QUARTERLY REPORT

CONFIDENTIAL
Studies on Production of Gluco c l~ :.;cra~ e

1 . Production of Arthrobacter Cell s

The results on 30-0allon fermentations that were conducted to produce

cells of Arthrobacter possessing glucose isomerase activity were as
recorded in Table 1 . Yields obtained were 800 to 1000 for twenty batches
and 700 to 800 }i units/ml for five batches .

Whole-culture fermented media (Nos . 219, 223, 234, and 244) were
submitted to Miss Long for cell-floc studies . Cells recovered from
batches 216, 218, 220, 222, 226, 235, 236, and 241 were frozen for future
use . Other fermentations (Nos . 245 through 257) were used to study lysis
of Arthrobacter cells by the activity of a mold, a species of Trichoderma .

TABLE I

DATA ON PRODUCTION OF CELLS OF ARTHROBACTE R

I'OSSESSING D-GLUCOSE ISOMERASE ACTIVIT Y

Complete* Cells _ Yiel d

Batch Medium (pH) GMS . We t Total GMS . Inoculum*** At 48 Hr s
No . _ Initial Final Wt ./100 ml Recovered** Percent Age U Units/m l

216 6 .85 5 .4 3 .55 3110 1 24 81 2

218 6 .85 5 .6 3 .07 2890 10 48 809
219 6 .85 5 .5 3 .53 -- 1 24 830
220 6 .85 5 .7 3 .80 3352 1.0 48 78 0
222 6 .85 5 .7 3 .97 3263 10 24 99 2
223 6 .80 5 .8 3 .43 -- 2 12 730
226 6 .85 5 .7 3 .93 3587 10 24 102 2
234 6 .85 5 .6 4 .43 -- 1 24 89 1
235 6 .85 5 .6 3 .56 3250 1 24 89 9
236 6 .85 5 .6 4 .42 3440 1 24 87 2
241 6 .80 5 .2 3 .80 3077 10 24 91 6
244 6 .80 5 .3 -- -- 1. 24 101 1
245 6 .80 5 .4 -- -- 1 24 948
246 6 .80 5 .5 -- -- 1 24 782
247 6 .80 5 .2 -- -- 1 24 91 2
248 6 .85 5 .7 -- -- 1 24 79 2
249 6 .90 5 .4 -- -- 1 24 899
250 6 .90 5 .7 -- -- 1 24 79 7
251 6 .90 5 .5 -- -- 1 24 84 1
252 6 .85 5 .5 -- -- 1 24 82 8
253 6 .90 5 .8 -- -- 1 24 87 4
254 6 .90 5 .4 -- -- 1 24 99 2
255 6 .80 5 .2 -- -- 1 24 87 6
256 6 .85 5 .4 -- -- 1 24 81 6
257 6 .85 5 .4 -- -- 1 24 102 3

*Complete medium = 0 .6 of O .M . HAP, 0 .1% BYF 100, 2% dextrose, 0 .20

1:1121"04, 0 .6% (NH4)2HPO4, 0 .0125% MgSO4 7H2O, and tap water .
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TABLE I (CONTINUED)

**jdhole-culture fermented media for Nos . 219, 223, 234 and 244 were
given to Miss Long for cell-floc studies . Fermentation Nos . 244
through 257 were used to study lysis of the Arthrobacter cells by
activity of a mold, a species of Trichoderma .

***Each 10% inoculum was prepared in 30-gal . fermentor .

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II . LYsis of Arthrobacte_r Cells by_the Activity of

a Mold, a_Species of 'L-richoderma

Studies were again made on lysis of Arthrobacter (A) cells by culturing

with a mold, a species of Trichoderma . As reported in August, the results
after culturing the mold with an A fermentation showed that the mold produced
factors on enzymes capable of releasing the isomerase enzyme . Assays of the
supernatants showed yield of 1050 to 1120 p units/ml .

The procedure as described in the August report was followed to conduct

No . 245 for lysis studies . Assays of the supernatants after culturing A
fermentation (Age :48 hrs) with the mold for 29-30 hrs showed yields of y
1190 to 1250 .

Other fermentations that were carried out to study lysis by the mold
indicated that it would be necessary to add constituents other than
inorganics at the time of inoculation with the mold to insure there would
be mold growth and therefore lysis of the A cells .

When conducting No . 257, assays of A cells at 48 hrs showed a yield

of 1023 p units/nil . At that time, the pH of 5 .4 was adjusted to 5 .15
with 113PO4 . The 1% mold inoculum was then added along with 0 .1% dextrose
0 .03% O .M . HAP, 0 .01% BYF 100 and 0 .008 of KH2P04 . Incubation was continued
using the same conditions as for culturing A with one exception . The baclc
p7essurz of 1 1/2 to 2 lbs was not employed . Assay of the supernatant after
23 hrs of culturing with the mold showed a yield of 1248 u units/ml (Table II) .

TABLE II

DATA ON LYSIS OF ARTHROBACTE R_ CELLS

_ _ _

! BY TRI CHODERMA ~

Incubation Time (Hrs)* Yield

After Inoculation U Units/ml
With Mold Cells Supernatant

23 101 1248
+ 109 1 .248
24 43 1254
+ + 1278
28 + 1290
+ + 1304
30 + 1268
+ + 1310
32 4- 1290
J 1 1258

*Arthro_bacter fermentation was 48 hrs at time of inoculation

with mold and addition of constituents .
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Other fermentations to study lysis indicated there would be growth

of the mold and lysis of the A cells at t .imp nf inonu?ation laith n,nlrl
if the pH was adjusted to about 5 .2 and constituents added for mold growth .

Yields of 800-826, ] .024-1160, 1000 and 982-1042 were obtained when

investigating lysis for Nos . 253, 254, 255 and 256 respectively .

III . Scale-up of Isomerase Fermentation

Work was done with personnel at the Dawe's Fermentation Laboratories

to study scale-up of isomerase fermentations to 120 and 1300-gal
quantities .

Data that have been obtained at the R .J .R . Laboratories on methods

employed to conduct isomerase fermentations were referred to and used
to assist with the scale-up fermentations .

Yields that were obtained when fermenting the 120 and 1300 gal
quantities were in the 800-1000 U units/ml range which were agreeable
with yields obtained at the P. .J .R . Laboratories .

Lawrence E . Hayes