01 HRS Vol 6 3 Sept 2017 min pdf

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ISO LA TION, IDEN TI FI CA TION AND CHAR AC TER IZA TION OF HEAVY MET ALS RE SIS TANT BAC TE RIA FROM ROOT OF Eichhornia crassipes

**Umesh Kumar* and A.P. Garg**

De part ment of Mi cro bi ol ogy, Ch. Charan Singh Uni ver sity Meerut 250 004 (In dia)

*Cor re spond ing Au thor’s E-mail:umesh_micro2015@ya hoo.com

ABSTRACT : The pollution of the environment with toxic heavy metals is spreading throughout the world along with industrial progress. Microorganism and microbial products can be highly efficient bioaccumulators of soluble and particulate forms of metals especially dilute external solution. Microbe related technologies may provide an alternative or addition to conventional method or metal recovery. The present study deals with isolation, identification and characterization of heavy metals resistant bacteria were isolated from roots Eichhornia crassipes in emerging pollutant drainage sites of industries at Unnao, Gajraulla, Hindon River Ghaziabad and Sobhapur village NH-58 Meerut. The eighteen bacterial strains were authentically identified as Pseudomonas aeruginosa, Pseudomonas fluoresencs, Escherichia coli, Micrococcus luteus, Kleibsella pneumonaie, Staphylococcus aureus, and Bacillus subtilis. The isolates showed optimum growth at alkaline pH 6.5 to 8.5 and optimum temperature for 37.0°C to 4.5 °C.The identified isolates are resistant to Arsenic trioxide (As O_{2 3}), Chromium tri-oxides (Cr O_{2 3}), Cadmium di oxides (CdO), Lead oxides (PbO), Zinc oxides (ZnO), Nickel oxides (NiO) and Copper Oxide (CuO). The identified heavy metals resistant bacteria could be useful for the bioremediation of heavy metal contaminated industrial effluent and waste water.

Keywords : Eichhornia sp., heavy met als, bac te rial strain, tol er ance con cen tra tion, The advent of industrial and technological

revolution economic indicators have been considered as the principal criteria for measuring the progress.

The industrial and technological progress, however has been accompanied by growing a negative impact on the environment in terms of heavy metal pollution throughout the world and more wide spread of the contaminants (Prasad, 8).

Industrialization has long been accepted as hallmark of civilization however the fact remains that industrial emanation has been adversely affecting the environment, industrial effluent containing toxic and heavy metals drain into the river which is often the source of drinking water for human population and including animals. Municipality water treatment facility in most of the developing countries at present are not well equipped to remove traces of heavy metals consequently exposing every consumers to unknown quantities of pollutant in the water they consume.

Metals are directly or indirectly involve in all aspect of growth metabolism and differentiation (Beveridge and Doyle, 1). Many metals are essential e.g. K, Na, Mg, Ca, Mn, Fe, Co, Ni, Co, Cu, Zn, and Mo, where as others have unknown essential biological function e.g. Al, Ag, Cd, Sn, Au, Sr, Hg, Tl, and Pb (Gadd, 6). Retaining suitable concentration of essential

metals such as copper and zinc while rejecting toxic metals such as arsenic, lead and cadmium was probably one of the toughest challenges of the living cells (Gatti et al., 7). Bacteria are among the most abundant organism that occur every where on earth.

Heavy metals are increasingly found in microbial habitats due to several natural and anthropogenic process; therefore microbes have evolved mechanism to tolerate the presence of heavy metals by either efflux, complexation or reduction of metals ions) or to use them as terminal electron acceptor in anaerobic respiration (Gadd, 5). Most of mechanism studied involved the efflux of metals ions outside of the cell and genes for tolerance mechanism have been found on both chromosomes and plasmid. Bacteria that are resistant to and grow on metals play an important role in the biogeochemical cycling of those metals ions (Gadd, 5). Conceding the importance of this tolerance mechanism. In the present study microorganism were isolated from contaminated sites of Unnao, Gajraulla, Hindon River Ghaziabad and polluted site of Sobhapur village of NH-58 Meerut.

MATERIALS AND METHODS

Sampling

The plant water hyacinths (Eichhornia crassipes) were collected from four emerging pollutant drained sites of industries of Uttar Pradesh (U.P) India. The samples were collected and brought to laboratory in

Vol. 6, Issue 3; 137-144 (September 2017) ISSN: 2250-2823

Article’s History:

Received: 29-12-16;Revised: 24-01-17; Accepted: 04-02-17

NAAS Rating : 3.78

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the Department of Microbiology and were processed for isolation of microorganisms from roots of Eicchornia crassipes.

Collection of test heavy metals

The tested heavy metals included Arsenic trioxide (As O₂ ₃), Chromium tri- oxides (Cr O₂ ₃), Cadmium di oxides (CdO), Lead oxides (PbO), Zinc oxides (ZnO), Nickel oxides (NiO) and Copper Oxide (CuO) all the reagents were analytical grade (fisher) and NEAST certified.

Preparation of stock solution of test heavy metals oxides

Analytical grade salt were dissolved in HPLC grade water to form 1.0 M stock solutions, it was sterilized through membrane filter (Millipore filters) using 0.22 ìm pore-size. The stock solutions were kept in refrigerator at 4ºC until used.

Media used

For isolation and purification and purification strains were routinely grown in nutrient agar medium (Hi-Media laboratories Pvt. Ltd, Mumbai, India) at 37.0C±0.5°C. For Maximum tolerance concentration (MTC) MH medium with supplement with different concentration of (1.0 to 0.0001 M) of metals oxides.

After autoclaving the medium, it was gently allowed to cooled down and suitable amount of membrane filtered stock solution of test heavy metals to obtain desired concentration of (1.0 to 0.0001 M). It was gently shaken by taking proper care to avoid any air bubbles and was poured aseptically into pre- sterilized Petri plates (9 cm dia) metal tolerance was analyzed on Muller Hinton agar (MHA) medium.

Isolation of microorganisms from roots of Eichhornia crassipes

The roots of Eichhornia crassipes were collected aseptically and washed with 250 ml of half strength Hogland solution. 5 g of fresh roots were transferred into sterile 250 ml Erlenmeyer flask containing 30 ml of sterilized (0.9% Nacl) saline solution, The flasks were plugged with non absorbent cotton and shaken at 200 rpm at 25ºC in temperature on B.O.D. incubator shaker (Remi). The plant roots were blended for 1 minute aseptically and repeated the procedure for 5 times.

Under aseptic conditions with serial dilution of 1/10, 1/100, and 1/1000 of roots suspension were prepared and the 100 µl of each diluted suspensions were inoculated onto nutrient agar plates with and without test heavy metals components. It was spread uniformly

onto nutrient agar plates and it was incubated at 25 ºC

±1.0ºC for 24– 48 h.

Colonial morphology

Isolated colonies of purified strains of grown on solidified agar plates and slants were observed and data was recorded regarding the form (circular, filamentous and irregular), elevation (flat, convex and umbonate), margins (entire, undulate, erose and filamentous) and optical features (opaque, translucent and transparent) of the colonies (Pelczar and Reid, 9).The size, margins, elevation, color and optical features (opaque, translucent and transparent) of the colonies were examined with the help of a stereoscopic binocular microscope (Zeiss Standards 20).

Cellular Morphology

Cells were observed with Gram’s staining (Duguid, 3) under stereoscopic binocular light microscope (Zeiss Standards 20). Shape of the cells (cocci, bacilli and cocco-bacilli) and arrangement of cells (scattered, bunches and chain) along with the Gram- reaction were observed and recorded.

Biochemical characterization

Biochemical tests were carried out according to standard methods (Pelczar and Reid, 9), Tests were recorded as “+” for positive and “–” for negative reactions.

Evaluation of growth of microbes under the influence of different concentration of heavy metals oxides

The pure forms of isolated strains were tested for their resistance under the influences of heavy metals (Arsenic trioxide (As O₂ ₃), Cadmium oxides, (CdO), Chromium tri-oxides (Cr O₂ ₃), Zinc oxides (ZnO), Lead oxides (PbO), Copper Oxide (CuO), and Nickel oxides (NiO). The 1000 ml of nutrient agar medium (Hi- media laboratories Pvt. Ltd, Mumbai, India) was prepared by dissolving 15 gram of mixed powder on hot plate with magnetic stirrer. pH of the medium was adjusted at 6.5 using testing pH strips (E.Merck) and 100 ml of liquid/

melted medium was poured into each 10 bottles (Scott) of 250 ml. The bottles containing medium were autoclaved at 15 Pa for 15 minutes. Desired concentrations (0.0001 to 1.0 M) of test heavy metals oxides was obtained by adding pre-calculated amounts of test heavy metals oxides which was sterilized through membrane filter (0.22 µm pore size). The contents were shaken gently to avoid formation of any air bubbles. The test medium was poured aseptically

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onto 9 cm dia sterile polystyrene disposable Petri dish (Torsen).

100 µl inoculums of Pseudomonas aeruginosa, Pseudomonas fluoresencs, Escherichia coli, Microco-

ccus luteus, Kleibsella pneumonaie, Staphylococcus aureus and Bacillus subtilis of test organism (104 CFU per ml – adjusted by measuring O.D. 600 nm by spectrophotometer (Sigma) was inoculated onto the plates and was spreaded uniformly by “L” shaped Table 1: Colony Morphology characteristics of strains isolates from roots of Eichhornia crassipes.

S. No. Strain code assigned Colour of colonies Form Appearance Optical features

1 ^CCSUPa1 ^{Grey white} ^{Short rods} ^Circular ^{Low convex}

2 CCSUPa2 Grey white Short rods Circular Low convex

3 CCSUPa3 Green or yellow Rods Circular Convex with entire margin

4 CCSUPf4 Grey white Short rods Circular Low convex

5 CCSUPf5 Grey white Short rods Circular Low convex

6 CCSUEc6 Moist grey Shiny Short rods Smooth Convex with entire margin

10 CCSUMl10 Creamy yellow Cocci Circular, Opaque Entire convex

11 CCSUMI1 Creamy yellow Cocci Circular, Opaque Entire convex

12. CCSUM12 Creamy yellow Cocci Circular, Opaque Entire convex

13. CCSUKp13 Off white Rods Circular, Slightly gummy Convex with entire margin 14. CCSUKp14 Off white Rods Circular, Slightly gummy Convex with entire margin

15. CCSUSa15 Gold yellow Cocci Moist Mucoid Convex

16. CCSUSa16 Gold yellow Cocci Moist Mucoid Convex

17. CCSUBs17 Ivory white ^Rods Circular Smooth Flat elevation Nontransparent 18. CCSUBs18 Ivory white Rods Circular Smooth Flat elevation Nontransparent Table 2 : Microscopic examination of strains isolated from roots of Eichhornia crassipes.

S.No. Strains code assigned

Shape Arrangement of cells

Staining behavior

Flagella Motility

1. CCSUPa1 Rods Single -ve +ve Motile

4. CCSUPf4 Rods Single -ve +ve Motile

5. CCSUPf5 Rods Single -ve +ve Motile

6. CCSUEc6 Rods Single/pair -ve -ve Non Motile

10. CCSUMI10 Cocci Irregular Cluster & tetrads +ve -ve Non Motile

13. CCSUkp13 Bacilli Single /pair -ve -ve Non Motile

14. CCSUkp14 Bacilli Single /pair -ve -ve Non Motile

15. CCSUSa15 Cocci Cluster +ve -ve Non Motile

16. CCSUSa16 Cocci Cluster +ve -ve Non Motile

17. CCSUBs17 Rod Single +ve +ve Motile

18. CCSUBs18 Rod Single +ve +ve Motile

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sterile glass spreader. The plates were incubated at 37

±1ºC temperature for 24 h-48 h in B.O.D. incubator.

Growth was recorded on specific concentrations of heavy metals oxides in terms of “+” or “-” and data were presented in table (1.8) the higher concentration which has no growth of viable colonies was termed as MTC.

Optimization of growth of heavy metals resistance isolates

For optimization of growth of resistant bacteria isolates mainly two parameter were observed.

Optimization of pH

5 ml of L.B. broth was added in cleaned sterilized test tube (Borosil) in 9 sets in triplicates. The pH of the medium was adjusted at 3.5, 4.5, and 6.5 using (E.Merck) testing pH strip. It was autoclaved at 15 Pa

for 15 min and inoculated with 20µl of freshly prepared culture of test organism (104 CFU per ml – adjusted by measuring O.D. 600 nm by spectrophotometer Systronics (AU2701)) of the test isolates into test tube.

The three sets of test tube were incubated at 3.5, 4.5, 6.5 and 8.5 separately for 24 h. There after the incubation 20 µl of culture was spread onto nutrient agar plates and colonies were counted. Data are expressed in number of CFU developed by test organism.

Optimization of temperature

5 ml of L.B. broth was added in cleaned sterilized test tube (Borosil) in 9 sets in triplicates and were autoclaved at 15 Pa for 15 min and inoculated with 20µl of freshly prepared culture of test organism (104 Table 3 : Biochemical characteristics of strains isolated from Roots of Eichhornia crassipes.

Isolated Strains From Heavy Metals Contaminated Sites from Eichhornia crassipesTotal isolates = 18 S.

No.

Biochemical characteristics

No. of Isolate

= 3

No. of Isolate = 2

No. of Isolate =

4

No. of Isolate

= 2

= 3

No. of Isolate =

2

= 2

1. Gram strain G⁺ ^G⁺ ^G⁺ ^G⁺ ^G⁺ ^G⁺ ^G⁺

2. Agar slant characteristics

Light brown negative rods

Rods, rough, light green

Colonies metallic

sheen

Cocco bacilli

Cocci Rods Coccus

rod shape 3. Fluorescence Fluorescent

Diffusible Yellow Pigment (Pscudo F Agar medium)

+ve

Fluorescent Diffusible Yellow Pigment (Pscudo F Agar medium)

-ve

4. Growth at temperature 37ºC ^37ºC ^37ºC ^37ºC ^37ºC ^37ºC ^37ºC

5. Growth at pH 5-7 ^5-7 ^5-7 ^5-7 ^5-11 ^5-8 ^5-7

6. Indole test -ve ^-ve ^+ve ^-ve ^-ve ^-ve ^-ve

7. Methyl red test -ve ^-ve ^+ve ^-ve ^-ve ^-ve ^+ve

8. VP test -ve ^-ve ^-ve ^+ve ^-ve ^-ve ^+ve

9. Citrate utilization test +ve ^+ve ^-ve ^+ve ^-ve ^+ve ^-ve

10. Starch hydrolysis +ve ^-ve ^-ve ^+ve ^-ve ^-ve ^-ve

11. Lipid hydrolysis -ve ^-ve ^-ve ^+ve ^-ve ^-ve ^+ve

12. Urease activity -ve ^-ve ^-ve ^+ve ^+ve ^+ve ^+ve

13. Oxidase test +ve ^+ve ^+ve ^+ve ^+ve ^+ve ^+ve

14. Nitrate reduction test -ve ^-ve ^+ve ^+ve ^-ve ^+ve ^+ve

15. H2S production +ve ^-ve ^+ve ^-ve ^-ve ^-ve ^-ve

16. Acid production +ve ^+ve ^+ve ^+ve ^-ve ^-ve ^+ve

17. Gelatin liquification +ve ^+ve ^-ve ^+ve ^-ve ^-ve ^+ve

Identification test Pseudomonas areuginosa

Pseudomonas fluorescens

Escheric hia coli

Bacillus subtilis

Microc occus luteus

Kelbsiella pmeumoni

ae

Staphyl ococcus

aureus Note :–ve (Negative), +ve (Positive), pH (Hydrogen concentration)

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CFU per ml – adjusted by measuring O.D. 600 nm by spectrophotometer Systronics (AU2701)) of the test isolates into test tube. The three sets of test tube were incubated at 25.0ºC, 37.0 ºC and 45.0 ºC separately for 24-96. There after the incubation 20 µl of culture was spread onto nutrient agar plates and colonies were counted, data are expressed in number of CFU developed by test organism at 4 different set of temperature.

RESULTS AND DISCUSSION

Isolation and purification of bacterial strains

18 bacterial strains were isolated from Eichhornia crassipes and designated as CCSUPa1, CCSUPa2, CCSUPa3, CCSUPf4, CCSUPf5, CCSUEc6, CCSUEc7, CCSUEc8, CCSUEc9, CCSUMl10, CCSUMl11, CCSUMl12, CCSUKp13, CCSUKp14, CCSUSa15, CCSUSa16, CCSUBs17 and CCSUBs18.

(Table 1).

Colonial morphology

Different species of isolated bacteria showed different color, shape and size of colonies which greatly

helped in the identification of isolated strains based on colony morphology which varied from grey white, moist grey, shiny, creamy yellow, off white, gold yellow and ivory white (Table 1).

Cellular morphology

On the basis of cellular morphology, stab culture and microscopic examination 61.1% of the isolates were rod shaped, 27.7% Cocci and 11.1% were Bacilli.

38% of bacteria were motile in nature while 61% were non motile. Gram’s staining reaction showed 61.1%

isolated as Gram (-ve) bacteria and 33.8% bacteria as Gram (+ve) (Table 1 and 2).

MTC of isolated strains

Maximum tolerance concentration (MTC) of all the strains against heavy metal oxides (Arsenic trioxide (As O₂ ₃), Cadmium di oxides, (CdO), Chromium tri-oxides (Cr O₂ ₃), Zinc oxides (ZnO), Lead oxides (PbO), Copper Oxide (CuO), and Nickel oxides (NiO) have shown that isolated strains were capable of growing at high concentration of heavy metals in NA medium (Table 3). CCSUPa3, CCSUEc9 and Table 4 : MTC of bacterial strains isolated from roots of Eichhornia crassipes.

CROSS RESISTANCE

Range of Concentration (in 0.0001M-1.0 M)

S.

No.

Strains & lab ID As2O3 CdO Cr2O3 NiO ZnO PbO CuO Control

1. P. aeruginosa (CCSUPa1) 0.01 0.05 0.05 0.1 0.5 0.5 0.5 ++++

4. P. fluoresencs (CCSUPf4) 0.5 0.5 0.1 0.1 0.1 0.1 0.5 ++++

5. P. fluoresencs (CCSUPf5) 0.5 0.05 0.05 0.05 0.05 0.05 0.1 ++++

6. Escherichia coli (CCSUEc6) 0.5 0.05 0.05 0.05 0.5 0.5 0.5 ++++

9. Escherichia coli (CCSUEC9) 1.0 0.1 0.5 0.1 0.1 0.5 0.5 ++++

10. M. luteus CCSUMl10) 0.5 0.1 0.1 0.05 0.5 0.1 0.1 ++++

11. M.luteus (CCSUMl11) 1.0 0.05 0.05 0.1 0.5 0.1 0.5 ++++

12. M.luteus (CCSUMl12) 0.1 0.1 0.001 0.01 0.1 0.5 0.5 ++++

13. K. pneumoniae (CCSUKp13) 0.5 0.05 0.05 0.05 1.0 0.05 0.1 ++++

14. K. pneumoniae (CCSUKp14) 0.1 0.05 0.1 0.05 0.5 0.5 0.05 ++++

15. S. aureus(CCSUSa15) 0.01 0.01 0.1 0.05 0.5 0.5 0.1 ++++

16. S.aureus (CCSUSa16) 0.1 0.05 0.05 0.01 0.1 0.5 0.5 ++++

17. B. subtilis (CCSUBs17) 0.5 0.5 0.1 0.1 0.5 0.1 1.0 ++++

18. B.subtilis (CCSUBs18) 0.05 0.1 0.1 0.05 0.5 0.1 0.5 ++++

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CCSUMl10 showed highest MTC for arsenic trioxide (As O₂ ₃). All isolated strains showed cross resistance against 7 test heavy metals (Table 3).

Determination of optimal growth condition

The optimal growth condition with reference to pH and temperature were determined. The isolates were grown in LB medium with different pH value 3.5, 4.5, 6.5 and 8.5 and incubation was carried out at temperature 25°C 37°C, 45°C and 60.0 °C. It was observed that except Escherichia coli (CCSUEc6) and Micrococcus luteus (CCSUMl10) all strains showed (Table 4) little or no difference in growth in range of pH 3.5 to 6.5. However at allow pH value at 8.5 all strains showed lesser growth in comparison to at acidic or neutral pH value, it shows that data further suggest that isolated strains can be better used in acidic or neutral pH in environment for bioremediation of heavy metals.

However in alkaline pH values of 8.5 the strains could be used for bisorption of heavy metals with lesser ability.

Table 5 reveals the effect of temperature on the growth of different strains on the comparison of growth at different isolates at 25. ±1.0 ºC 0C, 37.0±1.0ºC, 45.0±1.0 º C and 60.0±1.0ºC, it was found that all the 18 strains showed better growth at 37.0±1.0ºC and 45.0±1.0 º C while it was reduced at 60. ±1.0ºC temperature. It suggests that isolated strains can be better used between 37.0±1.0 ºC to 45. ±1.0 0ºC temperature of biosorption of heavy metals.

The pollutant drainage sites of industries that contain elevated concentration of heavy metals are a potential source of heavy metals resistant bacteria (Clausen, 2). For the present studies bacterial strains were isolated from roots of Eicchornia crassipes which were well grown at emerging drainage sites of industries like river, nala because most of small cottage industries are discharge their waste in River nala etc. it was expectation of isolating metals resistance bacteria from that metal contaminated sites. The ability of microbial strains to grow in the presence of heavy metals would be useful in the waste water treatment Table 5 : Optimum pH range for the growth of heavy metals resistance isolates from the roots of Eichhornia crassipes.

pH Tested

organism

3.5 Mean

of CFU/

ml

4.5 Mean

of CFU/

ML

6.5 Mean

of CFU/

ML

C₁ C₂ C₃ C₅ C₆ C₇ C₉ C₁₀ C₁₁

CCSU Pa1 ^4.8×10⁶ ^5.2×10⁶ ^5.9×10⁶ ^5.3×10⁶ ^5.2×10⁶ ^6.2×10⁶ ^6.9×10⁶ ^6.1×10⁶ ^5.0×10⁶ ^6.8×10⁶ ^7.2×10⁶ ^6.9×10⁶ CCSUPa2 ^4.7×10⁶ ^6.6×10⁶ ^5.8×10⁶ ^5.7×10⁶ ^5.5×10⁶ ^6.9×10⁶ ^6.3×10⁶ ^6.2×10⁶ ^4.9×10⁶ ^5.9×10⁶ ^7.1×10⁶ ^6.8×10⁶ CCSUPa3 ^5.5×10⁶ ^5.8×10⁶ ^6.9×10⁶ ^6.0×10⁶ ^7.6×10⁶ ^6.1×10⁶ ^6.0×10⁶ ^6.5×10⁶ ^8.9×10⁶ ^7.1×10⁶ ^8.6×10⁶ ^8.2×10⁶ CCSUPf4 ^5.4×10⁶ ^4.9×10⁶ ^5.8×10⁶ ^5.3×10⁶ ^8.5×10⁶ ^5.2×10⁶ ^7.9×10⁶ ^7.2×10⁶ ^7.9×10⁶ ^8.5×10⁶ ^7.3×10⁶ ^7.9×10⁶ CCSUPf5 ^4.6×10⁶ ^4.2×10⁶ ^3.9×10⁶ ^4.2×10⁶ ^4.6×10⁶ ^6.8×10⁶ ^5.7×10⁶ ^5.7×10⁶ ^5.6×10⁶ ^7.8×10⁶ ^7.1×10⁶ ^6.8×10⁶ CCSUEc6 ^2.3×10⁶ ^2.9×10⁶ ^2.6×10⁶ ^2.6×10⁶ ^5.6×10⁶ ^5.6×10⁶ ^5.9×10⁶ ^5.7×10⁶ ^6.4×10⁶ ^5.8×10⁶ ^6.3×10⁶ ^6.1^×10⁶ CCSU Ec7 ^4.5×10⁶ ^5.3×10⁶ ^5.5×10⁶ ^5.1×10⁶ ^6.2×10⁶ ^6.3×10⁶ ^6.6×10⁶ ^5.8×10⁶ ^6.5×10⁶ ^6.8×10⁶ ^7.1×10⁶ ^6.8×10⁶ CCSU Ec8 ^4.9×10⁶ ^4.8×10⁶ ^6.8×10⁶ ^5.5×10⁶ ^6.8×10⁶ ^5.9×10⁶ ^6.2×10⁶ ^6.3×10⁶ ^7.1×10⁶ ^6.8×10⁶ ^6.3×10⁶ ^6.7×10⁶ CCSU Ec9 ^5.9×10⁶ ^6.2×10⁶ ^6.3×10⁶ ^5.9×10⁶ ^8.5×10⁶ ^5.2×10⁶ ^7.9×10⁶ ^7.2×10⁶ ^8.9×10⁶ ^8.2

×10⁶

8.7×10⁶ 8.6×10⁶

CCSUMl10 ^5.4×10⁶ ^4.8×10⁶ ^5.6×10⁶ ^5.2×10⁶ ^7.1×10⁶ ^6.8×10⁶ ^6.3×10⁶ ^6.7×10⁶ ^8.5×10⁶ ^6.3×10⁶ ^7.3×10⁶ ^7.3×10⁶ CCSUMl11 ^6.8×10⁶ ^4.9×10⁶ ^5.9×10⁶ ^5.8×10⁶ ^6.2×10⁶ ^5.9×10⁶ ^6.2×10⁶ ^6.1×10⁶ ^6.8×10⁶ ^6.9×10⁶ ^8.7×10⁶ ^7.4×10⁶ CCSUMl12 ^5.7×10⁶ ^4.4×10⁶ ^4.8×10⁶ ^4.9×10⁶ ^5.0×10⁶ ^5.9×10⁶ ^6.6×10⁶ ^5.8×10⁶ ^7.0×10⁶ ^6.8×10⁶ ^6.6×10⁶ ^6.8×10⁶ CCSUKp13 ^5.9×10⁶ ^5.8×10⁶ ^6.8×10⁶ ^6.1×10⁶ ^5.9×10⁶ ^7.5×10⁶ ^6.9×10⁶ ^6.7×10⁶ ^8.8×10⁶ ^7.6×10⁶ ^8.8×10⁶ ^8.4×10⁶ CCSUKp14 ^5.6×10⁶ ^4.8×10⁶ ^8.2×10⁶ ^6.2×10⁶ ^6.8×10⁶ ^5.9×10⁶ ^6.2×10⁶ ^6.3×10⁶ ^6.8×10⁶ ^5.9×10⁶ ^6.2×10⁶ ^6.3×10⁶ CCSUSa15 ^4.8×10⁶ ^4.2×10⁶ ^4.6×10⁶ ^4.5×10⁶ ^4.7×10⁶ ^6.6×10⁶ ^5.8×10⁶ ^5.7×10⁶ ^6.4×10⁶ ^5.8×10⁶ ^6.3×10⁶ ^6.1^×10⁶ CCSUSa16 ^4.1×10⁶ ^4.2×10⁶ ^4.3×10⁶ ^4.2×10⁶ ^4.6×10⁶ ^5.5×10⁶ ^8.8×10⁶ ^6.3×10⁶ ^9.1×10⁶ ^8.9×10⁶ ^8.7×10⁶ ^8.9×10⁶ CCSUBs17 ^6.9×10⁶ ^6.8×10⁶ ^5.9×10⁶ ^6.5×10⁶ ^7.5×10⁶ ^7.6×10⁶ ^8.0×10⁶ ^7.7×10⁶ ^8.5×10⁶ ^5.2×10⁶ ^7.9×10⁶ ^7.2×10⁶ CCSUBs18 ^5.2×10⁶ ^5.1×10⁶ ^5.4×10⁶ ^5.2×10⁶ ^4.6×10⁶ ^5.5×10⁶ ^8.8×10⁶ ^6.3×10⁶ ^5.9×10⁶ ^7.5×10⁶ ^6.9×10⁶ ^6.7×10⁶ Note: CFU/ML – Colony forming unit per ml; pH-Hydrogen ion concentration.

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where microorganism are directly involved in the decomposition of organic matter in the biological process for waste water treatment, because often the inhibitory effect of heavy metals is common phenomenon that occurs in the biological treatment of waste water and industrial effluent (Filali et al., 4).

Initially several bacterial strains were purified out of which few showed resistance against heavy metals oxide were revived after being refrigerated for few weeks. Finally all those strains were selected for biochemical characterization which were consistent for their tolerance behavior; the isolated strains were designated as Pseudomonas aeruginosa (CCSUPa1), Pseudomonas aeruginosa (CCSUPa2), Pseudomonas aeruginosa (CCSUPa3), Pseudomonas fluorescens (CCSUPf4), Pseudomonas fluorescens (CCSUPf5), Escherichia coli (CCSUEc6), Escherichia coli (CCSUEc7), Escherichia coli (CCSUEc8), Escherichia coli (CCSUEc9), Micrococcus luteus (CCSUMl10),

Micrococcus luteus (CCSUMl11), Micrococcus luteus (CCSUMl12), Klebsiella pneumoniae (CCSUKp13), Klebsiella pneumoniae (CCSUKp14), Staphylococcus aureus (CCSUSa15), Staphylococcus aureus (CCSUSa16), Bacillus subtilis (CCSUBs17) and Bacillus subtilis (CCSUBs18).

Resistance system not only protects the organism in harsh environment but they also play an important role in the cycling of toxic metals in the biosphere. In some cases bacterial resistance have been shown to be due to difference in uptake and transport of the toxic metals. While in other cases the metals is enzymetically transformed by the oxidation, reduction, methylation or demethylation into chemical species which is either less toxic or more volatile than the parent compound (Williams and Silver, 10). Sampling sites were selected on the basis of heavy metal contamination due to anthropogenic industrial activities Table 6 : Optimum Temperature for the Growth of heavy metals resistance isolates from the Roots of Eichhornia crassipes.

Temperature

Tested organism

25.0ºC Mean

of CFU/

ml

37.0ºC Mean

of CFU/

ML

425.0ºC Mean

of CFU/

ML

C₁ C₂ C₃ C₅ C₆ C₇ C₉ C₁₀ C₁₁

CCSU Pa1 ^4.8×10⁶ ^5.2×10⁶ ^5.9×10⁶ ^5.3×10⁶ ^5.2×10⁶ ^6.2×10⁶ ^6.9×10⁶ ^6.1×10⁶ ^5.0×10⁶ ^6.8×10⁶ ^7.2×10⁶ ^6.9×10⁶ CCSU Pa1 ^4.3×10⁶ ^4.8×10⁶ ^5.0×10⁶ ^4.7×10⁶ ^5.8×10⁶ ^6.2×10⁶ ^6.1×10⁶ ^6.0×10⁶ ^5.9×10⁶ ^7.1×10⁶ ^6.8×10⁶ ^6.6×10⁶ CCSUPa2 ^5.2×10⁶ ^5.9×10⁶ ^5.8×10⁶ ^5.6×10⁶ ^5.5×10⁶ ^5.9×10⁶ ^6.2×10⁶ ^5.8×10⁶ ^5.9×10⁶ ^6.1×10⁶ ^6.2×10⁶ ^6.0×10⁶ CCSUPa3 ^5.4×10⁶ ^6.1×10⁶ ^4.8×10⁶ ^5.4×10⁶ ^6.4×10⁶ ^6.7×10⁶ ^5.8×10⁶ ^6.3×10⁶ ^8.2×10⁶ ^7.1×10⁶ ^7.9×10⁶ ^7.7×10⁶ CCSUPf4 ^5.8×10⁶ ^5.9×10⁶ ^3.8×10⁶ ^5.1×10⁶ ^8.5×10⁶ ^5.2×10⁶ ^7.9×10⁶ ^7.2×10⁶ ^7.9×10⁶ ^7.5×10⁶ ^8.1×10⁶ ^7.8×10⁶ CCSUPf5 ^3.6×10⁶ ^4.8×10⁶ ^5.9×10⁶ ^4.7×10⁶ ^3.6×10⁶ ^4.8×10⁶ ^5.9×10⁶ ^4.7×10⁶ ^5.5×10⁶ ^5.9×10⁶ ^6.1×10⁶ ^5.8×10⁶ CCSUEc6 ^2.3×10⁶ ^2.9×10⁶ ^2.6×10⁶ ^2.6×10⁶ ^3.6×10⁶ ^3.5×10⁶ ^3.9×10⁶ ^3.6×10⁶ ^4.2×10⁶ ^4.7×10⁶ ^5.1×10⁶ ^4.6×10⁶ CCSU Ec7 ^4.5×10⁶ ^5.3×10⁶ ^5.5×10⁶ ^5.1×10⁶ ^6.4×10⁶ ^5.8×10⁶ ^7.4×10⁶ ^6.5×10⁶ ^6.6×10⁶ ^6.5×10⁶ ^{6.9 10}⁶ ^6.6×10⁶ CCSU Ec8 ^4.9×10⁶ ^4.8×10⁶ ^6.8×10⁶ ^5.5×10⁶ ^6.4×10⁶ ^5.9×10⁶ ^6.2×10⁶ ^6.1×10⁶ ^6.2×10⁶ ^6.8×10⁶ ^7.4×10⁶ ^6.8×10⁶ CCSU Ec9 ^6.0×10⁶ ^6.2×10⁶ ^2.9×10⁶ ^5.0×10⁶ ^5.5×10⁶ ^6.6×10⁶ ^5.8×10⁶ ^5.9×10⁶ ^7.9×10⁶ ^7.1×10⁶ ^8.3×10⁶ ^7.7×10⁶ CCSUMl10 ^5.4×10⁶ ^4.8×10⁶ ^5.6×10⁶ ^5.2×10⁶ ^6.5×10⁶ ^6.0×10⁶ ^6.3×10⁶ ^6.2×10⁶ ^6.8×10⁶ ^6.9×10⁶ ^7.1×10⁶ ^6.9×10⁶ CCSUMl11 ^4.8×10⁶ ^4.9×10⁶ ^3.9×10⁶ ^4.5×10⁶ ^6.2×10⁶ ^5.9×10⁶ ^6.2×10⁶ ^6.1×10⁶ ^7.2×10⁶ ^7.6×10⁶ ^8.7×10⁶ ^7.8×10⁶ CCSUMl12 ^5.7×10⁶ ^4.4×10⁶ ^4.8×10⁶ ^4.9×10⁶ ^5.0×10⁶ ^6.2×10⁶ ^6.1×10⁶ ^5.7×10⁶ ^5.8×10⁶ ^5.9×10⁶ ^6.4×10⁶ ^6.0×10⁶ CCSUKp13 ^5.9×10⁶ ^5.8×10⁶ ^6.8×10⁶ ^6.1×10⁶ ^5.8×10⁶ ^7.6×10⁶ ^6.9×10⁶ ^6.7×10⁶ ^8.1×10⁶ ^8.3×10⁶ ^8.6×10⁶ ^8.3×10⁶ CCSUKp14 ^5.6×10⁶ ^4.8×10⁶ ^8.2×10⁶ ^6.2×10⁶ ^6.8×10⁶ ^5.9×10⁶ ^6.2×10⁶ ^6.3×10⁶ ^6.9×10⁶ ^6.6×10⁶ ^6.8×10⁶ ^6.7×10⁶ CCSUSa15 ^4.8×10⁶ ^4.2×10⁶ ^4.6×10⁶ ^4.5×10⁶ ^4.9₁₀6 5.9×10⁶ 6.9×10⁶ 5.9×10⁶ 5.9×10⁶ 6.1×10⁶ 6.2×10⁶ 6.0×10⁶

CCSUSa16 ^4.1×10⁶ ^4.2×10⁶ ^4.3×10⁶ ^4.2×10⁶ ^4.6×10⁶ ^5.5×10⁶ ^8.8×10⁶ ^6.3×10⁶ ^7.8×10⁶ ^7.4×10⁶ ^6.9×10⁶ ^7.3×10⁶ CCSUBs17 ^6.9×10⁶ ^6.8×10⁶ ^5.9×10⁶ ^6.5×10⁶ ^6.3×10⁶ ^6.7×10⁶ ^7.8×10⁶ ^6.9×10⁶ ^6.8×10⁶ ^6.2×10⁶ ^6.9×10⁶ ^6.6×10⁶ CCSUBs18 ^5.2×10⁶ ^5.1×10⁶ ^5.4×10⁶ ^5.2×10⁶ ^5.8×10⁶ ^6.2×10⁶ ^6.1×10⁶ ^6.0×10⁶ ^6.4×10⁶ ^6.9×10⁶ ^7.1×10⁶ ^6.8×10⁶ Note : CFU- Colony forming unit; ML- Mililiter.

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with aim to isolate heavy metals resistance bacteria for biological treatment of industrial effluent.

REFERENCES

1. Beveridge T. J. and Doyle R. J. (1989). Metals ions and bacteria. Wiley. New York.

2. Clausen C. A. (2000). Isolation metal- tolerant bacteria capable of removing copper, chromium, and arsenic from treated wood. Waste Manges.

Res., 18 : 261-268

3. Duguid J. P. (1989). Staining method. In JG.

College JG., JP Duguid. AG Eraser and BP Marmion. eds., Eds., Mackie & McCartney Practical method of microbiology. Churchill Livingstone, New York. 41-51.

4. Filali B.K., Taoufik, J., Zeroual Y., Dzairi F. Z., Talbi M. and Blaghen M (2000). Waste water bacteria resistant to heavy metals and antibiotics.

Curr. Microbiol., 41 : 151-156.

5. Gadd G. M. (1990). Microbial mineral recovery (Ehrlich, H.L. and Brierley, C.l., Eds) McGraw Hill.

New York. Pp. 249-275.

6. Gadd G.M. (1988). Biotechnology. A Comprehensive treatise (Rehm. HJ., Eds VCH Verlagsgesllschatti Weinheim Vol. 6b.pp 401- 433.

7. Gatti D., Mitra B. and Rosen B. P. (2000).

Minireview: E. coli soft metal ion translocating ATPase. The J. Biol.Chem.,. 275 (44) : 34009-34012.

8. Prasad M.N.V. (2011). A state of the art report on bioremediation, its application to contaminated sites in India. Ministry of Environment and forest, Government of India. New Delhi.

9. Pelczar M.J.J. and Reid R.D. (1958). Pure culture and growth characteristics. In: Microbiology.

McGraw Hill Book company New York. Pp.76-84.

10. Williams, J.W. and Silver S. (1984). Bacterial resistance and detoxification of heavy metals.

Enzyme Microb. Technol., 6 : 30-37

q

Citation : Kumar U. and Garg A.P. (2017). Iso la tion, iden ti fi ca tion and char ac ter iza tion of heavy met als re sis tant bac te ria from root of Eichhornia crassipes. HortFlora Res. Spectrum, 6(3) : 137-144.