DOI: 10.1002/rem.

21550

RESEARCH ARTICLE

Fungal biotransformation of 6:2 fluorotelomer alcohol

Nancy Merino Meng Wang Rocio Ambrocio Kimberly Mak Ellen O'Connor
An Gao Elisabeth L. Hawley Rula A. Deeb Linda Y. Tseng Shaily Mahendra

Correspondence
Shaily Mahendra, Department of Civil and
Environmental Engineering, 5732J Boelter
Hall, University of California, Los Angeles, CA
90095.
Email: mahendra@seas.ucla.edu
Abstract
Fungal degradation of 6:2 fluorotelomer alcohol (6:2 FTOH, C6 F13 CH2 CH2 OH) by two wood-
decaying fungal strains and six fungal isolates from a site contaminated with per- and polyfluo-
roalkyl substances (PFASs) was investigated. 6:2 FTOH is increasingly being used in FTOH-based
products, and previous reports on the microbial fate of 6:2 FTOH have focused on bacteria and
environmental microbial consortia. Prior to this study, one report demonstrated that the 6:2
FTOH biotransformation by the wood-decaying fungus, Phanerochaete chrysosporium, generated
more polyfluoroalkyl substances, such as 5:3 acid (F(CF2 )5 CH2 CH2 COOH), and diverted away
from producing the highly stable perfluorocarboxylic acids (PFCAs). Most of the fungi (Gloeophyl-
lum trabeum and isolates TW4-2, TW4-1, B79, and B76) examined in this study showed similar
degradation patterns, further demonstrating that fungi yield more 5:3 acid (up to 51 mol% of ini-
tial 6:2 FTOH dosed) relative to other metabolites (up to 12 mol% total PFCAs). However, medium
amendments can potentially improve 6:2 FTOH biotransformation rates and product profiles. The
six fungal isolates tolerated up to 100 or 1,000 milligrams per liter of perfluorooctanoic acid and
perfluorooctane sulfonic acid, and some isolates experienced increased growth with increasing
concentrations. This study proposes that fungal pathways must be considered for the biotransfor-
mation of potential PFAS precursors, such as 6:2 FTOH, and suggests the basis for selecting proper
microorganisms for remediation of fluoroalkyl-contaminated sites.

1 INTRODUCTION
6:2 Fluorotelomer alcohol (6:2 FTOH, C6 F13 CH2 CH2 OH) is part
of a larger group of compounds known as per- and polyfluoroalkyl
substances (PFASs), which are widely used in consumer, industrial,
and military applications due to their unique properties, including
resistance to water, oil, and stains (Baker & Rao, 1994). However,
long-chain perfluorocarboxylic acids (PFCAs), such as perfluorooc-
tanoic acid (PFOA), and their precursors, such as 8:2 FTOH, are being
phased out because of their toxicological and ecological impacts
(Ding & Peijnenburg, 2013; Paul & Jones, 2009; U.S. Enviromental
Protection Agency, 2006). 6:2 FTOH is one of the shorter-chain
substitutes (Ritter, 2010), but once released into the environment, it
can be transformed to PFCAs and other polyfluoroalkyl substances
by biological and physicochemical processes (Merino et al., 2016).
Biotransformation of 6:2 FTOH by bacteria and microbial consortia
has been previously reported (Butt, Muir, & Mabury, 2014). However,
one study examined the fungal biotransformation of 6:2 FTOH by the
wood-decaying fungus, Phanerochaete chrysosporium (Tseng, Wang,
Szostek, & Mahendra, 2014). Since fungi play an important role in
the ecosystem and account for up to 75 percent of the soil microbial
biomass (Ritz & Young, 2004), it is important to understand the various
6:2 FTOH fungal biotransformation pathways for predicting PFAS fate
in the environment and for devising remediation strategies.
In addition, 6:2 FTOH fungal biotransformation may be more favor-
able for the remediation of 6:2 FTOH-based products compared to
bacterial biotransformation pathways. The major metabolites of 6:2
FTOH biotransformation in soils, sediments, activated sludge, and
microbial consortia were PFCAs (e.g., perfluorobutanoic acid (PFBA)
[F(CF2 )3 COOH], perflouropentanoic acid (PFPeA) [F(CF2 )4 COOH],
and perfluorohexanoic acid (PFHxA) [F(CF2 )5 COOH]) and x:3 acids,
such as 5:3 acid and 4:3 acid [F(CF2 )4 CH2 CH2 COOH] (Liu et al., 2010b;
Zhao et al., 2013). For single bacterial strains, the major products were
two transient, initial intermediates: 6:2 fluorotelomer unsaturated car-
boxylic acid (FTUCA) (F(CF2 )5 CF = CHCOOH), and 6:2 fluorotelomer
carboxylic acid (FTCA) (F(CF2 )6 CH2 COOH; Kim, Wang, & Chu, 2013;
Kim, Wang, McDonald, & Chu, 2012). In contrast, fungal transforma-
tion of 6:2 FTOH by P. chrysosporium led to higher production of 5:3 acid
and 5:3 acid conjugates with minimal PFCA yields (Tseng et al., 2014).
Since polyfluoroalkyl substances do not contain a fully fluorinated

Remediation. 2018;28:59–70. wileyonlinelibrary.com/journal/rem 2018

c Wiley Periodicals, Inc. 59
60 MERINO ET AL .

carbon chain, these products can then be more easily transformed in zone (5–6 ft interval) with acetate liners. All equipment was rinsed
the environment by other processes. thoroughly first with tap water and then with deionized water before
While P. chrysosporium is the model white-rot basidiomycete for use. Samples were shipped and stored at 4 ◦ C and were used within
the degradation of a wide range of contaminants, the phylum Basid- one month of receiving the samples to isolate fungi capable of growing
iomycota accounts for about 34 percent of all described fungal species on high concentrations of PFOA and PFOS.
(Harms, Schlosser, & Wick, 2011). On the other hand, the largest fungal
group, Ascomycota, also includes species that can degrade pollutants, 2.3 Isolation of fungi
including toluene, aliphatic hydrocarbons, polycyclic aromatic hydro-
Culturable fungi were first enriched on nutrient-rich agar, followed
carbons (PAHs), pesticides, and chlorophenols (Harms et al., 2011).
by several transfers of morphologically different colonies in Kirk agar
Several studies have isolated soil fungi from contaminated sites to
(Tien & Kirk, 1988; Tseng et al., 2014) containing 10 milligrams (mg)
degrade organic chemicals, such as 𝛽-hexachlorocyclohexane (Ceci
PFOA or PFOS/L. Nutrient-rich agar consisted of either Yeast Pep-
et al., 2015), polyvinyl chloride (Ali et al., 2014), and dieldrin (Kataoka,
tone Dextrose (Becton Dickinson, Franklin Lakes, NJ), Malt Extract
Takagi, Kamei, Kiyota, & Sato, 2010). In order to better understand
(ME), or Potato Dextrose (Becton Dickinson, Franklin Lakes, NJ) agar.
the 6:2 FTOH fungal transformation pathways in the environment, it
ME agar contained (per L): 16 grams (g) glucose, 16 g ME, 1.8 g mal-
is important to examine various fungal taxonomic groups.
tose, 2 g peptone, 3.2 g yeast extract, 1 g asparagine, 2 g KH2 PO4 , 1 g
The objectives of this study were to determine the 6:2 FTOH bio-
MgSO4 ⋅7H2 O, 1 mg thiamine–HCl, and 15 g agar. Kirk agar was cho-
transformation potential of two fungal strains, Gloeophyllum trabeum
sen as the nutrient-defined medium to select for fungal isolates related
and Trametes versicolor, and six fungal isolates obtained from sites
to wood-decaying fungi, which are known to degrade contaminants,
historically contaminated with PFASs from the use of aqueous film-
such as pharmaceuticals, azo dyes, and explosives (Harms et al., 2011).
forming foam (AFFF). Additionally, this work aimed to identify whether
For soil samples, 10 g soil was immersed in 50 milliliters (mL) of filter
the six fungal isolates could tolerate exposure to PFOA and perfluo-
sterilized Phosphate Buffer Saline and wrist shaken for ∼30 minutes
rooctane sulfonic acid (PFOS).
(min) to resuspend soil-bound microorganisms. Afterwards, each sam-
ple was aseptically streaked onto nutrient-rich agar with a 10 micro-
liter (𝜇L) sterile inoculating loop. Groundwater samples were shaken
2 MATERIALS AND METHODS
and directly streaked onto nutrient-rich agar.

2.1 Chemicals and fungal pure cultures

The per- and polyfluoroalkyl chemicals described in this article are The objectives of this study
listed in Exhibit S1 with the chemical names, acronyms, and formulae.
The parent compound, 6:2 FTOH (99 percent purity) and standards
were to determine the 6:2
for the biotransformation products were provided by DuPont (Liu
et al., 2010b; Zhang et al., 2013). Internal standards 2H-perfluoro-[1,2-
FTOH biotransformation
13 C
2 ]-2-octenoic acid (M6:2 FTUCA,+98 percent; Wellington Labo- potential of two fungal
ratories, Ontario, Canada) and perfluoro-n-[1,2-13 C2 ] hexanoic acid
(MPFHxA, +98 percent; Wellington Laboratories, Ontario, Canada) strains, Gloeophyllum
were used for liquid chromatography-tandem mass spectrometry (LC-
MS/MS) quantitative analysis. Solvents for LC-MS/MS analysis were
trabeum and Trametes
HPLC grade or higher. All medium components had purities ≥98.0 per-
cent and were purchased from either Sigma-Aldrich (St. Louis, MO) or
versicolor, and six fungal
Thermo Fisher Scientific (Waltham, MA). isolates obtained from sites
G. trabeum was purchased from the American Type Culture Collec-
tion (ATCC 11539; Manassas, VA). Trametes versicolor (MAD697-R) was historically contaminated
generously provided by Professor Chad Jafvert of Purdue University.
with PFASs from the use of
2.2 Site description aqueous film-forming foam
Groundwater (4 liters [L] total) and soil samples (2-foot [ft] linear
core soil sample) were collected from two U.S. military bases by
(AFFF).
ARCADIS/Malcolm Pirnie Inc. Both sites utilized AFFF extensively
for fire training and other fire-related incidents. Detailed information, Triplicate nutrient-rich agar streak plates were prepared for each
including dissolved PFASs concentrations and other environmental groundwater and soil sample and incubated at 30◦ C for up to
parameters, are included in Exhibit S2. Groundwater was collected 4 weeks. Individual colonies were then selected and transferred to
from temporary wells and stored in polypropylene bottles with min- fresh nutrient-rich agar to isolate each morphologically different fun-
imal headspace. Soil samples were collected in the shallow vadose gal colony. All fungal isolates were then transferred to Kirk agar with
MERINO ET AL .
10 g glucose/L, followed by several transfers on Kirk agar without glu-
cose and with 10 to 100 mg PFOA or PFOS/L. All fungi were incu-
bated at 30◦ C to select for those that could tolerate these highly sta-
ble PFASs. Afterwards, each fungal isolate was incubated in liquid Kirk
medium with 10 g glucose/L and 10 to 100 mg PFOA or PFOS/L at
30 ◦ C with 150 revolutions per minute (rpm) orbital shaking. Glu-
cose was removed in future transfers to further isolate those fungi
that could grow in liquid medium on high PFAS concentrations. Among
several hundred initial fungi isolated on nutrient-rich agar, six were
chosen (three from each site) for biotransformation studies and DNA
sequencing.
2.4 6:2 FTOH biotransformation studies
All six fungal isolates were tested to biotransform 6:2 FTOH. Each iso-
late was grown on Potato Dextrose or Yeast Peptone Dextrose agar for
1 week at 30 ◦ C before collecting the spores, as described previously
(Tseng et al., 2014). Briefly, the spores on the agar plate were resus-
pended in 10 mL sterile deionized water and filtered through sterile
glass wool. The spores were then transferred to 1 L liquid Kirk medium
(Tien & Kirk, 1988) containing 10 g glucose/L and grown at 30 ◦ C with
150 rpm orbital shaking for 7 to 10 days and blended (ethanol and UV-
sterilized Hamilton Beach Power Elite Multi-Function Blender) for a
total of 2 min and 20 seconds (sec; 30 sec blend with 5 sec break inter-
vals) to obtain a starter culture. The fungal cultures, G. trabeum and
T. versicolor, were grown in a similar manner except that Newcombe
medium (Newcombe et al., 2002) and Tišma medium (Tišma, Znidarsic-
Plazl, Vasic-Racki, & Zelic, 2012) were used. Before starting each bio-
transformation study, the fungal starter cultures were blended a final
time and washed thrice by centrifuging at 7,000 rpm for 5 to 20 min
before decanting the supernatant and resuspending the pellet in sterile
deionized water. After the final washing step, the cell pellet was resus-
pended in 1 L volume of fresh Kirk, Newcombe, or Tišma medium with
0 g glucose/L.
Biotransformation studies were prepared as previously described
with a few modifications (Tseng et al., 2014). All experiments were con-
ducted in serum bottles (Wheaton, 160-mL), crimp-sealed with grey
butyl rubber stoppers, that contained 10 mL total volume of resus-
pended fungi, 40 mg activated C18 powder from Maxi-Clean C18 car-
tridges (Grace Davison Discovery Sciences; Deerfield, IL), and addi-
tional organic nutrient components. The C18 powder was added to
maintain 6:2 FTOH in the liquid phase since 6:2 FTOH is very volatile
(vapor pressure = 108 Pa at 35◦ C; Krusic et al., 2005) and was acti-
vated by eluting 10 mL acetonitrile through the cartridge, followed by
10 mL air, and let dry overnight. Two nutrient conditions were tested:
(1) addition of 10 mg glucose (G), 0.25 mg yeast extract (Y), and 2 mg
cellulose (C; +GYC) and (2) no added organic nutrients (–GYC). The
experiments were initiated on day 0 by adding 30 microliters of a 1 g
6:2 FTOH/L stock solution (final concentration 3 milligrams per liter
[mg/L]) made in 50 percent ethanol. Immediately after the addition of
6:2 FTOH, each bottle was crimp-sealed with butyl rubber stoppers
and two C18 cartridges were inserted into the headspace to capture
6:2 FTOH and any volatile intermediates (Tseng et al., 2014). The two
C18 cartridges also allowed for aeration, and when oxygen concen-
61
tration was ≤18 percent, as measured by headspace oxygen analyzer
(Model 902D, Quantek Instruments, Grafton, MA), all bottles were aer-
ated for 3 min with filter sterilized air at ∼150 cubic centimeters per
minute flow rate.
A sterile group and a PFAS-free (matrix) control group were
prepared to account for abiotic effects and for background PFAS
concentrations, respectively. The sterile control group consisted of
resuspended fungi autoclaved at 121 ◦ C for 30 min instead of live
fungi. The matrix control group was the same as the live group except
that an equivalent volume (30 𝜇L) of 50 percent ethanol solution was
added instead of the 6:2 FTOH stock solution. Each experimental
group consisted of triplicate samples sacrificed at each time point
(days 0, 7, 14, and 28).
2.5 Tolerance toward PFOA and PFOS
All fungal isolate starter cultures were grown and collected in the same
manner as in Section 2.4. PFOA and PFOS tolerance studies were con-
ducted in serum bottles (160 mL), crimp-sealed with butyl rubber stop-
pers containing 10 mL total volume of resuspended fungi in fresh Kirk
medium without glucose. The experiments were initiated on day 0 by
adding 0.1 mL of PFOA and PFOS stock solutions (1 to 100 g/L made
in 62 percent ethanol) to have a final concentration of 0 to 1,000 mg
PFOA and PFOS/L. Immediately after the addition of PFOA and PFOS,
each bottle was crimp-sealed with butyl rubber stoppers and two 0.22
micron (𝜇m)-pore sterile nylon filters were inserted into the headspace
to allow for aeration (measured headspace oxygen concentrations
remained ≥20 percent). A sterile control group and a PFAS-free con-
trol group were prepared as described in Section 2.4 to account for
abiotic effects and for background PFAS concentrations, respectively.
The matrix control group was the same as the live group except that
an equivalent volume of 62 percent ethanol stock solution was added
instead of the PFOA and PFOS stock solution. Each experimental group
consisted of duplicate samples sacrificed at each time point (days 0
and 14).
At each time point, each sample was filtered through a pre-weighed
glass fiber GF/C Whatman filter and placed in an oven at 50 ◦ C for
20 hours. The dried filter and fungal biomass was then weighed and the
pre-weighed mass of the filter was subtracted to obtain the dry weight
(mg) of the fungal biomass.
2.6 Collection and processing of samples
Triplicate bottles were analyzed at each time point from the bio-
transformation studies using a modified collection method described
in Tseng et al. (2014). Briefly, prior to sample collection, all bot-
tles were aerated with ∼1.9 L air (125 cubic centimeters per minute
for 15 min) through the C18 cartridges inserted into the headspace
to ensure that headspace 6:2 FTOH and volatile metabolites were
captured by the cartridges. Afterwards, the captured compounds
were eluted with 5 mL acetonitrile, followed by 5 mL air through
each cartridge and collected inside the bottle (“first and cartridge
extract”). The cartridges and the sample stopper were then removed,
and the bottle was re-crimped with a fresh butyl rubber stopper
62
and incubated horizontally overnight at 50◦ C with 150 rpm shak-
ing. After incubation, the bottles were stored at 4 ◦ C overnight to
allow for settling of the fungal mycelium and any other large par-
ticles. The supernatant was then filtered (0.22 𝜇m nylon) for LC-
MS/MS quantitative analysis, and a “second extract” on the bottle
was conducted by adding 200 𝜇L 1 molar (M) NaOH and 5 mL ace-
tonitrile. The second extract was then incubated and collected in the
same manner as the first extract. The extract from the sample stop-
per was obtained by incubating upright in a 20 mL scintillation vial
overnight at 50 ◦ C with 150 rpm shaking in 5 mL acetonitrile (“stop-
per extract”), and was then combined with the second extract. All sam-
ples were stored at –20 ◦ C before analysis and at –40 ◦ C for long-
term storage. Additional extractions were conducted, as described in
Discussion S1.
2.7 LC-MS/MS quantitative analysis
LC-MS/MS was performed to determine the concentrations of 6:2
FTOH and transformation products (Ruan et al., 2013; Zhang et al.,
2013). Briefly, 200 𝜇L filtered sample was spiked with 2 𝜇L inter-
nal standard containing (per mL) 1 nanogram (1,1-D; 1,2-13 C) 6:2
FTUCA and 1 nanogram (1,2-13 C) PFHxA. Quantitative analysis of 6:2
FTOH and transformation products were carried out with an Applied
Biosystems-MDS Sciex 4000 Qtrap equipped with a C8 column (ZOR-
BAX RX-C8, 5-𝜇m particle size, 2.1 × 150 millimeter) by Agilent (Santa
Clara, CA). The mass spectrometer was operated in negative electro-
spray ionization mode using scheduled multiple reaction monitoring.
Samples (11 𝜇L) were injected via an autosampler and eluted with
0.15 percent acetic acid in HPLC grade water (solvent A) and 0.15 per-
cent acetic acid in HPLC grade acetonitrile (solvent B) at a flow rate
of 400 microliters per minute. The gradient started with 90 percent
A and 10 percent B (1.50 min), followed by 45 percent A and 55 per-
cent B (1.60 to 2.50 min), 20 percent A and 80 percent B (2.50 to
8.00 min), 0 percent A and 100 percent B (8.10 to 10.10 min), and
ending with 90 percent A and 10 percent B (10.20 to 10.50 min).
Polytetrafluoroethylene (TeflonTM ) and other fluoropolymer materi-
als were avoided as much as possible to minimize background con-
tamination. More detailed information and ion transitions are listed in
Exhibit S3.
2.8 Identification of fungal isolates
All fungal isolates were identified based on the DNA sequence of the
Internal Transcribed Spacer (ITS) region encompassing the 5.8S rRNA
and 28S rRNA (Schoch et al., 2012). Total DNA (ZR Fungal/Bacterial
DNA MiniPrep kit, Zymo Research, Irvine, CA) was first extracted
from all fungal isolates grown in ME liquid medium at 30◦ C with
150 rpm orbital shaking for 4 days. Afterwards, polymerase chain
reaction (PCR) using the Promega GoTaq Kit (Promega, Madison, WI)
with ITS1-F and ITS4-R primers (Embong et al., 2008; Prewitt, Diehl,
McElroy, & Diehl, 2008) was conducted to amplify the ITS region.
The PCR conditions followed touchdown thermal cycling parameters
and contained 40 cycles: initial denaturation temperature 95 ◦C for
35 sec, melt temperature 95 ◦ C for 35 sec, annealing temperature
MERINO ET AL .
of (1) 64 ◦ C to 55 ◦ C (−1 ◦ C per cycle) for 55 sec (10 cycles) and
(2) 54 ◦ C for 55 sec (30 cycles), extending temperature of 72 ◦ C for
1 min, final extension temperature 72 ◦ C for 10 min, hold tempera-
ture 4 ◦ C. The PCR product was verified using gel electrophoresis and
cleaned using the UltraClean PCR Clean-Up Kit (Mo Bio Laboratories,
Carlsbad, CA). The cleaned PCR product was sequenced with ITS1-F
and ITS4-R primers (Schoch et al., 2012). Sequence data were subse-
quently aligned against the NCBI database using standard nucleotide
BLAST and were submitted to NCBI under the accession numbers
KU533844–KU533849.
The six fungal isolates
(TW1-3, TW4-2, TW4-1,
B76, B78, and B79) were
obtained from the culturable
microbial communities found
at two U.S. military bases
historically contaminated
with PFASs due to extensive
use of AFFF.
3 RESULTS AND DISCUSSION
3.1 Identification of fungal isolates
The six fungal isolates (TW1-3, TW4-2, TW4-1, B76, B78, and B79)
were obtained from the culturable microbial communities found at
two U.S. military bases historically contaminated with PFASs due
to extensive use of AFFF. Since these bases had been exposed to
several decades of PFASs and other contaminants, such as chlorinated
solvents and petroleum hydrocarbons, these fungal isolates were
part of a selected group of microorganisms that must have evolved
to tolerate or detoxify PFASs. In addition, these fungi were isolated
on nutrient-limited agar (Tien & Kirk, 1988) without glucose and high
concentrations (mg/L) of PFOA or PFOS. The ITS region of each fungus
was sequenced and compared against the NCBI database (Exhibit 1).
BLAST results indicated that four isolates, TW1-3, TW4-2, TW4-1, and
B79, could be identified as Fusarium sp., Penicillium sp., or Aspergillus sp.
(Exhibit 1). Species in these genera have been shown to degrade a wide
range of contaminants (Exhibit 1). The majority of the results for B76
and B78 were related to Fusarium sp. and Penicillium sp., respectively.
However, B76 may also be related to Lachnum sp. (BLAST Ident 99
percent), and B78 may be related to Aspergillus sp. (BLAST Ident 100
percent), Paecilomyces sp. (BLAST Ident 100 percent), or Hypocrea
sp. (BLAST Ident 100 percent). Species in Paecilomyces sp. have been
shown to degrade benzene, toluene, ethylbenzene, xylene, and phenol
(Estévez, Veiga, & Kennes, 2005; García-Peña, Ortiz, Hernández, &
MERINO ET AL . 63

EXHIBIT 1 BLAST identities of six fungal isolates

BLAST Closely related Biotransformation of other

Isolate Accession # ident genus contaminants Ref.
TW1-3 KU533849 100% Fusarium sp. Phenol, PAHs, lindane, plasticizers, Hidayat, Tachibana, and Itoh (2012);
B76a KU533845 99% dyes, polyamide-6 Luo et al. (2015); Sorkhoh,
Ghannoum, Ibrahim, Stretton, and
Radwan (1990)
B78b KU533847 100% Penicillium sp. PAHs, anthracene, endosulfan, Ceci et al. (2015); Hofrichter, Bublitz,
B79 KU533846 100% 𝛽-hexachlorocyclohexane, and Fritsche (1994); Jove et al.
halogenated phenols, imidazolium (2016); Mohsenzadeh, Rad, and
compounds, quaternary Akbari (2012); Song, Tian, Fan, and
ammonium compounds, dyes, He (2010)
polyethylene, polyamide-6,
aliphatic polyester resin Bionolle
TW4-2 KU533844 100% Aspergillus sp. PAHs, endosulfan, n-alkanes, Mohsenzadeh et al. (2012); Song
TW4-1 KU533848 99% dimethoate, dyes, et al. (2010)
carboxymethylchitosan-g-medium
chain length
polyhydroxyalkanoates
(mcl-PHA), polyamide-6, aliphatic
polyester resin Bionolle
a
B76 ITS rRNA region BLAST results contained one hit highly similar (Ident 99%) to Lachnum sp.
b
B78 ITS rRNA region BLAST results contained one hit each: Aspergillus sp (Ident 100%), Paecilomyces sp. (Ident 100%), and Hypocrea sp. (Ident 100%).

Revah, 2008; Wang et al., 2010), while species in Hypocrea sp. have
been shown to degrade PAHs, pyrene, biopolymers, and azo dyes
(Baldrian et al., 2010; Gajera, Bambharolia, Hirpara, Patel, & Golakiya,
2015; Hong, Park, & Gadd, 2010). Although few studies have reported
the biotransformation of organic pollutants by species in Lachnum
sp., one report demonstrated that Lachnum spartinae could produce
laccase, an extracellular fungal enzyme known to biotransform many
environmental pollutants (Desai & Nityanand, 2011) and may be able
to transform PFOA (Luo et al., 2015; Luo et al., 2017). These previous
reports and the ability of the six fungal isolates to tolerate high PFOA
and PFOS concentrations suggest that these isolates may be able to
transform fluorinated substances. Furthermore, previous research
utilizing forest soil suggested that fungi played an essential role in
the initial rapid breakdown of 8:2 fluorotelomer stearate monoester
(Dasu, Lee, Turco, & Nies, 2012).
3.2 Biotransformation of 6:2 FTOH by fungi
Previously, it was shown that P. chrysosporium, a white-rot fungus, could
transform 6:2 FTOH to 11 metabolites with 5:3 acid (43 mol%) as
the highest transformation product by day 14 (Tseng et al., 2014).
Exhibit S4 depicts the fungal 6:2 FTOH transformation pathway. Since
fungi play a large role in various ecosystems (Harms et al., 2011), this
study further evaluated 6:2 FTOH biotransformation by other fungi,
including a brown-rot fungus, G. trabeum; a soft-rot fungus, T. versicolor;
and six fungal isolates. G. trabeum and T. versicolor were observed to
transform 6:2 FTOH to 9 and 6 quantifiable transformation products,
respectively (Exhibits 2a, S4, S6, S7). All six fungal isolates were capable
of transforming 6:2 FTOH to 5 to 9 quantifiable transformation prod-
ucts within 28 days at various removal molar yields (mol%; Exhibits 2a,
S4, S8–S13) under the conditions tested. The biotransformation path-
ways and metabolite production profiles could change, for example,
with different media formulations (Merino, Wang, Wang, & Mahendra,
2018), initial 6:2 FTOH concentrations (Zhang, Merino, Wang, Ruan, &
Lu, 2017), or the concentrations of fungal inocula.
Total recovery of 6:2 FTOH and metabolites are shown in Exhibits
S5 to S13 and was around 38 to 133 mol% for all live fungal cultures.
The loss in recovery for certain fungi may indicate unknown metabo-
lites due to lack of authentic analytical standards or the formation
of bound-residues to fungal, cellular organic components (Liu et al.,
2010b; Tseng et al., 2014; Wang et al., 2009; Zhang et al., 2013). The
recovery of 6:2 FTOH in the sterile controls remained around 85 to
116 mol% of the initial 6:2 FTOH dosed for all fungi at most time
points. However, due to sacrificial sampling, instrumental variation, or
lower efficiency in some C18 solid phase extraction (SPE) cartridges,
the recovery for 4 out of 32 time points for sterile control bottles
was below 70 percent or above 120 percent. Similar ranges in recov-
ery for both the live and sterile control has been observed in previous
studies (Kim et al., 2013; Kim et al., 2012; Liu et al., 2010a; Liu et al.,
2010b; Zhang et al., 2013; Zhao et al., 2013). Furthermore, the lack of
metabolite formation in the sterile control and the satisfactory recov-
ery for most time points indicate the integrity of the fungal system and
extraction method.
From the biotransformation studies, the eight fungi could be
ranked from least to highest residual 6:2 FTOH: G. trabeum (16 ±
5 mol%) < TW4-2 (33 ± 4 mol%) < B76 (46 ± 16 mol%) < B78 (62 ±
6 mol%) < T. versicolor (63 ± 8 mol%) < B79 (65 ± 16 mol%) < TW4-
1 (82 ± 5 mol%) < TW1-3 (111 ± 7 mol%). While the amount of 6:2
FTOH in TW1-3 cultures was higher than the initial 6:2 FTOH dose,
increasing concentrations of 6:2 FTUCA (up to 8 mol% on day 28) were
observed over time. The lack of 6:2 FTOH transformation for some
fungi could have also resulted from the partitioning of 6:2 FTOH into
the headspace, where C18 SPE cartridges captured 6:2 FTOH, caus-
ing lower bioavailability. Since the extractions for the C18 SPE car-
tridge and the first liquid extraction were mixed, the recovery of 6:2
FTOH into the headspace versus the liquid medium could not be dis-
tinguished in this study. However, a previous report on 6:2 FTOH fun-
gal biotransformation demonstrated that after 28 days of incubation,
up to 46 mol% was observed in the headspace with only 2 to 16 mol%
remaining in the liquid culture (Tseng et al., 2014). In environmental
64
EXHIBIT 2 Transformation of 6:2 FTOH and major metabolites in nine fungal cultures
processes, 6:2 FTOH could still be bioavailable for transformation via
microbial processes due to sorption to soil and sediment, slowing the
rate of volatilization over time (Liu et al., 2010a; Zhao et al., 2013).
Previous studies have also shown that microbial processes in soil can
transform volatile, fluorinated parent compounds, such as 6:2 FTOH,
toward non-volatile intermediates and soil-bound residues (Liu, Lee,
Nies, Nakatsu, & Turco, 2007; Liu et al., 2010a; Liu et al., 2010b; Zhao
et al., 2013).
3.3 Polyfluoroalkyl acids were the major
transformation products
Each fungi could also be ranked by the sum total of detected metabo-
lites at the end of the experiment from highest to lowest: TW4-2 (86 ±
18 mol% total metabolites formed) > B79 (46 ± 5 mol%) > G. trabeum
(23 ± 4 mol%) > B76 (13 ± 4 mol%) > TW1-3 (9 ± 3 mol%) > B78 (9 ±
2 mol%) > TW4-1 (8 ± 4 mol%) > T. versicolor (6 ± 1 mol%; Exhibits 3,
S6–S14). For fungal isolates B76 and B78, slightly more transformation
products (23 ± 10 mol% and 12 ± 3 mol%, respectively) were observed
when the medium was supplemented with GYC (Exhibits S10B–S11B).
This suggests that B76 and B78 may need additional nutrients and
medium amendments to efficiently transform 6:2 FTOH. Indeed, the
aerobic transformation of 6:2 FTOH requires a reducing power, such
as NAD(P)H (Wang, Buck, Szostek, Sulecki, & Wolstenholme, 2012),
suggesting the medium for isolates B76 and B78 may be lacking nec-
essary nutrient components. Furthermore, variation in nutrients have
previously affected the biotransformation pathway of 6:2 FTOH by
MERINO ET AL .
a fungal culture, P. chrysosporium (Merino et al., 2018) and four bac-
terial cultures, Mycobacterium vaccae JOB5, Pseudomonas oleovorans,
Pseudomonas butanovora, and Pseudomonas fluorescens DSM 8341 (Kim
et al., 2013).
Among the eight fungi tested, five fungi, TW4-2, G. trabeum, TW4-
1, B79, and B76, transformed 6:2 FTOH toward more abundant
5:3 acid (9 to 51 mol%) relative to the other metabolites detected
(Exhibits 2b, 3, S4–S14). 5:3 Acid is one of the major downstream
intermediates that has only been observed during microbial 6:2 FTOH
transformation (Liu et al., 2010a; Liu et al., 2010b; Tseng et al., 2014;
Zhang et al., 2013; Zhao et al., 2013). The highest molar yields were
observed in TW4-2 cultures (51 ± 16 mol%) after 16 days of incu-
bation, which slightly increased to 62 ± 13 mol% after 63 days. The
reduced rate of 5:3 acid formation after 16 days suggests that neces-
sary nutrients were depleted, and TW4-2 could not produce enough
reducing power to further transform the transient intermediate, 6:2
FTUCA (Exhibits S4 and S8). More research is needed to develop a
substrate or nutrient supplement for long-term (>16 days) 6:2 FTOH
transformation.
Other fungi, including G. trabeum and isolates B79 and B76, may
also benefit from medium optimization to further transform 6:2 FTOH.
G. trabeum and isolates B79 and B76 had higher molar yields of 5:3
acid after 28 days of incubation (∼9 to 12 mol%) compared to total
PFCAs production (<1 to 6.7 mol%; Exhibit 3). Preliminary experi-
ments using G. trabeum also observed similar 5:3 acid (20 ± 4 mol%)
and PFCAs (<2 mol%) yields (Exhibit S14). This suggests that these
fungi are potential candidates to produce enough reducing power to
MERINO ET AL . 65

EXHIBIT 3 Yields of 6:2 FTOH transformation products in pure cultures and environmental matrices

Initial Transient
intermediates products toward Stable products and intermediates
(mol%) PFCAs (mol%) (mol%)
𝚺 6:2 FTCA, 6:2 𝚺 5:2 ketone, 5:2 𝚺 PFPeA, PFHxA, 𝚺 5:3 acid, 4:3
FTUCA sFTOH PFBA acida Ref.
Gloeophyllum trabeum 3.4 <1 6.7 12.0 This study
Trametes versicolor <1 2.6 <1 2.0 This study
TW1-3 7.9 <1 <1 <1 This study
TW4-2 22.5 4.5 4.6 51.4 This study
TW4-1b 1.1 nd 1.4 4.5 This study
B79b 30.1 nd 4.6 9.4 This study
B79 4.7 nd 2.1 12.6 This study
B76 4.4 nd <1 9.2 This study
b
B78 4.4 nd 1.9 4.8 This study
Phanerochaete chrysosporiumc <1 10.7 5.9 33.5 Tseng et al. (2014)
Aerobic sedimentd nd 21.7 20.3 25.1 Zhao et al. (2013)
Aerobic soil 30.7 17.0 6.1 5.5 Liu et al. (2010b)
Activated sludgee nd 49.4 15.4 15.4 Zhao et al. (2013a)
Anaerobic digester sludgef 42.9 2.5 <1 21.0 Zhang et al. (2013)
Mycobacterium vaccae JOB5g 49.0 21.1 1.4 3.8 Kim et al. (2013)
h
Pseudomonas oleovorans 26.9 42.0 2.7 2.1 Kim et al. (2013)
Pseudomonas oleovoransi 26.9 35.0 1.7 1.7 Kim et al. (2013)
Pseudomonas butanovoraj 18.6 22.0 1.5 7.3 Kim et al. (2013)
Pseudomonas fluorescens 50.0 52.0 1.9 5.7 Kim et al. (2013)
DSM 8341k
nd = not detected.
a 5:3 acid and 4:3 acid can be further transformed via the one-carbon removal pathway37 or 5:3 acid conjugation7 .
b Supplemented with (per 10 mL) 0.25 mg yeast extract, 2 mg cellulose, and 10 mg glucose.
c Modified Kirk medium without glucose and supplemented (per 10 mL) with 100 mg lignocellulosic powder, 0.25 mg yeast extract,

2 mg cellulose, or 10 mg glucose.
d Day 100 data in aerobic river sediment.
e Day 60 data in activated sludge.
f Day 90 data in anaerobic digester sludge.
g
Supplemented with 1-butanol+formate.
h
Supplemented with n-octane+formate.
i
Supplemented with n-octane+dicyclopropylketone.
j
Supplemented with 1-butanol+lactate.
k
Supplemented with sodium chloroacetate+yeast extract+format.

transform 6:2 FTOH toward 5:3 acid, but supplemental substrates may
be needed to increase 6:2 FTOH biotransformation. For example, the
amount of the initial intermediate 6:2 FTUCA observed on day 28
for B79 cultures increased from 4.7 ± 1.2 to 30.1 ± 1.3 mol% after
the removal of GYC, suggesting that GYC has some negative impact
on 6:2 FTOH biotransformation by B79 cultures. However, the same
nutrients were previously shown to improve the biotransformation
of 6:2 FTOH with P. chrysosporium (Merino et al., 2018; Tseng et al.,
2014).
Unknown transformation metabolites were likely produced by
some fungi tested since the total recovery of 6:2 FTOH and metabo-
lites could not be achieved. Additionally, when 3 mg 5:3 acid/L was
dosed as the parent compound, three fungal isolates, TW4-2, TW4-
1, and B79, may have transformed 5:3 acid to unquantifiable metabo-
lites (Exhibit S4). Further transformation of 5:3 acid could occur via
one-carbon removal pathways (Wang et al., 2012) or 5:3 acid conju-
gation with cellular organic compounds (Tseng et al., 2014). The one-
carbon removal pathway is dominant in bacterial transformation of 6:2
FTOH (Kim et al., 2013) and has also been observed for environmen-
tal microbial consortia (Liu et al., 2010b; Zhao et al., 2013). However,
since two of the major products of the one-carbon removal pathway,
4:3 acid and 3:3 acid, were not observed in any of the fungal systems
in this study, it is unlikely that this pathway played a major role in the
fungal transformation of 6:2 FTOH. Instead, 5:3 acid conjugation may
have occurred, especially for those fungi, such as G. trabeum and B76,
with more than 60 mol% loss in total recovery of the initial 6:2 FTOH
dosed. Similar losses were observed in preliminary experiments using
G. trabeum (Exhibit S14). The 5:3 acid conjugation pathway resulted in
the production of three new metabolites when 6:2 FTOH was biotrans-
formed by P. chrysosporium: 5:3 THPA, 5:3 TKE, and 5:3 TKHPA (Tseng
et al., 2014). Due to a lack of authentic analytical standards, these con-
jugates could not be quantified.
66
Other unknown fungal metabolites may have also occurred as some
of the fungi investigated in this study are likely to utilize different
biochemical pathways compared to P. chrysosporium. While G. trabeum
and T. versicolor belong to the same phylum (Basidiomycota) as P.
chrysosporium, the six fungal isolates belong to the phylum Ascomy-
cota. Furthermore, G. trabeum, T. versicolor, P. chrysosporium, Fusarium
sp., Penicillium sp, and Aspergillus sp. (Exhibit 1) employ different
strategies for the biotransformation of organic pollutants and produce
a different suite of intracellular and extracellular enzymes, including
cytochrome P450 monooxygenases and peroxidases (Harms et al.,
2011). The diversity of P450s in fungi are vast, with at least 150
P450 genes identified in P. chrysosporium (Yadav, Doddapaneni, &
Subramanian, 2006) and 250 P450 gene candidates in a brown-rot
fungi, Postia placenta (Ide, Ichinose, & Wariishi, 2012). Furthermore,
G. trabeum is capable of producing a hydroquinone-driven Fenton
system, generating hydroxyl radicals and recycling Fe2+ /Fe3+ at
low pH conditions (Hyde & Wood, 1997; Jensen, Houtman, Ryan, &
Hammel, 2001). While the Fenton reaction produced by G. trabeum
is not completely understood, these reactions may have influenced
the transformation of 6:2 FTOH in this study as FeSO4 ∙7H2 O (25 𝜇M
final concentration) was added to the medium (pH 4.5), and Fenton
reaction systems have already been extensively utilized to degrade
perfluoroalkyl acids (Merino et al., 2016).
The remaining three fungal cultures (fungal isolates TW1-3 and B78
and T. versicolor) did not transform 6:2 FTOH toward 5:3 acid under the
conditions tested. Fungal isolate TW1-3 could not transform 6:2 FTOH
beyond one of the initial metabolites, 6:2 FTUCA (7.9 ± 1.4 mol%), over
28 days of incubation (Exhibit 3). This is likely due to a lack of reduc-
ing power (Liu et al., 2010b; Wang et al., 2012) and may be rectified
by amending the medium (Kim et al., 2013; Merino et al., 2018). T. ver-
sicolor and fungal isolate B78 also minimally transformed 6:2 FTOH,
resulting in similar molar yields of 5:3 acid (∼2 and ∼5 mol%, respec-
tively) to the sum of 5:2 sFTOH and 5:2 ketone (∼3 and ∼4 mol%,
respectively), which are the direct precursors of the PFCAs, PFPeA,
and PFHxA (Exhibit S4; Liu et al., 2010b). However, production of
lower amounts of PFCAs and more transformation of 6:2 FTOH toward
5:3 acid and other polyfluoroalkyl substances may be achieved by
altering the medium. For example, the addition of 2,6-dimethoxy-1,4-
benzoquinone and chelated ferric ion (Fe3+ -oxalic acid) may improve
6:2 FTOH transformation by T. versicolor due to the production of
hydroxyl radicals (Harms et al., 2011; Marco-Urrea, Aranda, Caminal,
& Guillen, 2009; Vilaplana, García, Caminal, Guillén, & Sarrà, 2012).
3.4 Comparison of fungal cultures with other
microorganisms
Although 6:2 FTOH biotransformation by most fungi tested in this
study was low compared to previous reports using pure cultures and
environmental matrices (Exhibit 3; Butt et al., 2014; Tseng et al., 2014),
the amount of 5:3 acid observed was relatively higher than the produc-
tion of PFCAs and transient intermediates after 28 days of incubation.
In comparison, three Pseudomonas strains and M. vaccae JOB5 accu-
mulated either the initial intermediates, 6:2 FTCA and 6:2 FTUCA, or
the transient products, 5:2 ketone and 5:2 sFTOH (Kim et al., 2013;
MERINO ET AL .
Kim et al., 2012), which have the potential to be further transformed
to PFCAs by other microbial processes (Liu et al., 2010b). The effec-
tive transformation of 6:2 FTOH by the fungal isolate TW4-2 and G.
trabeum (Exhibit 2a) indicates that other fungi besides P. chrysosporium
are capable of oxidizing and reducing 6:2 FTOH. Furthermore, the 5:3
acid yield for both isolate TW4-2 and P. chrysosporium was higher than
that observed for environmental matrices (Liu et al., 2010b; Zhang
et al., 2013; Zhao et al., 2013), indicating these two fungi could employ
similar biotransformative mechanisms as entire microbial consortia.
However, for the other fungal cultures in this study, medium amend-
ments may be needed for enhanced 6:2 FTOH degradation toward
more degradable polyfluoroalkyl acids, preventing the accumulation
of PFCAs. The medium used in this study may have been less effec-
tive for the production of the necessary enzymes and reducing power
to degrade 6:2 FTOH. For example, replacing nitrilotriacetic acid with
ethylenediaminetetraacetic acid as the trace elements chelator might
enhance 6:2 FTOH biotransformation and increase 5:3 acid yields
(Merino et al., 2018).
Compared to bacterial cultures (Kim et al., 2013), the fungal cul-
tures were much slower to oxidize 6:2 FTOH to the initial intermedi-
ate, 6:2 FTCA. Within three days, P. fluorescens DSM 8431 transformed
64 mol% 6:2 FTOH, with the major products as 6:2 FTCA (55 mol%),
6:2 FTUCA (8.9 mol%), when sodium chloroacetate, yeast extract, and
formate were added (Kim et al., 2013). P. oleovorans also transformed
at least 95 mol% 6:2 FTOH within 0.5 day to 6:2 FTCA (6.5 mol%) and
6:2 FTUCA (25 mol%) when n-octane and dicyclopropylketone were
added (Kim et al., 2013). Similar results were observed for P. butanovora
and M. vaccae JOB5 (Kim et al., 2013). In contrast, fungi likely need at
least 7 to 14 days to achieve similar results when accounting for only
the bioavailable portion of 6:2 FTOH (Merino et al., 2018; Tseng et al.,
2014). Since the microbial consortia from soil, sediment, and sludge
achieved high molar yields of 5:3 acid (Liu et al., 2010b; Zhang et al.,
2013; Zhao et al., 2013), similar consortia made up of selected fun-
gal and bacterial cultures could be employed to efficiently degrade
6:2 FTOH and similar PFASs in remediation systems. These systems
may utilize bacteria, such as P. fluorescens DSM 8431, P. oleovorans,
and P. butanovora, to quickly transform 6:2 FTOH to 6:2 FTUCA. 6:2
FTUCA could then be further degraded by fungal degradation path-
ways toward 5:3 acid and 5:3 acid conjugates (Tseng et al., 2014) or
by one-carbon removal pathways to form x:3 acids (Wang et al., 2012).
Although the toxicity of polyfluoroalkyl acids, such as x:3 acids and
5:3 acid conjugates, is still unknown, these substances have the poten-
tial to be further biotransformed in the environment toward less toxic
compounds, unlike PFCAs that are environmentally persistent. Indeed,
more studies are needed to identify and describe the toxicological
properties of known and unknown polyfluoroalkyl acids before engi-
neering biological remediation systems.
3.5 Tolerance toward PFOA and PFOS
Since PFCAs are likely to be found with polyfluoroalkyl substances in
consumer products and industrial applications (Moody & Field, 1999)
and are also transformation end products in the aerobic degrada-
tion of polyfluoroalkyl substances (Butt et al., 2014), tolerance toward
MERINO ET AL .
EXHIBIT 4 Effect of PFOA and PFOS on growth of fungal isolates
PFOA and PFOS by the six fungal isolates was examined. Fungi were
exposed to increasing concentrations of PFOA and PFOS (0, 10, 100,
and 1,000 mg/L) to determine the approximate concentration that
would affect fungal growth over 14 days of incubation (Exhibit 4).
The isolates exhibited different growth behaviors based on their origi-
nal location. Those from site TW experienced increasing growth with
increasing concentrations of PFOA and PFOS, with maximum dry
weight observed at 1,000 mg PFOA and PFOS/L. In contrast, those
from site B were negatively impacted by 1,000 mg PFOA and PFOS/L,
and only B78 and B79 were positively affected by 100 mg PFOA
and PFOS/L. Generally, growth of all fungal isolates were not neg-
atively impacted by the lowest concentration dosed (10 mg/L), indi-
cating lower concentrations of PFOA, PFOS, and similar PFASs found
in the environment (Moody & Field, 1999) would not impact growth
of indigenous or bioaugmented fungi. Although all six fungal isolates
were unable to biotransform PFOA (Exhibits S15 and S16), tolerance
toward high concentrations of PFOA, PFOS, and similar PFASs sug-
gests that these fungi are potential candidates for PFASs remediation.
Furthermore, for sites contaminated with PFASs and metals, such as
chromium, most fungal isolates tested in this study were capable of
sorbing metals (Exhibit S17). However, the effects of perfluoroalkyl
substances on fungal stress responses and the fungal transformation
of 6:2 FTOH and related compounds has not yet been examined. Com-
paratively, bacteria (e.g., Dehalococcoides and Rhodococcus jostii RHA1)
67
were negatively impacted by perfluoroalkyl acids (Weathers, Harding-
Marjanovic, Higgins, Alvarez-Cohen, & Sharp, 2016; Weathers, Hig-
gins, & Sharp, 2015), and the bacterial community in aerobic river
sediment was observed to decrease in diversity with PFOA expo-
sure (>100 nanograms per gram dry weight in sediment; Sun, Wang,
Peng, Wang, & Lu, 2016). Hydrogenotrophic methanogens from anaer-
obic sludge were also inhibited by 500 mg PFOS/L, but other PFASs
tested, such as PFBS, PFPrA, and PFPeA, did not inhibit methanogene-
sis (Ochoa-Herrera, Field, Luna-Velasco, & Sierra-Alvarez, 2016).
The increased growth experienced by most fungal isolates up to
100 or 1,000 mg PFOA and PFOS/L may be attributed to increased
oxygen transfer. Other perfluorinated compounds have been shown
to improve the solubility of respiratory gases, including perfluo-
rotributylamine, perfluorodecalin, perfluorotripropylamine, and
perfluoro-octyl bromide (Lowe, Davey, & Power, 1998; Mitsuno,
Ohyanagi, & Naito, 1982). These compounds have also previously been
used to enhance the growth of prokaryotic and eukaryotic cell culture
systems (Lowe et al., 1998), including P. chrysosporium batch cultures
(Ntwampe, Williams, & Sheldon, 2010). Due to enhanced oxygen trans-
fer, improved growth and bioproductivity was sustained over longer
periods compared to cultures without perfluorinated compounds
(Lowe et al., 1998; Ntwampe et al., 2010). Perfluorinated compounds
have also been shown to impart other positive side effects, such as
decreased mechanical damage to biomass, increased nutrient uptake,
and increased enzyme productivity (Lowe et al., 1998; Ntwampe et al.,
2010). Furthermore, polytetrafluoroethylene (TeflonTM ) membranes
have also been used to oxygenate animal-hybridoma cells (Schneider,
Reymond, Marison, & Vonstockar, 1995) and plant cells (Lowe et al.,
1998).
4 CONCLUSIONS
Since 6:2 FTOH is increasingly being used to manufacture FTOH-based
products, it is important to understand the variety of microbial pro-
cesses that can transform 6:2 FTOH. This study examined the fun-
gal biotransformation of 6:2 FTOH by three well-characterized fungal
cultures, P. chrysosporium, G. trabeum, and T. versicolor, and six fungal
isolates. Among the nine fungal cultures, TW4-2 isolate readily bio-
transformed 6:2 FTOH and produced the highest yields of 5:3 acid.
Other fungal cultures transformed 6:2 FTOH at different removal
yields, and most cultures formed higher amounts of polyfluoroalkyl
substances, such as 5:3 acid, rather than terminal PFCAs. While more
studies are needed to characterize the environmental conditions to
improve 6:2 FTOH transformation rates and product profiles, the
diversion of the 6:2 FTOH biotransformation pathway away from ter-
minal PFCAs and toward PFAAs by fungal cultures suggests that fungi
possess favorable mechanisms for the biotransformation of PFCA pre-
cursors. Since PFCAs are still likely to be produced during aerobic 6:2
FTOH transformation processes and may be present at PFAS contam-
inated sites, the six fungal isolates were able to tolerate high con-
centrations of PFOA and PFOS, further demonstrating that fungi are
potential candidates for PFAS remediation strategies. These results
provide additional information on the role of bacteria and fungi on the
68
fate and transport of PFASs and could also be employed for developing
treatment trains involving biostimulation approaches or bioaugmenta-
tion cocultures containing fungi and bacteria to efficiently degrade 6:2
FTOH and other PFASs in waste streams and at contaminated sites.
ACKNOWLEDGMENTS
This work was supported by the Air Force Civil Engineer Center con-
tract #FA8903-11-C-8009 and performed in a renovated collabora-
tory funded by National Science Foundation Grant #0963183, which
was awarded under the American Recovery and Reinvestment Act
of 2009 (ARRA). The LC-MS/MS (UCLA Molecular Instrumentation
Center) was funded by the NSF Grant #S10RR024605. N. Merino
received U.S. Environmental Protection Agency Science to Achieve
Results (EPA-STAR) Fellowship, the UCLA Dissertation Year Fellow-
ship, SWE Scholarships, and the Earth-Life Science Institute Origin of
Life (EON) Postdoctoral Fellowship, which is supported by a grant from
the John Templeton Foundation. The opinions expressed in this pub-
lication are those of the author(s) and do not necessarily reflect the
views of the John Templeton Foundation. E. O'Connor received UCLA
Eugene V. Cota-Robles Fellowship and UCLA Competitive Edge Grant
(funded by a National Science Foundation Alliance for Graduate Educa-
tion and the Professoriate grant). S. Mahendra received UCLA Hellman
Fellowship, the DuPont Young Professor Award, and the NSF CAREER
Award. We also thank Dr. Ning Wang for providing advice on experi-
mental design, analytical measurements, and manuscript preparation.
REFERENCES
Ali, M. I., Ahmed, S., Robson, G., Javed, I., Ali, N., Atiq, N., & Hameed, A.
(2014). Isolation and molecular characterization of polyvinyl chloride
(PVC) plastic degrading fungal isolates. Journal of Basic Microbiology,
54(1), 18–27.
Baker, B. E., & Rao, N. S. (1994). Textile finishes and fluorosurfactants. In B. E.
Banks, B. E. Smart, & J. C. Tatlow (Eds.), Organofluorine chemistry: Princi-
ples and commercial applications (pp. 321–338). New York, NY: Springer.
Baldrian, P., Voříšková, J., Dobiášová, P., Merhautová, V., Lisá, L., & Valášková,
V. (2010). Production of extracellular enzymes and degradation of
biopolymers by saprotrophic microfungi from the upper layers of forest
soil. Plant and Soil, 338(1-2), 111–125.
Butt, C. M., Muir, D. C. G., & Mabury, S. A. (2014). Biotransformation
pathways of fluorotelomer-based polyfluoroalkyl substances: A review.
Environmental Toxicology and Chemistry, 33(2), 243–267.
Ceci, A., Pierro, L., Riccardi, C., Pinzari, F., Maggi, O., Persiani, A. M., … Papini,
M. (2015). Biotransformation of 𝛽-hexachlorocyclohexane by the sapro-
trophic soil fungus Penicillium griseofulvum. Chemosphere, 137, 101–107.
Dasu, K., Lee, L. S., Turco, R. F., & Nies, L. F. (2012). Aerobic biodegradation
of 8:2 fluorotelomer stearate monoester and 8:2 fluorotelomer citrate
triester in forest soil. Chemosphere, 91(3), 399–405.
Desai, S. S., & Nityanand, C. (2011). Microbial laccases and their applica-
tions: A review. Asian Journal of Biotechnology, 3(2), 98–124.
Ding, G., & Peijnenburg, W. J. G. M. (2013). Physicochemical properties and
aquatic toxicity of poly- and perfluorinated compounds. Critical Reviews
in Environmental Science and Technology, 43(6), 598–678.
Embong, Z., Hitam, W. H. W., Yean, C. Y., Rashid, N. H. A., Kamarudin, B.,
Abidin, S. K. Z., … Ravichandran, M. (2008). Specific detection of fungal
pathogens by 18S rRNA gene PCR in microbial keratitis. BMC Ophthal-
mology, 8. Retrieved from https://bmcophthalmol.biomedcentral.
MERINO ET AL .
com/track/pdf/10.1186/1471-2415-8-7?site=bmcophthalmol.
biomedcentral.com
Estévez, E., Veiga, M. C., & Kennes, C. (2005). Biodegradation of toluene by
the new fungal isolates Paecilomyces variotii and Exophiala oligosperma.
Journal of Industrial Microbiology and Biotechnology, 32(1), 33–37.
Gajera, H. P., Bambharolia, R. P., Hirpara, D. G., Patel, S. V., & Golakiya, B. A.
(2015). Molecular identification and characterization of novel Hypocrea
koningii associated with azo dyes decolorization and biodegradation of
textile dye effluents. Process Safety and Environmental Protection, 98,
406–416.
García-Peña, I., Ortiz, I., Hernández, S., & Revah, S. (2008). Biofiltration of
BTEX by the fungus Paecilomyces variotii. International Biodeterioration &
Biodegradation, 62(4), 442–447.
Harms, H., Schlosser, D., & Wick, L. Y. (2011). Untapped potential: Exploiting
fungi in bioremediation of hazardous chemicals. Nature Reviews Microbi-
ology, 9(3), 177–192.
Hidayat, A., Tachibana, S., & Itoh, K. (2012). Determination of chrysene
degradation under saline conditions by Fusarium sp F092, a fungus
screened from nature. Fungal Biology, 116(6), 706–714.
Hofrichter, M., Bublitz, F., & Fritsche, W. (1994). Unspecific degradation of
halogenated phenols by the soil fungus Penicillium frequentans Bi 7/2.
Journal of Basic Microbiology, 34(3), 163–172.
Hong, J. W., Park, J. Y., & Gadd, G. M. (2010). Pyrene degradation and
copper and zinc uptake by Fusarium solani and Hypocrea lixii isolated
from petrol station soil. Journal of Applied Microbiology, 108(6), 2030–
2040.
Hyde, S. M., & Wood, P. M. (1997). A mechanism for production of hydroxyl
radicals by the brown-rot fungus Coniophora puteana: Fe(III) reduction
by cellobiose dehydrogenase and Fe(II) oxidation at a distance from the
hyphae. Microbiology-UK, 143, 259–266.
Ide, M., Ichinose, H., & Wariishi, H. (2012). Molecular identification and
functional characterization of cytochrome P450 monooxygenases from
the brown-rot basidiomycete Postia placenta. Archives of Microbiology,
194(4), 243–253.
Jensen, K. A., Houtman, C. J., Ryan, Z. C., & Hammel, K. E. (2001). Path-
ways for extracellular fenton chemistry in the brown rot basidiomycete
Gloeophyllum trabeum. Applied and Environmental Microbiology, 67(6),
2705–2711.
Jove, P., Olivella, M. A., Camarero, S., Caixach, J., Planas, C., Cano, L., & De Las
Heras, F. X. (2016). Fungal biodegradation of anthracene-polluted cork:
A comparative study. Journal of Environmental Science and Health, Part A,
51(1), 70–77.
Kataoka, R., Takagi, K., Kamei, I., Kiyota, H., & Sato, Y. (2010). Biodegradation
of dieldrin by a soil fungus isolated from a soil with annual endosulfan
applications. Environmental Science & Technology, 44(16), 6343–6349.
Kim, M. H., Wang, N., & Chu, K. H. (2013). 6:2 Fluorotelomer alcohol (6:2
FTOH) biodegradation by multiple microbial species under different
physiological conditions. Applied Microbiology and Biotechnology, 98(4),
1831–1840.
Kim, M. H., Wang, N., McDonald, T., & Chu, K.-H. (2012). Biodeflu-
orination and biotransformation of fluorotelomer alcohols by two
alkane-degrading Pseudomonas strains. Biotechnology and Bioengineering,
109(12), 3041–3048.
Krusic, P. J., Marchione, A. A., Davidson, F., Kaiser, M. A., Kao, C.-P. C.,
Richardson, R. E., … Buck, R. C. (2005). Vapor pressure and intramolec-
ular hydrogen bonding in fluorotelomer alcohols. Journal of Physical
Chemistry A, 109(28), 6232–6241.
Liu, J., Lee, L. S., Nies, L. F., Nakatsu, C. H., & Turco, R. F. (2007). Biotransfor-
mation of 8:2 fluorotelomer alcohol in soil and by soil bacteria isolates.
Environmental Science & Technology, 41(23), 8024–8030.
MERINO ET AL .
Liu, J., Wang, N., Buck, R. C., Wolstenholme, B. W., Folsom, P. W., Sulecki,
L. M., & Bellin, C. A. (2010a). Aerobic biodegradation of [14 C] 6:2 flu-
orotelomer alcohol in a flow-through soil incubation system. Chemo-
sphere, 80(7), 716–723.
Liu, J., Wang, N., Szostek, B., Buck, R. C., Panciroli, P. K., Folsom, P. W., …
Bellin, C. A. (2010b). 6-2 Fluorotelomer alcohol aerobic biodegradation
in soil and mixed bacterial culture. Chemosphere, 78(4), 437–444.
Lowe, K. C., Davey, M. R., & Power, J. B. (1998). Perfluorochemicals: Their
applications and benefits to cell culture. Trends in Biotechnology, 16(6),
272–277.
Luo, Q., Lu, J., Zhang, H., Wang, Z., Feng, M., Chiang, S.-Y. D., … Huang, Q.
(2015). Laccase-catalyzed degradation of perfluorooctanoic acid. Envi-
ronmental Science & Technology Letters, 2(7), 198–203.
Luo, Q., Wang, Z., Feng, M., Chiang, D., Woodward, D., Liang, S., … Huang, Q.
(2017). Factors controlling the rate of perfluorooctanoic acid degrada-
tion in laccase-mediator systems: The impact of metal ions. Environmen-
tal Pollution, 224, 649–657.
Marco-Urrea, E., Aranda, E., Caminal, G., & Guillen, F. (2009). Induction of
hydroxyl radical production in Trametes versicolor to degrade recalci-
trant chlorinated hydrocarbons. Bioresource Technology, 100(23), 5757–
5762.
Merino, N., Qu, Y., Deeb, R. A., Hawley, E. L., Hoffmann, M. R., & Mahen-
dra, S. (2016). Degradation and removal methods for perfluoroalkyl and
polyfluoroalkyl substances in water. Environmental Engineering Science,
33(9), 615–649.
Merino, N., Wang, N., Wang, M., & Mahendra, S. (2018, In Review). Roles
of various nutrient components on the biotransformation of 6:2 fluo-
rotelomer alcohol (6:2 FTOH) on a wood-decaying fungi.
Mitsuno, T., Ohyanagi, H., & Naito, R. (1982). Clinical-studies of a perfluoro-
chemical whole-blood substitute (Fluosol-Da): Summary of 186 cases.
Annals of Surgery, 195(1), 60–69.
Mohsenzadeh, F., Rad, A. C., & Akbari, M. (2012). Evaluation of oil removal
efficiency and enzymatic activity in some fungal strains for bioremedia-
tion of petroleum-polluted soils. Iranian Journal of Environmental Health
Science & Engineering, 9. doi: 10.1186/1735-2746-9-26
Moody, C. A., & Field, J. A. (1999). Determination of perfluorocarboxylates
in groundwater impacted by fire-fighting activity. Environmental Science
& Technology, 33(16), 2800–2806.
Newcombe, D., Paszczynski, A., Gajewska, W., Kroger, M., Feis, G., & Craw-
ford, R. (2002). Production of small molecular weight catalysts and
the mechanism of trinitrotoluene degradation by several Gloeophyllum
species. Enzyme and Microbial Technology, 30(4), 506–517.
Ntwampe, S. K. O., Williams, C. C., & Sheldon, M. S. (2010). Influence of per-
fluorocarbons on Phanerochaete chrysosporium biomass development,
substrate consumption and enzyme production. Chemical and Biochemi-
cal Engineering Quarterly, 24(2), 187–194.
Ochoa-Herrera, V., Field, J. A., Luna-Velasco, A., & Sierra-Alvarez, R. (2016).
Microbial toxicity and biodegradability of perfluorooctane sulfonate
(PFOS) and shorter chain perfluoroalkyl and polyfluoroalkyl substances
(PFASs). Environmental Science Process Impacts, 18(9), 1236–1246.
Paul, A. G., & Jones, K. C. (2009). A first global production, emission, and
environmental inventory for perfluorooctane sulfonate. Environmental
Science & Technology, 43(2), 386–392.
Prewitt, M. L., Diehl, S. V., McElroy, T. C., & Diehl, W. J. (2008). Comparison of
general fungal and basidiomycete-specific ITS primers for identification
of wood decay fungi. Forest Products Journal, 58(4), 66–71.
Ritter, S. K. (2010). Fluorochemicals go short. Chemical Engineering News, 88,
12–17.
Ritz, K., & Young, I. M. (2004). Interactions between soil structure and fungi.
Mycologist, 18, 52–59.
69
Ruan, T., Szostek, B., Folsom, P., Wolstenholme, B. W., Liu, R., Liu, J.,
… Buck, R. C. (2013). Aerobic soil biotransformation of 6:2 fluo-
rotelomer iodide. Environmental Science & Technology, 47(20), 11504–
11511.
Schneider, M., Reymond, F., Marison, I. W., & Vonstockar, U. (1995). Bubble-
free oxygenation by means of hydrophobic porous membranes. Enzyme
and Microbial Technology, 17(9), 839–847.
Schoch, C. L., Seifert, K. A., Huhndorf, S., Robert, V., Spouge, J. L., Levesque, C.
A., … Schindel, D. (2012). Nuclear ribosomal internal transcribed spacer
(ITS) region as a universal DNA barcode marker for Fungi. Proceedings of
the National Academy of Sciences, 109(16), 6241–6246.
Song, F. Q., Tian, X. J., Fan, X. X., & He, X. B. (2010). Decomposing ability
of filamentous fungi on litter is involved in a subtropical mixed forest.
Mycologia, 102(1), 20–26.
Sorkhoh, N. A., Ghannoum, M. A., Ibrahim, A. S., Stretton, R. J., & Radwan,
S. S. (1990). Crude-oil and hydrocarbon-degrading strains of Rhodococ-
cus rhodochrous isolated from soil and marine environments in Kuwait.
Environmental Pollution, 65(1), 1–17.
Sun, Y., Wang, T., Peng, X., Wang, P., & Lu, Y. (2016). Bacterial community
compositions in sediment polluted by perfluoroalkyl acids (PFAAs) using
Illumina high-throughput sequencing. Environmental Science and Pollu-
tion Research, 23(11), 10556–10565.
Tien, M., & Kirk, T. K. (1988). Lignin peroxidase of Phanerochaete chrysospo-
rium. Methods in Enzymology, 161, 238–249.
Tišma, M., Znidarsic-Plazl, P., Vasic-Racki, D., & Zelic, B. (2012). Optimiza-
tion of laccase production by Trametes versicolor cultivated on industrial
waste. Applied Biochemistry and Biotechnology, 166(1), 36–46.
Tseng, N., Wang, N., Szostek, B., & Mahendra, S. (2014). Biotransformation
of 6:2 fluorotelomer alcohol (6:2 FTOH) by a wood-rotting fungus. Envi-
ronmental Science & Technology, 48(7), 4012–4020.
U.S. Enviromental Protection Agency. (2006). 2010/2015 PFOA stewardship
program. Washington, DC: Author.
Vilaplana, M., García, A. B., Caminal, G., Guillén, F., & Sarrà, M. (2012). Opti-
misation of the operational conditions of trichloroethylene degradation
using Trametes versicolor under quinone redox cycling conditions using
central composite design methodology. Biodegradation, 23(2), 333–
341.
Wang, L., Li, Y., Yu, P., Xie, Z., Luo, Y., & Lin, Y. (2010). Biodegradation of phe-
nol at high concentration by a novel fungal strain Paecilomyces variotii
JH6. Journal of Hazardous Materials, 183(1–3), 366–371.
Wang, N., Buck, R. C., Szostek, B., Sulecki, L. M., & Wolstenholme, B. W.
(2012). 5:3 Polyfluorinated acid aerobic biotransformation in activated
sludge via novel “one-carbon removal pathways”. Chemosphere, 87(5),
527–534.
Wang, N., Szostek, B., Buck, R. C., Folsom, P. W., Sulecki, L. M., & Gannon, J.
T. (2009). 8-2 Fluorotelomer alcohol aerobic soil biodegradation: Path-
ways, metabolites, and metabolite yields. Chemosphere, 75(8), 1089–
1096.
Weathers, T. S., Harding-Marjanovic, K., Higgins, C. P., Alvarez-Cohen, L., &
Sharp, J. O. (2016). Perfluoroalkyl acids inhibit reductive dechlorination
of trichloroethene by repressing Dehalococcoides. Environmental Science
& Technology, 50(1), 240–248.
Weathers, T. S., Higgins, C. P., & Sharp, J. O. (2015). Enhanced biofilm pro-
duction by a toluene-degrading Rhodococcus observed after exposure
to perfluoroalkyl acids. Environmental Science & Technology, 49(9), 5458–
5466.
Yadav, J. S., Doddapaneni, H., & Subramanian, V. (2006). P450ome of the
white rot fungus Phanerochaete chrysosporium: Structure, evolution and
regulation of expression of genomic P450 clusters. Biochemical Society
Transactions, 34(6), 1165–1169.
70
Zhang, S., Merino, N., Wang, N., Ruan, T., & Lu, X. (2017). Impact of 6:2 flu-
orotelomer alcohol aerobic biotransformation on a sediment microbial
community. Science of The Total Environment, 575, 1361–1368.
Zhang, S., Szostek, B., McCausland, P. K., Wolstenholme, B. W., Lu, X., Wang,
N., & Buck, R. C. (2013). 6:2 and 8:2 fluorotelomer alcohol anaerobic bio-
transformation in digester sludge from a WWTP under methanogenic
conditions. Environmental Science & Technology, 47, 4227–4235.
Zhao, L., Folsom, P. W., Wolstenholme, B. W., Sun, H., Wang, N., & Buck, R. C.
(2013). 6:2 Fluorotelomer alcohol biotransformation in an aerobic river
sediment system. Chemosphere, 90(2), 203–209.
AUTHOR'S BIOGRAPHIES
Nancy Merino, PhD, is currently a research fellow at the Earth Life
Science Institute and the University of Southern California analyz-
ing the microbial community of two terrestrial serpentinizing systems
(pH∼10.5 to 12). Nancy obtained her PhD from University of Califor-
nia, Los Angeles (UCLA) in 2016, and was working on the fungal bio-
transformation of per- and polyfluoroalkyl compounds in Dr. Shaily
Mahendra's laboratory.
Meng Wang is a PhD student in Dr. Shaily Mahendra's laboratory at
UCLA. His research interests include application of bionanomaterials
and enzymes in environmental remediation and water treatment.
Rocio Ambrocio was an undergraduate research assistant in Dr. Shaily
Mahendra's laboratory. She received her bachelor degree in civil and
environmental engineering from UCLA in 2016, and is currently a sci-
ence instructor for the Science Discovery Center in Orange County,
California.
Kimberly Mak was an undergraduate research assistant in Dr. Shaily
Mahendra's laboratory. She received her bachelor degree in environ-
mental science from UCLA in 2016, and is currently an independent
compliance monitor with the environmental consulting firm Environ-
mental Intelligence.
Ellen O'Connor is a graduate student obtaining her PhD in molecular
toxicology from UCLA where she did work on per- and polyfluoroalkyl
compounds during a rotation in Dr. Shaily Mahendra's laboratory. Ms.
O'Connor is now studying the role of oxidized lipids in the development
of pulmonary hypertension.
An Gao was an undergraduate research assistant under project “Grand
Challenges” in Dr. Shaily Mahendra's laboratory. She received her
bachelor degree in civil and environmental engineering from UCLA in
2016, and is currently a junior estimator at Bernards Bros Construc-
tion.
MERINO ET AL .
Elisabeth L. Hawley, PE, is a senior consultant at Geosyntec Consul-
tants in Oakland, California. She is a professional engineer with more
than 15 years of experience with environmental remediation, focusing
on PFASs and other emerging contaminants including applied research,
site characterization, fate and transport, and innovative treatment. She
earned an MS degree in civil and environmental engineering and a BS
in environmental engineering science from the University of California,
Berkeley.
Rula A. Deeb, PhD, is a senior principal at Geosyntec Consultants in
Oakland, California. She is a professional civil and environmental engi-
neer with more than 25 years of experience in private practice and
academia addressing the cross-media fate and transport of contami-
nants and remediation of complex soil and groundwater sites impacted
by non-aqueous phase liquids. She earned her PhD and postdoctoral
fellowship from the University of California, Berkeley.
Linda Y. Tseng, PhD, is an assistant professor at Colgate University in
the Environmental Studies Program and the Department of Physics
and Astronomy. She received her PhD in environmental engineering
from the University of California, Irvine, in 2012, and was a postdoc-
toral research associate in Dr. Shaily Mahendra's laboratory at UCLA.
Her research interests include extracellular polymeric substances, and
the fate and transport of emerging contaminants.
Shaily Mahendra, PhD, is an associate professor in the UCLA Depart-
ment of Civil and Environmental Engineering. She received PhD from
University of California, Berkeley, and postdoctoral fellowship from
Rice University. She recently won the Paul L. Busch Award, National
Science Foundation CAREER Award, DuPont Young Professor Award,
Northrop Grumman Excellence in Teaching Award, Samueli Fellowship,
Hellman Fellowship, Poptech Science and Public Leadership Fellow-
ship, and Environmental Science & Technology Excellence in Review
Award. Her research areas are microbial processes in natural and engi-
neered systems, applications of molecular and isotopic tools in envi-
ronmental microbiology, environmental applications of nanomaterials,
and biotransformation of water contaminants.
SUPPORTING INFORMATION
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How to cite this article: Merino N, Wang M, Ambrocio R, et al.
Fungal biotransformation of 6:2 fluorotelomer alcohol. Remedi-
ation. 2018;28:59–70. https://doi.org/10.1002/rem.21550