The massive development of bloom-forming cyanobacteria is causing serious problems in eutrophic water bodies worldwide. Many cyanobacterial species have been reported to be able to produce toxins, which threaten many aquatic ecosystems and cause serious and occasionally fatal human liver, digestive, neurological, and skin diseases. Despite the extensive researches in the areas of hydrometeorology, food chains, and genomics, the mechanism underlying the cyanobacterial bloom formation largely remained elusive. Microbial interaction with each other and their environment is known to play a significant role in maintaining the various components of the biological ecosystems at equilibrium. In cyanobacterial blooms, various microorganisms have been reported to interact with bloom-forming cyanobacterial species. To get insights into the mechanisms of cyanobacterial bloom formation, exploration of interaction among the bloom-forming cyanobacterial species and other co-existing species is indispensable. In this study, metagenomics, nutrient hydrology, meteorology and environment factor datasets based integrated approaches were utilized to reveal the mechanism of cyanobacterial bloom formation. Main findings of the present study are summarized below.
(1) An experimental platform method based on 16S rRNA amplicon for bacterial diversity
survey of Taihu Lake was established. Extraction of the DNA is a key step involved in identifying the bacterial diversity and community composition. To find the reliable DNA extraction method, four widely used DNA extraction methods, including CTAB, XS, commercial Mobio DNA solation kit and Omega DNA solation kit were evaluated using three different kinds of samples collected from Lake Taihu. To perform comprehensive assessments different factors including yield, purity, and integrity of Meta DNA were also considered along with “spectrum”. The results showed that XS is the most suitable for cyanobacterial aggregates (Attached bacteria), and Omega kit for origin water (Total bacteria) and filtered water (Bacterioplankton). Furthermore, to evaluate the performances of the V regions metrics including “coverage”, “specificity”, “spectrum”, and “POAOs” were considered and V4 was found as most prominent V region for achieving good domain specificity, higher coverage and a broader spectrum in the bacterial domain. S-D-Bact-0564-a-S-15/S-D-Bact-0785-b-A-18 was found as a promising primer set for surveying bacterial diversity in eutrophic lakes. Moreover, Greengenes was observed as a robust database for aligning sequences reads recovered from Lake Taihu, as compared to other available reference databases including SILVA and RDP.
(2) The bacterial succession pattern and dynamics in community structures, in Lake Taihu,
were also observed. Overall, Cyanobacteria, Proteobacteria, Verrucomicrobia, Actinobacteria and Planctomycetes were the dominant bacterial groups cross the four seasons in Lake Taihu.
Significant differences in community structure among the four seasons and an obvious seasonal succession pattern of bacteria were observed therein. The differences between two seasons succession were significantly observed, where main succession groups were Microcystis, ACK-M1, C111, Synechococcus, Actinomycetales, Pirellulaceae and Sphingobacteriaceae. Candida
-tus_Xiphinematobacter, Synechococcus, Flavobacterium were involved in winter-spring; Microcystis, Synechococcus, Planctomyces for spring-summer; Microcystis, Planctomyces, Opitutus for summer-autumn; Microcystis, Methylotenerar, Candidatus_Xiphinematobacte for autumn-winter. Moreover, phytoplankton, water temperature, and N were found as main driving factors for a succession of bacterial community.
(3) The community diversity of BACA was ascertained and temperature found playing key
roles in structuring BACA. Bacterial community succession was observed on long-term (Seasonal) and even short-term basis (Weekly) in Lake Taihu. Therefore, BACA might have an active role in the outbreak of cyanobacterial blooms and no significant difference between the floating and sinking BACA samples was observed. BACA of M. wesenbergii, M. panniformis, M. aeruginosa, and M. flos-aquea demonstrated the host specificity among the Microcystis. Importantly, BACA was found involved in carbon cycle and degradation of microcystin, nitrogen, and phosphorus, which affected their host from a functional perspective. Therefore, the classification of Microcystis, combined with morphology and its BACA could serve to differentiate the Microcystis spp. in their natural population.
(4) Complete genome sequence and genomic characterization of M. panniformis FACHB 1757.
A strain of Microcystis, M. panniformis FACHB1757, was isolated from Meiliang Bay of Lake Taihu in August 2011. The whole genome was sequenced using PacBio RS II sequencer with 48-fold coverage. The complete genome sequence with no gaps contained a 5,686,839 bp chromosome and a 38,683 bp plasmid, which coded for 6,519 and 49 proteins, respectively. Comparison with strains of M. aeruginosa and some other water bloom-forming cyanobacterial species revealed large-scale structure rearrangement and length variation at the genome level along with 36 genomic islands annotated genome-wide, which demonstrates high plasticity of the M. panniformis FACHB1757 genome and reveals that Microcystis has a flexible genome evolution. Therefore, high plasticity and flexibility in genome evolution might be the reason behind providing superiority to Microcystis in ecological competition and global distribution.
(5) Microbial profiles of MMCA in physiological and ecological functions. There were Signifi
-cant differences among the different phases of cyanobacterial blooms for the MBCA diversity and metabolic pathways were observed. Early phase of cyano-blooms (Jan. to Mar.) was characterized by low abundance of Rheinheimera, Pseudomonas, Phormidium, and exponential phase of cyanobacterial blooms (Apr. to Jun) was characterized by high abundance of Rheinheimera, Pseudomonas, Paucibacter, whereas, stationary phase of cyanobacterial blooms (Jul. to Oct.) was characterized by high abundance of Microcystis, Bdellovibrio, Bryobacter, and Rickettsia, respectively. DNRA, ANRA, and denitrification were the main processes occurring on MMCA, and nitrogen fixation proportion was not that high, and nitrification process was not found. DNRA and ANRA were observed in all the three phases and were particularly found dominate in N metabolic pathway, whereas no denitrification during the exponential and stationary phase of cyanobacterial blooms was observed. Notably, no significant difference in P metabolic pathway among the three phases of cyanobacterial blooms process was observed.
(6) The mechanism of the succession of Microcystis and Dolichospermum is revealed in Lake
Taihu. Host specificity was observed between Microcystis and Dolichospermum, which can be characterized by Gemmatimonas and Sediminibacterium, Saprospira. Meanwhile, DNRA, ANRA, and nitrogen fixation were observed in both Microcystis and Dolichospermum dominated blooms, whereas denitrification was absent in Microcystis blooms. Microcystis is non-N2 fixing cyanobacteria; however, MMCA can be a functional unit to fix nitrogen. This might be the possible explanation that non-N2 fixing genus (Microcystis) can maintain dominance in Lake Taihu. Additionally, the continuous decline of N load in Taihu Lake is considered to be the main reason for the occurrence of Dolichospermum blooms during winter and spring. It was further inferred from the analysis that N and water temperature are the main reasons for the succession of Microcystis and Dolichospermum during winter and spring in Lake Taihu.