PARALLEL MICRO-CHEMOSTATS IN AN AUTOMATED DROPLET MICROFLUIDIC SYSTEM 1
Sławomir Jakiela1, Tomasz S. Kaminski1, Olgierd Cybulski1 and Piotr Garstecki1
Institute of Physical Chemistry, Polish Academy of Sciences, Kasprzaka 44/52, 01-224 Warsaw, POLAND
ABSTRACT We report a microfluidic system supporting microliter chemostats in the form of droplets traveling in microchannels. The system monitors the growth of bacteria and controls the composition of media in each of the chemostats in time and independently. We use external valves to form droplets on demand, merge, route, and split them into subvolumes at arbitrary ratio between 0.1 and 0.9. We use these operations to prepare a sequence of drops containing bacteria and then iteratively culture bacteria monitoring their growth and use a fraction of the culture to seed droplets with fresh media. KEYWORDS: microdroplets, chemostat, bacteria, parallelization, automation INTRODUCTION One of the milestones in microbiology was the invention of a chemostat by Leo Shilard [1] and creation of a mathematical model of its dynamics [2]. This type of bioreactor has the unique feature and removing part of culture and feeding it with fresh media – thus enabling control of the concentration of microbes and nutrients. Macroscale chemostats were used to support continuous growth of bacteria, yeasts and algae [1], [3] allowing for research on ecology of microorganisms [4], dynamics of microbial prey-predator system [5], cross-feeding bacterial systems [6], and many other aspects of microbiology. The main drawback of macroscale chemostats is their cost and fact that they need large quantities of media and reagents. In recent years there were several attempts to miniaturize chemostats, these including integrated micro-systems [7], microchambers [8] parallel chip-based microreactors [9] or digital microfluidics [10]. Systems described in these reports needs only minute amounts of media, with the drawback that parallelization is still limited to few chambers. This limitation comes in part from the complexity of the devices. Another potential problem is the phenomenon of formation of biofilims causing some of these systems to be single-use [8] or requiring active washing [7]. THEORY An alternative that we follow in this work is to use droplets transported inside micrometer channels as individual chemostats. In this approach every droplet has the same volume and can serve as a distinct bioreactor. Thus this system theoretically can be easily scaled up to hundreds of even thousands of microchemostats processed sequentially. Additionally, droplets are isolated from the walls of channels prevents cross-contamination and formation of biofilms. The major challenge in constructing such a system supporting a large number of micro-chemostats is automation. Droplet microfluidic technology is still developing - up to date, there were several demonstrations of formation of droplets, break-up, merging and sorting. However, support for continuous processing of chemostats requires more complex operations of repeatable removal of grown cultures and feeding with fresh media; realization of this tasks using classical microdroplet technologies is impossible. Development of droplets-on-demands technology [11] together with the solutions for routing, splitting and merging of droplets that we describe here make the task possible. In this paper we demonstarte multiparalell droplet chemostat and several demonstration of bacteria growth under various dilution rates and different level of stress caused by presence of an antibiotic. EXPERIMENTAL We milled the chip (using CNC mill Ergwind MSG4025, Poland) into a slab of polycarbonate (Makroclear, Bayer, Germany) and assembled by 2 minutes exposure in plasma chamber and subsequent bonding in press with a pressure 0.4 MPa. The dimensions of channels were varied from 200 m to 800 m. Distilled water dyed by 12,5% solution red ink(Waterman, France) served as the droplet phase. The continuous liquid was 0.5 % w/w solution of Span80 in hexadecane. We used droplet on demand technology, developed in our group [11]. We used pressurized reservoirs of liquids interfaced with modified (as described previously by Churski et al. [11]) electromagnetic valve (V165, Sirai, Italy) Steel capillaries (I.D. 0.21 mm, Mifam, Poland) of high hydraulic resistance (2.95·1012 kg m-4 s-1) and of length equal to 80 cm served as connections between valve and polycarbonate chip and they were used in order to ensure stable formation of droplets. For connections between the capillaries the needles we used elastic Tygon tubing (inner diameter 0.25 mm, outer diameter 2 mm, Ismatec, Switzerland). Every channel began with round perpendicular hole fitted for 21 gauge needle of outer diameter of 0.82 mm. In all experiment we used Luria-Bertani (LB) Broth (BD Biosciences, Belgium), which is dedicated for testing of antibiotic susceptibility. As a tested organism we used E. coli strain ATCC25992. We made stock solution of cells in Luria-Bertani medium (BD Biosciences, Belgium) containing 30 % glycerol (Chempur, Poland) and froze a -80oC. Before experiment cells
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15th International Conference on Miniaturized Systems for Chemistry and Life Sciences October 2-6, 2011, Seattle, Washington, USA
were streaked on a LB agar plates and incubated overnight. Next we inoculated liquid broth and cultured at 37oC and 200 rpm overnight and then transferred to fresh media and grow until OD600 reached 0.1. We diluted culture 2x times prior to transfer to microfluidic device, so approximate staring cell concentration inside droplet was 107 cfu/ml. RESULTS AND DISCUSSION Figure 1 shows schematically the architecture and operation of the system. Droplet-on-demand generators [11] first produce a sequence of droplets, each with an individually predetermined composition and seeded with bacteria into a channel terminated on both ends with a valved source of oil and a valved drain, allowing to route the droplets back and forth through the on-chip spectrophotometer. After incubation each of the droplets is split into a „sample‟ (purged) and a „seed‟ droplet, subsequently merged with a droplet containing fresh media. Figure 1: Schematics of the microfluidic system supporting parallel microchemostats. a) a sequence of droplets seeded with bacteria is injected into the system, and cycled (b) for incubation and monitoring of growth of bacteria. After a chosen interval each droplet is split into a portion(sample) that is purged from the system and a small portion that is used to seed a freshly prepared droplet with medium (c). The legend explains the meaning of the symbols. The DOD generator comprises a T-junction and two external valves that control the flow of oil – one stream to push an aqueous sample deposited on the chip, the other supporting the flow of the continuous matrix liquid.
Figure 2. Normalized concentration of a red dye in a single droplet undergoing sequential splitting and additions of fresh liquids – illustrating the ability to control the composition of any particular droplet in time. We demonstrated that sequential splitting and dilution of droplets is very precise. The composition of the titrated media is controlled with the set of DOD generators allowing to vary the content of any particular droplet (chemostat) in time (Fig. 2). Figs. 3 shows the results of an highly reproducible incubation of 40 (4 L) droplets over 10 hours. After 5 hours each droplet was split into 2.5 and 1.5 L daughters and the latter was merged with 2.5 L drop of fresh medium. All 40 droplets contained degassed LB medium. 30 drops (6th-35th) were inoculated with ATCC 25922 E. coli.
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Figure 3. Optical density (600 nm) as a function of time illustrating two cycles of growth of bacteria in 30 droplets and lack of growth in ten control droplets.
Figure 4. Optical density (600 nm) showing the growth of colonies of bacteria in media containing different concentrations of tetracycline. Each concentration was tested in three individual microchemostats.
We demonstrated that chemostat can be used to grow bacteria in various conditions. Figs. 4 shows the results of an incubation of 30 (4 L) droplets containing 11 different concentrations of tetracycline (from 0.0 to 1.0 mg/L). Each concentration was tested in triplicates. CONCLUSION The system that we report i) has a simple and robust architecture that uses a minimal set of 11 valves, ii) performs formation, splitting and merging of droplets at single Hz frequencies, iii) can be used to control large sets of droplet chemostats, iv) provides a vista for arbitrary dilution ratios and changes in composition of the media in time, and v) avoids the problems associated with formation of a biofilms. This is first demonstration of microdloplets that serve as bacterial chemostats. We believe that this set of characteristics will prove useful in academic and industrial research in microbiology. ACKNOWLEDGEMENTS Project operated within the Foundation for Polish Science (FPS) Team Program (2008-1/1) co-financed by the EU European Regional Development Fund. REFERENCES [1] A. Novick and L. Szilard, "Description of the Chemostat," Science, vol. 112, pp. 715–716, (1950). [2] J. Monod, “La technique de culture continue theorie et applications,” Ann. Inst. Pasteur, 79, pp. 390–410, (1950). [3] T.W. James "Continuous Culture of Microorganisms," Annual Review of Microbiology vol. 15, pp. 27–46, (1961). [4] H.H. Topiwalaand C. Hamer, “Effect of wall growth in steady-state continuous cultures,” Biotechnology Bioengineering vol. 13, pp. 919-922, (1971). [5] L. Becks, F.M. Hilker, H. Malchow, K. Jürgens, H. Arndt, “Experimental demonstration of chaos in a microbial food web,” Nature, vol. 435, pp. 1226-1229, (2005). [6] D.E. Dykhuizen, A.M. Dean, “Evolution of specialists in an experimental microcosom,” Genetics, vol. 167, pp. 20152026, (2004). [7] F.K. Balagadde, L. You, C.L. Hansen, F.H. Arnold, S.R. Quake, Long-term monitoring of bacteria undergoing programmed population control in a microchemostat," Sciene, vol. 309, pp. 137-140 (2005) [8] A. Groisman, C. Lobo, H. Cho, J.K. Campbell, Y.S. Dufour, A.M. Stevens, A. Levchenko, “A microfluidic chemostat for experiments with bacterial and yeast cells,” Nat. Methods vol. 2, pp. 685–689, (2005). [9] N. Szita, P. Boccazzi, Z. Zhang , P. Boyle, A.J. Sinskey, K.F. Jensen “Development of a multiplexed microbioreactor system for high-throughput bioprocessing,” Lab Chip, vol. 5, pp. 819-826, (2005). [10] S.H. Au, S.C. Shih, A.R. Wheeler, “Integrated microbioreactor for culture and analysis of bacteria, algae and yeast,” Biomed Microdevices, vol. 13, pp. 41-50, (2011). [11] K. Churski, P.M. Korczyk and P. Garstecki, “High-throughput automated droplet microfluidic system for screening of reaction conditions,” Lab Chip vol. 10, pp. 816-818, (2010). CONTACT *Piotr Garstecki, email:
[email protected]
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