Current Research in Green and Sustainable Chemistry 4 (2021) 100059

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Current Research in Green and Sustainable Chemistry

journal homepage: www.elsevier.com/journals/
current-research-in-green-and-sustainable-chemistry/2666-0865

‘Lactobacillus sp. strain TERI-D3’, as microbial cell factory for fermentative

production of lactic acid’.
Dipti Verma, Sanjukta Subudhi, PhD *
DBT-TERI Centre of Excellence in Advanced Biofuels and Bio-commodities, Advanced Biofuels program, The Energy and Resources Institute, Darbari Seth Block, Habitat
Place, Lodhi Road, New Delhi, 110 003, India

A R T I C L E I N F O A B S T R A C T

Keywords: This study reports for lactic acid production from different carbohydrates; monosaccharide (glucose, galactose,
Lactobacillus sp lactose) & disaccharides (sucrose) and from lignocellulose biomass (rice straw) by a novel strain ‘Lactobacillus sp.
Strain TERI-D3’ strain TERI-D3’. ‘TERI-D3’ strain produced 19.9, 19.4, 18.1 and 15.8 g/L of lactic acid from glucose, sucrose,
Lactic acid
lactose and galactose, respectively. Maximum lactic acid yield efficiency (0.97 g/g) was observed with glucose
Monosaccharides
Disaccharides
(>95% of the theoretical maximum yield). Lactic acid titer from glucose was 0.41 g/L. The lactic acid titer and
Rice straw biomass yield from rice straw biomass sugar was; 11.58 g/L and 0.73 g/g (>80% of the theoretical maximum yield),
Anaerobic fermentation respectively. The novelty of this study is that the ‘TERI-D3’ strain is a promising microbe for green lactic acid as it
has potential to valorize cheese industry waste, algae biomass as well as to utilize inexpensive next generation
lignocellulose biomass that are abundant and do not compete with food chain supply. This approach of recycling
and or reuse of organic waste holds importance in the circular economy frame.

1. Introduction
Lactic acid, the simplest hydroxycarboxylic acid got widespread
application as an industry platform chemical in pharmaceutical in-
dustries and food industries [1,2]. Lactic acid is used as a food additive in
fermented foods; yoghurt, butter, canned vegetables and in this regard
the food industries account for 35% of total lactic acid demand. Lactic
acid and calcium lactate used for direct acidification of cheese, paneer,
yoghurt [3]. While poultry industries use lactic acid for increasing shelf
life to combat food-borne pathogens and beer making industries use
lactic acid for pH adjustment and flavor enhancement. In pharmaceutical
industries, lactic acid finds application in; implants, pills, dialysis, sur-
gical, controlled drug release systems. Lactic acid also got high demand
(39%) in polymer industries for production of biodegradable Poly Lactic
Acid (PLA) polymer [4]. In view of broad scale implication of lactic acid
in several industries, the demand for lactic acid is rising.
Lactic acid is a chiral molecule and has two optical enantiomers; L-(þ)
and D-(). Lactic acid is commercially synthesized by chemical route that
involves hydrolysis of lactonitrile using corrosive acid (HCl). The lacto-
nitrile is then purified to produce lactic acid. Major concern of chemical
process is the generation of racemic mixture of D and L forms of lactic
acid, which is not desirable by the industries. Chemical process involves
use of petroleum based feedstock [5]. With the global concern for climate
security issues, fermentative production of green lactic acid is preferred
over chemical synthesis, as these processes are simple, require mild
pressure and temperature, and fermentation process can utilize inex-
pensive renewable feedstock.
Microbial fermentation process is preferred over chemical synthesis
process as microbial fermentation processes can selectively lead to pro-
duction of one of its two enantiomers or the racemic mixture through
employment of select homo- or hetero fermentative Lactic Acid produc-
ing Bacteria (LAB). Several Lactobacillus sp. have been reported for pro-
duction of ‘L’ and ‘D’ form of lactic acid; L. delbrueckii, L. coryniformis, L.
jensenii, L. vitulinus, L. casei, L. paracasei, L. rhamnosus, L. pentosus, L.
plantarum, L. brevis, L. sake, and L. acidophilus [6,7]. Most of the
fermentation processes are targeted for production of L- (þ) form of
enantiomer, as D-() forms cannot be metabolized by animal cells and
hence restricted for use in food industries. Natural L (þ) form of LA
produced by certain microbes, gained importance in food industries as
acidulant, flavor enhancer, preservative and pH regulator [8,9]. The
polymer industries require high pure enantiomers of lactic acid for Poly
Lactic Acid (PLA) and are market drivers for lactic acid production [10].
Another advantage of the fermentation process is that this process can
utilize agriculture residue (lignocellulose biomass), third generation

* Corresponding author. DBT-TERI Centre of Excellence in Advanced Biofuels and Bio-commodities, Advanced Biofuels program, The Energy and Resources
Institute, Darbari Seth Block, Habitat Place, Lodhi Road, New Delhi, 110 003.
E-mail address: ssubudhi@teri.res.in (S. Subudhi).

https://doi.org/10.1016/j.crgsc.2021.100059
Received 31 August 2020; Received in revised form 6 January 2021; Accepted 11 January 2021
Available online 21 January 2021
2666-0865/© 2021 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
nc-nd/4.0/).
D. Verma, S. Subudhi
biomass and can valorize industry waste (cheese whey, glycerol, organic
waste).
Lignocellulose biomasses are available in abundant and do not
compete for the food chain supply. Use of these residue as low cost feed
for green lactic acid production can help in sustainable management of
agriculture residue. The algae biomass after oil extraction (through
transesterification) can also serve as feed for lactic acid in a bio-refinery
approach as major fraction of this biomass contain glucose, galactose.
The cheese industries generate substantial amount of cheese whey as
spent product. United states generate more than 325 gallon of cheese
whey annually, which is very high. Disposal of these wastes become a
major concern for these cheese making industries. These products are
either used as fertilizer and/or as animal feed. However, around 40–50%
of these byproducts are disposed as sewage. Cheese whey composed of
lactose (4.5–5%), lipids (4–0.5%) and soluble proteins (0.6–0.8% (w/v)
and few mineral salts. Lactose composition of cheese whey is very high
that can serve as very good raw material for production of green lactic
acid [11]. This can also help in safe disposal of the cheese whey that
holds significance in view of the circular bio-economy.
In this present study, we have selected ‘Lactobacillus sp. strain TERI-
D3’ due to its potential to feed on broad spectrum substrates including
monosaccharides, disaccharides as well as second generation lignocel-
lulose biomass to produce lactic acid.
2. Material & method
2.1. Sampling, enrichment and growth media
Dairy water samples were collected aseptically from local dairy farms
located in Gwal Pahari (Haryana). The samples were stored under
refrigerated condition till further use. Enrichment studies were carried
out by using MRS medium that was composed of (g/L); 10; peptone, 10;
beef extract, 5; yeast extract, 20; dextrose, 1; polysorbate 80, 2; ammo-
nium citrate, 5; sodium acetate, 0.1; magnesium sulphate, 0.05; man-
ganese sulphate, 2; dipotassium phosphate [12]. This medium was
prepared anaerobically by flushing with N2 gas. Media pH was set at 6.5
and sterilized at 120  C for 15 min and then cooled to room temperature.
Laboratory scale batch experiments were performed in 120 mL (30 mL
working volume) serum bottles. The dairy water samples (3 mL) were
enriched separately in 30 mL MRS medium and the enriched samples
were incubated at 37  C, 150 RPM for 120 h. Enrichments studies were
repeated for three generations.
2.2. Isolation, selection and purification of lactic acid producing bacteria
The lactic acid producing microbial consortia were screening and
then processed for purification and selection of maximum lactic acid
producing microbe. For purification, the cell suspension of select mi-
crobial consortium was diluted (to a scale of 104 dilution) and plated on
MRSþagar (1.5% agar W/V) plates and incubated at 37 C for 120 h.
Based on the different colony morphology, single colonies were picked
and streaked on solid MRS media to isolate pure single colonies. Purified
isolates were picked and cultured in fresh MRS broth and grown at 37  C,
150 RPM for 120 h. Purity of these isolates was monitored by observing
the gram stained cells under microscope (Olympus, Japan). The purified
bacterial isolates were screened to select maximum lactic acid producing
isolate. The select isolate was identified and processed for optimization
studies.
2.3. Morphological, biochemical and molecular characterization of the
purified lactic acid producing microbe
The cells were gram stained by using the gram staining kit (Hi-media,
India). Morphological characterization of the cells was done by observing
under the light microscope (Olympus, Japan) and Scanning Electron
Microscope (Zeiss, Japan). Biochemical characterization (catalase test,
2
Current Research in Green and Sustainable Chemistry 4 (2021) 100059
starch and casein hydrolysis test) was done by following the standard
protocols as reported earlier [13].
Molecular characterization was done by analyzing 16S rRNA gene
sequence. The RNA free genomic DNA was extracted by using the QI Amp
DNA mini kit (QIAGEN) [14]. PCR amplification of 16S rRNA gene was
carried out by using universal PCR primers. The PCR reaction mixture
was prepared in molecular grade water. PCR reaction mix contained;
forward and reverse primer (20 pmol/μl), 10 ng/μl DNA, 0.25 mmol
MgCl2, 10 Mm dNTPs, 10X PCR buffer and Taq polymerase.
Amplification was performed in a thermal cycler (Eppendorf, Ger-
many). PCR reaction was started with denaturation (96  C for 2 min)
followed by 35 cycles of amplification (94  C for 40 S, 55  C for 40 s, 72

C for 60 s). Final extension was performed at 72  C for 5 min [15]. The
PCR amplified product was purified using a gel extraction kit (Qiagen)
and purified PCR product was processed for 16S rRNA gene sequencing
(Macrogen, Korea). The 16S rRNA gene sequence was aligned with
reference nucleotide sequences in the NCBI database through employ-
ment of Basic Local Alignment Search Tool (BLAST).
Closely related sequences were aligned through employment of
CLUSTAL_W program. Phylogenetic tree was constructed by the
neighbour-joining analysis approach using the MEGA5.10 program.
Estimation of the confidence of the tree topologies was done by boot-
strapping analysis for 1000 replicates was used [15,16].
2.4. Nucleotide sequence accession number
The 16S rRNA gene sequence of the select lactic acid producing
bacterial isolate was deposited in NCBI/EMBL nucleotide sequence
database under the accession number ‘MT256258’.
2.5. Temperature optimization
Lactic acid production performance of the select isolate from glucose
was monitored at following different temperatures; 30, 37 and 42  C.
Experiments were performed in 120 mL serum bottles containing 50 mL
MRS broth supplemented with 2% glucose. The serum bottles were
inoculated with 2% starter culture and incubated at above mentioned
different temperatures at 150 RPM for 120 h. All the experiments were
performed in triplicates.
2.6. Lactic acid production from monosaccharide and disaccharide sugars
To monitor the lactic acid production potential of the select isolate
from simple and complex carbohydrates, separate experiments were
performed by using lactose, galactose and sucrose as substrate (2%) in
the MRS medium. Experimental bottles were incubated at 30  C, 150
p.m. for 96 h. Samples were then withdrawn at regular interval (24 h) to
monitor the cell growth, pH of the fermentation broth, lactic acid and
residual feed. All the experiments were performed in triplicates.
2.7. Second generation green lactic acid production from rice straw
biomass
The enzymatic hydrolysis of alkaline pre-treated rice straw biomass
(7.5% biomass loading) was carried out by using Cellic CTec2 enzyme.
The enzyme hydrolysis was carried out in 50 mM citrate buffer (pH 5).
The enzyme was added at a concentration of 20 units/1 g of biomass and
the hydrolysis reaction was carried out at 50  C, 150 RPM for 24 h. The
sugar concentration in the rice straw hydrolysate was monitored by High
Pressure Liquid Chromatographic (HPLC) analysis and used as feed in
following different proportions (v/v); 25%, 50%, 75% and undiluted
(100%), in the MRS medium. Batch fermentations were performed in 30
mL working volume scale. Inoculation was done with 2% pre-grown
starter culture. Experimental batch reactors were incubated at 30  C,
150 rpm for 96 h. All the experiments were done in triplicate.
D. Verma, S. Subudhi
Fig. 1. Scanning Electron Micrograph of ‘Lactobacillus sp. strain ‘TERI-D3’.
2.8. Scale up of lactic acid production in 500 mL scale
Scale up of lactic acid production was carried out in 1 L serum bottles
containing 500 mL growth medium supplemented with glucose (2%).
Initial pH of the medium was set at 6.5 and inoculated with starter cul-
tures. The batch reactors were incubated at 30  C, 150 rpm for 48 h.
Samples were withdrawn at regular intervals (24 h) to monitor the cell
growth profile, fermentation broth pH, residual sugar and lactic acid
productivity of the select isolate.
2.9. Analytical methods
Cell growth was monitored by measuring the optical density (OD) of
the cell culture at 600 nm in an UV visible spectrophotometer (UV-2450,
Shimadzu, Japan). High Performance Liquid Chromatography (HPLC,
Agilent 1100 series, USA) equipped with Aminex R HPX-87H, (300 mm
 7.8 mm) column (Agilent) and RID detector (optimum temperature 60
 D3’ strain
C) was used to detect the lactic acid, glucose, sucrose and galactose.
Sulphuric acid (0.005 M) was used as mobile phase at flow rate of 0.6
mL/min. Calibration was done by using the standards and the calibration
curve was prepared. R2 value of the calibration curve was 0.998.
Sugar concentration of the rice straw biomass hydrolysate was ana-
lysed using High Performance Liquid Chromatography (Agilent Tech-
nologies Inc. 1220 Infinity LC Series, USA) that was equipped with
Aminex-HPX87H column (Biorad, USA). 5 mM sulphuric acid was used
as the mobile phase at a flow rate of 0.6 mL/min and the column tem-
perature was maintained at 60  C.
All the experiments were executed in triplicates and the data repre-
sent the average of triplicate  standard deviation.
3. Results and discussion
3.1. Isolation, purification and lactic acid production performance of the
select bacterial isolate
Among four different microbial consortia enriched from dairy water
samples, the consortium designated as ‘TERI-D’, has demonstrated
maximum lactic acid production from glucose (2.3 g/L). This consortium
was selected for further purification and isolation of lactic acid producing
bacteria. Five morphologically different bacterial isolates were purified
from ‘TERI-D’ consortium, among which the strain designated as ‘TERI-
D3’ has demonstrated maximum lactic acid production; 15.49 g/L, from
2% glucose based fermentation medium. ‘TERI-D3’ strain was thus
selected for further research studies to monitor its potential to produce
lactic acid from different substrates; simple and complex carbohydrates,
lignocellulosic biomass sugars.
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Current Research in Green and Sustainable Chemistry 4 (2021) 100059
Fig. 2a. Fermentative lactic acid production profile of ‘Lactobacillus sp. strain
‘TERI-D3’ at different temperature.
Fig. 2b. Lactic acid yield of ‘Lactobacillus sp. strain ‘TERI-D3’ from glucose at
different temperature.
3.2. Morphological, biochemical and molecular characterization of ‘TERI-
Morphological characterization of ‘TERI-D3’ strain indicated that the
cells are gram positive, rod shaped, and non-spore forming. The colonies
were white in colour, small, circular, mucoid with elevated margin.
Biochemical analysis results indicated that the cells were catalase nega-
tive. The cells could not hydrolyse starch and casein. ‘TERI-D3’ cells could
grow in high salt concentrations (up to 10%). Scanning Electron Micro-
graph of ‘TERI D3’ revealed that the cells are non-spore forming, short,
straight and rod shaped with round ends (Fig. 1).
Comparative 16S rRNA gene sequence analysis of ‘TERI-D3’ strain
with closely related reference sequences of Gene Bank data base,
demonstrated maximum homology (99%) with Lactobacillus sp. and thus
this strain is identified as ‘Lactobacillus sp. strain TERI-D3’. Lactobacillus
sp. are reported for production of lactic acid from broad spectrum sub-
strates [17].
3.3. Temperature optimization
Fermentative lactic acid production performance of ‘TERI-D3’ strain
was monitored at different temperatures (30  C, 37  C, 42  C and 45  C).
All the experiments were performed using 2% glucose as the substrate.
Maximum lactic acid fermentation was observed after 96 h. Lactic acid
titre was maximum (17.51  0.2 g/L) at 30  C (Fig. 2a). Lactic acid titre
at 37  C and 42  C was; 17  0.2 and 15.5  0.2 g/L, respectively. While
lactic acid titre was minimum (8.6  0.2 g/L) at 45  C. The maximum
lactic acid yield was observed at 30  C (Fig. 2b); 0.875 g/g of glucose
(87.5% yield efficiency compared to maximum theoretical lactic acid
yield of 1 g/1 g of glucose). Lactic acid yield at 45  C reduced to 0.43 g/g
D. Verma, S. Subudhi Current Research in Green and Sustainable Chemistry 4 (2021) 100059

Fig. 5. Lactic acid production profile of Lactobacillus sp. strain ‘TERI-D3’ with
Fig. 3. Lactic acid production profile of Lactobacillus sp. strain ‘TERI-D3’ with respect to lactose utilization at different time interval.
respect to glucose utilization.

Fig. 4. Lactic acid production profile of Lactobacillus sp. strain ‘TERI-D3’ with Fig. 6. Lactic acid production profile of Lactobacillus sp. strain ‘TERI-D3’ with
respect to galactose utilization. respect to sucrose utilization at different time interval.

of glucose. Optimal temperature for lactic acid production by different yield efficiency observed with galactose (0.8 g/g, 80%). Complete sub-
Lactobacillus sp. varies from ‘25–45  C’ [18]. Lactobacillus casei G-0 strain strate utilization for glucose fermentation was observed after 48 h
produced maximum lactic acid at 41  C [19], whereas in case of L. casei (Fig. 3). While in case of galactose fermentation complete substrate uti-
M  15, L. casei NRRL B-441, L. rhamnosus NRRL B-445, Lactobacillus sp. lization was observed after 72 h (Fig. 4).
RKY optimum temperature for lactic acid production is reported at 37  C TERI-D3 strain could utilize both the sugars to produce lactic acid
[20,21]. All these studies imply that fermentation temperature plays a with high yield efficiency, which indicates that TERI-D3 strain has sig-
key role for fermentative lactic acid production. nificant potential to valorize cheese whey and to utilize algae biomass
(after oil extraction) to produce lactic acid. These results implied that
‘TERI-D3’ strain has broad spectrum substrate utilization potential to
3.4. Lactic acid production from different carbohydrates
produce lactic acid with high yield efficiency. Our studies are in consis-
tence with the earlier study that is reported for lactic acid production by
3.4.1. Lactic acid production profile of ‘TERI-D3’ strain from
L. paracasei strain MONGB-2 [23]. Lactic acid titre of MONGB-2 strain
monosaccharides (glucose and galactose)
from glucose (18.86 g/L) and galactose (18.23 g/L) is comparatively
Galactose is one of the major residual fraction of cheese industry
higher than that observed with TERI-D3 strain.
waste (cheese whey). There is a great concern for sustainable manage-
ment of this spent product as cheese industries generate substantial
3.4.2. Lactic acid production profile of ‘TERI-D3’ strain from disaccharides
cheese whey. Hence galactose fermentation by select Lactic Acid Bacteria
(lactose and sucrose)
(LAB) has been explored earlier [22]. With an aim to monitor potential of
With an aim to monitor disaccharide utilization potential of TERI D3
TERI-D3 strain to valorize spent product from cheese industry and 3rd
strain, lactose and sucrose were used separately as feed in fermentation
generation algae biomass containing galactose and glucose, we have
broth for lactic acid production. Lactose is composed of two monomers;
observed galactose fermentation by this strain for lactic acid production.
glucose & galactose and sucrose is composed of two monomers; glucose
Comparative lactic acid production from glucose and galactose by
and fructose. This strain could ferment both of these disaccharides to
TERI-D3 was observed at different time intervals at optimum temperature
produce lactic acid with significant yield efficiency. The results demon-
(30  C). Analysis of these results demonstrated that this strain could
strated that ‘TERI-D3’ strain produced 18.1  0.2 g/L lactic acid from
utilize galactose to produce lactic acid with significant yield efficiency,
lactose and lactose consumption rate was 0.27 g/L/h (Fig. 5). Lactic acid
close to the lactic acid yield observed with glucose (Figs. 3 and 4). Lactic
yield efficiency was 0.9 g/g of lactose while productivity was 0.25 g/L/h.
acid titre of this strain from glucose and galactose was; 17.5  0.2 g/L
In case of sucrose fermentation, consumption rate of this strain was
(Figs. 3) and 15.8  0.2 g/L (Fig. 4), respectively. The lactic acid yield
0.31 g/L/h and the lactic acid titer was 19.4  0.2 g/L (Fig. 6). Lactic acid
efficiency from glucose was higher (0.9 g/g, 90%) as compared to the

4
D. Verma, S. Subudhi Current Research in Green and Sustainable Chemistry 4 (2021) 100059

Fig. 7. Lactic acid production profile of Lactobacillus sp. strain TERI-D’3 with respect to its growth, glucose
utilization and variation in pH, at different time interval.

Table 1
Lactic acid performance of Lactobacillus sp. strain ‘TERI-D3’ from different mono-
and di-saccharide sugars.
S.No Substrate Working Lactic Yield Productivity (g/
(2%) volume (mL) acid (g/L) (g/g) L/h)

1 Glucose 50 17.51 0.96 0.36

2 Galactose 50 15.9 0.83 0.24
3 Lactose 50 18.2 0.9 0.25
4 Sucrose 50 18.9 0.86 0.26
5 Glucose 500 19.9 0.97 0.41

yield efficiency from sucrose was 0.86 g/g and productivity was 0.26 g/
L/h. These results demonstrated that ‘TERI-D3’ strain could feed on broad
spectrum substrates including simple as well complex carbohydrates to
produce lactic acid with high yield efficiency. This study implies Lacto-
bacillus TERI-D3 strain is a potential candidate for production of green
Fig. 8. Lactic acid production performance of Lactobacillus sp. strain ‘TERI-D3’
lactic acid from spent organic waste; cheese whey (composed of lactose), from rice straw biomass hydrolysed sugar.
molasses (composed of sucrose), spent product from sugar industries.
Algae biomass is composed of 20–25% carbohydrates that contains
mostly glucose, galactose and can serve as good feed (after oil extraction)
for lactic acid production by this strain. acid production became maximum. Complete substrate utilization was
observed at 48 h. Lactic acid yield efficiency was 0.97 g/g of glucose
(97% yield efficiency) and lactic acid productivity was; 0.41 g/L/h.
3.5. Scale-up of fermentative production of lactic acid in 500 mL scale
Lactic acid productivity of ‘TERI-D3’ strain from different carbohy-
drates demonstrated that this strain has significant potential to feed on
Among different carbohydrates explored as feed for fermentative
simple as well as complex carbohydrates for lactic acid production for
lactic acid production, maximum lactic acid tire and yield efficiency was
industrial applications. Table 1 presents comparative lactic acid pro-
observed with glucose. Thus further scale up studies are carried out using
ductivity, titre and yield efficiency of ‘TERI-D3’ strain from glucose,
glucose (2%) as feed. The process was up scaled from 30 mL to 500 mL
galactose, sucrose and lactose. The lactic acid yield efficiency from all
working volume scale in batch mode at 30  C and 6.5 pH. Growth and
these sugars was more than 80%. Highest yield was observed with
lactic acid production profile of this strain was monitored with respect to
glucose with an efficiency of 97%. Lactic acid yield efficiency from
its glucose utilization at different time interval. Variation in fermentation
lactose, sucrose and galactose was also significant; 90, 86 and 83%,
pH was also monitored. The cells exhibited shorter lag phase and lactic
respectively (Table 1).
acid production started during exponential growth phase. Maximum
Lactic acid productivity from glucose was 0.4 g/L/h (in 500 mL scale).
lactic acid production was observed after 48 h fermentation, when the
With increase in fermentation broth volume from 30 to 500 mL scale,
cells entered stationary phase. Maximum absorbance of the cells was
lactic acid productivity has increased from 0.36 to 0.4 g/L/h. These re-
observed at O. D600nm (2.99). During fermentation, the pH profile has
sults indicate that this strain is a potential candidate for scale up of the
revealed gradual downfall with respect to the production of lactic acid.
production of green lactic acid in pilot scale from renewable feed; algae
The final pH dropped down to 4.7. Lactic acid titre was 19.9 g/L (Fig. 7).
biomass and spent organic matter from dairy industries (rich in lactose
Maximum glucose utilization was observed after 6 h and production
and galactose), sugarcane industries (rich in sucrose).
has continued till 48 h fermentation, when the cell biomass and lactic

5
D. Verma, S. Subudhi
3.6. Second generation lactic acid production by ‘TERI-D3’ strain from rice
straw biomass sugar
In order to monitor the potential of this strain for lactic acid pro-
duction from second generation lignocellulose biomass, the enzyme
hydrolysed rice straw biomass hydrolysate was used as feed in the
fermentation broth. Total reduced sugar concentration of this hydroly-
sate was 11.58 g/L. This broth was used in the following proportions; 25,
50, 75 and 100% (v/v) in the fermentation medium. Lactic acid pro-
ductivity of ‘TERI-D3’ strain from rice straw biomass sugar was
monitored.
Lactic acid production was observed with all the fermentation broth
containing different proportion of (25, 50, 75%) rice straw biomass hy-
drolysate (Fig. 8). The results indicated for maximum lactic acid pro-
duction from 100% rice straw biomass hydrolysate based fermentation
broth (undiluted) and the titer was 11.16 g/L. Lactic acid yield was 0.95
g/g reduced sugar. The lactic acid yield observed with 75% hydrolysate
was 0.91 g/g (9.15 g/L titer) of reduced sugar. With decrease in feed
concentration in the fermentation broth the lactic acid titer also got
reduced to 6.43 and 3.49 g/L from 50% to 25% rice straw hydrolysate
based fermentation broth, respectively. Complete sugar utilization was
observed by ‘TERI-D3’ strain within 24 h fermentation. This study dem-
onstrates that the ‘TERI-D3’ strain could utilize second generation
lignocellulosic biomass sugar effectively to produce green lactic acid.
This holds importance in the context of circular bio economy frame.
Earlier studies demonstrated that the Lactobacillus genera has broad
spectrum substrate utilization capability. Lactic acid production has been
reported by several species of Lactobacillus from wide range of substrates;
wheat, wood, molasses, whey, rice bran, lignocellulose biomass sugars
[24–29]. Our study indicated that lactic acid yield efficiency of ‘Lacto-
bacillus sp. strain ‘TERI-D3’ from rice straw biomass hydrolysed sugar, is
higher than that has been reported for other sp. of Lactobacillus; Lacto-
bacillus pentosus, Lactobacillus bifermentans, Lactococcus lactis IO-1, from
different lignocellulosic biomass hydrolysed sugars [28,29]. Our study
demonstrated that the strain ‘TERI-D3’ encompasses broad spectrum
substrate utilization pathway to produce lactic acid with high yield ef-
ficiency from monosaccharaides, disaccharides, lignocellulose biomass
sugar. This strain has a great potential for lactic acid generation in in-
dustrial scale, in clean and green manner.
4. Conclusion
In this study we have appraised the ‘Lactobacillus sp. strain ‘TERI-D3’ a
novel strain isolated from spent dairy water for fermentative lactic acid
production from broad spectrum substrates; mon saccharides, di-
saccharides, lignocellulose biomass. The aim of this study was to monitor
the potential of this candidate to produce green lactic acid from 2nd and
3rd generation biomass and spent industrial waste in a bio-refinery
approach, as this holds importance in the perspectives of circular bio-
economy. Lactic acid titer and yield of this strain was monitored at op-
timum temperature from these substrates with respect to fermentation
time. This strain could utilize glucose, galactose, lactose, sucrose to
produce lactic acid with high yield efficiency (>85%). This strain also
valorized lignocellulosic biomass sugar to produce lactic acid with more
than 96% yield efficiency. These studies imply that ‘Lactobacillus sp.
strain TERI-D3’ is a promising candidate for production of green lactic
acid from spent industrial waste and lignocellulosic biomass in industrial
scale for downstream application in food and pharma industries.
CRediT authorship contribution statement
Dipti Verma: has contributed for execution of research activities.
Sanjukta Subudhi: has contributed for overall planning, experiment
designing, Supervision, Writing - review & editing.
6
Current Research in Green and Sustainable Chemistry 4 (2021) 100059
Declaration of competing interest
The authors declare that they do not have any competing interest for
preparation and submission of this manuscript.
Acknowledgement
The authors thank Director General, The Energy and Resources
Institute (TERI), New Delhi for providing the infrastructure support to
carry out this research study. The authors gratefully acknowledge
Department of Biotechnology, Ministry of Science and Technology,
Government of India, India for the financial support.
References
[1] Y. Qiu, P. Lei, Y. Zhang, Y. Sha, Y. Zhan, Z. Xu, S. Li, H. Xu, P. Ouyang, Recent
advances in bio-based multi-products of agricultural Jerusalem artichoke resources,
Biotechnol. Biofuels 11 (2018) 151.
[2] P. Tsapekos, M. Alvarado-Morales, S. Baladi, E.F. Bosma, I. Angelidaki,
Fermentative production of lactic acid as a sustainable approach to valorise
household bio-waste, Front. Sustain. 1 (2020) 4, https://doi.org/10.3389/
frsus.2020.00004.
[3] T. Ghaffar, M. Irshad, Z. Anwar, T. Aqil, Z. Zulifqar, A. Tariq, M. Kamran, N. Ehsan,
S. Mehmood, Recent trends in lactic acid biotechnology: a brief review on
production to purification, J of Radiation Research and Applied Sciences 7 (2014)
222–229.
[4] E. Cubas-Cano, C. Gonz
alez-Fern
andez, M. Ballesteros, E. Tom
as-Pej
o,
Biotechnological advances in lactic acid production by lactic acid bacteria:
lignocellulose as novel substrate, Biofuels, Bioprod. Bioref. 12 (2) (2018) 290–303.
[5] B.S. Krishna, G. Sarva, S. Nikhilesh, B. Tarun, K.V. Saibaba, R. Narayana, Gopinadh,
Industrial production of lactic acid and its applications, Int. J of Biotech Research. 1
(1) (2018) 42–54.
[6] S. Wuyts, W. Van Beeck, C.N. Allonsius, M. Fl van den Broek, S. Lebeer, Applications
of plant-based fermented foods and their microbes, Curr. Opin. Biotechnol. 61
(45–52) (2020).
[7] B.D. Ulery, L.S. Nair, C.T. Laurencin, Biomedical applications of biodegradable
polymers, J. Polym. Sci., Part B: Polym. Phys. 49 (2011) 832–864.
[8] Y.C. Kuo, S.F. Yuan, C.A. Wang, Y.J. Huang, G.L. Guo, W.S. Hwang, Production of
optically pure L-lactic acid from lignocellulosic hydrolysate by using a newly
isolated and D-lactate dehydrogenase gene-deficient Lactobacillus paracasei strain,
Bioresour. Technol. 198 (2015) 651–657.
[9] M.A. Abdel-Rahman, Y. Tashiro, K. Sonomoto, Lactic acid production from
lignocellulose-derived sugars using lactic acid bacteria: overview and limits,
J. Biotechnol. 156 (2011) 286–301.
~ez, J.L. Alonso, J.C. Paraj

[10] R. Y
an o, D-lactic acid production from waste cardboard,
J. Chem. Technol. Biotechnol. 80 (1) (2005) 76–84.
[11] M.P. Bernardo, L.F. Coelho, D.C. Sass, J. Contiero, l-(þ)- Lactic acid production by
Lactobacillus rhamnosus B103 from dairy industry waste, Braz. J. Microbiol. 47
(640–646) (2018).
[12] M.A. Abdel-Rahman, K. Sonomoto, Opportunities to overcome the current
limitations and challenges for efficient microbial production of optically pure lactic
acid, J. Biotechnol. 236 (2016) 176–192.
[13] B. Lanyi, Classical and rapid identification methods for medically important
bacteria, Methods Microbiol. 19 (1) (1987) 67.
[14] J. Sambrook, D.W. Russell, Molecular Cloning: a Laboratory Manual, third ed., Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001.
[15] J. Felsenstein, Confidance limit on phylogenies: an approach using the bootstrap,
Evolution (30) (1995) 783–791.
[16] N. Saitou, M. Nei, The neighbour-joining method: a new method for reconstructing
phylogenetic trees, Mol. Biol. Evol. 4 (1987) 405–425.
[17] M.Y. Ha, S.W. Kim, Y.W. Lee, M.J. Kim, S.J. Kim, Kinetics analysis of growth and
lactic acid production in pH-controlled batch cultures of Lactobacillus casei KH-1
using yeast extract/corn steep liquor/glucose medium, J. Biosci. Bioeng. 96
(134–140) (2003).
[18] H. Qin, S.S. Gong, X.Y. Ge, W.G. Zhang, The effect of temperature on L-lactic acid
production and metabolite distribution of Lactobacillus casei, Prep. Biochem.
Biotechnol. 42 (6) (2012) 564–573.
[19] K. Chaisu, A.L. Charles, Y.K. Guu, T.B. Yen, C.H. Chiu, Optimization of lactic acid
production from molasses renewable, raw material through response surface
methodology with lactobacillus casei M-15, APCBEE Procedia 8 (2014) 194–198.
[20] M. Hujanen, Y.-Y. Linko, Effect of temperature and various nitrogen sources on L (
þ) lactic acid production by Lactobacillus casei, Appl. Microbiol. Biotechnol. 45
(1996) 307–313.
[21] Y.J. Wee, J.N. Kim, J.S. Yun, H.W. Ryu, Optimum conditions for the biological
production of lactic acid by a newly isolated lactic acid bacterium, Lactobacillus sp,
RKY, Biotechnology and Bioprocess Engineering 10 (23) (2005).
[22] Q. Wu, C.K.W. Cheung, N.P. Shah, Towards galactose accumulation in dairy foods
fermented by conventional starter cultures: challenges and strategies, Trends Food
Sci. Technol. 41 (2015) 24–36.
[23] S.J. Kim, H.K. Seo, W.S. Kong, M.H. Yoon, Production of lactic acid by Lactobacillus
paracasei isolated from button mushroom bed, J. Mushroom Sci. Prod. 11 (4)
(2013) 187–193.
D. Verma, S. Subudhi
[24] K. Hofvendahl, B. Hahn-H€agerdal, L-lactic acid production from whole wheat flour
hydrolysate using strains of Lactobacilli and Lactococci, Enzym. Microb. Technol.
20 (4) (1997) 301–330, 1997.
[25] A.B. Moldes, J.L. Alonso, J.C. Paraj

o, Strategies to improve the bioconversion of
processed wood into lactic acid by simultaneous saccharification and fermentation,
J. Chem. Technol. Biotechnol. 76 (2001) 279–284.
[26] C. Kotzanmanidis, T. Roukas, G. Skaracis, Optimization of lactic acid production
from beet molasses Lactobacillus delbrueckii NCIMB 8130, World J. Microbiol.
Biotechnol. 18 (2002) 441–448.
Current Research in Green and Sustainable Chemistry 4 (2021) 100059
[27] T. Tanaka, M. Hoshina, S. Tanabe, K. Sakai, S. Ohtsubo, M. Taniguchi, Production of
D-lactic acid from defatted rice bran by simultaneous saccharification and
fermentation, Bioresour. Technol. 97 (2006) 211–217.
[28] A.B. Moldes, A. Torrado, A. Converti, J.M. Dominguez, Complete bioconversion of
hemicellulosic sugars from agricultural residues into lactic acid by Lactobacillus
pentosus, Appl. Biochem. Biotechnol. 135 (2006) 219–227.
[29] P. Laopaiboon, A. Thani, V. Leelavatcharamas, L. Laopaiboon, L, Acid hydrolysis of
sugarcane bagasse for lactic acid production, Bioresour. Technol. 101 (2010)
1036–1043.