Vol. 56, No. 9APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Sept. 1990, p. 2600-26050099-2240/90/092600-06$02.00/0Copyright C 1990, American Society for Microbiology

Hydrophobicity of Bacillus and Clostridium SporestK. MARK WIENCEK,1 N. ARLENE KLAPES,2 AND P. M. FOEGEDINGl.2*

Departments of Microbiology1 and Food Science,2 Box 7624, North Carolina State University,Raleigh, North Carolina 27695-7624

Received 30 January 1990/Accepted 10 June 1990

The hydrophobicities of spores and vegetative cells of several species of the genera Bacillus and Clostridiumwere measured by using the bacterial adherence to hexadecane assay and hydrophobic interaction chroma-tography. Although spore hydrophobicity varied among species and strains, the spores of each organism weremore hydrophobic than the vegetative cells. The relative hydrophobicities determined by the two methodsgenerally agreed. Sporulation media and conditions appeared to have little effect on spore hydrophobicity.However, exposure of spore suspensions to heat treatment caused a considerable increase in spore hydropho-bicity. The hydrophobic nature of Bacillus and Clostridium spores suggests that hydrophobic interactions mayplay a role in the adhesion of these spores to surfaces.

The role of hydrophobic interactions in the adhesion ofbacteria to the surfaces of inert materials has been addressedin recent studies (2, 22, 33). Both substratum hydrophobicityand bacterial cell surface hydrophobicity, as well as therelated parameters surface tension and surface free energy,mediate a nonspecific, reversible interaction which can leadto permanent adhesion. Effective colonization by the bacte-ria can condition the surface, allowing the attachment ofother organisms and the production of a complex biofilm (23,25). Bacterial adhesion is a beneficial phenomenon in fixed-film bioreactors, from which products are harvested withoutthe removal of bacteria. Conversely, adhesion has beenimplicated as a possible virulence factor for several patho-genic microorganisms that are important in the medical,pharmaceutical, and food industries. Hydrophobic interac-tions have been associated with the adhesion of bacteria tosurfaces in oral cavities (11, 34), contact lenses (26), surgicaland dental biomaterials (12, 28), polymers targeted for foodand pharmaceutical contact (39), and food (5).Although extensive data on vegetative cell hydrophobicity

and adhesion exist (2, 33, 38), relatively few studies havethoroughly examined the surface hydrophobicity of bacterialspores or the adhesion of spores to inanimate substrata (4, 6,9, 19, 31, 36). Bacillus and Clostridium spores are oftenimplicated in food spoilage and food-borne illnesses. Be-cause of their relative resistance to chemical and physicalsterilization agents, spores of these bacteria are used asindicators of sterilization efficiency for treatments involvingmoist and dry heat, UV irradiation, and hydrogen peroxide(14). Understanding the surface properties of bacterialspores and their interactions with inanimate substrata isimportant for selection of packaging materials and for eval-uation of the surface sterilization procedures used in thepackaging of food, pharmaceutical agents, and medical sup-plies.The hydrophobicity of bacterial spores can be determined

by applying methodologies used for measuring vegetativecell hydrophobicity. Established techniques for measuringsurface hydrophobicity include adherence to hydrocarbons(1, 32), hydrophobic interaction chromatography (HIC) (6,

* Corresponding author.t Paper number 12497 of the Journal Series of North Carolina

Agricultural Research Service, Raleigh, NC 27695-7643.

13), salt aggregation (36), and contact angle measurements(27). Because of the reported variability in the resultsobtained from these methods, several authors have sug-gested that more than one method should be used to studyhydrophobicity (18, 29, 33). In this study we used twodifferent methods, bacterial adherence to hexadecane(BATH) and HIC, to determine the relative hydrophobicitiesof spores of several Bacillus and Clostridium species and toevaluate the effects of heat and sporulation medium on sporehydrophobicity.

(This work was presented in part at the 89th AnnualMeeting of the American Society for Microbiology, NewOrleans, La., 14 to 18 May 1989.)

MATERIALS AND METHODSBacterial strains and cultivation. The strains of Bacillus

and Clostridium species used in this study are shown inTable 1, which also includes details concerning culturesources and sporulation conditions. Vegetative cell culturesof Bacillus spp. were grown in Trypticase soy broth (BBLMicrobiology Systems, Cockeysville, Md.). Clostridiumsporogenes ATCC 7955 and Clostridium putrefaciens ATCC25786 were cultured in reinforced clostridial medium (DifcoLaboratories, Detroit, Mich.). Clostridium botulinum 213Bwas grown in fluid thioglycolate medium (BBL). Overnightcultures of vegetative cells grown at the temperatures shownin Table 1 were used to inoculate sporulation media. Whenmaximum sporulation had occurred (at the times indicated inTable 1), spores were harvested, washed four to six times bycentrifugation (4,000 x g, 20 min, 4°C), and suspended indistilled water as described by Johnson et al. (15). Several ofthe spore preparations, including all Clostridium spp. prep-arations, required further cleaning until the concentration ofintact vegetative cells was less than 5%. To remove vegeta-tive cell debris, spore preparations in cold distilled waterwere sonicated with a probe sonicator (Branson S-110 Son-ifier; Heat Systems-Ultrasonics, Inc., Plainview, N.Y.),centrifuged through 55% sucrose, and washed three times incold distilled water.BATH assay. The relative hydrophobicities of bacterial

spores and vegetative cells were measured by using theBATH assay of Rosenberg (32). This method involves par-titioning a spore or vegetative cell suspension between anaqueous phase and the aqueous-hydrocarbon interface basedon the degree of bacterial surface hydrophobicity. Spore

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TABLE 1. Sources of cultures and sporulation conditions

Sporulation conditions

Strain Medium ~~~~~~~~~~~~~~~~~~~~~~~~~~CultureStrain Medium Temp Time sourcea

Composition Reference (OC) (days)

B. subtilis ATCC 6633 (crop I) Fortified nutrient agar 15 32 5 ATCCB. subtilis ATCC 6633 (crop II) Fortified nutrient broth (fortified nutrient 35 5 ATCC

agar without agar)B. subtilis ATCC 6633 (crop III) Glucose-salts 10 35 3 ATCCB. subtilis ATCC 6633 (crop IV) Modified fortified nutrient agar 7 35 4 ATCCB. subtilis ATCC 9372 (B. globigii)b Fortified nutrient agar 35 4 AMSCOB. subtilis ATCC 19221 Fortified nutrient agar 32 2 NCBSB. subtilis A Modified fortified nutrient agar 30 3 LindsayB. cereus T Fortified nutrient agar 30 2 NCSUB. stearothermophilus ATCC 7953 Supplemented nutrient agar 3 55 5 NCSUB. coagulans ATCC 8038 Brain heart infusion agar 19 43 3 ATCCB. coagulans FRR B666C Soya peptone agar 16 50 3 PflugB. megaterium ATCC 12872 Tryptone yeast agar 19 32 3 ATCCB. megaterium ATCC 33729 Tryptone yeast agar 32 3 ATCCC. botulinum 213Bcd Trypticase peptone medium 35 30 6 SwiftC. sporogenes ATCC 7955 Cooked meat mediume 37 3 K-VC. putrefaciens ATCC 25786 Cooked meat medium 25 7 ATCC

' ATCC, American Type Culture Collection, Rockville, Md.; AMSCO, American Sterilizer Co., Apex, N.C.; Lindsay, J. Lindsay, University of Florida,Gainesville; NCBS, North Carolina Biological Supply Co., Burlington, N.C.; NCSU, North Carolina State University, Raleigh; Pflug, I. J. Pflug, University ofMinnesota, Minneapolis; Swift, Swift and Co. Research Center, Oakbrook, Ill.; K-V, KabiVitrum, Clayton, N.C.

b This strain was identified as B. globigii by the American Sterilizer Co.Strain designation used by the culture source.

d Spores of this strain were prepared by P.M.F. in 1981 by using previously described methods (35) and were stored for 8 years in distilled water at 4'C.e Obtained from BBL Microbiology Systems.

suspensions or cell suspensions (overnight cell cultureswashed twice in 100 mM NaPO4 buffer, pH 6.8) at an A440 of0.8 to 1.0 were incubated for 15 min in a 35°C water bath.Spores were suspended in distilled water, and vegetativecells were suspended in 100 mM NaPO4 buffer (pH 6.8).Then 0.1, 0.2, 0.6, or 1.0 ml of hexadecane (Fisher ScientificCo., Dallas, Tex.) was added to 3.0 ml of each spore or cellsuspension. The mixture was vortexed (Vortex Genie 2;Fisher Scientific Co.; setting 5) for 1 min in round-bottomtest tubes (15 by 100 mm), and the hexadecane and aqueousphases were allowed to partition for 15 min. The aqueousphase was carefully removed with a Pasteur pipette. TheA440 of the aqueous suspension was measured with a modelUV-260 spectrophotometer (Shimadzu Corp., Kyoto, Ja-pan). The aqueous phase was monitored by phase-contrastmicroscopy for clumping or lysis of cells or spores that wasdue to hexadecane. The percent decrease in A440 for theaqueous suspension was calculated as follows: 100(AO -Af)/AO, where Ao and Af were the initial absorbance and finalabsorbance, respectively. For each trial, the index of hydro-phobicity was determined as the average percent decrease inabsorbance for the four hexadecane volumes given above.The hydrophobicities for the spores and vegetative cells ofeach organism are reported below as the means + standarddeviations for duplicate trials.

Standard curves relating A440 and spore concentration(phase-contrast direct microscopic counts) were preparedfor each strain of spores. These standard curves were usedto convert absorbance values to spore concentrations tocalculate the percentage of the spore population which washydrophobic.HIC. Duplicate columns of Sepharose CL-4B (Sigma

Chemical Co., St. Louis, Mo.) were prepared by usinglarge-volume Pasteur pipettes (Fisher Scientific Co.) pluggedwith glass wool and packed to a height of 25 mm (1.7 ml) asdescribed by Ismaeel et al. (13). Each column was washedseveral times with 4 M NaCl in 20 mM NaPO4 buffer (pH

6.85). The high ionic strength of the buffer was selected tomask the electrostatic repulsion between the spores and thecharged groups of the Sepharose CL-4B, thus allowinghydrophobic interactions to occur. Spores were centrifugedtwice and suspended in NaCl buffer at the desired concen-tration (A440, 0.3 to 0.6). Then 5 ml of the spore suspension,which was incubated at 35'C for 15 min, was passed throughthe column. The A440 of the eluent was measured. Thepercentage of adherence of spores to the hydrophobic gelwas calculated as described above for the BATH assay byusing averages from duplicate trials; these values are re-ported below as percent of hydrophobicity. The percentdecreases in spore concentration were derived from stan-dard curves of absorbance versus direct microscopic counts.

Effects of heat on spore hydrophobicity. Concentratedspore suspensions (ca. 109 spores per ml) of Bacillus subtilisA, B. subtilis ATCC 9372 (Bacillus globigii), and Bacillusstearothermophilus ATCC 7953 in distilled water wereheated in glass tubes at 75, 85, or 100°C for 10 min. Afterheating, the spores were rapidly cooled in an ice water bathand refrigerated overnight. The hydrophobicities of theheat-treated spores were measured by using the BATHassay described above and 0.1 ml of hexadecane; the valuesobtained were compared with the values obtained for theunheated controls.

RESULTS

Determination of spore and vegetative cell hydrophobicity.Figure 1 shows the percent hydrophobicity for spores andvegetative cells of each Bacillus and Clostridium strain ateach hexadecane volume tested. The resulting curves showthat spore hydrophobicity varied among species and strains.However, for each organism, the hydrophobicity of sporeswas greater than the vegetative cell hydrophobicity at eachof the four hexadecane volumes. Most vegetative cell andspore populations exhibited constant hydrophobicity at each

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2602 WIENCEK ET AL.

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-o

00 60-

40-

20

00.0 0.2 0.4 0.6 0.8 1.0 0.0 0.2 0.4 0.6 0.8 1.0

Hexadecane (ml)FIG. 1. Hydrophobicities of spores (closed symbols) and vegetative cells (open symbols) of Bacillus and Clostridium spp. as measured by

the BATH assay. Error bars indicate the standard deviations from duplicate trials. The percent hydrophobicity was the percent decrease inthe A440 of the aqueous phase that was due to partitioning with hexadecane. (A) Symbols: 1 and *, B. subtilis ATCC 6633 (crop I); A andA, B. megaterium ATCC 12872; 0 and 0, B. megaterium ATCC 33729. (B) Symbols: O and *, B. cereus T; A and A, B. stearothermophilusATCC 7953; 0 and 0, B. subtilis A. (C) Symbols: 0 and *, B. subtilis ATCC 19221; A and A, B. coagulans ATCC 8038; 0 and 0, B.coagulans FRR B666; V and V, B. subtilis ATCC 9372 (B. globigii). (D) Symbols: 0 and *, C. botulinum 213B; A and A, C. sporogenesATCC 7955; 0 and 0, C. putrefaciens ATCC 25786.

of the four hexadecane volumes. However, several vegeta-tive cell and spore populations had increased hydrophobic-ities at the higher volumes of hexadecane. The spores of B.stearothermophilus ATCC 7953, Bacillus megateriumATCC 33729, Bacillus coagulans ATCC 8038, B. coagulansFRR B666, and C. sporogenes ATCC 7955 increased at least40% in hydrophobicity as the hexadecane volume wasincreased from 0.1 to 1.0 ml (Fig. 1). The mean hydropho-bicity of each vegetative cell and spore population, asdetermined by the BATH method, is shown in Table 2. HICwith Sepharose CL-4B was also used to measure the hydro-phobicities of bacterial spores. Although phenyl-Sepharoseor octyl-Sepharose is commonly used as the gel matrix forHIC, the results of preliminary studies in which we usedphenyl-Sepharose as the gel matrix did not allow us todifferentiate among populations of spores with relativelyhigh hydrophobicities (data not shown). The percent hydro-phobicity as determined by HIC are shown in Table 2. Inmost cases, the spore hydrophobicity measured by HIC wassomewhat lower than the hydrophobicity determined byadherence to hexadecane. However, the two methods gen-erally produced similar results, especially with very hydro-phobic spores.

Effects of heat and sporulation conditions on bacterial sporehydrophobicity. The hydrophobicities of Bacillus sporeswere increased considerably by 10-min heat treatments in

distilled water (Fig. 2). Spores did not germinate during theheat treatment or the BATH assay as determined by phase-contrast microscopy. Each of the three strains testedshowed increased hydrophobicities at higher temperatures.No apparent effect of sporulation medium composition orsporulation temperature on spore hydrophobicity was ob-served (Tables 1 and 2). For example, the relative hydro-phobicities of four B. subtilis ATCC 6633 populations, eachsporulated in different types of media at different tempera-tures and for different lengths of time (Table 1), differed byless than 2% when they were measured by the BATHmethod and by less than 10% when the HIC assay was used.

DISCUSSION

The hydrophobicities of spores of both Bacillus and Clos-tridium spp. were considerably greater than the hydropho-bicities of the vegetative cells, as determined by the BATHassay. Several studies in which workers used adherence tohydrocarbons to examine bacterial spore hydrophobicityhave produced similar observations. Koshikawa et al. (19)used the BATH method to measure the spore hydrophobic-ities of several Bacillus spp. and reported that the spores ofB. megaterium QMB1551 were 80% more hydrophobic thanthe vegetative cells. The spores of Clostridium perfringensNCTC 8679 were found to be 82% hydrophobic, as measured

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TABLE 2. Hydrophobicities of spores and vegetative cells of Bacillus and Clostridium spp.

Spores

StrainBATH assay HIC% Hydrophobicity of

Strain BAHasyHCvegetative cells% Hydro- % Decrease % Hydro- % Decrease (BATH assay)aphobicitya in concnb phobicityc in concnb

B. subtilis ATCC 6633 (crop I) 94 (0.3)d 95 89 (1.5) 90 7 (1.6)B. subtilis ATCC 6633 (crop II) 95 (0.0) 96 93 (0.0) 94 8 (2.3)B. subtilis ATCC 6633 (cropII1) 95 (0.0) 96 83 (5.1) 84 5 (2.1)B. subtilis ATCC 6633 (crop IV) 92 (1.1) 93 88 (0.8) 89 NDeB. subtilis ATCC 19221 95 (0.2) 97 91(4.0) 92 5 (4.6)B. subtilis ATCC 9372 (B. globigii) 47 (2.4) 53 20 (3.2) 23 3 (1.4)B. subtilis A 19 (3.2) 23 23 (1.8) 27 10 (5.2)B. cereus T 95 (0.4) 95 98 (1.2) 98 3 (1.3)B. coagulans ATCC 8038 49 (1.1) 56 29 (2.0) 36 6 (5.1)B. coagulans FRR B666 65 (0.7) 71 50 (0.4) 57 17 (3.5)B. stearothermophilus ATCC 7953 53 (2.5) 57 41 (2.4) 46 1 (0.3)B. megaterium ATCC 12872 88 (0.0) 90 25 (2.5) 26 20 (0.7)B. megaterium ATCC 33729 30 (1.8) 33 41 (1.5) 45 9 (9.9)

C. botulinum 213B 50 (0.2) 56 35 (1.0) 41 11(3.4)C. sporogenes ATCC 7955 67 (1.1) 75 41(4.0) 50 24 (1.0)C. putrefaciens ATCC 25786 79 (2.6) 86 52 (4.4) 60 22 (4.6)

a Percent hydrophobicity is the average percent decrease in the A440 of the aqueous phases after partitioning with 0.1, 0.2, 0.6, and 1.0 ml of hexadecane.b Average percent decrease in the spore concentration of the aqueous phase that was due to hexadecane partitioning (BATH assay) or retention in the

Sepharose column (HIC).c Percent hydrophobicity is the average percent decrease in the A4,0 of the spore suspensions eluded from duplicate Sepharose columns.d The numbers in parentheses are standard deviations (n = 2).e ND, Not determined.

by percentage of adherence to 0.1 ml of toluene (4). Foege-ding and Fulp (8) reported a hydrophobic index of 67% forspores of Bacillus cereus T when the BATH assay was used.Doyle et al. (6) tested several Bacillus species by using theBATH method with 0.6 ml of hexadecane and found variablespore hydrophobicities, ranging from 13.2% for B. subtilis168 to 63.8% for B. cereus T. All vegetative cells of the

80 -

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20 -

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IO'

0La00I0"_

UNHEATEDCONTROL

8 0 9 0 1 00

TEMPERATURE (*C)FIG. 2. Effects of heat treatments on the hydrophobicities of

Bacillus spores. Spore suspensions were heated for 10 min, cooledrapidly in an ice bath, and refrigerated overnight. Hydrophobicitywas assayed with the BATH method by using 0.1 ml of hexadecane,as previously described. Error bars indicate the standard deviationsfrom duplicate trials. Symbols: *, B. subtilis A; A, B. subtilis ATCC9372 (B. globigii); 0, B. stearothermophilus ATCC 7953. Thepercent hydrophobicity was the percent decrease in the A4,0 of theaqueous phase that was due to partitioning with hexadecane.

Bacillus species tested were less than 6.0% adherent tohexadecane (6). Our observations indicate that the vegeta-tive cells of Bacillus and Clostridium spp. are generally nothydrophobic when they are measured by the BATH assay.However, several Bacillus and Clostridium vegetative cellpopulations exhibited elevated hydrophobicities at thehigher volumes of hexadecane (Fig. 1).HIC yielded results which indicated that there was in-

creased spore hydrophobicity when spores were suspendedin 4 M NaCl buffer. Without 4 M NaCl in the buffer, thespores were not retained in the Sepharose gel columns (datanot shown), indicating that high ionic strength was necessaryto overcome the electrostatic repulsion between the sporesand Sepharose. In the only previous study in which HIC wasused to measure bacterial spore hydrophobicity, Doyle et al.(6) measured the hydrophobicities of two Bacillus species byusing octyl-Sepharose columns in 0.15 M NaCl and foundthat both species were more than 67% adherent to thecolumns.There was a good correlation between the BATH and HIC

methods when they were used to measure bacterial sporehydrophobicities. While the absolute hydrophobicities for aspecific test organism did not agree well between the twomethods in several cases, the ranking of hydrophobic indicesfor species and strains of spores was consistent betweenmethods. One notable exception was the spore population ofB. megaterium ATCC 12872, which exhibited much greateraffinity for the hexadecane in the BATH assays than for theSepharose gel used in HIC.

It has been suggested that the increased hydrophobicity ofbacterial spores is due to the relative abundance of protein inthe outer coats and exosporium compared with peptidogly-can on gram-positive vegetative cell surfaces (6, 24, 37). Anassociation between spore hydrophobicity and the presenceof an exosporium has been reported recently for severalBacillus species (18). Kutima and Foegeding (20) noted a

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decrease in the hydrophobicity of spores of B. cereus Twhen spore coats were removed by chemical treatments.Takubo et al. (36) and Koshikawa et al. (19) reporteddecreased hydrophobicities in outer-coat-negative mutantsof B. megaterium QMB1551 (= ATCC 12872). The de-creased adherence to hexadecane of an outer-coat-negativespore mutant, B. megaterium ATCC 33729, compared withwild-type strain B. megaterium ATCC 12872 is shown inTable 2, suggesting that the outer coats or the exosporiumplays a role in spore hydrophobicity.We found evidence that heat applied to dormant spores

can raise their surface hydrophobicities. Doyle et al. (6)reported considerable increases in spore hydrophobicity fortwo Bacillus species after 15-min treatments in water at100°C, although the effects were not as pronounced as thosein our study. Craven and Blankenship (4) noted an increasein the relative hydrophobicities of spores of C. perfringensNCTC 8679 after treatment at 75°C for 20 min, but showedthat the effects could be negated by washing the spores afterthe heat treatment. Increases in the hydrophobicity of sporesbecause of heat treatment may result from the disruption ofouter coat or exosporium proteins (6). Li-Chan et al. (21)reported that increased temperatures can alter the structureof macromolecules, causing an unfolding of proteins andexposing internal hydrophobic moieties. Doyle et al. (6) havesuggested that hydrophobic interactions are important in theattachment of spores to environmental proteins. Increasedspore hydrophobicity because of heat activation might in-crease affinity of the spores for lipids or proteins, thusproviding a nutritional source for outgrowth of the vegeta-tive cells following germination of the spores. Therefore, thenet effect of heat to increase spore hydrophobicity mayrepresent a mechanism for increasing the chance for survivalfollowing germination of the spores.

Several studies have shown that the hydrophobic propertyof vegetative cells is dependent upon conditions such asgrowth medium and culture age (1, 30) and that sporulationconditions can affect spore properties such as heat andchemical resistance, structure, and germination (8, 10, 17).However, Koshikawa et al. (19) have reported recently thatsporulation medium does not affect Bacillus spore hydropho-bicity. Data presented here also indicate that sporulationmedium and sporulation temperature do not appear to affectBacillus spore hydrophobicity.The physical methods (e.g., differential centrifugation,

sonication) used in this study to produce spore crops whichwere free of vegetative cell material are recommended tominimize alteration of inherent spore hydrophobicity (datanot shown). Thermal treatment is not recommended for theremoval of vegetative cells from spore preparations whichare to be used in hydrophobicity assays because of observedincreases in spore hydrophobicity after sublethal heat treat-ment. In addition, the use of enzyme treatments, such aslysozyme or trypsin treatments, to clean spore preparationsis not recommended because of reported effects on sporehydrophobicity (6).Thus, the spores of Bacillus and Clostridium spp. are

relatively more hydrophobic than the vegetative cells. Thissuggests that hydrophobic interactions may play a role in theadhesion of spores to solid substrata. The increase in sporehydrophobicity because of exposure of spores to heat mayplay an important role in the increased adhesion of bacterialspores to materials following a sublethal thermal process.Further studies are needed to expand the base of knowledgein the area of bacterial spore hydrophobicity. Understandingthe role of hydrophobic interactions between bacterial

spores and substrata in the adhesion of spores to surfaces iscritical in the development of more efficient methods ofsurface sanitation or sterilization of equipment or packagingmaterials used in the medical, pharmaceutical, and foodindustries.

ACKNOWLEDGMENTS

This research was supported in part by the Center for AsepticProcessing and Packaging Studies and by North Carolina Agricul-tural Research Service project 2152.

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