Autotrophic Nitrification in the Ocean 75 



trate increased by 100 /xM. When nitrification did occur, appro- 

 priate amounts of ammonia were added to replace that lost 

 through aeration and oxidation. 



Ammonia-oxidizing bacteria were subcultured by inoculat- 

 ing one liter of sterile medium with 100 ml of a sample taken 

 when nitrites were increasing exponentially. The mediiun con- 

 tained, per liter of distilled water: NaCl, 25 g; (NH4)2S04, 3.0 g; 

 K.HPO., 1.0 g; MgSOrTH.O, 0.1 g; chelated iron ("Adas 

 EDTA"), 0.1 mg. In addition, 1 g of CaCO;; was included if solid 

 substrate was required. The pH was adjusted after sterilization 

 to 7.5 by adding 1 N NaOH. NaCl and CaCOa were added 

 to the water and sterilized by autoclaving. The remaining salts 

 were sterilized separately by Selas filtration and added asep- 

 tically. As ammonia was oxidized from the enriched cultures, 

 more was added aseptically. 



The marine bacteria that oxidized nitrite were subcultured 

 in the medium of Aleem and Alexander ( 1 ) with the addition of 

 2.5 per cent NaCl and, if solid substrate was wanted, 1 g/L 

 of CaCOa. 



Serial transfers were made using two methods: a 10 x^er 

 cent inoculum was transferred into a medium free of CaCO;!, or 

 a subculture containing CaCOs was filtered through an AA Milli- 

 pore filter and the residue transfeiTcd to fresh medium. The 

 latter method had two advantages: All undesirable end products, 

 such as nitrite and soluble organic matter, were eliminated from 

 the inoculum, and all the bacteria were retained. 



Terrestrial Nitrosomonas europaea (obtained from Dr. 

 David Pramer) and Nitwhacter agilis (obtained from Dr. Martin 

 Alexander ) were cultured, using the methods of Aleem and Alex- 

 ander (1) and Engle and Alexander (3). 



A polarographic oxygen electrode (4) was used for respira- 

 tion studies. Marine nitrifying bacteria grown in a medium with 

 CaCOa were filtered on a Millipore filter, washed with sterile 

 medium, and resuspended in sterile medium. The cultures were 

 diluted to give oxygen uptake values of about 5 ml/L/hr. Cul- 

 tures of terrestrial nitrifying bacteria grown in a soluble medium 

 were centrifuged at 34,800 x G and washed with 0.05 M K^HPO, 



