FIXATION AND TRANSLOCATION CF NITRCGEN
IN LICHENS WITH SPECIAL REFERENCE TO
PELTIGERA SPECIES
A thesis presented for the Degree of
Doctor of Philosophy
of the
University of London
by
C. J. B. Hitch, M. Sc.
Department of Botany
Imperial College of Science and Technology
London
November 1976 ACKNO";;LEDGEMENTS
I would like to express my gratitude to Professor Rutter., for allowing me to use .the facilities of the Botany Department, Imperial
College, freely in the furtherance of this Degree.
I would. also like to thank the members of the Department generally, for advice and to Dr. K. L. Alvin in particular, for the use of his phote-microscopic equipment, without which some of this work would not have been possible.
Further, I would like to thank the staff of the Workshop for their assistance in the development and construction of various pieces of apparatus and also to Hr. Rodney Dawes for his patience in making and mending glassware and to the Photographic Department, for help in the production of photographs and slides.
In conclusion I would like to express my sincere thanks to my supervisor, Dr. J. W. IIillbank, for his great generosity and thoughtful help and discussion during this period of research. TABLE CP CONTENTS
Section 1
1.1.1 ABSTRACT 1
1.2.1 GENERAL INTRODUCTION 3
1.2.2 MATERIALS AND COLLECTION SITES 7
Section 2
SCHE ASPECTS OP LICHEN STRUCTURE
THE PHOTOSYNTHETIC ALGAL CELL
2.1.1 INTRODUCTION 11
2.1.2 METHODS 13
2.1.3 ERRORS INVOLVED IN THE CALCULATICN a? ALGAL POPULATIONS 18
2.1.4 A COMPARISON OF CELL VOLUMES 28
THE HEqhROCYST
2.2.1 INTRODUCTION 30
2.2.2 METHODS 33
2.2.3 THE HETEROCYST FREQUENCY IN LICHENS WITH ONE PHYCOBICNT 35
2.2.4 THE HETEROCYST FREQUENCY IN LICHENS WITH TWO PHYCOBIONTS 44
Section 3
NITRCGEN FIXATION — USE (F ACETYLENE
3.1.1 INTRCDUCTICN 52
3.1.2 PMii0Ds IN NITRCGEN FIXATION 58
3.1.3 DISCUSSION ON AS.FLCTS CF NITRCGEN FIXATION 64 ,-
ASPECTS OF NITROGENASE ACTIVITY
3.2.1 INTRCDUCTICN 77
3.2.2 METHODS 79
3.2.3 VARIATIONS IN NITROGEN FIXING ACTIVITY OVER THE THALLUS AREA 80 --
Section 4 15 N NITRCGEN FIXATION — USE CF 2
4.1.1 INTRODUCTICU 98
4.1.2 METHODS 106
4.1.3 DISCUSSICU OF THE 15N —UPTAKE EXPERIMENTS 116 -
Section 5
SOME FACTORS AFFECTING NITROGEN FIXATION, PHOTOSYNTHESIS
AND RESPIRATION IN WHOLE 'PHALLI
5.1.1 INTRODUCTION 143
5.1.2 METHODS. 150 5.1.3 SOME INTER—RELATIONSHIPS AND EFFECTS OP CO2, pH, LIGHT AND
INHIBITORS ON NITROGEN FIXATION, PHOTOSYNTHESIS AND
RESPIRATION 154
5.1.3.1 Oxygen and its affect on nitrogen fixation 154
5.1.3.2 Light and darkness on nitrogen fixation 159
5.1.3.3 The effect of pH on nitrogen fixation 165 5.1.3.4 Effects of DCMU and CN— on nitrogen fixation,
photosynthesis and respiration 169
5.1.3.5 Variability of photosynthesis and respiration 173
CONCLUDING REMARKS 197 --- SUMMARY OF RESULTS 201 •APPENDICES 210
REFERENCES 219 1
Section 1
1.1.1 ABSTRACT 60 lichen species were examined in this investigation,
Peltigera canina and P. polydactyla in detail, the remainder to a much
lesser extent, for the purposes of comparison or survey.
Nostoc cell numbers in the two principal species were of the 7 order of 10 cells ( 1 sq cm thallus ) with a recorded maximum of
2.5 x 107 in P. canina. Heterocyst numbers were also examined from
many lichens with a blue—green algal phycobiont and photomicrographs
were prepared.
In lichens where the blue—green algae formed a layer over
the whole thallus, the heterocysts constituted about 5% of the algae,
whilst in species with cephalodia or internal islands of blue—green
•algal cells, the heterocyst ratio was of the order of 13 to 50%.
Nitrogen fixation was generally assayed by using the
15 was also acetylene reduction technique, though the heavy isotope N2 used. Fixation was only demonstrated in lichens where the phycobiont
had heterocysts. No British material with non—heterocystous phycobionts
was tested. Two lichens of this type from Israel were tested, using
the acetylene reduction technique and gave results which were only
indicative of minimal nitrogenase activity.
Fixation exhibited wide inter— and intra—thallic variation.
It was affected by light and dark, oxygen in the short term, CO2 in the
long term and by pH.
Using Peltigera polydactyla 15N2 was incorporated into the
thallus, a method being evolved to keep the samples active over 37 days.
see Scott (1957) 2
Immediate transfer of the fixed nitrogen occurred from the algal to the fungal zone. Each zone increased its degree of labelling consistently.
In neither case did the label reach a peak.
Oxygen turnover was measured using a Clark type oxygen electrode. The exchange was comparitively slow, with little increase
in evolution over the intensities 5,000 — 10,000 lux. Under those
conditions of augmented carbon dioxide ( HCO3 ) a "compensation point"
was noted and oxygen was evolved more rapidly. DCMU inhibited
photosystem II completely, but not nitrogen fixation. 3
1.2.1 GENERAL INTRODUCTION
Lichens are pioneers in plant succession, where they are
able to form long lived and stable communities. It is their adaptation
to xeric or mesic environments that has enabled them to dominate
habitats where competition is slight. They are remarkably specific to
different substrates and this characteristic is used in their
classification. However this is not entirely rigid and certain lichens
have such a wide distribution and little substrate specificity, that
they may become established in several communities. They are referred
to as corticolous when growing on trees with bark, saxicoluus when on
rocks and rock—like substances, terricolous when on soil, muscicolous
when on mosses and phyllicolous when on leaves.
These communities can be further subdivided, for example
some lichens are specific to certain types of trees, which may be rough
or smooth barked or have a distinct pH. Others to rocks, in which case
hardness and softness, acidity and alkalinity can affect their
distribution and much work has been carried out to examine the vertical
zonation of lichens on rock faces.
Hale (1955) has studied lichen frequency and cover on trees
in relation to pH, and Harris (1971, 1971a) has examined the distribution
throughout a tree canopy and related the variation of physiology to the
environment. Santesson (1939) in Sweden has discussed distribution in
relation to water submersion and similar zonation patterns have been
reported by Hale (1950), Raup (1930), Watson (1919) and Lewis (1964)
and various workers have investigated lichen colonisation of soil
communities. These are most vulnerable, since they are subjected to
rapid change and competition. Cladonia species often appear to form the
main biomass in these areas, with gradual zonation extending backwards,
from the most xeric regions, though other lichens may be more important 4 in certain habitats, where they are better adapted. This is true of the fast growing Peltigera species, but see the work of Brodo (1961), Looman
(1964), Rogers, Lange and Nicholas (1966) and Ahmadjian and Hale (1973).
Though lichens were first used by herbalists, they_were not studied in any detail till the eighteenth century and Acharius (1757 —
1819) a Swedish Medic first put lichenology on a sound scientific basis.
With the improvement of the microscope, advances were made, and DeBary and Schwendener were the first to understand the dual nature of lichens and the first to consider them on a physiological basis, though their findings were controversial and were not accepted for many years.
As Smith (1963) has said, "there has been much speculation about the nature of the association, between their constituent algae and fungi. Unfortunately this speculation has not been accompanied by much experimental investigation. A belief that they are 'difficult' material to vqe in laboratory experiments, has discouraged research into their physiology and difficulties in their taxonomy have discouraged botanists from becoming familiar with the group."
He went on to say that "lichens do not present any greater problems in physiological laboratory experiments compared with:other organisms, provided that proper procedures are adopted," As Hale- (1967) has shown, Smith has been in the forefront of lichen physiology, with his work on carbon metabolism, in which he developed the technique of disc dissection.
Both Quispel (1959) and Collins (1960)suggested that in order to discuss lichen physiology, they had to define the lichen association, though this was difficult due to the variability of other lichen—like associations present in nature. This leads to controversy as
Ahmadjian (1967) has pointed out. A true lichen cannot be described as a fairly permanent,_ association between algae and fungi, on the same
substrate, involving physical and physiological interractions, but should
be reserved for associations between algae and fungi, that have formed
a new morphological' unit, distinct from either of the two partners.
While Quispel and Collins are justified on a comparative
basis, their views do not affect the physiology of true lichens, which
have been summarised' by Ahmadjian (1967a), as follows:-
a) the growth of the individual symbionts is slow.
b) the net rate of assimilation is low.
c) the lichens exist on a nutrient-poor substrate
d) their exposure, often to extreme environmental conditions,
allows them only brief periods for optimum activity.
e) they are frequently wetted and dried.
f) they have low rates of protein synthesis.
Also, not mentioned by Ahmadjiant the rate of lltliciem of substances
•from external sources is very rapid.
It has been assumed that the algae synthesise organic
compounds autotrophically- which were obtained by the fungus by
haustorial penetration (Ahmadjian, 1965; Peveling, 1973), though Quispel
(1959) found that isolated algae behaved heterotrophically, but could be
stimulated to autotrophic growth by the addition of ascorbic acid. The
fungus does play the role of protector against desiccation and in providing water (the medulla of Peltigera alzh2Laa holds more water than the alga) and ,maybe mineral salts, which may or may not produce a
state of autotrophy. The lichen is a viable unit in nature and it is
probably a combination of these factors that has enabled them to remain
as morphological entities from generation to generation.
Lichens act as hydrophilic gels and can imbibe water equally
rapidly, whether alive or dead. This passive movement probably explains the findings of Smith (1962) with combined carbon and Bond and Scott
(1955) and Scott (1956) with combined nitrogen, that substances move
by diffusion and not active transport; it would also explain the
deleterious effect of the rapid accumulation of undesirable products,
such as pollutants and heavy metals, which lead to the development of
lichen deserts, in certain areas, particularly round cities (see Ferry,
Baddeley and Hawksworth 1973).
Most of the physiological studies that have been carried out,
deal exclusively with only a few lichen species; considering their
numbers and diversity, it is dangerous to extrapolate results except in
the broadest terms. Reservations should also be made when comparing
results using the isolated partners, with data derived from using whole
plants. Many attempts have been made to isolate the fungus and alga
and then resynthesise the association, but with little success and this
demonstrates that the maintenance of the symbiosis depends on a very
close interplay of the environmental conditions, such conditions being
adverse to the independent growth of the partners (Smith, 1962).
Individual aspects of the physiology will be dealt with in
greater detail in later sections. The work in this study was basically
concerned with observing the relationship between nitrogen fixation and
light, darkness, pH, oxygen and, by measuring photosynthesis and
respiration (by means of oxygen exchange), how fixation was also affected
by these processes. 7
1.2.2 MATERIALS AND COLLECTION SITES
Lichens were collected from many sites throughout the
country, Table 1. Normally they were placed in polythene bags as they
were collected from the field, either in the dry or the wet state,
brought back to the laboratory as quickly as possible and the contents
sorted and placed in the storage cabinet (see Kershaw and Milibank,
1969).
Material was also obtained from other areas due to the
kindness and generosity of Dr. M. Billington, Alaska, U.S.A.; Dr. M. Galun,
Israel; Dr. K. A. Kershaw, Ontario, Canada; Dr. P. B. Topham, Greenland
and Scotland; P. D. Crittenden, Iceland and P. W. James, British Museum
(Natural History). I am extremely grateful to them. In this instance
the material was always sent in the dry state and moistened at the
laboratory, either on arrival or prior to experimentation. (see Farrar,
1973, on lichen metabolism, when re-wetting a dry thallus).
Initially thalli in the cabinet were sprayed routinely with
glass distilled water and occasionally with NO3- solution (0.25g/1),
using a chromatogram spray, but subsequently after a suggestion from
Dr. K. A. Kershaw, spraying was done irregularly, with the thalli being
allowed to dry out at random intervals, as it was hoped that this method
would approach natural conditions more and maintain the material in a
viable condition for as long as possible.
Light was provided by four 3ft x tin "Warm White" flourescent
tubes and two 3ft x tin "Gro-lux" tubes (which provided extra red light),
to an overall maximum value of 2,500 lux at the thallus level. This
approximated to field conditions, where values of 1;500 to 2,500 lUx
were recorded. o Temperature was maintained at 10° - 12 C by a cooling unit
suspended in the cabinet and the air being stirred, by a small fan fixed 8
Table 1. Lichens used in the study and the collection site.
flkUil.lifq11:1. gRileM., "IiPII.ttilLq Haim. 16(NR)8772 Stonofiold Castle Hotel, Tarbert, Argyllshire. C. crls:.,um (Huds.) Web. 37(NO)4327 Newport-on-Tay, Fifoshire. C. crlstatum (L.) deb. _ 35(NY)13B2 Castlemilk, Lockerbie, Dumfriesshire. C. fluylltlie (Huds.) Steed. 37(NO)1748 Gorge by R. Ericht, Craighall, blairgowrio, Perthshire. C. furfuraceum (Arnold) Du Metz 16(NR)9794 Minard Castle, Lochgair, Argyllshire. C. cubfur7Im (M111. Arg.) Degel. 16(NR)9794 Minard Castle, Lochgair, Argyllshire. "DendrIscoca.11on umhausence (Auersw.) Zahlbr. 17(NM)7860 Strontian, Ardnamurchan Peninsula, Argyllshire. Ephehe Janata (L.) Vain. 27(NN)9328 River Almond, Buchanty, Perthshire. Lempholevl-A cladodes (Tuck.) Zahlbr. 34(SD)8967 Malham Tarn Nature Reserve, /lest Yorkshire. Lepte turn b,rgessil (L.) Mont. 16(NR)8772 Stonefield Castle Hotel, Tarbert, Argyllshire. L. vane::cens (Pers.) KErb. 27(NN)0300 Furnace, Loch Fyne, Argyllshire. L. lighen',Ides (L.) Zahlbr. (a) 27(NN)0200 Furnace, Loch Fyne, Argyllshire. (b)" 16(NR)8390 Crinan Canal, Cairnbaan, Lochgilphead. Argyllshire. (c) 37(NO)0141 The Hermitage, Dunkeld, Perthshire. (d) 16(NR)13148 North of Grogport, Kintyre, Argyllshire. L. schraderi (Ach.) Nyl. 35(NY)1382 Castlemilk, Lockerbie, Dumfriesshire. L. sinuate (Muds.) Massal. 35(NY)1382 Castlemilk, Lockerbie, Dumfriesshire. L. teretiwiculum (Nallr.) Arnold 16(NR)8148 North of Grogport, Kintyre, Argyllshire. L. tremelloides (L. fil.) Gray 27(NN)0300 Furnace, Loch Fyne, Argyllshire. Lichina confinis (0.F.M111.) C.Ag. 37(NO)6641 Arbroath, Angus. L. Pycraea (Lightf.) C.Ag. 37(NO)6948 Ethie Haven, Inverkeilor, Angus. *Lobaria a--lissima (Scop.) Forss. 17(NM)7860 Strontian, Ardnamurchan Peninsula, Argyllshire. L. laetevirens (Lightf.) Zahlbr. (a) 17(MM)7860 Strontian, Ardnamurchan Peninsula, Argyllshire. (b) 16(NR)9794 Minard Castle, Lochgair, Argyllshire. L. culmenaria (L.) Hoffm. (a) 27(NN)0300 Furnace, Loch Fyne, Argyllshire. (b) 16(NR)9794 Minard Castle, Lochgair, Argyllshire. L. scrobicilata (Scop.) DC. (a) 27(NN)0300 Furnace, Loch Fyne, Argyllshire. (b) 17(NM)7860 Strontian, Ardnamuchhan Peninsula, Argyllshire. Massalonoia carnosa (Dicks. arb. 16(NR)8772 Barmore Island, Tarbert, Argyllshire. Neohroma laevioatum Ach. (a) 37(NO)0141 The Hermitage, Dunkeld, Perthshire. (b) 27(NN)0300 Furnace, Loch Fyne, Argyllshire. 'N. parile (Ach.) Ach. 38(NJ)0052 Darnaway Forest, Forres, Morayshire. Pannaria microphylla var. cheilea (Mudd) A.L.Sm. 16(NR)8772 Barmore Island, Tarbert, Argyllshire. P. pezizoides (Web.) Trevis. 27(NN)6138 Ben Lawers, by Killin, Perthshire. P. pityrea (DC.) Degel. 27(NN)0909 Inveraray Castle, Inveraray, Argyllshire. P. robioinisa (Thunb. ex Ach.) Del. (a) 17(NM)7860 Strontian, Ardnamurchan Peninsula, Argyllshire. (b) 29(NC)4401 Strath Oykell, Ross and Cromarty. Parreliella atlantica Deoel. 27(NN)0807 West of Inveraray, Argyllshire. P. plumbea (Lightf.) Vain. 29(NC)4401 Strath Oykell, Ross and Cromarty. Peltigera aphthosa (L.) Wind. (a) 51(W)1752 Lodge Hill, Dorking, Surrey. (b) 37(NO)5971 The Burn, Edzell, (River North Esk) Kincardineshire. (c) 27(NN)0015 An-t-Inbhir, Loch Ave, Argyllshire. P. aohthosa var. varlolosa (Massal.) Thorns. 37(NO)2852 The River Isla, Alyth, Angus. P. canina (L.) Willd. (a) 38(NJ)0264 Culbin Forest, Forres, Morayshire. --- do --- (b) 28(NN)6001 Inch, Nr. Kingussie, Inverness-shire. (c) 37(NO)3316 Mount Hill, Cupar, Fifeshire. (d) 28(NH)5249 Muir of Ord, Ross and Cromarty. (e) 37(NO)5026 Tentsmuir Forest, Tayport, Fifeshire.
ErMia . Colloma tenax (Sw.) Ach. 16(NR)63e0 Crinan Canal, Cairnbaan, Nr. Lochgilphead, Argyllshire. Leptogium subtile (Sohrad.) Torus. 38(NJ)8366 Troup Roam, Ponnnit, Banff.
EIT.4So Dendrit:cocanlon umhausense and Lobarll ampltssIma wore parts of the same plant structure. " Sample (b) was 1,eptogium llchenoldes f. scoticum, the thallus of which forms a tight knit cushion. ' The material (mod came frOm the British Museum (Natural History). 1 The namplo of il(Iltigora onhthoa fran Loch Awo, had squamiform cephalodia.
9
Table 1. evitinued
Pe1f-L:19N! 1±91ThIqZ11. (Neck.) Hoffm. (a) 1600910 Asknish Forest, Road south of Lochgair, Argyllshire. (b) 27(111030) Furnace, forth side Loch Fyne, Argyllshire. (c) 37(110)4928 Tentsmuir Forest, Tayport, Fifeshire.
P. preetextata (Fl;irke ex Uommerf.) Zopf (a) .37(110)2252 River Isla, Alyth, Angus. (b) 270100200 Furnace, forth side Loch Fyne, Argyllshire.
P. veno-a (L.) Bauffl. 37(110)5771. The Burn, Edzell (River North Esk) Kincardineshire. Pilochoras distans (Holt.) Magnusson No ref Lichen from the British Museum (fat. fist.) ..,,:,dun. Placop:is qelida (L.) Nyl. (a) 27(11107964 Errochty Water, Glen Errochty, Perthshire. (b) 27(0)7270 Dalnacardoch Lodge, River Garry, Perthshire.
Placynthi.e% niqrum (Rads.) Gray (a) 27(0)7257 Foothills of Schichallion, Perthshire. (b) 37(10)0960 Limestone quarry, Kirkmichael, Perthshire.
P. mannariellum (Nyl.) Magnusson 27(1111)5435 Falls of Lochay, Killin, Perthshire. PolychiOiim muscicola (Sw.) Gray 37(10)5765 West Water, Edzell Castle, Edzell, Angus. Pseudosynellaria thouarsii (Del.) Degel. 16(NR)7615 Balnabraid Glen, South of Campbeltown, Argyllshire. Psoroma Lypnorom (Vahl) Gray 29(NC)5748 Foothills of Ben Loyal, Sutherland.
Solorina erocea (L.) Ach. 27(NN)9461 Foothills of Ben Vrackie, Pitlochry, Perthshire. S. sacce:a (L.) Ach. (a) 27(NN)7257 Foothills of Schichallion, Perthshire. (b) 34(50)7273 Footpath to Ingleborough Hill, :ngleton, Yorkshire.
S.. soorpaiosa (Sm.) Anzi 27(NN)7156 Foothills of Schichallion, Perthshire.
Stereaca.don vesuvianem Pers. 37(NO)2206 The Lomond Hills,'Falkland, Fifeshire. *Sticta canariensis Dory ex Del. 16(NR)7615 Coast by Balnabraid Glen, Campbeltown, Argyllshire.
*S. dufouril Del. 16(NR)7615 Coast by Balnabraid Glen, Campbeltown, Argyllshire. S. fulloinosa (Dicks.) Ach. 27(NN)0200 Furnace, North side Loch Fyne, Argyllshire. S. limbata (Sm.) Ach. (a) 16(NR)6-4- Gigha Island, off West Coast of Kintyre, Argyllshire. (b) 27(NN)6723 South shore Loch Earn, St. Fillans, Perthshire. Usnea sabfloridena Stirt. 16(NR)7574 south side Loch Caolisport, Ballyaurgain, Argyllshire.
NOTE.
* The S:Icta cenariensis/Sticte dufourii was a chimaeral association, growing as a single unit.
FOREIGN LICHENS.
Lobaria :finita (Ach,) Rebenh. (Dr. Billington) Beach Ridge, Barrow, Alaska, U.S.A. Nephrona arcticum (L.) Torss. (Dr. Kershaw) Hawley Lake, Nr. Hudson Bay, Canada. ."Peccania Dassal. ex Arnold nom. cons. (Dr. Calan) Negev Desert, Israel.
Pet.Lk12ra evansiana Gyeln. (Dr. Kershaw) • Nr. Hamilton, Ontario, Canada. P. venosa (L.) Baumg. (a) (Mr. Crittenden) Nr. SOlheimajaull, Iceland.
(b) (Dr. Topham) Mestersirg, North-cast Greenland. 'Peltula `;yl. (P. polyspera) (Dr. Gahm) Negev Desert, Israel. Placo ,sis oelida (L.) Nyl. (Dr. Topham) Mostersirg, North-cast Greenland. Solorina crocoa (L.) Ach. (Dr. Kershaw) Hawley Lake, Nr. Hudson Bay, Canada.,. Stietn Celix / nondriscoeanlen sp. (P.W.Jamee) Herb. R.K.(N.H.), New :ealand.
NOrKS.
' Tbo 1 THooYm of re to V°• 1-1:17.11 Is villKaLna. " Snooloa unknown.
The 1 ohe,.% mtc,erleinttivo i 1.1•01ti nrt. it ( 1. /16) ■Ulkt 611:10:111 1070)• 10 into the side wall.
Under these conditions, the lichen thalli were maintained in a usable state for three to six months. 11
Section 2
SOME ASPECTS a LICHEN STRUCTURE
THE PHOPOSYNTHETIC ALGAL CELL
2.1.1 INTRODUCTION
Lichens are symbiotic associations between algae and fungi,
the internal and external morphology of which remain more or less
constant and as such can be classified into families, genera and species
(Swinscowl 1964; Haynes, 1964).
Gross lichen morphology and anatomy has been well described
by Smith (1921). The fungus forms the gross morphological body of
lichens, except in a minority of cases and in each there is a different
species.
Two basic types of anatomical structure exist, the most
widespread is the heteromerous state, where the thallus is composed of
an upper layer of pseudoparenchymatous cells, the cortex, below which is
the algal rich zone and below this the medulla, a weft of loosely knit
fungal mycelium (see Smith, 1963), which may be contained by a lower
cortex, however this is not the case with those species attached to a
substrate, by rhizinae over the lower surface, e.g. Pel! era species.
In the heteromerous thallus, the fungal component forms about 85% of the
total volume. (Smith, 1963), excluding the algal—rich zone, which is also
mostly fungal in composition, as Bednar (1963) suggests, the algae in
Peltigera phthosa may form about 3 — 5% of the total volume.
In the hothoiomerous thallus, the structure is quite
different. The thallus is more or less uniform throughout, with the
fungal mycelium ramifying through the gelatinous matrix surrounding the
algae, though there is always more fungal tissue at the periphery of
the thallus. This type of structure is characteristic of the 12
Collemataceae, lichens with Nostoc as the phycobiont.
The algal partner is also important in helping to determine the lichen taxonomy. Haynes (1964) describes the criteria on which lichen algal classification is based and points out that the taxonomy of many of these algae is complex, based on such characters as the plane of division of the zoospores and for this reason Trebouxia has been regarded as the most widespread phycobiont.
Ahmadjian (1960) has shown that the same physiological strain of Trebouxia may be extracted from many different lichens, but he also reported (1965) two blue—green algal species as phycobionts in geographically separated samples of Lichina.
Lichenised algae and fungi do not normally exist freely, though closely related types do and they may only differ in their physiological requirements. Ahmadjian (1965) pointed out that the two
Calothrix species from Lichina are "both common in the free—living state".
The phycobiont is normally a member of the Chlorophyceae, though many lichens contain a blue—green algal symbiont (Cyanophyceae) and in one instance a member of the Xanthophyceae has been shown to be lichenised. For a full account of lichen algae see Ahmadjian (1958;
1967). 13
2.1.2 METHODS
2.1.2.1 Disintegration of the thallus by physical means
When grinding small samples, 0.5 — 1.0 sq cm areas, a modified
glass Potter homogenises was used. The plunger had the glass prongs
ground off, so that it could be pushed to the bottom of the glass tube.
Iml of grinding medium, see Appendix I, p210 was placed in the
tube with the lichen, which had been cleansed of adhering debris and
finely chopped. With the plunger pushed to its lower position, the
lichen and medium were forced upwards, between the walls of the plunger
and the tube. With careful rotation of the plunger in this position,
the material would disrupt fairly readily and give a fine particled
suspension.
When grinding large samples, of up to 50g fresh weight,the
wet thallus was cleansed of adhering debris and finely chopped as before.
Small portions were then taken and placed in a pestle and mortar, with
•1.0 — 2.0 ml grinding medium and ground very gently. The resulting
suspension was filtered through fine meshed muslin gauze, with additions
of medium and stirring and the resulting debris returned to the pestle
and mortar for further grinding. This grinding procedure was repeated
three or four times, until no appreciable thallus particles remained.
The resulting suspensions were bulked and centrifuged, using
a BTL bench centrifuge at 4,500rpm (3,500 x g) for 3 minutes. The
supernatant liquid was discarded and the pellet resuspended in a small
volume of grinding medium.
2.1.2.2 Pure whole cell preparations
Sufficient lichen thalli were ground to form 10m1 of a
relatively dense algal suspension, see above. Two centrifuge tubes were
prepared by pipetting 10m1 80% sucrose solution into each.
The lichen thalli were then layered, very carefully, onto the 14
sucrose with a specially ground wide—mouthed pipette. Extreme care was
needed during this process to ensure that the 10% / 80% sucrose
interface was not disrupted. The wide—mouthed pipette helped in• this
procedure, since the suspension was not even and due to the settling out
of the larger lichen fragments, with the time needed to layer the
suspension, clogging of the pipette orifice would occur. The two tubes
were then accurately reweighed, balanced with grinding medium if
necessary and centrifuged, using a swing—out head, at 4,500rpm
(3,500 x g) for 15 minutes.
Due to the differing densities of the algal cells and the
fungal mycelium,_ the algal cells remained at the 10% / 80%
interface, whilst the remaining debris passed through and formed a pellet
at the bottom of the tube.
Subsequently the algae were stirred up into the top layer
using a 10m1 pipette and removed. Small quantities of medium were added,
to enable as many cells to be collected as possible. This pure algal
suspension was spun and washed twice and made up to a required volume as
before. The grinding medium was used rather than distilled water for
all the processes, since it prevented lysis of the cells and loss of
chlorophyll.
In order to prove that 80% sucrose solution had the best
density for spinning and separation of the algal cells, a gradient was
tested, using 10%, 20%, 40%, 60% and 80% sucrose. Virtually all the
cells were held at the. 60% / 80% interface.
2.1.2.3 Counting cell numbers
A haemocytometer was used when the algal cell population in
a suspension was required. This could be referred back to the area of
thallus sampled.
2.1.2.4 Cell volumes
Small aliquots of pure algal suspension were placed on 15
a clean glass slide, a cover slip added and the algal cell dimensions
determined microscopically, using a calibrated eyepiece graticule.
2.1.2.5 Disintegration of the thallus b chemical means
-An adaptation of the method of Hill and Woolhouse (.1966)
was used. Chromic acidv better referred to as chromium trioxide or
chromic anhydride (Cr0 )is a crimson crystalline compound, which in 3 water forms an aqueous, extremely acid solution, probably containing
dichromic acid (H Cr0 in equilibrium with small quantities of chromic 2 7) acid (H2Cr04).
Treatment with a standard 10% solution, overnight (18 hours)
normally proved satlisfactory and softened the tissue so that it could
be completely macerated with a sterile Pasteur pipette. This method sixtxciu.a3 caused the fungal mycelium to disintegrate and leave the algal cells
intact, either in long or short chains, depending on the vigour with
which the pipetting was carried out. Cells were then counted with a
haemocytometer, see 2.1.2.3 above.
2.1.2.6 Pigment content of algal cells
In early experiments a lichen thallus suspension was
prepared, as for lichen cell counting, see 2.1.2.1 above, either using 3 1.0 sq cm discs as standard, or a small aliquot from a much larger suspension. The supernatant liquid was discarded and ethanol added to
the pellet to extract the chlorophyll. Iml ethanol was added to the
pellet, which,was agitated for 1 minute and centriguged at 41500rpm
(3,500 x g) for 3 minutes. This was repeated twice more with fresh
ethanol. The extracts were bulked and made up to 5m1, by weight using
a top pan balance. Triplicate samples and ethanol blanks were routinely
assayed.
In controls, where chopped 1 sq cm discs were used, 0.5ml
pyridine ("Analar" grade) was added to the disc at the start, to soften 16
the tissue and enable the ethanol to penetrate more easily (Hill, 1963).
In these cases stoppered centrifuge tubes and spectrophotometer tubes
were used and pryidine was also added to the blank control.
In later experiments the supernatant liquid was extracted.
The thallus was ground up normally, centrifuged and extracted. The
brown characteristically smelling supernatant liquid over the pellet,
was placed in centrifuge tubes and spun at 25,000rpm (59,000 x g), or
subsequently at 30,000rpm (85,000 x g) for 10 minutes to give a denser
pellet, using a MSE "Superspeed 65". This produced an almost colourless
supernatant liquid and a tiny pellet, brown in the centre and bright
green round the edge.
The pellet was extracted, using ethanol direct and
subsequent centrifugations at 4,500rpm (3,500 x g) for 3 minutes were
sufficient to compact the pellet enough to pour off the extracted
chlorophyll, since the addition of ethanol hardened the pellet
considerably. This method assumed that cell breakage was occurring and
enabled the percentage loss to be calculated.
Thirdly, complete analysis was undertaken. The thallus was
ground up and a macerated pellet was prepared normally. This was washed
and the washings plus the supernatant liquid were extracted, see "in
later experiments":above. The pellet was resuspended and made up to 10m1
and layered onto 80% sucrose. The resulting algal fraction after
centrifugation, was completely removed, washed and extracted. The
remaining pellet in the 80% layer was washed to remove the sucrose,
centrifuged and extracted.
2.1.2.7 Spectrophotometry
A unicam SP 500 spectrophotometer was routinely used, for
chlorophyll analysis. Initially the 01) (Optical Density) was read at
645mp and 663mp. Samples prepared normally and assayed by a colleague 17
using an automatic Optica spectrophotometer indicated that the OD at
645mji, was optically inaccurate as far as chlorophylls was concerned,
since part of the peak at this wavelength was due to other substances
than chlorophyll. For all subsequent readings therefore, the OD at
66391 was recorded, ( see Wilhelmsen, 1959).
2.1.2.8 Moisture content and dr,_might of the thallus
Thalli were taken, cleansed in the usual manner, tested for
acetylene reduction and dried downl .or dried down direct. Thalli so
treated, were blotted dry with absorbent paper tissue, weighed
immediately and then oven dried at 105 1- 5°C to determine the dry weight
and the water content. Some thalli were freeze dried when further
analysis was carried out, e.g. nitrogen determinations.
Normally thallus discs were dried, cooled in a vacuum
desiccator, weighed on an analytical balance and the process repeated till
a uniform weight was achieved, which did not vary by more than 0.2mg ,
a maximum error of 2%.
For weighing Peltigera aphthosa var. variolosa cephalodia,
the routine balance used, was not sensitive enough, as the samples tested
were less than 1.0mg in weight. A balance that measured down to 1.Ojig,
was therefore used and I am indebted to the Chemistry Department for the
use of their instrument. 18
2.1.3 ERRORS INVOLVED IN THE CALCULATION OF ALGAL POPULATIONS
During the assay .of discs of lichen thallus for nitrogen
fixation, a striking variation in activity was observed. The algal cell
density was thus determined, to see if it and ultimately the heterocyst
frequency could be used to explain this variability.
A simple technique was initially used. All the cell counts
were carried out on fresh thallus macerates, produced by grinding
procedures, either with a pestle and mortar, or a Potter homogeniser, 6 6 and by this method, counts of between 0.58 x 10 and 1.39 x 10 algal -1 cells (1 sq cm ) were recorded for Peltigera caning and up to 6 3.0 x 10 for P. polydactyla (Table 2). .However this method was both.
tedioun and time consuming and a more convenient approach was tried.
This was chlorophyll analysis, though absolute figures were not
calculated, as being unnecessary.
In early grinding experiments, it was wrongly assumed that
none of the algal cells were disrupted, since visual observations of the
supernatant liquid, after centrifuging the macerate, gave no indication
of chlorophylla. This was probably due to the dark brown pigmentation
of the liquid by phaeophytin compounds masking the chlorophyll.
When whole discs of lichen thallus were extracted, using
ethanol and pyridine (see Hill, 1963), the chlorophylla content per unit
area of thallus, was much greater than for the extract of the pellet of
the macerated discs. Therefore losses into the supernatant liquid had
occurred. The supernatant liquid was therefore analysed, to see if it
contained chlorophylla, assumed to be membrane bound. After high speed
centrifuging at 85,000 x g, in which a pellet was formed, see methods,
2.1,2.6andestimating of OD, it was found that the chlorophylla existing
in this supernatant fraction averaged 62% of the total, for Peltigera
Table 2. Mean total algal cell counts of two Peltigera species by homogenising fresh thalli.
PELTIGEHA CANINA * Thallus Suspending Mean Cells Calculated Mean % of Cells Counted Number of Std Standard Error Estimates :ample Volume per sq cm Total Mean Cells as a Proportion of the (sq cm) (ml) 6 6 Calculated Mean Deviation of the Mean x10 x10 6 10 1 0.62 8.98 6.9 0.40 x 106 0.13 x 10
* Percentage of Thallus Sample Suspending Total Cells Calculated Volume Mean Cells/ml Mean Cells/sq cm Cells Counted (sq cm) 6 6 Counted Total mean (ml) x10 x10 6 6 as a Proportion of x10 Cells x10 the Calculated Mean
3 3 1.39 1.39 4.17 26.9 15.5 6 3 2.60 1.30 7.80 53.9 14.5 6 3 2.01 1.00 6.03 53.9 11.2 10 10 1.18 0.59 11.80 89.8 13.1 50 50 0.58 0.58 29.00 449.0 6.5
PELTIGERA POLYDACTYLA
30** 8o 1.12 2.98 89.60 376.5 23.8 158** 200 1.72 2.17 344.00 1962.9 17.3
4 NOTES :- x10 6 Based on Cr0 analysis. For Peltigera canina = 8.98 x 10 3 cells/sq cm and for P. polydactyla = 12.5*ells/sq cm (Mean Values). ** Samples calculated from fresh and oven dried (to 105°C) weights of similar thalli. 20 canina, though considerably less for P. 221242.2tyla or Dendriscocaulon umhausense (Table 3).
For this reason it was assumed that the algal content of the suspension was comparable to the percentage chlorophyll partition in
Peltigera canina, i.e. the whole cells counted, formed 40% of the total.
Revised figures were calculated for the early cell counts, using this proportion as a basis. However even with this correction factor, the 6 highest cell counts obtained, 1.39 x 106, only became 3.5 x 10 and did not correlate with the results obtained for the pigment value of 6 extracted intact thallus. Table 4 shows that the OD of 1 x 10 cells is approximately 1/10th of the total OD of a disc of thallus, thus indicating 107 cells as a rough total cell density.
Originally the pellet containing unbroken algal cells had
been analysed; the results combined with the supernatant fraction and this assumed to be equal to the intact thallus, extracted with ethanol/
pyridine. It was not appreciated until.now, that the algal cells in the
pellet only formed a small proportion of the chlorophyll of that fraction,
a large amount of membranous material being carried down with the broken fungus mycelium.
A new comprehensive approach therefore had to be taken. In
all early experiments when the macerate was layered onto 80% sucrose,
the fraction passing through the 80% sucrose interface was discarded,
being assumed as entirely fungal, but this was now tested. Not only
were intact cells present in this fraction (Table 5), but chlorophyll
analysis showed that there was a far greater content of chlorophylla in
this sediment than that which could be accounted for by these cells and
by recalculating the results, it emerged that the intact algae recovered
at the interface, formed as little as 5% of the totalApopulation (Table
6). However this was a slight underestimate. Cell analysis based on
Table 3. A comparison of the chlorophylls pigment from lichen thalli, using optical density (CD) at 663mu of ethanol extracts of homogenised thallus. 07) Mean. Dry Volume . Mean Total etz-x per "S" as a CD/mg Number of Mt Thallus Thallus Area Fraction Mean VxCD percentage of Estimates (sq cm) (ml) - Mel OD sq cm Total Chl Dry Wt (mg) a
PELTICERA CANINA 0.83 ** 5 0.166 (0.05 0.02) 2.01 59 10.7 0.67 0.063 1.18 (0.10 0.0.05) range 0.394 (0.10 0.05) 55 - 63
0.099 0.51 66 (0.14 0.05) 1.48 10 10.7 3 0.98 (0.27 0.09) 0.49 range 0.046 3 0.325 (0.19 0.06) 59 - 75
PELTICERA POLYDACTYLA 0.219 1.10 5 1.54 "Si, 3 0.147 0.44 14.7 1.63 29 0.140 5 0.238 1.19 'ISIS 1.72 3 0.177 0.53
DENDRISCOCAULCU UMHAUSENSE
upft 1.22 30 *..* 5 0.347 35.0 (0.46 0.20) 1.73 0.056 5 (20.94 9.37) 0.51 (0.63 0.28) range (0.01 0.004) "Si' 3 0.173 (0.17 0.08) 24-33
"D" 12 0.570 6.84 1 127.0 13.98 51 0.102 "Si, 12 0.595 7.14
NOTES:- "D" refers to the pellet from the 3,500xg spin; "S" refers to the pellet from the 85,000xg spin. ** The figures in brackets are the Standard Deviation and the Standard Error of the Mean respectively. *** Two samples were taken from an homogenate of 200m1 volume containing 158 sq cm lichen thalli. **** The dry weight of the Dendriscocaulon umhausense thallus refers to the dendroid structures on the thallus of Lobaria amolissima and are 'cephalodia'. Table 4. The pigment (Chia) from Peltigera canina using the optical density at 663mp of ethanol extracts.
INTACT THALLUS CO Number of Thallus Area Volume Mean Mean Total Total Citi±ed....im Estimates (sq cm) (ml) OD' OD per sq cm 2.05 4 0.68 5 ' 3 5 0.410 (0.15 0.06)
INTACT CELLS
Estimated• 6 Cell Density x10 Volume 6 Tot al CD 6 Optical Density CD/10 Cells 6 Cells x10 x 10 Cells per ml Exiiaoted (ml) per sq cm
7.40 5 0.095 0.013 0.065 10.46
5.50 5 0.084 -1).015 0.075 9.07
15.60 5 0.157 0.010 0.050 13.60
6.36 5 0.123 0.019 0.097 7.01
NOTES:—
* The figures in brackets refer to the Standard Deviation and the Standard Error of the Mean.
Table 5. Assay of Peltirera polydactyla to determine the percentage of intact cells which are carried through the 60,;:' sucrose interface, when spinning at 3500g.
Mean Percentage Mean 0112/m1 Number of Standard Standard Error Fraction Total Cells of Cells in Estimates 6 Deviation of the Mean 6 m10 x10 Each Fraction
6 6 (i) 4 0.35 27.8 100 0.02 x 10 0.01 x 10 Total A. Macerate (ii) 2 1.72 344.0 100
6 6 (i) 4 4.18 16.7 60 0.06 x 10 0.03 x 10 Cells at B. 80% Sucrose Interface 6 6 (ii) 6 3.75 112.4 33 0.99 x 10 0.41 x 10
6 6 Debris Under (i) 4 0.22 4.4 16 0.06 x 10 0.03 x 10 C. the 80% Sucrose * (ii) n.d. n.d.
NOTES:—
Cells lost at the interface, B(i). (100 — B(i) + 0(i)) = 24% . B(ii) could not be calculated. * n.d. Not Determined. • Table 6. Mean total algal cell counts of Peltdpscra carina, using .hcmogenates of fresh and Cr03 treated thallus and
the relative partition of pigment.(Chla) of all fractions, as the CD663mp of the ethanol extract. from the same homogenate.
Fresh Thallus Cr03 Treated Thallus Number of Standard Standard Error Cell Density Cell Density Estimates 6 Deviation cf the Mean x 106 / ml x 10 /sq cm 6 4 0.58 0.07 x 10 0.04 x 106 6 6 8.25 0.55 x 10 0.28 x 10
NOTE:— The fresh thallus was assayed from an homogenate of 50 eq cm in 50 ml. The Cr03 treated thalli were 1 sq cm discs of similar material incubated separately.
Homogenate* Volume Number of Mean Mean Total 5 Partition Volume ' of the Various Fraction (ml) Samples CD x OD CD ' Fractions
Cells at A. eel Sucrose 3 2 0.254 0.76 1.54 5.0 Interface 32.8
Debris Under 3. the ear! 16 2 0.264 4.22 ' 8.44 27.8 Sucrose
Pellet of the 10 0.681 2.04 20.40 67.2 C. Supernatant 3 30.36
NODE:— * See methods page 13 — 14. 25
at gal Table' 6, shows that while theAcell density of the fresh thallus 6 suspension totalled 29 x 10 cells, 5% of the possible cell density, 6 6 i.e. 412.5 x 10 , based on Cr0 analysis, was equal to only 21 x 10 3 6 cells, so this difference of 8 x 10 cells lost at the interface and into the sediment (2%), should be added to the intact cell chlorophyll, which would therefore increase it to 7%.
It is of interest to note here that Drew- and Smith (1967) with Peltigera 221Elactil, using a grinding technique and a complex (434.1 centrifugation programme, also recovored. 6.7% of the totalAcell population, as calculated by chlorophyll analysis.
These considerably higher cell counts, though proved to be correct by chlorophyll analysis, had not been determined by counting.
With the use of Cr0 solution in which lichen discs were placed and which I" 4 A-%%.,,..ael 50,exe.. caused them oila-6.143.1=1 as a udhl method for the elucidation of lichenised algal cells (Hill and Woolhouse, 1966), there was virtually no cell loss and for the first time a realistic figure was obtained for counts of blue—green algal cells in lichens (Table 7) and may be compared with the results produced by Hill and Woolhouse, using
Xanthoria parietina, even though this lichen has a green phycobiont.
Neither the Cr0 technique, nor chlorophyll analysis, was a 3 speedy method to determine a basis for nitrogen fixation quickly, but they did ultimately show that an accurate determination of the phycobiont density was possible and the use of specifically Cr03' demonstrated the potential nitrogen fixing sites, see p.36.
Variability existed in the thalli tested. The chlorophylla of Peltigera canina and P. 222EILTlyla fell within reasonably narrow limits for small or large areas of thallus tested, as did the cell chlorophylla and knowing these factors it was possible to calculate the cell density. However, directly counting was found to be considerably Table 7. A comparison of the Mostoc content of three Peltigera species with two SticIa species using Cr03 treated thallus.
Mean Algal Mean Algal Mean Cephalodia Standard Standard Error Standard Standard Error Thallus Number of Cells / cc cm Cells/Cephalodia Lichen Samples Estimates per sq cm :eviations of .the Eet..n, leviations cf the Yean x 10 x 10° (a) (b) A 6 32 19.87 7.96 0.66 0.020 (a)
B 6 22 E.70 3.58 1.19 0.053 0.27 x 106 0.13 x 106 P. ashthosa var. variolosa c 6 17 9.33 3.81 1.25 0.076 (b) D 18 12.172 3 4.97 0.73 0.040 0.02 x 106 0.01 x 106
Mean Algal Cell Density Thallus Number of Standard Standard Error Lichen (Cells / sq cm Thallus) Samples Estimates 6 Deviations of the Mean x 10
* • 6 6 "Standard" 22 8.98 1.50 x 10 0.32 x 10 6 P. canina "Pale" 6 5.30 1.99 x 10 0.81 x 106 6 "Dark" 4 20.91 ' 3.95 x 10 0.96 x 106
6 6 "Standard" A* 4 12.55 2.06 x 10 1.03 x 10 6 6 "Standard" B 32 9.47 3.10 x 10 0.54 x 10 P. polydactyla 6 6 "Standard" C 22 9.89 . 2.99 x 10 0.64 x 10 6 6 "Pale" 4 6.38 0.57 x 10 0.29 x 10
6 6 S. fuliginosa A 4 10.07 0.54 x 10 0.27 x 10
S. limbata A 1 20.61
NO2BS:- Thalli from the field, were collected, which appeared different visually and were given an arbitrary corresponding classification. • 27 more accurate and it was possible to demonstrate wide variations in the 6 6 population, with extreme values of 3 x 10 to 25 x 10 being recorded.
There appears to be considerable variability between different lichens, using a similar technique. With Peltigera canina and -1 Dendriscocaulon umhausense, the OD (mg dry weight thallus) was equivalent, though Peltigera polydactyla had proportionately more chlorophylla, see Table 3. In relation to dry weight, area and cell numbers, there was- a 2.3—fold, 3.2—fold and 6.9—fold increase respectively.
Harris (1971b) has explained his variability in photosynthetic
and respiration studies at different levels of a tree canopy, as being 6 due in part to algal cell numbers and he quotes figures of 3.4 x 10
cells (1 sq cm)-1 for the tree top and 2.4 x 106 cells (1 sq cm) for the
base, though he also found an increase in numbers of cells of the order
of 1.2 to 1.7, between January and September.
In view of all these findings, the use of Cr0 analysis was 3 used for future work where necessary. 28
2.1.4 A COMPARISON OF CELL VOLUMES
The volume of an algal cell is calculated using the formula 2 derived for the oblate spheroid, i.e. Tr .AB where A is the long axis 6 and B is the short axis.
In an assay of the volumes of Nostoc cells in Peltigera
canina, the mean of the three most common "cell volume groups" was 94u3
and these formed 80% of the cells observed, with an upper limit of 473u3
and a lower limit of 78u3. Thus it was calculated that for 1 sq cm of 10 3 Peltigera canina thallus, with a volume of 9 x 10 u , the algae could
form about 1% of the total volume, based on a mean cell density of 6 9 x 10 cells. However, since the thickness of the thallus and the cell
population varies from sample to sample, this figure is somewhat
arbitrary and can only be a rough cross-check on the general validity of
cell number estimates.
With Coccomyxa from PeltimEa aphthosa var. variolosa, there
appears to be two "cell volume groups", one, with large cells, mean
volume 233u3, forming 50% and one with small cells, mean volume 580,
forming 30 f. Since the mean of these two groups (143u3) is approximately
1.5-fold greater than that for Peltigera canina and the unit weight of
thallus is approximately 1.5-fold greater, there could be approximately
3 to 5 times the number of cells which are found in P. canina, using
Bednar's data (Bodnar, 1963; see Bednar and Smith, 1965), i.e. 30 x 106 6 N -1 to 50 x 10 (1 sq cm) .
It was found that by carrying out Cr0 analysis, the cell 3 6 population of P. aphthosa var. variolosa was 44 x 10 (1 sq cm)-1 and was
comparable to the mean values obtained for P. canina and therefore
substantiates Bednar's work. The cell populations of the two lichens are
represented by histograms in Figure 1.
29 16
14
12
• 10
U P, r. PELTIGERA CARINA
with NOSTCO (Cyanophyceae)
S. ,0
7E 99 106 137 154 185 266 473
3 Volume p
16
1L1 FELTIGERA APHTHOSA var. VARICLOSA
12 with Coccomyxa (Chlorophyceae)
U
10 P . O LL
cr)
0
O .6 U 0
5t 72 86 97 129 162 194 345
3 Volume p
Figure 1. Rietograms of cell volumes of algal populations in two different Peltigera species. 30
THE HETEROCYST
2.2.1 INTRODUCTION
The heterocyst is a differentiated cell, found in filamentous
blue—green algae and is exceptional in that it does not appear to have
any contents, when viewed under the light microscope and has been
described by Fritsch (1951) as a "botanical enigma".
Much work has been carried out to elucidate its role in
nature. Lang and Fay (1971) and Kulasooriya, Lang and Fay (1972) have
examined it with the electron microscope and shown that it does in fact
have a complex internal structure. Wilcox (1970) and Mitchison and
Wilcox (1972) have discussed its method of differentiation. Singh and
Tiwari (1970) have shown that it may be a propagule under certain
circumstances and Fay, Stewart, Walsby and Fogg (1968) have tried to
determine its physiological status, without complete success, despite the
use of sophisticated techniques and its function is not completely known,
though it is almost certainly the site of nitrogen fixation. This is the
role that it is assumed to play in this study.
The heterocyst frequency of a number of lichens, both with
one phycobiont and those with two, the secondary one mostly in cephalodia,
have been examined, not only to determine the ratio to vegetative algal
cells, but also to see whether the population was related to nitrogen
fixation.
Lichens .with one phycobiont are simple and have already been
dealt with (see p 11 ). Cephalodia are however, more complex. The work of
Scott(1964)is important, since he showed that the blue—green alga may be
essential in forming the total partnership. Studies with Solorina
saccata indicated, that for the lichen to develop naturally, not only was
the green partner essential, but the blue—green alga too. A similar 31
requirement exists in the lichen Ealli= aphthosa. The apparently
obligate symbionts, could be described as parasymbionts and may have
originated as such, falling onto or being overgrown by the main thallus
and thus been incorporated. Their incorporation is well shown in the
studies of Jahns (1972), using the lichen Pilophorus, in which he showed
the fungal mycelium enclosing chains of Scytonema and forming a proper
cephalodium.
Jordan (1970), gives a systematic representation of three
types of cephalodial development, in an American species of Lobaria
(L. 2mgma), 1) a non—intrusive inferior cephalodium, 2) an intrusive
inferior cephalodium and 3) an intrusive superior cephalodium, all
derived from the incorporation of Nostoc on the lower surface of the
thallus. Jordan states that cephalodial development on the upper
surface of the lichens in this genus, is prevented by the mechanical
and physiological nature of the upper cortex in all species, 1) due to
the presence of several layers of thick walled paraplectenchyma and
2) mature cells lose their capacity for growth and are eroded away. He
has pointed out that it is thallus thickness, venation and the amount
of attachment to the substrate that determines the type of cephalodia
present.
Using these criteria, Jordan has quoted Lobaria pulmonaria
the only British representative in his study, as having inferior
cephalodia only. It was observed in both the British species Lobaria
pulmonaria and L. laetevirens, that the amount of intrusiveness varied.
Whether a cephalodium develops into an inferior or a superior type,
depends on the tissue thickness above the cephalodium and the algae once
incorporated will expand in the direction of least mechanical resistance.
Ozenda (1963) however, described a different form of
cephalodial development. In Lobaria amplissima, the "cephalodium" 32
Dendriscocaulon umhausense, a lichen in its own right under certain circumstances (Henssen, 1963), grows up through the thallus of Lobaria amplissima and emerges through the upper cortex, as a dendroid structure which is totally uncharacteristic of the other members of this genus.
Jahns (1973), discussing the whole subject of the relation- ship between the shrub—like structure Dendriscocaulon and Lobaria, the case of Sticta felix and the requirements of Dendriscocaulon to ensnare green algae in cortical hairs and thus form a compound structure, does not discuss the internal cephalodia of Lobaria amplissima and it remains to be seen whether they are comparable to those in other species of
Lobaria, or whether they are a stage of development, related to the pabsage of Dendriscocaulon through the thallus. 33
2.2.2 METHODS
2.2.2.1 Heterocyst counts using.21LO 3 For lichen thalli with a uni—algal layer, the samples were
cleansed of adherent plant material, sand, etc. and very small pieces,
though for foliose species 1 sq cm discs were cut out (No 7 cork borer),
soaked well in glass distilled water for 3 to 6 hours and incubated in
standard 10% Cr0 solution overnight, though longer periods of up to 3 42 hours were not detrimental. The thallus was disrupted with four
successive extrusions through a sterile Pasteur pipette and drops of the
suspension, placed on clean glass slides. A cover slip was added which
was ringed with petroleum jelly ("Vaseline") to prevent drying out. The
"Vaseline" was applied with a 5m1 glass hypodermic syringe and melted
round the cover slip with a hot innoculating needle. The cells were
then examined microscopically and counted.
For lichen thalli with external or internal cephalodia, which
• could be excised, the cephalodia were removed from the main thallus,
soaked and placed in the Cr0 solution. These cephalodia after 3 incubation were not macerated, but were picked up individually using a
sterile Pasteur pipette and placed on a glass slide, in a drop of the
incubating medium. If they were incubated for the correct length of
time, the weight of the cover slip when lowered gently onto the slide,
was enough to break the cephalodia open and release the chains of cells.
Some trials were carried out to ascertain the length of time
needed for this incubation period. It transpired that the cephalodia
behaved differently. From 5.5 to 30 hours was needed. If the incubation
period was too short, a slight increased pressure was needed to flatten
the cephalodium, while with excessive incubation, the cephalodium
flattened too readily. In either case the result was complete rupture,
resulting in a unicellular suspension and the impossibility of 34
calculating heterocyst ratios accurately.
2.2.2.2 Photography
Photomicrographs were obtained of chains of algal cells
released, using Cr0 treatment and also of untreated algal cells, 3 produced by physical grinding methods.
Ilford Pan F or Kodakrome XF daylight film was used. The
photographic equipment was made available by Dr. K. L. Alvin of the
Botany Department, Imperial College and was fully automatic. The light
meter control compensated for difference in illumination due to
magnification changes and material density on the slide. It also
indicated the exposure time that should be applied and this was
automatically set. The exposure time varied from 1/15th to 4 seconds,
according to the Conditions. Magnification was X100 and X400, by
microscopy. 35
2.2.3 THE HETEROCYST FREQUENCY IN LICHENS WITH ONE PHYCOBICNT
Despite the presence of a wealth of information on lichenised
blue—green algae from a taxonomic point of view, beginning with the work
of Bornet (1873) and the reviews of Ahmadjian (1958; 1967), reports of
the presence of heterocysts in lichens are very rare.
It was known (Linkola„ 1923) that Nostoc, a heterocystouS
genus, was the phycobiont of many Eallimlna species, though Durrell
(1967) stated that the algal partner in P. canina was Gloeocapsa, a non-
heterocystous genus, probably as a result of not seeing heterocysts.
Reports such as that of Shields, Mitchell and Drouet (1957)
and Watanabe and Kiyohara (1963), show the difficulties of recognising
the true status of the lichen phycobionts„ as in the lichenised state
these algae become markedly altered.
Drew and Smith (1967) studying Peltigera polydactyla used
electron microscopy, but did not see heterocysts in the many sections
' of Nostoc that they observed and Peat (1968) also using electron
microscopy, indicated that while they were present, they were difficult
to see and few in number.
More recently Griffiths, Greenwood and Millbank (1972),
again studied Peltigera (Peltigera canina), with a definite aim to assay
the frequency of heterocysts. They used electron microscopy and were
able to recognise heterocysts. From serial sections they suggested that
these cells formed about 3 to 4% of the algal population.
With macerated fresh tissue and using an homogeniser, it is
impossible to tell what any of the cells are with certainty, except for
a small proportion of vegetative cells which have survived in very short
chains of up to six cells maximum.
Cells were observed in the macerate that were considerably
bigger than the vegetative cells and could have been heterocysts, since 36 they appeared to have no cell contents. However they could well be the pseudo—parenchyma of the fungal cortex and were therefore disregarded.
When Cr0 solutions were used the disintegration of the 3 tissue was almost entierly chemical, noted by the thallus becoming quite transparent, so very little force, either with an homogeniser or a sterile Pasteur pipette was needed to disrupt the thallus. In fact this latter maceration technique was so gentle that it was sometimes possible to obtain very long chains of cells; a 250—celled chain from al-liana canina and a 364—celled chain from Collema subfervum have been recorded.
The obvious consequence of this was that heterocysts were immediately observable and ratios could be determined accurately, not only for Peltigera canina, which proved that the figure suggested by
Griffiths et al (1972) was correct, but it has been shown that this low frequency can be extended to all lichens with a mono—phycobionyic layer of blue—green algae (Table 8a —
It is not surprising that there has been so much controversy over the presence or absence of heterocysts in lichens with blue—green algae. Homogenisation as a grinding technique is a particularly vigorous method of disintegration, as the cells seem very fragile. In grinding fresh thallus all the heterocysts are lost and a great many of the vegetative cells as well, though they are at least clearly visible amongst the other debris, due to their colour. With incubation in Cr03 prior to grinding, at least some of the heterocysts remain intact, though here due to shrinkageof all the cells, it becomes increasingly difficult to determine counts accurately, when they are no longer in a single chain format.
It is not known how many of the vegetative cells are in fact lost by this method, and the work of Kershaw (1974) needs commenting on.
He homogenised thalli, that had been treated in Cr03, in a loose—fitting
Table ta. Heterccyst counts of score Collema species using Cr03 incubations.
Mean :eterccysts as Percentage Incubation Mean Vegetative Collection ”aterial Number of Heterocyst Percentage of Total Heterocyst Lichen Time Comments Estimates Cells Site Type (hr) Cells Mean Cells Range
Thallus easily Thallus 18 2.70 C. auriculatum disrupted 1 252 7
solution. - C. crisoum Thallus 48) No chemical disintegration of the thallus occurred when incubated with either 10% or 20% Cr03
Thallus easily Phallus 18 2 524 11 2.11 1.62 — 2.60 C. cristatum disrupted
fluviatile Phallus No chemical disintegration of the thallus occurred when incubated with either 10% or 20% Cr03 solution. C. 148
Nostoc in packets did Phallus 18 151 6 3.63 2.54 — 4.22 C. furfuraceum not disrupt easily 3
Total Thallus easily C. subfurvum 18 18 2.40 thallus disrupted 734
Thallus — Thallus easily 18 852 18 2.20 — do — isidia disrupted 3 1.73 — 2.43
Algal sacs hard to disrupt but 2.99 — do — Isidia 18 strands of algal cells emerged 3 496 14 2.48 — 3.96
C. tenax Phallus 148 No chemical disintegration of the thallus occurred when incubated with either 10% or 20% Cr03 solution.
liOTE:— * The collection site data are as stated in Table 1. Only one site was used for each of these lichen species. Table Eb. eterocyst• counts of some leotoium species using Cr03 incubations.
Incubation •Number of dean Vegetative Mean Heterocysts as Fercentae Collection* T:aterial Comments Percentage cf Total Lichen Time Estimates Cells Heterocyst Heterccyst Site Type (hr) Cells ' Mean Cells Range
Thallus Thallus easily 2 22 L. burressii 21.5 disrupted 447 5.11 4.69 — 5.52
Thallus easily L. c anescens Phallus 18 disrupted 2 465 14 2.83 2.70 — 2.95
Thallus easily disrupted — do — Thallus 27 Cells not swollen 1 542 15 2.69
Total Phallus easily lichenoides 18 L. A thallus disrupted 2 530 12 2.13 1.85 — 2.40
Total — do — 26 Algal chains very contorted 1 thallus and cells distorted 937 18 1.E8
Phallus — Thallus easily — do — 20 isidia disrupted 1 1775 34 1.88
Isidia easily — do — Isidia 20 2 disrupted 848 28 3.12 2.77 — 3.46
L. lichenoides B Thallus Cells very dilated 630 var. scoticum 26 1 19 2.93
18) L. schraderi Phallus No chemical disintegration of the thallus occurred when incubated with either 10% or 20% Cr03 solution. 42)
Thallus easily sinuatum — Thallus 2 L. 18 disrupted 645 14 2.17 1.86 — 2.48.
— do — — Thallus 42 Cells not swollen 2 498 13 2.45 2.44 — 2.46
Thallus easily tremellcides Phallus L. 18 disrupted 1 464 17 3.53 -
— do — — Thallus 42 Some swelling of cells 2 726 24 3.12 2.96 — 3.28
NOTE * The collection site data are as stated in Table 1. .A, B, C, and D, refer to the respective sites (a), (b), (c) and (d).
Table- €c. Heterocyst courts• of some Peltipera species using Cr03 incubations.
Incubation Mean Hetercusts as Percentage Collection* Material Number of Mean Vegetative Heterocyst Percentage of Total Heterocyst Lichen Time Comments Estimates Cells Site Type (hr) Cells Mean Cells Range
Phallus easily 21 6.29 P. canina A Thallus 18 disrupted 3 325 4.77 - 7.44
Phallus - 20 Thallus easily 2 28 6.06 5.44 - 6.68 P. evansiana isidia disrupted 429
- do - Isidia 20 1 408 12 2.86
Thallus - Thallus easily 2 818 51 6.01 5.05 - 6.96 - do - isidia 42 disrupted
Gentle disruption, sacs tough 12 - do - Isidia 42 Rougher treatment,chains shorter 2 284 4.13 3.83 - 4.42
Thallus easily 28 5.08 - 6.47 P. solydactyla C Thallus 18 disrupted 5 461 5.78 Thallus easily Thallus - 18 492 22 4.28 P. craetextata A isidia disrupted
Isidia easily 661 22 3.22 - - A Isidia 18 disrupted 1
- do - A Thallus 42 Thallus tough 2 570 17 2.91 2.74 - 3.07
- do - A Isidia 42 2 444 21 4.46 4.15 - 4.77
Thallus easily Total 1 326 14 4.12 o - thallus 18 disrupted
Thallus - 2 28 - do - B isidia 18 - 554 4.74 4.43 - 5.04
- do - B Isidia 18 - 2 631 35 5.26 5.15 - 5.37
Note :- * The collection site data are as stated in Table 1. A, B, C, refer to the respective sites (a), (b) and (c). Table Ed. Heterocyst counts of some Nephroma species, Pannaria species and Sticta species, using Cr03 incubations.
Incubation Mean Heterocysts as Percentage Collection* Material Number of Mean Vegetative Lichen Time Comments Heterocyst Percentage of Total Heterocyst Site Type Estimates Cells Cells Mean Cells Range (hr) •
Thallus easily — N. laevigatum ' A Phallus 19 disrupted 1 738 37 4.78
18 Thallus easily 2 620 26 3.14 - 5.08 — do — B Phallus disrupted 4.11
N. Thallus easily oarile — Phallus 18 disrupted 3 454 24 4.96 4.53 — 5.18
Thallus hard to break up, P. rubiginosa Thallus 20 1 172 9 A then algal chains very small 4.98
Thallus over incubated —do — Phallus 27 B no cells visible
P. microphylla Thallus too tough Thallus var.cheilea 18 to disrupt
A few algal Phallus — do — — 42 chains present 1 234 11 4.49
Chains very contorted and Thallus 22 2.63 - 3.87 P. rezizoides algal cells distorted 2 168 7 3.25
Thallus easily - S. fuliginosa Thallus 18 disrupted 1 140 9 6.05
No swelling of cells — do — Thallus 42 after this time 2 624 42 6.24 6.20 - 6.27
No swelling of cells 38 6.31 — 6.67 S. limbata A Thallus 42 after this time 2 535 6.49
No swelling of cells Thallus 1 572 30 — do — B 42 after this time 4.98
NOTE * The collection site data are as stated in Table 1. A and B refer to the respective sites (a) and (b).
Table Re. Heterocyst counts of lichens of the genera Ephebe, Lemcholemma, Lichina, 11.assalonria, Parmeliella, Placynthiun and Pseudocyphellariat using Cr0 incubations. 3
Mean Heterocysts as Percentage Incubation Number of. mean Vegetative Collection* Material Time Comments Heterocyst Percentage of Total Heterocyst Lichen Estimates Cells Site Type (hr) Cells Mean Cells Range
Ephebe Janata Thallus No chemical disintegration of the thallus occurred when incubated with either 10% or 20% Cr03 solution. 148)
18) Lemuholerma — Thallus No chemical disintegration of the thallus occurred when incubated with either 10% or 2 Cr03 solution. cladodes 48)
Thallus disrupted Algae 4* Lichina confinis — Thallus 18 in sacs Heterocysts paired 2 159 11 6.20 5.92 — 6.47
Thallus difficult to disrupt Phallus 18 84 L. ovgmaea Double heterocysts in places 1 4 4.40
Thallus easily Thallus 18 8 5.52 Massalongia carnosa disrupted 1 137
Parmeliella Lifficult to count Phallus 18 2 340 20 5.53 — 5.61 atlantica Cells triangular 5.53
Too vigourous pipetting — Thallus 18 1 7 10;77 - — do — Algal chains very short 58
le... Thallus disrupted 1 223 5.91 — P.plumbea — Phallus in 20% Cr03 solution 14
Placynthium *** 20% CrO solution used — Phallus 45 159 4 2.45 ElaY2 Algal chaies remained in sacs
Thallus disrupted in 10% Cr0 Thallus - 365 12 3.18 P. oannariellum 18 solution 20% excessive
Pseudocvnhellaria Phallus easily Thallus 18 300 20 6.25 thouarsii disrupted
NOTES :— The collection site data are as stated in Table 1. ** Paired heterocysts are characteristic of the genus Calothrix, the phycobiont of Lichina species. *** Using 10% Cr0, solution after 18 hours produced no effect. 42
glass homogeniser for 2 minutes, in order to carry out cell counts, which
at least on heterocysts is disastrous.
The use of Cr0 is not a fool-proof method and the type of 3 disintegration of the lichen thallus is most important. After overnight
treatment in the heterocyst frequency was lower in Peltigera Cr03' canina discs that were homogenised, as opposed to those that were not
(Table 9). However this did not apply to P. aphthosa var. variolosa
thallus (Table 9), so that although methodology is of vital importance
in making accurate counts, the lichen material will also affect the
results.
In making observations of the chains of cells, it was noted
that heterocysts were frequently found at the end of the chain and could
have indicated that this was the weak "link" (see Stewart, Haystead and
Pearson, 1969). If as appears, heterocysts are easily lost, at least in
some lichens, it could also explain the apparent non-existence of them
' in fresh thallus macerates, where the disrupting forces are that much
greater.
Plate 1 shows the result of grinding fresh thallus tissue in
a pestle and mortar and the use of when heterocysts become clearly Cr03' visible and are easily counted. Table 9. The percentage heterocyst ratio in relation to the grinding technique in two Peltigera species using Cr03 incubations.
Mean Mean Vegetative Heterocysts as Grinding Cells Heterocysts Percentage of Total Lichen Samples Techniques 6 Mean Cells x 10 Cells x 104
Peltigera canina 1 Homogenisation 9.99 9.78 0.97 2 — do — 7.66 2.61 0.34
3 - do — 8.99 4.24 0.47 4 - do — 8.52 5.31 0.62
Extrusions via a 50 5 Pasteur pipette 10.10 42. 4.04 6 — do — 7.96 47.29 5.61
Mean Heterocysts as Grinding Mean Vegetative Lichen Samples Heterocyst Percentage of Total Techniques Cells Cells Mean Cells
Peltigera anhthosa var. variolosa 1 Homogenisation 295 105 26.25
Extrusions via a 2 32.96 Pasteur pipette 537 177 3 — do — 578 172 29.75 4 —do — 352 84 23.85
43a
•
D
Algal cells from fresh thallus — no heterocysts present.
•
w0
Effect of Cr0 treatment. 3
. 1 •0111/ 9De'' ,4 0 • 04-ij 0 0 6— -.., ., . An.se .e. .4 J.o p) _ . et. - a c:D cA, .0... 4 4. , 0 : 5.0 6-3 dzo) ., to 0
s. a-0AP • ,
44io- 6 - .-_. ,...,.. o pop.. 0: , -,t% 41,11 ) r Cil, r. _ r6 -6 -
High powered magnification with heterocysts clearly visible.
Plate 1. The Nostoc phycobiont from Peltigera canina thallus. 44
2.2.4 THE HETEROCYST FREQUENCY IN LICHENS WITH TWO PHYCOBIONTS
The heterocyst ratio in lichens with either external or
internal cephalodia, or internal "islands", seen in Solorina species,
can be much higher than in mono—phycobiontic lichens, with blue—green
algae, and ranges from 7% up to about 55%.
In free—living blue—green algae the heterocyst ratio is low.
Kulasooriya, Lang and Fay (1972), give a mean basic heterocyst frequency
of approximately 4.8, but they point out that under anaerobic and
nitrogen deficient conditions, the percentage frequency rises to 7.8%
and Jewell and Kulasooriya (1970), using five blue—green algae with
heterocyst frequencies of between 2.5 and 4.9%, show that during growth,
i.e. loss of cellular nitrogen, the range of frequencies increases from 5.3% to 12%. It is well known that in the presence of ammonium ions, the
development of heterocysts is suppressed (Fogg, 1949), so the reverse,
i.e. an increase in the C:/1 ratio could well be a stimulent. Kulasooriya
et al (1972) showed that when the C:N ratio changed from 4.5:1 and
5.1:1 to 8:1, the heterocyst frequency increased.
Hill (1975), points out that in the Azolla/Anabaena symbiosis
the heterocyst frequency is related to capture time, that is the ratio
is very low, when the algae are first installed in a leaf cavity and
rises rapidly to 20 — 30% at leaf twelve from the apex and then remains
fairly constant, which might be related to nitrogen demand on the algae.
He has shown that plants grown with and without nitrate, had very similar
frequencies, so other factors must be involved.
Jordan (1970), discussing the importance of the nitrogen
balance in thalli of Lobaria.species, has indicated that there appears
to be a correlation between the size of the cephalodia and the degree of
substrate contact, in various species. In L. uuercizans, a species that 45
is in close contact with the bark and consequently inundated with run—off
water, rich in nitrogen, the cephalodia are poorly developed, though in
L. oregana, at the other extreme, the cephalodia are remarkably large,
in thalli which are loosely attached to horizontal branches.
Cephalodia are tiny nitrogen—fixing sites in a large area of
green phycobiontic non—nitrogen—fixing cells. Thus the drain of fixed
nitrogen might be so great that the heterocysts are stimulated to
develop to a far greater extent, than would normally be the case.
Kershaw and Millbank (1970), have shown that most of the
nitrogen fixed by the cephalodia of Peltigera aphthoses, is released to
the lichen mycobiont at a similar rate to that of fixation and Jordan • (1970) suggests that in species that form these associations with Nostoc,
the mycobionts have a higher nitrogen demand, than in lichens with green
algae only. However these factors alone are most unlikely to produce
the high frequencies measured.
In this study the small internal cephalodia of Lobaria
pulmonaria, L. laetevirens and L. amplissima, mostly had the highest
heterocyst frequencies (Table 10b), whilst the external cephalodia of.
Nephroma arcticum, Peltigera aphthosa var. variolosa and P. venosa,
Pilophorus distans, Placopsis gelida, Psoroma Lanaym and Stereocaulon vesuvianum (Tables 10a, 10b and 10c), generally had considerably lower
frequencies, which may be explained by an increased anaerobic state, in
the internal cephalodia and a further increase of the C:N ratio, since
the drain of nitrogen was from all sides. It is therefore less
explicable, why the heterocyst ratio of the internal "islands" of
Solorina crocea and S. saccata (Table 10b), is lower, and comparable to
the external cephalodial frequency.
It is suggested that in these latter cases, where there are
larger areas of blue—green algae, that the drain on individual cells must
Table 10a. Heterocyst counts of lichens with cephalodia in the genus Peltigera, using Cr03 incubations.
Incubation Mean Heterocysts as Percentage Collection* Material Number of Mean Vegetative Heterocyst Percentage of Total Heterocyst Lichen Time Comments Cells. Site Type (hr) Estimates Cells Mean Cells Range
Superior Disrupted by Pasteur pipette 18 1 89 32 26.44 P. arhthosa A Cephalodia Cells in balls, hard. to count
Superiorp 6 Squash preparation 52 26.85 24.92 — 27.84 — do — Cephalouia 3 147 Superior — Squamiform 18 Disrupted by Pasteur pipette 1 364 65 15.15 Cephalodia
•P. anhthosa Superior le Disrupted. by Pasteur pipette var. variolosa Cephalodia Incubation period too long
— do — Superior Squash preparation 2 185 27 12.71 12.56 — 12.86 Cephalodia 5
Superior — do — p Cephalodia 18 Squash preparation 326 74 18.54 17.57 — 19.50
Superior 30 Squash preparation 286 -82 — do — Cephalodia 1 22.49
** Inferior 2 P. venosa O. Cephalodia 7 Disrupted by Pasteur pipette 1 12 14.29
Inferior — do — Sc. Cephalodia 18 Disruptea by Pasteur pipette 384 74 16.16
*** Inferior — do — Squash preparation 1 36 Cr. Cephalodia 7 Cephalodia not broken up 460 7.25
Inferior Squash preparation ■ CLO — Cr. 8 1 7.02 Cephalodia Longer time beneficial 410 31
**** Inferior — do — lc. Cephalodia 20 Disrupted by Pasteur pipette 1 592 62 9.48
?JOKES * The collection site data are as stated in Table 1. A, B and C refer to the respective sites (a), (b) and (c).
** Sc. is Scotland. *4-11. is Greenland **** Ic. is Iceland.
Table lob. Heterocyst counts of lichens with cephalouia in the genera Lobaria, Nephroma ann Solorina using Cr03 incubations.
* Incubation CollectionMaterial Number of Mean Vegetative Mean Heterocysts as Percentage Lichen Time Comments Heterocyst Site Type Estimates Cells Percentage of Total Heterccyst (hr) Cells Mean Cells Range
Internal L. amplissima 8 Cephalodia Squash preparation 1 243 69 26.81
•— do — — Internal Cephalodia 29 Squash preparation 1 360 84 18.91
Internal L. laetevirens A 18 Cephalodia Squash preparation 1 10 11 52.38
— do — Internal H Cephalodia 5.5 Squash preparation 3 225 64 22.19 19.69 — 25.28
Internal L. pulmonaria Cephalodia 9 Squash preparation 2 131 78 37.06 31.86 — 42.25
Total L. scrobiculate A Cephalodia absent Thallus 18 in this species 362 14 3.72
Total Thallus tough — do — 18 10% no effect B Thallus Repeated with 20% Cr03 2 448 20 4.27 4.09 — 4.45
** Innate N. aroticum Ca. 18 The "cephalodial" areas were Islands excised and incubated 426 73 14.48 12.60 — 16.58
Internal Squash preparation S. crccea 18 Islands Knots of cells hard to count 3 226 42 17.50 13.50 — 24.52
Internal S. saccata A 18 Disrupted by Pasteur pipette 2 308 58 15.62 Islands 14.53 — 16.71
Internal — do— B 20 Disrupted by Pasteur pipette Islands Chains more easy to count 201 32 13.73
NOTE :— * The collection site data are as stated in Table 1. A and B refer to the respective sites (a) and (b). ** Cc. is Canada.
Table 10c. Heterocyst counts of lichens with cephalocia in the genera Pilophorus, Placopsis, Psoroma and Stereecaulon using Cr03 incubations.
Mean Heterocysts as Percentage Incubation Number of Mean Vegetative Collection* Material. Time Comments Heterocyst Percentage of Total Heterocyst Lichen Type Estimates, Cells Site (hr) Cells Mean Cells Range
Squash preparation Pxternal IC 2 4 7.27 — 7.69 Pilonhorus distans Cephalodia Sacs tough or disintegrated 44 7.4e
External Squash preparation 25 2 22 10.30 10.04 — 10.55 Psoroma hynnorum Cephalodia Sacs tough to disintegrate 193
Stereocaulon External 23.08 vesuvianum Cephalodia 18 Disrupted by Pasteur pipette 1 30 9
External — do — — Disrupted by Pasteur pipette 1 8 21.05 Cephalcdia 24 30
— do — — External 18 Squash preparation 1 32 21.95 Cephalonia 9
** auperior 6 Squash preparation*** 65 Placonsis relida Gr. Cephalodia 1 348 15.74
** Sueriorp Squash preparation 2 3E2 — do — Gr. Cephalodia 24 90 20.32 18.24 — 22.39
Superiorp — do — Cephalodia 18 Squash preparation 2 254 39 13.20 12.20 — 14.19
NOTES ;— * The collection site data are as stated in Table 1. B refers to the respective site (b). ** Gr. is Greenland.
. *** The blue—green phycobiont appears similar to Stigonema or Calothrix, but not Nostoc. Heterocysts were paired in places. 49
be less, as Jordan (1970) says, it is not unreasonable to assume that
larger cephalodia may contribute more nitrogen to the thallus than smaller
ones.
In this context, the investigation using three lichens, two
from Britain and, one from New Zealand, is most relevant. These are
1) Solorina =Lola, a form of S. saccata in which the green thallus is
reduced to a ring of tissue around the apothattecial found on a mat of
blue—green cephalodia, 2) the chimaera, between Sticta dufourii a mono-
phycobiontic foliose species with blue—green algae and S. canariensis a
mono—phycobiontic species with green algae, which sometimes become
attached, so that lobes of S. canariensis grow on the edge of S. dufourii
thallus and 3) the chimeara between Sticta felix, a foliose lichen with
a green phycobiont, which has occasionally been found on a fruiticose
lichen, a Dendriscocaulon sp., with a blue—green phycobiont.
In these instances, the heterocyst frequency at the
•interface was much higher (Table 11), but at a short distance from it,
i.e. about 2 — 3mm, the normal 5% frequency was re—established and it
would therefore appear that it is the green algae which are having the
stimulatory effect.
A further indication of the effect of the green phycobiont
on heterocyst frequency, rather than their position, comes from the assay
of different areas of mono—phycobiontic thalli. Lindahl (1960) had
suggested that the isidia of Peltigera evansiana are cephalodia as they
are easily dislodged, but they are not true cephalodia as they do not
have a different phycobiont from the main thallus.
Isidia and the main thallus were tested to establish whether
there was a variation in in the heterocyst frequency; this proved not to
be so. Two Peltigera species, P. evansiana and P. eraetext ata and
Collema subfL4rvum were tested. While the figures did vary slightly,
Table 11. Changes in the heterocyst ratio relative to the proximity of a green phycobiont in lichens which form chimeras, measured by Cr03 incubations.
Mean Heterocysts as Percentage Incubation Number of Mean Vegetative Heterocyst Percentage of Total Heterocyst Lichen Time Comments Estimates Cells (hr) Cells Mean Cells Range
Algae in sacs, do not aisrupt Iendriscocaulon 346 13 3.76 2.84 - 4.29 umhausense 30 easily using a Pasteur pipette . 3
Heterocyst count from the 6 - do - 18 base of a large old cephalodium 2 113 5.12 4.58 - 5.66
Heterocyst count from the - do - 18 apex of a large old cephalodium 2 250 5 2.18 1.86 - 2.50 • Heterocyst count from do - - 18 a smallish cephalodium 3 212 8 3.70 3.57 - 3.95
Heterocyst count from - co - 18 a tiny cephalodium 2 41 6 12.66 10.17 - 15.15
Polychidium muscicola 18 Total thallus 3 522 15 2.93 2.64 - 3.34
Solorina spongiosa 18 Disrupted by Pasteur pipette 1 175 10 5.41
Squash preparation Heterocyst count - do - 5.5 on cephalodia 1 sq cm from green algae 2 221 14 6.18 5.69 - 6.66
Squash preparation Heterocyst count - do - 8 on cephalodia touching green algae 3 62 47 45.45 34.53 - 55.55
18 Total thallus Sticta dufourii Disrupted by Pasteur pipette 578 22 3.62 3.12 - 4.12
S. dufourii + Algal chains are contorted Heterocyst 3 18 S. canariensis count of S. dufourii at interface 224 37 14.58 12.42 - 17.98
Sticta felix + Pasteur pipette preparation 18 1 152 31 16.93 Iendriscccaulon sp. Heterocyst count at the interface
Heterocyst count 226 le - do - 0.5cm from interface 1 7.38
Heterocyst count 15 3.09 - do - 2.0cm from interface 470
NC ES * Polychicdum muscicola is not part of a chimera. It was included here to compliment Dendriscoc::ulon umhausense, also known as Polychiditm umhausense. 51'
when ststistically tested using X2 analysis, the calculated result was
insignificantly different from the predicted value at 5% and therefore
the observed variation being due to the position of the isidia on the
thallus is unlikely.
Lichens are not the only organisms in which high heterocyst
frequencies are found. As has already been shown in Azolla (Hill, 1975),
the heterocyst frequency increases in the symbiotic state. Rodgers and
Stewart (1974) have demonstrated a high heterocyst ratio in Blasia and
Anthoceros and Silvester and McNamara (1976), have shown a similar effect
in Gunnera, where the frequency may be as high as 80%
Evidence of the C:N ratio being important, comes from the
work of Rodgers and Stewart (1974), who showed that fixed carbon is
supplied to the blue—green algae and there is indirect evidence from
Griffiths -.et al (1972), who showed that in Peltigera canina the blue- green algae are not nitrogen starved, as osmophilic structured granules,
•nitrogen storage bodies, are present in the vegetative cells and it would
be interesting to see if these granules were present in lichens with
high heterocyst frequencies.
The C:N ratio is probably not the only influence in these
symbioses, though whether other influences exist is not known. Recent
results show (Mitchison and Wilcox, 1973), that 7—azatryptophan has been
effectively used as a stimulent for increased heterocyst production and
it would be most interesting to see if a similar type of compound was
secreted from the green partner into the cephalodia.
Plate 2 shows the different heterocyst ratios in lichens
that have cephalodia.
• C) c": ye) • 6o. .-
VS :titk-i), i':1■ ca ■ N r' ' 0 ‘ ' t:))' ]?00 0; enC)
• ° %
(2) VI: n f- Pc,kc.- •
• 0 .„..„
11. st,
Phycobiont from Peltigera aphthosa var. variolosa.
r • .
•
• r . •% VAC, -•. 11••• 1, (54 4 ' • v
Phycobiont from Lobaria laetevirens.
Plate 2. Heterocyst ratios of Nostoc from lichens with cephalodia,
using CrC treatment. 3 52
Section 3
NITROGEN FIXATION —USE OF ACETYLENE
3.1.1 INTRODUCTION Whilst nitrogen fixation is a vast subject and has recently
been dealt with in many aspects (Mishustin and ShiltNikova, 1971;
Postgate, 1971; Carr and Whitton, 1973; Fogg, Stewart, Fay and Walsby,
1973; Quispel, 1974), the fixation of nitrogen by lichens is much less documented and this is clue almost entirely to their lack of economic
importance, when compared with leguminous crops, blue—green -algae,
- or even non—leguminous plants.
In no instance does pure green cellular tissue fix
atmospheric nitrogen (Stewart, 1966) and till recently it was thought
that blue—green algae with heterocysts (Fay et al, 1968) were only
capable of this process. However, there are several reports, where a
nitrogenase enzyme system can be induced, under laboratory conditions,
' in non—heterocystous species and in vegetative cells of heterocystous
species. Thus, Wyatt and Silvey (1969) have shown the effect in
Gloeocapsa; Stewart and Lex (1970) and Haystead, Robinson and Stewart
(1970) with Plectonema boryanum and Smith and Evans (1971) with an
Anabaena species, thoughwhetherthis phenomenon occurs naturally is not
proven.
It is suggested that approximately 85 of lichens contain
blue—green algae as the phycobiont (Fogg, 1956; Bond, 1959; Ahmadjian,
1967), which based.on a world population of between 15,000 and 20,000
Species ft licheng, depending on whether one is a lumper or a splitter
(Ahmadjian, 1965), is between 1,200 and 1,600, though not all these
contain heterocystous genera. The British flora has a similar composition
of which those with blue—green algae are shown in Table 12.
Smith (1963) states, that it is probable that most of those 53 Table 12. Established nitrogen-fixing species of British lichens containing blue-green algae.
Seterocysts ** Number of Number of Species Presence (+++) Phycobiont* Lichen Genus Species Tested Absence (---)
CO(X0ICUALES
Chroococcaceae
Chroococcus Pyrenonsidium 3 Glceocapca Buopsis -- 2 Psorotichia 2
Pyrenopsis 4 Synalissa 1 Chaemosiphonaceae
Hyena Arthopyrenia
11(1=NALES
na . +++ 2 2
Porocyphus +++ 1
Placynthium +++ 9 -
Stigonemataceae
Stigonema Ephebe ++1- - 2 -
Placonsis +++ 1 -
Sternocaulon +++ - - 19
Scytonemataceae
Scytonema Lendriscocaulon -1-1-+ 1 -
Massalongia +++ 1 -
Petractis . +++ 1 -
Polychidium +++ 2 -
Spilonema +++ 2 -
Thermutis +++ 1 -
•Nostocacene
•Nostoc Collema +++ 31 2
Hennia +++ 1 -
Lemnholemma +++ 8 -
Leptogium +++ 25 *** Lobaria +++ . 1+3 -
Nephroma +++ 4+1
Pennaria +++ 8 -
Parmeliella +++ 5
PeltiFera +++ 9+3 5
Pseudocyphellaria +++ 3+1 -
Solorina - +4+ 0+4 -
Oticta i-ri• 4+1
The lichen genera Vicroglaena,- Psoroma and Thyroa, all contain an unknown blue-green phycobiont and are untested. * Data from Newton(1931). . ** Data from James (1965). *** Numbers refer to species with the phycobiont in the main thalluo anu the cephalodia. 54
lichens which contain blue—green algae, can fix atmospheric nitrogen.
At the time that this was written, four species of the twenty six world
species now tested, had been shown to fix nitrogen, see Table 13 and it
seems that his conclusion is an overstatement, till much more data is
available.
Studies on bacterial nitrogen fixation in lichens are few and the bacteria do not substantially increase the nitrogen content of the thallus. Cengia—Sambo (1931) reported their presence in the sheaths of the
blue—green alga Nostoc of Peltigera aphthosa and suggested that they
were capable of fixation, but as the algae in the cephalodia is a genus
known to fix nitrogen, her results cannot be relied upon with any
certainty.
Azotobacter has been cultured from many lichens, Haynes
(1964), but none of these have been shown to fix nitrogen. There is no
question that free—living Azotobacter can fix nitrogen extremely rapidly
(10 — 20mg nitrogen fixed (1 gm carbohydrate)-1 utilised, Stewart, 1966),
but fixation is also rapidly inhibited below pH 6.0 (Burk, Lineweaver
and Horner, 1934) and this may be a reason why fixation by bacteria in
lichens is non—existent or very low. Hale (1967) reports that while all
lichen mycobionts, have an optimum pH for growth, with A few exceptions,
this is usually in the range pH 5.0 — 6.0
Panosyan and Nikogosyan (1966), who found Azotobacter in
lichens from Armenia, showed that their presence was governed by the
amount of glucose in the thallus, however they reported other bacteria
as being present, which could fix nitrogen and so regulate the nitrogen
availability to the thallus.
It has also been reported by Dodge (1964), that some lichens
from Antarctica appeared more luxuriant than others and this was
attributed to the presence of bacteria, "presumably Azotobacter". 55
Table 13. Retablinhed nitrogen—fixing opecios of lichens on a world basis.
Region from which Techniques Late Author Lichen species Lichens Collected Used 151 Collema granonum British Isles 1955 Bond and Scott Leptogiun lichenoides 2 15N 1956 Scott Peltigera praetextata British Isles 2 coccophorus Total nitrogen Collema New Mexico 1957 Shields Heppia despreauxii Leterminations
Collema coccophorus Collema nigrescens Heppia ruepini New Mexico 151, 1957 Shields at al Heppia denpreauxii ‘2 Solorina Baccata Solorina spongiosa
Collema subconyeniens* Nephroma tormentosum* 15N Peltigera polyaactyla Japan 1963 Uatanabe and Kiyohara Peltigera pruinosa 2 Peltigera virescens* Stereocaulon japonicum* 15N 1966 Rogers et al Collema coccophorus .Australia 2 Collema pulposum 15" 1962 Fogg and Stewart Peltigera venosa* Antarctica 2 Stereocaulon sp.
1969 Millbank and Kershaw Peltigera aphthosa British Isles 1.12
Lobaria pulmonaria British Isles C H 1970 Milbank and Kershaw Peltigera canina 2 2 15N2 Lichina confinis British Isles 1970 Stewart Lichina pygmaea
tuniforme Collema Sweden C H 1971 Henriksson and Simu Peltigera rufescens 2 2
Collema crispum Lichina confinis Lichina pygmaea Peltigera aphthosa Scotland 1971. Hitch Peltigera canina Peltigera polydactyla Peltigera rufescens Placopsis gelida
Collema pulposum 1972 Horne Peltigera sp. Antarctica Stereocaulon sp.*
Nephroma arcticum Finland C H 1972 Kallio et al Solorina crocea 2 2
-Peltigera canina Peltigera evansiana Canada 1974 Kershaw Peltigera polydactyla C2H2 Peltigera praetextata
( Peltigera aphthosa ( Feltigera caning Crittenden Plaocosis Iceland C H 1975 2 2 Stereocaulon alpinum Stereocaulon vesuvianum
NOTE * krazun -t,r-e,===pzzisiv af=ttm=ogen fiThba.ent bn4=id=vhttch it—we,e=13:0d=ciegket ■c in. 5 -eck cS \y‘‘‘, V-9?cc...\- eG . 1,9%. %V.V-Ory,t'w "."3- V`4)1( cVe wt. s 56
The question of whether polysymbionts affect fixation in lichens is further complicated by other reports, such as that of Brown and Metcalfe
(1957), who isolated a yeast—like organism, Pullularia from Cladonia uncialis and suggested that this was capable of nitrogen fixation.
However, Millbank (1969), has now shown that no yeast is capable of nitrogen fixation.
Till recently the only data available, regarding nitrogen metabolism in lichens, were the studies of Smith (1960, 1960a, 1960b) with combined nitrogen. It appears that lichens can use a wide range of nitrogen compounds and assimilate them rapidly, though protein synthesis appears to be remarkably slow. Smith (1961), has also shown that there is an annual increase of total nitrogen throughout the winter months, from November to April, with a steady fall during the summer when assimilation falls below utilisation. This will be discubsed further in the next section.
Of studies involving actual fixation, the early work carried out has already been mentioned and the heavy isotope of nitrogen was used.
This while accurate is very time consuming both in the incubation period and during the assay procedure. The whole concept of heavy isotope analysis will be dealt with in more detail in the next section.
In 1966, a new technique to assay nitrogen fixation, was reported. Both Dilworth (1966) and Schbllhorn and Burris (1966, 1967) independently showed that nitrogen—fixing cell—free extracts of
Clostridium pasteurianum, could reduce acetylene to ethylene and that the processes of acetylene reduction, as it was called and nitrogen fixation were the same on the basis of enzyme characteristics and also co—factor requirements.
Theoretically, the relationship between the two processes is as follows ;— 57
N + 3H ) 2 NH 2 2 3
C H + H2 C H 2 2 2 4
with a stoichiometric conversion factor of 3:1 on reductant analysis,
see also p 137. Acetylene is not the only substrate that the nitrogenase
enzyme complex will reduce. Hardy, Holsten, Jackson and Burns (1968),
have correlated the data from many workers and detailed other reactions
that can occur and Postgate. (1970), pointed out that all require anaerobic
conditions, a supply of ATP and an electron donor source (reductant), for
the reaction to take place.
The acetylene reduction technique is very widely used now and
has been described in detail by Hardy et al (1968). Stewart,
FitZgerald and Burris (1967), made the point that compared to 15N2
.analysis, acetylene reduction to ethylene is very rapid, measureable
amounts being produced after 5 seconds, is cheap in terms of gas and
equipment and many samples can be assayed over a short period of time.
The earliest work using this method was carried out by
Stewart, Fitzgerald and Burris (1967), on lake water samples and root
nodules but it was not used for nitrogen fixation studies with lichens,
till Millbank and Kershaw (1970), used it as an assay method and repotted fixation in Lobaria pulmonaria. 58
3.1.2 METHODS IN NITROGEN FIXATION
3.1.2.1 Acetylene reduction — incubation and assay technique Wet material was selected from the lichen cabinet, placed on
absorbant paper tissue ("Kleenex"), plant debris and sand removed,
washed in glass distilled water and areas of thallus from the central
region cut out using a No 39 5, 7 or 10 size cork borer, to give areas of
thallus, 0.25, 0.50, 1.00 and 1.50cm respectively. For non—foliose
species, small pieces of thallus were taken which had an approximate
dry weight of 30 — 50mg.
Serum bottles or McCarteney bottles were calibrated and those
were selected that fell within the range 6.25 0.25m1 and 26.50 ± 0.50m1
for the total gas volume under the serum cap.
Medium (Appendix 2), was added to the bottles, 0.50m1 for the
large discs and 0.25m1 for the smallest size, which maintained the discs
in the correct upside down position, relative to the light source on the
shaker. The bottles were made gas—tight with rubber serum liners
("Suba—seals") and pre—incubated.
Equilibration on the shaker (3Hz x 3cm stroke) was for up to
1h at 25°C and at a light intensity of 7,000 lux from below. Acetylene
was injected without the removal of air to 4% v/v and a gas sample
removed at TO (0.50m1) and stored in a plastic hypodermic syringe, with a
rubber wad over the needle till it could be assayed. This procedure was
repeated for other bottles at 30 second intervals. The bottles were then
placed back on the shaker and the TO samples analysed using the gas
chromatograph. Subsequently gas samples were removed at 30 second
intervals at time T1, T2, T3 etcetra, to achieve a plot of ethylene
evolution against time (Figure 2). The time interval between T1 and T2
assays was governed by the number of samples on the shaker and ultimately
on the period of analysis in the gas chromatograph (2 to 2.5 minutes per 59
12
10
10 20 30 40 50 70 8o (Minutes)
10
10 20 30 40 80 (Minutes)
Figure 2. The rate of C2H2 reduction with not shaking (A) and shaking (B), 1 sq cm discs of Peltigera zanina 02), P. polydactyla (C) and P. aphthosa var. varioloaa (W). Standard Deviation indicated by vertical lines. 60
sample). Figure 2A shows the effect of not shaking the samples during
the incubation period.
3.1.2.2 Acetylene reduction —. analysis on the gas chromatograph
The gas chromatograph used was a Pye Unicam 104 model, with
a 1.6m column of "Porapak R". The temperature was 50°C, with oxygen—free
nitrogen as the carrier gas, flowing at 100m1 minute-1. 3.1.2.3 EL2222f2222222iaLciwitlace-tlenereduc--assolubilit
Acetylene and to a much lesser extent ethylene, are very
soluble in water (see Merck Index), which will affect the partition of
the gases extracted for analysis. It was noticeable that the acetylene
peak on the pen trace of the gas chromatograph, dropped with subsequent
injections from a single bottle, however under the conditions of this
study, absolUte total ethylene evolution was not required and so this
solubility factor was not taken into account, as it was assumed that any
changes that might have been due to this, were negligible compared to
other parameters observed.
. 3.1.2.4 Problems associated with acetylene reduction — percentage sample
loss on removal of Elsirsaa bottle
In removing gas samples, the remaining gas volume decreases
non—linearly, which is coupled with an increase in the vacuum,
particularly with large samples from small test vials. To overcome this
a gas or liquid balancing medium could be added, to counteract the gas
removal, however a gas would alter the composition of subsequent samples,
but not the solubility, whilst a liquid would increase the solubility of
the acetylene and additions of a solid, though theoretically the best
method, is impossible in practice and while it was realised that these
physical interactions were competing, under the circumstances nothing
was done to correct them. 61
3.1.2.5 Problems associated with acetylene reduction — "Suba—seals"
These rubber liners have been used extensively in
acetylene reduction assays, since they are airtight and many extractions
can be made through a single cap without loss. Kavanagh and Postdate
(1970), suggested that these caps should be boiled and used only once,
a procedure followed by Millbank (1972).
Despite repeated observations, using caps that had been in
direct contact with high concentrations of ethylene and acetylene and
placed in fresh empty bottles, closed with unused serum caps, no
detectable acetylene or ethylene was measureable over 24 hours. For this
reason serum liners were used over again, in experiments where bottles
were assayed at 24h intervals over many days.
Fresh caps were used for each different experiment and old
caps were not re—sterilised as shrinkage in this process produced non-
gas—tight seals.
3.1.2.6 Problems associated with acetylene reduction — syringes
Possible carry—over of ethylene and acetylene, may occur
with dispdsable hypodermic syringes that are used more than once.
Observations showed that no ethylene remained adhering to the barrel of
the syringes and only small amounts of acetylene could be detected,
relative to the substrate concentration, i.e. 150'units compared to the 6 4% v/v value of 2.5 x 10 units and for this reason the same syringes
were used repeatedly, but not for different experiments.
3.1.2.7 Problems associated with acetylene reduction — terminators
Terminators, chemicals injected to end the reaction, were not
used in this study. Hitch (1971) and Thake and Rawle (1972); both found
that abiological ethylene was produced in some instances during their
applicatien. For this reason gas samples were removed by hypodermic
syringe and stored, till they could be assayed, at most 30 minutes after
62
their removal (this would have approximated to the time needed to analyse
12 — 15 samples), though it has been shown that longer periods are
possible and are not detrimental to the results, Hitch (1971).
3.1.2.8 Acetylene concentrations 4',/, acetylene over air was used for the majority of assays.
3.1.2.9 Total nitrogen analysis — preparation The lichen thalli or cephalodia were placed into boiling
tubes. Approximately 10 N H2304 with Kjeldahl catalyst added, see
Appendix 3, was pipetted into the tubes in the proportion of lml acid
mix to 1sq cm thallus, the volume not being critical, the essential only
being that there should be enough acid present to keep the charred remains
in a liquid state.
3.1.2.10 Total nitrogen analysis — heating the boiling tubes
An electrical heating apparatus was used. A "Variac"
transformer controlled the temperature. The voltages applied were,
1)120V during the removal of water,
2)160V during charring and
3)240v during clearing.
When the water had been driven off, noticeable as the tubes
became dry at the rim and white fumes started to be given off, glass
bubbles were placed on top of the tubes and the solution refluxed till
clear. After cooling the sides of the digestion tubes were rinsed with
glass distilled water (2 — 3m1), "Parafilm" placed over the orifices and
the tubes stored at 4°C till assayed.
3.1.2.11 Total nitrogen anal sis d assa of total nitrogen
The method follows that of Conway (1957), who gives a critical
analysis of the method. Duplicate "Conway Units" (9.0cm o.d) were taken
for each assay to ensure accuracy, the rim of the centre well and the
glass lid greased with petroleum jelly "Vaseline" and 17.0m1 of 2% boric 63
acid/1%alcohol indicator was pipetted into the centre well. The lids
were inverted and placed on the units upside down to prevent any stray
ammonia getting into the boric acid. Nitrogen analysis was postponed if
it was thought that ammonia was present in the atmosphere for any reason.
A small aliquot part of the cleared solution was pipetted
into the outer well, the glass lid reverted and placed almost in position,
leaving only a minute gap for the introduction of 1.5m1 of 40% NaCHI via
a 5.0m1 plastic hypodermic syringe. The minute gap left for the needle
entry, prevented loss of ammonia, should the two solutions mix, before
the lid could be completely slid on.
The units were then tipped very slightly, so that the two
solutions just moved in opposite directions, round the outer well and
mixed under their own forces. No further mixing was carried out at this
point, as this kept the strong exothermic reaction to a minimum and
prevented "spitting". Small quantities of dilute solutions should be used
•at all times. The need for excess H2SO4 in the digestion tubes, requires
excess alkali to neutralise it, giving rise to "spitting". This
contaminates the boric acid with strong acid, or alkali, or both and the
assay is destroyed.
As a precaution, weights were placed on the glass lids, to
counteract the upward lift, due to the expansion of the internal gas, by
the heat of neutralisation. The units were left in this position
overnight and in the morning the units were swirled round, to ensure that
the contents of the. outer well had mixed completely. The units were then
left a further 2 hours and the contents of the centre well, back titrated
with N/50 acid.
Calculation of the total nitrogen values were based on the
assumption that 1m1 of N/50 HC1 or is equivalent to 2E0ug nitrogen. H2SO4 3.1.2.12 Dry weight determinations (see methods 2.1.2.E p 17) 64
3.1.3 DISCUSSION ON ASPECTS CP NITROGEN FIXATICU There is now no doubt that some lichens are capable of
nitrogen fixation, though Watanabe and Kiyohara (1963) were not able
to demonstrate fixation, in a number of lichens potentially capable of
fixation and Fogg and Stewart (1968), could not demonstrate fixation in
Peltiera venosa.
A general survey of the British material was undertaken and
of the 33 species that were tested, all fixed nitrogen. Their optimal
rates were not observed and it is interesting to note therefore that
Peltigera and Sticta had the highest activities. Scholander, Flagg,
Walters and Irving (1952), studying respiration, found that Peltigera and
Sticta species had the highest rates, both from the tropics and the
Arctic at all temperatures tested. They compared 19 Arctic lichens and
10 tropical species of which 5 and 3 respectively, were Peltigeras and
Stictas.
As L'eltigaa, canina gave the highest activity of all the
lichens tested and because it is common in the field, it was studied in
detail. •
The lichens tested, Table 14, all contained phycobionts which
are members of the Heterocystineae ( Rivulariaceae, Stigonemataceae,
Scytonemataceae and Nostocaceae) and as reported by Fay, Stewart, Walsby
and Fogg (1968), there is unequivocal proof of fixation in this group.
None of the critical British species of lichen, containing
unicellular forms of blue—green algae (Chroococcus, Gloeocapsa, or Hyella)
have been tested so far and none were tested in this study, since they are
not at all common and no material was available.
Some material, however, was sent from the Negev Desert, Israel,
by Dr. Galun. Two lichens Heppia polyspora (Peltula polyspora) and a
Peccania sp., containing LITa2L1 and Gloeocapsa respectively
65
Table 14. A compLrimn of nitrogen fixation by lichene in an er-fielu condition, using total nitrogen end dgy weight detorminatione.
-1 -1 4 nV, 4 mgN 0 C 24 mg 0'. T. C;,11 2bviple Total Nitrogen Yean Percentage Oven Dry Weight C,Z Lichen 1 Nitrogen (mg) -1 -1 hr (mg) hp - hr 0.21' 19.29 0.69 f;ollema 5.40 3.6G 7.76 auriculatum 5.70 0.46 12.75 12.39 0.45
';ollema 1.56 4.37 35.00 fluviatile
26.41 1 .G0: 12.68 0.48 4.09 11.70 furfuracourr. 8.05 0.47 11.46 V,E3 0.77 4.08 0.95 24.70 5.13 0.20 0.92 1.22 3.85 31.72 7.31 0.28 subfurvum 11.22 1.27 33.02 8.84 0.34
21.30 2.57 73.30 8.29 G.29 Lendriscocaulon 1.35 4.27 38.42 7.7E C.27 unhausense 10.50 42.25 6.30 127.00 6.62 0.33 0.98 15.00 5.96 Lohebe 5.04 13.76 1.01 5.85 21.90 13.62 C.63 lanata 8.08 1.76 27.21 5.05 0.33
Lentoriiim 17.50 0.46 8.36 38.04 2.09 5.51 burressii 16.50 0.43 7.82 38.37 2.11 18.EC 0.45 11.61 41.7E 1.62 lettorim 14.7C 0.73 4.38 18.83 20.14 0.78 lichenoides 12.56 0.91 23.47 13.EG 0.54
••Lentorium 1.12 4.31. 26.00 sinuatum
Leotooium 7.0C 0.37 1.42. 25.93 18.72 C.27 subtile
Leetozium 0.80 1.47 4.5C 32.70 0.54 0.02 teretiusculum. 1.28 34.32 12.34 0.46 Lichina • 15.79 4.70 2.16 3.73 57.90 1.90 0.07 confinin 6.02 1.92 51.47 3. 4. 0.12
Lichina 3.81 5.26 55.70 pygmaea
0.30 15.08 • Lobaria 9.20 0.02 462.00. laeteyirons.. 2.14 107.00
lobaria 1.14 1.03 62.10 vul!ronaria 6.62 . 0.92 33.19 7.20 0.20 lobaria • 10.7 0.60 2.77 21.64 17.96 0.50 screOiculata 9.55 0.96 - 33.63 9.90 0.20 0.51 11.0e 19.05 0.05 7!assalonria 10.13 4.27 c:rnosa 13.0t3 0.75 17.47. 10.50 0.79. 0.71 18.47 0.65 'Leohroma 5.70 0.37 4.22 lr.eyirata 15.00 0.59 13.89 25.47 1.00
!W11,41 * Further analyuis or the Lento,r-ixm tcrotiunculum material wan carried out after incubation in Allen and Arnon'n I4e,iiva with the addition of 1g/1 MOT. Some compnrabla moult° with reltirera canina and. P. aththosa var. variclom at givon in FiGurea 2, 3 and 4.
** The high levels of nitrogen fixation in thin npocion aro duo entirely to tho provonce of caul:lac:dia. Calculaticna based on 19.4 pg, rather than 20 Cyt give a total value of 004.12 nM 0211 rA;;N hr-1. 4
66
Taeln 14. Continued I. nompraquon Gf nitrogen fixation by lichen') in an ex-field condition uning total nitrogen and dry weight determinationn. - 1 n7 Sample 1 nM 02114 mg (3■W 24 Total Nitrogen Mean Percentage Oven Lry Weight '171. 02n4 rgli Lichen N itrogen (m0 hr-1 (me) hr-1 hr-1 Pennaria microphylia 3.72 5.14 72.30 v. cheilea 13.33 1.12 0.07 0.94 0.85 6.36 r.ezizoidea 3.00 2.21 34.67 1.36 0.G9
Pansaria 0.27 0.09 3.(.o pityrea 91.07 12.99 0.29 Pannaria 26.75 2.06 2.27 rubiAnosa 44.00 3.73 164.91. 11.80 0.27
0.20 3.90 5.17 52.90 2.22 atlantic: 11.78 11.60 * 1.20 25.21 9.66 C.45 "armeliella 18.00 1.33 4.76 27.94 13.5c 0.64 nlumbea 12.20 2.60 54.62 4.92 0.23
Poltigera.. 23.75 0.09 6.92 1.30 263.88 18.25 anhthosa 22.59 0.09 4.60 2.00 984.33 44.30 v. varicicr:a 26.72 0.05 4.55 1.10 534.40 24.29 55.02 0.44 10.65 123.05 5.17 Peltigera... 4.13 carina 44.85 0.43 10.40 104.32 4.31 24.80 0.19 7.55 130.53 3.28 Peltigera"*. 15.20 0.17 2.52 6.76 89.41 2.25 colveactyla 21.07 0.19 7.55 110.89 2.79
Placynth:um 2.53 0.39 2.73 14.28 6.49 c.18 nigrum
Placvnthium 0.35 4.61 7.59 16.77 0.77 nannariellum 5.87 18.75 1.75 54.92 10.71 0.34 Polvchidium 17.25 1.57 3.19 49.27 10.99 0.35 ouscicola 15.19 1.54 48.33 9.86 0.31 8.27 1.38 34.96 5.99 0.21 Pseudocythe:laria 3.00 0.80 3.95 20.27 3.75 c.75 thouarsii 9.12 0.98 24.83 9.31 0.37 0.76 1.55 49.89 0.49 0.02 Solnrina". 1.52 1.53 3.11 49.25 0.99 0.23 crone 2.46 1.35 43.45 1.82 0.06
Solorina." 2.47 0.43 1.46 29.57 5.74 Baccata
Solnrina.. 4.40 0.17 25.83 sncngicsa 4.13 0.24 5.71 17.21 0.72 Sticta 16.00 0.96 4.19 22.65 16.67 0.70 fuligincoa 12.70 0.80 19.04 0.07 31.28 1.'47 Stints 61.74 1.22 3.93 50.61 limbata 33.98 0.82 21.02 41.44 1.02
Lichonn incubated in Allen and Arnon'n medium with added HCO (1g/1) chow a marked increase in t- 3 nitrogen fixation rmtnn. After 24 houro, the rate had trebled to 35.25 nM C,114 Sample ihr-1. lhono cronies have oephalodia *44 '!'bona nponion of foltigera only have one phynobiont and chow a rapid rate of nitrogen fixation. 68
(Ahmadjian, 1967; Snyder and Wullstein, 1973) were assayed. The results,
Table 15, showed rapid fixation initially, due to the fact that an epiphytic lichen, capable of fixation, was found situated on the thallus of the species being tested. However, when portions of the thallus of
Heppia and Peccania, were tested without the epiphyte present, the fixation measured after 24 hours in the light (in Allen and Arnon's
Medium, supplemented with 10mM HCO3) was very low indeed, although ' above background.
Two possibilites are suggested to explain the finding: fixation by contaminating or epiphytic bacteria, or fixation by the
phycobiont. It has been reported that bacteria are one of the symbionts
of lichens and there are conflicting reports by investigators who have examined'nitrogen fixation in lichens of soil crusts. Rogers, Lange and
Nicholas (1966), showed that Collema coccophorus, a prevalent desert 15 species, could fix but they also reported incorporation of N2' dinitrogen by Lecidea crystallifera (0.002 atom % excess) and by Parmelia conspersa (0.005 atom % excess), both of which have green phycobionts and
both of which live on soil. However, a rock species, Parmelia adhaerens,
showed no fixation and it may be a factor of soil crusts that enhance the
N-balance considerably.
Shields, Mitchell and Drouet (1957), also measured total nitrogen in various soil crusts, which included both nitrogen-fixing lichens and *algae together and showed conflicting results. Unfortunately their algal identification was not consistent in that they did not appear to be able to identify the phycobiont. In Collema coccophorus, they did
not cite the algal host, though they did cite Nostoc for C. nipTescens
and further they cited Dermatocarpon sduamellum as having Pleurococcus
and Heppia despreauxii "a blue-green alga, usually Pleurococcus", which
is the same as Protococcus, a member of the Chlorophyceae. They also Table 15. The relationship between nitrogen fixation in two lichens with unicellular blue-green algae.
A. Peltula polyspora ( Heppia polyspora ) Lichens B. Peccania sp.
EXPERIMENT I. In Conway Units, 30m1 capacity, 10% C2H2+Air. 0 hr Time Heppia polyspora Peccania SD. (mins) 30 "43) 2H4 sample-1 hr-1 1.91 ) nY C sample-1 hr 1 60 0.68) i C 2.73) 2 H4 72 hr 30 2.06) -1 3.65) 60 2.20) nM C H sample- hr nM C2H4 sample-1 hr-1 2 4 • 4.28)
EXPZRIMENT II. In Serum Bottles 6m1 capacity 10% C2H2+Air. Time 0 hr Heppia+Epiphyte Heppia-Epiphyte Peccania (mins) sp. 2.68) m 's -1 -1 -1 0.08) 0 n C2H4 ample hr (()):g46.))nM C2H4 sample hr 'nM)n]2 C2 HH4 sample-lhr-1 9 .50) 0 4 24 hr
120 1.79 )nM C H sample- r-1 0.17)nM C2H4 sample-1 r-1 0.04)nM C2H4 sample-lhr-1 2 4 70 quoted a similar phycobiont for H. amini, though it has been shown in the genus Hermia that the normal phycobiont is Anacystis. Further in
Lecidea decipiens form dealbata, they cited the algal host as Protococcus,
Gloeocapsa or Pleurococcus.
In all their total nitrogen measurements, which are consist- ently high, except for Dermatocarpon squamellum on moss and surprisingly
Collema nigrescens on sand, there is no difference between lichens with
and without blue—green algae, and since some of the free—living algal constituents of the samples are nitrogen—fixing species, this probably explains the high values obtained. Furthermore they were able to
isolate Azotobacter, a nitrogen—fixing species, from rather less than
half their samples.
The work of Snyder amiWullstein (1973) needs commenting on in
this context. They examined four lichens, two of which Pyrenopsis and
Peltula had Gloeocapsa and Anacystis as the blue—green phycobionts and two,
Sarcogyne and Parrnelia had green phycobionts. Their results shOwed very
slow rates of acetylene reduction and had large standard deviations,
indicating a poor correlation with nitrogenase activity. There was no
significant difference between lichens with blue—green or green
phycobionts.
Whether these results indicate fixation in lichens of this
type is highly questionable, as they incidentally showed that Azotobacter
were present as a soil constituent. The other possibility that non-
heterocystous fixation is occurring, cannot be ignored. Some analogy
can be drawn between these studies and that of Stewart and Lex (1970),
regarding inducement of fixation under anaerobic conditions. Are the
conditions within the thallus of these lichens micro—aerobic? Millbank
(pers comm), has found extremely rapid changes in oxygen tensions
within the cephalodia of Peltigera aphthosa var. variolosa. Using an 71
0 electrode probe, he has shown that the oxygen tension rises rapidly 2 to above atmospheric level in the light and drops very rapidly in the
dark, so these lichens could well have low oxygen sites, but according
to Stewart and Lex, the p02 has to be vanishingly small for at least
36 hours to develop a nitrogenase.
It is unlikely that the p02 would approach zero at any time
in a lichen, though it is not perhaps impossible. Since Wyatt and
Silvey (1969), found Gloeocapsa to fix nitrogen at atmospheric p02,
algal fixation in Pyrenopsis and Peccania could well be possible.
Whether the similar findings with Henpia can be explained in
this way has yet to be established, a great deal of work is needed on
this very interesting group of lichens.
The results obtained with the above nitrogen—fixing species,
were carried out on non—uniform areas of thallus and in order to produce
unification in the data, total nitrogen and dry weight determinations
' were carried out (Table 14).
The wide variability was also noted by Kershaw (1974) and he
suggested that in view of this variation "the nitrogenase activity is
best expressed in terms of thallus dry weight". Henriksson and Simu
(1971), have also based their results on dry weight. I consider this to
be an ambiguous method of representation of the data, since both the dry
weight and the total nitrogen (unit area of thallus)-1 can vary widely,
a feature particularly noticeable in Peltigera polydactyla.
Table 16 and 17 show this feature. In Table 17, all the
results are based on a standard dissection technique and not only do the
samples vary within a single group, but there is also wide variations at
different times of the year. Smith (1961) has shown a similar trend of
variation, though there was a general relationship between the two
parameters measured.
Table 16. The relationship between different fractions of the thallus of Peltigera aphthosa var. variolosa and P. nolydactyla based on dry weights and total nitrogen analyses.
?lumber of Nitrogen Content Cephalodial Nitrogen Cephalodial Iry Weight Thallts Fraction Dry Weight Percentage Nitrogen Estimates (mg). Content as a Percentage of the as a Percentage of the Total (pg) Green Thallus Nitrogen Thallus Dry Weight
FIRST EXPERIMENT. P. anhthosa var. variolosa using 6 x 1 sq cm discs of thallus.
Green thallus 1410 28.5 4.98 (2x3 discs) 1590 35.7 4.45
Cephalcdia ( Excised) 134.3 3.8 3.53 11.84 9.52 (2x3 discs) 95.9 3.4 2.82 8.56 6.03
SECCND EPERIEENT. P. aphthosa var. variolosa using 15 x 1 se cm discs cf thallus.
10.5E Green thallus 319.93 3.02 (64.55 37.26) (1.26 0.33)
(Yg) Cephalcdia per lag cm 15 28.47 544 5.23 8.89 4.93 ( Excised) (3.32 1.92) (156.7 40.98) (range 2.95 - 7.64)
PELTIGERA POLYLACTYLA
Mean Cells Mean Cell Nitrogen Total Cells Total Cell Thallus Nitrogen Total Thallus Dry Weight Total Thallus •Thallus Area Nitrbgen as a Percentage (sq cm) 106 6 (x 106 Cells)-1 Nitrogen Nitrogen Dry Weight x 10 mg (sq cm)-1 sq cm - 1 of the Dry Weight sq cm 1 (R) (mg) (mg) (mg) 3.37**** 157.6* 3.95 624.33 5.40 0.329 51.87 13.70 2,160 2.40
NCTES The thallus area was calculated from dry weight analysis, using the figure of 13.70 mg.
** The Nostoc cell density was measured by the use of Cr0 incubations. 3
* * * The mean of 4 discs assayed from the bulk of the thallus (*). The assay was carried out in November and showed a high level of nitrogen. The total cell nitrogen is equal to 6.50'!. of the total thallus nitrogen. -1 h.)
Table 17. Total nitrogen and dry weight analyses of Peltigera polydaetyla at different times of the year.
Total Nitrogen Dry Weight Percentage of the Percentage of the Total Thallus Nitrogen as a Collection Number of Thallus Date ( 1 sq cm )-1 Dry Weight ( 1 sq cm )-1 Nitrogen - Nitrogen Percentage Site Estimates Fractions in Each Fraction in Each Fraction . (pg) cf the Dry Weight (mg) (pg)
Algae* 94 57 16 +++ ( 17.40 4.30 ) ( Range 50 - 65 ) June Tentsmuir 163 1972 69 43 16 Fungi ( 10.74 2.69 ) ( Range 50 - 35 )
62 Algae - - 114 16 +++ ( 10 30 2.58 ) ( Range 50 - 71 ) Augut Tentomuir 185 1972 71 36 16 Fungi ( 12.50 3.13 ) ( Range 50 - 29 )
Algae 29 170 42 +++ 4.14 Tentsmuir 1 14.74 400 2.71 Fungi 10.60 71 230 58
December 1972 Algae 30 170 46 +++ 4.75 Furnace , 15.90 380 2.39 Fungi 11.15 70 210 54
Algae 142 39 +++ ( 15.36 7.70 ) ( Range 34 - 45 ) 13.70 363 2.61 Tentsmuir 6/8** ( 0.27 0.11 ) ( 2.72 1.11 ) 221 61 Fungi ( 10.90 5.40 ) ( R ge 66 - 55 ) April 1973 Algae 127 45 +++ ( 11.68 5.84 ) ( R ge 39 - 52 ) 2.42*** 13.10 282 Furnace 6/8" ( 0.77 0.31 ( 2.60 1.06 ) 155 Fungi 55 ( 5.80 2.90 ) ( Range 61 - 48 )
NOES Algae +++ is the algal rich zone. 6/8 refers to the estimates for dry weight and nitrogen analyses respectively. W * * * The percentage nitrogens for April 1973 are calculated from the dry weights and total nitrogens, which are comparable. 74
The use of dry weight might help to explain Kershaw's data
(Kershaw, 1974), since his results for P nolydactyla are lower than for
the other three species tested, all of which it is suggested have thinner
thalli and affect the mg dry weight analysis.
However, he also calculated his data on a cell numbers basis, 6 presumably total cells x 10 and there is a similar difference with this
method. It has been noted in this study that while P. polydactyla may
exhibit an equal activity as P. canina, it is normally less so and this
may be a characteristic feature.
Generally speaking the rate of nitrogen fixation in lichens
is high and Millbank and Kershaw (1969) were the first to report fixation
on a cell numbers basis in Peltigera aphthosa and they reported it as
being rapid, compared to free—living blue—green algae. Millbank (1972)
has since reported that P. canina shows similar rapid rates, again
compared to free—living blue—green algae. 6 His results were based on cell counts of 3 x 10 cells \ (1 sq cm)-1. This figure has since been revised by Hitch and Millbank 6 (1975), a figure of 9 x 10 being quoted. Rates of fixation were reported
as being of the same order as free—living cultures and there appeared to
be a broad correlation between fixation in Peltigera canina and 1,12111019.
aphthosa. The latter species although showing an 8—fold increase in
fixation compared to P. canina, had approximately an 8—fold increase in
the number of heterocysts (Table 18).
Millbank (1972) has shown that the normal nitrogenase activity -1 -1 -1 1 , of 50nM C H 0 sq cm) hr (equivalent to 3.8nM C H min mg protein ) 2 4 2 4 is almost the same as that for free—living algae (3.0nM C H min —1 2 4 -1 mg protein ). However, in free—living cultures, this is produced by a
12% heterocyst population, whilst in Peltigera canina, the population was
only 5%. In simple terms, either the nitrogenase activity in lichen 75
Table 1P. ' A comparison of various morphological and physiological parameters in the lichen genus Peltigera.
Peltigera Peltigera Peltigera aphthosa PARA"ETERS canina polydactyla v. variolosa ( 1 sq cm ) ( 1 eq cm ) ( 1 cephalodium )
Mean Total Algal Cells ( Unit of Thallus )71 x 106 (22)* E.89 w* (32)* 9.47 *, (3)* ( 1.50 0.32 ) ( 3.10 0.54 ) 0.047
Heterocysts as a Mean Percentage of the Total Cells (3) 6.29 (3)5.78 (3) 17.00
-1 6 Heterocysts ( Unit of •Thallus ) x 10 0.43 - 0.67 0.48 - 0.61 0.006 - 0.010
bean Dry Weight ( Unit of Thallus )71 (3) 10.7mg (4) 14.4mg (4) 17.38pg
*** *-* *** even Dry Weight of Algal Cells 592.711g 625.0pg 3.10 pg
Algal Cells as a Percentage of the Total thallus Weight 5.93 4.35 27.80
-1 C H ( Unit of Thallus r 2 4 50 24.80 0.509
nM C ( mg Thallus Oven Dry Weight )-1 hr 1 2'4 4.67 3.28 45.65
6 nM C H ( 1 x 10 Heterocysts )-1 2 4 87.72 70.80 63.63 yg Nitrogen mg Oven Dry :!eight Thallus )71 41.30 25.16 45.00
C ( mg Thallus Nitrogen )-1 hr-1 2-4 123.05 130.53 1014.44
Cephalodia Equivalent to 1 sq cm of Peltigera canina Thallus 959.64
Total Algal Cells ( 960 Cephalodia )71 45.12 x 1c6
Total Heterocysts ( 960 Cephalodia )71 7.67 x 106
Assumed Nitrogenase Activity Comparing 50 n11 C2H4 ( 1 sq cm )71hr71 and 6.29% Heterocysts 251.23
Assumed Nitrogenase Activity Comparing 50 nM C2H4 679.00 ( 1 sq cm )7111r71. and 17.00% Heterocysts nM C H ( Cephalodium 2 4 0.71
MOPES :- * Refers to the number cf estimates. Refers to the Standard Deviation ana Standard Error of the Mean Respectively. -1-; The oven dry weight of the algal cello wan based on the value, 66pg ( 1 x 106Cells )-1. 76 heterocysts is three times that in free—living material, or there is three times as much per heterocyst. This problem has not yet been resolved. 77
ASPECTS' OF NITROGENASE ACTIVITY
3.2.1 INTRODUCTION
In general lichens grow centrifugally and form orbicular
colonies. The exception to this rule being the fruiticose types, which
. grow terminally. Early growth activity increases in the surface area,
but lateral transport is restricted in old thallus regions, with the
result that they become thickened and energy is diverted into the
formation of propagule forming bodies. The older parts are said to
remain in a functional state throughout the life span of the lichen,
Hale (1967).
Lichen growth is very slow in some instances, with no
perceptible growth occurring over many decades and Beschel (1961), has
postulated that some Arctic lichens, e.g. Rhizocarpon geographicum, may
be several thousand years old.
Galan, Paran and Ben—shaul (1970), have studied the turnover
•of cells in an Aspicilia species and Squamarima crassa var. crassa and
shown that young cells are formed at the innermost part of the algal zone
and the perphery of the lobes and there is an aging process outwards,
with the senescing and decaying cells being extruded through the fungal
cortex to the exterior of the thallus.
Hill (1975), has indicated that in aging leaves of Azolla,
the algal symbiotic cells are becoming senescent and he stated that the
physiological state of such cells is not known. Hitch (1971) noted a
visible change in thallus appearance in Peltigera canina. The older
regions away from the growing edge, being dark and necrotic in places.
He found that the physiological activity of this region was very poor in
comparison to that of the actively growing cells. Physiological activity
over the thallus was investigated further, in greater detail.
Another factor that was investigated was the optimum 78 pC H concentration at the substrate level for nitrogenase activity and 2 2 various. percentages were tested. Dilworth (1966) and Shbllhorn and
Burris (1966), have both shown that a pC2H2 equivalent to 4mm Hg (0.50%). is sufficient to inhibit the fixation of atmospheric nitrogen by 50%.
10% is regarded as a standard concentration, though 20% in the presence of air is suggested by Stewart (pens comm) though this may not in fact be necessary.
Hardy, Holsten, Jackson and Burns (1968) studying the Km for acetylene reduction, indicated that soybean nitrogenase is saturated by
2.5 — 20% C2H2. There was no increase in activity using 40% and there was a definite inhibition when 50% was used.
Different systems appear to be effected in different ways.
Spiff and Odu (1973) with Beijerinckia, reported an increase in the rate of ethylene evolution, with increasing concentrations of acetylene, up to
75%, though with a reported fall off above this concentration.
Various concentrations of acetylene were applied to the lichens Peltigera canina and P. aphthosa var. varioloea. 79
3.2.2 METHODS
The lichens were tested for acetylene reduction in the normal
manner. A detailed analysis of the method used, is given in the previous
section (3.1.2.1 , p 58).' The acetylene gas was applied by hypodermic
syringe. When concentrations of 10% and above were used, an equivalent
part of the basic gas phase was removed prior to the injection. 80
3.2.3 VARIATION IN NITROGEN FIXING ACTIVITY OVER THE THALLUS AREA
Resulting from these studies are two general findings, 1)
sample discs cut from the edge of thalli, had much lower nitrogenase
activities than those from central regions and 2) there is evidently
very wide variations in the nitrogenase activity of different thalli.
Evidence for the variation in metabolic activity in young
and old thalli is scanty, though there have been some reports of
differences. Scholander, Flagg, Walters and Irving (1952), partly
explained the variation in their respiration studies as the result of
using young and old thalli. Reid (1960; see Hale, 1967) reported that
there was no difference in the respiration rates of very young or very
old thalli of Umbilicaria Imstulatal although there was a 2—fold
difference in the rates of photosynthesis. Similarly Ellee (1939)
examined three lichen species and found that the older thalli were the
less active photosynthetically.
Millbank and Kershaw (1969), explain variation in their
physiological data among replicate samples, as being due to age and Hale
(1967) studied the growth rates over a six month period (from September
to Febraury), in young and old thalli of Parmelia conspersa and found
that there was considerably better growth in the mature thalli, compared
to the young lobes.
In these latter instances, nitrogen fixation and growth, in
mature specimens was faster. It is necessary therefore to decide what
one means by young and old thalli and the physiological concept of lag
phase, exponential phase and stationary phase might be applicable here.
"If one considers the lag/exponential phase as equivalent to "young" and
"mature", then "mature" is more active than "young". However if the
exponential/stationary phases are considered, as "young" and "mature",
then the "young" stage would be expected to be more active. 81
Since in the majority of cases, under non—limiting
conditions, whole thalli appeared healthy, to quote age as a reason for
demonstrating variability, is dangerous unless critical measurements are
stated.
With Peltigera canina, a fast growing foliose lichen, the
cellular turnover is likely to be very rapid, with the complete cycle
occurring in 1 — 2 years, in lobes a few centimetres wide and long. For
this reason it was easy to assay whole lobes, by punching out discs
1 sq cm in areas and assaying them for acetylene reduction. The nitrogen
fixation rates of Peltigera canina and also the heterocyst numbers, were
greater in central portions of the thallus (Table 19).
It is likely that the heterocyst population would be higher
in the central regions of the thallus, since not only will some of the
filaments be that much older, but with the more rapid oxygen turnover,
see p 70, the possibility of anaerobic conditions favourable to
•heterocyst development could prevail and this indeed was so.
Griffiths, Greenwood and Millbank (1972), have shown that
there is a wide variation in the heterocyst frequency, in relation to
area, but they do not state from what area of the thallus their material
came.
The fact that only a very broad correlation between
heterocyst numbers and nitrogenase activity was demonstrated, using
Peltigera polydacty (Table 20),• though I would have no reason to
suppose that P. canina would be any different, would imply variation in
thallus material, probably reflecting its history before collection.
Jewell and Kulasooriya (1970), have looked at several free—living blue—
green algae and calculated a coefficient of acetylene reduction, based on
acetylene reduced and heterocyst numbers and they point out that although
the quantity of acetylene reduced varied more than 70—fold per volume of
Table 19. A comparison cf nitrogenase activity ( C2H2 Repuction ) over the thallus of Peltimera canina using 0.25 sc cm discs stamped cut.
C H * nM C284* Chlorophyll ** C 2 4 nM CH C H nM C2 -.4 ' 2-4 4 CC663 a ' 2 Total mg Thallue)-1 (mg Thallus)-1 -1 Cells ( sq cm )-1 ( eq cm )-1 ( sq cm )-1 (mg Thallus) Nitrogen ) ( Nitrogen ) ( 1 sq cm )-1 ( Mitrogen ) -1 . -1 106 hr hr 1 ( 5 ml )-1 nr hr 1 hr-1 hr-1
- - - THALLDS A - - - - THALLUS B THALLUS C
A 13.28 30.74 A 16.00 37.04 --- A 5.60 12.96 0.45 3 39.60 91.66 - B 8.75 20.25 0.35 B 20.60 47.69 C 30.12 69.72 - c 8.96 20.74 0.62 D 36.16 C3.70 D 19.53 45.21 0.44 • 19.04 44.07 E 29.32 67.87 ___ E 17.43 40.35 0.19 ® 0 F 30.92 71.57 --- F 17.92 41.48 0.26 • 20.24 46.85 0 37.96 27.87 --- G 11.97 27.71 0.17 THALLUS A 18.00 41.66 --- H 1.75 4.05 0.17 • 3.36 7.78 I 12.12 41.94 - oa J 19.92 46.11 F 5.60 12.96 Al 7.00 16.20 0.120 K 4.62 10.83 0.200 1 5.20 12.04 L 16.52 32.24 • 9.88 22.27 12.12 28.06 0.220 K 20.24 48.24 D1 12.40 22.70 0.232 N 23.68 H 16.20 37.50 E1 19.28 44.63 0.240 0 18.22 42.32 8 F1 28.64 66.30 0.244 P 1.64 3.20 13.00 30.09 --- Al 5.68 13.15 21.32 49.35 --- 3 12.60 29.17 THALLUS 3 1 29.40 68.06 c1 12.52 28.92 D1 18.06 41.85 E1 6.42 15.00 - 1.F. 6.02 14.07 - 01 12.24 2E.33 H1 20.02 46.48
NOTES * The n71 C ( mg Thallus Nitrogen )- hr; is based on 1 sq cm Felticera canina containing 2 H4 0.432 mg nitrogen. Earlier figures for Chlorophyll,, as 0D663 for 1 sq cm in 5 ml solvent, equalled 0.13 - C.15.
PHALLUS C 83
Ta')le 20. Variation in the nitrogenase activity ( C2H2 Reduction ) of Peltigera polydactyla thallus* using 1 sq cm discs.
nM C H nM C H Phycobiont Cells x 106 Total Phycobiont Percentage 2 4 2 4 6 7eretative Hetercoyst Cells x 10 Heterocysts 6 (eq cm)-1 hr-1 1x10 Heterocysts
9.04 0.10 10.02 1.80 1.20 6.66
5.58 0.20 5.78 3.46 3.47 17.35
12.12 0.44 12.62 3.49 3.79 2.61
9.82 0.28 10.10 2.77 3.81 13.61
8.30 0.32 8.70 3.68 3,87 12.09 14.20 0.40 14.60 2.74 4.11 10.28
12.08 0.30 13.18 2.40 4.27 14.23
12.60 0.20 12.00 1.56 4.56 22.80
11.12 0.60 11.72 5.12 5.12 8.53
6.92 0.40 7.32 5.46 5.15 12.27 F.44 0.22 8.66 2.54 5.20 23.64
7.22 0.32 7.54 4.24 5.60 17.50
1C.72 0.44 11.16 3.94 5.20 13.10
0.88 0.40 9.28 4.31 6.33 15.83
3.20 0.24 3.44 6.90 7.00 29.16
12.98 0.42 13.40 3.13 7.40 17.62
6.30 0.20 6.50 3.17 9.60 48.00
1i.44 0.36 10.00 3.33 14.40
5.30 0.72 10.10 7.13 15.20 21.11
15.16 0.46 15.72 2.95 16.00 34.78
11.30 0.46 11.76 3.91 21.07 45.80
12.14 0.40 12.54 3.19 24.80 62.00
::CTES * The thalli were all tested using a single gathering of material from Tentsmuir. Statistical Data. All zalculations are based cn 22 estimations. Mean Values Standard Deviations Standard Errors of the Mean 6 6 Vegetative Cells 9.54 x 10 2.93 x 10 0.62 x 106 6 6 6 Heterocyst Cells 0.35 x 10 0.13 x 10 0.03 x 10 6 6 Total Phycobiont Cells 9.29 x 10 2.99 x 10 0.64 x 106 nM C H 2 4 (sq cm)-1 hr -1 8.08 6.07 1.29 , ,-.1 nM C H 1x10( 6 Heterocystej 22.23 2 4 14.47 3.08 Percentage Heterocyst 3.61 ( Range 1.56 - 7.13 ) 84
culture, when related to heterocysts on a mg dry weight basis, the figures
became remarkably consistent.
Assuming the technique of estimating heterocyst numbers by
means of gives reliable results, it follows that there must be a Cr03' considerable physiological variation in the heterocyst population of any
given thallus specimen.
With lichens, growth is inordinately slow and the cells must
therefore remain in a viable state for a long time. Wilcox (1970) has
discussed heterocyst formation in rapidly dividing free—living cultures
of Anabaena and showed that a regular pattern was laid down in N—rich
cultures, which did not develop visibly till the algae were transferred
to a N—deficient medium and these N—rich proheterocyst cultures did not
fix nitrogen.
In this study it became obvious that in chains of cells in
which the heterocysts were spaced at fairly regular intervals (24, 25,
•28, 49, 26), a heterocyst could be expected to differentiate in the
middle of the block of 49 cells. In lichens it is impossible to follow
the conversion of a proheterocyst to a heterocyst. Growth would not be
affected, only differentiation and nitrogen—fixing capability, due to the
physiological cell state.
Wilcox, disagreed with Fogg's proposal, "that the formation
of a heterocyst from a vegetative cell occurs when the concentration
within the cell of a specific nitrogenous inhibitory substance, either
ammomia or a simple derivative, falls below a critical level", Fogg
(1949), since he observed heterocyst formation in 16 hours, prior to the
doubling time for the culture (24 hours), after transter to N—deficient
medium.
Kulasooriya Lang and Fay (1972) however, reported that 63
hours after initiation, was required to reach maximum cell activity, at 85 a constant 0:11 ratio. Whether these findings can be related to lichens is highly problematical.
It is now known that in many lichens, the heterocyst frequency is constant within narrow limits. Arising from the studies of
Galun, Paran and Ben—Shaul (1969), I suggest that at some stage a heterocyst will become inactive, though still clearly visible.
Variability in lichen metabolism makes comparisons extremely difficult. Smith (1963a), using Peltigera 221,yda2ILL.., and studying carbon fixation, cut out 7mm discs (38.3 sq mm area) and assayed as many as 40 — 70 in each sample. Kershaw (1974), measuring nitrogen fixation, suggested using "fairly extensive replication" to establish mean values, since he described variability as "this 'innate' factor in the 'system' which remains unexplained".
Variation in.heterocyst frequency and nitrogenase activity discussed above, would appear to answer the question as far as nitrogen fixation is concerned.
It has already been shown that single 1sq cm discs vary in nitrogen—fixing activity and when these are sub—divided, activity again varies from disc to disc (Table 21). Thus Smith (1960b), who selected uniform discs, cut them into quadrants and reselected the dissected quarters, may have achieved nothing. To achieve uniformity therefore, large samples may be the answer, though it can be envisaged that the results could be just as variable. In a comparison using large areas of
Peltigera polydactyla and P. aphthosa var. variolosa thallus, 10sq cm and
12sq cm respectively, no uniformity was imposed upon the system-(Table
22a, b). In view of this finding and the difficulty of obtaining
sufficient material to assay very large samples routinely, I decided to use single discs of thallus for most of the experiments.
It has already been suggested, that field conditions may have
Table 21.. Nitrogenase activity ( C2H2 Reduction) by a 1 sq cm disc of Peltigera carina and its subseqeent analysis.
nM C H Lichen Discs 2 4 ( sq cm )-1 hr-1 A 2.38
B 10.15
C 1.70
3 0.25 sq cm discs were cut out of Disc B above. Remainder of Disc B was put in to a fourth bottle. nM C H Disc B 2 4 (0.25 sq cm)—1 r-1
B1 0.81 ) ) ) B2 5.18 ) ). 9.81 ) B3 2.64 ) ) ) Remainder 1.18 ) Table 22a. Nitrogenase activity ( C2H2 Reduction ) by large samples of lichen thallus.
Peltieura nclydactyla
nM C2H4 ( 10 sq cm Thallus )-1 hr
Sample A Sample B* Sample C* Sample D* Sample E* Sample F* Sample G*
33.60 28.00 132.80 31.08 62.40 47.97 3.00
32.40 53.50 35.52 6.66 30.00 31.37 9.60
31.60 3e.00 99.20 111.00 . 60.00 44.28 2180
71.6o 36.00 94.40 50.32 67.50 95.02 164.00
12.20 64.50 52.8o 26.64 79.50 54.61 95.00
12.00 ; 42.00 49.60 111.00 81.00 3.51 9.20
51.60 27.00 62.42 56.24 42.00 56.67 32.20
49.29 27.5o 44.80 93.54 - 7.01 '94c
112.79 49.60
45.30 39.56 69.68 60.80 62.06 42.83 43.03 (29.73 9.91)** (12.62 4.46)** (30.45 10.15)*4 (37.46 13.25)** (16.74 6.33)** (27.63 9.77)** (53.84 19.04)**
NCTES :7 Each series.of samples is a different collection of thalli from the same site, (Tentsauir).
* * The figures in brackets refer tc the Standard .Leviation and Standard Error of the Mean, of the Mean Value immediately above.
Table 22b. ::itrop.nasc activity ( 02112 Reduction ) by large samples of lichen thallus.
Peltir-era aphthosa var. yariclosa
T%rTACT THALLUS E7CISED CEFIILCZIA Oven Dry Weight Oven Dry Weight nM C ,s nM C 11. Total Cephalodia nil' C2114 Total. Cephalodi: 2-4 2-4 Cephalodia (4mg on Cephalodia)-1 Cephalodia (4mg CIA/ Cephalodia) (4mg GLU Cephalodia)-1 (12 Discs) 1 - (6 Discs)-1 -1 -1 (mg) hr-1 (mg) hr hr Time TO hr (a) (b) Time TO hr Time T24 hr 2. 8 195 15.55 ) 29.82 ) . 4.1 3C0 ( 195+105 ) 41.14 . 32.C9 c6 -s6 1. 105 16.4 ) 36.54 ) --' .- 25.83 2.4 134 54.03 49.00 ) 1019 4.0 244 ( 134+110 ) 64.99 1.6 110 28,20 58.19 '
) 116.83 2.0 99 4C.17 e8.5954.EE 4.0 190 ( 99+91 2.0 91 55.38 95.55 143.47
190 ( 110+80 ) 107.89 2.4 110 79.23 126.16 E5.18 • 3.5 1.1 80 46.91 48.58 ) 151.5E
*** Oven Dry Height** nM C H nM C H 2 4 2 4 (Pa) ( 32.24 pg Cephalodia )-I ( 1COmg Cephalodia )-I 1 1 ( Cephalodium )-i hr hr At T24 hr At T24 hr 14.36) 12.38 ) 26.74 0.57 165.90
17.91 14.55 32.46 0.91 267.98
20 20) 21.97 ) 42.17 1.23 358.68
21.22) 1.30 378.95 13.75 ) 35'57 11017: S - Each disc was 1 sq cm in area. The mean oven dry weight of the Cephalodiumwas 32.24 pg, and was calculated from columns (a) and (b) above. *** The calculated nM 0 H ( 100 mg Cephalonia )-l is approximately equal to 10 sq cm of Pcltigera carina or P. nolydactyla on an area basis. 2 4 The letters CX.1 refer to Oven Dry '.:eight. 89
tt specific effect on lichen metabolism, since they are frequently subjected to rigouro-us changes, which prevent optimum physiological activity. Hill and Smith (1972) moistened air dry thalli for 24 hours prior to assay and Smith and Molesworth (1973) showed lichen metabolism after rewetting air- dry thalli, was governed by the prior field conditions that the lichen was subjected to. Kershaw (1974) considered that 4e hours was necessary to "remove any possible physiological variability from the lichen material, produced by previous and variable field conditions".
Initial studies showed that material taken from the 'storage cabinet' had an increased nitrogenase activity with time, either during continuous incubation periods of 2 — 3 hours (Table 23) , or by repeating the same experiment 3 times, using the same lichen discs in each case
(Table 24) and it is seen that under these conditions, a variable increase occurred of up to 14% over the initial rate.
Subsequently the three Peltigera species ( P. canina, P. polydactyla and P. aphthosa var. variolosa) were assayed in order to observe increases in nitrogenase activity over several days. 10mM HCO3 added to the Allen and Arnon's medium as a pH buffer, was thought to be causing the change and may have affected P. canine, but as similar medium without HCO had a comparable effect on P. aphthosa var. 3 variolosa, it became evident that it was the continuous moisture level that was affecting the nitrogenase activity (Figures 3, 4 and 5). The fact that nitrogen fixation was still increasing after 10 — 12 days, negates the ideas of Smith and Kershaw. Since the activity of a number of thalli, though increasing, did not reach a common value after this period, or could not be relied upon to do so, while it was known that this influence could occur, it was thought unnecessary to give these long pre—treatment periods prior to assay.
The third variable noted in this study, was the observed effect Table 23. Increase in nitrogenase activity ( C2H2 Reduction ) by different lichens without added HCO3 ions in short term'experiments.
Collema fluviatile Leptogium sinuatum Polychidium muscicola
nM C H nM C H nM C H nM C H Total Nitrogen 2 4 nM C H Total Nitrogen 2 4 nM C H Total Nitrogen 2 4 2 4 2 4 2 4 ( mg Thallus )-1 Time 1 ( mg Thallus )-1 Time -1 Time ( Sample )-1 ( Sample )- 1 ( Sample )-1 ( Sample )-' ( Sample )-4 ( Sample )-1 ( mg Thallus ) (m) ( Nitrogen ) (m) ( Nitrogen ) (m) ( Nitrogen ) 1 (mg) hr-1 (mg) -1 (mg) hr hi"1 hr-1 hr hr- -I
2.28 0.866 2.63 5.25 0.404 12.99 18.75 1.750 10.71 60 2.32 1.089 2.13 60 3.3o 0.398 8.29 60 17.25 1.570 10.98 1.88 1.027 1.83 5.85 0.378 15.48 15.19 1.540 9.86
3.58 4.13 5.95 14.72 42.85 24.49 120 3.40 - do - 3.12 120 3.60 - do - 9.05 120 38.81 - do- 24.72 2.65 2.58 7.00 18.52 38.10 24.74
3.30 3.81 6.93 17.15 180 4.10 - do - 3.77 180 3.73 do 9.37 3.10 3.02 7.07 18.70
Table 24. The effect on nitrogenase activity ( C2H4 Reduction ) by repeating the experiment twice.
------Experiment I ___------Experiment II Experiment III nM C H nM C H nM C H 2 4 Percentage 2 4 Percentage 2 4 Percentage ( 1 sq cm y-1 ( 1 sq cm )-1 ( 1 sq cm )-1 Increase hr-1 . Increase hr-1 Increase hr-1 •
29.12 0 32.32 0 27.54 0
31.84 9.34 36.18 11.19 30.82 11.19
29.90 1.03 36.86 14.05 29.54 7.26
values at the start and end of each experiment was 26.25 x 105 The NOTES — The mean of the 18 C2H2 . Standard Deviation was 1.46 x 105 and the Standard Error of the Mean 0.34 x 105 The discs of Peltigera canina were out out and assayed in 4% acetylene. The same discs were \ 0 used 3 times. The bottles were evacuated and given acetylene prior to each test. The tests lasted 60 minutes. 32
28
24
20
16
12
8.
4* iti
U 0 4 6 10 12 14 16 18 Ea'. 20 rays
12 •
8 •
4
00 1 2 • 3 4 Ave 5cooliCS. Figure 3. Variability ind4 Pel irera polydactyla thallus nitrogenaco activity ( C2H2 Reduction ),
with time, in the presence (A) and absence (H), of HCO3 — ions, 1g/l. (pH 8.4) Standard DeviatiOns indicated by vertical lines. 2 4 • Days sat4.-11112A Figure 4. Variability in^ Peltiger canina thallus nitrogenase activity ( C2H2 Reduction ), with time, in the presence (A) and absence (3), of HCO3 ions, 1g/l. (pH E.4). Standard Deviations indicated by vertical lines. 28 "
24
20
16
12
10 12 14 16 18 Days
20
16
12 Addition of HCO — ions 3
2 3 4 5 6 7 8 Days sam.ettg cf Figure 5. Variability in Peltivera aphthcsa var. variolopa thallus ( cephalodial ) nitrogenase nativity ( C2H2 RedUctien ), with time, in the presence (A) and the absence (B), of HCO3 ions, 1g/1. (pH 8.4). Standard Deviations indicated by vertical lines. 95
when using different concentrations of acetylene. Various concentrations
* were tested to see whether the 10% acetylene substrate concentration used
by other workers, was necessary and also to see what affect higher
concentrations had.
The nitrogenase activity of both Peltigera canina and P.
aphthosa var. variolosa, in 4% acetylene was rapid and an increase of up
to 10% or 20%, had no more effect on P. canina than had been achieved, by
repeating the experiment several times, i.e. approximately a 14% gain as
before, (Table 25a). However, with P. aphthosa var. variolosal large
increases of up to 80% were recorded (Table 25b), indicating that lichens
behave differently and it may well be that some, or all of the lichens
tested (Table 14), would have showed greater nitrogenase activity, with
higher concentrations of acetylene under the conditions applied.
As fixation was rapid when 4% acetylene was injected in the
presence of air, this method was used routinely for all assays. Table 25a. The effect on nitrogenase activity ( C2H2 Reduction ) by changing the acetylene concentration in 1 sq cm discs of Peltigera canina.
--- Evacuation and'4% C2H2 Air and 20% C2H2 Mean nM C H Mean nM C H 2 4 Mean 2 4 Mean Number of -1 Percentage cm )-1 Percentage Estimates ( 1 sq cm ) ( 1 sq -1 Increase 1 Increase hr hr- 18.61 21.80 16.61 5 ( 5.12 2.28 )r ( 6.15 2.75 )* ( 11.22 23.64 )**
••••■■•■•••■• Air and 4% C2.4.2 Air and 10% C2H2 Air and 20% C2H2 Mean nM C H Mean nM C H Mean nM C H 2 4 Mean 2 4 Mean 2 4 Mean. Number of sq cm )-1 Percentage ( 1 sq cm )-1 Percentage ( 1 sq cm )-1 Percentage Estimates ( 1 Increase Increase -1 Increase hr71 hr- 1 hr
11.31 12.84 14.56 13.93 15.39 •••••11011 ( 6.50 2.30 )* 6.82 2.41 f( -6.50 40.00 1* ( 8.09 3.11)*(-26.00 36.51
MOPES :- Refers to the Standard Deviation and the Standard Error of the Mean. ** Refers to the .limits of the Mean Percentage Increase.
Table 25b. The effect on nitrogenase activity ( C2H2 Reduction ) by changing the acetylene concentration in 1 sq cm discs of Peltigera aphthosa var. variolosa.
Air and 4% 0A2 ------Air and 17% C2E2 ------Air and 34% C2E2' Mean nM C H Mean nM C2H4 Mean nM C H 2 4 Mean Mean 2 4 Mean Time Number of cm )-1 ( 1 sq Percentage ( 1 sq cm )-1 Percentage ( 1 sq cm )-1 Percentage (Days) Estimate:: Increase -1 Increase _1 hr hr-1 hr
29.82 0 6 19.58 49.33 29.20 51.75 ( 6.05 2.47 )* ( 11.10 4.53 )* ( 32.14 63.16 )** ( 8.07 3.29 )* ( 27.70 76.39 )**
16.27 22.62 37.65 20.43 27.20 11 •••••• ( 2.14 0.87 )* ( 2.97 1.21 )* ( 18.00 63.41 )** ( 2.86 1.16 )* (-16.00 57.32 )**
Air and 41. C2:a2 Air and 10% C2E2 ------Air and 20% C2212,-----
Mean nM C H Mean nM C H Mean nM C H 2 4 2 4 Mean 2 4 Time Number of Mean Mean ( 1 sq cm )-1 Percentage ( 1 sq cm )-1 Percentage ( 1 sq cm )-1 Percentage (Days) Estimates Increase Increase -1 -1 1 Increase hr hr hr
18.90 34.13 83.47 33.70 79.48 0 ( 3.06 1.25 )* ( 3.47 1.42 )* ( 60.19 130.26 )** ( 4.71 1.92 )* ( 62.96 103.95 )**
NOTES :- * Refers to the Standard Deviation and the Standard Error of the Mean. ** Refers to the limits of the Mean Percentage Increase. 98
Section 4 NITROGEN FIXATION — USE CF 15N 2 4.1.1 INTRCDUCTICN
Biological fixation of nitrogen is a process of great
importance in agriculture. Donald (1960) has quoted a figure of 100 x 106 -1 ton annum which can only be approximate, but it probably far outweighs
that from other sources.
Interest in the process has increased recently for several
reasons and from the intensive studies, a number of fundamental advances
have been made, e.g. reliable enzyme preparations, simplified analytical
techniques and the realisation that there is a common enzymatic
mechanism amongst the wide diversity of nitrogen—fixing organisms.
Two indirect methods have been employed in the assay of
nitrogen as opposed to the nitrogen—fixing system and the earliest of
these was visual assessment. It was not quantitative, but was good fOr
screening organisms and is a suitable historical starting point. Complete
absence of combined nitrogen should have been essential. This was very
difficult to achieve and not always desirable. It has been found that
some organisms can make considerable growth, on undetectable traces of
nitrogen in the medium, or from combined nitrogen in the air and store
enough to continue growth in fresh culture.
However, some organisms appear incapable of adapting from a
non—fixing condition to a state where fixation is possible, directly.
Growth has to be started on a limited nitrogen source and when this is
exhausted, the organism adapts to fixing elemental nitrogen.
Another method that appeared to be reliable, but in fact can
give dubious results, was the direct estimate of an increase in total
nitrogen by Kjeldahl analysis. Many claims for fixation were made and it
has needed modern techniques to prove these claims false, for example see 99
Millbank (1969, 1970).
It has been suggested that increases of at least 1% of the total nitrogen, are necessary to show fixation and this may be only satisfactory in those systems that fix nitrogen rapidly. However, where there is a heterogeneous system, such as in seeds or root nodules, such increases may fall within experimental errors and it has been pointed out that much cross control should be introduced, together with statistical analysis, to establish what reliance can legitimately be placed on the results, (Wilson 1940, 1951).
A direct method of measuring fixation, that has been used in the past with some success, is manometry and it is still occasionally used. It was never widely accepted, but unlike the limitations of the total nitrogen method by Kjedahl analysis, it could overcome the presence of heterogeneous nitrogen products. It involves gas analysis at the beginning and end of an experiment, but is not very precise, since the amount of nitrogen used in relation to oxygen, by anaerobic organism is very small.
Some improvements have been made. Mortenson (1964) has shown that in certain circumstances, the method could compare favourably with the available isotopic method. He used a cell—free preparation, measured the total nitrogen taken up and showed that,
N 3H 2 NH 2 27 3
Another direct method which was initially developed between
1940 and 1950, uses the Mass Spectrometer. Burris (1972), stated that .
15N is a sensitive and specific tracer and furnishes a method to which others should be referred. There is a strong tendency to use hydrogen evolution, or acetylene reduction, as sole indices. Although the latter 100 are sensitive and useful, they are indirect and their use is to be viewed 15 with reserve. They should be compared with ammonia formation, or N2 fixation, to establish with the operator that his results are valid in terms of nitrogen fixation.
With the 15N method, great accuracy is achieved. It is at least 1,000 times as sensitive as Kjeldahl analysis and being a direct sampling method, heterogeneous materials may be assayed. If an organism is supplied with 60 atom % excess, the accumulation of 0.015 atom % excess, the accepted value, would require an increase of only 0.025% of the total nitrogen, compared to greater than 1% for the Kjeldahl method.
The use of stable isotopes (Table 26), i.e. those that do not give rise to radioactive emanations, for investigating biological problems in which they are employed as tracers, require an analytical procedure that should 1) be sensitive to small variations, 2) be capable of
Table 26. Natural abundance of the stable isotopes of bioligical importance.
Element Isotope Natural Abundance % 1H 99.98 Hydrogen 2H (D) 0.02
14N 99.63 Nitrogen
15N 0.37
160 99.76 Oxygen 18 0.24
handling small samples and 3) not be time consuming. The mass spectro- 101 meter is usually employed. Normally the element to be assayed, must be converted into a gaseous form I) which should be simple to produce from organic compounds, 2) which should have a simple structure, both from a molecular and isotopic viewpoint and 3) which should be readily pumped out of the mass spectrometer, i.e. it should not be adsorbed to the structural components of the machine.
The mass spectrometer separates, by an appropriate combination of electrical and magnetic fields, a beam of ions into a spectrum, dependant on their masses, the abundance of individual components being calculated by determining the magnitude of the current produced, by the positive ions falling onto a collector electrode (Figure 6).
The gas sample is introduced into the ionising chamber, from a resevoir, at a constant rate to maintain a pressure of 10-5mm Hg, which is achieved by a constriction (sintered silica) in the system,. through which the gas flows.
In the chamber a heated tungsten filament emits electrons, which bombard and thus ionise the sample molecules. The randomly moving non—ionised molecules are pumped away and the positively charged ions are directed towards the uniform magnetic field, by negative potentials on electrodes in the ion source.
In the magnetic field, the ions move in a circular path, the radius of which depends on the mass and energy of the ions. The path of an ion in the mass spectrometer is given by,
m 4.62 x 105 . B2 r2 V
where :— mass of the ion in atomic mass units
e = number of electronic charges lost in ionisation 102
ICV SWRCE
Gas sample inlet Trap Bombarding electrons Filament
Parallel ion beam
The whole apparatus
is connected to a
vacuum system and
the source, magnet MAGNET POLES and pre—amplifiers
are associated with
stabilised power units
Divergent ion beam
Lighter ions Heavier ions
Collector slit
Pre—amplifiers No2 Outputs from collectors
COLLECTOR
Figure 6. Schematic diagram of Mass Spectrometer. 103
B = magnetic field intensity in auss
r = radius of curvature of the ion path in cm
V = accelerating potential in volts and it is evident that if B and V are constant, the radius of curvature r, of a given ion, will be proportional to itsmass to charge m ratio. e Heavier ions will thus follow a path of greater radius than lighter ones,
and in this way the ion beam can be separated into components and by adjusting the accelerator voltage, or magnetic field, each can be focused to fall on the collector plate. If there is more than one collector
plate, then two mass species can be measured simultaneously and their ratios determined directly. With nitrogen, one plate collects ions of
mass 28 (14N — 14N) and the other, ions of mass 29 (14N — 155N). It is essential in a system of this type, that constancy and stability are
achieved. Millbank, (pers. comm.) states: "It will be seen that if the
sample gas is admitted at a constant rate to the ion source, bombarded by
a constant intensity beam of electrons, it will be ionised to a constant
proportion and if accelerated by an unvarying field, a constant beam of
positive ions will enter the tube. If this is then deflected by a
constant magnetic field, the intensity of the beam striking the collector,
will be constant and this ion current, if amplified by a stable amplifier,
will give a constant reading or display, proportional to the partial
pressure of the gas, in the original resevoir. Thus the entire
instrument depends on the stability of the electronic systems and
reliability of the vacuum pumps, for consistent operation."
Material to yield 0.1mg nitrogen is sufficient. Smaller
samples may give rise to spurious enrichments, due to molecular flow in
the mass spectrometer. However it depends on the instrument, the inlet
system fitted and the obvious though essential insurance against gas
leaks, particularly when using highly enriched nitrogen gas (contamination
104
15 by air of 95(/: enriched N, is far worse than that. to 30%. or 5%).
The sample is converted to ammonia by Kjeldahl analysis and then to nitrogen by lithium hypobromite (LiBrO), rather than NaBrO,
See Ross and Martin (1970) . In more complex systems, where it is required to see which N atom of a complex amino acid etc. is labelled, prodedures may be adopted to remove N atoms one at a time.
Estimation of abundance is calculated from the relative difference of the ion currents of the two species in nitrogen gas, produced when the ions are discharged at the collector plates. The currents are amplified and displayed, either on a galvonometer or 15 graphically. In all natural nitrogen, the N isotope occurs at a concentration of 0.36 — 0.37%, with the molecules being composed as follows :— 14 14N 15N 15N 14N N 5N due to random collisions given by the relationship, for 14 14 N N
14m 15N 2a(1 — a) where a is the fraction of 5N
15N 15N a 2
The atom % is therefore calculated from the following, i.e.
100 i 28 : 29 a ct = 2R + 1
200 29: 30 % R + 2
100 a of = 28 : 30 1 +N where R is the ratio of ion currents for two mass species.
The atom % excess is obtained by subtracting the normal abundance from the calculated value and in the case of nitrogen, it can be assumed to be a positive reading if it is 0.003 atom % excess above 105
background, though for all practical purposes, as has been stated before,
a conservative figure of 0.015 atom 5 excess, is generally accepted. Various workers have differed in their ideas as to the amount 15 of nitrogen gas necessary for satisfactory N experiments. According to
Burris and Wilson (1957) "it is not necessary to use enriched nitrogen
comparable to that in air. Nitrogen fixers, fix nitrogen nearly as
rapidly at a pN2 of 0.1 atm as at 0.8 atm" and they suggest the following
gas mixes. For anaerobes, 0.1 atm nitrogen and 0.9 atm helium and for
aerobes, 0.1 atm nitrogen, 0.2 atm oxygen and 0.7 atm helium.
However Burris (1972) points out that the work of Strandberg
and Wilson (1967) should be observed. The pN established in the vessel, 2 will be governed by the Michaelis constant (Km) for the nitrogen—fixing
system. For most aerobic systems, 0.3 atm nitrogen is suitable together
with 0.1 atm oxygen, though he suggests that a control should be run to
•see if 0 a higher pN2 is needed to affect the rate and if so apply a
' factor.
Stewart (1967) also found with his in vivo field experiments
that there was an increase in fixation with increased partial pressures
of nitrogen to approximately pN2 0.6 atm.
While a partial vacuum, as suggested by Burris, may not be
detrimental to the in vivo system, there must always be the questioable
possibility of an input of air and while this may be calculated, as
Burris (1972) has described with field samples, which cannot be
completely evacuated, any leakage which might occur, could be prevented
by the addition of helium or argon to 1 atmosphere, which cannot be
questionable and would be an advantage. 106
4.1.2 METHODS 15 Preparation of N gas 4.1.2.1 2 15 N gas was prepared, using the method whereby enriched 2 ammonium sulphate ( (15NH ) SO . 7H 0 ) is converted to nitrogen by 4 2 4 2 sodium hypobromite (NaBrO), for preparation see Appendix 4, in an all
glass apparatus (Figures 7a, 7b). The high vacuum ground glass joints were
greased with Apiezon M and a vacuum was applied, by a rotary pump (A) to
give a holding pressure of 0.001 mm Hg. Provided the vacuum held, the
procedure was continued to generate the gas. 1.2g of the 15N—salt (either
30 or 97 atom % excess) was routinely weighed out directly into the side-
arm of a special flask (B), glass distilled water was added, the joint
greased, the mixture frozen and the whole attached to the main body of the
flask, to which NaBrO and potassium iodide (KI) had been added.
Sufficient NaBrO was used to oxidise the salt: It had been
calculated that with 14NH solution, 17m1 of oxidant was required to 4 •liberate the nitrogen gas, therefore to give a 30% safety margin, 22m1 of
NaBrO was used. This saved time during the de—gassing procedure of
freezing, evacuating and thawing the solutions twice. The freezing was
carried out using liquid nitrogen. 15 Once the NH + solution and the NaBrO had been de—gassed and 4 the gas handling system was functioning, the ammonium solution was tipped
very gently into the NaBrO, liberating nitrogen. The gas was dried in
the.cold trap (M) and stored in the collection flask (L), over a solution
of 20% sodium sulphate (Na2SO4) in 5 volume % of H2SO4. This removed any 15 Laces of NH not oxidised to nitrogen. For the detailed procedure of 3 the method, see Appendix 5.
4.1.2.2 Incubation flasks (Peltigera alzaLstyla experiments) Two systems were initially tried, Thunberg tubes and Erlenmyer
side—arm flasks, but discarded, see discussion p 118. A different system Figure 7a and 7b. The all glass apparatus for the production of 15N2 gas.
Trap P205 C
B
Double oblique bore High Vacuum Stopcook
Figure 7b. Straight bore High Vacuum Stopcock 108
was therefore used. A vacuum desiccator lid was taken and a small volume
base constructed of two laminated sheets of "Perspex" (thickness 0.25"),
the upper having a hole of similar dimensions to the internal diameter of
the lid, to form the well. These two parts, lid and base, were held
together with wooden buttons, washers, bolts and wing—nuts and separated
by an 0.25" neoprene rubber gasket. A square gasket well in excess of
the size of the desiccator lid was essential, to prevent distortion under
vacuum. The vessel had a total internal volume of 600m1.
According to Sch011horn and Burris (1966), a pC2H2 equivalent
to 4 mm Hg is sufficient to inhibit nitrogen uptake by 50%. The
possibility that neoprene might give of acetylene was investigated,
subject to the work of a colleague, on the evolution of ethylene from
neoprene. A lump of neoprene was cut from the same sheet that had been
used for the gasket, total surface area, 1.125 sq in, placed in a
McCarteney bottle, volume 26.75m1 and incubated over 24 hours. 6 Acetylene was produced, equivalent to 0.001 x 10 units in the test flask 6 after 7 days, the maximum test period. This figure of 0.001 x 10 units
equalled a concentration of 0.002% (0.012 mm Hg), which whilst not zero, 6 is insignificantly small compared to 3.93 x 10 units (4 mm Hg) and may
be ignored, particularly in view of the fact that it was a constant factor
throughout the course of the 37 day experiment.
Acetylene reduction was used as an assay method before and 15 after N uptake to determine activity of the lichen nitrogenase. Table 2 26 shows that by evacuation, the substrate concentration is lowered below
that suggested by Schbllhorn and Burris and that this is not detrimental
to the nitrogenase activity, since re—incubation in 4% acetylene showed
that in two instances, there was an-increased reduction rate. 15 4.1.2.3 Incubation flasks (general N translocation experiments)
Standard 100m1Erlenmyer flasks, closed with "Suba—seals"
Table 26. Nitrogenase activity ( C2H2 Reduction ) in discs of Peltigera canina using 4% C2H2 in air and its inhibition due to removal of the gas by evacuation.
nM C H can nM C_H ** 2 4 4 Mean Mean Mean Number of ( 1 sq cm )-1 ( 1 sq cm )-1 Acetylene Percentage Percentage Treatment Estimates Concentration Concentration Inhibition h1,71 hr-1 14.40 No Evacuation 3 15.00 15.60 28.5 x 105 17.40 3 1st Evacuation 3 0.54 31.8 x 10 0.0570 96,50
2nd Evacuation 3 0.06 58.5 x 102 0.0084 99.60
2 3rd Evacuation 31.9 x 10 0.0045 100.00 16.15 Re-incubation 3 15.23 13-.-96* 31.5 x 105 4.00 10.51 10.52
5 No Evacuation 3 12.76 26.0 x 10 4.00
3 0.0380 90.20 1st Evacuation 1.24 24.5 x 10
Schallhorn and 3.98 x 105 0.530 50.00 Burris (1967)
-1 -1 NOTES :- * while the figure for Mean nM C2H4 ( 1sq cm ) hr after re-incubation is lower, the actual rates of activity in two instances show an increase. ** Although the 4% C2H2 value varies slightly as gas chromatograph peak height units, it reflects small variations in bottle size, solubility etc. 110
were used, containing large areas of uncleaned thalli.
4.1.2.4 Evacuation and subsequent testing
With the 'desiccator—lid vessels, thalli were incubated in an
ex—field condition, with adherent moss and other debris present, enough
lichen being used in each vessel to cover the base completely. The
thalli were maintaind in a moist state by spraying routinely with an
HCO — solution (10mM) from a chromatographic sprayer. 3 The vessels were assembled and evacuated via a hypodermic
needle, pushed through a "Suba—seal" in the outlet, to a pressure of
0.01 mm Hg. It was not possible to achieve a better vacuum, due to the
water vapour coming off the lichen material.
The gases were introduced by manometry through the "Suba—seal"
as :
pN 2 0.33 atm
p0 0.20 atm 2 pAr 0.47 atm (balancing gas)
pC0 no more CO was added than that from the HCO 2 2 3 solution Two vessels were set up in this manner and tested at weekly
intervals, fresh gas being added each time. Flask 1 was tested on days
0, 3, 9, 16, 23, 30 and 37, whilst Flask 2 was tested on days 1, 6, 13,
20, 27 and 34. Incubation of the flasks in both series of experiments,
was in the lichen storage cabinet, set at 10°C and a light intensity of
5,000 lux, during a 16 hour day.
4.1.2.5 Nitrogen assay (Peltigera polydactyla experiment)
At the end of each experimental period, 4 discs (0.5 sq cm)
were punched out and tested for acetylene reduction, which both 15 terminated the N uptake and gave a record of activity. On the result 2 of the acetylene/ethylene assay, the discs were paired to try and achieve
uniformity. The algal—rich zone of two discs was dissected off, with a
111
sharp scalpel under a binoccular dissecting microscope, a simple though
time consuming process, since the minute pieces of the relatively brittle
zone can be picked off and come away cleanly, fracturing at the interface
with the fungal medulla and leaving a nearly clean white fungal layer. 4.1.2.6 Total nitrogen determinations The separated portions of the lichen material were placed in
respective boiling tubes and digested. For a full account, see METHODS
3.1.2.9, p 62.
4.1.2.7 Indicator for titration 2% alcoholic boric acid indicator (Appendix 6), was used to
collect the ammonia, as the end—point was clearer, when alcohol was 15 present. In the N analysis method employed, see below, there was no 2 possibility of alcohol carryover, which would have given a spurious mass
29 peak.
4.1.2.8 Contamination
Bacterial contamination, on or in the lichen thalli, was not
suspected during the experiment and the only precaution taken was to wash
the discs in glass distilled water prior to dissection.
4.1.2.9 15N2 analysis
A method was used which followed that of Ross and Martin
(1970). Subsequent to titration in the Conway units, the centre well
solution was lowered from pH 5.0 (the end—point) to pH 4.0, using a glass
micro—electrode and monitoring the change on a pH meter. Dilute
sulphuric acid was the acidifying agent. The solution was then
transferred to 5m1 glass vials and dried down at 60°C.
The inlet system of the Mass Spectrometer (Figure 8a), was
built as described, but was modified in that a "Rotaflo" capillary—bore
valve assembly, replaced the "Springham" tpye, described by Ross and
Martin. 112
the O-ring is pulled into the cone and foms a seal.
to mass npectrcrneter
Pir;uI'C 81.1. !Ipparatuo for the t':(meration of 15)12 from a di,r:(wted lichen nronple, as 15N_ amrnonium borate complex. 113
Lithium hypobromite solution (more stable than sodium hypobromite and therefore does not require the addition of potassium iodide, Ni, to prevent the liberation of oxygen) was introduced under vacuum to the reservoir and stored under helium gas. For details of the operation of the inlet system, see p 115 and Figure 8b.
A freezing bath of ethanol/solid CO2.( — 80°C ), which gave 15 controlled cooling, surrounded the sample vials in which the N was 2 generated. Another cold trap of liquid nitrogen ( 196°C ) surrounded a glass tube in the inlet system, to collect water vapour, the presence of which in the Mass Spectrometer would have caused spurious results. 114
'Source'
To gas Generating plant
Figure eb. Inlet system to mass spectrometer. 1.15
OPERATIM OF THE INLET SYSTEM FOR THE INTRODUCTION CF 15 GENERATED N TO THE MASS SPECTROMETER 2
Between conversions, valve inlets 1, 2 and 3 are open, 4 is closed. Outlet of the high vacuum stopcock, marked with a spot, is at position A. (N.B. Always turn stopcock anticlockwise).
For operation, close valve inlet 2, attach sample vial and turn stopcock to position B. The vial sucks in. Check vacuum during this time and focus the mass spectrometer.
Open valve inlet 2, turn stopcock to position C.
Open the 'Rotaflol valve (see Figure 8a) to admit 0.5 — 1.0m1
(c. 20 drops) of lithium hypobromite solution.
Immerse the sample vial in a solid CO2 / ethanol freezing bath. Close valve inlet. 1; close valve inlet 3. Turn stopcock to position D. Admit the sample by opening valve inlet 4; check deflection and close valve inlet 4. Close valve inlet 2. Estimate the isotope ratio.
Remove the solid CO2 / ethanol freezing bath from around the sample vial. Turn the stopcock to position A.
Open inlet valve 3, open inlet valve 4.
Remove the sample vial by lifting the lithium hypobromite
vessel and thus breaking the vacuum.
Remove the lithium hypobromite vessel; clean the capilliary an
and capilliary outlet with vacuum line and distilled water, since it
might be contaminated and replace it on the inlet system.
Open inlet valve 2; open inlet valve 1 and close inlet valve 4. 116
15 4.1.3 DISCUSSION OF THE N-UPTAhE EXPERIMENTS The assay of nitrogenase activity by the uptake of the heavy
isotope of nitrogen, is not often used, because it is cumbersome, time
consuming and less sensitive, compared to the acetylene reduction
technique.
However, it is the only method for definitively demonstrating
nitrogen fixation. Bond and Scott (1955) were the first to use the method
for the assay of nitrogen fixation in lichens and Millbank (1972) and
Millbank and Kershaw (1973), showed that unlike the presupposed idea of
sluggish activity in these organisms, due to their slow growth rates,
some of their metabolic activities are rapid.
In determining translocation of fixed nitrogen, an experiment
was initially carried mit, using four different lichens, two foliose
species and two with cephalodia and the results (Table 27) show, that
nitrogen is fixed by the blue-green alga and transported either to the
fungus in the case of Sticta limbata and Pseudocyphellaria thouarsii, or
the main thallus in Lobaria anplissima and L. laetevirens. In the latter
case the green algae were not sepagted from the fungus, to see if the
15N had been translocated there as well. This has been considered by
Kershaw and Millbank (1970).
The experiment showed that nitrogen was fixed, but that
nitrogenase was substantially reduced in activity over the long test
periods, by assay with acetylene reduction.
Dissection of the foliose species was difficult, though the
method was considerably easier with Sticta limbata, due to the flatness
of the thallus, so of the Peltigera species, P. polydactyla was chosen,
as it was comparatively easy to aissect and fairly abundant in the field.
In this study, great difficulty was met at first, in trying
to maintain fixation rates over long periods. Discs of thalli which were
N uptake over 19 days. Table 27. Nitrogenase activity measured by acetylene reduction and 15 2
5N Analysis at Day 19 ** 2 nM C H Total Total pg Nitrogen 2 4 Mean Percentage Total Time Sample Nitrogen Nitrogen Lichen ( Sample )-1 Total Nitrogen Percentage Increase Increase (days) Size in Each Increase ( 1 sq cm ).-1 Fraction ( 1 sq cm )-1 hr-1 (pg) (pg)
247.1 A1/2 42.5 0.81 1.69 1.92* 0 21.53 F1/2 57.5 0.75 2.14 Pseudocyphellaria 3 sq cm thouarsii 1.29 3.12 n d A3/4 42.4 3.30* 19 F3/4 57.6 1.06 3.48
A1/2 46.1 1.18 1.55 1 . 48* 0 316.53 141.8 P1/2 53.9 0.91 1.39 Sticta Area of limbata Thallus 1.23 1.79 32.42 144.1 A3/4 50.3 1.67* 19 F3/4 49.7 1.07 1.54
od * Cephal ia 0.83 0.05 0 11.72 6.1 Lobaria 3 sq cm laetevirens Total Thallus 0.15 0.93 0.31 19 2.34 620.5
External Cephalodia -- 0.33 2.79 -- (a) 846.4 Lobaria*** 0.5 cm from (a) amplissima 0 26.44 -- 0.66 4.10 4.10 Area of 211.7 with Thallus 2 2.0 cm from (a) -- 0.92 2.91 2.91 Dendriscocaulon 19 5.50 316.1 umhausense 4.0 cm from (a) -- 0.26 0.56 0.56 216.3
Pseudocyphellaria thouarsii and Sticta limbata was carried out using 2 sq cm thallus,each being divided in half. NOTES :- The 15N2 analysis of ** The symbols A1/2, F1/2 and A3/4, F3/4 refer to the thallus fractions 1,2,3 and 4 and are the separated algal and fungal "zones". *** The internal cephalodia of Lobaria amplissima were not completely analysed, the Total Percentage Nitrogen Increase over 19 days was 1.509. **** The cephalodia of Lobaria laetevirens as with L. amplissima are internal 118 incubated in various gas mixtures, showed rapidly reduced activity over short periods of time, which was revealed by means of acetylene reduction assay. In the first two experiments, discs were cut out, assayed for acetylene reduction and incubated in Thunberg tubes at various 24
hour intervals, up to 26 days. In both cases the results were erratic
and in neither were the results the same. In the first experiment, there 15 was a rapid initial uptake of N over 4 days, with no further increase 15 over the next 21 days and in the second experiment, the uptake of N
was much more gradual, but basically continuous.
At the end of the second experiment, the inactive discs were
tested for recovery. The results show (Table 28), that there was slight
activity in three quarters of the discs after two days from the end of
the test period and after a further fourteen days, the thallus activity
was equal to the original rates.
This indicates that the loss of activity was due in some way
to the techniques used. Various aspects of the previous experiments
were therefore tested, to try and determine if they were the cause.
An experiment was prepared as before. Residual acetylene was
removed by evacuation (Table 26; p 109), which even if held for 24 hours
to 1 atm (Table 29), had no effect at all and could be ignored. It 15 gas was the toxic factor (Table 29); however, it alSo seemed that N2 emerged in further tests upon different discs of thallusu using the
identical gas, that, except for in one case, there was no toxic effect. 15 N was not the cause of trouble and It was therefore assumed that the 2 it was presumed that it could have been over saturation by the medium,
used to maintain high humidity; or light; or the physical disturbance of
cutting discs. Light was not seriously considered as deleterious. the
experiment was carried out under a light intensity of 5,000 lux, which
Table 28. The effect on the nitrogenase. activity of incubation in 15N2 gas using discs of Peltigera polydactyla thallus and recovery after removal to air.
■
Pre- and Post-incubation Post-incubation Recovery
111•1111■MOOMMIIMMOM in 15N2 gas in air
nM C H nM C H nM C H rim C H nM C H 2 4 2 4 2 4 2 4 2 4 Sample ( 1 sq ccm )71 ( 1 sq cm )-1 ( 1 sq cm )-1 ( 1 sq cm )-1 ( 1 sq cm )-1 hr-1 hr-1 hr-1 hr-1 hr-1
(Day 0) (Day 24) (Day 2) (Day 4) (Day 16)
(a) 19.88 0 0 0 19.44
14.90 0 0.96 1.39 11.10
6.56 0 0.34 0.81 6.14
4.20 0 0.14 0.23 1.02
NOTES * The thallus discs were maintained in a moist condition on filter paper in Petri dishes. ■ID The light regime and temperature were also maintained.
15 Table 29. Evacuation or N2 gas as possible reasons for the inhibition of nitrogenase activity as measured by acetylene reduction using Peltigera polydactyla thallus." nM C2H4 ( 1 sq cm )-1 hr-1
Gas Gas hr 72 hr 9¢ hr .144 hr Phase Phase
45.0 20.0 17.0 1.0 23.3 21.0 23.0 11.0 4.0 Air 34.5 34.0 31.0 48 hr in + 23.7 16.0 24.0 1.0 15N/02/Ar/CO2* 28.0 21.0 21.0 1.0 2H2 21.0 25.0 29.0 11.0 36.5 36.0 30.5 1.0
nM C2H4 1 sq cm )-1 hr-1
Gas Gas Gas hr hr 144 hr 240 hr 336 hr Phase 72 Phase Phase
16.0 10.4 9.0 9.0 96 hr in 16.0 4.0 8.8 11.5 16.0 22.0 14N2/02/Ar/CO2 Air 21.0 20.5 34.o 8.8 13.o 96 hr in 15-2/ 02/Ar/CO: 0.8 1.6 4.0 9.0 96 hr in 9.5 1.6 3.2 10.5 16.0 17.0 15N2/02/Ar/C0; 5.3 6.8 7.0 5.5 5.5
nM CH( 1 sq cm )-1 hr-1 24 Gas Gas Gas Gas 0 hr 72 hr 96 hr 144 hr 16e hr 240 hr 264 hr 312 hr Phase Phase Phase Phase (48 hr) Air 14.0 12.4 15.0 27.5 24 hr 19.0 Evacuation 17.0 17.0 14N2/02/Ar/CO2 18.5 + + 15 10.5 11.0 Evacuation 12.0 72 hr Air 8.5 11.0 N2/02/Ar/CO24. 1.0 C2E2 15.0 10.4
0 and was stored in a vial over the usual ammonia absorbant. NOTES :- All the 1 E2 was prepared from a single batch of (15NH4)2504.7H2 ON ** Different lichen discs were used for each experiment, but were all from a single gathering. 121 was comparable to that encountered in the field for this species.
In the fourth experiment, large areas of thallus were incubated in 100m1Erlenmyer flasks with side arms, again with similar loss of activity and it therefore does suggest that over saturation is the reason. This factor was also found to affect activity in other long term experiments, i.e. when incubating discs of thallus, with and without
and which was overcome by bedding the thallus on sheets of filter HCO3 paper, in the perti dishes containing the medium.
With Feltigera alohthosal using 8 sq cm areas of thallus,
Millbank and Kershaw (1969) had no apparent difficulty in maintaining the thallus in a viable state for up to 10, 13 and even 25 days and Kershaw
and Millbank (1970), maintained the thallus in a viable state for 55 days.
Bond and Scott (1955) and Watanabe and Kiyohara (1963), also used large
areas of thallus, Watanabe and Kiyohara using as much as 25 sq cm per
sample.
It had already been noted that lichen thalli could be
maintained at high temperatures (25°C) for a considerable period of time
(Figure 9), using acetylene reduction as the assay method. In the fifth
experiment (Figure 10), temperatures of 12°C and 25°C were used. There
appeared to be little correlation with the nitrogen-fixing activity of
the thallus, being little affected in this temperature range. This was
demonstrated by two series. In one series the rate at 25°C was 5-fold o that at 12 CI but in the other series, the data showed that the thalli
at 12°C were 3-fold more active.
In the final experiment, the experience obtained from the
previous trials was used and the thalli were tested in a natural state in
the large desiccator vessels (see METHODS 4.1.2.2 p 106). The thalli
were not disturbed beforehand and acetylene assays were only carried out
subsequently. It was assumed that the thalli which always looked 122
1 2 3 4 5 6 8 Days
Figure 9. The effect of continuous high temperature (25°C), 12mM MC03 and light, on nitrogenase activity ( C2H2 Reduction ), in disco of Peltigera canina (C) and P. polydactyla (0). 24
0 , 12 C. (A)
12°C.C (B)
10 12 14 16 18 '20 22 24 Days Figure 10. A comparison of different temperatures on 15N2 uptake by two samples of discs of Peltigera oolydactyla thallus. 124
perfectly healthy and dark blue—green, as they had done in all previous
tests, fixed nitrogen. This method proved satisfactory and discs with
approximately similar nitrogenase activity (see Figure 11 : Series I and
Series II), were used.
When one considers the various influential factors during the
total experimental period, they fall into two classes, those which are
not randomly variable, i.e. light intensity, temperature, moisture level,
gas composition, thallus size, day length and testing periodicity and
those which are either changing, or are inherently variable, i.e. total 15 combined nitrogen content, acetylene reduction activity, N 2 assimilation, cell activity, nitrogen transport rate, dissecting ability
and thallus composition, all of which can therefore affect the results.
Figure 11 : Series I and Series II, show the results of
duplicate flasks (desiccator vessels), with nominally identical lichen
material, given the same light intensity, moisture level, temperature and
' gas composition and it will be seen that the activity of one series of
discs (Figure11 : Series I), is about half that of the other series
(Figure11 : Series II), the results being based on 4 x 0.5 sq cm discs, -1 dissected, analysed and the means found (sq cm)
In order to minimise such variable factors as total nitrogen -1 content, or acetylene reduction, the results are related to activity mg 1 algal, fungal and thallus nitrogen and mg— thallus nitrogen (mean units N -1 of nitrogenase activity) but it is seen in Figure11 : Series I, Day 20,
that the high nitrogen value, while only slightly affecting the mean -1 percentage increase (sq cm) 1 has a marked affect on the nitrogen - assimilation (mg thallus nitrogen)N 1. A similar trend is noted in Figure
11: Series II, Day 30, but this is due to a general fall off of activity
and the result of calculating the mean of two discs, of which the
individuals vary. 125
0 3 6 9 12 is to 21 24 27 33 36 39 Days
Series I
(A) Mean increase in Algal Nitrogen (1 eq om)-1 (3) Xenn Inoreaee in Fialgal Nitrogen (1 eq em)-1 (D) (C) Mean Total Nitrogen Increase (t eq cs)-1
(A1) Algal Nitrogen incrusts, (1 eq cm)-4 (mg Algal Nitrogen) 1. (21) Fungal Nitrogen Increase (1 sq cm) 1 (mg Fungal Nitrogen) I 1 (C1) Absolute Total Nitrogen Inoreaee (1 sq em)-1 (mg Thallue Nitrogen)
-1 -1 (D) Absolute Total Nitrogen Increase (1 eq cm) (mg Menu. Nitrogen) (Mean Unite of Nitrogenase Activity)-1
a
16
9 12 15 18 21 Days
Figure 11. 15N2 uptake by discs of Peltigere polydactyla thallus.
20 126
16
12
0 3 6 9 12 15 le 21 24 27 30 33 36 39 Days 72
66 Series II
(A) Mean increase in Algal Nitrogen (1 sq am)-1
(B) Moan increase in Fungal Nitrogen (1 eq cm)-1 64 (C) Mean Total Nitrogen increase (1 eq cm)-1
(A1) Algal Nitrogen Increase (1 eq cm)-1 (mg Algal Nitrogen)-1
60 (B1) Fungal Nitrogen increaea (1 eq cm)-1 (mg Fungal Nitrogen)-1
(C1) Absolute Total Nitrogen increase (1 sq cm)-1 (mg Thallus Nitregen) 1
(ci) (D) Absolute Total Nitrogen Mcrae.. (1 eq cm)'i (mg Manuel Nitregen) 1 56 (Mean Unite of Nitrogenase Activity)-1
52
(NI)
44
4
36
32
2E
24
20
(c)
16
12
a
3 6 12 15 16 21 24 27 30 33 36 39 rays
Figure 11. 15112 uptake by discs of Feltigera thallus. 127
Nitrogen was assimilated throughout the whole test period in both series, though whereas in the first series (Figure 11 : Series I) there was low activity with increased uptake in latter samples, the opposite was the case in the second series (Figure 11 : Series II).
In neither instances did the observed increases, expressed as 15 N -1 percentage increase in N (sq cm) versus nM correlate with the 2 C2114 calculated expected value, based on the initial reading. Maximum assimilation rates occurred at the end of each total period, being 75% of the expected rate (Figure 12: Series I), between days 30 and 37 and 48% of the expected rate (Figure 12 : Series II), between days 20 and 27.
Variability in thallus nitrogenase activity has been noted by other workers. Watanabe and Kiyohara (1963), incubated various lichens 15 with N for 5 weeks and in only some of their samples, after this 2 period, did they measure uptake / as atom % excess, which though very low, was according to them above experimental error. Particularly odd are their results with Peltigera polydactyla, where they showed uptake in the thallus and none in the blue—green algal isolate (Nostoc punctiforme); none in the thallus of Peltigera virescens, but activity in the isolated phycobiont and no activity in either the thallus or the isolated algae in
Collema subconveniens, Nephroma tormentosum or Stereocaulon japonicum.
They do not record these species as having a blue—green phycobiont, though they do have blue—green algae in the thallus or cephalodia.
Burris and Wilson (1957) have shown in short term experiments 15 that after the initial uptake of N , there is a slowing down process, 2 though this will probably not be noticeable during long periods of
incubation, unless this lessening activity is continued. Whether the findings of Burris and Wilson can be used to explain the results of
Figure 12 : Series I and II, is not certain, because in this system, the
lichen thalli that were used, have proved to be extremely heterogeneous. 128
26 (A)
24 Series I
(A) Calculated Expeoted Increase (B) Observed Increase 22
20
18
16
2 14
Sr) 12 U U
10
8
6
(B)
4
2
4 8 12 16 20 24 28 32 36 Days
Figure 12. Observed and calculated expected total percentage increase in nitrogen.
( 1 eq cm )-1 (mean unite of nitrogenase activity )-1. 1 29
Series II
(A) Calculated Expected Increase (B) Observed Increase 44
(A)
40
36
(B)
4 12 16 20 24 28 32 36 Bays
Figure 12. Observed and calculated expected total percentage increase in nitrogen
( 1 eq cm )—1 (.mean units of nitrogenaoe activity )—1 130
Total nitrogen in the phycobiont and mycobiont fractions as a result of dissection, increased proportionally. There is normally a 40% to 60% disposal of nitrogen when the thallus is dissected (Table 30), which is not reflected in the dry weight of the two portions and in this
series of analyses the proportion was similar, with a bias in the direction of the fungal fraction. The nitrogen assimilated, increased in
both fractions, no saturation point was reached for the two fractions
and the fungus label was equivalent to or slightly higher than that in the algal zone.
Millbank and Kershaw (1969) and Kershaw and Millbank (1970)
have shown with Peltigera aphthosa, that a saturation point was reached,
but that there was a continual increase.in total thallus nitrogen, with
the major part being incorporated by the fungus and very little being
incorporated into the Coccomyxa, which they suggest was considerably
lower than the expected gain and might have arisen as uptake of
unabsorbed nitrogen in the intracellular solution.
There is no indication that there is a lag in the transfer of
fixed nitrogen from the alga to the fungus, though no really short time
course experiments were carried out. In this study, 24 hours was the 15 shortest period tested and in that time considerable amounts of N had 2 been incorporated and transferred to the mycobiont, indicating that rapid
transport of fixed nitrogen was occurring. Millbank and Kershaw (1969)
and Kershaw and Millbank (1970), have analysed their total uptake
experiments after only 2 — 4 hours and have indicated that, with measureable amounts being present then, there is immediate uptake of the
nitrogen gas.
Transport may well be by active secretion. Fogg (1952) and
Stewart (1963), have both shown with free—living blue—green algae, that
large amounts (15 — 30%) of fixed nitrogen are released into the medium Table 30. Total nitrogen and the total increase after incubation in 15N2 as a percentage, in dissected discs of Peltigera salydactyla thallus.
Population A Population B
Percentage Percentage Sampling Time Percentage •Sampling Time Percentage Total Nitrogen Total Nitrogen (Days) Total Nitrogen (Days) Total Nitrogen Increase Increase
Phycobiont Mycobiont Phycobiont Mycobiont Phycobiont Mycobiont Phycobiont Mycobiont Fraction Fraction Fraction Fraction Fraction Fraction Fraction Fraction
44 56 45 55 39 61 49 51 1 3 40 60 48 52 37 63 47 53
39 61 54 46 34 66 40 60 6 9 40 60 55 45 45 55 51 49 61 43 57 58 42 16 39 43 57 13 38 62 54 46 40 60 43 57
41 59 49 51 37 63 41 59 20 23 36 64 47 53 39 61 41 59
41 59 46 54 46 54 54 46 27 30 44 56 48 52 43 57 48 52
46 54 49 51 41 59 50 50 37 34 44 56 48 52 41 59 53 47
Mean Mean Mean Mean Percentage Percentage Percentage Percentage Total Nitrogen Total Nitrogen Total Nitrogen Total Nitrogen Increase Increase
41 59 50 50 40 60 47 53
Range of Percentages Range of Percentages
( 36 - 44 ) ( 64 - 56 ) ( 45 - 58) ( 55 - 42 ) ( 34 - 46 ) ( 66 - 54 ) ( 40 - 54 ) ( 6o - 46 )
Lo NUDES * Both populations came from a single gathering of material from Tentsmuir. 132 in which they were growing and Henriksson (1951, 1964) with Nostoc from
Collema tenax, has shown similar traits. Drew and Smith (1967) have shown that the capacity of Nostoc to secrete carbohydrates is soon lost, when the influence of the mycobiont is removed and this active secretion in Peltigera species is further substantiated by the observation that
Peat (1968) made, using electron microscopy, where he showed that there was no evidence of haustorial penetration by the fungus. Also, from the work of Peveling (1973) with Lichina pygmaea, it seems that haustoria are not essential for the removal of algal metabolites. Durrell (1961), shows electron micrographs where the intracellular spaces are filled with polysaccharides and also pictures which he indicates, show enzyme activity, in the vicinity of the hyphae (as the result of particular staining that he used) and a thinning of the algal cell walls. Peveling
(1973), discussed the possibility of vesicles in the phycobiont sheath of
Lichina pygmaea, as a possible transfer mechanism and discusses the whole concept of transfer in a general review (Peveling, 1973a).
As pointed out earlier, the concentration of nitrogen gas in the incubation vessel is most important and should be related to the
Michaelis constant (Km). In the final 15N2 uptake experiment with the 15 desiccator vessels, a pN2 of 0.4 atm (30 atom % excess N) was used.
Stewart (1967), found with Calothrix, in the field, that there was
considerable variation in fixation rates with the pN2 used, maximum uptake occurring with a pN2 of 0.4 — 0.6 atm. Bond and Scott (1955) used
90% N2; 10% 02 with added CO2, though Scott (1956) used less nitrogen. 755
for his assay of Cladonia and surprisingly 35% for his.assay of Peltigera,
the balance being replaced by argon and he explained his reason for doing 15 appeared unlikely to affect the this thus:"This step to conserve the N2 result since the value of 0.02 atm has been given by Burris and Wilson
(1946) for the pN2 for which half maximum rate of fixation by Nostoc 133
muscorum occurs." Watanabe and Kiyohara (1963) only used a pN2 of 0.05
atm and did not make any comment on this. Bergersen and Turner have also
indicated that the Km for nitrogen fixation varies according to the
nitrogen—fixing system used (Table 31). Their values are also all
consistently low.
If this figure of 2% nitrogen, quoted by Burris and Wilson is
correct, then it is likely that the 5% used by Watanabe and Kiyohara was
adequate, but in view of more recent experimentation it is very doubtful
and probably explains his labk of results.
The data for 15N uptake is very variable, with the most
rapid assimilation at the start of each experimental period, but compared
with other work on Peltigera spp. (Millbank and Kershaw, 1969), it is
considerably lower. They used Peltigera aphthosa, a species with a high
heterocyst ratio, known to fix nitrogen approximately four times as
rapidly as in P. canina. The maximum rate of fixation in P. canina is -1 -1 123.05 nM (mg thallus nitrogen) hr and though P. polydactyla is . capable of such fixation rates (Table 18, p 75), it is seen that in Table 32, the nitrogenase activity is only 39% of the maximum. Therefore
by multiplying the recorded. P. polydactyla rate 4—fold and converting the
figures to the maximum possible based on ethylene production, the
corrected figures are 4.0ug and 5.2ug and compare favourably with the
results of Millbank and Kershaw.
Considering the actual figures obtained, it would indicate
that based on a nitrogen content of 2.5%, the thallus would double its
weight in 290 or 225 days respectively (Table 33).
Other workers have reported much lower rates, e.g. Watanabe and Kiyohara (1963) and Scott (1956), with Peltigera 221xL21,11a and and P. praetextata respectively. Scott's reported activities (Scott,
1956) were extremely low, 0.113 atom % excess by 7 sq cm thallus in 5 values for various nitrogen—fixing systems. Table 31. Km and pN2 ( after Bergersen and Turner 1968 )
Km - Nitrogen—fixing pN Concentration 2 Systems (mm Hg) (atm)
Cell—free Extracts 62 - 118 0.09 — 0.17
Intact Nodules 60 0.07 — 0.09
Intact Bacteroids 20 0.03
Table 32. The relationship between nitrogenase activity ( C2H2 Reduction ) and thallus nitrogen content in Peltigera polydactyla.
Population A ( Day 1 )* Population B ( Day 3 )*
Total Thallus Total Phallus nM CH nM C H nM C H nM C H -e 4 1 Nitrogen 2 4 2 4 ...1 Nitrogen 2 4 ( 1 sq 0: )- ( mg Thallus Nitrogen )-1 ( 1 sq cm ) ( mg Thallus Nitrogen ) 1 ( 1 sq cm )-1 -1 -1 ( 1 sq cm )-1 —1 hr—1 hr hr hr (Pg) (Jig)
8.69** 5.63** 4.30 42.08 6.70 37.23 12.99 308.63 12.33 331.22
6.30** 4.65**
5.94 40.97 9.01 38.92
12.24 298.72 13.65 350.70
Mean nM C H 2 4 ) —1 ( mg Thallus Nitrogen hr
39.80
NOTES :— * Both populations came from a single gathering of material at Tentomuir. ** The acetylene reduction analyses were carried out cut on 0.5 eq cm discs of thallus.
Table 33. A comparison of nitrogen fixation rates in different lichens using 15N2 analysis.ysis.
Atom % Excess Mean pg Nitrogen Mean pg Nitrogen Oven Dry Weight Thallus Total Thallus Dry Weight Increase ' Thallus Nitrogen —1 Fixed Fixed , ( 1 sq cm )-1 Day Doubling Time Lichen ( 1 sq cm )-1 ( 100pg Thallus N )1 ( 1 sq cm )' as 2.5% of the ODW (Days) in 24 hr in 24 hr in 24 hr (mg) ( 1 sq cm )-1 (pz)
*** 0.756* 0.35 1.12 223 290 Poltirera 0.624 13 325 polydactyla 0.736** *** 0.44 •1.44 176 225 1.032
Peltigera**** 5.40 1200
aphthosa — 8.60 25°C
***** Peltirera 0.0035 oraetextata
NOTES : These samples were tested 1 day after the start of the experiment. ** These samples after 3 days. *** The experiment was carried out at 1200. **** The data come from Millbank and Kershaw (1969). ***** The data come from Scott (1956). rn 137
days. Comparing his results with those in this study (0.79 mean atom %
excess (sq cm)-1 day-1), the rate of doubling of his thalli would be
244 times as slow (181 years ?). However, Scott (1956) also measured
actual dry weight increases and found that the thalli doubled in 14
weeks, so it is evident that great difficulty is encountered in providing
suitable conditions for active nitrogenase activity and nitrogen
assimilation and the work of Burris and Wilson should therefore be
considered very seriously.
Stoichiometry of nitrogen fixation in the lichen Peltigera
polydactyly has been calculated and on an overall basis of 2Oug nitrogen
. fixed in 30 days with a 16 hour (day)-1 fixation period, the expected
conversion is not 3 : 1, but 12 : 1. This in vivo conversion factor for
lichens may not compare with other in vivo or in vitro studies.
Hardy, Burns and Holsten (1973), quote an average in vitro
• conversion factor of 3.7 for various extracts, relative to the
theoretical ratio of 3 : 1 and also qoute values of 3.8 for bacteria, 3.2 for blue—green algae, 3.9 for legumes, 2.4 for non—legumes and 4.3 for
soil and they point out "on a biochemical basis, it is difficult to
accept ratios outside the area of 3 or 4 in short term experimnets
unless there are unknown factors existing, differentially
affecting the two reductions.".
Table 34, shows that the relation between nitrogen fixed and
acetylene reduced, at any of the set assay times, may be greater or
lesser, indicated by (4-) or (—),' relative to the other assay of the pair.
It is also seen that by calculating the total possible percentage
assimilation, based on the initial assay, that subsequent observed
assays, in terms of percentage nitrogen assimilated, are not equivalent
and indicate that the nitrogenase activity, based on acetylene reduction,
has not been continually active at this rate, throughout the test period Calculation of the Total Assimilation at Day x
Relative to 12.99or 14.64nM 021.14
Sample Calculation :—
nM C H 4 Day 1 0.76 12.99
Day 6 1.62 20.16
If at Day 6 the acetylene was also 12.99 one could assume
that nitrogen uptake should be six times as much, as at Day 1,
i.e. 4.56%, but if the acetylene was 20.16 (recorded) at
Day 6, the percentage assimilation ought to be :—
4.56 x 20.16 12.99
which is 7.07%. The real reading is 1.62%, therefore the
_ acetylene reduction assay appears to have no connection with the
assimilated nitrogen as 15N 2. 139
The theoretical relationship between nitrogenase activity using C Table 34. 2H2 reduction and the uptake of 15N in discs of Peltigera polydactyla 2 thallus. Total .4 15 Incubation Sample Percentage nM C H Expected N2 Assimilation Calculation of the Time 2 4 Assuming the Relation Between Total Assimilation (Days) Dice Assimilation ( 1 sq cm )-' of -1 C2H4 (a) and Nitrogen (a) at Day x (m) Number Nitrogen hr to be unity Relative to 12.99 nM C2H4
1+ 2 0.76 (a) 12.99 (a) 1.00 0.76 1 o.76** 3+4 0.63 (b) 12.24 (b) 0.88 ( -) 0.72
1 +2 1.62 (a) 20.16 (a) 1.00 7.08 6 4.56 3+4 1.27 (b) 18.99 (b) 0.83 (-) 6.66
1 +2 1.23 (a) 13.45 (a) 1.00 10.23 13 9.88 3 +4 1.45 (b) 13.28 (b) 1.20 (+) 10.11
1 +2 1.07 (a) 10.94 (a) 1.00 12.80 20 15.20 3+4 0.93 (b) 12.57 (b) 0.77 (-) 14.71
1 + 2 3.56 (a) 11.eo (a) 1.00 18.64 27 20.52 3.+ 4 2.81 (b) 11.52 (b) 0.81 (-) 18.20
1 +2 4.21 (a) 11.20 (a) 1.00 22.28 34 25.84 3+4 4.43 (b) 9.92 (b) 1.19 (+) 19.74
Calculation as Above Relative to 14.64 nM C H 2 4 1 +2 2.21 (a) 14.64 (a) 1.00 2.21 3 2.21 3 +4 3.09 (b) 11.35 (b) 1.81 (+) 1.71
1 + 2 5.05 (a) 17.51 (a) 1.00 7.93 6.63 3 + 4 4.50 (b) 15.58 (b) 1.00 7.06
1 + 2 4.62 (a) 18.33 (a) 1.00 14.82 16 11.84 3 + 4 7.11 (b) 18.29 (b) 1.54 (+) 14.79
1 +2 7.87 (a) 20.32 (a) 1.00 23.62 23 17.02 3+4 9.53 (b) 20.61 (b) 1.19 (+) 23.96
1+2 7.72 (a) 16.42 (a) 1.00 24.90 30 22.20 3+4 10.21 (b) 17.19 (b) 1.26 (+) 26.07
1 +2 8.89 (a) 13.23 (a) 1.00 24.74 37 27.38 3 + 4 14.16 (b) 12.79 (b) 1.65 (+) 23.92
NOTES :- * See the calculation on opposite page. ** Total Percentage Assimilation of Nitrogen, value (a) Day 1, multiplied by the number of days passed. The ( -) or (+) sign in column 5 indicates the uptake of nitrogen, relative to the assumed acetylene' value (a). 140
and this must inevitably be reflected in the stoichiometry of the reaction.
Maximum relativity was therefore.calculated from Day 1 and Day
3 analyses. Table 35 shows that the observed ratio is between 5 : 1 and
6.5 : 1, except in one case where it was 2.97 : 1. No other data exists
for the C H / N ratio in lichens, since all stuAs observing nitrogenase 2 2 2 A activity have been carried out using either acetylene or nitrogen, but not
both methods in conjunction, see Kershaw and Millbank (1969, 1970), Millbank
and Kershaw (1970)1 Henriksson and Simu (1971), Kallio, Suhonen and Kallio
(1972),1\idllbank (1972), Hitch and Stewart (1973), Kershaw (1974) and
Crittenden (1975).
Other high figures quoted by Hardy et al (1973), are :—
Nitrogenase Systems Ratio Author
Azotobacter 6.0 — 1 Bergersen (1970)
Glycine max 6.6 — 1 Bergersen (1970) Fallow Soil 6.9 — 1 Steyn and Delwiche (1970)
Anaerobic Soil 25.0 — 1 Brouzes et al (1971, 1971a)
the latter being the result of incomplete diffusion of the nitrogen gas,
relative to the acetylene under the waterlogged conditions and also the
low pN2 used.
The in vivo ratio in lichens, follows that of other in vivo
systems initially, but the nitrogenase is not at maximum activity for
this lichen, based on acetylene reduction analyses and it would be very
interesting to see if highly active thallus could achieve a ratio, closer 1
to the theoretical value, in the short term. The single instance reported
•(Table 35), indicated that this is so, but the result was calculated on a
set 16 hour light period and it will be seen later that nitrogenase
activity (acetylene reduction) can take place in the dark.
The general finding in this set of experiments was, that
rapid nitrogenase activity does occur, but that laboratory conditions can •Table 35. The expected quantity of nitrogen fixed with a theoretical stoichiometric conversion factor of 3 and the observed ratio; by in vivo nitrogenase in Peltigera polydactyla thallus.
* Incubation nM C2H4 Total Nitrogen Fixed Expected Observed .1 Total Nitrogen Fixed Time ( 1 sq cm ) ( 1 sq cm )-1 Stoichiometric Ratio - (pg) (Days) hr 1 Olg) With a Conversion Factor of 3 N2/NH3 : yE2/C2H4
12.99 1.17 1.94 5.0 : 1.0 1 12.24 0.90 1.83 6.1 2 1.0
14.64 3.00 6.56 6.5 1.0
11.35 5.13 5.09 < 3.0 : 1.0
NOTE :- Based on a reduction period of 16 hours daylight in each 24hr period. 142
affect the rate considerably, when maintained over long periods of time.
Undisturbed thallus gave the best results and provided that all the factors that might affect the analyses, were taken into account, the
stoichiometry of acetylene reduced against nitrogen fixed, could achieve the theoretical value of 3 : 1. 143
Section 5
SOME FACTORS AFFECTING NITROGEN FIXATION, PHOTOSYNTHESIS
AND RESPIRATION IN WHOLE THALLI
5.1.1 INTRODUCTION
The physiology of lichens can be divided into that of the
intact thallus and that of the isolated partners, in culture. Because of
interest in re—synthesis and greater control with isolates, knowledge of
their responses is better known.
However, it is now being realised that these data cannot be
extrapolated to explain the physiology of the whole thallus on a simple
additive basis, but fresh experimentation must be carried out with the
thallus viewed as a separate functional unit, with quite distinct
properties.
Equally important is the realisation that lichens, basically
terrestrial organisms, are subjected to an environment which is one of
continual change and flux. Onto a basic rhythm of light and dark, are
superimposed changes in water availability, nutrient supply, temperature
and seasonal variation and all these parameters interact. They will also
effect the proceses of photosynthesis, respiration and nitrogen fixation,
in terms of energy formation, reductant supply and carbon, nitrogen and
oxygen utilisation and production.
The effects of light and darkness, oxygen concentration, pH
and metabolic inhibitors, have been observed in the short term and
discussed in relation to the free—living blue—green algae. In making
these comparisons, it is important to remember that the significance of
environmental fluctuations may have been underestimated. As Farrar
(1973) suggests, in carrying out physiological experiments, 1) the method
of assaying the responses must be suitable, 2) the simulation of
environmental variables in the laboratory must be realistic and 3) care 144
should be taken to differentiate between reversible and irreversible
changes in the lichen.
Fogg (1956) has pointed out that "there appear to be no features of photosynthesis in blue—green algae which might be
specifically related to their capacity for nitrogen fixation" and he
continues "the path of carbon in photosynthesis appears to be essentially
the same in these organisms as in green algae and higher plants."
Lyne and Stewart (1973) in an Emerson enhancement study,
pointed out that no competition was noted between the two processes of
nitrogen fixation and photosynthesis under light limiting conditions and
they suggest that the two processes depend on different pools of reductant
and ATP.
It has been shown in free—living blue—green algae and in
symbiotic forms that nitrogen fixation is impaired if the light intensity
is too low or too high. High light intensity may specifically inhibit
nitrogen fixation, the optimum intensities for nitrogen fixation and
photosynthesis are approximately 5,000 lux and 10,000 lux respectively
(Fogg and Than—Tun, 1960), with Anabaena cylindrica. This may in part be
due to the fact that at high light intensities the photosynthetic
activity decreased.
Ertl (1951) discussing the amount of light that falls on the
lichen phycobiont, showed that colouring matter in the fungal cortex,
could absorb 26 - 435 of the incident radiation, comported to 4 - 135 in
green leaves by the epidermis and the variation in the light absorbed is
also affected by whether the'thallus is wet or dry. It can easily be
shown when some lichens are wetted, that they become much more
transparent, e.g. Peltigera species, Lobaria scrobiculata and Physcia
species.
Lichens are adapted in many ways to receive light for 145 photosynthesis. Cyanophytic lichens, tend to live in moist shady habitats, as they can photOsynthesise at lower light intensities than lichens with green algae. Hamptom (1973) explains this as a reaction to green-rich light (in shade conditions) and he indicated that the quantity of phycoerythrin, a pigment which absorbs maximally in the green region of the spectrum, is increased. The quantity of light also affects the phycoerythrin level and in Nostoc, it is thought that it is the quantity of light which is important (Holm-Hansen, 1968).
Lichens with blue-green algae that are adapted to high light intensity habitats (Lichina, Placynthium, Lempholemma, Collema, etc.) are often pigmented black and Ellee (1939) has reported a relationship between light permeability and scotophily in Lobaria pulmonaria and
Peltigera rufescens.
Further, Barkman (1958) has shown that the effect is found in lichens with green algae too. In regions of high insolation, lichens become much more deeply pigmented (Xanthoria and Cladonia species), while similar species growing in the shade, are often green or grey-green, with no pigmentation at all.
The studies of Rao and LeBlanc (1965) have indicated that other adaptations exist. They examined the fluorescence spectra of a number of lichens, including Peltigera canina and found that atranorin, a lichen compound, not only absorbed unwanted light of high intensity, by quenching, but increased the use of light for photosynthesis, at low light intensities.
Wilhelmsen (1959), has indicated that the algal zone in lichens, in terms of active photosynthetic tissue, is only about 1/th to 4 - 1/10 01 of that of green leaves and further chlorophyll estimations vary, being much less in summer than in winter, probably as a result of drought.
For these reasons, photosynthesis in lichens does not compare favourably 146 with green leaves.
Respiration in photosynthetic organisms is a dark process, or at least it does not require light and f.5.:egred therefore should not be inhibited. Thomas (1939; see Kappen, 1973) stated that light intensity had no influence on the growth of mycobiont cultures. However, Kappen and Lange, using isolated mycobiont cultures of Cladonia rangiferina, found that in the light (10 K lux), the fungi released about half of the
CO that they evolved in the dark. 2 Haynes (1964), quoting the work of Stbafelt and of Butin, found that there was a correlation between habitat and light requirements for photosynthesis, in that in a tree canopy for example, Usnea aasyEaa on the trunk, had a compensation point at 400 lux, while Ramalina fraxinea,
on sunlit branches, had a compensation point at 2,000 lux, with the light
being 2,000 and 7,000 lux respectively, to enable half maximum
photosynthesis to proceed.
Hitch (1971), observing the effect of light on nitrogen
fixation in lichens, showed that similar intensities were more inhibitory
to some lichens than others, Lichina confinis being inhibited at a lower
light intensity than Peltigera rufescens, even though the light intensity
measured in the field, gave higher readings at the Lichina site.
This might be explained by the fact that, being on a rocky
shore, the thallus could dry out more quickly than in a sand—dune system,
so fixation could only be carried out when the light intensity and
temperature were low and the thallus moist. A similar point being made
by Lange (1969), who indicated that with some lichens, 805 saturated air
is sufficient to moisten the thalli enough, so that photosynthesis can
occur for a limited period (2 — 3 hours) after sunrise in the desert.
See also Kappen's review (Happen, 1973) of extremes affecting desert
lichens. 147
Stewart (1966), has shown a distinct relationship between the nitrogen—fixing ability of blue—green algae and the depth in the soil in which they were growing, fixation being much more rapid in the light in surface samples. A somewhat similar observation was noted by Bergersen,
Kennedy and Wittmann (1965), with endophytic algae of cycads, where they showed that coralloid roots of young plants, 2" — 3" under the surface, fixed more nitrogen than old opaque roots, 12" — 15" below ground level.
However, these findings could show an adaption to heterotrophy.
Light while indirectly important for nitrogen fixation, via the photophosphorylation system, producing energy (Stewart, 1973), is in itself not basically essential for nitrogen fixation. Whether direct photoreduction of nitrogen is possible, is not entirely clarified. Some bacteria are capable of the process and under certain experimental conditions, blue—green algae may photolyse water according to the equation,
3 H2O 2 NH 02 3 noted by the extra oxygen evolution in the absence of CO2. Hitch and
Stewart (1973), have postulated that light has little immediate affect on nitrogenase activity in the field and that direct photoreduction of nitrogen, is not the sole source of reductant. Though it may help to show that various pools of reductant exist in blue—green algae and could be used as a source for dark fixation.
The inter—relationship of photosynthesis and nitrogen fixation is further complicated by the evolution of oxygen, during the
photolysis of water and its possible affect on nitrogen fixation.
Nitrogen fixation must be separated from photosynthesis, though a lot of the early work on isolates, showed that the nitrogenase was associated 148
with the photosynthetic lamellae, a finding regarded as an artifact now,
caused by inexperience in preparatory techniques of the enzyme.
Nitrogen fixation is a reductive process and Stewart,
Haystead and Pearson (1969) have pointed out that blue—green algae were
some of the earliest organisms present on the earth and fixation was more
widespread in the reducing conditions then prevailing.
With the development of oxygen—evolving photosystems, it is
probable that nitrogenase activity in blue—green algae, is only possible
in aerobic conditions, in filamentous forms that have heterocysts. It
has been shown (Fay, Stewart, Walsby and Fogg, 1968) that photosystem II,
the oxygen—evolving pathway, by the photolysis of water, is absent in
heterocysts, since there is no phycocyanin present. Fay (1970) has
demomstrated photostimulation of nitrogen fixation in Anabaena cylindrica,
using monochromatic light and comparing action spectra of acetylene
reduction and photosynthetic oxygen evolution. Maximum nitrogenase
•activity was at a similar wavelength to that for optimum light absorption
by chlorophyll and indicated primary involvement with photosystem I. a Lyne and Stewart (1972) have also shown that light absorbed by
photosystem I, affected nitrogen fixation, but not carbon fixation.
It has been shown that heterocysts have a high respiratory
rate (Fay, Stewart, Walsby and Fogg, 1968) and are also highly reductive,
as formazan crystals developed in the heterocysts after an incubation
period in tri—phenyl tertazolium chloride (TTC), (Stewart, Haystead and
.Pearson, 1969). •
Fay et al (1968) state that of the 40 strains of blue—green
algae tested for fixation, all belonged to four filamentous orders. The
effect of oxygen on nitrogen fixation in unicellular whole organisms has
been studied by Drozd and Postgate (1970), with Azotobacter and Wyatt and
Silvey (1969), have found, that the unicellular bluegreen alga, 149
Gloeocapsa, is capable of fixing nitrogen in aerobic conditions, though earlier it was suggested that desert coccoid blue-green algae could fix nitrogen, (Cameron and Fuller, 1960).
Stewart and Lex (1970) and Kurz and LaRue (1971), with the non-heterooystous filamentous blue-green algae Plectonema borvanum strain o- sErtiii\ cyP 594 and Anabaena flos-aquae respectively, have shown that these can be / induced to fix nitrogen in the absence of oxygen.
Further indications that the heterocysts are oxygen depleted
sites, comes from the work of Stewart, Haystead and Pearson (1969), who
with sonication, and Fay and Kulasooriya (1972), with enzymatic
disruption, both proved that rupture of the vegetative cell-heterocyst
inter-connecting 'pore', was in direct relation to acetylene reduction
and - therefore the percentage of attached heterocysts. There is also much evidence to recently show that active
cell-free preparations, capable of nitrogen fixation, can be prepared
when the cellular tissue is disrupted under an atmosphere of argon,
(Haystead, Robinson and. Stewart, 1970; Smith and Evans, 1970 and 1971;
Gallon, LaRue and Kurz, 1972 and Haystead and Stewart, 1972).
Lichens can photosynthesise, respire and fix nitrogen
relatively rapidly and while it may not be possible to make comparisons,
as far as fundamentals are concerned, there is nothing to suggest that
the symbiotic algal partner is lacking in any of the features of free-
living forms.
150
5.1.2 METHODS
5.1.2.1 Effect of darkness Lichen thalli (1.0 sq cm) discs were incubated in McCarteney
bottles, since many gas samples had to be analysed. The bottles were
wrapped in black polythene (500 guage) to exclude light completely. This
material compared to aluminium foil, did not tear. No heating effects
were noted as the lights were turned off during dark periods, but the air
round the bottles was continually stirred by a fan.
5.1.2.2 Effect of pH
For neutral pH experiments. In all routine experiments,
Allen and Arnon's mineral salts medium was used, pH 7.2.
For high pH experiments. With the addition of 10mM HCO3-
(sodium bicarbonate) to unbuffered medium, to observe the effect of
concentrated CO on nitrogen fixation, there was an increase in pH to 2 8.4. Due to a mistake, a solution containing 100MM HCO - was also prepared and
gave rise to a pH 9.0.
For low pH experiments. In the preparation of anaerobic
medium, by sparging Allen and .Arnon's mineral salts medium with
"Argoshield"„ a comercially available gas mixture of Ar (95%) and CO2 (5),
3 orHEPES buffer, the CO depressed the pH despite the addition of P04 2 considerably from 7.2 to as low as 5.0;
5.1.2.3 Oxygen experiments - anaerobiosis Due to the above effect, (see:For low pH experiments),
Allen and Arnon's mineral salts medium with sodium dithionite added and
bubbled with pure argon was used. Lex, Sylvester and Stewart (1972),
have shown that bubbling nitrogen gas for only 2 - 3 minutes, would not
reduce the p02 / pCO2 enough to enable photorespiration to proceed.
5.1.2.4 Oxygen experiments - effect of varying the p07; with whole
thalli 151
Discs were assayed normally in serum bottles and for tests in
which the effect of high p02 was to be observed, oxygen was added finally
as part of the gas phase, using manometry.
5.1.2.5 Oxygen experiments — effect of oxygen on chopped, scraped and
ground—up thalli
In all the experiments the thalli were cleansed of debris
and washed free of sand in glass distilled water. Normally discs were
cut out.
Chopped and scraped thalli were usually treated in ordinary
air on the laboratory bench. In the low p02 experiments, a "Glove—box"
(Gallenkamp) was used, a rectangular metal box, with gas taps, a sloping
top with a "Perspex" window, detachable rubber gloves and a gas—tight
side compartment, all of which could be de—gassed. The whole box was
flushed with argon three times and it was found that due to big internal
pressure changes, that to evacuate and fill a polythene bag i the box
•was the quickest method, to ensure complete removal of the internal gas
in one operation. As a safety precaution this was repeated twice and the
final filler gas was passed through a paramagnetic oxygen analyser, to
ensure a zero p02. The gas tight side compartment could be de—gassed
separately and was used to load the lichen thalli, grinding apparatus and
medium. The grinding medium was sparged with pure argon and the lichen ed thalli were evacuati prior to maceration.
5.1.2.6 men experiments — measurement of oxygen exchange
A Clark oxygen electrode was used, of similar design to that
described by Delieu and Walker (1972). The body of the electrode was of
"Perspex", with silver and platinum electrodes. The electrolyte was
saturated KC1 solution, sepatrated from the centre well by a "Teflon"
membrane. The solution in the centre well was stirred by a glass coated
magnetic follower and changes in oxygen tension were reflected in changes 152
in the current passing between the electrodes. These were recorded
graphically by means 9f-a pen recorder.
Discs of lichen thallus were held in place in the centre well
in relation to the incident light, by a nichrome wire frame attached to
the plunger.
The whole apparatus was temperature controlled by an outer
water—jacket with water pumped from a thermostatically controlled water
bath.
Light was from a photographic slide projector. Its intensity
was varied by altering the distance between the source and the thallus,
the intensity at the lichen site, i.e. through two walls of the water—
jacket, being measured by means of a silicon photodiode, which was
calibrated using an EEL light meter.
The medium in the centre well was air saturated Allen and
Arnon's solution and metabolites or inhibitors were introduced into the
•centre well, via a capillary in the plunger. Small portions of the
medium were removed by inserting a long hypodermic needle through the
hole and replacing it with a similar volume of 10—fold strength. This
ensured that delays between assays were short and physical changes to the
system were as small as possible.
5.1.2.7 Chemicals
3'—(314—Dichloropheny1)-1'1'—dimethyl urea (DCMU). DCMU,
10-4M was made up in 25% ethanol and 75% glass distilled water. The final concentration was 105M at the thallus site. Potassium cyanide (KCN). KCN, 10 11 was made up in glass
distilled water. The final concentration was 10-5M at the thallus site.
Sodium dithionite (Na2S2040H20). A solution containing —1 5mg ml. was prepared. 100m1 of glass distilled water was bubbled with
hydrogen for 10 minutes and 0.5m1 N NaCH added. 25mg Na2S204.H20 was 153 weighed out into a Bijou bottle, evacuated filled with argon and 5m1 of the alkaline water injected. Final pH 7.0. Using 0.1m1 m1-1 of the —1 incubating medium is equivalent to 2.5umoles ml . 154
5.1.3 SOME INTER—RELATIONSHIPS AND EFFECTS OF CO2, OXYGEN, pH, LIGHT AND
INHIBITORS ON NITROGEN FIXATION,- PHOTOSYNTIEESIS AND RESPIRATION
5.1.3.1 Oxygen and its affect on nitrogen fixation
In the special circumstances of the blue—green algae,
photosynthesis and nitrogen fixation are potentially, mutually
conflicting, because the former gives rise to oxygen, whilst the latter
requires reducing conditions.
Nitrogen fixation in lichens appears to be rapid at a p02 of
0.2 atm. The p02 inside the thallus, relative to the outside, was not
measured, though Millbank (pers comm) has indicated that it is at the
atmospheric level or somewhat below, using a micro—oxygen probe.
Since the algal—rich zone is surrounded by respiring fungi,
it is not unlikely that the p02 would be lower than 0.2 atm. caPox99e1, The effect„on "hitrogenase activity was observed, when the
thallus was disturbed, either in air or under theoretically anaerobic
conditions (Table 36). This involved scraping the medulla off the back
of the thallus, chopping the thallus and macerating it in an homogeniser.
Peltigera canina showed considerable loss of activity when the medulla
was removed, 32% of full activity, due to its rather fragile nature,
though P. polydactyla, being tougher, was less affected, with a drop of
only 145%
Chopping the thallus had no inhibitory effect. With P.
canina, thallus nitrogenase activity increased, see the work of Smith
'(1963) with respiration on dissected discs. The increase in the absence
of air, being greater than in its presence. Peltigera uolvdactyla showed
a similar trend, when the bulk of the medulla was removed, see the notes
at the foot of Table 36.
Maceration in an homogeniser, caused loss of activity which
155
Tobin 36. The effect of maceration and oxygen on nitrognnase activity ( C H Seduction ) using lichen thallun. 2 2 Peltipers caning • ------Whole Thallus ------Stripped Thallun --
Mean nM C2I Mean nM C H -2 2 4 Nurber of 1 Percentage cm 1 Percentage Teat ( 1 eq cm )- ( 1 nq )- Estimates Activity Activity hr-1 • hr-1
A 3 4.19 100 1.36 32.46
Whole Thallus ----- Macerated Thallus --
nM C2H4 nM C H Number of Oxygen Percentage 2 4 -1 Percentage Teat ( 1 sq cm ) 1 ( 1 sq cm Estimates Gas Activity Activity hr hr-1 )
1 17.41 100 14.32 82.e5 B 5.63 100 5.22 92.71
Whole Thallus --- Macerated Thallus ----- Mean nM C,:ff Mean nM C H z 4 2 4 Number of OxYSert Percenta ge Percenta ge Teat ( 1 sq cm )-1 ( 1 sq cm )-1 Estimates Gas Activity Activity hr-1 hr -1 -0 4 2 15.55 100 0.23 (1.56 : 0.95-2.60) -0 4 2 22.11 100 1.61 (7.24 : 1.69-13.16)
Whole. Thallus ----- Chopped Thallus ---- Macerated Thallus ----
Mean nM C..0 Mean nM C2H4 Mean eM C H z 4 2 4 Ntmber of Percentage )-1 Percentage Test OxYgen ( 1 sq cm ( 1 sq cm cm )-1 Percentage Estimates Gas )-1 Activity Activity ( 1 sq -1 -1 Activity hr hr hr 6.07 5.92 ... 107 0.16 .. 2.33 6 +0 (3.33 100 2 2 ; 0.43) (2.67 ; 0.35) (87 - 136) (0.33 ; 0.04) ( 0 - 9 ) -02 7.40 100 9.42 (137 : 107-162) 1.21 ( 19 : 10-27 )
Whole Thallus Macerated Thallus Macerated Phallus
(a) Mean nM C2H4 (b) Mean nM C H (c) Mean nM C2H4 2 4 Activity (c} N,...mber of Percentage Test °Iygen ( 1 sq cm )-1 Percentage as a Fstimatos Gas Activity ( 1 sq cm )-1 -1 Activity of hr fo2hr-1 Activity(b) 2.80 0.76 66 ( 1 7111::: ( 2.10 - 3.50 ) ( 65 - 67 ) E disco -0 165 100 2 2.65 82 1.09 0.89 ( 2.50 - 2.60 ) -02 ( 76 - 88 )
N(YTES ' For experiment A, the tormentum was removed with no loon of nativity. The medulla was dissected from 'between the ribs on the lower side, these acted as strengthening and kept the thallus intact, though olightly fragmented. Discs were out from the tripped area, together with controls from the immediate vicinity. * The figures in brackets, asterieked, are the Standard Deviations and Standard Error of the Mean. Peltigera polydnotyla P. polydaotyla thallus showed very little lose of activity during dissection. Whole thallus 0.58 nM C2H4 (1 eq om )-1hr-1 ( 1(X, ). After initial dissection of fungal medulla the rate was 0.69 nM C2H4 ( 1 sq on ( 116.90:, ) After a secondary die:section to leave an intact algal zone, the rate was 0.50 nM C2H4 ( 1 eq om ( 86.1- ). No full scale analysin of Paltige rn Rolvdactyla was carried out. . 156 was related to subsequent particle size, compare Table 36 B and C, a similar effect being noted by Stewart, Haystead and Pearson (1969), when they tested nitrogenase activity in an algal culture after short periods of sonication. •
The presence of oxygen inhibited nitrogenase activity in macerated lichen thallus (Table 36 D), i.e. only 2.33% in the presence of oxygen and 19% of full activity in the absence of oxygen. A similar effect being noted in Table 36, E, where the thallus was macerated in argon, incubated in argon and then some of the samples being subjected to a p02 of 0.2 atm. The results show, that in air, the decrease to 66 I 1%, was greater than in argon 88 4- V. This small loss under argon was probably due to the fact that "pure" argon is not completely oxygen—free.
Higher oxygen tensions than a p02 of 0.2 atm (Table 37), begin to have a noticeable effect on acetylene reduction in lichens, with total inhibition at a p02 of 1.0 atm and it is only really marked at a p0 of 0.8 atm. Stewart and Pearson (1970), who have measured acetylene 2 reduction at various tensions, noted a similar fall off at high levels.
However, in their system, with free—living blue—green algae, they also noted an increase at lower oxygen levels than a p02 of 0.2 atm. Despite the work carried out by Millbank (1972), who postulated vegetative fixation to explain his rapid results, no increase could be achieved in the short term on the rapid results already obtained, when the effect of total anaerobiosis was studied, using anaerobic medium with sodium dithionite and argon as the gas.
After inhibition, activity rapidly recovered when the thallus of Peltigera canina was replaced in a p02 of 0.2 atm, except for those samples incubated in 80% and 100% oxygen. Here recovery, by extrapolation, took approximately 9 hours (Figure 13). It was also found that a second period of incubation in 100%. oxygen was less inhibitory
157
Table 37. The effect of different partial pressures of oxygen on nitrogenase activity ( C2H2 Reduction ) in discs of Peltigera carina thallus.
nM C H 2 4) ,* ( 1 sq cm -. -1 r-1 A nM C H ( 1 sq cm ) h hi: 2 4 Oxygen Sample (p02) To Establish Nitrogenase Time Time Time Activity 25 min 50 min 83 min Over 10 min p02 0.2 atm
1 16.80 0.20 16.90 20.49 19.04
2 15.60 0.20 17.88 21.91 21.90
7.80 0.40 5.34 6.90 7.48
4 13.20 0.60 8.28 9.39 9.50
18.00 0.80 0.62 0.93 1.00
6 13.80 1.00 0.06 0.03 0
The gas phase was evacuated and replaced by air, a p02 of 0.2 atm.
-1 nM C2H4 ( 1 sq cm )-1 hr
Sample Time Time Time Time
60 min 120 min 180 min 240 min
24.10 27.74 27.00 27.59
26.00 28.17 30.56 32.94
3 12.50 14.35 16.13 17.73
4 15.25 18.13 19.62 20.99
5 3.80 5.61 10.92
6 0.88 3.04 5.09 n d
Disc number 6 evacuated and oxygen introduced to a p02 of 1.0 atm.
nM C H ( 1 sq cm )-1 hr- 1 2 4
Sample Time Time Time 60 min 100 min 120 min
6 0.93 0.74 0.86
NOTE :- * Each incubation was carried out in Allen and Arnon's Medium plus 1gil HCO3. 18
16
14
12
4 5 6 7 8 9 10 Hours Figure 13. Rate of recovery of Peltigera °mina nitrogenase activity (C2H2 Reduction) in air (s02 0.2atm) after inhibition by oxygen (p02 1.0atm) for 1hr. The initial rate is also included (a). 159
than the first period, (Figure 14). Stewart and Pearson (1970) reported
that the nitrogenase of free—living blue—green algae recovered fully
after only 3 - 6 hours, from the time of withdrawal of the high concentrations of oxygen.
The fact that there was no increase in nitrogenase activity
at low oxygen tensions and that after exposure to a high p02 (0.8 — 1.0
atm), recovery took longer, suggests that the oxygen tension externally
and that at the algal site, may be very different, due to slow gas
diffusion and the subsequent decreased inhibition in 100% oxygen,
suggests that some metabolic process may have been stimulated, which
prevented the high concentration reaching the nitrogen—fixing site. This
might be either photorespiration, stimulated in high oxygen
concentrations, or, perhaps less likely, fungal respiration.
It has not been determined if there is a "switch—on / —off"
mechanism as in some bacteria (Drozd and Postgate, 1970; Yates, 1970), or
whether there is a regenerative pattern of new nitrogenase enzyme, The
delay suggests regeneration, however, various workers are not unanimous
that inhibitors of protein synthesis also inhibit recovery of nitrogenase
activity. This whole question is discussed by Stewart (1973).
The reason for the inhibition of nitrogenase in the presence
of high levels of oxygen are twofold; 1) Lex, Sylvester and Stewart
(1972), have shown that high oxygen concentrations, which enhance
photorespiration, cannot prevent seepage to the nitrogen—fixing site,
though it may retard the point at which inhibition occurs and 2) there is
a direct effect on the nitrogenase itself.
5.1.3.2 Light and darkness on nitrogen fixation
Ertl (1951) has pointed out that in some lichens, due to the
coloured pigment present the light intensity at the algal site might
be much lower than the external value. While Peltigera species do not 160
14
12 7 z
5
4
b.
N 3
2
.-.---A------.._._._9_..._ 20 40 60 80 100 120 140 160 180 tlinutes
Figure 14. Rates of peltipera canina nitrogenase activity (C2H2 Reduction) in oxygen (p02 1.0atm) (0), recovery in air (p02 0.2atm) (0) and a second incubation in oxygen (p02 1.0atm) (m). The initial rate is included (0). 161 have coloured pigments in the fungal cortex, they do mostly live in areas of low ambient light intensity and it could be that part of their metabolism is heterotrophic rather than phototrophic.
Quispel (1959), reviewing the carbon nutrition in lichenised algae, indicated that various workers had shown that sugars could often stimulate green algal growth, but light was necessary too. In Trebouxia
(a common lichenised alga), the effect is very pronounced in consequence of the negligible growth in purely autotrophic conditions, though this was not a generalisation that could be applied to all algae.
Referring to blue—green algae however, he quoted the work of
Henriksson (1951), who obtained a good growth of. Nostoc from Collema tenax on inorganic medium in the light and of Xratz and Myers (1955), who studying three related algae to lichenised forms, found that respiration was not increased on organic medium and that algae did not develop on these substrates in the dark. He concluded "Apparently these blue—green algae are obligate—autotrophic organisms."
Quispel (1943) tried to stimulate autotrophic growth in
Trebouxia and found that in the presence of ascorbic acid, development was comparable to that achieved on glucose. Ascorbic acid may affect chlorophylla production (Algeus, 1946, quoted by Quispel, 1959) and
Quispel (1959) questions whether lichen fungi excrete ascorbic acid, though in intact thalli, he questions the possibility of autotrophic growth by the excretion of ascorbic acid and postulates that photosynthesis may be too small to counterbalance the respiration of the surrounding fungus, either by lack of light or other ecological conditions.
Fogg (1956) quotes the work of Winter (1935), who showed that an endophytic speCies of alga fixed nitrogen best during heterotrophic growth, though Herriset (1952) could get no fixation of nitrogen in the 162 dark with sucrose. However, as Fogg pointed out, his technique was not
very sensitive and low endvto ted fixation might have been occurring.
Other carbohydrates could well have given better results, see the work of
Khdja and Whitton (1975).
Trifolium pratense has also been shown to fix nitrogen in the dark, at a decreasing rate with time, though if the pretreatment is 24
hours darkness, there was no nitrogen fixation at all.
Though general evidence from this study indicates that the
rate of nitrogen fixation by acetylene reduction in lichens in the light
is rapid, (see also Hitch (1971)), a possible suggestion that lichenised
blue—green algae may be capable of heterotrophic nitrogen fixation, comes
from the fact that unlike obligate phototrophic nitrogen—fixing organisms,
they are able to reduce acetylene and thus fix nitrogen (?) for a long,
period in the dark, though at a much reduced rate to that in the light,
probably related to the carbohydrate availability. Values (Table 38),
though very low, were measureable after 26 hours and Millbank (pers comm)
has indicated with Peltigera aphthosa var. variolosa that acetylene
reduction in the dark was still occurring after 4 days. The requirement of ATP for nitrogen fixation, is now
.confirmed. Up to 1962, various workers disproved the need for ATP (see
Stewart, 1966)), but in 1962 it was shown that arsenate inhibited ATP
formation and the fixation of nitrogen as well. Recently too, the need
for ATP and an ATP generating system has been demonstrated by many
workers, using cell—free preparations and the acetylene reduction
technique.
Cox and Fay (1969) have shown that ATP is derived from
photosystem I in heterocysts, since with CMU, the inhibitor of
photosystem II, there was an increase in acetylene reduction.
As with nitrogen fixation in the light, dark nitrogen fixation
Samples 3 2 6 5
1 sqcmr nM C- 28.46 36.26 Samples 19.36 14.66 4.96 6.06 hr -1 4 6 2 3 1 2 4
H 4
1 Dr. Millbank(pers.comm.) hasshownwiththisspecies,thatnitrogenaseactivity wasstillrapidafter4days.Ifheterotrophicdarkfixation is MOPES :- *** a C McCarteney ( 1sqcm)-' Bott len 25m1 nM 41.89 47.20 23.33 30 min
0.33 9.36 hr 1.41 33.92 37.94 15.80 25.70 6.34 7.23 Time -4 C
2 H
4 ,*
Table 38.Theeffectofdarkandlightonnitrogenaseactivity(C
The initial rateswere determinedto ensurethat thediscswere activeandcapable ofnitrogen fixation. This sampleof P. The ratesofactivity whilstlow,show Three out ofthe
** possible usingsugarsfromthe greenphycobiont,fixationmightcontinuetillthis supply wasexhausted.
110 min135159min•185220270 19.50 13.17 30.52 32.14 'd tH .... ,-■ 6.80 6.48 Time P 0. k a ..s4 E g k m d
a) mo
A A r... 0
six bottles canina 30.71 15.74 28.08 18.35 Dark Experiment 6.82 Time 6.11 60 min 0.02 Time 0.05 0.46 -- -- -
appearstoshow activityevenafter68hr inthedark.
30.78 19.01 16.23 27.95 were tested, afterthe darkperiod, toseeifthe nitrogenase wasstillactive. See(**) Time 6.78 6.24
**** 120 min DarkExperiment 0.01 0.04 0.67 Time Peltigera aphthosa a perceptible increase. 31.39 18.58 15.50 26.42 -- -
-- 6.69 6.36 Time
**
Peltigera nolydactyla
Peltigera 30.69 17.23 14.87 27.56 6.62 6.12 Time
1:1 I.. ,C1 El *el 1. 0.,.. A W al m canina
, csi ..-1 var. a a _
30.16 18.12 1 27.41 Time 7.29 6.51 5. 61 variolosa
2 155 min H ... f .5 •ri 6 0 .5> g g 2 ' tta 2 0.62 0.01 0.11 Time VI F-.+. Reduction)indiscsoflichenthallus. +. :4 4 0 0 0 o
o , i -1 4 4) "
43 min
0.09 Time 1.62 0.59 -- - - -
200 min 85 0.12 Time 0.77 -- 0.58 0.98 0 0.06 Time 0 0 0 . ------
min
LightExperiment 110 min 0.05 Time 0 0.49 .80 ------245 min
0.01 0.13 0.77
0 0
1- •5
+ eo ....■ 4 A g P .■-• t0 n a. , . 5 E.. , CV ..1 o E 4
* 140 min170230 0.380 0.048 0.690
Time 330 min 0.16 0.02 0.02 0. 0
above. 0 0 7
9 1 LightExperiment
0.600 0.031 0.410 Time
410 min 0.035 0.770 0.540 0.21 0.06 0.03 0.79 0.01 Time Time
*
290 min 0.037 0.860 1.700 Time
164 requires an energy source and electron donors. The production of ATP may also be indirect via the oxidative phosphorylation of carbohydrates, whether produced photosynthetically or exogenously andhas been demonstrated in blue—green algae (Stewart, 1973). -
The source of carbohydrate could come from the
'photosynthetically produced glucose of blue—green algae or ribitol of the green algae. It might explain why dark fixation went on so long in
Peltigera aphthosa var. variolosa, as ribitol could be diverted into the
pentose phosphate pathway, since relative to the cephalodia of Peltigera achthosa var. variolosa, the green cellular tissue is a vast storehouse
of sugars.
Lindstrom, Newton and Wilson (1952) have shown that
Rhodospirillum rubrum is able to grow heterotrophically in aerobic conditions and fix nitrogen in the dark, at the same rate as
photosynthetically grown cells do in the dark, so that they do not need
photochemicaaly produced substances for fixation.
The fact that oxygen is needed for nitrogen fixation, both by
bacteria and Anabaena cylindrical implicates oxidative phosphorylation.
It is unlikely that maximum fixation is achieved by this method, in fact the relative contribution of each method is unknown, as are all the factors, but up to 30 — 60% of the light rate might be obtained, see
Weare and Benemann (1973, 1973a).
Bradley and Carr (1971) have suggested that in the light
oxidative phosphorylation may give rise to a basal level of fixation, but
this is "topped up" with cyclic photophosphorylation to enable a much
faster rate of nitrogen fixation.
The requirement of oxygen for dark fixation, may have
resulted in inhibition in the experiment (Table 38), using Peltigera
canina, where fixation stopped after 26 hours, since the material was 165
enclosed in a relatively small volume, to ensure that it did not dry out
and where respiration would have been rapid. In this context, Millbank's
results may be relevant, for though enclosed, the much smaller quantity
of lichen used, cephalodia, did not deplete the oxygen supply so rapidly
and therefore fixation would not have been affected, for this reason, for
a long period.
The other requirement of nitrogen fixation is an electron
donor supply and there is no problem involved here as Stewart (1973) has
shown. Three possiblemechanisMs may be involved, the oxidation of
pyruvate, the pentose phosphate pathway and the TCA cycle and there is
evidence for and against each system. Much more work is needed to
ascertain whether the relevant enzymes are present in the organism tested.
Details of the various pathways are described by Stewart
(1973) and will not be discussed further here. Points which Stewart
makes, which may be mentioned for a dark generated reductant are 1) the
uncertainty concerning direct photoreduction, 2) some micro—organisms
fix nitrogen in the dark and 3) the very important finding, that the
respiratory inhibitor KCN affects nitrogen fixation more, than do
photosynthetic inhibitors.
5.1.3.3 The effect of pH on nitrogen fixation
Lichens vary considerably in their responses to pH and much
work has been carried out to assess the correlation between lichen cover
and speciation and bark acidity (see Hale, 1967).
Many lichens have an optimum in the range pH 5.0 — 6.0, for
mycobiont growth and Kari (1936), pouted by Hale (1967) showed that the
pH optimum of the alga does not correspond to that of the substrate, or to
the whole thallus and it is suggested that algae normally require a
higher pH than the fungi.
Hale (1961) has pointed out that in some lichens the pH 166 optimum may be very low as, for example, in Umbilicaria papulosa, the optimum pH for growth is 2.0. For a full account of the effect of pH on lichens, see Barkman (1958).
Effects of pH on the physiology of the lichens, have received little attention, but are probably as important as those brought about by changing the nitrogen or carbon supply.
pH has been shown to affect nitrogen fixation. In this study the effect of different pH on nitrogen fixation was observed. At pH 5.9, nitrogen fixation was completely inhibited (Figure 15), though as can be seen, nitrogenase activity was related to pH, since it could be started or stopped, depending in which solution the thallus was bathed. The idea of the nitrogenase being denatured or inactivated, has already been discussed with regard to oxygen tension. However, which process occurs under these conditions was not determined.
Approximately neutral pH, i.e. 7.2, was routinely used in all the studies with Allen. and Arnon's mineral salts medium and was not considered to have any inhibitory effect and the possible effect of using solutions with a pH of 8.4 has already been discussed (p 89).
Hill and Smith (1972) have pointed out that the pH of the solution and that at the physiological site, may be different, due to an efficient buffering capacity inside the lichen thallus, a postulate already suggested in relation to oxygen tension and it is only at the extreme limits of the range of oxygen or pH encountered in vivo, that this capaCity is over—ridden.
In the experiment in which exposure to a pH of 9.0 was examined (Table 39), the inhibition of nitrogen fixation observed in
Sticta fuliginosa and Nephroma laevigata, was greater than in the case of
Peltigera carina and P. polydacty].a. As Hill and Smith suggest, to bring different lichens to a common pH, may not be to give them uniform treatment. Allen and Arnon's Medium pH 8.4 •
pH 5.9 Allen and Arnon's Medium pH 8.4 0
pH 5.9 o pH 8.4 Allen and Arnon's Medium pH 5.9 o pH 8.4
0 1 2 3 4 5 7 9 10 Days
Figure 15. Rates of nitrogenase activity ( C2H2 Reduction ) in discs of Peltigera canina thallus influenced by different hydrogen ion concentrations. Table 39. The effect of high pH on nitrogenase activity ( C2H2 Reduction using discs of lichen thallus.
-1 -1 nM C H ( 1 sq cm ) hr Incubation 2 4 H2O Allen and Arnon's Allen and Arnon's Period H2O Lichen ( +HCO3- ) Medium Medium + HCO - (6.0)** 3 (Days) (9•0)** (7.2)** (9.0)**
0 10.07 8.37 14.08 11.82 Peltigera canine 1 24.70 6.68 25.55 14.75 7 6.63 4.03 25.73 16.28
0 12.10 13.50 8.95 8.45 Peltigera polydactyla 1 19.80 9.47 11.07 16.38 7 16.12 3.84 13.65 17.16
0 7.43 12.70 6.12 9.54 Sticta fuliginosa 1 30.14 0.09 12.38 0,93 7 48.19 0 27.19 0
0 1.76 1.70 0.57 2.63 Nephroma laevigata 1 19.00 1.59 9.95 1.28 7 28.0o 0 19.40 0
NOTES * Each result is the mean analysis of two 1 sq cm discs of thallus. CN CA ** pH values. 169
Studies on nitrogen fixation in relation to pH are few and
except for the work of Horne (1972), none have been carried out using
lichens. Stewart (1966), quoting the work of Allison and Hoover (1935),
points out that Nostoc muscorum grows between pH 7.0 and 8.5, with a
marked decrease below pH 6.5 and that nitrogen fixation may occur at pH 9.0 . Cobb and Myers (1964), using Anabaena cylindrica, demonstrated maximum nitrogenase activity at pH 7.5, with considerable
fixation continuing at 9.0 and Horne (197), showed in field experiments
in Antarctica, that high nitrogen fixation rates using lichens and
free—living Nostoc, were associated with a pH of 8.7 — 9.0 in wet
alkaline regions. He also stated that Nostoc was present in areas where
the-pH was only 6.6, though here fixation was low.
Horne's results showed that fixation was minimal at a pH
considerably higher than that demonstrated by Cobb and Myers and Haystead
•and Stewart (1972), with Anabaena cylindrica. They showed that the
enzyme was still active at a pH between 5.0 and 6.0 .
5.1.3.4 Effects of DCMU and CN— on nitrogen fixation, photosynthesis and
respiration
The effect of 10 5M DCMU was observed on nitrogen fixation and
photosynthesis. There was total inhibition of photosynthesis in
Peltigera canina, (Figure 16), though not with P. polydactyla and is in
accord with the work of Cox and Fay (1969) who showed that CMU is an
inhibitor of photosystem II, the oxygen producing photosystem, but not
inhibition of photosystem I. 3 x 10-5 CMU was used in inhibition studies
by Cox and Fay and the fact that P. polydactyla was not totally inhibited
suggests a greater number of active enzyme sites in this lichen. On a
unit area to weight basis there is more chlorophylla in P. polydactyla
than in P. canina. 12
170 10
8
6
4
2
2
4
6
10
12
14
16
18
20
22
Light Dark I Light 20,000 lux 24 20,000 . lux + DCMU Dark
• 16 32 48 64 80 96 112 128 Minutes
Figure 16. Oxygen evolution and uptake in the presence of 10-5M DCMU by dines of Peltigera canina thallus (o) and P. polydactyla thallus (n). 171
Wessels and van der Veen (1956), studying the effect of phenylureas on chlorophyll molecules, suggested, from their results, that only definite "active" chlorophyll molecules were affected, which were linked with vitamin K and it therefore follows that a uniform strength solution of DCMU would inhibit a. more concentrated suspension of chlorophyll less (see their values which produce 50% inhibition).
There was no evidence of DCMU inhibition of the nitrogenase in lichen thalli in an ex—field condition (Figure 17). Cox and Fay have shown that there was no effect of GNU on nitrogen fixation, using nitrogen starved cells, but in those.cells which were not starved, there was a 50% reduction in activity? Cox and Fay (1969).
Electron micrographs of Peltioera canina, show structured granules in the vegetative algal cells, which are nitrogen storage bodies
(Griffiths, Greenwood and Millbank, 1972), therefore it is possible that the nitrogen—fixing cells are not starved. -1 In thalli that were pre—treated with 0.25g 1 sodium nitrate solution (2.5mM), for 68 hours, a small reduction in activity was noted
(Figure 17), but did not compare with the results of Cox and Fay, Since Smith (1960, 1960a) used 5TrEsolution.s of nitrogenous compounds, for his uptake work with lichen discs, 5.0mM NO3— might have given a greater effect.
Lex, Sylvester and Stewart (1972) have shown that DCMU inhibits acetylene reduction under conditions which stimulate photorespiration, i.e. a low pC0 atm and a high p0 atm. Since the 2 2 experiment was carried out in the presence of 10mM HCO3 and at a p02 of 0.2 atm, there is little likelihood of this effect being shown.
The effect of CM— was also observed on photosynthesis and respiration, but not on nitrogen fixation, where it is known to be an alternative rathaiggigatia of the nitrogenase enzyme (Postgate1.1970). 172
40 eo 120 16o 200 240 280 Minutes
Figure 17. Rates of nitrogenase activity ( C2112 Reduction ) in discs of Peltigera carina thallus a) treated with 105M DCMU e3), b) treated with codium nitrate 3mM for 68hr (0) and c) treated with sodium nitrate 3mM for 68hr and 10-5M DCMU (s). 173
There was no effect on dark respiration at the concentration
used, 105M. However, photosynthesis was inhibited (Figure 18). Hill
and Whittingham (1961), state that HCN as opposed to CN, in the light,
may affect photosynthesis at lower concentrations than that needed to
inhibit respiration. Although KCN was made up in solution, HCN could
have been the molecular state according to the equation,
KCN H2O HCN KOH
Respiration was not altered and as Farrar (1973) states,
basal fungal respiration is cyanide insensitive and thought the blue—
green algae are cyanide sensitive, since these form a relatively small
proportion of the thallus, the effect from this fraction could well be
imperceptible.
5.1.3.5 Variability of photosynthesis and respiration Water is essential for all metabolic processes. With
photosynthesis, activity occurs at varying saturation levels for
different lichens, from moist air being sufficient for desert lichens, to
total saturation for those under water. In Peltigera canina, 905 of
saturation gives maximum activity, Smith (1960). The suggested reason
for this is, that at higher saturation levels, the water fills the
intracellular interstices and free gas exchange is not possible. Scott
(1960) found with P. praetextata however, that gas exchange was only
possible between the loosely woven hyphae at higher water saturation
levels.
The findings of Smyth (1934), Mee (1939) and Butin (1954) do not agree with Smith's and have all shown that in P. canina and other
Peltigera species with a loose thallus structure, that gas exchange can
take place when the thalli are fully saturated. 24
16 32 48 64 80 96 128 144 160 176 192 Minutes 112
Figure 18. Oxygen evolution and uptake, in the light (10,000 lux) and dark, by discs of Peltigera canine thallus; and the effect of 10-5.M CN—. 175
Respiration is affected by water as is photosynthesis. Smyth
(1934), has indicated that in Peltigera canina, there is more or less a
linear response to increased saturation, which appears to be a general
relationship, with a maximum rate at 85 — 90(70 full saturation, but unlike
photosynthesis, there appears to be no inhibition at higher levels.
In the oxygen electrode, the lichen thallus is completely
submerged in a closed system and water would enter during the
pre—incubation period. Pre incubation as carried out by Baddeley, Ferry
and Finegan (1971), after evacuation, as a means of ensuring comparable
results was not used since it was thought that it might offset the.
object of gas exchange. However, since all the treatments were the same
in this study, results are comparable.
Photosynthesis and respiration were assessed by measurement of
oxygen evolution and uptake and not by the more normal measurement of CO2
exchange, using infra red gas analysis. Therefore little comparison can
be made with the results of, e.g. Harris (1971), Kallio and Heinonen
(1971) or Kershaw (1972).
It has already been shown that young and old thalli have
differing nitrogenase activities and a similar effect can be found in
photosynthetic and respiratory rates in Peltigera canina, (Figures 19 and
20). Oxygen_ evolution in mature thallus, was more rapid in two out of
the three samples tested (Figures 19A and 19B), compared to young tissue,
with the third being less so (Figure 20). In the respiration studies,
two findings were recorded, 1) respiration was higher in all cases of
mature tisue, compared to young thallus and 2) out of the three samples
tested, respiration exceeded photosynthetic oxygen output in two (Figures
19A and 20).
The data in Figure 20, suggest that at relatively low light
intensities;, there may be continual carbon loss, even though there is a • 9
8 16
7,000 lux Dark I 7,000 lux 7,000 lux —I—Dark I 7,000 lux --I I
7 14
7,000 lux —I-- Dark ,000 lux --I -7,000 lux -F-- Dark —I 7,000 lux —I
6 12
a
lua
a 5 s 10 l ha a llu
A ha ' T
U ' P
U llus ture ha
. 49 'Ma 0 4 8 ture
4. ' P
F 'Ma
LL llus
ri ha
r-4 ON — 'Young — 4. ri ' T 4. O ke tion ta 3 6 lu
Up
0 'Young ke I Evo ta en en Up Oxyg
en 0 Oxyg 2 I 4 Oxyg
ti 0 lu o a Ev
en 1 g 2
16 32 64 eo p10 (1sq om)-1 16 32 64 80 2 p102 (lsq em)-1 Minutes hr Minutes hr— 1 Figure 19. Oxygen evolution and uptake by ,mature, (0) and 'young, (0) discs of Peltigera carina thallus in tho light and dark in distilled water at 17°C. 2 16
Dark 8,250 lux Dark 11,700 lux Dark 15,900 lux Dark
14
2 12
4 l NO
a 6 B 8 a
D 0.1 8 6
10 4 A) Oxygen Uptake 'Young' Thallus B) Oxygen Evolution — 'Young' Thallus C) Oxygen Uptake — 'Mature' Thallus D) Oxygen Evolution — 'Mature' Thallus 12 2
14 10 20 30 60 70 80 90 100 110 120 130 140 Minutes
Figure 20. Oxygen evolution and uptake by 'mature' (M) and 'young' (0) discs of Peltigera canina thallus in the light anddarkinAllenandArnon'smediuminthopresenoeeliC0.7ions (1g/1), at 20°C. 178
slight compensation by the increased carbon fixed ( assumed, by a change
in the rate of oxygen evolved) at the higher light intensities applied.
In an experiment with Peltigera polydactyla, not involving tissue age
(Figure 21), a similarity was noted. Under natural conditions, i.e. with
no added sodium bicarbonate 'but with intermittent dark and low light
cycles, there is a continuous carbon loss (oxygen uptake), which must be
to the detriment of the thallus. However, when the dark periods are
interspersed with high light cycles, there is a gradual increase
suggesting a very low rate of net carbon fixation and this may indicate
one reason for the slow growth rate of lichens.
Wilhelmsen (1959), has pointed out that chlorophyll is low
in lichen thalli in the summer and Smith (1961) suggests that most growth
occurs in the autumn and spring under more favourable conditions, but
during these periods of the year, the light will be relatively low and
the length of the dark periods will exceed those of the light. It is
' difficult to see in view of the results presented, how growth can occur
at all. It is assumed that fluctuations in light and temperature will be
critical to the growth of the thallus and show why lichens like
Peltigera species can be capable of relatively quick growth, but can also
deteriorate rapidly and seem to have a very short life—span.
Under field conditions, i.e. with no added sodium bicarbonate,
but with varying light intensities up to 60,000 lux in Peltigera canina
and 100,000 lux in P. polydactyla, the 'compensation point', when oxygen
evolution equates with oxygen uptake, was at 3,500 lux and 35,000 lux
for the two lichens (Figures 22A and 23A), which could probably be
explained as an adaptation to habitat. However, as with all other
studies on lichen physiology, individual areas of the thallus vary in
oxygen exchange and Figure 2213 and 22C shows that unlike the
'compensation points noted above for P. canina, values were also 0
4
6
8
10
1
16
18
20 Dark1,000 2,000 * 4,000 Dar 14,000 5.000i „ 10,000 I Dark 10,000 120,000 Dark2° 40,000 60,000 / 1 lux 1 lux k Dar:: Lark 100,060 lux 1 1 lux lux lux lux 1 lux I lux I luxIC41 lux 1 lux lux
24 48 72 96 120 144 168 192 216 240 264 288 312 336 360 Minutes
Figure 21. Oxygen evolution and uptake in th4: light ane dark by a disc of Peltiaera uolvdactvla thallus in Allen and Arnon's medium at 25°C. 15
10
(B) e(A) (C)
(2) Point of nil gas exchange ( 'compensation point' ) 3,5C0 lux (A) Inhibition of oxygen evolution above 25,000 lux.
(2) Point of nil gas exchange ( 'compensation point' ) 24,000 lux (B) No inhibition of oxygen evolution by light.
(2) Point of nil gas exchange ( 'compensation point' ) 25,CCO lux. (C) No inhibition of oxygen evolution by light.
(0) Point of nil gas exchange ( 'compensation point! ) 1,000 lux. Inhibition of oxygen evolution above 25,000 lux.
0 10 20 30 40 50 60 K lux
Figure 22. Oxygen evolution and uptake by Peltigera canina thallus at 15°C in the presence (0) and absence (0) of HCO3— ions (200mg/l), at different light intensities. Cb O 40
30
(A) 20
10
Point of nil gas exchange ( 'compensation point' ) 35,000 lux. No inhibition of oxygen evolution by light. 10
Point of nil gas exchange ( 'compensation point' ) 3,000 lux. (A) Inhibition of oxygen evoluton above 10,000 lux.
Point of nil gas exchange ( 'compensation point' ) 3,000 lux. (B) 20 Inhibition of oxygen evolution above 40,000 lux.
0 10 20 30 40 50 60 70 80 90 100 110 K lux
Co Figure 23. Oxygen evolution and uptake by Peltigera polydactyla thallus at 25°C in the presence (C) and absence (11) of HCO3— ions (1g/1), at different light intensities. --I' 182
determined at 24,000 lux and 25000 lux, more in accord with the data.for.
Peltigera paldL.actzla.
In conditions of very bright light, 60,000 lux to 100,000 lux
(Figures 22, 23 and 24), the rate of oxygen evolution is much lower than
the maximum rate, indicating that it is denaturation of the chlorophylla
as suggested by Fogg (1964), rather than a pure limitation of the system.
Figure 25 substantiates this idea. It shows the effect when oxygen
exchange was monitored at 12,000 lux in. medium with and without 20MM
sodium bicarbonate ions, but with an intervening period at 60,000 lux.
The subsequent period at 12,000 lux shows total inhibition of oxygen
evolution, when the sodium bicarbonate cannot have been depleted and is
most unlikely to have been inhibitory itself.
However, Figure 23A suggests, that if the 'compensation
. point' is very high due to habitat variability, as already proposed, then
considerably higher light intensities will be required to denature the
chlorophylla and in this instance 100,000 lux was not detrimental.
The 'compensation point' was assumed to be due to the
photosynthetic oxygen output, being equal to the uptake of oxygen, due to
both photorespiration and dark respiration. With the addition of sodium
bicarbonate, photorespiration was largely inhibited (Lex, Sylvester and
Stewart, 1972) and a true rate of photosynthesis could be estimated.
There is evidence to suggest that enhanced carbon fixation
occurs (a period of net oxygen evolution) when thalli are incubated in
medium without added bicarbonate ions, on illumination after a dark period,
during which time oxygen had been evolved. The oxygen output was
evidently related to the length of the dark period (Figures 26 and 27),
which suggests that photosynthesis greatly exceeded total respiration
(including photorespiration) until the 002 was depleted ;
photorespiration and fungal respiration then equated with photosynthetic Point of nil gas exchange ( 'compensation point' ) 4,000 lux. Inhibition of oxygen evolution above 10,000 lux.
Point of nil gas exchange ( 'compensation point' ) 2,L00 lux. Inhibition of oxygen evolution above 40,000 lux.
0 10 20 30 40 50 60 70 80 K lux
/ Coo Figure 24. Oxygen evolution and uptake by Peltivera polydactyla thallus at 15°C ka) and 5°C (DO in the presence of HCO3 ions (1g/1), at different light intensities. L41
le
16
14
12
10
Medium Minus HCO — 22114 HCO 10mM HCO — 20mM ECO — 3 3 3 3 8 12,000 60,000 lux 60,000 lux 60,000 lux 60,000 lux ---I 12,000 lux lux
6
4
2
,....---- . 20 40 60 80 100 120 140 160 180 200 220 Minutes
Cb
Figure 25. Oxygen evolution by Peltigera caning thallus at 25°C in different concentrations of mid HCO3 ions, at two light intensities. 14 Medium -HCO - ions Medium -HCO - ions 3 (B) 3
12 12 A1) 25,000 lux after dark period A) 1,000 lux after dark period A2) 10 10 B) 1,000 lux in fresh medium B) 25,000 lux
C) 0 lux in fresh medium C) 0 lux
(A)
C U U b (B) Meaium -HCO - ions 3
0 A) 25,000 lux after dark period ON
a. 5. B) 25,000 lux
0 lux
) 25,000 lux in medium +HCO - ions (1g/1) 3
10 10
12 12
14 14
16 16 (c) (c)
10 20 30 40 50 60 0 10 20 30 40 50 60 0 10 20 30 40 50 60 Minutes Minutes Minutes
Figure 26. Oxygen evolution and uptake in Peltigera carina thallus at 25°C in the absence of HCO3- ions J1 at two light intensities and the effect of darlaless on subsequent oxygen output. 186
6
4
2
0
2
E 0
cotr. 4
ON
6
0
8
10
0 lux 10,000 lux
Medium —HCO — i na 3 12
0 5 10 15 20 25 30 Minutes
Figure 27. Oxygen evolution and uptake in Peltigera polydactyly thallus at
o 25 C, at two different light intensities. 187
gas exchange.
Higher concentrations of CO2, i.e. higher than would normally
be found in in vivo conditions, were tested. Various solutions of mM
. sodium bicarbonate were supplied to thalli, to augment the data from the
in vivo conditions described above , in order to simulate varying periods
of darkness. Exactly analogous effects were obtained (Figure,28). This
concept is illustrated in Figure 29, but that when photosynthetic and
respiratory activity is rapid (Figure 30), the bicarbonate ions are used
up faster, though temperature and light will also affect the uptake.
Photosynthesis and respiration were both enhanced by the
addition of sodium bicarbonate ions. Fogg (1964) reported that 002 may
be limiting to photosynthesis, but Beevers (1960), reviewing the work of
many investigators indicated that CO2 was inhibitory to respiration in
high concentrations. In the light, sodium bicarbonate will remove the
limitation, though as Fogg (1956) points out, that while there is an
•expansion of the photosynthetic process, linked to nitrogen fixation
in obligate phototrophic nitrogen—fixing organisms, the competition for
substrates cannot be ignored and in this there is no equal sharing, since
photosynthesis benefits at the expense of nitrogen fixation.
In the presence of bicarbonate ions, the 'compensation
points' were much reduced, i.e. 1,000 lux and 3,000 lux for Peltigera
canina and P. polydactyla respectively (Figures 22D and 23B and 23C).
Table 40, shows the addition of different concentrations of
HCO - under different light intensities in Peltigera canina and P.
polydactyla. Respiration in both lichens is increased, but in the thalli
tested, was little affected in P. canina. Photosynthetic activity was
also stimulated in both lichens, but again the response was greater in
P. polydactyla. Each result is at least the mean of triplicate readings,
therefore it ismore likely to be an inherent characteristic of Peltigera 10 188 (A) 9
8
7
6
5
4 (B)
3
2 to
:NI 1 O (C) ri
(D) (A) 25,000 lux and 10mM HCO — ions 3
(B) 25,000 lux and 2mM HCO — ions 3
(C) 25,000 lux and 0.1mM HCO — ions 3 (D) 0 lux and —HCO3 ions
(E) 0 lux and 2mM HCO — ions 3
(E)
0 10 20 30 40 50 60 Minutes
Figure 28. Oxygen evolution and uptake by Peltigera canina thallus at 15°C in the presence
and absence of HCO3 ions at two light intensities. 6
4
2
Iavk CO3. a,lekc{ ex.ow.nonu,s1y Cog. 8,06.,a, medium 63 clozen. res.?rrol-ion
ri,c,t-efesvArokie., orevaktou...1 j"R" 5 2
0 r-t 4
to 14 ava.41 o.1, I e. yes?iva.k to-r.
6
8 T. 1.154
•
0 30 60 90 120 150 180 210 240 270 300 330 360 (Minutes)
Figure 29. A theoretical postulation of the relationships between photosynthesis and respiration in the presence and absence cf CC2, el:Aie-.(eljr Sa_vu...(a).es Lick(2.‘,
10 190
(A)
(B)
E tr
a (c)
o (D)
(E)
(A) 25°C 10mM HCO — 60,000 lux 3
(B) °C 10mM HCO3 60,000 lux 15
(c) 15°C 2mM HCO — 60,000 lux 3
(D) 2mM HCO 25,000 lux 15°C 3
(E) C 1mM HCO — 60,000 lux 15° 3 (F1) 15°C OmM HCO — 60,000 lux (F2) 3 (F2)
0 10 20 30 40 50 60 70 Minutes
Figure 30. Cmygen evolution and uptake by Peltigera cenina thallus at 15°C and 25°C in the
presence and absence of different concentrations cf HCO3 ions at two light intensities. Table 40. The effect of different concentrations of HCO - 3 ions on oxygen exchange ( photosynthesis and respiration ), at different light intensities, in two lichens.
Peltigera polydactyla
Light iL 02 ( 1 eq om )-1 hr-1 Intensity 0 2 5 10 (lux) mM HCO - mM HCO - mM HCO3 mM HCO - 3 3 3
0 -9.8 nd* nd -20.7
4,000 -2.6 -2.2 +2.2 +4.0
10,000 -2.5 +13.8 +21.0 +29.3
20,000 -1.5 +19.7 +23.3 +24.0
Peltigera canine
Light pl 02 ( 1 sq cm %) 1 hr- 1 Intensity 0 2 10 20 - (lux) mM HCO - mM HCO - mM HCO mM HCO3 3 3 3
0 -7.8 / -16.4 -9.7 nd nd
2,000 +2.6 +2.0 nd nd
6,000 +1.3 +5.0 nd
12,000 +1.1 +9.6 +14.8 +20.6
25,000 +5.8 +11.8 +16.3
NOTE :- * nd - not determined. 192
p olydactyl a.
These changes in the photosynthetic rate, shown by the relatively rapid oxygen liberationl in absolute terms, would hardly effect the p02 atm within the thallus and would thus be most unlikely to inhibit nitrogen fixation, particularly as it does not relate to in vivo field conditions.
The Q has been calculated for both Peltigera canina and P. o2 polydactyla, at 5°, 15° and.25°C (Table 41) and except for the inverted result for P. polydactyla at 15° and 25°C, shows that with increased temperature, oxygen uptake is more rapid.
Scholander, Flagg, Walters and Irving (1952), quote values for arctic and tropical lichens, the mean Q02 at 10°C (0.19), for arctic species, being the same as for tropical lichens at 20°C, though their figures at 20°C for arctic lichens are higher, 0.32. While all the lichens fall into the same range of activities, the mean Q02 values are not the same at a similar temperature, there being nearly a two—fold o difference with the' arctic lichens at high temperature (20 C)„ showing more activity than the tropical species. It is difficult to explain, if one considers that the arctic lichens may be cold adapted whilst the o tropical lichens live in a "steady temperature of 20° — 30 C" all the time.
Baddeley, Ferry and Finegan ('1971), also show respiration rates at various temperatures between 20°C and 40°C, for nine lichens, as
02 (g water saturated thallus)-1 minute-1. This gives a mean value at 20°C, assuming 200% water content (Smith, 1963) of 4.0 pl (mg dry -1 -1 weight thallus) hour , which is approximately a 3—fold increase over the results obtained in this study and approximately a 20—fold increase over the results of Scholander et al (1952).
Smith (1963), however, quotes figures as 111 oxygen uptake Table 41. Q02 values for two Peltigera species in the absence of HCO3— ions.
jil 02 ( mg dry weight thallus )_1 hr-1 Lichen 5 oc 15°C 25°C
Peltigera canina nd
Peltigera polydactyla 0.7 1.5 1.2
nd = not determined. 194
(100 mg dry weight)—/ hour 1 at 20°C of 121 pl for the algal rich zone
and 70 pl for the medulla, which is equivalent to 1.9 pl (mg dry weight)-1
hour-1 and is very close to the values in this study, though he makes the
point that "total oxygen uptake by tissue zones is 56% greater than with
undissected controls", so on this basis, Smith's results would become
1.1 pl (mg dry weight total thallus)-1 hour 1 , exactly analogous to the
figures obtained.
Baddeley et al (1971) suggest that evacuation of the thallus,
to give 100% saturation and therefore good gas exchange, is important,
but is unlikely to achieve such an effect. It does emphasise the need to
know environmental conditions, prior to the assay and also the fact that
different lichens do not behave in the same way.
The effect of temperature on oxygen exchange was observed, o o o using Peltigera canina and P. polydactyla, at 5 , 15 and 25 C iin 10mM
HCO3 Q10 (temperature coefficient), is the increase. in the rate of a
process (expressed as a multiple of the initial rate) produced by raising
the temperature 10°C (Abercrombie, Hickman and Johnston, 1951). There _ ) was a very small increase Q10 (15o 25°C) oxygen evolution by P. in polydactyla (1.1), though better than the Q10 (5° — 15°C), where it was
zero. However, respiration was affected more and a 0,10 of 2.3 was found o o between 5 and 15 CI but not 15° and 25°C. Mean activity was greater at
15°C and emphasises the variability of lichen thalli.
In Peltigera canina, temperature had more affect on oxygen
evolution and a 010 (15° — 25°C) of 2.2 was recorded. These values fell mostly between 2 and 3, usually noted for biological systems and it
appears that temperature had little affect in the thalli tested.
Scholander et al (1952) calculating the Q10 for tropical and
arctic lichens together, has shown a wide scatter of results, between 1.5
and 5.0 at 0°C, though the scatter is much reduced, with the majority of 195
results at 10°, 20° and 30°C, falling within the range 1.0 and 2.5 .
Baddeley et al (1971), quote respiration rates and the
calculated mean (20° — 30°C) was 1.4 with maximum and minimum values Q10 of 0.8 an 1.9 . It indicates that low temperatures are not inhibitory to
lichen metabolism.
Adaptation to low temperature is important and the lichens
which occupy an increasingly important role in the flora, with nearness
to the poles, may be particularly adapted to low temperatures. As many
lichens in the tropics can also withstand unfavourable low temperatures
(Lange, 1965), characteristic seems to have been fixed in this group of
plants in the early stages of evolution (Kallio and Heinonen, 1971).
Richardson (1973) has correlated the findings from various
studies and showed that alpine lichens could show net photosynthesis o down to —24 C, but were not harmed at much lower temperatures.
Scholander et al (1952), comparing the rates of tropical and arctic
lichens, though they did not use identical species for the two tests,
showed that while the spread of their data often varied by as much as
100%, using eight replicate samples, there was no total overall
difference between the two groups. Individual lichen species varied and
with Peltigera and Sticta species, those from the Arctic at 10°C,
respired as rapidly as the tropical species at 20°C and in this respect
appear to be cold adapted.
Baddeley et al (1971) also showed adaptation to high
temperatures. Of eight British species, four had temperature optima at
or above 40°C (the highest temperature tested), and all these species
were either from stable shingle banks, heathland or the tops of dry stone
walls, whilst the other four, which had optima at 35°C, were collected from tree boles. They point out that the periods were short at the high
temperatures tested and showed that when returned to lower temperatures 196 there was definite inhibition, indicating as Scholander, Flagg, Hock and
Irving (1953) have pointed out, that desiccation at high temperatures (or low temperatures) is an adaptation to preservation.
Despite rapid activity of arctic lichens at high temperatures, they can normally only be expected to metabolise at a fifth the rate of their counterparts, on a purely enzyme kinetic basis and because of other factors, i.e. the short arctic summer, low temperatures and drought periods, Scholander et al (1953) have stated that in fact they will not be likely to metabolise more than a twentieth to a thirtieth of the rate of tropical species.
It is interesting to speculate, that if as Richardson (1973) says, photosynthesis in arctic species can continue at low temperatures, whilst Scholander et al (1952) show that respiration is greatly reduced, then there would be a distinct advantage to lichens in these habitats, with fixed carbon not being immediately respired and thus for the short periods during. which metabolism was possible, a measureable increase in the biomass could be achieved. 197
cCd1CLTJTTIwG REMARKS
It is certain that lichens, as associations, can exist in
situations which the partners could not tolerate (Haynes, 1964). All
lichens exist in extreme ecological niches. They need specialised stable
conditions and no or little competition from excess shade forming
vegetation. Thomson (1972) says "the lichens are well known as being
supersensitive. Typically they occupy micro—habitats". However, they
are well adapted and have spread from the poles to the tropics.
In those extreme environments, where lichens exist, water
availability is still of principal importance and in hot dry conditions,
the lichen thallus is adversely affected. Smith (1961) has shown that
there are definite periodic variations in dry weight, nitrogen and
carbohydrate content and the variation in nitrogen was demonstrated in
the total thallus nitrogen recorded at different times of the year, for
Peltigera polydactyla, see p 73.
Their metabolism is also affected by the presence or absence
of water. Hitch (1971) found a relationship between water and nitrogen
fixation and Kershaw and Rouse (1971, a) and Kershaw (1972) measuring net
photosynthetic rates in Cladonia species, found that different species
showed optimal rates of activity at varying water saturation levels and
proposed an adaptive response to different ecological niches.
Variability within lichen species is very pronounced. Smith
(1961) has tried to overcome this by using large samples (350 — 400 discs
per experiment), but there is reason to think that large samples merely reduce this effect and not by any means eliminate it, see p E7. I have shown (1971) that whilst most samples of Peltigera
had different rates of nitrogen fixation (different thalli within a
single colony), it was possible by selection to achieve uniform maximum
rates in all three species tested, a finding also noted in this study, 198
with Peltigera canina and P. 2olydactzla.
Millbank and Kershaw (1969) have also indicated a similar
finding with Peltigera aphthosa. They assayed 15N uptake over a period 2 of 12 days and found that the saturation of the cephalodia, not only 15 varied in its holding capacity, in terms of N enrichment, but also in
the time it took to reach the saturation point and they said "throughout
the work, the problem of variability has been acute. The use of clonal
material is quite impractical, due to the slow growth rate It
has been necessary to rely finally on the use of experimental material
from a single site to avoid gross physiological variation found between
material from widely separated areas." This was the case with the
Peltigera samples used in this study and for this reason the majority of
Peltigera canina came from site (c) and P. polydactyla from site (c),
Table 1, though other samples were tested for comparison.
The problems associated with variability cannot be stressed
•t' strongly and while other organisms, may and do vary, because of the
slow growth rate of lichens, very little can be done to overcome this.
Kershaw (1974) noting this variability, pre—incubated his
lichen thalli for two days prior to analysis, to stabilise the metabolism
and he said "The lichen material was kept in a moist condition
for at least two days. This pre—treatment was de3igned to
remove any possible physiological variability from the lichen material,
induced by previous and variable field conditions to which it had been
exposed." However, considering the long term experiments in this study,
to achieve stability, it is doubtful whether Kershaw's treatment would
have had any effect at all.
In many of the studies on lichen physiology, the whole plant
has been tested, after removal from the field site, either directly or
after a period of storage and subsequent incubation, since as Millbank 199
. and Kershaw (1969) stated, clonal material cannot be prepared. The
conditions might well have a bearing on the experiment in hand and could
easily explain some of the variability encountered and contradictions
reported.
If the results are similar then it is probable that
comparisons are valid, but since lichens are so variable, with single
species being adapted to specific sites, such as the tops and bottoms of
trees, or sunny and shady rocks etc. , it would appear that to make
generalisations, or try to coerce different lichens to a uniform
laboratory regime, is very dangerous and in view of this much more
substantial evidence is needed from a greater variety of lichens and
different habitats.
To this end the field experiments of Lange, Schulze and Koch
(1968, 1970 and 1970a, see Kappen, 1973), are extremely worthwhile, with
as many parameters being measured, both during the experiments, but also
•beforehand.
Rapid nitrogen fixation in lichens is not compatible with
growth. Kratz and Myers (1955) showed that Nostoc muscorum could divide
every 191- hours and Haystead (pers comm) has indicated that during the
experiments of Haystead, Robinson and Stewart (1970), their algae could
divide in as little as 2 hours. Millbank and Kershaw (1969) showed that
with Peltigera aphthosa at a similar temperature, the rate of division of
the phycobiont could be every 11 hours, but they pointed out that growth
of the cephalodial Nostoc is extremely slow, with most of the nitrogen
being secreted to the mycobiont.
In 1 T studies, nitrogen uptake is slow compared to that
suggested from growth rates in the field. Peltime_Eas are some of the
fastest growing lichens, with annual increases in centimetres rather than
Millimetres. Nitrogen fixation does not appear to be of much value to 200
the 85 of lichens, that contain blue—green algae as the phycobiont, a
greater number than probably fix nitrogen. As Crittenden (1975) points
out, there seems to be no relation between nitrogen fixation and the size
of the thallus, if one compares Peltigera cenina with some of the bigger
Umbilicaria species, though one must consider the time taken to produce
such thalli.
It has been suggested that nitrogen—fixing lichens are some
of the first colonisers and primary producers (Fogg and Stewart, 1968)
and certainly they can provide their own nitrogen, which is non—existent
initially in these sites and thus enable other plants to become
established, but generally speaking they are not lichens of xeric
environments (Horne, 1972); however, note the exception of Collema
coccophorus, a lichen of desert regions (Shields, Mitchell and Drouet,
1957; Rogers, Lange and Nicholas, 1966).
On a biomass assay, there are many non—nitrogen—fixing
' lichens, mainly Cladonias, which in these habitats are much more abundant.
Kershaw and Rouse (1971) point out that the initiation of lichen cover,
is based on water availability and Cladonias in these ecosystems are able
to form a dense layer of vegetation that holds water, prevents desiccation
and initiates a succession to lichen enriched woodland.
I believe that within the vast range of lichen associations,
between algae and fungi, it is inevitable that some fungi would be
capable of having blue—green algae as the phycobiont partner and while it
may be beneficial to those that are nitrogen—fixing, the majority are
quite capable of obtaining sufficient nutrients, such as nitrogen etc. 1
by their inherent sponge—like structure, which passively absorbs them
from rainwater and dust that collects on the thallus surface. 201
SUMMARY CF RESULTS
Nitrogen fixation was measured, using acetylene reduction, in 60 lichens out of approximately 100 capable of the process in the
British flora. The majority of work however, was carried out using only two, Peltigera canina and P. polydactyla, both of which had a rapid nitrogenase, were relatively abundant in the field and easy to handle.
Various methods were tried to find a standard, against which nitrogen fixation could be monitored. In most experiments, whole thalli were used and the effects of oxygen, light/darkness and pH were demonstrated.
Nitrogen-15 / C H analyses showed that under suitable 2 2 conditions, a stoichiometric conversion factor for lichens of 3 : 1 was possible.
Sane work was carried out measuring oxygen exchange rates of photosynthesis and respiration:
From the above studiesl _it became abundantly clear that lichens show great and random variation, both in the morphology of the thallus and in their physiology, within wide, but fixed limits. Evidence of this variability was shown in the initial experiments in which a large number of different lichens were tested for nitrogen fixation, since they varied from species to species and within single species, (p 65). Even after imposing a standard regime on the system, it was impossible to achieve any real uniformity in the response of the thalli. In some instances, thalli were tested for periods of up'to three weeks with virtually no success in achieving uniformity, when nitrogen fixation rates were measured using a number of discs from a single lichen thallus,
(P 92 — 94). 202
Because of the unique properties of lichens such as their extremely slow growth rates and corresponding lack of cell turnover, no attempt was made to alter the structural characters. It became apparent that nitrogen and dry weight were the most satisfactory bases to express nitrogen fixation against, but even these were not entirely satisfactory, because the nitrogen content did vary at different times of the year, (p 73). Nitrogen and dry weight were not the only ones considered. Cell numbers and chlorophylls determinations were also tried.
It was found that the green algae from lichens such as P. aohthosa var. variolosa are extremely tough and could be homogenised in a Potter homogeniser, or pestle and mortar, virtually without loss, but it became clear that the blue—green algae from lichens such as P. canina, are much more fragile. Cell counts were carried out by grinding P. canina in a 6 pestle and mortar and figures of approximately 0.5 — 1.5 x 10 cells sq 1 cm were obtained. It was subsequently shown that these figures were not confirmed by chlorophyll analyses, when comparing undamaged discs of lichen thallus and cell suspensions of known composition. It was evident that the thallus of P. canina, contained 10 times the Nostoc population that estimates from counts of homogenised thalli indicated.
High speed centrifugation of the liquid over the cell macerate, showed that approximately 60% of the pigment was in this fraction, but even using this new factor to re—calculate the results, it still did not account for the total values expected.
The cell pellet was subsequently analysed. When homogenised thallus, made up in 10% sucrose, was layered onto high density sucrose
(80%) and centrifuged, the majority of the whole cells originally counted, were held at the suspension / 80% interface, but any broken particles passed through. Analysis of this bottom fraction showed that it contained far more chlorophylls than could be accounted for by the few 203
whole cells that had been carried through and it was confirmed with
fairly gentle homogenising, sufficient to disrupt the thallus, that only
about 5% of the blue—green algae remained intact, (p 24). Homogenates
were therefore of no use as a basis for comparing nitrogen fixation
rates, being not only complex, but causing a great loss of cells.
Further, there was strong evidence of a wide fluctuation in total cell
numbers.
This was subsequently confirmed by chemical disintegration of
the thallus, using a 10% solution of Cr03. The method was extremely easy
to use and confirmed the high total Nostoc content of lichen thalli that
was suspected. With the Cr0 solution treatment, algal cell counts of 3 6 between 3 x 10 and 2.5 x 107 sq cm-1 were recorded.
Another important finding from this technique was that
heterocysts could be freely observed in lichenised blue—green algae for
the first time without using electron microscopy, (p 43a and 51a). With
homogenisation of fresh thallus, no heterocysts were seen at all being
evidently even more fragile than the ordinary cells. The Cr0 solution 3 disintegrated the fungal mycelium, so that with very gentle extrusions
through a Pasteur pipette, or the weight of a cover slip in the case of
excised cephalodia, long chains of algal cells were readily prepared,
which was essential if heterocyst ratios were to be calculated, since at
least two heterocysts in a chain were needed to determine the
intermediary vegetative cell numbers.
Despite the ease with which these preparations could be made,
it was found that herteocyst numbers bore little or no relation to
nitrogenase activity within single species, (Table 20, p 83). It was
realised that while heterocysts were easily visible and countable, it was
virtually impossible to determine whether they were active and contained
nitrogenase enzyme in a viable state. So again the method was abandoned 204 as a means of comparing nitrogenase activities.
During the analysis of heterocyst ratios, it was conclusively shown that in lichens which had what I described as a monophycobiontic layer of blue—green algae, (as in Peltigeras, Stictas, Nephromas, etc.), the ratio of heterocysts to vegetative cells was of the order of 5%. On the other hand, however, lichens whose blue—green algae were located in a) external cephalodia, e.g. P. aphthosa var. variolosa, P. venosa,
Nephroma arcticum and Stereccaulons etc., b) internal islands, e.g.
Solorina crocea, or c) internal cephalodia, e.g. the Lobarias, and whose main phycobiont was a green species, the heterocyst ratio was increased to 10 — 30% in types a) and b) and up to 55% in type c).
It was postulated that this large increase in the latter case was due to an excessive drain of fixed nitrogen as a direct result of their proximity to the green phycobiont. In order to prove this point, various lichens were tested, which normally had a green phycobiont, but due to an aberration, sometimes produced a chimera, where folioles containing blue—green algae of similar or dissimilar lichens grew on the green thallus. It was found in all cases that at the interface between the two phycobionts, the heterocyst ratio was increased, but that at a few millimetres away, the normal ratio was re—established. This feature was not normally noted in cephalodial species, as the cephalodia are only about 1mm diameter.
In looking for standards against which fixation rates could be monitored, it was finally decided to see if large or small areas of thallus were more or less likely to show uniform nitrogenase activity.
Normally in all experiments, 1 sq cm discs of thallus were cut out with a cork borer. Larger areas of thallus, 10 sq cm sample-1 , did not confer greater constancy, neither did a single disc which was analysed and subsequently divided up. Each fraction showed wide variability, though 205 the total activity of the fractions nearly equalled the initial whole rate, consistent with loss of activity produced by damaging some of the heterocysts, during cutting. In view of all these variable factors, thallus discs were considered as separate entities, with subsequent analysis being related. back to the initial rate in each case.
Nitrogen fixation in lichens is very rapid, with maximum activity in P. canina and P. 22I Laaiyla being about three times that of free—living blue—green alae. Comparatively faster rates, assuming maximum activity, were observed in those species where cephalodia are present, roughly related to the heterocyst ratio, (p 74).
It was noted in the long term that large increases in activity could be achieved with and without the addition of bicarbonate ions, and in the case of P. aphthosa var. variolosa, large increases were also noted with increase in the acetylene concentration. This was much less noticeable with P. canina, (p 96). Other factors which caused an increase in nitrogenase activity, in the short term, over the initial rate recorded, were running the experiment more than once and partial removal of the fungal mycelium, which formed the bulk of the thallus.
This was noted with P. polydactyla which has a much thicker fungal medulla.
The effect of oxygen was tested on nitrogenase activity, using whole, chopped and ground thallus. Whole thalli appeared to give good protection (if it was necessary), which could have been due to respiration of the weft of fungal mycelium, completely surrounding the algal cells, but also of the algal cells themselves, c.f. free—living heterocystous filaments. Oxygen tensions up to 605 had relatively little inhibitory effect, E05 was more effectual and 1005 was immediately and totally inhibitory. It should be noted however, that after a recovery period in air, a second dosage at a p02 of 1 atm. was less harmful and 206
6q the reasons for this have been discussed, (p t6a).
In the experiments in which the thallus was chopped, scraped
and macerated, in some instances nitrogenase activity increased as has
been indicated earlier, though in most cases the effect was soon lost
and activity decreased rapidly. In one set of experiments, where the
thallus was ground up in an atmosphere of argon, tested, then exposed to
either oxygen or argon, the thallus that was maintained in argon had a
slightly higher activity, (Table 36E, p 155).
Nitrogenase activity was thought to be governed in the dark,
by the presence of respirable substrates, though it was postulated that
oxygen was an essential requirement as well, since the small cephalodia
of P. aphthosa var. variolosa continued for a much longer period,> 96
hours, in the dark, than larger discs of P. caninal < 24 hours, where it
was assumed that the carbohydrates and oxygen were limited, in a closed
system. Re—activation of the enzyme in the light, after this period,
with P. canina and P. polydactyla took several hours.
pH affected nitrogenase activity at high and low hydrogen
ion concentrations. All the routine experiments were run at pH 7.2, or
where bicarbonate ions were addedl at pH e.4, with no loss of activity.
At pH 9.0, P. canina and P. polydact,la were not, or only slightly
inhibited, though two other lichens, Nephroma laevigatum and Sticta
fElizinapa, showed complete loss of activity. At pH 5.9 however, the nitrogenase of P. canina was inactivated, though on return to pH 7.2
solution, the inhibition was removed.
Initial throughput experiments with different lichen species
. using15 N 2, showed that nitrogen fixed by the algae, rapidly reached the
fungal partner. For most of the experiments, P. polydactyla was used,
rather than P. canina or Sticta limbata, both of which showed rapid
nitrogen uptake, because it had relatively good nitrogenase activity, and .207 more important, the morphology enabled the thallus to be dissected easily, giving good partition of the different fractions.
The rate of uptake appeared to depend on careful treatment of the thallus, much more so than in experiments using acetylene reduction.
It was not the difference in the solubility of the gases in liquid, because recovery after supposed waterlogging, using acetylene, took several days. Waterlogging of the thallus caused total inhibition of 15 uptake of N and the only method which was successful, was to build a 2 special chamber, (p 106 et sea.), and place the lichen thalli in it, in an ex—field condition, with adhering moss etc, remaining undisturbed.
Using these conditions, it was shown that for up to 1 or 3 days, in a 37 day experiment, the rate was rapid and a ratio of 6 : 1 and in one instance 3 : 1 was achieved. However, calculations based on these results, only gave a doubling time of approximately 200 days, which in view of the fast growth rate of Peltigera species, showed that activity under the circumstances was not fast.
Measurements were made of the photosynthetic and respiration rates of both P. canina and P. polydactyla. Whole discs were suspended in a Clark type oxygen electrode and exchange rates were recorded at different light intensities, temperatures and in the presenCe of the inhibitors DCMU and potassium cyanide.
Under normal conditions, i.e artifically created field conditions, there was very little change in oxygen output with increase in light intensity and respiration in the dark was low as well. However, in the presence of increased CO2 levels, applied as mM bicarbonate solutions, a compensation point was recorded at approximately 4,000 lux, where the rate of oxygen exchange was nil. It was also found that the rates of oxygen output and uptake, were more rapid under these conditions. 208
Darkness could simulate the conditions of applied HCO ions, 3 since photosynthetic activity increased after periods of dark respiration
was re—fixed and the longer the dark period, in which the liberated CO2 the longer activity lasted. MM solutions of bicarbonate ions, in place
of dark respiration, had a similar effect, ( p 187).
As with all other experiments, discs of thalli showed
different oxygen exchange rates and therefore where possible, many
experiments were carried out on a single disc, so that comparisons could
be made. Differences in thalluS activity were also noted when high light
intensities were used. Kost results showed that in the presence of
bicarbonate ions, the compensation points were at 1,000 and 3,000 lux,
as has been noted earlier, with maximum photosynthetic oxygen output
occurring at 40,000 lux and definite inhibition in the region, 60,000 to
100,000 lux. However, in one instance there was no inhibition at the
higher intensities applied and correspondingly, the compensation point
' was much higher too.
Bright light however, was considered to be an essential for
the longevity of lichen thalli. An important finding was that when dark
periods were interspersed with low light periods, carbon loss by
respiration was considerably greater than that fixed by photosynthesis,
though this could be reversed by interspersing the dark periods with
bright light. It was assumed that in the winter months, fallen leaves or.
other vegetation could quickly kill off the lichen thalli, particularly
of those lichen species that inhabit semi—shaded ecological niches, such
as Peltigeras.
In terms of oxygen output in the preSence of added CO21 not
encountered in the field, bright light was sufficient to raise the p02
only just above the 205 level and would not have had any inhibitory
effect on the nitrogenase enzyme. 209
Temperatures in the range 50 25°C were tested on oxygen exchange to note the effects on photosynthesic and respiratory activity.
Q values were calculated and showed that under the conditions applied, 10 there was very little effect. Q0 values were also noted, (p 192). 2 DCMU totally inhibited photosynthetic activity in P. canina, but not in P. polydactyla, (p 169), and it was thought that since there is more chlorophylls in P. polydactyla, per unit area, not all the active sites were destroyed. Nitrogen fixation by discs of thallus in an ex—field condition were not inhibited with DCHU, but after the addition of 3mM NO solution, over 60 hours, there was a slight reduction in the 3 rate. CN was not used as an inhibitor in nitrogen fixation experiments, as it is a substrate for nitrogenase. Respiration and photosynthesis were affected to some extent in the dark and light and the reasons for this have been postulated, (p 173). 210
APPENDIX 1 Grinding medium
The grinding medium for the maceration of lichen thallus consisted of :-
Allen and Arnon,s mineral salts medium
10% sucrose (w/v)
80g sucrose taken and made up to 100m1 with glass distilled water. 25m1 diluted to 200m1 with Allen and Arnon's medium. The 805 sucrose was used for fractional centrifugation to recover clean algal cells. 211
APPENDIX 2 Allen and Amonis mineral salts medium
(see Allen and Amon 1955)
The medium contains the following :— Macronutrients. MgSO4 0.00117 CaC1 0.0005M 2 final nutrient solution NaCl 0.004m ) HPO 0.002M K2 4
Micronutrients, FeSC.711 0 2 ) (Fe) 4.00 ppm ) Na—EDTA ) ) 0 (Mn) 0.50 ppm ) MnSIC4.4H2 ) 1,4003 (o)M 0.10 ppm ) ) ZnS04 .4 N 2o (Zn) 0.05 ppm ) cuso4 . 5N 2o (Cu) 0.02 ppm final concentration H3B03 (B) 0.50 ppm of metals ) NN4vo3 (V) 0.10 ppm ) co(NO3)2.6H2o (co) 0.10 ppm ) ) NiS0 .6H 0 (Ni) 0.10 ppm ) 4 2 Cr2(SO4)3.K2SO4.241120 (Cr) 0.10 ppm 0 0.10 ppm Na2WO4.2112 (w) 0.10 ppm K2Ti(C204)2.2H20 (Ti)
Modified medium was used in this study. The concentration of HP0 was 0.001M and that of FeS0 .7H 0 (Fe) was 1.00 ppm. K2 4' 4 2 212
Practical details
1) Stocks of each of the macronutrients were made up at a x100 1 concentration. (Use 10m1 1 ).
2) A single stock solution was made up containing all of the trace
elements, except for iron, at a x1000 concentration. (Use 1m1 —1.1 ).
3) 10m1 of the macronutrient solutions and lml of the micronutrient solution were taken and made up to 11 with glass distilled water and —1 autoclaved at 151bs (sq in) for 15 minutes. Temperature 120°C.
4) The phosphate solution was autoclaved separately and added afterwards when cool. This prevented precipitation.
ALL THE STOCK SOLUTIONS WERE STORED AT 4°C IN THE DARK
Stock solution — 1
NaCl 23.4g 1 1
MgSO4.7H20 24.7g 1-1 (For MgSO4 use 12.03g 1-1)
Stock solution — 2
CaC1 .2H 0 1 2 2 7.3g 1 (For CaC12 use 5.6g 1"1)
Stock solution — 3 K2HPO4 17.4g 1-1
Trace element solution — 1
Na—EDTA 6.34g 1-1 FeSO4.7H20 4.98g 1-1 Leave to aerate overnight by bubbling with air. Use 1m1 1-1 of
culture medium.
Trace element solution — 2
NiS0 .6H 0 1.1964g in 250m1 4 2 213
Trace element solution — 3 (Make up in 0.1N Natal, needs 1-2 MoO3 3.7508g in 250m1 days to dissolve in the cold) Trace element solution — 4 Na WO .2H 0 0.4485g in 250m1 2 4 2 Trace element solution — 5
ZnSO4.7H20 5.4980g in 250m1 Trace element solution — 6
Cr2(SO4)3.K2SO4.241120 2.4006g in 250m1 Trace element solution — 7
K2Ti(C204)2.21120 1.8485g in 250m1 Trace element solution — 8
CuS0 .5H 1.9648g in 250m1 4 2 0 Trace element solution — 9
Co(NO3)2.6H20 1.2345g in 250m1 Trace element solution — 10
0.5741g in 250m1 NH4V0 3 Use 1m1 of each of the above solutions in 100m1 of trace element stock solution.
Trace element solution — 11
MnS0 .4H 0 5.0760g in 250 ml 4 2 Trace element solution — 12
H BO 7.1330g in 250 ml 3 3 Use 10m1 of each of the above solutions in 100m1 of trace element stock solution.
. When the stock solution has been made up from the above 11 solutions,
use 1m1 in 1000m1 medium. 214
APPENDIX 3 Kjeldahl catalyst for nitrogen determinations
The following catalytic mixtures have been suggested for
Kjeldahl digestions for the assay of nitrogen.
1) CuSO4 20g
K SO 80g 2 4 Na2Se04 9.34g
2) K SC 2 4 97g
Cu SO4 1.5g
Se 1.5g
For microKjeldahl analysis, see Umbreit, Burris and Stauffer.
CuS0 .5H 0 250mg 4 2 Na Se0 2 4 173mg
Method
Take 150m1 of glass distilled water and add 80m1 of nitrogen— free sulphuric acid (10N), slowly with cooling, as the reaction is extremely exothermic. add 37.5mg of mixture 3), which dissolves rapidly.
Make up to 250m1 when the solution is sufficiently cool. 215
APPENDIX 4 Preparation of sodium hypobromite and lithium hypobromite solutions.
15 Used for the preparation of N gas and the subsequent 2 analysis of the digested samples in the mass spectrometer.
255 NaCH solution was prepared. 25g NaOH pellets were weighed into a large flask. Glass distilled water was added to 100m1.
The flask was cooled under running water as the reaction is extremely exothermic. When cold the solution was placed in a 150m1 beaker, covered with 'Parafilm' and cooled in a refrigerator to 4°C.
An ice/water bath was set up at approximately 0°C. The cooled beaker was clamped in the bath and an electrically driven glass stirrer placed in the NaOH mixture. The whole operation being carried out in a fume cupboard.
) 10m1, was poured into a measuring cylinder. Bromine (Br2 ' It was added dropwise with constant stirring at a rate which enabled it to dissolve in the NaOH. 250mg potassium iodide was added to prevent th release of oxygen.
For analysis in the mass spectrometer, lithium hypobromite was used as it was more stable and did not require the addition of potassium iodide. (See Ross and Martin, 1970). 216
5 Preparation of 15N as (To be used in conjunction APPENDIX 2 g with Figures 7a and ,7b, p 107)
Ensure that the system will hold a vacuum to 0.001mm Hg.
This can be checked using the manometer in line.
gh out 1.2 H 2SO4.7H20 i to the side arm of the Wei g (15N 4) n generating flask and add 2m1 glass distilled water to dissolve it.
Add excess, 22m1, NaBrO to the main body of the generating vessel.
Freeze the side arm with liquid nitrogen (-196°C), the
NaBrO with a solid CO /ethanol mixture 2 (-eo°c). Freeze, evacuate and thaw each of the limbs twice.
Taps 0 and H are open, tap P is closed. Taps D and E are open. Close tap C and tap G.
Mix the two solutions in the generating vessel, when nitrogen is given off and flows through the cold trap N into the glass globes K and J.
Taps D and E are closed and the gas in the globes is pressurised by pumping air into chamber N by hand pump. Open tap G.
Raise the level of mercury through K and J with the hand pump, till it reaches just below tap G, close tap G. The 155N 2 gas is stored in globe L.
Evacuate mercury from J and K with a rotary vacuum pump, via tap F, till mercury at its original level.
Open tap El open tap D; close tap D, close tap E.
Pump gas into the globe L as before, with the hand pump.
Repeat the procedure, allowing the solution in the generating vessel B, just to boil, till the level in the capilliary of the manometer does not fall any further. The small volume of the capilliary avoids excess loss of 15N2. 217
Close taps G, H and 0. Remove the spring clips holding
globe L in place and remove it. AlloW the gas generating system to
return to atmospheric levels. 15 Pressurise the N gas in globe L by'introducing the 2 20% Na2SO4 in 5 volume % of H2SO4, via tap P. This is carried out
using apparatus Figure 7b. The glass conical flask is placed on
the ground glass joint, filled with Na2SO4 / H2SO4 and tap P opened. Ilhen the level in the flask is stationary, close tap P. N. B. Do not allow the liquid level in the conical flask to get too low or there may
be insufficient pressure to hold the gas in globe L.
In this way the gas is cleansed of any possible NH and 3 stored till used. 218
APPENDIX 6 Preparation of alcoholic boric acid indicator.
Boric acid is usually dissolved in alcohol, 20g,l,-1 to give a 2% solution, together with indicator in alcohol.
ForIni.L!-112.2..y±112L222. ...2111/LiaHapproj22.1.
2% boric acid solution was made up using glass distilled water. 1% mixed indicator in alcohol was added, 10m1,1-1.
NUDES
Mixed indicator
1.7mg bromocresol green (= bromocresol blue) ) — 11 alcohol 3.3mg methyl red
Characteristics of the indicators used
Colour pH- Range Acid Alkaline
Bromocresol green 3.6 — 5.2* Yellow Blue
Methyl red. 4.2 — 6.3* Red Yellow
* Data from 31)11 Chemicals (1973) United Kingdom Edition.
The indicator in the presence of ammonium — N is grass green and turns through grey to bright pink at the end—point pH 5.0 219
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NITROGEN METABOLISM IN LICHENS
VI. THE BLUE-GREEN PHYCOBIONT CONTENT, HETEROCYST FREQUENCY AND NITROGENASE ACTIVITY IN PELTIGERA SPECIES BY C. J. B. HITCH AND J. W. MILLBANK Department of Botany, Imperial College, London SW7 2AZ
(Received I October 1974)
SUMMARY The blue-green algal population and heterocyst frequency in Peltigera aphthosa, P. canina and P. polydactyla have been estimated by means of a chromium trioxide maceration technique followed by visual and photographic observation. Populations of the order of roe cells/sq cm thallus in P. canina and P. polydactyla, and rob/sq cm in P. aphthosa have been found. These figures are some threefold greater than previously reported; the earlier results are explained by unexpectedly high fragility of the algal cells. A heterocyst frequency of c. 3.5% in P. canina confirms previous estimates obtained by an- other technique; P. polydactyla is similar but P. aphthosa has a very high heterocyst frequency, c. 22% of the total cells. These findings are reflected in the nitrogenase activity and support the concept that the enzyme is only active in heterocysts.
INTRODUCTION Studies on the nitrogenase activity of lichens are comparatively few in number and understandably relate the results obtained to the dry weight of the thallus in the majority of cases. Millbank and Kershaw (1969), and Millbank (1972) related the rates of nitro- genase activity observed to the Nostoc population of the thallus and reported that the rates were very rapid, although the second report referred to previously suspected losses and amended the results significantly. An investigation of the heterocysts in lichen Nostoc filaments carried out by Griffiths, Greenwood and Millbank (1972) showed that the proportion (3.3%) was much lower than normally found in free-living cultures of the alga. When this finding was associated with the rates of nitrogenase activity observed, it could implicate the vegetative cells (Millbank, 1972). The technique for estimation of heterocyst numbers, although very reliable, was extremely laborious, and a less demand- ing technique suitable for a general survey of the heterocyst population in blue-green phycobionts was sought. Arising out of the technique developed, accurate and reliable estimates of the blue-green phycobiont cell population were obtained for all the species of Peltigera studied and these data are presented here with their implications on the rate of nitrogenase activity and its site.
MATERIALS AND METHODS Lichen material Collecting site Peltigera aphthosa var-variolosa Den of Airlie, Perthshire, Scotland. P. canina Culbin Forest, Morayshire, Scotland. P. polydactyla Tentsmuir Forest, Fife, Scotland. They were maintained in the air conditioned enclosure described previously (Kershaw and Millbank, 1969).
473
474 C. J. B. HITCH AND J. W. MILLBANK Technique for estimating heterocyst and algal cell number The procedure was essentially that due to. Hill and Woolhouse (1966). Specimens of thallus were thoroughly washed in tap and distilled water and all traces of soil and other plant debris removed. The cleaned thallus was then incubated for 48 h in a moist condition (c. 90% saturated) in the enclosure at 12° C and at a light intensity of 4000 lux, the latter with a regime of 16 h on, 8 h off. After this pretreatment thallus specimens were immersed in io% (W/V) chromium trioxide solution for 18 h. In the case of P. aphthosa, dissected cephalodia only were so treated. Depending upon whether the ratio of heterocysts to vegetative cells, or the total number of cells per unit area of thallus was required, the subsequent technique varied. Heterocyst proportion. The thalli, which still retained their gross morphology, were drawn very carefully into a Pasteur pipette and expelled up to four times. A drop of the suspension was then transferred to a microscope slide and examined in the normal way. With P. aphthosa, the cephalodia were transferred directly to a slide, and the weight of a cover glass was sufficient to disperse the mycobiont remnants. In all cases, the Nostoc remained as intact filaments, and it was particularly important that the conditions of exposure were such that the filaments did not disintegrate into discrete cells and heterocysts. In this way the proportions of heterocysts could be readily ascertained. Up to 500 cells were counted, usually in about 8--to filaments, for each thallus specimen. A typical long filament of Peltigera canina is shown in Plate i, No. 1. The convoluted form is very noticeable. A filament from P. aphthosa, with the typical high proportion of heterocysts, is shown in Plate 1, No. 2. Total cell number. The thallus specimens were drawn into and expelled from the Pasteur pipettes much more vigorously. The suspension was then made up to a known volume, and a drop transferred to a haemocytometer counting chamber, and the cell numbers estimated directly. Upwards of 500 cells were counted for each estimate.
Nitrogenase activity This was estimated by means of the acetylene reduction technique (Postgate, 1972). Thallus discs, i sq cm in area, were used in glass vials of 7 ml capacity, shaken vigorously and containing 0.5 ml of the nitrogen-free mineral salt medium, pH 7.2, described previously (Millbank, 1972). The vials were held at 25° C and the light in- tensity was 4000 lux, derived from 'warm-white' fluorescent tubes. An acetylene con- centration of 4%, in air, was normally used.
RESULTS The results of the estimates of algal numbers are shown in Table 1. It is clear that the previously reported (Millbank, 1972) figure of 2.99 x '06 cells/sq cm thallus in Peltigera
Table I. The Nostoc content of three Peltigera spp. Number of Mean algal cell density Standard Lichen estimates (cells/sq cm thallus) deviation Standard error P. canina 22 8.77 X 106 1.60 x 106 0.34 X 106 P. polydactyla 26 io.3o x io6 2.58 x io6 0.51 x io6 Nostoc Number of Total no. Total no. cells per Nostoc 1 sq cm of Dry wt of of Nostoc sq cm cells per discs cephalodia cephalodia cells thallus mg ceph. P. aphthosa 18 343 4.7 mgm 59 X ic>6 1.06 x 106 4.04 x 106 THE NEW PHYTOLOGIST, 74, 3 PLATE I
Nostoc filaments from the thalli of Peltigera spp. after treatment with chromium trioxide solution ; for experimental details see text. No. i. P. canina. No. a. P. aphthosa. C. J. B. HITCH AND 3. W. MILLBANK—HETEROCYST FREQUENCY IN PELTIGERA
(facing page 474)
Heterocyst frequency in Peltigera 475 canina was a serious underestimate; cell rupture was much greater than was estimated. Taking the estimates of nitrogenase activity reported previously (Millbank, 1972, Table 5) and substituting the figure of 8.8 x 106 for the Nostoc cell population, the mean rate of acetylene reduction becomes 3.16 nmoles/min/mg Nostoc protein, and the range 1.8-4.9. Results of the estimates of acetylene reduction by the thalli used in this investigation are presented in Table z. Two features are of particular note, the very high activity shown by the Nostoc of Peltigera aphthosa, and the comparative sluggishness and variability of P. polydactyla.
Table 2. The nitrogenase activity of the Nostoc phycobiont in three Peltigera spp. Nitrogenase activity Minimum Mean Maximum Number of Lichen A B A B A B estimates P. canina 2.0 1.6 3.5 2.7 5.6 4.3 35 P. polydactyla 0.3 0.2 0.9 0.7 2.2 1.7 27 P. aphthosa 4.4 3.4 10.7 8.3 24.o 18.6 18 Columns A: nmoles ethylene formed/h/to6 cells. Columns B: nmoles ethylene formed/min/mg Nostoc protein.
The outstanding nitrogenase activity of Nostoc from Peltigera aphthosa is reflected in the findings of heterocyst frequency. These are summarized in Table 3.
Table 3. The heterocyst frequency of three Peltigera spp. Heterocyst frequency (percentage of Number of Lichen total cells) estimates P. canina 3.50 35 P. polydactyla 3.48 27 P. aphthosa 2r.6o 18
DISCUSSION The finding that the population of Nostoc cells in the thallus of Peltigera canina was of the order of 9 x Io6 per sq cm thallus rather than 3 x lob, the latter figure being itself a revised estimate, requires some explanation. A reappraisal of the technique previously reported (Millbank, 1972) has established that the sediment that was referred to then as 'whole cells', being the sediment thrown down at i000 g and assumed to be composed of Nostoc cells and mycobiont mycelial fragments, was in fact considerably contami- nated with particulate material derived from ruptured phycobiont cells. The contami- nation represented a large proportion (approximately 67%) of the total algal pigment in the sediment, and was intimately associated with the ruptured mycelia. It is quite evi- dent that the lichenized' algal cells are extremely fragile compared with those in the free-living condition, and this, together with their very close contact with the myco- biont, renders their separation in an intact state extremely difficult, and liable to very large losses. The numbers of algal cells per unit area of thallus reported here are in close agreement with the findings of Kershaw (1974) in respect of Canadian specimens of Peltigera canina 476 C. J. B. HITCH AND J. W. MILLBANK and P. polydactyla. The lichens were also examined by the chromium trioxide technique followed by direct counting. It would seem, therefore, that the rate of acetylene re- duction by the Nostoc in Peltigera canina and P. polydactyla is of the same order as that in free-living cultures, and although the heterocyst proportion is low, the data no longer gives cause for considering that vegatative cell fixation is probable. This conclusion is supported by the finding of a very high proportion of hetero- cysts in algae from the cephalodia of P. aphthosa; the most rapid rate of acetylene reduction observed in that species was approximately five times that observed in P. canina; the proportion of heterocysts was about six times greater. Nonetheless, the possi- bility that the algal environment in lichen thalli is hypoaerobic still remains and is being investigated. The effects of conditions of low p02 and/or high p CO, could be very important to the metabolic pattern of lichen algae. The incidence of high proportions of heterocysts in symbiotic blue-green algae is quite widespread. In addition to certain lichens, the algae in Gunnera (Silvester, 1975), Blasia and Anthoceros (Rodgers and Stewart, 1974) and Azolla (Peters and Mayne, 1974), all exhibit this phenomenon. Kulasooriya, Lang and Fay (1972) have suggested that a high C/N ratio, associated with conditions of nitrogen starvation is a major factor in stimulating heterocyst differentiation. Whilst this could well apply in the associations mentioned above, the varying proportion in lichens, where the external environments of the thalli do not differ greatly, is an interesting question. The role played by the mycobiont is likely to be critical in this situation, and investigation of the internal thallus conditions would be particularly useful.
REFERENCES GRIFFITHS, H. B. GREENWOOD, A. D. & MILLBANK, J. W. (1972). The frequency of heterocysts in the Nostoc phycobiont of the lichen Peltigera canina, Willd. New Phytol., 75, HILL, D. H. & WOOLHOUSE, H. W. (1966). Aspects of the autecology of Xanthoria parietina agg. Licheno- logist, 3, 207. KERSHAW, K. A. & MILLBANK, J. W. (1969). A controlled environment lichen growth chamber. Licheno- logist, 4, 83. KERSHAW, K. A. (1974). Dependence of the level of nitrogenase activity on the water content of the thallus in Peltigera canina, P. evansiana, P. polydactyla and P. praetextata. Can. Bot., 52 1423. KULASOORIYA, S. A., LANG, N. J. & FAY, P. (1972). Heterocysts of blue-green algae III; Differentiation and nitrogenase activity. Proc. Roy. Soc. B., 181, 199. MILLBANK, J. W. & KERSHAW, K. A. (1969). Nitrogen metabolism in lichens. I. Nitrogen fixation in the cephalodia of Peltigera aphthosa. New Phylologist, 68, 721. MILLBANK, J. W. (1972). Nitrogen metabolism in lichens. IV. The nitrogenase activity of the Nostoc phycobiont in Peltigera canina. New Phytol., 71, I. PETERS, G. A. & MAYNE, B. C. (1974). The Azolla—Anabaena azollae relationship. 1. Initial characterisa- tion of the association. Plant Physiology, 53, 813. POSTGATE, J. R. (1972). The acetylene reduction test for nitrogen fixation. In: Methods in Microbiology 6B. (Ed. by J. R. Norris & D. W. Ribbons), pp. 343-356. Academic Press, London. RODGERS, A. & STEWART, W. D. P. (1974). Physiology and Interrelations of the blue-green algae Nostoc with the liverworts Anthoceros and Blasia. Brit. Phycol. J., 9, 223. SILVESTER, W. B. (1975). The Gunnera—Nostoc symbions. In: Symbiotic Nitrogen Fixation in Plants. (Ed. by P. S. Nutman). Cambridge University Press, 1975. New Phytol. (1975) 75, 239-244.
NITROGEN METABOLISM IN LICHENS
VII. NITROGENASE ACTIVITY AND HETEROCYST FREQUENCY IN LICHENS WITH BLUE-GREEN PHYCOBIONTS
BY C. J. B. HITCH AND J. W. MILLBANK
Department of Botany, Imperial College, London, SW7 2AZ
(Received 9 March 1975)
SUMMARY The N2(C2H2) fixing activity was surveyed in lichens of diverse structure and habitat, all containing a blue-green phycobiont; twenty-six species from fourteen genera are reported for the first time to fix nitrogen. Rates of N2(C2H2) fixation showed great variability between different lichen samples. The proportion of heterocysts, as the assumed site of N2 fixation, was estimated in all the above lichens together with fourteen additional species. In lichens with one phycobiont, heterocysts form about 4% of the total algal cell population; in those with two phycobionts the blue-green member is found in special structures and has a very high proportion of hetero- cysts, typically 20.-3o%; the N2(C2H2) activity appears to reflect this. The high proportion may be associated with the proximity of the green phycobiont.
INTRODUCTION Hitch and Stewart (1973) in summarizing the findings of a number of investigators, suggested that the fixation of nitrogen by lichens under natural conditions is a common phenomenon. Physiological studies of nitrogen fixation in lichens can often be aided by the choice of a species having morphological features which facilitate a specialized line of enquiry, and the work reported here provides information on the nitrogen-fixing capability of a variety of lichens of diverse structure and habitat. Nitrogen fixation is generally considered to take place in the differentiated heterocyst cells of filamentous blue-green algae. There are few estimates of heterocyst numbers in symbiotic blue-green algae, (see Greenwood, Griffiths and Millbank, 1972; Hitch and Millbank, 1975) and this report augments the available data.
MATERIALS AND METHODS Lichens Table 1 lists the species investigated, and shows the collecting sites (giving the British National Grid reference for U.K. material).
Estimation and heterocyst frequency This followed the procedure described by Hitch and Millbank (1975). Specimens were thoroughly cleansed of soil and other debris and washed in tap and distilled water. They were then incubated for 24 h in a moist condition in the air-conditioned enclosure previously described (Kershaw and Millbank, 1969) at r2° C and light intensity 2500 lux, 239
240 C. J. B. HITCH AND J. W. MILLBANK
Table I. Lichen materials and collecting sites Grid Lichen Collecting site reference Collema auriculatum Barmore Island NR 8671 C. cristatum Lockerbie NY 1577 C. fluviatile River Erricht, Blairgowrie NO 1748 C. furfuraceum Inverary Castle NN 0909 C. subfervum Minard Castle NR 9794 Dendriscocaulon umhausense Salen NM 7164 Ephebe lanata Glen Almond NN 9329 Leptogium burgessii Barmore Island NR 8671 L. cyanescens Furnace NN 0300 L. lichenoides Furnace NN 0300 L. sinuatum Lockerbie NY 1577 L. teretiusculum Chideock SY 3992 L. tremelloides Furnace NN o3oo Lichina confinis Arbroath NO 6641 L. pygmaea Lunan Bay NO 6949 Lobaria amplissima Salen NM 7164 L. laetevirens Ardnamurchan peninsula NM 6361 L. pulmonaria Furnace NN o3oo L. scrobiculata Furnace NN 0300 Massalongia carnosa Barmore Island NR 8671 Nephroma arcticum Pen Island, North Ontario Canada N. laevigatum Furnace NN o3oo N. parile Loch Awe NN 0014 Pannaria microphylla Barmore Island NR 8671 P. pezizoides Ben Lawers NN 6139 P. pityrea Inverary Castle NN 0909 P. rubiginosa Strath Oykel NC 440 Parmeliella atlantica Inverary NN 0908 P. plumbea Furnace NN o3oo Peltigera aphthosa Den of Airlie NO 2852 P. canina Mount Hill, Fife NO 3316 P. evansiana Waterdown, Ontario Canada P. polydactyly Tentsmuir Forest NO 4928 P. praetextata Den of Airlie NO 2852 P. venosa The Burn, Angus NO 5972 Placopsis gelida Glen Garry NH 727o Placynthium nigrum Mt. Schiehallion NH 7158 P. pannariellum Glen Lochay NN 5435 Polychidiuin muscicola Loch Sunart NM 8161 Psoroma hypnorum Ben Loyal NC 5749 Pseudocyphellaria thouarsii Balnabraid Glen NR 7615 Solorina crocea Ben Vrackie NH 9363 S. saccata Mt. Schiehallion NH 7158 S. spongiosa Mt. Schiehallion NH 7158 Stereocaulon vesuvianum Falkland NO 2406 Sticta canariensis Balnabraid Glen NR 7615 S. dufourii Balnabraid NR 7615 S. fuliginosa Ardnamurchan peninsula NM 6361 S. limbata Loch Tay NN 6635 with a 16 h 'day' period. After this pretreatment, the specimens were immersed in io% (w/v) chromium trioxide solution for up to 18 h. The proportion of heterocysts in the filamentous blue-green phycobiont was estimated by direct counting of intact chains of cells after suitable dispersal by means of a Pasteur pipette or a cover glass.
Estimation of nitrogenase activity This was by the acetylene reduction technique (see Postgate, 1972). Samples of thallus, after the normal pretreatment, were incubated in 7-ml glass vials containing 0.5 ml of a nitrogen-free mineral salts medium modified slightly from that of Allen and Arnon (1955) in the concentrations of phosphate and iron: io'm K2HPO4 and 1 pg/ml Nitrogenase activity and heterocyst frequency in lichens 241 of Fe (as EDTA complex). The pH of the medium was 7.2. The vials were held at 25° C, illuminated at 4000 lux provided by 'warm white' fluorescent tubes, and shaken vigorously (12o strokes/min, 3 cm) for 3o-6o min with air as the gas phase; acetylene was then introduced by syringe to a concentration of 4% (v/v), and the production of ethylene followed over the ensuing 6o min by analysis of sequential gas samples of 0.5 ml.
RESULTS Lichens with a single phycobiont Table 2 summarizes the results. Nitrogenase activities are expressed on the basis of both thallus nitrogen and thallus dry weight. The wide range of activities was very noticeable; the activity of individual thalli of a given species also exhibited very wide variations, the effect extending even to adjacent sample discs. Thus, values for nitrogen- fixing abilities of lichens in the field are only reliable if the number of samples taken and tested is very large. The highest activities were found in Peltigera species; that for P. canina was of the same order as has been reported previously (Hitch and Millbank, 1975). and it is evidently a most consistently effective fixer of nitrogen.
Table 2. Nitrogen content, nitrogenase activity, and heterocyst frequency in lichens with one blue-green phycobiont Nitrogenase activity Heterocyst population Number of nMC2H4/mg nMC2H4/mg thallus Thallus N thallus thallus (% total specimens Lichen content, % N/h dry wt/h algal cells) examined Collema auriculatum 3.6o 15.10 0.54 2.7 I C. cristatum 4.39 n.d. n.d. 2.1 i C. fluviatile 4.37 3.52 0.15 n.d. C. furfuraceum 4.09 22.67 0.93 4.0 2 C. subfervum 3.85 7.34 0.28 z.6 6 Ephebe lanata 5.8o 7.60 0.44 n.d. Leptogium burgessii 5.51 38.12 2.10 4.5 I L. cyanescens n.d. n.d. n.d. 2.7 I L. lichenoides 3.87 22.1 o.86 2.6 5 L. sinuatum 4.31 14.81 0.64 2.4 4 L. teretiusculum 4.50 2.22 0.10 n.d. L. tremelloides n.d. n.d. n.d. 3.5 I Lichinia confinis 3.73 4.17 0.16 2.4 1 L. pygmaea 6.49 2.93 0.19 4.6 I Lobaria scrobiculata 2.77 10.63 0.29 3.9 3 Massalongia carnosa 4.27 19.20 0.82 5.5 I Nephroma laevigaturn 4.22 23.89 1.01 4.1 3 N. parile n.d. n.d. n.d. 4.9 3 Pannaria microphylla 5.14 n.d. n.d. 4.5 I P. pezizoides 6.37 1.66 0.11 3.9 I P. rubiginosa 2.25 15.67 0.35 n.d. Parmeliella atlantica 3.90 58.31 2.27 7.3 3 P. plumbea 4.76 18.85 0.90 5.9 1 Peltigera canina 3.28 118.o 3.87 4.87 I P. evansiana n.d. n.d. n.d. 4.7 6 P. polydactyla 3.5 102.41 3.58 5.8 5 P. praetextata n.d. n.d. n.d. 4.4 to P. venosa n.d. n.d. n.d. 7.8 3 Placynthium nigrum 2.73 6.48 o.i8 2.0 I P. pannariellum 4.61 15.33 0.71 n.d. Polychidium muscicola 3.19 24.85 0.79 2.6 i Pseudocyphellaria thouarsii 3.90 5.89 0.23 6.7 I Sticta fuliginosa 4.19 16.34 o.68 6.o 3 S. limbata 3.93 1.35 4.9 7 n.d. = not determined.determined. 242 C. J. B. HITCH AND J. W. MILLBANK The ratio of heterocysts to total cells in the phycobiont filaments, of all the lichens was consistently and uniformly low, being of the order of 4%. Previous reports (Griffiths, Greenwood and Millbank, 1972; Hitch and Millbank, 1975) which referred only to Peltigera spp., seem to be of general applicability.
Lichens with two phycobionts In P. aphthosa, a lichen with two phycobionts, Coccomyxa and Nostoc, Hitch and Millbank (1975) reported a very high proportion of heterocysts in the Nostoc filaments in the cephalodia. Such a situation is usual in associations of blue-green algae and higher plants. Consequently, the heterocyst frequency in the blue-green component of a num- ber of other lichens containing two phycobionts was investigated (Table 3). The pro- portion of heterocysts in the blue-green algal filaments of these lichens is consistently
Table 3. The heterocyst frequency in the blue-green phycobiont filaments of cephalodiate lichens Mean heterocyst frequency (percentage Number of total of algal cells) estimates Dendriscocaulon umhausense 1.9-15.2 6 Lobaria amplissima 21.6 2 L. laetevirens 30.4 5 L. pulmonaria 35.6 2 Nephroma arcticum 14.1 r Peltigera aphthosa 21.1 13 Placopsis gelida 15.0 7 Psoroma hypnorum 10.1 2 Solorina crocea 17.8 6 S. saccata 54.7 3 S. spongiosa 5.8-54.7 12 Stereocaulon vesuvianum 20.5 2 very high and of the same order as reported in higher plant associations. It was about ten-fold greater than in lichens with one phycobiont. Whether a high proportion of heterocysts always reflects rapid nitrogenase activity is yet to be established. So far, Lobaria laetivirens and Placopsis gelida have been examined in addition to Peltigera aphthosa and nitrogenase activities of 200-1000 nmole C2H,/mg cephalodia N/h were found (Table 4). A high proportion of heterocysts therefore appears to be associated with rapid nitrogenase activity, though more detailed quantitative data on rates and algal content in these and other lichens is needed.
Table 4. Nitrogenase activity of dissected cephalodia nmoles C2H4/mg cephalodia N/hr Lobaria laetevirens 615 Peltigera aphthosa 1020 Placopsis gelida 200
Since a high proportion of heterocysts is consistently found in Nostoc filaments in cephalodiate lichens, it is possible that the green algal phycobiont was in some way con- cerned in the differentiation of the blue-green. Proximity of the two types of phyco-
Nitrogenase activity and heterocyst frequency in lichens 243 biont might be related to heterocyst differentiation, so lichens having a variable degree of proximity between the green and blue-green phycobionts were therefore examined. The proportion of heterocysts in areas containing blue-green algae taken from different positions relative to the green algal zone were estimated (Table 5). The relevant mor- phology of the lichens concerned was as follows.
Table 5. The relation between heterocyst frequency in the blue-green phycobiont and proximity to an associated green phycobiont Association Degree of proximity to green phycobiont Remote Adjacent (5-10 mm) (0mm)-2 Dendriscocaulon umhausense/ 1.9, 2.5, 4.0, 5.7 10.2, 15.2 Lobaria amplissima Solorina spongiosa 5.8, 4.9, 7.1 28.3, 43.1, 45.5, 54.7 Sticta dufourii/S. canariensis 3.1, 4.1 12.4, 13.3, 18.0 Figures indicate percentage of heterocysts in total algal cells.
(i) Solorina spongiosa. The green phycobiont (Coccomyxa) is the minor algal partner, occurring only in zones of very limited extent at the periphery of a few scattered apo- thecia. These are surrounded by many cephalodial structures containing the Nostoc phycobiont, forming the main body of the thallus. (2) The Sticta canariensis/S. dufourii heteromorphic association. In this rare 'com- pound', a foliole of one species (S. canariensis) sometimes becomes attached to lobes of the thallus of the other (S. dufourii); the latter has Nostoc as the phycobiont, the former Coccomyxa. (3) Dendriscocaulon umhausense. This lichen is a dendroid structure, with a Nostoc phycobiont and is most often found attached to the foliose lichen, Lobaria amplissima (Coccomyxa phycobiont). Table 5 suggests that the extent of heterocyst differentiation may be correlated with the degree of proximity to green phycobiont cells.
DISCUSSION The results described here, together with the previously reported findings of many other investigators, suggest that all lichens containing filamentous blue-green phyco- bionts are capable of fixing nitrogen, and many at a significant rate. Furthermore, in lichens with two phycobionts, the blue-green component has a high proportion of heterocysts and a notably rapid rate of nitrogenase activity. Hitch & Millbank (1975) noted that the proportion of heterocysts in symbiotic filamentous blue-green algae is generally high, lichens with a single blue-green phyco- biont being the only exception to this. The evidence has been recently augmented by Hill (1975) who has confirmed that the Anabaena associated with Azolla has a generally high proportion of heterocysts. He has also shown that the ratio varies, increasing with the age of the frond, i.e. distance from growing apex. This, together with our finding that the proportion appears to be related to the degree of proximity to the green phycobiont might indicate that an augmented supply of photosynthate stimulates the differentiation. Kulasooriya, Lang and Fay (1972) put forward the theory that the C/N ratio is a significant factor governing heterocyst differentiation, and the situation 244 C. J. B. HITCH AND J. W. MILLBANK in the three compound lichens tested could perhaps be accounted for on this basis, the release of fixed polyol by the relatively abundant green phycobiont modifying the C/N ratio and providing a stimulus for differentiation.
ACKNOWLEDGMENTS The authors wish to thank Dr P. B. Topham, S.H.R.I., Dundee; Professor K. A. Kershaw, McMaster University, Hamilton, Ontario; and Mr P. James of the B.M. (N.H.), London, S.W.7, for their assistance in collecting and supplying lichen material, and for helpful suggestions and discussions.
REFERENCES ALLEN, M. B. & ARNON, D. I. (1955). Studies on nitrogen fixing blue-green algae. I. Growth and nitrogen fixation by Anabaena cylindrica Lem. Pl. Physiol., Lancaster, 3o, 366. GRIFFITHS, H. B., GREENWOOD, A. D. & MILLBANK, J. W. (1972). The frequency of heterocysts in the Nostoc phycobiont of the lichen Peltigera canina Willd. New Phytol., 7x, 11. HILL, D. J. (1975). The pattern of development of Anabaena in the Azolla-Anabaena symbiosis. Planta (Berl.), (in press). HITCH, C. J. B. & MILLBANK, J. W. (1975). Nitrogen metabolism in lichens, VI. The blue-green phyco- biont content, heterocyst frequency and nitrogenase activity in Peltigera spp. New Phytol., 74, 473. HITCH, C. J. B. & STEWART, W. D. P. (1973). Nitrogen fixation by lichens in Scotland. New Phytol., 72, 509. KERSHAW, K. A. & MILLBANK, J. W. (1969). A controlled environment lichen growth chamber. The Lichenologist, 4, 83. KULASOORIYA, S. A., LANG, N. J. & FAY, P. (1972). Heterocysts of blue-green algae III ; Differentiation and nitrogenase activity. Proc. Roy. Soc. B., 18x, 199. POSTGATE, J. R. (1972). The acetylene reduction test for nitrogen fixation. In: Methods in Microbiology 6B. (Ed. by J. R. Norris & D. W. Ribbons), pp. 343-356. Academic Press, London.