Cellulose (2021) 28:4009–4024

https://doi.org/10.1007/s10570-021-03770-5 (0123456789().,-volV)
( 01234567
89().,-volV)

ORIGINAL RESEARCH

Cocoa pod husk valorization: alkaline-enzymatic pre-

treatment for propionic acid production
Zulma Sarmiento-Vásquez . Luciana Vandenberghe . Cristine Rodrigues .
Valcineide Oliveira A. Tanobe . Oranys Marı́n . Gilberto Vinicius de Melo Pereira .
Hervé Louis Ghislain Rogez . Aristóteles Góes-Neto . Carlos Ricardo Soccol

Received: 10 November 2019 / Accepted: 11 February 2021 / Published online: 11 March 2021
Ó The Author(s), under exclusive licence to Springer Nature B.V. 2021

Abstract Lately, cocoa pod husk (CPH) has
received some attention from researchers due to its
significant content of cellulose, hemicelluloses and
pectin, antioxidant capacity, potential for energy
generation, physicochemical characteristics and pos-
sibility to be used as adsorbent material or activated
carbon. In this work, alkaline (NaOH 2.3% w/v) and
enzymatic treatment [CellicÒ CTec 2 (2.4% v/v)]
were developed before propionic acid (PA) production
using fermentable sugars derived from cocoa pod husk
hydrolysate (CPHH). The physical, structural and
morphological characteristics of raw and treated
samples of CPH lignocellulosic matrix were assessed
using tools such as scanning electron microscopy,
Fourier transform infrared spectroscopy,
Z. Sarmiento-Vásquez  L. Vandenberghe (&) 
C. Rodrigues  V. O. A. Tanobe  O. Marı́n 
G. V. de Melo Pereira  C. R. Soccol
Department of Bioprocess Engineering and
Biotechnology, Federal University of Paraná, Curitiba,
PR CEP 81531-970, Brazil
e-mail: lvandenberghe@ufpr.br
thermogravimetry, and X-ray diffraction. The results
of these analyses revealed the effectiveness of CPH
sequential alkaline and enzymatic treatment for glu-
cose production, reaching maximum concentration of
60.5 gL-1 and a yield of 275 mg glucoseg-1 CPH.
Subsequently, a medium composed by glucose from
CPHH (7.5 gL-1), as low-cost feedstock, glycerol
(7.5 gL-1) and yeast extract (10 gL-1), was prepared
to obtain PA using Propionibacterium jensenii DSM
20274. PA concentration reached a maximum of
10.28 ± 1.05 gL-1 and a productivity of
0.08 ± 0.01 gL-1h-1 after 72 h of fermentation.
To the best of our knowledge, there is no literature
available on the bioconversion of CPH for the specific
production of PA or other organic acids. Thus, the
potential of CPH to be used as substrate for PA
production was demonstrated with good perspectives.

H. L. Ghislain Rogez
Centre for Valorisation of Amazonian Bioactive
Compounds (CVACBA), Federal University of Pará,
Belém, PA CEP 66075-750, Brazil

A. Góes-Neto
Institute of Biological Sciences, Federal University of
Minas Gerais, Belo Horizonte, MG CEP 31270-901,
Brazil

123
4010 Cellulose (2021) 28:4009–4024

Graphic abstract

Keywords Cocoa pod husk  Propionic acid  waste, which in most cases result in environmental and
Alkaline-enzymatic treatment  Batch fermentation phytosanitary issues within plantations. By using the
residual cocoa lignocellulosic material as a raw matter
in bioprocesses, it is possible to reduce production
Abbreviations
costs of many high-value-added components, empow-
Cocoa pod husk CPH
ering at the same time, the Bioeconomy and Bio-
Cocoa pod husk hydrolysate CPHH
refinery concepts, which are increasingly gaining
Fourier transform infrared FT-IR
importance in the agroindustrial field.
Propionic acid PA
Cocoa pod husk (CPH) is an example of a
Scanning electron microscopy SEM
lignocellulosic material that has been analyzed and
Thermogravimetry TG
assessed for the manufacture of some useful and
X-ray diffraction XRD
value-added components [e.g., pectin extract, xanthan
gum, dietary fiber, silver nanoparticles, ketones,
phenols (Chan and Choo 2013; Diniz et al. 2012;
Lateef et al. 2016; Lecumberri et al. 2007; Mansur
Introduction
et al. 2014; Vriesmann et al. 2012; Yapo et al. 2013)].
Currently, CPH is exported by almost 50 countries. In
Usually, in agricultural and agro-industrial production
2019, 220 thousand tonnes of CPH were exported for a
processes, lignocellulosic materials are produced in
value of US $ 206 million, where the main exporters
high quantities and volumes, requiring space to be
are Côte d’Ivoire (83%), Ghana (5%), and The
disposed and causing environmental and phytosani-
Netherlands (3%) (International Trade Center 2020a).
tary concerns. This is the case of the cocoa production
The main constituents of the CPH cell wall are
chain. During the 2018/19-crop period, the world
cellulose [20–26% (w/w)], hemicellulose [9–13% (w/
production of cocoa almonds was estimated at
w)] and lignin [14–28% (w/w)] (Lu et al., 2018).
4.7 million tons (The International Cocoa Organiza-
Lignin is a branched polymer mainly constituted of
tion 2020). Thus, it is calculated that approximately
aromatic compounds, which provides physical and
19 million tons of residual cocoa biomass were
chemical resistance towards adverse environmental
produced. These materials are considered as worthless

123
Cellulose (2021) 28:4009–4024
conditions or biological degradation caused by insects
and microorganisms (Zhang et al. 2017). Hemicellu-
lose is a copolymer of pentoses and hexoses. Finally,
cellulose is a glucose polymer that can be transformed
for the subsequent use of glucose monomers in
fermentation processes for the generation of various
value-added compounds (Brar et al. 2014). One option
to obtain this last fraction is to carry out a chemical
pretreatment for the separation of the main compo-
nents of lignocellulosic structure, followed by an
enzymatic saccharification.
Some chemical and biotechnological routes of CPH
conversion were reported (Campos-Vega et al. 2018;
Vásquez et al. 2019) and other works have previously
explored the use of CPH hydrolysate to produce value-
added molecules such as xylitol and ethanol (Mensah
et al. 2020; Santana et al. 2018; Shet et al. 2018).
However, to the best of our knowledge, there is no
literature available about the bioconversion of CPH
for propionic acid (PA) production. PA is a weak,
liquid-looking, monocarboxylic acid with a rancid,
colorless and corrosive odor (Ahmadi et al. 2017). It is
currently used in the food and feed industries because
of its antimicrobial and antifungal properties. Further-
more, the plastics, perfumery and pharmaceutical
industries use this substance as a promoter of reactions
within the manufacturing process (Schaechter and
Lederberg 2004). The price of PA depends on its
purity. Thus, its cost can vary between 1000 and
10,000 US$/Ton. During 2019, PA exports reached
$493 million dollars (International Trade Center
2020b).
Although this organic acid is commonly produced
from the petrochemical route, the research related to
its production through fermentation processes has
grown significantly during the last years (Ali et al.
2020; Ammar et al. 2020; Dahiya et al. 2020).
However, one of the main drawbacks of the imple-
mentation of fermentation at industrial scale is the
production cost, taking into account that when PA is
produced through fermentation its cost reaches up to
2000 US$/ton, whereas through the petrochemical
route it is reduced by half (Piwowarek et al. 2019).
Trying to reduce the production cost of PA, cheap
and alternative by-products (e.g., sugarcane molasses,
glycerol, corn steep liquor, whey) have been tested as
carbon and nitrogen sources in fermentation process
(Ranaei et al. 2020; Yang et al. 2018). Glycerol is an
undesirable by-product of biodiesel processing and
4011
counts with favorable physicochemical characteris-
tics, easy handling, and high compatibility with many
other substances. As a by-product, glycerol can be
used as substrate for biomolecules production, solving
an environmental problem.
In this study, the valorization of CPH for PA
production, a high/medium value-added molecule, is
proposed for the first time. A sequential alkaline/
enzymatic hydrolysis was assessed to obtain fer-
mentable sugars, which in turn served as a carbon
source, along with residual glycerol, to study the
viability of PA production, using P. jensenii DSM
20274 in a batch fermentation process.
Material and methods
Cocoa pod husk preparation
CPH was obtained from a local cultivation of Theo-
broma cacao L. (Malvaceae) in Pará State (02° 250
08‘‘ S; 48° 090 08’’W), Brazil. The husk was cleaned
and cut into small pieces (5–8 cm), and oven-dried for
72 h at 60 °C. The dried CPH was then grounded in a
mill (Marconi—MA580/E, Brazil) and sieved through
a 2 mm mesh for about 10–15 min. The dried biomass
was stored in glass bottles and kept at room temper-
ature until posterior use.
Alkaline and enzymatic treatments of CPH
Chemical pretreatment was carried out with sodium
hydroxide [NaOH, 2.3% (w/v)] in 100 mL Schott
bottles at 10% (w/v) of dried and milled CPH (particle
size 0.84–2.0 mm). The flasks were autoclaved at
121 °C for 30 min. Then, the material was washed,
filtered, and dried at 60 °C for 12 h. Subsequently, an
enzymatic treatment was conducted in a 125 mL
Erlenmeyer flask adding of 2.4% (v/v) of the enzyme
CellicÒ CTec 2 (Novozymes A/S, Denmark) in
100 mL of citrate buffer solution at pH 4.8 and 10%
(w/v) of the alkaline treated CPH for 24 h at 50 °C in a
water bath with constant stirring at 100 rpm. Finally,
the material was filtered and dried at 60 °C for 12 h.
Characterization of CPH structure
The structural characteristics of CPH (untreated and
alkaline-treated) were identified through optical
123
4012 Cellulose (2021) 28:4009–4024

microscopy (Leica Microsystems DM/LS, China) and Ic  Ia

CrI ð%Þ ¼  100 ð1Þ
histological staining techniques (with safranin 1% and Ic
Astra blue 1%) (Cutler et al. 2007).
where Ic is the total intensity of the (2 0 0) peak
between 21.5°–22.0° 2h (crystalline contribution) and,
Microscopic analyses using scanning electron
and Ia is the amorphous intensity between 18°–18.5°
microscopy (SEM)
2h (amorphous contribution).
Dried and milled CPH’ samples (raw and alkaline-
enzymatic treated) were analyzed for their structure by
PA production
Scanning Electronic Microscopy (SEM) that was
performed in a Tescan Mod Vega 3LMU device
Microorganism and media composition
(Oxford Instruments), operating at 15 keV. Before
examination, samples were vacuum dried and coated/
The stock culture of Propionibacterium jensenii strain
metallized with a layer of gold in a sputter coater and
DSM 20274 was cultivated anaerobically for 48 h at
then dried.
33 8C without shaking using the medium proposed by
Coral et al. (2008) composed of (per liter of deionized
Fourier-transform infrared spectroscopy (FT-IR)
water): 5.0 g sodium lactate, 5.0 g yeast extract, 2.0 g
(NH4)2 PO4, 1.0 g KH2PO4, 0.01 g MgSO47 H2O,
Functional groups of CPH samples (raw, alkaline and
0.01 g CaCl26 H2O, 0.01 g CoCl26 H2O, 0.005 g
alkaline-enzymatic treated) were characterized by FT-
FeSO4 7 H2O, 0.0025 g MnSO4H2O and 7.0 g agar,
IR spectroscopy in a Vertex 70 (Bruker) equipment,
in culture tubes with screw cap and stored at 4 °C.
with the accessory DRIFTS (diffuse reflectance) with
The inoculum was cultivated in the previously
64 scans, 4 cm-1 resolution, without the elimination
described medium, but with the addition of 40.0 g.L-1
of atmospheric compensation from 4000 to 400 cm-1.
lactose instead of sodium lactate, the augmentation of
Samples were previously dried at 45 °C for 24 h, then
yeast extract concentration to 10.0 g.L-1, and without
were ground and homogenized in KBr and placed in
agar (Goswami and Srivastava 2000). PA production
DRIFTS accessories for spectra acquisition. FT-IR
was conducted using the synthetic fermentation
spectral tables reported in literature (Silverstein et al.
medium with addition of 15.0 gL-1 of pure glycerol,
2005) were employed to analyze the results obtained
instead of lactose. The pH was adjusted to 7 and the
in this study.
medium was autoclaved at 121 °C for 15 min.
Fermentation assays were performed in 21 mL tubes,
Thermogravimetric analyses (TGA)
which were almost completely filled with the medium
and inoculum (15% (v/v), closed with screw caps and
Thermal stability of CPH samples (1 g) was evaluated
incubated without shaking for 48 h at 33 °C to create a
by thermogravimetry (TG) in a Setaram device (Setsys
reduced oxygen atmosphere).
Evolution TG/DTA/DSC), under N2 atmosphere from
Subsequently, PA production was carried out using
20 °C to 800 °C and gas flow 20 mLmin-1.
CPHH, instead of water, with a sugar concentration of
7.5 gL-1 and the addition of the components of the
X-ray diffraction (XRD)
previously mentioned synthetic medium, with the
exception of glycerol concentration that was reduced
The crystallinity index (CrI) of CPH samples was
to 7.5 gL-1. The pH was adjusted to 7.
obtained by measuring wide-angle X-ray scattering
patterns in a Shimadzu XRD 7000 Maxima diffrac-
Optimization of media composition
tometer and Ni-filtered copper radiation (CuKa,
k = 1.5418 Å). Samples were scanned in the 2h range
A two-step optimization of medium composition was
of 5 to 40 degrees (2 degrees per min), and the
performed. Initially, the effect of CPHH, glycerol and
generator was operated at 40 kV and 20 mA. The
salts concentration on PA production was evaluated
Segal crystallinity index (Segal et al. 1959) was
through a Plackett & Burman design experiment with
calculated by the following equation:

123
Cellulose (2021) 28:4009–4024 4013

7 variables with 8 runs and 3 central points. Thus, the
experiment was carried out with fixed concentrations
of glycerol (7.5 gL-1), glucose of CPHH (7.5 gL-1)
and yeast extract (10 gL-1) and presence or absence
of the following components (variables) in fermenta-
tion medium: (NH4)2HPO4, KH2PO4, MgSO4, CaCl2,
CoCl2, FeSO4 and MnSO4 (data not shown). The
second step of optimization was carried out to choose
the best levels of the previously screened significant
variables (CPHH, glycerol and yeast extract concen-
tration). A central composite design (CCD) (with 6
axial points and 5 centerpoint runs) was then per-
formed with 19 essays applying the Response Surface
Methodology (RSM) using the software StatisticaÒ
5.0. Essays and variable levels are shown in Table 1.
Kinetic studies of PA production
Fermentation assays were conducted in 100 mL
Schott bottles, with a work volume of 90 mL, which
were tightly sealed and incubated with low agitation
rate (50 rpm) to create a reduced oxygen atmosphere.
Assays were conducted in duplicate. Fermentation
kinetic was followed during 156 h and samples were
centrifuged at 6000 9 g for 20 min at room temper-
ature and the supernatant analyzed subsequently. The
data were expressed as mean ± standard error and
differences between means at the 5% of confidence
interval (p \ 0.05) were considered significant.
Analytical methods
After centrifugation and filtration (0.2 lm porosity
PVDF membranes) glucose, glycerol and organic
acids (i.e. propionic acid, succinic acid and, acetic
acid) were analyzed using the equipment Agilent 1260
Infinity HPLC. Hi-Plex H column (300 9 7.7 mm)
was used and operated at 60 °C, 0.005 M H2SO4 as the
mobile phase at 0.6 mLmin-1 flow rate. Anions and
cations were quantified by Ion Chromatography using
a Metrohm CH-9101. For the cation analysis the
Metrosep C3 250/4.0 column (250 9 4.0 mm ID; No.
5607002) was used, with the following analysis
conditions: eluent 5.0 mM HNO3; flow rate:
1.0 mLmin-1, detector: CD, temperature 40 °C,
injection volume: 20 lL. For the anion analysis the

Table 1 Central composite Central composite design levels Concentration (gL-1)

design matrix for the
experimental design for PA Run Glucose Glycerol Yeast Extract Glucose Glycerol Yeast Extract PA
production
1 -1 -1 -1 5.1 20.5 6.4 1.27
2 -1 -1 1 5.1 20.5 13.5 0.74
3 -1 1 -1 5.1 43.2 6.4 0.89
4 -1 1 1 5.1 43.2 13.5 0.91
5 1 -1 -1 10.8 20.5 6.4 0.70
6 1 -1 1 10.8 20.5 13.5 1.15
7 1 1 -1 10.8 43.2 6.4 0.68
8 1 1 1 10.8 43.2 13.5 1.28
9 - 1.68 0 0 3.2 31.9 10.0 0.86
10 1.68 0 0 12.8 31.9 10.0 0.97
11 0 - 1.68 0 8.0 12.8 10.0 0.84
12 0 1.68 0 8.0 51.0 10.0 0.94
13 0 0 - 1.68 8.0 31.9 4.0 0.81
14 0 0 1.68 8.0 31.9 15.9 1.14
15 0 0 0 8.0 31.9 10.0 2.15
16 0 0 0 8.0 31.9 10.0 1.86
17 0 0 0 8.0 31.9 10.0 2.08
18 0 0 0 8.0 31.9 10.0 2.19
19 0 0 0 8.0 31.9 10.0 2.30
Control – – – 7.5 7.5 10.0 1.90

123
4014
Metrosep A Supp 5 250/4.0 column (250 9 4.0 mm
ID; No. 7610789) was used, with the following
analysis conditions: eluent 3.2 mM Na2CO3-
? 1.0 mM NaHCO3, flow rate: 0.7 mLmin-1, detec-
tor: suppressed CD, room temperature (25 °C),
injection volume: 20 lL. Biomass quantification was
determined by dry weight method. Samples of 10 mL
of fermentation medium were centrifuged at
6000 9 g for 20 min in weighed pre-dried tube. The
cell pellet was then dried at 80 °C for 12 h until
respective measure of mass difference.
Results and discussion
Physicochemical characterization of CPH
Morphological analysis
Figures 1a and b show the morphological features that
were found in raw CPH and alkaline-treated CPH
samples, respectively. Figure 1a, displays an
unchanged group of spiral tracheal structures (Chung
et al. 2008). In Fig. 1b, a similar but rather disrupted
tracheal structure is found due to the alkaline
treatment. It is interesting to observe the arrangement
of this structure and its elongation capacity, to fulfill
the functions of transporting water, of mechanical
resistance and flexibility within the vegetal tissue,
which has ring, spiral, and reticulate or dotted
conformations. The presence of these structures was
corroborated via SEM analysis. Figure 1c shows the
distribution of plant cells of raw CPH and in Fig. 1d,
due to the alkaline pretreatment, which implicates in
solvation and saponification, the swelling of biomass
cells and internal surface alteration is evident, as it has
been shown in other lignocellulosic matrixes (Heise
et al. 2017).
Structural arrangements of CPH analyzed by SEM
Figure 2 a–f represents the SEM images of untreated
CPH samples (a–c) and (d–f) alkaline-enzymatic
treated CPH samples. Interesting changes in surface
texture, porosity and exposure of characteristic struc-
tures are observed. Figure 2a shows the 50 9 magni-
fication micrograph of raw CPH after mechanical
rupture. Irregular fragments with fibrous and moder-
ately porous appearance are observed. In Fig. 2b, the
123
Cellulose (2021) 28:4009–4024
same material (1000x) shows a compact, rigid and
rough outer surface with the presence of some
granules of particulate material, which is the result
of the grinding process. With an increase of 2000x, the
deposit of epicuticular waxes of the fruit (Fig. 2c) is
identified on the external and rough surface of the raw
CPH.
After treatments, the surface porosity of the parti-
cles increased as expected, when compared to the
original raw material (Fig. 2d). Figure 2e shows the
longitudinal view of the chemically and enzymatically
treated material, in which an exposed vascular bundle
is clearly observed due to the treatment action. In the
longitudinal view presented in Fig. 2f, the vascular
tissue of CPH was detected, showing a stitch and
reticulate configuration, which is characteristic of the
tracheal elements of the metaxylem and, in the same
way, a spiral structure conformation that is character-
istic of the tracheal elements of protoxilem, also
described by Chung et al., (2008) for CPH.
Determination of crystallinity index (CrI)
X-ray diffraction (XRD) was used to evaluate the
effect of NaOH treatment on CPH and the availability
of cellulose fraction, which is more accessible to
enzymatic hydrolysis. For the CrI calculation, the
Segal approach was used as an empirical measurement
for relative comparison. The CrI indicates a qualitative
or semi-quantitative evaluation of the amount of
cellulose that is in the crystalline state, and, conse-
quently, the cellulose in the amorphous state (Park
et al. 2010). The XRD profiles of untreated, alkaline
and alkaline-enzymatic treated CPH are shown in
Fig. 3. For the untreated CPH samples, the CrI was
46%, whereas, for CPH with alkaline treatment and
alkaline-enzymatic treatment, it was 55 and 44%,
respectively.
It could be assumed that the increase in CrI
observed after alkaline treatment is due to the
elimination of a large portion of the amorphous
fraction of the CPH, consisting mainly of lignin,
hemicellulose and non-structural components (i.e.,
nitrate/nitrites, protein, ash, chlorophyll, or waxes). As
described by Kim et al. (2016), the gross crystallinity
index of biomass with alkaline treatment often rises
after pretreatment, primarily, due to removal of
amorphous regions, rather than to structural change
in the cellulose fibers. In previous studies, the alkaline
Cellulose (2021) 28:4009–4024
Fig. 1 Comparison between raw and alkaline treated CPH fresh
samples analyzed by optical microscopy: a Native spiral
tracheal structure in raw CPH sample (10x); b Disrupted spiral
treatment was applied on a wide range of lignocellu-
losic agroindustrial materials (e.g., soybean straw and
husk, corn stover, oil palm empty fruit bunch) in
which, changes in CrI are similar to those obtained in
this work (Latip et al. 2019; Qing et al. 2017; Shi et al.
2021). Although cellulose was not structurally dam-
aged, it is assumed that its quality was affected by the
peeling reaction due to the alkaline treatment condi-
tions (Chen et al. 2017). This reaction stimulates the
generation of some formless regions in which enzy-
matic digestibility can be promoted with the conse-
quent recovery of sugars from cellulose, as well as was
observed in similar studies (Malik et al. 2020; Mota
4015
tracheal structure in CPH after alkaline treatment (10x); c Native
distribution of cells in raw CPH (10x); d Swollen cells in CPH
sample after alkaline treatment (10x)
et al. 2021). This suggests that through mild NaOH
solution treatment conditions was possible to remove
and/or partially degrade amorphous fraction and make
the cellulose of CPH sample, susceptible to enzymatic
hydrolysis and saccharification.
Functional groups of CPH
The spectra obtained by infrared spectroscopy showed
differences between treated (alkaline and alkaline-
enzymatic treatments) and raw CPH (Fig. 4). In the
region between 3600 and 3000 cm-1 (Fig. 4a) there
are signals that are attributed to stretching vibration of
123
4016
Fig. 2 Scanning electronic microscopic photos of CPH
samples: a Ground fragments of raw CPH (509); b Outer
surface of raw CPH (10009); c Outer surface of raw CPH with
presence of epicuticular waxes (2.0009); d Ground fragment of
CPH sample with alkaline-enzymatic treatment (509);
Fig. 3 X-ray spectra of CPH samples with different treatments
123
Cellulose (2021) 28:4009–4024
e Longitudinal view of vascular bundle of CPH sample with
alkaline-enzymatic treatment (5009); f Longitudinal view of a
tracheal element with a spiral pattern of CPH with alkaline-
enzymatic treatment (30009)
O–H bonds of the phenolic and alcoholic groups,
which are lignin components (Ahmad et al. 2012;
Morone et al. 2017; Rashid et al. 2016; Yang et al.
2007). The band was larger and intense for the raw
sample. Pre-treated samples suffered alterations that
acted directly in these groups, causing the decrease of
signal intensity. Between 2950—2890 cm-1, it is
possible to verify the presence of narrow peaks
attributed to the symmetric and asymmetric stretching
vibration of the C-H bonds of the CH3, CH2 and CH
groups (Coral Medina et al. 2015; Tinwala et al. 2015),
whose intensity was modified in the pre-treated
samples. As described by Ibrahim et al. (2013) the
main consequence of pre-treatment is the breakdown
of the cell wall matrix, where the bonds between
carbohydrates and lignin are cleaved (Chen et al.
Cellulose (2021) 28:4009–4024
Fig. 4 Infrared Spectroscopy of CPH samples: a Spectrum between 3800 and 500 cm-1; b Expanded spectrum between 1800 and
800 cm-1
2016; Geng et al. 2019), as well as the occurrence of
depolymerization and solubilization of hemicellulose.
In this case, the alkaline solution is applied to remove
lignin from the CPH without deteriorating carbohy-
drates and increasing the porosity of the biomass
surface augmenting the accessibility of enzymatic
cellulolytic complex (Kim et al. 2016). The subse-
quent use of ß-glucosidases assures the recovery of
hydrolysate with a high content in glucose prone for
fermentation.
For better analysis, the region between 1800 and
800 cm-1 was enlarged (Fig. 4b). In this region
(known as the ‘‘fingerprint’’) it was possible to identify
the most important differences between the functional
groups of the CPH samples. In 1735 cm-1, a narrow
band of medium intensity was observed for the raw
sample, which decreased after alkaline and enzymatic
treatments. Changes in this region are attributed to the
C = O stretching vibration of the carbonyl and acetyl
groups in the xylan component of hemicelluloses
(Ibrahim et al. 2013; Lionetto et al. 2012; Yang et al.
2007). When the raw sample is compared with the
treated samples, in 1700 and 1500 cm-1 region a
broad and intense band is observed, attributed to the
C = O with benzene ring (lignin) and to the stretching
vibration C = C bonds (Pua et al., 2013). In
1460–1184 cm-1 region, alterations that are origi-
nated from the treatments appear, and are attributed to
the deformation of the C-H, O–H groups of celluloses,
hemicelluloses, and lignin (Pua et al. 2013). Two
significant bands appeared for the alkaline-enzymatic
4017
sample, one of them in 1456 cm-1 that is associated to
C–H deformations in lignin (Ibrahim et al. 2013) and
the other one in 1319 cm-1 is probably attributed to
C-H vibration in cellulose (Chun et al. 2013). The
presence of C-O bonds of lignin (Chun et al. 2015) is
indicated between 1184 and 960 cm-1. That band is
much wider for the raw sample when compared to the
treated samples, suggesting the removal of lignin.
Finally, treated samples showed a defined peak of
medium intensity in the near region of 896 cm-1
(clearly absent in the raw sample), corresponding to
glycosidic linkages (Sun et al. 2000; Xu et al. 2015)
which suggest the presence of cellulose (Sribala et al.
2016) and the consequent effectiveness of treatments.
Thus, comparing the obtained spectra, it is possible
to ratify the effect of the CPH treatments, since the
specific adsorption bands that initially appeared in the
raw material were altered. According to the literature,
alkaline treatment acts on lignin and polysaccharide
structures of the lignocellulosic material (Bali et al.
2015), which is of great importance since the decrease
in the material’s recalcitrance facilitates the accessi-
bility of the enzyme to the cellulosic compounds.
Thermal stability of CPH
In TG curves, the thermal decomposition profile of the
material is observed in different stages and it is
directly related to specific compounds, which for the
case of vegetal biomass, would be classified as
hemicellulose, cellulose, and lignin. Figure 5 shows
123
4018
Fig. 5 Thermogravimetric curves of CPH samples with different treatments
the TG curves obtained for the samples of CPH
without treatment (black line), for the alkaline treated
CPH (red line) and for the alkaline-enzymatic treat-
ment CPH (blue line) and their respective derivatives
(dTG).
In Fig. 5 it is also possible to identify, through each
of the generated derivatives (discontinuous lines) for
the different samples, changes during the degradation
process. In the case of alkaline treatment with NaOH
(red discontinuous line), a ‘‘shoulder’’ appears
between 260 and 270 °C, which is supposed to
correspond to the degradation of the residual material
deposited in the CPH sample and related to residual
NaOH. In the case of the CPH sample with alkaline-
enzymatic treatment between 200 and 250 °C, the
same ‘‘shoulder’’ is observed and could possibly be
associated with the presence of residues from the
enzymatic formulation that was used for CPH enzy-
matic treatment.
In each TG curve of the CPH samples, it is possible
to identify three thermal events that are characteristic
of lignocellulosic material. In the first thermal event
(Zone 1) the mass loss is related to the evaporation of
volatile compounds (adsorbed water, absorbed water
and volatiles of low molar mass) (Soltani et al. 2015).
During the second and most important thermal event
123
Cellulose (2021) 28:4009–4024
(Zone 2), the degradation or depolymerization of the
hemicellulose, cellulose and lignin compounds hap-
pens and occurs the greatest mass loss (Chen et al.
2015). During the last thermal event (Zone 3) it is
possible to locate the total degradation of the material,
resulting in oxides or ashes of the inorganic elements
that constitute the material. Table 2 summarizes the
analyzed samples, the degradation temperature char-
acteristic of each zone and the respective percentages
of weight loss.
As identified in each TG curve and according to the
information presented in Table 2, the largest mass loss
corresponds to the sample without treatment. Com-
paring this sample with the treated material and as
expected, the mass loss is higher, since part of the
material was previously removed with the chemical
and biological processes carried out on the analyzed
Table 2 Thermogravimetric analysis of CPH
Sample of CPH Weight loss (%)
Raw 71.9
Alkaline treatment 65.1
Alkaline-enzymatic treatment 67.5
Cellulose (2021) 28:4009–4024
material. It is possible to conclude that the thermal
stability of the CPH samples was affected by both
alkaline and enzymatic treatments (Ndazi et al. 2007)
when compared to raw CPH, which exhibited higher
thermal stability, mainly due to the natural presence of
waxes and lignin. The lower thermal resistance of
treated samples is attributed to the effective degrada-
tion of lignin compounds by NaOH and, by the
subsequent enzymatic effect on the cellulosic mate-
rial, leaving the structures devoid of stability.
PA production
Optimization of medium composition
After consecutive alkaline [NaOH, 2.3% (w/v)] and
enzymatic [CellicÒ CTec 2 (2.4% v/v)] treatments of
CPH, a hydrolysate was obtained with maximum
concentration of 60.5 gL-1 glucose and 275 mg
glucoseg-1 CPH. Ion concentration of the obtained
hydrolysate were (per liter): F- 5.8 mg, Cl- 14.6 mg,
SO4-2 1.9 mg, PO4-3 2.9 mg, NH4? 645.9 mg, K?
25.4 mg, Mg ?2 41.8 mg, Ca?2 64.8 mg. Initial
fermentation process for PA production was carried
out using CPHH medium reaching 1.9 gL-1 PA.
The first step of medium optimization showed that
(NH4)2HPO4, CaCl2 and KH2PO4 were statistically
significant (P \ 0.05) for PA production by P. jensenii
DSM 20274. (NH4)2HPO4, and KH2PO4 had negative
coefficients, suggesting that the presence of ions
PO4-3, NH4?, K? in the hydrolysate, supplies the
requirements of the microorganism for PA production
and the addition of these salts is not necessary, which
could have a positive impact on production costs. On
the other hand, CaCl2 had positive effect and the
addition of this salt to the medium is required.
One of the main drawbacks that are faced in PA
production through fermentation is low the produc-
tivity when the batch fermentation system is imple-
mented: \ 1 gL-1h-1, as well as the low
concentrations of PA: \ 50 gL-1 (Parizzi et al.
2012). To understand and overcome this situation,
Wang and Yang (2013) studied the influence of
glycerol and glucose in PA production by P. freuden-
reichii subsp. shermanii, which were added as inde-
pendent or simultaneous carbon sources. They found
that despite a low PA yield, glucose promoted
considerable cell growth and acetate production,
4019
whereas glycerol allows higher propionate yield and
selectivity, but low productivity. In co-fermentation,
these carbon sources, enhanced PA productivity and
yield, and showed robust flux redistribution for redox
balance and cell growth. Following these findings, in
this work the co-fermentation strategy was imple-
mented, taking advantage of CPH and the glycerol
composition (as waste by-products) for value-added
molecules’ production.
The results of the optimization medium step are
presented in Fig. 6. R2 was 0.96908, indicating that the
model could explain * 97% of the response variabil-
ity. Response surface plot (Fig. 6a) showed the
interaction between glycerol and glucose concentra-
tions, indicating the presence of optimum point within
the established range, where the highest PA concen-
tration (2.3 gL-1), was reached using (per liter) 8 g
glucose from the CPHH and 32 g glycerol. It is known
that Propionibacterium metabolism is governed by the
dicarboxylic acid pathway where carbon sources are
oxidized to pyruvate via NADH-generating glycolysis
pathways (Yang et al. 2018). Thus, when PA is
produced from the simultaneous use of these carbon
sources, glycerol is predominantly consumed for PA
biosynthesis and glucose is used for cell biomass
synthesis, because it acts as a hydrogen donor
substrate for the supply of reducing equivalents and
ATP (Liu et al. 2011; Wang and Yang 2013).
Regarding the nitrogen demand, as it is observed in
the plotted response surface (Fig. 6b) the interaction
between glucose and yeast extract, 10 gL-1 of the
nitrogen source is the best concentration for the
optimal levels PA. Other studies have already suc-
cessfully used glycerol or glucose, along with yeast
extract (Dishisha et al. 2012; Zhang et al. 2015).
Glycerol and glucose concentration were not signif-
icant, individually, but only their interaction presented
significant influence on PA production in a certain
level. Contrarily, the quadratic interaction between
glycerol and glucose was statistically significant, but
with negative influence on PA production (Fig. 6c and
d). This means that a certain level of each carbon
source must be employed, not low or high concentra-
tions favor the synthesis of the organic acid, and both
may be employed at medium level with a medium
level of the nitrogen source (yeast extract) that leads to
a certain C/N ratio. Optimal conditions of PA accu-
mulation were observed with 8 gL-1 of glucose,
32 gL-1 of glycerol and 10 gL-1 of yeast extract.
123
4020
Fig. 6 Influence of carbon and nitrogen sources on PA
production: a Response surface plots for optimal concentrations
for PA production showing interactive effects of glycerol
(gL-1) vs. glucose (gL-1); b Response surface plots for
Kinetics of PA production
CPHH and glycerol were used as carbon sources for
PA production (Fig. 7). Both carbon sources were
consumed, along with the co-production of PA, acetic
and succinic acids. After 156 h, 89% of glucose and
39% of glycerol were consumed. PA concentration
achieved a maximum of 10.28 ± 1.05 gL-1 after
132 h of process representing a productivity of
0.08 ± 0.01 gL-1h-1 after 72 h. The highest yield
was 0.59 g PA/g carbon source after 108 h of process.
Using P. jensenii DSM 20274, Coral et al. (2008)
obtained PA concentration of 8.98 gL-1 and a
maximum productivity of 0.064 gL-1h-1 employing
glycerol and sugarcane molasses. These results
showed that, for the same strain, CPHH together with
glycerol are better carbon sources for PA production.
During fermentation, succinic and acetic acids,
which are common by-products of PA production
123
Cellulose (2021) 28:4009–4024
optimal concentrations for PA production showing interactive
effects of yeast extract (gL-1) vs. glucose (gL-1) c Pareto
chart of standardized effects on PA production; (p-value = 5%);
d Analysis of variance (ANOVA)
from glycerol and glucose (Ranaei et al. 2020), were
produced simultaneously at maximum concentrations
of 0.4 and 0.8 gL-1, respectively. It has been reported
that the generation of by-products (acetic, succinic,
lactic, pyruvic, malic, fumaric, isovaleric and formic
acids, propanol and CO2), when are produced in
considerable concentrations, hinders the purification
processes (Rodriguez et al., 2014), with significant
increase of production costs. The accumulation of
these organic acids affected the pH of the medium,
which started in 6.45 and reached 4, because no pH
control strategy was implemented during the process.
It should be emphasized that PA concentrations
could be still improved with the use of bioreactors and
different fermentation operations. Implementing dif-
ferent process strategies or using genetically engi-
neered microorganism, it would be possible to
enhance PA concentration, yield, and productivity.
As an example, when implementing fed-batch system,
Cellulose (2021) 28:4009–4024
Fig. 7 Kinetics of PA production in 100 mL glass Schott flask
Liu et al. (2012) produced 53.2 gL-1 of PA, with P.
acidipropionici ATCC 4875, using hemicelluloses as
carbon source. Liu et al. (2016) reached concentra-
tions of 26.9 gL-1 of PA after 228 h of process
(0.11 gL-1h-1), employing fed-batch operation and
a strain of P. jensenii ATCC 4868, with glycerol as
sole carbon source. Chen et al. (2012) implemented
the fed-batch system and cell immobilization of P.
freudenreichii strain CCTCCM 207015 with glucose
as a carbon source, reaching concentrations of up to
136.23 ± 6.77 gL-1 of PA after 240 h with a
productivity of 0.57 ± 0.03 gL-1h-1. On the other
hand, Zhuge et al. (2015) achieved a concentration of
21.38 gL-1 of PA after 156 h of process, using
engineered P. jensenii ATCC 4868 with glycerol as
carbon source. Considering these facts, future studies
of PA production may consider the fed-batch fermen-
tation system as a potential way to enhance the
productivity of the process using CPHH as carbon
source along with glycerol.
Conclusions
This work assesses an unexplored carbon source for an
industrially applied value-added molecule production.
Initially, the characterization of raw and pre-treated
CPH by different qualitative and quantitative
4021
techniques (optical microscopy, SEM, FT-IR, XRD,
and TG) allowed the analysis and understanding of the
physicochemical changes caused by the action of the
chemical and enzymatic compounds. It was revealed
that the alkaline pretreatment in CPH samples reduces
the content of lignin, promoting the increase of
porosity of the biomass surface and the swelling of
the cell wall matrix, which leads the increase of
cellulose accessibility by the enzymatic complex and
finally, enhancing the saccharification process effi-
ciency. CPH chemical and enzymatic hydrolysis
allowed the obtaining of a glucose-rich hydrolysate
of 60.5 gL-1 or 275 mg glucose g-1 CPH. The
highest yield of PA production was 0.59 g PA g-1
carbon source, after 108 h of process. It has been
shown that CPH has potential use as substrate in
biotechnological processes for high value biomole-
cules’ production.
Acknowledgments The authors thank the Coordenação de
Aperfeiçoamento de Pessoal de Nı́vel Superior (CAPES) and
Conselho Nacional de Desenvolvimento Cientı́fico e
Tecnológico do Brasil (CNPq) for the research scholarship.
Funding Authors Zulma Sarmiento Vásquez, Luciana Porto
de Souza Vandenberghe, Cristine Rodrigues, Valcineide O.
A. Tanobe, Oranys Marı́n, Gilberto V.M. Pereira, Carlos
Ricardo Soccol have received research funding from
Coordenação de Aperfeiçoamento de Pessoal de Nı́vel
Superior—CAPES, Brazil, projeto CAPES-PROCAD 2013.
123
4022
Compliance with ethical standards
Conflict of interest The authors declare that they have no
conflicts of interest.
References
Ahmad F, Daud WMAW, Ahmad MA, Radzi R (2012) Cocoa
(Theobroma cacao) shell-based activated carbon by CO 2
activation in removing of Cationic dye from aqueous
solution: Kinetics and equilibrium studies. Chem Eng Res
Des 90:1480–1490. https://doi.org/10.1016/j.cherd.2012.
01.017
Ahmadi N, Khosravi-Darani K, Mortazavian AM (2017) An
overview of biotechnological production of propionic acid:
From upstream to downstream processes. Electron J
Biotechnol 28:67–75. https://doi.org/10.1016/j.ejbt.2017.
04.004
Ali R, Saravia F, Hille-Reichel A, Härrer D, Gescher J, Horn H
(2020) Enhanced production of propionic acid through
acidic hydrolysis by choice of inoculum. J Chem Technol
Biotechnol. https://doi.org/10.1002/jctb.6529
Ammar EM, Martin J, Brabo-Catala L, Philippidis GP (2020)
Propionic acid production by Propionibacterium freuden-
reichii using sweet sorghum bagasse hydrolysate. Appl
Microbiol Biotechnol 104:9619–9629. https://doi.org/10.
1007/s00253-020-10953-w
Bali G, Meng X, Deneff JI, Sun Q, Ragauskas AJ (2015) The
effect of alkaline pretreatment methods on cellulose
structure and accessibility. Chemsuschem 8:275–279.
https://doi.org/10.1002/cssc.201402752
Brar SK, Dhillon GS, Soccol CR (2014). Biotransformation of
waste biomass into high value biochemicals, Springer,
USA. https://doi.org/10.1007/978-1-4614-8005-1
Campos-Vega R, Nieto-Figueroa KH, Oomah BD (2018) Cocoa
(Theobroma cacao L.) pod husk: renewable source of
bioactive compounds. Trends Food Sci. Technol.
81:172–184. https://doi.org/10.1016/j.tifs.2018.09.022
Chan SY, Choo WS (2013) Effect of extraction conditions on
the yield and chemical properties of pectin from cocoa
husks. Food Chem 141:3752–3758. https://doi.org/10.
1016/j.foodchem.2013.06.097
Chen F, Feng X, Xu H, Zhang D, Ouyang P (2012) Propionic
acid production in a plant fibrous-bed bioreactor with
immobilized Propionibacterium freudenreichii. J Biotech-
nol 164:202–210. https://doi.org/10.1016/j.jbiotec.2012.
08.025
Chen Z, Hu M, Zhu X, Guo D, Liu S, Hu Z, Xiao B, Wang J,
Laghari M (2015) Characteristics and kinetic study on
pyrolysis of five lignocellulosic biomass via thermogravi-
metric analysis. Bioresour Technol 192:441–450. https://
doi.org/10.1016/j.biortech.2015.05.062
Chen J-H, Xu J-K, Huang P-L, Sun R-C (2016) Effect of alka-
line pretreatment on the preparation of regenerated ligno-
cellulose fibers from bamboo stem. Cellulose. https://doi.
org/10.1007/s10570-016-0983-1
Chen W, Yang S, Zhang Y, Wang Y, Yuan T, Sun R (2017)
Effect of alkaline preswelling on the structure of lignins
123
Cellulose (2021) 28:4009–4024
from Eucalyptus. Sci Rep. https://doi.org/10.1038/
srep45752
Chun KS, Husseinsyah S, Osman H (2013) Modified cocoa pod
husk-filled polypropylene composites by using methacrylic
acid. BioResources. https://doi.org/10.15376/biores.8.3.
3260-3275
Chun KS, Husseinsyah S, Osman H (2015) Utilization of cocoa
pod husk as filler in polypropylene biocomposites: Effect
of maleated polypropylene. J Thermoplast Compos Mater
28:1507–1521. https://doi.org/10.1177/
0892705713513291
Chung BY, Cho JY, Lee SS, Nishiyama Y, Matsumoto Y,
Liyama K (2008) The relationship between lignin and
morphological characteristics of the tracheary elements
from cacao (Theobroma cacao L.) hulls. J Plant Biol
51:139–144. https://doi.org/10.1007/BF03030723
Coral J, Karp SG, De Souza P, Vandenberghe L, Parada JL,
Pandey A, Soccol CR (2008) Batch fermentation model of
propionic acid production by Propionibacterium acidipro-
pionici in different carbon sources. Appl Biochem
Biotechnol 151:333–341. https://doi.org/10.1007/s12010-
008-8196-1
Coral Medina DJ, Woiciechowski A, Zandona A, Noseda MD,
Satinder B, Soccol CR (2015) Lignin preparation from oil
palm empty fruit bunches by sequential acid / alkaline
treatment—A biorefinery approach. Bioresour Technol
194:172–178. https://doi.org/10.1016/j.biortech.2015.07.
018
Cutler DF, Botha CEJ, Stevenson DW (2007) Plant Anatomy -
An applied approach. Blackwell Publ. https://doi.org/10.
1002/9781119945734.ch7
Dahiya S, Lakshminarayanan S, Mohan VS (2020) Steering
acidogenesis towards selective propionic acid production
using co-factors and evaluating environmental sustain-
ability. Chem Eng J. https://doi.org/10.1016/j.cej.2019.
122135
de Diniz MD, Druzian JI, Audibert S (2012) Produção de goma
xantana por cepas nativas de Xanthomonas campestris a
partir de casca de cacau ou soro de leite. Polı́meros
22:278–281. https://doi.org/10.1590/S0104-
14282012005000032
Dishisha T, Alvarez MT, Hatti-Kaul R (2012) Batch- and con-
tinuous propionic acid production from glycerol using free
and immobilized cells of Propionibacterium acidipropi-
onici. Bioresour Technol 118:553–562. https://doi.org/10.
1016/j.biortech.2012.05.079
Geng W, Narron R, Jiang X, Pawlak JJ, Park S, Jameel H,
Venditti RA (2019) The influence of lignin content and
structure on hemicellulose alkaline extraction for non-
wood and hardwood lignocellulosic biomass. Cellulose
26(5):3219–3230
Goswami V, Srivastava AK (2000) Fed-batch propionic acid
production by Propionibacterium acidipropionici. Bio-
chem Eng J 4:121–128. https://doi.org/10.1016/S1369-
703X(99)00042-X
Heise K, Rossberg C, Strätz J, Bäurich C, Brendler E, Keller H,
Fischer S (2017) Impact of pre-treatments on properties of
lignocelluloses and their accessibility for a subsequent
carboxymethylation. Carbohydr Polym 161:82–89. https://
doi.org/10.1016/j.carbpol.2016.12.066
Cellulose (2021) 28:4009–4024
Ibrahim MM, El-zawawy WK, Jüttke Y, Koschella A, Heinze T
(2013) Cellulose and microcrystalline cellulose from rice
straw and banana plant waste: preparation and characteri-
zation. Cellulose 20(5):2403–2416. https://doi.org/10.
1007/s10570-013-9992-5
International Trade Center (2020a) List of exporters for the
product: 1802 Cocoa shells, husks, skins and other cocoa
waste. [WWW Document]. URL https://www.trademap.
org/ (accessed 7.10.20)
International Trade Center (2020b) List of exporters for the
product: 291550 Propionic acid, its salts and esters [WWW
Document]. URL https://www.trademap.org/ (accessed
10.7.20)
Kim JS, Lee YY, Kim TH (2016) A review on alkaline pre-
treatment technology for bioconversion of lignocellulosic
biomass. Bioresour Technol 199:42–48. https://doi.org/10.
1016/j.biortech.2015.08.085
Lateef A, Azeez MA, Asafa TB, Yekeen TA (2016) Cocoa pod
husk extract-mediated biosynthesis of silver nanoparticles:
its antimicrobial, antioxidant and larvicidal activities.
J Nanostruct Chem 6:159–169. https://doi.org/10.1007/
s40097-016-0191-4
Latip NA, Sofian AH, Ali MF, Ismail SN, Idris DMND (2019)
Structural and morphological studies on alkaline pre-
treatment of oil palm empty fruit bunch (OPEFB) fiber for
composite production, in: Materials Today: Proceedings.
Elsevier Ltd, pp. 1105–1111. Doi:https://doi.org/10.1016/
j.matpr.2019.06.529
Lecumberri E, Mateos R, Izquierdo-Pulido M, Rupérez P, Goya
L, Bravo L (2007) Dietary fibre composition, antioxidant
capacity and physico-chemical properties of a fibre-rich
product from cocoa (Theobroma cacao L.). Food Chem
104:948–954. https://doi.org/10.1016/j.foodchem.2006.
12.054
Lionetto F, Del Sole R, Cannoletta D, Vasapollo G, Maffezzoli
A (2012) Monitoring wood degradation during weathering
by cellulose crystallinity. Mater (Basel) 5:1910–1922.
https://doi.org/10.3390/ma5101910
Liu Y, Zhang YG, Zhang RB, Zhang F, Zhu J (2011) Glycerol/
glucose co-fermentation: one more proficient process to
produce propionic acid by Propionibacterium acidipropi-
onici. Curr Microbiol 62:152–158. https://doi.org/10.1007/
s00284-010-9683-5
Liu Z, Ma C, Gao C, Xu P (2012) Efficient utilization of
hemicellulose hydrolysate for propionic acid production
using Propionibacterium acidipropionici. Bioresour Tech-
nol 114:711–714. https://doi.org/10.1016/j.biortech.2012.
02.118
Liu L, Guan N, Zhu G, Li J, Shin HD, Du G, Chen J (2016)
Pathway engineering of Propionibacterium jensenii for
improved production of propionic acid. Sci Rep 6:1–9.
https://doi.org/10.1038/srep19963
Lu F, Rodriguez-Garcia J, Van Damme I, Westwood NJ, Shaw
L, Robinson JS, Warren G, Chatzifragkou A, McQueen
Mason S, Gomez L, Faas L, Balcombe K, Srinivasan C,
Picchioni F, Hadley P, Charalampopoulos D (2018)
Valorisation strategies for cocoa pod husk and its fractions.
Curr Opin Green Sustain Chem 14:80–88. https://doi.org/
10.1016/j.cogsc.2018.07.007
Malik K, Salama ES, Kim TH, Li X (2020) Enhanced ethanol
production by Saccharomyces cerevisiae fermentation post
4023
acidic and alkali chemical pretreatments of cotton stalk
lignocellulose. Int Biodeterior Biodegrad 147:104869.
https://doi.org/10.1016/j.ibiod.2019.104869
Mansur D, Tago T, Masuda T, Abimanyu H (2014) Conversion
of cacao pod husks by pyrolysis and catalytic reaction to
produce useful chemicals. Biomass Bioenerg 66:275–285.
https://doi.org/10.1016/j.biombioe.2014.03.065
Mensah M, Asiedu NY, Neba FA, Amaniampong PN, Boakye P,
Addo A (2020) Modeling, optimization and kinetic anal-
ysis of the hydrolysis process of waste cocoa pod husk to
reducing sugars. SN Appl Sci. https://doi.org/10.1007/
s42452-020-2966-y
Morone A, Chakrabarti T, Pandey RA (2017) Assessment of
alkaline peroxide-assisted wet air oxidation pretreatment
for rice straw and its effect on enzymatic hydrolysis. Cel-
lulose. https://doi.org/10.1007/s10570-017-1451-2
Mota TR, Oliveira DM, Simister R, Whitehead C, Lanot A, dos
Santos WD, Rezende CA, McQueen-Mason SJ, Gomez LD
(2021) Design of experiments driven optimization of
alkaline pretreatment and saccharification for sugarcane
bagasse. Bioresour Technol 321:124499. https://doi.org/
10.1016/j.biortech.2020.124499
Ndazi BS, Karlsson S, Tesha JV, Nyahumwa CW (2007)
Chemical and physical modifications of rice husks for use
as composite panels. Compos Part A Appl Sci Manuf
38:925–935. https://doi.org/10.1016/j.compositesa.2006.
07.004
Parizzi LP, Grassi MCB, Llerena LA, Carazzolle MF, Queiroz
VL, Lunardi I, Zeidler AF, Teixeira PJ, Mieczkowski P,
Rincones J, Pereira GA (2012) The genome sequence of
Propionibacterium acidipropionici provides insights into
its biotechnological and industrial potential. BMC Genom
13:562. https://doi.org/10.1186/1471-2164-13-562
Park S, Baker JO, Himmel ME, Parilla PA, Johnson DK (2010)
Cellulose crystallinity ı́ndex: measurement technique and
their impact n interpreting cellulase performance.
Biotechnol Fuels 3:10
Piwowarek K, Lipińska E, Hać-Szymańczuk E, Rudziak A,
Kieliszek M (2019) Optimization of propionic acid pro-
duction in apple pomace extract with Propionibacterium
freudenreichii. Prep Biochem Biotechnol 49:974–986.
https://doi.org/10.1080/10826068.2019.1650376
Pua FL, Sajab MS, Chia CH, Zakaria S, Rahman IA, Salit MS
(2013) Alkaline-treated cocoa pod husk as adsorbent for
removing methylene blue from aqueous solutions. J Envi-
ron Chem Eng 1:460–465. https://doi.org/10.1016/j.jece.
2013.06.012
Qing Q, Guo Q, Zhou L, Gao X, Lu X, Zhang Y (2017) Com-
parison of alkaline and acid pretreatments for enzymatic
hydrolysis of soybean hull and soybean straw to produce
fermentable sugars. Ind Crops Prod 109:391–397. https://
doi.org/10.1016/j.indcrop.2017.08.051
Ranaei V, Pilevar Z, Khaneghah AM, Hosseini H (2020) Pro-
pionic acid: method of production, current state and per-
spectives. Food Technol. Biotechnol 58:1–30. https://doi.
org/10.17113/FTB.58.02.20.6356
Rashid T, Kait CF, Murugesan T (2016) A ‘‘fourier transformed
infrared’’ compound study of lignin recovered from a for-
mic acid process. Procedia Eng 148:1312–1319. https://
doi.org/10.1016/j.proeng.2016.06.547
123
4024
Santana NB, Teixeira Dias JC, Rezende RP, Franco M, Silva
Oliveira LK, Souza LO (2018) Production of xylitol and
bio-detoxification of cocoa pod husk hemicellulose
hydrolysate by Candida boidinii XM02G. PLoS ONE
13:1–15. https://doi.org/10.1371/journal.pone.0195206
Schaechter M, Lederberg J (2004) The desk encyclopedia of
microbiology, 2nd edn. Elsevier Academic Press,
Netherlands
Segal L, Creely JJ, Martin EA, Conrad CM (1959) Empirical
method for estimating the degree of crystallinity of native
cellulose using the X-Ray diffractometer. Text. Res. J
29:786–794
Shet VB, Sanil N, Bhat M, Naik M, Mascarenhas LN, Goveas
LC, Rao CV, Ujwal P, Sandesh K, Aparna A (2018) Acid
hydrolysis optimization of cocoa pod shell using response
surface methodology approach toward ethanol production.
Agric. Nat. Resour 52:581–587. https://doi.org/10.1016/j.
anres.2018.11.022
Shi S, Guan W, Blersch D, Li J (2021) Improving the enzymatic
digestibility of alkaline-pretreated lignocellulosic biomass
using polyDADMAC. Ind Crops Prod 162:113244. https://
doi.org/10.1016/j.indcrop.2021.113244
Silverstein RM, Webster FX, Kiemle DJ (2005) Spectrometric
identification of organic compounds. J Chem Educ. https://
doi.org/10.1016/0022-2860(76)87024-X
Soltani N, Bahrami A, Pech-Canul MI, González LA (2015)
Review on the physicochemical treatments of rice husk for
production of advanced materials. Chem Eng J
264:899–935. https://doi.org/10.1016/j.cej.2014.11.056
Sribala G, Chennuru R, Mahapatra S, Vinu R (2016) Effect of
alkaline ultrasonic pretreatment on crystalline morphology
and enzymatic hydrolysis of cellulose. Cellulose. https://
doi.org/10.1007/s10570-016-0893-2
Sun RC, Tomkinson J, Wang YX, Xiao B (2000) Physico-
chemical and structural characterization of hemicelluloses
from wheat straw by alkaline peroxide extraction. Polym
(Guildf) 41:2647–2656. https://doi.org/10.1016/S0032-
3861(99)00436-X
The International Cocoa Organization (2020) ICCO Quarterly
Bulletin of Cocoa Statistics, vol. XLVI, No. 4, Cocoa year
2019/20. https://www.icco.org/november-2020-quarterly-
bulletin-of-cocoa-statistics/
Tinwala F, Mohanty P, Parmar S, Patel A, Pant KK (2015)
Intermediate pyrolysis of agro-industrial biomasses in
bench-scale pyrolyser: product yields and its characteri-
zation. Bioresour Technol 188:258–264. https://doi.org/10.
1016/j.biortech.2015.02.006
Vásquez ZS, Carvalho DPD, Pereira GVM, Vandenberghe LPS,
Oliveira PZD, Tiburcio PB, Rogez HLG, Góes A, Soccol
CR (2019) Biotechnological approaches for cocoa waste
123
Cellulose (2021) 28:4009–4024
management: a review. Waste Manage 90:72–83. https://
doi.org/10.1016/j.wasman.2019.04.030
Vriesmann LC, Teófilo RF, Petkowicz CLO (2012) Extraction
and characterization of pectin from cacao pod husks
(Theobroma cacao L.) with citric acid. LWT—Food Sci
Technol 49:108–116. https://doi.org/10.1016/j.lwt.2012.
04.018
Wang Z, Yang S (2013) Propionic acid production in glycerol /
glucose co-fermentation by Propionibacterium freudenre-
ichii subsp. shermanii. Bioresour Technol 137:116–123.
https://doi.org/10.1016/j.biortech.2013.03.012
Xu H, Yu G, Mu X, Zhang C, DeRoussel P, Liu C, Li B, Wang H
(2015) Effect and characterization of sodium lignosul-
fonate on alkali pretreatment for enhancing enzymatic
saccharification of corn stover. Ind Crops Prod
76:638–646. https://doi.org/10.1016/j.indcrop.2015.07.
057
Yang H, Yan R, Chen H, Lee DH, Zheng C (2007) Character-
istics of hemicellulose, cellulose and lignin pyrolysis. Fuel
86:1781–1788. https://doi.org/10.1016/j.fuel.2006.12.013
Yang H, Wang Z, Lin M, Yang ST (2018) Propionic acid pro-
duction from soy molasses by Propionibacterium
acidipropionici: fermentation kinetics and economic anal-
ysis. Bioresour Technol 250:1–9. https://doi.org/10.1016/j.
biortech.2017.11.016
Yapo BM, Besson V, Koubala BB, Koffi KL (2013) Adding
value to cacao pod husks as a potential antioxidant-dietary
fiber source. Am. J. Food Nutr 1:38–46. https://doi.org/10.
12691/AJFN-1-3-4
Zhang A, Sun J, Wang Z, Yang S-T, Zhou H (2015) Effects of
carbon dioxide on cell growth and propionic acid produc-
tion from glycerol and glucose by propionibacterium
acidipropionici. Bioresour. Technol 175:374–381. https://
doi.org/10.1016/j.biortech.2014.10.046
Zhang Y, Wu J, Li H, Yuan T, Wang Y, Sun R (2017) Heat
treatment of industrial alkaline lignin and its potential
application as an adhesive for green wood—lignin com-
posites. ACS Sustain Chem Eng. https://doi.org/10.1021/
acssuschemeng.7b01485
Zhuge X, Li J, Dong SH, Liu L, Du G, Chen J (2015) Improved
propionic acid production with metabolically engineered
Propionibacterium jensenii by an oxidoreduction potential-
shift control strategy. Bioresour. Technol 175:606–612.
https://doi.org/10.1016/j.biortech.2014.10.038
Publisher’s Note Springer Nature remains neutral with
regard to jurisdictional claims in published maps and
institutional affiliations.