Daniel Johnson. Department of Mechanical Engineering,. University of Michigan,. Ann Arbor, MI 48109. Roscoe Warner. Department of Pathology,. University of ...
Surface Roughness and Material Removal Rate in Machining Using Microorganisms Daniel Johnson Department of Mechanical Engineering, University of Michigan, Ann Arbor, MI 48109
Roscoe Warner Department of Pathology, University of Michigan, Ann Arbor, MI 48109
Albert J. Shih1 Department of Mechanical Engineering, University of Michigan, Ann Arbor, MI 48109
The use of bacteria as a tool for machining, also known as biomachining, is a novel material removal process. This study characterizes the surface changes and relates the material removal rate to quantified bacterial concentrations resulting from machining of pure polycrystalline Cu using the bacterium Acidithiobacillus ferrooxidans. Cu blocks, polished to four levels of surface roughness, were utilized to examine the surface effects of bacterial machining. The mass change in Cu foil was measured to find the material removal rate. The most probable number method, a statistical enumeration technique, was applied to estimate bacterial concentrations. Scanning electron microscope (SEM) micrographs demonstrate that bacterial machining is anisotropic, and roughness measurements of the polycrystalline Cu samples showed a deterioration of Ra values of 1.5– 2.5 m. Finally, suggestions for future work are presented that could potentially ameliorate current process problems. 关DOI: 10.1115/1.2401629兴
1
Introduction
The use of bacteria as the tool to remove metal from a workpiece, also known as biomachining, is a relatively new manufacturing technology with potential applications in the construction of micro-scale features. Microbiologists have discovered several bacterial species with applications in mining and recovery of radioactive waste, known as bioleaching and bioremediation, respectively 关1,2兴. These microorganisms are useful in these applications because the bacteria can dissolve 共oxidize兲 and/or deposit 共reduce兲 metals as part of their energy production cycle 关3–5兴. Furthermore, some of these microorganisms that dissolve materials can consume Fe or Cu, key industrial materials. Results have demonstrated definitively that this effect, known as biomachining, is different from traditional chemical etching or electrochemical corrosion. Acidithiobacillus ferrooxidans, formerly Thiobacillus ferrooxidans, was the species used in the preliminary biomachining work by Uno et al. 关6–9兴, Zhang and Li 关10,11兴, and Ting et al. 关12兴. After reviewing similar species, A. ferrooxidans was also chosen in this study for its documented ability to biomachine Cu. Two goals of this study are to characterize the surface roughness 1 Corresponding author. Contributed by the Manufacturing Engineering Division of ASME for publication in the JOURNAL OF MANUFACTURING SCIENCE AND ENGINEERING. Manuscript received December 8, 2005; final manuscript received August 27, 2006. Review conducted by Shreyes N. Melkote.
and to quantify the material removal rate and bacterial concentration in biomachining. Both topics are important to achieve the precision biomachining in the future. For manufacturing engineers, the concept of bacterial machining opens a new paradigm in micro-machining. Bacteria, measuring on the order of micrometers, can remove materials at the nm level. They exert negligible forces on the workpiece and produce no thermal damage. All of these characteristics suggest that bacteria could serve as a desirable tool in the machining of microscale features. Bacteria used in biomachining are commercially available and can be cultured continuously, and thus are selfrenewing tools. The process of material removal is environmentally benign and can be conducted with little energy input. Unlike other micromanufacturing processes that use a large amount of external energy to remove a small amount of material 共such as micro-EDM or laser-based processes兲, biomachining only requires enough energy to maintain environmental conditions for the bacteria. One potential application of biomachining is to replace the toxic FeCl3 currently employed for chemical etching of Cu in semiconductor manufacturing 关13兴. In this manuscript, the experimental setup, including bacteria and workpiece preparation, is first presented. The MPN technique for estimating bacterial concentrations is then explained. Finally, the surface roughness and visible surface characteristics of the workpiece before and after the biomachining experiments are compared.
2
Experiment Setup
Figure 1 shows the flow chart of bacterial machining procedures. The preparation of bacterial cultures and the Cu workpiece are discussed in the following two sections. 2.1 Culturing the Bacteria. A. ferrooxidans was acquired from the American Type Culture Collection 共ATCC兲 as a bacterial broth 共ATCC No. 21834兲. An environment with low pH and a metal to serve as the source of electrons in the respiration process is required for A. ferrooxidans to grow. About 2 ml of ATCC A. ferrooxidans broth was put onto the solid media 关14兴 and incubated at 35° C until bacterial growth became apparent as a bright yellow, semi-transparent haze 共rather than distinct colonies兲 on the surface of the media. Streaks were taken off the surface growth of the solid media plates using a sterile inoculating loop and mixed into 15 mL conical tubes of liquid 9K media, using the standard media recipe suggested by VKM 共the Russian Collection of Microorganisms兲. The bacteria were cultured at 35° C in the 15 mL tubes of the 9K media for 1 or 2 days, until a vivid color change in the media became apparent. Continuous cultures of A. ferrooxidans were subcultured by taking several mL from tubes and mixing them into 250 mL Pyrex flasks, each filled with 150 mL of sterile 9K media. Each flask was prepared with a vented top of tightly packed medical gauze and autoclaved, as shown in Fig. 1. All operations involving interaction with the bacteria and/or media were conducted under a bacterial hood with positive flow highefficiency particulate air 共HEPA兲 filter to avoid contamination. The inoculated flasks were then incubated at 35° C and shaken at 120 cycles per minute. Using this culturing protocol, populations grew for 4 to 7 days, after which a sample was aseptically removed and used to repeat the culturing. Each culture used in biomachining experiments was also incubated in this manner for 48 h prior to use. 2.2 Surface Study Procedures. To characterize surface finish effects after bacterial machining, 99.99% pure Alloy 101 oxygenfree electronic-grade Cu blocks, 12⫻ 12⫻ 12 mm3 in size, were used. The polycrystalline Cu blocks were polished using 320, 600, and 1200 ANSI grit SiC abrasive disk wheels as well as a diamond slurry 共600 nm particle size兲. Each was used in sequence until the desired initial finish was reached. The polished surfaces were examined using SEM both before and after each experiment.
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Fig. 1 Flow chart of biomachining procedure
A profilometer with a measurement length of 5.7 mm, 0.8 mm cutoff length, and Gaussian filter was used for roughness measurements. The arithmetic average roughness, Ra, was found to be representative and adequate to quantify the change of surface
roughness. For each experiment, the Cu blocks were mounted in plastic frames, all sanitized by soaking in 100% ethyl alcohol and then air dried under the bacterial hood, such that the polished surface was
Fig. 2 The change of Ra for Cu samples in 9 K media with and without the bacteria
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an oil-emersion optical microscope 共1000⫻ magnification兲 to observe and count the bacteria was attempted, but failed to yield accurate and reliable results. At high magnification, optical microscopy has limited resolution, which makes it difficult to distinguish individual cells, which are only 1 – 2 m long and 0.5 m wide. A. ferrooxidans appears in pairs or triples, reproducing in a line and then splitting off from one another. A more definitive statistical counting technique, the most probable number 共MPN兲 method 关15兴, was used instead of optical microscopy to determine bacterial concentrations. Previous work has shown that the MPN method is able to measure the bacterial concentration of A. ferrooxidans 关16兴 in a clear and definitive manner. For each experiment conducted, bacterial concentrations are recorded before and after each experimental run. The MPN technique involves two main steps: creating an initial series of dilutions of the original sample, and then using those dilutions to inoculate a collection of test tubes that are incubated to watch for growth. For the biomachining tests, the dilution series was carried to the 10−11 dilution. Each dilution from 10−4 to 10−11 was used to inoculate a corresponding set of three tubes, each filled with 9 mL of sterile media, resulting in a 3 by 8 array, or a total of 24 test tubes, as shown in Fig. 2. Each three-tube column is given a score corresponding to the number of tubes that show growth, ranging from +0 for no growth in any series to +3 for all three tubes showing growth. For the statistical analysis, the first column where no growth 共+0兲 occurs is noted. The sequence of scores of this column and the two preceding it, such as +3 +2 +0, is used to find a numerical value for the bacterial concentration, which comes from tabulated MPN tables 关15兴.
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Fig. 3 Diamond slurry polished surface after 48 h in sterile media: „a… 100Ã magnification and „b… 500Ã magnification close-up view showing the grain boundary erosion
upright and uncovered. These assemblies were placed into sealed, sterile cylindrical jars filled with approximately 150 mL of either sterile 9 K media or bacterial broth. The sealed jars were then incubated at 35° C for each specified time period, without agitation. At the conclusion of the incubation time, the Cu blocks were removed, rinsed with de-ionized water, and air dried. Roughness measurements and SEM micrographs were then taken again, with the same parameters as the preexperiment measurements. 2.3 Material Removal Rate Procedures. A 99.9% Alloy 110 electronic-grade Cu foil, 0.025 mm in thickness, was selected for the material removal rate study. The polycrystalline Cu foil was cut into 4 ⫻ 6 mm2 samples. The foil pieces were sanitized in the same manner as the Cu blocks, and then submerged in sterile media or bacterial broth under identical conditions to the copper blocks, then removed, rinsed, and air dried. The mass before and after each experiment was measured, and the change in mass was converted to the volumetric, and then dimensional, material removal rate using standard density values available in the literature.
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MPN Method for Bacteria Counting
Quantifying the bacterial concentration is an important step for consistency and quality control of the bacterial machining. Using Journal of Manufacturing Science and Engineering
Material Removal Rate
The change in mass of Cu alloy 110 foil in bacterial solutions of A. ferrooxidans is used to quantify the material removal rate. For a negative control, the Cu foil was placed in the media without bacteria for 24 and 48 h. Insignificant changes in mass, typically less than 0.004 g, were observed in the sample presumably due to the acidic nature of the media. At a bacterial concentration of 4.3⫻ 107 organisms/ mL, average material removal rates of 32 and 17 m / h were achieved in the 24 and 48 h bacterial machining tests, respectively. This demonstrates that the material removal rate is slow and appears to decline over time. The depletion of available oxygen might have slowed down the material removal rate because the bacteria are aerobic, and oxygen is required to be present for the metabolic processes involved in consumption of the workpiece to continue. Aside from the large mass differences exhibited by the foil pieces exposed to the bacteria, visible physical differences were also observed on the surface of bacterially machined workpiece. The characterization of surfaces is discussed in the following section.
5 Surface Roughness and SEM Micrographs of Bacteria Machined Surfaces The changes of arithmetic surface roughness, Ra, of the Cu blocks after submerging for 24 and 48 h in the media with and without bacteria are shown in Fig. 2. The initial Ra values for the 320, 600, and 1200 grit SiC and 600 nm diamond slurry polished surfaces fell into the ranges of greater than 0.4 µm, 0.25–0.4 µm, 0.1–0.25 µm, and below 0.1 µm, respectively. For the roughest surfaces, having an initial Ra larger than 0.4 m, submersion in sterile media slightly improves the surface roughness. Fine surfaces with initial Ra below 0.25 m, however, saw deterioration in their surface roughness. The change in surface roughness was particularly high 共0.43 m for the 48 h test兲 for both fine diamond slurry polished surfaces. Figure 3 shows SEM micrographs of the diamond polished surface after 48 h in sterile media. The lower magnification micrograph in Fig. 3共a兲 reveals the inconsistent nature of the surface. The brightness difference seen in the microFEBRUARY 2007, Vol. 129 / 225
Fig. 4 SEM micrographs of polished Cu surfaces before and after 48 h of bacteria machining; sample polished by „a… 320 grit SiC and „b… 600 nm diamond
graphs is due to surface roughness effects. The media unevenly erodes the surface, preferentially attacking grain boundaries. Grooves gouged in the polishing process are still recognizable. Minimal material removal was observed. The close-up view in Fig. 3共b兲 illustrates the eroded grain boundary. The media, with pH of 2.5–2.6, has the same effect on the polished surface as a mild chemical etchant. The exposure of the less organized, and thus prone to corrosion, grain boundaries contributed to the large increase in surface roughness values. The effects of 24 and 48 h of bacterial machining on Cu surface roughness are shown in the lower graph in Fig. 2. The surface roughness deteriorated in all bacteria machining tests. Initial surface roughness did not have a significant effect on the change in Ra. The range of change in Ra varied from 1.8 to 2.6 m for the 24 h test and 1.7 to 2.4 m for the 48 h test. A trend of larger change in Ra corresponding to a higher initial Ra is observed for the 24 h test. Submerging samples in the broth with bacteria longer 共48 h兲 slightly increased the surface roughness. SEM micrographs of two surfaces with different initial Ra before and after 48 h of bacteria machining are shown in Fig. 4. On the bacterially machined surfaces, grooves from polishing disappeared and grain boundaries and annealing twins are visible. The bacterial machining, however, is not uniform on polycrystalline Cu. Rather than preferentially attacking grain boundaries like the sterile media, seemingly random changes in topography appeared all over, not only at grain boundary regions. The surfaces became rougher due to such uneven material removal on the surface. Since such unevenness could be a result of preferential attacking of certain crystal orientations, future work could involve tests with single crystal or amorphous Cu. Under high magnification, traces of bacteria can be observed on the machined surface, as shown in Fig. 5. The rod-shaped black marks visible in the micrograph fit the scale and appearance of A. ferrooxidans. These traces, which are unlikely to be the bacteria due to the destructive effects of SEM, are likely places where 226 / Vol. 129, FEBRUARY 2007
individual bacteria once adhered to the copper surface. Before the 24 and 48 h machining tests, MPN tests showed concentrations of 2.4⫻ 107 and 4.3⫻ 107 organisms/ mL, respectively. The bacterial concentrations of four samples after the 24 and 48 h biomachining tests changed to 2.4– 4.3⫻ 107 and 2.4– 9.3⫻ 107 organisms/ mL, respectively. The populations showed no discernable pattern of growth or decline with exposure to the Cu workpiece.
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Concluding Remarks
This study presented the material removal rate and surface roughness for machining of pure Cu using the A. ferrooxidans
Fig. 5 Traces of bacteria on 600 grit SiC polished surface after 48 h of bacteria machining
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microorganism. The MPN method was applied to quantify the bacterial concentrations and correlate to experimental results. Bacteria have a slow material removal rate, which can be applied for precision machining applications. Independent of the initial workpiece surface roughness, the surface roughness deteriorates significantly to about 1.7 to 2.4 m Ra after machined for approximately 24 and 48 h. One of the potential applications of bacterial machining is to replace chemical etching of Cu in semiconductor and MEMS manufacturing. Future research will focus on bacterial machining of amorphous thin-film and single crystal Cu, the life-cycle design of a machining system using bacteria as renewable tools, and a system to maintain the bacteria concentration in machining.
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Acknowledgment We acknowledge the technical and laboratory assistance from Professor Steven Skerlos and Professor Kent Johnson of University of Michigan and Professor Ronald Scattergood of North Carolina State University.
References 关1兴 Liu, H., Chen, B., Lan, Y., and Cheng, Y., 2004, “Biosorption of Zn共II兲 and Cu共II兲 by the Indigenous Thiobacillus thiooxidans,” Chem. Eng. J., 97, pp. 195–201. 关2兴 Lovley, D. R., 2003, “Cleaning Up With Genomics: Applying Molecular Biology to Bioremediation,” Nat. Rev. Microbiol., 1, pp. 35–44. 关3兴 Lovley, D. R., Phillips, E. J. P., Gorby, Y. A., and Landa, E. R., 1991, “Microbial Reduction of Uranium,” Nature 共London兲, 350, pp. 413–416. 关4兴 Liu, C., Gorby, Y., Zachara, J., Fredrickson, J., and Brown, C., 2002, “Reduction Kinetics of Fe共III兲, Co共III兲, U共VI兲, Cr共VI兲, and Tc共VII兲 in Cultures of
Journal of Manufacturing Science and Engineering
关10兴 关11兴 关12兴 关13兴 关14兴 关15兴 关16兴
Dissimilatory Metal-Reducing Bacteria,” Biotechnol. Bioeng., 80共6兲, pp. 637– 649. Kurosaki, Y., Matsui, M., Nakamura, Y., Murai, K., and Kimura, T., 2003, “Material Processing Using Microorganisms 共An Investigation of Microbial Action on Metals兲,” JSME Int. J., Ser. C, 46, pp. 322–330. Uno, Y., Kaneeda, T., and Yokomizo, S., 1993, “Fundamental Study on Biomachining 共Machining of Metals by Thiobacillus Ferrooxidans兲,” Trans. Jpn. Soc. Mech. Eng., Ser. C, 59, pp. 3199–3204. Uno, Y., Kaneeda, T., and Yokomizo, S., 1996, “Fundamental Study on Biomachining 共Machining of Metals by Thiobacillus Ferrooxidans兲,” JSME Int. J., Ser. C, 39, pp. 837–842. Uno, Y., Kaneeda, T., Yokomizo, S., and Yoshimura, T., 1996, “Fundamental Study on Electric Field Assisted Biomachining,” J. Soc. Precis. Eng., 62, pp. 540–543. Kumada, M., Kawakado, T., Kobuchi, S., Uno, Y., Maeda, S., and Miyuki, H., 2001, “Investigations of Fine Biomachining of Metals by Using MicrobiallyInfluenced Corrosion Differences Between Steel and Copper in Metal Biomachining by Using Thiobacillus Ferrooxidans,” Zairyo? to Kankyo?, 50, pp. 411–417. Zhang, D., and Li, Y., 1998, “Possibility of Biological Micromachining Used for Metal Removal,” Sci. China, Ser. C: Life Sci., 41, pp. 151–156. Zhang, D., and Li, Y., 1999, “Studies on Kinetics and Thermodynamics of Biomachining Pure Copper,” Sci. China, Ser. C: Life Sci., 42, pp. 57–62. Ting, Y. P., Kumar, A. S., Rahman, M., and Chia, B. K., 2000, “Innovative Use of Thiobacillus ferrooxidans for the Biological Machining of Metals,” Acta Biotechnol., 20共2兲, pp. 87–96. Williams, K., Gupta, K., and Wasilik, M., 2003, “Etch Rates for Micromachining Processing-Part II,” J. Microelectromech. Syst., 12共6兲, pp. 761–778. Johnson, D. B., Macvicar, J. H. M., and Rolfe, S., 1987, “A New Solid Medium for the Isolation and Enumeration of Thiobacillus Ferrooxidans and Acidophilic Heterotrophic Bacteria,” J. Microbiol. Methods, 7共1兲, pp. 9–18. Oblinger, J. L., and Koburger, J. A., 1975, “Understanding and Teaching the Most Probable Number Technique,” J. Milk Food Technol., 38, pp. 540–545. Southam, G., and Beveridge, T. J., 1992, “Enumeration of Thiobacilli Within pH-Neutral and Acidic Mine Tailings and Their Role in the Development of Secondary Mineral Soil,” Appl. Environ. Microbiol., 58, pp. 1904–1912.
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