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International Journal of Chemical Engineering Research

ISSN 0975 6442Volume 1, Number 2 (2009), pp. 143154 Research India Publications

http://www.ripublication.com/ijcher.htm

Isolation and Characterization Two Novel Indigenous

Acidophilic Species Involved Copper Extraction

(Bioleaching) from the Aliabad Copper Mine in Iran

Jamshid Raheb

1,*, Ali Azimi

2, Mohammad Hadi Ardabili

2, Bahram Nasernejad

3,

Mahmood Arabnezhad2

and Mohammad Javad Hajipour1

1National Institute for genetic engineering and biotechnology, Tehran, Iran,

2National Iranian Copper industries Company, Tehran, Iran,

3Amirkabir University, Tehran, Iran

*Corresponding author E-mail: [email protected]

Abstract

We successfully isolated two novel gram-negative aerobic chemolithotrophic

sulfur-oxidizingbacterium, designated strain NICI1 and strain NICI2, from

mine ores growing under acidophilic conditions. Strain NICI2oxidized

elemental sulfur, thiosulfate, and sulfide tosulfate under oxic conditions and

cause to reduce pH. Strain NICI1 was able to oxidize the Fe++

to Fe+++

. The

rate of oxidation of elemental iron and sulfur from NICI1and NICI2 were

higher than those ofThiobacillus ferrooxidans DSM583 and Acidithiobacillus

thiooxidans DSM622 as standard strains respedtively. Further isolation 16S-

23S rRNA intergenic spacer gene sequence analysisrevealed that newly

isolated strains NICI1 and NICI2 were affiliated withmembers of the genus

Acidithiobacillus. These results demonstrated that NICI1 was

Acidithiobacillusferrooxidans and NICI2 was Acidithiobacillus thiooxidans

(Genbank accession number of 16S-23S intergenic spacer rRNA genefragment of NICI1 and NICI2 are AM404268 and AM404265 respectively ).

NICI1 and NICI2 were capable to extract the copper. Therefore the amount of

copper extraction of these bacteria were measured in shaking flasks and

showed that the efficiency of copper extraction in these bacteria were higher

than standard strains. According to results obtained from this study, the newly

indigenous NICI1 and NICI2 can be a promising candidate for industrial

application in copper extraction (Bioleaching).

Keyword: Acidithiobacillus ferrooxidans DSM583, Acidithiobacillus

thiooxidans DSM622, Bioleaching, chemolithotrophic, 16S-23S rRNA.

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144 Jamshid Raheb et al

IntroductionBioleaching, a general term refers to the conversion of an insoluble metal (e.g. CuS)

into a soluble form (usually CuSO4) by biological oxidation and by applying microbes

[1, 2, 3]. Metals for which this technique is mainly employed include copper, cobalt,

nickel, zinc and uranium. For recovery of gold and silver the activity of leaching

bacteria is applied only to remove interfering metal sulfides from ores bearing the

precious metals prior to cyanidation treatment [3]. The sulfur cycle, driven by sulfate

reduction and sulfide oxidation,is an important cycle of minerals in various sulfur and

sulfide-richenvironments and it is closely

linked with other cycles such as carbon,

oxygen, and nitrogen.For instance, up to 50% of organic matter can be mineralized

by

sulfate reduction in marine sediments [4, 5]. The use of microbes in bioleaching

process has some distinct advantage over traditional physicochemical methods. This

process is more environmental friendly, less consuming energy and useful for thelow-grade ores. And so, it is increasingly being used because of its economical

advantages [3, 6, 7]. Bioleaching is carried out by astonishingly diverse groups of

bacteria. Today, at least 11 putative prokaryote divisions can be related to this

phenomenon [8]. The most common microorganisms; belong to the genera

Thiobacillus and Leptosprillium which are mesophile, acidophile and

chemolithoautotrophe. They obtain energy from oxidation of either ferrous ion to

ferric or reduced sulfur compounds to sulfuric acid [9, 10]. The first step in

development of industrial bioleaching process in a metal mine is to investigate of its

bacterial population. One of the major obstacles encountered in studying the ecology

of these organisms is the difficulty involved in isolating, identifying and enumerating

individual species and strains from an environment which contains a plethora ofstrains with similar metabolic requirements [11]. Yates and his colleagues were the

first to suggest the use of genetic probes to identify different species of bioleaching

bacteria commonly isolated from biomining operation [12]. In PCR-mediated

detection of bioleaching bacteria, the DNA obtained from samples after laboratory

cultures was analyzed by PCR of the spacer regions between 16s-23s intergenic

spacer [13], 16s rDNA [14], or 23s rDNA [13]. In this study the composition of

bacterial populations in Aliabad copper mine was isolated and investigated by

electron microscopy. This analysis consisted of the characterization of the spacer

regions between the 16s and 23s genes in the bacterial rRNA genetic loci after PCR

amplification. The size of the spacer region amplified from DNAs obtained from

samples, were compared with the sizes of that obtained from culture of thebioleaching bacterial reference (Acidothiobacillus Ferrooxidans DSMZ 583) and the

16s-23s intergenic spacer regions sequences in database. Further identification of the

locale species was achieved by sequencing of the PCR product. In the next step the

elemental iron oxidation rate of NICI1 and Acidothiobacillus Ferrooxidans DSMZ

583 were compared. Then the sulfur oxidation rate of NICI2 measured using

elemental sulfur and the pH analysis. Afterward the capability of these two strain

(NICI1, NICI2) in copper extraction (Bioleachig) were evaluated and compared with

standard strains (Acidithiobacillus ferrooxidans DSM583 and Acidithiobacillus

thiooxidans DSM622).

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Isolation and Characterization Two Novel Indigenous 145

Material and MethodsStrains. The strains used in this study were Acidothiobacillus ferrooxidans DSM 583,

NICI1, NICI2 and Acidithiobacillus thiooxidans DSM622.

Sample collection.Ore samples were collected from various damp sites in Iranian

mine (Aliabad) in November 2008, using polypropylene flasks. Samples maintained

at room temperature.

Cultures

Acidothiobacillus ferrooxidans DSM 583 and NICI1 cultured in 9k medium contained

(per liter): KH2PO4 (0.4 gr), CaCl2.2H2O (0.2gr), MgSO4 . 7H2O (0.4

gr),(NH4)2SO4(0.4 gr),FeSO4 . 7H2O (33.3gr), PH: 1.5-2 at 30C and 180 rpm. Strain

NICI2 and Acidithiobacillus thiooxidans DSM622 cultured in 35 medium contained

(per liter): NH4Cl (0.1g), KH2PO4 (0.3g), MgCl2.6H2O (0.1g), CaCl2. 2H2O (0.14g),Sulfur (10g), pH=3 at 30C and 180 rpm. Ore samples treated with H2SO4 (0.5 N) for

three weeks, then, crushed into small particles (0.2-0.5 mm in diameter). 5 gram of

the sample incubated in 200ml of 9k and 35 medium. Cultures medium were

incubated for 1 to 8 weeks until growth was observed microscopically. In cultures the

growth of Acidithiobacillus ferrooxidans was accompanied by a characteristics ferric

precipitation and orange coloring of medium and the growth of Acidithiobacillus

thiooxidans was confirmed by pH changes.

DNA extraction200ml from the cultured media at the end of exponential phase was filtered with

through a Millipore filter (pore size, 0.22m), and washed twice with sterile distilledwater. Thepelletresuspended in 100lit TE (0.01M Tris,0.001M EDTA[PH:7.5]) and

DNA extracted by DNA extraction kit (Cinnagene Co. Cat. No. DN8115C).

PCR

The spacer region between 16s and 23s genes was amplified by PCR using 1.5ng of

DNA, G1 (GAAGTCGTAACAAGG) and L1 (CAAGGCATCCACCCGT)

primers.G1 oligonucleotide is located about 30 to 40 nucleotide upstream from the

spacer boundary and L1 oligonucleotide is located about 20 bases downstream from

the spacer boundary [15]. PCR was performed according to Pizzaro et al., including

25 cycles at 15s at 94C, 7 min at 55C and 30s at 72C and the one additional cycle

was run at 72C for 10 min. electrophoresis was performed on 1% agarose gel withTAE buffer and the DNA was visualize by staining the gel with ethidium bromide.

Scanning electron microscopy

Bacterial samples were fixed on specimen stubs, gold coated by auto spotter coater

(BioRad E5200) and examined by a SEM (Cambridje S-360) at 20kv.

Sequencing

The nucleotide sequence of the PCR bands was directly determined by automated

sequencing 3700ABI (Gene fanavaran, Macrogene Seoul,Korea).

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Iron analysisThe concentration of ferrous ions in solution was determined by titration with

potassium dichromate by using sodium diphenylamine sulfonate as the indicator[7].

pH analysis

NICI2 bacterium converted S0

to SO2-

4 and caused pH changed. The range of pH

changes were measured by pHmeter for 8 days.

Evaluation of copper extraction using NICI1 and NICI2 bacteriaIn this method four flask which contained 6g of copper ore sample powdered and 200

ml of 0.9K medium contained (per liter): KH2PO4 (0.4 gr), CaCl2.2H2O (0.2gr),

MgSO4 . 7H2O (0.4 gr), (NH4)2SO4 (0.4 gr), FeSO4. 7H2O (3.33gr), PH: 1.5-2 were

propagated. Afterward NICI1 bacterium added into the flask number 1, NICI2bacterium added into the flask number 2, NICI1 and NICI 2 bacteria added into the

flask number 3 and flask number 4 used as negative control. These samples were

incubated at 30C and 180 rpm. Then the amount of soluble copper was measured by

atomic absorption.

ResultsEnrichment, isolation and identification of strains NICI1 and NICI2.

For enrichment of strains NICI1, ores samples were gently washed, acidified,

homogenized, and incubated under oxic conditions in 9Kmedium. Iron oxidation

observed

after 3 days incubation. The culture produced constantly in 9K medium, andconcomitant

cell growth was confirmed. The use

of elemental sulfur as an electron

donor was effective for recoveringthe NICI1 population.

After 10 days culture in 35

medium, there was no cell growth of NICI1 as expected. However the NICI2 cell

growth was observed after several transfers in fresh 35 medium which is confirmed

by Light Microscopy.At this point, we examined the purity of the enrichment cultures

by 16S-23S rRNA-based molecular approaches (i.e., PCR amplification

andsequensing). PCR amplification withthe primer set specific for NICI1 clearly

showed the presenceof strain NICI1 in the cultures (Fig. 2). The partial sequence

(about500 bp) of the PCR-amplified 16S-23S intergenic spacer rRNA gene fragment

was approximately identicalto the sequence of NICI1 obtained previously, and PCR

amplification withthe designed primer for NICI2 clearly showed the presenceofstrain NICI2 in the cultures (Fig. 2). The partial sequence (about

400 bp) of the PCR-

amplified 16S-23S intergenic spacer rRNA gene fragment was approximately

identicalto the sequence of NICI2 obtained previously. The accession numbers of

16S-23S intergenic spacer rRNA gene fragment from NICI1 and NICI2 are

AM404268 and AM404265 respectively (GenBank). Furthermore, two novel strains

of thiobacillus were isolated from Aliabad mine copper ores using 9k medium. Since

thiobacillus strains are lithoautotrophe, they are not easily culturable in solid medium.

The samples were prepared and examined for light and scanning electron microscope

(Fig1). The primary examination of SEM as a microscopic result and applying the

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stringent condition of culture medium (pH: 1.5-2) demonstrated that the strain is

Acidithiobacillus species.

Iron oxidation of NICI1Analyses of the growths rate of NICI1 were applied using the Fe

+++production which

was compared with Acidothiobacillus ferrooxidans DSM583 and showed that the rate

of iron oxidation in NICI1 was higher than Acidothiobacillus ferrooxidans DSM583.

According to Figure 3, the amount of iron oxidation of NICI1 were 64% and 95%

after 3 and 5 days respectively. The amount of iron oxidation of Acidothiobacillus

ferrooxidans DSM583 were 32% and 82% after 3 and 5 days respectively.

Sulfur oxidation of NICI2

The rate of sulfur oxidation from NICI2 in 35 medium evaluated measuring the pHchanges. According to Figure 4, the sulfur oxidation activity of NICI2 increased after

3 days and caused to reduce pH of 35 medium from 3 to 1.7 after 5 days. In contrast

the pH of the 35 medium which has no NICI2 bacterium (Negative control) increased

after 5 days (Fig. 4).

Copper extraction using NICI1 and NICI2

The copper extraction of NICI1 was measured in 0.9K medium and showed that the

amount of copper extraction from NICI1 was higher than that ofAcidothiobacillus

ferrooxidans DSM583 up to 6 fold (Fig. 5). The copper extraction of NICI2 was

measured in 0.9K medium and compared with Acidothiobacillus thiooxidans

DSM622, this comparison demonstrated that the amount of copper extraction fromNICI2 was higher thanthat ofAcidothiobacillus thiooxidans DSM622 (Fig. 6). After

the comparison of copper extraction in NICI1 and NICI2, it is demonstrated that the

efficiency of copper extraction in NICI1 was higher than that of NICI2 (Fig. 7).

Finally the copper extraction activity of mixture of both strains NICI1 and NICI2 in

0.9K medium were compared with that of NICI1, NICI2, Acidothiobacillus

thiooxidans DSM622, Acidothiobacillus ferrooxidans DSM583 and control media

(0.9K medium) separately. The data obtained here showed that the efficiency of

copper extraction of mix culture (NICI1 and NICI2) was higher than that of other

cultures (Fig. 8).

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Figure 1: Light (b) and scanning electron microscopy photograph (a) (1000x and

10000x) of NICI1 bacterium.

Figure 2: PCR amplification product of spacer regions between the 16S and 23S

RNA genes of DNA from cultured sample. Lane 1 is NICI2, lane 2 is NICI1, lane 3 is

Acidothiobacillus Ferrooxidans DSM583 and lane 4 isMolecolar weight

marker100bp (Roche).

1a 1b

1 2 3 4

500

400

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Figure 3: The chart of Fe+++

production rate in 9k medium. Square is

Acidothiobacillus Ferrooxidans DSM 583, triangle is NICI1 and diamond is 9K

medium which has no bacteria.

0

0.5

1

1.5

2

2.5

3

3.5

0 2 4 6 8 10

Time(day)

pH

35 NICI2

Figure 4: The chart of pH changes. Square is 35 medium contain NICI2 and diamond

is 35 medium which has no bacteria.

0

10

20

30

40

50

60

70

80

90

100

0 1 2 3 4 5 6

Time (day)

Fe+++(%)

9k DSM583 NICI1

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Figure 5: Comparison of copper extraction from NICI1 and Acidothiobacillus

Ferrooxidans DSM583. Square is Acidothiobacillus Ferrooxidans DSM583, triangle

is NICI1 and diamond is 0.9K medium which has no bacteria.

Figure 6: Comparison of copper extraction from NICI2 and Acidothiobacillus

thiooxidans DSM622. Square is Acidothiobacillus thiooxidans DSM622, triangle is

NICI2 and diamond is 0.9K medium which has no bacteria.

0

5

10

15

20

25

30

3540

0 10 20 30 40Time(day)

Cu(ppm)

0.9k DSM583 NICI1

0

5

10

15

20

25

30

0 10 20 30 40

Time(day)

Cu(ppm)

0.9k DSM622 NICI2

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0

5

10

15

20

25

30

3540

0 10 20 30 40Time(day)

Cu(ppm)

0.9k NICI2 NICI1

Figure 7: Comparison of copper extraction from NICI1 and NICI2. Square is NICI2,

triangle is NICI1 and diamond is 0.9K medium which has no bacteria.

Figure 8: Comparison of copper extraction from mix culture, NICI1, NICI2,

Acidothiobacillus Ferrooxidans DSM583 and Acidothiobacillus thiooxidans

DSM622. Square is NICI2, triangle is NICI1, multiple is mix culture (NICI1 +

NICI2), stare is Acidothiobacillus Ferrooxidans DSM583, circle is Acidothiobacillus

thiooxidans DSM622 and diamond is 0.9K medium which has no bacteria.

0

5

10

15

20

25

30

35

40

45

50

0 5 10 15 20 25 30 35 40

Time(day)

Cu(ppm)

0.9k NICI2 NICI1 Mix (NICI1+NICI2) DSM583 DSM622

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DiscussionThe most microorganisms require suitable environmental factors for optimal growth

conditions. To isolate a specific strain of bacteria, from its habitat, it is necessary to

bring together all sufficient chemical and physical environmental factors in a selective

medium [16]. In this investigation variety of medium with the required factors were

used to isolate an important native microorganism acid mine drainage and bioleaching

environments Acidithiobacillus ferrooxidans from Iranian Aliabad ores. A modified

9K medium was specific in identification and isolation of this strain. The optimal

growth environmental factors for most bacteria arrange in specific range; however

there are broad variety range of environmental factors in a microorganism as a natural

habitat flora due to their structure and chemical combination of ores. The most

common type of bacteria grows well in a natural temperature between 25C and 40C,

while the range for a natural flora is quiet variable [17]. Although the optimaltemperature seems to be 30 C, but it must be modified in pilot plan particularly in

natural habitat conditions. The optimal pH for the most bacteria to grow best is

located between 6.5 and 2.5 [18] since the bacteria isolated from Aliabad ore are

acidophilic grow in pH range much lower than above range. In addition due to

interfere of organic acid with some media ingredients, the external buffer may needed

to maintain the correct pH. In addition to above primary isolation based on

environmental factors, PCR amplification of the 16s-23s intergenic spacer region by

using the highly conserved flanking sequence was applied. Sequence of 16s-23s

spacer regions was identical to the other bacteria of the same group using nucleotide

blast software [19]. The rRNA genes PCR have been extensively characterized by the

investigators such as Moriera and his colleagues (Moriera and Amils 1996). PCRamplification of the 16s, 23s (Moriera and Amils 1996) and 5s region were used to

bacterial identification, by investigators. On the basis of the results obtained, PCR

amplification of 16s-23s spacer region demonstrated that the significant promise as a

tool for the identification of strains belonging to a wider range of bacteria. In this

study for the first time we isolated and identified the newly indigenous NICI1 and

NICI2 bacteria from Aliabad ore in Iran which were involve in copper extraction.

Strains NICI1 is capable to oxidize Fe++

to Fe+++

and follow to this reaction leads to

oxidizing of the copper of minerals. NICI2 is capable to oxidize the sulfur of minerals

to SO42-

which cause to converting the insoluble oxidized copper to soluble CuSO4

compounds. The oxidation of sulfur leads to reduction of the pH in media provided

the optimum growth condition for NICI1 and NICI2 bacteria. The efficiency ofcopper extraction of NICI1 and NICI2 were higher than that ofAcidothiobacillus

ferrooxidans DSM583 and Acidothiobacillus thiooxidans DSM622 respectively. In

addition, the copper extraction activity of NICI2 was higher than that of

Acidithiobacillus ferrooxidans DSM583, however, Acidithiobacillus ferrooxidans is

known as the best bacteria in the bioleaching processes. The data obtained here

showed that the efficiency of copper extraction for both these newly indigenous

NICI1 and NICI2 bacteria were higer than that of standard strains. Furthermore, these

analyses demonstrated that mix culture of NICI1 and NICI2 extracted the highest

amount of copper from the ore. According to the results obtained here, two newly

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indigenous strains NICI1 and NICI2 could be a promising candidate for industrial

application in bioleaching processes.

AcknowledgementsThis work was funded by the National Iranian Copper industries Company.

Borgne, S.L., and Quintero, R., 2003, "Biotechnological process for the refining ofpetroleum,"Fuel. Process. Techno., 81, pp. 155-169.

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