Spatial and temporal variability in the potential of river water biofilms to degrade p-nitrophenol
Introduction
There is a vast diversity of chemicals in the environment, which arrive from industry, agriculture, medical treatment and common household products. Many of these chemicals have the potential to exert adverse human and ecological health effects (Mnif et al., 2011, Jones et al., 2003, Lapertot and Pulgarin, 2006). The persistence of chemicals entering the environment is controlled by a range of biotic and abiotic processes, which together determine environmental concentrations of the chemical and the extent to which it is transformed to metabolites (Kowalczyk et al., 2015a). For most chemicals, biodegradation, mediated by microorganisms, is the main factor controlling persistence. In order to predict chemical persistence the Organisation for Economic Cooperation and Development (OECD) has established a tiered series of biodegradation tests (Kowalczyk et al., 2015a).
The first tier ready biodegradability tests are intended as screening approaches to gauge if a chemical is rapidly degraded, or has potential to degrade, in the environment. These tests utilize a variety of environmental materials, such as water and sediments as inocula (Doi et al., 1996, Reuschenbach et al., 2003, Yuan et al., 2004). Guidelines provide flexibility for the inoculum that can be used and its method and time of collection. However, it is known that inoculum (i.e. microbial biomass) density is an important factor affecting the outcome of chemical biodegradation tests (Thouand et al., 1995, Godhead et al., 2014) and in particular, inconsistent test results may reflect differences in inoculum quality because of temporal and spatial variability of microbial community density and diversity within environmental compartments (Courtes et al., 1995, Mezzanotte et al., 2005).
Microbial communities are subject to seasonal variability due to changes in environmental conditions, which shape natural ecosystems and affect processes occurring in environmental compartments (Bertram et al., 2001, Yunus and Nakagoshi, 2004). In particular, changes in river water volume and flow rates (Naudin et al., 2001), temperature (LaPara et al., 2000), light penetration (Romaní and Sabater, 1999) and suspended matter (Cébron et al., 2007) can impact the composition of microbial communities inhabiting river water and sediment (Hudon, 1997, Hunt and Parry, 1998, Midwood and Chow-Fraser, 2012, LaPara et al., 2000). This can affect chemical bioavailability and biodegradation in situ. For example, seasonal variations in biodegradation of 2,4-dichlorophenoxyacetic acid (2,4-D) were reported by Watson (1977) in river water and sediment, with maximum breakdown observed during winter flood conditions, probably because of adsorption of the chemical onto suspended sediment, where microbial activity was high (Nesbit and Watson, 1980). Similarly, seasonal variation in degradation of hexadecane, associated with changes in nutrient availability and biofilm composition, was reported by Chénier et al. (2003).
As an alternative to the use of natural inoculum, there has been interest in generating standardised biofilms for use in chemical biodegradation tests (Kowalczyk et al., 2015a). Currently, biofilms are used as inocula in some OECD tests, such as the biodegradation simulation test guideline OECD 303 b (OECD, 2005), and bioreactors with biofilms comprising of defined bacterial strains and mixtures have been applied to study the bioremediation of a variety of chemicals, including chlorophenols (Puhakka et al., 1995, Kargi and Eker, 2005), herbicides (Oh and Tuovinen, 1994) and azo dyes (Zhang et al., 1995).
In natural habitats, biofilms typically have high species richness (Sabater et al., 2007) and density (Singh et al., 2006), show resistance to toxic chemicals e.g. antimicrobial agents (Mah and O'Toole, 2001) and recover quickly from perturbation (Morin et al., 2007, Proia et al., 2010). Furthermore, biofilms support synergistic interactions within communities (Elias and Banin, 2012), provide microhabitat heterogeneity (Donlan, 2002), and the potential for genetic exchange (Schwartz et al., 2003), all of which can promote chemical biodegradation.
For these reasons, use of biofilms in biodegradation tests could provide a means of reducing the chance of common test failures described by Thouand et al. (1995) and Godhead et al. (2014), by eliminating the ‘biodegradation lottery’ associated with low biomass in regulatory tests (Kowalczyk et al., 2015a). However, to date, investigations of the potential to use biofilm inoculum in regulatory tests has centered on laboratory generated inoculum.
In recent studies we have used a variety of culture dependent and independent approaches, including DNA-stable isotope probing and functional gene markers to identify para-nitrophenol (PNP) degrading communities in a UK river, establishing that species of Pseudomonas were associated with degradation in samples taken between 2010 and 2013 (Kowalczyk et al., 2015b). In the current study, we investigated temporal and spatial assembly of in situ river biofilm communities and relationships between community composition and biodegradation of PNP, addressing the following questions: (1) how does time affect the composition and biodegradation potential of river water biofilms? (2) does spatial location affect biofilm composition and degradation potential? and (3) does temporal and spatial variation in chemical biodegradation kinetics relate to dynamics of specific degraders within the biofilm community?
Section snippets
Biofilm preparation
Biofilms were prepared in the River Dene, in Warwickshire, UK (52°11′58.54″N and 1°36′44.94″W). The location is a rural catchment and there was no known previous exposure to industrial pollutants, including PNP. At each sampling time and location, Biofilms were generated on polysine glass slides (5.7 × 2.5 cm) (VWR, International) attached to a Hole Airbrick (21.0 × 13.5 × 5.0 cm) (Travis Perkins Trading Co. Ltd, UK) with cable ties (165 × 2.6 mm) (BHGS Ltd, UK). Each slide had a total surface
The biodegradation of PNP
DT50 ranged between 5.5 and 16 days across sampling times and locations. No PNP biodegradation (Fig. 1) was observed for upstream and downstream river water biofilms collected in November 2011, while two out of three replicates of effluent biofilm completed PNP biodegradation within nine days (Fig. 1). There was a significant difference in DT50 between effluent biofilms collected in November 2011 (16.11 days) and February 2012 (8.2 days) (Table 1). There were no significant differences in DT50
Discussion
In the current study we have demonstrated that the major factor determining microbial community composition of in situ biofilms and river water was sampling time, with minor differences between sampling locations. Biofilms taken at different sampling times had different potential for chemical biodegradation, with higher biodegradability potential observed for inocula collected in February and May 2012 in comparison with inocula sampled in November 2011. Significant differences in PNP
Acknowledgements
We thank the Safety and Environmental Assurance Centre, UK, Unilever for the financial support of this study.
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