Int. J. Biol. Sci. 2013, Vol.

9 598

Ivyspring
International Publisher
International Journal of Biological Sciences
2013; 9(6):598-612. doi: 10.7150/ijbs.6091
Review

Molecular Adaptation Mechanisms Employed by

Ethanologenic Bacteria in Response to Lignocellulose-
derived Inhibitory Compounds
Omodele Ibraheem and Bongani K. Ndimba
Research and Services Unit, Agricultural Research Council/Infruitech & The University of the Western Cape, Biotechnology Department,
Private Bag X17, Bellville, Cape Town, South Africa.

 Corresponding author: Professor Bongani K. Ndimba. Proteomics Research and Services Unit, Agricultural Research Council/Infruitech
& The University of the Western Cape, Biotechnology Department, Private Bag X17, Bellville, Cape Town, South Africa. Email:
[email protected], [email protected] Fax: +27 [0]21 959 1551.

© Ivyspring International Publisher. This is an open-access article distributed under the terms of the Creative Commons License (http://creativecommons.org/
licenses/by-nc-nd/3.0/). Reproduction is permitted for personal, noncommercial use, provided that the article is in whole, unmodified, and properly cited.

Received: 2013.02.18; Accepted: 2013.04.26; Published: 2013.06.28

Abstract
Current international interest in finding alternative sources of energy to the diminishing supplies of
fossil fuels has encouraged research efforts in improving biofuel production technologies. In
countries which lack sufficient food, the use of sustainable lignocellulosic feedstocks, for the
production of bioethanol, is an attractive option. In the pre-treatment of lignocellulosic feedstocks
for ethanol production, various chemicals and/or enzymatic processes are employed. These
methods generally result in a range of fermentable sugars, which are subjected to microbial fer-
mentation and distillation to produce bioethanol. However, these methods also produce com-
pounds that are inhibitory to the microbial fermentation process. These compounds include
products of sugar dehydration and lignin depolymerisation, such as organic acids, derivatised fur-
aldehydes and phenolic acids. These compounds are known to have a severe negative impact on
the ethanologenic microorganisms involved in the fermentation process by compromising the
integrity of their cell membranes, inhibiting essential enzymes and negatively interact with their
DNA/RNA. It is therefore important to understand the molecular mechanisms of these inhibitions,
and the mechanisms by which these microorganisms show increased adaptation to such inhibitors.
Presented here is a concise overview of the molecular adaptation mechanisms of ethanologenic
bacteria in response to lignocellulose-derived inhibitory compounds. These include general stress
response and tolerance mechanisms, which are typically those that maintain intracellular pH
homeostasis and cell membrane integrity, activation/regulation of global stress responses and in-
hibitor substrate-specific degradation pathways. We anticipate that understanding these adapta-
tion responses will be essential in the design of ‘intelligent’ metabolic engineering strategies for the
generation of hyper-tolerant fermentation bacteria strains.
Key words: Fermentation, Bioethanol, Lignocellulosic Inhibitors, Lignocellulolytic materials, Stress
Response, Microbial Physiology, Phenolics.

Introduction
Rapid world industrialization has resulted in an house gas emissions and global warming, pose severe
overburdening demand for refined fossil fuels. This socio-economic challenges [1]. There is thus an urgent
demand coupled with the continuous rise in cost of need for the development of environmentally sus-
refined fossil fuels, their high contribution to green- tainable and affordable energy sources. One such

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Int. J. Biol. Sci. 2013, Vol. 9 599

promising environmentally friendly, affordable and believed to be an attractive, affordable and sustaina-
sustainable alternative is bioethanol. One major ad- ble alternative.
vantage of bioethanol is that it results in far lower 2nd generation bioethanol production involves
toxic gas emissions in comparison to fossil fuels such several sub-processes, including a pre-treatment
as gasoline, diesel and kerosene [2]. With environ- phase, where cellulosic substrates are extracted from
mental protection laws in place in many countries for the raw lignocellulosic products, followed by cellu-
the implementation of bioethanol as an additive to lose hydrolysis or hydrolysis into fermentable pentose
fuel, the demand for bioethanol is rapidly increasing. and hexose sugars. Subsequently these sugars are
Currently, ethanol blended with gasoline (e.g. gashol fermented by ethanologenic microorganisms includ-
E5-E10 and gashol E80-85 containing 5-10% and ing bacteria, yeasts and fungi (Figure 1). Ethanolo-
15-20% ethanol, respectively) is marketed in many genic bacteria are of particular relevance as they have
developed economies including the USA and several higher growth rate than fungi, which allow them to
European countries [3]. produce more fermentative enzyme, and they can
Presently, most technologies for bioethanol utilize both pentose and hexose sugars (few exception
production make use of sugar cane juice and corn such as Zymomonas mobilis), while fungi rarely use
starch (1st generation). Around 10-15 billion gallons pentoses [6-11]. An important advantage of some
are currently produced per year, far short of the pro- bacterial strain, such as Clostridium thermocellum, is the
jected 60 billion gallons that would be required by the ability to ferment cellulose directly to ethanol [12, 13,
world economy [4]. Furthermore, 1st generation bio- 14]. This ability opens an opportunity to use them in
ethanol production places an extensive demand on the consolidating bioprocessing of biomass to pro-
the global food market for which these carbon sub- duce ethanol by 1 step (i.e without the need of break-
strates are destined, and the production costs may be ing down the cellulose into its components). The al-
as high as 40% of revenue derived from the bioethanol cohol by-products of microbial fermentation are sub-
[5]. 2nd generation bioethanol produced from inex- sequently distilled and dehydrated to produce an
pensive, renewable substrates such as lignocellulose is approximate 99.5% ethanol [15, 16, 17, 18].

Fig 1. A flow chart of lignocellulose biomass conversion to bioethanol. The lignocellulose biomass is degraded into its constituent sugars by various
pre-treatment methods and converted into ethanol using ethanologenic bacteria cells, which is distilled and ready for market.

There are several key challenges associated with microorganisms involved in sugar fermentation, and
the current 2nd generation bioethanol production these may include their capacity to adequately fer-
methods that need to be addressed in order to de- ment sugars into ethanol, their need for additional
velop sustainable lignocellulolytic material (LCM) nutritional requirements, their sensitivity/tolerance
bioconversion into ethanol. The key challenge is the towards ethanol and organic by-products of fermen-
development of a robust, sustainable, cost-effective, tation, as well as the high temperature and low pH
environmentally friendly and complementary alter- associated with the pre-treatment, cellulose hydroly-
native to 1st generation ethanol production and fossil sis and fermentation phases of the LCM bioconver-
fuels. These include many factors that relate to the sion process [6, 19-23]. Furthermore, one of the major

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challenges associated with the current biological ap- contributing towards improvement of LCM-based
proach for bioethanol production is that during the ethanol production.
pre-treatment and hydrolysis phases a number of
by-products are produced that could inhibit the Lignocellulose: A potential feedstock for
growth and metabolic capacity of ethanologenic mi- bioethanol production
croorganisms during the fermentation process [24-27]. Lignocellulose is an abundant natural biopoly-
A major goal of current research relating to LCM bi- mer which accounts for 50% of the world’s biomass.
oconversion is the development of effective means to An estimated 10 – 50 billion tons of lignocelluloses is
reduce or eliminate the fermentation inhibitors [28]. produced per annum [49]. It can be obtained easily
Several detoxification protocols have been proposed and inexpensively from various agricultural by
and introduced, including physical (e.g. adsorption products such as residual materials from grain crops,
with activated carbon or ion exchange resins), chem- seeds, peels and shells of fruits and vegetables, vege-
ical (e.g. lime or alkali treatment, ionic liquids; mix- table oils, industrial and municipal waste, forestry
tures of cationic and anionic salts that melt mostly residues and fast-growing energy grasses and trees [6,
below 100°C) or biological (e.g. laccase or peroxidise) 49-51]. One of the major advantages of using these
measures [26, 29-33]. However, these methods come wastes for bioethanol production, aside from their
at an additional cost and frequently introduce further renewable nature, will be the lower energy, environ-
toxic waste products [30, 34]. The screening and se- mental and economic costs associated with their dis-
lection of microorganisms which are highly tolerant posal, as they are still considered as waste products in
or resistant to fermentation inhibitors may represent a many parts of the world and often disposed of by
more sustainable and cost-effective strategy [35-38]. burning [52].
In this review, we discuss the fermentation in- Lignocellulolytic materials (LCMs) consist of
hibitors, as well as the means by which ethanologenic three main polymerized sugar components, cellulose,
bacteria may have adapted to tolerate or resist these hemicellulose and lignin (Figure 2). Cellulose ac-
lignocellulosic inhibitory compounds. Microbial fer- counts for 33 – 51%, hemicellulose for 19 – 34% and
mentation for ethanol production involves ethanolo- lignin for 21 – 32% of the dry weight of LCM, while
genic bacteria as well as fungi. Several publications proteins, oils and ash make up the remaining fraction.
covering and reviewing these topics with respect to Cellulose is the most abundant carbohydrate polymer
ethanologenic fungi are available [6-10, 16- 18, 20, 39- in nature. It is composed of linear chains of hundreds
45]. Microbial tolerance to organic solvents, other to thousands of β-1,4-linked D- glucose molecules
bio-products and chemicals from biorenewable fuels [53]. It is closely linked with proteins, lignin, hemi-
processes [46-48] were already discussed, however, celluloses and mineral elements, which makes it
not much of emphasis was laid on ethanolonogenic highly resistant to hydrolysis. However, it can be hy-
bacteria. Hence this review will focus on the effects of drolysed chemically (e.g. by acid treatment) or enzy-
and tolerance/adaptation to inhibitors in ethanolo- matically (e.g. microbial cellulases) [39, 54, 55]. Cel-
genic bacteria, for which reviews are currently lim- lulose is of high industrial value, being used in the
ited. Concise descriptions of lignocellulose as a po- production of various foods, chemicals, textiles ani-
tential source of biomass for bioethanol production mal feeds, pulp and paper [53].
and the 2nd generation bioconversion process are Hemicellulose is a branched polysaccharides
given. We discussed the inhibitory compounds gen- consisting of pentose (D-xylose and L-arabinose) and
erated during the pre-treatment and hydrolysis pro- hexose (D-glucose, D-mannose, D-galactose) sugars,
cesses, how they affect the cellular activities of etha- uronic acids (D-glucouronic, D-galactouronic) and
nologenic bacteria, as well as the mechanisms by various o-methylated sugars [56]. Its composition is
which ethanologenic bacteria may be able to with- primarily dependent on the plant source, and unlike
stand and survive in the presence of these inhibitors. cellulose it is more readily hydrolyzed into its com-
There have been a number of studies which have fo- ponent sugars [56]. However, some of these sugars
cused on the improvement of ethanol production can hamper the fermentation process as many micro-
through genetic engineering of ethanologenic bacte- organisms cannot utilize them as readily as glucose
ria. These have largely focused on yield, but not in [57]. Hemicellulose is also of high industrial value, as
means by which these bacteria can tolerate or resist a major source of xylose and xylitol. The latter can be
the LCM bioconversion inhibitors [7-10]. We antici- used as artificial sweetener and as antimicrobial agent
pate that the topics covered in this review will be in foods and other household products [58-60].
helpful in the future genetic engineering of ethanolo- Lignin, a complex aromatic macromolecule,
genic bacteria with enhanced tolerance adaptation forms an integral component of the secondary plant
strategies towards these inhibitory substrates, thus cell wall [47]. It is covalently linked with cellulose and

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Int. J. Biol. Sci. 2013, Vol. 9 601

hemicellulose and provides structural support to the lose-hemicellulose-lignin complex (CHLC) that then
plant cell, protects it from hydrolytic enzymes and allows possible access to hemicellulose and cellulose
pathogens entry, and plays a major role in the water for subsequent enzymatic depolymerisation into sim-
conductivity of vascular tissues [61]. It is formed by ple sugars (hexoses and pentoses) [19]. A number of
the polymerization and dehydration of three mono- different pre-treatment methods have been intro-
lignols (ρ-coumaryl, coniferyl and sinapyl alcohols) to duced, including acid/alkaline hydrolysis [40, 63],
form phenylpropanoids (ρ-hydroxyphenyl, guaiacyl steam/thermal explosion and hot water treatment
and syringal lignins, respectively) [62]. Products of [41-44] and ammonia fibre expansion [45, 64, 65]
lignin degradation such as ferulic acid, vanillin, and (Figure 1).
catechol are used in the production of herbicides, The type and quantity of these inhibitors is
pesticides, plastics, household products, food fla- greatly dependent on the source of the biomass since
vourants, anti-microbial and anti-foaming agents [39, lignin, one of the main sources of these inhibitory
61, 62]. compounds, has different structural qualities and de-
grees of bonding/interaction with cellulose and
Inhibitory compounds produced during hemicellulose depending on the plant, or waste LCM
LCM conversion resource used for ethanol production [39, 47, 61, 62].
The yield and productivity of LCM to ethanol Furthermore, the pre-treatment processes utilized as
bioconversion is greatly reduced due to the produc- well as fermentation variables (e.g. oxygen concen-
tion of cytotoxic-inhibitory compounds generated tration, pH of medium, etc.) can exacerbate the toxic
from lignin degradation and/or sugars dehydration effectors of the inhibitors [66]. Regardless of the
during the pre-treatment processes, which are sub- source or LCM preparation methods used, three main
sequently released into the hydrolysate along with the inhibitory compounds are produced during the
fermentable sugars. pre-treatment and hydrolysis steps, namely organic
The pre-treatment phase is vital for LCM bio- acids, furan derivatives and phenolic compounds [27,
conversion. It allows delignification of cellu- 30, 67, 68].

Fig 2. Main hydrolytic components of lignocellulose biomasses and generated inhibitory compounds. Biomasses are generated from wastes such as (A)
maize cobs (B) saw dust (C) sugar cane bagasse (D) fast growing grasses. Pre-treatment processes releases the sugars (C6/C5) and lignin, however these
processes also cause the breakdown of lignin and dehydration of the sugars, producing the inhibitory compounds that greatly reduces the overall efficiencies
of the ethanologenic cells in the lignocellulose hydrolysate.

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Int. J. Biol. Sci. 2013, Vol. 9 602

Organic acids the microorganismal cell growth and proliferation, it

does not lead to a reduction in the production of eth-
Among the major organic acids produced during
anol [72]. The cells tend to rapidly generate ATP in
the pre-treatment and hydrolysis processes are lac-
order to maintain the intracellular pH, forcing the
tate, succinate, formate and acetate [69]. The latter,
microorganisms to switch into anaerobic respiration,
most abundant organic acid generated during
consequently generating ethanol at the expense of
pre-treatment and hydrolysis processes, is formed
biomass formation [72]. Acetate concentrations as low
from the dehydration of released sugars and/or de-
as 0.5g/L were reported to inhibit Escherichia coli cell
composition of acetylxylan, a byproduct of hemicel-
growth by 50% in a batch and fermenter culture re-
lulose degradation (Figure 2) [63, 70].
spectively, but did not result in a reduction in the cell
Acetate is liposoluble and therefore diffuses
fermentation efficiency [73, 74]. This can be linked to
across the bacterial cell plasma membrane and disso-
the bacterial cells generating more ATP in order to
ciates into its anionic form, releasing protons into the
maintain the intracellular pH, forcing the bacteria to
cytoplasm (Figure 3) [71]. This result in a drop in the
switch to anaerobic respiration, thereby generating
intracellular pH, leading to disruption of the trans-
ethanol, while at the same time exhausting the proton
membrane pH potential, various damaging ani-
pumping capacity of the cell plasma membrane
on-specific effects on metabolism, protein/enzyme
ATPase, resulting in depletion of the ATP content,
activity/stability, and higher turgor pressure within
dissipation of the proton motive force and acidifica-
the cell [71]. The dissociation of this weak acid inside
tion of the cytoplasm (Figure 3) [75, 76]. The overall
the cytoplasm results from the higher intracellular pH
effect is a reduction in cell growth and proliferation
of ethanologenic bacteria (pH = ~7.8) compared to
[30].
that of acetate (pKa =4.75). While the dissociation of
acetate in the cytoplasm has a net negative effect on

Fig 3. A model of effects of inhibitors presence in ethanologenic bacteria cells. As depicted in the illustration, inhibitory effect could range from membrane
disruption, lowering of intracellular pH to interference with lots of cell metabolic targets/pathways.

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Int. J. Biol. Sci. 2013, Vol. 9 603

teins/enzymes, which results in their denaturation,

Furan derivatives
they damage cytoskeleton and other hydrophobic
2-furaldehyde (furfural) and 5-hydroxymethyl- intracellular targets, cause DNA mutagenesis, and
furfural (HMF) are dehydration products of pentose induce programmed cell death (Figure 3) [87]. Phe-
and hexose sugars, respectively, produced during nolic compounds have been reported to be more toxic,
acid pre-treatment and hydrolysis of LCMs (Figure 1 even at low concentrations, than furfural and HMF
and 2) [77]. The toxicity results from the inhibition of [88, 89].
glycolytic and fermentative enzymes essential to cen- While the mechanism and extent of cytotoxicity
tral metabolic pathways (such as pyruvate, acetalde- of lignocellulose inhibitory compounds generally
hyde and alcohol dehydrogenases) [78], pro- differ, they all result in gross physiological/metabolic
tein-protein cross linking and DNA degradation into changes in the ethanologenic microorganisms which
single strands (Figure 3) [79-82]. Their high hydro- concomitantly result in decreased cell viability and
phobicity allows furfural and HMF to compromise fermentation efficiency. One of the major determining
membrane integrity leading to extensive membrane factors of toxicity of these inhibitors is their hydro-
disruption/leakage, which eventually will cause re- phobicity potentials. Hydrophobicity shows the ex-
duction in cell replication rate, ATP production, and tent to which a compound can accumulate in the cell
consequently lower ethanol production [83]. In-vitro cytoplasm. Table 1 shows the hydrophobicity poten-
incubation of furfural with double stranded lambda tials of the aforementioned inhibitory compounds.
phage DNA led to single-strand breaks, primarily at Log P, which is the partition coefficient of solvent in
sequence sites with three or more adenine or thymine an equimolar mixture of octanol and water, is the
bases [79-82]. Furan derivatives are furthermore measure of hydrophobicity (i.e the rate of interaction
known to act synergistically with other inhibitors in- with non-polar molecules) [90, 91]. A high value of
cluding phenolic and aromatic compounds as well as Log P is an indication of high hydrophobicity (i.e the
acetic, formic and levulinic acids [30, 83]. The latter coumpound can readily translocate into the cell across
two acids also result as by-products of the acid deg- the non-polar cell membrane), and as a consequent,
radation of HMF [79]. Formic acid is more toxic than the high inhibitory the compound [90-94].
levulinic acid due to its smaller molecular size and
undissociated form which facilitates its higher mem- Bacterial LCM bioconversion inhibitor
brane permeability. Formic acid was shown to inhibit tolerance and adaptation mechanisms
the synthesis of macromolecules, as well as DNA
Efficient ethanologenic bacteria must be able to
synthesis and repair [84, 85].
ferment a variety of sugars (pentoses and hexoses)
Phenolic compounds and furthermore be capable of surviving, growing
Phenolic compounds formed during the degra- and replicating under the stressful conditions they
dation lignin and dehydration of sugars in the will encounter during the LCM bioconversion pro-
pre-treatment and hydrolysis stages are insoluble or cess. This includes the ability to tolerate or adapt to
partially soluble in the hydrolysate and include acids inhibitors introduced during the LCM pre-treatment
(ferulic acid, vanillic acids, 4-hydroxybenzoic acid phases [19, 20, 95, 96]. These factors form an integral
and syringic acid), alcohols (guaiacol, catechol and component in the screening and selection of effective
vanillyl alcohol) and aldehydes (vanillin, syringic ethanologenic microorganisms, as well as represent-
aldehyde and 4-hydroxylbenzaldehyde) [61, 62]. ing important selectable markers in the genetic engi-
These compounds are known to partition into bio- neering of ethanologenic microorganisms, be they
logical membranes altering the permeability and li- fungi, yeasts or bacteria. The molecular mechanisms
pid/protein ratio, which thus increases cell fluidity, employed by these ethanologenic bacteria to coun-
leading to cell membrane disruption, dissipation of teract these compounds are still largely unknown.
proton/ion gradients and compromising the ability of However, a number of specific and global stress re-
cellular membranes to act as selective barriers sponse mechanisms have been identified in bacteria
[86].This membrane disruption, allows the release of which could be used by ethanologenic bacteria to
proteins, RNAs, ATP, Ions, out of the cytoplasm, provide tolerance, resistance or protection from the
consequently causing reduced ATP levels, diminished many of the above-mentioned fermentation inhibitors
proton motive force and impaired protein function and these will be discussed below. These include
and nutrient transport [86]. Furthermore, they en- mechanisms for the maintenance of pH homeostasis
hance the generation of reactive oxygen species (ROS) and cell membrane integrity, the activation of global
such as hydrogen peroxide (H2O2), super oxides (O2-) stress responses and inhibitor degradation.
and super hydroxyl (OH-) that interact with pro-

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Table 1. Hydrophobicity potentials of organic acids, furans and phenolics on cell physiology.
Inhibitor IUPAC Name Molecular Formular Molecular Weight (g/L) Log P
Acetic acid Acetic acid C2H4O2 60.05 -0.32
Formic acid Formic acid CH2O2 46.03 -0.54
Levulenic acid 5-hydroxy-5-methyl-2-tetrahydrofuranone C5H8O3 116.12 1.34
HMF 5-(hydroxymethyl)-2-furaldehyde C6H6O3 126.11 -0.37
2-furaldehyde Furan-2-carbaldehyde C5H4O2 96.08 0.41
4-Hydroxybenzoic acid 4-Hydroxybenzoic acid C7H6O3 138.12 1.56
Ferulic acid (E)-3-(4-hydroxy-3-methoxy-phenyl)prop-2-enoic acid C10H10O4 194.18 1.641
Syringic acid 4-Hydroxy-3,5-dimethoxybenzoic acid C8H10O5 198.17 1.129
Vanillic acid 4-Hydroxy-3-methoxybenzoic acid C8H8O4 168.15 1.2014
4-Hydroxybenzaldehyde 4-Hydroxybenzaldehyde C7H6O2 122.12 1.392
Syringaldehyde 4-Hydroxy-3,5-dimethoxybenzaldehyde C9H10O4 182.17 0.863
Vanillin 4-Hydroxy-3-methoxybenzaldehyde C8H8O3 152.15 1.188
Catechol Pyrocatechol C6H6O2 110.10 0.88
4-methyl catechol 4-methyl catechol C7H8O2 124.14 1.37
Guaiacol 2-methoxyphenol C7H8O2 124.14 1.32
Vanillyl alcohol 4-Hydroxy-3-methoxybenzyl alcohol C8H10O3 154.16 0.003
Log P is the partition coefficient of solvent in an equimolar mixture of octanol and water. It is a measure of hydrophobicity, which is the rate of interaction with
non-polar molecules [90, 91]. A high value of Log P is an indication of high hydrophobicity, and means the compound can readily translocate into the cell across
the non-polar cell membrane, and as a consequent, the high inhibitory the compound. The Log P values of compounds were obtained from search on
ChemSpider [Free Chemical Identifier Data Base; http://www.chemspider.com/].

[97, 101]. In addition to this effect, malolactic enzyme

Maintenance of pH homeostasis
which converts mono-anionic malate into lactate by
One of the main effects of the LCM bioconver- the addition of 2H+ can also contribute towards re-
sion inhibitors is the intracellular acidification of ducing the intracellular H+ concentration (Figure 4C)
ethanologenic microorganisms [97]. The intracellular [97]. The results of these decarboxylation processes
pH can be maintained by several means in ethanolo- concurrently generate a proton motive force (PMF)
genic bacteria. One means to achieve pH homeostasis that is sufficient to drive ATP synthesis via F1-FO ATP
in response to cellular acidification is through the Synthases activity, as a result providing ATP for
increased production of ammonia (NH3) [98]. NH3 metabolic functions (Figure 4D) [97].
will combine with the excess H+ ions present in the
cell upon exposure to acids produced during LCM Maintenance of cell membrane integrity
pre-treatment to form ammonium (NH4+) ions, con- The hydrophobicity of the inhibitors results in
sequently raising the intracellular pH [98]. Several the interference with fluidity and rigidity and con-
enzymes have been identified which enable the pro- comitant instability of the bacterial cell membrane.
duction of ammonium, including intracellular ureases One means to cope with this instability is by increas-
and arginine deiminase. Urease converts urea into ing sterol production and altering phospholipid fatty
NH3 and CO2 while arginine deiminase converts acids through synthesis of more trans-monoenoic than
L-arginine into NH3 and L-citrulline (Figure 4A) cis-monoenoic unsaturated fatty acids [102-104]. This
[98-100]. enhances membrane restructuring, conferring higher
Furthermore, the activation of an amino acid rigidity and resistance to disruption by external fac-
decarboxylase coupled with an antiporter, pumps in tors such as LCM bioconversion inhibitors (Figure
amino acids (arginine, glutamate or lysine) and 4E). This has been demonstrated in Pseudomonas putida
pumps out decarboxylated products (agmatine, P8 as well as several other bacteria belonging to the
γ-amino butyrate or cadaverine) from the bacterial genera Pseudomonas and Vibrio which are resistant to
cell. This results in the expulsion of 2H+ molecules per high concentrations of phenolic compounds, and is
decarboxylated product and leads to increase in in- linked to the constitutively expressed periplasm-
tracellular pH (Figure 4B) [97, 100]. This mechanism localized enzyme cis-trans isomerase (Cti), which
was well studied in Escherichia coli where gluta- converts cis-unsaturated to trans-unsaturated fatty
mate-dependent decarboxylation was shown to be the acids. [105,106]. Sterols ensure that the bacterial cell
most robust, in terms of pH stabilization, while the membrane provide a greater hydrophobic barrier
lysine-dependent decarboxylation is the least effective against polar molecules and rigidity barrier against

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Int. J. Biol. Sci. 2013, Vol. 9 605

non-polar molecules, consequently blocking and subsequent translation. Activated response genes
non-specific translocation/permeation of toxic mole- include those encoding SOS response proteins such as
cules into the cell [104, 105, 107]. Bacterial Outer LexA and RecA which participate in various house-
Membrane Proteins (OMPs) located in the outer keeping functions including DNA repair and correc-
membranes of Gram-negative bacteria and cell enve- tion of mutation errors (Figure 4F) [ 118, 119], oxida-
lopes of Gram-positive bacteria may also play a vital tive stress response proteins such as superoxide dis-
role in providing a protective barrier against the in- mutase (which converts O2- to O2 and H2O2) and thi-
flux of LCM bioconversion inhibitory compounds oredoxin peroxidase (which converts H2O2 to H2O)
and/or facilitate their efflux through the plasma thus relieving oxidative stress (Figure 4G) [117, 120],
membrane, consequently protecting the cell [108-110]. and heat shock proteins/chaperones (DnaK and
The role of OMPs in the extrusion of and protection GroESL complex) which are involved in the folding,
against phenols has been described in both Pseudo- renaturation and stability of cellular proteins or re-
monas species and E. coli [111 - 113]. moval of damaged proteins during stress (Figure 4H)
[117, 120, 121]. Other regulators that could play simi-
Activation and regulation of global stress re-
lar role as the sigma factors are stress toler-
sponses
ance-related transcriptional factors such as Hfq, NhaA
Given the physiological stress introduced by and HimA [122-124]. These transcriptional factors
LCM bioconversion inhibitors on bacterial cells, an- were shown to be involved in the regulation of genes
other means by which bacteria can tolerate these in- involved in resistance to lignocellulosic pre-treatment
hibitors is through the activation of global stress re- inhibitory compounds in Zymomonas mobilis. Over
sponses. Sigma factors (σS and σB) that regulate the expression of the hfq, nhaA and himA genes resulted in
general stress responses in bacteria play a major role increased resistance to these inhibitory compounds,
in initiating the transcription of vital stress response while knock-out mutants were more sensitive
genes [114-117]. They form a complex with RNA [122-124].
polymerase that binds to the promoter regions of
these response genes resulting in their transcription

Fig 4. A model of tolerance and adaptation mechanisms which could be employed by ethanologenic bacteria against the effects of lignocellulose inhibitors
and which may involve maintenance of pH homeostasis and cell membrane integrity, activation and regulation of global cellular stress responses and
degradation of Inhibitors.

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Int. J. Biol. Sci. 2013, Vol. 9 606

Inhibitors degradation silencing of the NADPH-dependent oxidoreductase

The synthesis and activation of proteins such as genes yqhD and dkgA in E. coli EMFR9 resulted in
enzymes or co-enzymes in inhibitor-specific degrada- greater tolerance towards furfural and HMF [160,
tion pathways can contribute significantly towards 161]. This was attributed to the increased availability
alleviating the negative effects of the inhibitors on of NADPH for use by furfural reductase, as over ex-
ethanologenic bacteria (Figures 4I and 5). The degra- pression of glucose 6-phosphate dehydrogenase in S.
dation of these inhibitory compounds has been cerevisiae that produces NADPH, was found to en-
widely demonstrated in many bacterial species, par- hance tolerance to furfural [162]. Genetic manipula-
ticularly for phenolics, where meta/ortho-cleavage tion of four genetic traits (FucO, ucpA or pntAb and
and β-ketoadipate pathways are used for phenolic deletion of yqhD), were recently shown to increase
compound degradation [125-128]. Phenolic degrada- furfural tolerance in ethanol producing E. coli LY180
tion pathways have been previously described in (Strain W; ATCC derivative) and in succinate biocat-
Gram positive bacteria genera, such as Aerobacter [129] alyst E. coli KJ122 (Strain C; ATCC8739 derivative)
Arthrobacter [130], Bacillus [131-135], Lactobacillus [136, [163]. These strains were reported to be highly re-
137], Rhodococcus [138] and Paenibacillus [139], as well sistant to furfural and mixture of other hemicellulose
as in Gram negative bacteria genera such as Acineto- derived inhibitors, as equal yields of ethanol and suc-
bacter [140], Comamonas [141, 142], Enterobacter [143], cinate were produced when these strains were used in
Escherichia [144-148], Klebsiella [149], Pseudomonas hemicelluloses hydrolysates (which contain furfural,
[150-153] and Sphingomonas [154, 155]. It has been HMF, formic and acetic acids) and in laboratory con-
shown that these microorganisms have evolved with trolled fermentations [163].
abilities to degrade phenolics such as ferulic acid, va- The introduction of Laccase, a multicopper oxi-
nillic acid, protocatechuic acid, and catechol, and be dase enzyme, in willow plant hemicellulose hydroly-
able to break them down into simple products via sate has also been found to greatly reduce the effect of
series of enzymatic processes, which can then chan- lignocellulose inhibitory compounds (such as vanillic
nelled into the TCA and/or glyoxylate cycle(s) to acid, catechol and 4-hydroxybenzoic acid) on fer-
produce energy (Figure 5) [125-128, 131, 139, 147, 150]. mentation [164]. Laccases are produced by many
Mechanisms such as decarboxylation, demethylation, bacterial species, including Azospirillum lipoferum [165,
dehydroxylation, oxidation, ring-cleavage, reduction, 166], Bacillus subtilis [167, 168], Marinomonas mediter-
deacetylation are employed by several of the ranea [169, 170], Streptomyces griseus and Streptomyces
above-mentioned bacteria to degrade phenolic com- cyaneus [171-173], where it participates in the biodeg-
pounds. This degradation involves specific enzymes radation of polymers and ring cleavage of aromatic
such as ferulic acid decarboxylase, vanillate deme- compounds.
thylase, catechol-1,2-dioxygenase, maleylacetate re-
ductase, muconolactone isomerise, β-ketoadipate
Screening/engineering LCM inhibitor
enol-lactone hydrolase, γ-carboxymuconolactone de- tolerant ethanologenic bacteria
carboxylase (see Figure 5) [125-128, 131, 139, 147, 150]. The development of sustainable biofuel produc-
The conversion of the aldehyde and/or carbox- tion will require an efficient utilization of alternative
ylic acid groups of phenolic compounds to alcohol renewable and inexpensive biomass sources, such as
group compounds were shown to be more beneficial LCMs. Presently, the large-scale use of bioconversion
to the cell physiology [76, 86], probably due to the of LCMs to ethanol is hampered by the introduction
reduced toxicity of the alcohol functional groups. The of inhibitory compounds during the pre-treatment
enzyme carboxylic acid reductase in Nocardia sp. was steps which negatively affect the ethanologenic mi-
shown to convert the phenolic compound vanillic acid croorganisms used for downstream ethanol produc-
to vanillin aldehyde [156], while an aldehyde reduc- tion. There is thus an urgent need to improve and
tase in Gluconobacter oxydans can convert this aldehyde optimize the LCM pre-treatment and hydrolysis pro-
to vanillyl alcohol [157]. cesses in order to overcome this technical challenge
The enzyme furfural reductase produced by and thereby improve the fermentation efficiency [29,
many different ethanologenic bacterial species can 54, 174]. Existing chemical or physical strategies for
degrade furfural and HMF to the less toxic com- inhibitor elimination or reduction are expensive and
pounds furfuryl and hydroxymethyl furfuryl alcohol not entirely effective. Hence it has become important
respectively [158, 159]. It was shown that NADPH to look at means to improve the tolerance/resistance
concentration plays a vital role in the activity of this of ethanologenic microorganisms, including bacteria.
enzyme, and that NADPH-dependent reduction of This can be undertaken by screening for inhibitor tol-
the furan compounds competes with normal cell erant/resistant microorganisms or through genetic
metabolic biosyntheses that utilise NADPH [159]. The engineering.

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Int. J. Biol. Sci. 2013, Vol. 9 607

Fig 5. Inhibitor-specific degradative pathways in bacteria cells. Mechanisms often used by specific enzymes may involve decarboxylation, demethylation,
dehydroxylation, oxidation, ring-cleavage, reduction, deacetylation e.t.c. The products formed are often less toxic to cells physiology and could easily be
metabolised further to form products such as acetyl and succinyl COA that are easily assimilated by the cells.

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Int. J. Biol. Sci. 2013, Vol. 9 608

An important conventional biological approach rarely utilize C5 sugars. Over-expression of homolo-

which involves long-time course adaptation study gous and heterologous potentially beneficial genes of
could be employed. This could be done by culturing dehydrogenases and reductases enzymes that are
ethanologenic bacteria in specific medium containing involved in numerous detoxification reactions and
high concentrations of LCM inhibitory compounds which are shown to alter the levels of co-factor
with low carbon (sugar) source, allowing them to NADPH and NADH could also confer resistance to-
adapt and develop in the new environment. This ap- wards specific LCM inhibitory compounds [187, 188].
proach will lead to creation of inhibitor-tolerant bac- Furthermore, the use of genomics/metagenomic
terial strains, in a way that strains that have the ability approach in biomining varieties of environmental and
to survive, grow and adapt within this medium, industrial niches for lignocellulolytic bacteria with
would have activated specific proteins/enzymes and genes that confers resistance or ability to degrade
mechanisms that would have facilitated the trans- these inhibitory compounds, which could be cloned
formation of the LCM inhibitory compounds present and expressed in existing industrial strains (that have
in the hydrolysates, into less toxic compounds. This high ethanol production capabilities) could further
approach has resulted in increase in microbial biocat- enhance the current fermentation technologies [189,
alysts efficiency in production of ethanol with in- 190]. These bacteria are able to survive extreme habi-
creasing tolerance to LCM inhibitory compounds. For tats such as thermophilic, halophilic, acidophilic or
example, the Escherichia coli LY01 strain was found to alkaliphic environments as a result of their innate
show high tolerance to toxic aldehydes than its wild defence and adaptation mechanisms, and they often
type KO11, by expressing high levels of genes in- produce enzymes that are activity and potentially
volved in safeguarding osmolytic balance, stress re- stable at these harsh conditions, which are often sim-
sponse proteins and cell envelope components [76, ilar to extreme conditions present in lignocellulose
175]. Furthermore, Escherichia coli strain LY168 engi- degradation processes. Such bacteria could be used
neered from wild type K011 from was shown to pro- concurrently with the ethanologenic strains; helping
duce higher level of ethanol in a minimal nutritional to degrade the inhibitors while the ethanologenic
supplement from various lignocellulose biomass bacteria ferments the hydrolysate [191]. As example,
containing LCM inhibitory compounds [176]. It also the use of thermophilic bacterium, Ureibacillus ther-
shown that Methylobacterium extorquens, Pseudomonas mosphaercus was found to potentially increase ethanol
sp., Flavobacterium indologenes, Acinetobacter sp., Ar- production when used concomitantly with S. cerevisiae
throbacter aurescens could also degrade LCM inhibitory for ethanol production in waste house wood hydrol-
compounds when grown on them as the sole carbon ysate [22]. Phenolics and furan derivatives present in
and energy sources [177].The success of this approach the hydrolysate were confirmed to be degraded by
infers that it could be applied to other ethanologenic Chromatographic analysis, and the bacterium grows
bacteria to improve the overall tolerance to LCM in- fast and utilizes below 5% of the released fermentable
hibitory compounds and fermentation efficiencies. sugars.
Resequencing the genome/proteome of these strains
will also provide valuable information into the adap- Summary
tation mechanism that may have been employed by It is anticipated that in the near future green en-
these strains in adjusting to the new environment. ergy such as biofuels (bioethanol in particular) will
Another approach could be use of enzymatic gradually replace fossil fuels as a global energy source
detoxification. The use of laccase, phenoloxidase [192] for home and industrial use. However, more
and/or lignin peroxidase enzymes could be a great vigorous research and focused approaches must be
potential, since treatment of LCM hydrolysate with channelled into the development of robust and eco-
these enzymes have led to degradation of phenolic nomical technologies that will utilize lignocellulose
compounds and increase the ethanol yield with a biomasses on a larger scale and improve lignocellu-
negligible loss of total sugars [ 178-180]. Using ap- lose fermentation efficiencies. This could be achieved
propriate genetic engineering to over-express the ac- by overcome the existing challenges facing the fer-
tive production of these enzymes has been success- mentation processes through better understanding of
fully carried out in S. cerevisiae [181-186]. It will mechanisms of hydrolysate toxicity and engineering
therefore be of better advantage if the same technol- tolerance towards them. If this is actualized, ligno-
ogy could be used for ethanologenic bacteria cells, cellulolytic biomass fermentation will be able to meet
since they have the abilities to grow quickly, produc- and exceed the productivity of sugar/food crop-based
ing more of these enzymes and they also could suc- bioethanol bioprocesses that threatens world food
cessfully ferment both C6 and C5 sugars in the LCM security.
hydrolysate, unlike most fungi that ferment C6 and The further advancement of lignocellulose bio-

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Int. J. Biol. Sci. 2013, Vol. 9 609

technology will not only provide bioethanol for en- 18. Perez J, Muñoz-Dorado J, de la Rubia T, et al. Biodegradation and biological
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Acknowledgement and bacteria by degradation products produced during pre-treatment of bio-
mass. Appl Microbiol Biotechnol. 2004; 66: 10-26.
25. Mosier N, Wyman C, Dale B, et al. Features of promising technologies for
The authors wish to thank by the National Re- pretreatment of lignocellulosic biomass. Bioresour Technol. 2005; 96: 673-686.
search Foundation (NRF) South Africa, the University 26. Palmqvist E, Hahn-Hagerdal B. Fermentation of lignocellulosic hydrolysates.
II: Inhibitors and mechanisms of inhibition. Bioresour Technol. 2000; 74: 25-33.
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Council of South Africa, for their institutional and mechanisms in Escherichia coli. Biotechnol for Biofuels. 2009; 2 (1): 26-36.
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financial support. reveals the stress response and detoxification of yeast to combined inhibitors.
PLoS One. 2012; 7(8): 43474.
Competing Interests 29. Palmqvist E, Hahn-Hägerdal B. Fermentation of lignocellulose hydrolysates. I:
inhibition and detoxification. Biores Technol. 2000; 74(1): 17-24
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