Immobilization of cyanobacteria and microalgae on polyethylenimine-based sorbents

Immobilization of phototrophic microogranisms: microalgae (MA) and cyanobacteria (CB) on polyethylenimine (PEI)-based sorbents was studied. For this purpose, 3 insoluble porous polymeric materials were synthesized by cross-linking of PEI with epichlorohydrine and immobilization of PEI on the surface of styrene–divinylbenzene copolymer. The sorbent on the basis of cross-linked PEI was also alkylated with hexadecyl bromide to achieve hydrophobicity of its surface. The analysis of kinetics and efficiency of immobilization assessed for the model MA and CB cultures revealed the significant difference in the sorption activity of different types of sorbents depending on their synthesis procedure, chemical composition and hydrophilic-hydrophobic properties of polymeric surface. The hydrophobic sorbent obtained by immobilization of PEI on the surface of styrene–divinylbenzene copolymer characterized by very low sorption activity towards CB and MA cells. The highest immobilization efficiency of phototrophic cells was achieved for the hydrophilic sorbent on the basis of PEI cross-linked with epichlorohydrine, which provided the attachment of 50–70% of cells during 3 h of incubation. The hydrophobic sorbent based on alkylated cross-linked PEI effectively immobilized CB cells, while the colonization of the polymer surface by MA cells was very scarce. The noticed effect is explained by difference in prokaryotic (CB) and eukaryotic (MA) types of surface structures organization. Assessment of photosynthetic activity of immobilized MA cells by pulse-modulated fluorometry showed that hydrophobic sorbents had no toxic effect on the cells, while toxicity of hydrophilic cross-linked PEI-based sorbent was observed only after long-term cultivation ofphototrophic cells with this sorbent.     

The factor stabilizing the bioluminescence of PVA-immobilized photobacteria

Immobilization of photobacteria in the cryogel of polyvinyl alcohol (PVA) was carried out. Immobilization was found to result in increased intensity and stability of bioluminescence. The elements determining the stability of bioluminescence were investigated. Selection of the strain was found to be of the highest importance. Among immobilized cells, Photobacterium phosphoreum exhibited the most intense and prolonged light emission, while Vibrio harveyi showed the least one. The technological procedures for cryogenic immobilization of photobacteria were determined. The role of the environment of gel formation in the preservation of the bioluminescence activity was determined. In the gels formed in rich medium for submerged cultivation of photobacteria, almost 100% luminescence activity was preserved, while light emission was considerably prolonged. Bioluminescence intensity of the preparations was shown to depend significantly on pH of the incubation medium. The pH shift to acidic values during prolonged incubation of immobilized cells was shown to be one of the factors of bioluminescence quenching. The stress effects of cryogenic immobilization were found to have an insignificant effect on the temperature profile of bioluminescence. Decreased reduction rate of the luciferase flavin substrate was shown to be a possible reason for bioluminescence quenching.     

Inhibitory effects of phenolic ecotoxicants on photobacteria at various pH values

Kinetic characteristics of light emission by intact cells of the photobacteria Photobacterium phosphoreum and Vibrio harveyi at pH 5.5, 7.0, and 8.0 were studied as well as specific features of inhibitory effects of 2,4-di- and 2,4,5-triphenoxyacetic acids (2,4-D and 2,4,5-T), pentachlorophenol (PCP), and 2,6-dimethylphenol (2,6-DMP) at the same pH values. Nonstationarity of emission kinetics was observed at all the pH values studied. Exponential luminescence decay in a 60-sec range was observed at pH 5.5; a 5-min luminescence activation, at pH 7.0 and 8.0. The cell respiratory activity drops by over one order of magnitude at pH 5.5 compared with the activities at pH 7.0 and 8.0. The inhibitory effects of 2,4-D, 2,4,5-T, and PCP differ by one-two orders of magnitude depending on pH. The maximal cell sensitivity to these compounds appears at pH 5.5; the minimal, at pH 8.0. The effect of 2,6-DMP is independent of pH. As is demonstrated, it is hydrophobicity of the molecule and pK values of the toxicants that determine the inhibitory effect. Characteristic of the substrate-starved photobacterial cells are higher sensitivity to chlorophenolic compounds compared with the cells provided with high energy supply at all the pH values.     

Effect of anaesthetics on calcium-induced luminescence of aequorin

The effect of some general and local anaesthetics on the calcium-induced luminescence of aequorin was studied in vitro using a photomultiplier tube and recording technique. Purified aequorin (0.1 microliter) was injected into a 500 micron diameter porous cellulose acetate capillary tube containing 0.5 M KC1, 20 mM phosphate (pH 7.2) and calcium-EGTA buffers. The trapped aequorin was superfused with buffer solutions which sometimes contained anaesthetic (test) solutions. The results showed that some anaesthetics, e.g. urethane, etomidate and lignocaine, increased whereas others, e.g. methohexitone, thiopentone, decreased the light output (luminescence) of aequorin in constant ionized calcium and EGTA buffers. Similar results were produced by some non-anaesthetic drugs, e.g. glycerol, TEA, caffeine, etc. Concentration-response curves for calcium-dependent and -independent luminescence of aequorin showed that anaesthetics variously affected the aequorin response. Some anaesthetics, e.g. lignocaine, increased the maximum response while others, e.g. etomidate, increased the affinity (i.e. decreased EC50s) of aequorin to calcium ions without altering the slope, which remained at about 2. It was concluded that anaesthetics can either excite or depress aequorin luminescence, the effect being dependent on the type and the concentration used.     

Effect of propagators and inhibitors on the ultraweak luminescence from maize roots

This study has investigated the kinetics and mechanism of ultraweak luminescence in maize roots. Mannitol induced the second maximum and enhanced the main maximum of the relative intensity of luminescence from the roots. Hydroquinone and quinone enhanced the relative intensity of the luminescence. Catalase enhanced the maximum of the luminescence and changed the kinetics of the light emission. The effect of catalase on the kinetics was abolished by superoxide dismutase. Ascorbate in the presence of catalase reduced the luminescence maximum, but did not alter the kinetics. In the presence of catalase only, or in the combination with superoxide dismutase, or ascorbate, the luminescence intensity in the stationary phase was significantly lower compared to the control. The results support the participation of superoxide-radical, singlet oxygen, electron transfer and the role of peroxidase in the reactions generating ultraweak luminescence in the roots. Ascorbate, catalase and superoxide dismutase have a protective role in the luminescent reactions.     

Effect of cAMP on the growth and development of luminescence in Photobacterium belozerskii

The effect of cAMP on the parameters characterizing the development of luminescence in Photobacterium belozerskii is discussed. An addition of cAMP to the culture medium shortened the latent time of luminescence development by 3--6 hours. The intensity of bacterial luminescence increased with a varying rate. During luminescence enhancement the rate of luciferase synthesis increased by a factor of 10 to 10(3). The rate of luciferase synthesis and the maxiumum level of bacterial luminescence when cultivated in the glycerol medium containing arginine and proline increased under the influence of cAMP by 100 and 40 times, respectively. After an addition to cAMP into the glucose medium these parameters of luminescence development increased only when the medium contained arginine. After an addition of cAMP into the glycerol medium the rate of bacterial growth increased two-fold. Possible mechanisms regulating luminescence development and cAMP involvement in these processes are discussed.     

DNA immobilization on modified inorganic sorbents

Fragments of denaturated DNA having the length of about 500 nucleotides were immobilized, using silica carriers modified by organic polymers. It was shown that the organic coating allows to obtain sorbents, which practically exclude non-specific sorption with respect to DNA. The immobilization was carried out on hydroxyl-containing sorbents and on their phrosphorylated derivatives by means of water-soluble carbodiimide. The resulting preparations contained up to 60 units at A260 of DNA per 1 g of carrier. The effects of endonuclease A236, exonuclease A5 and pancreatic DNAse on the immobilized RNA were studied.     

Effect of calcium chelators on the Ca2+-dependent luminescence of aequorin.

The luminescence of aequorin, a useful tool for studying intracellular Ca2+, was recently found to be inhibited by the free EDTA and EGTA that are present in calcium buffers. In the present study we have examined the effect of the free forms of various chelators in the calibration of [Ca2+] with aequorin. Free EDTA and EGTA in low-ionic-strength solutions strongly inhibited the Ca2+-triggered luminescence of aequorin, causing large errors in the calibration of [Ca2+] (approx. 2 pCa units), whereas in solutions containing 150mM-KCl, errors were relatively small (0.2-0.3 pCa units). Citric acid in low-ionic-strength solutions and [(carbamoylmethyl)imino]diacetic acid in high-ionic-strength solutions showed no inhibition and did not cause detectable error in the calibration of [Ca2+], indicating that they are better chelators than EDTA and EGTA for use with aequorin.     

Effect of antibacterial factors and cell metabolism biostimulants on bacterial luminescence

Bioluminescence was used as an index of effects of a series of factors (differing in origin and structure) on cell metabolism. Luminescence of intact cells of Escherichia coli lum+ (a genetically modified strain) was measured on exposure to antibiotics, probiotics, phages, and biostimulators. The sensitivity of E. coli lum+ to antibiotics correlated with the antibiotic-induced luminescence quenching. Bioluminescence-based assessment of the antagonistic activity of the main probiotics (bificoll, bifidobacterin, acylact, colibacterin, and a composite probiotic preparation), each taken at a concentration of 1 dose/ml demonstrated that bacterial luminescence was inhibited by 75-99.9% (exposure time, 30 min). Bioluminescence changes reflected cell damage associated with phage infection. It was shown that bioluminescence stimulation could be used as an index of the effect of immunomodulators (olexin and vermin) on cell metabolism in bacterial cultures.     

Binding of Kunitz-Northrop inhibitor on some sorbents

This work is devoted to the problem of sorption and desorption of Kunitz--Northrop inhibitor on different sorbents. By passing through Dowex 1.10 column 0.05 M glycine-NaOH buffer, pH 10, two fractions with 100% inhibitor activity were identified, while different admixtures and inert proteins remained resin-bound. In trypsin-Spheron 300, trypsin-agarose and anhydrochymotrypsin-Spheron columns the contamination of sorbents-bound inhibitor complex was eliminated by washing out with 0.1 M NaCl, pH 8.0. The resin-bound inhibitor was released at pH 1. The specific activity of the preparation obtained was shown to increase in 200-240-fold, but in the case of anhydrochymotrypsin-Spheron 300 the inhibitors activity was detected both at pH 8.0 and pH 1.7. In this case the increase in specific activity was only 2 and 68-fold, respectively. The most effective inhibitor-peptide desalting was defined at application of dialysis membranes "Spectra/Por" (MWCo: 3000-5000 USA). While applying PAAG electrophoresis the standard SDS-system were shown to be ineffective. Therefore some modifications of this method were used. Being compared with molecular weight of Contrycal and other known peptides this preparation revealed the presence of protein contamination. Within 1-9 mg the peptides demonstrated a linear dependence in trypsin inhibition. The weight and molar ration of inhibitor: trypsin was found to be 1:1 and 3:1, respectively. It was calculated that IUE of the inhibitor had inhibited 0.73 mg of trypsin, IUE of Contrycal--0.15 of the enzyme, that was 4.6 fold less effective than the separated peptide.     

Effect of salts on luminescence of natural and recombinant luminescent bacterial biosensors

Effect of cations K+, Na+, Mg2+, and Ca2+ and anions SO4(2-), HCO3(-), and CO3(2-) on the luminescence intensity of the marine luminescent bacterium Photobacterium phorphoreum (Microbiosensor B-17 677f) and the recombinant strain Escherichia coli with cloned lux operon of P. leiognathi (Ekolyum-9). It is found that small concentrations of chlorides and sulfates of the cations studied had a concentration-dependent stimulatory effect on bacterial bioluminescence; as the concentration of agents increased, activation was succeeded by quenching. The strength of the inhibitory effect, which is characterized by EC50, decreased in the series Ca2+ > Na+ > Mg2+ > K+. Carbonates and hydrocarbonates had a pronounced inhibitory effect on the bioluminescence intensity, determined by an increase in pH. We showed that some types of highly mineralized water with a high hydrocarbonate content have a marked inhibitory effect on the luminescence intensity of microbial luminescent biosensors, mimicking the effect of chemical pollutants.     

Effect of salts on luminescence of natural and recombinant luminescent bacterial biosensors

Effect of cations K⁺, Na⁺, Mg²⁺, and Ca²⁺ and anions Cl^?, SO ₄ ^2? , HCO ₃ ^? , and CO ₃ ^2? on the luminescence intensity of the marine luminescent bacterium Photobacterium phorphoreum (Microbiosensor B-17 677f) and the recombinant strain Escherichia coli with cloned lux operon of P. leiognathi (Ecolum-9). It is found that small concentrations of chlorides and sulfates of the cations studied had a concentration-dependent stimulatory effect on bacterial bioluminescence; as the concentration of agents increased, activation was succeeded by quenching. The strength of the inhibitory effect, which is characterized by EC₅₀, decreased in the series Ca²⁺ > Na⁺ > Mg²⁺ > K⁺. Carbonates and hydrocarbonates had a pronounced inhibitory effect on the bioluminescence intensity, determined by an increase in pH. We showed that some types of highly mineralized water with a high hydrocarbonate content have a marked inhibitory effect on the luminescence intensity of microbial luminescent biosensors, mimicking the effect of chemical pollutants.     

Lumazine proteins from photobacteria: localization of the single ligand binding site to the N-terminal domain

Lumazine protein is believed to serve as an optical transponder in bioluminescence emission by certain marine bacteria. Sequence arguments suggest that the protein comprises two similarly folded riboflavin synthase-type domains, but earlier work also suggested that only one domain binds 6,7-dimethyl-8-ribityllumazine (DMRL). We show that the replacement of serine-48 or threonine-50 in the N-terminal domain of lumazine protein of Photobacterium leiognathi modulates the absorbance and fluorescence properties of bound DMRL or riboflavin. Moreover, the replacement of these amino acids is accompanied by reduced ligand affinity. Replacement of serine-48 by tryptophan shifts the (13)C NMR signal of the 6-methyl group in bound DMRL upfield by 2.9 ppm as compared to the wild-type protein complex. Replacement of threonine-50 causes a downfield shift of approximately 20 ppm for the (15)N NMR signal of N-5, as well as an upfield shift of 3 ppm for the (13)C NMR signal of C-7 in bound DMRL, respectively. The replacement of the topologically equivalent serine-144 and proline-146 in the C-terminal domain had no significant impact on optical properties, chemical shifts and apparent binding constants of bound DMRL. These data show that the N-terminal domain is the unique site for ligand binding in lumazine protein.     

Effect of organic and inorganic toxic compounds on luminescence of luminous fungi

The possibility of the development of the solid phase bioluminescent biotest using aerial mycelium of luminous fungi was investigated. Effect of organic and inorganic toxic compounds (TC) at concentrations from 10⁻⁶ to 1 mg/ml on luminescence of aerial mycelia of four species of luminous fungi—Armillaria borealis (Culture Collection of the Institute of Forest, Siberian Branch, Russian Academy of Sciences), A. mellea, A. gallica, and Lampteromyces japonicus (Fungi Collection of the Botanical Institute, Russian Academy of Sciences)—has been studied. Culture of A. mellea was shown to be most sensitive to solutions of the model TC. It was demonstrated that the sensitivity of the luminous fungi is comparable with the sensitivity of the bacteria that are used for environmental monitoring. Use of the aerial mycelium of luminous fungi on the solid support as a test object is a promising approach in biotesting for the development of continuous biosensors for air monitoring.     

The adhesion of pathogenic microflora on carbon sorbents

The adsorbing activity of granulated carbonic sorbents SKN and KAU, as well as their oxidated forms, containing protogenic carboxylic and phenolic groups with respect to Shigella flexneri, Salmonella typhimurium, Escherichia coli, Streptococcus aureus and Pseudomonas aeruginosa pathogenic strains has been studied. As shown in this study, the process of interaction between microorganisms and carbonic sorbents has two stages. At the first stage the main role is played by long-distance electrostatic forces and at the second stage, by Van der Waals short-distance forces, as well as bonds formed between cell structures and surface groupings of carbonaceous materials. In the mechanism of interaction between microbial cells and carbons the geometry of carbon surface plays an important role. KAU(0)-1 exhibits the highest degree of adhesion with respect to pathogenic bacteria.