HYDROCARBON ASSIMILATION BY YEASTS
p. BOS
QO
•JD
o N) »-O »-O U1 O OD U1
V£RViibii^
Voor Ina, Marieke, Reina en Foskea
BIBLIOTHEEK TU Delft P 1829 7236
HYDROCARBON ASSIMILATION BY YEASTS
PROEFSCHRIFT
TER VERKRUGING VAN DE GRAAD VAN DOCTOR IN DE TECHNISCHE WETENSCHAPPEN AAN DE TECHNISCHE HOGESCHOOL DELFT, OP GEZAG VAN DE RECTOR MAGNIFICUS IR. H. B. BOEREMA, HOOGLERAAR IN DE AFDELING DER ELECTRO-TECHNIEK, VOOR EEN COMMISSIE AANGEWEZEN
DOOR HET COLLEGE VAN DEKANEN TE VERDEDIGEN OP DONDERDAG 26 JUNI 1975
TE 16.00 UUR DOOR / d ' z o y i i ( , PIETER BOS SCHEIKUNDIG INGENIEUR GEBOREN TE 'S-GRAVENHAGE 1975
DE PROMOTOR PROFESSOR DR.T.O. WIKEN
DIT PROEFSCHRIFT WERD BEWERKT OP HET LABORATORIUM VOOR ALGEMENE EN TOEGEPASTE MICROBIOLOGIE VAN DE TECHNISCHE HOGE-SCHOOL TE DELFT
CONTENTS
VOORWOORD 7 CHAPTER I: Introduction 9
The aim of this thesis 9 The world food problem and single-cell protein 9
Historical background of hydrocarbon assimilation by
micro-organisms 11 Industrial production ofSCP on hydrocarbons 12
Biochemistry of hydrocarbon assimilation 13 The framework of the present in vestigations 17
CHAPTER II: Hydrocarbon assimUating yeasts 19
Introduction 19 Materials and methods 19
Results 20 Discussion 21 Conclusion 25
CHAPTER III: The growth of yeasts on lower hydrocarbons 27
Introduction 27 Materials and methods 27
Results 30 Discussion 39 Conclusion 41
CHAPTER IV: The uptake of hydrocarbons by yeasts 43
Introduction 43 Materials and methods 44
Results 46 Discussion 55 Conclusion 58
CHAPTER V: Manometric experiments with intact cells and
protoplasts 59
Introduction 59 Materials and methods 60
Conclusion 69
CHAPTER VI: Summary and general discussion 71
Summary 71 General discussion 73 SAMENVATTING 79 REFERENCES 83 ABBREVIATIONS 92 STELUNGEN 93
VOORWOORD
Dit proefschrift is mede tot stand gekomen door de inzet van anderen. In de eerste plaats zij genoemd mej. J.C. de Bruyn, die mij de afgelopen jaren op een uitstekende wijze heeft bijgestaan in het experimentele werk, en de laatste maanden veel energie besteed heeft aan het drukklaar maken van het manuscript. In het aanvangsstadium van het in dit proefschrift beschreven onderzoek werd ik geassisteerd door mevr. M. Kishonti-Brandwijk.
Tal van studenten hebben tijdens hun candidaatsstudie of daarna een bij-drage geleverd aan het onderzoek. Dit zijn o.a. mej. drs. C.C. Bakels, mevr. drs. F. van Veen-Feis, ir. P. Apontoweil, ir. E. Bakhuis, ir. A. van den Berg, ir. B. Boerman, de heren G.J.M. Hersbach en P.J. de Jong, dr. ir. V.F.M. Rijnierse en ir. B.C.J. Zoeteman. Tijdens hun studieverlof in Nederland, doorgebracht in het Laboratorium voor Algemene en Toegepaste Microbiologie, hebben dr. E. Celma Calamita (Madrid) en mevr. dr. L.I. Woino (Moskou) diverse aspecten van de koolwaterstof assimilatie door gisten bestudeerd. Diverse malen heb ik mogen profiteren van de kennis en vaardigheden van een aantal van mijn collega's, met name mej. drs. W.E. de Boer, mej. dr. R. Scheda, ir. P. Arntz, ir. J. van der Toorn en de heer D. Yarrow. Laatsgenoemde is werk-zaam hij het Centraalbureau voor Schimmelcultures, Afdeling Gisten, een insteUing die node gemist kon worden bij het in dit proefschrift beschreven onderzoek.
Bij het persklaar maken van het manuscript ondervond ik veel medewer-king van Mevr. W.H. Batenburg-van der Vegte en de heer J.A. Schuur voor de figuren en de foto's. De heren S.J.N, van Velzen en K.J.L. Bosman hebben de hteratuurlijst gecontroleerd. Tenslotte hebben mej. L.A. Robertson B.Sc. en de heer D. Yarrow aandacht besteed aan het gebruik van de Engelse taal. Mijn welgemeende dank gaat naar alien uit.
CHAPTER I
INTRODUCTION
The aim of this thesis
Since about 1960 the application of hydrocarbon assimilating yeasts in animal and human nutrition has been studied. Several commercial processes were developed. For the design of more sophisticated fermentors, attempts were made to approach the growth kinetics of yeasts, in hydrocarbon con-taining media, with mathematical models. The microbiologists and engi-neers engaged in the design of the fermentation equipment lack basic infor-mation. The physico-chemical mechanism of transfer and uptake of hydro-carbons and its practical implications for fermentor design are not yet sufficiently understood (Katinger, 1974). The main purpose of the investi-gations reported in this thesis is to contribute to a better insight into the uptake mechanism of hydrocarbons by yeasts. Before setting out the lines along which the investigations developed, some background information on microbial protein production in general, and hydrocarbon assimilation by yeasts in particular, will be given in this chapter.
The world food problem and single-cell protein
At the symposium "Proteins from hydrocarbons" (Aix en Provence, 1972) Senez discussed the world food problem in his paper on the present and potential role of yeasts grown on hydrocarbons. From reports from UNO and FAO he concluded that the world production of animal protein meets the requirements of the present world population. However, two thirds of this production are consumed by one third of the world population. In developing countries only 12 g of animal protein per head per day is available. Thus at this moment the world food problem is one of distribution. The birth-rate in developing countries is about twice that in developed nations. In the year 2000 the world population will be about 7,000 millions. At the same time the world protein production must be doubled. It does not seem probable that this increase in production may be effected only by improvements of the agricultural methods. Undoubtedly, the search for cereals etc. with a higher productivity, or for proteins with a higher biolog-ical value, will contribute in solving the world food problem. However, protein production by microbes on ?n industrial scale will probably gain in importance.
There is a great deal of experience available for the production of proteins of microbial origin, also called single-cell protein (SCP), and their application in human and animal feed-stuffs. In Germany, for example, during World Wars I and II fodder yeast was produced on the sulphite liquor from the cellulose industry. This yeast was also used in human nutrition and a production of 15,000 ton dry matter per year was reached (Peppier, 1970). However, during the first years after the war the process was uneconomical and the production was terminated. Since the charge for the treatment of effluents containing polluting constituents has become an important econom-ical factor, processes based on the simultaneous purification of wastes and the production of useful biomass are nowadays gaining in importance. In this respect the process of fodder yeast production on sulphite liquor, developed by the cellulose factory Attisholz (Switzerland), should be men-tioned. Some other processes based on the principle of waste water treatment and the production of yeasts are summarized by Wiken (1972). Numerous other processes were suggested, and developed to a greater or smaller extent, in which, besides yeasts, bacteria, fungi, and algae were involved and a large variety of substrates were used. Ribbons (1968) summarized the advantages of the cultivation of micro-organisms over the classical agricultural methods in the production of proteins. He mentioned, among other things, the high protein content of the microbial cell, the high growth-rates, pro-ductivity during 24 hours a day, and independency of climatological cir-cumstances. An important disadvantage of SCP must also be mentioned. The high nucleic acid content, especially in bacteria but also in yeasts, will offer problems if the product is to be used in human nutrition, because man lacks the enzyme uricase, which catalyzes the oxidation of uric acid to the more soluble allantoin. A high intake of purine derivatives will lead to an increased uric acid level in the blood, and this acid will precipitate and form crystals in the joints, for example, and contributes to the formation of stones in the urinary tract. Edozien et al. (1970) mention a daily maximum intake of 20 g yeast (dry weight) in young adults. Several methods have been developed to decrease the nucleic acid content in SCP. However, such treatments are often accompanied by a reduction of the biological value of the protein. Another disadvantage, especially in the case of SCP from bacteria and fungi, would be the psychological acceptance by the consumer.
Until recently the economical prospects of SCP production promised little, but during the last years the prices of fish meal and soy protein have increased considerably. Since 1973 a growing interest in SCP production has been noted (Anonymous, 1974). At this moment SCP production on an industrial scale using yeasts on hydrocarbons, in contrast to many other processes, has started. The knowledge of the nutritional value, and the safety of the product, justifies production for animal feeding.
Historical background of hydrocarbon assimilation by micro-organisms The biological role of hydrocarbons has been reviewed many times (ZoBell, 1946; Beerstecher, 1954; Fuhs, 1961; Foster, 1962; McKenna and Kallio, 1965; van der Linden and Thijsse, 1965; Davis, 1967; Wilkinson, 1971; Einsele and Fiechter, 1971; Hug and Markovetz, 1971). The most recent review dealing especially with the hydrocarbon assimilation by yeasts was compiled by Shennan and Levi (1974). In the scope of this thesis there is no need to pursue completeness in reviewing the literature on this subject, but a few points may be mentioned here. The literature up to 1945 has been reviewed by ZoBell (1946). At that time the ability of micro-organisms to attack hydrocarbons had already been known for 50 years. Miyoshi (1895) observed that mycelial threads ofBotrytis cinerea perforated paraffin blocks. Rahn (1906) reported on the ability of Penicillium glaucum to grow on paraffin. Sohngen (1906) isolated methane oxidizing bacteria. Independently, Kaserer (1906) enriched a mixed culture able to use methane as sole carbon and energy source. Sohngen (1913) gave a description of 17 bacterial strains able to attack paraffin, and also tested the ability of these strains to dissimilate petroleum ether and crude oil. Paraffins appeared to be the best substrates. Stormer (1908) observed the break-down of aromatic compounds, and Tausz and Peter (1919) the dissimilation of cyclic and naphthenic com-pounds.
From ZoBell's review (1946) one might conclude that until 1945 most research efforts were undertaken in the field of the physiology of hydrocar-bon assimilating micro-organisms. From the practical point of view attention was also paid to the harmful effects of these micro-organisms on stored petroleum products, and on mineral oil emulsions such as cutting oil. ZoBell mentioned the possible application of hydrocarbon assimilating micro-organisms in oil prospecting, and he discussed the role of these micro-organisms in oU polluted surface waters. ZoBell did not indicate the potentiality of these micro-organisms to produce protein for nutritional purposes. This important suggestion, previously made by Beckman (1926), remained undiscussed.
Beerstecher, the author of the first textbook on petroleum microbiology (1954), devoted a whole chapter (X) to the ideas of Beckman. In the opinion of Beerstecher, no serious efforts had been made to explore the possibilities concerned. Beerstecher supposed that it must be possible to replace the or-dinary substrates, such as carbohydrates, with hydrocarbons in the fermen-tative production, for example, of antibiotics and organic acids. He referred, in this respect, to a patent by Taggart (1946) in which a process to convert gaseous waste products from the petroleum industry into organic acids, alcohols and esters is described. Beerstecher was of the opinion that the process could not be realized because of the lack of detailed knowledge of
the products formed. In addition, he suggested that it looked worth-while to investigate the possibility of food production from petroleum products using micro-organisms. He did not mention the paper by Just and Schnabel (1948), in which a production of 200 g of bacterial cell mass (dry weight) in 4 - 5 1 enrichment cultures, with hydrocarbons as the sole carbon and energy source, is described. The cell mass was fed to rats. Just, Schnabel and Ullmann (1951) reported similar investigations with pure cultures of Candida lipolytica and Candida tropicalis. They found high yields, and they observed that the yeasts easily assimilated n-alkanes and n-alkenes containing more than 12 carbon atoms. Hoerburger (1955) grew Candida lipolytica in batch culture on an oil fraction rich in paraffins. He doubted the possibility of industrial application, because the necessary aeration rate was extremely high. Industrial production of SCP on hydrocarbons
Notwithstanding the pessimistic opinion of Hoerburger (1955), in 1957 the Societe Franfais de Pe'trole BP initiated research into the development of a process for the dewaxing of oil fractions, using micro-organisms. The production of SCP was initially an aspect of minor importance, but it would possibly contribute to the economics of the process. Six years later the first results of this work were published (Champagnat et al, 1963a,b). From these papers it was clear that the SCP production per se increased in impor-tance very rapidly irrespective of its r61e in the dewaxing process. The SCP obtained was rich in lysine and water soluble vitamins. The process, as it was developed in Lavera in the south of France, is fed with oil fractions. Not all the components of these fractions are used for growth. Some are reputed to be carcinogenic. The removal of the remaining compounds from the cell material is essential to obtain a safe product. The cell mass must be extracted with low-boiling hydrocarbons. Simultaneously the fat content is reduced sharply.
B P (British Petroleum) has developed another process in Grangemouth, Scotland. In this, the fermentors are fed with a paraffin mixture obtained from oil fractions by means of molecular sieves. The substrate is consumed completely and an extraction of the cell material is not necessary (Shephard, Fraissignes and Peet, 1974). The Grangemouth process can reach a production of 4,000 tons of protein per annum, and the Lavera process 16,000 tons of protein per annum.
Other oil firms, such as Esso, Gulf, Mobil Oil, and Sun, are also interested in the production of SCP on hydrocarbons. In Japan, the Kanegafuchi Chemical Industry had developed a process with a capacity for 60,000 tons of dry weight of yeast per annum which was ready to start in 1972 (Senez, 1972). However, public opinion in Japan prevented realisation of the project.
In 1973 licence for this process was given to an Italian firm and a factory is planned in Reggio Calabria. BP has also the mtention of starting a joint enter-pnse with the Italian Government on Sardinia (Abbott, 1974). In East-European countries, such as Russia and Czechoslovakia, microbial protein is also produced from hydrocarbons. In Russia, the production started in 1967 with 10,000 tons dry weight of yeast per annum. Within several years, an increase was programmed to 1,000,000 tons per annum (Senez, 1972) A process developed on Taiwan is an exception as bacteria are grown on hydrocarbons, instead of yeasts. The choice of yeasts in most processes is understandable because of the experience which has been gained with fodder yeasts, the lower nucleic acid content of the yeast cells and the greater difficulties in separating bactenal cells from the culture fluid.
Until now, SCP from hydrocarbons has been used only as a supplement m animal nutrition. Extensive research has been done on the nutritional value and safety of the products of the two BP processes Toprina L and G (Engel, 1972). In the nutritional tests, pigs and poultry were used Safety experiments were performed with rats and mice. It appeared that a replace-ment of 30% of the normal diet by these products is harmless. The research programme included subchronic (90 days), chronic (2 years), and multigene-ration studies. The possible carcinogenic, teratogenic, and mutagenic effects were also investigated. For example, the content of the carcinogenic com-pound benzpyrene was found to be extremely low.
Apart from the nutritional safety, attention must also be paid to the pathogenicity of the organisms used. In the Lave'ra-process a Candida
tropi-calis strain is used. Candida tropitropi-calis can cause diseases in man and animal
as a secondary pathogen or even as a primary pathogen in hosts lacking normal immunity (Emmons, 1969). One might expect that before a really important production of SCP on hydrocarbons is started, studies must prove the non-pathogenicity of the yeasts applied.
Biochemistry of hydrocarbon assimilation
From the biochemical point of view, the oxidation of hydrocarbons by micro-organisms has appeared to be of much interest since Stewart et al. (1959), using the isotope'* 0, found the incorporation of molecular oxygen into the substrate molecule, hy Micrococcus cerificans growing on n-alkanes. Probably an oxygenating enzyme system is present. Leadbetter and Foster (1960) postulated the formation, via a free radical mechanism, of alkyl-hydroperoxides as intermediary products in the oxidation of M-alkanes to the corresponding alcohols. A free radical equilibrium could explain why, besides terminal, subterminal oxidation takes place. There are some indica-tions which support the hydroperoxidation mechanism. For example, not
only are 1-alkyl-hydroperoxides oxidized by «-alkane grown cells (Stewart
et al., 1959; Finnerty et al., 1962), and by enzyme extracts of these cells
(McKenna and KaUio, 1965), but the same long chain fatty acid esters are also formed if the cells are grown on these hydroperoxides. However, the overall equation for the oxidation of «-alkane to the corresponding alcohol as determined experimentally by Peterson et al. (1969) does not fit with the hydroperoxidation mechanism.
Another mechanism is proposed by Peterson et al. (1967) based on experiments with cell free extracts of Pseudomonas oleovorans. The partially purified extract contained 3 separable protein components: rubredoxin, NADH rubredoxin reductase and an alkane 1-hydroxylase (Fig. 1 ). Kusunose
R - C H 3 + O , . /Rubredoxin fc ^ R e d u c t a s e , / NADH + H* (Fe^*) \ / (Ox)
Hydroxylase
Rubredoxin Reductase ^NAD* (Fe^*) (Red)
Fig. 1. Mechanism of the initial attack of n-alkanes in Pseudomonas oleovorans (Peterson etaL, 1961).
et al. (1967) confirmed the results of Peterson etal. (1967) and found that
an increase in hydroxylating activity was achieved by the addition of flavin-adenine dinucleotide to the enzyme system isolated from Pseudomonas
desmolytica. The overall equation according to Peterson et al. (1969) is:
NADH + H"^ + O2 + R - CH3 y NAD"^ + H2O + R - CH2OH The enzyme system of Pseudomonas oleovorans is insensitive to carbon monoxide (McKenna and Coon, 1970). This contrasts with the system from
Corynebacterium 7E1C (Cardini and Jurtshuk, 1968). Cytochrome P-450is
probably absent in Pseudomonas oleovorans, but functions in
Corynebacte-rium 7E1C. Lebeault, Lode and Coon (1971) found that in Candida tropicalis
cytochrome P-450 is present in the inducible «-alkane and fatty acid hydrox-ylating enzyme system. Here a NADPH dependency is found. The microso-mia! hydroxylase from rat liver also requires cytochrome P-450 and NADPH (Lu and Coon, 1968). The mechanism as it probably functions in higher organisms and in yeasts is summarized in Fig. 2.
R-CHj Cytochrome Non heme iron Flavoprotein NADP* P - 4 5 0 (Red) k 4 (Fe'+) \ / (Red) *^ "
0 . \ ( droxy Hydroxylase
Hj O Cytochrome * Non heme iron Flavoprotein NADPH + P - 4 5 0 (Ox) ( F e " ) (Ox) +
R-CH,OH H*
Fig. 2. Mechanism of the initial attack of n-alkanes as it probably functions in yeasts
and in higher organisms (Lu and Coon, 1968).
of hydrocarbons, as introduced by Senez and Azoulay (1961), has been invalidated. They had demonstrated that a cell suspension of a Pseudomonas sp. capable of oxidizing a n-alkane was also able to reduce NAD and pyocyanine under anaerobic conditions. Traces of the corresponding n-alkene were detected by Chouteau, Azoulay and Senez (1962). Azoulay, Chouteau and Davidovics (1963) postulated the following scheme:
«-alkane >• «-alkene *• epoxide —-»• primary alcohol In the formation of 1,2-epoxide, participation of molecular oxygen was supposed, and thus the dehydrogenation mechanism did not conflict with the results of the '^O-experiments of Stewart et al. (1959). The dehydro-genation theory was always met with scepticism. In 1964 Johnson stressed that such a mechanism is improbable for thermodynamic reasons. However, other scientists also reported on the existence of an alkane dehydrogenase (Wagner, Zahn and BUhring, 1967; lizuka, lida and Fujita, 1969). The formation of «-alkenes as intermediates seemed not only likely in bacteria, but also in yeasts. Lebeault et al. (1970) observed NAD -reduction in submitochondrial fractions of Candida tropicalis cells, grown on «-alkane. This reduction was activated by the addition of ATP. This paper conflicted with the results of Lebeault era/. (1971). They demonstrated a hydroxylating enzyme system in Candida tropicalis. This result forced Gallo et al. (1973) to re-examine the results of Lebeault et al. (1970). They found that the reduction of NAD was due to impurities present in the n-alkane used. In fact, this paper definitely disproved the dehydrogenation theory.
Besides the alkane hydroxylation, the enzyme system responsible for the following step in the dissimilation of n-alkanes, namely the oxidation of the alcohol to the corresponding aldehyde, was studied in detail. Van der Linden and van Ravenswaay Claasen (1971) found two inducible alcohol
dehydro-genases with a strong hydrophobic character. In contrast to the constitutive alcohol dehydrogenases, these enzymes are NADP-independent. The differ-ence between the inducible enzymes lies in their activity towards ethanol. The substrate molecules are attached to the enzyme surface by hydrophobic forces. Van Ravenswaay Claasen and van der Linden (1971) supposed that these forces also play a role in the n-alkane hydroxylase system. The specificity of the alkane hydroxylase and the inducible alcohol de-hydrogenases has been explained by assuming that the active site on the enzymes is like a hole with distinct dimensions, into which the substrate molecule fits like a key. Hydrocarbons and alcohols with an aplanar configuration cannot be oxidized because they cannot be bound on the active site.
The metabolic pathways for the oxidation of n-alkanes by yeasts are given in Fig. -3. This diagram was made up by Klug and Markovetz (1971) from hterature data based on experimental results with growing and resting cells. For most of the steps, enzymatical evidence is still lacking.
CHj —CHj —CHj —(CHj Jji^CHj —CHj —CHj n-alkane
major pathway minor pathway (Klug, 1969)
CHJ - C H , - C H , - ( C H , ) „ - C H , - C H , - C H , - C H , OH CH, -CH, -CHj -(CH, )n-CH, -CHOH-CH,
CH, -CH, -CH, (CH, )n-CH, -CH, -CHO
CH, -CH, -CH, -(CH, )n-CH, -CH, -COOH_
Oxidation (according to Knoop, 1904) I CH, -CH, -CH, -(CH, )n-COOH oxidation i etc HOH, C-CH, -CH, -(CH, )n-CH, -CH, -COOH OHC-CH, -CH, -(CH, )n-CH, -CH, -COOH 4-HOOC-CH, -CH, -(CH, )n-CH, -CH, -COOH ^Kjxidation etc.
Fig. 3. Dissimilatory pathways on n-alkanes by yeasts (according to Klug and Markovetz,
1971).
Cells growing on alipathic hydrocarbons probably incorporate the fatty acids formed as intermediates in their lipids. The inhibition of the de novo synthesis of fatty acids seems to be complete in Nocardia cells (Davis, 1964), whereas it is partially repressed in Candida tropicalis. Hug and Fiechter (1973b) found a stronger inhibition in the latter organism with increasing chain length of the hydrocarbon molecule used as substrate.
Van der Linden and Thijsse (1965) discussed extensively the question whether the n-alkane hydroxylase is constitutive or inducible. On the basis of data from the literature, they suggested that in bacteria the hydroxylase is induced by the hydrocarbon itself, or by other compounds. Their statement that: "It might well be that the adaptation to hydrocarbon oxidation is in fact merely the synthesis of a suitable permease or of a strategically located lipoid membrane, facilitating the operation of a mechanism already present in principle", is interesting. Although Klug and Markovetz (1971) asserted that the research on the metabolism of hydrocarbons is about to enter the age of molecular biology, in fact since the publication of van Eyk and Bartels (1968) on the induction and repression of paraffin oxidation in Pseudomonas
aeruginosa, little has been published on this subject. They found that a
number of compounds, which did not support growth, were found to be suitable substitutes for paraffins as inducers, for example cyclopropane and diethoxyme thane.
Nyns, Auquiere and Wiaux (1969) and Duvnjak, Roche and Azoulay (1970) assumed that in yeasts the hydroxylizing system is in general inducible. The latter authors were able to isolate a mutant with a constitutive hydrox-ylase. Hug, Blanch and Fiechter (1974) also supposed that the enzymes would be induced, but they attached great value to the increase found in lipid content during the transient state in which yeasts in continuous culture, con-suming glucose, adapt to growth on hydrocarbons. The increase in lipid content reflects, in their opinion, the formation of a lipoid structure essential for the functioning of the hydrophobic enzymes.
The framework of the present investigations
At the time when the investigations described in this thesis started, the knowledge of the occurrence of hydrocarbon assimilating ability was frag-mentary. Close co-operation with the Yeast Collection of the Centraalbureau voor Schimmelcultures (CBS) in the Laboratory of Microbiology of the Delft University of Technology enabled us to screen a large variety of yeasts for this ability. Preliminary results have already been published in 1966 (Scheda and Bos). Since that time a large number of new yeast species have been described, and also a new edition of the taxonomical study "The Yeasts" has appeared (Editor: Lodder, 1970). In view of this it seemed worth-while to bring the screening up to date, considering the new situation in yeast taxonomy. The results, also in part published by Bos and de Bruyn (1973) are presented in detail in Chapters II and III.
In the literature the view is often found that almost all yeasts are unable to assimilate n-alkanes with a chain length shorter than that of n-nonane (Shennan and Levi, 1974). However, from our results described in Chapter
III, it appears that a considerable number of yeast species can assimilate n-octane. The way in which the lower hydrocarbons are supplied to the culture has proved to be important. In batch cultures a yield of 17 g/1 was obtained if n-octane was supplied as a vapour to the culture (Celma Calamita, Arntz and Bos, 1971). A direct contact between the cells and the oil phase consisting of the lower hydrocarbons must be avoided, because such a contact will cause intoxication of the organism.
This finding conflicts with the idea, often found in the literature, that hydrocarbon utilizing yeasts need a direct contact with the oil phase. Only then are the high growth rates obtained on these substrates, with their extremely low solubility in water, conceivable. In fact, one must differen-tiate in this respect between the higher n-alkanes, such as n-hexadecane on one hand, and the lower n-alkanes, for example n-octane, on the other hand. This is also suggested by the calculations of Erdtsieck (1967).
In Chapters IV and V attempts are described to elucidate the way in which yeasts adapt to growth on hydrocarbons. The question arises as to whether only structural changes are needed to initiate hydrocarbon oxidation, or if the hydrocarbon hydroxylizing enzyme system must be induced too. In Chapter IV the cytological changes are described as revealed by light and electron microscopy. Special attention is paid to the cell wall, and the rSle of the wall in hydrocarbon uptake by the cells is discussed. The observations of the cellular structures are supported by lipid analysis of complete cells and cell walls. Experiments with protoplasts were also performed (Chapter V). In these investigations, apart from the higher n-alkanes, lower hydro-carbons such as n-octane and n-nonane were also studied. The results support the ideas developed in Chapter FV on the rOle of the yeast cell wall in the uptake of hydrocarbons by the yeast cell.
CHAPTER II
HYDROCARBON ASSIMILATING YEASTS
Introduction
The ability to use hydrocarbons as the sole carbon and energy source is widely spread among yeasts. In the literature it is often found that represen-tatives of the genus Candida are especially suited for hydrocarbon assimilation. However, in 1939 Tauson reported hydrocarbon assimilating strains in the
genevzDebaryomyces, Hansenula and Torulopsis. Just et al. (1951) observed
that, besides Candida lipolytica and Candida tropicalis, Torulopsis colliculosa also gave positive results. Markovetz and Kallio (1964) added to the list of positive geneia Rhodotorula and Trichosporon. Arima et al. (1965) were the first to show hydrocarbon assimilation by a Pichia species. Hydrocarbon utili-zation is occasionally also reported in a Saccharomyces strain (Nyns, Fruytier and Wiaux, 1968). Since the first publication on hydrocarbon assimilation by yeasts appeared, a great deal of work on the classification of yeasts has been done. It appeared worth-while to review critically the ability of yeasts to assimilate hydrocarbons. The possible application of our results in yeast identification will be discussed in this chapter.
Materials and methods
All yeast strains used in this study are from the collection of the Centraal-bureau voor Schimmelcultures, Yeast Division, Delft, The Netherlands. All type strains available of species mentioned in The Yeast (Editor: Lodder, 1970) were tested. In addition, strains of newly described yeast species, not mentioned in this monograph, were included (Table 1). In Table 2 the species are listed from which other strains, apart from the type strain, were tested. For the assimilation tests we used the method of Markovetz and Kallio (1964). The mineral medium contained 2 g NH4CI, 4 g KH2PO4, 6 g NaH2P04.2H20, 0.2 g MgS04.7H2 0, 1 mg FeS04.5H2 0, 0.1mgZnSO4, 0.1 mg CUSO4.5H2O, 0.04 mg H3BO3, 0.04 mg MnS04.7H2 0, 0.02mg M0O3.H2O in 1 Htre demineralized water, and was adjusted to pH 5.8 with KOH pellets. The medium was solidified by adding 1.75% Difco Bacto Agar. After autoclaving for 20 minutes at 120°C, and before solidification, 1 ml of the vitamin solution of van der Walt and van Kerken (1961) was added to 1 litre medium. After inoculation of the slants, 0.5 ml of one of the follow-ing n-alkanes was added, n-octane, n-decane, and n-hexadecane (more than
Table 1. Listof type strains tested, but not described in The Yeasts (1970, Ed J Lodder) Ambrosiozyma cicatncosa CBS 6157 Debaryomyces nepalensis CBS 5921 Hansenula dryadoides CBS 6154 - muscicola CBS 5800 - philodendra CBS 6075 - sydowtorum CBS 5995 Pichia ambrosiae CBS 6003 - casnllae CBS 6053 - spartinae CBS 6059 Saccharomyces cordubensis CBS 6007 - gaditensis CBS 6006 Bullera dendrophda CBS 6074 Sporobolomyces antarcticus CBS 5955 Brettanomyces abstmens CBS 6055 - naardenensis CBS 6042 Candida australis CBS 6304 - bombi CBS 5836 'Chilensis CBS 5719 - chiropterorum CBS 6064 - dendronema CBS 6270 - edax CBS 5657 -entomophila CBS 6160
- guilliermondii var japonica CBS 6021 - hylophila CBS 6226 - incommums CBS 5604 - ishiwadae CBS 6022 - obtusa var arabinosa CBS 5837
-obtusavii membranaefaciens CBS 5838
- parapsilosis var hokkai CBS 5605 - requinyii CBS 5687 Candida silvanomm CBS 6274 - steatolytica CBS 5839 -suecica CBS 5724 - f e p a e CBS 5115 terebra CBS 6023 -valdiviana CBS 5721 Rhodotorula araucanae CBS 6031 Stengmatomyces elviae CBS 5922 - poly boms CBS 6072 Sympodiomyces parvus CBS 6147 Torulopsis bombicola CBS 6009 -dendnca CBS 6151 humihs CBS 5658 - tnsectalens CBS 6149 karawaiewi CBS 5214 - A:extoni CBS 5674 - mannitofaciens CBS 5981 - nemodendra CBS 6280 - philyla CBS 6272 - psychrophila CBS 5956 - silvatica CBS 6277 xesfoftH CBS 5975 Trichosporon aquatile CBS 5973
- cutaneum var antarcticum CBS 5959 - erienxe CBS 5974 - fenmcum CBS 5928 - hellemcum CBS 4099 - mehbiosaceum CBS 6087 Selenottla intestmalis CBS 5946 - pe//flto CBS 5576
99% pure, Baker Chemicals) The cotton plugs of the tubes containing the two lower alkanes were covered with aluminium foil to prevent evaporation. The incubation temperature depended on the strain under study In most cases It was 25°C Over a period of 28 days the cultures were checked weekly for visible growth by comparing them with the blanks (cultures with no carbon source) and with controls containing a similar mineral medium with the addition of 2% glucose A strain growing in one of the three hydro-carbon containing tubes was considered to be positive in the hydrohydro-carbon assimilation test
Results
Our results are summarized in Table 3. Those obtained with n-octane will be discussed in more detail in Chapter III As is evident from Table 2, the ability to use hydrocarbons as the sole carbon and energy source is, in
Table 2. Species from which besides the type stram other strains were tested Debaryomyces hamenn - vanryi Lodderomyces elongisporus Metschmkowia pulchemma - reukaufii Pichia farmosa guilliermondii - haplophila - kudriavzevn - polymorpha - scolyti — vim Lipomyces kononenkoae - lipofer — starkeyi - tetrasporus Number of strains tested 45 4 7 17 7 22 37 2 4 3 7 5 2 4 6 U Positive strains 45 4 7 16 7 22 37 2 0 3 7 5 0 0 0 0 Saccharomycopsts lipolytica* Rhodospondium tomloides Candida catenulata - diddensii — intermedia - melmii - parapsilosis — ravautii - rhagii - sake - tenuis tropicalis - zeylanoides Torulopsis dattila famata - haemulonii Number of strains tested 12 2 5 12 10 6 U 5 4 29 7 24 15 2 12 2 Positive strains 12 2 5 10 10 1 11 5 4 29 7 24 14 2 12 2
This organism is better known in the literature on hydrocarbon fermentations as
Candida lipolytica. For practical reasons this name will be used for the orgamsm
in this thesis.
general, specific for the species. There are some exceptions, namely Candida
diddensii, Candida melinii, and Candida zeylanoides. The assimilation of
n-alkanes by the strains of one species may vary quantitatively. An extreme example is shown by the results obtained with 29 strains of Candida sake, where the ability varied from very weak to good. The type strain is a particularly poor hydrocarbon utilizer. Other strains, such as CBS 5611 and CBS 5612, originally described by Komagata, Nakase and Katsuya (1964) as
Candida maltosa and Candida cloacae respectively, are among the best
hydro-carbon assimilating yeasts found in this screening. From Table 3 it might be concluded that some genera, such as Hansenula, Saccharomyces,
Kluyvero-myces, LipoKluyvero-myces, and BrettanoKluyvero-myces, do not include any hydrocarbon
assimilating species. Discussion
The only representative of the genus Saccharomyces able to assimilate hydrocarbons according to our first screening (Scheda and Bos, 1966) was
Saccharomyces elongisporus. This species also differed from other species in
the genus by the shape of the ascospores. For these reasons van der Walt (1966) removed Saccharomyces elongisporus from the genus Saccharomyces and created the new genus Lodderomyces. Van der Walt was, in fact, the first worker to employ utilization of hydrocarbons as a criterion in yeast taxon-omy. In th»» definition of the genus Saccharomyces he also introduced the inability to assimilate hydrocarbons (van der Walt, 1970).
Table 3 Hydrocarbon assimilatmg yeast species Debaryomyces cantarellu - castelln - hansenii - mararrm - phaffii - vanri/i Endomyces ovetensis Lodderomyces elongisporus Metschnikowia pulchemma - reukaufii Pichia burtonii - castillae - etchelsii - farmosa - guilliermondn Leucospondmm scottii Spondiobolus /ohnsonii - ruinenn Sporobolomyces antarcticus - hispanicus - holsaticus - japonica - odorus - salmonicolor Candida aaseri - albicans - australis - beechu - blanku - cacaoi - catenulata - chiropterorum - ciferni cloacae** - conglobata - curvata - deformans - dendronema - diddensu - edax - entomophila - foliarum - friednchn - glaebosa - guilliermondii var - guilliermondn var - guilhermondu var - humicola carpophila ASCOMYCETES Pichia haplophila - media - oh men - polymorpha - pseudopolymorpha - scolyti - spartinae - stipitis - vini var. melibosi - Vint var vini
Saccharomycopsts lipolytica* Schwanniomyces alluvius - castelln - occidenCalis Wingea robertsn BASIDIOMYCETES Rhodospondium tomloides FUNGI IMPERFECTI Candida mogti - obtusa - oregonensis
- parapsilosis var hokkai - parapsilosis var parapsilosis - ravautu
- rhagii rugosa - sake
- santamariae var membranaefaciens
- santamariae var santamariae
shehatae - silvanomm tenuis - tropicalis - valdiviana - veronae zeylanoides Rhodotomla araucanae - glutims var mfusa - gramims - pilimanae Selenotila intestinalis - peltata Tomlopsis apis — bombicola - Candida - colliculosa - gropengiessen guilhermondu - kestoni lapomca ~ magnoliae - mans
Table 3. (continued)
Candida incommums Tomlopsis nitratophila - intermedia - norvegica
- javanica - pinus
- langaronii - torresii - lusitaniae - vanderwaltii - maltosa** - xestobii
- melibiosica Trichosporon aquatile - membranaefaciens - fennicum - mesenterica - melibiosaceum * See note Table 2.
** According to Meyer, Ahearn and Yarrow (1975) these species and Candida novellus are identical. In their opinion Candida maltosa is the correct name and Candida
cloacae and Candida novellus are synonyms.
The results of the hydrocarbon assimilation tests reflect, to some extent, the interrelations among yeasts as shown in Table 4, where a number of imperfect species are listed together with the forms considered as their perfect counterparts. These relationships are not only established by physio-logical and morphophysio-logical characteristics, but also made plausible by immu-nological studies (Tsuchiya, Fukazawa and Kawakita, 1965), DNA-homology and GC-percentage determinations (Meyer and Phaff, 1972; Nakase and Table 4. The behaviour of some Candida and Torulopsis species to hydrocarbons in
comparison with that of their perfect counterparts
Imperfect forms Perfect forms
Candida deformans + Saccharomycopsts lipolytica +
- krusei — Pichia kudriavzevii —
- lambica — - fermentans — - macedoniensis — Kluyveromyces marxianus —
- mycoderma — Pichia membranaefaciens — - pelliculosa — Hansenula anomala — - shehatae + Pichia stipitis +
- silvicola — Hansenula holstii —
- sorbosa — Pichia kudriavzevii — - utilis — Hansenula jadinii —
- valida — Pichia membranaefaciens —
Torulopsis bovina — Saccharomyces lelluris — - Candida + Debaryomyces hansenii +
- marama +
- colliculosa — Saccharomyces fermentati —
- globosa — Citeromyces matritensis — - holmii — Saccharomyces exiguus —
- humilis — - dairensis — - mogii — - rouxii — - molischiana - Hansenula capsulata —
Komagata, 1970 a, b, 1971 a,b,c,d,e,f), and by comparing the PMR spectra of glucomannans (Spencer and Gorin, 1969 a,b,c, 1970). Without exception the perfect and its supposed imperfect form behave similarly as regards hydrocarbon assimilation.
Some Candida species, such as Candida zeylanoides and Candida diddensii, are considered to be rather heterogeneous on the basis of the differences in the GC-percentages found. One strain (CBS 4077), originally described as
Candida vinaria by Ohara, Nonomura and Yamazaki (1960), does not
assimilate hydrocarbons and, according to the results of Nakase and Komagata (1971 f), is incorrectly grouped with Candida zeylanoides. This strain has a GC-percentage of 44.1, in contrast to the other strains of Candida zeylanoides which have a percentage varying from 54.7 to 55.9. The proposed synonymy
of Candida polymorpha (Ohara and Nonomura, 1954) with Candida diddensii
also seems questionable in view of the GC-percentages. Nakase and Komagata (1971 f) reported a percentage of 32.4 for Candida polymorpha. The other strains of Candida diddensii varied between 37.3 and 42.2. Our results con-firm this conclusion because, in contrast to the other Candida diddensii strains examined, this strain did not utilize hydrocarbons. In addition, out of six Candida melinii strains only one gave positive results in the test. This strain, CBS 2092, probably mislabelled, also fails to assimilate nitrate. Thus, if in a species the strains tested do not give the same result in the hydrocarbon assimilation test, or even if the ability varies from very weak to good, the heterogeneity of the species is suggested; for example Candida
sake. Nakase and Komagata (1971 f) considered Candida cloacae and Candida maltosa as separate species because of their deviating GC-percentages and
high maximal growth temperatures. This contrasts with the view of van Uden and Buckley (1970). The GC-percentages found were 39.5,36.3, and 42.2 respectively for Candida cloacae, Candida maltosa and the type strain of Candida sake.
The basis for the differentiation between the genera Candida and
Toru-lopsis, namely the ability to form pseudomycelium, is an artificial one (van
Uden and Buckley, 1970). Among the species of each of these imperfect genera, some assimilate hydrocarbons and others do not. Among the perfect genera, Saccharomyces, Kluyveromyces, and Hansenula are completely nega-tive in the hydrocarbon assimilation test. Because the imperfect counterparts of the species of these genera are to be found in the genera Candida and
Torulopsis, one can imagine that a natural grouping within these combined
genera might be based on the presence, or absence, of the ability to assimilate hydrocarbons in combination with other criteria such as nitrate assimilation and urea-splitting activity. For example, among those Candida and Torulopsis species which are hydrocarbon-negative, urea-negative and nitrate-positive, we may expect the imperfect counterparts of Hansenula
species, and among the hydrocarbon-, urea-, and nitrate-negative represen-tatives the imperfect forms of Kluyveromyces, Saccharomyces, and some
Pichia species.
In the genera Pichia and Debaryomyces, a number of hydrocarbon assimilating species are present. Several attempts were made to form natural groups within the genus Pichia. The proposals of Boidin, Pignal and Besson (1965) and Poncet (1967), both based on numerical classification of yeasts by a factor analysis method, and that of Spencer and Gorin (1969 a), based on the proton magnetic resonance spectra of the mannose containing polysaccharides, seem to be of little value in view of the GC-percentages found by Nakase and Komagata (1970 b). Within the proposed groups great differences are found in GC-percentages. However, there are no hydro-carbon assimilating species in the group B proposed by Poncet. Debaryomyces
tamarii, Debaryomyces nepalensis,and Debaryomyces coudertii are the only
negative species in the genus Debaryomyces.
The genera Trichosporon, Sporobolomyces, and Rhodotorula are very heterogeneous in the hydrocarbon assimilation test. It would be interesting to examine more strains of these genera and to check whether the hydro-carbon assimilation test might be of value in the differentiation of the species. Delimitation of the species belonging to these genera, by aid of the usual criteria in yeast identification, is hardly possible now, because the GC-percentages vary greatly among strains combined in one species (Storck, 1966; Nakase and Komagata, 1971 c).
There are only a few characteristics which all hydrocarbon assimilating species have in common. They are all petite negative according to the method of Bulder (1963). All can grow on the lysine medium according to Walters and Thiselton (1953). Yamada and Kondo (1972 a,b) reported on the nature of the coenzyme Q system in members of the genera Saccharomyces,
Endomycopsis, Rhodotorula, Cryptococcus, Sporobolomyces, Hansenula, Pichia, and Debaryomyces. All hydrocarbon assimilating species tested by
these investigators, with no exceptions, have the Q9 type. The authors suggest that analysis of the coenzyme Q system may give results of value in yeast taxonomy. In contrast to this time consuming analysis, the hydrocar-bon assimilation test is quite simple and suited for routine methods in yeast identification.
Conclusion
Hydrocarbon assimilation is a widely spread characteristic in the yeasts. However, in several genera of yeasts, hydrocarbon assimilating species are lacking. In addition, the presence or absence of the ability to assimilate hydro-carbons, in several cases, was found to coincide with the presence or absence
of other properties used as important characteristics in yeast identification. For these reasons the hydrocarbon assimilation test may be of value in yeast systematics.
As a result of the screening, Candida cloacae CBS 5612 was considered as the most promising strain for further studies as this strain assimilates hydro-carbons very well. Most of the experiments described in this thesis are per-formed with this organism. Candida cloacae is almost identical with Candida
novellus, the strain used by the Kanegafuchi (Themical Industry in their
CHAPTER III
THE GROWTH OF YEASTS ON LOWER HYDROCARBONS
Introduction
In the literature it is often stated that yeasts prefer unbranched aliphatic hydrocarbons with more than 10 carbon atoms and that, in general, no growth occurs on compounds with less than 9 carbon atoms (PClug and Markovetz, 1971; Shennan and Levi, 1974). However, Scheda (1966) re-ported growth of some yeast species on n-octane vapour. Kvasnikov, Solomko and Shchelokova (1971) even obtained poor growth with Candida tropicalis and a Torulopsis strain on a mixture of n-hexane and n-heptane vapour. In the studies of Scheda (1966) growth was inhibited if n-octane was added as a liquid to the culture. Similar results were obtained by Scheda and Bos (1966). Most hydrocarbon assimilating strains were able to grow on kerosene vapour, whereas growth was inhibited in the presence of liquid kerosene in the culture fluid.
The toxicity of lower n-alkanes and n-alkenes has already been known for a long time (unpublished work of Ishikura, cited by Foster, 1962; Hamilton, 1971). Johnson (1964) suggested that the toxicity of lower hydro-carbons might be explained by assuming an extraction of essential substances from the cells by these compounds, resulting in irreversible damage to the membrane structures. Other authors (Bell, 1971; Gill and Ratledge, 1972 and 1973; Atlas and Bartha, 1973) have suggested that the inhibitory action of lower n-alkanes may be due to the formation of intermediary products, such as the corresponding fatty acids.
In this chapter the attention will be focussed on the results of the screening of numerous yeasts for hydrocarbon assimilating ability, with n-octane as the substrate. In addition, attempts will be made to reveal under what circum-stances this substrate and other volatile hydrocarbons display their toxicity, and to find a plausible explanation of the inhibitory effects.
Materials and methods
Organisms and the screening for n-octane assimilating ability
Apart from the yeast strains from the culture collection of the CBS, baker's yeast from The Netherlands' Fermentation Industries Ltd. (Gist-Brocades), Delft, was also examined. Hydrocarbon assimilating strains, giving negative results with n-octane on application of the test tube method of
Markovetz and Kallio (1964), were retested on agar plates contaming the same mineral salts medium completed with B-vitamins as reported in Chapter II. These plates were incubated in a n-octane-ennched atmosphere. The incubation temperature depended on the strain concerned. In most cases it was 25° C. The plates were checked daily for visible growth by comparing the plates with the blanks.
Preparation of cell suspensions for toxicity tests and manometric experiments
Cells were grown in shaken cultures at 30° C in the basal mineral medium wathout agar-agar, and completed with B-vitamins (Chapter II) and a carbon source. Glucose was used in a 2%(w/v) concentration and n-hexadecane only m 0.2% (v/v), to prevent problems with harvesting of the cells. If, after mcubation, there was too much hydrocarbon left, the cells became attached firmly to the oil phase and this made it difficult to harvest and wash the cells. In order to obtain cells grown on n-octane, specially designed culture vessels were used (Fig. 4). After growth, the cells were harvested by centrif-ugation and washed twice with 0.067 M phosphate buffer, pH 5.8, and
[ )
filter paper
mineral base medium
Fig 4 Culture vessel in which the carbon source is supplied as a vapour to the growing
micro-organisms In the central cup a strip of filter paper, soaked in the volatile compound,is placed
resuspended in buffer or the basal mineral medium. For the toxicity tests, suspensions with about 5.10'' cells per ml were prepared. For manometric experiments the density of the cell suspensions was about 3 mg dry weight of cells per ml. In order to reduce the rate of endogeneous respiration, the suspensions of washed cells were submitted to a starvation of at least 2 hours duration, by shaking the suspension in buffer or basal mineral medium at 30° C.
Toxicity tests
To 25 ml of cell suspension in a 100 ml round flask, the hydrocarbon or other compound, the toxity of which was to be tested, was added. The flask was incubated on a shaking machine at 30° C. From time to time, counting plates on yeast extract-peptone-glucose agar were made via the appropriate dilutions. The death rate (K) was determined from the straight part of the survival curve, according to the following formula:
j ^ ^ -logN^/N,
t 2 - t i
(Ni = number of living cells at time t i ; Nj = number of living cells at time t2 ; time expressed in hours).
It was also possible to determine the toxicity of hydrocarbons in vapour phase by means of the culture vessel shown in Fig. 4.
Manometric experiments
Tlie main compartment of the Warburg-vessel contained 1 ml of cell suspension, the central cup a strip of filter paper with 0.1 ml of 20% KOH and the side bulb 0.1 ml of 5% glucose dissolved in the phosphate buffer or 0.1 ml of the hydrocarbon to be examined. When oxidation rates on hydro-carbon vapour were determined, the contents of the side bulb were not added to the main compartment.
Growth experiments
Growth was followed turbidimetrically by means of a Klett-Summerson colorimeter, and by dry weight determinations. The culture flasks were provided with a Klett tube as proposed by Tromp, Bonnet and Scheffers (1968; Fig. 1). If volatile hydrocarbons were used, the cotton plugs were covered with aluminium foil to prevent evaporation. Where the carbon source was supplied only as a vapour to the culture medium, flasks were used with a central cup as shown in Fig. 4 and with a Klett tube. This tube has been attached in such a way that there was no chance of the transfer of liquid hydrocarbon into the culture medium at the moment the cell densities were measured.
Chemicals
The chemicals used were: n-hexane (Baker Chemicals, Deventer, The Netheriands, 99% pure), n-heptane (Baker, 99%), n-octane (Baker, 99%), n-nonane (Baker, 99%), n-decane (Baker, 99%), n-hexadecane(Fluka, Buchs,
Switzeriand, 98%), 1-hexene (Fluka, 99%), 1-heptene (Fluka 99%), 1-octene (Fluka, 99%), 1-nonene (Fluka, 97%), 1-decene (Baker, 98%), 1-undecene (Fluka, 95%), 1-dodecene (Fluka, 98%), 1-hexanol (Fluka, 99%), 1-heptanol (Fluka, 99%), 1-octanol (Fluka, 99%), 1-nonanol (Fluka, 99%), 1-decanol (Fluka, 99%), n-hexanoic acid (Fluka, 99.5%), n-heptanoic acid (Fluka, 99%), n-octanoic acid (Fluka 99.5%), n-nonanoic acid (Fluka, 99%), n-decanoic acid (Fluka, 99%), pristane (2,6,10,14-tetramethylpentadecane, Koch-Light, Colnbrook, U.K., 97%), isooctane (Fluka, 99.7%), cyclohexane (Baker, 99%), cyclooctane (Fluka, 98%). All other chemicals were of the highest commercial grade available
Results
In Table 5 all those yeast species which are able to grow on n-octane as the sole source of carbon and energy, if tested according to the method of Markovetz and Kallio (1964), are listed. In Table 6 some species of yeasts are listed which give negative results in the tube test, but are positive on the plates incubated in an atmosphere enriched with n-octane vapour These results confirm that the way in which lower hydrocarbons are supplied often determines whether there will be growth or not. It was notable that, from strain to strain, the area in which colonies developed on the agar slants differed. Sometimes there was only growth far away from the n-octane level m the tube In a very few cases, colonies developed near this level. Occasion-ally we observed only a few isolated, well developed colonies on the slants, Table 5 Yeasts able to utilize «-octane as sole carbon and energy source, as tested by the
method of Markovetz and Kallio (1964)
ASCOMYCETES Debaryomyces hansenii - vannji Pichia etchellsu farmosa - guilhermondu - haplophila - media - oh men - polymorpha - spartinae - stipitis vini var mehbiosi vim var vini
Schwanniomyces castelln BASIDIOMYCETES Rhodospondium tomloides ± + + + + + + + + ± + ± + ± ±
Table 5. (continued) FUNGI IMPERFECTI Candida cacaoi - chiropteromm - cifemi - cloacae - conglobata - dendronema - diddensu - entomophila
- guilhermondu var carpophila - guilliermondn var guilhermondu - intermedia - lipolytica - lusitaniae - maltosa - melibiosica - obtusa - oregonensis
- parapsilosis var hokkai - mgosa
- santamariae var membranaefaciens - shehatae
- silvanomm - tenuis - valdiviana - veronae
Rhodotomla glutims var mfusa pilimanae Selenotila intestmalis Sporobolomyces antarcticus Tomlopsis colliculosa - kestoni - mans - nitratophila - torresu Trichosporon fennicum + + ± + ± + ± ± ± ± + ± ± + ± ± + + + ± ± ± ± ± + ± ± ± + ± ± ± ± + + + moderate to good growth, ± poor growth
Table 6 Yeasts givmg negative results with the method according to Markovetz and
Kalho (1964), but positive on the agar plates incubated in an atmosphere enriched with «-octane vapour
Pichia scolyti Wingea robertsn Candida ravautu
- rhagii
although these were heavily inoculated. This suggests a selection towards cells adapted to growth on n-octane.
Another illustration of the importance of the manner in which lower hydrocarbons are supplied to the culture, is shown by the results summarized in Table 7. Growth responses were measured, after 4 days of incubation, by dry weight determinations. Up to n-nonane the yeasts prefer the n-alkanes as a vapour Growth on the vapour of higher n-alkanes is always lower than if these compounds are added directly as a liquid to the culture medium. Because the vapour pressure and the solubility is decreasing with the increasing chain length of the hydrocarbon, it is conceivable that the biomass will decrease too if the yeasts depend only on the supply of the substrate as a vapour. Table 7 Comparison of the growth of Candida maltosa CBS 5611 on hydrocarbons
supplied as a liquid to the culture with the growth on the same hydrocarbons in the form of vapour
«-Heptane n-Octane «-Nonane n-Decane n-Undecane n-Dodecane 1-Heptene 1-Octene 1-Nonene 1-Decene 1-Undecene 1-Dodecene Liquid 0 0 0 23 0 28 0 38 0.20 0 0 0 0 0 14 0 18 Vapour 0.05 0.14 0.20 0.25 0.12 0.06 0 0 0.14 0.14 0.08 0 08 The growth is expressed in dry weight of cells (in gram) found in 50 ml cultures after 4 days of incubation The liquid hydrocarbons were added in 1% (v/v) concentration
In addition, experiments with n-alkenes were performed. No growth occurred on n-alkenes containing less than 9 carbon atoms. Here again, the hydro-carbons with the shorter carbon chains are metabolized more easily when added as a vapour to the culture From 1-undecene upwards, the highest yields are obtained if the substrate is supplied to the culture as a liquid. Obviously the presence of n-heptane, n-octane, 1-nonene, and 1-decene as oil droplets m the culture medium inhibits growth. To study these inhibitory effects we followed the growth of Candida cloacae CBS 5612 in several ex-periments, usmg the mineral base medium completed with glucose, a hydro-carbon, or a combination of both. The growth curves obtained with n-hexadecane, n-decane,and n-octane added in 2% (v/v) concentration are
?
300- 200- 100-5af 1
/ h / ' gy^
/ / — 1 --—y 10 20 30 time in hours Ol 40 50 60 70 120 300-?no 100 K u 5b ^ ^ - - ^ ^ ^r /^
IJ
]/
/ g + h / ^ / y + d ^ ^ " ^ g 10 20 30 40 50 60 70 hours 300-200 100 K uFig 5a,b,c Growth curves of Candida cloacae CBS 5612 m mineral base medium
completed with 0 25% (w/v) glucose and/or 2% (v/v) hydrocarbon. In some experiments n-octane was supplied as a vapour (g = glucose, o, = n-octane liquid, o = n-octane vapour, d = n-decane, h = n-hexadecane).
given in Fig. 5a The same figure contains the curves found with 0.25% (w/v) glucose and with octane vapour The latter part of the curve for n-octane (liq.) is dotted because growth did not always start in such cultures From Fig 5b it appears that the addition of 2% (v/v) n-hexadecane to a culture containing glucose does not influence the initial growth rate. After all of the glucose has been consumed, growth continues on the n-hexadecane This picture is different from the curves obtained when n-decane or n-octane are added. In the initial phase a delay in the growth rate on glucose appears, and a complete inhibition of growth, as demonstrated in Fig 5c, is obtained when n-octane is added as a liquid to the culture From this figure it is clear that n-octane vapour does not influence the growth rate in the presence of glucose
The results of the death rate determinations are summarized in Tables 8a, 8b, 9 and 10 The type of survival curves from which the death rates were calculated showed a "concave upwaids kinetical aspect" (Prokop and Humphrey, 1970) as is illustrated in Fig 6. We did not restrict our experi-ments to hydrocarbons, which can be metabolized, but we also included in our study such branched aliphatic hydrocarbons as isooctane and pristane which are both biologically mert, and some primary alcohols and fatty
time in hours » 1 2 3 4 5
I.,
-2-log - 3 - 4 - 5 - 6Fig 6 Survival curve (i e log N/N, versus time) for Candida cloacae CBS 5612, grown
on mineral base medium completed with 2% (w/v) glucose The cells tested were harvested from a 16 hours old culture To 25 ml washed cell suspension with a concentration of 5 10' cells per ml in the mineral base medium 2% (v/v) n-oc-tane was added The 100 ml flask was mcubated at 30° C on a reciprocal shaking machine Samples were taken and counting plates made every half hour
acids (Table 8a,b). In baker's yeast the toxicity of n-alkanes decreases with the increasing chain length of the compound tested. This is also the case in
Candida cloacae CBS 5612, for which the most toxic n-alkane was n-hexane.
As regards primary alcohols and monocarboxylic acids, the toxicity of the compounds with a greater chain length was higher than that of the com-pounds with a smaller chain length. In addition, it is evident that the most toxic alcohols and fatty acids do not have the same chain lengths as the n-alkanes showing similar toxicity. This is true for both baker's yeast and for
Candida cloacae CBS 5612. In the presence of n-octane, cells of Candida cloacae from the middle of the exponential growth phase (after 16 hours)
reach a death rate (K) of 1.8, while with cells from the stationary phase K falls to about 1. If the cells examined are grown on hydrocarbons instead of on glucose, the sensitivity is also lowered (Table 9). To study whether a relationship exists between the ability to use hydrocarbons and the inhibitory effects of these compounds, we determined the sensitivity of some yeasts utilizing hydrocarbons and others not having this ability. From the results
Table 8a. Death rates of baker's yeast (Netherlands' Fermentation Industries;
Gist-Brocades). Initial cell concentration 5.10' cells per ml. Total volume 25 mL Flasks incubated at 30° C on a shaking machine.
Concentration K* added v/v % n-Hexane n-Heptane n-Octane n-Nonane n-Decane 1-Hexanol 1-Heptanol 1-Octanol 1-NonanoI 1-Decanol Hexanoic acid Heptanoic acid Octanoic acid Nonanoic acid Decanoic acid Isooctane Cyclohexane Cyclooctane Pristane 2 2 2 2 2 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 2 2 2 2 0.5 0.2 0 0 0 0 4.9 6.3 > 8 > 8 0 0 0 > 8 > 8 0 3.8 0.5 0
Table 8b. Death rates of Candida cloacae CBS 5612. Imtial cell concentration 5.10'
cells per ml The tested cells originate from a 16 hours old culture. Cells grown on the mmeral base medium completed with 2% (w/v) glucose.
Concentration K* added v/v % n-Hexane n-Heptane n-Octane n-Nonane n-Decane 1-Hexanol 1-Heptanol 1-Octanol 1-Nonanol 1-Decanol Hexanoic acid Heptanoic acid Octanoic acid Nonanoic acid Decanoic acid Isooctane Cyclohexane Cyclooctane Pristane n-octane 2 2 2 2 2 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0 08 2 2 2 2 vapour 2.4 1.7 1.8 1.2 0.5 0 0.2 8 > 8 > 8 0.0 > 8 > 8 > 8 > 8 2 3.6 3.6 0 0
* See note Table 8a
Table 9. Death rates of cells of Candida cloacae CBS 5612
Cells grown on Age Compound Concn. K* added
Mineral base medium + 16 h n-octane 2% 1.8 2% (w/v) glucose
Mineral base medium + 16 h n-octane 2% 0.5 0.2% (v/v) n-hexadecane
Mineral base medium + 4 days n-octane 2% 0.4 n-octane vapour
was between 1 0 ' and 10' cells per ml. n-Octane* CBS no. oxidation Candida chiropterorum - cloacae - diddensu - edax - melinii - sake - zeylanoides Debaryomyces hansenii Pichia vim var. mehbiosi Torulopsis gropengiessen Candida diddensu - melimi - zeylanoides Hansenula anomala Pichia pinus Saccharomyces cerevisiae - fermentati Tomlopsis cantarelli - glabrata 6064 5612 4547 5657 2092 159 4077 767 5254 156 4094 601 947 5702 744 1171 818 4878 138 + + + + + -± + + _ -—
-* Results from manometric experiments, -*-* data from Chapter + : positive, - : negative; ± : weak.
Hydrocarbon** Inhibition of glucose oxidation*
assimilation by n-octane liquid K'
+ + + + + ± + + + + _ -_ -— -— -+ + -+ + + + + + -+ + + + -— + + + 1.0 1.8 2.8 0 6 4 9 0.0 5.8 3 8 1 6 0.2 3 7 1 6 0.7 0.8 1 2 0.7 0 6 0.0 0.5 ; *** see note on Table 8a.
hsted in Table 10 it appears that such a relationship does not exist. Also from Table 8b it is clear there is no correlation between the toxicity and degradability of hydrocarbons.
To elucidate not only the effects of lower hydrocarbons on the growth of yeasts, but also on the oxidative capabilities we performed some manomet-nc experiments. The results are given in Tables 10 and 11 Inhibition of the oxidation rate on glucose is found both in hydrocarbon assimilating yeasts and m those not having this ability, if n-octane is added to the cell suspension. The higher oxidation rate found with glucose-grown cells on n-octane vapour, as compared with that of similar cells in the presence of n-octane liquid, is remarkable In glucose-grown cells the oxidation rate on glucose is not inhibited by the presence of n-octane vapour However, the oxidation rate on glucose decreases sharply if n-octane is added as a liquid to the cell suspension From Table 11 one might conclude that the biologically non-degradable hydrocarbons such as isooctane and cyclooctane will show the same effects for the glucose oxidation by glucose-grown cells Hydrocarbon-grown cells are less sensitive to the action of these low molecular weight hydrocarbons on the oxidation of glucose. Here also, no correlation is found between the inhibitory effects and the degradability of the compounds tested.
Table IL Results of manometric experiments with cells of Candida cloacae CBS 5612
grown on the mmeral base medium with 2% (w/v) glucose or n-octane vapour
Substrate Maximum Oj uptake Maximum Oj uptake by glucose-grown
n-octane-vapour-Endogeneous respiration Glucose
n-Octane hquid n-Octane vapour Glucose + n-octane hquid Glucose + n-octane vapour Cyclooctane liquid
Glucose + cyclooctane hquid Isooctane liquid
Glucose + isooctane hquid
cells 0.1 1.7 0.7 1.1 0.5 2.4 0.1 0.3 0.1 0 4 grown ( 0 1 1 1 1 8 1 8 2 7 2 6 0 1 0 8 0 1 1 0
Oxygen uptake rate is expressed as p.[ O^ uptake per mg dry weight of yeast per mmute
Discussion
In contrast to statements in the literature (Klug and Markovetz, 1971; Shennan and Levi, 1974) there are a considerable number of yeast species which are able to grow on n-octane and even on n-heptane, although the growth on the latter alkane is poor. In our survey of positive species, repre-sentatives have been found of many different genera. Apart from Ascomycetes and Fungi Imperfecti, a Basidiomycete has also shown this ability to some extent. One may expect that if new methods are applied, more positive strains will be found. From our experiments it is obvious that the way in which the lower n-alkanes and n-alkenes are supplied determines whether growth will result or not. The direct contact between the cells and the lower hydrocarbons in liquid form seems to be fatal for the cells. If the culture medium does not contain oil droplets and the hydrocarbon is only present in the dissolved state, the cells loose neither viability nor activity. These phenomena contrast with those observed with higher alkanes. For good growth, contact between the cells and the oil phase seems to be essential (Chapter IV). The solubility of these compounds is so low that the growth rates actually reached in the experiments concerned are not con-ceivable by assuming an uptake of the dissolved hydrocarbons only from the water phase. In this connection the calculations of Erdtsieck (1967) should be mentioned. He found that the growth rate on n-alkanes containing 11 or 12 carbon atoms will depend also on the dissolved hydrocarbons. Yeasts growing on n-alkanes with more than 12 carbon atoms are, however, committed to a direct uptake from the oil phase.
The toxicity of the lower hydrocarbons has been discussed by several authors. Johnson's hypothesis (1964) concerning the role of the extractive properties of these compounds has already been mentioned in the Introduc-tion. In his opinion the phospholipid micelles of the cell membrane are partially destroyed or disorganized. Gill and Ratledge (1972) found a corre-lation between solubility and toxicity. They estimated the toxicity by measuring the reduction of the endogeneous respiration in a Beckman Laboratory Oxygen Analyzer. According to these authors the lower hydro-carbons display an inhibitory effect because their solubilities exceed a critical level. They found that their theory was supported by the observation that the addition of pristane, a biologically inert hydrocarbon, which is miscible with alkanes, diminished the toxicity of the lower n-alkanes, because their concentration in the aqueous phase is lowered to Henry's and Raoult's laws. Bell (1971), examining the inhibition of the growth of yeasts on n-alkanes by fatty acids, also found a correlation between the toxicity and solubility of these fatty acids. Growth was completely inhibited if the concentration of the fatty acids added exceeded a critical value. If the fatty
