Masters of Engineering Paper:
Growth Promoting Effects of a Bacterial Consortium on a Lipid Producing Algae
Strain
By
Stephen Menefee
Abstract
Photosynthetic microalgae offer promising opportunities for biodiesel production due to their
high growth rates and triacylglycerol (TAG) lipid synthesis. I am currently studying the growth
characteristics of one such marine algae, a Chlorella strain C596. Previously, it was found that
strain C596 is not axenic but contains a consortium of background bacteria. Upon removing the
majority of these bacteria, the algae showed a reduced growth rate compared to its co-culture
counterpart, indicating that the bacterial background could play a role in the increased growth
rate of the co-cultured Chlorella strain C596. The goal of my project was to study the
mechanisms by which this interaction is facilitating the increased growth rate of the Chlorella
strain C596. The recovery of growth was measured in resuspensions of partially purified
Chlorella strain C596 in media with the co-culture bacteria present, and in media containing
only the bacterially- produced growth factors. Results of both experimental scenarios indicated
that presence of growth factors alone could be sufficient to recover the maximal growth rates
associated with the original culture, but that the reintroduction of the microbial community
was necessary and sufficient for growth rate recovery. Lastly, as proper separation of the
bacteria from the algae is a significant step in understanding the interactions between the two,
a microfluidic H-Filter device was designed for potential separation of the algae from the
bacteria.
1
Introduction
Microalgae are viewed as a potentially fruitful source for biodiesel production. Due to
their high growth rates and lipid synthesis abilities, the need for understanding and improving
production and extraction processes has become a forefront of biofuel research. However, the
factors that influence the growth of algae are complicated and affected by many factors ranging
from genetic background, to nutrient availability and environmental stress conditions, to
syntrophic bacterial interactions. This last category is especially important due to the nature of
optimizing a wild organism, originally grown in a multi-specie and trophic level environment, for
isolated laboratory conditions. Syntrophic relationships between algae and bacteria are
common in nature and so it is not surprising that such a relationship might play an essential
role in the growth and overall lipid productivity of the organism. This paper explores two
mechanisms – bacterial mediated oxidative stress reduction and bacterial produced growth
factors – by which a particular microalgae, Chlorella strain C596, could be having its growth rate
affected by a background microbial community.
Theories as to syntrophic mediated metabolic processes are not novel in the field of
algal research. Previous work done by Krohn-Molt et al. showed evidence of both direct and
indirect bacteria-algae interaction [1]. The research studied the interaction between a
multitude of bacteria, including Alphaproteobactria, Betaproteobacteria, and Bacteroidetes,
and the microalgae Chlorella vulgaris and Scenedesmus obliquus. It was shown that bacteria
may act as a sink for lipids and fatty acids produced by the algae which can be used as carbon
sources. In addition, it was shown that some bacteria could bind directly to the algal cells and
2
produce a surrounding nano-filament structure. Further, genomic evidence supported that the
algae were benefited by bacterial production of B-group vitamins. All results indicated that
there existed a important interactions between the studied algae and its background bacteria.
There are other examples of bacteria-algae interactions which had growth promoting
effects, and were dependent on intercellular communication between the two organisms. Guo
et al. studied the role of extracellular organic carbon (EOC) production in the interactions
between Chlorella vulgaris and Pseudomonas sp. [2]. Evidence showed that algae benefitted
from bacterial EOC production as well as from bacterial mediated reduction of dissolved
oxygen, which could otherwise have been damaging to the algal cells. The algae cultures with
Pseudomonas present produced greater concentrations of EOC’s, indicating that there existed
some form of chemical communication between the algae and the bacteria which facilitated
their syntrophic relationship.
Similar growth promoting effects, which were due to bacteria-algae interactions, were
found by Watanabe et al. [3]. In their studies of Chlorella cultures, they found a consortium of
bacteria associated with Chlorella sorokiniana, some of which showed growth promoting
effects of the algae in co-culture. In addition, the various bacteria showed influence over
chlorophyll production, some increasing production with others reducing it.
Given the multitude of other cases in which algae and bacteria in nature exhibit
mutually beneficial interactions when grown in close proximity to one another, it was not a
surprise to find indications that a similar process could be occurring with the Chlorella strain
C596 isolated by collaborators working with Cellana, an algal-based products company
3
stationed in Kailua-Kona, Hawaii. Much of the research ongoing in the DOE-funded consortium
is involved in better understanding and optimizing metabolic and lipid synthesis pathways of
algal strains to increase lipid yields. The Chlorella strain C596, is a marine algae that exhibits
high growth rates and lipid production, making it valuable for potential biodiesel use [6].
However, genome sequencing data, generated at Cornell University, revealed that Chlorella
strain C596 contained a diverse but uncharacterized bacterial background [7]. Further study
found the predominant bacterial genus to be Rhodobacter in origin. Upon attempting to
remove the bacteria through repeated streaking methods, the partially purified Chlorella strain
C596 exhibited a reduced growth rate. This observation indicated that the microbial community
present in the original Chlorella strain C596 may be playing an essential role in growth
promotion of the algal cells.
Two hypotheses have been made as to the mechanism by which this interaction could
be occurring. The first is that the microbial community grown in co-culture with the Chlorella
strain C596 could be reducing the oxidative stress of the algae, and thereby increasing the algal
growth rate. In this scenario, the presence of the bacterial cells in co-culture with the Chlorella
strain C596 would be required for the reduction in oxidative stress and in turn increased growth
rate of the algae. The details of how this reduction in oxidative stress would occur were not
tested, but rather only whether the presence of the microbial community was required for
growth promotion in the algae. Experiments were designed to test this hypothesis by removing
the Chlorella strain C596 algal cells by filtration methods and resuspending the partially purified
Chlorella strain C596 algal cells into the filtrate media. If the partially purified Chlorella strain
C596 samples which grew in media containing bacteria from the original Chlorella strain C596
4
cultures showed an increased growth rate compared to the partially purified Chlorella strain
C596 samples grown in media without the bacteria, then this would show that the presence of
the original microbial community could be necessary or sufficient for growth promotion, and
that the syntrophic interaction could be related to oxidative stress reduction of the algae by
specific members of the microbial community.
The second hypothesis is that a necessary growth factor, not added to our synthetic
seawater, is provided by the bacterial co-culture. The bacteria grown in co-culture with the
Chlorella strain C596 might produce beneficial growth factors which the algal cells would take
up from the media and thereafter exhibit an increased growth rate. In this scenario, bacteria
from the Chlorella strain C596 cultures would be expected to produce algae beneficial growth
factors, which would be present in the media. In order to test this hypothesis, partially purified
Chlorella strain C596 cells were resuspended in media containing growth factors and media
void of growth factors. The growth factor present media was removed of any bacteria and algal
cells by autoclaving. If the partially purified Chlorella strain C596 samples grown in media
containing only growth factors and nutrients produced by the bacteria exhibited an increased
growth rate compared to the partially purified Chlorella strain C596 samples grown in media
void of these bacterial produced growth factors, then this would indicate that the bacterial
growth factors played a role in the algal growth rate. In addition, this experiment was designed
to further test the necessity of the microbial cell presence. By providing media containing both
bacteria and growth factors from the Chlorella strain C596 cultures to the purified Chlorella
strain C596 samples and measuring growth, the necessity of microbial cell presence could be
tested.
5
Methods
Media, Cultures, and Fluorometer Measurements
All cultures were initially grown on Aquil as the growth media [8]. Media was prepared
in 1000 mL acid washed and autoclaved polycarbonate bottles for each generation of each
experiment. Due to the short time span (1 week) of generations in experiments, bacterial
contamination was expected to be an insignificant factor in culture growth, and so the media
itself was not autoclaved. However, in an effort to still remove any small presence of bacterial
contamination, as well as reduce trace metal contamination, the media was microwave treated
to boil. This heat treatment was performed prior to the addition of filter-sterilized trace metals
and vitamin stock solutions so that these components would not degenerate during heating.
All cultures were grown in a constant 24 degree Celsius growth chamber. Samples from
Trial 1 and 2 of the Cell Presence Experiment and Trial 1 of the Growth Factor Experiment were
grown in samples were grown in 30 mL of Aquil in 1 inch diameter glass culture tubes. Samples
from Trial 2 of the Growth Factor Experiment were grown in 5 mL of Aquil in 0.5 inch diameter
glass culture tubes. Growth rates of samples were determined by fluorescent measurements on
a Turner Designs 10-AU Fluorometer made once a day for each of 3 replicates per experimental
condition. Growth rates were then calculated for each replicate by taking the slope of the
logarithmic linear regime of fluorescence over time, and then the individual replicate growth
rates were averaged together to compare statistic difference between treatments using a 2Tailed T-Test analysis. Generations were transferred once all replicates and conditions had
reached a stationary growth phase. Fluorescent measurements in Trial 1 and 2 of the Cell
6
Presence Experiment and Trial 1 of the Growth Factor Experiment were performed using the 1
inch diameter glass culture tubes filled with 30 mL of sample culture placed directly into the
fluorometer. Fluorescent measurements for Trial 2 of the Growth Factor Experiment were
made by transferring the 5 mL of culture into in 0.5 inch diameter glass fluorometer tubes.
Cell Presence Experiment
In Trial 1 of the Cell Presence Experiment, initial growth conditions were
unpurifedChlorella strain C596, Chlorella strain C596 Sacrificial, partially-purified Chlorella strain
C596, and partially-purified Chlorella strain C596 Test (Table 1 and Figure 1). One batch of the
Chlorella strain C596 replicates were grown to Exponential Phase, and then filtered through a 3
micron Nucleopore® polycarbonate filter to remove algal cells. The filtrate was then collected
and filtered through a 0.2 micron polycarbonate filters to collect bacteria. The used 0.2 micron
polycarbonate filters were then placed in their respective partially purified Chlorella strain C596
Test replicates overnight, which was in late lag to early exponential growth phase, vortexed,
and then removed. All samples were cultured in 30 mL volumes of Aquil. Two generations were
recorded so that any lag time in the effect of a growing bacterial population could be observed.
In Trial 2 of the Cell Presence Experiment, initial growth conditions were the same as in
Trial 1, however with an additional control to account for bacteria introduced from a clean
polycarbonate filter. In this control, a clean polycarbonate filter was placed in a set of partially
purified Chlorella strain C596 replicates overnight, vortexed, and removed on the same
timescale and process as the test conditions (Table 1 and Figure 1). Filtering occurred when the
partially purified Chlorella strain C596 replicates reached stationary phase. As with Trial 1, two
7
generations were recorded so that any lag time in the effect of a growing bacterial population
could be observed.
Growth Factor Experiment
In Trial 1 of the Growth Factor Experiment, batches of Chlorella strain C596 and partially
purified Chlorella strain C596 were initially grown to stationary phase (Table 2 and Figure 2).
Chlorella strain C596 was grown to stationary phase in 500 mL of Aquil in an acid washed
autoclaved 1000 mL polycarbonate container, while partially purified Chlorella strain C596 was
grown to stationary phase in 5 mL of Aquil in a 0.5 inch diameter glass culture tube. The 500 mL
of Chlorella strain C596 batch was then filtered with a 3 micron polycarbonate filter. Solutions
of 0.5 mL of 0.1 M Phosphate and 1.5 mL of 0.3 M Nitrate were added to the filtrate. The
filtrate was then split into two 250 mL volumes in acid-washed autoclaved polycarbonate
containers. Three 30 mL volumes of each media condition were then pipetted into 1 inch
diameter glass culture tubes.
A second batch of Chlorella strain C596 was grown in 20 mL of Aquil to stationary phase
and split into six 2 mL centrifuge tubes. The tubes were centrifuged at 8000 RPM for 1 minute
using an Eppendorf Centrifuge 5424, and the supernatant was removed down to 0.5 mL. The 30
mL of partially purified Chlorella strain C596 was split into nine 2 mL tubes, centrifuged at 8000
RPM for 1 minute using an Eppendorf Centrifuge 5424, and the supernatant was removed
down to 0.5 mL. All 15 tubes were then vortexed to resuspend the pellets and 30 µL of each
tube were inoculated into their respective 30 mL replicate conditions (Table 2).
8
In Trial 2 of the Growth Factor Experiment, batches of Chlorella strain C596 and partially
purified Chlorella strain C596 were initially grown to Stationary Phase (Table 2 and Figure 2).
The Chlorella strain C596 was grown in 250 mL of Aquil in an acid washed and autoclaved 500
mL polycarbonate container. The partially purified Chlorella strain C596 was grown in 30 mL of
Aquil in an autoclaved 1 inch diameter glass culture tube. The Chlorella strain C596 batch was
then split into five 50 mL falcon tubes with 30 mL in each and centrifuged at 7000 RPM for 15
minutes using an Eppendorf Centrifuge 5430. The supernatant of the centrifuged batch was
then collected in a new acid washed autoclaved 500 mL polycarbonate bottle and split into 100
mL and 150 mL volumes in new acid washed autoclaved 500 mL polycarbonate bottles.
Solutions of 0.1 mL of 0.1 M of Phosphate and 0.3 mL of 0.3 M Nitrate were added to the 100
mL batch. 0.15 mL of Phosphate solution and 0.45 mL of Nitrate solution was added to the 150
mL batch. The 100 mL batch of Chlorella strain C596 was then autoclaved, while the 150 mL
batch was not. 20 mL of the non-autoclaved 150 mL Chlorella strain C596 batch was then
filtered through a 0.2 micron polycarbonate filter as a negative control for bacterial presence in
the non-autoclaved condition. 5 mL of each condition was then pipetted into each of the
autoclaved 10 mL glass tube replicates (Table 2). The 30 mL of partially purified Chlorella strain
C596 was then pipetted into a 50 mL falcon tube and centrifuged at 7000 RPM for 15 minutes
using an Eppendorf Centrifuge 5430. The pellets were resuspended in 1 mL of Aquil, and 20 µL
of each inoculant was finally pipetted into the replicates of each media condition (Table 2).
Results and Discussion
Cell Presence Experiment
9
Trial 1 of the Cell Presence experiment showed a recovered growth rate in the partially
purified Chlorella strain C596 Test samples (Table 3 and Figure 3). These results suggest that the
presence of microbial background from the Chlorella strain C596 culture could be necessary or
sufficient to recover the partially purified Chlorella strain C596 growth rates. Generation 1 of
the purified Chlorella strain C596 Test samples showed a small growth rate increase of 5.6%,
and generation 2 showed a larger growth rate increase of 33.1% and were statistically
significant at the α = 0.1 and 0.05 level respectively. These results indicated that partial
recovery of purified Chlorella strain C596 growth rate increased with each generation toward
that of the Chlorella strain C596. However, this growth promotion could again be due to
bacterial growth factors which might be produced in co-culture. Further, it was suggested that
an additional control be added to account for any bacterial contamination which may have
been introduced to the purified Chlorella strain C596 test samples from the polycarbonate
filters themselves.
Trial 2 of the Cell Presence Experiment supported the results of Trial 1, showing a
recovered growth rate in the partially purified Chlorella strain C596 Test samples, which
contained the bacterial background from the Chlorella strain C596 culture (Table 4). Generation
1 of the partially purified Chlorella strain C596 Test samples showed a growth rate increase of
23.7%, and generation 2 showed a growth rate increase of 27.4%, both of which were
significantly at the α = 0.05 level. The partially purified Chlorella strain C596 with Filter showed
a small reduction in growth rate of -3.5% in generation 1 and a small increase in growth rate of
9.8% in generation 2. The results of the partially purified Chlorella strain C596 with Filter
samples showed a small (if any) effect of the polycarbonate filter’s background bacteria on the
10
growth rates of the partially purified Chlorella strain C596 samples. In addition, the
fluorescence of the partially purified Chlorella strain C596 Test cultures was measured before
and after the addition of the filter into each replicate. The algae carryover from the filter added
around 0.58 on a natural log scale to the initial fluorescent readings of the partially purified
Chlorella strain C596 Test cultures. This increase in fluorescence potentially caused by the
additional carry-over algae originating from the polycarbonate filter could indicate that a
significant portion of the algae added to the partially purified Chlorella strain C596 Test cultures
were of filter origin.
Increases in growth rates of the partially purified Chlorella strain C596 Test samples over
each subsequent generation was expected to be due to a lag in bacterial concentrations. Not all
bacteria from a Chlorella strain C596 culture can be captured through filtration, as some might
stay attached to the polycarbonate filter, while others could be forced through the pores into
the discarded filtrate. Therefore, if the bacterial presence was in fact playing a beneficial role in
promoting the algal growth, then it may take multiple generations for a recently inoculated
population of bacteria to reach its natural or optimal levels.
Additionally, assumptions were made that the Chlorella strain C596 cells were
genetically identical to the partially purified Chlorella strain C596 cells; however this
assumption has not been verified. If the two strains were actually dissimilar in genetic makeup,
then any Chlorella strain C596 which passed through the filter process could have also had an
effect on the recovered growth rates, especially over each subsequent generation.
Growth Factor Experiment
11
Trial 1 of the Growth Factor Experiment showed that the partially purified Chlorella
strain C596 grown in autoclaved media from the parent Chlorella strain C596 exhibited a
growth rate typical of the partially purified Chlorella strain C596 (0.7-0.8 day-1) (Table 5).
However, the growth rate of the partially purified Chlorella strain C596 grown in autoclaved
media was 9.0% lower than the growth rate of the partially purified Chlorella strain C596 grown
in fresh Aquil media and was significant at the α = 0.1 level. This further reduced growth rate
may have been caused by degeneration of any important growth factors present in the media
as a result of the autoclaving. However, if this effect were significant, then the parent Chlorella
strain C596 grown in autoclaved media would have shown a similar reduction in growth rate
from the unpurified Chlorella strain C596 grown in non-autoclaved media, which it did not.
Rather, the condition showed a slight increase in growth rate, indicating that autoclaving may
not have had a significant effect on the stability of important growth factors.
The partially purified Chlorella strain C596 grown in non-autoclaved media did however
show an increase in growth rate of 19.0% compared to the partially purified Chlorella strain
C596 grown in fresh media and was significant at the α = 0.05 level. The results indicate that the
presence of microbial background could be required for recovering the growth rate of the
partially purified Chlorella strain C596, further supporting the results of the Cell Presence
Experiments, and that bacterial growth factors alone would be insufficient to recover the
partially purified Chlorella strain C596 growth rate.
Trial 2 of the Growth Factor Experiment further replicated the findings of the previous
experiments, but the addition of a non-autoclaved 0.2 µM filtered experiment resulted in a
12
recovered growth of the partially purified Chlorella strain C596 samples grown in nonautoclaved media, suggesting that autoclaving may be damaging to important growth factors
(Table 6). The partially purified Chlorella strain C596 grown in autoclaved media showed a
decrease in growth rate of 20.7% compared to the partially purified Chlorella strain C596 fresh
Aquil media and was significant at the α = 0.05 level. These results indicated that bacterial
growth factors alone could be insufficient to recover the partially purified Chlorella strain C596
growth rate. However, the partially purified Chlorella strain C596 grown in non-autoclaved
media and 0.2 micron filtered also showed an increase in growth rate of 18.1% compared to the
partially purified Chlorella strain C596 grown in fresh Aquil media and was significant at the α =
0.05 level. In addition, the Chlorella strain C596 grown in autoclaved media showed a 0.9%
decrease in growth rate compared to the partially purified Chlorella strain C596 grown in fresh
media, significant at the α = 0.1 level. This could support the hypothesis that autoclaving the
cultures damages any important growth factors released into the medium by the bacteria,
thereby reducing the growth rate of such treated samples. The partially purified Chlorella strain
C596 grown in non-autoclaved media showed a 20.6% increase in growth rate compared to the
partially purified Chlorella strain C596 grown in fresh media, suggesting that microbial cell
presence may be sufficient and necessary for algal growth promotion.
In total, the results of all the experiments supported two findings – that bacterial
presence appeared to be necessary and sufficient for promoting Chlorella strain C596 growth
rates, and that autoclaving may destabilize important growth factors (Figure 4 and 5).
13
There were differences in initial fluorescence readings for all experiments that were
due to variations in sample algal cell concentrations for resuspensions and inoculum from stock
cultures. In addition, as transfers were made on the same day between all conditions and
cultures, there was an inherent difference in cell concentrations at the transfer time due to the
difference in growth rates. While transfers were attempted to be performed at fluorescent
reading saturation, differences in concentrations were still present.
H-Filter
A major step toward understanding the interactions between algae and its consortium
of bacteria is to separate the two organisms effectively. Our lab used repeated streaking
methods to separate algal cells from the bacterial background over a series of rounds.
However, this method was time and effort intensive. Other microfluidic methods have been
have been proven effective to separate microscale particles based on differences in diffusion
rates. For instance Schulte et al. analyzed the feasibility of using H-Filter designs in separating
ions, hormones, and drugs from larger blood cells based on differences in diffusion rates and
the non-turbulent properties of laminar flow [4].
An H-Filter design was similarly designed for the purpose of separating C596 algae cells
from its background bacteria (Figure 4 and Table 8). As the algal cells and bacterial cells are
different sizes, differences in diffusion rates were expected to exist. By flowing a mixture of the
cells through a channel of parallel laminar flows, the cells’ only ability to migrate perpendicular
to the flow would be by diffusion. Due to bacteria’s higher diffusion rate, bacteria could be
collected on one outflow, while an incrementally purified mixture of algae and bacteria can be
14
collected on the other. By placing the H-Filters in series, connecting the outflow (Q3) of the ith
H-Filter with the inflow (Q1) of the i+1th H-Filter, an increasing degree of purity of the algae can
be attained, each round removing more bacterial cells.
C596 algae are non-motile cells, and so an algal diffusion constant was determined by a
Stokes force approximation of a sphere in water, calculated by the equation: 𝐷 = 𝑓
𝑘𝑇
𝑠𝑝ℎ𝑒𝑟𝑒
where
𝑓𝑠𝑝ℎ𝑒𝑟𝑒 = 6𝜋𝜂𝑎, η is the viscosity of fluid (approximated as water), k is Boltzmann’s constant, T
is temperature in assumed to be 293 Kelvin, and α is the radius of the sphere approximated as
2.5 microns for an algal cell and 1 micron for a bacterial cell [5]. However, as the bacterial
consortium of the Chlorella strain C596 was composed of a mixed background of numerous
bacterial species, approximations for diffusion constants were made for both motile and nonmotile variants. The non-motile bacterial diffusion constant was determined by the same
Stokes method of force approximation on a sphere. The motile bacterial diffusion constant was
approximated as similar to that of an E. coli cell, which is a motile bacteria.
When designing the H-Filter, an acceptable operational time per filter run was first
determined – 15 minutes. Any times that were too long (on the order of hours) could be
problematic due to significant algal and bacterial growth during the filtration process. Diffusion
distances of the algae and bacterial cells were then calculated based on this time scale and the
following diffusion rate equation: 𝑡𝑑𝑖𝑓 =
<𝑥 2 >
2𝐷
=
ℎ𝑙𝑊5
𝑄5
(Table 7). It was assumed that the half
width of the H-filtered represented the tipping point that determined in which outflow the cells
would be collected. If a cell’s maximum diffusion distance was less than the width’s midway
point, then it would be collected in the left outflow. Similarly, if a cell’s diffused distance was
15
greater than the width’s midway point, then as a population of cells moves through the device
some fraction would be collected on the right outflow. By setting the width of the H-Filter to
double the distance an algal cell could diffuse in the allotted time, only bacterial cells would be
collected in the Q4 flow, while an incrementally purified mixture of algae and bacteria would be
collected in the Q3 flow.
Improved purity per round was determined by a uniform volume diffusion
approximation. If diffusion occurred as a wall moving in the x-direction, and not a parabola, and
a uniform distribution of cells existed behind this wall, then percentage of cells migrated could
be determined as the ratios of diffused lengths between the two organisms. For the motile
bacteria, this approximation was most likely valid, as the actual distance a motile bacteria could
diffuse in the 900 second allotted time was 950 µm, compared to the algal diffusion distance of
13 µm. Such a large difference between the diffusion distances would lead to a uniform
distribution of bacteria cells throughout the channel by the time the algal cells diffused to the
center of the channel. The case of the non-motile bacteria gave a bacterial diffusion distance of
21 µm by the time the algal cells diffused to the center of the channel; therefore such an
approximation would be expected to be less accurate.
Purity was then calculated from the percentage of distance the bacterial cells diffused
farther than the algal cells, representing a percentage of bacterial cells removed from the
𝑊5⁄
2
mixture collected by flow Q3, as shown by the equation: % 𝐹𝑖𝑙𝑡𝑒𝑟𝑒𝑑 = (1 − 𝑥
𝑏𝑎𝑐𝑡
) 100 where
W5 is the width of the center channel, and xbact is the maximum diffused distance of a bacterial
cell. It was found that for the motile bacteria condition, 50% purity could be attained with each
16
filter run, and therefore 7 filters in series would be needed for a 99% purity of algae (Table 7).
For the non-motile bacteria condition, 28.66% purity could be attained with each filter, and
therefore 14 filters would be needed in series for a 99% purity of algae (Table 7).
Dimensions and flow parameters were determined based initially on time sensitivity of
the experiment, and ultimately on pressure limitations. As decreasing the size of the
microfluidic channels increases the pressure inside the channel, dimensions for acceptable
pressures on both the cells and the device were needed. With the selected dimensions, the
maximum differential pressure across the channel was determined to be 5.15x10-10 Pa from the
equation: ∆𝑃 =
12𝑄𝐿𝜂
ℎ3
where Q is the flow rate, L is the length of the channel, η is the viscosity
of the fluid assumed to water, and h is the height of the channel. This pressure change is well
within an acceptable range for Poiseuille flow.
As the details of interactions between the bacterial and algal cells are unknown, the
filter could have scenarios which would prove it less useful. If for instance the bacterial cells
responsible for algal growth promotion are bound directly to the algal cell surface, then the
designed H-Filter would be unable to effectively separate the two cells based on their diffusion
rates. Still however, the design provides an opportunity for time efficient bio-separation of
some algal and bacterial mixtures. Moreover, the ease of printing these devices on agarose gels
could offer an inexpensive and effective means of cell separation for high throughput
purification algae from environmental samples.
Conclusion
17
Experimental results found that bacterial cell presence was necessary and possibly
sufficient for the growth promotion of Chlorella strain C596. The Cell Presence Experiments
showed that partially purified Chlorella strain C596 samples resuspended in media containing
bacteria from unpurified Chlorella strain C596 exhibited a recovered growth rates approaching
or reaching those of unpurified cultures. The Growth Factor Experiments showed that the
presence of bacterial growth factors could be promoting the Chlorella strain C596 growth rate,
but that autoclaving may destabilize the growth factors. However, the results also showed that
the presence of bacteria in non-autoclaved media from Chlorella strain C596, which contained
both bacterial cells and growth factors, was sufficient to recover the purified Chlorella strain
C596 growth rates, further supporting the results of Cell Presence Experiments. These results
together suggest evidence for the hypothesis that growth promotion in the Chlorella strain
C596 could be due to bacterial mediated oxidative stress reduction on the basis of algal growth
rate promotion mediated by microbial cell presence. However, some promotion of growth may
still be occurring from bacterial promoted growth factors. More in depth experiments to
further test this hypothesis are necessary for mechanistic details of the bacterial and algal
growth promoting interactions. As a next step toward this understanding, a microfluidic H-Filter
device was designed for the purpose of time effective separation of bacteria from algae. Future
experiments should be performed in testing the H-Filter device and in studying the specific role
of bacterial cell presence in promoting Chlorella strain C596 growth rates, as well as
determining the effect of autoclaving on important growth factors.
Acknowledgements
18
I thank Dr. Beth Ahner and Dr. Lubna Richter at Cornell University’s Department of Biological
and Environmental Engineering for their continued support in my project. I also thank Dr.
Cresten Manfeldt for his genome background on the Chlorella strain C596.
Figures and Tables
Figure 1. The Cell Presence Experiment flow chart shows the process flow for Trials 1
and 2. Chlorella strain C596 cells are removed with a 3 µm filter. The microbial
community is then collected using a 0.2 µm filter and placed into cultures of partially
purified Chlorella strain C596. The growth is then determined by measuring a fluorescence
intensity of chlorophyll over time.
Figure 2. The Growth Factor Experiment flow chart shows the process flows for Trials
1 and 2. Chlorella strain C596 cells are removed with a 3 µm filter, and a portion of
this filtrate is then autoclaved. Partially purified Chlorella strain C596 is then centrifuged
down to pellets and resuspended in the autoclaved and non-autoclaved filtrate. The
growth is then measured by taking fluorescent measurements.
19
Table 1. Cell Presence Experiments’ media conditions and inoculants. Sacrificial meant that the culture was grown for using
the media only and did not serve as a test or control scenario.
Condition
Aquil
Aquil (Sacrificial)
Aquil from Sacrificial
Aquil
Aquil with Clean Filter
Inoculant
Chlorella strain C596
Chlorella strain C596
Partially purified Chlorella
strain C596
Partially purified Chlorella
strain C596
Partially purified Chlorella
strain C596
Table 2. Growth Factor Experiments’ media conditions and inoculants.
Conditions
Autoclaved Aquil from Chlorella strain C596 culture
Autoclaved Aquil from Chlorella strain C596 culture
Non-autoclaved Aquil from Chlorella strain C596 culture
Non-autoclaved 0.2 micron Filtered Aquil from Chlorella
strain C596 culture (Trial 2)
Fresh Aquil
Inoculant
Chlorella strain C596
Partially purified Chlorella
strain C596
Chlorella strain C596
Partially purified Chlorella
strain C596
Partially purified Chlorella
strain C596
20
Table 3. Growth rates, percent differences, and statistical significance for each condition
compared to partially purified Chlorella strain C596 of the Cell Presence Experiment Trial 1.
21
Figure 3. Trial 1 of the Cell Presence Experiment growth curves showing the natural log of
fluorescence intensity of various conditions over time for generations 1 (top) and 2 (bottom).
One representative replicate is shown for clarity.
22
Table 4. Growth rates, percent differences, and statistical significance for each condition
compared to partially purified Chlorella strain C596 of the Cell Presence Experiment Trial 2.
Table 5. Growth rates, percent differences, and statistical significance for each condition
compared to partially purified Chlorella strain C596 of the Growth Factor Experiment Trial 1.
23
Table 6. Growth rates, percent differences, and statistical significance for each condition
compared to partially purified Chlorella strain C596 of the Growth Factor Experiment Trial 2.
Figure 4. Growth rates of the Cell Presence Experiment conditions normalized to the
partially purified Chlorella strain C596 growth rate.
24
Figure 5. Growth rates of the Growth Factor Experiment conditions normalized to the growth
rate of partially purified Chlorella strain C596 in fresh Aquil media.
Figure 6. H-Filter Schematic showing flows Q1-4, dimensions W1-5, L, and h, and the separation process
between diffusing algal and bacterial cells. Xbact represents the distance than an algal cell will diffuse
across the microfluidic channel.
25
Table 7. H-Filter constants for run time, pressure, algal diffusion, motile bacterial diffusion, and
non-motile bacterial diffusion; and solved values for the diffusion distances, percentage of
bacteria filtered per run, and the number of runs for 99% purity of algae.
Table 8. H-Filter parameter values.
W1-5 represent the widths of channels
1 through 5. Q1-5 represent the fluid
flows in channels 1 through 5. h
represents the height of the channels.
L represents the length of the device.
26
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