Method for testing microbial degradation of cellulose

Changes in the optical density and viscosity measurements of a water-soluble cellulose derivative which is enzyme depolymerizable can be used as a method to determine growth of cellulase-producing microorganisms and cellulose degradation. The method is particularly well suited to the measurement of the effect of pesticides on cellulose degradation.

BACKGROUND OF THE INVENTION 
The Environmental Protection Agency requires data on pesticidal effects 
upon microbial growth and function to support registration of a pesticide 
for commercial application. The growth of cellulase-producing 
microorganisms which degrade cellulose is particularly important for the 
eco-system. Therefore, a need exists for an accurate and economical method 
for testing a pesticide's potential inhibition of cellulase activity and 
cellulose degradation. 
PRIOR ART 
Several techniques have been developed for measuring cellulose degradation. 
One method involved the use of dye indicators. Cellulolytic fungi 
uncoupled blue dye from dye-bound cellulose powder during incubation, and 
the free dye diffused into the basal layer of medium. The amount of dye 
released, and the speed of release, appeared related to the degree of 
cellulolytic ability of individual cultures. See R. E. Smith, "Rapid Tube 
Test for Detecting Fungal Cellulase Production," 33 Applied and 
Environmental Microbiology 980 (1977). 
Another method involved measurement of the depth of clearing of opaque 
columns of an agar medium containing a partially crystalline cellulose 
preparation inoculated with fungi. As the organisms grew, they secreted 
cellulolytic enzymes which hydrolyzed the cellulose substrate. This 
created a sharply defined clear zone in the opaque medium beneath the 
growing culture. See G. S. Rautela and E. G. Cowling, "Simple Cultural 
Test for Relative Cellulolytic Activity of Fungi," 14 Applied Microbiology 
892 (1966). The method is better suited to measurement of relative 
activities of various fungi than to changes in the activity of a 
particular species under varying conditions. 
Filter papers and disks have also been used to assay cellulase activity. 
See "Antibiotic Disks--An Improvement in the Filter Paper Assay for 
Cullulose," 20 Biotechnology and Bioengineering 297 (1978). 
Common methods of measuring the cellulase inhibitory effects of herbicides 
have utilized the disintegration of cotton thread and calico in soil. See 
E. Grossbard and G. I. Wingfield, "The Effect of Herbicides on Cellulose 
Decomposition," Herbicides and Soil Microflora, 236-253. The limitation of 
these methods inheres in the insolubility of the cellulose which restricts 
quantitative measurement of its disintegration. 
SUMMARY OF THE INVENTION 
This invention provides a quantitative method for measuring growth of 
cellulase-producing microorganisms and degradation of cellulose. The 
method utilizes a water soluble cellulose derivative to measure microbial 
cellulose degradation as a reduction in viscosity. The method is 
particularly suited to testing the effect of pesticides on microbial 
growth and function. 
DESCRIPTION OF THE INVENTION 
This invention relates to a method for measuring the growth of 
cellulase-producing microorganisms and degradation of cellulose. It is 
superior to previous methods because it provides for precise quantitative 
measurement. 
The cellulose component used in the method of this invention must be a 
water-soluble colloidal cellulose derivative. It is used as a growth 
medium when supplied with nutrients and inoculated with a 
cellulase-producing microorganism. Growth of the microorganism or 
cellulase production is determined by measuring increases in the optical 
density of the medium. Microbial cellulose degradation is measured by 
reductions in viscosity. 
Although any water-soluble colloidal cellulose derivative, which is enzyme 
depolymerizable, may be used as a growth medium, the method was tested 
using cellulose sulfate ester derivatives. These cellulose derivatives and 
their preparation are described in U.S. Pat. Nos. 3,702,843 and 4,141,746, 
and an article by Richard G. Schweiger, "New Cellulose Sulfate Derivatives 
and Applications," 70 Carbohydrate Research 185-198 (1979). 
The sulfate ester groups of this cellulose derivative are homogeneously 
distributed among the polymer units. Preferably, the degree of 
substitution is less than one. When the degree of substitution is greater 
than one, the sulfates are relatively resistant to enzyme degradation. 
Since the viscous properties of the cellulose are critical to the method of 
this invention, it may be noted that the colloidal cellulose sulfate 
esters used herein were viscous at 1.0% of an aqueous solution. Reductions 
in viscosity during cellulose degradation can be quantitatively measured 
with a viscoso-meter. 
The colloidal cellulose sulfate ester derivative, described herein, can be 
prepared as a nutrient growth medium for the cellulase producing 
microorganisms as follows. An aqueous solution is prepared containing from 
approximately 0.1 to 10.0%, preferably 0.5 to 2.0% cellulose sulfate 
ester. To this solution a nutrient is added such as, from 0.01% to 20.0% 
yeast extract. Preferably, the yeast extract comprises between 0.1-2.0% of 
the solution. The solution may also contain up to 10.0%, but ideally up to 
2%, 2-amino-2-hydroxymethyl-1,3-propanediol. 
The medium is then inoculated with a cellulase-producing microorganism. 
Examples of such organisms include Trichodema viride and Cellulomonas 
biazotea. The latter microorganism was used to demonstrate the method of 
this invention.

The method of this invention for measuring the growth of the microorganism 
and cellulose degradation was tested according to the procedure described 
in Example 1. It was then tested to measure the effect of herbicides on 
cellulase growth and cellulose degradation as described in Examples 2 and 
3. 
EXAMPLE 1 
An aqueous medium was prepared containing 1.21% 
Tris(2-amino-2-hydroxymethyl-1,3-propanediol), 0.5% yeast extract, and 
1.2% colloidal cellulose sulfate ester having medium negative charge and 
viscosity of approximately 70-1600 cps. One-hundred milliliter (ml) 
portions of the medium were placed in 500 ml dented-bottomed flasks having 
gauze closures. The pH was adjusted to 7.5 by adding 38% hydrochloric 
acid. 
After sterilization by autoclaving for 20 minutes, each test flask of 
medium was inoculated with a 1 ml cell suspension of Cellulomonas 
biazotea. The cultures and 6 uninoculated control flasks were incubated at 
30.degree. C. and 150 rpm. 
Table I shows the viscosity and optical density measurements at 24, 48, and 
72 hour intervals. Assays of the uninoculated control flasks did not 
indicate a significant drop in viscosity. Assays of the inoculated flasks 
demonstrated degradation of cellulose by Cellulomonas biazotea as 
evidenced by the concommitant decrease in viscosity. Forty-eight hours 
after inoculation, viscosity dropped from an average of 155 centipoise to 
10 centipoise. After 54 hours it had dropped to 5 centipoise. 
Density increased from 40 Klett units 24 hours after inoculation to 264 at 
72 hours, indicating growth of the microorganism. 
TABLE I 
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Colloidal Cellulose Sulfate Ester 
Viscosity and Optical Density 
Hours 
0 24 30 48 54 72 
______________________________________ 
Viscosity (centipoise) 
Uninoculated cellulose 
155 -- 149 -- 140.5 
143 
Cellulose inoculated with 
-- 104 59 10 7 4 
Cellulomonas biazotea 
Density (Klett units) 
Cellulose inoculated with 
16 42 68 210 268 
Cellulomonas biazotea 
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EXAMPLE 2 
The method of this invention was used to test the effect of the herbicide 
S-ethyl diisobutylthiocarbamate on the growth of Cellulomonas biazotea and 
subsequent cellulose degradation of the colloidal cellulose sulfate ester. 
The herbicide can be prepared by the methods described in U.S. Pat. No. 
2,913,327. Inoculated batchs of the cellulose sulfate ester in 2% ethanol 
solvent were treated with 6, 12, and 60 parts per million (ppm) of the 
herbicide. The concentrations were considered equivalent to the maximum 
recommended 6, 12, and 60 pounds per acre (lb/A) application rates for the 
herbicide. 
Control groups contained (1) Cellulomonas biazotea inoculated cellulose 
alone, and (2) inoculated cellulose and 2% ethanol solvent. Test and 
control flasks were incubated under the same conditions as in Example 1. 
Viscosity measurements are shown in Table II. Optical density measurements 
are shown in Table III. At 66 hours after incubation the control cultures 
containing 2% ethanol and no herbicide showed a 70% loss in viscosity and 
a 30 Klett unit increase in optical density, indicating normal growth and 
function of the cellulase-producing microorganism. 
The viscosity and optical density of the test flakes containing herbicide 
at 6 ppm did not significantly deviate from the control group. 
Cultures with 12 and 60 ppm herbicide showed a decrease in optical density 
and less viscosity reduction. After 66 hours, cultures with 12 ppm 
herbicide had a viscosity reduction of only 85% of the control level and 
an optical density increase of only 47% of the control level. In cultures 
with 60 ppm herbicide the viscosity loss was further reduced to only 32% 
and the increase in optical density was only 12% of the control value. The 
results indicate that at elevated concentration levels the herbicide was 
inhibiting microorganism growth and cellulase production. 
TABLE II 
__________________________________________________________________________ 
Effect of Herbicide on Cellulose Degradation/Viscosity 
Viscosity (Centipoise)/Hours 
Medium 0 26.5 
42.5 
66 97.5 
118 
139 
__________________________________________________________________________ 
Uninoculated: 247 
-- -- -- -- -- 184 
Inoculated with Cellulomonas biazotea: 
No additives 175 
62 
8 -- -- -- 
2% ethanol added 195 
183 
70 -- -- -- 
2% ethanol and 6ppm S--ethyl 
203 
177 
69 18 11 -- 
diisobutylthiocarbamate added 
2% ethanol and 12 ppm S--ethyl 
205 
180 
97 30 15 -- 
diisobutylthiocarbamate added 
2% ethanol and 60 ppm S--ethyl 
203 
196 
178 
108 
-- 24 
diisobutylthiocarbamate added 
__________________________________________________________________________ 
TABLE III 
__________________________________________________________________________ 
Effect of Herbicide on Cellulomonas biazotea Growth 
Optical Density/Hours 
Medium 0 26.5 
42.5 
66 97.5 
118 
139 
__________________________________________________________________________ 
Uninoculated: 18 
-- -- -- -- -- 17 
Inoculated with Cellulomonas biazotea: 
No additives -- 
22 83 204 
-- -- -- 
2% ethanol added -- 
19 20 50 -- -- -- 
2% ethanol and 6 ppm S--ethyl 
-- 
17 19 49 114 
142 
-- 
diisobutylthiocarbamate added 
2% ethanol and 12 ppm S--ethyl 
-- 
19 23 33 75 118 
-- 
diisobutylthiocarbamate added 
2% ethanol and 60 ppm S--ethyl 
-- 
20 18 22 26 -- 109 
diisobutylthiocarbamate added 
__________________________________________________________________________ 
EXAMPLE 3 
The herbicide Naproamide, or 2-(.alpha.-naphthoxy)-N,N-diethylpropionamide, 
was also tested by the method of this invention to determine its effect on 
the growth of Cellulomonas biazotea and cellulose degradation. The 
herbicide which is commercially available as Devinol.RTM. can be prepared 
by the procedures described in U.S. Pat. No. 3,480,671. 
The measurements of cellulose degradation through viscosity reduction 
appear in Table IV. The measurements of the growth of Cellulomonas 
biazotea through optical density increases appear in Table V. 
TABLE IV 
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EFFECT OF NAPROPAMIDE ON CELLULOSE DEGRADATION (VISCOSITY) 
REDUCTION BY CELLULOMONAS BIAZOTEA IN COLLOID X-H2 BROTH 
Viscosity (Centipoise)/Time After Inoculation 
Colloid X-H2 Broth Additions 
Replicate 
Initial 
22.5 h 
29.5 h 
45 h 
52 h 
69 h 
__________________________________________________________________________ 
Uninoculated Sterile Medium 
A 149 -- -- -- -- 139.0 
0.1% ethanol B 154 -- -- -- -- 140.4 
Inoculated with Cellulomonas biazotea 
A -- 94 59 30 22 5 
0.1% ethanol B -- 94 61 29 18 7 
0.1% ethanol +3 ppm Napropamide 
A -- 109 59 29 -- 3 
B -- 92 64 29 -- -- 
0.1% ethanol +6 ppm Napropamide 
A -- 93 64 29 19 2 
B -- 105 69 34 18 2 
0.1% ethanol +30 ppm Napropamide 
A -- 120 63 35 19 8 
B -- 124 88 35 31 -- 
__________________________________________________________________________ 
TABLE V 
__________________________________________________________________________ 
EFFECT OF NAPROPAMDE ON THE GROWTH (OPTICAL DENSITY) OF 
CELLULOMONAS BIAZOTEA IN COLLOID X-H2 BROTH 
Klett Optical Density/Time After Inoculation 
Colloid X-H2 Broth Additions 
Replicate 
Initial 
22.5 h 
29.5 h 
45 h 
52 h 
69 h 
__________________________________________________________________________ 
Uninoculated Sterile Medium 
A 20 -- -- -- -- 20 
0.1% ethanol B 20 -- -- -- -- 20 
Inoculated with Cellulomonas biazotea 
A -- 65 120 220 
230 
250 
0.1% ethanol B -- 65 110 230 
236 
250 
0.1% ethanol +3 ppm Napropamide 
A -- 60 108 216 
-- 236 
B -- 63 101 218 
-- -- 
0.1% ethanol +6 ppm Napropamide 
A -- 65 100 198 
228 
236 
B -- 58 101 208 
228 
248 
0.1% ethanol +30 ppm Napropamide 
A -- 40 91 157 
195 
200 
B -- 48 66 150 
195 
-- 
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