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Impact of pretreatment methods on production of bioethanol and nanocrystalline


cellulose

Chun-Han Ko, Bing-Yuan Yang, Lang-Dong Lin, Fang-Chih Chang, Wen-Hua


Chen

PII: S0959-6526(19)34784-5
DOI: https://doi.org/10.1016/j.jclepro.2019.119914
Reference: JCLP 119914

To appear in: Journal of Cleaner Production

Received Date: 28 February 2019


Accepted Date: 27 December 2019

Please cite this article as: Chun-Han Ko, Bing-Yuan Yang, Lang-Dong Lin, Fang-Chih Chang, Wen-
Hua Chen, Impact of pretreatment methods on production of bioethanol and nanocrystalline
cellulose, Journal of Cleaner Production (2019), https://doi.org/10.1016/j.jclepro.2019.119914

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© 2019 Published by Elsevier.


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Impact of pretreatment methods on production of bioethanol and

nanocrystalline cellulose

Chun-Han Ko a, Bing-Yuan Yang a, Lang-Dong Lin b, Fang-Chih Chang c,*, Wen-Hua

Chen d

a School of Forest and Resources Conservation, National Taiwan University, Taipei

10617, Taiwan
b Department of Cultural Heritage Conservation, National Yunlin University of

Science and Technology, Yunlin, Taiwan


c The Experimental Forest, National Taiwan University, Nan-Tou 55750, Taiwan
d Chemistry Division, Institute of Nuclear Energy Research, AEC, Taoyuan, Taiwan

*Corresponding author.

Tel.: +886-2-33664615; fax: +886-2-23654520.

E-mail: d90541003@ntu.edu.tw (F.C. Chang)

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ABSTRACT

Cellulose, the most abundant terrestrial biopolymer, has immense potential for a range

of applications, such as textiles, biomedical applications, and sustainable building

components. Due to the recalcitrant nature of lignocellulosic materials, various

alkaline and acid pretreatment methods have been developed to recover mono sugars

for fermentation. Simultaneous production of bioethanol and nanocrystalline cellulose

(NCC) from pretreated Formosan alder biomass was conducted in two stages.

Formosan alder biomass was treated using unbleached kraft (UEK), acid steam

explosion (SEP), and bleached acid steam explosion (BSEP) and hydrolyzed using

cellulase. Filter paper (FP) and alpha-cellulose were used as positive controls.

Bioethanol fermentation using E. coli Ko11 was conducted. Residual biomass was

further recovered to produce NCC. After 96 h enzyme hydrolysis, the highest sugar

yields were 383.4 and 329.84 mg glucose/ g biomass for the FP and UEK samples.

The highest bioethanol fermentation yields were 4.18 and 3.62 g bioethanol/ g

biomass for FP and UEK samples. During enzyme hydrolysis of the biomass,

crystallinity increased. NCC yields were 613.93 nm (68%) and 1,583 nm (73%) for

the FP and UEK samples. The negative impact of lignin content was reflected in the

particle length of the final NCC. Further lignin removal is required for optimal NCC

production. This study showed that Formosan alder biomass could be used to produce

both bioethanol and NCC.

Keywords: Pretreatments, Nanocrystalline cellulose, Bioethanol, Enzyme hydrolysis,

Acid hydrolysis

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List of abbreviations

NCC Nanocrystalline cellulose NMMO N-methylmorpholine-N-oxide

UEK Unbleached kraft DLS Dynamic light scattering

SEP Acid steam explosion SEM Scanning electron microscope

BSEP Bleached acid steam explosion XRD X-ray diffraction

FP Filter paper LCA Life cycle assessment

CI Crystallinity index 5-HMF 5-hydroxymethylfurfural

SSF Simultaneous saccharification

and fermentation

1. Introduction

The biobased economy, from an agriculture perspective, can support

development. The bioeconomy is viewed as a response to climate challenges through

the use of renewable resources. Directing biomass for various needs (bulk/higher

value-added products) is a challenge (Näyhä, 2019). Biobased products, advanced

biofuels, and viable and efficient biorefinery concepts are being ever increasingly

explored for their potentials (Langeveld et al., 2010). Many non-fuel bioproducts (e.g.,

chemicals, pharmaceuticals, biopolymers) and presents basic principles and

development options for biorefineries that can be used to generate them alongside

biofuels, power, and by-products. Biobased products offer the most development

perspectives, combining small-scale, less endowed farmers and large market volumes

with medium to high price levels (Langeveld et al., 2010). A European Union (EU)

survey, conducted in 2011, provided an alternative definition of the bio-economy as

production paradigms that rely on biological processes and, as with natural

ecosystems, use natural inputs, expend minimum amounts of energy, and do not

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produce waste as all materials discarded by one process are inputs for another process

and are reused in the ecosystem (Nebe, 2011). Concepts of the circular economy,

green economy and bioeconomy are joined to reconcile economic, environmental, and

social goals, although with distinct assumptions and operationalization strategies

(D'Amato et al., 2017). For a more sustainable economy, renewable biomass such as

crop residues and dedicated energy crops are being increasingly used for the

production of fuels, chemicals, energy and materials; movement to a more sustainable

economy also composes one major challenge faced by the society nowadays.

Environmental concerns like climate change and the increased consumption of

petro-based resources as a consequence of the population growth, and the finite nature

of oil resources are serving reasons for the transition from the current fossil-based to a

biobased economy (Mussatto, 2017). In the context of circular bioeconomy, the global

bioenergy sector may face challenges and biomass conversion must retain a high

efficiency. Bioenergy can be integrated into the cascade biorefinery and/or offer waste

management solutions (Zabaniotou, 2018). A spatial approach quantifying the

residual biomass potential in the EU-27 plus Switzerland was conducted estimating

biomass residues stemming from 4 main activities: i) agriculture; ii) forestry residues;

iii) urban greenery management; and iv) agri-industrial food process waste and

municipal biodegradable waste (Hamelin et al., 2019). Esen and Yuksel (2013) also

evaluated the feasibility of greenhouse heating by renewable energy sources. Strategic

pathways for Malaysia had been analyzed by investigating roadmaps by optimum

bio-based systems, such as bioenergy and biorefinery, and their prospects in its

sustainable development. Natural resources e.g. forestry and agricultural resources

will attribute in co-production of bio-based products, food and pharmaceutical

ingredients, fine and specialty chemicals, polymers, alongside biofuel and bioenergy

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from biomass is possible to achieve overall sustainability by the replacement of fossil

resources (Sadhukhan et al., 2018). Increased urbanization worldwide has resulted in

a substantial increase in energy and consumption as well as anthropogenic waste

generation. Current global dependence on fossil fuels is increasingly detrimental

energy-environment nexus, and the above peril is being aggravated by increased

urbanization and sequential anthropogenic waste generation. There are various

bioprocess-based technological models that hypothesize a closed loop approach to

waste valorization contributing to the circular economy (Mohan et al., 2016).

Lignocelluloses are the most promising alternative energy resources that can be

utilized to produce bioethanol. A suitable pretreatment should not only largely

improve the digestibility of lignocelluloses but also facilitate the recovery of lignin

and hemicelluloses (Sun et al., 2016b). Advanced biorefining is still an emerging

sector, and further investigation is required into the choice of feedstocks,

cost-effective lignocellulosic pretreatment options, and the identification of viable end

products (Hassan et al., 2019). The recalcitrant nature of lignocellulosic materials that

include lignin, hemicelluloses and the high crystallinity index (CI) of cellulose are the

main barriers for ethanol production (Chundawat et al., 2011). Delignification

involves disrupting and removing the cross-linkages of lignin and hemicelluloses that

cover the cellulose fibers (Jahnavi et al., 2017). The potential for use of

agro-bioenergy co-products and agro-industrial residues, such as biofuel

co/by-products, agricultural residues, agricultural by-products and agro-wastes, is

receiving attention (De Corato et al., 2018), with a focus high-value-added products in

emerging markets. Such high-value-added products include succinic acid, cellulose,

glycerin, proteins, peptides, and enzymes (De Corato et al., 2018). Fan et al. (2018)

utilized a microwave biomass pre-processing system to produce solubilized

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carbohydrates and to ferment lignocellulosic waste resources to bio-based products

(such as 2-phenylethanol, chiral sugars, lipids, sterols, and terpenes).

Alkaline treatment has proven promising process in the complete utilization of

lignocellulosic materials without adverse effects on the environment. Using this

process, lignocellulosic materials are fractionated into alkali soluble lignin and

hemicelluloses, and residues, which can be used to generate valuable products

(Jahnavi et al., 2017). The application of alkaline pretreatment removed 73% of

hemicellulose and 50% of lignin from wood fiber leading to a more exposed cellulose

structure and increasing the production of nanocellulose (Bauli et al., 2019).

Lignocellulosic biomass, referred to as a second generation biofuel because it is

non-edible, is highly regarded in the sustainable production of fuels and has social,

economic, and environmental benefits (Liew et al., 2014). There are four main steps

for producing ethanol from lignocellulose: pretreatment, hydrolysis, fermentation, and

distillation (Mohapatra et al., 2017). Bauli et al. (2019) found that the impact of

enzymatic hydrolysis was lower for selected environmental impact categories. Among

these, pretreatment is the most challenging step for increasing the accessibility to

further hydrolysis and fermentation. Hydrolysis, fermentation, and distillation are

well-established processes.

Nanotechnology is gaining popularity, and innovative materials can be obtained

from renewable and abundant lignocellulosic materials, such as nanocrystalline

celluloses (NCC). Methods for isolating cellulose from wood or agriculture residues

have received a lot of attention (Brinchi et al., 2013). NCC is defined as cellulose in

terms of a nanometer in at least one dimension. There are several ways to isolate NCC

from raw plant material, such as grinding, acid hydrolysis, and steam explosion

(Brinchi et al., 2013). The renewable plant fibers can be used as a source of NCC to

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enhance the sustainability of the final composites (Sarasini and Fiore, 2018). NCC

improves the strength, density, uniformity, and biodegradability of biocomposites for

industrial application. When cellulose biomass is treated with an acid, the pulp will

start to degrade. The degradation will start with the most accessible parts of the fiber,

followed by the reducing end groups and the crystal surfaces. The properties of

nanocellulose are affected by time, temperature, acid concentration, and acid to pulp

ratio (Sun et al., 2016a). The types of acid also play a role in the final properties of

NCC, and sulfuric acid is the most commonly used (Liu et al., 2018). Cotton fibers

and bagasse were used to produce NCC through sulfuric acid hydrolysis, and a

decrease in the non-crystalline region was shown during acid hydrolysis (Sun et al.,

2016a). The enzymatic hydrolysis apparatus could process much greater volumes of

NC, optimizing the energy use (Bauli et al., 2019). Formosan alder (Alnus formosana)

is a fast-growing, adaptable, pioneer, native tree species in Taiwan, and it is

particularly suitable for reforestation. In this study, steam-exploded Formosan alder

(Alnus formosana) biomass was used to investigate lactic acid production by

simultaneous saccharification and fermentation (SSF). The impacts of different heavy

metals on saccharification were also investigated. A. formosana is commonly found at

land-slide or barren sites and has a wide altitude and latitude range (Liu et al., 1988).

A. formosana has nitrogen fixation ability with the assistance of Rhizobium flora.

Given the abundance of A. formosana, it has the potential for use as a raw material for

NCC and ethanol production.

Process Integration has been widely applied in industry to achieve substantial

reductions in industrial energy, water and utility use (Klemeš et al., 2018). Dorotić et

al. (2019) proposed an integrated carbon neutral energy plan for an island with

optimal utilization of renewable energy sources for power, heating, cooling and

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transport sectors. Tomić and Schneider (2018) combined the circular economy and

energy analysis approaches and sustainability assessment to investigate the impact of

closing the loop material- and energy-wise and to reduce the embodied energy of

recycled materials. Walmsley et al. (2018) also found that the substantial benefits

could be arised from combining and extending the scope of methods. The orange

waste was utilized to explore the production of nanocellulose and bioethanol

(Tsukamoto et al., 2013). These studies show the importance of integrated approaches

solving future problems of energy and material. There are few holistic studies

investigated the combined production of nanocellulose and bioethanol on native

broad-leaved trees. This study integrated sustainable energy with advanced

nanomaterial processing. In this study, the simultaneous production of bioethanol and

NCC from pretreated Formosan alder biomass was conducted in two stages.

Formosan alder biomass was pretreated using unbleached kraft (UEK), acid steam

explosion (SEP), and bleached acid steam explosion (BSEP) and then hydrolyzed

using cellulase. Filter paper and alpha-cellulose were used as positive controls. The

hydrolyzed biomass was subjected to bioethanol fermentation by E. coli Ko11.

Process parameters for the bioethanol and NCC processes were examined. The life

cycle impact assessment of nanocrystalline cellulose produced from various materials

has been also explored and compared.

2. Material and methods

The research framework for this study is shown in Fig. 1. In this study, Formosan

alder biomass (hardwoods) treated by unbleached kraft (UEK), acid steam explosion

(SEP), bleached acid steam explosion (BSEP), filter paper and alpha-cellulose were

hydrolyzed by cellulase in the first step. Hydrolysates were collected for bioethanol

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fermentation. Leftover solids after fermentation were collected and subject to acid

hydrolysis for NCC preparation.

2.1. Biomass pretreatment

Fifteen-year-old A. formosana wood chips were collected from the NTU

Experimental Forest of central Taiwan. The wood chips were subjected to pulping

processes. A batch reactor was loaded with 700 g of oven-dried wood chips with a

liquor-to-wood ratio of 4 (w/v). The cooking liquor contained NaOH and Na2S in

quantities sufficient to provide 25% of sulfidity and 17% active alkali concentration

(w/w as Na2S on wood basis). The cooking temperature ranged from 25 C to 160 C

with a heating ratio of 1.5 C per min. The cooking temperature was maintained

isothermally for 180 mins. The unbleached kraft pulp was denoted UEK.

The SEP was produced from wood chips of A. formosana, dried for 24 h and then

soaked in 1.5% sulfuric acid for 96 h. The optimal conditions for steam explosion

were based on Ko et al. (2012), using a 1:7 solid/liquid ratio, treated at 180oC for 10

mins. A component of the SEP was bleached using glacial acetic acid and sodium

chlorite (NaClO2) to remove the remaining lignin. This treatment was repeated until

the cellulose became white. The bleached SEP is denoted as BSEP.

α-cellulose in powder form was supplied by Sigma Aldrich (Product code:

101777401), Saint Louis, USA. TOYO Qualitative Filter paper (FP), Grade NO. 1

(Toyo, Tokyo, Japan) and was denoted as FP. All reagents were purchased from

commercial suppliers and used without further purification.

2.2. Chemical composition

All of the respective samples were oven-dried and crushed using a grinder and

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screened to a particle of 40-60 mesh before analysis of chemical composition.

Holocellulose content was measured using TAPPI T9 standards. The ground sample

was treated with sodium chlorite and glacial acetic acid in a hot water bath. After the

sample became white, the suspension was then cooled in an ice bath, filtered, and

washed with distilled water. The cellulose content was determined by TAPPI T203

standards. The ash content of the pulp was determined using a muffle furnace by

TAPPI T211 standards.

2.3. Enzyme hydrolysis

The hydrolyzing enzymes used in this experiment were cellulase complex

formulation 50013 (Novozyme, Denmark). Three enzyme loadings equivalent to 1.5,

4.5, and 9 IU endoglucanase (CMCase/mL) were used in the reaction solutions.

Hydrolysis was conducted in a total volume of 200 mL liquid with 0.1 M Tris-buffer

(pH 7), 2.5% (w/v) samples in a 250 mL conical flask. The flasks were water bathed

at 50oC, shaken at 100 rpm for 96 h, and the samples were analyzed every 12 h.

Activities for all the cellulases were measured using the dinitrosalicylic acid.

2.4. Ethanol fermentation

E. coli KO11 was purchased from the American Type Culture Collection (ATCC).

Before simultaneous saccharification and fermentation (SSF), the E. coli KO11 was

pre-cultured in a liquid medium (with 10 g/L tryptone, 5 g/L yeast extract,10 g/L

NaCl, and 20 g/L glucose dissolved in H2O) at 37 C for 24 h. Optical density

(OD600, Jasco V-530, Japan) was raised to approx. 1.4 prior to fermentation.

The flask and samples were autoclaved at 121C for 20 mins. Each flask

contained 0.5% w/v yeast extract,1% w/v tryptone, and 5 mL of E. coli KO11

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inoculums with the highest enzyme loading. The flasks were shaken slowly (100 rpm)

at 37C and samples were taken at every 12 h for 120 h and frozen until analysis.

The enzymatic hydrolysis samples were analyzed using high-performance liquid

chromatography (HPLC, Jasco PU-980, Japan) with an IC Sep ION-300 column

(Transgenomic, America) and a refractive index (Shodex RI-101, Japan) detector to

identify the alcohols and mono-sugars at 70C; sulfuric acid (0.0085 N) was used as

the mobile phase at 0.4 mL/min.

2.5. Preparation of NCC

Perspective biomass and fermentation residue were treated using sulfuric acid

(65%) to obtain NCC at different conditions. The acid hydrolysis was stopped

immediately using a 7-fold dilution with deionized water. The mixture was

centrifuged at 4C at a speed of 10,000 rpm for 10 mins. The centrifugation was

repeated three times, and the precipitate was then placed in regenerated cellulose

dialysis tubing with a molecular weight cutoff of 12,000 – 14,000 and dialyzed using

distilled water until the pH reached a value of 7.0.

Regenerated cellulose was prepared according to the following procedure. The

solution (around 1-3%) was prepared by dissolving the different kinds of cellulose

pulp (1-3 g) in commercial N-methylmorpholine-N-oxide (NMMO) solution under

magnetic stirring at 110oC in an oil bath until the cellulose had been dissolved

completely. In the procedure, propyl gallate was added as an antioxidant to prevent

oxidation and degradation during the dissolution of cellulose.The temperature did not

exceed 130oC to prevent degradation of the solution and generated byproducts. After

the cooking process, the samples were rinsed using deionized water owing to remove

the solvent, then freeze-dried and stored for the next experiment.

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2.6. Characterization of NCC

2.6.1. Particle size distribution

Particle size distributions of the biomass were measured using MorFi (TECHPAP,

Saint Martin d’Hères, France) at the Chung-Hwa Pulp Corporation in Hualien. More

detailed analysis of these methods is described in earlier work (Ko et al., 2019). The

samples were placed into a 1 L beaker then diluted with 1 L of RO water, to achieve a

uniform 0.3% solution. The beaker and fiber solution was then placed on the holder of

the MorFi analyzer. The particle size of samples after acid hydrolysis was measured

using dynamic light scattering (DLS, SZ-100, HORIBA Scientific, France) in the

range between 0.4 nm and 10 μm, under the following conditions: particle refractive

index 1.60, particle absorption coefficient 0.01, water refractive index 1.33,

temperature 25 C, and general calculation model for irregular particles. Each

measurement was repeated three times.

2.6.2 Scanning electron microscopy

The surface morphology of cellulosic fibers was observed using a scanning

electron microscope (SEM) model JSM-6510 (JEOL, Japan) with an accelerating

voltage of 15 kV. Prior to SEM examination, the samples were freeze-dried and then a

fine layer of gold was deposited on the samples by means of a plasma sputtering

apparatus (SPI-Module, Sputter Coater).

2.6.3 X-ray diffraction

X-ray diffraction (XRD) analysis of nanocellulose from different samples was

conducted using a powder X-ray diffractometer (D8, Bruker AXS Gmbh, Karlsruhe,

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Germany) equipped with a radiation source operating at 40 kV and 40 mA. The XRD

patterns were recorded over the angular range 2θ = 5-50.

The CI was calculated from the heights of the 200 peak (I200, 2θ= 22.6) and the

intensity minimum between the 200 and 110 peaks (Iam, 2θ= 18) based on Eq. (1).

I200 represents both crystalline and amorphous material, whereas Iam represents the

amorphous material.

CI (%) = (I200-Iam)*100/I200 (1)

2.7. Life cycle impact assessment

A previous study provided a broader evaluation of the use of alternative crops in

bioethanol production (Chang et al., 2017). SSF waste utilization will be suitable for

regions that rely on imported energy resources. A life cycle impact assessment of

fermentation waste was performed, using a functional unit of 1 g of NCC obtained

after 90 mins hydrolysis. This study used the IMPACT 2002+ model with midpoint

evaluation. This method analyzes four impacts: human health, ecosystem quality,

climate change, and resources. The energy inputs/outputs in terms of machinery usage,

chemicals, and water consumption were considered for different residues. This study

used the inventory data of the built-in database (Ecoinvent) of the SimaPro 8.2 Life

Cycle Assessment (LCA) software which were modified to reflect the actual situation

in Taiwan.

3 Results and discussions

3.1 Chemical composition of biomass

Chemical composition of the biomass is listed in Table 1. The results are based on

a triplicate proximate analysis. The α- cellulose content of FP, α- cellulose, BSEP,

and UEK is higher than SEP. The lignin content of SEP is higher than the other four
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materials. With respect to the extreme SEP pretreatment, the structures between

cellulose and lignin are degraded, but the lignin is not dissolved. As such, SEP is

responsible for partial hydrolysis of cellulose, so the content of α-cellulose is lower;

some holocellulose would degenerate to furfural, and hemicellulose is decreased

slightly. The results for BSEP showed that the bleaching treatment removed the lignin

efficiently, and the total lignin content decreased from 36.35% to 0.26% (Table 1).

Bauli et al. (2019) and Shanmugarajah et al. (2019) also found that the washing, alkali,

bleaching, and acid treatments were effective in the removal of hemicellulose, lignin,

and other impurities.

3.2 Enzymatic hydrolysis

Three dosages of 1.5, 4.5, and 9.0 CMCase/mL were used in the hydrolysis

processes. Fig. 2 depicts the 96 h enzymatic hydrolysis. Hydrolysis of the FP samples

using the three dosages yielded 140.03, 293.85, and 383.40 mg glucose /g dried

biomass; equivalent to 3.56, 7.47, and 10.63 g glucose /L. After enzymatic hydrolysis,

the remaining solid biomass 68.22, 57.22, and 43.86 (%, w/w), was recovered by

centrifugation and used for NCC production. Hydrolysis of the α-cellulose sample

using the above three dosages yielded 84.25, 195.49, and 241.92 mg glucose /g dried

biomass; equivalent to 2.11, 4.89, and 6.05 g glucose /L. After enzymatic hydrolysis,

the remaining solid biomass 72.31, 62.18, and 51.29 (%, w/w), was recovered by

centrifugation and used for NCC production. Hydrolysis of the UEK sample using the

three dosages yielded 121.11, 255.18, and 329.84 mg glucose /g dried biomass;

equivalent to 3.03, 6.38, and 8.25 g glucose /L. After enzymatic hydrolysis, the

remaining solid biomass 73.42, 57.76, and 45.89 (%, w/w), was recovered by

centrifugation and used for NCC production. Hydrolysis of the SEP sample using the

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three dosages yielded 74.91, 171.79, and 209.32 mg glucose/g dried biomass,;

equivalent to 1.87, 4.29, and 5.23 g glucose /L. After enzymatic hydrolysis, the

remaining solid biomass 72.71, 68.22, and 58.31 (%, w/w), was recovered by

centrifugation and used for NCC production. Hydrolysis of the BSEP sample using

the three dosages yielded 98.70, 223.86, and 277.44 mg glucose/g dried biomass;

equivalent to 2.47, 5.60, 6.94 g glucose /L. After enzymatic hydrolysis, remaining

76.10, 62.67, 49.69 (%, w/w) solid biomass, was recovered by centrifugation and

subjected for NCC production. The mass ratio of solid to liquid was 2.5%, (w/w). For

25 g cellulose in 1 L of reactant, the optimal yield of glucose was 27.75 g/L.

The maximum glucose yield (383.40 mg/g dried biomass) was obtained for FP

while UEK had a glucose yield of 329.84.40 mg/g dried biomass. Biomass with more

non-crystalline regions yields more glucose during hydrolysis.

At the end of incubation, the samples were centrifuged for 9,000 rpm at 4oC for 20

mins, and then the substrates and supernatant were separated and used for NCC

production.

3.3 Fermentation of hydrolysate

Fig. 3 depicts the 120 h fermentation of hydrolysate by E. coli KO11. Ethanol

concentrations reached the maximum after 96 h for three concentrations of starting

glucose. The ethanol fermentation curves are similar, regardless of concentrations of

starting glucose. The 96 h fermentation of FP hydrolysate by E. coli KO11 yielded

1.46, 3.04, and 4.18 g ethanol/L. The 96 h fermentation of α-cellulose hydrolysate by

E. coli KO11 yielded 0.86, 2.10, and 2.52 g ethanol/L. The 96 h fermentation of UEK

hydrolysate by E. coli KO11 yielded 1.28, 2.67, and 3.62 g ethanol/L. The 96 h

fermentation of SEP hydrolysate by E. coli KO11 yielded 0.75, 1.82, and 2.14 g

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ethanol/L. The 96 h fermentation of BSEP hydrolysate by E. coli KO11 yielded 1.04,

2.24, and 2.82 g ethanol/L.

The inhibitors such as 5-hydroxymethylfurfural (5-HMF) and furfural that were

generated during acid hydrolysis of wood chips were considered toxic to

microorganisms. Sugar degradation products such as furfural and 5-HMF can be

reduced using low severity acid pretreatment (Davis et al., 2013). In this study, the

concentrations of 5-HMF and furfural inhibitors were below the detection limit. The

results are similar with those from Maciel-Silva et al. (2019).

Optimal ethanol yields are from FP and UEK hydrolysates (4.18 and 3.62 g

glucose/L) using the highest CMCase dosages. The theoretical maximum ethanol

yield from E. coli KO 11 is 4.71 g ethanol/L from 10.63 g glucose/L hydrolysate

solution. The lower ethanol yields from α-cellulose and SEP hydrolysates than FP and

UEK ones are due to its high cellulose crystallinity and high lignin contents. Song et

al. (2019) also found that the production of bioethanol from hardwoods is difficult due

to the low level of enzymatic hydrolysis caused by the high lignin contents.

3.4 Crystallinity indexes (CIs) and Zeta potential

The CIs of five biomass materials were determined by X-ray diffraction; the XRD

pattern of (α-cellulose, FP, and SEP) is shown in Fig. 4. FP has the highest CI,

79.22%. The order of CI was FP > α-cellulose > UEK > BSEP > SEP. The bleaching

process results in more crystal regions in the cellulose and increases the CI from

61.19% for SEP to 65.58% for BSEP. The CI of SEP pulp was lower than UEK

owing to the pretreatments that leave more lignin in the SEP, resulting in lower

cellulose content. The CI values are higher from those obtained using alkaline

pretreatment and enzymatic hydrolysis, as presented by Bauli et al. (2019) and

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bleaching, alkali treatment, and acid hydrolysis, as presented by Shanmugarajah et al.

(2019). The SEP pretreatment only broke the bonds between the amorphous and

crystalline cellulose but did not remove the lignin. The CI and the presence of lignin

are the main factors which affect the reactivity and accessibility of cellulose (Lai,

2017).

The stability of colloidal dispersions can be determined by the zeta potential.

Nanoparticles in water with a higher zeta potential can generate electrostatic repulsion,

then form a stable suspension. All materials had negatively charged surfaces due to

the esterification of hydroxyl groups during the process of acid hydrolysis. As shown

in Fig. 5, the zeta potential value increased with the increased acid hydrolysis duration,

which means that the stability of the cellulose suspensions increased with an increase

in acid hydrolysis duration.

3.5 Particle size distribution

Fig. 6 (a) shows the fiber lengths of materials measured using MorFi. The average

fiber lengths of FP, α-cellulose, UEK, SEP, and BSEP were 1.004, 0.486, 0.712,

0.604, and 0.582, respectively. The average fiber length of FP was greater than the

other materials. Fig. 6 (b) shows the fiber widths of materials measured using MorFi.

The average fiber widths of FP, α-cellulose, UEK, SEP, and BSEP were 23.1, 16.8,

18.2, 43.3, and 34.7, respectively. SEP, UEK, and α-cellulose fibers are narrower than

the other materials. Fig. 7 shows the fiber fine lengths of materials measured using

MorFi. The average fiber lengths of FP, α-cellulose, UEK, SEP, and BSEP were 40,

40, 56, 37, and 45, respectively.

3.6 Mass balance for acid hydrolysis of biomass

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The mass balance for each sample is shown in Table 2. All samples show a

decrease in yield with an increase in hydrolysis duration. On the contrary, the yield of

sugar increased with an increase in hydrolysis duration.

The yields of UEK and SEP are higher than the other four samples due to the

presence of lignin which cannot be dissolved by acid and thus impedes the hydrolysis

process. Lignin affects not only the yield but also the particle size distribution which

will be discussed in the next section. Among the other three substrates, FP,

α-cellulose, and BSEP, the yield of FP is higher than α-cellulose and BSEP, because

the acid tends to attack the amorphous region, and the higher CI of FP makes

hydrolysis of the cellulose more difficult. The CI and the presence of lignin are the

main factors which affect the reactivity and accessibility of cellulose.

3.7 Change of particle size during hydrolysis

Changes in particle size distribution during FP hydrolysis were measured using

DLS as shown in Fig. 8(a). The results show that during FP hydrolysis, smaller

cellulosic particles could be obtained. After 4 h acid hydrolysis of FP, the results

show that particle sizes after FP hydrolysis ranged from 82 to 2,511 nm and the

average size is 396.2±13.4 nm.

Changes in particle size distribution during α-cellulose hydrolysis were measured

using DLS as shown in Fig. 8(b). The first peak of the particle sizes of α-cellulose is

between 39-105 nm, the second peak is between 118-655 nm; the average is

208.4±16.8 nm. After 1 h of stirring, the reaction became more uniform and the

cellulose degraded significantly. FP showed no significant difference after 1 h

hydrolysis and α-cellulose showed no significant difference after 1.5 h of hydrolysis.

FP and α-cellulose showed a bimodal size distribution.

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Changes in particle size distribution during BSEP hydrolysis were measured using

DLS as shown in Fig. 8(c). BSEP has sizes distributed from 72 to 118 nm and the

average is 96.4±14.3. In the statistical analysis, each sample showed a significant

difference between 0.5-1 h, as the initial solution was viscous which impeded the

reaction. BSEP showed no significant difference after 2 h of hydrolysis. These results

show that optimum hydrolysis time varies for different materials.

By comparing the CI of α-cellulose, FP, and BSEP, it is found that the lower of

the CI was, the higher the efficiency of hydrolysis and the smaller the particle size

was. There is no positive correlation between fiber length and particle size. The above

results show that the influence of the CI of the raw material on particle size is greater

than that of the fiber length. The CI of the raw material is a key factor affecting the

efficiency of hydrolysis.

3.8 Fiber morphology

Fig. 9 shows a comparison of the SEM images of the freeze-dried, acid

hydrolyzed cellulose fibers. Upon drying, all materials tend to be self-assembled into

micron width and micrometer length fibers. This may be due to the strong

inter-fibrillar attraction caused by hydrogen bonding among the surface hydroxyl

groups of cellulose during the drying process. The reduction in fiber diameter was

observed across all the fiber samples after acid hydrolysis due to the successful

removal of amorphous regions from the raw biomass materials.

3.9 Life cycle impact assessment of NCC

Table 3 lists the assessment results for the endpoint impact indicators for NCC

from BSEP, FP, and α-cellulose. The results show that the production of NCC from

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α-cellulose results in considerable environmental impacts, particularly due to the use

of dialysis water, acid, and drying. The production of NCC from FP has a lower

environmental impact in all four categories compared to the other materials. To

mitigate the impacts on human health, climate change, and resource depletion during

the production of NCC, there is a need for improvements to the hazardous processing

phases. As reported by Bauli et al. (2019), the most significant environmental impact

in NCC production relates to the energy expenditure during heating and drying

processes.

3.10 Comprehensive comparison with literature

Table 4 provides a comparison of NCC from different starting materials. Different

raw materials attributed different chemical constitution and cellulose structures which

influenced the effects of acid hydrolysis and varying pretreatments also play an

important role. Morais et al. (2013) reported that cotton linters provide higher purity

and crystallinity during acid hydrolysis and the properties of the NCC product are

similar to those in this study. The main differences in preparation were alkaline and

mechanical pre-treatment. The results of this study show that multiple pretreatments

could facilitate the production of NCC even when using complex raw material.

de Carvalho Benini et al. (2018) used Imperata brasiliensis, a type of herbaceous

plant containing less lignin with higher accessibility to acid hydrolysis. The NCC

produced through 75 mins acid hydrolysis of Imperata brasiliensis was similar this

study, indicating that pretreatments could shorten the reaction time and enhance the

suspension stability due to the increased zeta potential.

Tian et al. (2016) used a similar method for the production of NCC which

combined mechanical and chemical treatments and found that, as in this study,

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mechanical pretreatment could shorten the acid hydrolysis time effectively and

reaction time was curtailed to below 40 mins. The final properties of the NCC were

similar to this study although different raw materials were used.

Kargarzadeh et al. (2012) found that the native properties and chemical

composition of the raw materials influence the production of NCC. Kenaf bast is a

type of high-quality fiber with high cellulose content and high proportion of slender

fibers (Palle, 2008). Under similar acid hydrolysis conditions, NCC from kenaf bast

fibers showed higher recovery and more rod-like structures due to the fiber structure

and lower lignin content which act as a barrier during the NCC production. Yields and

sizes of NCC derived from corncob residue (Liu et al., 2016) were similar to that

found in this study.

4 Conclusions

In this study, five materials (α-cellulose, FP, SEP, UEK, and BSEP) were used to

produce NCC using different pretreatments and durations of acid hydrolysis, and the results

were analyzed and compared using DLS, XRD, and SEM techniques. Smaller cellulose

particles can be obtained with increasing hydrolysis time, and the average particle size after

4 h acid hydrolysis is SEP > UEK > FP > α-cellulose > BSEP. After 96 h enzyme

hydrolysis, the highest sugar yields were 383.4 and 329.84 mg glucose/ g biomass for FP

and UEK samples. The highest bioethanol fermentation yields were 4.18 and 3.62 g

bioethanol/ g biomass for FP and UEK samples. NCC yields were 68 and 73% for FP and

UEK samples. NCC yields were 613.93 and 1,583 nm for FP and UEK samples. The

results indicate that the influence of CI of raw materials on the particle size distribution is

greater than that of the fiber length of raw materials, so the CI of raw materials is a key

factor which affects the efficiency of acid hydrolysis. The zeta potential value for all

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materials increased with increased acid hydrolysis time because the esterification of

hydroxyl groups during the process of acid hydrolysis would form the negative charges on

the cellulose surface. Lignin constituents in biomass, such as UEK and SEP, would affect

the efficiency of acid hydrolysis. Compared with α-cellulose, FP, SEP, UEK, and BSEP,

the particle size distribution results of UEK and SEP show a wide range. The zeta potential

value of UEK and SEP are lower than the other materials, as particles of UEK and SEP

could not disperse well in solution. The delignification process (to separate the lignin and

cellulose after acid hydrolysis) is an important step during the process of NCC preparation.

Acknowledgements

The financial supports by the Ministry of Science and Technology, Taiwan for the

project MOST 105-2313-B-002-029, MOST 106-2313-B-002-008, MOST

107-2313-B-002-018, and MOST 107-2621-M-002-010 are gratefully acknowledged.

Kind donation of enzymes by Novozymes is also acknowledged.

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Figure captions:

Fig. 1. Research framework of this study.

Fig. 2. Kinetics of 96 h enzymatic hydrolysis of biomass samples by three dosages of

cellulase. Left panel: 1.5 CMCase/mL; middle panel: 4.5 CMCase/mL; right

panel: 9.0 CMCase/mL.

Fig. 3. Kinetics of 120 h fermentation of hydrolysate by E. coli KO11.

Fig. 4. XRD patterns of biomass materials.

Fig. 5. Effect of hydrolysis durations on the zeta potential of FP, α-cellulose, and

BSEP.

Fig. 6. Distribution of sample (a)fiber length and (b) fiber width.

Fig. 7. Size distribution of fines length.

Fig. 8. Effect of hydrolysis times on the size distribution of (a) FP, (b) α-cellulose, (c)

BSEP.

Fig. 9. SEM images of raw materials and acid hydrolyzed cellulosic biomass obtained

from (A) (a) FP, (B) (b) BSEP, and (C) (c) α-cellulose.

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Filter paper
Biomass
α-Cellulose
UEK
Pretreatment
SEP

Enzyme BSEP
Hydrolysis

Hydrolysate
Fermentation

Leftover
Solid Bioethanol
Acid
Hydrolysis

Nanocrystalline
Cellulose

Fig. 1.

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Fig. 2.

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Fig. 3.

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Fig. 4.

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Fig. 5.

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(a) (b)

Fig. 6.

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Fig. 7.

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(a)

(b)

(c)

Fig. 8.

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A a

B b
B

C c
Fig. 9.

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Table 1

Chemical composition of biomass and pulps.

Holocellulose α- cellulose Acid-insoluble Acid-soluble


Sample Ash (%)
(%) (%) lignin (%) lignin (%)
Formosan
69.89 42.62 28.5 1.87 0.51
alder chips
FP 95.18 92.12 0.02 N.D. 0.18
α- cellulose 94.12 91.92 0.28 0.01 0.31
UEK 91.65 87.15 3.45 0.12 2.54
SEP 60.60 51.66 33.79 2.56 0.35
BSEP 94.65 91.32 0.25 0.01 1.36

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Table 2

Mass balance for acid hydrolysis of biomass. Numbers denote hydrolysis times.

  Substrates Glucose TSS Total Other


FP- 30 6.96 ± 1.32 0.00 ± 0.00 0.03 ± 0.01 6.99 ±1.31 3.01±1.31
FP- 60 5.78 ± 0.88 0.62 ± 0.03 0.02 ± 0.01 6.42 ±0.87 3.58±0.87
FP- 90 5.49 ± 0.58 1.06 ± 0.29 0.02 ± 0.01 6.57 ±0.67 3.43±0.67
FP- 120 5.21 ± 0.36 1.33 ± 0.16 0.02 ± 0.01 6.54 ±0.58 3.46±0.58
FP- 240 4.81 ± 0.33 1.88 ± 0.21 0.02 ± 0.01 6.71 ±0.31 3.29±0.31
α-cellulose-30 5.73 ± 1.26 0.63 ± 0.02 - 6.37 ±1.10 3.63±1.10
α-cellulose-60 5.01 ± 0.79 1.15 ± 0.08 0.02 ± 0.02 6.18 ±0.98 3.82±0.98
α-cellulose-90 4.63 ± 0.57 1.34 ± 0.11 0.02 ± 0.01 5.99 ±0.73 4.01±0.73
α-cellulose-120 4.11 ± 0.62 1.31 ± 0.18 0.02 ± 0.01 5.44 ±0.58 4.56±0.58
α-cellulose-240 3.78 ± 0.46 2.88 ± 0.14 0.02 ± 0.01 6.68 ±0.55 4.32±0.55 
BSEP- 30 5.63 ± 0.82 0.46 ± 0.01 - 6.10 ±0.77 3.90±0.77
BSEP- 60 5.28 ± 0.41 0.53 ± 0.01 - 5.82 ±0.45 4.18±0.45
BSEP- 90 4.95 ± 0.24 0.79 ±0.02 - 5.75 ±0.30 4.25±0.30
BSEP- 120 4.66 ± 0.15 1.87 ±0.11 0.02 ± 0.01 6.54 ±0.23 4.19±0.23
BSEP- 240 4.31 ± 0.18 2.76 ±0.19 - 7.07 ±0.34 3.82±0.34
UEK- 30 6.16 ± 1.51 0.68± 0.03 0.04 ± 0.01 6.88 ±1.25 3.12±1.25
UEK- 60 5.97 ±1.12 0.80 ± 0.03 0.02 ± 0.01 6.79 ±1.01 3.21±1.01
UEK- 90 5.51 ± 0.46 0.88 ± 0.02 0.03 ± 0.01 6.42 ±0.57 3.58±0.57
UEK- 120 5.24 ± 0.66 0.77 ± 0.01 0.02 ± 0.02 6.03 ±0.81 3.97±0.81
UEK- 240 5.08 ± 0.23 1.00 ±0.02 0.02 ± 0.01 6.10 ±0.36 3.90±0.36
SEP- 30 6.81 ± 1.92 0.41 ± 0.04 0.16 ± 0.03 7.38 ±2.01 2.62±2.01
SEP- 60 6.26 ±1.46 0.39 ± 0.03 0.17 ± 0.02 6.83 ±1.21 3.17±1.21
SEP- 90 6.09 ± 0.89 1.41 ± 0.13 0.12 ± 0.01 7.62 ±0.72 2.38±0.72
SEP- 120 5.84 ±0.44 1.60 ± 0.11 0.14 ± 0.03 7.58 ±0.46 2.42±0.46
SEP- 240 5.56 ± 0.58 1.72 ± 0.09 0.14 ± 0.05 7.42 ±0.64 2.58±0.64

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Table 3

Assessment of the endpoint impact indicator for BSEP, FP, and α-cellulose.

Damage category Unit BSEP-90 FP-90 α-cellulose-90


Total mPt 2.507 2.261 2.682
Human health mPt 1.073 0.967 1.147
Ecosystem quality mPt 0.037 0.034 0.040
Climate change mPt 0.757 0.683 0.810
Resources mPt 0.640 0.577 0.685

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1

3 Table 4

4 Comparison of NCC from different starting materials.

Raw Material Procedures Particle Size (nm) Zeta ( mV) Recovery (%) Reference
1.Delignification
Corncob residue 2.Hydrochloric acid 198 ± 51 nm −33.8 ± 1.7 34.5% Palle, 2008
hydrolysis
1.Delignification Kargarzadeh et al.,
Kenaf bast fibers 124 ± 42 nm -8.77 ~ -95.3 55%
2. Sulfuric acid hydrolysis 2012
Cotton linters Sulfuric acid hydrolysis 179.3 nm -45.3 ± 1.4 - Morais et al., 2013
1. Sulfuric acid hydrolysis Length:100-300
Bleached eucalyptus wood pulp -13.3 ~ -44.5 - Tian et al., 2016
2.High-pressure homogenizer Width: 5-10
1.Milling machine
Cellulose fiber 300-400 nm - 59% Song et al., 2018
2. Oxalic acid hydrolysis
de Carvalho
Imperata brasiliensis Sulfuric acid hydrolysis 149.5 −30.83 ± 1.3 37-45%
Benini et al., 2018
1.Alkaline pretreatment
Pinus taeda 200 nm - 11.43 Bauli et al., 2019
2.Enzymatic hydrolysis
5

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Impact of pretreatment methods on production of bioethanol and

nanocrystalline cellulose

Chun-Han Ko a, Bing-Yuan Yang a, Lang-Dong Lin b, Fang-Chih Chang c,*, Wen-Hua

Chen d

a School of Forest and Resources Conservation, National Taiwan University, Taipei

10617, Taiwan (No. 1, Sec. 4, Roosevelt Rd., Taipei 10617, Taiwan)


b Department of Cultural Heritage Conservation, National Yunlin University of

Science and Technology, Yunlin, Taiwan (No. 123, Sec. 3, University Rd, Douliou,

Yunlin 64002, Taiwan)


c The Experimental Forest, National Taiwan University, Nan-Tou 55750, Taiwan

(No.12, Section 1, Chien-Shan Road, Chu-Shan, Nan-Tou 55750, Taiwan)


d Chemistry Division, Institute of Nuclear Energy Research, AEC, Taoyuan, Taiwan

(1000 Wenhua Rd., Longtan District, Taoyuan 32546, Taiwan)

Tel.: +886-2-33664615; fax: +886-2-23654520.

E-mail:

Chun-Han Ko: chunhank@ntu.edu.tw

Bing-Yuan Yang: r99625043@ntu.edu.tw

Lang-Dong Lin: ldannlin@yuntech.edu.tw

Fang-Chih Chang: d90541003@ntu.edu.tw

Wen-Hua Chen: wenhua@iner.gov.tw


Journal Pre-proof

*Corresponding author.

Tel.: +886-2-33664615; fax: +886-2-23654520.

E-mail: d90541003@ntu.edu.tw (F.C. Chang)


Journal Pre-proof

Research Highlights:

a. This study integrates sustainable energy with advanced nanomaterial

processing.

b. Prove that a native species is able to fulfill the future need.

c. Feedstock crystallinity is a key factor impacting acid hydrolysis efficiencies.

d. Degree of delignification process is dictating final bioethanol yields.

e. Nanocrystalline cellulose yields were 73% for unbleached kraft pulp as the

feedstock.

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