Running Head: TEAM BREATHE PROPOSAL

American Psychological Association, 6th Edition

Team BREATHE Proposal

Erica Brown, Gabrielle Enguillado, Robert McDermott, Nicole Palumbo, Jill Smith,
Michelle Stanley, Morgan Sulzbach, and Jaclyn Taylor
Mentors: Dr. Steven Cohan, Dr. Andrew Ristvey
Librarian: Dr. Svetla Baykoucheva

We pledge on our honor that we have not given or received any unauthorized assistance on this
assignment.

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Tables of Contents

Abstract............................................................................................................................................3
Introduction ....................................................................................................................................4
Volatile Organic Compounds .............................................................................................4
Existing Solutions ..............................................................................................................5
Literature Review.........................................................................................................................6
Wall Structure ....................................................................................................................6
Workplace Impact ..............................................................................................................9
Cost ...................................................................................................................................10
Plant Analysis ...................................................................................................................11
Growing Conditions ..........................................................................................................14
Past Methodologies ...........................................................................................................14
Methodology .................................................................................................................................16
Introduction .......................................................................................................................16
Phase 1A ...........................................................................................................................18
Air Sample Analysis .............................................................................................19
Existing Biowall Root Sample Collection ............................................................19
Bacterial Analysis .................................................................................................20
Phase 1B ............................................................................................................................21
Confounding Variables and Research Limitations ...........................................................25
Anticipated Results ...........................................................................................................26
References .....................................................................................................................................27
Appendix A - Equipment Images .................................................................................................33
Appendix B - Glossary..................................................................................................................34

Abstract

TEAM BREATHE PROPOSAL

Indoor biowalls present the opportunity to improve indoor air quality by removing harmful
volatile organic compounds (VOCs) from air. Hyphomicrobium spp. bacteria have been found to
thrive off VOCs, growing on the roots of plants in biowalls. Team BREATHE will research
which of these plants support this bacteria best. A novel biofiltration system will be developed,
using an industry-standard passive biowall and VOC filtration that captures these pollutants in
water and passes them through the biowall for amelioration. We hope this will present a market
option for a passive system that is not just aesthetic but is also effective at purifying indoor air.

Team BREATHE Proposal

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Introduction

Volatile Organic Compounds

Indoor air pollution is a frequently overlooked yet very important issue to the health and
wellbeing of all people. Poor indoor air quality (IAQ) can cause health problems, including
headaches, dizziness, nausea, fatigue, and eye irritation (Newkirk et al., 2015). The World
Health Organization has defined the side effects caused by poor indoor air quality as Sick
Building Syndrome (SBS) (Newkirk et al., 2015). Volatile organic compounds (VOCs) are
particularly dangerous and alarmingly abundant in indoor environments. On average, VOC
levels are ten times higher indoors than outdoors (Rehwagen et al., 2003). Volatile organic
compounds include a wide range of chemicals, many of which are carcinogenic and hazardous to
human health. Some of the most common indoor VOCs are formaldehyde, benzene, xylene,
toluene, and ethylbenzene (Mosaddegh et al., 2014).
In the spring of 2015, 60 Minutes reported that much of the laminated flooring sold by
the retail company Lumber Liquidators exhibited formaldehyde levels well over current
government safety standards (Cooper, 2015). This is especially disconcerting because
formaldehyde has been listed as a known carcinogen by the U.S. Department of Health and
Human Services (Substances Listed in the Thirteenth Report on Carcinogens, 2014). The
National Institute for Occupational Safety and Health also classified formaldehyde as
Immediately Dangerous to Life or Health Considerations, adding that exposure for five to ten
minutes at more than fifty parts per million (ppm) can cause serious injury to the lower
respiratory system (Chemical Listing and Documentation, 1994). Despite these dangers, the
United States Congress only recently enacted regulations on formaldehyde levels in wood

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products nationwide in 2010, which took effect in 2013 (S.1660 Formaldehyde Standards,
2010). Such a delayed application of regulatory power to a toxic chemical begs the question as
to how many other incidents of excessive levels of formaldehyde or other potentially harmful
VOCs have been taking place across the country. These findings highlight not only the obvious
need for more investigation of the causes of indoor air pollution, but also the need for
development of methods for processing and filtering indoor VOCs.
Existing Solutions
There are three common methods for treating indoor air pollution: controlling the
pollution at the source, improving ventilation, and using purification technologies (Luengas et
al., 2015). When source control or ventilation are not feasible, filtration technology is
implemented. There are a wide variety of filtration systems, the most popular of which are
mechanical filtration and electronic filtration (Luengas et al., 2015). Mechanical filtration, or the
use of physical filters, is a component in all air conditioning systems (Yu et al., 2009). However,
these filters become clogged over time and the old filters can add to the contamination, causing
further problems (Yu et al., 2009). Electronic filtration ionizes pollutant particles which are then
deflected or trapped in filters (Luengas et al., 2015). This method has a high efficiency rate,
between seventy five and ninety five percent, but can create hazardous charged particles or new
pollutants (Guieysse et al., 2008). Another widely commercialized system is adsorption, a
process where contaminants are retained onto an adsorbent material such as activated carbon or
silica gel (Luengas et al., 2015). The system is highly efficient on some compounds but does not
work on all contaminants and needs regular replacement (Luengas et al., 2015). Since all of

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these solutions exhibit major flaws, alternate solutions must be investigated, such as the
implementation of biowalls.
Literature Review
Wall Structure
Biowalls, also known as green walls or green facades, are systems of vegetation grown
on a vertical surface, either a buildings facade or a separate structural system (Loh, 2008). As
will be discussed later in this paper biowalls are preferable as a filtration system since there is a
relationship between the bacteria that exist on the roots of the plants and the VOC filtration
ability of the plants. The bacteria are able to filter the VOCs from the air only when there is no
soil to inhibit the process. Potted plants therefore have some air filtration qualities but a biowall
is the preferred method of biologic air filtration. Biowalls are commonly divided into three
categories: passive1, active2, and hybrid3 (Curtis & Stuart, 2010). These categories describe the
different technologies used to help biowall systems filter the air (Curtis & Stuart, 2010). Active
biowalls are integrated into heating, ventilation, and air conditioning (HVAC) systems while
passive biowalls are not incorporated into air handling systems (Curtis & Stuart, 2010). This
means that an active biowall structure is directly attached to the ventilation system of a building.
A hybrid system is a mixture of both the passive and active biowalls. Hybrid biowalls include a
negative air pressure system that draws polluted air through the wall while clean air is blown
out from the top and circulated into the existing HVAC system (Curtis & Stuart, 2010). Active
biowalls are more efficient in purifying the air within larger buildings, while passive systems are
more effective for smaller rooms (Curtis & Stuart, 2010). More research must be conducted to

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determine which type of living wall system would be the most cost effective for air purification
within a building.
According to a study done at George Brown College, the most feasible system is the
hybrid system which is generally just a passive system that includes a cavity for the roots
(Butkovich et al., 2008). Active systems are not as sustainable because they require more energy
to run and cost more to integrate into the HVAC system (Butkovich et al., 2008). Passive
systems, whose roots are embedded into the biowall do not purify the air as well because they
have not active air circulation for better air/root contact, necessary for pollutant amelioration
(Butkovich et al., 2008). The negative air pressure area within a hybrid system promotes more
air and root contact, improving purification (Butkovich et al., 2008). According to industry
expert Alan Darlington, the construction of a biowall alone would cost about $1700 per square
meter (Curtis and Stuart, 2010). However, this cost estimate does not include the cost of
integrating an active biowall into the HVAC system. Darlington estimated a return on investment
(ROI) of about 10 years with savings from utility costs (Knowles et al., 2002). However, since
passive systems dont integrate into air handling systems, the (ROI) could be reduced to less than
5 years (Knowles et al., 2002). This research investigates how to achieve the air purification
efficiency of an active biowall using the structure of a passive wall.
There are also several different types of biowall designs which differ in the way plants
are placed on the wall and the type of irrigation system used. Figure 1 includes diagrams of
three known biowall layouts called panel biowalls, felt biowalls, and trellis biowalls (Curtis &
Stuart, 2010). The panel system includes different sections in which plants are grown into the
growing media before placed on the wall (Curtis & Stuart, 2010). The felt system consists of a

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felt-like layer of growing media and several pockets on the wall for the plants to grow in (Curtis
& Stuart, 2010). Finally, the trellis system, commonly used for vine-like plants, contains
planters on the top and bottom of the wall for the plants to grow in the middle section (Curtis &
Stuart, 2010).

Figure 1. Three common designs typically utilized in biowalls (Curtis & Stuart, 2010).

The two most common irrigation systems, both hydroponic12, are the drip system and the
reservoir system (Curtis & Stuart, 2010). The drip system includes an automated technology in
which water drips down from the top of the wall and naturally disperses throughout the rest the
growing media using gravity. At the bottom of this system is a large water reservoir to collect
and recycle the water (Curtis & Stuart, 2010). The drip system is typically used for panel

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biowalls and is more conservative with water use (Curtis & Stuart, 2010). A second irrigation
type is the reservoir system that includes different channels behind the wall for the water to flow
through (Curtis & Stuart, 2010). Reservoir systems are most common in felt biowalls (Curtis &
Stuart, 2010). An alternate option for irrigation would be to manually irrrigate the biowall
similar to traditional indoor plants (Curtis & Stuart, 2010). The drip and reservoir systems are
central to running a biowall as a hydroponic system, in which the roots of the plants, located in
the felt matrix, are kept moist by the continual flow of water through the felt (Schwab et al.,
1998). A third type of irrigation system, though less common than a hydroponic system, is called
an aeroponic17 system. In an aeroponic biowall, the roots are suspended in air in a section behind
the main wall of plants, and are kept hydrated by a misting system constantly enveloping the root
space (Schwab et al., 1998). We will use a hydroponic system for the implementation of this
passive biowall research and development project.
In general, biowalls have numerous benefits including lowering energy consumption and
thereby lowering energy costs, improving health and wellbeing, and reducing the Urban Heat
Island (UHI) effect, which occurs when a city has a higher temperature than the surrounding area
because of human activity (Loh, 2008). The evapotranspiration from the plants on a biowall
lowers the air temperature within a building, reducing the need for air conditioning and
ultimately saving both energy and money (Alexandri & Jones, 2006). When biowalls are
constructed on the outside of buildings, the decrease in air temperature lowers the amount of heat
being reflected from the hard surfaces of a building, thus decreasing the UHI effect (Loh, 2008).
An indoor biowall can act as an air purification system by utilizing a variety of plant species and
naturally occurring microorganisms in their roots to remove VOCs from the air (Luengas et al.,

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2015). However, indoor biowalls have not been researched as fully as external biowalls
(Luengas et al., 2015). As shown above, existing biowall research focuses primarily on climate
control benefits rather than potential for air purification.
Workplace Impact
Studies have shown that poor IAQ can reduce human work productivity and overall
happiness and health. One experiment, performed at the International Centre for Indoor
Environment and Energy, found that poor indoor air quality can decrease office performance by
as much as nine percent (Wyon, 2004). Field experiments in the same study found that actual
productivity can drop even more drastically than theoretical laboratory values (Wyon, 2004).
The epidemic drop in productivity costs the United States approximately $125 billion each year
(Newkirk et al., 2015). Decreased productivity, along with previously mentioned health issues,
suggests that improving IAQ be a priority for employers.
Though any ventilation or air filtration system could help fix these issues, biowalls are an
especially efficient solution because they incorporate plants and therefore the psychological
benefits of green spaces indoors. A study investigating the relationship between employee
satisfaction and workplace environment found that employees in workplaces with more plants
and windows felt more satisfied with their job and reported higher overall quality-of-life
(Dravigne et al., 2008). Current research shows that biowalls have the potential to increase
worker productivity, happiness, and health due to their air filtration abilities and aesthetic appeal.
Cost
Biowalls are intricate structures that are often fairly expensive, making cost an important
factor for companies to consider. Biowalls can cost as much as $1,700 per square meter

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(Butkovich et al., 2008). Experts argue, however, that green buildings are a premium product
and an increased cost over standard buildings is expected (Roberts, 2014). Fortunately, an
increase in worker efficiency and a decrease in worker absence due to SBS symptoms can help to
significantly offset these problems, potentially increasing productivity and profitability. One
study, which examined various climates and office types, found that improved air quality
provides an annual benefit ten times greater than the cost of maintenance (Djukanovic et al.,
2002). Improving building ventilation decreases SBS symptoms by 5.3%, and can prevent 4.5
million sick days nationwide each year (Fisk et al., 2011). These studies show that the cost of
improving air quality can be earned back through increased productivity and fewer sick days in
as little as four months (Djukanovic et al., 2002). Such financial benefit is yet another reason
that biowalls are a viable solution for poor indoor air quality.
Plant Analysis
The leaves and roots are both vital to a plants ability to survive on a biowall and to
purify air. The biochemical process behind phytoremediation8, the use of plants to remove
hazardous substances from the environment, occurs in the leaves (Glick, 2003). The roots grow
into a permeable material to secure the plant in a vertical position. In addition to securing the
plant, roots also take in nutrients and absorb water which flows down the wall (Reece et al.,
2010). New research has also shown that microorganisms colonizing the roots play a role in
VOC remediation (Russell et al,. 2014).
A study performed utilizing Drexel Universitys biowall investigated the relationship
between these root bacterial communities and VOC remediation. The study found that these
microbes drive the purification ability of biowalls (Russell et al., 2014). Researchers utilized

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DNA sequencing to compare bacteria in roots of plants grown with and without VOC exposure
(Russell et al., 2014). It was determined that roots exposed to VOCs exhibited higher levels of
bacteria from the Hyphomicrobium genus, which are known to break down aromatic and
halogenated compounds (Russell et al., 2014). While the topic of VOC remediation in biowalls
through microorganisms in the roots is relatively new to this field, microbial biofilters have been
used to treat industrial waste gases containing VOCs since the 1950s (Devinny et al., 1999). In
these systems, contaminants are absorbed in water and then applied to the microbes for treatment
(Devinny et al., 1999). The most successful remediation in these systems occurs with pollutants
with a low molecular weight and high solubility in water (Devinny et al., 1999).
Currently, more is known about phytoremediation through a plants leaves. Leaves
contain dermal, ground and vascular tissue (Reece et al., 2010). The epidermis consists of a
waxy cuticle layer to protect the leaf interrupted by stomata, openings in the leaf to facilitate air
exchange (Reece et al., 2010). The ground tissue, or mesophyll, is split into two layers: a layer
of cylindrical cells containing many chloroplasts to take advantage of the sunlight, and a spongy
layer with more intercellular spaces to allow for diffusion of gases between the cells (Reece et
al., 2010). The vascular tissue carries nutrients to and from the leaves (Reece et al., 2010).
Plants remove VOCs by pulling the air through the open stomata on the leaf surface and into the
mesophyll layer (Seco et al., 2007). One study investigated formaldehyde removal rates of
Fatsia japonica and Ficus benjamaina in various lighting conditions (Kim et al., 2008). The
researchers found that formaldehyde removal rates in aerial parts of the plants, including leaves
and upper stem, increased significantly for the plants when they were exposed to light (Kim et
al., 2008). The significant change is most likely due to the fact that plants close their stomata

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when there is little to no light available for photosynthesis (Kim et al., 2008). In order to prevent
unnecessary water loss, plants close their stomata at night when carbon dioxide is not required
for cellular function (Zeiger et al., 1987). When plants are exposed to sunlight, they open the
stomata to facilitate photosynthesis (Zeiger et al., 1987). This information is especially relevant
to indoor biowalls, as lighting systems or placement with respect to natural light must be taken
into consideration.
In another study, a team of researchers exposed Epipremnum aureum and Ficus
benjamina to radioactively labeled formaldehyde to determine how exactly phytoremediation
occurs (Schmitz et al., 2000). After formaldehyde is absorbed by the plant, it is then processed
in two steps using formaldehyde dehydrogenase and formate dehydrogenase (Schmitz et al.,
2000). First, formaldehyde is oxidized into carbon dioxide which is less harmful and far more
useful to the plant. Afterward, the oxidized formaldehyde goes through the Calvin Cycle to
produce glucose (Schmitz et al., 2000). Between seventy five and ninety percent of oxidized
formaldehyde is converted to glucose, while five to fifteen percent becomes organic acids and
amino acids within the plant (Schmitz et al., 2000).
Formaldehyde is not the only VOC which can be absorbed and transformed. One study
tested both sides of plant leaves for their uptake ability with benzene and toluene, two common
aromatic hydrocarbons present in most indoor environments (Ugrekhelidze et al., 1997).
Benzene is a known carcinogen (Collins et al., 2000). Toluene, and its methylated derivative
xylene, are both known to be neurotoxic and hepatotoxic, meaning that they can damage the
nervous system and liver (Revilla et al., 2007). First, Ugrekhelidze et al., (1997) grew the plants
in sterile lab conditions to prevent any microorganisms from skewing the data. The plants were

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tested after being exposed to light and the VOCs for eight hours. The researchers found that
these aromatic hydrocarbons were absorbed through both the stomata and the cuticle, but that the
main form of absorption was via the stomata. The team then used radioactively tagged carbon
atoms to determine how benzene and toluene are transformed after being absorbed. It was
determined that the carbons from the benzene and toluene rings were mainly included in
nonvolatile organic compounds, such as amino acids, after their aromatic rings were cleaved
(Ugrekhelidze et al., 1997). In this way, harmful aromatic VOCs were filtered from the air and
included into organic molecules within the plant, thereby decreasing the concentration of indoor
air pollutants while also benefiting the plants intermolecular organic processes.
Growing Conditions
Biowall plant selection relies on the plants ability to grow in an indoor, soilless wall as
well as its compliance with design requirements. The primary problem when growing plants
indoors is light deficiency. Interior lighting is around forty times less intense than full summer
sunlight and is even less intense than sunlight levels during heavily overcast days, although the
difference is considerably less (Whiting et al., 2014). In order to be successfully grown in low
light conditions, the plant must have a high shade tolerance (Valladares & Niinemets, 2008).
Most plants can be grown without soil as long as the nutrient and support requirements are met
by the wall design (McCall & Nakagawa, 1970).
Another prominent problem when choosing plants for a biowall is whether or not the
plant can grow vertically. In most cases, this means that the plant needs to be of a small enough
size that its weight will not pull it down. Other design requirements that must be met during
plant selection are whether or not the plant is poisonous or contains a common allergen. The

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plants must also be aesthetically pleasing in order to be marketable in a commercial or residential

setting. While testing can be used to determine which plants could grow vertically on the
biowall, these preliminary factors must be considered in order to narrow down potential plant
species.
Past Methodologies
The first literature on VOC removal from indoor environments was in a study supported
by NASA, which demonstrated that plants could effectively reduce VOC levels in indoor spaces
(Wolverton et al., 1984). Since then, there have been few studies conducted on the removal of
VOCs in indoor air by plants, and research is varied and spread over a long time frame. A
review of this literature included articles ranging from 1984 to 2014, with each methodology
measuring VOC removal in a different way and very few plants tested in multiple studies. In a
review of eight articles testing plants for VOC removal, only five of twenty seven tested plants
appeared in more than one article. The plant mentioned in the most papers (four) was Hedera
helix, or English Ivy, which was tested by Cruz et al. (2014), Yang et al. (2009), Aydogan et al.
(2011), and Wolverton et al. (1993). However, the findings differed among these papers with
Cruz et al. (2014) finding that Hedera helix could remove toluene at a rate of 66.5 g/m2/h, and
Yang et al. (2009) finding that Hedera helix could remove toluene at a rate of 8.25 0.64 gm3

m-2 h-1 Other than English Ivy, the plants tested in these papers were common indoor plants,

many of which are not suitable for use on a biowall because they are too large or are small trees.
More research is needed to analyze these plants with consistent methodology while taking their
ability to grow on biowalls into consideration. Plants should be selected based on their ability to
survive indoors, and how many VOCs the plants themselves emit (Liu, 2007). This is necessary

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because in addition to removing VOCs from the air, plants can also release a wide range of
VOCs into the environment. Some of these VOCs have biological roles like protection against
pathogens, and some play a role in scent and flavor (Yang et al., 2009).
The most commonly tested VOCs in these papers are formaldehyde, benzene, and
toluene. A study conducted by Wolverton and Wolverton (1993) found Bostoniensis, or the
Boston Fern, to be the most effective house plant to remove formaldehyde. A similar study,
however, found Chrysanthemum morifolium to have the fastest formaldehyde uptake (Aydogan
& Montoya, 2011). Oputntia microdasys was determined by Mosaddegh et al. (2014) to have
the fastest benzene removal. Other researchers also tested a selection of plants for their ability to
remove benzene from an environment, from which they concluded Crassula portulacea had the
fastest removal rate (Liu et al., 2007). The wide range in plant species tested across multiple
papers with multiple testing procedures makes it difficult to ascertain what plant species are
actually the most effective in removing VOCs, especially when considering specifications for
plant survival on a biowall.
Methodology
Introduction
In the following section, we will describe our proposed experimental methodologies and
anticipated results of the experiments, along with potential confounding variables and research
limitations. Early research about plant-based airborne VOC amelioration focused on the plants
as the main degrading system (Collins et al., 2000; Schmitz et al., 2000). Recent research has
shown that certain kinds of bacteria, primarily Hyphomicrobium spp.14 which reside on plant root
systems, also play a very important role in the process (Russell et al., 2014; Kim et al., 2013).

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We have learned from Michael Furbish, an industry professional of Furbish Company,

that a biowalls effectiveness in degrading atmospheric VOCs in indoor environments greatly
differs between active2 and passive1 biowall systems (personal communication, Michael Furbish,
September 24, 2015). Active systems are much more efficient at degrading VOCs, but are
expensive, require significant amounts of energy, and are difficult to maintain. Active biowalls
circulate air directly through the root zone of the biowall, causing difficulty maintaining root
moisture and producing excessive noise (personal communication, Michael Furbish, September
24, 2015). Increased air circulation in the root zone makes it difficult for plants to thrive, causing
a much lower foliage density than is typical in a passive system (personal communication,
Michael Furbish, September 24, 2015). Passive systems1 require little energy, but they are not
nearly as effective at purifying air. Since there is no forced airflow over the roots, much of the
air in a large space does not come into contact with the roots, hindering purification abilities
(personal communication, Michael Furbish, September 24, 2015). An ideal biowall would
combine the ease of maintenance, lush foliage, and low cost of a passive system with the VOC
amelioration ability of an active system. We plan to bridge this functionality gap through two
segments of research. First, we will investigate the effectiveness of biowall plants and the
associated microorganism flora in degrading VOC pollutants. Second, we will develop a new
passive biowall system that can effectively clean indoor air without the added cost and
complications of an active system.
The first aspect of our research we hope to address is whether different plants species
host different amounts of Hyphomicrobium spp. bacteria on their roots. Hyphomicrobium spp. is
a genus that includes bacteria which live on the roots of many plants, thrive in environments with

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high VOC concentrations, and partake in the degradation of atmospheric VOCs (Russell et al.,
2014). Hyphomicrobium bacteria have been found on active biowalls, but we will be testing
passive walls for the same bacteria during our experimentation. We hypothesize that if bacterial
communities grow on the roots of plants, then different plant species will host different amounts
of Hyphomicrobium spp. bacteria. The null hypothesis is that different species of plants will not
host significantly different amounts of the bacteria. We plan to test this hypothesis during our
research by analyzing root samples of plants species the team will gather from existing passive
biowalls and plant species straight from greenhouses.
Both phases will use experimental lab research in order to accurately evaluate plant and
biowall system performance in a controlled setting. Phase 1 will be undertaken primarily by four
out of the eight members of our team, but all members will rotate laboratory shifts in order to
evenly distribute the heavy workload. The other four members of the team will focus mainly on
Phase 2. All team members are expected to understand the process of each phase of the research,
though individuals may focus more heavily on a particular portion of the research. The two
phases will occur mostly simultaneously, however the final part of Phase 2, which will test the
effectiveness of the dissolution system, will occur after the completion of Phase 1.
Phase 1A
Phase 1 will determine several aspects of airborne VOC degradation due to plant and
bacterial activity in biowall systems. Air samples from a building at the University of Maryland
will be analyzed to determine which VOCs will be used in the remainder of the research. We
will also sample roots taken from plants on local biowalls and determine their associated
bacterial populations. Hyphomicrobium spp. will be isolated from plant root tissue and

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quantified, potentially identifying plants that have a propensity to harbor more bacteria from this
genus. The purpose of this phase of research is to determine which VOCs and plants will be
used in later research.
Air sample analysis. We will begin research by collecting air samples from Stamp
Student Union at the University of Maryland, College Park in order to determine which VOCs
are most prevalent in indoor air. These samples will be collected at multiple times to reduce any
effect from other activities in the Stamp Student Union so that there is more information about
the VOC environment of the building. The samples will be taken from the Stamp Student Union
because we hope for the opportunity to apply our research at this location in the future, pending
university approval. It is not feasible for us to obtain an accurate analysis of the VOC content in
a large space using university equipment; instead, we will utilize a professional air quality
analysis service. We will collect an air sample in a SUMMA Canister16 by opening a valve and
allowing air to fill the canister, then sealing it. The canister will then be sent to EMSL
Analytical, Inc, a professional laboratory in Cinnaminson, New Jersey, for analysis (EMSL
Analyticals SUMMA, n.d.). These samples will be analyzed using EPA Method TO-155, and
the results will be returned to the team (EMSL Analyticals SUMMA, n.d.). We will use the
VOCs that are reported in the highest concentrations and that are also water soluble as
independent variables for the remainder of our research.
Existing biowall root sample collection. We will travel to at least two existing passive
biowalls constructed by Furbish Company or Jan Ferguson Interior Plantscaping in the
Washington and Baltimore areas. Those of us collecting samples of the root systems will all
wear sterile gloves while handling the roots to avoid contamination from any bacteria which may

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be present on our skin. Before taking any root samples, the scissors being used will be cleaned
with ethanol. Both gloves and scissors will be cleaned with ethanol between collecting each
sample to prevent cross-contamination (Russell et al., 2014). Samples of five individual plants
from each of six species will be taken from each biowall visited. We will excise root samples of
approximately 2 grams from each plant. While collecting the samples from each biowall, we
will record the location of the biowall, the location of the plant on the biowall, and the species of
plant being sampled. After the roots are removed from the wall, we will rinse them with
deionized water to remove only the loose particulates from the walls growing media; then, we
will place them in labeled sterile bags and refrigerate them until we are ready to test the samples
(Russell et al., 2014).
Bacterial analysis. To begin testing for the amount of Hyphomicrobium bacteria found
on each plant, we must isolate the bacteria from the rhizospheric area,11 which includes both the
rhizoplane9 and the rhizosphere10 (Barillot et al., 2012). Generally, the rhizospheric area
indicates the surface of roots and the volume of soil affected by the roots and what they excrete.
However, since the biowalls plants roots are in an environment without soil, the rhizospheric
area refers to the surface of the roots. We will cut each sample to be approximately one gram in
order to accurately compare bacteria quantities. Similar to the methodology for taking root
samples, the scissors will be rinsed with ethanol between samples to prevent crosscontamination. The team members working on this segment of research will wear gloves and
will rinse the gloves with ethanol between samples. Each root sample will be washed with the
same volume of deionized water, and the water will be collected. The water will remove and
collect the bacteria, and subsequent bacterial testing will be performed on the water.

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The deionized water containing the bacteria will be vacuum filtered through filters of
pore size 0.45 m in order to isolate the bacteria from the solution. The bacterial DNA will be
isolated by using a FastDNA SPIN kit for Soil (produced by MP Biomedicals). QuantitativePCR7 (qPCR) analysis will be performed on the extracted DNA on a Roche LightCycler 480
using primers for Hyphomicrobium spp. (Forward - 5-GGCTCAACCTCGGAACT-3, Reverse 5-CGAATTTCACCTCTACACTAGGAT-3 from Hayes et al., 2010). Computer software linked
to the LightCycler 480 will calculate the concentration of Hyphomicrobium in each sample. The
bacterial concentrations of the root samples will be compared to each other using an analysis of
variance (ANOVA) test in order to determine which plant species can host the most
Hyphomicrobium spp.
The concentration of Hyphomicrobium spp. from each sample will determine which
plants had the highest population of the Hyphomicrobium spp. colonies. According to Russell et
al.s research, a higher population of Hyphomicrobium spp. is correlated with the ability to filter
VOCs more effectively (2014). The four plants which have the highest population of the
Hyphomicrobium spp. colonies will be assumed to be the most effective plants for VOC
degradation, and will be selected for future testing with aeroponic17 chambers in Phase 1B of our
research.
Phase 1B
The purpose of this phase of research is to confirm previous research connecting
Hyphomicrobium spp. bacteria with VOC exposure as well as determine which plant species
remove the most VOCs from the air, which will be used in Phase 2. Using the information
gathered in Phase 1A regarding which specific plant species and VOCs we will be testing for, we

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will evaluate individual plants ability to degrade VOCs and the overall effectiveness of a passive
biowall.
To conduct this part of the research, we will use Clone King 25 Site Aeroponic Growth
Chambers (Figure A1) (Clone King, n.d.). This specific model has thirty sections in which to
grow plants (Clone King, n.d.). These aeroponic growth chambers allow the roots of plants to
be exposed to a VOC-laden airstream while keeping the chambers positively pressurized to
ensure that no air from the laboratory environment is able to infiltrate the root zone (Russell et
al., 2014). We will use the four species of plants determined in Phase 1A for the aeroponic
testing, as they are the best suited to sustain the bacteria. Plants used for these tests will be
received from the greenhouses of the teams industry partner, Michael Furbish. Each species will
be grown in its own individual aeroponic chamber. A total of four aeroponic chambers will be
used, one for each plant species with a total of twelve plants per species in each testing period.
Aeroponic chambers will be fitted with a closed air circulation system powered by a pump. The
VOCs will be injected into the closed airstream through a manifold and tube system on the side
of the aeroponic chambers. This testing will be replicated four times, introducing a different
VOC, chosen in Phase 1A, into the chambers for each of the tests.
The effectiveness of the plants and the bacterial root colonies in removing VOCs from
the air will be determined by evaluating VOC concentration levels and the concentration of
Hyphomicrobium spp. A baseline reading of the VOC concentrations in the air will be taken
with a Tiger Handheld VOC Gas Detector (Figure A2), a photoionization detector (PID) which
measures the VOC concentration levels in parts per billion (Tiger-Revolutionary, n.d.). Over a
24-hour test period, the team will record measurements of the VOC concentration levels in the

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aeroponic growth chambers using the PID. Once the test period is over, the final VOC
concentration levels will be recorded. The difference between the baseline and the ending VOC
concentration levels will be calculated and will be representative of the amount of VOCs
removed from the air by the root bacterial colonies. In order to test all four growth chambers
with access to only one PID, we will construct a manifold system with valves at each chamber to
connect multiple chambers to the same PID. Every two to three hours, we will close one valve
and open another to allow the PID to take readings from the next unit. In this manner, all units
will be able to be tested at the same time by alternating which VOC-laden air supply is being fed
to the PID for testing.
In addition to PID readings, qPCR for Hyphomicrobium spp, as described in Phase 1A,
will be conducted at the end of the testing period. We expect that more Hyphomicrobium spp.
will equate to more VOC degradation, which will confirm prior research (Russell et al., 2014).
The expected results from this phase of the project will affirm that the root bacterial colonies are
successful in removing VOCs from the air.
To test this with our newly designed passive biowall system we will build two different
model biowalls to test the new system. One with our new passive wall, and one with a standard
passive wall. Both will use felt growing media and a drip irrigation system used by Furbish
Company. The purpose of these mockup walls is to determine whether a greater quantity of
VOCs are removed using our system in comparison to a standard passive system. We will
determine this using the analysis techniques described below.
After construction of the two model systems, they will be analyzed in growth chambers at
the University of Maryland Greenhouses. Each system will be placed in a separate growth

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chamber that will be set to have the same growing conditions as one another. A VOC mixture
will be injected into each chamber, and we will analyze each systems ability to degrade the
VOCs over a 24-hour period. We will perform this analysis using two methods: PID analysis,
and bacterial counts. For our PID analysis we will take one PID reading with a Tiger Handheld
VOC Gas Detector at the start of testing to determine the baseline for VOCs in the air. Over a 24
hour period, PID readings will be collected. The results from these tests will allow for a better
understanding of how well each system removes VOCs from the air. The PID test will quantify
the VOCs that are being removed from the air in the growth chamber.
The second test will be bacterial counts. This research is based on previous research
performed at Drexel University, which shows that VOC degradation occurs in root microbial
communities (Russell et al., 2014). At the end of the 24-hour test period the Hyphobicrobium
spp. on the roots of the plants will be quantified using qPCR as described in Phase 1A with
samples taken from the roots of plants from each of the models. The anticipated result is that the
bacterial counts in the newly designed system will be equal to or higher than the bacterial counts
from the traditional passive system.
All three of these tests will be run on both the team-designed model and the model of a
traditional passive system. By running a multitude of tests, we will have several different ways
of analyzing the success of the new system. If VOC levels are lower in the modified system, this
design will be proven successful because it functions better than a traditional passive system.
We expect to find a higher number of Hyphomicrobium spp. bacteria in the better-performing
system, which will confirm the conclusion of Russell et al., that Hyphomicrobium spp. bacteria

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are responsible for VOC degradation (2014). The sequence of three tests will be repeated at least
three times in order to gather enough data to reach a valid conclusion.
Confounding Variables and Research Limitations
A possible confounding variable in our research could be the early growth environment
of the plants being used for testing. Differences in their early growth environments could lead to
different bacterial communities residing in the roots before the study even begins, which could
affect later analysis on differences between the root samples. Possible ways to combat this
confounding variable is to acquire all the plants from the same source, ensuring similar growth
conditions, or take preliminary root samples to normalize the differences later on.
A limitation of our research is the use of the PID. The PIDs readings are not always
accurate but they are precise, meaning that the VOC concentration levels recorded may not be
the actual levels present in an environment. This is acceptable for our project because the
analysis of the VOC concentrations is only concerned with the difference between the beginning
and ending levels. Another limitation with the PID is that the PID only measures total VOC
concentrations instead of reporting the levels of individual VOCs. This is acceptable for this
research because each VOC will be tested individually in Phase 1. The potential confounding
variables and limitations are being addressed in ways that should minimize their impact on this
particular research.
Anticipated Results
We anticipate that the research will show that different plants support different bacterial
communities, possibly supporting more or less of the Hyphomicrobium spp. bacteria. Once this
hypothesis has been tested, our work with the traditional passive system versus our system will

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determine if it improves the biowalls effectiveness at removing VOCs from the air. If the
research reveals these anticipated results, then the improved system could be implemented in the
current industry to improve a passive biowalls ability to filter VOCs from the air without the
additional costs and complications associated with an active wall.

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Appendix A - Equipment Images

Figure A1: Clone King 25 Site Aeroponic Growth Chambers (Clone King 25, n.d.)

Figure A2: Tiger Handheld VOC Gas Detector/ Photoionization detector (TigerRevolutionary, n.d.)

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Appendix B - Glossary

1. Passive System: A standalone biowall system that is not attached to a building's HVAC system.
2. Active System: A biowall system that is connected to a building's HVAC system in order to force
air over the plants.
3. Hybrid System: A biowall system that is a combination of a passive and an active system.
4. Analyte: A chemical substance that is the subject of chemical analysis.
5. EPA Method TO-15: A method established by the EPA for the measurement of subsets of the 97
volatile organic compounds (VOCs) that are included in the 189 hazardous air pollutants (HAPs)
listed in Title III of the Clean Air Act Amendments of 1990.
6. Inert: Chemically inactive.
7. Quantitative-PCR: A method of quantification that involves amplifying the DNA of a target
specimen and then measuring the fluorescence. The stronger the fluorescence, the more
amplified DNA present. Using a standard curve with known concentrations, unknown sample
concentrations can be found.
8. Phytoremediation: The use of green plants to remove pollutants from the environment or render
them harmless.
9. Rhizoplane: The thin layer of soil covering the roots and strongly adhering to them; it forms an
interface between the roots and the rhizosphere which corresponds to the rest of the rhizospheric
area.
10. Rhizosphere: The distal fraction of the rhizospheric area that is adjacent to the rhizoplane; it is
still under the roots influence but without direct contact to them.
11. Rhizospheric Area: The volume of soil influenced by the plant roots and their exudates.
12. Hydroponic: The process of growing plants in water without the use of soil or an aggregate
medium
13. Sorbent: A substance that has the property of collecting molecules of another substance by
sorption (adsorption, ions and molecules binding on another molecule, and absorption, the
incorporation of a substance in one state into another state).
14. Spp.: Plural of species (sp.), referring to multiple species within a genus

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15. Static Headspace Sampling: A separation technique in which volatile material may be extracted
from a heavier sample matrix.
16. Summa Canisters: Passivated stainless steel vacuum sampling devices used to collect air
samples for air quality analysis.
17. Aeroponic: The process of growing plants in an air or mist environment without the use of soil
or an aggregate medium