-17%. Viability of rhodococcal cells adsorptionally fixed in hydrophobized cryoPAAG was maintained at a level of 93-95% after a half-year period of storage. The results may be used for development of immobilized biocatalyst for directed transformation of hydrocarbon compounds and biological purification of oil-polluted water.     

Biosensors based on the luminous bacteria Photobaterium phosphoreum immobilized in polyvinyl alcohol cryogel for the monitoring of ecotoxicants

Immobilization of Photobacterium phosphoreum bacteria in polyvinyl alcohol cryogel was performed in order to develop biosensors used for ecotoxicant biomonitoring. The immobilization procedure, storage, and application of the immobilized cells for biomonitoring were optimized. It was shown that the immobilized cells demonstrate significantly higher stability and a longer duration of light emission than free bacteria. A discrete analysis of heavy metals and chlorophenols was conducted using the obtained biosensor samples.     

Microalgal cell immobilization for the long-term storage of the marine diatom Haslea ostrearia

To preserve the characteristics of the marine diatom Haslea ostrearia during long term storage, particularly size and shape, the algal cells were immobilized in alginate beads and stored at 4 ^∘C at reduced irradiance up to 4 months. Two clones of different size (Ho34, 63 μm and Ho40, 78 μm) were studied. With Ho34, a 10.4% decrease of the size was shown after 120 days, by using the conventional storage management, while it did not exceed 2.2% with immobilized cells. Consequently, H. ostrearia would have auxosporulated after 9 months compared to 52 months. At the same time, the rate of distortion (aberrant valve structure) free Ho34 cells reached 86% while no distorted immobilized cells were observed. Chorophyll content in cells showed that all the cells were alive up to 60 days and after this time cells immobilized in the core of the beads most probably suffered from the poor light diffusion. Culturability of the immobilized cells was tested immediately after their immobilization and after 60 and 120 days of storage. The delay (at least 5) before immobilized cells released from the beads decreased with the time of storage, because of the embrittlement of the beads during the storage. Once in fresh medium, the cells actively multiplied. We concluded that immobilization strongly slowed down the decrease in frustule size with time and allowed the storage of concentrated and calibrated inocula which could be inoculated directly in liquid culture medium without needing to dissolve the beads.     

A study of a bacterial immobilization substratum for use in the bioremediation of crude oil in asaltwater system

An immobilization matrix, Drizit, was examined for use in the bioremediation of oil in asaltwater system. The support was examined by scanning electron microscopy, cell-loadingcapacity, absorption of Ekofisk crude oil, the effect on cell recoverability of dryingDrizit-immobilized cells at room temperature, 55°C or freeze drying and storage for up to 3months, and the capability of immobilized Pseudomonas fluorescens to enhance biodegradation ofpetrol (Slovene diesel) in a saltwater system. Results showed that Drizit is an ideal immobilizationsubstratum for use in bioremediation, and the substratum was found to have a good cell-loadingcapacity (3·75 mg protein g⁻¹ substratum) and a high absorbency of oil(7·49 g Ekofisk g⁻¹ substratum). After drying the immobilized cells at roomtemperature, 55°C or freeze drying and subsequent storage, cells were not recoverable after 2weeks, 1 week or 2 months, respectively, and a significant decrease was seen in the number ofcells recovered after drying and 1 week's storage in all cases. Enhanced biodegradation of C₁₃–C₁₈ pristane and phytane, in petrol (Slovene diesel) occurred with theimmobilization of Ps. fluorescens to Drizit. An average degradation of 73·8%occurred in the immobilized system in comparison with the free system which resulted in anaverage degradation of 39·8%. When the two systems were supplemented with nitrates andphosphates, degradation in the free supplemented system increased to an average of 72·4%.However, no significant difference in biodegradation was found between the immobilized systemand the immobilized system supplemented with nitrates and phosphates which achieved anaverage degradation of 74.15%.     

Adsorptive immobilization of rhodococcal cells in hydrophobized derivatives of wide-pore poly(acrylamide) cryogel

Adsorption of Rhodococcus ruber cells on columns with poly(acrylamide) cryogel (cryoPAAG) partially hydrophobized by different quantities (0.2, 1, and 5, mol %) of chemically grafted n-dodecane residues has been studied. The adsorption capacity (1.1 × 10⁹ cells/g) of gel carrier for rhodococcal cells and the optimal content (1 mol %) of hydrophobizing groups were determined. The respirometric method showed the high catalytic activity and functional stability of immobilized bacterial cells. Respiratory activity of immobilized rhodococci in the presence of a model mixture of oil hydrocarbons exceeded the respective parameter for free cells by 12–17%. Viability of rhodococcal cells adsorptionally fixed in hydrophobized cryoPAAG was maintained at a level of 93–95% after a half-year period of storage. The results may be used for development of immobilized biocatalyst for directed transformation of hydrocarbon compounds and biological purification of oil-polluted water.     

Effect of frozen storage and aging on the Kashkaval cheese starter culture

Summary The changes in the number of the starter microorganisms Lb. delbrueckii subsp. bulgaricus and Str. thermophiluswere followed in frozen-stored Kashkaval cheese made from cow’s milk. Kashkaval samples of various aging times were produced industrially, frozen at T=−16 °C and stored at T=−10 to −12 °C for 12 months. It was found that the number of Lb. delbrueckiisubsp. bulgaricus and Str. thermophilusdecreased considerably during frozen storage. The decrease was more substantial for Lb. delbrueckiisubsp. bulgaricus, which was evidence for its greater sensitivity to the impact of low temperatures. The aging time of Kashkaval did not influence the changes in the starter culture during frozen storage but is important for its amount in the product aged after defrosting. There was an increase in the Str. thermophilus: Lb. delbrueckiisubsp. bulgaricus ratio in samples with shorter aging time subjected to frozen storage and aged after defrosting. The changes in the starter culture in frozen stored Kashkaval cheese can be controlled by an appropriate combination of the two factors: aging time and period of frozen storage.     

Monoclonal antibody production using a new supermacroporous cryogel bioreactor

A supermacroporous cryogel bioreactor has been developed to culture hybridoma cells for long-term continuous production of monoclonal antibodies (mAb). Hybridoma clone M2139, secreting antibodies against J1 epitope (GERGAAGIAGPK; amino acids, 551-564) of collagen type II, are immobilized in the porous bed matrix of a cryogel column (10 mL bed volume). The cells got attached to the matrix within 48 h after inoculation and grew as a confluent sheet inside the cryogel matrix. Cells were in the lag phase for 15 days and secreted mAb into the circulation medium. Glucose consumption and lactic acid production were also monitored, and during the exponential phase (approximately 20 days), the hybridoma cell line consumed 0.75 mM day-1 glucose, produced 2.48 mM day-1 lactic acid, and produced 6.5 microg mL-1 day-1 mAb during the exponential phase. The mAb concentration reached 130 microg mL-1 after continuous run of the cryogel column for 36 days. The yield of the mAb after purification was 67.5 mg L-1, which was three times greater than the mAb yield obtained from T-flask batch cultivation. Even after the exchange of medium reservoir, cells in the cryogel column were still active and had relatively stable mAb production for an extended period of time. The bioreactor was operated continuously for 55 days without any contamination. The results from ELISA as well as arthritis experiments demonstrate that the antibodies secreted by cells grown on the cryogel column did not differ from antibodies purified from the cells grown in commercial CL-1000 culture flasks. Thus, supermacroporous cryogels can be useful as a supporting material for productive hybridoma cell culture. Cells were found to be viable inside the porous matrix of the cryogel during the study period and secreted antibodies continuously. The antibodies thus obtained from the cryogel reactor were found to be functionally active in vivo, as demonstrated by their capacity to induce arthritis in mice.     

Boronate affinity chromatography of cells and biomacromolecules using cryogel matrices

Boronate affinity chromatography involves the interaction between cis-diol containing molecules and the hydroxyl group of boronate. Boronate affinity based cryogel chromatography matrices have been developed and the ligands were immobilized by two methods i.e., grafting of the boronate ligand on to the matrix and by copolymerization of monomer containing boronate with other co-monomer. The boronate grafted cryogel column was used to capture adherent and non-adherent cells and the captured cells were recovered at different fructose concentrations as an eluting agent, in chromatography mode. It was found that the adherent cells could be recovered at relatively higher fructose concentration (0.5 M) than non-adherent cells which could be recovered by using low fructose concentration (0.1 M). This might be due to the difference in the content of glycoprotein in adherent and non-adherent cells. In this way a new separation method can be devised for the fractionation of adherent and non-adherent cells. In another study, a copolymerized boronate cryogel column was developed for the separation of RNA from the bacterial crude extract without any pre-processing. RNA molecules were specifically retained in the cryogel column due to interaction between 2,3′ diol group of ribose sugar in RNA and the hydroxyl group of boronate. The DNA molecules were passed through the column uninteracted due to absence of 2′-hydroxyl group. Later, bound RNA molecules were recovered from the boronate affinity cryogel column.     

Methane production from wastewaters by immobilized methanogenic bacteria

A methanogenic population was immobilized onto agar gel, polyacrylamide gel, and collagen membrane. Agar-gel-entrapped methanogenic microorganisms gave the highest activity. The optimum agar concentration was between 1.5 and 3% (w/v), and the optimum microbial content was 20 mg wet cells/g gel. The optimum conditions for methane production by immobilized whole cells were pH 7.0–7.5 and 37–45°C. The rate of methane production was initially 1.8 μmol/g gel/hr. Methane productivity was gradually increased and reached a steady state (4.5μmol/g gel/hr) after 25 days of incubation. The immobilized methanogenic microbial population continuously evolved methane over a 90 day period. No difference in methane productivity was observed after three months of storage at 5°C. Methane was also produced by immobilized whole cells under aerobic conditions. Furthermore, carbohydrates, such as glucose, in wastewater completely decomposed by immobilized whole cells.     

Cryopreservation by slow cooling with DMSO diminished production of Oct-4 pluripotency marker in human embryonic stem cells

We tested a "standard" cryopreservation protocol (slow cooling with 10% DMSO) on the human embryonic stem cell (hESC) line H9 containing an Oct-4 (POU5F1) promoter-driven, enhanced green fluorescent protein (EGFP) reporter to monitor maintenance of pluripotency. Cells were cooled to -80 degrees C in cryovials and then transferred to a -80 degrees C freezer. Cells were held at -80 degrees C for 3 days ("short-term storage") or 3 months ("long-term storage"). Vials were thawed in a +36 degrees C water bath and cells were cultured for 3, 7, or 14 days. Propidium iodide (PI) was used to assess cell viability by flow cytometry. Control cells were passaged on the same day that the frozen cells were thawed. The majority of cells in control hESC cultures were Oct-4 positive and almost 99% of EGFP+ cells were alive as determined by exclusion of PI. In contrast, the frozen cells, even after 3 days of culture, contained only 50% live cells, and only 10% were EGFP-positive. After 7 days in culture, the proportion of dead cells decreased and there was an increase in the Oct-4-positive population but microscopic examination revealed large patches of EGFP-negative cells within clusters of colonies even after 14 days of culturing. After 3 months of storage at -80 degrees C the deleterious effect of freezing was even more pronounced: the samples regained a quantifiable number of EGFP-positive cells only after 7 days of culturing following thawing. It is concluded that new protocols and media are required for freezing hESC and safe storage at -80 degrees C as well as studies of the mechanisms of stress-related events associated with cell cryopreservation.     

Solubilization of fish proteins using immobilized microbial cells

Cells of Bacillus megaterium, Aeromonas hydrophila, and Pseudomonas marinoglutinosa were immobilized in calcium alginate. The immobilized cells secreted protease when held in fish meat suspension in water. The enzyme synthesis by the entrapped cells was supported by small amounts of soluble nutrients present in the meat. The secreted protease solubilized the fish meat, solubilization being optimum at pH range of 7.5 to 9.5 and at 50 degrees C. Under these conditions immobilized B. megaterium was most efficient giving 30% solubilization of the meat, followed by A. hydrophila (18%), while immobilized P. marinoglutinosa was less effective. The optimum ratio of fish meat to beads was about 4:3 for B. megaterium and A. hydrophila. The beads had a storage life of 30 days at 4 degrees C. The results suggested potential for use of immobilized microbial cells having extracellular protease activity to enhance solubility of waste proteins. A prototype reactor with beads holding assembly was fabricated which could recover the beads from the meat slurry after the treatment.     

Green biosynthesis of floxuridine by immobilized microorganisms

This work describes an efficient, simple, and green bioprocess for obtaining 5-halogenated pyrimidine nucleosides from thymidine by transglycosylation using whole cells. Biosynthesis of 5-fluoro-2'-deoxyuridine (floxuridine) was achieved by free and immobilized Aeromonas salmonicida ATCC 27013 with an 80% and 65% conversion occurring in 1 h, respectively. The immobilized biocatalyst was stable for more than 4 months in storage conditions (4 °C) and could be reused at least 30 times without loss of its activity. This microorganism was able to biosynthesize 2.0 mg L(-1) min(-1) (60%) of 5-chloro-2'-deoxyuridine in 3 h. These halogenated pyrimidine 2'-deoxynucleosides are used as antitumoral agents.     

Factors affecting the production of prednisolone by immobilization of Bacillus pumilus E601 cells in poly(vinyl alcohol) cryogels produced by radiation polymerization

To increase the yield percent of prednisolone from hydrocortisone (cortisol), Bacillus pumilus E601 (a radioresistant microorganism) was incorporated into poly(vinyl alcohol) (PVA) cryogel grafted with hydroxyethyl-methacrylate (HEMA) as a crosslinking agent. The polymer was prepared by a radiation polymerization technique at 20 kGy from Co-60 source. The optimum temperature for the biotransformation of hydrocortisone by free cells, poly(PVA)/HEMA, and poly(PVA)/HEMA /N-isopropylacrylamide (N-IPAAm) was 30 °C. The highest yield % of prednisolone was obtained by immobilization of the cells on poly(PVA/HEMA), the addition of N-IPAAm to poly(PVA/HEMA) protected the immobilized cells from temperatures above 35 °C during the fermentation process. The optimal pH (buffered pH) of the biotransformation of hydrocortisone by immobilized and free cells was 7.0, but the maximum yield of prednisolone (60%) was obtained by immobilized cells in comparison with free cells (42%) also at pH 7.0. The prednisolone yield reached 60–65% with 1,2-propanediol cosolvent containing media and 60–62% in the case of ethanediol cosolvent containing media at 1% (v/v) of both cosolvents. 10 mg/50 ml Tween 80 the medium increased the prednisolone yield by only 1.1-fold compared with the control. The maximum bioconversion efficiency was obtained at a substrate concentration of 20 mg/50 ml medium. Stability studies showed that the immobilized cells can be used for seven times without any significant decrease in activity.     

Immobilization of E. coli cells in polyacrylamide-based microporous cryogels

E. coli cells were immobilized in polyacrylamide cryogel by three ways: (1) introduction of cells in the reaction mixture followed by cryopolymerization; (2) the filling of the cryogel pores followed by cell fixation with diluted glutaric dialdehyde (GDA), and (3) the filling of the macropores of the polymeric matrix with modified surface. The ultrastructure of the gels and immobilized cells as well as distribution of attachment of the cells immobilized by different techniques were studied. The first type of immobilization was characterized by the highest quantity of the biomass in the gel (by protein) and by a sharp decrease of the cell viability. The second failed to retain the cells in the pores, and the GDA treatment significantly decreased the viability index. The latter technique was the mildest and completely maintained the viability of the population. However, the biomass content was lower as compared to the first type of immobilization, but could be considerably increased by the GDA treatment.     

Storage of Resting Spores of the Gypsy Moth Fungal Pathogen, Entomophaga maimaiga

The fungal pathogen, Entomophaga maimaiga causes epizootics in populations of the important North American forest defoliator gypsy moth ( Lymantria dispar ). Increasing use of this fungus for biological control is dependent on our ability to produce and manipulate the long-lived overwintering resting spores (azygospores). E. maimaiga resting spores undergo obligate dormancy before germination so we investigated conditions required for survival during dormancy as well as the dynamics of subsequent germination. After formation in the field during summer, resting spores were stored under various moisture levels, temperatures, and with and without soil in the laboratory and field. The following spring, for samples maintained in the field, germination was greatest among resting spores stored in plastic bags containing either moistened paper towels or sterile soil. Resting spores did not require light during storage to subsequently germinate. In the laboratory, only resting spores maintained with either sterile or unsterilized soil at 4°C (but not at 20 or -20°C) germinated the following spring, but at a much lower percentage than most field treatments. To further investigate the effects of relative humidity (RH) during storage, field-collected resting spores were placed at a range of humidities at 4°C. After 9.5 months, resting spore germination was highest at 58% RH and no resting spores stored at 88 or 100% RH germinated. To evaluate the dynamics of infections initiated by resting spores after storage, gypsy moth larvae were exposed to soil containing resting spores that had been collected in the field and stored at 4°C for varying lengths of time. No differences in infection occurred among larvae exposed to fall-collected soil samples stored at 4oC over the winter, versus soil samples collected from the same location the following spring. Springcollected resting spores stored at 4°C did not go into secondary dormancy. At the time that cold storage of soil containing resting spores began in spring, infection among exposed larvae was initiated within a few days after bringing the soil to 15°C. This same pattern was also found for spring-collected resting spore-bearing soil that was assayed after cold storage for 2-7 months. However, after 31-32 months in cold storage, infections started 14-18 days after soil was brought to 15°C, indicating a delay in resting spore activity after prolonged cold storage.     

Cell separation using cryogel-based affinity chromatography

In cell affinity chromatography, type-specific cell separation is based on the interaction between cell-surface receptors and an immobilized ligand on a stationary matrix. This protocol describes the preparation of monolithic polyacrylamide and polydimethylacrylamide cryogel affinity matrices that can be used as a generic type-specific cell separation approach. The supermacroporous monolithic cryogel has highly interconnected large pores (up to 100 μm) for convective migration of large particles such as mammalian cells. In this protocol, they are functionalized to immobilize a protein A ligand by a two-step derivatization of epoxy-containing cryogel monolith (reaction with ethylenediamine and glutaraldehyde). Target cells were labeled with specific antibodies and then they were captured in the cryogel through affinity with protein A. These specifically captured cells were recovered in high yields while retaining their viability by mechanical squeezing of the spongy and elastic cryogel matrices. The suggested cell separation protocol takes < 30 min for complete separation on a preprepared protein A-immobilized cryogel column.     

Optimising the viability during storage of freeze-dried cell preparations of Campylobacter jejuni

Freeze-dried cultures of Campylobacter jejuni are used in the food and microbiological industry for reference materials and culture collections. However, C. jejuni is very susceptible to damage during freeze-drying and subsequent storage and it would be useful to have longer-lasting cultures. The survival of C. jejuni during freeze-drying and subsequent storage was investigated with the aim of optimising survival. C. jejuni was freeze-dried using cultures of different age (24-120 h), various lyoprotectants (10% phytone peptone, proteose peptone, peptonized milk, trehalose, soytone and sorbitol), various storage (air, nitrogen and vacuum) and re-hydration (media, temperature and time) conditions. One-day-old cultures had significantly greater survival after freeze-drying than older cultures. The addition of trehalose to inositol broth as a lyoprotectant resulted in almost 2 log(10) increase in survival after 2 months storage at 4 degrees C. Storage in a vacuum atmosphere and re-hydration in inositol broth at 37 degrees C increased recovery by 1-2 log(10) survival compared to re-hydration in maximal recovery diluent (MRD) after storage at 4 degrees C. Survival during storage was optimal when a one-day-old culture was freeze-dried in inositol broth plus 10% (w/v) trehalose, stored under vacuum at 4 degrees C and re-hydrated at the same incubation temperature (37 degrees C) in inositol broth for 30 min. The results demonstrate that the survival of freeze-dried cells of C. jejuni during storage can be significantly increased by optimising the culture age, the lyoprotectant, and the storage and re-hydration conditions. The logarithmic rate of loss of viability (K) followed very well an inverse dependence on the absolute temperature, i.e., the Arrhenius rate law. Extrapolation of the results to a more typical storage temperature (4 degrees C) predicted a very low K value of 1.5 x 10(-3). These results will be useful to the development of improved reference materials and samples held in culture collections.     

Determination of free sulphite in wine using a microbial sensor

Summary A microbial sensor of immobilized Thiobacillus thiooxidans S3 cells was assembled to determine free sulphite in wine. Sulphite oxidation activity of the immobilized cells was sufficiently high for use even after 3 months storage at 4° C. The sensitivity of this sensor was 116 nA·1·mg^-1 for sulphur dioxide. The relationship between the current decrease and the sulphur dioxide concentration was linear up to 17 mg·1^-1. The sampling rate achieved was 10 min per sample including washing time. This sensor method needed no pretreatment of wine samples, and wines diluted with 5 mM sulphuric acid solution could be directly introduced in the computer-aided analysis system. The pigments in red wine did not disturbed the analysis.