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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.134, #2)
Comparative study of methods for extraction and purification of environmental DNA from soil and sludge samples by Changhyun Roh; Francois Villatte; Byung-Gee Kim; Rolf D. Schmid (pp. 97-112).
An important prerequisite for successful construction of a metagenome library is an efficient procedure for extracting DNA from environmental samples. We compared three indirect and four direct extraction methods, including a commercial kit, in terms of DNA yield, purity, and time requirement. A special focus was on methods that are appropriate for the extraction of environmental DNA (eDNA) from very limited sample sizes (0.1 g) to enable a highly parallel approach. Direct extraction procedures yielded on average 100-fold higher DNA amounts than indirect ones. A drawback of direct extraction was the small fragment sizeof approx 12 kb. The quality of the extracted DNA was evaluated by the ability of different restriction enzymes to digest the eDNA. Only the commercial kit and a direct extraction method using freeze-thaw cell lysis in combination with an in-gel patch electrophoresis with hydroxyapatite to remove humic acid substances yielded DNA, which was completely digested by all restriction enzymes. Moreover, only DNA extracted by these two procedures could be used as template for the amplification of fragments of several 16S rDNA, 18SrDNA groups under standard polymerase chain reaction conditions.
Keywords: Environmental DNA; metagenome; purification; direct extraction; indirect extraction; activated sludge
Element concentrations of dry-grind corn-processing streams by Ronald L. Belyea; Thomas E. Clevenger; Vijay Singh; M. E. Tumbleson; Kent D. Rausch (pp. 113-128).
The dry-grind corn process is one of two technologies used to convert corn into ethanol. In his process, all kernel components are processed through several sequential steps, including fermentation. Only one coproduct (distillers’ dried grains with solubles [DDGS]) is available for marketing. DDGS provide income to offset costs of processing; issues that affect marketing have implications in the economic viability of dry-grind plants. Two issues relate to elements in DDGS: high concentrations and excessive variation. Data on element concentrations in dry-grind processing streams could be helpful in addressing these concerns. The objective of this study was to determine element concentrations in primary process streams from dry-grind plants. Samples of corn, ground corn, beer, wet grains, syrup, and DDGS were obtained from nine dry-grind plants, and element concentrations were determinined. The concentrations of most elements in corn were not different among processing plants and were similar to published data. However, for the processing streams, there were differences in several element concentrations among processing plants. The concentrations of most elements in beer were about three times those of corn, due to the disappearance of starch during fermentation. Syrup had the highest element concentrations. Variations in element contents of DDGS and parent streams were due to processing conditions and not corn. Appropriate processing of thin stillage (the parent stream of syrup) could reduce the element content of DDGS.
Keywords: Distillers’ dried grains with solubles; ethanol; dry-grind processing; stillage; syrup; element concentrations
Production and properties of a biosurfactant applied to polycyclic aromatic hydrocarbon solubilization by B. Mahanty; K. Pakshirajan; V. V. Dasu (pp. 129-141).
Microorganisms isolated from a soil sample collected from a gasoline filling station (located in Guwahati) were tested for their pyrene- and anthracene-degrading potential. Preliminary studies showed the ability of the organism to grow on carbon-free mineral medium (CFMM) supplemented with pyrene as the sole source of carbon. The organisms were found to produce a bioemulsifier when grown on CFMM with glucose or glycerol and/or pyrene as the carbon source. The organisms could also utilize anthracene when grown on mineral salt medium along with 2% glycerol. Within 2 d, anthracene concentration dropped less than 30% of the original concentration. Approximately 100 mg of the emulsifier was isolated from 25 mL of the 5-d-grown culture. The emulsifier was tested to produce emulsion with both an aliphatic and an aromatic group of hydrocarbons and resulting emulsions were found to be stable for a long period of time when keptat 10–15°C. The emulsifier was also quite stable in a pH range of 3.0–11.0. In a concentration range of 0.5–10 mg/mL, it resulted in a linear increment of apparent pyrene and anthracene solubility in water.
Keywords: Soil microbes; biosurfactant; bioemulsifier; emulsifying activity; polycyclic aromatic hydrocarbons
Optimization of critical medium components for the maximal production of gentamicin by Micromonospora echinospora ATCC 15838 using response surface methodology by M. Himabindu; P. Ravichandra; K. Vishalakshi; Annapurna Jetty (pp. 143-154).
Optimization of the fermentation medium components for maximum gentamicin production by Micromonospora echinospora ATCC 15838 was carried out. Response surface methodology was applied to optimize the medium constituents. A 24full-factorial central composite design was chosen to explain the combined effects of the four medium constituents, viz. starch, soyabean meal, K2HPO4, and CoCl2 and to design a minimum number of experiments. A second order model was developed and fitted using least square method. The R 2 value of the model was 0.9723, which shows that model is best fit for the present studies. The results of analysis of variance and regression of a second order model showed that the linear effects of starch (p<0.001697) and CoCl2(p<7.99E-13), and cross product effects of starch and soyabean meal (p<0.029876) and soyabean meal and CoCl2 (p<0.008909) were more significant, suggesting that these were critical variables having the greatest effect on the production of gentamicin in the production medium. The optimized medium consisting of 9 g/L starch, 3 g/L soyabean meal, 0.9 g/L K2HPO4, and 0.01 g/L CoCL2 predicted 850 mg/L of gentamicin which was almost 110% higher than that of the unoptimized medium. The amounts of starch, soyabean meal, and K2HPO4 required were also reduced with RSM.
Keywords: Gentamicin; production medium; rotational central composite design (α=2); response surface methodology; optimization
Growth of Escherichia coli under extremely low-frequency electromagnetic fields by Oselys Rodriguez Justo; Victor Haber Pérez; David Chacon Alvarez; Ranulfo Monte Alegre (pp. 155-163).
The inflence of extremely low-frequency (ELF) electromagnetic fields on Escherichia coli cultures in submerse fermentation was studied. The fermentation processes were carried out recycling the culture medium externally through a stainless steel tube inserted in a magnetic field generator (solenoid). The exposure time and electromagnetic induction were varied in a range of 1 to 12 h and 0.010 to 0.10 T, respectively, according to a Box-Wilson Central Composite Designs of face centered with five central points. Growth of E. coli could be altered (stimulated or inhibited) under magnetic fieldinduced effects. E. coli culturesexposed at 0.1 T during 6.5 h exhibited changes in its viability compared to unexposed cells, which was 100 times higher than the control. The magnetic field generator associated with the cellular suspension recycle is a new way of magnetic treatment in fermentation processes and could be appropriate to industrial scale up.
Keywords: Escherichia coli ; growth; cell culture; electromagnetic fields; biological effects
Establishing mathematical and physical models for the adsorption of biomacromolecules by Songjun Li; Pingchu Chen; Wuke Li; Xiao Hao; Guangfu Yang (pp. 165-178).
In this article, a series of mathematical and physical models for the adsorption of biomacromolecules are established. As shown, the adsorption of biomacromolecules is actually considerably complicated and often involves various interactions, such as electrostatic, hydrophobic and hydrogen-bonding, etc. Considering this, these models show that if these interactions are heavily involved in the process, the plot of in Q (or Q) vs In C* is normally expected to be a straight-line. Otherwise, if the linearity exists between C*/Q vs C*, the adsorption would be an ideal process without the intervention of them. Meanwhile, this article also presents corresponding relationships for the adsorption in multisited binding and multilayer forms. Other aspects including ion-exchange systems are also discussed (C*, the equilibrium concentration of biomacromolecules; Q, the adsorbance).
Keywords: Biomacromolecule; adsorption; theory; interaction; ionexchange
Physicochemical characteristics of commercial lactases relevant to their application in the alleviation of lactose intolerance by S. O’Connell; G. Walsh (pp. 179-191).
Selected microbial lactases are used to treat lactose intolerance. A series of experiments were carried out in vitro in order to determine the likely relative suitability of four major commercial lactase products used in this regard. The lactases displayed between 55 and 61% of maximum activity at 37°C and significant acitvity between pH3.0 and 6.5. They retained between 0 and 65% of original activities after exposure to full simulated digestive tract conditions for 6 h. All four enzymes proved to be particularly acid sensitive and only two products were enteric coated.The products demonstrated varying ability to hydrolyze lactose under simulated digestive tract conditions. The most effective product hydrolyzed 2.7 g lactose per capsule, suggesting that consumption of several capsules, as opposed to the usually recommended one or two, would be required to hydrolyze the entire 12 g lactose load characteristic of a dairy-based meal. All enzymes were substantially pure and displayed similar kinetic properties and molecular weights. None appeared ideally suited for use in the alleviation of lactose intolerance. The findings may in part explain the variability and often disappointing results previously reported for lactase-based clinical trials and will provide comparative baseline data against which candidate second-generation lactases may be assessed.
Keywords: Lactase; β-galactosidase; lactose intolerance; gastrointestinal