(215c) Bioremediation Technology Development for Cleaning of Fracking Wastewater

Introduction

Hydraulic fracturing is a process used to recover natural
gas from deep shale formations. 
Large amounts of water, sand and chemical additives are pumped, under
high pressure, to create fractures, allowing the released gas to travel to the
surface.  While fracking increases
access to natural gas, the water is contaminated with a variety of chemicals,
some carcinogenic. 

In this study, we developed a biomaterial to be used for
biodegradation of chemicals found in fracking water.  We have shown active degradation of
several of these chemicals by Pseudomonas
putida NCIB 9816 and Mycyobacterium
vanbaalenii PYR-1 encapsulated in a silica/polymer matrix.  The porous matrix allows for diffusion
of nutrients into the cells for continued long-term viability and degradation
activity, while providing controlled isolation from the environment and
enhanced stability.

Materials &
Methods

Pseudomonas putida
NCIB 9816 was grown in modified Stanier's liquid minimal medium (1) with 18 mM
sodium pyruvate at 28°
C, with shaking at 250, rpm for all cell encapsulation experiments. Naphthalene
degradation pathway genes were induced by the addition of 500
µM anthranilate.  Mycobacterium
vanbaalenii PYR-1 was grown in phosphate-basal minimal medium (PBM) (2)
containing 5 g/L naphthalene at 28° C,
with shaking at 250 rpm, for all cell encapsulation experiments.  At the end of the log phase growth, NCIB
9816 and PYR-1 cultures were prepared for encapsulation by centrifugation at
9,800 x g at 4° C
for 20 min, washing twice with phosphate buffered saline (pH = 7.4). and weighing wet cell mass prior to suspending to 0.2 g/mL
in Stanier's or PBM medium, respectively.

The porous matrix was comprised of silica nanoparticles
Ludox TM40 (40%w/w) and the precursor Tetramethyl orthosilicate (TMOS, 98%),
along with polyethylene glycol (PEG, M_w = 600Da).  Porous gels were
formed by diluting silica nanoparticles in ultra-pure water.  PEG was added at a volume ratio of 1:4
and the solution was vortexed for 10min. 
Separately, TMOS was hydrolyzed by sonication in water and 0.01M HCl in
a volume ratio of 1:1:0.1, respectively. 
The hydrolyzed solution was mixed with the TM40-PEG solution at a volume ratio of 1:2. 
Finally a cell suspension (0.2g/ml) was added to the mixture at a ratio
of 1:1.  Using emulsion, the
solution was transferred to a syringe and added drop wise to mineral oil to
form beads.  Beads were rinsed
thrice in PBS prior to assaying and storage.  Encapsulated cells were stored at 4° C in
10 mL scintillation vials containing 1.0 mL minimal medium (Stanier's medium or
PBM) and 1.0 mL of silica-encapsulated cells.

Naphthalene degradation pathway gene activity was assayed
using biphenyl as a substrate and determining the formation of the yellow ring
cleavage product 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate  at 435 nm.  5 µL of saturated biphenyl in ethanol
was added per mL of sample before incubation
at room temperature with shaking at 200 rpm.  1mL of bead material was submerged in 5
mL of PBS prior to the addition of biphenyl, but due to the very low aqueous
solubility of biphenyl much of the material precipitates out of solution.  Despite this low solubility, biphenyl is
available to the encapsulated cells and linear degradation curves can be
observed (Fig. 1).

Aromatic ring dioxygenase activity in M. vanbaalenii PYR-1 encapsulated and free cell samples was
determined using dibenzothiophene as susbstrate (3). A 5.0 µL aliquot of a
dibenzothiophene solution in dimethylsulfoxide (33 mg/mL) was added per mL of
sample.  Samples were incubated at
room temperature, with shaking at 200 rpm, and absorbance was measured at 474
nm to detect the formation of the orange colored product trans-4-[2-(3-hydroxy)-thianaphthenyl]-2-oxo-3-butenoate described
by Kodama, et al. (4). To increase activity in samples stored for long periods,
samples were induced with 8 mM sodium benzoate and incubated at room temperature, with shaking at 200 rpm for 60 min. Samples
were then washed and resuspended in fresh medium prior to assay.

Results

Metabolically active cells are required for these complex biodegradation
pathways to be functional.  Silica
gel-encapsulated P. putida NCIB 9816
maintained approximately 60% of the original naphthalene degradation activity
after being stored for 7 days (Fig. 1) and 20% of original activity after 42
days of storage, at which point stability experiments were stopped (Fig 1.).  There was not a significant difference
in activity for encapsulated NCIB 9816 when naphthalene was added to the media
for storage (data not shown). 

Fig. 1. Biphenyl
Degradation by Silica Gel-Encapsulated Pseudomonas
putida NCIB 9816

In encapsulation experiments with M. vanbaalenii PYR-1, cells maintained approximately 50% of their
original activity after being stored for 4 weeks at 4° C (Fig. 2). In comparison, free
cell samples maintained approximately 35% of original activity after 4 weeks
(data not shown). The data suggests that silica encapsulation may play a role
in increasing the longevity of metabolic activity in M. vanbaalenii PYR-1 cells.

Fig. 2. Dibenzothiophene
Degradation by Silica Gel-Encapsulated M. vanbaalenii PYR-1 measured by
appearance of the degradation product trans-4-[2-(3-hydroxy)-thianaphthenyl]-2-oxo-3-butenoate.

Sodium benzoate, a common metabolite in the degradation of
many polycyclic aromatic hydrocarbons (PAHs), was used to maintain PAH
degradation pathway genes and "recharge" metabolic activity in silica
gel-encapsulated cells after long periods of storage.  After two weeks of storage, both free
and encapsulated cell activity was maintained by the addition of sodium
benzoate.  Encapsulated cell samples
showed a 2.5x increase in metabolic activity, while free cell activity
increased by 1.8x (data not shown).

Conclusion

Fracking fluid water is contaminated with potentially harmful
chemicals.  The biomaterial described
here demonstrated that PAH degradation pathways can be
active for up to 42 days.  The
silica/polymer matrix creates a confined environment while still allowing
nutrient delivery for continued activity of degradation pathways. We propose
further development for the use of degrading harmful fracking fluid
chemicals.   

References

1.     Turner,
K., S. Xu, P. Pasini, S. Deo, L. Bachas, and S. Daunert. 2007. Hydroxylated polychlorinated biphenyl
detection based on a genetically engineered bioluminescent whole-cell sensing
system. Anal. Chem. 79:5740–5745.

2.    
Kim, Y.,
K.H. Engesser and C.E. Cerniglia. 2003. Two polycyclic aromatic hydrocarbon
o-quinone reductases from a pyrene-degrading Mycobacterium. Arch. Biochem.
Biophys. 416:209-217.

3.    
Kweon,
O., S.J. Kim, J.P. Freeman, J. Song, S. Baek, C.E. Cerniglia. 2010. Substrate
specificity and structural characteristics of the novel Rieske nonheme iron
aromatic ring-hydroxylating oxygenases NidAB and NidA3B3 from Mycobacterium vanbaalenii PYR-1. MBio. 1: e00135-10.

4.    
Kodama,
K., K. Umehara, K. Shimizu, Y. Minoda and K. Yamada, 1973. Microbial conversion
of petro-sulfur compounds, final part, isolation of microbial products from dibenzothiophene
and its proposed oxidation pathway. Agr.
Biol. Chem. 37:45-50.