Org. Geochem. Vol, 6, pp. 769-778.1984 0146-6380/84 $(13.00+(X1.00 Printed in Great Britain. All rights reserved Copyright © 1984 Pergamon Press Ltd

Preliminary organic geochemical investigation of Upper-Jurassic dolomites of Plje~ivica (Yugoslavia)

M. ~ABAN*, L. GALEBOVI(~]', T. GLUMI(21C* and B. ~EBE(~IC~

*Faculty of Science, University of Belgrade, ¢Institute of Chemistry, Technology and Metallurgy, Belgrade,

~INA-projekt, Zagreb, Yugoslavia

Abstract--The amounts and compositions of the total and soluble organic matter were determined in 17 samples of Upper Jurassic dolomites from the slopes of the mountain Pljegivica (Lika, Yugoslavia; locations Vrelo-Mrsinj Grad, Vrelo--Draga Zubovida and Zavalje--Bela Greda). The content of the total organic matter was found to vary from 0.06 to 6.57%, with the exception of sample 8. containing a markedly higher amount of organic matter, 68.42%. The quantity of soluble organic matter in most of the samples, especially those from Zavalje, was higher than 10% of the total organic matter. The proportions of saturated and aromatic hydrocarbons and NSO constituents in the soluble organic matter were determined. In the n-alkane fractions homologous series members from C2~ to C2~ predominated with the CPIs being around 1. Antibiotic effects of some bitumen constituents of sample 8 were observed.

Key words: dolomites of Pljegivica (Yugoslavia), n-alkanes, aromatic hydrocarbons, polar NSO constituents, antimicrobial effects, ichthyol

INTRODUCTION

The occurrence of high contents of organic matter in Upper Jurassic dolomites of Lika (Yugoslavia) has been known since before World War II (Reitter, 1926). Sediments from this area were used in 1909 and in 1947 to obtain a commercial pyrolysis product, ichthyol, which has been employed for therapeutic purposes and as an insecticide. However, in the literature no data have been published on any recent organic geochemical study of these sediments.

Since, in general, organic-rich sediments are of interest as potential energy sources, and since the sediments from Lika are interesting as potential raw materials for the pharmaceutical industry, we have undertaken preliminary organic geochemical inves- tigations on several samples taken from different locations of Lika. These investigations are part of a larger programme of investigation of the sediments from Lika.

The geological prospecting involved in this work covered three areas: two locations in the vicinity of a small town Vrelo: Mrsinj Grad (samples 1-3) and Draga Zubovida (samples 4-8) and one location near a small place called Zavalje: Bela Greda (samples 9-12, 14-16, 18 and 19), The sediments examined were located on eastern and western slopes of the mountain Pljegivica (Fig. 1). There is a tectonic relationship between sediments at Vreolo and Zavalje, indicating possible migration of the bitu- men, i.e. hydrocarbons, along the fracture-fault system. The older fault running NW-SE gives the main structural-tectonic charactristic of the whole area, while the newer fault running NE-SW cuts across the older one and forms with it a system of blocks.

The inorganic portion of the samples was analysed using the methods described by Kolthoff and Sandell (1945).

Isolation and identification procedures of the orga- nic matter of the samples are shown in Scheme 1.

In order to establish whether and to what extent bitumen constituents of the sample 8, which is the richest in organic matter, exhibit any physiological activity, the authors have tested their action against some representatives of the most important types of micro-organisms, i.e. bacteria, actinomycetes, mold and yeast.

EXPERIMENTAL

Rock samples, after removal of the 1-2 mm surface layer, were washed with methanol and then crushed (jaw crusher) and powdered (disc mill). The soluble organic matter (bitumen) and the HCI/HF concen- trate were obtained from the powdered samples according to Scheme 1.

Both the bitumen and the HC1/HF concentrates were analysed for C, H, N and S (elemental mic- roanalysis). Uncorrected organic matter content was determined as weight loss of the HC1/HF concentrate after heating at 1000--ll00°C. The correction for the presence of pyrite was made on the basis of Fe(III) oxide content in the ash (Jovanovid and Vitorovid, 1952).

The bitumen was separated by A1203/Si02 column chromatography into an aliphatic hydrocarbon frac- tion (petroleum ether 40-70°C eluate) and an aroma- tic hydrocarbons fraction (cyclohexane-dichlor- omethane, 4 : 1 , eluate). Polar NSO compounds were eluted with a mixture of chloroform and methanol (1 : 1).

Aliphatic hydrocarbons were separated into n- alkanes and branched/cyclic hydrocarbons by using molecular sieves 5A. The n-alkane fractions were analysed by gas chromatography on a Varian 2440 FID gas chromatograph, using a 3 m x 0.15 cm

769

770 M. SABAN et al.

Mop Showing Locations of Anyolyzed Occurrences

Legend: I i i I I i

Fractures and faults

• I-19 Samples ~--" ( J - o • / Beograd -

\z o --\ - I \ avoljo . Z " , " o

vrelo ~- ~ .~.....I t \ -" \ - - ~ 5 ° 4 - 8 "~

0 5kin t J

0 150km

Fig. 1. Map showing locations of analysed occurrences.

Microscopy=

I HCI/HF

HCI/HF concentrate

) I Total organic

matter +

Ash

~ e t e r . Fe Corrected organic matter

Rock

Washing

I crush ng

=- Fisher essay

Powder

1 Soxhlet extraction dichloromethane/methanol

Bitumen - -

Elemental analysis Column chromato- graphy AIZ03/SiO 2

Petroleum I Cyclohexane ether 40-70°C [dichloromethane

"Aromatic HC's Aliphatic HC's

Molecular sieves 5#.

l Methanol chlo-oform

NSO compounds

) f n-AI kanes Branched

l cyclics

GLC

Scheme l. Separation and analysis scheme.

Upper Jurassic dolomites of Pljegivica 771

stainless steel column, packed with 5% Dexil 300 on 80/100 mesh Chromosorb W HP. The column temperature was programmed from 100 to 330°C at 4°C min- l . Nitrogen was used as carrier gas.

In an additional experiment, bitumen of the sam- ple 8 (15.51 g) was chromatographed on a silica-gel column with the following solvent systems of increas- ing polarity: petroleum ether (b.pt 40-70°C), pet- roleum ether-cyclohexane (9 : 1, 3 : 1, 1 : 1 and 1 : 9), cyclohexane, cyclohexane-toluene (9 : 1, 3 : 1, 1 : 1 and 1 : 9), toluene, toluene-methylene chloride (9 : 1, 3 : 1, 1 : 1 and 1 : 9 ) , methylene chloride, methylene chloride-methanol (9 : 1, 3 : 1, 1 : 1 and 1 : 9) and methanol. The chromatography was followed by weighing the dry residue of indi- vidual fractions, eluted with 500 cm 3 of each solvent system, as well as by recording their i.r. spectra as films on KBr disks (Perkin-Elmer 457). From the total of 94 fractions obtained (89.67% of bitumen), 18 fractions were tested for antibiotic properties.

The antibiotic action was investigated by the antibiogram method of diffusion into a double-layer agar, according to the following procedure: an agar layer (2 mm, i.e. 12.5 cm 3) is spread on a sterile Petri dish. After cooling in a refrigerator, a suspension of test micro-organism in physiological solution (0.5 cm 3) is added onto the layer, and a second layer of agar of the same thickness is spread over. After cooling, a round opening of 10 mm diameter is made in the double-layer agar. A chloroform solution (5 ~1) of the fraction to be tested is put into the opening and after cooling (20 min in a refrigerator), 50 Ixl of a physiological solution is added followed by repeated cooling (45 min). The samples thus prepared are kept in a thermostat at 37°C in tests with bacteria, and at 28°C in tests with other micro-organisms. The ther- mostatting time is adjusted to correspond to the age of the culture used. After that time, the diameter of the bactericidal and bacteriostatic zone were mea- sured, the opening diameter being included (Uhlik, 1972; WHO, 1977).

Table 1. shows the type and the name of the test-microorganisms, the age of the cultures used, the culture medium as well as the medium for antibio- gram test.

Two series of antibiogram tests were performed. In the first series the concentration of the chloroform solution of the bitumen and of its chromatographic fraction was 0.1 mg cm -3, in the second series it was 1 mg cm -3. Blank samples were run with pure chlor- oform.

RESULTS AND DISCUSSION

Analysis of the inorganic fraction

The samples examined represent predominantly dolomite and calcareous dolomite, and in some exceptional cases, dolomitic limestone (samples 12 and 14). The dolomites are predominantly lamin- ated, medium or finely crystalline. Sample 8 is not a carbonate rock: its SiO2 content is 20.56% and the content of dolomite only 8.75%, whereas the total mineral matter content is 31.58%.

Organic geochemical analysis

The data on the total organic matter content, soluble organic matter as well as the Fischer assay oil yields are shown in Table 2.

Only sample 8 (Vrelo-Draga Zubovi~a) was re- markably rich in organic matter: 68.42% relative to the sediment. The content of organic matter in the other samples was relatively low: three samples from Vrelo (Nos 1, 2 an 5) contained from 1.64 to 4.94%, and the other four less than 1% ; among nine samples from Zavalje-Bela Greda, five contained from 1.71 to 6.59% (Nos 9, 10, 11, 12 and 18) and the remaining ones less than 1%.

The bitumen yield of all samples from Vrelo and Zavalje, except of the samples 4 and 7, was higher than 400 p.p.m, of bitumen in the rock, i.e. it amounted to between 420 and 7260, with the excep- tion of samples 8 and 11, which gave very high yields of 53,630 and 40,050 p.p.m., respectively. The bitumen yields, expressed as weight percentage of the total organic matter, in samples from Vrelo were within the usual limits, i.e. up to 10%, but samples 3 and 7 had higher yields, 22.75 and 14.67%, respec- tively. However, in samples from Zavalje bitumen

Table 1. Test micro-organisms, their age and media for cultivation and antibiogram tests

Age Medium for Order Name (h) Medium antibiogram test

Bacteria Staphylococcus aureus ATCC 209 P 24 Nutrient agar Miieller-Hinton agar Escherichia coli 24 Nutrient agar Mi.ieller-Hinton agar

48 MiJeller-Hinton agar 48 Potato-dextrose agar

48 Malt agar 48 Agar for candida

Actinomycetes Streptomyces griseus ATCC 23345 Nutrient agar Actinomyces sp. 468 Potato-dextrose agar

Yeast Saccharomyces cerevisiae Madeira Malt agar Candida albicans Agar for candida

Mold Penicillium notatum 72 Malt agar Malt agar

772 M. LABAN et al.

Table 2. Total organic matter, soluble organic matter and oil yields

Sample no.

Total organic Soluble organic matter Fischer Assay matter (wt % of total oil yield

(wt % of (p.p.m. in organic (wt % of whole rock) rock) matter) whole rock)

1 4.28 3500 8.18 [).74 2 1.64 1300 7.93 3 0.51 1160 22.75 4 0.17 177 1(I.41 5 4.94 2300 4.66 1.46 6 0.52 434 8.35 7 0.06 88 14.67 8 68.42 53630 7.84 27.02 9 1.71 2131 12.47

10 1.75 4932 28.18 11 5.17 40050 77.47 3.42 12 6.57 3500 5.32 14 0.11 435 39.55 15 0.06 420 70.0t) 16 0.94 700 7.45 18 2.38 7260 30.5[) 1,20 19 0.84 970 11.55

yields were h igher than 10% and ranged from 11.55 to 77 .47%, except in samples 12 and 16. Because of these high conten ts of b i tumen , the organic ma t t e r of the major i ty of the samples f rom Zaval je behaves

r a the r like asphal t . The highest Fischer assay oil yield (27.02%) was

ob ta ined f rom the sample which was the richest in organic mat te r , i.e. sample 8. The rest of the samples examined yielded 0 .7 -3 .4% oil. The oil yield was ne i the r p ropor t iona l to the total organic ma t t e r con ten t nor to the b i tumen con ten t of the sediment .

The results of the e lementa l analyses are p resen ted in Table 3, Atomic H/C and O/C rat ios for the total organic ma t t e r (Fig. 2(a)) as well as for the soluble organic ma t t e r (Fig. 2(b)) were in t roduced in a van Krevelen diagram. Accord ing to the H/C vs O/C ratios, the total organic ma t t e r of most of the samples may be classified as of sapropel ic type. The somewha t high sulfur con ten t co r robora tes such an in terpre ta- t ion. The d iagram suggests tha t the organic ma t t e r of all samples is in the stage of diagenesis , except samples 8, 9 and 11. Accord ing to the H/C vs O/C

Table 3, Elemental analysis of HCI/HF concentrates and bitumens

HCI/HF concentrates Sample

no. C% H% N% S% 0%* Ash

Bitumens

C% H% N°/,, S% 0 % * Ash

1 68.41 7.20 2 66.12 7.06 3 68.92 6.89 4 34.72 5.47 5 59.63 6.25 6 66.61 6.67 7 52.98 6.65 8 69.40 8.01 9 72.13 7.74

l0 68.74 7.03 11 72.64 8.50 12 64.52 6.79 14 60.59 7.98 15 54.30 5.76 16 24.47 4.51 18 66.99 7.62 19 26.02 2.99

1.99 9.00 7.14 6.26 77.78 8.90 1.14 7.65 12.38 5.65 75.13 8.26 1.40 8.11 8.29 6.39 78.18 9.50 1.03 ND 12.69 46.09 66.47 8.46 1.35 5.95 16.71 10.11 72.96 8.47 1.37 7.20 13.29 4.86 53.08 6.13 1.44 6.19 12.27 20.47 12.38 1.95 2.69 10.08 3.94 5.88 71.92 8.27 2.65 9.49 4.65 3.34 76.67 8.41 1.99 8.96 8.79 4.49 76.08 8.56 2.34 9.39 1.82 2.57 76.64 8.74 1.42 8.61 9.62 9.04 74.42 8.[)7 ND 3.93 20.34 7.£6 70.94 10.16 1.60 7.77 24.98 5.59 73.2{) 8.2l 0.40 3.25 13.10 50.33 75.64 9.05 2.08 8.95 8.30 6.06 76.29 8.75 0.61 5.16 8.45 56.77 76.83 8.88

2.63 7.96 2.73 0.011 1.81 6.97 5.90 1.93 1.23 5.84 3.88 1.37 ND ND 14.38 10.69 1.48 5.81 9.57 1.71 0.88 3./15 11.23 25.63 (I.29 0.011 8.76 76.62 1.82 7.73 9.72 [1.54 1.87 7.64 3.97 1.44 2.20 8.65 3.22 1.29 1.70 8.77 4.15 0.00 3.26 8.15 4.33 1.77 1.02 1.30 13.75 2.83 1.90 7.01 2.27 7.41 1.63 8.13 4.27 1.19 2.(13 8.32 4.62 0.011 1.39 8.(11 1.97 2.92

*By difference. ND = not determined.

Upper Jurassic dolomites of Pljegivica 773

(a) Total organic mat te r

H/C

1.5

1.O

0.5

. 4 H/C

/ / I '~, / ~ ,14

I+ ~ I I 11 f"8",.._ +le ~ +19

~ -~ ~ ~ ~ o.5 I I 1 ) <

,'El',~

I I I I I 0.05 O.10 0.15 O. 20 0.25 O/C

( b ) B i tumen

*4

// +141

i( \ . -

j Z / -

I ~ /I

I I I I I 005 0.10 O.15 0.20 0.25 O/C

Legend

Vrelo-Mrsinj Grad (1 -3 ) Vrelo-Draga Zubovi~a ( 4 - 8 )

Zavalje-Bela Greda (9 -19)

Fig. 2. Elemental composition of the total organic matter (a) and of the bitumens (b) of the samples 1-19, introduced in the Van Krevelen diagram.

ratios samples 8 and 11 contain type I kerogen. The results of elemental analyses of the bitumens

introduced in the van Krevelen diagram show that the soluble organic matter in most of the samples was richer in saturated aliphatic compounds containing less heteroatoms compared to the total organic matter. The same result was obtained by introducing the data on C, H, N, O, S in a diagram of Leifman and Vassoevich (1979) (Fig. 3). According to this dia- gram, the total organic matter of most of the samples examined could be located between algal and fresh- water sapropels, peat, brown coal, cerites and asphalt (Fig. 3(a)). Nevertheless, the soluble organic matter (Fig. 3(b)) of most of the samples, especially from Zavalje and Vrelo-Mrsinj Grad, may be clas- sified as of asphaltic type.

Bitumen analysis

The results of the column chromatographic analy- sis of the bitumen are presented in Table 4. The total amount of hydrocarbons in the bitumen ranges from 11% up to about 30%, except in sample 7 where it is

OG 6:1/4-Y*

40.23%. The amount of saturated hydrocarbons in the bitumens ranges from 5 to 25%. Samples f rom Vrelo, which are low in organic matter (Nos 3, 4, 6 and 7), have a higher percentage of aliphatics 10- 25%, than the samples which are high in organic matter (Nos 1, 5 and 8), which contain 5-7%. Samples from Zavalje have a fairly narrow range, 6-12%, of saturated hydrocarbons in the bitumen. The percentage of aromatic hydrocarbons varies: in samples from Vrelo it is from about 5 to 15% and in samples from Zavalje from about 3 to 27%.

The ratio of saturated to aromatic hydrocarbons in all the samples tends to be lower, the higher the percentage of total organic matter of the sample. Thus, in samples with >2% of total organic matter, the ratio of alkanes to aromatics is from 0.2 : 1 to 1 : 1, with the exception of sample 12. In samples with <2% of total organic matter, the content of alkanes is 1.2-4.7 times that of aromatic hydrocar- bons, except in samples 15 and 19.

The NSO compounds represent the main compo- nents of all bitumens. In samples with <50% of NSO

774 M. SABAN et al.

1.6 "1.5 t.4 `1.3 `1.2

0.8

0.4

( o ) Tota l organic m a t t e r

o.4 o.8 ~.a 1̀.2 `14 ~.e 20 I I f I I I ~ . . . /

/ / , ~ " ' - . , ~ I v . i / I / I / /

~-- / / - - V - - +14 / / :F , o " D7 / / T T / ~ 18 / +1`1 /

// I e5 20~G1"+ 9 /

/ -.\ &,, v> "Z" v= . " g ! 7 /

"?y /---4" -- /

H - Z { O ÷ H -I" S)

H C

16 1.5 1,4 1.3 1,2

0.8

0.4

( b ) B i t u m e n

a 0.4 08 t a 1.2 1.4 16 2.0 [ : ] I I [ I

- ~ - - - I - z\16 " 2 > / - ~ . v t ~ e l / 3 / /

/ \ ; e ~p1.+o'// - / ~ ~ > r ~ . . .L+oI+,19/ _/ . - . 8 + ~ , ~ e

1. ! /K I I / / 9

~"--<.im iY

legend

~ - . , . . . I - o i l , T F ~ a s p h a l t e s - a s p h a l t i t e s TIT-cer i tes, I V - a l g a l saprope ls \

I - x / V - f r e s h w a t e r s a p r a p e l s , V I - p e a t ~ . t / V ~ - brawn coal , V m - h a r d coal

/ I X - s e r n i a n t h r a c i t e , X - a n t h r a c i t e o V r e l a - M r s i n j Grad ( I - 3 )

• V r e l o - D r a g a Zubovic ( 4 - 8 )

+ Z a v a l l e - B e l a Gredo ( 9 - 1 9 ]

H ~ 2 ( O + N + S )

Fig. 3. Elemental composition of the kerogen (a) and of the bitumen (b) of the samples 1-19. introduced in the Leifman-Vassoevich diagram.

compounds in the bitumen, the percentage of the most polar constituents, i.e. those which remain on the column is >20%.

The distribution of odd and even Cls~- n-alkanes is smooth, the CPIs being around 1. In the n-alkane fractions homologous series members from C22 to Czs predominated. According to the van Krevelen dia-

gram, the total organic matter is immature, so that it may be assumed that the smooth n-alkane distribu- tion is not due to maturation changes but rather to original precursor organic matter containing a similar n-alkane distribution, i.e. that the precursor organic matter consisted mainly of lower organisms. Howev- er, an epigenetic origin is also possible.

Upper Jurassic dolomites of Plje~ivica

Table 4. Bitumen analysis

775

Column chromatography Aliphatics Aromatics NSO compounds Normal

Sample (wt % crude (wt % crude (wt % crude no. bitumen) bitumen) bitumen) Range

Alkanes Branched/cyclic

(wt % in saturated CPI fraction)

1 5.49 5.48 47.00 CI6-C3~ 2 8.97 4.48 64.83 CI~-C3~ 3 20.50 11.22 59.03 Ct~-C~, 4 23.91 5.07 65.22 Cj7--C36 5 7.24 9.54 52.63 CI7-C3,~ 6 10.78 5.93 62.53 CI,-C3~ 7 25.29 14.94 59.77 C i r-C411 8 5.30 10.60 37.43 CI,-C34 9 11.79 9.93 68.40 C~,-C38

10 11.76 8.57 50.78 C i~,-C37 11 8.49 24.06 45.99 C 17-C 34 12 8.96 4.17 70.63 Cix-Css 14 12.02 2.53 49.05 C~8-C3_~ 15 8.42 10.26 57.37 CI,~-Cso 16 10.47 7.85 36.12 Cl~-Cso 18 6.19 27.18 50.26 C 17--C3~ 19 8.25 9.90 34.43 C i ~-Css

1.07 52.00 0.97 57.69 1.20 83.56 1.09 45.45 0.83 68.18 1.12 62.50 1.03 50.09 1.20 ND 0.99 61.36 1.07 32.43 1.05 25.(X1 1.06 92.59 1.03 92.10 I. 14 50.20 1.09 70.05 1.00 ND 0.96 ND

ANTIBIOGRAM TESTS

Several antibiogram tests were performed in order to check whether bitumen constituents of sample 8 exhibit any antimicrobial effect.

Antibiogram tests were carried out with raw bitu- men and with some bitumen fractions isolated by column chromatography. The results of chromatog- raphic separations of bitumen 8 are shown in the histogram, in Fig. 4.

The results of antibiogram tests are presented in Table 5. As seen from Table 5, the raw bitumen of sample 8 showed a positive antibiogram test, i.e. it was effective against only two of the micro-organisms tested, namely against Staphylococcus aureus ATCC 209 P and against Streptomyces aureus ATCC 23345, in experiments with solutions having bitumen con- centrations of 1.0 mg cm -3. However, numerous bitumen fractions exhibited antimicrobial action against not only the above-mentioned bacteria and actinomycetes, but also against yeasts Candida albi- cans and Saccharomyces cerevisiae Madeira, in most cases even with ten times less concentrated solutions (0.1 mg cm-3), except in the case of St. griseus. In the majority of tests, more concentrated solutions ex- erted a stronger antibiotic action than the less concentrated ones, but the effect was found not to be proportional to the concentration. A smaller number of fractions showed the same antimicrobial effect with both concentrations applied (fraction 2 against Candida albicans, fractions 50 and 51 against Sac- charomyces cerevisiae and fraction 75 against Staphy- lococcus aureus). On the other hand, some fractions showed a slightly stronger antimicrobial action in

diluted than in more concentrated solutions (frac- tions 51, 53 and 60 against Candida albicans) or, as in the case of fractions 64, 77 and 79, more concentrated solutions were found to exert no antimicrobial effect against Candida albicans.

Bacteriostatic action was observed only against Saccharomyces cerevisiae with dilute solutions of fractions 2 and 45.

The raw bitumen of the sample 8 as well as its chromatographic fractions showed a negative anti- biogram test against gram-negative bacterium Escherichia coli, actinomycetes Actinomyces sp. 468 and the mold Penicillium notatum, respectively. It is interesting to note that the bitumen fractions of the Aleksinac and Kimmeridge shale (~aban et al., 1983) showed positive antibiogram tests against Escher- ichia coli and Actinomyces sp. 468, whereas tests against Penicillium notatum and against yeasts Can- dida albicans and Saccharomyces cerevisiae were found to be negative.

Differences and similarities in antimicrobial action observed in the experiments performed, are beyond doubt due to the composition of bitumen fractions tested. The present paper does not comprise a detailed analysis of the composition of the fractions tested, but gives a rough picture of their constituents on the basis ofi .r , spectra, which are shown in Fig. 5. Infrared spectra show that the first fractions (1-13) containing only saturated and aromatic hydrocar- bons, are followed by fractions containing aliphatic and aromatic carbonyl compounds (39-47) including esters (50-57), whereas the final fractions (61-79) represent mixtures of aliphatic and aromatic car- boxylic acids.

776 M. SABAN et al.

rng

500

400

300

200

"100

552.8 mg :

706~3 mg

908.3 mg

731.1 rng

F~--Ether J R--IFther : 06H,2 I ~-~" C6H12 C6HsCH , !~IC, HsCH3:OH2OI2J~ICH2C[2;CH3OHJ C)

F r : c t i o n number

Eluent

Fig. 4. Yields of bitumen fractions from the sample 8.

Table 5. Diameters of bactericidal (B) and bacteriostatic (12]) zones, expressed in mm, the diameter of the opening not being included (x 0.5)

I , s , , , 8 05 6 , , 2 6 , , 5 Microorganism '~- ] 1 2 38 j 39 50 51 53 60 77 79

st__,p_,ytoooccu, o.1 I i - ! - - ~ • a u r e u s I _I !

A~--c-~-20gP 10 - j i i i - - " " Str eptornyces 0.1 I ' |

I~r,,,~, ! ! !-- ~ ' i

co°°,,o °1 .A_- ; i " - atbicans =, , . . . . . :

E

I11

When comparing the composition of fractions (Fig. 5) with the results of the antibiogram tests (Table 5) it is very difficult to correlate the chemical structure of fraction constituents with the antibiotic action observed. However, it is evident that the

fractions containing compounds with ester functions are effective against all micro-organisms tested. The antibiotic action of other fractions depends upon the test micro-organism as well as upon the concentra- tion of the fraction applied. The raw bitumen and

Upper Jurassic dolomites of Pljegivica

61 L • I ~ -

I IT....-,,/--v\) 3000 1700 1280 10O0 700

Wovenumber (era -1)

Fig. 5. Infra-red spectra of some bitumen fractions from the sample 8.

777

almost all fractions tested were found to be effective against St. aureus and St. griseus when 10 mg cm -3 solutions were tested. Different antimicrobial effects between St. aureus and St. griseus were obtained, however, with dilute (0.1 mg cm -3) solutions. These data suggest that the amounts of active substances producing bactericidal action against St. griseus were in all fractions tested so small that a concentration of 0.1 mg cm -3 is insufficient. In the case of St. aureus this statement is valid for fractions 1, 2, 38 and 39. Fractions containing non-polar or slightly polar com- pounds may be expected to effect only the cell membranes of micro-organisms, but fractions con- taining mixtures of more polar compounds having different types of carbonyl functions may interfere with the metabolic processes in the cell. In any case, it should be considered that the raw bitumen as well as its chromatographic fractions may contain minute

amounts of antibiotic constituents, not detected by i.r. spectroscopy.

Data obtained in this paper suggest that further investigations involving a more detailed analysis of bitumen constituents responsible for the antibiotic activity are worth making.

CONCLUSIONS

On the basis of the results of a preliminary investigation of the organic matter of Upper Jurassic dolomites taken from the locations Vrelo-Mrsinj Grad, Vrelo-Draga Zubovi~a and Zavalje-Bela Greda (on the slopes of the mountain Plje~ivica, Lika, Yugoslavia), the following conclusions may be drawn:

The organic matter content was found to vary from 0.06 to 6.57%. It did not depend greatly on the

778 M. SABAN et al.

provenance of the samples. The exception was sample 8 from Vrelo-Draga Zubovi6a which con- tained 68.42% of organic matter.

On the basis of the bitumen yields, expressed as wt % of the total organic matter, some differences were found between the samples from Vrelo and Zavalje: the bitumen yields from Vrelo samples were within the usual limits, i.e. up to 10%, with the exception of samples 3 and 7 which gave higher yields, 22.75 and 14.67%, respectively; the bitumen yields from Zavalje samples were higher than 10% and ranged from 11.55 to 77.47% except samples 12 and 16 (5.32 and 7.45%, respectively). Because of these high contents of the bitumen, the organic matter of the majority of samples from Zavaije behaves rather like asphalt.

According to the elemental composition (Van Krevelen and Leifman-Vassoevich diagrams), the organic matter of the sediments examined is imma- ture. The compositions of the total and soluble organic matter were different in most samples. The bitumens had a lower content of heteroatoms, i.e. lower atomic O/C and higher H - 2 ( O + N + S ) / C ratios, compared to the total organic matter, in spite of the fact that NSO fractions of the bitumens were more abundant than the sum of total hydrocarbons and the most polar constituents which were not eluted from the chromatographic column by a mix- ture of chloroform and methanol.

The distribution of odd and even members of the n-alkane fraction of all samples examined was smooth.

The bactericidal and bacteriostatic effects of the raw bitumen, as well as of the bitumen's fractions, of sample 8 against the gram-positive bacterium Staphy- lococcus aureus ATCC 209 P, the actinomycetes Streptomyces griseus ATCC 23345, yeast Candida albicans and Saccharomyces cerevisiae Madeira, sug- gests that this interesting property of the bitumen should be further investigated.

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