LIPOLYTIC MICROORGANISMS ASSOCIATED WITH COTTON-SEED

perature upon bacteriological and chemical changes infermenting cucumbers. N. Y. State Agr. Expt. Sta. Bull.No. 744, 33 pp.

PEDERSON, C. S., AND ALBURY, MI. N. 1953 Factors affectingthe bacterial flora in fermenting vegetables. Food Re-search, 18, 290-300.

PEDERSON, C. S., AND WARD, L. 1949 The effect of salt uponthe bacteriological and chemical changes in fermenting

cucumbers. N. Y. State Agr. Expt. Sta. Tech. Bull. No.288, 29 pp.

VAHLTEICH, H. W., HAURAND, C. H., AND PERRY, G. A. 1935Modern science applies itself to cucumber salting. FoodInds., 7, 334-336.

WUSTENFELD, H., AND KREIPE, H. 1933 Versuche ulber desweichwerden der Saueren Gurken. Deut. Essigind.,37 (10), 77-81.

Lipolytic Microorganisms Associated with Prime andDeteriorated Cottonseed

RUTH Y. MAYNE

Southern Regional Research Laboratory,' New Orleans, Louisiana

Received for publication May 7, 1956

Among the deteriorative processes occurring in thestorage of moist cottonseed, two types of production offree fatty acids have been noted in the oil of the seed.One is a slow development which seems to result fromthe action of the seed enzymes (Jensen et al., 1950); theother is a rapid development associated with the growthof microorganisms (vide supra; Christensen et al., 1949).In another seed, namely moldy corn, four of the fungiisolated were demonstrated by Goodman and Christen-sen (1952) to possess lipolytic abilities. However, therehas been no attempt to identify or enumerate the lipo-lytic organisms in a sample of cottonseed in order tocompare the numbers and kinds of such microorganismswith the fatty acid content of the oil of the seed. In thework reported here, a study was made of the conditionsof each of 28 cottonseed samples, as indicated by mois-ture, free fatty acid content, the general magnitude ofthe numbers of lipolytic and nonlipolytic microorgan-isms found in or on the cottonseed and able to grow onan oil emulsion medium, and the identities of the lipo-lytic organisms. In a later work (Mayne, 1956), thelipolytic activities of the identified or characterizedorganisms were measured quantitatively and grouped inrelation to the quality of the seed from which the organ-isms came.

MATERIALS AND METHODS

Cottonseed samples. Twenty-eight samples of cottonseed, mostly of the 1951 crop, were obtained from sixSouthern states. The history of each sample as receivedis shown in table 1. Seventeen of the samples had beenharvested recently; the other eleven samples had beenstored in various ways. It must be realized that even

I One of the laboratories of the Southern Utilization Re-search Branch, Agricultural Research Service, United StatesDepartment of Agriculture.

the samples in the newly harvested group had had anopportunity to undergo some change during the periodw-hile they were being mailed to the laboratory. Whenthe samples were analyzed and graded according to thegrading standards of the Federal Regulations (U.S.D.A.,1949) and the National Cottonseed Products Associ-ation (1955), the seeds fell into groups as follows: 7Prime Quality2, 14 Below Prime Quality, and 7 OffQuality. However, 12 of the samples contained less than1.8 per cent free fatty acids in the oil.The samples were placed, immediately after being

received, into a deep freeze box and held at -15 to-20 C until cultured. Because this step was expedientand there was a possibility that some organisms wouldbe killed by this temperature, counts are not acceptedas absolute. Comparison is made only of the generalmagnitude of the counts of a group of samples receivingone grade with the counts of another group of samplesassigned a different grade.Numbers of microorganisms. Flora from the whole

seed, rather than internal flora, were studied to avoidoverlooking any lipolytic organisms which might havesporulated outside the hull, yet might have sent theirmycelia and enzymes into the seed. Bacteria also werecollected, although they have been considered by manyworkers to be inactive at moisture contents which wouldbe in hygroscopic equilibrium wvith relative humidities

2 Prime Quality cottonseed contains not more than 1.0 percent of foreign matter, not more than 12.0 per cent of moisture,and not more than 1.8 per cent of free fatty acids in the oilin the seed. Below Prime Quality cottonseed contains 1.0 to10 per cent foreign matter, 12.0 to 20 per cent moisture, or 1.8to 12.5 per cent free fatty acids in the oil in the seed. Off Qualitycottonseed contains 12.5 per cent or more of free fatty acids,more than 10.0 per cent of foreign matter, or more than 20.0per cent of moisture. [N'ational Cottonseed Products Associa-tion (1955/56).]

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TABLE 1. Description of cottonseed samples as received

Sample Variety, Source ~~~~~~~~~~Storage Conditions Free Fatty MoistureSample Variety, Source Before Receipt Acids in the Content*

o 373 Delfos 9169, Mississippi Unknown or uncontrolled 0.5 7.2C 375 Empire, Mississippi Unknown or uncontrolled 0.6 7.2C 371 Stoneville 2B, Mississippi Unknown or uncontrolled 0.7 7.7C 374 Coker 100 Wilt, Mississippi Unknown or uncontrolled 0.9 7.3C 372 Deltapine 15, Mississippi Unknown or uncontrolled 0.9 7.7C 352 Plains, Tennessee Unknown or uncontrolled 0.4 13.7C 349 Paula, Missouri Unknown or uncontrolled 0.4 14.0C 351 Empire, Tennessee Unknown or uncontrolled 0.4 13.9C 350 Paula, Missouri Unknown or uncontrolled 0.4 17.0C 356 Coker 100 Wilt, South Carolina Unknown or uncontrolled 0.7 12.9C 354 Deltapine 15, Louisiana Unknown or uncontrolled 2.2 9.2C 357 Louisiana Unknown or uncontrolled 2.2 8.3C 355 Coker 100 Wilt, Louisiana Unknown or uncontrolled 3.3 7.2C 353 Coker 100, South Carolina Unknown or uncontrolled 3.4 10.4C 358 Louisiana Unknown or uncontrolled 3.6 9.6C 359 Louisiana Unknown or uncontrolled 4.7 9.4C 361 Louisiana Unknown or uncontrolled 5.7 8.7C 365 Storm Master, Texas delinted, sack storage (1 year) 0.9 7.0C 366 C. A. 119, Texas delinted, sack storage (1 year) 1.0 7.3C 385 Stoneville 2B, Mississippi.t Plant treated with 15% 100% R. H., 78 F (28 weeks) 5.2 14.2

maleic hydrazideC 388 Stoneville 2B, Mississippi.t Plant treated with 15% 100% R. H., 78 F (28 weeks) 8.9 14.5

maleic hydrazideC 382 Stoneville 2B, Mississippi.t Plant not treated 91% R. H., 78 F (28 weeks) 24.1 16.0C 386 Stoneville 2B, Mississippi.f Plant treated with 3% 91% R. H., 78 F (28 weeks) 26.1 16.9

maleic hydrazideC 381 Stoneville 2B, Mississippi.t Plant not treated 91% R. H., 78 F (28 weeks) 28.4 15.0C 383 Stoneville 2B, Mississippi.t Plant treated with 3% 91% R. H., 78 F (28 weeks) 29.0 16.0

maleic hydrazideC 380 Paula, Missouri 90% R. H., 76-78 F (5 months) 39.5 16.2C 378 Paula, Missouri 90% R. H., 78 F (3 months) 16.2C 379 Delfos, Mississippi 7 months cold room 76 F and 90% R. 47.7 19.1

H. (9 months)* Determined by Analytical, Physical-Chemical and Physics Section.t For complete description of these samples, see Lambou, Parker, and Carns (1956).

of less than 90 per cent. However, one of the samples didhave a sufficiently high moisture content (19.13 percent) to be in hygroscopic equilibrium with an at-mosphere of approximately 90 per cent relative hu-midity (Karon, 1947; Karon and Adams, 1948).

Before culturing, each sample was allowed to thaw,placed in a sterile, covered pan, mLxed and quarteredthree times. A 10-g portion of each sample was weighedaseptically, transferred to a mortar, and ground with apestle until it was observed that each seed was brokenand crushed to small bits. A mill could not be usedbecause the high moisture content of some samplescaused the oily seed and linters to adhere to the blades.The ground seed was transferred with 90 ml of a 0.05per cent aqueous solution of Tween 203 to a wide-mouthbottle and shaken mechanically for 20 minutes. Serialdilutions of the suspension were made as usual inprogressions of 10 up to a dilution of 1: 1 million; 1 mlof each dilution was used in duplicate pour plates. A

3 The use of trade names in this article is for identificationonly and implies no endorsement of manufacturer or product.

differential agar medium described by Goldman andRayman (1952) was selected to make it possible tocount and isolate the lipolytic microorganisms. Thismedium contained refined and bleached cottonseed oil(1.5 per cent), soybean phospholipid (Astec 41353),yeast extract, dipotassium phosphate, Nile blue sulfate,and agar, and was adjusted to a pH value of 7.2. In thepreparation of this medium, the oil, emulsifier, andwater were mixed in a Manton-Gaulin3 milk homogen-izer, and the emulsion was sterilized and added asep-tically to the other previously sterilized ingredients.The Goldman and Rayman medium had been com-

pared previously with the following media by culturingsix samples of cottonseed on each: tryptone glucoseextract agar; a modified Czapek's agar (Jensen et al.,1951) made with 1 per cent dextrose and primary potas-sium phosphate at pH 5.5; Goldman and Raymanmedium at a pH of 4.0; and Goldman and Raymanmedium at pH 7.2 but without oil. This comparisonindicated that the Goldman and Rayman medium atpH 7.2 was better than any of the other media except

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tryptone glucose extract agar for supporting the growthof different kinds of organisms, and either better or asgood for supporting the largest numbers of organisms.Tryptone glucose agar was slightly better for supportingthe growth of molds. Since the Goldman and Raymanmedium without oil supported growth the mere appear-ance of a colony on this medium was not an unqualifiedindication of lipolysis. It was considered that lipolysishad occurred wherever the color of medium, originallya salmon to purplish-pink, changed to blue, or whereverclearing of the oil emulsion was produced in a zonearound or under the colony.The cultures were incubated at 25 to 28 C for 7 days,

observations, counts, and transfers being made on thethird, fifth, and seventh days. Preliminary tests hadshown that counts in samples of the same lot of seedcould vary as much as 4- or 5-fold; therefore, onlydifferences greater than this order of magnitude shouldbe considered significant.

Lipolysis of microorganisms in pure cultures. Fromcultures of each cottonseed sample, two or three repre-sentative colonies of each type of lipolytic organism andoccasionally of nonlipolytic organisms were transferredfor isolation to other Goldman and Rayman plates, andfinally to glycerol agar slants. Notation was made as tothe intensity of lipolysis of the organisms in pure cultureand the cultural characteristics. An arbitrary scalebased on the size of the zone of blue color or of clearingof the emulsion, was devised to compare the activity ofthe organisms. The most lipolytic bacteria and moldswere chosen from isolates for a subsequent investigationin which their lipolytic activity was measured quantita-tively (Mayne, 1956).

Identifications of bacteria. All bacterial cultures weretaken from the mineral oil-covered stock tubes (Mortonand Pulaski, 1938) and streaked on nutrient agar plates.The colonies were described after incubation at 25 to26 C for one or two days, depending on their rates ofgrow-th. If more than one type were present, each wasidentified separately to determine whether it was avariant or contaminant. The usual descriptions andphysiologic reactions were determined for all cultures(Society of American Bacteriologists, Manual ofMethods for Pure Culture Study of Bacteria, 1936-54).For the aerobic sporeforming bacteria, the identifica-tion scheme of Smith et al., (1952) was followed. Forthe gram negative bacilli, which were possibly in thegenus Pseudomonas, the special characteristics notedin a publication and in an amended key by Haynes(1951, 1953) were observed. For those organisms whichwere thought to be Xanthomonas species, growth innutrient broth containing 5 per cent or 9 per centsodium chloride was determined also. Methods used,other than those mentioned above, include the follow-ing (incubation of cultures at 25 to 26 C):

Liquefaction of nutrient gelatin 3 days to 3 weeks at25.5 and 20.0 C.

Production of H2S in both peptone-iron agar and SIMagar (Difco Manual, 1953).

Production of indol in SIM agar and in tryptonewater tested with Kovac's solution.

Reduction of nitrate to nitrite tested after 1, 2, 4, 7,and 25 days, including a check for residual nitratewith zinc (Society of American Bacteriologists,Manual of M1ethods for Pure Culture Study ofBacteria, 1936-54), and also test in nitrate agarto observe possible gas formation.

Growth on Simmon's citrate 3 to 10 davs (DifcoManual).

Flagella stains were made according to Leifson's(1951) method.

Fermentation of sugars in Purple Broth (DifcoManual) and also in Smith's (1952) syntheticmedium.

Pigment production in Burton's medium (1948).Fluorescence production in King's medium (1948).Cellulose test in Omeliansky's medium (Fred andWaksman, 1928), and in the same medium plus 1per cent dextrose, and in Kellerman's medium(Kellerman and McBeth, 1912) with ground com-mercial filter paper.

Utilization of naphthalene according to Gray andThornton (1928) using ammonium sulfate in onemedium and potassium nitrate in another medium.

Fermentation of ethanol according to Berry et al.(1954).

Productioin of potassium-2-ketogluconate as de-scribed by Haynes (1951) but in still culture.

Identifications of molds. The molds were taken fromoil-covered stock cultures (Buell and Weston, 1947) andput onto potato dextrose and Czapek's sucrose agarplates. Slide cultures also were prepared according toLittman (1949). AMorphology of the various organismswere studied from these preparations.

RESULTS AND Discussiox

Niumbers of microorganisms. The numbers of lipo-lytic molds and bacteria and the total numbers of moldsand bacteria from each cottonseed sample are presentedin table 2. In several of the tests, complete counts werenot obtained because one or another type of organismgrew over the entire plate crowding out the rest of theflora. Notations also are made of the quality of the seedsamples as judged by moisture and free fatty acid con-tent. In comparing the presence of lipolytic microorgan-isms with content of free fatty acids in the oil of theseed, it may be seen that 8 of the 16 samples which hadover 1.8 per cent free fatty acids in the oil, containedover 10,000 lipolytic molds per gram of cottonseed;none of the other samples had as many of these molds.Ten of the samples with increased free fatty acids had

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TABLE 2. Numbers of microorganisms in cottonseedsamples

Sample No.

C 373C 375C 371C 374C 372C 365C 366C 352C 349C 351C 350C 356C 354C 357

C 355C 353C 358C 359C 361C 385C 388C 382C 386C 381C 383

C 380

C 378

C 379

Quality'

pppppppBtBtB$BtB$BBBBBBBBB0

0

0

0000

Total

Bacteria

thousands/g

337402506520014

140200

20,000180650

13,00010,00010,00011,00011,00010,0001,9002,7001,8001,000

15650490130<10§

Molds

thousands/g

62561260.1

<0.126652011070080

90060

3002,100

1446

6324

74,000130,00019,000

Lipolytic in MixedCulture

Bacteria Molds

thousands/gm

360130

30.70.6586

120160

2,6001,6001,9001,900

1206003009009160.528614? t? t? t

* P = Prime Quality; B = Below Prime Quality; 0 = OffQuality.

t Evidence of lipolysis not distinct-overgrown by otherorganisms.

t These samples have as low FFA content as prime seed.§ Overgrown by molds.

over 10,000 lipolytic bacteria per gram, but as many as

five of the other samples contained that many bacteria.On the other hand, only three of the samples with highfree fatty acids content had fewer than 10,000 of eitherlipolytic molds or bacteria per gram. In each sample,the percentage of bacteria which were lipolytic was

generally low, while the percentage of molds which were

lipolytic varied greatly, generally being more than 50per cent. Thus, the results indicate that the presence ofmany lipolytic organisms, especially lipolytic molds, isassociated with high free fatty acids content in the oilof the cottonseed.

Lipolysis tests of pure cultures. The activities in pure

culture of the isolates of organisms which had beenpicked from mixed cultures as appearing to be lipolyticwere rated according to an arbitrary scale devised forthe purpose. Of the 132 bacterial isolates, 16 were 2+,52 were 1+, 14 were i, and 50 were negative. Of the64 molds picked as lipolytic, 7 were 3+, 19 were 2+,

18 were 1+, 5 were i:, and 15 were negative. Four ofthe organisms picked as nonlipolytic were found to be1+. The reason for these differences of reactivity be-tween mixed and pure culture is not known. It is sug-gested that growth on artificial media may haveaffected organisms having weak or variable activity.The data of the counts were re-evaluated in terms of

these tests in pure culture since so many isolates werenegative. The eight samples with high free fatty acidscontent still had over 10,000 lipolytic molds per gram,and most samples had numbers close to the originalnumbers which were lipolytic in mixed culture. Oneother sample having high free fatty acids had at least21,000 molds which were lipolytic in pure culture butdid not appear so originally in the mixed culture. Amongthose 10 samples with high free fatty acids content andover 10,000 lipolytic bacteria per gram, all 10 had morethan 10,000 bacteria which were lipolytic in pureculture. However, among the five samples with low freefatty acids content and over 10,000 lipolytic bacteriaper gram in mixed culture, three had very many lessthan this number which were lipolytic in pure culture.

Therefore to recapitulate: of 16 samples with highfree fatty acids, 14 had more than 10,000 microorgan-isms, molds or bacteria or both, whose representativeswere lipolytic in pure culture; and of 12 samples withlow free fatty acid content, two had more than 10,000microorganisms whose representatives were lipolytic inpure culture. Only the most lipolytic isolates from eachsample of cottonseed were selected for identificationstudies.

Identification of lipolytic bacteria. Of 15 gram positivecultures originally isolated, one was found to be Bacillussubtilis, 12 were Bacillus pumilus, and two were notidentified. Two of the B. pumilus isolates were atypical,383B2a and variant b, and 388B4, in that they formeda pink pigment on gelatin and peptone iron agar. Threeothers of the original cultures of B. pumilus hadvariants, one (366B2b) of which was so different itcould not be identified. It differed from the other andmajor portion of the culture in these respects: (1) itformed punctiform, smooth, translucent colonies ratherthan rough and ridged; (2) the size of the rods was anaverage 0.5 x 1.4 to 3.0 , instead of 0.7 x 2.0 to 3.0 ,;(3) in the gram stain there was a suggestion of palisadeformation, but there were no metachromatic granules orpalisade formation shown in the methylene blue stain;(4) spores were absent; (5) in some media the culturewas yellow; (6) it did not grow well in nutrient broth;and (7) it was nonmotile. One of the unidentified grampositive bacilli, 386B5, a nonspore bearer, was similarto the B. pumilus cultures except for the lack of sporesand the lack of motility. No palisade formation wasnoted. The second unidentified culture, 357B2, formeda smooth punctiform colony, was almost coccoid, wasmotile with peritrichous flagella, had a fluorescent

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yellow color, and formed nitrite from nitrate. In allother tests, it gave negative reactions.Among the gram negative bacilli, 11 were in the genus

Pseudomonas, three of these having variants. Twocultures fit the key (Haynes, 1953) as Pseudomonasovalis. Other characteristics of these cultures were:

nitrate to nitrite reduction in one culture after longincubation; acid in glycerol and glucose, but not inmaltose, lactose, or sucrose. Four cultures, 349B6a andvariant b, 349B7a and b, 353B6, and 353B7, fit thenew key as Pseudomonas reptilivorous. They also were

similar to the description in Bergey's Manual of De-terminative Bacteriology (Breed, Murray, and Hitchens,1948). Since there may have been a question as towhether an organism having the reported habitat oflizards and Gila monsters would be in or on cottonseed,a close neighbor (Pseudomonas boreopolis) in the keywas considered. However, the organisms described heredid not attack naphthalene nor redden gelatin. Twoorganisms, 381B1 and 381B2, are considered to bePseudomonas aeruginosa because they grew at 42 C andproduced slime although they did not produce pigment.Three other cultures, considered unidentified as tospecies, 357B7, 375B5, and 385B2b, were also apyo-

cyanogenic. They grew well at 25 and 37 C but not at42 C. The last mentioned two cultures hydrolyzedstarch strongly, and produced acid in maltose but notin glucose. Although they fit the key as P. reptilivorous,and with only a few exceptions also fit the key as

Pseudomonas mephitica, they are not characteristic ofthe descriptions of either of these species in Bergqey'sManual of Determinative Bacteriology. Of the three setsof variants collected, two sets as mentioned above were

identified as P. reptilivorouts. Hoxvever, the other un-

identified set differed in some characteristics which are

important in species identification: 385B2a grew at42 C, produced only a slight slime, was only slightlyyellow, and utilized starch to a slight degree as com-

pared to its variant 385B2b which did not grow at42 C, was definitely yellow, and utilized starch strongly.The other 16 gram negative bacilli were very much

like members of the genus Xanthomonas. However, no

plant pathogenicity tests were made. These bacilli wvere

either pale yellowv or deep golden yellow, almost orange;

most of them had one polar flagellum, but some hadsome tufts of two to four polar flagella; all were prefer-ably aerobic. Eight of the isolates had rough andsmooth variants, making 24 cultures which could bedivided into six large groups according to their reac-

tions on starch, nitrate, and gelatin. Table 3 indicatesthe groups and their reactions. None of the organismswere exactly like the descriptions of the Xanthomonasspecies in Bergey's Manual, but some resembledXanthomonas pruni, Xanthomonas lactucae-scariolae,Xanthomonas beticola, or Xanthomonas papavericola.Although these bacteria fall into the category of

TABLE 3. Grouping of the Xanthomonas-like isolates

Group Reactions Isolates

I Gelatin liquefied, starch hydrolysis feeble, 3nitrate negative

II Gelatin liquefied, starch hydrolysis feeble, 1nitrate positive

III Gelatin liquefied, starch hydrolysis feeble, 11*nitrate very slightly positive

IV Gelatin liquefied, starch hydrolysis strong, 3nitrate late positive

V Gelatin liquefied, starch not hydrolyzed, 1nitrate slightly positive

VI Gelatin not liquefied, starch hydrolysis 1feeble, nitrate strongly positive

* One having a variant in Group IV and another having avariant in Group V.

Xanthomonas according to the key in Bergey's Manual,they are not considered to be in this genus by Dr. M. P.Starr (private communication) on the basis of color,slime production, and odor. Complete descriptions ofeach group wvil not be given here since all but twogroups had similar quantitative lipolytic reactions asnoted elsewhere (AMayne, 1956).

Identification of lipolytic molds. Seventeen of the 41mold cultures were A spergillus flavus, Link, one beingatypicallv floccose with radiate conidial heads and pre-dominately single sterigmata.

Other cultures that were characteristically identifi-able to the species were: one Aspergillus terretus Thom;one Aspergilluts terretts var. floccosus Shih; two Asper-gillus awamori Nakazawa; one Aspergillus lutchuensisInui; two Fusarium moniliforme (Sheld.) Snyder etHansen; two Fusarium nivale (Fr.) Snyder et Hansen;one Fusarium roseum (Lk.) Snyder et Hansen; oneFusarium oxysporum (Schl.) Snyder et Hansen; oneFusarium episphaeria (Tode) Snyder et Hansen; twoPenicillium citrinum; one Penicillium lanosuim; onePenicillium roseopurpureum; and one Penicilliumpusillum (all penicillia were identified by Drs. C. R.Benjamin and C. W. Hesseltine). The last two men-tioned were extremely interesting since their restrictedgrowth and light color resembled the appearance ofactinomycetes.Three cultures were identified only to the genera; one

Ceratostomella sp.; one Diplodia sp.; and one Hormo-dendrum sp.

Four cultures were not identified but can be de-scribed. Culture 349M6 was similar in gross appearanceto Diplodia with raised fluffy and woolly dark my-celium with some white hyphae; septate mycelium withsharply broken dark hyphae, some with dark knoblikegrowths or excrements on the outer walls; no sporesbut many chlamydospores, some in chains.

Culture 357M1 was mostly black, flat and submergedhyphae in bundles with some rising w4-hite hyphae. The

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colony has the appearance of a black woolly carpetwith carbonaceous selerotia. Slide culture shows theparallel hyphae anastomosing at various points to formcell bridges and hyaline hyphae turning dark withbrown chlamydospores.

Culture 354M13 had dark submerged or surface my-celium and very fluffy high aerial mycelium that seemedto avoid the agar in the center of the plate. On Czapek'sthese aerial hyphae are gray, white, faint yellow, andpale peach. On potato dextrose they are all gray andwhite. The giant colonies have the same appearance aspictures of giant colonies of Rhizoctonia. Microscopicexamination showed septate hyaline and brown hyphaewith some smooth, heavy walled, irregular brown cells,some large cells with inclusions; young branches ofhyphae were very thin walled and the older ones werethick and had many cell bridges between strands, somehad thicker walls on one side than the other giving theappearance of a twisted strand, others had wartlikeexeretions, no spores.

Culture 354M2 had white mycelium with red andyellow pigment in various places, the reverse was red

TABLE 4. Identities of microorganisms isolatedfrom various grades of cottonseed

NO. TotalName No .GSNo.Gmrades of SourcessoaeSources

Penicilliu1lm roseopuirpureum 1 1 0*Penicilliumin puesillum ........... 1 1 0Penicillium lanosum ............ 1 1 0Penicilliuim citrinumn ........... 2 1 0Ceratostomella sp ............... 1 1 0Aspergillius awamor2i ............ 2 2 0Pseudomonas aeruginosa........ 2 1 0Gram positive bacilli ........... 2 2 0, BtAspergillus fiavus ............... 17 9 2B, 6-0, 1PtDiplodia sp..................... 1 1 BAspergilluts lutchuensis .......... 1 1 BAspergilluts terr?*eus ...... ........ 22 BFuisarium?li roseutm ....... ........ 11 BFusarium nivale ................ 2 2 BFutsarium)lmtoniliforme .......... 2 2 BPseudomlonas ovalis ............. 2 2 2BBacillus subtilis ................ 1 1 BPseudomlonas reptilivorous .. 4 2 B, BP§Unidentified molds ............. 4 3 2B, BPHormodendrum)zsp .............. 1 1 BPFusariumnl episphaeria ........... 1 1 BPXanthonmonas-like bacteria.... ...16 9 8B, PPseudomiionas species ........... 3 3 2B, PBacillius pum?iluts ............... 12 7 3P, BP, B,

2-0Fuisariuio) oxysporum ........... 1 1 P

* Off Quality above 12.5 per cent free fatty acids, andabove 20 per cent moisture content.

t Below Prime Quality.t Prime Quality less than 1.8 per cent free fatty acids, and

12 per cent moisture content.§ Below Prime Quality because of high moisture content.

to rose. There were many chlamydospores, yellow-brown and rough, terminal and intercalary. There wereyellow thin walled cleistothecia, 17.5 ,, which lookedlike drops of liquid, hyaline asci 9.8 M, and hyaline,globose spores 2.1 Mu. Some chlamydospores were 10.5,u, with vacuoles of 1.4 or 2.1 A. Some vacuoles stainedblue (either fatty acid or protein) with Nile blue, othersdid not stain. None stained red with Nile blue or sudanII (neutral fat). In the mycelium, some large bodiesstained blue with the Nile blue. The knobs on thechlamydospores were of different sizes but mostly wereround. This culture, which has the general appearanceof a Fusarium, might be a lonascus.

Identities of organisms in various qualities of cotton-seed. The names of the most lipolytic organisms foundin the various types of seeds are listed in table 4. Mostof these organisms were found in seeds of high freefatty acid, appearing only occasionally in seeds of lowfree fatty acid. However, it was noted that the Bacillusspecies, B. subtilis and B. pumilus, were found in alltypes of seed. No one kind of lipolytic organism seemedto be associated exclusively with samples having highfree fatty acid content.

ACKNOWLEDGMENTS

The author is indebted to the following: (1) Dr. V. H.MIlcFarlane for suggesting the problem, (2) Dr. W. C.Haynes of N. U. R. B. for his advice on identificationof the pseudomonads, (3) Dr. M. P. Starr, Universityof California, for his observations concerning the yellowbacteria, (4) Drs. C. W. Hesseltine and C. R. Benjaminof N. U. R. B. for their identification and comments onthe Penicillium species, (5) Mrs. Vidabelle 0. Cirino,Miss Claire Lesslie, Miss Elizabeth R. McCall, Mrs.M. G. Lambou, and Mrs. N. S. Parker of S. U. R. B.for analyses of moisture and free fattv acids in thecottonseed, (6) Mr. James B. Dick, Mississippi; Dr.D. M. Simpson, Tennessee; Mrs. M. G. Lambou,Louisiana; Dr. C. H. Arndt, South Carolina; Dr. D. C.Neal, Louisiana; and Mr. Dalton Gandv, Louisiana, forsupplying samples of cottonseed, and (7) Dr. Ralph W.Planck for his aid in preparing the manuscript.

SUMMARY

The general quantity of microflora, the numbers oflipolytic microorganisms, and the identities of most ofthe lipolytic microorganisms contained in samples ofcottonseed with different amounts of free fatty acids inthe oil of the seed have been determined. Most (nine-tenths) of the numbers of cottonseed samples withlarge contents of free fatty acids in the oil containedlarge numbers, at least 10,000 per gram, of lipolyticmolds or lipolytic bacteria. None of the samples withlow amounts of free fatty acids in the oil containedlarge numbers of lipolytic molds, but some containedlarge numbers of lipolytic bacteria. Some of the lipo-

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LIPOLYTIC MICROORGANISMS ASSOCIATED WITH COTTONSEED

lytic bacteria in these good quality seed were includedin a group of lipolytic organisms which were eitherweak or variable in their activity.The lipolytic molds were identified as Penicillium

lanosum, P. citrinum, P. roseopurpureum, P. pusillum,Aspergillusflavus, A. awamori, A. luchuensis, A. terreustwo varieties, Fusarium moniliforme, F. nivale, F.roseum, F. oxysporum, F. episphaeria, a Diplodia sp.,a Hormodendrum sp., a Ceratostomella sp., and someunidentified species. The lipolytic bacteria wereXanthomonas-like organisms, Pseudomonas aeruginosa,P. ovalis, P. reptilivorous, other Pseudomonas species,Bacillus subtilis, B. pumilus, and a few unidentifiedgram positive bacilli.

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