1. Experiments have been conducted with aaporogenous
strains of Bacillus cereus and Bacillus megaterlum.
2. Methods end chemically defined media have been
developed for the bulk culture of the organisms.
Manganese was found to be an essential additive to the
3. A major constituent of the "lipid" granules In
these species Is a polymer of ß-hydroxybutyric acid (PHB).
The metabolism of PHB has been studied under various
4. PHBB was formed rapidly during growth and In
washed suspensions in conditions of carbon and energy
excess and dissimilated in the absence of a utilisable
substrate. The formation of PHB has not previously
been demonstrated In washed suspensions.
5. A high level of PHB In a cell conferred a high
rate of endogenous respiration and delayed autolysis.
6 The formation of PHB showed an optimum around
pH 7.5 and growth studies confirmed that It is unlikely
to be a "neutralisation mechanism".
7. It was concluded from the above (4 - 6) that PHB
acted primarily as a reserve of carbon and energy.
8. Washed suspension experiments showed that PHB was
dissimilated more rapidly aerobically than anaerobically.
More oxygen was absorbed than would have been required
for the complete oxidation of the PHB lost aerobically
and more acid was formed anaerobically than was
accounted for by a simple hydrolysis of the degraded PHB
to ß-hydroxybutyric acid.
9. Quantitative studies showed 90% of the
theoretical yield of ß-hydroxybutyric acid and
acetoacetic acid from the amount of PHB broken down
anaerobically. Little ß-hydroxybutyric acid or
acetoacetic acid accumulated aerobically and it
appeared that almost complete oxidation of the
degraded PHB took place.
10. Chromatographic analyses of the products of PHB
breakdown confirmed the presence of ß-hydroxybutyric and
11. The organisms formed PHB in the presence of
glucoses pyruvate or ß-hydroxybutyrate.
12. Acetate did not induce synthesis of PHB on its
own but caused an extensive increase in the amount
formed from the above substrates. With a fixed glucose
concentration (0.05 M.) and varied acetate concentrations,
PHB formation was proportional to the acetate
concentration up to 0.05 M.
13. Various other organic substances were tested as
substrates for PHB synthesis. No formation of PHB was
effected but several of the substances inhibited its
breakdown to some extent.
14. Neither a nitrogen nor a magnesium source was a
necessary additive for PHB synthesis.
15. PHB formation from glucose and acetate was
strongly inhibited by cyanide and dinitrophenol and less
strongly by the co-enzyme A antagonist pantoyl-tauryl
16. Low concentrations of fluoroacetete stimulated
PHB formation from glucose and acetate, the oxygen
consumption being slightly inhibited. The
ß-hydroxybutyrate analogue 2-hydroxy-1-propane
sulphonate caused a slight increase in PHB synthesis
without affecting the oxygen uptake.
17. Pure oxygen inhibited the formation of PHB,
though synthesis was optimal with 5% oxygen in nitrogen
as the gas phase. Neither B. cereus nor N. megaterium
formed PHB under nitrogen but the former organism did so
under hydrogen. Carbon dioxide did not stimulate PHB
18. While utilising ß-hydroxybutyrate for the
synthesis of PHB the organisms converted much of the
substrate to acetoacetate.
19. The hypochlorite method was unsuitable for the
estimation of PHB in lysates derived from lysozyme
treatment of whole cells. Evidence is led that the
ether soluble component from hypochlorite isolated
granules may be derived, at least partly, from other cell
20. Synthesis or degradation of PHB in lysozyme
prepared cell-free extracts could not be demonstrated.
21. Neither utilisation of ß-hydroxybutyrate nor
initiation of oxygen uptake upon its addition could be
shown with the extract.
22. A DPN-linked ß-hydroxybutyric dehydrogenase
could be demonstrated in carefully nrepared extracts
after a lag period which lengthened on storage.
Neither CoA, ATP nor KCN enhanced DPN reduction in these
23. Pyruvate was decarboxylated anaerobically with
the formation of quantitative amounts of acetaldehyde.
The addition of DPT accelerated this process but CoA
24. Studies using isotopically labelled substrates
showed that acetate was certainly incorporated into
PHB during synthesis in glucose ± acetate, and both
pyruvate and acetate during synthesis in pyruvate
25. Possible biochemical pathways of synthesis and
degradation of PHB are discussed in the light of these