Bioresource Technology 220 (2016) 208–214

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

Production of D-lactate from sugarcane bagasse and corn stover

hydrolysates using metabolic engineered Escherichia coli strains
José Utrilla 1,2, Alejandra Vargas-Tah 1, Berenice Trujillo-Martínez 1,3, Guillermo Gosset, Alfredo Martinez ⇑
Departamento de Ingeniería Celular y Biocatálisis, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Apdo. Postal 510-3, Cuernavaca, Mor. 62250, Mexico

h i g h l i g h t s

 D-Lactogenic Escherichia coli was engineered to reduce by-product formation.

 Laboratory simulated hydrolysates were used to evaluate strain performance.
 D-Lactate production with plant hydrolysates was evaluated.
 Sugar cane bagasse and corn stover hydrolysates were efficiently fermented.
 Lactate/sugar yields close to 0.95 g/g were obtained in all cases.

a r t i c l e i n f o a b s t r a c t

Article history: In this study, the lactogenic Escherichia coli strain JU15 was used and modified to produce D-lactate (D-LA)
Received 11 July 2016 from plant hydrolysates with a minimal nutrient addition in pH controlled fermenters. Results showed
Received in revised form 15 August 2016 that strain JU15 produces D-LA with high yield and productivity in laboratory simulated hydrolysate
Accepted 17 August 2016
media and actual sugar cane bagasse hemicellulosic hydrolysate. Strain JU15 showed sequential carbon
Available online 20 August 2016
source utilization and acetic acid production. The L-lactic and acetic acid production pathways were
deleted in JU15, resulting strain AV03 (JU15 DpoxB, DackA-pta, DmgsA), which showed simultaneous con-
Keywords:
sumption of glucose and xylose and no acetic acid production in the simulated hydrolysate. The D-LA
D-Lactate
Lactogenic Escherichia coli
yield from hydrolysate sugars was close to 0.95 gD-LA/gsugars in all cases. Our results show that D-LA can
Hemicellulosic hydrolysates be produced from plant hydrolysates in simple batch fermentation processes with a high productivity
Sugar cane bagasse using engineered E. coli strains at fermenter scales from 0.2 up to 10 L.
Corn stover Ó 2016 Elsevier Ltd. All rights reserved.

1. Introduction amounts of mannose and galactose) sugar mixtures obtained from

The production of optically pure D-lactate has become a key ele-
ment for the production of stereo complex polylactic acid (PLA)
with higher heat deflection temperature suitable for bioplastic
applications. (Li et al., 2016; Madhavan Nampoothiri et al., 2010).
The use of non-food carbon sources or agriculture wastes such as
sugarcane bagasse (SCB) or corn stover (CS), requires the develop-
ment of specific strains and processes with the ability to use the C5
(mainly xylose and arabinose) and C6 (mainly glucose, and lower
⇑ Corresponding author.
E-mail address: alfredo@ibt.unam.mx (A. Martinez).
1
These authors contributed equally to this work.
2
Current address: Laboratorio de Biología de Sistemas y Biología Sintética, Centro
de Ciencias Genómicas, Universidad Nacional Autónoma de México, Av. Universidad
S/N, Chamilpa, Cuernavaca, Mor. 62210, Mexico.
3
Current address: PROBIOMED, S.A. de C.V. Cruce de carreteras Acatzingo-
Zumpahuacán, Tenancingo, Estado de México 03020, Mexico.
plant hydrolysis process and to tolerate hydrolysate inhibitors
(Avci et al., 2013; Piotrowski et al., 2014; Vargas-Tah et al., 2015;
Zaldivar et al., 1999).
There are several metabolic engineered E. coli strains that pro-
duce D-lactate (D-LA) (Eiteman and Ramalingam, 2015; Orencio-
Trejo et al., 2010; Wang et al., 2013; Zhou et al., 2016; Zhu et al.,
2007) among those, strain JU15 shows several advantages when
using plant hydrolysates as feedstock (Utrilla et al., 2012). The
strain JU15 was engineered to produce D-LA from xylose by the
inactivation of the ATP-dependent xylose transporter (xylFGH)
and adaptive laboratory evolution in mineral media with 12%
xylose. This strain is able to convert xylose into D-LA with high pro-
ductivity and yield (0.8 gD-LA/L.h and 0.95 gD-LA/gXYL, respectively)
using mineral media (Utrilla et al., 2012). Remarkably, JU15 deriva-
tive strains are capable of tolerating up to 10 g/L of acetic acid, an
important hydrolysate toxic compound, without decreasing its
growth rate (Fernández-Sandoval et al., 2012). Although the

http://dx.doi.org/10.1016/j.biortech.2016.08.067
0960-8524/Ó 2016 Elsevier Ltd. All rights reserved.
 J. Utrilla et al. / Bioresource Technology 220 (2016) 208–214
D-LA/sugar yield is high in strain JU15, some acetic acid production
has been observed. Also, L-lactic acid contamination has been
reported in D-LA production strains after adaptive laboratory evo-
lution (Grabar et al., 2006).
In the present work, strain JU15 was used and further engi-
neered to convert the hemicellulosic hydrolysates of sugarcane
bagasse and lignocellulosic hydrolysates from corn stover into D-
LA. In order to reduce acetic acid and L-lactic acid formation, the
strain JU15 was metabolically engineered by removing the poxB,
mgsA, and ackA-pta genes to generate strain AV03. Laboratory sim-
ulated hydrolysates were used to evaluate strain performance and
by-product formation. JU15 strain was used to produce D-LA from
SCB hemicellulosic hydrolysates and scaled up to a 10 L fermenta-
tion and strain AV03 was used to produce D-LA from CS hydroly-
sate, both with minimal media supplements. This work shows
the production of D-LA from plant hydrolysates with high yield
using metabolic engineered E. coli strains in a batch simple fermen-
tation process.
2. Materials and methods
2.1. Organisms and culture conditions
The strain JU15 (MG1655, DpflB, DadhE, DfrdA, DxylFGH, gatC-
S184L, DmidarpA, Dreg 27.3 kb) was used (Utrilla et al., 2012) and
genetically modified to delete the pathways for the production of
acetic acid and L-lactic acid, leading to the generation of strain
AV03 (JU15 DpoxB, DackA-pta, DmgsA). The elimination of genes
on JU15 was performed by homologous recombination using the
method reported by Datsenko and Wanner (Datsenko and
Wanner, 2000). To block the pathway leading to the production
of acetic acid from pyruvic acid the poxB gene was deleted. The pri-
mers used to perform this mutation were poxBDFw (TCA GAT GAA
CTA AAC TTG TTA CCG TTA TCA CAT TCA GGA GAT GGA GAA CCG
TGT AGG CTG GAG CTG CTT CG) and poxBDRv (TCA GAT GAA CTA
AAC TTG TTA CCG TTA TCA CAT TCA GGA GAT GGA GAA CCG TGT
AGG CTG GAG CTG CTT CG). The deletion was confirmed by PCR
using the following primers: poxBFw (GTG AGC AGC ACA ATG
ACG CTG A) and poxBRv (AGT GAC TGA GCA GAG CGA CCA G).
The reversible production of acetic acid from acetyl-CoA acid was
blocked deleting in tandem the ackA and pta genes. For such a pur-
pose, the ackADFw (CTC TAT GGC TCC CTG ACG TTT TTT TAG CCA
CGT ATC AAT TAT AGG TAC TTC C GT GTA GGC TGG AGC TGC TTC
G) and ptaDRv (GAT TAT TTC CGG TTC AGA TAT CCG CAG CGC AAA
GCT GCG GAT GAT GAC GAG ACA TAT GAA TAT CCT CCT TAG TTC)
primers were used to perform the homologous recombination. This
deletion was confirmed by PCR using the following primers: ack-
FWf (GCA GCC TGA AGG CCT AAG TAG) and pta-RVf (CGG GCA
TTG CCC ATC TTC TTG). The gene mgsA was deleted to avoid the
production of L-lactic acid trough the methylglyoxal pathway. For
this deletion, the primers mgsADFw (GCA GCG ATA AGT GCT TAC
AGT AAT CTG TAG GAA AGT TAA CTA CGG ATG TAC ATT GTG
TAG GCT GGA GCT GCT TCG) and mgsADRv (CGG TGG CGA GAA
AAC CGT AAG AAA CAG GTG GCG TTT GCC ACC TGT GCA ATA
CAT ATG AAT ATC CTC CTT AGT TCC) were used. The primers
mgsAFw (GGA TAT TCT CGC CAT TAC CTC), and mgsARv (AGC
CTG GAA AAA GCG GCA ACA) were used to confirm the deletion
of mgsA by PCR.
Several fermentations were performed with AM1 medium
(Martinez et al., 2007). The composition of AM1 medium was:
2.63 g/L (NH4)2HPO4, 0.87 g/L NH4H2PO4, 1.0 mL/L MgSO4
7H2O
(1 M), 1.5 mL/L trace elements, 1.0 mL/L KCl (2 M), 1.0 mL/L
betaine HCl (1 M). The medium was supplemented with 0.1 g/L
of sodium citrate and different concentrations of xylose, glucose,
209
arabinose and sodium acetate. The trace elements solution con-
tains per liter: 1.6 g FeCl3, 0.2 g CoCl2
6H2O, 0.1 g CuCl2, 0.2 g
ZnCl2
4H2O, 0.2 g Na2MoO4, 0.05 g H3BO3 and 0.33 g MnCl2
4H2O.
The inocula for experiments with strain JU15 were prepared by
transferring the content of a cryovial (1 mL of glycerol 80% and
1 mL of cells grown in mineral medium with xylose) to a mini fer-
menter (Martinez et al., 2007) with 200 mL of mineral medium
(xylose 20 g/L). The inoculum was incubated for 24 h reaching
1.5–2 OD600. The cultures for D-LA production were inoculated by
re-suspending a cell pellet of inoculum centrifuged at 1610g
(10 min, room temperature) (Sorvall ST16R Thermo Scientific, Ger-
many). The cultures were started at 0.037 gDCW/L.
The pre-inocula for strain AV03 was grown in test tubes con-
taining 3 mL of Luria-Bertani medium by transferring the cryopre-
served strain (800 lL of strain AV03 at OD600 3.0 grown in
mineral media with xylose and mixed with 800 lL of glycerol at
80% v/v for cryopreservation), at 37 °C for 2 h and 300 rpm
(Shaker-Incubator Series 25, New Brunswick Scientific, New Jersey,
USA). The inocula were grown in a pH controlled 3-L bioreactor
(Applikon ADI 1010/ADI 1025, Delft, The Netherlands) with 2.2 L
of AM1 medium supplemented with 20 g/L of xylose (37 °C, pH
7, 480 rpm and 0.22 Lair/min), until it reached 1.48 gDWC/L. The cells
were harvested at 1610g (10 min, room temperature) to inocu-
late 0.2 L mini-fermenters with an initial cell mass of 3.7 gDCW/L.
2.2. Sugar cane bagasse hemicellulosic hydrolysate
The hemicellulosic fraction of SCB from Emiliano Zapata’s sugar
mill in Zacatepec (Morelos, Mexico) was hydrolyzed using a dilute
acid thermochemical treatment. The hydrolysis was performed in a
batch reactor with 2% w/w sulfuric acid at 121 °C for 1.5 h with a
2:1 liquid:solid ratio. Two batches containing a load of 20 kg dry
SCB each were used to perform the thermochemical hydrolysis in
a home-made 80 L stainless steel reactor. After cooling down to
room temperature, the syrup was squeezed manually with the
aid of an endless screw. This syrup contained in (g/L): glucose:
3.0 (±0.14), xylose 23.1 (±1.84), arabinose 1.55 (±0.07), and acetate
0.495 (±0.05). The hydrolysate was detoxified with Ca(OH)2 at
room temperature to reach pH 10 (Martinez et al., 2000). To chal-
lenge strain JU15 with a high concentration of sugars, mainly
xylose, from a plant hydrolysate syrup, the detoxified SCB syrup
was concentrated in a rotary evaporator (Buchi 185 Ex, Fawil-
Schweiz, Germany) at 60 °C under vacuum (540 mm Hg) to reach
70 g/L of total sugars. Solids were separated in a tubular centrifuge
(Mini-Sharples CLI-1, New Brunswick Scientific, New Jersey, USA)
at 13,010g and at a feeding flow rate of 0.15 L/min.
2.3. Corn stover hydrolysate
A load of 15% w/w of CS, with particles sieved and retained
between meshes 20–80 (0.18–0.85 mm), was pretreated with 2%
sulfuric acid at 130 °C, 8 min and 90 rpm in a high-pressure chem-
ical reactor (Model 4530/Controller 4848, Parr Instruments, IL,
USA). The slurry was cooled down to room temperature, the pH
was adjusted to 4.8 with a concentrated KOH and sodium citrate
was added to a concentration of 50 mM. The saccharification of
the slurry from the thermochemical hydrolysis, was performed at
50 °C for 24 h and 150 rpm (Vargas-Tah et al., 2015), in a system
of 200 mL reactors fitted with peg mixers (Caspeta et al., 2014)
and adding 15 PFU/gglucan of a commercial enzymatic cocktail
(NS22086, Novozymes, Brazil). The fermentation of the CS hydroly-
sate was carried out without detoxification and with all the solids
presents in the slurry.
210 J. Utrilla et al. / Bioresource Technology 220 (2016) 208–214
Table 1
Reduction of phosphate ammonium salts, MgSO4
7H2O, KCl and trace elements in AM1 medium to complement SCB hydrolysates. Maximum number of cells (as Colony Forming
Units: CFU), the specific growth rate (l), D-LA volumetric productivity (at 72 h) and D-LA yield on sugars are shown in the bottom part of the table. In all fermentations, betaine
(1 mM) and sodium citrate (0.1 g/L) were used.
Components Medium
(A) (B)
Ammonia phosphate salts 0.25X 0.25X
KCl 0.25X 0.25X
MgSO4
7H2O 0.25X 0.25X
Trace elements 0.25X – 0.25X
Xmax (CFU/mL) 5.9  1010 5.5  1010
l (h 1) 0.19 0.21
QD-LA (gD-LA/Lh) 0.77 0.77
YD-LA (gD-LA/gSugars) 1.14 1.14
2.4. Medium reduction for hydrolysate fermentations
The hydrolysates from SCB and CS were supplemented with
some compounds of AM1 medium to perform the fermentation.
As hydrolysates may contain some minerals present in the culture
medium, a study to reduce the amount of salts present in AM1 was
performed using the SCB hydrolysate and strain JU15. Betaine, an
osmoprotectant for E. coli fermentations (Zhou et al., 2006) was
maintained constant (at 1 mM) in all cultivations and sodium
citrate was also maintained at 0.1 g/L to avoid salts precipitation.
A combination of tests was performed by reducing the addition
of phosphate ammonium salts, MgSO4
7H2O, KCl and trace ele-
ments to one-quarter (0.25X) of the amount used in AM1 (Table 1).
The test was validated using the maximum number of cells per mL
(measured as colony forming units in solid media), the specific
growth rate, the D-LA volumetric productivity and the D-LA yield
on consumed glucose, xylose, and arabinose.
2.5. Culture conditions
Anaerobic cultures in fleakers (mini-fermenters) (Beall et al.,
1991) containing 0.2 L of AM1 medium were performed at 37 °C,
100 rpm and pH 7. The pH was controlled with the automatic addi-
tion of KOH (2 N). AM1 media for strain characterization was supple-
mented with 40 g/L of sugar (glucose or xylose) and sodium acetate
(2 g/L for JU15 and 4 g/L for AV03) and using an initial cell mass of
0.037 gDCW/L. To test the strain JU15 the laboratory simulated
hydrolysate was composed of AM1 media plus 6.7, 51.9 and 3.4 g/
L of glucose, xylose, and arabinose, respectively, and 1.7 g/L of
sodium acetate. To test the strain AV03 the laboratory simulated
hydrolysate was composed of AM1 media plus 42, 32 and 4 g/L of
glucose, xylose and arabinose respectively, and 4 g/L of sodium acet-
ate. The fermentations in 0.2 L mini-fermenters were started with
0.037 gDCW/L for strain JU15 and 3.7 gDCW/L for strain AV03. The
scale-up of fermentations with strain JU15 were carried out in a
10 L pilot scale fermenter (Microferm, New Brunswick, New Jersey,
USA). The pH, temperature, and stirring speed conditions were
6.6–7.0, 37 °C and 240 rpm, respectively. The pH was controlled
with the automatic addition of KOH (4 N). The D-lactate fermenta-
tions with strain JU15 and SCB hydrolysates in the 10 L fermenters
and with strain AV03 and CS hydrolysates in the 0.2 L mini-
fermenters, respectively, were supplemented with betaine (1 mM),
citrate (0.1 g/L) and phosphate salts (0.66 g/L (NH4)2HPO4, 0.22 g/L
NH4H2PO4,). Most experiments were carried out in triplicate and
the average and standard deviation is shown in figures and tables.
2.6. Analytic methods
For cultivations in AM1 medium and simulated hydrolysates,
cell concentration was measured as optical density at 600 nm
(C) (D) (E) (F)
0.25X 0.25X 0.125X –
0.25X – – –
– – – –
– – –
4.9  1010 7.9  109 7.7  109 6.1  109
0.20 0.23 0.23 0.22
0.75 0.77 0.70 0.45
1.11 1.09 0.98 0.66
(OD600) with a spectrophotometer (DU-70, Beckman instrument,
Inc. Fullerton, CA, USA). Metabolite concentrations were deter-
mined with an HPLC system (600E quaternary bomb, 717 auto-
matic injector, 2410 refraction index detector, Waters, Milford,
MA). An Aminex HPX-87H column (Bio-Rad, Hercules, CA) was
used for determination of glucose, xylose, arabinose and acetate
using the following conditions: 5 mM H2SO4 as mobile phase at a
flow of 0.5 mL/min and 50 °C. Metabolite concentrations were cor-
rected by the amount of added base to the initial working volume
during fermentations. For some experiments, L-lactic acid was
quantified with a biochemical analyzer (YSI model 2700, YSI Inc.,
Yellow Springs, OH, USA). The enzymatic determination using this
system is stereospecific for L-lactic acid.
The specific growth rate (l) was calculated during the exponen-
tial growth phase of each experiment. The product/substrate yield
(YD-LA/Sugars) was calculated taking into account the whole fermen-
tation elapsed time for each experiment and for a fair comparison,
the volumetric productivity of D-LA (QD-LA) was calculated at 48 h
for the simulated and actual hydrolysates.
3. Results and discussion
3.1. Fermentation performance of lactogenic E. coli JU15 in minimal
medium with glucose or xylose and simulated hydrolysates
The lactogenic strain JU15 was previously developed by meta-
bolic engineering and adaptive evolution for the efficient conver-
sion of xylose into D-LA with the aim of using xylose-rich
hydrolysates as a fermentation feedstock. Previously it was
demonstrated that JU15 shows a good performance in terms of
growth rate, D-LA productivity and yield in mineral media with
xylose under non-aerated conditions (Utrilla et al., 2012). Never-
theless, for a fair comparison in this work E. coli JU15 was evalu-
ated in AM1 mineral media with 40 g/L of glucose or xylose and
2 g/L of sodium acetate. The results shown in Fig. 1 indicate that
the specific growth rates, sugar consumption, and the D-LA volu-
metric productivity are about two-fold higher when glucose was
used as carbon source compared to xylose; however, the D-LA/
sugar yields were similar between both sugars in mineral media.
From a 40 g/L xylose fermentation with JU15 we found 0.060 g/L
of L-lactic acid and 0.028 g/L with AV03. The strain JU15 produced
38 g/L of total LA, therefore a 99.84% L-LA optical purity was
obtained. With strain AV03, 36.96 g/L of total LA were produced.
Hence the L-LA optical purity was increased to 99.92%.
The strain JU15 has not been previously evaluated with sugar
mixtures resembling plant hydrolysates or with actual ones. To
test strain performance in sugar mixtures, similar to plant hemicel-
lulosic hydrolysates containing a high amount of xylose, the AM1
mineral medium was supplemented with: 51.9 g/L xylose, 6.7 g/L
glucose, 3.3 g/L arabinose, and 2 g/L sodium acetate. This sugar
 J. Utrilla et al. / Bioresource Technology 220 (2016) 208–214 211

(A) (B)
8 8
0.4 0.4

qS (gXYL / gDCW h)
qS (gGLC / gDCW h)
6 6
0.3 0.3

µ (h-1)
µ (h-1)

4 4
0.2 0.2

2 0.1 2
0.1

0.0 0 0.0 0
JU15 AV03 JU15 AV03 JU15 AV03 JU15 AV03

1.0 1.0
1.6 1.6
YD-LA (48 h) (gD-LA / gGLC)

YD-LA (48 h) (gD-LA / gXYL)

QD-LA (24 h) (gD-LA / L h)

QD-LA (24 h) (gD-LA / L h)

0.8 0.8
1.2 1.2
0.6 0.6
0.8 0.8
0.4 0.4

0.4 0.4
0.2 0.2

0.0 0.0 0.0 0.0

JU15 AV03 JU15 AV03 JU15 AV03 JU15 AV03

Fig. 1. Comparison of JU15 and AV03 strain performance in A) glucose (40 g/L) and B) xylose (40 g/L) 0.2 L fermentations in AM1 mineral media supplemented with 2 g/L
sodium acetate. Specific growth rate (l), specific substrate consumption rate (qs), D-lactate yield (YD-LA/GLC or YD-LA/XYL), D-lactate volumetric productivity (QD-LA).

composition is similar to the one found in some diluted acid hydro- been reported to require low amounts of nutrients added; Wang
lysates of the SCB hemicellulosic fraction (Martinez et al., 2000, et al. (2013) found that only corn steep liquor was necessary to
2001) (Fig. 2). Results shown in Fig. 2 indicate that strain JU15 is add to achieve a L-lactic acid yield of 0.85 g/g of sugars from sugar-
not inhibited by 2 g/L of sodium acetate since the specific growth cane molasses.
rate and D-LA productivity values were similar to those previously Concentrated syrups from overliming treated (Martinez et al.,
found in mineral media containing 40 g/L xylose (0.19 h 1 and 2001) hemicellulosic hydrolysate from SCB (see Section 2 for
0.79 gD-LA/Lh respectively; Fig. 1B). In accordance with previous details) were fermented by JU15 in a 10 L fermenter. Most of the
findings with strain JU15 (Utrilla et al., 2012, 2009), acetate was operating parameters were the same as those used in the mini-
produced (2.3 g/L) during the exponential and stationary growth fermenters (pH 7, 37 °C and an initial inoculum of 0.037 gDCW/L).
phases. Also, after glucose was depleted, a simultaneous consump- The agitation rate was set at 240 rpm in order to achieve a homo-
tion of pentoses (xylose and arabinose) was observed, but no dia- geneous mixing of the hydrolysate in the bioreactor. The main sug-
uxic growth was evident. All sugars were totally consumed after ars in the hydrolysate were xylose, glucose, and arabinose, at initial
72 h with a high D-LA conversion yield (Fig. 2). These results point concentrations of 37.1, 4.34 and 1.83 g/L, respectively. Fermenta-
out that JU15 strain is a good biocatalyst for the production of D-LA tion results are shown in Fig. 3. In these fermentations the D-LA
from xylose-rich syrups generated from the hydrolysis of the hemi- yield (based on the consumption of xylose, glucose, and arabinose)
cellulosic fraction of lignocellulose. was higher than the theoretical maximum, YD-LA/Sugars 1.3 gD-LA/
gSugars. The metabolism of non-quantified sugars in the SCB hydro-
3.2. Sugar cane bagasse hydrolysate fermentation by JU15 lysates to D-LA resulted in an apparent yield 30% above the theoret-
ical maximum. At 96 h of cultivation, the most abundant sugar
To develop the best conditions of growth and fermentation in a (xylose) was not completely consumed, but the glucose and arabi-
SCB hemicellulosic hydrolysate based medium, different propor- nose were depleted before 60 h. Also, after glucose depletion, the
tions of the components of the AM2 medium were added (Table 1). simultaneous consumption of pentoses (xylose and arabinose)
The addition of 1 mM betaine, 0.1 g/L sodium citrate and 0.85 g/L was observed. As a result of the concentration step (see Section 2),
of ammonium phosphate salts were enough to sustain fermenta- the initial acetate concentration was 12.4 g/L and in average
tion with the same specific growth rate, number of cells and D-LA 17.4 g/L of total acetate was found at 96 h. Such amount of initial
volumetric productivity found with complete AM1 media and lab- acid and its additional accumulation could delay the strain meta-
oratory simulated hydrolysates containing high amounts of xylose bolism (Fernández-Sandoval et al., 2012). The volumetric produc-
(see Fig. 1 and 2A). Those nutrients were selected to supplement all tivity attained was 0.51 gD-LA/Lh, which was lower in comparison
further experiments with hydrolysates. Fig. 2B shows the results to fermentations carried out in mini-fermenters under similar con-
obtained with the test D shown in Table 1 (1 mM betaine, 0.1 g/L ditions (0.98 gD-LA/Lh; Fig. 2). This low productivity can be attribu-
sodium citrate and 0.88 g/L ammonium phosphate); the three most ted to different factors: a high initial concentration of acetate
abundant sugars (xylose, glucose, and arabinose) were depleted at (above 12 g/L); the presence of unquantified furans or phenolic
72 h, and a volumetric productivity of 0.98 gD-LA/Lh was observed compounds (Martinez et al., 2000) and the low initial cell density,
(calculated at 48 h of fermentation elapsed time). D-LA yield was among others. Therefore, further experiments with CS hydrolysates
1.11 gD-LA/gSugars based on measured consumption of xylose, glu- were started at a higher cell density.
cose, and arabinose. As shown below, other non-measured sugars Even though the productivity was reduced compared to other
account for the yield above the theoretical maximum. Other experiments presented here (Figs. 2 and 4), the D-LA titer was very
metabolically engineered E. coli lactic acid producing strains have close to 50 g/L (at 96 h of elapsed fermentation time) using a
212 J. Utrilla et al. / Bioresource Technology 220 (2016) 208–214

7 7
50
(A) 6
50 (B) 6
40

Arabinose (g/L)
40

Glucose (g/L)
5 5

Xylose (g/L)
Xylose (g/L)

Glucose (g/L)
Arabinose (g/L)
30 4 30 4

3 3
20 20
2 2
10 10
1 1

0 0 0 0

6 60 60 60
Cell mass (g/L)
Acetate (g/L)

Acetate (g/L)

D-LA (g/L)
D-LA (g/L)
4 40 40 40

2 20 20 20

0 0 0 0
0 12 24 36 48 60 72 84 96 0 12 24 36 48 60 72
Time (h) Time (h)

YD-LA (gD-LA/gSugars) = 0.94 ± 0.048 YD-LA (gD-LA/gSugars) = 1.11 ± 0.030

QD-LA (gD-LA/L h) = 1.11 ± 0.035 QD-LA (gD-LA/L h) = 0.98 ± 0.095

Fig. 2. Fermentation kinetics with strain JU15. A) SCB simulated hemicellulosic hydrolysate: AM1 mineral medium plus xylose, glucose, arabinose and sodium acetate: 51.9,
6.7, 3.4 and 1.7 g/L, respectively. B) Actual SCB hemicellulosic hydrolysate containing xylose, glucose, arabinose and acetate: 43.65, 5.34, 2.15 g/L and 14.25, respectively. The
SCB hemicellulosic hydrolysate was supplemented with 0.25X ammonia phosphate salts, betaine (1 mM) and sodium citrate (0.1 g/L). These fermentations were performed in
mini-fermenters with 0.2 L of medium at pH 7, 37 °C and 100 rpm, the initial inoculum was 0.037 gDCW/L.

7 of a high xylose concentration, the main sugar in the hemicellu-

50 losic fraction of SCB hydrolysates. The acetic acid tolerance of
6
JU15 and its derivatives, previously assessed by our group
Arabinose (g/L)

40
Glucose (g/L)

5
Xylose (g/L)

(Fernández-Sandoval et al., 2012), is of special relevance to cope

4 with the acetic acid released by the hydrolysis of acetylated sugars
30
in lignocellulosic hydrolysate production (Martinez et al., 2001;
3 Trček et al., 2015). Therefore, this process shows a potential for fur-
20
2 ther scale-up and development to produce bioplastic building
10 blocks from lignocellulosic biomass feedstocks. Other studies,
1
using laboratory glucose and the metabolically engineered E. coli
0 0 strain HBUT-D, has shown that similar fermentation processes
can be scaled-up to 6 ton fermenter vessels (Liu et al., 2014).
60 60
Acetate (g/L)

3.3. Engineering of JU15 to reduce acetic acid formation, racemic

D-LA (g/L)

40 40 lactate and simultaneous use of sugar mixtures

Similar to other E. coli strains, JU15 produces acetic acid as fer-

20
0 0
0 12 24 36 48 60
Time (h)
YD-LA (gD-LA/gSugars) = 1.30 ± 0.009
QD-LA (gD-LA/L h) = 0.51 ± 0.011
Fig. 3. Lactate production at 10 L scale by strain JU15 with SCB hemicellulosic
hydrolysate, containing xylose, glucose, arabinose, and acetate: 37.1, 4.34, 1.83 and
12.44 g/L, respectively. The SCB hemicellulosic hydrolysate was supplemented with
0.25X ammonia phosphate salts, betaine (1 mM) and sodium citrate (0.1 g/L). The
fermentations were started with 0.037 gDCW/L and performed in 10 L fermenters
controlled at pH 6.6–7.0, 37 °C and 240 rpm.
simple batch fermentation process with minimal nutrient addition.
It is worth to mention that strain JU15 was obtained by adaptive
evolution and its mutations confer advantages for the fermentation
20 mentation by-product and contains the methyl glyoxalate pathway
that can produce L-lactic acid. Furthermore, it has been shown that
the elimination of mgsA (the gene encoding the enzyme that cat-
72 84 96 alyzes the conversion of methylglyoxal into L-lactic acid) generates
E. coli strains with the ability to consume simultaneously pentoses
and hexoses (Yomano et al., 2009). Hence, with the aim of improv-
ing JU15, the strain was further engineered to remove acetic acid
and racemic lactic acid production pathways. The E. coli strain
AV03 was constructed by deleting the genes that code for acetic
acid production enzymes: pyruvate oxidase (poxB), acetate kinase
(ack) and phosphotransacetilase (pta); and the gene coding for
the enzyme involved in racemic lactate production: the methylgly-
oxal synthase (mgsA) (Grabar et al., 2006). The resulting strain,
named AV03 (JU15 DpoxB, DackA-pta, DmgsA), was first character-
ized in AM1 mineral medium with 40 g/L of glucose or xylose and
4 g/L of sodium acetate. The results showed that the growth rates
were 0.197 and 0.189 h 1 from glucose and xylose respectively
(Fig. 1). The growth rate in xylose 40 g/L was roughly the same
 J. Utrilla et al. / Bioresource Technology 220 (2016) 208–214 213

7 7
50 (A) 6
50 (B) 6

Glucose (g/L)
Glucose (g/L)

Arabinose (g/L)
40

Arabinose (g/L)
5 40 5
Xylose (g/L)

Xylose (g/L)
30 4 30 4
3 3
20 20
2 2
10 10
1 1
0 0 0 0

6 60 6 60
Cell mass (g/L)

Acetate (g/L)
Acetate (g/L)

D-LA (g/L)
D-LA (g/L)
4 40 4 40

2 20 2 20

0 0 0 0
0 12 24 36 48 0 12 24 36 48
Time (h) Time (h)
YD-LA (gD-LA/gSugars) = 0.95 ± 0.010 YD-LA (gD-LA/gSugars) = 1.11 ± 0.064
QD-LA (gD-LA/L h) = 1.32 ± 0.025 QD-LA (gD-LA/L h) = 1.21 ± 0.050

Fig. 4. Fermentation kinetics with strain AV03. A) CS simulated hydrolysate: AM1 mineral medium plus glucose, xylose, arabinose and sodium acetate: 42, 32, 4 and 4 g/L,
respectively. B) Actual CS hydrolysate containing glucose, xylose, arabinose and acetate: 30.51, 24.37, 4.42 and 3.68 g/L, respectively. The CS hydrolysate was supplemented
with 0.25X ammonia phosphate salts, betaine (1 mM) and sodium citrate (0.1 g/L). These fermentations were performed in mini-fermenters with 0.2 L of medium at pH 7,
37 °C and 100 rpm, the initial inoculum was 3.7 gDCW/L.

between JU15 and AV03 strains (Fig. 1), the D-LA/sugar yield and
volumetric productivity were also similar between the two strains
in xylose mineral media. These results suggest that consistent with
what was observed for the parental strain JU15, AV03 showed no
acetic acid inhibition. In contrast to what was obtained with
xylose, in glucose, the cellular growth for AV03 strain was reduced
and it showed a specific growth rate 49% lower compared to JU15,
concomitantly the specific glucose consumption rate was also
lower with AV03. The lower growth rate of AV03 in glucose
affected the D-LA productivity, resulting in a 41% reduction in
AV03 compared to the productivity of JU15 in the same conditions.
It is worth noting that the D-LA/glucose yield was similar between
AV03 and JU15, either using glucose or xylose.
These results show that the deletion of acetic acid production
pathway, that may reduce ATP yield, does not have detrimental
effects when using xylose. However, in medium containing glucose
and in comparison to JU15, strain AV03 showed a reduction in the
specific rates of growth and sugar consumption and hence the D-LA
volumetric productivity was also decreased. Several authors have
reported that mutants in the mgsA gene showed a reduction in glu-
cose consumption by an increase in the regulatory protein CRP,
which in turn reduces catabolic repression and increases the
simultaneous use of alternate carbon sources (Yao et al., 2011;
Yomano et al., 2009). The co-utilization of carbon sources is a
desired trait in strains aimed to use lignocellulosic hydrolysates
as feedstocks (Kim et al., 2010, 2015).
3.4. Production of D-LA with AV03 strain in laboratory simulated corn
stover hydrolysate
To observe the performance of AV03 strain in sugar mixtures
with similar composition to CS hydrolysates (Vargas-Tah et al.,
2015), a laboratory simulated CS hydrolysate (LSHCS) fermentation
was evaluated using 3.7 gDCW/L as inoculum. The mineral medium
sugar mixture composition was: glucose (42 g/L), xylose (32 g/L)
and arabinose (4 g/L), the media also contained sodium acetate
(4 g/L). During fermentation no significant increase in cell mass
was observed (Fig. 4A), due to the initial high load used, ammonia
phosphate contained in AM1 media limits the amount of biomass
that can be generated (Martinez et al., 2007). Nevertheless, in con-
trast to what was shown above for strain JU15, strain AV03 con-
sumed the three sugars simultaneously (Fig. 4A), as mentioned
above this effect has been previously observed for DmgsA strains
(Yao et al., 2011; Yomano et al., 2009). The yield of D-LA, based
on consumed sugars, in the simulated hydrolyzed was 0.95 gD-LA/
gSugars, a similar yield obtained with strain JU15 in LSHSCB. After
10 h of fermentation elapsed time, xylose and arabinose were con-
sumed more slowly, and after 48 h 6.19 of xylose and 2.05 g/L of
arabinose remained in the media (Fig. 4A). Glucose was the only
depleted sugar in the fermentation. These results show that it
was possible to use sugar mixtures to produce D-LA without acetic
acid production, obtaining a simultaneous consumption of pen-
toses and hexoses and reaching high conversion yields such as
the ones attained with JU15 using individual or sugar mixtures.
3.5. Corn stover hydrolysate fermentation by AV03 strain
Corn stover hydrolysates were generated using a load of 15% w/
w and 2% sulfuric acid and further saccharification as previously
reported (Vargas-Tah et al., 2015). The syrup was not detoxified
and the solids were not removed. In order to test the performance
of the AV03 strain to ferment the CS hydrolysate, cultures were
performed with the same nutrient addition as SCB hydrolysate fer-
mentation and using, as initial cell load, 3.7 gDCW/L. Initially mea-
sured concentrations of sugars in the CS hydrolysate were: 30.51,
24.37 and 4.42 g/L of glucose, xylose and arabinose, respectively,
and it contained 3.68 g/L of acetate. Results show that AV03 strain
produced D-LA (Fig. 4B) with a 1.112 gD-LA/gSugars yield similar as
JU15 for the SCB hydrolysates. As cited above, the conversion yield
is higher than the theoretical maximum (from the measured sug-
214 J. Utrilla et al. / Bioresource Technology 220 (2016) 208–214
ars) and may be due to non-quantified carbon sources present in
the hydrolysates, such as mannose and galactose. The sugar
consumption measurements show that glucose was totally utilized
in 24 h, but 2.7 of xylose and 1.2 g/L of arabinose remained after
96 h of fermentation (Fig. 4A). A volumetric productivity of
1.21 gD-LA/L.h was observed; an 8.4% reduction compared with
what obtained in laboratory simulated hydrolysate
(Fig. 4A and B). The AV03 strain did not show acetic acid produc-
tion. Furthermore, as shown in the LSHCS fermentation, the AV03
strain simultaneously consumed glucose, xylose, and arabinose.
These results show that the nutrients present in CS hydrolysates
are beneficial to E. coli D-LA producing strains JU15 and AV03.
In this work, it was demonstrated that D-LA can be produced
from sugars obtained from the thermochemical hydrolysis of ligno-
cellulosic materials. These hydrolysates contain mixtures rich in C5
and C6 sugars, acetic acid and may contain other bacterial growth
inhibitors such as furfural, hydroxymethylfurfural, and phenolic
compounds. Also, we showed that the acetic acid present in the
hydrolysates did not affect the growth and the metabolism of
strains JU15 and AV03 and that it was possible to construct a strain
able to reduce by-product formation and simultaneous use of sugar
mixtures. Some lactic acid bacteria can also ferment hemicellulosic
hydrolysates but complex nutrients have been used and low yields
achieved (Garde et al., 2002; Laopaiboon et al., 2010). Altogether
these results show that strains JU15 and AV03 perform well in
terms of growth rate and D-LA yield and productivity with the
addition of small amounts of ammonia phosphates to the hydroly-
sates in a very simple batch process. These results encourage the
use of metabolic engineered strains as a platform to develop
derivative strains for the production of other commodity chemicals
from lignocellulosic feedstocks (Fernández-Sandoval et al., 2012).
4. Conclusions
The strain JU15 was capable of converting xylose-rich syrups
obtained by diluted acid thermochemical hydrolysis of SCB to D-
LA with yields close to 95% of the theoretical. Furthermore, E. coli
AV03 was shown to simultaneously ferment pentoses and glucose
from CS hydrolysates without detoxification to D-LA without acetic
acid production and maintaining the same yield as JU15. Using a
few medium supplements, the hemicellulosic fraction of SCB
hydrolysates and the whole slurry of total hydrolysis of CS were
converted to D-LA by strains JU15 and AV03. The 10 L test showed
potential for achieving pilot and production scales.
Acknowledgements
We thank Georgina Hernández Chávez, Mario A. Caro-
Bermudez, Mercedes Enzaldo-Cruz, Martín Patiño Vera, Mario
Trejo Loyo and Servando Aguirre Cruz for technical assistance dur-
ing the project. This work was supported by the Mexican National
Council for Science and Technology (CONACYT), FONCICYT
ERANet-LAC Grant C0013-248192. Enzymatic preparation
NS22086 was kindly provided by Novozymes, Brazil.
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