טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentShlafman Emilia
SubjectWhat is the Substrate for Ammonia Oxidizing Bacteria:
Ammonia NH3 or Ammonium Ion NH4+?
DepartmentDepartment of Biotechnology
Supervisor Professor Emeritus Michal Green


Abstract

In autotrophic ammonia oxidizing bacteria (AOB), free ammonia is considered to be the substrate for the primary enzyme, ammonia mono oxygenase (AMO). Transport of free ammonia into the cells, unlike ammonium ions, is by passive diffusion. Previous experiments at the Technion showed that high rate nitrification by common AOB in both biofilm and suspended biomass reactors can occur at low pH where concentrations free ammonia are negligible. The hypothesis of this research work is that although free ammonia is the substrate to AMO, the transported species to AOB can be not only free ammonia by nonspecific diffusion, but also ammonium ions. Microbiological work was carried out using pure cultures of the model AOB Nitrosomonas europaea.

In the first part of the research, pure cultures of N. europaea were used to determine the kinetic parameters Ks and qmax, at different pH values of 6 to 8.5. The experiments were carried out under two different conditions of salinity, one typical of microbiological work (higher salinity) and the other typical to nitrifying reactors (lower salinity). Under high salinity conditions, results showed that KS value based on free ammonia concentration remained constant throughout the pH range tested. Under low salinity conditions, KS values based on free ammonia concentration were not constant throughout the pH range tested. These results suggest that ammonia is the substrate for N. europaea, but only under conditions of higher salinity.

In the second part of this research, the ability of Nitrosomonas europaea to adapt to low pH under low salinity conditions was tested. A biofilm reactor was inoculated with Nitrosomonas europaea and first operated to high nitrification rates under conditions favorable to N. europaea (pH>7; high ammonium concentrations). To eliminate inhibitory concentrations of nitrite at low pH, an enriched culture of Nitrospira (a nitrite oxidizing bacteria) was then added. The transition from neutral to acidic conditions was attempted by sharply lowering the nitrification rate and by using a feeding solution containing insufficient buffer for complete nitrification. Unlike other transitions to low pH, the reactor reached pH 4.5 only after four weeks. FISH results showed that while the percent of AOB and Nitrospira remained relatively constant, the AOB community changed completely in 60 days from 100% N. europaea to 100% Nitrosomonas oligotropha. Even though N.oligotropha was not intentionally introduced into the reactor, it is apparently much better than N. europaea adapted to conditions of low pH.