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Advancements in Instrumentation
Development of an Automatic Phase-Contrast Microscopic System Capable of Determining the Microbial Density and Distribution inside an Immobilized Carrier Yong-Woo LEE,* Jong-Kwang LEE,* Young-Kun MIN,** Hiro-o HAMAGUCHI,** and Jinwook CHUNG*† *R&D Center, Samsung Engineering Co. Ltd., 415-10 Woncheon-Dong, Youngtong-Gu, Suwon, Gyeonggi-Do 443-823, Korea **Department of Chemistry, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo 113–8656, Japan
The cell-immobilization technique is used for the physical and chemical fixation of cells onto a solid support in order to increase their stability and capacity of substrate uptake. However, there is no apparatus to observe the microbial community’s structure inside cell immobilizing polymeric carrier. In order to satisfy the demand of monitoring for the microbial distribution inside the carrier, we developed an automatic phase contrast microscopic monitoring system capable of determining the microbial density and distribution inside a cell-entrapped carrier automatically. (Received December 26, 2007; Accepted February 1, 2008; Published April 10, 2008)
Introduction Industrial wastewater reuse can provide alternative sources of water and reduce the pollution load to the water environment by less discharged wastewater. It has a big potential to bring about environmental, economic and financial benefits. Especially, the electronics industry requires a high quality and large quantity of water, such as ultrapure water for washing circuit boards and other electronic compounds. The reuse of electronic wastewater is seriously complicated. Low-concentrated electronic wastewater contains isopropyl alcohol (IPA), dimethyl sulfoxide (DMSO), tetramethylammonium hydroxide (TMAH), acetone, and acetic acid. Among these compounds, TMAH, which is utilized in etching the surface of silicone chips in the semiconductor manufacturing process, is toxic to humans and resistant to biodegradation. Its concentration in organic wastewater is relatively high. Detoxification technology, such as the biological removal of TMAH from wastewater to be re-circulated in plants again, has become a very important issue in order to reuse low concentrated electronic wastewater. Cell-entrapping immobilization is an ideal technology to improve reaction rates by a specific microorganism separated from the reaction mixture, and to minimize the inhibition of other toxic chemicals.1–3 Research on immobilizing technologies of microorganisms has been directed towards the immobilization by acrylamide,4 polyvinyl alcohol,5 alginate1 and polyethylene glycol (PEG).6 In a previous study,7 we developed a new method for the immobilization of enriched microorganisms capable of degrading TMAH by PEG prepolymer to reuse lowconcentrated electronic wastewater, according to a modified method of Sumino et al.6 The developed carrier is a so-called To whom correspondence should be addressed. E-mail:
[email protected] †
selected microbe immobilized carrier (SMIC), which is a soft swollen polymeric gel type. Generally, information about the microbial community in a biological wastewater treatment process provides a way to evaluate the present status of microbial activity, and can be applied to forecast the performance and stability of the system. Therefore, an investigation of the microbial density and distribution as an indicator is significantly important to operate and manage a biological treatment system. Until now, the microbial activity and microbial distribution of a microbial community inside a cell-immobilized carrier were observed by applying various molecular biotechnologies, such as 4′-6diamidino-2-phenylindole (DAPI)8 and fluorescent in-situ hybridization (FISH),9 as well as electron microscopic observations, such a scanning electron microscopy (SEM),10 and transmission electron microscopy (TEM).10 These approaches can only provide the local information about the biomass inside a carrier, and have some drawbacks, such as an unfeasibility in analyzing the microbial density and distribution in the whole carrier. Therefore, in order to apply cell-immobilization technology to a practical process, monitoring of the microbial density and distribution inside the carrier is required. The purpose of this study is to develop a new system to determine the microbial density and distribution inside SMIC. This system is based on phase-contrast microscopy, which can clearly show microbial shape without staining (Fig. 1 and Table 1). A reconstructed whole cross-sectional image to view the microbial distribution and density is obtained within 2 s by an auto-positioning stage and newly developed software.
Experimental For this experiment, round-bottomed cylindrical SMICs (4 mmD × 4 mm-H) were prepared by immobilizing Mycobacterium sp.
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Fig. 1
Table 1
Schematic diagram of the automatic phase-contrast microscopic system.
Detail specification of the automatic phase-contrast microscopic system
Vibration microtome
Probe/lenses
Camera Software
Automated stand (for auto-focusing) Automated X-Y stage
Light source
Cutting frequency Amplitude Cutting speed Speed of return travel Max. specimen size Method Objective lens Dimension of view-region Condenser lens CCD sensor Stage control Image tiling Auto-focusing Height control Travel Stage dimension Resolution Straightness (H/V) Repeatability Light source Power
(average size: 0.4 × 5 μm) in PEG gel. The determining the microbial distribution and density inside SMIC consisted of six parts, including slicing of the carrier, sample preparation, scanning of a unit image, data calculation, image processing, and resultant image display, as shown in Fig. 2. The first step is cutting into 15 μm slices by a vibration microtome (HM 650V, MICROM, Germany) from a SMIC stuck on a sample plate immersed in a water container. In order to successfully prepare a smooth surface of a slice, it is essential to properly adjust the knife attack angle, passing speed and vibrating frequency. A slice was put on a slide glass and covered with a cover glass, and then sealed by sealants to prevent from drying. The sliced sample was loaded onto the X, Y, and Z-axis controllable stage of a microscope.
30 – 100 Hz 0.1 – 1.2 mm 0 – 5 mm/s 5 mm/s 45 × 45 mm Transparent, phase-contrast, Zernike method Phase-contrast, 40× PH-DM lens, NA0.65, w.d. = 1 mm Horizontal 115 μm × vertical 88 μm Abbe condensers, NA1.25 CCD, 1/3”, 1.3M pixel USB camera For X-, Y-, and Z-control Image acquisition and tiling of shrunk image Auto-focusing at 3 points of the sample Controlled by 5 phase stepping motor (0.3 μm step) ±10 mm 155 mm × 155 mm 0.001 mm 0.008 mm ±0.002 mm High brightness LED DC 12 V
Before starting image acquisition, in order to keep the same distance between the objective lens and the whole sample surface, auto-focusing was conducted at 3 points of the sample, and the data were fed back to the stage controller. A total of 2000 unit images (40 × 50, a unit image size (125 × 100 μm) with 40× PH-DM lens) were sequentially accumulated within 180 s by using phase-contrast microscopy. After shrinking the sizes of 2000 real images, we assembled them into a complete whole image, as shown in Figs. 3a and 3b. Each unit image was sequentially converted to a numerical value representing the microbial concentration as an occupied area by image analysis. The morphological recognition standard of the microbial density in each pixel is regulated by the brightness as a parameter function in the software. Based on the
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Fig. 2 Procedure for determining the microbial density and distribution in carriers using the automatic phase-contrast microscopic system.
microbial density determined at the calculation step, the computed image considering microbial density can be obtained (Figs. 3c and 3d). Finally, an image display can show the actual and computed image completed through an image-processing step on the screen. When a unit pixel of the reconstructed images is clicked, the pop-up window shows each detailed and magnified image (unit pixel). A bench-scale continuous-flow reactor was filled with a cellimmobilizing carrier with a packing rate of 75% (v/v), and operated to investigate the biodegradation of TMAH using lowconcentrated electronic wastewater during 70 days. The carriers were circulated by air blown up from the bottom of air stone placed at the middle of the reactor. The carrier sample after operating for 70 days and control without operation were prepared.
Results and Discussion Figure 3 shows the microbial density and the distribution inside the carrier using the newly developed automatic phase-contrast microscopic system. Figures 3a and 3b demonstrate a reconstructed one whole image by 2000 size-adjusted 40× images of a cross-section of the carrier immediately after polymerization, and after operating for 70 days, respectively. Figures 3c and 3d show reconstructed images representing the microbial density inside two carriers after the above-mentioned operation. Figures 3e and 3f show the original images of an inner and an outer pixel in the carrier operated for 70 days, indicating that the microbial density of the outer part was relatively higher than that of the inner part. The microbial density inside the cell-entrapped carrier during the early stage of operation is a significant constant. As the operation begins, transport of the substrate in the bulk liquid due to advection, dispersion and diffusion occurs inside the saturated carrier along with biological transformation. Among the microorganisms inside the carrier, microorganisms located near to the carrier’s surface can easily contact with a relatively higher level of the substrate, but that located at the carrier’s center is subjected to an inhibition of growth due to the substrate limitation. It is supposed that the substrate limitation and contact spot (easiness) of the carrier has a significant effect on the microbial
Fig. 3 Image analysis in carriers using the automatic phase-contrast microscopic system; black spots indicate microorganisms at (e) and (f) images.
proliferation and the density. Figure 4 shows the distribution of microorganisms in the cross-section of the carriers quantitatively. The communities of
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ANALYTICAL SCIENCES APRIL 2008, VOL. 24 As shown in Figs. 3 and 4, the newly developed automatic phase-contrast microscopic system was proven to be an effective tool for determining the microbial density and distribution in the carriers. In the future, if Raman microspectroscopy is adopted, this system can be ever more improved so as to be able to not only determine the distribution of microbial communities, but also to estimate the existence and distribution of specific species in a complex microorganism system.
Acknowledgements This research was supported by a grant (I2WATERTECH 04-06) from I2WaterTech of Eco-STAR Project funded by Ministry of Environment, Korea.
Fig. 4 Microbial distribution in the cross section of the carrier. a, 0 day; G, 70 days.
microorganisms immediately after synthesis were in the range of 0.58 – 0.85% (average 0.74%), and their communities were shown to be evenly distributed over the whole cross-sectional area of the carriers (Fig. 3c). However, after operating for 70 days, the microorganism communities inside of carriers were shown to be in the range of 0.45 – 0.95% (average 0.57%), meaning that 0.17% of the microbial communities were reduced compared to the immediately polymerized carriers. In contrast to inside the carriers, it was observed in outside carriers that the microbial communities on the surface of the carriers grew toward the inner carriers within up to 250 μm, as shown in Fig. 4. This means that the microorganisms significantly proliferated on the surface of the carriers when they were contacted with electronic wastewater containing low level of TMAH in the bench-scale continuous-flow reactor during 70 days of operation. The relatively higher permeability of the substrate and oxygen on the surface of the carriers might induce microorganisms to grow more towards the outer carrier.
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