APPLIED MICROBIOLOGY, Jan. 1973, p. 62-69Copyright 0 1973 American Society for Microbiology

Vol. 25, No. 1Printed in U.SA.

Cellulolytic Bacteria Associated with SloughingSpoilage of California Ripe Olives

ISHWARLAL B. PATEL AND REESE H. VAUGHN

Department of Food Science and Technology, University of California, Davis, California 95616

Received for publication 22 December 1971

Sloughing spoilage of California ripe olives during processing is characterizedby severe softening, skin rupture, and flesh sloughing. It was assumed thatcellulolytic activity was responsible for skin rupture and sloughing of flesh, andso a deliberate search was made for cellulolytic bacteria from olivesundergoing sloughing spoilage. A bacterium identified as Cellulomonasflavigena was highly cellulolytic, attacking filter paper, carboxymethyl cellulose(CMC) gel, and olive tissue. Other bacteria attacking CMC, but not filterpaper, enhanced the activity of the Cellulomonas strain when grown in mixedculture, although they did not, in pure culture, have any effect on filter paper.

These latter cultures (all degraded olive tissue) represented the genera

Xanthomonas, Aerobacter, and Escherichia. Other noncellulolytic bacteriabelonging to the genera Alcaligenes, Kurthia, and Micrococcus also were usedfor study of mixed culture fermentation of cellulose by C. flavigena. Cellobioseaccumulation at levels of 1.0% (w/v) and above suppressed growth of C.flavigena.

In a previous study (11), gram-negativepectinolytic bacteria were reported to be as-sociated with the sloughing spoilage of Cali-fornia ripe olives during processing. However,in spite of rapid softening, rupture of the skinand sloughing of the flesh were not observed.Because skin rupture and flesh sloughing aswell as generalized tissue softening are typicalsymptoms of this spoilage, it was postulatedthat free-living, cellulase-producing microbesmight also be associated with the deterioration.Therefore a deliberate search was made forcellulolytic bacteria from olives undergoingsloughing. This report describes the results ofthat search and clearly associates cellulolyticbacteria with sloughing spoilage of olives.

MATERIALS AND METHODSIsolation and culture media. The basal medium

used for growth and isolation of cellulose-decompos-ing organisms was the one described by Han andSrinivasan (4). The cellulosic substrates were What-man no. 1 filter paper and carboxymethyl cellulose(CMC) (type 7HF, Hercules Inc.). The latter wasused at a concentration of 2.0% as recommended byGoto and Okabe (3).

Enrichment, isolation, and purification. Thesloughed olives investigated were of commercialorigin and represented the Mission, Manzanilla, andSevillano varieties. The covering liquid was a diluteaqueous solution of lye, or salt, or both. About 1 ml of

this solution from the sloughed olives was inoculatedinto a test tube containing approximately 10 ml ofthe basal medium and a strip of filter paper so placedthat its top portion projected about 3/4 inch (ca. 1.9cm) above the surface of the liquid. After 5 to 7 daysat 30 C on a Rollor drum machine (model TC-5, NewBrunswick Scientific Co.), if cellulolytic bacteriawere present, a patch of yellow-pigmented materialappeared at the air-liquid interface of the paper.Then a small piece of the paper showing slightdisintegration at the air-liquid interface was trans-ferred aseptically with sterile forceps to a new tube ofthe same medium. This process was repeated sevenor eight times to enrich the cellulolytic organisms.Then the paper from the last enrichment was re-moved, macerated in a small amount of sterilephysiological saline solution, and streaked ontoplates containing nutrient agar, CMC agar (0.5%CMC in basal medium) plus 1.5% agar, and filterpaper agar (a plate of nutrient agar covered with afilter paper disc), respectively.

Representative colonies which developed on eachof these agar media were picked and inoculated intofresh filter paper medium and into CMC gel medium.Cellulolytic cultures were further purified by alter-nately plating to test purity and enriching in thefilter paper medium and CMC gel until pure cultureswere obtained.Two filter paper decomposing cultures were ob-

tained. On the basis of morphology and colonycharacteristics they appeared to be identical, so onlyone was retained for further study. Four cultureswhich attacked CMC gel but not filter paper were

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CELLULOLYTIC BACTERIA AND SLOUGHING OF OLIVES

also retained for further study. Three noncellulolyticcultures were isolated for mixed-culture fermentationstudies.

Identification of the bacteria. The culturesretained for further study were identified by conven-tional methods used for generic and species alloca-tion. General references included Breed et al. (1),Society of American Bacteriologists (9), and Sker-man (8). When necessary the original literature wasconsulted.

Preparation of crude cell-free cellulolytic en-zyme solutions. The one filter paper decomposingisolate was grown in the basal medium containing0.25% filter paper (w/v) or 1% CMC (w/v). Afterincubation for 5 days at 30 C with continuousshaking, cell-free preparations were obtained bycentrifugation in a Sorvall, Super Speed R.C.-2,automatic refrigerated centrifuge at 9,500 rev/min for15 min at 3 to 4 C. The clear supernatant fluid whichcontained the cellulolytic enzyme(s) was collected. A1-ml amount of 1% Merthiolate (thimerosal powder,N.F.) was added per 100 ml of solution. The solutionswere stored in a refrigerator until used.The four cellulolytic cultures unable to macerate

filter paper were grown in the basal medium contain-ing 1% CMC (type 7MF, Hercules Inc.). After incu-bation for 10 days at 30 C with continuous shaking,cell-free solutions were prepared by centrifugation at9,500 rev/min for 20 min at 3 to 4 C. The clearsupernatant fluid was collected and preserved asdescribed above.

Gravimetric determination of cellulolyticactivity. The growing 18- to 24-hr cultures wereinoculated into 250-ml Erlenmeyer flasks containing100 ml of basal medium and a limited amount offilter paper (110 to 120 mg per flask). After 5 days ofincubation at 30 C on a continuous shaker, the flaskswere removed, and their contents were filtered,washed, and dried in Gooch crucibles of 30-mlcapacity and medium porosity according to theprocedure of Lembeck and Colmer (5).

Viscosimetric determination of cellulolytic en-zyme activity. The activity of the crude enzymepreparations was determined by measurement of thechanges in viscosity of the CMC (type 7MF) sub-strates that were induced by the enzymes with anOstwald viscosimeter as described by Nortje andVaughn (7).The volume of crude enzyme solution was always

10 ml. The volume of 1% CMC (T7MF) was always 20ml. The reaction time was always 30 min. The effectofpH was determined at 30 C. The effect of tempera-ture was studied at pH 6.0 with the enzyme producedin the presence of filter paper and at pH 6.5 withCMC as the substrate.

Softening of olives by crude enzymepreparations. The possibility exists that cellulolyticdegradation of olive tissue can occur during theirstorage in salt brine as well as during the final stagesof processing when leaching with water is used toremove the lye used to destroy the bitter glucosideoeluropein. It was not possible in the laboratory toexactly duplicate the conditions existing in industry.However, the in vitro tests were designed to duplicateall but the physical pressure produced by 3- to 4-ft

depths of olives.Ability of the crude enzyme preparations to soften

olive tissue was done with Manzanilla ripe processfruit and with Sevillano variety olives from salt brinestorage with preparations adjusted to various pHvalues (range 4.5 to 8.0) with McIlvaine (6) bufferand preserved with Merthiolate. Controls, regardlessof the variety, consisted of olives, buffer solution ofthe desired pH, and Merthiolate to preserve themixture. Both of the varieties were submerged in theenzyme solutions for 5 days at 30 C. The ripe processolives were observed daily for signs of softening, skinrupture, and flesh sloughing by sight and feel. After 5days the Sevillano olives were put through the ripeprocesses (see Vaughn [101 and Cruess [2] for de-tails). After the lye had penetrated to the pit, theolives were leached with three to four changes ofwater each day for 5 days. Signs of disintegrationwere observed daily during the 15 days of processing.The Manzanilla ripe olives were desalted so they

would simulate olives during the washing periodused to leach the residual lye from the fruits duringthe final stages of processing prior to canning (seeabove). It is during the 4- to 5-day washing periodthat sloughing spoilage becomes apparent.The brine storage (7.0% salt, 0.35% total acidity as

lactic acid, and pH 4.85) Sevillano fruits during the15 days required for processing would lose all of thesalt and total acidity because of leaching, and thefinal pH would be in the range of 7.0 to 8.0 during thefinal washing period.

RESULTSSolely on the basis of the tests shown in

Tables 1 and 2, the eight isolates describedappeared to be in seven different genera andeight species of bacteria. The salient features ofeach follow.Cellulomonas flavigena. The C. flavigena

culture (DS) was the only strongly cellulolyticbacterium isolated. It disintegrated filter paperand CMC readily and caused skin rupture andflesh sloughing of olives. The characteristicsare identical to the description of C. flavigenafound in Bergey's Manual, 7th ed. (1).The genus Xanthomonas. The two cultures

representing the genus Xanthomonas causedliquefaction of CMC gel and disintegrated olivetissue, but did not attack filter paper. Theculture DB has been allocated to X. stewartii,although plant pathogenicity was not tested.The other culture (98C) was not allocated to aspecific species, although it resembled thedescriptions of X. pruni and X.maculifoliigardeniae found in Bergey's Manual(1), the only difference being that culture 98Cdid not digest milk.The coliform bacteria. The coliform bac-

teria culture 98A was able to liquefy CMC geland degrade olive tissue but not filter paper. Ithad all of the characteristics of Aerobacter

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PATEL AND VAUGHN

cloacae and was so allocated. The other culture(4A) also was cellulolytic to a degree, but was

the slowest of the five cellulolytic culturesstudied. Even though culture 4A utilized ci-trate slowly and formed only acid from lactose,it was allocated to the Escherichia intermediagroup. An alternative would be to call itParacolobactrum intermedium on the basis ofits anomalous lactose fermentation.Kurthia bessonii. Culture CA was not cel-

lulolytic, but was the only pectolytic organismfound in this study. This culture differs onlyslightly from the descriptions found in Bergey'sManual (1) and given by Skerman (8) for K.bessonii. The main differences are the variabil-ity of the Gram stain and the slight amount ofacid produced from carbohydrates.The Micrococcus. Culture CW was placed

in the genus Micrococcus solely on morphologi-cal grounds, but it could not be identifiedsatisfactorily even as to genus because of manyvariations in morphological and physiologicalcharacteristics. These characteristics, however,best match those of the genera Micrococcus or

Gaffkya as found in Bergey's Manual (1) or

Skerman (8).Alcaligenes faecalis. The other noncel-

lulolytic culture (AW) had all of the character-istics of A. faecalis as found in Bergey's Manual(1) and was so named. It is felt that neither A.faecalis nor the unidentified micrococcus playany important role in mixed fermentation ac-

celeration of cellulolytic activity as will beshown below.

All of the isolates involved in this study couldgrow in the presence of 8% sodium chloride(w/v) in nutrient glucose, tryptone, and yeastextract broth.

Cellulolytic activity. The results given inTable 3 compare the attack of C. flavigena on

filter paper in pure culture and in mixedculture with the other isolates. The degree ofdigestion (percent of solubilized cellulose) was

32.35% when C. flavigena was grown alone.Digestion increased about 25 to 50% when thebacterium was grown in mixed culture with theisolates able to use CMC. The noncellulolyticA. faecalis and Micrococcus sp. had little effecton increasing filter paper digestion.

Five of the eight cultures involved in thisstudy grew well on CMC-gel and liquefied it atvarying rates. However, because of the slow-ness of their reaction rates, C. flavigena was

used to produce crude enzyme to study theeffect of pH and temperature on cellulolyticactivity as measured by viscosimetry. Data on

the effect of pH are shown in Fig. 1; those on

the effect of temperature are shown in Fig. 2.

TABLE 3. Gravimetric determination of cellulolyticactivity

Wt of Final Degree of

Cultures filter wt of digestionpaper residues bilized(mg) (mg) cellulose)a

C. flavigena 115.3 78.0 32.35C. flavigena plus X. 118.3 21.5 81.82

stewartiiC. flavigena plus Xan- 119.7 22.1 81.54thomonas sp.

C. flavigena plus A. 113.2 24.8 78.09cloaeae

C. flavigena plus E. 117.5 50.9 56.68intermedia

C. flavigena plus K. 116.9 42.0 64.07bessonii

C. flavigena plus Mi- 115.6 70.6 38.92crococcus Sp.

C. flavigena plus A. 114.8 73.4 36.06faecalis

aDegree of digestion of cellulose was measuredgravimetrically after the organisms were inoculatedinto 100 ml of media containing filter paper (What-man no. 1) as a sole source of carbon. Inoculum was1.0 ml of cell suspension for single-culture and 0.5 mlof each for mixed-culture fermentation. Incubationwas for 5 days at 30 C on a continuous shaker.

The cell-free crude-enzyme filter paper prepar-ation was most active at about pH 6.0 and hadgood activity in the range of pH 5.0 to 7.0,whereas the preparation obtained with CMC asthe substrate was most active at about pH 6.5and had good activity in the range of pH 5.0 to7.5. As shown, the optimum temperature foractivity of the crude enzymes was 50 C regard-less of the substrate.Degradation of olive tissue. Crude, cell-

free enzymes prepared from the cellulolyticbacteria all softened desalted Manzanilla ripe-olive tissue as shown in Table 4. In the range ofpH 7.0 to 8.0 the softening was pronounced,and there was skin rupture and flesh sloughingtypical of the spoilage as observed under com-mercial conditions.Nearly identical results were obtained with

brine storage Sevillano olives that were treatedwith crude, cell-free enzymes and then wereput through the ripe-pickling process. Figure 3shows in vitro sloughing spoilage.

Softening and skin rupture of olives sub-jected to the crude enzymes was consistent andinvolved all of the olives tested to a greater orlesser degree. In contrast, the control olivesshowed no evidence of softening or sloughing.

Cellulolytic activity of pectolytic bacteriapreviously studied. Seventeen of the 19 cul-

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VOL. 25, 1973 CELLULOLYTIC BACTERIA AND SLOUGHING OF OLIVES

TABLE 4. Softening of California ripe olives by crudebacterial enzyme preparations

Crude enzyme produced by:

pHofEenzyme X. ste- Xantho- A. clo- E.prepar-

in wartii monas sp. acae mediaation media____

Aa Bb A A - A A

4.5 _C _5.0 _ _ _ _ _ _5.5 c 4 4 -_6.0 +cC + + ± ±6.5 + + + + + +7.0 ++C + + + + + + + +7.5 ++ ++ ++ ++ ++ ++8.0 ++ ++ ++ ++ ++ ++

a Enzymes produced in the basal medium with 1%CMC (type 7MF).

'Enzymes produced in the basal medium with40 5I0 60 7-0 80 90 0.25% filter paper (Whatman No. 1).

pH c -, Indicates no softening after being submergedin the enzyme preparations for five days at 30 C; ±,

FIG. 1. The effect of pH on the activity of the indicates slight softening at ends; +, indicates mod-cellulolytic enzymes produced by C. flavigena. 0, erate softening; ++, indicates characteristic soften-Filter paper substrate; A, carboxymethyl cellulose ing with tissue breakdown.su bstrate.

tures described in 1969 (11) were still availablein the laboratory culture collection. Thesecultures were examined for their ability to

50-_ / 8 \attack CMC gel, degrade cellobiose, and dis-integrate filter paper.

A/8 \ None of the cultures could disintegrate filterpaper but, surprisingly, 11 of the cultures could

40-_ Hecompletely liquefy CMC gel, and 14 decom-

posed cellobiose with acid or acid and gas>_ / \production, depending on the genus. Thein Lo\ \Achromobacter species culture 178, previouslyQ 0 shown to be extremely pectolytic, caused com-30_ plete liquefaction of CMC gel in 3 days, the

> r X X same time required by C. flavigena.Z 0 Five of the cultures including AerobacterX) aerogenes 43, Aeromonas liquefaciens 46,°n 2>Achromobacter sp. 178, E. intermedia 177 and82 / \Paracolobactrum aerogenoides 38 were growne inoin mixed culture with C. flavigena in the basal

medium with filter paper strips for 5 days at 30C on the Rollor drum agitator. The mixed

10-_ \ l cultures had the same pronounced effect inincreasing solubilization of filter paper cel-lulose over that produced by C. flavigena inpure culture as was observed with the cultures

I I I I I isolated in this study.0 10 20 30 40 50 60 70

TEMPERATURE (CC) DISCUSSIONFIG. 2. The effect of temperature on the activity of

the cellulolytic enzymes produced by C. flavigena. O, Association of cellulolytic bacteria withFilter paper substrate; A, carboxymethyl cellulose sloughing spoilage of olives confirms the specu-substrate. lation by Vaughn et al. (11) that cellulolytic

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FIG. 3. In vitro softening and sloughing of olives.

microbes might cause the skin rupture andflesh sloughing so characteristic of this spoil-age. Some of the pectolytic bacteria previouslyassociated with softening, but not sloughing,were also found to be cellulolytic and alsoincreased cellulose solubilization when grownin mixed culture with C. flavigena. The cul-tures, pectolytic or not, that degraded CMC alldecomposed cellobiose. A concentration of 1.0%cellobiose (w/v) or more was found to inhibit C.flavigena. It is possible that the other bacteriafavor the activity of C. flavigena by keeping thecellobiose at a low level. However, noncel-lulolytic, cellobiose-degrading yeasts grownwith C. flavigena did not increase solubiliza-tion of the filter paper so this explanation issuspect. Another possibility is that the bacteriaunable to decompose filter paper cellulosesupply micronutrients to C. flavigena. Thislatter speculation is suspect, because the twononcellulolytic, cellobiose-negative bacteriaused in this study did not materially increasecellulose degradation when grown in mixedculture with C. flavigena. It is also possiblethat, once C. flavigena has degraded the filter

paper to a certain degree of polimerization, theCMC-degrading bacteria can attack the lesserdegree of polimerization cellulose and thusincrease the total solubilization of the filterpaper cellulose. A concise explanation obvi-ously is dependent on further research. In anyevent, both cellulolytic and pectolytic enzymesproduced by various bacteria are involved insloughing spoilage of olives.

C. flavigena and the other cellulolytic cul-tures also caused marked tissue destructionwhen grown in sterile olives in brine.

ACKNOWLEDGMENTS

This investigation was supported by the Olive Adminis-trative Committee administering Federal Olive Order no.932.We thank H. J. Phaff of this Department for cellobiose-

degrading yeast cultures.

LITERATURE CITED

1. Breed, R. S., E. G. D. Murray, and N. R. Smith. 1957.Bergey's manual of determinative bacteriology, 7thed. The Williams & Wilkins Co., Baltimore.

2. Cruess, W. V. 1958. Commercial fruit and vegetableproducts, 4th ed. McGraw-Hill Book Co., Inc., NewYork.

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3. Goto, M., and N. Okabe. 1958. Celluloytic activity ofphytopathogenic bacteria. Nature (London) 182:1516.

4. Han, Y. W., and V. R. Srinivasan. 1968. Isolation andcharacterization of a cellulose-utilizing bacterium.Appl. Microbiol. 16:1140-1145.

5. Lembeck, W. J., and A. R. Colmer. 1967. Effect ofherbicides on cellulose decomposition by Sporocyto-phaga myxococcoides. Appl. Microbiol. 15:300-303.

6. McIlvaine, T. C. 1921. A buffer solution for calorimetriccomparison. J. Biol. Chem. 49:183-186.

7. Nortje, B. K., and R. H. Vaughn. 1952. The pectolyticactivity of species of the genus Bacillus: qualitativestudies with Bacillus subtilis and Bacillus pumilus inrelation to the softening of olives and pickles. FoodRes. 18: 57-69.

8. Skerman, V. B. D. 1967. A guide to the identification ofthe genera of bacteria, 2nd ed. The Williams &Wilkins Co., Baltimore.

9. Society of American Bacteriologists. 1957. Manual ofmicrobiological methods. McGraw-Hill Book Co.,Inc., New York.

10. Vaughn, R. H. 1946. Olives, p. 1-73. In Processed foods,vol. 4, part 2. Quartermaster Food and ContainerInstitute for the Armed Forces, Military PlanningDivision, Office of the Quartermaster General.

11. Vaughn, R. H., A. D. King, C. W. Nagel, Henry Ng, R.E. Levin, J. D. MacMillan, and G. K. York II. 1969.Gram negative bacteria associated with sloughing, a

softening of California ripe olives. J. Food Sci.34:224-227.

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