| Nitrogen pollution is an increasingly serious problem around the world, thus it is very urgent to be treated. The traditional denitrification, which uses organics as electron donor, is efficient but high-cost. Autotrophic denitrification attracts more and more attentions as it is cost effective and the C/N ratio in wastewater is decreasing recently resulting from the organic pollution being controlled. Compared with the H2-dependent and S-dependent autotrophic denitrification, Fe-dependent autotrophic denitrification is safe, cheap and environment-friendly.Moreover, the product, namely ferric iron, could be used to precipitate or absorb phosphate or heavy metals in wastewaters. In the present study, aiming at a stably and highly efficient ferrous-dependent autotrophic denitrification technology, the reactor was operated and improved,the granular sludge was characterized and studied, the pure culture was achieved and identified,the microbial mechanism was explored and analyzed.1) Bioaugmentation by DeS (Denitrification Sludge) to FeNiR (Ferrous-dependent Nitrate Reduction) efficiency was determined, and DeS was proved to be a better seeding sludge for FeNiR.A FeNiR reactor was performed stably in lab with DiS (Digested Sludge) as seeding sludge.The VRRs of nitrate and ferrous iron were 0.08±0.004 kg-N/(m3·d) and 0.74±0.009 kg-Fe/(m3 d)respectively. by dosing DeS of 6% (V/V) into FeNiR reactor, the VRRs of nitrate and ferrous increased to 0.18±0.013 kg-N/(m3 d) and 1.62±0.062 kg-Fe/(m3 d),which were 2.4 times and 2.2 times of primaryone respectively. The bioaugmentation by DeS was proved to be reproducible. Tracing the change of DeS in FeNiR reactor by detecting the live bacteria percentage and alkaline phosphatase (ALP) activity. Combining with the performance of FeNiR reactor and the specific activity of DeS, it was found that the added DeS, which only occupied 6% in the whole sludge volume of reactor, contributed 82.3% of the total nitrogen removal and 84.7% of the total iron removal.This indicates that bioaugmentation by adding DeS was attributed to the high FeNiR activity of DeS. Batch tests were conducted to compare the FeNiR activity of both sludges (DiS and DeS). The specific FeNiR activity of DeS was 11.36 times higher than that of DiS. The predominant bacteria of DeS were Thauera, Hyphomicrobium and Azoarcus. DeS could be used as a better seeding sludge for FeNiR.2) FeNiR-Pro (Ferrous-dependent Nitrate Reduction by "Professional Sludgef")technology was developed, and its N & Fe-removal efficiencies along with the sludge characteristics were studied.With DeS as seeding sludge, FeNiR-Pro technology was developed and FeNiR-Pro reactor was operated. Influent nitrate and ferrous iron concentrations were set at 130 mg-N/L and 2600 mg-Fe/L. With HRT at 4.4 h and VSS content at 0.11 g/g sludge, the removal efficiency of nitrogen and ferrous iron was 98.00% and 80.00%, and the VRRs were 0.70 kg-N/(m3·d) and 9.29 kg-Fe/(m3 d) respectively. FeNiR-Pro reactor possessed a high efficiency. Under its best operation condition,the live bacteria percentage in FeNiR-Pro reactor maintained above 60%,the ALP activity maintained above 0.45 mmol/(g VSS min),the specific nitrate reduction rate was above 0.2 mg-N/(g VSS h) and the specific ferrous iron oxidation rate was above 2.2 mg-Fe/(g VSS h). The high specific activity of FeNiR-Pro sludge ensured the high effieicncy of FeNiR-Pro reactor. Ferric iron, the FeNiR product, was precipitated on the cell surface, which formed the iron encrustation. The iron encrustation impeded substrate transportation and thus resulted in the activity decrease and the biomass loss. The specific activity of FeNiR-Pro sludge recovered after dissolved by chaotropic agents, indicating that the iron encrustation was the main reason to limit the sludge activity. How to remove the iron encrustation or to prevent the formation of iron encrustation is the key for developing FeNiR-Pro technology, and how to maintain the sludge activity in reactor is the direction for improving FeNiR-Pro technology.3) A novel FeNiR-Pro-Plus (the advanced Ferrous-dependent Nitrate Reduction by"Professional Sludge")technology was developed, the operation parameters of FeNiR-Pro-Plus reactor were determined and the working mechanism of FeNiR-Pro-Plus technology was revealed.Sequencing sludge-adding technology was used to operate a novel FeNiR-Pro-Plus reactor.Influent nitrate and ferrous iron concentrations were set at 128.96±12.65 mg-N/L and 1211.99±126.96 mg-Fe/L. With HRT at 4.5 h and VSS content at 0.12 g/g sludge, the removal efficiency of nitrogen and ferrous iron was 70.00% and 88.22%, the VRRs were 0.70 kg-N/(m3·d) and 8.69 kg-Fe/(m3·d) with relative standard deviations at 5.7% (N) and 8.1% (Fe).FeNiR-Pro-Plus technology possessed high and stable efficiency. Sequencing sludge-adding operation was the key to FeNiR-Pro-Plus technology. The volumetric ratio of added sludge to the whole sludge in reactor was 10.6% (V/V). The period of sludge addition was 4 days. The iron encrustation was the limit factor of sludge activity. The iron encrustation of granular sludge became thicker and thicker along with the operation. By replacing "old sludge" with "fresh sludge", the activity decrease and the biomass lose in reactor were compensated and thus the reactor efficiency maintained at a high level.4) The pure FeNiR culture was acchieved and identified, its morphology,physio-biochemical characteristics and specific activity of FeNiR strain were studied, and the FeNiR mechanism was revealed and the model of FeNiR reaction was estiblished.A FeNiR strain was isolated from the FeNiR reactor and was named as strain FI. Strain F1 was in rod shape and Gram negtive with a size of (0.3～0.5) μm ×(0.8～1.0) μm. It was classified as Shewanella sp. basing on bio-chemical characteristics and 16S rRNA sequencing analysis.Strain F1 was demonstrated to have the high FeNiR activity with the maximum nitrate reduction rate at 3.24 mg-N/(L·h) and the maximum ferrous iron oxidation rate at 94.68 mg-Fe/(L·h). The optimal temperature for nitrate reduction and ferrous iron oxidation was 35.54 ℃ and 36.66 ℃respectively, while the optimal pH for nitrate reduction and ferrous iron oxidation was 7.50 and 6.35. By the results of morphology and activity measurement, the FeNiR mechanisms were supposed to be (1) iron oxidation happened extracellularly without iron taking-in which resulted in humps on the cell surface; (2) iron oxidation happened intracellularly with iron taking-in which resulted in an iron shell in the periplasmic space. |
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