Enhanced production of microbial cellulose de coco Center for NanoCellulosics Plant cellulose vs. microbial cellulose Center for NanoCellulosics U.S. Department of Energy Genome Programs,

Enhanced production of microbial cellulose

Jeffrey Catchmark, Kuan‐Chen Cheng and Ali DemirciDepartment of Agricultural and Biological Engineering

Pennsylvania State University

2009 International Conference on Nanotechnology for the Forest Products

IndustryJune 23‐26, 2009

Center for NanoCellulosics

1) Introduction2) Difference between plant and microbial cellulose3) Microbial cellulose production methods4) Approaches for improving the production yield of microbial cellulose:

Culture additivesPlastic composite supports (PCS)

5) Conclusions

Overview


The forest products industry generates more than $200 billion in sales per year, employs ~1.0 million people (many in rural America), and ranks among gthe top 10 domestic manufacturing sectors in contribution to the GDP.Uses 400‐500 million acres of land (U.S.).Consumes ~ 4 billion trees per year (globally).This industry produces many renewable and sustainable products which are essential to our daily life: – Paper, packaging, wood, wood chip and fiber composite materials.

The Forest Products Industry


Wood is composed of 3 major compounds:Wood is 40%‐50% Cellulose, 25%‐35% Hemicellulose and 20%‐25% Lignin.Cellulose: high molecular weight linear chain polysaccharide, β‐linked 1,4 glucan (glucose) residues (10’s of thousands of units long).Hemicellulose: lower molecular weight branched chain polysaccharides produced from other 6‐carbon sugars including galactose and mannose, as well as five carbon sugars including xylose and arabinose (hundreds of units long).Lignin: complex high molecular weight polymer built upon phenylpropane units. Lignin is phenolic (aromatic compound derived from the 6 carbon compound benzene ring with at least 1 hydroxyl group per ring). Lignin serves as a binding agent and provides wood with its stiffness. Cellulose without lignin and hemicellulose is like cotton fabric!


Cellulose used in the FPI is purified from wood

Microbial cellulose

Pure, no hemicellulose or lignin needed to be removed and biomass/culture media relatively simple to remove. Longer fiber length (200,000 glucose units) and higher crystallinity.Can be grown to any shape.

Advantages

DisadvantagesExpensive: sugar and culture media.Production scale‐up difficult.Insolubility and aggregation of cellulose product limits the bioreactor design and yield.


Microbial cellulose products

Ultrafiltration membranes

Klemm et al, 2001 Svensson et al., 2005

Czaja, 2006

Gyre, 2008

Materials for wound care and tissue engineering Artificial blood vessels

Diet foods and dessertsCellulose nanowhiskers

Future products:

Nata de coco


Plant cellulose vs. microbial cellulose


U.S. Department of Energy Genome Programs, http://genomics.energy.gov. “Genomics:GTL Transforming Cellulosic Biomass,ʺ U.S. Department of Energy Office of Science and Office of Energy Efficiency and Renewable Energy, June 2006, http://genomicsgtl.energy.gov/biofuels/ and U.S. DOE


http://genomics.energy.gov/

http://genomicsgtl.energy.gov/biofuels/





Plant cellulose synthesis

Takao Itoh, Satoshi Kimura and R. Malcolm Brown, Jr., Theoretical considerations of immunogold labeling of cellulose synthesizing terminal complexes, Cellulose 11: 385–394, 2004.

6 cellulose synthase enzymes per feature for a total of 36 in the 25nm diameter complex

A single cellulose synthase enzyme

66

66

66


Microbial cellulose synthesis

Takao Itoh, Satoshi Kimura and R. Malcolm Brown, Jr., Theoretical considerations of immunogold labeling of cellulose synthesizing terminal complexes, Cellulose 11: 385–394, 2004.R. Malcolm Brown, et. al., Cellulose Biosynthesis in Acetobacter xylinum: Visualization of the Site of Synthesis and Direct Measurement of the in vivo Process, PNAS 73:4565‐4569, 1976.

Not clear if each feature represents one or more cellulose synthase enzymes.


Plant vs. microbial cellulose

36 joined cellulose synthase proteins (rosette) produce cellulose nanofibers in plants.

~28nm

Bacteria have 1‐6 cellulose synthase proteins.


Implications: cellulose nanowhiskers

Hydrolyzed Acetobacter xylinum celluloseFiber size: ~10‐15nm × ~1‐3microns

Concentrated hydrolyzed Whatman CF11 cellulose (cotton)Fiber size: ~25nm+ × 0.2‐0.5 microns

Cellulose hydrolyzed in 63% H2SO4 at 47C for 130min.


Cellulose synthase complexes

Why have cellulose synthase complexes formed?Create a crystalline cellulose fibrilAchieve mechanical strengthAchieve resistance to enzymatic attackOthers…


Microbial cellulose production processes


Microbial cellulose production

Most common production processes are static cultures and submerged agitated cultures.Oxygen delivery to bacteria a major factor.

Pellicle (Static)

Bioreactor (agitated)

Flask (agitated)


Bioreactor production

Unlike static and flask cultivation, bioreactors offer additional control over process parameters:• Agitation• Temperature• Dissolved oxygen• pH


Rotating disc reactors (Serafica, 1997) Rotating plates pass through the media providing oxygen and nutrients.Can control environment and introduce additives.

Other production approaches


Silicone membrane (Yoshino et al., 1996)

Microbial cellulose

Spray chamber (Hornung, et al., 2006)

Other production approaches


Research objectives

Enhance production of microbial cellulose in submerged culture by:

Incorporating additivesIncorporating a solid nutrient support

Evaluate the properties of cellulose produced.


Dynamic Dynamic mechanic mechanic analysisanalysis

Material strength

Thermo Thermo Gravimetric Gravimetric

(TGA)(TGA)

Thermostability

FESEMFESEM

BC structure

TGA XRD

FE‐SEMDMA

XX--ray ray diffractiondiffraction

Crystallinity;Crystal size

Characterization


AdditivesMicrocrystalline cellulose

Sodium alginateSodium

carboxymethylcellulose (CMC)Agar

0.2, 0.5% (w/v)

5 day cultivation in 250 ml flasks

Harvest cellulose and analyze

Removal of cells and media (0.1 N, NaOH )

Experimental design: effect of additives

Bacterial strain: Acetobacter xylinum(ATCC 700178) Medium:CSL‐Fru medium (Kouda et al. 1997)

Cellulose producing bacteria first reported by Adrian Brown while working with Bacterium aceti in 1886.


0

1

2

3

4

56

7

8

9

10

Control

0.2% avic

el0.5

% avicel

0.2% C

MC

0.5% C

MC

0.2% sodiu

m alginate

0.5% sodiu

m alginate

0.2% A

gar

Types and concentration of additives

Wei

ght o

f BC

(g/L

)

Bacterial cellulose production by A. xylinum in CSL-Fru medium containing Microcrystalline cellulose, CMC, sodium alginate or agar in 250 ml flasks.

7.3 g/L

Results: cellulose yield


1.3 g/L

5.6×improvement

0

2

4

6

8

10

0.0 0.2 0.5 0.8 1.0

CMC concentration (%; w/v)

Wei

ght o

f BC

(g/L

)

Bacterial cellulose production by A. xylinum in CSL-Fru medium containing different concentration of CMC in 250 ml flasks.

8.2 g/L

Results: cellulose yield with CMC

6.3×improvement

1.3 g/L


FESEM images of freeze dried microbial cellulose produced . (a) Control; (b) 0.2% CMC; (c) 0.5% CMC; (d) 0.8% CMC; and (e) 1% CMC addition.

Results: FESEM of cellulose‐CMC materials


(a) (b) (c)

(d) (e)

Results: cellulose crystallinity

• The decrease in crystallinity is responsible for the increased cellulose yield, as shown by Haigler (J Cell Biol 94(1):64‐69, 1982), where cellulose crystallization is shown to be a rate limiting step in cellulose production.






Microbial cellulose synthesis

Takao Itoh, Satoshi Kimura and R. Malcolm Brown, Jr., Theoretical considerations of immunogold labeling of cellulose synthesizing terminal complexes, Cellulose 11: 385–394, 2004.R. Malcolm Brown, et. al., Cellulose Biosynthesis in Acetobacter xylinum: Visualization of the Site of Synthesis and Direct Measurement of the in vivo Process, PNAS 73:4565‐4569, 1976.

Not clear if each feature represents one or more cellulose synthase enzymes.


Derivative of the TGA curves of control BC and CMC-altered BC.

Results: thermogravimetric analysis

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

0 100 200 300 400 500 600 700

Temperature (oC)

Der

iv. W

eigh

t (%

/o C)

ControlCMC 0.2%CMC 0.5%CMc 0.8%CMC 1.0%


Temp. ramp 10°C/min.

Results of tensile test of BC. (A) Stress at break; (B) Strain at break; (C) Young’s modulus.

(A)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

Pellicle 0% CMC 0.2% CMC 0.5% CMC 0.8% CMC 1.0% CMCTypes of BC sample

Stra

in a

t bre

ak (%

)

(B)

(C)


Results: mechanical analysis

Results: aggregation of product

0.8% CMC No CMC

Addition of CMC in bioreactor culture may prevent aggregation

while also substantially improving yield


Conclusions: effects of additives

The optimal CMC concentration is around 0.8% (w/v), where cellulose production reached 8.2 g/L, a 6.3×improvement over the control.

The crystallinity and crystal size of the cellulose decrease when cultured in 0.8% CMC.

The aggregation of cellulose is prevented when cultured in 0.8% CMC.

The decrease in crystallinity and/or aggregation is believed to be responsible for the substantial improvement in cellulose production yield.


Experiment: solid nutrient support

Biofilm reactor cultivation uses a solid nutrient support to form a stable biofilm (bacteria colonies).

PCS –Plastic composite (nutrient) support

Advantages:Release nutrients locally.Cells grow on the solid surface.Increase biomass in the reactor.Reduces the risk of washing out cells during continuous fermentation.Eliminating need for re‐inoculation during repeated‐batch fermentation.


Experiment: solid nutrient support

Biofilm reactor cultivation uses a solid nutrient support to form a stable biofilm (bacteria colonies).

PCS –Plastic composite (nutrient) support

Applications: production ofNisinEthanolLactic acidCellulases Amylases Lipaseslignin peroxidases

and wastewater treatmentCenter for NanoCellulosics

PCS solid nutrient support fabrication

Extruder

PCS

Mix


PCS solid nutrient support fabrication

Experiment: PCS solid nutrient support

Selection of suitable PCS with different nutrition composition (flask study)

Production of cellulose in a PCS biofilm reactor

Harvest cellulose and analyze

Removal of cells and media (0.1 N, NaOH )


Results: selection of PCS composition

0.00.51.01.52.02.53.03.54.04.5

Contro

l SSB+

SF+ SFB SFR SFYSFY

B+SFY

R+SR+

SY+SYB+SFY

BRSFY

B

Types of PCS

Bio

mas

s(x1

03 g/g

PC

S)

02468

1012141618

Contro

lSup

portSB+

SF+ SFB SFR SFYSFY

B+SFY

R+SR+

SY+SYB+SFY

BRSFY

B

Type of PCS

Cel

lulo

se(g

/L)

Results of Biomass and cellulose production with 13 different PCS materials.

Based on the availability and nutrient leaching rate, we chose SFYR + as optimal PCS for Biofilmreactor study.

S: dried, ground, soybean hulls

F: defatted soybean flour

Y: yeast extract

R: dried bovine red blood cell

+: w/ mineralsCenter for NanoCellulosics


0.00.51.01.52.02.53.03.54.04.5

Contro

l SSB+

SF+ SFB SFR SFYSFY

B+SFY

R+SR+

SY+SYB+SFY

BRSFY

B

Types of PCS

Bio

mas

s(x1

03 g/g

PC

S)

02468

1012141618

Contro

lSup

portSB+

SF+ SFB SFR SFYSFY

B+SFY

R+SR+

SY+SYB+SFY

BRSFY

B

Type of PCS

Cel

lulo

se(g

/L)





Y: yeast extract




0.00.51.01.52.02.53.03.54.04.5

Contro

l SSB+

SF+ SFB SFR SFYSFY

B+SFY

R+SR+

SY+SYB+SFY

BRSFY

B

Types of PCS

Bio

mas

s(x1

03 g/g

PC

S)

02468

1012141618

Contro

lSup

portSB+

SF+ SFB SFR SFYSFY

B+SFY

R+SR+

SY+SYB+SFY

BRSFY

B

Type of PCS

Cel

lulo

se(g

/L)





Y: yeast extract



Results: yield and crystallinity

The PCS biofilm reactor yielded BC production (7.05 g/L) that was 2.5‐fold greater than the control (2.82 g/L).


Cellulose grown on the PCS shaft after 120 hr cultivation.


Results: FESEM

Bars are 1 micron (top right) and 50 microns (bottom right)

0.00.20.40.60.81.01.21.41.6

0 200 400 600 800Tempertature (oC)

Der

iv. W

eigh

t (%

/o C)

ControlPCS grown BC

Derivative TGA patterns of BC from PCS biofilm and suspended-cell reactor.


Results: thermogravimetric analysis

Temp. ramp 10°C/min.

0

5

10

15

20

25

30

35

40

PCS grow n BC Cont rolType of BC pell icle

Stre

ss a

t br

eak

(MPa

)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

PCS grown BC Control

Type of BC

Stra

in a

t br

eak

(%)

0

500

1000

1500

2000

2500

3000

PCS grow n BC Cont rolType of BC

Youn

g's

mod

ulus

(MPa

)

Results of tensile test of BC. (A) Stress at break; (B) Strain at break; (C) Young’s modulus.

(A)

(C)

(B)


Results: mechanical analysis

Conclusions: PCS solid nutrient supportThe PCS biofilm reactor using SFYR+ type PCS yielded cellulose production of 7.05 g/L, which was 2.5‐fold greater than the control.

XRD results demonstrated that PCS‐grown cellulose exhibited higher crystallinity (93%) and similar crystal size (5.2 nm) to the control.

TGA results indicated that PCS‐grown cellulose exhibited higher decomposition temperature compared to the control but PCS support material incorporation is expected to be a contributing factor.

DMA results showed that cellulose from the PCS biofilm reactor increased its mechanical property values, i.e., stress at break and Young’s modulus when compared to the control cellulose but PCS support material incorporation is expected to be a contributing factor.


Acknowledgments

This work was supported in part by a seed grant from the collegeof Agricultural sciences at the Pennsylvania State University and the Pennsylvania Experiment Station.

Students and collaborators:

Kuan‐Chen ChengPh.D. StudentAgricultural and Biological Engineering, Penn State University

Dr. Ali DemirciAssociate ProfessorAgricultural and Biological Engineering, Penn State University


Thank You!


Documents

Enhanced production of microbial cellulose de coco Center for NanoCellulosics Plant cellulose vs. microbial cellulose Center for NanoCellulosics U.S. Department of Energy Genome Programs,