Modeling and Analysis

Integrated enzyme production lowers

the cost of cellulosic ethanol
Eric Johnson, Atlantic Consulting, Gattikon, Switzerland

Received September 8, 2015; revised November 20, 2015; and accepted December 15, 2015
View online February 18, 2016 at Wiley Online Library (wileyonlinelibrary.com);
DOI: 10.1002/bbb.1634; Biofuels. Bioprod. Bioref 10:164–174 (2016)

Abstract: Previous studies of cellulosic-ethanol production have shown that the cost of producing
cellulase is surprisingly significant, and that reducing this cost is key to making cellulosic-ethanol
economically viable. This study confirms that finding, and compares the costs of the three approaches
for producing cellulase: off-site, on-site, and integrated. It finds that the integrated method is the
lowest cost, primarily because it substitutes an inexpensive feedstock, biomass, for a relatively expen-
sive one, glucose. This substitution also makes the ethanol a 100% second-generation biofuel, i.e., it
uses no ‘food for fuel’. This study also compares the activity of cellulase produced by the integrated
method versus that produced by the off-site method. Laboratory trials of the two show the ‘integrated’
cellulase to be better or equal to commercially available ‘off-site’ cellulase in converting cellulose
to sugar. © 2016 The Authors. Biofuels, Bioproducts, Biorefining published by Society of Chemical
Industry and John Wiley & Sons, Ltd.

Keywords: cellulosic ethanol; cellulase; bioethanol; cost; integrated cellulase; food for fuel

Introduction Cellulase is produced in an industrial fermentation

process. Fungi feed on a stream of incoming nutrients,
ellulose and hemi-cellulose are the most abundant converting them to outgoing cellulase. This production

C types of biomass on Earth. As interest in renew-

able energy has surged over the past decade, so
too has interest in developing a process for converting
process is typically characterized by two main variables:

• Location: Traditional cellulase production is off-site to

this biomass to ethanol at a cost that could compete with
that of conventional gasoline (for which the ethanol can
substitute).
One of the key challenges to cost-competitiveness,1–3 is
the cost of the enzyme, the cellulase that converts poly-
meric cellulose into single molecules of sugar which are
then fermented into ethanol. This challenge has been
broadly recognized only recently,4 and to most observers
it came as a surprise, because most prior studies ‘signifi-
cantly underestimated the contribution of enzyme costs to
biofuel production’. Therefore, ‘a significant effort is still
required to lower the contribution of enzymes to biofuel
production costs’.4
the ethanol plant, i.e., at a central factory that supplies
numerous cellulosic ethanol operations. On-site pro-
duction of cellulase, adjacent to the ethanol plant, has
more recently been presented as a potentially lower-
cost option, by researchers.4–9
• Feedstock: Both off-site and on-site cellulase produc-
tion use glucose as the primary feedstock. However,
in an on-site configuration, it is possible to substitute
glucose with much-cheaper cellulosic biomass – the
same material that at the same site is being converted
to ethanol. By using the same feedstock and location,
it is possible to integrate cellulase and ethanol produc-
tion – so this approach is called ‘integrated’.

Correspondence to: Eric Johnson, Atlantic Consulting Obstgartenstrasse 14 CH-8136 Gattikon, Switzerland.
E-mail: ejohnson@ecosite.co.uk

© 2016 The Authors. Biofuels, Bioproducts, Biorefining published by Society of Chemical Industry and John Wiley & Sons, Ltd.
164 This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution
and reproduction in any medium, provided the original work is properly cited.
Modeling and Analysis: Integrated enzyme production lowers the cost of cellulosic ethanol
This paper examines these three approaches to cellulase
production: off-site, on-site, and integrated. First the
approaches are described, then the mass balances are
reviewed, and from that the costs are estimated and
compared. In a final section, the paper examines another
question regarding the cost of cellulase production: the
activity of the cellulase, i.e., its ability to convert cellulose
and hemi-cellulose from polymers into monomolecular
sugars. Based on laboratory experiments, this paper
compares the activity of cellulases that are produced from
glucose and from biomass.
Process descriptions of cellulase
production approaches
Based on a literature review and discussions with
researchers and operators, descriptions have been com-
piled for the three approaches to cellulase production: off-
site, on-site, and integrated (Fig. 1).
In all cases, the basic process is as follows. Feedstocks are
fed to micro-organisms that produce an enzyme complex,
cellulase. The cellulase is used to catalyze the breakdown
of cellulose and hemi-cellulose into monosaccharides,
which are then fermented into ethanol.
Off-site (glucose-fed)
In this approach, cellulase is produced off-site, at a central
plant, from which it is shipped out to ethanol plants. The
primary feedstock is glucose. Secondary feedstocks are a
cocktail containing mainly nitrogen, phosphate and potas-
sium compounds. Corn steep liquor can also be added as a
source of protein.8
Figure 1. The three approaches to cellulase production.
© 2016 The Authors. Biofuels, Bioproducts, Biorefining published by Society of Chemical Industry and John Wiley & Sons, Ltd.
| Biofuels, Bioprod. Bioref. 10:164–174 (2016); DOI: 10.1002/bbb
E Johnson
The protein coming out of the fermenter contains a mix-
ture of cellulase and other proteins. This is then formu-
lated, i.e., purified to cellulase. Formulation is required in
order to stabilize the cellulase for shipment, i.e., to keep it
viable and active during transport. Post-shipment, the for-
mulated cellulase is then fed to the ethanol plant.
On-site (glucose-fed)
This approach is essentially the same as that of off-site pro-
duction, except that the cellulase plant is located on-site,
adjacent to the ethanol plant. Both plants are stand-alone.
Being ‘next door’ eliminates the need for formulation; the
protein mixture is fed directly to the ethanol plant with-
out purification and stabilization. The scale of cellulase
production is larger in an off-site than in an on-site plant,
which is sized to accommodate one ethanol plant rather
than many.
Integrated (cellulose-fed)
This approach is on-site, but not stand-alone. Instead, the
cellulase production is:
• Integrated, by both process and energy flows, directly
into the ethanol plant.
• Fed by cellulose, not glucose. The same, pre-treated cel-
lulosic feedstock is used for both cellulase and ethanol.
A fraction of the cellulose going into the ethanol plant
is diverted to cellulase production; the cellulose substi-
tutes glucose as the primary feedstock for cellulase.
The volume of cellulase production is similar to that of
an on-site, glucose-fed plant, and is sized to fit the produc-
tion capacity of ethanol. Yields are assumed to be similar,
because of the ability of process designers and operators
to optimize the interaction of microorganism, substrate
and product. This can be done with native or recombinant
strains,10 and is a focus of ongoing research.11–14
With integrated enzyme production, it is possible to
use complex substrates such as straw to produce enzymes
with high activity, compared to those grown on glucose
or sucrose. Enzyme production from a complex substrate
is comparable to that of the simple substrate like sugars, if
the process is engineered specifically for the chosen micro-
organism. Delabona et al.15 have shown that with selected
micro-organisms, steam pre-treated bagasse provides
higher FPase, xylanase, β-glucosidase than do sucrose,
glycerol and lactose. Pullan et al.16 have shown that the
inducing substrate used for the production of enzymes has
marked effects on the enzyme cocktail achieved. These
findings highlight the limitations of using simple inducing
165
E Johnson
substrates to generate complex mixtures of enzymes.
Using simple substrates with de-repressed fungal strains
denies the benefits of fungi’s enzymatic capability for com-
plex substrates.
Neither human nor animal food is used as a feedstock,
making this process the only 100% second-generation pro-
cess for bioethanol.
Mass balances of cellulase and
cellulosic ethanol production
Published cost estimates of cellulase production are almost
always expressed with respect to ethanol output, i.e.,
US$ per unit of ethanol. Moreover, feedstock inputs and
product yields are critical factors in determining cellulase
costs. So to analyze the costs transparently, it is necessary
to clarify the mass balances involved. Of these, there are
three primary ones:
• Biomass:ethanol – the amount of biomass feedstock
coming into the ethanol plant relative to the amount of
ethanol going out.
Figure 2. Simplified flow sheet of the entire process (adapted from Humbird et al.6)
© 2016 The Authors. Biofuels, Bioproducts, Biorefining published by Society of Chemical Industry and John Wiley & Sons, Ltd.
166 | Biofuels, Bioprod. Bioref. 10:164–174 (2016); DOI: 10.1002/bbb
Modeling and Analysis: Integrated enzyme production lowers the cost of cellulosic ethanol
• Feedstocks:cellulase – the amount of sugar or biomass
into the enzyme plant relative to the cellulase product.
• Cellulase:biomass – the ‘enzyme loading’ of the ethanol
plant, the amount of cellulase charged to the ethanol
plant, relative to the amount of biomass feedstock
charged to it.
Biomass:ethanol
Biomass in, ethanol out – this is the basic transforma-
tion behind cellulosic ethanol. A schematic is presented
in Fig. 1, and a more-detailed flowsheet6 of the on-site
process with ethanol production is also presented (Fig. 2).
Based on an examination of the literature, particularly
6,8,17–19
and some discussions with operators, we derived
a base-case mass balance of 1000 kg of dry biomass to
225 kg of pure ethanol (Table 1). Th is yield of 22.5% is
representative of the reported range, lower than some of
the studies,6,8 but higher than others20 as well as industry
estimates.18
In the literature, two of these bases are somewhat
unclear:
Modeling and Analysis: Integrated enzyme production lowers the cost of cellulosic ethanol E Johnson

Table 1. Cellulose to ethanol, mass balance, base Table 2. Feedstocks to cellulase, mass balance,
case. base case (weight).
Line item Amount Unit Comment Cellulase production approach
Biomass, dry 1000 kg Straw Off-site On-site Integrated
Biomass, dry, 65% weight (21) adjusted Input
carbohydrate content down slightly
Biomass, dry 1000
Carbohydrate, dry 650 kg
Carbohydrate content in 650
Sugar/carbohydrate yield 80% weight biomass
Sugar to fermentation, of 520 kg Glucose/Dextrose 703.2 703.2
which §
Glycerol/sucrose 220.1
Glucose 60% weight
Ammonium sulphate 12.63 12.63 11.67
Xylose & Arabinose 40% weight
Monopotassium phosphate 18.05 18.05 16.68
EtOH/sugar yield, 51% weight
Magnesium sulphate 2.71 2.71 2.50
theoretical
Calcium chloride 3.61 3.61 3.34
EtOH/sugar yield, % of 85% weight
theoretical Sodium chloride 27.5 27.5 25.42

EtOH, mass 225 kg Ammonia 32.83 32.83 30.35

EtOH density 0.789 kg/litre Antifoam 54.27 54.27 50.16

EtOH, volume 286 litres SO2 3.73 3.73 3.45

EtOH/Biomass, dry: 23% weight Corn steep liquor 99.62 99.62 92.08
calculated Output
Cellulase, protein 110 128.6 118.871
Yield
• Dry or wet biomass – Most papers refer to biomass
input on a dry basis, but some are not explicit about it. Protein/glucose or 15.6% 18.3% 18.3%
Carbohydrate
If biomass is measured as wet, usually the assumption
appears to be that moisture is 20% of the incoming Protein/biomass 11.9%
§
mass. As a stabilizer in the formulation.
• Biomass in its entirety or only as its carbohydrate con-
tent – Some papers are unclear as to their basis. We
have assumed a carbohydrate content of 65%. An often- yield from glucose or carbohydrate, because the biomass
cited source21 suggests 67%, but review and discussions itself is only 65% carbohydrate.
with producers suggest that actual carbohydrate con- Other authors,20,24,25 support this general picture, but
tent tends to be lower than this, so we adjusted it down they do not provide quantitative mass balances. Both
slightly. Hong et al.8 and Nielsen et al.25 report the use of stabilizer
in off-site production. The former quotes the stabilizer as
Feedstocks:cellulase glycerol, the latter as sucrose.
The most detailed mass balance for this in the literature
is presented by Hong et al. (Supporting Information,
Cellulase:biomass
Table S3), which was derived in part Humbird et al.6 Some This mass balance is often referred to as ‘enzyme loading’,
detail is also presented by Dunn et al.22 i.e., the amount of enzyme that needs to be charged to
Protein yields on glucose or carbohydrate are higher for the ethanol plant, relative to the amount of biomass being
the on-site and integrated approaches than for the offsite charged to the same plant. Based on Hong et al.,8 MacLean
approach (Table 2), because there are ‘protein losses dur- and Spatari,20 and Dunn et al.,22 we specified a base case
ing the purification process, which is only required for off- loading of 1% weight cellulase to substrate, i.e., 10 g enzyme
site production, whereas on-site production leads to dosing protein to 1 kg of biomass input to the ethanol plant.
a whole fermentation broth. Thus, more protein/cellulase In the literature, there are two aspects of this mass
is available from an on-site [or integrated] production balance that can lead to miscalculations or inaccurate
strategy.’23 The protein/biomass yield is lower than the comparisons:
© 2016 The Authors. Biofuels, Bioproducts, Biorefining published by Society of Chemical Industry and John Wiley & Sons, Ltd.
| Biofuels, Bioprod. Bioref. 10:164–174 (2016); DOI: 10.1002/bbb 167
E Johnson Modeling and Analysis: Integrated enzyme production lowers the cost of cellulosic ethanol

Table 3. Existing, commercial-scale cellulosic-ethanol plants.**

Annual capacity
Operator Location Volume Weight
††
Abengoa Hugoton, Kansas, USA 25 million gallons 74,677 tonnes
DuPont Nevada, Iowa, USA 30 million gallons 89,601 tonnes
POET-DSM Emmetsburg, Iowa, USA 25 million gallons 74,677 tonnes
Ineos Vero Beach, Florida, UA 8 million gallons 23,894 tonnes
GranBio Alagoas, Brazil 21 million gallons 62,721 tonnes
Mossi & Ghisolfi Crescentino, Italy 75 million litres 59,175 tonnes
**Sourced from company and press reports.
††
106

• Biomass in its entirety, or only as its carbohydrate con- Table 4. Overall mass balance – biomass
tent – This is the same issue mentioned above. Some and cellulase to ethanol, base case, tonnes
papers express enzyme loading per unit of substrate per annum.
(the entire dry biomass), while others express it per Cellulase production approach
unit of carbohydrate in the biomass. Off-site On-site Integrated
• Units of enzyme – Most studies refer to mass of ‘pro-
Input
tein’, which, as Humbird et al. 6 point out, ‘refers to the
Biomass, dry, for EtOH 299,042‡‡ 310,531 310,531
total amount of high molecular weight protein in the
Biomass, dry, for cellulase 26,123
enzyme broth as determined by assay; not all of this
protein is active cellulase’. This can be complicated, Glucose/dextrose,
for cellulase 19,117 16,980
in that different assay methods give different protein
§§
weights from the same sample. Some studies refer to Glycerol/sucrose , for EtOH 5,984

fi lter paper units (FPUs) as a measure of enzyme load- Cellulase, protein 2,990 3,105 3,105
ing. When off-site cellulase is used, it is not always Output
clear if the enzyme loading refers to the protein alone Ethanol 70,000 70,000 70,000
or to the entire cocktail of protein plus stabilizer. ‡‡
Adjusted for the glycerol/sucrose feedstock.
§§
Added as stabilizer to the cellulase
Overall, annualized mass balance
(base case) Cost estimates
With the exception of a plant operated by Ineos, commer-
cial-scale cellulosic ethanol plants range around the size of Costs of the three production methods have been
70 000-tonne per year capacity (Table 3). So, this was cho- estimated. The bases of this are presented in this sec-
sen as the size for the base-case mass balance.* tion. In the next section, the costs are compiled and
Using that capacity, and then combining the mass bal- compared.
ances presented above, an overall mass balance, from bio-
mass to ethanol, was calculated for the three approaches Operating costs
to cellulase production (Table 4). The off-site approach As a base case, the mass balances presented in the previous
requires less input biomass than the on-site one, because section (Tables 1, 2, and 4) were applied and normalized to
the glycerol/sucrose stabilizer is converted to ethanol, an ethanol output of 70 000 tonnes per year, equivalent to
reducing the need for input biomass (and for input cel- 88.720 million liters or 23.437 million gallons.
lulase). The integrated approach obviously requires more Prices for variable inputs (Table 5) were taken mainly
biomass, because some of it is converted to cellulase. from Humbird et al.,6 except for electricity and steam,
which were sourced from the SRI Consulting Process
*One reviewer of this paper noted that such plants might be limited by
feedstock availability. This is a valid point. For comparitive purposes, Economics Program (SRI PEP), and cellulase transport,
nonetheless, the subsequent cost analysis is done on an equivalent- which was sourced from an industry estimate. For the
capacity basis, as is common in practice and in the literature. cellulase plant, the same utility prices were used, while
© 2016 The Authors. Biofuels, Bioproducts, Biorefining published by Society of Chemical Industry and John Wiley & Sons, Ltd.
168 | Biofuels, Bioprod. Bioref. 10:164–174 (2016); DOI: 10.1002/bbb
Modeling and Analysis: Integrated enzyme production lowers the cost of cellulosic ethanol E Johnson

Table 5. Prices of variable inputs to the cellulase Capital costs

and ethanol plants.
A base capital cost for each production approach was esti-
Raw materials & consumables
mated according to Humbird et al.,6 then this was adjusted
Biomass, dry 58.5 $/t for scale (at a 0.65 exponent). Scales for each approach
Ammonia 439.38 $/t are slightly different (Table 4), because the biomass input
Ammonium sulfate 152.00 $/t required per unit of ethanol output varies according to the
Antifoam 800.00 $/t production approach. Off-site and on-site use sugar as a
Biomass, dry 58.50 $/t feedstock for cellulase; off-site uses sugar to stabilize cel-
Calcium chloride 125.00 $/t lulase. Integrated requires no sugar as feedstock – this is
Caustic 149.47 $/t substituted by biomass.
Cellulase calculated in this work In addition to the 0.65-exponent scaling, other adjust-
Cellulase transport*** 52 $/t ments were made:
Corn steep liquor 55.56 $/t • The biomass-cellulase process is sized larger than the
DAP 966.73 $/t sugar-cellulase alternatives, to accommodate the bulk-
Glucose/Dextrose 568.57 $/t ier feedstock.
Glycerol/sucrose 440.92 $/t • A cellulase formulation plant is included in the off-site
Lime 199.30 $/t approach, whereas cellulase formulation is not needed
Magnesium sulfate 150.00 $/t
in the other two approaches.
Monopotassium phosphate 800.00 $/t There are no significant economy-of-scale differences
SO2 303.80 $/t between the approaches. Cellulase production in all three
Sodium chloride 100.00 $/t cases is at maximum size reported for industrial cellulase
Sorbitol 1103.41 $/t production.
Sulphuric acid 87.96 $/t This was then added to the capital cost of the ethanol
Water 0.22 $/t
plant, estimated from Humbird et al.,6 to come up with a
full plant installed cost (Table 6). Straight-line depreciation
Utilities
was assumed, and this is taken as an annual charge – that
Electricity 0.05 $/kWh
does not include finance charges for debt or debt service.
Steam 1.5 $/t
Waste 28.86 $/t
***Applies only to the off-site production approach.
Cost comparison
First the costs of cellulase production are presented, then
raw material prices were taken from a variety of sources,
those of the entire chain through to ethanol. Finally, these
including Humbird et al.6 for the glucose/dextrose,
are compared to estimates from previous studies.
Index Mundi† for glycerol/sucrose and the rest from
SRI PEP.
Production of cellulase
Fixed costs for labor, overheads and insurance were
taken from Humbird et al.6 For the on-site and integrated The biggest cost differences between the three produc-
approaches, it was assumed that the ethanol and the cel- tion approaches are in the raw materials and consumables
lulase plants are operated as one unit, i.e., the fi xed costs (Table 7). Primarily this comes down to the huge gap between
are spread across both plants. For the off-site approach, the cost of biomass in the integrated plant and the cost of
these were split among the ethanol and cellulase plants. sugars in the off-site and on-site ones. The varying yields of
However, total labor, overhead, and insurance costs are the cellulase (Table 2) also play a role, but far more modestly.
same for all three approaches. We have not assumed that All of this rolls through to the cash cost of cellulase
the off-site plant is run any less efficiently; presumably it is (Table 9), which is the cost used to calculate the overall cost
part of a larger chemical-processing complex where costs of ethanol production (presented in the next subsection).
are integrated and optimized.
Production of ethanol
†
http://www.indexmundi.com/commodities/?commodity=sugar& The cash cost of cellulase (Table 8) rolls through to the
months=60 cash cost of ethanol production (Table 9), where it is the
© 2016 The Authors. Biofuels, Bioproducts, Biorefining published by Society of Chemical Industry and John Wiley & Sons, Ltd.
| Biofuels, Bioprod. Bioref. 10:164–174 (2016); DOI: 10.1002/bbb 169
E Johnson Modeling and Analysis: Integrated enzyme production lowers the cost of cellulosic ethanol

Table 6. Capital costs, cellulase plant, and full EtOH/cellulase plant, US$.
Cellulase production approach
Off-site On-site Integrated
Cellulase capacity cost 9,319,261 8,166,476 8,166,476
EtOH full plant installed cost 214,319,261 213,166,476 216,203,445
Plant life, years 15 15 15
Capital charge, annual 14,287,951 14,211,098 14,413,563

Table 7. Cellulase raw materials Table 9. Cellulosic ethanol annual cash costs,
and consumables annual cost, US$.††† US$.§§§
Cellulase production approach Cellulase production approach
Off-site On-site Integrated Cost item Off-site On-site Integrated
Raw materials & consumables Raw materials & consumables
Biomass, dry 0 0 1,528,220
Biomass, dry 17,493,945 18,166,090 19,694,310
Glucose/Dextrose 10,869,361 9,654,492 0
Sulphuric acid 625,322 649,348 703,974
Glycerol/sucrose 2,638,298 0 0
Ammonia 1,657,129 1,720,799 1,865,561
Chemicals and 2,296,535 2,039,852 2,039,852
process aids Corn steep liquor 230,864 239,734 259,901

SUM 15,804,194 11,694,344 3,568,072 DAP 492,611 511,538 554,571

††† Sorbitol 174,222 180,916 196,135
For 70 000-tonne/year ethanol production
Cellulase 18,239,988 13,557,533 5,431,261

Table 8. Cellulase annual cash costs, US$.‡‡‡ Cellulase transport 466,647

Cellulase production approach Caustic 1,207,937 1,254,348 1,359,870

Cost item Off-site On-site Integrated Lime 640,085 664,678 720,594
Raw materials & 15,804,194 11,694,344 3,568,072 Water 116,406 120,879 131,048
consumables Utilities
Utilities 1,794,251 1,863,189 1,863,189 Electricity 287,000 287,000 287,000
Fixed costs
Steam 451,500 451,500 451,500
Labor 385,221 included in included in
Waste 592,903 615,683 667,477
ethanol ethanol
Fixed costs
Overheads & insurance 256,322 included in included in
ethanol ethanol Labor 2,114,779 2,500,000 2,500,000
SUM 18,239,988 13,557,533 5,431,261 Overheads & insurance 6,000,000 6,000,000 6,000,000
‡‡‡
For 70 000-tonne/year ethanol production SUM 50,791,338 46,920,046 40,823,204
§§§
For 70 000-tonne/year ethanol production
major cost difference between the three approaches. The
bases of raw materials and consumables costs are pre-
sented in the previous two sections of this paper. Utilities for a bit more than 80% of full costs: overheads & insur-
consumptions are taken from Humbird et al.6 and prices ance, capital/finance charges, biomass feedstock and cel-
from (Table 5). Fixed costs are taken from Humbird et al.6 lulase production. The cost of cellulase is by far the most
with the off-site labor costs adjusted for the labor that variable by production approach. With off-site, it accounts
occurs off-site (Table 8). of 28% of full costs, with on-site it reduces to 22% and
The cash costs (Table 9) and the capital costs (Table 6) of with integrated it falls to 10%.
course roll through to full costs for cellulosic ethanol pro-
Cellulase costs are significant,
duction (Table 10).
When full costs are itemized and normalized on the
and variable by production approach
basis of per-gallon ethanol output (Table 11), it becomes Following the findings of Cherry and Fidantsef,1 Fang
clear that there are four items that when compiled account et al.,2 and Brijwani et al.,3 Klein-Marcushamer et al.4
© 2016 The Authors. Biofuels, Bioproducts, Biorefining published by Society of Chemical Industry and John Wiley & Sons, Ltd.
170 | Biofuels, Bioprod. Bioref. 10:164–174 (2016); DOI: 10.1002/bbb
Modeling and Analysis: Integrated enzyme production lowers the cost of cellulosic ethanol E Johnson

Table 10. Cellulosic ethanol annual full costs, presented by other authors that range from $0.10 to 0.40
US$.**** per gallon of ethanol; and it presents its own estimate of
Cellulase production approach $0.68–1.47 per gallon. This study confirms that cellulase is
indeed a significant cost (Tables 9 and 11).
Cost Off-site Off-site Off-site
This study models the current, available process
Cash costs, $/year 50,791,338 46,920,046 40,823,204
approaches, finding that they have relatively comparable
Capital/finance costs, 14,287,951 14,211,098 14,413,563
capital and operating costs, with the exception of the cost/
$/year
price of feedstock for the cellulase. This study then quanti-
Sum, $/year 65,079,289 61,131,144 55,236,767
fies the differences, showing that this cellulase cost can
be significantly reduced, from 0.78 to 0.58 to 0.23 $/gal-
Sum, $/tonne ethanol 930 873 789 lon (Table 11), by shifting from the off-site to the on-site
Sum, $/litre ethanol 0.73 0.69 0.62 to the integrated approach of cellulase production. These
Sum, $/gallon ethanol 2.78 2.61 2.36 are, respectively, 8% and 20% reductions in cash costs, and
****For 70 000-tonne/year ethanol production 7% and 19% reductions in full costs of cellulosic ethanol
production.

Table 11. Cellulosic ethanol annual full costs,

itemized per gallon, US$.†††† Activity comparison of glucose- and
Cellulase production approach cellulose-based cellulase
Cost Off-site Off-site Off-site
Raw materials & consumables The preceding analysis finds integrated enzyme produc-
Biomass, dry 0.75 0.78 0.84 tion to be lower cost than off-site or on-site. It also raises
Sulphuric acid 0.03 0.03 0.03 the question: How do the performances compare? Based
Ammonia 0.07 0.07 0.08 on a blinded, benchmarked experiment (section on
Corn steep liquor 0.01 0.01 0.01 Experimental details), performance – in converting cel-
DAP 0.02 0.02 0.02
lulose to monomers glucose and xylose – was compared of
Sorbitol 0.01 0.01 0.01
integrated enzyme versus off-site enzyme.
Sugar yields of integrated enzyme are better to or equal
Cellulase 0.78 0.58 0.23
to that of off-site enzyme, across enzyme loadings ranging
Cellulase transport 0.02 0.00 0.00
from 0.05 to 0.5%.‡ With neutral pre-treatment and 3-day
Caustic 0.05 0.05 0.06
enzyme exposure the performance is clearly better (Fig. 3);
Lime 0.03 0.03 0.03
with acid pre-treatment it is about the same (Fig. 4).
Water 0.00 0.01 0.01
A reason for the superior performance might result from
Subtotal 1.76 1.58 1.32 the fact that the integrated enzymes are already produced
Utilities on the feedstock that they are than used to hydrolyze.
Electricity 0.01 0.01 0.01 Hence, these enzymes are feedstock and process specific in
Steam 0.02 0.02 0.02 comparison to the off-site enzyme which provides a com-
Waste 0.03 0.03 0.03 promise applicable for all processes and feedstocks, but for
Subtotal 0.06 0.06 0.06 the price of a somewhat lower sugar yield.
Fixed costs
Labor 0.09 0.11 0.11 Extra time does not help very-low
Overheads & insurance 0.26 0.26 0.26 loadings
Capital/finance 0.61 0.61 0.61
One reaction to the high cost of enzyme could be to lower
Subtotal 0.96 0.97 0.98
enzyme loadings and to raise enzyme exposure times, i.e.,
SUM 2.78 2.61 2.36 charge less enzyme to depolymerization but give it longer
††††
For 70 000-tonne/year ethanol production to react. So, how low can go we go? Based on a blinded,

declare that for cellulosic ethanol production, ‘the cost

of producing enzymes was much higher than that com- ‡
The enzyme loading in the preceding economic analysis was 1%.
monly assumed in the literature.’ Their paper4 cites costs Analyses reported in the literature use loadings of 1–3%.

© 2016 The Authors. Biofuels, Bioproducts, Biorefining published by Society of Chemical Industry and John Wiley & Sons, Ltd.
| Biofuels, Bioprod. Bioref. 10:164–174 (2016); DOI: 10.1002/bbb 171
E Johnson Modeling and Analysis: Integrated enzyme production lowers the cost of cellulosic ethanol

Figure 3. Sugar yield of integrated vs off-site enzymes (neutral pre-treatment).

Figure 4. Sugar yield of integrated vs off-site enzymes (acid pre-treatment).

benchmarked experiment (Experimental details section),

0.05–0.1% loadings are too low.
Very-low enzyme loadings – namely of 0.05% and 0.1% –
improve their yields over time only modestly at best. Only
in one of four cases, 0.1% enzyme after 7 days (Fig. 5), does
the very-low loading even begin to reach the xylose yield
achieved with a 0.5% loading. In all other cases (Figs 6–8)
yields do not reach those of 0.5% loadings, even after 10
days of exposure.

Experimental detail
Figure 5. Xylose yields, longer exposure, 0.1% enzyme
In the first experiment, cellulase was used to hydrolyze
loading, neutral pre-treatment.
wheat straw pre-treated one of two ways: in pH neutral

© 2016 The Authors. Biofuels, Bioproducts, Biorefining published by Society of Chemical Industry and John Wiley & Sons, Ltd.
172 | Biofuels, Bioprod. Bioref. 10:164–174 (2016); DOI: 10.1002/bbb
Modeling and Analysis: Integrated enzyme production lowers the cost of cellulosic ethanol
Figure 6. Glucose yields, longer exposure, 0.1% enzyme
loadings, neutral pre-treatment.
Figure 7. Xylose yields, longer exposure, 0.05% enzyme
loadings, acid pre-treatment.
Figure 8. Glucose yields, longer exposure, 0.05% enzyme
loadings, acid pre-treatment.
conditions and in acidic conditions. Samples of 200 mg
(dry mass) of pre-treated straw were mixed with 1–0.1 mg
enzyme (0.5–0.05% enzyme/substrate loading). Sodium
acetate was added 100 mM up to 1 mL. The mixture was
incubated for 3 days at 50°C. The supernatant was then ana-
lyzed by high-performance liquid chromatography (HPLC)
© 2016 The Authors. Biofuels, Bioproducts, Biorefining published by Society of Chemical Industry and John Wiley & Sons, Ltd.
| Biofuels, Bioprod. Bioref. 10:164–174 (2016); DOI: 10.1002/bbb
E Johnson
to determine its glucose und xylose content. The second
experiment was similar, except that enzyme loading was
0.2–0.1 mg, and the incubation was for 3–10 days.
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Eric Johnson
Eric Johnson is Managing Director of
Atlantic Consulting, Editor-in-Chief
Emeritus of Environmental Impact
Assessment Review, an editor of the
Journal of Health and Pollution, and a
Technical Advisor to two NGOs, Green
Cross and the Blacksmith Institute. He
serves on the CEN/ISO Committees that are developing
‘sustainability’ criteria for biofuels. Technology, econom-
ics and environmental impacts of fuels, chemicals and
plastics – these are key areas of his expertise. He is a
chemist, who began his career as an editor of Chemi-
cal Engineering and Chemical Week magazines. He has
been nominated by Switzerland’s federal government as
an IPCC inventory assessor.