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R E S E A R C H A R T I C L E Open Access
Designer synthetic media for studying microbial-
catalyzed biofuel production
Xiaoyu Tang
1*
, Leonardo da Costa Sousa
2
, Mingjie Jin
2
, Shishir PS Chundawat
2,3
, Charles Kevin Chambliss
4
,
Ming W Lau
2
, Zeyi Xiao
5
, Bruce E Dale
2
and Venkatesh Balan
2*
Abstract
Background: The fermentation inhibition of yeast or bacteria by lignocellulose-derived degradation products,
during hexose/pentose co-fermentation, is a major bottleneck for cost-effective lignocellulosic biorefineries. To
engineer microbial strains for improved performance, it is critical to understand the mechanisms of inhibition that affect
fermentative organisms in the presence of major components of a lignocellulosic hydrolysate. The development of a
synthetic lignocellulosic hydrolysate (SH) media with a composition similar to the actual biomass hydrolysate will be an
important advancement to facilitate these studies. In this work, we characterized the nutrients and plant-derived
decomposition products present in AFEX™pretreated corn stover hydrolysate (ACH). The SH was formulated based on
the ACH composition and was further used to evaluate the inhibitory effects of various families of decomposition
products during Saccharomyces cerevisiae 424A (LNH-ST) fermentation.
Results: The ACH contained high levels of nitrogenous compounds, notably amides, pyrazines, and imidazoles. In
contrast, a relatively low content of furans and aromatic and aliphatic acids were found in the ACH. Though most of
the families of decomposition products were inhibitory to xylose fermentation, due to their abundance, the
nitrogenous compounds showed the most inhibition. From these compounds, amides (products of the ammonolysis
reaction) contributed the most to the reduction of the fermentation performance. However, this result is associated to
a concentration effect, as the corresponding carboxylic acids (products of hydrolysis) promoted greater inhibition when
present at the same molar concentration as the amides.
Due to its complexity, the formulated SH did not perfectly match the fermentation profile of the actual hydrolysate,
especially the growth curve. However, the SH formulation was effective for studying the inhibitory effect of various
compounds on yeast fermentation.
Conclusions: The formulation of SHs is an important advancement for future multi-omics studies and for better
understanding the mechanisms of fermentation inhibition in lignocellulosic hydrolysates. The SH formulated in
this work was instrumental for defining the most important inhibitors in the ACH. Major AFEX decomposition products
are less inhibitory to yeast fermentation than the products of dilute acid or steam explosion pretreatments; thus, ACH is
readily fermentable by yeast without any detoxification.
Keywords: Synthetic hydrolysate, Lignocellulose, AFEX, Yeast fermentation inhibition, Amides inhibition, Carboxylic
acids inhibition, Pretreatment decomposition products, Hydrolysate composition
* Correspondence: tangxiaoyu@caas.cn;balan@msu.edu
1
Biogas Institute of Ministry of Agriculture, Section 4-13 Remin South Road,
Chengdu 610041, P. R. China
2
DOE Great Lakes Bioenergy Research Center, Biomass Conversion Research
Lab (BCRL), Chemical Engineering and Materials Science, Michigan State
University, 3815 Technology Boulevard, Suite 1045, Lansing 48910, USA
Full list of author information is available at the end of the article
© 2015 Tang et al.; licensee BioMed Central. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain
Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article,
unless otherwise stated.
Tang et al. Biotechnology for Biofuels (2015) 8:1
DOI 10.1186/s13068-014-0179-6
Background
Increasing fossil fuel utilization by industrialized econ-
omies has been a subject of intense debate within the
scientific and political communities worldwide. Increas-
ing energy demand, depleting petroleum reserves, the
negative environmental repercussions due to increased
greenhouse gas (GHG) emissions, and the control of fos-
sil fuel production by a limited number of nations are
among the main reasons why there is an ongoing effort
to restructure our energy sector towards greater sustain-
ability via utilization of renewable sources of energy [1].
Lignocellulosic biofuels are projected to play a sub-
stantial role in the replacement of current-generation
fossil-derived liquid fuels such as gasoline and diesel
[2,3]. In second generation biorefineries, ethanol produc-
tion from lignocellulosic substrates involves enzymatic
digestion of cellulose and hemicellulose sugar polymers
into fermentable sugars, which can be converted to etha-
nol during microbial fermentation. However, the plant
cell wall structure has naturally evolved to be highly re-
calcitrant to enzymatic deconstruction by fungi and bac-
teria. In order to improve enzyme accessibility to the
polysaccharides embedded in plant cell walls, some form
of pretreatment is necessary to reduce biomass recalci-
trance to enzymatic hydrolysis.
Among the pretreatment technologies available today,
thermochemical pretreatments are considered to be the
most promising [4]. Most of these pretreatment pro-
cesses use either acids (such as sulfuric acid, phosphoric
acid, and maleic acid) or bases (such as ammonium hy-
droxide, sodium hydroxide, and potassium hydroxide) to
pretreat plant cell walls, often resulting in the formation
of cell wall-derived decomposition products that can in-
hibit both enzymes and microbes [5-7].
Ammonia Fiber Expansion (AFEX™)
a
is a well-established
pretreatment technology that utilizes concentrated ammo-
nia at relatively low temperatures (60 to 140°C) and short
residence times (5 to 45 min) to pretreat biomass [4]. AFEX
has proven to be particularly effective on monocot-based
grasses(forexample,cornstover),improvingcellulosehy-
drolysis rates by up to fivefold and generating highly fer-
mentable hydrolysates [8]. Moreover, AFEX produces
much lower concentrations of sugar-derived decomposition
products compared to acidic pretreatments, while preserv-
ing the native nutrient content for more efficient fermenta-
tion [9-11]. Therefore, AFEX-based biomass hydrolysates
do not require detoxification, exogenous nutrient supple-
mentation, and extensive water washing of the pretreated
substrate for efficient glucose fermentation by yeast or bac-
teria [10,12]. However, the efficiency of xylose consumption
during co-fermentation of AFEX pretreated biomass hydro-
lysates (enriched in both pentoses and hexoses) still re-
quires improvement. Some of the issues faced during
mixed hexose/pentose fermentation are the low xylose
consumption rate and the lower ability of yeast and
bacteria to co-ferment hexose/pentose mixtures [10,12].
Our recent work in Escherichia coli KO11 and Saccha-
romyces cerevisiae 424A (LNH-ST) demonstrated that
the xylose consumption rate is related to the presence
of pretreatment-derived biomass decomposition prod-
ucts, ethanol, and other fermentation metabolites [13].
In the case of E. coli KO11, the ability to consume xy-
lose from AFEX hydrolysate was severely affected by
the presence of pretreatment-derived biomass degrad-
ation products in combination with high concentrations
of ethanol. On the other hand, a 22% reduction of cell
growth and 13% reduction of specific xylose consump-
tion rate was observed for S. cerevisiae 424A (LNH-ST)
due to the presence of AFEX decomposition products
in the hydrolysate. However, very little is known about
the nature of pretreatment-based biomass decompos-
ition products that inhibit xylose consumption, their
mechanism of action, and their overall effect on the
metabolism of sugars by yeast and bacteria. Answering
these questions is an important step toward developing
new microbial strains with improved performance on
lignocellulosic hydrolysates, and hence increasing the
economic competitiveness of liquid biofuels as a viable
substitute to conventional gasoline and diesel.
One approach for gaining a deeper understanding of the
interactions between inhibitory components present in
biomass hydrolysates and microorganisms, including in-
hibition synergies, levels of inhibition, and metabolic ef-
fects, involves using a synthetic medium that mimics the
composition of authentic lignocellulosic hydrolysates, that
is, a synthetic hydrolysate (SH). The importance of such
SHs for these studies is supported by the work published
by Lau and Dale (2009) [10], who observed that the inhib-
ition of xylose fermentation is closely dependent on the
nutrient availability in the culture medium. The formula-
tion of an SH will enable the inclusion of precisely defined
positive and negative controls in experimental designs,
which represent a current limitation of directly using com-
plex lignocellulosic hydrolysates. Also, using an SH will
allow the manipulation of relative concentrations and ra-
tios between the different components of the hydrolysate,
according to the objective of each study. Furthermore, the
SH will facilitate the integration of isotope-labeled compo-
nents in the medium (for example,
13
C-labeled xylose or
glucose) to conduct metabolomics-based experiments,
aiming to trace potential deviations in the metabolic flux
during xylose consumption in the presence and absence of
compounds of interest.
In this work, we have attempted to establish a platform
for conducting the above-mentioned studies, by character-
izing a highly complex lignocellulosic hydrolysate derived
from AFEX pretreated corn stover (AFEX-CS) and formu-
lating a well-defined SH using both commercially available
Tang et al. Biotechnology for Biofuels (2015) 8:1 Page 2 of 17
and custom-synthesized reagents/chemicals. This SH
platform was also implemented here to screen the ef-
fect of different classes of AFEX pretreatment-based
biomass decomposition products on xylose fermenta-
tion using a recombinant S. cerevisiae 424A (LNH-ST)
strain.
Methods
Biomass
Corn stover (CS) was harvested at Field 570-C Arlington
Research Station, University of Wisconsin, in the year
2008. Pioneer 36H56 (triple stack - corn borer/rootworm/
Roundup Ready) seeds were used for planting. The CS
sample containing leaves, stem, and cobs was dried to <
8% moisture (dry weight basis) using a 60°C oven and
milled to 4-mesh size and stored in sealed polythene bags
at room temperature until further use. The composition
of the untreated corn stover (UT-CS) was 35.7% glucan,
21.2% xylan, 2.6% arabinan, 17.4% lignin, 5.9% ash, and
2.4% acetyl content. AFEX pretreatment was carried out
using the procedure reported by Balan et al. [14]. The pre-
treatment condition in this study was 1:1 ammonia to bio-
mass ratio (dry weight), 60% moisture loading, and 140°C
for 15 min total residence time. After pretreatment, the re-
sidual ammonia was allowed to evaporate in the hood
overnight, before being bagged and stored at 4°C prior to
further usage. The composition of the pretreated biomass
did not change appreciably as a result of the AFEX pre-
treatment [15].
Chemicals
Feruloyl amide
Feruloyl amide was synthesized via a one-step ammonoly-
sis reaction, using ethyl 4-hydroxy-3-methoxycinnamate
(AK Scientific, Inc., Mountain View, CA, USA) as the start-
ing reagent. For the reaction, 3 g of ethyl 4-hydroxy-3-
methoxycinnamate was dissolved in 150 mL of 28 to 30%
ammonium hydroxide (EMD, Gibbstown, NJ, USA) solu-
tion in a high pressure reactor (HEL, Inc., Lawrenceville,
NJ, USA). The reaction was carried out at 100°C for 5 h at
300 rpm mixing speed. Under these conditions feruloyl
amide was the major product, followed by ferulic acid. The
purification and recovery of feruloyl amide were conducted
by preparative-scale HPLC using a Waters XBridgeTM
Prep C18 column (5.0 μm, 10 mm × 100 mm; Waters Co.,
Milford, MA, USA). The HPLC system was equipped
with a Waters 600 Controller, Waters Delta 600 pump,
and a Shimadzu SPD-M10A VP Diode Array Detector
and connected to a Waters Fraction Collector (Waters
Co., Milford, MA, USA). The solution was diluted to
about 5 g/L of feruloyl amide in methanol before injec-
tion. The injection volume was 5 mL, and the HPLC
flow rate was 0.25 mL/min using the gradient shown in
Table 1. The feruloyl amide enriched fractions were
freeze dried to a powder and stored in a desiccator.
Fractions with purity >95% (as determined by LC-MS
and described in Chundawat et al. [15]) were pooled to-
gether before utilization in fermentation experiments.
Coumaroyl amide
Coumaroyl amide was also synthesized using the same
methodology as for feruloyl amide. However, in this case
the ammonolysis reaction was carried out on methyl 4-
hydroxycinnamate (Frinton Laboratories, Inc., Vineland,
NJ, USA). The purification and recovery of coumaroyl
amide were performed using the same methodology as
for feruloyl amide. Fractions with purity >95% (as deter-
mined by LC-MS and described in Chundawat et al.
[15]) were pooled together before utilization in fermen-
tation experiments.
Xylo-oligomers
The xylo-oligomer mixture was produced by enzymatic
hydrolysis of Birchwood xylan (Sigma-Aldrich, St. Louis,
MO, USA) using an NS50014 series endoxylanase en-
zyme (5 mg/mL protein concentration estimated by the
Kjeldahl method) provided by Novozymes (Davis, CA,
USA). The enzyme composition and substrate specificity
for this cocktail have been provided elsewhere [16]. The
reaction was carried out at 6% (w/v) solids loading in a
250-mL flask at pH 4.8 (0.05 M phosphate buffer),
150 rpm, and 50°C for 48 h, with an enzyme loading of
2 mL/g xylan. The supernatant was separated from the
undissolved solids by centrifugation to further isolate the
soluble xylo-oligomers. A Thermo Scientific Hypersep
Hypercarb PGC 453 column (Thermo Scientific, Bellefonte,
PA, USA) was employed to separate the xylo-oligomers
from other soluble products. The column was first condi-
tioned with 30 mL of methanol followed by 30 mL of dis-
tilled water. A sample volume of 3.5 mL of hydrolysate was
then added to the column. After washing the column with
45 mL of water, 60 mL of methanol was added to elute
xylo-oligomers from the sorbent. Methanol was removed
using a rotary evaporator (BUCHI, Switzerland) and the
Table 1 HPLC mobile phase and gradient used for
isolation of phenolic amides
Time 0.1% formic acid 100% methanol
(min) (% Solvent A) (% Solvent B)
095 5
1.00 95 5
10.00 70 30
18.00 50 50
25.00 50 50
25.01 95 5
30.00 95 5
Tang et al. Biotechnology for Biofuels (2015) 8:1 Page 3 of 17
xylo-oligomer solution was adjusted to appropriate concen-
trations in distilled water.
Other chemicals
With the exception of the compounds described above,
all other chemicals used in the SH were purchased
from various commercial vendors: formic acid, 4-
hydroxybenzaldehyde, trans-aconitic acid, vanillic acid,
and vanillin were obtained from Fluka (St. Louis, MO,
USA), and all other chemicals were purchased from
Sigma-Aldrich (St. Louis, MO, USA). Cellobiose was
usedastheonlygluco-oligomericcomponent.
Preparation of AFEX-CS hydrolysate (ACH)
AFEX-CS was enzymatically hydrolyzed with a commer-
cial enzyme mixture as previously described [10]. The
enzyme mixture was composed of Spezyme™CP
(79.6 mL/kg CS; protein concentration: 88 mg/mL),
Novozyme™188 (40.1 mL/kg CS; protein concentration:
150 mg/mL), Multifect Xylanase (11.6 mL/kg CS; pro-
tein concentration: 35 mg/mL), and Multifect Pectinase
(8.2 mL/kg CS; protein concentration: 90 mg/mL). Spe-
zyme™CP and Multifect enzyme cocktails were obtained
from Genencor Inc., while Novozyme™188 was procured
from Sigma-Aldrich Co. The glucan loading used for
biomass hydrolysis was 6% by weight, which was equiva-
lent to about 19% solids loading. The enzymatic hydroly-
sis was performed in a 3-L glass autoclavable bioreactor
equipped with ez-Control (Applikon Biotechnology B.V.,
Schiedam, Netherlands) at 50°C and 1,000 rpm for 96 h.
A total of 2.5 kg of reaction contents (biomass, water,
enzymes, and antibiotics) was loaded into the reactor
with biomass added in two batches separated by 3 h in-
tervals. The pH was maintained at 4.8 with 6 M KOH
during the course of hydrolysis. Chloramphenicol
(Sigma-Aldrich, St. Louis, MO, USA) was added at a
final concentration of 50 mg/L to minimize the risk of
microbial contamination. The hydrolyzed mixture was
separated by centrifugation at 8,000 rpm for 30 min, and
the separated supernatant was heat-deactivated by heat-
ing the hydrolysate for 15 min at 90°C in a water bath
and filtered with a 0.22-μm sterile filter (Millipore
Stericup®, Millipore™, Billerica, MA, USA). The filtrate
was collected and stored in the freezer until further use.
Compositional analysis of the ACH
Glucose, xylose, arabinose, acetate, formate, and lactate in
this hydrolysate mixture were analyzed using an HPLC sys-
tem equipped with a Bio-Rad Aminex HPX-87H column
(Bio-Rad Co., Hercules, CA, USA) as previously described
[12]. The mobile phase was 5 mM H
2
SO4 at a flow rate of
0.6 mL/min, and the column temperature was maintained
at 50°C. Oligosaccharides were determined by acid hydroly-
sis following the NREL protocol (LAP-014; www.nrel.gov/
biomass/analytical_procedures.html), and the monomeric
sugars produced after acid hydrolysis were quantified using
HPLC (LAP-002).
Protein-derived amino acids quantification was con-
ducted on an LC-MS system in the Department of
Biochemistry and Molecular Biology at Michigan State
University. The analytical methodology details have been
reported elsewhere [17]. For total amino acids analysis,
100 μL of CS hydrolysate was hydrolyzed with 1 mL 6 M
HCl at 110°C overnight and then dried under vacuum
(SpeedVac, Eppendorf, Germany). The hydrolyzed dry sam-
ple was solubilized in 10 mL of water. Valine-d8 (1 μM)
was added into the solution as an internal standard. For
free amino acid analysis, the same procedure was followed
with the exception of the 6 M HCl hydrolysis step.
Protein and ammonium nitrogen contents in the bio-
mass were determined using Kjeldahl assays and a Tim-
berline TL-1800 ammonia analyzer, respectively, at Dairy
One Cooperative Inc. (Ithaca, NY). Nitrogen incorpo-
rated in the biomass during ammonolysis reactions was
estimated by subtracting the total nitrogen (w/w) present
in AFEX-CS from the nitrogen in UT-CS, as described
previously [15].
Trace element analysis was carried out with induct-
ively coupled plasma mass spectrometry (ICP-MS) in the
ICPMS & XRF Laboratory at Michigan State University
[18]. Approximately 1 mL of liquid sample was digested
on a hot plate, sub-boiling in acid-cleaned Teflon Savillex
beakers using 1.9 mL Optima nitric acid and 0.1 mL trace
metal clean hydrofluoric acid for 24 h. After digestion,
250 μL of trace metal clean 30% hydrogen peroxide was
added, and the sample was evaporated to near dryness on
a hotplate. Samples were then brought up to final volume
with 5 mL of 2% Optima nitric acid: visual inspection
showed a complete digestion of all samples. This solution
was run in the ICP-MS for full mass scan analyses. For
the major element analysis, potassium (K), magnesium
(Mg), calcium (Ca), phosphorus (P), and sodium (Na)
samples were diluted 1:300 prior to analysis. For the
trace element analysis, cobalt (Co), copper (Cu), man-
ganese (Mn), zinc (Zn), and iron (Fe) samples were run
without dilution.
Organic acids and aromatic aldehyde/ketone analyses
were conducted by LC-MS/MS at Baylor University.
Instrumentation and details of the applied methodology
have been published elsewhere [19]. The nitrogenous
compounds were identified and quantified by LC-MS/
MS and GC-MS for AFEX-CS hot water extracts as re-
ported previously by Chundawat et al. [15]. The com-
position of the ACH is presented in Table 2.
Microorganism and seed culture
S. cerevisiae 424A (LNH-ST) [20], a xylose-fermenting
yeast strain obtained from Purdue University, was used
Tang et al. Biotechnology for Biofuels (2015) 8:1 Page 4 of 17
in this study. The seed culture was prepared by inoculat-
ing a frozen glycerol stock into 50 mL of synthetic
medium (described below) using 50 g/L glucose as the
sole carbon source in a 125 mL flask. The culture typic-
ally reached a cell density of 6.0 to 6.2 OD
600
(optical
density at 600 nm wavelength) after 18 h incubation at
30°C and 150 rpm. The cells were then harvested by
centrifugation at 4,000 rpm for 5 min at room
temperature and used as the inoculum for all reported
experiments.
Synthetic medium and fermentation
A synthetic medium (SM) with a well-defined composition
was used as a seed culture and fermentation medium. The
composition of the SM, as shown in Table 3, was designed
to closely match the nutrient composition of AFEX-CS hy-
drolysate (ACH) at 6% (w/w) glucan loading (Table 2).
Concentrated stock solutions of sugars, peptone (BD
Bacto™Tryptone, Franklin Lakes, NJ, USA), vitamins
(Sigma-Aldrich, St. Louis, MO, USA), ammonium sulfate,
and mineral salts were prepared separately and sterilized
by vacuum filtration (Millipore Stericup®, 0.2 μm). The
medium was adjusted to an initial pH of 5.5 with KOH
and sterile filtered after the addition of all relevant
components.
Fermentations were performed in a 25 mL Erlenmeyer
flask with a working volume of 10 mL. The flasks were
capped with rubber stoppers, which were pierced with a
needle to vent CO
2
. Fermentations were not performed
under strict anaerobic conditions; however, the air initially
present in the head space of the fermentation flasks was
displaced by the CO
2
generated during fermentation. The
seed was inoculated into the medium at an initial OD
600
of 0.5, corresponding to 0.24 g/L cell mass concentration
(dry weight). All fermentations were conducted in tripli-
cate at 30°C and 150 rpm in a shaking incubator and the
pH was maintained around 5.5 by periodic manual addi-
tions of 6 M KOH. As the fermentation media did not
contain buffer, the pH had a tendency to decrease in the
first 24 h. Therefore, we adjusted the pH to 5.5 before
every sampling time. Approximately 300 μL of samples
were withdrawn at designated times (0, 4, 8, 18, 24, 48,
and 72 h) and frozen immediately for subsequent analysis.
Cell mass was estimated using a UV/Vis spectropho-
tometer (Beckman Coulter, Brea, CA) at 600-nm wave-
length. One unit of absorbance is approximately equal to
0.48 g/L yeast cell biomass (dry weight). Sugars, ethanol,
organic acids, glycerol, and xylitol were determined by
HPLC, using the method described for compositional
analysis of the hydrolysate.
The ethanol productivities for the various fermenta-
tion experiments were calculated for 24 h and/or 48 h
Table 2 Nutrient content of AFEX-CS hydrolysate (ACH)
Category Nutrients Concentration Unit
Carbohydrates Glucose 60 g/L
Xylose 26
Nitrogen Ammonia 1.44 g/L
Amino acids 1.44
Vitamins* Pantothenic acid 3.01 μM
Pyridoxine 2.14
Nicotinic acid 26.78
Biotin 0.1
Thiamine 0.4
Macro-elements P 829.38 mg/L
K 3886.50
Mg 292.86
Na 498.86
Ca 120.72
Trace elements Mn 3.67 mg/L
Co 0.02
Cu 0.13
Zn 1.21
Fe 0.93
*Data derived from previous study [18].
Table 3 Nutrient content of synthetic medium mimicking
AFEX-CS hydrolysate (ACH)
Category Nutrients Concentration Unit
Carbon sources Glucose 60 g/L
Xylose 26
Nitrogen sources (NH
4
)
2
SO
4
※
5.23 g/L
Peptone
#
4.35
Vitamins Pantothenic acid 3.01 μM
Pyridoxine 2.14
Nicotinic acid 26.78
Biotin 0.1
Thiamine 0.4
Mineral salts KH
2
PO
4
3319.28 mg/L
K
2
HPO
4
415.66
KCl 4341.80
MgCl
2
·6H
2
0 2449.73
Na
2
CO
3
574.96
NaCl 634.08
Ca(NO
3
)
2
711.30
MnCl
2
·4H
2
O 13.23
CoCl
2
·6H
2
O 0.06
CuCl
2
0.27
ZnCl
2
2.53
FeCl
3
2.69
※
(NH
4
)
2
SO
4
equivalent of ammonia content in the hydrolysate.
#
Peptone equivalent to total amino acid content in the hydrolysate.
Tang et al. Biotechnology for Biofuels (2015) 8:1 Page 5 of 17
time points, dividing the produced ethanol concentra-
tion by the respective residence time. Fermentation
product yields were calculated for the first 48 h period,
dividing the mass of product formed by the mass of total
sugar consumed during that period. Statistical analyses
for the fermentation results included standard deviations
(shown in the respective tables) and a two-tailed t-test
analysis (see Additional file 1) performed in Microsoft
Excel (Microsoft, Seattle, WA).
Effect of ACH components on fermentation
To simplify this study, all characterized AFEX pre-
treatment-derived biomass decomposition products
were divided into five groups (Table 4): 1) nitrogenous
compounds, 2) furans, 3) aliphatic acids, 4) aromatic
compounds, and 5) carbohydrates.
The effect of these five groups of compounds on xy-
lose fermentation was tested individually and in combin-
ation (five groups in combination) in order to investigate
their synergistic inhibitory effect. The fermentations
were conducted in SM supplemented with 60 g/L glu-
cose and 26 g/L xylose. The decomposition products in
each group and their concentrations are given in Table 2,
and matched their absolute abundance as found in 6%
glucan loading-based ACHs. To make stock solutions of
decomposition products, all compounds were dissolved
in water according to the categories of aliphatic acids,
aromatic acids, aromatic aldehyde/ketones, furans, imid-
azoles, and pyrazines at 50-fold higher concentrations
and the stock solutions were sterile filtered prior to their
addition into the SM. Ferulic acid, p-coumaric acid, am-
ides, and carbohydrates were directly added to the fer-
mentation media at the desired concentrations (Table 2)
due to their lower solubility in water. Fermentations of
SM without any decomposition products (blank) and
ACHs were used as negative and positive controls, re-
spectively. The ACH was adjusted to pH 5.5 before in-
oculum addition.
Effect of nitrogenous compounds on fermentation
Fermentations were carried out in SM supplemented
with 60 g/L glucose and 26 g/L xylose, respectively. The
nitrogenous compounds investigated are classified into
three subgroups: 1) amides, 2) pyrazines, and 3) imidaz-
oles. All compounds in each subgroup and their concen-
trations tested are listed in Table 2. Fermentation of SM
without any decomposition products was the control ex-
periment (blank).
Effect of amides and corresponding acids on
fermentation
In order to compare the effect of amides and their corre-
sponding acids on hexose/pentose sugars co-fermentation
performance, feruloyl amide (6.2 mM), ferulic acid
(6.2 mM), p-coumaroyl amide (7.5 mM), p-coumaric acid
(7.5 mM), acetamide (28.8 mM), and acetic acid
(28.8 mM) were selected for this study. The molar
Table 4 Plant cell wall-derived decomposition products
and water-soluble extractives present in AFEX-CS
hydrolysate (ACH)
Category Compound Concentration
(mg/L)
Nitrogenous
compounds
¶
Feruloyl amide 1065
p-Coumaroyl amide 886
Acetamide 5674
2-Methylpyrazine 10
2,5-Dimethylpyrazine 1
2,6-Dimethylpyrazine 4
2,4-Dimethyl-1 H-
imidazole
24
4-Methyl-1 H-imidazole 95
Furan
¶
5-Hydroxymethyl
furfural
145
Aliphatic acids Malonic acid 33
Lactic acid 181
cis-Aconitic acid 111
Succinic acid 60
Fumaric acid 30
trans-Aconitic acid 329
Levulinic acid 2.5
Itaconic acid 8.2
Acetic acid 1958
Formic acid 517
Aromatic compounds Vanillic acid 15
Syringic acid 15
Benzoic acid 59
p-Coumaric acid 345
Ferulic acid 137
Cinnamic acid 14
Caffeic acid 2
Vanillin 20
Syringaldehyde 29.5
4-Hydroxybenzaldehyde 24
4-Hydroxyacetophenone 3.4
Carbohydrates Glucose 60 g/L
Xylose 26 g/L
Arabinose 5 g/L
Gluco-oligomers 12 g/L
Xylo-oligomers 18 g/L
¶
The concentration of nitrogenous compounds and furan were calculated
from the content of the analyte in dry pretreated biomass [15] based on 18%
solids loading (w/v) assuming 100% solubilization into the liquid phase.
Tang et al. Biotechnology for Biofuels (2015) 8:1 Page 6 of 17
concentration of each compound was the total sum of the
amide and its corresponding acid found in ACH at 18%
solids loading (Table 2). The compound was directly dis-
solved in SM and the initial pH of the medium was ad-
justed to 5.5 before filter sterilization. The fermentation
media and control experiments were as described above.
Results and discussion
The major objective of this work is the formulation of a
synthetic lignocellulosic hydrolysate (SH), as a tool to
understand the effect of various components from pre-
treated biomass on microbial fermentation. This work
provides guidelines and a methodology to formulate a
detailed SH, based on the composition of industrially
relevant lignocellulosic hydrolysates. The SH described
in this work was designed based on the composition of
ACH and was used to determine the impact of various
major biomass-derived products on the performance of
S. cerevisiae 424A (LNH-ST) fermentation. The details
concerning 1) characterization of the AFEX-CS hydrol-
ysate, 2) formulation of an SH, and 3) the impact of
major hydrolysate components on yeast fermentation
will be discussed here.
Characterization of the ACH
Characterization of the ACH involves identification and
quantification of 1) natively available microbial nutrients,
2) plant-derived chemicals, and 3) pretreatment-specific
decomposition products. The nutrients available in the
ACH are listed in Table 2 and comprise various forms of
carbohydrates, nitrogenous compounds, vitamins, and
minerals. The carbohydrates which could be consumed
by S. cerevisiae 424A (LNH-ST) as a carbon source were
glucose (60 g/L) and xylose (26 g/L). Other carbohy-
drates were found in ACH at lower abundances, includ-
ing arabinose (5 g/L), glucan-derived oligomers (12 g/L),
and xylan-derived oligomers (18 g/L). However, these
were not categorized as nutrients, as S. cerevisiae 424A
(LNH-ST) is not capable of using these sugars as a pri-
mary carbon source [21].
A total of 1.44 g/L of protein was estimated by LC-MS
during the amino acid analysis of the ACH. Individual
amino acid concentrations are shown in Additional file 1:
Table S1. While the total protein concentration is fairly
similar to the results of a previous study [18], the relative
proportions of individual amino acids were significantly
different. In this work, aspartate, valine, and proline were
the most abundant amino acids, as opposed to glutamate,
glycine, and alanine reported by Lau et al. [18]. These dif-
ferences are likely related to the fact that, in this study, en-
zymatic hydrolysis was performed on a different source of
corn stover, using different commercial enzymes. How-
ever, these changes in amino acid proportions, due to dif-
ferences in feedstock and enzyme sources, did not affect
the overall fermentation profiles of S. cerevisiae 424A
(LNH-ST) grown on the hydrolysate, as the results ob-
tained in this study are comparable to those of our previ-
ous work [10].
The free ammonium concentration found in the hydrol-
ysate was the same as the total protein (1.44 g/L) and sig-
nificantly different from the value reported previously
(0.75 g/L) [18]. The concentration of free ammonium in
the hydrolysate is dependent on the levels of residual am-
monia left adsorbed on the biomass after AFEX pretreat-
ment, which may vary due to differences in the relative
organic acid content of the feedstock and the efficiency of
ammonia removal by evaporation in the fume hood fol-
lowing pretreatment. However, these variations in ammo-
nium concentration between pretreatment batches did not
have significant effects on the fermentation profiles of S.
cerevisiae 424A (LNH-ST) compared to previous studies
[10,13]. This observation suggests that nitrogen is not a
limiting factor for efficient fermentation of ACH. Vitamin
concentrations reported in this manuscript (Table 2) were
based on results previously reported by our laboratory
[18]. A sensitivity analysis was carried out to evaluate the
impact of vitamin concentrations on the fermentability of
SH and verify if using previously reported values is a rea-
sonable assumption for this work. For this purpose, exper-
iments using ACH with and without 50% vitamin
supplementation (based on values from Table 2) were per-
formed. As no significant differences were observed on
the fermentation profiles (data not shown) during this sen-
sitivity analysis, it was reasonable to assume that the
values obtained in the previous study could be used to es-
timate the vitamin composition of the SH (Table 2). It is
also important to note that the goal of this study is to for-
mulate a SH based on a typical composition of an indus-
trially relevant biomass hydrolysate, which can vary
significantly depending on the origin of the feedstock and
enzymes used. Therefore, using values from our previous
study was a reasonable assumption for achieving the
aforementioned goals of the current study.
The mineral content of ACH was quantified by ICP-
MS. Macro-elements such as P, K, and Mg were present
in concentrations above the optimum range required for
yeast growth defined by Jones and Greenfield (1984)
[22]. These minerals are essential to all yeast and must
be present in millimolar concentrations for optimal cell
growth [22]. From Table 2, it is possible to observe an
extremely high level of K (3886.50 mg/L), which resulted
from the utilization of KOH for pH adjustment to 4.8
during enzymatic hydrolysis. Unfortunately, pH main-
tenance is essential to maximize enzymatic hydrolysis
conversions; therefore, little could be done to avoid the
accumulation of this macro-element.
Elements such as Na, Ca, and Mn are also available in
concentrations above the optimum for yeast growth
Tang et al. Biotechnology for Biofuels (2015) 8:1 Page 7 of 17
[22]. However, the effect of high levels of trace elements
in combination with other components found in ACHs
is not yet understood. Chelation effects and ionic inter-
actions with hydrolysate components may affect the
optimum range of these minerals for yeast growth [23].
Characterization of pretreatment-derived decompos-
ition products and potential plant- derived inhibitory
compounds present in the hydrolysate was performed
using targeted LC- and GC-MS analysis. In our previous
work, the most abundant compounds produced during
AFEX pretreatment of CS were identified and quantified
[15]. That work served as the platform to characterize
the hydrolysate composition described here. In Table 4,
these products were categorized into nitrogenous com-
pounds, furans, aliphatic acids, aromatic compounds,
and carbohydrates.
The concentration of nitrogenous compounds and fu-
rans was calculated based on the amounts present in
AFEX-CS, as previously reported by Chundawat et al.
[15]. High levels of sugars in the hydrolysate interfered
with the direct quantification of these products by GC-
MS, including acetamide, pyrazines, imidazoles, and fu-
rans. We considered removing those monomeric sugars
from the hydrolysate prior to GC-MS analysis; however,
this would have required extensive sample preparation
and would thus affect the accuracy of absolute quantifi-
cation of each target compound (without extensive
method development, see previous study for issues en-
countered during typical GC-MS analysis in presence of
high soluble sugar background [24]). For achieving the
major goals of our current study, we have assumed that
all nitrogenous compounds and furans found in AFEX-
CS were totally solubilized (with 100% recovery) in the
supernatant during enzymatic hydrolysis, as they are
highly soluble at those concentration levels (see Table 4
for details). All other compounds presented in Table 4
were directly quantified in the hydrolysate using HPLC
and LC-MS analysis.
Carbohydrates are by far the most abundant com-
pounds in the hydrolysate, where 60 g/L glucose, 26 g/L
xylose, and 5 g/L arabinose were quantified as the major
carbohydrate monomers (Table 4). The concentration of
carbohydrates (and other compounds) depends on the
solids loading used during enzymatic hydrolysis of the
pretreated biomass. In this work, 18% solids loading en-
zymatic hydrolysis was performed to create the ACH,
giving a sufficient concentration of sugars to produce
approximately 4 wt% ethanol after fermentation. This
enzymatic hydrolysis condition is considered to be in-
dustrially relevant; therefore, results from this study have
practical industrial relevance. However, typically under
such high solids loadings, the enzymes are inhibited by
high concentrations of soluble sugars [25,26], leading to
the progressive accumulation of sugar oligomers derived
from xylan and glucan. This likely explains the presence
of 12 g/L and 18 g/L of gluco- and xylo-oligomers, re-
spectively, in the ACH.
From Table 4, it is clear that, besides carbohydrates, the
major water soluble plant-derived compounds present in
ACH are feruloyl amide, p-coumaroyl amide, acetamide,
acetic acid, trans-aconitic acid, formic acid, and p-couma-
ric acid. All these components are present in the hydrolys-
ate in concentrations above 300 mg/L and, therefore, their
presence at such levels can potentially impact the per-
formance of yeast fermentation during biofuel production.
The nitrogenous compounds presented in Table 4 are
products of reactions between plant cell wall components
and ammonia, which are produced during AFEX pretreat-
ment [15]. For example, acetamide, feruloyl amide, and p-
coumaroyl amide are products of ammonolysis reactions
that cleave ester-bound acetates, coumarates, and feru-
lates, which are abundantly present in the plant cell wall
of CS [27,28]. These reactions are thought to be important
for the efficacy of the pretreatment, by disrupting the ester
cross-links between carbohydrates and lignin, or by deace-
tylating the xylan backbone of hemicellulose [15,28,29].
The acid counterparts of these amides, that is, acetic acid,
ferulic acid, and p-coumaric acid, are products of hydroly-
sis of the same esters, which also occur during AFEX due
to the presence of hydroxyl ions in the pretreatment
media [15]. Similarly to dilute acid pretreatment, formic
acid is also widely produced during AFEX; however, it is
formed by a different mechanism, likely via alkaline peel-
ing reactions of polysaccharides [15,30,31]. On the other
hand, trans-aconitic acid is not regarded as a typical AFEX
pretreatment-derived decomposition product, but it is a
well-known plant metabolite that is particularly abundant
in grasses, including maize [15,32,33]. Therefore, its pres-
ence in a CS-derived hydrolysate at these levels is
expected.
Other less abundant products present in the hydrolysate,
also listed in Table 4, are included in various categories
such as nitrogenous compounds, furans, aliphatic acids,
and aromatic compounds. Though they are present in
lower amounts in the hydrolysate, their inclusion in the
SH is important because their cumulative and synergistic
inhibitory effects may be significant during microbial fer-
mentation [34].
Formulation of a control synthetic medium
As mentioned above, ACH contains nutrients, as well as
plant-derived compounds that are potentially inhibitory
to microorganisms. The control synthetic medium was
formulated to contain a similar level of nutrients as the
biomass-derived hydrolysate, without the plant-derived
inhibitory components. Table 3 summarizes the nutrient
formulation of the control synthetic medium used in this
work. Specifically, (NH
4
)
2
SO
4,
peptone, and vitamins
Tang et al. Biotechnology for Biofuels (2015) 8:1 Page 8 of 17
were used to match the concentrations of ammonia, pro-
tein, and vitamins, respectively, present in the hydrolysate.
The concentrations of mineral elements added to the con-
trol synthetic medium were largely matched by adding a
selection of salts as described in Table 3. The salts were
carefully selected to avoid solubility problems during media
preparation. In general, chlorine-based salts showed higher
solubility in the synthetic hydrolysate than the sulfate,
phosphate, or carbonate counterparts. However, a recent
studybyCaseyet al. [35] revealed that chloride salts can
be more detrimental to the specific xylose consumption
rate of S. cerevisiae 424 A (LNH-ST) compared to their
sulfate counterparts, for example. Therefore, high concen-
trations of chlorine anions in solution could negatively
affect xylose fermentation. To avoid the presence of high
levels of chlorine-based salts in the synthetic medium, we
selected potassium salts with three different anion pairs
and sodium salts with two different anion pairs (Table 3).
For the same reason, we also chose to use Ca(NO
3
)
2
in-
stead of CaCl
2
.
In this synthetic control medium, S. cerevisiae 424A
completely consumed glucose and xylose in 18 and 72 h,
respectively, generating ethanol at a concentration of
around 35 g/L (about 80% metabolic yield) and a cell
density (OD 600 nm) of approximately 12 (Figure 1).
These results suggest that ACH is not limited by nitrogen,
protein, or micronutrients for consuming glucose and xy-
lose during ethanol production (though the rate of xylose
uptake is significantly slower than that of glucose). How-
ever, determining the nutrient composition of AFEX pre-
treated biomass hydrolysates and formulating a control
synthetic medium is critical to further improving micro-
bial co-fermentations for more efficient and rapid conver-
sion of lignocellulosic hydrolysates in the presence of
inhibitory compounds. This is highlighted by the fact that
xylose fermentation is affected by the nutrients level in the
medium and that individual decomposition products in
ACH are not very inhibitory for robust yeast species such
as S. cerevisiae [10]. Thus, it is likely that the inhibitory ef-
fect of many of the plant-derived compounds present in
the hydrolysate would not be observed in nutrient-rich
media (such as Yeast Extract Peptone, YEP medium).
Though the control synthetic medium formulated in this
work is not exactly comparable with the actual biomass
hydrolysate due to experimental limitations, the nutrient
value is close enough to be considered acceptable for
studying the effect of different inhibitors on strain per-
formance. A sensitivity analysis was performed to deter-
mine how the concentration of amino acids affects cell
growth and the fermentation performance. It was found
that a variation of the amino acid concentration by up to
two times the amount detected in the hydrolysate did not
affect cell growth; however, it did improve the xylose con-
sumption rate (data not shown).
Inhibitory effect of different classes of compounds from
ACH on S. cerevisiae 424A fermentation
As previously mentioned, the plant-derived compounds
and pretreatment decomposition products present in
ACH were divided into five groups: nitrogenous com-
pounds, organic acids, aromatic compounds, carbohy-
drates, and furans (Table 4). The effect of the different
groups of lignocellulose decomposition products on S.
cerevisiae 424A fermentation was investigated and com-
pared with the control synthetic medium (blank) formu-
lated in this work (Table 5). The various classes of
compounds identified in ACH were added to the blank
medium at an abundance comparable to that in the ac-
tual hydrolysate to determine their individual and com-
binatorial inhibitory contribution to yeast fermentation.
From the results presented in Table 5, the nitrogenous
compounds caused a significant decrease in cell biomass
yield, xylose consumption rate, and 24 h ethanol prod-
uctivity compared to the blank control SM (Additional
file 1: Table S2-1, S2-2, and S2-4). Though this class of
compounds is not usually found in most lignocellulosic
hydrolysates, certain amides are produced by a variety of
plants and are known to have anti-fungal effects [36]. As
nitrogenous compounds are quite abundant in AFEX
biomass-derived hydrolysates and limited information
about their inhibitory effect on microbes is currently
available in the literature, we will discuss this in more
detail in the subsequent section.
Similarly to the effect of nitrogenous compounds, the
xylose consumption rate and cell biomass yields were
also negatively affected by the addition of aliphatic acids
and aromatic compounds (Table 5). On the other hand,
the ethanol metabolic yield was enhanced by the pres-
ence of these two classes of compounds, which is con-
sistent with earlier findings in the literature [37]. As
these weak acids will be present in the hydrolysate solu-
tion predominantly in their non-dissociated form, they
will be permeable through the yeast cell membrane [38].
Once they enter the cytosol, the acids will dissociate and
the cell will be forced to pump excess protons through
the membrane to maintain homeostasis. Though low
concentrations of organic acid have been observed to in-
crease ethanol yields and fermentation rates, this benefit
is lost at higher acid concentrations [39-42]. High levels
of anionic acid species are also toxic to the cell and can
result in cessation of growth or cell death [41,43], which
does not seem to be the case for ACH. Lignin-derived
aromatic compounds, such as phenols, are also known
to inhibit S. cerevisiae growth, especially lower molecular
weight phenolics. The toxicity of these compounds is
dependent on the relative position (ortho, meta, or para)
of the functional group in the benzene ring [44] and also
on the type of functional group (for example, aldehydes,
ketones, or acids). The phenolic compounds may interact
Tang et al. Biotechnology for Biofuels (2015) 8:1 Page 9 of 17
with biological membranes, interfering with their function.
However, the inhibition mechanism of this family of com-
pounds is not well understood [45].
The oligomeric carbohydrates (particularly xylo-
oligomers) also negatively affected xylose consump-
tion rate in the first 24 h (18% of the control xylose
consumption was reduced in the first 24 h). However,
at the 48 h time point this difference was reduced to
2.7% of the control xylose consumption. As a result,
the 48 h ethanol metabolic yield was only reduced by
2.5% of the control in the first 48 h. To our know-
ledge, xylose consumption inhibition by oligomeric
carbohydrates has never been reported in the litera-
ture, and it would be interesting to determine the pos-
sible reason for this observation in a future study.
Addition of furans did not affect fermentation kinetics
in great extent compared to the control (blank). The re-
sults from Table 5 show that there were no significant
differences in biomass yield, 24 h ethanol productivity,
and 48 h acetate yields compared to the blank SM (see
Additional file 1: S2). Although the other parameters
shown in Table 5 related to furan addition were statistically
different from those of the blank SM, the observed differ-
ence was not very pronounced. The inhibitory effects of fu-
rans (such as furfural and hydroxymethyl furfural) on
cellular metabolism have been thoroughly studied by sev-
eral researchers [37,46]. These effects include oxidative
damage of yeast cells by lower abundance of reducing
agent concentrations (such as NADPH and NADH) and
reduced activities of enzymes involved in the glycolysis
pathway. From the most common furans found in lignocel-
lulosic hydrolysates, furfural seems to be more inhibitory
when compared to 5-HMF, at equivalent concentrations
[47]. As AFEX pretreatment produces a low level of 5-
HMF (Table 4) and practically no furfural, the concentra-
tion of this class of compounds in the hydrolysate appears
to be low enough to avoid oxidative damage during yeast
fermentation.
The synergistic inhibitory effect of the various classes
of decomposition products (DP) was observed on xylose
fermentation. The combination of all compounds (blank +
DP in combination) showed a higher inhibitory effect than
the aggregate value of individually added products (48 h
data). This result agrees with previous reports that also
observed synergies on the inhibitory effect of different com-
pounds during yeast fermentation [47].
Among all the classes of decomposition products
tested herein, nitrogenous compounds were the most in-
hibitory to xylose fermentation (Table 5), which could
potentially be explained by their relatively higher con-
centration in the hydrolysate.
In the presence of aliphatic acids, about 70% decrease in
acetate production was observed compared to the blank
SM (Table 5). This result may be related to end-product
(acetate) inhibition of the acetate synthesis pathway in
yeast. Moreover, when all the decomposition products were
added together, acetate was consumed by the yeast after
48 h fermentation, instead of being produced. It is possible
that the yeast cells consume acetate to equilibrate the redox
imbalance caused by the xylose metabolic pathway and due
to the presence of high concentrations of other inhibitory
compounds [48]. However, to better understand this find-
ing, more detailed metabolomic experiments will need to
be carried out in the future using SHs.
The carbon mass balance closures for the various syn-
thetic media evaluated in Table 5 are approximately
020406080
0
10
20
30
40
50
60
OD 600nm
Concentration (g/L)
Time (h)
Glucose
Xylose
Ethanol
0
5
10
15
20
25
OD
A
Blank
Figure 1 Fermentation profile of the control synthetic medium (blank) without addition of nutrients.
Tang et al. Biotechnology for Biofuels (2015) 8:1 Page 10 of 17
Table 5 Fermentation parameters for synthetic media (SM) in presence of various groups of lignocellulose decomposition products (DP)
e
Biomass yield
b
(g/g) Xylose consumption (%) Ethanol productivity (g/L/h) Ethanol yield
c
(g/g)
Glycerol yield (g/g) Xylitol yield
(g/g)
Acetate yield
(g/g)
Carbon
balance
closure
18 h 24 h 48 h 24 h 48 h 48 h 48 h 48 h 48 h
Blank SM
a
0.078 ± 0.002 66.92 ± 0.18 97.08 ± 0.04 1.29 ± 0.01 0.71 ± 0.01 0.406 ± 0.001 0.058 ± 0.000 0.053 ± 0.002 0.007 ± 0.000 1.00
Blank + Nitrogenous compounds 0.067 ± 0.003 44.69 ± 0.68 86.92 ± 0.61 1.21 ± 0.00 0.69 ± 0.01 0.402 ± 0.007 0.060 ± 0.001 0.055 ± 0.003 0.007 ± 0.000 0.98
Blank + Aliphatic acids
d
0.068 ± 0.002 58.95 ± 0.16 94.17 ± 0.04 1.23 ± 0.01 0.73 ± 0.01 0.427 ± 0.001 0.051 ± 0.000 0.042 ± 0.001 0.002 ± 0.000 1.01
Blank + Aromatic compounds 0.072 ± 0.002 68.70 ± 0.19 88.34 ± 0.04 1.29 ± 0.00 0.72 ± 0.01 0.421 ± 0.001 0.051 ± 0.000 0.032 ± 0.001 0.007 ± 0.000 1.00
Blank + Carbohydrates (oligos) 0.074 ± 0.002 54.68 ± 0.98 94.49 ± 0.50 1.28 ± 0.00 0.73 ± 0.00 0.416 ± 0.001 0.059 ± 0.000 0.043 ± 0.000 0.007 ± 0.000 1.01
Blank + Furans 0.078 ± 0.002 69.25 ± 0.19 96.90 ± 0.04 1.29 ± 0.00 0.72 ± 0.01 0.410 ± 0.001 0.059 ± 0.000 0.052 ± 0.002 0.007 ± 0.000 1.01
Blank + DP in combination
d
0.059 ± 0.002 21.17 ± 0.09 40.05 ± 1.28 1.20 ± 0.01 0.64 ± 0.00 0.440 ± 0.000 0.045 ± 0.000 0.024 ± 0.000 −0.002 ± 0.000 0.99
Actual Hydrolysate
d
0.065 ± 0.001 14.91 ± 0.53 43.31 ± 0.47 1.26 ± 0.01 0.73 ± 0.00 0.474 ± 0.002 0.048 ± 0.001 0.018 ± 0.000 −0.003 ± 0.001 1.06
a
The blank was the synthetic medium without the addition of decomposition products (DP).
b
Biomass yield was based on both glucose and xylose consumed at 18 h fermentation, when the cell density reached the maximum. One unit of absorbance at 600 nm is approximately equal to 0.48 g dry cell wt/L.
c
Theoretical metabolic yield of ethanol for both sugars was 0.51 g EtOH/g consumed sugar.
d
The initial concentration of acetate in the hydrolysate and synthetic medium with the addition of aliphatic acids and DP in combination was 1.9 g/L.
e
The t-test results for determining statistically significant differences between the different results are presented in Additional file 1, S2, Tables S2-1 - S2-9.
Tang et al. Biotechnology for Biofuels (2015) 8:1 Page 11 of 17
equal to 1. However, for the actual hydrolysate the car-
bon mass balance closes at 1.06, which means that there
is 6% more carbon being formed than the carbon con-
sumed. This observation may suggest that there are
other carbon sources present in small quantities in the
actual hydrolysate, which were not detected or analyzed
in this study. More in-depth characterization is required
to determine the minor carbon sources that contribute
to this carbon mass balance closure.
Inhibitory effect of individual families of nitrogenous
compounds
Since the effects of nitrogenous products on fermenta-
tion are particularly less well understood than the
remaining categories of compounds, and because these
products are specifically linked to ammonia-based pre-
treatment, we decided to further investigate their indi-
vidual effect on the fermentation profile of S. cerevisiae
424A. Here, we evaluated in more detail the effect of
various 1) pyrazines, 2) imidazoles, and 3) amides on xy-
lose consumption and ethanol production rates (Table 6
and Figure 2). The results show that the addition of pyr-
azines or imidazoles to a well-defined SM did not sig-
nificantly affect the kinetics of xylose consumption and
ethanol production (P-value ≥0.05, see Additional file 1,
S3, Tables S3-2 - S3-4). These two families of com-
pounds are not present in the hydrolysate at high con-
centrations and therefore are likely not to have any
major inhibitory effect on yeast fermentation. However,
amides are present at much higher concentration in the
hydrolysate, and their addition to the blank media re-
sulted in decrease of biomass yield, xylose consumption
rate, and ethanol productivity. Specifically, with the addition
of amides the biomass yield, xylose consumption, and etha-
nol productivity were reduced from 0.068 g/g, 95%, and
0.71 g/L/h to 0.061 g/g, 80%, and 0.65 g/L/h, respectively.
Though we see some level of inhibition on xylose con-
sumption and ethanol production, the mechanisms of
amide inhibition are not well understood. It is possible that
phenolic amides have a similar mechanism of inhibition to
the lignin-derived phenolic compounds, which tend to im-
pact the integrity of the cell membranes when present at
high concentrations [44,45].
Comparison between the inhibitory effects of amides and
the corresponding carboxylic acids
Three amides (feruloyl amide, coumaroyl amide, and
acetamide) present in the hydrolysate were further stud-
ied individually and their inhibition profiles were com-
pared to their corresponding acid forms (ferulic acid,
coumaric acid, and acetic acid) in the blank synthetic
medium (Figure 3). Unlike the previous experiments re-
ported herein, the concentration of amides and acids
chosen for this study was not based on their actual
amount in the ACH. In this case, it was assumed that all
the reacting esters present in the biomass were cleaved
by ammonolysis or hydrolysis reactions, respectively. As
a result, the same exact molar concentrations of the acid
and amide counterparts were used for each comparative
inhibition experiment.
In contrast to our previous results for when the am-
ides were added together, the individual amides did not
show a substantial inhibitory effect on fermentation
compared to the control. Xylose was completely con-
sumed to undetectable levels within 72 h with maximum
OD
600
of around 12 for all the amides tested in this
work (Figure 3). Therefore, the inhibitory behavior of
amides is likely a synergistic effect, coupled with the fact
that the total concentration of amides was higher than
when present individually. The corresponding acid forms
of those amide compounds, however, all showed sub-
stantial inhibition on cell growth and xylose fermenta-
tion. Among all acids, ferulic acid showed the highest
inhibitory effect followed by acetic acid and coumaric
acid, which was consistent with their relative abundance
in the ACH (Table 4). Furthermore, ferulic acid is known
to be a more potent inhibitor of yeast growth than cou-
maric acid, when present at similar concentrations.
From the results presented in Figure 3B, the presence of
ferulic acid in the fermentation media reduced the cell
density by 45%. The average xylose consumption rate
decreased to a very low 0.09 g/L/h (0 to 24 h), a much
larger decrease than that caused by feruloyl amide (re-
duced to 0.55 g/L/h). Ferulic acid even affected the glu-
cose consumption rate, which was not observed for any
other decomposition product tested herein. Complete
glucose consumption was only achieved after 48 h fer-
mentation instead of 18 h, as it was in the case of the
control blank medium.
From these results, it is evident that amides are less in-
hibitory than their corresponding acid forms, based on
the same molar concentration, on yeast fermentations.
Carboxylic acids permeate into the cytosol in their un-
dissociated form when performing fermentations at
pH 5.5. While in the cytosol, the acids dissociate due to
the near-neutral conditions of the cytosol, decreasing
the intracellular pH [38]. This effect will not be observed
for amides, which typically have pKa values greater than
10. This could partially explain why AFEX pretreated
biomass has greater fermentability compared to dilute
acid pretreated biomass [9]. Ester hydrolysis reactions
that occur during dilute acid and steam explosion pre-
treatments result in the formation of the organic acids
studied herein, probably at similar concentrations to the
ones used in this study. However, in the case of AFEX
pretreatment (under the presently employed conditions)
only about one third of the total available esters are hydro-
lyzed to yield acids, while the remaining are ammonolyzed
Tang et al. Biotechnology for Biofuels (2015) 8:1 Page 12 of 17
tothelessinhibitoryamides.Onepossiblewaytoenhance
the fermentability of AFEX pretreated biomass is to further
reduce the hydrolysis reaction products during pretreat-
ment and promote conditions that improve the selectivity
toward the less inhibitory ammonolysis reaction-derived
products.
Comparison between SH and ACH
The fermentation profile of S. cerevisiae 424A in SH was
compared side by side to the actual ACH as shown in
Figure 4. The cell growth during fermentation in the
control synthetic medium (blank) was comparable to
that of the actual hydrolysate, achieving a cell density of
OD
600
11.5 after 18 h (Figure 4D). However, cell growth
in the SH, in the presence of all the decomposition
products from Table 4, was greatly reduced, showing a
cell density of around OD
600
8 after 18 h fermentation.
This value represents just 68% of the cell density ob-
tained using the blank medium.
As expected, xylose was almost completely consumed
after 48 h fermentation in the control synthetic medium.
The average xylose consumption rate was 0.70 g/L/h (0
to 24 h). However, the xylose consumption rates in the
SH and ACH were 0.23 g/L/h and 0.28 g/L/h,
respectively, which were much lower than the control
rate. The lower cell density in the SH was one of the
possible causes of the decreased xylose consumption
rates. DP inhibition of specific xylose consumption rate
and decreasing viable cell density were probably the
other two reasons for the slow xylose fermentation [13].
Regarding the ethanol yield, SH and ACH results were
statistically different (0.439 g/g and 0.486 g/g, respect-
ively) and this difference represents about 10% of the
ACH ethanol yield. In both these cases, the ethanol
yields were significantly higher than the control
(0.405 g/g). This increased ethanol metabolic yield in the
presence of AFEX pretreatment-derived decomposition
products is consistent with our previous observations
and other reports [10,13,49]. The final ethanol concen-
trations achieved in the ACH, control synthetic medium,
and SH were 38 g/L, 35 g/L, and 32 g/L, respectively.
During the first 18 h period, glucose and xylose con-
sumption were equivalent for both media, and only after
18 h fermentation was it possible to observe significant
differences in xylose consumption and, consequently, in
ethanol production. Therefore, the higher ethanol yields
observed for the actual biomass hydrolysate seem to be
related to better xylose fermentation.
Table 6 Fermentation parameters of synthetic media (SM) with/without the addition of various nitrogenous
compounds commonly found in AFEX-CS hydrolysates (ACHs)
Biomass yield
(g/g)
Xylose consumption
a
(%)
Ethanol productivity
a
(g/L/h)
Ethanol yield
(g/g)
Glycerol yield
(g/g)
Xylitol yield
(g/g)
Acetate yield
(g/g)
Blank (SM)
b
0.068 ± 0.001 95 ± 1 0.71 ± 0.01 0.411 ± 0.005 0.059 ± 0.001 0.060 ± 0.004 0.010 ± 0.001
Blank + pyrazines 0.070 ± 0.002 95 ± 0 0.72 ± 0.00 0.416 ± 0.002 0.062 ± 0.003 0.061 ± 0.001 0.010 ± 0.004
Blank +
imidazoles
0.070 ± 0.002 95 ± 0 0.72 ± 0.00 0.414 ± 0.001 0.062 ± 0.003 0.060 ± 0.003 0.012 ± 0.000
Blank + amides 0.061 ± 0.000 80 ± 1 0.65 ± 0.01 0.390 ± 0.005 0.069 ± 0.001 0.056 ± 0.003 0.013 ± 0.001
a
Both xylose consumption and ethanol volumetric productivity are shown at 48 h.
b
Except for biomass yield, the differences between all other blank (SM) results from Tables 5and 6are not statistically significant (P> 0.05).
020406080
0
10
20
30
Xylose concentration (g/L)
Time (h)
Blank
Pyrazines
Imidazoles
Amides
020406080
0
10
20
30
40
Ethanol concentration (g/L)
Time (h)
AB
Figure 2 Time course profile for xylose uptake (A) and ethanol production (B) during fermentation by Saccharomyces cerevisiae 424A
(LNH-ST) in a defined minimal synthetic medium (or blank) with addition of pyrazines, imidazoles, and amides.
Tang et al. Biotechnology for Biofuels (2015) 8:1 Page 13 of 17
0 20406080
0
10
20
30
40
50
60
Concentration (g/L)
OD 600nm
Time (h)
0
5
10
15
20
25
Ferulic acid Feruloyl amide
020406080
0
10
20
30
40
50
60
OD 600nm
Concentration (g/L)
Time (h)
Glucose
Xylose
Ethanol
0
5
10
15
20
25
OD
020406080
0
10
20
30
40
50
60
OD 600nm
Concentration (g/L)
Time (h)
0
5
10
15
20
25
020406080
0
10
20
30
40
50
60
OD 600nm
Concentration (g/L)
Time (h)
0
5
10
15
20
25
Coumaric acid Coumaroyl
amide
Acetic acid Acetamide
AB
CD
Blank
Figure 3 Time course profile of co-fermentation in a minimal synthetic medium without the addition of pretreatment-based decomposition
products (blank) (A) and with the addition of 6.2 mM feruloyl amide and ferulic acid (B), 7.5 mM coumaroyl amide and coumaric acid (C),
and 28.8 mM acetamide and acetic acid (D) mimicking a dilute acid or ammonia pretreated lignocellulosic hydrolysate. Solid lines depict
acids; dashed lines depict amides.
Figure 4 Time course profile of co-fermentation in minimal synthetic medium (SM) with or without the addition of plant cell wall
decomposition products (DP) compared to hydrolysate (ACH). (A) and (B) depict glucose and xylose uptake, respectively; (C) depicts ethanol
concentration produced; and (D) depicts cell density as OD 600 nm.
Tang et al. Biotechnology for Biofuels (2015) 8:1 Page 14 of 17
For an ideal SH, one would expect identical cell growth
behavior, sugar consumption rates and ethanol yields to
those observed for the actual hydrolysate. The differences
in cell growth profile between the SH and the ACH may
be due to incomplete evaluation of the composition of the
actual hydrolysate, which is very complex and presents
various analytical challenges. Possible improvements for
future versions of the SH may include the analysis of
redox co-factors present in plant biomass (for example,
NAD(P)H), which could potentially help the yeast cells to
improve their fermentation performance. Also, the higher
concentration of chlorine-containing salts in the SH might
be another possible factor that could have caused such a
negative impact (Tables 2 and 3). Therefore, optimizing
the choice of salts to closely match the mineral content of
the ACH would help improve the performance of the SH.
Nevertheless, the SH presented in this study was success-
fully used to evaluate the relative levels of inhibition asso-
ciated with the various classes of compounds that are
present in the actual ACH. Moreover, as we performed a
detailed characterization of the amino acids present in the
ACH (Additional file 1: Table S1), it is possible to formu-
late a well-defined synthetic medium by the addition of in-
dividual amino acids, at the respective concentrations, in
contrast to peptone. The utilization of defined synthetic
media will be important for future multi-omics studies
that will help us understand the mechanisms of inhibition
under well-controlled experimental conditions.
Conclusion
In this work, nutrients and decomposition products
present in ACH were characterized with the goal of for-
mulating a synthetic hydrolysate, which will be used in
multi-omics analysis for understanding the inhibition
mechanisms of the lignocellulosic hydrolysate. This work
also provides an example showing how synthetic ligno-
cellulosic hydrolysates derived from other pretreatment
technologies (such as dilute acid and steam explosion)
can be formulated.
The ACH contained high levels of nitrogenous com-
pounds, notably phenolic amides and acetamide. Due to
their presence at high concentrations, their observed in-
hibitory effect on xylose consumption and ethanol pro-
duction was the most significant among all the families
of compounds tested herein, which included aliphatic
acids, furans, lignin-derived phenolic compounds, and
oligomeric carbohydrates. However, when comparing the
inhibition due to amides at the same molar concentra-
tions as their acid counterparts, we observed that amides
are significantly less inhibitory to both glucose and xy-
lose fermentation than the acids. The reduced inhibitory
effect of amides is a major advantage of AFEX- and
ammonia-based pretreatments over other pretreatment
technologies that mainly produce carboxylic acids as
decomposition products. Because of the reduced pro-
duction of carboxylic acids and furans, notably furfural
and 5-HMF, the ACH is easily fermentable without any
detoxification.
Although we were able to identify the major groups of
inhibitory compounds present in the ACH, the SH did
not exactly match the performance of the actual hydrol-
ysate. The cell density in SH was considerably lower
than in the actual hydrolysate and, as a consequence, the
xylose consumption rate was also slightly reduced. How-
ever, the proposed SH was instrumental in identifying
the inhibitory effect of various classes of compounds
present in the hydrolysate and their relative contribution
to the overall inhibition. Due to the complexity of the lig-
nocellulosic hydrolysate composition, we will likely develop
more representative versions of the SH as we learn more
about the composition of actual hydrolysates. The SH for-
mulation will be instrumental in future multi-omics studies
to understand the nature of AFEX pretreatment-specific
decomposition products and how they inhibit yeast and
bacteria, so that we can engineer better strains to maximize
biofuel yields and productivity.
Endnote
a
TM - AFEX is a trademark of MBI International,
Lansing, Michigan.
Additional file
Additional file 1: S1. Amino acid content of AFEX-CS hydrolysate (ACH)
and Peptone. Table S1. Analysis of amino acid content in AFEX-CS
hydrolysate (ACH) and peptone. Calculation of the peptone equivalent
concentration to meet the total amino acid value present in ACH. S2.
Statistical analysis for Table 5. Table S2-1. Map of P-value range from
t-test for biomass yield results. Table S2-2. Map of P-value range from
t-test for 24 h xylose consumption results. Table S2-3. Map of P-value
range from t-test for 48 h xylose consumption results. Table S2-4. Map
of P-value range from t-test for 24 h ethanol productivity results. Table
S2-5. Map of P-value range from t-test for 48 h ethanol productivity
results.S3. Statistical analysis for Table 6. Table S3-1. Map of P-value from
t-test for 18 h biomass yield results. Table S3-2. Map of P-value from
t-test for 48 h xylose consumption results. Table S3-3. Map of P-value
from t-test for 48 h ethanol productivity results. Table S3-4. Map of
P-value from t-test for 48 h ethanol yield results. Table S3-5. Map of
P-value from t-test for 48 h glycerol yield results. Table S3-6. Map of
P-value from t-test for 48 h xylitol yield results. Table S3-7. Map of P-value
from t-test for 48 h acetate yield results.
Abbreviations
AA: amino acid; ACH: AFEX™pretreated corn stover hydrolysate;
AFEX: Ammonia Fiber Expansion; CS: corn stover; DP: decomposition
products; 5-HMF: 5-hydroxymethylfurfural; SH: synthetic lignocellulosic
hydrolysate; SM: synthetic medium; UT-CS: untreated corn stover; YEP: Yeast
extract peptone.
Competing interests
The authors declare that they have no competing interests.
Authors’contributions
XT produced the ACH and performed the fermentation studies, purification
of synthesized compounds, and sample preparation for amino acid, mineral,
Tang et al. Biotechnology for Biofuels (2015) 8:1 Page 15 of 17
and pretreatment decomposition product analyses. XT also contributed to
the formulation of the SH, the experimental design, and the preparation of
the manuscript. LDCS contributed in the synthesis of the amides used in the
SH, the experimental design, formulation of the SH, data interpretation, and
preparation of the manuscript. MJ contributed to the design of the
fermentation experiments, data interpretation, and preparation of the
manuscript. SPSC contributed to the methodology development, data
interpretation, and preparation of the manuscript. CKC contributed by
analyzing/quantifying the pretreatment decomposition products in ACH and
provided important insight during the preparation of the manuscript. MWL
helped with the methodology development and contributed by providing
guidelines to formulate the SH. ZX and BED participated on the identification
of the problem and helped with the preparation of the manuscript. VB also
participated in the identification of the problem, helped with the
experimental design, and coordinated this work. All authors read and
approved the final manuscript.
Acknowledgements
We would like to acknowledge Novozymes and Genencor for kindly
providing the enzymes used in the production of the lignocellulosic
hydrolysate and oligomeric xylan. We thank Professor Nancy Ho from Purdue
University for kindly providing the recombinant Saccharomyces cerevisiae
424A (LNH-ST) strain. We would like to acknowledge Professor Dan Jones for
making available some of the analytical instrumentation at the MSU mass
spectrometry facility for hydrolysate analysis. We also want to thank Charles
Donald, Christa Gunawan, and Jeffrey Halim for their assistance in sample
preparation and analysis. This work was partially funded by the DOE Great
Lakes Bioenergy Research Center (GLBRC), grant number DE‐FC02‐
07ER64494. Leonardo Sousa was funded by Fundação para a Ciência e a
Tecnologia and European Social Fund, grant number SFRH/BD/62517/ 2009.
Xiaoyu Tang was supported by the China Scholarship Council, grant number
2009101936.
Author details
1
Biogas Institute of Ministry of Agriculture, Section 4-13 Remin South Road,
Chengdu 610041, P. R. China.
2
DOE Great Lakes Bioenergy Research Center,
Biomass Conversion Research Lab (BCRL), Chemical Engineering and
Materials Science, Michigan State University, 3815 Technology Boulevard,
Suite 1045, Lansing 48910, USA.
3
Department of Chemical & Biochemical
Engineering, Rutgers, The State University of New Jersey, 98 Brett Road,
Room C-150A, Piscataway, NJ 08854, USA.
4
Department of Chemistry and
Biochemistry, Baylor University, Waco, TX 76798, USA.
5
School of Chemical
Engineering, Sichuan University, No. 24 South Section 1, Yihuan Road,
Chengdu 610065, P. R. China.
Received: 22 April 2014 Accepted: 4 December 2014
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