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Vitamin requirements of hydrocarbon-utilizing soil bacteria
Microbiol. Res. (200 I) 155, 301- 307 http://www.urbanfischer.de/joumals/microbiolres ©
Urban & Fischer Verlag
Vitamin requirements of hydrocarbon-utilizing soil bacteria Samir S. Radwan, Awatif S. AI-Muteirie Department of Biological Sciences, Faculty of Science, Kuwait University, P. O. Box 5969, Safat 13060, Kuwait Accepted: May 6, 2000
Abstract The numbers of oil-utilizing bacteria in several samples of clean and oil-polluted soils counted on vitamin-containing media were severalfold higher than the numbers counted on vitamin-free media. Colonies that grew on a medium containing a vitamin mixture were tested for growth on the same medium lacking any vitamins. More than 90% of the total colonies failed to grow. The remaining 10% grew, yet their growth was enhanced, when vitamins were added. The predominant oil-utilizing bacteria in one of the test desert soil samples were various strains of Cellulomonas flavigena and Rhodococcus erythropolis. Minor organisms belonged to the genera Pseudomonas, Bacillus and Arthrobacter. Two vitamin-requiring biovars of C. flavigena and R. erythropolis were selected for further study. Their growth on n-octadecane and phenanthrene as sole sources of carbon and energy as well as their potential for hydrocarbon consumption were enhanced by added vitamins, e.g. folic acid, pyridoxine, vitamin B 12, biotin and others. In a field experiment, it was confirmed that vitamin fertilization of an oil-polluted sand sample enhanced the biodegradation of constituent hydrocarbons of that sample.
Key words: bioremediation - oil-biodegradation - vitamin fertilization
Introduction The subject of hydrocarbon-degrading microorganisms and their role in cleaning oil-polluted environments has been repeatedly reviewed (e.g. Klug and Markovetz 1971 ; Levi et ai. 1979; Atlas 1981 ; Leahy and Colwell Corresponding author: S. S. Radwan e-mail: [email protected] 0944-5013/01/155/04-301
$15.00/0
1990; Radwan and Sorkhoh 1993; Lehmann 1998). Hydrocarbons can be utilized by a wide scope of soil and aquatic microorganisms including various bacteria, yeasts, filamentous fungi and even phototrophic microorganisms. In addition to soil and water, oil-utilizers were found in large numbers associated with coastal cyanobacterial mats (Sorkhoh et ai. 1992), roots of higher plants (Radwan et al. 1995, 1998) and coastal epilithic biomass (Radwan et ai. 1999). The physiology and biochemistry of microbial hydrocarbon utilization are now rather well understood (for reviews see e.g. (Rehm and Reiff 1981; Boulton and Ratledge 1984)). The initial attack by hydrocarbon-utilizing microorganisms on these substrates involves the introduction of one (or two) oxygen atom(s) into aliphatic (or aromatic) hydrocarbons via the activities of mono- (or di-) oxygenase systems which are key enzymes in such microorganisms. The alcohols produced are then oxidized up to the fatty acids which could then be used as carbon and/or energy sources. The growth factor requirements of conventional soil microorganisms were the subject of several earlier reviews (e.g. (Guirard and Snell 1962; Fries 1965; Koster 1968; Harris 1970)). On the other hand, there is an information gap regarding the vitamin requirements of hydrocarbon-utilizing soil microorganisms. We have recently found that the rhizosphere effect dramatically enhances hydrocarbon utilization of bacteria (Radwan et al. 1998). Vitamins are known to be among the microbial growth-enhancing exudates produced by plant roots. These facts stimulated our interest in studying the vitamin requirements of hydrocarbon-utilizing microorganisms. The main objective of this paper is to contribute to filling that information gap, and to demonstrate whether or not, added vitamins may affect the potential of microorganisms for growth and hydrocarbon consumption. Microbial. Res. ISS (200 I) 4
301
Materials and methods
ed and expressed as numbers of hydrocarbon-utilizing bacteria per gram dry soil.
Soil samples. Two desert soil samples, one compost sample, one garden soil sample supporting peanut, and one garden soil sample supporting corn were used in this study. Both, clean and oil-polluted samples were investigated. The polluted samples were prepared by mixing the clean samples with 10% (w/w) weathered crude oil and keeping them under open field conditions for 6 weeks before they were analyzed.
Isolation and identification. Individual representative colonies of the bacteria were isolated and purified by repeated streaking on the same solid medium. Pure cultures were identified by consulting "Bergey's Manual of Systematic Bacteriology", and the identification was confirmed by comparing the cultures with strains of the local culture collection that had been previously identified by the DSM (Deutsche Sammlung von Mikroorganismen und Zellkulturen = German Collection of Microorganisms and Cell Cultures, Germany). The two strains that were used for detailed study were identified by the NCIMB LTD (The National Collection of Industrial and Marine Bacteria Limited), Aberdeen Scotland, UK, as Cellulomonas jlavigena and Rhodococcus erythropoles.
Nutrient media. Two nutrient media were used, one conventional and one specific for hydrocarbon-utilizing microorganism. The conventional medium was nutrient broth (Difco, USA); it consisted of (g/I) 3.0 Bacto-beef extract and 5.0 Bacto-peptone, pH 7.0. The medium specific for hydrocarbon-utilizing microorganisms was a basal inorganic medium supplemented with hydrocarbons as sole sources of carbon and energy. The basal inorganic medium had the following composition (gil): 0.85 NaN0 3 , 0.56 KH 2P04 , 0.86 Na 2HP0 4 , 0.17 K2S04 , 0.37 MgS04 . 7 H20, 0.007 CaCI 2 . 2 H20 and 2.5 ml of a trace element solution consisting of (gil): 2.32 ZnS04 . 7H 20, 1.78 MnS04 . 4Hp, 0.56 H3 B0 3 , 1.0 CuS04 ·5 H20, 0.39 NaMo04 . 2Hp, 0.42 CoCI 2 . 6H 20, 0.66 KI, 1.0 EDTA, 0.4 FeS04 • 7Hp, 0.0004 NiCI 2 • 6H 20 (Sorkhoh et al. 1991). The pH of the media was adjusted to 7.0, and 1.5% agar was used for solidification. Vitamins. The following vitamins (purchased from Sigma, USA) were added to the media in the following standard concentrations (designated as 1 x concentrations) : thiamine, 5 mg; pyridoxine, 10 mg; vitamin B 12, 0.1 mg; biotin, 2 mg; riboflavin, 5 mg and folic acid 2 mgll medium. Vitamin solutions were filter-sterilized before they were added to the autoclaved media. Counting of soil bacteria. In all soil samples, total bacteria capable of utilizing crude oil as a sole source of carbon and energy were counted using the standard dilution-plating method. For this, basal solid inorganic medium aliquots with and without added vitamins (at 1x concentrations) were prepared and poured in Petri dishes. Aliquots, 1.0 g of each soil sample were suspended in 99 ml sterile water, each, and down series of dilutions were prepared, using sterile water. Aliquots, 0.5 ml each, of the various dilutions were spread on the surfaces of the solid inorganic medium in Petri dishes. Three replicates were made for each dilution. Sterile filter papers inserted in the dish covers were impregnated with 0.5 ml sterile crude oil each, and the inoculated dish-bottoms were inverted on the lids. The Petri dishes were sealed and kept inverted so that the cells sticking to the solid inorganic medium might make use of the volatile hydrocarbons. Cultures were incubated at 30°C for two weeks. The resulting colonies were count302
Microbiol. Res. 155 (200 I) 4
Hydrocarbon consumption. Fresh biomass samples (0.5 g) of the test bacterium (autoclaved, for control samples) were suspended in 25 ml ofthe basal inorganic medium with and without added vitamins, along with 10 mg of the test hydrocarbon n-octadecane or phenanthrene. Care was taken that organic solvents were aseptically volatilized before the biomass samples were suspended in the medium aliquots. The containers were sealed and incubated on an electric shaker, 120 rpm, at 30°C for 24 h. The cultures were centrifuged to remove the cells, which were washed with boiling inorganic medium to recover adhering hydrocarbons and the wash aliquots were added to the supernatant. The residual hydrocarbons were recovered from the supernatant by three extractions with diethyl ether. The solvent was volatilized, and the residue dissolved in 1.0 ml of hexane and 1.0 !AI aliquots were analyzed by GLC using a Chrompack CP-9000 instrument equipped with a flame ionization detector, a WCOT fuse silica capillary column and a temperature program 45-31O°C, raising the temperature 10°C/min. The peak areas for the hydrocarbons were measured, and the percent decreases were calculated based on the areas of the control peak. Field experiment. A field experiment was conducted to study the effect of fertilizing an oil-polluted desert sample with vitamins on the self-cleaning potential of that sample under field conditions. The experiment was started in June and took 4 weeks. Two holes, 40 x 40 x 30 cm 3 , were dug in a naked area of the Botanical Garden, Kuwait University, AI-Khaldiya, and filled each with 30 kg of clean sand that had been polluted with 10% (w/w) of crude oil. The control core was irrigated weekly with tap water, 125 ml/kg sand, whereas the test core was irrigated weekly with the same amount of water containing a mixture of the following vitamins
(mg/I): 5.0 thiamine, 1.0 pyridoxine, 0.1 cyanocobalamin, 2.0 folic acid. Sand samples were collected at zero time and after 4 weeks, and used for hydrocarbon extraction and analysis. Each gram of sand was extracted thrice with diethyl ether. The solvent was volatilized and the total hydrocarbons fractionated by column chromatography. A I cm x 10 cm column, packed with silica gel-69, 70-230 mesh (ASTM), was used and the total aliphatic hydrocarbons were eluted with hexane (10 ml). The solvents were volatilized and the residues redissolved in 1.0 ml hexane, each. The aliphatic fractions were analyzed by GLC as described above, and the total peak areas were taken as a quantitative measure of the total extractable alkanes from each sample.
Results Numbers of vitamin-requiring bacteria Table I shows that the numbers of oil-utilizing bacteria counted for four different soil samples on vitamincontaining media were significantly higher than the corresponding numbers counted on vitamin-free media. The data in Table 2 also show that the lowest numbers of oil-utilizing bacteria in another desert soil sample were counted on the vitamin free medium (control). This was true both for the clean as well as the oil-polluted soil samples. For the oily soil samples, the fold increase values in numbers in response to the vitamin addition to the medium were obviously higher than the corresponding values for the clean samples. Bacteria needing folic acid, pyridoxine and biotin exhibited the highest foldincrease values. The addition of the vitamin mixture resulted in a 9-fold increase in bacterial numbers. All colonies that grew on the vitamin mixture-containing
medium were tested for growth on the vitamin-free medium. For this experiment, the oily soil sample was used. The results showed that 94.2 % of the total colonies failed to grow. The results presented in Table 3 show that Cellulomonasflavigena and Rhodococcus erythropolis were the most dominant oil-utilizing bacteria in the desert soil sample studied. In addition, smaller proportions of Pseudomonas spp, Bacillus spp and Arthrobacter spp (designated "others" in Table 3) were also identified. The data show that oil pollution resulted in an increase in the proportions of C. flavigena and, albeit to a lesser extent, of R. erythropolis on the expense of the other bacteria, whose proportions became minimal in the oily soil sample. Another experiment showed that colonies of C. flavigena and R. erythropolis consisted each of two biovars, as far as the strict requirement for vitamins is concerned. For this experiment the oily soil sample was used. All individual colonies of C. flavigena and R. erythropolis that grew on the vitamin mixture-containing medium were tested for growth on the vitamin-free medium. It was found that 97.3% of C. flavigena and 93.9% of R. erythropolis failed to grow. It was thus apparent that the great majority of the strains show a strict requirement for vitamins. It was also noticed that even the minor biovars with no strict requirement for vitamins grew considerably better in the presence of vitamins than in their absence.
Effect of vitamins on growth The two biovars C. flavigena KCCBW 201 and R. erythropolis KCCBO 202 were used for this and the next experiments. (KCC = Kuwaiti Culture Collection). Both strains, although minor among all isolates were
Table 1. Numbers of oil-utilizing bacteria in various soil samples as determined by the standard dilution-plating method using media without and with added vitamins. Soil Samples
Clean Vitamin-free medium
Oil-polluted Medium with vitamins (p-value)
Vitamin-free medium
Medium with vitamins (p-value)
Compost
20.2
92.9 (0.03)
98.9
425.8 (0.04)
Garden soil supporting peanut
22.7
121.0 (0.008)
80.5
680.4 (0.002)
Garden soil supporting com
20.1
114.5 (0.01)
41.0
410.1 (0.04)
Desert soil, sample A
ND
ND
0.5
4.7 (0.001)
Data are bacterial numbers in millions and are means of three determinations, each. p- Values < 0.05 indicate significant increases in numbers on vitamin-containing media compared to numbers on vitamin-free media. The vitamin mixture used contained: 5 mg thiamin, 10 mg pyridoxine, 0.1 mg B 12, 2 mg biotin, 5 mg riboflavin and 2 mg folic acid/I medium. ND = Not determined. Microbiol. Res. 155 (2001) 4
303
Table 2. Numbers of oil-utilizing bacteria in the desert soil sample B, as determined by the standard dilution-plating method using media without and with added vitamins. Vitamins (mgfl)
Number x None Control) Thiamin (5) Pyridoxine ( 10) B I2 (0.1) Biotin (2) Riboflavin (5) Folic acid (2) Mixture
Oily desert soil
Clean desert soil 106
0.8 ±O.I 3.4 ± 0.4 0.9±0.1 2.8 ±0.2 1.7 ± 0.2 2.1 ±0.6 0.9±0.1 2.3 ±0.7
Fold increase"
Numbers x 106
Fold increase"
4.3 I.I 3.5 2.1 2.6 I.I 2.9
0.6±0.1 4.1 ± 0.1 5.6 ±0.2 3.4 ± 0.2 5.5 ±O.I 2.7 ±0.5 8.3 ±0.2 5.4 ± 0.3
6.8 9.3 5.7 9.2 4.5 14.0 9.0
Bacterial numbers are means of three determinations, each, ± standard deviation, and were calculated per g soil; " the fold increase was calculated by dividing the number obtained on the vitamin-containing media by those obtained on the vitamin-free medium (control). Table 3. Composition of the oil-utilizing bacteria from the desert soil sample B as determined by the standard dilution-plating method using media without and with added vitamins. Vitamins (mgfl)
None Control) Thiamin (5) Pyridoxine (10) B I2 (0.1) Biotin (2) Riboflavin (5) Folic acid (2) Mixture
Oily desert soil
Clean desert soil
Cellulomonas flavigena
Rhodococcus erythropolis
Others"
Cellulomonas flavigena
Rhodococcus erythropolis
Others"
(%)
(%)
(%)
(%)
(%)
(%)
54.2 62.1 69.4 45.7 53.6 48.9 71.4 21.8
12.1 22.7 29.0 22.9 36.3 29.8 14.0 42.2
33.7 15.2 1.6 31.3 10.1 21.3 14.6 36.0
65.2 93.5 79.9 87.6 80.2 81.0 96.0 87.3
30.5 5.9 12.2 12.0 16.2 18.1 3.0 12.1
4.4 0.6 7.9 0.4 3.6 0.9 1.0 0.6
Data are expressed in % of the total numbers recorded in Table 1. " Predominantly strains of Pseudomonos spp., Bacillus spp, and Arthrobacter spp.
selected for this experiment because they offered the advantage over the predominant strict vitamin auxotrophs that they were capable of growth, albeit weakly, in the control vitamin-free medium. At first the potential of the two biovars for growth on individual hydrocarbons as sole sources of carbon and energy was tested. The results showed that both biovars were capable of utilizing n-alkanes of various chain lengths as well as aromatic hydrocarbons including polynuclear compounds. Thus, C. flavigena KCCBW 20 I showed fair growth (compared to the growth on conventional nutrient agar) with n-tetradecane, n-hexadocane, n-octadecane, benzene, toluene, xylene, naphthalene, phenanthrene and pyrene, and weak growth with n-eicosane, n-octacosane, n-triacontane, n-dotriacontane, n-hexatriacontane, n-tetracontane, and biphenyl, as sole sources of carbon and energy. R. erythropolis KCCBO 202 showed fair growth with n-tetradecane, n-hexadecane, n-octadecane, benzene, toluene, xylene, 304
Microbial. Res. 155 (200 I) 4
naphthalene, phenanthrene and pyrene, and weak growth with n-eicosane, n-octacosane, n-triacontane, n-dotriacontane, n-hexatriacontane, n-tetracontane, and biphenyl, as sole sources of carbon and energy. It is to be noted in this context that the above tests were done using vitamin-free media. Figure I shows the effects of added vitamins on the growth of the two test biovars in media containing hydrocarbons as sole sources of carbon and energy. Although both organisms, as expected, grew in the absence of any added vitamins, their growth was optimally enhanced by all vitamins added at their standard concentrations nonnally used (I x) or at one tenth of these concentrations. Increasing the concentrations of the added vitamins above these values inhibited the growth substantially. The vitamins which led to the best growth results depended not only on the test organism but also on the hydrocarbon substrate. For C. flavigena KCCBW 201,
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Fig. 1. Effect of vitamins on the growth of Cellulomonas flavigena KCCBW 201 (upper) and Rhodococcus erythropolis KCCBO 202 (lower) on n-octadecane (left) and phenanthrene (right) as sole sources of carbon and energy. Thiamin (I x = 5 mg/!), open circles; Pyridoxin (I x = 10 mg/!), filled triangles; Biotin (I x = 2 mg/!), open square; Folic acid (I x = 2 mg/!), filled circles.
Fig.2. Effect of vitamins on the consumption of n-octadecane (left) and phenanthrene (right) by Cellulomonas flavigena KCCBW 201 (upper) and Rhodococcus erythropolis KCCBO 202 (lower). Symbols as in Fig. I.
the test vitamins may be arranged in the following order of growth-enhancing effects, irrespective of the hydrocarbon substrate: folic acid> pyridoxine> vitamin B 12 > biotin> thiamine. For R. erythropolis KCCBO 202
growing on n-octadecane, the sequence was: vitamin B 12, thiamine and biotin> pyridoxine> folic acid, and on phenanthrene: thiamine and folic acid> biotin and vitamin B 12 > pyridoxine. Microbiol. Res. 155 (200 1) 4
305
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Fig.3. Typical GLC profiles of total extractable alkanes from oil-polluted soil. Upper =at zero time; Middle =after 4 weeks, non-treated; Bottom = after 4 weeks, fertilized with vitamins.
Effect of vitamins on hydrocarbon consumption The results presented in Fig. 2 show that added vitamins, did not only enhance bacterial growth but also the utilization of individual hydrocarbons. For C. flavigena KCCBW 201, one of the vitamins that enhanced growth best, namely pyridoxine, also enhanced the consumption of the hydrocarbons best. Similarly, for R. erythropolis KCCBO 202, vitamin B12 which was optimal for growth on n-octadecane as a sole source of carbon and energy was also optimal for the consumption of this alkane. However, the vitamin which was associated with the best consumption of phenanthrene by this organism was biotin, which was only second in the order of growth promotion potential. It is also noted that all 306
Microbiol. Res. 155 (200 I) 4
vitamins enhanced, yet to varying degrees, the microbial hydrocarbon consumption when added at the standard concentrations normally used (I x), or one tenth of those concentrations; higher concentrations were inhibitory. The above results were confirmed in the field experiment which showed that the fertilization of an oilpolluted soil sample with a vitamin mixture at the standard concentrations dramatically enhanced the hydrocarbon attenuation in that sample (Fig. 3). The quantitative analysis of the GLC profiles shows that the total extractable alkanes in the non-treated soil decreased after 4 weeks by only 14.1 % of the values at zero time. By contrast, the total extractable alkanes in the vitamin-fertilized soil sample decreased after 4 weeks by 42.2%. The profiles show further that shorter-chain alkanes, as expected, were more readily utilized than longer-chain alkanes.
Discussion Similar to the conventional soil bacteria (Fries 1965; Koster 1968; Harris 1970; Alexander 1977), most of the oil-utilizing soil microorganisms need to be provided with one or more vitamins for optimal growth and activity. This similarity was to be expected in view of the fact that oil-utilizing bacteria are actually a part of the indigenous conventional bacteria in soil, that in the absence of hydrocarbons are capable of utilizing conventional organic carbon sources (Atlas 1981; Radwan and Sorkhoh 1993). In the available literature, there are no records of soil bacteria that are absolutely hydrocarbon-specific in their nutrition. It is interesting that more than 90% of the hydrocarbon-utilizing bacteria are absolutely vitamin-auxotrophs. The growth and hydrocarbon degradation potential of even the remaining minor fraction of bacteria capable of growth without vitamins was obviously enhanced by added vitamins. These results consolidate the recommendation that vitamins, especially of the B group, should be included in media prepared for counting hydrocarbon-utilizing microorganisms. Vitamin-free media would allow for the growth of only less than 10% of the total hydrocarbon-utilizing microflora actually present. Another important conclusion of this study is that vitamin fertilization appears to be a promising practice for enhancing the bioremediation potential of oil-polluted soils. This does not necessarily mean that pure vitamin preparations may be recommended; rather more, vitamin-rich wastes or agricultural by-products may be used as vitamin sources. Alternatively, vitaminproducing bacterial strains may be inoculated in oilpolluted environments, as demonstrated in a few earlier
studies on the biodehalogenation of halogenated hydrocarbons (Jensen 1957; Slater and Lovatt 1984; Van den Wijngaard et al. 1993).
Acknowledgement This work has been supported by Kuwait University, research grants number SO 067 and SO 062.
References Alexander, M. (1977): Introduction to soil microbiology. John Wiley and Sons, Inc. New York. Atlas, R. M. (1981): Microbial degradation of petroleum hydrocarbons: an environmental perspective. Microbiol. Rev.45,180-209. Boulton, C. A., Ratledge, C. (1984): The physiology of hydrocarbon-utilizing microorganisms. In: Topics in Fermentation and Enzyme Technology Vol. 9 (Ed. Wiseman, A.). Ellis Horwood, Chichester, U.K. Fries, N. (1965): The chemical environment for fungal growth. 3. Vitamins and other organic growth factors. In: The Fungi, Vol. 2 (Eds: Ainsworth, G. c., Sussman, A. S.). Academic Press, New York, 491-523. Guirard, B. M., Snell, E. E. (1962): Nutritional requirements of microorganisms. In: The Bacteria, Vol. 4 (Eds: Gunsalus, I. c., Stanier, R.Y.). Academic Press, New York, 33-93. Harris, I. J. (1970): The discovery of vitamins. In: The chemistry of life (Ed: Needham, J.). Cambridge University Press, New York, 156-170. Jensen, H. L. (1957): Decomposition of chloro-substituted aliphatic acids by soil bacteria. Can. 1. Microbiol. 3, 151-164. Klug, M. J., Markovetz, A. J. (1971): Utilization of aliphatic hydrocarbons by microorganisms. Adv. Microb. Physiol. 5, 1-43. Koster, S. A. (1968): Vitamin requirements of bacteria and yeast. Thomas, Springfield, Illinois, 663.
Leahy, J. G. Colwell, R. R. (1990): Microbial degradation of hydrocarbons in the environment. Microbiol. Rev. 54, 305-315. Lehmann, V. (1998): Bioremediation: A solution for polluted soils in the south? Biotechnol. Develop. Monitor 4, 13-17. Levi, J. D., Shennan, J. L., Ebbon, G. P. (1979): Biomass from n-alkanes. In: Economic Microbiology, Vol. 4 (Eds: Rose, A. H.). Academic Press, London. Radwan, S. S., AI-Awadhi, H., Sorkhoh, N. A., EI-Nemr, I. M. (1998): Rhizospheric hydrocarbon-utilizing microorganisms as potential contributors to phytoremediation for the oily Kuwaiti desert. Microbiol. Res. 153,247-251. Radwan, S. S., AI-Hasan, R. H., AI-Awadhi, H., Salamah, S., Abdullah, H. (1999): Higher oil biodegradation potential at the Arabian Gulf Coast than in the water body. Mar. BioI. 135,741-745. Radwan, S. S., Sorkhoh, N. A. (1993): Lipids of n-alkaneutilizing, microorganisms and their application potential. Adv. Appl. Microbiol. 39, 29-90. Radwan, S. S., Sorkhoh, N. A. and EI-Nemr, I. M. (1995): Oil biodegradation around roots. Nature 376, 302. Rehm, H.-J., Reiff, I. (1981): Mechanisms and occurrence of microbial oxidation of long-chain alkanes. Adv. Biochem. Eng. 19, 175-216. Slater, J. H., Lovatt, D. (1984): Biodegradation and the significance of microbial communities. In: Microbial degradation of organic compounds (Ed: Gibson, I., David, T.). Marcel Deckker, Inc., New York, 439-485. Sorkhoh, N., AI-Hassan, R., Radwan, S., Hopner, T. (1992): Self-cleaning of the gulf. Nature 359, 109. Sorkhoh, N. A., Ghannoum, M. A., Ibrahim, A. S., Stretton, R. J., Radwan, S.S. (1991): Growth of Candida albicans in the presence of hydrocarbons: a correlation between sterol concentration and hydrocarbon uptake. Appl. Microbiol. Biotechnol. 34,509-512. Van den Wijngaard, A., Van Del' Kleij, R. G., Doomweerd, R. E., Janssen, D.B. (1993): Influence of organic nutrients and cocultures on the competitive behavior of 1,2-dichloroethane-degrading bacteria. Appl. Environ. Microbiol. 59, 3400-3405.
Microbiol. Res. ISS (200 1) 4
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Vitamin requirements of hydrocarbon-utilizing soil bacteria Samir S. Radwan, Awatif S. AI-Muteirie Department of Biological Sciences, Faculty of Science, Kuwait University, P. O. Box 5969, Safat 13060, Kuwait Accepted: May 6, 2000
Abstract The numbers of oil-utilizing bacteria in several samples of clean and oil-polluted soils counted on vitamin-containing media were severalfold higher than the numbers counted on vitamin-free media. Colonies that grew on a medium containing a vitamin mixture were tested for growth on the same medium lacking any vitamins. More than 90% of the total colonies failed to grow. The remaining 10% grew, yet their growth was enhanced, when vitamins were added. The predominant oil-utilizing bacteria in one of the test desert soil samples were various strains of Cellulomonas flavigena and Rhodococcus erythropolis. Minor organisms belonged to the genera Pseudomonas, Bacillus and Arthrobacter. Two vitamin-requiring biovars of C. flavigena and R. erythropolis were selected for further study. Their growth on n-octadecane and phenanthrene as sole sources of carbon and energy as well as their potential for hydrocarbon consumption were enhanced by added vitamins, e.g. folic acid, pyridoxine, vitamin B 12, biotin and others. In a field experiment, it was confirmed that vitamin fertilization of an oil-polluted sand sample enhanced the biodegradation of constituent hydrocarbons of that sample.
Key words: bioremediation - oil-biodegradation - vitamin fertilization
Introduction The subject of hydrocarbon-degrading microorganisms and their role in cleaning oil-polluted environments has been repeatedly reviewed (e.g. Klug and Markovetz 1971 ; Levi et ai. 1979; Atlas 1981 ; Leahy and Colwell Corresponding author: S. S. Radwan e-mail: [email protected] 0944-5013/01/155/04-301
$15.00/0
1990; Radwan and Sorkhoh 1993; Lehmann 1998). Hydrocarbons can be utilized by a wide scope of soil and aquatic microorganisms including various bacteria, yeasts, filamentous fungi and even phototrophic microorganisms. In addition to soil and water, oil-utilizers were found in large numbers associated with coastal cyanobacterial mats (Sorkhoh et ai. 1992), roots of higher plants (Radwan et al. 1995, 1998) and coastal epilithic biomass (Radwan et ai. 1999). The physiology and biochemistry of microbial hydrocarbon utilization are now rather well understood (for reviews see e.g. (Rehm and Reiff 1981; Boulton and Ratledge 1984)). The initial attack by hydrocarbon-utilizing microorganisms on these substrates involves the introduction of one (or two) oxygen atom(s) into aliphatic (or aromatic) hydrocarbons via the activities of mono- (or di-) oxygenase systems which are key enzymes in such microorganisms. The alcohols produced are then oxidized up to the fatty acids which could then be used as carbon and/or energy sources. The growth factor requirements of conventional soil microorganisms were the subject of several earlier reviews (e.g. (Guirard and Snell 1962; Fries 1965; Koster 1968; Harris 1970)). On the other hand, there is an information gap regarding the vitamin requirements of hydrocarbon-utilizing soil microorganisms. We have recently found that the rhizosphere effect dramatically enhances hydrocarbon utilization of bacteria (Radwan et al. 1998). Vitamins are known to be among the microbial growth-enhancing exudates produced by plant roots. These facts stimulated our interest in studying the vitamin requirements of hydrocarbon-utilizing microorganisms. The main objective of this paper is to contribute to filling that information gap, and to demonstrate whether or not, added vitamins may affect the potential of microorganisms for growth and hydrocarbon consumption. Microbial. Res. ISS (200 I) 4
301
Materials and methods
ed and expressed as numbers of hydrocarbon-utilizing bacteria per gram dry soil.
Soil samples. Two desert soil samples, one compost sample, one garden soil sample supporting peanut, and one garden soil sample supporting corn were used in this study. Both, clean and oil-polluted samples were investigated. The polluted samples were prepared by mixing the clean samples with 10% (w/w) weathered crude oil and keeping them under open field conditions for 6 weeks before they were analyzed.
Isolation and identification. Individual representative colonies of the bacteria were isolated and purified by repeated streaking on the same solid medium. Pure cultures were identified by consulting "Bergey's Manual of Systematic Bacteriology", and the identification was confirmed by comparing the cultures with strains of the local culture collection that had been previously identified by the DSM (Deutsche Sammlung von Mikroorganismen und Zellkulturen = German Collection of Microorganisms and Cell Cultures, Germany). The two strains that were used for detailed study were identified by the NCIMB LTD (The National Collection of Industrial and Marine Bacteria Limited), Aberdeen Scotland, UK, as Cellulomonas jlavigena and Rhodococcus erythropoles.
Nutrient media. Two nutrient media were used, one conventional and one specific for hydrocarbon-utilizing microorganism. The conventional medium was nutrient broth (Difco, USA); it consisted of (g/I) 3.0 Bacto-beef extract and 5.0 Bacto-peptone, pH 7.0. The medium specific for hydrocarbon-utilizing microorganisms was a basal inorganic medium supplemented with hydrocarbons as sole sources of carbon and energy. The basal inorganic medium had the following composition (gil): 0.85 NaN0 3 , 0.56 KH 2P04 , 0.86 Na 2HP0 4 , 0.17 K2S04 , 0.37 MgS04 . 7 H20, 0.007 CaCI 2 . 2 H20 and 2.5 ml of a trace element solution consisting of (gil): 2.32 ZnS04 . 7H 20, 1.78 MnS04 . 4Hp, 0.56 H3 B0 3 , 1.0 CuS04 ·5 H20, 0.39 NaMo04 . 2Hp, 0.42 CoCI 2 . 6H 20, 0.66 KI, 1.0 EDTA, 0.4 FeS04 • 7Hp, 0.0004 NiCI 2 • 6H 20 (Sorkhoh et al. 1991). The pH of the media was adjusted to 7.0, and 1.5% agar was used for solidification. Vitamins. The following vitamins (purchased from Sigma, USA) were added to the media in the following standard concentrations (designated as 1 x concentrations) : thiamine, 5 mg; pyridoxine, 10 mg; vitamin B 12, 0.1 mg; biotin, 2 mg; riboflavin, 5 mg and folic acid 2 mgll medium. Vitamin solutions were filter-sterilized before they were added to the autoclaved media. Counting of soil bacteria. In all soil samples, total bacteria capable of utilizing crude oil as a sole source of carbon and energy were counted using the standard dilution-plating method. For this, basal solid inorganic medium aliquots with and without added vitamins (at 1x concentrations) were prepared and poured in Petri dishes. Aliquots, 1.0 g of each soil sample were suspended in 99 ml sterile water, each, and down series of dilutions were prepared, using sterile water. Aliquots, 0.5 ml each, of the various dilutions were spread on the surfaces of the solid inorganic medium in Petri dishes. Three replicates were made for each dilution. Sterile filter papers inserted in the dish covers were impregnated with 0.5 ml sterile crude oil each, and the inoculated dish-bottoms were inverted on the lids. The Petri dishes were sealed and kept inverted so that the cells sticking to the solid inorganic medium might make use of the volatile hydrocarbons. Cultures were incubated at 30°C for two weeks. The resulting colonies were count302
Microbiol. Res. 155 (200 I) 4
Hydrocarbon consumption. Fresh biomass samples (0.5 g) of the test bacterium (autoclaved, for control samples) were suspended in 25 ml ofthe basal inorganic medium with and without added vitamins, along with 10 mg of the test hydrocarbon n-octadecane or phenanthrene. Care was taken that organic solvents were aseptically volatilized before the biomass samples were suspended in the medium aliquots. The containers were sealed and incubated on an electric shaker, 120 rpm, at 30°C for 24 h. The cultures were centrifuged to remove the cells, which were washed with boiling inorganic medium to recover adhering hydrocarbons and the wash aliquots were added to the supernatant. The residual hydrocarbons were recovered from the supernatant by three extractions with diethyl ether. The solvent was volatilized, and the residue dissolved in 1.0 ml of hexane and 1.0 !AI aliquots were analyzed by GLC using a Chrompack CP-9000 instrument equipped with a flame ionization detector, a WCOT fuse silica capillary column and a temperature program 45-31O°C, raising the temperature 10°C/min. The peak areas for the hydrocarbons were measured, and the percent decreases were calculated based on the areas of the control peak. Field experiment. A field experiment was conducted to study the effect of fertilizing an oil-polluted desert sample with vitamins on the self-cleaning potential of that sample under field conditions. The experiment was started in June and took 4 weeks. Two holes, 40 x 40 x 30 cm 3 , were dug in a naked area of the Botanical Garden, Kuwait University, AI-Khaldiya, and filled each with 30 kg of clean sand that had been polluted with 10% (w/w) of crude oil. The control core was irrigated weekly with tap water, 125 ml/kg sand, whereas the test core was irrigated weekly with the same amount of water containing a mixture of the following vitamins
(mg/I): 5.0 thiamine, 1.0 pyridoxine, 0.1 cyanocobalamin, 2.0 folic acid. Sand samples were collected at zero time and after 4 weeks, and used for hydrocarbon extraction and analysis. Each gram of sand was extracted thrice with diethyl ether. The solvent was volatilized and the total hydrocarbons fractionated by column chromatography. A I cm x 10 cm column, packed with silica gel-69, 70-230 mesh (ASTM), was used and the total aliphatic hydrocarbons were eluted with hexane (10 ml). The solvents were volatilized and the residues redissolved in 1.0 ml hexane, each. The aliphatic fractions were analyzed by GLC as described above, and the total peak areas were taken as a quantitative measure of the total extractable alkanes from each sample.
Results Numbers of vitamin-requiring bacteria Table I shows that the numbers of oil-utilizing bacteria counted for four different soil samples on vitamincontaining media were significantly higher than the corresponding numbers counted on vitamin-free media. The data in Table 2 also show that the lowest numbers of oil-utilizing bacteria in another desert soil sample were counted on the vitamin free medium (control). This was true both for the clean as well as the oil-polluted soil samples. For the oily soil samples, the fold increase values in numbers in response to the vitamin addition to the medium were obviously higher than the corresponding values for the clean samples. Bacteria needing folic acid, pyridoxine and biotin exhibited the highest foldincrease values. The addition of the vitamin mixture resulted in a 9-fold increase in bacterial numbers. All colonies that grew on the vitamin mixture-containing
medium were tested for growth on the vitamin-free medium. For this experiment, the oily soil sample was used. The results showed that 94.2 % of the total colonies failed to grow. The results presented in Table 3 show that Cellulomonasflavigena and Rhodococcus erythropolis were the most dominant oil-utilizing bacteria in the desert soil sample studied. In addition, smaller proportions of Pseudomonas spp, Bacillus spp and Arthrobacter spp (designated "others" in Table 3) were also identified. The data show that oil pollution resulted in an increase in the proportions of C. flavigena and, albeit to a lesser extent, of R. erythropolis on the expense of the other bacteria, whose proportions became minimal in the oily soil sample. Another experiment showed that colonies of C. flavigena and R. erythropolis consisted each of two biovars, as far as the strict requirement for vitamins is concerned. For this experiment the oily soil sample was used. All individual colonies of C. flavigena and R. erythropolis that grew on the vitamin mixture-containing medium were tested for growth on the vitamin-free medium. It was found that 97.3% of C. flavigena and 93.9% of R. erythropolis failed to grow. It was thus apparent that the great majority of the strains show a strict requirement for vitamins. It was also noticed that even the minor biovars with no strict requirement for vitamins grew considerably better in the presence of vitamins than in their absence.
Effect of vitamins on growth The two biovars C. flavigena KCCBW 201 and R. erythropolis KCCBO 202 were used for this and the next experiments. (KCC = Kuwaiti Culture Collection). Both strains, although minor among all isolates were
Table 1. Numbers of oil-utilizing bacteria in various soil samples as determined by the standard dilution-plating method using media without and with added vitamins. Soil Samples
Clean Vitamin-free medium
Oil-polluted Medium with vitamins (p-value)
Vitamin-free medium
Medium with vitamins (p-value)
Compost
20.2
92.9 (0.03)
98.9
425.8 (0.04)
Garden soil supporting peanut
22.7
121.0 (0.008)
80.5
680.4 (0.002)
Garden soil supporting com
20.1
114.5 (0.01)
41.0
410.1 (0.04)
Desert soil, sample A
ND
ND
0.5
4.7 (0.001)
Data are bacterial numbers in millions and are means of three determinations, each. p- Values < 0.05 indicate significant increases in numbers on vitamin-containing media compared to numbers on vitamin-free media. The vitamin mixture used contained: 5 mg thiamin, 10 mg pyridoxine, 0.1 mg B 12, 2 mg biotin, 5 mg riboflavin and 2 mg folic acid/I medium. ND = Not determined. Microbiol. Res. 155 (2001) 4
303
Table 2. Numbers of oil-utilizing bacteria in the desert soil sample B, as determined by the standard dilution-plating method using media without and with added vitamins. Vitamins (mgfl)
Number x None Control) Thiamin (5) Pyridoxine ( 10) B I2 (0.1) Biotin (2) Riboflavin (5) Folic acid (2) Mixture
Oily desert soil
Clean desert soil 106
0.8 ±O.I 3.4 ± 0.4 0.9±0.1 2.8 ±0.2 1.7 ± 0.2 2.1 ±0.6 0.9±0.1 2.3 ±0.7
Fold increase"
Numbers x 106
Fold increase"
4.3 I.I 3.5 2.1 2.6 I.I 2.9
0.6±0.1 4.1 ± 0.1 5.6 ±0.2 3.4 ± 0.2 5.5 ±O.I 2.7 ±0.5 8.3 ±0.2 5.4 ± 0.3
6.8 9.3 5.7 9.2 4.5 14.0 9.0
Bacterial numbers are means of three determinations, each, ± standard deviation, and were calculated per g soil; " the fold increase was calculated by dividing the number obtained on the vitamin-containing media by those obtained on the vitamin-free medium (control). Table 3. Composition of the oil-utilizing bacteria from the desert soil sample B as determined by the standard dilution-plating method using media without and with added vitamins. Vitamins (mgfl)
None Control) Thiamin (5) Pyridoxine (10) B I2 (0.1) Biotin (2) Riboflavin (5) Folic acid (2) Mixture
Oily desert soil
Clean desert soil
Cellulomonas flavigena
Rhodococcus erythropolis
Others"
Cellulomonas flavigena
Rhodococcus erythropolis
Others"
(%)
(%)
(%)
(%)
(%)
(%)
54.2 62.1 69.4 45.7 53.6 48.9 71.4 21.8
12.1 22.7 29.0 22.9 36.3 29.8 14.0 42.2
33.7 15.2 1.6 31.3 10.1 21.3 14.6 36.0
65.2 93.5 79.9 87.6 80.2 81.0 96.0 87.3
30.5 5.9 12.2 12.0 16.2 18.1 3.0 12.1
4.4 0.6 7.9 0.4 3.6 0.9 1.0 0.6
Data are expressed in % of the total numbers recorded in Table 1. " Predominantly strains of Pseudomonos spp., Bacillus spp, and Arthrobacter spp.
selected for this experiment because they offered the advantage over the predominant strict vitamin auxotrophs that they were capable of growth, albeit weakly, in the control vitamin-free medium. At first the potential of the two biovars for growth on individual hydrocarbons as sole sources of carbon and energy was tested. The results showed that both biovars were capable of utilizing n-alkanes of various chain lengths as well as aromatic hydrocarbons including polynuclear compounds. Thus, C. flavigena KCCBW 20 I showed fair growth (compared to the growth on conventional nutrient agar) with n-tetradecane, n-hexadocane, n-octadecane, benzene, toluene, xylene, naphthalene, phenanthrene and pyrene, and weak growth with n-eicosane, n-octacosane, n-triacontane, n-dotriacontane, n-hexatriacontane, n-tetracontane, and biphenyl, as sole sources of carbon and energy. R. erythropolis KCCBO 202 showed fair growth with n-tetradecane, n-hexadecane, n-octadecane, benzene, toluene, xylene, 304
Microbial. Res. 155 (200 I) 4
naphthalene, phenanthrene and pyrene, and weak growth with n-eicosane, n-octacosane, n-triacontane, n-dotriacontane, n-hexatriacontane, n-tetracontane, and biphenyl, as sole sources of carbon and energy. It is to be noted in this context that the above tests were done using vitamin-free media. Figure I shows the effects of added vitamins on the growth of the two test biovars in media containing hydrocarbons as sole sources of carbon and energy. Although both organisms, as expected, grew in the absence of any added vitamins, their growth was optimally enhanced by all vitamins added at their standard concentrations nonnally used (I x) or at one tenth of these concentrations. Increasing the concentrations of the added vitamins above these values inhibited the growth substantially. The vitamins which led to the best growth results depended not only on the test organism but also on the hydrocarbon substrate. For C. flavigena KCCBW 201,
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Fig. 1. Effect of vitamins on the growth of Cellulomonas flavigena KCCBW 201 (upper) and Rhodococcus erythropolis KCCBO 202 (lower) on n-octadecane (left) and phenanthrene (right) as sole sources of carbon and energy. Thiamin (I x = 5 mg/!), open circles; Pyridoxin (I x = 10 mg/!), filled triangles; Biotin (I x = 2 mg/!), open square; Folic acid (I x = 2 mg/!), filled circles.
Fig.2. Effect of vitamins on the consumption of n-octadecane (left) and phenanthrene (right) by Cellulomonas flavigena KCCBW 201 (upper) and Rhodococcus erythropolis KCCBO 202 (lower). Symbols as in Fig. I.
the test vitamins may be arranged in the following order of growth-enhancing effects, irrespective of the hydrocarbon substrate: folic acid> pyridoxine> vitamin B 12 > biotin> thiamine. For R. erythropolis KCCBO 202
growing on n-octadecane, the sequence was: vitamin B 12, thiamine and biotin> pyridoxine> folic acid, and on phenanthrene: thiamine and folic acid> biotin and vitamin B 12 > pyridoxine. Microbiol. Res. 155 (200 1) 4
305
C14
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Fig.3. Typical GLC profiles of total extractable alkanes from oil-polluted soil. Upper =at zero time; Middle =after 4 weeks, non-treated; Bottom = after 4 weeks, fertilized with vitamins.
Effect of vitamins on hydrocarbon consumption The results presented in Fig. 2 show that added vitamins, did not only enhance bacterial growth but also the utilization of individual hydrocarbons. For C. flavigena KCCBW 201, one of the vitamins that enhanced growth best, namely pyridoxine, also enhanced the consumption of the hydrocarbons best. Similarly, for R. erythropolis KCCBO 202, vitamin B12 which was optimal for growth on n-octadecane as a sole source of carbon and energy was also optimal for the consumption of this alkane. However, the vitamin which was associated with the best consumption of phenanthrene by this organism was biotin, which was only second in the order of growth promotion potential. It is also noted that all 306
Microbiol. Res. 155 (200 I) 4
vitamins enhanced, yet to varying degrees, the microbial hydrocarbon consumption when added at the standard concentrations normally used (I x), or one tenth of those concentrations; higher concentrations were inhibitory. The above results were confirmed in the field experiment which showed that the fertilization of an oilpolluted soil sample with a vitamin mixture at the standard concentrations dramatically enhanced the hydrocarbon attenuation in that sample (Fig. 3). The quantitative analysis of the GLC profiles shows that the total extractable alkanes in the non-treated soil decreased after 4 weeks by only 14.1 % of the values at zero time. By contrast, the total extractable alkanes in the vitamin-fertilized soil sample decreased after 4 weeks by 42.2%. The profiles show further that shorter-chain alkanes, as expected, were more readily utilized than longer-chain alkanes.
Discussion Similar to the conventional soil bacteria (Fries 1965; Koster 1968; Harris 1970; Alexander 1977), most of the oil-utilizing soil microorganisms need to be provided with one or more vitamins for optimal growth and activity. This similarity was to be expected in view of the fact that oil-utilizing bacteria are actually a part of the indigenous conventional bacteria in soil, that in the absence of hydrocarbons are capable of utilizing conventional organic carbon sources (Atlas 1981; Radwan and Sorkhoh 1993). In the available literature, there are no records of soil bacteria that are absolutely hydrocarbon-specific in their nutrition. It is interesting that more than 90% of the hydrocarbon-utilizing bacteria are absolutely vitamin-auxotrophs. The growth and hydrocarbon degradation potential of even the remaining minor fraction of bacteria capable of growth without vitamins was obviously enhanced by added vitamins. These results consolidate the recommendation that vitamins, especially of the B group, should be included in media prepared for counting hydrocarbon-utilizing microorganisms. Vitamin-free media would allow for the growth of only less than 10% of the total hydrocarbon-utilizing microflora actually present. Another important conclusion of this study is that vitamin fertilization appears to be a promising practice for enhancing the bioremediation potential of oil-polluted soils. This does not necessarily mean that pure vitamin preparations may be recommended; rather more, vitamin-rich wastes or agricultural by-products may be used as vitamin sources. Alternatively, vitaminproducing bacterial strains may be inoculated in oilpolluted environments, as demonstrated in a few earlier
studies on the biodehalogenation of halogenated hydrocarbons (Jensen 1957; Slater and Lovatt 1984; Van den Wijngaard et al. 1993).
Acknowledgement This work has been supported by Kuwait University, research grants number SO 067 and SO 062.
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