Culturing oil sands microbes as mixed species communities enhances ex situ model naphthenic acid degradation

Abstract

Oil sands surface mining for bitumen results in the formation of oil sands process water (OSPW), containing acutely toxic naphthenic acids (NAs). Potential exists for OSPW toxicity to be mitigated by aerobic degradation of the NAs by microorganisms indigenous to the oil sands tailings ponds, the success of which is dependent on the methods used to exploit the metabolisms of the environmental microbial community. Having hypothesized that the xenobiotic tolerant biofilm mode-of-life may represent a feasible way to harness environmental microbes for ex situ treatment of OSPW NAs, we aerobically grew OSPW microbes as single and mixed species biofilm and planktonic cultures under various conditions for the purpose of assaying their ability to tolerate and degrade NAs. The NAs evaluated were a diverse mixture of eight commercially available model compounds. Confocal microscopy confirmed the ability of mixed and single species OSPW cultures to grow as biofilms in the presence of the NAs evaluated. qPCR enumeration demonstrated that the addition of supplemental nutrients at concentrations of 1 g L(-1) resulted in a more numerous population than 0.001 g L(-1) supplementation by approximately 1 order of magnitude. GC-FID analysis revealed that mixed species cultures (regardless of the mode of growth) are the most effective at degrading the NAs tested. All constituent NAs evaluated were degraded below detectable limits with the exception of 1-adamantane carboxylic acid (ACA); subsequent experimentation with ACA as the sole NA also failed to exhibit degradation of this compound. Single species cultures degraded select few NA compounds. The degradation trends highlighted many structure-persistence relationships among the eight NAs tested, demonstrating the effect of side chain configuration and alkyl branching on compound recalcitrance. Of all the isolates, the Rhodococcus spp. degraded the greatest number of NA compounds, although still less than the mixed species cultures. Overall, these observations lend support to the notion that harnessing a community of microorganisms as opposed to targeted isolates can enhance NA degradation ex situ. Moreover, the variable success caused by NA structure related persistence emphasized the difficulties associated with employing bioremediation to treat complex, undefined mixtures of toxicants such as OSPW NAs.

Keywords: biofilms; bioreactor; bioremediation; naphthenic acids; oil sands.

FIGURE 1

Confocal laser scanning microscopy (CLSM) of OSPW multispecies biofilms grown on the polystyrene pegs of the CBD for 14 days at 25°C, and 125 rpm. TSB rich medium was inoculated with OSPW to serve as a control for inoculum viability (A). The remaining biofilms were grown in the presence of 8χNA, using BH minimal media supplemented with 0.001 g L^-1 molasses (B), 1 g L^-1 yeast extract (C), 0.001 g L^-1 yeast extract (D), 1 g L^-1 peptone (E), and 0.001 g L^-1 peptone (F).

FIGURE 2

Quantitative polymerase chain reaction (qPCR) 16S rRNA gene enumeration of mixed species biofilm and planktonic cultures grown for 14 days on BH minimal medium in the presence of 8χNA and either 1 g L^-1 supplemental carbon (A), or 0.001 g L^-1 supplemental carbon (B). Enumeration of biofilms is reported as 16S gene copies per peg, enumeration of planktonic microbes reported as copies per well (n = 3). Error bars represent standard error of the mean.

FIGURE 3

Confocal laser scanning microscopy 3D renderings of OSPW single species Rhodococcus sp. (Y3; A), and Cyanobacteria (G1; B) biofilms grown on the polystyrene pegs of the CBD for 14 days at 25°C, 125 rpm using BH minimal medium supplemented with either yeast extract or glucose (at 1 g L^-1) respectively.

FIGURE 4

qPCR 16S rRNA gene enumeration of single species biofilm and planktonic isolates grown for 14 days on BH minimal medium in the presence of 8χNA and either 1 g L^-1 supplemental carbon (A), or 0.001 g L^-1 supplemental carbon (B). Enumeration of biofilms is reported as 16S gene copies per peg, enumeration of planktonic microbes reported as copies per well (n = 3). Error bars represent standard error of the mean. Asterisks denote samples whose qPCR copy number threshold was less than or equivalent to non-template controls.

FIGURE 5

Abundance of individual components of the 8χNA mixture following 14 days incubation with OSPW multispecies biofilm and planktonic cultures grown in BH minimal medium with either 1 g L^-1 supplemental carbon (A), or 0.001 g L^-1 supplemental carbon (B). GC-FID was used to determine normalized abundance values relative to an internal standard (n = 2). Error bars represent standard error of the mean. A normalized abundance value of ‘1.0’ represents starting NA levels.

FIGURE 6

Abundance of individual components of the 8χNA mixture following 14 days incubation with OSPW single species biofilm and planktonic isolates grown in BH minimal medium with either 1 g L^-1 supplemental carbon (A), or 0.001 g L^-1 supplemental carbon (B). GC-FID was used to determine normalized abundance values relative to an internal standard (n = 2). Error bars represent standard error of the mean. A normalized abundance value of ‘1.0’ represents starting NA levels.

FIGURE 7

Abundance of the tricyclic, diamondoid ACA following incubation with OSPW multispecies biofilms grown in BH minimal medium and 100 mg L^-1 ACA with either 1 g L^-1 supplemental carbon (A), or 0.001 g L^-1 supplemental carbon (B). GC-FID was used to determine normalized abundance values relative to an internal standard at 14, and 28 days time points (n = 2). Error bars represent standard error of the mean. A normalized abundance value of ‘1.0’ represents starting ACA levels.