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Growth of Nitrosomonas europaea and Nitrobacter winogradskyi in a chemostat


The method allows growing the strains in batch until the NH4+ and NO2- are consumed. The cells can be harvested or kept growing feeding fresh medium and drawing cells in spent medium at equivalent rates. A steady state a chemostat can produce up to 50% of the volume/day.

THE MEDIUM

For a chemostat culture, the medium consists of 60 mM NH4Cl, 0.75 mM MgSO4, 0.1 mM CaCl2, 5 mM phosphate, 0.34 mM Na2CO3 and trace metals (9.9 µM FeCl3, 10.0 µM CuSO4 0.6 µM Na2Mo4O4, 1.59 µM MnCl2, 0.6 µM CoCl2, 0.096 µM ZnCl2). The medium is prepared as described @ Growing Nitrosomonas europaea

In chemostat co-culture conditions, growth of N. winogradskyi is dependent on the conversion of NH3 to NO2- by N. europaea.


THE CHEMOSTAT

This chemostat design is a fraction of the cost of those commercially available. The chemostat containing the medium can be autoclaved fully assembled.

1. The chemostat is a glass vessel (e.g. Bellco Bio-probe Ca No. 1965-97001) (1 liter culture media) that can be used to establish a steady state at a hydraulic retention time of ~5 days (dilution rate 0.008 h-1 or 200ml/day) at 30°C.

2. Aeration (70% O2 saturation) is provided by stirring with an impeller at ~100 rpm and by gentle bubbling of filtered air (e.g. from an aquarium pump).

3. A pH of 7.5 +/‑0.1 is maintained with a pH controller (e.g. Alpha pH 560; Eutech Instruments/Thermo Scientific) and a pH probe (e.g. pH Fermprobe; Bradley-James Corp) to supply automatic additions of an aqueous solution of Na2CO3 (10% w/v) as necessary through a solenoid-operated pinch valve (e.g. Takasago Electric PK-0802–NC3 12 VDC.)


CO-CULTURE ESTABLISHMENT

1. The chemostat vessel is inoculated with both N. europaea and N. winogradskyi (3% vol/vol of each) in medium containing 60 mM NH4+ and left in batch mode until N. europaea produces approximately 45 to 50 mM NO2-.

2. At approximately day 4, N. winogradskyi begins to consume NO2-. A flow rate of growth medium (60 mM NH4Cl) is set to 200ml/day (D = 0.008 h-1 or cell doubling time of 86 h) by the use of a double channel peristaltic pump (inflow and outflow at equivalent rates).

3. A chemostat culture typically requires ~ 12 days to reach steady state (e.g constant concentration of NO3- (53 to 59 mM) in the outflow, and a stable OD600 that usually ranges between 0.2 to 0.29).


Luis Sayavedra-Soto
Botany and Plant Pathology
Oregon State University, Corvallis OR 97331, USA