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Baraka GitariMaste rs T hes i s Proposa l Pa r t 1 o f 3

BACTERIAL CELLULOSE: THE PERFECT MEDIUM

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-Goal -History (Literature) - Subcellular processes - Hestrin Schramm Medium with modifications - Extracting cellulose -Plan -Synthesizing -Processing (Extracting, electrospinning) -Characterizing (DSC, SEM, XRD, TGA, OM) -Hurdles -Aseptic procedure

OUTLINE

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GOAL

K.I. Uhlin, R.H. Atalla, N.S. Thompson, Influence of hemicelluloses on the aggregation patterns of bacterial cellulose, Cellulose. 2 (1995) 129–144. doi:10.1007/bf00816385.

P. Wambua, J. Ivens, I. Verpoest, Natural fibres: can they replace glass in fibre reinforced plastics?, Composites Science and Technology. 63 (2003) 1259–1264. doi:10.1016/s0266-3538(03)00096-4.

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- 1,4 β glycosidic bonds- Most abundant natural polymer (115.55 m redwood) (chart from assignment 2)- Biodegradable- Sustainable*

- ht tp : / /p u b s . r s c . o r g / s e r v i c e s / i m a g e s / R S C p u b s . e P l a t f o r m . S e r v i c e . F r e e C o n t e n t . I m a g e S e r v i c e . s v c / I m a g e S e r v i c e / A r t i c l e i m a ge / 2 0 0 6 / C S / b 6 0 1 8 7 2 f / b 6 0 1 8 7 2 f - f 2 . g i f

- h t t p : / / w w w. r i s h . k y o t o - u . a c . j p / W / L B M I / r e s e a r c h / I a i b _ S . j p g

CELLULOSE

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-Intracellular polymerization, extracellular crystallization- Gluconacetobacter xylinus - Bacterial Cellulose Synthase 5 - activated by ci-di-GMP - Substrate UDP-Glucose - BcsA/B/C/D subunits from BcsI and BcsII opersons - Mutations6

- G. xyl inus NBRC 3288,(frameshif t bcsBI and transposon insert ion bcsCII) - K. hansenii ATCC 23769 (IS cel lulose synthase) - Acetobacter tropical is SKU1100 (R to S via single C delet ion or insert ion polE) - complete genome sequencing - Basics of protein function and mechanisms

5 . J . L . W. M o rg a n , J . T. M c n a m a r a , J . Z i m m e r, M e c h a n i s m o f a c t i v a t i o n o f b a c t e r i a l c e l l u l o s e s y n t h a s e b y c y c l i c d i -G M P, N a t S t r u c t M o l B i o l N a t u r e S t r u c t u r a l & A m p ; M o l e c u l a r B i o l o g y. 2 1 ( 2 0 1 4 ) 4 8 9 – 4 9 6 . d o i : 1 0 . 1 0 3 8 / n s m b . 2 8 0 3 .6 . M . M a t s u t a n i , K . I t o , Y. A z u m a , H . O g i n o , M . S h i r a i , T. Ya k u s h i , e t a l . , A d a p t i v e m u t a t i o n r e l a t e d t o c e l l u l o s e p r o d u c i b i l i t y i n K o m a g a t a e i b a c t e r m e d e l l i n e n s i s ( G l u c o n a c e t o b a c t e r x y l i n u s ) N B R C 3 2 8 8 , A p p l M i c r o b i o l B i o t e c h n o l A p p l i e d M i c r o b i o l o g y a n d B i o t e c h n o l o g y. 9 9 ( 2 0 1 5 ) 7 2 2 9 – 7 2 4 0 . d o i : 1 0 . 1 0 0 7 / s 0 0 2 5 3 - 0 1 5 - 6 5 9 8 - x .

HISTORY: SUBCELLULAR

ci-di-GMP UDP-Glucose

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7 . K . J i , W. Wa n g , B . Z e n g , S . C h e n , Q . Z h a o , Y. C h e n , e t a l . , B a c t e r i a l c e l l u l o s e s y n t h e s i s m e c h a n i s m o f f a c u l t a t i v e a n a e r o b e E n t e r o b a c t e r s p . F Y- 0 7 , S c i . R e p . S c i e n t i f i c R e p o r t s . 6 ( 2 0 1 6 ) 2 1 8 6 3 . d o i : 1 0 . 1 0 3 8 / s r e p 2 1 8 6 3 .

HISTORY: SUBCELLULAR

75 . J .L .W. Morgan , J .T. Mcnamara , J . Z immer, Mechanism of ac t iva t ion of bac te r ia l ce l lu lose syn thase by cyc l ic d i -GMP, Na t S t ruc t Mol Bio l Na ture S t ruc tura l &Amp; Molecu la r Bio logy. 21 (2014) 489–496 . do i :10 .1038/nsmb.2803

HISTORY: SUBCELLULAR

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- Hestrin Schramm Medium 8 (1954) (% w/v)

- glucose 2 .0 - bactopeptone 0 .5 - yeas t ex t ract 0 .5 - d i -sodium hydrogen phosphate * Na 2 HPO 4 0 .27 - ci t r i c ac id* 0 .12 - s ta r t ing pH 6 .0- Modifications -sugars (sucrose , f ruc tose) - Alcohols -micronut r ien ts (P, S , K, Mg, Ca , Fe) - an t ioxidants ( l ignosul fonate) - pH 8 . M . S c h r a m m , S . H e s t r i n , Fa c t o r s a ff e c t i n g P ro d u c t i o n o f C e l l u l o s e a t t h e A i r / L i q u i d I n t e r f a c e o f a C u l t u re o f Ac e t o b a c t e r x y l i n u m , J o u rn a l o f G e n e r a l M i c ro b i o l o g y. 11 (1 954 ) 123– 129 . d o i : 10 . 109 9 / 0022 1287 - 11 - 1 - 123 .

h t t p s : / / n d b . n a l . u s d a . g o v / n d b / f o o d s / s h o w / 873 4? f o rm a t = Fu l l & re p o r t f m t = p d f & p d f Q vs = % 7B % 7D

HISTORY: GROWTH MEDIUM

9A. Kurosumi, C. Sasaki, Y. Yamashita, Y. Nakamura, Utilization of various fruit juices as carbon source for production of bacterial cellulose by Acetobacter xylinum NBRC 13693, Carbohydrate Polymers. 76 (2009) 333–335. doi:10.1016/j.carbpol.2008.11.009.

Micronutrient Orange Juice Pineapple Juice

Calcium 10.89 mg 13 mgIron 0.20 mg 0.29 mgMagnesium 10.89 mg 12 mgPotassium 200 mg 109 mg

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Cellulose Specialty Uses

https://phy214uhart.wikispaces.com/Capacitors

1. K.-Y. Lee, J.J. Blaker, R. Murakami, J.Y.Y. Heng, A. Bismarck, Phase Behavior of Medium and High Internal Phase Water-in-Oil Emulsions Stabilized Solely by Hydrophobized Bacterial Cellulose Nanofibrils, Langmuir. 30 (2014) 452–460. doi:10.1021/la4032514.

2. M.Ö. Seydibeyoğlu, M. Misra, A. Mohanty, J.J. Blaker, K.-Y. Lee, A. Bismarck, et al., Green polyurethane nanocomposites from soy polyol and bacterial cellulose, J Mater Sci Journal of Materials Science. 48 (2012) 2167–2175. doi:10.1007/s10853-012-6992-z.

3. K.-Y. Lee, H. Qian, F.H. Tay, J.J. Blaker, S.G. Kazarian, A. Bismarck, Bacterial cellulose as source for activated nanosized carbon for electric double layer capacitors, J Mater Sci Journal of Materials Science. 48 (2012) 367–376. doi:10.1007/s10853-012-6754-y.

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http://www.rubber-silanes.com/product/rubber-silanes/en/effects/reinforcement/pages/default.aspx

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TA B L E 2 . 0 I N I T I A L I N V E S T M E N T

Material Cost (per experiment)peptone 68971-500G-F $128 (0.64)

yeast extract Y1625-250G $60.70 (0.61)

di-sodium hydrogen phosphate 71640-250G

$31.00 (0.17)

Resazurin R7017-1G $33.90 (0.03)

Ammonium sulfate A4418-500G $87.60 (0.219)

Gluconacetobacter xylinus $554.00 (once*)

Bunsen Bruner $55.00 (once)

Maple Syrup (Grade B) $38.77 (0.116)

Inoculation loop $145 (once)70% ethanol 02877-1L $62.10 (1.5525)Total $1 133.97 (3.34)

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TA B L E 1 .0 GR OWT H MEDIUM SOU R CE AN D B AC TE R IA L FUNC T IONElement Medium Source Function

Carbon -Grade B Maple Syrup-peptone, yeast extract

- Backbone of organic molecules

Oxygen -Oxygen in air- organic compounds

- Last acceptor in ETC- Organic molecules

Nitrogen - Peptone,- Yeast extract - Ammonium sulfate

- Proteins- Nucleic acids- Coenzymes

Hydrogen -organic compounds- H2 in air

-Organic molecules

Phosphorous - Na2HPO4   (di-sodium hydrogen phosphate)

-Nucleic acids-Phospholipids- Lipopolysaccharides (LPS)

Sulfur NH4SO4 - Amino Acids with Sulfur- Coenzyme

Potassium -Grade B Maple Syrup -intracellular cation-cytoplasmic signaling-incorporation of sulfur into protein- Cofactor (pyruvate kinase)

Magnesium -Grade B Maple Syrup - Cofactor (pyruvate kinase)- Stabilizes (ATP, nucleic acids, RNA, membrane)

Calcium -Grade B Maple Syrup - Intracellular signaling hypothesized

Iron -Grade B Maple Syrup - Cytochrome ETC

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PLAN: EXPERIMENTAL PROCEDURE

autoclave Bunsen burner

Shaker ΔT

53524

centrifuge

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- Aseptic procedure - c l ea n b en c h -Au t oc l a v e ( 12 1 - 14 0 o C 2 - 3 0 m in u t e s 1 5 - 3 0 ps i ) - 7 0 % e th a no l - B u n se n bu r n e r - c on v e c t i o n cu r ren t - h ea t i n g i n oc u l a t i o n l o op , n ec k s o f b o t t l e s

-Monitor ing - c e l l n u mbe r - p H - g lu c o se , g l u co n i c ac id , b y p r o du c t c on c e n t ra t i o n , c e l l u lo s e p r od u c t i on

-P in p o in t i n g me d iu m co mp o s i t i o n / c o nc e n t ra t i on

h t t p : / / w w w. e x p l a i n t h a t s t u f f . c o m / a u t o c l a v e s . h t m l h t t p : / /w w w. s t e r i l i z e r s . c o m / a u t o c l a v e - t i m e - t e m p e r a t u r e - p r e s s u r e - c h a r t . h t m l

Ta b l e 1 . 0 X . Z e n g , D . P. S m a l l , W. Wa n , S t a t i s t i c a l o p t i m i z a t i o n o f c u l t u re c o n d i t i o n s f o r b a c t e r i a l c e l l u l o s e p ro d u c t i o n b y A c e t o b a c t e r x y l i n u m B P R 2 0 0 1 f ro m m a p l e s y r u p , C a r b o h y d r a t e Po l y m e r s . 8 5 ( 2 0 1 1 ) 5 0 6 – 5 1 3 . d o i : 1 0 . 1 0 1 6 / j . c a r b p o l . 2 0 1 1 . 0 2 . 0 3 4 .

HURDLES