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Screening of Exopolysaccharides Producing Bacteria Strains

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1.0 Introduction
Among the microbial products, exopolysaccharides (EPSs) play a significant role in main physiological functions and applications. The increased demand for current and natural polymeric materials by several industrial fields such as pharmaceutical, food and others has moved the interest to the polysaccharides produced by microorganism during the past years. Polysaccharide is a macromolecule comprising more than about ten monosaccharides residues linked glycosidically in branch or unbranched chains arbitrarily(Lackie, 2013; Singleton & Sainsbury, 2006). Several external polysaccharides obtained from microorganisms have important commercial uses such as Alginate, Curdlan, Dextran, Gellan Gum, Xanthan Gum and etc.
1.1 Objectives
This purpose of this research study is to design a novel rapid screening assay for isolation of excessive exopolysaccharides producing bacteria strains. Besides that, this research study also aimed to isolate bacteria strains that produce excessive amount of exopolysaccharides from low cost sugar substrates through laboratory scale experiments. The morphology and molecular features of exopolysaccharides also will be identified and characterized in this research.
1.2 Scope of Study
This research study gives a wide range of discovery on novel exopolysaccharides producing bacteria strains.
1.3 Problem Statement
Different bacteria strains produce different types of exopolysaccharides in different rates from varied types of sources. There are possibilities in discovering novel exopolysaccharides that can be utilized for different applications. Bacterial exopolysaccharides production is affected by the growth conditions.
2.0 Literature Review
Microbial Polysaccharides
Microbial polysaccharides have multiple functions and can be classified into 3 integral parts which are intracellular polysaccharides, cellular polysaccharides and extracellular polysaccharides/exopolysaccharides(EPSs).
2.2 Extracellular Polymeric Substances (EPS)
Microbial aggregates consisting of microbial polymers by means of biopolymer composed of different classes of macromolecules such as polysaccharides, proteins, nucleic acids, (phospho)lipids and other polymeric compounds. EPS make up a three-dimensional, gel-like, highly hydrated and often charge the biofilm matrix in which the microorganisms are embedded. The proportional of EPS in biofilms can vary between approximately 50-90% of the total organic matter.(Fleming & Wingender, 2002)
2.2.1 Composition
The composition of EPS analysed largely depend on the isolation methods but there are no method had been developed to yield complete extraction of EPS without contamination from intracellular components currently.

Component Contents in EPS
Polysaccharides 40-95%
Protein <1-60%
Nucleic Acids <1-10%
Lipids <1-40%

Table 2.2.0 Composition of Extracted EPS and Concentration Range of Component
Source: (Fleming & Wingender, 2002)
2.2.4 Biopolymer
Biopolymers have a high amount of functional groups: hydroxyl and negatively charged carboxyl groups. Thus, biopolymers could bind through specific protein-polysaccharides interactions, hydrophobic interactions, hydrogen bonding and ionic interactions. Extracellular biopolymers are produced by bacteria and typically can be attached to the cell as capsule or excreted into the surrounding medium as slime.
2.2.3 Biofilms
An adherent layer of microbial cells encircled in a mucilaginous polymer matrix which is attached to surfaces exposed to biological fluids or water (Lackie, 2013; Singleton & Sainsbury, 2006). The accumulation of a significant amount of inactive or inert solids include extracellular polymeric substances (EPS) that hold the bacterial-fungal biofilm together near the substratum because low substrate concentration and endogenous decay lead to a residue of ‘dead’ or ‘dormant’ biomass(Ritmann & Laspidou, 2002). The biofilm is found in the case of mineral materials on the surface and in the pore system especially the pores with a diameter between 0.5 and 10μm are optimally suited for microbial growth. This is due to these large pores allow the invasion of microbial cells for their internal growth resulting in a filled spores. (Sand & Jozsa, 2002)
2.3 Exopolysaccharides (EPSs)
Classification and Functionality
Biosynthesis
Health Benefits
Probiotic Effects
Immunostimulatory Activity
Anti-Tumoral Activity
EPS & Blood Chloresterol-lowering effects
Genetic Engineering: microbial polymers
2.1 Exopolysaccharides producing Microorganisms
2.1.1 Extremophiles
Thermophiles
Various thermophilic ecosystems such as both deep and shallow marine hot spring, and terrestrial hot spring have served as sources for isolation of microbial producers of EPSs. Microbial communities associated with deep-sea hydrothermal vents were found to produce EPSs. These ecosystems are characterized by extremely high pressure and temperature with high level of toxic elements such as sulphur and heavy metal, and the EPSs act as enhancers for bacterial survival.
Psychrophiles
Fresh and marine waters, polar and high alphine soils, waters and glacies are examples of cold environments dominate the biosphere. Bacterial EPSs provide protection and ecosystem stability in marine environment. The enhanced production of a high molecular weight polyanionic EPS at suboptimal incubation temperatures lends support to theories that EPS may serve as a cryo-protectant both for organisms and their enzymes. (Nicolaus, Kambourova, & Oner, 2010)
Halophilic bacteria
Acidophilic bacteria
Alkaliphilic bacteria
2.1.2 Soil Bacteria
Agrobacteria sp.
Xanthomonas sp.
Pseudomonas sp.
Pseudomonas members are among the most commonly encountered environmental microorganisms which can survive under extremely low-nutrient conditions such as distilled water and have a high tolerance to variety of extreme temperatures (high temperatures of 43áµ’C and low temperature of 4áµ’C) (Toranzos, 2002).
2.1.2 Probiotic Bacteria
LAB
Propionic bacteria
Bifidobacteria
2.1.3 Marine Bacteria
2.5 Exopolysaccharides Commercial Production
Alginate
Alginate is a salt of alginic acid commonly synthesised in strains of Pseudomonas aeruginosa and commercially important in food processing, swabs, some filters, fire-retardants amongst other(Lackie, 2013; Singleton & Sainsbury, 2006). It is composed of 1,4-β-linked D-mannuronic acid residues and 1,4-α-linked L-guluronic acid residues(Singleton & Sainsbury, 2006).
Cellulose
It is a straight chain polysaccharide comprises β-1-4-linked glucose subunit through the process of polymerization(Lackie, 2013). Extracellular cellulose in bacteria strains are responsible for the formation of the characteristic packets of the cells and involved in the adhesion of bacteria to plants(Singleton & Sainsbury, 2006).
Collanid Acid
Curdlan
Curdlan is a homopolymer of D-glucose linked in β-1-3-linked with an average degree of polymerization (DP) of approximately 450 and is unbranched(Lee, 2005). Curdlan is normally produced by strains of Agrobacterium and Rhizobium(Singleton & Sainsbury, 2006).
Dextran
It is a high molecular polysaccharides consist of D-glucose linked b α-1,6-bonds and a few α-1,4-bonds synthesised by microorganisms(Lackie, 2013). The size of molecular, the nature and extent of branching depend on the source of dextran(Singleton & Sainsbury, 2006).
Gellan Gum
Gellan gum is a linear polymer consisting residues of glucose, rhamnose and glucoronic acid connected with 1,4-β-linkages produced by ‘Pseudomonas elodea’(Singleton & Sainsbury, 2006).
Hyaluronic Acid
Hyaluronic acid is an extremely high molecular weight (3-4 million daltons) composed of repeating disaccharide units of glucoronic acid and N-acetyl-glucosamine linked by 1,4-β-linkages which can forms the core of complex proteoglycan aggregates found in extracellular marix(Lackie, 2013). Hyaluronic acid exist in the capsules of certain group streptococci to form highly viscous solution in water and readily form gels(Singleton & Sainsbury, 2006).
Xanthan/Xanthan Gum
Xanthan is comprises a linear backbone chain of 1,4-linked β-D-glucosyl residues in which alternate residues are substituted at the O-3 position with a trisaccharide side-chain(Singleton & Sainsbury, 2006).
2.5 Methods for the Identification of Microbial Isolates
Obtaining Isolates
Sampling Techniques
Single Isolation
Serial Dilution
Primary Screening
Secondary Screening
2.6 Approaches to the isolates identification
Visual Traits
Structural Components
Gram Staining
Physiological/Metabolic Tests
Biochemical Tests
Genetic Analysis
DNA extraction
Agarose Gel Electrophoresis
Polymerase Chain Reaction (PCR)
PCR Product Purification and sequencing
BLAST analysis of 16s rDNA gene sequence
3.0 Methodology
Soil sampling
The starting samples used for the isolation of bacteria strains will collect from soil of two rainfed area of UMK, Kelantan which are the Agropark tropical forest region and lakeside region. The soil will take from 6 inches (10-15cm) depth(Naseem & Bano, 2014). All soil samples collected will send to the laboratory and store at 10áµ’C until further progressing(Lamovsek, Stare, & Urek, 2014).
Bacteria isolates
The isolation of bacteria strains will start within one week time after collecting of soil samples and done by serial dilution method(Lamovsek et al., 2014). By using this method, 10g of soil samples will suspend in 9ml of distilled water and centrifuge at 3000rpm for 10-15 minutes(Naseem & Bano, 2014).
Staining Tests
Simple Stain
Stain the bacterial isolates with methylene blue for one minute. Briefly wash off the stain slide with water. Blot off the water drops slide carefully with bibulous paper.
Gram Stain
Cover the smear with crystal violet and stand for 20 seconds. Briefly wash off the stain using a wash bottle of distilled water. Drain off excess water. Cover the smear with Gram’s Iodine solution and stand for one minute. Wash off the Gram’s iodine. Hold the slide at a 45-degree angle and allow the 95% alcohol to flow down the surface of the slide. Do this until the alcohol is colorless as it flows from the smear down the surface of the slide. Stop decolorization by washing the slide with a gentle stream of water. Cover the smear with safranin for one minute. Wash gently for a few seconds and blot dry with bibulous paper to air-dry it. Examine the slide under oil immersion.
Acid Fast Stain
Capsule Stain
Mix two loopfuls of the bacterial isolates in a small drop of india ink. Spread the ink suspension of bacterial isolates over slide and air-dry. Heat and dry the slide gently to fix the microbes on the slide. Stain the smear with crystal violet for one minute. Wash off the crystal violet gently with water. Blot dry the slide with bibulous paper and examine with oil immersion objective.
Spore Stain
Physiological/Metabolic Tests
Starch hydrolysis
Litmus Milk Test
Oxidase Test
Small loop of each bacteria isolates will rub on a filter paper with drops of tetramethyl–ρ-phenylenediamine dihydrochloride solution. The isolates turn into purple color within 10 seconds will record as positive whereas the isolates turn into purple color within 10-60 seconds will record as negative to oxidase test.
Catalase Test
Methyl Red Test
Inoculate each experimental organism into its appropriately labelled tube of medium by means of a loop inoculation by using sterile technique. The last tube will serve as a control. Incubate all cultures for 24 to 48 hours at 37áµ’C.
Voges-Proskauer Test
Genetic Analysis
DNA Extraction
Agarose Gel Electrophoresis
Polymerase Chain Reaction (PCR)
PCR product Purification and Sequencing
BLAST analysis of 16s rDNA gene sequence
 
6.0 References
Fleming, H.-C., & Wingender, J. (2002). Extracellular Polymeric Substances(EPS): Structural, Ecological and Technical Aspects. In G. Bitton, D. L.Balkwill, R. S. Burlage, D. Capone, T. L. Crisman, S. E. Dowd, H.-C. Flemming, C. P. Gerba, M. W. L. Gerba, M. W. L. Chevallier, A. Leis, E. Madsen, J. A. Nienow, K. Scow, R. J. Sevious, L. D. Stetzenach, M. H. Stewart, & D. C. White (Eds.), Encyclepodia of ENVIRONMENTAL MICROBIOLOGICAL (Vol. 3, pp. 1223-1231). Canada: John Wiley & Sons, Inc.
Lackie, J. M. (Ed.) (2013) CELL AND MOLECULAR BIOLOGY (5 ed.). Oxford: Elsevier Ltd.
Lamovsek, J., Stare, B. G., & Urek, G. (2014). Isolation of non-pathogenic Agrobacterium spp. biovar 1 from agricultural soils in Slovenia. Phytopathologia Mediterranea, 53(1), 130-139.
Lee, D. I.-Y. (2005). Curdlan Biopolymers Online (pp. 135-144). Korea: Wiley-VCH Verlag GmbH & Co. KGaA.
Naseem, H., & Bano, A. (2014). Role of plant growth-promoting rhizobacteria and their exopolysaccharide in drought tolerance of maize. Journal of Plant Interactions, 9(1), 689-701. doi: Doi 10.1080/17429145.2014.902125
Nicolaus, B., Kambourova, M., & Oner, E. T. (2010). Exopolysaccharides from extremophiles: from fundamentals to biotechnology. Environmental Technology, 31(10), 1145-1158. doi: Pii 923113253 Doi 10.1080/09593330903552094
Ritmann, B. E., & Laspidou, C. S. (2002). Biofilm Dettachment In G. Bitton, D. L.Balkwill, R. S. Burlage, D. Capone, T. L. Crisman, S. E. Dowd, H.-C. Flemming, C. P. Gerba, M. W. L. Gerba, M. W. L. Chevallier, A. Leis, E. Madsen, J. A. Nienow, K. Scow, R. J. Sevious, L. D. Stetzenach, M. H. Stewart, & D. C. White (Eds.), Encyclepodia of ENVIRONMENTAL MICROBIOLOGICAL (Vol. 1, pp. 544-550). Canada: John Wiley & Sons, Inc.
Sand, W., & Jozsa, P.-G. (2002). Weathering, Microbiol. In G. Bitton, D. L.Balkwill, R. S. Burlage, D. Capone, T. L. Crisman, S. E. Dowd, H.-C. Flemming, C. P. Gerba, M. W. L. Gerba, M. W. L. Chevallier, A. Leis, E. Madsen, J. A. Nienow, K. Scow, R. J. Sevious, L. D. Stetzenach, M. H. Stewart, & D. C. White (Eds.), Encyclepodia of ENVIRONMENTAL MICROBIOLOGICAL (Vol. 6, pp. 3364-3375). Canada John Wiley & Sons, Inc.
Singleton, P., & Sainsbury, D. (2006). Dictionary of Microbiology and Molecular Biology (3 ed.). Chichester: John Wiley & Sons Ltd.
Toranzos, G. A. (2002). Pseudomonas. In G. Bitton, D. L.Balkwill, R. S. Burlage, D. Capone, T. L. Crisman, S. E. Dowd, H.-C. Flemming, C. P. Gerba, M. W. L. Gerba, M. W. L. Chevallier, A. Leis, E. Madsen, J. A. Nienow, K. Scow, R. J. Sevious, L. D. Stetzenach, M. H. Stewart, & D. C. White (Eds.), Encyclepodia of ENVIRONMENTAL MICROBIOLOGICAL (Vol. 5, pp. 2632-2639). Canada
John Wiley & Sons, Inc.


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