Abstract: High-throughput sequencing technology was used to study the changes in the diversity of culturable bacteria in solid and liquid beef extract peptone soil during serial enrichment, and to quantify the proportion of culturable bacteria in the background microbial community of a paddy soil. Serial passages were performed by setting up treatments of conventional nutrient and 1/10 low nutrient treatments, including conventional beef extract peptone solid (NA) and liquid (NB) cultures, as well as low nutrient solid (1/10 NA) and low nutrient liquid (1/10 NB) cultures. The cultures were enriched 10 times to obtain the 1^st, 3^rd, 5^th, 7^th, and 10^th generation of bacterial cultures and then to extract their DNAs. At the same time, all microbial genomic DNAs in the paddy soil were directly extracted and high-throughput sequencing 16S rRNA gene was used to analyze the change of culturable microorganisms communities in the paddy soil and their proportions to the background indigenous microorganisms. The results show that the background Chao index of the microbial diversity of the paddy soil was 4 806, with a maximum reduction of 98.9% in 10 consecutive generations. Among them, the Chao index of solid and liquid culturable microorganisms was the lowest in the first generation, which were 49.7 and 142, respectively. Under 1/10 solid and 1/10 liquid cultures, the Chao index was 75.1 and 531, respectively. High-throughput sequencing of 16S rRNA genes revealed a total of 713 genera of background indigenous microorganisms in paddy soil. During the enrichment culture process of 10 consecutive passages, 52 and 600 genera were detected in solid and liquid conventional culture, respectively. However, 62 and 597 genera were detected in 1/10 solid and 1/10 liquid cultures, respectively. The highest proportion of culturable bacteria was 8.7% and 83.7%, respectively. During the 1^st, 3^rd, 5^th, 7^th and 10^th consecutive subcultures, 7, 2, 3, 4 and 3 genera were unique to each passage in solid medium, while the unique genera in liquid culture medium were 5, 1, 102, 44, and 24 genera, respectively, and similar results were observed in low-nutrient (1/10) cultures. Pseudomonas was an absolute dominant group in paddy soil during 10 consecutive passages, especially in solid medium, the relative abundance range was 97.70%-99.47%, which is 74 times higher than that of the background in the paddy soil. Under the low-nutrient 1/10 solids, Bacillus and Delftia became the dominant taxa with the increase of passage times, and the highest relative abundance in the 10^th generation was 18.07% and 13.82%, respectively. During the 10 consecutive liquid passages, Pseudomonas was also an absolutely dominant group, but compared with solid culturable bacteria, it decreased slightly to 45.48%-55.99%, while Clostridium was the second most dominant group, with a relative abundance ranged from 23.58% to 42.40%, while the genus Lysinibacillus only observed in the 5^th, 7^th and 10^th generations, and its abundance ranged from 1.46% to 6.74%. On the contrary, under the condition of low nutrient 1/10 NB liquid, Clostridium and Lysinibacillus decreased sharply with the increase of culture generation, Pseudomonas became the absolute dominant bacteria, while Paenibacillus increased with increasing passage number. These results show that the background microbial diversity of paddy soil can be up to 96.7 times of culturable bacteria. During the continuous enrichment culture, the Chao index of culturable microorganisms in paddy soil first increased and then decreased, and 74 and 662 genera were obtained by solid and liquid cultures, respectively. The proportion of solid and liquid cultured bacteria in all microorganisms in paddy soil was 10.4% and 92.8%, respectively. In the process of solid and liquid subculture, Pseudomonas was the dominant group, and the diversity of culturable bacteria in liquid was significantly higher than that in solid culture. Under low-nutrient 1/10 solid and 1/10 liquid culture conditions, the culturable microbial groups increased significantly, and some of the microbial groups that appeared in the first generation were gradually replaced by some dominant bacteria, indicating that in the process of continuous enrichment culture, the average number of bacteria in different generations increased. The groups with obvious differences in physiological and metabolic characteristics were enriched, and serial enrichment did not lead to homogenization and simplification of the population structure.

Key words: culturable method of microorganism, continuous passage, solid liquid medium, nitrogen nutrition, high-throughput sequencing