Selection of oil-tolerant cyanobacteria on oil polluted sediments

eschweizerbartxxx author

Selection of oil-tolerant cyanobacteria on oil polluted sediments

Raeid M. M. abed1,* and Stjepko Golubic2

1Sultan Qaboos University, College of Science, Biology Department, Muscat, Sultanate of Oman2 Biological Science Center, Boston University, Boston, MA, USA

With 3 figures and 1 table

Abstract: An experimental approach was used to explore the identity of oil-tolerant cyanobacteria combining direct microscopy and Denaturing Gradient Gel Elec-trophoresis (DGGE). Microbial mat inhabiting cyanobacteria were grown over sediment in an open mesocosm setting (aquarium). Cultures were started from a mixed inoculum of mats from three different geographic locations, exposed to con-ditions imitating oil spill, with an oil-free control. The analyses by microscopy and denaturing gradient gel electrophoresis were performed at the start of the experi-ment and after 2, 4 and 8 months of growth. Oil contamination resulted in devel-opment of a compact cohesive mat, different from the fluffy, loosely attached mat developed over the oil-free sediment. Both, direct microscopy and DGGE, docu-mented a significant shift in composition of cyanobacterial community over time, favouring morphotypes with narrow trichomes; however, the change in oil-free and oil-contaminated cultures took different directions. The relatively large Oscillato-ria-like cyanobacteria of the initial phase of the mat development yielded Lep-tolyngbya-like morphotypes, with bands that clustered with Halomicronema excen-tricum and a sequence identified as Leptolyngbya sp. respectively, which persisted over 8 months of incubation. The oil-exposed mats were dominated by organisms from the same Halomicronema excentricum cluster, which shared the dominance with two different phylotypes, one related to Phormidium minutum D5 sequence, and another one close to Phormidium ambiguum M71. After 8 months, two cyano-bacteria phylogenetically close to Halomicronmena excentricum and Phormidium minutum overtook all others. DGGE showed clearly the replacement of all original community members as well as changes in the composition of cyanobacterial com-munity over time in both oil-exposed and oil-free settings. The results indicate that thin filamentous cyanobacteria contain a group of opportunistic microorganisms that tend to prevail after perturbations in ecosystems. This group may include oil-tolerant cyanobacteria that dominate the mats immediately after oil spill events.

Key words: cyanobacteria, microbial mats, oil pollution, PCR-DGGE, phylogeny

* corresponding author

Algological Studies 130 69–79 Stuttgart, October 2009

DOI: 10.1127/1864-1318/2009/0130-0069 1864-1318/09/0130-069 $ 2.75

© 2009 E. Schweizerbart’sche Verlagsbuchhandlung, D-70176 Stuttgart


70 R. M. M. abed and S. Golubic

Introduction

Cyanobacterial mats are dense, vertically structured associations of different groups of aerobic and anaerobic microorganisms with cyanobacteria as the main primary producers (van Gemerden 1993). They have been observed to colonize oil-polluted sediments and their growth was even stimulated af-ter oil spills (Hoffmann 1996, Höpner et al. 1996). Laboratory experiments (slurries and intact mats) demonstrated the potential of mat microbial com-munities to degrade various aliphatic and aromatic petroleum compounds (abed et al. 2002, GrötzscHel et al. 2002). Although it has been shown that cyanobacteria within these communities lack the potential to degrade oil compounds (abed et al. 2002, radwan et al. 2002), they did support oil bio-degradation by providing oil-degrading bacteria with required oxygen, fixed nitrogen and organics (abed & Köster 2005, musat et al. 2006).

Crude oils have different compositions, but irrespective of their origin, they contain a number of fundamental chemical classes namely, saturated hydrocarbons, aromatic hydrocarbons, asphaltenes and resins (Hunt 1979, tissot & welte 1984). Upon oil spills, the toxicity of some of these com-pounds to certain bacterial populations leads to a significant loss of the original microbial community. In contrast, the growth of oil-degrading bac-teria is stimulated due to the direct utilization of aliphatic and aromatic hy-drocarbons. Other bacterial populations can tolerate oil pollution although they are not directly involved in oil biodegradation. Although the support-ing role of cyanobacteria in oil polluted sediments has been postulated, it is still unclear why cyanobacteria colonize and dominate oil polluted ecosys-tems and what is the identity of oil-tolerant cyanobacterial types.

In this study, we followed changes in cyanobacterial communities in oil-polluted sediments with the aim to identify oil-tolerant types. Oil spill was simulated in glass aquaria filled with marine sediments and inoculated with microbial mat suspensions. Oil-tolerant cyanobaceria were identified by comparing cyanobacterial composition in oil and oil-free sediments using direct microscopy and Denaturing Gradient Gel Electrophoresis (DGGE).

Materials and methods

Experimental setting. A detailed description of the method used to estab-lish the experimentally oil-polluted sediment used in this study was given by musat et al. (2004). Briefly, two glass aquaria were maintained (38 x 23 x 14 cm), one subjected to oil contamination, and the other kept oil-free as control. Both aquaria were filled with 4 cm thick seawater-saturated pristine sediment collected freshly from the tidal area of the Wadden Sea (Horumersiel, Wilhelmshaven, Germany). In the experimental aquarium this sediment layer was covered with oil-sediment slurry, while the control


Oil-tolerant cyanobacteria 71

aquarium was kept oil-free. The oil-sediment slurry was prepared by soak-ing dried and homogenized sediment in Maya crude oil (ca. 0.2 ml oil per cm2 sediment) and leaving the mixture under a fume hood for four hours to allow the formation of viscous slurry. Both aquaria were then inoculated with equal quantities of a mixture of oxic and anoxic cyanobacterial mats collected from one polluted (Etang de Berre, France) and two pristine har-bours (L’Ampolla, Spain and Horumersiel, near Wilhelmshaven, Germany). The sediments were carefully covered with seawater (3.5 % salinity) so that floating of oil or inoculum was avoided. The aquaria were incubated in a greenhouse and illuminated using mercury lamps (200 W, Osram) under a 12 h light/12 h dark regime (light intensity was ca. 1,000 µE m–2 s–1) and the temperature was controlled (ca. 30 °C) using a cooling unit.Microscopic analysis. The upper mat layer was cut with a sterile blade and suspended in sterile medium. An aliquot of this suspension was filtered through 0.2 µm GTTP filter (Millipore), to achieve uniform distribution of the cyanobacterial filaments. Counting and size measurements were per-formed under a fluorescent and phase contrast microscope considering only the focused autofluorescent cyanobacteria. For the purpose of quan-tification, cyanobacteria were distinguished in three groups as unicellular, thick- filamentous and thin-filamentous.Molecular analysis. Three mat cores (ca. 300–500 mg each) from each aquarium at separate points were taken for nucleic acid extraction. The DNA extracts from the three samples were pooled together and subjected to polymerase chain reaction (PCR) and Denaturing Gradient Gel Elec-trophoresis (DGGE) as previously described (abed et al. 2001). PCR for the amplification of 16S rRNA gene was carried out using the cyanobacte-ria-specific primers CYA359F (with 40 nucleotide GC clamp at the 5' end) and CYA781R. A hot start program was performed as described by nübel et al. (1997). DGGE was run at 60 °C and at a constant voltage of 200 V for 3.5 hours (abed et al. 2001). The DGGE bands were excised manually, the DNA let to diffuse out in buffer overnight, and PCR re-amplified. Products were then sequenced.Phylogenetic reconstruction. Cyanobacterial phylogenetic trees were con-structed based on long 16S rRNA sequences (more than 1,300 bp) by ap-plying different methods integrated in the ARB software (ludwiG et al. 1998) such as maximum likelihood, maximum parsimony and neighbour joining (a maximum likelihood tree is presented in Fig. 3). The 16S rRNA gene sequences of Escherichia coli and Bacillus subtilis were included in the calculations as outgroup sequences. Cyanobacterial partial sequences (ca. 400 bp) obtained in this study were aligned to the sequences in the ARB database using the alignment tool of the ARB software package. These sequences were then inserted into the pre-established tree using the parsimony ARB tool and maintaining the overall tree topology without changes.



ResultsMacro- and microscopic differences between oil-free and oil-polluted matsClear textural differences between the oil and the oil-free sediments were no-ticed after four weeks of the experiment. In both aquaria, a green-brownish layer of ca. 1-2 mm was developed due to the growth of cyanobacteria. This layer was quite loose and fluffy in case of the pristine sediment while it ad-hered firmly on the top of the oil layer in case of the polluted sediment. The production of extrapolymeric substances (EPS) was notably more expressed in oil-contaminated sediments, which rendered the surface of the sediment a gelatinous appearance. Microscopic examination revealed the presence of 10 different cyanobacterial morphotypes that belonged to thin filamentous, thick filamentous and unicellular cyanobacteria (Fig. 1). Leptolyngbya-type thin filamentous morphotypes (A-C) tended to dominate the oil-polluted mats. Four identified unicellular cyanobacterial morphotypes (D-G) showed a tendency to form dense clusters in pristine mat, but form loose aggrega-tions in the oil polluted mat. The relatively large Oscillatoria-like morphot-ypes (H-J) dominated in the oil-free mat, especially in the initial stages of the experiment and were rare in the oil-polluted mat (Table 1).Comparison of cyanobacterial communities by DGGE analysis. The in-oculum used at the beginning of the experiment displayed five dominant DGGE bands using cyanobacteria-specific primers. Their sequences were

A B C

D E F G

H I J

Fig. 1. Photomicrographs of cyanobacteria observed in the oil-free and oil-con-taminated microbial mats: A–C – filamentous morphotypes with narrow trichomes, D–G – coccoid morphotypes, H–J – filamentous morphotypes with wide trichomes. The letter designations are the same as in Table 1. Scale bar is 10 µm for all pic-tures.



affiliated to Oscillatoria terebriformis, Spirulina sp. and to the diatom Skel-etonema pseudocostatum, whose 16S rRNA sequence was also amplified by the same primers. This community underwent dramatic shifts in its compo-sition already after 2 months of incubation but in different directions in the oil-free and the oil-polluted sediments. In the oil-free sediment, all original community members were replaced by three new populations that were phylogenetically closely affiliated to the recently described moderately

Table 1: Distribution of cyanobacterial morphotypes encountered in the oil-free and oil contaminated microbial mats, as determined by light and epifluorescence microscopy (see Fig. 1)Morphotype Description cell width (µm) oil mat control matLeptolyngbya -likeMorphotype A filamentous 1 +++ ++Morphotype B filamentous 1,5-2 +++ +Morphotype C filamentous 2-4 ++ +Unicellular Morphotype D coccoid cell, no clusters 1.5-4 ++ -Morphotype E coccoid cells in clusters 1.5-4 - +++Morphotype F rod-shaped 1.5-5 - ++Morphotype G coccoid cells, no clusters 2-4 ++ -Oscillatoria -likeMorphotype H filamentous 8-10 + ++Morphotype I filamentous 10-12 + +Morphotype J filamentous 12-16 - +++++ dominant, ++ subdominant, + rare, - absent

With oilWithout oilC0 C2 C5 C8 C0 O2 O5 O8

1

3

2

4

5

1

2

3

1 1

223

4

1

2

Fig. 2. DGGE banding patterns of PCR-amplified 16S rRNA fragments obtained from averaged triplicate samples of the oil-free (C) and the oil-polluted (O) mats at the begin-ning of the experiment (0) and after 2, 5 and 8 months. Letter-number combinations identify the columns, the numbers in the gel strips identify the bands in each column.



halotolerant, moderately thermotolerant Halomicronema excentricum (abed et al. 2002). After 5 months of incubation, a cyanobacterium, whose 16S rRNA was phylogenetically close to a sequence marked as Leptolyn-gbya sp. appeared in addition to Halomicronema excentricum. These two cyanobacteria persisted to dominate the oil-free mats even after one and half year (data not shown).

Marinepicoplankton

C8-Band 2C2-Band 2O2-band 3

C2-band 1C2-band 3

Halomicronema excentricumO8 band-1

Phormidium “ectocarpi” PCC 7375O2-band 1

Phormidium minutum D5Oscillatoria neglecta M-82Oscillatoria sp.

LPP-group cyanobacterium QSSC8cya Synechococcus sp. PCC 7335

Plectonema sp. F3C8-band 1

Leptolyngbya sp.Oscillatoria sp.

Heterocystous cyanobacteria

O2-band 4 Spirulina sp.

Spirulina subsalsaHalospirulina sp.

Halospirulina tapeticolaCyanothece PCC741

Euhalothece 'MPI 95AH13Dactylococcopsis PCC830

Halothece 'MPI 96P605Oscillatoria terebriformis OH-93-ot

C0-band 3C0-band 4

Lyngbya sp.Symploca sp. VP377

Microcoleus PCC 7420“Oscillatoria corallinae” CJ1 SAG8.92

Spirulina speciesC0-band 5

Synechocystis sp.Oscillatoria roseSynechococcus PCC700

Tichodesmium cluster

Oscillatoria limneticaPseudanabaenas

Phormidium sp.Pseudanabaena sp. PCC 7367

Phormidium mucicola M-221O2-band 2

Phormidium ambiguum M-71C0-band 2

C0-band 1Skeletonema pseudocostatum

Emiliania huxleyiZea mays

Bacillus subtilis Escherichia coli

10%

Fig. 3. Phylogenetic affiliation of the cyanobacteria and plastids (maximum likeli-hood) obtained from experimental oil-contaminated and oil-free mats in relation to the publicly available, almost complete display of 16S rRNA cyanobacterial genes, using E. coli and B. subtilis as outgroups. The next phylogenetic placement of 400 bp long 16S rRNA sequences from our excised DGGE bands (Fig. 2) was carried out using parsimony criteria without changing the topology of the pre-es-tablished tree using the tool in the ARB software. The bar indicates 10% sequence divergence.



After two months of exposure to oil pollution, the cyanobacterial com-munity shifted in favour of four different populations as inferred from the DGGE pattern. Three cyanobacterial populations were phylogenetically related to the thin filamentous cyanobacteria: Halomicronema excentricum, Phormidium minutum and Phormidium ambiguum, whereas one popu-lation was close to Spirulina sp. After 8 months of incubation, the above organisms were outcompeted by a different population that was also phy-logenetically related to Halomicronema excentricum. The DGGE bands representing diatoms persisted in the oil-polluted sediment but not in the oil-free sediment.

Discussion

Why cyanobacteria colonize and dominate polluted sediments?

Our results are consistent with the observations made after the Gulf War in 1991 that oil-contamination promotes the growth of cyanobacteria and the development of compact microbial mats. The oil-free sediment used in our experiment, following 2 months of incubation supported a loose mat, whereas the cyanobacteria in the oil-rich sediment formed a dense mat. The viscous oil apparently solidified initially the loose sediment and provided a favourable substratum for the attachment and growth of cyanobacteria. Furthermore, the remarkable excessive production of cyanobacterial exo-polymeric substances (EPS), in response to oil, contributed to the stabiliza-tion of the sediment. EPS production by phototrophs was shown to be stim-ulated under environmental stress (liu & busKey 2000, abdullaHi et al. 2006). It has also been shown that cyanobacterial exopolymeric substances facilitate oil degradation by contributing significantly to the emulsification of oil (boucHotrocH et al. 2000, iwabucHi et al. 2002). A possible conse-quence is a reduction of oil toxicity and an increase in the accessibility of its components for bacterial biodegradation. The stimulation of cyanobac-terial growth in oil-polluted sites might also be triggered by the increased activity of oil-degrading aerobic heterotrophs in the top mat layer, which provide nutrients and locally release CO2, which may be immediately used for photosynthesis. Conversely, the oxygen produced by photosynthesis can be taken up by the aerobic organotrophs and stimulate the degradation of oil compounds. The reduction of oxygen around microaerophilic cyanobac-teria is essential for their growth while high oxygen concentrations can be toxic. Oxygen acts as a competitive inhibitor of RubisCO carboxylase activ-ity and high oxygen levels result in photooxidation in the mat-forming cya-nobacteria (Garcia-picHel et al. 1999). Aerobic organotrophs may also utilize the cyanobacterial fixed nitrogen in addition to organics produced by photosynthesis (bateson et al. 1988, Glud et al. 1992, paerl et al. 1993). Fixed nitrogen and easily assimilated photosynthates have been proven to



facilitate aerobic degradation in cyanobacterial mats (musat et al. 2006). The physically close association and complementary growth requirements render cyanobacteria and oil-degrading aerobic heterotrophic bacteria an ideal consortium for the degradation of petroleum compounds (radwan et al. 2002, abed & Köster 2005, sáncHez et al. 2005). This conclusion is consistent with the observation that cyanobacteria colonize sediments and grow vigorously immediately after oil spills.Identity of oil-tolerant cyanobacteria. DGGE analysis showed remark-able shifts in the cyanobacterial community composition in the course of 2 months of incubation, starting from a mixed broad-spectrum inoculum. The entire community was replaced by other cyanobacteria, which were appar-ently less dominant in the original inoculum but favoured the new condi-tions (see also abed et al. 2003, ricHert et al. 2006)

The differences in cyanobacterial composition that developed in the oil-free and the oil-polluted mats, respectively, suggested that certain cya-nobacteria were sensitive to oil while others were tolerant. It is now well established that components of crude oils, particularly aromatics are toxic to cyanobacteria (narro 1987, radwan & al-Hasan 2000 and references therein). Previous studies have demonstrated that contamination with crude oils and aromatic hydrocarbons may lead to reduced growth (bat-terton et al. 1978), inhibited photosynthesis (vandermeulen & aHem 1976, winters et al. 1977), reduced nitrogen-fixing ability (o’brien & dixon 1976, vandermeulen & aHem 1976) and reduced activity of other enzymes (meGHaraj et al. 2000), resulting in reduced growth of oxygenic phototrophs. In contrast, other reports have shown a stimulatory effect of n-alkanes to the photosynthesis and growth of cyanobacteria (scHröder & reHm 1981, Gaur & sinGH 1990). Our experiments demonstrated that in the process of experimental oil exposure, the sensitive cyanobacteria were outcompeted by more tolerant species that grew well in the presence of oil, thus demonstrating specific differences among cyanobacteria in relation to oil pollution.

Oscillatoria-like cyanobacteria observed by microscopy in the early mats were apparently most sensitive both to the environmental change and to the presence of oil, being outcompeted by other cyanobacteria. DGGE showed 2 bands in the inoculum at time 0 that could be related to these cya-nobacteria, as they clustered close to a sequence identified in the Genbank as Oscillatoria terebriformis. These bands did not show in later DGGE gels.

Most of the oil tolerant cyanobacteria belonged to thin filamentous Lep-tolyngbya-types corresponding to the genera Halomicronema and Phormi-dium of the Genbank. Some of them were also detected in the oil-free mats. Halomicronema-related cyanobacteria were shown to become enriched in microbial mats from Wadi Gaza upon pollution with petroleum compounds suggesting their tolerance and competitiveness under these conditions (abed et al. 2002). However, experiments with the axenic Halomicronema



excentricum isolated from the same mats showed that this cyanobacterium was not able to degrade any of the tested aliphatic and aromatic compounds (abed & Köster 2005). Since some cyanobacteria are sensitive and others are tolerant to pollution, any alteration in their community composition could be used as a bio-indicator of pollution (meGHaraj et al. 2000).

Dominance of thin filamentous cyanobacteria in perturbated sites

In general, thin filamentous cyanobacteria have been observed to overtake other types of cyanobacteria when ecosystems are perturbated or environ-mental conditions are altered. Overgrowth of cyanobacteria belonging to this group has been recorded independently in several studies that tried to isolate the field dominant types (abed et al. 2001, 2003, ricHert et al. 2006). The transfer of a microbial mat from its original habitat in Solar Lake to an artificial pond in Eilat resulted in the replacement of the dominant Microcoleus chthonoplastes by Halomicronmena excentricum (abed et al. 2001). This suggests that the thin filamentous group of cyanobacteria in-cludes many opportunistic species, which may be present in minor quantities or even be dormant in many ecosystems, but take advantage of any changes in the environmental settings that affect negatively the dominant forms.

Acknowledgements

We would like to thank florin musat for setting up the aquaria and Prof. frie-dricH widdel for fruitful discussions and support. This work was supported by the European project MATBIOPOL (EVK3-CT1999-00010).

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Manuscript received January 13, 2008, accepted November 12, 2008.

The authors’ addresses:

raeid m. m. abedSultan Qaboos University, College of ScienceBiology Department, P.O. Box 36Al Khod, postal code 123Muscat, Sultanate of OmanE-mail: [email protected]

stjepKo GolubicBiological Science Center, Boston University5 Cummington StreetBoston, MA 02215, USA


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Selection of oil-tolerant cyanobacteria on oil polluted sediments