AbbreviatedForm

Full Form

APHA American Public Health AssociationBM Basal MediumCPT Camptothecin

CS-137 Cesium-137DCW Dry Cell Weight

DMSO Dimethyl Sulfoxide-PGA Gamma-Polyglutamic acidGC Gas Chromatography

H-NMR Proton-Nuclear Magnetic ResonanceIR Infra Red

KDa Kilo DaltonLB Luria-Bertani

MW Molecular WeightO/F Oxidation-FermentationPG Polyglutamic acid

PG-CPT Polyglutamic acid-CamptothecinPG-TXL Polyglutamic acid-Paclitaxel

Ra-86 Radium-86Rf Specific movement

Sr-137 Strontium-137TLC Thin Layer ChromatographyTXL Paclitaxel

ACKNOWLEDGEMENT

My gratitude and indebtedness is extended to my supervisors Prof.

Dr. Amin A. Al-Sulami and Prof. Dr. Gheyath H. Majeed (College of

Agriculture).

My thanks go to Prof. Dr. Malik H. Ali (Director of Marine Science

Centre) for the logistic support in finishing the experimental parts of this

thesis.

My deep appreciation to my colleagues in Biology Department and

Marine Environmental Chemistry in Marine Science Centre.

Special thanks are to Mr. Faris Jasim for his efforts in analyzing H-

NMR and to Prof. Dr. Kahtan Adnan (Physics Department) for treating

the samples by irradiation.

Sixty-eight isolates have been purified from sugarcane fields,

seawater and marshes water. They were identified as Bacillus subtilis

according to their morphological and biochemical characteristics. The

bacteria were tested for their abilities to produce polyglutamic acid. Out

of sixty-eight only twenty-one were found to produce PGA, 16 isolates

from sugarcane fields and 5 isolates from seawater.

To obtain high production of PGA, media of various concentrations

of different ingredients have been tested at different pHs, temperatures,

and incubation periods. These ingredients included sodium chloride,

carbon source, nitrogen source, and manganese sulphate. Six mineral and

one basal media have been modified to enhance PGA production.

The highest yields of 18.34 g/l in liquid state and 16.81 g/l in solid

state were produced by isolate SC10 from sugarcane fields. While the

highest yields of 16.77 g/l in liquid state and 14.95 g/l in solid state were

produced by isolate SW5 from seawater, using a modified PGA producing

medium supplemented with 2% DL-alanine, 2% D-glucose and 1.2%

citric acid as carbon sources and 0.7% ammonium chloride as nitrogen

source at 30 C°, pH 7 and 120 r.p.m aeration rate. This is the first time to

produce PGA from DL-alanine instead of L- glutamic acid.

The isolates SC10 and SW5 also have the ability to produce PGA with

the presence of L-glutamic acid. Maximum yields reached at 14.07 g/l in

liquid state and 13.81 g/l in solid state by isolate SC10 and 13.93 g/l in

liquid state and 13.67 g/l in solid state by SW5 in a modified PGA

producing medium supplemented with 2% L-glutamic acid, 2% D-glucose

and 1.2% citric acid as carbon sources and 0.7% ammonium chloride as

nitrogen source at 30

productions whether by isolate SC10 or SW5 was 2-3 times higher than

that at the original conditions.

The PGA production was purified by several steps including

methanol precipitation, centrifugation, dialysis, and lyophilization.

The identification of PGA included the use of ninhydrin test to detect

the presence of single molecule of L-glutamic acid. Phenol-sulphuric acid

method showed the clearance of PGA from any by-product

polysaccharide. Also, thin layer chromatography showed that the

structural units of PGA were only D-glutamic acid. The estimation of

PGA molecular weights ranged between 103055-132500 Dalton for

isolate SC10 and 97631-103055 Dalton for isolate SW5. Gas

chromatography, infrared, and nuclear magnetic resonance spectra

analyses have been used for PGA identification.

Furthermore, the solubility of PGA in 25 different organic and

inorganic solvents has been tested. The solubility of PGA in organic

solvents ranged between 40.4%-90.6%, while in inorganic acid such as

deuterium oxide was 78.2% and water 100%.

Hydrogels were prepared from 2% and 5% of PGA in sterilized

distilled water, using four types of irradiation at different dosages

including alpha, beta, gamma rays and neutron. The PGA hydrogels have

been synthesized by gamma ray at 2.7 and 3.37 Mrad and by neutron at

3.4 X 1015 n/cm2 Sec. only from PGA of the isolate SC10.

1.1. Introduction

Gamma- -PGA, PGA or PG), an amino acid

polymer, consists of only D-glutamic acid, L-glutamic acid or DL-

glutamic acid. Units are connected by amide linkages between -amino

- Carboxylic acid groups.

PGA was first discovered as a capsule of Bacillus anthracis which

was released into the medium upon autoclaving or upon aging and

autolysis of the cells (Ivanovics and Bruckner, 1937; Ivanovics and Erdos,

1937).

Different microorganisms produce -PGA, so there are different

mechanistic systems for PGA production in different bacteria. Beside -

PGA gives us some physiological information on ribosome independent

synthesis of the structurally unusual polypeptides and optical isomers of

macromolecules (Cheng et al., 1989; Weber, 1990; Goto and Kunioka,

1992).

Attractive properties of - PGA are that it is a novel water-soluble,

biodegradable, edible and non-toxic toward humans and environment.

These and other features make it of interest for applications in the fields

of medicines, foods, plastics and many others (Lu et al., 2004; Drysdale et

al., 2004, 2005; Koncianova et al., 2005). PGA is a large polymer which

consists of 1000-10000 monomers (Ashiuchi et al., 2001).

The aim of this study

1. Isolation and identification of local strains of Bacillus subtilis

from the soil, marshes water, and seawater are capable of

producing gamma-polyglutamic acid.

2. Production of -PGA by batch culture method using a modified

medium with optimum conditions.

3. Purification and identification of -PGA.

4. Obtaining a hydrogel structure of PGA through treatment with

gamma, alpha, beta rays and neutrons.

1.2. Literature Review

1.2.1. Distribution of Polyglutamic Acid in Organisms

There are only three different poly (amino acids) known to occur in

nature. One of them is -polyglutamic acid (Opperman-Sanio and

Steinbüchel, 2002), whose chemical structure is described in Figure 1.

Figure 1 - PGA (Chibnall et al., 1958)

-PGA was first detected as component of the cell capsule of the

highly pathogenic Gram-positive bacterium B. anthracis (Ivanovics and

Erdos, 1937). Later the polymer was found in the cell surrounding of

other non-pathogenic Gram-positive strains of the genus Bacillus (B.

licheniformis, B. megaterium, B. subtilis, B. mesentericus and B.

amyloliquefaciens) (Ivanovics and Bruckner, 1937; Bovarnick, 1942;

Thorne et al., 1954; Aumayr et al., 1981 ; Kambourova et al., 2001).

At the beginning of the last century a Bacillus strain able to produce

high amount of PGA was isolated from fermented soybeans, a traditional

food in Japan "natto" (Sawamura, 1913). In addition to the endospore-

forming Bacillus Spp., two halophilic eubacteria, Sporosarcina halophila

and planococcus halophila (Kandler et al., 1983) and the halophilic

archaebacterium Natrialba aegyptiaca Sp. Nov. Str. 40 (Hezayen et al.,

2000) have been shown to be capable of excreting PGA. In addition to

prokaryotes, PGA was also detected in significant amounts in the

nematocysts of Cnidaria [e.g. Hydra (Weber, 1990)].

1.2.2. -PGA by Microorganisms

1.2.2.1. Genes R -PGA Synthesis

-PGA is unusual and it is different structurally and functionally from

proteins in that glutamate is polymerized via the gamma-amide linkages,

and thus should be synthesized by a ribosome-independent manner

(Opperman-Sanio and Steinbüchel, 2002).

The genes related to -PGA synthesis in B. anthracis lie on a large

plasmid DNA (Vietri et al., 1995). In contrast, it was reported that B.

subtilis TAM-4 and B. subtilis IFO 3336 (natto) had no plasmid and the

gene coding for the formation of -PGA lies on the genomic DNA

(Onodera et al., 1994; Nagai et al., 1997). Several attempts have been

made to isolate the genes responsible of PGA production (Ashiuchi et al.,

1998, 1999). They screened Escherichia coli clones consisting of the

DNA genomic library of B. subtilis IFO 3336 for PGA production, and

found a positive clone that produce -PGA extracellulary. This clone

identified as harboring three genes (newly designated as pgs BCA) were

highly homologous with cap BCA genes of B. anthracis, which were

presumed to encode a membrane-associated enzyme complex important

for the encapsulation of poly ( -D-glutamic acid) (Makino et al., 1989).

1.2.2.2. Enzymes R -PGA Synthesis

The enzyme transglutaminase has been reported to be related to PGA

polymerization with B. subtilis NR-1 (Ogawa et al., 1991). Thorne and

Co-workers (Willams and Thorne, 1954; Willams et al., 1955; Thorne et

al., 1955) have reported that a crude extracellular preparation from B.

subtilis 9945A contained a glutamyl transamidase which catalyzed the

transfer of the glutamyl group from L-glutamine to either optical isomer

of glutamic acid or glutamyl dipeptide, resulting in the formation of

glutamyl di-and tripeptides, respectively. They further showed that a

transpeptidation reaction between gamma-glutamyl dipeptides forming

gamma-glutamyl peptides of longer chain length was catalyzed by a

glutamyl trans peptidase present in the same enzyme preparation.

Biochemical investigations on the polymerization of PGA have been done

with B. anthracis (Meynell and Meynell, 1966) and in more detail with B.

licheniformis ATCC 9945 A (Leonard and Housewright, 1963; Troy

1973a, b; 1985; Gardner and Troy, 1979), which led to the identification

of the membrane-bound PGA synthetase system in the latter. This enzyme

system consists of at least three enzyme components. The presence of

these enzymes is shown to be involved in the indirect conversion of L-

glutamic acid to the D-isomer by the reactions shown in Fig 2. The same

indirect conversion of L-glutamic acid to the D-isomer was also shown in

B. subtilis (natto) (Hara et al., 1982 a, b).

for - PGA production most notably are B. subtilis 5E (Murao,

1969), B. subtilis TAM-4 (Ito et al., 1996), and B.

licheniformis A35 (Cheng et al., 1989).

B. subtilis 5E can produce -PGA from L-proline as the sole carbon

and nitrogen source in mineral medium. B. licheniformis A35 can

produce -PGA from glucose and ammonium chloride under denitrifying

conditions, and B. subtilis TAM-4 produces a large amount of -PGA

when grown in a culture medium containing ammonium salt and sugar as

sources of nitrogen and carbon.

Besides carbon and nitrogen sources, factors such as ionic strength,

aeration, medium pH also effect in the production of PGA (Ko and Gross,

1998; Perez-Camero et al., 1999; Yang et al., 2001; Zanuy and Aleman,

2001; Do et al., 2001; Richard and Margaritis, 2003; Ashiuchi et al.,

2004). Some -PGA producing bacteria are listed in Table 1, in which the

nutrient requirement, cultivation conditions, productivity and molecular

weight are summarized.

Table 1: Table 1 -PGA- producing bacteria

1.2.2.5. Biodegradation

All PGA-producing bacteria can use this polymer as sole carbon and

nitrogen source (Birrer et al., 1994; Kunioka and Goto 1994). Cells of B.

licheniformis ATCC 9945A grown on PGA express a depolymerase

(Birrer et al., 1994), which seems to be physically associated with the

PGA in the surrounding of the cell (King et al., 2000). A PGA hydrolase

was isolated from a filamentous fungus, which was shown to be specific

for the release of L-glutamic acid oligomers from PGA consisting of both

amino acid enantiomers (Tanaka et al., 1993). To investigate the ability of

microbial community to degrade PGA, a mineral salts medium containing

PGA as sole carbon source was inoculated with sewage sludge. Twelve

bacterial strains, which were able to use PGA as carbon source for

growth, were finally isolated from this habitat and identified (Oppermann

et al., 1998).

1.2.3. -PGA

1.2.3.1. -PGA in the Environment

According to shih et al., (2001), flocculating agents are categorized

into three major groups:

1. Inorganic flocculants: aluminum sulfate and polyaluminium

chloride.

2. Organic synthetic polymer flocculants: polyacrylamide

derivatives, polyacrylic acids and polyethylene imine.

3. Naturally occurring biopolymer flocculants: chitosan algin and

microbial flocculants.

These flocculants have been widely used in wastewater treatment and

in a wide range of industrial downstream processes (Nakamura et al.,

1976; Gutcho, 1977; Kurane et al., 1986). The organic synthetic polymer

flocculants are mostly used because they are economical and highly

effective. However, their uses often give rise to environmental and health

problems in that some of them are not readily biodegradable and some of

their degraded monomers, such as acrylamide are neuro-toxic and even

strong human carcinogens (Vanhorick and Moens, 1983; Dearfield and

Abermathy, 1988). Thus, the development of safe biodegradable

flocculants that will minimize environmental and health risks is urgently

required.

The use of biopolymers produced by microorganisms has been

investigated. These microbial polymers are expected to be useful

flocculating agents due to their bio-degradability and the harmlessness of

their degradation intermediates toward humans and the environment (Shih

et al., 2001). Several bioflocculants from different microorganisms have

been reported that one of them is - PGA produced by Bacillus sp. PY-90

(Yokoi et al., 1995), B. subtilis IFO 3335 (Yokoi et al., 1996), and B.

licheniformis CCRC 12826 (Shih et al., 2001), and which had shown

high flocculating activities. For the production of -PGA flocculants by

strain PY-90, a medium containing 2-5% L-glutamic acid was used. In

kaolin suspensions, the -PGA flocculant attained the highest flocculating

activity at the concentration of 20 mg/l, and the flocculating activity

increased by the addition of metal cations such as Ca+2 , Mg+2 , Fe+2. The

flocculating activity was high in an acidic pH range 3.0-5.0, and

decreased upon heating at 100 C (Yokoi et al., 1995). For the production

of -PGA by B. subtilis IFO 3335 and B. licheniformis CCRC 12826

media containing 2-3% L-glutamic acid were used (Yokoi et al., 1996;

Shih et al., 2001).

Both flocculants were shown to flocculate various inorganic (active

carbon, acid clay, solid soil, calcium and magnesium compounds) and

organic (cellulose and yeast) suspensions. In kaolin suspensions, the

flocculating activities of both flocculants were stimulated by the addition

of Ca+2, Mg+2, Fe+2 to the suspension. High flocculating activities of strain

PY-90 and both flocculants were induced in a kaolin suspension by the

addition of Al+3 and Fe+3 and pH adjustment to the neutral range. It is

anticipated that -PGA will be utilized not only in the areas of wastewater

treatment but also drinking water processing and downstream processing

in the food and fermentation industry (Shih and Van, 2001).

Due to accumulation in the environment of many heavy metals and

radionuclides which threaten the public health, there has been an increase

in research and development aimed at environmental remediation. The

remediation of contaminated soils, sediments, and water is a tremendous

challenge facing us today (Macaskie and Basnakova, 1998). There is an

increasing research interest in the use of biogeochemical processes to

reduce the environmental risks of heavy metals. McLean et al.,

(1990, 1992), studied the metal-binding affinity of the anionic - PGA of

B. licheniformis and found that it binds a variety of metals including Ni+2,

Cu+2, Mn+2, Al+3, and Cr+3. Bhattacharrya and co-workers (Bhattacharrya

et al., 1998; Ritchie et al., 1999) reported that, some PGA-functionalized

cellulosic membranes showed capacities as high as 1.3 - 1.5 g/g

membrane with Pb and about 0.8 mol metal sorbed/mol repeat unit for Cd

and Ni.

Ritchie et al., (2001) found that PGA is high capacity sorbents (0.3-

3.7 mg/cm2) with excellent accessibility and selectivity for heavy metals,

such as Hg, Pb and Cd over nontoxic components such as calcium. PGA-

functionalized membranes have been found to selectively sorb Pb versus

Cd. The high capacity, site accessibility, and ease of regeneration of PGA-

functionalized membrane make them ideal for environmental separations

when volume reduction or selective recovery is required (Ritchie et al.,

2001).

1.2.3.2. -PGA in Medicine

Paclitaxel (Taxol, TXL) a natural antimicrotubule agent, is a chemo-

therapeutic agent with potent antitumor activity against various human

malignancies, including breast and ovarian tumors (Rowinsky and

Donehower, 1995; Holmes et al., 1995). The major difficulty in the

clinical use of paclitaxel has been its insolubility in water. Polyglutamic

acid paclitaxel (PG-TXL) is a water- soluble paclitaxel conjugated made

between TXL and PG via ester bonds and exhibited greater antitumor

activity against murine tumors and human tumor xenografts than TXL (Li

et al., 1999; Auzenne et al., 2002). Polyglutamic acid can be used with

other cancer drug, camptothecin. The therapeutic efficacy of camptothecin

(CPT) is limited in humans by the instability of the active lactone form

due to preferential binding of the carboxylate to serum albumin and by

difficulty in formulation (Singer et al., 2001).

Formation of a water-soluble CPT derivative by coupling PGA via

the hydroxyl group at carbon 20 of CPT stabilizes the lactone. Linking

CPT to a high molecular weight anionic polymer enhances solubility and

improves distribution to the tumor through enhanced permeability and

retention (Singer et al., 2000, 2001). In vitro, PG-CPT was less potent in

inhibiting cell growth than free CPT in all human tumor cell lines tested.

PG-CPT showed better antitumor activity and tolerability than did CPT in

vivo (Zou et al., 2001).

Biological adhesives are commonly used in surgical either synthetic

or semi-synthetic, such as cynoacrylate (Tseng et al., 1990 a, b), urethane

prepolymers (Kobayashi et al., 1991), and fibrin glue (Spotniz, 1996).

These adhesives have problems such as cytotoxicity, low degradation

rates, its adhesion property to tissues is poor and mechanical strength is

low.

A new biological adhesive, formed by chemical cross linking of

gelatin and PGA, both of which are biodegradable (Otani et al., 1996 a,

b), showed much higher bonding strength to soft tissue. A new biological

adhesive made from porcine collagen and PGA is superior to fibrin in

sealing air leakage from the lungs (Sekine et al., 2000).

1.2.3.3. Thermoplastic and Hydrogels

Ester derivatives of PGA have the ability to form biodegradable

fibers and films that can replace currently used non-biodegradable

polymers. It was found that the estrified PGA served as an excellent

thermoplastic (Giannos et al., 1990) and that poly ( -glutamic acid- -

benzyl ester) could be processed into fibers or membranes with excellent

strength, transparency and elasticity by the standard methods used in

polymer processing (Kubota et al., 1992; Yahata et al., 1992).

Development of new hydrogels which have a variety of applications

such as controlled release devices, super absorbent materials and

biomaterials like enzyme immobilization (Han and Choi, 1999),

irradiation (Kunioka, 1993, 2003), repetitive freezing (Han and Choi,

1999), and chemical cross-linking (Kunioka and Furusawa, 1997;

Markland et al., 1999) have been representative techniques to develop

new hydrogels. A novel -PGA-based hydrogel prepared by - irradiation

of aqueous solution of microbial PGA showed high water sorption ability

and various equilibrium swelling behaviors responding to the conditions

of surrounding medium (Han and Choi, 1999).

1.2.3.4. Food Application

- PGA is an effective cryoprotectant for frozen food, because it has a

weaker taste than the commonly used lower molecular weight

cryoprotectant such as saccharides, inorganic salts and amino acids. Thus,

it can be added to foods in larger quantities without a serious change in

the taste. Furthermore, it has been shown that the addition of -PGA

and/or its decomposition products in high mineral food preparations

accelerates the absorption of minerals in the small intestine while it masks

the extraneous taste of enriched minerals (Tanimoto et al., 1995).

- PGA can be used also as a bitterness- relieving agent (Sakai et al.,

1995). It has been shown that adding PGA to one or more substances

having a bitter taste (amino acids, peptides, quinine, Caffeine, minerals,

etc.) greatly relieved bitterness. The addition of PGA or an edible salt in

the manufacture of starch foods (Kunno et al., 1988 a, b), mainly bakery

products and noodles results in the prevention of aging and the

improvement of textures, and contributes to the shape retention of food.

PGA is used as an ice-cream stabilizer (Daninippon, 1972), a thickener

for fruit juice beverages, or is added to sports drinks to improve taste and

drink ability (Yamanaka, 1991).

2.1. Materials

2.1.1. Chemicals

All chemicals were analar and they included: ethanol, methanol,

acetone, n-butanol, acetonitrile, glycerin, toluene, benzene, n-hexane,

petroleum ether, di-ethyl ether, carbon tetrachloride, di-methyl foramide,

di-methyl sulfoxide (DMSO) , 1,1,1-trichloroethane, c-hexane, pyridine,

acetic acid, hydrochloric acid, hydrogen peroxide, L-glutamic acid, citric

acid, ammonium chloride (NH4Cl), potassium di-hydrogen

orthophosphate (KH2PO4), di-sodium hydrogen orthophosphate

(Na2HPO4), sodium chloride (NaCl), magnesium sulphate

(MgSO4.7H2O), di-ammonium sulphate [(NH4)2 SO4] , sucrose, gelatin,

starch, D-glucose, D-mannitol, salicin, manganese sulphate

(MnSO4.H2O), potassium hydroxide (KOH), sodium hydroxide (NaOH),

-naphthol, ferric chloride (FeCl3.6H2O), potassium bromide (KBr),

mercuric chloride, 4-aminobenzaldehyde, and tetra methyl-Para-

phenylene diamine dihydroxide. All of them were purchased from BDH

chemical Ltd, Pool, England.

Amyl alcohol, 1,4-dioxane, deuterium oxide (D2O), benzene bromo-

d5, L-alanine, DL-alanine, L-arabinose, D-xylose, D-mannose, xylitol, D-

ribose, and sorbitol were purchased from Fluka AG, CH, 9470 Buchs,

Switzerland.

Formaldehyde, ethyl acetate, di-chloro methane, ninhydrin, biotin, di-

potassium hydrogen orthophosphate (K2HPO4), and calcium carbonate

(CaCO3) were purchased from Riedel De Haen AG, Seelze, Hanover,

Germany.

Crystal violet, safranine, and malachite green were purchased from

Maknur Lab. Ottawa, Canada.

Calcium chloride (CaCl2.2H2O), iodine, and potassium iodide were

purchased from Hopkins & Williams Chadwell health Essex, England.

Bacto-Tryptone, Bacto- Peptone, Beef Extract, Yeast Extract, and

Skim milk were purchased from Difco Laboratories Detroit, Michigan,

USA.

2.1.2. Sampling

Samples were collected from

1. Marshes water, south of Iraq.

2. Seawater, Khor-Al-Zubair.

3. Sugarcane fields in Missan.

The water samples were collected in sterile 250 ml Nalgene

polycarbonate conical flasks, while the soil samples were collected in

sterile plastic bags. The samples were placed in ice until return to the

laboratory.

2.1.3. Media

2.1.3.1. Isolation Medium

It was purchased from Lab M, Wash Lane, Bury, BL 9, AV. England.

2.1.3.2. Identification Media

It consists of 1g of Bacto-Tryptone (Difco), 0.1 g of Bacto- Yeast

Extract (Difco), 1 g of D-glucose, 0.5 g of NaCl, and 1.5 g of Bacto-agar

(Difco) per 100 ml distilled water. This medium was autoclaved for 15

minutes (APHA, 1984). It used for catalase test.

It was purchased from Lab M. Wash Lane, Bury, BL 9, AV. England

and used for indole test.

It was purchased from Oxoid LTD, Basingstoke Hampshire, England

and used for Voges-Proskauer test.

It was purchased from Oxoid and used for citrate utilization.

It was purchased from Difco Laboratories Detroit, Michigan. USA

and used with the addition of 1 g of suitable sugar for acid production.

It was purchased from Difco and used for deamination of

phenylalanine.

It contains 3 g of Beef Extract (Difco), 5 g of peptone (Difco), 10 g of

skim milk (Difco) and 2 g of Bacto-agar (Difco) per 100 ml of distilled

water. This medium was autoclaved for 5 min and used for hydrolysis of

casein.

It was purchased from Difco and used for liquification of gelatin.

Similar to 2.1.3.2.1, it was supplemented with 1 g of starch instead of

1 g of glucose and used for starch hydrolysis.

2.1.3.3. Polyglutamic Acid Production Media

It was purchased from Lab M with the addition of 0.5 g of

MgSO4.7H2O per 100 ml, pH 7.

It was composed of (per 100 ml, pH 7) 1 g of Tryptone (Difco), 0.5 g

of Bacto-Yeast Extract (Difco), 0.5 g of NaCl, and 0.5 g of MgSO4.7H2O

It was purchased from Difco

It was composed of (per 100 ml, pH 7.2) 1.8 g of NH4Cl, 7.5 g of

glucose, 0.15 g of K2HPO4, 0.035 g of MgSO4.7H2O, 0.005 g of

MnSO4.H2O, 0.004 g of FeCl3.6H2O, 2 g of CaCO3, and 1.5 g of agar

(Cheng et al., 1989)

1. Type A

It was composed of (per 100 ml, pH 6.5) 2 g of L-glutamic acid, 1.2 g

of citric acid, 8 g of glycerin, 7 g of NH4Cl, 0.05 g of K2HPO4, 0.05 g of

MgSO4.7H2O, 0.004 g of FeCl3.6H2O, 0.015 g of CaCl2.2H2O, 0.0104 g

of MnSO4.H2O, and 1.5 g of agar (Yoon et al., 2000)

1. Type B

It was composed of (per 100 ml, pH 7) 2 g of L-glutamic acid, 5 g of

sucrose, 0.27 g of KH2PO4, 0.42 g of Na2HPO4, 5 g of NaCl, 0.5 g of

MgSO4.7H2O, and 1.5 g of agar (Ashiuchi et al., 2001).

It was composed of (per 100 ml, pH 7) 5 g of sucrose, 2 g of (NH4)2

SO4, 0.27 g of KH2PO4, 0.42 g of Na2HPO4. 0.05 g of NaCl, and 0.5 g

MgSO4.7H2O (Ashiuchi et al., 2001).

2.1.3.4. Modified Polyglutamic Acid Media

The following media are modifications made by Al-Taee at the

Marine Science Centre.

The medium 2.1.3.3.5b was modified by decreasing NaCl to 0.2 g

(per 100 ml, pH 7).

The medium 2.1.3.4.1 was modified by the addition of 1.2 g of citric

acid, 0.7 g of NH4Cl, 0.0114 g of MnSO4.H2O, and 2 g of D-glucose

instead of sucrose (per 100 ml, pH 7).

The medium 2.1.3.4.1 was modified by the addition of 0.0114 g of

MnSO4.H2O (per 100 ml, pH 7).

It was composed of (per 100 ml, pH 7) 2 g of L-alanine, 1.2 g of

citric acid, 2 g of glucose, 0.7 g of NH4Cl, 0.27 g of KH2PO4, 0.42 g of

Na2HPO4, 0.2 g of NaCl, 0.5 g of MgSO4.7H2O, 0.0114 g of MnSO4.H2O,

and 1.6 g of agar.

Composed of (per 100 ml, pH 7) 2 g of DL-alanine, 1.2 g of citric

acid, 0.7 g of NH4Cl, 2 g of glucose, 0.27 g of KH2PO4, 0.42 g of

Na2HPO4, 0.2 g of NaCl, 0.5 g of MgSO4.7H2O, 0.0114 g of MnSO4.H2O,

and 1.6 g of agar.

It was composed of (per 100 ml, pH 7) 2 g of L-alanine, 2 g of

glucose , 0.0011 g of biotin, 1.2 g of citric acid, 0.7 g of NH4Cl, 0.27 g of

KH2PO4, 0.42 g of Na2HPO4, 0.2 g of NaCl, 0.5 g of MgSO4.7H2O,

0.0114 g of MnSO4.H2O, and 1.6 g of agar.

It was composed of (per 100 ml, pH 7) 2 g of DL-alanine, 2 g of

glucose, 0.0011 g of biotin , 1.2 g of citric acid , 0.7 g of NH4Cl, 0.27 g of

KH2PO4, 0.42 g of Na2HPO4, 0.2 g of NaCl, 0.5 g of MgSO4.7H2O,

0.0114 g of MnSO4.H2O, and 1.6 g of agar.

The medium 2.1.3.3.6 was modified by the addition of 2 g of glucose

instead of 5 g of sucrose.

2.2. Methods

2.2.1. Isolation of Bacteria

2.2.1.1. Water Samples

The water samples were heated at 60 C for 60 min. in water bath

(Aslim et al., 2002), then cooled and filtered (APHA, 1998) by passing

25ml in duplicates from each sample through 0.45µ Millipore NCS type

filters (Albet, Corp., Germany). The filter papers were incubated on

2.1.3.1.1 type medi

2.2.1.2. Soil Samples

Each one gram of the soil sample was suspended in 99 ml of sterile

distilled water and shaken vigorously for 2 min. The samples were heated

at 60 C for 60 min in water bath (Aslim et al., 2002). The liquid was

serially decimal diluted supernatant in sterile distilled water of 10-1 to 10-4

and was plated on 2.1.3.1.1for 24 h.

2.2.2. Identification of Bacteria

Figure (3) describes the procedures used for the isolation and

identification of bacteria obtained in this study.

Figure 3:Diagram of identification of Bacillus subtilis

2.2.3. Colony Purification

The white, middle or big, and flat colonies on 2.1.3.1.1 type medium

were selected and purified for several times to secure pure cultures.

2.2.4. Stock Cultures

Two types of maintenance were used

1) One Month

The bacteria were cultured on 2.1.3.1.1 type medium and incubated at

30 C for 24 h, and then stored in refrigerator.

2) Long Period

The bacteria were cultured on 2.1.3.3.2 type medium, incubated at 30

C for 24 h, and then lyophilized by freeze dryer (Edwards, UK). The

powder was stored in deep freezer (-18 C )

2.2.5. Optimum Condition for PGA Production

Media of various concentrations of different ingredients have been

used to obtain high production of PGA. These ingredients including NaCl,

carbon source, nitrogen source, MnSO4, pH, temperature and time of

fermentation were also kinds of modifications subjected to variation.

2.2.5.1. Sodium Chloride

Five concentrations of NaCl were optimized through 2.1.3.4.1 type

medium as (4, 2.5, 1, 0.5, 0.2) g/100 ml.

2.2.5.2. Carbon Source

Concentrations of five media ingredients (glutamic acid, citric acid,

glucose, L-alanine, and DL-alanine) were optimized to predict the

production of PGA in production medium 2.1.3.4.1.The pH was adjusted

to 7.0 with 6M NaOH solution.

Four concentrations were used (1, 2, 3, 4) g/100 ml.

Five concentrations of L-alanine were used instead of L-glutamic

acid (1, 2, 3, 4, 5) g/100 ml to produce PGA.

Five concentrations of DL- alanine have been used (1, 2, 3, 4, 5)

g/100 ml instead of L-glutamic acid.

It was used as carbon source in four concentrations (0.8, 1.2. 1.6, 2) g

/100 ml.

2.2.5.3. Nitrogen Source

Only NH4Cl was used as nitrogen source in four concentrations (0.5,

0.7, 1, 1.3) g/ 100 ml.

2.2.5.4. Manganese Sulphate

Five concentrations of this salt were used to induce the production of

PGA as (0.001, 0.005, 0.007, 0.009, 0.0114) g/100 ml.

2.2.5.5. Optimum pH

Production media were selected in different initial pH 5.5, 6, 6.5, 7,

7.2 and 7.5 to determine the optimum pH.

2.2.5.6. Optimum Temperature

Different incubation temperature have been used for fermentation in

production media as (20, 25, 30, 35, 37, 40, 60) C .

2.2.5.7. Time of Fermentation

Six periods have been used for fermentation production media as (12,

24, 48, 72, 96, and 120) h.

2.2.5.8. Estimation of High Productivity isolate

The isolates were grown on 2.1.3.3.1 type medium as starter medium

and then grown on modified media at 30 C for 72 h as production media.

2.2.5.9. Comparison between Liquid and Solid Fermentations

The media 2.1.3.4.2 and 2.1.3.4.5 were used with and without agar as

production media at pH 7.0 and incubated for 72 h at 30 C .

2.2.5.10. Comparison between 2.1.3.3.2 and 2.1.3.3.3 as Starter

Media

These media were used as starter media to induce spores and obtain

high inocula needed in production media. The bacteria were cultured at 30

C for 24 h.

2.2.6. Polyglutamic Acid Production

Figure (4) shows the processes of PGA production.

2.2.6.1. The Growth of Bacteria

Bacterial cells were grown on a medium 2.1.3.3.1 at 30 C for 24 h

(pH 7.0). After that they were transferred to one of production E media

(including modified media) . A bacterium was

isolated from highly mucous colony, inoculated into 50 ml of medium

2.1.3.3.2, and incubated at 30 C . When the turbidity of culture at 600 nm

(spectrophotomer Cintra 5, Australia) reached 2.1 (5 109 cell ml-1,

containing mainly cells in stationary phase), cells were harvested by

centrifugation (Heroeus Christ GMBH Osterode, Germany) at 15.000

r.p.m. for 20 min at 4 C , washed with 50 ml of 0.85% NaCl solution, and

then suspended in 5 ml of 0.85% NaCl solution. The cell suspension was

transferred to 45 ml of 2.1.3.3.6 and 2.1.3.4.8 and incubated aerobically at

30 C for 72 h. with stirring at 120 r.p.m.

Figure 4:The processes of PGA production by B. subtilis isolates

2.2.6.2. Estimation of Biomass

After every production of PGA, dry cell weight (DCW gl-1) was

determined by centrifuging at 20.000 r.p.m. for 40 min at 4 C , washing

with distilled water and drying

at 95 C to a constant weight (Yoon et al., 2000).

2.2.6.3. PGA Purification

PGA was purified by the methanol precipitation method (Yoon et al.,

2000). Cells were separated from the culture broth by centrifugation at

20.000 r.p.m for 40 min and 4 C . The supernatant containing PGA was

poured into 4 volumes of methanol and left overnight with gentle stirring

(40 r.p.m). The resulting precipitate containing crude PGA was collected

by centrifugation at 20.000 r.p.m. for 60 min and 4 C . Then, crude PGA

was dissolved in distilled water and any insoluble contaminants were

removed by centrifugation at 20.000 r.p.m for 40 min and 4 C .

The aqueous PGA solution was desalted by dialysis against 1l of

distilled water for 12 h with three water exchanges (Kambourova et al.,

2001).

After dialysis, the PGA solutions were lyophilized by freeze dryer

(Edwards, UK) to prepare pure PGA.

2.2.6.4. Identification of PGA

Test was adopted for L-glutamic acid dependent bacteria to confirm

the absence of free L-glutamic acid (Kambourova et al., 2001)

Reagents used: 0.2g Ninhydrin dissolved in 100ml of acetone

Procedures:-

In a test tube put 1g of PGA

Add 10ml of 0.2% ninhydrin

Allow the tube to stand for 3 min

The presence of blue colour indicates the presence of L-

glutamic acid in PGA samples.

The total sugars were determined according to Dubois et al., (1956).

Reagents used:

1. 5% aqueous solution of phenol

2. 95.5% of sulphuric acid

3. Analytes: 10 mg of PGA dissolved in 10 ml of distilled water

Procedure:

1. In a test tube, put 2 ml of analyte solution

2. Add 1.0 ml of phenol solution

3. Add 5.0 ml of sulphuric acid

4. Shake the tubes vigorously for 2 min and then allow to cool at

room temperature

5. The presence of orange- yellow colour indicates the presence

of sugar in PGA samples.

To hydrolyze PGA to its constituent amino acids, 0.01g of PGA was

dissolved in 1 ml of distilled water with addition of 10 ml of 6N HCl at

100 C overnight. The hydrolysate was neutralized with 6N NaOH. The

solution was washed with distilled water and filtered through paper type

The analysis was done in the physiology Department, College of

Medicine, Basrah University.

0.1g of PGA product from media containing L-alanine or DL-alanine

was dissolved in 10 ml n-hexane. 100µl of this solute was compared with

authentic L-glutamic acid and analyzed in gas chromatography type

Sigma 300 capillary gas chromatography Perkins Elmer, U.S.A. in Marine

Environmental Chemistry Department, Marine Science Centre, Basrah

University. The condition of analysis was: Initial ,final

temperature 200 C , Rate 8 C /min

IR analysis was done by using Shimadzu FT-IR (Creon Lab. AG

Shimadzu. Japan) in Petrochemical Company, Basrah. KBr disc for the

PGA produced by B. subtilis and for free amino acids as follows:

1. L-glutamic acid

2. L-alanine

3. DL-alanine

4. PGA produced from L-glutamic acid

5. PGA produced from L-alanine

6. PGA produced from DL- alanine

H-NMR was done by using JNM-LA 300, 300MHz , 7.1T (FT NMR,

Jeol, Japan) in central Laboratories Unit, United Arab Emirates

University, U.A.E. by Dr. Rao, M.V. and MSc El Sayed H. Awad .

Solvents used were Deuterium oxide (D2O) and di-methyl sulfoxide

(DMSO) at 21 C and 35 C .

2.2.6.5. Further Characterization of PGA

Twenty-five different solvents including water were used to solve

PGA at room temperature (g/l). According to their dielectric constant: n-

hexane, c-hexane, carbon tetrachloride, ethanol, methanol, acetone, di-

ethyl ether, petroleum ether, n-butanol, benzene, formaldehyde, toluene,

glycerin, N,N di-methyl foramide, di-methyl sulfoxide, 1,1,1-trichloro

ethane, acetic acid, 1,4-dioxane, pyridine, acetonitrile, deuterium oxide,

benzene bromo-d5, ethyl acetate, and di-chloro methane. The dielectric

constant values (also called static values) were obtained from Weast and

Astle (1979).

2% and 5% of PGA in sterile distilled water were exposed to four

sources of irradiation.

1. Alpha Source ( ) activity 10 mrad/Sec

Source: Ra-86 Amersham UK

2. activity 120 mrad/min

Source: Sr-90 Amersham UK

3. Gamma Source ( ) activity 67 rad/min

Source: Cs-137 Shepherd USA

4. Neutron Source activity 20Gi Ø = 5 108 n/Cm2.Sec

Source: Am-Be Amersham UK

3.1. Isolation and Identification

According to Sneath et al., (1986); Ivanova et al., (1999); Ashiuchi et

al., (2001); Keawachi and Prasertsan (2002), sixty-eight isolates of B.

subtilis were isolated from marshes water (30), seawater (18) and

sugarcane fields (20). The bacteria were identified depending on colonies

morphology of grown isolates on LB agar (2.1.3.1.1) type medium.

Colony size appear middle to big and has cream colour, while the shape is

round and convex.

The cells of B. subtilis are Gram positive, and may occur singly or in

chains. Rod shape may be small or large.

B. subtilis is able to form endospore after the end of exponential cell

growth or if vegetative cells are transferred from a rich to a poor medium.

The endospore of B. subtilis is cylindrical and central. All isolates of B.

subtilis tested in this study, were oxidase negative and catalase positive.

Table (2) showed the biochemical tests for identification of B. subtilis and

five tests used for the first time for B. subtilis.

3.2. Production of Polyglutamic Acid

Sixty-eight isolates of B. subtilis have been tested for their ability to

produce PGA. At first the bacteria have been cultivated in LB agar

medium (2.1.3.3.1) at 30 C for 24 h and then were transferred to E-

medium (2.1.3.3.5b) and cultured for 72 h at 30 C (Ashiuchi et al., 2001).

As in table (3), only 16 isolates of B. subtilis from sugarcane and 5

isolates from seawater (Table 4) have the ability to produce PGA. From

the 21 isolates that have the ability to produce PGA, two isolates were

chosen (SC10 from sugarcane fields and SW5 from seawater) for

additional tests.

3.2.1. Comparison between SC10 and SW5

The two isolates were cultured in media 2.1.3.3.4, 2.1.3.3.5a and

2.1.3.3.5b in order to confirm their ability to produce PGA in different

media and different conditions (Table 5 and 6).

3.3. Optimum Conditions for PGA Production

3.3.1. Sodium Chloride

It has been shown that the concentration of NaCl in media has an

effect on the PGA production. Figure (5) shows that at low concentration

of NaCl the amount of PGA reached maximum.

3.3.2. Carbon Sources

3.3.2.1. L-Glutamic Acid

By using different concentrations of L-glutamic acid as one of the

carbon sources in the medium 2.1.3.4.1 the production of PGA by SC10

and SW5 differ but the best production was at 2% concentration (Fig. 6).

3.3.2.2. L-Alanine

Searching for enhancers of PGA production, L-glutamic acid has

been substituted by L-alanine and as in figure (7), the amount of PGA

increased at 1% and 2%, and decreased with the increasing of the

concentrations.

Table 2:Taxonomical characteristics of Bacillus subtilis isolated from sugarcanefields, marshes water, and seawater.

Table 3:The amounts of PGA produced by B. subtilis isolated from sugarcane fields

Table 4:The amounts of PGA produced by B. subtilis isolated from seawaterand

Table 5:The amounts of PGA produced by SC10 grown in differentmedia,temperature, and period of fermentation.

Table 6:The amounts of PGA produced by SW5¬ grown in different media,temperatureand period of fermentation.

Figure 5:The effect of NaCl concentrations on the PGA production by SC10 and

25

1025

4050

Sw5

SC10

11.63311.367

10.35810.295

10.2119.966

10.93210.791

9.6129.488

8.85

6.226

0

2

4

6

8

10

12

NaCl g/l

PGA g/l

Sw5SC10

Figure 6:The amounts of PGA produced by SC10 and SW5 in medium 2.1.3.4.1 atdifferent concentrations of L- glut

1020

3040

SW5

SC10

9.875

11.633

10.621

8.945

9.542

10.932

10.267

8.353

0

2

4

6

8

10

12

L-glutamic acid g/l

PGA g/l

SW5SC10

Figure 8:The amounts of PGA produced by SC10 and SW5 in medium 2.1.3.4.1 atdifferent concentrations of DL-

1020

3040

50

SW5

SC10

9.67

11.35

8.6

8

6.16

8.93 9.129.01

8.53

7.83

0

2

4

6

8

10

12

DL-alanine g/l

PGA g/l

SW5SC10

3.3.6. Optimum Temperature

The incubation temperature of B. subtilis has an effect on its ability

to produce PGA. From figure (14), it seems that 30 C is suitable for the

PGA production.

3.3.7. Time of Fermentation

The period of incubation have affected the amount of PGA produced.

Figure (15) shows that when the period of incubation is short, the amount

of PGA is little, and also, when it is long, the amount of PGA is little. But

the best period is 72h.

Figure 1372 h and 120 r.p.m.

5.56

6.57

7.27.5

SW5

SC10

4.6

6.45

10.01

13.2513.12

11.85

5.16

6.29

9.99

12.8912.69

12.14

0

2

4

6

8

10

12

14

pH

PGA g/l

SW5SC10

Figure 14:The effect of incubation temperature at the PGA production by SC10 andSW5 for 72 h and 120 r.p.m.

2025

3035

3740

60

SW5

SC10

8.49

10

13.1212.98 12.96

9.13

6.03

9.53

11.16

12.6912.35 12.29

10.95

7.81

0

2

4

6

8

10

12

14

PGA g/l

SW5SC10

Figure 15:The effect of incubation period on the PGA production by SC10 and SW5

3.4. Modified Media

Six media have been modified according to the nutrient requirements

of B. subtilis isolates producing PGA. These bacteria are able to produce

PGA with the addition of L-glutamic acid to the medium and also have

the ability to produce PGA without the addition of L-glutamic acid. After

the growth of SC10 and SW5 in the modified media, they were transferred

into 2.1.3.3.6 type medium and modified medium 2.1.3.4.8 which were

used to induce PGA production. From table (7), the amount of PGA in

medium 2.1.3.4.8 was more than that in medium 2.1.3.3.6.

1224

4872

96120

SW5

SC10

0.6

2.81

8.56

13.25

12.34

8.19

0.59

2.47

7.81

12.89

11.1

9.51

0

2

4

6

8

10

12

14

Inc. period h.

PGA g/l

SW5SC10

3.5. Estimation of Biomass

The dry cell weight of bacteria SC10 and SW5 were determined after

every production of PGA in media 2.1.3.3.6 and 2.1.3.4.8. Table (8)

shows that the medium 2.1.3.3.6 gives to biomass more than the medium

2.1.3.4.8 which gives better production of PGA.

Table 7and 120r.p.m.

Table 8:The amounts of biomass produced in 2.1.3.3.6 and 2.1.3.4.8 media.

3.6. Comparison between Liquid and Solid

Fermentations

In an attempt to enhance the amount of PGA, the media 2.1.3.4.2 and

2.1.3.4.5 were used either in solid or liquid state. From table (9), the

amount of PGA and biomass either in medium 2.1.3.4.2 or 2.1.3.4.5 for

each of the two isolates in liquid state were greater than in solid state.

Table 9:Productivity of PGA and biomass in media 2.1.3.4.2 and 2.1.3.4.5 in liquidand solid state.

3.7. Comparison between 2.1.3.3.2 and 2.1.3.3.3 as

Starter Media

In all experiments we used LB agar as starter medium. But in this

experiment, two media were used as starter media LB broth (2.1.3.3.2)

and nutrient broth (2.1.3.3.3). After the incubation of bacteria in either of

these media for 24 h at 30 C and 120 r.p.m, the cells were harvested by

centrifuge at 20000 r.p.m for 15 min at 4 C . It was transferred to medium

2.1.3.4.5 and incubated for 72 h at 30 C and 120 r.p.m. After that, the

cells were harvested as above and re-incubated in medium 2.1.3.4.5 and

2.1.3.4.8 for 72 h at 30 C and 120 r.p.m without using the medium

2.1.3.3.2 as middle step and without washing the cells by 0.85% NaCl

solution. The amounts of PGA in the three media were measured and the

results as in Table (10).

Table 10:The use of nutrient broth and LB broth as starter media to produce PGA bySC10 at 30 Co and 120 r.p.m.

Starter media 2.1.3.4.5.1st

step PGA g/l2.1.3.4.5 2nd step

PGA g/l2.1.3.4.8PGA g/l

2.1.3.3.2 6.144 3.585 17.00

2.1.3.3.3 5.981 7.003 7.829

3.8. Purification and Identification of PGA

The purification of PGA involves three steps:

1. The removal of cells by centrifugation.

2. Precipitation of the product from cell-free medium by

methanol.

3. The dialysis of low-molecular weight impurities below a

nominal molecular weight limit.

The purified PGA is usually identified first by the absence of free

amino acids and polysaccharide, using ninhydrin test and phenol-

sulphuric acid method.

3.8.1. Thin Layer Chromatography (TLC)

Figure (16) shows the specific movement of glutamic acid from PGA

used L-glutamic acid, L-alanine and DL-alanine compared with authentic

L-glutamic acid was not identical (Table 11) and this indicates that the

structure units of PGA are not L-type of glutamic acid.

first time and second time by SC10 has been measured as 154583 Dalton

and 142692 Dalton respectively.

Table 12:The molecular weight of PGA produced by SC10¬ and SW5 at differentsources of carbon

IsolateM.W of PGA from

L-glutamic acid L-alanine DL-alanineSC10 132500 103055 103055

SW5 103055 103055 97631

3.8.3. Gas Chromatography Analysis

PGA produced from L-alanine and DL-alanine by SC10 and SW5 has

been separated by GC. Figures (17a, 18a) for PGA produced from L-

alanine by SC10 and SW5 show the retention time 4.26 min. and 4.43 min.

respectively. Figures (17b, 18b) show the retention time 4.6 min and 4.52

min respectively. While the retention time of authentic L-glutamic acid

was 6.81 min (Fig. 17c, 18c).

3.8.4. Infra Red Spectra Analysis

All amino acids have two infra red spectral bands at 1590-1660 cm-1

(amide I stretch) and 1480-1550 cm-1 (amide II stretch) in addition to a

band from NH3+ at 3030-3130 (Fig. 19, 20, 21). The PGA produced from

L-glutamic acid, L-alanine and DL-alanine either by SC10 or SW5 as

shown in figures (19, 20, 21) has only one band at 1517.1-1538 ( amide II

stretch) and a band at 3087.9-3110 for NH3+ (Table 13).

Figure 17:Gas chromatography separation of PGA produced by SC10 from A- L-alanine B- DL-alanine C- Authentic L- glutamic acid.

Fig. 18: Figure 18:Gas Chromatographic separation of PGA produced by SW5from A-L- alanine B-DL-alanine as compared with C-Authentic L-glutamic

Figure 19:IR analysis of PGA produced by SC10 and SW5 from L-glutamic acid as compared with authentic L-glutamic acid.

Figure 20:IR analysis of PGA produced by SC10 and SW5 from L-alanine ascompared with authentic L- alanine

Figure 21:IR analysis of PGA produced by SC10 and SW5 from DL-alanineascompared with authentic DL-alanine

Table 13:The site of functional groups of PGA from L-glutamic acid. L-alanine andDL-alanine analyzed by IR for SC10 and SW5.

IsolatePGA From (Cm-1)

Functional groupsL-glutamic acid L-alanine DL-alanine

SC101531 1525.8 1523.3 Amide II stretch

3104 3110 3103.4 NH3+ band

SW51518.2 1538 1517.1 Amide II stretch

3098.9 3108 3087.9 NH3+ band

3.8.5. H- NMR Analysis

The H-NMR spectra have been recorded by using two different

solvents D2O and DMSO. From figures (22 a, b) for PGA from DL-

alanine for isolate SC10 and figures (23a, b) for PGA from DL-alanine for

isolate SW5, only one peak was shown at the site of aliphatic proton while

the amide and carboxylic groups were not shown.

Figure 22:22a: H-NMR analysis of PGA produced by SC10 from DL-alanine usingD2O.

Figure 23:23a: H-NMR analysis of PGA produced by SW5 from DL-alanine usingD2O.

Figure:23b: H-NMR analysis of PGA produced by SW5 from DL-alanine usingDMSO.

Table (14) shows that the solubility of PGA in the solvents were

variable from 100% soluble in water at all concentrations, very soluble in

formaldehyde and D2O, more than 50% soluble in most solvents and

slightly soluble in others.

Table 14:Solubility of PGA in 25 differeng/10ml solvents.

SolventsDielectricConstant

Weight of Soluble PGA

Weight of InsolublePGA

n-Hexane 1.890 0.0649 0.0351

c-hexane 2.015 0.0530 0.0470

1,4- Dioxane 2.209 0.0530 0.0470

Carbon tetra Chloride 2.228 0.0468 0.0532

Benzene 2.274 0.0513 0.0487

Toluene 2.379 0.0602 0.0398

1,1,1-Trichloro ethane 3.400 0.0523 0.0477Diethyl ether 4.335 0.0409 0.0591Ethyl acetate 6.020 0.0543 0.0457

Acetic acid 6.150 0.0435 0.0565

Dichloro methane 8.930 0.0561 0.0439

Pyridine 12.3000 0.0495 0.0505

n-Butanol 17.100 0.0495 0.0505

Acetone 20.700 0.0559 0.0441

Ethanol 24.300 0.0644 0.0356

Methanol 32.630 0.0404 0.0596

N,N-Dimethyl foramide 36.710 0.0455 0.0545

Acetonitrile 37.500 0.0473 0.0527

Glycerin 42.500 0.0523 0.0477

Dimethyl Sulfoxide 46.680 0.0687 0.0313

Deuterium oxide 78.250 0.0781 0.0219

Water (H2O) 78.540 0.1000 0.0000

Formaldehyde - 0.0906 0.0094

Petroleum ether 40-60Co - 0.0509 0.0491

Benzene bromo-d5 - 0.0651 0.0349

3.9. Hydrogel Prepared by Irradiation

Two concentrations of PGA were exposed to four types of

irradiations at different concentrations in order to enhance hydrogel

formation. From table (15) three concentrations for each source of

irradiation were used. Only gamma source at concentrations 2.7 Mrad and

3.37 Mrad and neutron at concentration 3.4 X 1015 n/Cm2.Sec give

positive PGA hydrogel for PGA produced by isolate SC10 while the PGA

produced from SW5 gives negative results.

Table 15:Types of irradiations at different concentrations used to produce hydrogelfrom PGA.

Sixty eight isolates have been obtained from sugarcane fields,

marshes water and seawater. They were identified as Bacillus subtilis

according to their phenotypic characteristics (Table 2), which agree with

Sneath et al., (1986); Ivanova et al., (1999); Ashiuchi et al., (2001); and

Keawachi and Prasertsan (2002).

In this study, we succeeded in isolating and identifying local isolates

of B. subtilis which have the ability to produce PGA either in the presence

or absence of L-glutamic acid. Also these isolates have properties differ

from other strains which are used for PGA synthesis.

At the beginning, we found only twenty one out of sixty eight isolates

of B. subtilis which could produce PGA in various amounts ranging

between 4.536-9.968 g/l for isolates from sugarcane fields (Table 3) and

6.074-8.226 g/1 for isolates from seawater (Table 4) in medium

containing L-glutamic acid and sucrose as sources of carbon and nitrogen.

The isolates SC10 and SW5 have been cultured in three different

media to confirm and measure their yields of PGA (Tables 5 and 6). In

medium 2.1.3.3.4, the productions of PGA by SC10 and SW5 were 6.443

g/l and 6.193 g/l respectively. While Cheng et al., (1989) found that the

maximum production of PGA was 8-12 g/l by B. licheniformis A35. In

medium type 2.1.3.3.5a, the maximum yields of PGA by the isolates SC10

and SW5 were 6.001 g/l and 7.269 g/l respectively. In contrast Yoon et al.,

(2000) produced 35 g/l of PGA in fed culture by B. licheniformis ATCC

9945 A. In medium type 2.1.3.3.5b, the amounts of PGA produced by

SC10 and SW5 were 9.968 g/l and 8.226 g/l respectively. But Ashiuchi et

al., (2001) found that the yields of PGA reached 13.5 g/l by B. subtilis

(chungkookjang).

Thorne et al., (1954) suggested that the different environmental

conditions for production of PGA may be responsible for variations to the

relative amounts of PGA. This suggestion is confirmed by our results

which showed that the same isolates of SC10 and SW5 produced different

amounts of PGA under different conditions.

4.1. Optimum Conditions for PGA Production

Sodium chloride was significant effectors of PGA production, as

shown in Figure 5. In general, the culture medium becomes highly

viscous upon PGA production results in limitation of the volumetric

oxygen mass transfer leading to insufficient cell growth and decrease in

PGA yields. Ogawa et al., (1997) found that the addition of 3% NaCl can

decrease the viscosity and stable cultivation can be obtained. Also at high

salt concentration, the bacteria cannot sufficiently grow and this agrees

with Ashiuchi et al., (2001) who found that B. subtilis (natto) cannot

grow under high saline conditions.

4.1.1. Carbon Sources

In this study, the work has been carried out on the nutritional

requirements of cell growth and improving conditions for PGA

productivity by SC10 and SW5. From Figure 6 the highest yields of PGA

produced by isolates SC10 and SW5 were 11.633 g/l and 10.932 g/l

respectively at a concentration of 2% L-glutamic acid, while the lowest

yields were 8.945 g/l and 8.353 g/l respectively at concentration of 4% L-

glutamic acid. These results are in accordance with Troy (1973a) who

found that the maximum production of PGA by B. licheniformis ATCC

9945 reached 17.23 g/l in a medium containing 2% of L-glutamic acid,

while Kunioka and Goto (1994) had only 10-20 g/l of PGA by B. subtilis

IFO 3335 in a medium containing 3% of L-glutamic acid. The repression

in PGA production of high concentration of L-glutamic acid is related to

several reasons as mentioned by Gardner and Troy (1979). At high

concentrations of L-glutamate there was decrease in glutamyl

hydroxymate and this may be related to the presence of relatively high

concentrations of L-glutamate present endogenously in the membranous

enzyme as these cells were grown. Birrer et al., (1994) found that the drop

in the pH of the medium has an effect in the PGA production and they

noticed that cell grows mainly during the first 24 h. The largest PGA

volumetric productivity is between 2 and 4 days and reduces pH from 7.4

to 5 by 42 h cultivation.

The researchers differ in their explanation about the role or the

benefit of the L-glutamic acid in the synthesis of PGA. Thorne et al.,

(1954) indicated that B. licheniformis ATCC 9945 does not require L-

glutamic acid for high levels of PGA production. Therefore, they

-iminoglutaric acid or a derivative as a possible intermediate

in PGA synthesis. While Troy (1973a) used the same strain and he found

that this bacterium catalyzes the polymerization of L-glutamic acid to

form a high molecular weight polymer of D-glutamic acid. Kunioka and

Goto (1994) used B. subtilis IFO 3335 in a medium containing L-

glutamic acid, citric acid, and ammonium sulfate and found that the

addition of L-glutamic acid is merely an activator for enzymes in the

pathway of PGA synthesis. Ashiuchi et al., (2001) reached high yields of

PGA by B. subtilis (chungkookjang) in a medium containing L-glutamic

acid. Gardner and Troy (1979) failed to detect any soluble L-glutamic

acid racemase in B. licheniformis and therefore postulated that D-

glutamic acid was found as a result of a transamination reaction involving

D- - Ketoglutaric acid.

Production of PGA was most extensively studied in L-glutamic acid

dependent bacteria. In contrast, little is known about L-glutamic acid

independent bacteria as the study of Murao (1969) on B. subtilis 5E

which can produce PGA from L-proline as the sole carbon and nitrogen

source in mineral medium. Ito et al., (1996) found that B. subtilis TAM4

has the ability to produce PGA from a medium containing ammonium

chloride and fructose as nitrogen and carbon sources. While Cheng et al.,

(1989) observed that B. licheniformis A35 produces PGA in a medium

containing ammonium chloride, glucose and nitric acid and incubated for

5 days under nitrate-respiration conditions.

We succeeded for the first time, in increasing PGA production by

using L-alanine or DL-alanine as sources of carbon instead of L-glutamic

acid. In Figure 7, the highest amounts of PGA produced by SC10 and SW5

in a medium containing L-alanine at concentration of 2% were 12.722 g/l

and 10.893 g/l respectively. While the lowest yields at 5% L-alanine were

7.513 g/l and 7.753 g/l respectively. From Figure 8, the maximum yields

of PGA at concentration of 2% DL-alanine were 11.355 g/l and 9.12 g/l

by isolates SC10 and SW5 respectively and the minimum yields at

concentration of 5% were 6.16 g/l and 7.831 g/l respectively.

From the results above, the isolates SC10 and SW5 behaved as L-

glutamic acid-dependent bacteria with the presence of L-glutamate which

means it may have the ability to convert accumulated L-glutamate in the

cells by glutamate racemase to D-glutamate the best substrate for PGA

synthesis. With the absence of L-glutamate, they act as L-glutamic acid-

independent bacteria which exploit any other source of carbon such as L-

alanine or DL-alanine. In this case, they use another pathway to supply D-

glutamate, in which L-alanine is racemized into D-alanine by alanine

racemase. Then D- - ketoglutarate are converted into

pyruvate and D-glutamate by D-amino acid transferase (Kunioka, 1997;

Ashiuchi et al., 2001). So, L-alanine in this pathway promotes PGA

production. No other researchers have used these two substrates to

produce PGA.

The isolates SC10 and SW5 also have the ability to exploit citric acid

in the medium. From Figure 9, the maximum productions of PGA were

11.86 g/l and 11.056 g/l respectively at concentration of 1.2% and lower

yields were 8.81 g/l and 8.47 g/l respectively at concentration of 2%.

These results agree with Thorne et al., (1954) who found that the

maximum production of PGA in medium containing 12 g/l citric acid

reached 15 mg/ml, but when citric acid was omitted, the yields of PGA

were low. Goto and Kunioka (1992) found that the citrate is precursor

substrate for polymer production. Yoon et al., (2000) demonstrated that

when citric acid concentration was reduced to 1g/l, PGA was hardly

produced.

Kambourova et al., (2001) showed that the strains of B. licheniformis

9945 and B. subtilis are required citrate to enhance the production -

ketoglutarate which is the direct precursor for glutamate and PGA

production.

Glucose can be used as a carbon source for cell growth and PGA

production by the isolates SC10 and SW5. High yields of PGA reached

12.68 g/l and 11.885 g/l respectively at concentration of 2% and low

yields were 8.95 g/l and 8.77 g/l respectively at 5% of glucose (Fig. 10).

Many researchers reported that the concentration of 2% glucose

could, in principle, be used as a primary source of carbon for both cell

growth and increasing

PGA production (Potter et al., 2001; Kambourova et al., 2001;

Hoppensack et al., 2003). Other researchers such as Ito et al., (1996)

showed that B. subtilis TAM4 produced less than 1% of polysaccharide

by-product with PGA. While Kunioka (1997) found that the strain B.

subtilis IFO 3335 produced PGA together with polysaccharide by-product

when it is grown in a medium containing 3% of glucose. In contrast the

PGA produced by SC10 and SW5 was tested for polysaccharide by-product

by phenol-sulfuric method but no polysaccharide was detected.

The production of PGA from isolates SC10 and SW5 was increased

with the addition of 2% glucose to a medium containing citric acid and

glutamate. This agreed with Ko and Gross (1998) who found that the

glucose was a better carbon source than glycerol and its utilization was

faster than glycerol, citrate and glutamate. Also they found that the

conversion of glucose to PGA by the strain B. licheniformis 9945 was

believed to occur by glycolysis of glucose to acetyl-CoA and TCA to

for - ketoglutarate which is a direct precursor for glutamate and PGA.

From these studies, we can conclude that the addition of citric acid

and glucose as sources of carbon are important for PGA synthesis.

4.1.2. Nitrogen Source

Among the three carbon sources used in medium 2.1.3.4.1,

ammonium chloride has been the sole source of nitrogen. As illustrated in

Fig. 11 the maximum yields of PGA were 12.68 g/l and 11.885 g/l by

isolates SC10 and SW5 respectively at concentration of 0.7% NH4Cl while

the minimum yields were 11.813 g/l at concentration of 0.5% NH4Cl for

SW5 and 12.018 g/l at concentration of 1.3% NH4Cl for SC10. The benefit

of NH4Cl for bacteria is increasing the yields of PGA and cells growth.

Many workers (Thorne et al., 1954; Troy, 1973a; Cheng et al., 1989; Ito

et al., 1996; Potter et al., 2001; Kambourova et al., 2001; Hoppensack et

al., 2003) believed that the ability of Bacillus sp. to grow rapidly in

mineral salts medium with ammonium

as sole nitrogen source is an indication that ammonium is a suitable

nitrogen source of this bacteria as it is rapidly metabolized and allowed

high growth rates. Also they found that the ammonium is directly

incorporated into L-glutamate by glutamate dehydrogenase.

4.2. Manganese Sulphate

From Figure 12, the highest yields of PGA were 13.256 g/l and

12.895 g/l by isolates SC10 and SW5 respectively at concentration of

0.0114%. While the lowest yields were 12.056 g/l and 11.651 g/l

respectively at concentration of 0.001%. The results reported here showed

that by varying the concentration of Mn+2 in the medium it was possible to

increase the amount of PGA produced. These findings are in agreement

with the results of Leonard et al., (1958) who found that low

concentration of Mn+2 was needed for maximum cell growth, and

increasing the concentration of Mn+2 to 6.15 X 10-4 M resulted in

maximum yield of PGA. Also, they found that the D-glutamic acid in

PGA increased gradually with the increasing of Mn+2 concentrations.

Cromwick and Gross (1995) found that the number of viable cells

increased for all concentrations of MnSO4, while the PGA yield increased

for corresponding increases in MnSO4. Also they found the streochemical

(enantiomeric) content of PGA varies inversely with Mn+2 concentrations

from L- to D-glutamic acid. Similar results have been obtained by Perez-

Camero et al., (1999) that the enantiomeric composition of PGA was

increased with the increasing of Mn+2 concentrations.

4.3. Optimum pH

The initial pH value of media has been controlled at 5.5, 6.0, 6.5, 7.0,

7.2, and 7.5. Among these values, pH 7.0 was found to be preferred for

PGA production (Fig. 13). The PGA yield was highest by isolates SC10

and SW5 (13.256 g/l and 12.895 g/l respectively, 72 h time growth) at pH

7.0, but decreased at pH values of 5.5 and 7.5. These results are in

agreement with Ashiuchi et al., (2001) who found that the PGA yields

reached maximum at pH 7.0. While other researchers found that there are

different values of pH for PGA production in different bacteria, indicating

that PGA production is diverse in microorganisms (Yoon et al., 2000;

Urushibata et al., 2002; Lu et al., 2004).

4.4. Optimum Temperature

The optimum incubation temperature for PGA production of isolates

SC10 and SW5 was 30 C (13.124 g/l and 12.699 g/l respectively pH 7.0,

72 h). As shown in Figure 14, the yields of PGA were reduced at low and

high temperature. This might be related to that of the optimum

temperature growth for these bacteria at 30 C . These results are in

correspondence with the results of Ashiuchi et al., (2001).

4.5. Time of Fermentation

PGA accumulation was determined at the stationary phase (72 h)

when the yields were maximal by isolates SC10 and SW5 (13.256 g/l and

12.895 g/l respectively, pH 7.0, 30 C ) (Fig. 15). This is a considerably

less incubation period than that used by other researchers which varied

between 96 and 120 h (Troy, 1973a; Cheng et al., 1989; Ashiuchi et al.,

2001).

4.6. Modified Media

In this study, after investigating the factors affecting the production

of PGA and the favorable conditions for high yields, the mineral and basal

media have been modified, as illustrated in Table (7). The isolates SC10

and SW5 were grown at six modified media which differ in their carbon

sources and with or without nitrogen source. The purpose of such

culturing as noticed by Ashiuchi et al., (1998), is to accumulate L-

glutamate in the cells which is further converted into D-glutamate, the

best substrate of PGA.

After that, bacteria were transferred into either basal medium or

modified basal medium in which the polymer was released. The best

modified medium for SC10 and SW5 was 2.1.3.4.5 type medium

containing DL-alanine, citric acid and glucose as carbon sources and

ammonium chloride as nitrogen source. The effects of these carbon and

nitrogen sources on PGA production were synergistic. The highest yields

of PGA after the cultivation of SC10 in basal medium (2.1.3.3.6 type

medium) were 14.361 g/l and 16.815 g/l in modified basal medium

(2.1.3.4.8 type medium). For SW5, the highest yields of PGA were 13.416

g/l in 2.1.3.3.6 type medium and 14.951 g/l in 2.1.3.4.8 type medium. The

lowest yields of PGA were harvested in a medium type 2.1.3.4.3 which

contained L-glutamic acid and sucrose either by SC10 or SW5. In contrast,

the amount of PGA in medium 2.1.3.4.2 was more than in 2.1.3.4.3 in

spite of having L-glutamate but also glucose instead of sucrose. From

these results, we can conclude that the isolates SC10 and SW5 can exploit

glucose for PGA production better than what sucrose and L-glutamate

may be used as activator for enzymes in PGA pathway, as mentioned by

Kunioka and Goto (1994). On the other hand, the PGA productivity in

medium type 2.1.3.4.8 is more than that in medium type 2.1.3.3.6 for SC10

and SW5 and vice versa for biomass production (Table 8).

The addition of biotin in media 2.1.3.4.6 and 2.1.3.4.7 reduced the

amount of PGA in both media 2.1.3.3.6 and 2.1.3.4.8 by SC10 and SW5.

So, the biotin is not necessary for PGA synthesis but the bacteria may

require biotin for growth. This is in agreement with Inatsu et al., (2002)

who found that about 20% of B. subtilis (natto) strains require 10 µg/ml

of biotin for growth.

4.7. Comparison between Liquid and Solid

Fermentation

In all the previous experiments, the mineral media (including

modified media) were used in a solid state. In this experiment, SC10 and

SW5 were grown in two modified media 2.1.3.4.2 and 2.1.3.4.5 in both

solid and liquid states and then cultured in medium 2.1.3.4.8. From Table

(9), the amounts of PGA and biomass for SC10 and SW5 in liquid state

were more than a solid state. This might have resulted from that, in the

solid state, the significant increase in viscosity of the accumulated

biomass results in the limitation of the volumetric oxygen mass transfer

and a shortage of nutrients within growth zone leading to insufficient cell

growth and decrease in PGA yields. While in the liquid state, the bacteria

were growing with a shaking at 120 r.p.m which permits better supply of

oxygen and accessibility to nutrients leading to increase cell growth and

PGA yields.

4.8. Comparison between Medium Type 2.1.3.3.2 and

2.1.3.3.3

Most of the researchers (Yoon et al., 2000; Ashiuchi et al., 2001;

Ornek et al., 2002; Minami et al., 2003, Lu et al., 2004) used LB agar as

starter medium for the growing of Bacillus sp. Others used either nutrient

broth or nutrient agar (Shih et al., 2001; Aslim et al., 2002; Inatsu et al.,

2002) to induce sporulation or germination of spores. In this experiment,

we used LB broth (2.1.3.3.2) and nutrient broth (2.1.3.3.3) as starter

media. Evidently, the biomass production in LB broth was more than in

nutrient broth. This may be related to the presence of Mg+2 ions in LB

broth which has certain effects on PGA synthesis (Troy, 1973a; Gardner

and Troy, 1979; Yang et al., 2001; Ashiuchi et al., 2004). To observe the

effect of starter media on PGA production and also to evaluate the

procedure of PGA production, the middle steps of the original procedure

have been omitted. We found that if the bacteria were incubated in

medium 2.1.3.4.5 only, the yields of PGA after 72 h were 6.144 g/l for

medium type 2.1.3.3.2 and 5.981 g/l for medium 2.1.3.3.3. If the bacteria

were transferred from medium 2.1.3.4.5 and re-incubated either at the

same medium for further 72 h, the yields of PGA were 3.585 g/l for

medium 2.1.3.3.2 and 7.003 g/l for medium 2.1.3.3.3 medium type, or at

medium 2.1.3.4.8 directly without any middle steps, the yields of PGA

increased in the case of medium type 2.1.3.3.2 to 17.00 g/l but no change

to medium 2.1.3.3.3.

This may have an indication that the incubation of bacteria in

medium 2.1.3.4.5 results to the accumulation of PGA in the cells. So the

re-incubation in the same medium did not lead to the release of the PGA

and this is the reason to low PGA. When the bacteria incubated in

medium 2.1.3.4.8, the PGA released and the yields were increased.

4.9. Purification and Identification of PGA

The steps of purification used in this study almost approach other

researchers

(Troy, 1973a; Gardner and Troy, 1979; Yoon et al., 2000;

Kambourova et al., 2001; Ashiuchi et al., 2001).

4.9.1. Thin Layer Chromatography

From Figure 16 and Table (11), the specific movement (Rf) of

glutamic acid was extracted from pure PGA by SC10 which used L-

glutamic acid, L-alanine and DL-alanine as sources of carbon differ from

the Rf of authentic L-glutamic acid. So, the building units of the PGA

consist of only D-glutamic acid.

4.9.2. Estimation of Molecular Weight

The molecular weight of microbial PGA is an important feature, that

polymers with different molecular weights are needed for different

purposes.

The molecular weights of PGA produced by SC10 from L-glutamic

acid were higher than that from L- and DL-alanine and also more than

that produced by SW5 (Table 12). On the other hand, the molecular

weight of PGA produced by SC10 at experiment (4.8) using medium

2.1.3.3.2 and medium 2.1.3.4.5 at first step of cultivation was higher than

at medium 2.1.3.4.8. The variations in molecular weight may have

resulted from the alteration of medium composition. These results are in

agreement with the findings of several researchers (Troy, 1973a; Cheng et

al., 1989; Kunioka and Goto, 1994; Ito et al., 1996; Cromwick and Gross,

1996) who found that several methods such as alkaline hydrolysis,

ultrasonic degradation, microbial or enzymatic degradation and alteration

of medium composition have been used to obtain microbial PGA with

various molecular weights. Also, the researchers (Troy, 1973a; Birrer et

al., 1994) observed that a decrease in the viscosity in media, suggesting

the existence of an enzyme called depolymerase responsible for the

breakdown of - PGA located either within the cells or bound to the

membrane.

King et al., (2000) identified a polyglutamyl -hydrolase enzyme

which catalyzes the hydrolytic breakdown of PGA from B. licheniformis.

Shih and Van (2001) reported that the variation of molecular weights is

important for different purposes. Such a high molecular weight polymer is

useful as a viscosity- adding agent. While the investigation of drug

delivery system or polymeric drugs, polymers of different molecular sizes

will be required to control their delivery in tissues.

4.9.3. Gas Chromatography Separation

The retention time of PGA produced either from L-alanine or DL-

alanine by SC10 or SW5 (Fig. 17, 18) in comparison with authentic L-

glutamic acid was different. So, this result gave another confirmation that

the structural units of PGA were D-glutamic acid. This is in agreement

with Troy (1973b) who found that more than 90% of the glutamyl

residues from the enzymatically synthesized PGA was of the D-

configuration as obtained by gas-liquid chromatographic separation.

4.9.4. Infra Red Spectra Analysis

The bacterial PGA has thre - -sheet

and random coil, when distinguished by IR spectroscopy (Cantor and

Schimmel, 1980). Also they found that the two infra red spectral bands at

1655 Cm-1 (amide I stretch) and 1550 Cm-1 (amide II stretch) are typical

fo -helical structure, while the amide I band shifts to 1630 Cm-1 for

-sheet structure and the amide II band shifts to 1535 Cm-1 for random

coils. The IR analysis of PGA produced from L-glutamic acid, L-alanine

and DL-alanine either by SC10 or SW5 (Fig. 19, 20, 21 and Table 13)

shows the shifting in amide II bands between 1523.3-1531 for SC10 and

1517.1-1538 for SW5. From these results, we can conclude that the

conformation of their PGA has random coils. He et al., (2000) observed

that bacterially produced exopolymers can exist in various configurations

depending on solution conditions, solution temperature, pH, polymer

concentration and ionic strength. At low pH, PGA exhibited a helical

conformation. At neutral to high pH is suggesting a structural shift to -

form. At low concentration and pH greater than 7, PGA assumed an

elongated shift conformation. At low ionic strength, the PGA of B.

licheniformis adopts a helical conformation. As ionic strength increases,

the structure -helix, and -sheet.

4.9.5. H-NMR Analysis

From Figure (22a, b, 23a, b) for PGA produced from DL-alanine

either by SC10 or SW5 only one peak has been shown at aliphatic proton

site and it is very clear at DMSO solvent rather than in D2O solvent, while

the amide and carboxylic groups were not shown. This is in agreement

with Borbely et al., (1994) who found that the aliphatic group ranged

between 1.9-4.27 and the carboxylic and amide groups were not shown in

D2O solvent. While the aliphatic group in DMSO ranged between 1.73-

4.12 amide group may appear at 8.1, and the carboxylic group was not

shown.

4.10. Solubility in Solvents

Most researchers (Yoon et al., 2000; Shih and Van, 2001; Potter et

al., 2001; Kambourova et al., 2001) reported that PGA is water soluble,

but there was no report about the ability of PGA to dissolve in other types

of solvent. In this study, we used 25 solvents including water (Table 14)

to dissolve PGA, twenty-three of them are organic solvents and two are

inorganic solvents. The organic solvents were different in their dielectric

constants and in number of carbons. The percentage of PGA solubility in

these solvents ranged between 40.4%- 90.6%. On the other hand, the

percentage of its solubility in the inorganic solvents, including D2O, is

about 78.2% and in water 100%.

4.11. Hydrogel Prepared by Irradiation

PGA hydrogel has been prepared by irradiation of PGA from SC10 or

SW5 in sterilized distilled water. Four types of irradiation in three

concentrations for each have been used (Table 15). Only gamma source at

two concentrations and neutron at one concentration produced PGA

hydrogel for PGA from SC10 at concentrations of 2% and 5%.

The effect of gamma irradiation has been studied by Han and Choi

(1999) and Kunioka (2003) intensively. They reported that gamma

irradiation on aqueous solution in general produce hydroxyl radicals

which mediate radical reaction. Han and Choi (1999) used 13C NMR to

study the characterization of cross linking and bond scission in PGA

hydrogels produced by gamma irradiation. They found that most possible

cross links and hydrolysis by radical reactions would be chemical bond

formation of CH2 -; -CH2 -; -CH N-; -CH -; in

addition to carbonyl carbon involved in the cross links and hydrolysis.

The effects of other irradiation were not observed in any articles, so the

- -

irradiations known as weak sources, have no ability for cross-links in

aqueous solution. The effects of pH and salt concentration in PGA

hydrogel were studied by Shih and Van (2001) and Kunioka (2003) and

they found that under acidic condition or in addition of electrolytes, PGA

hydrogel was decreased.

5.1. Conclusions

1. Isolation of local strains of Bacillus subtilis which have

the ability to produce gamma-polyglutamic acid either

with the presence or absence of L-glutamic acid using

batch culture from sugarcane fields and seawater,

represent new finding.

2. Producing high yields of gamma-polyglutamic acid in

modified medium containing DL-alanine, D-glucose,

and citric acid as carbon sources and ammonium

chloride as nitrogen source. The productivity reached

about 18.34 g/l in liquid state and 16.81 g/l in solid state

by an isolate from sugarcane fields. While the

productivity reached about 16.77 g/l in liquid state and

14.95 g/l in solid state by isolate from seawater. All this

point out to a successful approach of optimization for

better productivity.

3. Producing high amounts of -PGA 17 g/l by isolate from

sugarcane fields using a liquid medium and omitting the

middle steps of the original procedure, is another

approach for optimization.

4. Preparing PGA hydrogel by gamma ray and neutron

from PGA produced by isolate from sugarcane fields

indicates successful attempts despite the very low source

of radiation available.

5.2. Recommendations

1. Increasing the production of -PGA by Bacillus subtilis by

using fed-culture method.

2. Improving the genetic feature of Bacillus subtilis for

increasing the productivity of -PGA.

3. Increasing the production of PGA hydrogel as suitable water

sorption materials for agriculture.

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