276 
Journal of Agricultural Research voi. xxx, no. a 
nitrogen was reassimilated by the 
organism and, after all the dextrose 
was used up, ammonia began to accum¬ 
ulate in the medium; only a small part 
of the nitrogen was present in this case 
in the form of polypeptides. 
Similar results were obtained with 
the Trichoderma. In the absence of 
dextrose, the organism produced a 
more abundant growth than the Zy- 
gorhynchus, and it also changed about 
40 per cent of the nitrogen in solution 
to amino nitrogen and ammonia. The 
fact that Trichoderma is capable of 
breaking down proteins more vigor¬ 
ously than Zygorhynchus is further in¬ 
dicated by repeated experiments on 
casein decomposition: After 11 days, 
there was present in the medium 40.02 
mg. of amino nitrogen and 24,29 mg. of 
ammonia nitrogen, or nearly 60 per 
cent of the total nitrogen. Dextrose 
exerts a protective action, both on the 
hydrolysis of the protein and upon 
ammonia formation, similar to the 
phenomena elucidated above. As long 
as there is dextrose left in the medium, 
the reaction will be acid (causing a 
precipitation of the casein, followed by 
its dissolution as soon as the dextrose 
is used up), and very little ammonia 
will accumulate. 
The Actinomyces behaved in as imilar 
way. There was, however, an increase 
in the amino nitrogen content in the 
dextrose containing media; the ammo¬ 
nia accumulated largely in the dextrose- 
free media. 
B. cereus attacked casein more vig” 
orously in the absence of dextrose 
than in its presence. There was at 
first a rapid increase in the amino 
nitrogen content in the absence of 
dextrose followed by a decrease, prob¬ 
ably owing to the fact that, after some 
time, the increase in the amino nitrogen 
content resulting from the hydrolysis of 
the protein, then of the protein deriva¬ 
tives, was balanced and finally exceeded 
by the increase in the ammonia result¬ 
ing from the decomposition of the amino 
compounds. It is interesting to note 
that there was a decided increase in the 
amino nitrogen content even in the 
presence of dextrose, confirming the 
observations of DeBord ( 5 ), although in 
this case it was lower than in the ab¬ 
sence of dextrose. The ammonia ac¬ 
cumulation was insignificant in the 
presence of dextrose. This would 
indicate that, although the protein 
molecule may be hydrolyzed by B. 
cereus even in the presence of dextrose, 
the hydrolysis does not go much 
further than the amino acid stage, so 
that no ammonia can accumulate. 
In view of the fact that B. cereus 
does not attack readily simple amino 
acids, at least not those that were 
tested, one would expect that the 
hydrolysis of casein by B. cereus 
would lead to an accumulation of cer¬ 
tain amino acids; since ammonia 
results chiefly when the amino acids 
are used as sources of energy (compara¬ 
tively little energy being liberated in the 
hydrolysis of the protein to the amino 
acids), no ammonia would accumulate 
in the presence of an available carbohy¬ 
drate. In the absence of dextrose, the 
organism has to utilize the amino acids 
as sources of energy, and the results 
seem to indicate that B. cereus is cap¬ 
able of decomposing at least certain 
fractions of the protein molecule, when 
ammonia is formed and allowed to 
accumulate in the medium. 
Bad. fluorescens was found unable to 
utilize casein, either as a source of 
carbon or of nitrogen; the fact that 
small amounts of dextrose were used 
would lead one to think there were 
certain substances present in the casein 
which were utilized by the organisms 
as sources of nitrogen. 
The two bacteria behaved thus in a 
distinctly different manner toward a 
protein and its derivatives: B. cereus 
is typically proteolytic, .capable of de¬ 
composing native proteins (the same 
was found to hold true in the case of 
purified vegetable proteins, as will be 
shown elsewhere), while Bad. fluores- 
cens is nonproteolytic but can utilize 
amino acids (and probably also poly¬ 
peptides) as sources of energy and nitro¬ 
gen. By combining the two organ¬ 
isms, one would logically expect that 
the course of transformation of the 
protein molecule would take place in a 
distinctly different manner than by 
either organism alone. This was actu¬ 
ally found to be the case, as shown in 
Table VIII and Figure 1. 
B. cereus alone hydrolyzed the casein 
with a gradual accumulation of amino 
nitrogen and ammonia, so that, in 6 
days, 35 per cent of the nitrogen in 
solution was in the form of amino ni¬ 
trogen and 12 per cent in the form of 
ammonia. After that the total con¬ 
tent of amino nitrogen decreased, owing 
to its transformation to ammonia, so 
that, in 15 days, the total amino nitro¬ 
gen dropped from 44.89 mg. (6 days) 
to 24.62 mg. (about 30 per cent of the 
nitrogen in solution); the ammonia rose 
in that period of time from 16.62 mg. 
to 46.52 mg., or from 12.5 per cent to 
over 50 per cent of the nitrogen in* 
solution. This indicates definitely that 
the hydrolysis of the polypeptides con¬ 
tinues even after the maximum amino 
nitrogen has been reached; only more 
of it is changed to ammonia. 
