Identification Of Bacterial Community Associated With Nannochloropsis By Culture-dependent Approach And 454 Pyrosequencing Technology

Nannochloropsis sp. has been widely accepted as one of the most promising oleaginous microalgae for biofuel production, which is known to grow rapidly, produce substantial amount of oil, and grow well in the outdoor systems for large-scale biofuel production. The prevalent interactions between microalgae and microbial community have been found not only in natue but also in the artifical system for large-scale cultivation. The composition and function of bacterial community could affect the growth of microalgae and the productivity in mass cultivation. In this study, we characterized the bacterial community associated with Nannochloropsis by both culture-dependent approach and culture-independent 454 pyrosequencing technology. This study should be the fundamental for the establishment of the coculture system of microalga and probiotic bacteria and the development of methods for rapid microbial monitoring in production settings. The major results include as below:1. Four bacterial strains identified as FG-2, FG-3, FG-4 and FG-5 were isolated from a laboratory culture of Nannochloropsis sp. OZ-1 for better understanding of the interactions between them. Phylogenetic analysis of 16S rDNA sequence revealed that strains FG-2, FG-3, FG-4 and FG-5 were close to Paracoccus sp. JAM-AL07, Planomicrobium sp. EP-20, Bacillus sp. YKJ-11, and Stappia stellulata, respectively. In addition to the growth promotion of Nannochloropsis sp. OZ-1 by FG-3, the growth of strains FG-3, FG-4, FG-5 was also significantly enhanced by co-culturing with extracellular organic matter from Nannochloropsis sp. OZ-1, indicating a symbiotic relationship between the FG-3 and Nannochloropsis sp. OZ-1. In addition, the first observation of symbiosis between Nannochloropsis sp. OZ-1 and bacteria on 2216 agar plate was reported here. 2. The computational pipeline for 454 reads analysis was customized in this tudy for alga-associated baterial community analysis. In this customized pipeline, we incorporated the functionality of one comprehensive software package, several powerful previous software tools and specific perl scripts for this study. In addition to improve the data quality, we eliminated all reads assigned to―Caynobacteria‖for further analysis.3. The alga-associated bacterial communities in lab culture were preliminarily characterized by 454 pyrosequencing approach. This deep-sequencing technique revealed tremendous diversity in the variable and complex microbiotas associated with microalgae. We found that there should be a vast number of bacterial groups in―axenic‖culture of Nannochloropsis after antibiotic treatments for purification. Moreover, the diversity and richness of bacterial taxa in Nannochloropsis culture at stationary phase were both much higher than those in parent cultures. These indicated that the interactive relationship could be potentially existed between Nannochloropsis and bacterial community.4. Here we also focus on the bacterial components of microalgal production systems and provide the metagenomic characterization of bacterial diversity in industrial cultures of an oleaginous alga, Nannochloropsis salina. While scaling up, we found a significant increase in the phylogentic diversity and richness of OTU number. The core microbiome were identified among the microbiomes in different production settings. The shared bacterial groups among samples could provide us a new insight into discovering the novel symbiotic relationship between algal host and bacteria.