I
SIGMUND BERK, HELEN EBERT’, and LEONARD TEITELL Pitman-Dunn Laboratories, Frankford Arsenal, Philadelphia, Pa.
Utilization of Plasticizers and Related Organic Compounds by Fungi
.
M A N Y FUNGI and bacteria are able to use as organic nutrients the plasticizers contained in resin formulations. Berk (7) showed that poly(viny1 chloride) films plasticized with dibutyl sebacate were embrittled by mold attack, while those plasticized with dioctyl phthalate or butadiene-acrylonitrile were not affected. Brown ( 5 ) in an OSRD report on tropical deterioration, reviewed early literature on plasticizer attack by microorganisms; Stahl and Pessen (75, 76) covered more recent references. The liquid plasticizers, which usually have a considerably lower molecular weight than the plastic to which they are added, have been grouped (72) according to structure in two broad categories : linear ester plasticizers, which include the alkyl esters of phosphoric and certain dibasic and monobasic fatty acids; and polymeric plasticizers, which include polyesters of dicarboxylic acids and the polyglycols and their esters. Di(2-ethylhexyl) phthalate (DOP), the most widely used plasticizer, belongs to the first group, which also includes the sebacates, adipates, and ricinoleates, all of which impart low-temperature flexibility to plastic formulations. The sebacic acid polyesters and the poly(ethy1ene glycols) belong to the second group. When plasticizer or stabilizer is removedfrom resin films, as aresultof microbiological attack or otherwise, the films may become stiff and brittle (9,75), and increase in tensile strength and decrease in per cent elongation may occur (7). Although Brown’s report (5) rates a number of plasticizers according to susceptibility to fungus attack, there is a definite need to evaluate the new plasticizers which are being continually introduced. In the present report a number of compounds are rated as to their ability to serve as carbon sources for 24 species of fungi. Included are a large group of commercial plasticizers, a homologous series of adipates, and related compounds. Stahl and Pessen (75) recently reported the effects of chemical structure of a homologous series of sebacates on the ability of a fungus, Aspergillus versicolor, and a bacterium, Pseudomonas aeruginosa, to utilize the compounds as carbon sources. In the present work an attempt 1 Present address, Smith, Kline a n d French Laboratories, Philadelphia, Pa.
Table 1.
Ability of Fungi Used as Test Organisms to Utilize 90 Esters as Carbon Sources FA Culture Culture Av. Test Organism
Paecilomyces varioti Trichoderma sp. Aspergillus flavus Penicillium funiculosum Aspergillus ustus Aspergillus terreus Fusarium sp. Mucor sp. Aspergillus ustus Aspergillus ustus Curvularia genicutata Aspergi&~eoryzae Stemphylium consortiale Aspergillus niger Glomerella cingutata Penicillium citrinum Penicillium frequentans Alternaria solani Penicillium fuhiculosum Myrothecium verrucaria Penicillium chrysogenum Aspergillus versicolor Stachybotrys atra Pullularia pullulans
N0.O
Mediumb
GrowthC
72 69 70 71 467 473 21 34 491 493 408 456 45 1 81 419 402 457 406 492 29 468 483 240 482
3 3 4 3 4 4 4 3
3.4 3.3 3.2 3.1 3.1 3.0 2.9 2.9 2.7 2.7 2.7 2.6 2.4 2.2 2.2 2.0 2.0 1.8 1.7 1.7 1.6 1.3 1.1 1.0
3
4 3 4 3 3 3 4 4 3 3 3 4
1 3 3
Frankford Arsenal Culture Collection number. 3. Bacto potato dextrose agar. Ciapek’s medium (IS). Used to grow inoculum. Sum of diameters of colony growth (cm.) on esters tested number of esters O
* 1. Bacto Sabouraud’s dextrose agar.
is made to correlate the extent of fungus growth and the chemical structure of the compounds. Stahl and Pessen (75) used the weight of mycelial pellets in shake flasks as a criterion of the degree of susceptibility of the plasticizer to microbiological degradation. In the present work the plasticizers are dispersed in mineral salts agar? and the extent of
b
b b b
4 . Modifiration of
growth on the agar is used as a measure of plasticizer utilization ( 2 ) by the fungi.
Materials
Methods
Table I lists the fungi used and the culture media on which the inocula were grown for approximately 1 month. A complete list of the plasticizers and
Diesters of saturated, aliphatic dibasic acids can be utilized by fungi, if they contain 12 or more carbon atoms. The maleates are fairly fungus-resistant and the alkyl derivatives of phosphoric and phthalic acids do not serve a s carbon sources for fungi. The polyhydric alcohols can be used by fungi, if the hydroxyl groups are on adjacent or end carbon atoms. An ether linkage into the carbon chain decreases ability to support fungus growth. VOL. 49,
NO. 7
JULY 1957
1 115
Table II.
Ability of Fungi to Utilize Plasticizers and Related Organic Compounds as Carbon Sources
(Compounds incorporated in mineral salts agar, inoculated, and incubated for 2 weeks at 29' =t1' C.) Diameter of Colony Growth of Test Organisms (FA Culture No. Shown), Cm.a Aspergillus Penicillium Code No.
Compounds
ustus Sourceb 467
ustus 491
ustus 493
versicolor 483
terreus niger 473 81
flavus 70
junicfrequen- cri- chryso- funiculo- ulooryzae tans tinunz genum sum sum 456 457 402 468 71 492
Acids and Their Esters
1 1A 2 3 4
Adipic acid (pH = 2.5) Adipic acid alkalid Methyl adipate Diethyl adipate n-Propyl adipate
R
1
2
2
0
3
0
R
7 O
O
R
O
0 0 0
3 0 0 0
0 0
H
0 0 0 0
5 6 7
H
le 3
2"
Ze
9
Di-(1-methylethyl) adipate n-Butyl adipate n-Pentyl adipate Di-(1-methylbutyl) adipate Di-(2-methylbutyl) adipate
H
6 6"
3 3e 5 4e
2 4 5 3e
10 11 12 13 14
Di-(3-methylbutyl) adipate Di-(1-ethylpropyl) adipate Di-n-hexyl adipate Dihexyl adipate Di-(2-methylpentyl) adipate
H
4e
H H
6 6
38 4 3e
c
5
0
R
5
1.5 16
Di-(2-ethylbutyl) adipate Di-(l,3-dimethylbutyl) adipate Dibutoxyethyl adipate Dicapryl adipate Dioctyl adipate
R
8
17 18
19 20 21 22
23 24
+
Dioctyl adipate Diiso-octyl adipate Diiso-octyl adipate Diiso-octyl adipate Dinonyl adipate
25 26 27
Didecyl adipate n-Octyl decyl adipate Mixed n-octyl, n-decyl adipate 28 Azelaic acid (pH = 3.3) 28A Azelaic acid alkalid
+
29 30 31 32 33
Diethyl azelate Di-(I-ethylpropyl) azelate Di-(2-ethylbuty1)azelate Di-(Z-ethylbutyl) azelate Di-(2-ethylhexyl) azelate
Di-(2-ethylhexyl) azelate Esters of azelaic and pelargonic acids 36 Esters of azelaic and pelargonic acids 37 Citrazinic acid (pH = 3.5) 37A Citrazinic acid alkalid
34 35
+ 38 Fumaric acid (pH = 2.1) 38A Fumaric acid + alkalid 39 Glutaric acid (pH = 2.8) 39A Glutaric acid + alkalid 40 Itaconic acid (pH = 2.5) 40A Itaconic acid + alkalid n-Butyl itaconate 42 Maleic acid (pH = 1.6) 42A Maleic acid alkalid 43 Diethyl maleate
41
+
0 0
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.,
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R
2 5 1
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c
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c
R R
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4
2
4 5 0
0
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6 0 0
1 0 0 0 0
0 0
4s
..
5 8 6
5 0 0 0 0
Values for a single culture plate. C. Commercial plasticizer. E. Synthesized at Frankford Arsenal. R. From supplier of laboratory chemicals. sum of diameters of colony growth (cm.) c Average growth = number of fungi used d pH adjusted to 6.4 by addition of 10N NaOH. e Formation of clear zone in agar medium during incubation. f Data not available. Supported from 1 to 5 cm. of growth upon prolonged incubation- -i.e., from 3 to 13 weeks.
a
b
1 1 16
5 4 0 0 0
0 0
0
R H
0
I 0
INDUSTRIAL AND ENGINEERING CHEMISTRY
26 06 06
2 p
26
26
2 2"
16
2s
2
3e
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1
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1
2
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3
3
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5
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5 1 5
3 5 2
0
6 3
0 0
1 0
3
0 0 0
0 0 0
0 0 0
0 3 0
PLASTICIZER U T I L I Z A T I O N BY F U N G I Table II.
Ability of Fungi to Utilize Plasticizers and Related Organic Compounds as Carbon Sources (Continued) (Compounds incorporated in mineral salts agar, inoculated and incubated for 2 weeks a t 20' C.) # Diameter of Colony Growth of Test Organisms (FA Culture No. Shown), Cm.a Other Fungi
Fusarium Code
No. 1 1A 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 1s 19 20 21 22 23 24 25 26 27 28 28A 29 30 31 32 33 34 35 36 37 37A 38 38A 39 39A 40 40A 41 42 42A 43
Trichoderma
sp. 21
SP.
0 1 0 0 1' 2' 4 2' 7 3 6 3 4
0 0 0 0 2' 0 6 9 9 6 9 9 9 0
7 1' 0' 4 0' 9 0' 0e
0' 08
0' 0 0 8
5 0 9 3 5 0" 06 0.3 0'
0 4 0
4 5 6 0 7 1
2 0 0 0 0
69
0'
0 0 0' 9 0
0 0 0 0 0 0 9 9 0
..
3 9 0 0 0 0 9 9 0 0 8 0 0
0 5 0 0 0 0 0
St emphylMyroAlternaria Curvularia ium Pullularia Glomerella thecium geniculata consortiale pullulans cingulata verrucaria solani 419 29 406 408 451 482 0 0 0 0 0 0 0 0 0 9 0 0 0 0 0 0 0 0 0
0
0 0 1' 2' 2' 2' 1' 3 26 3' 2' 2' 4 1' 2' 0'
0 0 2 2e 5 4
0 6
0 6
4 5 7 3 0
3 2' 5 2 0
1' 1' 20
0' 0'
0
0
0
0
0
1
0
0
0
2' 3 3 2' 2' 2 4 4 2 2 3 1' 7 0 26
2' 26 0 1 1' 2 28 2 1 2
1' 1' 2 0 1' 0 2' 2 4' 0'
4 3 3 4' 3' 2 9 5 4 4
0
0' 1'
2 3 3 3 3' 1 4 0 4 4' 0' 26
0'
1' 1'
2' 1' 2e le
..
0'
0
0
0
4
6 9 0
6
1' 1 0
7
2 7 5 2 5 5 7 8
0 5 1' 4 2' 3 0 0' 5 7
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0
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0
0
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le
6 6 26 06
0
.... *.
2
.. .. 0 2 1 0 0 0
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.... 0
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1
0 0 6 0
0 0 0
0 0 0
e .
0
0
Oe
..
0
1"
0 0
*.
0
0
3 0 0
3'
0
Paecilomyces varioti 72 3 7
1
2 l e
3 2' 2' 1' 3' 2 2
6 0
.. 4 .. 0 le
0 4
06
..
0' 08
0'
2' 0' 0
4
28 8 4 4 20 2e 0 4 0 9
1'
0
0 5 0
0
0
0
0
3
0
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3
.. 0
1 0
0
0
0 0
0 0
..
0 0 0 0
e .
.... 0' ..
0
0 3
..
0
0'
0 0 4
1.1
..
1.2 0.7
..
0.1 5.5 5.2
0 9 4 7 5
9 .
..
4
......
06 06
..
..
2 2
1
7
.... ..
.. .. 8 .. 0 9
5 0 0 0 0
0.9 2.1 0 0 0.6 0.8 2.6 3.2 3.6 3.1 3.1 2.9 4.3 2.8 2.5 2.3 2.1 1.5 4.6 1.0 1.8
0
.. .. 8
0
0 8 9
1 1 0 0 0
.. ..
1' 0' 0' 0' 0
0
0 0
..
0'
2 2 2 5 2 2 6 6 0
4 4 5 8
0'
0
5
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le
.... 0
0
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1 4 3 6 6 3 5
1' 0' 1'
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5
4 46
2'
9
0' 0 0' 5
0 0
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0
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9
le
..
AverageC Growth
0
..
0
0' 0'
sp. 34
0 0 0 4
3
..
Mucor
0
5 4
0 0
0
Stachybotrys atra 240 0
0.4
0.5 6.3 2.2 4.8 3.4 3.1 2.0 1.5 5.5 5.9 0.2 0.4 3.0 3.1 1.8 3.2 1.6 3.4 0.1 0
0
0
0
0
(Continued on pages 7 118-19)
The organic compound was added to organic compounds evaluated as carbon sources appears in Table 11, together 300-ml. portions of sterile, melted (SO0 with experimental results. The comto 60' C.) FA No. 5 mineral salts pounds were used as received from the agar medium and blended for 3 minutes supplier without further purification. in a sterile Waring Blendor. The organic The compounds tested are arranged in compounds were not sterilized. The Table I1 in three major groups: acids carbon content of the medium for each (listed alphabetically) and their esters (in compound was maintained a t 0.8%. Apparently most of the diesters were order of increasing chain length and insterile, as very few culture plates had creased branching of side chains), polycontaminating organisms. Approxihydric alcohols and their esters, and mately 30-ml. portions of the dispersed miscellaneous compounds. The second and third groups are arranged accord- ' medium were poured into 9-cm. sterile ing to increasing chain length and chemiPetri dishes and allowed to solidify at cal complexity. room temperature.
FA No. 5 Mineral Salts Agar Medium Grams KHzPO4, 0.7 KzHPOd, 0.7
MgS04.7R~0,0.7 NHrNOs, 1.0
NaC1, 0.005 BeSO4.7Hz0, 0.002 ZnS04.7H20, 0.002 M n S 0 4 . 7 H ~ 0 ,0.001 Agar (Difco), 15.0 dissolved in 1000 ml. distilled water
The mineral salts agar medium had a p H of 6.4,which was not altered appreciably by addition of esters and alcohols. However, the organic acids produced a considerable decrease in the p H of the medium and these values are listed in Table I1 below the chemical name of VOL. 49, NO. 7
JULY 1957
1 1 17
Table II.
Ability of Fungi to Utilize Plasticizers and Related Organic Compounds u s Carbon Sources (Continued) (Compounds incorporated in mineral salts agar, inoculated, and incubated for 2 weeks at 29'
Code NO.
ustus
Compounds
Sourceb 467
Dibutyl maleate Di-(2-ethylhexyl) maleate 46 dl-Malic acid (pH = 2.2) 46A dl-Malic acid alkalid 47 Malonic acid (pH = 2.1) 44 45
+
+-
47A Malonic acid alkalid 48 Diethyl malonate 49 Methoxyethyl oleate 50 Tetrahydrofurfuryl oleate 51 Oxalic (pH = 1.2)
51A Oxalic acid
+
alkalid Ethyl oxalate 53 Isoamyl oxalate 54 Pelargonic acid (pH = 4.7) 54A Pelargonic acid alkalid 52
+
55
Ethyl phosphate %-Butyl phosphate Tributoxyethyl phosphate Trioctyl phosphate An alkyl aryl phosphate
56 57 58 59
Dibutyl phthalate Dioctyl phthalate Dioctyl phthalate Diiso-octyl phthalate Dinonyl phthalate
60 61 62 63 64
o
C R
R
0 0 4 0
R C C R
5 O 6 1 O
0 6
0
0
R
O
0
R
1
a.
R
0
0
0
0
0 0 1 0 0
R
O
R
O
C
0
0 0 0 0 0
0 0 0 0 0
0 0 0 0
0 0
on
ofl
0
0
0
0
Didecyl phthalate Mixed n-octyl, n-decyl phthalate Mixed iso-octyl, n-octyl, ndecyl phthalate Mixed phthalates Pimelic acid (pH = 3.2) R
65 66 67
68 69
70 71 72 73
o o o o o o o o o o
og
..
74
n-Hexyl pinate Methyl ricinoleate Methyl acetyl ricinoleate Methyl celloso'lve acetyl ricinoleate Butyl ricinoleate
75 76 77 78 78A
Butyl acetyl polyricinoleate Barium ricinoleate Zinc ricinoleate Sebacic acid (pH = 4.0) Sebacic acid f alkalid
C C C
79
c R
83
Dimethyl sebacate Diethyl sebacate n-Butyl sebacate Dibutyl sebacate Dioctyl sebacate
84 85 86 87
Dioctyl sebacate Diiso-octyl sebacate Dibenzyl sebacate Sebacic acid polyester
c
48
c c C
4 3 8
80
81 82
a
e f 0
Diameter of Colony Growth of Test Organisms (FA Culture N o . Shown), Cm.a ____ Aspergillus Penicillium . funicversifrequen- cri- chrgso- funiculo- uloustus ustus color terreus niger jlavus oryzae tans tinum genuna dum sum 491 493 483 473 81 70 456 457 402 468 71 492
c
c c c c c c c c c c c
0
0 0 3
0 0 3
0 1 0
6 Q
4 0
3 0
2
1 0
2 0 3 3 0
7 0
*.
7 0
0
0 0
0
0
0
0
2
4 4
9
1
1 1
5
2
4
2 0
4 0
2
1 0 9 9 0
7 0
1 0 8 8 0
0 0 0 0 0
0
0 7 5 0
0
18
10
0 0 0
0
0
0 0
0 0
0
0
0
0 0 1 0
0
0
0 0 0
0
0
0 0 0 0 0
0
2 0 0 0 0
ofl
0
0
0 0
6
.. 0
0 0 0 0 0 0
0
I,
0 0 1 2
3
0
1. 0 4 0
3
0
3 0
. a
5
6 0
1
2
0
0 1 0 0
0
0 0
5
0
0
0
0 0 0
0 0 0
0
0 0
0
4
2 3
I.
4
0
0
0
4
0
0
0
0
4
7
3
5 0
4
8 0
3 0
0 0
0 0
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1 0
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0
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1
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0
0
0
0 0
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0 0 0
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0
1 0 0
0
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0 0
0
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0 0 1 0
0
0
0
0
0
x
0 0
0
0 0 1
0
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0 08
0 0 0 0
0
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0
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0
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2
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0
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4
2
0 7 2
0
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0
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0
1
0 1
2 1
08
0 88
0
0
0
0
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8
4
4
4 4
8
9
0 3 3
3 4
9 9
3 3
3
9
2 8
2 7 5 5
3 2
06
0
2
5 8
6
7
4
0 9
6
7
3 3
8
9
9
8
3
5
C C
8 8
6 6
6 6
3 3
9
9
4
8
8 8
6
8
7" 8
4
5
8 8 8 8
6
3 6
8 8
9 5
a
9
3
9
8
9
8
4 6
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6" 8
9
7
46 4
6 8 8
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8 4 9 46 6
8
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1
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5
c
7
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5
4
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3'
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5 4 3
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4
3"
26
38 2 6 7
38 2
2e 0 7 2
4
3e 0
36
8
9 9
5 9
5"
6 6
Values for a single culture plate. C. Commercial plasticizer. H. Synthesized at Frankford Arsenal. R. From supplier of laboratory chemicals. sum of diameters of colony growth (cm.) Average growth = number of fungi used p H adjusted t o 6.4 by addition of 10N NaOH. Formation of clear zone in agar medium during incubation. Data not available. Supported from 1 to 5 om. of growth upon prolonged incubation-Le.,
1 1 18
0 0
09
c C
R
C.)
lNDUSTRlAL AND ENGINEERING CHEMISTRY
6
7 8
*.
9
5
from 3 to 13 weeks.
5 3 3 2
5
..
3 4
0 3 4
4
2 36 3 4
0
18
38
2 0 6
38
9
5
0 4
26 0 6 5
1e
1
0
4
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6 0 4 3
2 0
0 2 7 8 3"
2 0
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1
3e
0 3
4 7
4
9
4
2
4 5
P L A S T I C I Z E R U T I L I Z A T I O N BY FUNGI *
Table 11.
Ability of Fungi to Utilize Plasticizers and Related Organic Compounds as Carbon Sources (Continued) (Compounds incorporated in mineral salts agar, inoculated, and incubated for 2 weeks a t 29' C.) Diameter of Colony Growth of Test Organisms (FA Culture No. Shown), Cm.=
Fusarium Code NO.
44 45 46 46A 47 47A 48 49 50 51 51A 52 53 54 54A 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 78A 79 80 81 82 83 84 85 86 87
Trichoderma
SP. 21 0 0 5 8 0 9 0 1 9 0 0 0 4 0 0 0 0
SP. 69
0 0
0 0 0
0
0 0 0 0 0 0 0 0 0 0
0 9 9 9 9 9 2 9
..
0 18
4 6 5 0 ' 16
0 9 9
0 0
9 1 0
7 0 9
9 0 0 0 16
0 0
0 0
0 0 0 0
0 0 0 0
1 0
0
9 9 9 9 9 9 9 9 9 2 6 9 9 0 0 0 9 9
Other Fungi StemphylMyro~. Alternaria Curvularia ium Pullularia Glornerella thecium solani oeniculata consortiale wullulans cingulat a verrucaria 406 408 451 48 1 419 29 0 08 0 0 0 0 0 0 1 0 0 0 0 a 0 1 0 0 1 7 4 5 6 1 0 1 0 0 0 1 1 1 7 4 4 0 0 0 0 0 0 1 8 8 2 6 6 5 8 2 8 8 6 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 1' 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0s 0 0 0 0 0 0 0 0 a . 0 0 0 0 0 0 1 5 0 0 0 0 0 2 0 a 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 OQ 0 0 1 0 0 2 a *. 0 0 0 1 0 0 1 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 2 8 7 6 8 4 7 3 7 7 4 7 8 8 6 3 8 6 2 6 6 7 6 8' 8 2 6 6 6 0 6 4 4 6 3 6 5 6 8 9 6 6 ' 2' 26 0 2 6 0 1 0 0 16 0 1 0 0 0 26 2 2 26 2 2 ' 2' 4 24 2 1' 1 2 3 4 1 2' 0 3e 1* 2 0 3' 5 3' 16 3' 3 4 5 0 1 0 2 5 2 ' 6 4 4 7 7 6 2
..
..
..
..
..
.. ..
..
..
..
..
..
..
..
Paecilomyces varioti 72 0 0
7 0 0 0
0 9 9 0 0 0
Stachybotrys
atra 240 0
..
0 5
0
2 0 5 7 0 0
Mucor SP.
34 0 0 1
.. .. .. 0 .... 0 0 0 0
.. 0
0
0
0
.. ..
..
0 0
0 0
.. 0 ..
0
0
1
0 0
0
0 0
1 09
0 0 0 0 0 2
,. 8 8 8 6
4 5 8
7 6
2' 4 9
0 0 0 0 0 0 0 0
0 0 0
4 6 6 5 6 0 0 0 0
y* ..
.I
9 0 0
3'
0
0
9
..
..
16
..
AverageC Growth
0 0.4 2.3 3.6 0.4 2.9 0
5.6 6.2 0
0.2 0
0.9 0 0 0
0 0
.. 0 ..
0.1 0
1
0.4 0.1 0.2
.. ..
.... .... .. ..
0
.. .. ..
.... .. .... 9 9
4 3 6 8
..
4 4 6 9
0
0 0 0.4
0.3 0.7 0.9
0.1 6.0 6.4 6.5 6.1 6.5 4.5 6.8 4.8 4.6 1.0 2.9 3.9 4.4 1.7 2.6 1.6 5.5 6.8
(Concluded on pages 7 720- 7 )
x
the compound. The organic acids were evaluated also in a medium adjusted with sodium hydroxide to p H 6.4. The medium containing each compound was distributed in 24 culture dishes which were inoculated with spore suspensions of the 24 test organisms. The spores were harvested and freed from mycelial fragments by filtering through sterile glass wool. Each spore load was suspended in sterile distilled water and washed three times by centrifugation. The solidified agar plates were inoculated with the spore suspensions, by using a 2-mm. platinum loop. The
inoculated plates were incubated in an inverted position for 2 or more weeks in a room maintained at constant temperature (29' f1O C.).
Results and Discussion Table I1 gives the diameter of colony growth to the nearest centimeter for 127 chemical compounds dispersed in mineral salts agar and inoculated with 24 species of fungi. The values shown are for a 2-week incubation period, although many of the plates were kept for a longer time.
Table I1 also includes compounds which had no growth after a 2-week period but showed 1 to 5 cm. of growth after additional incubation of 1 to 13 weeks. I n certain .cases, such as the growth of a strain of Aspergillus ustus (FA 493) upon n-butyl succinate, there was definite but slight growth a t the end of 2-week incubation. However, the radial growth attained a diameter of 7 cm. on prolonged incubation. Evaluation of the fungus resistance of plasticizers may require an incubation of 12 weeks. Davis and Solowey (6) worked on the utilization of some organic compounds by VOL. 49, NO. 7
JULY 1957
1 1 19
Table II.
Ability of Fungi to Utilize Plasticizers and Related Organic Compounds as Carbon Sources (Confinued) (Compounds incorporated in mineral salts agar, inoculated, and incubated for 2 weeks at 29" C.) Diameter of Colony Growth of Test Organisms (FA Culture No. Shown), Cm.a Aspergillus Penicillium
Code
No.
Compounds
2LStUS Sourceb 467
ustus 491
ustus 493
wersicolor 483
terreus niger 473 81
-
junicfrequen- cri- chrvso- funiculo- uloflavus oryzae tans tinum genum aum sum 70 456 457 402 468 71 492
88
Polypropylene sebacate
c
7
6
7
3
7
6
6
7
5
45
5
7
3
89 90 91 92 93
Sebacic acid polyester Polyester Polyester Butyl stearate Butoxyethyl stearate
c
7
5@
0 4 5 8
1
8
8
5
4e 4
8
5
4
8
5
7 0
8 5
56 5 5
7
5
7 4 6 8 1
I
6 7 6
3 3 3 3 2
8
C c C
6 6 6 6 1
5 1
5 4
4 1
8 1
3e 3 3 3 1
94 94A 95 96 97 98 99
Succinic acid (pH = 2.4) Succinic acid alkalid Diethyl succinate n-Butyl succinate n-Amyl succinate Phenyl succinate Benzyl succinate
4 7 0
0 2 0 0
5
7 8
4 8
4
6
7
2 3
2 2
0
0
0
3 0
0
0
7 8 0
3 7 0
0
3 4 1 6
2 0 6
5
2 8 4 6
2 3
+
c
R
1
1
6 6 6
7
4 7
R R
O
0
R R R
4 9 7
le
le
18
16
26 2 6
2 6
le
0 2
7 0 4 3 1
3
3 2 2
1
5
2 5 4
1
*.
16
0
16
3 0 4
3
3 2 5
3 3 3
2 1 1 0
3 6 6 7 0
2 2 2 0
1
3
Polyhydric Alcohols and Esters
100 101 102 103 104
Ethylene glycol Tetramethylene glycol 1,3-Butanediol 2,3-Butanediol Diethylene glycol
105
Diethylene glycol dipelargonate Pentamethylene glycol 2,4-Pentanediol 2-Methyl-1,3-pentanediol 2-Methyl-2,4-pentanediol
106 107 108 109 110 111 112 113 114
115 116 117 118
119 120
121 122 123
124
2,5-Hexanediol Triethylene glycol Triethylene glycol diacetate Triethylene glycol di-(2ethylhexoate) Octylene glycol
2,5-Dimethyl-3-hexyne-2,5diol 3,6-Dimethyl-4-octyne-3,6diol Polyglycol Polyglycol Glycerol Pentaerythritol ester of caproic acid Dextrose Methyl phthalyl ethyl glycollate Ethyl phthalyl ethyl glycollate Butyl phthalyl butyl glycollate
R R
3 5
R
7 4
R
0
3 4 7
.. 0
3 4 4
..
0
..
3 1 3
.. 0 3 1
2 7 4 6 0
4 4 1 2 1
4
.. 0 0 0
5
..
1
6 2 2 2
0
0
4 4 4 4 0
6 3 0 0
3 2 1 1 0
3
4
1 0
2 0
0
4 2 0 1 0
0
0
0 0
0 1
0
0
1
6
4
4
0 0 3
1 5
0 1
1
1
2
C
6
R R
O O
R
2
6 2 0 2
R
O
1
2 0 2 1
R R R
O O 5
1 0 5
1 1 5
1 1 2
0
*.
0 6
c
o
0
0
0 0
0 0
0
0 0
0 0
0 0
0
O
0 0
0
R
2 0
0
3 0
0
R
O
0
0
0
0
0
0
0
0
0
0
0
0
R
O
0
0
0 0 0
0 0 0
0
1
0 0 0
0
0 0
8
6
1 1 3
0 0 0 7
0
1
0 3 0 2
08
0 0 5
9
8
6
2
2
0 0 8
0 0 2 3
c c
R
o o
0
0
1 0
1 4
2 0 0
0 0 0
6 7 .*
3 I 0
1
1
x
1
2 2 0
1 0
1
c
3
4
2 5
0 9
2
4
I
7
3 8
2
7
2 3
1
8
5 7
0
R
7
3
4
7
4
c c c
26
2e
20
0
16
0
1
0
0
26
18
2e
0
oe
26
2"
0
2
0
2
0
0
0
1'
06
le
o
3
0
0
0
0
0
0
0
0
0
0
0
c
o
0
0
0
OQ
0
0
0
0
0
0
0
0
c c
o o
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
2
Miscellaneous Compounds
125 126
127
Organic phosphate 0and p-toluene sulfonamides N-Ethyl, 0- and p-toluene sulfonamides
Values for a single culture plate. C. Commercial plasticizer. H. Synthesized a t Frankford Arsenal. R. From supplier of laboratory chemicals. sum of diameters of colony growth (em.). Average growth = number of fungi used pH adjusted t o 6.3 by addition of 10N NaOW. Formation of clear zone in agar medium during incubation. f Data not available. Supported from 1 to 5 cm. of growth upon prolonged incubation-i.e., a
Q
1 120
INDUSTRIAL AND ENGINEERING CH€MISTRY
from 3 to 13 weeks.
P L A S T I C I Z E R U T I L I Z A T I O N BY FUNGI Table II.
Ability of Fungi to Utilize Plasticizers and Related Organic Compounds as Carbon Sources (Concluded)
(Compounds incorporated in mineral salts agar, ipoculated, and incubated for 2 weeks a t 29' C.) Diameter of Colony Growth of Test Organisms (FA Culture No. Shown), C I ~ . ~
Fusarium
Trichoderma
sp.
SP.
21
69
88
9
9
89 90 91 92 93
9 9 9 9 0
94 94A 95 96 97 98 99
5
Code No.
Other Fungi StemphvlMyroAlternaria Curvularia ium Pullularia Glomerella thecium solani geniculata consortiale pullulans cingulata verrucaria 406 408 451 481 419 29
..
7
8
.. ..
8 8
8
8 0
7 *.
3
3
7
.. 8 8 2
7 9 9
100
9
5
9
0
6 5 6
7 7
5
6
0 5
18
18
6 0 9
2e 0 26
0
0 6 0 0 3 0 5
6
2 2
0
0
0
0 2 0 5
18
2
4
1
.. 1
0 5
0 0 0
110 111 112 113 114
2 1 8
0 2
0
0
115 116
0 0 0
0 08 0
0 9
0 9 0 .9 0
119
120 121 122 123 124 125 126 127
5 '
9 0 0 0
0 0 0
0
..
0
117 118
..
b
0 0 0 0
0 0
0
0
0
9 3 6 1 3
0 2
3 1
9
.. ..
I.
0 0 0
7
4
4
1 28 0
0 08
16
0 0 6 1 4 0
26
Oe
9 0 0
0
0
0
0
0
0
0 0
0 0
0 0 0
0
0
I=
0
.. .. ....
4
0
0
0
0
0
0
..
0
0
0 0 0 0 9 6
1
0
.. ..
0
0
0
5
3 0
..
5 0
..
1 8 0 0
..
..
0
..
0
0
0
2.9 4.1 3.9 4.3 0.3
8 0 2
1 4
..
.. ..
0
0 0 0
8
3.7 5.9
.... 4
..
1 0
....
3 9 8 9 3
1
0 0
8
0
7 8 0
1 3 0 0
..
5.1 5.6 6.0 6.2 2.2
1.3 3.1 2.2 4.4
1 8
1 3 9 4 1
.. .... .. 0
4
105 106 107 108 109
AverageC Growth
4 4 1
..
le
..
34
0
le
4
sp.
..
0
..
1
4
0
8
0 0
..
,. 0 2
8 0
..
40 6 6
le
2 1 6 8 3
5
..
....
5
2
..
4
,
Mucor
5.9
2" 2
8 6 8
101 102 103 104
e
9 8 0
Stachybotrys atra 240
4
c
8 0 2
Paecilomyces varioti 72
08
0 0
..
..
0 0 0
0
0 0
0
0
0
.. 2 1
.o 0 0
....
2 1 9
.. .... ..
3.6 2.0 1.0 1.1 0.5 0.4 0.6 4.3 0.3 0
0.1 0 0.4 0.2 6.0 2.4 6.3
0.7 0.8 0.3 0 0 0.1
h
Salmonella and concluded that prolonged
incubation may determine whether a particular carbon source can be utilized. Some fungi, after a period of time, can attack certain substrates that are not readily utilized by them (77, 78). This adjustment to the new environment is attributed to development of adaptive enzymes by the mold. Average growth (Table 11) varied from 0 to 6.8 cm., the latter value for zinc ricinoleate. Dextrose (No. 121 in Table 11) had an average growth value of 6.3. If all the organisms filled the dishes, the compound would have a maximum average growth value of 9 (the diameter
of the Petri dish is 9 cm.). Where one or more rapidly growing organisms filled the dish within the 2-week period, the average growth value is nominal as the mold could have produced a larger diameter colony if the dish had been larger. Only one plate was used for each plasticizer with each test organism. Experience at this laboratory has shown that replicate plates poured from the same batch of culture medium and inoculated with the same spore suspension the same day agreed within 0.2 to 0.3 cm. Other investigators have found the same reproducibility. Brancato and Golding ( 2 ) ran quintuplicate plates and reported
average diameters of colony growth in 0.01 cm. Even when the size of the spore inoculum was varied, the diameter of the colony varied no more than several tenths of a centimeter. As the results in Table I1 are reported only to 1 cm., it was not felt necessary to use several replicate plates. Instead, a greater number of organisms were tested. However, the reproducibility is not so good when replication occurs with different batches of culture medium inoculated with different spore suspensions on different days. Dextrose was incorporated in the mineral salts medium and the diameters of colony growth were measured at VOL. 49, NO. 7
JULY 1 9 5 7 .
1121
different times, with each group of plasticizers evaluated. The over-all standard deviation for all the organisms was 0.8 cm. Because most of the compounds evaluated were not soluble in the agar medium at room temperature, the culture media were opaque or turbid. A clear zone advanced before the mycelial growth in some of these opaque plates; these zones varied from a narrow peripheral band around a colony to almost a n entire plate. I n some plates with large cleared areas, the mycelial growth was confined to a small colony in the center of the dish. All of the fungi tested showed this clear zone on occasion, but Alternaria solani and Pullaria pullulans were outstanding in this respect. An illustration of a clear zone is given in Figure 1, which shows the utilization of diethyl azelate by Alternaria solani. At first the clear zones were observed on esters only. They were considered zones of hydrolysis produced by esterase activity of certain fungi, where the hydrolyzates could not be utilized as fast as they were formed. However, similar clear zones were observed on dibasic acids of higher molecular weight and less soluble dibasic acids such as azelaic and sebacic acids. T h e average growth values for the homologous series of aliphatic dibasic acids, with and without the addition of sodium hydroxide, are summarized in Figure 2. Free acids a t low pH values may be toxic (8) and low pH may influence the rate of growth. These effects were removed by adjusting the p H of the medium to 6.4 (the pH of the basal medium). The susceptibility of these compounds to fungus growth was thereby increased. Most of the fungi tested show growth passing through two maxima as the number of carbon atoms in the dibasic acid is increased from two to ten. The first occurs among acids having three to five carbon atoms. All organisms supported a t least 2 cm. of growth on succinic acid, The data are incomplete for acids beyond adipic in the series, but where pH was adjusted, there appears to be a second maximum in the vicinity of azelaic acid, which contains nine carbon atoms. In all cases except oxalic acid, the acids supported an average growth of at least 2 cm. when the p H was adjusted to 6.4. Fergus (7) showed that Penicillzum digitaturn was not able to utilize oxalic acid, and reported poor growth with media adjusted to p H 4.4 and containing fumaric and succinic acids. However, with the five species of Penicillia used in this work and with no adjustment of p H for the two acids, Table I1 shows that growth on succinic acid ranged from 2 to 7 cm., and on fumaric acid, from 1 to 5 cm.
1 122
Figure 1. Production of a clear zone b y Alternaria solani (FA 4 0 6 ) on agar medium containing dispersed diethyl azelate. Plate incubated 5 weeks
The ability of a n ester to support fungus growth, according to the results of the test, depends upon the structure of both the alcohol and acid portions of the molecule. In general, short-chain dibasic acids are poor carbon sources for fungus growth. Increasing the carbon content of the ester-either the acid or the alcohol portion-improves it as a carbon source. Isoamyl oxalate (Table
11) shows some mold growth, even though oxalic acid itself and ethyl oxalate support no growth. Usually the ethyl esters of the dibasic acids are not utilized as carbon sources, but lengthening the carbon chain of the acids results in the use of diethyl azelate and dimethyl sebacate as carbon sources by the fungi. From Table I1 it appears that a minimum of 12 carbon atoms is required be-
NO. OF CARBON ATOMS
ACID
2
OXALIC
3
MALONIC
4
SUCCINIC
5
GLUTARIC
6
ADIPIC
9
AZELAIC
IO
SEBACIC
n
ADJUSTED TO 6.4 H NOT ADJUSTED
0 Figure 2. tion
INDUSTRIAL AND ENGINEERING CHEMISTRY
'
1
2 3 4 5 A V E R A G E G R O W T H (cm,)
6
7
Average growth of 2 4 fungi upon dibasic acids after 2-week incuba-
PLASTICIZER U T I L I Z A T I O N BY F U N G I METHYL ETHYL n- PROPYL
I- METHY LETHYL n-BUTYL
-
n PENTYL I-METHYLBUTYL n-HEXYL I-METHYLHEPTYL
8- METHYLBUTYL 3- METHYLBUTYL
I- ETHYLPROPYL 2- METHYLPENTYL
2- ETHYLBUTYL 1,3- DIMETHYLBUTYL 2- ETHYLHEXYL ISOOCTYL NONYL DECYL
0
I
2
3
AVERAGE GROWTH
4
(crn)
Figure 3. Average growth of 24 fungi on dialkyl adipates after 2-week incubation Upper. lower.
Average growth on normal and 1 -methyl derivatives Average growth on more highiy branched derivatives
n- PENTYL I-METHY LBUTYL
2- METHY LBUTY L
I
3-METHYLBUTYL
I
I-ETHYLPROPYL
n-HEXYL
2- METHYLPENTYL 2-ETHYLBUTYL I ,3-01ME THY LBUTYL
AVERAGE GROWTH (cm)
Figure 4. Effect of branching on utilization of isomeric dialkyl adipates by 2 4 fungi after 2-week incubation Upper. Average growth on isomeric dipentyl adipates lower. Average growth on Isomeric dihexyl adipates
fore the aliphatic diesters can support fungus growth. Presumably, this is due to a decreasing extent of hydrolysis as the molecular weight of the ester increases. Thus, the concentration of alcohol in the medium is kept below the toxic level. There are cases where esterification, especially with short-chain alcohols, decreases the ability ,of a n acid to support fungus growth. Succinic acid and its esters are the outstanding examples. Apparently, the lower alcohols released by hydrolysis inhibit growth. Diethyl succinate, for instance, supports no growth, whereas even the free acid supports moderate growth. Brian, Grove, and McGowan (4)stated that living cells have a greater permeability to esters than to acids. In this case hydrolysis inside the cell may be the important factor. A homologous series of adipates was tested. I n Figures 3 and 4 IO-methylheptyl and 2-ethylhexyl adipates are used for dicapryl and dioctyl adipates, respectively, to denote the structure of the commercial compounds. When the alkyl derivatives of adipic acid are arranged in order of increased complexity of structure, the average growth of the 24 fungi tested increases with molecular weight for the normal and 1-methyl derivatives. Branching of the chain appeared to have little effect on utilization of butyl and pentyl adipates. Branching decreased growth on the hexyl adipates and on the octyl adipates. The iso-octyl, nonyl, and decyl adipates were commercial plasticizers, but the lower adipates were products of laboratory syntheses. Apparently, for fungi in general, the normal and 1-methylalkyl derivatives most readily support fungus growth. This finding is in agreement with that of Stahl and Pessen (75), who observed that Astergillus versicolor converted the n-alkyl sebacates into cellular material more readily than the branched-chain isomers. As the longer, aliphatic side chains of the ester-type plasticizers should approach hydrocarbons in nature. it is appropriate to compare here the effect of structure of the esters and hydrocarbons upon their utilization as carbon sources. The effect of hydrocarbon branching seems to be controversial. Zobell (79,20) reports that iso-octane (2,2,4-trimethylpentane)is utilized much more rapidly than n-octane by mixed cultures of soil and marine bacteria, and that, in general, the branched-chain hydrocarbons are more susceptible to microbial oxidation than normal or straightchain compounds. Other investigators (70, 77)have found that the reverse relationship holds for certain organisms. The effect of molecular weight is more VOL. 49, NO. 7
JULY 1957
1123
decreased the ability of the organic compound to serve as a carbon source for fungi. , The following six species of fungi produced good visual growth on ester-type plasticizers : Paecilomyces variotz, Trichoderma sp., Aspergillus Jlavus, Penicillium funiculosum, Aspergillus ustus, and Asfier-
ETHYLENE GLYCOL DIETHYLENE GLYCOL 2,3-BUTANE D I O L 14-BUTANEDIOL ( ’ T E T R A M E T H Y L E N E GLYCOL) I,3-BUTANEDIOL
gillus terreus.
I,5-PE N T A N ED10 L ( P E N T A M E T H Y L E N E GLYCOL)
Acknowledgment
2,4-PE N T A N E 0 I O L 2,5-H E X ANE D I O L T R I E T H Y L E N E GLYCOL 0
2
I
3
4
5
AVERAGE GROWTH ( c m )
Figure 5. Average growth of 24 fungi on diols after 2-week incubation definite. I t is a general observation (79) that the susceptibility of hydrocarbons to oxidation by microorganisms increases with chain length up to 15 to 20 carbon atoms. In the diols tested (Figure 5), the position of the hydroxyl groups has a significant effect upon ability to be utilized. Utilization seems to be favored by having the hydroxyl groups on adjacent or end carbon atoms. Although ethylene glycol is considered toxic to fungi (3),appreciable growth is obtained a t the concentration used in these experiments, 2% by weight. The introduction of ether linkages, as in the formation of polyglycols, considerably reduced the fungus growth supported by the diols tested. In turn, esterification of these polyglycols with certain acids improved them as carbon sources. As the ability to grow upon a chemical compound varies with the fungus, a carbon source may have a low average growth (Table 11), yet be very susceptible to fungus attack by a few organisms. From Table I1 it can be seen that the alkyl derivatives of phthalic and phosphoric acids do not serve as carbon sources for fungi. Although trioctyl phosphate and dibutyl and dioctyl phthalates show evidence of supporting fungus growth, the growth is not conclusive enough to class them as carbon sources. The maleates tested are not very susceptible to fungus attack. The fungi were not able to utilize maleic, oxalic, and pelargonic acids. Table I compares the 2 4 species of fungi according to their ability to utilize the esters tested. An average diameter for the growth of the fungus colony upon the esters is used to classify the organisms. Reese, Cravetz, and Mandels (14) screened 358 organisms from the Quartermaster Culture Collection to determine the range of organisms capable of growing on three esters. Assuming extracellular hydrolysis of the ester linkage to be the most probable mechanism 1 124
for the microbiological attack of esters, they reported the ability to utilize esters and fatty materials to be widespread among fungi. The present work includes a wide range of both fungi and plasticizers. Two fungi, Aspergillus versicolor and Aspergillus niger, commonly used in tests of the fungus resistance of plastic materials, do not produce as much visual growth upon the esters tested as many of the other fungi. From Table I1 it is seen that Aspergillus versicolor and Aspergillus niger were not able to utilize some of the plasticizers on which other species of fungi, such as Aspergillus terreus, Aspergillus j a v u s , Mucor sp., and Trichoderma sp., showed moderate to heavy growth. Therefore, conclusions concerning fungus resistance, which are based upon studies using one organism, may be misleading. Alternaria solani and Pullularia pulluluns, two organisms outstanding in their production of clear zones during incubation, appear near the end of the list. Conclusions
In the homologous series of aliphatic dibasic acids from oxalic to sebacic acid (pimelic and suberic acids not tested), all but oxalic were utilized as sources of carbon by fungi. All the diesters of the saturated, aliphatic dibasic acids tested, which contain 12 or more carbon atoms, supported fungus growth. As molecular weight is increased, the ability of the 1-methyl and normal alkyl adipates to support fungus growth is increased. I n a series of dihexyl adipates, the normal hexyl adipate served as a better carbon source than the branched-chain isomers. Of the diols tested, those which are unbranched and have hydroxyl groups on adjacent carbon atoms, or on the end carbon atoms, supported fungus growth best. Introduction of a n ether linkage, in the case of the polyglycols, considerably
INDUSTRIAL AND ENGINEERING CHEMISTRY
Appreciation is expressed to the Ordnance Corps, Department of Defense, for permission to publish this report. Special thanks are due Henry Gisser for supplying some of the diesters and to E. G. Simmons and associates of the Quartermaster Research and Development Center, Natick, Mass., for identification of some of the fungus cultures. Liferafure Cited (1) Berk, S., ASTM Bull. 168, 53-5 (September 1950). (2) Brancato, F. P., Golding, N. S., Mycologia 45, 848-64 (1953). (3) Ibid., 46,442-56 (1954). (4) Brian, P. W., Grove, J. F., McGowan, J. C., Nature 158 (4024), 876 ( 1946). (5) Brown, A. E., “Problem of Fungal
Growth on Synthetic Resins, Plastics, and Plasticizers,” Office of Scientific Research & Development, OSRD 6067 (October 1945). (6) Davis, F., Solowey, M., J. Bacterid. 59, 361-6 (1950).
( 7 ) Fergus, C. L., Mycologia 44, 183-99 (1952). (8) Ibid., 46, 543-55 (1954). ( 9 ) Harvey, J. V., Meloro, F. J., “Studies
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RECEIVED for review April 7, 1956 ACCEPTED January 2, 1957
