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Fundamental Study on Biomachining : Machining of Metals by Thiobacillus

ferrooxidans

Article  in  JSME International Journal · December 1996

DOI: 10.1299/jsmec1993.39.837

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 ∂J7

Fundamental Study on Biomachining*

(Machining of Metals by Thiobacillus ferrooxidans)

Yoshiyuki UNO*ち ToShiaki KANEEDA***

and Seiichi YOKOⅣ IIZO****

In this work, we investigate the possibility of biological machining by bacteria as

a new technique for metal removal. Some kinds of chemolithotrophic bacteria gain
energy from inorganic matter and fix carbon dioxide from the air. One of them, the
bacterium Thiobaciilus ferrooxidans, "eats" metals. Machining of grooves on pure iron
and pure copper by Thiobacillus ferrooxidans is experimentally investigated. The
experimental analysis showed that biomachining was possible, since the depth of the
grooves generated on the workpiece increased almost linearly with the machining
time. F'urthermore, electric field-assisted biomachining proved to be even more
effective, because the removed rate became much higher under an eiectric field.

Key Words: Nontraditional Machining, Chemolithotrophic Bacteria, Biomachining,

Thiobacillus ferrooxidans, Removed Rate, Electric Field-Assisted
Biomachining

processing. There exist many kinds of microorgan-

1. Introduction
The various material processing techniques pres-
ently in use can be broadly classified into two cate-
gories according to the energy used in the technique,
that is, physical processing and chemical processing.
Natural science or engineering systems are, generally
speaking, classified into three categories
- physical,
chemical and biological. However, no biological tech-
nique has ever been developed for material processing.
The recent advances in biotechnology have led to
widespread application of its techniques in many
flelds. Thus, it appears desirable to investigate the
possibility of biological techniques for material
* Received 10th March, 1995. Japanese original: Trans.
Jpn. Soc. Mech. Eng., Vo1.59, No. 566, C(i993),
pp. 3199-3204 (Received l8th January, 1993)
** Faculty of Engineering, Okayama University, 3-1
Tsushima-Naka, Okayama 700, Japan
*** Department of Mechanical Engineering, Okayama
University of Science, 1-1 Ridai-cho, Okayama
Japan .
**** Industriai Technology Center of Okayama Prefec-
ture, 5301 Haga, Okayama 701-13, Japan
isms, some of whose actions can be considered as a
kind of materiai processing. If it is possible for
microorganisms such as bacteria to machine the desir-
ed part to the required depth, they can be useful as
tools for micromachining.
From the above point of view, the possibility of
biological machining of metals by a kind of lithotroph,
Thiobacillus ferrooxidans which lives in a specific
environment is investigated.
2. Definition of Biomachining
As shown in Table 1, material processing is gener-
ally classified into physical, chemical or biological
processing, according to the energy used and volume
change in the process. Conventional material process-
ing can be classified further into physical or chemical
categories, then each category can be classified fur-
1
ther into removal, addition or deformation processing.
A new material processing technique using biolog-
700,
ical energy is introduced here as biomachining, biode-
position and bioforming corresponding to three kinds
of conventional material processing. Biomachining in
this study is defined as biological removal processing.

JSME International Journal Series C,V0139,N04ぅ 1996

838

Table 1 Classification of material processing from the Table 3 Components of 9 K medium(fOr 10 L)

viewpoint of energy used and volume change
Removal-Cutting, Grinding, EDM
。(NH4)2S04 30g
-
Addition-Wetding, Spraying, pVD
;Physical t ・ K2HP° 4 5g
- Defomation Casting, Forging, Press Foming
] o MgS(ム
I r Removal-Photo Etching, Chemical Milling 5g
Addition-
rocessrnS f I Chemicat IL
Yill'lil--
'
Plating. cvD
Defomarion-ExplosionForming
・ KCl lg
| -
Removal- Biomachining ・Ca(N03)2 01g
L giotoei"ut - l -
L
Addrtron r B iodeposirion r
・ Delonizcd Watcr loL
Deformation-(Biofoming)
- ・ AttustmCnt Of pH t0 2.5 with H2S° 4
・ FeS04 3 vol%
Table 2 Classification of organisms from the viewpoint
of carbon source and energy source
Carbon Energy

¨
￨
¨
Chemonthotrc Ph_― C02 MI話
Inorgmi(
Thiobaci‖ us Thi∞ xidalls

Photdれ hotroph― ―― co2-― ―bght Plants,CmOrena,cyanophta

I緊 Q…

祀
諄 nOlroph_躙
mC鍛
phocl。 劉 。Юph― °rgttc Lgh Rh“
に 嗣 ヽ血 り Bmtta

os,rlllκeac 9K Medium

3. Thiobacillus ferrooxidans
Organisms are classified into many groups from
the viewpoint of the energy source necessary for
Culturc fluid
viability and the carbon source used for nutrition as
shown in Table 2('). Organisms which obtain carbon Fig. 1 Schematic diagram of culture of bacteria
from organic matter are called organotrophs, and
include animals, and many kinds of bacteria. On the need be concentrated at the machining point, and so
other hand, organisms which directly utilize carbon neither a damaged layer nor a heat-affected zone in
dioxide in air as a carbon source are called lith- the machined surface is generated.
otrophs. Plants, chlorella and cyanophyceae are
classifled into this category. From the viewpoint of
4. Experimental Procedures
energy source, organotrophs are divided into chemoor- 4.1 Culture of Thiobacillus ferrooxidans
ganotrophs which utilize organic matter, and photoor- There are seven known types of Thiobacillus
ganotrophs which use 1ight. Similarly, lithotrophs are ferrooxidans, which live in such speciflc environments
divided into chemolithotrophs which utilize inorganic as waste solution from ore, registered by the Amer-
matter and photolithotrophs which utilize light. ican Type Culture Collection('). Three of them,
Among the chemolithotrophs there are some bacteria ATCC 13598, ATCC 13661 and ATCC 33020 were used
that "eat" metal. Thiobacillus ferrooxidans and in this study. These bacteria were collected from
Thiobacillus thiooxidans are included in this category. different places, but it has not been determined
These bacteria utilize energy generated by oxidation whether they differ from each other genetically. First,
of iron or sulfur to fix carbon dioxide in the air. For it is necessary to obtain a pure culture of the bacteria.
example, Thiobaciilus ferrooxidans can gain energy Figure I shows a schematic diagram of the experi-
by oxidation of bivalent iron as follows. mental apparatus used in the culture of the bacteria.
4Fe++02+4H+→ 4Fe3++2H20 (1) The components of the culture fluid called 9 K
In this reaction, about 8 kcal per mol is generated(I). medium, employed in this experiment, are shown in
In this study, a metal removal process using the Table 3. As mentioned before, Thiobacillus ferroox-
chemolithotropic bacterium, Thiobacillus ferroox- idans utilizes energy generated in the oxidation of
idans as a tool, is investigated. This bacterium is a bivalent iron to trivalent iron. However, bivalent iron
kind of short bacillus, about 0.5 pm in diameter and I is oxidized spontaneously to trivalent iron at pH
pm in length. If it is possible to apply the bacterium values lighter than 3. Therefore Thiobacillus ferroox-
to material removal, it will be very advantageous as a idans survives only in a strong acid solution with pH
tool for micromachining because of its small removal less than 3. The culture of the bacteria was carried
volume. Moreover, since the metabolic function of the out in the foilowing stages. First, an appropriate
bacterium is utilized, no physical or chemical energy volume of 9 K medium was sterilized in an autoclave

Series C, Vo]. il9, No. 4, 1996 JSME InternationaL Journal

 839

at 132"C for 12 minutes. Next, 3 volume percent of
undiluted culture fluid was inoculated into 9 K medium
in a clean bench. Then this medium was shaken in an
incubator at 28"C, at a shaking rate of 160 cpm and
with shaking width of 25 mm for both X and Y
directions. The growth rate of Thiobacillus ferroox-
idans is low, actually lower than that of colibacillus,
since it takes about a week for the density of bacteria
in 9 K medium to reach a constant value. The number
of bacteria was counted using a haemacytometer
under a microscope.
Figure 2 shows Thiobacillus ferrooxidans on the
haemacytometer, where each black spot corresponds
to a single bacterium.
Figure 3 shows the variations of the number of
bacteria with the culture time for the three different
日 ミ ｏい
kinds of bacteria. The number of bacteria increased
exponentially with culture time after 25 hours, reach-
ing 10'/mL at about 100 hours, then increased more
gradually and reach stationary phase after about a
week. Marked differences in growth among the three
types of bacteria cultured were not found. The fluid
cultured for a week, in which the bacteria had reached
stationary phase, was utilized in the metal removal
experiment.
4.2 Metal Removal Experiment
The metal removal experiments were conducted
using the apparatus shown in Fig.4. The mask pat-
tern used to form the grooves was previously prepared
on the workpiece by the photolithography process.
Iron of 99 .95% purity and copper of 99.9% purity were
adopted as the workpiece, because these bacteria have
been employed to leach iron, copper and uranium(3)
from ore. The machined groove contour on the
workpiece was measured by a surface profile meter.

The mean depth of grooves was defined as the

removed amount. Figure 5 shows a SEM micrograph
and an example of the measurement of machined
grooves. As can be seen in these figures, the grooves
were machined with an approximately uniform depth.

Fig. 2 Thiobacillus ferrooxidans on haemacytometer

口日＼ ｏ

Temperature : 28'C
９ × 輌ｌ ｏ

Shaking rate :160 cpm

週ち︼

Bio-machining
ｏＯ日●Ｚ

50 100 150 200

Time(hr)
Fig. 4 Schematic diagram of the metal removal
FiS. 3 Variations in number of bacteria with culture time experiment

Fig. 5 SEM micrograph and results of groove measurement

JSME International Journal Series C,V。 1.39,NO.4,1996

840

depth achieved using the culture fluid containing

5.
Figure 6 shows the variations of groove depth
with the machining time for pure iron. The culture
fluids containing the bacteria were shaken at 28"C, 160
cpm in this experiment. The solid circles in the figure
show the removed amount in 9 K medium without
bacteria, which was negligible even after 10 hours.
This indicates that the amount of material removed
by chemical etching was minute even in a strong acid
solution of pH 2.5. On the other hand, the groove
depth increased almost linearly with the machining
time using the culture fluid with the bacteria. This
suggests that Thiobacillus ferrooxidans can be used
for machining iron and the removed amount can be
controlled by the machining time. As shown in the
figure, there were no marked differences among the
three types of bacteria employed here, and their iron
machining abilities appeared similar. The mean
removed rate for iron reached about 14 pm per hour.
Figure 7 shows the variations of the groove depth
with the machining time for pure copper. The groove
︵口︻ミ ︶ Ｏ＞〇〇﹄∞ ︺〇  〓一ＱＯ︵Ｈ
Metal Removal Experiment
Thiobacillus ferrooxidans increased linearly with the
machining time, while that achieved using the 9 K
medium was negligible as in the case of pure iron.
This suggests that Thiobacillus ferrooxidans can also
be used for machining copper. The mean removed
rate for pure copper was about 20 pm per hour, which
is higher than that for pure iron.
Figure B shows the effects of the shaking rate on
the mean removed rates of pure iron and pure copper
using bacteria ATCC 13598. The data shown here-
after are all obtained using ATCC 13598.
The mean removed rate increased with the shak-
ing rate, and reached a constant value at 160 cpm.
This indicates that it is necessary for bacteria to
make contact many times with the workpiece surface
due to the shaking motion in order to reach a constant
removed rate. The mean removed rate of pure copper
is higher than that of pure iron for any shaking rate.
Figure 9 shows the effects of the machining tem-
perature on the mean removed rates of pure iron and
pure copper. Machining temperatures selected in this
experiment ranged from 18 to 43'C, which were also
used for culturing the bacteria. The center of range is

Workpiece : Fe
28"C. As shown in the figure, the variation trend for
Temperature : 28"C
Shaking rate :160 cpm iron differs from that for copper. That is to say, the
含く日 、︶０
３         ２         １
０      ０      ０

Bacteria : ATCC 13598

Temperature :28"C
１ Ｂち日ｅ層Ｏ

0246 810
Σ

Time (hr)
Fis 6 Variations of groove depth with machining time
for pure iron 0 50 100 150 200
Shaking rate ("ptn)
３
０         ０         ０
０

Fig. 8 Effects of shaking rate on mean removed rate

︵口︻ミ ︶ 〇＞〇〇︺“ ］〇   〓一ＱＯ∩︻

Workpiece : Cu
→〓ヽ

Temperature : 28'C
日 ミ︶ ０

Bacteria : ATCC13598
Shaking rate :160 cpm
０
２

Shaking rate :160 cpm

20
起﹁Ｂ ＞ｏ日ｅ ●Ｓ Σ
０
１

10

0
0246810 10 20 30 40 50
Time (hr) Temperature (C)
Fig. 7 Variations of groove depth with machining time Fig. 9 Effects of machining temperature on mean
for pure copper removed rate

Series C, Vo].39, No.4, 1996 JSME lruternational Journal

 ∂イI

′
０      ０      ０
０      ０      ０
０           ４           つ４
mean removed rate for pure copper reached its maxi-

︵
日 、︶ｏ
Bacteria : ATCC13598, Workpiece : Cu
mum around 30"C, while that for iron at around 40'C. Temperature : 28"C, Shaking rate :160 cpm

These bacteria are optimally active around 30'C, Bio machining 0 5V(+

＞ｏ
which agrees with the result in the case of pure Bio-machining

２如︺
copper. However, in the case of pure iron, another 9K Medium 0 5V(+)
factor should be considered; chemical reactions 9K Mcdium 05V(― )

Ｏ ｆＱ
which increase the removed rate with increase in the
machining temperature may have occur in addition to

ｏ∩
the bacterial activity, which would produce such a
variation trend as mentioned above.
Some mineralogists have been investigating the
possibility of using Thiobacillus ferrooxidans for
TimC(hr)
bacteria leaching of 1ow-grade ore('), particularly
copper and uranium ore(3). Two bacteria leaching Fig 10 ヽrariations of gr00ve depth with nlachining tinle
in electric― neld― assisted biomachining fOr pure
mechanisms have been proposed(u). One is the direct
copper
leaching mechanism, in which the bacteria stick to the
surface of ore and oxidize the sulfur component to

︵
Bacteria : ATCC13598, Workpiece : Fe

日 、︶０＞ｏＯ︼
generate sulfuric acid. Another is the indirect-leach- Temperature : 28t , Shaking rate :160 cpm
ing mechanism, in which chemical oxidation of Fe ○ Bio‐ machlnlng o 5V(+)
0 Bio― machining 0 5V(― )

allows the metal component in the ore to leach as □

metal sulfuric salt. For example, the leaching mecha- △ 9K Mcdium 05V(十 )
“ ︺〇 〓一

▲ 9K Medium UOV(―
nism for uranium ore is presented as follows(u):
CaU(PO,), t F e,(SO,)3 + H,SO, * 2H,O-
UOzSO+*2treSOa*2H:POa-f CaSO, (2)
ＱＯ∩

In this mechanism Thiobacillus ferrooxidans has the

effect of oxidizing Fe2* to Fe3* as shown in Eq. ( 1 ). It
is very difficult to identify the actual leaching mecha- 0
nism in practical leaching processes; both of them --0 z 4 6 B 10
appear to
operate simultaneously(5). The leaching Time (hr)
mechanism of pure metal has never been investigated Fig. 11 Variations of groove depth with machining time
before, therefore the mechanisrn of metal removal has in electric-fleld-assisted biomachir.ring for pure
not been clarifled. However, considering the compo- iron
nents of the workpiece, it is reasonable to assume that
the direct leaching mechanism predominates for pure chemically machined, as shown by " a" in Fig. 10. If a
iron, while the indirect leaching mechanism predomi- simple addition rule can be applied to these results,
nates for pure copper. the removed amount after machining for 10 hours
The effect of electric fie1ds on biomachining has under the electric fie1d should be " (a l &)". However,
also been investigated since oxidation or reduction is the removed amount in this case, c, satisfied :

based on the transfer of electrons. Figure 10 shows θ>α tt b (3)

the variations of groove depth with the machining
time when an electric field, DC 0.5 V, was applied
between two workpieces of pure copper. The current
was maintained at 0.01 A in order to suppress electro-
chemical machining. The dotted line in the flgure
represents the groove depth in biomachining without
the electric fleld, and "&" shows the groove depth after
10 hours. When the electric field was applied between
two workpieces, the depth of grooves generated on the
anodic workpiece(shown as "c" for 10 hours in Fig.
10)was about twice that in normal biomachining,
while that on the cathodic workpiece became
extremely sma11. On the other hand, when the same
electric field was applied in 9 K medium without
bacteria, the anodic workpiece was slightly electro-
and was much larger than the amount calculated
assuming the addition rule. This result indicates that
the electric field can produce a kind of rnultiplication
effect between biomachining and electrochemical
machining. We therefore call this method "electric-
fleld-assisted biomachining".
Figure 11 shows the variations of the groove
depth with the machining tirne in electric-field assist-
ed biomachining of pure iron. A similar multiplication
effect can be observed, although the increase of the
groove depth is less than that for pure copper.
However, in the case of 9 K rnedium r-rot containing
the bacteria metal deposition could be found on the
cathodic workpiece surface due to electrodeposition.

JSME International Journal Series C, Vol. 39, No. 4, 1996

 842

6. Conclusions Acknowledgments
A new metal removal process called "biomachin- The authors would like to thank Professor T.
ing" using a lithotrophic bacterium, Thiobacillus fer- Sugio, Okayama University for supplying bacteria as
rooxidans which is employed for bacteria leaching has well as useful information on the culture of bacteria.
been investigated. The performance and machining The authors also thank Mr. S. Terao, Ms. C.
variables have also been determined. The main con- Akamoto, Mr. M. Hiruta and Mr. A. Miwa for help
clusions obtained in this work are as follows. with the experiments. This work was funded through
Thiobacillus ferrooxidans in culture fluid
(1) Grants-in-Aid of the Science Research Fund, General
showed the ability to machine pure iron and pure Research Category C from the Ministry of Education.
copper and the removed depth was approximately
References
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rate for copper was larger than for iron. ( 1 ) Imai, K., Lithotrophic Bacteria (in Japanese),
(2) Differences in the biomachining ability for Kagaku Dojin, (1984), p. 4.
pure iron and pure copper were not found among the (2) American Type Culture Collection, Catalog of
Bacteria and Bacteriophages, 17th ed. (1989), p. 250.
three kinds of Thiobacillus ferrooxidans employed.
( 3 ) The removed rate increased with an increase
(3 ) Yamanaka, T., Energy Metabolism of Micro-
organisms (in Japanese), Gakkai Shuppan Center,
of the shaking rate, and reached a constant value (1986), p. 148.
which yielded at around 160 cpm. (4 ) Sugio, T. and Tano, T., Bacteria Leaching (in
( 4 ) The culture fluid temperature which yielded Japanese), Heredity, Yo1. 42, No. 8(1988), p. 28.
at the maximum removed rate was around 30'C for (5 ) Takahashi, T. and Kai, T., Bacteria Leaching at
pure copper and 40"C for pure iron. Present and in the Future (in Japanese), BIO
( 5 ) In electric-field-assisted biomachining, i.e. INDUSTRY, Vol. 5, No. 11(1988), p. 789.
biomachining under an electric field, the removed rate
(6 ) Tomizuka, T., In Place Bacteria Leaching from
Uranium Ore, Chemistry and Organisms (in
at the anodic workpiece became much higher than (1977), p. 714.
Japanese), VoI. 14, No. 11
that in normal biomachining, while the removed
amount at the cathodic workpiece was minute.

Series C, Vol.39, No.4, 1996 JSME International Journal

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