HiHIHHIHIWMl HHIL I \ 1 HM“ 1W 1““ 'm-hN \‘ A STUDY ON THE EEOLOGICAL ACTIVITY 61F TWQ fifiERMC'QHELiC ACTlNC'MYCETES [WEI SPECLAL REFERENCE TQ EQROTEOLYTK ACTWETY Time-is fee“ (fie. MM 69 Pin. DE. [Mm-{EGAN STATE UNWEREI‘E‘Y Lawrence Lee Read E9545 This is to certify that the thesis entitled A Study on the Biological Activity of two Thermophilic ' Actinomycetes with special reference to Proteolytic presented by Lawrence Lee Reed has been accepted towards fulfillment of the requirements for Doctor of Philosoggz degree in Microbiology and Public Health Major professor Date May 18L1956 0-169 A STUDY ON THE BIOLOGICAL ACTIVITY OF TWO THERMOPHILIC ACTINOMYCETES WITH SPECIAL REFERENCE TO PROTEOLYTIC ACTIVITY By LAWRENCE LEE REED A THESIS Submitted to the School for Advanced Graduate Studies of Michigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of MicrobiOIOgy and Public Health 1956 Approved by: ‘”“(“(1\’W"Y”}(3315Lpryququc\ f/J-‘S/a"? j'zsbék AN ABSTRACT Two thermophilic actinomycetes were isolated at 45 to 50 C from an active vegetative compost. One Species produces spores in chain-like fashion at the terminal ends of hyphae and is classified as a thermophilic §tg;p- tonges species. The other species produces single spores at the tips of extremely short sporophores that are side branches of the hyphae. This type of spore formation places this species either in the genus Micromonogpora (Bergey) or in the proposed genus Thermoactinomyceg (Waksman and Corks). The latter genus is more valid for the thermophilic forms of this type of actinomycete. Both of the organisms are gram positive and non~acid fast. Temperature studies showed that neither of the species could develops growth at temperatures below 35 C or above 59 C and the optimum temperature is 50 to 52 C. The organisms develope Optimum growth be- tween pH 7.0 to 8.5 and very little, if any, growth occurs below pH 6.0 or above pH 9.0. Growth characteristics were observed on 21 dif- ferent media. These thermophilic actinomycetes are aerobic since they grow only at the surface of sta- tionary liquid and stab cultures. 0n media support- ing good growth, the Therm0§ctinomyceg sp. produces a very light cream colored vegetative mycelium and a white to light grey aerial mycelium that appears as. dry and fragile. The Streptomygeg sp. also has a cream colored vegetative mycelium with white aerial mycelium which becomes grey with the production of spores. No pigmentation is produced on any of the media by either of the organisms. Both of the species liquefy gelatin and cause proteolysis of milk after the formation of a curd. Cellulolytic activity is negative. When inoculated into solutions of maltose, manni- tol, dextrin, sorbose, cellobiose, fructose, rhamnose, lactose, sucrose, and glucose, the Streptomzceg species ferments all but rhamnose, sorbose, and lactose whereas the Ihgrmoggtigomyceg species utilizes only glucose, fructose, and sucrose. 0f the following organic acids; malic, fumaric, succinic, pyruvic, citric, lactic, ox- alic, and tartaric, only pyruvic acid is utilized and then only slowly by Streptomyces sp. Proteolytic activity was studied using casein, gel- atin, and phytone in a basal salts solution. Each organ- ism was inoculated into each type of protein medium con- tained in a series of flasks and growth was developed in shake culture at 50 C. Samples were removed at various times during a 72 hour incubation period and from each sample, mycelial weights and ammonia were determined. By means of paper chromatography, free amino acids were determined in growth samples of casein and gelatin. My- celial weights, when plotted against incubation time, produce curves very similar to bacterial growth curves but the area of the curve that corresponds to deceler- ated growth in bacteria is actually an autolysis in the actinomycetes. Ammonia was produced from the proteins in amounts considerably in excess of the physiological requirements of the organisms. Graphical interpreta- tion of the data produced sigmoid-type curves which show ammonia production continues even during the peri- od of autolysis. The ammonia is apparently released by deamination during proteolysis. A total of seventeen amino acids and two amines were identified in the culture filtrates. The number of amino acids occurred in groups of ten or less for each incubation increment of the total 72 hours. Larg- er numbers of amino acids were found in cultured casein medium than in the cultured gelatin medium. The iden- tified compounds were: cystine, cysteine, lysine, as- paragine, histidine, arginine, glutamine, glutamic acid, aspartic acid, serine, hydroxyproline, threonine, ala- nine, proline, tryptophane, methionine, valine, phené ylalanine, and isoleucine. The number of amino acids found, the amount of growth produced, and the amount of ammonia produced from proteins indicate that these thermophilic actinomycetes are strongly proteolytic and possess a very active deaminase system. ‘l «a. REFERENCES Breed, R.S., Murray, E.G.D., and Hitchens, A. Parker 1948 Bergey's manual of determinative bacteri- ology. Sixth Edition The William and Wilkins 00., Baltimore, Md. Waksman, S.A., and Corks, C.T. 1953 Thermogctino- myceg Tsiklinsky, a genus of thermophilic ac- tinomycetes. J. Bact. pg, 377. BIOGRAPHY Lawrence L. Reed was born November 4, 1921, in Grand Rapids, Michigan. After completion of high school, he was a member of the United States Navy for nearly four years during World War II. He enrolled in college in 1946 and in 1950 received a Bachelor of Arts degree from Wayne University, Detroit, Michigan. In 1951, he was granted a Master of Science degree from Michigan State College and since that time he has been engaged in studies lead- ing to the degree of Doctor of Philosophy. During the period of advanced studies he has been employed as a research assistant. ACKNOWLEDGMENT The author expresses his sincere appreciation to Professor W.L. Mallmann for counsel and guidance dur- ing the years of graduate study which has culminated in the successful completion of this thesis. TABLE OF CONTENTS Introduction Historical Materials and Methods Isolation and Maintenance of Cultures Taxonomic Studies Utilization of Carbohydrates and Organic Acids Proteolytic Activity Experimental Results Taxonomy Mycelial Weights and pH Ammonia Determination Paper ChromatOgraphy Discussion Summary Appendix Formulae of Media References 55 58 Table Table Table Table Table Table Table Table Table Table Table Table Table Table 9. 10. 11. 12. 13. 140 LIST OF TABLES Growth temperature range of Thermoacting- myces sp. and gtrgptomycgg sp. Growth of Thermoactinomyces sp. in carbo- hydrate and organic acid media. Growth of Streptomyces sp. in carbohy- drate and organic acid media. Mycelial weights of Ibermoactinomzceg sp. grown in protein media. Mycelial weights of Streptomyces sp. grown in protein media. Changes of pH in protein media during growth of Thermogctinomgceg sp. Changes of pH in protein media during growth of §treptomxce§ sp. Page 17 20 24 25 27 29 30 Ammonia released by growth of Thermoactino- myces sp. in protein media. 32 Ammonia released by growth of Strgptomyceg- sp. in protein media. Rf values of known amino acids on paper chromatograms. Amino acids and amines identified in case- in medium after growth of Thermoacting- arses sp. Amino acids and amines identified in gel- atin medium after growth of Thermggctino- myceg SP. Amino acids and amines identified in case- in medium after growth of Streptomyceg sp. Amino acids and amines identified in gel- atin medium after growth of Streptomyces Sp. 34 36 37 38 39 40 Figure Figure Figure Figure 2. 3. 4. LIST OF FIGURES Growth of Thermoactinomyces sp. in protein media. Mycelial weights vs. incubation time. Growth of Strentomyces sp. in protein media. Mycelial weights vs. incubation time. Ammonia produced by Thermoactinomyces sp. in protein media. Ammonia vs. incubation time. Ammonia produced by Streptomvces sp. in protein media. Ammonia vs. incubation time. Page 26 28 33 35 In moderate climates, the majority of forms of life have Optimum growth temperatures between 15 and 37 C but there are some forms which are capable of development at temperatures below and above these limits. Those forms which have optimum growth temperatures above 45 C are usually termed thermOphilic organisms. Among these lat- ter forms of organisms is a group of microorganisms which have received little consideration. These are the thermo- philic actinomycetes. Most of the available information concerning this group was published between 1888 and 1912 and is mainly concerned with the occurrence of these or- ganisms in natural substrates and with their possible action in self—heating plant residues. This study was undertaken with the hope that more knowledge might be ac- quired in regard to the biological activity of the thermo- philic actinomycetes. The first reported isolation of thermophilic actino- mycetes was made by Globig in 1888. He isolated these or- ganisms from soil by developing the culture on potato slic- es held at 52 to 65 0. He extended his investigations to the examination of animal feces, canal water, tap water, and dust of the floors. Early reports about these organ- isms described them as thread-like "bacteria". Thus, it is probable that investigators before this time had ob- served these microorganisms and considered them to be bac- teria. Globig's publication created enough interest to promote further investigations. Rabinowitsch (1895) found thermophilic actinomycetes in the feces of the horse, cow, dog, guinea pig, mouse, and fish. His method of isolation consisted of suspend- ing the fecal material in water for 18 to 24 hours at 62 C and then inoculating agar plates with the suspension. The agar plates, incubated at elevated temperatures, showed colonies in 16 to 24 hours. Kedzier (1896) carried out a more detailed study of a thermophilic actinomycete which he had isolated from sew- age. He described the growth characteristics of this or- ganism on various types of media. Temperature studies in- dicated that the actinomycete could develops over a temper- ature range from 35 to 65 C and its Optimum growth temper- ature was 55 C. He also made studies on the resistance Of the spores to heat, desiccation, and 5% phenol solution. Tsiklinsky (1899,1903) isolated thermophilic actino- mycetes from composts, soils, and feces. He inoculated potato with the material being examined and incubated it at 53 to 55 C. Isolations were made on agar plates at 55 to 57 0. Two actinomycetes, differing in type of spore formation, were isolated. The one organism produced chains of spores at the ends of hyphae and thus was classified as a true Agtipomzcgg. The morphology Of this thermOphilic form agreed with descriptions given by other investigators. The other actinomycete produced single round or ovoid spores at the end of short side branches. This organism had an optimum growth temperature at 57 C but could also develope in the range of 48 to 68 C. It exhibited prominent pro- teolytic behavior but not amylolytic. The name Thgrm - actinomyggtgs 1313331: was given to this organism because it was believed to be widely distributed in nature. Several strains Of a thermophilic actinomycete were isolated by Gilbert (1904) from various soil types. He designated these strains as Actinomycgs thermgphilus. These strains, when grown on potato, produced a folded white growth which later became grey on the surface. The optimum temperature was 55 C with the growth Of most strains ceasing at 45 C but some strains could be adapt- ed tO grow at 37 C and even 22 C. The liquefaction of gelatin was slow. Miehe (1907) isolated thermophilic actinomycetes from self-heated hay and considered these organisms to be characteristic of decomposing plant masses since the spores would survive on hay particles but rapidly lost their viability on other media, especially agar media. One of his cultures produced single spores on side bran- ches and this organism was designated Actigongeg thegmg- philug (Berestnev). It grew best between 40 and 50 C and not at all at 25 or 60 C. He apparently had another type present also since he reported that one form produced spores in chains. Representatives of the two spore-bearing types of thermOphilic actinomycetes were isolated by Schutze (1908) from decomposing clover hay. One of these was designated as Actipogzges thgrmophilug (Berestnev) and the other as ‘gtipgmyggg mogogpogga (Lehman and Schutze). The latter organism produced single spores at the end Of small side branches. Noack (1912) also isolated a number Of thermo- philig strains of actinomycetes from moist hay held at 45 to 46 C. Bernstein and Morton (1934) in studies Of pasteur- ized cheeses repeatedly isolated a thermophilic actino- mycete which could withstand temperatures Of 140 to 160 F (60 to 71 C). The Optimum growth temperature for this organism was found to be 56 C. They suggested that this species be designated as Agtinomycgg ggggi since its characteristics were apparently different from species. previously described. Waksman gt _1. (1939), reporting on studies Of soils and composts, found that thermOphilic actinomycetes were present in all seasons in all types of soils examined and were especially numerous where soils had received stable manure. Even in frozen soils these organisms were found to average 10,000 to 15,000 per gram. In moist composts held at 50 C for ten days, counts Of thermophilic actino- mycetes on egg albumen agar were found to be as high as 12,000,000,000 per gram of compost. The greatest number of actinomycetes in horse manure compost occurred at tem- peratures between 50 and 65 C and also the greatest amount of decomposition was observed in this temperature range. Katznelson (1940) found that a thermophilic actino- 4 mycete which he had isolated from horse manure autolyzed on a starch-ammonium sulfate agar after a certain incu- bation time at 50 C. Investigating this phenomenon, he found that the autolytic process was initiated when the pH of the medium reached 6.0 to 6.5 and the agent of 1y- sis was non-transmissible. Erikson (1952) isolated an actinomycete from com- posts of lawn cuttings. He designated this organism as Migrgmggggppgg Iglggrig (Tsiklinsky). The Optimum tem- perature range for growth was 45 to 60 C. Spores Of this culture germinated more readily if they were given a five minute preliminary heat treatment at 75 to 90 C. The spores resisted 100 C for 45 minutes when suspended in a 1% sucrose solution and retained their viability after storage at 2 C for six months. Theseobservations indi- cate the probable role of spores in surviving unfavorable growth conditions. The classification of the thermophilic actinomycetes has long been in a state of confusion primarily due tO the incomplete descriptions given in the early reports on stud- ies Of these microorganisms. From the time of early stud- ies on these organisms, it has been recognized that two distinct types exist. One type produces true aerial my- celium, and spores are formed in either straight or spi- ral chains with some strains producing whorls of spore- phores. Members of this type definitely belong to the -genus recognized as Streptomycea. The other type pro- 5 duces single spores on short sporophores arising from the mycelium. Tsiklinsky (1899) first described a member of this form and proposed the genus Thermoactinomyceg for organisms with this characteristic. Orskov (1923)lintro- duced the generic name Micromogospora for single-spore- producing forms and because of a more careful descrip- tion of this group and insufficient differentiation of Ihgzmgactigomygeg, the tendency has been to include the thermOphilic forms in this genus (Waksman £3 51. 1939). The organism described by Miehs (1907) and desig- nated as A. thermoghilug belongs to the Thermoactinomyces- Migromonogpora group but the organism described by Krassil- nikov (1941)2 and Mishustin (1950)3 as A. thermOphilus appears to be in the Streptomxceg group since they des- cribe a spiral producing form. Waksman and Corks (1953), after comparative studies of Micgomogogpoga and Thegmogctinomyceg, proposed that Thermoactinomyceg (Tsiklinsky) be established as a genus of thermophilic actinomycetes. The differences between the two genera being mainly that members of Micromonosngrg do not produce true aerial mycelium and grow readily at 35 to 37 C whereas members of Thermoactinomvces do pro- duce true aerial mycelium and are only thermophilic forms. They isolated a form which produces a rose colored pig- ment on certain media and suggested the name Thermoactino- myceg thalpOphilus for it. 1,2,3 as cited in Waksman & Corks (1953), Jour. Bact.éé Schuurmans (1954) cultured a green pigmented thermo- philic actinomycete which has an Optimum temperature for growth at 55 C and which produces a bactericidal agent. He suggested that this organism be designated as Thermo- actinomygeg viridia and the antibiotic substance has been named Thermoviridin. In the study to be presented, two thermophilic actino- mycetes have been used, one being a representative of the Streptomygeg group and the other from the Thermogctino- myggg- igrgmonogpora group. MATERIALS AND METHODS Isolation and Maintenance of Cultures. The organisms used in this study were isolated from a garbage compost- ing project at Michigan State University, East Lansing, Michigan. Isolation was accomplished by plating appro- priate dilutions of water suspensions of garbage compost on pryptoneoglucose-extract agar (Difco) and incubating the plates at 45 to 50 C for 36 to 48 hours. After suf- ficient incubation time, typical actinomycete colonies were picked and seeded into nutrient broth tubes. These tubes were vigorously shaken and placed at 45 to 50 C for 12 hours prior to diluting and replating. The procedure for repleting was the same as that used for the primary isolation. After development of mycelium on the plates, colonies were picked and inoculated onto slants of tryp- tone-glucose-extract agar. .Stock cultures Of the isolated thermophilic actino- mycetes were maintained on slants of the isolation medium. This medium was chosen because it readily produced a lux- uriant growth. A number of slants were prepared from the isolated actinomycetes and after spore formation occurred, these slants were placed in a refrigerator at a temper- ature Of 2 tO 4 C and no transfers were made except to de- termine viability and comparison of these stored cultures with repeatedly transferred cultures. Other slants were prepared and monthly transfers were made; these cultures were the working stock for the studies described in the following pages. Taxonomic Studies. Before developing studies upon these cultures, it was necessary to determine the Optimum temperature for their growth. This was accomplished by inoculating tryptons-glucose-extract agar slants and tryp- tone-glucose-extract broth with the actinomycetes and in- cubating them at the following temperatures: 25, 30, 35, 37, 40, 42, 45, 47, 5o, 52, 55, 57, and 60 c. The cri-5 terion used for optimum growth temperature was the tem- perature at which first visible growth occurred. Incu- bation was carried out in a hot air incubator containing a reservoir Of water to give a high humidity and thus prevent desiccation. The hydrogen ion concentration for optimum growth of these actinomycetes was determined by Observing their de- velopment in a medium beffered at various levels of pH. Nutrient broth was buffered over a pH range of 5.5 to 8.5 at two-tenths intervals using mixtures of sodium monohydrogen phosphate and potassium dihydrogen phos- phate. For pH values above 8.5, the pH of the medium was adjusted with sodium hydroxide solutions. Repli- cats tubes at the various pH levels were inoculated with the actinomycetes and incubated at 50 C. The cri- terion used for Optimum pH level was the pH at which first visible growth appeared. The presence or absence Of growth and the character- istics Of growth on various types of media have been es- tablished as part of the system of classification of ac- tinomycetes. Following this system, the following media were used for growth observations: corn meal agar, corn steep agar, Czapek’s agar, Emerson's agar, glucose-as- paragine agar, glucose-peptone A agar and broth, glucose- peptone B agar and broth, nutrient agar and broth, nut- rient glycerol agar, potato slants, Sabouraud's agar, starch agar, tryptone-glucose-extract agar, and yeast- glucoseagar. In the case of solid media, plates were prepared and replicate plates of each medium were streak- ed with the cultures of the thermOphilic actinomycetes. These plates were incubated at 50 C for varying lengths of time depending upon the organisms ability to develope growth on a particular medium. In the case where good growth occurred, the plates were incubated until spore formation develOped which usually occurred in 48 to 72 hours. Where little or no growth occurred, the plates incubated up to 7 days. Broth cultures were also incuba- ted at 50 C and for varying periods of time using the same criteria as those used for solid media. Formulas for the above named media are given in the appendix. The following biochemical activities were observed: proteolysis, gelatin liquefaction, nitrate reduction, starch hydrolysis, and cellulolytic activity. Litmus milk was used for the observation of proteolysis. Two methods were used to determine liquefaction Of gelatin because of the high temperature of incubation. The or- dinary tube method was used with 4% gelatin and after varying times of incubation at 50 C the tubes were re- frigerated to determine liquefaction. The other method was the plate method of Smith (1946) in which 0.4% gel- atin is incorporated in nutrient agar. After sufficient incubation at the desired temperature, liquefaction is de- termined by flooding the plate with a mercuric chloride— hydrochloric acid solution and observing whether or not clear zones appear around areas of growth. Unchanged gelatin reacts with the reagent to produce opaque areas whereas areas of degradated gelatin remain clear. Nitrate reduction tests were performed following the usual qualitative method for identification of microorgan- isms. Nutrient broth containing 0.1% potassium nitrate was inoculated and tested for the presence of nitrite us- ing sulphanilic acid and alpha—naphthylamine reagents. Tests were made at regular intervals during the total 10 incubation period to insure the detection of the nitrite ion before it may have been further reduced to the ammo- nium ion. The ability of these organisms to hydrolize starch was tested by growing the actinomycetes on a starch agar and then flooding the plate with iodine solution at vary- ing times during the growth of the culture. The presence of a clear zone about an area of growth is indicative of starch hydrolysis. Cellulolytic activity of these organisms was qual- itatively examined by inoculating strips of filter paper immersed in Dubos cellulose medium and in a basal salts solution, and also by inoculating a suspension of cell- ulose in this basal solution. The basal medium has the following composition: ammonium monohydrogen phosphate, 1.0g; potassium chloride, 0.2g; magnesium sulfate (hepta- hydrate), 0.2g; and water, lOOOml.‘ All the above biochemical tests were carried out using replicate tubes or plates. Incubation in all cases was at 50 C for varying periods of time but in no case did the total time exceed 8 days. For microscopic examination of the cultures two staining procedures were used: (1) the Burke modi- fication of Gram's stain, and (2) the Ziehl-Neelson method of acid fast staining. Growth of mycelium and spores for this examination were obtained by using the agar block method of Riddell (1950). ll Utilization of Carbohydrates and Organic Acids. Using the basal medium previously mentioned in cellulolytic ac- tivity, 1% solutions were prepared using the following car- bohydrates: lactose, sucrose, glucose, maltose, mannitol, dextrin, cellobiose, rhamnose, sorbose, and fructose. 0r- ganic acid solutions of 0.5% concentration were also pre- pared with this basal medium. The acids used were malic, fumaric, citric, succinic, pyruvic, lactic, oxalic, and tartaric. Replicate tubes of each of these resulting so- lutions were inoculated with the actinomycete cultures and incubated at 50 C for periods up to 96 hours. Abil- ity or inability to grow in these media was Observed. Proteolytic Activity. Preliminary experiments show- ed that the thermophilic actinomycetes used in this study were capable of utilizing the following proteins for de- velopment of growth: keratin, sein, gluten, gelatin, case- in, and phytonel. Of this group keratin, zein, and gluten are either insoluble or soluble only after treatment with acid or bass and because of this undesirable property only gelatin, casein, and phytone were used for further studies. A medium was prepared of each of these using 1.5g of the protein material per liter of the salts of Czapek's solu- tion. Each medium was dispensed into 125ml Erlenmeyer flasks in the volume of 50ml per flask and autoclaved. Twelve flasks of each medium were inoculated with the 1 A peptone preparation of Baltimore Biological Laboratory, Inc. 12 desired culture, placed on a Burrell wrist-action shaker, and incubated at 50 C. A flask was removed for analysis according to the following schedule of hours of incuba- tion: 0, 3, 6, 9, 12, 15, 18, 24, 30, 36, 48, and 72. Replicate series were run for each organism. The inoculum of each culture was prepared by inoc- ulating spores of the culture into 50ml of the medium to be used for study. The culture was develOped on the Burr- ell shaker at 50 C for 12 to 18 hours at which time it was blended in a sterile Waring Blender and the Erlenmeyer flasks were inoculated with lml per flask of the result- ing mycelial suspension. The amount of growth occurring during the incubation period was determined by weighing the mycelium present in each sample removed at the specified times. The sample was filtered through tared filter paper which had been dried in a 105 C oven for 12 to 14 hours. After filter- ing, the mycelium and filter paper were again dried at 105 C for 12 to 14 hours and weighed. The difference in weight between the filter paper alone and the filter pa- per plus the mycelium gave the dry mycelial weight per sample. The filtrate from each sample was divided into two equal portions. One portion was immediately frozen to be used for amino acid studies by paper chromatography. The other portion was used for ammonia determinations. The amount of ammonia in the filtrate for each cul- l3 ture sample removed at the designated incubation periods was determined by the modified aeration method of Van Slyke and Cullen. A 25ml filtrate sample treated with a saturated solution of potassium carbonate was-aerated for 3 hours with air washed by dilute (1:10) sulfuric acid. The freed ammonia was trapped in a 2% boric acid receiver which was then titrated with standard sulfuric acid (0.02 N). Ammonia free water was used for prepar- ing all solutions and for rinsing glassware. One dimensional paper chromatOgrams were made on the culture filtrates to determine the presence of free amino acids. Whatman No. 1 filter paper for chromato- graphy (18%" x 22%") was marked Off in twelve divisions so that the various samples from 0 to 72 hours could be spotted on the same sheet of paper. Fifty microliters of each culture filtrate was applied at each spot. Chromatograms were developed by the ascending method at 25-1 0 in an insulated Chromatocabl using a solvent sys- tem of n-butanOl-acetic acid-water (4:1:1). After devel- opment of the chromatOgrams, the papers were dried at 100 C in an air circulating oven. Color develOpment of the amino acid spots was Obtained by spraying the dried chromatograms with a ninhydrin solution composed of 1g ninhydrin and 500ml of n-butanol. The ninhydrin sprayed papers were dried at 100 C and placed in the dark for 18 to 24 hours to allow full color development. Known amino 1 Product of Research Equipment Corporation, Oakland, California. 14 acids and amines were chromatographed at the same time and in the same manner as the unknowns. EXPERIMENTAL RESULTS Several actinomycete cultures were isolated at 50 C from the composting waste material over a period of two weeks. The cultures could be separated into two groups by gross examination of their growth on tryp- tone—glucose-extract agar. One group produced a light colored aerial mycelium which gave the appearance of fragility whereas the other group produced a grey aer- ial mycelium which was compact and appeared more hardy. One specimen from each group was selected for further study. The cultures selected were those which produced the most rapid and abundant growth on the isolation med- ium. Taxonomic Studies. Microscopic examination reveal- ed these thermophilic actinomycetes to be of two differ- ent genera, ThermOQOtipomycea (Waksman and Corks, 1953) and Streptomyceg (Bergey). Thegmoggtipomyggg sp. Microscopic examination of this thermophilic actinomycete showed that conidia were produced singly on extremely short conidiOphores branch- ing from the hyphae. The conidiOphores are so short that Iin the majority of Observations the conidium appears to be resting directly upon the hypha. The spores are round but occasionally a spore appears to be slightly elongate. The diameter of the spore is between 0.8 and 1.0 micron 15 which is slightly larger than the average hypha diameter of 0.5 to 0.8 micron. Stained preparations of this organism showed it to be non-acid fast and gram positive in reaction. However, in- stances were noted when, in stained preparations of older cultures, the mycelium was gram negative but the spores were always gram positive whether in young or old cul- tures. The temperature range for growth (Table l) Of this organism is 37 to 59 C with the optimum temperature be- ing 50 to 52 C. NO growth is obtained at temperatures below 37 C at which temperature only slight growth oc- curs after 48 to 72 hours. NO growth is observed at 60 C. A vigorous culture produces visible vegetative grow- th in 7 to 8 hours when incubated at its Optimum temper- ature. The hydrogen ion concentration range for good grow- th of this Ibermoactinomyces g2. is slightly alkaline and it appears that equal amounts of growth can be ob- tained over the range of pH 7.0 to 8.5. Mycelial de- velOpment occurs above and below this range but in di- minishing quantities as the medium becomes more alka- line or more acid. In the acid range, little growth appears below pH 5.8, and in the alkaline range grow- th ceases at pH 9.0. The cultural and biochemical characteristics of this actinomycete are given below. In general, the 16 Table 1 Growth Temperatures Temp, C, 303:3 incubated until yisible grogth, Streptomzceg Thermoactinomyceg 25 No growth No growth 35 24 e No growth 37 18 48 40 10 24 42 9 20 45 8 12 47 7 9 5O 6 8 52 6 7 55 9 8 57 12 11 59 24 20 60 No growth No growth 17 organism is a strict aerobe producing mycelium only at the surface of broth or stab cultures. 0n media where growth occurs, the vegetative mycelium is a very light cream color and the aerial mycelium varies from white to very light grey and has a characteristic dry appear- ance. No pigment either of the mycelium or the water- soluble type is produced on any of the media used in this study. The cultural and biochemical character- istics are: TGE Large, slightly raised colony of dry white aerial mycelium and light cream colored vege- tative mycelium. Center of colony wrinkled. Nutrient Agar Growth very similar to that on TGE agar with the exception that there is less sporogen- ous development. Emerson's Agar Flat, rough, dry colony with striations from center to edge. Vegetative mycelium of light cream. Aerial mycelium has the appearance of white and light grey concentric rings. Nutrient-glycerol Agar Flat, rough, dry colony of light cream vegetative mycelium and white aerial mycelium. Colony not as large as on the above media. Yeast-glucose Agar Colony similar to that on TGE agar only more compact and wrinkled. Glucose-asparagine Agar No growth. Glucose-peptone A Agar Very small colony formation of light cream vegetative mycelium and no aerial mycelium. Glucose-peptone B Agar Very small colonies of vegetative mycelium only. 18 Czapek's Agar Sabouraud's Agar Corn Meal Agar Corn Steep Agar Starch Agar Nutrient Broth Glucose-peptone A Broth Glucose-peptone B Broth Potato Slant Litmus Milk Gelatin Nitrate Cellulose NO growth. NO growth. No growth. No growth. Small white colonies with slight zone of hydrolysis. White, surface pellicle-type grow- th. Some floccules settle when disturbed. Very slight white growth at sur- faces Very slight growth at surface. NO growth. Coagulation followed by nearly complete digestion in 3 days. Alkaline reaction. Liquefaction positive but slow. Reduction to nitrite negative. No growth. The ability of this organism to use various sugars and organic acids as energy sources is summarized in Table 2. We as. Microscopic examination of this thermophilic actinomycete reveals that the spores are borne in the typical manner of members of the genus Strep- tomyceg, The spores are produced in chain—like formation at the terminal ends of hyphal elements. round and have a diameter of 0.5 to 0.7 micron. These spores are The ter- minal ends of those hyphae which produce spores become slightly larger in diameter than the hyphal element which 19 a 1‘ of. )s o . ,. , . t I . . . . . o 4 . . . . _ A o .. I _ \ . . O 7 .L t. 4\, r c . o J v.. r . c .w. e O a _ a .s O .n x 4 fix I I o. . . J r I ( . . .l. C Illlllil I11 ill. Table 2 Growth of ThermOggtingmyggg sp. in various sugar and organic acid liquid media. fingggg Gro t h r t r Amt, Growth Eigal 2H1 Maltose No growth - 7.0 Mannitol No growth - 7.0 Dextrin NO growth - 7.0 Sorbose No growth - 7.0 Cellobiose No growth - 7.0 Fructose White surface clump f 6.8 Rhamnose No growth - 7.0 Lactose NO growth - 7.0 Sucrose White surface clump + 6.9 Glucose White surface clump + 6.4 A2191 Malic NO growth - 7.4 Fumaric No growth - 7.4 Succinic NO growth - 7.3 Pyruvic No growth - 7.4 Citric No growth - 7.5 Lactic No growth - 7.3 Oxalic NO growth - 7.3 Tartaric No growth - 7.4 1. Original pH of sugar media was 7.0. 1. Original pH of organic acid media varied from 7.3 to 7.5. a. 20 has an average diameter of 0.4 to 0.6 micron. Some of the hyphae appear to form whorls at their terminal ends. This characteristic is also Observed in certain species of the mesoPhilic members of this genus. Staining procedures show this species to be gram positive and non-acid fast. Growth temperature studies (Table l) disclosed that this species can develOpe more growth over a wider temper- ature range than can the Thermogctigomyceg species. No growth is developed at 25 C but slow growth occurs at 35 C. The optimum temperature like that of the ThgrmOggtigg- nyggg species is 50 to 52 C and no growth developed at 60 C. This fitreptomxggg grows best in a slightly alkaline medium. Development of growth appears equally well in a range of pH 7.0 to 8.5. On the alkaline end of this range decreasing amounts of growth occur up to pH 9.2 and cease beyond this point. In the range below neutrality, develOp- ment occurs in decreasing quantities to pH 5.8 below which no growth developss. Growth characteristics of this organism on various media are as follows: TGE Agar Large raised colony with light cream vegetative mycelium. Aerial mycelium is white chang- ing to grey with production of spores. Abundant growth. Nutrient Agar Growth good with large raised colony having light cream color- ed vegetative mycelium and white aerial mycelium turning to grey. 21 Emerson's Agar Nutrient-glycerol Agar Yeast-glucose Agar Glucose-asparagine Agar Glucose-peptone A Agar Glucose-peptone B Agar Czapek's Agar Sabouraud's Agar Corn Meal Agar Corn Steep Agar Starch Agar Nutrient Broth Glucose-peptone A Broth Glucose-peptone B Broth Potato Slant Litmus Milk Gelatin Growth good with slightly raised colony. Colorations similar to above media. Abundant growth with raised colony which is considerably wrinkled. Colorations similar to above media. Abundant growth with raised, wrin- kled colony with extensive sporu- lation giving a dark grey appear- ance. Poor growth, consisting mostly of vegetative mycelium. Aerial my- celium very sparse with little sporulation. Small white colony with no sporu- lation occurring. Small colony formation with sparse aerial mycelium but some sporula- tion occurring. Slight vegetative growth only. No growth. Slight growth of light cream vege- tative mycelium and some white aerial mycelium. No growth. No growth. Abundant surface growth with pel- licle formation. Abundant sporu- lation. White surface growth but not abun- dante Slight amount of white surface grow- th. No growth. Soft curd formation with slow di- gestion. Alkaline reaction. Positive liquefaction. 22 - j“, e I 0 e a a . e 4 . O I e C a b e , . Nitrate Positive nitrite reaction. Cellulose No growth. On media which produce good mycelial development of this organism, the general appearance of the growth con- sists of a light cream colored vegetative mycelium which gives rise to a white aerial mycelium. The appearance of the colony is dry and wrinkled and becomes decidedly grey with the advent of sporulation. Development of growth of this organism in various sugar and organic acid solutions is given in table 3. Mycelial Weights and pH. Growth of the two thermo— philic actinomycetes in the protein solutions of phytone, gelatin, and casein was followed by determining the amount of dry mycelial weight for each sample removed at the vari- ous periods of incubation. Results are recorded as milli- grams Of dry mycelium per 50ml of medium and are given in tables 4 and 5 for both organisms. These mycelial weights have also been plotted against incubation time to produce the curves found in figures 1 and 2. Changes in pH value during growth of each organism in the protein media were followed by determining the pH of each sample removed at the specified times of incubation. The pH values for respective samples of replicate series of each medium were found to be very similar and the aver- age of these values for each medium have been recorded in tables 6 and 7 for each organism. Ammonia Determination. The date collected from the 23 Table 3 Growth of Streptomyces sp. in various sugar and organic acid liquid media. Suggrg Growth thrgcter Amt, Grogth Final 2H1 Maltose White pellicle ++ 6.5 Mannitol White pellicle 4 6.1 Dextrin White pellicle + 6.3 Sorbose No growth - 7.0 Cellobiose White pellicle ++ 6.5 Fructose Grey pellicle +++ 6.2 Rhamnose No growth V - 7.0 Lactose No growth - 7.0 Sucrose White pellicle ++ 6.8 Glucose Grey pellicle . 4+++ 6.1 £21.92 Malic No growth - 7.4 Fumaric NO growth - 7.4 Succinic No growth - 7.3 Pyruvic Light grey pellicle -- 8.6 Citric No growth - 7.5 Lactic No growth - 7.3 Oxalic NO growth - 7.3 Tartaric No growth - 7.4 1. Original pH of sugar media was 7.0. 1. Original pH of organic acid media varied from 7.3 to 7.5. 24 «.H 0.H N.N 0.N «.m N.m «.0 «.0 m.0 H.0 «.0H 0.0a m.HH 0.HH 0.0a b.0H 0.0 0.0 0.0 0.0 «.0 «.0 madam. 25 H.H 0.H 0.H 0.H 0.H «.N b.N 0.0 «.N Nb N.H n.n 0.0 «.m 0.0 0.n N.m 0.0 0.0 00 H.N v.0 «.0 0.0 5.0 b.« 0.« 0.« b.« 0m 0.N 0.5 b.b 0.0 0.b 0.6 0.5 m.b N.b on 0.0 «.0 0.0 5.0 «.0 0.0a H.HH «.oa 0.0a 0m 0.« 0.0 0.0 H.0 0.b 0.HH b.HH «.HH «.Hd 0H «.0 v.0 «.0 0.0 0.0 0.0 0.0 5.0 0.0 «H 0.HH 5.0 0.0 0.0 «.0 0.b 0.b ¢.b «.b NH 0.0a 0.0 0.0 0.0 0.0 N.N n.N 0.0 N.N o «.0 0.0 0.0 0.0 «.0 «.H b.H 0.H «.H 0 «.0 «.0 «.0 «.0 0.0 0.0 0.0 «.0 0.0 m N.0 m.0 m.0 «.0 0.0 «.0 0.0 0.0 0.0 0 .obd .o>< e "a. E as a panama .esavos canvass Ho Ha 0«\ma a“ commOAQNo .nm NMMHmmmdeflmmMMMH no magmas: Headache hum . e .Hoea 54 l I l l l ,' m .’ - J'Es—e 5 / a: — 2 I, “3' .4 5;: fu’ I : fr’ ‘9 I ,e g L. . 5?], ‘fi . --:E: I: / I .. z / ,’ m ._. 2g :,4‘ 1V __ 5; 55 o F- g// '1’ z ._ }/ . ’0'. _ g 2 1’ E . .g' ._ 2 a \\ .oo‘.. . . 2 g I'd‘~'- ’ '- —.. ~~O~~ ° ---1 9., O.‘ ~~~~ .00...-..-.. \Q . r— ..-""'eV‘ 0 \- * 1 1 1 1 1 1 \, o S 9 0° ‘0 - V N O )ISV'IJ '83:! Wfll'IBOAW SNVHSIT‘IIW Figure 1. Dry mycelial weights of Thermoactinomyces sp. grown in 50 m1 portions of protein media. 26 m.« «.0 «.0 m.m o.aa ~.oH e.ea H.o~ «.mH e.na m.o «.0 .o>d 0.« «.« 0.0 0.0 0.0 5.0 0.0 H.0H 0.0a <.HH o.«a «.0H H.5H 0.bH b.0H 0.00 ”.0H «.0H «.ma 0.nH 0.0 0.0 0.0 «.0 mmmmwmm m.« «.0 «.0 v.0 0.HH «.0H 0.5a N.0N 0.0a 0.0a 0.0 0.0 ~.o m.m o.o m.oa m.~H o.se 0.HH «.0 0.0 H.N 0.H 0.0 .o>< .ssacos canvass Ho Ha 0«\ma ca 0.0 «.0 0.0 0.0 0.0 0.0 «.0H b.0H 0.NH «.NH 0.«H «.0H 0.HH <.HH 0.0 «.0 «.0 0.« 0.0 0.H H.N 0.H «.0 «.0 mflmmdmw 0.0 «.0 «.0 n.0H H.NH b.0H 0.HH «.0 0.0 0.0 m.H «.0 v.0H «.0H «.ma N.0N «.wm 0.0m 0.bN H.N~ m.b 5.0 0.0 N.0 .o>< 0.0a 0.0a 0.0a 0.HH N.MH 0.0H 0.00 b.0H as as H.bN 0.0m H.0N H.bN N.NN b.d~ H.0 b.b 0.0 0.0 0.0 «.0 0.0 N.0 mammmm vcuooumxo .0. dddflmmmmmmmw no uvnmaos Headache ban a oaoee b.0H 0.HH 0.0H «.00 0.00 0.0m 0.b~ 0.NN 0.5 0.0 0.0 N.0 Nb 0% 0m on «m 0H «H NH addmmqmmmfl easom 27 l l ' I I l I i —- e e. — 3 l E I : L— : — / : / .' n—- .0. —" ‘0 g o '0 :3 / : g ./ i ._ m“ E / 2 - 3. / ._ g i: - 9' - l— l I.’ '5 < - \ o' m N a __ ‘e.§~~ (9 _ z s... - 1 l l o a a 2 ° XSV'IJ '83:] Will-EGAN SWVHOI'I'IIW Figure 2. Dry mycelial weights of Streptomyceg sp. grown in 50 ml portions of protein media. 28 Average pH values occurring during growth ‘Table 6 of Thgrmogctigomxces sp. in liquid protein media. Hours lnggbated gaggip 0 8.4 3 8.3 6 8.3 9 8.1 12 7.8 15 7.7 18 7.6 24 7.8 30 7.6 36 8.0 48 7.3 72 7.6 Gelgtig Phytong 7.5 7.7 7.4 7.65 7.35 7.5 7.2 7.3 7.2 7.2 7.2 7.06 7.45 7.4 7.02 7.6 7.2 7.1 7.2 7.0 7.8 6.9 7.1 6.85 Table 7 Average pH values occurring during growth of Streptomycgg sp. in liquid protein media. rngfififiiee Casein Gelgtin Phytone o 7.5 7.45 7.6 3 7.7 7.5 7.5 o 7.6 7.55 7.2 9 7.2 7.7 7.3 12 7.2 7.6 7.9 15 7.4 7.48 8.0 18 7.6 7.68 8.0 24 8.4 7.85 8.4 30 8.1 7.2 8.9 36 8.7 7.3 8.7 48 8.8 8.4 8.7 72 8.5 7.55 9.0 30 titration of ammonia in each culture sample were used to calculate the amount of ammonia present in each milliliter of culture medium after growth of one of the actinomycetes for a designated time. The amount of ammonia per milli- liter is so small that calculations were made on the basis. of milligrams of ammonia per liter of medium. The results of the replicate determinations are given in table 8 for the Ihermoactinomyceg sp. and in table 9 for the Streptg- £1521 sp. The average values of the determinations of replicate samples for each incubation period have been plotted as milligrams of ammonia per liter against in- cubation time so that the amount of ammonia produced from each protein by growth of the organisms can be fol- lowed graphically. These graphs are represented in fig- ures 3 and 4. Paper Chromatography. ChromatOgrams were made of 18 l-amino acids and 2 amines. The names of these compounds and their corresponding Rf values are given in table 10. ChromatOgrams of the culture filtrate samples from each protein medium of casein and gelatin were made at the same time that the known compounds were being chromato- graphed. The Rf values of unknown spots were determined and comparison of these values with the known Rf values identified the unknown spots. The identified amino acids and amines for each sample chromatographed are given in tables 11, 12, 13, and 14. 31 0.0« 0.0« «.H« «.0« 0.00 0.00 «.00 0.0a «.0 .o>< at; RR. 0.0« 0.0« 0.3 RR 0.0« 0.0« 0.00 0.00 0.00 «.00 «.0m 0.00 0.3 To «.0 «.0 mug asfivoa musvaso 0.? RR 0.3 0.3. HS 93 0.0m es .3 «.«00 0.«0H 0.«0H 0.00H 0.00H 0.00 «.00 «.00 0.00 0.00 «.0 0.« .o>< H.00H «.00H 0.00H 0.00 0.00 «.00 0.00 0.00 0.m0 0.00 0.00 «.00 0.00 «.0 0.0 «.« 0.« 0000000 0.00 0.00 0.00 0.00 0.H0 m.0 0.« 0 capsa 0.00 0.«0 0.00 0.00 0.0« 0.00 0.0a 0.« 0.m .o>4 .nopaa\mmz assumfiaaas ms womanhQNo .0» ummflmdmflflmmmmwmmH Mo essence seasosad 0.00 0.00 0.«0 0.00 0.00 0.00 0.00 0.00 0.0« 0.0« 0.00 0.mm 0.0a H.0H 0.« 0.« «.m «.0 and 0.00 0.00 0.00 0.H0 0.H« 0.00 0.MH 0.« 0.m .0opoeuppsm seen me: maoupcoo copsasoonfims Mo accuses easoasd e 00 00 00 on 00 0H 0H 0000000000 mason 32 1000 -- GELATIN n: m t .l ‘—-“""'-' m‘ 75'- /" '- 33 ~/r CASEIN U) 2 A < a: .. ‘9 ---- . ......... 3 50 PHYTONE '- i 5’ z 0 2 _ 2 25 < l l l 0 l2 24 36 48 60 INCUBATION TIME, HOURS Figure 3. Ammonia produced during growth of Thermoactino- mxceg 8p. in protein media. 33 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.0H 0.0 .o>< KER 0:: v.3 Hi 93 $3 1.3 93 0.3 «.3 QR 3.. SK 1% «33.. «.3 n4 “:0 g 0.00 0.00 0.00 0.00 H.00 H.00 0.00 0.0a 0.0 0.H0 0.00 0.H0 0.00 0.00 0.00 0.00 0.HH 0.0 .obd .0mpounpndm moon was maonvzoo voaaasoonans 0.H0 0.H0 0.00 0.00 0.H0 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.HH 0.HH H.0 H.0 343.00. 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.0a 0.0 H.00 0.00 0.00 0.00 0.00 0.00 0.00 0.0 0.0 .m>< 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 n.00 0.00 0.00 0.00 0.00 0.0 0.0 0.0 H.0 g .hop«H\0mz naunmfiflaaa no umuuounuo finance chapHUO umoHaopmonpw Mo 0:00:00 taaqoasd 0 manna 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.0 0.0 no pampnoo manoaad * N0 00 on on 00 ma 2 0 o g unsom 34 '°° 1 . I l l I CASEIN‘ n: ’“flflfl m /- GELATIN L'. .1 . m. 75"“ I\ . ——1‘ m . a. o co // i ’ PHYTONE m ' .o"---- 0", g 500— / ,..-—" — :a ’ .A’ z .’ < I I, '— I z o 2 25*- -w 2 I < "I C .‘I o l l l l l 0 l2 24 36 48 60 INCUBATION TIME, HOURS Figure 4. Ammonia produced during growth of Streptomyceg sp. in protein media. 35 Table 10 Rf values for known L-amino acids and amines chromatod graphed in n-butanol-acetic acid-water .solvent (48181) at 25-1 C. g; 1313; Comgound 3; Iglgg Qompoung .036 Cyatine .265 Hydroxyproline .074 Cysteine .283 Threonine .09 Lysine .351 Alanine .098 Asparagine .412 Proline .109 Histidine .525 Tryptophane .115 Arginine .592 Methionine .126 Glutamine .627 Valine .139 Glutamic acid .665 Phenylalanine .15 Aspartic acid .747 Leucins .185 Serine .754 Isoleucine 36 Table 11 Amino acids and amines identified in liquid casein medium after growth of ThermOQctinomyceg sp. Hours Ingubated 3 12 15 Compound Cystine Histidine Glutamic acid Serine Alanine Proline Valine Cystine Cysteine Histidine Aspartic acid Serine Alanine Cystine Cysteine Lysine Histidine Glutamine Aspartic acid Hydroxyproline Alanine Proline Valine Lysine Histidine Glutamine Aspartic acid Hydroxyproline Alanine Proline Valine Isoleucine Cysteine Lysine Histidine Glutamic acid Hydroxyproline Alanine TryptOphane Phenylalanine 37 Hours Incubate; 18 24 30 36 48 72 Comgound Cysteine Lysine Histidine Glutamine Hydroxyproline Alanine Tryptophane Methionine *Phenylalanine Isoleucine Cysteine Lysine Glutamine Aspartic acid Methionine Phenylalanine Isoleucine Cysteine Lysine Histidine Aspartic acid Hydroxyproline Isoleucine Cysteine Lysine Aspartic acid Hydroxyproline TryptOphane Cysteine Lysine Histidine Glutamine Alanine Tryptophane Cysteine Lysine Histidine Tryptophane Table 12 Amino acids and amines identified in liquid gelatin medium after growth of Ebermogctinomyceg sp. Hours Incubated 3 12 15 18 Comgougd Cysteine Lysine Glutamine Cysteine Lysine Arginine Cysteine Lysine Arginine Cysteine Arginine Serine Hydroxyproline Cystine Lysine Histidine Glutamine Hydroxyproline Valine Cystine Cysteine Asparagine Glutamine Hydroxyproline 38 Hours Incubated 24 30 36 48 72 W Cysteine Lysine Arginine Glutamine Threonine Cysteine Lysine Aspartic acid Serine Tryptophane Cysteine Lysine Arginine Glutamic acid Serine Cysteine Lysine Arginine Serine Hydroxyproline Threonine Cysteine Lysine Serine Hydroxyproline Threonine Table 13 Amino acids and amines identified in liquid casein medium after growth of Streptomyces sp. Hours Incu 3 12 15 e Compound Cystine Cysteine Lysine Histidine Arginine Glutamic acid Hydroxyproline Threonine Alanine Proline Cystine Lysine Asparagine Arginine Glutamic acid Proline Valine Lysine Asparagine Arginine Glutamic acid Aspartic acid Cystine Asparagine Glutamic acid Aspartic acid Threonine Alanine Cystine Cysteine Asparagine Aspartic acid Threonine Alanine 39 Hours Miss; 18 24 30 36 48 72 Comgound Aspartic acid Threonine Alanine Lysine Histidine Glutamic acid Aspartic acid Threonine Alanine Cysteine Lysine Histidine Glutamic acid Aspartic acid Threonine Alanine Cysteine Asparagine Glutamic acid Threonine Alanine Cysteine Asparagine Glutamine Aspartic acid Threonine Alanine Lysine Glutamine Aspartic acid Serine Threonine Alanine Table 14 Amino acids and amines identified in liquid gelatin medium after growth of Streptomyceg sp. Hours Incubated 3 12 15 18 Compound Lysine Glutamine Lysine Glutamine Threonine Lysine Glutamine Threonine Lysine Glutamine Threonine Lysine Arginine Threonine Lysine Arginine Threonine Hours Incubated 24 30 36 48 72 40 Compound Lysine Arginine Hydroxyproline Threonine Lysine Histidine Lysine Histidine Aspartic acid Asparagine Aspartic acid Lysine Arginine DISCUSSION The classification of the two thermophilic actino- mycetes used in this study has been limited to genue be- cause of the confusion in descriptions given by past in- vestigators and because of the possible variations which have been demonstrated to occur in other cultures of ac- tinomycetes. Although few thermophilic actinomycetes have been recorded in literature, they have been described under seven different genera, two of which are bacteria and one a fungus. The generic names which have been used are: glgdophrig, Streppothrix, Therpom c , Actinomycgg, Strep— pppypgg, Thgrp0gctinomyces, and MicrOQOpogpor . The last three generic names are those commonly used of recent times. The general characteristics of the Strgptomyceg and the Therpogcpinomzces-Mipromonosporg groups have been describ- edTearlier in the paper. The generic name Thgppogcpin - 313;; has been used to designate one of the organisms in this study because it fulfills the requirements set forth for this proposed genus by Waksman and Corks (1953). Since there has been a lack of interest in this group of microorganisms, no pattern for identification proc- edures has been established and if further work is to be done, standards must be established to reduce or elimin- ate the confusion resulting from past descriptions. The results of the investigations on the isolated Ihgrmogctipomyceg species indicate that it most nearly resembles the established species ThermOQctinomyces Eu;- 41 33;;1. The characteristics of the species used in this study do not agree with the recognized description in that growth does occur in sucrose medium, there is no growth on potato, and the temperature range and optimum temperature for growth is lower by several degrees. Al- though these differences make identification doubtful, they are not significantly great to establish a new species especially when the possibility of variation is considered. The Sprgptomyces species used in this study most nearly resembles §tr§ptomyc§§ thermophilgs described by Gilbert (1904). It differs from the recognized species in that there is no growth on potato, starch agar, or Czapek's agar and it is unable to develope growth at 28 C. The description given for fitpeptomzpeg phepppppilug is lacking in many respects and thus a true comparison cannot be established. An examination of table 2 will indicate that the Ihgrponppipomyceg species does not produce the necessary enzyme systems for the utilization of sugars. Only the three sugars sucrose, glucose, and fructose were util- ized and these were done so with difficulty since the amount of growth after three days of incubation was slight. The inability to use sugars is in agreement with the investigations of Bernstein and Morton (1934). They found that a thermophilic actinomycete isolated ‘ from pasteurized cheese was unable to ferment maltose, 42_ mannitol, lactose, sucrose, glucose, dextrin, inulin, xylose, and cellobiose. Schuurmans also found that the Ihermoactinomycpg he isolated did not utilize car- bohydrates readily but grew well on media containing proteins or peptones. Similar results were obtained in this study. In contrast to the ThermOQctipomypep species, the Spreptomypea species utilizes all but three of the sug- ars used for study. The three not fermented are sor- bose, rhamnose, and lactose. Sorbose and rhamnose are the only hexoses in the group which are not utilized. With the exception of lactose, it appears that this organism is able to utilize the complex sugars but only up to a certain point of complexity since it was unable to hydrolize starch or cellulose. The Streptomycep spe- cies, therefore, has the capacity to produce a number of enzymes effective against several sugars and derivitives of sugars but the Ihe;poacpipomype§ species is quite lim- ited in its capacity to produce such enzymes. In those sugar media which support growth, the re- action of any particular sugar medium becomes more acidic with increased growth. It seems apparent that the metab- olism of the sugars by the thermophilic actinomycete pro- duces an acid or acids. It is interesting to note that the carbohydrates that are utilized contain glucose or fructose molecules, or molecules which can be converted to either of these sugars. Numerous microorganisms, 43 including mesophilic Strgptomypep, are known to produce various acids from the metabolism of glucose and fruc- tose. Thermoacpipopyceg sp. was not able to grow in any of the organic acid media and Spreptomyceg sp. was able to grow only in the pyruvic acid medium. These acids are commonly found in vegetative matter and malic, fu- maric, succinic, pyruvic, and citric acids are implica- ted in the tricarboxylic acid cycle of glucose metabo- lisim. If this cycle is operative in these organisms, it is expected that the intermediates of the cycle would be utilized. However, this was found not to be true with the exception of pyruvic acid in the case of the §trpptomyc§§ species. Succinic acid and pyruvic acid might also be expected to be utilized since they can be formed by the deamination of the aspartic acid and alanine respectively and deamination is a primary function when the actinomycetes are grown in protein or amino acid media. The average mycelial weights which were determined at various periods of incubation when plotted graph- ically result in curves which bear a close resemblance to growth curves of bacterial cultures. There is a lag phase during the early hours of incubation where the my- celial weight increases but slightly. This occurs even though a vigorous culture is used for the inoculum. This lag phase which lasts for 3 to 9 hours is follow- 44 ed by a rapid acceleration in growth. With both species, the most accelerated growth is observed when phytone is used as the medium. This would be expected since phytone is a partially hydrolized protein preparation. The slow- est acceleration is found with gelatin and growth accel- eration on casein is found to be intermediate between phytone and gelatin. The largest amount of mycelium was obtained from the casein medium and this was especially true in the case of the Streptogypes. Casein contains more amino acids than the other two proteins so that it is possible that it furnishes more of the constituents needed for the growth of these organisms. The peak of growth is reached in 12 to 24 hours depending upon the medium and the species of organism. After the maximum amount of growth occurs, there is a rather rapid decrease is the amount of mycelium obtain- ed. This decrease must be due to an autolysis because without such a phenomenon the weight of mycelium would at least remain at the maximum level. Katznelson (1940) also observed autolysis in a thermophilic actinomycete which he had isolated. The deceleration of growth or decrease in mycelial weight continues until about 36 hours at which time it begins to level off and produces only a very slight decrease between 48 and 72 hours. It is apparent that autolysis of the culture occurs at a more rapid rate than growth of new cells. Subcultures can be made of the organisms during the rapid autolytic 45 period and also between 48 and 72 hours when the curves indicate that the cultures are essentially stabilized. During the growth of the actinomycetes on a protein medium there is a considerable amount of ammonia releas- ed into the medium. Graphical interpretation of the data collected from ammonia determinations show that a sigmoid type curve is produced. Up to the point of maximum grow- th, the release of ammonia into the medium closely paral- lels tha amount of mycelium produced. Beyond this point the amount of ammonia in the medium continues to increase but at a steadily decreasing rate until there is only a slight increase from 48 to 72 hours. The continued in- crease in ammonia during the time of decreased mycelial weights or autolysis is further evidence that metabolism is occurring during this phase. The amount of ammonia released into the phytone med- ium by both species is very similar and it is also much less than that released from the other two proteins. How- ever, since phytone is already partially hydrolized and has a smaller total nitrogen content less amino groups are present for deamination. The ammonia in the casein and gelatin media of the Spreptomyceg species are essen- tially the same throughout the entire incubation period. However, nearly twice as much growth, as indicated by mycelial weight, was produced from the casein as from the gelatin. This indicates that more deamination oc- curred in the casein medium but a great amount of re- 46 leased ammonia was utilized in the propagation of new cellular material. In the Thermogptinomzceg culture a- bout 25 per cent more ammonia was produced from the gel- atin than from the casein but from the standpoint of growth about 25 per cent more mycelium was produced from the casein medium. In both cultures more growth was obtained from the phytone medium than from the gel- atin but considerably less ammonia was produced from the phytone. It becomes obvious that although there is a positive relationship between growth and ammonia pro- duction the amount of ammonia found in a medium does not indicate the amount of mycelium produced. The pH of the three protein media during growth of the cultures shows some variations within each medium but in general there is a definite trend in pH produced by each organism on all three media. The Thermoaptin - pypgp species has a tendency to lower the pH or increase the hydrogen ion concentration whereas the fitreptomyceg species tends to increase the pH or decrease the hydro- gen ion concentration. The difference in pH trend for each of these organisms suggests that their metabolic products are different and thus their metabolic path- ways would also be different. Although there is a difference in pH trend for each organism, the pH remains in the alkaline range during the total incubation period. Investigations on the desirable pH for these organisms revealed that 47 they prefer a slightly alkaline medium. It was also ob- served that these thermophiles prefer a protein or pro- tein-like medium for good growth. When grown in a pro- tein, these cultures produce a quantity of ammonia which is greatly in excess of their requirements and which is released by deamination. Gale (1940) demonstrated that the deaminase system of Epcherichig ppli functions be- tween pH 6.0 and 8.5 and the Optimum pH for the system is 7.5 to 8.0. From the data gathered in this study, it appears evident that the two thermophilic actino— mycetes have a strong deaminase system and the desir- able conditions of growth and metabolic activity center about this enzyme system. Even when supplied with a fermentable sugar and a usable nitrogen source, the growth is not as abundant as that produced on a desir- able protein medium. A number of free amino acids were identified in the culture filtrates of the two organisms grown in casein and gelatin media. There were also a large num- ber of unidentifiable ninhydrin reactive substances. The majority of these were observed in the early hours of incubation and they were of low Rf values which sug- gests that they may have been small fragments of pro- tein hydrolysis, e.g. small peptides. These low Rf value spots diminished in number with increasing incu- bation time which further indicates that they were pro- tein fragments. The amino acids vary somewhat depend- 48 ing upon the protein source. In general, most of the identified amino acids and amines appeared more than once throughout the entire incubation period. In the Thgrmoacpip0pyce§ culture of the casein medium, glutamine and the following amino acids were identified: cystine, cysteine, lysine, histidine, glutamic acid, serine, alanine, proline, valine, as- partic acid, phdroxyproline, isoleucine, tryptophane, phenylalanine, and methionine. Essentially the same amino acids with the addition of arginine, threonine, and asparagine were identified in the gelatin medium but in much smaller groups per incubation period. In both of these protein media cystine, cysteine, lysine, hydroxyproline, and histidine appear very frequently if not in every sample throughout the total growth period. Such predominance in both media suggests that these amino acids are metabolized sparingly or not at all by this species of Therm0§ctinom1ce§. Some of the other amino acids appear often but not contin- uously while others appear only once or twice. These amino acids are probably utilized by the growing organ- ism. The amino acids and amines identified in the casein medium cultured with the §treptomyceg species are: cys- tine, cysteine, lysine, histidine, arginine, glutamic acid, hydroxyproline, threonine, alanine, proline, va- line, aspartic acid, glutamine, and asparagine. In the 49 gelatin medium, lysine, glutamine, threonine, arginine, hydroxyproline, histidine, and aspartic acid appear but mainly only in groups of two or three for each incubation period. Lysine appears very frequently in both of these media which indicates that this amino acid is not util- ized to any extent by the §treptomyce§ species. Glu- tamic acid also appears frequently in the culture fil- trates but it is felt that its appearance is due to the large amount of the amino acid that is contained in case- in. In consideration of both cultures of the actinomy- cetes, the largest groups of amino acids were produced from the casein medium. At times as many as ten amino acids were identified in a culture filtrate. The abun- dance of different amino acids found in the culture fil- trates and the amount of growth obtained from casein in- dicates that the proteolytic enzymes of these two organ- isms are well adapted to the degradation of casein. The gelatin medium contained smaller groups of amino acids and the Streptomyces culture produced the least number of different amino acids from gelatin. The consistent appearance of only a few amino acids in this culture is reflected by the lesser amount of growth of the §trgpt - 31233 on gelatin. The numbers of amino acids identified in these cul- ture media of gelatin and casein cannot be considered as being entirely due to the hydrolytic derivatives of these 50 two proteins. The culture organisms undergo autolysis and may release amino acids into the culture medium. That such a process occurs is indicated by the pres- ence of asparagine and glutamine early in the incuba- tion period even while the organism is undergoing rap- id growth. However, the numbers of amino acids releas- ed by autolysis cannot be too pronounced since the num- ber of identified amino acids in the culture filtrates during the period of decreasing mycelial weights does not increase to any extent. This is especially noted in the §preptomype§ cultures where the number of amino acids remain the same or decrease. The presence of free amino acids in the culture filtrates during the growth of these organisms indicates that during the hydrolytic process of proteolysis individual amino ac- ids are split-off. It is probable that these amino acids are then deaminated to produce ammonia as a ni- trogen source for metabolism and the remaining residue provides the carbon source for metabolic processes. SUMMARY From a number of thermophilic actinomycetes iso- lated from an active vegetative compost, two species, each representing a different group, were selected for further study. Microscopic examination revealed that one species produces spores in chains and thus is a thermophilic member of the genus Sprpptopyceg. The other species produces single spores on extremely 51 short sporOphores branching directly from the hypha. This organism may be included in the genus Mipropon05pora (Berg- ey) or the proposed genus Thermogctinomyceg (Waksman and Corks). The latter genus is preferred by the author since it has more validity for thermOphilic forms. Both of the organisms are gram positive and non-acid fast. Temperature studies revealed that neither of the or- ganisms could develope growth at temperatures below 35 C or above 59 C and the optimum temperature is 50 to 52 0. Optimum growth of the organisms occurs when the pH of the medium is between 7.0 and 8.5. Very little if any growth is obtained below pH 6.0 or above 9.0. Observations on growth characteristics of these actino- mycetes were made using 21 different media. Both of the species are strict aerobes producing only surface growth in broth and stab cultures. On agar media which produc- ed good growth, the Thermoappinopzpea sp. produces a very light cream colored vegetative mycelium and a white to very light grey aerial mycelium which appears dry and fragile. The Streptomyceg sp. also has a cream color- ed vegetative mycelium which produces a white aerial my- celium that becomes grey with the production of spores. Neither of the species produces any pigmentation on the media used. Both of the organisms liquefy gelatin and cause proteolysis of milk after the formation of a curd but they differ in nitrate reaction; the Streptomyceg sp. producing a positive nitrite test whereas the Therppgctino- 52 pyppp ap. has a negative nitrite reaction. No cellulo- lytic activity was observed for either species but both [developed growth in protein solutions of keratin, gluten, zein, casein, gelatin, and phytone. Each of the organisms was inoculated into the follow- ing carbohydrate solutions of pH 7.0: maltose, mannitol, dextrin, sorbose, cellobiose, fructose, rhamnose, sucrose, and glucose. The §treptomyce§ species is able to ferment all except sorbose, rhamnose, and lactose. The Thermo- gptinomycep is able to ferment only fructose, glucose, and sucrose and then only slightly. Malic, fumaric, suc— cinic, pyruvic, lactin, oxalic, and tartaric acids buffer- ed at pH 7.3 to 7.5 were also inoculated but only pyruvic acid is utilized and then only slightly by Streptomycep sp. The proteolytic activity of these thermophilic ac- tinomycetes was studied using casein, gelatin, and phy- tone in a basal salts solution. Each organism was inoc- ulated into individual series of flasks with each series containing one of the three protein media. Individual flasks were removed at various periods of incubation up to 72 hours. From each sample flask mycelial weights were determined, amount of ammonia was determined, and paper chromatograms were made to identify free amino acids present in the culture filtrate. Mycelial weights plotted against incubation time gave curves similar to -bacterial growth curves but the decelerated growth is 53 actually due to autolysis of the mycelium. A consider- able amount of ammonia in excess of physiological re- quirements was released into the protein medium. The ammonia is the result of the deamination of amino acids released by the proteolytic processes of the actino- mycetes. Seventeen amino acids and two amines were identified in the culture filtrates. The identified compounds were: cystine, cysteine, lysine, asparagine, histidine, argin- ine, glutamine, glutamic acid, aspartic acid, serine, hydroxyproline, threonine, alanine, proline, trypto- phane, methionine, valine, phenylalanine, and isoleu- cine. Larger numbers of amino acids were found in cul- tured casein filtrates than in cultured gelatin filtrates. The number of amino acids found, the amount of growth produced on these proteins, and the amount of ammonia produced indicate that these organisms have a strong deaminase system and their proteolytic activity centers about this system. 54 APPENDIX Formulae of Media Corn Meal Agar Corn meal, infusion from Glucose Agar Water, distilled Corn Steep Medium Peptone Corn steep Sodium chloride Glucose Water, distilled Czapek's Agar Sodium nitrate Potassium monohydrogen phosphate Magnesium sulfate Potassium chloride Ferrous sulfate Sucrose Agar Water, distilled Dubos' Cellulose Medium Sodium nitrate Dipotassium phosphate Magnesium sulfate Potassium chloride Ferric sulfate Water, distilled Emerson's Agar Beef extract Peptone Sodium chloride Yeast extract Glucose Agar Water, distilled Glucose-Asparagine Agar Glucose Asparagine 55 50.0g 2.0g 15.0g lOO0.0ml 5.0g 15.0g 5.0g 10.03 lOO0.0ml 2.0g 1.0g 0.5g 0.53 OeOlg 30.0g l5e0g lOO0.0ml 0.5g 1.08 0.53 . 0.53 trace 1000.0ml 4.08 4.0g 2.5g 1.0g 10.0g 20.0g 1000.0ml 10.0g 0.5g Dipotassium phosphate Agar Water, distilled Glucose—Peptone A Agar Peptone Glucose Sodium chloride Agar Water, distilled Glucose-Peptone B Agar Peptone Glucose Potassium dihydrogen phosphate Magnesium sulfate Agar Water, distilled Glycerol Agar Glycerol Sodium asparaginate Dipotassium phosphate Agar Tapwater Nutrient Agar Beef extract Peptone Sodium chloride Agar Water, distilled Sabouraud's Dextrose Agar Peptone Dextrose Agar Hater, distilled Starch Agar Starch (Potato or Corn) Dipotassium phosphate Magnesium carbonate Sodium chloride Sodium nitrate Agar Water, distilled Neutralize and autoclave for 30 minutes at 110 C. 56 0.5g 15.0g 1000.0m1 5.0g 20.0g 5.0g 15.0g 1000.0ml 5.0g lO.Cg 1.0g 5.0g 15e0g 1000.0ml 10.0g 1.0g 1.0g 15e0g 1000.0m1 3.0g 5.0g 5.03 15.0g lOO0.0ml 10.0g 40.0g 15e0g 1000.0ml 10.0g 0.3g 1.0g 0.5g 1.0g 15.0g 1000.0ml Yeast Glucose Agar Yeast extract 10.0g Glucose 10.0g Sodium chloride 5.0g Magnesium sulfate 0.25g Ferrous sulfate 0.0lg Agar 15.0g Water, distilled 1000.0ml 57 REFERENCES Balaton, J.N. and Talbot, B.F. 1952 A guide to filter paper and cellulose powder chromatography. 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