Journal of Cleaner Production 369 (2022) 133340

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Journal of Cleaner Production

journal homepage: www.elsevier.com/locate/jclepro

Techno-economic evaluation of second-generation ethanol from sugarcane

bagasse: Commercial versus on-site produced enzymes and use of the xylose
liquor
R.R. Carpio a ,∗, S.G. Secchi a,b , R.O. Barros b , R.A. Oliveira b , S. Queiroz b , R.S.S. Teixeira b ,
E.P.S. Bon b , A.R. Secchi a
a
Chemical Engineering Program, Universidade Federal do Rio de Janeiro (UFRJ), Brazil
b
Bioethanol Laboratory, Universidade Federal do Rio de Janeiro (UFRJ), Brazil

ARTICLE INFO ABSTRACT

Handling Editor: Zhen Leng Biomass ethanol production presents shortcomings related to enzymes cost and efficiency of xylose fermen-
tation. Commercial enzymes used in second-generation ethanol (2GE) plants are produced off-site/offshore,
Keywords:
with the additional formulation, transportation and storage costs. Alternatively, lower cost cellulases can be
Second generation ethanol
Modeling and simulation
produced on-site. This study focused on the techno-economic evaluation of enzymes produced on-site versus
Techno-economic analysis commercial. Another limiting feature of the 2GE technology is the use of the hemicellulose sugars for ethanol
On-site cellulases production fermentation. Thus, this work addressed its use for biogas production. Process systems engineering tools were
C5 biodigestion used to evaluate four different configurations of a 2GE plant annexed to an already existent autonomous
distillery. The application of the methodology allowed useful perceptions of the ethanol and biogas production,
water and electricity consumption, and economic profitability. The need of a second fungus strain for the on-
site enzyme production was identified as the main bottleneck of this cellulase production process, which is an
important insight that could guide the future research efforts.

1. Introduction
In the past few years, the use of renewable lignocellulosic biomass
feedstock to produce fuel ethanol was the subject of intensive re-
search worldwide, fueling the inauguration of industrial-scale second-
generation ethanol (2GE) plants that use off-site produced enzymes.
Although commercial cellulases have improved significantly over the
past decade, the contribution of enzyme costs is still a challenge to the
economics of 2GE production (Johnson, 2016; Madhavan et al., 2021).
Nevertheless there is a consensus that the final enzyme cost con-
tribution should be around $0.40 per ethanol gallon (Valdivia et al.,
2016; Chandel et al., 2019), this is a quite uncharted territory because
the enzyme producing companies do not disclose information on the
enzymes cost contribution and sign confidential contracts directly with
2GE producers.
Alternatively, on-site production of cellulases has been presented as
a strategy to lower cellulase production cost and by extension the 2GE
enzymes cost contribution (Johnson, 2016; Ellilä et al., 2017). Johnson
(2016) showed that the use of cellulosic biomass to produce enzymes,
in an integrated cellulase and ethanol scenario, further decreases the
cost of cellulosic ethanol. These authors reported that cellulase costs
were reduced from $0.80 per ethanol gallon in an off-site approach
to $0.58 and $0.23 per ethanol gallon using an on-site or integrated
approach, respectively.
Recently, Ferreira et al. (2021) reviewed the techno-economic anal-
yses published since the year 2000 for the production of cellulases
and other lignocellulose degrading enzymes. These authors concluded
that the enzymes downstream processing, which is closely related to
the choice between on-site and off-site production, holds a signifi-
cant impact on enzyme cost. Indeed, in the on-site production, some
downstream processing steps such as primary recovery and stabilization
would not be required allowing cost decrease.
Moreover, the cost scenario does not relate solely to the biotech-
nological process for enzymes production and processing as enzymes,
usually purchased from an off-site and even offshore manufacturer,
requires transportation and storage under refrigerated conditions to
keep the biocatalyst stability for significant lengths of time that sig-
nificantly add to the overall enzymes cost. On the other hand, on-site
cellulase production not only circumvents the transportation and long
storage costs but is also strategically advantageous due to the flex-
ibility and reliability of enzyme supply. On-site cellulase production

∗ Correspondence to: Av. Horácio Macedo no 2030, Bloco G, Sala 116, Cidade Universitária, Rio de Janeiro, RJ, Brazil.
E-mail address: roymel@peq.coppe.ufrj.br (R.R. Carpio).

https://doi.org/10.1016/j.jclepro.2022.133340
Received 2 April 2022; Received in revised form 1 July 2022; Accepted 24 July 2022
Available online 3 August 2022
0959-6526/© 2022 Elsevier Ltd. All rights reserved.
R.R. Carpio et al.
has also shown a high potential for further greenhouse gases emission
reduction (Olofsson et al., 2017; Chandel et al., 2019).
Thus, to contribute to this cost-availability decisive conundrum, the
on-site cellulases production has been the subject of various studies
that have dealt with strain development, production processes and
the growth medium formulation (Ellilä et al., 2017; Li et al., 2022;
Singhania et al., 2021), the cost contribution of enzymes on 2GE
production and the techno-economic analysis via modeling and sim-
ulation (Junqueira et al., 2017; Bechara et al., 2018; Longati et al.,
2018).
Among the microorganisms that produce cellulolytic enzymes, the
filamentous fungus Trichoderma reesei RUT C-30 is an industrially im-
portant cellulolytic fungus and a reference in the production of cellu-
lases. However, this strain secretes a low activity of 𝛽-glucosidase (Sing-
hania et al., 2013; Pryor and Nahar, 2015), requiring strategies such as
supplementation with 𝛽-glycosidases and accessory enzymes from other
strains, as such Aspergillus awamori (Silva et al., 2009; Gottschalk et al.,
2010, 2013). Silva et al. (2020) found that enzymes produced by A.
awamori associated with enzymes produced by T. reesei or other com-
mercial enzymes promoted greater liquefaction and reduced viscosity
in the hydrolysis of lignocellulosic biomass and by extension increased
the release of fermentable sugar.
The production of biomass sugar syrups (the C6 sugars from the
cellulose and the C5 sugars from the hemicellulose) is followed by
ethanol fermentation to produce the 2GE. Nevertheless, the C6 sugars
are readily fermentable using the industrial Saccharomyces yeast strains
this does not hold true for the C5 sugars adding another layer of
difficulty to the 2GE technology. The main challenge regarding the
use of hemicellulose hydrolyzate (C5 sugars) is to obtain strains of
recombinant Saccharomyces that achieve high conversion of xylose and
tolerate high levels of inhibitors (Pereira et al., 2021).
Several reports have focused on the C5 ethanol fermentation us-
ing GMO yeasts (Kim et al., 2021; Sharma and Arora, 2020) and
bacteria (Baeyens et al., 2015). Nevertheless, the wealth of sound
scientific results that have been published and the development of C5
fermentations at industrial scale, the technology lacks robustness and
productivity in comparison to the use of the industrial yeast strains able
to efficiently ferment C6 sugars to ethanol.
In this scenario, a worthwhile alternative for the use of the C5 sugars
is the anaerobically digestion to produce biogas that was evaluated
by Bechara et al. (2016). In this study, the authors explored the xylose
biodigestion for producing biogas which is used in the cogeneration
system. The alternatives of xylose fermentation or biodigestion were
also assessed by Longati et al. (2018) and Carpio et al. (2019). More
recently, Fonseca et al. (2020) and Hilares et al. (2022) also evaluated
the use of the xylose liquor for fermentation and biodigestion processes,
respectively.
The Bioethanol Laboratory (LaBio, 2019) developed a proprietary
technology to obtain an enzyme cocktail able to faster hydrolyze sugar-
cane bagasse at high solids loads and industrial conditions (Silva et al.,
2009, 2016; Espinheira et al., 2022), which is a desired target of the
second-generation ethanol industry. Despite of the demonstrated effec-
tiveness and robustness of this enzyme cocktail, a suitability assessment
that considers the technical and economic aspects of the on-site produc-
tion and use of this enzyme cocktail, as an alternative to the current
standard, which is the purchase of commercial enzymes, had not been
conducted yet and was carried out in this study. In this context, the
C5 (xylose liquor) was considered for the production of either biogas
or ethanol. Process systems engineering tools were used to evaluate
four different configurations of a 2GE plant annexed to an already
existent autonomous distillery. The application of the methodology
allowed achieving useful insights regarding ethanol and biogas produc-
tion, water and electricity consumption, economic feasibility, and main
bottlenecks identification as a guide for future research efforts.
This paper is organized as follows. The methods applied for the
process modeling, simulation and economic analysis are detailed in
Section 2. The results of the numerical simulation and discussion are
presented in Section 3. Concluding remarks are presented in Sections
Section 4.
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Journal of Cleaner Production 369 (2022) 133340
2. Materials and methods
A detailed description of the methods used for the modeling, simu-
lation and economic analysis of the studied process is included in this
section.
2.1. General description of the process
The second-generation ethanol (2GE) plant was projected as an
annexed facility to an already existent standard autonomous distillery
as described by Carpio et al. (2019, 2020, 2021). The sugarcane bagasse
generated in the autonomous distillery is used as raw material for the
production of 2GE. The utilities (vapor, water and electricity) for the
2GE plant were also considered as supplied by the autonomous dis-
tillery. For the integration of both plants, the glucose syrup produced in
the 2GE plant is mixed with the sugarcane juice used for the production
of first-generation ethanol in the autonomous distillery.
The bagasse used for 2GE production is milled for particle size re-
duction before being submitted to a liquid hot water
pretreatment (Sousa Paredes et al., 2015; Longati et al., 2018). This
pretreatment was selected because it is attractive from a cost-savings
perspective (neither require the addition of chemicals or catalysts
nor high-cost materials and maintenance for reactors due to its low-
corrosion potential) (da Silva et al., 2013), it does not alter the
biomass glucan content (a glucose recovery rate of 97% was ob-
served for sugarcane bagasse pretreated using this method) (Laser
et al., 2002), it achieved suitable results in bench scale experimental
tests (LaBio, 2019) and has been adopted in commercial 2GE plants in
Brazil (GranBio, 2022; Raizen, 2022).
After this stage, a belt filter is used to separate the solid phase from
the liquid one. The extracted cellulose is in the solid fraction, whereas
the liquid phase contains the xylose and xylooligosaccharides. At this
point the solid and liquid phases take different process pathways. The
solid fraction is submitted to the enzymatic hydrolysis for converting
the cellulose into glucose, which is sent to the autonomous distillery
for ethanol production (Bechara et al., 2016). On the other hand, the
liquid phase may be fermented alone to produce ethanol or biodigested
to obtain methane biogas.
Four different process configurations were evaluated in this work.
The steps described above are the same for all configurations, which
differ in the source of enzymes and destination of xylose liquor. The
four configurations are defined below:
Conf. 1: On-site enzyme production and xylose biodigestion
(Fig. 1(a))
Conf. 2: On-site enzyme production and xylose fermentation
(Fig. 1(b))
Conf. 3: Commercial enzyme purchasing and xylose biodigestion
(Fig. 2(a))
Conf. 4: Commercial enzyme purchasing and xylose fermentation
(Fig. 2(b))
For the on-site production of enzymes, the patented process pro-
posed by Silva et al. (2009) was considered. This process describes
the production of a cellulolytic enzymes blend, which is cocktail of
enzymes secreted by the fungus Trichoderma reesei RUT C-30, able to
secrete around 1,000 FPU/L, and by the fungus Aspergillus awamori,
which produces 𝛽-glucosidase that is not secreted by Trichoderma ree-
sei (Pryor and Nahar, 2015; Singhania et al., 2013). In addition to
producing high levels of 𝛽-glucosidase, the fungus Aspergillus awamori
produces accessory enzymes that enhance the efficiency of enzymatic
hydrolysis (Gottschalk et al., 2010).
Therefore, the on-site enzyme production plant is composed by 2
units: the production unit from T. reesei and the production unit from
A. awamori. This units have the following procedure steps: preparation
R.R. Carpio et al.
Fig. 1. Process Flow Diagram in EMSO of: (a) Conf. 1 and (b) Conf. 2.
and sterilization of the culture medium; inoculum activation; cell prop-
agation; enzyme production; centrifugation, aiming at the separation of
cell biomass and enzymatic supernatant; and microfiltration to remove
microorganisms and insoluble solids remaining from the centrifugation
step. In the production unit from T. reesei, there is also an ultrafiltration
step, in order to concentrate the enzymatic supernatant.
The enzymatic preparations from both units are mixed to achieved
an activity exoglucanase and 𝛽-glucosidase ratio of 1:4, and the result-
ing blend is sent to the enzymatic hydrolysis reactor of the 2GE plant.
The PFD of this production process is shown in Fig. 3.
Regarding the xylose liquor, it could be used for biogas or ethanol
production. Both options are considered in this study. In the first
case, xylose liquor is sent to an anaerobic biodigester process (Ribeiro
et al., 2017). The produced methane is used as complementary fuel
to increase steam production. The second alternative considers the
concentration and fermentation of xylose liquor as described by Longati
et al. (2018). After that, the produced xylose wine is sent to the
distillation columns of the autonomous distillery, together with the
glucose wine.
2.2. Process model and simulation
The second-generation ethanol plant was built as an annexed facility
to an already existent first-generation autonomous distillery with a
processing capacity of 500 ton/h of sugarcane (Bechara et al., 2016).
Consequently, part of the sugarcane bagasse generated in the first-
generation autonomous distillery could be used as raw material for the
second-generation process.
Considering an average 14% of fibers in the sugarcane, approxi-
mately 70 t/h of dry bagasse (or 140 t/h of humid bagasse) are obtained
after the milling stage (Carpio et al., 2019). Despite some studies
consider the hydrolysis of more than 50% of the sugarcane bagasse
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Journal of Cleaner Production 369 (2022) 133340
obtained in first-generation ethanol (1GE) process (Bechara et al., 2016;
Longati et al., 2018; Carpio et al., 2021), in this work just 40% of the
generated bagasse (56 t/h of humid bagasse) is diverted to the 2GE
plant, in order fulfill the capacity of the distillation system of the al-
ready existent first-generation autonomous distillery. The composition
of the bagasse fed to the 2GE plant was set as the average observed
in experimental characterization of bagasse received from an industrial
plant located in São Paulo, Brazil.
The process was modeled using mainly mass balances, energy bal-
ances and fundamental equations. Nevertheless, some process parame-
ter such as yields, efficiencies and loss were obtained from experimental
results developed at the Bioethanol laboratory of Universidade Federal
do Rio de Janeiro.
The pretreatment stage was modeled as a yield reactor considering
the experimental results. On the other hand, the kinetic model of
the hydrolysis reactor proposed by Angarita et al. (2015) was used
in the hydrolysis stage. All the produced hexoses were simulated as
glucose (Dias et al., 2009). The parameters of this kinetic model were
estimated to fit the experimental results as shown in Fig. 4.
Regarding the on-site enzyme production process, the kinetic mod-
els proposed by Velkovska et al. (1997) and by Villena and Gutiérrez-
Correa (2012) were used to describe the production of enzymes by
Trichoderma reesei and by Aspergillus awamori, respectively. As this
enzyme production process works in batch operation, dynamic models
were implemented. However, it was adapted to continuous operation
through a global model, which describes a control volume around the
entire enzyme production plant to evaluate the inlet and outlet streams
of this plant.
The equation-oriented simulator EMSO (Soares and Secchi, 2003)
was used for modeling and simulation of the 2GE plant. The thermo-
dynamic models and components database used for this study were the
same of several previous studies (Longati et al., 2018; Oliveira et al.,
R.R. Carpio et al. Journal of Cleaner Production 369 (2022) 133340

Fig. 2. Process Flow Diagram in EMSO of: (a) Conf. 3 and (b) Conf. 4.

Fig. 3. PFD in EMSO of the on-site enzymes production.

2018; Elias et al., 2019) and are detailed in Tables S1 and S2 of the
Supplementary Material of Carpio et al. (2021). Both process and eco-
nomic models were coded using this software, allowing simultaneous
calculation of the Key Performance Indicators of the process (KPIs) and
economic indices, without needing auxiliary spreadsheet.
The parameters and variables specifications used in this study
were primarily obtained from real application of the patented enzyme
cocktail in several bench scale experimental tests involving pretreat-
ment and hydrolysis of sugarcane bagasse. It took years of previous
studies in the Bioethanol Laboratory of Federal University of Rio de
Janeiro (LaBio, 2019) to achieve the feasible operational conditions
used in this study. The main data used in the simulation of the global
process and in the on-site enzyme production process are shown in
Tables 1 and 2, respectively.
2.3. Economic analysis
The economic analysis involved the estimation of capital costs
(CAPEX), operating costs (OPEX) and cash flows (CF) by using the
methodology detailed on Peters et al. (2003).
The base equipment purchase costs were obtained from Humbird
et al. (2011) and online tools (Peters et al., 2019; Matches, 2019). These
base costs were transformed to final equipment costs by considering

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R.R. Carpio et al. Journal of Cleaner Production 369 (2022) 133340

Table 2
Main data used in the simulation of on-site enzyme production process.
Source: Experimental data from Bioethanol Laboratory of Universidade
Federal do Rio de Janeiro (LaBio, 2019).
Description Value
Trichoderma reesei
Temperature (o C) 30
Pressure (atm) 1
Reaction time (h) 96
Exoglucanase activity (FPU/L) 1,000
Aspergillus awamori
Temperature (o C) 30
Pressure (atm) 1
Reaction time (h) 168
𝛽-glucosidase activity (IU/L) 15,000

Plant Cost Index of update year and original purchase year; and 𝑓𝑖𝑛𝑠𝑡 is
the installation factor.
The operating costs (OPEX) and cash flow (CF) were estimated
based on the raw material and utilities consumption and the ethanol
production (which are also results of the simulation). Other premises
considered for the profitability estimation are reported in Table 3.

3. Results and discussion

Table 4 summarizes the main simulation results of each configura-

tion.
The highest values of ethanol production and yield are achieved in
Conf. 2 and Conf. 4, since they exploit the xylose liquor to produce
additional ethanol via fermentation of xyloses. Consequently, the use
of this alternative allows increasing the bagasse-to-ethanol yield on
approximately 27%. Unfortunately, this fact has a negative impact on
the capital and operating cost as will be discussed later.
Fig. 4. Comparison of experimental results and kinetic model prediction of the On the contrary, Conf. 1 and Conf. 3 use the xylose liquor for
hydrolysis stage for (a) glucose and (b) xylose. biogas generation. Conf. 1 produces the higher volume of biogas due
to the use of not only xylose liquor but also the residuals of the on-
Table 1 site enzyme production process. Surprisingly, Conf. 2 produces more
Main data used in the simulation of the global process.
Source: Experimental data from Bioethanol Laboratory of Universidade
biogas than Conf. 3, demonstrating that most of biogas generation is
Federal do Rio de Janeiro (LaBio, 2019). due to residuals of on-site enzyme production instead of xylose liquor.
Description Value Notice that Conf. 4 does not produce biogas because the xylose is used
Bagasse composition
for ethanol production and the commercial enzyme is supplied, thus
Cellulose (% w/w) 22.50 there are no residuals of the on-site enzyme production process.
Hemicellulose (% w/w) 14.37 It can be observed that Conf. 2 and Conf. 4 present a lower spe-
Lignin (% w/w) 12.49 cific water consumption. This is because part of the process water is
Ash (% w/w) 0.64
Water (% w/w) 50.00
recovered by evaporation in the xylose liquor concentration step. It may
Hydrothermal Pretreatment also be noticed that Conf. 1 consumes more water than Conf. 3, and
Temperature (◦ C) 190 that Conf. 2 consumes more water than Conf. 4, as the on-site enzyme
Pressure (atm) 25 production plant demands a considerable amount of additional water.
Solids fraction (% w/w) 10
The highest consumption of vapor is presented in Conf 2 and Conf.
Residence time (min.) 10
Hemicellulose to xylose yield (% w/w) 31.83 4. These results are expected since they involve the utilization of
Cellulose to glucose yield (% w/w) 4.37 evaporators for concentrating the xylose liquor before the fermentation
Enzymatic Hydrolysis stage. Conf. 2 has the highest value because of the use of additional
Temperature (◦ C) 50
vapor on the on-site enzyme production process. This is the same reason
Pressure (atm) 1
Solids fraction (% w/w) 15 for Conf. 1 having higher vapor consumption than Conf. 3.
Residence time (h) 48 Regarding the electricity consumption, its higher demand on the
Enzyme loading (FPU/g of biomass) 9 on-site enzyme production section compared to the rest of the process
is notable. This results from the large size of the on-site enzyme
production plant, whose high consumption of electricity occurs mainly
scaling exponents, update index and installation factors using Eq. (1). in the bioreactors agitation and aeration operations. Furthermore, it is
( ) ( ) observed that the specific electricity consumption of Conf. 2 is lower
𝑆𝑖𝑧𝑒𝑎𝑐𝑡𝑢𝑎𝑙 𝑠𝑒 𝐶𝐸𝑃 𝐶𝐼 𝑝𝑟𝑒𝑠𝑒𝑛𝑡 𝑦𝑒𝑎𝑟 than that of Conf. 1, despite having a larger number of equipment that
𝐶𝑜𝑠𝑡𝑐𝑜𝑟𝑟𝑒𝑐𝑡𝑒𝑑 = 𝐶𝑜𝑠𝑡𝑏𝑎𝑠𝑒 𝑓𝑖𝑛𝑠𝑡 (1)
𝑆𝑖𝑧𝑒𝑏𝑎𝑠𝑒 𝐶𝐸𝑃 𝐶𝐼 𝑏𝑎𝑠𝑒 𝑦𝑒𝑎𝑟 demand electricity. This is justified by the higher production of 2GE at
were 𝐶𝑜𝑠𝑡𝑐𝑜𝑟𝑟𝑒𝑐𝑡𝑒𝑑 and 𝐶𝑜𝑠𝑡𝑏𝑎𝑠𝑒 are the final and base equipment costs; Conf. 2, that makes the consumption of electricity per liter of ethanol
𝑆𝑖𝑧𝑒𝑎𝑐𝑡𝑢𝑎𝑙 and 𝑆𝑖𝑧𝑒𝑏𝑎𝑠𝑒 represent the actual (which is a simulation out- produced to be lower.
put) and original equipment size, 𝑠𝑒 is the scaling exponent; The capital and operating costs of the four configurations are de-
𝐶𝐸𝑃 𝐶𝐼 𝑝𝑟𝑒𝑠𝑒𝑛𝑡 𝑦𝑒𝑎𝑟 and 𝐶𝐸𝑃 𝐶𝐼 𝑏𝑎𝑠𝑒 𝑦𝑒𝑎𝑟 are the Chemical Engineering tailed in Table 5.

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R.R. Carpio et al. Journal of Cleaner Production 369 (2022) 133340

Table 3
Main premises considered in the economic analysis.
Description Value Reference
Ethanol selling price (US$/m3 ) 529.72 MAPA (2019)
Electricity selling price (US$/MWh) 46.23 CCEE (2019)
Commercial Enzyme cost (US$/kg) 2.75 Novozymes Cellic CTec3
Fresh water cost (US$/t) 0.06 Bechara et al. (2016)
Refrigeration cost (US$/kW) 0.04 Bechara et al. (2016)
Operation time (h/year) 6,120 Personal communication
Tax Rate (%) 34 Carpio et al. (2021)
Discount rate (%) 8 Carpio et al. (2021)
Depreciation strategy (%/year) 10 Carpio et al. (2021)
Maintenance costs (% of Fixed Capital/year) 5 Bechara et al. (2016)
Overhead costs (% of Fixed Capital/year) 1 Bechara et al. (2016)

Table 4
Simulation results of the four configurations.
Description Conf. 1 Conf. 2 Conf. 3 Conf. 4
Ethanol production (m3 /year) 29,730.5 37,718.6 29,730.6 37,718.8
Ethanol yield (L/t dry bagasse) 173.50 220.11 173.50 220.11
Biogas production (t/year) 62,009.8 48,710.3 20,797.8 0
Lignin production (t/year) 131,262 131,262 123,449 123,449
Water consumption (kg/L) 61.01 21.35 52.33 13.92
Vapor consumption (kg/L) 24.05 37.80 21.95 32.16
Electricity consumption (kWh/L) 7.59 6.26 0.91 0.99

Table 5 Trichoderma strain able to excrete around 10 FPU/mL on the fermented

Capital and operating costs of the four configurations.
Description Conf. 1 Conf. 2 Conf. 3
Capital Cost (MM US$) 253.007 261.298 69.321
Operating Cost (MM US$) 268.944 270.374 32.461
It is noticed that the operating costs of Conf. 1 and Conf. 2 are
about 88% higher than the operating costs of Conf. 3 and Conf. 4.
Therefore, in order to identify the sources of the high operating cost of
these configurations, it is worth to open the relative weight of the dif-
ferent elements that constitute the operating cost of each configuration
(Fig. 5).
Analyzing Figs. 5(a) and 5(b), it is possible to realize that more than
80% of the operating cost of Conf. 1 and Conf. 2 refers to the raw
materials and consumables cost from the on-site enzyme production
process, with 62.73% of this cost from the enzyme production unit
from Aspergillus awamori and 37.27% from the cellulase production unit
from Trichoderma reesei. The operating costs that weigh the most for
configurations 3 and 4 are the cost of commercial enzymes, followed
by the cost of bagasse, which corresponds to approximately 43% and
36% of the operating cost, respectively.
The results presented in Table 5 and Fig. 5 indicate that the on-site
enzyme production process has a large impact on the total production
cost of the 2GE, caused by raw materials and capital costs, due to the
large size of the tanks, related to the low enzyme activities achieved
in the Trichoderma reesei fermented broth. For this reason, an analysis
was carried out to verify the impact of the exoglucanase activity in
the fermented broth on the enzymes production cost, as well as the
impact of using two strains of cellulase-producing fungi (T. reesei and
A. awamori) compared to the use of a single hypothetical strain, based
on Trichoderma reesei (same culture medium and reaction time), but
able to produce all the necessary cellulolytic enzymes. For making this
analysis, the parameter related to the exoglucanase activity achieved
on the fermentation broth was fixed at several different values (from
1 to 20 FPU/ml) and the simulation was repeatedly executed, being
possible to obtain the enzyme production cost, which is an output of
the simulation, for each case. The results of this analysis are shown in
Fig. 6.
From Fig. 6, it is possible to infer that the ideal scenario for this
process would be: (i) use of a single fungus strain to produce the
enzyme blend and (ii) to use a high exoglucanase activity producing
broth. In this scenario, the reduction in production cost compared to
Conf. 4
the current scenario (use of two strains and an activity of 1 FPU/mL)
71.756 would be over 20 times, that is a challenging scenario. However, the
33.012
use of a single strain of fungus would already allow a very significant
cost reduction compared to the current cost of the process, even with
low exoglucanase activity in the broth. This indicates that the need
to supplement the Trichoderma reesei enzymes with enzymes secreted
from a second fungus strain is the main bottleneck in this cellulase
production process. Therefore, advanced molecular biology and genetic
engineering techniques can be great allies in the pursue of a strain
capable of producing all the cellulolytic enzymes required to hydrolyze
lignocellulosic biomass, which has not been an easy task. Besides, the
Trichoderma enzymes supplementation by the Aspergillus enzymes does
not relate solely to the contribution of 𝛽-glucosidase but also to an
array of hemicellulolytic enzymes and accessory proteins that play an
important role on the synergy of biomass enzymatic hydrolysis.
The operating cash flow of the four configurations are detailed in
Table 6. Notice that the Operating Cash Flow of all configurations is
negative, thus the process is unfeasible from the economic point of
view at the current technological status. It is worth mentioning that
the economic analysis did not consider alternative incomes that could
be earned from the carbon credit market due to the second-generation
ethanol production. Nevertheless, the current economic unprofitability
reinforces the need of continuing the research for more profitable
enzymes and the accomplishment of optimization studies of the process
as a whole.
4. Conclusions
In this work, process systems engineering tools were used to evalu-
ate the technical and economical aspects of a second-generation ethanol
plant annexed to an already existent standard autonomous distillery.
Alternative configurations of the 2GE plant, which consider the on-site
production of the patented enzyme cocktail of Bioethanol Laboratory or
the buying of commercial one and the fermentation or the biodigestion
of xylose, were evaluated from the technical and economic point of
views. The results of the techno-economic assessment supported some
conclusions.
The configurations that exploit the xylose liquor to produce ad-
ditional ethanol via fermentation of xyloses (Conf. 2 and Conf. 4)

6
R.R. Carpio et al.
Fig. 5. Operating cost composition of each configuration: (a) Conf. 1, (b) Conf. 2, (c) Conf. 3 and (d) Conf. 4.
Table 6
Operating cash flow of each configuration.
Description
Incomes (MM US$)
Operating Cost (MM US$)
Operating Cash Flow (MM US$)
Fig. 6. Enzymes production cost in relation to exoglucanase activity on the broth,
using one and two strains of cellulase-producing fungi.
achieve the highest values of ethanol production, allowing to increase
the bagasse-to-ethanol yield on approximately 27%.
The residuals of the on-site enzyme production process have a great
potential for biogas production, but this process demands a consid-
erable amount of additional water and electricity. Besides, the low
FPU concentration of the Trichoderma fermentation and the use of two
fungus strains lead to high raw materials and capital costs of the on-site
enzyme production process.
A Hypothetical scenario considering the use of a single fungus strain
which produces exoglucanase activity on the fermented broth of around
Journal of Cleaner Production 369 (2022) 133340
Conf. 1 Conf. 2 Conf. 3 Conf. 4
24.653 27.979 21.872 24.687
268.944 270.374 32.461 33.012
−244.290 −242.395 −10.589 −8.324
10 FPU/mL would lead an enzyme cost reduction of over 20 times
compared to the current scenario. Particularly, the use of a single
strain of fungus would already allow a very significant cost reduction,
even keeping the low exoglucanase activity in the broth. This indicates
that the need of a second fungus strain is the main bottleneck of this
cellulase production process, which is an important insight that could
guide the future research efforts.
The process is unfeasible from the economic point of view at the
current technological status. This fact reinforces the need of continuing
the research for more profitable enzymes and the accomplishment of
optimization studies of the process as a whole.
CRediT authorship contribution statement
R.R. Carpio: Conceptualization, Methodology, Software, Data cura-
tion, Writing – original draft, Writing – review & editing, Visualization.
S.G. Secchi: Conceptualization, Methodology, Software, Data curation,
Writing – original draft, Writing – review & editing, Visualization.
R.O. Barros: Conceptualization, Investigation, Data curation, Writing –
review & editing. R.A. Oliveira: Conceptualization, Investigation, Data
curation, Writing – review & editing. S. Queiroz: Conceptualization, In-
vestigation, Data curation, Writing – review & editing. R.S.S. Teixeira:
Conceptualization, Methodology, Validation, Writing – original draft,
Writing – review & editing, Supervision. E.P.S. Bon: Conceptualization,
Methodology, Validation, Writing – original draft, Writing – review &
editing, Supervision, Project administration, Funding acquisition. A.R.
Secchi: Conceptualization, Methodology, Validation, Resources, Writ-
ing – original draft, Writing – review & editing, Supervision, Project
administration.
7
R.R. Carpio et al.
Declaration of competing interest
The authors declare that they have no known competing finan-
cial interests or personal relationships that could have appeared to
influence the work reported in this paper.
Data availability
Data will be made available on request.
Acknowledgment
Financial support from FINEP (grant No 01.09.0566.001421/08) are
gratefully acknowledged.
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