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)
Extracellular lipase
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)
Extracellular lipase
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
Intracellular lipase
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
Intracellular lipase
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.
H. Fukuda et al., Trends in Biotechnology Vol.26 No.12, 2008
Intracellular lipase
• 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.
H. Fukuda et al., Trends in Biotechnology Vol.26 No.12, 2008
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)
Transesterification (alcoholysis)
Supercritical fluid
Chemicalcatalysis
Enzyme
Acid-catalysisAlkali-catalysis
ExtracellularIntracellular
Alkali-catalysis
High conversion Short reaction time
cheapness
Water & free fatty acid
Transesterification (alcoholysis)
Supercritical fluid
Chemicalcatalysis
Enzyme
Acid-catalysisAlkali-catalysis
ExtracellularIntracellular
Alkali-catalysis vs. Acid-catalysis
• Lower conversion and rate• Low grade material such as sulphur
oil
Transesterification (alcoholysis)
Supercritical fluid
Chemicalcatalysis
Enzyme
Acid-catalysisAlkali-catalysis
ExtracellularIntracellular
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.
Transesterification (alcoholysis)
Supercritical fluid
Chemicalcatalysis
Enzyme
Acid-catalysisAlkali-catalysis
ExtracellularIntracellular
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
Transesterification (alcoholysis)
Supercritical fluid
Chemicalcatalysis
Enzyme
Acid-catalysisAlkali-catalysis
ExtracellularIntracellular
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.