PLoS One. 2016 Nov 3;11(11):e0165839. doi: 10.1371/journal.pone.0165839.

Fabrication of Bacteria Environment Cubes with Dry Lift-Off Fabrication Process for Enhanced Nitrification

Po-Jung Huang1, S. A. P. L. Samarasinghe2, Yiru Shao3, Michael Pishk4, Kung-Hui Chu3, Jun Kameoka1,2

1Department of Materials Science and Engineering, Texas A&M University, College Station, Texas, United States of America

2Department of Electrical & Computer Engineering, Texas A&M University, College Station, Texas, United States of America,

3Zachry Department of Civil Engineering, Texas A&M University, College Station, Texas, United States of America,

4Department of Biomedical Engineering, Texas A&M University, College Station, Texas, United States of America

Correspondence should be addressed to Jun Kameoka, Department of Electrical Engineering, Texas A&M University, College Station, USA, E.mail:




Waste water management process includes non-biological and biological process. Non-biological process is simply for removing solid debris, and biological treatment is to digest organic chemicals. In biological process, ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) play a critical role to metabolize and convert toxic waste to non-toxic salt. AOB convert ammonia to nitrite and NOB metabolizes nitrite to nitrate. Because AOB and NOB are sensitive to environmental factors such as pH and temperature, the nitrification process is currently inefficient and is difficult to be used widely. Moreover, the uneven distribution and concentration of AOB, and NOB lead to incomplete nitrification. Herein, we develop a dry lift-off fabrication process to co-culture AOB, and NOB by encapsulating close range to reduce the incomplete nitrification process.

Dry lift-off process can encapsulate multiple types of bacteria three dimensionally in Poly(ethylene) glycol diacrylate (PEGDA). Figure 1 shows the schematic diagram about the dry lift-off fabrication process. Figure 1a presents DLO process, which indicates three-step fabrication. The layers of PDMS DLO masks are fabricated by the standard soft lithographic process. A PDMS solution is poured on the first- (300 μm thickness), second-(200 μm thickness), and third-layer (300 μm thickness) DLO mask molds. The masks are peeled from master molds after cross-linked under proper condition. The first layer is placed and fixed on silicon wafer and then dispersed 30 wt% PEGDA contained AOB in each square and the square size is 1mm x 1mm. After UV exposing for 30 seconds, the second-layer of DLO mask is placed above first layer and PEGDA containing NOB is dispersed into four small squares to localize NOB after alignment. Figure 1b shows the microscopic image of the PEGDA cubes after the second-layer patterning. Four small PEGDA squares are patterned precisely above the first layer of the PEGDA. The third DLO mask, the top layer, is aligned and attached to the surface of the second PEGDA layer. A PEGDA solution mixed with AOB is then dispensed and solidified after UV curing. As the final step, the third layer of DLO mask can be peeled off and the PEGDA cubes detached from silicon surface. Figure 1c presents the microscopic image of PEGDA cubes in culture media. Figure 1d demonstrates the schematic diagram for the final 3D PEGDA cubes containing AOB and NOB at specific locations. Ammonia can be digested by AOB located surface of PEGDA cubes and converted to nitrite that can diffuse into NOB portion. NOB can oxidizes nitrite to nitrate. The conversion efficiency by encapsulated bacteria is 30-fold better than that by suspended bacteria.



Figure 1 Schematic diagram of the 3D dry lift-off (DLO) process and 3D structure of PEGDA cubes containing Ammonia Oxidation Bacteria (AOB) and Nitrite Oxidizing Bacteria (NOB) (a) DLO process contains 3 steps process to make PEGDA cubes. A PEGDA solution mixed with AOB disperses on 1st DLO mask and cross-linked by 30 seconds of 365nm UV exposure. The 2nd layer of DLO mask attaches on 1st layer and makes alignment. A PEGDA solution mixed with NOB disperses on 2nd DLO mask and cross-linked. The 3rd layer of DLO mask attaches on 1st layer and aligns the pattern on 1st layer after peeling off 2nd layer. A PEGDA solution mixed with AOB disperses on 3rd DLO mask and cross-linked. (b) Fluorescent image of PEGDA cubes with localized AOB and NOB (c) microscopic image for PEGDA cubes through DLO process (Scale bar: 500 µm) (d) Schematic diagram of function of AOB and NOB in PEGDA particles.