Fermentative Production of Ethanol Using Pinus patula as Raw Material: Economic and Energy Assessment

Abstract

The production of cellulosic ethanol has been gaining attention in the industry sector because of the high availability of lignocellulosic biomass from agricultural and forestry activities. Pinus patula is one of the most typical softwood species in Colombia. The aim of this work is to evaluate the production of ethanol using Pinus patula as raw material using dilute acid pretreatment and enzymatic hydrolysis to produce sugars able to be used as substrate for the strain Saccharomyces cerevisiae. Three fermentation configurations were selected to evaluate the performance of the microorganism: configurations 1 and 2 used glucose in a percentage of 80%w/v and 70%w/v, respectively, as substrate to establish the adaptation requirements of the microorganism. The configuration 3 considered the use of concentrated P. patula hydrolysate. An experimental yield of 0.364 ± 0.009 g ethanol/g sugar (73% of the theoretical) was obtained. Additionally, the economic and energetic comparison between the biochemical (ethanol production through fermentation) and thermochemical (synthesis gas through gasification) pathways to produce bioenergy was performed through simulation approaches. As main results, a higher ethanol production cost (1.53 USD/L) was obtained in comparison to the market price (0.77 USD/L) and a low energy efficiency (20%). Different alternatives such as waste integration and energy incentives must be considered in order to produce ethanol in a feasible way.

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Waste and Biomass Valorization
https://doi.org/10.1007/s12649-018-0494-4
ORIGINAL PAPER
Fermentative Production ofEthanol Using Pinus patula asRaw
Material: Economic andEnergy Assessment
CarlosA.García‑Velásquez1· EstefannyCarmona‑Garcia1· AshleySthefaníaCaballero1· JuanC.Solarte‑Toro1 ·
JimmyA.Martínez‑Ruano1,2· CarlosA.Cardona1
Received: 6 February 2018 / Accepted: 24 October 2018
© Springer Nature B.V. 2018
Abstract
The production of cellulosic ethanol has been gaining attention in the industry sector because of the high availability of
lignocellulosic biomass from agricultural and forestry activities. Pinus patula is one of the most typical softwood species in
Colombia. The aim of this work is to evaluate the production of ethanol using Pinus patula as raw material using dilute acid
pretreatment and enzymatic hydrolysis to produce sugars able to be used as substrate for the strain Saccharomyces cerevisiae.
Three fermentation configurations were selected to evaluate the performance of the microorganism: configurations 1 and 2
used glucose in a percentage of 80%w/v and 70%w/v, respectively, as substrate to establish the adaptation requirements of
the microorganism. The configuration 3 considered the use of concentrated P. patula hydrolysate. An experimental yield of
0.364 ± 0.009g ethanol/g sugar (73% of the theoretical) was obtained. Additionally, the economic and energetic comparison
between the biochemical (ethanol production through fermentation) and thermochemical (synthesis gas through gasification)
pathways to produce bioenergy was performed through simulation approaches. As main results, a higher ethanol production
cost (1.53USD/L) was obtained in comparison to the market price (0.77USD/L) and a low energy efficiency (20%). Different
alternatives such as waste integration and energy incentives must be considered in order to produce ethanol in a feasible way.
Keywords Ethanolic fermentation· Energy and economic assessment· Biochemical versus thermochemical routes
State ofNovelty
Pinus patula is a softwood that is highly available in Colom-
bia and other countries. Nevertheless, the application of this
raw material has been limited to furniture making and direct
energy production through combustion. The high cellulose
and hemicellulose content of P. patula as well as its process-
ing residues (having the same composition) evidences the
possibility to obtain different added-value products through
different platforms (i.e. sugar extraction and gasification).
Therefore, this work presents a technical, economic and
energetic assessment of the ethanol production using P. pat-
ula as raw material in order to determine the main technical
concerns (experimental procedure), economic bottlenecks
(processing costs) and energy constraints (valorization of
residues).
Introduction
Pinus patula (PP) is one of the types of softwood with high
production in Colombia because of its fast growth and high
crop yield among the forest plantations [1]. Pinus patula has
a high content of cellulose and hemicellulose, between 40
and 50% on dry basis [2]. However, one of the disadvantages
of most softwoods is the high lignin content (approximately,
25% on dry basis) that hinders the application of this type
of raw material to obtain bio-based materials. Consequently,
the use of softwood as feedstock for the direct production
of energy (thermochemical route) is very common due to
the easy treatment requirements [3]. Thermochemical routes
Electronic supplementary material The online version of this
article (https ://doi.org/10.1007/s1264 9-018-0494-4) contains
supplementary material, which is available to authorized users.
* Carlos A. Cardona
ccardonaal@unal.edu.co
1 Instituto de Biotecnología y Agroindustria, Departamento
de Ingeniería Química, Universidad Nacional de Colombia
Sede Manizales, Cra. 27 No. 64-60, Manizales, Colombia
2 School ofBiochemical Engineering, Pontificia Universidad
Catolica de Valparaíso, Av. Brasil, 2085Valparaíso, Chile

Waste and Biomass Valorization
1 3
involve different processes such as pyrolysis, combustion,
and gasification in order to produce different species that
can be used as platform for different products such as elec-
tricity, steam and liquid fuels through the Fischer–Tropsch
process [4]. On the other hand, the growing interest in the
production of cellulosic ethanol is related to the advances
in different pretreatment methods (chemical, enzymatic and
physical) that allow the production of fermentable sugars,
which are considered a promising substrate for microorgan-
isms to produce different added-value products, such as
ethanol [5–7]. The most studied application of softwood in
fermentative processes is the production of ethanol on labo-
ratory [8, 9] and commercial scales [10] due to the content
of cellulose, hemicellulose and lignin. In fact, softwoods
contain around 43–45% cellulose, 20–23% hemicellulose
and 25–28% lignin. Theoretically, around 410L of ethanol
can be produced per metric ton of dry raw material using
only the hexose fraction and 455L if all carbohydrates are
considered [8].
In this context, Söderström et al. [11] investi-
gated the effect of the two-step steam pretreatment of
SO2-impregnated softwood (Picea abies.) on the ethanol
yield at different severities. Additionally, the effectiveness
of the developed pretreatment was assessed by both enzy-
matic hydrolysis of the solids and simultaneous sacchari-
fication and fermentation (SSF) of the whole slurry. From
the enzymatic hydrolysis, an overall sugar yield of 80% was
reached. In the SSF configuration, an overall ethanol yield of
69% was achieved. Hoyer etal. [9] studied the production of
ethanol using spruce as raw material. Based on the content
of fermentable sugars in the fermenter, a theoretical yield
of 81% was reached using a water-insoluble solids (WIS)
content of 12% and SSF. Furthermore, Cellunolix® plant
located in Kajaan (Finland) produces 10million L of ethanol
per year on an industrial scale using sawdust and recycled
wood as raw material [12].
On the other hand, the thermochemical pathway com-
prises the direct conversion of the raw material into inter-
mediate products (i.e. synthesis gas and bio-oil) that can
be used as platform for the production of energy (heat and/
or electricity). The most important thermochemical pro-
cesses that have been used to evaluate the energy potential
of softwood are pyrolysis, combustion, and gasification.
Pyrolysis includes the conversion of biomass into char,
gas and a liquid composed of a mixture of hundreds of
oxygenated organic compounds, which is known as bio-oil
[13–15]. Combustion involves three phenomena: evapora-
tion of the water, volatilization at 200°C and combustion
at 500°C [16, 17]. Instead, gasification converts biomass
into a gaseous mixture (i.e., hydrogen, methane, carbon
monoxide and carbon dioxide), small quantities of char
and condensable compounds [18–20]. The use of softwood
for one of these pathways will depend on different factors
such as environmental friendliness, energy efficiency and
economic profitability. In this paper, the main goal of the
production of energy using P. patula will be the economic
and energetic comparison of two technological routes
(biochemical and thermochemical). Different authors
have evaluated the economic performance of the ethanol
and electricity production using softwood[21]. Moncada
etal. [2] performed the techno-economic analysis of the
ethanol production using P. patula as raw material, obtain-
ing a production cost of 1.08USD/L. Furthermore, other
authors have evaluated the production cost of electricity
through gasification (5USD/kWh) and direct combustion
(7.2USD/kWh) using as raw material softwood [22].
The aim of this work is to evaluate the potential use of
P. patula for the production of ethanol using two different
approaches: experiments and simulation. First, a series of
experiments were carried out involving dilute acid pre-
treatment followed by an enzymatic saccharification. Then,
C6-sugar fermentation was carried out using the strain Sac-
charomyses cerevisiae to obtain ethanol. Additionally, the
simulation of the ethanol production was performed using
the software Aspen Plus v9.0. Subsequently, the economic
assessment of the fermentative production of ethanol was
evaluated at different process scales. Finally, the energy
comparison between the biochemical and thermochemi-
cal pathways for energy production using P. patula as
raw material was performed based on the energy poten-
tial of the produced ethanol and the electricity generation
through gasification.
Materials andMethods
In this section, two procedures are presented aiming to
evaluate the production of ethanol using P. patula as raw
material. The characterization of the raw material, the
pretreatment (i.e. dilute acid hydrolysis and enzymatic
saccharification) and the ethanol production (i.e. fermen-
tation) are categorized in “Experimental Procedure” sec-
tion. The main purpose of this section is to summarize the
methods, protocols, and conditions used in each one of the
experimental procedures. On the other hand, the simula-
tion process (“Simulation Procedure” section) involves the
use of the obtained results from the experimental assays
as well as the reported data in the literature for ethanolic
fermentation of softwood as input for the economic and
energy assessment of the biochemical route for ethanol
production. Additionally, the gasification of P. patula was
simulated in order to compare the ethanol fermentation
from the energetic point of view.

Waste and Biomass Valorization
1 3
Experimental Procedure
Raw Material
Pinus patula (PP) was collected from a farm located
in the central western region of Colombia (5°03′58″N
75°29′05″W) and dried at room temperature (18–23°C).
The wood was milled with an upper vibratory disk mill
(Retsch SR 200) and sieved to a particle diameter of 400µm.
The chemical characterization of P. patula was taken from a
previous work García etal. [23].
Methods
Reagents For the pretreatment of P. patula, sulfuric acid
96% (MERCK) and distillate water were used. In the
enzymatic hydrolysis, cellulases (Celluclast® 1.5 L),
multienzyme complex (Viscozyme® L), citric acid (Dis-
proalquimicos) and sodium citrate (CARLO ERBA) for
the buffer solution, and sodium azide solution 2% were
used. Freeze-dried Saccharomyces cerevisiae strain, d(+)-
glucose (MERCK), ammonium sulfate (Panreac), potas-
sium dihydrogen phosphate (MERCK), magnesium sulfate
(MERCK), calcium chloride (Panreac) and distillate water
were used in the fermentation. For the determination of sug-
ars, 3,5-dinitrosalicylic acid (MERCK), sodium hydroxide
(MERCK), potassium sodium tartrate (Panreac) and distil-
late water were employed.
Dilute‑Acid Pretreatment ofP. patula The raw material was
mixed with a dilute solution of sulfuric acid (2%v/v) in a
solid to liquid (S/L) ratio of 1:10. The procedure was carried
out in an autoclave at 121°C and 15 psig during 90 min,
which are typical operation conditions that ensures a high
hemicellulose solubilization and low inhibitors production
[24]. After the acid pretreatment, a vacuum filtration was
performed aiming to separate the solid and liquid fraction
(i.e., hydrolysate). The solid fraction was washed using tap
water until reach a pH near to 4.8, which is the pH condi-
tion of the enzymatic hydrolysis. This fraction was not dried
to avoid re-crystallization of amorphous cellulose and to
ensure a high sugars yield from this process [25]. Instead,
the liquid fraction from the dilute acid pretreatment was
cooled to room temperature and stored until sugars analysis.
Enzymatic Saccharification Enzymatic hydrolysis was car-
ried out following the protocol NREL/TP-510-42629 [26]
with a commercial cellulase (Celluclast® 1.5L) and multi-
enzyme complex (Viscozyme® L). The enzymatic hydroly-
sis was carried out using 30g of the solid fraction from the
dilute acid pretreatment. For this, it was used an enzyme
dosage of 64 filter paper units (FPU) per g of cellulose and
60 p-nitrophenyl-β-glucoside units (pNPGU) per g of cellu-
lose for Celluclast 1.5L and Viscozyme L, respectively. The
reaction volume was 2L, the pH was adjusted using a 0.1M
citrate buffer solution (pH 4.8–5.0). Finally, a 2%v/v sodium
azide solution was added to prevent the growth of organ-
isms during the saccharification. The operative conditions
for the enzymatic hydrolysis process were 72h at 50°C and
100rpm. Afterward, the liquid C6-rich hydrolysate was sep-
arated by vacuum filtration. Then, this hydrolysate was used
as substrate for the fermentation process.
Ethanolic Fermentation Freeze-dried S. cerevisiae was
used for the production of ethanol. The microorganism was
able to intake C6 sugars from the saccharification process.
Different fermentative configurations (three mediums) were
prepared in 200 mL Erlenmeyer flasks (100mL working
volume): sugar concentration of 15g/L (80% glucose and
20% hydrolysate), sugar concentration of 10g/L (70% glu-
cose and 30% hydrolysate) and total (100%) hydrolysate.
The mediums were supplemented with minerals: 1.5 g/L
(
NH
4)2
SO
4
, 1 g/L
KH2PO4
, 0.3 g/L
MgSO4
and 0.05g/L
CaCl2
. Experiments were carried out at 32°C and 150rpm
in an incubator (BINDER Incubator BD056) with a shaker.
Analytical Quantification The amount of sugar in the sam-
ple was determined using the dinitrosalicylic acid (DNS)
method, where the absorbance was measured at a wave-
length of 540nm according to the procedure proposed by
Miller [27]. The biomass concentration (g/L) was deter-
mined by dry weight of yeast. The concentration of ethanol
was determined using a GC-2014 (Shimadzu) gas chroma-
tograph equipped with a flame ionization detector (FID) and
a capillary column Stabilwax. The operating conditions of
the gas chromatograph were: injection volume 10µL, injec-
tor temperature 220°C, detector temperature 270°C, nitro-
gen as carrier gas with a flow in the column of 0.5mL/min
and running time of 21.75min.
Simulation Procedure
The production of ethanol using P. patula as raw material
was simulated based on the results from the experimen-
tal section and complemented with data reported in the
literature. For this purpose, the dilute acid pretreatment,
enzymatic saccharification and ethanolic fermentation
were conceptually designed using main simulation tools as
software Aspen Plus V9.0 (Aspen Technology, Inc, USA),
which allows calculating the mass and energy balances of
the process scheme. The behavior of the pretreatment, sac-
charification and fermentation were described based on data
reported in the literature from different authors (see, “Eth-
anolic Fermentation” section) [28, 29]. Then, the kinetic
models of these stages were modeled using the computa-
tional tool Matlab (MathWorks, USA). The properties of

Waste and Biomass Valorization
1 3
missing components in the Aspen Plus Database were taken
from external databases, especially the work developed by
Wooley and Putsche [30]. The selection of the thermody-
namic method was based on previous works [31], where
the Non-Random Two Liquid (NRTL) model was used to
describe the behavior of the liquid phase. For calculations,
a mass flow rate of 3300 ton/day of P. patula (wet basis)
and a moisture content of 40% were used in the simulation
procedure.
The results from the simulation procedure were used as
starting point for the economic and energetic analysis. In
the economic analysis, the contribution of the total capital
costs and the operating costs (variable and fixed) to the total
production cost of the process was assessed. Furthermore,
the ethanol production cost from the evaluated process was
compared with other studies that deal with the production of
ethanol using different pretreatment configurations and raw
materials [32, 33]. Since most forest materials (hardwood
and softwood) are commonly used for the direct produc-
tion of energy through thermochemical processes (i.e. com-
bustion and gasification), the energy analysis was focused
on the potential energy used from P. patula for bioenergy
production through a biochemical pathway (fermentation)
and its comparison with a common thermochemical route
(i.e. gasification). Therefore, the gasification of P. patula
was also simulated but only for the energy comparison. The
energy efficiency of both processes was calculated based
on the relation between the energy content of the products
(ethanol and synthesis gas) and the energy content of the
feedstock [3]. A detailed description of the economic and
energy assessment is presented in “Economic Assessment”
and “Energy Analysis” section, respectively.
Ethanolic Fermentation
Pinus patula is initially chipped and dried in order to achieve
the particle size and moisture content required for the fer-
mentation (see, “Ethanolic Fermentation” section). Subse-
quently, the physical treated raw material is submitted to a
two-step hydrolysis process in order to achieve the extraction
of sugars from the lignocellulosic matrix. In the first stage,
the hemicellulose fraction is hydrolyzed with dilute sulfuric
acid (2% by weight) at a temperature of 121°C. This stage
was modeled using the kinetic model and the parameters
reported by Rafiqul etal. [34] (see, Supplementary material
TableS1). Moreover, the kinetic model was solved using the
residence time specified in the experimental section (i.e.,
90min) aiming to obtain similar concentrations in compari-
son with those quantified in the experimental dilute acid pre-
treatment. Once the model was solved using the experimen-
tal conditions, the process was modeled in the Aspen Plus
V9.0 simulation software and the flowsheet was completed.
From this pretreatment, a non-converted solid fraction and
a rich-pentose liquor are obtained. This stream is separated
by filtration. The liquor is mainly composed of xylose and
glucose. Although, this stream was not considered for fer-
mentation since the strain S. cerevisiae used in this study
was not able to degrade xylose. The solid fraction, rich in
cellulose and lignin, is sent to an enzymatic saccharification
process at 50°C using cellulase as enzyme, which is able to
convert the cellulose to glucose for further use as substrate
in the ethanolic fermentation. The enzymatic hydrolysis
was modeled based on the experimental yields and using
the kinetic model and parameters reported by Khodaverdi
etal. [35] and Kadam etal. [36] (see, Supplementary mate-
rial TableS2). As in the case of the dilute acid pretreatment,
the experimental conditions applied to the enzymatic sac-
charification were used as input data in the kinetic model.
In fact, the cellulose, cellobiose and glucose concentration
profiles were calculated up to a residence time of 72h. Once
the final yields of the saccharification process were obtained,
these results were used to simulate the enzymatic hydroly-
sis in the Aspen Plus V9.0 software employing a RYield
model. Then, the fermentation process is simulated using
the S. cerevisiae as microorganism at 32°C. The yields of
the ethanolic fermentation were taken from the results of the
experimental procedure previously described in “Gasifica-
tion” section. Afterward, the cell biomass is separated from
the culture broth through a centrifuge. The culture broth
with an ethanol concentration of 7–10wt% is taken to the
separation stage that consists of two distillation columns and
molecular sieves. In the first column, ethanol is concentrated
nearly to 50–55% by weight[37]. This distillation column is
designed with 20 trays, a reflux ratio of 1.5 and a distillate-
to-feed ratio of 0.03. In the second column, the liquor is con-
centrated until the azeotropic point (96wt%) using a 20-tray
column with a reflux ratio of 1.5 and a distillate-to-feed ratio
of 0.3. Subsequently, the azeotropic mixture is sent to the
dehydration zone with molecular sieves to obtain ethanol at
99.7wt% [38]. The process scheme for the ethanol produc-
tion from P. patula is presented in Fig.1.
Gasification
The simulation of the gasification was previously described
by García etal. [39]. The raw material is first conditioned
to the particle size and moisture content required for the
gasification. For this purpose, chipping and drying processes
are simulated in order to obtain a particle size between 0.5
and 1cm and a moisture content of 20%. Subsequently, the
chipped and dried raw material is submitted to a downdraft
gasifier where different chemical reactions (devolatilization,
combustion and reduction) take place. For simulation pur-
poses, the downdraft gasification is divided into two steps:
first, the devolatilization (pyrolysis) of the raw material is
carried out at 600°C in absence of air in order to obtain as

Waste and Biomass Valorization
1 3
main components carbon, hydrogen, oxygen, nitrogen, char
and ash. The elemental analysis (raw material characteriza-
tion) was used to determine the yields of carbon, hydrogen,
oxygen and nitrogen; whereas the char and ash content were
taken from the proximate analysis (raw material characteri-
zation). Then, the combustion and reduction are simulated
using the Free Gibbs Energy minimization method to predict
the composition of H2, CO2, CO, CH4, N2 in the synthesis
gas [40, 41]. Ash and particulate matter are separated from
the synthesis gas using a cyclone. This syngas can be used as
fuel for the production of electricity using gas engines. The
simulation of the engine was done based on the combustion
reaction between the syngas and air, but also considering the
electric efficiency of gas engines, which can vary between
40 and 45%.
Economic Assessment
The results of the mass and energy balances from the simula-
tion procedure were used as starting point for the economic
analysis. The total capital investment of the ethanolic fermen-
tation was calculated based on the data provided by the com-
plementary software Aspen Economic Analyzer v9.0 (Aspen
Technologies, Inc., USA), which performs the “mapping” (siz-
ing and costing) of the equipment. The operating costs were
divided into two types: fixed and variable. The fixed operat-
ing costs are those related to the operation of equipment in
the process such as labor, maintenance, fixed and general and
plant overhead costs. These costs were calculated based on the
estimation method reported by Peters and Timmerhaus [42].
Maintenance costs were estimated as the 6% of the total capital
investment [42]. The labor costs were determined based on
the number of employees-hour/day required for the operation
of a 3300 ton/day facility according to the data reported by
Peters and Timmerhaus [42]. Fixed charges were calculated
as the 3% of the total capital investment, whereas the plant
overhead and general costs were estimated as the 60% and
20% of the labor costs, respectively [42]. On the other hand,
the variable operating costs are those associated with the raw
material, reagents and utilities (heating and cooling) purchase
costs. The raw material and reagent costs were calculated
from the inflows of the process and the purchase prices (see,
Table1). The utility costs were calculated using the comple-
mentary software Aspen Energy Analyzer and data about the
market prices (see, Table1). This analysis was estimated in
US dollars for a 10-year period at an annual interest rate of
17% (typical for the Colombian economy), considering the
H2SO4
(1)
(2)
(3)(4)
Ca(OH)2
Enzyme
CaSO4
(7)
(9)(10)
Water
(6)(12)(13)
(14)
ETHANOL
(11)
Nutrients
S. cerevisiae
CO2
Biomass
Glucose
rich-liquor
Stillage
Water
Residual Solid Fraction
Pinus Patula
Cellulose-rich
solid Water
Xylose-rich
liquid
(5)
(8)
Fig. 1 Scheme of the ethanol production from P. patula using S. cer-
evisiae as microorganism. Units (1) Acid Hydrolysis, (2) Filter, (3)
Detoxification, (4) Filter, (5) Dewatering, (6) Enzymatic Saccharifica-
tion, (7) Filter, (8) Dewatering, (9) Autoclave, (10) Ethanol Fermen-
tation, (11) Centrifuge, (12) Distillation column, (13) Rectification
column and (14) Ethanol dehydration. Repoduced with permission
from Referemce [31]
Table 1 Market prices of the raw material, utilities, and products
a Price of 1 ton of wood with a moisture content of 40% [43]. Update
2018
b Price taken from Kemcore Supplier [44]. Updated 2018
c Price adapted from Colombian conditions. Update 2018
d Enzyme price calculated based on literature reports [45]
e Price taken from Federación Nacional de Biocombustibles [46].
Update 2018
f Price estimated using the correlations from [47]
Compound Value Unit
P. patula 60aUSD/ton
Sulfuric acid 275bUSD/ton
Process water 0.28fUSD/m3
Enzyme 110dUSD/ton EtOH
Ethanol 0.77eUSD/L
Electricity 0.016aUSD/MJ
Cooling water 0.07fUSD/m3
High-pressure steam 9.86cUSD/ton
Mid-pressure steam 8.18cUSD/ton
Low-pressure steam 7.56cUSD/ton

Waste and Biomass Valorization
1 3
straight-line depreciation method and an income tax of 25%.
Prices and economic data used in this analysis such as the costs
of the raw materials and utilities, income tax, labor salaries,
among others are summarized in Table1.
The results from the software Aspen Process Economic
Analyzer and the estimation method reported by Peters and
Timmerhaus [42] were used to determine the production cost
of 1L of ethanol using the dilute-acid hydrolysis coupled to
the ethanolic fermentation and P. patula as raw material. The
calculated production costs of the proposed process scheme
were compared to the reported data in the literature from dif-
ferent authors that have evaluated the production of ethanol
using different raw materials and pretreatments.
Energy Analysis
As mentioned in the methodology (“Materials and Meth-
ods” section), the energy comparison of the biochemical and
thermochemical pathways was carried out using ethanol and
syngas as energy carriers, respectively. The criteria selected
for this purpose was the energy efficiency of both processes,
which was calculated following the methodology proposed by
García etal. [3]. The energy efficiency was evaluated consid-
ering the gross energy content of the P. patula
(
E
biomass)
and
the main products
(
E
products)
of each pathway, as presented in
Eq.1.
The energy content of the biomass was calculated based on
the mass flow (
̇m
) and the lower heating value (
LHVB
) of the
P. patula reported by García etal. [23] (see, Eq.2).
In the same way, the energy content of the products was
calculated based on the mass flow rates and their respective
heating values (see, Eq.3, 4). The flow rates of ethanol and
syngas were obtained from the simulation procedure in the
software Aspen Plus. On the other hand, the heating values
were taken from different sources: the lower heating value
(LHV) of ethanol was 26.95MJ/kg [48], whereas the lower
heating value (LHV) of the synthesis gas was calculated
considering the mass composition and the heating content
of the main gaseous species in the synthesis gas (hydrogen,
methane and carbon monoxide). The LHV of the hydro-
gen, methane and carbon monoxide were 119.96, 50 and
10.11MJ/kg, respectively [48]. The calculated value for the
synthesis gas was 6.77MJ/kg.
(1)
𝜂
=
E
products
E
biomass
(2)
EBiomass
=
̇m
⋅
LHVB
(3)
EEtOH
=
̇mEtOH
⋅
LHVEtOH
(4)
Esyngas
=
̇msyngas
⋅
LHVsyngas
Results andDiscussion
Dilute Acid Pretreatment andEnzymatic
Saccharification
Dilute-acid hydrolysis was used as the first stage for the pre-
treatment of the lignocellulosic biomass (P. patula) in order
to improve the efficiency of the enzymatic hydrolysis. The
quantification of the total reducing sugars was 25.37g/L,
which is higher than the results reported by Moncada etal.
[2] (20g/L) using P. patula bark. Additionally, Kim [49]
and Bösch etal. [50] reported yields of 39.3g of sugars per
100g of dry hemlock sawdust and 26.3g of sugars per 100g
of softwood spruce, respectively. The results obtained in this
work are acceptable in comparison to those reported in the
literature, despite the use of one-step dilute acid hydrolysis.
The solid fraction from the dilute acid hydrolysis was
used as input for the saccharification process. The highest
amount of reducing sugars in the liquid fraction from this
process was 20g of reducing sugars per 100g of dry feed
(3.02g/L). The results obtained in this work are in agree-
ment with those reported by other authors such as Söder-
ström etal. [11] (17g of sugars per 100g of Picea abies)
and Shinozaki etal. [51] (14g of reducing sugars per 100
f of ensiled rice straw). From the enzymatic saccharifica-
tion, a cellulose conversion of 67% (w/w) was achieved.
The enzymatic hydrolysate was used as carbon substrate
for the production of ethanol using S. cerevisiae as micro-
organism. The low concentration of reducing sugars in
the hydrolysate can be explained due to the high amount
of water used in the enzymatic hydrolysis. In this sense,
the hydrolysate was concentrated until a concentration of
13.5g/L.
Ethanolic Fermentation
The main objective of the experimental procedure was to
evaluate the potential use of P. patula for the production
of ethanol. For this purpose, the ability of the microor-
ganism to directly degrade the hydrolysate was evaluated.
Microorganisms can adapt to environmental perturbations,
such as changes in the osmotic pressure, temperature, and
depletion of nutrients [52]. Nevertheless, if the changes are
not progressive, the microorganism is submitted to stress,
which leads to a decrease in the cell growth or prolonged
lag phases. In order to reduce the stress of the microorgan-
ism, three fermentation configurations were proposed to
evaluate the effect of the substrate concentration and type
in the ethanol production (see, Table2).
Figure2 presents the consumption of total reducing
sugars during 69h of fermentation. As a result, a substrate

Waste and Biomass Valorization
1 3
consumption of 8.185, 7.996 and 6.295g/L was obtained
from the fermentation configurations 1, 2 and 3, respec-
tively. In the fermentation configuration 1, a relatively
high consumption of substrate was achieved; whereas the
final concentration of sugars in the configurations 2 and
3 were 50% of the initial concentration. It is important
to clarify that in the present study, inhibitors were not
quantified. However, previous studies have demonstrated
that lignocellulosic biomass (i.e. wood wastes) generates
hydrolysates that contain inhibitors such as furfural, HMF
and aromatic-lignin compounds that limit the microbial
growth, microorganism metabolism in the fermentation
and therefore, the consumption of the substrate [53]. In
this context, Qian etal. evaluated the dilute-acid hydroly-
sis of the softwood aiming to obtain a hydrolysate com-
posed of glucose and xylose, and its further fermentation
to ethanol by co-cultures [54]. In this study, two fermen-
tation configurations were studied: (i) detoxified hydro-
lysate by a combined method and (ii) hydrolysate without
detoxification. The use of the softwood hydrolysate with-
out detoxification evidenced the same behavior as the pre-
sent study, where 50% of the substrate was not consumed,
which indicates the possible presence of inhibitors in the
hydrolysate. Therefore, the hydrolysate should be detoxi-
fied in order to increase the substrate consumption and
ethanol production. However, the dilute-acid pretreatment
generates lower degradation products than the use of con-
centrated-acid pretreatments. Additionally, high hydrolysis
yields have been reported when pretreating lignocellulosic
materials with diluted H2SO4 [53]. Fermentation configu-
ration 3 showed a substrate consumption similar to con-
figuration 2, which was rich in glucose (15g/L). Conse-
quently, the microorganism does not require an adaptation
stage, so it is possible to use the microorganism in the
hydrolysate.
Additionally, Table2 presents the ethanol concen-
tration in each fermentation. An experimental yield of
0.364 ± 0.009g ethanol/g sugar (73% of the theoretical)
was obtained, which it is similar to the yields reported by
other authors Chacha [55] and Hawkins and Doran-Peterson
[56]. Nguyen etal. [57] used softwood chips through direct
impregnation with sulfuric acid and steam explosion as pre-
treatment to produce ethanol using S. cerevisiae. As a result,
ethanol yields between 74 and 89% of the theoretical were
obtained. Qian etal. [54] studied the fermentation of the
dilute-acid softwood hydrolysate to produce ethanol using
co-cultures of adapted S. cerevisiae and Escherichia coli.
The highest ethanol yield from the experimental procedure
was 0.45g/g total sugar. The low concentration of ethanol in
this work can be explained due to the use of substrates with
low sugar content, which suggests that the hydrolysate must
be concentrated to obtain a higher ethanol concentration.
Furthermore, fermentation 1 evidenced the lowest con-
centration of biomass (3.34g/L), as shown in Fig.3. While
Fermentation 2 (highest concentration of glucose) produced
a high concentration of biomass (4.51g/L) after 69h of fer-
mentation. The biomass concentration in the concentrated
hydrolysate showed an intermediate value between the two
Table 2 Fermentation
configurations for the ethanol
production
Description Total reducing sugars
(g/L)
Ethanol
concentration
(g/L)
Fermentation 1 Glucose (70%) + hydrolysate (30%) 10 3.678
Fermentation 2 Glucose (80%) + hydrolysate (20%) 15 5.563
Fermentation 3 Concentrated hydrolysate 13.5 4.790
Fig. 2 Substrate uptake in the
different fermentation configu-
rations
0
2
4
6
8
10
12
14
16
010203040506
070
Total reducing sugar(g/l)
Time (hour)

Waste and Biomass Valorization
1 3
previous experiments (3.7g/L). It should be noted that it is
very important to guarantee aseptic conditions (sterilization
of the medium and sampling) since the microorganism is
susceptible to external agents. This contamination can cause
reduction in the cell growth and undesirable products such as
lactic and acetic acid. In this work, these products were not
detected during the quantification of the samples.
Techno‑Eeconomic Analysis (TEA) oftheEthanolic
Fermentation
The total production costs of the ethanolic fermentation
were classified into four categories: raw material costs, util-
ity costs, fixed operating costs and total investment costs.
Figure4 presents the share distribution of these costs, evi-
dencing the effect of the raw material costs (63.4%) in the
total ethanol production cost. The utility costs account for
22.7% of the total production costs due to the high heating
requirements of the downstream processing of the fermenta-
tion broth. Besides, the purchase of equipment contributes
up to 12% of the total production costs. These results are in
agreement with the data reported by Mesa etal. [58] who
evaluated the production of ethanol through a dilute-acid
pretreatment using sugarcane straw as raw material. The
authors reported that the costs of raw materials (sugarcane
straw and enzymes) had the most significant impact on the
total production costs. The same behavior was evidenced in
this work as observed in Fig.5, where the share contribution
of the main raw materials costs is presented. In this case,
the P. patula and enzyme costs account for 59% of the raw
materials costs, whereas sulfuric acid accounts for 34%.
The moisture content of the P. patula has a significant
impact on the purchase price of this feedstock. According
to Forest Fuels [43], raw materials with high moisture con-
tent (40%) have a market price of 60 USD/ton, whereas raw
materials with low moisture (20%) content have a higher
market price 160 USD/ton.
Several authors have studied different methods to improve
the economic feasibility of the cellulosic ethanol production
by means of the on-site enzyme production. Liu etal. [45]
studied the influence of the enzyme cost on the cellulosic
ethanol production considering different scenarios where
the enzyme was commercially purchased or on-site pro-
duced. The authors concluded that the on-site enzyme pro-
duction can significantly reduce the enzyme cost, providing
Fig. 3 Biomass growth in the
ethanol fermentation
0
1
2
3
4
5
010203040506
070
Cell concentration (g/l)
Time (hour)
Fig. 4 Share distribution of the total production costs of ethanol from
P. patula
Fig. 5 Share distribution of the raw material costs

Waste and Biomass Valorization
1 3
a promising option for the large-scale cellulosic ethanol
production.
On the other hand, the high contribution to the total costs
of the sulfuric acid is related to the high liquid-to-solid ratio
used in the acid hydrolysis. From the experimental proce-
dure, a dilute sulfuric acid solution (2%v/v) in a liquid-to-
solid ratio of 1:10 was used. Considering the large process-
ing capacity of the evaluated process (3300 ton/day), the
required amount of sulfuric acid is too high and thus, it has
a great influence in the economic performance of the ethanol
production. Therefore, the amount of sulfuric acid used for
the pretreatment of P. patula should be reduced in order
to increase the profits of the process. One of the possibili-
ties is to reduce the concentration of sulfuric acid and the
liquid-to-solid ratio. However, these changes can affect the
sugar yields of the process and thus other variables must
be considered such as temperature and pretreatment time.
Parajó etal. [59] have evaluated these parameters in order to
determine the best conditions for the dilute acid hydrolysis
in order to use low amount of reagents and relative high
pretreatment times.
Based on the information provided by the economic
assessment, the production costs of 1L of ethanol were
calculated and compared with literature reports of differ-
ent raw materials that use the dilute-acid hydrolysis as pre-
treatment. Figure6 presents the comparison between the
ethanol production costs of the evaluated process, some
literature reports, and the market price. In this work, the
ethanol production cost was 1.53USD/L, which is higher
than the market price (0.77USD/L). However, the source
of the raw material has an important effect on the feasibility
of the ethanol production as evidenced by Daystar etal. [60]
who evaluated the ethanol production cost from different
raw materials (i.e. pine, eucalyptus, switchgrass and sweet
sorghum) using the dilute-acid pretreatment. When forest
biomass (pine) was used as raw material, the ethanol produc-
tion costs was 2.25USD/L, but if agricultural crops such as
sweet sorghum are used as feedstock, the ethanol production
cost drops to 0.5USD/L. Zhao etal. [61] studied the effect
of the dilute acid pretreatment in the ethanol production
using corn stover. As a result, an ethanol production cost of
1.6USD/L was obtained if no incentives were considered;
however, the ethanol cost can be reduced up to 1.24USD/L
if tax exemptions and Feed-in-tariff are considered.
Energetic Comparison ofBiochemical
andThermochemical Pathways forEnergy
Production Using P. patula
Table3 presents the results from the energy analysis of both
biochemical and thermochemical processes for bioenergy
production. It is evidenced that the energy yield of the etha-
nolic fermentation is lower than the gasification of P. patula.
As a result, the net energy efficiency of both processes was
20% and 42% for the ethanol and synthesis gas production,
respectively. The energy efficiency of the ethanolic fermen-
tation is lower than the data reported in the literature for
different raw materials [62–64] since the valorization of the
xylose rich-liquor and the lignin was not considered in this
work. Additionally, the stillage from the first distillation col-
umn can be used as input for cogeneration systems, which
can reduce the heating requirements (steam) of the process
and thus, improve the overall energy efficiency. Despite the
high pretreatment requirements of P. patula for sugar extrac-
tion and the relatively low energy efficiency, the production
of ethanol through fermentation can promote the valoriza-
tion of the lignocellulosic matrix (cellulose, hemicellulose
and lignin) of this raw material to obtain different platforms
in comparison to the direct conversion to electricity. Elec-
tricity cannot be considered an added-value product due to
the low market price and the diversity of methods to produce
it, especially in Colombia where hydropower plants are used
to produce more than 60% of the electricity required in the
country [65]. On the other hand, the increment of the earth
temperature has increased the concern of using fossil fuels
as energy carriers due to the global warming. In this sense,
ethanol is gaining an important place as a future alternative
to fossil fuels, especially, in the automotive sector. In order
Fig. 6 Comparison of the ethanol production cost (USD/L) from dif-
ferent feedstocks
Table 3 Comparison of the energy potential of biochemical and ther-
mochemical pathways for energy production
a Calculation based on 3300 ton per day of P. patula
Biochemical pathway Thermo-
chemical
pathway
Process Fermentation Gasification
Main product Ethanol Synthesis gas
Energetic potential
Yield (MJ per kg PP)a3.91 7.68
Energy efficiency 22% 42%

Waste and Biomass Valorization
1 3
to fulfill the agreement in the COP21, blends between fossil
fuels and ethanol have been implemented aiming to reduce
the emissions from their combustion. This scenario provides
an opportunity not only to evaluate different technologies
to produce energy but also to consider other raw materials
that can be transformed into added-value products such as
ethanol.
Conclusions
It was evidenced that the P. patula hydrolysate can be used as
substrate for the production of ethanol. The microorganism
did not require a prior adaptation period to the hydrolysate.
Despite the low substrate consumption, an experimental
yield of 0.364 ± 0.009gethanol/g sugar (73% theoreti-
cal yield) was obtained. Nevertheless, it is highly recom-
mended to concentrate the hydrolysate due to the low solid
concentration in the enzymatic hydrolysis (15g/L). From
the simulation procedure, the costs associated with the raw
materials (P. patula, enzymes and sulfuric acid) have a sig-
nificant influence in the ethanol production costs and thus,
different scenarios should be analyzed in order to reduce the
ethanol production costs such as on-site enzyme production
and low sulfuric acid concentration. The calculated ethanol
production costs (1.55USD/L) were higher than the market
price (0.77USD/L); however, different alternatives can be
considered in the future aiming to improve the feasibility
of the ethanol production from forest biomass: integration
of waste streams and incentives for bioenergy production.
The lower energy efficiency (22%) of the biochemical route
in comparison to the thermochemical pathway endorse the
necessity to implement the previous alternatives in order to
consider the P. patula as a potential raw material to produce
added-value products such as ethanol.
Acknowledgements The authors express their acknowledgments to
the Centro de Bioinformática y Biología Computacional (BIOS) for
the financial support through the project entitled “Fortalecimiento
de CTEI en biotecnologia para el departamento de Caldas apoyado
por infraestructura computacional avanzada y trabajo colaborativo
(CALDAS BIOREGION)” Grant No. 08112013-0621. The authors
also express their gratitude to the Universidad Nacional de Colombia
Sede Manizales through the Projects entitled “Development of modu-
lar small-scale integrated biorefineries to produce an optimal range
of bioproducts from a variety of rural agricultural and agroindus-
trial residues/wastes with a minimum consumption of fossil energy—
SMIBIO” from ERANET LAC 2015 Grant No. 202010011331 and the
Project “Techno-economic and environmental evaluation of a biorefin-
ery using the residues from the Coffee Crop” Grant No. 202010014230.
References
1. Ford, C.M., Jones, N.B., Chirwa, P.W.: Pinus patula and pine
hybrid hedge productivity in South Africa: a comparison
between two vegetative propagation systems exposed to natural
infection by Fusarium circinatum. J. For. Sci. 2620, 1–9 (2014).
https ://doi.org/10.2989/20702 620.2014.91650 1
2. Moncada, J., Cardona, C.A., Higuita, J.C., Vélez, J.J., López-
Suarez, F.E.: Wood residue (Pinus patula bark) as an alterna-
tive feedstock for producing ethanol and furfural in Colombia:
experimental, techno-economic and environmental assessments.
Chem. Eng. Sci. 140, 309–318 (2016). https ://doi.org/10.1016/j.
ces.2015.10.027
3. García, C.A., Betancourt, R., Cardona, C.A.: Stand-alone and
biorefinery pathways to produce hydrogen through gasification
and dark fermentation using Pinus patula. J. Environ. Manage.
(2015). https ://doi.org/10.1016/j.jenvm an.2016.04.001
4. Damartzis, T., Zabaniotou, A.: Thermochemical conversion
of biomass to second generation biofuels through integrated
process design—a review. Renew. Sustain. Energy Rev. 15,
366–378 (2011). https ://doi.org/10.1016/J.RSER.2010.08.003
5. Johnson, E.: Integrated enzyme production lowers the cost of
cellulosic ethanol. Biofuels Bioprod. Biorefin. 10, 164–174
(2016). https ://doi.org/10.1002/bbb
6. Soudham, V.P., Raut, D.G., Anugwom, I., Brandberg, T., Lars-
son, C., Mikkola, J.P.: Coupled enzymatic hydrolysis and etha-
nol fermentation: ionic liquid pretreatment for enhanced yields.
Biotechnol. Biofuels. 8, 135 (2015). https ://doi.org/10.1186/
s1306 8-015-0310-3
7. Daza Serna, L.V., Orrego Alzate, C.E., Cardona Alzate, C.A.:
Supercritical fluids as a green technology for the pretreatment
of lignocellulosic biomass. Bioresour. Technol. 199, 113–120
(2016)
8. Galbe, M., Zacchi, G.: A review of the production of ethanol from
softwood. Appl. Microbiol. Biotechnol. 59, 618–628 (2002). https
://doi.org/10.1007/s0025 3-002-1058-9
9. Hoyer, K., Galbe, M., Zacchi, G.: Production of fuel ethanol from
softwood by simultaneous saccharification and fermentation at
high dry matter content. J. Chem. Technol. Biotechnol. 84, 570–
577 (2009). https ://doi.org/10.1002/jctb.2082
10. St1 Biofuels Oy: Cellunolix® ethanol plant to be built in Finland.
https ://www.st1.eu/cellu nolix -ethan olpla nt-to-be-built -in-finla nd
11. Söderström, J., Pilcher, L., Galbe, M., Zacchi, G.: Two-step pre-
treatment of softwood with SO2 two-step steam pretreatment of
softwood with SO2 impregnation for ethanol production. Appl.
Biochem. Biotechnol. 98–100, 5–21 (2002)
12. St1 Biofuels Oy: St1´s and SOK´s joint venture NEB plans
50-million-litre Cellunolix® bioethanol plant in Pietarsaari. https
://www.st1.eu/st1s-and-soks-joint -ventu re-neb-plans -50-milli on-
litre -cellu nolix -bioet hanol pla
13. Aho, A., Kumar, N., Eränen, K., Holmbom, B., Hupa, M., Salmi,
T., Murzin, D.Y.: Pyrolysis of softwood carbohydrates in a fluid-
ized bed reactor. Int. J. Mol. Sci. 9(9), 1665–1675 (2008)
14. Garcìa-Pérez, M., Chaala, A., Pakdel, H., Kretschmer, D., Roy,
C.: Vacuum pyrolysis of softwood and hardwood biomass: com-
parison between product yields and bio-oil properties. J. Anal.
Appl. Pyrolysis. 78, 104–116 (2007). https ://doi.org/10.1016/J.
JAAP.2006.05.003
15. Oasmaa, A., Solantausta, Y., Arpiainen, V., Kuoppala, E., Sipilä,
K.: Fast pyrolysis bio-oils from wood and agricultural residues.
Energy Fuels. 24, 1380–1388 (2010). https ://doi.org/10.1021/
ef901 107f
16. Amaral, S., De Carvalho Junior, A., Costa, M.A.M., Neto, T.G.S.,
Dellani, R., Leite, L.H.S.: Comparative study for hardwood and
softwood forest biomass: chemical characterization, combus-
tion phases and gas and particulate matter emissions. Biore-
sour. Technol. 164, 55–63 (2014). https ://doi.org/10.1016/j.biort
ech.2014.04.060
17. Roy, M.M., Corscadden, K.W.: An experimental study of combus-
tion and emissions of biomass briquettes in a domestic wood stove.

Waste and Biomass Valorization
1 3
Appl. Energy. 99, 206–212 (2012). https ://doi.org/10.1016/J.
APENE RGY.2012.05.003
18. García, C.A., Morales, M., Quintero, J., Aroca, G., Cardona, C.A.:
Environmental assessment of hydrogen production based on Pinus
patula plantations in Colombia. Energy. 139, 606–616 (2017).
https ://doi.org/10.1016/j.energ y.2017.08.012
19. Waldner, M.H., Vogel, F.: Renewable production of methane from
woody biomass by catalytic hydrothermal gasification. Ind. Eng.
Chem. Res. 44, 4543–4551 (2005). https ://doi.org/10.1021/ie050
161h
20. Mandl, C., Obernberger, I., Scharler, I.R.: Characterisation of fuel
bound nitrogen in the gasification process and the staged combus-
tion of producer gas from the updraft gasification of softwood
pellets. Biomass Bioenergy 35, 4595–4604 (2011). https ://doi.
org/10.1016/J.BIOMB IOE.2011.09.001
21. Moncada, J., Tamayo, J., Cardona, C.A.: Evolution from biofuels
to integrated biorefineries: techno-economic and environmental
assessment of oil palm in Colombia. J. Clean. Prod. 81, 51–59
(2014)
22. Chum, H., Faaij, A., Moreira, J.: Bioenergy. In: Edenhofer, O.,
Pichs-Madruga, R., Sokona, Y. (eds.) Renewable Energy Sources
and Climate Change Mitigation. pp.46–55. Intergovernmental
Panel on Climate Change (IPCC), Geneva (2011)
23. García-Velásquez, C.A.: Hydrogen production through gasifica-
tion and dark fermentation. (2016). http://www.bdigi tal.unal.edu.
co/54321 /
24. Timung, R., Mohan, M., Chilukoti, B., Sasmal, S., Banerjee, T.,
Goud, V.V.: Optimization of dilute acid and hot water pretreat-
ment of different lignocellulosic biomass: a comparative study.
Biomass Bioenergy 81, 9–18 (2015). https ://doi.org/10.1016/j.
biomb ioe.2015.05.006
25. Karimi, K., Taherzadeh, M.J.: A critical review of analytical meth-
ods in pretreatment of lignocelluloses: composition, imaging, and
crystallinity. Bioresour. Technol. 200, 1008–1018 (2016). https ://
doi.org/10.1016/j.biort ech.2015.11.022
26. Selig, M., Weiss, N., Ji, Y.: Enzymatic Saccharification of Ligno-
cellulosic Biomass, pp. 1–5. National Renewable Energy Labora-
tory, Golden (2008)
27. Miller, G.L.: Use of dinitrosalicylic acid reagent for determination
of reducing sugar. Anal. Chem. 31, 426–428 (1959). https ://doi.
org/10.1021/ac601 47a03 0
28. Jensen, J., Morinelly, J., Aglan, A., Mix, A., Shonnard, D.: Kinetic
characterization of biomass dilute sulfuric acid hydrolysis: mix-
tures of hardwoods, softwood, and switchgrass. Environ. Energy
Eng. 54, 1637–1645 (2008). https ://doi.org/10.1002/aic
29. Esteghlalian, A., Hashimoto, A.G., Fenske, J.J., Penner, M.H.:
Modeling and optimization of the dilute-sulfuric-acid pretreatment
of corn stover, poplar and switchgrass. Bioresour. Technol. 59,
129–136 (1997). https ://doi.org/10.1016/S0960 -8524(97)81606 -9
30. Wooley, R.J., Putsche, V.: Development of an ASPEN PLUS phys-
ical property database for biofuels components, pp.1–38. National
Renewable Energy Laboratory, Golden (1996)
31. García, C.A., Peña, Á, Betancourt, R., Cardona, C.A.: Energetic
and environmental assessment of thermochemical and biochemi-
cal ways for producing energy from agricultural solid residues:
coffee cut-stems case. J. Environ. Manage. (2017). https ://doi.
org/10.1016/j.jenvm an.2017.04.029
32. Quintero, J.a., Moncada, J., Cardona, C.a.: Techno-economic
analysis of bioethanol production from lignocellulosic resi-
dues in Colombia: a process simulation approach. Bioresour.
Technol. 139, 300–307 (2013). https ://doi.org/10.1016/j.biort
ech.2013.04.048
33. Moncada, J., Tamayo, J.A., Cardona, C.A.: Integrating first, sec-
ond, and third generation biorefineries: incorporating microalgae
into the sugarcane biorefinery. Chem. Eng. Sci. 118, 126–140
(2014). https ://doi.org/10.1016/j.ces.2014.07.035
34. Rafiqul, I.S.M., Mimi Sakinah, aM.: Kinetic studies on acid
hydrolysis of Meranti wood sawdust for xylose production.
Chem. Eng. Sci. 71, 431–437 (2012). https ://doi.org/10.1016/j.
ces.2011.11.007
35. Khodaverdi, M., Karimi, K., Jeihanipour, A., Taherzadeh, M.J.:
Kinetic modeling of rapid enzymatic hydrolysis of crystal-
line cellulose after pretreatment by NMMO. J. Ind. Microbiol.
Biotechnol. 39, 429–438 (2012). https ://doi.org/10.1007/s1029
5-011-1048-y
36. Kadam, K.L., Rydholm, E.C., McMillan, J.D.: Development and
validation of a kinetic model for enzymatic saccharification of
lignocellulosic biomass. Biotechnol. Prog. 20, 698–705 (2004).
https ://doi.org/10.1021/bp034 316x
37. Quintero, J.A., Cardona, C.A.: Process Simulation of Fuel Ethanol
Production from Lignocellulosics using Aspen Plus. Ind. Eng.
Chem. Res. 50(10), 6205–6212 (2011)
38. Pitt, W.W., Haag, G.L., Lee, D.D.: Recovery of ethanol from fer-
mentation broths using selective sorption-desorption. Biotechnol.
Bioeng. 25, 123–131 (1983). https ://doi.org/10.1002/bit.26025
0110
39. García, C.A., Moncada, J., Aristizábal Marulanda, V., Cardona,
C.A.: Techno-economic and energetic assessment of hydrogen
production through gasification in the Colombian context: coffee
cut-stems. Int. J. Hydrogen Energy. 42, 5849–5864 (2017). https
://doi.org/10.1016/j.ijhea tmass trans fer.2017.09.038
40. Melgar, A., Pérez, J.F., Laget, H., Horillo, A.: Thermochemi-
cal equilibrium modelling of a gasifying process. Energy Con-
vers. Manag. 48, 59–67 (2007). https ://doi.org/10.1016/j.encon
man.2006.05.004
41. Jarungthammachote, S., Dutta, A.: Equilibrium modeling of
gasification: Gibbs free energy minimization approach and its
application to spouted bed and spout-fluid bed gasifiers. Energy
Convers. Manag. 49, 1345–1356 (2008). https ://doi.or g/ 10.1016/j.
encon man.2008.01.006
42. Peters, M.S., Timmerhaus, K.D., West, R.E.: Plant Design and
Economics for Chemical Engineers. McGraw-Hill, New York
(2004)
43. Fuels, F.: Wood chip and wood pellet price comparison. https ://
www.fores tfuel s.co.uk/wood-fuel-price compa rison /
44. Kemcore: Reagents Market Price. https ://www.kemco re.com/
sulph uric-acid-98.html
45. Liu, G., Zhang, J., Bao, J.: Cost evaluation of cellulase enzyme
for industrial-scale cellulosic ethanol production based on rigor-
ous Aspen Plus modeling. Bioprocess. Biosyst. Eng. 39, 133–140
(2016). https ://doi.org/10.1007/s0044 9-015-1497-1
46. Federación Nacional de Biocombustibles de Colombia—Fedebio-
combustibles: Biofuel prices in Colombia 2016–2017
47. Ulrich, G.D., Vasudevan, P.T.: How to estimate utility costs.
Chem. Eng. 113, 66–69 (2006)
48. Boundy, B.: Biomass Energy Data Book, 4 ed, p. 254. Deparment
of Energy, Washington DC, (2011)
49. Kim, K.H.: Two-stage dilute acid-catalyzed hydrolytic conversion
of softwood sawdust into sugars fermentable by ethanologenic
microorganisms. J. Sci. Food Agric. 85, 2461–2467 (2005). https
://doi.org/10.1002/jsfa.2268
50. Bösch, P., Wallberg, O., Joelsson, E., Galbe, M., Zacchi, G.:
Research impact of dual temperature profile in dilute acid hydroly-
sis of spruce for ethanol production. Biotechnol. Biofuels. 3, 15
(2010). https ://doi.org/10.1186/1754-6834-3-15
51. Shinozaki, Y., Kitamoto, H.K.: Ethanol production from ensiled
rice straw and whole-crop silage by the simultaneous enzymatic
saccharification and fermentation process. J. Biosci. Bioeng. 111,
320–325 (2011). https ://doi.org/10.1016/j.jbios c.2010.11.003
52. Dinh, T.N., Nagahisa, K., Hirasawa, T., Furusawa, C., Shimizu,
H.: Adaptation of Saccharomyces cerevisiae cells to high etha-
nol concentration and changes in fatty acid composition of

Waste and Biomass Valorization
1 3
membrane and cell size. PLoS ONE. 3, e2623 (2008). https ://doi.
org/10.1371/journ al.pone.00026 23
53. Alvira, P., Tomás-Pejó, E., Ballesteros, M., Negro, M.J.: Pretreat-
ment technologies for an efficient bioethanol production process
based on enzymatic hydrolysis: a review. Bioresour. Technol. 101,
4851–4861 (2010). https ://doi.org/10.1016/j.biort ech.2009.11.093
54. Qian, M., Tian, S., Li, X., Zhang, J., Pan, Y., Yang, X.: Ethanol
production from dilute-acid softwood hydrolysate by co-culture.
Appl. Biochem. Biotechnol. (2006). https ://doi.org/10.1385/
ABAB:134:3:273
55. Nyangi Chacha: Comparison of Escherichia coli KO11 and Sac-
charomyces cerevisiae ATCC 96581 in fermenting Pinus patula
hydrolysate pretreated at different steam explosion severity. Afr.
J. Biotechnol. (2012). https ://doi.org/10.5897/AJB11 .3498
56. Hawkins, G.M., Doran-Peterson, J.: A strain of Saccharo-
myces cerevisiae evolved for fermentation of lignocellulosic
biomass displays improved growth and fermentative ability
in high solids concentrations and in the presence of inhibitory
compounds. Biotechnol. Biofuels. 4, 49 (2011). https ://doi.
org/10.1186/1754-6834-4-49
57. Nguyen, Q.A., Tucker, M.P., Keller, F.A., Beaty, D.A., Con-
nors, K.M., Eddy, F.P.: Dilute acid hydrolysis of softwoods.
Appl. Biochem. Biotechnol. (1999). https ://doi.org/10.1385/
ABAB:77:1-3:133
58. Mesa, L., Martínez, Y., Barrio, E., González, E.: Desirability
function for optimization of dilute acid pretreatment of sugarcane
straw for ethanol production and preliminary economic analysis
based in three fermentation configurations. Appl. Energy. 198,
299–311 (2017). https ://doi.org/10.1016/j.apene rgy.2017.03.018
59. Parajó, J.C., Vázquez, D., Alonso, J.L., Santos, V., Dominguez,
H.: Prehydrolysis of Eucalyptus wood with dilute sulphuric acid:
operation at atmospheric pressure. Holz als Roh- und Werkst. 51,
357–363 (1993). https ://doi.org/10.1007/BF026 63809
60. Daystar, J., Treasure, T., Gonzalez, R., Reeb, C., Venditti, R.,
Kelley, S.: The NREL biochemical and thermochemical etha-
nol conversion processes: financial and environmental analysis
comparison. BioResources. 10, 5096–5116 (2015). https ://doi.
org/10.15376 /biore s.10.3.5096-5116
61. Zhao, L., Zhang, X., Xu, J., Ou, X., Chang, S., Wu, M.: Techno-
economic analysis of bioethanol production from lignocellulosic
biomass in china: dilute-acid pretreatment and enzymatic hydrol-
ysis of corn stover. Energies. 8, 4096–4117 (2015). https ://doi.
org/10.3390/en805 4096
62. Foust, T.D., Aden, A., Dutta, A., Phillips, S.: An economic and
environmental comparison of a biochemical and a thermochemi-
cal lignocellulosic ethanol conversion processes. Cellulose. 16,
547–565 (2009). https ://doi.org/10.1007/s1057 0-009-9317-x
63. Piccolo, C., Bezzo, F.: A techno-economic comparison between
two technologies for bioethanol production from lignocellulose.
Biomass Bioenergy 33, 478–491 (2009). https ://doi.org/10.1016/j.
biomb ioe.2008.08.008
64. Wingren, A., Söderström, J., Galbe, M., Zacchi, G.: Process con-
siderations and economic evaluation of two-step steam pretreat-
ment for production of fuel ethanol from softwood. Biotechnol.
Prog. 20, 1421–1429 (2004). https ://doi.org/10.1021/bp049 931v
65. Unidad de Planeación Energética (UPME): Informe Mensual De
Variables De Generación Y Del Mercado Eléctrico Colombiano.,
Bogotá, Colombia (2015)