Free Cell Culture Research Articles

Comparative proteomic analysis of planktonic and immobilized Pseudomonas aeruginosa cells: a multivariate statistical approach

The protein maps of Pseudomonas aeruginosa cells from two natural (attached) and one artificial (gel-entrapped) immobilized-cell (IC) systems, together with their free (suspended) counterparts, were compared after incubation for 18 or 48 h in a minimal salt medium. Principal component analysis (PCA) was used to interpret the variations in protein spot densities that were observed on electropherogram obtained by two-dimensional electrophoresis (2-DE). PCA of the 2-DE data, a matrix of 933 rows (observations, i.e., spot density values) and 12 columns (variables, i.e., incubation conditions), in which observations were standardized horizontally, extracted four principal components (PCs) accounting for 78.75% of the variability in the protein expression profiles. PC1 opposed the two modes of growth (planktonic and immobilized) while PC2 discriminated between the incubation times of free cell cultures. The incubation conditions of ICs, including the immobilization procedure (entrapment vs attachment) and the nature of the biofilm substratum, were fairly separated in PC3 × PC4. The dependence of the protein patterns on the cell immobilization process was further illustrated by the identification of a number of peptides whose amount remained unchanged or was altered in ICs compared to free bacteria. These results reinforce the topical assertion that bacteria in the immobilized state display a specific physiological behavior but also question the existence of a unique IC phenotype.

Growth and nutrient removal in free and immobilized planktonic green algae isolated from pig manure

Laboratory experiments were performed to study the growth rate and phosphorus (P) and nitrogen (N) uptake of two nanoplanktonic algal species, Scenedesmus intermedius Chod. and Nannochloris sp. that were isolated from different sources of pig manure. Experiments were carried out for immobilized and free cell cultures simultaneously, with three replicates for each type of culture. Algal immobilization was prepared in calcium alginate beads. The growth rate (measured as chlorophyll increase per hour) and the P and N uptake rate (measured as P and N increase per hour) were estimated by linearization of the logistic growth and nutrient uptake curves, which allowed us to apply proper statistical comparative analyses. P and N uptake rates for S. intermedius were 0.014 and 0.012 mg P h −1 and 0.022 and 0.009 mg N h −1 for free and immobilized cells. Rates for Nannochloris sp. were 0.006 and 0.009 mg P h −1 and 0.011 and 0.006 mg N h −1 for free and immobilized cells. P and N removal rates obtained in the present study, which used species isolated from wastewater, are markedly higher than rates described in experiments that used commercial species, probably because the former species are better adapted to high nutrient concentrations. Utilizing autochthonous species for wastewater treatment may be a simple method to increase nutrient removal efficiency.

Oestrogenic compounds and oxidative stress (in human sperm and lymphocytes in the Comet assay).

Reactive oxygen species (ROS) are produced by a wide variety of chemicals and physiological processes in which enzymes catalyse the transfer of electrons from a substrate to molecular oxygen. The immediate products of such reactions, superoxide anion radicals and hydrogen peroxide can be metabolised by enzymes such as superoxide dismutase (SOD) and catalase (CAT), respectively, and depending on its concentration by Vitamin C (Vit C). Under certain circumstances the ROS form highly reactive hydroxyl radicals. We examined human sperm and lymphocytes after treatment with six oestrogenic compounds in the Comet assay, which measures DNA damage, and observed that all caused damage in both cell types. The damage was diminished in nearly all cases by catalase, and in some instances by SOD and Vit C. This response pattern was also seen with hydrogen peroxide. This similarity suggests that the oestrogen-mediated effects could be acting via the production of hydrogen peroxide since catalase always markedly reduced the response. The variable responses with SOD indicate a lesser involvement of superoxide anion radicals due to SOD-mediated conversion of superoxide to hydrogen peroxide generally causing a lower level of DNA damage than other ROS. The variable Vit C responses are explained by a reduction of hydrogen peroxide at low Vit C concentrations and a pro-oxidant activity at higher concentrations. Together these data provide evidence that inappropriate exposure to oestrogenic compounds could lead to free-radical mediated damage. It is believed that the observed activities were not generated by cell free cell culture conditions because increased responses were observed over and above control values when the compounds were added, and also increasing dose-response relationships have been found after treatment with such oestrogenic compounds in previously reported studies.

Study of the potential cytotoxicity of dental impression materials

The aim of this study was to assess the cytotoxicity of two types of impression dental materials: polyethers (Impregum Penta, Permadyne Penta Heavy and Light) and vinyl polysiloxanes (Elite Mono Tray, Medium, Low viscosity and Elite H-D Putty). Their cytotoxic effects were studied by indirect and direct tests. The indirect tests were performed by incubating impression materials in serum free cell culture medium to prepare the soluble extracts. Balb/c 3T3 cells were incubated with extract dilutions (25, 50, 75 and 100%) for 24 h. The extracts of polyether materials caused a decrease of cellular viability, evaluated by light microscopy, by cell counting and by MTT test. The extracts of vinyl polysiloxanes materials induced a slight effect on cellular number and viability. The direct tests were performed by placing the impression materials in the centre of Petri dishes while Balb/c 3T3 were settling. The cellular proliferation was drastically reduced by polyethers and it was unaffected by the presence of vinyl polysiloxanes. These results show that: (a) the polyether materials are more toxic than vinyl polysiloxanes in our experimental conditions, (b) the impression materials are cytotoxic to the same degree in all assay methods.

Transformation of high concentrations of chlorophenols by the white-rot basidiomycete Trametes versicolor immobilized on nylon mesh

Free-cell cultures of Trametes versicolor were compared with cultures immobilized on nylon mesh in a 2-litre bioreactor for transformation of pentachlorophenol (PCP) and 2,4-dichlorophenol (2,4-DCP), added at intervals to the liquid culture medium over a period of 816 hrs. Increasing amounts of PCP from 200 ppm to 2000 ppm added batchwise to cultures permitted acclimatization of the fungus to these toxic pollutants. A total addition of 2000 ppm of 2,4-DCP and 3400 ppm PCP were removed from the immobilized cultures with 85% of 2,4-DCP and 70% of PCP transformed by enzymes (laccase and Mn-peroxidase), 5% 2,4-DCP and 28% PCP adsorbed by the biomass and 10% 2,4-DCP and 2% PCP retained in the medium at the termination of the fermentation after 1020 hrs. In contrast free-cell cultures in the same medium with the same addition regime of PCP and 2,4-DCP, transformed 20% 2,4-DCP and 12% PCP by enzyme action, adsorbed 58% 2,4-DCP and 80% PCP by the biomass, and retained 22% 2,4-DCP and 8% PCP in the medium. The use of nylon mesh as an immobilization matrix for removal of PCP and 2,4-DCP facilitates more efficient removal of chlorophenols and can be adapted to scale-up for application of large volumes of chlorophenol-containing aqueous effluents.

Transformation of high concentrations of chlorophenols by the white-rot basidiomycete Trametes versicolor immobilized on nylon mesh

Free-cell cultures of Trametes versicolor were compared with cultures immobilized on nylon mesh in a 2-litre bioreactor for transformation of pentachlorophenol (PCP) and 2,4-dichlorophenol (2,4-DCP), added at intervals to the liquid culture medium over a period of 816 hrs. Increasing amounts of PCP from 200 ppm to 2000 ppm added batchwise to cultures permitted acclimatization of the fungus to these toxic pollutants. A total addition of 2000 ppm of 2,4-DCP and 3400 ppm PCP were removed from the immobilized cultures with 85% of 2,4-DCP and 70% of PCP transformed by enzymes (laccase and Mn-peroxidase), 5% 2,4-DCP and 28% PCP adsorbed by the biomass and 10% 2,4-DCP and 2% PCP retained in the medium at the termination of the fermentation after 1020 hrs. In contrast free-cell cultures in the same medium with the same addition regime of PCP and 2,4-DCP, transformed 20% 2,4-DCP and 12% PCP by enzyme action, adsorbed 58% 2,4-DCP and 80% PCP by the biomass, and retained 22% 2,4-DCP and 8% PCP in the medium. The use of nylon mesh as an immobilization matrix for removal of PCP and 2,4-DCP facilitates more efficient removal of chlorophenols and can be adapted to scale-up for application of large volumes of chlorophenol-containing aqueous effluents.

Monitoring the growth and stress responses of yeast cells by two-dimensional fluorescence spectroscopy: first results.

S. cerevisiae growth and responses to different treatments were monitored by two-dimensional fluorescence spectroscopy, which simultaneously detects the fluorescence of a number of cells' own fluorophores. Growth curves of cultures of free cells were measured by means of tryptophan fluorescence in nonfluorescent culture medium and a flow-through system at a suitable excitation/emission beam geometry. Fast responses of the cells to anaerobic-aerobic transition or addition of glucose, methanol or cyanide, which could not be measured in this system because of the time delay inherent in transporting the cells from the culture flask to the cuvette, were monitored with cells immobilized in alginate. The major fluorescence changes caused by these treatments belonged to NAD(P)H which is a good indicator of the redox state of the cells.

The culture of single plant cells: a historical view.

Plant tissue culture, introduced unsuccessfully at the beginning of the twentieth century by Haberlandt, received full confirmation in the late Thirties by the works of Gautheret and Nobécourt, thanks to the discovery of auxin. A further special improvement--the free cell culture--, already fore-told by Haberlandt, was successfully achieved towards the mid-1950s by several physiologists thanks to coconut milk (cytokinin). The English physiologist Frederick Steward (who grouped an excellent American team of research during his twenty years stay at the Cornell University in Ithaca) was able to obtain complete cell differentiation from single cells cultured in vitro and demonstrate the totipotence of plant cells at any stage of development. The historical meaning of the research of Steward's team, accomplished between 1958 and 1970, rests on the concept of plant hormones as regulators of gene activity. In other terms, organogenesis was conceived as an epigenetically controlled series of events in which plant genes were "switched on" or "switched off" by special biomolecules. Steward's research paved the way for molecular plant physiology and inspired future research on the relation between cell receptors and specific hormones.

Biotransformation of phenylpropanoid compounds to vanilla flavor metabolites in cultures of Haematococcus pluvialis

Biotransformation of phenylpropanoid intermediates-ferulic acid, coniferyl aldehyde and p-coumaric acid was studied in free and immobilized cell cultures of Haematococcus pluvialis. H. pluvialis cultures accumulated vanilla flavor metabolites-vanillin, vanillic acid, vanillyl alcohol, protocatechuic acid, p-hydroxybenzoic acid, p-hydroxybenzaldehyde and p-coumaric acid when treated with these precursors. The accumulation of these metabolites increased gradually and reached a maximum on the third day in 1.0 mM ferulic acid fed immobilized Haematococcus cultures. The immobilized H. pluvialis cultures showed maximum vanillin (10.6 mg l −1), vanillic acid (5.4 mg l −1), vanillyl alcohol (3.3 mg l −1), protocatechuic acid (1.6 mg l −1), p-hydroxybenzoic acid (1.4 mg l −1) and p-coumaric acid (1.1 mg l −1) on the third day of the experimental period and these were 2.1, 1.8, 1.3, 1.14, 1.1 and 1.1 folds higher over free cell cultures of Haematococcus fed with 1.0 mM ferulic acid. Coniferyl aldehyde fed H. pluvialis cultures showed 2.3 and 2.2 folds higher vanillin and vanillyl alcohol accumulation when compared with 0.5 mM fed cultures. p-Coumaric acid fed Haematococcus cultures showed accumulation of p-hydroxybenzoic acid, p-hydoxybenzaldehyde and protocatechuic acid. These results demonstrated that cultures of Haematococcus can bioconvert externally fed phenylpropanoid precursors to a range of vanilla flavor metabolites.

Growth characteristics and acidifying activity of Lactobacillus rhamnosus in alginate/starch liquid-core capsules

Lactobacillus rhamnosus cells were encapsulated in alginate/starch liquid-core capsules and cultured in a whey-based growth medium supplemented with yeast extract, Tryptone, and calcium carbonate to enable concentrated biomass production in an immobilized matrix without centrifugation or filtration. The characteristics of the encapsulated cell culture were compared with those of freely suspended cell fermentation under the same bioreactor conditions. The population of encapsulated viable cells reached a concentration of 4.8×10 10 cfu cm −3 of capsule volume, while the population of free cells cultured at the same conditions during control fermentation was 5.7×10 9 cfu cm −3 of growth medium. Despite the higher cell concentration in the capsules, the overall cell productivity of encapsulated culture was lower than that obtained in free-cell fermentation. The effects of lactic acid inhibition on cell growth in the capsules caused by mass transfer limitations were evidenced by premature growth cessation and earlier uncoupling between growth and acid production as compared to free-cell culture. The capsules remained stable and no cell release was observed during 60 h of fermentation.

Cultivation and modelling of encapsulated Saccharomyces cerevisiae in NaCS-PDMDAAC polyelectrolyte complexes

The cultivation of encapsulated S. cerevisiae in NaCS-PDMDAAC polyelectrolyte complexes was studied. The results showed that the encapsulated microorganisms had the same growth trends as in its free cell culture and, thus, NaCS-PDMDAAC microcapsules were suitable for the encapsulation of these biological substances. The encapsulated S. cerevisiae cells were fermented sequentially for 16 batches. The highest cell density in the capsules reached 2.64 #50 10 10 cells mL -1 and the ethanol concentration was 47.0 g L -1 . A model of the cultivation of the encapsulated S. cerevisiae was developed.

The production of freeze-dried immobilized cultures of Streptococcus thermophilus and their acidification properties in milk.

The aim of this study was to prepare alginate-immobilized freeze-dried cultures of Streptococcus thermophilus and to compare the acidifying activities of these rehydrated cultures with classical free cell liquid inoculants. Streptococcus thermophilus BT1 grew in alginate beads and the population reached 10(10) cfu g(-1) after 6 h incubation. Re-inoculation of the beads in fresh medium with a further 6 h incubation did not improve the biomass level, but extending the incubation at 42 degrees C to 24 h caused significant death. The rehydrated immobilized cell technology (ICT) starter contained 13% free cells. In acidifying activity tests, the ICT culture had a similar acidification curve to that of a classical milk-grown free cell culture, except that it reached lower final pH values. Although the differences between the ICT and liquid cultures were not important, there were significant effects of inoculation level on lag time, maximum acidification rate and on the pH and time at which the acidification rate was at its highest.

Production and molecular weight characteristics of alginate from free and immobilized-cell cultures of Azotobacter vinelandii

Viable Azotobacter vinelandii cells were immobilized in composite agar gel layer/microporous membrane structures and tested for alginate production from sucrose during batch incubation. Microporous membranes with varying pore sizes (0.22–3.0 μm) were used in the composite structures. Whatever the membrane pore size (MPS), the duration of the production period was short (ca. 100 h), most sucrose remaining unconsumed by immobilized organisms. The amount of alginate recovered (0.5–0.9 g dm −3) and volumetric productivity increased slightly with the MPS, but the average production yield was fairly stable with a mean value of 0.24 g alginate produced g −1 sucrose consumed, i.e. noticeably higher than that of free-cell counterparts (0.09 g g −1). Furthermore, immobilization was shown to favour the production of higher molecular weight (MW) polysaccharide macromolecules and to stabilize the MW distribution of alginate produced as compared to conventional free-cell cultures grown in shake flasks.

The potential of positively-charged cellulose sponge for malolactic fermentation of wine, using Oenococcus oeni.

Malolactic fermentation (MLF) is a secondary bioconversion developed in some wines involving malic acid decarboxylation. The induction of MLF in wine by cultures of free and immobilized Oenococcus oeni cells was investigated. This work reports on the effect of surface charges in the immobilization material, a recently described fibrous sponge, as well as the pH and the composition of the media where cells are suspended. A chemical treatment provided positive charge to the sponges (DE or DEAE) and gave the highest cell loadings and subsequent resistance to removal. Preculture media to grow the malolactic bacteria before the immobilization procedure were also evaluated. We have established favorable conditions for growth (Medium of Preculture), suspension solution (Tartrate-Phosphate Buffer), suspension pH (3.5-5.5) and immobilization matrix (DE or DEAE cellulose sponge) to induce MLF in red wine. The use of a semi-continuous system permitted a high-efficiency malic acid conversion by 2 x 10(9) cfu sponge(-)(1) in at least four subsequent batch fermentations.

Biotransformation of oleic acid into (E)-10-hydroxy-8-octadecenoic acid and (E)-7,10-dihydroxy-8-octadecenoic acid by Pseudomonas sp. 42A2 in an immobilized system

Oleic acid was transformed into 10-hydroxy-8E-octadecenoic acid and 7,10-dihydroxy-8E-octadecenoic acid using Pseudomonas sp. 42A2 NCIMB 40045 in different immobilised supports. Celite R633 gave highest conversions in 48 h: the cellular yield (Yp/x) g products g−1 of cellular protein in the immobilised culture was 5 compared with the free-cell culture Yp/x of 3.8. Conversion of oleic acid in the immobilised cell culture was 50% of the carbon source supplied.

Metabolic differences between attached and free-living marine bacteria: inadequacy of liquid cultures for describing in situ bacterial activity

Marinobacter sp. strain CAB was cultivated with or without porous glass beads as solid support. Two substrates were used: the hydrophilic sodium lactate and a hydrophobic C 18-isoprenoid ketone (6,10,14-trimethylpentadecan-2-one (TMP)). The substrate adsorption onto the beads was measured. Bacterial adhesion was determined by a direct count technique and amounted to 70% of total cells. In the immobilised cell cultures (ICC), generation times were 1.5 and 1.8 times shorter than in the planktonic cultures (FCC) with sodium lactate and with TMP, respectively. In ICC, the growth yields were lower (15.3 FCC×10 9 and 0.8 ICC×10 9 bacteria mg −1 of sodium lactate; 50 FCC×10 9 and 35 ICC×10 9 bacteria mg −1 of TMP). The mineralisation of substrates was estimated after mass spectrometric determination of the CO 2 production rates of both free and immobilised cell cultures. The results indicated a higher specific CO 2 production rate in the ICC with sodium lactate (3.1 FCC±0.2 and 3.5 ICC±0.3 nmol CO 2 mg −1 protein min −1) but not in the ICC with TMP (1.9 FCC±0.7 and 0.5 ICC±0.3 nmol CO 2 mg −1 protein min −1). The affinities for the two substrates were lower in the presence of the solid support ( K m,ICC=18.2±0.2 μM and 37.1±2.0 μM, for sodium lactate and TMP, respectively) than without support ( K m,FCC=8.5±1.5 μM and 8.4±1.2 μM, for sodium lactate and TMP, respectively). Moreover, the presence of a solid support showed a lower inhibition by the TMP ( K i,FCC=3.8±1.0 μM and K i,ICC=12.2±2.5 μM) which may explain why the immobilised cell cultures degraded hydrophobic TMP more efficiently than the planktonic cultures.

Aerobic bacterial metabolism of phytol in seawater: effect of particle association on an abiotic intermediate step and its biogeochemical consequences

The biodegradation of phytol by a bacterial community isolated from the oxic zone of a Recent temperate marine sediment was studied in the presence, and in the absence, of a solid support. Analyses of isoprenoid wax esters produced by condensation of bacterial metabolites with themselves, or with phytol, showed that bacterial metabolism of phytol was strongly modified in the presence of the solid support. Aerobic metabolism of phytol involves the transient production of (E)-phytenal, which in turn can be quickly abiotically converted to 6,10,14-trimethylpentadecan-2-one or (E)-phytenic acid. Sorption on mineral particles appears to hinder the addition of water to the activated double bond of (E)-phytenal, which constitutes the first step of its transformation to the C18 isoprenoid ketone. Consequently, it is concluded that in surface temperate sediments phytol must be metabolised mainly via (E)-phytenic acid and that results obtained from in vitro experiments with free cell cultures may not be comparable to processes occuring in marine sedimentary environments.

Induction of polymethylgalacturonase biosynthesis by immobilized cells of Aspergillus niger 26

The involvement of the regulatory mechanism induction in the biosynthesis of polymethylgalacturonase (PMG) by immobilized cells of Aspergillus niger 26 was studied. The immobilization altered the spectrum of pectin-degrading enzymes. The free cell cultures produced four pectinolytic enzyme activities, namely PMG, polygalacturonase (PG), pectinesterase (PE) and pectinylase (PL), while the entrapped mycelium synthesized PMG and PG only. The best inducer for PMG synthesis was highly esterified apple pectin. The effects of various pectin concentrations and time of incubation with inducer showed the induction model of PMG biosynthesis. The deinduction experiments provided evidence that the presence of the inducer in every cycle was obligatory for the PMG biosynthesis during repeated replacement cultivation.

Chromatographic Processing Scheme for Continuous Harvesting of rec-Prourokinase from Serum Free Medium Cell Cultures

Continuous perfusion cell culture, using albumin containing medium, offers the potential advantages of higher recombinant Prourokinase (r-ProUK) yields, higher initial product purity and increased throughput compared to batch culture technology using medium supplemented with fetal bovine serum. We have characterized the production of r-ProUK in medium supplemented with a lipid rich bovine serum albumin (Albumax) in a perfusion system. The results of these studies showed that it was necessary to modify the r-ProUK batch recovery scheme to process r-ProUK from a perfusion system. To accommodate large volumes of perfusate harvested over a ten to fourteen day production cycle, cation exchange and hydrophobic interaction chromatography (HIC) resins were identified that had increased product binding capacity, better flow characteristics and wider pH ranges which allowed caustic cleaning. The mobile phase composition, pH and ionic strength were modified to improve r-ProUK yields from the identified resins, and procedures were developed to eliminate r-ProUK degradation products. Strategies were defined for processing continuous harvest, which contained four to seven times the amount of r-ProUK of batch harvests.

The characteristics of encapsulated whole cell β-galactosidase

We prepared encapsulated whole cell β-galactosidase using E. coli. The cell culture was divided into two steps for the cell accumulation inside the capsule and enzyme production in the cell. Growth and production media were used individually for this purpose. The dry cell weight of the free cell culture was increased 2.8 times by controlling the pH of the growth medium during cultivation. However, the weight of cells accumulated in the capsule reduced 40% with pH control. The dry cell weight increased with lactose concentration of the production medium for both cases of free and capsule cultures. The dry cell weights were 1.5 g/l for free culture and 100 g/l in the capsule when the lactose concentration of the production medium was 10 g/l. The dry cell weight increased about 60% for both cases as the lactose concentration increased from 10 to 50 g/l. The specific activity of whole cell enzyme decreased with lactose concentration from 5 to 1.4 unit/g dry cell for free culture and from 1.1 to 0.65 unit/g dry cell in the capsule. The value of Michaelis constant, Km, of whole cell enzyme increased 3 times because of the resistance of mass transfer through the capsule membrane. The constants of Michaelis-Menten equation for the whole cell enzyme in the capsule were Vm: 0.0479 mM/min and Km: 44.86 mM. These constants of the membrane-free cells were Vm: 0.0464 mM/min and Km: 15.64 mM. To increase the whole cell enzyme activity, we treated encapsulated cells with organic solvents. The activity of encapsulated whole cell enzyme was increased 3.5 times with the treatment of chloroform and ethanol. The activity of the encapsulated whole cell enzymes was reserved after repeating the process 30 times.