Microorganisms Used and Process Principles

Every organism can be classified based on how it obtains its energy to run all metabolic processes for growth. Those organisms that obtain carbon from inorganic compounds (including carbon dioxide) are known as lithotrophs. On the other hand, organotrophs obtain all the carbon they use from organic compounds, including those from other organisms. Each of these groups can be further subdivided based on how included organisms obtain energy. The chemolithotrophs typically process inorganic matter. The chemoorganotrophs process organic matter for their growth. Many animals (including humans) and bacteria are classified as chemoorganotrophs. The organisms that use light as an energy source are called as phototrophic organisms. Plants are the most common examples. A summary of the classifications, species, is displayed in Fig. 1.

Organisms fitting into the category of chemolithotrophs have been the subject of intensive research lately for their unique ability to either oxidize or reduce certain inorganic compounds, especially heavy metals. Such organisms have been applied for bioremediation process. The natural reduction reactions of the Shewanella oneidensis have been used for removal of toxic chemicals from the groundwater (Viamajala et al. 2003). Species such as Geobacter metallireducens and Rhodoferax ferrireducens have shown potential application for biologically based fuel cells (Bond et al. 2002; Chaudhuri and Lovley 2003).
It is a need of time that the biological processes have to be integrated into future industrial processes. This is important to meet environmental regulations and to establish eco-friendly, energy-saving processes. Hence, such processes are characterized by an application of biocatalysts (e.g., microorganisms, enzymes) in an industrial process and the substitution of existing processes (Liang et al. 2010). It was reported that certain microorganisms can dissolve metal compounds (Wang et al. 2009). At. ferrooxidans and At. thiooxidans are the principal microorganisms involved in the above process. These bacteria can grow by obtaining energy from oxidation of ferrous iron (Fe2+) or elemental sulfur (S0) and thereby carrying metal solubilization (Ubaldini et al. 2003; Bosecker 1997). Such metal bioleaching technique has been used in biomining on industrial scale (Ewart and Hughes 1991; Rawlings and Silver 1995). Moreover, these bacteria were also used to recycle metals from various wastes (Wang et al. 2009; Zhao et al. 2008; Xin et al. 2009b; Ishigaki et al. 2005; Vestola et al. 2010). This metal-extraction property of microorganism was exploited for machining of metals. At. ferrooxidans was the species used in the preliminary biomachining work (Uno et al. 1996; Uno 2002). Uno et al. (1996; Uno 2002) applied three At. ferrooxidans strains (ATCC 13598, 13661, and 33020) for biomachining of copper and iron. They used basal 9 K medium for growth of At. ferrooxidans. The composition for medium was 3.0 g (NH4)2 SO4, 0.5 g K2 HPO4, 0.5 g MgSO4.7H2O, 0.1 g KCl, and 0.01 g Ca(NO3)2 in 1 l distilled water. The pH was adjusted to 2.5 with 1.0% sulfuric acid and sterilized by autoclave. The FeSO4 is used as energy source for growth of At. ferrooxidans (Uno et al. 1996). In another study, Hocheng et al. (2012a) used 510 medium for growth of At. ferrooxidans. The basal 510 medium composed of solutions A and B. Solution A contains the following substances per 800 ml of glass-distilled water: 0.8 g (NH4)2 SO4, 2.0 g MgSO4.7H2O, 0.4 g K2HPO4, and 5 ml of 0.22 mm filtersterilized Wolfe’s mineral solution. Wolfe’s mineral solution contains the following substances: 1.5 g nitrilotriacetic acid, 3.0 g MgSO4.7H2O, 0.5 g MnSO4.H2O, 1.0 g NaCl, 0.1 g FeSO4.7H2O, 0.1 g CoCl2.6H2O, 0.1 g CaCl2, 0.1 g ZnSO4.7H2O, 0.01 g CuSO4.5H2O, 0.01 g A1K(SO4)2.12H2O, 0.01 g H3BO3, and 0.01 g Na2MoO4.2H2O in 1 l distilled water. The pH was adjusted to 2.5 with 1.0 % (weight/volume) sulfuric acid and autoclaved for sterilization. The presterilized solution B was added to solution A. The freshly prepared solution B contained 20.0 g FeSO4.7H2O per 200 ml of glass-distilled water (Hocheng et al. 2012a). Chang et al. (2008) and Hocheng et al. (2012b) used At. thiooxidans for the biomachining of metals. They used basal 317 medium for growth of bacteria. The basal 317 medium contained the following per liter of glass-distilled water: 0.3 g (NH4)2SO4, 3.5 g K2HPO4, 0.5 g MgSO4.7H2O, and 0.25 g CaCl2. The pH was adjusted to 4.5 with sulfuric acid. 1.0 % (weight/volume) elemental sulfur was presterilized and added to 317 medium for growth and maintenance of At. thiooxidans. Shikata et al. (2009) used Staphylococcus spp. for biomachining process. Staphylococcus spp. will grow on any media. They are salt tolerant, as most organisms are killed by high NaCl. “Salt agar” is 7–10 % salt containing broth and agar or salt cooked meat broth. Shikata et al. (2009) revealed the possibility of using bacteria to drill metallic surfaces (Shikata et al. 2009). Many species of bacteria are reported to be involved in the corrosion of copper. They used Staphylococcus sp. for biomachining experiments. Staphylococcus sp. is a facultative anaerobic bacterium. They isolated it from corroded copper piping installed in groundwater environment in Japan. An experiment involved exposure of copper coupons (25 mm x 15 mm x 3 mm) to a culture of Staphylococcus sp. for a maximum period of 7 days. The total pit area and volume on these coupons were determined using image analysis. They showed that both the biomachined area and volume increased with the duration of coupon exposure. In the drilling experiment, a copper thin film (2 μm thick) was perforated by this bacterium within a period of 7 days (Shikata et al. 2009).
There are two main principles behind the biomachining process. They are oxidation and acidolysis. At. ferrooxidans use mainly oxidation process, while At. thiooxidans use acidolysis process for metal removal. Again an oxidation process can be further divided into direct and indirect oxidation of metals (Uno et al. 1996; Rohwerder et al. 2003; Istiyanto et al. 2011).

Oxidation Process

The direct mechanism is based on an attachment of bacterial cell on metal surface (e.g., copper) (Fig. 2). At. ferrooxidans contains iron-oxidizing enzyme in the periplasmic space and inner membrane (Zhang and Li 1999; Istiyanto et al. 2011). The bacterial growth medium contains Fe2+ which is the main energy source for At. ferrooxidans (Uno et al. 1996; Rohwerder et al. 2003). This Fe2+ radical is then transported to the periplasmic space from the culture fluid. Here it loses an electron through the catalysis of iron oxidization. This electron is then transported to oxygen with an electron transport chain, through a set of mediating biochemical reactions (Istiyanto et al. 2011). The overall reaction is as follows:

This reaction generates energy. Also during this reaction Fe2+ is converted into Fe3+. This Fe3+ is then expelled from the cell. The Fe3+ is a strong oxidant. It has an ability to oxidize pure copper (Cu0) into Cu2+. Hence, a metal workpiece can be machined by Fe3+ that is produced by At. ferrooxidans:

During the machining process the Fe3+ produced by At. ferrooxidans is reduced to Fe2+. Again this Fe2+ can enter At. ferrooxidans cell and get reoxidized to Fe3+ by oxygen. Thus, a redox cycle can be formed (Uno et al. 1996; Zhang and Li 1999; Rohwerder et al. 2003; Istiyanto et al. 2011).
Recently, it is found that an indirect mechanism of bioleaching of metals also works well during metal-extraction process. Liang et al. (2010) found that copper in printed circuit boards (PCBs) can be continuously leached out by applying an indirect mechanism. During this process a cycle between Fe3+ and Fe2+ is built. In a similar way, Wang et al. (2009) divided the metal dissolution process into two continuous periods. First, the dissolution of metals such as copper is due to an attack of printed wire boards (PWBs) by Fe3+. In the first stage of attack, Fe3+ is reduced to Fe2+ and causes the increase of Fe2+ concentration in leaching solution. Then in second stage, bacteria use Fe2+ as an energy source, which leads to a rapid increase in bacterial population and the regeneration of Fe3+ upon Fe2+ oxidation. They proposed that the role of bacteria is just to oxidize Fe2+ into Fe3+. It is now known that this is applicable to biomachining process as well. The machining of copper, nickel, and aluminum was experimentally investigated using both the cells and the culture supernatant of At. ferrooxidans.
Higher metal removal rate was achieved for copper by using the culture supernatant (Hocheng et al. 2012c). This is done to find out actual mechanism behind biomachining process. It has been shown that an initial concentration of Fe2+ decreased from 0.072 to 0.0024 M after 48 h incubation. At the same time concentration of Fe3+ increased with time. Also an effect of cell number on conversion of Fe2+ into Fe3+ was studied. Optimization of process parameters for oxidation of Fe2+ is an important aspect because the produced ferric ions play key role in the biomachining process. The results indicate that all the cell concentrations used were able to transform Fe2+ into Fe3+ but the rate by which the transformation occurred was different for each cell concentration. The cell concentration of 5 x 108 for copper, 2.0 x 108 for nickel, and 1.0 x 108 cells/ml for aluminum were found optimal for oxidation of Fe2+ into Fe3+ (Fig. 3).

Acidolysis Process

At. thiooxidans use acidolysis process for metal removal. During the growth At. thiooxidans produce sulfuric acid. This acid is further used for metal dissolution (Hocheng et al. 2012b). The overall reaction is as follows:

where M is a metal.

Material Removal Rate

The key criterion to characterize any industrial machining process is metal removal rate. Uno et al. (1996) determined an amount of metal removed during machining process. They also studied the effects of the initial conditions on metal removal. They found that the removed depth was approximately proportional to machining time. For Cu, the material removal rate in depth was about 20 μm/h and for Fe was 14 μm/h. The experimental conditions were a temperature of 28o C and a shaking speed of 160 cycles per minute. They also run a control in parallel by using the 9 K medium. They showed that the bacteria are responsible for the material removal effect despite the corrosive effect of medium (Fig. 4).

They incubated one strain (ATCC 13598) at various temperatures and observed the mean material removal rate. The peak material removal rate for Cu (~23 μm/h) is near 30o C, while for Fe, the max material removal rate (~20 μm/h) occurs near 40o C (Fig. 5).
The temperature proves to be a relatively key variable in the efficiency of biomachining. There is a need to control temperature to keep the material removal rate near its theoretical maximum. These authors also studied the effect of electric field on biomachining rate. They reported that the depth of grooves generated on an anodic workpiece was about twice than in a normal biomachining. Hence, an applied electric field has pronounced effect on biomachining (Uno et al. 1996). If positive applied voltages are used, the material removal rate is accelerated over baseline (Fig. 6). For iron, if the voltage applied is negative (0.5 V in this case), the material removal rate is actually negative, indicating the material is deposited. Thus, an applied voltage can have an accelerating effect on the biomachining rate. They found that there is a need of further research to determine if there is a maximum voltage which yields a benefit to the process.

Zhang and Li (1998) showed that machining of pure iron, pure copper, and constantan was possible by using At. ferrooxidans. They biomachined a microgear and grooves on pure copper workpiece (Fig. 7).
They found that the depth of the groove biomachined was directly dependent on the machining time. In addition to this, Zhang and Li (1998) explained the Fe oxidizing and reducing reactions involved. They also speculated about what parts of the cell (periplasmic space, cytochromes, etc.) are used in the reactions. They found that the biological recreation of Fe3+ has a major role in the biomachining process. Zhang and Li (1999) also analyzed the kinetics and thermodynamics of biomachining of pure copper. They studied the kinetic effect of bacteria on the ion cycle between Fe2+ and Fe3+. Also they studied the kinetics of each sub-reaction and how experimental concentrations affected each of these reaction rates. The first reaction relates the consumption of Fe3+ to the mass change of a Cu sample. Equilibrium equations of generating and consuming Fe3+ show that these quantities are directly proportional to the Fe2+ and Fe3+ concentrations, respectively. They revealed the thermodynamic effect of hydrolysis on the changes of pH in biomachining process. In their study Zhang and Li (1999) found that biomachining rate depends directly on bacteria concentration. Also the necessary conditions for maintaining the stable equilibrium of the biomachining system were the supply of H+ and the removal of Cu2+. Their study does not show the presence of any maximum bacterial concentration at which biomachining can occur. The bacterial count cannot increase indefinitely. Thus, their study does not explain how to overcome this problem. Also there is a need to keep the pH around 2 so that there will be no negative impact on the process. Finally, Zhang and Li (1999) concluded that the biomachining process slowed down due to the buildup of Cu ions in the solution. They bubbled H2S gas into the solution to solve this problem. This forms precipitate of CuS, thus removing the Cu ions from solution.
Ting et al. (2000) applied At. ferrooxidans (ATCC 13598 and ATCC 33020) for the biomachining of mild steel and copper (Ting et al. 2000). They did not find any difference in the mean removal rate of copper and mild steel between ATCC 13598 and ATCC 33020.
They showed that the mass removed increased proportionately with machining time and shaking speed. Like Uno et al. (1996) Ting et al. (2000) also found that an increase in temperature led to an increase in the mean removal rates. An optimum temperature for biomachining found between 30o C and 35o C. Ting et al. (2000) compared the mass removal rate and undercutting occurred during biomachining and chemical machining. They reported that the mass removal rate was significantly higher in chemical machining than biomachining. When the width of the machined groove extended into the coated region, an undercutting occurred. An undercutting was more severe in chemically machined workpieces (Fig. 8). It was also found that during biomachining, undercutting occurred to a greater extent for mild steel. Nonetheless, an undercutting was not as significant (compared with chemical machining), although an extent increased with machining time, as expected. They feel that there is a need of further research to control this effect during the biomachining process. Ting et al. (2000) also manufactured the microstructure by using organic photoresistive materials to mask. In their experiments they were able to biomachine unmasked regions of the metals. They found that the final machined profile was similar to the coating image on the original metal (Fig. 9). Also an undesired leaching of the metal in the region under the masked area (termed undercutting) was not severely encountered during the biomachining process.

Kumada et al. (2001) also used the microbially influenced corrosion for the fine biomachining of steel and copper. They found that loss in mass of SS 400 was larger than that of copper. Also the surface of SS 400 was rougher as compared to copper. Yang et al. (2009) used At. ferrooxidans in the machining process of metal components (Yang et al. 2009). They studied the change in machining depth with machining time and found linear relationship. An etching rate of brass is about 9.20 μm/h; etching bronze is about 10.82 μm/h and etching copper is 13.62 μm/h (Fig. 10).

Besides this the researchers studied the factors that influence the biomachining, including temperature, pH, kinds of the culture medium, etc. They stated that the pH is an important influencing factor during biomachining process. Yang et al. (2009) showed that pH value of etching solution increased gradually (before 40 h) along with the etching process (Fig. 11). The growth of microorganism was affected by increase in the pH. This affected the machining process and the metals removal decreased gradually. To overcome this problem they used H2SO4 to maintain the pH in desired range.
Hocheng et al. (2012c) found that the SMRR was different for different metals. 2.0, 1.6, and 0.55 mg/h.cm2 SMRR were achieved for copper, nickel, and aluminum, in 48, 49, and 41.5 h, respectively. Now it is also known that the material removal rate is slow and appears to decline over time. This happened because the bacteria are aerobic and the depletion of available oxygen might have slowed down the material removal rate. It is also known that the SMRR is independent on the cell concentration, except when the cell concentration is too high. It has been proved that during biomachining process, the metals are found dissolved by the chemical action of iron ions (Fe3+) (Hocheng et al. 2012a). These primary iron ions are supplied by the bacteria At. ferrooxidans and are proposed to be oxidizing agents for the dissolution and removal of metals. Therefore, there is no absolute requirement for the bacteria to attach to the surface of metals. Unattached bacteria are also able to catalyze mineral dissolution through a noncontact mechanism. The culture supernatant is an effective medium for metal removal. Hocheng et al. (2012c) found that large amount of metal removal is achieved by using culture supernatant as compared to bacterial cells. The SMRR obtained were 5.5, 4.2, and 0.7 mg/h.cm2 in 6 h for copper, nickel, and aluminum, respectively (Hocheng et al. 2012c). All these experimental results support an indirect mechanism during the process of biomachining.

Surface Integrity

Now the method of biomachining is well established. Various researchers successfully fabricated parts by using biomachining (Yang et al. 2009; Ting et al. 2000; Hocheng et al. 2012a; Zhang and Li 1999). During a manufacturing process, the quality of the surface produced is a very important aspect. Every manufacturing process, however, has limitations regarding this characteristic. It is therefore important to explore the surface finish characteristic of the biomachining process (Istiyanto et al. 2011). Recently, many studies were carried out considering these objectives. Johnson et al. (2007) reported an increase in overall roughness after the exposure of workpiece to the bacteria. They stated that an initial surface roughness did not have a significant effect on the change in Ra. The researchers studied an effect of biomachining on surface roughness for 24 and 48 h. The change in Ra obtained for 24 h was 1.8–2.6 μm while for 48 h 1.7–2.4 μm. There was larger change in Ra corresponding to a higher initial Ra for the 24 h test. Even if the machining time was increased up to 48 h, the surface roughness increased slightly. They found that bacterial machining is not uniform on polycrystalline Cu.
The surfaces became rougher due to such uneven material removal on the surface (Fig. 12). On the bacterially machined surfaces, grooves from polishing disappeared and grain boundaries and annealing twins are visible. Rather than preferentially attacking grain boundaries like the sterile media, seemingly random changes in topography appeared all over, not only at grain boundary regions. 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 μm.

Yang et al. (2009) studied the surface roughness effects during biomachining process. They found that the surface roughness increased rapidly with time, from an initial less than 0.2 μm to the final near to 2 μm (Fig. 13). They observed that in the first 2 h, the surface quality deteriorates rapidly and at end of the 2 h the surface roughness of the three kinds of materials reach the worst. After 2 h biomachining, the surface roughness no longer increased significantly, but fluctuated inside certain scope and then reached the stable stage (Fig. 13).
Istiyanto et al. (2010) studied the changes in Ra for 6, 12, and 18 h of machining processes. They observed an increase in Ra from 6 to 12 h of machining was less than those from 12 to 18 h. These changes occurred with reference to initial Ra values before machining of about 0.4 μm. The randomized surface profile might be responsible for this effect. The longer machining time will result in a more randomized surface profile. They carried out a second experiment, in which the workpieces were polished using a 220-grit SiC abrasive wheel and had initial Ra values of about 0.54 μm. In this experiment also, the surface appearance significantly changed after biomachining. The straight-line pattern of the surface changed to random patterns after biomachining. In this experiment, the change of Ra also varied significantly after 12 h of machining time, similar to the behavior in the first experiment. The increase in Ra from 12 to 18 h of machining was higher than that from 6 to 12 h. An increase in machining time did not linearly affect the change of Ra. This explains the relationship between Ra and machining time. They also suggested that phenomenon of increasing Ra could be a limitation of the biomachining process, if one wants to conduct biomachining for fine surface roughness.
Istiyanto et al. (2011) further studied the final machined part by using scanning electron micrograph and a noncontact 3-D microsurface profiler. They revealed that the profile form of copper was U-shaped (Fig. 14). Also they found that the width of the groove profile as well as the depth increased with time. An increase was relatively linear for both width and depth from 12 to 48 h. Thus, the required width and depth of the groove can be obtained by selecting an appropriate machining time (Fig. 15).

It is now known that the metal removal rate and surface roughness can be controlled by using various physicochemical parameters. In previous section it has been discussed that At. ferrooxidans oxidizes Fe2+ to Fe3+ and derives energy required for its growth. The Fe3+ generated by this process can be used for dissolution of metal. A study was carried out to explore an effect of Fe3+ on metal dissolution. An increasing concentration of Fe3+ in culture supernatants was obtained by oxidation of increasing concentrations FeSO4. These culture supernatants were used for machining of copper metal workpieces for 1 h. At 10 g/l FeSO4 concentration, the SMRR was 15.91 mg/h.cm2 with 1.07 μm surface roughness. With increase in FeSO4 concentration, the SMRR increased linearly with rapid deterioration in surface quality. The 40.2 mg/h.cm2 SMRR with 5.18 μm surface roughness was achieved by using 40 g/l FeSO4. Therefore, the further experiment efforts were taken to control the quality of the surface produced by varying different parameters. Effect of shaking speed on biomachining was studied. The results for this study showed that the SMRR increased with an increase in shaking speed. For initial three shaking speeds, SMRR was very low. The SMRRs for 0, 50, and 100 rpm shaking speeds were 0.5, 1.88, and 4.96 mg/h.cm2, respectively. It is found that at 150 and 200 rpm shaking speed, the SMRR increased significantly. The surface roughness was found increased in parallel with increase in shaking speed from 0.38 μm (0 rpm) to 1.46 μm (200 rpm). Effect of volume of culture supernatant on biomachining was studied. The copper metal workpieces were covered with variable volume (50–200 ml) of culture supernatant, and the flasks were incubated at 150 rpm. It is found that the SMRR increased up to 100 ml and then decreased. The surface roughness also showed similar pattern. The parameters, volume of supernatant, and shaking speed showed pronounced effect on SMRR and surface roughness. To further control the surface roughness below 1.0 μm, the same study was carried out at 50 rpm. Again the SMRR increased up to 100 ml and then decreased, while the surface roughness decreased with increase in volume of supernatant at 50 rpm shaking speed. Large volume of the working medium facilitates the transport of ionic product from metal removal process off the machined surface. This effect saturates beyond a certain amount of the supplied volume. Effect of incubation temperature on biomachining was studied. Uno et al. (1996) showed that incubation temperature has pronounced effect on metal removal rate. Recently, it has been found that no such effect on SMRR and surface roughness was observed when culture supernatant was used for biomachining process. The 2.39 mg/h.cm2 SMRR with 0.38 μm surface roughness was achieved by applying 10 g/l FeSO4, 100 ml volume of culture supernatant, 50 rpm shaking speed and 30o C temperature. For all the temperatures studied, similar SMRR and surface roughness were obtained. This might be due to the use of culture supernatant. In previous studies bacterial cells were used for biomachining. At. ferrooxidans growth was affected by incubation temperature which resulted in change in metal removal rate (Uno et al. 1996). In the culture supernatant the bacterial cells were not present during machining process. Therefore, no effect of incubation temperature on SMRR and surface roughness was found (Jadhav et al. 2013).
While various researchers extensively used At. ferrooxidans for biomachining of metals, the potential of At. thiooxidans for biomachining remain unused until Chang et al. (2008) tried it for biomachining of metals. At. thiooxidansis an extremely acidophilic bacterium. It is remarkably tolerant of an acidic environment at a pH of 1 or below and thus differs from At. ferrooxidans (Chen and Lin 2004). The metabolite, mainly sulfuric acid, plays a major role in bioleaching (Wong and Henry 1998). Chang et al. (2008) reported that biomachining of copper, nickel, and aluminum can be carried out by using At. thiooxidans (Chang et al. 2008). They found that nickel is the most readily solubilized metal, while aluminum was poorly solubilized. Also the cell concentration enhanced the machining rate.
They put forth a two-dimensional machining process for metal removal from an exposed workpiece (Fig. 16). The machining depth was approximately 15 μm per day. An amount of undercut was very small and influences the degree of biomachining during an overall machining process. An aspect ratio under the biomachining condition was around 1.85. Further Hocheng et al. (2012b) showed that an indirect mechanism predominates during the biomachining of metals by using At. thiooxidans. An extracellular supernatant showed higher machining rate than the cells in solution. They studied aspect ratio in micropattern machining of copper and nickel and found that the behavior of aluminum in micromachining was unsatisfactory.

Authors tried to explore the surface finish characteristics during machining of copper by using At. thiooxidans culture supernatant. During an oxidation of copper, the changes in surface appearance and surface roughness were observed (Figs. 17 and 18).

Examples of Microsize Features

The feasibility of biomachining processes in fabricating microsize features has been studied by various researchers. Yang et al. (2009) fabricated several shims with a thickness of 0.1 mm and a diameter of only 2 mm and with a thickness of 0.07 mm and a diameter of 15 and 16 mm (Fig. 19).
Hocheng et al. (2012a) applied indirect mechanism for the formation of patterned copper films (Fig. 20)

Istianto et al. (2012) investigated the feasibility of biomachining processes in fabricating microsize features on pure copper. They produced several complex features such as lines, circles, and rectangles, as well as combination features. The dimension of 25 μm for these circular-island and gear-shaped features was obtained. A small feature down to 3 μm was successfully manufactured by using single crystal copper. They biomachined the line feature after 12 h machining time. The widths of lines were about 110 μm spaced apart by about 300 μm. The lengths of the lines were 770, 850, and 920 μm. Rounded ends were produced. The cutting rate or etching rate (terms commonly used in chemical etching) was reduced with the increase of machining time (Fig. 21).

In another experiment they developed a circular pattern (Fig. 22a). The circles of various diameters were perfectly developed after 24 h of machining.
The diameters of 245, 348, and 450 μm were obtained corresponded to the mask diameters of 200, 300, and 400 μm, respectively. They observed that the lateral cutting rates were 0.96, 1, and 1.04 μm/h, respectively. These cutting rates did not significantly increase as the diameter increased. They used a 3D microsurface profiler to measure the depth. The mean maximum depth for all circles was 48 μm, equivalent to a depth formed at 2 μm/h cutting rate. The vertical rate of the circular feature was similar to that of the line feature for the same machining time. However, the lateral rate of the circular feature was slightly lower than that of the line feature. Istianto et al. (2012) also fabricated different size rectangular features (200–450 μm) by using biomachining (Fig. 22b). They found that inside surfaces of the features were flat. These authors also showed that other combination features can be produced by using the biomachining process (Fig. 23).

Figure 23a represents the multiline feature and Fig. 23b represents the multicircular feature, the so-called circular-island feature, whose island diameter was 25 μm. The gear-shaped island feature, which is a more complex feature, with tooth depth of 45 μm, was obtained, as shown in Fig. 24.
Abovementioned biomachining studies clearly show an application of microorganisms in machining of various metals and in fabrication of machined parts. Biomachining is based on the natural ability of microorganisms to transform solid compounds to a soluble form. This involves an attack on the solid compound by metabolic products of microorganisms. The main thrust of work has been done with sulfur-oxidizing microorganisms, such as At. thiooxidans and At. ferrooxidans. In case of At. ferrooxidans, Fe3+ acts as a metabolic product, while in case of At. thiooxidans, sulfuric acid acts as a metabolic product. It is now known that organic acids produced by heterotrophic fungi can be used for biomachining of various metals (Jadhav and Hocheng 2014). Hence, biomachining approaches are generally considered a “green technology” with low-cost and low-energy requirement.