Microcosm experiments of oil degradation by microbial mats. II. The changes in microbial species

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Abstract

The influence of microbial mats on the degradation of two crude oils (Casablanca and Maya) and the effect of oil pollution on the mat structure were assessed using model ecosystems, prepared under laboratory conditions subject to tidal movements, from pristine Ebro Delta microbial-mat ecosystems. Both selected oils are examples of those currently used for commercial purposes. Casablanca crude oil is aliphatic with a low viscosity; Maya represents a sulphur-rich heavy crude oil that is predominantly aromatic. In the unpolluted microcosms, Microcoleus chthonoplastes-, Phormidium- and Oscillatoria-like were the dominant filamentous cyanobacterial morphotypes, whilst Synechoccocus-, Synechocystis- and Gloeocapsa-like were the most abundant unicellular cyanobacteria. After oil contamination, no significant changes of chlorophyll a and protein concentrations were observed, though cyanobacterial diversity shifts were monitored. Among filamentous cyanobacteria, M. chthonoplastes-like morphotype was the most resistant for both oils, unlike the other cyanobacteria, which tolerated Casablanca but not Maya. Unicellular cyanobacteria seemed to be resistant to pollution with both essayed oils, with the exception of the morphotype resembling Gloeocapsa, which was sensitive to both oils. The crude-oil addition also had a significant effect on certain components of the heterotrophic microbial community. Casablanca oil induced an increase in anaerobic heterotrophic bacteria, whereas the opposite effect was observed in those heterotrophs when polluted with Maya oil. The overall results, microbiological and crude-oil transformation analysis, indicate that the indigenous community has a considerable potential to degrade oil components by means of the metabolic cooperation of phototrophic and heterotrophic populations.

Introduction

Marine pollution due to oil spills is one of the most prevalent environmental and economic concerns worldwide. The impact of such episodes on open sea and coastal areas has been evaluated in several works, which suggest that crude oil accumulates in the natural environment for long periods (Readman et al., 1992, Fowler et al., 1993, Sauer et al., 1998). Chemical pollution by petroleum hydrocarbons has a negative effect on natural marine ecosystems. Changes in the diversity, abundance, and activity of autochthonous populations, as a consequence of oil spills have been reported (Macnaughton et al., 1999, Megharaj et al., 2000, Saul et al., 2005, Bode et al., 2006). As a result, these polluted communities are typically less diverse than those in non-stressed systems. However, the extent of the damage may depend on the amount and quality of spilled oil, the length of time that the populations have been exposed and their different tolerances to hydrocarbon compounds.

Different natural processes may be involved in the hydrocarbon removal of these polluted areas, including evaporation, photo-oxidation and microbial degradation. However, among these, the application of biological degradation strategies for this purpose is of particular interest (Viñas et al., 2002, Medina-Bellver et al., 2005, Fernández-Álvarez et al., 2006, Gallego, 2006, Jiménez, 2006, Gallego et al., 2007). Observations carried out in chronically polluted areas provide evidence that microbial populations within these contaminated ecosystems tend to be dominated by organisms capable of utilizing and/or surviving toxic contamination (Abed et al., 2006, Chaillan et al., 2006, Al-Thukair et al., 2007). Understanding of crude-oil biodegradation is still limited and is drawn from consortia or microorganisms isolated from these highly polluted environments (Al-Hasan et al., 1994, Al-Hasan et al., 2001, Sorkhoh et al., 1995, Kanaly et al., 2000, Rios-Hernandez et al., 2003, Abed and Köster, 2005, Radwan et al., 2005). The establishment of a relationship between the presence of specific populations after an episode of crude-oil pollution and the disappearance of some of its components is therefore of paramount interest in understanding the role played by indigenous organisms, and as a first step towards the development of useful bioremediation strategies.

Cyanobacterial microbial mats have been deemed promising bioremediation agents due to their metabolic complexity in a few millimetres of depth and because of their capacity to colonise crude-oil polluted coastal zones (Sorkhoh et al., 1992, Sorkhoh et al., 1995, Al-Hasan et al., 1994, Grötzchel et al., 2002). In fact, several studies have been carried out aimed at evaluating the importance of microbial-mat ecosystems for the bioremediation of polluted zones and also to determine the relationship between the degradation of certain crude-oil constituents and the presence of specific microbial-mat inhabitants (Sorkhoh et al., 1992, Sorkhoh et al., 1995, Kuritz and Wolk, 1995, Radwan et al., 1999, Al-Hasan et al., 1998, Raghukumar et al., 2001, Abed, 2002, Abed and Köster, 2005, García de Oteyza and Grimalt, 2006). There is no information on the role played by the whole body of mat inhabitants. Comprehensive studies on the oil components that are refractory or amenable to microbial mat activity are therefore needed.

Studies on the environmental impact of different pollutants have been carried out directly on polluted ecosystems, which are already modified (Abed, 2002, Chaillan et al., 2006). These investigations are always characterised for a lack of information on the pristine situation, which is essential to studying the impact of a contamination event on natural habitats. Another possibility is the use of small-scale model ecosystems (microcosms) in an effort to correctly reproduce the original ecosystem under laboratory conditions (Leff et al., 1997, Fenchel and Kühl, 2000). This latter approach has been widely used for the risk assessment in liberating various pollutants (Bachoon et al., 2001, Evans, 2004, Suderman and Thistle, 2004, Yoshida, 2006). Llirós et al. (2003) and Martínez-Alonso et al. (2004) described a new laboratory-scale device for growing hypersaline microbial mats to study the effect of a crude-oil contamination on the growth and developmental patterns of microbial mats. With this approach, a substitution pattern in cyanobacterial populations can be observed. In such experiments, however, a link between those shifts and the disappearance of petroleum hydrocarbons could not be clearly established, probably because the effect of a black tide could not be properly reproduced.

The aim of the present work was to determine the effect of two types of crude oils, Casablanca and Maya, on cyanobacterial-mat populations, and their influence on hydrocarbon transformations, by using model ecosystems (microcosms). With the purpose of reproducing the natural habitat as much as possible, the microcosms were designed through simulating a black tide, with a daily exposure of mat inhabitants to crude oil. A first report on the results of these experiments was described in García de Oteyza et al. (2006) where the qualitative and quantitative aspects of transformation of these two oils were investigated. The present study is focussed on the description of the changes in the relative abundance of cyanobacteria under crude oil exposure. These cyanobacterial populations were analysed by using phase-contrast light microscopy and Confocal Laser Scanning Microscopy (CLSM). Chlorophyll a (Chl a) and protein concentrations were also measured and used as biomass indicators.

Section snippets

Microcosms design

The model laboratory ecosystems (microcosms) were prepared from Ebro Delta microbial mats, pristine marine benthic ecosystems described previously (Mir et al., 1991). Briefly, phototrophic and heterotrophic populations constitute these vertically stratified ecosystems, developed at the water–sediment interface of sand flats. Cyanobacteria are the dominant phototrophic populations located on the upper green layers, which are underlain by red laminations dominated by purple sulphur bacteria.

Changes in the unpolluted microbial community

In the unpolluted microcosm (microcosm 1), microscopic analysis did not show any noticeable qualitative change in cyanobacterial diversity over time (Table 1, Fig. 2A and B). Samples observed under the microscope revealed the presence of at least seven different cyanobacterial morphotypes, which could be distinguished in terms of their shape and size. Morphotypes A and B, resembling Microcoleus chthonoplastes and Phormidium, respectively, were the predominant filamentous oxygenic phototrophic

Discussion

Microbial mats are widespread ecosystems along the shoreline. This area is particularly damaged upon oil spills. These communities encompass a high microbial diversity with different metabolic pathways that are arranged in a few millimetres of depth, and which can initially be considered of high potential for crude-oil bioremediation. Changes in bacterial and cyanobacterial diversity upon crude-oil exposure have been previously described in these model ecosystems (Abed, 2002, Grötzchel et al.,

Conclusions

Experiments performed on microbial mats grown under controlled laboratory conditions show that different types of crude oil have distinct effects on the microorganisms inhabiting these ecosystems, including shifts in species composition, bacterial counts and biomass concentration. Tidal movements have the effect of increasing the contact between crude oil and microbial mats, increasing the biodegradation capabilities of certain mat inhabitants. Looking at total-biomass content, no drastic

Acknowledgements

We would like to thank the Salines de la Trinitat and Parc Natural del Delta de l’Ebre (Generalitat de Catalunya) for permission to access the sampling site, and REPSOL-YPF for providing samples of Casablanca and Maya crude oils. We also thank members of the Servei de Microscòpia at the Autonomous University of Barcelona (UAB) for support with CLSM. We are grateful to Lidia Hernandez and Carol Egea for technical assistance with the microcosms and sample processing. This research was funded by

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