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 requiruents for an advanced degree at the University of Arizona and
is deposit in the University Library to be made available to borrowers under rules of the Library.
Brief quotations from this thesis are allowable without
special permission, provided that accurate acknowledgment of source
is made. Requests for permission for extended quotatiQn from or
reproduction 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
the interests of scholarship. In ail other instances, howe-er, permission must be obtained from the author.
material is in
SIGNED:
(C
APPROVAL BY THESIS DIRECTOR
This thesis has been approved on the date shown below:
W. H. FULLER
Head, 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
1952-195)4
Attended the State College of Washington, Pullman, WashingB. S. in Bacterio1or and
ton. B. S. in Agriculture 1953.
Public 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. Kuykendali, 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 .
Importance of Soil Algae in Agriculture
Methodology
.
EXPERI1'UNTAL METHODS AND MATERIALS
Collection of Samples and Isolation for Purpose of
Identification of Microorganisms
Nitrogen Fixation in Pure and Mixed Culture
Preliminary Procedure
Culture Solutions and Conditions for Nitrogen
Fixation
Apparatus for Nitrogen Fixation
Nitrogen Fixation by Soil Crusts
Analyses for Nitrogen Fixation in Nutrient
Solutions
3
U
23
32
32
39
39
Io
Li3
I8
50
53
RESULTS AND DISCJSSION
Isolation and Identification
Description of Blue-Green Algae Observed
.
.
Nitrogen Fixation with Pure and Mixed Cultures
Organisms Not Demonstrating Nitrogen Fixation .
Organisms Demonstrating Nitrogen Fixation . . .
Chemical Analyses of Soil Crusts
Nitrogen Fixation by Soil Crusts
53
65
83
83
85
96
99
SUWIA.RY
103
BIELIOGRkPHY
io6
iii
LIST OF TABLES, FIGURES, AND PLATES
Thbles
Number
Page
Culture Media for Algae ,
37
Total Micronutrient Solution for Algae.
38
. .
Conditions for Nitrogen Fixation in Pure Culture
Conditions for Nitrogen Fixation in Mixed Culture
14.
1414
.
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 Algae
in Pure Culture
87
Nitrogen Fixation in Aqueous Solution by Blue-Green Algae
in Mixed Culture
88
Nitrogen Fixation in Aqueous Solution by Blue-Green Algae
in Mixed Culture
89
Summary of Nitrogen Fixation in Culture Solutions
91
.
.
.
.
.
Chemical Analyses of Samples of Soil Crusts Containing
Algae and/or Lichens
Nitrogen Fixation by Soil Crusts in Moist Chambers
Figures
Number
Nitrogen Fixation by Soil Crusts in Moist Chambers
1.
Plates
Number
i,
Typical Sample Collection Area
iv
.
97
.
.
101
Following
Page
,
.
.
101
Following
Page
32
Following
Page
Number
32
Close-up of Algal Crusts
Close-up ol' Conspicuously Raised Lichen Crusts
14.
.
32
.
Incubation of Soil Crusts in Moist Chambers
314
Growth of Nostoc sp. in Nitrogen Fixation Experiment,
314
Experiment on Nitrogen Fixation with Lyngbya
.
. . . . . . .
.
Diguetti . . . . . . .
8
.
.
314
Nitrogen Fixation Urder Conditions of Controlled Temperature, Light, and Use of Filtered Air . . . . .
143
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
V
.
81
INTRODUCTION
Nitrogen is one of the elements essential for plant growth.
is also the least abundant major essential element in the soil.
It
In aria
and semi-arid regions of Southwestern United States there is very littie 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 maintain 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 culturing 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 investigation 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 associations 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.
jerinck
(18)
In
1901 Bei-
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 limited the value of early work on nitrogen fixation.
3
However, the
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.,
(157)
Moore and Webster
gae.
in 1920, also worked with impure cultures of a1-
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,
19l1..
Kruger and Schneidewind (]Jo) in 1900, and Schramm (206) in
Even as late as 1927 Bristol-Roach
(37)
questioned the reli-
ability of results obtained for nitrogen fixation by algae in pure cultures.
It was decided that positive results obtained for nitrogen
fixa-
tion were due either to the presence of other organisms or faulty chernical 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. variabilis 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 evidence 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-fixing 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 forthcoming 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
Bortels (30),
Singh (216),
ied from 0.3 to
2.0
and Fogg (80).
(58)
Rate of algal growth var-
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 investigating a number of soils.
He and his co-workers found
13 species that
fixed nitrogen, including the organisms Tolypothrix teni,s
brevissima, Anàbaenopsis, Schizothrix, and Plectonema.
Watanabe's publication
(253)
Oslo thrix
Unfortunately,
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
This
Schizothrix and Plectonema is based on impure culture studies,
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
These included the following algae:
NlS.
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-fixers.
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
blue-green algae of three families, i.e. Nostocaceae (Nostoc
common
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
light was first used by Allison
(7)
and Bortels (30).
and Morris in
mating bacteria in algal cultures.
Ultra-violet
1930 to destroy contain-
Subsequent workers also used this
method to obtain bacteria-free cultures without destroying the algae,
i.e. Gerloff, Fitzgerald,
Henrjksson
(US) 1951,
and
Skoog (105), 1950, Fogg
(83) 1951,
and
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.
recommended plating out in agar in 1912
later (192).
Skinner (218)
in 1932
Pringsheim (186) used and
and still does forty years
published a lucid description of
similar methods employed in isolating 50 strains of soil algae.
ca-gel has also been used by such investigators as Pringsheim
19114, Schramm (206) 19114, De (58) 1939, and Singh
(216) 19142.
Sili-
(187)
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 nitrogenous 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
pounds.
in organic com-
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,
19142, but no quantitative data are given.
.
(145) in
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
dence can be obtained as to whether or
can fix atmospheric nitrogen.
not a
using
NiS, conclusive evi-
certain biological systn
irJhen air is enriched with N, atom per-
cent of NiS in cells may be determined and
be present in nitrogen-fixing algae.
significant amounts shown
to
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.
195.5,
On an international
basis, Bond
and Scott (26) in
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
century (235).
workers throughout the world since the middle of the 18th
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, 13otrydiopsis, 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 Carter
(159)
Moore and Karrer (160) and
found a definite subterranean
the Missouri Botanical Garden.
algal
flora in
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,
son and Forest
(259) in 1957
Wil..
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
ations or soil, fauna.
layers
or else carried down by tillage oper-
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
.6,219).
Some bluegreen algae grow in the dark
(l,36,39,1.O,55,56,
and fix
nitrogen.
Ac-
cording to Allen (1), this was first reported by Bouilhac in 1897 when
he grew Nostoc punctiforme with sugars.
N. punctifoxne in the dark.
AllisOn,
Harder (in)
in 1917
also grew
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
Others who grew nitrogen-fixing algae
per grain of glucose utilized.
were Winter
and Wolfe
(265) in 1935,
(87) in 1951i..
De
(58) in 1939,
in 1952 Herisset found
According to Fogg (86),
that another species of Nostoc, N. commune,
when grown in the dark.
Allen (1) in 1952, and Fogg
(86)
Fogg
not
used by Herisset were probably
fixed
nitrogen, but not
believes that analytical methods
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, Troe1cia, 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 fixVogel (2143) observed that xero-
ing nitrogen in Russian desert areas.
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.
also in Australia, found
14
Moewus (1S14)
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, Microcoleus, Nostoc, and several members of the family Chroococcaceae.
Others had found that rain crusts reduced water infiltration and promoted 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
served
and control of erosion.
Shields, et al.(212,213) in
the stabilizing influence of algae
and lichen surface crusts in
semi-arid habitats of New Mexico and other areas.
(66)
Drouet
l97 ob-
Piercy
(182) and
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.),
concentration
(5,1L9,81,217),
extreme temperatures (2,51i.,83), a high salt
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 capacity of certain organisms (81,261), and the algae, especially the bluegz'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,
zona, Fletcher and Martin (78) found increases of
In Ari-
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 semidesert 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
Martin
(78)
in Arizona
also noted by Fletcher and
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,
(153) has shown that losses of nitrogen
may be due to algae.
Mitsui
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.
f or rice soils, in
As
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.
as Ayyanger
(ilL),
noted that
Indian workers such
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 Cyanophyceae 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.
(57)
year, further research by De
In that same
suggested that nitrogen fixation
ifl
water-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 acre.
lbs. of nitrogen per
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 hemisphere.
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 nitrogenfixing 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, occurred 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
loss of nitrogen from flooded soils.
(257,258)
have reported the
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 equivalent 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).
a]..
(9)
Allison, et
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 contributes 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).
information on sampling is oftentimes not mentioned
l68,23),
For the most part
(8,67,78,98,l66,J.67,
or is fragmentary (l,73,l7L,l7,l99,220,230,259).
(l2)
Mitra
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.
sterile knife whereas Tchan
Lund (l1i3,1I) collected samples with a
(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
sheim
Beijerinck, Detmer, Bristol
23
Knop Molisch3 Pring-
and others (23,189).
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 contaminants (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.
successfully used lately by others
This method has been
(213,259).
As mentioned previously, agar was used with success for growth
and isolation of algae.
(Si).
Moore
(158)
used flasks of agar, as did Ohodat
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 cultures
(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.
inck (17) in 1898
and Moore
(158) in 1903
Beijer-
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)
Bold (23)
in 1898.
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) discloses 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, potassium, magnesium, phosphorus, sulfur, nitrogen, iron, copper, manganese,
boron, and zinc (22,136).
Carbon, although supplied in the form of
(8S)
carbonates in media, is obtained from the air as CO2 by most algae
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 nitrogen 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.
ed by vanadium
(3)
It can not be replac-
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 possib].e required microelements.
Recently essential microelements for algal cultures
added in the chelate form (233).
have
been
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 alkaline 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 dipotassium 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 influence 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).
20 -
2S°C (i),
The usual temperature for research purposes is
although higher temperatures may be used (9,714).
The influence of light on the growth of algae has also received 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 exceed 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 conditions, 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 dioxide into the air stream as a stinulant to photosynthesis (23).
percent carbon dioxide is commonly administered
even required for dense laboratory cultures
Five
(138,161,163), and
(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
They also found that aeration was not a satisfactory substi-
shaken.
tute for shaking.
Krauss (138) found that a greater photosynthetic
rate could be achieved in natural or laboratory cultures either by
agitation or turbulence.
shaking
Allen and Arnon (Li)did not believe that
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,
mentioned previously, various filtering devices may be used in
As
31
experiments on nitrogen fixation (9,80), but others
(U5,116,2S3)
them in individual flasks exposed to unfiltered laboratory air.
grow
If an
apparatus is used, the choice of culture vessels may be the most jimportarit 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
Erlerimeyer flasks, or Pyrex glass washing bottles (9).
(l31.)
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 determined 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.
of samples were collected from the Tucson area.
The majority
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
The surface crusts, or surface soIls, were placed in
expedition.
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,
letter
110W
also represented surface
32
rusts from the same area, but
The
P1. 1
Typical collection
area, with vegetation
of dry grass, weeds, and
cacti. Also shown are
crust samples on sack
used for subsoil and cyllndrical paper containers for crust samples.
Fl. 2
Close-up of algal
crusts shown in P1,1.
Algal crusts are several inches in diameter. Crusts in place
are shown in the area
surrounding the displayed crusts (sample
18a).
P1. 3
Close-up of conspicuously raised lichen
crusts which occurred
on rocky soil in Catalina foothills. (sample 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 conditions, 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
cases passed through a 2 mm. sieve.
Weight of rocks above 2 mm. was
noted and the sample weight determined.
the subsurface samples, but only 200
used,
1l
Total weight was taken for
n. of the surface CIIIStS were
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.
b
Incubation of soil
crusts in moist. chambers.
Some growth has
spread out on moist
filter paper. (Also
see P1. 8, extreme
right for height of
fluorescent light
above chambers),
P1. 5
Growth of Nostoc sp. in
a nitrogen fixation exper-.
linent (see Fl. 7),
Some
growth adhered to the vessel walls and extended
several inches above the
surface of the medium.
(Incubation period of 35
days, 7-21i.-57 -8-2847).
p
P1. 6
Experiment on nitrogen
fixation with Lyngbya
Diguetii. Flasks 1-6,Nfree salts and tap water.
Flasks 7 and 8, NaNO3
added. Flask 9, uninoculated.
Nitrogen was not
fixed 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 phototactic 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.
lined by Bold (23)
Weighed agar was washed at first by the method outA 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).
likewise be toxic (192).
The former also contained chlorine which may
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 -
iron source was also added in the chelate form.
Table 1, were also used with l.
percent agar and
liter of a total micronutrient, solution.
1.0 ppm.
The
Later on other media
O.S - 2.0
See Table
cc. per
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
satisfactory.
Deionized water was found to be
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)
miscellaneous papers
tifying organisms.
(67,68,79,166,167,168)
were used as
as well as
aids
in iden.-
Measurements were taken for some of the organisms
by means of an occular micrometer, drawings were made, and a description
37
TABLE 1.
edium
CULTURE KEDIA FOR ALGAE
Salts
Concentration
gm./10
II
K2HPO
MgSO . 7H0
NaC].
CaSOI4 . 2H0
CaCO3
hA
K2HP0j
CaSOb , 2HO
0.5
0.2
0.2
0.1
CaCO3
0.5
K2HP01
0.5
0.2
0.2
0.1
2.5
MgSO)4
. 7HO
Nal
113
MgS0 . 7H0
NaC1
CaSOj . 2H0
CaCO3
III
K2HP0j
MgSO . 7H0
0.55
Na2C3
oJ.5
CaC12 . 2H0
'V
K2HP0j
0.12
0.15
Na2CO3
CaCO3
0.35
0.25
0.20
0.20
CaC12 . 2HO
0.15
K2HPO1
0.55
MgS0. . 7H0
0.25
0.20
0.30
NgS01. .
7H0
Na2CO3
CaC12 . 2320
VI
0.5
0.2
0.2
0.1
0.2
K2HP0j
0.55
MgSO . 7H0
Na2CO3
CaC12 . 2H0
0,12
0.20
0.30
38
TABLE 2.
TOTAl MICRONr.TTRIENT SOLUTION FOR ALGAE
Mieronutrient
Source
Goncentration
gm./18 l
Boron
Manganese
Sodium
Iron
Molybdenum
Zinc
Copper
Cobalt
Vanadium
A1th1inum
Nickel
Silicon
Lithium
Iodine
Bromide
Tin
Uranium
Tungsten
Ohiomium
H3B03
MnSO)
Na citrate
FeC].3
MoO3
ZnS0j
OuSOl1. . 5H20
CoC12
6H20
Na3VOI1.
il2(so)3 , 18HO
Ni0) . 6HO
9H0
Na2siO3
LiC1
KI
KBr
SnCl2
2HO
1J02 acetate
P205 . 2LWO3 . 2H0
K Cr20?
11.0
7 0
7.0
5.0
5.0
1.0
1.0
1.0
1.0
1.0
1.0
0.5
0.5
0.5
0.5
0.5
0,5
0.5
0.5
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 icieritification 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 streakedout on artificial media as above, except for the substitution of deionized 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,
tures were easily obtained on streak plates.
Unialgal cul-
A few pure cultures
could be procured after 10-20 plates were prepared per organism.
1 Personal ccmmiunication with Dr. Walter S. Phillips, Head of
Department 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 appeared 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.
total of
A
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 filtrate 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 constitute 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 solution.
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),
fixation experiments.
and an
alkaline medium was used in nitrogen
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 appropriate concentrations to the culture solutions.
The stock solutions
were resterilized after use when it was found that fungus grew proliuically 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 slightly 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 preliminary 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 fluctuating 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.
tion was the only means of' agitation.
Fcr this research, aera-
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 calcium chloride in order to remove water vapor, and next bubbled through
a container o± concentrated sulfuric acid to remove possible contamin-
P1, 7
Nitrogen fixation under
conditions of controlled
temperature, light, and
use of filtered air, Air
was pulled through the
system from left to right
by means of a vacuum pump.
P1. 8
Nitrogen fixation conducted at room temperature using natural (window) light and filtered
aire Air was forced
through the system by
means of compressed air.
Fl. 9
Nitrogen fixation by
soil crusts in desiccators using controlled
temperature, light, filtered and non-filtered
air. At lower right are
flasks of algae in nitrogen-free media.
37
9-7-57
1O-2l.-57
Anabaena.
Levanderi
7-2LL-57
8-28 -57
35
35
7-2t 57
8-28-57
35
8-28-57
7 -21-57
35
7-2 - 7
9-7-57
Scytonna
Archangel 1 i
Soytonema sp. 7-2 - 7
8-28-57
Nostoc sp.
Nostoc sp.
Anabaena
spiroides
Noatoc sp.
Nostoc sp.
7-16-57
8-22-57
Anabaena
spiroides
52
7-10-57
8-31-57
52
7-10-57
8-31-57
52
Length of
Expt,
days
Culture
1
2
1
2
3
1
2
VI
III
IV
LI
V
V
V
IV
Cultures Medium
No. of
0,5
1.0
0.5
1.0
1.5
1.0
1,0
1.0
2.0
1.
Volume
7.5
6.2
7.1
6,1
-
8.6
7.6
7.6
7,6
-
7.2
7.2
7.2
7.1
6.8
7.0
7.0
7.L.
7.0
7.3
8.5
9J
9.2
pH
Final
7.0
7.1
7.0
7.0
7.0
Begin
CONDITIONS FOR NITROGEN FIXATION IN PURE CULTURE
Organisms
TABLE 3,
fluor,
fluor,
fluor.
fluor.
fluor.
natural
natural
natural
fluor.
Light
Source
25
25
25
25
25
fluct.
fluct,
fluct0
25
ature
OC
flask
Container
vacuum
vacuum
vacuum
vacuum
vacuum
jug
jug
jug
jug
jug
diaphrafl jar
diaphragmjar
diaphraginjar
vacuum
Strewn
Temper- Air
Expt.
days
52
10-29-57
plus 9- 7-57
9- 7-57
7-214-57
15
9-7-57
7-2)4-57
'45
9- 7-57
7-2)4-57
3
8-28-57
7-2)4-57
52
6-31-57
55
14-19-57
52
7-10-57
8-31-57
52
7-10-57
8-31-57
52
10-29-57
*3*
Anaerobic conditL ona
pH
ii
II
It
Ii
II
H
Ii
It
ii
It
it
II
a
It
6.8
7.0
7.0
7.0
7.0
7.0
6,7
7.0
9,14
9:2
9,0
8.2
79
6.9
7.2
9.0
8.6
8.14
9.].
7.5
7.6
7.6
7.2
7.2
7.0
7.5
7.6
7.0
7.0
]..0 (Approx) 8.6
8.6
7.6
1.0
1.0
1.0
1.0
1.0
1.0
0.5
9.5
0.5
0.5
1.0
U
II*
IV
IV
V
V
V
V
IV
hA
ha.
(+ 0.18 gm.
niannitol)
VI
V
it
1
1
1
1
1
1
1
1
1
1
2
Culture
Medium Volume Begin Final
1
* Culture not bacteria-free
** The tap water used contained 1.3 mgrn. N/i,
soil bacteria
Nostoc sp.* plus
above
Same organisms as
fungus
algal inoc, with
Scytonema sp. uni-
unialgal
Anabaena Levanderi* 9..7..57
Aphanocapsa grevil-9- 7-57
lei* and Lyngbya
10-29-57
Diguetli
52
bling Gloeocapsa &
Mynnecia) unialgal
Chroococcus rufes-
9- 7-57
cens* (also resem- 10-29-57
.Anabaena sp.*
Scytonema sp.
Scytonenia sp.*
Same organisms
as above
Nostoc sp. plus
Nicrocoleus sp.
as above
Same organisms
Dicothi'ix Orsiril-
Cultures
Length of No. of
CONDITIONS FOR IITROGEN FIXATION IN MIXED CULTURE
7-2-57
ana* plus Lyngbya 8--57
Organisms
TABLE 14.
it
It
II
It
It
natural
natural
natural
natural
natural
natural
natural
fluor,
fluor.
fluor.
fluor.
fluor.
Source
Light
Temper-
diaphragm
vacuum
vacuum
vacuum
vacuum
vacuum
Stream
Air
it
ft
It
ii
Ii
H
fluct.
fluct.
fluct.
fluct.
fluct.
a
U
H
It
ii
II
diaphragm
diaphragm
diaphragm
diaphragni
diaphram
f].uct, diaphragm
fluct.
25
25
25
25
25
ature
11
It
It
It
II
jar
jar
jar
jar
jar
jug
jug
jug
jug
jug
Container
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 contaminants in the air stream.
containing deionized water
Air was next bubbled through a hydrator
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.
in a sthiilar manner.
The tubes of glass wool and cotton were treated
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 vessels 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 sources as previously mentioned.
Insofar as possible sterile technique
was used throughout the entire procedure of inoculation and determination 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.
in actual operation.
See Plate 9 for the apparatus used and system
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.
sterilized open petri dishes of
dejccators as show
in Plate 9.
lb
These were then placed in
millimeters diameter and placed in
Four of the desiccators were inserted
in a system whereby the air was filtered through calcium chloride, concentrated 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.
up as demonstrated in Plate 9.
Four other desiccators were also set
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.
was thermostatically controlled at approximately 25°C.
Temperature
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,
crusts.
Only nitrogen was determined on the incubated
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.
contaminants,
This served as one means for identification of
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 contaminants 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
using an angle head. ôentrifuge.
and centrifuged for 10 minutes
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 supernatant. 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 microlCjeldahl 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
The analyses for
dete.uriined by trial.
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.
each sample was ignited in
weight in a tared crucible.
a muffle
A weighed proportion of
furance at S00°C to a constant
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 microflora.
Frequently observed fauna included protozoa such as the cili-
ates and flagellates, as well as amoebic cysts and trophozoites.
fauna included soil nematodes and microscopic insect eggs.
Other
The pro-
tozoa were more numerous when dilution methods were first used in attempts 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.
o
These organisms contained many globular fragments,
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
54
'O
H
H
Os0,
O0I
0,
Os
0
P.
g
4
0
en
U
11
Os
441
0,
ii
IoI
Os
0
H
I
40
+4
h
-
0
.4
40.00, 0
H
en
p
4
Os
.40,
'-''I
P
11
-
1101
0g
-I
-14
-
a
0,
0-
I
0,
H
H
H
'A
'c'
c0
'A
en
a
en
'0
a
-0
'A
'A
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I
'0
'A
a
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'A
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.4
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 colorless and light pink to deep orange and brown.
bacteria were present.
Some photosynthetic
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.
in some of these se crusts.
Radiobacter were found
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 culture, 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.
heterocysts and cells
The heterocysts would be more abundant, with
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
(23L).
method is needed to obtain growth and isolation of such organisms
These organisms would sometimes appear on crusts incubated for an extended 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).
occurred whenever the myxobacteria formed fruiting bodies.
This also
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
57
S
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 state
of the lichen.
The more common genera
of green algae in the lichens
included Trebouxia, Chlorococcum, Palniellococcus, and Myrmecia.
Table 6.
See
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-.
* Occurred in lichens.
Rhabdodenna
Nodularia
Syneehococcus
Aphanothece
Die othrix
Porphyrosiphon
Schizothrix
Aphanocapsa
Scytonerna*
Tolypothrix
Spirulina
Phonnidium
Mierochaete
Oscillatoria
Microcoleus
Synploca
Lyngbya
Nostoc*
Anabaena
Euglena
Euglenophy-ta
Pyrrophyta
Urococcus
Peroniella
Botrydiuni
Monocilia
Chroiiulina
Chrysophyta
Protosiphon
Trebouxia*
Gloeocystis
Chlorococcum*
Chlorefla*
Palmellococcus*
Myrmecia*
Sphaerocystis
Planktosphaeria
Protocoocus*
Kentrosphaera
Desmids
Plectonema
Chroococeus*
Calothrix
Gloeocapsa
Trichodesmiuxa
Chlorophrba
Cyanophyta
Stauroneis
Dpiloneis
Pinnularia
Melosira
Synedra
Amphora
Nitz schia
Diatoms
Navicula
Epithemia
Scytonema
Dicothrix
Aphanocapsa
Symploea
Lyngbya
Porphyrosiphon
Schizothrix
M±croc oleus
Gloeocapsa
Spirulina
Phonnidium
Oscillatoria
Anabaeria
Pie ctonema
Nostoc
Blue-Greens
probably not
fixing N
Chroococ cus
Blue-Greens
Fixing N
GENERA. OF ALGAE FOUND IN ARIZONA SOILS
Genera of Algae Identified in
Soil Crusts
TABLE 6
Tolypothrix
Rhabdoderma
Microchaete
Nodularia
Synechococeus
Aphanothece
Trichodesinium
Calothrix
Blue-Greens
not tested
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
Algae identified from aD. crusts examined are included in Table
5.
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.
as unicellular forms.
This applies to filamentous as well
In an aquatic environment, filaments of Nostoc
app., without heterocysts, resembled Trichodesxniuin ap.
hibited movement characteristic for this genus.
and even ex-
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 environment was provided.
Other filamentous species were also observed which
were ensheathed until provided with an aqueous environment.
isms were then observed to move out of their sheaths.
The organ-
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 without 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.
tinetly Lilamentous forms were observed.
Very few dis-
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.
algae were not found.
Previously known nitrogen-fixing blue-green
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.
pH values of
5.6
Soil samples 1a and )46a had
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 observed in the field.
This phenomenon was exhibited when the orgaEin was
grown in unialgal culture on filter paper moistened with Bristols medium (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
62
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.
Fritach (96). and Bristol
(3)
As noted by
as well as others (212,213) such an in-
vestment is characteristic for surface forms and prevents loss of inoisture during drought.
change.
Such cells need not show any appreciable outward
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 identification 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 provisions 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
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 required 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 isolation 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 culture 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 contaxainating 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.
single easy method has been devised to obtain pure cultures.
No
Bacter-
ial streak-plates finally were resorted to as a dependable, but tedious method.
Nhen isolated areas of algal growth could be found in which
there was no microscopic evidence of contaminants, then a few organisms 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 contamination, 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 germinate, 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 identifiable 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 yellowish-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.
movement.
Such filaments exhibited definite direction of
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 colorful in aqueous media.
membrane.
On soil crusts it appeared as a black, warty
Within the membrane individual filaments were observed,
some with wide definite sheaths, others with rather diffluent or Indistinct 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 membranous mass,
Cells were approximately 3-,4 in diameter, spherical, bar-
rel-shaped or cylindric, up to 6,i in length.
or less globose.
Reterocysts were more
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.
the colonia1
Filaments within
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 exhibited 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.
pull apart.
The mass also was very mucoid and difficult to
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 granules 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:
soft, gelatinous layer.
On filter paper the plant mass was in a thin,
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.
gestive osciUatori-like movement.
Trichonies then exhibited a sug-
The cells were usually barrel-
shaped, although sometimes truncate-globose, slightly constricted at
the cell walls, contained granules and pseudovacuoles, and were palegreen in color,
The apical cell of the
trichomes was sometimes slight-
ly tapered, but always rounded at the end.
round or slightly ovate, not punctate.
A. sphaerica:
Heterocysts were usually
No akinetes were observed.
In nitrogen-free aqueous media, trichomes were
solitary and flexed, not coiled; sheaths were inconspicuous.
were
b - S4MIn
Cells
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 vacuolated.
Heterocysts and akinetes were infrequent, but when present,
they were adjacent.
in length.
Heterocysts were
Akinetes were
A. flos-aquae:
5-
5 - 6.Sj.t in
diameter and 6 - 10)1
614n diameter and 6 - ó.Sp in length.
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,
were usually ovoid in shape, less frequently spherical.
Cells
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 investments.
A heterocyst occurred at a narrowed end of the honnogonium.
A. spiroides: Adjacent regularly spiraled trichomes were observed in pale, brownish masses on agar plates.
Trichomes in solitary,
broad spirals were observed in nitrogen-free aqueous media.
well as heterocysts
Cells as
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 similar nature.
usually
In aqueous media, trichomes were sometimes solitary, but
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.
laxaeflated.
The sheath was firm, definite, and
conspicuously
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.
more species were observed than were identified.
present resembled the following species:
Many
Some of the 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.
to 3.Ee in
length.
CeUs,were 2 to 39uin diameter and 1
Filaments of this organism were found attached or
in association with filamentous-blue-green and unicellular algae.
aqueous media the plants formed a much entangled blue-green masse
L. epiphytica:
Plants were similar to L. Diguetti, bt of
In
71
slightly smaller dimensions,
They were somethnes found spirally
twisted about or in close association with larger filamentous bluegreen 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,
cultivated than in virgin soils.
They were more apparent in
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 frequently observed in growth on soil.
orga
Under microscopic observation, these
sins were observed to be in an apparent dormant state in desiccated
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.
application of moisture to desiccated crusts, these organism
evidence of macroscopic growth within 1 to 12 hours.
Upon
gave
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 degenerated, gave way to other algae, and under continued application of moisture, 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.
Microcoleus were noted in the crusts examined.
Several species. of
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.
and.
Cells were distinctly cylindrical, Li.
8 - 1S,a in
length.
-
.5,Min diameter
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.
3-
Cells were
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 upright 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 Microcoleus; 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 diameter, 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 3S. purpurascens:
in length.
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 Schizothrix.
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 appeared in shades of gray and brown.
This growth was often noted in
dense to thin tangled masses, aerial or subaerial in nature, but subaerial growth was not always apparent.
Microscopically, growth from desic-
cated crusts showed sheaths of yellowish-brown color enclosing hormogorila 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 Schizothrix 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:
aqueous media.
green.
The plant mass appeared tufted and may float in
The color of the mass ranged from blue-green to olive-
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.
cells became yellow and vacuolated.
9-21M'
Older
Sheaths ranged in diameter from
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.
long and branches not frequently observed.
Trichonies were
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 commonly 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 extensive, 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 tapered 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 nitrogen-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,
time the resemblance to Gloeocapsa was very pronounced.
With
At this stage
the organism was inoculated into media for the nitrogen fixation experiment.
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 investments.
Size of cells at this stage was
and the color ranged from
cells was
green to blue-green.
The shape of the
lipsoid to pyriforn.
An opaque "central body" with or without "pyrenoid"
was apparent in many of the cells.
from globose and el-
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 similarto 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
within
color.
a gelatinous matrix which was hyaline to
The cells were usually
cells
yellowish-brown in
in diameter, not including the sheaths,
P1. 10
Photoinicrograph of
Chroococcus rufescens
30) as it appeared
in N-free media. Colonies arid single cells
Nuceli and
are shown.
protuberance from cells
can be observed in
some cells,
P1. 11
Photomicrograph of
Anabaena sp. (X930)
as it appeared in Nfree media. Filaments
of Anabaena sp. are
shown with variousshaped heterocysts.
P1. 12
Photomicrograph of
Aphanocapsa grevillei
(X930) as it was observed in N-free media,
Cells and membraneous
colonies are shown as
they appeared in the
gelatinous matrix of
the plant mass.
82
and ranged in color from a deep blue-green to yellow or yellowishbrown.
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 several 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 olivegreen 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 ellipsiod 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 aggregated, 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 purpose 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 favorable 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 association is recognized and understood, an exhaustive search may be neces
sary in order to find the conditions necessary for growth in pure cultures,
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 culture 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.
tained nitrogen as sodium nitrate,
Flasks 7 and 8 also con-
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 substitution of deionized water for tap water, the alga was cultured in a
system under conditions of controlled temperature and light and provisions 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 algae
and/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.
possible.
These were noted and controlled as much as
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
Scytonema Archangelli
Scytonema sp.
Anabaena spiroides
Nostoc sp.
Nostoc sp.
oc sp.
Nostoc sp.
Anabaena Levanderi
62,1
O . SILO?
045767
0.5337
O 0828
52.7
89.IL
86,)4
1.1)450
o. li.692
0.S31
0.3266
o.IL771
O.01i36
0.3917
0.2325
0.3636
0.8530
1.3366
0.3581
o.)4662
gms.
Weight lost
upon ignition
Percent
Total
76,9
73.7
71.2
77.5
71i..5
0.6063
gnis.
salts
algae +
Dry Wte
2.IL
3.1
9.IL
0.3
9.3
3,,?
15.0
0.0
mgin.
natant
Super..
Nitrogen
72.3
10.2
21.3
1,9
50.0
20.3
li..)4
26.0
30.0
mgiu.
Wt.
26.3.
30.0
18.1
20.6
59.3
81.7
13.3
mgm.
±lxed
Dry Total N
NITROGEN FIXATION IN AQUEOUS SOLUTION BY BLUE-GREEN ALGAE IN PURE CULTURE
Anabaena spiroides
Organisms
TABLE 70
26.9
18.1
10.1
13,9
13.1
12 8
17,7
ganic matter
to N fixed
Ratio of or-
No growth
0.9836
0.1176
O.ISOIL
Same organism as above
Dicothrix Orsiniana plus Lyngbya sp., neither culture recovered bacteria-free
Scytonema sp.
bacteria-free
Probably not
Same organisms as above
Nostoc sp. plus Microcoleus
sp, No growth of Microcoleus
s.. observed
Same organisms as above
0.1159
0.3699
not fungus-free. No growth
of fungus observed
rtoneita sp., unialgal but
Anabaena Levanderi, unialgal,
but not bacteria-free
bya Diguetti, not bacteria-free
Aphanocapsa reviUei plus Lyng-10060
of Scytonema sp. observed
Chroococcus rufescens also resembling Gloeocapsa and Myrme- 0.2O3L
cia) unialgai, not bacteria-free
plus Scytonenia ap.
gins.
Dry wt.
algae +
salts
0.2536
1.50
o.LiO
o.O6OI
S1.IL
56.3
0.30
0.00
0.00
0.00
0.00
0.0517
O.236L
0.7223
0.1168
0.8390
lti..O
2.8
2.7
JJ4.)4
32.0
5.9
25.9
Nitrogen
Dry
Super
Nt.
natant
mgm.
mgxn.
L.h.6
63.9
71.8
57J.L
85.3
gnis.
Weight lost
upon ignition
Percent
Total
15.5
3.2
2.7
)J4.1.
32.0
5.9
25.9
Total N
fixed
mgm.
NITROGEN FIXATION IN AQUEOUS SOLUTION BY BLUE-GREEN ALGAE IN MIXED CULTURE
Anabaena sp. not bacteria-free
Or anisms
TABLE 8.
16.Li.
18.9
19.1
22.6
19.8
32.I
Ratio of
organic matter
to N fixed
14.3
15.2
16.3
15.5
12.2
10.2
mgni.
27.5
2.2
0.1085
0.U514
3.0
2.7
2.85
2,7
3.0
26.5
28.3
28.6
25.3
22.5
29.7
mgm.
39.35
38.5
110.2
16.0
16.1
1.)4.7
16.14
16.9
15.9
Ratio of
organic
Total N matter to
N fixed
fixed
This amount was subtracted from the
0.0
0.0
214.11
22.11
27 2
28.6
mgm.
1.1
0.0
0,9
0.1
Anaerobic Conditions
0.3318
O.!78O
0.14159
O.ti785
0.14559
* The tap water used contained 1.3 mgm. nitrogen per liter,
values reported above.
Average
Nostoc sp. plus soil bac2.14652
teria. Bacteria did not
2,681J.
appear to grow on N-free agar
Average
14,6859
2.7970
2,6835
2.7195
3.11i77
gms.
gms.
Nitrogen
SuperDry
wt,
natant*
Aerobic Conditions
Weight lost
upon ignition
Percent
Total
algae
plus
salts
Drr
NITROGEN FIXATION IN AQUEOUS SOLUTION BY BLUE-GREEN ALGAE IN MIXED CULTURE
Nostoc sp. plus soil bacteria. Bacteria did not
appear to grow on N-free
agar.
Qganisms
TABLE 9,
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 condition, 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.
trogen was found.
No ammonia ni-
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 considerable 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 anaerobically.
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
91
TABLE 10,
SUTYiMARY OF NITROGEN FIliTION IN CULTURE SOLUTIONS
Organisms
Wt0 Lost
Upon
Ignition
us.
S. Archangefli
A. spiroides
A. Levander2.
Dicothrix Orsiniana + Lyngbya sp.
Nostoc sp.
Scytonema sp.
Chroocoecus rufescens
Aphanocapsa grevillei + Lyngbya
Diguetti
Anabaena sp.
O.Ob36
1.3192
0.6281
O.35I1
5.i65I
0.6161
0.1168
0.7223
0,8390
N
Fixed
Ratio of
O.M. to
N Fixed
N as
% of
Dry Wt,
mgni.
1.3
100.3
10.1
.13.1
9.9
7.7
lLi.1
7.].
22.3
310.5
35.1
15.9
6.3
5,9
19.8
32.0
25.9
16.6
17.6
6.0
5.7
5.1
22.6
32,LL
3.1
92
cultures of any one kind of alga or association regardless of the conditions 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
plus Lyngbya
,
Nostoc sp.
,
,
A. levanderi ), Dicothrix Orsiniana
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 nitrogen-fixing blue-green algae, and species used in this research may not
have been those previously known to fix nitrogen.
sp.
As shown by the rank-
did not demonstrate the
ing of nitrogen-fixing organisms,
Iost
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 association 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 investigated, 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.
literature review, members of a related genus,
As given in the
are powerful
nitrogen-fixers, and it is therefore rather surprising that members
of such a closely related genus have been neglected in nitrogen fixation 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.
a blue-green color and fixed nitrogen well.
It later developed
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 sigriificant that no nitrogen appeared in the supernatant.
Upon reinocu-
lation on nitrogen-free agar, microscopic examination showed the bacteria 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
Pure cultures of this
alga
5.
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 fixation study also presented a complicated picture in that nitrogen was
fixed in the presence of associated organisms.
were not obtained.
Pure culture conditions
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 medium showed L. Diguetti and bacteria scattered through ie matrix of an
extensive growth of Ae grevillei,
See Plate 12.
nitrogen-free agar, this association persisted.
when inoculated on
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 investigation.
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, rulescens,
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,
obtained.
For two sanles, Li.3a and 96e, a value as high as 8,0 was
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
Sam.le No.
35
35a
35b
37
ANALYSES OF SOIL CRUSTS CONTAINING ALWE AND/OR LICINS
Rocks> 2
8.0
9.7
-9.0
,
.H
Carbon
Nitro:en
CrN Ratio
paste
7.3
7.0
-8.0
8.2
7.7
8.7
7.7
O.13
0.26
1.37
0.33
0.20
0.80
0.08
0.75
0.17
0.53
0.11
0.62
0.19
0.38
0.19
.072
5.2
6.0
.oli.o
6,5
S.
6.2
0,514.
0.25
.0141
5.3
146a
6.5
9.3
8.5
7.8
8.2
5.6
3.6
6.6
.250
.053
.029
.107
.018
.120
.026
.077
.015
.086
.021
.061
.036
.050
.032
.065
0.147
.061
7..?
Ii.6b
114.0
14.5
0.22
7.8
1.114
8.14
0.26
149b
11,2
20.5
7.1
22.0
.0141
.0614
5.14
IL8a
149c
5,14.
149d
SOa
Sob
Sla
51b
19,5
13.6
52.2
9.9
19.5
Sic
]J.L.)4
51d
52a
52b
63a
63b
63c
82a
82b
82c
82d
82e
83a
83b
96a
96b
26.1
5.2
21.9
37a
37b
39a
39t
)40b
141g.
hlb
b2a
b2b
11.3
11.0
11.2
12.0
20.9
io,5
182
5.5
Lth.2
iOta
6.0
25.9
9.5
14kb
iLp..Lt.
ti5a
14.3a
b3b
liSb
148b
149a
96c
96e
96f
2.2
5.6
8.14.
7,9
8.7
7.7
8.2
8.0
8.L
7.9
8.3
7,9
8.5
7.14
7.9
7.0
6.5
5,6
6.0
7.9
8,1
7.8
8.7
8.14
6.6
18.3
9.2
7.9
7.2
7.0
5.3
6.7
5.7
2,6
L.8
6.8
2,14
6.9
14.0
9,5
3.2
7.7
7.8
8.2
7.8
8,3
7.9
8.2
8.0
8.1
0.714
0.11
7.5
6,3.
65
6,9
7.3
7.2
9.O,
6.,2.
5.3.
3j
8.3
0.77
0.33
.081
O.11.L
.015
0.87
0.11
1.23
.081
.015
17.3
7.9
9,5
7.8
10.7
6.1
.2614.
14,7
.055
.065
.025
.026
.016
.113
.0314.
.095
.012
.026
.072
.030
.096
.025
.073
7.8
8,2
8.0
7,3
6.9
9,5
6.8
8.6
5.9
7,3
8.8
8,3
7,3
7.6
7,5
8.0
0.143
0.53
0.20
0.19
0.11
1.07
0.23
0,82
0.07
0.19
0.63
0.25
0.70
0.19
0.55
1,16
0.17
1,014
0.114
0.87
0.16
0.75
o.i6
.1145
.023
.122
.020
.109
.0214
.091
.023
7,14.
8,5
7.0
8.0
6.7
8.2
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, sampie 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
50b, which had the highest percent carbon.
on which growth occurred.
soil, 50a and
Plate 3 shows the rocky soil
Only five other samples of the virgin soils
contained more than 0.]. percent nitrogen.
52a,
same
Two of these samples, bOa and
contained lichens, but the other three samples,
were lichen-free algal crusts.
39a, 83a, and 96a,
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
values were 0.300 and 0.2S0 percent respectively.
and
37.
These
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 relationship (1b6).
from about
The ratios ranged from about
.3 to
O for the subsoils.
to 18 for the crusts and
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,
Nitrogen_Fixation
sample 50a,
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.
Tab1
This was shown by the 96 series of cmsts.
See
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.
algal crusts and the thi'd contained lichens
The first two soils were
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
101
ThBLE 12.
NITROGEN FIXATION BY SOIL CRUSTS IN MOIST CHAMBERS
Total Nitrogen
Soil
xed
Begin
2
14
Incubation
Weeks
Weeks
Tucson Sandy
Increase
0.09
Q107
0.026
(63a)O.09S
0.117
O.U9
0.0214
0.096
0.118
0.136
0.0140
Gila Fine
Sandy Loam
Gila Sandy
Loam (82c)
.10
0
.07
LJ.08
4:.
LU
'
z.II
F-
(3
jJ3
Z
J4
4
0
2
63a
4
C11a Fine Sandy Loam
INCUBATION TIME
2
TUCSOfl Sandy Loam
IN
2
WEEKS
0
82c
Gila Sandy Loam
CRUSTS IN MOIST CHAMBERS
NITROGEN FIXATION BY SOIL
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 organisms.
Particular attention was given to the blue-green algae which
were investigated in detail.
blue-green algae observed.
Descriptions were given for some of the
Fifty-one genera of algae were identified
belonging to the orders Cyanophyta, Chiorophyta, Chrysophyta, Pyrophyta,
Euglenophyta, and Diatoms.
green algae.
Of the genera identified, 21 were blue-
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 biological, 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.
103
These are ranked
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
grevillei plus Lyngbya
, and Anabaena sp.
fixed ranged from about
)4
, Aphanocapsa
The amount of nitrogen
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 cultures 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.
gen values ranged from o.OSO to 0.26)4 percent for the
0.029 to O.05 percent for the subsoils.
Nitro-
rusts and from
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
tion in pure culture experiments.
nitrogen fixa-
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