Biocatalysis and Agricultural Biotechnology 15 (2018) 185–191

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Biocatalysis and Agricultural Biotechnology

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Use of wheat straw biomass in production of L-lactic acid applying T

biocatalysis and combined lactic acid bacteria strains belonging to the genus
Lactobacillus
⁎
Dalia Cizeikiene , Grazina Juodeikiene, Jonas Damasius
Kaunas University of Technology, Department of Food Science and Technology, Radvilenu rd. 19, 50254 Kaunas, Lithuania

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

Keywords: The aim of the study was to investigate the usability of wheat straw in the production of L-lactic acid via
Biocatalysis fermentation applying by newly isolated lactic acid bacteria (LAB) strains belonging to the genus Lactobacillus
Lactobacillus and its combinations. Biotreatment of wheat straw was carried out through a three-step procedure consisting of:
Lactobacillus delbrueckii subsp. bulgaricus (i) physical pre-treatment, (ii) enzymatic hydrolysis and (iii) fermentation with LAB strains under laboratory
Lactic acid
conditions. Lactic acid production, residual sugar, cell biomass, pH medium and the influence of ions such as
Lactic acid bacteria
magnesium, calcium and sodium on efficiency of enzymatic hydrolysis of wheat straw were the main features
Wheat straw
examined. Moreover, optimal parameters for enzymatic hydrolysis were selected. Increased lactic acid pro-
duction was observed, when mixed LAB cultures were used in comparison to individual ones. The results con-
firmed that tested wheat straw could be used for lactic acid and L-lactic acid production using selected enzymes
and combined LAB strains.

1. Introduction
Lactic acid or 2-hydroxypropionic acid is a platform chemical, and
its salts have a long history of commercial uses and wide applications
(Wee et al., 2006). The interest in lactic acid is related to many aspects,
among which is its relatively high added-value. In particular, the
pharmaceutical and food industries have a preference for the L(+)
lactic acid isomer, the only one that can be metabolized by the human
body. However, the chemical industry requires one of the pure isomers
or a mixture of both, according to the application (Martineza et al.,
2013). This chemical has a market with great growth potential, can be
alternatively produced by fermentation or chemical synthesis and can
employ a large variety of different waste materials as substrates. The
economics of lactic acid production by fermentation is dependent on
many factors, of which the cost of the raw materials is very significant.
It is very expensive when sugars, e.g., glucose, sucrose, starch and other
etc., are used as the feedstock for lactic acid production (Abdel-
Rahmana et al., 2011a). Therefore, lignocellulosic biomass is a pro-
mising feedstock for lactic acid production considering its great sus-
tainability, availability, and low cost compared to refined sugars. Ac-
cording to the worldwide economic and environmental pollution issues
the research interest in the recycling of lignocellulosic biomass into the
various valuable chemicals such as lactic acid and other have been
increased (Abdel-Rahman et al., 2011b; Anwar et al., 2014). Despite its
advantages, commercial use of lignocellulose in lactic acid production
is still under research. Cheap cellulosic materials such as agricultural
waste, industrial solid waste are regarded as economically attractive
feedstocks for lactic acid fermentation, which allow the utilization of
agro-industrial waste as source of carbohydrate (Tang et al., 2011;
Juodeikiene et al., 2016). Higher economical effect has been de-
termined applying biotechnological conversion of wheat biomass to
lactic acid vs chemical synthesis (Juodeikiene et al., 2015). The che-
mical route produces a racemic mixture of DL-lactic acid, while opti-
cally pure L(+)- or D(−)-lactic acid can be obtained by microbial
fermentation. Since elevated levels of D-isomer are harmful to humans,
L-(+)-lactic acid is the preferred isomer in food and pharmaceutical
industries (Hofvendahl and Hahn-Hagerdal, 2000). Therefore, the
search for microorganisms producing high content of L-lactic acid from
lignocellulosic material is of outstanding importance. Many species
have been used for lactic acid production. However lactic acid bacteria
(LAB) belonging to the genus Lactobacillus are the most frequently used
bacteria (Abdel-Rahmana et al., 2011a). LAB can be classified into 2
groups on the basis of the end product of their fermentation: homo-
fermentative and heterofermentative. Homofermentative LABs virtually
produce only lactic acid, whereas other products are generated by
heterofermentative LABs along with lactic acid (Hofvendahl and Hahn-

⁎
Corresponding author.
E-mail address: dalia.cizeikiene@ktu.lt (D. Cizeikiene).

https://doi.org/10.1016/j.bcab.2018.06.015
Received 24 October 2017; Received in revised form 28 February 2018; Accepted 20 June 2018
Available online 21 June 2018
1878-8181/ © 2018 Elsevier Ltd. All rights reserved.
D. Cizeikiene et al.
Fig. 1. A general flow chart of the “conventional” process for lactic acid production from wheat straw biomass.
Hagerdal, 2000). Recently synergistic effects of LABs have been re-
ported regarding enhanced lactic acid production (KiBeom et al., 2005;
Plessas et al., 2008). Lignocellulosic material requires pre-treatment
applying physicochemical or enzymatic methods prior to lactic acid
fermentation because the microorganisms cannot directly convert this
material (Okano et al., 2010) into lactic acid. Therefore, the selection of
specific enzymes and positive enzyme effectors for degree of hydrolysis
increasing is necessary to obtain high content of bioproduct.
The aim of the study was to investigate the usability of wheat straw
in the production of L-lactic acid via fermentation through a three-step
procedure consisting of: (i) physical pre-treatment, (ii) enzymatic hy-
drolysis and (iii) fermentation with single LAB strains and their com-
binations under laboratory conditions.
2. Material and methods
2.1. Enzymes and microorganisms
Commercial enzyme preparation CeluStar XL containing xylanase,
cellulase, β-glucanase and additional side activities such as pectinase,
mannanase, xyloglucanase, laminarase, β-glucosidase, β-xylosidase, α-
L-arabinofuranosidase, amylase and protease, a branded product of
Dyadic International, Inc, was used.
Lactic acid bacteria (LAB) were previously isolated from wheat and
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Biocatalysis and Agricultural Biotechnology 15 (2018) 185–191
rye sourdough and dairy environments. LABs were cultivated in a MRS
broth (CM0359; Oxoid Ltd., Hampshire, UK) at optimal temperatures
(for Lactobacillus sanfranciscensis MR29, Lactobacillus frumenti H10,
Lactobacillus rossiae M2, Lactobacillus crustorum W19 and Lactobacillus
sanfranciscensis MW15 at 25 °C; for Lactobacillus rossiae GL14 strain at
37 °C; for Lactobacillus helveticus DSM 20075, Lactobacillus delbrueckii
subsp. bulgaricus MI, Lactobacillus delbrueckii subsp. bulgaricus DSM
20081 at 42 °C) for 24 h until further use.
After preliminary LAB screening for further experiments to evaluate
synergetic activity of LABs three LAB mixtures ((i) L. sanfranciscensis
MW15, L. crustorum W19 and L. sanfranciscensis MR29; (ii) L. delbrueckii
subsp. bulgaricus DSM 20081 and L. delbrueckii subsp. bulgaricus MI; (iii)
L. crustorum W19 and L. sanfranciscensis MR29) were prepared by
mixing freshly grown single LAB strains. Equal quantity of mixed LAB
strains was used for wheat straw fermentation at 25 °C in case of me-
sophilic strains and at 42 °C in case of thermophilic strains.
2.2. Plant material
The wheat was grown in experimental field located in Kaunas
(Lithuania) in 2016. Wheat straw was dried to the level where it con-
tained approx. 7% of water and was stored in dark environment at
ambient temperature.
D. Cizeikiene et al.
2.3. Determination of the influence of hydrolysis duration and addition of
ions on efficiency of wheat straw hydrolysis
The influence of (i) hydrolysis duration and (ii) addition of ions on
efficiency of wheat straw hydrolysis were evaluated by measuring the
content of reducing sugars. The influence of hydrolysis duration for 1, 2
and 3 h on reducing sugar content was examined at 55 °C during the
first step of analysis. Optimal hydrolysis duration (2 h) was selected for
further experiment. The influence of ion additives such as MgSO4·7H2O,
CaCl2 and NaCl in range of concentration 0 – 175 g/l on reducing sugar
content was examined during the second step of analysis. Enzymatic
hydrolysis of wheat straw samples using ion additives was carried out
for 2 h at 55 °C. The selected optimal amount of ions was added to
improve enzymatic hydrolysis for further experiments. The content of
reducing sugars after enzymatic hydrolysis in wheat straw medium was
carried out using DNS reagent as described below.
2.4. Mechanical and physical treatments of biomass
A laboratory scale impact mill MIAG (Bühler-Miag, Brunswick,
Germany) was used for physical pre-treatment of wheat straw, i.e. for
breaking down the structure of the lignocellulosic matrix by grinding to
a particle size fraction of less than 2 mm. The grounded straw samples
were mixed with a distilled water (water ratio of the sample 1:20) in
glass bottles and covered with a cap. The samples were pre-treated
under 1.1 atm pressure for 30 min at 121 °C. Wheat biomass has been
treated according to the scheme presented in Fig. 1.
2.5. Biotreatment of wheat straw for lactic acid production
After heat and pressure pre-treatment the samples were cooled to
55 °C and mixed with enzymatic preparation CeluStar XL (250 g/ metric
ton of treated substrate). The selected amounts of MgSO4·7H2O
(100 mg/l) and CaCl2·(50 mg/l) were added to improve enzymatic hy-
drolysis. Enzymatic hydrolysis was carried out as described above. After
enzymatic hydrolysis the samples were sterilised at 121 °C for 15 min,
then cooled to
25 °C and mixed with fresh single LAB strains or their mixtures
(0.01% v/w). The fermentation was carried out at temperatures that are
optimal for LABs (as described above)., The growth of LABs, pH, utili-
zation of reducing sugars and content of lactic acid isomers in wheat
straw medium during 120 h of fermentation with LABs were analysed.
2.6. pH determination
In order to determine pH, 5 g of fermented sample were taken out
from chamber under aseptic conditions using a sterile spoon, the
sample was homogenised with 50 ml of distilled water and then was
filtered through a Whatman's filter paper No. 1. The pH was then
measured in filtrate using a pH meter (PP-15; Sartorius, Goettingen,
Germany).
2.7. Determination of LAB growth
The growth of LABs in wheat straw medium during fermentation
was evaluated using standard counting techniques. Ten grams of fer-
mented sample were homogenised with 90 ml of 0.9% NaCl sterile so-
lution. LAB counts were determined on MRS agar (Liofilchem, Italy)
using standard plate count techniques. The plates were incubated under
anaerobic conditions for 72 h at the temperatures that are optimal for
each LAB (as given above) in a jar (SigmaeAldrich, Broendby,
Denmark) using anaerobic atmosphere generation bags (SigmaeAldrich,
Broendby, Denmark). The number of viable LAB cells was expressed as
a decimal logarithmic value of colony-forming units (cfu) per gram.
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Biocatalysis and Agricultural Biotechnology 15 (2018) 185–191
2.8. Reducing sugars evaluation
Dinitrosalicylic acid reagent was used for determination of reducing
sugar according to Miller (1959) with some modifications. In order to
evaluate reducing sugars, 5 g of fermented sample were taken out from
chamber under aseptic conditions using a sterile spoon, the sample was
homogenised with 50 ml of distilled water and then was filtered
through a Whatman's filter paper No. 1. The filtrate (0.5 ml) was mixed
with 3.5-dinitro salicylic acid (DNS) regent (0.5 ml). The mixture was
boiled for 5 min, then cooled on ice and diluted with 10 ml of distilled
water. Its optical density at 540 nm was determined. Glucose solutions
in range 0.33 – 6.66 µmol/ml were used for a standard curve.
2.9. Determination of lactic acid
A sample of fermented wheat straw (5 g) was mixed with 100 ml of
distilled water and centrifuged (5000g, 10 min) for the purpose of lactic
acid determination analysis. After centrifugation supernatant (1 ml)
was diluted with a distilled water up to 100 ml before analysis. An
enzymatic test K-DLATE 08/11 (Megazyme Int. Ireland, Wicklow,
Ireland) was used for determination of total lactic acid and D/L-lactate
according to the manufacturer's recommendations. The yield of lactic
acid produced (g) in comparison with the substrate consumed (kg) was
calculated.
2.10. Statistical analysis
Statistical data analysis was conducted using a Microsoft Excel
spreadsheet, and the statistical program for Windows (SPSS for
Windows, Ver. 16.0., SPSS Inc., Chicago, IL, USA) was used for com-
parison of the means by one-way analysis of variance (ANOVA). The
significance of the results from the data analysis was considered by
P < 0.05.
3. Results and discussion
3.1. Effect of the added magnesium, calcium and sodium ions on enzymatic
hydrolysis of wheat straw
The influence of ions on efficiency of enzymatic hydrolysis was in-
vestigated during this study. The content of reducing sugars after en-
zymatic hydrolysis in wheat straw medium prepared with and without
added ions of magnesium, calcium, and sodium is presented in Fig. 2.
The results show that 50 mg/l of CaCl2, 100 mg/l of MgSO4·7H2O added
in wheat straw medium significantly increased enzymatic hydrolysis
efficiency by 22.7% and 55.4%, whereas the addition of NaCl in range
Fig. 2. The content of reducing sugars after enzymatic hydrolysis in wheat
straw medium prepared with and without added ions of magnesium, calcium
and sodium (straw sample to water ratio = 1:20, 55 °C, 120 min). Letters a, b, c,
and d indicate substantial differences compared with the same type of ions at
95% probability level.
D. Cizeikiene et al. Biocatalysis and Agricultural Biotechnology 15 (2018) 185–191

50–175 mg/l decreased the content of reducing sugars by 20.2%. Si-

milar results on negative Na+ influence on cellulase activity was re-
ported by Wang et al. (2012). Moreover, our study is compatible with
statements of Zeng et al. (2016) and Wang et al. (2012) who have re-
ported that Ca2+ could remarkably promote the cellulase activity.
Whereas the inhibition pattern of cellulase by Mg2+ differed from that
reported by Wang et al. (2012). Ion influence on enzyme activity could
depend on the source of enzymes. Enzymatic hydrolysis is the most
promising means to yield fermentable sugars from pre-treated lig-
nocellulosic biomass, and is necessary to allow LABs to utilize poly-
saccharides as a carbon source (Lin et al., 2011). The purpose of en-
zymatic hydrolysis is to depolymerize the polysaccharides in the water-
insoluble solid fraction that remains after pre-treatment (Abdel-
Rahmana et al., 2011a). In order to maximize enzymatic hydrolysis,
mixtures of cellulases and hemicellulases should be used to increase
hemicellulose hydrolysis and thus increase the access of cellulase,
leading to a decreased hydrolysis time and process cost (Tu and
Saddler, 2010; Zhang et al., 2010). Unlike cellulose, xylans are che-
mically quite complex, and their degradation requires multiple en-
zymes. Enzymatic hydrolysis of hemicellulose requires endo-1,4–xyla-
nase, -xylosidase, - glucuronidase, -l-arabinofuranosidase, and
acetylxylan esterase, which act on xylan degradation and sacchar-
ification (Carvalheiro et al., 2008), and -mannanase and -mannosidase,
which cleave the glucomannan polymer backbone (Kumar et al., 2008).
According to manufacturers, CeluStar XL preparation contains the
aforementioned enzymes. Although more enzymes are required for
xylan hydrolysis than for cellulose hydrolysis, the substrate is more
easily accessible because xylan does not form tight crystalline structures
(Keshwani and Cheng, 2009).

3.2. The ability of lactic acid bacteria to produce lactic acid in wheat straw
medium
Fig. 3. The pH changes during 120 h of fermentation in wheat straw medium
using single LABs (a): Ls1 (L. sanfranciscensis MW15), Lc (L. crustorum W19), Ls2
Lactic acid production from wheat straw using different LABs be-
(L. sanfranciscensis MR29), Ld1 (L. delbrueckii subsp. bulgaricus MI), Ld2 (L.
longing to the genus Lactobacillus is presented in Table 1. The highest
delbrueckii subsp. bulgaricus DSM 20081) and their mixtures (b): 1)
content of lactic acid was produced by L. sanfranciscensis MW15, L. Ls1 ×Lc×Ls2 – L. sanfranciscensis MW15, L. crustorum W19 and L. san-
crustorum W19, L. sanfranciscensis MR29, L. delbrueckii subsp. bulgaricus franciscensis MR29; 2) Ld1 ×Ld2 – L. delbrueckii subsp. bulgaricus DSM 20081
MI, L. delbrueckii subsp. bulgaricus DSM 20081. Therefore, these strains and L. delbrueckii subsp. bulgaricus MI; 3) Lc×Ls2 – L. crustorum W19 and L.
were selected for further experiments as single strains and their com- sanfranciscensis MR29.
binations were prepared. According to literature, many species have
been used for lactic acid production (Abdel-Rahmana et al., 2011a).
strains in wheat straw medium are presented in Fig. 3. The results
However, to the best of our knowledge, it is the first report describing
showed that the pH values decreased using all tested LABs with in-
lactic acid production from lignocellulosic biomass by L. crustorum and
creased time of fermentation. The most intensive decrease of pH from
L. sanfranciscensis isolated from sourdough.
initial value (5.45) was observed after 72 h of fermentation, i.e. 4.52,
4.72, 4.69, 4.78 and 4.77 respectively when L. delbrueckii subsp. bul-
3.3. pH changes during lactic acid fermentation of wheat straw medium garicus DSM 20081, L. sanfranciscensis MW15, L. delbrueckii subsp. bul-
garicus MI, L. crustorum W19 and L. sanfranciscensis MR29 strains for
The pH changes during 120 h of fermentation with selected five LAB pre-treated wheat straw fermentation were used. Significantly higher
decrease of pH was observed during 24 and 48 h of wheat straw fer-
Table 1 mentation when LAB mixtures were used. The decrease of pH is cor-
Lactic acid produced after 72 h of fermentation using different LABs from en- related to the organic acids production from sugars and it is an im-
zymatically pre-treated wheat straw. portant factor indicating fermentation process. According to
LAB g/L Yielda g/kg Hofvendahl and Hahn-Hagerdal (2000) titration using base solutions or
electrodialysis of lactic acid could be applied for control of pH during
D(+) L(+) Total D(+) L(+) Total
fermentation. According to the literature, the low pH values of 3.8
L. sanfranciscensis MR29 0 2.85 2.85 0 57.0 57.0 could be reached in wheat medium during fermentation with LAB
L. rossiae GL14 0.03 0.93 0.96 0.6 18.6 19.2 (Hansen and Hansen, 1994; Juodeikiene et al., 2016). Coda et al.
L. frumenti H10 0.45 1.45 1.90 9 29 38 (2012) managed to achieve pH values from 3.72 to 4.03 after 8 h of
L. rossiae M2 0.41 1.13 1.54 8.2 22.6 30.8 fermentation of rice, barely, oat or soy flour with L. plantarum strains.
L. crustorum W19 0 2.94 2.94 0 58.8 58.8
L. sanfranciscensis MW15 0.38 4.56 4.94 7.6 91.2 98.8
Low pH values of 3.4–3.6 and 3.5–3.8 were reached during 24 h of
L. helveticus DSM 20075 0.19 1.84 2.03 3.8 36.8 40.6 fermentation of rye flour and rye bran, respectively, with Lactobacillus
L. delbrueckii subsp. bulgaricus MI 0.15 4.59 4.74 3.0 91.8 94.8 spp. (Muller et al., 2001). According to Juodeikiene et al. (2016), pH of
L. delbrueckii subsp. bulgaricus DSM 0.32 4.49 4.81 6.4 89.8 96.2 the wheat bran decreased only to 4.2 after 24 h of fermentation using P.
20081
pentosaceus KTU05–9 strain, whereas after 24 h of fermentation of
a
Calculated as grams of lactic acid produced per 1 kg of wheat straw. whole grain wheat flour (starchy raw material) with the same LAB the

188
D. Cizeikiene et al.
Table 2
Viable LAB cell counts in wheat straw medium after 24, 48 and 72 h of fermentation.
Viable LAB cell counts, log10 cfu/g
Fermentation duration, h
LAB strain 0 24
L. sanfranciscensis MW15 7.26 ± 0.16a
L. crustorum W19 7.13 ± 0.21a
L. sanfranciscensis MR29 7.21 ± 0.24a
L. delbrueckii subsp. bulgaricus MI 7.32 ± 0.19a
L. delbrueckii subsp. bulgaricus DSM 20081 7.25 ± 0.17a
* Letters a, b, c, and d indicate substantial differences compared with different strain at 95% probability level.
pH of medium of the value 3.5 was reached (Cizeikiene et al., 2015).
3.4. The growth of LAB and utilization of reducing sugars in wheat straw
medium
Concentration of viable LAB cells during 120 h of fermentation in
wheat straw medium after enzymatic hydrolysis is presented in Table 2.
The content of viable LAB cells in wheat medium after 48 h of fer-
mentation depended on LAB species and strains used for wheat straw
fermentation (P < 0.05), whereas no significant differences of LAB
count between different species and strains were observed after 24 h of
fermentation. Significantly higher content of L. sanfranciscensis MW15
(9.34 log10 cfu/ml) was observed in comparison with L. sanfranciscensis
MR29 (8.78 log10 cfu/ml) after 48 h of fermentation. Decrease of pH
resulted in increase in the number of lactic acid bacteria (R2 = 0.8982)
(Fig. 3). Similar count of viable LABs (109 cfu/ml) was reported by
Djukic-Vukovic et al. (2013) after 54 h of distillery stillage fermentation
with Lactobacillus rhamnosus. Comparable contents of LAB in starchy
media prepared from brewer's spent grain were reported by
Juodeikiene et al. (2016), whereas, as reported by the authors, higher
content of LAB was detected in the medium of wheat bran (from 10.2 to
10.8 log10 cfu/ml depending on LAB strain). According to
Charalampopoulos et al. (2002) the maximum LAB cell population of
8.1–10.1 log10 cfu/ml was determined in the malt medium after 48 h of
fermentation with various LAB species such as Lactobacillus fermentum,
Lactobacillus reuteri, Lactobacillus acidophilus and Lactobacillus plan-
tarum. Nutrient concentration in the medium is one of the important
factors affecting the growth of LABs, which usually requires complex
nutrients such as carbon, nitrogen, minerals and vitamins. Carbon
source is used for lactic acid production via fermentation. LABs can
usually ferment disaccharides and monosaccharides such as glucose and
xylose the content of which increases after enzymatic hydrolysis from
lignocellulosic material. The most often used substrates for LAB are
sugars occurring in by-products such as wheat straw hexoses and pen-
toses (Abdel-Rahman et al., 2011a). Hexoses other than glucose, such as
mannose, galactose, and fructose are also fermented by many LABs. The
tissue of wheat straw is rich in rhamnose, fructose, arabinose, xylose,
mannose, galactose, glucose and other valuable compounds which can
befermented by LABs (Halasz, 2009; Collins et al., 2014). Reducing
sugars in enzymatically hydrolysed wheat straw medium (control) and
after 120 h of fermentation with LAB is presented in Fig. 4. The content
of reducing sugars decreased by 38.0 – 47.5% during fermentation with
single LAB depending on LAB strain used. A more pronounced decrease
in a content of reducing sugars was observed in cases when mixtures of
LAB strains were used for wheat straw fermentation (57.2–62.8%). The
correlation (R2 = 0.9719) between the content of reducing sugars and
lactic acid content was observed. Moreover, the correlation (R2
= 0.9462) between the content of reducing sugars and pH was ob-
served too.
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Biocatalysis and Agricultural Biotechnology 15 (2018) 185–191
48 72
8.43 ± 0.19a 9.34 ± 0.11b 9.24 ± 0.13b
8.31 ± 0.21a 8.85 ± 0.12c 8.74 ± 0.22c
8.30 ± 0.17a 8.78 ± 0.23c 8.85 ± 0.19c
8.45 ± 0.14a 9.23 ± 0.19ab 9.30 ± 0.12b
8.45 ± 0.25a 9.02 ± 0.12ad 9.08 ± 0.11b
3.5. Lactic acid production during wheat straw fermentation
Lactic acid is the end product of carbohydrates fermentation under
normal conditions (Abdel-Rahmana et al., 2011a). Lactic acid produc-
tion from wheat straw was studied during 120 h of fermentation
(Fig. 5). The results of the study showed that lactic acid production was
significantly (P < 0.05) dependent on the LAB strain used. The highest
content of total lactic acid after 120 h of fermentation, respectively
101.8, 99.4 and 97.4 g/kg of raw material, was produced by L. san-
franciscensis MW15, L. delbrueckii subsp. bulgaricus MI and L. delbrueckii
subsp. bulgaricus DSM 20081, whereas L. crustorum W19 and L. san-
franciscensis MR29 were responsible for significantly less production of
total lactic acid content, respectively 66.4 and 64.2 g/kg. L. san-
franciscensis MW15, L. delbrueckii subsp. bulgaricus MI and L. delbrueckii
subsp. bulgaricus DSM 20081 produced high L-lactic acid content after
72 h of fermentation, respectively 91.2, 91,8 and 89.8 g/kg of raw
material, whereas L-lactic acid content after 120 h of fermentation was
higher only by 1.9–2.8% in comparison with 72 h of fermentation. L.
crustorum W19 and L. sanfranciscensis MR29 produced significantly less
L-lactic acid content in wheat straw medium. Moreover, no undesirable
D-lactic acid isomer was produced by these strains during 120 h of
fermentation. Production of D-lactic acid with help of L. sanfranciscensis
MW15, L. delbrueckii subsp. bulgaricus MI and L. delbrueckii subsp. bul-
garicus DSM 20081 strains represented only 8.6%, 5.0% and 5.9%, re-
spectively, of total lactic acid produced after 72 h fermentation and
7.7%, 3.2% and 6.7%, respectively, of total lactic acid produced after
120 h of fermentation. Selection of the bacteria strains producing pure
L-lactic acid isomer is one of the most important parameters for suc-
cessful lactic acid production. However, high yield of productivity is not
the only goal in the industry of lactic acid production. Another goal is
the ability to rework cheap raw materials into favourable bioproducts.
The bioconversion efficiency of rich in cellulose agro-industrial by-
products such as wheat bran, spent distiller's grain with solids, brewer's
spent grain and lupine wholemeal to lactic acid using acid tolerant LAB
strains were studied by Juodeikiene et al. (2016). It was reported by the
authors that the highest concentration of L-lactic acid, i.e. 86.11 g/kg,
was reached after the bioconversion of hydrolysed wheat brans in
combination with 48 h of fermentation with Pediococcus pentosaceus
KTU05–9 strain. Whereas, as our studies revealed, the significantly
higher content of L-lactic acid was produced from wheat straw by L.
sanfranciscensis MW15, L. delbrueckii subsp. bulgaricus MI and L. del-
brueckii subsp. bulgaricus DSM 20081 strains.
It has generally been observed that pH, nutrient concentration,
substrate concentration, end product concentration and temperature
significantly affect the growth of LABs and lactic acid production
(Martineza et al., 2013; Ghaffar et al., 2014). According to literature
high lactic acid production has been obtained with batch fermentation
of LABs where 150 g/l of L-lactate were produced from glucose (Bai
et al., 2004), 87.2 g/L of D-lactate were produced from glucose (Tashiro
et al., 2011), and 119 g/l of L-lactate (Abdel-Rahman et al., 2011a) and
80 g/l of D-lactate (Joshi et al., 2010) were produced from cellobiose.
D. Cizeikiene et al.
Fig. 5. L-lactic acid (a) and D-lactic acid (b) production in wheat straw medium
during 120 h of fermentation using different LABs: Ls1 (L. sanfranciscensis
MW15), Lc (L. crustorum W19), Ls2 (L. sanfranciscensis MR29), Ld1 (L. del-
brueckii subsp. bulgaricus MI), Ld2 (L. delbrueckii subsp. bulgaricus DSM 20081).
Whereas a significantly lower content of lactic acid could be obtained
from lignocelulosic material. Garde et al. (2002) managed to obtain
7.1 g/l of lactic acid from wheat straw hemicellulose with help of
Lactobacillus brevis and Lactobacillus pentosus during batch process. Wee
and Ryu (2009) managed to obtain 27.0 g/l of lactic acid from lig-
nocellulosic hydrolysates during continuous with cell-recycle fermen-
tation process using Lactobacillus spp. The stereospecificity and optical
purity of lactic acid depends on the LAB and its enzyme used in the
production. The yield of lactic acid obtained from the fermented lig-
nocellulosic substrates depended on substrate and the LAB strain used
and varied from 0.53 to 0.97 g/g (Mazzoli et al., 2014).
The possible synergetic effect on lactic acid production using the
mixtures of selected LABs for fermentation of wheat straw was eval-
uated (Fig. 6). The significantly higher lactic acid content was observed
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Biocatalysis and Agricultural Biotechnology 15 (2018) 185–191
Fig. 4. Reducing sugars in enzymatically hydrolysed
wheat straw medium (control) and after 120 h of fer-
mentation with single LABs: Ls1 (L. sanfranciscensis
MW15), Lc (L. crustorum W19), Ls2 (L. sanfranciscensis
MR29), Ld1 (L. delbrueckii subsp. bulgaricus MI), Ld2 (L.
delbrueckii subsp. bulgaricus DSM 20081) and their mix-
tures: 1) Ls1 ×Lc×Ls2 – L. sanfranciscensis MW15, L.
crustorum W19 and L. sanfranciscensis MR29; 2) Ld1 ×Ld2
– L. delbrueckii subsp. bulgaricus DSM 20081 and L. del-
brueckii subsp. bulgaricus MI; 3) Lc×Ls2 – L. crustorum W19
and L. sanfranciscensis MR29.
Fig. 6. L(+) and D(-) lactic acid production in wheat straw medium during
120 h of fermentation using combined LAB strains: 1) Ls1 ×Lc×Ls2 – L. san-
franciscensis MW15, L. crustorum W19 and L. sanfranciscensis MR29; 2)
Ld1 ×Ld2 – L. delbrueckii subsp. bulgaricus DSM 20081 and L. delbrueckii subsp.
bulgaricus MI; 3) Lc×Ls2 – L. crustorum W19 and L. sanfranciscensis MR29.
when LAB mixes were used for fermentation of wheat straw. This re-
vealed possible synergistic effects between the LABs tested. The highest
amount of L-lactic acid was obtained using a mixture of L. san-
franciscensis MW15, L. crustorum W19 and L. sanfranciscensis MR29
strains (125.2 g/kg of raw material) and the mixture of L. delbrueckii
subsp. bulgaricus DSM 20081 and L. delbrueckii subsp. bulgaricus MI
strains (125.8 g/kg of raw material) after 120 h of fermentation. The
mixture of thermophilic LABs (L. delbrueckii subsp. bulgaricus DSM
20081 and L. delbrueckii subsp. bulgaricus MI) increased a content of L-
lactic acid from 19% to 59% depending on duration of fermentation in
comparison with pure L. delbrueckii subsp. bulgaricus MI strain which
produced the highest content of lactic acid as a single strain. The
mixture of mesophilic LABs increased a content of L-lactic acid from
24% to 107% depending on duration of fermentation in comparison
with pure L. sanfranciscensis MW15 strain which produced the highest
content of lactic acid as a single strain among the mesophilic LABs
tested. A mixture of L. crustorum W19 and L. sanfranciscensis MR29 and
their pure strains produced a pure L-lactic acid in wheat straw medium
during 120 h of fermentation. Positive synergetic effect of mixture of
cultures on lactic acid production was published by Plessas et al.
(2008). Increased concentrations of lactic acid were obtained by the
authors using a mix of cultures of L. delbrueckii ssp. bulgaricus and K.
marxianus yeast in comparison with the cases when the individual ones
were used for cheese whey fermentation. However, no such synergistic
effect was observed when combination of 2 LABs was used during their
study. Meanwhile the results obtained by KiBeom (2005) showed that
mixed LAB cell cultures used during the study that was carried out by
the author could effectively improve lactic acid production. The author
related the synergetic effect of LABs to symbiotic associations in a
mixed culture that not only overcome but also overcompensate
D. Cizeikiene et al.
nutritional limitations in the substrate via amino acid production. Our
study is compatible with the statements of KiBeom (2005) as we found
out that a positive synergetic effect could be obtained through combi-
nation of few LAB strains for fermentation of wheat straw and this could
be explained by a positive symbiosis between these cultures. The results
of the study confirm a significant economic advantage of mixed cultures
over single cultures for lactic acid production on the basis of pre-treated
industrial wheat straw medium as such production allows saving the
cost of expensive organic supplements.
4. Conclusion
The results of the study show that the proposed mixes of lactic acid
bacteria (LAB) starter cultures, namely mesophilic L. sanfranciscensis
MW15, L. crustorum W19 and L. sanfranciscensis MR29 strains and
combination of thermophilic L. delbrueckii subsp. bulgaricus DSM 20081
and L. delbrueckii subsp. bulgaricus MI strains can be successfully used to
enhance lactic acid production from bio-treated wheat straw. Moreover,
L. crustorum W19 and L. sanfranciscensis MR29 strains as well as their
mixture could be used for production of pure L-lactic acid isomer from
wheat straw medium. This suggests that combination of different LABs
has the possible synergistic effect on L-lactic acid production.
Conflict of interest
The authors declare that there is no conflict of interest.
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