COMPARATIVE PHYSIOLOGY OF FATTY ACID UTILIZATION BY SPECIES by GENE K. ESTES, B.S. A THESIS IN MICROBIOLOGY

Submitted to the graduate faculty of Texas Technological College in Partial Fulfillment of the Requirements for the Degree of

MASTER OF SCIENCE

Approved

Director

Accepted

May, 19G9 AC So? T3

^Jo.49

ACKNOWLEDGEMJJIJTS

I am deeply indebted to Dr. Harold L. Lewis for his direction of tliis thesis and to the other mcirtbers of my committee, Drs. Arthur M. Elliot, Lylc C. liuhnley, Robert

W. Gorden, Ira C. Felkner, Murray v;. Coulter and Earl D.

Carap for their lielpful criticism. I am also indebted to

Drs. C. W. Hesseltine and Chester M. Rowel1 for their cul­ tures and photographic equipment v.'hich v.'ere so vital to my researcii.

11 COr^TENTS

ACKNOV;LLDGL!:M,"rf' ii

LIST OF TAliJ.l.S iv

LIST OF FIGUKi.S V

I. HISTORICAL Ri:vii:\; Araj INTKODUCTIO:.' 1

II. MATERIALS AND MLTHODf; 6

III. RESbi/ri; Ai:i) ohr.],r,VATJo:,'S 9

Grc)\.'th on Fatty Acids 9

Fatty Acid Composition 21

IV. DISCUSSIO:; Al.'i) CONCLUSIONS 27

V. LiTi::i;/i'urj-: ciTi-D 35

111 LIST OF TABLllS

TABLE PAGE

I Cani)arison of the ratios of saturated to

un-sdturatcd bound fatty acids in four

species of Cunn i nglianiolla 23

IV LIST OF FIGURi^S

FIGURE PAGE

1 Grcvth of C. cchinulnta on even-chain, ali­

phatic, saturated fatty acidf. at eclectcd

pH valuer 10 2 GrowLli of C. hertholloti ao on evcn-ch.jin, aliphatic, saturated ftitty acidfj at selected pH values 11

3 Growtli of C. blaker>leeana on even-cliain,

aliphatic, saturated fatty acids at

selected pll values 12

4 Growth of C. elegans on even-chaii., aliphatic,

saturated fatty acids at selected pH values . . 13

5 Growtii of C. vesiculosa on even-chain, ali­

phatic, saturated fatty acids at selected

pH values 14

6 Grov.th of C. echinulnta on even-chain,

alij^hatic, saturated fatty acids at

selected pH values 16

7 Growth of C. bertholJetiat- on even-chain,

aliphatic, saturalcc; fatty acids at

selected pU values 17 8 Grov.'th of C. bla^oslc eana on cvcn-cliaiii, aliphatic, .saturated fatty acids at selected pH values 18 v LIST OF FIGURES

FIGURE PAGE

9 Growth of C. elegans on even-chain, ali­

phatic, saturated fatty acids at selected

pU values 19

10 Grov.'th of C. vesiculosa on even-chain,

aliphatic, saturtited fatty acids at

selected pH values 20

11 Comparison of major qualitative differences

in gro\>/th on fatty acids 29

12 Comparison of major qualitative differences

in grov.'th on fatty acids 30

13 Key to the species of the Genus

CunninghrjTiella 31

VI CHAPTER I INTRODUCTIUN AND HTiJTORICAL REVIEW

Oodocephalum cchinulaturn was described by Thaxter (1891) and placed in the clar.s because of its failure to manifest the perfect stage. Matruchot, in 1903, established the genus Cunninghnine 11a based on hif; descrip­ tion of Cunninghamella £fri^<-:aira which he collected in the French Sudan (Cutter, 1940). Later in the same year, Thaxter claimed that C. africana merely represented a redescription of his Oedocephalum echinulaturn and he subsequently intro­ duced the nomenclatural combination, Cunninghamella echinu- laj^a (Thaxter) Thaxter (Mil'ko and Delya);ova, 1966). Matru­ chot believed that the genus Cunninghamella should be placed in the order despite its purely conidial reproduc­ tion and supported this claim v;ith a series of tests showing that members of Cunninghamella v/ere vulnerable to attack by species of Piptoccphalis which are obligately parasitic upon the Kucoralcs. Based on this history, Matruchot is given credit for tiic establisliruont of genus Cunninghamella, and the type species is Cunninghanc>lla echinulata (Thaxter) Thaxter.

Since 1893, many additioiial species have been described: Cunninghamella echinata (Saccardo) Matruchot, 1903; Actino- cephalum japonicum Saito, 1905; C. elegans Landner, 1907; C. bertholletiae Stadel, 1911; Muratella elegans Bainier and Sartory, 1913; Cunninghamella "A" Blakeslee, Cartledge and Welch, 1921; C. blakeslocana Lendner, 1927; C. verticillata Paine, 1927; C. dalmitica Pispek, 1929; C. ramor>a Pispek, 1929; C. polymori^iia Pispek, 1929; Muratella sp. Ling-Young, 1930; C. albida Zycha, 1931>; C. bainieri Naumov, 1939; C. homothallica Kominami, Kobayasi and Tubaki, 1952; C. phaeo- spora Uoedijn, 191^8; C. vesiculosa Misra, 1966 (Cutter, 1946, Mil'ko and Belyakova, 1966 and Misra, 1966). Comprehensive surveys and monographs have been published by Zycha in 1935, Naumov in 1939 and Cutter (1946) . Of the fifteen species described up to 1946, Cutter (1946) recognized only five, based on their c,rowth and mor­ phology on Mucorales dextrose asparagine agar. These five species and their synonyms are as follows: C. echinulata (Oedocephalum echinulaturn, C. africana), C. bainieri ("ura- tella elegans, Muratella sp.), C. blakesleeana (C. "A", C. verticillata, C. echinata), C. elegans, and C. bertholletiae. He considered Actinocephalum japonicum, C. dalmitica, C. ramosa, C. polymorpha and C. albida to be doubtful species. Up to the time of Mil'ko and Bclyakova's publication in 1966, only two additional species had been described. These workers recognized four sjucirs based upon gelatin liquifac- tion and their grov;th and morphology on C/apck's agar and wort agar. These four species and their synonyms are: C. echinulata (Oedocephalum echinulatu.a, C. af ricana, Muratella elegans) C. verticillata, C. echinata, C. bainieri), C. blakes­ leeana (Cunninghamella "A") , C. elegans (Actinoccphaluia japonicum, C. bertholletiae, C. ['haespora) , and C. homotlial-

lica. They considered C. alj,^Aiil' 9.' d'llmitica, C. rarnosa,

9.* poly'»orpba and Muratella sp. to bu doubtful si*ecies. At about the same time of the work of Mil'ko and belyakova anotlior species, C. vesiculo:>a, wa.s described by Misra (1966) and this appears to be a valid specie.s based upon morpholo­ gical criteria. Tlius, at the i>resent there are five recog­ nized species uring Mil'ko and Belyal:ova*s key and seven species using Cutter's key.

The systematic position of the genus has been treated

in various ways by different authors. It has been placed in

the tribe Choanephorae and in the families Chaetocladiaceae,

Choanephoraceae and Cunninghtniollaceac (Cutter, 1946) . Most

current autliorities are in agreciacnt v;ith Naumov's creation of the family Cunninghcimellaceae since included genera all

produce globose, cchinulate conidia in the complete absence

of sporangia. According to Hesseltine (1955), the Cunning-

hamellaceae along with tlie Thc-Mnidiaceae comprise one of six major evolutionary lines in the Mucc)rales.

A feature of this which ir as characteristic as

its morphology, is its marked affinity for fatty substrates.

In this respect, it is interesting to note that Reese, et al.

(1955) reported that out of 381> specicn of fungi tested for

their ability to degrade natural fats, three species of Cun- ninghamfella v/ere the most active. Subsequently, Lev/is and Johnson (1967) demonstrated that C. echinulata cou.ld utilize each of the saturated fatty acids from acetate through stearatc as sole sources of carbon and energy. Prior to this work no fungus had been shown to grov; on all of these sub­ strates, thus Cunninghamella species possess the ability to utilize the breakdown products of natural fals as v/ell as the fats thciuselves.

Beginning with the earliest inve:itigations of the effects of fatty acids on fungi, there have been various reports con­ cerning their fungicidal and fungistatic properties. Kiesel (1913) first demonstrated the phenomenon of fatty acid toxi­ city. He reported that short chain fatty acids (C2 through C6) inhibited the germination of spores and the production of mycelium in Aspergillus nigor. Wyss, et al. (1945) re­ ported the fungicidal and fungistatic action of intermediate chain length fatty acids C8 through C14. Bushnell (1941) and Baechler (1939) conducted studies in which they found that toxicity increased with increasing chain length. Tetsu- moto (1933) confirmed Kiesel's findings and reported that unsaturated acids v;ere more activi' v.ith rega.rd to toxicity than the corresponding saturated acidr.. Dicarboxylic acids were aliiOst v.ithout activity. The confusion which exists jn tlie literature over the mode of action of various fatty acids as toxic agents was greatly'clarified by Lcv.'is and Johnson (1967). Their studies of the grov/th of C. echinulata on even-chain fatty acids showed that the toxicity of thci.e acids vas variable v;ith changes in the pH of the medium v.hich resulted in a corres­ ponding change in tlie concentration of undissociated acid and fatty anion. In these experiment.-, all saturated even- chain fatty acids (C2 - C18) supported grov/th at one pH value or another. Viiey therefore deduced that since the inhibition by even-chain fatty acids was dependent upon pH, the mech­ anism of toxicity was correlated with the pujiaeant form of the molecule.

Since the genus Cunni ughajnella is knov/n to have a strong affinity for fatty substrates, it is surprising that attempts have not been made to establish relationships among species based upon comparative physiology using fatty acids as sole sourccG of carbon and energy. This research v.as designed to compare physiological responses of the genus Cunninghamella to fatty acids (C2 - C18). CHAPTER II

MATERIALS AND MLTHOUS

Isolates of Cunninghamella used in this study were:

C. echinulata (Arkaniar. isolate) , C. cclunulata (Minnesota isolate), C. bertholletiae (Texas isolate), C. bainieri

ATCC 8987, C. elegans ATCC 10326 (+), C. blakerlceana ATCC

8688a ( + ), C. Makerlecana ATCC 8C88b (-) , and C. vesiculosa

NRRL 3009. There isolates v.-cre maintained on Mucorales dex- trore asparagine agar slants (dextrose, 40 g; asparagine,

2.0 g; KH2P0^, 0.5 g; MgSO^, 0.2 5 g; thiamine hydrochloride,

0.5 ng; Difco agar, 15 g; distilled water, 1000 ml adjusted to ph* 4.2 by addition of 0.1 N HCl) as described by Cutter

(1946) .

All species v.cre exaiaincd microscopically by mounting bits of mycelium in a solution of equal parts of phenol crys­ tals, lactic acid, glycerin and dirtilled v/ater to v/hich

"Tergitol" penetrant 7 and Sudan III were added at concentra­ tions of 0.3 ajid 5% respectively (Cutter, 1946). From these exciminations, isolates v.cre identified according to the morphological criteria established by Cutter (1946).

Growth studies v.ore conducted using 50 ml test tubes with metal closures, each containing 20 ml of medium. The medium v/as Czapek miriC.rcil salts solution (L'aNOs, 2.0 g;

KH2P0^ or K2HP0M, 1.0 g; KCl, 0.5 g; l\qSOi,'IU2O, 0.5 g;

6 FoSO^•7H20, 0.01 g; distilled water, 1000 ml) containing

0.5% of a particular fatty acid. Saturated aliphatic even- chain fatty acids (C2 - C18) were used at pH levels 5.5, 6.5,

7.2,and 8.0. The pH of each medium v;as adjusted to the de­ sired value using 20%, 1 N ,or 0.1 N potar.sium hydroxide and

1 N or 0.1 N hydrocliloric acid. All media were sterilized in an autoclave at 121**C for 15 minutes. Inocula consisted of spore suspensions harvestcxl with 10 ml of sterile saline

(0.85u NaCl) from 6 day old culture.^. Spores v/ere counted using a standard he;nacytometer and each suspension v.as diluted with sterile saline to contain 1.0 x 10^ spores per ml. One ml of tlic spore suspen.sion from each species v/as added to the appropriate grov/th tube and the cultures were incubated under stationary conditions at 25 or 40**C. After 14 days incubation, the cultures v;ere heated to 80®C and the mycelia were separated from the supernatant solutions by filtration while the media v.-ere still hot and v/ashed two times v/ith boiling distilled v/ater. The mycelia v/ere then dried for 24 hours in a hot air oven at 80°C and v/eighed on a Metier ana­ lytical balance to deterr.iine dry cell weight.

Mycelia for lipid analy: is were grov/n in Mucorales dex- troxe asparagine (M.D.A.) mediuu (Cutter, 1946) in 2800 ml

Fernbach flasks at 25**C. Spore inocula were prepared as de­ scribed above for the fatty acid grov.'th experiments. After

6 days of incubation, mycelia v.'cre harvested by filtration. 8 dried at BO'^C for 24 hr, and ground to a fine pov/der v/ith a mortar and pestle. Extraction, i>urifictition, .saponification, esterification, and gas-liquid chrornatogi .i))liic

(1967). All gas-liquid clu oiatography v/as done v/itli a model A-600-D (Varinn At^rograpli Corp.) chromatograph. The carrier gas v;as nitrc>gon at 20 ml per minute and \.'ari introduced into the colu.Mn aL an inlet pre.sj.ure of 4 0 psi and an outlet pressure ev]uivalenL to abuosphcric. A hydro;jen flame ioniza­ tion detector v;as employed v/ith an air flov/ of 30 ml })er min­ ute aiiu a hydro^jon flov; of 20 ml per minute. The teriperature of tl.c detector and the colu.in v/as 205*'C. Methyl enters of fatty acids v.erc separated on a 5 ft by 1/8 inch (internal diameter) stainless steel column packed v.ith 5^ SE-30 sili­ cone on Chroriasorb V.' (Varicin Aerograph Corp.) 60/80 U.S. mesh. Methyl esters of fatty acids v.*ere identified by com­ parison of their retention tiiaes to those of known standards. CHAPTHK III

RESULTS AND OBSnRVA'J'lON.S

Grov/th on Patty Acid;;

The species testod for their iil^ility to grow on fatty acids are as follov/s: C. echinulata, C. bertholletiae, C. elegans, C. blakesleojina and C. vesiculosa. Tliree + strains ®^ ?.• echinulvita and llic + and - strains of C. blakesleeana were tested. Tlie data presented for each temperature are

averages of v.viglits obtained from three replications.

Growth experiments at 2i;**C revealed that all five species

tested failed to utilize short-chain fatty acids C4, C6, and

C8 at pH 5.5 (Figs. 1-5). At pH 6.5, C. vesiculosa was unique

by virtue of its inability to utilize C6 (Fig. 5). At pH

7.2, C. blakesleeana and C. elegans shov/ed similar responses

in their inability to grow on C8, CIO, and C12 (Figs. 3 and

4), and C. echinulata and C. bertholletiae were exclusively

unable to utilize C8 (Figs. 1 and 2). C. vesiculosa failed

to respond to C8 and CIO but showed a slight response on C12

(Fig. 5). At pH 8.0, all species failed to grow on C4 with

the exception of C. vesiculosa (Figs. 1-5). C. elegans and

C. blakesleeana once again shov/ed .similar responses by fail­ ure to utilize CIO, C12, and C14 (Figs. 3 and 4). C. berthol­ letiae and C. echinulata failed to use CIO and C12, and C12, respectively (Figs. 1 and 2). Replication of these experi­ ments resulted in differences in quantitative amounts of

(0 'O •H U <0

^^^^^^^^^^^^^^H fatt y ^^^^^^^^^^^^^^H r •0 ^^^^^^^^^^^^^^^^^^^K 0 4J

BHgI.'An.3 >• 3 •P

CO MBECS3QCSHHIHHI o R3 ' 4J •• ph a y - ^

1- * f3 •- i«x .<: « f C 1 ' f « 1 ,G • » I • *

o n- c m o • • t 1 • • > • - o o ft ni • • dfli <^ < c • « f * 1 0 « I K.\ • • d BE3 • • '', c 3 • (j 1 r O C-s 1 o • r c • ' J 0 W 1 V ' 1 ' fH 0 M :3 O —« 1 c:, > f -' • r-l J3

%i'^ -*.*.. . oi'^mj • a • t * -_—^^- - . ITj 0 " v;-t -M t:-~_xa 0 u r-^ci ' sol e

ggmpgjmL T-- ~—• owt h V^ -)-» 1 O rj 1- 1 •

• • II t y • 1 ^ d ___^ 1 % IP ^ % •H [X4 ul.i: a O TJ o x: 4J -p u IS o O r-i I iTiiiiiM 'Baaa u o

^

•H I."

15 growth, but at no time, were differences observed in qualita­ tive responses. Based on this finding, qualitative growth was used as the major criterion for comparative physiology among i.pecios.

Examination of growth responses at 40*'C (Figs. 6-10) revealed that all five species failed to utilize fatty acids C2 through C8 at pH 5.5, C6 through CIO at pH 6.5, C8 through C12 at pH 7.2 and C8 through C16 at pH 8.0. Once again, C6 was toxic for C. vesiculosa at pH 6.5 but supported growth for the otiier species at this pH and temi)erature. Cunning­ hamella elegans and C. blakesleeaiia responded similarly at 40'C, hov.ever, at this temperature a qualitative difference was observed at pH 7.2. failed to utilize C16 but C. blakesleeana responded well to this fatty acid (Figs. 8 and 9). Cunninghamella bertholletiae and C. echinulata responded very much alike at this teraperature, but at ph 6.5 C. bertholletiae v/as different by failing to uti­ lize Ch (Figs. 6 and 7). A comparison of growth at 25**C and 40*C clearly shows that: (1) . Througliout the genus a greater number of fatty acids inhibit grovth at the higher than at the lov/er temper­ ature (Figs. 1-10). (2). The plienomenon of increasing toxicity v/ith decrea.;ing pH of short-chain fatty acids and increasing toxicity with increasing pH of long-chain fatty acids as reported by Lewis and Johnson (1967) is v/ell

21 manifested at 40«C. (3). There is a marked difference in the growth response of each species at the tv/o temperatures.

Comparative phy.siology of fatty acid utilization by these organisms appears to be a species specific phenomenon.

Growth studies v.ere conducted on the Arkansas and Minnesota isolates of C. echinulata and the (+) and (-) strains of C. blakesleeana to see if tliere were differences in grov/th re­ sponses of strainr. of the same si>ecics. Since C. bainieri has been rei)orted to be synonomous v/ith C. echinulata (Mil'ko and Belyakova, 1966), it was also tested. The (+) and (-) strains of C. blakesleeana responded identically under all conditions. Tiie two isolates of C. echinulata and the ATCC strain of C. bainieri were reiaarkably similar in their grov/th responses on fatty acids (C2 - C18).

Fatty Acid Composition

Since grov/th studies revealed prominent differences be­ tween the species of Cunninghamella, the possibility that there might be differences in their fatty acid composition was considered. Clasr.if ication of microorganisms based on fatty acjd coiaposition is a lelatively ncv; conce])t associated with Liocher.iical sysLei.iatics. Tlie advar.tages and disadvan­ tages of this type of sy.stematics are outlined by Alston and

Turner (1967). Abel, Scluaertzing,.and Peterson (19G2) con­ ducted studies of this nature in v.'hich they v.ere able to resolve major differences betv;een four families of bacteria. 22

These studies, however, failed to sliow any qualitative dif­ ferences between specie:; of one genus. MacLeod and Brown

(1962) were able to distingui.sh between Strej-)tococcus lactij and Streptococcus cremori.n based on fatty acid composition.

Differentiation between species in this study war. m^ide pos­

sible by critical experimental tecluiiques in whicli even-chain

and odd-chain, saturated and un-saturuted, aliphatic and

aromatic fatty acid er.terr. were analy/ed.

It was decided that an analysis of the even-chain fatty

acid composition of various species of Cunningha:.iolla might

be helpful when com])ared v/ith the growth responses. Percent

composition of various even-chain saturated and un-saturated

fatty acids of four specie:; of Cunningheime 11a are compared

in Table 1. The data in Table 1 indicate that the minus

strain of C. bla};csleoana has a higher perceiitage of un-sat­

urated fatty acids than tlie plus strain. C. elegans (also

a plus strain) shov/s about the same ratio of saturated to

un-saturated fatty acids as the plu.«^. strain of C^. blakesleeana

It also ai^^.cars that as the cultivation temperature is in­

creased the degree of unsaturation increares. This is best

illustrated by C. echii;ulata grov.n at 25* and 40**C (Table 1) . 23 TABLL I COMPARISON OF THE RATIOS OF SATURATLD TO UK-SATURATED BOUND FATTY ACIDS IN FOUR SPLCIi;S OF CUNNINGHAMELLA

Percent Percent Species Saturated Acid Un-saturated Acid *. - ».•<,

C. echinulata 25*C 75.30 23. ,20

C. echinulata 40*C 66.90 28, .21

C. blakesleeana (+) ?5°C 86.15 12. .70

C. blakesleeana (-) 25°C 76.50 23. .20

C. elegans (+) 25°C 86.88 12. .84

C. bertholletiae 40°C 89.30 10. .70 CHAPTER IV DISCUSSION AND CONCLUSIONS

Qualitative differences in growth on fatty acids at various pH levels are numerous when the data are viewed col­ lectively, but if comparisons of the fatty acid utilization among species are made, distinct similarities can be detected Furtliennore, these similarities in growth responses do not necessarily coincide with morphological similarities. In this connection it should be noted that Lendner and Zycha considered C. bertholletiae and C. elegans to be synonyhious species based on their morphology (Cutter, 1946). Cutter (1946) retained C. bertholletiae as a valid species but em­ phasized tile close morphological similarity to C. elegans, and only recently, Mil'ko and Belyakova (1966) conducted studies in which they reduced these tv;o species to synonomy. A comparison of the fatty acid utilization of these tv/o species at 25**C reveals qualitative differences at pH 7.2 and 8.0 (Figs. 2 and 4) and even more distinct differences at all pH levels at 40**C (Figs. 7 and 9). Although C. ber­ tholletiae and C. elegans are morphologically very similar, they appear to be physio!ojically distinct.

Conversely, C. elegans appears to bo physiologically related to C. blakesleeana. A comparison of the grov/th re­ sponses of these two species reveals only tv/o qualitative 25 differences at 25*C (Figs. 3 and 4) and one qualitative dif­ ference at 40»C (Figs. 8 and 9). Thus, although these two species are morphologically distinct, they are very similar with respect to fatty acid utilization. Cunninghame11a vesiculosa, a new species described by Misra in 1966, is morphologically distinct by virtue of the constricted vesicles, tlic unusually long conidial spines, and the persistent pure v/hite color of its colonies. A com­ parison of fatty acid utilization by this species with all the others indicates that it is also physiologically distinct At 25*C, C. vesiculosa is inhibited by C6 at pH 6.5 and by C14 at pH 7.2 (Figs. 1-5). Under the same conditions, these fatty acids support growth for all of the other species. A comparison among species at 40**C shov/s C. vesiculosa to be unique by virtue of its inability to grow on C6 at pH 7.2 (Figs. 5-10). A comparison of grov/th at 25*C v/ith that at 40®C shows that each species of Cunninghamella responds differently v/ith respect to fatty acid utilization at the higher temperature. Since these fungi respond differently at 40**C, it might be expected that there are differences in their fatty acid com­ position at this temperature. Marr and Ingraham (1962) re­ ported that an increase in cultivation temperature caused a corresponding increase in the saturation of the intracellular fatty acids of Escherichia coli. Tiber and Herodek (1964) found the reverse relationship to be true in the case of 26 Crustacean plankton, it is not known what effect an increase or decrease in saturation of intracellular fatty acids in species of Cunnijiajii^^ would have on their uptake of fatty acids from the culture medium, but the possibility that this phenomenon plays a role in altering metabolism should not be overlooked. Data from experiments on fatty acid utilization as well as analysis of bound fatty acid extracts (Table 1) show that marked metabolic changes take place at the higher cultivation temperature. In this connection, the fatty acid fraction of C. echinulata grown at 40**C contained 11% lino- leic acid, whereas extracts from the same species grov/n at 25® were devoid of this un-saturated fatty acid. These data are therefore comparable to the findings of Tiber and Hero­ dek (1964). It appears that with C. echinulata an increase in the cultivation temperature causes a corresponding in- jj crease in the degree of un-saturation of intracellular fatty ' acids. I This research might be helpful in elucidating some of • I I the problei.is in teixonomy of the genus Cunninghamella. Using I fatty acids as sole sources of carbon for grov/th by species of Cunninghamella has shov.n that such a method, v/hen used as a teixonomic tool, aids in the identification of isolates. These findings may also help to clarify confusion v/hich exists over the validity of doubtful species. Host authori­ ties on the of this genus are in agreement on C. echinulata, C. elegans, C. blakesleeana and C. vesiculosa 27 as valid species. There is considerable disagreement as to the validity of C. bainieri and C. bertholletiae. Cutter

(194b) points out that C. bertholletiae is considered to be synonyn\ous with C. elegans by Lendner and Zycha, whereas

Naumov, and Alcorn and Yeager (1938) retain C. bertholletiae as a valid species. Cutter's studies in 1946 strongly sug­ gest that C. bertholletiae is indeed a valid species, but there is still contradiction over this matter (Mil'ko and

Belyakova, 1966). Morphological descriptions have clearly revealed tlie close relationship of these tv/o organisms, hov/- ever, analysis of fatty acid growth responses reveal marked differences. Tiiese data support the views of Maujnov, Alcorn and Yeager, and Cutter that C. bertholletiae is a valid species.

Morphologically, C. bainieri is very closely related to

C. echinulata. Cutter (1946) distinguishes betv/een them at the cellular level by the presence of giant conidia in the former. Mil'ko and Belyakova (1966) in their recent survey of the taxono:.iy of the Cunninghamellaceae consider C. echin­ ulata and C. bainieri to be synonymous. Growth studies con­ ducted on these tv/o organisms resulted in fev;er qualitative differences bctv.'een theni than those between the tv/o isolates of C. echinulata. These findings on the morphology and phy­ siology of C. bainieri tend to support the vie\.s of Mil'ko and Belyakova (1966) that C. bainieri is i.ierely a redescrip­ tion of C. echinulata. 28 Cunninghamella hoinothallica may be a valid species, how­ ever, little mention of this organism has been made in the literature since its initial description which accounts pri­ marily for our failure to obtain a culture for these studies. The application of a systematic approach to this re­ search can be made by a comparison of the major differences in growth responses among species at one cultivation tempera­ ture and pH. A comparison of this nature is made in Fig. 11. Cunninghamella elegans and C. bla):esleeana fail to utilize fatty acid carbon chainlengths C8, CIO, and C12 under these conditions. Cunningheimella bertholletiae, C. echinulata and C. vesiculosa however, did show a growth response on one or more of tliese intermediate chain-length fatty acids. At pH 6.5 (Fig. 12), C. bertholletiae, C. echinulata and C. vesicu­ losa are physiologically different by their lack of growth on C8; C8 and CIO; and C6, C8, and CIO, respectively. Cunning­ hamella blakesleeana and C. elegans are different by their growth response to C16 at pll 7.2 at 40°C. This information can be organized into a dichotomous key such as the one shown in Figure 13.

Lewis and Johnson (196 7) proposed that the ability of a fatty acid to inhibit growth and respiration of C. echinu- lata was dependent upoii its chain-length, and the concentra­ tion of the permeant forjn of the compound. The relative concentrations of the undissociatcd fatty acid and the fatty anion in an aqueous fatty acid solution or suspension is a

•|t" OZ / 5M

32 function of Uie pH value of the solution. Thus, as the pH of a fatty acid solution is lowered, the concentration of the undissociatcd acid increases and the concentration of tlie fatty anion decreases. On the other hand, as the pH of the solution increases, the reverse concentration relation­ ship between undissociatcd acid and fatty anion occurs. In tlic case of the short chain fatty acids, the factors correlated with increasing fungal toxicity (increasing chain lengtli, decreasing pH) are also factors which increase the lipophilic properties of tlie molecule. Thus, the growth re­ sponses of Cunninghamella spp. on these compounds correlate nicely witli the extensive data supporting Overton's (1899) "lipoid solubility" theory of permeability for such substances Toxicity data for the longer chain length acids are not ex- pliceible on tlie same basis, since these compounds become less inhibitory as the pH was lov/ered and also as the chain-length was increased. Concerning this point, Collander and Barlund (1933) dejnonstrated, through studies on the rates of penetra­ tion of several types of compounds into cells of Chara ccra- tophylla, that the rates of penetration of related substances into the cell v/ere proportional to cliain-length only up to a certain molecular voluiae. In the case of compounds v/ith molecular volumes above a given threshold value, the rates of penetration into the cell v.-ere inversely related to the size of the molecules. These observations led these workers to support a different meclianism of permeal.ility for these 33 substances, in which the plasma membrane was held to act as a "molecular sieve" with reference to them. The growth responses of Cunningliamella spp. on the intermediate- to long-chain acids are in agreement with this concept. An objection which may be raised to the above interpre­ tation of tlie growth responses of Cunninghamella spp. on fatty acids is that the longer-chain-length compounds are rather insoluble in water at 25*C and their activities could be controlled by this parameter rather than the concentra­ tion of a specific permeant form at different pH values, however, when the growtli experiments v/ere repeated at 40**C, (the interir.ediate-chain-length acids are quite soluble at 40*C) even more impressive evidence for the fatty anion being the permeant form of longer-chain acids and the undissoci­ atcd acid the penueant form of short-chain acids v/as obtained (Figs. 6-10). Thus these findings support the hypothesis that short and long-chain acids have different permeant forras as proposed by Lewis and Johnson (19 67) . Based upon the "lipoid solubility" theory of membrane transport (Overton, 1899), differences in grov.th responses of Cunninghamella spp. on fatty acids could be due to dif­ ferences in the fatty acid content of their cytoplasmic mem­ branes. Comparisons of the ratio of saturated to un-saturated fatty acids of several of there organisms (Table I) with their growth responses on fatty acids (I'igs. 1-10 ) suggest that such an explanation is feasible. Hov.-ever, analysis of 34 isolated cytoi)lasmic membranes would have to be done before this could be established.

According to Ainsworth and Sussman (1965), the chief function of the cytoplasmic membrane is regulation of cellu­ lar composition with respect to diffusable substances. A distinct cytoplasmic membrane is present in fungi and studies with isolated yeast membranes have shown that 40% of the membrane is lipid and 40% protein. An apparent function of lipids is to provide a structural basis for a membrane con­ sisting of a double layer with the hydrophobic parts end to end and tiie liydrophilic ones associated with a layer of pro­ tein on either side. In addition, differences in the fatty acid composition of the lipids in the plasma membrane could alter the selective permeability of fungal cells. Since the structure of the plasma meimbrane is controlled by the cell's genome, differences in grov/th responses on fatty acids among Cunninghcunella spp. may reflect a basic genetic difference in these fungi. LITERATURE CITED

Abel, K. H., D. Schmertzing, and J. J. Peterson, 1962. Classification of microorganisms by analysis of chemical coiaposition. I. Fcasability of utilizing gas chromatography, j. Bacterid. 85: 1039-1044. Ainsworth, G. C. and A. S. Sussman. 1965. The fungi. Vol. 1. Academic Press, New York. 430-435. Alston, R. E. and B. L. Turner. 1967. Biochemical System- atics. Prentice Hall Inc., Englewood Cliffs, N. J. 119-131. Alcorn, G. D. and C. C. Yeager. 19 38. A monograph of the genus Cunninghamella with additional descriptions of several common species. Mycologie 30: 653-658. Baechler, R. H. 1939. Toxicity of normal aliphatic alco­ hols, acids and sodium salts, hm. Wood-preservers Assoc. 1-8. Bushnell, L. D. and H. F. Haas. 1941. The utilization of certain hydrocarbons by microbes. J. Bacterid. 41: 653-673. Collander, R., and H. Barlund. 1933. Permeability in Chara ceratophylla. II. Permeability to non-electrolytes. Acta Botan. Fennica 11:1-114. Cutter, V. M., Jr. 1946. The genus Cunninghamella (Muco­ rales). Farlowia 2(3): 321-343. Hesseltine, C. W. and Patricia Anderson. 1956. The genus Thcuunidiam and a study of the formation of its zygo­ spores, /v'.ter. J. Bot. 43: 696-702. Hesseltine, C. W. 1955. Genera of Mucorales with notes on their synonomy. Mycdogia 47: 225-229. Hammarstrand, K. 1967. Gas-liquid chromatographic analysis of fatty acids. Tech. publication. Varian-Aerograph Corp. Walnut Creed, California. Kiesel, A. 1913. Reserches r.ur I'action de divers et sels acids sur le developement de 1 'Aspergillus niger. Ann. I'Inst. Pasteur 27: 391-^i20. Lewis, H. L. and G. T. Johnson. 1966. Intermediates of fatty acid metabolism by Cunningliamella echinulata. Mycdogia 58: 137-147. 35 36 Lewis, H. L. and G. T. Johnson. 1967. Growth and oxygen uptake responses of Cunninghamella echinulata on even-chain fatty acicls. My'coTogTa 5Srr""iJ78^^'7. MacLeod, Patricia and J. Brown. 1962. Fatty acid composi­ tion of lipids from Streptococcus lactis var. maltigenes. J. Bacterid. 85: 1056-1060". Marr, A. G. and J. L. Ingraham. 1962. Effect of tempera­ ture on the fatty acids in Escherichia coli. J. Bacterid. 84: 1260-1267. "" Mil'ko, A. A. and L. A. Belyakova. 1966. Genus Cunning- hamella Matruchot and taxonomy of the cunninghamel­ laceae. Mikrobidogya 36(4): 684-690. English translation. Misra, P. C. 1966. A new species of Cunnincjhcmiella. Can. J. Botany 44: 441-444. Overton, E. 1899. Uber die allgemeinen osmotischen Eigen- schaften der Zelle, ihre vermutlichen Ursachen und ihre Bedeutung fiir die Physiologic. Vierteiljahrs- schr. naturforsch. Ges. Zurich 44:88-135. Paine, F. S. 1927. Studies of the fungus flora of virgin soils. Mycdogia 19: 248-397. Peterson, G. E., H. L. Lev/is, and J. R. Davis. 1962. Pre­ paration of uniform dispersions of cholesterol and other water-insoluble carbon sources in agar media. J. Lipid Res. 3(12): 275-276. Tetsumoto, S. 1933. Sterilization action of acids. II. Sterilization action of saturated monobasic fatty acids. J. Agr. Chem. Soc. 9: 388-397. Thaxter, R. 1891. On certain new or peculiar North Ameri­ can Hypho: tycetes. I. Oedocephalum, Rhopalomyces and SigmoidcomycGS. Bot. Gaz. 16: 14-26. Tiber, F. and S. Herodek. 1964. The effect of environmental temperature on the fatty acid coi ,i)osition of Crus­ tacean plankton. J. Lipid Res. 5(3): 369-373. Wyss, D., B. J. Ludwig, and R. Joiner. 19''i2. The fungista­ tic and fungicidal action of fatty acids and related compounds. Arch. }>iochcm. Biophys. 7: 415-42 3.