SELECTED PHYSIOLOGICAL AND BIOCHEMICAL STUDIES

ON BLUE-GREEN ALGAE

APPROVED:

Graduate Committeea

Major Professor

Minor Professor

it Committee Member

'A>f Committee Member \J V^lIUU-i- \J \J O O vJ X

Commttee Member

Directorof the Department of Biology

Dean or the Graduate School

SELECTED PHYSIOLOGICAL Ml) BIOCHEMICAL STUDIES

ON BLUE-GREEN ALGAE

DISSERTATION

Presented to the Graduate Council of the

North Texas State University in Partial

Fulfillment of the Requirements

For the Degree of

DOCTOR OF PHILOSOPHY

By

Jimmy T. Wyatt, B, S., M.'S,

Denton, Texas

August, 1969

TABLE OF CONTENTS

Page LIST OF TABLES . . . . . . . . . . . . . iv

LIST OF ILLUSTRATIONS . . . . . . . . . . . . v

Chapter

I. INTRODUCTION 1

II. MATERIALS AND METHODS 17

Isolation Purification Culture Growth Rate Heterotrophy Odor Production Nitrogen Fixation Respiration and Photosynthesis

III. RESULTS AND DISCUSSION . . . . . 35

IV. CONCLUSION 69

LITERATURE CITED 74

LIST OF TABLES

6.

Table

1. Composition of ASM-1 in g/l

2. List of Blue-Green Algal Species Investigated

3. Free-Living Nitrogen-Fixing Bacteria Utilized

4. Environmental Distribution of Isolates . . .

5. Axenic Cultures

Ethylene Production in jf795 Gloeocapsa sp. and Reference Organisms

7. Acetylene Reduction Rates in Selected Nitrogen Fixing Species

Page

, 24

, 25

. 32

• 36

, 41

• 55

8. Respiration and Photosynthesis

60

65

IV

LIST OF ILLUSTRATIONS

Figure Page

1. Growth of #625 Anacystis nldulans, #4$6 Nostoc museorum and #NT6B-$ Oscillatoria sp. . . . '46

• 2. Growth of $617 Symploca muscorum, #5$4 Nostoc commune and"#484 Oscillatoria tenuis . . . . 47

3. Acetylene Reduction Rates after Nitrogenase Initiation 56

4. Acetylene Reduction Rates after Nitrogenase Initiation 57

5. "Dark Bottle" Acetylene Reduction Rate Decrease by Pre-Dark Treatment . . . . . 6l

6. "Dark Bottle" Acetylene Reduction Rate Decrease by Pre-Dark Treatment 62

v

CHAPTER I

INTRODUCTION

Most of the published information on the physiology and

biochemistry of blue-green algae has been derived from a mere

handful of species. For example, a somewhat selective, yet

comprehensive survey of literature directly or indirectly

concerning the metabolism of blue-green algae indicated that

most of the papers refer to eight organisms. These are

Anabaena variabilis (14 papers), Anabaena cylindrlca (31 pa-

pers) , Anabaena flos-aquae (13 papers), Nostoc museorum

(25 papers), Chlorogloea frltschii |~= Nostoc sp. , (Fay et al.,

19640 (& papers), Tolypothrlx tenuis (15 papers), Anacystis

nidulans (15 papers), and Microcystis aeruginosa £= Anacystis

cyanea, (Drouet and Daily, 1956) 1 (18 papers). • Of these,

all but the latter two are nitrogen fixers. The ability to

fix nitrogen, and other traits to be discussed later, have

played an extremely important role in the selection of this

group as experimental organisms.

The literature survey covered a span of about eighty

years, but the majority of the papers reviewed are relatively

recent, because advances in the study of blue-green algal

physiology and biochemistry have closely paralleled the vastly

improved bacteriological and sterile techniques that developed

during the latter part of the nineteenth century. .These

developments provided the necessary technical improvements

in isolation, culture, and purification which were necessary

for meaningful investigations of blue-green algal metabolism.

The fact that certain algae grew and maintained them-

selves in situations which seemed to be devoid of combined

nitrogen was noted prior to 1900. This observation provoked

a great deal of interest among early investigators, and in

1$$9> Frank suggested that some blue-green algae were able

to utilize atmospheric nitrogen. Because Beijerinck (1901)

used impure cultures when he obtained evidence of nitrogen

fixation by blue-green algae, he was unable to establish his

observations unequivocally. Pringsheim (1913, 1914) was one

of the first to successfully maintain experimental cultures

of blue-green over extended periods of time, and probably

achieved the first pure cultures of blue-green algae. Devel-

opment of axenic culture techniques continued, and two inde-

pendent investigations (Drewes, 1928; Allison and Morris,

1930) established irrevocable proof of atmospheric fixation

in blue-green algae. For the next decade most of the work'

concerning blue-green algal physiology was either directly

or indirectly concerned with nitrogen fixation (for example,

see Allison and Morris, 1932; Allison and Hoover, 1935;

Winter, 1935; Allison et al., 1937; De, 1939)> and to some

extent this trend continues today.

The influence of experimental inquiries into nitrogen

fixation by blue-green algae has strongly determined the

choice of experimental material and the resultant course of

investigation followed. Even a cursory literature survey

reveals that the choice of experimental organisms has closely

followed publication of stimulating research relative to that

organism,or, what is perhaps unfortunate, the chance purifi-

cation of an organism by some investigator who was willing

to make the purified organism widely available. Since most

of the papers dealing with the aforementioned species con-

cern strains that were more or less random isolates of what

must be literally hundreds or thousands of different strains,

one cannot keep from speculating as to how different the

information might be today if the original isolator had

selected another sample or clone. As will be discussed

later, this paper will note results of moderate inquiries

into this subject. Research upon these previously mentioned

species has truly exerted a disproportionate influence upon

what is known today of the physiology and biochemistry of

blue-green algae. Perhaps this might best be illustrated

by a brief resume of the work published about each organism.

Anabaena variabilis: In spite of being a relatively

•inconspicuous and common soil dweller, Anabaena variabilis

has enjoyed a rather favorable position in scientific en-

deavour. It has the distinction of being one of the organ-

isms used for the first conclusive demonstration of nitrogen

fixation in blue-green algae (Drewes, 1928). Study of its

role in relation to soil fertility (De, 1939), and its exten-

sive use as a test organism to demonstrate molybdenum re-

quirements in blue-green algae (Bortels, 1940) have produced

an early and continuing interest in this species.

Its respiratory characteristics have been studied (Brown

and Webster, 1953; Webster and Frenkel, 1953), and it has

been widely used in organic uptake experiments (Hoare and

Moore, 1965; Pearce and Carr, 1966, 1967; Carr and Pearce,

1966). Some selected aspects of its phosphate metabolism

have also been investigated (Petrack, 1959; Carr and Sandhu,

1966). Finally, it has been used in such widely diverse

studies as chromatin distribution (Leak and Wilson, i960),

extracellular production of amino acids (Venkataraman and

Saxena, 1963) and adaptability to hydroxylamine (Jain, 1967).

5

Anabaena cylindrica: Conspicuously absent from species

accounts in some popular phycological manuals (Smith, 1950;

Desikachary, 1959; Prescott, 1962) but listed by Geitler,

(1932), Anabaena cylindrica is one of the most vigorous of

all blue-green algal species tested to date. It was isolated

by S. P. Chu from a garden pond and laboriously purified by

G. E. Fogg (Fogg, 1942). Fogg's several intensively illumi-

nating and exacting investigations (1942, 1944, 1949, 1951a,

1952) have perhaps made Anabaena cylindrica the most widely

familiar of all blue-green algae.

This alga's nearly unique adaptability to almost any

remotely suitable growth condition and its tendency-to grow

as hormogones without extensive clumping have made it perhaps

the most studied of all blue-green algae. Its responses to

varied mineral and nutritional levels have been investigated

(Wolfe, 1954a, 1954b; Allen and Arnon, 1955a, 1955b) and its

effective responses to different light intensities have been

utilized in photometabolic investigations (Fogg and Than-Tun,

195$) I960; Cobb and Myers, 1961, 1964).- A superficial ex-

amination of an aged culture will reveal that it has been

an efficient producer of extracellular and cellular products.

This production has been studied by Fowden (1951), Fogg (1952),

Bishop et al,, (1954)> Whitton (1965), and Walsby (1965).

cylindrica has been widely used to produce cell-free

preparations {Schneider et al,, I960; Cox et al., 1964;

Fujita et al., 1964; Fujita and Myers, 1964; Fay and Cox,

1967) for diverse types of enzymic or particulate-fraction

investigations. Its glucose metabolism has been investi-

gated by Wildon and ap Rees (1964)? and its nuclear fine-

structure has been studied by Hopwood and Glauert (i960).

Since it can be induced to produce akinetes and hetero-

cysts in great quantities, A. cylindrica has been used in

studies hopefully designed to solve the "heterocyst enigma"

{Talpasayi, 1962, 1967; Fay and Walsby, 1966) and has been

used as the subject of Wolkfs (1965, 1966, 1967, 1963) ex-

tensive studies of growth, sporulation, and heterocyst devel-

opment .

Anabaena flos-aquae: This planktonic "bloom"-former

was probably first studied in conjunction with toxic water-

blooms (Deem and Thorp, 1939; Rose, 1953; Olson, I960), and

resultant investigations into its toxicity (Gorham, 1962,

1964; Gorham et al., 1964) provided much of the initial

stimulation and interest in the physiology and biochemistry

of this algae. Anabaena flos-aquae was probably initially

purified by Tischer (1965), who studied its extracellular

polysaccharides (Moore and Tischer, 1964, 1965) and its

7

ability to cause photoreduction of nitrogen (Davis and

Tiseher, 1966). Anabaena fios-aquae has also been investi-

gated with respect to its nitrogen nutrition (Davis et al.,

1966; Mickelson et al., 196?) and its response to various

levels of sodium and potassium in the growth medium (Bostwick

et al., 1968).

Nostoc muscorum: Although originally thought to be

Anabaena variabilis (Allison and Morris, 1930), Nostoc

muscorum (Allison et al., 1937) was first isolated into

unialgal culture in the late 1920's, eventually purified,

and shown to fix nitrogen. It was used as the experimental

organism in much of the original work on nitrogen fixation

here in the United States (Allison and Morris, 1930, 1932;

Allison and Hoover, 1935; Allison et al., 1937). After

!!• muscorum's nitrogen-fixing qualities had been studied,

it was investigated with respect to its nitrogen-fixing

enzyme system (Burris and Wilson, 1946) and in regard to its

nitrogen nutrition in a later study (Magee and Burris, 1954).

Allison et al. (1953, 1954) conducted organic feeding

experiments; and in a widely quoted and perhaps very signif-

icant paper, Linko et al. (1957) described, its uptake.

Its mineral nutrition has been investigated extensively by

Eyster (1952, 1958, 1959). Nostoc muscorum seldom exhibits

a r

the continuously elevated metabolic rates of some other

blue-green species investigated, but certain of its photo-

synthetic aspects have been studied (Clendenning et al.,

1956; Clendenning and Brown, 1956), and its extracellular

products have been the subject of studies by Jakob (1954,

1957, 1961), Rzhanova (1967), and. Biswas (1957). Finally,

its developmental cycles have been the subject of rather

lengthy and involved experiments (Lazaroff, 1955, 1966;

Lazaroff and Vishniac, 1961, 1962; Lazaroff and Schiff,

1962). Its ornithine-cycle enzym.es were studied by Holm-

Hansen and Brown (1963).

Chlorogloea fritschii: Originally thought to have been

a member of the Chroococcales (Mitra, 1950) and later shown

to fix atmospheric nitrogen (Fay and Fogg, 1962), Chlorogloea

fritschii should have created much interest except for the

observation that it producted heterocysts and short filaments

in certain growth stages. That it is probably an atypical

Nostoc was confirmed by Fay et al. (1964). Since this alga

has a relatively slow rate of growth and is difficult to

work with (Fay and Fogg, 1962), perhaps its only attractive

feature is a capability for slow heterotrophic growth (Fay,

1965; Holton et al., 1968). The metabolism of this alga is

discussed in such excellent papers as those of Carr (1966),

Peat and Whitton, (1967), and Fay and Cox (1967).

Tolypothrix tenuis: The widespread interest in this

alga today is the direct result of exhaustive searches for

nitrogen-fixing blue-green algae throughout Southeast Asia

(Watanabe, 1959a). Of 643 samples tested, Tolypothrix tenuis

is one of 13 blue-green algal species shown to fix nitrogen

(Watanabe, 1951), and has proved to be the most vigorous of

these in nitrogen fixation. Because of its high potential

in artificial maintenance of nitrogen fertility in rice

paddy agriculture, this alga is one of the most intensively

investigated forms today.

Originally isolated and purified by Watanabe (1951),

Tolypothrix tenuis was then tested as a 'means of increasing J

the fertility of soils for rice plants (Watanabe et al.,

1951; Watanabe, 1962). Its growth characteristics were

investigated (Ukai et al., 195$; Watanabe, 1959b), and the

various relationships between light and phycobilin pigment

production were studied (Hattori and Fujita, 1959a, 1959b,

1959c; Fujita and Hattori, I960, 1962). Since it is one of

the few blue-green algae shown to make some heterotrophic

growth (Kiyohara et al., I960), it has been extensively used

in dark and light organic-uptake studies (Kiyohara et al.,

1962; Cheung et al., 1964; Cheung and Gibbs, 1966).

10

Anaeystis nidulans: Originally isolated by W. A. Kratz,

purified by M. B. Allen (Kratz and Myers, 1955a), and sub-

sequently deposited in the Indiana University Culture Collec-

tion, Anaeystis nidulans has become perhaps the most widely

used experimental blue-green alga. Some of the prime factors

for this are the organism's very sensitive responses to envi-

ronmental stimuli, its rapid rate of growth with increased

temperature and light incidence (Kratz and Myers, 1955a,

1955b), and its characteristic growth as unicells in culture.

This latter condition highly facilitates growth measurements

and makes the alga very attractive as a subject for investi-

gation. In addition, Anaeystis nidulans has been used by

1

many investigators somewhat as an internal standard of com-

parison with the response of some other species (for example,

Stewart et al., 196$; Weber and Bock, 196$).

Several investigations of selected enzymic reactions

and subcellular fractions of A. nidulans have been published

(Richter, 1961a; Willard et al., 1965; Norton and Roth, 1967;

Hirayama, 1967; Capesius and Richter, 1967), and the alga

has also been used to some extent in experimental uptake

studies (Kandler, 1961; Kindel and Gibbs, 1963). Its ex-

tremely high photosynthetic rate has made it very attractive

for respiratory and photosynthetic experiments (Jones

11

and Myers, 1963; Gerhardt and Trebst, 1965). Of the litera-

ture surveyed, only two mineral deficiency or nutritional

studies using Anacystis nidulans exclusively have been pub-

lished to date: one on manganese (Richter, 1961b) and one

on phosphate (Batterton and Van Baalen, 1968).

Microcystis aeruginosa: Probably no other non-nitrogen-

fixing blue-green alga is such a common component of water-

blooms (Borgstrom, 1935; Brandenburg and Shigley, 1947;

Wurtz, 1949; Komarek, 1957) nor is as likely to be associated

with toxic conditions (Ashworth and Mason, 1946; Hughes et al.,

1955, 1958; Bishop et al., 1959; Gorham, I960, 1962).

This alga's repeated occurrence as-a nuisance has.led t

to extensive investigations into its mineral nutrition

(Gerloff et al., 1952; Gerloff and Skoog, 1954> 1957a, 1957b).

Significant investigations concerning its growth responses

and characteristics (Zehnder and Gorham, I960; McLachlan and

Gorham, 1961, 1962) have produced much of what is known con-

cerning planktonic blue-green algae. Under natural condi-

tions Microcystis aeruginosa exists in the form of large

clathrate colonies but becomes unicellular upon artificial

culture; therefore, much laboratory data may not be appli-

cable to natural requirements. Although such studies as

Vance (1966) on its antibiotic sensitivity continue to

12

add to the slowly accumulating knowledge, active work is

still needed.

Other species: This survey reveals only about twenty-

five reports dealing extensively with the physiology or

biochemistry of other blue-green algal species. By far the

majority of papers are concerned with those organisms that

have the ability to fix nitrogen. For example, nitrogen

fixation is the central theme in studies involving twelve

species: Mastlgocladus laminosus (Fogg, 1951b), Cylindrospermum

sphaerica (Venkataraman et al., 1959), Stigonema dendrideum

(Venkataraman, 1961), Anabaena azollae (Venkataraman, 1962),

Calothrix scopulorum and Nostoc entophytum (Stewart, 1962),

Nostoc commune (Taha and El Refae, 1962a, 1962b), Fischerella

musciocola and F. major (Pankow, 1964), Westiellopsis prolifica

{Pattnaik, 1966), Cylindrospermum muscicola (Venkataraman and

Neelakantan, 196?). Because of its tendency to form toxic

"blooms," Aphanizomenon flos-aquae has attracted attention,

resulting in recent studies of its growth and toxin production

(McLachlan et al., 1963; Jackim and Gentile, 1963; Sawyer et al.,

1963; Gentile and Maloney, 1969).

Only nine papers, restricted to eight species, are

available on species unable to fix nitrogen. Photosyn-

thetic experiments have been conducted with Chroococcus

(Emerson and Lewis, 1942; Arnold and Oppenheimer, 1950),

13 (T

whereas extensive growth and cultivation studies were con-

ducted with Phormidium persicinum (Pinter and Provasoli,

195$) and Oscillatoria rubescens (Staub, 1961). Independent

studies on growth and pigment changes were carried out with

Plectonema nostocorum (Kingsbury, 1955) and the nitrogen-

fixing species, Scytonema tenue (Venkataraman, i960). Such

diverse investigations as the effect of temperature on growth

and photosynthesis in Oscillatoria subbrevis (Moyse et al.,

1957),"the production of antibacterial substances by

Oscillatoria formosa (Davidson, 1961), and the amino acid

composition of Spirulina maxima (Clement et al., 1967) com-

plete this survey.

- The following statements of eminent investigators attest

to the limited state of studies on blue-green algal physiol-

ogy. Recently, Pringsheim (1967) stated with regard to the

blue-green algae:

Only a few species have been investigated under diverse, reasonably well-controlled conditions. Iden-tification is in many cases problematical; this is due to there being many more taxa than realized. The ver-satility of the class is so extraordinary that a better understanding may only be expected if many strains are compared, e.g., in respect to salt and pH tolerances, organic nutrients, etc. Descriptions in floras are often misleading because diagnoses of species contain observations on forms not identical in dimensions and ecological adaptations.

14

Concurring with this opinion, Holm-Hansen (196$) states,

Although the blue-green algae comprise a wide assemblage of free-living and symbiotic forms, the bulk of laboratory investigations have been done with only a few dozen species. As the species chosen for labora-tory studies are generally those which are easy to cultivate, it is possible that many deviations from the generalizations presented in this review may emerge from future studies on some of the lesser-known genera.

Bold (1968) perhaps delivers the most stirring summary:

I am repeatedly impressed that relatively few algae have been studied physiologically and biochemi-cally and that we have as a result made many extrapola-tions on the basis of a very small sample. We might perhaps paraphrase the British by saying "Never have so many broad conclusions been based on so few data."

For several years, studies in this laboratory, although

not wholly or directly concerned with blue-green algae,

nonetheless have been subject to possible influence from these

organisms. Two of these research problems continually assoc-

iated with blue-green algae merit mention at this time: the

suspected participation of blue-green algae in the production

of tastes and odors in potable water supplies, and the poorly

understood growth and developmental, or "bloom", characteris-

tics, which cause great difficulty in the implementation of

standard bioassay procedures for eutrophication measurements.

The need for such procedures is highly evident, and

proposals for such have been initiated (Khan, 1964; Skulberg,

1966; Oswald and Golueke, 1966). In tests such as those

15

proposed, either natural or cultural algal populations will

have to be employed, and in many of these populations, blue-

green algae seem to play a dominant role. In regard to the

second problem, certain blue-green algal species have been

implicated in taste and odor outbreaks in potable water

supplies (Rochlich and Sarles, 1949), and there have been

several recent investigations concerning actual taste and

odor compounds produced by blue-green algae (Safferman et al.,

1967; Jenkins et al., 1967; Medsker et al., 196$). Much of

the work undertaken and reported in this paper was initiated

in the hope of providing additional background information of

blue-green algae physiology and biochemistry, for forthcoming

comprehensive investigations of the two aforementioned problems,

This research has consisted of two investigative phases.

The first is a test of the validity of certain specific gen-

eralizations regarding blue-green algae by comparing phys-

iological and biochemical measurements obtained from experi-

ments employing three different groups of organisms:

conventional "test" blue-green species, those having been in

culture several years but little used for experimental pur-

poses, and newly isolated organisms from the natural environ-

ment. The successful interpretation of these measurements"

should almost certainly provide some answers to the following

16

questions: (1) What physiological type of blue-green

algae can normally be expected to be isolated from South-

western reservoirs or other habitats, and how much influence

will different isolation techniques have upon the type of

isolates so obtained? (2) What degree of difficulty is

encountered in these isolation and purification procedures

when facilitated by modern technology? (3) How would the

metabolic patterns of these recent isolates compare to the

patterns of cultured blue-green algae upon which most phys-

iological information is based? (4) Would the adaptations

(unnatural selection?) that have surely occurred in con-

ventional experimental blue-green algal species during

their several years in artificial culture result in detect-

able metabolic differences? (5) Do only heterocystic

filamentous blue-green algae fix nitrogen? (6) Can blue-

green algae actually grow as heterotrophs? (7) What blue-

green algae apparently produce taste and odor compounds, and

is this production grossly affected by their nitrogen nutri-

tion? Secondly, this work is specifically designed to

determine and compare the respiratory and photosynthetic

rates of an ecologically extensive variety of blue-green

algae, with the hope that this information may provide

insight into the metabolic behavior of each organism.

CHAPTER II

MATERIAL AND METHODS

Isolation

In order to obtain new planktonic blue-green algal

isolates, water samples from two Southwestern reservoirs,

Garza-Little Elm Reservoir, Denton County, Texas, and Lake

Hefner, Oklahoma County, Oklahoma, were taken at random at

various times and depths. In each case, ,50 to 100 ml of the

water sample was filtered through a .45-p Millipore filter.

The filter was placed in a small volume - of distilled water

and examined with the aid of a stereoscopic microscope for

filaments, trichomes, and colonies.

Those structures resembling blue-green algae were picked

up by means of a micro-pipette and plated on 1.5$ agar dis-

solved in algal culture medium. In instances when individual

groups of organisms appeared unialgal, inoculation was also

made directly into liquid culture medium, which was then-

incubated in subdued light and shaken gently at about 100

cycles per min at 24 C. Excessive development of diatoms

and green algae as contaminants in both plate and liquid culture

usually necessitated treatment with 200 ppm of the antibiotic,

17

IS

acti-dione (cycloheximide), which restricts diatomic and

green algal development but has little effect on blue-green

algae (Zehnder and Hughes, 1958). The growth of diatoms and

green algae was also restricted in several cases by incuba-

tion of the isolation cultures at a temperature of 35 C

(Allen and Stainer, 1968). This elevated temperature also

seems to promote faster blue-green algal development. In

cases of severe contamination, both methods were employed

concurrently. Each time samples were obtained, plates and

liquid cultures free of combined nitrogen were prepared for

the selective isolation of nitrogen-fixing blue-green algae

(Taha, 1963). i

After 14 to 28 days, developing blue-green algal colonies

which appeared to be mostly free of other algal and fungal

contaminants were picked off the agar plates with a wire

loop or microscopically separated on a glass slide from the

liquid culture medium, blended in a sterile Waring Blendor,

and plated in dilution series by means of the spread plate

technique. Following development, the plates were examined,

and in many cases, this separation process had to be repeated

several times before a unialgal isolate could be obtained.

In order to obtain experimental organisms with extremely

variable physiological and biochemical processes, collections

were also made from such highly diverse environments as

19

puddles, ponds, soil-crusts, effluent basins, and slime coat-

ings of deep water-well pumps. Visible colonies of blue-green

algae from these habitats were collected into sterile con-

tainers, usually as large masses of a particularly dominant

species. In the laboratory, the sample was blended, plated,

and subjected to the previously described isolation procedures,

Purification

In order to achieve maximum growth rate and the least

possible build-up of extracellular products (i.e., the cul-

tural state which seems to favor the chances of successful

purification), organisms were repeatedly subcultured on a

shaker at 100 cycles/min before purification attempts. Those /

blue-green algal species forming mats, clumps, or visible

colonies were thoroughly blended in a sterile Waring Blendor

as many hours prior to treatment as possible. The tendency

to reform unevenly suspended cultures is usually rapid,and

generally never allows a post-blending recovery time in

excess of 48 hours. This is somewhat unfortunate, because

the blending process can exert a physiological stress on'the

alga that may not be effectively overcome. Thus the organism

may succumb during treatments that it might otherwise have

survived. Other than repeated subculturing, species growing

as unicells or hormogones were not subjected to special

20

treatment. In any case, those cultures selected for purifi-

cation experiments were always young, uniform, dilute, and

growing at maximum rate under the conditions provided.

Ultraviolet radiation.—The methods employed were

essentially those of Allison and Morris (1930), and Krat-z and

Myers (1955a). Enough algal suspension to cover the bottom,

approximately 2 to 3 mm deep, was poured into a sterile unit

consisting of a petri dish with a magnetic bar. The un-

covered dish was placed on a magnetic stirrer, with the

cultural solution to be treated 10 cm below the radiation

source. During treatment the bar was slowly rotated and

0.1 ml portions pipetted in triplicate to fresh, sterile I

medium at timed, 5-minute intervals, 0 through 60 minutes.

The radiation source was a Westinghouse "Sterilamp" No. 7&2H--30,

with delivery of 1.45 x 10^ ergs/cm^ at the surface of the

irradiated suspension.

X-ray irradiation.—This treatment is a modification of

that proposed by Kraus (1966). A dilute cell suspension of

the alga was poured to a depth of 1-2 mm in a 4.5~cm petri

dish,which was then placed 10 cm from the source of a

G. E. Model XK 10 X-ray unit. Voltage was maintained at

87-5 ,Kvp with a current of 5 ma,allowing the unit to produce

13 Kr/min at the distance being irradiated. Exposure time

21

was 21 minutes, producing a total dosage of about 274 Kr.

This dosage has allowed the greatest percent survival of

blue-green algae, together with almost maximum bacterial kill

(Kraus, 1966). Five 0.1-ml portions were removed after treat-

ment to fresh sterile medium, and observed for growth.

Elevated temperature.—Wieringa (196$) reported that

prolonged exposure to temperatures ranging from 45 C to 60 C

was frequently fatal to non-sporeforming bacteria. Since

the most common bacterial contaminants of' blue-green algae

fall into this category, this method of treatment was pro-

posed for bacterial purification. For each alga to be treated,

three recently autoclaved 250-ml culture flasks containing

160 ml medium were placed in a 48 C water bath and allowed

to stabilize. Using a 1-ml suspension of cells from the

previously described algal cultures, the tip of the delivery

pipette was placed just below the surface of the heated

culture vessel, its contents were released and the pipette

was withdrawn carefully without touching the sides of the

vessel. After a treatment period of 1 hour, the triplicate

vessels were removed, allowed to cool at room temperature,

and incubated for further observation.

Antibiotic treatment.—Streptomycin sulfate, tetracycline

hydrochloride, erythromycin ethyl succinate, and acti-dione

22

(cycloheximide) were freshly dissolved in culture medium at

concentrations of 1.0, 6.0, 1.0, and 200.0 mg/l, respectively,

and sterilized via filtration through ,22-p Millipore filters.

Following the method of Droop (1967), six parallel tube

cultures of each alga to be treated were prepared by equally

dividing (blending if necessary) a vigorously growing ISO-mi

stock culture into 6 sterile nylon centrifuge tubes with

caps. When the supernate was removed after centrifugation,

tube §1 received 20 ml of the antibiotic culture solution,

and tubes #2-#6, the same volume of sterile culture medium.

Each tube was thoroughly mixed on a Vortex mixer, and the

contents of tubes #1 and $2 poured together, mixed, and

equally divided. Tube §1 was then set aside and the same

process repeated throughout the series. Final dilution of

tube §6 with an equal volume of sterile medium completed the

process and resulted in a total antibiotic concentration of

104} 52, 26, 13, 6.5} and 3-25 mg/l? respectively. Upon

completion of the 24-hour incubation period, 0.1-ml portions

were pipetted in triplicate from each antibiotic concentra-

tion into fresh medium and observed for growth.

Final purity check.—Those algal cultures exhibiting

growth after purification treatments were tested for bacterial

contaminants by use of a combination of those methods proposed

23

by Fogg (1942) and Gerloff. et al. (1950b). For preliminary-

testing, three types of organic substrates were dissolved

in algal cultural medium, 0.01 M glucose, 0.01 M sucrose,

and casamino acids at a concentration of 5 g/l- If turbidity

was not detected in any of these, further tests were made

with dextrose-peptone broth {Vfo each of dextrose and peptone),

1.5% sodium casinate agar, Burk's medium (Wilson and Knight,

1952), and Brewer's medium (Brewer, 1939). If no bacterial

growth occurred in any of the latter four test media, the

culture was presumed to be axenic.

Culture

Unless otherwise stated, all organisms grown and experi-

ments undertaken took place under the following cultural

conditions:

Culture vessel 300-ml Delong flask

Volume of culture medium 160 ml

Temperature 24 C, + 1°

Gentle shaking at 100 cycles/min

Continuous "cool white"

fluorescent lighting of 300-ft candles (3228 Lux)

Four different algal culture media were initially used:

Chu No. 10 (Chu, 1942), with iron citrate modification as

originally suggested by Rodhe (194$) and presented by

. 2 4

Gerloff et al. (1950a), Hughes et al. No. 11 (Hughes et al.,

1958), Modified Synthetic Sewage (Davis and Wilcomb, 3.967) >

and ASM-l (Gorham et al., 1964) • ASM-1 (Table 1) eventually-

exhibited the greatest utility, and all blue-green algal,

cultures listed in Table 2 were grown on such unless other-

wise noted. Those species capable of using atmospheric

nitrogen were cultivated routinely on ASM-1 minus nitrate.

TABLE 1. Composition of ASM-1 in g/l

NaN03* .1700

MgSO^ * 7H20 .0493

MgCl2 • 6H20 . 0406

CaCl2 .0222

K2HPO4 .0174

Na2HP04 • 7H20 .0268

FeCl3 • 6H20 .0011

H3BO3 .0025

MnCl2 • 4H20 .0014

ZnC3„2 .0004

CoCl2 * 6H20 .000019

CuCl2 ' 2H20 .00000013

Na2EDTA .0074

*In nitrogen fixation experiments, NaNOo was omitted and ,0005 g/l Na2Mo04 • 2H20 (.0002 g/l Mo++) added.

25

TABLE 2. List of blue-green algal species investigated

a. Commonly used experimental organisms. All obtained from the Indiana University Culture Collection. (Starr, 1964)

Number Organism

629 Anabaena cylindrica 1444 Anabaena flos-aquae 625 Anacystis nidulans

*466 Nostoc museorurn

Isolater

G. E. Fogg R. G. Tischer ¥. A. Kratz F. E. Allison

b. Organisms having been in artificial culture for some time but seldom used as experimental subjects. All obtained from the IUCC.

Number Organism Number Organism

379 Calothrix membranacea 756 Nostoc sp. 942 Cylindrospermum sp. 366 Oscillatoria chalybea 1519 Eucapsis sp. 390 Oscillatoria formosa 1301 Fischerella musicola *426 Oscillatoria tenuis 461 Fremyella diplosiphon 427 Phormidium faveolarum 569 Gloeocapsa apicola 561 Plectonema boryanum 1596 Gloeocapsa apicola 1163 Scytonema sp. 795 Gloeocapsa sp. *617 Symploca muscorum 941 Gloeotrichia sp. 1191 Synechococcus cedrorum 466 Lyngbya sp. 424 Tolypothrix distorta *564 Nostoc commune

c. Recently isolated organisms.

Number Organism

NT69-6 Anabaena Bornetiana

NT69-2 Anabaena circlnalis

NT66-2 Anabaena subcylindrica •

NT66-1 Anacystis cyanea

NT66-3 Arthrospira sp.

Source

Garza Little Elm Reservoir, Denton County, Texas

Deep water-well pump, Texas High Plains

Aquatic beetle tank, NTSU Campus, Denton, Texas

Aquatic beetle washing, NTSU Campus, Denton, Texas

26

Number

NT68-4

NT68-5

NT68-18

NT6S-6

NT63-7

*NT68-8

*NT68-19

•NT68-20

NT68-9

NT68-11

NT63-12

NT63-13

NT63-16

NT68-10

NT68-17

TABLE 2.—Continued

Organism

Chroococcus sp.

Source

Chroococcus sp.

Nostoc sphaericum

Oscillatoria sp.

Oscillatoria sp.

Oscillatoria sp.

Oscillatoria sp.

Oscillatoria sp.

Phormidium sp.

Phormidium sp.

Phormidium sp.

Phormidium sp.

Aquatic beetle washing, NTSU Campus, Denton, Texas

Deep water-well pump, Texas High Plains

Puddle, NTSU Campus, Denton, Texas

Deep water-well pump, Texas High Plains

Sewage disposal mat, Paris, Texas

Aquatic beetle washing, NTSU Campus, Denton, Texas

Small pond, Paris, Texas

Small East Texas spring, Starrville, Texas

Lake Hefner, Oklahoma County, Oklahoma.

Paper mill effluent basin, Orange, Texas

Elwood Lake, Gladewater, Texas

Deep water-well pump, Texas High Plains

Phormidium purpurascens Lake Hefner, Oklahoma City, Oklahoma

Pseudanabaena catenata "

Pseudanabaena sp, Small pond, Denton County, Texas

27

TABLE 2.—Continued

Number Organism Source

NT63-14 Scytonema sp. Spring, Piatt National Park, Oklahoma

NT68-15 Symploca sp. East Texas soil-crust, Gladewater, Texas

^Odor producing species.

All blue-green algal experimental cultures were unialgal

clonal isolates,and were axenic, or nearly so. The cultures

that were axenic had been taken through so many purification

steps and repeated transfers in purely mineral medium that

their contaminant population must have been near zero. At

no time was there a sufficient bacterial population present

to cause detectable turbidity in the culture medium, even

after 30 daysT incubation.

Growth Rate

Several hundred clean 50-ml Erlenmeyer flasks containing

40 ml of sterile ASM-1 were each inoculated with 0.1 ml of

dilute algal suspension of the species to be tested. After

the inoculated cultures were grown under the conditions pre-

viously specified, triplicates were randomly selected and

removed daily. Each was diluted to 100 ml with double-glass-

distilled water, blended in a Waring Blendor for 10 seconds,

2g

and its optical density read at approximately 650 mp. with' a

Klett-Summerson photoelectric colorimeter Model No. 9QO-3

with filter #66. The mean of the three O.D. 650 readings

was then determined and the resultant growth curves plotted

from these data.

Heterotrophy

All heterotrophic studies were conducted using 125-ml

Erlenmeyer flasks containing SO ml ASM-1 supplemented with

an organic substrate, generally following the methods of

Kiyohara et al. (I960) and Fay (1965). The organic substrates

were 0.01 M concentrations of glucose, sucrose, acetate,

citrate, and inositol, along with casamino acids at 5 g/l«

Because the cultures were not agitated, they remained stag-

nant throughout the test period. The growth flasks were

either wrapped in aluminum, foil or placed in a completely

darkened cabinet during the period of examination.

Odor Production

The "Threshold Odor Test" (Staff Report, i960) was used

to determine odor production levels in those blue-green algal

species capable of producing such. In these organisms, odor

production was tested using all four previously mentioned

algal culture media: Chu's No. 10 modified, Hughes et al.

No. 11, Modified Synthetic Sewage, and ASM-1. In addition,

29

the effect of different inorganic nitrogen sources on gross

odor production was investigated using ASM-1 (NO^). NH^ and

NO2 were tested by the substitution of the nitrogenous com-

ponent of ASM-1 (NaNO^) with equal moles of combined nitrogen

in the form of NH _C1 and NaNC^, respectively- The effect of

the absence of combined nitrogen on gross odor production

was also studied by Its omission from the medium. Sodium

nitrate was replaced by 0.2 ppm Mo++ in the form of

NagMoO^ * 2^0, a molybdenum concentration which has been

shown (Wolfe, 1954a) to most effectively promote growth in

media free of combined nitrogen.

Experimental algal inoculation with odor-producing

actinomycetes was effected with two species—Streptomyces sp.

(North Texas Strain No. 62) and Streptomyces sp. (isolated

as a contaminant along with a blue-green alga from a local

puddle). Both these organisms were grown prior to inocula-

tion on a medium containing 4 g/l NH^NO^, 40 g/l brown sugar,

& g/l peptone (Difco), with the pH adjusted to $.3 with KOH

prior to autoclaving. In order to insure ample carryover of-

actinomycete and organic substrate, 5 ml of each streptomycete

suspension was pipetted into an experimental algal culture

which previously produced no odor. The mixed culture was

then incubated normally and tested-for odor production.

30

Nitrogen Fixation

Unless otherwise noted, organisms tested for the ability

to fix nitrogen were cultured under the previously described

conditions but were adapted to ASM--1 medium minus its normal

source of combined nitrogen. Obtained from vigorous 7~ to

14-day-old cultures, 5~ml suspensions of thickly concentrated

cells were removed and added to 15 ml of sterile nitrate-free

media in 40-ml serum bottles capped by rubber stoppers. For

each alga tested, samples were prepared in triplicate for

each time period to be measured. Using the acetylene reduc-

tion method (Stewart et al., 1967, 1968; Hardy, 1963), the

samples were flushed (by adding and venting through separate

needles) for 2 minutes with a mixture of 7&% argon, .05% carbon

dioxide, and 22% oxygen. The needles were then removed and

0.2 ml of the gas mixture was withdrawn and replaced with an

equal volume of acetylene at spaced intervals, allowing ample

time periods for measurement of ethylene production and sub-

sequent treatment of the sample. The resultant pre-incubation

gas phase is approximately 10% acetylene, 70% argon, .04% car-

bon dioxide, and 20% oxygen.

The cultures were then incubated horizontally under

normal cultural conditions,for the prescribed time period.

Upon termination of the incubation period, the gas phase was

31

analyzed for ethylene by injecting 1-jul samples into a

MicroTek GC-1600 gas chroraatograph equipped with a hydrogen-

flame ionization detector and a 9~ft column of Porapak R

(Stewart et al., 1968). Nitrogen was used as the carrier

gas,with the flow rate adjusted to approximately 30 ml/min.

The oven temperature was 60 C. Upon measurement, each sample

was immediately diluted to 100 ml with distilled water,

blended for 30 sec in a Waring Blendor, and its optical

density taken,as in growth measurements. It was then filtered,

dried at 50 C, and weighed. Gassed and incubated controls

always included uninoculated media, dark bottles (serum

bottles wrapped in aluminum foil which have received a pre-

dark treatment), and suspensions of known non-fixers. All

conditions were the same for nitrogenase induction rate

measurements,except that the algal cells used were grown on

complete ASM-1. Immediately prior to use, the cells were

centrifuged, suspended, and washed three times in double-

glass-distilled water.

Establishment of experimental commensalism or mutualism

between non-nitrogen fixing blue-green algae and free-living,

nitrogen fixing bacteria was attempted. The bacterial organ-

isms used in these tests are listed in Table 3.

32

TABLE 3• Free-living nitrogen-fixing bacteria utilized

NTS-U Number Organism Source or Strain

255 Azotobacter chroococcum UT (75-1)

253 Azotobacter vinelandii ATCC 12837

2&1 • Azotobacter vinelandii op U. of Wise.

109 Rhodospirillum rubrum MC

The Azotobactors were grown on Burk's medium (Wilson

and Knight, 1952), and Rhodospirillum was grown on Brewer's

medium (Brewer, 1939). Heavy 5-ml inocula of each bacterium,

along with a dense 10-ml suspension of each non-nitrogen

fixing blue-green alga tested, were made into sterile ASM-l,

free of combined nitrogen. The liberal amounts of inoculum

were introduced to insure ample carryover of nutrients from

the parent cultures—carbohydrates for the bacteria and

nitrates for the blue-green alga. Incubation was as pre-

viously described.

Respiration and Photosynthesis

The amount and rate of carbon dioxide change in both

light and dark cultures was monitored continuously by record-

ing the cultural pH with a Leeds and North2"up Expanded Scale

pH meter, Model 7403, and a Hitachi Perkin Elmer Recorder,

Model I65. Algal cultures with random weights and in various

growth phases were tested, but very densely growing cultures

33

were usually diluted with fresh medium if the heavy cell

concentration was detected to be self-shading, and therefore

light-limited. In all blue-green algal cultures tested,

measurement of the carbon dioxide change for both light and

dark periods was repeated at least once. Continuous exposure

periods to both darkness and light, ranging from 4 to 24 hours,

were tested. Measurements were recorded under the cultural

conditions previously described, except that the dark period

was provided by wrapping the culture vessel in heavy black

plastic. The medium was 200 ml of ASM-1, unless otherwise

noted. At the termination of measurements, the culture was

filtered on dried and pre-weighed Gelman glass-fiber filters,

Type A, dried to constant weight at 60 C, and weighed. The

200 ml of filtrate was poured into a 250-ml graduated cylinder

and stripped of carbon dioxide by bubbling nitrogen gas for

about 30 minutes through an air stone placed at the bottom

of the cylinder. The "stripped" medium was then gently

poured into' a 250-ml beaker and slowly stirred with a mag-

netic stirrer while being titrated with carbon-dioxide-satu-

rated distilled water with the aid of a Tonometer (see

Trotter, 1969 for a detailed description and exact titrametric

directions). The resultant pH change was recorded on the

Hitachi Perkin Elmer unit by allowing a slow constant flow

34

and noting . 5~ml increment points along the pH change ciirve

marked by the instrument. This entire process is but a

slight modification of that described by Beyers et al. (1963).

These authors also presented exact directions for the deter-

mination of the molar concentration of carbon dioxide in

the saturated distilled water used for titration at given

temperatures and barometric pressures. This determination

permits easy computation of carbon dioxide uptake or release

per time per amount of algal material present, by referring

the pH change curves previously recorded during respiratory

and photosynthetic measurements to the standard pH change

curve produced in the actual cultural medium by known incre-i

ments of carbon dioxide. Data reported in this paper were

compiled under the assumption that respiratory release of

carbon dioxide in light approximately equals that in darkness.

CHAPTER III

RESULTS AND DISCUSSION

Twenty-two different unialgal clonal Isolates have been

obtained at random for experimental purposes over a period

of about one year. Also, during this period, at least 12

other species or strains have been isolated into unialgal

cultures which had not yet been identified and/or signifi-

cantly cleared of heterotrophic contaminants.

The 22 former organisms can be conveniently classified

into four generalized groups (Table 4): < Group 1, those

from true planktonic environments; Group 2, those from

diverse aquatic habitats; Group 3, organisms from undeter-

mined habitat; Group 4> isolates from terrestrial or semi-

aquatic environments.

Group 1. Both Anabaena Bornetiana and Anabaena circinalis

have been obtained via selective isolation in combined-

nitrogen-free medium. Each continually occurs with great

frequency in plankton samples from area reservoirs. Micro-

scopic examination of five different plankton samples taken

from Garza Little Elm Reservoir over a two-month period from

mid-April through mid-June reveals for A. Bornetiana an

36

TABLE 4. Environmental distribution of isolates

Group 1. Euplanktonic organisms

NT69-6 Anabaena Bornetiana

NT69-2 Anabaena circinalis

NT68-9 Phormidium sp.

NT68-12 Phormidium sp.

NT6S-16 Phormidium purpurascens

NT66-10 Pseudanabaena catenata

NT6S-17 Pseudanabaena sp.

Group 3. Organisms from unde-termined habitat.

NT68-3 Arthorospira sp.

NT68-4 Chroococcus sp.

NT68-8 Oscillatoria sp.

Group 2. Organisms from diverse aquatic environments.

NT6$-1 Anacystis cyanea

NT6&-2 Anabaena subcylindrica

NT6S-5 Chroococcus sp.-

NT68-6 Oscillatoria sp.

NT6S-19 Oscillatoria sp.

NT6$-20 0sci3.1atoria sp.

NT6S-11 Phormidium sp.

NT6&-13 Phormidium sp.

NT6S-14 Scytonema sp.

Group 4. Terrestrial or semi-aquatic organisms.

NT6S-1S Nostoc sphaericum

NT68-7 Oscillatoria sp.

NY6S-15 Symploca sp.

average heterocyst frequency of about 40 cells/heterocyst,

whereas A. circinalis tends to be virtually free of hetero-

cysts. Should heterocyst frequency reflect the tendency of

the organism to fix atmospheric nitrogen (Fogg, 1944, 1949,

1951a; Mickelson et al., 1967; Stewart et al., 1963; Fay

et al., 1968), one might assume that under the prevailing

. 3 7

reservoir conditions, the former is more likely to be a

nitrogen fixer in nature. Both maintain an average hetero-

cyst frequency of about 7 cells/heterocyst when laboratory-

cultivated in nitrate-free ASM-1. A. circinalis tends to

become much less tightly coiled in culture.

In this study, efforts to isolate Aphanizomenon f los-

aquae, which also occurs in great abundance in area reser-

voirs, have thus far failed. So far, it has continually

been overgrown in complete medium and has not survived in

nitrate-free medium despite having been reported as a nitro-

gen fixer (Stewart et al., 1968; Gentile and Maloney, 1969).

Group 1 organisms belonging to the genus Phormidium

an^"Pseudanabaena are nearly always the dominant blue-green

algal forms when plankton samples are grown on complete

medium. They are tiny (cell width usually 1 to 2 p with the

length seldom exceeding twice the width), and, with the excep-

tion of Phormidium purpurascens, tend to grow in short

hormogones; their developmental rate is high and they will

rapidly overgrow all other blue-green algal forms. When

they are found as contaminants in isolation culture, no

other blue-green algal species has been freed of these

organisms, in spite of literally hundreds of attempts.

In culture, Phormidium purpurascens adheres with great

tenacity to the sides of the culture vessel and, if rubbed

33

free, usually winds into a ball. This alga also has another

distinctive characteristic: sometimes after even moderate

blending, resuspension of this alga in fresh medium will

result in the new medium's assuming a bright clear pink color,

which normally fades after a few hours. The reasons for the

inconsistency of this color phenomenon are not apparent.

The generally high metabolic rates exhibited by all

other Group 1 organisms and their tendency to grow as evenly

dispersed hormogonia make these organisms highly favorable

experimental subjects.

Groups 2, 3, and 4 $NT6§-1 Anacystis cyanea and $NT6S~11

Ph.ormi.dlum sp. exhibit much the same tendencies and seem to

respond to environmental stimuli in a manner similar to that

of the planktonic Group 1 organisms. #NT68~3 Arthrospira sp.,

T63--6 Oscillatoria sp., $NT6S~13 Phormidium sp., #NT63-15

Symploca sp. all have the tendency to grow in liquid culture

as large, free-floating, fluffy, entangled masses. With most

of the algal colony self-shaded and unresponsive, development

of new investigative procedure would appear necessary before

these organisms can be conveniently used for experimental

species.

#NT6$--8 Oscillatoria sp., $NT63-19 Oscillatoria sp., and

#NT68-20 Oscill atoria sp. are all. "large forms producing

earthy or musty odors. #NT6S~7 is similar in morphology and

39

growth habits but produces a strong, sour, acrid odor.

#NT69-20 Oscillatoria sp. is unique in that when cultured

on a shaker, it does not clump or form mats,but instead

remains as long,evenly dispersed filaments. This trait

makes it an attractive experimental subject. Also excellent

prospects for future experimental work are $NT6$-4 Chroococcus

sp. and $NT6$~5 Chroococcus sp., because of their rapid growth

and even distribution in the culture medium. These algae

are quite similar in appearance but exhibit extremely dif-

ferent cultural characteristics.

Of the three nitrogen-fixing organisms of these groups,

#NT6S-2 Anabaena subcylindrica is by far the best suited to

biological experimentation. Its appearafice is similar to

the well-known Anabaena cyllndrica, and its gross cultural

characteristics closely resemble the substantially investi-

gated strain of Anabaena flos-aquae (IUCC No. 1444). $NT6$-l£

Nostoc sphaerlcurn exhibits a very low rate of growth in

"shake" culture. It grows in perfectly round spheres which

continually roll and float across the bottom of the culture

vessel; the size of these spheres ranges from microscopic

dimensions up to 10 cm in diameter. The interior of the

colony becomes less dense with aging,and finally the older

or larger colonies become generally hollow,

40

r

#NT68-14 Scytonema sp. represents something of an

enigma. It has yet to exhibit any sort of repeatable cul-

tural characteristic but inconsistency. Occasionally it has

remained vigorous and bright green in stock culture for

periods up to six months; at other times, it fades and

appears exhausted within a week. This alga has even failed

to grow on that universally dependable medium, soil-water

extract. Needless to say that the same is true for each of

the four previously described mineral media. One-unit

increments in cultural pH from pH 5 through pH 10; photo-

periods of 24, IS, and 12 hours; temperatures of 20, 24, 30,

and 35 C; N0^, NH^, and N2 as nitrogen sources; light inten-

sities of 50, 200, 300, and 600 ft candles; cultural agita-

tion or stagnation; and supplementation with all known growth

factors and vitamins have all been tested, and no combination

has consistently produced growth and development. Although

the solution to these seemingly perplexing inconsistencies

may be quite simple, it is unapparent at this writing.

Of all problems in experimental work with blue-green

algae, that of purification is perhaps most apparent. Despite

many repeated attempts at purification of most of the 47

species listed in Table 2, only nine species (Table 5)

have been able to pass rigorous tests of purity.

41

TABLE 5• Axenic cultures

Method Organisms

Ultraviolet radiation #1444 Anabaena flos-aquae

#NT68-10 Pseudanabaena catenata

X-ray irradiation #4^1 Fremyella diplosiphon

#795 Gloeocapsa sp.

#NT6$~4 Chroococcus sp.

Elevated temperature to 48 C #1519 Eucapsis sp.

#756 Nostoc sp.

#NT6S-1 Anacystis cyanea

#NT6$-l8 Nostoc sphaericum

Antibiotics None

After purification treatments, #629 Anabaena cylindrica,

#941 Gloeotrichia sp., #NT6$-9 Phormidium sp., and #NT68-12

Phormidium sp. have been initially tested as pure cultures

but have been subsequently recontaminated or had never been

axenic despite negative bacterial tests.

U. v. radiation treatment has proved most disappointing

so far. It is perhaps the most widely used purification

method (Allison and Morris, 1930; Allison et al., 1937;

Gerloff et al., 1950b; Kratz and Myers, 1955a; Yenkataraman

et al., 1959; Venkataraman, 1961, 1962) but has repeatedly

failed to purify a single odor-producing species despite a

42

great many hours devoted to its' perfection.' I-ray irradia-

tion for blue-green algal purification has not been exten-

sively used but it may eventually prove more effective that

the results listed in Table 4 suggest. Although the use of

elevated temperatures as a purification measure (Wieringa,

1968) was originally suggested only for nitrogen fixers, it

seems to serve equally well for non-fixers. Preliminary

evidence not reported here indicates that repeating the pro-

cedure daily for at least three days may prove quite effective

Even though the antibiotic purification method of Droop (1967)

has been used without success, it has not been adequately

tested because of the limited antibiotic combinations employed,

Whenever a species has been determined to be axenic

throughout the course of these experiments, fresh subcultures

of both axenic and "ultra-clean" but non-axenic samples have

been effected for comparison purposes. While nonetheless

inconclusive, a great deal of preliminary observation has not

revealed gross differences between the two types of cultures.

In fact, it is generally impossible to detect any bacterial

contamination except by the addition of organic substrate.

As an example, if the non-axenic experimental blue-green

algal cultures are cultivated in a well-aerated, purely

mineral medium which is changed regularly (to guard against

the accumulation of extracellular products) and the organisms

43

sr

maintained in vigorous growth condition, no apparently sig-

nificant heterotroph populations exist. Many microscopic

examinations of cultures grown in the above manner have

never revealed a bacterial population that exceeded an

estimate of lCT^ Qf the algal mass.

The argument here is not against use of axenic cultures;

rather it is to suggest strongly that after a newly isolated

culture (which initially is almost always grossly contami-

nated) has been extensively plated, subcultured, treated,

and otherwise laboriously "cleaned-up," step by step, it

can still be of value even if unable to pass purity tests.

Since the absence of bacteria is established solely by nega-

tive evidence and theoretically impossible to prove (Tischer,

1965), many of the so-called "pure cultures" reported in the

literature may be questionable.

For many years blue-green algae were generally regarded

as extremely difficult to culture (Pringsheim, 1946), and it

was not until around the mid-1940's that these organisms,

particularly the planktonic species, became cultivated com-

monly in scientific laboratories (Gerloff et al.,'1950a).

Even as late as 195$) Fogg suggested that few true planktonic

forms had actually been successfully cultured, and it was not

until relatively recently (for example: Drews et al., 1961;

44

Zehnder, 1963; Eberly, .1965-, 1966, 1967) that planktonic

species have been routinely cultivated.

With the exception of $NT68-14 Scytonema sp., all 22

recent isolates, including the nine probable euplankters,

presented no apparent cultivation problems. Continual im-

provement in cultural media design, culminating in such an

excellent example as ASM-1 (Gorham et al., 1964), probably

represents the most important recent development in Cyanophy-

cean research.

Results from this study indicate at least one other

point regarding culture that should be mentioned; even highly

improved culture media of today cannot always nullify the

apparent and somewhat inconsistent natural sensitivity of

blue-green algae to environmental change. Often colonies

which seem most healthy and vigorous abruptly die—even when

placed in an artificial culture specifically designed to

mimic the natural environment; at other times profuse devel-

opment occurs despite apparently unfavorable conditions." The

success or failure of an isolation attempt may not be known

for some time. Illustrating this is the fact that upon

initial removal from the natural environment, many of the

previously mentioned isolates developed quite slowly in arti-

ficial culture, particularly if the isolation had been made

45

in nitrate-free medium. Often 30 days, and in extreme cases,

up to two months of development were necessary before appear-

ance of visible colonies. Pearce and Carr (1966) suggested

that this patent "sluggishness" may be due to lack' of adap-

tive control of enzyme activity.

Characteristic growth curves obtained under the condi-

tions provided are presented in Figure 1 and 2 for #625

Anacystis nidulans and five odor-producing species. The

markedly greater growth rate of A. nidulans was typical of

other non-filamentous forms such as #1191 Synechoccus cedrorum,

#5$9 Gloeocapsa apicola, #1598 Gloeocapsa apicola, #NT68~1

Anacystis cyanea and those highly active hormogonal-like

species typified by #629 Anabaena cylindrica, #NT63-10

Pseudanabaena catenata and #NT68-12 Phormidium sp. The

slower growth rates exhibited by #NT68-3 Oscillatoria sp.,

#42$ Oscillatoria tenuis, and #617 Symploca museorum were

similar to those of most large filamentous species which

have the tendency to form clumps, mats, or balls.

Although not apparent upon superficial visual examina-

tion, most species of Nostoc form very tightly compact

colonies, resulting in the shading of most of the cglls

within. Under the growth procedures used in these experi-

ments, low growth rates were recorded (for example, Figures 1

and 2) for all Nostoc cultures. It is highly possible that

46

300

o o o

X o in to

d o

200

100

- # 625 Anqcystis nidulcmn

$ 4BS fc!ostec muscorum

——— # NT68-8 Oscillatorio st>.

Days

F i g . 1 — G r o w t h of $625 A n a c y s t . i s n i d u l a n s , $ 4 $ 6 N o s t o c muscorum and $NT68~$ O s c i i l a t o r i a s p . ~

47

# 5 8 4 Nostoc comune

* 4 8 4 Osciilatorio tenuis

# 6 1 7 Svmpioca muscorum

- 100

Days

Fig. 2—Growth of $617 Symploca muscorum, Nostoc commune and #4$4 Oscillatoria tenuis.

43

under normal laboratory conditions these organisms are usually

light-limited because an increased light intensity of about

1500 ft candles promoted a striking increase in growth rate

(estimated at 10 fold) when #5$4 Nostoc commune was grown on

a fermenter in vessels of 7 liter capacity. The suggestion

that light increase is a major factor is tentative since it

was obvious that other conditions had also been altered.

Of the 47 species listed in Table 2, 36 (the 11 excep-

tions being Nos. 379, 1519, 1301, 5^9, 1593, 531, 1163,

NT69-6, NT69-2, NT63-19, and NT63-20) were tested for hetero-

trophic ability on six different organic substrates: acetate,

citrate, glucose, sucrose, inositol, and casamino acids.

However, because several of the blue-green cultures used

were non-axenic, some substrates were overgrown with con-

taminants, and hence, were not reportable. At least one

substrate/non-axenic culture was seemingly free of visible

contaminants in all species but $942 Cylindrospermum sp.,

#NT63-12 Phormidium sp., and #NT63-17 Pseudanabaena sp.

Despite several reports (Allison et al,, 1937; Kiyohara

et al., I960; Fay, 1965; Watanabe and Yamamoto, 1967) of

heterotrophy in blue-green algae, these experiments reveal

only a single instance that might be interpreted as hetero-.

trophic growth. Observations were made at the end of 30, 60,

49

and 90 days on all cultures; in all cases where small clumps,

colonies, or mat fragments.had been used to inoculate cultures,

a marked loss in size was noted. Even though growth was not

noted, the 90-day survival rate was high, being nearly 100%

in all substrates except casamino acids. Those blue-green

species which normally develop dark pigments to produce

colors such as black, grey-green, red, or brown faded to a

characteristic light aqua-green. In no case was color com-

pletely absent.

By the end of the first 30-day observation period, it

was apparent that $461 Fremyella diplosiphon in sucrose had

produced a barely visible layer over most of the culture

vessel bottom; however, no subsequent increase could be de-

tected 60 days later. In order to verify this apparent

heterotrophic ability in §L$1 Fremyella. diplosiphon, somewhat

more favorable growth conditions were employed. Six 250-ml

Erlenmeyer culture vessels containing 160 ml of .01 M sucrose

in ASM-1 were each inoculated with 0.1 ml cell suspensions

from the questionable heterotrophic culture. Three of these

were wrapped in aluminum foil and all were placed on a shaker

at 100 cycles/min. At the end of a 30-day growth period, the

light grown cultures averaged about 50 mg dry wt/vessel

whereas the dark grown cultures produced less than 0.1 mg dry

wt each.

50

The failure of #4$1 Frernyella dlploslphon to show any

heterotrophic growth upon transfer from dark culture seems

to lend support to the theory that most autotrophically

grown blue-green algae may only be able to grow as hetero--

trophs for short periods of time before some light dependent

energy source is exhausted,. In fact, the inability of any

of the five isolates from deep water-well pump slimes (obvi-

ously, areas devoid of light) to show any sort of heterotrophic

response and seemingly obligate autotrophy inherent in all of

the remaining species tested, supports Holm-Hansen's (1967)

contention that most evidence of heterotrophic growth in

blue-green algae may be questionable.

Apparent production of earthy or musty odors occurs in

healthy and growing cultures of #486 Nostoc muscorum, #5^4

Nostoc commune, #617 Symploca muscorum, #42$ Oscillatoria

tenuis, #NT6$~B Oscillatoria sp., #NT63-19 Oscillatoria sp.,

and $NT68-20 Oscillatoria sp. A survey of the organisms

3.istecl (Table 2) and 12 additional isolates not yet cleared

for experimental purposes indicates this ability may be con-

fined to two families of the Nostocales--the Nostocaceae and

Oscillatoriaceae. If this experimental sample, which is re-

stricted to about 60 randomly selected species or strains of

blue-green algae, can be considered statistically valid and

51

perhaps generally representative of the Cyanophyceae, then

odor production seems to appear less frequently in the genus

Nostoc (2 of 9 different cultures are odor producers) than

in the genus Oscillatorla (with 6 of 10 cultural types pro-

ducing odor).

Early testing on different mineral media (Chu's, MSS,

Hughes, and ASM-1) had demonstrated no significant differ-

ence in the level of odor production within those species

possessing such capacity, as long as significant growth occurs.

However, odor production abruptly ceased, when odor producers

were transferred from complete to nitrate-free medium, even

though growth continued, with a gradual decline to zero as

the nitrate supply was exhausted and nitrogenous reserves

depleted. Since odor production appeared to accompany growth,

the inference was that its termination should have followed

the same type gradual decline. Since odor production abruptly

ceased in the two Nostoc odor producers (nitrogen fixers),

this observation initially suggested that odor production and

nitrate metabolism might be linked; however, no "hard" sup-

portive evidence was obtained..

Upon cultivation in nitrate-free medium over extended

periods of time, the two Nostoc cultures slowly began to

resume odor production at rates considerably less than when

grown on complete medium. Some evidence obtained later and

52

reported elsewhere in this paper suggested that this observed

"lag" in odor production was more likely due to metabolic

sluggishness brought about by a slow rate of nitrogenase

induction.

Inconclusive tests indicated that all odor producers

apparently yielded odors when cultivated in a medium initially

containing either of the following ions: NO^ . NOg, as the

only source of combined nitrogen. Odor production on ammo-

nium would seem to rule out involvement in nitrate metabolism.

Techniques for measuring odor production are inexact; there-

fore, these data must be considered presumptive and not

confirmatory. Technical improvement in odor production

measurement is prerequisite to the presentation of unequivo-

cal data.

Five b.!ue-green algal species which did not produce

odors were selected for inoculation with two notably strong

odor-producing actinomycetes as has been previously described.

The algae selected were #756 Nostoc sp., #NT63-1$ Nostoc •

sphaericum, #3^6 Oscillatoria chalybea, #390 Oscillatoria

formosa, and #NT6&-6 Oscillatoria sp. Intermittent observa-

tions over three subculture periods ending at 90 days failed

to confirm the presence of odors in any of the test mixtures

except immediately after the initial inoculation. Since

53

substantial amounts of odor materials were probably "carried

over?T in the intentional].1/ heavy exordial inoculum, these

results were assumed to be negative.

Despite the general agreement that atmospheric nitrogen

utilization by blue-green algae was solely confined to fila-

mentous genera with heterocysts (Fogg and Wolfe, 1954; Singh,

1961; Stewart, 1966, 1969; Pringsheim, 196$), there have

been occasional reports that this ability may be present in

other genera: Gloeocapsa minor (Odintzova, 1941)> Chroococcus

ruf esc ens (Cameron and Fuller, I960) , Oscil.lato.ria subbrevis

(Moyse et al,, 1957)> Lyngbya aestuarii (Van Baalen, 1961) .

Since non-fixing strains of species normally using atmospheric

nitrogen had also been reported (Allen, 1952; Kratz and

Myers 1955a) , clonal isolates of each blue-green algal species

used in these studies were tested for this ability. The

isolated clone was placed in nitrate-free medium (ASM-1) and

observed through three consecutive subcultures of 30 days

each. If apparent growth developed, the isolates were then

subjected to further testing.

The discovery (Dilworth, 1966; Sch'ollhorn and Burris,

1966, 1967) that the nitrogen-fixing enzyme, nitrogenase, not

only reduces nitrogen to ammonia but also acetylene to ethy-

lene makes it practical to measure indirectly the ability of

54

nitrogen .fixation in any suspect culture.- Reduction o.f N2

to ammonia requires six electrons (three electrons/NH^ mol-

ecule produced) and reduction of acetylene to ethylene

requires two electrons per molecule. This theoretical rela-

tionship results in a possible 1.5 to 1.0 production ratio

of ethylene to ammonia.

The extensively developed techniques of Stewart et al.

(1967, 1963) and Hardy et al. (1968) in acetylene reduction

as an indirect measurement of nitrogen fixation were used to

obtain all data reported herein relative to nitrogen fixation.

The detection (Wyatt and Silvey, 1969) of nitrogen fix-

ing ability in #795 Gloeocapsa sp. (Table 6.) has produced,

perhaps for the first time, valid evidence of atmospheric-

nitrogen fixing ability in a member of the Chroococcales.

Also, since #795 Gloeocapsa. sp. is a non-heterocystic alga,

this ability eliminates the suggested possibility that the

heterocyst might be the site of nitrogen fixation (Fay et al.,

1968; Ogawa and Carr, 1969), at least for this species of

alga.

Most blue-green algae lose the ability to utilize atmos-

pheric nitrogen (usually attributed to "dilution out" of the

nitrogenase system) when grown in ample supply of combined

nitrogen. An investigation of comparative duration and rate

55

TABLE 6. Ethylene production in #795 Gloeocapsa sp. and. reference organisms

Organisms Mean Mean mg Mean mp moles O.D.^JQ cells ethylene/mg

cells/min

#795 Gloeocapsa sp. .072 19.3 .112

#942 Cylindrospermum sp. .114 8.8 .578

#436 Nostoc muscorum .068 7.7 .246

y#5$4 Nostoc commune .158 30.0 .042

#424 Tolypothrix distorta .05$ 10.0 .173

#427 Phcrmidium faveolarum .090 8.2 .0

Uninoculated controls .0 .0 .0

#795 Gloeocapsa sp. controls:

*dark bottles .068 '16.4 .0

**predark treatments .032 20.5 .0

^-^nitrate repressed .104 24.7 .0

*Serum bottles wrapped in aluminum foil.

-^Samples also receiving 8 hours predark treatment but light incubated.

***Samples removed from complete ASM-1 only 18 hours prioj to gasing.

of decline of nitrogen fixing ability in selected blue-green

algal species upon transferral to complete medium is incom-

plete at this writing; however, studies concerning the reverse

process (induction rates for the nitrogenase system) are pre-

sented in Figures 3 and 4.

56

— — #NT68-£ Anobaenq subcvlindrica

#1444 Anahaena flos-oauGO

- 2 0 0

Fig. 3-initiation,

Hours of nifrogenase induction

Acetylene reduction rates after nitrogenase

57

120

no

o o o

X c E v. m "U o

E

© c © »>

•*-©

Vt & o E ± E

# 629 Anobaona cylindrica

# 424 Tolypothri* tenuis

# 941 Colcfhrix membranacea

# 4 8 6 Nostcc muscorum

24 36 49 6 0 72

Hours of nitrogonase induction

Fig. 4—Acetylene reduction rates after nitrogenase initiation.

58

In four of seven species studied, a brief spurt of ethy-

lene production after about three to six hours was recorded.

A simple explanation for this phenomenon is that since these

organisms were briskly growing and undergoing rapid metabolic

activity in complete medium, some "store or pool" of an ac-

tive metabolic product was probably present in the experi-

mental cultures; until its exhaustion, this "pool" permitted

limited nitrogenase synthesis. This early-produced nitrogenase

was not in sufficient amounts to maintain continual protein

synthesis and overall metabolism at pre-transfer levels;

thus the decline and gradual resumption of acetylene reduction

ability as normal physiological processes adjusted to the new

substrate.

There seems to be a wide range in effective nitrogenase

induction times; jf379 Calothrix membranacea (not shown) did

not start to reduce acetylene by the end of a 96-hour period,

and #4^6 Nostoc muscorum's reduction rate was almost non-

existent at the same induction period.

Nitrogen fixation by free living heterotrophic bacteria

in aerobic, non-organic media is probably very slight

(Delwiche and Wijler, 1956; Kuznetsov, 1959; Dilworth and

Kennedy, 1963; Chang and Knowles, 1965); however, when nitro-

gen fixation in blue-green algae is being studied, there is

59

still a marked need for control methods which enable the

investigator to be certain that a measured response is due

solely to the species being considered. The reason for this

is that he may have to use non-axenic cultures, or if not,

absence of bacterial contaminants remains theoretically im-

possible to prove (Tischer, 1965), since proof is supported

only by negative evidence.

One seemingly effective control is dark-bottle fixation.

Since evidence presented elsewhere in this paper demonstrates

almost universal obligate autotrophy in the blue-green species

tested and lengthy heterotrophic responses (Allison et al.,

1937; Fay? 1965; Watanabe and Yamamoto, 1967) are question-

able (Holm-Hansen, 1967), the following experiments have been

designed to determine the length of pre-dark treatment neces-

sary for dark-bottle controls. The duration and rate at

which selected cultures of light grown blue-green algae can

continue to fix nitrogen (as measured by acetylene reduction)

in darkness are measured. The normal photosynthetic acetylene

reduction rates of randomly selected culture stocks (Table 7)

are measured in triplicate and each mean becomes the initial

point of the dark-bottle ethylene production studies pre-

sented in Figures 5 and 6. After selected pre-dark treatments,

the rate of acetylene reduction is recorded and plotted.

60

TABLE 7. Acety.1 ene reduction rates in selected, nitrogen fixing species

Organisms moles ethylene/mg cells/min

#NT69-6 Anabaena Bornetiana .785 1.400 (.943) . 644

#1444 Anabaena flos-aquae .096 •09S (.086) . 066

#379 Calothrix membranacea .084 .072 (.097) .135

#942 Cylindrospermum sp. .606 .801 (,730} .784

§195 Gloeocapsa sp. • .027 .027 (.027) .028

$486 Nostoc museorum .019 .012 ( .014) .011

#1301 Fischerella musicola .257 .269 ( .322) # 442

The data generally indicate a dramatic fall in the ability

to produce ethylene within a six-to eighth hour period; those

species having the lowest rates decline to undetectable levels,

but some of those with higher rates persist in low-level

acetylene reduction at least 24 hours. In any case, marked

photodependence is apparent in all cultures tested.

61

150

o o o X

E \ w *5! o a* E m c © *>»

0> (0 © O £ d. E

100

# 1301 Fischarelia musicola

SO O CM •<r to Cvi cn f*~ k 4

# ©42 Cyhndrospermi'in s&

# NT69-6 Anctba&na BornstiGfla

Hours of pre-dork treatment

Fig. 5—"Dark Bottle" acetelene reduction rate decrease by pre-dark treatment.

62

— # 379 Colof lirix membronccea

# (444 Anobaeno flos-aauoo

# 795 Gloeocagsa sg.

# 436 Nostoc rnuscorum

Hours of pre-dark treatment

Fig, 6 !,i)arK .riott ie" acetylene reduction rat© decrease by pre-dark treatment.

63

In order to test experimental establishment of commensal

or mutualistic relationships between species of blue-green

algae unable to utilize atmospheric nitrogen and free-living

nitrogen-fixing bacteria, 10 mixed cultures were prepared in

the manner previously described. Controls included flasks

of nitrate-free medium, each inoculated only with a blue-green

species, and culture vessels containing complete ASM-1 which

were inoculated with the bacterial-algal mix. The blue-green

algal species used were #625 Anacystis nidulans, #1519 Eucapsis

sp., #4$8 Lyngbya sp., #390 Oscillatoria formosa, #42& Oscil-

latoria tenuis, #427 Phormidium faveolarum, #617 Symploca

muscorum, #1191 Synechococcus cedrorum, #NT63~4 Chroococcus

sp., and #NT6S~5 Chroococcus sp.

In no case was survival (retention of color and continu-

ous growth) observed except in all the complete medium con-

trols. In the nitrate-free medium, the rate of vitality

decline (as seen by yellowing or lack of green coloring) in

the "bacterial-algal" and "algae only" cultures was indistin-

guishable. This failure to establish a relationship between

the presence of nitrogen-fixing bacteria and survival of non-

fixing blue-green algae should suggest that continuous growth

by a blue-green alga, whether axenic or not, over extended

periods of time in nitrate-free medium almost certainly should

establish it as a nitrogen fixer.

64

Since the metabolic behavior of laboratory cultures is

not unvarying, the respiratory and photosynthetic rates

listed in Table 8 are not presented as absolute values;

rather, these are rates generally reflecting the metabolic

nature of those species when cultured in the laboratory.

Nonetheless, in order to insure as much uniformity as possible

under the conditions employed, duplicate recording periods,

and in many cases, replicates from different parent cultures

were used to establish these values.

Twenty-one-to twenty-eight-day-old, seemingly vigorous

and healthy stocks were usually selected; the exception was

the use of #NT69-6 Anabaena Bornetiana and $NT69~2 Anabaena

circinalis in log growth phase. Selection of cultures was

directed toward the "normally metabolizing" cultures at the

top of the log phase, or in the early stationary phase because,

if a culture is growing rapidly, the total cell mass changes

drastically during the course of the experiment if several

different light and dark periods are monitored for the same

culture. Since low-intensity light was lised throughout the

course of these tests, only a maximum of about 20 to 40 mg

cells could effectively be employed in the 200 ml of medium

measured,because of self-shading.

65

TABLE 3. Respiration and Photosynthesis

Organism mean mp moles CC^/mg cells/min

a, Planktonic isolates Fixed Respired Net

#NT69-6 Anabaena Bornetina 51.75 15.91 35.34

#NT69~2 Anabaena circinalis 17.63 7.4S 10.15

#NT63~9 Phormidium sp. 6.59 2.66 3.93

#NT63--12 Phormidium sp. 6.5S 1.43 5.16

#NT63~10 Pseudanabaena catenata 10.69 2.06 3.49

b. Species with generally high tory culture

metabolic activity in labora-

#629 Anabaena cylindrica 4.60 2.50 2.10

$625 Anacystis nidulans 6-20 2. SO 5.40

§ 1519 Eucapsis sp. 5.13 2.12 3 .01

$531 Plectonema boryanum 6.47 1.35 5.12

#1191 Synechococcus cedrorum 9.26 3.29 5.93

c. Species generally exhibiting moderate metabolic rates

#1444 Anabaena flos-aquae 4 • 42 2.06 • 2.36

$NT63~3 Arthrospira sp. 3.49 1.56 1.93

#433 Lyngbya sp. 5.34 0.39 4.95

$NT63-13 Phormidium sp. 4.17 0.89 3.27

$NT63~20 Oscillatoria sp. 3.43 1.90 1.52

66

TABLE 0. — Continued

d. • Species generally metabolieally inactive in laboratory culture

mean mp moles C02/mg cells/min

Organism Fixed Respired Net

#379 Calothrix membranacea 0.30 0.21 0.09

#1301 Fischerella musicola 0.58 0.35 0.23

#466 Nostoc muscorura 1-95 1.15 0.80

#NT68-l8 Nostoc sphaericum 0.56 0.21 0.35

#424 Tolypothrix tenuis 0.43 0.35 0.13

The respiratory and phccosynthetic rates expressed in

Table $ demonstrate that, once adapted to laboratory condi-j

tions, blue-green algae produce at rates comparable to or

greater than those of other algal groups (Norris et al., 1954;

Eppley and Sloan, 1965; Nalewaiko, 1966; Nalewajko and Marin,

1968).

Rates of photosynthesis and respiration in the species

tested for this series of experiments remarkably reflect

other laboratory observations—namely, that those organisms

which generally appear to have the highest metabolic rates

have high photosynthetic and respiratory rates, and vice

versa. Overall, the net production rate was about 60 per

cent: Group a averaged about 70, Groups b and c about 65,

and Group d, 40 per cent. Generally, the more "active"

67

organisms were more efficient producers, even if their

respiratory rates were also higher.

Probably one of the more significant factors detected

in these series of experiments was the wide variation in

respiratory efficiency among different species. In conduct-

ing several replicate measurements, it was noted that the

repeatability of this feature was much better than the actual

value of the total amount of gross photosynthesis, respira-

tion, or net production. For example, almost any species

nurtured into log rate growth that can be kept evenly sus-

pended in culture medium at low cell density (about 10 mg

cells/200 ml medium) can be induced to fix 10 to 20 mp moles

CO2 per mg cells per minute; whereas", under normal laboratory

conditions, the gross productivity never approached these

levels. Regardless of the total fixation and respiration

rates, the respiratory efficiency of each species tends to

remain much the same in several replicate measurements. How-

ever, since the cultures were invariably selected for uniform-

ity, some reservations must be maintained regarding this

seemingly stable respiratory efficiency.

After several hours of darkness, ranging from $ to 13

hours, and perhaps depending upon different physiological

states of cultures being measured, there is a period in which

63

the rates of gross photosynthesis and respiration are equal.

The duration of this period, arbitrarily defined as "net

production lag phase" is apparently dependent upon the length

of the dark period. It was seldom noted after dark periods

shorter than 8' hours, but has lasted up to 4 hours in a ,

culture which had been in darkness 1$ hours. Upon light

initiation, the "net production lag phase" starts almost

instantly; its termination is as abrupt as its inception,

with the net photosynthesis curve assuming its characteristic

shape immediately.

After a long dark period, there seems to be either some

marked cellular deficiency, with all production immediately ?

oxidized for replenishment energy, or there is a possible

depletion of some component or enzyme system necessary for

production in excess of respiration. Since the "net produc-

tion lag phase" curve is a straight line, the former explana-

tion may be best suited. Investigation of this phenomenon

is continuing.

CHAPTER IV

CONCLUSION

The results of this study indicate that use of modern

methods and technological advances have not removed much of

the element of chance from determining which individual

blue-green alga might be successfully isolated. Several

species obtained in this study are not listed (and presum-

ably absent) in some of the world's major blue-green algal

culture collections (Indiana University, University of

Wisconsin, Cambridge University), and this may be the first

time that any aspect of their physiological nature has been

investigated. On the other hand, such well known and com-

monly occurring forms as Anabaena variabilis, Aphanizomenon

flos-aquae, Nostoc commune, and Nostoc muscorum are conspic-

uously absent from the list of isolates.

These studies indicate that a particular species can

usually be relatively easily isolated if it happens to occupy-

some special ecological niche (e.g., #NT6$~6 Oscillatoria sp.

was the only alga in a slime sample from a deep water-well

pump) or- if it has some special characteristic or structure

(e.g., nitrogen fixing ability; endospores or akinetes which

6Q

70

resist heating or drying). Possession of highly adaptive

traits which enable a species to develop rapidly in culture

will sometimes free it from other organisms.

Fortunately, except in soil crusts where several differ-

ent Nostocs commonly occur, different species of the same

genus usually do not occupy the same ecological niche at the

same time; however, other combinations which have extremely

similar cultural characteristics (e.g., Oscillatoria and

Phormidium) have often been encountered and are almost; insep-

arable. Another very difficult and perhaps completely un-

predictable task is the isolation of a pre-determined,

innocuous blue-green algal form which has no special selective i

feature. For example, during the past two years, many attempts

have been made, but without success, to isolate the incon-

spicuous but ever present planktonic genus, Gomphosphaeria.

The length of time spent attempting to isolate this species

does not approach record status, however, for Taha (1964)

spent eight years isolating Haplosiphon fontinalis, Anabaena

variabilis,and Calothrix elenklnli.

The contention of Bowyer and Skerman (196$) that chance

plays a major role in successful purification of blue-green

algae is certainly supported by the data presented in this

paper. Other than a somewhat limited success with elevated

71

temperatures, little or no improvement over the u. v„ radia-

tion treatment initiated forty years ago by Allison and

Morris (1930) is demonstrated in this study. It is perhaps

fortunate for mankind that few other biological endeavors

suffer this ignominy.

Among generic species, euplanktonic blue-green algae

seem to generally express an overall metabolic rate in excess

of that found in similar forms from other environments.

Individual species tested have generally shown wide variation

in particular physiological and biochemical responses; how-

ever, little "hard" evidence for strikingly different metabolic

patterns (e.g., the failure of deep water-well isolates to

exhibit an expected heterotrophic response) between recent

isolates and commonly cultured forms has yet to be revealed

by these inquiries. Nonetheless, many subtile dissimilarities

which merit further study are apparent.

Comparisons of blue-green algal growth rates are usually

regarded as reliable indices of general metabolic behaviour.

Evidence was sought, but not found, that significant metabolic

changes have occurred in long-cultured forms because of the

"unnatural" selection of artificial cultivation. The litera-

ture does reveal significant growth rate differences in forms

that have been laboratory cultivated for several years; for

72

example, the early growth rates reported for Anabaena

cylindrica (Fogg, 1944)> Anabaena variabilis (Drewes, 192$),

and Nostoc museorurn (Allison and Morris, 1930) are about

half those reported by Browne11 and Nicholas (196?), Ogawa

and Carr (1969), and Rzhanova (1967) in recent papers which

investigated each species respectively. However, the data

reported in this investigation support the contention that

this increased rate is much more likely due to improved

culture media rather than to changes resulting from selected

physiological adaptations during the years in culture.

The detection of the ability to fix atmospheric nitrogen

in a member of the Chroococcales {§195 Qloeocapsa sp.) t

reveals that this ability is not confined solely to fila-

mentous genera with heterocysts. Since the latter view has

enjoyed rather wide support in the past, some reservation

should be maintained regarding widespread generalizations

now in vogue.

Taste and odor production by blue-green algae does not

seem to be a common trait. Of about 60 species tested, only

seven have this trait. Odor production has always accompanied

growth, but there is no evidence that manufacture of odor

compounds is directly affected by nitrogen nutrition.

73 r

The inability to establish mutualistic or symbiotic

relationships between free-living nitrogen-fixing bacteria

and non-fixing blue-green algae in nitrate-free medium, and

failure to promote-odor producing systems consisting of

actinomycetes and non-odor forming blue-green algae in purely

mineral medium strongly suggest that neither combination of

organisms can solely support each other. Therefore, when a

non-axenic blue-green algal culture survives and grows through

repeated transfers in a medium free of combined nitrogen, it

is almost certainly a nitrogen fixer; by the same token,

continuous odor production by a blue-green algal culture is

highly indicative that the alga is solely responsible for the

odor.

Absolutely no evidence of heterotrophic growth in blue-

green algae is revealed by this study. While it may be

possible that this method of life support occasionally occurs

in blue-green algae, obligate autotrophy is the rule in all

species tested. One hundred percent survival without growth

was noted for all species investigated during a 90-day test

period.

Measurements of respiratory and photosynthetic rates

indicate that at least one-half of the species tested are

highly efficient in organic production. The euplanktonic

74

forms tend to have the highest production rates, and true

soil dwellers the lowest. Perhaps the two most interesting

features revealed in these studies are the widely varying

"respiratory efficiency rates" in the different species and

the commonly occurring "net production lag phase" after

dark periods of several hours' duration.

LITERATURE CITED

Allen, M. B. 1952. The cultivation of Myxophyceae. Arch. Mikrobiol. 17:34-53.

Allen M. B. and D. I. Arnon. 1955a. Studies on nitrogen--iixing o.lue—green algae. I. Growth and nitrogen fixation by Anafcaena cyli.ndri.ca Lemm. Plant Physiol. 30:366-372.

Allen, M. B. ana D. I, Arnon. 1955b. The sodium requirement o f Anabaena cylindrica. Physiol. Plant. 5:653-660.

Allen,_Mt M. and R. Y. Stainer. 1963. Selective isolation of blue—green algae from water and soil. J. Gen. Microbiol 51:203-209. '

Allison, I-. E. and S. R. Hoover. 1935- Conditions which favor nitrogen-fixation by a blue-green alga. Proc. 3rd. Int. Congr. Soil Sci. 1:145-147.

Allison, ?. E., S. R. Hoover, and H. oT. Morris. 1937. Phys-iological studies with the nitrogen-fixing alga, Nostoc museorum. Bot. Gaz. 98:433-463.

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