在微生物領域，細菌培養是一個基本的實驗技術，並在多方面有著各種應用，像是發酵（fermentation）、微生物感染診斷（bacteria infection diagnostics）、人造生物學（synthetic biology），由於經濟成本的考量，微生物生長室（growth chamber）的微小化是必要的，微小化可以帶來許多的優點，像是藥劑消耗的減少、快速檢測。在納升（nanoliter）尺度下流體有著低雷諾常數（Reynold number）的特性，所以我們可以將一個完整的生長室分割成N個離散的隔室，因此可在微流體晶片中達成連續性的恆化器（chemostat），意思是連續的供給養分和稀釋微生物，而我們做的是最簡化的恆化器，即選擇最少的隔室（N=2）和最少的稀釋步驟，本晶片可自動化的驅動，可以在納升尺度下進行長時間（~100小時）的培養以及監控，由於有較高的面積體積比（surface to volume ratio），此晶片也可做為生物膜流動反應室，並可與恆化器數學模型擬合(fitting)的非常好，我們期望此晶片可應用在微生物學的許多領域，如微生物生態學（microbial ecology）、抗藥性（antibiotic drug resistance）研究、人類微生物（human microbiome）和生物膜（biofilm）研究。

Bacteria culture is a basic technique in both fundamental and applied microbiology. The excessive reagent consumption and difficulty to maintain bulk bioreactors for microbial culture has prompted the development of the miniaturized on chip bioreactors. At microscopic scale, the low Reynold number fluidics justifies the design of a class of discrete chemostats of N compartment growth chamber to recapitulate two mechanistic aspects of a chemostat, namely continual supply of nutrient and dilution of microbial population. With the minimal choice of two compartment (N=2) and discrete time, periodic dilution steps, we realize a microfluidic chemostat that mimics the serial dilution transfer culture at macroscopic scale. This device supports automated, long term (~100 hr) microbial culture with nanoliter scale working volume and real time monitoring of microbial population at single cell resolution. Due to high surface to volume ratio, the device also serves an effective biofilm flow reactor to support a planktonic and biofilm ecology with proper choice of plumbing protocol. In the dilute biofilm limit, a simple chemostat model with wall growth fits well with the growth curves. We expect such devices will open opportunities in many fields of microbiology such as microbial ecology, antibiotic drug resistance study, and human microbiome, and biofilm study.

1. Unger, M., et al. Monolithic microfabricated valves and pumps by multilayer soft lithography. Science 288, 113-116, (2000).
2. Melin, J. & Quake, S. R. Microfluidic large-scale integration: the evolution of design rules for Biological Automation. Annu. Rev. Biophys. Biomol. Struct., 36, 213-231, (2007).
3. Streets, A. M., & Huang, Y. Chip in a lab: Microfluidics for next generation life science research. Biomicrofluidics, 7, 011302, (2013).
4. Novick and Szilard. Description of Chemostat. Science 1950
5. de Crécy, E. et al. Development of a novel continuous culture device for experimental evolution of bacterial populations. Appl. Microbiol. Biotechnol. 77, 489–496 (2007)
6. Balagadde F. K, You, L. C, Hansen, C. L, Arnold, F. H., & Quake, S. R. Long-term monitoring of bacteria undergoing programmed population control in a microchemostat. Science 309: 137–140, (2005).
7. Groisman, A., Lobo, C., Cho, H., Campbell, J. K., Dufour, Y. S., Stevens A., M., & Levchenko, A. A microfluidic chemostat for experiments with bacterial and yeast cells. Nature Method 9, 685-689, (2005).
8. Long, Z., Nugent, E., Javer, A., Cicuta, P., Sclavi, B., Lagomarsino, M. C., & Dorfman, K. D. Microfluidic chemostat for measuring single cell dynamics in bacteria. Lab Chip 13, 947-954, (2013).
9. O’Toole, Kaplan, Roberto Kolter,biofilm formation as microbial development, Annu. Rev. Microbiol. 2000. 54:49–79
10. Unger, M. et al. Monolithic microfabricated valves and pumps by multilayer soft lithography. Science 288, 113–116 (2000).
11. Melin, J. & Quake, S. R. Microfluidic large-scale integration: the evolution of design rules for Biological Automation. Annu. Rev. Biophys. Biomol. Struct., 36, 213-231, (2007).
12. Balagadde, F. K. et al. A synthetic Escherichia coli predator-prey ecosystem. Mol. Syst. Biol. 4, 187 (2008).
13. de Crecy-Lagard V. A., Bellalou, J. M., Mutzel, R. & Marliae, P. Long term adaptation of a microbial population to a permanent metabolic constraint: overcoming thymineless death by experimental evolution of Escherichia coli. BMC Biotechnol. 1 10 (2001).
14. Unger, M., et al. Monolithic microfabricated valves and pumps by multilayer soft lithography. Science 288, 113-116, (2000).
15. Melin, J. & Quake, S. R. Microfluidic large-scale integration: the evolution of design rules for Biological Automation. Annu. Rev. Biophys. Biomol. Struct., 36, 213-231, (2007).
16. Balagadde F. K, You, L. C, Hansen, C. L, Arnold, F. H. & Quake, S. R. Long-term monitoring of bacteria undergoing programmed population control in a microchemostat. Science 309, 137– 140 (2005).
17. de Crécy, E. et al. Development of a novel continuous culture device for experimental evolution of bacterial populations. Appl. Microbiol. Biotechnol. 77, 489–496 (2007)
18. Ghannoum, M., & O’Toole, G. A. (eds) in Microbial Biofilm. (ASM Press, 2004).
19. PIlyugin, S. S., & Waltman, P. The simple chemostat with wall growth. S.I.A.M. J. Appl. Math. 5, 1552-1572, (1999).
20. Ghannoum, M., & O’Toole, G. A. (eds) in Microbial Biofilm. (ASM Press, 2004).
21. Smith, H. L. & Waltman, P. in The theory of the Chemostat (Cambridge University Press, 1995).
22. Mohan, R. et al. A multiplexed microfluidic platform for rapid antibiotic susceptibility testing. Biosens. Bioelectron. 49, 118–125 (2013).
23. Clark, D. S. & Blanch, H. W. Biochemical Engineering (2nd edition, CRC Press, 1996).
24. LeClerc, J. E., Li, B., Payne, W. L., & Cebula, T. High mutation frequencies among Escherichia coli and Salmonella pathogens. Science 274, 1208-1211, (1996).
25. Kaeberlein, T., Lewis, K., & Epstein, S. S. Uncultivable microorganism in a pure culture in a simulated natural environment. Science 296, 1127-1129, 2002.
26. Smith, H. L. Bacteria competition in serial transfer culture Mathematical Bioscience 229, 149-159, (2011).