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Materials Today: Proceedings 5 (2018) 4225–4234 www.materialstoday.com/proceedings

ICMPC 2017

Experimental Investigations and Surface Morphology of Bio-

Micromachining on Copper
Abhishek Singha, N. Arul Manikandanb, M. Ravi Sankarb*, K. Pakshirajanb, L. Royc
a
Department of Mechanical Engineering, Indian Institute of Technology,Guwahati, Guwahati-781039, Assam, India a
b
Department of Biotechnology , Indian Institute of Technology,Guwahati, Guwahati-781039, Assam, India
c
Department of Mechanical Engineering, National Institute of Technology Silchar-788010, Assam, India

Abstract

Bio-micromachining is an emerging latest non-traditional manufacturing process and has various advantages. In this
manufacturing process micro-organisms are used for material removal in desired shape and size. Acidithiobacillus ferrooxidans
micro-organism are used as a tool for machining the copper workpiece and the effects of various parameters like pH,
temperature, shaking speed, machining time are being calculated and there optimized value is being purposed. Vickers hardness
test was also being performed and comparative study between bio-micromachining and photochemical machining is done in this
paper.
© 2017 Elsevier Ltd. All rights reserved.
Selection and/or Peer-review under responsibility of 7th International Conference of Materials Processing and Characterization.

Keywords: Bio-micro-machiniung; Vickers hardness test ; Acidithiobacillus ferrooxidans.

1. Introduction

Since the beginning of human race stone and metals were used for different purpose and they were casted in
different structure, shape and size according to their purpose. As time passed on the size has reduced and shape
became complicated. As of now the shape has become more intricate and the product size are reaching in micro or
even up to Nano levels. Bio-micromachining is a non-traditional manufacturing process used for the removal of
metals from workpiece surface which have potential application in the construction of micro-scale spaces [1]. With
the advancement in the field of manufacturing technology, various micro-machining method have been developed
for making contracted devices with the help of techniques like Chemical Machining, EDM, Ultrasonic Machining
and many other processes. Although these machining technique is capable of making devices micro levels or even
up to Nano levels.
* Corresponding author.
E-mail address: evmrs@iitg.ernet.in

2214-7853 © 2017 Elsevier Ltd. All rights reserved.

Selection and/or Peer-review under responsibility of 7th International Conference of Materials Processing and Characterization.
4226 Ramanaiah et al./ Materials Today: Proceedings 5 (2018) 4225–4234

Unfortunately, these all manufacturing techniques have some drawbacks and even some techniques are not even fit
for environment and have antagonistic effect on environment. Some of their drawbacks are: - heat affected zone,
recast layer, porosity and damage to the metallurgical properties of workpiece and many others process. These
machining techniques also have environmental issues. For example, chemical used in Chemical Machining should
not be discharge in ecosystem because its strong acid and same in the case of Di-electric used in EDM, Wire EDM.
The answer to these problem lies in nature itself.
In environment, more than 50+ metallic matters are bio-processed by organisms for withdrawing energy and
nutrients for their own existence. By using this property of micro-organisms they can be used as a tool of machining
the metals and it can be new non-traditional manufacturing techniques. In environment, generally micro-organism
are of 0.1-5.0 µm in size but they are able to extract metal from workpiece surface in sub-microns. Thus, this novel
micromachining process has potential applications in machining of nano to micro scale features. Thus, bio-
micromachining can be defined as the process of removing material from workpiece surface in required shape and
dimensions with the help of micro-organism. In bio-micromachining mainly Chemolithotrophic bacteria such as
Acidithiobacillus ferrooxidans, Thiooxidans etc. are used for machining various metals like Iron, Copper, Nickel,
Aluminium, Zinc, Tin and many other.
Uno et al [2] carried out state-of-art work and showed the future opportunity of Acidithiobacillus ferrooxidans in
material processing. They performed set of experiments and showed the effects of varying some of the parameters
which are involved in the bio-machining process and also concluded a way to accelerate the process with the
application of electric field. Shows that if positive applied voltage are used, then MRR will be accelerated. Zhang
and Li [3] perform similar experiment as Uno et al [2]. There experimental results show that machining of pure iron,
pure copper and they also fabricated micro gear and grooves on pure copper piece with the help of bio-machining.
There results concluded that grooves which were bio-machined were directly prepositional on the machining time.
They also analysed the bio-machining mechanism i.e electron-transport chain (ETC) in the Thiobacillus
ferrooxidans membrane and also proposed the biological activity to regenerate the intermediate oxidant which is
Fe3+.
Zhang and Li [4] showed the thermodynamics and kinetic of bio-machining on pure copper by Thiobacillus
ferrooxidans micro-organism. They illustrates the model of ion-cycle in the bio-machining and proposed the method
for maintaining the stable equilibrium of the ion exchange cycles and also concluded that the machining rate
depends on bacteria amount in the sample. The main conditions for maintaining the stable equilibrium in this
process is to supply of H+ ion. Yasuyuki et al [5] suggested the MRR at micro levels in the bio-machining process of
controlled structure in low carbon steel using Acidithiobacillus ferrooxidans. Kurosaki et al [6] also performed the
corrosive action of anaerobes and aerobes for the bio-machining on the metals. Johnson et al [7] performed series of
experiments to characterize the surface roughness of workpiece and also quantified the MRR in bio-machining but
they didn’t describe the relations and fashion of relationship between machining time and surface roughness.
Istiyanto et al [8] purposed the MRR and surface roughness characteristics in the bio-machining on copper for
various machining time interval. Their result shows that MRR during bio-machining was inversely dependent on the
machining time.
Hocheng and Jadhav [9] conducted an experiment to study the effect of biologically ferric sulphate on MRR and
also suggested the green recycling process that can be done for extraction of metals from electronic waste like Ni-Cd
batteries by using biologically produced ferric sulphate. Istiyanto et al [10] studied the feasibility of bio-machining
in making of micro-sized features, such as circles, rectangles and lines as well as complex shape on pure copper.
Hocheng et al [11] concluded that the indirect mechanism is mainly responsible for the oxidation of elements and
their results showed that the Cu demonstrates the higher MRR followed by Ni, Al. Jadhav et al [12] conducted
experiments that the concentration of ferrous sulfate, shaking speed and amount of culture supernatant showed the
effect on SMRR and surface roughness. They also found out that by varying temperature range there is no effect on
surface roughness as well as on SMRR. Jadhav and Hocheng [13] concluded the effect of shaking speed, volume of
culture supernatant and temperature. An experimental studies for a new manufacturing material processing
technique using Acidithiobacillus ferrooxidans was carried out and according to the finding the optimum range of
various parameters like pH, temperature, machining time and shaking speed are being suggested. Vickers hardness
test was conducted before and after the machining process. A comparative study between photo-chemical machining
and bio-micromachining was also being performed.
 Ramanaiah et al./ Materials Today: Proceedings 5 (2018) 4225–4234 4227

2. Experimental Details

2.1. Micro-organism culturing

Acidithiobacillus ferrooxidans was being acquired from the Council of Scientific & Industrial Research-Institute
of Minerals and Materials Technology, Bhubaneswar and is used in this study. A natural environment with low pH
and a metal to serve as the source of electrons for their growth is required for Acidithiobacillus ferrooxidans to
grow. About 5 ml of Acidithiobacillus ferrooxidans was put into the solid media [14] and it was incubated at 350C
until micro-organism growth became apparent as light yellow, semi-transparent on the surface of media. The 9K+
media (Silverman & Lundgren, 1959) containing KH2PO4-0.5 g\l, MgSO4.7H2O-0.5g\l KCl-0.1g\l, (NH4)2 SO4 -3g\l,
FeSO4-44.2g\l. and pH was adjusted to 2.5 with 1N H2SO4. After receiving the culture inoculated 100 ml of freshly
prepared 9K+ medium with half of the cultured. Incubate at 350C with 180 rpm, till the medium become red (3-4
days). Continuously culture of Acidithiobacillus ferrooxidans were sub-cultured and by taking several ml from
conical flask and mixing them. All operation were conducted under the bacterial hood with positive flow high
efficiency particular air (HEPA) filter in order to avoid contamination.
After that the inoculated flasks were incubated at 350 C and shaken at 180 rpm and using culturing protocol,
populations grew from 5 to 12 days. After that samples were removed and used to repeat the culturing for future
experiments.

2.2. Sample preparation

For experiment, first sample of 99.99% pure Copper were prepared. The copper were cut into dimension of 10 x
10 x 2 mm buy using Wire EDM and hand hex-saw. One face was chosen and ground to the desired level of surface
finish. Polishing of the chosen side was carried out using grinding wheels fitted with paper containing SiC of
various grit sizes. The grit size which were chosen were 320, 600 and 1200, each used in sequence until the desired
finish level of surface finish was reached. Once polished, every sample was contained in a sealed plastic bag to
reduce surface oxidation before use in the experiment. A total of 120 samples were prepared and used.
Then the figure of the final product was designed using Auto CAD 2012 Student Version by Auto Desk and the
product design was printed on the transparent sheet. Then, the workpiece was coated with the photoresist making
which LPR E-1020 was supplied by Cadmosil Chemical Pvt. Ltd. The workpiece was dipped in photo resist for
about 10 seconds and after taking out from the photo resist the workpiece is dried out in hot air chamber for about 4
minutes. Then workpiece is exposed to UV light for about 2 minutes in UV chamber. After that the workpiece was
dipped in developer for about 1 minute and dried in an open environment.
Finally, an image of micro-mixer was developed. Before beginning the experiment, each sample was subjected to
surface roughness measurement using a profilometer as shown in fig. 1. On the uncovered area of the workpiece
which is going to be bio-machined throughout the whole profile. The initial Ra and depth of the image was
measured. Simultaneously, the workpiece was also observed by an optical microscope and also initial mass was
measured. Due to the similarities of surfaces of this specimen, only representative micrographs are included for the
pre-experiment portion of the data.

3. Results and Discussion

The workpiece was prepared for bio-micro machining experiments. In order to study the effect of various parameters
like pH, shaking speed, temperature and machining time on material removal experiments were conducted. The
4228 Ramanaiah et al./ Materials Today: Proceedings 5 (2018) 4225–4234

surface roughness measurement was taken at four different portions in workpiece with the help of profilometer at
same location before and after experiments.

Photoresist
masked region

Photoresist un-
masked region

Fig. 1: Overview of photoresist masked and un-masked regions of micro mixture on copper workpiece surface

3.1.1. Effect of pH range

The pH play an important role in the growth of Acidithiobacillus ferrooxidans. An experiment was run in order to
find out which pH range is most suitable for growth of A. ferrooxidans and also to find out on which range
maximum material removal rate can be obtained. At different pH values like 2.5, 4.0 and 7.0 experiments were
conducted and respective material removal rate was being calculated. But it can be concluded that the pH range 2.5
is best for the growth of microorganism and higher the growth of microorganism higher will result in higher material
removal rate as shown in fig. 2.

3.1.2. Effect of temperature

The temperature play a vital role in the growth of Acidithiobacillus ferrooxidans and the temperature range and
also manipulate the material removal rate. Experiment were run on the copper metal workpiece and they were
placed under 100 mL of culture supernatant in conical flask. In order to study the temperature effects the conical
flask were incubated at various temperature range i.e. 25-450C in a shaker incubator at 180 rpm. Fig.3 shows the
variation in temperature and its corresponding material removal rate for copper. The material removal rate reached
to the maximum point at temperature range of 350 C and after that again it started decreasing. At 350C the material
 Ramanaiah et al./ Materials Today: Proceedings 5 (2018) 4225–4234 4229

removal of copper was found to be 0.7 mg/ hours. It can be concluded that A. ferrooxidans organism are optimally
active in range of 300C to 350C and suitable temperature for machining is at 350C.

0.06 0.8
pH=2.5
0.7
0.05
pH=4
0.6

MRR (mg/hr)
0.04 pH=7
0.5
MRR (mg/hr)

0.03 0.4
0.3
0.02
0.2
0.01 0.1

0 0
0 1 2 3 20 25 30 35 40 45
Time (hours) Temperature (0C)
Fig. 2: Effect of pH on material removal rate Fig. 3: Effect of temperature on material removal rateR

3.1.3. Effect of shaking speed

Variable shaking speed between 0-200 rpm were varied. Acidithiobacillus ferrooxidans micro-organism was
inoculated in 9K+. The copper workpiece were placed inside 100 mL of culture in conical flask respectively. These
flask were incubated at shaking speed varying from 0-200 rpm at 35 0C. Fig. 4 shows the effect of shaking speed on
MRR. Result indicate that the MRR first almost remain constant in shaking speed between 0-50 rpm and after 50
rpm the material removal rate seems to increase rapidly till 140 rpm. After 140 rpm MRR seems to linearly increase
with shaking speed but after 200 rpm it remains constant because it affect the contact between micro-organism and
workpiece exposed surface due to centrifugal forces.

12 400
350
10
300
MRR(mg/hr)

8
MRR (mg/hr)

250
200
6
150
4 100
50
2
0
0 24 48 72 96 120 144 168 192 216 240 264 288
Machining Time (hr)
0 50 100 150 200 250
Shaking Rate (rpm)
Fig. 5: Effect of machining time on material removal rate
Fig. 4: Variation of material removal rate with shaking speed

3.1.4. Effect of machining time

This is another important factor which affect the material removal rate. In order to find out the effect of this
parameter the copper metal workpiece were placed under 100 mL of culture supernatant in conical flask respectively
and the initial mass was measured for every workpiece. The experiment was then run for 24 hours and at interval 24
4230 Ramanaiah et al./ Materials Today: Proceedings 5 (2018) 4225–4234

hours. The finial mass of workpiece was measured and reduction in mass was calculated. Fig. 5 illustrate the effect
of machining time on MRR and it can be concluded that the MRR increase when time increases also. It was
observed that increases and but there is direct increase in material removal rate upto 120 hours and after that there is
unexpected increase in MRR upto 192 hours but after 240 hours the material removal rate become more or less
stable. In other words, MRR becomes saturated.

3.2. Effect of inoculum age duration on specific metal removal rate and material removal rate

Fig.6 show the relationship between the specific metal removal rate (SMRR) and material removal rate (MRR)
for copper workpiece. It is observed that there is inverse relationship between SMRR and MRR. As MRR increases
the SMRR decrease this is due to increase in cell density of Acidithiobacillus ferrooxidans because initially the cell
density of microorganism is very less in inoculum volume and as the machining process goes on the cell density or
development of new daughter cell takes place with result in increase in population of microorganism and higher
amount of material removal is achieved as process goes on.

400

350 SMRR MRR

300
M R R (mg/hr)

250

200

150

100

50

0
1 2 3 4 5 6 7 8 9 10 11
Inoculum age duration (Days)
Fig. 6: Effect of inoculum age duration on material removal rate and specific metal removal rate

3.3. Vickers hardness test

Vickers hardness test was conducted on bio-micro-machined surface in order to compare the hardness of
workpiece before and after the machining process. But the result shows that there is no change in hardness of
workpiece and hardness remains same i.e. 89.55HV/20 for copper workpiece as shown in Fig. 7 (a-b).
 Ramanaiah et al./ Materials Today: Proceedings 5 (2018) 4225–4234 4231

(a) (b)

Figure 7: Surface micrograph of indentation (a) before bio-micromachining and (b) after bio-micromachining

3.4. Colour change test

As the machining progress the colour of culture/media keeps on changining and this colour change indicates that
machining is in progress and it is found that the initial colour of media was light yellow or off white and as the
machining progress after 24 hours the colour became light yellow which then turn to dark yellow and after that it
was observed that colour became organce and later on its turn to wine red the change in colour as shown in Fig. 8.

(a) (b) (c) (d) (e)

Fig. 8. Variation in colour of media after certain time interval were (a) initial colour solution (b) after 24 hours (c) after 48 hours (d) after 120
hours (e) after 168 hours

4. Comparison between Bio-micromachining and Photo-chemical Machining

A comparison was made between bio-micromachining using Acidithiobacillus ferrooxidans and photo chemical
machining via-a-vis the effects on the mass removal rate and undercutting. Fig.9 represent the image in which
copper workpiece was bio-micro-machined. The micro-organism were cultured in 9K+ media and experiments were
carried out at 2.5 pH, 350C, 180 rpm and machining time was 32 hours. After machining, the depth of workpiece
was measured by using profilometer and it was found to be 200 µm. As shown in Above Fig 8 (a) (b) and (c) (d) (e)
Fig. 10 shows the graph between machining time and material removal for both bio-micromachining and
photochemical machining. It was observed that the material removal for photochemical machining of copper was
high in comparison with bio-micromachining of copper. In the same machining time the photochemical machining
leach out more material than the bio-micromachining. In this ferric chloride was used in case of photochemical
machining and Acidithiobacillus ferrooxidans was used in case of bio-micromachining and same workpiece i.e.
copper was used.
4232 Ramanaiah et al./ Materials Today: Proceedings 5 (2018) 4225–4234

Similarly, the Fig.11 represents the image of photo-chemical machined copper workpiece in which ferric chloride
was used as the tool for machining. The experiment was carried out for about 20 minutes at temperature range of 45-
55oC. After machining the depth of micro-mixer was found to be around 200 µm.
For the comparative study although the depth of workpiece were kept the same but in case of PCM undercut was
observed more in comparison to bio-micromachining. Fig. 9 show the profile and for this horizontal distance was
found to be 346.57 µm which was initial gap. Fig. 11 represent the profile curve of bio-machined copper workpiece.
The gap or horizontal distance was found to be 422.97 µm and difference in horizontal distance between original
and after machining was found to be 76.4 µm. Fig.10 represent the profile curve of bio-machined copper workpiece.
The gap or horizontal distance was found to be 463.96 µm and difference in horizontal distance between original
and after machining is found to be 117.39 µm. Thus it is clear that undercut in case of PCM is more than in case of
Bio-micromachining and as shown in below Fig 12.

Fig.10. Surface micrograph and 2-D profile of bio-micromachined copper workpiece

 Ramanaiah et al./ Materials Today: Proceedings 5 (2018) 4225–4234 4233

0.50

Material Removal (gm) 0.45 Biomachining of Copper

0.40 PCM of Copper

0.35

0.30

0.25

0.20

0.15

0.10

0.05

0.00
0 2 4 6 8
Machining Time (hr)
Fig.11. Comparison of machining rates between photo-chemical machining and bio-micromachining

Fig. 12. Surface micrograph and 2-D profile of photo-chemical machining of copper workpiece
4234 Ramanaiah et al./ Materials Today: Proceedings 5 (2018) 4225–4234

5. Conclusion

According to the experimental results it is concluded that the pH 2.5, shaking speed 180 rpm, 350C temperate and
32.71 hours machining time are the optimal parameters for bio-micromachining of copper workpiece because at
these parameters the micro-organism growth is maximum and therefore material removal rate is also maximum. By
the results of Vickers hardness test it is concluded that the hardness of workpiece doesn’t get affected by this
machining process and it remains same before and after the machining process and there is no change in
metallurgical properties of workpiece material. The comparative study between the PCM and bio-micromachining
process show as that the undercut in case of PCM is more but in case of bio-micromachining the machining time is
more.
One possible way to make the material removal rate more uniform with time is to constantly supply fresh,
aerated media to and flush water compounds from the whole working fluid environment. Bio-micromachining can
be new future non machining process. But still some more variation and control over this process yet to be
achieved. In short green manufacturing of metals can be done with this process and sustainable development can be
achieve with the help of this process.

Acknowledgment

The authors are thankful to the Council of Scientific & Industrial Research-Institute of Minerals and Materials
Technology, Bhubaneswar for providing the Acidithiobacillus ferrooxidans micro-organism and they are also
thankful to Indian Institute of Technology, Guwahati for providing their laboratory facilities.

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