Accepted Manuscript

Title: Hemicellulose based biorefinery from pineapple peel

waste: Xylan extraction and its conversion into
xylooligosaccharides

Authors: Shivali Banerjee, Antonio F. Patti, Vijayaraghavan

Ranganathan, Amit Arora

PII: S0960-3085(19)30083-5
DOI: https://doi.org/10.1016/j.fbp.2019.06.012
Reference: FBP 1095

To appear in: Food and Bioproducts Processing

Received date: 29 January 2019

Revised date: 17 May 2019
Accepted date: 19 June 2019

Please cite this article as: Banerjee S, Patti AF, Ranganathan V, Arora A,
Hemicellulose based biorefinery from pineapple peel waste: Xylan extraction and
its conversion into xylooligosaccharides, Food and Bioproducts Processing (2019),
https://doi.org/10.1016/j.fbp.2019.06.012

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 Hemicellulose based biorefinery from pineapple peel waste: Xylan extraction and its
conversion into xylooligosaccharides

Shivali Banerjee1,2, Antonio F. Patti3, Vijayaraghavan Ranganathan3, Amit Arora1,2,*

1
IITB- Monash Research Academy, Indian Institute of Technology Bombay, Powai, Mumbai

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400076, India

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2
Bio- Processing Laboratory, Centre for Technology Alternatives for Rural Areas, Indian
Institute of Technology Bombay, Powai, Mumbai 400076, India

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3

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School of Chemistry, Green Chemical Futures, Monash University, Wellington Road,
Clayton, Victoria 3800, Australia

* Corresponding Author
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Email: aarora@iitb.ac.in
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Graphical abstract
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Highlights

 Pineapple peel emerged out to be a rich source of hemicellulose.

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 Maximum hemicellulose was recovered at 45˚C with pre-incubation of 16h.

 The hydrothermal process gave maximum hemicellulose yield without pre-incubation.
 XOS and xylose were final products of the hemicellulose bio-refinery.
 XOS mixture was composed of xylobiose (~83% of XOS) and xylotriose (~16% of
XOS).
Abstract

Pineapple peel waste was utilized as an unexplored source of hemicellulose (31.8 ± 1.9%) for
value addition. Hemicellulose was extracted by an alkali-based method, where the peels were
incubated at different alkali concentrations (5%, 10% and 15% w/v) at temperatures ranging
from 35˚C to a maximum of 65˚C for a fixed period of 16 h. A maximum recovery of
hemicellulose (95.9 ± 2.0%) was observed after incubating extractive-free pineapple peels in

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15% (w/v) alkali solution for 16 h at 45˚C. Higher incubation temperatures (65˚C) for 16 h,

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resulted in a lower yield of hemicellulose (81.7 ± 3.7%) which can be attributed to the
disintegration of the hemicellulose structure due to large severity factor (temperature- time

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combination). With low severity factor, it was noted that higher yields (96.6 ± 0.3%) were

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obtained 65˚C, 4 h). Hydrothermal-assisted alkali extraction was also evaluated for
maximizing the recovery of pineapple peel hemicellulose. The maximum relative recovery of

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~87.6% was obtained with 10% (w/v) alkali at the end of 1.5 h of hydrothermal pretreatment
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(121˚C and 15 psi pressure). The hemicellulose extracted by hydrothermal-assisted alkali
pretreatment was enzymatically hydrolyzed to produce XOS and the process was optimized
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in terms of enzyme dose (U), temperature (˚C), pH and time (h). Direct hydrolysis of
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pineapple peels with dilute nitric acid produced xylose-rich liquor (~91% xylose yield) at
0.5% nitric acid, reaction time of 1h and solid-liquid ratio of 1:20. The xylose-rich liquor
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could be converted to potential chemicals such as xylitol.

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Keywords: pineapple peels, biorefinery, hemicellulose, xylooligosaccharides, xylose.

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1. Introduction

Lignocellulosic biomass residues are generated as waste from the agricultural residues and
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are gaining interest from the point of view of a biorefinery (Mikkonen & Tenkanen, 2012;
Anwar et al., 2014; Isikgor & Becer, 2015; Machado et al., 2016; Banerjee et al., 2018). The
concept of a biorefinery is similar to that of the traditional petroleum refinery where a
substrate is completely utilized to obtain biofuels, bio-based chemicals and other biomass-
derived products (Cherubini, 2010). Cellulose, hemicellulose and lignin are the major
components of the lignocellulosic biomass along with pectin, wax, protein and extractives as
the minor components. The relative proportion of cellulose, hemicellulose and lignin varies
with type of biomass. Hemicellulose is one of the most abundant components of
lignocellulosic biomass after cellulose and makes about 15 - 35% of lignocellulosic biomass
(Limayem & Ricke, 2012). The amorphous and highly branched nature of hemicellulose
allows easy conversion into different value-added compounds. It is more unstable in
comparison to cellulose and lignin and hence, involves less harsh pre-treatment conditions for
its degradation (Luo, et al. 2018). The utilization of the hemicellulose fraction helps in
justifying the overall economics of the production of biofuels and bio-chemicals from

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lignocellulosic biomass. The hemicellulose proportion varies with type of feedstock. They

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can be categorized into four major groups, namely, (1) hardwood such as poplar (26.2 -

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28.7%) and oak (35.9%); (2) softwood such as pine (24.0 - 27.0%) and spruce (22.9%); (3)
agricultural waste such as barley hull (36.0%), corn cob (31.9 - 36.0%) and sugarcane

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bagasse (28.0 - 32.0%); and (4) grasses such as switch grass (25.0 - 50.0%) (Isikgor & Becer,
2015). Xylan is the most abundant hemicellulose occurring in nature and consists of β-D-

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xylopyranosyl (xylose) residues that are linked via β-1-4glycosidic bonds. Other chemical
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units such as arabinose or glucuronic acid might also be attached to the xylan backbone and
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results into sub categories of xylan namely, homoxylan, arabinoxylan, glucuronoxylan and
arabinoglucuronoxylan (Van Dyk & Pletschke, 2012; Koukiekolo, et al. 2005).
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Hemicellulose, mainly xylan, finds potential application as bio-composite, bio-films which

could act as barrier towards oil and fats (Stevanic, et al. 2011). Xylan extracted from barley
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husk has been found to form films even without plasticizers which suggest that the structure
of xylan influences its film-forming properties (Hoije et al. 2005). The hydrophilic character
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of xylan is responsible for its non-affinity towards oils and fats (Stevanic et al. 2012;
Stevanic, et al. 2011). Other properties such as high light transmittance, low oxygen
permeability and aroma permeability, makes xylan a suitable substrate for packaging
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applications (Mikkonen & Tenkanen, 2012). The extracted xylan can be converted into a
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wide range of high-value chemicals such as xylitol, furfural or ethanol via chemical or bio-
chemical routes (Naidu, Hlangothi & John, 2018; Singh et al. 2017; Machado, et al. 2016;
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Zabed et al. 2016; Rafiqul & Mimi Sakinah, 2012). It can also be converted into
xylooligosaccharides (XOS) with different degree of polymerization (DP). The XOS with
(DP)n=2,3 is reported to have maximum prebiotic potential (Aachary & Prapulla, 2009). The
xylan component can also be directly converted into xylose which can further be converted
into xylitol which could be used as an alternative low-calorie sweetener. Xylitol is also used
in the pharmaceutical, food and beverage industries.
In a biorefinery, the hemicellulose component of the biomass can be extracted by employing
a variety of physical, chemical and biological pretreatment approaches to overcome the
recalcitrant nature of associated cellulose (Chaturvedi & Verma, 2013) and preserve extracted
xylan for further valorization. Hydrothermal assisted alkali pretreatment has been reported for
the extraction of hemicellulose (90-96%) from corn cobs using 16% alkali (Samanta, et al.
2012). The conventional alkali-based extraction of hemicellulose from arecanut husk gives
maximum yield after an incubation period of 16 h whereas the time of extraction of
hemicellulose is reduced to half in the case of steam-assisted alkali pretreatment (8 h of

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incubation) (Singh et al. 2018). Recent studies have shown an increase in the accessibility of

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the cellulose and hemicellulose using microwave irradiation (Kumar & Sharma, 2017; Singh

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et al., 2017). However, it is imperative to overcome practical challenges, such as high capital
cost with this sophisticated technology.

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The current work was carried out within the biorefinery approach with xylan, XOS and
xylose as the major products along with cellulose-rich residue from pineapple peels. The

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global production of pineapple was estimated to be 25.8 million tonnes for the year 2014-15
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(FAOSTAT, 2016). Pineapple is one of the most widely processed fruit which generates a
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huge quantity of processing and on-farm waste (Banerjee et al., 2018). Pineapple peels are
reported to have 22 - 35% of hemicellulose on dry basis (Pardo et al. 2014) and is also rich in
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other valuable components such as bromelain which have a huge value in the food and
therapeutic sector (Banerjee et al., 2018). To the best of our knowledge this paper describes
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the first detailed study on the extraction and characterisation of hemicellulose from pineapple
peel waste. The Hydrothermal-assisted alkali-based method was compared with conventional
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process for the extraction of hemicellulose. The major emphasis has been made to reduce the
alkali concentration and extraction time. The high recovery of hemicellulose in the form of
xylan opens the avenues to generate a variety of bio-based chemicals such as xylose, XOS,
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xylitol and other value-added products (hemicellulose-based edible films) (Banerjee et al.,
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2018). In this study, the hemicellulose extracted under the optimum conditions was
enzymatically hydrolysed to produce XOS. In addition, pineapple peels were hydrolysed
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using dilute nitric acid (0.5% v/v) to maximize xylose production which can further be
converted into value added products such as xylitol and ethanol (via xylulose).

2. Materials and Methods

2.1. Materials
 The pineapples (Indian Kew variety) used in this study were procured from a local market
and kept at 4˚C till further use. All the analytical grade chemicals were used as received.
Sodium hydroxide was obtained from Merck Millipore, USA while Glacial acetic acid was
procured from Sigma Aldrich, USA. Double distilled water was used for preparation of
other reagents.

2.2. Composition of Pineapple Peels and extracted hemicellulose

2.2.1. Proximate Analysis of pineapple peels

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The pineapple fruit was crushed, and the pulp was completely removed from the peels and

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dried in a hot-air oven at 60˚C until constant weight was obtained. Total extractives and ash
content were estimated by the National Renewable Energy Laboratory (NREL) analytical

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protocols TP 510-42619 and TP-510-42622 respectively (Sluiter et al. 2008 a,b). The

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cellulose and hemicellulose content of pineapple peel was obtained by determining neutral
detergent fiber (NDF). The acid detergent fiber (ADF) (Van Soest et al. 1991) and acid

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insoluble lignin (AIL) was determined following NREL protocol for structural
carbohydrates and lignin (Sluiter et al. 2008 a,b). The crude protein content in the peels was
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estimated through the conventional Kjeldahl method (AOAC, 2000).
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2.2.2. Chemical composition of extracted hemicellulose
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The constituent sugar units present in the extracted pineapple peel hemicellulose were
determined according to the acid hydrolysis procedure from NREL. Briefly, hemicellulose
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samples were mixed with 4% H2SO4 in a ratio 1:30 and was hydrothermally pretreated for 1
h. After acid hydrolysis, samples were centrifuged at 5000 rpm for 20 min and analyzed by
HPLC for monomeric sugars and acetic acid.
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2.2.3. Ultimate Analysis

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The elemental analysis (C, H, N and O) of dried pineapple peels and extracted hemicellulose
was determined using CHNSO elemental analyser (Flash EA 1112 series, Thermo Finnigan,
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Italy). The elemental composition of the hemicellulose extracted by both the methods was
compared with the elemental composition of dried pineapple peels and extractive-free dried
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pineapple peels.

2.3. Extraction of hemicellulose

Figure 1(a) represents the experimental design for the extraction of xylan from pineapple
peels. The experimental design includes the conventional and hydrothermal-assisted alkali-
based method of extraction of xylan. The actual and relative recovery of hemicellulose is
given by equation 1 and 2 respectively.

Actual recovery (%) = (A / S) x 100% ---------(1)

Relative recovery (%) = (A / H) x 100% ---------(2)

where A is dry weight of extracted hemicellulose (g), S is the dry weight of the sample
biomass (g) and H is the dry weight of hemicellulose present in the sample biomass (g).

2.3.1. Conventional method of alkaline extraction

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Extractives were removed from the oven-dried pineapple peel sample using a Soxhlet

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extractor separately with hexane, deionised water and ethanol as the extracting solvents. 5 g

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of extractive-free biomass (EFB) and 50 mL of the corresponding NaOH solution (5%, 10%
and 15%) were incubated in a shaker flask at 35˚C, 45˚C, 55˚C and 65˚C for 16 h at 100

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rpm. The solid residue was filtered after incubation. The residue was rinsed thoroughly with
100 ml of hot deionised water. The residue was then dried at 45˚C and kept in air-tight

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containers for further analysis. The filtrate was centrifuged at 5000 rpm for 15 minutes to
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obtain clear supernatant. The supernatant was decanted, and its pH was adjusted to 5 using
glacial acetic acid. Hemicellulose was precipitated using 95% ice-cold ethanol. The
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precipitate was allowed to settle down overnight. The precipitate was isolated by
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centrifugation followed by washing with ethanol and deionised water. The washed
precipitate pellet was then dried at 50˚C and stored in air-tight containers. It was further
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analysed to determine its characteristic features.

The different extraction temperatures chosen for this study were compared using severity
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factor, Ro which combines the effect of extraction time and temperature (Eqn. 3).

Ro = t x e T-100/14.75 ---------(3)
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Where t is the time (in min) and T is the extraction temperature (in ˚C) (Overend et al.,
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1987). The reference temperature is 100˚C and 14.75 is an empirical factor related to
temperature and activation energy assuming first order kinetics (Carvalheiro et al., 2009).
The eqn. 3 considers neutral pH levels only. An extension of the severity factor has been
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given for acid catalysed reactions and is known as the combined severity factor R o’
(Abatzoglou et al., 1992; Chum et al., 1990). It depends upon the pH of the extraction media
and the simplified form of the combined severity factor is given in Eqn. 4.

log (Ro’) = log (Ro) – pH ----------(4)

Eqn. 4 was found to be applicable to alkali catalysed extractions as well (Svärd et al., 2017).
 2.3.2. Hydrothermal assisted alkaline extraction

The hydrothermal assisted extraction of hemicellulose involved extraction at 121˚C (without

any previous incubation) with different concentration of alkali (5, 10 and 15% w/v) and
solid to liquid ratio of 1:10 for 30, 60 and 90 minutes respectively. The extractive-free
pineapple peel powder was mixed with alkali solution of desired concentration at 121˚C in
the reactor. After the desired reaction time, the reactor was depressurized and solution was
filtered. The filtered residue was washed with hot water involving precipitation of the

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hemicellulose from the solution after acidifying to pH 5 with glacial acetic acid followed by

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the addition of 95% ice-cold ethanol.

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2.4. Physicochemical and morphological characterization of extracted xylan

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2.4.1. Fourier Transformed Infrared Spectroscopy (FT-IR)

FT-IR spectra were recorded for the hemicellulose pellet extracted by the conventional

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alkali-based method and the hydrothermal-assisted alkali-based method. These spectra were
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compared with standard corn cob xylan to analyze the peculiar peaks for xylan. The spectra
were recorded over a range of 400 - 4000 cm-1 on 3000 Hyperion Microscope with Vertex
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80 FTIR system, Bruker, Germany.
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2.4.2. Scanning Electron Microscopy (SEM)

Morphological features of the extracted hemicellulose and the biomass after pretreatment
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were analyzed using a Hitachi SU8030 Scanning Electron Microscope. Powdered samples
were coated over carbon tape and mounted over the sample holder. The cellulosic residue left
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after pre-treatment was sputtered with gold plasma to improve the SEM images by reducing
electrical charging of the samples.
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2.4.3. Thermogravimetric analysis (TGA)

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The thermal behavior of the extracted hemicellulose was examined using Diamond TG/DTA
(Perking Elmer, USA) at a heating rate of 10˚C/min with temperature range of 0-600˚C
under nitrogen atmosphere (200 ml/min). The TGA curve obtained for the extracted
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hemicellulose (pineapple peel) was compared with standard corn cob xylan to correlate the
thermal stability of the two.

2.5. Enzymatic production of XOS

The enzymatic hydrolysis of the extracted pineapple peel xylan was carried out using endo-
1, 4- β-Xylanase M1 obtained from Trichoderma viridea (Megazyme, USA). The dried xylan
pellet, ground into fine particles in 50 mM acetate buffer (1% w/v) was incubated with the
desired dose of the enzyme at different temperatures. Aliquots were taken out of the reaction
mixture at fixed intervals of time (24 h, 48 h and 72 h) to monitor the products. The
enzymatic reaction was quenched by heating the aliquots in a boiling water bath for 10
minutes. The aliquot was then filtered through a 0.2 μ cellulose nitrate membrane to obtain a
clear supernatant which is used for the analysis. The constituent sugars and XOS produced by

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enzymatic hydrolysis were analysed using HPLC (Agilent, USA) method. The most active

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oligosaccharides (xylobiose and xylotriose) responsible for prebiotic activity along with

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monosaccharides (glucose, xylose, arabinose) and acetic acid were eluted using 5mM
sulphuric acid in HPLC grade water as mobile phase at a flow rate of 0.7 ml/min. The

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samples were analysed using Hi-Plex H column (300×7.7 mm) attached to a refractive index
detector (RID), operating at 65˚C and 50˚C, respectively.

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2.6. Production of xylose
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To maximize the xylose yield, pineapple peels were hydrolysed using dilute nitric acid
(Manaf et al. 2018). Variables such as nitric acid concentration (0.1% - 4%), liquid-solid ratio
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(1:10-1:30 v/w), and reaction time (0.5h, 1 h) were optimized for maximum xylose recovery.
The reaction mixture was hydrothermally pretreated at 121˚C for 1h. The residue was filtered
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and washed with 100 mL of deionised water. The residue was stored at 4˚C while the filtrate
was analysed for xylose and other sugar monomers by HPLC after being neutralized with
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anhydrous calcium carbonate. The xylose-rich liquor could further be converted into potential
chemicals such as xylitol.
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2.7. Statistical Analysis

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All experiments were performed in triplicates and results have been indicated as mean ±
standard deviation. General full factorial design was used for XOS production. Analysis of
variance (ANOVA) was conducted to determine the statistical significance of the response (p
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< 0.05). Experiments were conducted in a randomized order as suggested by the run order in
the table to prevent any bias and nuisance factors affecting the output. The data were
analysed using Minitab Statistical software version 16 (Pennsylvania State University, USA).

3. Results and Discussion

 3.1. Composition of Pineapple peels and extracted hemicellulose

The chemical composition of the dried pineapple peels was categorized into proximate and
ultimate analysis. The chemical composition of extracted hemicellulose was determined by
its acid hydrolysis and the elemental analysis.

3.1.1. Proximate analysis

The cellulose, hemicellulose and lignin content of pineapple peels were found to be in the
range reported in the literature (Rani & Nand, 2004; Pardo, Cassellis, Escobedo, & Garcia,

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2014; Roda, De Faveri, Dordoni, & Lambri, 2014; Romelle, Ashwini, & Manohar, 2016)

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(Table 1). The hemicellulose content of pineapple peels was found to be higher than that of
wheat straw (20-25%), rice straw (18%) (Prasad, Singh, & Joshi, 2007), corn stover (18-21%)

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(Weiss, Farmer, & Schell, 2010), sugarcane bagasse (25%) (Kim & Day, 2011) and arecanut

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husk (24-25%) (Singh et al. 2017). The cellulose content of the pineapple peels was in the
range of 19-21% which is quite lower when compared to wheat straw, rice straw, bagasse,

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switchgrass, hardwood, soft wood, arecanut husk (27-55%) (McKendry, 2002; Prasad et al.
2007; Anwar et al, 2014; Singh et al. 2017). The lignin content of pineapple peels was found
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to be less in comparison to that of other conventional biomass sources such as corn cob
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(15%) (Prasad et al. 2007), corn stover (14-15%) (Weiss et al. 2010), bagasse (20%) (Kim &
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Day, 2011), rice straw (18%) (Prasad et al. 2007), wheat straw (15-20%) (McKendry, 2002),
sweet sorghum (21%) (Kim & Day, 2011) and arecanut husk (22-26%) (Singh et al. 2017).
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Pectin, reducing sugars and non-reducing sugar content were major contributor of total
extractives of pineapple peel.
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3.1.2. Chemical composition of extracted hemicellulose

Unlike cellulose, hemicelluloses consist of various sugar units arranged in different
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proportions. Xylose, glucose, arabinose, mannose, glucouronic acid, rhamnose, fucose and
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other methylated neutral sugars constitute hemicellulose (Chaturvedi & Verma, 2013). The
extracted hemicellulose contained primarily xylan, as the acid hydrolysis of the extracted
hemicellulose yielded glucose (7.1 ± 0.7%), xylose (73.5 ± 1.7%) and arabinose (5.3 ± 0.4%)
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as the monomeric sugar components and acetic acid (9.8 ± 0.5%) as the organic acid due to
the hydrolysis of acetyl linkages present in hemicellulose.

3.1.3. Ultimate analysis

The carbon content of dried pineapple peel was found to be 43.9% which is similar to 43-
48% as reported for arecanut husk (Singh et al. 2017); wheat straw, timothy grass &
 pinewood bark (Nanda, et al. 2013); and napier grass (Mohammed, et al. 2015). However,
the carbon content of pineapple peel was found to be higher than that reported for finger
millet (33.5%), and rice bran (34.8%). The total nitrogen content was found to be 0.6%
which is quite low as compared to that in finger millet (2.7%) (Palaniappan et al. 2017).
This is mainly due to low protein content in pineapple peels. The elemental composition of
the xylan extracted by the conventional alkali extraction and the hydrothermal method was
similar in terms of carbon and hydrogen (36.1% and 36.7% for Carbon & 5.70% and 5.18%
for Hydrogen respectively) (Table 2). However, nitrogen and sulphur were not present in

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any of the xylan samples.

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3.2. Hemicellulose yields from conventional alkaline extraction

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In conventional alkaline extraction process, hemicellulose was dissolved in three different
alkali concentrations (5%, 10%, 15%) and incubated for 16 h. For each of the incubation

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temperatures, it was observed that the highest concentration of alkali resulted in the
maximum recovery of hemicellulose. Incubation of dried pineapple peels in 15% (w/v) alkali
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solution for 16 h at 45˚C resulted in a recovery of 95.9 ± 2.0% of hemicellulose, which
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matches with results reported for arecanut husk (Singh et al., 2017) and corn cob (Samanta, et
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al. 2012). However, the temperature required in the case of pineapple peels was lower when
compared with other substrates. The selective dissolution of hemicelluloses in alkali is widely
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known. It is mainly caused by the disruption of cell wall by the hydrolysis of the ester
linkages between hemicelluloses and lignin and decreases cellulose crystallinity (Chaturvedi
& Verma, 2013). In addition, in an alkali environment, ionization of lignin’s phenolic groups
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and carboxylic moieties of hemicellulose would increase their solubility. In this process,
cellulose fibers swollen their crystallinity decreased which enhanced the release of
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hemicelluloses and residual lignin It has been reported that higher the concentration of alkali,
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the greater the dissolution of hemicelluloses and an associated higher delignification of the
biomass (Samanta, et al. 2012). In case of pineapple peel, lower lignin content (10-15%) and
more concentrated xylan (after extractives were removed) still required higher dosage of
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alkali for maximum recovery of hemicelluloses. Dissolution of hemicellulose and lignin

varied significantly depending on the pretreatment parameters. The statistical analysis
indicates interaction of all three variables have a significant effect on lignin removal and
hemicellulose recovery. Interestingly, with lower intrinsic lignin content in pineapple peel,
required alkali dosage was not proportionately low even when biomass was incubated at
highest temperature i.e. 65˚C. This shows that delignification and hemicellulose dissolution
from biomass are alkali limiting steps. Depolymerization of hemicellulose is significantly
affected by the all three parameters with alkali loading having the greatest effect (Table 3).

Severity factor calculations show that the removal of hemicellulose from the solid phase is
dependent upon the combined severity factor i.e. log Ro’. The calculated combined severity
factor values provided information about the extraction conditions (pH, temperature and
time) and the hemicellulose recovery. The recovery of hemicellulose increased with the

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increasing severity factor until it reached the optimum severity for alkali-assisted
hemicellulose extraction. However, higher incubation temperatures (65˚C) resulted in a lower

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yield of hemicellulose which can be attributed to the disintegration of the hemicellulose

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structure on increasing the severity factor further (Table 3). The higher incubation

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temperature resulted in a significantly higher yield of hemicellulose (95.9 ± 2.0%) at a shorter
incubation period (4h) for which the severity factor value coincides with the optimum value.

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3.3. Hemicellulose yields from hydrothermal-assisted alkaline extraction
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Hydrothermal assisted alkali extraction was evaluated for maximizing the recovery of
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pineapple peel hemicellulose in a shorter period. The hydrothermal pretreatment involved
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zero incubation time, which is one of the main highlighting points compared to the
conventional alkali-based method. The maximum relative recovery of 91.5 ± 1.9% was
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obtained with 15% alkali at the end of 1.5 h of hydrothermal pretreatment (121˚C and 15
psi pressure) (Table 4). A significant yield of hemicellulose (87.4 ± 0.7%) was obtained
with 10% alkali at the end of 1.5 h. The concentration of alkali is one of the key
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components in the alkaline pretreatment as it significantly affects the economics of the

process due to the downstream processing costs involved with high alkali concentrations.
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More than 94% recovery of xylan from arecanut husk has been reported with 10% alkali
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(w/v) with 8h of incubation before 1 h of hydrothermal pretreatment (Singh et al. 2018)

whereas in this study ~87% of hemicellulose was recovered with 10% alkali in 1.5 h with
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zero pre-incubation in alkali environment.

The statistical model contains two main effects, viz, Time (h) and alkali concentration (%)
with their interaction term. Statistical results of the model showed that the individual
parameters and interaction terms were significant (Table S1). The adjusted R2 for this
model was 98.8% which indicates a good fit of the model.
In Figure S1, it is evident that the concentration of alkali was the most influential factor in
the recovery of hemicellulose from pineapple peel waste. Increase in the concentration of
alkali from 5% to 10% (w/v) significantly improved the yield of hemicellulose across all
the hydrothermal pretreatment times. However, no significant increase was observed in the
hemicellulose yields when the alkali concentration was further increased from 10% to
15% (w/v) at hydrothermal pretreatment times of 1 and 1.5 h, respectively. This clearly
shows the synergistic effect of time-alkali concentration combination on hemicellulose
recovery. In addition to higher costs and downstream challenges associated with higher

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alkali concentration, another disadvantage with high alkali dosage is substantial loss of

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carbohydrates when alkali concentration crosses a certain threshold during pretreatment

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which offsets increased substrate digestibility. Therefore, we selected combination of 10%
alkali and 1.5 h duration to pretreat biomass for hemicellulose recovery for further

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processing into XOS.

3.4. Physicochemical and morphological characterization

3.4.1. Fourier Transformed Infrared Spectroscopy (FT-IR) U
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The FT-IR spectra of extracted pineapple peel xylan and standard corn cob xylan revealed a
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high degree of similarity indicating the presence of common functional groups shown in
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Figure 2. Most of the peaks present in all the three spectra (a, b and c), correspond to O-H
and C=O vibrations which are the kind of bonds present in hemicelluloses (xylan)
(Palaniappan et al. 2017). It is clear from the spectra that a broad band (in region of 3600-
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3100 cm-1) can be attributed to the hydroxyl O-H group, which is similar to the FT-IR spectra
for xylan extracted from different biomass (Singh et al. 2017). Appearance of peaks in the
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spectra at 2919 cm-1 and 2910 cm-1 were assigned to the C-H vibration. The presence of
acetyl groups in the extracted pineapple peel xylan was confirmed by the adsorption band at
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1641cm-1 and 1619 cm-1 (C=O, aldehyde absorption, which mainly occurs in the amorphous
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state) (Palaniappan et al. 2017). However, the presence of an absorption band at 1576 cm-1 in
(b) could be attributed to bound lignin as an impurity (Maziero et al. 2012). The absorption
bands at 1382 cm-1, 1418 cm-1 and 1419 cm-1 are assignable to the C-H bending vibration
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indicating the presence of hemicellulose. The peculiar absorption bands at 1042 cm-1, 1041
cm-1 and 1044 cm-1 are the characteristic feature bands for xylan (Palaniappan et al. 2017). It
was also observed that a peak at 1042 cm-1 arises mainly because of C-O, C-C stretching, or
C-OH bending of hemicellulose (Singh et al. 2017). The peaks arising at 895 cm-1, 896 cm-1
and 897 cm-1 were characteristic of β- glycosidic linkages between sugar molecules (Buslov
et al. 2010).

3.4.2. Scanning Electron Microscopy (SEM)

The dried pineapple peel powder was observed under scanning electron microscope before
alkali pretreatment (Figure 3 (a)). The obtained micrographs were compared to visualize the
morphological changes that occurred due to pretreatment process. Figure 3 (b) shows the
changes in the structure of pineapple peel waste due to delignification and removal of

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hemicellulose. It indicates that the alkali pretreatment resulted in a more open structure in

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pineapple peels. Figure 3 (c) represents the scanning electron micrograph for the
hemicellulose pellet obtained after the alkali pretreatment of pineapple peel waste. The

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micrographs showed some irregular morphologies with large particle size and rough surface.

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3.4.3. Thermogravimetric analysis (TGA)

The study of thermal behaviour of the extracted xylan was made to understand the

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degradation pattern of xylan. It is known that hemicelluloses (xylan) are thermally more
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unstable in contrast to celluloses and lignin due to their amorphous nature (Palaniappan,
Yuvaraj, Sonaimuthu, & Antony, 2017) and it decomposes at around 225-325˚C. Due to the
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presence of branched chain, hemicellulose structure is quite complex than cellulose and
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lignin. Cellulose is a polymer composed of linear chains of glucose a degree of

polymerisation of ten to one hundred times higher than that of hemicellulose. Lignin, on the
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other hand, is a highly cross-linked polymer consisting of aromatic rings with multiple
branches and functional groups, whose bonds cleave along a wide range of temperatures
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(Chaturvedi & Verma, 2013).

The TGA curve of pineapple peel xylan showed significantly higher decomposition,
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compared to standard corn cob xylan. The loss in weight was 20% in the temperature range
50˚C - 200˚C which is attributed to loss of water and some organic moieties attached to
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hemicellulose. A significant loss in weight (30-40%) of pineapple peel xylan occurs from
210˚C - 320˚C which is attributed to the degradation of amorphous hemicellulose into CO,
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CO2 and some hydrocarbons (Yang et al., 2007). The third stage of decomposition occurs in
the temperature range 320˚C - 620˚C where 15% and 10% loss in weight is observed for
pineapple peel xylan and standard corn cob xylan respectively. Even at 600˚C, around 40%
carbon is left which can be useful as electrocatalysts. The behavior of decomposition of pure
xylan differs from lignocellulosic biomass which is fairly complex in nature. In biomass,
 interactions with other components is expects which hinders the reactions. In order to get
more clarity on this aspect, a detailed investigation is needed.

3.5. Production of XOS

The xylan extracted from pineapple peel waste was enzymatically hydrolyzed to produce
XOS. Enzymatic hydrolysis is one of the important methods to produce oligosaccharides
from polysaccharides. Since enzymatic hydrolysis does not require harsh operating
conditions and controlled rate of product formation is possible with avoided risk of

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producing inhibitory compounds such as furfurals and hydroxyl methyl furfurals. In this

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work, the effects of enzyme digestion time, pH, enzyme dosage and temperature on the
production of XOS from extracted hemicellulose were explored.

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Experimental design in Table 6 shows production of xylobiose (DP2), xylotriose (DP3) and

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total XOS (Sum of DP2 and DP3 sugars) from pineapple peels at different operating
parameters. From the results, XOS obtained was ranging from 5.1 ± 1.7 to 25.6 ± 0.3 g/100g

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hemicellulose. Variation in XOS production was influenced by process conditions.
Experimental conditions with Enzyme dosage: 15 U, time: 24 h, Temp: 50ºC, pH: 5
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successfully yielded the highest amount of XOS while Enzyme dosage: 15 U, time: 72 h,
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Temp: 40ºC, pH: 6 resulted into the lowest amount of XOS. The R2 and adjusted R2 for this
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model were found to be 95.9% and 91.9%, respectively which proved the adequacy of model
equation.
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Overall, by comparing the highest and lowest result of XOS produced, enzyme dosage and
pH have shown variability in determining the yield which may illustrate the importance of
these factors to influence enzymatic hydrolysis. However, significance of contributing factors
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was understood by statistical analysis using general linear model by considering the variation
of the all experimental runs. The significance of model terms was determined by p-value.
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In this study, all four main factors viz. Time (h); Enzyme Dose (U); Temp. (˚C) and pH were
CC

found to be significant (p<0.05). Similarly, the squared interaction effect terms, namely,
Enzyme Dose (U) * pH; Temp (˚C) * pH; Enzyme Dose (U)*Temp (˚C); Temp (˚C)* Time
A

(h) and pH*Time (h) were also significant. Some of the higher order terms, even though
significant, were not considered as they don’t have practical significance. This was confirmed
by systematically removing those interaction terms and new R2 and R2(adjusted) were
analyzed (Table S2) and it is very clear that new R2 and R2(adjusted) values did not change
(91% to 86%) much after 3 way and 4 way interaction terms were removed from the model.
The main effect is a difference between the mean responses of a factor measured in the
specified levels. In other words, it is the change in XOS content when the levels of predictor
variables are changed. Interaction effects, however, show the changes in the values of the
response when one factor is held constant at a time, and the other factors were changed. From
the main effect and interaction plots, it can be observed that pH was the most influential
parameter in the enzymatic production of XOS from pineapple peel xylan. Increase in the pH
to an optimum value of 5.0 gave the maximum yield of XOS across all the incubation
temperatures, time and enzyme dosage (Figure 5). This is mainly attributed to the behavior of

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the enzyme xylanase with the change in pH of the reaction media. The optimum pH for

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xylanase is in the range of 5.0 - 5.5. However, at optimal pH (5.0), increase in enzyme dosage

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did not improve XOS yields. Reaching plateau at high enzyme dosage could be attributed to
the binding of the enzyme protein with the substrate, resulting in no net gain in product yield.

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For given operating conditions, the incubation time for enzymatic hydrolysis did not have a
prominent effect on XOS yields. The interaction plot for temperature (˚C) and pH clearly

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indicates that at pH 5.0, a higher temperature (50˚C) gives a better yield of XOS. Other two-
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way interaction terms were not significant (Table 5). To summarize, the optimum conditions
for the maximum production of XOS were: 50˚C, pH 5.0 and 15 U enzyme dose. Under this
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condition, a total of 25.7 ± 0.4 g of XOS/100g of xylan was produced with degree of
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polymerization (DP)n; n=2 and 3 which are reported to have maximum prebiotic potential
(Aachary & Prapulla, 2009).
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Contribution of DP2 obtained in this study varied from 54 to 100% of total low DP XOS. For
maximum XOS recovery, i.e. 25.6 g/100g hemicelluose, xylobiose and xylotriose
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contribution were found to be about 83.7% and 16%, respectively. Infact, in all experimental
runs where XOS yields were more than 20 g/100g biomass, DP2 contribution was more than
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80% (Table 6).

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3.6 Xylose Production

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Operating parameters (nitric acid concentration, reaction time and solid-liquid ratio) were
evaluated to obtain maximum xylose yields (Figures S3, S4 and S5) and highest xylose yield
(38.5±0.4 %) was obtained at 0.5% nitric acid, with solid/liquid ratio of 1:20 and the reaction
time of 1 hour at 121˚C. The recovery of xylose was about 91.3% of total xylose present in
the biomass which is significantly high and could be utilized in the production of xylitol with
further purification. Dilute nitric acid assisted hydrolysis also led to the formation of
xylotriose (1.9±0.01 g/100 g), glucose (0.5±0.01 g/100 g), arabinose (3.2±0.03 g/100 g) and
acetic acid (3.9±0.01 g/100g) as the minor products (Figure S2). Further increase in acid
concentrations resulted in reduced xylose yields which might be due to degradation of xylose
into furfurals. Researchers have established that biomass can directly be fractionated into
monomeric sugars with good efficiency using dilute nitric acid (Abdul Manaf et al., 2018;
Skiba et al., 2017; Bensah & Mensah, 2013; Zhang et al., 2011). In this study, since
hemicellulose forms a significant portion of pineapple peel composition with low lignin
content, product recovery was found to be high with moderate reaction conditions. Being

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amorphous in nature, hemicellulose hydrolyses well in presence of nitric acid (a strong

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oxidizing agent). On the other hand, cellulose structure did not get affected in the given range

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of operating conditions. Acetic acid concentration was found to be 3.9±0.01 g/100g at chosen
operating conditions. This is due to hydrolysis of the acetyl groups on the side chain of

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hemicellulose when exposed to severe conditions (Singh et al., 2018; Abdul Manaf et al.,
2018; Jönsson & Martín, 2016). The strategy for downstream purification of xylose will be

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influenced by feed (biomass liquor) composition, ionic strength of the feed solution and
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desired purity of xylose. A techno-economic analysis would reveal whether value-added
A
products such as xylitol production via acid treatment followed by set of separation
techniques, for example membrane separation could be a viable alternative justifying the
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implementation of new process steps.

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4. Conclusion

Pineapple peels were found to be rich source of hemicellulose which can compete well with
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another hemicellulose-rich biomass. The current hydrothermal-assisted process involves

zero pre-incubation time, which is the highlight of the present study, in contrast to the
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conventional process. A maximum yield of hemicellulose was obtained at 45˚C. XOS (25.7
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± 0.4 g /100g of xylan) and xylose (~91.3% yield) were the final products of the
hemicellulose bio-refinery which is expected to improve the profitability of the production
of bio-fuel from lignocellulosic waste along with the production of high value chemicals.
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Acknowledgement

AA and SB are grateful to Prof. Eckart Ruehl, Freie University, Berlin for the SEM studies.
SB is grateful to Mr. Ramkrishna Singh, IITB, for providing the xylobiose and xylotriose
standards. AA is grateful to IRCC, IIT Bombay for providing partial funding through seed
 grant (13IRCCSG003). AA and SB are grateful to IITB-Monash Research Academy to
provide financial assistance to SB for her doctoral study (IMURA 0453).

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Figure 1: Schematic for extraction of hemicellulose from Pineapple Peel Waste (PPW)
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and its valorization into XOS
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A
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Figure 2: FT-IR Spectra of (a) standard corn cob xylan; (b) hemicellulose extracted via
hydrothermal pretreatment of PPW and (c) hemicellulose extracted via conventional alkali
process.
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Figure 3: Scanning Electron Micrographs of (a) Pineapple peel waste (PPW) before
pretreatment; (b) PPW after hydrothermal assisted alkaline pretreatment; (c) hemicellulose

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from pineapple peel waste; and (d) hemicellulose from pineapple peel waste (higher
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magnification)
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M
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Figure 4: Thermogravimetric analysis of standard corn cob xylan and hemicellulose extracted
from PPW

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(a)

Data Means

Enzyme Dose (U) Time (hrs)

20

18

16

14

12
Mean

5 10 15 24 48 72
Temp. (° C) pH
20

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18

16

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14

12

R
40 50 4 5 6

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(b)

Data Means
24 48 72 40 50 4

U
5 6

20
Enzyme
Dose (U)
N
15
5
Enzyme Dose (U)
10
10 15
Enzyme
Time
A
20 Dose (U)
(hrs)
15
24 5
Time (hrs)
48 10
72 15
M

10
Enzyme
Time
Temp.
Dose (U)
20 (hrs)
(°C) 5
24
40
Temp. (°C) 15
4850 10
72 15
ED

10

pH
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Figure 5: (a) Main effects and (b) interaction plots for enzymatic hydrolysis of extracted
hemicellulose to produce XOS
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U
(a) 15.0 (b) 70 XOS

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XOS < 5
< 5 5 – 10
5 – 10 10 – 15
10 – 15 15 – 20

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12.5 15 – 20 60 20 – 25
20 – 25 > 25
> 25
Enzyme (U)

Time (h)
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50
10.0

ED 40
7.5
30

5.0
4.0 4.5 5.0 5.5 6.0 4.0 4.5 5.0 5.5 6.0
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pH pH

(c) 50 (d) 15.0

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XOS XOS
< 5 < 5
5 – 10 5 – 10
10 – 15
48 10 – 15
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15 – 20
20 – 25 12.5 15 – 20
20 – 25
> 25 > 25
Enzyme (U)

46
Temp (C)

10.0
A

44

7.5
42

40 5.0
4.0 4.5 5.0 5.5 6.0 40 42 44 46 48 50
pH Temp (C)

(e) 15.0
(f) 70
XOS
(e) XOS
< 5
5
< 5
– 10
5 – 10
10– 15
10 – 15
15– 20
12.5 15 – 20 60 20– 25
20 – 25 > 25
> 25
yme (U)

ime (h)

50
10.0
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Figure 6: Contour plots (a-f) showing influence of parameters on enzymatic hydrolysis of extracted hemicellulose for XOS production.

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Table1: Composition of dried Pineapple Peel Waste (PPW)

Biomass Component Value (% dry basis)

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Cellulose 20.9 ± 0.6

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Hemicellulose 31.8 ± 1.9

M
Lignin 10.4 ± 1.0

ED Crude Protein 3.9 ± 0.2

Total Extractives 28.1 ± 2.5

Ash 5.9 ± 0.06

PT

Organic matter 94.04 ± 0.06

E
CC

Table2: Elemental analysis of dried Pineapple Peel Waste (PPW) and extracted xylan
A

Element Pineapple Extractive- Xylan Xylan Standard xylan

peel Biomass free Biomass conventional hydrothermal from corn-cob
(%)

Carbon 43.9 45.1 36.1 36.7 40.8

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Hydrogen 5.7 5.7 5.4 5.3 6.4

U
Nitrogen 0.6 0.3 - - -

N
A
M
ED
Table 3: Extraction conditions explored for recovery of hemicellulose via conventional alkali incubation method and calculated severity factors
PT

log (Ro) and log (Ro’) for each extraction.

E

S.No. Temp. Time Alkali Alkali log Ro pHa at log Ro' Relative Absolute
(T˚C) (h) conc. conc. ext. Recovery Recovery
CC

(%) (M) temp. Hemicellulose Hemicellulose

(%) b (%) b
A

1 35 16 5 1.25 1.06 13.8 -12.73 33.4 (0.01) 15.1 (0.01)

2 35 16 10 2.5 1.06 14.1 -13.03 66.2 (2.9) 29.9 (1.3)
3 35 16 15 3.75 1.06 14.3 -13.23 68.3 (2.6) 30.9 (2.1)
4 45 16 5 1.25 1.36 13.5 -12.13 38.8 (0.6) 17.6 (0.3)
5 45 16 10 2.5 1.36 13.8 -12.43 82.5 (0.2) 37.3 (0.1)
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6 45 16 15 3.75 1.36 14 -12.63 95.9 (2.0) 43.4 (1.8)
7 55 16 5 1.25 1.65 13.2 -11.54 29.5 (1.1) 13.4 (0.4)

U
8 55 16 10 2.5 1.65 13.5 -11.84 62.7 (1.6) 28.4 (0.7)

N
9 55 16 15 3.75 1.65 13.7 -12.04 79.5 (0.4) 36.2 (0.2)

A
10 65 16 5 1.25 1.95 13 -11.04 36.4 (2.4) 16.5 (1.1)
11 65 16 10 2.5 1.95 13.3 -11.34 61.1 (2.9) 27.6 (1.3)

M
12 65 16 15 3.75 1.951 13.5 -11.54 81.7 (3.7) 37 (2.6)
13 65 8 ED 15 3.75 1.65 13.5 -11.85 86.2 (1.2) 38.8 (0.1)
14 65 4 15 3.75 1.35 13.5 -12.15 96.6 (0.3) 43.5 (0.02)
PT
a
Calculated pH values
b
Values in parenthesis are standard errors
E
CC

Table 4: Percentage hemicellulose recovered from dried Pineapple peel waste at different alkali concentrations and hydrothermal pretreatment
conditions
A

Process Alkali concentration Hemicellulose Absolute Relative

(%) recovery (%) recovery (%)
Hydrothermal (30 min.) 5 15.9 ± 0.4g 35.1 ± 0.9
121˚C 10 26.0 ± 1.2e 57.5 ± 2.8

15 30.0 ± 1.8d 65.9 ± 4.6

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Hydrothermal (60 min.) 5 18.4 ± 1.1f 40.7 ± 2.6
121˚C 10 35.4 ± 0.3c 78.3 ± 0.7

U
15 37.8 ± 0.1b 83.6 ± 0.2

N
Hydrothermal (90 min.) 5 20.7 ± 1.7f 45.7 ± 3.7

A
121˚C 10 39.8 ± 0.3a,b 87.4 ± 0.7
15 41.4 ± 0.8a 91.5 ± 1.9

M
*absolute recovery values sharing the same alphabet are not significantly different (p<0.05)
ED
EPT
CC
A
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Table 5a: Selected parameters and their levels for the full factorial design of experiment

U
Factor Levels Values

N
Enzymes (U) 3 5 10 15
Time (h) 3 24 48 72

A
Temp (C) 2 40 50

M
pH 3 4 5 6

ED
Table 5b: Analysis of variance (ANOVA) test for experimental response for enzymatic hydrolysis of pineapple peel xylan to XOS
PT

Source DF Seq SS Adj SS Adj MS F P

E

Enzyme (U) 2 313.580 313.580 156.790 65.06 < 0.0001

Time (h) 2 30.298 30.298 15.149 6.29 0.004
CC

Temp. (˚C) 1 534.223 534.223 534.223 221.69 < 0.0001

pH 2 1580.525 1580.525 790.263 327.93 < 0.0001
A

Enzyme (U)*Time (h) 4 7.456 7.546 1.864 0.77 0.547

Enzyme (U)*Temp. (˚C) 2 15.161 15.161 7.581 3.15 0.051
Enzyme (U)*pH 4 146.650 146.650 36.663 15.21 < 0.0001
Time (h)*Temp. (˚C) 2 23.928 23.928 11.964 4.96 0.010
Time (h)*pH 4 48.631 48.631 12.158 5.05 0.002
Temp.(˚C)*pH 2 183.690 183.690 91.845 38.11 < 0.0001
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Enzyme (U)*Time (h)*Temp. 4 3.858 3.858 0.965 0.40 0.808
(˚C)

U
Enzyme (U)*Time (h)*pH 8 37.249 37.249 4.656 1.93 0.074
Enzyme (U)*Temp.(˚C)*pH 4 43.268 43.268 10.817 4.49 0.003

N
Time (h)*Temp.(˚C)*pH 4 70.826 70.826 17.707 7.35 < 0.0001

A
Enzyme (U)*Time (h)* 8 43.651 43.651 5.456 2.26 0.036
Temp.(˚C)*pH

M
Error 54 130.130 130.130 2.410
Total ED 107 3213.124
R2 = 95.9%, R2 (adj) = 91.9%
EPT
CC
A
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Table 6: Full Factorial experimental design layout of enzymatic hydrolysis and average yields of oligomers

U
Enzyme Time Temp. pH Xylobiose Xylotriose XOS

N
Dose (U) (h) (˚C)
5 24 40 4 3.4 ± 0.3 2.2 ± 0.7 5.6 ± 0.9

A
5 24 40 5 8.0 ± 2.5 3.3 ± 0.5 11.3 ± 3.0

M
5 24 40 6 8.3 ± 1.3 1.2 ± 0.1 9.5 ± 1.2
5 24 50 4 8.7 ± 0.9 3.6 ± 0.4 12.3 ± 1.4
5 24
ED
50 5 14.0 ± 2.6 4.5 ± 0.1 18.5 ± 2.7
5 24 50 6 7.3 ± 0.4 3.3 ± 0.1 10.6 ± 0.5
5 48 40 4 3.5 ± 1.4 2.3 ± 0.7 5.8 ± 2.1
PT

5 48 40 5 10.8 ± 2.5 4.5 ± 0.5 15.3 ± 3.1

5 48 40 6 9.1 ± 1.7 2.2 ± 0.3 11.3 ± 2.1
E

5 48 50 4 9.9 ± 0.7 4.0 ± 0.4 13.9 ± 1.1

CC

5 48 50 5 14.6 ± 1.4 4.3 ± 0.1 18.9 ± 1.5

5 48 50 6 7.2 ± 0.8 3.3 ± 0.2 10.5 ± 1.1
A

5 72 40 4 3.5 ± 1.6 2.6 ± 0.7 6.1 ± 2.4

5 72 40 5 10.0 ± 2.5 1.5 ± 0.3 11.5 ± 2.8
5 72 40 6 11.5 ± 2.7 1.7 ± 0.1 13.2 ± 2.8
5 72 50 4 9.4 ± 0.2 4.0 ± 0.4 13.4 ± 0.7
5 72 50 5 14.1 ± 2.4 4.3 ± 0.1 18.4 ± 2.5
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5 72 50 6 7.4 ± 0.5 3.4 ± 0.2 10.8 ± 0.7
10 24 40 4 5.3 ± 1.6 2.8 ± 0.4 8.1 ± 2.1

U
10 24 40 5 12.4 ± 1.2 3.3 ± 0.1 15.7 ± 1.3

N
10 24 40 6 11.8 ± 1.4 1.1 ± 0.1 12.9 ± 1.3

A
10 24 50 4 10.9 ± 1.2 3.9 ± 0.2 14.8 ± 1.4
10 24 50 5 20.7 ± 0.1 4.5 ± 0.1 25.2 ± 0.1

M
10 24 50 6 8.3 ± 0.7 3.3 ± 0.3 11.6 ± 1.1
10 48 40
ED 4 6.5 ± 2.3 2.9 ± 0.9 9.4 ± 3.3
10 48 40 5 18.5 ± 0.7 4.3 ± 0.1 22.8 ± 0.8
10 48 40 6 10.1 ± 1.1 1.9 ± 0.3 12.0 ± 1.4
PT

10 48 50 4 12.2 ± 0.6 4.1 ± 0.2 16.3 ± 0.8

10 48 50 5 19.4 ± 1.2 4.2 ± 0.2 23.6 ± 1.1
E

10 48 50 6 7.5 ± 0.7 3.1 ± 0.4 10.6 ± 1.2

CC

10 72 40 4 7.1 ± 0.2 3.2 ± 0.1 10.3 ± 0.1

10 72 40 5 17.9 ± 1.3 0.0 ± 0.0 17.9 ± 1.3
10 72 40 6 10.9 ± 2.8 1.9 ± 0.0 12.8 ± 2.8
A

10 72 50 4 11.5 ± 0.6 4.0 ± 0.2 15.5 ± 0.8

10 72 50 5 19.6 ± 0.4 4.3 ± 0.2 23.9 ± 0.7
10 72 50 6 8.8 ± 0.2 3.5 ± 0.0 12.3 ± 0.2
15 24 40 4 6.0 ± 0.5 2.2 ± 0.1 8.2 ± 0.6
15 24 40 5 12.2 ± 1.7 3.3 ± 0.5 15.5 ± 2.2
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15 24 40 6 10.9 ± 0.2 1.1 ± 0.0 12.0 ± 0.2
15 24 50 4 12.6 ± 0.7 3.8 ± 0.4 16.4 ± 1.2

U
15 24 50 5 21.4 ± 0.3 4.2 ± 0.0 25.6 ± 0.3

N
15 24 50 6 10.7 ± 0.5 3.4 ± 0.0 14.1 ± 0.5

A
15 48 40 4 8.1 ± 1.5 3.2 ± 0.5 11.3 ± 2.1
15 48 40 5 19.3 ± 0.8 4.8 ± 0.3 24.1 ± 1.2

M
15 48 40 6 9.1 ± 0.8 1.2 ± 0.3 10.3 ± 0.5
15 48 50
ED 4 15.2 ± 0.1 4.3 ± 0.0 19.5 ± 0.1
15 48 50 5 20.7 ± 0.7 3.9 ± 0.1 24.6 ± 0.8
15 48 50 6 9.4 ± 0.0 3.2 ± 0.2 12.6 ± 0.2
PT

15 72 40 4 9.2 ± 1.3 3.4 ± 0.4 12.6 ± 1.8

15 72 40 5 18.8 ± 1.3 1.2 ± 0.2 20.0 ± 1.1
E

15 72 40 6 4.3 ± 1.6 0.8 ± 0.1 5.1 ± 1.7

CC

15 72 50 4 13.5 ± 0.9 4.1 ± 0.4 17.6 ± 1.3

15 72 50 5 20.0 ± 0.5 3.9 ± 0.1 23.9 ± 0.3
15 72 50 6 10.6 ± 0.0 3.4 ± 0.2 14.0 ± 0.2
A