High levels of nitrifying bacteria in intermittently

aerated reactors treating high ammonia wastewater

Xian Gu12/17/2010

Introduction

• The most common method of nitrogen removal from wastewater combines nitrification and anoxic denitrification.

• Nitrifying systems have traditionally been monitored using chemical parameters rather than microbiological data.

• The monitoring of key microorganisms by molecular methods might permit better prediction and control of process performance and effciency.

• In this study, we assessed a limited set of ANA （ aeration to non-aeration ） time ratios at different influent nitrogen loads and determined their effects on nitrogen removal performance and nitrifier populations.

• The fractions of nitrifying bacteria (AOB ：the aerobic oxidation of ammonia to nitrite by ammonia-oxidizing bacteria ， and NOB ： oxidation of nitrite to nitrate by nitrite-oxidizing bacteria) were monitored at the genus level using slot-blot hybridization and FISH with 16S rRNA-targeted probes, and the results were compared with values from mathematical modeling.

Materials and methods

• Reactor design and operation

• Stage I: relatively high ammonia concentrations (average of 115 mg/L) with ANA time ratio of 1:1.

• Stage II: higher ammonia concentrations (average of 146 mg/L).

• Stage IIa: biomass subjected to long (overnight) non-aerated periods;

• StageIIb: biomass subjected to ANA of 1:1.• Stage III: relatively high ammonia concentrations

(average of 115 mg/L) and biomass subjected to ANA of 1:2.

• Stage IV: higher ammonia concentrations (average of 146 mg/L) and biomass subjected to ANA of 1:3.

• Analytical methods TKN, NH3-N, NO3-N, NO2-N, soluble COD,TOC,pH, TSS,VSS, DO

• Bacterial cultures Nitrosomonas europaea (ATCC25978, ATCC medium 2265), Nitrosospira

multiformis (ATCC 25196, ATCC medium 929), and Nitrobacter agilis (ATCC 25384, ATCC medium 480)

• Nucleic acids extraction• PCR, cloning, and in vitro transcription 16 S rRNA ,

• Oligonucleotide probes targeting the 16S rRNA of AOB and NOB. FISH

• Mathematical modeling Modeling analysis was performed as described by Rittmann et al.

with the objective of estimating the theoretical biomass fractions of nitrifiers at each stage and comparing the results with the measured data.

θ= hydraulic retention time (days)Yhet = true yield coeffcient for heterotrophs (0.45 kg VSS/kg BODL )bhet = endogenous decay coeffcient for heterotrophs (0.1 day-1), ΔBODL = removal of BODL across the system (mg BODL/L), Ynit = true yield for all nitrifiers (0.45 kg VSS/kg N), bnit = endogenous decay coeffcient for nitrifiers (0.15 day-1), ΔTKN = TKN nitrified(mg N/L), fd = fraction of newly synthesized biomass that is degradable by endogenous decay (0.8).

Xv ： mixed liquor volatile suspended solids

(Xao)a/Xa ： The ratio of active ammonia oxidizers to active biomass

Yao = 0.34 kg VSS/kg NΔBOD = the influent soluble COD ΔTKN = the difference between influent and effuent TKN values less the ammonia assimilated into aerobic heterotrophs and not available for nitrification. The typical value of 0.124 kg N/kg VSS was assumed for the nitrogen content of heterotrophic biomass.

Results• Performance of reactors

• Fraction of nitrifiers in the biomass

(a) Average fractions of total betaproteobacterial AOB, Nitrosomonas and Nitrosospira;

(b) Average fractions of Nitrospira and Nitrobacter for reactor A (stages I and II).

correspond to FISH data for the same day (expressed as % DAPI area), with respective error bar.

correspond to average theoretical AOB biomass fractions.

(a) Average fractions of total betaproteobacterial AOB,Nitrosomonas and Nitrosospira;

(b) Average fractions of Nitrospiraand Nitrobacter for reactor B (stages III and IV).

correspond to FISH data for the same day (expressed as % DAPI area), with respective error bar.

correspond to average theoretical AOB biomass fractions.

Discussion

• Novel AOB members might be present in the reactors.

• The low levels of AOB measured, as compared to NOB levels, could indicate the presence of novel AOB in the reactors that were undetected by the probes used.

• Factors selecting for Nitrospira or Nitrobacter remain unknown and deserve further investigation.

• It has not been determined whether these relatively low levels of nitrifiers can sustain the same performance for extended periods.

Thank you!

• FISH (fluorescence in situ hybridization) is a cytogenetic technique developed by Christoph Lengauer that is used to detect and localize the presence or absence of specific DNA sequences on chromosomes. FISH uses fluorescent probes that bind to only those parts of the chromosome with which they show a high degree of sequence similarity. Fluorescence microscopy can be used to find out where the fluorescent probe bound to the chromosomes. FISH is often used for finding specific features in DNA for use in genetic counselling, medicine, and species identification. FISH can also be used to detect and localize specific mRNAs within tissue samples. In this context, it can help define the spatial-temporal patterns of gene expression within cells and tissues. 幻灯片 3

• 16S ribosomal RNA (or 16S rRNA) is a component of the 30S subunit of prokaryotic ribosomes. It is 1,542 nts in length. Multiple sequences of 16S rRNA can exist within a single bacterium.

• Functions 1.Like the large (23S) ribosomal RNA, it has a structural

role, acting as a scaffold defining the positions of the ribosomal protein

2.The 3' end contains the anti-Shine-Dalgarno sequence, which binds upstream to the AUG start codon on the mRNA

3.Interacts with 23S, aiding in the binding of the two ribosomal subunits (50S+30S)

4.Stabilizes correct codon-anticodon pairing in the A site, via a hydrogen bond formation between the N1 atom of Adenine (see image of Purine chemical structure) residues 1492 and 1493 and the 2'OH group of the mRNA backbone 幻灯片 3