Bioresource Technology 299 (2020) 122633

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Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Review

A critical review on current strategies and trends employed for removal of T

inhibitors and toxic materials generated during biomass pretreatment
⁎
Vinod Kumara, Sudesh K. Yadava, Jitendra Kumarb, Vivek Ahluwaliab,
a
Centre of Innovative and Applied Bioprocessing, Mohali, Punjab 160 071, India
b
Institute of Pesticide Formulation Technology, Gurugram, Haryana 122 016, India

G R A P H I C A L A B S T R A C T

A R T I C LE I N FO A B S T R A C T

Keywords: The main objective of biomass pretreatment is to separate biomass components and provide easier access with
Lignocellulosic biomass ultimate aim for lignin removal, hemicellulose protection and cellulose crystallinity reduction. Effective bio-
Pretreatment conversion with least inhibitory compound production would play a considerable role in economic practicability
Fermentation of the process in order to achieve economic sustainability. In this regard, detoxification is an important condition
Inhibitors
to make biomass hydrolysate acquiescent to bioconversion; also, understanding of inhibitors effect on growth
Detoxifications
and fermentation are necessary requirements for system detoxification. A number of physical, chemical and
Physical methods
Biological methods biological methods like feedstock selection, membrane selection, neutralization, use of activated charcoal etc
Chemical methods have been recommended and developed for removal or minimizing the inhibitory compounds effect. This work
reviews various inhibitory compounds produced during pretreatment methods and their removal by various
processes.

1. Introduction aromatic and amorphous nature constituting 18–35% of plant biomass.

Lignocellulosic biomass is made of lignin and polysaccharides such
as cellulose, hemicellulose. The cellulose is a chain of monomers of d-
glucose linked to each other by linear β (1–4) linkage, while hemi-
cellulose is composed of different polymers of short chains like d-xy-
lose, l-arabinose, d-galactose, d-mannose, and d-glucose. Lignin has an
A bountiful accessibility of lignocellulosic materials boosts the inven-
tion for plentiful products and an attempt to foods, chemicals, textiles,
and biofuel sources. The researchers involved in renewable and sus-
tainable energy are continuously investigating various methods for ef-
fective pretreatment methods for effective release of sugars (Kim,
2018).

⁎
Corresponding author.
E-mail address: ahluwalia.vivek@gmail.com (V. Ahluwalia).

https://doi.org/10.1016/j.biortech.2019.122633
Received 30 October 2019; Received in revised form 12 December 2019; Accepted 15 December 2019
Available online 17 December 2019
0960-8524/ © 2019 Elsevier Ltd. All rights reserved.
V. Kumar, et al.
Lignocellulosic materials contain polysaccharides which are not
readily accessible for bioconversion and pretreatment is a necessary
stage for making cellulose more accessible to the enzymes. A good
pretreatment process includes hydrolysis process with ability to release
maximum amount of sugars, least formation of inhibitory products and
cost effectiveness. The pretreatment process is mainly performed by
four methodologies i.e. mechanical, chemical, mechanic-chemical and
biological pretreatments. In mechanical pretreatment, the lig-
nocellulosic biomass is grinded/ milled by either milling or ultrasonic
treatment. Mechanical pretreatment increases the surface/volume
ratio, making hydrolysis process more feasible. Chemical treatment
mainly involves steps where the biomass is disintegrated by chemical
reactions and is done by processes like acid hydrolysis, alkaline hy-
drolysis, organosolv treatment, oxidative delignification and ionic li-
quids etc. (Jönsson and Martín, 2016). Both chemical and mechanical
pretreatment processes have some limitations and these limitations can
be reduced to a large extent by joining the mechanical methodology
with a chemical treatment (Kamarludin et al., 2014; Kundu et al.,
2018).
The ionic liquids have also been considered as evolving solvent
technique for pretreatment processes. There are two different methods
for using ionic liquids. Using ionic liquids in pretreatment process
earlier to enzymatic hydrolysis is first method, while in second method
simultaneous pretreatment and saccharification of biomass in ionic li-
quids is performed. This technique is considered as “green”, but opti-
mizing solvent cost for cost effective process is still a challenge
(Elgharbawy et al., 2016; George et al., 2015). Recently, one pot con-
version of lignocellulose to levulinic acid was studied with good se-
lectivity under hydrothermal conditions and results established that
very good yield was obtained based on the quantity of C6-sugars (Liu
et al., 2018). Among various methods of pretreatment, dilute ammonia
pretreatment is also considered good because of its effectiveness in
removing lignin and enhancing enzymatic digestibility. But, harsh
conditions promote formation of inhibitors which restrict downstream
process. Recently, a study was conducted to investigate the effective-
ness of flocculation treatment in the detoxification process produced
during dilute ammonia pretreatment of energy cane bagasse. The re-
sults of the study endorsed that polyelectrolyte flocculants have the
potential to eliminate compounds like organic acids, phenolic com-
pounds, and furan derivatives with much reduced sugar losses. Floc-
culation is a method used for improvement of solid-liquid separations
and inorganic salts, organic polyelectrolytes can be used as flocculants
(Deng and Aita, 2018).
To increase cellulose hydrolysis, it is desirable to remove maximum
lignin in the pretreatment step. While the idea behind acid catalyzed
pretreatments is to remove hemicellulose to the maximum content,
alkali based pretreatments relies on solubilizing lignin from biomass
keeping cellulose and most of the hemicellulose as solid part. Steam
explosion is a physico-chemical method used for the pretreatment
process which is cost effective and require lower energy. The steam
produced during this process expands polysaccharides of the cell wall,
thus increasing the effective accessibility of the enzyme. Parameters
like biomass source, particulate dimension, temperature and moisture
content plays an important role in this process.
In biological pretreatment, different microbial organism are used for
degradation of lignocellulosic biomass. Pretreatment process by biolo-
gical means is considered promising and safe, being an eco-friendly
route. The biological pretreatment is carried out in very mild condi-
tions, so there is production of very less inhibitory products (Sindhu
et al., 2016). Microbes are grown directly into biomass for microbial-
based pretreatment and pilot scale biological pretreatment require a
longer incubation period as compared to conventional pretreatment
strategies (Kim, 2018).
Formation of inhibitors like phenolic compounds and other aro-
matics like furan, aldehydes and bioalcohols etc. during pretreatment is
a limiting factor in production of valuable chemicals from biomass.
2
Bioresource Technology 299 (2020) 122633
Inhibitors negatively affect yield by creating unfriendly environments
for fermentative microbes, increasing the length of lag phase, causing
loss of cell density and lowering growth rates of fermenting microbes
(Zabed et al., 2019).
2. Inhibitors production
2.1. Cellulose based inhibitors and their effects
Physical and chemical pretreatment have shown promise in en-
hancing biomass digestibility. Inhibitors’ productions from cellulose
residue depend on various pretreatment techniques used. The main
objective of pretreatment step is to eliminate the lignin and hemi-
cellulosic content from lignocellulosic biomass and to keep the cellulose
fibers intact. Still cellulose depending on pretreatment method em-
ployed degrades to hexoses to different extent. Different acids used in
pretreatment process disrupts its internal structure by breaking intra
and inter hydrogen bonds along with glycosidic bonds paying way for
its degradation into sugars. Hexoses formed from degradation of cel-
lulose are dehydrated to 5-hydroxymethyl furfural. Chemically, 5-HMF
is composed of a furan ring attached with aldehydic group. As furan
ring is more stable comparative to aldehydic group, the chemical
transformations occurs at aldehydic group and HMF is further dehy-
drated to form levulinic acid and formic acid. 5-HMF is known to be
toxic to the growth of ethanolegens. Inhibitory effect of HMF has been
addressed by several studies (Kumar et al., 2018a; Zabed et al., 2019).
2.2. Hemicellulose and its degradation products
Hemicellulose is the second most plentiful polymer found in lig-
nocellulosic biomass made up of different components like pentoses,
hexoses and sugar acids. In hardwood and agricultural plants dominant
component is xylan, while galactoglucomannan is major portion for
softwood. The cellulose can be made handier to microbes and enzymes
treatment in subsequent steps by solubilizing hemicellulose. For this
step, biomass is pretreated prior to its incorporation. One of the effi-
cient ways to improve hemicellulose solubility is acid treatment.
Among various acid used for pretreatment, hydrochloric acid, phos-
phoric acid, nitric acid, and sulphuric acid are most commonly em-
ployed (Das et al., 2015; Kumar et al., 2018b). The hemicellulose after
pretreatment degrades into inhibitor products like sugars, sugar acids,
aliphatic acids, and furan aldehydes. Among all inhibitors, furfural is
the most abundant and potent inhibitor. Dehydration of acetyl group in
hemicellulose leads to the formation of acetic acid in all hydrolyzates
from all lignocellulosic biomass without any exception. Since its for-
mation cannot be prevented, biomass needs to be detoxified or neu-
tralized before further processing. Formic acid, acrylic acid and levu-
linic acid are other carboxylic acids found in hemicellulose
hydrolyzates (Wang et al., 2018a; Zabed et al., 2019).
2.3. Lignin and its degradation products
After cellulose and hemicellulose, lignin is the third most abundant
polymer and the most abundant non polysaccharide found in lig-
nocellulosic biomass. Softwood possess highest content of lignin while
lowest content being in herbaceous plants. The lignin is made up of
different phenylpropane units that are bonded by different linkages.
After breakdown, monolignols contribute to p-hydroxyphenyl (H),
guaiacyl (G), and syringyl (S) units. The H/G/S ratio of lignin and type
of pretreatment are two factors determining formation of the type of
aromatic by-products. Limited breakdown of lignin at the p-hydro-
xyphenyl, guaiacyl and syringyl residue leads to formation of 4-hy-
droxybenzoic acid, vanillin and syringaldehyde respectively. Studies
have concluded that 4-hydroxybenzoic acid, 4-hydroxybenzaldehyde,
vanillin, dihydroconiferyl alcohol, coniferyl aldehyde, syringaldehyde
and syringic acid as most common phenols formed from lignin
V. Kumar, et al. Bioresource Technology 299 (2020) 122633

degradation. The molecular weight of phenols show inverse relation-
ship with subsequent toxicity on yeast. Several studies have acknowl-
edged that the phenols are more toxic compounds than furans and
carboxylic acids as phenols being low molecular weight compounds are
able to penetrate cell membrane easily (Kumar et al., 2018b).
3. Removal methods of inhibitors
The occurrence of inhibitory compounds and simultaneously re-
covery of sugar at low concentration are main challenges in efficient
bioconversion of feedstock. Efficient and effective methods are re-
quirement of the day for concurrently separating inhibitors and con-
centrated sugars. Inhibitors removal from hydrolysate is an essential
step before performing microbial fermentation. The majority of pre-
treatment techniques result in the development/production of in-
hibitory by-products (Cunha et al., 2019; Zhang et al., 2016). When
hydrolysed with different types of acids, the feedstock produces dif-
ferent inhibitors and sugars depending on severity of the process used.
The microbial growth is affected by presence of inhibitors in reaction
process, which also impact fermentation process. Phenolics are con-
sidered as one of the major inhibitors released upon the lignin break-
down leading to loss in unity of biological membranes (Jung and Kim,
2017). Literature reports showed that phenolics compounds having
higher carbonyl group showed toughest inhibitory outcome on cellulase
hydrolysis and exo-cellulase activity (Li et al., 2017; Rajan and Carrier,
2016; Zhai et al., 2018). Lignin also plays an important part in enzymes
hydrolysis and different lignin follows different mechanism. Organosolv
lignin adsorbs cellulose and decrease accessibility of enzymes leading to
reduced sugar return while kraft lignin precipitate on exterior of cel-
lulose and obstruct its interaction with the enzymes (Li et al., 2018).
Other than phenolics by products, furfural is also reported as one of key
inhibitor in acid-pretreated lignocellulosic substrates, while in alkaline-
pretreated lignocellulosic materials these are formic acid, coumaric
acid, and acetic acid (van der Pol et al., 2016). Inhibition problem is
one of the major trouble in fermentation process and it can be reduced
by detoxification or conditioning of lignocellulosic hydrolysates and
slurries (Tables 1 and 2). There are reviews where different strategies
have been described to reduce the effect of inhibitors (Jönsson et al.,
2013; Jönsson and Martín, 2016; Ko et al., 2015). At industrial scale,
three processes are mostly preferred, i.e. kraft process (using alkaline
solution); organo-solv processes (using organic solvents) and hydrolysis
(using dilute inorganic acids) (Zhang et al., 2016). There can be dif-
ferent strategies to effectively reduce inhibitory effect of various com-
pounds on fermentation process by microbes. These strategies can be
physicochemical detoxification of hydrolysate, microbial engineering
and plant improvements for reduced lignin content (Table 2).
3.1. Physical methods
3.1.1. Feedstock selection
Feedstock selection plays a pivotal role in efficient bioconversion of
lignocellulisic biomass to value added chemicals. The low resistant
feedstock where pretreatment can be achieved with mild conditions
will be commercially more viable. Miscanthus grass and wheat straw
are examples where acid catalysts was not used in pretreatment and
very low concentration of furan aldehyde and phenols were reported
(Chiaramonti et al., 2012; Larsen et al., 2012). Varieties with low re-
calcitrant like Populus trichocarpa for bioconversion using a sugar
platform theory can be of attention (Studer et al., 2011).
3.1.2. Membrane separation
Membrane separation technology has some favourable properties
like less energy consumption, extensive flexibility of operation and
maintainable processing which make it more suitable for use in various
biorefinery fields (Wei et al., 2014). Membrane separation technique
includes processes like microfiltration (MF), ultrafiltration (UF), nano-
filtration (NF) and reverse osmosis (RO) (Fig. 1). Due to unique cap-
ability of separation and purification, these techniques are also used in
hydrolysates detoxification and sugar concentration. Usually inhibitors
have small molecular weight and allowed to pass through membrane
while molecules with high molecular weight like sugars are not allowed
to pass through membrane and retained in the retentate portion (Pan
et al., 2019; Wang et al., 2018b).
Study was conducted to access the influences of pH, pressure,
temperature and feed concentration on solute retention along with low
concentration of acetic acid on fermentation process. Comparative
study was also conducted to access the capability of NF and RO mem-
brane for simultaneous separation of acetic acid and sugars and it was
suggested that RO membrane has better results (Zhou et al., 2013a;
Zhou et al., 2013b). The nanofiltration under diafiltration mode was
used for elimination of inhibitory compounds from diluted acid hy-
drolysate of olive pomace substrate to increase its xylose fermentability.
NF270 and NF90 membranes were also explored by the authors for
detoxification process. NF270 showed lowest toxic compounds rejec-
tion and highest permeate reflux, so this was used in the diananofil-
tration, also it was reported that membrane can be used for effective
removal of acetic acid, formic acid and furfural (Brás et al., 2014).
The separation of phenolic acids from monosaccharides was skill-
fully done by a single NF at alkaline pH or by an combined enzymatic
membrane reactor (EMR) at acidic pH, where the monosaccharides
effortlessly cross the NF membranes at a low working pressure. The
retaining capability of phenolic acids by NF improved at pH 9.55 for
NTR7450 (86–88%) and NF270 (90–94%) and proved that charge re-
pulsion, hydrophobic adsorption and size exclusion were principal

Table 1
Summary of detoxification process for lignocellulose hydrolysates.
S.No. Biomass residue Detoxification methods Removed of inhibitors Products References

1 Alamo-I switchgrass
(Panicum virgatumis)
2 Olive-tree pruning
3 Biomass hydrolysate
4 Cassava stem
5 Sugarcane bagasse
6 Cocoa pod husk
7 Hollow-structured porous aromatic
8 Sugar cane Bagasse
Activated carbon
NaBH4
Microporous hyper-cross-linked
adsorption resin (SY-01)
Overliming and activated carbon
Methyl-isobutyl-ketone (MIBK)
(1) Activated charcoal
(2) Ion exchange resins
polymer
(1) Adsorption resin CX-6
(2) Overliming
(3) Charcoal
(1) HMF & Furfural (50–60%) Acetone, butanol and ethanol Wang et al., 2019
(2) Total phenolic compounds (TPC) (50%) Fermentation
(1) Furans (2) Phenolic compounds (40%) Ethanol Peinado et al.,
2019
(1) Formic Acid (2) 5- Levulinic acid (LA) Zheng et al., 2018
Hydroxymethylfurfural
Phenolic contents such as Furfural Bioethanol Tanaka et al., 2019
(1) Global acetic acid (85.4%) Ethanol Roque et al., 2019
(2) Phenolics (69.0%)
(1) Total phenolic compounds (2) Furfural Xylitol Santana et al.,
(3) HMF 2018
5-Hydroxymethylfurfural Zhang et al., 2019
Acid soluble lignin (ASL) Lipid Chen et al., 2019

3
V. Kumar, et al. Bioresource Technology 299 (2020) 122633

Kumar et al., 2018c; Kumar et al., 2019;

Anburajan et al., 2019; Shah et al., 2019

Al-Shorgani et al., 2019; Vallejos et al.,

Hong et al., 2019; Li et al., 2019; Mun,

Sankaran et al., 2019; Yan et al., 2016

Deshavath et al., 2019; Nguyen et al.,

Kadier et al., 2016; Pant et al., 2016;

Zeng et al., 2017; Zhao et al., 2016

Oliva-Taravilla et al., 2016

Pooja and Padmaja, 2015
Nguyen et al., 2015

Nguyen et al., 2019

Roque et al., 2019

Reference

2016

2019

2019
Precipitation of phenolics and large molecular weight inhibitors

Utilization of the hydrolysate as the substrate without

Level reduction of furfural, 5-HMF , vanillic acid, 4-
hydroxybenzaldehyde and 4-hydroxyacetophenone.
Organic acids and phenolics compounds removal

Fig. 1. Overview of separation of sugars from acid hydrolyzed through mem-

Removal of salt, acid and phenolic contents

brane process.
Productivity and purity enhancement

reasons for phenolic acids retention by different NF membranes (Luo

Molecular weight based separatin

et al., 2015). In another study, ten nanofiltration (NF) and reverse os-
Removal of phenolic contents

Removal of phenolic content

Enzymatic saccharification

mosis (RO) membranes with low molecular weight cut-off were eval-
Phase separation for sugar

uated for the detoxification process and it was concluded that NF

membranes were more suitable for detoxification process (Nguyen
et al., 2015).
detoxification.

Recently, researchers studied an effective filtration method where

inhibitors separation and sugar concentration processes were simulta-
Effect

neously performed with the nanofiltration (NF) and reverse osmosis

(RO) membranes by lignocellulosic hydrolysate in batch recycling
method. In this study, other factors like pH and anionic polymer con-
Ethyl acetate, Supercritical fluid extraction, Trialkylamine, 1-butanol,

Recombinant Escherichia coli BL21 (DE3), hybrid immobilized beads.

centration were also studied and it was found that bidding with sodium
tripolyphosphate during NF/RO enhance the inhibitor separation. Also,
the inhibitory separation was found much significant in case of formic
DOWEX MONOSPHERE™ 99/310 resin ionized with K+

NaOH, CaCO3, Ba(OH)2, CaOH2, Mg(OH)2, KOH, CaSO4

acid and acetic acid (Pan et al., 2019).

Microfiltration, Ultrafiltration and Nanofiltration
Activated Charcoal, Zeolite, Bentonite, diatomite

3.1.3. Ion-exchange resins

H+ resin and OH– resin, polyethyleneimine

Researchers are working on development of different methods to

isobutyl acetate and methyl-isobutyl-ketone

decrease effects of the inhibitory compounds. Different methods and

parameters have been studied and researchers are still developing dif-
Detoxification of lignocellulose hydrolysates and slurries using different techniques.

ferent methods/parameters for effective hydrolysate detoxification

Acetonitrile, Isopropanol, water,

Microbial electrochemical cells

procedures. Use of ion-exchange resins for removal of inhibitory com-

pounds are one of most efficient and economical methods, as they can
be regenerated and reused (De Carvalho et al., 2004). The chemical
effects of detoxification approaches on the hydrolysates were assessed
Laccase enzymes

by anion-exchange resins and found to affect phenolics, furan aldehydes

and aliphatic acids. Anion exchange resins were also affecting fer-
Procedure

mentable sugars concentration in the hydrolysates (Nilvebrant et al.,

2001). The effect of XAD-4 (non-polar hydrophobic adsorbent) treat-
ment was evaluated for removal of fermentation inhibitors like HMF,
Simulated moving bed separations

furfural, vanillin etc. and found effective. Study showed inhibitors re-
moval was more than 99% while sugars like xylose, glucose and ara-
Sugaring - out extraction and

binose were retained in good amount (Hatano et al., 2013). The weakly
Microbial fuel cell (MFC)
Liquid–liquid extraction

basic resin Amberlite™ IRA-67 has tertiary amine functional groups and
Salting-out extraction

Enzymatic treatment
Membrane filtration

Microbial treatment
Ion-exchange resins

due to the acid/base neutralization reaction it show good adsorption

ability. It efficiently removed lactic acid in situ for sustained frame-up of
Neutralization

lactic acid fermentation with a mixed culture fermenting corn stover

Adsorption
Technique

sugars. The effect of resin bed reuse was evaluated for Amberlite™ IRA-
67 resin and no substantial effect was found on lactic acid loading, but
change was observed for acetic acid loading (Garrett et al., 2015). The
resins like IRA-400 (Cl−), IRA-400 (OH−) and IRA-958 (Cl−) are strong
Chemical methods

Biological method
Physical Methods

anion-exchange resins containing tertiary amine functional groups; also

IRA-958 (Cl−) is with polyacrylate/DVB matrix that confers it a polar
Methods

and hydrophilic nature. These resins along with XAD-4 were evaluated
Table 2

for removal of acid soluble lignin from poplar hydrolysate. IRA-400

(OH−) was found with highest capacity for ASL adsorption and removal

4
V. Kumar, et al.
of inhibitors like phenolic and HMF (Yu and Christopher, 2017). The
resin was also evaluated for removal of nitrate salts, phenolic com-
pounds and 5-HMF and study showed that it effectively removed in-
hibitors (Kumar et al., 2018c; Kumar et al., 2019).
3.1.4. Simulated moving bed separations:
SMB (Simulated moving bed) is a sophisticated continuous chro-
matography and widely used in separation of petroleum chemicals,
sugar industry and pharmaceutical industry. Recently, this technique
was used for the separation of sugars and sulfuric acid. Different resins
like Dowex 1X4, Dowex 1X8 and Diaion MA03SS were evaluated for
this purpose and it was proposed that results of Dowex 1X8 and Diaion
MA03SS were almost similar. With Dowex 1X4 recoveries for sulfuric
acid (95.0–95.5%) glucose (100–101%) and xylose (86.1–91.7%) were
more (Sun et al., 2016). Similarly, this technique was evaluated for
competent retrieval of xylobiose from xylooligosaccharides (Choi et al.,
2016). A recent study showed that Amberchrom-CG71C resin is good
adsorbent for separation of fumaric acid and acetic acid with recovery
of fumaric acid in high yield and purity through SMB chromatographic
process (Choi et al., 2017).
3.1.5. Activated charcoal:
Use of activated charcoal is a well-established and low-cost detox-
ification method since centuries. Literature has many reports where
activated charcoal has been used for different purification processes.
Inhibitors like furan and phenolic content were successfully removed
using activated charcoal. Authors suggested that inhibitors removal is
related to component hydrophobicity in the simulated hydrolysates and
strong hydrophobic nature of activated charcoal (Lee and Park, 2016).
Increased xylitol yield was obtained after detoxification of sago trunk
hydrolysate with activated charcoal. Also, there was reported reduction
of 53% and 78% of furfural and phenolic compounds respectively after
detoxification process. This study proposed that detoxification method
using activated charcoal has a significant impact on xylitol production
(Kamal et al., 2011). Recently, a study showed that charcoal can be
used as cost effective detoxification agent for the corn cob acid hy-
drolyzed with least loss of xylose content at 1% (Kumar et al., 2019). In
another study charcoal was used to remove 5-hydroxymethylfurfural
from waste hand paper hydrolysate. The authors evaluated different
parameters like concentration, temperature, and agitation speed for this
study and found that activated carbon can be a good detoxifying agent
(Onaran et al., 2019).
3.2. Chemical methods
3.2.1. Neutralization
Before pentose sugars are purified, the inorganic acids are neu-
tralized (partially or completely) in industrial methods. The main aim is
to precipitate lignins, proteins and finally metal residues by using
caustic soda or lime. The purification process involves ion-exchange
chromatography, adsorption, chromatography or crystallization
(Heikkila and Hyoky, 1992; Heikkilä et al., 2001; Heikkila et al., 2015).
Acid hydrolysate process produces sulphuric acid which is generally
neutralized with calcium hydroxide or calcium carbonate. Process
produces insoluble calcium sulphate (gypsum) which is utilized in
board manufacturing, cement, and medicine industries (Seong et al.,
2016; Yoon et al., 2014). Researchers also worked on neutralization of
acid hydrolysates using different types of base like calcium hydroxide,
potassium hydroxide or sodium hydroxide and suggested that among
studied, potassium hydroxide is a good neutralizing agent compare to
other with high yield of sugar (Seong et al., 2016). Working on a similar
type of hypothesis, Deshavath and his team used sorghum biomass as
model substrate for the making of xylulosic ethanol. The biomass was
treated with sulphuric acid and then hydrolysate was neutralized with
alkaline agents like calcium hydroxide (Ca(OH)2), potassium hydroxide
(KOH), magnesium hydroxide (Mg(OH)2), sodium hydroxide (NaOH)
5
Bioresource Technology 299 (2020) 122633
and ammonia (NH3). Study found that Ca(OH)2 was best neutralizing
agent among the studied (Deshavath et al., 2019).
3.2.2. Liquid–liquid extraction
Liquid–liquid extraction (LLE) is an important separation method
used to separate two miscible compounds using a solvent that specially
dissolves one of them with properties like low mutual solubility, fast
demixing and low toxicity and used in different chemical industries,
mining industries and downstream recovery of fermentation products.
The choice of solvent in this extraction process is mostly based on its
polarity as generally, polar solvents extract polar analytes better than
apolar or nonpolar analytes. This technique was effectively used in
parting and refining xylitol using ethyl acetate with reported yield of
33.26% (Misra et al., 2011; Mun et al., 2016). N-butanol had also been
reported as efficient solvent for the inhibitor removals (Sukhbaatar
et al., 2014). Recently, solvent like ethyl acetate (EtOAc), tri-n-octyl-
phosphine oxide (TOPO), tri-n-octylamine (TOA), and tri-n-alkylpho-
sphine oxide (TAPO) were evaluated to regain acetic acid from a syn-
thetic ethanol fermentation broth with finding that TAPO was best
solvent (Pham et al., 2015).
3.2.3. Sugaring – out extraction
During fermentation process, microbes produces bio-based chemi-
cals like n-butanol, 1,3-propanediol, 2,3-butanediol, lactic acid, suc-
cinic acid etc. from sugars. Separation of these compounds from fer-
mentation is an important purification step. Sugaring-out extraction
(SOE) is a phase separation method used firstly for separation of acet-
onitrile and sugar. Until now, this method has been used in different
systems acetonitrile, metal ions, biomolecules, antibiotics and drugs
(Dai et al., 2015; Dai et al., 2017).
Lactic acid is broadly used in innumerable kinds of industries like
food, non-food, cosmetic, pharmaceutical industries etc. Recently, this
chemical has received attention because of its use in manufacture of
biodegradable poly-lactic acid (PLA). Separation and purification are
two most important steps in economical production of any chemical
including lactic acid. In the traditional methods, sulfuric acid is con-
sumed to precipitate lactic acid as calcium lactate. In this process large
amount of calcium sulfate is also produced as a by-product. Recently,
sugaring-out extraction (SOE) method was evaluated including influ-
ence of different sugars and organic solvents on the partition and ab-
straction of lactic acid from the fermentation broth. In this investiga-
tion, acetone, isopropanol, n-propanol, tert-butanol, isobutanol, n-
butanol, n-pentanol, ethyl acetate and butyl acetate were used as or-
ganic solvents. Choice of sugars also plays a pivotal role in sugaring out
extraction experiments. The xylose, glucose, fructose and saccharose
were used as sugars in this investigation. Experiments showed that
isopropanol and glucose were best solvent systems for the recovery of
the lactic acid from fermentation broth and yield of lactic acid was
reported at 84.27% (Yan et al., 2016).
3.2.4. Salting-out extraction
The fermentation broth contains a large amount of impurities, thus
considered as bottleneck in industrial production for bio-based chemi-
cals. Removal of impurities is important step in direction of production
for biomass based chemicals. And salting-out extraction process is im-
portant step in direction as this process is built on the compounds
partition between two phases which can be formed of salts and poly-
mers or hydrophilic solvents, hydrophobic solvents, and amphipathic
chemicals. Low cost, easy retrieval of phase-forming components, short
phase separation time, easy scale-up and option of continuous opera-
tion are advantages of this separation process. Recently a review was
published with keeping focus on separation of 1,3-propanediol, 2,3-
butanediol, acetoin, and lactic acid. The review discussed separation of
bio-based chemicals from fermentation broths by salting-out extraction
process (Dai et al., 2014). Study was conducted to develop economic-
ally viable technology for retrieval of different carboxylic acids with
V. Kumar, et al.
aim to study partition behavior of carboxylic acids in the ethanol/am-
monium sulfate. The different parameters like tie line length, phase
volume ratio, temperature, system pH and concentration of acids were
also studies (Fu et al., 2015). In a similar type of study, Fu et al. found
that ammonium sulfate, monosodium phosphate and calcium chloride
were best salts among for separation of butyric acid while employing
salting-out extraction (Fu et al., 2017).
3.3. Biological methods
Biofuel industry is a growing industry and complex and costly
physical and chemical detoxification methods are used by the industry.
The use of microorganisms has been explored as efficient and en-
vironment friendly method to succeed in dealing with toxicity of the
hemicellulosic hydrolysates or inhibitory compounds. Biological de-
toxification of different hemicellulosic hydrolysates like sugarcane ba-
gasse, sugarcane straw, coffee husk, and corn fiber using laccases are
gaining attention as detoxification enzymes (De La Torre et al., 2017).
Laccase eliminates phenols from the slurry, thus making use of whole
slurry possible but its use has limitations also. Some studies showed that
its use affect the glucose recovery. Hybrid hydrolysis and fermentation
can be an option to overcome adverse effects of laccase treatment
(Oliva-Taravilla et al., 2016). The Bordetella sp. BTIITR, a bacterium
was isolated from soil that was able to degrade furfural (100%), HMF
(94%) and acetic acid (82%) from sugarcane bagasse hydrolysate liquor
(Singh et al., 2017).
Also, plant genetic engineering to change the composition and ratio
of Lignin syringyl/guaiacyl (S/G) ratio exploration is also a curious
methodology. The change in ratio of lignin S/G can lead to decrease in
the recalcitrance of syringyl-rich lignin, leading to easy hydrolysis and
fewer inhibitor production (Fu et al., 2011). Recently, researchers used
genetically modified sorghum traits to achieve maximum hemicellulose
hydrolysis with low energy input. The chemical composition of ge-
netically modified sorghum showed 11.6–17.7% of lignin content and
pretreatment process produced low level of sugar decomposition pro-
ducts (Deshavath et al., 2018). These approaches are of attentiveness
primarily with respect to short-rotation crops devoted to biorefining -
through a sugar platform route (Table 2).
3.3.1. Membrane-assisted cell retention
The capacity of the membrane to resourcefully separate two or more
than two compounds and/or cells/particles is basis for this technique.
The membranes have a unique skill of selecting permeate and retentate.
The compounds that pass through membrane are called as permeate
while those retained are termed as retentate. This selective behavior is
because of membrane pore size, morphology, membrane charge, affi-
nity or hydrophobicity. Immobilization technique for cell retention can
be used to escalate volumetric production of biological processes as it
carries benefit of easier cell reuse at high biomass concentration. Cell
encapsulation methodology can be a solution to the inhibitor tolerance
problem as it differs from bead immobilization (Westman et al., 2014).
As a technique, it also carries some drawbacks. Major drawbacks car-
ried by this technique are time consuming, laborious, flaw in capsule
preparation and agitation which can lead to capsule disintegration and
rupture (Ishola et al., 2015a; Ishola et al., 2015b; Ylitervo et al., 2011).
4. Future perspectives
In the future, the market is expected for sharp growth for lig-
nocellulosic derived products. The promising products like ethanol,
xylitol, organic acids and 2,3-BD may play a pivotal role in this direc-
tion. Technologies for enhanced yield of different value-added products
need to be developed from hemicellulose fraction. Cellulosic derived
hexose sugars fermentation is an easy step as compared to pentose
sugars and there is lack of established bioconversion process for pentose
sugars. New processes need to be developed for hemicellulosic
6
Bioresource Technology 299 (2020) 122633
degradation including study for limiting the role of fermentation in-
hibitors. In recent years, literature has reports related to hemicellulose
hydrolysis using different methodologies with different microorgan-
isms, but many reports have used same microorganisms. Despite pro-
gress in hemicellulose hydrolysate, different and newer approach needs
to be developed for recovery of highly purified sugars monomers, along
with inhibitor removal and eventually converting them to value-added
products. Microorganism screening and isolation for hemicellulose
fermentation need to be stressed for better process development for the
production and yields of the hemicellulose derived products in the
market.
5. Conclusion
The present review describes the inhibitor effects, effect of detox-
ification and effective removal of the inhibitors in pretreatment
methods. The suitable choice of the pretreatment processes and in-
hibitor removal is a challenging step which ultimately effect final yield
of the end product. The inhibitory compounds produced during pre-
treatment are noxious to fermenting micro-organisms. Inhibitor pro-
duction and their effect on enzyme and microbial activities still need
further studies. The detoxification process with enzymes and microbes
is action specific and the efficacy is comparable to other physical and
chemical methods. The microbes or micro-organisms can be explored
for adaption to acid pre-treated lignocellulosic hydrolysates and there
are some literature reports for the same. Genetic engineering is also an
emerging approach in this field where tolerance can be increased
against specific inhibitor.
CRediT authorship contribution statement
Vinod Kumar: Resources. Sudesh K. Yadav: Conceptualization.
Jitendra Kumar: Supervision. Vivek Ahluwalia: Writing - original
draft, Writing - review & editing.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledgments
Authors are thankful to Director, Institute of Pesticide Formulation
Technology, Gurugram and CEO, CIAB, Mohali for continuous support
and encouragement.
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