Long-Term Low DO Enriches and Shifts Nitrifier Community in Activated Sludge

In the activated sludge process, reducing the operational dissolved oxygen (DO) concentration can improve oxygen transfer efficiency, thereby reducing energy use. The low DO, however, may result in incomplete nitrification. This research investigated the long-term effect of low DO on the nitrification performance of activated sludge. Results indicated that, for reactors with 10 and 40 day solids retention times (SRTs), complete nitrification was accomplished after a long-term operation with a DO of 0.37 and 0.16 mg/L, respectively. Under long-term low DO conditions, nitrite oxidizing bacteria (NOB) became a better oxygen competitor than ammonia oxidizing bacteria (AOB) and, as a result, no nitrite accumulated. Real-time PCR assays indicated that the long-term low DO enriched both AOB and NOB in activated sludge, increasing the sludge nitrification capacity and diminishing the adverse effect of low DO on the overall nitrification performance. The increase in the population size of nitrifiers was likely resulted from the reduced nitrifier endogenous decay rate by a low DO. Under long-term low DO conditions, <i>Nitrosomonas europaea/eutropha</i> remained as the dominant AOB, whereas the number of <i>Nitrospira</i>-like NOB became much greater than <i>Nitrobacter</i>-like NOB, especially for the 40 day SRT sludge. The enrichment and shift of the nitrifier community reduced the adverse effect of low DO on nitrification; therefore, low DO operation of a complete nitrification process is feasible

siRNA sequences used in this study.

<p>siRNA sequences used in this study.</p

Overexpression of Arf proteins could not rescue EV71 replication from BFA exposure.

<p>(A) All the Arf proteins tested are expressed at high levels in RD cells. (B) Overexpression of Arf proteins did not rescue viral replication under BFA exposure (<i>P</i>>0.05).</p

GBF1 interacts with viral 3A protein.

<p>(A) Immunoprecipitation was conducted with Protein G agarose plus anti-FLAG antibody. Western blots were probed with specific antibodies, as indicated. (B) Immunoprecipitation was conducted with Protein G agarose plus anti-GFP antibody. Western blots were probed with specific antibodies, as indicated.</p

Quantitative real-time PCR primers.

<p>Quantitative real-time PCR primers.</p

COPI depletion protects RD cells from EV71 infection.

<p>RD cells were transfected with αCOP and ζ1COP siRNA and infected with EV71. At 24 hpi, cells were fixed with 4% paraformaldehyde and permeabilized. EV71 infection was determined by immunofluorescence staining on VP1. RD cells were observed by DAPI.</p

GBF1 is required for EV71 replication.

<p>(A) Targeted siRNAs could effectively knockdown the expression of each GEF (<i>P</i><0.05). (B) Knockdown of GBF1, but not BIG1 or BIG2, inhibited EV71 replication in cells (<i>P</i><0.05). (C) GBF1-EGFP is overexpressed in RD cells by transfection. (D) Overexpression of GBF1 rescued viral replication from BFA exposure (<i>P</i><0.05).</p

COPI is required for EV71 production.

<p>(A) αCOP, (B) ζ1COP silencing with two individual siRNA duplexes blocked EV71 viral protein VP1 expression by Western blotting with the indicated antibodies (<i>P</i><0.05). Quantification analysis of EV71 VP1 synthesis was performed by Image J. Standard deviations of three independent experiments are shown. (C) αCOP and ζ1COP depletion by siRNA inhibited EV71 virions production (<i>P</i><0.05). Values were obtained from triplicate wells and are mean ± SD.</p

COPI but not COPII is essential for EV71 replication.

<p>COPI subunits (A) αCOP and (B) ζ1COP silencing with two individual siRNA duplexes blocked replication of EV71 (<i>P</i><0.05). COPII subunits (C) Sec13p and (D) Sec23p depletion by siRNA was dispensable to EV71 replication (<i>P</i>>0.05). Values were obtained from triplicate wells and are mean ± SD.</p

COPI is required downstream of viral entry.

<p>(<b>A</b>) Cells were pretreated with siRNA, infected at 4°C to allow surface binding, followed by 3 hr at 25°C to resume endocytosis. Viral entry was measured by determining viral genome copies, which was almost unchanged between COPI knocked down group and control group (<i>P</i>>0.05). (<b>B</b>) BFA and GCA inhibited EV71 replication (<i>P</i><0.05), when added up to 6 hpi. RD cells were infected with at 0.1 MOI and harvested at 24 hpi. The experiment was performed in triplicate, and the bars represent means ± SD.</p