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Iraqi Journal of Biotechnology, 2016, Vol. 15, No. 1 , 83-96

 

 

 

Levan Production using Pseudomonas Brassicacearum Isolated from Rhizosphere Soil of Cowpea Farm in Iraq

 

Safaa AL-Deen Ahmed Shanter AL-Qaysi1, Zaid Akram Thabit2, Sanaa Basheer Kadhem1

 

1Department of Biology, College of Science for Women, University of Baghdad

2Biotechnology Research Center, University of AL-Nahrian

 

Received: November 1, 2015 / Accepted: January 28, 2016

 

 

Abstract: The present investigation, was designed in order to isolate bacteria from rhizosphere soil of Cowpea farm in Iraq which is capable of levan production. The selected levan producing bacterium was identified as Pseudomonas brassicacearum based on the phenotypic identification by Bergey’s Manual of Systematic Bacteriology and confirmed with the 16S rRNA gene sequencing. Acid hydrolyzed levan sample was subjected to TLC technique which revealed that, levan is composed of only one sugar which is fructose. Rf values of levan produced by P. brassicacearum was 0.42 which is identical or close to Rf values of fructose of 0.42. The effect of some factors on levan production by P. brassicacearum was investigated. The results showed that the best carbon source for levan production was sucrose 7.77 g/l, and casein was the best nitrogen source for levan production which gave 8.56 g/l, followed by ammonium sulfate and corn steep liquor which they gave 8.52, 8.09 g/l respectively. The highest levan yield was at the sucrose concentration of 300 g/l which gave 7.95 g/l. At initial pH of 7.8, P. brassicacearum gave their highest levan production that was 7.77 g/l. Levan production by P. brassicacearum continued increasing until it reached its maximum production at 40ºC which was 8.24 g/l. The optimal incubation period for levan production, was estimated at 8.70 g/l after 72 h of incubation.

 

 

Key words: rhizosphere soil, P. brassicacearum, levan production, carbon sources, 16S rRNA gene sequencing.

 

Corresponding author: should be addressed (Email: Safaa.ahmed.bio@gmail.com)

 

 


Introduction

 

Levan is a homo-polysaccharides (Poly- fructan, biopolymer) with β- 2,6- linked branches that consist of β- 2,1- linked side branched, The enzyme responsible for levan synthesis generally is called as levansucrase, from sucrose- based substrate that catalases biosynthesis of levan by converting fructose on the sucrose donor to an acceptor molecule


(1). In nature levan is derived from several sources such as  Bacteria, Yeasts, molds and plants (2, 3, 4, 5). The unique properties of levan like odorless, nontoxic, biologically active, tasteless, non-mutagenic, made it suitable to use in more than one site of potential applications in food, pharmaceutical, Immunostimulant, cosmetics, antimicrobial and antitumor. There are many researchers work in the


 

 


field of medicine and pharmaceutical applications, such as antipathogenic bacteria and antiviral (7, 8). Levan also used as anticancer and antitumor agent (9). Furthermore, levan is used as Immune modulator (10), anti- inflammatory (11) and as immuno- stimulant by the activation of non- specific defense mechanisms to protect fish against pathogens (12). The earlier levan production from bacteria has been carried out by using of different bacterial species such as P. fluorescencs (13). P. syringae (14), Leuconostoc kimchi (15). (16) Reported that there are several bacterial species producing extracellular levan, like Zymomonas mobilis, Bacillus subtilis, Bacillus polymyxa and Acetobacter xylinum. However, Bacterial production of levan and other exopolysaccharides in rhizosphere soil play an important role in the regulation of carbon source diffusion in addition to its role in the increasing of water retention in the microbial and plants root environment. One of the rhizosphere bacterial species that capable to produce levan in the soil is Pseudomonas spp. which their production to levan is increased during desiccation (17), the increase in production leads to increase the aggregation of soil, and thus improve growth conditions of rhizosphere microbes and plants (18). Pseudomonas brassicacearum is a gram negative, motile bacterium. It's aerobic, oxidase and DNase positive and levan producing bacterium that have a rod shape with about 1.0-1.5 µm in length and 0.5 µm in width. These bacteria form a mucoid colonies with entire edges when grown on TSA. At the late stationary phase, these bacteria produce a diffusible, brown-orange colored pigment on TSA and TSB media. In addition to that, these bacteria produce a


fluorescent pigment when grown on CAA media (19). Bacteria in young cultures form small, mucous colonies and this represents phase I. In the present work, the aim was the isolation and the identification of new P. brassicacearum which are able to produce levan. It also aimed to optimize levan production by P. brassicacearum isolate.

 

Materials and Methods Samples Collection

A levan producing P. brassicacearum used in this study was isolated from rhizosphere soil of cowpea (Vinga unguiculata L.), sample of soil were collected carefully from different fields of cowpea in Iraq by uprooting the root system, and placed them in clean sterile nylon bags for transport to a laboratory. The samples were stored at 4 °C for bacterial isolation.

 

Isolation of Bacteria

 

Ten grams of soil had been mixed in 90 ml of sterilized peptone water and then the soil suspension serially diluted. The 10-1, 10-2 and 10-3 dilutions were streaked on nutrient agar plates (HIMEDIA-INDIA), the pH adjusted to 7.5, and was sterilized by autoclaving at 121 °C for 20 min, after that, all the plates  were  incubated  anaerobically at

37 °C for 24-48 h. Morphologically different growing colonies were picked up and subcultured by streaking method on sucrose nutrient agar media (which composed of 200g/l sucrose and 28g/l nutrient agar, this media was sterilized by Autoclaving at 121 °C for 20 min.) and then incubated anaerobically for 24- 48  h  at  37  °C.  Mucoid  colonies were


 

 


chosen for identification and characterization (20).

 

Phenotypic           and           Genotypic Identification

 

Biochemical and physiological characterization of selected bacteria were performed according to Berge’s Manual of Systematic Bacteriology (21), bacterial isolates were characterized according to both their phenotypic traits: such as shape, size, margin, surface, elevation, color, pigmentation and staining by Gram stain, and also Biochemical test. The later was performed using standard biochemical and physiological testes that included, catalase test (3% H2O2), oxidase reaction (Kovacs method) and diffusible pigment production, In addation carbohydrate fermentation was accomplished by using medium containing specific carbohydrate source (sucrose, glucose, lactose, mannitol, maltose, rhaminose) and starch hydrolysis was achieved by culture isolation on Starch nitrate agar medium, After incubation for 24 h, at 37 °C, the plate was covered with iodine, and a clear zone around the growth of colonies was detected on the medium plate. Gelatin hydrolysis was achieved by stab tubes of nutrient agar containing gelatin, after inoculation with tested bacteria, liquefaction of gelatin was observed (22). The Identification was then confirmed by 16S rRNA gene sequence. Genomic DNA from the isolates was extracted using of Gene JETTM genomic DNA purification Kit (Thermo). After DNA extraction, PCR amplification reaction was performed by using PCR master mix (Thermo) in

50 µl final volume, consist of 25 µl maximum hot start PCR master mix (2X),  using  universal  bacterial  primer


sets: 1µl (20 µM 16S RNA Forward primer            5`-

AGAGTTTGATCCTGGCTCAG-3`)  1

µl (20 µM 16S RNA Revere primer 5`- GGTTACCTTGTTACGACTT-3`),   18

µl deionized water, nuclease-free and 5 µl DNA template. The PCR reaction was incubated in a thermocycler under following conditions, the initial denaturation at 95°C for 5 min. followed by 30 cycles of denaturation at 95°C for 30 sec, annealing at 60°C for 30sec extension at 70 °C for 2 min and final extension at 72°C for 10 min. Finally, the reaction was hold at 4°C hold. The PCR products were verified by agarose gel electrophoresis and purified with the genomic DNA purification Kit (Qiagen Science, MD), the product sequences were aligned with specific public databases that are available in the Genbank resources using  NCBI  site (http://www.ncbi.nlm.nih.gov/blast / Blast. cgi). In addition, Phylogenetic tree was generated by alignment of the obtained sequences of 16s rDNA of the tested bacteria with the data base sequences in the gene bank.

 

Levan Production

 

Inoculum           Preparation           and Fermentation Conduction

 

The first step for bacterial biopolymer (levan) production was done by preparation of standard inoculum (1ml contained 7 x 106 viable cell according to McFarland tube) for fermentation, which prepared by inoculating a loopful of pure bacterial culture into Erlenmeyer conical flask (250 ml volume) that contains 50 ml nutrient broth medium (HIMEDIA-India), and incubated at 36 °C for 24 h. The productive medium that modified by


 

 


(23) was used as a fermentation  medium to produce levan, and is composed of (g/l): commercial sucrose, 200; MgSO4.7H2O, 0.2; K2HPO4, 5.5. The pH was adjusted to 7.8. The fermentation process was carried out by dispering of this medium into 250 ml Erlenmeyer conical flasks which then sterilized by autoclaving at 121 °C for 20 min. The flasks were inoculated with

2.5 ml (5%) of standard inoculum to each flask, and incubated (fermented) statically at 37°C for 24 h.

 

Levan Extraction

 

At the end of fermentation period, samples 10 ml were centrifuged at 10000 rpm for 10 min to remove bacterial cell free supernatant was used to precipitate levan by the addition 1.5 volumes of absolute ethanol, and incubated for one hour at 37°C. Then the levan were collected by centrifugation at 10000 rpm for 10 min. The precipitated pellets were hydrolyzed by HCl 5% (v/v) for one hour at 100 °C. Levan was estimated corresponding to fructose units by using Glucose oxidase kit (SPINREACT) and the absorbance was read at wavelength 505 nm. (38).

 

Characterization of Levan by Thin- Layer Chromatography

 

Equal weights (1 mg) of glucose, sucrose and fructose were dissolved in 1 ml of 1% ethanol. Then, a 10 µL of hydrolyzed levan and other sugar suspensions were spotted by capillary tubes at equal distances and about 2 cm of the lower edge of the plate. Then, TLC plate was placed in a closed jar that contains the mobile phase which is composed   of   chloroform   /acetic acid

/water  [v/v]  (6:7:1).  Levan  and   other


sugars were diffused through a silica gel plate. After drying, the plate was sprayed by a mixture of H2SO4 and ethanol at [v/v] (9:1) and placed in oven for 5-10 min, at 90 ºC. Levan components were appeared as dark colored spots (24).

 

Optimization of Levan Production Effect of Carbon Sources

This experiment was conducted to study the effect of various carbon sources on levan production and the possibility of using cheap and available carbon source alternatives in its production. Fructose and agricultural materials like Date syrup and sugarcane molasses were used. Dates syrup was obtained from Baghdad markets and sugarcane molasses was obtained from a sugar factory in Al-Hawamdiya city, Cairo, Egypt. These materials were used by replacing sucrose in the productive medium with the equivalent amounts of alternatives (38). All flasks were incubated at 37 °C for 24 h. Levan amount was estimated as described above.

 

Effect of Nitrogen Sources

 

This experiment was conducted in order to investigate the effect of various nitrogen sources and to determine the most suitable nitrogen source on levan production. Organic (Corn Steep Liquor and Casein) and inorganic (Ammonium sulfate and Ammonium phosphate) nitrogen sources were used as alternatives for the original nitrogen source in the productive medium. These materials were used by replacing yeast extract in the productive medium with equivalent amounts of alternatives. Flasks were incubated and levan


 

 


amount was estimated as described above (38).

 

Effect of Sucrose Concentrations

 

This experiment was conducted to identify the effect of various sucrose concentrations on levan production. Different sucrose concentrations (50g, 100g, 150g and 300g) were used in productive medium preparation. Flasks were incubated, and levan amount was estimated as described above (38).

 

Effect of Initial pH

 

This experiment were performed to study the effect of various pH values on levan production. Four groups of productive medium flasks were prepared, each group was adjusted to a particular pH value (7, 7.5, 8, and 8.5) by adding 1 N HCL and 1 N NaOH. Flasks were incubated and levan amount was estimated as described above (38).

 

Effect of Temperature

 

To study the effect of temperature on levan production, different incubation temperatures were used (30, 33, 40 °C) for 24 hr. At the end of fermentation period, levan amount was estimated as described above (38).

 

Effect of the Incubation Period

 

To study the effect of the various incubation times, production medium was incubated for (24, 48, 72 and 96) hr. at 37 °C. This experiment was conducted to determine the effect of incubation time on bacterial growth and levan production. At the end of


fermentation period, levan amount was estimated as described above (38).

 

Statistical Analysis

 

The effect of different factors on levan production were tested statistically by using Duncan’s test multiple range test at the level of P ˂0.05. Least significant difference –LSD test was used to significantly compared between means in this study (25).

 

Results and Discussion

 

1.          Bacterial Identification

 

The isolate was identified through molecular characterization by 16S rRNA sequencing and phenotypic characterization by using standard physiological and biochemical tests. The isolate showed small and mucus, yellow pigmented colonies which released a fluorescence pigment in the growth medium. Microscopic examinations revealed that the  isolate was Gram negative rods, single or paired. The isolate showed different biochemical results shown in the table (1).


 

 

Table (1) Biochemical and Physiological tests of the isolate

 

 

No. of Test

Test

Result

1

Colony texture

Mucoid

2

Gram stain

Negative (-ve)

3

Growth on MacConkey medium

Positive (+ve)

4

Catalase

Positive (+ve)

5

Oxidase

Positive (+ve)

6

Blood Hemolysis

Negative (-ve)

7

Oxidation test

A/K*

8

Urea hydrolysis

Negative (-ve)

9

Starch hydrolysis

Positive (+ve)

10

Gelatin hydrolysis

Positive (+ve)

11

Production of byoverdine (Green fluorescent pigment)

Positive (+ve)

Fermentation of Carbohydrates

1

Sucrose

Positive (+ve)

2

glucose

Positive (+ve)

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