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Tuatara aims to stimulate and widen interest in the natural sciences in New Zealand, by publishing articles which (a), review recent advances of broad interest; or (b), give clear, illustrated, and readily understood keys to the identification of New Zealand plants and animals; or (c), relate New Zealand biological problems to a broader Pacific or Southern Hemisphere context. Authors are asked to explain any special terminology required by their topic. Address for contributions: Editor of Tuatara, c/o Victoria University of Wellington, Box 196, Wellington, New Zealand. Enquiries about subscriptions should be sent to: Business Manager of Tuatara, c/o Victoria University of Wellington, Box 196, Wellington, New Zealand.
is the journal of the Biological Society, Victoria University of Wellington, New Zealand and is published three times a year. Editor:
Hitherto all enquiries concerning William Bell, one of our earlier students and collectors of the New Zealand mosses, have failed to bring to light any information concerning either the man or the location of his moss collections. Mrs. Audrey Larsen, who is compiling a history of Pine Hill in Dunedin where Bell resided, has unearthed the missing information, and at my suggestion she has made a precis of the facts for publication in ‘Tuatara’.
—W. Martin
Born in Dumfriesshire, Scotland, about 1833, William Bell was one of a family of eight children, his mother widowed early in life. He trained as a journeyman gardener at the Royal Botanic Garden, Edinburgh, where he developed an interest in alpine mosses and because of this was granted the privilege of attending lectures and field studies under Professor Hutton Balfour. In September of 1860 he accompanied W. Keddie and Professor Balfour on an ascent of Schihillion (Scotland), ‘commissioned from the Botanic Garden to collect ferns’, and later presented some alpine mosses to the Botanical Society herbarium — possibly his first recorded moss collection. In December of 1861 he was elected an Associate of the Botanical Society of Edinburgh which in subsequent years published several items by him regarding Scottish mosses and other plants. In May of 1862, at the age of twenty-nine years, he took up the position of head gardener at the Botanic Garden of Saharanpur in North West India, which was conducting early experiments in tea and cinchona culture. He remained there during the wild, speculative period among the tea companies both in Britain and India, which reached its climax in 1865, returning slowly to a stable position in 1869, by which time Bell was back at the Royal Botanic Garden, Edinburgh, as assistant to J. Sadler, curator of the herbarium. While in Saharanpur (spelt at the time Saharanpore) he botanised around the district, sending specimens back to Edinburgh in 1863. An orchid which Bell
Eulophia campanulata by Duthie, in punning reference to his name. He explored the forest regions of Dehra Dun on the northern side of the Siwalik Hills, sending a description to Professor Balfour (which was published in Trans. Bot. Soc Edinburgh IX, 274-92: 1869), and also the hot springs at Jumnotri, which he communicated to the Botanical Society personally in Edinburgh and which was later published by them. Although his term of work with the Botanic Garden at Saharanpur had been set for four years, he did not return home until 1868, collecting specimens from Gibraltar on his journey back to Edinburgh. In 1869, Bell and Sadler made several excursions around Edinburgh collecting mosses, notice of which was published by the Botanical Society, who also published two items by Bell regarding tea: ‘Notes on the varieties of tea cultivated in India’ and ‘Remarks on tea manufacture in north west provinces of India’ (Trans. Bot. Soc. Edinb X, 162-64 (1869) and XI, 174-87 (1871).
The next venue for Bell was New Zealand, with Dunedin as his mailing address. Here he spent nine weeks in the autumn of 1872, working at harvest and other miscellaneous jobs, travelling as far as Timaru where a three-week-old telegram recalled him to Dunedin to receive a letter telling him to start at once for Dehra Dhoon where the tea company had appointed a temporary manager, the position to be kept open for Bell. However, travel between New Zealand and India was not fast in 1872 and by the time William Bell arrived in Dehra Dhoon the temporary manager had made ‘such a mess of things’ that the company had given the job to Nelson of Saharanpore. Bell was given charge of a china grass (Boehmeria nivea) plantation by a Colonel Thelwell, on the Markham Grant Estate, an industry that was experiencing difficulties with the processing of the fibre for manufacture, and was at that time holding competitive trials at Saharanpur for suitable processing machinery. Bell finally did obtain the position of manager to the Dehra Dhoon Tea Company as a letter to Professor Balfour dated November 25, 1873, records, and remained with it until some time in the 1880's, punctuated by visits to New Zealand on sick leave.
It was during one of these periods of sick leave (congestion of the liver) that the incident occurred that decided Bell, who had presented his collection of Indian mosses to the Otago (then the Dunedin) Museum, not to offer his New Zealand collections to that institution, but to donate the 3,000 New Zealand mosses in his private herbarium to Brotherus in Finland when the time came. He spent two six-month periods of sick leave in New Zealand, based in Dunedin, the first in 1873-74, then again in late 1880-81, but the time of actual residence on retirement from the tea company has not yet been established. However photostat copies from the Botanical Museum, Helsinki University, Finland, of Psilopilum bellii specimens annotated in Bell's own hand give a first date of February, 1887,
Some time before 1900, Bell returned to India and spent a period as superintendent at the Saharanpur Botanic Garden before final retirement in Christchurch, where in December of 1902 he wrote again to Dr. Brotherus, ‘On account of failing sight I have been unable to do anything amongst the mosses for some time past, hence I have resolved on disposing of the whole of my collection. Should you care to accept it as a donation to your Herbarium, I shall give it with pleasure,’ and on May 4, 1903, this left Christchurch (per Messrs. Heywood and Coy.) for Finland along with a small bundle of Hepaticae from the late Mr. Buchanan, who had given specimens to Bell at a time when W.B. had the intention of making a collection of these.
In all, Bell sent Brotherus 3,000 New Zealand mosses, an account of which was published by A. Palmgren (1927: 126 (Prof. V. F. Brotherus’ mossherbarium forvarfvas till Helsingfors Universitet.—Memoranda Soc. Fauna Flora Fennica 1: 120-27). Not all Bell's mosses, however, left New Zealand, as he was a very large contributor of specimens in the Beckett herbarium lodged with the Canterbury Museum. It would be interesting to know just how many mosses in Scotland, India and New Zealand William Bell collected over a lifetime of interest before his death on March 17, 1916, in Christ-church, the result of an attack of pneumonia in his eighty-fourth year.
The photostat copies of Psilopilum beliii (sent by courtesy of Dr. Pekka Isoviita, Acting Associate Curator of Cryptogams, Botanical Museum, University of Helsinki) show a much wider distribution in the Pine Hill/Mt. Cargill region than was previously thought, no one else other than Bell having at that period located it here. The annotations in Bell's own hand show:
If the words ‘upper bound.’ in the 1887 Pine Hill annotation mean upper boundary of Bell's own property, then the figures given at the end of the first four annotations refer to altitude and are correct. The Mt. Cargill road altitudes could be around 1,100 or 1,200 feet, depending on location. This is an area that Bell would have explored thoroughly, his home on Campbells Road being situated close to a sizeable tract of virgin bush, ravined with creeks and waterfall that extended from approximately 300 feet at the northern end of North East Valley to 2,000 feet near the peak of Mt. Cargill, an area now conserved by the Dunedin City Council as ‘Bethunes’ Gully Reserve’. It is not impossible that specimens of Psilopilum bellii could still be discovered here. The last habitat of this moss in the Mt. Cargill area known to
The list of altitudes in Bell's letter to Dr. Brotherus of January 12, 1895, to be used in conjunction with the specimens sent to Brotherus give a good indication of the areas of collection covered by Bell up to that date but should not be taken as exclusive as he sent only duplicate specimens. As far as can be deciphered from Bell's crabbed handwriting (result of an accident to hand) these areas are:
The name of William Bell is perpetuated in the endemic moss genus Bellia and in three South Island endemic moss species: Anoectangium bellii, Psilopilum bellii, and
A great deal of deep interest still remains to be discovered of Bell's life and work, but the main structure can now be seen, thanks to Mr. B. L. Burtt of the Royal Botanic Garden, Edinburgh; Dr. Pekka Isoviita of the Botanical Museum, University of Helsinki; Mr. L. J. Metcalf of Christchurch Botanic Garden; Mrs. Florence Hawke (William Bell's neice) of Christchurch and late of Pine Hill; Mr. W. Martin and Mr. W. H. Davidson, both of Dunedin. Research into the life of William Bell has proved as difficult, fascinating and rewarding as, I am told, is that of the four endemic New Zealand mosses which perpetuate his name, but the day is not too distant when his ‘song’ can rise out of obscurity and be sung with full accompaniment. Then at long last can the man and his work be recognised, appreciated and understood in the three lands he loved.
Over Much of the Earth the genus Cladonia is represented on heaths, bogs, and sterile soils, or on decaying logs, at elevations ranging from sea-level to alpine habitats. The majority of some seventy species indigenous to New Zealand occur at elevations under 1,200 metres. Endemic species are few; some are cosmopolitan, and most are wide-ranging. Some species are easy to recognise; but as Bruce Fink has pointed out when referring to the lichen flora of North America. ‘Cladonias are the most variable of all lichens, and the most difficult to describe accurately. Nothing but the most careful observation will enable anyone to determine a Cladonia with any degree of certainty even with the best descriptions.’
Since these observations were recorded, however, a great deal of research has been devoted to the genus Cladonia by Asahina, Evans, Ahti, Sandstede, Des Abbrayes, Culberson, and others, all of whom have studied the chemical as well as the morphological attributes of the various species. A majority of lichens produce ‘lichen acids’, most of which are found only in lichens, and for the recognition of many of which simple microchemical tests have been devised. Usnic acid and atronorine are located in the cortex but most of the others form an incrustation on the hyphae of the medulla. The most commonly used reagents are (1) an aqueous solution of potassium hydrate (KOH); (2) a saturated aqueous solution of calcium hypo-chlorite (CaOCl2) or alternatively one of the commercial bleaching fluids; and (3) an alcoholic solution of para-phenyline-diamine freshly prepared. Each of these produces a distinctive colour change when applied to the cortex or medulla when certain acids are present, and none in their absence.
Many species of Cladonia constantly produce the same acid or acids which in consequence are termed ‘diagnostic’ for that species. Other accompanying acids not invariably present are termed ‘accessory’. If therefore a specimen believed to be a particular species is found not to contain an acid diagnostic for that species then the determination is wrong and must be revised. The above reagents are indicated as a rule by the letters K, C, and P or Pd. KC indicates a drop of K is immediately followed by a drop of C on the same area. A + sign following indicates a colour change results and this is usually followed by the first letter of the colour resulting. Thus K + y indicates a colour change to yellow, while K — symbolises no colour change. The identification of acids that produce no colour reaction can best be determined by the form of their crystals or crystals of their salts or by means of paper chromatography.
Lichen Handbook by Mason E. Hale — a Smithsonian Institute publication.
Every lichen represents a symbiotic association of a fungus, and a colony of algal cells on which the fungus is dependent for food and enzymes, as all fungi are devoid of chlorophyll. The relationship is more than simple symbiosis, however, as the lichen acquires its own distinctive form and often produces structures such as soredia, isidia, cephalodia, or cyphellae unknown save on lichens. The fruiting bodies (apothecia) are those of the fungus only and the contained spores are of course fungus spores, and can only reproduce a new generation of lichens when on germination they fortuitously make contact with the appropriate algal cells living free in the area. It is believed, however, that propagation in Cladonia is rarely from spores but from such diaspores’ as soredia, isidia, or thallus fragments in which both symbionts are already associated.
The germinating diaspore develops into a primary thallus either squamulose or crustose in form. The crustose form is characteristic of sections Unciales and Cladinae and the squamulose form of most other species. The largest squamules 2-3cm. long are found in Sections Foliosae and Podostelides. After a time a secondary fruticose thallus may form on the surface or margin of the primary thallus, known as a podetium. The apothecia in Cladonia are always borne on the tips of the podetia or of their branches. A cross-section of a typical podetium displays a central hollow cylinder of compact chondroid tissue surrounded by a medulla of fungal hyphae, then by an algal zone, and commonly by an outer cortex which may be more or less continuous, or areolate, and often sorediate.
Weise has demonstrated experimentally that only the chondroid cylinder arises from the tissue of the primary thallus, the outer layers being derived from windborne soredia that lodge on its surface. In the absence of an algal layer in the podetial wall, the central cylinder often atrophies for lack of the supplementary nourishment these algae are able to provide.
The podetia of the more primitive species are short and simple or few branched, and the pycnidia and apothecia may occur on the primary thallus. In the Cladinae the podetia are often densely branched and both pycnidia and apothecia are confined to the branch tips of fertile plants. The primary thallus may be persistent or ephemeral, and soon disappears when crustose. Some species have podetia that die at the base but continue growth at the apex. Some species rarely or never produce apothecia or spores.
A problem not yet finally resolved relates to the so-called ‘chemical species’. Do chemotypes have true taxonomic status? Most botanists recognise them, but not all. It has been suggested that when two or more lichens morphologically alike but each with a different acid content occupy different regions, they should be
Cladonia chlorophaea, Cladonia grayi, Cladonia merochlorophaea, and Cladonia cryptochlorophaea as four chemical strains of Cladonia chlorophaea. Three additional species closely resembling them, viz. Cl. fimbriata, Cl. conista, and Cl. pyxidata, can be distinguished by morphological features and are deemed valid species by all.
A number of species supposedly indigenous have been reported so seldom or once only that further corroboration is desirable. Even expert specialists can and do make wrong determinations. Thus New Zealand members of Section Cladinae were long regarded as Cl. rangiferina, Cl. sylvestris, Cl. alpestris, and Cl. pycnoclada — not one of which was correctly determined. This section today comprises Cl. mitis, Cl. alpestroides, and Cl. leptoclada, all P —, though three of the former species are P +. Species collected only once and listed as indigenous include C. chondrotypa, Cl. Krempelhuber, Cl. metalepla, Cl. oceanica, and Cl. solida. Other doubtful indigens include Cl. decorticata, Cl. medusina, and Cl. ventricosa. The supposed endemics are Cl. neozelandica, Cl. southlandica, Cl. murrayi, and Cl. enantia. The first two seem amply distinct but Cl. enantia scarcely differs from Cl. cariosa, and, though morphologically distinct, its chemistry has suggested the possibility that Cl. murrayi may be a monstrous form of Cl. deformis.
A key to the indigenous Cladoniae may be consulted in Trans. Roy. Soc. N.Z., vol. 85, part 4, pp. 603-22 (1958). The diagnostic lichen acids accompanying each Cladonia species together with their chemical reactions to K, KC, and P are listed below. It may be noted that all New Zealand species in Sections Unciales, Cladinae, and Foliosae contain usnic acid, and Podostelides, Thallostelides and Chasmaria species are devoid of usnic acid. All Thallostelides species other than Cl. grayi and Cl. nernoxyna contain fumarprotocetraric acid which is absent from all New Zealand Cladinae and Unciales. The lichen acids bellidiflorine, didymic acid, and zeorine are confined to the Cocciferae.
The following lichen acids give no colour reactions with K, KC, or P — viz. Barbatic, Cervicornic, Didymic, Fimbriatic, Rangiformic, Grayanic, Perlatolic, and Squamatic acids — but can be identified from the form of crystals of the acid or of its salts. For illustrations of these crystals and the technique of obtaining them see Hale's Lichen Handbook, chapter 6, pp. 66-75.
(Note: f = faintly; ± = sometimes +, sometimes -; br = brown, r = red, y = yellow; sq = squamules)
To identify a species of Cladonia first consult the key to the indigenous species in Vol. 85, part 4, of the Trans. Roy. Soc. of N.Z., and make a tentative determination both of the species and of the section of Cladonia to which it belongs. Now apply the reagents K, KC, and P to the cortex or medulla as the case may require, and note the reactions. Compare these with those of the presumed species in the above table. If they differ your tentative determination is probably incorrect, but if they agree you are probably but not necessarily correct. Final corroboration requires comparison of the specimen with a full description of the species; but if this is not available, compare the plant with authentic specimens in some herbarium,
Some species show their reactions best when the contained acids are extracted by placing a portion of the plant in the bottom of a test-tube and covering with acetone. The specimen is now removed and the solution evaporated to a small quantity which is then transferred to a microscopic slide to complete the evaporation. The solid residue is now divided into separate portions to which the reagents are separately applied, and any colour changes noted. Many botanists apply this technique to all specimens.
The First Report in the open scientific literature of the occurrence of Cladosporium resinae (Lindau) de Vries in hydrocarbon fuels appears to be that of Hendey in 1964. Hazzard (1963) positively identified C. resinae in aviation kerosene in Australia in 1961 but his results are contained in a technical report which has not been widely circulated. In the same year Prince (1961) reported finding a Cladosporium sp. in jet fuel in the United States of America and his photographs indicate that it was probably C. resinae. Contamination of aviation fuels with C. resinae has subsequently been shown to be widespread. The fungus has been isolated from aviation fuels, aircraft tanks or filters in Australia (Hazzard, 1963), Brazil (Gutheil, 1966), Denmark, England, India, Japan, Nigeria, New Zealand, Syria (Anon., 1968) and U.S.A. (Engel and Swatek, 1966).
Parbery (1967) first reported consistently isolating C. resinae from Australian soils in 1967. He was later (1969) able to show that this fungus is widespread in soils in Australia, Europe and Britain. The results of a soil survey in New Zealand support his findings (Sheridan, Steel and Knox. 1971).
Four forms of C. resinae are known to exist (De Vries, 1955) but only one, f. avellaneum, has apparently been recovered from kerosene-type fuels (Hazzard, 1963; Hendey, 1964). C. resinae f. resinae has arisen in culture from f. avellaneum (Hendey, 1964) and been isolated occasionally from soil (Parbery, 1968). An albino, f. albidum, has also arisen in culture from f. avellaneum (Parbery, 1968; Sheridan, Steel and Knox, 1971). The only form isolated from kerosene fuels and soils in New Zealand is C. resinae f. avellaneum (Sheridan, Steel and Knox, 1971). The other two forms have been found here as saltants in culture — the fourth form (f. sterile) has not been seen by us (Sheridan — unpublished). All of the above forms (except f. sterile) have been tested in our laboratory for ability to grow in kerosene. Parbery's (1968) isolates of f. resinae from soils are reported by him as not growing in kerosene. As far as we are aware no one has tested isolates of C. resinae from the air for their ability to grow in kerosene.
This paper compares the growth of New Zealand soil, fuel and air isolates of the ‘kerosene fungus’ C. resinae in aviation turbine and lighting kerosene.
Isolations were made from soil by the creosoted matchstick method (Sheridan, Steel and Knox, 1971).
Isolations were made from aviation fuels by passing 250ml. of the fuel through a sterile millipore membrane filter, pore size 0.45 micron, in a manner similar to that employed by Hazzard and Kuster (1962). The filter membrane was then placed inside a sterile Petri dish, V-8 juice agar at 50° C. poured gently over its surface, and the plate incubated at 25° C. for five days. A number of filters carrying C. resinae and a number of isolates of the fungus on agar media were kindly sent to us by Mr. W. Johnston of B.P. New Zealand Ltd.
Isolations were made from the air using the methods described by Sheridan and Nelson (1971).
All isolates were grown on V-8 juice agar (Sheridan, Steel and Knox, 1971) until required for testing on kerosene.
The ability of isolates to grow in kerosene was tested in 16oz. medicine flat bottles (Fig. 1). Each bottle contained 200ml. of Bushnell-Haas (B/H) mineral salts medium (Sheridan, Steel and Knox, 1971) sterilised by autoclaving. One loopful (platinumindium loop, 5mm. diam.) of a distilled water suspension of spores of each isolate was inoculated into the B/H medium and 50ml. of kerosene (sterilised by Millipore filtration) was layered on top. The aluminium cap, with the rubber liner removed, was screwed on loosely and the bottles incubated in the laboratory, usually for six
The mycelial mat produced by the fungus was harvested after six weeks by carefully removing it with a bent nichrome wire. In general the complete growth could be removed in this way, thus obviating the necessity for filtration. The growth was then pressed gently between a few thicknesses of blotting paper to remove excess kerosene, placed in a pre-weighed aluminium cup and dried to constant weight at 80° C. in a hot-air oven. Constant weight was achieved in two days in the case of growths from aviation turbine kerosene. Growths from lighting kerosene required somewhat longer.
The fungus grew at the interface between the mineral salts medium and kerosene (Fig. 1) producing a dark brown fungal mat of mycelium in the case of f. avellaneum and a white fungal mat in the case of f. albidum (Fig. 2). After six weeks when the growths were harvested, black sclerotial-type bodies were present in some cases adhering firmly to the side of the bottle. These were possibly immature ascocarps. The fungus produced asexual spores within the kerosene.
All isolates tested were able to grow on both aviation turbine and lighting kerosene. Table 1 shows the results of three separate
C. resinae was compared under similar conditions. The figures in the table refer to dry weight of growth harvested from two bottles. Before harvesting the fungus in the first test, V-8 juice agar plates were inoculated with each isolate. The resulting growth was used to inoculate kerosene in the second test. The process was repeated for the third test (note that in the third test 8oz. bottles were used containing 100ml. of mineral salts medium and 25ml. kerosene). Thus the requirements of ‘Koch's Postulates’ were satisfied.
In each test soil and air isolates grew better than fuel isolates. The amount of growth of soil and fuel isolates tended to decrease on successive transfers through kerosene but that of the air isolates increased. In Fig. 3 growth of soil, fuel and air isolates in aviation turbine kerosene in the first test is compared in the form of a histogram. This shows the large difference in amount of growth, particularly between soil and fuel isolates. In Fig. 4 means of the growth of the isolates in aviation turbine and lighting kerosene in the same test are compared. The amount of growth of soil and fuel
In Third Test used 8oz. medicine flats with 100ml. B/H + 25ml. kerosene.
isolates was greater in lighting kerosene than in aviation turbine kerosene. The opposite was true of the air isolates. However, in the second test (see Table 1) the amount of growth produced by air isolates in aviation turbine and lighting kerosene was very similar. Visual observation from the time of inoculation indicated that the growth of all isolates was initially slower in lighting kerosene. By the third or fourth week it had caught up and overtaken that in aviation turbine kerosene.
The indications from the above results are that soil and air isolates of C. resinae grow better and faster in kerosene than do fuel isolates. Because of the small number of isolates involved it was decided to include a greater number of soil and fuel isolates in another experiment. Results are shown in Fig. 5. The greatest amount of growth was produced by a soil isolate; some fuel isolates, although producing visible growth, did not produce enough for harvesting. It is obvious from Fig. 5 that there is a wide range of ability of isolates of the fungus to grow in aviation turbine kerosene.
Some isolates of f. avellaneum produced a reddish brown pigmentation of the mineral salt medium. The nature of this pigment is being investigated.
C. resinae f. resinae has been shown to grow in aviation turbine kerosene but has not yet been tested in lighting kerosene. The white form, f. albidum, was recovered from two bottles containing aviation turbine kerosene which had been inoculated with the fuel isolates of f. avellaneum (C94, C95) but it was not tested further.
All soil and air isolates and the majority of fuel isolates tested by us were able to grow in aviation turbine and lighting kerosene in the absence of any other source of carbon. Isolates varied considerably in the amount of growth produced after six weeks, indicating that many strains of the fungus exist, differing in their ability to grow in and utilise kerosene. All isolates grew well on V-8 juice agar without creosote, no difference being noted in the rate of growth. Further evidence for the existence of a number of strains of this fungus is obtained from the amount of reddish brown pigment produced by isolates and present in the aqueous phase when growing in kerosene. Some isolates produced an appreciable amount of this pigment, others none at all.
The amount of growth of soil and fuel isolates in general decreased on successive transfers through kerosene while that of air isolates increased. The reason for this is not known. Further work is necessary in relation to air isolates of the fungus because contamination of fuel by airborne spores is likely to occur.
All three forms of C. resinae tested, i.e. f. avellaneum, f. albidum and f. resinae, were able to grow in kerosene. The question may be
avellaneum is isolated from samples of aviation fuel. The reason for this appears to be related to the predominance of this form in the soil (Parbery, 1968; Sheridan, Steel and Knox, 1971) and in the air (Sheridan, 1971). We have not so far isolated f. albidum or f. resinae directly from soil or air. C. resinae f. resinae does not sporulate as profusely as f. avellaneum in culture and the spores are much more difficult to dislodge. In two instances we recovered f. albidum from kerosene after inoculating with f. avellaneum
C. resinae.
The authors wish to thank Mr. W. Freitag and Mr. W. Johnston of B.P. New Zealand Ltd. for their help and interest in this work, particularly for supplying us with samples of kerosene. Financial assistance provided by the Victoria University of Wellington, Internal Research Committee, is gratefully acknowledged. Finally, our thanks are due to all those within the Botany Department of this university, and elsewhere, who have assisted us in so many ways.
Introduction.
Background to the problem.
Method of attack by micro-organisms.
The relative importance of bacteria and fungi in fuel contamination and corrosion.
The occurrence of Cladosporium resinae in aviation fuels.
Methods of control.
Methods for monitoring fuels and testing resistance of tank sealants and linings.
Summary and conclusions.
The ‘Kerosene Fungus’ has become of importance because of its association with aviation fuels. It was Hendey in 1964 who coined the name ‘kerosene fungus’ but prior to this the fungus had been known as the ‘creosote fungus’ (Marsden, 1954) because of its association with creosoted timbers. This fungus exists in two states; the imperfect or asexual state Cladosporium resinae (Lindau) de Vries (de Vries, 1955), and the perfect or sexual state Amorphotheca resinae Parbery (Parbery, 1969a). It is usually referred to as Cladosporium resinae because this is the state in which it normally occurs in kerosene and soil. To further complicate matters four forms of the imperfect state have been described. Intermediate forms exist and most workers find the fungus to be very variable indeed (see Parbery, 1969a).
Interest in this fungus was first aroused by reports of its occurrence in storage and aircraft fuel tanks containing aviation fuel in the early 1960's. Until the mid-1960's, however, a certain amount of secrecy or mystery surrounded the problem of microbial growth in fuels because much of the work was done at defence establishments or in technical laboratories and most reports were difficult of access.
Publicity could, perhaps, have resulted in public panic. The first significant publication in the open scientific literature which drew the attention of mycologists to the problem was Hendey's paper (he was working at the Admiralty Materials Laboratory, Poole, Dorset, England) published in 1964 which dealt with the distribution of the ‘kerosene fungus’ in kerosene-type fuels, its identification and the conditions under which growth occurs. The utilisation of hydrocarbons by micro-organisms had been known for some time previously and Bushnell and Haas (1941) showed that gasoline (= petrol), kerosene, light and heavy mineral oils and paraffin wax could be used as sources of carbon. They examined the ‘water-bottoms’ from various petroleum storage tanks but recorded only bacteria (Pseudomonas and Corynebacterium).
Lansdown, writing in the Royal Aeronautical Society's Journal in 1965, has listed specific problems associated with microbial growth in aviation gasoline and kerosene. These include fuel pump failures and corrosion, filter clogging and fuel tank corrosion. Both bacteria and fungi were implicated. He said at that time (1965), ‘It has now become apparent that microbial contamination is widespread in aircraft fuel supply systems, both on land and in aircraft carriers, where serious clogging of fuel system filters has occurred.’ The problem, although worse in the tropics, appeared to be world wide. It has now been shown that among the fungi the ‘kerosene fungus’ is the organism most frequently implicated, e.g. it was present in 78% of all fuel samples from aircraft tanks examinated in Australia (Hazzard, 1963) and in 80% of all fuel samples examined in California (Engel and Swatek, 1966). It is probably the most important micro-organism in contamination of fuels and in corrosion at the present time (Parbery, 1968).
In addition to its presence in aviation fuel the ‘kerosene fungus’ has been found in other hydrocarbon fuels and hydraulic fluids (Nicot and Zakartchenko, 1966), on creosoted timbers such as telegraph poles and railway sleepers (Marsden, 1954), on tarred woods, asphalt and resins (Christensen, Kaufert, Schmitz and Allison, 1942), bituminised cardboard (Anon, 1968), a cosmetic face cream (de Vries, 1952), the female sex hormone, progesterone (Fonken, Murray and Reineke, 1960) and methyl-p-hydroxybenzoate (Solkolski, Chidester and Honeywell, 1965).
Very recently it has been isolated from soil in Australia and Europe (Parbery, 1968a, 1969b) and in New Zealand (Sheridan, Steel and Knox, 1971) and it appears to be widespread.
Both Hazzard and Hendey have indicated that a complete study of this fungus is an immense task (Hazzard, 1963). Our knowledge can be expected to increase only gradually as more mycologists become interested in the fungus and initiate studies on its biology. In our opinion the three most significant contributions of recent research to our knowledge of this fungus are: (1) the development
et al., 1942; Hendey, 1964) and it possibly enters the fuel via the air, but they were unable to isolate it from the air. In our laboratory we are currently studying isolates from fuels, soils and air, a number of which produce the perfect state under mineral oil.
We became interested in the ‘kerosene fungus’ as a result of finding it in a local soil in November, 1969, Our studies have been concerned with the biology of New Zealand isolates of the fungus, their distribution in soils and occurrence in air and aviation fuels. The two questions which are put to us most frequently are (1) how widespread is contamination of aviation fuels with C. resinae at the present time and (2) how does the fungus get into the fuels? These are obviously important questions. The former is difficult to answer because of inaccessibility of reports as mentioned earlier and the expression of conflicting opinions, the latter because of the failure of earlier workers to isolate the fungus from soil or air. These questions, and others, have prompted us to make a thorough search of the literature in an attempt to gather together in one paper a body of information on the ‘kerosene fungus’ and its activities. In so far as our own work here in New Zealand contributes to these aims we refer to it. The paper is published in three parts. Part I is concerned with the problem of microbial contamination of aviation fuels, Part II with the natural habitat of C. resinae and Part III with morphology and physiology of C. resinae.
For many years now micro-organisms have been known to grow in hydrocrabons (Miyoshi, 1895; Kaserer, 1906; Stormer, 1908; Sohngen, 1913), but it is only relatively recently that their growth has caused concern. It is the consequences of their growth that raises problems, not just their presence. Fuel pumps and filters may be clogged, capacitance gauges may not function properly and tanks and pumps may be corroded resulting in engine failure (Rogers and Kaplan, 1967).
The problem of microbial contamination of jet fuels was first observed in the American Air Force during 1956 (Leathen and
Parbery (1968) has noted that except for one report made in 1944 most reports of the microbial corrosion of aluminium have been published since 1960. These deal almost exclusively with the corrosion of alloys in fuel tanks and wing components of aircraft using jet fuel. Integral fuel tank corrosion was first observed in the American Air Force in 1960. About the same time similar problems were encountered in several other military and commercial airlines and in the Australian Air Force and Australian airlines (Churchill, 1963). However, it was not until 1960 and 1961 when the American Air Force began its modification programme on the relatively new ‘wet wing’ aircraft (B-52 G, KC-135 and C-130) that the seriousness of integral fuel tank contamination and associated corrosion problems was recognised (Ward, 1963). Large amounts of sludge and bacterial growth were found in the tank bottoms and sides — corrosion was widespread in the areas covered by this contamination. The problem was greatest in tropical environments. Ward (1963) reports that the C-130 fleet has had aircraft with holes corroded completely through the lower skin from integral fuel tank corrosion. Corrosion ranged from isolated pits and small areas of exfoliation to extensive pitting and exfoliation covering complete panels. The wing tank skin was made of aluminium plates and stringers of extruded, integrally stiffened aluminium alloy panels. No corrosion-resistant coatings were provided for the tanks at that time. The shape of the tanks made it virtually impossible to drain off water and sludge.
In addition to the above there are a number of other reports of problems associated with microbial contamination of aircraft fuels. These have been gathered together by Lansdown (1965) and include: fuel pump failures in Valetta and Hastings aircraft operating from the Suez Canal Zone in 1952; heavy fuel pump corrosion in Canberra aircraft operating from Butterworth in Malaya in 1956; fuel filter clogging in Boeing B-47 and KC-97 aircraft in 1958 and fuel tank corrosion in North American Fury (FJ-3 and FJ-4) in 1961.
Hazzard (1961) has reported fuel tank corrosion in Lockheed Hercules and Electras in Australia. The problem has also been reported from Brazil (Gutheil, 1966).
From these reports it is clear that damage has occurred to Australian, American and British aircraft. Most trouble is encountered where aircraft are operating in tropical regions and in aircraft with integral fuel tanks. Damage to British-built and British-operated aircraft, according to Lansdown (1965) has been extremely low and in all cases where it occurred aircraft had been operating in the Far East. The damage was disclosed during maintenance without any operating difficulties arising. He suggests as a possible reason the temperate climate of Britain which gives a lower daily temperature variation and thus a lower water condensation (water is essential for growth of micro-organisms in fuels) and temperatures below the optimum for microbial growth. Also the tank drains, to remove water and sludge, appear properly located in British aircraft. There is another possibility worth considering, that is the possibility that different strains of micro-organisms may be involved. This aspect has not yet been studied as far as we are aware although we have found some evidence for the existence of strains of the ‘kerosene fungus’ differing in their growth rate in kerosene (see Part III).
Aviation fuel consists almost entirely of hydrocarbons (compounds containing only carbon and hydrogen) but will also contain traces of additives and contaminants. In high-octane aviation gasoline the major additive is a lead compound, usually lead-tetraethyl with an organic bromide to prevent lead fouling. Other additives present in much smaller quantities in aviation fuels are anti-oxidants, metal de-activators and possibly corrosion inhibitors, anti-icing additives or anti-static additives (see Lansdown, 1965). Modern jet aircraft use aviation turbine kerosene or kerosene-type fuels. In its natural state kerosene can dissolve up to 75 parts per million (ppm) of water, the actual amount depending on temperature and atmospheric humidity. Free water will be present in fuels subject to temperature variations due to condensation. This water will extract components from the fuel and might, for example, contain a few ppm of hydrocarbons and several per cent of anti-icing additive. Both hydrocarbon and water phases will contain dissolved air (Hill, 1970) and materials leached from tank coatings or produced by corrosion of the metals present.
Sea water has often been deliberately introduced to fuel tanks to seal off leaks at the bottom of the tank, to flush out the fuel from less accessible parts of the system and to act as ballast in tanks. For example, it has recently been claimed that at least 20% of the world's tanker fleet discharges 600,000 tons of oil during tanker cleaning — the equivalent of five Torrey Canyons (Anon., 1970a). The sea water in the tanks will provide inorganic salts and possibly biological contaminants.
Even in the absence of sea water fuels may contain fungal spores and bacteria presumably derived from the air or soil as contaminants during handling. These are not removed by normal filtration (but see under ‘Methods of Control’). Some of these organisms will arrive at the interface between fuel and water: the fuel provides the source of energy, the water provides dissolved oxygen and trace materials. In vented aircraft and fuel tanks air is drawn in to replace fuel used and airborne fungi and bacteria can thus enter and may settle in the fuel. In gasometer-type storage tanks (with a floating roof) rain water carrying microbial contaminants may enter the tanks (Prince, 1961).
Different classes of hydrocarbons are preferentially attacked by different micro-organisms. Hendey (1964) has shown, for example, that the ‘kerosene fungus’ Cladosporium resinae can use kerosene as its sole carbon source. This has been confirmed in France (Nicot and Zakartchenko, 1966) and New Zealand (Sheridan, Steel and Knox, 1971). Between 20% and 50% of the carbon assimilated by bacteria and fungi is converted into cell substance (Zobell, 1946), the proportion being higher in the earlier stages of growth.
The remainder of the carbon is converted to more highly oxidised compounds including carbon dioxide, organic acids, alcohols and esters. According to Lansdown (1965) these products will themselves alter the environment: e.g. lower molecular weight fatty acids will lower the pH of the aqueous phase making it more corrosive to metals. Reactions with specific mineral salts may produce more corrosive acids. Corrosion of metals gives rise to more available ions in solution (De Gray and Killian, 1960). Higher molecular weight fatty acids are surface active and by concentrating at the fuel/water interface will reduce the interfacial tension and permit ready emulsi-fication of fuel and water. Alcohols and esters will increase the solubility of fuel in the aqueous phase. All this results in extension of the zone for optimum microbial growth. Other micro-organisms which hitherto had remained dormant may now find conditions suitable for their growth. It may be that growth is now rapid and oxygen is all consumed leading to anaerobic conditions. If sulphate is present (from direct contamination or oxidation of sulphur-compounds in fuel by aerobic micro-organisms) conditions will be favourable for the growth of sulphate-reducing bacteria. These produce fuel-soluble corrosive sulphide (e.g. hydrogen sulphide) which can be carried with the fuel and cause corrosion of components of an aircraft fuel system (Hill, 1970).
Although it has been claimed that saline water, rust, sulphates and ‘dirt’ will themselves cause corrosion even in the absence of micro-organisms (Calvelli, 1963), a strong microbial growth will most probably enhance corrosion (Lansdown, 1965). London, Finefrock
Corrosion of aircraft integral fuel tanks has been studied by a number of workers (see Parbery, 1968) and without doubt has been very serious. In some cases wing planks have been corroded through (Lansdown, 1965). Where tanks were lined with rubber coatings it appeared that fungi were able to penetrate these and there is also the possibility that they could do the same to the metal walls of the tanks. Lansdown (1965) points out that although there have been indications that mechanical penetration of aluminium foil can occur, it is very difficult to show that the effect is not a simple chemical attack associated with the corrosive products of metabolism around the growing fungal hyphae. Experiments by Hazzard (1963) have shown that the fungus Cladosporium resinae can penetrate rubber linings (see details under ‘Methods of Control’). It is well known that many plant pathogens can mechanically penetrate thin films of chemically inert materials by the formation of special attachment organs (appressoria) (Wood, 1967). C. resinae has been observed by us to attach itself firmly to the glass walls of culture bottles: the attachment organs closely resemble appressoria, so purely mechanical penetration would not be surprising. No work has yet been done on this aspect in our laboratory.
Microbial attack is also shown by the formation of sludge or solid matter which may clog downstream parts of the fuel system, particularly filters and screens. There is some doubt as to whether bacterial slime has sufficient mechanical strength to block filters (Bakanauskas, 1958) but there is little doubt that fungal mycelium can block filters, screens and other small apertures, perhaps even the drain points of fuel tanks. Pump screens partly blocked by fungal growth have been found by Hazzard (1963).
Fungal growth may also become attached to the fuel tank walls and prove difficult to dislodge during cleaning operations.
De Gray and Killian (1960) report that some rust inhibitors appear to function as nutrients for bacteria. They have found stimulation of growth at normal levels of such inhibitors (25 to 50 ppm) in both gasoline and kerosene. In a kerosene, essentially the same as a JP-4 fuel, they found the bacterial count after 48 hours without rust inhibitor to be 50,000/ml.; with 25 ppm inhibitor it was 2,200,000/ml. The surface-active rust inhibitors apparently reduce the interfacial tension of water and hydrocarbon, and so increase the availability of hydrocarbon to bacteria. ‘Bacteria encourage rust and make slimes, which hold rust in suspension, which encourages bacteria, which encourage rust and make slime …’ is a very apt description of the vicious circle (De Gray and Killian, 1960). Anti-icing additives may in low concentrations also prove nutritional to micro-organisms (Hill, 1970).
The following organisms are reported as having been isolated from jet fuel and jet fuel-water samples:
Among the fungi Cladosporium was the most prevalent and among the bacteria Pseudomonas aeruginosa.
Parbery (1968b) has reviewed the literature comparing the roles of bacteria and fungi in corroding aluminium and concludes that bacteria were generally believed to be more important although he considers that there is no acceptable basis for this belief. Numerous difficulties are encountered in attempting to equate growth of bacteria and fungi because the former are unicellular and readily counted, the latter are mycelial (thread-like) and tend to clump. Reports of much of the work done in an attempt to elucidate the problem are not available to other research workers because they have not been published in the open scientific literature, a situation which may, in effect, mean that ‘they are to all intents and purposes not published’ (Parbery, 1968c). It is clear, however, that masses of bacterial growth are more easily broken up than fungal growth and so are less likely to cause blockages. Fungal hyphae can readily attach them-selves to solid objects such as fuel tank walls and may exert a mechanical pressure which may allow penetration of the linings (see under ‘3. Method of attack by micro-organisms). Bacteria cannot do this. The microbial growth sludge removed from corroded fuel tanks invariably contains a mixture of fungal and bacterial species (Parbery, 1968b). The bacterium Ps. acruginosa and the fungus C. resinae have been found often in association with corroded alloys and each can induce corrosion in aluminium plates in culture (Hedrick, Carrol, Owen and Pritchard, 1963; Davis, 1967).
Parbery (1968b) has studied growth of C. resinae in relation to corrosion of aluminium and found a correlation between amount of mycelium produced and the percentage loss in weight of aluminium pieces. Hendey (1964) showed that all samples of aluminium in his experiments which were in contact with C. resinae were perforated and had lost considerable weight: controls were unaffected and had lost no weight.
Other organisms less commonly found, which also apparently cause corrosion, are a gram-ve bacterium identified as ‘17-11’ (Hosteller and Powers, 1963), Desulfovibrio desulfuricans (Hedrick, Carroll, Owen and Pritchard, 1963; Iverson, 1967) and A. niger (Hedrick, Carroll, Owen and Pritchard, 1963).
Many organisms, other than the above, found in fuel may possibly be dormant and do not grow — some of the fungi listed at the beginning of this section possibly arise as contaminants during sampling.
Hazzard (1963) has surveyed 45 airfields in Australia and the Far East and examined 599 samples of aviation turbine kerosene. He found C. resinae in 218, Paecilomyces in 55, A. niger in 48 and Helminthosporium in 27.
Rogers and Kaplan (1964) studied the growth of over 800 isolates of fungi from fuels in relation to their ability to grow in the presence of aviation fuel. Best growth occurred with isolates of C. resinae, Fusarium sp. Stysanus sp. (mis-spelled as Stypanus) and
Alternaria sp. A group classed as having moderate ability to utilise fuel included several Cephalosporium spp.; other Cladosporium spp. and a few penicillia, aspergilli and an Helminthosporium. Basidiomy-cetes, A. niger, Trichoderma sp., Pullularia sp., yeasts and actinomy-cetes grew very little or not at all. Nicot and Zakartchenko (1966) have inoculated sterile minimal medium with a mixture of fungi isolated from jet fuel and covered the medium with a layer of fuel. Only C. resinae grew.
Engel and Swatek (1966) report that Ustilago (a smut fungus) was isolated from a number of fuel samples (but not from soil). They report that all their isolates of Ustilago grew readily with fuel as their carbon source, sporulating profusely. Ustilago is a plant pathogen and its ability to grow in aviation fuel appears rather unusual.
We have examined a number of fungi isolated from aviation fuels in New Zealand for ability to grow in creosote and in aviation kerosene (unpublished). These were identified by us as Cephalo-sporium sp., Paecilomyces sp., Penicillium spp., Trichoderma viride, Stemphylium sp. and Cladosporium resinae. All except Stemphylium and T. viride grew on an agar medium containing 0.1% creosote; only C. resinae grew on the medium containing 1% creosote and it was the only fungus to grow in aviation kerosene. (Fig. 1.)
Isolates of C. resinae from soil and air also grew readily in kerosene. None of the yeasts isolated by us and only two of the bacteria (belonging to the genus Pseudomonas) grew in kerosene. We are currently studying corrosion.
It seems clear, therefore, that the fungus C. resinae is undoubtedly the most important and dangerous contaminant of aviation fuels at the present time. However, as Hedrick, Reynolds and Crum (1968) point out, there is an urgent need for intensive studies on fungi (moulds and yeasts) and bacteria in connection with their growth in jet fuels because our knowledge is still very small.
Figures for the occurrence of C. resinae in aviation fuel in aircraft and storage tanks are not readily available. However, a summary of those available to us is given here.
Engel and Swatek (1966) studied the micro-organisms isolated from 59 jet aircraft integral fuel tanks in California, U.S.A., and found the most prevalent fungus to be C. resinae (= Hormodendrum resinae). It was present in 80% of the samples. They mention that of 9 soil samples taken from strategic positions at the overhaul base, none yielded this organism. This is rather surprising since the fungus has subsequently been shown to be widespread in soils in Australia (Parbery, 1969b) and New Zealand (Sheridan, Steel and Knox, 1971), and would be expected to enter fuel tanks from this source via the air.
In an examination of fuel tanks of Australian aircraft during 1961 and 1962 Hazzard (1963) found fungi in all but one (a relatively new Boeing 707) of 21 aircraft. C. resinae was the predominant fungus isolated: it was present in 78% of all samples. His results for 157 samples from tanks of the 21 aircraft are shown below.
He reports that Shell examined 65 samples of fuel from 2 Comet IV, 6 Boeing 707 and 4 Electra aircraft and found C. resinae in 63 of them (97%). Contamination was heavy with counts of 100-300/litre (see ‘6. Methods for monitoring fuel’).
In a later survey of fuels (1963) delivered to civil aircraft in Australia and Pacific Islands, carried out by private companies,
C. resinae was reported in only 21 samples out of 194. This represents 39 airfields. The counts were less than 10/litre. The survey was carried out after attempts had been made to control the problem.
Because of the high incidence and levels of contamination originally found in military fuel systems and aircraft, the R.A.A.F. arranged overhaul and where necessary replacement of all fuel installations at its bases. Subsequent testing (in 1963) showed a very much lower level of contamination: C. resinae was found in only 9 out of 88 samples taken from 10 bases. Previous figures were 47 out of 115. Fungal counts were now down to 3/litre.
These results indicate a considerable reduction in observations of the occurrence of C. resinae consequent on the application of measures designed to reduce the problem.
Reliable figures for the occurrence of C. resinae in aviation fuels are not yet available for New Zealand. Some indication of the present situation in this country may, perhaps, be reflected in our recent examination of 13 samples of aviation turbine kerosene, 6 of which were found to be contaminated with C. resinae. The highest count was 24/litre.
The catalogue of the culture collection of the Commonwealth Mycological Institute, Kew, (Anon., 1968) lists C. resinae from kerosene filters from Brazil, Japan, Syria, Nigeria, Denmark, England and India. It is also listed from aircraft fuel tanks from New Zealand.
Present methods of control are aimed mainly at preventing or destroying growth of micro-organisms in the fuel. This can be tackled in a number of ways: (1) regular removal of water, which is essential for growth, and periodic washing out of tanks to remove microbial growth; (2) addition of a biostat which prevents reproduction, or biocide which kills micro-organisms; (3) use of resistant tank coatings so that the metal walls of the tanks cannot be reached by the micro-organisms or their metabolic products; (4) filtration to remove all bacterial and fungal growth and (5) removal from fuels of fractions essential for microbial growth.
The first approach constitutes what is commonly known as ‘good housekeeping’ and is the usual method of control, as far as aviation kerosene is concerned, applied at present. Where applied rigorously it works satisfactorily. But difficulties arise, for example in aircraft carriers, or in oil tankers, where fuel is stored for long periods. It is impossible to eliminate water entirely from aircraft fuel tanks because condensation provides a constant source of free water. The best that can be done is to remove this water by frequent and careful draining. Tanks are now designed to make this possible. Filters and filter separators have been designed to remove solid contaminants and water from the fuel supply. In order to kill
C. resinae (see 4. The occurrence of C. resinae in aviation fuels). Their value lies in constant and careful application.
The second approach, i.e. the addition of biostats which prevent reproduction or biocides which kill micro-organisms, is not so easy to apply. The biostat or biocide must be active chiefly in the aqueous phase yet must not be removed from the system by the periodic drain checks which are essential to remove condensed water. In a modern jet aircraft such as the Boeing 727 it is estimated that between one pint and one quart of water is produced for each vent in the fuel tank every time an aircraft lands (personal communication — Shell (N.Z.)). Hence best control is achieved by using water-soluble/non fuel-soluble biocides in fuel storage installations. Compounds such as silver wool, borates, chlorides and methyl violet (Lansdown, 1965) have been tried for water bottoms where sulphate reducing bacteria occur, and a boron compound, Biobor JF, has been used in some United States executive aircraft.
An anti-icing inhibitor containing 99.6% methyl cellosolve and 0.4% glycerol (= ethylene glycol monomethyl ether or EGME) introduced by the U.S.A.F. in 1962 appears to act as a biocide (Lansdown, 1965; Hill, 1970). Neither methyl cellosolve nor glycerol are active on their own. Hill (1970) has shown EGME to be an effective biocide but concentration of EGME and degree of contamination of fuels by micro-organisms is important. He found that at 10% concentration in mineral salts medium EGME required 7 days to effect sterilisation. On the other hand an organo-borate at 1% produced sterility within 24 hours. He found that in low concentrations EGME may even be actively nutritional. He further describes experiments in which mixtures of para hydroxy-benzoates dissolved in EGME are used as a fuel biocide, and sterility achieved within a few hours. Another drawback to the use of inhibitors is their high cost (Hill, personal communication).
The only figures available to us are as follows. The price of Biobor, marketed in the United States by Boron and Chemical Corp., is given as 60 cents/lb. in bulk, $1.21/lb. in case lots (Anon., 1970b). It has been estimated that initial treatment at 270 ppm would cost $2.19/1,000 gal. fuel. Maintenance levels of 135 ppm would run at $1.10/1,000 gal.
Rogers and Kaplan (1967) have screened a number of prospective biocides, tabulated the requirements for the ideal biocide and discussed two commonly in use which meet most of the criteria. These are EGME and a mixture of boron compounds.
From the beginning of 1970 all the R.A.F. fuels have contained EGME (Hill, personal communication). The Australian services and the R.N.Z.A.F. use an anti-icing agent which contains EGME and has apparently eliminated the problem of microbial contamination in their aircraft (personal communications).
In the third approach, the use of resistant fuel tank linings, much work has been done. According to Ward (1963) a polyurethane coating has successfully withstood many tests devised to simulate microbiological environments within fuel tanks. Hazzard (1963) compared a number of tank lining materials, recommended by airframe manufacturers: these included Buna-N rubber, polyurethane and epoxy top-coatings. Fungal penetration (by C. resinae) of Buna-N occurred within 4 to 6 days but no penetration of epoxy or polyurethane had occurred after 148 or 180 days respectively. The incorporation of fungicides into Buna-N films delays penetration. He recommended the use of polyurethane in integral tank protection. On the other hand, Miller, Herron, Krigrens, Cameron and Terry (1964) found that a polyurethane film was penetrated in 8 days by a mixed culture of micro-organisms obtained from a JP-4 fuel tank. They suggest that the polyester type of polyurethane may be less resistant than the polyether type. Hazzard was working in Australia, Miller et al. in America: the discrepancy of results may also be possibly due to the existence of species or strains of fungus differing in their penetrating ability. This aspect has not been studied by anyone as far as we are aware. A ‘furane’ lining has been claimed by McGregor (1963) to give protection.
In the fourth approach, i.e. filtration to remove all bacterial and fungal growth, Hedrick, Owen, Carroll, Pritchard, Albrecht and Martel (1964) have concluded from their studies that fuels treated with ultrafine mechanical filtration can be made free of microbial contamination. They used 0.35 and 0.45 micron pore-diameter filters. Approximately 13.8 square feet of filter surface was required to filter 600 gallons/minute of JP-4 fuel at 50 p.s.i. filter differential pressure. About 20 square feet would be required to filter JP-5 under the same conditions. These figures are for fuel with no free water — the presence of water slows down the rate of filtration considerably and may affect sterility. They concluded that sterility could only be obtained providing all water is removed from the fuel prior to filtration. Fuel additives may affect the efficiency of the filter membrane. Normal filling (not using an ultrafine filter) of a VC-10, for example, is at the rate of 1,000 gals./min. Even after removal of all micro-organisms from fuel, contamination may subsequently occur from air sucked into tanks through vents during flight. Recent work (Sheridan and Nelson, 1971) has shown that C. resinae, the most serious fungal contaminant, is air-borne.
Before the fifth line of attack can be applied (removal of fractions of fuel required for growth of micro-organisms) it is necessary to
C. resinae using gas chromatography. It appears that this fungus utilises many components of the kerosene and it appears unlikely, therefore, that removal of a specific fraction from the fuel would have much effect. Further work is necessary.
Other methods of control which have been tried include gamma ray sterilisation and radio frequency (Kivel, Chatterton and Palchak 1967; Hedrick, Carroll and Owen, 1964).
In conclusion, therefore, it can be said that ‘good housekeeping’ applied rigorously has proved an effective method of controlling microbial contamination of aviation fuels and consequent problems. It seems unlikely, however, that this method will succeed in eliminating the fungus C. resinae from fuels because of the widespread occurrence of the fungus in soils and in the atmosphere. Indeed, now that strains of the fungus, present naturally in soils in New Zealand, have been shown to grow more vigorously than many of those isolated from fuels (see Part III) a constant watch must be kept for any increase in the incidence and levels of C. resinae in aviation kerosene.
The problem of microbiological fuel contamination and corrosion is now generally considered to be under control. Continuing care is necessary, however, to maintain this control. The search for effective biocides still continues although to date there has not been much success. A combination of ‘good housekeeping’ with good aircraft design ensuring well-located drain points, tank linings which are resistant to fungal penetration, and an endeavour to keep fuel as clean and dry as possible is still absolutely essential if trouble is to be avoided in the future.
The methods commonly used by the petroleum industry for monitoring fuels for the presence of micro-organisms are those developed by Hazzard and Kuster (1962: Report 252, Fungal growths in aviation fuel systems. Part 2. Test Methods), and Hazzard (1967), where a known quantity of the fuel sample is passed through a sterile membrane filter of 0.45 micron pore size and the membrane placed on a nutrient medium for incubation, or where large quantities of fuel in the field can be passed through a field monitor and level of microbial contamination determined in the laboratory. The former method is suitable for tank-drainings, water bottoms, etc., where the level of contamination is high, the latter for fuels in systems under pressure where the level of contamination is low. More recently Hill (1970) has described a microbiological test for aircraft fuel which has the advantage of simplicity and speed. Hazzard's methods appear to be applied world-wide: we must wait and see whether
A known quantity of the sample is passed through a sterile membrane of 0.45 micron pore size (47mm. diameter) and the membrane placed on a nutrient medium for incubation. The number of microbial growths observable at the end of four days’ incubation is reported as the ‘fungal count’. The growths are identified by normal microbiological techniques.
The filtration apparatus (Millipore) is sterilised and assembled, and the sterile filter membrane is placed in position on the fritted glass or metal plate.
The fuel sample is agitated and filtered through the membrane using the minimum of suction necessary to achieve transfer through the membrane. The top of the filtration apparatus is kept covered except when adding the sample. The size of sample is selected by experience and can vary from 1ml. for highly contaminated samples to several litres for samples containing only a few fungal spores. Where an anti-icing additive, which is also a biocide, is present in aviation turbine kerosene, the bowl and filter are washed with 2 x 10ml. sterile water containing 0.1% detergent.
Suction is applied for 30 seconds after the last drops of the sample have passed through the membrane. The pump is then disconnected and the membrane transferred, using sterile forceps, to the centre of a Petri dish of agar medium (agar 15g., malt extract 25g., yeast extract 0.5g., water 1 litre. 0.5g. penicillin is added to suppress bacteria. The medium is sterilised at 15 psi (103 kNm-2) for 20 minutes).
The Petri dish is placed in an incubator at 85-90° F. (29-32° C.). After 96 hours’ incubation the number of growths (= colonies) on the membrane are counted.
The final report gives the number of colonies as ‘fungal count’ in the given sample size. With experience the identity and number of each type of fungus can be reported. (Fig. 2.)
This method, although primarily designed for detecting fungi, can be adapted for bacteria. It is then best to use a 0.25 micron pore size membrane, because some bacteria may pass through the 0.45 micron pores, and a special bacteriological nutrient agar medium. In our experience when testing specimens for fungi, bacteria often grow on
C. resinae (e.g. Trichoderma viride and Penicillium spp. sometimes do so) we then either filter another sample of the specimen and cover the filter with V-8 juice agar containing 0.1% creosote or simply pour this selective medium over the original filter with its fungal colonies. Only C. resinae grows readily on this medium. Also, C. resinae can be isolated from mixed cultures by this method. In a comparison of methods for detection of C. resinae in fuel-water samples involving placing the membrane on agar surface or pouring agar over the membrane we obtained highest results by using the latter.
One problem which we have not been able to overcome concerns the difficulties experienced in filtering mixtures of fuel and water. After shaking and pouring into filter apparatus problems arise because of the high pressure necessary to suck the mixture through. It appears that kerosene is reluctant to pass through the membrane filter if the filter is first wet with water and vice versa. Our water pump sometimes fails to effect filtration and we resort to an electric suction pump. The effect of these higher suction pressures on the membrane filter is not known.
This method of sampling describes a procedure by which samples may be obtained from any point in a fuel system, at which the static head exceeds 5 feet, for the detection of viable microbiological material. A bomb sampler fitted with a sterile field monitor (supplied by Millipore Filter Corporation) is attached to the system and a suitable amount of sample is passed through the monitor. The membrane is then transferred to the surface of a nutrient medium for the cultivation of microbiological material.
The bomb sampler is sterilised by washing the interior with petroleum ether, then sterile distilled water and finally methanol. It is dried in a current of filtered air. Details of assembly and procedure are given by Hazzard and Kuster in Report 252, part 2, page 8, 1962.
The field monitor is sent to the laboratory, the filter membrane is transferred aseptically to the surface of a nutrient agar plate and incubated at 85-90° F. (29-32° C.) for 96 hours. The number of colonies is counted and reported as ‘fungal count’ in 850ml. or whatever quantity of fuel is sampled.
This test is designed to provide a rapid method of determining whether micro-organisms can penetrate thin films of organic coating materials either in the form of a continuous self-supporting film, or as a continuous coating applied to a nylon fabric substrate. It has been specifically designed for C. resinae.
A membrane of the coating material, sealed across the mouth of a tube, is immersed at the interface of kerosene/Bushnell-Haas medium in a test jar. Sterile kerosene is introduced to this tube and the upper surface is inoculated with a suspension of viable fungal spores or hyphae. The assembly is placed in an incubator at 85-90° F. (29-32° C.). Visual observation of the underside is made at intervals to detect the first appearance of fungal growth through the film. ‘Penetration time, days, (Cladosporium resinae)’ is reported as the number of days elapsing between inoculation and the first positive appearance of fungal growth on the underside of the film.
This method depends on the detection of phosphatases, a group of enzymes very widely distributed in micro-organisms. These enzymes split off phosphate from organic phosphate compounds and can therefore be readily detected. The acid phosphatases occur particularly in fungi and yeasts and the alkaline phosphatases predominate in bacteria (Hill, 1970). Hill used pure cultures of Pseudomonas spp., Candida spp and C. resinae all previously isolated from aircraft
The standardised test is as follows:
5ml. of aqueous sample is added to 0.051g. potassium hydrogen phthalate and 0.0075g. p-nitrophenyl phosphate in a bijou bottle ‘A’.
The bottle is shaken and incubated at 37° C. (trouser pocket if necessary) for one hour.
The contents of bottle ‘A’ are tipped into bijou bottle ‘B’ containing 0.2g. tribasic sodium phosphate.
The colour is graded 0-5 by comparison with a standard and/or the contents centrifuged and the optical density at 420mmu. noted.
A possible disadvantage lies in the fact that both living and dead micro-organisms give positive results, and additives (e.g. biocides) may influence results. The greatest advantage of the method lies in its speed — results in one hour as against five days by existing methods. We have not tried Hill's method here.
There have been a number of reports concerning problems raised by the growth of micro-organisms in aviation fuels. These include filter clogging and corrosion of pumps and fuel tanks. If left unchecked, there is little doubt that the consequences of microbial growth in fuels would be very serious. Both bacteria and fungi have been isolated and some have been shown to use the hydrocarbons as a food and to cause corrosion. Among the former Pseudomonas aeruginosa is the most prevalent, and among the latter the ‘kerosene fungus’, Cladosporium resinae. The incidence and level of C. resinae in fuel tanks and systems was high in the early 1960's but have fallen considerably in recent years as a result of the vigorous application of control measures, particularly ‘good housekeeping’. Few serious problems occur at present. Nevertheless there is no reason for complacency. Some people appear to believe that because the biological population is concentrated in the aqueous phase, the elimination of water or the suppression of life in the water will eliminate the micro-organisms. Hazzard (1967) has found that this view is incorrect. Some organisms, notably C. resinae, can introduce into the hydrocarbon phase material which will remain viable for several years in essentially water-free petroleum products. The application of ‘good housekeeping’ procedures will not, therefore, eliminate the micro-organisms from fuel. The demonstration that many soil and air isolates of this fungus grow more vigorously in kerosene than those normally isolated from this source further illustrates the seriousness of the problem (see Part III).
There is an urgent need for research aimed at developing better methods of detecting microbial growth in fuels and better methods of controlling it. An intensive study of all those organisms capable of utilising hydrocarbons is needed. The work of Parbery in Australia and our own work here in New Zealand is directed towards obtaining a better understanding of the biology of the ‘kerosene fungus’, C. resinae. Parts II and III of this paper are concerned with this work.
The authors are grateful to the Chief Superintendent of the Defence Standards Laboratories, Australia, for permission to quote data contained in D.S.L. Report No. 252, and to the Chief Chemist, B.P. (N.Z.) Ltd., for supplying samples of aviation kerosene. Thanks are due to Mr. Herbert Christophers for photographic assistance and to Mrs. Mary Sheridan for reading and criticising the manuscript.
The plant cell wall is a product of protoplasmic activity and in the higher plants its development begins with the formation of the cell plate, immediately after nuclear division. This thin cell plate quickly acquires the form of a primary cell wall, which is defined by Wardrop (1962) ‘as the structure which encloses the protoplasts during the period of cell enlargement’. Once the period of cell enlargement is over the cell wall becomes thickened to become the secondary wall. The secondary wall is regarded as the structural component of the plant (that is the plant skeleton). Such walls are also the major components of the conducting vessels.
The interrelationships of the protoplast and the cell wall, and the processes whereby the former maintains and extends the structure of the latter, thus present fundamental botanical problems which have been of interest for more than a hundred years.
The principal constituents of the cell wall are cellulose, hemi-cellulose, pectic substances, lignin and proteins. Waxes, cutin, suberin and sporopollenin are also found.
Cellulose: This is the most abundant substance in the plant kingdom, and is a polymer of -b-glucose residues joined in long chains by 1-4 links. Over certain parts of their length these chains lie parallel to each other and are very regularly spaced, to form long crystalline microfibrils. These microfibrils may be arranged randomly or in a regular fashion (Probine, 1963). Meyer and Misch (1937) proposed the ‘unit cell’ of cellulose. They considered that the cellulose chains lie antiparallel in such a way that alternate chains point in opposite directions. Within the microfibrils themselves are smaller units, the micelles, which are small aggregations of cellulose molecules that lie parallel to one another and thus confer a crystalline structure upon the microfibrils. More recently it has been claimed that the ultimate structural units of the cell wall are elementary fibrils about 35 A in diameter which are not aggregated into larger strands (Muhlethaler, 1967). The microfibrils are necessary to bear the stress in the wall due to turgor pressure.
The Pectic Substances: These consist of polymers of d-galacturonic acid, l-arabinose, d-galactose and l-rhamnose. These substances are found mainly in the middle lamella of primary walls. (Stace, 1963.)
Northcote (1969) has studied the metabolic changes which occur in the pectic substances deposited in the wall. He showed that the strongly acidic polygalacturonic acid was formed first. He found that pectin in the cell walls of a mature tissue such as in apple fruit was composed mainly of neutral and weakly acidic material and only traces of the strongly acidic polymer could be detected.
Hemi Celluloses: These are amorphous and consist of linear or branched polymers of d-xyloses, d-galactose, d-mannose, l-arabinose, and l-rhamnose. In contrast to cellulose the hemicelluloses are not crystalline in their natural condition although they have been found in a crystalline state after extraction (Roelofsen, 1959).
The distinction between the hemicelluloses and the pectins is not absolutely one of chemical constituents, but rather of physical properties, mainly solubility, which depends on their degree of methylation, cross-linkage and the extent to which different sugars are joined in the same molecule.
Lignin: This is an amorphous substance which occurs as an incrustation between cellulose microfibrils. The concentration is highest in the middle lamella and falls off towards the lumen. It is an important structural material and it is this substance that gives strength to wood.
Protein: Recent work has demonstrated the occurrence of a group of proteins containing hydroxyproline in the primary walls of various tissues. The amount present increases during growth and it is thought that the protein may serve enzymatic as well as structural functions.
The primary and secondary walls differ in their chemical composition and in fine structure. In most cases the secondary walls have a higher percentage of cellulose and lignin while the pectic substances are present only in trace quantities as compared to the primary walls.
Thus the general cell wall consists of the structural components of the wall, the cellulose microfibrils, orientated in the amorphous component comprising the pectic substances, hemicelluloses, lignin and the protein substances.
The endoplasmic reticulum, Golgi bodies and microtubules have all been implicated in the synthesis and organised deposition of the material of the plant cell wall on the basis of their association in electron micrographs with developing cell walls (Abersheim, 1965). Direct chemical evidence of a role for these organelles in the synthesis of wall materials is almost entirely lacking except for a demonstration by Northcote and Picket-Heaps (1966) that pectic substances are formed in the Golgi apparatus and are transported to the cell wall in the Golgi vesicles.
The possible role that the endoplasmic reticulum plays in the mechanism for laying down cell walls has been investigated but as yet ribosomes have not been identified in electron micrographs of the cell walls. However, Kivilaon et al. (1959) have detected trace amounts of ribosomal RNA (rRNA) in a preparation of cell walls of Zea mays coleoptiles. Phetheon et al. (1968) considered that the rRNA found in Zea mays was bacterial contamination, but they have isolated highly purified preparations of cell walls from a number of plants and have found that RNA was invariably present.
The possible role of rRNA in the cell wall may be concerned with the synthesis of protein in situ (Albersheim, 1965). However, Phetheon et al. (1968) consider that protein synthesis requires such an elaborate system of enzymes and nucleic acids that it is unlikely that such a synthesis would occur in the wall. The process of biosynthesis and orientated deposition of cellulose fibrils is considered by Ben-Hayyim and Ohad (1965) to consist of four steps: (a) polymerisation (of the activated monomeric precursor) to form a cellulose molecule of high molecular weight; (b) transport of the molecule from the site of synthesis to that of crystallisation; (c) crystallisation or fibril formation; (d) orientation of fibrils during deposition.
These steps are not necessarily isolated from one another. Frei and Preston (1961) believe the polymerisation, fibril formation and orientation occur simultaneously in plants. They propose that a catalytically active protein moves around the cell membrane and deposits the formed cellulose fibrils in pre-determined directions, the whole process being under genetic control.
Preston and Goodman (1968) considered the hypothesis of end synthesis of microfibrils through ordered enzyme-complex granules as a satisfactory explanation for the organisation and biosynthesis of the microfibrils. They suggested that the cytoplasmic surface is covered by layers of granules in cubic packing each containing cellulose synthesising enzymes. When these granules come into contact with a microfibril end they are stimulated and start synthesising the microfibril. The microfibrils are maintained straight both by intra-chain hydrogen bonding and mutual orientation of the granules. The packing of the granules are such that they would restrict the direction of growth of the microfibrils in such a way that only two directions at right angles to another and an occasional departure along the diagonals to these directions would be possible. It is considered that this hypothesis may explain the observations of Setterfield and Bailey (1958) that cellulose synthesis can occur in outer wall layers away from the cytoplasm, as the granules which come to be buried in the wall could continue to synthesise cellulose. Where or how the microfibril ends are produced is not known; however it is proposed that they could be produced by enzyme degradation of the cellulose or by stresses in the wall during cell elongation.
Frey-Wyssling (1962) has suggested that extrusions of the plasmolemma inside the cell wall are responsible for the synthesis and orientation of cellulose fibrils in plants.
Opposed to this is evidence that the synthesis, transport and crystallisation may be separate processes in bacterial systems (Ohad et al., 1962) and probably in some plants (Setterfield and Bayley, 1968). Many workers still regard the process of cellulose fibril orientation in the secondary wall of plant cells as a passive one, rearrangement being the result of mechanical strain or stress (Roelofsen and Houwinh, 1953). Northcoate (1963) noted that the synthesis of material and the extent of enlargement of the cell wall during development can be influenced by nutrition of the growing cell. Factors affecting growth are thus important in affecting the structure and growth of the cell wall.
The microfibrils are orientated in various ways in the cell wall, usually more regularly in the secondary wall. In the primary wall the microfibrils are often orientated in a direction transverse to the
Allium cepa the cell walls of the apical initials show a loosely woven mesh of microfibrils; in slightly older cells the microfibrils are mainly aligned horizontally, and this is the case during active elongation.
Jensen (1961) revealed that in the onion root these changes in cell structure can apparently be correlated with changes in the selective amount of cell wall components both in cells of different stages of development, i.e. at different distances from the root tip and in cells of different tissues at any one level. In particular the transitional region between radial enlargement and rapid elongation of the root is characterised by changing relationships between the wall components.
Ben Hayyim and Ohad (1965) have suggested a possible model for the orientation of microfibrils in the cell wall. They introduced a synthetic cellulose called Ma-carboxymethyl cellulose (CMC) into the microfibrils. This, they considered, played a similar role to that of charged polysaccharides. The parallel orientation of fibrils containing CMC could be explained partially in terms of charged interactions and mechanical stress. Non-charged cellulose is considered to form a random mesh, the rigidity of which would depend on the number of interfibriller cross links, i.e. H bonds. Electrostatic repulsion between fibrils containing CMC can weaken the interfibriller links occurring in the vicinity of adjacent carboxyls. Stress or strain applied to this kind of mesh could result in an easier deformation of the matrix in the direction of the applied force. The most stable state would be achieved when the fibrils are parallel and the overall numbers of links formed between fibrils exceed the number of weak bonds due to electrostatic repulsion. From a similar consideration it is conceivable that a second layer of fibrils formed above the first would reach a more stable position when oriented at an angle with the first, when the repulsion between the layers would be minimal.
Formerly two theories were held regarding the method of cell wall growth in thickness: that of growth by intussusception, where new microfibrils were held to be laid down between existing microfibrils and that of growth by apposition where new microfibrils were laid down on top of the existing ones, forming a new layer.
Fig 1.
It is now considered that the formation of both primary and secondary cell walls occurs principally by the mechanism of apposition (Muhlethaler, 1961). It is probable, however, that some growth by intussusception does occur. Deposition of cellulose uniformly over the whole surface of the cell has been demonstrated by the use of the radio-active isotope C14. This was incorporated into the whole length of the primary cell wall (Roelofsen, 1959).
With respect to longitudinal growth the theory now most widely held is the multi-net theory of cell growth (first proposed by Roelofsen and Houwinh, 1953). This accounts also for the observed orientation of the microfibrils in successive layers of the wall. In this theory microfibrils are first deposited transversely to the long axis of the cell, and this layer is later pushed outwards as a result of the formation by apposition of a layer internal to it. During cell elongation the first formed layers of microfibrils are stretched and thus become orientated in a progressively more longitudinal plane.
Recent studies of cell wall formation in fibres and tracheids using the electron microscope and also the technique of autoradiography whereby the path of a radio-active isotope is followed are consistent with the multi-net theory of cell wall growth (Wardrop et al., 1965).
As in early work with the primary cell walls, labelled carbon was found to be deposited more or less uniformly over the secondary wall fibres and tracheids. In fibres, however, the formation of the secondary wall may begin near the centre of the cell and progress towards the tips. The wall is thus thicker near the centre (Cutter, 1969). In some cells, e.g. root hairs, pollen tubes, tracheids and fibres, growth occurs only at the tip (Roelofsen, 1965). This tip growth is regarded as a localised type of multi-net growth. The role of determining whether the wall of the whole cell will grow or only a localised part of it, as for example in root hairs or stellate parenchyma cells, is attributed to the cytoplasm (Roelofsen, 1965).
These are formed after cell expansion has stopped. The secondary walls, particularly of fibres and tracheids, show microscopic layering. The microscopic layers are commonly known as the S1 (outer), S2 (middle) and S3 (inner). The S3 layer is usually thinner than either the S1 or S2 and may be absent altogether. The S1 layer normally consists of four submicroscopic lamellae, alternate ones having microfibrils in opposed helices. The S2 layer, the middle of the secondary wall, consists of numerous lamellae in which the orientation of the microfibrils is at only a small angle to the long axis of the cell. There seems to be a tendency for the microfibrils of this very thick and conspicuous wall layer to be aggregated into macrofibrils. The S3 layer is always poorly developed in contrast to the S2 layers and there is evidence too that the S3 layer may differ chemically in some way from S1 and S2 (Clowes and Juniper, 1968).
The hypothesis that the Golgi apparatus is involved in the bio-synthesis of the amorphous components of the cell wall and the ordered granules in the biosynthesis and orientation of the cellulose microfibrils is now supported by a great deal of evidence and accords well with the known geometrical requirements of the cell wall.
Here is an attempt to describe the anatomy of grassland ecology; it is not intended to be a review of the subject. The author believes there is an increasing need to achieve a synthesis of ecology and agriculture and that this is particularly so in the field of grassland ecology. He attempts to unify the components of the system although he believes a synthesis will be achieved only by the use of mathematical models.
The introduction, where legumes are included in grassland, is followed by seven chapters describing grasses and legumes as individual plants and as populations; their morphology and growth habit; their mineral nutrition; photosynthesis; their rate and pattern of growth and reproduction; and the cycle of vegetative parts. The efficiency of production (the ratio of output of products to the input of resources) and the factors affecting the efficiency of production are discussed in terms of land area, light, nitrogen, carbon and water. It is pointed out that most of the world's grasslands are natural plant communities and are ‘exploited’ because production is derived with the minimum of input. Natural grasslands are characterised by their varied species composition and this is related to competition and productivity. The significance of ecological principles to the maintenance of grass as an agricultural crop are outlined and the limitations of grasses as food for animals; nutrient cycles and the effect of grazing on grassland. Factors altering the efficiency of utilisation (yield relative to amount grown) are outlined, and the concept of efficiency in secondary production with regard to such factors as animal size and climate are discussed. Three chapters describe the production of milk, meat, wool and hides. The final chapter deals with the contribution of grasslands to man. Here it is suggested that their possible future role may be altered taking into account changes in their economic, cultural and social setting. There is a very good appendix giving an example of mathematical simulation of a biological system; pasture contamination by the excreta of grazing animals being the case in point.
The text makes very easy reading and there is a pleasing absence of unqualified technical terms. Quantified descriptions involving formulae are used to describe some of the relationships and there are about seventy figures and tables. Each chapter is followed by a comprehensive reference list. The book is fully indexed both by authors and subjects.
It is easy to criticise anything so wide as the scope of this book, on grounds of incompleteness. However, in view of the plea in the
New Zealanders will feel a little let down; snow tussock grassland is not mentioned and the index contains mohair and partridge but neither mountain nor phosphorus. Nevertheless, as an ‘ecologist’ I found the book interesting and easy to assimilate, despite its somewhat dry appearance. It will no doubt find a place in under-graduate levels of agricultural curricula, and it would do no harm if it found its way into the ivory tower.