IBSB – Industrial Biotechnology and Systems Biology Research Group

Workshop BioGlueVienna – Nov 2010

Marmara University, Department of Bioengineering, Istanbul, Turkey

Ebru Toksoy Öner

IBSB

Extremophiles

Levan by Bacillus sp.

Extremophilic Levan

Industrial Biotechnology and Systems Biology Research Group

Marmara University

Department of Bioengineering

Istanbul, Turkeyhttp://ibsb. marmara.edu.tr

Ebru Toksoy Oner, Assoc. Prof.

Kazım Yalcın Arga, Assist. Prof.

1 Post-Doc

8 pHD students

2 MS students

Industrial Biotechnology and Systems Biology Research Group

Marmara University

Department of Bioengineering

Istanbul, Turkey

http://ibsb. marmara.edu.tr

Betül Kırdar, Prof.

Barbara Nicolaus, Prof.

Stephen G. Oliver, Prof.

Robert Dekker, Prof.

interpret and optimize production capabilities

of yeast, bacteria and extremophiles via systems

based approach

optimization of fermentation processes to

design high-yield production lines

production of biopolymers form extremophiles

isolation and identification of extremophiles

strain improvement for bioethanol production

Ion N. Mihailescu, Prof.

Extreme conditions can refer to physical

extremes (temperature, pressure or

radiation) or geochemical extremes

(salinity, desiccation, oxygen tension

and pH).

Extremophiles are microorganisms that

not only tolerate such extreme

conditions, but usually require such

environmental extremes for their

survival and growth.

ecological systems such as hot

springs, salt and soda lakes, deserts

and ocean beds that are not

compatible with human life are

considered as being extreme.

Anoxybacillus amylolyticus MR3CT

Isolated from M. Rittmann (Antarctica) T 60°C

Geobacillus thermoleovorans subsp. stromboliensis sbsp.novIsolated from the geothermal volcanic environment (Italy)

T 70°C

Halomonas sp. AAD6 Isolated from Camaltı Saltern Area (Turkey)

Starchy agro-

industrial wastes

Bioethanol…

to successfully engineer

Biopolymer properties and

improve production

Systems Biology Aprroach

Protein-encoding gene models 584

Metabolites 1389

Intracellular metabolites 1020

Extracellular metabolites 369

Reactions 1080

Enzymatic reactions 870

Transport fluxes 210

Metabolic Model of Halomonas sp.

Microbial Production

Nanostructured Coatings for

Biomedical Applications Thin, high-quality and uniform films

produced by the MAPLE (Matrix Assisted

Pulsed Laser Evaporation) technique

Microbial Production

Chemical media

Low cost substrates

Levan – based micro/nano particles

may be a potential delivery system for

macromolecules.

Drug Delivery Systems

Environmental Applications

Emulsifying Agent

Bioflocculating activity

Functional Biofilms

Edible Food Packaging

FRUCTAN

a homopolimer of fructose units (polyfructose, fructan)

a crucial component of drought and freeze protection in plants

ability to stabilize membranes in dry and cold environments

Levan

Inulin

Graminancomplex and branched

linear β-(2,6)-linked

fructofuranosyl units

linear β-(2,1)-linked

fructofuranosyl units

Blood plasma extender

Hypocholesterolaemic agent

Anti-AIDS agent

Immunostimulating agent

Tablet binder

Add sweetness to foods

prebiotics

Filler

Bulking agent

Substitute for gums

Animal feed

Edible food packaging

Carrier for flavor and

fragrances

bioadhesive

Provide viscosity

Holding capacity for water

and chemicals

Selective plugging agent

Source for pure fructose

cryoprotectant

Emulsifier

Formulation aid

Stabilizer and thickener

Surface-finishing agent

Encapsulating agent

Cosmetic

….

Medical Pharmaceutical OthersIndustrialFoods

BENEFITS :

Produced from renewable resource, sugar.

No petroleum or natural gas derivatives in product.

No VOCs, HAPs or other toxic emissions.

Water-based with no solvents.

No health issues for users.

No dermal irritation.

No allergic contact sensitization.

Biodegradable.

Reduce regulatory burden.

Long term storage as powder.

produced by Bacillus sp.

has strong bioadhesive properties

hydroxyl groups in its structure form adhesive bonds with various substrates.

the most promising commercial polysaccharide based adhesives are actually made from levan

•Combie, J. (2005) Properties of Levan and Potential Medical Uses, Polysaccharides for Drug Delivery and Pharmaceutical Applications, June 22, 2006. Vol.934, 263-269.

•Mancuso Nichols et al. (2009) 'Screening Microalgal Cultures in Search of Microbial Exopolysaccharides with Potential as Adhesives', The Journal of Adhesion, 85: 2, 97 — 125.

http://specialtybiopolymers.com

Dr. Joan Combie

Montana Polysaccharides Corp. (USA)

Natural Polymer Tensile Strength

psi

Levan 991

Carboxymethylcellulose 193

Inulin 124

Guar gum 63

Xanthan gum 33

Very limited information and literature

Not produced at large scale

Production conditions depend on the microbial system used

MW and degree of branching depend on the production conditions

Biological activity and physicochemical properties depend on production

Strict control over process parameters is necessary

Difficult to purify

Expensive

an optimal cost-effective production process is a must!

Plant

•Grasses (Dactylis glomerata, Poa secunda, Agropyron cristatum)

Microorganisms

•Zymomonas mobilis

•Bacillus sp.

•Erwinia herbicola

•Gluconoacetobacter xylinus

•Microbacterium laevaniformans

•Rahnella aquatilis

•Serratia levanicum

•Pseudomonas syringae

•Halomonas sp.

* Poli A. et al.. 2009. Carbohydrate Polymers, 78, 651-657.

the first and only levan-producer extremophile *

Highest production yield on available substrate

Microbial Production by Halomonas sp.

Chemical media + sucrose

Sugar beet molasses

10 X increase in

uronic acid content !!

Biocompatibility studies showed that levan produced by Halomonas sp.

did not affect cellular viability and proliferation of osteoblasts and

murine macrophages suggesting the high biocompatibility of this EPS.

Brine Shrimp Test. The inhibition of avarol toxic activity on brine shrimp

(Artemia salina) test was performed in artificial sea water. With

decreasing doses of levan solution (500, 50, 5 ppm), protective effect

against the toxic activity of avarol increased. Halomonas levan was

found to increase the LD50 value of avarol from 0.18 ppm up to 10

ppm. The protective effect of the polymer against the toxic activity of

Avarol implied its potential use as an anti-cytotoxic agent.

MTT cell proliferation assay was

employed to assess the cell

viability. Levan by Halomonas sp.

showed high biocompatibility and

affinity against both cell lines.

cancerous cell line He La (human cervical cancer cells)

non-cancerous cell line L929 (mouse fibroblast cells)

mouse monocyte/macrophage cell line J774 osteoblast cells isolated from

the calvaria of Wistar rats

0

0.2

0.4

0.6

0.8

1

1.2

Control EPS

via

bil

ity/p

roli

fera

tio

n (

OD

595n

m)

AL

P s

ecre

tio

n (

μg

/ml)

viability/proliferation

ALP secretion

Idea: extreme conditions (salinity) at which levan is

microbially produced may also confer it some unique

properties enhancing its adhesive strength !

current research efforts on levan from

Halomonas sp. are now focused on

elucidating its potential to be used as a

commercially useful adhesive by

Developing new formulations with the

polymer and its modified forms

Understanding its mechanism of action

http://ibsb. marmara.edu.tr

Thank you for your listening !

http://ibsb. marmara.edu.tr