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Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

Aims: The objective of this work was to study picocyanobacteria in the Arabian Gulf water in relation to oil pollution.

Methods and Results: Epifluorescent microscopic counting showed that offshore water samples along the Kuwaiti coast of the Arabian Gulf were rich in picocyanobacteria which ranged in numbers between about 1 × 105 and 6 × 105 ml−1. Most dominant was the genus Synechococcus; less dominant genera were Synechocystis, Pleurocapsa and Dermocarpella. All isolates grew well in an inorganic medium containing up to 0·1% crude oil (w/v) and could survive in the presence of up to 1% crude oil. Hydrocarbon analysis by gas liquid chromatography (GLC) showed that representative strains of the four genera had the potential for the accumulation of hydrocarbons (the aliphatic n-hexadecane, aromatic phenanthrene and crude oil hydrocarbons) from aqueous media. Electron microscopy showed that the cells of these strains appeared to store hydrocarbons in their inter thylakoid spaces. Analysis by GLC of constituent fatty acids of total lipids and individual lipid classes from representative picoplankton strains grown in the absence and presence of hydrocarbons showed, however, that the fatty acid patterns were not markedly affected by the hydrocabon substrates, meaning that the test strains could not oxidize the accumulated hydrocarbons.

Conclusions: The Arabian Gulf is among the water bodies of the world richest in picocyanobacteria. These micro-organisms accumulate hydrocarbons from the water body, but do not biodegrade these compounds. It is assumed that hydrocarbon-utilizing bacteria that were always found associated with all picocyanobacteria in nature may carry out the biodegradation of these compounds.

Significance and Importance of the Study: The results shed light on the potential role of picocyanobacteria in controlling marine oil pollution.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

Legal activities (e.g. production and transport of crude oil) as well as illegal actions (e.g. disposal of oily wastes) charge the marine environment worldwide with huge amounts of petroleum hydrocarbons. It was estimated a couple of decades ago that 6–10 million tons of petroleum pollute the marine ecosystem yearly (Blumer et al. 1971; National Academy of Science 1975). Since then, this has probably grown steadily, given the increasing oil production and transport activities. The problem of oil pollution is especially acute in an oil-producing area such as the Arabian Gulf, where about 60% of the marine-transported oil in the world is produced (British Petroleum Co. 1980) and transported through this rather shallow and enclosed sea (Hunter 1982). It has been estimated that the Arabian Gulf has, in the past, had petroleum hydrocarbon concentrations of 1·2–546 μg ml−1 compared with 0·4–66·8 μg ml−1 in the Gulf of Mexico (Sen Gupta and Kureishy 1981; Marchand et al. 1982; El Samra et al. 1986). The situation for this environment became even worse during the occupation of Kuwait (2 August 1990–26 February 1991) after the Iraqi forces released about 500 000 tons of crude oil into the Gulf from the Mina Al-Ahmadi terminal.

The present group has published studies on the bioremediation of the oily Arabian Gulf environment (Sorkhoh et al. 1990; Sorkhoh et al. 1992; Al-Hasan et al. 1994; Al-Hasan et al. 1998; Radwan et al. 1995; Radwan et al. 1999; Radwan and Al-Hasan 2000). One of the findings was that the Gulf coast contained far higher numbers of hydrocarbon-utilizing bacteria than the main water body (Radwan et al. 1999). In view of the fact that picoplanktonic cyanobacteria are recognized to be of cosmopolitan occurrence in seas and oceans, it was proposed to study these micro-organisms in the Arabian Gulf water in relation to oil pollution. The results of this study are summarized in this paper.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

Sampling, counting and isolation

Basically the techniques described by Waterbury et al. (1986) were followed. Water samples were taken during 1998 and 1999 from seven different stations along the Kuwait coast, stations 1 and 7 being at the extreme north and south of the country, respectively. Each two adjacent stations were roughly 30 km apart. Sampling was conducted using a water sampler (Widco, Saginaw, MI, USA) provided with sterile plastic bottles, 2–3 m below the water surface and 2–3 km offshore.

For counting, a known volume of each water sample was filtered through a 0·45-μm membrane filter (Millipore, Bedford, MA, USA) and the picocyanobacteria directly viewed and counted using an epifluorescent microscope (Carlzeiss Company, Oberkochen, Germany) equipped with a green excitation filter (510–560 nm). In each of three parallel samples a total of at least 500 epifluorescent cells were counted in a known area of the filter surface and the total cell numbers per ml of water sample were calculated.

For the isolation of picocyanobacteria, each water sample was differentially filtered first through a 5·0-μm glass filter to remove large organisms and particles and then through a 1·0-μm cellulose nitrate membrane filter. The final filtrate was used to isolate Synechococcus and Synechocystis, whereas material trapped on the cellulose nitrate membrane filter was used for the isolation of the larger cyanobacteria Pleurocapsa and Dermocarpella. Final filtrate samples and whole membrane filters were inoculated into sterile MN medium (Rippka 1988) and the culture flasks incubated overnight in a growth chamber at 25°C, in the dark, and then under 12 h light (14 μE m–2 s−1)/12 h dark cycles until growth was recognized, which usually occurred after 6–12 weeks. For purification, loopfuls were streaked on solid MN medium and cell masses at the most diluted end of the streak isolated. This process was repeated several times. The antibiotics ampicillin (1 mg ml−1) (Rippka 1988), streptomycin sulphate (0·2 mg ml−1), neomycin sulphate (0·8 mg l−1) and polymyxin B (95 units ml−1) (Whitton 1968) were included in medium aliquots in which cyanobacterial isolates were repeatedly subcultured in an attempt to rid them of associated heterotrophic bacteria. The cells were examined by bright field and phase contrast microscopy, as well as an epifluorescence microscope equipped with a green excitation filter (510–560 nm) and a barrier filter LP 590.

The counting and isolation of hydrocarbon-utilizing bacteria associated with picocyanobacteria was undertaken by the standard dilution plate method using an inorganic solid medium containing 1% crude oil as a sole source of carbon and energy (Sorkhoh et al. 1990).

Hydrocarbon accumulation potential

The hydrocarbon accumulation potential of picocyanobacteria was measured by incubation of the cells with known amounts of crude oil or pure hydrocarbons in MN medium, after which the residual hydrocarbons were recovered and analysed quantitatively by gas liquid chromatography (GLC).

Aliquots of 0·5 g fresh 15 d cells were suspended in 25 ml aliquots of MN medium containing 10 mg of the test hydrocarbon (crude oil, n-hexadecane or phenanthrene). The cultures were incubated in the dark and under illumination on an electric shaker at 100 rev min−1 at 25°C for 12 h. Cells were recovered by centrifugation at 4500 rev min−1 for 10 min and used for cell lipid and hydrocarbon extraction and analysis. The residual hydrocarbons in the supernatant fluid were quantitatively recovered by three extractions with 10 ml aliquots of diethyl ether and the combined extract was concentrated and made up to 10 ml. Aliquots of 1 μl were injected in a GLC instrument (CP 9000; Chrompack Middle East, Dubai, UAE) equipped with a Wall-coated Open Tubular Column (WCOT) fused silica capillary column and a Flame Ionization Chamber (FID) with a temperature programme of 110–230°C at 10°C min−1. The total peak areas were used to calculate the hydrocarbon concentration.

Cell hydrocarbons and fatty acids

The cells harvested by centrifugation were used for extraction of total lipids, including cell hydrocarbons, by chloroform methanol (Folch et al. 1957). The lipid samples were subjected to methanolysis (Christie 1973) by heating 10 mg under N2 gas with 5 ml 2% H2SO4 in absolute methanol at 90°C for 90 min. An equal volume of water was then added and the mixture of fatty acid methyl esters and total cell hydrocarbons recovered by three extractions with 3-ml aliquots of diethyl ether. The fatty acid methyl esters were separated from the cell hydrocarbons by preparative thin layer chromatography on silicic acid plates, 1-mm thick, using 80 : 20 (v/v) hexane : diethyl ether as the developing solvent (Mangold and Malins 1960). The cell hydrocarbons were analysed by GLC as described above, whereas the fatty acid methyl esters were analysed by a GLC equipped with a WCOT fused silica capillary column, a FID and a temperature programme of 180°C for 2 min, + 5°C min−1 to 190°C, 190°C for 5 min followed by + 10°C min−1 to 225°C.

Electron microscopy

Minute solid medium pieces supporting cyanobacterial growth were removed and the cells fixed with 2·5% glutaraldehyde in N-2-hydroxyethylpiperazine-N-2-ethanol sulfonic acid (HEBES) buffer for 24 h. The cubes were washed three times with HEBES buffer, post fixed with 1% osmium tetraoxide for 1 h and finally washed three times with HEBES buffer. For scanning electron microscopy, the cubes were dehydrated in an ascending concentration series of acetone, starting with 30% and ending with 100% acetone. The dry samples were coated and examined. For transmission electron microscopy, the cubes were dried in a concentration series of ethanol, starting with 50% and ending with 100% ethanol, and embedded in epoxy resin. Thin sections were stained with uranyl acetate and examined.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

Table 1 shows the total numbers of picocyanoplankton recovered from offshore waters at seven stations along the whole Kuwait coast. Counting covered two successive years and was performed in the summer and winter. The numbers ranged roughly between 1 × 105 and 1 × 106 cells ml−1.

Table 1.   Profiles of abundance of picocyanobacteria in the water body along the Kuwait coast Thumbnail image of

A total of 19 strains of picocyanobacteria were isolated from offshore water samples of the different stations. These strains are listed in Table 2 together with some of their main properties. Most dominant was the genus Synechococcus followed by the genera Synechocystis, Pleurocapsa and Dermocarpella. Strains of the first two genera consisted of single cells or aggregates of two to a few cells, whereas the other two genera consisted of colonial forms. All strains grew well on MN medium containing up to 0·1% crude oil (w/v) and survived in the presence of up to 1% crude oil (Table 2).

Table 2.   Properties of 19 strains of picocyanobacteria from the Arabian Gulf Thumbnail image of

The data in Table 3 show that inoculating cells of representative strains of the four genera of picocyanobacteria in mineral media containing either n-hexadecane or phenanthrene led to the attenuation of those compounds, with hydrocarbon attenuation occurring in both light and dark conditions. All attempts to produce axenic cultures were unsuccessful and heterotrophic bacteria could still be detected in picocyanobacterial inocula, with total numbers of 2·4 × 106–4·6 × 106 cells g−1 of the fresh inoculum. All experiments showed that heterotrophic bacteria associated with the test picocyanobacteria (mainly the genera Caulobacter, 54–61%, Acinetobacter, 20–23% and Pseudomonas, 19–23%) could utilize hydrocarbons as sole sources of carbon and energy. These heterotrophs were found to live in close association with the cyanobacterial cells (Fig. 1). In order to limit the role of these heterotrophic bacteria in the experiment we used only 0·2 g fresh picocyanobacteria. This contained only several hundred thousand heterotrophic bacteria and this level of heterotrophic bacteria did not result in any significant hydrocarbon attenuation during the given incubation period (12 h) (Table 3).

Table 3.   Hydrocarbon attenuation in media supplemented with n-hexadecane or phenanthrene Thumbnail image of
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Figure 1.  A scanning electron micrograph showing bacteria in close association with a Synechococcus cell. Pole to pole contact is shown and was frequent in the preparations

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The typical representative GLC profiles demonstrate that pure hydrocarbons (e.g. phenanthrene and crude oil hydrocarbons) added to the medium appeared in the cell hydrocarbon fractions (Fig. 2). The transmission electron micrographs in Fig. 3 show that the interthylakoid spaces of hydrocarbon-treated picocyanobacteria were much larger than those of non-treated cells. Measurements of the total interthylakoid space of phenanthrene-treated Synechococcus 3007 cells showed that this space occupied 45·7% (mean of 10 cells) of the whole section area, whereas the corresponding value for the non-treated cells was only 17·4%.

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Figure 2.  Typical gas liquid chromatography profiles of cell hydrocarbons. (a) Synechococcus 3003 cells incubated without any hydrocarbons added to the medium (lower profile) and with added phenanthrene (middle profile) and crude oil (upper profile). (b) Synechocystis 3009 cells without any added hydrocarbons (lower profile) and with phenanthrene (upper profile) added to the medium

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Figure 3.  Transmission electron micrographs of Synechococcus cells incubated (a) without any hydrocarbons and (b) with crude oil added to the medium. Bars=0·2 μm

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The GLC analysis of the total fatty acids of picocyanobacteria revealed that the fatty acid profiles were not dramatically affected by inclusion of hydrocarbons into the medium. Typical profiles of total fatty acids from Dermocarpella 3016 are shown in Fig. 4 and Table 4 presents the fatty acid composition of total lipids from four test picocyanobacteria incubated with and without pure hydrocarbons. Table 5 presents the fatty acid composition of major lipid classes of Synechococcus 3007 grown in the presence and absence of crude oil. The major saturated fatty acids in all samples were palmitic (16 : 0) and myristic (14 : 0) acids and the major unsaturated acid was palmitoleic acid (16 : 1). Fatty acids with chains shorter than 14 or longer than 18 carbon atoms were not detected.

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Figure 4.  Typical gas liquid chromatography profiles of total fatty acids of Dermocarpella 3016 cells incubated in a medium without any added hydrocarbons (lower profile) and with added n-hexadecane (middle profile) and crude oil (upper profile)

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Table 4.   Constituent fatty acids of total lipids from picocyanobacteria incubated without and with n-hexadecane or phenanthrene Thumbnail image of
Table 5.   Patterns of constituent fatty acids of major lipid classes from cells of Synechococcus 3007 incubated without and with 0·1% crude oil Thumbnail image of

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

The results of this investigation reveal that picocyanobacteria are abundant in the Arabian Gulf water body, ranging in number from 1 × 105 to 1 × 106 cells ml−1. The concentrations of these organisms in the marine ecosystem in other parts of the world range from a few cells ml−1 in some areas to approximately 106 cells ml−1 in others (Waterbury et al. 1986). Thus, the Arabian Gulf is among the water bodies richest in picocyanobacteria worldwide.

It is interesting that all strains isolated could grow well in the presence of up to 0·1% crude oil and survived even in the presence of 1% oil. In this context, the highest concentration of petroleum hydrocarbons recorded for the Arabian Gulf water is only 546 μg ml−1 (about 0·05%) (Marchand et al. 1982; El Samra et al. 1986). However, oil slicks in nature appear to kill picocyanobacteria, which are not as tolerant of crude oil as filamentous cyanobacteria (Al-Hasan et al. 1994; Sorkhoh et al. 1992).

This study offers experimental evidence that cyanopicoplankton in the Arabian Gulf have a potential for the accumulation of hydrocarbons and thus, possibly, contribute to the removal of hydrocarbon pollutants from this water body. Typical hydrocarbon-utilizing bacteria accumulate these compounds as special inclusions in the cytoplasm prior to their catabolism (Radwan and Sorkhoh 1993). Such inclusions have not been observed in electron micrographs of hydrocarbon-incubated picocyanobacteria. Instead, the interthylakoid spaces were found to have enlarged markedly, indicating that they may be the site of hydrocarbon accumulation in these organisms.

Typically, hydrocarbon-utilizing micro-organisms oxidize such substrates, producing the corresponding fatty acids which are then further degraded into acetyl CoA units, ready for production of energy and cell material (Klug and Markovetz 1971; Radwan and Sorkhoh 1993). Some of these fatty acids are usually incorporated into the cell lipids, so that the fatty acid patterns of the cell lipids normally reflect the chain lengths of the hydrocarbon substrate (Klug and Markovetz 1971). However, the analyses in this study revealed that the fatty acid profiles of hydrocarbon- and crude oil-incubated picocyanobacteria did not change markedly from the profiles of the untreated cells. This result implies that, although picocyanobacteria accumulate hydrocarbons from the environment, they do not possess the complement of enzymes to catalyse their mineralization. This leads to an important question about the fate of these accumulated hydrocarbons which, unless biodegraded, will still remain as potential pollutants in the water body. This study does not give a rigorous answer to that question. However, it has been observed that all the strains of picocyanobacteria in culture were associated with hydrocarbon-utilizing bacteria (Fig. 1). Thus, it is probable that this microbial consortium could be efficient in nature in accumulating and biodegrading oil pollutants present in the water body. The precise nature of the interaction among members of this microbial consortium is currently under investigation in our laboratory.

In conclusion, the Arabian Gulf appears to be among the richest water bodies of the world in picocyanobacteria. These micro-organisms accumulate hydrocarbons from the water body, but do not biodegrade these compounds. Associated heterotropic bacteria are possibly involved in the biodegradation of the hydrocarbons.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

This work was supported by the Kuwait Foundation for Advancement of Science, research grant KFAS 950803.

References

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References
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