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Chapter 9 Nitrification 9.0 Nitrification - The microbiological oxidation of NH4+-N to NO2--N and NO3--N. NH4+-N removal is a mandated process for some wastewaters because i) NH4+ consumes oxygen up to 4.57 g O2 / g NH4+-N. ii) NH4+ is toxic to aquatic macroorganisms In addition, wastewater treatment that involves denitrification of NO3- -N frequently requires nitrification to convert the input NH4+-N to NO3--N. 9. 0 Nitrification to remove NH4+-N from drinking-water supplies also is practiced i) to make the water biologically stable ii) to eliminate the free-chlorine demand that produces chloramines when free chlorine is desired. NH3 + HOCl NH2Cl (monochloroamine) + H2O 9.1 Biochemistry and Physiology of Nitrifying Bacteria The nitrifying bacteria are autotrophs, chemolithotrophs, and obligate aerobes. Energy source: light ---- phototroph Chemical ---- chemotroph chemolithotroph chemoorganotroph Carbon source: CO2 ---- autotroph organic carbon ----- heterotroph Photoautotroph: cyanobacteria, some purple and green bacteria Photoheterotroph: some purple and green bacteria autotrophs, Being autotrophs, the nitrifiers must fix and reduce inorganic carbon. Chemoheterotroph = chemoorganotroph: most bacteria, some archaea Chemoautotroph = chemolithotroph: a few bacteria and many archaea Methylotroph: 1 carbon compound as carbon source 9.1 Biochemistry and Physiology of Nitrifying Bacteria chemolithotrophs, Their chemolithotrophic nature makes fso and Y still smaller, because their nitrogen e-donors release less energy per electron equivalent than do organic e-donors, H2, or reduced sulfur. 9.1 Biochemistry and Physiology of Nitrifying Bacteria chemolithotrophs, Of course, the low Y value translate into a small ∧ maximum specific growth rate ( µ) and a large , Θx min . Therefore, nitrifiers are slow growers. ∧ ∧ µ = qY θ χmin K + S0 = 0 S (Yqˆ − b ) − bK [3.7] [3.26] 9.1 Biochemistry and Physiology of Nitrifying Bacteria obligate aerobes - They use O2 1) for respiration (energy), and 2) as a direct reactant for the initial monooxygenation of NH4+ to NH2OH (hydroxylamine) - The latter use of oxygen may be the reason why nitrifiers are relatively intolerant of low dissolved-oxygen concentrations; nitrifier catabolism is slowed by oxygen limitation at concentration that have no effect on many heterotrophs. 9.1 Biochemistry and Physiology of Nitrifying Bacteria - Nitrification is a two-step process. - In the first step, NH4+ is oxidized to NO2- according to the following energy-yielding rxn. (The most famous genus; Nitrosomonas) 1 1 1 1 1 + NH 4 + O2 = NO2− + H + + H 2O 6 4 6 3 6 [9.1] ∆G o ' = − 45.79 kJ per e − eq - the second stage of the nitrification reaction is the oxidation of NO2to NO3- :(The most famous genus; Nitrobacter, Nitrospira) 1 1 1 NO2− + O2 = NO3− 2 4 2 ∆G o ' = − 37.07 kj per e − eq [ 9.2] - Since nitrifiers exist in environment in which organic compounds are present, It might be curious that nitrifiers have not evolved to use organic molecules as their carbon source. ~ probably related to their evolutionary link to photosynthetic microorganisms. 9.1 Biochemistry and Physiology of Nitrifying Bacteria For ammonium oxidizers Heterotrophs 0.6-0.7 20 (mgBODL/mgVSSa-d) For nitrite oxidizers fso: very low for each group, compared to the typical fos value of 0.6-0.7 for aerobic heterotrophs Nitrifiers conserve very few electrons in biomass The low fso values translate directly to low Y values (Caution: Unit is different form each other !!). In units of g VSSa/ g OD, Y = 0.1 for ammonium oxidizer, Y =0.45 for heterotrophs 9.1 Biochemistry and Physiology of Nitrifying Bacteria For ammonium oxidizers Heterotrophs 9 d-1 For nitrite oxidizers Maximum specific growth rate of both organisms are low : less than 1 d –1 at 20 oC. The limiting value of [θ χmin ]lim is large. [θ χmin ]lim = 1 Yqˆ − b [3.27] The relatively high values of Ko quantify that nitrifiers are not tolerant of low DO concentrations 9.1 Biochemistry and Physiology of Nitrifying Bacteria For ammonium oxidizers Heterotrophs 0.11 d 0.17 For nitrite oxidizers mgBODL/L(complex substrates) [Θxmin] lim must be large, greater than 1 d 1 [θ χmin ]lim = [3.27] ˆ Yq − b Smin < 1 mg/L Effluent NH4+, NO2- can be very low levels Thus, as long as the SRT is maintained well above θ χ and sufficient min dissolved oxygen is present, the nitrification can be highly efficient Primary consumers of organic waste: heterotrophic bacteria (Generic parameters at 20 0C). Limiting substrate BODL Y (true yield for synthesis) 0.45 mg VSSa/mg BODL (substrate utilization) 20 mg BODL /mg VSSa- d (maximum specific growth rate) 9 d-1 K (conc. Giving one-half the maximum rate) simple substrate : 1 mg BODL /L complex substrate : > 10 mg BODL /L b (endogenous decay coefficient) 0.15 d q̂ µˆ = Yqˆ fd (biodegradable biomass fraction) θ xmin (value at which washout begins) Smin (the minimum substrate conc. capable of supporting steady-state biomass) 0.8 0.11 d safety factor 100 11 d safety factor 36 4 d simple substrate : 0.017 mg BODL /L complex substrate : > 0.17 mg BODL /L 9.1 Temperature effect - The temperature effecs are very important -Nitrification is sometimes considered impossible for low-water temperatures. -However, in fact, stable nitrification can be maintained at 5 oC or lower, as long as the SRT remains high enough For example, for 5 oC, a safety factor of only 5 requires that Θx min be 3.6 x 5 = 18 d (quite large). Thus SRT should be larger than 18 days. -One problem with low-temperature nitrification is that û becomes quite small, making recovery of nitrification after a wash out a very slow process. Thus, avoiding nitrifier washout due to excess sludge wasting, low DO, or inhibition must be an absolute priority, particularly for low temperatures. 9.1 Biochemistry and Physiology of Nitrifying Bacteria min The directly comparable parameters: fso, µ̂ , θ χ , Smin -Very similar for two types of nitrifiers. -This circumstance is completely logical, since both are aerobic chemolithoauthotrophs oxidizing N. -They almost always coexist in the same habits, experiencing the same SRT and oxygen concentrations. -Reflects their biochemical and ecological similarities 9.1 Biochemistry and Physiology of Nitrifying Bacteria Overall, balanced equation for the complete oxidation of NH4+ to NO3-N by nitrifiers having Θx = 15 d , f s = 0.067 NH 4+ + 1.815 O2 + 0.1304 CO2 = 0.0261C5 H 7O2 N + 0.973 NO3− + 0.921 H 2O + 1.973 H + [9.3] - The low net formation of nitrifier biomass : Ynet = 0.21 g VSSa/g N refer to next slide - Nitrification creates a major oxygen demand. it is 1.815 x 32 / 14 = 4.14 g O2 / g NH4+-N consumed - Produces almost two (1.973) strong-acid equivalents (1.973) per mole of NH4+ removed Thus the alkalinity consumption is 1.973 x 50/14 =7.05 g as CaCO3/g NH4+-N - The first step, ammonium oxidation is responsible for the acid production 2.5 Overall Reactions for Biological Growth 2) Obtain the overall reaction (R) including energy and synthesis using portions of electrons, fe (= 0.6) and fs (= 0.4) fe*Re: 0.02 C6H5COO- + 0.12 NO3- + 0.12 H+ -> 0.12 CO2 + 0.06 N2 + 0.002 HCO3+ 0.1 H2O fs*Rs: 0.0133 C6H5COO- + 0.02 NH4+ + 0.0067 HCO3- -> 0.02 C5H7O2N + 0.0067 H2O R : 0.0333 C6H5COO- + 0.12 NO3- + 0.02 NH4+ + 0.12 H+ -> 0.02 C5H7O2N + 0.12 CO2 + 0.06 N2 + 0.0133 HCO3- + 0.1067 H2O [2.34] The overall reaction for net synthesis of bacteria that are using benzoate as an e- donor, nitrate as an e- acceptor, and ammonium as nitrogen source. 9.1 Biochemistry and Physiology of Nitrifying Bacteria Nitrifiers produce SMPs which can be consumed by heterotrophic bacteria - - Most of the nitrifier-produced SMP are BAP (biomass-associated products) - SMPs are part of the decay process of the nitrifiers and reduce the net synthesis of the nitrifiers - A way in which nitrifiers create e-donors for heterotrophs and increase the heterotrophic biomass 9.1 Biochemistry and Physiology of Nitrifying Bacteria Chemical inhibition - Highly sensitive to chemical inhibition. - The very slow growth rate of nitrifiers magnifies the negative impacts of inhibition and, in part, makes it appear that nitrifiers are more sensitive than are faster growing bacteria. - Furthermore, some apparent inhibitors are e-donors whose oxidation depletes the DO and may cause oxygen limitation. - Most relevant inhibitors are: unionized NH3 (at higher pH), undissociated HNO2 (usually at low pH), anionic surfactants, heavy metals, chlorinated organic chemicals, and low pH 9.2 Common Process Considerations Even with the relatively long SRT of extended aeration, nitrifying process often have relatively small safety factors. - for economic reasons - [Θx min ]lim is 1-3 d, the reactor volume is too great when the safety factor is greater than about 10, - Because the Θx / Θ ratio cannot be increased indefinitely to compensate. - Of course, operating with a small safety factor increases the risk of washout due to solids loss or inhibition and increases the needs for operator attention. - Unfortunately, the risk is high, and instability in nitrification is a common problem in treatment operations 9.2 Common Process Considerations A successful nitrification process – suspended growth or biofilmmust account for the reality that - heterotrophic bacteria always are present - and competing with the nitrifiers for dissolved oxygen and space. two disadvantages - The nitrifiers’ relatively high Ko value puts them at a disadvantage in the competition for oxygen. - Their slow growth rate is a disadvantage when competing for any space that requires a high growth rate overcome by ensuring that - the nitrifiers have a long SRT, typically greater than 15 d, - larger value may be needed in the presence of toxic materials, a low D.O. concentration, or low temperature. 9.3 Activated Sludge Nitrification : One-sludge Versus Two-Sludge 9.3 Activated Sludge Nitrification : One-sludge Versus Two-Sludge • One-sludge nitrification - One-sludge nitrification is the process configuration in which heterophic and nitrifying bacteria coexist in a single mixed liquor that simultaneously oxidizes ammonium and organic BOD - Use the term one sludge to emphasize the ecological relationship between the nitrifiers and hetrotrophs 9.3 Activated Sludge Nitrification : One-sludge Versus Two-Sludge • Two-sludge nitrification - Two-sludge nitrification is an attempt to reduce the competition between heterotrophs and nitrifiers by oxidizing most of the organic BOD in a first stage - The first sludge is essentially free of nitrifiers, while the second sludge has a major fraction of nitrifiers • One-sludge nitrification can be carried out in sequencing batch reactors (SBR) - involve sequential periods of filling, aerobic reaction, settling, and effluent draw-off in one tank - In most ways, SBR nitrification resembles any other one-sludge system. 9.7 The ANAMMOX Process - Recently (1999), a novel bacterium in the planctomycetes group has been discovered for its ability to anaerobically oxidize NH4+-N to N2, not NO2-. - It is called the ANAMMOX microorganism, Anaerobic Ammonium Oxidation. - The discovery of ANAMMOX is one of the most startling ones in environmental biotechnology. Ammonium: electron donor Nitrite: electron acceptor NH 4+ + NO2− = N 2 + 2 H 2O Autotrophs: the reduction of inorganic carbon via oxidation of nitrite to nitrate, nitrite as the nitrogen source 5CO2 + 14 NO2− + 3H 2O + H + → C5 H 7 O2 N + 2 NO3− 9.7 The ANAMMOX Process The yield and specific growth rate are low. 0.14 g VSSa/g NH4+-N, 0.065/d This gives an overall stoichiometry of approximately: NH 4+ + 1.26 NO2− + 0.085CO2 + 0.02 H + → N 2 + 0.017C5 H 7 O2 N + 0.024 NO3− + 1.95 H 2O ANAMMOX’s favoring condition - exceptional biomass retention: a very long SRT - stable operation - the presence of nitrite - lack of oxygen - lack of donor: cause the reduction of nitrite via denitrification