Objectives We investigated the in the physical, chemical and biological properties of the topsoil with different biogas slurry application years.

Methods The field experiment of replacing chemical fertilizer with biogas slurry was conducted in Huzhou, Zhejiang Province. The control plots were applied with only chemical fertilizer, while the treatment plots were applied with only biogas slurry for 1yr, 2 yr and 3yr. All the four treatments were applied with the same amount of N each year. Biogas slurry was applied two times during each rice season, in June and September of each year, and each application amount was about 300 t/hm². Plant and soil samples were collected after rice harvest every year. Rice yields and relevant physicochemical properties were investigated, and resident microbial community was analyzed through Miseq high-throughput sequencing.

Results The rice yields of 1, 2 and 3 years of biogas slurry application were 9670 kg/hm², 9765 kg/hm² and 9823 kg/hm². The rice yields of the three application treatments were similar, but all significantly higher than the chemical fertilizer control (7800 kg/hm²). Besides, the biogas slurry application increased the content of protein and NPK, not affected the amylose content in grains. Compared with CK, biogas slurry application improved soil chemical properties significantly. 2yr treatment was recorded lower pH but 3yr was recorded similar pH with CK. All the 1yr, 2yr and 3yr treatment were recorded similar but higher available P, total N and organic matter content. The NH₄⁺-N and NO₃^--N content were significantly decreased in 2yr and 3yr treatment than in 1yr treatment. Available K and the activities of C-, N- and P-acquiring enzymes kept increasing with the continue application of biogas slurry. The results of soil microbial community analysis showed that the longer the biogas slurry was applied, the higher the microbial markers abundance (most of which were nitrogen cycle-related microorganisms) would be. In addition, short-term (1 year or 2 years in this study) biogas slurry application increased microbial community abundance and phylogenetic diversity, while α diversity recovered back to the initial status (P>0.05) after long-term biogas slurry application. Besides, β diversity of microbial community also showed a significant recovering tendency.

Conclusions Biogas slurry application could effectively increase the yield and nutrition quality of rice, without decreasing the taste of rice. Biogas slurry application could improve and maintain a high and stable soil organic matter, total N and available P, accumulate available K, but decrease inorganic N contents. Short-term (1 year or 2 years in this study) application of biogas slurry will increase the soil microbial diversity, however, the soil microbial diversity may decrease gradually with the increasing application period.