Lactic Acid Production from Sugarcane Bagasse Hydrolysates by Lactobacillus

pentosus: Integrating Xylose and Glucose Fermentation

Daiana Wischral
Laboratórios de Desenvolvimento de Bioprocessos, Escola de Química, Departamento de Engenharia Bioquímica, Universidade Federal do
Rio de Janeiro, Rio de Janeiro, RJ, Brazil

Johanna Méndez Arias

Laboratórios de Desenvolvimento de Bioprocessos, Escola de Química, Departamento de Engenharia Bioquímica, Universidade Federal do
Rio de Janeiro, Rio de Janeiro, RJ, Brazil
Escuela Ingeniería Industrial, Instituto de Investigaciones en Ingeniería, Universidad de Costa Rica. Ciudad Universitaria Rodrigo Facio,
San Pedro, Montes de Oca, Costa Rica

Luiz Felipe Modesto

Laboratórios de Desenvolvimento de Bioprocessos, Escola de Química, Departamento de Engenharia Bioquímica, Universidade Federal do
Rio de Janeiro, Rio de Janeiro, RJ, Brazil

Douglas de França Passos

Laboratórios de Desenvolvimento de Bioprocessos, Escola de Química, Departamento de Engenharia Bioquímica, Universidade Federal do
Rio de Janeiro, Rio de Janeiro, RJ, Brazil

Nei Pereira Jr
Laboratórios de Desenvolvimento de Bioprocessos, Escola de Química, Departamento de Engenharia Bioquímica, Universidade Federal do
Rio de Janeiro, Rio de Janeiro, RJ, Brazil

DOI 10.1002/btpr.2718
Published online October 9, 2018 in Wiley Online Library (wileyonlinelibrary.com)

Lactic acid, traditionally obtained through fermentation process, presents numerous applica-
tions in different industrial segments, including production of biodegradable polylactic acid
(PLA). Development of low cost substrate fermentations could improve economic viability of
lactic acid production, through the use of agricultural residues as lignocellulosic biomass.
Studies regarding the use of sugarcane bagasse hydrolysates for lactic acid production by Lac-
tobacillus spp. are reported. First, five strains of Lactobacillus spp. were investigated for one
that had the ability to consume xylose efficiently. Subsequently, biomass fractionation was per-
formed by dilute acid and alkaline pretreatments, and the hemicellulose hydrolysate
(HH) fermentability by the selected strain was carried out in bioreactor. Maximum lactic acid
concentration and productivity achieved in HH batch were 42.5 g/L and 1.02 g/L h, respec-
tively. Hydrolyses of partially delignified cellulignin (PDCL) by two different enzymatic cock-
tails were compared. Finally, fermentation of HH and PDCL hydrolysate together was carried
out in bioreactor in a hybrid process: saccharification and co-fermentation with an initial
enzymatic hydrolysis. The high fermentability of these process herein developed was demon-
strated by the total consumption of xylose and glucose by Lactobacillus pentosus, reaching at
65.0 g/L of lactic acid, 0.93 g/g of yield, and 1.01 g/L h of productivity. © 2018 American
Institute of Chemical Engineers Biotechnol. Prog., 35: e2718, 2019
Keywords: enzymatic hydrolysis, lactic acid, Lactobacillus pentosus, lignocellulosic biomass,
lignocellulosic pretreatment

Introduction materials, biodegradable agro-based products that are more

Lactic acid is a valuable chemical platform that presents
extensive applications in food, cosmetics, textile, pharmaceuti-
cal, and chemical industries. Demand for lactic acid has grown
substantially in recent years, owing to its great potential as a
building block for the production of polylactic acid (PLA)
Correspondence concerning this article should be addressed to
N. Pereira Jr at nei@eq.ufrj.br
environmentally friendly, and alternative to petroleum-based
plastics.1–4 In 2013, global demands for lactic acids and PLA
were 714.2 and 360.8 kilo tons. Considering an expected
annual growth of 15.5% and 18.8%, demands could reach
1,960 and 1,205 kilo tons, respectively, by 2020. 5
Lactic acid has optical isomers: L-lactic acid and D-lactic
acid, which can be produced by chemical synthesis (DL-lactic)
or microbial fermentation (L-lactic, D-lactic, or DL-lactic).
Between both, microbial fermentation processes present more

© 2018 American Institute of Chemical Engineers 1 of9

2 of9
advantages, since they make use of renewable substrates for
lactic acid bacteria, and require mild production conditions and
low energy consumption.6 Lactic acid bacteria are capable of
producing lactic acid by homo- or heterofermentative degrada-
tion of sugars. During anaerobic growth of obligatory homofer-
mentative lactic acid bacteria, in conditions of excess substrate,
energy sources such as glucose are converted into pyruvate via
Embden–Meyerhoff–Parnas pathway (EMP-P), and pyruvate is
further metabolized to lactate. Throughout the heterofermenta-
tive pathway, sugars metabolism is catabolized via phosphoke-
tolase pathway (PK-P), resulting in equimolar amounts of CO2,
lactate, and acetate or ethanol. On the other hand, heterofermen-
tative lactic acid bacteria can be divided into obligatory hetero-
fermentative species, which ferment both hexoses and pentoses
through PK-P, and facultative heterofermentative organisms,
which degrade hexoses via EMP-P and pentoses via PK-P.
Final products and proportions may vary, depending on the
presence of other proton and electron acceptors.7
Lignocellulosic materials stand out as cheap, abundant, and
renewable sources for the production of biofuels, biomolecules,
biomaterials, and energy, and represent a more sustainable alterna-
tive within the biorefinery context. This biomass is mainly com-
posed of cellulose, hemicellulose, and lignin, and, after going
through pretreatment and hydrolysis steps, polysaccharides from
its cell wall can be converted to fermentable sugars.8 Also, enzy-
matic hydrolysis presents advantages over chemical hydrolysis,
since much less severe conditions are used and negligible amounts
of fermentation inhibitors (furfural, 5-hydroxymethylfurfural, some
organic acids, and lignin derivatives) are generated.9,10
Brazil, a country that presents an agriculture-based econ-
omy, presents a high potential for the utilization of lignocellu-
losic materials, mostly from sugarcane. In the 2015–2016
sugarcane crop, 666 millions of tons of sugarcane were pro-
duced, and for each ton approximately 250–270 kg of bagasse
are generated. This abundant residue is an important feedstock
for the production of second-generation bioproducts.11
Thus, the present work reports the investigation of
sugarcane bagasse hydrolysates (HH and PDCL) as sources of
substrates (xylose, glucose) for lactic acid production by
Lactobacillus spp.
Materials and Methods
Pretreatment of sugarcane bagasse
Acid pretreatment of sugarcane bagasse was performed,
using a solid:liquid ratio of 1:2.8 (1 g of bagasse: 2.8 mL of
1% v/v sulfuric acid solution) and at 121 ○C during 27 min,
as previously described by Betancur and Pereira Jr. 12 The liq-
uid fraction obtained after acid pretreatment, denominated
hemicellulose hydrolysate (HH), had its pH adjusted to 6.5
(fermentation condition) through addition of CaCO 3. Then, it
was subjected to vacuum filtration and analyzed by high per-
formance liquid chromatography (HPLC), using H 2SO4
(0.5 mM) as mobile phase at a flow rate of 0.6 mL/min
at 60 ○C.
The remaining solid fraction from acid pretreatment was
submitted to alkaline pretreatment through addition of 4%
(w/v) sodium hydroxide solution at a solid:liquid ratio of
1:20 at 121 ○C for 30 min. The process was performed as
described by Vásquez et al.13 The solid fraction obtained after
alkaline pretreatment is named partially delignified cellulignin
(PDCL). Compositions of in natura sugarcane bagasse and
Biotechnol. Prog., 2019, Vol. 35, No. 1
PDCL were determined by chemical hydrolysis as previously
described by Sluiter et al.14
Enzymatic hydrolysis
In order to compare the performance between the optimized
enzymatic cocktail, previously obtained by Mendéz et al.15
(Ladebio cocktail 13 FPU/mL, xylanase activity of 64 U/mL),
and commercial cocktail (Cellic CTec2® 326 FPU/mL, xyla-
nase activity of 5,805 U/mL). Enzymatic hydrolysis was car-
ried out at 50 ○C, pH 5.0, 200 rpm for 24 h in bioreactor
containing 550 mL of total reaction volume. Solids (substrate)
and total protein loadings were, respectively, 100 g/L (dry
weight) and 30 mg/g of cellulose. Sugars were analyzed by
HPLC using H2SO4 (0.5 mM) as mobile phase at a flow rate
of 0.6 mL/min at 60 ○C.
Bacterial strains and culture media
Xylose consumption and lactic acid production were evalu-
ated for the following strains of Lactobacillus spp.: Lactoba-
cillus coryniformis torquens ATCC 25600, L. coryniformis
coryniformis ATCC 25602, Lactobacillus helveticus ATCC
15009, Lactobacillus pentosus ATCC 8041, and
L. delbrueckii lactis DSM 20076, all of them obtained from
Deutsche Sammlung von Mikroorganismen und Zellkulturen
GmbH (DSMZ) and American Type Culture Collection
(ATCC). Culture media applied in this work were based on
Man, Rogosa, Sharpe medium (MRS), traditionally used for
Lactobacillus spp. fermentations, which composition is as fol-
lows: 20 g/L glucose, 10 g/L peptone, 5 g/L yeast extract,
10 g/L beef extract, 1 mL/L Tween 80, 2 g/L ammonium cit-
rate, 5 g/L sodium citrate, 0.1 g/L MgSO4, 0.05 g/L MnSO4,
and 2 g/L K2HPO4.16
Fermentation
During the screening step of Lactobacillus spp., 20 g/L
of xylose were added to culture media (MRS composition
except glucose) in order to evaluate xylose consumption and
lactic acid production by different strains. All of these
assays were held in duplicate, in serum bottles (100 mL
containing 70 mL of total reaction volume) with 20 g/L of
calcium carbonate as buffering agent. Studies regarding con-
sumption of HH and PDCL hydrolysates were carried out
in bioreactor inoculated with the Lactobacillus spp., previ-
ously selected as the best xylose consumer and lactic acid
producer, and to which were added the proper culture media
nutrients.
All of the fermentations systems were previously sterilized
(121 ○C for 20 min) and, with the purpose of achieving anaer-
obic conditions, nitrogen was sparged during 10 min in case
of serum bottles, and 20 min in bioreactors. Fermentations
were inoculated with 10% (v/v) of a cell suspension with the
selected strain, which had been precultured in MRS medium,
and were carried out at 37 ○C and 120 rpm. For experiments
carried out in bioreactor, a 1 L-stirred tank, model BioFlo III,
manufactured by New Brunswick, the pH was kept at 6.5
through addition of NaOH (2 N) and samples were taken at
regular intervals.
For assays concerning evaluation of HH consumption,
hydrolysate concentrations varied from 40% to 100% (v/v) in
different batches in bioreactor. HH and PDCL hydrolysates
Biotechnol. Prog., 2019, Vol. 35, No. 1 3 of9

consumption was evaluated in bioreactor through saccharifica- addition, depending on the pretreatment technique, lignin
tion and co-fermentation with an initial enzymatic hydrolysis structure can suffer distinct changes and interact with
(SSCF*), which was performed to initiate the SSCF* process enzymes, be it through nonspecific bindings, hydrophobic or
with higher concentrations of glucose. Once PDCL was electrostatic interactions, or hydrogen bonds.17
hydrolyzed, 175 mL of HH (adding MRS nutrients, except Acid and alkaline pretreatments were applied to in natura
glucose: 10 g/L peptone, 5 g/L yeast extract, 10 g/L beef sugarcane bagasse, HH (Table 1) and PDCL (Table 2) were
extract, 1 mL/L Tween 80, 2 g/L ammonium citrate, 5 g/L obtained, both to be later fermented to lactic acid. The pre-
sodium citrate, 0.1 g/L MgSO4, 0.05 g/L MnSO4, and 2 g/L treatments were efficient since cellulose increased from 34.7%
K2HPO4) were added, totalizing 350 mL of fermentation to 60.9% (w/w), while hemicellulose and lignin content
medium in bioreactor. Then, fermentation conditions were set decreased, respectively, from 25.2% to 11.6% (w/w) and from
(pH 6.5, 120 rpm, 37 ○C, and anaerobic conditions) and the 19.2% to 8.1% (w/w) in PDCL. It is widely known that lignin
precultured selected strain was inoculated (10% v/v). In addi- hinders the direct utilization of lignocellulosic materials. This
tion, to evaluate the amount of lactic acid produced per dry polyphenolic macromolecule offers a physical barrier for the
weight of sugarcane bagasse, the efficiencies were calculated use of native cellulose.18 These results from Tables 1 and 2
thought mass balance, based on volume recovered and solid: corroborating with previously reported by Betancur and Per-
liquid relation. eira Jr,12 Méndez et al.,15 and Maeda et al.18
Glucose, xylose, D-lactic acid, L-lactic acid and acetic acid
were analyzed in duplicate by HPLC (Shimadzu) using exter-
nal standards for identification and quantification of peak
Lactic acid production from HH
areas. Glucose, xylose, and acetic acid were quantified at PL
Hi-Plex column H 8 μm (300 × 7.7 mm), with 20 μL of injec- Scientific literature regarding lactic acid production from
tion volume and H2SO40.005 mol/L as mobile phase at lignocellulosic materials mainly reports utilization of the cellu-
0.6 mL/min, 60 ○C, and using RID and UV (210 nm) detec- losic fraction, since just few strains are capable of consuming
tors. D- and L-lactic acid were quantified at Chirex 3126 (D)- hemicellulose-derived sugars.19–22 The investigation of HH
penicillamine column (150 × 4.6 mm), with 10 μL of injection fermentation to lactic acid began with the screening of Lacto-
volume and CuSO4.5H2O 0.001 mol/L as mobile phase at bacillus spp. These assays were carried out in serum bottles
1.0 mL/min, 50 ○C and using UV detector (254 nm). and the results are shown in Table 3. L. pentosus ATCC 8041
was the strain that exhibited the best results for lactic acid pro-
duction (16.2 g/L), yield (0.94 g/g), percentage reduction of
substrate (71%), and productivity (0.34 g/L h). The selection
Results and Discussion
of L. pentosus ATCC 8041 (as shown in Table 3) corroborated
Pretreatment of sugarcane bagasse the results reported by Moldes et al.,21 who also investigated
Acid and alkaline pretreatments were implemented aimed at this strain for fermenting hemicellulose-derived sugars from
fractioning the lignocellulosic complex, by disarranging it and agro-industrial residues. Once the proper Lactobacillus strain
removing part of the hemicellulose and lignin fractions, which was selected, consumption of different HH percentages was
represent physical barriers for enzyme access to cellulose. In evaluated in bioreactor. To that end, assays were held in biore-
actor starting with 40% of HH (Figure 1). Fermentation profile
indicates that after 19 h of fermentation, 21.7 g/L of lactic
Table 1. Composition of sugarcane bagasse hemicelluloses hydrolysate
after acid pretreatment acid was produced (12.5 g/L of D-lactic acid isomer) and that
Component Concentration (g/L) xylose was totally consumed after 24 h. These results are bet-
ter than those observed with assays held in serum bottles
Glucose 3.9 T 1.7
Xylose 55.3 T 3.2
(Table 3, in which only 71% of substrate was consumed) most
Cellobiose 2.3 T 1.7 likely due to the effective pH control in the bioreactor.
Acetic acid 12.1 T 2.5 The HH obtained after acid pretreatment (Table 1) presents
HMF 0.06 T 0.01 in its compositions substances, such as hydroxymethylfurfural
Furfural 0.19 T 0.03 (HMF), furfural, and acetic acid, which have been reported as
fermentation inhibitors.12 These compounds can affect cell
Table 2. Sugarcane bagasse composition in natura and after acid and growth, fermentation rate, and intracellular levels of ATP and
alkaline pretreatments NAD(P)H, lowering them. Pol et al.23 reported that among the
Acid and Alkaline investigated lactic acid-producing microorganisms concerning
Fraction In Natura Pretreated (PDCL) the inhibition by furfural, the main inhibitor arisen from the
Cellulose (%) 34.7 T 2.1 60.9 T 4.5 hemicellulose diluted acid hydrolysate, Lactobacillus casei
Hemicellulose (%) 25.2 T 0.8 11.6 T 2.7 and Lactococcus lactis were only mildly inhibited, Bulbus
Lignin (%) 19.2 T 0.2 8.1 T 0.3 smithii was significantly inhibited and Bacillus coagulans was
Ashes (%) 1.0 T 0.1 0.6 T 0.1
fully inhibited.24 HMF and furfural, which are generated by

Table 3. Screening of Lactobacillus spp. for lactic acid production in serum bottles, 37 ○C, 48 h, MRS medium with ca. 20 g/L of xylose
Strain Lactic Acid (g/L) Yield (g/g) Productivity (g/L h) PRS (%)
L. coryniformis torquens ATCC 25600 3.27 T 0.03 0.50 0.07 10
L. coryniformis coryniformis ATCC 25602 1.97 T 0.04 0.16 0.04 3
L. helveticus ATCC 15009 2.67 T 0.07 0.39 0.06 7
L. pentosus ATCC 8041 16.19 T 0.05 0.94 0.34 71
L. delbrueckii lactis DSM 20076 12.36 T 0.09 0.82 0.26 62
PRS, percentage reduction of substrate.
4 of9 Biotechnol. Prog., 2019, Vol. 35, No. 1

Figure 1. Batch fermentation kinetics of lactic acid from hemicellulose hydrolysate (40%) by L. pentosus ATCC 8041 in bioreactor at pH
6.5, anaerobiosis, 37 ○C and 120 rpm.

Figure 2. Batch fermentation kinetics of lactic acid from hemicellulose hydrolysate (100%) by L. pentosus ATCC 8041 in bioreactor at
pH 6.5, anaerobiosis, 37 ○C and 120 rpm.

sugar dehydration caused by the degree of severity imposed
by the synergy of temperature, exposition time, and acid con-
centration during dilute acid pretreatment. Acetic acid and acid
lignin derivatives are also generated during dilute acid pre-
treatment, increasing the toxicity of the HH. However, for
100% of HH in culture medium (Figure 2), 32.5 g/L of lactic
acid (19.5 g/L of D-lactic acid) was produced after 33 h. In
addition, as the total consumption of xylose occurred after
42 h, the presence of inhibitors (HMF, furfural, and acetic
acid) did not affect production of lactic acid by L. pentosus
ATCC 8041 from HH. Thus, it was unnecessary to detoxify
the HH prior to the fermentation step, which means that
L. pentosus ATCC 8041 stands as a robust strain and an inter-
esting option related mainly with xylose consumption, with
potential application in the industrial production of lactic acid.
Moldes et al.21 studied xylose and arabinose fermentation by
L. pentosus CECT-4023T (ATCC 8041) and achieved
excellent lactic acid productivity and yield, regardless of resid-
ual glucose amount in culture media. HHs from trimming vine
shoots, barley bran husks, or corncobs and Eucalyptus globu-
lus (detoxified with activated charcoal) were used as carbon
sources. Maximum production (33 g/L) and productivity
Biotechnol. Prog., 2019, Vol. 35, No. 1
Figure 3. Glucose released from sugarcane bagasse enzymatic
hydrolysis at 9 h, 12 h, and 24 h, 50 ○C, using differ-
ent enzymatic cocktails (Ladebio cocktail and Cellic
CTec2®). Solids loading: 100 g/L; enzymatic cocktail
protein loading: 30 mg/g cellulose.
(0.60 g/L h) were reached using barley bran husks, while
maximum product yield from pentoses (0.76 g/g) was
achieved using hydrolysate from trimming vine shoots, fol-
lowed by hydrolysates from E. globulus (0.70 g/g), barley
bran (0.57 g/g), and corncob (0.53 g/g).21
Enzymatic hydrolysis
Usually, cells of Lactobacillus spp. that are able to ferment
hexose cannot metabolize pentose as carbon source. In order
to overcome this issue, research groups around the world have
been investigating new strains and using genetic engineering
to find a microorganism that is able to efficiently metabolize
both hexose and pentose.7 Méndez et al.15 developed an opti-
mized enzymatic cocktail (dedicated enzymatic preparation),
coded by Ladebio, which provided promising results for sug-
arcane bagasse hydrolysis. In the current work, enzymatic
hydrolysis of PDCL from sugarcane bagasse was carried out
in bioreactor, and HH were added to the bioreactor with the
purpose to integrate hexose and pentose consumption by
L. pentosus.
First, enzymatic hydrolysis of PDCL was investigated in
bioreactor, comparing the Ladebio enzymatic cocktail and the
commercial enzymatic cocktail (Cellic CTec2®). Figure 3 dis-
plays the performance of both cocktails, in which can be seen
that the Ladebio cocktail resulted in higher release of glucose
than Cellic CTec2® at the end of the process, despite the lower
results until the first 9 h of enzymatic hydrolysis. Maximum
glucose concentrations reached for each cocktail were 62 g/L
(Ladebio cocktail) and 45 g/L (Cellic CTec2®), corresponding
to hydrolysis efficiencies of 92% and 67%, respectively. The
Cellic CTec2® efficiency from this work was similar to that
reported by Ramos et al.,25 who also used same commercial
enzymatic cocktail to accomplish sugarcane bagasse hydroly-
sis, reaching 69% of efficiency after 96 h.
The use of the commercial cocktail Cellic CTec2® promoted
higher initial hydrolysis rate than the Ladebio optimized mix-
ture. Possibly, this occurred due to the presence of stabilizing
agents and accessory proteins in the commercial cocktail that
take part in the amorphogenesis of cellulose, which influenced
5 of9
on the enzyme access to cellulose, mainly during the first
hours of the hydrolysis process. Scientific literature has
reported that Cellic CTec2® contains LPMO (lytic polysaccha-
ride monooxygenases) proteins that act synergistically with
cellulase in the hydrolysis cellulose through an oxidative
mechanism.26,27 These enzymes might be responsible for
increasing enzyme access to the substrate, therefore leading to
higher initial hydrolysis rates observed.
However, when using Cellic CTec2® an earlier stabilization
in the hydrolysis efficiency was observed, which may be
related to changes in the composition of the lignocellulosic
material along the hydrolysis process, since, as cellulose is
hydrolyzed, lignin proportion is increased. It is well documen-
ted that depending on the origin and composition of the ligno-
cellulosic feedstock as well as the pretreatment, this
polyphenol macromolecule may hinder the access of the
enzymes to cellulose; also, some enzyme activities might
establish unproductive linkages with lignin. Haven and
Jørgensen28 studied in detail the adsorption of β-glucosidase
present in Cellic CTec2® and concluded that this enzyme
adsorbs more significantly to lignin than β-glucosidase pro-
duced from other sources (Aspergillus niger, for example). In
addition, the optimized mixture (Ladebio cocktail) presents in
its compositions 35% of enzymes from A. niger, mainly β-
glucosidase,15 which was produced using PDCL as carbon
source and as inductor for cellulase production. Both studies
give support for explaining the differences observed in the
hydrolysis rates between both cocktails (Figure 3).
Co-fermentation of PDCL and HHs to lactic acid
Since the Ladebio cocktail presented a better performance
than the Cellic CTec2® (ca. 40% more glucose), it was
selected to be used in a SSCF* process (Figure 4A), which
beforehand was submitted to a previous enzymatic hydrolysis
of cellulose from PDCL. Once PDCL prehydrolysis was car-
ried out, HH with culture medium nutrients was added to the
same bioreactor and suitable fermentation conditions for
L. pentosus ATCC 8041 were set.
This hybrid system, that is SSCF*, was adopted for maxi-
mizing glucose concentration, since the enzymatic cocktail
could act previously in its optimum conditions of pH and tem-
perature. The enzymatic cocktail kept on acting during co-
fermentation (after bacterium inoculation), although with
reduced catalytic activity. Fermentation kinetics (Figure 4B)
indicate that L. pentosus consumed concomitantly glucose and
xylose. Production of 65.0 g/L of total lactic acid (38.0 g/L of
D-lactic acid) was achieved, corresponding to a yield of
0.93 g/g, and xylose and glucose were completely consumed.
L. pentosus is heterofermentative facultative, which means it
is able to metabolize hexose via EMP pathway and pentose
through PK-P. According to Gobbetti et al.,29 when different
carbon sources are available, bacteria choose the one that pro-
motes maximum growth yield, which explain that L. pentosus
firstly consumed glucose and then xylose.
In all fermentations carried out using L. pentosus, only
acetic acid was formed as by-product. Its maximum concentra-
tions reached 13.0 g/L in HH and PDCL co-fermentation
(Figure 4B). It is likely that acetic acid formed during the
bagasse acid pretreatment changes redox balance, favoring
acid production in detriment to ethanol production, reason
why this alcohol was absent in all fermented media. More-
over, during glucose and xylose co-fermentation, acetic acid
6 of9 Biotechnol. Prog., 2019, Vol. 35, No. 1

Figure 4. Saccharification and co-fermentation with an initial enzymatic hydrolysis (SSCF*) in bioreactor. Ladebio enzymatic cocktail
was used for PDCL hydrolysis, HH and medium components add in the end of saccharification. Fermentation of PDCL and
HH hydrolysates were performed by L. pentosus at pH 6.5, anaerobiosis, 37 ○C and 120 rpm. Scheme (a) and fermentation
profile (b).

was probably formed from xylose and glucose consumption
by L. pentosus ATCC 8041.
Compared to other naturally occurring lactic acid producers
in batch fermentation (Table 4), L. pentosus ATCC 8041 pre-
sents higher results for acid concentration (65.0 g/L), similar
results of yield (0.93 g/g) and similar productivity (1.01 g/
L h), when using xylose and glucose as carbon sources alto-
gether. However, when considering the use of other carbon
sources (galactose, lactose, fructose, maltose) and the adoption
of acclimation and ultraviolet mutagenesis, a mutant strain of
Lactobacillus delbrueckii was able to produce a high lactic
acid concentration (135 g/L), corresponding a volumetric
productivity of 2.81 g/L h and 0.90 g/g lactic acid of yield,
according to Kadam et al.37 studies.
More recently, Kuo et al.40 reported the production of opti-
cally pure L-lactic acid from lignocellulosic hydrolysates of
waste wood chips and rice straw by using a newly isolated
and D-lactate dehydrogenase gene-deficient Lactobacillus
paracasei strain. The isolated microorganism showed to be a
very efficient glucose-fermenting strain, producing L-lactic
acid in concentrations of approximately 100 g/L with high
yield (0.96 g/g); nonetheless, the xylose uptake was not effi-
cient as glucose. Percentage reduction of xylose was not supe-
rior to 38%. Moraes et al.16 obtained 57.0 g/L of D-lactic acid
Biotechnol. Prog., 2019, Vol. 35, No. 1 7 of9

Table 4. Lactic acid production from different sources reported in the literature
Titer Yield Productivity
Strain Carbon Source, System (g/L) (g/g) (g/L h) References
L. casei ATCC 393 Residual yoghurt whey, batch 41.5 0.91 0.86 30
L. coryniformis subsp. torquens Green microalgae (Hydrodictyon reticulatum), batch 36.6 0.46 1.02 31
L. delbrueckii NRRL B445 Pulp mill, batch 25.1 0.93 0.13 32
L. paracaseiLA104 Green microalgae (Hydrodictyon reticulatum), batch 36.1 0.46 1.03 33
L. pentosus ATCC 8041 HH from sugarcane bagasse, batch 42.4 0.71 1.02 This work
L. pentosus ATCC 8041 HH and PDCL from sugarcane bagasse (SSCF*), batch 64.8 0.93 1.01 This work
L. pentosus CECT-4023T Vine shoots, batch 46.0 0.78 0.93 7
(ATCC 8041)
L. pentosus ATCC 8041 Microalgae (Nannochloropsis salina), batch 23.0 0.93 0.45 34
L. pentosus CECT-4023T Hemicellulosic fraction form barley bran husks, batch 33.7 0.57 0.60 21
(ATCC 8041) Hemicellulosic fraction form corn cobs, batch 24.7 0.53 0.34
Hemicellulosic fraction form vine shoots, batch 26.5 0.76 0.51
Hemicellulosic fraction form E. globulus chips, batch 14.5 0.70 0.28
Lactobacillus pentosus Sugarcane bagasse (SSF), fed-batch 72.7 0.61 1.01 35
Lactobacillus delbrueckii Uc-3a Sugarcane bagasse cellulose (SSF), batch 67.0 0.83 0.93 36
Lactobacillus delbrueckii Sugarcane bagasse hydrolysate, batch 135.0 0.90 2.81 37
NCIM 2365a
Bacillus coagulans DSM2314 Acid pretreated sugarcane bagasse (SSF), batch 74.6 0.94 0.92 38
Acid pretreated sugarcane bagasse (SSF), two stage 58.2 0.90 1.81
Bacillus sp. P38 Sugarcane bagasse hydrolysate, fed-batch 185.0 0.99 1.93 39

HH, hemicelluloses hydrolysate; PDCL, partially delignified cellulignin; SSCF*, saccharification and co-fermentation with an initial enzymatic
hydrolysis.
a
Mutant isolated by UV mutagenesis.

Table 5. Material balance for the production of lactic acid from
sugarcane bagasse
Step Input Output Efficiency
Pretreatment 1,000 g 1.6 L + 289
51%
SB + 2.8 L g PDCL
1.1%
v/v H2SO4 +
1:20 4% m/v
NaOH
SSCF* 1.6 L + 289 g 290.8 g 93%
PDCL lactic acid
SSCF*, saccharification and co-fermentation with an initial enzy-
matic hydrolysis; SB, sugarcane bagasse; PDCL, partially delignified
cellulignin.
and 0.97 g/g of yield by L. coryniformis subsp. torquens with
PDCL from sugarcane consumption in bioreactor (only glu-
cose as carbon source).
A very interesting aspect of the present work is that
L. pentosus ATCC 8041 presented the ability of consuming
efficiently the two most abundant sugars of the lignocellulosic
hydrolysates—xylose and glucose—both individually or
simultaneously, demonstrating that they are promising carbon
sources for lactic acid production, within the context of
second-generation Biorefinery. Previously, Patel et al.41 stud-
ied the fermentation of sugarcane bagasse HH by Bacillus
sp. strain 17C5, reaching 617 mM of lactic acid and 89%
yield considering both xylose and glucose from HH. Also, Pol
et al.,38 who applied acid pretreatment, steam explosion, and
simultaneous saccharification fermentation (SSF) using
B. coagulans DSM2314; published similar results regarding
lactic acid production in terms of titer from lignocellulosic
hydrolysates (xylose and glucose simultaneously): 64.1 g/L of
lactic acid (with 80% of yield and 0.78 g/L h of productivity).
The stereo-specificity and optical purity of lactic acid pro-
duced from microbial cultures mainly depend on the particular
microbe selected and the specificity of its lactate dehydroge-
nase (LDH). Lactic acid is produced from pyruvate via stereo-
specific NAD-dependent enzymes of L- or D-LDH, thereby
affecting the lactic acid configuration. Optically pure lactic
acid (L- or D-isomer) is more valuable than racemic DL-lactic
acid, and each isomer presents specific applications. Depend-
ing on microbial source, lactic fermentation can produce D-
lactic acid or L-lactic acid with high optical purity, or a mix-
ture of these.5 Recently, Awasthi et al.2 reported results with a
genetic engineered Bacillus subtilis strain with D-LDH from
L. delbrueckii subspecies bulgaricus, using glucose as carbon
source, they reported a production of lactic acid of 87 g/L,
along with a yield of 0.96 g/g.
Optical purity influence on several proprieties of produced
PLA: melting point, mechanical strength, and crystallinity
depend on the proportions of L, D, or meso-lactide.1 This way
both isomers are important to produce PLA with different
properties. One of the methods to produce high molecular
mass PLA concerns polymerization through lactide formation,
which was developed by Cargill Inc. in 1992.42 In this
method, either D-lactic acid, L-lactic acid, or a mixture of the
two are prepolymerized to obtain an intermediate low molecu-
lar mass PLA, which is then depolymerized into a mixture of
lactide stereoisomers. Lactide is formed through condensation
of two lactic acid molecules as it follows: L-lactide (two L-
lactic acid molecules), D-lactide (two D-lactic acid molecules),
and meso-lactide (one L-lactic acid and one D-lactic acid mol-
ecule). After purification, lactides are polymerized into high
molecular mass PLA.1,42 It is in line with the work published
by Liu et al.,43 which concerns production of polymers using
racemic mixture of DL- Lactic acid, in which the lignin-graft-
PLA has been synthesized via ring-opening polymerization of
D-, DL-, and L-lactic acid.
In addition, a material balance considering the results
obtained in the current work was calculated for the SSCF*
process (Figure 4A). The purpose was to evaluate the amount
of lactic acid produced per dry weight of sugarcane bagasse.
Table 5 shows the inputs and outputs for each step: pretreat-
ments (acid and alkaline) and SSCF*. According to that, the
potential production of 290 g of lactic acid from 1,000 g of
sugarcane bagasse is estimated, corresponding to efficiencies
of 51% and 93% with respect to pretreatment and SSCF*
steps, respectively. Although these are good results, there is
still room for improvement, that is, it is possible that feedings
8 of9
with PDCL and/or with enzymatic cocktail during enzymatic
hydrolysis or lactic acid fermentation steps could increase glu-
cose concentration, which in turn would result in a higher pro-
duction of lactic acid.
Conclusions
The findings of this work indicate that HH obtained from
dilute acid pretreatment, as well as cellulose hydrolysate
obtained via enzymatic hydrolysis, stand as promising feed-
stocks for lactic acid production by L. pentosus ATCC 8041,
with complete xylose and/or glucose consumption. Also, com-
pared to commercial cocktail Cellic CTec2®, use of an opti-
mized enzymatic cocktail (Ladebio) promoted an increase of
38% in the levels of released glucose from PDCL. Consider-
ing an integrated SSCF* process, cellulose and HHs can be
used even in cases which inhibitors from pretreatment are pre-
sent. Hence, such findings contribute to the development of
more efficient bio-based technologies, within the context of
second-generation biorefinery.
Acknowledgments
The authors would like to thank the Brazilian Petroleum
Company for financial support; the Brazilian Council for
Research for scholarship to the first, fourth, and fifth authors
and the Universidad de Costa Rica for scholarship to the sec-
ond author.
Conflict of Interest
The authors declare that they have no conflicts of interest.
Ethical statement
This article does not contain any studies with human partici-
pants or animals performed by any of the authors. The authors
confirm that principles of ethical and professional conduct
have been followed in this research and in the preparation of
this article.
Literature Cited
1. Auras RA, Harte B, Selke S, Hernandez R. Mechanical, physical,
and barrier properties of poly (lactide) films. J Plast Film Sheet.
2003;19:123–135.
2. Awasthi D, Wang L, Rhee MS, Wang Q, Chauliac D,
Ingram LO, Shanmugam KT. Metabolic engineering of Bacillus
subtilis for production of D-lactic acid. Biotechnol Bioeng. 2018;
115:453–463.
3. Abdel-Rahman MA, Tashiro Y, Sonomoto K. Recent advances in
lactic acid production by microbial fermentation processes. Bio-
technol Adv. 2013;31:877–902.
4. Karamanlioglu M, Preziosi R, Robson GD. Abiotic and biotic
environmental degradation of the bioplastic polymer poly (lactic
acid): a review. Polym Degrad Stab. 2017;137:122–130.
5. Abdel-Rahman MA, Sonomoto K. Opportunities to overcome the
current limitations and challenges for efficient microbial produc-
tion of optically pure lactic acid. J Biotechnol. 2016;236: 176–
192.
6. Reddy LV, Park JH, Wee YJ. Homofermentative production of
optically pure L lactic acid from sucrose and mixed sugars by
batch fermentation of Enterococcus faecalis RKY1. Biotechnol
Bioproc Eng. 2015;20:1099–1105.
Biotechnol. Prog., 2019, Vol. 35, No. 1
7. Bustos G, Moldes AB, Cruz JM, Domínguez JM. Influence of
metabolism pathway on lactic acid production from hemicellulo-
sic trimming vine shoots hydrolysates using Lactobacillus pento-
sus. Biotechnol Prog. 2005;21:793–798.
8. Van Dyk JS, Pletschke BI. A review of lignocellulose bioconver-
sion using enzymatic hydrolysis and synergistic cooperation
between enzymes—factors affecting enzymes, conversion and
synergy. Biotechnol Adv. 2012;30:1458–1480.
9. Maitan-Alfenas GP, Visser EM, Guimarães VM. Enzymatic
hydrolysis of lignocellulosic biomass: converting food waste in
valuable products. Curr Opin Food Sci. 2015;1:44–49.
10. Jönsson LJ, Martín C. Pretreatment of lignocellulose: formation
of inhibitory byproducts and strategies for minimizing their
effects. Bioresour Technol. 2016;199:103–112.
11. Única, Brazilian Sugarcane Industry Association, Sugarcane Pro-
duction and Processing, 2017. http://english.unica.com.br/,
[accessed March 22, 2018].
12. Betancur GJ, Pereira N Jr. Sugar cane bagasse as feedstock for
second generation ethanol production: part I: diluted acid pre-
treatment optimization. Electron J Biotechnol. 2010;13:10–11.
13. Vásquez MP, Silva JNC, Souza MB, Pereira N. Enzymatic
hydrolysis optimization to ethanol production by simultaneous
saccharification and fermentation. Appl. Biochem. Biotechnol.
2007;137/140:141–154.
14. A. Sluiter, B. Hames, R. Ruiz, C. Scarlata, J. Sluiter,
D. Templeton, Determination of structural carbohydrates and lig-
nin in biomass, LaboratoryAnalytical Procedure (LAP), Technical
Report NREL/TP-510-42618, National Renew Energy Lab
(NREL) 1–18, 2008. https://www.nrel.gov/docs/gen/fy13/42618.
pdf (accessed March 22, 2018).
15. Méndez JA, Modesto LF, Polikarpov I, Pereira N. Design of an
enzyme cocktail consisting of different fungal platforms for effi-
cient hydrolysis of sugarcane bagasse: optimization and syner-
gism studies. Biotechnol Prog. 2016;32:1222–1229.
16. Moraes AO, Ramirez NI, Pereira N. Evaluation of the fermenta-
tion potential of pulp mill residue to produce D (−)-lactic acid by
separate hydrolysis and fermentation using Lactobacillus coryni-
formis subsp. torquens. Appl BiochemBiotechnol. 2016;180:
1574–1585.
17. Liu H, Sun J, Leu SY, Chen S. Toward a fundamental understand-
ing of cellulase-lignin interactions in the whole slurry enzymatic
saccharification process. Biofuels Bioprod Biorefin. 2016;1–16.
18. Maeda RN, da Silva MMP, Santa Anna LMM, Pereira N. Nitro-
gen source optimization for cellulase production by Penicillium
funiculosum, using a sequential experimental design methodology
and the desirability function. Appl Biochem Biotechnol. 2010;
161:411–422.
19. Bustos G, Moldes AB, Cruz JM, Domínguez JM. Revalorization
of hemicellulosic trimming vine shoots hydrolizates trough con-
tinuous production lactic acid and biosurfactants by L. pentosus.
J Food Eng. 2007;78:405–412.
20. Rivera OMP, Moldes AB, Torrado AM, Domínguez JM. Lactic
acid and biosurfactants production from hydrolyzed distilled
grape marc. Process Biochem. 2007;42:1010–1020.
21. Moldes AB, Torrado A, Converti A, Domínguez JM. Complete
bioconversion of hemicellulosic sugars from agricultural residues
into lactic acid byLactobacillus pentosus. Appl Biochem Biotech-
nol. 2006;135:219–227.
22. Hofvendahl K, Hahn-Hägerdal B. Factors affecting the fermenta-
tive lactic acid production from renewable resources. Enz Microb
Techonol. 2000;26:87–107.
23. Pol EC, Vaessen E, Weusthuis RA, Eggink G. Identifying inhibi-
tory effects of lignocellulosic by-products on growth of lactic
acid producing micro-organisms using a rapid small-scale screen-
ing method. Bioresour Technol. 2016;209:297–304.
24. Zhang L, Li X, Yong Q, Yang ST, Ouyang J, S Y. Impacts of
lignocellulose-derived inhibitors on L-lactic acid fermentation by
Rhizopus oryzae. Bioresour Technol. 2016;203:173–180.
25. Ramos LP, da Silva L, Ballem AC, Pitarelo AP, Chiarello LM,
Silveira MHL. Enzymatic hydrolysis of steam-exploded sugar-
cane bagasse using high total solids and low enzyme loadings.
Bioresour Technol. 2015;175:195–202.
26. Cannella D, Chia-Wen CH, Felby C, Jørgensen H. Production
and effect of aldonic acids during enzymatic hydrolysis of
Biotechnol. Prog., 2019, Vol. 35, No. 1
lignocellulose at high dry matter content. Biotechnol Biofuels.
2012;5:1.
27. Cannella D, Jørgensen H. Do new cellulolytic enzyme prepara-
tions affect the industrial strategies for high solids lignocellulosic
ethanol production. Biotechnol Bioeng. 2014;111:59–68.
28. Haven MØ, Jørgensen H. Adsorption of β-glucosidases in two
commercial preparations onto pretreated biomass and lignin. Bio-
technol Biofuels. 2013;6:16.
29. Gobbetti M, de Angelis M, Corsetti A, di Cagno R. Biochemistry
and physiology of sourdough lactic acid bacteria. Trends Food
Sci Technol. 2005;16:57–69.
30. Alonso S, Herrero M, Rendueles M, Díaz M. Physiological het-
erogeneity in Lactobacillus casei fermentations on residual
yoghurt whey. Process Biochem. 2014;49:732–739.
31. Nguyen CM, Kim JS, Song JK, Choi GJ, Choi YH, Jang KS,
Kim JC. D-lactic acid production from dry biomass of Hydrodict-
yon reticulatum by simultaneous saccharification and co-
fermentation using Lactobacillus coryniformis subsp. tor- quens.
Biotechnol Lett. 2012;34:2235–2240.
32. Thomas S. Production of lactic acid from pulp mill solid waste
and xylose using Lactobacillus delbrueckii (NRRL B445). Appl
Biochem Biotechnol. 2000;84:455–468.
33. Nguyen CM, Kim JS, Hwang HJ, Park MS, Choi GJ, Choi YH,
Jang KS, Kim JC. Production of L-lactic acid from a green
microalga, Hydrodictyon reticulum, by Lactobacillus paracasei
LA104 isolated from the traditional Korean food makgeolli. Bior-
esour Technol. 2012;30:552–559.
34. Talukder MM, Das P, Wu JC. Microalgae (Nannochloropsis salina)
biomass to lactic acid and lipid. Biochem Eng J. 2012;68:109–113.
35. Unrean P. Optimized feeding schemes of simultaneous saccharifi-
cation and fermentation process for high lactic acid titer from
sugarcane bagasse. Ind Crops Prod. 2018;111:660–666.
9 of9
36. Adsul MG, Varma AJ, Gokhale DV. Lactic acid production from
waste sugarcane bagasse derived cellulose. Green Chem. 2007;9:
58–62.
37. Kadam SR, Patil SS, Bastawde KB, Khire JM, Gokhale DV.
Strain improvement of Lactobacillus delbrueckii NCIM2365 for
lactic acid production. Process Biochem. 2006;41:120–126.
38. Pol EC, Eggink G, Weusthuis RA. Production of L(+)-lactic acid
from acid pretreated sugarcane bagasse using Bacillus coagulans
DSM2314 in a simultaneous saccharification and fermentation
strategy. Biotechnol Biofuels. 2016;9:1–12.
39. Peng L, Xie N, Guo L, Wang L, Yu B, Ma Y. Efficient open fer-
mentative production of polymer-grade L-lactate from sugarcane
bagasse hydrolysate by thermotolerant Bacillus sp. strain P38.
PLoS One. 2014;9:e107143.
40. Kuo YC, Yuan SF, Wang CA, Huang YJ, Guo GL, Hwang WS.
Production of optically pure L-lactic acid from lignocellulosic
hydrolysate by using a newly isolated and D-lactate dehydroge-
nase gene-deficient Lactobacillus paracasei strain. Bioresour
Technol. 2015;198:651–657.
41. Patel M, Ou M, Ingram LO, Shanmugam KT. Fermentation of
sugar cane bagasse hemicellulose hydrolysate to L(+)-lactic acid
by a thermotolerant acidophilic Bacillus sp. Biotechnol Lett.
2004;26:865–868.
42. Hartmann MH. High molecular weight polylactic acid polymers.
In: Kaplan DL, editor. Biopolymers from Renewable Resources,
Macromolecular Systems — Materials Approach. Berlin,
Heidelberg: Springer; 1998:367–411.
43. Liu R, Dai L, Hu LQ, Zhou WQ, Si CL. Fabrication of high-
performance poly (L-lactic acid)/lignin-graft-poly (D-lactic acid)
stereocomplex films. Mater Sci Eng. 2017;80:397–403.
Manuscript received Jun. 8, 2018, revision received Aug. 16, 2018.