Faculty of Science
GORDONIA SP. NB4-1Y: MICROBIAL DEGRADATION OF 6:2 FLUOROTELOMER
SULFONAMIDE ALKYL BETAINE (FTAB) AND 6:2 FLUOROTELOMER SULFONATE
(FTSA)
2017 | DAYTON MATTHEW JORDAN SHAW

B.Sc. Honours thesis – Chemical Biology

Gordonia sp. NB4-1Y: Microbial Degradation of 6:2 Fluorotelomer Sulfonamide Alkyl
Betaine (FTAB) and 6:2 Fluorotelomer Sulfonate (FTSA)

by

DAYTON MATTHEW JORDAN SHAW

A THESIS SUBMITTED IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF

BACHELOR OF SCIENCE (HONS.)
in the
DEPARTMENTS OF BIOLOGICAL AND PHYSICAL SCIENCES
(Chemical Biology)

This thesis has been accepted as conforming to the required standards by:
Jonathan Van Hamme (Ph.D.), Thesis Supervisor, Dept. Biological Sciences
Sharon Brewer (Ph.D.), Co-supervisor, Dept. Physical Sciences
Bruno Cinel (Ph.D.), Examining Committee Member, Dept. Physical Sciences

Dated this 2nd day of May, 2017, in Kamloops, British Columbia, Canada

© Dayton Matthew Jordan Shaw, 2017

ABSTRACT
The purpose of this study was to identify and quantify breakdown products of the
environmental contaminants 6:2 fluorotelomer sulfonamide alkyl betaine (FTAB) and 6:2
fluorotelomer sulfonate (FTSA), when added as the sole-sulfur source to cultures of the soil
bacterium, Gordonia sp. NB4-1Y. FTAB is used in aqueous film-forming firefighting foams and
it is known that FTSA will appear, upon the initial degradation of FTAB, in soil, aquatic
ecosystems, and ground water with suspected adverse health effects in animals and humans. The
majority of existing data on FTAB biodegradation is from analysis of contaminated soil and water
samples, and metabolism by mixed microbial communities in sewage treatment plants, rather than
from studies involving pure cultures. In an effort to understand the fundamental biochemical
processes driving bacterial FTAB and FTSA metabolism, sulfur-limited pure cultures of NB4-1Y
were analyzed for both volatile and water-soluble breakdown products using a Dionex Ultimate
3000 UHPLC chain coupled to a Thermo Q-Exactive Orbitrap mass spectrometer over 7 days.
Over 168 h, 99.97 % of 60-µM FTSA was degraded into eleven major breakdown products, with
a mol % recovery of 88.22, while 70.42 % of 60-µM FTAB was degraded into ten major
breakdown products, with a mol % recovery of 84.65. The products detected indicate that NB41Y may be using two different approaches for FTSA desulfonation, with approximately 55 mol %
of breakdown products being assigned to a major pathway that begins with the conversion of FTSA
to 5:2-FT-ketone, with 5:2-FT-ketone being the major product at 43.62 mol %. The minor
pathway, hypothesized to begin with conversion of FTSA to 5:3-FTCA, accounted for less than
1.0 mol % of products after 168-hrs of incubation. FTSA is a proposed intermediate of FTAB
breakdown by NB4-1Y, but was not detected, possibly due to rapid utilization under sulfurlimiting conditions. As with FTSA breakdown, 20.07 % of FTAB products were assigned to the

ii

major pathway, while less than 1.0 mol % were assigned to the minor pathway. Future research
will involve isolating enzymes involved in FTAB and FTSA metabolism, verifying their function
in vitro, and designing molecular tools for monitoring their metabolism in the environment by
diverse microbial communities.

iii

ACKNOWLEDGEMENTS
I would like to thank my supervisor, Jonathan Van Hamme (Ph.D) for the opportunity to
conduct research with him, his continued guidance and support, and for making the whole
experience an enjoyable one. I would like to thank my second supervisor Sharon Brewer (Ph.D)
for introducing me to research during my third year and for her positive support and leadership. I
would also like to thank Bruno Cinel (Ph.D) for agreeing to be my third committee member.
Finally, I would like to thank Breanne McAmmond for all her help with my honours and making
my life a little more manageable.

iv

TABLE OF CONTENTS
1

2

INTRODUCTION ........................................................................................................ 1
1.1

Gordonia spp. ........................................................................................................ 1

1.2

Per- and Poly-fluorinated Contaminants ............................................................... 2

1.3

6:2-FTOH Degradation Pathway in Diverse Microbial Communities.................. 4

1.4

FTSA Degradation Pathway in Gordonia sp. NB4-1Y Pure Cultures .................. 6

1.5

Goals of this Study ................................................................................................ 8

MATERIALS AND METHODS ................................................................................. 9
2.1

Chemicals, Media, and Stock Solution Preparation .............................................. 9

2.1.1 Chemicals ........................................................................................................ 9
2.1.2 Media and Stock Solution Preparation ............................................................ 9
2.2

Gordonia sp. NB4-1Y Growth Optimization...................................................... 10

2.2.1 Ethanol Toxicity Test .................................................................................... 10
2.2.2 Optimal FTSA & FTAB Concentration for Growth ..................................... 11
2.3

Time-Course Experiment .................................................................................... 11

2.3.1 Inoculum Cultures ......................................................................................... 11
2.3.2 Experimental Cultures ................................................................................... 12
2.3.3 Purging Schedule........................................................................................... 12
2.3.4 Sample Extraction ......................................................................................... 13
2.4

Analysis of Metabolites ....................................................................................... 14
v

2.4.1 Internal Standards and Instrumental Controls ............................................... 14
2.4.2 Analyte Identification and Quantification ..................................................... 14
2.4.3 Sample Preparation ....................................................................................... 15
2.4.4 Instrumental Analysis .................................................................................... 15
2.4.5 Quantitative Process ...................................................................................... 16
3

RESULTS ................................................................................................................... 17
3.1

Gordonia sp. NB4-1Y Growth Optimization...................................................... 17

3.1.1 Ethanol Toxicity ............................................................................................ 17
3.1.2 Optimum FTSA Concentration ..................................................................... 18
3.1.3 Optimum FTAB Concentration ..................................................................... 19
3.2

Experimental Controls......................................................................................... 21

3.2.1 Method Validation......................................................................................... 21
3.2.2 Quality Control .............................................................................................. 22
3.3

Experimental Results........................................................................................... 27

3.3.1 FTSA Liquid Cultures and C18 Eluates ........................................................ 27
3.3.2 FTAB Liquid Cultures and C18 Eluates ....................................................... 31
4

DISCUSSION ............................................................................................................. 36
4.1

Gordonia sp. NB4-1Y Growth Optimization...................................................... 36

4.1.1 Ethanol Toxicity ............................................................................................ 36
4.1.2 Optimum FTSA Concentration ..................................................................... 36
vi

4.1.3 Optimum FTAB Concentration ..................................................................... 37
4.2

FTSA Liquid Cultures and C18 Eluates .............................................................. 38

4.2.1 Analysis of FTSA Liquid Cultures and C18 Eluates .................................... 39
4.2.2 Postulated FTSA Degradation Pathway by NB4-1Y .................................... 41
4.3

FTAB Liquid Cultures and C18 Eluates ............................................................. 42

4.3.1 Analysis of FTAB Liquid Cultures and C18 Eluates .................................... 42
4.3.2 Postulated FTAB Degradation Pathway by NB4-1Y.................................... 44
5

CONCLUSION AND FUTURE WORK ................................................................... 47

6

LITERATURE CITED ............................................................................................... 49

7

APPENDIX ................................................................................................................ 52
7.1

LC-MS Method Parameters Summary ................................................................ 52

7.2

Control Charts – Absolute Area .......................................................................... 53

7.3

Control Charts – Analyte/IS Ratio ...................................................................... 56

7.4

PFAS Summary ................................................................................................... 58

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LIST OF FIGURES
Figure 1-1: Aerobic biodegradation pathways of 6:2-FTOH in activated sludge and aerobic soil
using diverse microbial cultures. ........................................................................................ 5
Figure 1-2: Proposed metabolic pathway for 6:2-FTSA in NB4-1Y pure cultures. ....................... 7
Figure 1-3: Proposed aerobic biotransformation pathway for 6:2-FTSA in activated sludge. ....... 8
Figure 2-1: Experimental culture setup......................................................................................... 12
Figure 3-1: Determination of ethanol tolerance in Gordonia sp. NB4-1Y between 0.5-2.5 % v/v
ethanol. .............................................................................................................................. 17
Figure 3-2: Determination of ethanol tolerance of Gordonia NB4-1Y between 3.0-5.0 % v/v
ethanol. .............................................................................................................................. 18
Figure 3-3: Determination of an optimal concentration of FTSA to be used as a sole-sulfur source
for Gordonia sp. NB4-1Y. ................................................................................................ 19
Figure 3-4: Determination of an optimal concentration of FTAB to be used as a sole-sulfur source
for Gordonia sp. NB4-1Y. ................................................................................................ 20
Figure 3-5: Illustration of quality control approach along the analytical sequence; control chart
used to monitor the signal received upon the detection of 5-ng mL-1 PFBA, PFPeA,
PFHxA, and PFHpA. ........................................................................................................ 23
Figure 3-6: Illustration of quality control approach along the analytical sequence; control chart
used to monitor the signal received upon the detection of 5-ng mL-1 PFBA, PFPeA,
PFHxA, and PFHpA. ........................................................................................................ 24
Figure 3-7: Illustration of quality control approach along the analytical sequence; average retention
time and standard deviation (n = 15) with an average relative standard deviation (RSD) of
0.8 % of monitored PFAs throughout analytical analysis................................................. 25
Figure 3-8: Time course of FTSA degradation in NB4-1Y cultures and production of breakdown
products 6:2-FTOH, 6:2-FTCA, 6:2-FTUA in aqueous samples and their respective C18
eluates over a 168-hr time period...................................................................................... 28
Figure 3-9: Major degradation pathway products of inoculated FTSA cultures and C18 eluate over
a 168-hr time period. ......................................................................................................... 29
Figure 3-10: Minor degradation pathway products of inoculated FTSA cultures over a 168-hr time
period. ............................................................................................................................... 30

viii

Figure 3-11: FTAB degradation in NB4-1Y cultures and production of 6:2-FTOH, 6:2-FTCA, 6:2FTUA in aqueous samples and their respective C18 eluates over a 168-hr time period. . 32
Figure 3-12: Major degradation pathway products of inoculated FTAB cultures and C18 eluate
over a 168-hr time period. ................................................................................................. 33
Figure 3-13: Minor degradation pathway products of inoculated FTAB cultures over a 168-hr time
period. ............................................................................................................................... 34
Figure 4-1: Proposed metabolic pathway utilized by Gordonia sp. NB4-1Y for the desulfonation
of FTSA. ........................................................................................................................... 41
Figure 4-2: Proposed metabolic pathway utilized by Gordonia sp. NB4-1Y for the degradation of
FTAB. ............................................................................................................................... 45
Figure 7-1: Illustration of quality control approach along the analytical sequence; control chart
used to monitor the signal received upon the detection of 5-ng mL-1 3:3-FTCA, 4:3-FTCA,
5:3-FTCA, and 6:2-FTCA. ............................................................................................... 53
Figure 7-2: Illustration of quality control approach along the analytical sequence; control chart
used to monitor the signal received upon the detection of 5-ng mL-1 6:2-FTUA, 6:2-FTSA,
and 87.5-ng mL-1 5:2-FT-ketone....................................................................................... 54
Figure 7-3: Illustration of quality control approach along the analytical sequence; control chart
used to monitor the signal received upon the detection of 87.5-ng mL-1 5:2-sFTOH. ..... 54
Figure 7-4: Illustration of quality control approach along the analytical sequence; control chart
used to monitor the signal received upon the detection of 5-ng mL-1 FTAB. .................. 55
Figure 7-5: Illustration of quality control approach along the analytical sequence; control chart
used to monitor the signal received upon the detection of 87.5-ng mL-1 6:2-FTOH........ 55
Figure 7-6: Illustration of quality control approach along the analytical sequence; control chart
used to monitor the signal received upon the detection of 5-ng mL-1 3:3-FTCA, 4:3-FTCA,
5:3-FTCA, and 6:2-FTCA. ............................................................................................... 56
Figure 7-7: Illustration of quality control approach along the analytical sequence; control chart
used to monitor the signal received upon the detection of 5-ng mL-1 6:2-FTUA, 6:2-FTSA,
and 87.5 ng mL-1 5:2-FT-ketone. ...................................................................................... 56
Figure 7-8: Illustration of quality control approach along the analytical sequence; control chart
used to monitor the signal received upon the detection of 87.5-ng mL-1 5:2-sFTOH and
6:2-FTOH. ......................................................................................................................... 57
ix

Figure 7-9: Illustration of quality control approach along the analytical sequence; control chart
used to monitor the signal received upon the detection of 5-ng mL-1 FTAB. .................. 57

x

LIST OF TABLES
Table 3-1: Linearity and instrumental detection limits for the expected fluorinated metabolites,
FTSA, and FTAB. ............................................................................................................. 21
Table 3-2: Procedural blank analysis conducted during the months of October and November
throughout experimental analysis. .................................................................................... 26
Table 3-3: Percent recovery of fluorinated analytes and concentration of analytes identified in the
aqueous and volatile phase in inoculated FTSA cultures at specific time points. ............ 31
Table 3-4: Percent recovery of fluorinated analytes and proportion of analytes identified in the
aqueous and volatile phase in inoculated FTAB cultures at specific time points. ............ 35
Table 7-1: Summary of LC-MS method parameters .................................................................... 52
Table 7-2: PFAS analytes in the study, their acronym, and molecular structure.......................... 58

xi

1

INTRODUCTION

1.1

Gordonia spp.
The genus of microorganisms known as Gordonia of the family Gordoniaceae, are mycolic

acid-containing, Gram-positive, soil bacteria of the order Actinomycetales (Drzyzga 2012). As of
2012, the Gordonia genus comprised of 32 species isolated from a wide-range of environmental
habitats, much like their mycolic acid-containing relatives Mycobacteria and Rhodococcus
(Drzyzga 2012). Strains have been isolated from contaminated soil and wastewater, estuary sand,
mangrove rhizosphere, oil-producing wells, biofilters, activated sludge, and clinical samples
(Drzyzga 2012). The specific strain of Gordonia discussed in this thesis, NB4-1Y, was isolated
from enriched vermicompost by Van Hamme et al. (2013) using bis-(3-pentafluorophenylpropyl)sulfide (PFPS) as a novel sulfur source. Unusual compounds, such as PFPS or other non-naturally
occurring compounds, have been used previously to isolate unique bacterial strains (Arenskotter
et al. 2004), an example being Rhodococcus sp. JVH1, also isolated for its C—S bond cleaving
ability using PFPS (Van Hamme et al. 2004).
Gordonia have received much interest from researchers in the environmental, industrial,
biotechnological, and medical fields (Drzyzga 2012). Gordonia possess unique abilities to degrade
xenobiotics, toxic environmental pollutants, hydrocarbons, and natural compounds that are not
readily biodegradable (Arenskotter et al. 2004). The plethora of chemical compounds that the
Gordonia genus is capable of transforming, degrading, and synthesizing make them a strong
candidate for a variety of environmental and industrial applications (Drzyzga 2012). In fact, as of
October 2011, over 65 patents have been registered in relation to the Gordonia species (Drzyzga
2012).

1

This study focusses on the preliminary steps involved in developing potential
bioremediation technologies for anthropogenic fluorinated chemicals. Here, the bacterial strain
NB4-1Y of the Gordonia genus is examined for its capability to metabolize the heavily fluorinated
environmental contaminants, 6:2 fluorotelomer sulfonamide alkyl betaine (FTAB) and 6:2
fluorotelomer sulfonate (FTSA).

1.2

Per- and Poly-fluorinated Contaminants
Incorporating fluorine into organic compounds can increase stability and alter chemical

properties such as lipophilicity and acidity (Murphy 2010). Specific to this thesis, per- and polyfluoroalkyl substances (PFASs) are a group of aliphatic fluorinated chemicals in which all (per-)
or nearly all (poly-) carbons are fluorinated, exclusive of the terminal functional group (Boiteux et
al. 2017) (Table 7-2). Consequently, PFASs exhibit unique characteristics that have been imparted
into various products to increase qualities like oil and water repellency, and reduction in surface
tension such as in fire-fighting foams (Houtz et al. 2016).
The manufacturing of PFAS-based surfactants and polymers has been occurring at large
scale since the 1950’s (Butt et al. 2014; Houtz et al. 2016), although environmental concerns have
emerged only due to their global detection in the environment, biota, and humans (Boiteux et al.
2017). Concerns about PFASs in the environment are related to:
1. The strength of the carbon-fluorine bond and associated resistance to hydrolysis,
photolysis, and biodegradation resulting in their persistence and bioaccumulation
in water, soil, and the environment (Boiteux et al. 2017);

2

2. Suspected adverse health effects (Krafft and Riess 2015) and toxicity (Phillips et al.
2007; Newsted et al. 2008);
3. Their incomplete removal during sewage treatment (Higgins et al. 2005;
Loganathan et al. 2007);
4. Their widespread use in surfactants, grease and water repellents, lubricants, and of
particular interest to this study, aqueous film-forming foams (AFFFs) (Clarke and
Smith 2011).
AFFF products containing fluorinated surfactants are used in industrial and military
firefighting to extinguish hydrocarbon-fuel fires (Zhang et al. 2016). These products typically
contain a per- or poly-fluoroalkylthiol carbon chain appended with an alkylamidosulfonate or
alkylbetaine terminal group (e.g. FTAB found in Table 7-2) (Zhang et al. 2016). Upon the release
of AFFF products such as Forafac® 1157, which contains FTAB (Moe et al. 2012), biodegradation
studies have demonstrated that FTAB is degraded into 6:2 fluorotelomer sulfonamide (6:2FTSAOA) in blue mussels (Moe et al. 2012) and FTSA in fire-fighting areas and laboratory
microcosms (Schultz et al. 2004). FTSA is listed under the Unregulated Contaminant Monitoring
Regulation (UCMR 3; US EPA enforces health standards for contaminants in drinking water) (Park
et al. 2016) and has been observed in concentrations as high as 14,600-µg/L at military sites
(Schultz et al. 2004). Degradation of FTSA begins with desulfonation to form 6:2 fluorotelomer
aldehyde (FTAL) and the release of HSO3-, bypassing the formation of 6:2-FTOH in both activated
sludge (Wang et al. 2011; Liu and Avendaño 2013) and in pure culture (Van Hamme et al. 2013).
In activated sludge, the continued degradation of 6:2-FTAL continues along the well characterized
pathways observed in 6:2-FTOH degradation (Wang et al. 2011).

3

1.3

6:2-FTOH Degradation Pathway in Diverse Microbial Communities
The aerobic biodegradation of fluorotelomer alcohols (FTOHs) such as 8:2-FTOH and 6:2-

FTOH have been well studied (Dinglasan et al. 2004; Liu et al. 2010). In a review by Liu and
Avendaño (2013), the aerobic biodegradation pathways of 6:2-FTOH demonstrated in both
activated sludge and aerobic soils (Figure 1-1) are summarized. Analysis of 6:2-FTOH aerobic
degradation using diverse microbial communities revealed that 6:2-FTOH was degraded into five
terminal metabolites: PFBA, PFPeA, PFHxA, 5:3-FTCA, and 4:3-FTCA (Liu et al. 2010).

4

Figure 1-1: Aerobic biodegradation pathways of 6:2-FTOH in activated sludge and aerobic soil
using diverse microbial cultures.
Double arrows indicate multiple transformation steps. Blue pathways have only been seen in soil
(Liu et al. 2010), while red pathways have only been observed in sludge (Wang et al. 2012). Stable
and semi-stable compounds are in dashed boxes. Figure from Liu and Avendaño (2013).
5

Van Hamme et al. (2013) indicate that as of April 2013, the majority of research has
focused on the microbial degradation of PFASs utilizing mass loads in sewage treatment plants
(Sinclair and Kannan 2006; Loganathan et al. 2007), in vitro using mixed sewage inocula
(Dinglasan et al. 2004; Rhoads et al. 2008), or in soil microcosms (Russell et al. 2008). Further
studies employing pure cultures are necessary in beginning to understand the biochemistry and
molecular biology utilized by bacteria in PFAS degradation (Van Hamme et al. 2013).
As previously noted, FTSA degradation has been described to bypass 6:2-FTOH
production and convert straight into 6:2-FTAL (Wang et al. 2011; Van Hamme et al. 2013). The
metabolites described in Figure 1-1 from 6:2-FTAL degradation, provide a reference point for
analysis of FTSA degradation using pure cultures of Gordonia sp. NB4-1Y.

1.4

FTSA Degradation Pathway in Gordonia sp. NB4-1Y Pure Cultures
Desulfonation is the initial step required for FTSA to be biologically and chemically

transformed into downstream products (Zhang et al. 2016). Desulfonation of FTSA by microbes
was determined to be the rate-limiting step for further biodegradation (Wang et al. 2011). Van
Hamme et al. (2013) describe a potential biotransformation pathway for FTSA using pure cultures
of Gordonia sp. NB4-1Y (Figure 1-2). Wang et al. (2011) propose a similar pathway using mixed
bacterial cultures in closed bottles of activated sludge from waste water treatment plants (Figure
1-3). However, Wang et al. (2011) propose that 5:3-acid is a minor product of FTSA degradation,

6

and the volatile compounds 5:2-ketone and 5:2sFTOH

comprise

the

majority

of

transformation products. For the present study,
culture vessels were designed to ensure volatile
metabolites would be captured if NB4-1Y
could produce them.

Figure 1-2: Proposed metabolic pathway for 6:2-FTSA in NB4-1Y pure cultures.
Figure from Van Hamme et al. (2013).

7

Figure 1-3: Proposed aerobic biotransformation pathway for 6:2-FTSA in activated sludge.
Double arrows indicate multiple transofrmation steps. The analyte 5:3-acid (5:3-FTCA) is
proposed to be part of a ‘minor’ pathway, with 5:2-ketone (5:2-FT-ketone) being inferred as the
‘major’ pathway. Figure from Wang et al. (2011).

1.5

Goals of this Study
This study set out with three goals in mind. First, we wanted to identify the majority of the

metabolites produced by Gordonia sp. NB4-1Y when 6:2 fluorotelomer sulfonate or 6:2
fluorotelomer sulfonamide alkyl betaine are provided as the sole-sulfur source. Second, we wanted
to quantify all metabolites produced during this degradation in order to generate a mass balance.
The last goal of this study was to understand the degradation pathways Gordonia sp. NB4-1Y
utilizes to metabolize FTSA and FTAB.

8

2

MATERIALS AND METHODS

2.1

Chemicals, Media, and Stock Solution Preparation

2.1.1 Chemicals
The following compounds were purchased from Fischer Chemical: KH2PO4
(Crystalline/Certified ACS, ≥ 99 % pure; 0.003 % sulfate max), K2HPO4 (Crystalline
powder/Certified ACS, ≥ 98.0 % pure; 0.005 % sulfate max), NH4Cl (Crystalline/Certified ACS,
≥ 99.5 % pure; 0.002 % sulfate max), NaCH3COO- (Fused crystals/Certified ACS, ≥ 99 % pure;
0.003 % sulfate max), and acetonitrile (HPLC grade, submicron filtered, ≥ 99.9 % pure). MgSO4
from J.T. Baker (anhydrous, powder; ≥ 99.0 % pure) was used in this experiment. De-ionized
water (18-MΩ) for media and stock solutions was generated with a Barnstead Nanopure Diamond
system. Anhydrous ethyl alcohol from Commercial Alcohols (100 % v/v) was used.
The ammonium salt of 6:2-FTSA was obtained from Synquest Laboratories (certified 98.0
% pure by titration with NaOH). A 0.27-g mL-1 solution of FTAB was provided by McGill University.
Calibration standards for PFBA, PFPeA, PFHxA, PFHPa, 3:3-FTCA. 4:3-FTCA, 5:3-FTCA, 6:2FTCA, 6:2-FTOH. 5:2-FT-ketone, 5:2-sFTOH, 6:2-FTSA, and 6:2-FTAB and labelled internal
standards MPFBA, MPFPeA, MPFHxA, MPFOA, M62-FTCA, M62-FTS, and M62-FTUA came
from Wellington Laboratories (Guelph ON (CAN)). Maxi-Clean 600-mg SPE C18 cartridges were
purchased from GRACE (Deerfield IL (USA)).

2.1.2 Media and Stock Solution Preparation
Sulfur-free acetate (SFA) media was prepared according to Van Hamme et al. (2004) with
the following composition per liter: 0.40-g potassium dibasic phosphate (KH2PO4), 1.60-g

9

potassium monobasic phosphate (K2HPO4), 1.55-g ammonium chloride (NH4Cl), 5.00-g sodium
acetate (NaCH3COO), 0.165-g magnesium chloride (MgCl2), 0.090-g calcium chloride dihydrate
(CaCl2 • 2H2O), 5.00-mL Wolfe’s minerals, and 1.00-mL Pfenning’s vitamin solution. The
appropriate masses for KH2PO4, K2HPO4, NH4Cl, and NaCH3COO were added to a specific
volume before being autoclaved at 121oC for 20 minutes. The solution was allowed to cool to room
temperature before MgCl2, CaCl2 • H2O, Wolfe’s minerals, and Pfenning’s vitamin solution were
added aseptically in a flowhood. Wolfe’s minerals and Pfenning’s vitamins solution were added
as a 0.22-µM pore sized filter sterilized solution to cooled medium after autoclaving. Carbon-free
media was prepared the same as sulfur-free media except for the addition of acetate. Stock
solutions of MgSO4, FTSA, and FTAB were prepared at 4-mM in a 50:50 mixture of anhydrous
ethanol and 18-MΩ water. The FTSA solution was heated to 55oC and stirred overnight to fully
dissolve the solution.

2.2

Gordonia sp. NB4-1Y Growth Optimization

2.2.1 Ethanol Toxicity Test
Gordonia sp. NB4-1Y cultures were grown using various concentrations of ethanol to
determine an appropriate volume of ethanol to solubilize FTSA and FTAB in and henceforth,
optimize growth conditions. A liquid inoculum culture containing 100-µL of anhydrous ethanol
(1.0 % v/v), 25-µL of MgSO4 stock, 375-µL of sterilized 18 MΩ water, and 9.50-mL of carbonfree media was inoculated from fresh plate culture and incubated for 92-hrs at 30oC with constant
mixing. The range 0.5-5.0 % v/v of ethanol in a total culture volume of 15-mL was tested. Two
replicates of each concentration contained 150-µL of liquid inoculum culture, 25-µL of MgSO4

10

stock, 75-750 µL of anhydrous ethanol, 0-675 µL of sterilized 18 MΩ water, and 14.1-mL of
carbon-free media. Optical density was measured at 600 nm every 24-hrs for 168-hrs.

2.2.2 Optimal FTSA & FTAB Concentration for Growth
Gordonia sp. NB4-1Y cultures were grown using various concentrations of FTSA or FTAB
as the sole-sulfur source, to determine an optimal concentration for growth. A liquid inoculum
culture containing 125-µL of FTSA or FTAB stock (50-µM of FTSA/FTAB), 125-µL of 50 %
ethanol solution, and 9.75-mL of SFA medium was inoculated from plate culture and incubated
for 92-hrs at 30oC in a 50-mL rolling tube. Over a concentration range of 4 to 100-µM, two
replicates for each specified concentration of FTSA or FTAB was tested over a 168-hr period.
Rolling tube cultures contained 150-µL of inoculum, 15.0 to 375-µL of FTSA or FTAB stock, 0
to 360-µL of 50 % ethanol solution, and 14.475-mL of SFA media. Each culture had a starting
ethanol concentration of 1.25 % v/v. Optical density readings were measured every 24-hrs of
growth using a wavelength of 600 nm.
2.3

Time-Course Experiment

2.3.1 Inoculum Cultures
Inoculum cultures were prepared aseptically in sterilized 100-mL serum bottles with the
following composition: 150-µL of their respective stock solution and 9850-µL of SFA. Two
replicates were made for each condition: FTSA, FTAB, and MgSO4. Two replicates for sterile
FTSA, FTAB, MgSO4 cultures were also made. Two replicates of a negative control were also
made and contained 150-µL of 50 % ethanol solution and 9850-µL of SFA. Cultures were sealed
air-tight with a butyl rubber septum and aluminum crimp cap. Cultures were grown for 72-hrs. at
30oC and 150 rotations per minute.
11

2.3.2 Experimental Cultures
Experimental cultures were prepared in sterilized 100-mL serum bottles aseptically in a
flowhood with the following composition: 200-µL of their respective inoculum culture, 300-µL of
their respective chemical stock, and 19.50-mL of SFA media. Each
culture contained 60-µM of target sulfur compound. Prior to
inoculation, experimental cultures were sealed using a foam plug and
incubated for 48-hrs. at 150 rotations per minute. After inoculation,
bottles were sealed air-tight with a butyl rubber septum and aluminum
crimp cap. Butyl rubber septa were sterilized using isopropanol prior
to inserting two 20 G needles (Becton Dickinson and Company,
Franklin NJ (USA)). A solid-phase C18 cartridge (GRACE 600-mg)
Figure 2-1: Experimental
was inserted into one needle while the other needle was inserted culture setup.
temporarily for purging cultures with sterilized air to replenish oxygen levels and capture volatile
metabolites. A total of 25 replicates were produced for each condition, yielding 175 cultures total.

2.3.3 Purging Schedule
Oxygen levels for each culture condition were monitored using a Firesting O2 Fibre-optic
Oxygen Meter and Pyro Oxygen Logger software (Ohio Lumex Company Inc., Solon OH (USA)).
One culture for each condition contained an oxygen sensor enabling the measurement of the
depletion of oxygen levels. To ensure each culture was purged for the same length of time, the
culture condition whose oxygen levels were depleted the greatest determined the length of time
each culture was purged to fully replenish their oxygen levels.
12

Prior to purging, the surface of the butyl rubber septa were sterilized with isopropanol and
a fresh 20G needle was inserted. An AquaClear 50 aquarium air pump (Rolf C. Hagen Corp.,
Mansfield MA (U.S.A)) customized with a HEPA-CAPTM glass microfiber filter with
polypropylene housing, two 0.30-µM membrane filters, and one 0.40-µM membrane filter was
used to purge air into each culture. The pump had the following parameters: 120 Volts AC/60
Hz/3.5 watts, 2 x 2 500 cc/min, and a pounds per square inch (psi) of 2.5 lb/po2. Purging time
varied from 20 – 120 seconds and after purging, the purge needle was discarded.

2.3.4 Sample Extraction
After purging, four replicates from each condition (28 total) were taken for plating and
extraction. Two replicates for each condition were plated aseptically on nutrient agar and incubated
for 72-hrs at 30oC in a INFORS HT Multitron Pro incubator. After plating, each replicate was
treated with three volumes of acetonitrile (60-mL) and incubated for one hour at 30oC, 150 rpm.
A 10-mL aliquot of the supernatant was placed in a 50-mL polypropylene centrifuge tube and
centrifuged in a Thermo Scientific ST 16R centrifuge at 4000  g for ten minutes. Centrifuged
samples were filtered using a syringe and 0.40-µM attachable membrane filter into a
polypropylene sample vial. Aqueous samples were stored at -20oC until analysis. Solid-phase C18
cartridges were removed after purging and eluted with 3-mL of acetonitrile into sample vials
during the 1-hr incubation. C18 eluate samples were stored in polypropylene sample vials at -20oC
until analysis.

13

2.4

Analysis of Metabolites

2.4.1 Internal Standards and Instrumental Controls
Solutions

spiked

with

5-ppb

of

unlabeled

perfluorobutanoic

acid

(PFBA),

perfluoropentanoic acid (PFPeA), perfluorohexanoic acid (PFHxA), perfluoroheptanoic acid
(PFHpA), 3:3 fluorotelomer carboxylic acid (3:3-FTCA), 4:3 fluorotelomer carboxylic acid (4:3FTCA), 5:3 fluorotelomer carboxylic acid (5:3-FTCA), 6:2 fluorotelomer carboxylic acid (6:2FTCA), 6:2 fluorotelomer unsaturated acid (6:2-FTUA), FTSA, and FTAB, as well as 87.7-ppb of
5:2 secondary fluorotelomer alcohol (5:2-sFTOH), 5:2 fluorotelomer ketone (5:2-FT-ketone), and
6:2 fluorotelomer alcohol (6:2-FTOH) were used for quality control analysis. A linear range and
instrument limit of detection (iLOD) for PFBA, PFPeA, PFHxA, PFHpA, 3:3-FTCA, 4:3-FTCA,
5:3-FTCA, 6:2-FTCA, 5:2-sFTOH, 5:2-FT-ketone, 6:2-FTOH, FTSA and FTAB was determined
(Table 3-1).
Quality control samples were injected every 20 samples along the analytical sequence.
Absolute area, the analyte to internal standard (IS) response ratio, retention time, and procedural
blanks were monitored for each of the expected metabolites. Quality control is essential for
ensuring the production of reliable data with a minimum amount of error, and to produce
consistent, reliable data.

2.4.2 Analyte Identification and Quantification
Due to the lack of analytical standards for some of the investigated compounds, isotopelabelled ISs with similar chemical structure were used to quantify some compounds. Proposed by
Backe et al. (2013), analytes can be classified into three categories based on the availability of

14

isotope-labelled ISs: quantitative (Qn) analytes, semi-quantitative (Sq), and qualitative (Ql). All
of the analytes for this project were quantitative. Quantitative analytes are those of which the native
analyte has a suitable corresponding IS available (Munoz et al. 2016). For example, MPFOA is
used to quantify PFHpA as they are both similar in chemical structure (perfluoroalkyl
carboxylates), a method optimized and validated by Munoz et al. (2016). All of the expected
metabolites in this experiment are considered to be quantitative analytes, as ISs with similar
molecular structure were available. An IS solution containing MPFBA, MPFPeA, MPFHxA,
MPFOA, M62-FTS, M62-FTCA, and M62-FTUA was used to quantify the expected metabolites.
Each component of the IS solution corresponds to one or several analytes. A summary of the
quantitative analytes and their corresponding IS can be found in the Appendix: Table 7-2.

2.4.3 Sample Preparation
C18 eluate samples were diluted by a factor of 1.25 and contained 160-µL of eluate sample
in ACN and 40-µL of an IS solution at 25-ng mL-1. Aqueous samples were diluted one to two
times. Dilution level 1 was a factor of 16 containing 50-µL of the extracted sample, 110-µL of
ACN, and 40-µL of IS solution at 25-ng mL-1. Dilution level 2 was a factor of 400 containing 40µL of the diluted sample, 120-µL of ACN, and 40-µL of IS solution at 25-ng mL-1.

2.4.4 Instrumental Analysis
Chromatographic operating conditions were adapted from a procedure developed by
Munoz et al. (2016). The Dionex Ultimate 3000 LC chain was operated out of the University of
Montreal. A Thermo Hypersil Gold aQ column (100 × 2.1 mm; dp = 1.9-μm) was used for analyte
15

separation and a Thermo Hypercarb column (20 x 2.1 mm; dp = 7 µM) was the delay column used.
The aqueous mobile phase consisted of HPLC-water with 0.15 % acetic acid (v/v) and the organic
phase consisted of acetonitrile with 0.15 % acetic acid (v/v). Flow rate was set at 0.55-mL min-1,
injection volume at 15-µL, and the column inlet temperature at 40oC. Details on chromatographic
gradient elution conditions are supplied in the Appendix: Table 7-2. The heated electrospray
ionization source was used with the following conditions: sheath gas flow rate was 45 arbitrary
units (a.u.), auxiliary gas flow rate was 10 a.u., sweep gas flow rate was 10 a.u., and capillary
temperature was 320oC. Spray voltage was either -3-kV or +3-kV operating in fast polarityswitching mode. Analyte detection was performed using a Q-Exactive Orbitrap mass spectrometer.
Orbitrap parameters were set as follows: AGC was set at 5x106, maximum injection time at 50ms, and resolution at 70,000. The mass scan range was set at 150-700 m/z (full scan MS mode). A
summary of the method parameters can be found in the Appendix: Table 7-2.

2.4.5 Quantitative Process
Analytes were quantified in the final diluted aqueous and C18 eluate samples using the
analyte/IS ratios and worked back through dilution factors to determine the amounts in the original
aqueous samples and C18 eluate. These ng mL-1 values were then converted to mol % to represent
concentrations collected from the aqueous phase and C18 eluates.

16

3

RESULTS

3.1

Gordonia sp. NB4-1Y Growth Optimization

3.1.1 Ethanol Toxicity
Given that PFAS stock solutions were prepared in ethanol, the concentration of ethanol
that Gordonia sp. NB4-1Y could tolerate was determined by exposing cultures to ethanol
concentrations ranging from 0.5 to 2.5 % v/v and monitoring OD600 nm. At ethanol concentrations
of 0.5 and 1.0 %, NB4-1Y grew to an OD600 nm of 0.387 and 0.410 after 24-hrs, and 0.517 and
0.544 after 144-hrs, respectively. Cultures grown at an ethanol concentration of 2.5 % had an
average OD600

nm

of 0.136 after 24-hrs and 0.620 after 144-hrs (Figure 3-1).

1.000

OD600 nm

0.5%
1.0%
1.5%
2.0%
2.5%

0.100
0

24

48

72

96

120

144

168

Time Incubated (Hours)

Figure 3-1: Determination of ethanol tolerance in Gordonia sp. NB4-1Y between 0.5-2.5 % v/v
ethanol.
Exponential growth of NB4-1Y is delayed upon increasing ethanol concentration from 0.5-2.5 %.

17

Given that NB4-1Y appeared to tolerate 2.5 % v/v ethanol, the experiment was repeated
with concentrations between 3.0 and 5.0 % v/v. At 3.0 % v/v ethanol, OD600 nm values of 0.073 and
0.712 were observed after 24 and 144-hrs of incubation, respectively. Cultures grown at an ethanol
concentration of 5.0 % v/v had an average absorbance of 0.008 and 0.079 after 24 and 144-hrs of
incubation (Figure 3-2), an order of magnitude lower than at concentrations below 4.0 % v/v.
1.000

0.100

OD600 nm

3.0%
3.5%
4.0%
4.5%

0.010

5.0%

0.001
0

24

48

72

96

120

144

168

Time Incubated (Hours)

Figure 3-2: Determination of ethanol tolerance of Gordonia NB4-1Y between 3.0-5.0 % v/v
ethanol.
Exponential growth is further delayed upon increasing ethanol concentration past 2.5 %.
Toxicological effects are experience in ethanol concentrations greater than 4.0 %.

3.1.2 Optimum FTSA Concentration
In order to optimize conditions for detecting FTSA metabolites in NB4-1Y cultures, the
organism was exposed to FTSA concentrations ranging from 4 to 100-µM. At 4-µM, NB4-1Y
grew to an OD600 nm of 0.039 after 24-hrs and 0.249 after 168-hrs of incubation. When supplied
18

with 100-µM FTSA, cultures grew to OD600 nm 0.028 after 24-hrs and 0.565 after 168-hrs (Figure
3-3). There was a 52.8 % increase in overall growth by increasing the concentration of FTSA from
20-µM (0.207 after 168-hrs) to 40-µM (0.392 after 168-hrs) (Figure 3-3). A FTSA concentration
of

60-µM

was

utilized

in

the

growing

of

NB4-1Y

cultures

in

this

study.

1.000

OD600 nm

100 µM
80 µM
60 µM

0.100

40 µM
20 µM
10 µM
4 µM

0.010
0

20

40

60

80

100

120

140

160

180

Time Incubated (Hours)

Figure 3-3: Determination of an optimal concentration of FTSA to be used as a sole-sulfur source
for Gordonia sp. NB4-1Y.
No apparent toxicological effects are exhibited by NB4-1Y in the 4-100-µM range. An increase in
overall growth is observed when increasing the concentration of FTSA from 20 to 40-µM.
3.1.3 Optimum FTAB Concentration
In order to optimize conditions for detecting FTAB metabolites in NB4-1Y cultures, the
organism was exposed to FTAB concentrations ranging from 4 to 100-µM. At 4-µM, NB4-1Y
grew to an OD600 nm of 0.023 after 24-hrs and 0.095 after 168-hrs of incubation. When supplied
19

with 100-µM FTAB, cultures grew to an OD600 nm of 0.035 after 24-hrs and 0.316 after 168-hrs
(Figure 3-4). There was a 57.7 % increase in overall growth between increasing the concentration
of FTAB from 20-µM (0.126 after 168-hrs) to 40-µM (0.218 after 168-hrs) (Figure 3-4). At a
concentration of 80-µM, NB4-1Y exhibited increased growth (0.333), while at 100-µM NB4-1Y
showed

a

slight

decrease

(0.316).

1.000

OD600 nm

100 µM
80 µM
60 µM

0.100

40 µM
20 µM
10 µM
4 µM

0.010
0

20

40

60

80

100

120

140

160

180

Time Incubated

Figure 3-4: Determination of an optimal concentration of FTAB to be used as a sole-sulfur source
for Gordonia sp. NB4-1Y.
No apparent toxicological effects are exhibited by NB4-1Y in the 4-100-µM range tested. A
significant increase in overall growth is seen when increasing the concentration of FTAB from 20
to 40-µM.

20

3.2

Experimental Controls

3.2.1 Method Validation
3.2.1.1 Linearity and Instrumental Detection Limits
A linear range and instrumental limit of detection for FTSA, FTAB, and the twelve
expected metabolites was determined prior to sample analysis. The coefficient of determination
(R2) for each analyte was >0.9920 and the instrumental limit of detection for the compounds
ranged from 0.04 to 8-ng mL-1 (Table 3-1).
Table 3-1: Linearity and instrumental detection limits for the expected fluorinated metabolites,
FTSA, and FTAB.
Compound

R2

Linear Range (ng mL-1)

iLOD (ng mL-1)

PFBA

0.9962

0.29-34

0.09

PFPeA

0.9959

0.29-34

0.09

PFHxA

0.9970

0.06-34

0.04

PFHpA

0.9986

0.29-34

0.08

6:2-FTAB

0.9994

0.29-34

0.18

6:2-FTSA

0.9928

0.29-34

0.17

3:3-FTCA

0.9927

0.29-34

0.2

4:3-FTCA

0.9970

0.29-34

0.16

5:3-FTCA

0.9973

0.29-34

0.27

6:2-FTCA

0.9966

0.06-34

0.07

6:2-FTUA

0.9965

0.06-34

0.06

6:2-FTOH

0.9931

10-600

8

5:2-sFTOH

0.9938

1-600

0.9

5:2-FT-ketone

0.9956

1-600

1

21

3.2.2 Quality Control
3.2.2.1 Absolute Area
In this study, control charts for absolute area, analyte/IS response ratio, and retention time
were utilized to evaluate the performance of the LC-MS/MS and ensure precise and accurate
measurements of each analyte. Control charts are a visual representation of the confidence interval
of some important parameter which represents the quality assurance or the integrity of the study.
Confidence intervals are a range of values so defined that there is a specified probability that the
value of a parameter will lie within it. For example, analysis of a solution containing 5-ng mL-1 of
PFBA should yield a constant response every time a solution with 5-ng mL-1 of PFBA is analyzed.
PFBA, PFPeA, PFHxA, PFHxA, 3:3-FTCA, 4:3-FTCA, 5:3-FTCA, 6:2-FTUA, and 6:2FTSA showed relatively constant values for absolute area throughout quality control while 6:2FTCA, 5:2-FT-ketone, 5:2-sFTOH, 6:2-FTAB, and 6:2-FTOH fluctuated. The control chart for

22

PFBA, PFPeA, PFHxA, and PFHpA is presented here (Figure 3-5), and others can be found in the
Appendix: Figure 7-1 to Figure 7-4.
30000000

Absolute Area

25000000

20000000
15000000

PFBA
PFPeA

10000000

PFHxA
PFHpA

5000000
0

Figure 3-5: Illustration of quality control approach along the analytical sequence; control chart
used to monitor the signal received upon the detection of 5-ng mL-1 PFBA, PFPeA, PFHxA, and
PFHpA.

3.2.2.2 Analyte to Internal Standard Response Ratio
Control charts for the analyte/IS response ratio were monitored throughout the experiment
to ensure accurate quantification of each analyte. PFBA, PFPeA, PFHxA, PFHpA, 3:3-FTCA, 4:3FTCA, 5:3-FTCA, 6:2-FTUA, 5:2-FT-ketone, 6:2-FTSA, 5:2-sFTOH, 6:2-FTOH showed
relatively constant analyte/IS ratios throughout quality control analysis, while 6:2-FTCA and 6:2FTAB fluctuated. The control chart for PFBA, PFPeA, PFHxA, and PFHpA is presented here

23

(Figure 3-6) and the remaining control charts can be found in the Appendix: Figure 7-5 to Figure
7-9.
Figure 3-6: Illustration of quality control approach along the analytical sequence; control chart
used to monitor the signal received upon the detection of 5-ng mL-1 PFBA, PFPeA, PFHxA, and
PFHpA.

3.2.2.3 Retention Time
The retention time among the 14 analytes in the quality control samples varied very little (Figure
3-7).
7.000

Average Retention Time (minutes)

6.000

5.000

4.000

3.000

2.000

1.000

0.000

Figure 3-7: Illustration of quality control approach along the analytical sequence; average retention
time and standard deviation (n = 15) with an average relative standard deviation (RSD) of 0.8 %
of monitored PFAs throughout analytical analysis.
24

25

3.2.2.4 Procedural Blanks
Procedural blanks were conducted throughout experimental analysis to monitor for trace
amounts of carryover and for method and procedure validation.
Table 3-2: Procedural blank analysis conducted during the months of October and November
throughout experimental analysis.

26

3.3

Experimental Results

3.3.1 FTSA Liquid Cultures and C18 Eluates
3.3.1.1 Initial Steps in FTSA Degradation
The initial breakdown products of FTSA (6:2-FTCA, 6:2-FTUA, 6:2-FTOH) were
quantified over a 168-hr incubation period, during which time NB4-1Y degraded the FTSA from
100.55 to 0.03 mol % (Figure 3-8). The metabolites 6:2-FTCA and 6:2-FTUA were present at the
beginning of incubation (0-hr) and their concentrations increased in the aqueous phase from 0.02
to 4.28 mol % and 0.45 to 13.68 mol %, respectively, over the 168-hr incubation period. 6:2-FTOH
was found to increase in concentration in the eluate of the C18 cartridges from 0.17 to 4.14 mol %
between 96 and 168-hrs of incubation (Figure 3-8). In sterile FTSA cultures, the concentration of
FTSA ranged from 90.27 mol % at 0-hr to 129.75 mol % after 72-hrs, to 105.11 mol % after 168hrs of incubation (Figure 3-8), averaging 103.53 mol %. Additional degradation products were

27

quantified and split into two groups, major and minor degradation pathways, as presented below.
16.0%

125.0%
12.0%
100.0%
75.0%

8.0%

50.0%
4.0%
25.0%

Mol % of 6:2-FTOH, FTCA, & FTUA

Mol % of 6:2-FTSA & Sterile FTSA

150.0%

Sterile FTSA

6:2-FTSA

6:2-FTOH

6:2-FTCA

6:2-FTUA

0.0%

0.0%
0

24
48
72
96
Time Incubated (Hours)

168

Figure 3-8: Time course of FTSA degradation in NB4-1Y cultures and production of breakdown
products 6:2-FTOH, 6:2-FTCA, 6:2-FTUA in aqueous samples and their respective C18 eluates
over a 168-hr time period.
FTSA, 6:2-FTCA, and 6:2-FTUA are present in the aqueous phase of inoculated FTSA cultures,
while 6:2-FTOH (red) was identified in the C18 eluate of inoculated FTSA cultures. Sterile FTSA
(green) represents the quantification of FTSA present in sterile FTSA cultures. Four replicates for
each time point were analyzed (n=4).

3.3.1.2 Major Degradation Pathway Products
NB4-1Y converted 43.68 mol % of total FTSA into the major metabolite 5:2-FT-ketone,
which was recovered from C18 cartridges (Figure 3-9). 5:2-FT-ketone first appeared after 24-hrs
of incubation. Approximately 8.97 mol % of total FTSA was converted into 5:2-sFTOH after 168hrs of incubation and first appeared after 96-hrs. PFHxA was present as 0.097 mol % beginning at
0-hr and increased to 0.534 mol % after 168-hrs. In the aqueous phase, PFPeA appeared after 7228

hrs yielding 0.002 mol % and increased to 0.075 mol % after 168-hrs (Figure 3-9).
0.80%

40.0%

0.60%

30.0%
0.40%
20.0%
0.20%

10.0%

0.0%

Mol % of PFPeA & PFHxA

Mol % of 5:2-ketone & sFTOH

50.0%

5:2-ketone

5:2-s-FTOH
PFPeA
PFHxA

0.00%
0

24
48
72
96
Time Incubated (Hours)

168

Figure 3-9: Major degradation pathway products of inoculated FTSA cultures and C18 eluate over
a 168-hr time period.
5:2-FT-ketone and 5:2-sFTOH are present in C18 eluate while PFPeA and PFHxA are present in
inoculated FTSA cultures. Four replicates for each time point were analyzed (n=4).

3.3.1.3 Minor Degradation Pathway Products
The metabolite 5:3-FTCA appeared after 48-hrs yielding 0.099 mol %, peaked at 0.726
mol % after 96-hrs, and finished at 0.351 mol % after 168-hrs of incubation. The metabolites PFBA
and 4:3-FTCA were first identified after 168-hrs yielding 0.050 and 0.020 mol %, respectively

29

(Figure

3-10).
0.80%

0.60%

Mol %

5:3-FTCA
0.40%
4:3-FTCA
PFBA

0.20%

0.00%
0

24

48
72
96
Time Incubated (Hours)

168

Figure 3-10: Minor degradation pathway products of inoculated FTSA cultures over a 168-hr time
period.
5:3-FTCA, 4:3-FTCA, and PFBA were identified in the aqueous phase in extremely small
quantities. Four replicates for each time point were analyzed (n=4).

3.3.1.4 Percent Recovery
After 168-hrs, more than 50 % of the metabolites produced from FTSA degradation were
found in the volatile phase (eluted from C18 cartridges), while approximately 1/5 of the total FTSA
was converted to water-soluble metabolites. The percent recovery for FTSA and the expected
metabolites ranged from 74.09-105.92 % (Table 3-3), yielding an average recovery rate of 88.21
%.

30

Table 3-3: Percent recovery of fluorinated analytes and concentration of analytes identified in the
aqueous and volatile phase in inoculated FTSA cultures at specific time points.
HOURS
INCUBATED

AQUEOUS TOTAL
(µm)

C18 ELUATE
TOTAL (µm)

TOTAL (µM)

% RECOVERY

0

60.76

0.00

60.76

101.27

24

57.57

0.00

57.58

95.96

48

44.52

0.32

44.84

74.73

72

61.86

1.69

63.55

105.92

96

40.61

3.84

44.45

74.09

168

12.26

34.13

46.39

77.31

3.3.2 FTAB Liquid Cultures and C18 Eluates
3.3.2.1 Initial Steps in FTAB Degradation
Over the 168-hr incubation period, Gordonia sp. NB4-1Y converted more than 50 % of the
FTAB present in the culture. Degradation of FTAB appeared to occur between 96 and 168-hrs of
incubation (Figure 3-11). FTSA was present in the initial solution at a concentration of 0.93 mol
% and was completely degraded after 72-hrs. The volatile metabolite 6:2-FTOH increased from
0.197 – 2.421 mol % from the time it appeared at 96-hrs to 168-hrs. 6:2-FTCA was initially present
at 0.009 mol % and increased to 7.467 mol % after 168-hrs. 6:2-FTUA was initially present at
0.015 mol % and increased to yield 10.791 mol % after 168-hrs. In sterile FTAB cultures, the

31

12.0%

100.0%

10.0%

Mol % of 6:2-FTAB & Sterile FTAB

120.0%

80.0%

8.0%

60.0%

6.0%

40.0%

4.0%

20.0%

2.0%

0.0%

0.0%
0

24

48
72
96
Time Incubated (Hours)

Mol % of 6:2-FTSA, FTOH, FTCA, & FTUA

concentration of FTAB ranged from 65.42 – 109.73 mol % (Figure 3-11), averaging 83.05 mol %.

Sterile FTAB
6:2-FTAB
6:2-FTSA
6:2-FTOH
6:2-FTCA
6:2-FTUA

168

Figure 3-11: FTAB degradation in NB4-1Y cultures and production of 6:2-FTOH, 6:2-FTCA, 6:2FTUA in aqueous samples and their respective C18 eluates over a 168-hr time period.
FTAB, FTSA, 6:2-FTCA, and 6:2-FTUA are present in the aqueous phase of inoculated FTAB
cultures, while 6:2-FTOH (red) was identified in C18 eluate. Sterile FTAB (dark blue) represents
the quantification of FTAB present in sterile FTAB cultures. Four replicates for each time point
were analyzed (n=4).

3.3.2.2 Major Degradation Pathway Products
Gordonia sp. NB4-1Y converted 18.349 mol % of FTAB into volatile 5:2-ketone after 168hrs of incubation. 5:2-ketone was first eluted from C18 cartridges after 24-hrs of incubation,
yielding 0.008 mol %. The metabolite 5:2-sFTOH first appeared after 96-hrs and increased from
0.024 – 1.247 mol % after 168-hrs (Figure 3-12). PFPeA and PFHxA were detected at low

32

concentrations, yielding 0.001 and 0.026 mol %, respectively, after 168-hrs (Figure 3-12).
1.6%

20.0%

Mol % of 5:2-ketone

1.2%
16.0%
12.0%

0.8%

8.0%
0.4%
4.0%
0.0%

Mol % of 5:2-sFTOH, PFPeA & PFHxA

24.0%

5:2-ketone
5:2-s-FTOH
PFPeA
PFHxA

0.0%
0

24

48
72
Axis Title

96

168

Figure 3-12: Major degradation pathway products of inoculated FTAB cultures and C18 eluate
over a 168-hr time period.
PFPeA and PFHxA were identified in the aqueous phase, while 5:2-FT-ketone and 5:2-sFTOH
were eluted from C18 cartridges. Four replicates for each time point were analyzed (n=4).

3.3.2.3 Minor Degradation Pathway
The metabolite 5:3-FTCA slowly increased 10-fold from 0.040 mol % at 48-hrs to 0.413
mol % at 168-hrs (Figure 3-13). The metabolites 4:3-FTCA and PFBA were both identified after

33

Mol %

168-hrs of incubation and were quantified at 0.018 mol % of the total FTAB present in the cultures.
0.50%

0.03%

0.40%

0.02%

0.30%

0.02%
5:3-FTCA

0.20%

0.01%

4:3-FTCA
PFBA

0.10%

0.01%

0.00%

0.00%
0

24

48

72

96

168

Time Incubated (Hours)

Figure 3-13: Minor degradation pathway products of inoculated FTAB cultures over a 168-hr time
period.
5:3-FTCA, 4:3-FTCA, and PFBA were identified in the aqueous phase in extremely small
quantities. Four replicates for each time point were analyzed (n=4).

3.3.2.4 Percent Recovery
After 168-hrs, 13.22 of the total 60-µM present was in the volatile phase and eluted from
C18 cartridges, while 29.24-µM was identified in the aqueous phase (Table 3-4). The percent
recovery of FTAB and the expected metabolites ranged from 70.77 – 99.09 % (Table 3-4), yielding
an average recovery rate of 84.64 %.

34

Table 3-4: Percent recovery of fluorinated analytes and proportion of analytes identified in the
aqueous and volatile phase in inoculated FTAB cultures at specific time points.
HOURS INCUBATED

AQUEOUS TOTAL (µm)

C18 TOTAL (µm)

TOTAL (µm)

% RECOVERY

0

48.24

0.00

48.24

80.39

24

51.91

0.01

51.92

86.53

48

46.53

0.12

46.65

77.75

72

58.50

0.96

59.46

99.09

96

53.47

2.52

55.99

93.32

168

29.24

13.23

42.47

70.78

35

4

DISCUSSION

4.1

Gordonia sp. NB4-1Y Growth Optimization

4.1.1 Ethanol Toxicity
Prior to investigating the degradation of specific fluorochemicals by NB4-1Y, the
concentration of ethanol found to be toxic to the microorganisms needed to be determined, as both
FTSA and FTAB were dissolved in a 50 % ethanol solution. Analysis indicated that no significant
hindrance to growth occurred until 4. 5 % v/v ethanol. In fact, an ethanol concentration as high as
4.0 % increased the total amount of growth after 168-hrs (OD600 nm of 0.720) (Figure 3-1). While
it was safe to grow NB4-1Y in media containing up to 4.0 % v/v ethanol, a reduction in the initial
growth rate was observed at higher concentrations. After 24-hrs, cultures grown in 0.5 % v/v
ethanol exhibited an OD600 nm of 0.387, while cultures grown at 4.0 % v/v ethanol had an OD600 nm
of 0.023 (Figure 3-1). The first sign of this decline can be seen in cultures grown with as little as
1.5 % v/v ethanol, and a decrease in initial growth rate is seen at 2.5 % v/v ethanol (Figure 3-1).
These results are indicative that Gordonia sp. NB4-1Y are capable of growing at ethanol
concentrations of up to 4.0 % v/v, but as ethanol concentration increases, NB4-1Y may take longer
to adjust to their environment before proliferation can occur. With these results, an ethanol
concentration of 1.25 % v/v was utilized to ensure cultures experienced limited ethanol toxicity.
4.1.2 Optimum FTSA Concentration
Determining an optimum concentration of FTSA and FTAB was necessary to access the
toxicity of the heavily fluorinated compounds towards NB4-1Y and to identify a suitable
concentration for quantification of breakdown products. NB4-1Y did not appear to suffer any toxic
effects when exposed to FTSA concentration as high as 100-µM, and total growth of the cultures
seemed to profit from the increased presence of FTSA. Additionally, based on Figure 3-2, the
36

growth rate of NB4-1Y in the exponential phase (between 24-48-hrs) appeared to increase as the
concentration of FTSA increased. It is also important to note the increase in total growth NB4-1Y
exhibits upon increasing the concentration of FTSA from 20-µM to 40-µM (Figure 3-3).
Analysis indicated that Gordonia sp. NB4-1Y was capable of tolerating a relatively high
concentration of FTSA, which bodes well for analytical analysis. It is also important to consider
the large growth increase between 20 and 40-µM, as utilizing a concentration of 40-µM or greater
will not only help benefit the NB4-1Y population, but will increase the quantity of metabolites
produced over a shorter period of time. Past studies have utilized concentrations of FTSA ranging
from 1.3-mg L-1 (~3.0-µM) in their analysis of aerobic/anaerobic degradation

of FTSA in

sediment (Zhang et al. 2016) to 40-µM of FTSA in monitoring degradation using pure culture
(Van Hamme et al. 2013). However, it should be noted that the latter concentration may not be
relevant to environmental conditions.

4.1.3 Optimum FTAB Concentration
NB4-1Y did not appear to exude any significant toxicological characteristics from
increased FTAB exposure, however this cannot be definitively stated without comparison to a
MgSO4 control (not conducted). Again, much like what was seen with FTSA, NB4-1Y seemed to
benefit from increased FTAB concentration in terms of overall growth of the cultures, with the
one exception being a slight decrease in growth rate during the exponential phase and overall
growth when increasing concentration from 80-µM to 100-µM (Figure 3-4). Furthermore, as the
concentration of FTAB was increased from 20-µM to 40-µM an increase in growth rate and a 52.8
% increase in overall growth was seen, with OD600 nm readings of 0.126 and 0.218, respectively.

37

Analysis demonstrated that Gordonia sp. NB4-1Y can tolerate a relatively high
concentration of FTAB. Much like FTSA, it is important to consider the large growth increase
between 20 and 40-µM, as utilizing a concentration of 40-µM or greater will not only help benefit
the NB4-1Y population, but will increase the quantity of metabolites produced over a shorter
period of time. However, concentrations 40-µM or greater may not be representative of
environmental conditions.
Gordonia sp. NB4-1Y exhibited fairly similar growth curves when FTSA or FTAB were
provided as the sole-sulfur source. It is not overly surprising due to the resiliency of
microorganisms and to the fact that both compounds are extremely similar themselves (Appendix:
Table 7-2). It is important to note however, that NB4-1Y was not able to utilize FTAB as well as
it was able to exploit FTSA. While both sets of cultures exhibited similar growth early on (e.g. the
24-hr time point), NB4-1Y experienced decreased overall growth after 168-hrs at all
concentrations. Overall growth for 100-µM FTSA cultures plateaued around 0.550 (Figure 3-3),
while 100-µM FTAB cultures plateaued around 0.300 (Figure 3-4).

4.2

FTSA Liquid Cultures and C18 Eluates
In this study, Gordonia sp. NB4-1Y were grown in sulfur-limiting conditions with 60-µM

of FTSA, FTAB or MgSO4 as the sole-sulfur source. A total of 168 cultures and 168 C18 cartridges
under seven different conditions were incubated over a 168-hr time period and were analyzed using
an ultra-high pressure liquid chromatography chain coupled to an Orbitrap mass spectrometer.
Analysis of FTSA/FTAB cultures and their respective C18 eluate identified and quantified a total
of eleven metabolites: PFBA, PFPeA, PFHxA, PFHpA, 4:3-FTCA, 5:3-FTCA, 6:2-FTCA, 6:2FTUA, 5:2-sFTOH, 5:2-FT-ketone, and 6:2-FTOH. However, PFHpA was not identified in FTAB
38

analysis and was not found to increase in concentration over FTSA degradation. It is believed that
PFHpA is an impurity in the FTSA stock solution, as it was seen in sterile FTSA cultures. Analysis
of sterile FTSA/FTAB cultures indicates that very little, if any, degradation of FTSA/FTAB occurs
naturally (Figure 3-8; Figure 3-11). In combination with the analysis done on inoculated
FTSA/FTAB cultures, sterile FTSA/FTAB cultures indicate that FTSA/FTAB degradation is
occurring due to microbial activity. With all culture purity checks coming back positive for only
Gordonia sp. NB4-1Y, it is evident that NB4-1Y is responsible for the degradation of FTSA, as
previously determined by Van Hamme et al. (2013), and responsible for the degradation of FTAB.
4.2.1 Analysis of FTSA Liquid Cultures and C18 Eluates
With FTSA as their sole-sulfur source, Gordonia sp. NB4-1Y degraded 99.97 % of the
environmental contaminant after 168-hrs (Figure 3-8). Significant degradation of FTSA did not
appear to occur until after 72-hrs of incubation. This may be attributed to NB4-1Y adjusting to its
new environment (lag phase) or there is enough sulfur already present in the media for NB4-1Y to
survive for the first 72-hrs (therefore, has no need to use energy to extract the sulfur from FTSA),
or a combination of both. However, with the appearance of the first metabolites after 24-hrs, it is
clear that FTSA desulfonation is occurring.
The first analytes detected were 6:2-FTUA and 6:2-FTCA in the aqueous phase, after 24hrs (Figure 3-8), 5:3-FTCA in the aqueous phase, after 48-hrs (Figure 3-10), and 5:2-FT-ketone
the volatile phase, after 24-hrs (Figure 3-9). The early production of 6:2-FTCA and 6:2-FTUA
were expected based upon the previously proposed degradation pathways (Wang et al. 2011; Van
Hamme et al. 2013) (Figure 1-2; Figure 1-3). Production of 6:2-FTCA and 6:2-FTUA increases
relatively slow, with the most significant increase occurring between the 96 and 168-hr time points
(Figure 3-8). This is supportive of NB4-1Y utilizing contaminating sulfate already present in the
39

media combined with minor degradation of FTSA before sulfur must be extracted and significant
degradation occurs by NB4-1Y.
The majority of metabolites produced after 168-hrs were identified in the volatile phase
(Table 3-3), with those analytes being 5:2-FT-ketone (43.68 mol %), 5:2-sFTOH (8.97 mol %)
(Figure 3-9), and 6:2-FTOH (4.14 mol %) (Figure 3-8). It is important to note, however, that
although 5:2-FT-ketone was the most abundant metabolite, further degradation of this analyte is
strongly dependent upon its solubility into the aqueous phase. If 5:2-FT-ketone has low solubility,
most of the analyte would exist in the gaseous phase above the liquid culture, as opposed to being
dissolved in the liquid phase and within ‘reach’ of NB4-1Y. Therefore, the analyte would not be
in close enough proximity for NB4-1Y cells to further degrade the metabolite. Additionally, once
the cultures are purged, all volatile compounds become unavailable for further biotransformation.
Based on Liu and Avendaño's (2013) proposed degradation pathway, the identification of
5:3-FTCA and 5:2-FT-ketone indicates the use of two potential degradation pathways by NB41Y. These two analytes have been identified as two separate degradation products arising from
6:2-FTUA. Additionally, with 5:2-FT-ketone comprising the vast majority of metabolites
produced (Figure 3-9), there is strong evidence that NB4-1Y preferentially utilizes one pathway
over the other. In this case, conversion of 6:2-FTUA into 5:2-FT-ketone is preferred opposed to
conversion of 6:2-FTUA into 5:3-FTCA (Error! Reference source not found.). Greater than 50
mol % of the metabolites produced after 168-hrs belong to the 5:2-FT-ketone pathway, termed the
‘major’ degradation pathway in this report. Less than 1.0 mol % of the metabolites produced
belong to the 5:3-FTCA pathway, here termed the ‘minor’ degradation pathway (Error!
Reference source not found.).

40

4.2.2 Postulated FTSA Degradation Pathway by NB4-1Y
Regarding past hypothesized degradation pathways for FTSA (Wang et al. 2011; Van
Hamme et al. 2013), 6:2-FTOH (Liu and Avendaño 2013), and with the eleven metabolites
detected in NB4-1Y cultures, two metabolic pathways can be proposed (Error! Reference source
not

found.).

Figure 4-1: Proposed metabolic pathway utilized by Gordonia sp. NB4-1Y for the desulfonation
of FTSA.
Orange arrows are processes involved in the initial desulfonation of FTSA. The dotted orange line
indicates the identification of an alternative pathway not previously suggested by Zhang et al.
(2016). Blue arrows indicate the ‘major’ pathway utilized by NB4-1Y and the green arrows

41

indicate the ‘minor’ pathway. Steps with two arrows indicate that two or more steps occur between
analytes.

It is important to note that previous experiments had not implicated 6:2-FTOH as part of
the degradation pathway utilized by Gordonia sp. NB4-1Y (Van Hamme et al. 2013) or mixed
bacterial cultures (Wang et al. 2011). However, a more recent study has detected 6:2-FTOH in the
degradation of 6:2-FTSA in aerobic sediment mixture (Zhang et al. 2016). With the generation
0.173 mol % of 6:2-FTOH at 96-hrs and an increase to 4.144 mol % at 168-hrs (Figure 3-8), it is
more apparent that NB4-1Y may not generate 6:2-fluortelomer aldehyde (6:2-FTAL) by skipping
the conversion of FTSA to 6:2-FTOH (Error! Reference source not found.). Alkane metabolism
by bacteria typically converts alkanes to alcohols, then aldehydes, followed by carboxylic acids
(Van Hamme et al. 2003). NB4-1Y may follow a similar process during FTSA degradation by
converting FTSA to 6:2-FTOH (alcohol), then 6:2-FTAL (aldehyde), followed by 6:2-FTCA
(carboxylic acid). Furthermore, the biotransformation of FTSA to 6:2-FTOH to 6:2-FTAL may be
a rapid process such that detectable amounts of 6:2-FTOH may not accumulate until a high level
of FTSA degradation has occurred.

4.3

FTAB Liquid Cultures and C18 Eluates

4.3.1 Analysis of FTAB Liquid Cultures and C18 Eluates
Analysis of inoculated FTAB cultures identified the same ten metabolites produced in
NB4-1Y cultures during FTSA degradation, but in much lower quantities. For example, in
comparison to the 43.63 mol % of 5:2-FT-ketone produced in FTSA degradation (Figure 3-9),
FTAB degradation only produced 18.35 mol % over 168-hrs (Figure 3-12). This may be a result

42

of NB4-1Y taking longer to utilize FTAB or NB4-1Y is incapable of utilizing FTAB to the same
extent as it utilizes FTSA. However, the latter may not be the case, as NB4-1Y degraded over 50
% of the FTAB provided (Figure 3-11). An alternative explanation is the degradation pathway that
NB4-1Y must utilize for FTAB metabolism requires additional steps prior to releasing the
sulfonate from the molecule, at minimum one if the organism is able to cleave the sulfur-nitrogen
bond directly.
Significant degradation of FTAB did not occur until after 96-hrs of incubation, where
approximately 80 mol % of FTAB was removed to a concentration of 29.58 mol % at 168-hrs
(Figure 3-11). It is interesting to note that upon the increased degradation of FTAB, there appeared
to be no production of FTSA. Previous research has postulated that initial degradation of FTAB
primarily results in the production of FTSA (Zhang et al. 2016), however, these observations were
made in groundwater samples from regions where AFFFs were used (Schultz et al. 2004). There
are two possibilities that may account for this result. First, there was a small amount of FTSA (<
1.0 mol %) already present in in the cultures which was completely degraded within 72-hrs. Given
this, the enzymes responsible for FTSA desulfonation may already be present before FTAB
degradation started. Upon the degradation of FTAB, rapid utilization of FTSA could occur,
effectively masking the biotransformation of FTAB into FTSA. Otherwise, NB4-1Y may convert
FTAB into an alternative intermediate, which can then be transformed into FTSA or it may bypass
FTSA production completely (Error! Reference source not found.). Understanding the initial
degradation process will require the production of mutants, isolation and in vitro analysis of the
enzymes responsible, or the use of isotopically-labelled compounds to better track intermediates.
It is clear that FTAB biotransformation does follow the same lower degradation pathway
as FTSA. This is evident in the quantity (mol %) of major and minor pathway metabolites
43

produced, as they far surpass the amount of FTSA originally present in the culture. For example,
the initial concentration of FTSA in FTAB cultures peaked below 1.0 mol % (Figure 3-11), while
FTSA metabolites such as 5:2-FT-ketone peaked around 18.0 mol % (Figure 3-12).

4.3.2 Postulated FTAB Degradation Pathway by NB4-1Y
With the identification of the same ten metabolites produced during FTSA degradation, a
modified version of the previously postulated metabolic pathway for FTSA can be proposed for
FTAB degradation. The modifications accommodate the unknown initial degradation processes of
FTAB (Error! Reference source not found.). As mentioned previously (Section 1.2), it has also
been reported that 6:2 fluorotelomer sulfonamide [F(CF2)6CH2CH2SO2NH2] (6:2-FTSAOA) is
initially produced in FTAB degradation by blue mussels (Moe et al. 2012). They also report that
four other metabolites were produced in FTAB degradation (Moe et al. 2012). Furthermore, their
analysis of soil samples known to contain AFFF products identified the same metabolites (Moe et
al. 2012). These metabolites may be part of the degradation pathway NB4-1Y utilizes when

44

degrading

FTAB.

Figure 4-2: Proposed metabolic pathway utilized by Gordonia sp. NB4-1Y for the degradation of
FTAB.
Orange arrows are processes involved in the initial desulfonation of FTSA. The dotted orange line
indicates the identification of an alternative pathway previously suggested by Zhang et al (2016).
Blue arrows indicate the ‘major’ pathway utilized by NB4-1Y and the green arrows indicate the
‘minor’ pathway. Steps with two arrows indicate that two or more steps occur between analytes.
Purple arrows are proposed pathways that Gordonia sp. NB4-1Y may use to degrade FTAB.
45

While our experiment demonstrated that both FTSA and FTAB are utilized by NB4-1Y, it
is not clear which steps are biologically or chemically-mediated. Referring to Figure 1-2, Van
Hamme et al. (2013) report that the transformation of 6:2-FTS to 6:2-FTAL is biologicallymediated through desulfonation genes ISGA 1218 & 1222, while the conversion of 6:2-FTCA to
6:2-FTUCA may be chemically-mediated through the spontaneous loss of hydrogen fluoride. In
other words, each transformation step throughout degradation may be a result of an enzymatic
reaction or natural chemical degradation. For example, 6:2-FTCA may be relatively unstable,
resulting in the spontaneous loss of HF, producing 6:2-FTUCA.

46

5

CONCLUSION AND FUTURE WORK
Gordonia sp. NB4-1Y is capable of complete desulfonation of FTSA, degradation of

FTAB, and use of both compounds as the primary sulfur source under sulfur-limiting conditions.
A total of eleven metabolites for FTSA and ten metabolites for FTAB have been identified and
quantified, with additional analyses for new intermediates currently in progress. NB4-1Y appears
to utilize two degradation pathways –major (5:2-FT-ketone) & minor (5:3-FTCA)– diverging from
6:2-FTUA. Conversion of 6:2-FTUA into 5:2-FT-ketone was the metabolic pathway primarily
used by NB4-1Y with more than 50 % of metabolites being 5:2-FT-ketone, 5:2-sFTOH, PFPeA,
and PFHxA. Although FTAB degradation did not reach the same extent as FTSA after 168-hrs,
results indicate that the lower degradation pathway is similar to what is seen with FTSA, and that
both major and minor metabolic routes are present.
The metabolite 6:2-FTOH was identified as a biotransformation product of FTSA
degradation. Previous studies had not detected 6:2-FTOH and hypothesized that FTSA
transformation bypassed 6:2-FTOH production, producing 6:2-FTAL directly (Wang et al. 2011;
Van Hamme et al. 2013). However, recently Zhang et al. (2016) detected 6:2-FTOH (<2.5 mol %)
in an aerobic sediment mixture over 90 days, suggesting that 6:2-FTOH was an initial degradation
product of 6:2-FTSA (Zhang et al. 2016). The initial degradation of FTAB may not involve the
production of FTSA. NB4-1Y may bypass production of FTSA by conversion to an alternative
intermediate, otherwise FTSA is rapidly utilized keeping levels below detectable limits. Lastly, it
would be interesting to determine how the sulfonamide alkyl betaine portion of FTAB is
metabolized. Additional analysis scanning for this portion of FTAB may provide some insight into
this process. Isolating and utilizing this segment of FTAB as a supply of sulfur, nitrogen, and
carbon while repeating the experiment is an intriguing aspect.

47

Attempts to extract mRNA from Gordonia sp. NB4-1Y cultures under the same sulfurlimiting conditions is underway. Once sufficient mRNA is recovered, the transcriptome can be
sequenced in order to better understand gene expression changes when NB4-1Y metabolizes FTSA
and FTAB. These experiments will facilitate future research isolating enzymes involved in FTAB
and FTSA metabolism, verifying their function in vitro, and designing molecular tools for
monitoring their metabolism by diverse microbial communities in the environment. Additionally,
further analysis of stored samples is being conducted in hopes of identifying new intermediates
and degradation products, as it is not completely clear which degradation steps are biologicallymediated, and which are chemically-mediated.

48

6

LITERATURE CITED

Arenskotter M, Broker D, Steinbuchel A. 2004. Biology of the metabolically diverse genus
Gordonia. Appl. Environ. Microbiol. 70:3195–3204.
Backe WJ, Day TC, Field JA. 2013. Zwitterionic, cationic, and anionic fluorinated chemicals in
aqueous film forming foam formulations and groundwater from US military bases by nonaqueous
large-volume injection HPLC-MS/MS. Environ. Sci. Technol. 47:5226–5234.
Boiteux V, Dauchy X, Bach C, Colin A, Hemard J, Sagres V, Rosin C, Munoz J-F. 2017.
Concentrations and patterns of perfluoroalkyl and polyfluoroalkyl substances in a river and three
drinking water treatment plants near and far from a major production source. Sci. Total Environ.
583:393–400.
Butt CM, Muir DC, Mabury SA. 2014. Biotransformation pathways of fluorotelomer-based
polyfluoroalkyl substances: A review. Environ. Toxicol. Chem. 33:243–267.
Clarke BO, Smith SR. 2011. Review of “emerging”organic contaminants in biosolids and
assessment of international research priorities for the agricultural use of biosolids. Environ. Int.
37:226–247.
Dinglasan MJA, Ye Y, Edwards EA, Mabury SA. 2004. Fluorotelomer alcohol biodegradation yields
poly- and perfluorinated acids. Environ. Sci. Technol. 38:2857–2864.
Drzyzga O. 2012. The strengths and weaknesses of Gordonia: a review of an emerging genus with
increasing biotechnological potential. Crit. Rev. Microbiol. 38:300–316.
Higgins C, Field J, Criddle C, Luthy R. 2005. Quantitative determination of perfluorochemicals in
sediments and domestic sludge. Environ. Sci. Technol. 39:3946–3956.
Houtz EF, Sutton R, Park J-S, Sedlak M. 2016. Poly-and perfluoroalkyl substances in wastewater:
Significance of unknown precursors, manufacturing shifts, and likely AFFF impacts. Water Res.
95:142–149.
Krafft MP, Riess JG. 2015. Per-and polyfluorinated substances (PFASs): Environmental challenges.
Curr. Opin. Colloid Interface Sci. 20:192–212.
Liu J, Avendaño SM. 2013. Microbial degradation of polyfluoroalkyl chemicals in the
environment: a review. Environ. Int. 61:98–114.
Liu J, Wang N, Szostek B, Buck RC, Panciroli PK, Folsom PW, Sulecki LM, Bellin CA. 2010. 6-2
Fluorotelomer alcohol aerobic biodegradation in soil and mixed bacterial culture. Chemosphere
78:437–444.

49

Loganathan BG, Sajwan KS, Sinclair E, Senthil Kumar K, Kannan K. 2007. Perfluoroalkyl sulfonates
and perfluorocarboxylates in two wastewater treatment facilities in Kentucky and Georgia. Water
Res. 41:4611–4620.
Moe MK, Huber S, Svenson J, Hagenaars A, Pabon M, Trümper M, Berger U, Knapen D, Herzke D.
2012. The structure of the fire fighting foam surfactant Forafac® 1157 and its biological and
photolytic transformation products. Chemosphere 89:869–875.
Munoz G, Duy SV, Labadie P, Botta F, Budzinski H, Lestremau F, Liu J, Sauvé S. 2016. Analysis of
zwitterionic, cationic, and anionic poly-and perfluoroalkyl surfactants in sediments by liquid
chromatography polarity-switching electrospray ionization coupled to high resolution mass
spectrometry. Talanta 152:447–456.
Murphy CD. 2010. Biodegradation and biotransformation of organofluorine compounds.
Biotechnol. Lett. 32:351–359.
Newsted J, Beach S, Gallagher S, Giesy J. 2008. Acute and chronic effects of perfluorobutane
sulfonate (PFBS) on the mallard and northern bobwhite quail. Arch. Environ. Contam. Toxicol.
54:535–545.
Park S, Lee LS, Medina VF, Zull A, Waisner S. 2016. Heat-activated persulfate oxidation of PFOA,
6: 2 fluorotelomer sulfonate, and PFOS under conditions suitable for in-situ groundwater
remediation. Chemosphere 145:376–383.
Phillips M, Dinglasan-Panlilio M, Mabury S, Solomon K, Sibley P. 2007. Fluorotelomer acids are
more toxic than perfluorinated acids. Environ. Sci. Technol. 41:7159–7163.
Rhoads KR, Janssen EM-L, Luthy RG, Criddle CS. 2008. Aerobic biotransformation and fate of Nethyl perfluorooctane sulfonamidoethanol (N-EtFOSE) in activated sludge. Environ. Sci. Technol.
42:2873–2878.
Russell MH, Berti WR, Szostek B, Buck RC. 2008. Investigation of the biodegradation potential of
a fluoroacrylate polymer product in aerobic soils. Environ. Sci. Technol. 42:800–807.
Schultz MM, Barofsky DF, Field JA. 2004. Quantitative determination of fluorotelomer sulfonates
in groundwater by LC MS/MS. Environ. Sci. Technol. 38:1828–1835.
Sinclair E, Kannan K. 2006. Mass loading and fate of perfluoroalkyl surfactants in wastewater
treatment plants. Environ. Sci. Technol. 40:1408–1414.
Van Hamme JD, Bottos EM, Bilbey NJ, Brewer SE. 2013. Genomic and proteomic characterization
of Gordonia sp. NB4-1Y in relation to 6: 2 fluorotelomer sulfonate biodegradation. Microbiology
159:1618–1628.

50

Van Hamme JD, Fedorak PM, Foght JM, Gray MR, Dettman HD. 2004. Use of a novel fluorinated
organosulfur compound to isolate bacteria capable of carbon-sulfur bond cleavage. Appl.
Environ. Microbiol. 70:1487–1493.
Van Hamme JD, Singh A, Ward OP. 2003. Recent advances in petroleum microbiology. Microbiol.
Mol. Biol. Rev. 67:503–549.
Wang N, Buck RC, Szostek B, Sulecki LM, Wolstenholme BW. 2012. 5: 3 Polyfluorinated acid
aerobic biotransformation in activated sludge via novel “one-carbon removal pathways.”
Chemosphere 87:527–534.
Wang N, Liu J, Buck RC, Korzeniowski SH, Wolstenholme BW, Folsom PW, Sulecki LM. 2011. 6:2
Fluorotelomer sulfonate aerobic biotransformation in activated sludge of waste water treatment
plants. Chemosphere 82:853–858.
Zhang S, Lu X, Wang N, Buck RC. 2016. Biotransformation potential of 6: 2 fluorotelomer
sulfonate (6:2 FTSA) in aerobic and anaerobic sediment. Chemosphere 154:224–230.

51

7
7.1

APPENDIX
LC-MS Method Parameters Summary

Table 7-1: Summary of LC-MS method parameters
Instrument
Thermo Q-Exactive Orbitrap mass spectrometer
Dionex Ultimate 3000 UHPLC chain
Ionization

Electrospray ionization source operated in fast polarity switching mode

Acquisition mode

Full Scan MS mode

Analytical column

Thermo Hypersil Gold aQ column (100 × 2.1 mm; 1.9 μm)

Delay column

Thermo Hypercarb column (20 × 2.1 mm; 7 μm)

Column
Temperature

40°C

Mobile Phases

A: 0.15% acetic acid in HPLC-water
B: 0.15% acetic acid in acetonitrile
Flow rate (mL/min) 0.55

Gradient Profile

Time (min)
0.0
6.5
7.0
9.5
9.6
12

Injection Volume

15 μL

Source/gas

Sheath gas flow rate 45
Aux gas flow rate 10
Sweep gas flow rate 10
Spray voltage (|kV|) 3
Capillary temperature (°C) 320
S-lens RF level 55

Orbitrap
parameters

Resolution 70,000
AGC target 5e6
Maximum Inject Time (ms) 50
Scan range (m/z) 150–700

Quantitative
analytes
and
correspondence
with ISs

Analyte
PFBA
PFPeA
PFHxA
PFHpA

%B
10
72.5
100
100
10
10

m/z
212.97947
262.97669
312.97335
362.97013

IS
MPFBA
MPFPeA
MPFHxA
MPFOA
52

6:2 FTAB
6:2 FTSA
3:3 FTCA
4:3 FTCA
5:3 FTCA
6:2 FTCA
6:2 FTUA
6:2 FTOH
5:2 sFTOH
5:2 Ketone
MPFBA
MPFPeA
MPFHxA
MPFOA
M62-FTCA
M62-FTS
M62-FTUA

7.2

571.09362
426.96866
241.00995
291.00676
341.00356
376.98472
356.97849
423.02658
373.02978
310.99299
216.99177
265.98674
314.98039
416.97975
378.99142
428.97537
358.98520

M62-FTS
M62-FTS
M62-FTCA
M62-FTCA
M62-FTCA
M62-FTCA
M62-FTUA
MPFOA
MPFOA
MPFOA
-

Control Charts – Absolute Area

16000000
14000000

Absolute Area

12000000
10000000
8000000
6000000

3:3-FTCA
4:3-FTCA
5:3-FTCA

4000000

6:2-FTCA

2000000
0

Figure 7-1: Illustration of quality control approach along the analytical sequence; control chart
used to monitor the signal received upon the detection of 5-ng mL-1 3:3-FTCA, 4:3-FTCA, 5:3FTCA, and 6:2-FTCA.
53

31000000
29000000

Absolute Area

27000000
25000000
23000000

6:2-FTUA

21000000

5:2-FT-ketone

19000000

6:2-FTSA

17000000
15000000

Figure 7-2: Illustration of quality control approach along the analytical sequence; control chart
used to monitor the signal received upon the detection of 5-ng mL-1 6:2-FTUA, 6:2-FTSA, and
87.5-ng mL-1 5:2-FT-ketone.
4600000
4400000

Absolute Area

4200000
4000000
3800000
3600000

5:2-s-FTOH

3400000
3200000
3000000

Figure 7-3: Illustration of quality control approach along the analytical sequence; control chart
used to monitor the signal received upon the detection of 87.5-ng mL-1 5:2-sFTOH.
54

46000000
44000000

Absolute Area

42000000
40000000
38000000
36000000

6:2-FTAB

34000000
32000000
30000000

Figure 7-4: Illustration of quality control approach along the analytical sequence; control chart
used to monitor the signal received upon the detection of 5-ng mL-1 FTAB.

550000
500000

Absolute Area

450000
400000
350000
6:2-FTOH
300000
250000
200000

Figure 7-5: Illustration of quality control approach along the analytical sequence; control chart
used to monitor the signal received upon the detection of 87.5-ng mL-1 6:2-FTOH.

55

7.3

Control Charts – Analyte/IS Ratio

1.600
1.500

Analyte/IS ratio

1.400
1.300
1.200
1.100
1.000

3:3-FTCA
4:3-FTCA
5:3-FTCA
6:2-FTCA

0.900
0.800

Figure 7-6: Illustration of quality control approach along the analytical sequence; control chart
used to monitor the signal received upon the detection of 5-ng mL-1 3:3-FTCA, 4:3-FTCA, 5:3FTCA, and 6:2-FTCA.

1.800

Analyte/IS ratio

1.600
1.400
1.200
1.000

6:2-FTUA
5:2-FT-ketone
6:2-FTSA

0.800
0.600

Figure 7-7: Illustration of quality control approach along the analytical sequence; control chart
used to monitor the signal received upon the detection of 5-ng mL-1 6:2-FTUA, 6:2-FTSA, and
87.5 ng mL-1 5:2-FT-ketone.
56

0.140

Analyte/IS ratio

0.120
0.100
0.080
0.060

5:2-s-FTOH
6:2-FTOH

0.040
0.020
0.000

Figure 7-8: Illustration of quality control approach along the analytical sequence; control chart
used to monitor the signal received upon the detection of 87.5-ng mL-1 5:2-sFTOH and 6:2-FTOH.

3.400
3.300

Analyte/IS ratio

3.200
3.100
3.000
2.900

6:2-FTAB

2.800
2.700
2.600

Figure 7-9: Illustration of quality control approach along the analytical sequence; control chart
used to monitor the signal received upon the detection of 5-ng mL-1 FTAB.
57

7.4

PFAS Summary

Table 7-2: PFAS analytes in the study, their acronym, and molecular structure.
Chemical Name

Acronym

Perfluorobutyric acid

PFBA

Perfluoropentanoic acid

PFPeA

Perfluorohexanoic acid

PFHxA

Perfluoroheptanoic acid

PFHpA

3:3 fluorotelomer carboxylic acid

3:3-FTCA

4:3 fluorotelomer carboxylic acid

4:3-FTCA

5:3 fluortelomer carboxylic acid

5:3-FTCA

6:2 fluorotelomer carboxylic acid

6:2-FTCA

Molecular Structure

58

Chemical Name

Acronym

6:2 fluorotelomer unsaturated carboxylic acid

6:2-FTUA

5:2 secondary fluorotelomer alcohol

5:2-sFTOH

5:2 fluorotelomer ketone

5:2-FT-ketone

6:2 fluorotelomer alcohol

6:2-FTOH

6:2 fluortelomer sulfonate

6:2-FTSA

6:2 fluortelomer sulfonamide alkyl betaine

6:2-FTAB

Molecular Structure

59