Feng Maurer Wilson 1

Team B

Using Novel Magnetic Nanoparticles to Increase Batch Fermentation O2 Transport

1. Abstract

The effect of novel magnetic nanoparticles upon increasing the dissolved oxygen

concentration during a batch fermentation reaction will be measured at low and high cell

densities. Although it has already been proven in laboratory work conducted in the

fermentation module of 10.28 that the addition of these nanoparticles can indeed increase

the concentration of dissolved oxygen in a cell-free system, their effect upon bacterial

cell culture has not yet been determined. However, having measured a marked increase

in kLa upon nanoparticle addition using the sodium sulfite oxidation method in

laboratory, and given other literature supporting this conclusion (Olle et al. 2005), we

hypothesize that the measured kLa should increase during this fermentation experiment.

This research could be useful for recombinant DNA applications and biopharmaceutical

research, as an increased kLa would permit cells to be grown at higher densities, and thus

allow target proteins and enzymes to be synthesized more rapidly and at a lower cost.

2. Background and Significance

By providing large samples of diverse host organisms, microbial and animal cell

culture has played an important role in the development of vaccines, antibiotics, drugs,

and other biopharmaceutical products in the bioengineering field. Recombinant DNA

technology, developed only as recently as the 1984, uses bacteria such as Escherichia

coli to generate numerous R-DNA products such as insulin, growth hormones, and

stimulation factors. Sales from mammalian cell products alone constitute about a $25

billion industry (Wang 2005). Thus, from an economic standpoint, there exists a strong
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demand for these host cell protein products and thus also for procedures that will allow

these cells to be cultured to high densities.

Currently, the most widespread method for the large-scale production of

recombinant products is the use of a stirred-tank reactor (STR) with a cell cultures in

suspension. However, the use of an STR for this application is strongly limited by the

transfer of oxygen and nutrients to the cells, particularly at large reactor volumes. The

solubility of oxygen in water is low and dependent upon a number of factors, most

significantly the agitation and aeration rates. While high agitation and air sparging

conditions can be used to increase the concentration of dissolved oxygen within a reactor,

these conditions introduce strains upon the cells. Cell damage can occur either as a result

of the shear stress created by high agitation rates or when surrounding air bubbles burst at

the liquid surface, thus decreasing cell viability and overall protein production.

Maintaining high agitation and aeration rates also requires increased power and gas

inputs, which can become costly on a larger scale.

The oxygen transfer rate (OTR) to cells is determined from the oxygen mass

transfer coefficient kLa, which is dependent upon oxygen concentration, the oxygen

uptake rate (OUR), and aeration and agitation conditions. Some of the key limitations

upon oxygen transfer are gas-liquid interfacial area and oxygen solubility

(Parakulsuksatid, 2000). The development of novel nanoparticles which can be dispersed

in the bioreactor media has potential to resolve current issues regarding these two

parameters. These nanoparticles, which are biocompatible and nontoxic, contain a

magnetic core surrounded by a hydrocarbon coating that helps to increase the solubility

of oxygen. The presence of nanoparticles also introduces a larger interfacial area and
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Team B

allows for better oxygen uptake rates at bubble boundary layers (Olle et al. 2005).

Current research using sodium sulfite oxidation methods has found that nanoparticles can

increase the kLa of a cell-free system as much as 3 to 4 times (Olle et al. 2005). In

addition, the magnetic nature of the nanoparticle core creates efficient removal conditions

from media after fermentation by running the cells through a magnetic field.

The enhancement of oxygen transfer in sodium sulfite oxidation methods shows potential

for the application of nanoparticles to real bacterial fermentation processes. The objective

of this study is to determine the kLa enhancement associated with the inclusion of

nanoparticles in fermentation experiments with E. coli. One potential advantage of using

nanoparticles in fermentation processes is oxygen mass transfer into cells, which could

potentially lead to greater cell viability and productivity within a bioreactor. Additionally,

the use of nanoparticles might allow operation at lower agitation and aeration rates to

achieve the same level of oxygen transfer as that without nanoparticles, thus reducing

shear damage to cells and lowering the power cost of maintaining high agitation rates.

3. Materials and Methods

3.1 Materials

BioFlo 110 reactor controllers, manufactured by New Brunswick Scientific, will be used

in conjunction with 1-L reaction vessels in both experiments. The CSH36 E. coli strain

will be cultured in LB medium to carry out the fermentation. The nanoparticles used will

be novel magnetic core nanoparticles with hydrocarbon coats prepared by the Hamel

group. A Perkin-Elmer gas analyzer, which operates on the principle of mass

spectrometry, will be used to determine the chemical composition of gas samples. A

YSI2700 analyzer will be used to determine glucose concentration of samples.

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Team B

3.2 Methods

The dynamic method described in the 10.28 Biological Engineering Laboratory Manual

(Hamel et al., 2005). will be used to determine kLa values at low cell densities. At

higher cell densities, the method of off-gas analysis (Whittemore, 2005) will be used

instead. Fermentation reactions will be conducted approximately according to the

method outlined for Day 5 in the laboratory manual. Dry cell weight (DCW) assays will

be performed according to this description as well.

4. Technical Approach

The purpose of these experiments is to test the magnitude of increased oxygen transfer

when magnetic core nanoparticles are added to the fermentation broth. Because the

nanoparticles we are using have already been demonstrated to promote increased oxygen

transfer in cell-free environments, we hypothesize that these nanoparticles will provide a

significant increase in oxygen mass transfer both at high and low cell densities. To test

this hypothesis, we will run four fermentations in 1-liter bioreactors.

4.1 Dynamic Method at Low Cell Density

Two identical bioreactors will be operated at agitation and aeration rates of 500 rpm and

1 vvm to determine the oxygen mass transfer and optical density profiles by the dynamic

method at low cell densities. Magnetic core nanoparticles will be added to the

fermentation broth of one bioreactor. The other will be operated without nanoparticles.

To obtain data, we will remove a 2-mL sample from the sample port and measure its

optical density at 600 nm via spectrophotometery and its pH with the offline pH meter.

The oxygen supply to the reactor will then be turned off and the dissolved oxygen

concentration will be recorded every 30 seconds as it drops. When at least five declining
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dissolved oxygen transfer data points have been taken or when the dissolved oxygen

concentration reaches 20%, the oxygen will be turned back on and the dissolved oxygen

concentration will continue to be recorded until it is constant. Sampling will continue in

this manner once every 30 minutes until the optical density at 600 nm reaches 4.0 or until

the DO begins to drop so quickly upon turning off the oxygen supply that 5 data points

cannot be taken before it reaches 20%. The dissolved oxygen data will be graphed and

analyzed as discussed in Appendix B of the 10.28 Fermentation Lab Manual to determine

the oxygen mass transfer coefficients for each case. To compare the cases with and

without nanoparticles, plots of OD against time and kLa will be analyzed to determine

the effect of nanoparticles upon on the growth and oxygen transfer rates under the two

reaction conditions. The parameters will thus be the presence or absence of magnetic

nanoparticles and time. The response variables will be the oxygen mass transfer

coefficient and optical density. Scheduling details are included in Appendix 1.

4.2 Off-Gas Analysis at High Cell Densities

Because the dissolved oxygen concentration drops too quickly to use the dynamic method

at high cell densities, off-gas analysis will be used to determine the oxygen mass transfer

coefficient. Two fermentations will be performed in identical 1-liter bioreactors at 500

rpm and 1 vvm as off-gas compositions are be monitored by mass spectrophotometry.

Every 30 minutes, a 5-mL sample will be taken to measure the optical density and dry

cell weight and the off-gas composition will be recorded for kLa calculation. The

fermentation will be continued over approximately 18 continuous hours or until the

optical density becomes constant, indicating that the cells are no longer growing. As in

the dynamic method above, the optical density over time will be compared in the two
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Team B

reactors, as will the oxygen mass transfer compared to the optical density. The

parameters will be the presence of magnetic nanoparticles and time. The response

variables will be the oxygen mass transfer coefficient and optical density. Scheduling

details are included in Appendix 1.

4.3. Challenges and Strategies

Using these two methods, we may develop two slightly different oxygen mass transfer

profiles for the low cell density case, but we will use the duplicity of the data to help

eliminate sources of error between the two models. Other challenges will include the

response time of the off-gas analyzer and finding a suitable method for analyzing the

optical density in the presence of nanoparticles. To control the variation that may occur

due to the response time of the off-gas analyzer, samples will only be taken from the

reactor immediately after a gas sample is analyzed. To adjust for the optical density of

the nanoparticles, the blank that is used to calibrate the spectrophotometer will contain

nanoparticles in the same concentration as the reactor medium.

5. Safety Considerations

Safety will be of particular importance during the grueling 18-hour fermentation reaction,

particularly since we will be occupying the lab beyond its normal hours of operation.

Although a teaching assistant or instructor is expected be present in lab at all times, we

will ensure that two students from our group are present in lab at all times in case of

emergency. Other safety precautions typical for bacterial cell culture and fermentation

will also be considered—in particular, inoculation will be performed in a sterile

environment inside a biosafety hood.

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6. References

Hamel, JF, et al. 2005. 10.28: Biological Engineering Laboratory Manual,

Fermentation Module.

Olle B, Bromberg L, Hatton TA, Wang DIC. “Enhancement of Oxygen Transfer in

Bioreactors by Use of Magnetic Nanoparticles.” 12th International Biotechnology
Symposium. Santiago, Chile. 20 Oct 2004.

Parakulsuksatid P. “Utilization of a Microbubble Dispersion to Increase Oxygen

Transfer in Pilot-Scale Baker’s Yeast Fermentation Unit.” Thesis. VPISU, 2000.

Wang DIC. “Biopharmaceuticals from Recombinant DNA Technology.” 10.28:

Biological Engineering Laboratory Lecture. 66-0042, Cambridge. 9 Sept 2005.

Wang DIC. “Effect of Sparging and Shear in Mammalian Cell Culture and Strategies to
Increase Oxygen Transfer Rate.” 10.28: Biological Engineering Laboratory
Lecture. 66-154, Cambridge. 10 Oct 2005.

Whittmore BA. “Recombinant Collagen Production Optimization in Escherichia coli."

Thesis. MIT, 2005.
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Appendix A – Scheduling

11/1 – Medium Preparation and E. coli inoculation (Christina)

Bioreactor setup and calibration (Cynthia and Sam)

11/3 – Dynamic Method Experiment (Christina, Sam)

Setup, inoculation, and medium prep for High cell density experiment (Cynthia)

As Scheduled with staff: 6 AM to approximately midnight long experiment in shifts

Time Reactor with Nanoparticles Reactor without Nanoparticles

6 am Cynthia Christina
9 am Cynthia Sam
12 noon Christina Sam
3 pm Christina Cynthia
6 pm Sam Cynthia
9 pm Sam Christina
If the experiment does not take as long as expected, we will clean up on the day of the

experiment. If that is not possible, we will come in on the next appropriate lab day to

complete the clean-up.