An automatic microturbidostat for bacterial culture at constant density
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- Luo, X., Shen, K., Luo, C. et al. Biomed Microdevices (2010) 12: 499. doi:10.1007/s10544-010-9406-5
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We have developed a microturbidostat for long time bacterial culture at constant density controlled by optical detection and integrated pneumatic valves. The device was fabricated by multilayer soft lithography and in-situ formation of an agarose filter. The culture chamber of bacteria was connected in one side to a single bacterial input-output channel and in another side to a nutrient channel in which the agarose filter was formed to ensure the diffusion of nutrients and metabolites without bacterial loss. The bacterial number in the culture chamber was determined by measuring the fluorescence intensity of GFP proteins of the bacteria and the redundant bacteria could be exported automatically through the input-output channel with integrated micro-valves. In order to optimize the operation performance, we investigated the bacterial exportation efficiency with different input-output channel widths. As expected, the bacterial sorting coefficient was proportional to the input-output channel width. The results also showed that with a 20 µm channel-width, a long time culture was possible with a constant bacterial number in the chamber in the range from 400 to 700.
There are several reasons to be interested in the development of new tools and technologies for improved microorganism cultivation. First, the current cultivation and bio-processing are mostly relying on shake flasks, which are not flexible for the microenvironment control and easy to keep the concentration of chemical components in constant (Akgun et al. 2007). Comparing to the batch operation, continuous-flow culture systems offer the advantages of constant environmental conditions (Calcott 1981) but both conventional chemostat (Novick and Szilard 1950) and turbidostat (Martin and Hempfling 1976) have the drawback of complex step-up and hard handling. Second, the consummation for large volume systems is remarkably high, which is often not necessary for experimentations such as cultivation parameter determination. Third, it is difficult for the commonly used systems to perform parallel operation. Finally, many microorganisms found in nature are still unculturable using current techniques or conditions (Maharbiz et al. 2004) and it is therefore important to develop new systems with an improved parameter control, reduced consumption, and increased parallelism and automatism.
Lab chip technology offers a unique solution to these problems. Indeed, many groups have successfully demonstrated the relevance of fabricated microsystems in microorganism cultivation. Zanzotto et al. (2004) have developed a membrane-aerated microbireactor with integrated sensors for optical density (OD), dissolved oxygen (DO), and pH measurements. Puskeiler et al. (2005a, b), Weuster-Botz et al. (2005) and Zhang et al. (2005) have reported the integration of magnetic mixers in their microbioreactors systems. Whereas Balagadde et al. (2005) have demonstrated the operation of a microchemostat with integrated peristaltic pumps and micro valves for parallel and long-term culture of microorganisms, Zhang et al. (2005) have presented a microchemostat which is capable of OD, DO and pH real-time measurements for continuous cultivation of microorganisms. In addition, Groisman et al. (2005) have applied cell trapping technique in similar microchemostat devices.
We report in this work the design and fabrication of an automatic microturbidostat which can be used to control the biomass concentration by feedback system and thus to provide the microorganisms with constant physiochemical environment. Our device consisted of integrated culture chamber, nutrient channel, bacterial input-output channel and pneumatic valves. An agarose filter integrated between the nutrient channel and the culture chamber allowed the diffusion of nutrients and metabolites without bacterial losing (Beebe et al. 2000; Wu et al. 2006; Cheng et al. 2007; Kim and Beebe 2007). To evaluate the bacterial exportation efficiency, five devices have been fabricated with different input-output channel-widths. Experimental data were then analyzed and compared with a simple bacterial exporting model. For turbidostat culture, the bacterial number was calculated by image-processing algorithm and then changed with a feedback control system.
2 Materials and methods
2.1 Device design and fabrication
2.2 Experimental setup
2.3 Strain and culture
E.coli MG1655 (wt), kindly provided by Dr. Francois Taddei of University Rene Descartes, was transformed with plasmid I7001 expressing ampicillin resistance and GFP proteins. Luria-Bertani (LB) medium used consisted of 5 g/L yeast extract, 10 g/L NaCl and 10 g/L tryptone. Single colony of E.coli MG1655 (I7001, Amp) on LB plate (containing 2%(wt/vol) agar and 250 µg/mL ampicillin) were cultured overnight in 2 mL LB medium with 250 µg/L ampicillin on a shaker at 180 rpm in 37°C. Then, 10 µL of culture medium was transferred to a test tube containing 1 ml of fresh, which was also incubated on a shaker at 180 rpm in 37°C until the optical density (OD600 nm) reached 0.6. The culture medium was injected into the microfluidic device at normal temperature, which had been sterilized by injecting 75% ethanol for 2 h and rinsed with filtered deionized water to remove the ethanol.
2.4 Cell loading
The PDMS device was first degassed in a vacuum desiccator for 5 min. Then, filtered deionized water was injected into both of the nutrient channel and the air-liquid channel. Since the degassed PDMS absorbs the gas, water could be introduced into the bacterial culture chamber (Luo et al. 2008). Afterward, the microfluidic channels were sterilized by 75% ethanol for 2 h and rinsed with filtered deionized water. The sterilized LB medium was finally injected into the nutrient channel. When the bacteria were injected into the air-liquid channel, they swam into the culture chamber through the bacterial input-output channel, due to bacterial chemotaxis.
3 Results and discussion
The fitting curves in Fig. 5 were obtained from data in the range of 0–300 min. For more than 300 min, the bacterial number is much smaller and there appear large deviation from the exporting model because of the following two reasons. First, the bacteria located in the culture chamber closer to the nutrient channel can not be easily exported. In fact, these bacteria undergo the attraction of nutrients from both sides. Thus, the migration capacity of bacteria toward the input-output channel is much reduced so that the reduction of the bacterial number is less rapid with time. Second, when the exporting time becomes long, the influence of cell growth on exporting effect increases, resulting in the deviation from the model. More laborious are expected for a more quantitative interpretation of the present results.
We designed and realized an automatic microturbidostat for bacterial culture in constant density. The results showed that the bacterial sorting coefficient in our devices was proportional to the width of the input-output channel which connected the culture chamber and the input-output channel. Under optimal conditions, the designed system can be used for bacterial culture in a well-controlled micro-environment. We believe that such a microturbidostat has unique advantages of easy and robust sample handling with much reduced reagent consumption. Furthermore, both device fabrication technology and operation strategy proposed in this work can be extended for high throughput screening by considering either parallel or sequential system integration.
The authors would like to thank F Taddei, C.B. Lou, L.P. Xu and M. Ni for useful discussion and help. This work is partially supported by the Chinese Natural Science Foundation (NO. 10704002, 10634010, 10721403) and National 973 project (NO. 2003CB715900).