Effectiveness of Shewanella oneidensis bioaugmentation in the bioremediation of phenanthrene-contaminated sediments and possible consortia with omnivore-carnivore meiobenthic nematodes

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Highlights

  • Univariate indices of meiobenthic nematodes decreased after exposure to phenanthrene.

  • Chromadorella and Mesacanthion are negative bioindicative taxa to phenanthrene.

  • Proportions of carnivores-omnivores decreased after exposure to phenanthrene.

  • Bioaugmentation with Shewanellea oneidensis was efficient to remediate 10 μg phenanthrene.kg−1.

Abstract

This study was conducted to assess the impact and efficiency of the bioaugmentation as a bioremediation technique in annoying effects of a polycyclic aromatic hydrocarbon (phenanthrene) on a community of free-living nematodes from Bizerte bay (Tunisia). For this purpose, closed microcosms were exposed to three doses of phananthrene (0.1 μg kg−1, 1 μg kg−1 and 10 μg kg−1), in combination or not with a strain of Shewanella oneidensis. After 40 days of the exposure, results were obtained at the numerical, taxonomic and feeding levels. The results of univariate analyses revealed significant decreases in most univariate indices for phenanthrene treated communities compared to controls, with a discernible increase in the proportion of epistrate feeders. After bioaugmentation, similar patterns were observed for univariate and multivariate analyses, with the exception of the highest treatment, which showed no difference from the controls. The results obtained showed that the bioaugmentation with Shewanellea oneidensis was highly effective in reducing the negative impact of the highest dose of phenanthrene (10 μg kg−1 Dry Weight) tested on meiobenthic nematodes. Furthermore, a combination of Shewanellea oneidensis and four omnivore-carnivore nematode taxa could be suggested as an effective method in the bioremediation of phenanthrene-contaminated sediment.

Introduction

In the marine environment, the main sources of hydrocarbon contamination are oil spills, degassing and offshore crude oil production (Kennish, 1992; Louati et al., 2001; Mahmoudi et., 2005; Allouche et al., 2020a; Hedfi et al., 2021). Polycyclic aromatic hydrocarbons (PAHs) comprise organic compounds with combined benzene rings. Their presence in the environment is usually derived from the incomplete combustion of organic compounds such as wood and fossil fuels (INERIS, 2006). The phenanthrene (three cycles) is mainly used in the coloring, explosive and pharmaceutical industries (Verschueren, 1996). It is also stable with a relatively large absorption on substrates (Allouche et al., 2020a). Phenanthrene has chronic and acute toxic effects on marine biota (Swartz et al., 1997; INERIS, 2006). In humans, the studies on the level of absorption of the phenanthrene from the skin are limited, however traces of this substance were found in the blood of individuals after the application of phenanthrene on their skin for two days (Storer et al., 1984).

Phenanthrene degrades partially in aqueous medium; after 4 weeks, its degradation is equal to 54% (OECDE301C method) (CITI, 1992) and its half-life ranges from 64 to 800 days (Howard et al., 1991). The transformation and/or the elimination of PAHs in the marine environment appears to be slower due their resistance to degradation by indigenous bacteria (Maciorowski et al., 1980). The natural attenuation of PAHs is driven by autochthonous microbs’ intrinsic degradation (El-Alawi et al., 2002). In order to enhance the biodegradation efficiency, the bioaugmentation (i.e., inoculation of microorganisms with recognized degradation ability) was proposed previously (Ben Said et al., 2012). This approach showed to be a promising alternative over other potential physical and chemical treatments, due to its lower costs and environmental impact (Venkata et al., 2006). Several bacterial strains were isolated from contaminated soils and tested for their ability to mineralize low molecular weight hydrocarbons (i.e. 2 to 3 benzene cycles) (Forsyth et al., 1995; Mrozik et al., 2003; Ben Saïd et al., 2010) as well as their efficiency in biodegradation of PAHs, whether in aerobic (Geisel-Brecht et al., 1998; Kanaly and Harayama, 2000; Melcher et al., 2002) or anaerobic conditions (Coates et al., 1997). However, there were few studies undertaken on the bioremediation of marine sediments (Schratzberger et al., 2003; Miyasaka et al., 2006; Bai et al., 2021). Two such studies that we are aware of were conducted by Ben Said et al. (2012, 2015) in Tunisia. The authors used Acinetobacter sp. and Staphylococcus sp., isolated from Bizerte lagoon, to remediate contaminated sediments with 5.5 and 10 μg anthracene g−1, respectively. These authors reported that the process of bioremediation based on this method is a lengthy process, and recommended that combining PAHs degraders could result in faster removal of PAHs from sediments compared to using single microorganisms.

Herein, an experimental study was carried out to assess the responses of a community of marine nematodes to the addition of Shewanella oneidensis (bioaugmentaion) in phenanthrene contaminated sediment. This strain belonging to γ-protobacteria and was previously isolated from the Bizerte lagoon in Tunisia (Ben Saïd et al., 2008) with a great potential to degrade a wide range of PAHs. Because of their ability to grow on hydrocarbon contaminated media, these bacteria may play an important role in natural attenuation of pollution. In the current study, tiny organisms belonging to the so called ‘small food web’ (i.e. microbs, protists and meiofauna) were preferred over macrobenthic invertebrates to assess the efficacy of bioaugmentation as a method to eleminate the phenanthrene contamination and to ensure the precocity of subsequent management actions and environmental regulations (Schratzberger et al., 2000; Hedfi et al., 2021). Free-living nematodes are the dominant meiofaua group (Mahmoudi et al., 2005). They are considered ideal bioindicators in field-based studies and bioassays given they ensure and/or enhance the mineralization of organic matter, spend their short life cycle (days to weeks) in sediment, are small (1–5 mm in average length), abundant (up to 20 million individuals m−2) and easy to maintain under controlled laboratory conditions (Suderman and Thistle, 2003).

Until recently, few attempts were made to combine meiobenthic nematodes with bacteria for efficient bioindicators combined with potential bioremediation purposes. We believe that the application of integrated tools would be more efficient and profitable for small and closed to partially closed aquatic biotopes, such as ports or even for larger ones, as the Mediterranean Sea and the Black Sea. In the current study we aimed finding the nematode species that could be used in combination with Shewanellea oneidensis for an effective bioremediation of phenanthrene-contaminated areas. Promising axes can be envisaged since nematodes can be preserved inactive at low temperatures for long periods of time (Smith et al., 2008). The current work aims to fill this gap by providing answers to the following questions: (1) how do nematode taxa respond following their exposure to phenanthrene, both alone and associated with Shewanellea oneidensis? (2) if discernible different responses are observed, at which threshold does the bioaugmentation becomes efficient in bioremediation and what are the best combinations of marine nematodes and Shewanellea oneidensis with bioremediation potential?

Section snippets

Sampling

Sediment with its natural meiofauna was collected on February 22, 2011 from Rimel beach (37° 15′590″ N, 9° 54′730′ E), Tunisia. The depth at this collecting site was 1.2 m. The temperature and salinity were measured at the sediment-water interface using a Microprocessor Conductivity meter (LF.196). Dissolved oxygen and pH levels were also measured with the aid of portable Oximeter (C G 867) and pH-meter (WTW pH 196). This location was previously considered by Zrafi et al. (2010), Boufahja et

Sediment loads in viable heterotrophic aerobic bacteria after incubation time (40d)

The bacterial loads prior to bioaugmentation are summarized in Fig. 1A. The results of the Scheffé test indicate that the reduction of the bacterial loads following the phenanthrene contamination was significant for two treatments: phe-0.1 and phe-10 (Fig. 1A, p < 0.05).

The pattern of bacterial loads changed after the inoculation with Shewanella oneidensis (Fig. 1A, ANOVA: p < 0.05). Thus, the treatments phe-0.1 + Bc and phe-1+Bc were significantly higher than controls C2 (Fig. 1A, Scheffé

Discussion

The meiobenthic nematodes' abundance and taxonomic diversity were negatively by the enrichment in phenanthrene, excepting the treatments contaminated with the lowest dose of phenanthrene (phe-0.1). The harmful effects of hydrocarbons on nematofauna were previously reported (Coull and Chandler, 1992; Peterson et al., 1996; Carman et al., 1997, 2000; Beyrem and Aïssa, 2000; Mahmoudi et al., 2003, 2005; Hedfi et al., 2007; Louati et al., 2014; Ben Said., 2015; Allouche et al., 2020a; Hedfi et al.,

Conclusions

In the microcosms contaminated with increasing concentrations of phenanthrene but not inoculated with the bacterial strain, significant reductions in the most abundant nematode community parameters were observed compared to control treatments. The results of this experiment confirm the low tolerance of indigenous bacteria, given the slight reduction (by 10–15%) in phenanthrene concentration in sediment by the end of the experiment.

After bioaugmentation, there was also observed a significant

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This work was supported by the Tunisian Ministry of the High Education and Scientific Research. The authors extend their appreciation to the deanship of scientific research for funding this article by Taif University Research Supporting Project number (TURSP-2020/225), Taif University, Taif, Saudi Arabia. Octavian Pacioglu was funded by the National Core Program - Romanian Ministry of Research and Innovation Program, project 25 N/2019 BIODIVERS 19270103.

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