| The microorganism is ubiquitous in the nature, and it participates in and is undertaking the important role in nature biogeochemical cycling, such as carbon, nitrogen cycling, mineral decency and formation. However, since 99% of microorganisms in nature are unable to culture, the detection, description and quantitation of environmental microorganism and its relationship between environmental factors are big issue in environmental microbial ecology. The purpose of this study is that using microarray based GeoChip to investigate the whole microbial communities under two typical environments (extreme acid environment and global warming affecting soil) and the relationship between microbial communities to environmental factors. This research mainly based on the analysis with GeoChip which developed by the Institute for Environmental Genomics of University of Oklahoma. It is a robust and comprehensive tool to investigate environmental microbial community and it includes GeoChip 2.0 and the GeoChip 3.0 versions. GeoChip 2.0 contains 24,000 nucleotide probe (50 mer), including larger than 150 functional gene categories and 10,000 genes, and has covered the nitrogen, carbon, sulfur and phosphorus cycling, and metal resistance and organic contaminant degradation. The GeoChip 3.0 version contains approximately 28,000 probes and covers about 57,000 gene sequences in more than 292 gene families. The total environment microbial DNA were extracted, purified, amplified, randomly labeled and hybridized for further data analysis. Other methods, such as clone library method, BIOLOG, PLFA and soil respiration meter were also performed to exam the activity of microbial community. Correlation and correspondence analysis were used for analyzing the relationship between microbial communities and environmental factors.The results showed that, firstly, At least 50 to 60% of the crude DNA could be recovered from the low melting point (LMP) gel purification with high A26o/2so and A26o/23o ratios, approximately 1.6-1.8 and 1.7-2.0, respectively, than the other three commercial purification kits tested: Wizard(?) Plus Midipreps DNA purification system from Promega (Promega kit), QIAEXⅡgel extraction kit (QIAEX kit), and E.Z.N.A.(?) gel purification kit from Omega (Omega kit). Purified DNA was successfully tested for PCR amplification of 16S rDNA. whole community genome DNA amplification using Phi 29 DNA polymerase, and DNA labeling with Cy dyes using klenow fragment. A larger set of samples,136 soil samples, containing a variety of agricultural and forest soils were then tested and high quality DNA was successfully obtained. Our results showed that the improved procedure yields better results than other available extraction and purification procedures and will provide a new way to obtain high quality DNA from soils. Secondly, for community analysis in acid mine drainage (AMD), clone library method revealed total 1691 clones were obtained from three copper mine in southeast of China and majority (97.7%) of the clones were classified intoα-,β, andγ-Proteobacteria, Acidobacteria, Actinobacteria, Bacteriodetes, Cyanobacteria, Firmicutes, Nitrospirae, and Planctomycetes. GeoChip 2.0 analysis revealed that these microbial communities were functionally heterogeneous as measured by the number of detected, overlapping,and unique genes and diversity indices. Almost all key functional genes targeted by GeoChip 2.0 were detected in the AMD microbial communities including carbon fixation, carbon degradation, methane generation, nitrogen fixation, nitrification, denitrification, ammonification, nitrogen reduction, sulfur metabolism, metal resistance, and organic contaminant degradation, suggesting that the functional gene diversity may be higher than was previously thought. Mantel test results indicated that AMD microbial communities might be largely shaped by surrounding environmental factors. For global warming analysis, the results showed that warming altered soil microbial communities by DCA and cluster analyses, and even caused a higher microbial biomass, helping us to reject the substrate depletion for explaining respiration acclimation, but accept microbial adaptation which may diminish positive feedback. The treatment of clipping (mimicking biofuel feedstock harvest) exaggerated the increase of temperature, but decreased the divergence at community (structure, diversity, richness and bacteria abundance) and functional gene (abundance of genes in C, N, P and S cycles) levels, indicating that the direct effect of elevated temperature (T) was not the major factor affecting microorganisms. Other than soil temperature, the soil and plant variables could explain 79.4% of total variation, suggesting the indirect effects of elevated T on these variables were more important in shaping microbial community structure. A few possibly negative mechanisms under warming were observed by the abundance of functional genes in GeoChip, including increased C fixation, N fixation and methane oxidation without clipping, while positive mechanisms might exist due to increased methane production but decreased oxidation with clipping, and increased denitrification without clipping. However, the importance of these mechanisms on determining the overall direction of feedback need to be future investigated.Overall, this study showed that GeoChip is a powerful tool to investigate environmental microbial communities’functional genes structure and it can imply ecological potential functions and widen our understanding for exploring the mechanisms of environmental microbial community response for environmental factor. |
PDF Full Text Request