project etb-2012-26 optisolv - development, optimization and scale-up of biological solvent...

UNIVERSITÀ DEGLI STUDI DI NAPOLI FEDERICO II

DIPARTIMENTO DI INGEGNERIA CHIMICA DEI MATERIALI E DELLA PRODUZIONE INDUSTRIALE

Project ETB-2012-26

OPTISOLV - Development, optimization and scale-up of biological solvent production

3nd International Meeting

PortoMantovano, December 01, 2014

F. Raganati

SUMMARY

Biofilm reactors Start Up Glucose/Lactose Lactose Scheduled Activity

The ABE fermentation process by adopting renewable resources. Characterization in terms of kinetics and yields.

simple sugars (glucose, fructose and sucrose) typically present in high sugar content beverages;

High Sugar Content Beverages Scheduled Activity

Characterization of the ABE fermentation process. Dynamic kinetic modelling Solventogenic Kinetics Scheduled Activity

Series of4 PBR: Butanol

Production

MEDIUM

TANK

Nutritional Factors

Concentration [g/L]

Sugar 60

Yeast Extract 5

NH4Cl 2

K2HPO4 0.25

KH2PO4 0.25

MgSO4 0.2

FeSO4 0.01

MnSO4 0.01

1 2 3 4

TASK 1.2: SET-UP OF A CONTINUOUS REACTOR IN LAB-SCALE

Voverall = 160 mL

Porto Mantovano – December 1, 2014

BIOFILM REACTORS: CONTINUOUS BUTANOL PRODUCTION. GLUCOSE/LACTOSE

1 2 3 4

0 1 2 3 4 50

1

2

3

4

5

6

7

8

9

10

11

12

13

14G 75% - L 25%

G 50% - L 50%

G 25% - L 75%

L 100%

n° reactor

Bg/L

0 1 2 3 4 50

10

20

30

40

50

60G (G 75%- L 25% )

G (G 50% - L 50%)

G (G 25% - L 75%)

G (L 100%)

L (G 75% - L 25%)

L (G 50% - L 50%)

L (G 25% - L 75%)

L (L 100%)

n° reactor

Sugarg/L

Total Sugar60 g/L

D = 0.15 1/h

Specific lactose production:4 stages @ D = 0.15 1/h: 0.9 g/Lh1 stage @ D = 1 1/h: 4.5 g/Lh

1 stage


4 stages

0 1 2 3 4 50

1

2

3

4

5

6

7

8

9

10

11

12

13

n° reattore

Bg/L

0 1 2 3 4 50

10

20

30

40

50

60

Latg/L

BIOFILM REACTORS: CONTINUOUS BUTANOL PRODUCTION. LACTOSE

1 2 3 4

0 1 2 3 4 50

1

2

3

4

5

6

7

n° reactor

ABg/L

0 1 2 3 4 50

1

2

3

4

5

Dtot=0.15

Dtot=0.2

Dtot=0.25

Dtot=0.35

Dtot=0.45

Dtot=0.55

Dtot=0.65

Dtot=0.75

Dtot=0.85

Dtot=0.9

AAg/L

1 stage

4 stages

BIOFILM REACTORS: CONTINUOUS BUTANOL PRODUCTION. LACTOSE

1 2 3 4


0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.000

1

2

3

4

5

6

7

8

9

10

D - h-1

Buta

nol P

roduct

ivit

y -

g/L

h

1 stage

4 stages

BIOFILM REACTORS: SCHEDULED ACTIVITY

1 2 3 4


Series of 4 packed bed reactors Continuous additional in line feeding between bioreactors

addition of glucose to the 2nd or 3rd bioreactor at D= 0.1 h -1.

addition of AA, AB, glucose or combination of those to the 2nd bioreactor at

D=0.15-0.2 h-1 in concentrations that simulated conditions in the flow from the

1st to 2nd bioreactor at high dilution rates.

addition of butanol at concentrations of around 0.5 g/to the feed to the 1st

bioreactor at D= 0.1-0.15 h-1.

operation of the system at different glucose concentration in the feeding stream.

SUMMARY



simple sugars (glucose, fructose and sucrose) typically present in high sugar content beverages;



THE ABE FERMENTATION PROCESS BY ADOPTING RENEWABLE RESOURCES: HSCB

Batch tests using single representative sugars

Batch tests using a mixture of the 3 sugars

GFS

Kinetic characterization

Nutritional Factors

Concentration [g/L]

Sugar 60

YE 5

NH4Cl 2

K2HPO4 0.25

KH2PO4 0.25

MgSO4 0.2

FeSO4 0.01

MnSO4 0.01

CaCO3 5

Syrups:Lemon & Almond

Fruit juices:Pineapple & Pear

Soft drinks:Coca cola & Sprite

Sport drinks:Powerade

COMPLEX MEDIUM



THE ABE FERMENTATION PROCESS BY ADOPTING RENEWABLE RESOURCES: HSCB

Unsupplemented

Medium (HSCB)

Supplemented

Medium (HSCB+)

Hydrolized

Supplemented

Medium (HHSCB+)NO

GROWTH

Lack of some indispensable

nutrients

• Glucose conversion: complete • Fructose conversion: almost complete

about 10 g/L of butanol were produced

a significant amount of sucrose was unconverted

tests with hydrolysed HSCB+ (HHSCB+) were performed

improved solvents production and sugar conversion degree

13 g/L

THE ABE FERMENTATION PROCESS BY ADOPTING RENEWABLE RESOURCES: SCHEDULED ACTIVITY

HSCB as complex substrate to feed the 4 PBR in series.

1 2 3 4


SUMMARY



Simple sugars (glucose, fructose and sucrose) typically present in high sugar content beverages;



CHARACTERIZATION OF THE ABE FERMENTATION PROCESS: DYNAMIC KINETIC MODEL

A kinetic dynamic model of acetone–butanol–ethanol (ABE) production by Clostridium acetobutylicum DSM 792 was developped according to the biochemical networks simulator COPASI

Substrate effects investigation: • glucose, mannose, fructose• sucrose, lactose• xylose, and arabinose

• The Embden-Meyerhof-Parnas (EMP) pathway for hexoses and disaccharides

• The pentose phosphate (PP) pathway for pentoses.


The proposed model was an update of the model by Shinto et al. (2007, 2008)*

Kinetics Shinto et al. Proposed Model

Substrate Inhibition+

Non Competitive Butanol Inhibition

Substrate Inhibition+

Complete Butanol Inhibition

BA Activation +


BA Activation+

CompleteButanol Inhibition


Multi ProductInhibition

Mass ActionSpecific Butanol Activation


Glu Man Fru Suc Lac Ara Xyl

ProposedModel

0.855 0.812 0.820 0.800 0.904 0.848 0.830

Shinto et al.(2007-2008)

0.894 0.887 0.870 0.880 0.925 0.904 0.890

The r2 increased with respect to that calculated for Shinto’s simulation, whatever the tested sugar

The results confirmed that the structure of the present model improved the simulation results.

The soundeness of the model has been tested according to two procedures:

the assessment of the average squared correlation coefficients (r2) between the simulation results and the experimental data.

the comparison of the results of the presenet model with those reported by Shinto et al. (2007-2008).

r2


CHARACTERIZATION OF THE SOLVENTOGENESIS KINETICS: THEORETICAL FRAMEWORK

R = 0.14, 0.54 and 0.88

D = 0.02 - 0.15 h-1.

The system was described by the equation set:

BIOMASS BALANCE

CELL TRANSFORMATION PATHS

REACTION SET


CHARACTERIZATION OF THE SOLVENTOGENESIS KINETICS: RESULTS

R=0.88

The concentration of acidogenic cells increases linearly with D while the spore concentration decreases exponentially with D

The concentration of solventogenic cells is almost constant with D except for a little increase at low D

As D increases, the progressively shift toward a less harsh conditions – low concentration of solvents and acids – promotes the presence of acidogenic cells at spore’s expense


The agreement between the model prediction and experimental data is satisfactory.


rBMAX

gB/gDMhKL

g/LKAA

g/LKBA

g/LKB

g/L

5 0 0.8 0.05 0.48

The production rate of butanol referred to the mass unit of solventogenic cells was calculated for all tests.

Lactose, acetic acid and butyric acid were considered as substrate and butanol as the inhibition product (Monod-Boulton model)


CHARACTERIZATION OF THE ABE FERMENTATION PROCESS: SCHEDULED ACTIVITY

Model of a Biofilm PBR

Model based on:


Dynamic Model

Acidogenic Kintics

Solventogenic Kintics

List of contributions

Raganati, F., Olivieri, G., Procentese, A., Russo, M. E., Salatino, P., Marzocchella, A. (2013). Butanol production by bioconversion of cheese whey in a continuous packed bed reactor. Bioresource Technology, 138, 259–265

Raganati, F., Procentese, A., Olivieri, G., Russo, M. E., Marzocchella, A.. MFA of Clostridium acetobutylicum pathway: the role of glucose and xylose on the acid formation/uptake. Chemical Engineering Transactions. 2014 V. 38, p.193-198

A. Procentese, T. Guida, F. Raganati, G. Olivieri, P. Salatino, A. Marzocchella. Process Simulation of Biobutanol Production from Lignocellulosic Feedstocks. Chemical Engineering Transactions. 2014 V. 38, p.343-438

A. Procentese, F. Raganati, G. Olivieri, M.E. Russo, P. Salatino, A. Marzocchella. Butanol production by fermentation of Clostridium acetobutylicum: solventogenic kinetics. Submitted to Bioresource Technology

F. Raganati, A. Procentese, G. Olivieri, P. Gotz, P. Salatino, A. Marzocchella. Kinetic study of butanol production from various sugars by Clostridium acetobutylicum using dynamic model . Submitted to Biochemical Engineering Journal

F. Raganati, A. Procentese, F. Montagnaro, G. Olivieri, A. Marzocchella. Butanol Production from Leftover Beverages and Sport Drinks. BioEnergy Research. 2014 - DOI 10.1007/s12155-014-9531-8Porto Mantovano – December 1, 2014

UNIVERSITÀ DEGLI STUDI DI NAPOLI FEDERICO II

DIPARTIMENTO DI INGEGNERIA CHIMICA DEI MATERIALI E DELLA PRODUZIONE INDUSTRIALE

Teresa GuidaAntonio MarzocchellaGiuseppe OlivieriAlessandra ProcenteseFrancesca RaganatiMaria Elena Russo (IRC – CNR)Piero Salatino

WORKING PLAN & PROJECT SCHEDULE


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36WP1Task 1.1 D1.1Task 1.2 D1.2Task 1.3 D1.3Task 1.4 D1.4Task 1.5 D1.5Task 1.6 D1.6WP2Task 2.1 D2.1Task 2.2 D2.2Task 2.3 D2.3Task 2.4 D2.4Task 2.5 D2.5WP3Task 3.1 D3.1Task 3.2 D3.2Task 3.3 D3.3WP4Task 4.1 D4.1Task 4.2 D4.2Task 4.3 D4.3Task 4.4 D4.4WP5Task 5.1 D5.1/M5.1Task 5.2 D5.2/M5.2Task 5.3 D5.3

Italy: Jan 1, 2013Germany: May 20, 2013K.O. meeting: May 27, 2013

December 1, 2014

INTERRELATION BETWEEN ENGINEERING AND NATURAL SCIENCE WORK PACKAGES (WP 1,2 AND 3)



R=0.88

The lactose conversion and the concentration of products (cells and metabolites) decrease with the D

The butanol selectivity increased with D and it approached a constant value of about 0.90 g/g

Butanol and ABE productivities increased with D.• A double slope may be observed in the productivity vs. D

data with a discontinuity at D≈0.1 1/h• the slope at lower D is higher than that at higher D


The µ was typically smaller than D and larger than DOUT

the accumulation of acidogenic cells - µ>DOUT - was prevented by the establishment of a cell population controlled by the equilibrium among acidogenic cells, solventogenic cells and spores.

The analysis of µS and of concentration of acids and solvents suggests that acids promote the solventogenic cell formation while solvents inhibit the formation.

The study carried out during the present Ph.D. program aimed at investigating the Acetone-Butanol-Ethanol (ABE) production process by fermentation of renewable feedstocks

The activities were articulated along three paths

The characterization of the ABE fermentation process as regards kinetics and yields using different renewable resources

sugars representative of hydrolized lignocelluloe (glucose, mannose, arabinose and xylose)

sugars representative of high sugar content beverages (glucose, fructose and sucrose)

Characterization of the ABE fermentation process through MFA and dynamic kinetic models

the MFA was adopted to investigate the role of the main reaction steps of the C. acetobutylicum metabolic pathway to convert reference sugars

A kinetic dynamic model of acetone–butanol–ethanol (ABE) production by Clostridium acetobutylicum DSM 792 was proposed using the biochemical networks simulator COPASI.

Development of innovative continuous reactor for the ABE production

High sugar content beverages & Cheese Whey

CONCLUSIONS

Biofilm Packed Bed Reactor

Assessment of the model parameters

The maximum reaction rate of a reaction step depends on the sugar because it depends on the enzyme concentration

The “affinity” constants do not depend on the sugar because they depend on the enzyme responsible of the reaction step but not on its concentration

Parameters of the sugar uptake reactions have been assessed for each sugar according to Servinsky et al. (2010): C. acetobutylicum has sugar-specific mechanisms for the transport and metabolism genes.

*Servinsky et al., (2010). Microbiol. 156:3478–3491

The soundeness of the model has been tested according to two procedures:

the assessment of the average squared correlation coefficients (r2) between the simulation results and the experimental data.

the comparison of the results of the presenet model with those reported by Shinto et al. (2007-2008).

project etb-2012-26 optisolv - development, optimization and scale-up of biological solvent...

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