MIXED CULTURE OF CHLQRELLA PYRENOIDOSA TX71105

AND A VARIANT STRAIN OP BACILLUS MEGATERIUM

APPROVED:

' J Major Professor

I

Minor PS»oTe(s br

~\A '' \VA-A \XiU \

Director of the Department o$ Biological Sciences

'.T Dean of the Graduate School

MIXED CULTURE OP CHLORBLLA FYRENOIDOSA TX71105

AND A VARIANT STRAIN OP BACILLUS MEGATERIUM

THESIS

Presented to the Graduate Council of the

North Texas State University in Partial

Fulfillment of the Requirements

For the Degree of

MASTER OP ARTS

By

Raymond C. Yao, B. A.

Denton, Texas

August, 1970

TABLE OF CONTENTS

*

Page

LIST OP ILLUSTRATIONS j.v

Chapter

I. INTRODUCTION 1

II. MATERIALS AND METHODS 9

III. RESULTS. . . . ; 19

IV, DISCUSSION 38

V. SUMMARY kS

BIBLIOGRAPHY 1 8

iii

LIST OP ILLUSTRATIONS

Figure Page

1. Outline of the Original Schraidt-Thannhauser Procedure for Determination of RNA and DNA li*

2. Mixed Culture of Bacterial and Algae on Algal Culture Agar Containing One Per Cent Glucose. . . . . . . . . . . . 16

3. Comparative Cell Counts of Algal and Bacterial Controls 23

1-. Comparative Cell Counts of Mixed Culture of Thirty-Hour Bacterial Culture and Algal Culture 25

Comparative Cell Counts of Mixed Culture of Sixty-Hour Bacterial Culture and Algal Culture. . . . . . . , 26

6. Comparative Cell Counts of Mixed Culture of Seventy-Hour Bacterial Culture and Algal Culture. . . . . . . . . . . 28

7. Comparative Determinations of the Amount of Reducing Sugars Present in the Cell-Free Media of the Algal Control, Bacterial Control, and Mixed Culture . . . . . . . . . 29

8. Comparative Determinations of the Amount of Protein Present in the Cell-Free Media of the Algal Control, Bacterial Control, and Mixed Culture. 33

9. Comparative Determinations of Nucleic Acids in the Cell-Free Media of Algal Control, Bacterial Control, and Mixed Culture . . . . 35

10. Comparative Determinations of the Amount of Lipid in the Cell-Free Media of Algal Control, Bacterial Control, and Mixed Culture 36

iv

CHAPTER I

INTRODUCTION

In the past twenty years, extensive studies have been

conducted on algae as a potential source of food for man

and animals (3, 5» 8, 15). This concern is enhanced because

it is well established that algal cells contain adequate

quantities of protein, fats, carbohydrates, and vitamins.

Krauss (8) has claimed that unicellular green algae, es-

pecially Chlorella, should be an excellent source of food

for both man and animals. The work of Podor, Raoz, and Be do

(5) showed that albino rats fed an experimental diet of

algae for a period of thirty days had a 25.6 per cent gain

in body weight, but control animal3 on a conventional diet

only gained 11.9 per cent of body weight. They also reported

that rats feeding on algae powder had a better appetite than

the control animals. On the other hand, the use of algae as

a sole source of food may have drastic limits. Szekely (20,

21) has shown that air dried algae were recoverable, from the

feces of chickens, and that large numbers of organisms passed

through the digestive tract without being degraded., Krauss

(8) also indicated the presence of digestive tract difficulties

in chickens fed algae.

1L

Recent developments in the space program of the United

States emphasize the possibility of employing algae in gas

exchange systems and as a food source for man in space

travel (2, 3, 11, lij., 22, 26), Research in this area indi-

cates the value of human wastes as algal nutrients in closed

ecological systems (2, 10, lij., 22, 25) and, at the same time,

the value of algae as removers of carbon dioxide from the

environment, as a source of food, and as producers of oxygen.

The feasibility of closed ecological systems using men and

alga© as symbionts is theoretically possible (I}.) and its use

is considered necessary for extraterrestrial colonies in the

future.

Bacterial contamination of mass cultures of algae is

now being studied as a result of the above interests and

studies of bacterial contaminants in mass cultures of algae

have been reported by previous investigators (1, 11, 17, 23,

2lj.). Further, several researchers have stated the fact that

bacterial proliferation is a function of algal growth and

bacterial growth occurs as a result of the excretion of

organic substances into the culture medium, by rapidly dividing

algae. According to Blasco (1), the number of bacteria in-

creases with increased algal density, and bacteria are found

imbedded in the surface of algal cells when the cultures of

algae are characterized by subnormal rates of growth and

photosynthetic gas exchange. Because bacterial isolates

failed to grow on the sterile algal medium but grow well in

algal cultures, it is established that the bacteria were

actually dependent on the algae for a source of energy and

nutrients. Krauss and Thomas (9) also reported that bacterial

populations are nourished by the organic cell wall materials

cast off during active algal cell division.

Further investigation on population dynamics of mixed

cultures of bacteria and algae established that a greater

number of bacteria are present in the slowly growing micro-

nutrient-deficient cultures, suggesting that the secretion

of metabolites by the cells is more important than cell

materials as nutrient for the bacteria.. This theory was

further substantiated by Ward, Moyer and Vela (2i|) who claimed

that algal excretory products serve as the primary carbon

source for bacterial contaminants and. that certain bacteria

are able to grow in the effluent from axenic culture of

Chlorella pyrenoldoaa TX71105. A1X the above evidence indi-

cates that bacteria are dependent on the algae for growth.

Wardr Moyer and Vela (2ij.) reported a decrease of as much

as twenty per cent in algal cell yield as a result of heavy

contamination by selected bacteria.. Vela and Guerra (23) sub-

sequently reported that only a few bacteria isolated at random

could grow extensively in cultures^ of: Chlorella pyrenoidosa

TX7110f> and none of the bacteria had the capacity to inhibit

the algal growth aignificantly. Thus,, Fogg, Krauss, and

Thomas (6, 9) and Vela and Guerra (23) theorized that the

bacterial growth results from the utilization of algal meta-

bolites. Vela and Guerra (23) further showed that oxidizable

metabolites are dialyzable into distilled water when placed

in Visking membranes and that different bacteria utilize

different metabolites. Only a small fraction of the soil and

air isolates tested were able to grow extensively in algal

cultures.

As a result of the investigation of Jorgensen (7), an

antibacterial substance which would inhibit bacterial growth

to some extent was discovered in the extracts of certain

algae. These substances were then purified and classified as

a type of allomerized chlorophllide. This photoaynthetic

pigment, in a concentrated form, caused complete growth inhi-

bition of a wide range of bacteria. Also* in. nmaynchronized

cultures, the antibacterial substance varied in. concentration

in the medium as a function of culture time and was capable

of preventing the growth of a wide variety of bacterial

contaminants.

Very little work has been done on bacteria capable of

significantly inhibiting algal growth. This thesis reports

the research on mixed cultures of a high-temperature strain

of algae, Chlorella pyrenoidosa TX71105, and an organism

isolated from the air and tentatively identified as a variant

strain of Bacillus megaterium. These bacteria inhibit algal

growth, discolor algal chlorophyl, and cause the lysis of the

algal cell walls.

CHAPTER BIBLIOGRAPHY

1. Blasco, R. J., "Nature and Role of Bacterial Contaminants in Mass Culture of Thermophilic Chlorella pyrenoidosa," Applied Microbiology, 13 (September, 1963), 4 7 3 - 4 7 5 7 ^

2. Brown, L. R., M. V. Kenedy, snd G, Tischer, "An Algal Medium Produced from Human Wastes," Developments in Industrial Microbiology, 6 (August^ 1964), 2^5-249.

3. Casida, L. R. Jr., Industrial Microbiology, New York, John Wiley and Sons, Inc., 1968.

4* Eley, J. H. and J. Myers, "Study of a Photosynthetic Gas Exchanger, a Quantitative Repetition of the Priestley Experiment," Texas Journal of Science, 16 (July, 1964), 296.

5. Fodor, G. Y., G. Racz, and K. Bedo, "Determination of the Nutritional Value of Green Algse," Proceedings of the Fourth Congress of the Hungarian Association of Microbiology, 61; (Spring, 1964), 405#

6. Fogg,. G. E,, "Extracellular. Products," Physiology and Biochemistry of Algae, edited by Ralph A. Lewin, New York, Academic.Press, 1962.

7. Jorgensen, E. G., "Antibiotic Substances from Cells and Culture Solutions of Unicellular Algae with Special Reference to Some Chlorophyll Derivatives," Physiologia Plantarum, 15 (May, 1962), 530-545.

8. Erauss, R. W#, "Mass Culture of Algae for Food and Other Inorganic Compounds," American Journal of Botany. 49 (November, 1962), 425-433.

Erauss,. R. W# and W. H. ..Thomas, "The Growth and Inorganic Nutrition of Scenedesmus obliquus in Mass Culture," Plant Physiology,. 29 (May. 1954). 205-214.

10, Lancaster, J. H., R. G. Tischer, and R. Z. Long, "Human Feces as a Nitrogen Source for some Green Algae," Developments in Industrial Microbiology, 3 (August, 1961), 25-34.

11. Mayers, A., M. V. Zuri, Y. Shain, and H. Ginzburg, "Problem of Design and Ecological Considerations in Mass Culture of Algae," Biotechnical Bioengineering, 6 (May, 196I4J, 173-179.

12. Myers, J., J. N. Philips Jr. and J. R. Graham, "On the Mass Culture of Algae," Plant Physiology, 26 (June, 1961), 539-^9.

13. Myers, J., "Physiology of tho Algae," Annual Review of Microbiology, 5 (March, 1951), 157-180",

ll|. Philips, J. N. Jr., "Closed Ecological System for Space and Extraterrestrial Habitation," Developments in Industrial Microbiology, 3 (August, 1961), 5-13.

15. Schmidt, W# E. and J. Verduin, "Mass-Culture-Produced Algae as a Pood Source," Symposium: Microorganisms as Potential Food Sources, 177 (January, I960), 250.

16. Smith. H. C., H. E. Brown, J„ E. Moyer, and C. H. Ward, Utilization of Excretory Products of Chlorella pyrenoidosa," Developments in Industrial Microbio-logy , 9 (August, 19W;, 3^27

17. Smith. H. C., H. E. Brown, J„ E. Moyer, and C. H. Ward, Some Excretory Products of Chlorella pyrenoid osa by Selected Bacteria," Developments in Industrial Microbiology, 9 (August, 1967), 363-3^9.

18. Sorokin, C. and J. Myers, "A High-temperature Strain of Chlorella." Science,. 117 (March, 1953), 33P-331-#

19. Szekely, K., Semiautomatic Equipment for Mass Cultivation of Protophytic Fresh Water Algae, Proceedings of the Fourth Congress of the Hungarian Association of Microbiology, Budapest,, 1964.

t» 20. Szekely, K., A. Eperjessy, M. Kerebes, and M. Bedo, Studies on the Digestability of Unicellular Algae II; In Vitro Effects of Proteolytic Enzymes on EriedTlgae, Proceedings of. the Fourth Congress-of the Hungarian Assoc la t.i on. of Microbiology^, Budapest, 1961*.

8

21. Szekely, K., S. Bedo, B. Sedo, and G. Y. Fodor, Studies on the Digestability of Protophytic Algae 1j Effects of Digestive enzymes on Dried Algae in Vivo, Proceedings of the Fourth Congress of the Hungarian Association of Microbiology, Budapest, 1961*.

22. Tischer, G. R., B. B. Tischer, and D. Cook, "Human Feces as a Nitrogen for Algae in Closed Space Ecologies," Developments in Industrial Microbiology, 3 (August, 1961),72-86.

23. Vela, G. R. and C. N. Guerra, "On the Nature of Mixed Cultures of Chiore11a pyrenoidosa TX 71105 and Various Bacteria," Journal of General Microbiology, lj.2 (June, 1966), 123-131.

?1|. Ward, C. H,t J. E. Moyer, and G. R# Vela, "Studies on Bacteria Associated with Chlorella pyrenoidosa TX 71105 in Mass Culture,"11 Developments in Industrial Microbiology, 6 (August, 1961}.), 213-222.

25. Winders, W. H. and R. G, Tischer, "The Nutritive Value for Algae of Fecal Pyrolysis Gasis," Developments in Industrial Microbiology, 3 (August, 1961), Hf-EE

26. Zuraw, E. A., "Algae-Primate Gas Exchange in a Closed Gas System," Developments in Industrial Microbiology, 3 (August, 1961), 1^0 11*9..

CHAPTER II

MATERIALS AND METHODS

Organisms

All research was conducted with a thermotolerant alga

designated Chlorella pyrenoidosa TX 71105 by Sorokin and

Myers (12) which has an optimal growth temperature of 39°C.

The bacterium utilized was an aerobic spore-forming bacillus

isolated from the air by Vela and Harrel (15). It was tenta-

tively identified as a variant strain of Bacillus megaterium

according to Burgey's Manual of Determinative Bacteriology,

Since the optimal growth temperatures for both organisms are

similar, all experiments were conducted at a temperature of

37°G. in a reciprocal incubator-shaker.

Media

Stock cultures of Chlorella pyrenoidosa TX 71105 were

grown and maintained in Bristol's solution as modified by

Bold (2). It was prepared as indicated below:

Six stock solutions, I4.OO ml* in volume were employed,

Each contains one of the following salts in the amounts

listed:

HaNO^ 10.0 gra

CaCl^ 1,0 gra

MgS0^.7H20 3.0 gm

9

10

K2HP0u . 3.0 gm

KH2P0^ 7.0 gm

NaCl 1.0 gm

10 ml of each stock solution were added to

9l(.0 ml distilled water. To this was added a

drop of 1.0 % FeCl^ solution. Two ml. of trace

elements solution were also added. When neces-

sary solid media were prepared by adding 15 gm.

of agar to the liquid medium. All media were

sterilized in the autoclave at 121°C. for

fifteen minutes.

Cultural Conditions and Growth Determinations

The slgae were grown in Bristol's salt medium in flasks

placed in an environmental chamber at 37°C., illuminated with

1,000 foot-candle flourescent li girts and aerated contin-

uously by shaking at ninety strokes per minute. The air in

the shaker-incubator was enrich with C02 to a concentration

of approximately five per cent. The bacillus was grown in.

Bacto Tryptic Soy broth (Difco 0370-01) also at 37°C. in the

same shaker-incubator. Pure cultures of the algae were- ob-

tained by transferring growth onto Bacto Algal Culture Agar:

(Difco 051+5-01) every four days until bacteria-free clones

were isolated. The pure cultures were then kept in s.cr.ew-cap

11

tubes on agar slants and stored in the refrigerator for

later work.

Algal cultures at the early stationary phase of growth

were mixed in equal volumes with bacterial cultures at

various stages of growth. The result of the algal-bacteria

population equilibria was expressed in terms of total cell

counts by using the Petroff-Hausser Cell Counting Chamber

(made by C. A. Hausser and Con, Incorporated). The counting

chamber was used to evaluate the population equilibria of

the mixed cultures, to learn the effects of both organisms

on one another, and to find the total number of algal cells,

bacteria, and bacterial spores. These population constituents

were determined at regular intervals after mixing. Control

flasks of axenic bacteria and axenic algae were also prepared

and studied under the same conditions in order to establish

algal and bacterial growth craves. Control flasks. containing

equal volumes of uninnoculated tryptic soy broth and" algal

cultures were also set up for comparison.

Quantitative Biochemical Analysis

In order to determine the amount of different organic

compounds released into the medium during algal cell lysis,

quantitative analysis of carbohydrates, protein,, lipids, and

nucleic acids (AHA and RNA) were performed. The analyses

were also performed on the bacterial and algal control flasks

12

for comparison. Culture to be analyzed were centrifuged ill a

Sorvall Superspeed RC2-B Automatic Refrigerated Centrifuge

and the culture supernatent was used for the following deter-

minations: carbohydrates, protein, lipid, and nucleic acid

determinations.

Carbohydrates De te.vmination

Nelson's alkaline copper reagent was used. It was pre-

pared by mixing 15.2 milliliters of Nelson's reagent A with

0.5 milliliters of Nelson's reagent B. The Nelson's A and B

reagents were prepared as follows:

Nelson's A reagent: dissolve 12.5 g

Na^CO^ (anhydrous), 12.5 g potassium sodium

tartate (anhydrous), 10 g NaHCO^ and 100 g

Na SO (anhydrous) in 350 ml* H O and dilute 2 k 2

to 500 ml.

Nelson1s B reagent: dissolve 7«5 g

CuSO >, . 5H~0 in 50 ml. BL0 and add one drop Jj. C. <L

of concentrated H_S0. . 2 lj.

One milliliter of Nelson's alkaline copper reagent was

added to each sample to be tested and the optical densities

were determined colorime trie ally on a~ Bausch and Lomb

"Spectronic Twenty" (Bausch and Lomb Company) at 51+0 milli-

microns (wave-length). Glucose was used to construct reference

curves of concentration-optical density correlations..

13

Protein Determination

Biuret reagent (Weichselbaum Original Formula, Harle

Company) was used for quantitative determination of protein.

Eight milliliters of Biuret reagent was added to each sample

to be tested and after thirty minutes the optical density

was read at 550 milli-microns. Bovine albumin was used to

construct a standard reference curve.

Lipid Determination

The total lipid content was determined by a gravimetric

method. Lipid extractions of culture fluids were effected

with methanol and chloroform in the ratio of two to one.

After extraction, purification, filtration, and evaporation

to dryness, the amount of lipids was determined gravimetrically.

Nucleic Acid Determination

DNA and RNA were isolated and purified according to the

procedure of Schmidt-Thannhauser (8). Before nucleic acid

estimations could be run, preliminary procedures for the re-

moval of compounds that interfere with the estimation were

necessary. Nucleic acid were extracted in a partially

purified state and finally measured by colorimetry. Figure

One shows a flow-chart of the Schmidt-Thannhauser procedure.

111.

Culture Supernatent

Cold acid extraction

Ac id-s'oluble fraction (small molecules) Lipid solvents

(ethanol : ether)

Lipid fraction (phospholipids)

Acid-soluble

RNA fraction

RNA by phosphorus estimation

Digestion in alkali followed by acidification

Precipitate of DNA and protein

DNA by phosphorus estimation

Pig, 1—Outline of the original Schmidt-Thannhauser pro-cedure for determination of RNA and DNA.

15

Sporulation Studies

Since many observations indicated that algae were

hydrolyzed more rapidly when the bacteria released spores

into the medium, it was thought possible that an enzyme or

an antibiotic substance was present or released from the

bacterial cells and that it was this substance which was re-

sponsible for algal cell lysis. An attempt was made to deter-

mine whether this substance, a bacterial metabolite, was

excreted into the culture medium or whether it was a part of

the intact cells.

The effect of bacterial cell lysates and cell debris on

algal growth was determined by sonifying a sporulating bac-

terial culture with a "Sonifier Coll Disrupter" Model Wl85>D

(Heat Systems-Ultrasonics, Incorporated, Plainsview, New York)

and centrifuging in a Sorvall Superspeed RC2-B Automatic

Refrigerated Centrifuge. The clear supernatent was filtered

through a 0.1 5 micron, millipore grid membrane filter (Nagle

Sybron Company, Rochester, New York) under aseptic conditions,

and it was then mixed at equal volumes with algal cells in

a stationary phase of growth. The sedimented fraction collected

in the centrifuge tube contained whole cells and cell debris.

It was resuspended in distilled water and mixed with algal cells

under the same experimental conditions.

16

Agar Plate Studies

Another attempt was made to elucidate the possible

mechanism by which algal cell hydrolysis was brought about

by bacterial action. In order to determine if some diffusible

bacterial metabolite lyzed algal cells, a streak of algae was

grown across the center of an agar plate with Bacto Algal

Culture Agar (Difco O^hS-01) containing one per cent glucose.

After about four days when the algal cells were growing

luxuriantly on the solid medium, two streaks of bacterial

cells were innoculated by a sterilized cotton swab on both

sides of the algal cells approaching from far to near until

they met the algal cells at the end of the plate as indicated

in Figure Two. This was designed to detect diffusible bac-

terial metabolites whieh might cause algal cell lysis.

Algal

Bacterial Cells

Cells

Bacterial Cells

Pig. 2—Mixed culture of bacterial and algae on Algal culture agar containing one per cent glucose.

CHAPTER BIEL10GRAPHY

1. Breed, R. G., T. D. Murray, and N. R. Smith, Burgey's Manual of Determinative Bacteriology, 7th ed., SaTHiJiore, The Williams and Wilkins Company, 1957.

2. Bold, H. C., Culturing of Algae. Bulletin of Torrey Botanical Club, New York, 19l}-9.

3. Brunei J., G. W. Prescott, and L. H. Tiffany, The Oul-turing of Algae, Washington, The Charles P. Kettering Foundation, 1950*

lj.. Clark, J. M., Jr., Experimental Biochemistry, San Francisco, W. H. Freeman and Company, 1965.

5. Florkin, H. and E. H. Stotz, "Carbohydrates," Comprehensive Biochemistry, Vol. V, New York, Elsevier Publishing Company, 19o3.

6. Florkin, II. and E. H. Stotz,, "Protein/' Comprehensive Biochemistry, Vol. VII, New York, Elsevier Publishing Company, 19o3.

7. Glick. D.. Method of Biochemical Analysis, Vol. II, New York, Interscience Publisher, Incorporated, 1955.

8. Glick, D., Method of Biochemical Analysis, Vol. XIV, New York, Interscience Pub1isher, Incorporated, 1966.

9. Barrel, Steve K., "Growth Inhibition of Chlorella Pyrenoldosa TX71105 "by an Unknown Soil Bacillus, unpublished master's thesis, Department of Biology, North Texas State University, 1968.

10. Kereluk, K., T. Eng, and B. Banishek, "Growth Measurements of Unicellular Algae," Developments in Indus trial Microbiology, 3(August, 1961), 98.

11. Sherman, V. B. D., A Guide to the Identification of the Genera of Bacteria, 2nd ed., Baltimore, The Williams and tfilklns, Company, 1957.

17

1 8

12. Smillie, R. M. and G. Krotkov, "The Estimation of Nucleic Acids in Some Algae and Higher Plants," Canadian Journal of Botany, 38 (January, I960), 31-i+9.

13. Sorokin, G, and J. Myers, "A High-Temperature Strain of Chi ore11a," Science, 117 (March, 1953)# 330-331*

U[. Snell, P. D. and C. T. Snell, Colorimetric Methods of Analysis, Vol. Ill, New Jersey, D. Van NostrancT" Company, Incorporated, 1965.

15. Snell, P. D. and C. T. Snell, Colorimetric Methods of Analysis, Vol. IV, New Jersey, D. Van Nostrand CompanyJ Incorporated, 1965.

16. Wied, G. L., Introduction to Quantitative Cytochemistry, New York, Academic Press, 1966".

CHAPTER III

RESULTS

The aerobic spore-forming bacterium, used in this inves-

tigation, belonged to the genus Bacillus, It was isolated

from the air and identified according to Bergey's Manual of

Determinative Bacteriology, However, the morphological des-

criptions and biochemical properties failed to coincide with

the species descriptions available. The organism was highly

motile in young cultures. It was a large, rod-shaped organism

which formed oval subterminal spores with rather thin spore

walls. Sporangia were not definitely swollen. It formed

rough, radially ridged, elevated mucous colonies on Tryptic

Soy Agar. No acid was formed from mannitol with ammonium ni-

trate as the source of nitrogen and acetylmethylcarbinal was

not produced from glucose. Together with all other biochemical

properties, it was tentatively identified as a variant strain

of Bacillus mageterlum.

As indicated from the results obtained, this bacterium

was found to have an inhibitory effect on algal growth. The

algal cell count decreased as much as fifty to seventy per

cent when algal cell were mixed with bacterial vegetative

cells. At the time of sporulation, algal cell numbers were

19

20

found to drop abruptly within the first fifteen hours and then

decreased to less than five per cent of the original cell

number. Eventually total algal cell lysis occurred accompanied

by gradual decolorization of the culture due to the breaking

down of chlorophyll. Microscopic observations further revealed

that algal cells were destroyed beginning with the loss of

arrangement of the pyrenoids and the loss of other internal

structures. There were large numbers of ruptured cell walls

and much cell debris in the mixed cultures of bacteria and algae.

An attempt was made to determine the population equilibria

of algae and the bacteria in mixed culture. The first work per-

formed was designed to detect the effects of one organism on

the other. This was done by mixing equal volumes of bacteria

in Tryptic Soy Broth at different stages of growth with algal

cells in Bristol's medium at early stationary phases of growth.

The mixed cultures were grown in £00 milliliter Erlenmyer

Flasks on a reciprocal shaker under the experimental conditions

described previously.

The mixed cultures were observed microscopically at con-

stant time intervals and at the same time quantitative esti-

mation of both the vegetative bacteria cells, bacterial spores,

and algal cells were performed by using the Petroff-Hausser

Cell Counting Chamber. Algal and bacterial control flasks

21

served to establish normal growth curves of algae and bacteria

in axenic culture. Control flasks with algal culture mixed

with uninnoculated Tryptic Soy Broth were also set up together

with the mixed cultures. Results obtained indicated that

through the action of the bacteria, definite inhibition on

algal growth occurred as compared with the control flasks.

The algal inhibition increased in amount as a function of in-

cubation time until approximately twenty hours later when the

amount of inhibition leveled off and became constant. It was

also found that more drastic inhibition occurred during the

time when the bacteria formed spores.. This was indicated by

an abrupt drop in the total algal cell count. Microscopic

observations also revealed that algal cell lysis took place

and at the same time algal cell wall debris and decolorized

algal ghost cells were found in the culture medium. The lysis

of cells together with the breaking down of chlorophyll: subse-

quently brought about the ffoloar change in the mixed culture,

which was green at the beginning and: finally turned yellow..

More than ninety-five per cent of the total algal cells were

hydrolysed degraded by the baaterial. action. On the other: hand,

bacterial populations were found to increase by twenty to

thirty per cent as a result of the breaking of algal cells..

This strongly suggested that organic compounds were released

into the medkum as a result of algal, hydrolysis. These, organic

22

molecules were in turn degraded and utilized by bacteria as a

source of energy and nutrients for growth. The first attempt

for series of experiments was designed to detect the time re-

quired for the complete breaking down of algal cells by bac-

terial cultures of different physiological conditions. All

cultures were mixed with equal volumes of algal cells in an

early stationary phase (seventy-hour culture) and bacterial

cells in various stages of growth. The general results of

these experiments were as follows.

Algal and Bacterial Controls

Growth curves were established to indicate the population

dynamics of the algae and bacteria without mixing as. indicated

in Figure Three. Controls were also set up with algal cultures

mixed in equal volumes with uninnoculated Tryptic Soy Broth

and also bacterial cultures mixed in equal amounts with unin-

noculated Bristolrs medium. Results in control algal" mixed

cultures indicated a slight but. no significant rise ih_algal

population. While the control bacterial cultures became in-

active, vegetative bacterial cells declined slowly with a

slow formation of spores.

23

o H

X

•a u <D P, GO rH H <D O <H o u <D •i &

501-

72 96 Time in Hours

Pig, 3—Comparative cell counts of algal' and bacterial controls. Symbols: Bacterial: con-trol; - - - Algal control.

21+

Thirty-Hoar Bacterial Culture

Bacterial culture was grown for thirty hours before

being mixed with algae. In Figure Pour algal cell counts

were found to decline at a slow rate to approximately fifty

per cent of the roiginal amount after twenty hours. Then the

algal cell population began to level offy eventually becoming

fairly constant. Bacterial cells were actively motile. They

were in log phase of growth and finally reached a peak

approximately twenty-five hours after mixing. The total

number of bacterial cells was found to be higher than the

control bacterial cultures. The bacterial cells probably in-

creased due to algal cell substances excreted into the medium

due to algal metabolism or cell lysis. At this time algal

ghost cells were seldom found. The total number of algal

cells was approximately half of that present at the time of

mixing. The color of the medium remained green due to: the

presence of chlorophyll in. surviving algal cells.

Sixty-Hour Bacterial Culture

Bacterial culture was grown for sixty hours before being

mixed with algae. In this stage of bacterial growth, the

mixing of cultures results in. greater cell lysis of: algal

cells. Algal cells as indicated in Figure Five decreased as

a function of time until twenty-five hours after mixing.

There were only about one-third of the original algal: cells

2$

o H X

*6 u o P. m r~4 H <D O

O

0>

Time in Hours

p l g. ^.comparative " l l o ^ of t h i r t y - h o u r ^ a o t e r i ^ A l g a l C e l l s .

Symbols • -

26

O o H X

CO H H <D O CH 0 u <D

1

60

50

r-j s i+o ®

cu 30

20

10

20 30 1+0 Time in Hours

Fig« 5—1Comparative cell counts of mixed culture of sixty-hour bacterial culture and algal culture. Symbols: Bacterial Cells; - - - Algal Cells;

— - Bacterial Spores.

27

left in the cultures. The Inhibition in algal cell numbers

increased to approximately seventy per cent as indicated in

Figure Five. Small amounts of algal cell wall debris and

empty ghost cells started to accumulate. The remaining algal

cells became pleomorphic and there was a loss of sharp de-

lineation of internal structure. At the same time bacterial

cells were less motile and reached stationary phase of growth

after ten hours, which then declined while a few spores be-

gan to form. The formation of spores increased at a constant

rate. More inhibition was found at this time than at the

previous time,

Seventy-Hour Bacterial Culture

Bacterial culture was grown for seventy hours before being

mixed with algae. At this time bacterial spores started to

accumulate and about ten per cent of the cells showed spores

as indicated in Figure Six. Vegetative cells of bacteria were

found to decrease correspondingly. During this time of sporu-

lation, there was an abrupt drop in the number of algal cells

as shown in Figure Seven. This drastic drop was found to take

place in the first ten to fifteen hours after mixing. Approxi-

mately ten per cent of the original number of algal cells

remained in the medium. The scope of this work was not such

that it was felt necessary to determine whether or not these

cells were viable. These last surviving cells disappeared on

50

30

°o

W

£ ©

P< ra H r - i ©

O

o Jn © •Q

20

10 \

/

/

/

\ / /

/ \ \ \ \

28

/

/

4 -0 10 20 30 k0

Time In Hours

50

Pig. 6--Comparative cell counts of mixed culture of seventy-hour bacterial culture and algal culture. Symbols: Bacterial Cells; Algal Cells;

Bacterial Spores.

29

r-i e & d> a to 3

ca & GS bO 3

W a •H O

'U <D C£ <M o

a o *H 4> at Sh -P CJ <D O C o o

100

90

80

70

60

50

^0

30

20

10

/ A % \ \

\

/ - A ' »

0 10 $ 1±= 0 30 i+'o ^jcr-\ . Time in Houjsa- ' "

T o

Pig« 7—Comparative determinations of the amount of reducing sugars present in the cell-free media of the algal control, bacterial control, and mixed culture. Symbols: Bacterial control; - - - Algal control; Mixed culture; — . -Net curve.

30

prolonged incubation, that is, twenty-four or more hours.

More decolorized algal ghost cells and cell wall debris were

observed in these cultures than in the ones described pre-

viously. The hydrolysis of algal cells was perfectly corre-

lated with the changed of color of the cultures from green to

pale atraw yellow. This suggested the degradation of

chlorophyll by the actions of the bacterial cells. The great

majority of algal cells were hydrolyzed within fifteen hours.

It was believed that either an enzyme or some antibiotic

substance was formed during the sporulation process and that

this substance was capable of inhibition algal growth. It

was also assumed that this or some other substance eventually

brought about algal cell lysis.

Eighty-Six Hour Bacterial Culture

Bacterial culture was grown for eighty-six hours before

being mixed with algae. At this time of mixing more than sixty

per cent of the bacteria had formed spores and some of the

spores were released into the medium together with the ruptured

vegetative cell wall materials. Algal cell destruction and

population were found to be less than fifty per cent of the

original amount. We infer from this that the active substance

in cell lysis is formed at the time of sporulation and that

it does not last long in active form in the bacterial culture.

31

Biochemical Analysis

It was assumed that if algal cells were being broken

down in the mixed cultures, organic substances contained in

the algal cells would be released into the culture medium.

An attempt was made to detect the organic compounds present

in the culture fluid as a result of lysis. Quantitative

measurements of carbohydrates, protein, lipids, and nucleic

acids (DNA and RNA) were made in cell-free filtrates of

mixed cultures. The same determinations were also performed

in the control flasks of bacteria and algae and used as a

basis for comparison.

Carbohydrate Determinations

The amount of carbohydrate was r.nalyzed in terms of re-

ducing sugars in the culture fluid of Nelson's reagent. The

results indicated in Figure Seven show the carbohydrate con-

tent on the mixed culture, bacterial control, and algal control.

In order to correlate the relationships between the mixed cul-

ture and the controls, it was necessary to draw a curve showing

the net change. This is done by substracting half the sum of

the sugar content of the bacterial and algal controls from that

of the mixed culture at each interval, because the mixed cul-

ture contained bacterial and algal cultures in equal amounts.

Prom the net curve, the amount of reducing sugars was found

to increase constantly from zero hour onward reaching a peak

32

at fifteen hours, after which it decreased at a fast rate.

The net amount of reducing sugars decreased below the zero

point as indicated in the net curve showed that more reducing

sugars were detected in the bacterial and algal controls than

in the mixed culture. When compared to the population curves

in Figure Six, the time at which carbohydrates were released

during algal cell lysis corresponded closely to the time when

there was an exponential increase in bacterial population.

This strongly suggested that during algal cell lysis sugars

were released into the medium, which then were utilized by

the bacteria as a source of energy and carbon for growth.

It was possible that a greater amount of the sugars released

were being utilised for tho support of bacterial growth as

indicated by a twenty-thirty per cent increase in bacterial

vegetative cells in comparison to the controls.

Protein Determination

Similar results were obtained when proteins were measured,

A net curve as shown in Figure Eight indicated that the amount

of protein increased constantly from the fifth hour onward

reaching its peak at fifteen hours. This further suggested

the algal hydrolysis brought the release of protein molecules

into the medium.

33

E5 •H <D •P O Pn

o d c -P cd & -p C © o d o o

1.0

H s ©

0.8

0.6

o.k

0.2

\

/•

0^7

/

/ W

\

N TO 30

Time in Hours •fe i 0 60

Pig. 8--Comparative determination of the amount of protein present in the cell-free media of the algal control, bacterial control, and mixed culture. Symbols: — Bacterial con~ trol; - - - Algal control; Mixed cul-ture;— Net curve.

%

Nucleic Acid Determination DMA and RNA

In Figure Nine no detectable DNA or RNA were found in

either bacterial or algal control cultures. On the other

hand, mixed cultures showed small amounts of RNA in the

supernatant fluid at approximately fifteen hours after mixing,

and increased until twenty hours. No DNA was detected in

these mixed cultures. This indicated to the author that RNA

was released as a result of algal cell lysis. This RNA was

probably degraded or metabolized by the bacteria since it

eventually disappeared from the medium.

Lipid Determination

No detectable lipid was found in tho flasks used as algal

control, while the net curve indicated that lipid was released

and accumulated in the medium at a constant rate after mixing

with bacteria as seen in Figure Ten. The amount of lipid in-

creased and reached a peak at twenty hours after mixing,, which

again decreased at a constant rate. The curve indicated that

lipid was released as a result of algal hydrolysis and then

being metabolized for bacterial growth.

The next experiment, in which sporulating bacterial

cultures were centrifuged, washed, and sonified,, was designed

to show whether algal lysis was caused by the bacterial

metabolites released into the medium or by particulate bac-

terial materials. The supernatant fluid and the sediraented:

35

1.0

<D Pi w 3L

0#8

m 3 U O A P* CQ O A SU u o d o •H •P 0$ •P d <D O a o o

0*6

0#i;

0.2

0 10 20 30

Time in Hours

o 60 70

Pig. 9—Comparative determination of nucleic acids in the cell-free media of algal control, bacterial control, and mixed culture. No detectable DNA in all determinations. No detectable RNA in control cultures * Symbols:. Phosphorus in RNA of mixed culture.

3&

<D 94

& •H

*0 *r4 Pk a

o d o •r-t -P a$ u -p d o & o o

0*2

\

0.1

/ \

V /

20 30 Time in Hours

Pi^ 10—-Comparative determination of the amount of litod*in the cell-free media of algal c°n^r0J>

control, and mixed culture. Ho detectable

. e g i 1 i s : r k i t r ; - l s - - ^ = ^ e t

37

cell materials were mixed separately with algal cultures.

Results indicated that the particulate fraction contained the

active substance which was responsible for algal cell hydrol-

ysis. Cell counts revealed that algal cells decreased to

less than ten per cent of the original cell number in twelve

hours time after mixing with the sedimented cell debris. The

remaining algal cells were found to be damaged as indicated

by degradation of internal arrangement of cell organelles.

Empty algal ghost cells were also present exactly as seen in

all mixed cultures. On the other hand, only a very slight

inhibitory effect for algal cells was found in the supernatant

fraction.

The result of experiments on mixed cultures on a solid

medium showed that no inhibitory material was secreted and

diffused through the agar to cause algal cell hydrolysis. How-

ever, at the place of contact where the bacterial and algal

cells were growing together, it was plainly evident that the

algal cells were hydralyzed in. the same manner as in .mixed

cultures. The algal cells became yellow and abnormal. Micro-

scopic observations also indicated' that the chlorophyll

materials of the algal cells were being degraded and empty

algal ghost cells were found.. This zone of inhibition was

manifested by a region of yellow algal cells which could be

easily observed by visual appearance.

CHAPTER IV

DISCUSSION

The most pertinent fact which was revealed by this work

is the fact that this variant strain of Bacillus megaterium

is capable of significantly inhibiting algal growth during

the course of its growth and bringing about complete algal

cell lysis as a result of aporulation. Ward, Moyer, and

Vela (5) reported that up to a twenty per cent loss in the

production of algal cell mass was caused by specific bac-

teriaj however, this loss was slight when compared with the

nearly one-hundred per cent growth inhibition caused by the

bacterium described here.

As a result of algal degradation by bacterial action,

it was thought possible that algal structural and functional

raacromolecules were released into the culture medium. It

was, thus, not surprising to detect the presence of these

organic materials as carbohydrates, protein, lipids, and

nucleic acids. These macromolecules, in turn, served as

substrate leading to an exponential increase in bacterial

population. It was very possible that a greater- amount of

these organic materials was released than what could be de-

tected by the experiments described here. It-is reasonable

38

39

to assume that these substances were consumed and degraded by

the bacteria immediately after being released from algal cells.

If there is some method by which these algal degradation pro-

ducts can be protected from immediate consumption by the

bacteria, it is postulated that these organic rich materials

may be made to serve as a source of food for animals,

The problems and techniques of mass culture of algae have

been under active study in recent years because of the possible

role of these algae as a food supply for the expanding popu-

lation of today. Other than the technical problems of mass

production of algal cells in which scientists are interested

today, the problem of indigestability is a major concern.

Since algal cells are excellent sources of food in that they

are rich in carbohydrates, protein, and lipid, the major pro-

blem for feeding animals lies in the difficulty of digestion

of algal cell walls, chlorophyl, and nucleic acids. If there

is some way that these macromolecules of algal components can

be partially degraded, then animals may be able to utilize

the remaining organic compounds as a source of energy for

growth. Attempts were made to investigate the role of the

bacteria in causing inhibition, and also the possible mechanism

in which algal hydrolysis was caused. It is interesting to

note that as the result of the sporulating activities of these

bacteria, algal cells were being hydrolyzed and degraded to

2*0

smaller molecules. As Indicated from the results, it is

possible that a particle bound enzyme or antibiotic substance

which is responsible for algal cell destruction is formed

during the sporulating process. The experiment in which the

sedimented cell fragments of the bacteria were mixed with

algal cells indicated that this enzyme in antibiotic sub-

stances is located mainly on the intact cells as a cell

component as indicated by its biological activity. We

postulate that a whole enzyme system is involved because of

the multiplicity of activities observed. A slight inhibitory

effect was also found in the supernatant fraction which

showed: (1) small amounts of this substance in a soluble

form, or (2) the incomplete removal of particulate materials

by centrifugation. The latter is more likely to occur. The

results on mixed cultures on agar plates further supported

this observation. No detectable inhibitory substance was

found to diffuse across the gel medium to cause algal inhi-

bition and hydrolysis. Yet, at the place where bacterial and

algal cells met, drastic algal cell lysis was observed. Thus,

concluding from the above results, it is probable that this

algal cell-lysis substance is formed during the metabolic

changes that takes place during the sporulation process of

the bacterium, and that it remains as a structural part of the

cell component. Sporogenesis is accompanied by major shifts

in the number and kinds of proteins synthesized by the cell.

la

Some of these are associated with the mechanism governing

the production of the spores and some are components of the

spores themselves.

It is also reported that certain enzymes are found pre-

sent in spores, but undetectable in vegetative cells (i+).

These enzymes are induced during sporulation. It is also

possible that the vegative cells of this variant strain,

Bacillus megaterlum, contains an enzyme which is responsible

for algal hydrolysis. The enzyme locates in a part of the

cell wall material as a structural component. Then this

enzyme is set free along with the vegetative cell component

as a result of rupture of the sporangia during sporulation.

Thus, it was thought logical that the filuered supernatant

extract of the bacterial culture did not contain this inhi-

bitory substance, while the sedimented cell materials con-

tained this enzyme system and, thus, brought about algal cell

hydrolysis.

Prom the light of this experiment, it is possible to

postulate the pre-treatment of algal cells through degradation

by bacterial enzyme action before it is fed to animals. Of.

course, further investigation and research is needed in order

to understand the nature and. role of this enzyme before it can

be of any practical value.

L L 2

There are several useful applications to which these

criteria can be used, A productive use for this bacteria is

its possible role in controlling the algal bloom in water

supplies, stock tanks, or any other body of water in which

prolific algal growth is a problem. Since this bacteria,

which was found to have a detrimental effect on algal growth,

is an air isolate, it is very possible that there is a. great

chance of transmission of spores through the air and conco-

mitant region-wide control of algal bloom. Algal bloom can

be a serious problem causing utidersirable odors in water

supply systems. It also can distrub the ecoligical equilibria

of a given environment. These bacteria are capable of con-

trolling algal growth through natural contamination or by

artificial means of introduction to the system.

Since it took quite a large number of bacteria in order

to cause complete algal cell lysis in an open environment,

the bacterial population is not liable to increase to an ex-

tent to cause complete algae degradation and subsequently in-

duce the upset of the equilibria of the ecological system.

On the other hand, an undesirable effect may be brought

about by the contamination of algal cultures in "closed

ecological systems" by this bacterium., The success of- algae,

as gas exchangers and as a source of food depends on their

ability to grow and reproduce• If this is affected they

U3

cease to function. Such systems are usually considered in

the design for the support of human life in space craft or

in nuclear submarines. The work which has been performed

to date seems to indicate that in a closed environmental

system, which makes use of Chlorella, there is a definite

possibility of bacterial inhibition of algal growth by this

bacterium or related organisms. Since it is an aerobic,

spore-forming organism found in the air, it is highly capable

of contaminating algal cultures. This fact would seem to

necessitate strict control of the environment in life support

systems which make use of algae as a precaution against

bacterial contamination.

CHAPTER BIBLIOGRAPHY

1. Campbell, L. L, and H. 0. Halvorson, Spores IIIt A Report of the Symposium of the American Society for Microbiology, Allerton Park, Illinois, University of Illinois and Office of Naval Research, 1961*.

2. Holvorson, H. 0., Spores II, A Report of the Symposium of the American Society for Microbiology, Allerton Park, Illinois, University of Illinois and Office of Naval Research, I960.

3. Mandelstam, J, and K. McQ,uillen, Biochemistry of Bacterial Growth, 1st ed., Edinburgh, Blackwell scientific Publications, 1968.

Ij.. Rose, A. H. and J. P. Wilkinson, Advances in Microbial Physiology. Vol. I, New York, Academic Press, 1967.

5. Ward, C. II., J. E. Meyer, and G«. Rs Vol*, "Studies on Bacteria with Chlorella pyrenoidosa TX71105 in Mass Culturet" Developments in Industrial Microbiology. 6 (August, 196ll),^l>2'52.~--

\

TX. ~-r: - ° ;-j • ' " 0.;vv:,/s 6aiC^W!y5

1 ;7/ / > " *'-''' l»'' * ' " / V V'

i ' J < ; D's fc?i '(.">/•'''! -•' {.u ) ^

x-> "-'ni*

CHAPTER V

SUMMARY

Studies of bacterial contamination in mass cultures of

Chlorella pyrenoidosa TX71105 have been reported by previous

investigators. Especially noteworthy are the works of

Sorokin and Myers (5)» Krauss and Thomas (3), Ward, Moyer,

and Vela (7), Vela and Guerra (6), and Blasco (1). These

reports revealed that bacterial growth has only slight or

no significant effect on algal growth. On the other hand,

Ward, Moyer, and Vela (7) reported a decrease of as much as

twenty per cent in algal cell yield as a result of heavy con-

tamination by selected bacteria. Vela and Guerra (6) subse-

quently reported that only a very few bacteria isolated at

random can grow extensively In cultures of Chlorella

pyrenoidoaa TX71105, and none of these had the capacity to

inhibit the algal growth significantly. The work of: Fogg (2),

Krauss and Thomas (3)» and also that of Vela and Guerra (6)

showed that bacterial growth results from the utilization of

algal metabolites excreted into the medium. This paper is a

report of a study of mixed cultures of Chlorella pyrenoidosa

TX71105 and an organism, isolated from the air. and tentatively

identified as a variant strain, of Bacillus me^aterium. This

145

k-6

bacterium appears to inhibit algal growth and bringjs about

algal cell lysis when it forms spores.

Bacterial cultures at different stages of growth were

mixed with algal cells at stationary phase. Microscopic

observations and population dynamics indicated algal cells

were inhibited significantly by vegetative cells of the

bacteria. A drastic effect in which algal cell lysis was

obsered during the sporulating process of the bacteria.

Biochemical analys s were performed to determine the

amount of carbohydrates, protein, lipid, and nucleic acids

being released as a result of algal hydrolysis. Results in-

dicated that these organic materials were being released and

consumed immediately as substrates for bacterial growth.

An enzyme or antibiotic substance found associated

closely to particulate cell components, is believed to be

responsible for causing the algal hydrolysis.. This enzyme

may be present either on the vegetative cells and released

together with cell materials during the r upture of sporangium

in sporulation, or an adaptive enzyme formed during the

metabolic changes which occurred during sporulation. It is

felt, however, that further investigation, and research are

needed to elucidate the possible mechanisms involved.

CHAPTER BIBLIOGRAPHY

1. Blasco, R. J., "Nature and Role of Bacterial Contaminants in Mass Culture of Thermophilic Chlorella pyrenoidosa," Applied Microbiology, 13 ("September, 1963), 473-U75.

2. Fogg, G. E., "Extracellular Products," Physiology and Biochemistry of Algae, edited by Ralph A. Lewin, New York, Academic Press, 1962,

3. Rrauss, R. W#, "Mass Culture of Algae for Pood and Other Inorganic Compounds," American Journal of Botany, i|9 (November, 1962), l\.25-k3>$»

I}.. Lewin, R. A., Physiology of Algae, New York, Academic Press, 19&2.

5. Sorokin, C. and J, Myers, "A High-Temperature Strain of Chlorella," Science, 117 (March, 1953), 330-331.

6. Vela, G, R. and C. N. Guerra, "On the Nature of Mixed Cultures of Chlorella pyrenoidosa TX71105> and Various Bacteri'aT" Journal of General Microbiology, I4.2 (June, 1966), 123-131.

7. Ward, C. H,, J. E. Moyer, and G» R, Vela, "Studies on Bacteria Associated with Chlorella pyrenoidosa TX71105 in Mass Culture," Developments in Industrial Microbiology, 6 (August, 196i|), 213-222,

BIBLIOGRAPHY"

Books

Breed, R. G., T. D. Murray, and N. R. Smith, Burgey* s Manual of Determinative Bacteriology, Jth e d . , Baltimore, iiie wixxxtuiiis anu ui.lri.iua Company, 1957»

Casida, L. R., Jr., Industrial Microbiology, New York, John Wiley and Sons, Inc., i960.

Clark, J. M,, Jr., Experimental Biochemistry, San Francisco, W. H. Freeman and Company, 1965.

Fogg, G. E., "Extracellular Products," Physiology and Biochemistry of Algae, edited by Ralph A. Lewin, New York, Academic Press, 1962.

Mandelstam, J. and K. McQuillen, Biochemlstry of Bacterial Growth, 1st ed., Edinburgh, Blackwell Scientific Publications, '1966,

Sherman, V. B. D., A Guide t_o the Identification of the Genera of Bacteria, 2nd ed., Baltimore, The Williams and Wilkins, Company, 1957.

Wied, G, L., Introduction to Quantitative Cytochemistry, New York, Academic Press, 1966,

Articles

Blasco, R. J., "Nature and Role of Bacterial Contaminants in Mass Culture of Thermophilic Chlorella pyrenoidosa," Applied Microbiology. lfTSeptember. 1963) , \ 73 - l+75 .

Brown, L. R., M. V. Kenedy, and R^ G. Tischer, "An Algal Medium Produced from Human Wastes," Developments in Industrial Microbiology. 6 (August, 1964), 255-2^9.

1*8

49

Eley, J. H. and J. Myers, "Study of a Photosynthetic Gas Exchanger, a Quantitative Repetition of the Priestley Experiment," Texas Journal of Science, 16 (July, 1961+), 296.

Jorgensen, E. G., "Antibiotic Substances from Cells and Culture Solutions of Unicellular Algae with Special Reference to Some Chlorophyll Derivatives," Physiologia Plantarum, 15 (May, 1962), 530-545.

Kereluk, K., T. Eng, and B. Banishek, "Growth Measurements of Unicellular Algae,11 Developments in Industrial Microbiology, 3 (August, 1961), 9B,

Krauss, R. W., "Mass Culture of Algae for Pood and Other Inorganic Compounds," American Journal of Botany, i|9 (November, 1962), l|25-i|33«

Krauss, R„ ¥. and W. H. Thomas, "The Growth and Inorganic Nutrition of Scenedesmus obliquus in Mass Culture," Plant Physiology, 29 (May, 195U), 205-21i|.

Lancaster, J. H., R. G. Tischer, and R. Z. Long, "Human Feces as a Nitrogen Source for some Green Algae," Develonments in Industrial Microbiology,, 3 (August, 1^)71^-34. *.

Mayers, A., M. V. Zuri, Y. Shain, and H. Ginzburg, "Problem of Design and Ecological Considerations in Mass Culture of Algae," Biotechnlcal Bioengineering, 6 (May, 1961}.), 173-179.

Myers, J., "Physiology of the Algae," Annual Review of Microbiology, 5 (March, 1951)> 157-THO.

Myers, J., J. N, Philips, Jr. and J. R, Graham, "On the Mass Culture of Algae," Plant Physiology, 26 (June, 1961), 539-549.

Philips, J. N,, Jr., "Closed Ecological System for Space and Extraterrestrial Habitation," Developments in Industrial Microbiology, 3 (August, 1961), 5-13.

Schmidt, W, E. and J. Verduin, "Mass-Culture-Produced Algae as a Pood Source," Symposium; Microorganisms as Potential Food Sources, 177 [January, I960), 2f?07

5o

Smillie, R. M. and G. Krotkov, "The Estimation of Nucleic Acids in Some Algae and Higher Plants," Canadian Journal of Botany, 38 (January, I960), 31—i+9•

Smith. H. C,, H. E. Brown, J. E. Moyer, and C. H. Ward, "Some Excretory Products of Chlorella pyrenoid osa by Selected Bacteria," Developments in Industrial Microbiology, 9 (August, 1967), 363-3^9.

Smith. H. C., H. E. Brown, J. E. Moyer, and C. H, Ward, "Utilization of Excretory Products of Chlorella pyrenoidosa,11 Developments in muusoriai i-iiui-uulo--logy, 9 (August, 1967j, 3T5* 362T

Sorokin, C. and J, Myers, "A High-temperature Strain of Chlorella," Science, 117 (March, 1953), 330-331.

Tischer, G. R., B, B. Tischer, and D, Cook, "Human Feces as a Nitrogen for Algae in Closed Space Ecologies," Developments in Industrial Microbiology, 3 (August, T961),^2T55.

Vela, G. R» and C, N. Guerra, "On the Nature of Mixed Cultures of Chlorella pyrenoidosa TX 71105 and Various Bacterid," Joi*T»nn1 of General Microbiology, lj.2 (June, 1966), 123-131;

Ward, C, H., J, E. Moyer, and G. R. Vela, "Studies on Bacteria Associated with Qhlore11a pyrenoidosa TX 71105 in Mass Culture," Developments in Industrial Micro-biology, 6 (August, 1961).), 213-25?.

Winders, W. H, and R. G, Tischer, "The Nutritive Value for Algae of Pecal Pyrolysis Gas is," Developments in Industrial Microbiology, 3 (August, 1961), II4.-2I47

Zuraw, E. A., "Algae-Primate Gas Exchange in a Closed Gas System, Developments in Industrial Microbiology, 3 (August, 1961), 11+0-11+9.

Reports

Bold, H. C., Culturing of Algae, Bulletin of Torrey Botanical Club, New York, 19l|9.

51

Brunei, J., G. W. Prescott, and L. H. Tiffany, The Cult tiring of Algae, Washington, The Charles P. Kettering Foundation, 135^7

Campbell, L. L. and H. 0, Halvorson, Spores III, A Report of the Symposium of the American Society For Microbiology, Allerton Park, Illinois, University of Illinois and Office of Naval Research, I96I4..

Podor, G. Y., G. Racz, and K. Bedo, Determination of the Nutritional Value of Green Algae, Proceedings o7 the Fouriii uongr"esT"of"~vne"Hungarian Association 01 wicro-biology, Budapest, 19614-.

Holvorson, H. 0,, Spores II, A Report of the Symposium of the American Society for~Hicrobiology, Allerton Park, Illinois, University of Illinois and Office of Naval Research, I960.

Szekely, K., Semiautomatic Equipment for Mass Cultivation of Protophytic Pres~h V / a t e r Algae, Proceedings of The Fourth Congress of the Hungarian Association of Microbiology, Budapest, 196l|.

Szekely, K., S. Bedo, B. Sedo, and G. Y. Podor, Studies on the .Olgfestabiliuy or Pr-otophytic Algae i.; afreets of Digestive Enzymes on pried Algae in Vivo, Proceedings of the Fourth Congress of the Hungarian Association of Microbiology, Budapest, 196^.

Szekely, K., A, Eperjessy, M. Kerebes, and M. Bedo, Studies on the Digestability of Unicellular Algae II; In Vitro ETfects of Proteolytic Enzymes' on Dried 'Algae, "Pro-ceedings of" the Fourth Congress of the Hungarian Association of Microbiology, Budapest, 1961+.

Encyclopedia Articles

Glick, D., Method of Biochemical Analysis, Vol. II, New York, Interscience Publisher, Incorporated, 1955.

Glick, D., Method of Biochemical Analysis, Vol. XIV, New York, Interscience Publisher, Incorporated, 1966.

Rose, A. H., and J. P. Wilkinson, Advances in Microbial Physiology. Vol. I, New York, Academic Press, 1967.

£2

Snell, P. D. and C. T. Snell, Colorimetrlc Methods of Analysis, Vol. Ill, New Jersey, D. Van Nostrand Company, Incorporated, 1965.

Snell, P. D. and C. T. Snell, Colorimetrie Methods of Analysis, Vol. IV, New Jersey, D. Van Nostrand Company, Incorporated, 196£.

Stotz, E. H. and H. Plorkin, "Carbohydrates," Comprehensive Biochemistry, Vol. V, New York, Elsevier Publishing Company, 19o3.

Stotz, E. H. and H. Plorkin, "Protein," Comprehensive Biochemistry, Vol. Vii, New York, Elsevier Publishing Company, 19&3.

Unpublished Materials

Harrel, Steve K., "Growth Inhibition of Chlorella pyrenoidosa TX71105 by an Unknown Soil Bacillus," unpublished master's thesis, Department of Biology, North Texas State University, 1968.