On the Precipitation of Calcium Carbonate. 1 1 



contents of sea-water exists, and hence it is impossible to correlate quantita- 

 tive plankton observations with direct analyses of the amount of combined 

 nitrogen present in sea-water in different localities. Much valuable work 

 on this subject has been done by Raben (15), but he states that his error 

 in control experiments averages over 30 per cent. An exhaustive study 

 (as yet unpublished) of all the methods of estimating combined nitrogen in 

 sea-water as given by various investigators has been made by Mr. D. J. 

 Matthews, hydrographer to the Marine Biological Association of the 

 United Kingdom and to the Fishery Branch of the Department of Agri- 

 culture, etc., for Ireland, and he has come to the conclusion that the limits 

 of error in all these methods are so large as to make them unsuitable for 

 investigations such as these. Since chemical methods are at present inade- 

 quate to give evidence on this hypothetical deficiency of nitrates in warmer 

 seas, it seemed of interest to investigate the distribution and relative activity 

 of denitrifying bacteria in tropical waters in comparison to those found in 

 temperate seas, and it was with this primary object that the present work 

 was undertaken. 



The previous researches most closely related to these investigations are 

 those of Gran (9), who isolated a number of species of denitrifying bacteria 

 from the inshore waters of the Dutch coast. He made use of solutions of 

 nitrates, nitrites, or ammonium salts as the sole source of nitrogen in his 

 culture media, which contained only a dilute solution of calcium malate as 

 organic nutrient material for the bacteria. He classifies the bacteria into 

 four groups, according to their reactions in pure cultures towards nitrates 

 or nitrites. 



1. Those which reduce nitrates and nitrites to free nitrogen without any 



ammonia formation. 



2. Those which readily reduce nitrates to nitrites. The nitrite disappears 



slowly without perceptible formation of free nitrogen and some 

 ammonia is formed. 



3. Those which can not reduce nitrates to nitrites, but which are capable 



of slowly removing the nitrite without perceptible formation of 

 free nitrogen. Though the nitrites are not reduced, yet they can 

 serve as the sole source of nitrogen for the growth of the bacteria. 



4. Those which can not reduce and are not capable of assimilating either 



nitrates or nitrites, but will flourish when ammonium salts are 

 present. 



In investigations on samples of water taken in the English Channel some 

 10 miles off Plymouth, I was able to recognize species belonging to the 

 second group of Gran's classification, but could not detect the presence of 

 species belonging to any of the other groups, and it would seem probable 

 that these other groups are chiefly composed of littoral forms. 



In fluid culture media inoculated with samples of sea-water and kept 

 at a temperature of 28° C., Gran found that the formation of nitrite was 

 detectible in from i to 2 days, and that eventually all the nitrate and nitrite 

 was destroyed in the majority of cases, especially if the cultures were 



