Characterization of sulfate-reducing bacteria in yeast industry waste by microcalorimetry and PCR amplification

Sulfate-reducing bacteria (SRB) were isolated from anaerobic sludge of yeast factory wastewater treatment plant by cultivation on Postgate C medium. Microcalorimetry was used to monitor the anaerobic digestion processes and to measure the growth rates of sulfate-reducing bacteria. The maximum growth rates determined by microcalorimetry and ATP analysis were different—μ_max(dQ/dt) = 0.165 ± 0.008 h⁻¹ and μ_max(N_ATP) = 0.207 ± 0.013 h⁻¹. Experiments on the cultivation of SRB from yeast industry wastewater treatment plant in batch culture showed that during the first 20 h the concentration of sulfate decreased from 78.3 mM down to 62.2 mM while the increase of sulfide production was negligible. Perceptible amount of sulfide (7.82 mM) appeared on the 33.5 h of fermentation together with a peak on the power–time curve and considerable increase in the cell count (1.26 × 10⁹). First steps of sulfate metabolism (activation of sulfate by ATP sulfurylase, production of H₂) are accompanied by endothermic heat effects, therefore the values of thermal power remain moderate until the evolution of sulfide starts. The influence of green microalgae Chlorococcum sp. (preparation Biotreat 100) on the growth characteristics of microorganisms was also studied. Identification of one SRB strain was started by sequencing of PCR-amplified 16S rRNA gene. Two sets of primers were used for PCR amplification, both specific for domain Bacteria but giving different gene fragments. PCR-products were purified with JETQUICK kit according to the manufacturer instructions.     

Deposition of Oral Bacteria and Polystyrene Particles to Quartz and Dental Enamel in a Parallel Plate and Stagnation Point Flow Chamber

The aim of this paper is to determine to what extent (i) deposition of oral bacteria and polystyrene particles, (ii) onto quartz and dental enamel with and without a salivary conditioning film, (iii) in a parallel plate (PP) and stagnation point (SP) flow chamber and at common Peclet numbers are comparable. All three bacterial strains showed different adhesion behaviors, and even Streptococcus mitis BMS, possessing a similar cell surface hydrophobicity as polystyrene particles, did not mimic polystyrene particles in its adhesion behavior, possibly as a result of the more negative ζ potentials of the polystyrene particles. The stationary endpoint adhesion of all strains, including polystyrene particles, was lower in the presence of a salivary conditioning film, while also desorption probabilities under flow were higher in the presence of a conditioning film than in its absence. Deposition onto quartz and enamel surfaces was different, but without a consistent trend valid for all strains and polystyrene particles. It is concluded that differences in experimental results exist, and the process of bacterial deposition to enamel surfaces cannot be modeled by using polystyrene particles and quartz collector surfaces.     

Engineering cytochrome P-450s

Cytochrome P-450 isozymes represent a critical component of nature’s spectrum of detoxification catalysts that could be exploited for bioremediation. The ethanol-inducible human cytochrome P-450 2E1 serves as a model eukaryotic P-450 that complements the bacterial P-450 cam in dehalogenation and detoxification of environmental pollutants. We explored the construction of novel chimeric P-450s using cytochrome P-450 camC and 2E1 genes. For construction of chimera 1 (478 amino acids, 55.14 kDa), 145 amino acids from the N-terminus of P-450 2E1 protein (493 amino acids, 56.84 kDa) were replaced with 130 amino acids from the N-terminus of P-450 camC protein (415 amino acids, 46.66 kDa). In chimera 2 (525 amino acids, 60.24 kDa) the strategy involves replacement of 28 amino acids in the C-terminus of chimera 1 with 75 amino acids from the C-terminus of P-450 camC gene. Homology models of both the chimeric proteins were developed using SWISS-MODEL based on the known crystal structure of cytochrome P-450 camC, BM-3, 1DT6A, and 2C17A. The models indicated that the proposed heme-binding site was intact, which is inevitable for catalytic activity of cytochrome P-450s. The expression of chimera 1 and 2 genes in Escherichia coli DH5α was evident from light-pink cell pellets, protein band in sodium dodecyl sulfate polyacrylamide gel electrophoresis, and diagnostic carbon monoxide-difference spectra. Our studies show that strategies can be developed to exploit the natural diversity of the P-450 superfamily to generate chimeric biocatalysts that would provide new templates amenable to directed evolution.     

Purification of a marine bacterial glucose dehydrogenase fromCytophaga marinoflava and its application for measurement of 1,5-anhydro-d-glucitol

A novel glucose dehydrogenase (GDH) from a marine bacteriumCytophaga marinoflava IFO 14170 was isolated from its membrane fraction. This GDH catalyzes the oxidation of a hydroxy group of glucose, but does not react in its C-l position. This enzyme is composed of a single peptide with a mol wt of 67,000. The GDH can react under high salinity. The optimum pH is around 8.0, showing a typical property of marine bacterial enzymes. Using this novel enzyme, an enzymatic determination of 1,5-anhydro-D-glucitol (1,5AG) utilizing 2,6-dichrolophenolindophenol (DCIP) and phenazine methosulfate (PMS) as electron mediators was caried out. A good linear correlation was observed from 0.5 mM to 4 mM of 1,5AG.     

Effect of growth conditions on the rheological properties and chemical composition ofVolcaniella eurihalina exopolysaccharide

The exopolysaccharide produced byVolcaniella eurihalina, an halophilic eubacterium, under different environmental and nutritional conditions, is studied.V. eurihalina synthesizes an acidic heteropolysaccharide, composed by rhamnose, glucose, and mannose, as well as amino sugars, uronic acids, and acetyl and sulphate residues. This composition varies depending on the nutrients of culture medium. Viscosity and pseudoplasticity of the polymer solutions are also influenced by the nutritional conditions in which the microorganism was grown.     

Multiple-locus variable number tandem repeats analysis for genetic fingerprinting of pathogenic bacteria

DNA fingerprinting has attracted considerable interest as means for identifying, tracing and preventing the dissemination of infectious agents. Various methods have been developed for typing of pathogenic bacteria, which differ in discriminative power, reproducibility and ease of interpretation. During recent years a typing method, which uses the information provided by whole genome sequencing of bacterial species, has gained increased attention. Short sequence repeat (SSR) motifs are known to undergo frequent variation in the number of repeated units through cellular mechanisms most commonly active during chromosome replication. A class of SSRs, named variable number of tandem repeats (VNTRs), has proven to be a suitable target for assessing genetic polymorphisms within bacterial species. This review attempts to give an overview of bacterial agents where VNTR-based typing, or multiple-locus variant-repeat analysis (MLVA) has been developed for typing purposes, together with addressing advantages and drawbacks associated with the use of tandem repeated DNA motifs as targets for bacterial typing and identification.     

Evaluation of epifluorescence image analysis of biofilm growth on stainless steel surfaces

Biofilm growth of Bacillus subtilis, Pseudomonas fragi, Pediococcus inopinatus and Listeria monocytogenes was studied on stainless steel surfaces at room and low temperatures to evaluate the results of traditional hygiene measures. The results were compared with those of image analysis of stainless steel surfaces in an epifluorescence microscope. Statistical analyses were carried out to determine the variations between the conventional cultivation swab method, the glycocalyx amount obtained using swabbing, and the values of the areas of the biofilm, slime and cells. As a general rule, old biofilms showed total counts at approximately the same levels as the young biofilm. The results showed that temperature affected the results for all strains except B. subtilis. The strains of Pe. inopinatus and Ps. fragi showed increased attachment at 6°C and L. monocytogenes at 25°C. The biofilm slime was more easily detached than the cells. The results indicated that the traditional swab method is not reliable for the measurement of biofilm formation on surfaces.     

Biofuel cell anode based on the Gluconobacter oxydans bacteria cells and 2,6-dichlorophenolindophenol as an electron transport mediator

A basic scheme of the use of the Gluconobacter oxydans bacteria cells as a biocatalyst at an anode of a biofuel cell with air-based cathode is raised up. The anode and cathode of the cell are made of graphite; 2,6-dichlorophenolindophenol serves as an electron transport mediator; and glucose is the substrate to be oxidized. The open-circuit voltage is 55 mV, for the bacteria cell, the mediator, and glucose concentrations of 3 mg/ml (raw weight), 34 mM, and 10 mM, respectively. The voltage and current of the biofuel cell loaded with an external resistance of 10 kohm are 5.6 mV and 0.56 mA. The cell’s internal resistance is 88 kohm.     

Anaerobic reduction of a sulfonated azo dye, Congo Red, by sulfate-reducing bacteria

The capacity for anaerobic decolorization of a sulfonated azo dye, Congo Red, by a strain of a sulfate-reducing bacterium was evaluated. After optimizing the growth rate of the bacteria on a simple carbon source and terminal electron acceptor pair, lactate and sulfate, respectively, the effect of the dye concentration on their growth rate was analyzed. The decolorization rate was affected by the dye concentration in the growth medium. The azo-bond cleavage mechanism of reductive decolorization with the formation of benzidine was consistent with the results, as this metabolite was identified by high-performance liquid chromatography. Several fractions of the culture medium, including lysed cell extracts, were examined for the capacity to reduce the azo dye. This reduction capacity was found in the culture medium in which the cells had previously grown. The results showed that the mechanism of reductive decolorization of this sulfonated azo dye was extracellular and nonenzymatic, consistent with the production of sulfide anion by the microorganisms while growing on lactate and sulfate. The sulfide anions were the cause of the reduction leading to the disappearance of color in the medium. To increase the rate of decolorization, the presence of ferrous ion was also necessary together with the lactate and sulfate substrates.     

Comparison on Protein Bioaccessibility of Soymilk Gels Induced by Glucono-δ-Lactone and Lactic Acid Bacteria

In this study, the protein bioaccessibility of soymilk gels produced by the addition of glu-cono-δ-lactone (GDL) and fermentation with lactic acid bacteria (LAB) was examined using an in vitro gastrointestinal simulated digestion model. The in vitro protein digestibility, soluble protein content, free amino acids contents, degree of hydrolysis, electrophoretic patterns, and peptide content were measured. The results suggested that acid-induced soymilk gel generated by GDL (SG) showed considerably reduced in vitro protein digestibility of 75.33 ± 1.00% compared to the soymilk gel induced by LAB (SL) of 80.57 ± 1.53% (p < 0.05). During the gastric digestion stage, dramatically higher (p < 0.05) soluble protein contents were observed in the SG (4.79–5.05 mg/mL) than that of SL (4.31–4.35 mg/mL). However, during the later intestinal digestion phase, the results were the opposite. At the end of the gastrointestinal digestion phase, the content of small peptides was not significantly different (p > 0.05) between the SL (2.15 ± 0.03 mg/mL) and SG (2.17 ± 0.01 mg/mL), but SL showed higher content of free amino acids (20.637 g/L) than that of SG (19.851 g/L). In general, soymilk gel induced by LAB had a higher protein bioaccessibility than the soymilk gel coagulated by GDL.