ZoBell — 156 — Marine Microbiology 



Nitrogen fixation: — Benecke (1933) relates that Keutner was the 

 first to make an extensive search for nitrogen-fixing bacteria in the sea, 

 this being the subject of his doctorate dissertation at Kiel University. 

 Species of the aerobic nitrogen fixer, Azotohacter chroococcum, and also 

 the anaerobic Clostridium pastorianum were found (Benecke and Keut- 

 ner, 1903). Azotohacter occurred primarily associated with the surface 

 slime of algae, and CI. pastorianum occurred chiefiy in bottom water and 

 ooze. After finding both types of nitrogen-fixers in the Baltic Sea, the 

 North Sea, off the African coast, and in the Malay Archipelago, Keutner 

 (1905) concluded that nitrogen-fixing bacteria are normal inhabitants of 

 the sea. Azotohacter chroococcum was found to tolerate up to 8 per cent 

 NaCl. 



Reinke (1903), believed Azotohacter to occur as an epiphyte on 

 phytoplankton and larger marine algae, symbiotically obtaining utilizable 

 organic matter therefrom while furnishing fixed nitrogen in return. Sev- 

 eral other investigators have noted the occurrence of Azotohacter on marine 

 as well as fresh-water algae, but a definite symbiotic relationship has never 

 been established. 



Fischer (1904) demonstrated that nitrogen fixers may obtain their 

 energy from the hydrolytic products of agar liquefiers. This observation 

 was confirmed by the Pringsheim brothers (1910) who inoculated nitro- 

 gen-free agar media with cultures of Pseudomonas gelatica and Clostrid- 

 ium pastorianum (also known as Bacillus amylohacter) . Good growth of 

 both organisms occurred. Presumably Ps. gelatica broke the agar down 

 into substances which CI. pastorianum could utilize. 



The strains of Azotohacter, which Keding (1906) found on the surface 

 slime of algae near shore, appeared to be identical with those found in 

 soil. The strains of Azotohacter and Clostridium, which Issatchenko (1914, 

 1926) found associated with marine algae, required enough salt for their 

 development to lead him to believe that these nitrogen fixers were spe- 

 cifically adapted to sea water. 



According to Korinek (1932), there are species in the ocean which 

 closely resemble Azotohacter morphologically, but they do not assimilate 

 free nitrogen. He found that Azotohacter chroococcum grew only poorly in 

 sea- water media. He admits that there may be marine strains of Azoto- 

 hacter which fix nitrogen but he failed to find one. Lloyd (1930) likewise 

 regarded the existence of active marine nitrogen-fixing bacteria as 

 problematical. 



Azotohacter cells were only occasionally found by Bavendamm (1932) 

 in enrichment cultures of calcareous mud from around the Bahama 

 Islands. CI. pastorianum was generally present. 



The occurrence in the sea of an abundant population of nitrogen- 

 fixing Azotohacter and Clostridium is reported by Waksman et al. (19336), 

 In the presence of a favorable source of energy, the bacteria were capable 

 of fixing appreciable quantities of nitrogen. However, the extent to 

 which such a process actually takes place in the sea remains to be deter- 

 mined, von Brand et al. (1942) concluded that except for the sporadic 

 presence of nitrogen-fixing bacteria, there is no evidence of nitrogen 

 fixation in the sea. 



Nitrogen fixation is an endothermic reaction which requires consider- 

 able energy. Nitrogen-fixing bacteria obtain this energy from the oxida- 

 tion of organic matter. Only in the presence of readily available organic 

 matter, therefore, can nitrogen fixers be expected to be functional. With- 



