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New Window Opens on the Secret Life of Microbes: Scientists Develop First Microbial Profiles of Ecosystems
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Applied Biochemistry and Microbiology (v.48, #2)
Bioprospecting microalgae as potential sources of “Green Energy”—challenges and perspectives (Review) by S. K. Ratha; R. Prasanna (pp. 109-125).
Microalgae and cyanobacteria are potential food and feed, sources of high-value bioactive molecules and biofuels, and find tremendous applications in bioremediation and agriculture. Although few efforts have been undertaken to index the microalgal germplasm available in terms of lipid content, information on suitability of strains for mass multiplication and advances in development of methods for extraction and generating biofuel are scarce. Our review summarizes the potential of microalgae, latest developments in the field and analyzes the “pitfalls” in oversimplification of their promise in the years to come. Microalgae represent “green gold mines” for generating energy; however, the path to success is long and winding and needs tremendous and concerted efforts from science and industry, besides political will and social acceptance for overcoming the limitations. The major advantages of second generation biofuels based on microalgal systems, include their higher photon conversion efficiency, growth all around the year, even in wastewaters, and production of environment friendly biodegradable biofuels.
Microalgae biofuel potentials (Review) by Y. Ghasemi; S. Rasoul-Amini; A. T. Naseri; N. Montazeri-Najafabady; M. A. Mobasher; F. Dabbagh (pp. 126-144).
With the decrease of fossil based fuels and the environmental impact of them over the planet, it seems necessary to seek the sustainable sources of clean energy. Biofuels, is becoming a worldwide leader in the development of renewable energy resources. It is worthwhile to say that algal biofuel production is thought to help stabilize the concentration of carbon dioxide in the atmosphere and decrease global warming impacts. Also, among algal fuels’ attractive characteristics, algal biodiesel is non toxic, with no sulfur, highly biodegradable and relatively harmless to the environment if spilled. Algae are capable of producing in excess of 30 times more oil per acre than corn and soybean crops. Currently, algal biofuel production has not been commercialized due to high costs associated with production, harvesting and oil extraction but the technology is progressing. Extensive research was conducted to determine the utilization of microalgae as an energy source and make algae oil production commercially viable.
Enzymatic synthesis of electroconductive biocomposites based on DNA and optically active polyaniline by Yu. S. Zeifman; I. O. Maiboroda; Yu. V. Grishchenko; O. V. Morozova; I. S. Vasil’eva; G. P. Shumakovich; A. I. Yaropolov (pp. 145-150).
Electroconductive interpolymer polyaniline complexes are synthesized on the DNA matrix, using the method of oxidative polymerization of aniline with two different biocatalyzers: horseradish root peroxidase and micropiroxidase-11 biomimetic. The spectral characteristics and morphology of the acquired biocomposites have been studied. The stereospecificity of the acquired samples of interpolymer complexes is shown, depending on the biocatalyzers used. The results acquired indicate the important role of a biocatalyzer in the formation of the twist direction of an electroconductive polymer spiral on the DNA matrix; i.e., the optical activity of the polymer samples acquired is apparently associated with the biocatalyzer properties.
Enzymatic modification of chitosan with quercetin and its application as antioxidant edible films by E. Torres; V. Marín; J. Aburto; H. I. Beltrán; K. Shirai; S. Villanueva; G. Sandoval (pp. 151-158).
Quercetin, rutin, naringin, hesperidin and chrysin were tested as substrates for cloroperoxidase to produce reactive quinones to graft onto chitosan. Quercetin and rutin quinones were successfully chemically attached to low molecular weight chitosan. The quercetin-modified chitosan showed an enhancement of plastic, antioxidant and antimicrobial properties as well as of thermal degradability. Finally, chitosan-quercetin films visibly decreased enzymatic oxidation when applied to Opuntia ficus indica cladodes.
Enzymatic synthesis of L-tryptophan from D,L-2-amino-Δ2-thiazoline-4-carboxylic acid and indole by Pseudomonas sp. TS1138 L-2-amino-Δ2-thiazoline-4-carboxylic acid hydrolase, S-carbamyl-L-cysteine amidohydrolase and Escherichia coli L-tryptophanase by J. Du; J. J. Duan; Q. Zhang; J. Hou; F. Bai; N. Chen; G. Bai (pp. 159-166).
L-Tryptophan (L-Trp) is an essential amino acid. It is widely used in medical, health and food products, so a low-cost supply is needed. There are 4 methods for L-Trp production: chemical synthesis, extraction, enzymatic synthesis, and fermentation. In this study, we produced a recombinant bacterial strain pET-tnaA of Escherichia coli which has the L-tryptophanase gene. Using the pET-tnaA E. coli and the strain TS1138 of Pseudomonas sp., a one-pot enzymatic synthesis of L-Trp was developed. Pseudomonas sp. TS1138 was added to a solution of D,L-2-amino-Δ2-thiazoline-4-carboxylic acid (DL-ATC) to convert it to L-cysteine (L-Cys). After concentration, E. coli BL21 (DE 3) cells including plasmid pET-tnaA, indole, and pyridoxal 5’-phosphate were added. At the optimum conditions, the conversion rates of DL-ATC and L-Cys were 95.4% and 92.1%, respectively. After purifying using macroporous resin S8 and NKA-II, 10.32 g of L-Trp of 98.3% purity was obtained. This study established methods for one-pot enzymatic synthesis and separation of L-Trp. This method of producing L-Trp is more environmentally sound than methods using chemical synthesis, and it lays the foundations for industrial production of L-Trp from dl-ATC and indole.
Proline dehydrogenase from Pseudomonas fluorescens: Gene cloning, purification, characterization and homology modeling by H. Shahbaz Mohammadi; E. Omidinia (pp. 167-174).
The gene encoding proline dehydrogenase (ProDH) from Pseudomonas fluorescens was isolated using PCR amplification and cloned into pET23a expression vector. The expression of the recombinant target enzyme was induced by addition of IPTG. The produced His-fusion enzyme was purified and its kinetic properties were studied. The 3D structure modeling was also performed to identify key amino acids involved in FAD-binding and catalysis. The PCR product contained a 1033 bp open reading frame encoding 345 amino acid residue polypeptide chain. SDS-PAGE analysis revealed a MW of 40 kDa, whereas the native enzyme exhibited a MW of 40 kDa suggesting a monomeric protein. The K m and V max values of the P. fluorescens ProDH were estimated to be 35 mM and 116 μmol/min, respectively. ProDH activity was stable at alkaline pH and the highest activity was observed at 30°C and pH 8.5. The modeling analysis of the three dimensional structure elucidated that Lys-173 and Asp-202, which were oriented near the hydroxyl group of the substrate, were essential residues for the ProDH activity. This study, to our knowledge, is the first data on the cloning and biochemical and structural properties of P. fluorescens ProDH.
Isolation and characterization of feather degrading enzymes from Bacillus megaterium SN1 isolated from Ghazipur poultry waste site by S. Agrahari; N. Wadhwa (pp. 175-181).
The SN1 strain of Bacillus megaterium, isolated from soil of Ghazipur poultry waste site (India) produced extracellular caseinolytic and keratinolytic enzymes in basal media at 30°C, 160 rpm in the presence of 10% feather. Feathers were completely degraded after 72 h of incubation. The caesinolytic enzyme was separated from the basal media following ammonium sulphate precipitation and ion exchange chromatography. We report 29.3-fold purification of protease after Q Sepharose chromatography. The molecular weight of this enzyme was estimated to be 30 kDa as shown by SDS-PAGE and zymography studies. Protease activity increased by 2-fold in presence of 10 mM Mn2+ whereas Ba2+ and Hg2+ inhibited it. Ratio of milk clotting activity to caseinolytic activity was found to be 520.8 for the 30–60% ammonium sulphate fraction in presence of Mn2+ ion suggesting potential application in dairy industry. Keratinase was purified to 655.64 fold with specific activity of 544.7 U/mg protein and 12.4% recovery. We adopted the strategy of isolating the keratinolytic and caesinolytic producing microorganism by its selective growing in enriched media and found that feather protein can be metabolized for production of animal feed protein concentrates.
Effect of partial pressure of CO2 on the production of thermostable α-amylase and neutral protease by Bacillus caldolyticus by J. Bader; L. Skelac; S. Wewetzer; M. Senz; M. K. Popović; R. Bajpai (pp. 182-187).
Controlling the concentration of dissolved oxygen is a standard feature in aerobic fermentation processes but the measurement of dissolved CO2 concentrations is often neglected in spite of its influence on the cellular metabolism. In this work room air and room air supplemented with 5 and 10% carbon dioxide were used for aeration during the cultivation of the thermophilic microorganism Bacillus caldolyticus (DSM 405) on starch to produce α-amylase (E.C. 3.2.1.1) and neutral protease (E.C. 3.4.24.27/28). The increased CO2 concentrations resulted in a 22% raise in activity of secreted α-amylase and a 43% raise in protease activity when compared with aeration with un-supplemented room air. There was no effect on the final biomass concentration. Furthermore, the lag-phase of fermentation was reduced by 30%, further increasing the productivity of α-amylase production. Determinations of dissolved CO2 in the culture broth were conducted both in situ with a probe as well as using exhaust gas analysis and both the methods of quantification showed good qualitative congruence.
Rhamnolipid production by Pseudomonas aeruginosa engineered with the Vitreoscilla hemoglobin gene by H. Kahraman; S. O. Erenler (pp. 188-193).
The potential of Pseudomonas aeruginosa expressing the Vitreoscilla hemoglobin gene (vgb) for rhamnolipid production was studied. P. aeruginosa (NRRL B-771) and its transposon mediated vgb transferred recombinant strain, PaJC, were used in the research. The optimization of rhamnolipid production was carried out in the different conditions of cultivation (agitation rate, the composition of culture medium and temperature) in a time-course manner. The nutrient source, especially the carbon type, had a dramatic effect on rhamnolipid production. The PaJC strain and the wild type cells of P. aeruginosa started producing biosurfactant at the stationary phase and its concentration reached maximum at 24 h (838 mg/l−1) and at 72 h (751 mg l−1) of the incubation respectively. Rhamnolipid production was optimal in batch cultures when the temperature and agitation rate were controlled at 30°C and 100 rpm. It reached 8373 mg l−1 when the PaJC cells were grown in 1.0% glucose supplemented minimal media. Genetic engineering of biosurfactant producing strains with vgb may be an effective method to increase its production.
Prolonged cultivation of an anaerobic bacterial community producing hydrogen by B. F. Belokopytov; Ya. V. Ryzhmanova; K. S. Laurinavichyus; V. A. Shcherbakova (pp. 194-199).
This paper studies various methods of long-term maintenance of the process of hydrogen evolution during the growth of an anaerobic bacterial community on a starch-containing medium. Continuos fermentation with periodic feeding and effluent removal for 72 days, allow to obtain from 0.10 to 0.23 H2/l of medium/day. The regime of regular transfer lasted more than 100 days, forming an average of 0.81 l H2/l of medium/day. The advantages and disadvantages of different methods of microbial hydrogen production during a dark starch fermentation process are presented. From the obtained H2-producing microbial community, we isolated an anaerobic spore-forming bacterium (strain BF). Phylogenetic analysis of the 16S PNA gene sequence of the new strain showed that according to its genotype it belongs to the Clostridium butyricum species.
Effect of exogenous fatty acids on the growth and production of exopolysaccharides of obligately methylotrophic bacterium Methylophilus quaylei by S. A. M. Otman; A. B. Pshenichnikova; V. I. Shvets (pp. 200-205).
Accelerating growth and increasing exopolysaccharide production in obligate methylotrophic bacterium Methylophilus quaylei were observed in the presence of C12–C18 fatty acids added to the growth media. Sodium oleate was the best growth factor. Based on data on the composition of the free fatty acids fraction in the cells and the values of the ξ-potential and fluorescence anisotropy of whole cells, we suggested that fatty acids were incorporated in the outer membrane of M. quaylei.
Ratio [13C]/[12C] as an index for express estimation of hydrocarbon-oxidizing potential of microbiota in soil polluted with crude oil by A. M. Zyakun; A. M. Boronin; V. V. Kochetkov; B. P. Baskunov; K. S. Laurinavichus; V. N. Zakharchenko; V. P. Peshenko; T. O. Anokhina; T. V. Siunova (pp. 206-215).
The hydrocarbon-oxidizing potential of soil microbiota and hydrocarbon-oxidizing microorganisms introduced into soil was studied based on the quantitative and isotopic characteristics of carbon in products formed in microbial degradation of oil hydrocarbons. Comparison of CO2 production rates in native soil and that polluted with crude oil showed the intensity of microbial mineralization of soil organic matter (SOM) in the presence of oil hydrocarbons to be higher as compared with non-polluted soil, that is, revealed a priming effect of oil. The amount of carbon of newly synthesized organic products (cell biomass and exometabolites) due to consumed petroleum was shown to significantly exceed that of SOM consumed for production of CO2. The result of microbial processes in oil-polluted soil was found to be a potent release of carbon dioxide to the atmosphere.
Construction of the industrial ethanol-producing strain of Saccharomyces cerevisiae able to ferment cellobiose and melibiose by L. Zhang; Z. -P. Guo; Z. -Y. Ding; Z. -X. Wang; G. -Y. Shi (pp. 216-221).
The gene mel1, encoding α-galactosidase in Schizosaccharomyces pombe, and the gene bgl2, encoding and α-glucosidase in Trichoderma reesei, were isolated and co-expressed in the industrial ethanolproducing strain of Saccharomyces cerevisiae. The resulting strains were able to grow on cellobiose and melibiose through simultaneous production of sufficient extracellular α-galactosidase and β-glucosidase activity. Under aerobic conditions, the growth rate of the recombinant strain GC1 co-expressing 2 genes could achieve 0.29 OD600 h−1 and a biomass yield up to 7.8 g l−1 dry cell weight on medium containing 10.0 g l−1 cellobiose and 10.0 g l−1 melibiose as sole carbohydrate source. Meanwhile, the new strain of S. cerevisiae CG1 demonstrated the ability to directly produce ethanol from microcrystalline cellulose during simultaneous saccharification and fermentation process. Approximately 36.5 g l−1 ethanol was produced from 100 g of cellulose supplied with 5 g l−1 melibose within 60 h. The yield (g of ethanol produced/g of carbohydrate consumed) was 0.44 g/g, which corresponds to 88.0% of the theoretical yield.
Development and optimization of immunoassays for the detection of botulinum toxins by A. A. Titov; I. V. Shilenko; A. A. Morozov; S. P. Yarkov; V. N. Zlobin (pp. 222-228).
Monoparametric immunoassay tests for detecting botulinum toxins types A and B and multiparametric assays for simultaneous detection of botulinum toxins type A and B have been developed. It is shown that the sensitivity of assays is affected by the size of nanoparticles of colloidal gold used as a marker of antibodies, load intensity of antibodies of colloidal gold in conjugates, the type of analytical membranes, as well as the chemical composition of buffer solutions used for the storage of conjugates and immunoassay analysis. The detection limit of monoparametric immunoassay tests is 0.5 ng/ml; that of multiparametric assays, 5.0 ng/ml. The developed immunoassay can be used for rapid assay of product quality, for grade control of botulinum toxins in pharmaceuticals, and environmental monitoring.