Development Of A Microbial Community Microarray To Evaluate The Activated Sludge

Activated sludge consisting of bacteria, protozoan, metazoan, got more and more attentions for its characterics of diversal microbial community and complex function. Above all, it will be great significant to study bacteria population, bacteria function and their relationship in activated sludge, to understand the community structure and its dynamics, to earn key bacterium and indicative microorganisms closely related to quality of activated sludge. However, there are no effective means to study it.Here we developed and validated an oligonucleotide prokaryotic community microarray targeting environmentally occurring bacterial and archaeal genotypes, followed by investigation of bacterial population of activated sludge in lab-scale Sequenced Batch Reactor(SBR) through analysis of clone libraries generated by random cloning of amplified 16S rRNA genes. Our approach allows the rapid detection of a large range of activated sludge bacteria at various taxonomic hierarchies and give more information about sample populations. It also can monitor microbial changes during the process of activated sludge bulking and get indicative bacteria by comparatively hybridized with a set of RT-PCR products of sludge samples, which was validated by FISH.Establish of a rapid method for extracting high-quality RNA from activated sludge: An effective and fast RNA isolation method of activated sludge was established and five different methods were compared based on RNA yield, purity, integrity, RT-PCR amplification of 16S rRNA genes and subsequent terminal restriction fragment length polymorphism (T-RFLP) analysis. That is, the precipated activated sludge was washed with TENP and PBS buffer, followed by using lysozyme and TRIzol to directly lyse of microbial cells, chloroform to remove protein and most of the DNA from bacterial lysate, isopropanol to precipitate nucleic acid and DNase I to hydrolyze residual DNA. To further purify RNA, RNA purifing column was utilized. The results demonstrated that the extraction method, with the aid of TRIzol and RNA purification kit, could effectively extract high-quality RNA. It not only means low degradability and high quantity, purity and diversity, but also the genes of 16S rRNA and amoA could be amplified by RT-PCR. Compared with other methods, it showed a great advantage of low cost and high efficiency and could be applied to RNA extraction from activated sludge in a large number. Furthermore, T-RFLP results indicated that the community composition as well as the abundance of individual members was affected by the kind of RNA extraction methods.Analysis of bacteria diversity in SBR activated sludge:Living bacterial communities in lab-scale activated sludge reactors after a period of several months of steady operation were examined by construction of 16S rRNA libraries and RFLP analysis of clones. Sequence analysis of representative clones revealed that 16S rRNA library was clearly dominated by clones ofβ-Proteobacteria(49.9%), followed by Bacteroidetes(20.4%),α-Proteobacteria(18.9%) and Firmicutes. Others including Planctomycetacia, Acidobacteria, Verrucomicrobiae and Cyanobacteria were also found in samples. At least 30 genus dominated by genus Zoogloea, Thauera, Rhodobacter were observed in activated sludge, which also included genus Mesorhizobium, Paracoccus, Methylobacterium and so on.Development of a microbial community microarray and its evaluation: An oligonucleotide microbial community microarray consisting of 64 probes and targetting 57 operational taxonomy units(OTUs) in the 16S rRNA library were developed. Two pairs of universal primers and specific oligonucleotide probes targeted to V3, V5, V6 regions of 16S rRNA genes were designed basing on phylogenetic analysis of 16S rRNA sequences from 16S rRNA clone library of samples. The probes were tested for their specificity against the corresponding OTU clones by microarray hybridization using PCR-amplified fluorescent DNA of the 16S rRNA genes. And it showed that the microarray system could successfully discriminate among the OTUs at various taxonomic hierarchies with high specificity and stability. Furthermore, 100 CFU bacteria under pure culture condition could be detected by PCR amplification and crude PCR amplification products should be applied in order to have a strong fluorescence signal.Applications of microbial community microarray: In order to verify its function to evaluate activated sludge, the microbial community microarray was applied to hybridize with RT-PCR products of different samples coming from the process of bulking. Data analysis results showed that with the occurrence of non-filamentous activated sludge bulking, Streptococcaceae and Flavobacterium continued to grow and the symbiotic bacteria of ciliate, some bacteria in family Bacteroidetes, genus Runella were significantly reduced. It was also validated by FISH that Flavobacterium could be indicative bacteria of non-filamentous activated sludge bulking.