Metagenomics Of BIOLAK And A~2O Activated Sludges Based On Next Generation Sequencing Technology

BIOLAK(BIOLAK) and A~2O(Anaerobic/ Anoxic/ Oxic) are two common processes in numerous biological treatment methods of wastewater. Activated sludge is the core component in biological wastewater treatment technology. The biological composition and function of activated sludge decide treatment efficiency fundamentally. In the actual operation of activated sludge systems, the input water quality, quantity and environmental conditions can affect the treatment efficiency of activated sludge system. Sometimes it is difficult to achieve the best running status and play the greatest role. Therefore, we should understand the community structure and function of microorganisms in activated sludge fully and systematically, which has great significance to improve the treatment efficiency of activated sludge. Meanwhile, it is particularly important to comparative analysis of species and functional genes contained in these two kinds of sludges.This paper summarized the progress of the next-generation of sequencing technologies, metagenomics and microbial studies of activated sludge. Then, the community structure and function of activated sludge microorganisms in BIOLAK and A~2O processes were revealed fully and systematically by the next-generation high-throughput Illumina sequencing technology, with the principle of metagenomics independent amplification. At the same time, biological community characteristics and functional classification were compared between BIOLAK and A~2O activated sludge metagenomes.The main conclusions of the study are as follows:1. Amazing biodiversity was revealed in the BIOLAK activated sludge, which includes 47 phyla, 872 genera, 1351 species and a considerable number of non matching high quality sequences(136133 sequences), which is far beyond the previously reported biological diversity of BIOLAK activated sludge. Most of the sequences detected in BIOLAK activated sludge belong to the domain Bacteria. There are 33 phyla and 748 genera identified in the Bacteria domain. Proteobacteria is the most abundant phylum, followed by Bacteroidetes, Nitrospirae and Planctomycetes, suggesting that these groups play a key role in the BIOLAK wastewater treatment system. Nitrospira is the most prevalent genus, which is a key group in the nitrogen cycle. Followed by Gemmatimonas, which is an important genus in biological phosphorus removal process. The rest of the sequences were assigned to the Eukaryota domain(including 10 phyla and 60 genera), the Archaea domain(including 3 phyla and 39 genera) and the Virus domain(448 viral sequences dominated by bacteriophages). Most of the viruses detected belong to the order Caudovirales. The presence of these viruses may have a potential impact on the effectiveness of sewage treatment and the receiving water.2. There are abundant and widespread metabolic genes in BIOLAK activated sludge metagenome. Carbohydrates, virulence, amino acids, protein metabolism, respiration and DNA metabolism related functional genes are closely related to the growth and survival of biological groups. The proportions of nitrogen, phosphorus, sulfur and aromatic compounds metabolism in the BIOLAK sludge are 2.4978%, 1.5606%, 1.2932% and 2.2781%, respectively. In the subsystems of nitrogen metabolism, the vast majority of genes are associated with ammoniation and denitrification, which are the important critical pathways in the nitrogen removal. In the phosphorus metabolism, the number of function genes involved phosphate metabolism is the largest. In sulfur metabolism, the function genes involved in organic sulfur assimilation accounted for the highest proportion. The number of genes involved in the peripheral pathways for catabolism is the largest in aromatic compounds metabolism. The above key functional genes were dominant in nitrogen, phosphorus, sulfur and aromatic compounds metabolism, which play critical roles in the effectiveness of the treatment of BIOLAK activated sludge. There are 13886 planctomycetes sequences detected in the BIOLAK sludge, which belong to 14 genera involved in the ANAMMOX process, among them 8 genera belong to the top 100 most abundant genera. In addition, among four processes of nitrogen metabolism, denitrification related genes are most abundant. Therefore, the functional genes related to denitrification are important in the process of nitrogen metabolism, which play an key role in the removal of nitrogen from wastewater.3. Compared with the previously reported A~2O activated sludge, this study revealed a higher biological diversity of A~2O activated sludge. There are 51 phyla, nearly 900 genera and a considerable number of non matching high quality sequences identified from the A~2O activated sludge ecosystem. Among the 742 bacteria genera, Nitrospira, Thauera, Dechloromonas and Ignavibacterium are most prevalent genera, which have abilities to metabolize nitrogen and aromatic compounds. The sequence numbers of genera Paracoccus, Alcaligenes and Nitrosospira are 105, 39 and 353, however, which were not detected in the previous A~2O sludge. The genus Nitrosospira is ranked as one of the top 100 most abundant genera in the current A~2O sludge metagenome. Meanwhile, in the domain Archaea, there were four phyla and 50 genera detected in the A~2O sludge metagenome. In addition, there were 77 eukaryotes genera detected in the A~2O sludge ecosystem, which belong to 12 phyla. In addition, viruses in the A~2O sludge metagenome were dominated by bacteriophages.4. The functional genes related to carbohydrate, amino acid and protein metabolism were detected in the metagenome of A~2O activated sludge, which is closely related to the growth and development of microorganisms. At the same time, nitrogen, phosphorus, sulfur and aromatic compounds metabolism related genes were detected, and the proportions are 2.72%, 1.48%, 1.15% and 2.24%, respectively. Functional genes involved in the denitrification play a dominant role in the process of nitrogen metabolism. These functional genes related to metabolism are directly involved in the biological degradation or absorption of related compounds, and they are closely related to the treatment performance of wastewater treatment plants. By the analysis of SEED Level 2 subsystems, 9756 functional gene sequences associated with nitrogen metabolism and Candidatus Accumulibacter(accounted for 1.37%) were identified, which play a key roles on the removal of nitrogen and phosphate in wastewater. Through function analysis of A~2O activated sludge metagenome sequences, four planctomycetes genera(Planctomyces, Pirellula, Gemmata and Singulisphaera) play an important role in nitrogen metabolism.5. The core microbial functional groups were revealed in two different activated sludges. Among the top 100 most abundant genera, 88 genera are identical, which are dominant microorganisms in two activated sludges, and play a crucial role in the stability of activated sludge system and effective removal of pollutants. In addition, function genes involved denitrification and ammonification are dominant in these two kinds of sludges, which play a key role on the removal of nitrogen in wastewater. At the same time, there are significant differences on the level of phylum and the genus in the two kinds of activated sludge. The proportions of all phyla are different in the two kinds of sludges, especially the bacterial phylum Planctomycetes. The proportion of the phylum in the BIOLAK sludge is 4.7625%, more than 60% higher than that in the A~2O sludge(2.9835%), indicating that ANAMMOX process plays a more important role in the nitrogen metabolism of BIOLAK than in the A~2O sludge. There are 167 genera only detected in the A~2O sludge, which were not found in BIOLAK sludge. On the other hand, 50 genera identified in the BIOLAK sludge were not detected in the A~2O sludge. In the two sludges, 24 major abundant genera were identified, each of which accounted for more than 1% in at least one of the metagenomes. Among them, only the proportions of genus Sorangium in two sludges are similar. The proportions of other 23 genera are different in two sludges. As for the SEED Level 2 subsystems, the proportions of nitrogen, phosphorus, sulfur and aromatic compounds metabolic processes are different in BIOLAK and A~2O sludge. Thus, in the real wastewater treatment, we should optimize the composition and proportion of functional microorganisms based on the biological communities and functions of BIOLAK and A~2O sludge metagenome revealed by this research, which will help to improve the treatment efficiency of activated sludge.