Using Ribosomal RNA Pyrosequencing To Explore The Microbial Community Structure

Microbial community diversity is an essential issue in both microbial ecology and environmental microbiology. The features and characteristics of uncultured microorganisms, also known as the majority population in environments, could be accomplished because of the introduction of molecular methods. Ribosomal RNA (rRNA), is the most widely used and useful molecular marker for molecular ecological researches, which caused by its highly functional conservation and diverse mutation rates in different positions. Phylogenetic analysis at different taxonomic level could be done by the comparison of rRNA sequences. Nowadays, PCR amplification of whole length or partial small subunit (SSU) rRNA is the most common used method for the investigation of microbial community diversity.However, the PCR bias caused by PCR amplification especially by primer mismatches usually makes quantitative analysis of microbial communities inaccessible and hampered the analysis of "rare" biosphere even with massive sequencing. When compared with the rRNA received from the metagenomic projects, the "universal primers" cannot cover the majority organisms that comprise the "rare" biosphere especially when using the degenerate "universal" primers. Here, a new method is presented in this study, which achieved by sequencing SSU rRNA without specific PCR amplification. The three domains (Bacteria, Archaea and Eukaryota) microorganisms in the complex environmental samples can be quantificationally analyzed simultaneously without specific PCR amplification, so the microbial community can be reconstructed more integrally. The rRNA sequences from reverse transcription reaction by random primer distributed in different region and concentrated in V3 hypervariable region in the 16S rRNA sequences. To test if this sequences set is suitable to analyze the community diversity and taxonomic annotations, nearly full length of sequences from the rRNA database (RDP and Silva) are extracted and simulated the reverse transcription reaction to form a 400 bp sequences set. That 400 bp sequences are annotated the taxonomy information and compared with the nearly full length sequences and more than 99%of sequences can be annotated correctly at all taxonomic levels.This result showed that the sequences obtained by this method are appropriate for the community diversity and taxonomic annotations analysis. Two complex environmental samples (activated sludge and anaerobic sludge) are analyzed in this study. Most of the microorganisms in the activated sludge sample are Bacteria and Eukaryota while that in the anaerobic sludge sample are Bacteria and Archaea. The microbial community diversities in both samples are very high. In the activated sludge sample, the dominant Bacteria are from the family Rhodocyclaceae, the phylum Proteobacteria and the dominant Eukaryota are the phyla Metazoa and Alveolata. In the anaerobic sludge sample, the dominant Bacteria are the families Desulfovibrionaceae and Synergistaceae in the phyla Proteobacteria and Synergistetes and the dominant Archaea are the family Methanosaeta in the phylum Euryarchaeota. In addition, the sequences which cannot be assigned to any microorganisms are abundant, which suggests that the higher microbial community diversity. When compared the sequences obtained from PCR amplification and our methods, difference are detected in the communities. The diversity index calculated from the the PCR independent rRNA sequences and the PCR amplification sequences show that the PCR independent rRNA sequences is a powerful technique to determine more broad range of microbial populations. The primer sets used in this study are compared with the PCR independent rRNA sequences and many types of mismathches are detected. Remarkable, no site in the primers can match all the PCR independent rRNA sequences. The method in this study will contribute significantly to our understanding of the diversity of complex microbial communities and expand our knowledge of the "rare" biosphere.Microbial molecular methods have been widely used to monitor the microbial community diversity during the production process. The barcoded primers for pyrosequencing can be used to process many samples in a single pyrosequencing run making it possible to monitor the microbial community diversity in many samples. In this study, the starter production and the fermentation process of one of the most renowned "light-fragrance" liquors in China Fen liquor were analyzed using different strategies aiming at different microbial types. Bacterial diversity was analyzed using the 16S rRNA gene clone library and sequencing. Fungal diversity was analyzed using the ITS1 region via pyrosequencing. Quantitative real-time PCR (qPCR) helped us compare the quantity of bacteria and fungi during the starter production and fermentation process. To the best of our knowledge, this is the first report to reveal the fungal diversity within the food- fermentation process by using the pyrosequencing. In bacteria, the abundant families were Lactobacillaceae (35.4%) and Bacillaceae (28.1%) in the starter samples. However, during the fermentation process, Lactobacillaceae (90.8%) dominated nearly all the samples. As to fungi, Saccharomycetaceae (96.9%) dominated the starter samples while Saccharomycetaceae (60.1%) and Saccharomycopsidaceae (28.8%) coexisting in the fermentation process samples. The results of qPCR showed that the content of bacteria was increased while that of fungi was more stable in the fermentation process and both of bacteria and fungi decreased after the starters had produced.