Relationship between the extracellular polymeric substances and surface characteristics of Rhodopseudomonas acidophila

The relationship between the extracellular polymeric substances (EPS) and surface characteristics of Rhodopseudomonas acidophila in its different growth phases was established. The equilibrium constant of partition (K _par) and the Gibbs energies of partition (△G _par) between hexadecane and aqueous phases were also calculated according to the microbial adhesion to hexadecane (MATH) testing. The EPS content decreased with cultivation time at the logarithmic phase, but kept almost unchanged around 22.9 mg g⁻¹ dry cell at the stationary phase. The EPS production of R. acidophila had a significant effect on its surface characteristics. The relative hydrophobicity and the K _par values of R. acidophila before EPS extraction were both lower than those after extraction. Both EPS content and ratio of proteins to carbohydrates had a negative effect on the water contact angle of the bacterium, but had a positive influence on the bacterial surface free energy and its polar component. On the other hand, the EPS were not related with MATH% or the Gibbs energy of partition between hexadecane and aqueous phase.     

Extracellular polysaccharides of a copper-sensitive and a copper-resistant Pseudomonas aeruginosa strain: synthesis,chemical nature and copper binding

Extracellular polysaccharides (EPS) of a copper-sensitive (Cu^s) and a copper-resistant (Cu^r) Pseudomonas aeruginosa strain were investigated in terms of their production, chemical nature and response towards copper exposure. The extent of EPS synthesis by the resistant strain (4.78 mg mg^–1 cell dry wt.) was considerably higher over its sensitive counterpart (2.78 mg mg^–1 dry wt.). FTIR-spectroscopy and gas chromatography revealed that both the polymers were acidic in nature, containing alginate as the major component along with various neutral- and amino-sugars. Acid content in the Cu^r EPS (480.54 mg g^–1) was greater than that in the Cu^s EPS (442.0 mg g^–1). Presence of Cu²⁺ in the growth medium caused a dramatic stimulation (approximately 4-fold) in EPS synthesis by the Cu^r strain, while in a similar condition, the Cu^s failed to exhibit such response. The polymer of the resistant strain showed elevated Cu²⁺ binding (320 mg g^–1 EPS) compared to that of the sensitive type (270 mg g^–1). The overall observations show the potential of the Cu^r EPS for its deployment in metal bioremediation.     

Enhanced extraction of extracellular polymeric substances from biofilms by alternating current

The extraction of extracellular polymeric substances (EPS) including polysaccharides and proteins from aerobic biofilms using either EDTA or NaOH was enhanced by alternating current especially within the first 0.5 h of current application. At 60 mA and 50 Hz, the average extraction rate of polysaccharides within the first 0.5 h using EDTA was 8.3 mg h^–1 g TS^–1 (TS: total solid, dry wt), 1.6 times of that without current, and that of proteins was 2.5 mg h^–1 g TS^–1, 1.2 times higher. The extraction of polysaccharides was maximal at around 500 Hz while that of proteins was less affected by increasing the current frequency.     

Extraction of squalene from yeast by supercritical carbon dioxide

Squalene produced under anaerobic conditions, by a strain of Torulaspora delbrueckii was extracted from the biomass using supercritical carbon dioxide. Minimum use of solvent, lower time of isolation and a higher selectivity of extraction merit use of supercritical fluid extraction (SFE) technique over solvent extraction of squalene, as optimized and reported previously. A maximum squalene yield of 11.12 g g^–1 (dry weight) of yeast cells was obtained at a temperature of 60 °C and pressure of 250–255 bar at a constant flow rate of 0.2l min^–1 of carbon dioxide. Lyophilization prior to SFE increased the squalene yield to 430.52 g g^–1 dry weight of yeast cells, an amount that is far greater than that obtained by (2:1) chloroform–methanol solvent extraction.     

Composition and Distribution of Extracellular Polymeric Substances in Aerobic Flocs and Granular Sludge

Extracellular polymeric substances (EPS) were quantified in flocculent and aerobic granular sludge developed in two sequencing batch reactors with the same shear force but different settling times. Several EPS extraction methods were compared to investigate how different methods affect EPS chemical characterization, and fluorescent stains were used to visualize EPS in intact samples and 20-μm cryosections. Reactor 1 (operated with a 10-min settle) enriched predominantly flocculent sludge with a sludge volume index (SVI) of 120 ± 12 ml g⁻¹, and reactor 2 (2-min settle time) formed compact aerobic granules with an SVI of 50 ± 2 ml g⁻¹. EPS extraction by using a cation-exchange resin showed that proteins were more dominant than polysaccharides in all samples, and the protein content was 50% more in granular EPS than flocculent EPS. NaOH and heat extraction produced a higher protein and polysaccharide content from cell lysis. In situ EPS staining of granules showed that cells and polysaccharides were localized to the outer edge of granules, whereas the center was comprised mostly of proteins. These observations confirm the chemical extraction data and indicate that granule formation and stability are dependent on a noncellular, protein core. The comparison of EPS methods explains how significant cell lysis and contamination by dead biomass leads to different and opposing conclusions.     

Anions effects on biosorption of Mn(II) by extracellular polymeric substance (EPS) from Rhizobium etli

Microbial extracellular polymeric substances (EPS) are potential biosorbents for metal remediation and recovery. The Langmuir and Freundlich kinetics of Mn(II) binding by the EPS from a novel Mn(II) oxidising strain of Rhizobium etli were determined. Maximum manganese specific adsorptions (q _max) decreased in the sequence: sulphate (62 mg Mn per g EPS) > nitrate (53 mg g^–1) > chloride (21 mg g^–1). Consideration of the anion during kinetic studies is usually neglected but is important in providing more practical and comparable data between different biosorbent systems.     

Improvement of phenylethanoid glycosides production by a fungal elicitor in cell suspension culture of Cistanche deserticola

When, on the 15th day of growth, an elicitor from Fusarium solani was added at 40 mg l^–1 to Cistanche deserticola cell suspension cultures, the contents of echinacoside, acteoside and total phenylethanoid glycosides (PeGs) in cultured cells all increased over the next 27 d by over 100% to 15 mg g^–1 dry wt, 9 mg g^–1 dry wt and 57 mg g^–1 dry wt, respectively. The final biomass (1.3 mg dry wt ml^–1) was not affected.     

Determination of cell lysis and death kinetics in continuous hybridoma cultures from the measurement of lactate dehydrogenase release

The death of the hybridoma VO 208 in a continuous culture at pH 7 and 6.8 was investigated by measuring both the appearance of visible dead cells which do not exclude the trypan blue dye and the release of lactate dehydrogenase (LDH) in the culture medium. The intracellular LDH was found to be completely released either when live cells lysed or when they were transformed into visible dead cells. No significant lysis of blue dead cells could be observed at the two different pH. Using a LDH balance over the culture system, cell lysis was found negligible at pH 7, but accounted for 20% of the total cell death at pH 6.8. A methodology is proposed to evaluate the rate constants of hybridoma lysis and total death. For the investigated cell line in continuous culture, the calculated total cell death rate constant was found to increase from 0.002 h^–1 to 0.01 h^–1 when decreasing the pH from 7 to 6.8.Abbreviations D dilution rate (h^–1) - k_b specific trypan-blue dead cells appearance rate (h^–1) - k_L specific lysis rate of viable cells (h^–1) - k_d specific death rate (h-1) - LDH₀ lactate dehydrogenase activity in the feed culture medium (IU.l^–1) - LDH lactate dehydrogenase activity in the outlet culture medium (IU.l^–1) - LDH_i intracellular lactate dehydrogenase activity of viable cells (IU.10^–9 cells) - r_LDH total rate of LDH release (IU.h^–1.L^–1) - r_b transformation rate of viable cells into blue dead cells (10⁹ cells.h^–1.L^–1) - x_v viable cell concentration (10⁹ cells.l^–1) - x_b trypan-blue dead cell concentration (10⁹ cells.l^–1)     

Characterization of an exopolysaccharide mutant of Nostoc spongiaeforme: Zn2+-sorption and uptake

Exposure of the exopolysaccharide (EPS)-synthesizing cyanobacterium Nostoc spongiaeforme to Zn²⁺ (20 M) transformed the biomass into white debris. However, a few blue–green pin-heads emerged after 2 weeks in the same Zn²⁺-containing medium and formed less mucoid microcolonies (1–2 mm) relative to the protruding colonies (2–4 mm) of the parent strain on nutrient agar. One of such survivors (designated as Zn₂₀) that was stable through 10 successive transfers in Zn²⁺-lacking medium has been adopted for further characterization. The parent strain retained almost 88% of the total EPS synthesized, the rest being released into the ambient medium, while for Zn₂₀, the EPS retained approximated to 74%. Although the Zn²⁺-sensitivity of the mutant was comparable with that of the parent (LD₅₀, 7 M), Zn²⁺ uptake was still 5-fold higher in the former (2 g mg^–1 biomass dry wt., 20 M, external concentration). Also, both the strains showed insignificant difference in Zn²⁺-sorption onto their isolated EPS. The mutant was characterized by having higher cell carbohydrate content (642.8 g mg^–1 dry wt.) than its parent (513.6 g). The X-ray diffraction pattern revealed Zn²⁺ deposition on EPS from the parent mainly as zinc hypophosphite monohydrate [Zn(H₂PO₂)₂·H₂O], whereas there was a lack of distinct peaks in similar samples from Zn₂₀, thus confirming the amorphous nature. There was participation in Zn²⁺ binding of only COO^–, N=O, NO₂, SO₂ groups in the parent while participation of P—O and C=O groups in mutant EPS was evident in IR spectra. The observations suggest that the mutant could be deployed to achieve sustained EPS synthesis, its release and metal sorption/desorption in repeated cycles.     

Induction of astaxanthin formation by reactive oxygen species in mixotrophic culture of Chlorococcum sp.

The induction of astaxanthin formation by reactive oxygen species in mixotrophic culture of Chlorococcum sp. was investigated. H₂O₂ (0.1 mM) enhanced the total astaxanthin formation from 5.8 to 6.5 mg g^–1 cell dry wt. Fe²⁺ (0.5 mM) added to the medium with H₂O₂ (0.1 mM) further promoted astaxanthin formation to 7.1 mg g^–1 cell dry wt. Similarly, Fe²⁺ (0.5 mM) together with methyl viologen (0.01 mM) promoted astaxanthin formation to 6.3 mg g^–1 cell dry wt. In contrast, an addition of KI (1 mM), a specific scavenger for hydroxyl radicals (OH), together with H₂O₂ (0.1 mM) and Fe²⁺ (0.5 mM), to the medium decreased astaxanthin formation to 1.8 mg g^–1 cell dry wt. KI (1 mM) also inhibited the enhancement of carotenogenesis by superoxide anion radicals (O₂ ^–), with a decrease of astaxanthin formation to 1.7 mg g^–1 cell dry wt. This suggested that O₂ ^– might be transformed to OH before promoting carotenogenesis in Chlorococcum sp.     

Optimization of a Propionibacterium acidipropionici continuous culture utilizing sucrose

To produce propionic acid and vitamin B₁₂ from sucrose, the strain Propionibacterium acidipropionici NRRL B3569 was selected by screening a number of Propionibacterium strains. The nutrient composition and the fermentation conditions for this strain were optimized in continuous culture. The investigations show that within a concentration range of 30–170 g l^–1 of sucrose in the fermentation medium, no significant substrate inhibition occurred. For the production of propionic acid and vitamin B₁₂, concentrations of 1.5 mg FeSO₄·7H₂O g^–1 dry biomass, 0.75 mg cobalt ions g^–1 dry biomass, 0.3 mg 5,6-dimethylbenzimidazole g^–1 dry biomass, and 12 g yeast extract 1^–1 were necessary additions to the sources of nitrogen, phosphate, and magnesium ions. The extra addition of up to 2.8 g betaine g^–1 dry biomass significantly increases the production of vitamin B₁₂. In the optimization of the pH value, temperature, and aeration, it was established that the conditions for propionic acid production and vitamin B₁₂ production are different. Whereas the optimal production of propionic acid took place under completely anaerobic conditions with a pH value of 6.5 and a temperature of 37°C, optimal vitamin B₁₂ production required a temperature of 40°C and aerobic conditions (0.5 vvm aeration at 100 rpm) with a pH value of 6.5.     

Production of extracellular polymeric substances from Rhodopseudomonas acidophila in the presence of toxic substances

A hydrogen-producing photosynthetic bacteria strain, Rhodopseudomonas acidophila, was used to investigate the production of extracellular polymeric substances (EPS) in the presence of toxic substances and the effect of toxicants on bacterial surface characteristics. Addition of the toxic substances including Cu(II), Cr(VI), Cd(II) and 2,4-dichlorophenol (2,4-DCP) stimulated the production of EPS but reduced the cell dry weight. At concentrations of 30 mg l⁻¹ Cu(II), 40 mg l⁻¹ Cr(VI), 5 mg l⁻¹ Cd(II) and 100 mg l⁻¹ 2,4-DCP, the EPS content increased by 5.5, 2.5, 4.0 and 1.4 times, respectively, than the control. These toxic substances also greatly influenced the proteins/carbohydrates ratio of EPS. The ratios in the presence of toxic substances were always higher than that of control. Furthermore, under toxic conditions, the increase in the protein content far exceeded than that of others in EPS, suggesting that extracellular proteins could protect cells against toxic substances. The toxic substances significantly changed the surface characteristics and flocculation ability of R. acidophila, such as surface energy, relative hydrophobicity and free energy of adhesion.     

In vitro culture of Montanoa tomentosa for the production of diterpenic acids

Following a solid phase extraction, GC-MS and GC-FID procedures, the production of three kaurane derivatives (grandiflorenic, kaurenoic and monoginoic acids) was detected in callus and cell suspension batch cultures of Montanoa tomentosa. From different hormonal combinations, the addition of 0.5 mg 2,4-dichlorophenoxyacetic acid l^–1 + 2 mg kinetin l^–1 increased the accumulation of total kaurenoids in 6 months old calluses to 2.1 mg g^–1 dry weight and in cell suspensions cultures up to 0.76 mg g^–1 dry weight. Monoginoic acid, which has not been detected before in leaves of wild plants, accumulated in both in vitro systems.     

Nitrogen and phosphorus uptake by the Brazilian kelp Laminaria abyssalis (Phaeophyta) in culture

The uptake of ammonium, nitrate and phosphate by laboratory-grown young sporophytes of Laminaria abyssalis was measured in a perturbed system (batch mode) at 18 °C and 35 ± 5 µE m^–2 s^–1 photon flux density. Uptake of all appeared to follow saturation-type nutrient uptake kinetics. The NO _inf3 ^sup– (K _s = 14.0 µM, V _max = 5.0 µmol h^–1 g^–1 dry wt) and NH _inf4 ^sup+ (K _s = 4.6 µM, V _max= 2.0 µmol h^–1 g^–1 dry wt) were taken up simultaneously, although NH _inf4 ^sup+ was taken up more rapidly. Values of K ₃ and V _max for phosphate were, respectively, 2.21 µM and 0.83 µmol h^–1 g^–1 dry wt. Nitrate and phosphate were both consumed in similar rates (V _max /Ks 0.37) at low concentrations. NH _inf4 ^sup+ , thus, might be a more efficient form of N fertilizer if artificial enrichment of seawater is used.     

A Study on the Synthetic Characteristics of the Extracellular Polysaccharide (EPS) of Ganoderma lucidum Cultured in Batch Fermentation Using a Kinetic Model

The synthetic characteristics of the extracellular polysaccharide (EPS) of Ganoderma lucidum in batch fermentation were studied. The result showed that the production of EPS was partially growth-associated. The cell dry weight (CDW) and EPS reached 15.56 g·L⁻¹ and 3.02 g·L⁻¹, respectively. The yield of EPS to cell dry weight (Y_p/x) was 0.19. On the basis of the test results of batch fermentation, a kinetic model was proposed by using the Logistic equation for cell growth, the Luedeking–Piret equation for EPS production, and the Luedeking-piret-like equation for the consumption of glucose as substrate. The calculated results using these models were satisfactorily compared with the experimental data under various concentrations of glucose, and the average of relative errors was found to be not more than 5%. The kinetic model had practical guidance interesting in producing PES by Ganoderma lucidum.     

Astragaloside IV and polysaccharide production by hairy roots of Astragalus membranaceus in bioreactors

Hairy roots of Astragalus membranaceus were grown in bioreactors up to 30 l for 20 d. Cultures from a 30 l airlift bioreactor gave 11.5 g l dry wt with 1.4 mg g^–1 astragaloside IV, similar to cultures from 250 ml and 1 l flasks, but greater than yields from a 10 l bioreactor (dry wt 9.4 g l^–1, astragaloside IV 0.9 mg g^–1). Polysaccharide yields were similar amongst the different bioreactors (range 25–32 mg g^–1). The active constituent content of the cells approached that of plant extracts, indicating that large scale hairy root cultures of A. membranaceus has the potential to provide an alternative to plant crops without compromising yield or pharmacological potential.     

Influence of physiological conditions on EDTA degradation

Aerobic biodegradation of the chelating agent EDTA by a mixed bacterial culture was investigated. Bacterial growth and degradation of the substrate required the presence of sufficient metal ions in the culture fluid. Uncomplexed EDTA interacted negatively with the cell walls of the bacteria and completely inhibited bacterial growth, whereas Mg(II)/Ca(II)-EDTA was degraded up to an initial concentration of 4.7 g/l. Therefore, concentrations of metal ions must be stoichiometric to that of EDTA or higher. Specific degradation rates ranged between 120 mg EDTA g^–1 (cell dry weight) h^–1 and 285 mg EDTA g^–1 h^–1. In contrast, complexes with high thermodynamic stability constants such as Fe(III)-EDTA remained as inert compounds in the solution. Specific growth rates of the mixed culture were found to vary between 0.03 h^–1 and 0.07 h^–1, which could be explained by population dynamics within the synergistic mixed community. Growth was significantly accelerated by the addition of vitamins.     

Effects of autoclave-induced carbohydrate hydrolysis on the growth ofBeta vulgaris cells in suspension

Media of de Greef & Jacobs (1979) were autoclaved either with all the nutrient components in a single vessel (medium 1) or with the following components in separate vessels: FeNa–EDTA+CaCl₂ (medium 2), FeNa–EDTA+NaH₂PO₄ (medium 3) or sucrose (medium 4). Medium 5 was prepared by autoclaving FeNa–EDTA+NaH₂PO₄ and sucrose in two separate vessels. It was found that the dry mass yield of cell suspensions ofBeta vulgaris was lowest in medium 1, followed by media 2 and 3. There was no significant difference among media 3, 4, and 5.The plot of dry mass yield of the cell suspensions against the rates of cyanide-initiated oxygen consumption which indicate the extent of carbohydrate hydrolysis of the media during autoclaving, indicated the presence of a threshold rate of about 17–20 nmol ml^–1 min^–1. Dry mass yield of the suspensions decreased rapidly when the rate exceeded this value.For media with glucose as the source of carbohydrate, the rate of cyanide-initiated oxygen consumption exceeded the threshold value by a factor of 1.5 to 2, depending on the volume of the media autoclaved.Abbreviations FeNa-EDTA ferric monosodium ethylenediamine-tetraacetic acid     

Optimized isolation procedure for obtaining strongly actinide binding exopolymeric substances (EPS) from two bacteria (Sagittula stellata and Pseudomonas fluorescens Biovar II)

Different chemical extractants (NaCl, EDTA, HCl and NaOH) and physical methods (ultrasonication and heating) were examined by their efficacies of extracting “attached” exopolymeric substances (EPS) secreted by marine bacterium Sagittula stellata (SS) and terrestrial bacterium Pseudomonas fluorescens Biovar II (PF). Extraction by 0.5 N HCl for 3 h was best for SS while extraction by 0.05 N NaCl for 3–5 h was regarded as optimal for PF. Improvements in EPS purification included a pre-diafiltration step to remove the broth material and reduce the solution volume, thus the usage of ethanol, and time. The EPS harvested at the optimal time and purified by the improved method were enriched in polysaccharides, with smaller amounts of proteins, thus having amphiphilic properties. Isoelectric focusing of ²³⁴Th or ²⁴⁰Pu labeled EPS showed both actinides were strongly bound to macromolecules with low pI, similar to reported marine or soil colloidal natural organic matter (NOM).     

Production of ajmalicine and ajmaline in hairy root cultures of Rauvolfia micrantha Hook f., a rare and endemic medicinal plant

Hairy roots of Rauvolfia micrantha were induced from hypocotyl explants of 2–3 weeks old aseptic seedlings using Agrobacterium rhizogenes ATCC 15834. Hairy roots grown in half-strength Murashige & Skoog (MS) medium with 0.2 mg indole 3-butyric acid l^–1 and 0.1 mg -naphthaleneacetic acid l^–1 produced more ajmaline (0.01 mg g^–1 dry wt) and ajmalicine (0.006 mg g^–1 dry wt) than roots grown in auxin-free medium. Ajmaline (0.003 mg g^–1 dry wt) and ajmalicine (0.0007 mg g^–1 dry wt) were also produced in normal root cultures. This is the first report of production of ajmaline and ajmalicine in hairy root cultures of Rauvolfia micrantha.