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Temporal alterations of Nannochloropsis salina (Eustigmatophyceae) grown under aqueous diesel fuel stress. Nagwa Gamal-EI Din Mohammady1,∗.

Journal of Applied Phycology (2005) 17: 161–170 DOI: 10.1007/s10811-005-5510-y

 C Springer 2005

Temporal alterations of Nannochloropsis salina (Eustigmatophyceae) grown under aqueous diesel fuel stress Nagwa Gamal-EI Din Mohammady1,∗ , Yean-Chang Chen2 , Abd-El-Ruhman Aly El-Mahdy3 & Rania Farag Mohammad4 1

Department of Botany, Faculty of Science, Muharram Beck, Alexandria University, Alexandria, Egypt; Department of Aquaculture, National Taiwan Ocean University, Keelung, Taiwan; 3 Department of Botany, Faculty of Agriculture, Nile Valley University, Atbara, Sudan; 4 Department of Biology, Faculty of Science, Omar El-Mokhtar University, El Baidaa, Libya

2



Author for correspondence e-mail: nagwa [email protected]

Received 21 July 2004; revised and accepted 13 October 2004

Key words: cell viability, growth, infrared analysis, light microscopy, mucilage, Nannochloropsis salina, proteome analysis, stress, transmission electron microscopy Abstract The influence of an aqueous extract of diesel fuel was tested on growth of the marine eustigmatophyte Nannochloropsis (Monallantus) salina Hibberd. An increase in the concentration of the pollutant led to a decrease in growth rate as measured by optical density, with maximum effect observed (33% of control) at 100% aqueous pollutant. Spectrophotometric examination of cell viability (using Evan’s blue dye) showed a significant negative effect of the diesel extract ( p ≤ 0.05, r = −0.92). Infrared spectra showed a slight change in the absorbance of contaminated compared with controlled cells. Proteome analysis (sodium dodecyl sulfate polyacrylamide gel electrophoresis – “SDS-PAGE”) indicated that cell protein profiles depended on the pollutant concentration. Some of the resultant bands were characteristic to the pollutant concentration applied, indicating a distinct effect of the pollutant on the proteome structure. Iodine and toluidine blue dyes were applied using light microscopy to detect starch and mucilage, respectively. This indicated the presence of starch during all treatments, while the mucilage has been reduced. Transmission electron microscopy showed alterations to cell walls and membranes with different degrees of plasmolysis leading to a gradual increase in cell volume. However, the nucleus, the nucleolus and the pyrenoid remained unaffected. Similar results were obtained when the alga was cultured for 25 days in the 100% aqueous diesel extract indicating that long-term culture does not affect the degree of pollutant stress. Further, these cells recovered their normal appearance and characteristics within two days of being transferred to culture medium free of extract, indicating that N. salina shows a high tolerance to aqueous diesel fuel pollution. Introduction Nannochloropsis, which is the sole marine genus of the Eustigmatophyceae (Hibberd, 1981), is frequently used in commercial aquaculture due to its nutritional values such as sterols (V´eron et al., 1998) and polyunsaturated fatty acids (Rocha et al., 2003). These fatty acids, especially C20:5ω3, are also the cause for the good growth of young fish fed on this alga (Zittelli et al., 1999). The interest in Nannochloropsis

as a source of valuable pigments is related to the availability of a range of pigments, such as zeaxanthin, canthaxanthin and astaxanthin, each with high production levels (Lubi´an et al., 2000). Diesel fuel is a fraction of crude oil, consisting mostly of linear and branched alkanes with carbon chain lengths of between “C10–C20” (Whyte et al., 1998). Since this fraction is used in motor vehicles, therefore it is one of the causes of seawater pollution. Furthermore, it is common in Arabic coastal waters and

162 has been shown to have impacts on native microorganisms (Piehler et al., 1997). The effect of the petroleum pollution and its derivatives on the marine microalgae is well studied. Nechev et al. (2002) postulated that oil pollutants could penetrate at least partially into the cells, instead of covering only the outer surface of the marine organism. However, Wodzinski and Coyle (1974) showed that microorganisms utilize only water-soluble molecules of the oil that are dissolved in the aqueous phase. The effect of growing microbial cells in a culture medium contaminated by hydrocarbon pollutant, including diesel fuel, accompanied by metabolic and structural alterations of the cell (Hommel, 1990; Piehler et al., 2003), at the molecular and ultrastructural levels (Falkowski, 1992). Other alterations may be detected by specific histological staining (Lobban et al., 1988). Observations of optical properties (Gitelson et al., 2000), growth (Ansari et al., 1997), pigment composition (Gentile & Blanch, 2001), production of exopolysaccharides (Shepherd et al., 1999) and microscopic examination of cells (Soto et al., 1979a,b) will also reveal stress induced alterations. Some recent studies have also attempted to discriminate between controlled and stressed algal cells by spectrometric tools such as resonance Raman spectroscopy (Wu et al., 1998) and fluorescence spectroscopy (Henrion et al., 1997). Infrared spectroscopy is a routine chemical tool for the study of molecular structure of algal cells, but when applied to a large collection of intact cells, the resulting spectra reflect the total biochemical composition of the cells (Kansiz et al., 1999) and can thus be considered as a total and simultaneous chemical analysis. Moreover, proteomic techniques are also helpful in studying responses to environmental stress as well as to improve our understanding of mechanisms that control many physiological processes (Diez et al., 2001; Yu et al., 2004). Previous studies have been described Nannochloropsis as a tolerant species to different stress conditions, which was obtained by Lubi´an (1982a,b) concluded that Nannochloropsis gaditana is characterized by a hard cell wall. It has a high resistance to drastic treatments (Moreno-Garrido et al., 1998). A similar conclusion was shown by Gonen-Zurgil et al. (1996) in an examination of the herbicidal compound DCMU on Nannochloropsis sp. The aim of this study was to consider the possible alterations of Nannochloropsis salina as an important component of the nutritional food chain of the marine habitat, when cultivated under stress of aqueous

diesel fuel pollutant. Various features were chosen to assess the effects, including growth rate, cell viability, infrared spectra, proteome structure and microscopy observations. Materials and methods Source and maintenance of culture Culture material of Nannochloropsis (Monallantus) salina Hibberd was obtained from the Mariculture Center in Eilat, Israel, originating from the Solar Energy Research Institute (SERI) Culture Collection in Golden, Colorado, USA. The alga was grown axenically in Boussiba’s Enriched Seawater (BES, Boussiba et al., 1987). Preparation of aqueous extract of diesel fuel The stock pollutant of contaminated culture medium was prepared following the method of Boylan & Tripp (1971). One litre of BES was shaken overnight with 50 mL diesel fuel and the aqueous fraction was then recovered using a separating funnel. Dilutions of this aqueous fraction were prepared with BES (control) to form polluted culture medium, using 25, 50, 75 and 100% levels of contamination. Culture conditions An Innoculum of 106 logarithmic-phase cells per millilitre was added to 100 mL of contaminated BES medium (and to BES as a control) in triplicate 500-mL flasks equipped with inlet and outlet tubes for aeration in a temperature controlled room at 25 ± 1 ◦ C. Cultures were continuously agitated by bubbling with sterile air (by passing through sterilized Gamma 12 filter units to remove contaminants), which was also enriched with 0.5% CO2 . Illumination was provided by fluorescent lamps with an irradiance of 300 µmol m−2 s−1 at the surface of the cultures under a 16 h/8 h light/dark regime. At the mid logarithmic growth phase (day) 8, the cells were harvested by centrifugation at 1000 × g for 5 min. Growth measurement Growth was monitored spectrophotometrically as the optical density of the cell suspension at 560 nm (Wetherell, 1961) at the mid of the logarithmic growth

163 phase (at eighth day). The effect of the different concentrations of the pollutant on growth was expressed as percentage of control culture and the standard error of the mean of three replicates was obtained. Cell viability The cell viability (physiological state) was examined after the methods of Crippen & Perrier (1974) and Gallagher (1984). One millilitre of a 1% (w/v) stock solution of Evan’s blue dye was added, before centrifugation, to 20 ml culture to yield a final concentration of 5 × 10−7 g stain per ml seawater. The dye stains the dead parts of the cells blue, including exopolysaccharide materials outside the cell wall. Measurements of cell viability were then made as absorbance at 468 nm (Franklin et al., 2001) using a Perkin Elmer spectrophotometer (Lambadal). According to Agarwal (1988) the absorbance data were subjected to standard one way analysis of variance (ANOVA), using COSTAT 2.0 statistical analysis software. Means were tested with least square difference (LSD), where the difference of p ≤ 0.05 was significant. Least squares linear regression (r) analysis was also applied. Infrared (IR) spectra Different biochemical profiles of the cell were scanned using Perkin Elmer 1430 ratio recording infrared spectroscopy according to Kansiz et al. (1999). Preliminary trials were performed to determine the density of cells in suspension necessary to produce spectra with a good signal. Lugol’s iodine solution was added to a known weight of algal pellet. The dried cells were used for spectral acquisition and the absorbance spectra were collected between 3650 and 700 cm−1 as the mean of 10 sequential scans. Protein analysis Following the method of Laemmli (1970), cultures were centrifuged and the media aspirated, each pellet was resuspended in 5× sample buffer and denatured for 5 min in a boiling water bath. Proteins were separated on 10% of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and 5% of the stacking gel. Samples were applied to the slab gel along with molecular weight markers (BioRad, prestained-SDS markers). The gels were run at 150 V for about 2 h and was then stained with Coomassie Brilliant Blue R-250 (0.06% Coomassie Brilliant Blue R-250 in 50%

methanol, 10% acetic acid) for 1 h with gentle agitation at room temperature. The gel was destained using a destain solution (60 ml methanol, 40 ml acetic and 800 ml distilled water). the resultant protein profiles were compared using the similarity coefficient of Czekanowski (1913). Microscopic examinations Light microscopy (LM): To determine changes in some aspects of the biochemical composition of N. salina, including starch and crude mucilage, two separate samples of the algal cells were centrifuged and stained individually using iodine solution (KI/I2 , 15%, w/v) to detect starch (Lobban et al., 1988) and Toluidine blue dye to detect crude mucilage (0.5% (w/v), Shepherd et al., 1999). Transmission electron microscopy (TEM): The algal cells were prepared from the centrifuged cultures according to Chen (2003). Cells were collected in 15 ml centrifuge tubes, followed by separate pre-fixation in modified BES medium with 2% glutaraldehyde at 4 ◦ C for 2 h, and then fixed in a 0.1 M sucrose solution containing 4% glutaraldehyde and 0.1 M sodium cacodylate buffer (pH 7.0) at 4 ◦ C for 4 h. They were then rinsed twice with a 0.1 M sodium cacodylate buffer containing 10 mM CaCl2 , and the sucrose concentration was successively reduced to 0.05 M. This was followed by two rinses in sucrose free 0.1 M sodium cacodylate buffer containing 10 mM CaCl2 . Post-fixation was performed with 2% OsO4 in 0.1 M sodium cacodylate buffer containing 10 mM CaCl2 for 1 h at 4 ◦ C. All materials were then rinsed four times with a sodium cacodylate buffer containing 10 mM CaCl2 , three times with aqueous ethanol (50%) and then serially dehydrated in ethanol to absolute. The dehydrated material was rinsed in propylene oxide (three times, 30 min each), followed by infiltration in propylene oxide-Spurr’s resin in a decreasing ratio from 2:1 (2 parts propylene oxide:1 part Spurr’s resin) to 1:1 each for 4 h. Samples were then suspended in pure Spurr’s resin for two days at 4 ◦ C in darkness before embedding in Spurr’s resin (Spurr, 1969). The thin sections were stained according to Smith and Croft (1991).

Results Assessment of the effect of aqueous diesel fuel on N. salina indicated a decrease in growth, with respect to the control in the cultures exposed to all applied

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Figure 1. Effect of aqueous diesel fuel extract concentration on growth of N. salina (expressed as fraction of control culture). Standard error = ±0.2; n = 3. Figure 3. Infrared spectra of N. salina growing under control and aqueous diesel fuel extract concentration.

Figure 2. Cell viability and the regression line of N. salina growing under control and aqueous diesel fuel extract concentration.

concentrations at the eighth day. The growth of the alga, as regarded by the optical density, was affected by the application of the different concentrations of the pollutant as shown in Figure 1. The inhibition increased by increasing the concentration of the pollutant since it reached the maximum value of 33% at 100% aqueous diesel concentration. The effect of aqueous diesel fuel extract on the viability of cells (Figure 2) revealed a significant deleterious effect of the pollutant ( p ≤ 0.05) on the algal cells, while the linear regression (r ) = −0.92 indicating that cell viability is inversely proportional with pollutant concentration. Infrared (IR) spectra Infrared spectra (IR) of the algal cells growing under different concentrations of aqueous diesel extract show the recognized band assignments over the bandwidth of

3650–700 cm−1 , and differences between the biochemical profiles of control and treated cells are identifiable (Figure 3). Spectral regions that correspond to the biochemical composition of the cells are common to all treatments. The bandwidth of 1200–900 cm−1 represents absorption from the C–O–C stretching vibrations of polysaccharides, together with the symmetric and asymmetric stretching vibrations of PO− 2 functional groups at a frequency of about 1080 cm−1 . There is also a distinct absorption peak at approximately 1740 cm−1 which arises from the stretching vibration of the ester C O group of lipids and fatty acids. Identification of the major features of the spectra obtained from the culture alga are shown in Table 1. The SDS-PAGE electrophoresis of soluble protein (Figure 4) revealed protein bands with molecular weights between >6.4 and 198 kDa. Twelve protein bands appeared in controlled cells followed by 5, 8, 10 and 11 protein bands in treated cells, respectively. Some bands were common to all treatments, while other bands were found either only in cells from the control cultures or only in cells from the polluted cultures. The similarity matrices between the protein profiles (Table 2) obtained from cells grown in the control and in the 25% diesel extract cultures was 0.58. The addition of the pollutant to the culture resulted in the loss of all the low molecular weight protein bands, without the addition of any new bands. The highest similarity (0.70) was observed between the protein profiles of cells cultured in 50 and 75% diesel extract. In the latter two treatments new protein bands

165 Table 1. IR analysis of cell composition of N. salina growing under control and four aqueous diesel fuel extract concentrations (list of band assignment). Frequency (cm−1 )

Assignment

Comments

∼2929 ∼2850 ∼1740

ν as C H of methylene groups (Nelson, 1991) ν s C H of methylene groups (Nelson, 1991) νC O of ester functional groups primarily from lipids and fatty acids (Hedrick et al., 1991; Williams & Fleming, 1996) νC O of amides associated with proteins (Nelson, 1991; Williams & Fleming, 1996)

– –

∼1650



Usually called amide I band, may also contain contribution from C C of olefinic and aromatic compounds

∼1540

δN H of amides associated with proteins (Nelson, 1991; Williams & Fleming, 1996)

Usually called amide II band, may also contain contributions from C N

∼1455

δ as CH3 and δ as CH2 of proteins (Kansiz et al., 1999) δ s CH3 and δ s CH2 of proteins and vs C O of COO-groups (Nelson, 1991) ν s P O of the phosphodiester backbone of nucleic acids (DNA and RNA) (Nelson, 1991; Wong et al., 1991)

Positions of these assignments vary in the literature Positions of these assignments vary in the literature

νC O C of polysaccharides (Wong et al., 1991)

Predominant polysaccharide in Eustigmatophyceae is glucan

∼1398 ∼1080 ∼1200 900

May be due to presence of phosphorylated proteins and polyphosphate storage

νas : Asymmetric stretch. νs : Symmetric stretch. δas : Asymmetric deformation (band). δs : Symmetric deformation (band).

Table 2. Influence of aqueous diesel fuel extract concentration on similarity matrix of protein profile of N. salina. Aqueous diesel fuel extract concentration

Control 25% 50% 75% 100% s

Control

25%

50%

75%

100%

1.0 0.58 0.26 0.34 0.41

1.0 0.15 0.26 0.50

1.0 0.70 0.44

1.0 0.47

1.0

Light microscopy Figure 4. Electrophoretic analysis (SDS-PAGE) of N. salina growing under control and aqueous diesel fuel extract concentration.

appeared, most of them with low molecular weights, which were not present in either the control or the 25% extract profiles. But, in both of treatments, the protein bands that were lost were of high molecular weight. However, the protein profile of cells grown in 100% diesel extract was markedly different. Additional high molecular weight protein bands were observed together with the disappearance of most of the low molecular weight bands that were present in the cells from lower concentration cultures.

The results of the histological approach to the detection of pollution stress related effects to the cultured cells are shown in Figures 5: 1–5. Iodine staining showed the characteristic blue color indicated the presence of starch within cells from all the cultures (Figures 5: 1B, 2A, 3– 5). Toluidine blue stained a layer of exopolysaccharides on cells from the control cultures (Figure 5: 1C) but the thickness of the layer appeared reduced in cells from the polluted culture (Figure 5: 2B). Transmission electron microscopy N. salina is a unicellular coccoid with a multilayered cell wall, which is covered by an irregular network of

166 subtle ribs. The cell has a large anterior nucleus and a single large parietal chloroplast. Thylakoids are found in groups of three, but there are no girdle lamellae. The stalked pyrenoid is a compound internal type, containing two outside sheaths. A set of thylakoids run penetrating the core of pyrenoid. Fat granules were observed inside the plastid. The eye spot (eustigma) is outside the plastid and has a characteristic basal swelling. In the polluted cultures, cell volume and the percentage of plasmolysed cells increased with increasing proportions of diesel extract. In 25% extract Figures 6: 1A–1C, a cell volume increased by about 25% with few plasmolysed cells but in the 50% (Figures 6: 2A–2B) and 75% (Figures 6: 3A–3B) cultures there was an increased irregularity of the cell walls and plasmalemma, however, at 75% many cells showed apparent plasmolysis. Cells cultured in 100% extract (Figures 6: 4A– 4B) were double the volume of the control cells and most showed full plasmolysis. The nucleus, nucleolus and pyrenoid remained intact (although at the highest concentrations indistinct due to the contraction of the cytoplasm) in all the polluted cultures. There was some evidence of disruption to the lipid membranes of the chloroplast at lower concentrations but in the 100% culture, the thylakoids appeared less compact, despite the extent of the apparent plasmolysis. Cells that were cultured for 25 days in the 100% extract (Figures 6: 5A–5B) showed the same degree of distortion as those cultured only until they were in exponential phase. However, these cells recovered their normal appearance and characteristics within two days of being transferred to a diesel-extract-free culture medium (Figures 6: 6A–6B).

Discussion Figure 5. LM of N. salina growing under various concentrations of diesel extract: (1) control, (2) 25%, (3) 50%, (4) 75%, (5) 100%. (1A) Without stain. Scale bar: 10 µm. (1B) Stained with iodine. Scale bar: 8 µm. (1C) Stained with toluidine blue. Scale bar: 10 µm. (2A) Stained with iodine. Scale bar: 8 µm. (2B) Stained with toluidine blue. Scale bar: 8 µm. Note the disappearance of mucilage). (3–5) All stained with iodine and scale bar: 8 µm.

The effect of growing N. salina in a medium contaminated by aqueous diesel fuel was temporary, although a reduction effect of the pollutant on the algal growth and vitality was obviously shown. The reduction of growth by diesel has been considered in many other strains

−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−→

Figure 6. Ultrastructure of N. salina growing under various concentrations of diesel extract: (1) 25%, (2) 50%, (3) 75%, (4) 100%. (Note that the numbering for the various concentrations is not the same as in Figure 5.) (1A) CW: cell wall, C: chloroplast, F: fat granules. Scale bar: 0.33 µm. (1B) The chloroplast with stalked pyrenoid (P), T: thylakoids, scale bar: 0.2 µm. (1C) Nucleus (N) and nucleolus (nu). Scale bar: 0.2 µm. (2A) Scale bar 0.33 µm; (2B) nucleus and nucleolus, scale bar: 0.33 µm. (3A) Scale bar 0.33 µm. (3B) The chloroplast with stalked pyrenoid. Scale bar: 0.2 µm. (4A) S: empty space. PL: plasmalemma. Scale bar: 0.5 µm. (4B) The chloroplast with stalked pyrenoid, scale bar: 0.25 µm. Ultrastructure after long-term culture (25 days) under 100% aqueous diesel fuel extract concentration: (5A) scale bar: 0.66 µm. (5B) The chloroplast with stalked pyrenoid, scale bar: 0.66 µm. Ultrastructures of recovered N. salina after transfer to diese-free medium: (6A) resting cell. Scale bar: 0.4 µm. (6B) Dividing cell. Scale bar: 0.5 µm.

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168 such as Scenedesmus quadricauda (Dennington et al., 1975) and Isochrysis sp. (Ansari et al., 1997). As demonstrated by Piehler et al. (2003), several days’ exposure to weathered diesel fuel provided information about effects on microalgae that may result from spills through possibly cell divisions. However, Lai and Khanna (1996) and Tongpim and Pickard (1996) suggested the reduction of growth of Acinetobacter calcoaceticus and Rhodococcus sp. by diesel fuel metabolic intermediates octadecane and anthracene was attributed to strain’s decrease bioavailability, the results which completely confirmed with us since a negative significant effect of the pollutant on the cell viability, using Evan’s blue dye, was obviously shown. IR analysis revealed the safety of the essential biochemical composition (between 900 and 1650 cm−1 ) of the treated cells, however, a slight raising of the absorbance spectra were observed in contaminated cells compared with those of control. As demonstrated by Kansiz et al. (1999), the raise of the absorbance spectra about the normalized one has been correlated with increasing in the absorption in the C–H stretching due to the long carbon chains containing relatively large numbers of CH2 groups. So upon comparison, the variability between the absorbance spectra between different cultures is directly proportional to the pollutant concentration and this overall increase of the absorbance spectra of the polluted cells is correlated to the increase of CH2 groups containing molecules of the pollutant. In this investigation, a distinct effect of the pollutant on the banding pattern of the soluble proteins was observed, since each protein profile was characteristic of the extract concentration used in the culture. Some bands were created during the treatments and others disappeared. However, some bands lost and then seemed to come back (as with ones at about 44, 100 and 150 kDa). The newly synthesized proteins may help cells to defend themselves against stress conditions (Chrisman et al., 1985) such as those having molecular weights ranging between 42.18 and 49.47 kDa which were recorded to act as protein receptors to enhance the cell division-machinery and operating the mechanism of gene regulation efficiently resulting in production of certain types of functional proteins having the potentiality to overcome the inimical effects of various pollutants and resume the normal growth (Hieks et al., 1989). These new protein bands with this molecular weights, in our results, were detected within the contaminated cells with 50, 75, and 100% aqueous diesel.

However, Mullar and Gottschalk (1973) found the disappearance of some protein bands could be attributed to the occurrence of mutational events or defensive mechanisms that protect cells from external stress. The disappearance or reappearance of some bands after stress treatment could be attributed to gene recombination produced by the induction of chromosome exchanges (El-Ghamery et al., 2002). In the present work, aqueous diesel pollution induce alteration in the proteome structure of N. salina and may exert its effects through a specialized general mechanism, even if the inhibition of the expression of some proteins might be involved through a specific effect on the metabolism. The light microscopy revealed a positive result of iodine test in all contaminated cells indicating a permanent production of starch and hence the viability of pyrenoid. However, toluidine blue test indicated the disappearance of exopolysaccharides in the polluted cells of N. salina. As demonstrated by Baldi et al. (1999), exopolysaccharides can be dissolved in diesel fuel containing alkanes. Our ultrastructure study showed a significant effect of aqueous diesel pollution on the cell wall and membranes of the investigated alga with various levels of plasmolysis which increased with increasing pollutant concentration, although the nucleus, the nucleolus and the pyrenoid remained intact. The same results were obtained when the alga was cultivated for a long period in the aqueous extract of diesel (25 days), indicating that culture time does not affect the degree of the pollutant stress. However, the algal cells regained their normal shape and vitality within two days when re-cultured in pollution free medium, indicating that N. salina shows a high tolerance to diesel fuel pollution. This result demonstrated a deplasmolysis of N. salina since the microalga was observed in both resting and dividing states. This finding proved that aqueous diesel fuel pollution is less toxic to N. salina, only affects the cell membranes. That is, the microalga is still alive when it suffered from strong pollutant stress. The hydrophobicity of diesel causes dissolution and disruption of lipid containing structures of algal cells (Krauss & Hutchinson, 1975) such as the disruption of membranes; membranes of nuclei, mitochondria and chloroplast (Reynolds, 1987). Some microalgae have been affected by diesel treatments such as the chlorophyte Scenedesmus species (Tukaj & Bohdanowicz, 1995), the dinophyte Ceratium sp. (Hutchinson, 1967), chrysophytes (Sandgren, 1988), cyanobacteria and diatoms (Trainor et al., 1971). However, our results appeared to be similar to

169 those obtained by Soto et al. (1979a,b) when they examined the effect of crude oil and naphthalene on the green alga Chlamydomonas angulosa, since phenomena that resemble plasmolysis was observed. Cells of C. angulosa increased in size, with an increase in the dimension of the cell wall and the formation of a wide space between the cell wall and the plasmalemma (the protoplast decreasing in size). The cells treated with the crude oil recovered from the induced abnormalities within three days. However, no recovery of naphthalene treated cells was noticed but since naphthalene represents one of the most toxic components of crude oil this was not unexpected. The recovery of diesel fuel pollution has been demonstrated by Piehler et al. (2003), who found the amount of chlorophyll a produced by five-day recovery of Anabaena sp. and some pinnate diatoms from diesel fuel stress, is the same as the produced by control. The result indicated the resilient of the examined species in the face of diesel fuel stress. In conclusion, Nannochloropsis salina shows a high tolerance to pollution by aqueous diesel fuel extract. The effect of pollution seemed to be similar to an osmotic shock, as only the cell wall and membranes were affected. When the microalga was allowed to be recovered from the pollutant, it regained its normal conformation and vitality. It is suggested that the microalga has a defence mechanism that can protect the cells against aqueous diesel stress and this mechanism was expressed by the appearance or disappearance of the unique protein bands during the experimental culture. Acknowledgments The first author is grateful to colleagues in the Central Laboratory of the Faculty of Science Alexandria University, for the infrared analysis and microscopic examinations. Many thanks to Dr. Y. Gohar, professor of microbiology, for his advice and proteome analysis in his lab. Also to Dr. S. El Darer, professor of ecology, for his help with the statistical analysis. Special thanks to Dr. Daryl Anne Birkett for her suggestions and linguistic help. References Agarwal BL (1988) Basic Statistics. Wiley Eastern Limited, New Delhi, 748 pp. Ansari ZA, Saldanha MC, Rajkumar R (1997) Effects of petroleum hydrocarbons on the growth of microalga Iosocrysis sp. (Chrysophyta). Indian J. Mar. Sci. 26: 372–376.

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