Spectroscopy Letters, 42:296–304, 2009

Copyright # Taylor & Francis Group, LLC

ISSN: 0038-7010 print=1532-2289 online
DOI: 10.1080/00387010903178632

1
H NMR Quantitative Assessment of Lactic
Acid Produced by Biofermentation
of Cane Sugar Juice
Avin Ramanjooloo1,
Archana Bhaw-Luximon1, ABSTRACT The demand for lactic acid as a raw material for the chemical
Dhanjay Jhurry1, industry is on the rise. Various lactic acid bacteria (LAB) have been tested
and Frederic Cadet2 for the fermentation of sugar-cane juice and molasses into lactic acid. Indust-
1
Department of Chemistry, rially, the most commonly used producer strains are from the Lactobacillus
Faculty of Science, University
genus. We report here on the fermentation of sugar-cane juice and syrup
of Mauritius, Réduit, Mauritius
2 into L-lactic acid using Lactobacillus delbrueckii subsp delbrueckii (NCIM
La Réunion, St. Dennis, France
2365). We present a reliable method to quantitatively assess the amount
of L-lactic acid produced during fermentation using 1H NMR spectroscopy.
This analytical method offers various advantages over existing ones and can
be extended to the on-line monitoring of fermentation of sugars into lactic
acid in industry.

1
KEYWORDS H NMR spectroscopy, lactic acid, quantitative assessment

INTRODUCTION
The green bio-refinery concept presents itself as an interesting industrial
opportunity and long-term sustainability for many countries worldwide,
including Mauritius. A bio-refinery is a facility that integrates biomass con-
version processes and equipment to produce fuels, power, and chemicals
from biomass. The bio-refinery concept is analogous to today’s petroleum
refineries, which produce multiple fuels and products from petroleum.
An invited paper submitted to a Instead of refining petroleum to make hydrocarbon derivatives, the technol-
special issue on Green Spectroscopy
and Analytical Techniques, organized ogy refines biomass mainly into sugars, fibers, fuel, and energy. The sugars
by Professor Miguel de la Guardia, of are raw materials that can be used for making many further products.
the Department of Chemistry,
University of Valencia, Spain, and
The sugar industry the world over is presently faced with major challenges on
Professor Arabinda Kumar Das, of the account of the liberalization of the world market and abolition of established
Department of Chemistry, University sugar protocols, resulting in a 36% cut in the price of sugar. Given this perspec-
of Burdwan, West Bengal, India.
tive, sugar may no longer be the end product but rather be considered as a raw
Received 17 June 2008;
accepted 1 October 2008. material for the local manufacture of value-added chemicals and products.
Address correspondence to One such niche product is lactic acid, which can be commercialized as
Dhanjay Jhurry, Faculty of Science, such or after conversion into other products. Present global demand of lactic
Department of Chemistry, University
of Mauritius, Réduit, Mauritius. acid as a raw material for the chemical industry is estimated at 90,000 MT
E-mail: [email protected] p.a. Various studies point to a major boost in demand of lactic acid and
296
related products such as polylactic acid (bioplastics) lactic acid production. In general, the desirable
and lactate esters. characteristics for industrial LAB are their abilities
to rapidly and completely convert cheap raw materi-
als into lactic acid with minimal nutritional require-
Biofermentation Process ments and to provide high yields of preferred
Lactic acid is mainly produced via industrial fer- stereoisomer without by-product formation. For
mentation of sugars from sugar cane and molasses, instance, molasses is a waste product from the sugar
corn, and so on. Microorganisms that can produce manufacturing process and usually contains a large
lactic acid can be divided into two groups: bacteria amount of sucrose. Shukla et al.[6] previously engi-
and fungi. However, most investigations have used neered Escherichia coli W3110 derivatives, strains
lactic acid bacteria (LAB) for fermentation. In indus- SZ63 and SZ85, to produce optically pure D(
)
try, the most commonly used producer strains come and L(þ)-lactate from hexose and pentose sugars.
from the genus Lactobacillus, of which the most Timbuntam et al.[7] used a newly isolated Lactoba-
widely used strain for producing lactic acid is Lacto- cillus sp. strain FCP2 for the production of lactic acid
bacillus delbrueckii NCIB 8130. Alternative produ- from sugar-cane juice with good yield (96%) and
cers of lactic acid include strains of the Bacillus productivity (2.8 gl
1h
1). The Lactobacillus strain
family.[1–3] Only a few lactic acid bacteria, such as FCP2 can use both disaccharides and monosacchar-
Lactobacillus brevis, helveticus, and delbrueckii can ides in the sugar-cane juice as carbon sources for lac-
produce optically pure lactic acid.[4] tic acid fermentation. Kadam et al.[8] have used a
Homo-fermentative strains generally lead to mutant strain of Lactobacillus delbrueckii NCIM
higher yields of lactic acid as compared to the 2365 for the fermentation of hydrolyzed cane sugar
hetero-fermentative ones.[5] Indeed, with the latter and have obtained 90% lactic acid yield with
strains considerable amounts of by-products such 150 g=l of cane sugar in batch fermentation and a
as ethanol, acetic acid, and carbon dioxide are lactic acid concentration of 135 g=l.
also formed. Temperature and pH are reported to Table 1 summarizes the experimental conditions
be important factors influencing LAB growth and used and the productivity of lactic acid obtained

TABLE 1 Lactic Acid Production by Fermentation of Glucose and Molasses

Productivity
Glucose of lactic acid
LAB Temp ( C) pH (gl
1) (gl
1h
1)
Glucose, Batch fermentation
Lactococcus lactis subsp lactis[9] 32 6.0 20 1.00
60 0.67
No control 20 0.29
60 0.21
Sporolactobacillus cellulosolvens[10] 40 6.5 5% (w=v) 0.34
Lactobacillus Mon4þ[10] 37 6.5 4% gl
1 30 gl
1
Lactobacillus amylophilus[11] 30 6.0 20 1.56
Lactobacillus casei LA-04-1[12] 42 6.25 140 1.34
Lactobacillus delbrueckii NRRL B445[6] 49 5.8–6.0 150 0.94–1.25
Enterococcus Faecalis[13] 38 7.0 30 5–6
Lactobacillus[14] — 6.5 — 3.00

Glucose, Continuous fermentation

Lactobaccilus casei–rhamnosus 1828[15] 39 0.07 (Residual 38.2
sugar)
Lactobacillus delbrueckii NRRL B445[6] 42 6.0 50 8.93

Molasses, Batch fermentation

Sporolactobacillus cellulosolvens (NCIMB 12173)[10] 40 6.5 5 24.2

297 Lactic Acid Produced by Biofermentation of Cane Sugar Juice

from fermentation of glucose and molasses using D-Fructose was obtained from Himedia Laboratories
various bacteria. Batch, fed-batch, repeated batch, Pvt. Ltd.
and continuous fermentations are the most Sugar-cane mixed-juice and syrup were obtained
frequently used methods for lactic acid production. from Beau Champ Sugar Estate (Mauritius).
Lactobacillus delbrueckii subsp delbrueckii (NCIM
Analytical Determination of Lactic 2365) was purchased from National Chemical
Laboratories (Pune, India). Yeast extract was from
Acid Concentration
Sigma. MRS Broth Agar (CM0361) and MRS Broth
The classical analysis of lactic acid is a combined (CM0359) were obtained from Oxoid.
enzymatic-colorimetric method which makes use of
an enzymatic test kit.[16,17] The enzyme L(þ) - or Microorganisms and Culture
D(
)-lactate dehydrogenase catalyze the oxidation
Conditions
of L(þ)- or D(
)-lactate in the presence of nicotina-
mide adenine dinucleotide (NADþ) followed by col- Microorganisms were maintained in liquid MRS
orimetric analysis of the lactic acid formed. L-lactic medium in screwed cap test tubes at 15 C in a refrig-
acid has also been analyzed using an amperometric erator and every three weeks the cultures were trans-
graphite-Teflon biosensor.[18] The correlation ferred to fresh liquid MRS medium to maintain live
between the bienzyme sensor method and classical bacteria. These bacteria were used as stock cultures
colorimetric enzymatic method was quite good, of for preparation of inoculum for fermentation.
the order of 0.95.
The quantitative analysis and separation of LA Sub-Culture Preparation
optical isomers by liquid chromatography has also MRS Broth (5.22 g) was placed in distilled water
been reported.[19] (100 ml). The mixture was stirred to give a homoge-
In this article, we demonstrate the feasibility of the neous solution. Aliquots of solution (30 ml) were
fermentation process of sugar-cane juice from transferred to multiple test tubes, which were steri-
Mauritian sugar factories into L-lactic acid using one lized at 121 C for 15 min. After sterilization, the solu-
specific Lactobacillus strain, namely Lactobacillus tions were cooled to room temperature and a loopful
delbrueckii subsp delbrueckii NCIM 2365. To our of the culture medium was transferred to the MRS
knowledge, we report here for the first time the media near a flame to avoid contamination by
on-line quantitative determination of the amount of atmospheric bacteria.
lactic acid produced during biofermentation using
1
H NMR spectroscopy as a green analytical chemistry Inoculum Preparation
alternative to existing methods. Cells from stock cultures (1 ml) were transferred to
100 ml sterile growth medium in 250 ml screw cap
MATERIALS AND METHODS conical flask containing carbohydrates (10 g), cal-
cium carbonate (5 g), and yeast extract (1 g). Flasks
Apparatus were incubated at 41 C for 24 hr under stationary
A Berthold Hermle AG (Germany), type ‘‘Z420’’ conditions. After 24 hr, these cultures (2 ml) were
centrifuge with a maximum speed of 10,000 rpm transferred to another sterile growth medium of the
þ=
5% was used. same composition. The flask was incubated at 41 C
A Portaclave size 4 model AAJ 040=6 (ASTELL for another 24 hr, under stationary conditions. This
SCIENTIFIC) with digital temperature control was used. culture was used as the inoculum to be transferred
1
H-NMR spectra were recorded in D2O at room tem- to the fermentation medium.
perature using a Bruker FT Spectrometer at 250 MHz.
Fermentation of Glucose, Fructose,
Materials and Reagents and Sucrose
Sucrose was purchased from Saarchem. Anhy- Glucose or fructose or sucrose (1 g or 10 g) was
drous D-(þ) Glucose was purchased from Labosi. measured and transferred to separate conical flasks

A. Ramanjooloo et al. 298

to which yeast extract (1 g), calcium carbonate (5 g), for 2 days to allow bacterial growth. After 2 days,
and distilled water (100 ml) were added. The the number of colonies formed was counted. Plates
solutions were sterilized before the addition of having 30–300 colonies were chosen, as this range
bacteria from the inoculum medium (5 ml). The is considered statistically significant.
flasks were screwed and fermentation was carried
out at 41 C in a shaking orbital with shaking speed 1
H NMR Determination of the
of 150 rpm over a period of 5 days.
Amount of Lactic Acid in the
The fermentation of sucrose was also studied after
hydrolysis. Sucrose (1 g or 10 g) was measured and Fermented Solutions Using Mandelic
transferred to a conical flask and distilled water Acid as Internal Standard
(100 ml) added. Sulfuric acid (20%, 1 ml) was then
Model Studies
added. The acidified sugar solution was heated in
boiling water bath for 20 min before the addition of NMR spectra were recorded for varying concentra-
yeast extract (1 g) and calcium carbonate (5 g). The tions of commercial L-lactic acid solutions in the
medium was sterilized and bacteria added. Fermen- presence of a fixed amount of mandelic acid.
tation was performed under the same conditions as Calibration curve of experimental ratios of lactic acid
above. to mandelic acid as determined by 1H NMR were
In the case of sugar-cane juice and syrup, 10% plotted against the theoretical ratios in the range
(w=v) sucrose solutions were first prepared. Calcium 0.5 to 2.5. NMR spectra of solutions containing mix-
carbonate (5%) and yeast extract (5%) were added tures of lactic acid, mandelic acid, and a fixed con-
and fermentation allowed to proceed under similar centration of sucrose were analyzed under
conditions. conditions preliminarily established and the corre-
sponding calibration curve plotted.
Treatment of Fermented Solutions NMR analysis was run in the presence of
DL-Mandelic acid as an internal standard. A known
After fermentation the solutions were centrifuged amount of the latter was added to the solutions after
or filtered to separate excess calcium carbonate and fermentation had been stopped.
were boiled to stop fermentation. Then the solutions
were acidified with sulfuric acid (1 M) to pH 1.6. Determination of Lactic Acid
The precipitate from the solutions was filtered and
A known mass of DL-mandelic acid and a known
concentrated before further analysis.
volume of fermented solution were dissolved in D2O
and mixed in a 5 mm NMR tube. The 1H-NMR spectra
Purification of Fermented Solution
of the fermented solutions were used to calculate the
Purification of the fermented solution was per- amount of lactic acid present in the concentrated
formed by extraction with diisopropyl ether (n vols) solutions.
and re-extraction of the solution with an equal
volume of distilled water.
RESULTS AND DISCUSSION
Enumeration of Microorganisms Preliminary Fermentation Studies on
after Fermentation D(þ)-glucose, D-fructose, Sucrose,
After fermentation of sugar-cane juice and syrup, a
and Hydrolyzed Sucrose
sample of the fermented solution (1.0 ml) was trans- Initial fermentation studies were conducted on
ferred to a clean test tube. Distilled water (9.0 ml) model compounds D(þ)-glucose, D-fructose,
was added and the solution homogenized. This sucrose, and hydrolyzed sucrose using Lactobacillus
ten-fold dilution was repeated a further six times in delbrueckii subsp delbruekii at two different concen-
separate test tubes. Then, an aliquot from the seven trations, namely 1% and 10% (w=v). Fermentation
different test tubes (1 ml) was transferred to agar was carried out in the presence of calcium carbonate
plates. The plates were placed in an oven (41 C) to avoid significant drop in pH, which was

299 Lactic Acid Produced by Biofermentation of Cane Sugar Juice

TABLE 2 pH Before and After Fermentation m ¼ brix of solution obtained directly from a
Concentration=% pH before pH after refractometer;
(w=v) fermentation fermentation t ¼ 22 C (temperature of the invert solution);
C is obtained using the brix value, C ¼ 142.80.
Glucose 1 6.65 5.30
10 6.80 5.80
Fructose 1 6.94 5.91 1
10 6.60 5.20
H NMR Analysis of Fermentation
Sucrose 1 6.74 5.40 Medium
10 6.77 5.20
Hydrolysed 1 6.86 6.50 The fermented products from glucose, fructose,
sucrose 10 6.74 5.30 sucrose, and hydrolyzed sucrose obtained at 1%
and 10% (w=v) were characterized by 1H-NMR.
The spectrum recorded for a 1% hydrolyzed sucrose
monitored before and after fermentation (Table 2).
solution after fermentation is shown in Fig. 1a. As
The presence of yeast extract is important to provide
can be seen, the solutions contain only L-lactic acid,
vitamins and trace elements essential for lactic acid
characterized by a doublet centered at 1.3 ppm and a
biosynthesis. After the required fermentation period,
quartet at 4.2 ppm corresponding to the methyl
the solutions were acidified with sulfuric acid,
(CH3a) and methine (CHb) protons of lactic acid,
filtered, and concentrated before being subjected to
1 respectively. It is to be noted that commercial L-lactic
H NMR analysis.
acid (Fig. 1b) also contains oligomers as depicted by
0
a quartet at 5.1 ppm corresponding to -CHb of the
Fermentation of Sugar-Cane Mixed main chain and quartet at 4.3 ppm representing –
Juice and Syrup CHb’’ of the ultimate unit of the oligomers. Moreover,
The sucrose content and brix of juice and syrup in in the spectra of 10% solutions, signals correspond-
the freshly collected sugar-cane juice and syrup were ing to unfermented saccharides are also observed
characterized prior to fermentation (Table 3). The in the range 2.9–3.7 ppm. The percentage ratio of
solutions were diluted with distilled water to provide L-lactic acid to unfermented saccharides was deter-
a 10% (w=v) sucrose solution and fermentation mined by comparing proton intensities in the region
allowed to proceed during a period of 5 days using 2.9–3.7 ppm to those at 1.3 ppm. The results are
similar conditions as for the model studies. shown in Table 4.
The sucrose content[20] in sugar-cane juice and The fermentation of hydrolyzed sucrose was
syrup was calculated according to Eq. (1). found to be more efficient than that of unhydrolyzed
sucrose (Table 4). Indeed, after hydrolysis a 1:1 mix-
½2ðp
p0 Þ100pol factor ture of glucose and fructose is obtained, which lends
ð1Þ itself more readily to fermentation. The percentage
C þ 0:0794ðm
13Þ
0:53t
composition of lactic acid is also shown to increase
p ¼ direct pol; p0 ¼ invert pol; as a function of time (Fig. 2).
p and p0 of the solution were obtained from a
saccharimeter; Quantitative Assessment of L-Lactic
TABLE 3 Pol, Brix, and Sucrose Content of Sugar-Cane Juice Acid Produced from Sugar Syrups
and Syrup
by 1H NMR
Juice Syrup
Model Studies
Brix ( ) 16.0 70.0 (undiluted)
14.0 (diluted)
As described in the previous section, the ratio of
Sucrose (%) 14.6 60.8 (undiluted) L-lactic acid to unfermented sugars can be estimated
12.15 (diluted) by 1H NMR. However, the simple comparison of pro-
Pol factor 0.24473 0.24671 ton intensities does not enable a quantitative deter-
P 29.4 24.8 mination of the amount of L-lactic acid formed
P’
9.7
7.5
since the signals due to the unfermented sugars

A. Ramanjooloo et al. 300

FIGURE 1 1
H NMR (D2O) spectra of fermented (a) 1% hydrolyzed sucrose; (b) commercial L-lactic acid.

probably correspond to a mixture of sucrose, in water; and (3) the aromatic protons of the acid are
glucose, and fructose. To overcome this difficulty, centered at 7.45 ppm and the methine proton reso-
the NMR analysis was run in the presence of an inter- nate at 5.29 ppm and therefore do not overlap with
nal standard, namely DL-mandelic acid. The latter the protons of either lactic acid or unfermented
was added to the solutions after fermentation had sugars.
been stopped. This acid was chosen for several In the first instance, NMR spectra were recorded
reasons: (1) it is a solid available at low cost and for varying concentrations of commercial L-lactic
can be handled with accuracy; (2) it is readily soluble acid solutions in the presence of a fixed amount of

301 Lactic Acid Produced by Biofermentation of Cane Sugar Juice

TABLE 4 Molar Percentage Compositions of Lactic Acid
and Unfermented Saccharides as Determined by 1H NMR in 10%
Fermented Solutions

Unfermented
Saccharides Lactic acid (%) saccharides (%)
Glucose 77.8 22.2
Fructose 77.3 22.7
Sucrose 58.9 41.1
Hydrolyzed sucrose 78.4 21.6

mandelic acid. By comparing the intensity of

aromatic protons of mandelic acid to that of aliphatic
protons (CH3) of lactic acid, the ratio of lactic acid to
FIGURE 3 Graph of experimental ratio against theoretical ratio
mandelic acid was determined. The relaxation delay of L-lactic acid to DL-mandelic acid.
(RD) used for each NMR pulse was found to be
determinant in the correlation between experimental
and theoretical ratios. Indeed, at low RD values (2 s), accuracy of the determination of lactic acid. NMR
there result significant errors (average 60%) between spectra of solutions containing mixtures of lactic
experimental and theoretical ratios. The best results acid, mandelic acid, and a fixed concentration of
were obtained at a relaxation delay of 10 s and this sucrose were analyzed under conditions prelimina-
value has been used throughout the study. Calibra- rily established and the corresponding calibration
tion curves of experimental ratios of lactic acid to curve plotted (Fig. 4). Again the accuracy of the
mandelic acid as determined by 1H NMR were determination was found to be excellent for ratios
plotted against the theoretical ratios in the range of lactic acid to mandelic acid between 0 and 2 as
0.5 to 2.5 (Fig. 3). As can be seen, a very good corre- noted previously but deviations occur as the concen-
lation is obtained under our conditions presenting a tration of lactic acid compared to mandelic acid
typical equation y ¼ 0.9978x (R2 ¼ 0.9901) where y increases. To conclude, the presence of sugar
corresponds to the experimental and x to the theore- does not perturb the accuracy of the determination
tical molar ratio of lactic acid to mandelic acid. and it is important to ensure that the amount of
Next, it was important to verify whether the mandelic acid used as internal standard is within
presence of sucrose would or would not affect the acceptable ratios.

FIGURE 2 Molar percentage composition of lactic acid and

unfermented saccharides after 24 hr, 48 hr, 72 hr, 96 hr, and FIGURE 4 Calibration plot for mixture of lactic acid, mandelic
144 hr as determined by 1H NMR. acid, and sucrose.

A. Ramanjooloo et al. 302

FIGURE 5 1
H-NMR (D2O) spectrum of fermented juice with D,L-mandelic acid as internal standard.

Application to Fermented Solutions fermentation medium and this analysis can be

extended to on-line monitoring.
of Sugar-Cane Mixed Juice and Syrup
Pure lactic acid was obtained upon further extrac-
A typical 1H NMR spectrum of a fermented tion with diisopropyl ether and purification with
sugar-cane juice solution to which mandelic acid water and verified by 1H NMR. Negative optical
has been added is depicted in Fig. 5. As can be seen, rotatory values confirmed also that the pure
signals characteristic of L-lactic acid and residual L-enantiomer was obtained.
sugars (3.1–4.1 ppm) are present. The amount of lac-
tic acid formed is calculated according to Eq. (2) and
corresponds to a value of 25 g=l (productivity ¼ 0.21
CONCLUSIONS
gl
1h
1). We have shown that the biofermentation of
 a     sugar-cane mixed juice and syrup proceeds with
ICH3 mMA 1000 reasonable yields. Direct analysis of fermentation
mLA ¼ dI
 1:67 90 ð2Þ
C6 H5 152 Vsoln solutions by 1H NMR enables the ratio of L-lactic acid
to unfermented sugar to be determined. To assess
a
ICH3 ¼ intensity of methyl protons of lactic acid the amount of L-lactic acid formed quantitatively,
d
IC6 H5 ¼ intensity of phenyl protons of mandelic acid NMR was run in the presence of DL-mandelic acid
mMA ¼ mass of mandelic acid as an internal standard. NMR parameters such as
Vsoln ¼ volume of solution analysed the relaxation delay as well as the ratio of lactic acid
to mandelic acid were found to be key factors for
NMR indeed proves to be quite an interesting techni- accurate determination of lactic acid content. We
que because it gives quick results with a high level of thus demonstrated that 1H NMR spectroscopy can
accuracy under predetermined conditions. In addi- be used as a fast, easy, reliable, and valuable techni-
tion, the small volumes of solution used for analysis que to monitor the on-line fermentation of sugar
does not perturb the overall concentration of the to lactic acid as compared to existing methods.

303 Lactic Acid Produced by Biofermentation of Cane Sugar Juice

The NMR technique does not require sample 4. Benthin, S.; Villadsen, J. Production of optically pure D-lactate by
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