Isolation And Characterization Of Halophilic Bacteria Biology Essay

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Extremophiles are organisms that thrive in extreme conditions. Among the extremophiles, halophiles are organisms that thrive in hypersaline environments with very high concentrations of salt. In this study halophilic bacteria were obtained from sediments of mangroves on the western coast of India. The samples were obtained and processed within two hours in a media containing 50% marine water to obtain maximum population of halophilic bacteria. Morphological and biochemical characterization of the isolates were carried out. The broth was amended with 1000 mg L-1 zinc metal to assess the solubilisation and it was confirmed that within 120 hours the zinc was completely solubilised. The concentration of heavy metal was assessed by AAS. FT-IR was performed to analyse the carboxylic acid group. HPLC was performed to identify the organic acid causing solubilization of heavy metal. The morphological variation in the bacterial culture with zinc was observed using atomic force microscope. Phylogenetic analysis and nucleotide sequencing will be performed for the effective bacterial isolate using bioinformatic tools such as Clustal W, Phylip and Treeview.

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Keywords: Halophiles, Extremophiles, Mangroves, FT-IR, HPLC, AAS, AFM.

Introduction

Mangroves are woody plants that grow at the interface between land and sea in tropical and subtropical latitudes (Chapman 1984). These plants and the associated microbes, fungi, plants and animals constitute the mangrove forest community or Mangal (Spalding 1997). One of the studies, (Kathiresan 2002) shows that the mangrove habitat is found to be adversely affected due to poor microbial counts. Microorganisms in mangroves play an important role in the recycling of nutrients and establishment of mangrove seedlings through the mineralization and also by the production of phytohormones (Ravikumar et al. 2002a and b; Ravikumar et al. 2004). Reduction in the total counts of microorganisms in mangroves affects the growth of the mangrove fauna and flora (Kathiresan 2001). The presence of zinc metal in living systems is considered to be equivocal. Zinc performs the activity of a cofactor in all six classes of enzymes, at trace concentrations, as per IUBMB nomenclature (Valle et al. 1993) and in DNA binding proteins as well (Ho 2004). Organic acid production, production of polysaccharides and proton activity mediate the process of zinc solubilization. In bioleaching, the commercial applications of solubilization of zinc are vast, involving autotrophic Acidithiobacillus thiooxidans and Acidithiobacillus ferroxidans for solubilizing zinc from various ores (Rawalings et al. 2002). Many organic acids like gluconic acid, citric acid and malic acid are produced, for solubilizing zinc, phosphate and other heavy metals, by halophilic bacteria's like Pseudomonas spp. Ability to solubilize essential metals may mirror specific strategies to maintain metal homeostasis in soils either poor or rich in available essential metal ions (Jacqueline et al. 1997) .

In industrial areas these heavy metals are present in their salt forms which result in change in pH, homeostasis and normal flora of the soil affecting plant growth (Martino et al. 2003). The concentration of zinc in the soil may affect the biodiversity of these ecosystems which is due to the dual character of this element. Zinc, can also exhibit toxic effects at low concentrations even though it is required in adequate quantities by living organisms. Many harmful effects of zinc like, nitrogen fixation, microbial biomass production and cyanobacterial proliferation, have been observed in sewage contaminated soils (Di Simine et al. 1998). The present study focuses on the isolation of bacteria from mangroves and their ability in solubilizing zinc.

Materials and methods:

Sampling procedure:

Mangrove sediment samples were collected from Pichavaram, (51km North East of Chidambaram between latitude 11°20'-11°30' N and longitudes 79°45'-79°55' E) (Figure 1) TamilNadu , in sterile screw capped bottle. The samples were then processed within 2 h.

Figure 1. Pichavaram map (Site of sample collection)

Isolation of halophilic bacteria:

For the isolation of halophilic bacterial cultures, 0.1% mangrove sediment was spread on an enriched halophilic medium which composed of 50% marine water and trace elements (such as CuSO4, CoCl2, ZnSO4 etc). The plates were incubated at room temperature for 24 h (Atlas et al. 2005).

Preparation of seed culture:

The isolated colonies were further inoculated in nutrient broth and OD was set to 0.5. The seed culture was inoculated in fresh nutrient broth containing 50% marine water and was kept on shaker (Suthindiran et al. 2009).

Characterization techniques:

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The bacterial isolates were characterized based on morphological and biochemical analysis, the results were compared with Bergey's manual for the identification of genus and species of the isolate.

Morphological characterization:

Morphological characterization of the cultures were done by Gram staining using modified Brown and Bren method (1979) and hanging drop method was performed to check for motility (Swift et al. 1963).

Biochemical characterization:

The bacterial isolates were characterized by various biochemical tests (IMViC Test) i.e. Indole, Methyl red, Voges Proskauer, Citrate, and TSI. Production of catalase enzyme was determined by mixing a single colony with 3% v/v H2O2. Presence of cytochrome c oxidases enzyme was also checked using 1% w/v N, N, N, N tetra methyl p-phenylenediamine reagent which was already impregnated in the disc (Jurtshuk et al. 1983) (Sherman and Cappuccino 2010).

Antibiotic sensitivity of the isolates:

Antibiotic sensitivity test was carried out for the selected isolates using Kirby-Bauer's Method. The isolates were swabbed on Mueller Hinton agar and antibiotic discs like gentamycin (10µg), nalidixic acid (30µg), ciprofloxacin (10µg) and ceftazadime (30µg) were selected and placed on it. The plates were incubated at room temperature for 24h. The zone of inhibition was measured by comparing it with standard NCCLS chart (Mishra et al. 2012).

Atomic absorption spectroscopy (AAS) analysis of zinc solublization by the isolates:

For analyzing the zinc solubilizing ability of the isolates, the halophilic broth was supplemented with 0.1 g of zinc metal powder and was kept on shaking condition at 120 rpm for 5 days. 10ml of suspension culture was taken after every 24h which was centrifuged at 10,000 rpm for 10 minutes at 4°C to separate supernatant from the cells. The pellet was acid digested and both the supernatant and the pellet were given for AAS analysis. The concentration of zinc metal ions present in the pellet and supernatants were estimated using AAS. The concentrations of the standards used were 50, 100, 150 and 200 mg L-1 respectively (Mishra et al. 2012).

Fourier transform infrared spectroscopy (FT-IR) analysis for functional groups:

After 120h, for the FT-IR analysis, Zn-amended and control flask culture suspensions (5 ml) were assessed for the functional groups. The culture suspension was dried in hot air oven at 40°C and given for FT-IR analysis. The infrared spectra of the dried sample was recorded using a Nicolet FT-IR (AVATAR-330) equipped with a narrow-band mercury/cadmium/telluride liquid nitrogen-cooled IR detector. The optical bench was continuously purged with dry air, and the acquisition parameters were 4 cm-1 resolution, 32 co-added interferograms, within a range of 4,000-400 cm-1 (Saravanan et al. 2007).

Organic acid analysis by high performance liquid chromatography (HPLC):

5ml of 120h old culture was centrifuged at 10,000 rpm for 10 minutes at 4ËšC and 20µl of the supernatant was subjected to HPLC (C18 reverse phase column-UV detector) using 0.1% phosphoric acid as mobile phase for 30 minutes and the peaks were observed at 210 nm. The standards used were gluconic acid and malic acid (Pujeri et al. 2012).

Microscopical studies on the morphological variation by AFM:

The Zn-amended and control culture broths were both subjected to AFM analysis. For the AFM, samples (10 ml) were taken from 120 h broth cultures of the Zn-amended and control treatments. A smear was prepared on a clean glass slide and was heat fixed. The slide was positioned on the head holder of AFM, and the tip was allowed to approach the glass slide perpendicularly. Scanning was performed immediately on dried smear at 30°C using AFM (Shimadzu SPM 9500-2J). The cantilever used for studying the structure was Olympus-type OMCL-TR 800 PSA: DC contact mode, 200µm lever length, 800nm lever thickness, 0.15 N/m, 2.0µm tip height and 24 KHz resonance frequency. Different scan sizes (800-12000nm) were used to obtain images at 0 V set point voltage at 1-4 Hz scan speed and data was obtained 400 lines in a constant height mode and 400 points per lines (Saravanan et al. 2007).

Results and Discussion

Isolation:

After incubation for 24h eight different colonies were seen which was purified and maintained in glycerol stock ( Figure 2).

a. b.

c. d.

Figure 2. Pure cultures of JPAN1 and JPAN2 - 2(a), JPAN3 and JPAN4 - 2(b) , JPAN5 and JPAN6 - 2(c) , JPAN7 and JPAN8 - 2(d).

Characterization:

Morphological and Biochemical Analysis of the isolates:

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The morphological analysis showed by Gram's showed that all the eight isolates were gram negative with four coccobacilli and two cocci and two bacilli (Table 1) (Figure 3).

Table 1 Biochemical Test Results (Incubation condition: RT/24 hours)

Test

JPAN1

JPAN2

JPAN3

JPAN4

JPAN5

JPAN6

JPAN7

JPAN8

Control

Indole

-

-

-

-

+

-

-

-

-

Methyl Red

-

+

+

+

+

-

-

-

-

Voges- Proskauer

-

-

-

-

-

-

-

-

-

Citrate

-

-

-

-

-

-

-

-

-

TSI-Slant

K

A

A

A

A

K

A

A

-

Butt

A

A

A

A

A

A

A

A

-

Acid

-

-

-

-

-

-

-

-

-

Gas

-

-

-

-

-

-

-

-

-

Oxidase

-

+

+

+

+

-

+

+

-

Catalase

-

-

-

+

+

+

-

+

-

key: negative : (-) positive ( +), K-Alkaline, A-Acid

Figure 3. IMViC results

Antibiotic Sensitivity Results of the isolates:

Antibiotic sensitivity test was performed using Kirby-Bauer method and was incubated for 24h at room temperature. JPAN 1 showed resistance to nalidixic Acid, ciprofloxacin and ceftazidime whereas intermediate to gentamycin. JPAN 4 showed resistance to all the antibiotics used for this test. JPAN 6 was found to be resistant to nalidixic acid and ceftazidime whereas intermediate to ciprofloxacin and sensitive to gentamycin. JPAN 8 was resistant to gentamycin and ceftazidime, and intermediate to nalidixic acid and ciprofloxacin, on comparing with NCCLS chart (Table 2) (Figure4a, 4b, 4c). On comparing the work done previously, it was observed that gram negative bacteria showed sensitivity to gentamycin and highly sensitive to nalidixic acid (Coronado et al. 1995).

Table 2. Antibiotic Sensitivity Test of the isolates

Colonies

Zone of Inhibition (mm)

Gentamycin

Nalidixic Acid

Ciprofloxacin

JPAN1

13

10

-

JPAN4

11

12

9

JPAN6

16

9

18

JPAN8

11

15

18

a. b.

c. d.

Figure 4. Antibiotic sensitivity results as shown by 4(a) - JPAN8,4(b) - JPAN1 and 4(c) - JPAN4 and 4(d) - JPAN6.

Organic acid analysis by high performance liquid chromatography (HPLC) :

After HPLC analysis of standards-gluconic acid and malic acid (Figure 5a, 5b) peaks were obtained at 15.049 and 14.653 respectively. On comparing the peaks obtained in the test (Figure 5c) with the standard peaks, it was observed that the test peak (14.767) was corresponding to malic acid (Leonora et al. 2009).

a. b.

c.

Figure 5. HPLC analysis results of (a) gluconic acid standard, (b) malic acid standard,

(c) zinc amended JPAN8.

Fourier transform infrared spectroscopy (FT-IR) analysis for functional groups:

The components of the culture broth were analyzed using FT-IR. Figure 6a shows the IR spectra of the dried control and the absorption was obtained at 1631.78cm-1, which typically denotes the presence of COOH group. For the 120h culture broth amended with zinc (Figure 6b), there was a slight shift in the spectrum , which was recorded at 1641.42 cm-1, probably due to the chelation of some Zn ions in the carboxyl terminal (Murugappan et al. 2011).

a. b.

Figure 6. FT-IR absorption spectra obtained from (a) control and (b) zinc amended dried culture sample of JPAN8.

Atomic Force Microscopy (AFM)

The cells were analyzed using AFM to check for any deformation which might have occurred due to metal stress. It was found that the small spherical shaped cocci in the broth without zinc (Figure 7a) became deformed, disorganized, large-sized (approximately double the normal size) in the test sample with zinc (Figure 7b) (Chang et al. 2012).

a. b.

Figure 7. AFM images of (a) normal cocci in control flask and (b) deformed, larger cells in test flask.

Atomic Absorption Spectroscopy (AAS) for determining concentration of Zinc

On performing AAS, it was observed that the concentration of zinc decreased in pellet after every 24h when sample was collected for five consecutive days from 450µg to 31µg ,signifying that the organism was converting insoluble zinc metal into soluble zinc which was present in supernatant (Figure 8) (Table 3) . The solubilization rate of zinc for the organism dealt in our study was found similar to work done on Gluconacetobacter diazotrophicus. (Saravanan et al. 2006).

Table 3. Determination of concentration of Zinc by AAS analysis

Time(h)

Conc. of Zinc in

Supernatant

Pellet

24 h

568

450

48 h

710

250

72 h

872

107

96 h

943

42

120h

956

31

Figure 8. Comparison of conc. of zinc in supernatant and pellet

Conclusion

In our study we conclude that the bacterial isolates of mangrove sediments are highly resistant to antibiotics and also they are capable of solubilizing zinc metal powder. The mechanism involved in the process of solubilization was also studied and it was proved that malic acid aids in the solubilization of zinc metal powder. This was further supported by the FT-IR results where peaks were obtained corresponding to the presence of carboxylic acid. The morphological variation was also assessed and it was found that the zinc treated cells were 10 times larger than the normal cells. Hence we prove in our study that the bacterial isolates of mangroves could be effectively used in the fields for solubilization and uptake of zinc by plants.

Acknowledgments

The Authors would like to thank the lab technicians of The School of Biomedical Sciences and Technology, VIT University and also the VIT University Management for providing the financial support.