Exploring The Enhancement Of Wastewater Treatment Using A Culture Supernatant From Micrococcus Luteus Containing Resuscitation Promoting Factor(SRpf)in SBR Process

In recent years, the quantity of wastewater has been increasing with the rapid development of social economy. Since most waste water treatment plants (WWTPs) are facing increasingly stringent effluent limitations, removing more and more complex pollutants from wastewater is becoming a acute problem. Therefore, there is an urgent need for technique solutions to enhance nutrients removal. Conventional activated sludge systems are widespread in Chinese WWTPs, which will face enormous challenges for modifying treatment systems due to the limitation of land and capital, creating an acute demand for more cost-efficient and less land-occupied technology. Activated sludge is the essential functional element of biological wastewater treatment. It is well known that prokaryotic microorganisms in activated sludge catalyze the most important transformations in WWTPs, and the efficient and stable operation of activated sludge process relies upon bacterial communities. Thus, improving the biological activity of functional bacterial communities in activated sludge could be a promising way to enhance wastewater treatment.In this study, we used resuscitation-promoting factor (Rpf) secreted by Micrococcus luteus to explore the potential environmental functions of viable but none-culturable (VBNC) or uncultured bacteria in activated sludge. We used SRpf, known as the culture supernatant from Micrococcus luteus containing Rpf. The wastewater treatment efficiencies of three different sequencing batch reactors (SBRs) (control reactor R1 with inactivated SRpf addition, treatment reactor R2 with SRpf addition and blank reactor R3 without SRpf) were compared and the bacteria community compositions in these sludge samples were assessed by high throughput sequencing. Main results of this study are as follows.(1) SRpf accelerated the start-up process and significantly enhanced the biological nutrients removal in SBRs. PO43--P removal efficiency increased by over 12% and total nitrogen removal efficiency increased by over 8% in treatment reactor acclimated by SRpf compared with those without SRpf addition. It was indicated that the promoting effects could last for several days even without SRpf constantly addition, and it could quickly regain previous high level of nutrients removal after restarting SRpf acclimation. Therefore, SRpf might be regarded as a promising way to not only enhance removal capability, but also improve the activity of activated sludge from deterioration. SRpf acclimation also enhanced the phosphorus release and uptake, as well as enhanced the nitrification process and denitrification process. Results of SOUR experiment suggests that SRpf successfully enhanced the strength of microbial metabolism on substrate utilization in activated sludge, which to some extent, explained the promotion of the better efficiency on COD and nutrients removal in R2.(2) The high-throughput sequencing analysis showed that SRpf played an essential role in shifts in the composition and diversity of bacterial community. The phyla of Proteobacteria and Actinobacteria, which were closely related to biological nutrients removal, were greatly abundant after SRpf addition. Specifically, SRpf promoted Nitrosomonas and Nitrospira for nitrification. SRpf promoted Dechloromonas and Pseudomonas for denitrification. SRpf also promoted Acinetobacter, Pseudomonas and Rhodocyclus for phosphorus removal, some of which were reported as the VBNC state bacteria. It demonstrates that SRpf acclimation significantly promoted functional bacteria in activated sludge for nutrients removal.(3) SBR with SRpf acclimation presented significantly better ability for phenol and nutrients removal under phenol stress in the long run. Phenol inhibition has taken place in the biological process of phosphorus release and uptake, as well as the nitrification process and denitrification process. NO2--N accumulation occurred in typical cycle because the inhibition effect towards Nitrite Oxidizing Bacteria (NOB) was stronger than that towards Ammonia Oxidizing Bacteria (AOB). SRpf acclimation could significantly reduce the inhibition effect of phenol, so that R2 showed best performance of nutrients and phenol treating phenol containing wastewater.(4) The Illumina high-throughput sequencing analysis showed that SRpf played an essential role in boosting bacteria diversity in activated sludge, which led to better system stability under phenol stress. The phyla of Proteobacteria and Actinobacteria, which were closely related to biological nutrients removal, were greatly abundant after SRpf addition. Specifically, SRpf promoted Nitrosomonas and Nitrospira for nitrification. SRpf promoted family Comamonadaceae, genus Dechloromonas and Pseudomonas for denitrification. SRpf also promoted Acinetobacter, Pseudomonas and Rhodocyclus for phosphorus removal. Moreover, the abundance of Bacillus and Klebsiella were observed to be significantly increased after SRpf acclimation. They were responsible for phenol removal as well as Dechloromonas and Pseudomonas. It explained why SBR with SRpf acclimatin performed significantly better in nutrients and phenol removal.