Luận văn bioprospecting of haloarchaea for the development and expression of potent novel antioxidant enzymes

Isolation and screening of Haloarchaea The water samples were serially diluted by taking 1 ml from the mixed water dilution was used for plating on Zobell marine agar plates by spread pl

BIOPROSPECTING OF HALOARCHAEA FOR THE DEVELOPMENT AND EXPRESSION OF POTENT

NOVEL ANTIOXIDANT ENZYMES

A THESIS

Submitted by

S MURUGAN

(Reg No 8177) BIOCHEMISTRY

in partial fulfillment of the requirements for the degree of

DOCTOR OF PHILOSOPHY

MANONMANIAM SUNDARANAR UNIVERSITY

TIRUNELVELI - 627 012

APRIL - 2018

MANONMANIAM SUNDARANAR UNIVERSITY

TIRUNELVELI - 627 012

CERTIFICATE

The research work embodied in the present thesis entitled

“BIOPROSPECTING OF HALOARCHAEA FOR THE PRODUCTION

has been carried out in the Department of Biochemistry of ManonmaniamSundaranaruniversity in Tirunelveli The work reported herein is original and does not form part of any other thesis or dissertation on the basis

of which a degree or award was conferred on an earlier occasion or to any other scholar

I understand the University’s policy on plagiarism and declare that the thesis and publications are my own work, except where specifically acknowledged and has not been copied from other sources or been previously submitted for award or assessment

MURUGAN S

RESEARCH SCHOLAR

Dr R.D STEVENS JONES, Dr K.R.T ASHA,

Co- SUPERVISOR, SUPERVISOR,

Associate professor, Assistant Professor

Department of Zoology, Department of Biochemistry
Scott Christian college, Govt Arts College,

ACKNOWLEDGEMENT

I am extremely thankful to GOD ALMIGHTY for giving me strength, health

and support for successfully completing this work at the right time

I wish to record my deep sense of gratitude and profound thanks to my

research supervisor Dr K.R.T ASHA, Assistant Professor, Department of

Biochemistry, Government Arts College, Paramakudi, Ramanathapuram District, Tamil Nadu,for her keen interest, inspiring guidance, constant encouragement with

my work during all stages, to bring this thesis into fruition

I am extremely indebted to my joint supervisor Dr STEVENS JONES,

Professor Department of Zoology, Scott Christian College (Autonomous) for his valuable suggestions and support during the course of my research work

I express my sincere thanks to Dr Pamaela Oliver, Department of English, Lekshmipuram College of Arts and Science, Neyyoor, Dr A RamalingomPillai,

Department of Chemistry, Lekshmipuram College of Arts and Science, Neyyoor for their valuable encouragement throughout the study

I am extremely indebted to Dr A Tamil Selvi, Dr C Sunithra,

Dr P NandhaKumari, Department of Zoology, Lekshmipuram College of Arts and Science, Neyyoor,Dr N.C.J PackiyaLekshmi, Department of Microbiology,

Udhaya College of Arts and Science, Vellamadi, for theirvaluable suggestions and support during the course of my research work

I wish to express my heartfelt thanks to The Management, Principal and the Staff members (Teaching and Non-Teaching) of Biochemistry Department of Lekshmipuram College of Arts and Science, Neyyoor

I am deeply indebted to my beloved Parents, Wife, Children and my family members for their moral support and constant encouragement at every stage of this

work

S MURUGAN

2.3.14 Growth in various N2 sources 45

3.2.6.3 Estimation of catalase by dichromoacetic

3.2.8.2 Purification by chromatography 79

3.3.3.3 Effect of enzyme concentration in SOD

4.2.1 Sample preparation 127

4.2.4 Gene ontology by DAVID software and Protein

5.2.2 Determination of invitro cardiac protective activity of

5.3.1.1 Antiproliferative effect of SOD on cultured

5.3.1.2 Invitroantiproliferative effect of SOD against

5.3.2.4.Determination of invitroantiproliferative effect

Table

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Figure

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No

Figure

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CHAPTER - I 2.0 ISOLATION AND CHARACTERIZATION OF

HALOARCHAEAL STRAIN

2.1 INTRODUCTION

An extremophile is an organism that thrives in physiologically or geochemically extreme conditions that are detrimental to most life on earth (Rampelotto, 2010) In the 1980s and 1990s, biologists found that microbial life has

an amazing flexibility for surviving in extreme environments-niches that are extraordinarily hot or acidic Some scientists concluded that life may have begun on

scientists reported that bacteria were found living in the cold and dark in a lake buried

researchers reported data that suggested microbial life forms thrive in the Mariana

Trench, the deepest spot on the earth (Choi et al., 2013; Glud et al., 2013) Other

researchers reported related studies that microbes thrive inside rocks upto 1900 feet below the sea floor under 8500 feet of ocean off the coast of the northwestern United

States (Choi et al., 2013; Oskin et al., 2013) According to one of the researchers, we

can find microbes everywhere; they are extremely adaptable to conditions and survive

wherever they are (Choi et al., 2013)

Different types of halophiles have solved the problem how to hope with salt stress (and often with other forms of stress as well) in different ways, so that the study

of microbial life at high salt concentrations can answer many basic questions on the adaptation of microorganisms to their environments Most known halophiles are

relatively easy to grow, and genera such as Halobacterium, Haloferax, and

Haloarcula have become popular models for studies of the archaeal domain as they are much simpler to handle than methanogenic and hyperthermophilic Archaea Haloarchaea belongs to order Halobacteriales and family Halobacteriaceae are

dominant microorganisms requiring hypersaline environment for their growth This

family contains 130 species of organisms (Oren et al., 2012a, 2012b; Tindall et al.,

1992) distinguished as pigmented organisms because of the presence of ruberins and

A wide variety of media are used for the cultivation of halophiles and halotolerant bacteria Choosing appropriate media and growth conditions is important and published media are typically associated with a particular microbial genus or species Extreme halophiles are predominantly archaea and are cultured at warmer

temperatures (37˚C) with salinities of 20% or more (Schneegurt et al., 2004)

Hypersaline media can be divided into complex media that include organic components for which exact chemical formulae are not known and defined media where all components can be described by chemical formulae The media for extreme halophiles have elevated levels of magnesium and the source of water used for preparing hypersaline media varies (Yu and Kawamura, 1987; Wais, 1988; Bertrand

et al., 1990) Filtered sea water or salt plains brine were effective bases for growing halotolerant Cyanobacteria and algae (Henley et al., 2002; Kirkwood and Henley,

2006)

Many halophilic and halotolerant microbes isolated to date are neutrophiles,

growing best in media with pHs from 6.8 to 7.5 (Vreeland et al., 2002; Caton et al.,

2004) Anaerobes grow over a similar pH range of 6-7 (Oren 1983a, 1986; Yu and Kawamura, 1987) Alkaliphilic organisms are grown at pHs of 8-10 (Soliman and Truper, 1982)

Therefore, in this era researchers are turning their attention towards this less explored and useful kingdom The southern coast of Kanyakumari harbors a variety of ecosystems for such studies; some of the areas have been already excavated while some are underway Hence this chapter deals with the isolation, optimization and characterization of Haloarchaeal strain with potent antioxidant enzymes

2.2 MATERIALS AND METHODS

2.2.1 Sampling site

The study site was Puthalam saltpans of Kanyakumari district The total area

of the salt pan is 150 acres This area is under the influence of very low tides and surrounded by marshy land The climate is generally warm and humid, fluctuating from a minimum of 20˚C in the month of December and 42˚C in May The salt producing season is normally during the non-monsoon months Each pan is surrounded by ‘bandhs’ or mud borders on all four sides which prevent siltation and facilitate to regulate the flow of water Water gets heated up and evaporates with crude salt In the peak salt producing season, salt is harvested and collected daily The physico-chemical characterization of water samples were analyzed according to Abbasi, 1988

2.2.2 Collection of samples

Water samples were collected from the saltpan at 3 different sites The collected samples were transferred to sterile polythene bags to prevent direct contact with air and were transported to the laboratory in an ice box for further examination

2.2.3 Isolation and screening of Haloarchaea

The water samples were serially diluted by taking 1 ml from the mixed water

dilution was used for plating on Zobell marine agar plates by spread plate method

bacterial isolates were obtained by repeated sub culturing on marine agar plates, and were stored in agar slants with the help of 15% glycerol

2.2.4 Screening of SOD activity

The isolated halobacterial strains were inoculated in conicalflask containing marine broth supplemented with 2% pyrogallol and incubated the flask in water bath

phase culture by centrifuging the culture broth at 3000 rpm for 10 minutes The supernatant obtained was used as enzyme source

The 2% pyrogallol in 0.05M Tris HCl buffer at pH 7.5 acts as the substrate The SOD activity was assayed by using Marklund and Marklund method (1974) The halobacteria with maximum SOD activity was screened using the assay procedure

2.2.5 Screening of catalase activity

The isolated halobacterial strains were inoculated in concical flask containing marine broth with 2% hydrogen peroxide The flask was incubated in water bath with shaker at 37ºC for 12 days The early stationary phase culture was taken and centrifuged at 3000 rpm for 10 minutes The supernatant obtained was used as

substrate The catalase activity was assayed by using dichromoacetic acid method (Arsu K Sinha, 1972)

2.2.6 Biochemical characterization

The culture characterization was identified by performing the biochemical tests such as gelatinase production test, cellulase production test, catalase test, oxidase

carbohydrate test, nitrate reduction test, citrate utilization test, Gram staining and motility test

2.2.6.1 Gelatinase production test

The organism was inoculated in the gelatin agar (Himedia) and incubated for

12 days at 37˚C After incubation, 0.1 % mercuric chloride solution was added and allowed to stand for 5-10 minutes Formation of clear zone around the colony indicated positive result

2.2.6.2 Cellulase production test

The isolates were inoculated in the Czapek medium (Himedia) (CM) supplemented with NaCl After incubation, the plates were flooded with 1ml solution

of hexaethyltrimethyl sulphate The plates, which showed clear zone around the growth, were considered as cellulase positive

2.2.6.3 Catalase test

Agar slant was inoculated with the test culture and was incubated at 37˚C for

12 days, 1ml of 2% hydrogen peroxide was added to flow down the slant and was examined immediately and after 5 minutes for the evolution of bubbles which indicated positive test

2.2.6.4 Oxidase test

Strips of whatman No.1 filter paper was soaked in a freshly prepared 1% solution of tetramethyl paraphenyl diamine dihydrochloride and dried in oven at 40˚C The isolate was streaked on it with sterile platinum loop An intense deep purple colour appearance within 5 – 10 seconds indicated positive reaction, and a negative reaction indicated by the absence of colouration

2.2.6.5 Urease test

Urease is a hydrolytic enzyme that attacks the nitrogen and carbon bonds in amide compounds like ammonia and urea and releases alkaline end product such as ammonia The bacterial culture was inoculated in Christensen’s agar slant and incubated at 37˚C and the reaction was recorded after 4, 8, and 12 days of incubation The production of enzyme urease was detected by a change in colour of the medium from yellow to purple

2.2.6.6 H 2 S production test

TSI (Triple Sugar Iron Test) was used to differentiate different group of

Enterobacteriaceae from other gram negative bacilli The TSI slants contain 1% each

of lactose, sucrose and glucose 0.1% The phenol red acid base indicator was incorporated in the medium to detect carbohydrate fermentation

The bacterial isolate was inoculated in the TSI agar slant by stabbing the butt down to the bottom and then streaking the surface of the slant, incubated at 37˚C for

7 – 12 days The result would be either of following three types

a Acid butt, alkaline slant (yellow butt, red slant) – glucose has been fermented but either lactose or sucrose

b Acid butt, acid slant (yellow butt, red slant) – lactose and / or sucrose has been fermented

c Alkaline butt, alkaline slant (red butt, red slant) – either glucose or lactose or sucrose has been fermented

Gas production : indicated by bubbles in the butt or the agar may be broken or

pushed upwards

2.2.6.7 Indole production test

Tryptone broth tubes were prepared and sterilized at 121˚C for 15 minutes The culture was then inoculated and incubated at 37˚C for 7 – 12 days After the incubation, a few drops of (0.2 ml) Kovac’s reagent was added into the tubes and the result was observed The development of bright red colour at the interface of reagent and medium indicated the positive result The absence of colour change at this interface indicated the negative test

2.2.6.8 Carbohydrate test

The bacterial species isolated were checked for their ability to ferment various carbohydrates (lactose, mannitol, sucrose and dextrose) Oxidation – fermentation basal medium was prepared with 1% of different sugars each and sterilized Inoculated for 2 or 3 loopful of 7 – 12 days old broth culture into sterile fermentation medium and incubated for 24 – 48 h at 30˚C Change in the colour of the medium to yellow indicates acid reaction

2.2.6.9 Nitrate reduction test

Nitrate broth (HiMedia) was prepared and 5ml was dispensed in the test tubes The isolates were inoculated into nitrate broth The tubes were then incubated at 37˚C for 96 hours After incubation, 0.1 ml of sulphanilic acid and alpha naphthalamine was added and mixed well Finally, the results were observed

2.2.6.10 Citrate utilization test

Simmon’s citrate agar was prepared and dispensed on test tubes and sterilized

at 121˚C for 15 minutes and allowed to set as slants The cultures were then inoculated into the tubes containing Simmons citrate agar slants (Stabbed into the bud and streaked on the surface of slants) and incubated at 37˚C for 7 – 12 days After the incubation period, the development of intense blue colour from the original green colour of the medium indicated the ability of the organism to utilize citrate as carbon source and constituted the positive reaction The absence of blue colour indicates the negative reaction

2.2.7 Determination of halophily

In order to determine the halophily, the broth culture was supplemented with NaCl of different concentrations (g/dL) at 25%, 26%, 27%, 28%, 29%, 30% and

obtained by recording O.D values at 540 nm Based on the growth characteristics, the optimum salinity requirement for the strain was arrived by extrapolating the graph

2.2.8 Determination of temperature in the growth of isolate

In order to find out the optimum temperature requirement of strain, the isolate

was obtained by recording O.D values at 540 nm The temperature required for the strain was calculated by extrapolating the graph

2.2.9 Determination of optimum pH

Optimization of pH value for the growth rate of specific strain was determined

at different pH values 8.0, 8.4, 8.8 and 9.2 The isolate was inoculated and incubated

recording O.D values at 540 nm Based on the observed growth characteristics, the optimum pH required for the strain was calculated by extrapolating the graph

2.2.10 Determination of growth curve and generation time

isolate in marine broth at 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12 days at 37˚C and kept in rotary shaker at 120 rpm The growth rate was obtained by recording O.D values at 540 nm Based on the growth characteristics, the generation time was calculted from the extrapolation of the graph

2.2.11 Growth of isolate in different agars

The isolated halobacterial strain was inoculated in different agars such as casein hydrolysate agar, starch agar, potato dextrose agar, Rose Bengal agar, Macconkey agar, Zobell marine agar, Blood agar, Gelatin agar and EMB agar to analyze its effective growth All the agars were supplemented with 25% NaCl The

2.2.12 Growth in various carbon sources

In order to determine the growth rate, the isolated strain was inoculated on Zobell marine agar plates supplemented with various carbon sources such as glucose, fructose, lactose, starch, maltose, galactose and sucrose separately The plates were

strain into Zobell marine broth along with the above sources in different concentrations (glucose: 1-10%, fructose: 1-6%, lactose: 1-5%, starch: 1-11%, maltose: 1-5%, galactose: 1-7% and sucrose: 1-6%) The growth rate of the strain was obtained by recording OD values at 540nm

2.2.13 Growth in various N 2 sources

In order to increase the growth rate the isolated strain was inoculated in Zobell

arginine, cysteine, methionine, histidine, glycine and proline (w/v) individually The

individually by inoculating the strain into Zobell marine broth along with the above sources (tryptophan: 0.5-2.0%, tyrosine: 0.5-2%, arginine: 0.2-1.0%, cysteine: 0.5-2.0%, histidine: 0.2-1.2% and glycine: 0.2-1.0%) The growth rate of the strain was obtained by recording OD values at 540nm

2.2.14 Growth in various inorganic sources

The growth rate of isolated strain was studied by supplementing the marine

a concentration of 0.5gm (w/v) were used The plates were incubated at 37˚C to evaluate the optimum growth by recording OD values at 540nm

2.2.15 Growth in various organic solvents

In order to determine the efficacy of organic solvent, the isolated strain was inoculated in Zobell marine agar plates and broth supplemented with different concentrations of organic solvents such as, methanol (1-5% (v/v)), ethanol (1-7% (v/v)), chloroform (1-6% (v/v)), diethyl ether (1-7% (v/v)) and acetone The results were recorded by taking OD values at 540nm

2.2.16 Media formulation

A semi synthetic medium was used for the general cultivation of the isolated strain.The selective medium containing marine broth (4gm), NaCl (29gm), Fructose (1gm), Glucose (1gm), Sucrose (1gm), Galactose (1gm), Maltose (1gm), Lactose (1gm), Cysteine (0.5gm), Histidine (0.5gm), Arginine (0.5gm), Glycine (0.5gm), Tryptophan (0.5gm), pH (8.8), agar (15gm) in 100ml of distilled water

2.2.17 Antimicrobial analysis

The supernatant liquid collected from the broth was subjected to antimicrobial

activity against E.coli, Staphylococcus aureus, Klebsiella sps, Pseudomonas aeroginosa, Enterobacter sp., and Proteus vulgaris by standard paper disc assay

method (Acar, 1980) Whatman No.1 filter paper discs (5 mm diameter) were autoclaved for 15 minutes at 121˚C The sterile discs were loaded with crude culture supernatant (50µl/disc) Agar plates were spreaded uniformly with the suspension of bacterial strains The discs with supernatant were placed on the agar plate A disc loaded with Chloramphenicol was used as control The agar plates were incubated at 37˚C for 48 hours The diameter of bacterial growth inhibition zone around each disc was examined under transmitted light source

2.2.18 16S rRNA Sequencing

DNA purification kit (PureFast® Bacterial Genomic DNA purification kit), PCR Master Mix, Agarose gel electrophoresis consumables and Primers purchased from HELINI Biomolecules, Chennai, India

2.2.18.1 2X Master Mix

It contains 2U of Taq DNA polymerase, 10X Taq reaction buffer, 2mM

2.2.18.2 Agarose gel electrophoresis

Agarose, 50X TAE buffer, 6X gel loading buffer and Ethidium bromide are purchased from HELINI Biomolecules, Chennai

2.2.18.2.1 Procedure

Pellet was suspended in 200μl of PBS, added 50μl of lysozyme and incubated

at 37ºC for 15min 400μl of Lysis buffer and 40μl of Proteinase K [10mg/ml] were added and gently mixed well, incubated in water bath at 70°C for 10 min Transferred the whole lysate into purefast spin column and centrifuged at 10000 rpm for 1min Discarded the flow through and added 500μl of wash buffer and centrifuged at 10000rpm for 1 min Discarded the flow through and added 500μl of wash buffer-2 and centrifuged at 10000rpm for 1min Repeated the same procedure for one more time Discarded the flow through and centrifuged the column for 2 minutes more to remove the residual ethanol Eluted the DNA by adding 100μl of elution buffer and centrifuged for 1min Qualitative and quantitative examination of extracted DNA was carried out by loading in 1% agarose gel and 1μl of extracted DNA was used for PCR amplification

2.2.18.3 PCR Procedure

mix, and 2U proof reading Taq DNA polymerase

2.2.18.3.1 Components Quantity

In PCR vial

Initial Denaturation : 94ºC for 3 min

Denaturation : 94ºC for 1 min

Annealing : 60ºC for 1min 30 cycles

Extension : 72ºC for 1min

Final extension : 72º C for 5 min

2.2.18.3.2 Loading

Prepared 2% agarose gel [2gm of agarose in 100ml of 1X TAE buffer] Mixed 8μl 6X Gel loading dye to each PCR vial and loaded 5μl of PCR sample Run electrophoresis at 50V till the dye reaches three fourth distances and observe the bands in UV transilluminator

2.2.18.3.3 Agarose gel electrophoresis

Prepared 2% agarose (2gm agarose in 100ml of 1X TAE buffer and melted using Micro oven).When the agarose gel temperature was around 60ºC, added 5μl of

Ethidium bromide Poured warm agarose solution slowly into the gel platform and kept the gel set undisturbed till the agarose solidifies Poured 1X TAE buffer into submarine gel tank, carefully placed the gel platform into tank The tank buffer level was maintined at 0.5cm above the gel PCR Samples are loaded after mixing with gel loading dye along with 10μl HELINI QuickRef 250+bp DNA Ladder Run electrophoresis at 50V till the dye reaches three fourth distance of the gel Gel viewed

in UV Transilluminator and observed the bands pattern Sample load in well 1 and 3: ~1500bp PCR product QuickRef® DNA Ladder: 100bp, 250bp, 500bp, 750bp, 1000bp, 2000bp, 3000bp, and 4000bp

Phylogenetic tree was constructed from evolutionary distances with the help of

neighbour-joining method of MEGA 6 program package (Kumar et al., 2004; Suchitra B Bhorgave et al., 2012) The 16S rRNA sequence of the isolated bacterium

was submitted to NCBI Gene Bank Database

2.3 RESULTS

2.3.1 Description of sampling site

The study site was Puthalam saltpan located at extreme south of Kanyakumari, the peninsular tip of southern part of India The total area of salt pan is 150 acres This area is under the influence of very low tides and surrounding marshy land supports rich mangrove vegetation The salt producing season is normally during non-monsoon months Water gets heated up and evaporated with crude salt In the peak salt producing season, salt is harvested and collected daily The information about the production of salt was collected from the salt department of Government of India near Puthalam From the saltern land, three sites were selected and are shown in Plates (2.1-2.3)

Plate 2.1 : Water in the saltpan becomes reddish pink in color, where the salt

started to form from sea water by evaporation in site 1

Plate 2.2: Water sample from the salt depositing area of salt pan in site 2

Plate 2.3: Sediment sample mixed with sea water in site 3

Water samples mixed with sea water in site 1 The arrow mark indicates the location where the sea water comes and mixes with salt pan (Plate 2.3) Samples were collected from the salt depositing area that is the salt pan itself represented as site 1 The arrowmark indicates the area of salt deposition at different pans (Plates 2.3)

formation began from sea water by evaporation Arrow mark indicates that reddish pink colour of the water (Plates 2.1)

2.3.2 Physico-chemical analysis

The average temperature at the sampling sites was 27˚C in the morning and 32˚C at noon Colour of the water collected from three sites were observed as reddish pink in site 1 (Plate 2.1), light pink in site 2 (Plate 2.2) and white in site 3 (Plate 2.3) Lower dissolved oxygen was observed (2mg/l) in water collected from site 3 and was

content was measured in the water samples The value was found to be 278 ppm at site 1 and 220 ppm at site 2 respectively The water sample collected nearby location

sample from site 3 (Plate 2.3) was found to be 10 ppm The bicarbonate content was found to be 60 ppm, 56 ppm and 24 ppm in site 1, site 2 and site 3 respectively

The chloride content of the seawater was found to be more In the present study, chloride content of the water sample collected from site 3 was high (24.82 mg/l), when compared with sites 1 and 2 Certain samples of water containing 250 gm Cl/l are salty in taste, when the main cation is sodium In general, the sodium and chloride ion are dominant in sea water while in fresh water carbonated calcium ions

are predominant (Asha et al., 2004)

When compared to other water sampling sites, the salinity of the water sample present along seawater (Plate 2.1) was found to be 40.78 ppm, which was higher than

the other samples Haloarchaea thrive in environments with salt concentrations

approaching saturation, the water turning to reddish purple in colour indicated the massive growth

Generally the hardness of water (respective soil sample) is due to the presence

of divalent metallic cations like calcium, magnesium, strontium, manganese, etc The water sample in site 1 had hardness of about 220 mg/l and it is due to the presence of the above said minerals The conductivity of the water sample nearby the collected salt was found to be 0.08 million /mho

Magnesium content was found to be more (1.5g/Kg) in site 1 (Plate 2.1) compared to other sites Nitrogen content of water sample was maximum (32 ppm) at site 1, whereas iron content was more at site 2 and site 3 (4.6 and 5 ppm) Copper content was more at site 1 (1.0 ppm) and zinc was found to be more (0.924 ppm) at site 3 Calcium content was high at site 2 (4.3 ppm) compared to other sites The details of the results are presented in Table 2.1

Table 2.1 : Physicochemical characteristics of solar saltern located at Puthalam

near Kanyakumari coast of India

Characteristics Sample site 1 Sample site 2 Sample site 3

2.3.3 Isolation and screening

for plating by spread plate method The inoculated plates were incubated at 37˚C in an

Different shades of red, yellow and white coloured colonies were seen (Plate 2.4) The observed colonies were sub cultured individually on Zobell marine agar plates

Plate 2.4: Growth of isolate on Zobell marine agar plates

2.3.4 Screening of SOD producing Halobacterium

The isolated strains were screened for SOD activity and the results are as given in table (2.2)

Table 2.2: Activity of SOD in different strains

I (Red)

II (yellow)

III (White)

0.792 0.07 0.13

All the three isolates were screened for SOD Among the three strains, strain 1 red pigmented bacteria has the highest SOD activity (0.792 U/ml) Hence the red pigmented strain was used for further study

2.3.5 Screening of catalase producing strain

The isolated strains were screened for catalase activity and the results are as in table (2.3)

Table 2.3: Activity of catalase in different strains

I (Red)

II (yellow)

III (White)

2.826 0.716 0.458

Among the three strains, strain 1 red pigmented bacteria has the highest catalase activity (2.826 U/ml) Hence the red pigmented strain was used for further study

2.3.6 Biochemical characterization of isolate

The isolated pure culture was subjected to various biochemical

characterizations (Kamekura et al., 1995) and the results are as in the Table 2.4

Table 2.4: Characterization of isolated strain

The results indicated that the strain is red coloured, Gram negative rod with evenly spread colonies It shows motility and positive reaction in catalase, oxidase, gelatin liquefaction, starch hydrolysate, casein production, glucose, sucrose, dextrose, arabinose and mannitol tests

2.3.7 Determination of halophily

Growth of isolate in the presence of varying concentrations of NaCl was determined, and the results are given in Table 2.5

The results clearly indicate that the strain exhibited significant growth at 29% (w/v), where as delayed growth response was observed above 29% NaCl (0.95±0.48), excluding the concentration of NaCl in marine broth and marine water, as represented

in figure 2.1 The total salinity was calculated as 34.45% (concentration of NaCl in marine water = 3.5% + NaCl in marine broth = 1.95% + Added NaCl = 29%)

Figure 2.1: Effect of NaCl in the growth of isolate

2.3.8 Effect of temperature in the growth of isolate

The effect of temperature in the growth of isolate was recorded and the results are shown in table 2.6

Figure 2.2: Effect of temperature in growth of isolate

The present study clearly noticed that a gradual increase in the growth of strain took place when the temperature was gradually raised The optimum growth was found at 42ºC (0.89±0.20) Above this temperature the growth rate started to decline (fig.2.2) This may be due to denaturation of proteins responsible for growth

at higher temperature

2.3.9 Effect of pH in the growth of strain

Growth of isolate in the presence of varied pH was analyzed and the observations were recorded and shown in Table 2.7

Figure 2.3: Effect of pH in the growth of strain

Based on the observed results the biomass production was found to be increased when increasing the pH of culture medium as shown in figure 2.3 The strain attained well established growth at pH 8.8 (0.70±0.18), beyond that the growth rate was found to be decreased substantially

2.3.10 Growth curve and generation time

at 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12 days by keeping in rotary shaker at 120 rpm under 37˚C The results were recorded and tabulated in Table 2.8

Table 2.8: Growth curve of isolate