30 Papers from the Marine Biological Laboratory at Tortugas. 



From these experiments it appears that the bacteria from subcultures 

 from the Marquesas have a much greater denitrifying power than those 

 from subcultures from a point 70 miles west of Ushant, and that as the 

 bacteria from the Marquesas appear to be of the same species as those 

 investigated at the Dry Tortugas, their power of causing complete denitri- 

 fication in Gran's medium has been lost by successive cultivations on 

 peptone agar. 



The presence of the thick layers of fine chalky mud within the Marquesas 

 Keys, and elsewhere in many places near the Florida coast, led to a con- 

 sideration of the possibility of its precipitation by bacterial agency. 



Since these bacteria grow freely in Gran's medium, the calcium salt of 

 a simple organic acid is a sufficient source of organic food for them, and it 

 seems probable that they would thrive in sea-water containing the products 

 of decomposing vegetable matter, provided that the nitrate supply and 

 conditions of light and temperature were suitable. Such conditions should 

 be especially well fulfilled by the drainage into the sea of a well-wooded 

 country with a calcareous subsoil, and the soluble organic calcium salts 

 would be precipitated as calcium carbonate by the action of the bacteria. 

 In addition, the elimination of the acid radicle from the nitrate in the process 

 of denitrification, by whatever stages it may occur, must leave the alkaline 

 base free to destroy the normal equilibrium of the salts in sea-water, and by 

 increasing the alkalinity would also result in the precipitation of calcium 

 carbonate. 



To test this theory cultures were made in a medium having the following 

 composition: 



Calcium succinate 2.5 grams 



Potassium nitrate 0.5 gram 



Sea-water 1,000.0 c.c. 



Calcium succinate is soluble in these proportions and the medium is 

 quite clear. Free growth was manifested by the cloudiness of the medium 

 48 hours after inoculation, and nitrite formation was apparent. 



After 96 hours the medium appeared quite milky and this milkiness was 

 due to the presence of exceedingly fine particles of a substance which was 

 soluble in dilute hydrochloric acid with evolution of gas, and was presumably 

 calcium carbonate. In some cultures these particles settled as a definite 

 sediment, but in others the particles were so minute that they showed little 

 tendency to settle and could only be separated with difficulty by centrifugal- 

 ization. The conditions determining the size of the particles formed could 

 not be ascertained, as the size varied in cultures which were apparently made 

 and grown under identical conditions. 



The addition to cultures in which the particles of calcium carbonate were 

 so small as to remain in suspension of any foreign substance, such as finely 

 poAvdered calcium sulphate or of larger particles of sand, resulted in the 

 aggregation around them of the particles of calcium carbonate, forming a 

 concretion around a central nucleus. These concretions were hard and of 



