de;partme;nt of marine bioi^ogy. 139 



at first in a solution of 0.5 per cent potassium malate, and 0.05 per cent 

 potassium nitrate in sea-water, but in this medium growth apparently ceased 

 after a few days and denitrification was never complete. A slight precipita- 

 tion occurred, and the solution was found to have very definitely increased 

 in alkalinity. It was this increase in alkalinity and consequent cessation of 

 growth of the bacteria that led Gran to employ a calcium instead of a potas- 

 sium salt in his culture medium, as in this way the calcium carbonate formed 

 by the bacteria from the organic calcium salt is removed from the sphere of 

 action by precipitation. 



In its cultural reactions this bacrerium is very similar to certain denitrify- 

 ing forms found in the English Channel ; the chief points of difference con- 

 sist in its much greater denitrifying power and the relatively higher tempera- 

 ture necessary for its growth, by which it would be prevented from spreading 

 into more temperate waters, unless it is capable of acquiring an adaptation 

 to altered conditions of temperature. 



Since the calcium salt of a simple organic acid is a sufficient source of or- 

 ganic food for these denitrifying bacteria, it would seem probable that they 

 could thrive in sea-water containing the products of decomposing vegetable 

 life, 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 composed of calcareous rock, 

 and the soluble organic calcium salts would be precipitated as calcium car- 

 bonate 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 equi- 

 librium of the salts in sea-water, and by increasing the alkalinity would also 

 result in the precipitation of calcium carbonate. 



The formation of beds of fine unorganized chalky mud in certain places 

 off the southern Florida Keys may possibly be explained in this w^ay, and it 

 is conceivable that some such bacterial action may have played a part in the 

 formation of some chalk and oolitic limestone beds in geologic times. 



The characteristics of the scarcer non-denitrifying form of bacterium 

 found on the agar plates are as follows : 



Growth on the potassium malate agar medium is very slow and indefinite. 

 On peptone agar growth is somewhat slower than in the case of the denitri- 

 fying form. On the surface circular, cream-colored colonies are formed, 

 having a brownish center ; the edges are smooth and regular, and the colony 

 remains discrete and does not tend to spread over the surface. The deep 

 colonies are smaller and usually ovoid in shape, and of a somewhat darker 

 color than those on the surface. 



No growth was obtained on gelatin media. 



Acid formation occurs in dextrose and levulose, but not in cane sugar, 

 lactose, or mannite media. 



Growth takes place slowly at 10° C. No visible growth occurred at 0° C, 

 but cultures were not killed by 24 hours' exposure to this temperature. 



Growth is retarded by light, and cultures are killed by 4 hours' exposure 

 to bright sunlight. 



The bacterium is a strict aerobe. 



Free growth takes place in Gran's medium, but is much slower than in the 

 case of the denitrifying form ; no growth occurs if the potassium nitrate be 

 omitted, though no denitrification results if it be present. 



Many attempts were made to discover whether this bacterium had any 

 nitrifying or denitrifying action in cultures, but uniformly negative results 

 were obtained. Nitrites were neither oxidized to nitrates nor reduced to 



