discussion of 3 journal papers on nitrification, microbiological ammonia oxidation

1. Role of Bacteria in Nitrogen
Biogeochemical Cycling
Carlos Loyola | May 2nd, 2013
UCR, Environmental Science 301

2. Outline of this talk
 A quick note on the Nitrogen biogeochemical cycle
 Hagopian & Riley 1998. A closer look at the bacteriology
of nitrification
 Ruiz et al. 2003. Nitrification with high nitrite
accumulation for the treatment of wastewater with high
ammonia concentration.
 Fdz-Polanco et al. 2000. Spatial distribution of
heterotrophs and nitrifiers in a submerged biofilter
for nitrification

3. Biogeochemical Cycle: The cycling of a chemical element or
compound along the biosphere and geosphere:
http://www.h2ou.com/h2nitrogencycle.htm

4. http://nett21.gec.jp/gesap/themes/themes4_8_2.html
The Nitrogen Cycle

5. Hagopian & Riley 1998. A closer look at the
bacteriology of nitrification
 Review from an aquaculture perspective
Sorry, this paper has
too much text!

6. Hagopian & Riley 1998. A closer look at the
bacteriology of nitrification
 Nitrogen: Proteins, nucleic acids, adenosine
phosphates, nucleotides, pigments
 Fish N waste: urine, feces. (In aquaculture, uneaten
feed also contributes to nitrogenous waste loadin.)
 Ammonia and ammonium: 60–90% of the total N
excreted
 Urea: 9–27% of the soluble N excreted

7. A closer look at the bacteriology of nitrification
 If food/fecal matter accumulate in water, organic
compounds are proteolyzed and deaminated to
ammonia.
 A process called ammonification.
 Further mineralization takes place by nitrification.
 Both unionized ammonia and nitrite (NO2−) are toxic
to fish at low concentrations.

8.  Acute NH3 toxicity in salmonids: 0.2 mg l−1
 Recommended NH3 maximum permissible level:
0.002 mg l−1 (Haywood, 1983)
 Nitrite toxicity varies greatly between species and life
stages, a concentration as low as 1.8 mg l−1 NO2− has
been documented to be lethal to rainbow trout
(Oncorhynchus mykiss) within 24 h
 The lethal concentration of nitrate is 6200 mg l−1 for
channel catfish (Ictalurus punctatus)
A closer look at the bacteriology of nitrification

9.  Nitrification is performed by two phylogenetically
distinct groups of bacteria:
 Ammonia-oxidizing bacteria (or nitrite bacteria)
obtain energy by catabolizing un-ionized ammonia
to nitrite
 NH3+ 1.5O2  NO2
− + H2O + H+ + 84 kcal mol−1
 nitrite-oxidizing bacteria (or nitrate bacteria)
mineralize nitrite to nitrate
 NO2
− + 0.5O2  NO3
− + 17.8 kcal mol−1
A closer look at the bacteriology of nitrification

10.  Nitrobacteraceae: Gram-negative chemoautotrophs,
or lithotrophs.
 Nitrite bacteria: Nitrosomonas, Nitrosococcus,
Nitrosospira, Nitrosolobus, and Nitrosovibrio.
 Nitrate bacteria: Nitrobacter, Nitrococcus,
Nitrospira, and Nitrospina
 Chemoautotrophic bacteria are characterized by the
ability to utilize an inorganic chemical substrate (e.g.
NH3, H2, Fe2+) as a source of electrons for the
immobilization of inorganic carbon (i.e. CO2 (aq) or
HCO3−) into biomass
A closer look at the bacteriology of nitrification

11.  This sole energy source drives carbon fixation, the
assimilation of monomers into precursor
metabolites, and the subsequent polymerization of
building blocks and macromolecules
 Chemoautotrophs are aerobic, usually employing
dioxygen gas (O2) as the final or terminal electron
acceptor
 In contrast to photoautotrophic cyanobacteria and
algae, there is no net production of oxygen.
A closer look at the bacteriology of nitrification

12.  Nitrosomonas europaea is the most abundantly cited and
extensively studied nitrifier. The bacteria are short rods
(0.8×1–2 mm), typically non-motile, and ubiquitous in soils
(Watson, 1971). Other Nitrosomonas species have one or two
subpolar flagella and inhabit freshwater and marine
sediments
A closer look at the bacteriology of nitrification

13.  As autotrophs, nitrifiers are independently capable of
creating the entirety of their biomass through a full
complement of biosynthetic pathways
 Urea and methane oxidation and the co-metabolism of a
wide variety of hydrocarbons is common
 Cells may be maintained under starvation conditions
(i.e. ammonia or nitrite deprivation) through the low-
level endogenous respiration of cytoplasmic
compounds, while anabolic processes are lowered to
undetectable levels
A closer look at the bacteriology of nitrification

14.  At low DO levels, ammonia-oxidizers use nitrite as an
artificial electron acceptor and generate nitrous oxide
(N2O) gas. Nitric oxide (NO) is produced by ammonia-
oxidizers, but less sensitive to DO
 Nitrifying bacteria are photoinhibited and are especially
sensitive to disturbances away from optimal alkaline
and mesophilic conditions
 A commonly cited optimum pH for nitrifiers is 7.8
 For temperature, an overall optimum of 25°C has been
suggested
A closer look at the bacteriology of nitrification

15.  The maximum specific growth rate of nitrifying bacteria is
uncommonly slow, owing to the low yield of their energy
producing pathways and the burden of precursor formation
through the incorporation of inorganic carbon
 A doubling time of 7–8 h is possible under ideal conditions
 Nitrifiers normally make up a very low percentage of the total
microflora in soils, sediments, and naturally derived waste
streams
 Nitrifying biofilms are heavily overgrown by heterotrophs (pH
and oxygen concentration gradient problems are exacerbated)
A closer look at the bacteriology of nitrification

16.  In natural waters nitrifiers are associated with suspended and
settled particles, rather than free, unattached flotation in
the water column
 70 and 95% of suspended nitrifiers will cling to a fine inert
media within 30 min of its introduction
 If the detention time of a chemostat is not longer than the
generation time of a suspended microorganism, it will rapidly
be flushed out with the effluent.
 Even in a flow-through system that is designed for slow
growing nitrifiers, the steady-state balance may be upset by a
change in the condition of the medium
A closer look at the bacteriology of nitrification
End of Hagopian’s paper

17. Nitrification with high nitrite accumulation for the
treatment of wastewater with high ammonia
concentration. Ruiz et al., 2003
 Authors studied the effects of pH and dissolved oxygen
(DO) on ammonia oxidation.
 Aim was to determine the conditions for saving oxygen.
Aeration is a costly supply in wastewater treatment
plants.
 Assayed oxygen at intervals from 0.5 to 5.5 mg/L
 They identified that as low as 0.7 mg/L allows the
conversion of up to 98 % ammonia, accumulating up to
65% as nitrite.
Ruiz’s paper starts here

18. Nitrification with high nitrite accumulation for the
treatment of wastewater with high ammonia
 Parameters that are necessary to determine the
bacteria specific growth rate μ

19. Nitrification with high nitrite accumulation for the
treatment of wastewater with high ammonia

20. Nitrification with high nitrite accumulation for the
treatment of wastewater with high ammonia
 VSS: Volatile suspended solids, max ≅ 6.3 g/L
 NLR: Nitrogen loading rate, max ≅ 3.5 kg/m3 d
 Ammonia, max ≅ 780 mg N/L

21. Nitrification with high nitrite accumulation for the
treatment of wastewater with high ammonia
 Tipical removal efficiency plot (Inlet/Outlet
concentrations)

22. Nitrification with high nitrite accumulation for the
treatment of wastewater with high ammonia
 Ammonia concentration at inlet (feed) 610 mg/L
 DO 5.5 mg/L
 pH is the dotted line

23. Nitrification with high nitrite accumulation for the
treatment of wastewater with high ammonia
 Effect of dissolved oxygen (DO): the dotted line.
NO3
-
out NO2
-
out
NH4
+
out
End of Ruiz’s paper

24. Spatial distribution of heterotrophs and nitrifiers
in a submerged biofilter for nitrification

25. Spatial distribution of heterotrophs and nitrifiers
in a submerged biofilter for nitrification
 This paper describes the changes in biofilm density and
specific activities of carbon, ammonia and nitrite
oxidizers
 Experimental setup was a nitrifying upflow biological
aerated filter (UBAF)
 Biochemical pathways develop as a function of the C:N
ratio in the (synthetic) wastewater entering the filter.
 The reactor resisted the entrance of up 200 mg COD/l
without losing nitrification efficiency. This limit
corresponds to a COD:NH+4 -N ratio of four.

26. Spatial distribution of heterotrophs and nitrifiers
in a submerged biofilter for nitrification
 Organic content measured as TOC, Total Organic Carbon
and COD, Chemical oxygen demand, the amount oxygen
needed to chemically oxidize organic matter (a
parameter describing the organic matter content)
 The “entrance” zone of the filter removed
3.85 kg TOC/m3 day and 0.19 kg N/m3 day
 The second zone of the filter removed
0.42 kg TOC/m3 day and 0.96 kg N/m3 day.

27. Spatial distribution of heterotrophs and nitrifiers
 The spatial distribution of heterotrophic and
nitrifying populations was quantified in terms of:
 Oxygen uptake rates (OUR) or specific activities at
different filter heights and
 For increasing COD concentrations entering the reactor.
 Specific activities of three microbial groups,
ammonia oxidizers, nitrite oxidizers and
heterotrophs aerobes revealed a clear microbial
segregation along the filter depending on the COD
concentration entering the reactor.

28. Spatial distribution of heterotrophs and nitrifiers

29. Spatial distribution of heterotrophs and nitrifiers
 Head loss: Increasing pressure inside the reactor due to
friction (“pressure drop” also used)

30. Spatial distribution of heterotrophs and nitrifiers

31. Spatial distribution of heterotrophs and nitrifiers

32. Spatial distribution of heterotrophs and nitrifiers
At 0.5 m heigth
At 2 m heigth

33. Spatial distribution of heterotrophs and nitrifiers
in a submerged biofilter for nitrification
 Remarks
 Critical parameters to control: DO, COD, (pH, Temp).
 Configuration: DO gradient concentration along the
reactor
 Competition among heterotrophs/nitrifiers
 Light inhibition

34. Questions, comments, suggestions