Fixation of nitrogen by algae and associatedorganisms in semi- arid soils: identification

and characterization of soil organisms

Item Type Thesis-Reproduction (electronic); text

Authors Cameron, R. E.(Roy E.)

Publisher The University of Arizona.

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FIXATION OF NITROGEN BY ALGAE AND ASSOCIATED ORGANISMS

IN SEMI..ARID SOILS: IDENTIFCATEON AND

CHARACTERIZATION OF SOIL ORGANISMS

by

Roy Eugene Cameron

A Thesis Submitted to the Faculty of the

DEPARENT OF AGRICULT(JRAL CHEMISTRY AND SOILS

In Partial Fulfillment of the Requirements'or the Degree of

MASTER OF SCIENCE

In the Graduate College

UNIVERSITY OF ARIZONA

19S8

STATET BY AUTHOR

This thesis has been sunitted in partial fu.lfiThnent of re-quiruents for an advanced degree at the University of Arizona andis deposit in the University Library to be made available to borrow-ers under rules of the Library.

Brief quotations from this thesis are allowable withoutspecial permission, provided that accurate acknowledgment of sourceis made. Requests for permission for extended quotatiQn from orreproduction of this manuscript in whole or in part may be granted'by the head of the major department or the Dean of the Graduate Go].-].ege when in their judgment the proposed use of the material is inthe interests of scholarship. In ail other instances, howe-er, per-mission must be obtained from the author.

SIGNED:(C

APPROVAL BY THESIS DIRECTOR

This thesis has been approved on the date shown below:

W. H. FULLERHead, Department of Agricultural

Chemistry and Soils

1929 Born July 16th, Denver, Colorado

19)47 Graduated from Lincoln High School, Tacoma, Washington

19)47-19)48 Attended the University of Washington, Seattle, Washington

195)4-1955

1950-1952 U. S. Army Medical Laboratory Technician

19)48-1950 Attended the State College of Washington, Pullman, Washing-

1952-195)4 ton. B. S. in Agriculture 1953. B. S. in Bacterio1or andPublic Health 195)4.

1955-1956 Research Laboratory Analyst, Hughes Aircraft, Thcson, Arizona

1956-1958 Graduate Student in Soils, University of Arizona

May 1955 M. S. University of Arizona

Honor Societies: Sigma Alpha Omicron (Bact.), Pi Tan Iota (Pre-Med.),Beta Beta Beth, and Phi Lambda Upsilon, Sigma Xi (Assoc. Member)

1

ACKNOWLEDGMENT

The author wishes to express his appreciation to the staff of

the Department of Agricultural Chemistry and Soils, University of An--

zorn for their cooperation during the progress of this research. To

Dr. Fuller, the research director, the author is indebted for the basic

idea of this research. His willing expenditure of time, stimulating

suggestions, constructive criticism and over-all supervision greatly

contributed to the production of this research.

Gratitude is also expressed to other departments and individuals

whose cooperation was necessary due to the nature of the thesis topic.

Sincere thanks are here extended to a few of these individuals: Dr.

W. S. Phillips, Head of the Botany Department,for invaluable time spent

with the author on algal identifications; Dr. K, F. Wertauan, Head of

the Bacteriology Department, for the use of facilities; Dr. R. Kuyken-

dali, Assistant Professor of Horticulture, for the use of experimental

chelates; and to Mr. J. E. Fletcher, USDA Soil Conservation Service, for

advice, suggestions, and invaluable information0

The author also wishes to express his indebtedness to the U. S.

Atomic Energy Commission for funds which made this work possible0

TABLE OF CONTENTS

INTRODUCTION 1

LITERAJRE REVIEW 3

Laboratory Research on Nitrogen Fixation by Algae . . 3Importance of Soil Algae in Agriculture UMethodology 23

EXPERI1'UNTAL METHODS AND MATERIALS 32

SUWIA.RY 103

BIELIOGRkPHY io6

iii

Collection of Samples and Isolation for Purpose ofIdentification of Microorganisms

Nitrogen Fixation in Pure and Mixed CulturePreliminary ProcedureCulture Solutions and Conditions for Nitrogen

32

3939

Fixation IoApparatus for Nitrogen Fixation Li3

Nitrogen Fixation by Soil Crusts I8Analyses for Nitrogen Fixation in Nutrient

Solutions 50

RESULTS AND DISCJSSION 53

Isolation and Identification 53Description of Blue-Green Algae Observed 65Nitrogen Fixation with Pure and Mixed Cultures . . 83

Organisms Not Demonstrating Nitrogen Fixation . 83Organisms Demonstrating Nitrogen Fixation . . . 85Chemical Analyses of Soil Crusts 96Nitrogen Fixation by Soil Crusts 99

LIST OF TABLES, FIGURES, AND PLATES

ThblesNumber Page

Culture Media for Algae , 37

Total Micronutrient Solution for Algae. 38

Conditions for Nitrogen Fixation in Pure Culture . . 1414

14. Conditions for Nitrogen Fixation in Mixed Culture . . . 145

Description of Samples and Genera of Algae Within Them. 514

Genera of Algae Found in Arizona Soils 58

Nitrogen Fixation in Aqueous Solution by Blue-Green Algaein Pure Culture 87

Nitrogen Fixation in Aqueous Solution by Blue-Green Algaein Mixed Culture 88

Nitrogen Fixation in Aqueous Solution by Blue-Green Algaein Mixed Culture 89

Summary of Nitrogen Fixation in Culture Solutions . . . 91

Chemical Analyses of Samples of Soil Crusts ContainingAlgae and/or Lichens 97

Nitrogen Fixation by Soil Crusts in Moist Chambers . . . 101

Figures FollowingNumber Page

1. Nitrogen Fixation by Soil Crusts in Moist Chambers , . . 101

Plates FollowingNumber Page

i, Typical Sample Collection Area 32

iv

Following

NumberPage

Close-up of Algal Crusts 32

Close-up ol' Conspicuously Raised Lichen Crusts . . 32

14. Incubation of Soil Crusts in Moist Chambers 314

. Growth of Nostoc sp. in Nitrogen Fixation Experiment, 314

Experiment on Nitrogen Fixation with Lyngbya

Diguetti . . . . . . . . . . . . . . . . . . 314

Nitrogen Fixation Urder Conditions of Controlled Tem-

perature, Light, and Use of Filtered Air . . . . . 143

8 Nitrogen Fixation Conducted at Room Temperature Using

Natural Light and Filtered Air . . . 143

Nitrogen Fixation by Soil Crusts 143

Photoinicrograph of Chroococcus rufescens 81

Photoinicrograph of Anabaena sp. 81

Photomicrograph of Aphanocapsa grevillei . 81

V

INTRODUCTION

Nitrogen is one of the elements essential for plant growth. It

is also the least abundant major essential element in the soil. In aria

and semi-arid regions of Southwestern United States there is very lit-

tie organic matter in the soil, and therefore very little nitrogen

(1b9), since soil nitrogen usually is almost wholly in the organic

form. Amounts of organic matter in virgin Arizona soils usually do not

exceed 0.1 to 1.0 percent (103).

Soils lose nitrogen by one or more processes. Since Arizona

soils are low in nitrogen, constant replenishment is required to main-

tain maximum crop production. Nitrogen losses may be balanced by re-

plenishuients through the addition of animal and green manures, crop

residues, artificial fertilizers, or by fixation processes involving

symbiotic or nonsymbiotic microorganisms.

In temperate regions agricultural soils left fallow for some

years are found to gain in nitrogen and organic matter, even though no

manures, crop residues, or artificial fertilizers are applied (200).

This increase is believed to be brought about by fixation of atmospheric

nitrogen by microorganisms. Nitrogen fixation is considered as an Ira-

portant means for gaining soil nitrogen, and of economic importance in

the maintenance of soil fertility. Although nonsymbiotic fixation has

been reported to be especially important in desert soils (lit8), it has

received little attention compared with the vast amount of research on

1

symbiotic nitrogen fixation.

To better evaluate the importance of nonsymbiotic nitrogen

fixation in soil fertility, it is essential to gain information about

the characteristics of the organisms involved, their habits, a.nd the

factors influencing their vital processes. The problem involved in

this research considers the identification of soil algae, particularly

the blue-green algae, and the determination as to which of these algae

are nitrogenfixers. A study of their habits is necessary for cultur-

ing purposes and to obtain quantitative -i nfonnation on nitrogen fixation

by the organisms in question. The general method used to solve these

problems consisted of the procurement of soil crusts and the isolation

and identification of organisms within the crusts0 The organisms ob-

tained were then cultured and nitrogeh fixation determined in pure and

mixed cultures, using available laboratory facilities and equinent1

LI1RATURE REVIEW

Laboratory Research on Nitrogen Fixation by Algae

Frank, in 1859 (89), was the first to receive attention for in-

vestigation of fixation of free atmospheric nitrogen by blue-green

algae in soil cultures, He found that there were gains in soil nitro-

gen but mistakenly proposed, on the basis of his impure culture experi-

ments, that all algae possessed the ability to fix atmospheric nitrogen,

In 1891 Schloesing and Laurent (202) suggested that algae probably sup-

plied carbohydrates to nitrogen-fixing bacteria, and in 189k Kosso-

witsch (133) concluded that algae did not fix nitrogen even though

some of his flasks of mineral salt solutions gained in nitrogen fixed

with the organism Nostoc present. Bouilhac (3), Bouilhac and Giustini-

ani (32, 33) further investigated the fixation of nitrogen with associ-

ations of algae and bacteria. They inoculated sand cultures of buck-

wheat, mustard, corn and cress with impure cultures of blue-green algae.

Not only were they able to grow plants under these conditions, but they

also found an increase in nitrogen in the sand cultures. In 1901 Bei-

jerinck (18) obtained nitrogen fixation in initially nitrogen-free media

using a small amount of soil and a generous inoculuin, principally of

Anabaena catenula and other Anabaena spy,

Difficulties in isolating blue-green algae in pure culture limi-

ted the value of early work on nitrogen fixation. However, the

3

bacteriological methods of Robert Koch (8) were used with increasing

success in isolating bacteria-free cultures. Use of gelatin plates en-

couraged research arid the development of bacteriological techniques, such

as repeated plating and streaking out, provided a means for removing con-

tiinating bacteria from gelatinous coverings of algae. Tischutkin (238)

in 1897 and Beijerinck (17) in 1898 are credited with being the first to

use agar for cultivating algae. Pringsheim (166) and Chodat (51) also

led the way in research by using agar. to obtain bacteria-free cultures of

algae.

According to Pringsheim (189), Zumstein (1900) was probably the

first to obtain bacteria-free cultures of algae, but in 1912 Pringsheini

(187) may have been the first to isolate blue-green algae (Oscillatoria

and Nostoc species) in bacteria-free cultures. Neither Pringaheim (187)

nor his students, Glade (107) and Maertens (150) in l9lL could show

nitrogen fixation for species isolated. Nitrogen fixation still could

not be shawn to occur with algae without associated bacteria according

to Nakano (165) 1917, Emerson (72) 1918, and Lipman and Teakie (lLi2) 1925.,

Moore and Webster (157) in 1920, also worked with impure cultures of a1-

gae. Nitrogen fixation occurred and was attributed to the algae, but no

attempt was made to obtain cultures free of bacteria and other organisms.

In 1917 Harder (111) obtained a pure culture of Nostoc punctiforme frcmi

Gunnera scabra. Harder observed growth in the absence of combined

sources of nitrogen, but attributed growth to nitrogenous impurities in

the agar rather than to nitrogen fixation.

In 1921, Wann (219) reported nitrogen fixation in pure cultures

by some members of the Chlorophyceae, but results for nitrogen fixation

S

could not be confirmed (!41, 7, 8). Fixation by impure cultures of

green algae had been previously reported negative by Kossowitsch (133)

in 189I, Kruger and Schneidewind (]Jo) in 1900, and Schramm (206) in

19l1.. Even as late as 1927 Bristol-Roach (37) questioned the reli-

ability of results obtained for nitrogen fixation by algae in pure cul-

tures. It was decided that positive results obtained for nitrogen fixa-

tion were due either to the presence of other organisms or faulty chern-

ical methods. She criticized Wann for his methods in determining ni-

trogen fixation, even though he had used pure cultures of algae.

In 1928 Drewes (65) obtained the first bacteria-free cultures

of blue-green algae shown to fix atmospheric nitrogen. Two organisms,

Nostoc punctiforme and Anabaena variabilis, were shown to be able to

fix 2-3 mgra. of nitrogen in a nitrogen-free medium after a fifty day

incubation period, Allison and Morris (7) in 1929 partially confirmed

Drewes' work in that they had also independently isolated An. varia-

bilis in pure culture from soil, They found that this organism fixed

an average of 5 mgm. of atmospheric nitrogen, or 8.5 mgm. with sucrose

added, per 100 cc. of originally nitrogen-free medium during an incu-

bation period of 75 days. In 1932 they claimed fixation of 11.6 mgm.

of nitrogen per 100 cc. of medium for this organism (8). Copeland (53)

in 1932 then claimed that species of Oscillatoriaceae and Chroococcales

fixed nitrogen, but there was no adequate evidence presented for the

purity of the cultures used.

There were still reports of nitrogen-fixing bacteria being

present in the gelatinous coverings of blue-green algae (129), but evi-

dence of nitrogen fixation for pure cultures of blue-green algae

6

became more and more substantiated. Some doubts were raised about the

supposed role of algae in relationship with bacteria. For example,

Allison and Morris (8) could find no further increase in nitrogen by

growth of Azotobacter vinelandii alone, than when this organism was

grown in the presence of Chiorella, Chlamydoinonas, Scenedesmus, and

Pleurococcus (obtained from Wann). According to previous theories,

there should have been a mutually beneficial interrelationship between

algae and bacteria, with algae supplying carbohydrates to nitrogen-fix-

ing bacteria (3l,76,UL,.,l29,l33,lL10,l65,l99,202).

Further evidence was gained for nitrogen fixation by blue-green

algae in pure culture by Winter (265) in 1935, who had isolated strains

of Nostoc punctiforne from Cycas and Gunnera. Allison, Hoover, and

Morris (9) in 1937 published additional results and conditions for

growth and isolation of Notoc muscoruin, They found the amount of ni-

trogen fixed to be 10 mgni. in L5 days or 18 mgin. in 85 days per 100 cc,

of a medium originally nitrogen free and containing no carbohydrates.

A nunber of reports on nitrogen fixation by blue-green algae were forth-

coming within the next few years.

In 1939 De (58) of India reported nitrogen fixation in pure

cultures for the blue-green algae Anabaena gelatinosa, An. naviculoides,

and An. variabilis. In 191i.0, Bortels (30) obtained nitrogen fixation

for Anabaena cylindrica, An. huinicola, An, variabilis, Nostoc paludosum,

and two newpecies, Cylindrosperuin lichenfonue and C, maius, In l9I.2

Singh (216) obtained nitrogen fixation by blue-green algae isolated from

paddy soil8 (as bad De ilL 1939), These were identified as Anabaena

ambigua, An. fertilissima, Cylindrospermum gorakporense, and a new

7

species, Aulosira 'ertilissiina. Also in 19142, Fogg (80) confinned

nitrogen fixation by a strain of An, cylindric*. Conclusive evidence

was then presented for fixation of atmospheric nitrogen by blue-green

algae in pure culture by Drewes (65), Allison t al. (o,9,l0,), De (58)

Bortels (30), Singh (216), and Fogg (80). Rate of algal growth var-

ied from 0.3 to 2.0 gms. (dry weight) of cell material and amount of

nitrogen fixed varied from 2.0 - 180.0 mgnl. per liter for incubation

periods of 20 - 85 days.

Watanabe, et al. (253) in 1951 made a significant contribution

to the research on nitrogen fixation by blue-green algae after investi-

gating a number of soils. He and his co-workers found 13 species that

fixed nitrogen, including the organisms Tolypothrix teni,s Oslo thrix

brevissima, Anàbaenopsis, Schizothrix, and Plectonema. Unfortunately,

Watanabe's publication (253) does not reveal the use of filtered air;

cotton-stoppered flasks containing nitrogen-free media were incubated

in the laboratory. A review of the literature does not show support-

ing evidence for fixation by members of the genera Schizothrix or

Plectonenia. Allen (1) could not show fixation for Plectonema notatum.

Fogg and Wolfe (87) state that Watanabe's claim for fixation by

Schizothrix and Plectonema is based on impure culture studies, This

may explain the 4earth of supporting evidence in the literature.

Williams and Burrs (255) in 1952 obtained negative evidence

for a. number of blue-green algae using the radioactive isotope NlS.

These included the following algae: Coccochioris peniocystis, Diplo-

cystic aeruginosa, (lloeocapsa membranina, Aphanizomenon flos-aquae,

p].ectonema nostocoruin, Phorinidiuin tenue, and Gloeocapsa dimidiata.

8

Positive fixation was obtained for Nostoc muscorum, Nostoc ap., Gale-.

thrix parietina, and an impure culture of Gloeotrichia. Fogg and

Wolfe (87) in 1951 also attribute nitrogen fixation to impure ciii-

tures of Gloeotrichia, Odintzova (171) claimed nitrogen fixation by

the organism Gloeocapsa minor, but evidence in support of this was

unsatisfactory because pure cultures of the organism were not isolated.

Herisset (117) in 19b6 and again in 19S2 (86) has shown that

the organism Nostoc commune must be included among the nitrogen-fix-

ers. Fogg (83) also claims demonstration of nitrogen fixation by a

blue-green alga, Mastigocladus laminosus, which was isolated from a

hot springs. Recently, Okuda and Yamaguchi (l7Li.,l75) reported nitro-

gen fixation for Tolypothrix tenuis, Nostoc songiafonue, Nostoc

spongiaforme plus Phormidium crossbyaniuzn, and Chroococcus dispersus,

but do not mention the use of pure cultures. The weight of evidence

has been previously negative for nitrogen fixation by a Chroococcus

(86,87). Nitrogen fixation has now been reasonably established for

common blue-green algae of three families, i.e. Nostocaceae (Nostoc

and Anabaena), .Ftivulariaceae (Galothrix), and Scjytonemataceae (Toly-.

pothrix). Few additional algae have probably been found to fix nitro-

gen, and a few strains of previously determined nitrogen-fixing genera

have been found not to fix nitrogen (l,l3l.i). Some changes in identi-

fication of organisms may also be necessary from time to time (oh.,86,

131). A good review of the distribution of nitrogen-fixing blue-green

algae is given by Fogg and Wolfe (87), and again by Fogg (86) in 196.

No green algae are as yet positively known to fix nitrogen, although

there are still occasional reports. For example, Fernandes and Bhat

9

(?5) in l915 reported good growth o± Chiorococcum humicolum in a ni-

trogen-free medium. Spoehr and Milner (255) also reservedly reported

that Chiorella may be able to fix nitrogen under special conditions,

but Fogg (86) believes that the gains of nitrogen were probably due

to absorption of traces of a,imnonia and oxides of nitrogen from the air.

However, a difficulty may be found in that certain algae may not be

readily classified as "blue-green" or "green", e. g. Allen's work on

a blue-green Chlorefla (1,2).

Pure culture techniques were developed further as a result of

research by Allison and Morris (7) and Bortels (30). Ultra-violet

light was first used by Allison and Morris in 1930 to destroy contain-

mating bacteria in algal cultures. Subsequent workers also used this

method to obtain bacteria-free cultures without destroying the algae,

i.e. Gerloff, Fitzgerald, and Skoog (105), 1950, Fogg (83) 1951, and

Henrjksson (US) 1951, Allen (1) also used ultra-violet light, but

not for routine purification of cultures. Beijerinek (16) in 1890 had

developed plating out in gelatin, then in agar (17), and this has been

a method used by many subsequent workers. Pringsheim (186) used and

recommended plating out in agar in 1912 and still does forty years

later (192). Skinner (218) in 1932 published a lucid description of

similar methods employed in isolating 50 strains of soil algae. Sili-

ca-gel has also been used by such investigators as Pringsheim (187)

19114, Schramm (206) 19114, De (58) 1939, and Singh (216) 19142. Other

methods that have been used to obtain pure cultures are given in the

following references (1,23,106,192). Fogg (80) used chlorine water.

Antibiotics have been the latest innovation in obtaining pure cu1ture

10

of algae (77,lOB,191L,195,l96).

Agar is still a preferred medium for isolation (192) but agar

is not always found in a pure condition and often must be carefully

washed to remove impurities (23). A recent report shows that agar mai

contain significant amounts of heavy metals (15).

Wann (2I9) and Allison and Morris (7) were among the first to

observe the precautions of Beijerinck (18) in regards to removing ni-

trogenous impurities from air used in experimehts on nitrogen fixation.

This precaution was again set forth by Fogg (80) in 191i.2, but is still

not observed by many workers, some as late as Watanabe et al. (253)

1951, and Sen (210) 1956.

The lCjeldahl method is used to determine the amount of nitrogen

fixed. This method has reoeived much criticism by some research work-

ers, i.e. Bristol-Roach and Page 4l) 1923, Bristol-Roach (37) 1927,

Wilson (260,261) 1937and 19l0, Hiller.et al. (118) 191i8, Fry (102)

19%, and De and Mandal (60) 1956.

An analytical technique whic1 has proved to be of vast impor..

tance in biological nitrogen studies is that described by Rittenberg

et al. (198). In 1938 Rittenberg and his co-workers constructed a mass

spectràgraph for determining the nitrogen isotope in organic com-

pounds. This technique was first developed for studies on protein

metabolism in animals (2oL.), then used in 19140 to study the assimilation

of ammonia by tobacco plants (2142). In 19141 Burns and Miller (147)

indicated the application of N15 for the study of biological nitrogen

fixation. In 19142 Burns (1414) established imporlant techniques in the

use of in a study of the distribution of isotopic nitrogen in

11

Azotobacter. First mention of the use of for studying nitrogen

fixation by blue-green algae is in a paper by Burns, . (145) in

19142, but no quantitative data are given. Since then, Burns, Wilson,

and associates have used &- many times to study nitrogen fixation by

blue-green algae (146,148,1147,255,263,2614.).

By means of a technique now developed using NiS, conclusive evi-

dence can be obtained as to whether or not a certain biological systn

can fix atmospheric nitrogen. irJhen air is enriched with N, atom per-

cent of NiS in cells may be determined and significant amounts shown to

be present in nitrogen-fixing algae. As demonstrated by Schoenheimer

and Rittenberg (2014) in 1938, an increase of 0.003% N above the normal

0.368% in less than one mgrn. of nitrogen may be determined using the

mass spectrograph. On an international basis, Bond and Scott (26) in

195.5, and Scott (207) in 1956, in Scotland, have used N15 to study ni-

trogen fixation in lichens and liverworts containing Nostoc. Use of N15

combined with newly developed rapid culture techniques by Allen and

Arnon (3,14) should greatly facilitate research on nitrogen fixation by

blue-green algae. Unfortunately, however, equipment and facilities

necessary to obtain and analyze Nl.5 are costly and limit the use of this

method' (263).

'Importance of Soil Alg.e in Agriculture

Soil algae have been described since very early historical times

and studied by workers throughout the world since the middle of the 18th

century (235). There appears to be a rather definite algal flora in soils,

members including the Gyanophyceae or blue-green, Chlorophyceae or green,

12

Xanthophyceae or yellow-green, and the BaclUariophyceas or diatoms.

Although many species may occur in aquatic environments, some apparent..

ly occur only on soils (235). These may include Botyridium, Proto-

siphon, and certain species of Zygneinia, Zygogonium, Oedogoniurn, 13o-

trydiopsis, Vaucheria, and Microcoleus, Algae are found not only on

the surface of the soil, but under the surface as well, to depths of

several inches to a few feet. Bristol-Roach (38) found them growing

heterotrophically below the soil surface. Moore and Karrer (160) and

Moore and Carter (159) found a definite subterranean algal flora in

the Missouri Botanical Garden. Smith (220) investigated the algal flora

of Florida and found Chiorococcuni abundant to a depth of two feet and

Stichococcus to a depth of thirty inches in a Norfolk fine sand, Wil..

son and Forest (259) in 1957 found many algae present to a depth of

six inches in soils in central Oklahoma. Fritsch (95), however, ex-

pressed the view that the majority of subterranean algae had 'either

been washed down to lower layers or else carried down by tillage oper-

ations or soil, fauna. Stokes (227) did not believe that algae possesse

any significant activity in the subsurface soil.

Considering that there is no noticeable penetration of light

beneath the soil surface, it is noteworthy that many algae can be

grown in the dark (23). This phenomenon was first investigated by

Artari (13) in 1906 and by many workers since then (l,36,39,1.O,55,56,

.6,219). Some bluegreen algae grow in the dark and fix nitrogen. Ac-

cording to Allen (1), this was first reported by Bouilhac in 1897 when

he grew Nostoc punctiforme with sugars. Harder (in) in 1917 also grew

N. punctifoxne in the dark. AllisOn, Hoover, and Morris (9) grew

13

N. muscoruin in the dark as did Stokes (227). Allison, et al. (9) found

that when N. muscorum grew in the dark, 10-12 mg. of nitrogen waa fixed

per grain of glucose utilized. Others who grew nitrogen-fixing algae

were Winter (265) in 1935, De (58) in 1939, Allen (1) in 1952, and Fogg

and Wolfe (87) in 1951i.. According to Fogg (86), in 1952 Herisset found

that another species of Nostoc, N. commune, fixed nitrogen, but not

when grown in the dark. Fogg (86) believes that analytical methods

used by Herisset were probably not sensitive enough to have detected

small amounts of nitrogen fixed.

The importance of algae in colonizing bared areas of rock and

soil can not be denied (81). This function of algae was particularly

exnplified in a publication by Treu.b (2LLO). He noted that after the

volcanic explosion of Krakatoa in 1883 the island was denuded of all

visible plant life, but several years later the first plants to appear

were blue-green algae which formed a dark green gelatinous layer over

the pumice and volcanic rock. Algal succession in colonizing a rocky

island was also recently noted in India by Parija and Parija (180), In

1907 Fritsch (91L) portrayed the colonization of new ground by algal

growth and in 1915 Fritsch and Salisbury (101) published an account of

algal colonization of burned-over heath land in England. The algal

growth was found to be composed of Gloeocystis, Cystococcus, Troe1-

cia, and Dactylococcus. Bews (20) found a blue-green alga, Gloeoapsa

sanguines, to be the earliest colonizer of bare rock surfaces and cliffs

in South Africa.

Algae have received recognition by a number of workers as being

important agents in stabilizing soil which has lost vegetation due to

114

erosion. According to Russell (200) Bolyshev and Evdolthnova noted the

importance of algal surface crusts in Russia, as had Shtina (2114) in

197. Odintzova (171) found blue-green algae colonizing rock and fix-

ing nitrogen in Russian desert areas. Vogel (2143) observed that xero-

philou5 Qyanophyceae grew on and under the soil surface and retarded

erosion in the South African desert. Jones (129) in England referred

to the frequent observation of prolific growths of blue-green algae in

sandy wastes where higher plants failed to grow. In Australia, Tchan

and Beadle (231) found nitrogen-fixing blue-green algae, Nostoc and

Anabaena as well as members of the Chlorophyceae, in a number of' rocky

ridges, bare soil, stony land, dunes, and scalded areas. Moewus (1S14)

also in Australia, found 14 algal species in semi-desert areas, 26 of

them being species of Gyanophyceae. Bright blue-green algal films oc-

curred underneath quartz stones.

In our own country, Elwell, et al. (71) noted the importance

of algae in control of erosion. Booth (27) observed that algae were

not only important in erosion control, but had an influence on water

infiltration. Jn Arizona, Fletcher and Martin (78) observed the in.-

fluence of algae and. molds in rain crusts on the stability of soil.

The algae found were all Cyanophyceae: Oscillatoria, Nodularia, Micro-

coleus, Nostoc, and several members of the family Chroococcaceae.

Others had found that rain crusts reduced water infiltration and pro-

moted erosion (28,70,201), but Fletcher and Martin (78) believed that

the inicroflora of rain crusts imprQved infiltration, decreased erosion,

and aided in establishment of seed plants under harsh desert conditions.

Since their observations, Osburn (177) had also noted that soil algae

were important in range land because of their stabilizing influence on

rainfall and control of erosion. Shields, et al.(212,213) in l97 ob-served the stabilizing influence of algae and lichen surface crusts in

semi-arid habitats of New Mexico and other areas. Piercy (182) and

Drouet (66) found that soil algae were important colonizers of land

denuded of visible vegetation by drought and this is, of course, of

great importance in any arid and semi-arid area.

The success of algae as colonizers and stabilizers of soil pro-

bably depends a great deal on factors such as the ability to withstand

desiccation (3S,96,99,111j.), extreme temperatures (2,51i.,83), a high salt

concentration (5,1L9,81,217), and the ability of certain algae to fix

nitrogen on diverse substrates and to foxm symbiotic associations with

other organisms such as lichens (26,81,8,86,uo,uS,2O7,212,213).Without doubt, the earth depends on the nitrogen-fixing capa-

city of certain organisms (81,261), and the algae, especially the blue-

gz'een algae, play an important part in the nitrogen economy of the

soil (211). In this respect, algae are again of importance in arid and

semi-arid areas, Robbins (199) believed that "niter areas" in Colorado

soils were due to nitrogen-fixing organisms and found a number of algae

present. Odintzova (171) found that certain blue-green algae fixed ni-

trogen under desert conditions and were responsible for an accumulation

of nitrate. Shtina (21h) also attributed increased nitrogen and fer-

tility of soil as due to the growth of algae. Fogg (81) in 19LL7 re-

viewed thepart played by blue-green algae and noted that blue-green

algae were important in the nitrogen nutrition of the desert, In Ari-

zona, Fletcher and Martin (78) found increases of OO percent in nitrogen

16

content as well as 300 percent increases in organic carbon content in

rain crusts with extensive algal growth.

In Australia, Tchan and Beadle (231) estinated that algae pre-

sent in a desert soil were responsible for one pound ol' nitrogen per

year added to the soil. Recently, Shields, et al (213) found lh algal

species in alga- and lichen-stabilized soil crusts from several semi-

desert substrates in New Mexico. Amino nitrogen for 9 lichen crusts

from lava soil averaged 1985 ppm. and for 50 corresponding algal crust,

ppm, The amino nitrogen averaged 866 ppm. for lava surface soil

lacking algal or lichen growth.

Algae, since most of them obtain carbon from the air, are also

noted for their role in adding organic matter to the soil. Robbins

(199) noted that algae contributed to the organic matter in arid areas

of Colorado; as nientioned before, this was also noted by Fletcher and

Martin (78) in Arizona It has also been noted by Fletcher, as quoted

by Fuller and Rogers (io1.), that three tons of carbon or six tons of

organic matter may be added to an acre of soil. What additional roles

algae may play in desert soils remain to be investigated. Active

growth of algae is responsible for the tying-up of nutrients, but after

death and decay, nutrients may be lost or returned to the soil, Mitsui

(153) has shown that losses of nitrogen may be due to algae. Fuller

and Rogers (iOu.) have shown that algal phosphorus may be utilized for

crop growth.

Agriculturally, algae are becoming of increasing importance in

ri.ce soils, although the importance of algae in soils has been doubted

by some workers. Stokes (226,227,228) believed that algae had only a

17

limited value in soil, although he did not deny that b1ue.green algae

were direct participants in the fixation of nitrogen in the soil. As

f or rice soils, in l91O Chaudhari (So) of India believed that bacteria

instead of algae were chiefly responsible for nitrogen fixation; dead

algae supposedly functioned to improve the growth of Azotobacter.

Algae have probably been known to be present in rice $0115 for

centuries, even before their worth was realized. Indian workers such

as Ayyanger (ilL), noted that scum" increased fertility and delayed

ripening of rice, but Harrison and Aiyer (13) in l9llL were probably

the first to suggest that nitrogen fixation occurred among algae and

bacteria in algal films on Indian paddy fields. Howard (122) in 192l.L

also suggested that nitrogen used to replenish rice soils was obtained

from the atmosphere and, "the most probable seat of thi.s fixation is

in the submerged algal film on the surface of the mud" Howard (122)

was also one of the first to observe that rice crops were produced

year after year in India on the same land and without addition of

fertilizer (manure). This same observation has been noted by others

According to Singh (215), Banerji in 1935 was one of the first

to investigate the algal flora of a paddy field, but investigated only

the surface soils. Singh (215) should probably be credited for his

notably thorough investigation of the surface as well as subsurface

algal flora of paddy soils from samples collected in 1936. He found b3

species of algae in collections taken from paddy fields of four disc-

tricts of the United Provinces. A good many members of the Cyanophy-

ceae were observed to be growing on these soils. It was not until 191L2

18

that Singh (216) investigated the fixation of nitrogen by blue-green

algae in paddy soils and showed that all of the algae investigated were

active in fixation of atmospheric nitrogen. One alga, Aulosira fertil-

issixna, was found to Lix 8 mgm. of nitrogen per 100 cc. of nitrogen-

free medium after an incubation period of 15 days. De and Pain (61)

noted in 1936 that there was a definite algal growth in paddy fields,

but in laboratory research, they could not show any significant increase

of nitrogen for soils exposed to sunlight for a month. In that same

year, further research by De (57) suggested that nitrogen fixation iflwater-logged soils was probably not a bacterial process, and that the

addion of calcium stimulated algal growth and increased nitrogen

fixation. Unfortunately, the only alga identified in his soils was

Phormidium orientale and this by Fritsch in England,

In 1938 De and Bose (58) studied the microbiological conditions

existing in rice soils, but made no mention of algae. De next went

to England to work under the highly recognized algologist, F. B.

Fritch (21). Fritech and De (98) put forth a paper on the fixation

of nitrogen by pure cultures of three species of Anabaena and a species

of Phormidium isolated from rice soils, The Phorinidiuxn sp, was not

found to fix nitrogen, but the three species of .Anabaena fixed 3-5 nigm.

of nitrogen per 100 cc. of culture medium for an incubation period of

two months. An important finding was that the amount of nitrogen fixed

by algae in the presence of Azotobacter and other bacteria was the same

as in pure cultures of algae. It was therefore concluded that bacteria

played a relatively unimportant part in nitrogen fixation in rice fields

In 1939, De (5) published a more detailed paper on the role of

19

blue-green algae in rice fields and stated that nitrogen fixation is

brought about mainly by algae and that bacteria may even play no part

in nitrogen fixation in rice soils. It was also reported that blue-

green algae, Anabaena spp., were able to fix 5.3 - 5.8 mgm. of nitrogen

per 15 gm. of soil. De and his associates have published several

papers since then, noting the influences between algal growth and rice

crops. In 1956 De and Mandal (60) were able to show that cropped, but

unfertilized, soils were able to fix 13,8 - lbs. of nitrogen per

acre. Both growth and fixation were considerably increased in the pre

sence of the crop (60,62,63).

Another worker in India, Prasad (183), has also contributed to

the knowledge of nitrogen fixation by algae in rice fields and found

that 12.9 lbs. of nitrogen per acre were added to rice fields after

harvest as a result of fixation by algae.

In the Far East, rice is also an important crop, if not the

most important crop (200) and nitrogen fixation in rice soils is of

consequent importance. In Japan, Nakano (165) investigated the associ

ation between algal flora and Azotobacter, and when Mo].isch (155)

visited Japan, he found numbers of blue-green algae growing on exten..

sive tracts of ground. Watanabe, et al. (253) did extensive work with

blue-green algae, not only in Japan but in much of the Eastern hemis-

phere. Of 6b3 samples of blue-green algae coflected from the Far East

and South Seas, 13 species were found to be nitrogen-fixers. These

species were abundant in the tropics, but not in Japan and other parts

of northern Asia. Watanabe and his associates found that by means of

pot cultures cropped with rice plants and inoculated with Tolypothrix

20

tenuis, fixation of nitrogen was obtained corresponding to 20 lbs.

per acre.

In 1956 Watanabe (252) reported on the results of experiments

conducted at eight experiment stations in which T. tenuis was applied

to rice plants in paddy fields. A5 a result of inoculations with

this blue-green alga, the yield of rice increased by 2.7 percent the

first year, 8.14 percent the second, 19.1 percent the third, and 21.8

percent the fourth year, based on an average of eleven fields. Calo-

thrix also exerted a positive influence on rice plants, almost equal

to that of T. tenixLs, although it did not possess the same nitrogen-

fixing ability (251). Hirano, et a].. (U9) obtained similar results

with inoculations of T, tenuis.

Other workers in Japan, Okuda and Yaxnaguchi (172,1714,175),

have also studied nitrogen-fixing blue-green algae in paddy soils.

They found that blue-green algae were widely distributed in Japan, oc-

curred more frequently in paddy soils than in ordinary farm soils and

uncultivated soils, but were more plentiful in the latter soils than

reported by Stokes (226). Nitrogen fixation occurred under all water-

logged conditions and was attributed to the blue-green algae and photo-

synthetic bacteria (173,175). Nonsuiphur purple bacteria were believ-

ed to have nitrogen-fixing ability and to be important in the nitrogen

economy of paddy soils, since they occurred frequently in paddy soils

but were absent in ordinary farm soils (175,176).

In this country Willis and Sturgis (257,258) have reported the

loss of nitrogen from flooded soils. Willis and Green (256) also have

reported the loss of nitrogen by downward movement in flooded soils

2].

planted to rice. By means of' pot cultures, Willis and Green (256) were

able to demonstra,te that nitrogen fixed by blue-green algae was enough

to support a good crop of rice and provided nitrogen left over equival-

ent to 70 or more pounds per acre. Results of their study indicated

that gains of fixed nitrogen in growing rice fields may be equivalent

to greater than the nitrogen utilized by the crop.

In this countz'y nitrogen is cmonly the only nutrient element

applied to rice (229), but Watanabes experiments (252,253) have shown

economical means of applying nitrogen to rice with inoculations of

Tolypothrix tennis. Also of importance is that Allen (3) has recently

shown in this country that plants may be grown at the expense of

molecular nitrogen when inoculated with Anabaena cylindrica. Allen (3)

envisioned the possibility of fertilizing other crops by a prelimLnary

flooding of a field and consequent growth of a mass of nitrogen-fixing

blue-green algae.

There is little doubt now that nitrogen fixed by blue-green

algae is available to non-nitrogen-fixing organisms (81). Allison, et

a].. (9) found nitrogenous products in the external culture medium of

Nostoc muscoruin, Winter (265) found this was true for N. punctifonte

(or Anabaena cycadeae). Both De (58) and Fogg (8h) have shown that

healthy cultures of blue-green algae may contain up to ho percent of

the nitrogen in soluble organic forn. Henriksson (115) showed that a

pure culture of Nostoc isolated from the lichen Collema tenax contri-

butes about one-fourth of its fixed nitrogen to the external medium.

Later experiments showed that up to 15 percent of the nitrogen was

extracellular (116). Watanabe (251) found that Caiothrix secreted"

22

free amino acids, and Magee and Burns (lL7) identified amino acids

excreted by Anabaena cylindnica and Nostoc muscorrni. Approximately S

percent of the total nitrogen fixed was found to be excreted.

In desert soils Shields, et al.(213) have found that algae and

lichens in soil crusts, through death and decomposition, release amino

and other nitrogen compounds. The surface growth of algae and lichens

contribute a continually renewable supply of soil nitrogen.

ItETHODOL0GY

Diverse procedures have been used to solve the problems such as

those involved with the research presented here. In view of this, the

following review is presented which may be useful to others involved in

s.inilar research,

Any number of methods could have been utilized to secure samples,

methods of sampling being as diverse as the individuals performing the

sampling (l00,l1O,l27,l2S,lLi3,li4IL,lI,l2,l6O,2l2). For the most part

information on sampling is oftentimes not mentioned (8,67,78,98,l66,J.67,

l68,23), or is fragmentary (l,73,l7L,l7,l99,220,230,259). Mitra (l2)for example, was extremely cautious in the coflection of samples. He

used a steel plate that was flamed each time it was pushed into the soil,

a hot scapula for scraping off samples, and a sterilized cork borer for

the actual sample coflection. Lund (l1i3,1I) collected samples with a

sterile knife whereas Tchan (230), Tohan and Beadle (231) made no men-

tion of sterile technique, but did have sterilized containers.

Several methods have been used for the purpose of isolation

and growth of algae. Fred and Waksmari (90) list a method whereby soil

may be introduced into flasks of inorganic salts. This method was also

used by Moore and Karrer (160), Pringsheim (190,191), and Beijerinck

(17). Mineral solutions used may include Chu #l0 Knop Molisch3 Pring-

sheim Beijerinck, Detmer, Bristol and others (23,189).

23

214

Various dilutions may be used in order to isolate the algae,

but the use of any one medium is not encouraged since it may be too

selective, Problems of this nature were noted by Moore (158) as early

as 1903 when he found that there was no one method or medium which is

equally well adapted to all algae.

Others were also aware of this problem and. a number of media

for culturing algae became established. After extensive investi-

gation, Allen (i) listed a summary of nutrient solutions which had

been used to cultivate members of the Hyxophyceae. It is also real-

ized that small quantities of various organic substances may enhance

growth (1,105,189,192), but they also enhance the growth of contamin-

ants (1). Fogg (86) states that there has been no demonstration of

organic growth factors for nitrogen-fixing blue-green algae. Nutrient

solutions are unfavorable for growth in some cases because some algae

are terrestrial rather than aquatic (235) and grow poorly, if at all,

in an aquatic environment.

Inoculation into sand plates was attempted as early as 1893

by Koch and Kossowitsch (132) and by subsequent workers (159,160).

This method was an improvement over that of nutrient solutions in

flasks in that a broad, relatively flat, solid substrate was accessible

for growth, Allen (1) in 1952 also noted the influence of physical

conditions for growth and added Pyxex glass wool to nutrient solutions

in order to provide a more or less solid substrate,

Esmarch (73) in 19114 was one of the first to use a moist chain-

ber method for the study of blue-green algae in the soil. He intro-

duced soil into a petri dish, moistened it with sterile water, placed

25

a piece of filter paper on the soil surface, and set the chamber in

light in order to develop the growth of algae on the filter paper.

Fletcher and Martin (78) used an innovation of this method when they

placed the filter paper under the soil sample. This method has been

successfully used lately by others (213,259).

As mentioned previously, agar was used with success for growth

and isolation of algae. Moore (158) used flasks of agar, as did Ohodat

(Si). Plating out was developed for isolating algae based on Beijer-

inckts experience with bacteria (23). Pringsheini (188) in 1926 recom-

mended plating out, but also advocated the expediency of streak cul-

tures (188). Allen (1) made repeated transfers on agar in order to

obtain pure cultures of filamentous blue-green algae. However, it

is not recommended that agar be used unless it is purified. Beijer-

inck (17) in 1898 and Moore (158) in 1903 were early workers who main-

tained that agar should be washed when used for algal cultures, and

Pringsheim and Pringsheim (193) in 1910 even demonstrated that agar

could be used as a source of energy for nitrogen-fixing organisms.

A smp1e method of washing agar was first suggested by Beijerinok (17)

in 1898. Bold (23) reviews the various methods of purifying agar.

One method consists of washing previously weighed agar in a confining

cheesecloth bag. It is washed for several days in tap water and then

soaked for several days in changes of distilled water before adding

nutrients.

A review of nutrient solution media for algae (23,189) dis-

closes that elements included are most of the essential elements re-

quired by higher plants, but not necessarily in the same quantities.

26

4Undebatab1eessentja1 elements are known to be carbon, hydrogen, oxygen,

phosphorus, potassium, nitrogen, sulfur, magnesium, and iron, most of

which can be supplied in mineral form (162). Analyses on the ash of

algae show the usual 'essential' plant minerals to be calcium, potas-

sium, magnesium, phosphorus, sulfur, nitrogen, iron, copper, manganese,

boron, and zinc (22,136). Carbon, although supplied in the form of

carbonates in media, is obtained from the air as CO2 by most algae (8S)

and nitrogen may be assimilated by blue-green algae as the element, as

nitrate, nitrite, ammonia, or in various organic forms (87) It is ad-

visable not to include nitrogen in the medium for experiments on nitro-

gen fixation (Ll,1S9).

Calcium may or may not be a required element (1), and Pringsheim

(192) states that, "Calcium is either not needed by many lower algae

or is required in emounts as to be always present", and applies the sane

statement to manganese. However, Allen and Arnon ()) have shown that

calcium is essential for Anabaena cylindrica arid Walker (2L) has

shown that calcium or strontium is required by Ohlorefla pyrenoidosa.

Walker (2)) has also shown that manganese is required by Chlorella

pyrenoidosa. Boron is necessary for the growth of Nostoc muscorimi (7L)

and cobalt is an essential element for this alga as well as for other

blue-green algae (3,120),

Sodium is essential for growth of Anabaena cylindrica (3,),

and JCratz and Myers (l3l) have shown that both sodium and potassium are

required for maximum growth of three other species of blue-green algae.

Sodium is required for growth of algae regardless of the potassium

status (214) and other alkali metals can not be substituted for it (3).

27

I4olybdenum was one of the first microelements shown to be

required by nitrogen-fixing organisms (29) and also was shown to be

necessary for nitrogen-fixing blue-green algae. It can not be replac-

ed by vanadium (3) and appears to be needed by algae for nitrate re-

duction as well as nitrogen fixation (3,14,82,a,87,266,267). Vanadium

has been established as an essential element for Scenedesmus obliquus

(12). Two other elements, copper and zinc, may be required by some

algae (2LLi,2l.i5), and silicon is necessary for normal valve formation

by diatoms (22).

Pringsheim (192) would include very low concentrations of the

following elements to cultures for algae: lithium, copper, zinc,

boron, altmrinum, tin, manganese, cobalt, nickel, titanium, iodine,

and silicon. No medium for algae can be considered adequate without

provision for micronutrients (136) but the amount necessary is some-

times debatable and usu.afly the microelements of all media for algae

are provided by 'shotgun' procedure (1,l3Li.), For this reason, a "total'

micronutrient solution may be prepared in order to include all possi-

b].e required microelements.

Recently essential microelements for algal cultures have been

added in the chelate form (233). The iron source for algal cultures

has received considerable attention in this respect and several differ..

ent iron sources were used in this research. In early research on

nitrogen-fixing blue-green algae, iron was supplied in the form of

ferric chloride (9) and is still listed for use in prepared algal

culture solutions (131,189), However, when ferric chloride is compared

with ferric citrate in culture solutions, the ferric citrate is found

28

to give higher yields (los). It is to be noted that the stock solu-

tion must be autoclaved separately, otherwise it is hydrolysed to

ferric hydroxide (12). This precautionary measure was observed in the

research reported here. Hutner, et al. (l2) next made a contribution

toward a better culture solution by investigating new complexing agents

collectively termed u chelates'1 Chelates, usually EDTA., have now

been used extensively in algal research (i&,ll,19,l3lt,136,137,170,233,

214h). Kratz and Myers (l3L) claim that citrate is as good as EDTA.

for algal cultures, but Waris (2O) states that EDTA is a far better

complexing agent than citric acid. Krauss (136) recommends the use

of chelates for alga]. øiltures since it helps to prevent precipitation

and will release enough ions through mass action to provide for the

need of cells. It is claimed that a chelating agent is necessary in

order to obtain reproducible and maximum growth rates of blue-green

algae (131i). 'Blue-green algae prefer a slightly alkaline medium (1,

81,189), so it may be advisable to use chelates recommended for alka-

line conditions (208,209).

Son blue-green algae may have a very high optimum pH (lOS),

however, values of pH 7.0 - 8.5 have been used to grow nitrogen-fixing

blue-green algae (9,88,l15,1l6,13t). An alkaline shift in pH may be

expected in time, but this may not be altogether undesirable (105). A

weakly alkaline reaction is usually obtained by the addition of di-

potassium hydrogen phosphate (1,23), but calcium or sodium carbonate

may be added as a buffer (9,23,105). The pH of a nediuin changes after

sterilization (23), which may be due to the later effects of aeration

(9,23). The initial and final pH can then be determined by the proper

29

choice of salts. If a solution of dipotassium hydrogen phosphate is

used, it is recommended that it be sterilized separately and added to

the medium after cooling in order to prevent precipitation (14).

There has not been much extensive investigation on the influ-

ence of temperature for culturing blue-green algae (1). Many algae

appear to have a certain temperature range beyond which they do not

survive, but blue-green algae have been found in frigid Antartic lakes

(97), while one blue-green nitrogen-fixing alga has been isolated from

a hot springs (83). The usual temperature for research purposes is

20 - 2S°C (i), although higher temperatures may be used (9,714).

The influence of light on the growth of algae has also re-

ceived little extensive investigation Ci). Allen and Arnon (14) state

that algae may be grown at rather low intensities of 300-3000 lux, but

found that the yield of cells increased with intensity up to at least

16,000 lux. Laboratory cultures of algae can develop with an inten-

sity of l0 foot-candles (88,9].), or less (714,92,163,178). For alga].

cultures, the light source used may be sunlight (23,1014,1614), tungsten-

filament lamps (93,13S,189), fluoresent lights (12,91,116,2SS), or

both (l3b,l3). Fluorescent light is, convenient because a cooling

fan or water bath is not necessary and is probably satisfactory for

all algal cultures (178), although it is low in red rays (178,181).

If the light source is provided by sunlight, certain precautionary

measures are necessary since it may be too intense for best growth

(9,189) and in small culture vessels the concentration of heat may ex-

ceed the tolerance of most algae (23).

Aeration and agitation are two additional important factors to

30

be considered in culturing of algae, not only to provide aerobic con-

ditions, but in order to keep the cells in suspension (131). Bold (23)

believed that aeration was beneficial and in some cases obligatory.

For aeration purposes, it is usual to introduce additional carbon di-

oxide into the air stream as a stinulant to photosynthesis (23). Five

percent carbon dioxide is commonly administered (138,161,163), and

even required for dense laboratory cultures (178). Lesser amounts of

carbon dioxide have also been used; the range may vary from 0J - 3

percent (9,l0,131i,221t,22S). It has been shown, however, that 0.03

percent carbon dioxide normally found in air provides sufficient carbon

dioxide to pennit an optimum rate of photosynthesis in algae (138,169).

A condition is that this amount may be sufficient only. when a very strong

air stream is driven through a small culture (138).

Agitatior of cultures is maintained to be necessary according

to Myers (161). Miller and Fogg (i1) found that they could obtain

considerably more algal growth with cultures that were continuously

shaken. They also found that aeration was not a satisfactory substi-

tute for shaking. Krauss (138) found that a greater photosynthetic

rate could be achieved in natural or laboratory cultures either by

agitation or turbulence. Allen and Arnon (Li)did not believe that

shaking was strictly necessary for good growth when a large liquid-gas

interface was available.

AU of the above factors are to be considered in the culturing

of algae. Additional factors which may receive attention relate to

the apparatus to be used in a system and the proper culture vessels, As

mentioned previously, various filtering devices may be used in

31

experiments on nitrogen fixation (9,80), but others (U5,116,2S3) grow

them in individual flasks exposed to unfiltered laboratory air. If an

apparatus is used, the choice of culture vessels may be the most jim-

portarit and troublesome engineering aspect 3). Culture vessels may

vary in algal research from polyetbylene lined vats (139) and gallon

carboys (l6I) to large test tubes (12), Pyrex Roux culture bottles (l31.)

Erlerimeyer flasks, or Pyrex glass washing bottles (9). In considering

the composition of culture vessels, Pyrex containers may be preferable

(23,189,192), and it is emphasized that the glassware not have been

in use for tOo long a period of time (23).

As a final stage in the research, nitrogen fixation may be de-

termined by analytical methods involving the micro-Kjeldah3. method

(121) on aliquots of algae and/or medium (L1,9,116). Algal growth may

be separated from the medium by means of filtration (116), or the use

of a centrifuge (LL,130) when it is desired to determine nitrogen in

either fraction.

EXPERDIENTâL METHODS A1D MA.T3RIALS

Collection of Samples and isolation for Purpose of

Identification of Microorganisms

Soils were óollected in Arizona from areas ranging as far

north as Phoenix to as far Bouth as Benson and Nogales. The majority

of samples were collected from the Tucson area. Sampling was begun

in October 1956, with the collection of soil crusts, and concluded in

September ]..9S7. Over 100 samples were collected, of which some were

selected for specific experiments, and the majority examined for the

presence of algae. See Plate 1 for a typical collection area and

Plates 2 and 3 for samples collected,

Samples were collected using ólean, but not sterile, spatulas;

one spatula was used per sample collected during any one collecting

expedition. The surface crusts, or surface soIls, were placed in

new pint or quart cylindrical, heavy paper cartons with lids, and the

underlying soil into clean paper sacks using a gardnert s hand trowel.

Sterile equipment was not used, but certain precautionary technique

was observed. Soils collected from any given area were assigned an

identification number and further subdivided by means of letters,

The letter "a" was used to characterize particular surface crusts, or

surface samples of a few millimeters to as much as - inch, and ?lbll for

the soil collected to a depth of 6 inches beneath the flaU crusts, The

letter 110W also represented surface rusts from the same area, but

32

Fl. 2Close-up of algal

crusts shown in P1,1.Algal crusts are sev-eral inches in diame-ter. Crusts in placeare shown in the areasurrounding the dis-played crusts (sample18a).

P1. 1Typical collection

area, with vegetationof dry grass, weeds, andcacti. Also shown arecrust samples on sackused for subsoil and cy-llndrical paper contain-ers for crust samples.

P1. 3Close-up of conspic-

uously raised lichencrusts which occurredon rocky soil in Cata-lina foothills. (sam-ple 50a).

33

different in some respect, e.g. time of collection, and "d" was used

to designate the soil removed down to a depth of 6 inches beneath

A brief description was also made of the location from which the

samples were collected and notations made as to obvious moisture con-

ditions, evidence of fresh (active), or desiccated growth of algae,

and any other possibly pertinent information influencing the growth of

algae, such as proximinty of higher plants.

Upon conclusion of each collecting expedition, the soil samples

were brought to the laboratory, allowed to air dry if collected in

a moist state, and then prepared for various analyses, treatnents, and

identification procedures, Samples for chemical analyses were in 1l

cases passed through a 2 mm. sieve. Weight of rocks above 2 mm. was

noted and the sample weight determined. Total weight was taken for

the subsurface samples, but only 200 n. of the surface CIIIStS were

used, The samples were then ground in a mechanical grinder in order

to achieve a more homogeneous mixture. Alternate surface and subsur-

face sanles were ground and the grinding apparatus brushed after each

grinding to reduce contamination of successively ground samples. Heavy

coarse materials (rocks) were not discarded from the samples, primarilT

because the growth present was observed to adhere to such materials in

some samples.

The chemical analyses performed on the crusts and subsurface

samples included determinations of pH, total nitrogen, and organic

carbon. The tests for pH were determined ith the BecInan glass elec-

trode on the saturated soil pastes according to the method given in

the Salinity Laboratory Manual (197). The analyses for total nitrogen

3b

were performed on duplicate samples according to the KjeldahJ. method

(156). Organic carbon was determined titrametrically by the Walkley

and Black method (2I6,21i.7), except that orthophenanthroline indicator

was used to aid in deteniünation of the endpoint.

Several methods were used for the purpose of isolation and

identification of algae, especially the blue-rreen algae. One method

consisted of the introduction of soil into 250 cc. Erlenmayer flasks

containing 100 cc. of nutrient solution of various composition as

suggested by Moore and Karrer (i6, Fred and Wakaman (90), Bold (23),

Pringsheu (191), Allen (1) and others (i2). Solutions used included

Ohu #10, Molisch calcium sulfate solution, Detuter calcium nitrate

solution, Knop calcium and potassium nitrate solution, Bristol sodium

nitrate solution, Beijerinck sodium nitrate solution, and Winogradsky's

Azotobacter medium (dthout agar). Various dilutions of the above

mineral media as well as dilutions of soil were employed in attempts

to secure the growth and isolation of organisms.

A second method used to obtain growth of algae was essentially

that used by Fletcher and Martin (78). See Plate 1. It was also used

as an aid in primary isolation and identification of the algae. As

the soil was incubated under the light source, fluorescent or natural,

various algae 'were found to grow on the soil crusts and to spread out

over the surrounding substrate. A sterile inoculating ncedle was then

used to transfer some of the growth (a single organism or colony if

possible) to a plate of agar where it was streaked-out in order to

further isolate the organisms.

Another means of obtaining growth and isolation of algae

P1. 5Growth of Nostoc sp. in

a nitrogen fixation exper-.linent (see Fl. 7), Somegrowth adhered to the ves-sel walls and extendedseveral inches above thesurface of the medium.(Incubation period of 35days, 7-21i.-57 -8-2847).

P1. bIncubation of soil

crusts in moist. cham-bers. Some growth hasspread out on moistfilter paper. (Alsosee P1. 8, extremeright for height offluorescent lightabove chambers),

p

P1. 6Experiment on nitrogen

fixation with LyngbyaDiguetii. Flasks 1-6,N-free salts and tap water.Flasks 7 and 8, NaNO3added. Flask 9, uninocu-lated. Nitrogen was notfixed by this alga.

3S

consisted of placing a smu11 amount of soil or a soil crust in a

sterile petri dish and partially sithnerging the soil with cooled agar,

1,S percent, made with tap water or deionized water. The soil was

maintained in a moist state by the surrounding semisolid medium arid

the constitution of the agar medium was of such a restricted nature

that contamination by organisms other than algae was reduced as photo-

tactic algae spread across the plate. This method, although satis-

factory for the most part, possessed a disadvantage in that the agar

and soil became desiccated with time. The method of plating out in

agar was tried but not found promising for isolation of any alga,

The agar used in the beginning of this research was not washed

to remove impurities, nor was any water used except tap water. How-

ever, it was found that there was less growth of contaminating organ-

isms if the agar was dialyzed for t8 hours in tap water, washed several

times with distilled water, and washing completed by several rinses of

deionized water. Weighed agar was washed at first by the method out-

lined by Bold (23) A method later used consisted in washing agar

in a large flask with an inlet tube near the bottom of the flask and

an outlet tube near the neck. After I5 hours washing in tap water,

the tap water was poured off,distilled water admitted, and finally

deionized water admitted by means of gravity flow.

Tap water was used in the first stages of this research in

the hope that it would serve as a micronutrient source for algae.

However, the tap water was found to give a positive qualitative test

for nitrates using diphenylamine indicator. Tap water, as well as

distilled water, may be toxic to algae if it contains heavy metals,

36

i.e. copper ions (23). The former also contained chlorine which may

likewise be toxic (192). The mineral medium used in the first few

months of this research consisted of the constituents and amounts

listed for medium IIB, Table 1, plus micronutrients as molybdenum,

iron, manganese, and boron added at the rate of 0.5 - 1.0 ppm. The

iron source was also added in the chelate form. Later on other media

Table 1, were also used with l. percent agar and O.S - 2.0 cc. per

liter of a total micronutrient, solution. See Table 2,

usuially growth was satisfactory for identification purposes

after completion of one plating of the organism in question. Often-

times satisfactory organisms for identification purposes could be

obtained from growth on the soil crust or on moist filter paper. In

order to identify the organisms,. the macroscopic or gross appearance

of growth was noted on culture media and the individual organisms

examined microscopically in a fresh mount on a slide with a cover

glass. In order to provide a favorable environment for cells, Prings-

helm (189) recommended the bathing of organisms in soil extract, but

this was not found to be necessary0 Deionized water was found to be

satisfactory. Specific chemical tests, e.g. iodine-starch test, were

used as an aid in the identification of organisms, but particular

attention was directed to characteristics of morphology, reproduction,

locomotion, differences in translucency, or other conspicuous features.

General texts (18I,l85,22l,222,223,236,237,2i,268,269) as well as

miscellaneous papers (67,68,79,166,167,168) were used as aids in iden.-

tifying organisms. Measurements were taken for some of the organisms

by means of an occular micrometer, drawings were made, and a description

TABLE 1. CULTURE KEDIA FOR ALGAE

37

edium Salts Concentration

gm./10

II K2HPO 0.5MgSO . 7H0 0.2NaC]. 0.2CaSOI4 . 2H0 0.1CaCO3 0.2

hA K2HP0j 0.5MgSO)4 . 7HO 0.2Nal 0.2CaSOb , 2HO 0.1CaCO3 0.5

113 K2HP01 0.5MgS0 . 7H0 0.2NaC1 0.2CaSOj . 2H0 0.1CaCO3 2.5

III K2HP0j 0.55MgSO . 7H0 0.12Na2C3 oJ.5CaC12 . 2H0 0.15

VI K2HP0j 0.55MgSO . 7H0 0,12Na2CO3 0.20CaC12 . 2H0 0.30

'V K2HP0j 0.35NgS01. . 7H0 0.25Na2CO3 0.20CaCO3 0.20CaC12 . 2HO 0.15

K2HPO1 0.55MgS0. . 7H0 0.25Na2CO3 0.20CaC12 . 2320 0.30

TABLE 2. TOTAl MICRONr.TTRIENT SOLUTION FOR ALGAE

Goncen-Mieronutrient Source tration

gm./18 l

Boron H3B03 11.0Manganese MnSO) 7 0Sodium Na citrate 7.0Iron FeC].3 5.0Molybdenum MoO3 5.0Zinc ZnS0j 1.0Copper OuSOl1. . 5H20 1.0Cobalt CoC12 6H20 1.0Vanadium Na3VOI1. 1.0A1th1inum il2(so)3 , 18HO 1.0Nickel Ni0) . 6HO 1.0Silicon Na2siO3 9H0 0.5Lithium LiC1 0.5Iodine KI 0.5Bromide KBr 0.5Tin SnCl2 2HO 0.5Uranium 1J02 acetate 0,5Tungsten P205 . 2LWO3 . 2H0 0.5Ohiomium K Cr20? 0.5

38

39

of the organisms set down for reference purposes. No text has so

far been published on the algae of this area but nsu1tation was oh.'

tamed from time to time with an individual experienced in the icieri-

tification of algae1,

Nitrogen Fixation in Pure and Mixed Culture

Preliminary Procedure

After a reasonable period of incubation, usually a few days

to several weeks, promising growth of possible nitrogen-fixing algae

was observed either on the soil crusts proper or on the rnediirni adjacent

to the crusts, The general procedure then consisted in streaking-out

some of this growth on artificial media prepared from various salts,

tap water, and washed agar. Mineral agar is recommended in order to

exclude heterotrophic organisms (121,l89). Algae which exhibited

favorable growth and proliferation on this medium were next streaked-

out on artificial media as above, except for the substitution of deion-

ized water for tap water. Further streak plates were prepared for

isolation and purification purposes when necessary. Streak plates

were used in preference to other methods, e.g. the micromanipulator

method of Pringsheiin (192), after it was ascertained that reasonable

success could be acquired by means of streak plates. Plating-out was

attempted, but not found to be a very desirable method, Unialgal cul-

tures were easily obtained on streak plates. A few pure cultures

could be procured after 10-20 plates were prepared per organism.

1 Personal ccmmiunication with Dr. Walter S. Phillips, Head ofDepartment of Botany, University of Arizona.

140

In order to find a satisfactory inoculum for nitrogen fixation

experiments, certain areas of selected plates were inspected under

a stereomicroscope and a small amount of growth removed by means of a

sterile inoculating needle or small loop. The organisms were then ob-

served microscopically using low, high, and oil immersion objectives.

If the algae were apparently healthy and there was no evidence of

contaminants, i.e. bacteria or fungi, in the vicinity of the algae

(such as on occasional bits of agar) or adhering to the gelatinous

covering of algae, the growth was considered satisfactory for pure

culture experiments. Slides stained with carbol erythrosin (6) were

also observed in order to detect contaminants. If the algae in ques-

tion could not be separated from the contaminant, but the algae ap-

peared useful for experimental purposes, notation was made as to the

presence and nature of the contaminant. Finally, with observance of

sterile technique, by means of an inoculating needle or small wire

loop, algal growth was inoculated into glass containers in order to

determine nitrogen fixation in pure or mixed culture experiments

Culture Solutions and Conditions for Nitrogen Fixation

As indicated by Tables 1 and 2, a number of elnents were used

in the preparation of nutrient solutions for growth of culres. For

the experiment on nitrogen fixation, no nitrogen was added in mineral

or organic form (except for the first set of experiments when tap water

was used). Carbon, other than from the atmosphere, was usually sup-

plied in the I ona of carbonates. Some blue-green algae may, of course,

be considered as strict autotropha, A "total" raicronutrient solutjon

as a composite of many inicroelements was prepared from nitrogen-free

chemicals. Method of preparing the solution was based on experience

with contaminants in salt baths used to harden and anneal metals2. A

total of 6 grains of salts plus 20 grams of Na2003 were used in the

formation of the micronutrient solution, The salts were first dis-

solved in hot water, filtered, and then diluted to 1 liter to consti-

tute solution A. Twenty grams of Na2CO3 were then fused with the

water insoluble precipitate, dissolved to a considerable extent in

hot water, filtered, and diluted to 1 liter to constitute solution B.

The remaining precipitate was washed with hot, dilute HC1 and this fil-

trate also diluted to 1 liter which constituted the third solution,

The remaining insoluble precipitate consisted of 0.S2 grams and this

was discarded,

One cc. of each stock solution, A, B, and C, was then made

into a working solution diluted to volume with distilled water to con-

stitute a fairly soluble solution of ions. The concentration of the

working solution was similar to that given by Pringsheim (189). One

half to two cc. of this solution was added per liter of nutrient solu-

tion.

Two new chelates were used in this research, Sequesterene 330

Fe and Chel 138 HFe, They are recommended for their stability in alk-

aline soil (2O8,209,2)8), and an alkaline medium was used in nitrogen

fixation experiments. No information was available on the use or pre-

paration of these two chelates for alga]. cultures, but potassium hy-

droxide is used to p±'epare. theEDTA cnplex (126), and for this reason

2 Hughes Aircraft, Glassified Laboratory Methods

3 Dr. R. Kuykendall, Dept. of Horticulture, Univ. of Arizona

I2

potassium hydroxide was used to dissolve the above complexes. The

dry complex was first dissolved in 10 cc. of dilute (0.2 N) potassium

hydroxide, then diluted to volume with deionized water so that 10 cc.

of Sequesterene 330 Fe stook solution contained S ppiron and 10 cc.

of Che]. 138 liFe contained 1 p. iron. Both stock solutions were

sterilized separately by use of an autoclave and then added in appro-

priate concentrations to the culture solutions. The stock solutions

were resterilized after use when it was found that fungus grew pro-

liuically in the solution of Sequesterene 330 Fe,

The primary factors, other than the nutrient supply, to be

considered in the culturing of algae included pH, temperature, light,

aeration, and agitation of cultures. As mentioned before, an alkaline

pH has been used to grow nitrogen-fixing blue-green algae and for this

research an attempt was made to achieve a pH near neutrality or slight-

ly alkaline for most of the cultures at the beginning of each nitrogen

fixation experiment. If the pH was not satisfactory upon the addition

of the nutrients, the pH was adjusted with dilute hydrochloric acid

using the Beckman glass electrcde. UsuaLr the solutions were made

acid before autoclaving and through ta1 it was detenrilned what pre-

liminary pH was necessary in order to achieve a satisfactory pH after

autoclaving.

The temperature was not controlled for some of the experiments

in nitrogen fixation. The culture vessels were not placed in water

baths and were therefore influenced to a considerable extent by fluc-

tuating room temperatures. Other experiments were conducted in a therm

ostatically controlled room 'with a temperature of approximately 25°C0

b3

For some experiments the light source consisted of' diffuse

south window light, see Plate 8. For other experiments (those conducted.

in the thermostatically controlled rocw) daylight-tpe fluorescent tubes

were used. These provided a continuous light source of lO - 3O foot-

candles over the incubation area, see Plate 7

Aeration and a certain degree of agitation were provided for

the cultures by an air stream that was either driven through the system

by means of two diaphram-type aerators or compressed air, or pulled

thrQugh the system by means of a vacuum pinup. By either means, it was

possible to aerate and agitate the culture vessels with several hundred

cc. of air per minute, or more, if necessary. Fcr this research, aera-

tion was the only means of' agitation. The aerator tubes were constructed

with an opening at a depth of several inches beneath the culture medium

and usually within one half inch of the botti of the vessel. Vigorous

aeration was used in most cases in an attempt to promote a more uniform

growth and suspension of' cells. See Tables 3 and L for culture con-

ditions.

Apparatus for Nitrogen Fixation

The apparatus was constructed after consideration of proper

precautionary measures regarding contaminants and the environmental

requirements necessary to culture algae in question. A general pre-

caution to be observed was the use of' filtered air. Therefore1 air.,

or compressed air was first rnade to enter

a blank trap, filtered through one or two containers of anhydrous cal-

cium chloride in order to remove water vapor, and next bubbled through

a container o± concentrated sulfuric acid to remove possible contamin-

P1. 8Nitrogen fixation con-

ducted at room tempera-ture using natural (win-dow) light and filteredaire Air was forcedthrough the system bymeans of compressed air.

P1, 7Nitrogen fixation under

conditions of controlledtemperature, light, anduse of filtered air, Airwas pulled through thesystem from left to rightby means of a vacuum pump.

Fl. 9Nitrogen fixation by

soil crusts in desicca-tors using controlledtemperature, light, fil-tered and non-filteredair. At lower right areflasks of algae in ni-trogen-free media.

TABLE 3,

CONDITIONS FOR NITROGEN FIXATION IN PURE CULTURE

Length of

No. of

Culture

pH

Light

Temper- Air

Organisms

Expt,

Cultures Medium

Volume

Begin

Final

Source

ature

Strewn

Container

days

1.

OC

Anabaena

7-16-57

2IV

2.0

7.0

9.2

fluor.

25

vacuum

flask

spiroides

8-22-57

7.0

9J

37

Anabaena.

9-7-57

Levanderi

1O-2l.-57

V1.0

7.0

8.5

natural

fluc

t0diaphraginjar

52

Nostoc sp.

7-10-57

8-31-57

1V

1,0

7.1

7.3

natural

fluct,

diaphragmjar

52

Noatoc sp.

7-10-57

8-31-57

V1.0

7.0

7.0

natural

fluct.

diaphrafl jar

52

Anabaena

7 -21-57

6.8

7.1

spiroides

8-28-57

3LI

1.5

7.0

7.L.

fluor.

25

vacuum

jug

35

7.0

7,6

Nostoc sp.

7-2t 57

21.0

7.2

7.6

fluor.

25

vacuum

jug

8-28-57

35

7.2

7.6

Nostoc sp.

7-2LL-57

1IV

0.5

7.2

8.6

fluor.

25

vacuum

jug

8-28 -57

35

Soytonema sp. 7-2 - 7

8-28-57

35

2III

1.0

-6,1

-6.2

fluor,

25

vacuum

jug

Scytonna

7-2 - 7

Archangel

1 i

9-7-57

1VI

0,5

7.1

7.5

fluor,

25

vacuum

jug

TA

BL

E 1

4.C

ON

DIT

ION

S FO

R I

ITR

OG

EN

FIX

AT

ION

IN

MIX

ED

CU

LT

UR

E

Len

gth

of N

o. o

fC

ultu

repH

Lig

htT

empe

r-A

irOrganisms

Expt.

Cultures

Med

ium

Vol

ume

Begin Final

Source

ature

Stream

Container

days

fluor.

25va

cuum

jug

fluor.

25va

cuum

jug

fluo

r.25

vacu

umju

g

fluor.

25va

cuum

jug

fluor,

25va

cuum

jug

natural

fluct.

diaphragm

jar

natu

ral

f].u

ct,

diap

hrag

m

natu

ral

fluct.

diaphram

natural

fluct.

diaphragni

jar

natural

fluct.

diaphragm

jar

natural

fluct.

diaphragm

jar

natural

fluct.

diaphragm

jar

ItH

IIII

Iiii

ItIt

iiIt

ItII

ItH

ItIt

ftU

11

itit

a

9- 7

-57

.Anabaena sp.*

plus

9-

7-57

Scytonema sp.

10-2

9-57

52

Chroococcus rufes-

9- 7

-57

cens

* (a

lso

rese

m-

10-2

9-57

bling Gloeocapsa &

52

Myn

neci

a)unialgal

Aphanocapsa grevil-9-

7-57

lei* and Lyngbya

10-29-57

Diguetli

52

Ana

baen

aLevanderi*

9..7

..57

unia

lgal

10-2

9-57

52

* Culture not bacteria-free

** The tap water used contained 1.3

mgrn. N/i,

*3*Anaerobic conditL ona

Scytonema sp. uni-

7-10

-57

alga

l ino

c, w

ith8-

31-5

7fungus

52

Same organisms as

7-10

-57

abov

e8-

31-5

752

Nos

toc

sp.*

plu

s14

-19-

57so

il ba

cter

ia6-

31-5

755

1

2V

1.0

7.0

7.5

7.0

7.6

1V

I0.

57.

27.

2(+

0.1

8 gm

.ni

anni

tol)

1ha

.0.

57.

07.

6

1hA

9.5

7.0

7.6

1IV

0.5

6,7

7.5

1V

1.0

7.0

9.].

1V

1.0

7.0

8.14

1V

1.0

7.0

8.6

V1.

07.

09.

0

1IV

1.0

7.0

7.2

1IV

1.0

6.8

6.9

1II

*].

.0(A

ppro

x)8.

67.

68.

6it

UIt

II9,

0a

it8.

2Ii

IIIt

ii7

9H

Itii

9:2

ItIi

II9,

14

Dicothi'ix Orsiril-

7-2-

57an

a* p

lus

Lyn

gbya

8--

57

Same organisms

7-2)

4-57

as a

bove

8-28

-57

3

Nos

toc

sp. p

lus

7-2)

4-57

Nic

roco

leus

sp.

9- 7

-57

'45

Sam

e or

gani

sms

7-2)

4-57

as above

9-7-

5715

Scyt

onen

ia s

p.*

7-21

4-57

L6

ating vapors of nitrogen such as ammonia. The air stream then flowed

through a large tube of tightly packed glass wool before passing

through a subsequent filter composed of tightly packed non-adsorbent

cotton which was used to riove any bacteria or other biological con-

taminants in the air stream. Air was next bubbled through a hydrator

containing deionized water and then either passed through a sterile

central distributing system via capillaries into the culture vessels or

passed in sequence through all of the culture vessels. Concentrated

sulfuric acid was used to filter the air stream at both ends of the

system in the early stages of this research, but not for the later

experinierrb s The air stream for any particular systn was controlled

to a certain extent by means of a control valve or valves (for com-

pressed air or vacuum) as well as by glass capillaries and screw clamps

used to regulate the air flow into each culture vessel,

Culture vessels were chosen which would suit the conditions

of the experiment as well as satisfy the requirements of the organisms

for a favorable environment. Pyrex glassware was used in much of this

research as was other heat-resistant glass. Wide mouthed, gallon

capacity, cylindrical pickle jars were used for some of the experiments.

Other glassware consisted of new, Pyrex, 2k-liter capacity, flat-bottom

culture vessels, and new, liter capacity, narrow-mouthed cylindrical

jugs. All culture vessels were fitted with air-tight, thick, rubber

stoppers. They were also fitted with an inoculation tube (except for

the one-liter jugs), an inlet tube which opened several inches beneath

the culture medium, and an exhaust tube exit beginning several inches

beneath the rubber stoppers. AU connections were of glass or tygon

Li.7

tubing, See Plates 7 and 8.

As a preliminary step to the actual experiment, a "dry-run"

was conducted. This consisted of assembly and operation of the appar-

atus and culture vessels in the complete system as it was to be used

under experimental conditions. Water was substituted for the nutri-

ent solution, but used in the same volume as the nutrient solution

would be used in the culture vessels in order to duplicate pressures

within the system. All connections were made as secure as possible

and the air stream then passed through the entire system. The flow of

air was increased to a point which would exceed that in actual operation

and the entire system examined for defects such as leaks. Upon cor-

rection of any flaws, the system was finally run for a period of time

ranging from a few hours for a system with positive pressure to at

least a day for a system with negative pressure. Upon demonstration

of satisfactory operation, the culture vessels were removed from the

system along with the tubes of glass wool and cotton.

For experimentaL purposes, all culture vessels were washed

carefully, first with soap or detergent, rinsed at least several times

in tap water, and then several times more in distifled water. A clean-

ing mixture of sulZuric acid-potassium dichromate was not used at any

time since residual traces of the mixture may be detrimental to the

growth of microorganisms (203). Stock solutions of dry salts were next

pipetted or weighed into each culture vessel and the salts diluted to

volume with deionized water. A culture solution volume of one liter

was chosen for the pickle jars as well as for the larger flat-bottom

culture flasks, and 500 cc. of solution was used in the liter jugs0

After preparation of the nutrient solutions was completed,

the connections between vessels were tightly wrapped in brown paper

and tied with string. The tubes of glass wool and cotton were treated

in a sthiilar manner. Culture vessels and tubes were subsequently

placed in an autoclave and sterilized for approximately an hour at

230°F. Individually prepared stock nutrient solutions were also

sterilized before arid after use, but usually for a shorter period of

time.

Upon completion of the sterilization period, the culture ves-

sels were allowed to cool, pH determined on aliquots of the medium,

and corrected if necessary with dilute hydrochloric acid. The vessels

and tubes were then reinserted into the system and the air stream

passed throuithe system as for the "dry run". Additional steel wire

was used to tighten connections if necessary and several thick coats

of rubber glue were used around connections or other places in the

apparatus in order to insure an air-tiglt system.

After satisfactory demonstration of an operable system, the

selected inoculum was placed into each culture vessel by means of

a pin-point inoculation or small loop of organisms from selected sour-

ces as previously mentioned. Insofar as possible sterile technique

was used throughout the entire procedure of inoculation and determin-

ation of pH, as well as in the reassembly of the complete system for

the experiment. Asa final step, the entire system was again put into

operation and observed for irregularities. These were duly corrected,

but difficulties were seldom encountered in the system after the first

2)4 hours of' operation.

Nitroen Fixation by Soil Crusts

A second method for demonstrating nitrogen fixation involved

the use of soil crusts gathered from the field. This experiment was

conducted so as to simulate field conditions, but with certain refine-

ments or modifications. See Plate 9 for the apparatus used and system

in actual operation. The experimental procedure and method is given

as follows.

Lichen and algal soil crusts gathered from the field were air-

dried ad 50 gm. of crusts weighed out on two pieces of #30 filter

paper with a diameter of 12.5 millimeters. These were then placed in

sterilized open petri dishes of lb millimeters diameter and placed in

dejccators as show in Plate 9. Four of the desiccators were inserted

in a system whereby the air was filtered through calcium chloride, con-

centrated sulfuric acid, a tube of glass wool and hydrator containing

deionized water. A constant air supply was pulled through the system

by means of a vacuum pump. Petri dishes in these desiccators were

designated as the check plates. Four other desiccators were also set

up as demonstrated in Plate 9. The petri dishes in these desiccators

did not receive filtered air nor a moving air stream.

For each petri dish of crusts placed in the desiccators in the

system, a duplicate was placed in a desiccator not connected into an

aeration system. Sterile deionized water was used to moisten the crusts.

The nutrients were provided by the soil and light by means of daylight

fluorescent tubes to give approximately 350 foot-candles. Temperature

was thermostatically controlled at approximately 25°C. No chemical

analyses of any nature were conducted in this or surrounding rooms and

so

therefore contaminating vapors were eliminated to a great extent.

Incubation periods varied from 1 to L. weeks, one dish of

crusts being removed each week from a desiccator in the system and

from a control desiccator. A new dish of crusts replaced each removed

dish so that the system was in continual operation. Growth in the

removed dishes was then examined for identification of organisms and

the crusts arid growth dried at 1O.-5O°C. Dried material, not including

filter paper, was ground in a mechanical grinder and then further

ground by hand using a mortar and pestle in order to obtain a good

mixture. Determinations were carried out as mentioned previously on

the original crusts, Only nitrogen was determined on the incubated

crusts. It was determined by the Kjeldahl method on duplicate 5-10

grain samples.

In addition to chemical analyses carried out on the original

soil crusts, all crusts were investigated for Azotobacter, Winograd-

sky's Azotobacter medium was used as previously mentioned; 50 mgm,

of soil sprin'.c ed over the agar surface according to a method given

by Martin (l).i.8); and the plates incubated for a period of 14 weeks.

The plates were then investigated at the end of one week periods for

typical growth of Azotobacter.

Analyses for Nitrogen Fixation in Nutrient Solutions

Upon completion of the incubation period as determined by the

appearance of macroscopic growth, the culture vessels were removed

from the system with due consideration of sterile technique. Connect-

ing tubes to each culture vessel were clamped tight by means of screw

clamps and the culture vessels prepared for examination,

5].

Before any chemical analyses were performed on the contents

of the culture vessels, a loopful of growth was removed from the vessels

and streaked-out, on agar plates of Winogradsky a Azotobacter medium pre-

pared with tap water. This served as one means for identification of

contaminants, Several more loopfuls of fresh material were examined

under the microscope, the pertinent features of the algae noted, and

the slide examined for contaminants. Fixed slides of material were also

stained with carbol erythrosin and examined microscopically for contamin-

ants using the oil immersion objective,

The first chemical determination performed was that of pH value

which was done as soon as possible after examination of organisms. The

pH determination was made on an aliquot of the culture vessel contents -

algae plus medium, Total volume of the algae plus medium was measured

and like organisms and medium were combined in some cases,

The technique for separating alga]. growth from the culture medium

involved the use of filtration and centrifugation. Adhering algal growth

was first scraped free from the sides of culture vessels by means of

rubber-tipped glass rods fitted with a policeman. The glasé rods were

constructed so as to suit the vessel in question. Algae and medium

were poured into plastic, balanced tubes and centrifuged for 10 minutes

using an angle head. ôentrifuge. The 'supernatant solution was carefully

poured from the centrifuge tubes through #30 Whatman filter paper in a

buchner funnel attached to suction and the supernatant obtained, promptly

acidified with a few drops of concentrated sulfuric acid, The algae

were washed at least three times with a small stream of deionized water,

The algae and culture medium again were centrifuged, filtered, and washed

as before. The supernatant obtained was finally diluted or decreased

in volume for analyses and as many con;ainers as possible stored ma

refrigerator if not analysed immediately. The total algal growth ob-

tained was placed in tared crucibles and dried at a temperature below

LO°C. The total dry weight of material was then noted and the dried

material ground to a powder by means of a mort.ar and pestle. The

powder was then stored in glass tubes for chemical analyses.

Analyses performed on the supernatant included spot checks

for ammonia by means of Nessler's Reagent and for nitrates by means of

diphenylamine indicator. If the qualitative test for nitrates was

positive, nitrates were determined quantitatively using a modified

version of Rarpert& phenol-disulfonic acid method (112) for the super-

natant. Chlorides in' 0 - 100 cc. of supernatant were precipitated

with the silver sulfate solution. The amount of silver sulfate was

based on the amount of precipitating anions plus a slight excess, Total

nitrogen was determined on aliquots of the supernatant using the micro-

lCjeldahl method (121). The aliquots of the supernatant were addition-

ally acidified with several drops of concentrated sulfuric acid and

the volume reduced before digestion and distillation were completed.

The amount of aliquot sufficient for analysis was dete.uriined by trial.

The analyses for nitrogen were also performed on the dried material

using the micro-Kjeldahl method (121). The amount of organic matter in

the dried material was determined by ignition. A weighed proportion of

each sample was ignited in a muffle furance at S00°C to a constant

weight in a tared crucible.

RESULTS AND DISCUSSIOI

Isolation and Identification

A number of different kinds of organisms were exsmined as com-

munities within the soil crusts. A brief description of some of the

more extensively investigated crusts is given in Table S. Organisms

in these, as wefl as other crusts, included both microfauna and micro-

flora. Frequently observed fauna included protozoa such as the cili-

ates and flagellates, as well as amoebic cysts and trophozoites. Other

fauna included soil nematodes and microscopic insect eggs. The pro-

tozoa were more numerous when dilution methods were first used in at-

tempts to secure the growth and isolation of organisms. Some of the

protozoa evidently fed on the algae, since they contained partially

digested franents of algae.

Some of the orgrn4 sins observed could not be catagorized either

as plants or animals, and may have been chlorophyfl-containing animals,

or animal-like plants. As an example, microscopic organisms of

spheroidal shape existed in loose but Independent colonies and exhibit-

ed frequent vertical movement in an aqueous enviromnent much like a

ball on a string. These organisms contained many globular fragments,

o what appeared to be blue-green algae, in a translucent membrane.

Observation of these organisms poses the question as to whether the

organisms were plant or animal, or a mutualistic relationship of the two.

These organisms could not be isolated for culture purposes,

53

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en

a

0

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0-

aen

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54

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c0'0

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en

441

Os

11

0

1101

H'A

g

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Os0,

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-11+0 '0 0,,+0 .4

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40+4

h 0.4 40.00, 0H

- en

The flora, except for the algae, was not investigated extensive-

ly, but organisms other than algae were quite evident. Bacteria were

always present, many of thipiiented, with colors ranging from color-

less and light pink to deep orange and brown. Some photosynthetic

bacteria were present. Cytophagas were also present as evidenced by the

a:biiost complete digestion of filter paper on which some crusts were

placed. From a cursory survey, Azotobacter were isolated from only 1

out of 20 crusts examined for this organisms. Radiobacter were found

in some of these se crusts. No Azotobacter were found in the crusts

used for nitrogen balance research.

Actinomycetes were present and molds were common. Extensive

hyphac existed in the crusts but the organisms were not extensively

identified. Some of the molds noted were Trichoderma, various members

of the Ascomycetes (e.g. Penicihium, Aspergiflus, Alternaria), and

Fu.n,i Imperfecti, Sometimes such molds made isolation of algae diffi-

cult and one alga, a Scytonema, although obtained in a unialgal cul-

ture, was not obtained in a pure culture for this reason. See Table Li..

A curious aspect of this particular association was that the presence

of fungus accentuated the color of the alga in question, giving it a

pronounced blue to deep bluish-green color. When apparently alone,

the alga would appear more yellowish-brown or green. Microscopically

the alga would appear more like the blue-green alga Tolypothrix rather

than a Scytonema when in association with the fungus. Algal filaments

associated with the fungus also would be more frequently singly

branched at a heterocyst. The heterocysts would be more abundant, with

heterocysts and cells pearing more or less subglobose. In either

case, branches were erect and more or less parallel.

Nyxomycetes, some conspicuously pigmented, were not uncommon in

the crusts and occasionally spread from the crusts to filter paper or

agar where they could be advantageously investigated. Appearance of

these organisms suggests that they may be more common in soil than is

noted. They are difficult to obtain from the soil, however. A good

method is needed to obtain growth and isolation of such organisms (23L).

These organisms would sometimes appear on crusts incubated for an ex-

tended period of time and after a decline in healthy alga]. growth.

They were frequently noted on decaying lichens0

Myxobacteria, such as Myxococcus xanthu, were apparent in some

of the crusts. Occasionally they would spread to the filter paper or

agar surrounding the crusts during incubation. In one case a myxo-

bacteria and alga, Protocoecus, were transferred to agar plates. On

agar the alga was carried along by the pseudo-plasmodium of the myxo-

bacteria and appeared in concentric rings of growth (69). This also

occurred whenever the myxobacteria formed fruiting bodies. Neither

this alga nor the myxobacteria is known to be a nitrogen-fixer. The

alga appeared to have a deeper green color when in association with

this organism, even in the aark.

Soil lichens were prominent in some of the crusts obtained and

in some cases constituted the most conspicuous growth whether in the

desiccated or apparently active condition. The lichens were not iden-

tified as such, but an attempt was made to identify the algae within

them. Such algae included unicellular green algae and/or filamentous

and non-filamentous blue-green algae. Bacteria were also evident in

S 57

in these lichens. Attptà to culture soil lichens as such were not

successful. Lichens collected from desert rocks also could not be

cultured. The new environment either promoted the dominant growth of

the algal component of the lichen or else the fungus, and no balance

of growth could be obtained resemblizg the original or natural stateof the lichen. The more common genera of green algae in the lichens

included Trebouxia, Chlorococcum, Palniellococcus, and Myrmecia. See

Table 6. The mQst common blue-green components of lichens were of the

genera Nostoc, Chroococeus, and Scytonna0 Any one lichen examined

did not necessarily contain only one alga; various combinations were

noted. Algae within the lichens were often difficult to identify un-

less they could be isolated from the lichen in question. Within the

lichen the association of alga and fungus often tended to give the

alga a bizarre appearance. In some lichens funga]. hyphae appeared to

have concentrated granules of blue-green pigment. In some cases, this

concentration of blue-green pigment was intensified in a second green

algal component of the lichen. Trebouxia Cladoniae was the only alga

isolated in pure culture from a lichen. The other green algae were

isolated, but not in pure culture.

Organisms resembling liverworts were obtained in some of the

crusts. In aU cases such organisms were found to contain an alga.

A Nostoc, resembling N. muscorum, was obtained from platings of one of

these growths.

Moss and higher plants were in evidence on some of the crusts.

The presence of moss further complicated the identification of some

algae. For example, moss protonema could be confused with growth of'

fjlamentous green algae. Also present in the crusts, but not investi-.

TABLE

6GENERA. OF ALGAE FOUND IN ARIZONA SOILS

Genera of Algae Identified in

Blue-Greens

Soil Crusts

Fixing N

Cyanophyta

Plectonema

Chroococeus*

Calothrix

Gloeocapsa

Trichodesmiuxa

Tolypothrix

Spirulina

Phonnidium

Mierochaete

Oscillatoria

Microcoleus

Synploca

Lyngbya

Nostoc*

Anabaena

Scytonerna*

Porphyrosiphon

Schizothrix

Aphanocapsa

Die othrix

Rhabdodenna

Nodularia

Syneehococcus

Aphanothece

* Occurred in lichens.

Chlorophrba

Protosiphon

Trebouxia*

Gloeocystis

Chlorococcum*

Chlorefla*

Palmellococcus*

Myrmecia*

Sphaerocystis

Planktosphaeria

Protocoocus*

Kentrosphaera

Desmids

Chrysophyta

Chroiiulina

Monocilia

Botrydiuni

Peroniella

Pyrrophyta

Urococcus

Euglenophy-ta

Euglena

Chroococ cus

Nostoc

Anabaeria

Scytonema

Dicothrix

Aphanocapsa

Diatoms

Navicula

Epithemia

Nitz schia

Stauroneis

Dpiloneis

Pinnularia

Melosira

Synedra

Amphora

Blue-Greens

probably not

fixing N

Pie ctonema

Gloeocapsa

Spirulina

Phonnidium

Oscillatoria

M±croc oleus

Symploea

Lyngbya

Porphyrosiphon

Schizothrix

Blue-Greens

not tested

Calothrix

Trichodesinium

Tolypothrix

Rhabdoderma

Microchaete

Nodularia

Synechococeus

Aphanothece

59

gated, were various higher plants, the seeds of which amnetiines ger-

minated to produce seedlings which soon succumbed to fungus or adverse

environmental conditions.

Algae identified within specific crusts are given in Table 5.

Algae identified from aD. crusts examined are included in Table 6. A

detailed description of some of the blue-green algae observed is given

in pages 65 to 82. Identification of these algae was rendered diffi-

cult in eome cases if not more than one or two observations from a

single environment were depended upon for identification purposes.

This difficulty has been frequently noted by others (2L,97,l23,l14h,l79,

235,239,21l,259).

Care must therefore be taken in assuming that any one organism

can be identified with finality. This applies to filamentous as well

as unicellular forms. In an aquatic environment, filaments of Nostoc

app., without heterocysts, resembled Trichodesxniuin ap. and even ex-

hibited movement characteristic for this genus. Species of Nostoc

as well as Anabaena were sometimes observed to unflex and move out of

gelatinous colonies into the aqueous environment whèn. such an environ-

ment was provided. Other filamentous species were also observed which

were ensheathed until provided with an aqueous environment. The organ-

isms were then observed to move out of their sheaths. Members ol' the

genera exhibiting this phenomenon included Plectonema, Tolypothrix,

Scytonema, Phox'midi.um, Symploca, Lyngbya, Porphyrosiphon, Schizothrix,

Nicrocoleus, and Microchaete. Under microscopic observation movement

out of the sheath may be rapid and occur within minutes, as in the case

of Microcoleus app., require up to several hours as for Schizothrix app,,

60

or even up to several days as in the case of Scytonema spp. In such

cases care must be taken not to classify the trichomes of such organ-

isms with that of Oscilatorja app., which is characteristically with-

out sheaths.

The identification of unicellular blue-green algae as described

for the genera Chroococcus and .Aphanocapsa can be particularly diffi-

cult since the organism varies greatly with time and the conditions of

the environment Obviously, unicellular algae as well as filamentous

species vary according to whether or not the environment is aqueous.

This points out the fallacy of classLfying an organism merely on its

occurrence at any one time from a single environment, although such

information is at times useful to a certain extent for classification

purposes.

Green algae and members of other orders of algae were not inves-

tigated in detail, but some of them were identified. Very few dis-

tinetly Lilamentous forms were observed. Unicellular green algae,

when present in virgin soils, usu)1y occurred in lichens as was noted

for blue-green unicellular forms. They also were difficult to iden-

tify on the basis of a single stage observed. In cultivated soils

green algae and diatoms appeared more abundant than the blue-green

algae.

The areas of the Citrus Farm which appeared to have a reddish

growth were found to contain algae which were rusty-red in color when

observed microscopically. Previously known nitrogen-fixing blue-green

algae were not found. The ttred algae were Botrydium sp. Protococcus

61

viridis arid Protosiphon botryoides. Both Botrydium sp. and P.botry-

oldes rapidly changed from the mature vegetative stage to other stages

in the life cycle when soil was plated in moist chambers in the labora'-

tory. Change in color of these organisms on soil may have been due to

desiccation, a decrease in pH, or both. Soil samples 1a and )46a had

pH values of 5.6 and 6.6 respectively, and growth on these samples was

noted for being rusty in color. Sulfur had recently been applied to

these soils as was evidenced by the presence of undissolved sulfur

powder on the soil surface. However, sample L3a had rust- as well as

green-colored growth, but the pH was 8.0. The application of a phos-

phorus fertilizer may have had an influence in the latter case, but

desiccation could not be ruled out for either case. In the laboratory

P. viridis was found to exhibit the same rusty-red color as was obser-

ved in the field. This phenomenon was exhibited when the orgaEin was

grown in unialgal culture on filter paper moistened with Bristols med-

ium (acid pH) and then allowed to dry. When moistened again, the rust-

colored organisms did not become green again, but new growth was green

in color. The factor of light was not investigated, but may also be

a factor in regard to change in color (96).

The influence of pH on the algal growt.t in soil crusts was not

thoroughly determined. As mentioned previously, blue-green algae pre-

fer an alkaline pH, and a review of pH values, see Table U, shows

that the majority of pH values are on the alkaline side of neutrality.

This may help explain the presence of blue-green algae in these soils.

It was also noted that the pH values of the subsoils were usually, but

not always, more alkaline than the corresponding surface crusts. During

a rain, the pH of the surface crusts tended to corns to equilibrirn

with the subsoil. This was shown by the 96 series of samples, see

Table U.

Except for the diatoms, which have a siliceous cell wall,

it may be noted that nearly all of the other algae observed have one

thing in common. This is the presence of a sheath or investment of a

gelatinous or mucoid nature. Smaller filamentous algae were sometimes

adherent to these sheaths and made isolation difficult. As noted by

Fritach (96). and Bristol (3) as well as others (212,213) such an in-

vestment is characteristic for surface forms and prevents loss of inois-

ture during drought. Such cells need not show any appreciable outward

change. This is said to be distinctive for surface forms (96). Under

conditions of desiccation or other adverse conditions, green algae may

store fat as well as starch, a condition which is confusing for identi-

fication purposes. Botx7dium, for example, is said to be distingushed

fran Protosiphon in that the former organism never contains starch

whereas the latter organism does (223). In some cases, tests for starch

as well as fat were doubtful on some larger species and therefore of

no help in identification. The presence of accumulations of granules

was frequently noted in desiccated crusts, especially in the spepies

of filamentous blue-green algae such as Nostoc and Scytonema. These

granules were noted in hormogonia as well as apparently normal cells.

There was usually a decrease in the granulated condition under provi-

sions for adequate moisture, but not for all species observed. A char-

acteristic for some species of algae for identification purposes is

based to some extent on the presence, density, and size of granules, It

62

63

is belieYed that the development of granules is related to drought

resistance, but the relationship is not clear (96). Presence of gran

ales would help to explain the drought resistance of N. vaginatus anl

N. laclustris but not N, paludosus.

As mentioned previously, most of the filamentous blue-green

algae were observed to move out of their investments under adequate

conditions of moisture, Fritsch (96) has noted that a characteristic

feature of surface forms is that the change from the active to resting

state or vice-versa may occur in a very short space of time. This

statement has been shown to be true according to this research. During

or inmediately foUodng a sudden downpour, algae were observed to

cover the ground, even within as little as 15 minutes. Species of

Microcoleus were the first to appear and were soon followed by species

of Schizothrix. Light and temperature are, of course, usually decreas-

ed to some eictent during these periods.

For some of the other algae, the relatively short time re-

quired to change from the dormant to active state may help to explain

the dominance of reproduction by vegetative means. Spores such as

gonidia were rarely observed and this further complicated identifica-.

tion when species of some genera were noted to be classified on the

basis of this characteristic alone. However, a rapid rate of repro-

duction may be used advantageously for the purpose of isolating the

alga in question. Preliminary growth was easily obtained using the

moist chamber method of Fletcher and Martin (78) since growth could

be obtained from the crusts or surrounding medium of agar or moist

filter per. In fact, growth could be obtained in some plates for

6L.

over a 'year by the occasional addition of agar or tap water. However,

the presence of nematodes and insects or mites oftentimes made isola-

tion of algae difficult and usually necessitated the discarding of plates

of crusts containing these organisms. The hatching of insect eggs not

only contaminated the crusts in which they hatched, but other plates

as well. The insects would then spread to other plates. Such insects

were voracious feeders of algae and could eliminate a promising cul-

ture within a few days. Spraying of insecticide on arid between plates

was helpful to some extent as a control measure, as was the alternate

drying and wetting of crusts.

For any method of isolation, once the mecLium bad been chosen

the problem of obtaining a pure culture also was difficult due to con-

taxainating organisms. The gelatinous or mucoid investments of the algae

provided a harbor for contaminating bacteria and fungi. These organ-

isms adhered to the algae and were harbored by them as they grew. No

single easy method has been devised to obtain pure cultures. Bacter-

ial streak-plates finally were resorted to as a dependable, but tedi-

ous method. Nhen isolated areas of algal growth could be found in which

there was no microscopic evidence of contaminants, then a few organ-

isms were used for inoculations into culture vessels for the nitrogen

fixation experiments. Use of such a small amount of inoculum was ad-

vantageous from the standpoint o± reducing or eliminating contamina-

tion, but necessitated a longer period of incubation in order to obtain

sufficient growth in the nitrogen fixation experiments,

65

Description of Blue-Green Algae Observed

Nostoc app.

Several species were observed. In the dormant state, the

plant mass was confluent and irregular, or globose, with colonies

usually yellowish-brown in color, and of various dimensions within

hard, gelatinous investments. Occasionally individual hormogon.ia were

present, but more often a colony would contain agglomerated honnogonia

or individual trichomes in short, thick, gelatinous investments. Upon

application of moisture the plant mass would soften, honnogonia ger-

minate, and the alga in question would then produce more distinctive

individuals, At various stages of growth and in various environments

similarities in appearance made species identif cation very difficult.

Various species which may have been observed include N. Linckia, N.

muscorum, and N. verrucosuxn,

N. Linckia: This orgmism possessed characteristics identifi-

able with this species when observed in aqueous solution in a nitrogen

fixation experiment, but may have been the stage of another species.

Many colonies were finn, globular and independent when roung, with many

twisted entangled filaments of a blue-green color. Apparently older

colonies were clathrate, in membranous sheets of a dirty green to yel-

lowish-brown color and contained individual cells surrounded by a

similar membrane, With time more and more raight to slightly flexed

trichomes with sub-spherical to ovate-shaped heterocysts were observed

in the aqueous medium. Such filaments exhibited definite direction of

movement. Direction of movement may be exhibited in the opposite direction

even by adjacent trichomes, Some trichomes were several to many cells

6o

in length arid did not possess heterocysts. Cells were of no dominant

shape, but all were constricted at the cross-walls,

N. verrucosum: This organism was observed on agar plates, moist

filter paper, crusts and in aqueous solution for a nitrogen fixation

experiment. The colony formed was firm and leathery on a solid sub-

strate, firm, colorless to yellowish-brown, but not as tough or color-

ful in aqueous media. On soil crusts it appeared as a black, warty

membrane. Within the membrane individual filaments were observed,

some with wide definite sheaths, others with rather diffluent or Indis-

tinct sheaths. Filaments were usually LLexuously twisted and densely

entangled, particularly near the surface. The colonies were often

olive-black when young, but became brownish-green to brownish-yellow

with age. Cells were compressed to depressed globose, 3-)jin diameter,

in length, and oftentimes contained granules, particularly when

older. I-Ieterocysts were usually spherical and oftentimes observed at

the end of a wide sheath, either within or outside the sheath. mdi-

vidual trichomes in aqueous solution were loosely twisted and without

evident sheaths.

N. muscorum: This organism was frequently observed in soil

crusts, usuafly within small (approximately 1OOt), globose colonies,

appearing bright blue-green when young and later becoming confluent

into an irregularly expanded, nodulated, dark green or olive membran-

ous mass, Cells were approximately 3-,4 in diameter, spherical, bar-

rel-shaped or cylindric, up to 6,i in length. Reterocysts were more

or less globose. Trichomes were densely entangled within the colony,

but filaments were free or in colonies in aqueous media.

67

Anabaena spp.

This organism may be represented by a half-dozen species or

more hi SOIl Ci'Usts On desiccated crusts, the organisms are not read-

ily apparent, but colonies may fonn rapidly upon application of moisture.

to crusts. The colonies were usually very soft and mucoid and did not

easily retain their shape when probed; colonies were difficult to secure

from a surface by means of an inoculating needle. Filaments within

the colonia1 mucilage were usuaily].00se3.y flexed, but coiled and con-

torted in some colonies. Some filaments exhibited a definite sheath,

usually when old; others did not. In aqueous solution, filaments

within the colony sometimes rapidly escaped, unfiexed and then exhibit-

ed definite direction of movement. Colors of colonies varied from

light yellow-green to olive-green and shades of brown. Organisms which

were noted resembled the following species; A. levanderi, A. flos-aquae,

A. oscillarojdes, A. circinalis, A. spiroides, and A. sphaerica.

A. levanderi: The plant mass was dark brown, elevated, warty,

and confluent on agar. The mass also was very mucoid and difficult to

pull apart. Triehomes within the colonies were sometimes enclosed in

a rather indistinct sheath, and slightly flexed; they were not densely

entangled. Cells were cy].indric in shape, usually 2 to 3 times as long

as wide, and constricted at the cross-walls, which were rounded. The

cells were light brown in color, contained conspicuous refractile gran-

ules and frequent pseudovacuoles. Heterocysts were either spherical or

ellipsoid and of the same dimensions or larger than the vegetative

cells. In aqueous nitrogen-free media, the trichornes were solitary, not

in flakes, straight, flexed, or in globose colonies.

68

A. oscillaroides: On filter paper the plant mass was in a thin,

soft, gelatinous layer. Filaments were first observed to be entangled

within the plant mass, but while under microscopic observation, the

filaments tended to unflex, gradually straighten and move out of the

plant mass into the aqueous medium. Trichonies then exhibited a sug-

gestive osciUatori-like movement. The cells were usually barrel-

shaped, although sometimes truncate-globose, slightly constricted at

the cell walls, contained granules and pseudovacuoles, and were pale-

green in color, The apical cell of the trichomes was sometimes slight-

ly tapered, but always rounded at the end. Heterocysts were usually

round or slightly ovate, not punctate. No akinetes were observed.

A. sphaerica: In nitrogen-free aqueous media, trichomes were

solitary and flexed, not coiled; sheaths were inconspicuous. Cells

were b - S4MIn diameter to 2.S - 7.S,qin length, barrel-shaped to

short cylindric and constricted slightly at the cross-walls. Cells

also contained granules, were light olive-brown in color and not vacu-

olated. Heterocysts and akinetes were infrequent, but when present,

they were adjacent. Heterocysts were 5 - 6.Sj.t in diameter and 6 - 10)1

in length. Akinetes were 5 - 614n diameter and 6 - ó.Sp in length.

A. flos-aquae: This species also occurred in growth on soil

crusts; trichomes and hormogonia were observed. Trichornes were either

very flexuous to contorted and solitary, or twisted into a mass, Cells

were usually ovoid in shape, less frequently spherical. Cell contents

were slightly granular, light green in color, and cnspciuous with

pseudovacuoles, usually at one end of the cell. Heterocysts were ovate,

slightly wider in diameter than the vegetative cells, but not longer.

69

Hormogonia contained single, contorted filaments within expansive in-

vestments. A heterocyst occurred at a narrowed end of the honnogonium.

A. spiroides: Adjacent regularly spiraled trichomes were ob-

served in pale, brownish masses on agar plates. Trichomes in solitary,

broad spirals were observed in nitrogen-free aqueous media. Cells as

well as heterocysts ere rounded and of the same dimensions; cells were

granular and with pseudovacuoles,

A. circinales: A colony isolated from growth on soil crusts

was quite muoid, lacking definite shape, and was pale brown in color.

Vegetative cells were spherical or oblate; heterocysts were of a simi-

lar nature. In aqueous media, trichomes were sometimes solitary, but

usually twisted into floccose, torn or ragged aggregates.

Microchaete robusta

Trichomes were observed from growth on soil crusts. Trichomes

w:re uniseriate with a single basal heterocyst as well as intercalary

heterocysts. The basal heterocyst was globose, but incercalary hetero-

cysts were ovate. The sheath was firm, definite, and conspicuously

laxaeflated. The cells were barrel-shaped, shorter than wide, arid

faintly granular. Under microscopic observation, active filaments

were observed to move out of the open end of the encasing sheath and

into the surrounding aqueous medium.

Lyngbya spp.

Species of this organism were frequently observed in growth

on soil crusts, on filter paper, on agar and in aqueous media. These

organisms are uniseriate, filaruentous, unbranched, and enclosed by a

70

more or less conspicuous and flim sheath, The empty sheath may usu1 ly

be observed to extend some distance beyond the enclosed cells. A row

of cells or a few cells within the sheath may be separated from another

row of cells by an expanse of empty sheath to give the appearance of

horniogonia. Under adverse conditions, expanses of adjoining cells lose

their identity, cell walls become indistinct and the cells become

confluent into an apparently dormant state also resembiinj hormogonia

in which many granules may be observed. Under favorable conditions,

filaments of cells of the larger species were observed to move slowly

out of their sheaths and into the surrounding aqueous medium. Many

more species were observed than were identified. Some of the species

present resembled the following species: L, epiphytica, L. Diguetni,

L. Lagerheimii, L. contorta, L. Martensiana, and L. Birgeii. The

organism L. Birgeii was not frequently observed in soil crusts. In

plant masses, organisms resembling L. Martensiana were often observed,

Organisms resembling L. epiphytica aixi L. Diguetii were frequently ob-

served in association with other filamentous blue-green algae such as

species of Nostoc, Schizothrix, and Microcoleus. Tt.o of the Lyngbya are

described below.

L. Diguetti:. Trichomes were unconstricted at the cross-walls,

about 2.5k in diameter. Filaments were only slightly larger, approxi-

mately 3.5,ecwith their sheaths. CeUs,were 2 to 39uin diameter and 1

to 3.Ee in length. Filaments of this organism were found attached or

in association with filamentous-blue-green and unicellular algae. In

aqueous media the plants formed a much entangled blue-green masse

L. epiphytica: Plants were similar to L. Diguetti, bt of

slightly smaller dimensions, They were somethnes found spirally

twisted about or in close association with larger filamentous blue-

green algae throughout their entire length,

Oseillatoria app.

These organisms may have been present more frequently than

was realized. They were not particularly evident except when suffi-

cient moisture was available. If the organisms were not isolated in

an aqueous medium, an extended period of microscopic observation was

necessary to debexiiine their presence, They were more apparent in

cultivated than in virgin soils. These organisms may be easi1r con-

fused with trichomes of other blue-green algae when such algae have

separated from their filaments, On agar plates these organisms rapid-.

ly covered the entire plate, even to the extent of burrowing slightly

beneath the surface of the agar. In aqueous media, the organisms were

solitary, never agglutinatec, arid demonstrated the oscillating move-

ment characteristic for the genus. Species: present included 0. angus-

tissna with extremely fine thread-like filaments, less than

0. amphibia, 0. animalis, 0. formosa, 0. tennis, and 0. Lemmezmannii,

No large species were observed, The species named above did not differ

noticeably from those species commonly described in the literature,

except for the occasional presence of many granules.

Microcoleus app,

Members of this genus, along with Scytonema, were most frequent-

ly observed in growth on soil. Under microscopic observation, these

orga sins were observed to be in an apparent dormant state in desiccated

71

72

soil snples. The entwined filaments of cells usually appeared normal

within wide, but firm, translucent or yellowish-brown sheaths. Occa-

sionally there 1were sheaths twisted about one another and even sheaths

twisted together giving the over-all appearance of a microscopic plant

showing the outline of a "priinary root" with "secondary roots". How-

ever, the sheaths themselves were not branched.

Under microscopic observation, application of moisture to ap-

parently dormant colonies of ensheathed organisms gave rise to activity.

Within as little tiine as iS minutes filaments of organisms within the

sheaths gave evidence of characteristic slithering motion and began

emerging from the wide enclosing sheaths as single trichonies. Upon

application of moisture to desiccated crusts, these organism gave

evidence of macroscopic growth within 1 to 12 hours. Within the first

few days, prolific growth of these organisms frequently over-shadowed

growth of other algae. However, after extensive rapid' growth, some-

'times with the formation of suggestive tufts, these brganisms degener-

ated, gave way to other algae, and under continued application of mois-

ture, soon decreased in number as to make their presence difficult to

detect. These organisms alsp made rapid growth on agar plates, but in

aqueous media they did not proliferate extensively. Several species. of

Microcoleus were noted in the crusts examined. These included species

resembling N. paiudosue, M. iaclustris, and M. vaginatus.

N. paludosus: Most of the filaments observed were straight or

slightly twisted, with the apex gently tapered, apical cefl slightly

rounded to acute; trichomes were not capitate nor constricted at the

cross-walls. The cells were quadrate to oblong, up to twice the

73

diameter in length, nongranular, and blue-green in color, Sheaths were

colorless in young colonies.

M, laclustris: Organisms of this species differed from the

above species in that the cells possessed granules and were paler in

color. Cells were distinctly cylindrical, Li. - .5,Min diameter

and. 8 - 1S,a in length. The trichomes frequently possessed a longer,

bluntly- to gently-tapered apical cell which lacked a calyptra. Cells

of the trichomes were slightly constricted at the cross-walls.

M. vaginatus: This species differed from both of the other

two species in that the apical cell possessed a calyptra. Cells were

3 - in diameter and 2 - 7,J.c in length, pale blue-green to olive-

green in color, nongranular to granular, and not constricted at the

cross-walls. Sheaths were uneven and colorless when young.

Phorinidium sp.

Organisms of this genus were found occurring in the plant mass

on crusts particularly in association with other filamentous blue-green

algae such as species of Lyiigbya, Microcoleus, Schizothrix, and

p].oca. The sheaths of the filaments were not extensive, and not too

evident unless the organisms were adjacent in parallel or densely inter-

woven trichomes. The plant mass on crusts was often a dense expanded

stratum but was sub-aerial to some extent. Interwoven trichomes some-

times extended a few millimeters above the surface as a flexed and

coiled mass having a broad base of mucoid filaments and narrowed to a

few filaments at the proximal end. More species were noted than there

were attempts made at identification. According to descriptions given

for this genus, organisms were present resembling the following species:

7b

P. angustissium, P. tenue, P. ainbigum, P. inundatum, and P. incrusta..

twa. No capitate or hooked species were noted.

Syraploca muscorum

This alga sometimes occurred on soil crusts as tufts of up-

right or horizontal, closely associated filaments with evident sheaths,

macroscopicafly resembling Scytonema spp, On desiccated crusts the

plant mass gave the appearance of a cottony mass of a dark bluish-green,

grayish-brom, or black color. Microscopically, the filaments were

seldom branched and much entangled in the basal stratum. Sheaths were

sometimes dark yellow and the cells appeared coarsely granular.

Schizothrix spp.

Members of this genus gave the same general appearance as that

of Microcoleus on soil crusts. They sometimes occurred with Microco].eus

spp. as well as with other filamentous blue-green algae, but not always.

Careful microscopic observation is necessary in order to identify the

genus. The plant mass may be aerial or subaerial, These organisms

were contained within more definite, delimiting sheaths than the Micro-

coleus; sometimes the sheaths were definitely lamellated. Color of the

sheaths ranged from colorless to yellow, yellowish-brown and shades of

red and purple; older sheaths may be more colorful. Sheaths were eithe

smooth, or gave a rough, splintered appearance and may be branched or

unbranched. There were usually only one to a few trichomes within the

sheaths; on occasions there were more. In contrast to Microcoleus, the

triches were usually loosely aggregated and either motionless or

exhibited only very slight movement. With time, trichomes may, on

7

occasion, move out of their investing sheaths.

More species of this genus were present than were identified

by the literature available. Some of the organisms resembled those

given for descriptions of S. Friesil, S. stricklandii, and S. puijuras-

cens.

S. Friesii:. One to six trichomes occurred within a fixrn, color-

less, lamellated sheath. Cells were granular, blue-green to olive-green

in color, not constricted at the cross-walls, quadrate, 3-6in diame-

ter, and 6-l9 in length. The apical cell was bluntly to gently taper-

ed, but not capitate.

S. stricklandli: One to six trichomes occurred within a firm,

lamellated, and somewhat rough, colorless sheath. Trichomes were twist-

ed, sometimes preseuting a woven appearance, and were blue-green to

olive-green in color. The apex was slightly tapered with the apical

cell truncate or slightly so. The cells were more or less quadrate,

slightly granular, scarcely constricted at the cross-walls, approximate-

ly in diameter and 3- in length.

S. purpurascens: Sheaths were very rough, lamellated, and color-

ed brownish- to purplish-red when old (colorless when young). One to

many trichomes with conical apical cells occurred within the sheaths.

Cells were granular, pale blue-green, slightly constricted at the cross-

walls, 6-8 in width, 3-4 in length.

Sohizothrix, sp.: Sheaths were colorless, confining and branched,

containing 2 or more trichomes. The apical cell was not capitate and

was gently tapered to bluntly rounded, Cells were quadrate to oblong,

not constricted at the cross-walls, blue-green to olive-green in color,

76

- 7.5,itin diameter arid 8-1)#in length (apicaJ. cell longer).

Schizothrix sp.: This organism, although present in a number

of soil erasts could not be identified with certainty. It does belong

in the family Osciflatoriaceae and may be a member of the genus Schizo-

thrix. Growth of this organism was observed on filter paper, on

nitrogen-free agar and in nitrogen-free nutrient solution; however,

growth was not rapid and no quantitative data were obtained,

On solid media or in aqueous solution, species appeared in

densely entangled masses of' a greenish-black or black color. Filaments

could not be easily separated or pulled apart from the plant mass.

Sheaths of the filaments were thin, firm, and rather indistinct when

young, but up to O, in thickness when old. Older sheaths were also

very firm, confining, and colored shades of violet. Sheaths did not

extend an appreciable distance beyond the trichoines although young

trichomes sometimes extended beyond old, colored sheaths. Filaments

were usually loose, sometimes loosely spiraled, sometimes twisted in

the plant mass, and if branched, then not frequently so. Trichomes

were not often definite, and the cell walls not usually distinct. The

apical cell was bluntly to gently rounded, the apex being slightly to

not at all tapered. Cells were not constricted at the cross-walls,

were 2-3,i in diameter and 2-6 in length.

Scytonema app.

Members of' this genus were observed in most of the soil crusts

collected from virgin soils. Oftentimes it was the most conspicuous

growth in desiccated crusts, giving the soil a ftburnedfl appearance0

77

The desiccated growth was frequently black in color, but it also ap-

peared in shades of gray and brown. This growth was often noted in

dense to thin tangled masses, aerial or subaerial in nature, but subaer-

ial growth was not always apparent. Microscopically, growth from desic-

cated crusts showed sheaths of yellowish-brown color enclosing hormo-

gorila or granular cells; the apical cells were more deeply colored and!

or shortened, Heterocysts were often frequent and quite varied in the

desiccated growth. Short, false branches were also present and method

of branching was sometimes more charácteris tic for the genus Tolypo-

thrix than for Scytonna,

After application of moisture, new growth was slower to obtain

than for other ensheathed blue-green algae such as species of Schizo-

thrix and Micro coleus. New growth usually disrupted and grew from the

distal ends of the plant mass. Less frequently, hormogonia were ob-

served to move out of the old sheaths and into the surrounding aqueous

medium. Members of this genus were also observed on filter paper,

nitrogen-free agar plates, and nitrogen-free nutrient solutions, Growth

was usually slow but eventually luxuriant on the latter two media.

More species of this genus were observed than could be identified with

the literature available. Some of the species identified resembled

S. ocellatum, S. mirabile,S. tolypothricoides, and S. Archangelii.

As was noted for species of Nostoc, at various stages of growth similar

appearances in growth made species identification difficult.

S. oceflatuin: The plant mass appeared tufted and may float in

aqueous media. The color of the mass ranged from blue-green to olive-

green. Microscopically, many entangled branches were observed, up to

78

a few millimeters in length, but sometimes with short false branches

of a few to a dozen cells. Sheaths appeared firm and lamellose; the

color ranged from colorless to yellow. Occasional doubly concave

"patches" of deep-blue to blue-green piaent, separated trichonies with-

in the sheaths. Cells were quadrate, with none to very slight constric-

tions at the cross-walls, S_l8Min diameter, 5-2],Min length. Older

cells became yellow and vacuolated. Sheaths ranged in diameter from

9-21M' Heterocysts were nearly quadrate, about the same diameter as

the vegetative cells and yellow in color. From crust samples, hetero-

cysts were nearly always quadrate, but in aqueous media they were

sometimes cylindric, especially in older filaments.

S. mirabile: This alga at times resembled the alga Tolypoth.rix,

particularly in the manner of branching. On soil crusts and agar plates

branches arose singly, sometimes at the heterocyst. Trichonies were

long and branches not frequently observed. Heterocysts were not fre-

quent, but varied from quadrate-globose to cylindric in shape. Sheaths

were thin and close in the branches, thicker in the main filaments,

yellow in older or desiccated filaments. Cells ranged in shape from

quadrate-globose to quadrate and cylindric, similar to S. ocellatum

in the upper size range. Organisms resembling this species were most

often uped in nitrogen-fixation experiments,

S. tolyothricoides: This species differed from the previous

organisms in that the cells were densely granulose and the cell walls

were indistinct; frequently so in growth in the desiccated crusts.

Heterocysts as well as cell content rarely appeared rose-colored0

Sheaths became brown to brownish-orange with age or when desiccated,

79

False branches were nusierous. The plant mass was definitely tufted.

On some crusts in moist chambers this organism grew prolifically after

some months when virtually all other algae had ceased active growth.

S. Archangelli: The alga resembling this species foniied

cu.shiony, attached, plant masses, even in aqueous nitrogen-free media.

The trichomes were long and gracefully curved, and usually arose com-

monly in pairs between the heterocysts. The cells were quadrate and

without constrictions at the cross-walls, except occasion11y just

after the point of branching, Heterocysts were quadrate to cylimIrical,

sheaths thin, close and hyaline. They were not colored.

Dicothrix spp.

Nnbers of this genus were relatively infrequently observed

in soil crusts, and could not easily be distinguished from species of

Calothrix. They grew in association with other filainentous blue-green

algae and were apparently overwhelmed by more prolific algae. For this

reason it was found to be more feasible to isolate this organism from

dominant growth on a cat's dish than from the soil.

Dicothrix Orsiniana: On agar this organism grew well, and

even covered the surface of nitrogen-free agar plates, fonning exten-

sive, fuzzy, olive-green mats of growth. Prolific growth was not ob-

tained in aqueous media, and that obtained was usually adherent to the

sides or bottom of the container. On agar plates the sheath enclosing

the two filaments, even for part of their length, was frequently im-.

perceptible. Usually a number of single filaments radiated from the

basal portion of a cTnnon filament. In aqueous media, the presence

80

of several, usul1y 2, trichoxnes within a sheath became apparent and

the filsments then branched freely. Vegetative cells within the sheath

were frequently subglobose near the basal heterocyst, longer and taper-

ed distally, and olive-green in color. Heterocysts were usually sub-

globose to hemi-spherjca]. in shape.

Chroococcus app.

Of all the organisms observed, this one was the most confus-

ing due to different stages of the organism which resemble descriptions

for other genera. This organism was not infrequently found in soil

crusts, but identification was difficult due to unfamiliarity with the

stages of the life cycle. The organism for this research was first

isolated on filter paper, cultured on nitrogen-free agar, then in ni-

trogen-free aqueous media and used in experiments on nitrogen fixation,

On agar plates it formed clumped, gelatinous colonies, blue-green in

color, but under conditions of desiccation or age the colonies became

massed, were colored yellow or broim as well as green to deep blue, and

became powdery in appearance.

At various stages in the life cycle of Chroococcus, it resexnbl&.

Synechocy3tis, Myrnecia, and Glbeocapsa and could be classified as any

of the above genera as well as Chroococcus. It may be placed in the

genus Chroococcus when it is realized that sedentary species of this

genus are laiown to have stages of development similar to Gloeocapsa (l8h).When first isolated, the cells were found in small clumpsof

individual cells. No colonial matrix was in evidence and a sheath was

not conspicuous or else homogeneous and not very thick. Cells were

usually spherical, pale blue-green, and with a dense to faintly granular

81

"central body" which appeared rather opaque arid with a suggestion of

a reddish tinge. On the basis of these characteristics the organism

could be identified as Synechocystis.

On agar plates the organism was found as cells which possessed

lamellated sheaths, either singly, in groups of two or three, in chains

of up to 25 cells in length, and as many closely packed spherical to

ovoid cells within ovoid to globose gelatinous membranes. At this stage

some of the small isolated clumps of cells resembled Gloeocapsa, With

time the resemblance to Gloeocapsa was very pronounced. At this stage

the organism was inoculated into media for the nitrogen fixation ex-

periment. After 52 days some of the organisms were removed from their

attachments in vessels of aqueous nitrogen-free media and again examined0

Cells were found free and independent, in clumps of single cells, or a

densely packed colony of cells within globose gelatinous hyaline invest-

ments. Size of cells at this stage was and the color ranged from

green to blue-green. The shape of the cells was from globose and el-

lipsoid to pyriforn. An opaque "central body" with or without "pyrenoid"

was apparent in many of the cells. Many cells also had a manillate

thickening at one side of the wall, oftentimes laterally expanded. Ac-

cording to the latter description, this stage of the organism is simi-

larto that for My-rrnecia (223). See Plate 10.

Inoculated again on nitrogen-free agar the cells were identified

as to the genus Chroococcus. The cells were spherical, hsmispherical

or angular, forming variously shaped colonies of 2 to 50 or more cells

within a gelatinous matrix which was hyaline to yellowish-brown in

color. The cells were usually in diameter, not including the sheaths,

P1. 11Photomicrograph of

Anabaena sp. (X930)as it appeared in N-free media. Filamentsof Anabaena sp. areshown with various-shaped heterocysts.

P1. 10Photoinicrograph of

Chroococcus rufescens30) as it appeared

in N-free media. Colo-nies arid single cellsare shown. Nuceli andprotuberance from cellscan be observed insome cells,

P1. 12Photomicrograph of

Aphanocapsa grevillei(X930) as it was ob-served in N-free media,Cells and membraneouscolonies are shown asthey appeared in thegelatinous matrix ofthe plant mass.

82

and ranged in color from a deep blue-green to yellow or yellowish-

brown. The species closely resembled that given for C. rufescens (236).

Aphanocapsa spp.

This organism was first observed attached to filaments of a

Scytoneina sp. in soil crusts and. at this stage it resembled the genus

Gloeocapsa, However, after the organism had been streaked-out on sever-

al nitrogen-free agar plates, the organism began to resemble hanothece,

The cells were then scattered throughout the homcgeneous colonial

matrix or else the cell sheaths were indistinct. The shape of the cells

ranged from ovate to short cylindric with rounded ends. The cell con-

tents were gray or grayish-yellow, and the plant mass appeared olive-

green to yellow. The plant mass easily broke apart when disturbed.

In aqueous nitrogen-free media this organism most closely re-

sembled the genus Aphanocapsa, The cells were then spherical to ellip-

siod in shape, 3. - diameter, frequently averaged when

spherical, were yellowish-green to blue-green in color, and slightly

granular or homogeneous; sheaths were lacking. Within the hyaline

homogeneous gelatinous matrix, cells were scattered or closely aggre-

gated, but not densely so. The colonies occurred attached and not free

within the aqueous medium. The organism as described here closely

resembles A. grevillei. See Plate 12,

83

Nitrogen Fixation with Pure nr Mixed Cultures

Orgni sms Not Demonstrating Nitrogen Fixation

As many blue-green algae as possible were isolated for the pur-

pose of determining nitrogen fixation. Some blue-green algae could not

be isolated in a pure culture for test purposes. These included the

algae listed under "Blue-Greens Not Tested" in Table 6.' Frequency of

occurrence of these organisms was either limited, they could not be

separated from other algae, or a medium was not found which was favor-

able for growth and isolation. Species of two genera listed, Tolypo-

thrix and Calothrix have been reported as nitrogen-fixers by other

workers.

The blue-green algae which did not present favorable evidence

for nitrogen fixation are also listed in Table 6. Some of these algae

were investigated in detail, others were eliminated on the basis of

poor growth on agar plates made with nitrogen-free salts and tap water.

It is. to be realized that this list may include nitrogen-fixers, Time

did. not allow for a detailed investigation into the cultivation of each

and every organism identified. A detailed search for the proper en-

vironment, particularly as to organic constituents in the culture media,

use of various soil cultures, or associated organisms may show in the

future that some of these organisms do fix nitrogen. The investigation

of nitrogen fixation with associated organisms, except for Azotobacter,

has been neglected by workers on nitrogen fixation by algae, the emphasis

being placed on pure cultures. Pringsheim (190) believed that use of

pure cultures had been over-emphasized and that associations of organisms

are useful for various purposes, especially for culturing. Pure cultures

8)4

have not been found in nature and unless the relationship of any asso-

ciation is recognized and understood, an exhaustive search may be neces

sary in order to find the conditions necessary for growth in pure cul-

tures, For example, Goryunova (109) reported that Oscillatoria grew

poorly in the absence of bacteria and that this organism is probably

predacious on bacteria, In this investigation an Oscillatoria sp.

which was isolated from Arizona soil also was found to grow well in

association with other organisms such as bacteria and algae. In fact,

it grew as the dominant organism in a soil culture for as long as a

year. The Oscilatoria sp. in unialgal culture, however, also showed

reasonably good growth on agar made with nitrogen-free salts and tap

water, although growth was never obtained upon transfer of a single

filament to nitrogen-free agar. This organism was not investigated

further for the possibility of nitrogen fixation. The growth of this

or other algae for the purpose of demonstrating nitrogen fixation was

not undertaken by means of soil cultures because the composition of

such. a medium would be relatively unknown ithout extensive investiga-.

tion, Secondly, the introduction of a fixed" source of nitrogen was

not deemed advisable for nitrogen fixation experiments (Ll).

A species of Lyngbya, L. Diguetti, showed promising growth on

agar made qith nitrogen-free salts and tap water, but not in pure cul-

ture on nitrogen-free agar. This organism made very limited, poor, chlo-

rotic growth in nutrient solution made from nitrogen-free salts and tap

water. Plate 6 shows flasks of this organism as used in an experiment.

Flasks 1-6 contained the medium given abcve. Flasks 7 and 8 also con-

tained nitrogen as sodium nitrate, Flask 9 was uninoculated, Flasks

8S

7 and 8 contained a much greater amount of growth of healthy alga.

In a second experiment using the same medium, except for the substitu-

tion of deionized water for tap water, the alga was cultured in a

system under conditions of controlled temperature and light and pro-

visions for exclusion of nitrogenous contaminants. The alga iriade

even less growth, but was not dead, since it fluorished to produce

extensive normal growth upon the addition of nitrogen as sodium nitrate.

Sodium was not a limiting factor for growth.

An organism resembling Plectonenia Wollei was also investigated

in regards to nitrogen fixation. This organism grew extensively on

agar made from nitrogen-free salts and tap water, but did not have the

characteristic dark blue-green color. Microscopic examination did not

show what could be interpreted as abnormal or unhealthy cells, so the

organism was inoculated into a culture designed for nitrogen fixation

study. Even after several inoculations no growth of this organism

could be obtained in aqueous nitrogen-free media. Either this organ-

ism does not fix nitrogeil or the proper conditions were not found for

nitrogen fixation.

Organisms Demonstxating Nitrogen Fixation

Nitrogen fixation was determined with pure cultures of blue-

green algae, Table 7, as well as with mixed cultures, Tables L and 9.

A mixed culture was noted as such (a) when the associated organism was

bpure culture - a unialgal culture; a culture containing one kind

of algae and no associated organisms such as bacteria or fungi.

SNixed culture - a culture containing one or more kinds of algaeand/or associated organisms.

86

inoculated into the culture vessel but failed to grow or could not be

recovered again when restreaked on agar plates, (b) when any culture

could not be recovered in the pure state, or (c) when the culture was

contaminated during the course of the research. The associated organ-

ism was noted and listed under ttQrganjsfl for all mixed cultures.

In order to evaluate the extent of growth of the algae while

fixing nitrogen, the cultures were concentrated and ignited to about

OO°C at the termination of the experinent. The loss in weight upon

ignition was used as an indication of the amount of organic matter

produced. It was noted that the percent of weight lost upon ignition

was quite variable for mixed cultures. The amount of solids in the

supernatant solution was dependent to a great extent upon the solu-

bility of the salts in the media. The cultures which contained a con-

siderable amount of calcium carbonate lost little weight upon ignition,

especially those grown under anaerobic conditions. The balance of the

weight was reported as the weight of insoluble salts plus the ash of

the organism. A combination o± cultures was made in certain instances.

The total weight lost of these cultures varied extensively, depending

on the number of cultures combined as well as on the nature of the

nitrogen-fixing organism and its associates, Other conditions under

which nitrogen fixation took place also were found to determine the

organic matter produced. These were noted and controlled as much as

possible. See Tables 3 and it for environmental conditions.

The amount of nitrogen in the supernatant varied from 0.0 to

as much as l.O mgm. In general more nitrogen occurred in the super-

natants of the pure cultures than in the mixed cultures. Possibly the

TA

BL

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alga

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salts

gnis

.

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ost

upon

igni

tion

Perc

ent

Tot

algm

s.

Nitr

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Tot

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mgm

.

Rat

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mat

ter

to N

fix

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Ana

baen

a sp

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des

0.60

6376,9

o.)4662

15.0

26.0

Ana

baen

a L

evan

deri

0.S31

73.7

0.3917

0.0

30.0

30.0

13.1

Nostoc sp.

0.3266

71.2

0.2325

3,,?

li..)4

18.1

12

8oc

sp.

o. li

.692

77.5

0.3636

0.3

20.3

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17,7

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450

71i..

50.8530

9.3

50.0

59.3

Nos

toc

sp.

O .SILO?

86,)4

1.3366

9.IL

72.3

81.7

13,9

Nos

toc

sp.

045767

62,1

0.3581

3.1

10.2

13.3

26.9

Scytonema sp.

0.5337

89.IL

o.IL771

21.3

26.3.

18.1

Scyt

onem

a A

rcha

ngel

liO

0828

52.7

O.01i36

2.IL

1,9

10.1

TABLE 8.

NITROGEN FIXATION IN AQUEOUS SOLUTION BY BLUE-GREEN ALGAE IN MIXED CULTURE

Or anisms

Anabaena sp. not bacteria-free

plus

Scy

tone

nia

ap.

No

grow

th0.

9836

of Scytonema sp. observed

Chroococcus rufescens

also re-

sembling Gloeocapsa and Myrme-

0.2O3L

cia) unialgai, not bacteria-free

0.1159

Same organism as above

0.1176

Dicothrix Orsiniana plus Lyng-

bya sp., neither culture re-

O.ISOIL

covered bacteria-free

Aphanocapsa reviUei plus Lyng-10060

bya Diguetti, not bacteria-free

Anabaena Levanderi, unialgal,

0.3699

but not bacteria-free

rtoneita sp., unialgal but

not fungus-free.

No growth

of fungus observed

Same organisms as above

Nostoc sp. plus Microcoleus

sp, No growth of Microcoleus

s.. observed

Same organisms as above

Scytonema sp.

Probably not

bacteria-free

Dry wt.

algae +

salts

gins.

Weight lost

upon ignition

Nitrogen

Total N

fixed

Ratio

oforganic matter

to N fixed

Super

natant

Dry

Nt.

Percent

Total

gnis

.m

gm.

mgx

n.m

gm.

85.3

0.83

900.

0025.9

25.9

32.I

57J.L

0.11

680.

005.

95.

919

.8

71.8

0.7223

0.00

32.0

32.0

22.6

63.9

O.2

36L

0.00

JJ4.

)4)J

4.1.

L.h.6

0.0517

0.30

2.7

2.7

19.1

S1.I

Lo.

O6O

Io.

LiO

2.8

3.2

18.9

56.3

0.25

361.

50lti

..O15

.516

.Li.

TABLE 9,

NITROGEN FIXATION IN AQUEOUS SOLUTION BY BLUE-GREEN ALGAE IN MIXED CULTURE

Nostoc sp. plus soil bac-

teria.

Bacteria did not

appear to grow on N-free

agar.

Average

3.11i77

2.7970

2,6835

2.7195

14,6859

* The tap water used contained 1.3 mgm. nitrogen per liter,

This amount was subtracted from the

values reported above.

Aerobic Conditions

15.2

O.ti785

1.1

27 2

28.3

16.9

16.3

0.14559

0.0

28.6

28.6

15.9

15.5

0.14159

0,9

214.11

25.3

16.14

12.2

0.3318

0.1

22.11

22.5

1.)4.7

10.2

O.!78O

2.2

27.5

29.7

16.1

26.5

16.0

Anaerobic Conditions

0.1085

0.0

2.7

2,7

110.2

14.3

0.U514

0.0

3.0

3.0

38.5

2.85

39.35

Ratio of

Weight lost

Nitrogen

organic

upon ignition

Super-

Dry

Total N matter to

Percent

Total

natant*

wt,

fixed

N fixed

gms.

mgni.

mgm.

mgm.

Drr

algae

plus

Qganisms

salts

gms.

Nostoc sp. plus soil bac-

2.14652

teria.

Bacteria did not

2,681J.

appear to grow on N-free agar

Average

90

Soluble nitrogen in mixed cultures contributed to the nitrogen supply

of non-nitrogen-fixing associates and consequently was fixed in their

cells in an insoluble form. However, the difference may have been

due to the particular nature of the nitrogen-fixing alga, its condi-

tion, or its environment. The nitrogen in the su.pernatant could pro-

bably be considered as nitrogen available to other plants, although the

chemical nature of this nitrogen was not detenained. No ammonia ni-

trogen was found. An attempt was made to determine the amount of

nitrate-nitrogen present at the termination of some of the experiments.

The amount present was too minute for accurate detemination. Nitrate-

nitrogen could not be determined in cultures containing chelates, since

there was interference with color formations

The amount of nitrogen in the dried material also had a con-

siderable range, depndirig on factors mentioned previously. However,

it was possible to determine the ratio of total nitrogen fixed to

weight-loss upon ignition. This is given in Tables 7, 8, and 9, as

the ratio of organic matter to nitrogen fixed and. is useful for com-

parative purposes. For nitrogen fixation in pure culture, the ratio of

organic matter to nitrogen fixed ranged from about 10 to 27 For

mixed cultures, the range was from about 9 to 32, but was somewhat

higher for the two cultures of Nostoc sp. which were grown anaerobical-

ly. To what extent aeration was a factor in the other cultures could

not be determined. Heavy growths of algae sometimes formed around the

openi.rigs of the aerator tube and could have reduced aeration to some

extent.

In Table 10 a summary of nitrogen values is given for all

TABLE 10, SUTYiMARY OF NITROGEN FIliTION IN CULTURE SOLUTIONS

91

Organisms

Wt0 LostUponIgnition

NFixed

Ratio ofO.M. toN Fixed

N as% ofDry Wt,

us. mgni.

S. Archangefli O.Ob36 1.3 10.1 9.9A. spiroides 1.3192 100.3 .13.1 7.7A. Levander2. 0.6281 lLi.1 7.].

Dicothrix Orsiniana + Lyngbya sp. O.35I1 22.3 15.9 6.3Nostoc sp. 5.i65I 310.5 16.6 6.0Scytonema sp. 0.6161 35.1 17.6 5.7Chroocoecus rufescens 0.1168 5,9 19.8 5.1Aphanocapsa grevillei + Lyngbya

0.7223 32.0 22.6DiguettiAnabaena sp. 0,8390 25.9 32,LL 3.1

92

cultures of any one kind of alga or association regardless of the con-

ditions under which the experiments were carried out, except for deliber-

ate anaerobic conditions for two cultures of Nostoc sp, The amount of

organic matter produced for different organisms ranged from O.OL36 to

S,3893 gms,, depending on the total nount of growth obtained. The

amount of nitrogen fixed ranged from Lj..3 to 326.2 mgin. per cultures

These values also depended considerably on the total amount of growth

obtained. The ratio of organic matter to nitrogen fixed ranged from

about 10 to 32, which is significant for ranking the organisms as to

their ability to fix nitrogen, These values may also be compared in

terms of the amount of nitrogen as percentage of dry weight - organic

matter. This is, of course, the inverse of the ratios. As shown by

these figures the ability to fix nitrogen ranks the organisms as foflows:

S. Archangefli> , A. spiroides , A. levanderi ), Dicothrix Orsiniana

plus Lyngbya , Nostoc sp. , Scytonema sp.', Chroococcus rufescens,

Aphanocapsa grevillei plus Lyngbya , and Anabaena sp.

Of significance is the fact that only the organisms belonging

to the genera Nostoc and Anabaena were previously known prominent nitro-

gen-fixing blue-green algae, and species used in this research may not

have been those previously known to fix nitrogen. As shown by the rank-

ing of nitrogen-fixing organisms, Iost sp. did not demonstrate the

greatest ability to fix nitrogen. Of the Anabaefla, two species ranked

high in the ability to fix nitrogen, but one species ranked the lowest

in comparison with the other nitrogen-fixers cultured.

The amount of nitrogen fixed by the association of Dicothrix

0jniana and Lyngbya ap. is unique in several ways. The Lngbya sp.

93

was found not to fix nitrogen alone as determined by one experiment i

Unialgal culture and one experiment in pure culture, see Plate 6. The

D. Orsiniana grew luxuriantly on nitrogen-free agar, but poorly in

aqueous media of the sane composition. Growth of sufficient quantity

could not be obtained for the purpose of analysis even after several

inoculations. However, the D. Orsinianma and Lygbya sp, grew reason-

ably well when in association and nitrogen was fixed. It may be sig-

nificant that the two organisms were oiginaUy found in this asso-

ciation in nature. Unfortunately, the influence of a contaminating

bacterium could not be ruled out, even though this organii failed

to grow on nitrogen-free agar.

The growth and nitrogen fixation of the species of Scytonema

is to be noted in that no species of this genera have as yet been re-

ported as demonstrating nitrogen fixation. It should also be noted

that this orgn sm occurred in many of the virgin soil crusts inves-

tigated, although not in the cultivated soils examined. In studying

nitrogen fixation by Scytonema spp. difficulty was encountered because

the organism habitm1 ly became attached to the walls of the containers,

The growth obtained was apparently quite healthy and prolific for any

one colony or plant mass, but it was obvious that vigorous agitation

of the medium was needed in order to promote an even distribution of

growth. The growth of the alga identified as S. Archangelli was

quite local, but for the amount of growth obtained, the ratio of

organic matter to nitrogen fixed and the amount of nitrogen as per-

centage of dry weight were the highest of all organisms investigated

in this research. These values as shom in Table 10 were 10.1 and 9.9

9)3

respectively. On the basis of this information, it could be predicted

that for every grain of weight of this alga obtained, one-tenth of its

weight would be nitrogen.. A photornicrograph of Anabaena ap. from

a nitrogen fixation experiment is shown in Plate 11. As given in the

literature review, members of a related genus, are powerful

nitrogen-fixers, and it is therefore rather surprising that members

of such a closely related genus have been neglected in nitrogen fixa-

tion studies.

Of further significance in this research was the demonstration

of nitrogen fixation by two unicellular members of the blue-green algae.

So far nitrogen fixation has been reported in the literature only for

filaxnentous blue-green algae. hy these organisms have not been pre-

viously reported as nitrogen-fixers is not known, but may have been due

to a failure to recognise the organisms at various stages of the life

cycle, or perhaps a "key formula" was not found for growth of these

organisms under conditions whereby nitrogen fixation could take place.

Adaption could not, of course, be entirely ruled out, For example,

when Chroococcus rufescens was first isolated it lacked a blue-green

color and made poor growth on nitrogen-free agar. It later developed

a blue-green color and fixed nitrogen well. Jhether an adaptive species

developed or not is difficult to ascertain since this was the first of

a series of plates on which the organism was isolated, A photomicro-

graph of this organism as it appeared in nitrogen-free aqueous media is

shown lfl Plate 10.

C. rufescens also grew well on nitrogen-free agar.. It is believed

to have been a pure culture when inoculated into the culture vessel for

95

nitrogen fixation. However, bacteria were found in association with

the alga at the conclusion of the experiinent. Although the contri-

bution of the associated bacteria were not determined, it may be sig-

riificant that no nitrogen appeared in the supernatant. Upon reinocu-

lation on nitrogen-free agar, microscopic examination showed the bac-

teria to be adherent to the gelatinous coverings of the alga. On the

basis of quantitative data obtained from nitrogen fixation, the ratio

of organic matter to nitrogen fixed was about 23 and the amount of

nitrogen as percent of dry weight was about 5.

Pure cultures of this alga also grew in aqueous nitrogen-free

media, but no quantitative data were taken, Healthy growth was ob-

tained in such media, but added nitrogen as an]moniunl nitrate appeared

to increase the rate of growth slightly. Microscopically, the appear-

ance of growth under conditions of combined nitrogen as amm.onium

nitrate did not seem different from that where free nitrogen was the

source of supply.

The relationship of Aphanocapsa grevillei in a nitrogen fixa-

tion study also presented a complicated picture in that nitrogen was

fixed in the presence of associated organisms. Pure culture conditions

were not obtained. A photomicrograph of this organism as it appeared

in aqueous nitrogen-free media is shown in Plate 12. As shown in

Table 8, 71.8 percent o± the dried growth was organic matter. Much

green growth of a flocculant nature was obtained in pure culture. This

was used to obtain quantitative information in the final experiment on

nftrogen fixation rather than for pure culture studies. The organism

made growth On agar plates containing nitrogen-free salts and tap

96

water, but the growth was not constant in appearance. Sometimes it

appeared dark green in color and at other times it appeared rather

chlorotic. Contamination with Lyngbya Diguetti, which occurred ac-

cidentially, served to promote the growth of the former alga. Nicro-

Scopic examination of growth taken from the aqueous nitrogen-free med-

ium showed L. Diguetti and bacteria scattered through ie matrix of an

extensive growth of Ae grevillei, See Plate 12. when inoculated on

nitrogen-free agar, this association persisted. The growth of the A.

grevillel was again dominant, but doubts as to the nitrogen-fixing

ability of this organism can not be settled without rurther investiga-

tion. As was noted for C. ru.fescens, no nitrogen was found in the super-

natant The ratio of organic matter to nitrogen fixed and the per-

centage of nitrogen as dry weight were comparable to that for C, rules-

cens, These vlues were about 23 and Li., respectively,

Chemical Analyses of Soil Crusts

To determine the extent to which nitrogen as well as carbon had

been fixed in the field, some of the crust samples were analyzed for

carbon and nitrogen, the CtN ratio calculated, and the pH value determined,

These valies are given in Table 11. In the majority of cases, except as

mentioned previously for the treated citrus plots, the samples had an

alkaline pH, For two sanles, Li.3a and 96e, a value as high as 8,0 was

obtained. Usually a slightly higher alkaline pH was obtained for sub-

soils of'corresponding surface crusts. As stated before, neither an

alkaline pH nor high salt content should interfere with the growth of

blue-green algae, since they have a preference for the former and a

tolerance for the latter, Brannon (3t) believed that pH was a dominant

97

jç ANALYSES OF SOIL CRUSTS CONTAINING ALWE AND/OR LICINS

Sam.le No. Rocks> 2 , .H Carbon Nitro:en CrN Ratio

35paste

5.2-

35a 8.0 7.3 O.13 .072 6.035b 9.7 7.0 0.26 .oli.o 6,537 -- -- 1.37 .250 S.37a 9.0 8.0 0.33 .053 6.237b 11.3 8.2 0.20 .02939a 11.0 7.7 0.80 .107 7.539t 11.2 8.7 0.08 .018

12.0 7.7 0.75 .120 6,3.)40b 20.9 8.14. 0.17 .026 65141g. io,5 7,9 0.53 .077 6,9hlb 182 8.7 0.11 .015 7.3b2a 5.5 7.7 0.62 .086 7.2b2b Lth.2 8.2 0.19 .021 9.O,

14.3a 6.0 8.0 0.38 .061 6.,2.

b3b 25.9 8.L 0.19 .036 5.3.

iOta 9.5 7.8 0.714 .05014kb iLp..Lt. 8.2 0.11 .032 3jti5a 6.5 5.6 0,514. .065 8.3

liSb 9.3 3.6 0.25 .0141 5.3146a 8.5 6.6 0.147 .061 7..?

Ii.6b 114.0 14.5 0.22 .0141 5.14

IL8a 11,2 7.8 1.114 .0614 17.3148b 20.5 8.14 0.26 0.33 7.9149a 7.1 7.9 0.77 .081 9,5149b 22.0 8.3 O.11.L .015 7.8149c 5,14. 7,9 0.87 .081 10.7149d 19,5 8.5 0.11 .015 6.1

SOa 13.6 7.14 1.23 .2614. 14,7

Sob 52.2 7.9 0.143 .055 7.8

Sla 9.9 7.0 0.53 .065 8,2

51b 19.5 6.5 0.20 .025 8.0

Sic ]J.L.)4 5,6 0.19 .026 7,3

51d 26.1 6.0 0.11 .016 6.9

52a 5.2 7.9 1.07 .113 9,552b 21.9 8,1 0.23 .0314. 6.8

63a 2.2 7.8 0,82 .095 8.6

63b 5.6 8.7 0.07 .012 5.9

63c 8.14 0.19 .026 7,382a 6.6 7.9 0.63 .072 8.8

82b 18.3 7.2 0.25 .030 8,3

82c 9.2 7.0 0.70 .096 7,3

82d 5.3 6.7 0.19 .025 7.6

82e 5.7 6.8 0.55 .073 7,5

83a 2,6 7.8 1,16 .1145 8.0

83b L.8 8.2 0.17 .023 7,14.

96a 2,14 7.8 1,014 .122 8,596b 6.9 8,3 0.114 .020 7.0

96c 14.0 7.9 0.87 .109 8.0

9,5 8.2 0.16 .0214 6.7

96e 3.2 8.0 0.75 .091 8.2

96f 7.7 8.1 o.i6 .023 7.0

96

factor in the distribution of blue-green algae in Florida and Tsr

Meulen (232) found blue-green algae common in neutral or alkaline soils,

but rarely present under acid conditions.

Values for the percent of carbon are shown in Table U. For

virgin soils these values ranged from 0.38 to 1.23 percent for the

surface crusts, and from 0.07 to 0.I.3 percent for the subsoils. The

highest value for carbon was obtained for a soil having lichens, sam-

pie SOa. This same soil also had the highest value for the subsoil and

this value probably would have been higher except for the presence of

52.2 percent rocks, see Plate 3

The carbon percentages of samples from the citrus plots did not

show as wide a range of values as for the virgin soils. The values

obtained were from 0.33 to 0.7L. percent for the surface soils and from

0.11 to 0.26 percent for the subsoils. Scrapings from surface growth

on citrus plot samples yielded a considerably higher percent of carbon.

Values detennined for two of these samples, 35 and 37, were 1.37 and

1.57 percent, respectively.

Analyses for the percent of nitrogen showed that these values

ranged from O.061i. to 0.261. percent for the crusts of virgin soils and

from 0.012 to 0.55 percent for the ubsoils. The highest percent of

nitrogen for a surface crust was obtained for the same soil, 50a and

50b, which had the highest percent carbon. Plate 3 shows the rocky soil

on which growth occurred. Only five other samples of the virgin soils

contained more than 0.]. percent nitrogen. Two of these samples, bOa and

52a, contained lichens, but the other three samples, 39a, 83a, and 96a,

were lichen-free algal crusts. Of significance may be the fact that all

99

of these samples, except 96a, contained recognizable Scytonema sp.

Which was a nitrogen-fixing organism as determined by this research,

Samples 39a and )40a contained no previously known algae reputed. to be

nitrogen-fixers. The level of nitrogen obtained for sample 96a can not

be accounted for on the basis of the diversity of algal species nor

the presence of Scytonema sp., although the latter could have been

overlooked if it were not prominent in the crusts observed,

The values for percent of nitrogen for the citrus plot samples

ranged from 0.00 to 0.072 percent fo2' the surface soils and from

0,029 to 0.017 percent for the subsoils. The highest values were ob-

tained for scrapings from surface growth of samples 3 and 37. These

values were 0.300 and 0.2S0 percent respectively. An Oscillatoria sp.

was the only prcmdnent blue-green alga identified and isolated from

these scrapings. As was previously mentioned, this organism could

not be grown in pure culture on a nitrogen-free medium.

The CN ratios were reasonable for sailes collected from

virgin soils as well as samples collected from the citrus plots as

representative cultivated soils. In general, the ratios were wider

for surface crusts than for the subsoils, which is the usual relation-

ship (1b6). The ratios ranged from about to 18 for the crusts and

from about .3 to O for the subsoils. As may be expected, the C:N

ratios tend to be lower in ari&ic soils than in humid areas where the

temperature is Comparable (lL6). It may be significant that the low-

est value for a crust sample was for one containing lichens, sample 50a,

Nitrogen_Fixation By Soil Crusts

An attempt was made to deteimine nitrogen fixation in the field

Unv1 of Arizona Library

100

Under natural conditions. However, natural precipitation could not be

relied upon as a constant source of moisture. Even for a one week per-

iod, the amount of carbon as well as nitrogen was found to decrease in

the surface crusts. This was shown by the 96 series of cmsts. See

Tab1 and U for description of the crusts, time of collection, and

results of chemical analyses,

In order to determine nitrogen fixation by soil crusts within

a reasonable period of time, crusts were placed in desiccators for the

purpose of simulating field conditions. To provide a substitute for

rain, deionized water was applied to the filter paper of moist ehainbers

A more restricted condition consisted in the filtration of the air

source for a duplicate sample as a precaution against contamination

with nitrogenous impurities. Under conditions of the experiment no

significant difference could be shown for duplicate samples and they

were therefore combined to give the values shown in Table 12. These

values are shown graphically in Figure 1,

Analyses were carried out after each week of incubation, After

combining values for each two weeks of incubation, significant increases

could be shown in nitrogen fixation. After L weeks the Tucson sandy

loam had increased in nitrogen by 0.026 percent, the Gila fine sandy

loam by 0.02L percent, and the Gila sandy loam by 0.0LO percent nitrogen.

.jj. three of these soils contained Nostoc sp. , two contained Anabaena

sp., and all three contained Scytonema sp. The first two soils were

algal crusts and the thi'd contained lichens The third soil contained

16 genera of algae, which was twice as many genera. of algae as the first

soil. It contained 8 more genera of algae than the second soil and many

Total Nitrogen xedBegin 2 14

Soil Incubation Weeks Weeks Increase

101

ThBLE 12. NITROGEN FIXATION BY SOIL CRUSTS IN MOIST CHAMBERS

Tucson Sandy0.09 Q107 0.026

Gila FineSandy Loam (63a)O.09S 0.117 O.U9 0.0214

Gila SandyLoam (82c) 0.096 0.118 0.136 0.0140

J4N

ITR

OG

EN

FIX

AT

ION

BY

SO

ILZ

CR

US

TS

IN M

OIS

T C

HA

MB

ER

SjJ

3T

UC

SOfl

San

dy L

oam

(3 F- z.II '.1

0

LU 4:.

LJ.0

8

.07 0

C11

a Fi

ne S

andy

Loa

m63

a82

c

24

02

4IN

CU

BA

TIO

N T

IME

INW

EE

KS

Gila

San

dy L

oam

02

4

102

more recognizable species than either soil. This may account for a

greater increase in nitogen with the third soil than by the other

two.

SUNMA.RI

Both microfauna and microflora were examined in the semi-arid

soils collected. The soil fauna included cilicates, flagellates,

amoebae, neivatodes, and various insects. The flora included algae,

lichens, higher and lower bacteria, fungi, myxomycetes, actinoinycetes,

moss, and several higher plants.

The algae were investigated more extensively than the other or-

ganisms. Particular attention was given to the blue-green algae which

were investigated in detail. Descriptions were given for some of the

blue-green algae observed. Fifty-one genera of algae were identified

belonging to the orders Cyanophyta, Chiorophyta, Chrysophyta, Pyrophyta,

Euglenophyta, and Diatoms. Of the genera identified, 21 were blue-

green algae. These included the following genera: Plectonenia, Chroococ-

cus, Calothrix, Gloeocapsa, Triohodesmium, Tolypothrix, Spirulina,

Phorinidium, Microchaete, Osciflatoria, ?Iicrocoleus, Symploca, Lyngbya,

Nos toc, Anabaena, Scytonema, Porphyrosiphon, S chizothrix, Aphanocapsa,

Dicothrix, Rhabdoderma, Nodularia, Synechococcis, and Aphanothece.

Characteristics of the organisms were found to vary depending on biologi-

cal, chemical and physical conditions of the habitat. All of the algae

encountered possessed the ability to resist desiccation and to change

from the resting to active state in a relatively short space of time.

For the purpose of deterzwiriing nitrogen fixation, as many blue-

green algae as possible were isolated. Some species were found to fix

nitrogen when in pure or mixed culture experiments. These are ranked

103

10)4

according to their ability to fix nitrogen: S. Archangefli ?' A,

Spiroides,), A. levanderi ), Dicothrix Orsiniana plus Lyngbya),

Nostoc sp.), Scytonema HP.'?, Chroococcus rufescens , Aphanocapsa

grevillei plus Lyngbya , and Anabaena sp. The amount of nitrogen

fixed ranged from about )4 to 326 mgm. per total culture. The ratio

of org'nc matter to nitrogen fixed ranged from about 10 to 32. The

ratio was higher under anaerobic conditions.

Only members belonging to the genera Nostoc and Anabaena were

previously known nitrogen-fixtng blue-green algae. Unicellular blue-

green algae have not been previously shown to fix nitrogen, but cul-

tures of C. rufescens and A. grevillei fixed nitrogen.

Under simulated field conditions nitrogen fixation was obtained

by associated organisms in soil crusts. After )4 weeks the percentage

nitrogen in an algal crust increased from 0.081 to 0.107 percent and

in a lichen crust from 0.096 to 0,136 percent.

Chemical analyses of soils showed that algal and/or lichen

crusts contained carbon percentages ranging from 0.33 to 1.23 percent

for the crusts and fxom 0.07 to O.)43 percent for the subsoils. Nitro-

gen values ranged from o.OSO to 0.26)4 percent for the rusts and from

0.029 to O.05 percent for the subsoils. The C:N ratios varied from

about 5 to 18 for the crusts and from about 3 to 10 for the subsoils.

Higher values were obtained for carbon and nitrogen detenninations on

the virgin soils than on soil from the citrus faim plots,

In conclusion it has been shown that nitrogen-fixing blue-green

algae occur in the semi-arid soils of Arizona and that they contribute

significantly to the nitrogen status of the soil, Species of Soytonenta

loS

also are believed to be important organisms in nitrogen fixation,

since they occur in many of the crusts and dvionstrate nitrogen fixa-

tion in pure culture experiments.

BIBLIOGRapHY

A].1en, N. E.

The cultivation of Myxophyceae. Arch, Mikrobiol. l7:31.-3.l92.

Allen, L 3.Studies on a blue-green cborella. Cong. Internatl. Bot.

Comm, 8 (Sect. 17):Lil-h2. l914.,

3 Allen, N. B.Photosynthetic nitrogen fixation by blue-green algae. Sci.

Mon. 83:100-106. 1956.

Al1erL B. and Arnon, D. I.Studies on nitrogen-fixing blue-green algae. I. Growth andnitrogen fixation by Anabaena cylindrica Lemm. Plant Physiol.

30:366-372. 1955.

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