This document proposes creating an in vitro gut biofilm model capable of sustaining multiple bacterial species. The researchers will 3D print Lactobacillus and Bifidobacterium bacteria onto a porous mucin membrane within a device consisting of two layers separated by the membrane. Nutrients, food, and waste can diffuse through the membrane, allowing the bacteria to communicate via quorum sensing while being physically separated. Experiments will manipulate environmental factors like pH, temperature, and nutrients to determine optimal conditions for the long-term coexistence of both bacterial species, overcoming the "winner takes all" phenomenon seen in previous single-species models. If successful, this model could be used to study probiotics and gut bacteria responses without human testing.

1. H4 Angelica Du, Jiatao Liang, Connor Thompson
7/22/14
SAAST Biotechnology
Simulating Gut Biofilm In Vitro
Abstract
A biofilm is an aggregate of microorganisms attached to a biotic or abiotic surface. One of the
most important biofilms people encounter is that within our gut, which aids in digestion, immune
response, and food regulation. In vitro biofilms are produced currently via technology such as a
drip flow biofilm fermenter. However, the drip flow model is limited in its ability to represent a
true biofilm because it can only grow one type of bacteria, due to the “winner takes all”
phenomenon. The “winner takes all” phenomenon is when one species of bacteria shows a
greater affinity for the given nutrients, causing the other species to starve and die-out. The goal
of our research proposal is to overcome the “winner takes all” phenomenon by creating a gut
biofilm in vitro, capable of supporting the coexistence of multiple species of bacteria. To
accomplish this, Lactobacillus and Bifidobacterium will be 3d printed separately onto a porous,
mucin-based membrane. The membrane limits the mobility of the bacteria, while still allowing
them to quorum sense, the process by which bacteria communicate with one another via
chemical sensing which aids in the growth and health of the biofilm. Our device will consist of
two layers separated by a porous membrane, through which chemicals, food, and waste can be
diffused. The bacteria will be engineered with green fluorescent protein so that we can identify
colonies and analyze bacterial growth under a UV light. Since the device will preserve the
chemical interconnectivity of the bacteria, we expect to see an increase in the number of
colonies, which will indicate a successful biofilm, capable of sustaining multi-species bacteria
growth. Later on, this model can be used to observe the reaction of the human gut bacteria to
various drugs and treatments without resorting to risky, costly human testing. Ultimately, this
model can serve as a platform for future testing of probiotics and further study on the mechanics
of the human GIT.
Introduction
A biofilm is an aggregate of microorganisms attached to a biotic or abiotic surface. One
of the most important places biofilms are found is within the human gut, or gastrointestinal tract
(GIT), which functions as a complex ecosystem harboring a group of coexisting microbial
populations living in synergy and eubiosis1. This ecosystem aids its host with digestion, immune
response, and food regulation. Also, modern diseases such as obesity and diabetes have show
strong links to the composition of the gut flora.2 The dynamics of gut flora are crucial to
maintaining a healthy body, aiding in the protection against invasive pathogens and harmful
chemical compounds.
The gut lining secretes a mucin layer about 400 micrometers thick along the intestinal
walls3, which regulates the movement of the bacteria and provides the foundation for biofilm
1
Holzapfel, Wilhelm and Haberer, Petra, et al. “Overview of Gut Flora and Probiotics.” International Journal of
Food Microbiology, Volume 41, 85-10, 1998.
2
Kotzampassi, Katerina, Evangelos J. Giamarellos-Bourboulis, and George Stavrou. "Obesity as a Consequence of
Gut Bacteria and Diet Interaction."ISRN Obesity 2014 (2014): n. pag. Web.
3
Matsumoto,Mitsuharu and Tani, Hisanori, et al. “Adhesive Property of Bifidobacterium lactis LKM512 and
Predominant Bacteria of Intestinal Microflora to Human Intestinal Mucin,” Current Microbiology, Volume 44, 212-
215, 2008. DOI: 10.1007/s00284-001-0087-4.

2. growth4. The ability of the gut flora to coexist relies heavily on quorum sensing, which is
bacterial communication through chemical signaling5. Quorum sensing is essential in the growth
of the biofilm as a whole, allowing the bacteria to exist in eubiosis, the balance between the
beneficial and the harmful bacteria.
Communication between the multiple species is imperative, for they exist within a very
hostile environment and must rely on each other for mutual survival. Characteristics of the GIT
include a pH range of 2.5-3.5 and the secretion of aggressive intestinal fluids. The amount of
nutrients fluctuates with the individual’s diet, which places a lot of stress on the bioflora as a
whole. However, the interactions between separate bacteria, as well as with their environment,
make them more robust and resistant to calamity.
Statement of Need
The unique environment of the gut is hard to replicate in laboratory-grown multi-species
cultures. One dominant species of bacteria is often more equipped for the given environment,
creating a lopsided competition and pushing out the other species6. This is known as the “winner
takes all” phenomenon and is the crux of the issue with in vitro biofilm. Because of this, attempts
to study biofilm have been mainly concerned with single species studies, which cannot give an
accurate representation of the entire gut biofilm.
A model of the gut biofilm in vitro will allow scientists to systematically study the
bacterial interactions that are key to comprehending our digestive system. Previous methods for
constructing laboratory biofilm include using a drip flow biofilm reactor (Figure 1), in which a
thin film of bacteria is maintained by a steady drip of nutrients flowing over the slide of biofilm
with waste elimination on the other side. However, this device is limited in its ability to study
bacterial interactions of a biofilm, because it focuses on a single type of immobile bacteria with
limited potential for growth. Replication of the gut requires multiple species of bacteria, along
with specific conditions concerning the pH, temperature, and flow of nutrients.
Objectives
There are many variables concerning the homeostasis of the gut biofilm, including
individual dietary factors and unattainable bacterial diversity that makes the gut difficult to
exactly replicate in all its complexity. To overcome the “winner takes all” phenomenon, we seek
to make our own device consisting of multiple species of bacteria, with specific environmental
conditions that mimic that of the gut. For our own model, we will focus primarily on the chemical
communication that occurs between the bacteria, the most significant factor for the success of the
gut ecosystem. We hypothesize that an environment mimicking that of our gut will be the most
successful in growing a stable biofilm.
4
Holzapfel, 86.
5
Hooper, Lora and Gordon, Jeffrey. “Commensal Host-Bacterial Relationships in the Gut.” Science Magazine,
Volume 292, 2001.
6
Kim HJ, Boedicker JQ, Choi JW, Ismagilov RF. “Defined spatial structure stabilizes a synthetic multispecies
bacterial community.” Proc. Natl Acad. Sci. USA 105, 18 188–18 193. 2008.

3. Figure 1: Drip Flow Biofilm Reactor. The biofilm is grown on the
substratum, while food is entered through the inlet and waste is
eliminated through the effluent outlet. Source: (Coenye, Tom and
Hans Nelis, “In vitro and in vivo model systems to study microbial
biofilm formation,” Journal of Microbiological Methods, Volume
83, 89-105,2010. )
Approach
I. The Device
To overcome this challenge,
we plan to incorporate some of the
known techniques in biotechnology,
specifically the drip flow biofilm
reactor (Figure 1) and 3D printing
(Figure 2), into our own biofilm
model. However, we can build upon
them to represent the dynamics of a
gut biofilm, including multiple
species of bacteria, an efficient
nutrient delivery and waste removal
system, and a way to arrange the
bacteria for optimal living
conditions. After building our device,
our goal is to sustain the survival of
the different bacteria species for as
long as possible by experimenting
with dynamic conditions and
discovering the combination that will optimize the bacteria lifetime length.
The spatial arrangement of the bacteria in the biofilm plays an important role in their
social behavior in terms of reproduction and mobility in a cooperative relationship. Using a
recent microscopic 3D printing method (Figure 2), we will construct a bacterial community that
allows for the organization of multiple populations of bacteria in a geometric arrangement with
each colony 200 micrometers apart. This separates the two bacterial species just enough to avoid
competition for nutrients, while still allowing them to communicate using quorum sensing
signals.7 This is important for it allows for the cooperation between species that exists within the
gut to be replicated, while avoiding the “winner takes all” phenomenon that exists in previous in
vitro models. In this manner, we can arrange the bacteria in adjacent or nested colonies, held in
place by a highly porous, cross-linked gelatin material, to mimic the biofilm structure of the gut.
In this shared
microenvironment,
the bacteria will be
suspended within
the set gel while
still being able to
communicate with
each other through
the porous
material. While
this departs from
an exact model of
7
Connel, Jodi et al. “3D Printing of Microscopic Bacterial Communities.” Proceedings of the National Academy of
Sciences of the United States of America. Vol. 110, 2013, DOI: 10.1073/pnas.1309729110.
Figure 2: These are the steps taken to create the enclosed cell structure in our biofilm
reactor.(Source: Connel5
)

4. the gut biofilm, it preserves the chemical interconnectivity between the bacteria, an important
factor for biofilm formation through bacteria working in harmony 8.
The two bacterial species we will choose are Lactobacillus and Bifidobacterium (Figure
4). Both strains have been studied in-depth by scientists for their presence in the gut and
probiotic properties. We will engineer the bacteria with strains of pGLO, which will give us an
easy indicator of the colony size, number, and growth.
To print the bacteria onto the porous surface, we will mix the Lactobacillus and
Bifidobacterium with a fabrication precursor containing mucin instead of gelatin to better
emulate the gut. This cellular suspension will be moved to a sterile well to be cooled, resulting in
a gelled matrix. The geometric configurations are fabricated around one or more bacteria by
scanning a highly focused pulsed laser beam in three dimensions. We will design a computer
model of the bacterial colonies’ spatial orientations of the 200 microns distances between each
colony forming unit, and the technology will follow this plan with sub-micrometer 3D
resolution, creating an artificial biofilm that will mimic the gut on a small-scale.
In order to preserve the realistic conditions of the gut, we will control the pH level and
temperature to mirror that of the human body’s. Our gut (Figure 3) will be molded using
polydimethylsiloxane (PDMS). PDMS is a commonly used silicon-based polymer that is clear,
non-toxic, and resistant to strong acids and bases9. This is ideal because even though this portion
of the model will not come in contact with bacteria directly, it needs to be able to withstand
strong acid that simulates the fluid in the gut.
The mold has two levels: an upper, cylindrical area where the bacteria and the mucin
layer are, and a lower cavity that allows for the flow of simple sugars, yeast extract, and proteins
as nutrients for the growing bacteria. On top of the half-pipe will be a coverslip, through which
we can observe the growth of the
biofilm.
The structure resembles a
half pipe to simulate the tubes of
the small intestine, specifically the
duodenum, and to eliminate
corners in which bacteria can
aggregate. The mucin layer and
the cavity will be exposed to one
another through the pores, through
which nutrients, as well as control
factors such as acids and bases,
can diffuse upwards to the
bacteria. The bacteria
continuously communicate via
quorum sensing by sending
chemical messages through the
pores (Figure 3).
8
Kim HJ, Boedicker JQ, Choi JW, Ismagilov RF. “Defined spatial structure stabilizes a synthetic multispecies
bacterial community.” Proc. Natl Acad. Sci. USA 105, 18 188–18 193. 2008.
9
Lotters, J. C, "The mechanical properties of the rubber elastic polymer polydimethylsiloxane for sensor
applications" Journal of Micromechanics and Microengineering, 147-153. 2007
Figure 3: Our model of the gut. It will have a ultraviolet light on the
top to reveal the glow of the recombinant bacteria, a porous layer
that allows the bacteria to communicate with each other, and a
cavity through which food, mucin, and waste can flow through and
diffuse either way.

5. II. Experiments
We will overcome the
“winner takes all” phenomenon by
both limiting mobility via the
mucin layer and forcing the
bacteria to rely on each other by
imposing harsh conditions in
which no single species of bacteria
can survive alone. We will exploit
the fact that bacteria in biofilm
have greater resistance to harsher
conditions than planktonic
bacteria. By mirroring such
conditions like those in the gut, we
expose the bacteria to the same
unsympathetic environment that they are used to surviving and thriving in by working in
harmony with the bacteria group.
The bacteria will be used to assess the effectiveness of our in vitro biofilm model in
mimicking these conditions of the gut. Colony growth will be measured by counting the
fluorescent colonies of pGLO modified bacteria with the UV light built into our device (Figure
3). An increase in the amount of colonies of both the Lactobacillus and the Bifidobacteria will
indicate a successful biofilm, capable of housing and accommodating growth.
Our hypothesis states that the optimal conditions for culturing the bacteria are those that
mimic the harsh habitat of the gut to the closest extent. Once our biofilm model is tested
successfully for growing multi-species ecosystems, we want to test this hypothesis by changing
various environmental factors and observing the colony in response to each adjustment. We can
experiment by increasing and decreasing specific variables to observe how the bacteria act as in
response. For each experiment, the bacteria will be counted using the same GFP method, so that
we can discover the optimal conditions and evaluate our hypothesis accordingly.
First, the pH of the system can be adjusted using the bottom cavity, which we will use as
our primary control system. High concentrations of hydrochloric acid (HCl) will flow in the
lower cavity and diffuse upwards to the bacteria, lowering the pH. If the solution becomes too
acidic, we can flow a basic solution of sodium hydroxide (NaOH) to neutralize some of the acid.
We will experiment with various ranges of pH and observe the degree to which the bacteria are
able to survive under certain chemical circumstances. The ideal pH is known to be between 2.5
and 3.5. Thus, we will start at 3.5, record the growth, and gradually lower it. This will reveal the
optimal pH for the growth of both Bifidobacterium and Lactobacillus.
We will maintain a constant external temperature of 37°C, the average temperature of the
human gut, to ensure the accurate replication of the gut environment with the first few trials.
Similarly to the pH modification, we can adjust the temperature in the biofilm to see how the
bacteria react to the change in a favorable or unfavorable manner. For example, under cold
temperatures, we expect to see that the bacteria will grow very slowly due to slower rate of
molecular motion and biological reaction10. We will first change the temperature up and down by
increments 5°C order to observe the effects and possible growth trends.
10
Erdal, Ufuk G. and Randall, Clifford W. “Thermal Adaptation of Bacteria to Cold Temperatures in an EBPR
System.” Department of Civil and Environmental Engineering, Virginia Polytechnic Institute and
Figure 4: Lactobacillus and Bifidobacterium. Each as applications in
probiotics and are found in a human gut Source: (Weiner, Melinda. "A
Cultured Response to HIV." Nature Medicine. Nature, 2009.Web. 17 July
2014,Holm, Carl. "Scientists Unravel Probiotics Gut Defence › News in
Science (ABC Science)." ABC Science. ABC, 27 Jan. 2011.Web. 17 July
2014.)

6. Lastly, we will conduct an experiment with increasing and decreasing amount of
nutrients provided so we can see whether competition over these nutrients arises. We plan to
flow simple sugars, which are easiest to break down in the digestive system. Using the UV light
to observe the growth of colonies, we will be able to determine the optimal amount of nutrients
to provide our bacteria to ensure a prolonged period of growth. If one grows quicker than the
other, it would show one species dominating the other. To avoid this scenario, we will adjust the
kind of nutrients we provide the bacteria, preventing the “winner takes all” phenomenon to
occurring, ensuring our bionic gut’s success.
Ultimately, we will find the best combination of all of these factors to see whether they
line up with the conditions of the gut, therefore verifying our original hypothesis.
Significance
We present this model as a proof of concept. The actual human gut has a myriad of
factors that our current model does not take into account: for instance, salt balance and the
thousands of other bacteria we must include. If this method of cultivating gut flora on a
microscale with only two different strains of bacteria works, we can expand it to encompass
more different strains of bacteria, eventually culminating to the cultivation of the entire
microbiota of the gut, as well as the more complex systems of the gut.
When completed, pharmaceutical companies can use our model to test the effects of
drugs on gut biota, without resorting to human subjects that can be both expensive and risky.
Moreover, doctors can use the model for individualized diagnosis. The gut of a single patient can
be replicated in vitro by adjusting the model to their specific pH and bacterial-composition.
Testing drugs and treatments on the bionic gut allows the doctor to observe whether or not their
treatment kills gut biota, prompting them to adjust their dosages accordingly, avoiding
ineffective treatment and patient injury. Additionally, an in vitro model of the gut will allow us
to study bacteria that have never been cultured before because of their interdependence on others
in the gut flora11. We will eventually be able to culture those bacteria outside of a living person
and systematically study all of the bacteria of the gut.
10
State University, 2002.
11
Flint, Harry J., Paul W. O'Toole, and Alan W. Walker. "The Human Intestinal Microbiota." Microbiology 156
(2010): 3203-204. Web.