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Greatly Enhanced Removal of Volatile Organic Carcinogens by a Genetically Modified Houseplant, Pothos Ivy ( Epipremnum aureum ) Expressing the Mammalian Cytochrome P450 2e1 Gene

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The indoor air in urban homes of developed countries is usually contaminated with significant levels of volatile organic carcinogens (VOCs), such as formaldehyde, benzene, and chloroform. There is a need for a practical, sustainable technology for the removal of VOCs in homes. Here we show that a detoxifying transgene, mammalian cytochrome P450 2e1 can be expressed in a houseplant, Epipremnum aureum, pothos ivy, and that the resulting genetically modified plant has sufficient detoxifying activity against benzene and chloroform to suggest that biofilters using transgenic plants could remove VOCs from home air at useful rates.
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Greatly Enhanced Removal of Volatile Organic Carcinogens by a
Genetically Modied Houseplant, Pothos Ivy (Epipremnum aureum)
Expressing the Mammalian Cytochrome P450 2e1 Gene
Long Zhang, Ryan Routsong, and Stuart E. Strand*
Department of Civil and Environmental Engineering, University of Washington, Box 355014, Seattle, Washington 98195-5014,
United States
*
SSupporting Information
ABSTRACT: The indoor air in urban homes of developed countries is usually
contaminated with signicant levels of volatile organic carcinogens (VOCs), such
as formaldehyde, benzene, and chloroform. There is a need for a practical,
sustainable technology for the removal of VOCs in homes. Here we show that a
detoxifying transgene, mammalian cytochrome P450 2e1 can be expressed in a
houseplant, Epipremnum aureum, pothos ivy, and that the resulting genetically
modied plant has sucient detoxifying activity against benzene and chloroform
to suggest that biolters using transgenic plants could remove VOCs from home
air at useful rates.
INTRODUCTION
Household air is more polluted than oce air and school air,
and those who spend much of their time at home, such as
children and home workers,
1
receive a proportionately higher
dose of home air carcinogens
2
than the general population.
Infants are particularly susceptible to indoor air pollution due
to their low body weight and continuous exposure to indoor
air. Loh et al.
3
ranked the cancer risks of indoor air volatile
organic carcinogens (VOCs). The highest risk VOCs were
benzene, formaldehyde, 1,3-butadiene, carbon tetrachloride,
acetaldehyde, 1,4-dichlorobenzene (PDCB), naphthalene,
perchloroethylene, chloroform, and ethylene dichloride.
VOCs that exceeded acute exposure standards were acrolein
and formaldehyde (during cooking)
4
and chloroform (during
showering).
3
Some sources of these chemicals can be eliminated or
reduced. For example, PDCB could be greatly reduced by
eliminating products containing it from the home. Form-
aldehyde in household air can be reduced by changing
construction and upholstery material compositions, but
formaldehyde is also emitted from other sources, including
cooking,
4
which are not easily eliminated. Other carcinogens
with multiple sources are more dicult to eliminate, such as
benzene, which originates from fuel storage in attached
garages, outside air, and environmental tobacco smoke.
Physical-chemical methods for VOC removal include
adsorption on activated carbon, activated alumina, zeolites or
other surfaces and photocatalytic oxidation.
5
Adsorption
methods are not well suited for formaldehyde and other
polar compounds. Low molecular weight compounds may be
desorbed in competition with higher molecular weight
pollutants. Adsorption methods are not destructive, and the
sorbents must be periodically regenerated, usually remotely
using energy intensive methods. Low temperature in-place
methods achieved energy ecient regeneration but would
require exterior ducting.
6
Oxidation methods use photo-
catalysized redox destruction of VOCs on catalytic materials,
such as TiO2. Photocatalytic oxidation methods result in
complete mineralization of most pollutants, but they are
ineective with chlorinated VOCs such as chloroform. Further,
photocatalytic oxidation methods may introduce ozone into
the home air, and they are energy intensive.
5
Indoor plants have been widely touted as having the ability
to remove air pollutants from indoor air. This approach is
known as the green liverconcept and is a central idea of the
eld of phytoremediation, the use of plants to remove
xenobiotic pollutants from the environment.
7
Early studies of
air detoxication by household plants found that formaldehyde
was removed from the air of chambers containing spider
plants.
8,9
Other researchers reported that soil or water alone
could explain the removal.
10
Subsequently, controlled pure
culture plant experiments showed that plants can assimilate
and metabolize formaldehyde from the air.
1113
However, the
formaldehyde uptake rate through the leaf surface of typical
house plants appears to be insucient to remove formaldehyde
Received: August 27, 2018
Revised: November 3, 2018
Accepted: November 26, 2018
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from a typical room without an excessive number of plants.
12,14
Several studies have found that common plants can remove
VOCs such as formaldehyde and benzene from air, but those
studies produced highly variable estimates of the rate that a
particular plant species removes a given pollutant from air. The
concentrations used in these tests were several orders of
magnitude greater that those typical of home air (e.g., 17μg
m3).
15
For example, ve dierent laboratories found that
seven plant species removed benzene from the air at rates that
varied by 7 orders of magnitude for the same plants.
16
These conicting data notwithstanding, plants do have many
attractive features as a platform for metabolism of organic
pollutants. Unlike most bacteria, cultivated plants have excess
energy available to support cometabolic catalysis. Plants have
high surface areas that facilitate mass transfer of trace gases
from the air. Plants are self-sustaining and do not require the
high maintenance typical of bacterial systems. There is
certainty of the genetic and enzymatic composition of the
cultivated plant compared to a soil bacterial community.
The mammalian cytochrome P450 2E1 (2E1) oxidizes a
wide range of important VOCs found in home air, such as
benzene, chloroform, trichloroethylene, and carbon tetra-
chloride.
17
The CYP2e1 (2e1) gene has been introduced into
several plants, including trees, resulting in signicantly
increased degradation of the VOCs.
1720
Plants have been genetically modied to overexpress native
plant formaldehyde dehydrogenase activity, but the rate of
formaldehyde removal was increased by only 25% over
unmodied plants.
13
Expression in transformed tobacco plants
of the transgene for formaldehyde dehydrogenase, faldh, from
Brevibacillus brevis increased formaldehyde removal by 3-fold.
21
But, to date, no detoxifying genes have been expressed in
houseplants.
Thus, our objective in this study was to increase the
detoxication of indoor air by adding the ability to metabolize
VOCs to a common houseplant by transgene modication.
Our approach was to introduce the mammalian cytochrome
P450 2e1 gene into the common houseplant, pothos ivy
(Epipremnum aureum). Pothos ivy has several advantages over
other houseplants for this purpose: it is robust and grows well
in low light and a method for the transformation of pothos ivy
has been published.
22,23
Pothos ivy does not ower in indoor
cultivation or outdoors in the U.S., which is an advantage for
biosafety considerations regarding the release of the transgenes
into the environment. In order to provide additional biosafety
assurances, we added egfp, the gene for the enhanced green
uorescent protein,
24
EGFP, to the cassette of genes used to
transform the plant.
MATERIALS AND METHODS
Preparation of Pothos Ivy. Golden pothos ivy plants,
obtained from a retail horticulture store, were grown under 50
μEm
2s1illumination with a 16 h day/8 h night cycle at 25
°C in a plant room. The stem fragments were excised, surface-
sterilized with 15% sodium hypochlorite and then washed with
sterile deionized water three times. The sterilized stem
fragments were cultured on solid Murashige and Skoogs
(MS) basic medium
25
in culture vessels. After 12 months
culture under light, new leaves and roots developed from stems
and these sterile plants were used for infection with engineered
agrobacteria for genetic modication.
Vector Construction and Genetic Transformation. In
order to genetically modify pothos ivy we constructed a genetic
vector containing the transgenes 2e1, egfp, and hpt, each
anked by promoter and terminator sequences suitable for
pothos ivy. The hpt gene coded for hygromycin B
phosphotransferase, which confers resistance to hygromycin.
Hygromycin was used to select for transformed cells since it
kills wild-type pothos. These three genes were integrated into a
transformation vector (binary vector) based on a system of
cloning vectors called pSAT containing insertion sites for use
with specic restriction enzymes.
26
Then the binary vector was
introduced into the modied Agrobacterium strain EHA105,
which was used to infect pothos ivy callus cultures.
The rabbit cytochrome P450 2e1 gene was amplied by
polymerase chain reaction (PCR) from the plasmid pSLD50
6 (the sequences of the primers are listed in Supporting
Information (SI) Table S1), a kind gift from S. L. Doty
(University of Washington) and double digested with
restriction enzymes Hind III and KpnI. Then 2e1 DNA was
inserted into cloning vector pNSAT3a to produce pNSAT3a-
2E1. After insertion into pNSAT3a, the 2e1 gene was
integrated between promoter and terminator sequence to
produce an expression cassette to drive the expression of 2e1 in
plant cells. The egfp gene was cloned by PCR from vector
pGH00.0126
28
and inserted into pNSAT6a as a Hind III-PstI
fragment to produce pNSAT6a-EGFP. The expression
cassettes of hpt, 2e1,andegfp genes were cut from
pNSAT1a-HPT,
27
pNSAT3a-2E1, and pNSAT6a-EGFP vec-
tors using restriction enzymes Asc I, I-Ppo I, and PI-Psp I
separately and inserted into the pRCS2 binary vector to
produce pRCS22E1-EGFP.
The binary vector pRCS22E1-EGFP was transferred into
Agrobacterium strain EHA105 by the freezethaw method
29
and the resulting strain, EHA105 (pRCS22E1-EGFP) was
grown in LB medium (lysogeny broth) with 50 mg L1
rifampicin, 100 mg L1spectinomycin, and 300 mg L1
streptomycin for infection of pothos ivy. EHA105 (pRCS2
2E1-EGFP) was initiated in 100 mL LB medium with
rifampicin at 50 mg L1, spectinomycin at 100 mg L1and
cultured overnight at 28 °C on a rotary shaker at 200 rpm. The
bacteria were centrifuged at 4000 rpm for 10 min and
resuspended in liquid E medium (MS medium with 2 mg L1
thidiazuron (TDZ) and 0.2 mg L11-naphthaleneacetic acid
(NAA)) with 100 μM acetosyringone (AS) and cultured under
the same conditions until OD600 (absorbance of bacteria
suspension at 600 nm) of 0.81.0 was reached.
The following method for transformation of pothos ivy was
adapted from that of Zhao et al.
22
Leaf discs and petiole
segments from sterile pothos plants were immersed in the
agrobacterium culture at 25 °C for 20 min and then transferred
to double-layered lter paper moistened with liquid E medium
with AS at 100 μM in Petri dish for 5-day coculture at 25 °C.
The leaf discs and petiole fragments were washed with sterile
water and transferred to E medium with 100 mg L1
cefotaxime, 100 mg L1carbenicillin (PhytoTechnology
Laboratories) and 20 mg L1hygromycin for screening. The
explants were subcultured to fresh selection medium every 3
weeks.
After 23 months selection, the somatic embryos that
developed from explants on selection medium were transferred
to fresh medium for another month and then transferred to G
medium (MS medium with 2 mg L16-benzylaminopurine (6-
BA) and 0.2 mg L1NAA) with 100 mg L1cefotaxime, 100
mg L1carbenicillin and 20 mg L1hygromycin and cultured
under light for regeneration. After culture for two months, the
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regenerated plantlets were transferred to MS medium with 100
mg L1cefotaxime, 100 mg L1carbenicillin and 20 mg L1
hygromycin for rooting and growth, which required two
additional months of culture.
Molecular Analysis of Transformed Plants. For
polymerase chain reaction (PCR) analysis, the DNeasy plant
mini kit (Qiagen, Valencia, CA) was used to purify DNA from
hygromycin-resistant plants. PCR reactions were carried out
with primers specicto2e1 and egfp cassettes (SI Table S1).
Total RNA was extracted from the leaves of plants using the
RNeasy plant mini kit (Qiagen, Valencia, CA). For real-time
quantitative RT-PCR analysis, 1 μg of total RNA was reverse
transcribed to cDNA using M-MLV reverse transcriptase
(Promega, Madison, WI). Real-time quantitative PCR was
performed using the SensiFAST SYBR No-ROX kit (Bioline)
on a uorometric thermal cycler, Light Cycler (Roche), and
data were analyzed with Light Cycler 3 software (Roche). The
standard curve was constructed from the plasmid DNA of
pRCS2-2E1-EGFP. The values of transcripts measured using
Figure 1. Structure of binary vector pRCS22E1-EGFP used to transform pothos ivy. T35s, terminator of CaMV 35s gene; hpt, hygromycin
phosphotransferase gene, provides hygromycin resistance; OsActin, promoter of actin gene of Oryza sativa; Tmas, terminator of mannopine
synthase gene; 2e1, cytochrome P450 2E1 gene from rabbit; ZmUbi, promoter of ubiquitin of Zea mays;PvUbi, promoter of ubiquitin gene of
Panicum virgatum (switchgrass); egfp, enhanced green uorescent protein; Trbc, terminator of rubisco small subunit gene; LB, left border of T-
DNA region; RB, right border of T-DNA region.
Figure 2. Genetic transformation of pothos ivy with 2e1 gene via Agrobacterium infection. Leaf discs and fragments of petiole of pothos ivy were
infected with EHA105 harboring pRCS22E1-EGFP. The explants were cultured on somatic embryo induction medium with hygromycin at 20 mg
L1for selection. (A) Callus developed from explants after 34 months screening. (B) Hygromycin resistant callus was transferred to regeneration
medium supplied with hygromycin at 20 mg L1for 24 months to induce development of new plantlets. (C) Regenerated plants were transferred
to MS medium with hygromycin at 20 mg L1for rooting and growth. (D) PCR- and RT-PCR-positive transformed plants were cultured on callus
induction medium with hygromycin for callus induction and propagation.
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C
RT-qPCR were normalized to the pothos ivy 5.8S gene and
presented relative to the level of the transcript in clone VD1.
Benzene and Chloroform Uptake by Transformed
Pothos Ivy. Sterile plantlets of pothos ivy clones (1 g) were
incubated in 40 mL volatile organic analysis (VOA) vials
(Fisher Scientic, 14823-213), closed with septum valves
(Mininert, Valco Instruments Co. Inc., 614163), and
containing 5 mL half-strength Hoaglands solution (Caisson
Laboratories, HOP0110LT.1). Wild-type untransformed
plantlets and no-plant controls were incubated in parallel
with clone VD3 and each treatment was repeated in
quadruplicate.
Benzene gas was injected into the vials using gastight glass
syringes to achieve a headspace concentration of 1850 ±160
mg m3, taking into account gas liquid partitioning by Henrys
Law. The vials were incubated for 9 days with rotary shaking at
80 rpm. The concentration of benzene was determined by
manually injecting 100 μL of the headspace into a GCFID
(ame ionization detector) (PerkinElmer AutoSystem XL).
Chromatographic parameters were: oven temperature 60 °C,
injector temperature 250 °C, and detector temperature 250
°C, 1.33 mL min1nitrogen carrier gas, using a ResTek RTX-1
microcapillary column (ResTek, 10121).
Similarly, chloroform was introduced into VOA vials using
gastight glass syringes from sealed aqueous dilutions of
chloroform (Acros Organics, 423550010). Transformed
plantlets, wild type (WT), and no plant controls were
incubated in quadruplicate, and headspace samples taken for
analysis of chloroform levels by gas chromatography with an
electron capture detector (GCECD) (PerkinElmer AutoSys-
tem XL) with a VOCOL capillary column 60 m ×0.53 mm
(Sigma). Chromatographic parameters were detector temper-
ature at 325 °C, nitrogen carrier gas at 1.76 mL min1, with a
100 mL min1split, the oven at 100 °C, and the injection port
at 300 °C.
EGFP Fluorescence. The EGFP signal of epidermal cells
of pothos leaf was observed by uorescent microscopy using
the LSM 5 PASCAL system (ZEISS). The EGFP signal was
excited by blue light and a FITI lter was used to collect
uorescent light. Axiocam 503 mono camera and software
ZEN 2.3 lite were used to capture pictures.
Data Analysis. Data were analyzed for statistical
signicance using ANOVA in Microsoft Excel software
(Microsoft Excel 2016 MSO). When ANOVA analysis gave a
signicant dierence, Fishers Least Signicant Dierence
(LSD)methodwasperformedtocomparethemeans.
Groupings diering by statistical signicance (p< 0.05) are
labeled by letters in the gures.
RESULTS
Vector Construction and Generation of Transgenic
Pothos Ivy. The structure of the plasmid pRCS22E1-EGFP
used to transform pothos ivy is shown in Figure 1. In order to
achieve constant, high levels of expression, all of the transgenes
were driven by constitutive monocot promoters. The
hygromycin resistance gene, hpt, was driven by the actin
promoter from rice (Oryza sativa),
30
the 2e1 gene was driven
by the ubiquitin promoter of corn (Zea mays),
31
and the egfp
gene was driven by the ubiquitin promoter from switchgrass
(Panicum virgatum).
32
The explants of pothos ivy were infected with EHA105
containing the vector pRCS22E1-EGFP and then screened
on callus induction medium with hygromycin as selection
agent for 23 months. Capitate somatic embryos developed
from cut edges of leaf discs and petiole fragments (Figure 2A).
During subsequent culture, calli formed at the base and more
cluster somatic embryos developed from the calli. After 34
months culture, the hygromycin-resistant calli were transferred
to regeneration medium for induction of plantlets. After
another 23 months culture, plantlets developed with both
shoots and roots from the somatic embryos. Some plantlets
developed only with shoots (Figure 2B). These plants were
transferred to MS medium with hygromycin for further growth
and rooting (Figure 2C). The leaf discs of PCR and RT-qPCR
positive lines were cultured on E medium with 15 mg L1
hygromycin to induce somatic embryos for propagation while
still under selection (Figure 2D).
Molecular Analysis to Conrm the Transformation of
Hygromycin-Resistant Lines. PCR primed by primer pairs
annealing to promoter and terminator regions of 2e1 and egfp
cassettes conrmed the integration of target genes into the
genome of pothos ivy (data not shown). To measure the
transcript abundance of 2e1 and egfp genes RT-qPCR was
performed for eight transgenic lines, VD1-VD8. The
expression levels of egfp were lower than that of 2e1, and
were separated into two groups, with signicant dierences
between VD3 and VD2 or VD7 (p< 0.01, Figure 3). The
expression levels of the 2e1 gene between dierent transformed
lines were dierent with high signicance (p = 0.00001). The
clonal lines VD3, VD7, and VD8 had much higher expression
levels of 2e1 compared to other lines. None of the transformed
clonal lines had observable changes in morphology or growth
compared to wild types.
Figure 3. Transcript abundance measured using quantitative RT-PCR
on pothos ivy lines transformed with 2e1 and egfp genes. The y-axis
shows values that were normalized to the pothos ivy 5.8s rRNA gene
and relative to the level of VD1 (n=3±SE). Letters indicate means
that were not signicantly dierent (p= 0.05, ANOVA).
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EGFP Observation. Using a uorescent microscope, EGFP
uorescence was observed near the plasma membrane and
around the nucleus (Figure 4A) due to the presence of
vacuoles in the epidermal cells of pothos ivy leaf. The
emissions were marginally greater than emissions from the wild
type, but weak. The wild-type cells were weakly autouor-
escent generally, but not specically from the cytosol. Green
uorescence was not visible to the eye in the transformed
pothos ivy with hand-held UV lamp illumination.
Benzene Uptake by Transformed Pothos Ivy. To
determine the ability of 2e1-egfp transformed pothos ivy to
take up benzene, we incubated plants in closed vials with the
VOC. Benzene (144 μg) was injected into 40 mL VOA vials
containing transformed and wild-type pothos ivy to achieve a
nal headspace concentration of 2500 mg m3benzene. After 3
days culture, the benzene concentration in vials with VD3
plants had fallen dramatically (Figure 5). After 8 days, the
benzene concentration in no-plant vials had fallen by about
10%. The benzene concentrations in the vials containing VD3
plants were signicantly dierent compared to vials containing
wild-type plants after 3 days culture (p= 0.039), p= 0.012 at
day 4, and p= 0.0008 at day 8.
The time course of the benzene concentration in the vials
with transformed pothos ivy clone VD3 was plotted on
semilogarithmic axes and t by linear regression with a rst-
order rate constant equal to (0.249 d1,SI Figure S1), or
0.115 d1(g fresh biomass)1(SI Table S2). Since small
pothos plants have 29 cm2leaf area (g fresh biomass)1, this
kinetic constant is equivalent to 39.8 d1(m2leaf area)1,
normalized to leaf area. The slope of the best linear t to the
semilogarithmic plot of the time course of benzene
concentration for wild-type plants (0.044 d1,SI Figure
S2)wassignicantly dierent from zero (p= 0.015),
suggesting that the wild-type plants did take up some benzene.
The wild-type pothos took up benzene at a rst-order rate
normalized to biomass equivalent to- 0.024 d1(g biomass)1,
or 8.5 d1(m2leaf area)1, normalized to leaf area. The
normalized rate constant for uptake of benzene uptake by
transformed clone VD3 was 4.7 times that of the wild-type.
Chloroform Uptake by Transformed Pothos Ivy. The
concentration of chloroform in the headspace of vials
incubated with VD3 plants fell rapidly, while chloroform
concentrations in incubations with wild-type plantlets and no-
plant controls did not change signicantly (Figure 6). The
concentration of chloroform decreased by 82% during the rst
3 days in the vials containing clone VD3 plants and chloroform
was barely detectable after 6 days. Linear regression of the
semilogarithmic plot of the chloroform data yielded a rst-
order degradation constant equal to 0.549 d1(SI Figure
S3). The slope of the best linear t to the semilogarithmic plot
of the time course of chloroform concentration for wild-type
plants (SI Figure S4) was not signicantly dierent from zero
(p= 0.22), suggesting that the wild-type plants did not take up
chloroform. The rate constant for the VD3 transformed pothos
ivy normalized to biomass was 0.552 d1(g fresh biomass)1,
equivalent to 180 d1(m2leaf area)1, normalized to leaf area
(SI Table S3).
Figure 4. Observation of EGFP signal in the epidermal cells of pothos ivy clone VD3 using uorescence microscopy. The green uorescence signal
of EGFP was observed in cytosol in the epidermal cells of leaf of pothos ivy clone VD3 (A). The emissions in the wild-type pothos ivy (B) were due
to autouorescence.
Figure 5. Uptake of benzene by 2e1-egfp transformed pothos ivy
grown in liquid culture. The concentration of benzene in headspace
during eight-day culture of VD3 (2e1), wild-type plants (WT), and
no-plant controls (NPC). N= 4. Averages ±SE.
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E
DISCUSSION
VOCs in indoor air pose signicant cancer risks to vulnerable
populations, such as children, yet there are no practical,
sustainable technologies available for their removal in the
home. Physical-chemical methods based on sorbents and
oxidation methods are energy intensive and of limited use for
the removal of formaldehyde and chloroform, respectively.
Various houseplants have been touted as having the ability to
remove VOCs from air, but plant uptake rates vary
exponentially from one study to another. Many studies appear
to be aected by artifactual enhancement of soil bacterial
activities by high VOC concentrations.
16
In this study we also
used high VOC concentrations to facilitate analysis by hand
injection of headspace samples onto GCFID in the case of
benzene, but we performed the assays in axenic conditions,
without bacterial activity. As can be seen in Figures 5 and 6
there was little or no loss of benzene or chloroform in the vials
containing wild-type pothos ivy, while most of the benzene and
all of the chloroform was removed in 6 days in the vials with
2E1-expressing clone VD3. These results show the eective-
ness of genetically modied pothos for VOC removal
compared to wild-type pothos.
As we have shown for 2e1-transformed tobacco, other VOC
substrates of 2E1 may also be removed by the transformed
ivy.
33
Future work will determine whether other indoor air
pollutants that are known to be substrates of 2E1 in
mammalian cultures, such as PDCB, toluene, naphthalene,
and methyl chloroform, are removed by 2e1-transformed
pothos.
Expression of green uorescent protein was intended as a
visible indication that the pothos ivy was transformed, but
uorescence of transformed clone VD3 was too weak to be
visible without microscopy. Other variants of GFP, such as
mGFP-ER
34
and the use of a stronger monocot promoter may
provide stronger uorescence.
Additional improvements for removal of VOCs from home
air using transgenic houseplants could be made by combining
expression of 2e1 with other detoxifying genes. Formaldehyde,
the other VOC that poses most risk in home air will be of
prime interest. Overexpression of faldh gene from Brevibacillus
brevis in tobacco conferred plants a high tolerance to HCHO
and increased the ability to take up formaldehyde 23 times
faster than wild-type plants.
21
The faldh gene could be stacked
with 2e1 and other detoxifying genes in vectors that are used to
genetically modify pothos ivy and other houseplants, resulting
in plants that could degrade most of the important indoor air
VOCs.
Since 2e1 gene expression in the transformed pothos ivy is
under constitutive promoters the level of 2E1 expression is
expected to be independent of benzene or chloroform
concentration. Therefore, the kinetic parameters of pollutant
degradation are expected to be invariant with pollutant
concentration. However, we have not conrmed this
assumption empirically.
We calculated the performance of an enclosed, forced-air
biolter (see SI) using the same rst-order degradation
constant observed in the batch experiments with chloroform,
0.52 d1(g biomass)1. For the case of a completely mixed
biolter with a volume of 0.7 m3and an airow rate of 300 m3
h1, 10 kg of pothos ivy clone VD3 could remove 34% of the
chloroform in one pass. This hypothetical biolter would have
a clean air delivery rate (CADR) of 100 m3h1, comparable to
CADRs of current commercial home particulate lters.
35
This
calculation, while tentative, suggests that genetically modied
plants may have practical utility for sustainable phytoremedia-
tion of home air.
Compared to current chemical/physical methods for
removal of VOCs from indoor air biolters using transgenic
plants oer the advantages of low energy use and decreased
need for maintenance. All of the removal methods require a
means for moving the air through the apparatus, but adsorptive
methods also require signicant energy expenditure to
regenerate the media and photooxidative methods require
high energy inputs to oxidize the pollutants, making those
methods less sustainable. Transgenic phytoremediation
requires very little additional energy beyond that required for
air movement. Pothos ivy is well adapted to medium- and low-
light levels so articial lighting would usually not be required,
giving phytoremediation an intrinsic sustainability advantage.
More work is needed to conrm these ndings and to
establish the practical usefulness of transgenic biolters. It is
necessary to determine the removal rates at low concentrations
of indoor air pollutants, the eectiveness of the formaldehyde
dehydrogenase gene expressed in pothos, the eects of light
and dark and photoperiod on removal, the eects of increased
mixing and air ow rate in the biolter, and whether increased
VOC removal eciencies can be achieved through biological
manipulations such as increased transgene copy numbers.
ASSOCIATED CONTENT
*
SSupporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.est.8b04811.
The biolter model, four gures and three tables (PDF)
AUTHOR INFORMATION
Corresponding Author
*Phone: 206-543-5350; fax: 206-685-9996; e-mail: sstrand@
uw.edu.
ORCID
Stuart E. Strand: 0000-0002-6700-3498
Notes
The authors declare no competing nancial interest.
Figure 6. Uptake of chloroform by 2e1-egfptransformed pothos ivy
grown in liquid culture. The concentration of chloroform in
headspace during 11-day culture of VD3, wild-type plants (WT),
and no-plant controls (NPC). N=4±SE.
Environmental Science & Technology Article
DOI: 10.1021/acs.est.8b04811
Environ. Sci. Technol. XXXX, XXX, XXXXXX
F
ACKNOWLEDGMENTS
This work was funded by NSF CBET-1438266, Amazon
Catalyst grant UW 100631088, and NIEHS grant 2P42-
ES004696-19.
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