PNNL-13501 - Pacific Northwest National Laboratory

Malt extract broth cultures were allowed to grow 3 days
before an aliquot of each was used to inoculate 200 mL to
300 mL 10% glucose solution (Sigma #G-8270 D +
Glucose [Dextrose; corn sugar, minimum 99.5%]).
Figure 1. Glucose fermentation setup for 250 mL bubbler
flasks: spherical growth (top); bubbler flask setup in the
laminar flow hood
Glucose cultures were sampled starting at 4 days after
inoculation and at ~2- to 3-day intervals thereafter for a
total of 21 to 28 days. Sampling consisted of removal of
1.5 mL aliquots under sterile conditions in a laminar flow
hood using the bubbler pipette as the transfer pipette. The
aliquots were centrifuged at 5585g for 5 minutes to
provide cell-free extract. Subsequently, each 1 mL
supernatant was decanted into a labeled 1.5 mL
polypropylene centrifuge tube. The samples were frozen
and stored until analysis by liquid chromatography at our
Laboratory. The remaining 0.5 mL of each aliquot was
used for pH measurement.
Results and Accomplishments
Table 1 is a summary of the production of organic acids
and solvents by 45 fungal strains (one strain lost due to
contamination). Approximately 24 fungal strains
produced malonic acid; of these, 20 met criteria of
increasing or high-peak yield of malonic acid and were
then selected for follow-on studies.
Acidity and alkalinity measurements were taken on all
liquid fungal cultures used in the screening study. The
74 FY 2000 Laboratory Directed Research and Development Annual Report
Table 1. Summary of organic byproducts of fungal
fermentation of glucose (g/L)
Species
Name Oxalate Citrate Malonic Sorbitol Glycerol
D 2.5 – 2.6 0.30
E 2.2 – 2.9 3.35 0.3 – 0.9 0.20
F 1.50
J 1.0 – 1.1 0.50
Y 1.7 – 2.6 1.30 0.4 – 0.5 0.40
Po 1.4 – 13.2 0.30
V 0.20 – 0.32 0.3 – 1.5 1.20
ZZ 0.20 0.90
13 0.1 – 4.0 0.7 – 2.5 1.1 - 2.1 1.00 0.7 – 1.7
I 2.4 – 3.2 1.3 – 2.8 1.3 – 2.5 2.40 1.70
A 0.2 – 2.3 1.1 – 1.5
K 2.3 – 2.8 0.2 – 2.2 1.90 0.20
Gf 2.2 – 2.7 1.0 – 1.5 0.10
M 1.9 – 3.1 1.6 – 3.5 1.70 1.00 1.1 – 2.9
O 1.8 – 2.4 1.0 – 1.8 2.0 – 2.6
P 1.9 – 2.6 1.2 – 1.8
ZZZ 2.3 – 3.2 1.2 – 2.1 0.20
R 2.1 – 2.7 1.3 – 3.1
8 1.7 – 2.8 1.5 – 1.9 2.10
20 0.1 – 2.7
H 2.3 – 3.1 1.00 0.6 – 1.0
N 2..5 – 3.0
Q 2.5 – 3.0 2.00 1.60
S 2.1 – 2.8 1.0 1.9 – 2.1 3.40
W 2.1 – 2.9 0.70
Z 2.1 – 2.6 1.5 – 2.3 1.30
ZZZZ 2.0 – 3.1 0.8 – 1.1 1.50 0.2 – 3.3
ZZZZZZ 2.4 – 3.2 1.40
3 0.1 – 2.8 0.40
10 0.3 – 3.2 0.4 – 1.8
Ab1 0.1 – 3.2 0.3 – 0.4 0.20
CC2 0.2 – 3.2 0.40 0.10 0.40
CV 0.1 – 3.7 0.3 – 0.4 0.70
FV 0.2 – 2.4 0.3 – 0.7 0.20
GFO 0.1 – 3.5 0.60
results showed that at least 11 strains were growing at or
below pH 4 by day 15 of testing. The fungal strains that
performed on low pH conditions (