g2 J. T. HOLDEN 
grams. In animal tissues the D-amino acids probably occur in small amounts or more 
likely not at all, and this has not been a serious defect of such studies. However, a 
number of D-amino acids are now known to occur in sizeable quantities in bacterial 
cells, particularly in the cell wall, and examination of cell extracts has revealed their 
presence in this fraction as well§. §4; 88. HOLDEN AND HOLMAN have shown that the 
freely-extractable pool in freshly harvested cells of L. avabinosus can contain as 
much as 5 wmoles of D-glutamic acid per 100 mg dry weight of cells and that this 
can increase to 37 wmoles/Ioo mg when resting cells accumulate L-glutamic acid 
from the external buffer. A systematic study of the occurrence of D-amino acids in 
microbial pools appears not to have been reported. 
Extracellular amino acids. A detailed survey of this subject is beyond the scope of 
this review and only a few reports will be mentioned as a guide to the reader. These 
are largely empirical observations which so far have provided little insight to the 
factors which control the relative distribution of amino acids between intra- and extra- 
cellular fluid. Generally, there are two types of studies, those in which cells are grown 
in a complex medium and the removal of amino acids monitored at intervals through- 
out the growth phase, and those in which cells are grown in a simpler, carbohydrate— 
salts medium and the appearance in the extracellular fluid described of amino acids 
synthesized internally during cultivation. Many of the studies on the internal pool 
cited previously have included such chromatographic investigation of culture filtrates 
(scewfomexample prefs hn 52ers er2ON4n, WABIEDA 76 TACs 7 SESS) 
Among the earliest studies are those of PRooM AND Worwoop who examined the 
culture filtrates of 300 bacterial strains® for the most part describing the removal 
of amino acids from the medium. Subsequently!8’, the release of newly-synthesized 
ninhydrin-reactive substances to the medium also was described. DAGLEY AND 
JOHNSON® grew E. coli, Ps. aeruginosa and a vibrio in mineral-salts medium, and 
followed the appearance of amino acids extracellularly. Of some interest was the 
finding that EF. coli released amino acids even in the early exponential phase when 
protein degradation could be expected to be minimal, and that this release followed 
an ordered pattern in which an increasing number of amino acids appeared at pro- 
gressively later stages of the exponential growth phase. The appearance of amino 
acids in the growth medium during cultivation of E. coli and Ps. aeruginosa also has 
been studied respectively by KAwANOo* and CESAIRE et al.28, 
Recently, a large number of reports have appeared dealing with the production of 
amino acids by microbial fermentation (cf. refs. 29, 102, 103). In some instances, a 
50% yield of glutamic acid based on the amount of glucose used has been obtained. 
It can be expected that future studies with these organisms may provide clues to the 
factors which control intracellular synthesis and retention of amino acids. 
CONDITIONS WHICH MODIFY POOL SIZE AND COMPOSITION 
Having described the composition of the free amino acid pools in various commonly 
studied groups of microorganisms, we shall now consider what is known about the 
conditions which control pool composition. As indicated previously, many investi- 
gators have understood that the pool can have a variable composition and have 
attempted to identify some of the factors which contribute to this variability. In the 
References p. 105/108 
