Biotechnology in mining

Microorganisms as tiny miners

Laboratory report

Submitted to

Prof. Simone Schopf

Submitted by

Shine-Od.M
Jargalmaa.O
Junior at GMIT

26 November 2017
Introduction:
Bioleaching is the extraction of metals such as copper, zinc, arsenic, antimony, nickel
and molybdenum etc. from their ores using living organisms. This is much cleaner
than the outdated heap leaching with cyanide. Bioleaching is one of several
applications within biohydrometallurgy.
Bioleaching can contain numerous ferrous iron and sulfur oxidizing bacteria,
including Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans. As a
general principle, Fe3+ ions are used to oxidize the ore. This stage is completely
independent of microbes. We should know how to sterile samples to run bioleaching
process.
Sterilization is the killing or removal of all microorganisms, including bacterial spores
which are highly resistant. There are 4 types of sterilization method such as heat,
chemicals, irradiation, high pressure and filtration. Sterilization is used for food,
medicine and surgical instruments.
Task No 1: Enrichment of iron oxidizing bacteria in liquid media
1. Preparation of FeSO4- Solution
Lab preparation:
FeSO4
H2SO4
Gas burner
Membrane filter
Procedure:
First, we calculate the amount of ferrous sulfate (MFeSO4*7H2O=55,6g/mol) that is
needed for 200ml of solution. After that 200ml deionized water is adjusted to pH 1.8
with sulfuric acid by universal paper indicator. Add the ferrous sulfate to acidified
water to dissolve completely. Use sterile filtration by gas burner sterilization and
membrane filter with pore size 0.22um into a sterile bottle. FeSO4 liquid media was
Fe rich solution, which was main food for bacteria. Finally, solution stored in
refrigerator at 4oC.
2. Liquid medium iFe
Lab preparation:
Na2SO4 - 1.5g MgSO4*7H2O - 5g
(NH)2SO4 - 4.5g KH2PO4 - 0.5g
KCl - 0.5g
 Ca(NO3)2*4H2O - 0.14g Autoclave
1L cylinder
Procedure:

First, 400ml HBS solution was prepared in 1L measurement cylinder. This solution
prepared for environmental condition for bacteria.
Weighted all compounds then add it to 300ml deionized water and dissolve on a
magnetic stirrer. Then fill up to 400ml with deionized water. Finally, solution was
sterilized in autoclave at 121oC for 20min.

Next liquid medium was prepared:

Solution A: 50xHBS-Solution 20ml, trace element solution 1ml, dH2O 954ml.
Adjusted pH at 2-2.5 with sulfuric acid by universal paper indicator.
Solution B: 1M FeSO4 (pH=1.8) 25ml (=25mM)
Solution A and B prepared separately. Then mix solutions in a 1L sterile Schott flask.

Figure 1. Sterilization by heat and filtration

Discussion:

Bacteria need to grow 2 solutions, which were one for food another for one for
environment. Both solutions supposed to adjust pH=1.8 for ferrous sulfate and pH=2
for iFe by laboratory indicator machine, which does not work. So, solution adjusted
universal indicator, which means adjust result was not accurate as possible. Also,
another important thing was sterilization for present to contain any other bacteria. In
this task three different sterilization methods used. Sterilization by heat, that one was
gas burn other one was steam (autoclave at 121oC for 20min) and last one was
filtration. We learned how to sterile or cultivate iron oxidizers on liquid and solid
media.
Task 2: Enrichment/Isolation of iron oxidizing bacteria on several types of liquid
media.

Materials:

NB plates: recipe for agar plates

Yeast extract 1.6g Agar Agar 12g

Peptone 4g H2O ad 800ml
NaCl 4g
Procedure:

Firstly, weight all chemicals in 1L cylinder and add 800ml water. Stir by magnetic
stirrer. Bottle was filled by aluminum sheet. Made a small hole for breathe. Put
autoclave at 121oC. After heat sterilization the solution was cooled at 45oC. Mix the
solution then pour it to petri dishes.

Figure 2. NB plate
 For inoculation with airborne germs on NB-plates

Open NB-plates and put four locations each person.

Result:

Table 1. Result of NB plate in different spots

Different
Number of
Location Color types of
Colonies
colony

2nd floor experiment room 1 Yellowish white 1

2nd floor womans toilet 3 Yellowish white 2

Library 2 Yellowish white 2

2nd floor corridor 4 Yellowish white 3

Natural occurrence of bacteria/ influence of disinfection of hands on NB-plate

On one NB-plate divided four sections by permanent marker for each member.
Section1: put unwashed finger print on plate
Section 2: wash hands with soap and dry it on air
Section 3: wash hands with soap and rub with towel
Section 4: wash hands with soap and rub your finger on laboratory cloth
After that plate was closed and sealed with parafilm and incubate with upside down at
30oC for several days.
 Figure 3. Finger test on NB plate
Figure 4. One of the different place's test

Number of colonies on finger print

Section 1 Section 2 Section 3 Section4

Shine-Od 30 21 17 24

Do not open the plates while colonies were counted. Because of prevent different
bacteria from the air.

Discussion:

Bacteria are microorganisms that grow everywhere. We can collect and grow them in
specially prepared petri dishes.

NB stands for Nutrient Broth that is typically made of yeast extract and peptone.
Broth is convenient, as most bacteria will grow in this type of medium, even those
with widely different oxygen requirement. When we prepared NB plate, we pour agar
agar which solidify the media. You can test the effectiveness of treating different petri
dishes with dirty hands before washing and drying cleaned hands after washing.

According to the result, dirty hands bacterias colonies grew mostly and most
effective way of drying hand after washing is rub with towels. And we testified four
different places to know how much bacteria in the air by using NB plate. NB and
bacteria need oxygen. Therefore, we placed the plates different spots openly.
Unfortunately, this was located for 20 minutes during the experiment. That means
bacteria could not grow sufficiently. And it was proven from data. To sum up, we
learned how iron oxidizing bacteria look under the microscope and on plates.

Task 3. Copper quantification

Introduction:

Copper quantification was determined in all 16 samples. Firstly, 16 samples

determined by AAC cuvette. From the behind following formula exist.

Cu2=[mmol/l]=0.0607*A595-0.0004

Materials:

Erlenmeyer flask 250ml Sulfur, 0.5

Universal pH indicator Bacteria, 25 ml
Water Sodium hydroxide
Chalcopyrite, 0.5g Burette, 50 ml
Chalcocite, 0.5g Beaker, 25 and 100 ml
Covelline, 1g Alkaline sodium borate buffer

Procedure:

Prepare 5 x 250 ml Erlenmeyer flask

Add per flask 0.5g chalcopyrite (CuFeS2), 0.5g chalcocite (Cu2S), 0.5g
covelline (CuS), 0.5g covelline (CuS) and 0.5g sulfur (S)
Add per flask 5 ml bacteria from Reiche Zeche
Check pH
Add water until sample is reached to 400l
All frozen samples must be liquified and spiked with 200 l 10 % NH3. After
strong mixing they are incubated for at least 10 min.
Centrifuge sample rapidly for 5 min. 100 l of the clear supernatant are
transported into a clean Eppendorf tube and diluted with 100 l water.
Add 200 l ammonium citrate buffer solution, 10 l previous solution, 120 l
alkaline sodium borate buffer and water into new Eppendorf tube.
Mix carefully and wait for 5 min.
 Put the sample into AAS cuvette.

Figure 6. Samples

Figure 5. Bioleaching bacteria

Figure 7. After taking out from centrifugal machine Figure 8. Samples with copper content
Figure 9. Centrifugal machine

Result:

Table 2. pH result

pH universal paper indicator

Oct 6 Oct 9 Oct 12 Oct 16

CuFeS2 1.5 1 1 1

Cu2S 1.8 2 1 1

CuS* 1.8 2 1 1

S 1.75 1 1 1

CuS 3 3 3 3

Table 3. Titration result

NaOH (ml)

Oct 6 Oct 9 Oct 12 Oct 16

CuS - 9,6 12 13.7

Table 4. AAS result

AAS cuvette result

Oct 6 Oct 9 Oct 12 Oct 16

CuFeS2
0.018 -0.067 -0.065 -0.037

Cu2S -0.004 -0.018 0.024 0.146

CuS
0.016 -0.068 -0.05 -0.062

CuS*
-0.048 -0.023 -0.046 0.007

Discussion:
5 samples pH amount almost decrease except 2nd days result. In that case sample
used universal paper indicator, which means it was not so exactly.
Two CuS were had, one with copper CuS* other one without copper CuS. For the one
without copper amount we measured how much NaOH required. Idea behind that
describes following formulas.
MS+2Fe3+=> M2++S0+2Fe2+

S0+1,5O2+H2O=>2H++SO42-

Sulfuric acid affected to decrease pH value, because of increasing acidic condition.

To get to know sodium hydroxide amount, which measured from pH. From the
titration result sodium hydroxide were increased.
Bioleaching is the extraction of a metal from sulfide ores or concentrates using the
materials found natural to the environment such as water, air and microorganisms. In
other words, bioleaching is the commercialization of the ability of certain bacteria and
archaea, found in nature, to catalyze the oxidation of sulfide minerals. According to
the result, the most leached compound was chalcocite in our experiment. During the
experiment, we should precipitate ferrous iron ions by using centrifugal machine and
aqueous ammonia. Copper ions are in liquid part and then we can define copper
quantification using atomic absorption spectrophotometry. Our calibration media was
distilled water. Unfortunately, many of the result were minus which means we may
had errors that includes random and systematic errors. It is caused by unknown,
unpredictable changes, measuring instruments that is wrongly used by us in the
experiment. On other hand, we could say there has copper or not because of the
blueish color of sample. Finally, we understood the principle of bioleaching
mechanisms and to set up and monitor bioleaching experiment.

Task 4 Microscope
Solid iFe plates with gelrite: our group made solution C and D.
Solution C: 1M FeSO4(pH1,8) 10ml
Solution D: 1M MgSO4x7H2O 1622ul
Cool 60C, mixed both solutions and added 19ml of sterile-filtered solution C and
1622ul solution D. Pour it to plate. That plate used for finger print, locate different
spots and cultivate media.
Bacteria did not grow for the finger print and several spot.
On the plate we tried to grow bacteria from Freiberg. Plate used cultivation Media for
bacteria. We draw curved shape by plastic long object with bacteria. But some of
bacteria did not grow. After that we saw plate on microscope.

Figure 10. Acidithiobacillus test on plate

Figure 11. Microscope

Conclusion:
From the laboratory work we studied microorganisms growth. This laboratory work
helps us to understand practical knowledge of bioleaching process, copper
quantification, bacterial growth on sulfur, type of several liquid and solid media.
This laboratory work gave us to apply theory to practical way. This study gave us
study in depth of bioleaching process.
Reference: Biotechnology in mining Microorganisms as tiny miners paper.