ABSTRACT
Single-Strand Conformation Polymorphism(SSCP) is an effect method for investigating environment microbial genetic polymorphism, with its characterization of rapidness, simplicity, and sensitivity. However, many factors can influence the results of SSCP in the analysis of complex environment samples, and its optimization is highly needed. In this paper, optimal PCR-SSCP conditions were discussed based on PAGE concentration, formamide deionized in denaturing loading buffer, electrophoresis time and temperature. The resluts showed that the optimal conditions were as follows: 16S rDNA V1~V3 was selected as the targeted gene, the ratio of acrylamide to N, N-dimethylacrylamide in 12% polyacrylamide gel electrophoresis(PAGE)gel was 49:1, the ratio of formamide deionized in denaturing loading buffer was 1:3, running the SSCP gel at 300 V for 18 h (under 4 ℃). Aside from this, the validations using samples from a simultaneous desulfurification and denitrification bioreactor were conducted under this optimal conditions.
ABSTRACT
Molecular technologies are sensitive, fast, and cheap in the study on microbial ecology. However, these methods do not provide information about morphology, number, and spatial distribution of the microorganisms. In contrast, Fluorescence in situ hybridization (FISH) combines the precision of molecular biology with the visual information from microscope, and permits visualization and identification of individual miciobial cells within their natural habitat. FISH not only allows the detection of slow growing microorganisms, but also of yet-to-be cultured. It is useful for many applications in diagnosis and assessment of the population structure of complex microbial communities, and is a powerful tool for molecular ecology studies in microbiology, In this paper, major techniques and progresses of FTSH were described. Its application in microbial ecology, as well as problems , pitfalls, and perspectives of FISH are discussed.
ABSTRACT
A anaerobic hydrogen-producing strain HR-1 was isolated from compost. Phylogenetic analysis based on 16S rRNA sequence similarity indicates that strain HR-1 is the closest relative to Clostridium ace- tobutylicum ATCC 824, with the similarity of 96%. Biological characteristics and phylogenetic analysis of 16S rRNA gene indicate that HR-1 is a new species named Clostridium sp. HR-1. Cells are Gram-positive, mobile rod-shaped. Spores and flagellums were no observed. Temperature range for growth is 10?C to 45?C (optimum temperature 37?C~39?C), and range pH for growth is 4.0 to 10.0 (optimum pH 7.5~8.0). H2, CO2, acetate, butyrate and a little ethanol are the end products of PYG fermentation. Strain HR-1 has the ability to use organic nitrogen and inorganic nitrogen sources for growth and hydrogen production, and yeast extract is the optimum nitrogen source for hydrogen production. Strain HR-1 produces hydrogen from xylose (3 g/L) at 37?C and initial pH 6.5, the hydrogen yields and maximal hydrogen production rate are 1.84 mol-H2/mol-xylose and 10.52 mmol-H2/h?g-cdw, respectively. Strain HR-1 is able to utilize glucose, galactose, fructose, mannose and cellobiose for hydrogen production and the hydrogen yields from glucose is 2.36 mol-H2/mol-glucose.
ABSTRACT
Designed the degenerate primers of alcohol dehydeogenase and L-lactate dehydrogenase to aug- ment Ethanoligenens harbinense B49 genomic DNA, and obtained about 780 bp and 610 bp PCR product respectively. Augmented flank sequences of the two PCR fragments with the Cassette PCR method. Similar- ity alignment showed that the products of the cloned DNA were very high similar to those of alcohol dehy- drogenase genes and L-lactate dehydrogenase genes respectively. One of the two sequences was 1902 bp long, and the ORF of adh was 1101 bp long and encoded 366 amino acids. Its putative molecular weight was about 39.71 kD, its calculational isoionic point was pH 5.93. The maximal identity and positive was 51% and 73% with Clostridium thermocellum ATCC 27405 adh. The other one was 2490 bp long, and the ORF of adh was 951 bp long and encoded 316 amino acids. Its putative molecular weight was 34.23 kD, its calcula-tional isoionic point was pH 6.09. The maximal identity and positive was 55% and 74% with Bacillus megaterium L-ldh. Successfully cloning these two genes would not only enrich the gene resources of L-lactate dehydrogenase and alcohol dehydrogenase genes, but also give the scientific warrant for the meta- bolic engineering research and the construction of the gene-engineering bacteria.