lecture 43 laboratory experiments in metals biotechnology -...

Lecture 43: Laboratory Experiments In Metals Biotechnology – I NPTEL Web Course

1 Course Title: Metals Biotechnology

Course Co-ordinator: Prof. K. A. Natarajan, IISc Bangalore

Lecture 43

Laboratory Experiments In Metals Biotechnology - I

Keywords: Microscope, Petroff Hausser Counter, Bacterial Isolation

The subject matter of METALS BIOTECHNOLOGY is interdisciplinary dealing with

microbiological, chemical and metallurgical aspects. It thus becomes essential to familiarize

with microbiological aspects such as identification, growth and culturing of relevant

microorganisms, enumeration and growth protocols, bioleaching techniques,

bioremediation strategies as well as other laboratory procedures for culturing mining

bacteria and evaluation of their metabolites. In lectures 43-45, laboratory techniques and

research strategies in metals biotechnology are illustrated [259-266].

Microbiological techniques

Microscopy

Microscope is an optical instrument consisting of a combination of lenses to view magnified

images.

Phase - contrast microscope

Unpigmented living cells are not visible clearly in bright field due to little difference in contrast

between cells and water. A phase contrast microscope adjusts small differences in refractive

indices and cell density into easily detectable changes in light intensity. Living cells can be very

clearly viewed at different magnifications. A photograph of phase contrast microscope is given

in fig.43.1.

Phase contrast microscope has two additional plates - annular diaphragm and phase shifting

plate. Such a modification permits only a ring of light to pass through the condenser and

subsequently to the objective. When incident light falls on a specimen, two rays emerge. When




direct and defracted beams join, phase differences are apparent and contrast in the image is

obtained.

Phase contrast microscopy is useful to assess microbial motility, endospores, shape, and

microbial encrustations or inclusions. For bacterial cultures generally used in

biohydrometallurgy, such as Acidithiobacillus sp. phase contrast microscopic examination is

very useful.




Cell Count using Petroff Hausser counter

Bacterial cell counts can to be made using Petroff Hausser counter which is depicted in fig. 43.2.

Cell count of bacteria has to be taken by directly putting a drop of culture on to the counter slide

and cell number measured under a phase contrast microscope. The counter consists of ruling

covering a square millimeter. The center square millimeter is ruled into 25 groups, each

consisting of 16 squares. All the 25 groups are separated with triple ruling where as each of the

single squares of 16 square are singly ruled. The height of the ruling wires is 0.02 mm in height.

The area of each squares are 1/400 mm2. The bacterial cells have to be counted in this center

square.

Typical calculations are given below:

Depth of small square = 1/50 mm

Area of small square = 1/400 mm2

Fig. 43.1: Phase Contrast Microscope




Volume of small square = 1/50 x 1/400 mm3 = 1/50 x 1/400 x 10

-3 cm

3.

No of cells per ml = Average no. of cells counted per small square/volume in cm3

No of cells per ml = Average no. of cells counted per small square x 20.000 x 1000

No of cells per ml = Number of cells x 20 x 106 / 16

No of cells per ml = Number of cells x 1.25 x 106

Fig 43.2: (a) A photograph and (b) Schematic diagram of a Petroff Hausser counting chamber

A

B




Isolation, evaluation and characterisation of mining microorganisms

Sources for isolation of Acidithiobacillus, Leptospirillum and Sulfate Reducing Bacteria are the

following:

Mine water collected at sulfide ore sites

Acidic mine water drainage

Mine sediments with moisture

Ore samples from open cast and underground mines of coal and sulfide mineral ore deposits

Solid and liquid samples from tailing dams.

The above samples are inoculated in enrichment media; to obtain enrichment cultures. One can

also use desired prescribed media for various acidophiles, neutrophilic and anaerobic SRB.

Isolation of acidophiles

Enrichment in 9K medium – Appearance of brownish colour and ferric-salt precipitation on

incubation. For adaptation, desired metal ions or metal ores and concentrates can be present

during bacterial subculturing and growth.

Isolation of pure culture using solid agar 9K medium - silica gel plates can also be used

impregnated with the medium. From the colonies grown on solid medium, inoculations are

made into liquid nutrient media. Purity of cultures is verified.

Method of end-point dilution can be used to isolate pure cultures of At.ferrooxidans.

Purity check for possible contamination by other acidophiles.

The isolated culture are to be maintained in 9K medium (in the presence of metal ions or ore

concentrates, if necessary) in a refrigerator at 40C. Transfers should be made atleast once in two

weeks.

Various isolated strains of At.ferrooxidans can be classified with respect to their respective iron

oxidation rates (or iron oxidation index).

For At.thiooxidans, rate of sulfate production from sulfur can be used for similar classification.

For Sulfate Reducing Bacteria, sulfate reduction rates can be used for classification.




Media composition

The media composition for At.ferrooxidans, At.thiooxidans and Leptospirillum ferrooxidans are

given in table 43.1, 43.2 and 43.3 respectively.

Table 43.1: Silverman and Lundgren medium (9K medium) for Acidithiobacillus ferrooxidans

Components g / L

(NH4)2SO4 3.0

KCl 0.1

K2HPO4 0.5

MgSO4 0.5

Ca(NO3)2 0.01

Distilled water 1000 ml

The above constituents have to be dissolved in 1000 ml distilled water and the pH has to be

adjusted to 1.9-2.0 with 10M H2SO4 and sterilize by autoclaving (Solution A). 44.8 g of

FeSO4.7H2O should be dissolved in 100 ml of medium and filter sterilize (Solution B). The

media has to be prepared by mixing the two solutions.

Table 43.2: Basal media for Acidithiobacillus thiooxidans

Components g / L

(NH4)2SO4 2.0

MgSO4 0.5

K2HPO4 0.25

Sulphur powder 10.00

H2O 1000 ml

pH 2.4

The medium should be sterilized without Sulphur powder by autoclaving. Later Sulphur powder

should be added and sterilize by tyndallization.




Table 43.3: Silverman and Lundgren medium (9K medium) for Leptospirillum ferrooxidans

Components g / L

(NH4)2SO4 3.0

KCl 0.1

K2HPO4 0.5

MgSO4 0.5

Ca(NO3)2 0.01

Distilled water 1000 ml

pH 1.2

The above constituents have to be dissolved in 1000 ml distilled water and the pH has to be

adjusted with 10M H2SO4 and sterilize by autoclaving (Solution A). 44.8 g of FeSO4.7H2O

should be dissolved in 100 ml of medium and filter sterilize (Solution B). The media has to be

prepared by mixing the two solutions.

Growth of chemolithotrophs on solid media

Special techniques are used to grow autotrophic bacteria on solid media. Isolated colonies grown

on plates have to be transferred to liquid medium and cultures should be maintained only in

liquid media for preservation.

Solid medium for the growth of Acidithiobacillus ferrooxidans

The composition of solid or agar medium for Acidithiobacillus ferrooxidans is shown in table

43.4.

Solution A: FeSO4.7H2O (33.4g/L)

Table 43.4: Solution B: Basal salts

Components g/ L

(NH4)2SO4 6.0

KCl 0.2

MgSO4.7H2O 1.0

Ca(NO3)2 0.02

pH has to be adjusted to 2.5




Solution C

Purified agar has to be added at a concentration of 0.7%. Agar should be added to distilled water

and stirred for 20 min and the supernatant discarded. The same process has to be repeated thrice

to remove all the organic matter.

Solution A has to be filter sterilized. Solutions B and C are autoclaved separately, cooled and all

the three solutions have to be mixed. Later the medium should be poured into petriplates.

Identification Studies

The characteristic features of genus Acidithiobacillus (At.ferrooxidans, At.thiooxidans)

Strictly aerobic

Gram-negative

Motile ,Mono flagellated

Rod shaped

Cells are 0.3 x1 to 3 µm in size

Non-spore forming; best growth at 25-35°

Able to oxidise iron, sulfide, elemental sulfur, thiosulphate.

T.thioparus , grows at neutral pH

Responsible for acid mine drainage, due to production of sulfuric acid

The characteristic features Leptospirillum ferrooxidans

Vibrious, spiral-shaped, pseudococci

0.1µm in diameter and 6µm to 12µm in length

Gram negative, obligate chemolithoautotrophs




Oxidises ferrous ions to ferric

Motile (mono flagellated), obligately aerobic, with a generation time of 6-16h

Oxidase positive, catalase or peroxidase positive

Optimum temperature is 28 to 30°C

On solid media, colonies appear in small red-brown colour

Some phenotypic and biochemical characteristics of At.ferrooxidans, At.thiooxidans and

Leptospirillum ferrooxidans are given in table 43.5.

Table 43.5: Phenotypic and biochemical characteristics

Phenotypic

characteristics At. Ferrooxidans L.ferrooxidans At.thiooxidans

Cell morphology Rods 0.5-1.5 m

Vibrios 1 m

Spirilla

(2-5 turns)

Rods 0.5-1 m

Endospores - - -

Motility (+) ++ (+)

Reaction to Gram’s

stain - - -

Fe2+

oxidation + + -

S oxidation + - +

Biochemical characteristics

Oxidase test + + -

Catalase test + + -

Symbols: +, positive; -, negative; ++, strongly motile; (+), limited motility

Typical scanning electron micrographs and digital photographs of At.ferrooxidans,

At.thiooxidans and Leptospirillum ferrooxidans are illustrated in fig.43.3.




L. ferrooxidans

At. ferrooxidans

At. thiooxidans

Growth of At. ferrooxidans and

L. ferrooxidans in liquid media

Growth of At. thiooxidans in liquid

medium

Growth of At. ferrooxidans and

L. ferrooxidans on agar plates

Fig. 43.3: Scanning electron micrographs and digital photographs of At.ferrooxidans, At.thiooxidans and

Leptospirillum ferrooxidans




A typical growth curve of bacteria is depicted in fig. 43.4 which shows the initial lag,

logarithmic, stationary and decline or death phase of bacteria. Bacteria are cultured in

Erlenmeyer flasks and placed in a shaking incubator to attain full growth. (fig. 43.5).

Growth kinetics

Growth kinetics of Acidithiobacillus ferrooxidans

Figure 43.6 shows the typical growth curve of Acidithiobacillus ferrooxidans. As can be

observed, the lag phase for this strain extends up to 12h. This is followed by the exponential

growth phase up to 38h. The maximum cell number corresponds to 2x108 cells /mL. The redox

potential values continuously increase from 280 to 540mV with increase in time. This is in

agreement with the increase in the ferric concentration and decrease in the ferrous concentration.

The pH increases initially from 1.9 to 2.5 and then drops to about 2.2. The decrease in pH may

be attributed to the formation of acidic ferric sulphate. There was also a simultaneous decrease in

Fig. 43.4: Bacterial growth in batch mode Fig. 43.5: Shaking incubator




dissolved ferric iron concentration observed, presumably due to its precipitation either as ferric

hydroxide or jarosite or both.

The change in the ferrous and ferric concentrations as a function of time during growth of

At.ferrooxidas shown in Figure 43.6. The time taken for the complete decrease in ferrous iron

concentration is about 40h. A steep increase in ferric concentration is observed from 10 to 40h.

The trends with respect to increase in the ferric concentration complement the decrease in the

ferrous concentration as a function of time. This further testifies to enhanced bacterial activity.

Growth kinetics of Leptospirillum ferrooxidans

Growth curve of Leptospirillum ferrooxidans is similar to that of Acidithiobacillus ferrooxidans

except for the pH. For Leptospirillum ferrooxidans growth pH is 1.2.

Growth kinetics of Acidithiobacillus thiooxidans

From Figure 43.7 it is evident that the lag period of growth extends up to 24h, beyond which the

exponential growth phase can be observed up to 130h. The maximum growth attained was 8 x

108 cells/ml.

0 10 20 30 40 50 60 70

4.0x107

8.0x107

1.2x108

1.6x108

2.0x108

No

.of

ce

lls

/ m

L

Time (hours)

Cell count

250

300

350

400

450

500

550

ESCE

ES

CE

in m

V

1.9

2.0

2.1

2.2

2.3

2.4

2.5

pH

pH

0 10 20 30 40 50 60 70

0

2

4

6

8

10

Fe

2+ a

nd

Fe3+

co

nc

(g /

L)

Time (hours)

Fe3+

Fe2+

Fig43.6: (A) Cell number, pH, ESCE as a function of

time during growth of At. ferrooxidans (B) Ferrous and ferric concentration as a function of

time during growth of At.ferrooxidans

A B




The variation in the pH values for the sample as a function of time is shown in Figure 43.7. As

the cell concentration increases, the pH of the solution decreases from 2.0 to 0.5 by 220h. Such a

significant pH decrease during bacterial growth is due to production of sulphurous and sulphuric

acid, by oxidation of the sulphur present in the medium.

From Fig. 43.7 it is also evident that the sulphate concentration continuously increases as a

function of time. The sulphate concentration increases from 1.6g to 28g/L in 240h.

Fig. 43.7: (A) Cell number as a function of time during

growth of Acidithiobacillus thiooxidans (B) pH & SO4 conc. as a function of time during

growth of Acidithiobacillus thiooxidans

A B




Estimation of Iron

Iron can be estimated spectrophotometrically by 1, 10-phenanthroline method. Iron (II) reacts

with 1, 10-phenanthroline to form an orange-red complex, [(C12H8N2)3 Fe]2+

. Iron (III) should

be reduced with hydroxylamine chloride and then reacted with 1, 10-phenanthroline. The color

intensity of iron-1, 10-phenanthroline complex is independent of acidity in the pH range 2-9 and

is stable for long periods. 1mL of known concentrations of iron (II) has to be taken and buffered

with 2 ml of 0.2M potassium hydrogen phthalate solution at pH 3.9. To this, 10 ml of 0.15%

solution of 1, 10-phenanthroline should be added and made up to 25 ml using pH 2 solution. The

absorbance is measured using UV- visible spectrophotometer. The absorbance need be measured

against a reagent blank at 512 nm wavelength. A similar procedure should be adopted for total

iron (as ferrous) after reduction of the sample with 4mL of 10% hydroxylamine hydrochloride

solution for half an hour. The calibration graph is shown in Fig. 43.8 using which, the

concentration of iron in the samples can be determined.

Concentrations of total iron, iron as ferric and iron as ferrous can be determined.

Fig. 43.8: Calibration curve for Iron




Estimation of Sulphate

Sulphate has to be estimated spectrophotometrically after precipitation as barium sulphate. 1ml

of the sulphate sample should be added to 5 ml of conditioning reagent and left undisturbed for

30 min. For the preparation of conditioning reagent, dissolve 75g of NaCl in 30 ml concentrated

HCl, 50 ml glycerol, 100 ml isopropanol, and the solution should be made up to 1000 ml using

Milli-Q water. Then the solution has to be made up to 100 ml using Milli-Q water. To this 0.3g

of barium chloride should be added with continuous stirring. The barium sulphate precipitate so

formed is allowed to stand for 5min and then the concentration of sulphate has to be determined

in the spectrophotometer at 420 nm wavelength using against a reagent blank. A typical

calibration graph is shown in fig.43.9.

Calibration curve for sulphate

Fig. 43.9: Calibration curve for Sulphate




Estimation of Copper

Copper can be estimated using atomic absorption spectrophotometer in air acetylene oxidizing

flame at 327.4 nm wavelength. Stock solution to be prepared by dissolving 1g of copper metal in

1:1 HNO3 and making it up to 1000 ml using Milli-Q water. Various solutions for calibration

ranging from 1-4 mg/l of copper have to be prepared from the stock solution. The calibration

curve is shown in fig. 43.10.

Fig. 43.10: Calibration curve for Copper

lecture 43 laboratory experiments in metals biotechnology -...

Documents