Review

On the production cost of

lignocellulose-degrading enzymes
Rafael G Ferreira, Departamento de Engenharia Química, Escola Politécnica, Universidade de São
Paulo, São Paulo, Brazil; Intelligen Brasil, São Paulo, Brazil
Adriano R Azzoni, Departamento de Engenharia Química, Escola Politécnica da Universidade de São
Paulo Brazil
Sindelia Freitas , Faculdade de Engenharia Química, Universidade Estadual de Campinas,
Campinas, Brazil; Programa Integrado de Pós-Graduação em Bioenergia, Faculdade de Engenharia de
Alimentos, Universidade Estadual de Campinas (UNICAMP), Campinas, Brazil

Received February 12 2020; Revised July 07 2020; Accepted July 27 2020;

View online at Wiley Online Library (wileyonlinelibrary.com);
DOI: 10.1002/bbb.2142; Biofuels. Bioprod. Bioref. (2020)

Abstract: Lignocellulose is the most abundant renewable material on Earth and the primary component
of agricultural wastes such as sugarcane bagasse and wheat straw. It consists of a composite material
made of cellulose, hemicellulose, and lignin. Cellulose and hemicellulose can be broken down into
monomers by a set of appropriate enzymes, and the resulting monomers may be used to produce a
variety of fuels or chemicals through either biological or chemical routes. However, the high production
cost of these lignocellulose-degrading enzymes remains a major challenge for the use of lignocellulosic
biomass as raw material. In this context, this article reviews techno-economic analyses concerning
the production of cellulases and other lignocellulose-degrading enzymes published over the last two
decades. The major characteristics of each enzyme production process are described, underscoring
the similarities and differences across the various process designs. Moreover, the enzyme production
costs derived from these process designs and their composition in terms of raw materials, capital-related
factors, utilities, labor costs, etc., are compared. First, this analysis reveals that most techno-economic
evaluations in the literature address either cellulase production by submerged culture with Trichoderma
reesei or enzyme production by solid-state culture with filamentous fungi. Second, this analysis shows
wide cost variations across process designs but it indicates that raw materials and capital-related costs
are generally the main drivers of the enzyme production cost. Furthermore, this assessment corroborates
the importance of process parameters, such as product yield, production titer, and volumetric
productivity, in the process economics of enzyme production. © 2020 Society of Chemical Industry and
John Wiley & Sons, Ltd

Key words: enzymes; techno-economic analysis; cellulases; lignocellulose breakdown, production cost

Correspondence to: Adriano R. Azzoni, Departamento de Engenharia Química, Escola Politécnica da Universidade de São
Paulo., Avenida Prof. Luciano Gualberto, Travessa do Politécnico, Nº 380, CEP 05508-010 - São Paulo, SP, Brazil.
E-mail: adriano.azzoni@usp.br; Sindelia Freitas, Faculdade de Engenharia Química, Universidade Estadual de Campinas,
Avenida Albert Einstein, 500, CEP 13083-852 Campinas, SP, Brazil. E-mail: sfazzoni@unicamp.br

© 2020 Society of Chemical Industry and John Wiley & Sons, Ltd 1
 R Gama Ferreira et al.
Introduction
I
n 2017, the global market for industrial enzymes was
estimated at US$ 5.4 billion.1 Industrial enzymes have
found numerous applications in diverse sectors such
as detergents, textiles, food and beverages, animal feed,
pulp and paper, and, more recently, in the production of
biobased fuels and chemicals. Enzymes that break down
lignocelluloses compose a significant share of this market,2
especially in the textile, animal feed, pulp and paper, and
biofuel sectors; in fact, only three of the lignocellulose-
degrading enzymes (cellulases, xylanases and pectinases)
accounted for approximately 20% of all industrial enzyme
sales in the late 1990s.3 Moreover, the use of lignocellulose-
degrading enzymes has grown rapidly over the last two
decades and is expected to continue growing over the next
few years because of strong government and market efforts
to produce renewable fuels and chemicals by means of
enzymatic hydrolysis of lignocellulosic biomass. In effect,
enzymes related to biofuel production (which include
amylases and lipases other than lignocellulose-degrading
enzymes) are expected to be the fastest growing segment of
industrial enzymes, with a compound annual growth rate
(CAGR) of 7.3% from 2018 to 2024.1 These developments fit
into a broader transition towards a circular economy based
on carbon from plant cell walls instead of carbon from fossil
sources.4,5
In this review, we revisit the main characteristics, mode of
action, and microbial sources of the major lignocellulose-
degrading enzymes of industrial interest. We review
the techno-economic analyses (TEAs) concerning the
production of these enzymes, with a clear emphasis on
biofuel applications. The major process design characteristics
considered in these TEAs are described and compared. The
enzyme production costs estimated in these works are also
evaluated. This assessment includes a comparison of enzyme
cost composition in terms of raw materials, utilities, labor,
etc., and a comparison of important process parameters
such as product yield, production titer, and volumetric
productivity.
Enzymes for lignocellulose
hydrolysis
Cellulases
The cellulose fraction of biomass can be broken down by
two basic enzymatic systems: either a set of free extracellular
enzymes, produced by bacteria or fungi, or a multienzymatic
complex attached to the surface of the cell, called a
2 © 2020 Society of Chemical Industry and John Wiley & Sons, Ltd | Biofuels, Bioprod. Bioref. (2020); DOI: 10.1002/bbb.2142
Review: On the cost of lignocellulose-degrading enzymes
cellulosome, produced by certain anaerobic bacteria.6–8
In any case, multiple enzymatic activities are required to
hydrolyze cellulose into soluble monosaccharides that can be
assimilated by cells. The classic model of enzymatic hydrolysis
consists of three major types of glycosyl-hydrolases (GHs):
(i) endoglucanases (EGs; EC 3.2.1.4), which randomly
cleave inner bonds of cellulose chains, preferentially in
amorphous regions; (ii) cellobiohydrolases (CBHs; also
called exoglucanases), which cleave bonds at a distance of
two glucose units from the end of the chain in a processive
manner (there are two subtypes of CBHs: those acting on
reducing ends, CBH1 (EC 3.2.1.176), and those acting on
nonreducing ends, CBH2), thus generating a disaccharide
of glucose (cellobiose); and (iii) β-glucosidases (BGLs;
EC 3.2.1.21; also called cellobiases), which convert short
oligosaccharides, especially the cellobiose generated by CBHs,
into glucose; collectively, these three GH classes are called
cellulases.7–12 Cellulases act on cellulose synergistically: EGs
generate new chain ends on which CBHs can act, and the
activity of CBHs exposes new amorphous regions to the
EGs.13,14 Furthermore, BGL eliminates cellobiose, which
is a strong inhibitor of EGs and CBHs, from the reaction
mixture.15–17
Recently, it was discovered that a different type of enzyme,
called lytic polysaccharide monooxygenase (LPMO), also
takes part in cellulose hydrolysis, acting synergistically
with conventional cellulases. Lytic polysaccharide
monooxygenases (AA9 or AA10 in the Carbohydrate-
Active Enzymes database)18 are copper-dependent
enzymes that oxidatively cleave internal glycosidic bonds
of cellulose at C1 and/or C4 positions, even on crystalline
regions.9–11,19–21 Moreover, they have been shown to act on
other polysaccharides, such as chitin, xylan, xyloglucans,
β-glucans, glucomannans, and starch.11,19 They are thought
to boost cellulose hydrolysis primarily by making new
chain ends available to the other enzymes, particularly in
crystalline regions that would be otherwise highly resistant to
cellulases.9,11,19–21 Lytic polysaccharide monooxygenases are
already present in the latest commercial cellulase cocktails.9,11
Many cellulases (and hemicellulases) that act on insoluble
substrates have a modular structure: they are constituted
by a catalytic domain connected by a flexible peptide linker
to a so-called carbohydrate-binding module (CBM), which
anchors the enzyme to the solid substrate. Carbohydrate-
binding modules assist biomass hydrolysis by effectively
increasing the concentration of their enzymes near the
substrate surface and, depending on their amino acid
sequence and resulting shape, provide specificity to a
certain substrate or substrate region (such as reducing or
nonreducing ends). The concerted action of cellulases,
 Review: On the cost of lignocellulose-degrading enzymes R Gama Ferreira et al.

Figure 1. Enzymatic breakdown of lignocellulose by a set of extracellular enzymes. Lytic polysaccharide monooxygenases
(LMPOs) cleave crystalline chains, generating amorphous regions (red dots represents C=O linkage). Endoglucanases randomly
break bonds in the middle of cellulose chains, thereby creating reducing and nonreducing ends. Cellobiohydrolases of type 1
(CBH1) and 2 (CBH2) cleave on the reducing and nonreducing ends, respectively, generating cellobiose. β-Glucosidases (BGL)
split cellobiose into two glucose molecules. Products from lignin breakdown by ligninases are represented in brown.

hemicellulases, and auxiliary enzymes is schematically
presented in Fig. 1.
Hemicellulases
Complete hydrolysis of hemicellulose requires another set of
enzymes, collectively called hemicellulases, which depend
on the nature of the hemicellulose in question. In general,
endo-1,4-β-xylanases (which randomly hydrolyze xylan
chains at internal positions – EC 3.2.1.8), exo-β-1,4-xylanases
(which hydrolyze reducing ends of xylan, releasing xylose
and xylobiose units – without EC number), 1,4-β-xylosidases
(which hydrolyze xylose units from the nonreducing ends
of xylan – EC 3.2.1.37), α-l-arabinofuranosidases (which
hydrolyze arabinose units hanging on the xylan chain – EC
3.2.1.55), α-glucuronidases (which hydrolyze glucuronic
acid units – EC 3.2.1.139), acetyl xylanoesterases (which
deacetylate xylan chains – EC 3.1.1.72) and feruloyl/
cumaroyl esterases (which hydrolyze ester bonds between
coniferic or p-coumaric acid from lignin and sugars from
hemicellulose – EC 3.1.1.73) are useful enzyme activities for
hemicellulose deconstruction.12,22–27 In the case of sugarcane
bagasse, whose hemicellulosic fraction consists mainly of
xylans,28 xylanases and feruloyl esterases are particularly
useful because they digest hemicellulose and disentangle
hemicellulose from lignin, respectively.29
Ligninases
The enzymatic degradation of lignin constitutes a distinct
challenge because lignin biosynthesis involves the oxidative
coupling of monolignols. This produces a heterogeneous
and irregular polymer network, cross-linked through aryl
ether and C-C bonds that are less reactive than most bonds
present in other biological polymers.30–32 Consequently,
the depolymerization of lignin is complex, requiring a
variety of nonhydrolytic, oxidoreductase enzymes, notably
lignin peroxidases (EC 1.11.1.14), manganese peroxidases
(EC 1.11.1.13), versatile peroxidases (EC 1.11.1.16) and
laccases (EC 1.10.3.2), to be accomplished.10,31,33–36 Other
relevant enzyme activities for lignin degradation are glyoxal
oxidase (EC 1.2.3.5), aryl-alcohol oxidase (EC 1.1.3.7),34,37,38
cellobiose dehydrogenase (EC 1.1.99.18),37,38 manganese-
independent peroxidase (EC 1.1.1.7), pyranose 2-oxidase (EC

© 2020 Society of Chemical Industry and John Wiley & Sons, Ltd | Biofuels, Bioprod. Bioref. (2020); DOI: 10.1002/bbb.2142 3
 R Gama Ferreira et al.
1.1.3.4), cellobiose/quinone oxidoreductase (EC 1.1.5.1),38
and feruloyl esterase (EC 3.1.1.73).34
Expansin-like proteins
Over the last decade, proteins that exhibit no catalytic
activity but nonetheless help break down lignocellulose have
been discovered. They act by weakening and disrupting
intermolecular interactions among the cellulose chains, thus
making the cellulose microfibrils swell and disperse, which
renders the lignocellulosic biomass more accessible to the
catalytic proteins. The first proteins identified with such
properties were discovered in plants and called expansins.
By loosening the lignocellulosic structure of the plant cell
wall, expansins allow the plant to grow.39–41 Later, proteins
with similar properties were found in fungi and bacteria,
notably swollenin, in Trichoderma reesei,9–11,21,40 loosenin, in
Bjerkanderaadusta,40,42,43 and protein BsEXLX1, in Bacillus
subtilis.44 Moreover, it has been found that supplementing
cellulases with microbial expansin-like proteins enhances
the efficiency of lignocellulose hydrolysis.9,40,41 Thus, the
use of expansins or functionally similar microbial proteins
may be regarded as a mild biological pretreatment process,
presumably reducing the intensity of the chemical /
physicochemical pretreatment needed for hydrolysis.
Microorganisms for producing
lignocellulose-degrading enzymes
The microorganisms most commonly used for producing
cellulases, in both industrial and academic settings, are
filamentous fungi of the genus Trichoderma, particularly T.
reesei.7,8,11,45,46 Trichoderma fungi can secrete high levels of
cellulase cocktails while using a variety of cellulosic materials
as carbon sources, such as agri-food wastes,47 streams from
the pulp and paper industry, 48 and domestic wastewater.49
In fact, substrate characteristics have been recognized as an
important factor affecting not only enzyme titers but also the
composition of the enzymatic cocktail produced by T. reesei,
as recently reviewed.50
The T. reesei species was first isolated from rotting US
Army equipment in the Solomon Islands during World
War II. Its ability to produce large amounts of cellulose-
degrading enzymes, at first regarded as a problem, was soon
recognized as a potentially useful trait, and since then, the
species has been modified to secrete ever increasing levels
of cellulases,21,51 with recent reports of industrial strains
capable of secreting up to 100 g of protein L–1.8,45,52,53
Trichoderma reesei produces at least two types of CBH
(CBH-I and CBH-II), five types of EG, and two types of BGL;
4 © 2020 Society of Chemical Industry and John Wiley & Sons, Ltd | Biofuels, Bioprod. Bioref. (2020); DOI: 10.1002/bbb.2142
Review: On the cost of lignocellulose-degrading enzymes
however, CBHs and EGs account for approximately 65–78%
and 21–32% of the total secreted protein, respectively,54
while the amount of BGL and other hydrolytic enzymes is
small.54,55 Consequently, the enzymatic cocktail from T. reesei
is often supplemented with BGL from other sources.16,17
Although BGL can be produced by various yeasts and
bacteria, including recombinant strains of Escherichia coli
and Saccharomyces cerevisiae, it is usually produced by
filamentous fungi, particularly Aspergillus sp. and Penicillium
sp.16,46 Hemicellulases and ligninases are also synthesized by
a wide variety of bacteria and fungi. Industrially, however,
hemicellulases are mainly produced by filamentous fungi
from genera Trichoderma and Aspergillus,23,24,28,56 whereas
ligninases are most often produced by white-rot fungi such as
Phanerochaete chrysosporium.10,31,57
Clearly, there is no single microorganism in nature capable
of producing a complete and balanced set of enzymes that
efficiently degrades all types of lignocellulosic biomass.37,58–60
This is to be expected, given that, in the natural environment,
plant biomass is degraded by an entire community of
organisms (in fact, developing a single organism capable
of decomposing lignocellulose into sugars alone is a major
goal of consolidated bioprocessing). For this reason,
various strategies for improving the industrial process of
lignocellulose degradation have been proposed. These include
supplementing the fungal cellulase mixture to compensate
for missing enzyme activity and / or adding accessory
proteins;12,16,17,29,60–66 supplementing cellulolytic organisms
such as T. reesei or Clostridium to compensate for inadequate
enzyme activity or providing better enzymes through
genetic and protein engineering;7,16,21,23,26–28,46,55,59,67–73
coculturing T. reesei with good producers of lacking
enzymes, such as Aspergillus sp.;16,26,28,60,66,67,74 or
endowing cellulolytic enzymes with fermenting,
noncellulolytic organisms such as E. coli or S. cerevisiae
(e.g., adding BGLs to S. cerevisiae to consume cellobiose
­directly).7,8,21,23,26–28,55,70,72,75,76’7,8,21,23,26–28,55,70,72,75,76
Techno-economic analyses of
the production of lignocellulose-
degrading enzymes
The cost of cellulases and other lignocellulose-degrading
enzymes remains one of the major bottlenecks for
establishing cost-effective production processes for fuels and
chemicals from biomass. Nevertheless, relatively few TEAs of
cellulases or other industrial enzymes have been published in
the literature over the last 20 years, as shown in Table 1.
First, we observe that more than half of the economic
evaluations of enzyme production published during that
 Table 1. Cost of industrial enzymes found in the literature from 1999 to 2019.
Ref. Enzyme Enzyme cost Production scale Microorganism Production mode Process simulator
77
Cellulase Offsite (glucose-fed) 3105 ton year−1 Trichoderma reesei SmC/on- and offsite None
0.78 US$ gal−1 EtOH (inferred)
Onsite (glucose-fed)
0.58 US$ gal−1 EtOH
Integrated (cellulose-fed) 0.23 US$
gal−1 EtOH
78
Cellulase 4.24 US$ kg−1 Seven bioreactors of 300 m3 each T. reesei SmC/onsite (NREL- Aspen plus
(inferred) based)
79
Cellulase 0.078 EUR L−1 EtOH (offsite) Unclear T. reesei SmC/on- and offsite Aspen plus
80 −1 −1
Cellulase 3.8–6.7 US$ kg (onsite) 690 kg h (onsite, high demand T. reesei SmC/on- and offsite Aspen plus
4.0–8.8 US$ kg−1 (offsite) scenario) (NREL-based)
Review: On the cost of lignocellulose-degrading enzymes

15–75 ton year−1 (offsite)

81
Cellulase 10.14 US$ kg−1 Twelve bioreactors of 300 m3 each T. reesei (inferred) SmC/offsite SuperPro designer
82 −1
Cellulase 0.42–0.53 SEK L EtOH Twenty-four bioreactors with a total T. reesei SmC/onsite Aspen plus
MESP 4.71–4.82 SEK L−1 volume of 37–121 m3
83
Cellulase 16 US$ kg−1 (SSC) Fifteen bioreactors of 938 m3 each Clostridium SmC and SSC/on- and SuperPro designer
40 US$ kg−1 (SmC) (SmC) thermocellum offsite
>90 US$ kg−1 (market price) One bioreactor of 2404 m3 (SSC)
84
Cellulase 0.12 ₤ L−1 EtOH 385 kg h−1 T. reesei SmC/onsite (NREL- SuperPro designer,
based) MATLAB
85
Cellulase 5.38 US$ kg−1 Seven bioreactors of 300 m3 each T. reesei SmC/onsite (NREL) Aspen plus
86 −1 3
Cellulase 4.23 US$ kg Seven bioreactors of 300 m each T. reesei SmC/onsite (NREL) Aspen plus
87
Cellulase 5.38 US$ kg−1 Seven bioreactors of 300 m3 each T. reesei SmC/onsite (NREL) Aspen plus
88 −1 3
Cellulase 4.24 US$ kg Seven bioreactors of 300 m each T. reesei SmC/onsite (NREL) Aspen plus
89
Cellulase 6.16 US$ kg−1 Seven bioreactors of 300 m3 each T. reesei SmC/onsite (NREL) Aspen plus

© 2020 Society of Chemical Industry and John Wiley & Sons, Ltd | Biofuels, Bioprod. Bioref. (2020); DOI: 10.1002/bbb.2142
90
β-glucosidase 316 US$ kg−1 100 m3 Escherichia coli SmC SuperPro designer
(recombinant)
91
Manganese 2.27 EU kU−1 30 L White-rot fungi SmC None
peroxidase and 1.08 EUR kU−1 (Irpexlacteus and
laccase (only operating cost) Ganoderma lucidum)
92
Lipase 1.06 × 106 IDR kg−1 4.3 ton year−1 Aspergillus niger SSC None
Approx. 15 US$ kg−1
R Gama Ferreira et al.

5
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R Gama Ferreira et al.

Table 1. (Continued).
Ref. Enzyme Enzyme cost Production scale Microorganism Production mode Process simulator
93 −1 −1
Protease 1.66 US$ kg (50% moisture) 30.6 ton year (50% moisture) Brevibacteriumluteolum SSC None
=3.32 US$ kg−1 (dry basis)
94
Pectinase 858–1605 INR L−1 30 m3 year−1 of concentrate Aspergillus carbonarius SmC & SSC None
(poly- 3000 U L−1 75 L reactor in SmC case
galacturonase) Approx. 23 US$ mg−1
95
Laccase 0.40–70 EUR kU−1 Shake flasks and trays White-rot fungi SmC & SSC None
(Trametespubescens)
96
Lipase 42–131 US$ L−1 100 m3 of concentrate year−1 Penicillium restrictum SmC & SSC Own program
97
Amylases, 10.4 US$ kg−1 644 kg year−1 (50 wt% enzymes) Aspergillus awamori SSC SuperPro designer
cellulases,
xylanases,
proteases
98
Amylases, 70–217 US$ kg−1 100 ton year−1 A. awamori SSC SuperPro designer
cellulases, (no coproduct revenues)
xylanases, 57–139 US$ kg−1
proteases (with coproduct revenues)
99
Laccase 0.16–0.25 EUR kU−1 200 L Pichia pastoris SmC SuperPro designer
100
Laccase, 0.107 US$ cm−3 (~2 g/L 100 m3 of fungal broth per batch Aspergillus iizukae SmC SuperPro designer
manganese of crude enzyme protein)
peroxidase,
lignin
peroxidase
The production costs of industrial enzymes were obtained from techno-economic analyses published in the literature from 1999 to 2019. For each reference, the type of enzyme and

© 2020 Society of Chemical Industry and John Wiley & Sons, Ltd | Biofuels, Bioprod. Bioref. (2020); DOI: 10.1002/bbb.2142
its production cost are indicated, as well as the production scale, the microorganism employed for enzyme expression, the process mode (submerged culture or solid-state culture,
abbreviated as SmC and SSC, respectively) and the process simulator used (if any).
Review: On the cost of lignocellulose-degrading enzymes
 Review: On the cost of lignocellulose-degrading enzymes
period concern Trichoderma cellulase production. Second,
studies concerning enzymes other than cellulases are mostly
based on the solid-state cultivation of filamentous fungi.
A number of these reports consider very small production
scales, such as shake flasks and trays, which makes them
of limited use for cost projections on an industrial scale.
Several articles also present cost results in terms of enzymatic
activity only, without indicating enzyme specific activity,
which renders comparisons between different enzymes
virtually unfeasible. Some reports also present their cost
results in local currencies, without indicating their exchange
rate to the US dollar at the time of the study, which further
complicates direct comparisons. Moreover, certain works
concerning cellulase production report the enzyme cost in
terms of dollars per gallon (or liter) of ethanol, given that
the assessment of cellulase cost is often a minor part of an
extensive economic evaluation of lignocellulosic ethanol (or
another end product), assuming that cellulases are produced
onsite. This unit of cellulase cost makes it difficult to compare
values from different sources, given that it depends not only
on the enzyme cost itself but also on many other factors
such as the choice of feedstock, enzyme loading and process
yield.81 In any case, the enzyme cost estimated in those works
that consider industrially significant scales varies widely, from
3.3 US$ kg−1 of protein93 to 316 US$ kg−1 of protein.90
As mentioned above, cellulase production is often studied
in the context of lignocellulosic ethanol production. In that
setting, cellulase production can be carried out either offsite
or onsite.77 In offsite production, the enzyme is considered
a purchased reagent, and the ethanol production cost is
always dependent on the commercial cellulase price, which
has been reported to be a significant uncertainty factor in
TEAs of cellulosic ethanol.78,79,82 In onsite production, by
contrast, the enzyme is produced in a facility integrated
with the ethanol plant, thus eliminating the dependence of
the ethanol producer on external suppliers. Moreover, in
this case, a fraction of the lignocellulosic feedstock can be
used as an inexpensive carbon source for microbial enzyme
production. Some downstream processing steps such as
primary recovery and stabilization are not also required,
nor are the transport logistics to the customer’s site. All
these factors can contribute to reduce the final cost and
environmental impact of lignocellulosic ethanol production.
Indeed, lifecycle assessment studies have indicated that
onsite production generates lower greenhouse gas emissions,
thereby contributing to reduce the environmental impact
of the entire lignocellulosic ethanol production.79,80 With
respect to the economics of cellulase production, however,
some works have shown that the offsite approach can actually
be superior due to the possibility of developing a more
© 2020 Society of Chemical Industry and John Wiley & Sons, Ltd | Biofuels, Bioprod. Bioref. (2020); DOI: 10.1002/bbb.2142
R Gama Ferreira et al.
productive process and of using larger production scales.
In fact, an onsite enzyme plant is usually sized to meet the
demands of the single ethanol plant to which it belongs,
whereas an offsite plant may serve multiple lignocellulosic
ethanol producers.79,80 With regard to process productivity,
enzyme productivity and yield have been identified as key
parameters to improve cellulase production and, in particular,
to make onsite cellulase production economically feasible for
ethanol production.79
Process design of cellulase production
With regard to the analyses of cellulase production,
most studies assume synthesis by the filamentous fungus
Trichoderma reesei through submerged cultivation, under
fed-batch mode and aerobic conditions, within up to 12 large
bioreactors (usually 300 m3), and using Aspen Plus (Aspen
Technology, Massachusetts, USA) or SuperPro Designer
(Intelligen, New Jersey, USA) to model and simulate the
entire bioprocess. It is worth noting that a considerable
number of bioreactors is often necessary because, as the
enzyme production scale increases and bioreactor volumes
become too large, significant mass and heat transfer
limitations may affect cultivation.101
A good number of these TEAs were performed by the
National Renewable Energy Laboratory (NREL)86–89 or
were based on the Aspen Plus model made freely available
by NREL, in which T. reesei is cultivated aerobically in a
300 m3 bioreactor using a submerged fed-batch process for
120 h. For inoculum production, a series of seed bioreactors
are employed, with an expansion factor of 10; all these seed
bioreactors, as well as the main bioreactor, are made of grade
316 stainless steel (SS316). The main carbon source of all
these cultures is glucose, and cellulase expression in the main
bioreactor is induced by sophorose. This sugar is produced
from glucose and the cellulase enzyme itself by diverting a
small amount of the enzyme produced. Finally, in this model,
the enzymatic cocktail is neither concentrated nor purified,
since the authors assume that the plant is located onsite;
i.e., all the enzyme produced is rapidly used in a hydrolysis
reactor nearby. As such, the NREL design considers that all
fungal broth will be directly introduced into the hydrolysis
reactor. Hong et al.80 created two process designs starting
from the NREL model – one for onsite production and the
other for offsite production. In the latter case, NREL includes
a microfilter and an ultrafilter to recover and concentrate the
cellulase mixture. Barta et al.82 also designed a process similar
to the NREL model on Aspen Plus; however, the process
uses spruce wood chips as the main carbon source and
enzyme inducer. They also considered a shorter cultivation
7
 R Gama Ferreira et al.
time (108 h), lower production titers (11–35 g/L), and
smaller process scales (24 production fermentors with a total
volume of 37–121 m3). In this case, the reactor size used for
enzyme production could be solved by back calculation from
productivity in the enzyme reactor, enzyme dose needed, and
ethanol fermenter volume. These authors also adopted a seed
train expansion factor of 20×, instead of 10×, and an inferior
grade of stainless steel for the bioreactors (SS304). Figure 2
presents a generic process design of cellulase production with
filamentous fungi.
Klein-Marcuschamer et al.81 also developed a cellulase
production process, somewhat similar to the NREL model,
on SuperPro Designer. As in the NREL model, T. reesei is
subjected to submerged, fed-batch cultivation in an 80 000
gal (300 m3) bioreactor, but poplar rather than sophorose
is employed to induce enzyme expression. Moreover, after
cultivation, the enzyme is isolated through a rotary vacuum
filter and concentrated through an ultrafilter. The expansion
factor stipulated for the seed train is also 20×, as in the Barta
et al.82 model, and the cultivation time is slightly longer
(168 h).
Zhuang and Marchant83 also designed a microbial
process for cellulase production with SuperPro Designer;
however, they employed a bacterial platform, Clostridium
thermocellum, rather than the fungal (T. reesei) paradigm.
They also developed two distinct processes: one based on
submerged cultivation (SmC) and the other based on solid-
state cultivation (SSC). Both processes are radically different
from those described earlier; both are anaerobic, and their
main substrate is paper pulp, which is assumed to have no
cost. The main reactor in the SmC case has a volume of 938
m3 and, in the SSC case, 2741 m3. Moreover, in the SmC
process, the liquid broth is concentrated in a multi-effect
Figure 2. Flowsheet of a generic cellulase production process with filamentous fungi.
8 © 2020 Society of Chemical Industry and John Wiley & Sons, Ltd | Biofuels, Bioprod. Bioref. (2020); DOI: 10.1002/bbb.2142
Review: On the cost of lignocellulose-degrading enzymes
evaporator and freeze-dried afterwards. For both SmC
and SSC, the authors developed an inoculum production
scheme based on SmC, consisting of a seed train with a large
expansion factor equal to 100×.
Ferreira et al.90 described a bacterial process to produce
a cellulolytic enzyme; however, their process relies on a
recombinant E. coli platform to produce a single enzyme,
β-glucosidase. In their baseline scenario, the process is
conducted as a fed-batch, aerobic liquid culture, with
glucose as the main carbon source, in a 100 m3 bioreactor.
Cultivation lasts 22 h, enzyme production is intracellular,
and enzyme expression requires induction with isopropyl
β-d-1-thiogalactopyranoside (IPTG), an expensive chemical.
The enzyme titer at the point of harvest is equal to 5 g L–1,
substantially lower than those of other (multienzymatic)
processes. As in other models, a seed train is required for
inoculum production; in that work, the expansion factor was
set to 20×. After the main cultivation, cells are disrupted in a
high-pressure homogenizer; the liquid phase is recovered by
centrifugation, and the enzyme is concentrated and stabilized
through diafiltration. It should be mentioned, however, that
these authors simulated many scenarios in which the various
parameters mentioned above were changed; in some cases,
for instance, the enzyme was extracellularly produced or did
not go through diafiltration at all.
Process models of industrial enzymes
other than cellulases
The cost analyses of enzymes other than cellulases primarily
concern SSCs of filamentous fungi, as presented in Table 1.
The enzyme product is often a ligninase (particularly laccase)
or a multienzymatic mixture (e.g., a combination of amylases,
 Review: On the cost of lignocellulose-degrading enzymes
cellulases, xylanases and proteases) produced extracellularly.
The main substrate for cultivation is usually agroindustrial
waste, such as babassu cake, wheat bran, or rice bran.
Moreover, the main cultivation is typically carried out in
tray bioreactors, and at the end of the process, enzymes are
extracted by adding water or a buffer solution. Next, a coarse
solid–liquid separation is employed to recover the liquid
phase (such as rotary drum filtration or hydrocycloning),
followed by finer solid–liquid separation such as
microfiltration. Finally, the enzyme solution is generally
concentrated by ultrafiltration. With regard to the upstream
section, inoculum propagation may be performed either
through solid-state or submerged culture; in fact, de Castro
et al.98 compared both strategies. It is worth noting that, in
several of these works, the authors assign a certain market
value to the solid material that remains after cultivation (e.g.,
as supplementation for animal feed); that is, they consider it a
coproduct, which ultimately reduces the final enzyme cost.
Cost analysis of enzyme production
The most relevant characteristics of selected enzyme
production processes are collected in Table 2. In addition to
basic process characteristics, such as the microbial platform
or mode of cultivation, three process parameters are included
in this table: product yield (g product g−1 substrate), product
concentration (g product L−1), and volumetric productivity
(g product L−1 h−1). These process metrics are vital to
evaluate the performance of any biotechnological process:
the product yield indicates the efficiency of conversion
(substrate into product) remembering that the substrate is
usually significant in the final enzyme cost composition;
the product concentration gives a sense of the downstream
operating and capital costs; and the volumetric productivity
is a parameter of efficiency and will ultimately reflect on
the size of the industrial plant102 and the overall production
cost. Although these three metrics could not be found (or
deduced) in every TEA of industrial enzyme production, the
data available in Table 2 suggest that the low cost of cellulases
from T. reesei results from a combination of exceptionally
high enzyme yields, high enzyme titers and high volumetric
productivities. The advantage of T. reesei processes for
cellulase production is particularly striking in terms of
product yield: whereas T. reesei processes exhibit values in
the range of 21–26%, other enzyme production processes
fail to surpass 2.0–2.5%. In other words, the yields of T. reesei
processes are approximately one order of magnitude higher
than those of other processes. The fact that, in nature, the
cellulolytic enzymes produced by T. reesei are crucial for its
primary metabolism may partially explain its extraordinary
product yields. Nonetheless, it is worth underscoring that the
© 2020 Society of Chemical Industry and John Wiley & Sons, Ltd | Biofuels, Bioprod. Bioref. (2020); DOI: 10.1002/bbb.2142
R Gama Ferreira et al.
cellulases produced by T. reesei actually consist of multiple
enzymes (mostly cellobiohydrolases and endoglucanases);
similarly, the enzymatic product of Clostridium thermocellum
in the work of Zhuang and Marchant83 and that of Aspergillus
awamori in the work of de Castro et al.97 each comprises
various enzymatic activities. The recombinant production
of β-glucosidase by E. coli, in contrast, essentially delivers a
single enzyme. These differences should be considered when
comparing enzyme production processes.
Table 2 also provides the production cost of each enzyme
and its composition in terms of raw materials, utilities,
facility-dependent cost, etc. At first, we observe that in all
analyses, except for the SmC of Zhuang and Marchant,83
the facility-dependent / capital-related cost and the cost of
raw materials are the factors that most strongly impact the
enzyme cost, together accounting for 70% or more of the
total cost. In most cases, the facility-dependent / capital-
related cost is the main culprit, excepting the assessments
of NREL and Hong et al., in which the raw materials took
first place. Also notable is the significant contribution of
labor costs in the solid-state processes developed by Zhuang
and Marchant83 and de Castro et al.,98 equal to 26% and
18%, respectively. These results corroborate the common
perception that SSC is more labor-intensive.
Parameter sensitivity
The values indicated in Table 2 refer to the baseline scenarios
analyzed in each study. In reality, all those works performed
sensitivity analyses on key variables to evaluate their impact
on the final enzyme production cost. For instance, Ferreira
et al.,90 Hong et al.,80 Zhuang and Marchant,83 and de Castro
et al.98 detected the considerable impact of the enzyme
production scale on the enzyme cost (the larger the scale, the
lower the cost). Klein-Marcuschamer et al.,81 Ferreira et al.,90
and de Castro et al.98 evaluated the effect of the purchasing
price of the main fermentation substrate (poplar, glucose, and
babassu cake, respectively) on the enzyme cost and found this
parameter to be significant, too, which agrees with the large
contribution of raw materials to the enzyme cost shown in
most works listed in Table 2. Barta et al.,82 de Castro et al.,98
Ferreira et al.,90 and Hong et al.80 also analyzed the influence
of the enzyme yield on the enzyme cost, concluding that this
variable had a dramatic effect on the process economics.
As the sensitivity analyses discussed above indicate, many
intrinsic characteristics of each enzyme production process
explain the sharp disparities in value and composition of
enzyme costs across the works under consideration. For
instance, process designs that consider enzyme isolation
and concentration obviously tend to produce higher prices
than those that do not because of additional equipment, raw
9
10
Table 2. Process characteristics and enzyme cost composition of selected techno-economic analyses of industrial enzyme production.
Reference Klein- Humbird et al. Barta et al.82 Hong et al.80 Zhuang and Marchant83 Ferreira, Azzoni de Castro
Marcuschamer (NREL)88 and Freitas90 et al.98
et al.81
Process characteristics
R Gama Ferreira et al.

Enzyme product Cellulase mix Cellulase mix Cellulase mix Cellulase mix Cellulase mix β-glucosidase Multienzyme
Microbial platform Trichoderma Trichoderma Trichoderma Trichoderma Clostridium thermocellum Escherichia coli Aspergillus
reesei reesei reesei reesei (recombinant) awamori
Cultivation mode SmC SmC SmC SmC SmC SSC SmC SSC
3 3a
Reactor size (m ) 300 300 1.5–5.0 m ? 938 2431 100 ?
Number of reactors 12 7 24 ? 15 1 1 ?
Reactor material SS316 SS316 SS304 ? SS304 SS304 SS316 ?
Reactor price (103 US$) 510 400 2440b ? 205 2194 1460 ?
Inoculum volume 5% 10% 5% ? 1% 5% ?
Downstream processing Yes No No No (onsite)/yes Yes No Yes Yes
(offsite)
Culture duration (h) 192 120 108 ? 96 144 22 ?
Production titer (g/L) 50 50 11–35 ? 19 4 5 ?
Productivity (g∙L−1∙h−1) 0.26 0.42 0.10–0.32 ? 0.20 0.03 0.21 ?
Product yield (g protein/g 20% 24% 26% 21% 2.0% 2.5% 2.4%
carbon source)
Carbon source Glucose/poplar Glucose Spruce/ Glucose Paper pulp Glucose Babassu waste
Molasses
Inducer Poplar Sophorose Spruce ? Paper pulp IPTG Babassu waste
(from glucose)
Enzyme cost composition
Facility-dependent/capital-related 48% 21% +++ 20% 22% 63% 45% 43%
Raw materials/nutrients 28% 62% ++ 60% 12% 6% 25% 31%
Utilities/electricity 10% 13% + 15% 55% 2% 2% 4%
Consumables 4% 0% 0% 0% 0% 23% 5%

© 2020 Society of Chemical Industry and John Wiley & Sons, Ltd | Biofuels, Bioprod. Bioref. (2020); DOI: 10.1002/bbb.2142
Labor/fixed cost 7% 4% + 5% 9% 26% 4% 18%
Other costs 3% 0% 0% 1% 4% 1% 0%
Enzyme cost (US$ kg−1) 10 5 4 5 16 40 316 59
a
Size inferred from the total volume of 37–121 m3 divided into 24 vessels.
b
This value was found by converting 2.35 × 107 Swedish krona (SEK) to US dollars with the current (February 2020) exchange rate. Moreover, it was obtained from the work of
Olofsson et al.,79 which employed the same process model as Barta et al82.
Major process characteristics such as microbial platform, reactor size, and volumetric productivity for selected economic analyses of industrial enzyme production. The total
enzyme cost estimated in each work is also provided, as well as the cost composition. Note that the cost components have slightly different definitions depending on the
work under consideration; as such, some references80,82,88 break down the cost in terms of capital-related cost, cost of nutrients, electricity cost, and fixed cost. The other
references81,83,90,98 do so in terms of facility-dependent cost, cost of raw materials, cost of utilities, cost of consumables, labor cost, and other costs.
Review: On the cost of lignocellulose-degrading enzymes
 Review: On the cost of lignocellulose-degrading enzymes
material, labor, and utility costs. In the same vein, the use of
efficient microorganisms that display high enzyme yields and
volumetric productivities tends to rescue production costs.
Certain extrinsic characteristics, such as the process scale
and price data sources, also have a significant effect on both
the value and composition of the enzyme cost. Indeed, the
differences in orders of magnitude between the enzyme costs
of the smallest and largest processes in Table 1 are likely due
to differences in process scale, for the most part. With respect
to price data sources, there is also great variation across
the studies in question, especially in the case of equipment
prices. In general, prices may be quoted with manufacturers,
provided by process simulators (such as Aspen Icarus or
SuperPro Designer), obtained on governmental databases
or private websites, found in past peer-reviewed articles,
etc. Furthermore, prices also depend on the geographical
region under consideration, the year of analysis and the local
currency. In many instances, prices require adjustments to the
process scale as well (this is usually the case for equipment).
Consequently, it comes as no surprise that price estimations
vary widely from one work to another. This is especially
evident in the case of the main bioreactor, which is normally
the most expensive piece of equipment in a bioprocess and, as
a result, significantly impacts the capital-related contribution
of the enzyme cost. For instance, Klein-Marcuschamer et al.81
and Humbird et al.88 estimated US$ 510 k and 400 k for their
SS316, 300-m3 bioreactors, respectively, from manufacturer
quotes; Olofsson et al.,79 on the other hand, used Aspen Icarus
to price their 24 SS304 production bioreactors at a total value
of 2.35 × 107 SEK, which translates to approximately US$
2440 k – i.e., approximately US$ 100 k per fermenter. Ferreira
et al.90 estimated their SS316, 100-m3 bioreactor at US$1460 k
using SuperPro Designer, and Zhuang and Marchant83
assigned the values of US$205 k and 2194 k to their 938 m3
SmC reactor and 2431 m3 SSC reactor, respectively, obtained
through manufacturer quotes. These largely contradictory
figures suggest that the facility-dependent contribution
of enzyme cost should be evaluated with caution and that
uncertainty analyses that consider the price variation of key
pieces of equipment may be useful in future works.
Coproducts and cogeneration
It is worth mentioning that some works achieved noticeable cost
reductions by taking advantage of coproducts. In some process
simulations based on SSC, for instance, the solid material that
remains after cultivation is sold as a supplement for animal
feed.97,98 Additionally, in the designs of Klein-Marcuschamer
et al.81 and Barta et al.,82 residual biomass (particularly lignin)
is burned for energy generation (in the form of steam). These
examples support a growing consensus in the field of biobased
© 2020 Society of Chemical Industry and John Wiley & Sons, Ltd | Biofuels, Bioprod. Bioref. (2020); DOI: 10.1002/bbb.2142
R Gama Ferreira et al.
production of fuels and chemicals in which it is indispensable
to maximize process revenues by drawing upon the value of
coproducts and cogeneration of energy. In other words, a true
biorefinery approach is called for.103–105
Conclusions
Relatively few TEAs of the production of industrial enzymes
have been published in peer-reviewed literature over the last
two decades, despite the growing importance of industrial
enzymes for establishing the production of biobased
chemicals and fuels in the future. Moreover, the published
analyses are dominated by a single process: the production
of cellulases by T. reesei through submerged cultivation.
Assessments of enzyme production based on SSC, especially
ligninases, are also comparatively common.
Nevertheless, we observe quite diverse process assumptions
and characteristics across the works mentioned above, which
render comparisons complicated. In any case, it appears that
the facility-dependent cost and the cost of raw materials are
generally the main factors in the composition of the enzyme
cost, especially in processes based on submerged cultivation.
In solid-state processes, the labor cost becomes relevant as
well. Given that the facility-dependent cost strongly depends
on the equipment cost and that equipment prices show wide
variations across analyses, special attention should be given to
these price estimations. The employment of low-cost media,
such as those based on agricultural or forestry residues, may
also be beneficial, given that they entail lower costs of raw
materials. In fact, sensitivity analyses performed in several
studies indicate that the cost of the main fermentation
substrate, as well as the enzyme yield and production scale,
has a significant impact on the enzyme cost. The dramatic
effect of the enzyme yield on the enzyme cost, in particular,
demonstrates the importance of developing microorganisms
and processes that lead to high production yields. The extent
of the downstream section, which is closely related to the
choice between onsite and offsite production, also appears
to have a significant impact on enzyme cost. Finally, the
production cost of enzymes can be mitigated by taking
advantage of waste streams that can be converted into
valuable co-products or energy sources while simultaneously
improving their environmental impact.
Acknowledgements
The authors thank the Fundação de Amparo à Pesquisa do
Estado de São Paulo (FAPESP) (São Paulo, Brazil, grant
2014/13974-6) and Conselho Nacional de Desenvolvimento
Científico e Tecnológico – CNPq (Brazil, grants 165075/2017-
11
 R Gama Ferreira et al.
1, 307739/2015-5, and 304125/2018-0) for financial support.
The authors also thank the Coordenação de Aperfeiçoamento
de Pessoal de Nível Superior – Brazil (CAPES/PROEX) –
Finance Code 001 – for support. The authors acknowledge
Bianca C. Bussamra for providing the first version of Fig. 1.
Conflict of interests
The authors declare that they have no conflict of interests.
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386:121954 (2020). Adriano R. Azzoni
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in Industrial Biotechnology: Products and Processes, 1st
Chemical Engineering (2002), with
edn, ed. by Wittmann C, Liao JC, Lee SY, Nielsen J and
a 3 years period as a postdoctoral
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Germany, p. 85 (2016). researcher at the Institute for
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production of chemical products. Comput Chem Eng Portugal (2004-2007). He has 18 years
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MT et al., The techno-economic basis for coproduct professor at the Polytechnic School, University of São
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Sindelia F. Azzoni
coupled to bioenergies with potential to enhance business Sindelia F. Azzoni is a chemical
development of sustainable biorefineries. Renew Sustain engineer with a PhD in biotechnology
Energy Rev 70:793–804 (2017).
from the Technical University of
Lisbon. She has around 10 years
of experience in bioprocess
Rafael G. Ferreira development, focused on cellulolytic
enzymes and 2G biofuel production.
Rafael G. Ferreira received a PhD
She is currently with the Advanced Second Generation
in chemical engineering from the
Biofuels Laboratory at the University of Campinas, Brazil.
University of São Paulo in 2019,
working on process design and
economics of recombinant enzymes
for biomass hydrolysis. He currently
works as an applications engineer
for Intelligen Inc., which develops process simulation
software.

© 2020 Society of Chemical Industry and John Wiley & Sons, Ltd | Biofuels, Bioprod. Bioref. (2020); DOI: 10.1002/bbb.2142 15