48 NITROGEN METABOLISM 



growth on a N-free media, these techniques involve gaso- 

 metric analysis, nitrogen estimations by Kjeldahl proce- 

 dures, manometry or the use of the nitrogen isotope N^^. 



Gasometric analysis is used to detect whether there has 

 been a decrease in the amount of gaseous Ng in a closed 

 system, whilst increases in fixed nitrogen can be determined 

 by the use of a Kjeldahl technique. Although often used 

 successfully [22], gasometric analysis is tedious and the 

 elimination of experimental errors is not easy. The Kjeldahl 

 method, which at first sight appears to be an ideal and 

 technically simple procedure, has yielded many erroneous 

 results, the reasons for which have been cogently assessed 

 by Wilson [41]. The samples taken for analysis do not have 

 a homogenous composition, and whilst a particular Kjeldahl 

 procedure will estimate with precision the nitrogen content 

 of one or more related compounds, it does not necessarily 

 follow that the nitrogen in other compounds is estimated 

 with a comparable degree of accuracy; e.g. the mere addition 

 of water to dry seeds appeared to increase their nitrogen 

 content as measured by the Kjeldahl procedure being used. 

 None the less, provided such sources of error are borne in 

 mind, the Kjeldahl technique has been, and is, of great 

 value. 



A more convenient, though indirect, method for study- 

 ing Ng-fixation by free-living organisms is based on the use 

 of the Warburg manometer, organisms being grown in the 

 flask of the apparatus and the gas changes consequent on 

 growth being followed in the usual manner. An obvious com- 

 plication with aerobic organisms is that superimposed on 

 any uptake of Ng , there is a concomitant uptake of Og due 

 to respiration. Indeed, Azotobacter and Rhizohium possess the 

 highest respiration rates known, the Qq^ on glucose being 

 of the order 1,000 to 2,000. Where large O2 uptakes are 

 expected, the usual Brodie manometric fluid is often re- 

 placed by one of greater density [e.g. Hg] in order that the 

 manometer readings will be of convenient dimensions. If 

 the rate of Ng-fixation is the factor limiting growth, then 

 fixation by aerobic organisms can be followed in terms of an 

 increase in the respiration rate, since the latter is directly 



