Enzyme Engineering 9. Applications(3): Energy & Environment 9.1 Biodiesel Production 9.2 Bioethanol Production 9.3 Enzymatic Denitrification

Enzyme Engineering

9. Applications(3): Energy & Environment

9.1 Biodiesel Production

9.2 Bioethanol Production

9.3 Enzymatic Denitrification

9.1 Biodiesel Production

Biodiesel• Mixture of mono-alkyl esters

(fatty acid alkyl esters)

• Typically made by transesterification of vegetable oils or animal fats.

• Advantages– Renewable resource– Biodegradable and less toxic– Favourable combustion emission profile

• Low carbon monoxide, sulphur oxide, and nitrogen oxide– High flash point

• Less volatile and safer to transport

• Conversion of triglyceride to fatty acid alkyl esters

• Bottlenecks for lipase-catalyzed biodiesel production Enzyme inhibitions by methanol and glycerol Decrease of enzyme activity High cost of lipase

Transesterification process

Transesterification by lipase

• Enzymatic production is possible using both extracellular and intracellular lipases.

• In both the cases the enzyme is immobilized and used which eliminates downstream operations like separation and recycling.

• Both the processes are reported to be highly efficient compared to using free enzymes.

Comparison of steps involved in the immobilization of extracellular and intracellular enzymes: (a) extarcellular lipase and (b) intracellular lipase.

S.V. Ranganathan et al., Bioresource Technology 99 (2008) 3975–3981

Biodiesel production

S.V. Ranganathan et al., Bioresource Technology 99 (2008) 3975–3981

Continuous reactor

Author Oil Enzyme Reactor type Remarks Conversi

on Year

S. Halim et al.Waste cooking

palm oilNovozyme

435Packed bed

reactortert-butanol

80% for 120hr

2009

Royon et al. Cottonseed oilNovoayme

435Fixed bed

reactortert-butanol 95% 2007

Watanabe et al. Waste edible oilNovozyme

435Fixed bed

reactorThree step reaction 90.9% 2006

Shimada et al.Vegetable, tuna and waste edible

oil

Novozyme 435

Packed bed reactor

3 packed bed reactor in series

90% for 100 days

2002

Watanabe et al. Vegetable oilNovozyme

435Packed bed

reactor2 and 3 packed bed

reactors in series93% for

100 days2000

Extracellular lipase

• Such enzyme-catalyzed processes in entirely nonaqueous solvents were described by Klibanov et al. (1984).

• Candida antarctica lipase

– Novozyme 435 (CALB immobilized on acrylic resin)– The most effective lipase among any of the lipases tested for

methanolysis.

Y. Shimada et al., JAOCS, Vol. 77, no. 7 (1999)


FIG.1. Effect of methanol content on methanolysis of vegetable oil. A mixture of 10 g oil/methanol and 0.4 g immobilized lipase was shaken at 30°C for 24 h.

FIG. 2. Time courses of two- and three-step methanolyses of vegetable oil. Two-step reaction (○): after 7-h reaction, 2.10 g methanol was added in the reaction mixture , Three-step reaction (●): 1.05 g methanol was added after 7- and 17-h reactions

Y. Watanabe et al., JAOCS, Vol. 77, no. 4 (2000) Y. Shimada et al., JAOCS, Vol. 77, no. 7 (1999)


Candida antarctica B-lipase (CALB); Novozym 435 Thermomyces lanuginosa (TLL); Lipozyme TL IM

P. M. Nielsen et al., Eur. J. Lipid Sci. Technol. 2008, 110, 692–700

Intracellular lipase

• Bacteria, yeast and filamentous fungi that would serve as whole-cell biocatalysts based on their ability of immobilization and the display of functional proteins of interest on their cell surface.

FIG. Micrographs of surface (a) and cross-section (b) of a BSP

• Developed by Matsumoto et al. (2001)– R. oryzae cell– Stepwise methanolysis of plant oil– In solvent free and water containing system– 71% after a 165 hr at 37 ℃

K. Ban et al. / Biochemical Engineering Journal 8 (2001) 39–43


I. Addition of substrate-related compounds to the culture medium improved the lipase activity.

II. Gluteraldehyde cross-linking of membrane bound lipase

• High lipase activity could be maintained over several cycles.

III. Fatty acid membrane composition• The membrane permeability and stability is significantly affected by the

fatty acid composition.

IV. Yatalase treatment; An enzyme that partially degrades fungal cell wall and allows the exposure of membrane-bound lipase to the substrate, thereby enhancing its activity.

H. Fukuda et al., Trends in Biotechnology Vol.26 No.12, 2008


Secretory mechanism and localization

• In the immobilized culture, the fungal mycelium is immobilized into BSPs and large amounts of lipase are retained within the cells.

• Cleavage of pro-region from the precursor lipase Pre-pro ROL gives rise to two lipases.

a. Cell membrane; ROL 31 b. Cell wall; ROL 34

• Cell immobilization strongly inhibited the secretion of ROL 31 into the culture medium.



• A yeast cell surface display system for lipase from R. oryzae was developed.

• Schematic diagram of a yeast whole-cell biocatalyst displaying ROL via an FOL 1 anchor.

• Lipase displaying system– the N-terminus of ROL including

a pro-sequence Pro-ROL has been fused to the flocculation functional domain of FLO p, a lectin-like cell-wall protein of yeast.


Extracellular lipase vs. Intracellular lipase

• An outstanding reaction rate was obtained with Novozym 435 in continuous operation (7 h with 92–94% ME content) and fed-batch operations (3.5 h with 87% ME content) respectively.

• Considerable reduction in cost can be achieve with intracellular lipase.

• The reaction rate of intracellular lipase is very slow in a batch reaction and more than 70 h are required to obtain 80–90% ME content.

CalB Expression in E.coli Materials

Cell: E.coli 1) DH5α 2) Rosettagami 3) Novablue Vector: pCold Ⅰ

Cold shock promoter N-terminal 6-His tag Selection : Ampicillin

Chaperone plasmid map

CalB Expression in E.coli Results

(1) Confirmation of lipase activity

(2) Halo test

(3) SDS-PAGE Co-expression of Cal B and Chaperone

DH5α Rosettagami Novablue

DH5α Rosettagami Novablue

CalB Expression in E.coli E. coli 를 이용하여 Candida antarctica 에서 유래된 lipase B (Cal B) 의

발현여부를 빠르게 확인하고 , 특성을 분석하여 효율적인 발현시스템을구축하며 , 대량발현 시스템에 적용이 가능한지를 확인하였다 .

발현 효율이 높고 활성이 좋은 재조합 lipase 개량에 있어서 빠른 확인을 할 수 있는 시스템을 확보하였다 .

E. coli 에서 lipase 를 효율적으로 발현하기 위해서는 숙주세포 DH5α 에Cal B 유전자가 재조합된 pCold Ι vector 와 pGro7(groES/groEL) 을 공동

형질전환 시킨 재조합 균주가 Cal B 발현시스템에 가장 효율이 좋았다 .

CalB Expression in Pichia pastoris

Materials Cell: Pichia pastoris X-33 Vector: pPICZα B

α-peptide: protein secretion AOX1 promoter: induction by

methanol Selection: Zeocin

Clone gene of interest into the pPICαA

Linearize construct with Sac I

Transform X33 using EasyCompTM method

Plate transformants on medium containing Zeocin

All transformants integrate at 5’ AOX1 locus by single crossover

Select 6-10 colonies for small-scale expression

Choose highest expressers for large-scale expression

Object : 생촉매 대량생산

CalB Expression in Pichia pastoris Results

(1) Confirmation of lipase activity

(2) Halo test Halo formation around colony by oil

degradation due to lipase secretion

Normal colony

Halo formation by methanol induction

(3) SDS-PAGE Confirmation of CalB expression and secretion

Non-concentrated media Concentrated media

CalB Expression in Pichia pastoris (4) Cal B Zymogram Confirmation of CalB expression and secretion

(5) Activity test Measurement of Cal B activity using p-NPP

Time (days) OD400 Unit/ml

3 0.057 62.71

4 0.066 72.44

5 0.077 84.34

6 0.092 100.55

7 0.104 113.53

8 0.116 126.50

9 0.129 140.56

10 0.117 127.58

Summary The pellet sample's band was much clearer than that of the supernatant

sample on the SDS-PAGE gel when the CalB expression was checked in the Pichia pastoris X-33 cell culture that was electroporated with the pPICZαB vector recombining a CalB gene.

From the thickness of the bands the expression level can be confirmed. As the culture time increased, CalB secreted to the media accumulated thereby thickening the bands.

When the activity of the enzyme was measured using p-NPP, the maximum value of 140,560U/L was reached on the day 9 of the culture. During the cell culture the cell growth reached 57.54~71.64g/L when BMGY→BMMY media was used.

Biocatalyst immobilization methods

Immobilization methods

Covalent binding

on beads

Covalent binding

on mesoporous silicaSol-gel entrapment

Advantages• Easiness of

substrate binding on biocatalyst

• Protection of biocatalyst from

solvents

• Large surface area to maximize enzyme

loading amount

• Protection of biocatalyst from

solvents

• Maintenance of enzyme native

structure

• Simple process

• Immobilization under mild condition

Disadvantages• Deactivation by

solvent attack• Diffusion limitation • Diffusion limitation

SNU : Sol-gel 방법을 사용

Sol-gel entrapment

Use of hydrophobic precursors Known as CalB maintains higher activity in hydrophobic circumstance Easiness of oil feeding to enzyme Prevention of enzyme deactivation by hydrophilic methanol

ETMS(Ethyltrimethoxysilane)

MTMS(Methyltrimethoxysilane)

iso-BTMS(iso-Buthyltrimethoxysilane)

PTMS(Propyltrimethoxysilane)

TMOS(Tetramethoxysilane)

Enzyme

지금까지의 방법 : Hydrophobic precursor 사용

Selection of sol-gel matrix

Comparison of the activity in various matrix TMOS (hydrophilic) MTMS, ETMS, PTMS, iso-BTMS (hydrophobic)

matrix TMOS MTMS* ETMS* PTMS*iso-

BTMS*

Activity (U/g support) 0.144 0.482 0.579 0.267 0.460Specific activity (U/g

enzyme) 9.71 13.4 21.03 7.54 5.13

* Mixed with TMOS (TMOS: hydrophobic precursor= 1:4)

ETMS/TMOS

Lipase immobilized in ETMS mixed with TMOS showed the highest activity and specific activity.

IDEA : Hydrophobic-hydrophilic 한 precursor 의 비율을 조정 및 최적화

Pretreatment of biocatalyst

Activity maintenance and acquisition of stability of CalB Methods

Olive oil pretreatment for 1hr at 30 ℃ steric hindrance around active site during immobilization

GA pretreatment for 1hr at 25 ℃

Result : Development of new biocatalyst having the same activity compared with Novozym435

IDEA : Oil 전처리 방법 도입

Matrix UnpretreatedGA

pretreated Oil

pretreatedOil and GApretreated

Novozym 435

Activity

(U/g support)0.579 0.498 0.896 0.595 0.916

Specific activity

(U/g enzyme)21.03 17.65 47.23 33.81 -

Activity of Immobilized lipase

Comparison of the activity Lipase type

Candida antarctica lipase B Rhizomucor miehei lipase Thermomyces lanuginosus lipase

Particle size 100-300 μm and 300-500 μm

Sol-gel 제조방법 개선 및 particle 사이즈 변경 & 다른 lipase 도 고정화

Stability of immobilized enzymes

Time (hr)

0 5 10 15 20 25 30

Res

idu

al a

ctiv

ity

(%)

0

20

40

60

80

100

120

140Free CALBNo pretreated lipasePretreated lipase by oilPretreated lipase by oil & GA

Time (hr)

0 5 10 15 20 25

Bio

dies

el c

onve

rsio

n (%

)

0

20

40

60

80

100Free lipaseNo pretreated lipasePretreated lipase by oil Pretreated lipase by oil & GA

Thermostability (60℃) Methanol tolerance

(Oil : MeOH = 1:3)

Acquisition of thermostability and methanol tolerance via immobilization of pretreated CALB

FAME production by immobilized lipases

FAME conversion using free lipase and immobilized lipase Meterials

Free lipase : Lipozyme (CalB) Sol-gel entrapped CalB using ETMS precursor

Time (hr)

0 5 10 15 20 25

Bio

dies

el c

onve

rsio

n (

%)

0

20

40

60

80

100Free lipaseNo pretreated lipasePretreated lipase by oilPretreated lipase by oil & GA

Results Over 90% of conversion

rate in oil pretreated biocatalyst

Methods

Pre-incubation of oil with

methanol for 18 hrs, 300

rpm, at 50℃

immobilized lipase: 25%

(w/w) of substrate Methanol stepwise

addition

CalB immobilization

• CalB immobilization onto nanocarbon materials• High loading of enzyme• Enhancement of CalB stability in organic solvent• Support materials: functionalized nanocarbon material

EDC

CalB

Result_ Immobilization of CalB• 1ml of Lipozyme CALB L were immobilized with 10mg of

nanoparticle. • Single layer immobilization was formed around 6mg enzyme/mg

support.• Maximum ammount : 17.4 mg enzyme/ mg support• Unusually higher amount repeat experiment again.

Methanol inhibition

• Methanol has a strong inhibitory effect on the lipase.– conversion decreased when methanol and oil molar ratio is more then 1.5.

• To prevent the methanol inhibition, step addition was suggested.

FIG 1. Effect of methanol content on methanolysis of vegetable oil. FIG. 2. Time courses of two- and three-step methanolyses of vegetable oil. Two-step reaction (○), Three-step reaction (●)

Y. Shimada et al., JAOCS, Vol. 77, no. 7 (1999), Y. Watanabe et al., JAOCS, Vol. 77, no. 4 (2000)

A deposit of glycerol coating the immobilized catalyst is formed during the process, which reduces the enzymes activity .

• Glycerol removal by membrane was proposed.• The two phase is separated by cellulose acetate membrane.

• Primary phase: oil-biodiesel-glycerol mixture

• Secondary phase: aqueous phase

Hydrophobic molecules(FAME and oil) can not pass this membrane.

Only hydrophilic ones, glycerol can pass this membrane.

The continuous inhibition of glycerol can be decreased.

Glycerol inhibition

K.Belafi-Bako et al., Biocatalysis and Biotransformation, 2002 Vol. 20 (6), pp. 437–439

• The packed bed reactor has been investigated by several researchers for biodiesel production.

– But step addition reactions were needed to prevent methanol inhibition.– And it needs down streams for glycerol separation.

• To overcome these disadvantages, flat sheet membrane reactor which was used for glyceride synthesis can be applicable for biodiesel production.

• Using hydrophilic membrane, the inhibition of continuously produced glycerol can be decreased and methanol can be fed continuously to improve the productivity during short operation time.

Research objective

MeOH & Glycerol

MeOH

Oil

FAME

Continuous system

Magnetic stirrer

Product (FAME)

MeOH &Glycerol

Water Bath

Water Bath

Immobilized enzyme

Membrane

OilMeOH

Experiment without enzyme

MeOH

MeOH

Oil

Oil&MeOH

Result_ Methanol permeability

Methanol permeation was tested without enzyme.

• Without stirring, 130 mM of methanol was maintained after 2hrs.• With stirring;

• Methanol concentration was increased steadily.• Maximum concentration of methanol was 400mM.

Experiment with Novozyme 435

MeOH & Glycerol

MeOH

Oil

FAME

Result _Biodiesel production

• Biodiesel production was tested with continuous system.• 25% conversion was achieved at 30 min.• After 1 hr conversion rate was dramatically decreased and this low conversion

rate (below 6%) was maintained during 10hr.• This low conversion rate was based on enzyme deactivation by the methanol

inhibition which is caused by high methanol concentration and mixing problem.

9.2 Bioethanol Production

9.3 Enzymatic Denitrification

Biological Denitrification

Economically and environmental friendly

Microorganisms use nitrate as a final electron acceptor

Occur under O2 limitation condition

General process

Oxic Anoxic SedimentInfluent

Return sludge Waste sludge

Effluent

Enzyme Reactions

NO3- NO2

- NO N2O

N2

NADH NAD+ + H+

NADHdehydrogenase

NaR NiR NoR No2R

e-

NaR: nitrate reductase

NiR: nitrite reductase

NoR: nitric oxide reductase

No2R: nitrous oxide reductase

Screening of Denitrifying Bacteria

MicroorganismsDenitrification activity

(nmol/min mg-cell)References

Pseudomonas stutzeri 90.2Carlson and

Igrshsm (1983)

Pseudomonas aeruginosa 45.2Carlson and

Igrshsm (1983)

Paracoccus denitrificans 34.2Carlson and

Igrshsm (1983)

Flexibacter canadensis 23.4Wu and Knowles

(1994)

Pseudomonas stutzeri 67.2Nussinovitch et al.

(1996)

Shewanella putrefaciens 41.0Krause et al.

(1997)

Ochrobactrum anthropi SY509 104 This study

Packed-bed reactor

④

⑥

⑤

③

②①

③

④

⑦

⑧

① Packed-bed reactor (100 ml)

② ORP/pH meter ORP probe pH probe③ ④ ⑤ monitor feeding water pump ⑥ ⑦ ⑧ nitrogen gas purging

Time (hour)

0 3 6 9 12 15 18 21 24 27

Con

cent

ratio

n (N

O3- -N

mg/

l)

0

20

40

60

80

100

120

212 mV120 mV80 mV

- Flow rate : 5ml/min

effluent

influent

Bioelectrochemical Denitrification

electrod

e

e- Mox

Mred

NO3-

NO2-

cellcell

N2…

Cell Permeabilization

Control

Permeabilized cell

chloroform (%v/v)

0 1 2 3 4

Cel

l via

bilit

y (

mol

e/m

in/m

g-ce

ll)

0.0

0.2

0.4

0.6

0.8

1.0

Act

ivity

of n

itrat

e re

duct

ase

(uni

t/mg-

cell)

0.00

0.25

0.30

0.35

0.40

0.45

0.50

Immobilization of Biocatalyst and Mediator on Electrode Surface

Carbon nanopowder-Neutral red

Polymer matrix (Ca-alginate)

Permeabilized cell

Reactor System for Bioelectrochemical Denitrification

Work

ing

ele

ctrode

Refe

rence

ele

ctrode

v

Counte

r

ele

ctrode

Power supply

S

P 2H2O

O2+4H+

+4e-

Time(min)

0 20 40 60 80 100 120 140 160 180

NO

3-N

(ppm

)

0

20

40

60

80

100

120

140

C-NR + 5g/l cellC-NR + 10g/l cellC-sp-NR + 5g/l cellC-sp-NR + 10g/l cellC-sp-NR + 15g/l cell

Denitrification Efficiency

Time(min)

0 20 40 60 80 100 120 140 160 180

NO

3-N

(ppm

)

0

20

40

60

80

100

120

140

1mM C-sp-NR + 5g/l cell2mM C-sp-NR + 5g/l cell5mM C-sp-NR + 5g/l cell

Figure. The residual nitrate level (a)for various cell concentrations and (b)for various concentrations of mediator

(a) (b)

The cell concentration is rate-limiting compared to mediator concentration.

Conductive powder was mixed with the cells and alginate for electrode.

Novel Biocatalytic Electrode

permeabilized cell

alginate

conductive powder

Figure. SEM image (x3500) of novel electrode

Denitrification Efficiency

Time (min)

0 20 40 60 80 100 120 140 160 180

NO

3-N

(p

pm)

0

20

40

60

80

100

120

140

10g/l permeabilized cell with immobilized neutral red(2mM)10g/l permeabilized cell with copper powder(1g)

Continuous Reactor

influent

effluent

anode: carbon felt

cathode combined with biocatalyst

Time / h

0 5 10 15 20 25 30

NO

3-N

/ p

pm

0

20

40

60

80

100

120

140

160

1st generation: mixed population

oxic, anoix tanks

2nd generation: single microorganism (enzymes)

immobilized packed-bed reactor

3rd generation: whole cell biocatalyst

enzymatic-electrochemical reactor

Biological Nitrate Removal Technology

Biodiesel Fuel Production by Transesterification of Oils

Fukuda H, Kondo A, and Noda H, Kobe Univ., Japan

Enzyme Engineering

Minsuk Kim2012.06.04.

Review

Triglycerides!!

•Hold promise as alternative diesel engine fuel

•Vegetable oils / Oil blends / Oil waste▫Too viscous

Transform triglycerides to lower viscosity

Pyrolysis – unacceptable in terms of ash, pour point etc.

Micro-emulsification - incomplete combustion etc.

Transesterification (alcoholysis)

Supercritical fluid

Chemicalcatalysis

Enzyme

Acid-catalysisAlkali-catalysis

ExtracellularIntracellular

Biodiesel fuel

•Product from alcoholysis of triglycerides.

•Attractive features▫Plant-derived, carbon neutral ▫Lower dependence on petroleum▫Biodegradable▫Reduced levels of pollutants (CO, NO, SOx)


Supercritical fluid

Chemicalcatalysis

Enzyme



Alkali-catalysis

High conversion Short reaction time

cheapness

Water & free fatty acid


Supercritical fluid

Chemicalcatalysis

Enzyme



Alkali-catalysis vs. Acid-catalysis

• Lower conversion and rate• Low grade material such as sulphur

oil


Supercritical fluid

Chemicalcatalysis

Enzyme



Alkali-catalysis vs. Enzymatic process

+ waste water treatment

• Methanol & ethanol are utilized frequently.▫ Low cost and physical and chemical advantages.

• Lipases are known to have a propensity to act on long-chain fatty alcohols.▫Conversion : Methanol < Ethanol

• Methanol can inactivate enzyme.▫ Stepwise addition of methanol.▫ Methanol should dissolve completely.


Supercritical fluid

Chemicalcatalysis

Enzyme



Extracellular vs. Intracellular

• Purification of enzyme is too expensive

• Not easy to recover enzyme

Porous biomass support particles (BSPs)

•No chemical additives•No need to

preproduction of cells•No aseptic handling of

particle•Large mass transfer

rate•Easy scale-up•Low cost

Cell : Ryzopus oryzaeLipase induction by olive oil or oleic acid

Stabilization


Supercritical fluid

Chemicalcatalysis

Enzyme



Alkali-catalysis vs. Supercritical fluid

• Without any catalyst▫ Supercritical methanol - 350℃ & 240s & 45MPa

• Supercritical methanol is hydrophobic▫ Fast reaction rate

• Much simpler purification & environmental friendly

Conclusions and Future Prospects

•Alkali-catalysis is generally employed in actual biodiesel production.

•However, enzymatic transesterification is also attractive.

•Further improvement by genetic engineering.▫High level of expression of lipase.▫Stabilize toward methanol.

Comments

•Why fatty acid methyl ester? i.e. Why not fatty acid ethyl ester?▫Lipases are unstable in methanol▫Methanol is toxic to cells▫Many lipases has higher activity toward ethanol

than methanol

•Vegetable oils biodiesel fuel vs. Cellulosic biomass biodiesel fuel▫By metabolic engineering.▫Which source has bigger potential?

The Challenge of Enzyme Cost in the The Challenge of Enzyme Cost in the Production of Lignocellulosic Biofuels Production of Lignocellulosic Biofuels

2012.06.11 Enzyme engineering (2012, Spring semester)

Jeong Eun Shin

IntroductionIntroduction

Understanding the contribution of enzymes to the cost of lignocellulosic biofuels.

Ethanol Fungal cellulases

Some authors… The cost of enzyme is a major barrier for

biofuel productionOthers… The cost is relatively low or can be decreased

with technological innovation or other advances

1) Techno-economic model 2) Sensitivity analysis

Techno-economic modelTechno-economic model The cost of enzymes [dollars per gallon of biofuel]: a “top-down” measure that depends on many

factors besides the cost of enzymes themselves, including the choice of feedstock, the enzyme loading, and the overall biofuel yield The inconsistency in the cost of enzymes !!!

• The development of a techno-economic model for the production of cellulases

The determination of “bottom-up” enzyme costs based on process characteristics

The refinement of costs as better information

• Hard to perform robust techno-economic analysis of biofuel production processes

• Adding uncertainty in decision – making at many levels(From researchers & policymakers to investors & industry players)

Techno-economic modelTechno-economic model JBEI’s techno-economic analysis

wiki site(http://econ.jbei.org) The process model includes all unit

operations required for the production of cellulases from stream-exploded poplar by Trichoderma reesei

Feed stock transport, steam explosion, fungal growth, cellulase production and down stream processing (biomass filtration and incineration)

Using model for Corn stover ethanol production with dilute acid pretreatment (Klein-Marcuschamber et al., 2010) to contextulaize the costs of enzymes based on typical ethanol yields.

Techno-economic modelTechno-economic model The costs of enzymes on a $/gal basis depend on the yield of

ethanol.(1)The theoretical maximum yield based on conversion of all C5 and

C6 sugars present in corn stover 111.4 gal/DT(dry tonne)

(2)The yield based on conversion of C6 sugars at a 95% efficiency, but not C5 sugars 67.13 gal/DT

(3)The yield based on conversion of all C5 and C6 sugars after a saccharification cellulose conversion of 70% 88.9gal/DT

(4)The yield based on conversion of C5 and C6 sugars expected from a typical saccharification and a typical fermentation using engineered yeast 51.6 gal/DT

Comparing (1)&(3) to (2)&(4) , the main technological bottleneck for improving the overall yield is associated with fermentation

Efficient fermentation does not impact the cost of enzyme production

Saccharification efficiency is very high, but fermentation efficiency is remained.

Saccharification efficiency does not improved, but fermentation efficiency is high. Merely shift the burden for improvement to fermentation technologies

Unattainable scenario

Techno-economic modelTechno-economic model The cost of one of the cheapest proteins available

today : Soy protein (~$1.25/kg of protein , ~$500/MT) If cellulases could be produced at this cost,

One can analyze the cost of production of biotechnologically derived enzymes using the process model presented here.

scenario Cost contribution

1 $0.08/gal

2 $0.14/gal

3 $0.11/gal

4 $0.18/gal

The cost contribution in the literatureCellulases is cheap as soy protein

Ethanol yield is close to the theoretical maximum

Unrealistic!!!

scenario Cost contribution

1 $0.68/gal

2 $1.13/gal

3 $0.85/gal

4 $1.47/gal

The baseline production cost of enzyme was found to be $10.14/kg.

Techno-economic modelTechno-economic model It is not possible to produce enzymes cheaper than

plant-derived proteins. It is quite expensive simply to purchase the

equipment for the production facility.

Enzyme

Sensitivity analysisSensitivity analysis Sensitivity analysis for exploring the effect of

parameter choices on the cost of enzymes1) The fermentation residence time - Using carbon source simpler than poplar, such as

amorphous cellulose - Using Trichoderma engineered for faster growth rates Shorter fermentation residence time

2) The costs of raw materials- The chosen baseline cost for poplar ($60/MT) is low

compared to projections of the cost of biomass(~$140/MT)

- The choices of carbon sources and inducers used commercially are not publicly discussed.

The costs of raw materials are uncertain.

Sensitivity analysisSensitivity analysis

The sensitivity of the contribution of enzyme costs to poplar price and fermentation residence time.

The change of poplar feedstock prices does not help. Decreasing the growth and fermentation time would help through lowering the capital cost associated with fermentation.

Sensitivity analysisSensitivity analysis

Lowering enzyme loadings required for saccharification would be an obvious target for improvement.Including more stable cellulasesBetter pretreatment technologies that enable high saccharification yields at lower enzyme loadingsReducing the presence of phenolic compounds, which inhibit and deactivate cellulases

10FPU/g cellulases 5FPU/g cellulases(1) $0.68/gal (2) $1.13/gal (3) $0.85/gal (4) $1.47/gal(1) $0.34/gal (2) $0.55/gal (3) $0.42/gal (4) $0.73/gal

Improving the economics of enzyme production is a

significant hurdle!!!

ConclusionConclusion This research constructed techno-economic model for the

production of fungal cellulases. The cost of producing enzymes was much higher than that

commonly assumed in the literature. This model would be helpful to decrease the enzyme production

cost

The sensitivity analysis was performed to study the effect of feedstock prices and fermentation times on the cost contribution of enzymes to ethanol price.

The contribution can be lowered by shifting to lower cost feedstocks, reducing the fermentation times, and reducing the complexity of the process to drive down capital costs.

Achieving high overall biofuel yields at low enzyme loadings is much more promising by improved pretreatment and enzyme technologies, which is associated with biorefinery capital costs.

A significant effort is still required to lower the contribution of enzymes to biofuel production costs.

Documents

Enzyme Engineering 9. Applications(3): Energy & Environment 9.1 Biodiesel Production 9.2 Bioethanol Production 9.3 Enzymatic Denitrification