OBLIGA'J!ELY THERMOPHILIC KITROG&I-FJLXATION

IN 30MJ3 SOIL BACTERIA

APPROVED:

Major Proxessor

Mi nor to¥es sor

L £ i

-dL

Miner Professor

P\ , ! \ r \

,, ,/l K

c

V-.PS,vXnrtJii Director of the Department of*[Biological Sciences

Dean of the Graduate School"

ABSTRACT

Soil bacteria were isolated which grew at 55 C in nitrogen-free

media. They were found to be obligatory theraiophiles in nitrogen-free

media and facultative therraophiles in media containing organically bound

nitrogen. Identification procedures gave results indicating that all

isolates are Bacillus and that one is very closely related to

B._ stearothermophilus > While this isolate -was s lib cultured 50 times

in nitrogen-free medium, the ATCC strain of B._ stearothermophilus

could not grovj oven in a primary culture in nitrogen-free medium.

Even though the increase in turbidity of cultures in nitrogen-free

medium at 55 C is slight, viable cell counts showed a large increase

in cell numbers within 20 hours of incubation at this temperature.

OBLIGATE!.Y THERMOPHILIC MTUCGEW-FIXATCOH

IN SOME SOIL BACTERIA

THESIS

Presented to the Graduate Council of the

North Texas State University in Partial

Fulfilment of the Requirements

For the Degree of

MASTER OF SCIENCE

By

Mary Milam, B. S.

Denton, Texas

August, 1971

TABLE OF CONTENTS

Page LIST OP TABLES , iv

LIST OP ILLUSTRATIONS v

Chapter

I. INTRODUCTION . . . 1

II. MATERIALS AND METHODS 6

III. RESULTS 12

IV. DISCUSSION 17

v.. SUMMARY 21

TABLES . 22

ILLUSTRATIONS • 2?

BIBLIOGRAPHY 33

LIST OP TABLES

Table Page

1. Cellular and colonial morphology of ten isolates 22

2. Results of growth curves of NT-15 at various temperatures. . . 23

3. Results of growth curves of NT-10 at various temperatures. . . 24

4. Results of growth curves of NT-l6 at various temperatures. . . 25

5. Results of growth carves of HT-4 at various temperatures . . . 26

LIST OF ILLUSTRATIONS

Figure Page

1. Growth carves of NT-15 &t 55 C on TSB, Burk's + 1TO.EO,, and Burk's N-free media 2J

2. Growth curves of NT-10 at 55 C on TSB, Burk's + NH. N0_, and Burk's N-free media 28

3. Growth curves of NT-l6 at 55 C on TSB, Burk's + HHj N0_, and Burk's N-free media 29

4. Growth curves of NT-U at 55 C on TSB, Burk's + Mj.N0-,

and Burk's N-free media,. 30

5. Growth curve of NT-l6 in Burk's nitrogen-free medium at 55 C . 31

6. Growth curve of NT-15 in Burk's nitrogen-free medium at 55 C . 32

CHAPTER X

INTRODUCTION

The biological principle of life at high temperatures has not been

thoroughly and systematically studied. However, knowledge of the

metabolism of thermophilic organisms provides insight- into the determinants

of the heat stability of protoplasm, especially of proteins and enzymes.

Obligate thermophiles. grow at temperatures from s.n approximate minimum

of hO C to an approximate maximum of 80 C for blue-green algae and 95 C

for some bacteria (3). While obligate thermophiles do not grow at 37 C,

facultative thermophiles grow at both 37 and 55 C (7).

A wide variety of organisms is capable of growing in the thermophilic

range. Crustaceans, molluscs, and insects are .reported at temperatures

up to 50 C, and nematodes may grow at 0 C or higher. Among the plants

and microorganisms, many species of fungi have a maximum temperature for

growth between 50 and 60 C (8), and some unusual blue-green algae are

found above 80 C (ll).

The thermophilic bacteria have been the object of scientific interest

since the isolation of the first one by Miquel in 1879 (l). Even though

thermophilic microorganisms had been known since the early years of the

nineteenth century, investigations concerning them vera limited to

observations of more or less spectacular growths in hot springs. . The

bacterium which Miquel isolated, however, was interesting in that it

could grow at 73 C; and it was capable of growth at low temperature* (l).

Curing the foil lowing decades, thermophilic bacteria were isolated

from almost every sample of soilj mud, or water which was examined. Not

only were they found in tropical soils and desert sands, bat also in air,

freshly fallen snow, sea water, the feces of man and various animals,

cultivated soils, and accumulations of decaying plant materials. While

the great majority of the bacteria isolated at high temperatures were

proved to be aerobic sporeforcing bacilli (l), thermophilic nonsporeforxcers

such as Thiobaciilus have occasionally been isolated (8).

The probable mechanism of thermophilic metabolism has been explained

by showing that the enzymes of many thermophilic organisms actually operate

best at elevated temperatures (10). It must be remembered, however, that

the thermal denaturation point of most enzymes is about 50 C or slightly

higher. Thus the optimum temperature for enzyme activity in thermophiles

is about the same as the thermal denaturation temperature of most enzymes.

This is in contrast to the majority of mesophilic systems, such as in

mammals, where enzyme activity usually reaches a maximum about 37 C, and

then declines before the thermal denaturation temperature is reached.

Nevertheless, several enzymes of the thermophilic bacterium Bacillus

stcarothermophilus, such as malic dehydrogenase, cytochrome oxidase,

and an enzyme capable of dephosphorylating ATP, have been found to exhibit

marked resistance to thermal inactivation. However, the cell-free pyruvic

oxidase of this same organism is heat stable only in the presence of its

substrate pyruvate, magnesium ions, and oxygen (1).

. It has also been found that protein synthesis occurs at maximal rates

at k3~55 C, but that the enzymes are not particularly heat stable; Thus

while there is a high rate of enayme inactivation by heat, there is a

3

concomitant high rate of enzyme synthesis. Accordingly, it has been

suggested that thermophily may be the consequence of heat stable respiratory

enzymes or the consequence of rapid protein synthesis or both (l).

Among the thermophilic bacteria, several species of Bacillus require

biotin for growth (U), and some require other vitamins such as niacin and

thiamine (5). Baker and co-workers in i960 tested the growth requirements

of 9" strains of thermophilic bacteria of the genus Bacillus. All those

tested required methionine and/or other amino acids or vitamins (2).

Studies like Baker's have shown that all thermophilic bacteria require

complex growth factors. In a review article in 1953 > Allen (l) pointed

out that thermophilic bacteria do not need such extensive vitamin and

amino acid supplements, and that much past difficulty in growing these

organisms in defined media must be traced to the use of an inadequate

mineral base and, possibly, of unsuitable or insufficient carbon sources.

Many thermophilic bacteria have extensive proteolytic properties,

and some are able to decompose starch- and cellulose. In contrast, Allen's

paper (l) pointed out that in 1909 DeKruijff observed the growth of

thermophilic bacteria in nitrogen-free media but did not obtain sufficient

nitrogen fixation for the increase in nitrogen content to be detectable by

Kjeldahl analysis. Also, according to Allen (l), Pringsheim in 19U

reported nitrogen fixation by mixed cultures of thermophilic bacteria.

Three to six milligrams of nitrogen were fixed per gram of sugar utilized,

an amount appreciably less than that fixed by Azotobacter at ordinary

temperatures. He reported no attempt to isolate trie nitrogen-fixers in

pure culture nor did he give adequate descriptions of his cultures.

These reports of early investigators contradict the more modern concept

of nutritional requirements of thermophiles. At the present time, it is

assumed that all thermophiles are fastidious in their nutritional requirements.

However, in 196$, Epstein and Grossowicz (9) reported a thermophilic

Bacillus which was capable of growing in a minimal medium consisting of

glucose, ammonium salts, phosphate buffer, and inorganic salts with optimal

growth at 55-59 C, minimum growth temperature of 1 C, and maximum growth

temperature of 65 C.

Thus the previous investigators of thermophilic microorganisms concluded

that bacteria had to be fastidious in order to grow at elevated temperatures.

Seme showed that thermophiles had an obligatory requirement for certain

vitamins and amino acids. In recent years, however, the concept has changed

with the report of the thermophile which could grow utilizing ammonium salts

as its sole nitrogen source. Since the publication of this paper in 1969>

no other articles have been found concerning prototrophic thermophilic

bacteria. In the entire search of the literature no papers were found

on nitrogen-fixation at elevated temperatures. In the work presented here,

it is claimed that bacteria have been isolated which are capable of growth

at high temperatures utilizing molecular nitrogen as their sole nitrogen

source.

CHAPTER BIBLIOGRAPHY

1. Allen, M. B. 1953. The thermophilic sporefortr.ing bacteria. Bacterid. Rev. 17:125-173.

2. Baker, H., 0. Frank, I. Pasher, B. Black, S. H. Hunter, and H. Sobotka. i960. Growth requirements of $k strains of thermophilic bacteria. Can. J. Microbiol. 6:557-563.

3. Brock, T. D. 1567. Life at high temperatures. Science. 158:1012-1020.

4. Campbell, L. L., Jr., and 0. B. Williams. 1953. Observations on the biotin requirement of thermophilic bacteria. J. Bacterid. 65:1^1-1^5.

5. Campbell, L. L., Jr., and 0, B. Williams,, 1953« The effect of temperature on the nutritional requirements of facultative and obligate thermophilic bacteria. J. Baeteriol. 65:1^6-1^7.

6. Carpenter, P. L. 1961. Microbiology. W. B. Saunders and Co., Philadelphia, p. 2h2.

7. Carpenter, P. L. 1967. Microbiology. W. B. Saunders and Co., Philadelphia, p. 192.

8. Cooney, D. G., and R. Emerson. 1964. Thermophilic fungi. W. H. Freeman and Co., San Francisco, p. k.

9. Epstein, I., and EL Grossowicz. 1969. Prototrophic thermophilic bacillus: isolation, properties, and kinetics of growth. J. Bacterid. 99:lnM«L7.

10. Oginsky, E. L., and W. W. Umbreit. 1959* An introduction to bacterial physiology. W. H. Freeman arid Co., San Francisco, pp. 119-120.

11. Thinann, K. V. 1963. The life of bacteria. The Macmillan Co., Hew York. p. 177.

CHAPTER II

MATERIALS AED MSTHCDS

Soil samples were taken from fields near Paris, Texas, and were

inoculated into 125-ml Erlenrseyer flasks containing 12 ml of Bur It's

nitrogen-free medium of the following composition:

Grains Per Liter

KJtjPO . . . 0.2

KpIEPO 0.8

ksSe^TH^o 0.2

CaClp 0.085

FeSO^'^I^O 0.05

Na^MoO^ • SHgO . 0.0Q3

glucose . . . . . . . 10.0

distilled ELO . . . . . . . . . . . . . . . . . . . C.

Incubations were carried out on the rotary shaker at 55 C. After turbidity

was noted in these primary enrichment cultures, subcultures were made in

Bark's medium. The secondary enrichment cultures were incubated in the

seine manner as the primary ones. After they became turbid, subcultures

were again made and also a loopful from each secondary culture was streaked

onto plates of Bark's medium containing 2$ agar as the solidifying agent.

After three days of incubation at 55 C, the mornholcgy of colonies on all

the plates -was noted. Growth from tertiary, etc., cultures was also plated

out, and colonies were subcultured for further study.

Because one of the organisms Isolated matched the taxononiic criteria

of Bacillus sfcearothetir.ophil'is, a certified cultui-e (strain .12980) of this'

organism was obtained from the American Type Culture Collection for direct

comparison. This organism requires methionine and thiamine for growth.

To test possible nitrogen fixing properties of this organism, it was

inoculated into Burk's nitrogen-free modi lira containing 1 mg of methionine

and 1 mg of thiamine per liter of culture medium. Cultures were incubated

at 55 G on the rotary shaker and were examined for growth by direct

microscopic observation for a period of 5 days. This experiment was

repeated, and comparison was made with the same medium to which nitrogen

was added,.

In order to ascertain the nitrogen-fixing ability of the organisms

isolated, they were transferred serially for twenty or more transfers in

liquid Burk's medium. The adequacy of this test was proven because

Bacillus stearothermophilus was incapable of surviving two transfers,

although it would do so when nitrogen was added to the basal medium.

A confirmation of this was obtained by showing that the Isolates were

not obtaining nitrogen in the form of nitrogenous contaminants in the

air, but must indeed be fixing molecular nitrogen. This was accomplished

by growing four of the isolates in liquid cultures in flasks placed-in

desiccators. Again the control organism was B, stearothermophilus. The

desiccators were evacuated and then filled with air which had bubbled

through a dilute suliuric acid and then through a water trap. After

repetition of the evacuation-replacement procedure three times, the air

in the desiccators was presumed to be free of nitrogenous contaminants

such as ninhydri'n, ammonia, cigarette smoke, nitrous oxides, and other

air contaminants commonly found in bacteriology laboratories. The

desiccators containing these flask cultt.res were incubated at 55 C for

h8 hours in a stationary condition.

In order to deteimine if the organisms wore utilizing endogenous

nitrogen compounds rather than molecular nitrogen, the ATCC strain of

B. stearothermophilus and the isolate thought to he B. stearothsrmophilus

were grown in Burk's Medium, and this was incubated as before. This

procedure was repeated until the test organisms failed to grow or until

a reasonable number of transfers had been made to make tenable this

assumption that they would grow in the absence of fixed nitrogen.

Growth curves ware obtained for four of the ten organisms. Cultures

of each organism were grown in Tryptic Soy Broth, Burk's medium plus

0.3$ NH^NO^, and Burk's nitrogen-free medium on the rotary shaker, using

250-ml side arm flasks with 25 inl of culture per flask. The Klett-

Summerson Photoe.l ectric Colorimeter with green filter was used to measure

turbidity, which was used as the index of growth. . Growth curves of all

organisms were obtained at five different temperatures: 25, 35, h5, 55,

and 65 C,

Because turbidity increase in the. nitrogen-free medium at 55 C was

so slight, microscopic examination of the cultures was made. When it was

noted that large numbers of cells could be seen microscopically in cultures

giving very slight increases in turbidity, growth curves were obtained,

using viable cell counts as the growth indicator. Two organisms were used,

and the incubations were carried out in Buck's medium at 55 C.

. In order to see if ten isolates were facultative or obligate thermophlles,

each organism was inoculated onto three plates of Burk's medium and onto

three plates of Tryptic Soy Agar. One iroculated plate of each medium

was incubated at 30 C, another at 37 <'? and the 'final set at 55 C. The

plates were examined daily for five days for the presence of colonies.

Identification procedures were then carried out. Following the

sequence of criteria for identification of species of Bacillus in Bergey* s

l«Ianual of Determinative Bacteriology, spore stains were performed on each

culture by the Sehaeffer and Fulton method (l), employing malachite green

and a safranin counterstain. Once the shape and position of the spores

had been established, the organisms were inoculated into Purple Broth Base

containing 1$ glucose and a, Durham t>/be to test for gas production from

glucose. Then the organisms were inoculated onto plates of starch agar

(peptone 1$, EaCl 0.05$, starch 0.2<j£, and agar 2 $), and after 2h hours of

incubation, the plates were flooded with iodine solution to test for the

hydrolysis of starch.

In farther ceapliance with the -key, one of the ten cult ares NT-IT

was inoculated into SIM medium and into MR-VP broth. After 8 hours of

incubatj.op, 1 b1 of chloroform ana 1 ml of Kova.cs reagent were added to

the SIM medium to test for indol production. One milliliter of the MR-VP

broth culture was decanted into a small test tube. To it were added 8 drops

of alpha-naphthol and 8 drops of hG% KOH containing 0.3$ creatine to test

for the presence of acetylmethylcarbinol. Another criterion needed.for

identification was the organism's ability to grow at 65 C. Cultures were

inoculated onto plates of Burk's agar and onto plates of 'ISA. and these

were incubated at 65 C and were examined daily for three days to see whether

the'organism could grow at this temperature.

The other nine organisms were each inoculated into two tubes of glucose

broth. One set of tubes was incubated aeroWeally, and the other set

10

anaerobicelly, The pH of the aerobic cultures vas determined periodically.

All incubations were carried out at 55 C, After 5 <3ays of incubation, the

anaerobic cultures ware examined for the presence or absence of gx'owth.

Knowing that none of the experiments conclusively rpoved that these

organisms utilized molecular nitrogen as their nitrogen source, an isotope

tracer experiment was attempted. With, the help of Dr. Tom Gray and his

students in the NTSU Physics Department, an experiment was designed'to

produce nitrogen-13. Beuterons were produced in the Van de Graaff generator

and were accelerated down an evacuated tube to bombard the carbon-12 atoms

in a target of mylar plastic. A neutron was emitted, and nitrogen-13 was •

produced, A carrier gas of 5 a r g o n and 50$ air was used to move the

1 3

N from the target into a gas bottle containing the ATCC strain of

B» stearothermoph:ilu.s and to a second gas bottle containing isolate NT-17>

the organism which met the taxonomic criteria of B»_ stearothermophilus. •

At ten-minute time intervals, 10-rnl samples were removed from each gas

bottle and were transferred to a test tube. Nitrogen-lU was bubbled through 13

each sample for five minutes to flush out any N which was in the medium.

Then the samples were assayed for radioactivity by pouring the sample

directly into a plastic petri dish and setting this dish directly on a

radicacti /e counter. All procedures had to be performed quickly, since

the half-life of " i s ten minutes.

Thermophilic nitrogen-fixing bacteria were isolated from the soils

from the Paris, Texas, location by others in the laboratory. In order to

establish the distribution of these bacteria in soil, sixty different

soils were obtained from different localities in Texas, Oklahoma, and

Massachusetts. Each soil, was examined by the methods described above.

CHAPTER BIBLIOGRAPHY

1. Eklund, C., and C. E. Lankford, I9&7. Laboratory manual for general microbiology. Prentice-Hall, Inc., Eaglewood Cliffs, N. J. p. 292.

CT;AFTER III

RESULTS

All. thermophiles isolated were sporeforming, gram variable rods.

Their cellular and colonial morphology on Bark's agar incubated at 55 C

is described in Table 1.

In the experiment designed to test the nitrogen-fixing abilities of

the ATCC strain of stearothermophilus, microscopic observation of the

culture after 5 days of incubation showed cells present but in no greater

numbers than when first inoculated into the Burk's medium containing 1 rr.g

methionine and 1 rng thiamine per liter. On transfer of a small inoculum

of this culture to fresh medium, there was no evidence of any cells after

5 days of incubation. The same results were obtained when this experiment

was repeated. Apparently the organisms vrore surviving in the primary culture

utilizing the nitrogen carried over from the Tryptic Soy Agar on which they

were previously growing,. The culture was serially transferred ten times

in this same medium to which nitrogen was added.

In the experiment designed to determine whether the isolates were

i tixizing molecular niurogen rather than nitrogenous contaminants, growth

as evidenced by an increase in turbidity and microscopic observation of

the culture was noted in all four cultures after three days of incubation

nn the desiccators containing the scrubbed air. Thus it appears from

these data that these bacteria are utilising molecular nitrogen. Bacillus

stearotherraophilus, which served as the control, failed to grow in all

cultures prepared,.

13

The experiment intending to show that IIT--17'was not utilizing endogenous

nitrogen was performed twice. Both times this organism was transferred

serially 50 times from one flask containing nitrogen-free medium to

another, using a small inoculum. In the case of the ATCC culture, the

cells never grew past the primary culture. Thus it appears that NT-IT

utilizes molecular nitrogen rather than an endogenous supply.

The growth curves at various temperatures shows well the thermophilic

nature of these organisms. Isolate NT-15 grew quite well in Tryptic Soy

Broth (TSB) at 25, 35, 5, 55, and 65 C (Table 2). However, the length

of time required to reach maximal turbidity decreased as the temperature

increased, giving evidence that the organism is truly thermophilic.

Additional information leading to the verification of nitrogen-fixation

by this organism is the increase :i n turbidity in nitrogen-free medium.

Moreover, since this increase occurs only at 55 C and higher temperatures,

it appears that the organism has an obligatory requirement .for high

temperatures if it is to fix nitrogen. HT-10 (Table 3), NT-l6 (Table 4),

and NT-4 (Table 5) show similar results. Apparently, the optimum growth

temperature for the four organisms is 55 C. Growth curves of these organisms

in the three media at 55 C (Fig. 1-4) serve to further illustrate the

observations mentioned 3,bove.

Because the increase in tsirbidity in nitrogen-free medium is so slight

compared to the increase in TSB, the growth carves with viable cell counts

at 55 C were necessary. The results of these growth curves show that there

is a three log increase in the number of viable cells in 20 hours for

NT-16 (Fig. 5) and a 2.6 log increase in 16 hours for NT-15 (Fig. 6).

.1*4

In the experiment designed to show whether the thermophilic nature

of these organisms is obligatory or facultative, the results were

unequivocal. All cultures grew luxuriantly on Tryptic Soy Agar at 30, 37,

and 55 C. None of the organisms were able to grow on Burk's agar at 3^

or at 37 C. They did, of course, grow on Burk's agar at 55 C. I infer

from these data that the organisms are obligately thermophilic on nitrogen-

free medium and facultative with respect to temperature requirements when

cultured on media containing fixed nitrogen.

In the tests performed to identify the species of the thermophiles,

it was found that after 5 days of incubation none of the organisms had

produced gas from glucose. While one organism—I1T -17 - - hydroxy zed starch,

as indicated by clear zones surrounding the colonies with the rest of the

agar being stained dark blue after flooding the plate with iodine, the

other nine did not.' Because no red color developed at the interface between

the chloroform and ICovacs reagent when the two were added to the culture

of UT-17 growing in SIM medium, the organism is said to have not produced

indol. Since no pink color developed in the culture of the same organism

growing in MR-VP broth after the alpha-naphthoi and potassium hydroxide

were added, this particular thermophile apparently did not produce

acetylmethylcarbinol. This organism also grew at 65 C on both Burk's

agar and TSA. The reactions which NT-17 gave on all these tests match

the results which Bergey's Manual uses to characterize B^ stearothermophilus.

Since the other nine organisms did not hydrolyze starch, as indicated

by the absence of any clear zones around colonies when the plates were

flooded with iodine, they•followed a different branch of the key to the

species of Bacillus. However, at this point some discrepancy concerning

15

the identification process occurs, For an organism to be classified as

B. brevis, it must not possess the ability to grow in glucose broth

anaerobically, and the pH of glucose broth cultures must be 8.0 or higher.

If, on the other hand, the pH of glucose broth cultures is less than 8.0,

and the organism can grow in glucose broth under anaerobic conditions,

then it is L laterosporus or L pulvifaciens.

After five days of incubation, however, there was no growth in the

anaerobic glucose broth of any of the re,mining nine organisms. Every

time the pH of the aerobic glucose broth cultures was determined, it was

less than 8.0. Since B._ pulvifaciens cannot grow on carbohydrates with

ammonium salts as the nitrogen source, these nine organisms are probably

not a variant of this species because the growth curves showed that these

thermophiles can grow in glucose broth with NH^KO^ as the nitrogen source.

Thus the other nine might be variants of brevis or laterosporus.

Unfortunately the experiments with the nitrogen-13 were not successful.

These experiments were attempted several times. At first there was a

problem of the cultures foaming extensively, even though they had been

washed three times with phosphate buffer prior to putting them in gas

bottles. (Addition of an anti-foam agent corrected this problem). The

obstacle, however, which led to the termination of the experiment had to

do with focusing the deuteron beam on the target. At first the beam was

1?

not centered on the target. While N was being produced, as evidenced

by determination of its half life by counting radioactivity through

several half life intervals, its quantity was not sufficient for the

purposes of the experiment. When the beam was increased to produce a

13 greater flux of N, it burned holes in the mylar, causing the vacuum

16

in the Van de Graaff to be broken and. the K generating reaction to be

terminated. This problem could not be solved during the summer of 1970.

Other laboratory workers were able to isolate nitrogen-fixing thermophiles

from the Paris, Texas, soils. The attempt to isolate nitrogen-fixing

thermophiles from the 60 random soil samples was unsuccessful. While

several primary cultures shewed turbidity and the presence of microorganisms

as evidenced by microscopic examination, no growth occurred in any of the

secondary cultures. Since these nitrogen-fixing thermophiles do not

appear to be ubiquitous as thermophiles in general are, there may be

something peculiar about the soils from which these organisms were isolated

which is conducive to their growth.

CHAPTER IV

DISCUSSION

On the basis of the above data, it can be concluded that the organisms

isolated are nitrogen-fixing therrnophiles. They are facu3.tat.ive thermophilos

when grown on media containing organic nitrogen, but they are obligate

therrnophiles in .media containing only EHj.WO or in nitrogen-free media.

While arguments against nitrogen-fixation might be raised because of the

low turbidities of the cultures in nitrogen-free media, the viable cell

counts showing a 2.6 to 3-0 log increase in viable cells in the first

20 hours of incubation tend to dispel doubt. The number of viable cells

ir ".ich culture decreased gradually as spores were produced.

Because of their spore formation, gra?r> reaction, and aerobic metabolism,

these organisms were classified as members of the genus Bacillus. Isolate

NT-.l? classified as a variant of B._ steafotherreoph 11 us because it is

gram variable with terminal spores, hydrolyzes starch, doss not produce

acetylmethylearbinol nor indol, and grows at 65 C. The discrepancies

encountered in assigning a species to the other nine organisms make it

impossible to say for certain that one, some, or all of them are .one species

or another. The location of their spores would lead one to believe, however,

that they are different species. Because of their inability to hydrolyze

starch, they are apparently not the same species as NT-17. While it appears

that some of the remaining nJne might be B_._ brevis or B. laterosporus, the

data are not definite enough to draw a conclusion. The fa'ct that Sergey's

Manual does not use the criteria of thermophily and rdtrogen-fixation

l8

together made classification of these organisms difficult and probably

impossible. Because Bscillus are generally thought to be large rods,

it might be pointed out that these calls when grown in Tryptie Roy Broth

are approximately times larger than they are vhen grown in Burk's

medium.

The ATCC strain of B^ stearothermophilu s apparently is not a nitrogen-

fixer because of its inability to grew in Burk's medium, even though the

medium was supplemented with growth-factor amounts of methionine and thiamine.

Survival in the primary culture can be attributed to utilization of

nitrogenous products carried over from the Tryptie Soy Agar on which they

were growing when inoculated into Burk's, but in no case was there evidence

of even slight amounts of growth.

The failure to isolate similar organisms from the sixty various soil

samples gives evidence that organisms such as the ones described here are

not ubiquitous in nature. If this is true, then the question of their

origin and their .role in the soil microflora is raised.

Evidence against the possibility that these organisms utilize

nitrogenous products such as amines in the air as their nitrogen source

was obtained by the scrubbed air experiment. To discount the possibility

that the organisms utilize endogenous nitrogen, the fifty successive

transfers on two different occasions make it possible to conclude'that

endogenous nitrogen is not the supply that these organisms are utilizing.

Regrettably nothing can be concluded from the nitrogen-13 experiments.

Correction of the major technical difficulties encountered would have

required extensive trial-and-error methods and a great amount of time

which the physicists were not able to rpovide because of their own research.

19

Of course, none of the results obtained conclusively prove that these

organisms fix nitrogen. An isotope tracer experiment utilizing riitrogen-15

must be performed before it can definitely be stated that these organisms

utilize molecular"nitrogen. Acetylene reduction, the standard test for

the nitrogenase enzyme which is essential in the nitrogen-fixation process,

could not be demonstrated because of the insolubility of the ga.s in water

at 35 C.

Prom these data, however, it can be inferred that the rates of the

metabolic reactions concerned with KH^NO^ assimilation and nitrogen-fixation

are temperature dependent. In the same organism the NHj.NO assimilation

system has a lower energy of activation than the nitrogen-fixing system.

It is obivious that the products of these reactions, essential amino acids

and other nitrogenous precursors, can be synthesized from or

molecular nitrogen only at elevated temperatures. In these bacteria,

these biosynthetic pathways are not operative in the raecophilic range.

As well as presenting this interesting phenomenon of temperature

dependent nitrogen assimilation, these data also show a new aspect of

the nutritional requirements of thermophilic bacteria. While they were

thought to be fastidious when first observed, this assumption has gradually

been cast aside because of reports such as Baker's (1), which stated that

thermophiles could grow when provided with only one or two amino acids

or vitamins rather than a full complement. Epstein and Grossowicz's

report (2) of two years ago of a thermophile which could utilize ammonium

salts as its sole nitrogen source gave more evidence to discount the complex

nutritional requirements idea. Then the organisms discussed in this paper

go one step further by being able to grow in the absence of fixed nitrogen

at high temperatures.

CHAPTER BIBLIOGRAPHY

1. Baker, H., 0. Frank, I. Pasher, B. Black, S. IL Hunter, and H. Sobotka. i960. Growth requirements of 9*+ strains of thermophilic bacteria. Can. J. Microbiol. 6:557-563.

2. Epstein, I., and N. Grossowicz. 1969. Prototrophic thermophilic bacillus: isolation, properties, and kinetics of growth. J. Bacteriol. 99:^l4-Ul7.

CHAPTER V

SUMMARY

Soil bacteria were isolated which grew at 55 C in nitrogen-free

media. They were found to be obligatory theraiophiles in nitrogen-free

media and facultative t'nermcphiles in media containing organically bound

nitrogen. Identification procedures gave results * indicating that all

isolates are Bacillus and that one is very closely related to

5* stearothermophilus. While this isolate was subcultured 50 times

in nitrogen-free medium, the ATCC strain of stearothermophilus could

not grow even in a primary culture in nitrogen-free medium. Even though

the increase in turbidity of cultures in nitrogen-free medium at 55 C is

slight, viable cell counts showed a large increase in cell numbers within

20 hours of incubation at this temperature'.

2?

TABLE 1. Cellular aa-3 colonial morphology of ten isolates

Organism

NT-4

Relative length of cell

short

Position of spore

central

Description of colony

small convex colorless

NT-7 short central small convex white

NT-8 medium terminal• small convex colorles£

NT-9 medium ; ubterminal small flat colorless

NT-10 >hort terminal larger mucoid white

NT-13 short terminal small convex colorless

MT-15 short central larger spreading colorless

NT-16 short terminal larger convex white

NT-IT short terminal larger flat colorless

HT-18 short • terminal larger convex white

-3

TABLE 2. Results of growth curves of NT-15 • at various temperatures

Maximal Turbidity

Klett Units/hours

Temp.

25

35

55

65

TSB

700/120

400/150

520/75

580/25

550/15

Burk's +

mij+i©3

8/150

20/150

20/75

160/65

60/45

N-free Burk's

5/150

10/150

5/1 0

60/140

65/45

2h

TABIiE 3. Results of growth curves of IIT-IO at various temperatures

Maximal Turbidity

Klett Units/hours

Burk's + II-free Peaip. TSB NII^NO^ Burk's

25 5J+0/50 9 8 / 1 2 0 5 / 1 2 0

35 6k0/k0 60/TO . 1 0 / 1 2 0

^5 550/27 6 0 / 6 5 1 5 / 9 3

55 6 8 0 / 2 4 8 0 / 4 0 3 0 / 7 0

65 6 5 0 / 1 8 7 0 / 3 0 3 5 / 3 8

TABLE U. Results of growth curves of NT~l6 at various temperatures

Maximal Turbidity

Kiett Units/hours

Temp.

25

35

1+5

55

65

TSB

500/55

Gko/ko

550/27

680/25

680/15

Burk's +

5/120

60 /70

60 /65

110/25

80 /20

M-free Burk* s

2/120

10/120

lH/93

Ho/so

60/25

26

"CABLE 5. Results of growth carves of li'T-U at various temperatures

Maximal Turbidity

Kletc Units/hours

Temp.

25

35

1+5

55

65

TSB

50/120

515/125

500/35

680/20

360/18

Burk's -f RII WO

7/120

50/130

65/35

115/75

80/70

N-free Burk's

3/120

10/130

10/65

30/90

40/30

8?

FIG. 1. G.twth curves of NT-15 at 55 C on TSB,

V ^ 3 Burk5 s 4- and Purkf s N-free media.

02 4»

• - H

B -p p 0 d

hours

?00

ICO

.0 TSB

Bark's + NH^NO^

Burk's N-free JO

100

28

FIG* 2, Growth curves of WT-XO at 55 C on TSB, J3urkfs * KH^NG^ and Burkf s N-freo media.

to -p •H £>

-P P 0) H

700

600

500

400

300

200

100

p - " ' Wpte!^ ,#w _0 TSB

Burk',-5 + KH|,K0. © 4 3

— e r p 3 ^ - "

Burk's If-free - G - - - ©

J_ 20 ijO 6o

hours

8o 3.00

29

700

600

500

w 00 -p •rl ,0 £>

0)

300

200

100

FIG. 3. Growth curves of NI~l6 at 55 C on TSB, Burk's + and Burk's N-free media.

TSB

0

IlOlLVS

Burk's + NHJ(Nq^

Burk's H-free o

J 100

FIG. Growth curves of at 55 C on TSB, Buck's -J- NH NO * and Bark's If-free media.

30

700

600

500

w p •H & :o •p

Id d

Uoo

300

O

— © ~ -0 TSB

200

100

20- 1*0

-a-

. 6o

hoars

Burk'a + MHNO

• Burk's H-free

jO

80 100 J

31

FIG# 5. Growth ca rve of I i r - l 6 i n B t i r k f s n i t r o g e n - f r e e medium at 55 C*

7

w H H 0) o <L> rH *3 •H >

0

1 S3 fM O -b3 O H

20 i*0 60

hours

SO 100

FIG* 6, Growth curve of KT-15 in Bark's nitrogen-free • medium at 55 0.

ca H HI a> a a> rH %

J>

o & Cl>

O t*o o H

hours

BIBLIOGRAPHY

Books

Carpenter, P. L. 1961. Microbiology. W. B. Saunders and Co., Philadelphia.

Carpenter, P. L. 19&7. Microbiology. W. B. Saunders and Co., Philadelphia.

Cooney, D. G., and R. Emerson. l$6h. Thermophilic fungi. W. H. Freeman and Co., San Francisco.

Eklund, C., and C. E. Lankford. 1967. Laboratory manual for general microbiology. Prentice-Hall, Inc., Englewood Cliffs, N. J.

Oginsky, E. L., and W. W. Um'oreit. 1959* An introduction to bacterial physiology. W. H. Freeman and Co., San Francisco.

Thimarm, K. V. .1963. The life of bacteria. The Macmillan Co., New York.

Articles

Allen,, M. B. 1953. The thermophilic spore forming bacteria. Bacteriol. Rev. 17:125-173.

Baker, H., 0. Frank, I. Pasher, B. Black, S. H. Hunter, and H. Sobotka. i960. Growth requirements of strains of thermophilic bacteria. Can. J. MieroMol. 6:557-563.

Brock, T. D. 1967. Life at high temperatures. Science. 158:1012-1020.

Campbell, L. L., Jr., and 0. B. Williams. 1953. Observations on the biotin requirement of thermophilic bacteria. J. Bacteriol. 65:lUl-l!*5.

Campbell, L. L., Jr., and 0. B. Williams. 1953 > The effect of temperature on the nutritional requirements of facultative and obligate thermophilic bacteria. J. Bacteriol. 65:1^6-1^7.

Epstein, I.., and N. Grossowicz. 1969* Prototrophic thermophilic bacillus: isolation, properties, and kinetics of growth. J. Bacte.rio]. . 99:^1^-^17.