Research articleInfluence of bioaugmentation and biostimulation on PAH degradation in aged contaminated soils: Response and dynamics of the bacterial community
Introduction
The ubiquitous occurrence and associated carcinogenic and mutagenic properties of polycyclic aromatic hydrocarbons (PAHs) has raised significant concerns for both, human and ecosystem health (Xiong et al., 2017). Bioremediation is often considered as the most advantageous remediation technology over other conventional technologies to restore the ecosystem (Raquel et al., 2013). Microbial degradation of PAHs is widespread and has proven to be environmental friendly, reliable, effective and economical in the removal of PAHs from contaminated soils (Haleyur et al., 2018). Remediation of PAHs are performed by various organisms, including bacteria, fungi and algae (Haritash and Kaushik, 2009). There are abundant bacteria capable of degrading PAHs in chronically polluted sites by using them as their sole carbon and energy sources (Taketani et al., 2010). Apart from elimination and transformation of PAHs, soil microorganisms are indicators of soil quality in the presence of organic or inorganic pollutants and are helpful in the assessment of different soils (Haritash and Kaushik, 2009). Nevertheless, only 1 per cent of the total microbial communities are adapted to degrade PAHs, which prolongs the removal of PAHs in soils (Lors et al., 2012).
For successful bioremediation, the overall degradation and removal rate of PAHs must be more rapid than the natural attenuation processes (Mohan et al., 2008). The development of a successful remediation strategy for PAHs involves microbial degradation and is considered as the most sustainable approach in terms of decontamination of PAHs-polluted soils (Mohan et al., 2008). As a result, specific strains capable of degrading PAHs have been isolated and applied in different soil systems. Assessing microorganisms for their potential degradation properties in the natural environment (non-sterile conditions) in the presence and absence of various biotic (Sipilä et al., 2008) and abiotic (Simarro et al., 2011) factors is very important (Pandey et al., 2009). However, the bioavailability of PAHs, especially high molecular weight (HMW) PAHs in aged contaminated soils can be extremely low resulting in only limited PAHs degradation (Zielińska and Oleszczuk, 2016). Despite drawbacks such as the additional costs and the technical complexity of bioaugmentation, the use of bacteria for effective bioaugmentation of PAHs has received significant attention in recent years (Innemanová et al., 2018; Wu et al., 2017a, 2017b).
In addition to bioaugmentation, biostimulation (BS) can be developed as a management option for contaminated soils. This strategy is usually centred around the addition of nutrients to the soil (Koshlaf et al., 2016). Most research focuses on the addition of nutrients during bioremediation as the BS strategy (Koshlaf et al., 2016; Bento et al., 2005). However various other factors that affect the rate of degradation of PAHs, such as moisture content, pH, and oxygen concentration (Molina-Barahona et al., 2004) can also be manipulated as part of a BS strategy. To date, to the best of the authors’ knowledge there have been no reports in which these parameters have been used as a successful BS strategy for the remediation of aged PAH contaminated soils.
In recent years, PAH-degrading microbial communities have been widely studied through various molecular techniques opening new frontiers in microbial community analysis. However, the PAH degradation potential exhibited by indigenous microbiota remains poorly understood (Ren et al., 2015). Various factors that influence the composition and abundance of total or degradative bacterial populations in PAH-contaminated soils include the size fraction of soil (Uyttebroek et al., 2006) and type of PAHs (Singleton et al., 2005).
The application of Next Generation Sequencing (NGS) methods to analyse natural ecological communities in PAH contaminated soils are now widely practiced (Guazzaroni et al., 2013; Duarte et al., 2017; Riesenfeld et al., 2004; Myrold et al., 2014). NGS approaches help in the determination of organisms actually carrying out specific function (e.g. PAH degradation) in contaminated soils. Various studies have investigated PAH-degrading microbial communities in soils (Koshlaf et al., 2016; Festa et al., 2016a, 2016b). In one study, soil samples contaminated from PAHs for several decades were analysed for the presence of biodegradation capabilities in soils; the results showed that biostimulated communities were capable of greater rates of naphthalene utilisation compared to non-stimulated communities (Guazzaroni et al., 2013). However, to date there are no reports examining the impact of bioremediation on the community structure for chronically contaminated, aged Australian soils. This is an area for further study as following remediation, there is increasing pressure for the reuse of these soils. It is crucial to determine that these soils are fit for reuse in terms of the soil microbial ecology.
In the current study, soils obtained from a former gas manufacturing site and contaminated with PAHs for more than 50 years were subjected to bioaugmentation, using single and mixed bacterial isolates and biostimulation, through the maintenance of soil aeration, temperature and moisture content in mesocosms incubation experiments. Real-time PCR and Illumina sequencing techniques were employed to investigate the bacterial community changes. The results explored: (1) the extent of PAHs degradation in bioaugmentation and biostimulation treatments by added isolates; (2) the abundance of PAHs-degrading bacteria using qPCR assays; and (3) how the native bacterial community can degrade different fractions of PAHs.
Section snippets
Sample collection and characterization
Soil samples were collected from an aged stockpile of soil with a history of long term contamination of PAHs from a former gas work site located in Cootamundra, New South Wales, Australia (34°31′26.6″S 147°35′56.6″E). These samples were collected, labelled and transported to the laboratory in sterile plastic bags and stored at 4 °C prior to further analysis. Soil samples were air dried at room temperature for one day before use. To remove any larger particles including gravels and debris
Impact of bioaugmentation and biostimulation on PAH degradation
The initial PAH concentration was 1483 ± 46.27 mg kg−1 (Supplementary Table 2). The effects of different treatments on PAHs-degradation after 56 days are shown in Fig. 1. In control mesocosms, 94% of PAHs remained at the end of the incubation. In treated mesocosms no significant differences in PAH-degradation were observed among the BS and other treatments at any observed time point; around 56 ± 9% of PAHs were reduced within the first 14 days in all treatments. Mesocosms inoculated with Pantoea
Impact of bioaugmentation and BS on PAHs degradation
Various studies have shown that biostimulation and bioaugmentation can successfully remediate PAHs (Koshlaf et al., 2016; Festa et al., 2016a, 2016b; Teng et al., 2010; Andreolli et al., 2015). However, in most studies the soils were artificially spiked by PAHs and then assessed for bioremediation. To the author's knowledge, only two studies (Kuppusamy et al., 2015; Thavamani et al., 2012) employed aged, weathered PAH-contaminated soils from Australia for PAHs degradation at the mesocosm scale.
Conclusion
In the present study, a 99% reduction of PAHs in aged, weathered contaminated soils was demonstrated using BS; bioaugmentation did not further enhance the degradation of PAHs. During BS, metabolic activities of the indigenous soil microflora were stimulated for greater PAHs degradation by providing appropriate conditions and therefore BS can serve as an important approach for the bioremediation of PAH contaminated soils, especially when aged and weathered. Gram-positive bacteria were dominant
Conflicts of interest
The authors declare that they have no conflict of interest.
Acknowledgements
We would like to thank Environmental Earth Sciences for providing the samples. A special thanks to Arturo Aburto-Medina, Tanvi H. Makadia, Sneha Shikha and Warren Blyth for their support during Illumina Miseq sequencing. This research was also supported by RMIT University under the Research Training Scheme.
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A. Mark Osborn and Andrew S Ball contributed equally to this work.