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Degradation of Sterols, Triacylglycerol, and Phospholipids by Soil Microorganisms
Zbl. B akt. II. Abt. 136 (1981), 420 -426 [D epartmen t of Botany, F acul t y of Scie n ces, Ain Shams Univ ersity, Cairo , Egypt)
Degradation of Sterols, Triacylglycerol, and Phospholipids by Soil Microorganisms ARWA H. SOLI'MAN and S. S. RADWAN
Summary Cholesterol , p ·sitost erol, triolein , and a mixture of phospholipids wer e adde d to aliquots of garden soil and reco ver ed b y extra cti on a fter 11, 17, and 23 weeks. Degradation of these lipids occurred in fresh aliquots of soil but not in t hose that h ad b een sterilized by aut ocla vi ng . The halflife t imes of t he differ en t lipid com po u nds in soil com pr ised a bout 4 to 6 months. In response to the t reatme nt of soil with lipids, bo th the numbers an d the id entities of b acteria and fungi therein changed dramatically. We isol ated and id en tified sev eral microorganisms, in cluding on e mesophilic S treptom yces sp ., t wo thermophilic B acillu s sp p ., an d two thermophilic Mycobacterium spp., as well as seven sp ecies of fungi belonging t o the gen era Penicillium, Aspergillus, an d Cladosporium, that all were a ble to utilize lipids as sole sou rces of car bon in their nutrition .
Zusammenfassung Choles terin , P-Sitosterin, Triolein und ein Gemisch von Phospholipiden wurden jeweils Gartenerde zugegebe n und nach 11, 17 und 23 Wo ch en wieder extrahiert, Zer setzung diesel' Lipide er folg te in Irischer, jedo ch ni cht in vorher autoklavierter Erde. Di e Halbwer tz eiten der versehiedenen Verbindungen betrugen 4 - 6 Monate. N ach Zugabe d el' Lipide variier t en di e Bakterien und Pilze in der Garten erde quantitat iv und qualitativ. Eine mesophile Str eptomy ces' Art, zwei thermophile B acillus-Art en, zwei thermophile My cobacterium·Arten und sieben Arten von Pilzen der Gat tungen P enicill iu m, Aspergillus und Cladosporiu m, di e in der Lage war en, Lipide als alleinige Kohlenst off quelle zu ver wert.en , wurden id en tifiziert.
Industrial wastes and natural product s that contribute to environmental pollution contain both dige stible and undigestible compounds. Although microorganisms are known to play essential roles in pro cesses such as sludge stabilization and sewage purification etc ., sys t ematic research on the microbial degradation of particular compounds is still lacking. Animal and plant debris a nd excretions that accumulat e in soil contain considerable a mount s of lipids. Th e subject of lipids in soil has been reviewed (BREGER 1966, STEVENSON 1966, AM1I10S0VA et al. 1973, BRAIDS and MILLER 1975), but major emphasis ha s been placed on t he origin of such compounds, their chemical composition and t he experimental problems associated with their extraction. The fate of such water-in soluble substances and the possible role of soil-mioroflora in their decomposition hav e received, so far, a much more limited attention (e.g. TURFITT 1943, CASALICCIUO et al. 1974, BENGTSSON et al, 1977). The aim of the present communication is to provide experimental evidence , indicating the microbial degradation of lipids in soil and the occurrence, therein, of microorganisms that can utilize such compounds as sources of carbon in their nutrition.
Materials and Methods a) Lipids Cholesterol and p-sitost er ol wer e purch ased from E. Merck A. G., Darmstadt, Germany. A com me rcia l preparation of t riole in was purified by chr om at og raph y on a silicic a cid column
D egradation of Sterols
421
(CHRISTIE 1973) with hexane, d iethyl et her , 4: 1 b y vol. , as elut ing solven t. Egg phospholipids were ext r ac ted from hen's egg-yolk (F LOCH et al, 1957) an d purified b y repeated pre cipitation in a cetone until t he preparation proved to be pure by the cr iter ion of thin-la yor chrom at og r a p hy. This phospholipid mixture contained di a cylglycerophosphocholines and sm aller proportions of diacylglycerophosphoethanolamines. All solvents had been redistilled prior to u se, an d the lipids wer e extracted , purified, and k ept in an oxygen-free nitrogen atmosp here as far as feasible.
b) Addition of lipids to soil A sample of soil was obtained from the Botanical Garden of the Faculty of Science, Ain Sham s University, Ca ir o, Egypt. The sam ple was t aken under asept ic cond it ions a fter r emoval of a 5 em la yer on t he surfa ce. The pH o f the sa m ple was 6.3 a nd its water con tent wa s 8.3 %. Each 50 g aliquot of soi l was sus pen de d in 25 m l sterile t ap wat er in a 250 "ml con ical flask an d supplied with 450 mg lipid t hroug ho ut 9 successive wee ks , 50 m g wee k ly. For each of t he four lipid samples, both fresh al iquots of so il and aliquots, t hat had been au toclaved at 150 °C for two hours, wer e u sed. Fresh soil aliquots t hat h ad not been pro vided with lipids were k ept as cont r ol. The different flasks were in cuba t ed at 28 °C. Flask s wer e t aken for lipid recovery an d analysis 11, 17, an d 23 weeks a fte r t he beginning of t he exper im ent. For the m icrobiologi cal analyses soil a liquots were taken a fter 23 weeks.
c) Recovery and analysis of lipids To determine the most e ffic ient m ethod for recove r y of lipids from soil, known weights of these com pou nd s wer e mixed wi th soi l an d extracted with differ ent solvents. H exane, di ethyl et he r , chl oro form , an d ch loroform-methan ol, 2: 1 b y vol., wer e tested. In eac h case t he extract ion was r epeated three t imes. The li pids were r ecover ed from the var ious a li qu ot s of soil , an d aliqu ots that h ad not been provided wit h lipid s were also extr a ct ed . The lipid extr ac ts wer e purified (FLOCHet al. 1957), weighed, a nd a nalysed b y t h in -layer chr om a t ogr a p h y (MANGOLD an d MALINS 196 0, NICHOLS 1964), following es t ablished p roc ed ures.
d) Analyses of soil-microflora Bacteria and fungi in the differ en t aliqu ots of soil were cou n ted using the pl ate dilution method. Mesophilic and thermophilic b a ct eria that utilize con ven t ion a l sources of carbon were counted on glycerol-peptone- ag ar medium that had been su p plied with yeast ex t r a ct as a source of vitamins. The medium con tain ed per 1,000 ml tap water 20 m l glycerol, 2.5 g peptone, 2 g yeast extra ct, 1 g KlI:iPOt, 3 g N a Cl, 0.2 5 g MgSO t, 70 m g F eSO t , 40 mg CaC 0 3 , a n d 15 g ag ar. The pH was adjusted t o 7.0. Mesop h ilic b a ct eri a wer e in cubated at 28 °C, wher e as t he r m oph ilic b a ct eri a wer e kept at 50 °C. F or cou nting meso ph ilic an d ther m ophilic b a ct eri a t hat utilize lipids as sole source of carb on we used t he same n u t rien t med ium , but with 250 m gj l each of the correspon d in g lipids in stead of gl ycerol , pe ptone, an d yeast extr act. F ungi that u tilize conven t ional sou rces of carb on were counted on Czapek's m edium , which containe d per 1,000 m l tap wa ter 10 g gluc ose , 3 g NaN 0 3 , 1 g K~POt , 0.5 g x ci, 0.5 g MgS0 4 , and 15 g agar. T he pH wa s adj us ted to 5. 8. The same medium was used for cou n t in g fu ngi t hat utilize lipids as sole sources of carb on , but with a d d it ion of 250 m g /l eac h of t he correspon d ing lipids, ex clu d ing glucose. Fungi wer e in cubated at 28 ce. Bacterial genera were id en ti fied a ccor d ing t o their morpholo gi cal, stain reaction, and physiological charact eri st ics, followin g estab lished sy st ems (BERGEY 1975). Fungal species were ch ar ac t erized an d com pa red with standard cultu re s.
Results a) D egrad a tion of lipid s
III
soil
It was found t hat both hexane and diethyl ether could recover cholest erol, fi-sito. sterol, and tri olein quantitatively from soil. Hence, hexane was used in the present study for the extraction of these compounds. For the recovery of phospholipids these solvents were not efficient, but chloroform-methanol, 2 : 1 by vol., gave 100 % recovery.
422
A. H.
SOLIMAN
and
S. S. RADWAN
The garden soil studied contained only 0.15 % total lipids of the soil dry weight. Table 1 presents data on the degradation of lipids in fresh garden soil. After 17 to 23 weeks only about one half of the different lipids were recovered. The various lipid compounds were extracted almost quantitatively from soil aliquots that had been sterilized by autoclaving. Thin-layer chromatography of the various extracts showed that they contained the respective lipids as major fractions. Table 1. Degradation of lipids added to soil Incubation period in weeks 11
17 23
cholesterol
P-sitosterol
triolein
phospholipids
71.1 66.7 62.2
88.9 45.6 40.0
% Lipid recovered 58.9 53.5 48.9
66.7 55.6 55.5
b) Quantitative effects of lipids on soil-microflora Table 2 shows the effects of lipids on the numbers of microorganisms that utilize conventional sources of carbon. Table 2. Effect of lipids added to soil on the numbers of microorganisms that utilize conventional sources of carbon Microorganisms
Untreated soil
Lipids added to soil cholesterol
p-sitosterol
triolein
phospholipids
Numbers in thousands per gram dry soil Mesophilic bacterial} Thermophilic bacterias) Fungi'')
3300.0 26.0 4.0
6800.0 52.0 4.0
4100.0 50.0 4.0
9800.0 80.0 7.0
1000.0 56.0 4.0
1) Counts on modified glycerol-peptone modrum at 28 DC. 2} Counts on modified glycerol-peptone medium at 50 DC. 3) Counts on Czapek's medium at 28 DC. All counts have been made 23 weeks after the beginning of the experiment.
The lipids caused increases in the numbers of mesophilic and thermophilic bacteria in soil, whereas the numbers of fungi remained almost constant. Exceptions were phospholipids that effected a decrease in the numbers of mesophilic bacteria, and triolein which caused an increase in the numbers of fungi. The effects of lipids on the numbers of microorganisms that utilized these compounds as sole sources of carbon are presented in Table 3. The results show that the numbers of bacteria and fungi in aliquots of soil to which lipids had been added were higher than those in the untreated soils. Only the numbers of thermophilic bacteria that utilize P-sitosterol as source of carbon were slightly lower in the P-sitosterol-supplied soil than in the untreated one. c) Qualitative effects of lipids on soil-microflora Table 4 presents the patterns of microbial genera and species that utilize conventional sources of carbon. The predominant mesophilic bacterium in the untreated soil was Bacillus mycoides. This species was either absent or present only as a minor bacterium in aliquots of soil
Degradation of Sterols
423
Table 3. Effect of lipids added to soil on the numbers of microorganisms that utilize such compounds as sole sources of carbon p.sitosterol triolein Microorganisms cholesterol (days of ineuuntreated treated untreated treated untreated treated bation of the soil soil soil soil soil soil plates) Numbers in thousands per gram dry soil Mesophilic bacteria (22) Thermophilic bacteria (4) Fungi (6)
phospholipids untreated treated soil soil
1,400.0
2,200.0
400.0
970.0
240.0
320.0
880.0
1,300.0
22.0
29.0
26.0
25.0
6.0
10.0
4.0
7.0
6.0
9.0
6.0
27.0
3.0
11.0
2.0
3.0
All counts have been done on media, contining the corresponding lipids as sole sources of carbon, 23 weeks after the beginning of the experiment.
that had been provided with lipids. Various other species of Bacillus predominated in aliquots of soil that had been supplied with cholesterol or phospholipids. In soil aliquots that had received ~.sitosterol or triolein, an unidentified mesophilic bacterium was predominent, whose cells were Gram-negative, non-spore-forming rods. The major thermophilic bacteria in soil belonged to the genus Mycobacterium, with one and the same species predominating in the untreated soil and in the soil to which p.sitosterol, triolein, or phospholipids had been added. A different Mycobacterium species predominanted in the cholesterol-supplied soil. The major fungus in the untreated soil was Penicillium cyclopium, but in the lipidsupplied soils this species was absent or present only in low proportions. Alternaria alternata, Cladosporium sphaerospermum, and Aspergillus niger predominanted, respectively, in cholesterol-, triolein- and phospholipid- supplied soils. In the soil that had received p.sitosterol, Penicillium cyclopium, Aspergillus niger, and Fusarium moniliforme were present in equal proportions. The patterns of microbial genera that utilize lipids as sole sources of carbon are shown in Table 5. Only one mesophilic bacterium that belonged to the genus Streptomyces was isolated from the different soils, using media which contained the various lipids as sole sources of carbon. A Bacillus sp. was the predominant thermophilic bacterium in the untreated soil as well as in cholesterol- or phospholipid-supplied soils on media that contained these lipids as sole sources of carbon. In P-sitosterol- or triolein-supplied soils, as well as in the untreated soils, an unidentified genus predominated, whose cells were Grampositive, non-spore-forming rods. On media that contained cholesterol as a sole source of carbon, the untreated soil contained Penicillium cyclopium as the predominant fungus, whereas the major one in the cholesterol-supplied soil was Aspergillus niger. In the case of P-sitosterol, Penicillium funiculosum and Penicillium cyclopium predominated in the untreated soil and in the lipid-supplied soil, respectively. For triolein, Penicillium martensii and Aspergillus tamarii predominated in the untreated and the lipid.supplied soils, and for phospholipids the major fungi were Penicillium martensii and Aspergillus niger, respectively. Discussion Although a lot of information is available about the origin and composition of lipids in soil (BREGER 1966, STEVENSON 1966, AMMosovA et al. 1973, CAsALICcmo and LERCKER 1974, BRAIDS and MILLER 1975), only a few studies have shown that
424
A . H. SOLIMAN and S. S . RADWAN
T able 4. Effe ct of lipids a d ded t o soil on the pa t t erns of mi cr oorganism s that utilize co nventional sources of ca rbo n Micr oorga nism s
Untreated soil
L ipids a dde d to soil ch olest er ol
{1-sitostero l
t rio lein
phospholipids
0.0 73 .0 0.0 0.0 0.0 0.0 0.0 25.0 0.0
0.0 0.0 0.0 10.0 0.0 0.0 0.0 0.0 90.0
% of tota l mi cr oorganism s Mesophilic ba cte ria: Coun ts on modified gly cero l-peptone m ed ium a t 28 °C
Bacill us m ycoides Unidentified (A ) B acillus sp. (I) Unidentified (B) B aeillu« sp. (II) Baeillu « sp. (III) P seudomonas sp . Un ide n t ified (C) Bacillu« sp . (IV)
40.0 20.0 10.0 9.0 8.0 0.0 0.0 0.0 0.0
0.0 0.0 0.0 0.0 0.0 60 .0 38.0 0.0 0.0
8.0 80.0 0.0 0.0 0.0 0.0 0.0 10.0 0.0
Thermophilic bacteria: Counts on mo dified glycerol-pept one medium at 50 00
Mycobacterium sp . (i) Bacillus sp. Mycobacterium sp . (ii) Mycobacteriu m sp. (iii)
90.0 10.0 0.0 0.0
0.0 14.0 85.0 0.0
90.0 0.0 0.0 10.0
81. 0 0.0 18.0 0.0
70.0 0.0 0_0 27. 0
Fungi: Counts on Czapek's me di um at 28 00
P enicilliu m cy clopium A sperg illu8 tamarii A lternaria aliernota Asperqillu« niq er F usarium moniliforme Cladosporium sp haero8permum Aspergillus flaoue
80.0 20.0 0.0 0.0 0.0 0.0
10. 0 0.0 90.0 0.0 0.0 0.0
33.0 0.0 0.0 33. 0 33.0 0.0
0.0 10.0 0.0 0.0 0.0 90 .0
25.0 0.0 0.0 50 .0 0.0 0.0
0.0
0.0
0.0
0.0
25.0
Characte rist ics of bacterial isola te s: A = Colonies punctiform ; cells Gram -negative long r od s, single or in pair. B = Colo nies regular, gl istening, smooth; cells Gram-posit ive long r od s, usually in chains . C = Coloni es like B ; cells minute Gra m -posit ive r od s, usually in pairs. I = Colonies regul ar , grey ; cells usually in pairs. II = Colon ies spreading, irregu la r ; cells long r ods, single or in pairs. III = Colon ies reg ular, re ddish , sm all ; cells us ually in pairs. IV = Colonies wide-spreading, very irregular; cells in chains or in pairs. i = Colonies small , flat, regular, white; cells ve r y long, single or in pairs, r ich in meta chromatic granules. ii = Colon ies m inu t e, regular; cells u sually in l ong chain s, poor in m etach rom a ti c granules. iii = Colonies reddish , eleva t ed , small; cells ri ch in me tachroma t ic granules.
some lipid compounds may be decomposed by microorganisms therein. Earlier investigators believed that certain lipids per sist in soil. The long-term preservation of butter by burying in peat, a practice that had been widespread in Ireland until the end of the eighteenth century (BERGMANN 1963), should demon strate clearly how persistent triacylglycerols were believed to be. In a few cases exp erimental evidence has been provided for t he degradation of a few lipids, for exa mple cholesterol (TURFITT 1943) that had been added to soil. E ven lipids with relati vely high molecular weights t hat had been believed to persist in soil are now believed to be digesti ble (BRAID S and MILLER 1975). Our result s contribute to t he limited knowledge in t his field and provide experimental evidence for t he degradati on of cholesterol, P-sitosterol, triolein, and mixtures
Degradation of Sterols
425
Table 5. Effect of lipids added to soil on the patterns of microorganisms that utilize such compounds as sole sources of carbon Microorganisms cholesterol untreated treated soil soil
P-sitosterol
triolein
phospholipids
untreated treated soil soil
untreated treated soil soil
untreated treated soil soil
% of total microorganisms Mesophilic bacteria: Streptomyces sp. 100.0 Thermophilic bacteria: Bacillus sp. (1) 90.0 Mycobacterium 8.0 sp. (i) Mycobacterium 0.0 sp. (ii) Unidentified (A) 0.0 Bacillus sp. (II) 0.0 Fungi: P. cyclopium 60.0 Ai flaou« 30.0 A. niger 10.0 P. martensii 0.0 P. funiculosum 0.0 C.oxysporum 0.0 A. tamarii 0.0 A. versicolor 0.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
98.0 0.0
7.0 0.0
0.0 0.0
30.0 0.0
20.0 7.0
67.0 0.0
80.0 0.0
2.0
0.0
3.0
0.0
0.0
12.0
0.0
0.0 0.0
89.0 0.0
96.0 0.0
50.0 15.0
70.0 0.0
20.0 0.0
18.0 0.0
0.0 6.0 88.0 8.0 0.0 0.0 0.0 0.0
0.0 15.0 0.0 0.0 70.0 10.0 0.0 0.0
88.0 7.0 5.0 0.0 0.0 0.0 0.0 0.0
0.0 0.0 25.0 75.0 0.0 0.0 0.0 0.0
0.0 0.0 0.0 25.0 0.0 0.0 50.0 25.0
0.0 15.0 10.0 75.0 0.0 0.0 0.0 0.0
0.0 29.0 70.0 1.0 0.0 0.0 0.0 0.0
Characteristics of bacterial isolates: 1 = Colonies punctiform, II = Colonies spreading, irregular, i and ii identical with i and ii in Table 4. A = Colonies irregular, smooth; cells Gram-positive rods. single or in pairs, rarely long chains. P = Penicillium; A. = Aspergillus; C. = Cladosporium.
of phospholipids in soil. Under normal conditions this process appears to proceed relatively slow. Proper conditions for enhanced degradation can be studied with a view to applying this process in controlling pollution by lipids, such as fats, oils and steroidal compounds. It is believed that aeration, pH, and other factors influence the rates of microbial degradation of lipids in soil (STEVENSON 1966). That the degradation of lipids is due to activities of microorganisms is evident from our result which indicates that degradation ceased in autoclaved soil. Earlier investigators found that the proportions of lipids extracted from forest soil were highest in winter and attributed this phenomenon to a decreased bacterial activity at low temperature (CASALICCHIO et al. 1974). We have found that both the quantitative and qualitative equilibria of the microflora in soil were dramatically disturbed by the addition of lipids. With a few exceptions the numbers of various microorganisms increased in response to this treatment. Since, as a rule, any medium used for counting soil microorganisms has a rather selective property, the data in Tables 2 to 5 comprise only a portion of the microflora actually present in soil. The numbers of bacteria and fungi that utilize 'lipids as a sole source of carbon, as presented in Table 3, include only microorganisms that are capable of growing in the absence of amino acids, vitamins, or any other growth factor. We had excluded such substances from the nutrient media in order to avoid confusions by microorganims that can utilize them also as sources of carbon.
426
A . H. SOLIMAN and S. S. RADWAN, Degradation of Sterols
The identi ties of microbial genera were modified drastically by the addition of lipids to soil. Exceptional were t he mesophilic bacteria that utilize lipids as sole sour ce of carbon (Table 5). Irrespective of t he lipids supplied, only one bacterium, a Streptomyces sp., was found. Of course t his Streptomyces sp. should not be considered the single mesophilic bacterium in soil t hat can utilize lipids. In t his connection it is to be not ed t hat some microorganisms have been reported to decompose lipids in vitro. Thu s, P roactinomyces erythropolis (TURFITT 1948), Mycobacterium mw;osum (BRUST and BEVERINA 1973), and several species of bacteria isolated from urine (GRAEF et al. 1975) could degrade sterols t hat had been added to t he nu trient media. Apparently, there is no stric t specificity bet ween t he microorganisms and t he lipid classes they can decompose. It seems that several different systematic groups can be involved in t he degrad at ion of the sam e lipid class. Ack no w l ed geme nt Thanks are du to Professor Y. A. Y OUSSEF, Ain Shams U n iversit y, for hi s valuable help in id en tifying fungi.
References A MMosovA, YA. M., ORLOV, D. S., and SADOVNISKOVA, L. K.: Soil lipi ds in a system of humic substances. Gu m in o vye Udobr. 4. (1973) , 39 . B ENGTSSON, LARS P., BRORSON, T ., F LEISCHER, S., an d ASTROM, C. : Variation of seston quality in lakes a ft er sedi me n t at ion . Acta Hydroch im. H ydrobiol, 5 (1977) , 153. B ERGEY'S Manu al of D eter m in at ive B a ct eriolog y, 8t h Ed. The Williams an d W ilkings Com pan y , B al ti more 1975. BE RG)[AS N, ' V. : Geochem istry of lipid s. In: MUELLER, G. (ed .) , Or gani c cosm oc h em ist r y : BREGER, 1. A. (ed.), Inte rn . Ser. Monogr aphs E arth Sci. Vol. Iii, P- 503, P er gam on Press , Oxford 1963 . B RAIDS, O. C., and MiLLER, R . H . : Fats, waxes an d resins in soil. In : GIESEKING , J. E . (ed.], Soil com pone nt s. Vol. 1, p. 343, Springer -Verlag, Berlin, H eidelberg, New Y ork 1975. BREGER, I . A.: Geo che m istry of lip ids. J. Am er. Oil Che rn , Soc. 43 (1966), 197. CASALICC}[JO, G. , and L ERCKER, G. : Com pos ition of t he soil lip id fr a ct ion. VI. Status an d dynamics of some st erols in pro files of n atural soils and in arb oreal vegetat ion . Ag roc him ica 18 (1974) , 52 8. - and CAPELLA, P.: Study on the cons tituents of soil lipi d fra ction s. VI. Seas onal var iations in the lipid content in forest te rra in profile and its vegetat ion (twinge and lea ves). Ann . A cca d. N az . Agri c, 94 (1974), 293 . CHRISTIE, 'V . , Y. : Lipid an alysis. P ergamon P ress, New York, Toronto, Sydney, P aris, Braunschweig 1973. F OLCH, ,J., LEES, M., and SLOANE STANI,EY, G. H . : A simple m ethod for the isolation and purification of total lipi ds from animal tissue s. J. Bi ol, Chern. 22ii (1957), 497. GRAEF, V ., FU CHS, M. , and B ALKE, E. : B acterial degradation of steroids in urine. Z. Klin, Chern. K lin . Bio chem . 13 (1975), 41. MANGOr,D, H . K., an d MALINS, D. C.: Fractionat ion of fats, oils an d waxes on thin-layers of sili cic a cid. J. Amer. Oil Chern, Soc. 37 (1960), 383. NICHOLS, B . W .: Separation of plant phospholipids and glycolipid. In : J AMES, A. T., and MORRIS, L . ,J. (eds .), N ew biochemical separat ions , p. 322. Van Nostrand Comp any, L ondon 1964. SRUST, S. M., and SEVER!", A, L. 0 .: Sp ecificit y of the action of intact Mycobacterium mUC08um 12 10 cells and t heir extr act s on som e st ero id s. I zv . Akad. Nauk. SSSR , Ser. BioI. 6 (1973), 896 . STEVENSON, F . J. : Lipids in soil. J . Am er , Oil Chern . Soc. 43 (1966), 203. TURFITT, G. E. : Microbi ological age n cies in t he deg rad at ion of steroids. (I). Chole sterol decomposing organisms of soils. Bi och em . J . 37 (1943), 115. - Micr obiologi ca l agen cies in the degradation of stero ids . (II). Steroid utilization b y t he m icroflora of soils. B ioche m . J . 42 (1948) , 376. Authors' address : D r. ARWA H . SOLJ]\[AN an d Dr. S. S. R ADWAN, D epartment of B ot an y, Facul t y of Scien ce, Ain Shams University, Cairo, Egypt .
Degradation of Sterols, Triacylglycerol, and Phospholipids by Soil Microorganisms ARWA H. SOLI'MAN and S. S. RADWAN
Summary Cholesterol , p ·sitost erol, triolein , and a mixture of phospholipids wer e adde d to aliquots of garden soil and reco ver ed b y extra cti on a fter 11, 17, and 23 weeks. Degradation of these lipids occurred in fresh aliquots of soil but not in t hose that h ad b een sterilized by aut ocla vi ng . The halflife t imes of t he differ en t lipid com po u nds in soil com pr ised a bout 4 to 6 months. In response to the t reatme nt of soil with lipids, bo th the numbers an d the id entities of b acteria and fungi therein changed dramatically. We isol ated and id en tified sev eral microorganisms, in cluding on e mesophilic S treptom yces sp ., t wo thermophilic B acillu s sp p ., an d two thermophilic Mycobacterium spp., as well as seven sp ecies of fungi belonging t o the gen era Penicillium, Aspergillus, an d Cladosporium, that all were a ble to utilize lipids as sole sou rces of car bon in their nutrition .
Zusammenfassung Choles terin , P-Sitosterin, Triolein und ein Gemisch von Phospholipiden wurden jeweils Gartenerde zugegebe n und nach 11, 17 und 23 Wo ch en wieder extrahiert, Zer setzung diesel' Lipide er folg te in Irischer, jedo ch ni cht in vorher autoklavierter Erde. Di e Halbwer tz eiten der versehiedenen Verbindungen betrugen 4 - 6 Monate. N ach Zugabe d el' Lipide variier t en di e Bakterien und Pilze in der Garten erde quantitat iv und qualitativ. Eine mesophile Str eptomy ces' Art, zwei thermophile B acillus-Art en, zwei thermophile My cobacterium·Arten und sieben Arten von Pilzen der Gat tungen P enicill iu m, Aspergillus und Cladosporiu m, di e in der Lage war en, Lipide als alleinige Kohlenst off quelle zu ver wert.en , wurden id en tifiziert.
Industrial wastes and natural product s that contribute to environmental pollution contain both dige stible and undigestible compounds. Although microorganisms are known to play essential roles in pro cesses such as sludge stabilization and sewage purification etc ., sys t ematic research on the microbial degradation of particular compounds is still lacking. Animal and plant debris a nd excretions that accumulat e in soil contain considerable a mount s of lipids. Th e subject of lipids in soil has been reviewed (BREGER 1966, STEVENSON 1966, AM1I10S0VA et al. 1973, BRAIDS and MILLER 1975), but major emphasis ha s been placed on t he origin of such compounds, their chemical composition and t he experimental problems associated with their extraction. The fate of such water-in soluble substances and the possible role of soil-mioroflora in their decomposition hav e received, so far, a much more limited attention (e.g. TURFITT 1943, CASALICCIUO et al. 1974, BENGTSSON et al, 1977). The aim of the present communication is to provide experimental evidence , indicating the microbial degradation of lipids in soil and the occurrence, therein, of microorganisms that can utilize such compounds as sources of carbon in their nutrition.
Materials and Methods a) Lipids Cholesterol and p-sitost er ol wer e purch ased from E. Merck A. G., Darmstadt, Germany. A com me rcia l preparation of t riole in was purified by chr om at og raph y on a silicic a cid column
D egradation of Sterols
421
(CHRISTIE 1973) with hexane, d iethyl et her , 4: 1 b y vol. , as elut ing solven t. Egg phospholipids were ext r ac ted from hen's egg-yolk (F LOCH et al, 1957) an d purified b y repeated pre cipitation in a cetone until t he preparation proved to be pure by the cr iter ion of thin-la yor chrom at og r a p hy. This phospholipid mixture contained di a cylglycerophosphocholines and sm aller proportions of diacylglycerophosphoethanolamines. All solvents had been redistilled prior to u se, an d the lipids wer e extracted , purified, and k ept in an oxygen-free nitrogen atmosp here as far as feasible.
b) Addition of lipids to soil A sample of soil was obtained from the Botanical Garden of the Faculty of Science, Ain Sham s University, Ca ir o, Egypt. The sam ple was t aken under asept ic cond it ions a fter r emoval of a 5 em la yer on t he surfa ce. The pH o f the sa m ple was 6.3 a nd its water con tent wa s 8.3 %. Each 50 g aliquot of soi l was sus pen de d in 25 m l sterile t ap wat er in a 250 "ml con ical flask an d supplied with 450 mg lipid t hroug ho ut 9 successive wee ks , 50 m g wee k ly. For each of t he four lipid samples, both fresh al iquots of so il and aliquots, t hat had been au toclaved at 150 °C for two hours, wer e u sed. Fresh soil aliquots t hat h ad not been pro vided with lipids were k ept as cont r ol. The different flasks were in cuba t ed at 28 °C. Flask s wer e t aken for lipid recovery an d analysis 11, 17, an d 23 weeks a fte r t he beginning of t he exper im ent. For the m icrobiologi cal analyses soil a liquots were taken a fter 23 weeks.
c) Recovery and analysis of lipids To determine the most e ffic ient m ethod for recove r y of lipids from soil, known weights of these com pou nd s wer e mixed wi th soi l an d extracted with differ ent solvents. H exane, di ethyl et he r , chl oro form , an d ch loroform-methan ol, 2: 1 b y vol., wer e tested. In eac h case t he extract ion was r epeated three t imes. The li pids were r ecover ed from the var ious a li qu ot s of soil , an d aliqu ots that h ad not been provided wit h lipid s were also extr a ct ed . The lipid extr ac ts wer e purified (FLOCHet al. 1957), weighed, a nd a nalysed b y t h in -layer chr om a t ogr a p h y (MANGOLD an d MALINS 196 0, NICHOLS 1964), following es t ablished p roc ed ures.
d) Analyses of soil-microflora Bacteria and fungi in the differ en t aliqu ots of soil were cou n ted using the pl ate dilution method. Mesophilic and thermophilic b a ct eria that utilize con ven t ion a l sources of carbon were counted on glycerol-peptone- ag ar medium that had been su p plied with yeast ex t r a ct as a source of vitamins. The medium con tain ed per 1,000 ml tap water 20 m l glycerol, 2.5 g peptone, 2 g yeast extra ct, 1 g KlI:iPOt, 3 g N a Cl, 0.2 5 g MgSO t, 70 m g F eSO t , 40 mg CaC 0 3 , a n d 15 g ag ar. The pH was adjusted t o 7.0. Mesop h ilic b a ct eri a wer e in cubated at 28 °C, wher e as t he r m oph ilic b a ct eri a wer e kept at 50 °C. F or cou nting meso ph ilic an d ther m ophilic b a ct eri a t hat utilize lipids as sole source of carb on we used t he same n u t rien t med ium , but with 250 m gj l each of the correspon d in g lipids in stead of gl ycerol , pe ptone, an d yeast extr act. F ungi that u tilize conven t ional sou rces of carb on were counted on Czapek's m edium , which containe d per 1,000 m l tap wa ter 10 g gluc ose , 3 g NaN 0 3 , 1 g K~POt , 0.5 g x ci, 0.5 g MgS0 4 , and 15 g agar. T he pH wa s adj us ted to 5. 8. The same medium was used for cou n t in g fu ngi t hat utilize lipids as sole sources of carb on , but with a d d it ion of 250 m g /l eac h of t he correspon d ing lipids, ex clu d ing glucose. Fungi wer e in cubated at 28 ce. Bacterial genera were id en ti fied a ccor d ing t o their morpholo gi cal, stain reaction, and physiological charact eri st ics, followin g estab lished sy st ems (BERGEY 1975). Fungal species were ch ar ac t erized an d com pa red with standard cultu re s.
Results a) D egrad a tion of lipid s
III
soil
It was found t hat both hexane and diethyl ether could recover cholest erol, fi-sito. sterol, and tri olein quantitatively from soil. Hence, hexane was used in the present study for the extraction of these compounds. For the recovery of phospholipids these solvents were not efficient, but chloroform-methanol, 2 : 1 by vol., gave 100 % recovery.
422
A. H.
SOLIMAN
and
S. S. RADWAN
The garden soil studied contained only 0.15 % total lipids of the soil dry weight. Table 1 presents data on the degradation of lipids in fresh garden soil. After 17 to 23 weeks only about one half of the different lipids were recovered. The various lipid compounds were extracted almost quantitatively from soil aliquots that had been sterilized by autoclaving. Thin-layer chromatography of the various extracts showed that they contained the respective lipids as major fractions. Table 1. Degradation of lipids added to soil Incubation period in weeks 11
17 23
cholesterol
P-sitosterol
triolein
phospholipids
71.1 66.7 62.2
88.9 45.6 40.0
% Lipid recovered 58.9 53.5 48.9
66.7 55.6 55.5
b) Quantitative effects of lipids on soil-microflora Table 2 shows the effects of lipids on the numbers of microorganisms that utilize conventional sources of carbon. Table 2. Effect of lipids added to soil on the numbers of microorganisms that utilize conventional sources of carbon Microorganisms
Untreated soil
Lipids added to soil cholesterol
p-sitosterol
triolein
phospholipids
Numbers in thousands per gram dry soil Mesophilic bacterial} Thermophilic bacterias) Fungi'')
3300.0 26.0 4.0
6800.0 52.0 4.0
4100.0 50.0 4.0
9800.0 80.0 7.0
1000.0 56.0 4.0
1) Counts on modified glycerol-peptone modrum at 28 DC. 2} Counts on modified glycerol-peptone medium at 50 DC. 3) Counts on Czapek's medium at 28 DC. All counts have been made 23 weeks after the beginning of the experiment.
The lipids caused increases in the numbers of mesophilic and thermophilic bacteria in soil, whereas the numbers of fungi remained almost constant. Exceptions were phospholipids that effected a decrease in the numbers of mesophilic bacteria, and triolein which caused an increase in the numbers of fungi. The effects of lipids on the numbers of microorganisms that utilized these compounds as sole sources of carbon are presented in Table 3. The results show that the numbers of bacteria and fungi in aliquots of soil to which lipids had been added were higher than those in the untreated soils. Only the numbers of thermophilic bacteria that utilize P-sitosterol as source of carbon were slightly lower in the P-sitosterol-supplied soil than in the untreated one. c) Qualitative effects of lipids on soil-microflora Table 4 presents the patterns of microbial genera and species that utilize conventional sources of carbon. The predominant mesophilic bacterium in the untreated soil was Bacillus mycoides. This species was either absent or present only as a minor bacterium in aliquots of soil
Degradation of Sterols
423
Table 3. Effect of lipids added to soil on the numbers of microorganisms that utilize such compounds as sole sources of carbon p.sitosterol triolein Microorganisms cholesterol (days of ineuuntreated treated untreated treated untreated treated bation of the soil soil soil soil soil soil plates) Numbers in thousands per gram dry soil Mesophilic bacteria (22) Thermophilic bacteria (4) Fungi (6)
phospholipids untreated treated soil soil
1,400.0
2,200.0
400.0
970.0
240.0
320.0
880.0
1,300.0
22.0
29.0
26.0
25.0
6.0
10.0
4.0
7.0
6.0
9.0
6.0
27.0
3.0
11.0
2.0
3.0
All counts have been done on media, contining the corresponding lipids as sole sources of carbon, 23 weeks after the beginning of the experiment.
that had been provided with lipids. Various other species of Bacillus predominated in aliquots of soil that had been supplied with cholesterol or phospholipids. In soil aliquots that had received ~.sitosterol or triolein, an unidentified mesophilic bacterium was predominent, whose cells were Gram-negative, non-spore-forming rods. The major thermophilic bacteria in soil belonged to the genus Mycobacterium, with one and the same species predominating in the untreated soil and in the soil to which p.sitosterol, triolein, or phospholipids had been added. A different Mycobacterium species predominanted in the cholesterol-supplied soil. The major fungus in the untreated soil was Penicillium cyclopium, but in the lipidsupplied soils this species was absent or present only in low proportions. Alternaria alternata, Cladosporium sphaerospermum, and Aspergillus niger predominanted, respectively, in cholesterol-, triolein- and phospholipid- supplied soils. In the soil that had received p.sitosterol, Penicillium cyclopium, Aspergillus niger, and Fusarium moniliforme were present in equal proportions. The patterns of microbial genera that utilize lipids as sole sources of carbon are shown in Table 5. Only one mesophilic bacterium that belonged to the genus Streptomyces was isolated from the different soils, using media which contained the various lipids as sole sources of carbon. A Bacillus sp. was the predominant thermophilic bacterium in the untreated soil as well as in cholesterol- or phospholipid-supplied soils on media that contained these lipids as sole sources of carbon. In P-sitosterol- or triolein-supplied soils, as well as in the untreated soils, an unidentified genus predominated, whose cells were Grampositive, non-spore-forming rods. On media that contained cholesterol as a sole source of carbon, the untreated soil contained Penicillium cyclopium as the predominant fungus, whereas the major one in the cholesterol-supplied soil was Aspergillus niger. In the case of P-sitosterol, Penicillium funiculosum and Penicillium cyclopium predominated in the untreated soil and in the lipid-supplied soil, respectively. For triolein, Penicillium martensii and Aspergillus tamarii predominated in the untreated and the lipid.supplied soils, and for phospholipids the major fungi were Penicillium martensii and Aspergillus niger, respectively. Discussion Although a lot of information is available about the origin and composition of lipids in soil (BREGER 1966, STEVENSON 1966, AMMosovA et al. 1973, CAsALICcmo and LERCKER 1974, BRAIDS and MILLER 1975), only a few studies have shown that
424
A . H. SOLIMAN and S. S . RADWAN
T able 4. Effe ct of lipids a d ded t o soil on the pa t t erns of mi cr oorganism s that utilize co nventional sources of ca rbo n Micr oorga nism s
Untreated soil
L ipids a dde d to soil ch olest er ol
{1-sitostero l
t rio lein
phospholipids
0.0 73 .0 0.0 0.0 0.0 0.0 0.0 25.0 0.0
0.0 0.0 0.0 10.0 0.0 0.0 0.0 0.0 90.0
% of tota l mi cr oorganism s Mesophilic ba cte ria: Coun ts on modified gly cero l-peptone m ed ium a t 28 °C
Bacill us m ycoides Unidentified (A ) B acillus sp. (I) Unidentified (B) B aeillu« sp. (II) Baeillu « sp. (III) P seudomonas sp . Un ide n t ified (C) Bacillu« sp . (IV)
40.0 20.0 10.0 9.0 8.0 0.0 0.0 0.0 0.0
0.0 0.0 0.0 0.0 0.0 60 .0 38.0 0.0 0.0
8.0 80.0 0.0 0.0 0.0 0.0 0.0 10.0 0.0
Thermophilic bacteria: Counts on mo dified glycerol-pept one medium at 50 00
Mycobacterium sp . (i) Bacillus sp. Mycobacterium sp . (ii) Mycobacteriu m sp. (iii)
90.0 10.0 0.0 0.0
0.0 14.0 85.0 0.0
90.0 0.0 0.0 10.0
81. 0 0.0 18.0 0.0
70.0 0.0 0_0 27. 0
Fungi: Counts on Czapek's me di um at 28 00
P enicilliu m cy clopium A sperg illu8 tamarii A lternaria aliernota Asperqillu« niq er F usarium moniliforme Cladosporium sp haero8permum Aspergillus flaoue
80.0 20.0 0.0 0.0 0.0 0.0
10. 0 0.0 90.0 0.0 0.0 0.0
33.0 0.0 0.0 33. 0 33.0 0.0
0.0 10.0 0.0 0.0 0.0 90 .0
25.0 0.0 0.0 50 .0 0.0 0.0
0.0
0.0
0.0
0.0
25.0
Characte rist ics of bacterial isola te s: A = Colonies punctiform ; cells Gram -negative long r od s, single or in pair. B = Colo nies regular, gl istening, smooth; cells Gram-posit ive long r od s, usually in chains . C = Coloni es like B ; cells minute Gra m -posit ive r od s, usually in pairs. I = Colonies regul ar , grey ; cells usually in pairs. II = Colon ies spreading, irregu la r ; cells long r ods, single or in pairs. III = Colon ies reg ular, re ddish , sm all ; cells us ually in pairs. IV = Colonies wide-spreading, very irregular; cells in chains or in pairs. i = Colonies small , flat, regular, white; cells ve r y long, single or in pairs, r ich in meta chromatic granules. ii = Colon ies m inu t e, regular; cells u sually in l ong chain s, poor in m etach rom a ti c granules. iii = Colonies reddish , eleva t ed , small; cells ri ch in me tachroma t ic granules.
some lipid compounds may be decomposed by microorganisms therein. Earlier investigators believed that certain lipids per sist in soil. The long-term preservation of butter by burying in peat, a practice that had been widespread in Ireland until the end of the eighteenth century (BERGMANN 1963), should demon strate clearly how persistent triacylglycerols were believed to be. In a few cases exp erimental evidence has been provided for t he degradation of a few lipids, for exa mple cholesterol (TURFITT 1943) that had been added to soil. E ven lipids with relati vely high molecular weights t hat had been believed to persist in soil are now believed to be digesti ble (BRAID S and MILLER 1975). Our result s contribute to t he limited knowledge in t his field and provide experimental evidence for t he degradati on of cholesterol, P-sitosterol, triolein, and mixtures
Degradation of Sterols
425
Table 5. Effect of lipids added to soil on the patterns of microorganisms that utilize such compounds as sole sources of carbon Microorganisms cholesterol untreated treated soil soil
P-sitosterol
triolein
phospholipids
untreated treated soil soil
untreated treated soil soil
untreated treated soil soil
% of total microorganisms Mesophilic bacteria: Streptomyces sp. 100.0 Thermophilic bacteria: Bacillus sp. (1) 90.0 Mycobacterium 8.0 sp. (i) Mycobacterium 0.0 sp. (ii) Unidentified (A) 0.0 Bacillus sp. (II) 0.0 Fungi: P. cyclopium 60.0 Ai flaou« 30.0 A. niger 10.0 P. martensii 0.0 P. funiculosum 0.0 C.oxysporum 0.0 A. tamarii 0.0 A. versicolor 0.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
98.0 0.0
7.0 0.0
0.0 0.0
30.0 0.0
20.0 7.0
67.0 0.0
80.0 0.0
2.0
0.0
3.0
0.0
0.0
12.0
0.0
0.0 0.0
89.0 0.0
96.0 0.0
50.0 15.0
70.0 0.0
20.0 0.0
18.0 0.0
0.0 6.0 88.0 8.0 0.0 0.0 0.0 0.0
0.0 15.0 0.0 0.0 70.0 10.0 0.0 0.0
88.0 7.0 5.0 0.0 0.0 0.0 0.0 0.0
0.0 0.0 25.0 75.0 0.0 0.0 0.0 0.0
0.0 0.0 0.0 25.0 0.0 0.0 50.0 25.0
0.0 15.0 10.0 75.0 0.0 0.0 0.0 0.0
0.0 29.0 70.0 1.0 0.0 0.0 0.0 0.0
Characteristics of bacterial isolates: 1 = Colonies punctiform, II = Colonies spreading, irregular, i and ii identical with i and ii in Table 4. A = Colonies irregular, smooth; cells Gram-positive rods. single or in pairs, rarely long chains. P = Penicillium; A. = Aspergillus; C. = Cladosporium.
of phospholipids in soil. Under normal conditions this process appears to proceed relatively slow. Proper conditions for enhanced degradation can be studied with a view to applying this process in controlling pollution by lipids, such as fats, oils and steroidal compounds. It is believed that aeration, pH, and other factors influence the rates of microbial degradation of lipids in soil (STEVENSON 1966). That the degradation of lipids is due to activities of microorganisms is evident from our result which indicates that degradation ceased in autoclaved soil. Earlier investigators found that the proportions of lipids extracted from forest soil were highest in winter and attributed this phenomenon to a decreased bacterial activity at low temperature (CASALICCHIO et al. 1974). We have found that both the quantitative and qualitative equilibria of the microflora in soil were dramatically disturbed by the addition of lipids. With a few exceptions the numbers of various microorganisms increased in response to this treatment. Since, as a rule, any medium used for counting soil microorganisms has a rather selective property, the data in Tables 2 to 5 comprise only a portion of the microflora actually present in soil. The numbers of bacteria and fungi that utilize 'lipids as a sole source of carbon, as presented in Table 3, include only microorganisms that are capable of growing in the absence of amino acids, vitamins, or any other growth factor. We had excluded such substances from the nutrient media in order to avoid confusions by microorganims that can utilize them also as sources of carbon.
426
A . H. SOLIMAN and S. S. RADWAN, Degradation of Sterols
The identi ties of microbial genera were modified drastically by the addition of lipids to soil. Exceptional were t he mesophilic bacteria that utilize lipids as sole sour ce of carbon (Table 5). Irrespective of t he lipids supplied, only one bacterium, a Streptomyces sp., was found. Of course t his Streptomyces sp. should not be considered the single mesophilic bacterium in soil t hat can utilize lipids. In t his connection it is to be not ed t hat some microorganisms have been reported to decompose lipids in vitro. Thu s, P roactinomyces erythropolis (TURFITT 1948), Mycobacterium mw;osum (BRUST and BEVERINA 1973), and several species of bacteria isolated from urine (GRAEF et al. 1975) could degrade sterols t hat had been added to t he nu trient media. Apparently, there is no stric t specificity bet ween t he microorganisms and t he lipid classes they can decompose. It seems that several different systematic groups can be involved in t he degrad at ion of the sam e lipid class. Ack no w l ed geme nt Thanks are du to Professor Y. A. Y OUSSEF, Ain Shams U n iversit y, for hi s valuable help in id en tifying fungi.
References A MMosovA, YA. M., ORLOV, D. S., and SADOVNISKOVA, L. K.: Soil lipi ds in a system of humic substances. Gu m in o vye Udobr. 4. (1973) , 39 . B ENGTSSON, LARS P., BRORSON, T ., F LEISCHER, S., an d ASTROM, C. : Variation of seston quality in lakes a ft er sedi me n t at ion . Acta Hydroch im. H ydrobiol, 5 (1977) , 153. B ERGEY'S Manu al of D eter m in at ive B a ct eriolog y, 8t h Ed. The Williams an d W ilkings Com pan y , B al ti more 1975. BE RG)[AS N, ' V. : Geochem istry of lipid s. In: MUELLER, G. (ed .) , Or gani c cosm oc h em ist r y : BREGER, 1. A. (ed.), Inte rn . Ser. Monogr aphs E arth Sci. Vol. Iii, P- 503, P er gam on Press , Oxford 1963 . B RAIDS, O. C., and MiLLER, R . H . : Fats, waxes an d resins in soil. In : GIESEKING , J. E . (ed.], Soil com pone nt s. Vol. 1, p. 343, Springer -Verlag, Berlin, H eidelberg, New Y ork 1975. BREGER, I . A.: Geo che m istry of lip ids. J. Am er. Oil Che rn , Soc. 43 (1966), 197. CASALICC}[JO, G. , and L ERCKER, G. : Com pos ition of t he soil lip id fr a ct ion. VI. Status an d dynamics of some st erols in pro files of n atural soils and in arb oreal vegetat ion . Ag roc him ica 18 (1974) , 52 8. - and CAPELLA, P.: Study on the cons tituents of soil lipi d fra ction s. VI. Seas onal var iations in the lipid content in forest te rra in profile and its vegetat ion (twinge and lea ves). Ann . A cca d. N az . Agri c, 94 (1974), 293 . CHRISTIE, 'V . , Y. : Lipid an alysis. P ergamon P ress, New York, Toronto, Sydney, P aris, Braunschweig 1973. F OLCH, ,J., LEES, M., and SLOANE STANI,EY, G. H . : A simple m ethod for the isolation and purification of total lipi ds from animal tissue s. J. Bi ol, Chern. 22ii (1957), 497. GRAEF, V ., FU CHS, M. , and B ALKE, E. : B acterial degradation of steroids in urine. Z. Klin, Chern. K lin . Bio chem . 13 (1975), 41. MANGOr,D, H . K., an d MALINS, D. C.: Fractionat ion of fats, oils an d waxes on thin-layers of sili cic a cid. J. Amer. Oil Chern, Soc. 37 (1960), 383. NICHOLS, B . W .: Separation of plant phospholipids and glycolipid. In : J AMES, A. T., and MORRIS, L . ,J. (eds .), N ew biochemical separat ions , p. 322. Van Nostrand Comp any, L ondon 1964. SRUST, S. M., and SEVER!", A, L. 0 .: Sp ecificit y of the action of intact Mycobacterium mUC08um 12 10 cells and t heir extr act s on som e st ero id s. I zv . Akad. Nauk. SSSR , Ser. BioI. 6 (1973), 896 . STEVENSON, F . J. : Lipids in soil. J . Am er , Oil Chern . Soc. 43 (1966), 203. TURFITT, G. E. : Microbi ological age n cies in t he deg rad at ion of steroids. (I). Chole sterol decomposing organisms of soils. Bi och em . J . 37 (1943), 115. - Micr obiologi ca l agen cies in the degradation of stero ids . (II). Steroid utilization b y t he m icroflora of soils. B ioche m . J . 42 (1948) , 376. Authors' address : D r. ARWA H . SOLJ]\[AN an d Dr. S. S. R ADWAN, D epartment of B ot an y, Facul t y of Scien ce, Ain Shams University, Cairo, Egypt .