Nitrification Inhibition to Nitrifying Bacteria and Activated Sludge by Silver Nanoparticles.docx

1、Nitrification Inhibition to Nitrifying Bacteria and Activated Sludge by Silver NanoparticlesNitrification Inhibition to Nitrifying Bacteria and Activated Sludge by Silver NanoparticlesKey words: nitrification inhibition; silver nanoparticles; Nitrosomonas; Nitrobacter; SBR; activated sludgeABSTRACTT

2、his study focused on the nitrification inhibition on nitrifiers and activated sludge treatment process by silver nanoparticles (AgNPs). AgNPs with the diameters of 9, 13, 15 and 23 nm and concentrations range from 0.12 to 4.82 mg/L were used to test the inhibition effect of Nitrosomonas and Nitrobac

3、ter. It was found that the NH4+-N removal efficiency of Nitrosomonas was decreased by nearly 70% by the addition of nanoparticles with size of 23 nm when the silver concentration reached 4.82 mg/L, while NO2-N removal efficiency was merely affected. Comparison showed that particles with the size of

4、9 nm had the most severe toxicity to Nitrosomonas. Nitrification inhibition by silver nanoparticles on activated sludge process was studied with bench scale sequencing batch reactor (SBR) with four different sizes and four different concentrations of AgNPs. The inhibition effect was 30% of reduction

5、 of nitrogen removal was monitored in SBR process. INTRODUCTIONWith the increasing utility of nanotechnology and nano-materials, the environmental effects from nanoparticles have attracted more and more attention. With remarkably different physiochemical characteristics such as increased optical, el

6、ectromagnetic and catalytic properties from the bulk counterpart, silver nanoparticle, the nano-size particle of silver element, becomes one of the most commonly used material in consumers products (Wenseleers, et al, 2002; Kelly, et al, 2003). As of 2007, the Project on Emerging Nanotechnologies at

7、 the Woodrow Wilson International Center for Scholars had compiled a list of more than 500 consumer products that claim to include some form of engineered nanoparticle (Woodrow Wilson International Center for Scholars, 2007). Of these products, about 20% contain silver nanoparticles. Socks, paint, b

8、andage, food container, washing machine, feeding bottle, cosmetic, and soap incorporate silver nanoparticles to exploit its antimicrobial properties.Like as many other nanoparticles, silver nanoparticles would also enter wastewater treatment plants (WWTPs) (Mueller and Nowack, 2008). It was reported

9、 that socks containing silver nanopartilces would likely to release silver after several times washing (Benn and Westerhoff, 2008), and as much as 1.3 mg Ag/L (650 g silver in 500 mL distilled water) would be released in ideal condition. Silver nanoparticles disposed in the sewer pipe may aggregate

10、depending on pH, redox potential, and ionic strength in tap water (Yakutik and Shevchenko, 2004; Zhang, et al, 2008). It was reported that these particles would likely be oxidized to silver ions which were capability to complex with anions in water easily (Lok, et al, 2007). There were considerable

11、amount of silver nanoparticles entering WWTPs last few years and the amount of silver is increasing gradually, however, not much about their adverse effect to wastewater treatment was known. As it was known, nitrification was a key process in biological nitrogen removal. Because of its sensitivity t

12、o external conditions such as pH, dissolved oxygen concentration, and toxic chemicals (Blum and Speece, 1991; Hu, et al, 2003), nitrification was known as the controlling step in wastewater treatment process. It was reported that nitrification process, especially the ammonia oxidation step, was sens

13、itive to silver nanoparticles (Choi and Hu1, 2008; Choi, et al, 2008; Choi and Hu2, 2008; Choi and Hu, 2009). In synthesis wastewater, nitrification would be inhibited by nearly 90% when silver concentration achieved 1 mg/L in batch extant respirometric assay. Therefore, nitrification process and ni

14、trifying bacteria were chosen to evaluate the toxicity of silver nanoparticles in this research. This study focused on the inhibition effect by silver nanoparticles to nitrifying bacteria, both to the Nitrosomonas and Nitrobacter, and activated sludge in municipal wastewater treatment.MATERIALS AND

15、METHODSNitrifying BacteriaNitrifying bacteria used in this research were isolated from the activated sludge from local wastewater treatment plant. Activated sludge from WWTP was put into cultures of Nitrosomonas (ammonia-nitrogen oxidation bacteria, short for AOB) and Nitrobacter (nitrite-nitrogen o

16、xidation bacteria, short for NOB), respectively. The compositions of both cultures are shown as Table 2.1.Table 2.1 Composition of Two CulturesAOB CultureNOB CultureCompoundWeight (g)CompoundWeight (g)(NH4)2SO42.0NaNO21.0NaH2PO40.25Na2CO31.0MnSO44H2O0.01NaH2PO40.25K2HPO40.75CaCO31.0MgSO47H2O0.03K2HP

17、O40.75CaCO35.0MnSO40.01MgSO44H2O0.03Diluted to 1 000 mL with distilling water, adjusted pH to 7.2 with 1.0 M HCl or NaOH, then sterilized under 121C for 30 min.Cultures had been sterilized under 121 C, 0.1 MPa before use. All cultures were then placed in shaking table (180 rpm) under 35 C. The bacte

18、ria solution were tested by Griess agent and diphenylamine everyday, respectively. After the AOB cultures appeared red after the addition of Griess agent, and the NOB cultures blue after the addition of concentrated sulfuric acid and diphenylamine, Nitrosomonas and Nitrobacter were enriched. After e

19、nrichment, all bacteria solutions were then kept under 4 C.Activated Sludge and Bench ReactorActivated sludge was from the local WWTP and then cultivated by municipal wastewater from local wastewater system in a reactor with the total volume of 20 L. Characteristics of wastewater were shown as Table

20、 2.2.Table 2.2 Characteristics of Local Municipal WastewaterInfluentmg/LCODTotal NitrogenAmmonia NitrogenTotal Phosphorus1802253240212835The bench scale reactor was divided into 5 zones and each zone had the volume of 4 L, respectively. Aliquots of dried sludge were added into 5 zones and then culti

21、vated by 4 L municipal wastewater (Figure 2.1). The reactor was operated as the style of sequence batch reactor (SBR), with solid retention time (SRT) of 10 d and hydraulic retention time (HRT) of 8 h, contained 0.5 hour filling time, 6 hours of reaction time, 1 hour of settling time, 0.5 hour draw

22、and idle time. The quality of influent and effluent was tested every 2 days, including total nitrogen, ammonia nitrogen, COD and MLSS. MLSS of each zone was controlled to be around 2,500 mg/L by discharging exceeding sludge. Activated sludge was cultivated to achieve steady state with stable ammonia

23、 nitrogen removal rate and steady MLSS value.SRT = 10 d, HRT = 8 hInfluent (20 L totally and 4 L each)Figure 2.1 Schematic of the Bench ReactorSilver NanoparticlesAg nanoparticles were prepared by the sodium borohydride (NaBH4) reduction process (Choi and Hu1, 2008), in which silver nitrate was redu

24、ced with sodium borohydride and adding polyvinyl alcohol (PVA) as the capping agent to control the growth of nanocrystals and agglomeration of nanoparticles. To dissolve PVA, a solution containing 0.06% (wt) PVA was heated to 100 C, then 1 mL silver nitrate with the concentration of 14 mM was added

25、into 20 mL PVA solution and then the mixture was boiled. Silver particles were prepared by rapidly injecting 0.1 mL of 10 mM NaBH4 into the boiled PVA solution. With the addition of NaBH4, the solution became yellow immediately, indicating the production of silver nanoparticles. After 5 min of stirr

26、ing, the reaction mixture was stored at 4 C before use.It was reported that adding different concentration of sodium borohydride, made the molar ratios of BH4-/Ag+ become 0.1, 0.2, 0.38, 0.6, 1.2 in the mixture, could synthesize silver nanoparticles with the average sizes from 9 to 21 nm (Choi and H

27、u2, 2008). In this study, 4 BH4-/Ag+ ratios were adapted, including 0.1, 0.3, 0.7, 1.0, to synthesize suspension with different concentrations and different sizes particles. Inhibition ExperimentsInhibition Experiment to NitrifierIt was reported that the inhibition effect of Nitrosomonas and Nitroba

28、cter by toxic substances can be observed in pure cultures and the experiment can be carried in shaking table using tubes (Svenson, et al, 1999; Grunditzm and Dalhammar, 2001). In this study, the enriched Nitrosomonas and Nitrobacter were exposed to silver nanoparticles in wastewater treatment and th

29、e experiments were carried out in mixed cultures containing municipal wastewater and nitrifier solution. All tubes (after sterilizing) were divided into two groups, one for Nitrosomonas and the other for Nitrobacter. Then, aliquots of 10 mL municipal wastewater were added into each group of tubes, r

30、espectively. In this research, a gradient of 4 concentrations was chosen, and so was a gradient of 4 different sizes. Every concentration or size had 3 parallel in this experiment.Aliquots of 1 mL Nitrosomonas and Nitrobacter solutions were inoculated into two groups of tubes, respectively. In each

31、group, three tubes of mixed liquid were set aside to test the initial NH4+-N value of the mixture with bacteria solution and municipal wastewater. After dosing, both groups of tubes were packed and placed into shaking table, the mixtures would have reaction under 351 C at an oscillation speed of 200

32、 rpm in shaking table. Nitrosomonas and Nitrobacter would have a reaction time of 48 hours and 120 hours, respectively. As the group of tubes for Nitrosomonas was stopped reaction after 48 hours in mixture, while the one for Nitrobacter was stopped after 120 hours, blank samples of both groups should be tested to get the initial NH4+-N values and NO2-N values for Nitrosomonas and Nitrobacter. After sampling, both of these two groups of tubes were then divided into 8 groups (A11A14, A21A24, A31A34, A41