3 materials and methods.pdf

Biosynthesis, characterization, optimization, Partial purification and application of poly-β-hydroxybutyrate from Bacillus sps

30 Materials and methods

3. MATERIALS AND METHODS

This chapter deals with methods used for the isolation, qualitative and quantitative

screening, extraction and estimation of PHB from soil isolates. Also the detailed methods involved

for optimization of physical and chemical methods for the PHB production, compared with standard

strains are discussed. The cheap substrates are utilized for the PHB production. The experimental

procedure followed for mass cultivation of PHB under optimized condition, the physical and

chemical characterisation of PHB extracted powder and stability studies of PHB extracted powder

are discussed. The molecular identification processed for the higher PHB producing strains. The

steps involved in the preparation of PHB coated textiles laminates, tests conducted to check the

functional properties of the coated laminates, physical and chemical characterisation of the

developed laminates, functional properties of the laminates for medical and health care application

are given in detail.

3.1 Collection of Soil Samples

The microbial isolates were screened for PHB production from three soil samples

collected from three different geographical zones.

Zone I: Soil samples collected from Western Ghats (Latitude 10.87708 and Longitude 76.90158)

near Madukarai, Coimbatore, Tamilnadu, India.

Zone II: Soil samples collected from Agro waste storage compost at Kulathupalayam, (Latitude

10.93688 and Longitude 76.94658) Kovaipudur, Coimbatore, Tamilnadu, India.

Zone III: Agriculture and vegetable waste dumped soil samples collected from field area,

Agriculture University, (Latitude 11.01553 and Longitude 76.93693) Coimbatore, Tamilnadu,

India.

The soil samples were collected from various zones in and around Coimbatore district

(zone I, zone II and zone III). About 15–20g of soil samples were collected by scrapping to a depth

of 5–8 cm with a sterile spatula. The samples were placed in sterile plastic bags and stored at 4ºC

and transported to the laboratory.

PDF created with pdfFactory trial version www.pdffactory.com

http://www.pdffactory.com



3.1.1 Isolation of PHB Producing Organisms from Soil Samples (Yilmaz et al., 2005)

One gram of soil sample was suspended in 99 ml sterile distilled water and shaken

vigorously for 2 min. The diluted soil samples were heated at 60ºC for 30 min in water bath. Then

the liquid was serially diluted and plated on nutrient agar medium. The plates were incubated at

37°C for 24–48 hrs. The isolated colonies were selected and sub cultured on minimal agar medium

for further studies (Cappuccino, 1992).

Composition of Minimal agar (pH 7)

Dipotassium hydrogen phosphate - 7 gm

Di ammonium sulphate - 1 gm

Magnesium sulphate - 0.5 gm

Glucose - 20 gm

Agar - 20 gm

Distilled water - 1000 ml

Standard Cultures used for Comparison

The standard cultures such as Bacillus subtilis MTCC 441 and Bacillus megaterium

MTCC 453 (Microbial Type Culture Collection and Gene Bank-MTCC) collected from the Institute

of Microbial Technology (IMTECH), Chandigarh, India, were used for optimization studies. The

standard cultures were selected based on the criteria to compare the physiochemical characteristics

of the isolated strains and PHB production rate under standardised conditions.

3.2 Qualitative Screening for the Production of PHB using Sudan Black Staining Technique

(Williamson and Wilkinson, 1958).

The isolated bacterial strains were screened for PHB production. As a preliminary step,

screening of PHB producers was carried out using viable colony staining technique. The cultures

were grown on minimal media supplemented with glucose (2%) as a sole carbon source, incubated

at 40ºC for 48hrs. After incubation, the plates were flooded with Sudan black B solution for the





detection of microbial intracellular lipid granules and kept undisturbed for 20 minutes. The excess

of Sudan black solution was drained off. Viable colony staining technique was selected in order to

reveal the different pattern of sudan black absorption seen on the agar plates such as Maximum,

Moderate and Minimum absorption.

Preparation of Sudan Black B Solution (Parshad et al., 2001)

The Sudan Black B solution was prepared by dissolving 0.3 gm of Sudan black B

powder in 75 ml of 95% ethanol. The solution was made up to 100 ml with distilled water and

mixed thoroughly and the filtrate was stored for further use.

The effective PHB producers were selected from qualitative analysis and subjected to

quantitative analysis.

3.2.1 Quantitative Screening for PHB Producing Isolates

The selected strains were grown on minimal broth (pH 7) under standard conditions and

incubated at 37°C. During incubation, samples were retrieved after every 24 hrs for 4 days (24-72

hrs) to quantify the production of PHB (µg/ml) by chloroform extraction method.

3.3 Extraction of Poly-β-hydroxybutyrate (Extracted from Ishizaki and Tanaka, 1991)

PHB produced from the selected and standard isolates were extracted by the following

procedure. About 10ml of the bacterial cultures (24-96 hours) grown in minimal broth was retrieved

at an interval of 24 hrs and centrifuged at 10,000 rpm for 10 minutes. The supernatant was

discarded and the pellet was suspended with 2.5ml of 4% sodium hypochlorite solution and 2.5ml

of chloroform. The pellet suspension was incubated at 30ºC for 1 hour. After incubation, the

suspension was centrifuged at 1,500 rpm for 10 minutes. After centrifugation, three phases were

obtained. The upper phase consisted of hypochlorite solution which was removed and the middle

phase (chloroform containing undisturbed cells) was separated by filtration from the bottom phase

(chloroform with PHB). The extracted chloroform phase was used to quantify the production of

PHB by measuring the absorbance at 230nm.





3.3.1 Estimation of Poly-β -hydroxybutyrate by Spectrophotometric Method

According to Khanafari et al., (2006) the standard curve was derived by preparing the

PHB standard solution at different concentration (100-1000µg/ml). About 2ml of concentrated

sulphuric acid was added to all the tubes, and kept in boiling water bath for 10 minutes for the

conversion of PHB into crotonic acid. After cooling, the absorbance was measured at 230 nm using

UV spectrophotometer (Systronics 180) and standard graph was plotted. About 2ml of concentrated

sulphuric acid was used as blank. Similar procedure was carried out for all the samples. The

readings were plotted in standard graph of crotonic acid and concentrations of PHB in the sample

were determined.

The best PHB producing strains were selected based on standardised conditions by

quantitative method and were used for further analysis.

3.4 Identification of PHB Producing Bacterial Isolates

The isolates screened out from three different zones were subjected for identification

based on the Morphological and Biochemical characteristics.

3.4.1 Morphological Characterisation

Morphological characters such as form, shape, size, colour and texture of the selected

isolates were investigated according to Bergey’s manual of Determinative Bacteriology (Holt et al.,

1993) by growing them on minimal agar medium.

3.4.2 Microscopic Characterisation

3.4.2.1 Gram’s Staining

The microscopic characteristics of the isolates were studied based on their reactions to

Gram’s test. In Gram staining procedure, Crystal violet was used as primary stain, alcohol as

decolourising agent, Grams iodine as mordant and Saffranin as counter stain. In Gram positive

cells, due to low lipid concentration in the cell wall, the action of alcohol created small pores. So

CV-I complex was retained inside and not removed by the alcohol, the cell appeared purple in

colour. In Gram negative cell wall, due to high lipid concentration in the cell wall the action of

alcohol created large pores in the cell wall. CV-I complex was removed out through the large pore.





So the counter stain Saffranin was taken by the cell wall and appeared pink in colour. A thin smear

of the isolate was prepared on slide. The procedure was carried out for all isolates. The prepared

smear was flooded with crystal violet for a minute and washed with running water. Then, Gram’s

iodine was added to the slide and kept for a minute. It was de-stained with few drops of alcohol and

counter stained with Saffranin. The slide after washing was blot dried and observed under oil

immersion objective (100 X oil immersion).

3.4.2.2 Endospore Staining

The test was performed to determine the presence of bacterial endospore by Schaeffer-

Fulton method. Organisms have the capacity to exist as metabolically active, vegetative cells or

metabolically inactive highly resistant cells called spores. The endospores are intracellular structure

formed during sporegenesis under unfavourable environmental conditions with the exhaustion of

nutritional carbon source. The Primary stain, malachite green was taken up by spore under the

application of heat, which helps in the penetration of the stain into the impervious coat. The counter

stain (Saffranin) was used to stain the vegetative cell which will appear in red colour, in contrast to

endospore which will appear in green colour.

3.4.3 Biochemical Test

All the isolates were subjected to biochemical analysis. Biochemical tests such as

Indole, Methyl red, Voges proskauer, Citrate utilization, Carbohydrate fermentation, Triple Sugar

Iron Agar test, Starch utilization and catalase tests were carried out (Cappuccino, 1992). All the

cultures were subjected to grow on the selective media (blood agar).

3.4.3.1 Blood Agar Test

Blood agar test was performed to determine the ability of microorganism to exhibit

haemolysis on blood agar plate. The isolates were inoculated on blood agar and incubated at 37°C

for 24-48 hrs. Diffuse zone of β-haemolysis surrounding the colonies indicated as positive result

and absence of haemolysis indicated as negative result.

The qualitative and quantitative analysis and physiochemical characterisation of

selected isolates were done for the selected isolates.





3.5 Optimization of Physical Parameters (pH and Temperature)

3.5.1 Optimization of pH for the Production of PHB

To standardise the optimum pH for the production of PHB, the bacterial cultures were

inoculated, in minimal broth at different pH (5, 7 and 9) and incubated at constant temperature 37ºC

for the optimum incubation time. All the isolates were incubated under selected conditions for

determining the optimum pH. Then the isolates were subjected to extraction of PHB by crotonic

acid method. The extracted PHB was quantified by measuring the absorbance at 230 nm. The

optimum pH was determined based on the amount of PHB produced. In each trial, the production

rate of PHB was compared with standard strain Bacillus subtilis MTCC 441 and Bacillus

megaterium MTCC 453.

3.5.2 Optimization of Temperature for the Production of PHB

In order to optimize the temperature for the production of PHB, the bacterial cultures

were inoculated in minimal broth (pH 7) and incubated at temperature 30°C, 37°C and 45°C for 48

hours. All the isolates were incubated under standardised conditions for the optimization of

temperature. After incubation, the broth culture were subjected to PHB extraction by crotonic acid

method and in each trial, the production rate of PHB was compared with standard strains Bacillus

subtilis MTCC 441 and Bacillus megaterium MTCC 453.

The optimization of physical parameters such as pH and temperature were compared

with the standard strains and the growth studies were done.

3.6 Studies on the Growth and Production of Poly-β-hydroxybutyrate

In order to investigate the growth curve and intracellular PHB accumulation, the growth

and the production of PHB at different stages were studied under optimized temperature and pH. As

a periodic analysis of growth, the Dry Cell Weight was measured in (mg/ml). About 50 ml of

sample was centrifuged at 5000 rpm for 15 minutes. The supernatant was discarded, the pellet was

resuspended with distilled water and recentrifuged, the supernatant was discarded and pellet was

dried at 110°C until a constant weight was obtained. The cells were harvested at an interval of 8

hours (0-72 hours) by centrifugation; the weight of dried pellet was calculated (mg/ml). The

concentration of PHB was calculated as µg/ml at an interval of 8 hours (0-72 hours) by crotonic

acid method.





The physical parameters such as temperature, pH and duration, were standardised. The

effect of chemical supplementation of carbon sources, nitrogen sources and vitamin sources were

studied and optimised.

3.7 Effect of Chemical Supplementation on the Production of PHB (Poly-β -hydroxybutyrate).

The effect of chemical supplementation such as carbon sources, nitrogen sources,

vitamin sources and cheap substrates such as paper mill effluent and dairy effluent were studied for

the higher production of PHB under optimized conditions.

3.7.1 Effect of Different Carbon Sources Supplementation on PHB Production

Different carbon sources such as glucose, lactose, mannose, maltose, mannitol,

galactose, fructose and raffinose in varying concentration of 0.2%, 0.4%, 0.8%, 1.0% and 2% were

sterilized by Millipore filter with pore size of 0.45μg/ml and added to the production media

(minimal broth) and the selected isolates were inoculated to the production medium, maintained at

pH 7 and incubated at temperature 37ºC for optimized duration (48 hr). The production of PHB was

determined by extracting the PHB and estimating the absorbance at 230 nm.

3.7.2 Effect of Nitrogen Supplementation on PHB Production

Different Nitrogen sources such as protease peptone, ammonium chloride, ammonium

nitrate, L-cysteine, ammonium sulphate, peptone, L-glycine and yeast extract were used for the

study. These nitrogen sources were added at different concentrations (0.2%, 0.4%, 0.8% 1% and

2%) to the production medium and incubated at optimized temperature for optimized duration of 48

hrs. After incubation, the extraction of PHB was done to determine the rate of PHB production by

measuring the absorbance at 230nm using spectrophotometer.

3.7.3 Effect of Vitamin Supplementation on Production of PHB

Vitamin sources such as amino benzoic acid, biotin, pyridoxine and thiamine were used

as supplement for the production of poly-β-hydroxybutyrate at the concentration of 1mg/L. The

stock solution of low concentration was prepared and added to the production medium. The isolated

strains were inoculated to the production medium and were incubated at optimized temperature at

37ºC and pH 7 for 48 hrs and PHB production was quantified spectrophotometrically.





3.7.4 Production of PHB using Cheap Substrate

Due to the high cost for the production of PHB, low cost substrates are used for the

study. According to Tian et al., (2009), the cheaper substrate the Paper mill effluent was used at

various concentrations (4%, 6%, 8% and 10%). According to Haywood et al., (1988), dairy effluent

was used at various concentrations (10%, 20%, 30%, 40%, 50% and 60%). As the preliminary step,

the physiochemical and biological characteristics analysis of effluent samples are done.

3.8 Analysis of Effluent Samples (Dairy and Paper Mill Effluent)

Physical, chemical and biological characteristics of the effluent samples were analysed

as per as the standard methods for the estimation of water and waste water, APHA (1980).

Physico-Chemical Parameters of Dairy and Paper mill Effluent Tested in the Individual Series of

the Experiment (Senthilkumar et al., 2011).

3.8a Collection of effluent samples

The effluent sample (dairy effluent) was collected from Aavin dairy industry

Pachapalayam, Coimbatore district and paper mill effluent was collected from Pallipalayam Seshai

papermill, Erode district and subjected for physical and biochemical analysis.

Samples for physical examination were collected in sterile bottles. The sampling bottles

were closed with a round glass stopper having an overlapping rim. The stopper was relaxed by an

intervening strip of paper to prevent breakage. The bottles were protected by covering with aluminium

foil and sterilized in an autoclave at 20 psi for 15 minutes. The bottles were opened only at the time of

sampling.

3.8b Physical properties

Colour : Using spectrophotometer the colour was measured and expressed in Platium-

coblat colour units.

Suspended solids : A known quantity of the effluent was filtered using Whatman No.1 filter

paper and the residue was dried at 105°C and weighed.





Dissolved solids : The filtrate obtained from the suspended solids was evaporated, dried at

105°C and weighed.

Total solids dried : A known quantity of the effluent was evaporated at 105°C and weighed.

3.8.1 Total Suspended Solids

Suspended solids are the portions of solids (from effluent sample) that are retained on a

filter of standard specified size (generally 2.0 µ) under specific conditions.

A well mixed sample was filtered through a weighed standard glass fibre filter. The

residue was retained on the filter and it was dried to a constant weight at 103-105 o C. The increase

in the weight of the filter was determined by the total suspended solids.

A known volume of vigorously shaken sample (50ml) was filtered through a pre-

weighed glass fibre filter disk fitted to suction pump, and washed successively with distilled water.

The filter was carefully removed from the filtration apparatus and dried for an hour at 103-105 o C

in an oven, cooled in desicator and weighed.

Calculation

Total Suspended Solids (mg/L) = (W1-W2) (1000) / Sample volume (ml)

W1 = Weight of dried glass fibre filter + residue (g)

W2 = Weight of glass fibre filter disk before filtering (g)

3.8.2 Total Dissolved Solids

Dissolved solids are solids that are in dissolved state in solution. Waters with high

dissolved solids generally are of inferior palatability and may induce an unfavourable physiological

reaction in the transient consumer. The difference in the weights of Total Solids (W1) and Total

Suspended Solids (W2) expressed in the same units gave Total Dissolved Solids (TDS).





Calculation

Total Dissolved Solids (mg/L) = (W1-W2) (1000) / Sample volume (ml)

W1 = Weight of total solids + dish (g)

W2 = Weight of total suspended solids (g)

3.8.3 Determination of pH (Jackson, 1973)

The pH is defined as the negative logarithm of hydrogen ion concentration or simply the

log of the reciprocal of the hydrogen ion concentration. A glass electrode in contact with H ions of

the solution acquires an electrode potential which depends on the concentration of H ions and

measured potentially against some reference electrode which was usually a calomel electrode and

expressed in pH units. Two electrodes are used in the determination of pH. One was the reference

electrode which provides a standard voltage and the outer electrode was glass electrode that consists

of a tube enclosing a lead wire made of Ag coated with AgCl.

pH measurement

About 20ml of the sample was taken in 100ml beaker, it was stirred constantly. The

electrodes were immersed into the beaker containing sample and recorded the meter reading.

3.8.4 Biological Oxygen Demand (BOD) APHA (1980)

Biological Oxygen Demand is the measurement of oxygen required by microorganisms

in stabilizing the biodegradable organic matter under aerobic conditions. The dissolved oxygen

content of the sample was determined before and after five days of incubation at 20ºC. The amount

of oxygen depleted is BOD.

Reagents used

1.Calcium chloride solution

About 2.75 g of hydrated calcium chloride was dissolved in 1000 ml of distilled water.





2.Magnesium sulfate solution

About 25 g of hydrated magnesium sulfate was dissolved in 1000 ml distilled water.

3.Phosphate buffer solution

About 8.5 g potassium hydrogen phosphate, 21.1g of di-potassium hydrogen phosphate,

33.4g of disodium-hydrogen phosphate and 1.7g ammonium chloride were dissolved in 500 ml of

distilled water and made up to 1000 ml.

The required volume of distilled water was aerated and 1ml of calcium chloride,

magnesium sulfate, ferric chloride and phosphate buffer solution were added per litre of aerated

distilled water and mixed thoroughly.

Method

A glass container was taken containing about 5 litres of pure water and bubbled with

compressed air for 2 days to attain saturation. About 1 ml of manganese sulphate, phosphate

buffer, ferric chloride and chloride solution was added for each litre of distilled water and the

sample was neutralized to pH 7. The dilutions of the sample were made such that about 50 %

depletion of dissolved oxygen took place and residual dissolved oxygen after incubation for 5 days

will not less than 1 mg/L.

The following dilutions were suggested

0.1 – 1.0 percent -- Strong wastes

1.0 – 5.0 percent -- Raw sewage

5.0 – 25.0 percent -- Treated effluent

In 6 BOD bottles, 2 bottles were served as blank for determination of initial dissolved

oxygen and the remaining 4 bottles were kept in the incubator at 20 ºC for 5days. One blank and

sample (dairy effluent and treated paper mill effluent) was taken to determine its initial dissolved

oxygen content by addition of manganese sulphate, alkaline iodide azide agent, Conc.H2SO4 and

titrated with 0.025 N sodium thio-sulphate solution using starch as indicator and the burette reading





was noted. After 5 days, the dissolved oxygen in the incubator bottles (blank and sample) were

determined in the similar way.

Finally, the titrated value was recorded and calculated.

3.8.5 Chemical oxygen demand (COD) APHA (1980)

The chemical oxygen demand is the measurement of oxygen equivalent of that portion

of the organic matter in the sample that was susceptible to oxidation by strong chemical oxidant.

The test was an important and quickly measured parameter for pollution studies of streams and

industrial waste water. Most of the organic matter was destroyed in boiling mixture of chromic and

sulphuric acid. The purpose of running the blank was to compensate for any error that may result

because of presence of extraneous organic matter in the reagents.

Reagents

1. Standard 0.25N potassium dichromate solution: About 12.259 g of pure potassium dichromate

was dissolved in distilled water and diluted to 1 litre.

2. Sulphuric acid – silver sulphate reagent: About 5.5 g of silver sulphate was dissolved with 1 litre

of conc. sulphuric acid and kept for overnight.

3. Standard 0.1N ferrous ammonium sulphate solution: About 30 g of pure ferrous ammonium

sulphate solution was dissolved and diluted with distilled water. About 20 ml of conc. sulphuric

acid was added and diluted to 1 litre.

4. Ferroin indicator: About 1.485 g of phenanthraline monohydrate was dissolved with 0.695 g pure

ferrous ammonium sulphate and diluted with 100ml of distilled water.

5. Concentrated sulphuric acid.

Procedure

About 20 ml of sample was taken in a reflux flask and 0.4 g of sulphuric acid was

added, with 10 ml of 0.25 N potassium dichromate solutions. Some pumice stones were dropped

slowly and 30 ml of conc sulphuric acid was added with silver sulphate reagent. The contents were

mixed thoroughly and connected with the flask to a condenser and refluxed for 2 hours. After the

reflux the condenser was cooled and washed. The mixture was diluted to 150 ml by distilled water.





About 3 drops of ferroin indicator was added and titrated with N/10 ferrous ammonium sulphate

solution, till the colour changed from green to wine red. The same procedure was performed using

distilled water as a blank.

Calculation

COD ( mg / l ) = (Burette reading X Normality of ferrous ammonium sulphate X 8000) Volume of sample (ml)

3.8.6 Total Hardness of Effluent for Calcium and Magnesium APHA (1980)

Total hardness was predominantly caused by divalent cations such as calcium,

magnesium, alkaline earth metal such as iron, manganese, strontium, etc. The total hardness is

defined as the sum of calcium and magnesium concentrations, both expressed as CaCO3 in mg/L.

Carbonates and bicarbonates of calcium and magnesium cause temporary hardness. Sulphates and

chlorides cause permanent hardness.

The total hardness of effluent was due to calcium and magnesium expressed as calcium

carbonate. The calcium and magnesium content of the effluent sample was estimated by titrating

with a known volume with 0.02N EDTA solution in the presence of ammonium chloride-

ammonium hydroxide buffer and eriochrome black-T indicator. About 25ml of the sample was

pipette out into a porcelain basin and added about 10ml of ammonium chloride- ammonium

hydroxide buffer and few drops of eriochrome black-T indicator. Both the samples (dairy effluent

and paper mill effluent) were titrated against 0.02 N EDTA, the endpoint was reported as colour

change from wine red to sky blue. From the volume of 0.02 N EDTA consumed, the total hardness

was calculated.

Calculation

Volume of effluent sample taken = 25ml

Volume of 0.02N EDTA consumed = X ml

1ml of 0.02N EDTA contains = 0.001g of CaCO3

A ml of 0.02N EDTA contains = 0.001x X g of CaCO3





This is present in 25ml of water sample

Total hardness of water = 0.001 x (X) x106 ppm

25

3.8.7 Estimation of Total Soluble Salts APHA (1980)

A known volume of water was evaporated to dryness and the amount of soluble salts

present in the water was estimated gravimetrically. 100 ml of water was pipette out into a cleaned,

weighed and dry potash basin. Basin was dried by evaporating water in the water bath. In order to

remove the water content, the outside of the basin was wiped and dried in an air oven at 105º C for

an hour. The difference in the weights is the weight of total soluble salts. The results were

expressed in ppm.

Calculation

Volume of effluent taken = 100ml

Weight of empty basin = X g

Weight of basin + total soluble salts = Y g

Weight of total soluble salts alone = Y- X

This is present in 100ml of water

Total soluble salts content of water = (Y-X) ×106 ppm.

100

3.8.8 Estimation of Total Alkalinity, APHA (1980)

The alkalinity of water was normally due to the presence of carbonates, bicarbonates

and hydroxides of calcium, magnesium, sodium, and potassium. Borates, phosphate and silicates

also contribute to the alkalinity. However, due to the abundance of carbonate minerals, most water

contain carbonate and bicarbonate only. Since OH and HCO3 alkalinities cannot co exist, the CO3

estimation with Phenolpthalein represents the CO3 alkalinity while the estimation with a methyl

orange represents the alkalinity due to CO3 and HCO3 together.





Alkalinity was determined by titration with (standard) sulphuric acid using

phenolphthalein and methyl orange indicators

2CaCO3 + H2SO4 CaSO4 + Ca (HCO3)2

Ca (HCO3)2 +H2SO4 CaSO4 +2CO2+2H2O

Procedure

About 50ml of the effluent water was taken to determine the CO3 and HCO3 content by

titration with standard H2SO4 using and methyl orange indicators. The CO3 represents CO3

alkalinity or phenopthalein alkalinity and the HCO3 content represents the HCO3 alkalinity or

methyl orange alkalinity. The sum of these two alkalinities represents the total alkalinity.

Effluents (paper mill effluent and dairy effluent) are selected as cheap substrates for the

production of PHB, were subjected to chemical and biological parameters to evaluate the waste water

pollution. For the quantitative analysis of effluents, total suspended solids, total dissolved solids, total

solids, pH, BOD, COD, total hardness, total soluble salts and total alkalinity were analysed.

The PHB production was carried using cheap substrates, estimation of Biological

Oxygen Demand (BOD) and Chemical Oxygen Demand (COD) for the effluents were performed

before and after the production. Both the effluent in different concentrations (4%, 6%, 8% and 10%)

paper mill effluent and dairy effluent (10%, 20%, 30%, 40%, 50% and 60%) was added to the

production medium. The screened isolates were inoculated and incubated at optimized temperature

37 ºC for 48hrs.

The growth pattern and the production of PHB were done for the selected isolates. The

extraction of PHB was done by chloroform extraction method. The physical optimization, chemical

supplementation studies were done using different carbon, nitrogen and vitamin sources. The

production of PHB using cheap substrates such as paper mill effluent and dairy effluent was

discussed.

3.9 Statistical Analysis

Statistical analysis was performed by ANOVA (Analysis Of Variance) for the

production of PHB in different sources. The comparison studies were carried out by LSD method





(Least Significant Difference). The highest and lowest productions of PHB from the selected

isolates were determined according to the F-value.

3.10 Mass Cultivation

Optimum conditions were maintained as all the physical and chemical parameters

influenced the production of PHB for the selected isolates. All these conditions were applied for the

mass production of PHB.

3.11 Molecular Identification

Genetic approaches for the classification of bacteria were aimed at identifying the Genera

and species of maximum PHB producer.

Steps followed

1. Genomic DNA was isolated from the culture

2. The DNA fragment was amplified using PCR polymerase.

3. The PCR product was sequenced bi-directionally using the forward and reverse primer.

4. The sequence data was aligned and analyzed to identify the bacterium.

3.11.1 Procedure for Genomic DNA Isolation (Chromous Genomic DNA Isolation KIT-RKT09)

About 1.5 ml of overnight broth culture of the best strains were transferred into 2ml

micro centrifuge tubes and centrifuged at 8000 rpm for 5 minutes. After centrifugation, the

supernatant was discarded and the pellet was collected. The pellet was suspended in 200 µl of 1X TE

buffer and 100µl of 10% SDS. The contents were mixed by vortexing and the tubes were heated in

water bath at 60oC for 20 minutes. 300 µl of Phenol: Chloroform: Isoamyl alcohol mixture were

added to extract the DNA and mixed completely by vortexing. The aqueous phase containing the

DNA was carefully removed and transferred to new tubes. Equal volume of Isopropanol was added

to the tubes containing the aqueous phase. Then the tubes were mixed by inverting the tubes 3 to 4

times. The tubes were centrifuged at 10,000 rpm for 10 minutes. The supernatant was discarded and

pellet containing the DNA was collected. 200 µl of 70% ethanol was added and centrifuged at 10,000





rpm for 10 minutes. Then ethanol was decanted completely and the pellet was air dried. The dried

DNA pellet was re-suspended in 20 µl of TE buffer and dissolved by tapping.

3.11.2 Polymerised Chain Reaction (PCR)

The PCR mix was prepared in thin walled PCR tube in a sterile laminar air flow. The

following reagents were added in to the mix.

DNA from selected isolate - 1 µl

10X Taq DNA Polymerase Assay buffer - 10 µl

Taq DNA Polymerase Enzyme - 1 µl

dNTP (2.5mM each) - 4 µl

16s Forward Primer - 400 ng

16s Reverse Primer - 400 ng

Molecular grade water - 13 µl

Total reaction volume - 100 µl

Table 2 Reaction profile for PCR condition

Conditions Initial denaturation Denaturation Hybridization Elongation Temperature 94°C 94°C 55°C 72 °C Duration 55min 30 sec 30 sec 22 min No of cycles 35 35 35 35

PCR product was sequenced bi-directionally using the forward and reverse primer.

16s rRNA specific primers

16s Forward Primer : 5' - AAG TCG AGC GGA CAG ATG G -3'

16s Reverse Primer : 5' - CCA G I T CCA ATG ACC CTC CCC -3'

3.11.3 16S rRNA Sequencing

The cycle sequencing reaction was performed using BigDye terminator V3.1 cycle

sequencing Kit containing AmpliTac DNA polymerase (from Applied Biosystems, P/N: 4337457).





The sequencing reaction - mix was prepared by adding 1μl of Big Dye v3.1, 2 μl of 5x sequencing

buffer and 1 μl of 50% DMSO. To 4 μl of Sequencing reaction –mix, added 4 Pico moles of primer

(2ul) and sufficient amount of plasmid. The constituted reaction was denatured at 95°C for 5

minutes. Cycling began with denaturing at 95°C for 30 seconds, annealing at 52°C for 30 seconds

and extension for 4 minutes at 60°C and cycle repeated for a total of 30 cycles in a MWG

cthermocycler. The reaction was then purified on sepheadex plate (Edge Biosystems) by

centrifugation to remove unbound labelled and unlabelled nucleotides and salts. The purified

reaction was loaded on to the 96 capillary ABI 3700 DNA analyzer and electrophoresis was carried

out for 4 hours. DNA Sequencing report was computed using DNA analyzer.

3.12 Culture Maintenance

The PHB producing bacterial isolates were used for the further studies was inoculated on

nutrient agar medium and incubated at 25± 2 o C for 48 hours and maintained at 4oC. The selected

isolates from molecular identification were subjected for extraction of PHB by the standard

chloroform extraction method (Extracted from Ishizaki and Tanaka, 1991).

3.13 Characterization of Extracted PHB by Chemical and Stability Studies

Purification of PHB by High Performance Liquid Chromatography (HPLC)

The PHB from hot chloroform phase extracted from the potent PHB producer, was

checked for its purity by HPLC analysis (High Performance Liquid Chromatography).

The C18 column, organic solvent, 50/50 acetonitrile and sulphuric acid (mobile phase)

were used for the HPLC analysis for the chloroform extracted phase with PHB content. The

wavelength used was 260nm and the flow rate of 1ml/min. The standard (PHB powder with

chloroform solution-1mg/ml) was analysed for comparison of PHB extracted from the selected

samples. Under these conditions, the concentration was analysed and purification of PHB was

analysed. The PHB content of selected isolates were calculated according to the following formula,





PHB content (%) = Area of sample × Weight of standard × 98

Area of standard Weight of sample

Whereas,

the weight of the standard = 1mg/ml (0.001)

the weight of the sample = 10ml/50ml (0.2)

98 is the purity of the standard (%)

3.14 FTIR (Fourier Transform Infra-red Analysis)

FTIR Spectroscopy is a form of vibrational spectroscopy, the sample was irradiated

with infrared radiation from an infrared source, and absorption of the radiation stimulates

vibrational motions by depositing quanta of energy into vibrational modes. The functional groups of

PHB were identified and compared with the standard references. The analysis was done from the

Department of Biotechnology, PSG college of Arts and Science, Coimbatore.

The IR spectrum of the sample represented the total chemical composition, because

every chemical compound in the sample made its own distinct contribution to the absorbance

spectrum. The distinction of an individual spectrum, which was determined by the chemical

structure of each component and the degree to which each component contributes to the spectrum is

directly related to the concentrations of the component of the sample. The chloroform phase

containing PHB was subjected to FTIR spectroscopic analysis. In order to know the functional

groups present in PHB, 1mg of extracted sample of PHB was dissolved in 5 ml of chloroform. The

chloroform was allowed to evaporate to get PHB powder, which was subjected to FTIR analysis

using FTIR spectrophotometer. Spectra were recorded in 4000 cm-1 to 400 cm-1 range.

3.15 Biodegradation Studies of PHB using Nitric Acid

PHB was extracted using chloroform extraction method. The extracted PHB was spread

on the x-ray sheet and kept in water bath to vaporise the chloroform (80°C for half an hour). After

complete vaporisation, PHB crystals were formed on x-ray sheet as an irregular thick film. PHB

crystals were observed as crystalline white powder on the sheet. Initially the weight of PHB powder

was checked. Different concentrations of nitric acid were prepared (0.1%, 0.3%, 0.5%, 0.8% and

1%). About 2ml of each concentration of nitric acid was sprayed on PHB crystals for alternate days.

The weight of PHB crystalline powder was checked after 28 days, the amount of PHB degradation

was calculated by comparing with the initial weight.





3.16 Development of PHB Coated Textile as Laminates

As a preliminary step, the selection of textile binder and the concentration of the binder

were standardised. The extracted PHB powder was coated onto the cotton fabrics by pad-dry-cure

method for medical and health care applications.

3.16.1 Standardisation of Binder Concentration with the PHB Powder on the Textile Material

(40’s Count Cloth)

3gm of binder [poly (acrylamide-co-acrylic acid)] was dissolved in 100ml of distilled

water. About 7.5% of PHB powder (0.7gm in 10 ml of binder solution) was dissolved with binder

and mixed it properly. De-starched 40’s count cloth (15×15 cm) was dipped in the PHB with binder

solution for half an hour. The cloth was allowed to dry in oven (50°C for half an hour) or dried

under sunlight.

3.16.2 Finishing of Fabrics (Yadav et al., 2006)

The coating of PHB powder on to the 40’s count cloth was performed by Pad Dry Cure

method. The fabric cut to the size of 30 X 30 cm was immersed in 10gms of binder [poly

(acrylamide-co-acrylic acid)] dissolved in 1000 ml of distilled water. 7.5% (75gms in 1000ml of

binder) of PHB powder was added to binder solution and mixed properly till the PHB get

completely dissolved in it. Then it was passed through a padding mangle (R.B.Electronic and

Engineering, Mumbai), running at a speed of 15 m/min with a pressure of 1kgf/cm2 to remove

excess solution. A 100% wet pick-up was maintained for all of the treatments. The cloth was then

kept in oven at 50°C for half an hour for drying and curing process was done under 110º C for 1

hour.

3.17 Characterization of the Textile Laminates

3.17.1 Physical Characterization of Textile Laminates-SEM

Scanning electron microscopy (SEM) was used to examine the morphology of the PHB

coated fabric and the control fabric. Both the samples were sonicated at 20 KHZ for 3cycles of 5

minutes each. Fracture surfaces of both the samples were made into fragments by breaking the

extruded tapes in liquid nitrogen. The fragments with fracture surfaces were glued on metal holder

and covered by platinum layer in normal atmosphere. After sample preparation, the photographs of





the sample were taken by Scanning Electron Microscope, Model-JEOL-6390, magnification - 5 to

300, 000, accelerating - 0.5 to 30 kv.

3.17.2 Chemical Characterization of Coated Fabric - FTIR

Mass cultivation of PHB under optimized conditions, extracted, standardised and

purified PHB was developed as the textile laminates was subjected to FTIR Spectroscopic analysis.

3.18.1 Physical and Functional Analysis of the PHB Coated Textile Laminates

3.18.2 Water Repellency Test for Textile Laminates (AATCC 22-2005)

This test was performed to measure the resistance of fabric to wetting process by water

and helps to find out the water repellency efficacy of extracted PHB powder applied onto fabric

material and to the non coated fabric material.

Method

The ability of fabric to wet or penetration of liquid into fabric was determined as water

repellency behaviour. The water was allowed to spray on the fabric material and the spray time

must be between 25-30 sec. The surface of the specimen should be smooth without wrinkles. About

250 ml of distilled water at 27 ± 1°C (80 ± 2°F) was taken and poured into the funnel and allowed

to spray onto the test specimen (Coated fabric with PHB and uncoated fabric).

Fig 1e Standard Spray Test Ratings





Table 3 Spray Test Rating Chart

3.18.2 Absorbency Test for Textile Laminates – (Wicking, Wetting, Warping) AATCC 79-2000

The absorbency test for the coated fabric and the control fabric was calculated by the

time required to collect certain amount of water, which passes through the fabrics. The test was

performed in order to measure the propensity of a material to take in and retain a liquid, usually the

water, in to the pores and the intensities of the material to absorb water. The completeness and the

uniformity of the PHB coated fabric depends upon the absorbency.

A drop of water was allowed to fall from a fixed height on to the taut surface of a test

specimen. The time required for the specular reflection of the water drop disappeared was measured

and recorded as melting time. The time required was calculated, using stopwatch which raises to 60

sec maximum for the surface of the liquid to lose its specular reflectance, the point was determined

between the observer and a source of light such as a laboratory spot light at such an angle that the

specular reflectance of light from the surface of the flattened drop. The time required was calculated

for the drop to be absorbed, depends upon the area of tiny mirror diminishes and finally vanishes

entirely or only a dull wet spot was observed.

3.18.3 Measurement of Abrasion Resistance (AATCC 119-2004)

The abrasion resistance property was tested with the help of Martindale abrasion tester.

The fabric sample was prepared and the initial weight was measured. Then the fabric was abraded

Values Spray test rating

Value 100 No sticking or wetting of specimen face.

Value 90 Slight random sticking or wetting of specimen face.

Value 80 Wetting of the specimen face at spray points.

Value 70 Partial wetting of the entire specimen face beyond the spray points.

Value 50 Complete wetting of the entire specimen face beyond the spray points.

Value 0 Complete wetting of the entire face of the specimen.





for 50 cycles. After these cycles, the fabric sample was weighed again. The difference in the two

weights (initial and final) was calculated and the percentage weight loss was calculated.

Martindale abrasion tester

The design of the instrument makes use of the principle of two simple harmonic

motions working at right angles. The instrument can be used for getting circular or linear motion.

The pressure of abrasion and type of abrading can be changed. The Martindale abrasion tester can

be used for testing abrasion resistance and also pilling.

Working

The machine consisted of a top plate supported by three pillars. On top of the 3 pillars, a

ball caster was fixed at each point. These ball casters were allowed the plates to slide easily in the

horizontal directions and determined by 3 supported points. The arrangement of drives for the plates

consisted of a mechanical device of worm and worm wheel, 3 circular cam discs. On the cams, the

pins were provided with bush bearings and the pins engaged with the slots into same line of the

centre pin worked in the slot at right angles. As the cam discs were carried the pins to rotate the

plate. Hence the result of these motions would be two simple harmonic motions at right angles.

The top plate consisted of 4 holes to carry the circular sample holders and they can be

clamped to the top plate. Four specimens of 38 mm diameter are cut and fixed on the sample

holders. The sample holders touch the table surface and it would be flat and move in the same plane

when the top plate slides. Because of these movements, the cloth was rubbed against the cloth

surface in harmonic pattern. At one stage it would be circular and then changes to a curve of an

ellipse until the line becomes a straight line along the diagonal of the circle. The type of motion has

the advantage that the specimen would be rubbed in all portions that both in warped and wefted

ways and superior to flex abrasion.

3.18.4 Measurement of Air Permeability and Resistance (ASTM-D-737-2004)

The air permeability of the cotton fabrics were analyzed using Kawabata Evaluation

System (KES-F8-API) automatic air permeability tester which works on the air flow principle. The

air permeability of a fabric was very sensitive indicator of the fabric construction and type of fibers

and yarn used. There are several factors, which influence the air permeability. Among which, the





type of fabric, construction, bulk density, thickness, and air porosity in the yarn are important

factors. The air permeability of a fabric is the volume of air measured in cubic centimeters passed /

second through 1 cm2 area of the fabric at pressure of 1 cm3 of air.

Air resistance

The air resistance of the fabric was measured by the time in seconds for 1 cm3 of air to

pass through 1 cm2 area of a fabric under a pressure head of 1 cm3 of air. Air resistance was the

reciprocal of air permeability.

Air porosity

The porosity of the fabric was the ratio of air space to the total volume of the fabric

expressed in percentage.

3.18.5 Tensile Strength - Grab Test (ASTM D 5034–95-2001)

A tensile testing machine was used to determine the breaking strength and elongation of

PHB coated fabric and uncoated fabric as control. The specimens were conditioned to moisture at

equilibrium as directed in ASTM D 1776. The test was performed either wet or dry and samples were

cut in both the warp and weft directions. The specimen was mounted in the clamp of the testing

machine. Care was taken that the specimen was centrally located and the long dimension was as nearly

parallel as possible to the direction of force application. It was made sure that the tension on the

specimen was uniform across the clamped width. Uniform and equal tension was achieved by

attaching an auxillary clamp to the bottom of the specimen and at the point below the lower clamp of

the testing machine. The lower clamp was tightened and auxillary clamp was removed. The machine

was operated to break the specimen. The breaking force was read from the testing machine indicating

mechanism.

3.18.6 Tear Strength (ASTM D2261-96)

The tearing strength of the treated (PHB coated) fabric and untreated fabric (non coated)

was measured by the tongue (slip rip) procedure using constant rate of tensile testing. The specimens

were conditioned in the standard atmosphere for testing textiles, which were 21 ± 1ºC (70 ± 2ºF) and

65 ± 2% of relative humidity. A rectangular specimen was cut in the center of a short edge to form a

two-tongued (trouser shaped) specimen, in which one tongue of the specimen was gripped in the upper





jaw and the other tongue was gripped in the lower jaw of the tensile testing machine. The separation of

the jaws was continuously increased to apply a force to propagate the tear. At the same time the force

developed was recorded. The force continued to tear the samples (PHB coated and non coated fabric)

was calculated from autographic chart recorders or microprocessor data collection systems.

3.18.7 Washing Shrinkage (ASTM D751-06)

To determine the ability of PHB coated fabrics to resist wash shrinkage. Three

specimens 10 x 10 inch (250 x 250 mm) accurately measured in each direction to the nearest 1/32

in. (0.5 mm). The specimens were soaked for 30 minutes in distilled water at room temperature,

removed and dried at 200°F (87°C) for 30 minutes. The specimens were conditioned at the standard

conditions as provided in ASTM Specification D751-06 for a minimum of 8 hours prior to re

measuring. The percent shrinkage in each direction was calculated using the following formula:

% Shrinkage = A - B x 100 A

Where A = Length before test

B = Length after test

3.18.8 Stain Resistance (ASTM D1308-02-2007)

To determine the resistance of the surface of coated fabrics to staining by common

household chemicals and/or different staining compounds.

A piece of PHB coated fabric and uncoated fabric was taken for stain testing (8.5 x 11

inch) and passed through the opening, in a continuous line across the width. The cotton swab was

used to apply the stain on the fabric and distributed along the opening. Cleaning was recommended

to eliminate cross contamination, the specific sample preparations under the CFFA (Chemical fabric

and film association) Standard Test Conditions for 2 and 24 hours. Allow the staining agents to set

at normal atmospheric conditions and the excess of the staining material was removed. Clean the

stains using a dry, clean cotton swab. The application of stains on the coated fabric and the control

fabric, followed by the cleaning process was done for 10 cycles.





The ratings for stain resistance are based on the following scale:

10 =Excellent cleanability, no stain mark in the material.

5 = Good cleanability, slight stain.

3 = Poor cleanability, stain is almost intact.

1 = Non cleanable, no stain removed.

3.18.9 UV Protection Test (AATCC 183-1999)

UV Protection testing for fabrics can provide excellent protection against the hazards of

sunlight. UV protection factor of textile material increases the stretch, wetness and degradation. The

standard method used for determining the UPF was AATCC 183 – 1999 (Transmittance or Blocking of

Erythemally weighted Ultraviolet Radiation through fabrics).

UV Transmittance of sample using UV Spectrophotometer

The standard method used for determining the UPF was AATCC 183 – 1999

(Transmittance or Blocking of Erythemally weighted Ultraviolet Radiation through fabrics). Percent

blocked was expressed in units, in order to reveal the fabric property and the ability of the fabric to

block UV depends upon its passage. The higher percent of block, the better the fabric was kept at

UV radiation and away from direct skin exposure, UV block was expressed as UVA block and as

UVB block.

The UPF rating provides information on how much amount of UV radiation would pass

through the unstretched, dried material. The PHB coated fabric and control fabric was placed into a

spectrophotometer. UV transmittance through the fabric samples was determined within a wave

length range from of 280 to 400 nm using a Shimadzu UV/Vis Spectrophotometer. Many values are

collected as a transmittance value was taken at five-nanometer increments along the UV spectrum

(UVA and UVB).

Formula for UPF: Step 1: 100 – UVR Block = A

Step 2: 100/ A = UPF





Table 4 Ultraviolet Protective Factor ratings for the fabrics

UPF Range UPF Rating Protection Category % UV Radiation Blocked

15-24 15,20 Good 6.7-4.2

25-39 25,30,35 Very Good 4.1-2.6

40-50,50+ 40,45,50,50+ Excellent Less than 2.5

UPF Rating was to specifically formulated to stop sun fading and soiling and for the

water proofing fabric winnings, tents, tarps, boat covers, umbrellas, flags, beach bags and golf bags

or to weather proof any of an outdoor fabrics.

3.19 Tissue Response of Chick Chorioallantoic Membranes (CAM) to PHB Treated Cotton

Fabrics

The avian chorioallantoic membrane (CAM) was the outer most extra embryonic

membrane lining the non cellular egg shell membrane. The CAM was formed by fusion of the

splanchnic mesoderm of the allantois and the somatic mesoderm of the chorion. The fused CAM

developed as a cover to the entire surface of the inner shell membrane at 12th day of incubation and

the chick hatched at 21th day of incubation. The CAM served as a support for the extra embryonic

respiratory capillaries actively transports sodium and chloride from the allantoic sac and calcium

form the egg shell into the embryonic vasculature and formed as a part of the wall of the allantoic

sac.

Egg Windowing

Fertilized chicken eggs were received from the poultry farm (Kovaipudur) and

incubated at 37°C with approximately 60% humidity. After 11th day of incubation, the eggs were

gently cleaned with a 70% ethanol solution. Using a 5-cc syringe and 18-gauge needle, 2.5 ml of

albumen was extracted from the egg. By extracting the albumen, the CAM of the fertilized egg was

separated from the top part of the shell, which allows for a small, 1.5-cm2 window to be cut in the

shell of the egg, without damaging the embryonic structures. The PHB treated swatch was placed

on the CAM, the window was then sealed using a tape and the egg was placed back in the incubator

until 7th day of incubation as shown in plate no 5a.





CAM Implants

The CAM model used for the study of tissue reactions to PHB treated swatch, both the

acute and chronic inflammatory responses of the chick embryo chorio allantoic membrane were

examined. Acute inflammation was evaluated with the PHB treated implants placed on top of the

CAM for 24 h. The chronic inflammation to the PHB coated thread and the silastic tubing was

evaluated after 1 week (and also at 11 days for the coated thread). At these time points, the implants

and surrounding tissues were retrieved.

Fixation of CA Membrane

The CA membrane were carefully dissected out and immediately placed in 10 percent

formal-saline solution. After 24 hours of fixation, the implant materials were teased away from the

underlying CA membranes, paraffin-embedded and prepared for staining by hematoxylin and eosin

and viewed under microscope as shown in plate no 5a.

3.20 Preparation of Polymer Solution

3.20.1 PHB Polymer Solution (Yuan et al., 2009)

A polymer solution was prepared by dissolving PHB in isopropanol with sufficient

stirring at room temperature.

3.20.2 Fabrication of PHB Nanofibrous Scaffolds by Electrospinning (M 98-D01)

Electrospinning Process (Li et al., 2008)

The blended polymer solution (PHB) was stocked in a 5 ml glass syringe per hour. A

metal needle (18 G) was used as a nozzle. A piece of aluminium sheet was used as a screen

collector placed opposite to the metal nozzle. Upon the high voltage, the polymer solutions were

electrospun with 2.0 ml/hr of a mass flow rate of about 7 kv of voltage and at a 12 cm of the

distance between the nozzle tip and the aluminium collector. The electrospinning was continued for

10-12 days and the developed nanofibers were placed in a vacuum drying oven at room temperature

for another five days for drying.





3.20.3 Characterisation of Developed PHB Nanofibrous Scaffolds

Physical Characterisation of Developed PHB Nanofibrous Scaffolds

The morphological appearance of the spun product (nanofiber) was observed by

scanning electron microscope (SEM). The specimen for SEM observation was prepared by cutting

an aluminium sheet covered with the spun products and the cut section was carefully affixed on a

copper stub. After sample preparation, the photographs of the sample were taken by Scanning

Electron Microscope (Model-JEOL-6390 under the magnification 5X to 300,000X, accelerating at

the voltage 0.5 to 30 kv).



3 materials and methods.pdf

Documents