PRESENCE OF KREBS CYCLE INTERMEDIATES IN PRIMARY

MYCELIA OP AN ACTINOMYCETE

APPROVED:

Ma jor*5j?rof essor

y \J O -' V"t XL' Minor Professor.

I

ry^\}AAk Director of the Depart mentvjof Biology

an or the Graduate School Dean

PRESENCE OP KREBS CYCLE INTERMEDIATES IN

PRIMARY MYCELIA OF AN ACTINOMYCES '

THESIS it •1

Presented to the Graduate Council of the

North Texas State University in Partial

Fulfillment of the Requirements

L For the Degree of L

MASTER OF ARTS

By

Joe Raymon Callaway, B. A.

Denton, Texas

August, 1968

TABLE OP CONTENTS

Page

LIST OF TABLES iv

Chapter

X. INTRODUCTION 1

History of Odoriferous Compounds Produced by Actinomycetes

Classification of the Streptomycetes Physiology and Metabolism in the

Streptomycetes

II. MATERIALS AND METHODS . . . . 15

III. RESULTS 21

IV. DISCUSSION 27

V. SUMMARY AND CONCLUSIONS 32

BIBLIOGRAPHY . . . . . . 33

lii

LIST OF TABLES

Table Page

I. Rf Values of Compounds Detected 23

II. Radioactivity Detected in Compounds from Soni-cated Mycelia 24

III, Radioactivity of Compounds Detected In Radio-active Media 25

lv

CHAPTER I

INTRODUCTION

Prior to 1940, studies of the physiology of the strep-

tomycetes were primarily concerned with a) the degradation

of organic matter in the soil, b) vitamin production, and

c) the role of these organisms in certain infections in man

and animals. Since 1940, most of the investigations' related

to this group of microorganisms have been directed toward

the discovery of antibiotics. Waksman (46) gave this period

a certain historical significance by listing the "antibiotic

period5' (1940 to the present) as a new era in the study of

the actinomycetes. His ideas have been largely confirmed by

recent reviews (3, 29, 47) In which the authors list exten-

sive references that demonstrate the widespread extent of

work devoted to the search for antibiotic-producing actino-

mycetes and the production and characterization of these an-

tibiotics. Other investigations were prompted by the ability

of certain actinomycetes to excrete repugnant metabolic by-

products into the environments in which they exist. This

phenomenon is especially evident where these repugnant odor-

iferous compounds are Incorporated into surface water sup-

plies when these waters support large populations of certain

apecies of the Streptomyces. It was noted by this author

that comparatively few references could be found in the sci-

entific literature concerning actinomycetes associated with

aquatic environments. Further evidence of this fact is noted

in that Bergey1s Manual (4) lists no members of the Strepto-

myces as being Isolated from aquatic habitats.

History of Odoriferous Compounds Produced by Actinomycetes

Before the beginning of this century (1895), Rullman

(37) reported a type of actinomycete that produced a pungent

odor which he described as "earthy." Biejerinck (6) con-

firmed this observation and suggested that some of the odors

from newly tilled soils were produced by the actinomycetes

growing therein. Berthelot and Andre (5) also regarded the

odors produced by actinomycete cultures to be very much like

those of freshly plowed soils. Although these odors from

terrestrial actinomycetes were often offensive, they presented

little or no problem in that they were quickly dissipated

into the atmosphere. On the other hand, this was not the

case where surface water supplies impregnated with these

odoriferous compounds were used for human consumption. Prob-

ably the first report to appear in the literature of an

"earthy taste" in potable water was made by Adams (l, 2) in

1929. He,suggested that the odor was due to contamination

of the water by actinomycetes; and also, in 1929, Rushton (38)

reported that the "earthy or muddy tastes" in water were due

to diatoms. The nature of the causative agent of unpleasant

tastes and odors in water supplies remained unresolved for

a period of some thirty years. Burger and Thomas (8) asserted

that the earthy taste in the waters'studied by them was the

result of complex microbial decomposition of organic matter.

They did not deny that actinomycetes could impart "earthy

tastes" to water, but concluded that the taste resulted from

the activities of a mixture of organisms. Thaysen (44) found

in 1936 that earthy or muddy tastes and odors both in the

water and in the fish found in those waters were caused by

actinomycetes.

Umbreit and McCoy (45) and Erikson (17) reported the

occurrence of actinomycetes of the genus Micromonospora in

certain northern lakes in the United States, but made no men-

tion that these organisms could elaborate substances capable

of imparting tastes and odors to the water in which they

were found. Issatchenko and Egorova (27) were able to show

that the actinomycetes which multiplied in silt and which

got into the waters of the Moscow River in.relatively large

quantities were important causes of the earthy and unplea-

sant flavors in this water. They assumed that the actinomy-

cetes were terrestrial microorganisms which had adapted them-

selves to an aquatic mode of existence. Silvey and Roach

(42) isolated members of the genus Micromonospora from a

Southwestern reservoir, but found that these organisms

did not produce odors. At the same time, however, they did

isolate some members of the genus Streptomyces that produced

strong odors in these reservoirs.

Silvey et al. (43) reported a number of actinomycetes

isolated from an aquatic environment that produced the char-

acteristic earthy, woody, musty, potato-bin tastes and odors

associated with certain members of this group of microorgan-

isms. These isolates were identified as Streptomyces spp.

by Waksman at Rutgers University. Roach and Silvey (36)

later proposed these aquatic organisms to be a separate

group since they seemed to possess an isogamous pattern of

sexual sporulation. Further investigations by Higgins and

Silvey (25) disproved this sexual pattern and verified the

isolates as members of the genus Streptomyces.

Classification of the Streptomycetes

The genus Streptomyces is a member of the family Strep-

tomycetaceae of the order Actinomycetales (46). This genus

has been described as a saprophytic, aerobic group of the

Actinomycetales characterized by highly branched mycelia

whose terminal ends consist of spore chains borne on aerial

mycelia (48). Orskov (32) was the first to observe that

members of the genus Streptomyces produced two separate growth

phases—the substrate, vegetative mycelia, and the aerial,

sporogenous mycelia. Klieneberger-Nobel (28) later renamed

these phases the primary and secondary mycelia respectively.

Physiology and Metabolism In the Streptomycetes

Classical taxonomy has defined the streptomycetes as

aerobic organisms f 33) • Recent Investigators (20, 25, 34)

have shown that members of this group can carry out fer-

mentations under microaerophillc conditions. Bisset (7)

and Erikson (18) suggested that the primary and secondary

mycelia of the streptomycetes have distinctly different phys-

iological as well as morphological characteristics. Davis

(l4) and Higgins (24) concluded that the primary mycelia were

facultatively aerobic, whereas the secondary mycelia were

obligatorily aerobic. Henssen (23) described two species of

streptomycetes as facultatively aerobic.

Changes in the growth pattern and physiology of Strep-

tomyces griseus were described by several investigators.

Waksman, Schartz, and Reilly (49) noted that S. griseus

grown in stationary and submerged cultures would reach a

maximum amount of growth and then a gradual loss of cellu-

lar elements due to slow lysis of cell material. This was

shown by the fact that the ash and nitrogen content of the

mycelia tended to be higher (per cell) during the early

stages of growth. Tests after attainment of maximal growth

showed a decrease In ash and nitrogen content. Gottlieb and

Anderson (22) demonstrated a decreased oxygen consumption

during the early growth phase of this organism. This was

attributed to a depletion of sugar in the medium with a

concomitant reduction in metabolism and an inherent change

in respiration of the mycella as a function of age. They

demonstrated increases in oxygen consumption upon the addi-

tion of more dextrose at various time intervals, but the

oxygen consumption never reached the level of that observed

in young cultures. Dulaney and Perlman (l6) recognized two

phases of metabolic activity in S. grlseus. The first phase,

the growth phase, was accompanied by a reduction in soluble

constituents of the medium, fermentation of the available

carbohydrate, and a high oxygen demand. This was followed

by an autolytic phase in which the mycelial weight decreased

markedly, inorganic phosphorus and soluble nitrogen were re-

leased into the medium, and the oxygen demand dropped.

There are certain inconsistencies in their descriptions which

may be attributed to a failure to distinguish between pri-

mary and secondary mycelia.

Several Investigators have presented evidence for some

of the well known metabolic pathways In the streptomycetes.

Cochrane (10) reported the existence of the glycolytic

scheme in Streptomyces coellcolor. Wang et al. (50) con-

cluded that this metabolic pathway was responsible for the

7

major portion of glucose breakdown with a minor part going

via the pentose shunt in Streptomyces griseus. The ability

of some species of Streptomyces to form pyruvic acid aero-

bically was demonstrated by Sahay (39). Hockenhull et al.

(26) showed that glucose was converted primarily to struc-

tural material and carbon dioxide under highly aerobic con-

ditions, but under restricted aeration lactic acid was

formed. They also showed that pyruvic acid was formed dur-

ing these stages of most rapid growth. Other investigators

(ll, 13, 20, 30, 4l) also concluded that the pentose shunt

was present and operative in the streptomycetes. The find-

ings of Hockenhull _et al. (26) supported the existence of

the pentose shunt by demonstrating that the utilization of

glucose" was controlled by mechanisms involving oxidative

phosphorylation.

Evidence of the presence of the Krebs cycle in Strep-

tomyces spp. came primarily from studies on crude extracts

and from isotope distribution experiments. Cochrane and

Peck (12) showed that cell-free extracts of Streptomyces

coelicolor were able to oxidize (with a requirement for

adenosine triphosphate) most of the Krebs cycle intermedi-

ates. No distinction was made between primary and second-

ary mycelial components in the extracts. They also demon-

strated the slow incorporation of carbon-14 from acetate-

2-C-14 into alpha-ketoglutarate. Prom these results they

8

concluded that Streptomyces coellcolor was able to carry out

the reactions of the citric acid cycle. Ganguly and Roy

(19) reported that the oxidation of the Krebs cycle inter-

mediates indicated the existence of that cycle in Strepto-

myces griseus. Again, no indication was given of the rela-

tive abundance of primary and secondary mycelia in their

preparations. Gilmour et al. (21) were able to demonstrate

the incorporation of more than twice as much carbon-14 from

acetate-l~C-l4 into the gamma carboxyl atom as was present

in the alpha carboxyl atom of glutamic acid. They reasoned

that this particular distribution was indicative of exten-

sive cyclic activity by way of asymmetric citric acid and

the other acids of the Krebs cycle. At approximately the

same time, Butterworth, Gilmour, and Wang (9) showed a

C 3—condensation product coupled with Krebs cycle activ-

ity as a major pathway of carbon dioxide fixation by Strep-

tomyces griseus.

More recently, Prave (35) demonstrated that extracts

of Streptomyces spp. oxidized the known major acids of the

Krebs cycle. Douglas and San Clemente (15) were able to

show malonate inhibition of succinate oxidation in .intact

mycelia, although the physiological types of the mycelia

were not indicated. Hockenhull et_ al. (26) reported that

Streptomyces griseus metabolized compounds of the Krebs

cycle, although citrate and alpha-ketoglutarate substrates

showed much less oxygen consumption at pH 7.3 than pyruvate,

acetate, succinate, fumarate, and malate. They demonstrated

the fact that ketoacids were produced in the presence of

arsenite from fumarate, malate, glucose, lactate, acetate,

succinate, glutamate, and citrate in descending order of

yield. Again, these workers made no mention of the active

morphological stages of the mycelia which they used. The

mediation of terminal respiration by cytochromes in the

streptomycetes was presented by several investigators

(31, 40, 39).

Statement of the Problem

The articles reviewed above have presented conclusive

evidence for the existence of the Krebs cycle in the strep-

tomycetes, but no distinction was made by any of the authors

between the primary and the secondary mycelia. Therefore,

it is the purpose of this study to determine the presence

or absence of a typical Krebs cycle metabolism in the pri-

mary mycelia of an aquatic streptomycete.

CHAPTER BIBLIOGRAPHY

1.. Adams, B. A., "Odors in Water of the Nile River," Water and Water Engineering, XXXI (1929), 309.

2, /"The Role of Actinomycetes in Producing Earthy Tastes and Smells in Potable Water," Paper 14, Department of Public Works, Roads, and Transport Con-gress, London, 1933.

3. Baldacci, E., "The Classification of Actinomycetes in Relation ot Their Antibiotic Activity," Advances in Applied Microbiology, III (1961), 257~27<3̂

Bergey, D. H., Bergey's Manual of Determinative Bacter-iology, 7th ed., Baltimore, Williams and Wilklns Company, 1957.

5. Berthelot, M. and G. Andre, "Sur I'odeur propre de la terre," Comptes-Rendus, CXII (1891), 598-602.

6. Biejerinck, M. W., "Uber Chinonbildung durch Strepto-thrix chromogena und Lebensweise dieses Mikroben, Zentralblatt Ttlr Bakteriologle, Abtellung II (Allge-meine Landwirtschaftlische Bakteriologle), YI (1900), 2-12.

7. Bisset, K. A., "The Morphology and Natural Relationships of Saprophytic Actinomycetes," Progress in Industrial Microbiology, I (1959), 29-^2.

8. Burger, J. W, and S. Thomas, "Tastes and Odors in the Delaware River," Journal of the American Water Works Association, XXIV U934), 120-127.

9. Butterworth, E. M., C. M. Gilmour and C. H. Wang, "Studies on the Biochemistry of the Streptomyces. II. Fixation of C1402 by Intact Cells of Streptomyces ' grlseus," Journal of Bacteriology, LXIX (1955), 725-727.

10, Cochrane, V. W., "The Metabolism of Species of Strepto-myces, VIII. Reactions of the Embden-Meyerhof-Parnas Sequence In Streptomyces coelicolor," Journal of Bac-teriology, LXIX U955), 256-263.

10

11

11. Cochrane, V. W. and P. L. Hawley, "The Metabolism of Species of Streptornyces. IX. Metabolism of Pentose and Hexose Phosphates,^ Journal of Bacteriology, LXXI (1956), 308-314.

12. Cochrane, V. W. and H. D. Peck, Jr., "The Metabolism of Species of Streptornyces. VI. Tricarboxylic Acid Cycle Reactions in Streptornyces coellcolor," Journal of Bacteriology, LXV~Tl9537, 37-W. ~ '

13. Cochrane, V. W., H. D. Peck, Jr., and A. Harrison, "The Metabolism of Species of Streptornyces. VII. The Hexo-semonophosphate Shunt and Associated Reactions," Jour-nal of Bacteriology, LXVT (1953), 17-23.

14. Davis, Ernst, "Assimilation of Inorganic Nitrogen by Actinomycetes," unpublished Master's thesis, Depart-ment of Biology, North Texas State University, Denton, Texas, 1962.

15. Douglas, R. J. and C. L. San Clemente, "Respiration of Scab-Producing Strains of Actinomycetes," Canadian Journal of Microbiology, II (1956), 407-415.

16. Dulaney, E. L. and D. Perlman, "Observations on Strepto-myces griseus. I. Chemical Changes Occurring During Submerged Streptomycin Fermentations," Bulletin of the Torrey Botanical Club, LXXIV (1947),"50^-511.

17. Erikson, D., "Studies on Some Lake-Mud Strains of Mlcro-monospora," Journal of Bacteriology, IXL (1941), 277-300.

18. , "The Morphology, Cytology, and Taxonomy of the Actinomycetes," Annual Review of Microbiology, III (1949), 23-54!

19. Ganguly, S. and S. C. Roy, "Oxidation of Substrates by Streptornyces griseus," Archives of Biochemistry and Biophysics, LIX (l955),~1f5-51.

20. , "Utilization of Phosphorus by,Streptornyces griseus During Its Aerobic and Anaero-bic Growth," Archives of Biochemistry and Biophysics, LXIII (1956), 26-31. "

21. Gilmour, C. M., E. M. Butterworth, E. P. Noble and C. H. Wang, Studies on the Biochemistry of the Strep-tomycetes. I. Terminal Oxidative Metabolism in Strep-tomyces griseus," Journal of Bacteriology, LXIX (1955), 719-724.

12

22. Gottlieb, D. and H, W. Anderson, "The Respiration of Streptomyces griseus," Science, CVII-(1948), 172-173.

23. Henssen, A., "Beltrage zur Morphologie und Systematik der thermophilen Actinomyceten," Archiv fur Mikrobi-ologle, XXVI (1957), 373-414.

24. Hlggins, Michael Lee, "The Life Cycle of an Aquatic Actinomycete," unpublished Master's thesis, Depart-ment of Biology, North Texas State University, Denton, Texas, 1964.

25. Higgins, M. L. and J. K. G. Silvey, "Slide Culture Ob-servations of Two Freshwater Actinomycetes," Trans-actions of the American Microscopical Society, LXXXY TT9F5T, 390-398.

26. Hockenhull, D. J. D., K. H. Fantes, M. Herbert, and B. Whitehead, "Glucose Utilization by Streptomyces griseus," Journal of General Microbiology, X (1954), 353-370.

27. Issatchenko, B. and A. Egorova, "Actinomycetes in Reser-voirs as One of the Causes Responsible for Earthy Smell of Their Waters," Microbiology, XIII (1944), 224-230.

28. Klieneberger-Nobel, E., "The Life Cycle of Sporing Actinomyces as Revealed by a Study of Their Structure and Septation," Journal of General Microbiology, I (1949), 22-32.

29. Lechevalier, H. A. and M. P. Lechevalier, "Biology of Actinomycetes," Annual Review of Microbiology, XXI (1967), 71-100.

30. Maitra, P. K. and S. C. Roy, "Pathways of Glucose Dis-similation by Streptomyces olivaceus," Journal of Bio-logical Chemistry, CCXXXIV (1959), 2497-2503.

31. Niederpruem, D. J. and D. P. Hackett, "Respiratory Chain of Streptomyces," Journal of Bacteriology, LXXXI (1961), 557-563.

32. Orskov, J., Investigations into the Morphology of the Ray Fungi, Copenhagen, Levin and Nunksquard, 1923.

33. Perlman, D., "Physiological Studies on the Actinomycetes," Botanical Review, XIX (1953), 49-97.

13

34. Prave, P.', "Stoffwechsel und Actinomycinbildung von Streptomyceten," Archiv fur MIkrobiologie, XXXII (1958), 278-285.

35. f "Citronensaurecyclus und Actinomycinbildung," Archly fur MIkrobiologie, XXXII (1958), 286-295.

36. Roach, A. W. and J. K. G. Sllvey, "The Morphology and Life Cycle of Fresh-Water Actinomycetes," Transactions of the American Microscopical Society, LXXVII (1958), 35-¥77

37» Rullman, W., "Ohemisch-bakteriologische Untersuchungeri von Zwishendeckenfullungen mit besonderer Berucksich-tigung von Cladothrix odorlfera," Zentrablatt fur Bakteriologle, Parasitenkunde, "Abteilung I, TMedi-zlnlsch-Hygienische Bakteriol'ogleTT XV"II (189577 884-8B5.

38, Rushton, W., "The Purity of Drinking Water from a Bio-logical Aspect," Paper 12, Report of Public Works, Roads, and Transport Congress, London, 1929.

39* Sahay, B. N. "Untersuchungen an farblosen, Farbstoff und Antibiotica bildenden Streptomyceten," Archiv fiir MIkrobiologie, XXXVII (i960), 327-340.

40. Sato, S.. "Cytochromes in Bacteria Especially Actinomy-cetes, Kltasata Archives of Experimental Medicine (in English)", XVII (1940), 2.

41. Silverman, M. and S. V. Rieder, "Formation of N-methyl-L-glucosamlne from D-glucose by Streptomyces griseus," Journal of Biological Chemistry, CCXXXV (i960), 1251-1254.

42. Sllvey, J. K. G. and A. W. Roach, "Actinomycetes in the Oklahoma Water Supply," Journal of the American Water Works Association, VL (195377^09-415.

43. Sllvey, J. K. G., J. C. Russell, D. R. Redden and W. C. McCormick, "Actinomycetes and Common Taste and Odors," Journal of the "American Water Works Association, VIIIL (195077 1018-1025.

44. Thaysen, A. C., ''The Origin of an Earthy or Muddy Taint in Fish. I. The Nature and Isolation of the Taint," Annuals of Applied Biology, XXXIII (1936), 99-104.

45. Umbreit, W. ¥. and E. McCoy, "The Occurrence of Actino-mycetes of the Genus Mlcromonospora In Inland Lakes," Symposium on Hydrobiology, University of Wisconsin, Madison, l"9¥l.

46. Waksman, S. A., The Actinomycetes—Nature, Occurrence, and Activities, Vol. I, Baltimore, Williams and Wilkins Company, 1959.

47. , "The Actinomycetes and Their Antibiotics," Advances in Applied Microbiology, V (1963), 235-315.

48. Waksman, S. A. and A. T. Henrici, "The Nomenclature and Classification of the Actinomycetes," Journal of Bacteriology, XLVI (1943), 337-341.

49. Waksman, S. A., A. Schartz, and H. C. Reilly, "Metabolism and the Chemical Nature of Streptomyces griseus," Journal of Bacteriology, LI (1946), 753-759.

50. Wang, C. H., J, J. Bialy, S. Klungsoyr and C. M. Gil-mour, "Studies on the Biochemistry of Streptomyces. III. Glucose Catabolism in Streptomyces griseus," Journal of Bacteriology, LXXV U957), 31-37.

CHAPTER II

MATERIALS AND METHODS

The organism used in this study was one designated num-

ber 62 from the North Texas State University Actinomycete

Collection, This organism was selected because many other

investigations (2, 3, 4, 6) had "been performed with this

culture. Higgins and Silvey (5) showed this strain to be a

member of the genus Streptomyces. The organism was orig-

inally isolated from a Southwestern reservoir in which re-

pugnant tastes and odors were detected.

Classification of isolate number 62 was attempted using

serological techniques currently under development at North

Texas State University. Computerized data analysis indi-

cated that this organism closely resembles Streptomyces antl-

blotlcus (7). This author knows of no other attempts to

classify isolate number 62.

The experimental stock culture of isolate number 62 was

maintained on modified starch agar slants consisting of solu-

ble starch 10 g.# Tryptone 5 g.# dipotassium phosphate 0.5

g., sodium chloride 0.5 gferrous sulfate 0.1 g., Noble

agar (Difco) 12 g., and distilled water 1000 ml. On this

medium growth was rapid, and sporulatlon took place in seven

days.

15

16

Spore suspensions were made by flooding seven day-old

modified starch agar slants with a sterile, 0,05 per cent

aqueous detergent solution (Joy, Procter and Gamble) and agi-

tating gently. The detergent was found necessary as a wet-

ting agent for making homogeneous spore suspensions since the

spores are very hydrophobic. One tenth milliliter aliquots

of these suspensions were used to inoculate flasks contain-

ing 20 ml. of an enriched broth medium consisting of Trypti-

case Soy Broth 5 g., Nutrient Broth 4 g., Emerson's Broth

8 g,, brown sugar 8 g., Neopeptone (Difco 4 g., ammonium ni-

trate 1 g,, soil water extract 100 ml., and distilled water

900 ml.). This medium was adjusted to pH 6.8 with dilute

phosphoric acid before autoclaving. This medium gave rapid

germination of the spores and abundant mycelial development.

After a 24 hour growth period in the enriched medium, micro-

scopic examinations were utilized to determine the presence

of primary mycelia.

The primary mycelia were separated from the above en-

riched medium in a Sorvall RC2-B centrifuge. Two separate

mycelial preparations were prepared simultaneously, and each

was washed three times with 10 ml. portions of sterile 0.85

per cent sodium chloride solution. Wet weights of the pri-

mary mycelial preparations were determined at this time. . A

10 ml. solution of radioactive sodium-l,2-C-l4-acetate con-

taining 250 microcuries (specific activity 23.4 millicuries

17

per millimole) was prepared and added to the first mycelial

preparation; a 10 ml. solution of radioactive succinic acid-

2,3--C-l4 containing 100 microcuries (specific activity 10.2

millicuries per millimole) was prepared and added as previ-

ously described.

The above preparations containing the radioactive sub-

strates were immediately placed in a Torbal jar and closed.

Laboratory air was passed through the jar, and the effluent

air was bubbled through a strong sodium hydroxide trap to

eliminate the discharge of radioactive carbon dioxide into

the laboratory atmosphere. This apparatus was placed on a

reciprocating shaker at 80 oscillations per minute. The my-

celia were exposed to the radioactive material for two hours

at room temperature (25-26 degrees C,). At the end of this

time, the mycelia were again centrifuged and washed as be-

fore in order to remove the unincorporated radioactive sub-

strates. The mycelia were immediately suspended in distilled

water and sonicated in three five-second intervals using a

Branson Instruments Model LS-75 sonicator.

The disrupted mycelial suspensions were centrifuged in

order to eliminate fragments, and the soluble supernatants

were chromatographed on Whatman Number 1 chromatographic pa-

per. Each chromatogram was prepared by spotting and drying

repeatedly until 100 microliters of solution had been applied

to the paper. Two-dimensional chromatography was employed,

18

utilizing solvents described by Benson et _al, (l). Descend-

ing chromatography was used in the first separation with

water-saturated phenol (approximately 88 per cent phenol from

Mallinckrodt) as the solvent. Ascending chromatography was

employed for the second separation (dimension) with freshly

prepared solvent made by mixing equal volumes of solution A

(1246 ml, of n-butanol and 84 ml. of water) and solution B

(620 ml, of propionic acid and 790 ml, of water). The sol-

vents' were evaporated from the chromatograms by drying over-

night in the hood at room temperature by drying in a hot air

oven (100 degrees C.) for one hour. A 0,04 per cent eth-

anolic solution of bromcresol green was used to locate the

acidic Intermediates of the Krebs cycle. Known standards of

the Krebs cycle compounds (alpha-ketoglutarate, citrate, fu-

marate, malate, oxalacetate, and succinate) were used to re-

inforce the original spots containing the trace quantities

of labeled Krebs cycle Intermediates obtained from the or-

ganism. This procedure facilitated identification of the

chromatographic spots.

The amino acids were identified on another chromatogram

(prepared and developed as previously described) by develop-

ment with a 0.2 per cent ethanolic nlnhydrin. These chromato-

grams did not require reinforcement. The spots of the de-

tected compounds were cut out in 20 mm, circles and placed on

25 mm. planohettes. Beta-emissions of the radioactive

19

compounds were determined by counting the number of dis-

integrations on a Tracerlab Scalar Model SC-63 proportional

gas flow counter. All counts were corrected for background.

CHAPTER BIBLIOGRAPHY

1. Benson, A. A., J. A. Bassham. M. Calvin, T. C. Goodale, V. A. Haas and ¥. Stepka, "The Path of Carbon in Photo-synthesis, V, Paper Chromatography and Radioautography of the Products," Journal of the American Chemical So- • olety, LXXII (1950), 1710-171^7"

2. Davis, Ernst, "Assimilation of Inorganic Nitrogen by Actinomycetes," unpublished Master's thesis, Depart-ment of Biology, North Texas State University, Denton, Texas, 1962.

3. Francisco, Donald E., "The Effect of Carbon Monoxide on the Growth of an Aquatic Streptomycete," unpublished Master's thesis, Department of Biology, North Texas State University, Denton, Texas, 1966.

4. Hlggins, Michael Lee, "The Life Cycle of an Aquatic Ac-tinomycete," unpublished Master's thesis, Department of Biology. North Texas State University, Denton, Texas, 1964,

5. Higglns, M. L. and J. K. G. Silvey, "Slide Culture Ob-servations of Two Freshwater Actinomycetes," Trans-actions of the American Microscopical Society, LXXXV U9bb), 390-398.

6. Shao, Yi-min, "Assimilation of Organic Carbon by Aquatic Actinomycetes," unpublished Master's thesis, Depart-ment of Biology, North Texas State University, Denton, Texas, 1963.

7. Taylor, Gerald R., unpublished data, Department of Bi-ology, North Texas State University, Denton, Texas, 1967.

20

CHAPTER III

RESULTS

Microscopic examination of growth after 24 hours of in-

cubation showed that only primary mycelia were present.

This observation was in accordance with the findings of Hig-

gins and Silvey (3) and Francisco (l). These investigators

showed that the organism used in this study required 36 or

more hours of sustained growth after germina'tion of the

spores before secondary mycelia developed in the same or

similar media. The enriched medium used in this study sup-

ported rapid germination of the spores and abundant growth

of the primary mycelia in" a short period of time.

Wet weights of the two mycelial preparations described

above were determined. It was found that 0.7418 g. and

1.0064 g. were used for the radioactive acetate and suc-

cinate solutions respectively. A 0.1 ml, aliquot of each

of the radioactive solutions was taken before and after ex-

posure to the mycelia. Comparison of the relative radio-

activity of these aliquots showed that approximately 19 per

cent of the radioactive acetate and 60 per cent of the radio-

active succinate were utilized by the respective mycelial

preparations,

21

22

The Rf value, as given In Table I, on the following

page, for each compound, represents an average of the values

from 8 chromatograms for Krebs cycle intermediates and 14

chromatograms for the amino acids detected. Although Rf

values for any given compound varied, the relative position

of the compound did not change in relationship to the other

compounds. Therefore, it was possible to locate and identify

each of the compounds. The Rf values listed in Table I are

averages of the Rf values calculated from all the chromato-

graphic preparations.

The relative radioactive labeling of the various com-

pounds detected in the supernatant from the sonicated mycelia

are listed in Table II (page 24). It was observed that the

largest amount of radioactivity was accumulated in glutamic

acid in both preparations. The labeling of the other amino

acids in decreasing order were aspartie acid, serine, and

phenylalanine. No labeling was found in alanine and gly-

cine, although these substances were detected.

Comparatively, the labeling of the Intermediates of

the Krebs cycle was low (Table II). This was attributed to

low concentrations of these intermediates, as. the radioactive

labeling was the only evidence of the existence of these

substances in this organism. Identification of the inter-

mediate compounds by standard paper chromatographic methods

could not be accomplished without reinforcement of the spots

with known standards.

23

TABLE'I

Rf VALUES OF COMPOUNDS DETECTED

*Water saturated phenol (approximately llnckrodt),

Compounds Rf Values for Solvent A*

Rf Values for Solvent B**

Alanine 0.74 0.43

Aspartic acid 0.19 0.27

Glutamic acid 0.27 0.36

Glycine 0.36 0.28

Phenylalanine 0.94 0.24

a-ketoglutarate 0.58 0.44

Citrate 0.29 0.36

Fumarate 0.31 0.83

Malate 0.34 0.38

Oxalacetate 0.27 0.31

Succinate 0.46 0.56

phenol; Mai.

**Prepared fresh each time by mixing equal volumes of solution A (1246 ml. of n-butanol and 84 ml. of water) and solution B (620 ml. of propionic acid and 790 ml. of water).

24

TABLE II

RADIOACTIVITY DETECTED IN COMPOUNDS PROM SONICATED MYCELIA*

Compounds Acetate Medium

cts/min** Succinate Medium

cts/min**

Alanine none none

Aspartic acid 323 634

Glutamic acid 11,435 6,324

Glycine none none

Phenylalanine - 107 57

Serine 213 157

a~ketoglutarate 91 84

Citrate 3^ . 27

Fumarate 176 217

Malate 163 187

Oxalacetate 157 325

Succinate 122 4l8

*Chromatographed 100 microliters from 5 ml, superna* tent decanted from sonicated mycelia.

**Counts per minute—corrected for background.

25

Reference to Table III shows the presence of labeled

glutamic acid in the radioactive solutions after exposure

to the mycella used in this study. This was in agreement

with the findings of Gilmour et al. (2) and' Perlman and

O'Brien (4). These investigators demonstrated the accumu-

lation of glutamic acid in various media by members of the

streptomycetes.

TABLE III

RADIOACTIVITY OF COMPOUNDS DETECTED IN RADIOACTIVE MEDIA*

Compound Acetate Medium Succinate Medium

Compound cts/min** cts/min**

Glutamic acid 2,394 1,415

Succinic acid none 72,068

*Chromatographed 100 microliters,

**Counts per minute—corrected for background.

CHAPTER BIBLIOGRAPHY

1. Francisco, Donald E., "The Effect of Carbon Monoxide on the Growth of an Aquatic Streptomycete," unpublished Master13 thesis, Department of Biology, North Texas State University, Denton, Texas, i960.

2. Gilmour, C. M.. E. M. Butterworth, E. P. Noble and C. H. Wang, Studies on the Biochemistry of the Strep-tomycetes. I. Terminal Oxidative Metabolism in Strep-tomyces griseus," Journal of Bacteriology, LXIX (1955)* 719-72?.

3. Higgins, M. L. and J. K. G. Silvey, "Slide Culture 0b~ servations of Two Freshwater Actinomycetes," Trans-actions of the American Microscopical Society, LXXXV (1966), 390-398.

4. Perlman, D, and E. OiBrien, "Production of Glutamic Acid by Streptomycetes," Journal of Bacteriology, LXXV (1958), 611.

26

CHAPTER IV

DISCUSSION

The streptomycetes have been generally described as an

aerobic group (8) of microorganisms, but several workers

have shown that some members of this group carry out fer-

mentations under anaerobic or, more aptly, microaerophilic

conditions (4, J, 10). No consideration was given by any

of these investigators to the active morphological stages

under microaerophilic conditions. Higgins (6), Francisco

(3), and Davis (2) demonstrated that the primary mycella could

develop under microaerophilic conditions whereas the second-

ary mycelia could not develop. From these observations, it

was questionable as to whether a typical Krebs cycle existed

in the primary mycelia or only fermentative metabolism in

the primary, substrate mycelia, and later, aerobic metabolism

after development of the secondary, aerial mycelia.

Radioactive compounds were used to demonstrate the pres-

ence of the Krebs cycle. It was felt that the demonstration

of the specific intermediates of the Krebs cycle or certain

analogues of these intermediates was necessary to rule out

alternative pathways of oxidative metabolism. The relative

labeling of the Krebs cycle intermediates (Table II) indi-

cates that these substances are present and active but in

27

28

comparatively small concentrations. This does not necessar-t

ily reduce the Importance of this pathway In the overall

metabolism of this organism, but It may Indicate a rapid

utilization of these Intermediates, The significance of

the reduced seemingly concentrations of the Krebs cycle

intermediates may lie in the fact that these substances may

occur in approximately the same concentrations as their spe-

cific enzymes.

The mycelial preparation for the radioactive succinate

weighed more than the mycelial preparation for the radio-

active acetate. This weight difference (approximately 26

per cent more for the succinate) should not account for the

larger utilization of the succinate (60 per cent) as opposed

to the acetate (19 per cent). This may possibly be explained

by the access of succinate to an alternative metabolic path-

way (such as the-pentose shunt). In the general scheme of

the Krebs cycle, succinate is converted to fumarate by suc-

cinic dehydrogenase. Fumarate, in turn, is transformed into

malate by fumarase. At this point in the general scheme of

Krebs cycle, malate could have been decarboxalated by the

malic enzyme to form pyruvate. Subsequent reversal of the

glycolytic scheme could have converted pyruvate to phospho-

glyceraldehyde, which is a component of the pentose shunt.

This procedure would have placed a portion of the original

radioactive succinate molecule Into substances that were

29

not investigated. Using this same reasoning, additional

metabolic pathways could be presented by which the radio-

activity in the succinate could have been incorporated into

other mycelial components besides those of the Krebs cycle. a"

The larger relative labeling of aspartic acid and

especially glutamic acid (Table II) was perhaps the most

significant evidence for the existence of a typical Krebs

cycle. Basch and Baltrush (l) concluded that labeled glu-

tamic acid derived from using radioactive acetate was mainly

by way of Krebs cycle and alpha-ketoglutarate in all systems.

By the same reasoning, aspartic acid arises primarily from

oxalacetate which is the analogue of aspartic acid in the

Krebs cycle,. The labeling of aspartic acid from the radio-

active succinate was greater than from the radioactive

acetate (Table II). This would be expected as the succinate

was more readily accessible for aspartic acid formation.

Evidence for a complete Krebs cycle comes primarily

from detection of labeled compounds derived from the radio-

active succinate. The decarboxylation of alpha-ketogluta-

rate by coenzyme-A and the subsequent formation of succinyl-

coenzyme~A is irreversible. Therefore, radioactive succinate

could not have been incorporated into alpha-ketoglutarate

and them into glutamic acid by reversal of the Krebs cycle

succinyl-coenzyme-A reaction.. Consequently, it is reason-

able to assume that the labeled glutamic acid (Table II and

30

III) derived from the radioactive succinate (or a portion

thereof) came mainly from utilization of the typical Krebs

cycle intermediates.

Previous workers (5, 9) have demonstrated the accumula-

tion of glutamic acid in the various media used in cultur- .

ing the streptomycetes. This investigation concurred with

these findings since radioactive glutamic acid was detected

in both the radioactive acetate and succinate media (Table

III). The data do not indicate the significance of the

relatively large quantities of this amino acid formed by

the organism used in this study. Further investigations

are needed to explain this phenomenon.

Although previous workers have shown that the primary

mycelia can develop under microaerophilic conditions with

energy derived from fermentative reactions, the data in this

study suggest the existence and utilization of a typical

Krebs cycle. The data do not, however, indicate the quan-

titative importance of this cycle in the organism used in

this study.

CHAPTER BIBLIOGRAPHY

1. Basch, H. and ,H. A. Baltrush, "Isotopic Equilibration between the Citric Acid Cycle and Glutamic Acid," A Symposium on Amino Acid Metabolism, edited by W. D, McElroy and H. 33. Glass, Baltimore, Johns Hopkins Press, 1955? PP. 291-300.

2. Davis, Ernst, "Assimilation of Inorganic Nitrogen by Actinomycetes," unpublished Master's thesis, Depart-ment of Biology, North Texas State University, Denton, Texas, 1962.

3. Francisco, Donald E., "The Effect of Carbon Monoxide on the Growth of an Aquatic Streptomycete," unpub-lished Master's thesis, Department of Biology, North Texas State University, Denton, Texas, 1966.

4. Ganguly, S. and S. C. Roy, "Oxidation of Substrates by Streptornyces grlseus," Archives of Biochemistry and Biophysics, LIX (1955)* 45-51.

5. Gilmour, C. M. . E. M. Butterworth, E. P. Noble, and C. H. Wang, Studies on the Biochemistry of the Streptomycetes. I. Terminal Oxidative Metabolism

' in Streptornyces griseus," Journal of Bacteriology, LXIX (1955) , 719-724"

6. Higgins, Michael L., "The Life Cycle of an Aquatic Actinomycete," unpublished Master's thesis, Depart-ment of Biology, North Texas State University, Denton, Texas, 1964.

7. Hockenhull, D. J. D., K. H. Fantes, M. Herbert and B. Whitehead, "Glucose Utilization by Streptornyces grlseus," Journal of General Microbiology, X (1954), 353-370.

8. Perlman, D., "Physiological Studies on the Actinomy-cetes," Botanical Review, XIX (1953), 49-97.

9. Perlman, D. and E. O'Brien, "Production of Glutamic Acid by Streptomycetes," Journal of Bacteriology, LXXV (1958), 611.

10. Prave, P., "Stoffwechsel und Actinomyclnbildung von Streptomyceten," Archiv fur Mikroblologie, XXXII (1958), 278-285.

31

CHAPTER V

SUMMARY AND CONCLUSIONS

1. Radioactive compounds were employed to demonstrate

the presence of the Intermediates of the Krebs cycle.

2. Radioactive succinate apparently was utilized by

the organism investigated to a greater extent than the

radioactive acetate. A plausible explanation of this phe-

nomenon was the incorporation of the succinate into alter-

native metabolic pathways.

3. The amino acid analogues (aspartic acid and glu-

tamic acid) of two intermediates (oxalacetate and alpha-

ketoglutarate respectively) of the Krebs cycle were the

recipitants of the largest amount of labeling in both the

radioactive acetate and succinate preparations.

4. The labeling of the glutamic acid In the radio-

active succinate preparation was the most significant evi-

dence for the existence of a typical Krebs cycle.

5. Extramycellal radioactive glutamic acid was de-

tected in both the radioactive acetate and succinate media.

The data do not indicate the significance of this phenomenon.

The data indicate the presence of typical Krebs cycle

intermediates, but do not demonstrate the quantitative im-

portance of this metabolic pathway.

32

BIBLIOGRAPHY

Books

Sergey, D. H., Bergey1s Manual of Determinative Baterlol-ogy, ,'th ed"., Baltimore,""Williams and Wllkins Jomp&ny, 1957.

McElroy, W. D, and H. B. Glass, editors, A Symposium on Amino Acid Metabolism, Baltimore, Johns Hopkins Press, 1955.

Orskov, J., Investigations into the Morphology'of the Ray Fungi, Copenhagen, Levin and Nunksquard, 1923.

Umbreit, W. W. and E. McCoy, Symposium on Hydrobiology, Madison, University of Wisconsin, 19^1.

Waksman, S. A., The Actinomycetes--Nature, Occurrence, and Activities, Vol. I*, Baltimore, Williams and Wilklns Company, 1959.

Articles

Adams, B. A., "Odors in Water of the Nile River," Water and and Water Engineering, XXXI (1929), 309.

Baldacci, E., "The Classification of Actinomycetes in Re-lation to Their Antibiotic Activity," Advances in Ap-plied Microbiology, III (1961), 25J-27B~. '

Benson, A. A., J, A. Bassham, M, Calvin, T. C. Goodale, V. A. Haas and W. Stepka, "The Path of Carbon in Photosynthesis. V. Paper Chromatography and Radio-autography of the Products," Journal of the American Chemical Society, LXXII (1950J, 1710-171H7"

Berthelot, M. and G. Andre, "Sur 11odeur propre de la terre," Comptes-Rendus, CXII (1891), 598-602.

Biejerinck, M. W., "Uber Chinonbildung durch Streptothrix chromogena und Lebensw'eise dieses Mlkroben, Zentral-blatt fur Bakteriologle, Abteilung II (Allgemelne Land-wlrtschaftlische Bakteriologle), VII (1900), 2-12.

33

3^

Blsset, K. Q., "The Morphology and Natural Relationships of' Saprophytic Actlnomycetes." Progress In Industrial Mi-crobiology, I (1959), 29-42.

Burger, J. W. and S. Thomas, "Tastes and Odors In the Dela-ware River," Journal of the American Water Works Asso-clatlon, XXIV (l93IF7, 120-127.

* *

Butterworth, E. M., C. M. Gllmour, and C. H. Wang, " Studies on the Biochemistry of the Streptomyces. II. Fixation of C 1 40 2 by Intact Cells of Streptomyces gr'iseus," Journal of Bacteriology, LXIX (1955), 725-727.

Cochrane, V. W., "The Metabolism of Species of Streptomyces. YIII. Reactions of the Bnbden-Meyerhof-Parnas Sequence in Streptomyces coelicolor," Journal of Bacteriology, LXIX U955), 256-263.

Cochrane, V. W. and P. L. Hawley, "The Metabolism of Species of Streptomyces. IX. Metabolism of Pentose and Hexose Phosphates," Journal of Bacteriology, LXXI (1956), 308-314,

Cochrane, V. W. and H. D. Peck, Jr., "The Metabolism of Species of•Streptomyces. VI. Tricarboxylic Acid Cycle Reactions in Streptomvces coelicolor," Journal of Bac-teriology;, LXV (1953•), 3 7 - W

Cochrane, V. W., H. D, Peck, Jr. and A. Harrison, "The Metabolism of Species of Streptomyces. VII. The Hexo-semonophosphate Shunt and Associated Reactions," Jour-nal of Bacteriology, LXVI (1953), 17-23.

Douglas, R. J. and C. L. San Clemente, "Respiration of Scab-Producing Strains of Actlnomycetes,"' Canadian Journal of Microbiology, II (1956), 407-415.

Dulaney, E. L. and D. Perlman, "Observations on Streptomyces griseus. I. Chemical Changes Occurring During Submerged Streptomycin Fermentations," Bulletin of the Torrey Bo-tanlcal Club, LXXIV (19^7), 504-511.

Erikson, D.f "Studies on Some Lake-Mud Strains of Micromono-spora," Journal of Bacteriology, IXL (l94l), 277-300.

, "The Morphology, Cytology, and Taxonomy of the Actlnomycetes," Annual Review of Microbiology, III (1949), 23-54!

35

Ganguly, S. and S. C. Roy, "Oxidation of Substrates by Strep-tomyces griseus," Archives of Biochemistry and Bio-physics, Lix~Ti955), 45-51.

"Utilization of Phosphorus by Streptomyces griseus During Its Aerobic and Anaerobic Growth, Archives of Biochemistry and Biophysics, LXIII (1956), 2F-31.

Gilmour, C. M., E. M. Butterworth, E, P. Noble and C. H. Wang, "Studies on the Biochemistry of the Streptomycetes. I. Terminal Oxidative Metabolism in Streptomyces griseus," Journal of Bacteriology, LXIX (1955), 719-724,

Gottlieb, D. and H. W. Anderson, "The Respiration of Strep-tomyces griseus," Science, CYII (1948), 172-173.

Henssen, A., "Beitrage zur Morphologie und Systematik der thermophilen Actinomyceten," A'rchiv fur Microbiologie, XXVI (1957), 373-414.

Hlggins, M. L. and J. K. G. Silvey, "Slide Culture Observa-tions of Two Freshwater Actinomycetes," Transactions of the American Microscopical Society, LXXXV (1966), 390-398.

Hockenhull, D. J. D., K. -H, Pantes, M. Herbert and B. White-head, "Glucose Utilization by Streptomyces griseus," Journal of General Microbiology"^ I (1954), 353-370.

Issatchenko, B. and A. Egorova, "Actinomycetes in Reservoirs as One of the Causes Responsible for Earthy Smell of Their Waters," Microbiology, XIII (1944), 224-230.

Klieneberger-Nobel, E., "The Life Cycle of Sporing Actino-myces as Revealed by a Study of Their Structure and Septation," Journal of General Microbiology, I (1949) 22-32.

Lechevalier, H. A. and M. P. Lechevalier, "Biology of Actino-mycetes," Annual Review of Microbiology, XXI (1967), 71-100.

Maitra, P. K. and S. C, Roy, "Pathways of Glucose Dissimi-lation by Streptomyces olivaceus," Journal of Biologi-cal Chemistry, CCXXXIY (1959), 2497-2503:

Niederpruem, D. J. and D, P, Hackett, "Respiratory Chain of Streptomyces," Journal of Bacteriology, LXXXI (1961), 557-563.

36

Perlman, D., "Physiological Studies on the Actinomycetes," Botanical Review, XIX (1953), 49-97.

Perlman, D. and E. O'Brien, "Production of Glutamic Acid by Streptomycetes," Journal of Bacteriology, LXXV (1958), 611.

Prave, P., "Stoffwechsel und Actinomycinbildung von Streptomy-ceten," Archly fttr Microbiologic, XXXII (1958), 278-285 .

, "Citronensaurecyclus und Actinomycinbildung," Arc'hiv fur Microbiologic, XXXII (1958), 286-295.

Roach, A. W, and J. K. G. Silvey, "The Morphology and Life Cycle of Fresh-Water Actinomycetes," Transactions of the American Microscopical Society, LXXVII (1958), 36-Tf.

Rullman, W., "Chemisch-bakteriologische Untersuchungen von Zwishendeckenfullungen mit besonderer Berucksichtigung von Cladothrix odorifera," Zentrablatt fur Bakteriolo-gie, Parasitenkunde, Abteilung I, (Medizinisch-Hygien-ische Bakteriologie), XVII TT895), ««4-««5.

Sahay, B. N., "Untersuchungen an farblosen. Farbstoff und Antibiotica bildenden Streptomyceten, Archiv fur Mikrobiologie, XXXVII ( i 9 6 0 ) , 327-340.

Sato, S., "Cytochromes in Bacteria Especially'Actinomycetes." Kitasata Archives of Experimental Medicine (in English), x v n ~ ( T 9 4 o 7 , 2 .

Silverman, M. and S. V. Rieder, "Formation of N-methyl-L-glucosamene from D-glucose by Streptomyces griseus," Jour-nal of Biological Chemistry, CCXXXVT1950J, 1251-1254.

Silvey, J. K. G. and A. W. Roach, "Actinomycetes in the Ok-lahoma Water Supply," Journal of the American Water Works Association, VL (1953), 409-4157

Silvey, J. K. T. . J. C. Russell, D. R. Redden, and W. C. McCormick, Actinomycetes and Common Taste and Odors," Journal of the American Water Works Association, VIIIL (1950;, 101B-1025.

Thaysen, A. C., "The Origin of an Earthy or Muddy Taint in Fish. I. The Nature and Isolation of the Taint," An-nuals of Applied Biology, XXXIII (1936), 99-104,.

Waksman, S. A., "The Actinomycetes and' Their Antibiotics," Advances in Applied Microbiology, V ( 1 9 6 3 ) , 235-315.

37

Waksman, S. A. and A. T. Henrlci, "The Nomenclature and Clas-sification of'the Actinomycetes," Journal of Bacteriology, XLVI (19^3), 337-341.

Waksman, S. A., A. Schartz, and H. C. Reilly, "Metabolism and the Chemical Nature of Streptomyces griseus," Jour-nal of Bacteriology, LI (1946), 753-759.

Wang, C. H., J. J. Bialy, S. Klungsoyr, and C. M. Gilmour, "Studies on the Biochemistry of Streptomyces. III. Glucose Catabolism in Streptomyces griseus," Journal °L Bacteriology, LXXV Il957), 31-37-

Public Documents

Adams, B. A., "The Role of Actinomycetes in Producing Earthy Tastes and Smells in Potable Water," Paper 14, Depart-ment of Public Works, Roads, and Transport Congress, London, 1933.

Rushton, W., "The Purity of Drinking Water from a Biological Aspect," Paper 12, Report of Public Works, Roads, and Transport Congress, London, 1929.

Unpublished Materials

Davis, Ernst, "Assimilation of Inorganic Nitrogen by Actino-mycetes," unpublished Masterfs thesis, Department of Biology, North Texas State University, Denton, Texas, 1962.

Francisco, Donald E., "The Effect of Carbon Monoxide on the Growth of an Aquatic Streptomycete," unpublished Mas-ter's thesis, Department of Biology, North Texas State University, Denton, Texas, 1966.

Higgins, Michael Lee, "The Life Cycle of an Aquatic Actino-mycete," unpublished Master's thesis, Department of Biology, North Texas State University, Denton, Texas, 1964.

Shao, Yi-min, "Assimilation of Organic Carbon by Aquatic Actinomycetes," unpublished Master's thesis, Department of Biology, North Texas State University, Denton, Texas, 1963.

Taylor, Gerald R., unpublished data, Department of Biology, North Texas State University, Denton, Texas, 1967.