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US 20100062503Al

(19) United States

(12) Patent Application Publication (10) Pub. No.: US 2010/0062503 A1
Visser et al. (43) Pub. Date: Mar. 11, 2010

(54) LACTIC ACID PRODUCTION FROM Publication Classi?cation

(51) Int. Cl.
C12P 7/62 (2006.01)
(75) Inventors: Diana Visser, Gorinchem (NL);
C12P 7/56 (2006.01)
Jan Van Breugel, Woudrichem
(NL); Johannes Martinus De (52) US. Cl. ....................................... .. 435/135; 435/139
Bruijn, Eys (NL); Paul A’Campo,
Eemnes (NL)
Correspondence Address:
The present invention is in the ?eld of the preparation of lactic
PO. BOX 320850 acid by means of fermentation on industrial scale Wherein a
concentrated raW beet juice having a Brix of at least 60 is used
ALEXANDRIA, VA 22320-4850 (US)
as fermentation substrate. After dilution to the desired initial
(73) Assignee: PURAC BIOCHEM BV, sugar concentration and addition of nutrients, the juice is
GORINCHEM (NL) fermented to lactic acid and/or lactate by means of a lactic
acid-producing microorganism. Said concentrated raW beet
(21) Appl. No.: 12/308,464 juice is prepared by: Washing and cutting sugar beet and
extracting the cossettes in Water, removing the beet pulp from
(22) PCT Filed: Jun. 22, 2007 the resulting raW beet juice, and heat treating the raW beet
juice at a temperature between 50 and 90 degrees Celsius, and
(86) PCT No.: PCT/EP2007/056256 concentrating the raW beet juice to at least 60 Brix.

§ 371 (0X1)’ It Was found that concentrated beet juice having a Brix of at
(2), (4) Date: Jan. 29, 2009 least 60 is storage-stable, is not very sensitive to infections,
and can be used as fermentation substrate for lactic acid
(30) Foreign Application Priority Data production on industrial scale With the same yield, chemical
purity, optical purity, clarity and taste as lactic acid obtained
Jun. 22, 2006 (EP) ................................ .. 061158853 from fermenting White sugar.
Patent Application Publication Mar. 11, 2010 US 2010/0062503 A1

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US 2010/0062503 A1 Mar. 11, 2010

LACTIC ACID PRODUCTION FROM 1), 57-66, also the fermentation of raW sugar beet juice is
CONCENTRATED RAW SUGAR BEET JUICE described. Zakharov reports that the heating or sterilization of
the sugar beet juice has a detrimental effect on the fermenta
tion. The beet syrup, prepared by evaporation of beet juice to
[0001] The present invention is in the ?eld of the prepara a sugar content of 51% Was diluted and fermentation Was
tion of lactic acid by means of fermentation. tried: virtually no fermentation took place both With and
[0002] Lactic acid, its salts and esters have long been used Without chalk (probably calcium hydroxide is meant here)
as food additive and in various chemical and pharmaceutical
addition. Zakharov concluded that a medium of such syrup
applications. More recently, lactic acid has been used in the
making of biodegradable polymers both as a replacement for proved unsuitable for the fermentation to lactic acid.
present plastic materials as Well as various neW uses Where [0007] All in all, it must be concluded that the fermentation
biodegradability is needed or desired such as for medical of these crude substrates such as raW sugar beet juice to lactic
implants, solvable sutures and controlled release drugs. The acid on industrial scale proves to be much more complicated
production of lactic acid is commonly carried out by fermen than a comparable fermentation to ethanol. The same can be
tation by means of a micro-organism such as bacteria, yeasts said of the puri?cation (i.e. the doWnstream processing) of
and fungi. The fermentation medium consists of a carbohy lactic acid prepared by fermentation compared to the puri?
drate substrate together With suitable mineral and proteina cation of fermentatively prepared ethanol.
ceous nutrients. A commonly used fermentation substrate is
White sugar. Sugar is the most important contributor to the [0008] We have found that concentrated raW beet juice
manufacturing cost price of lactic acid. Major reductions in having a Brix of at least 60 (i.e. amount of sugar in Weight per
the manufacturing cost price of lactic acid can therefore be 100 grams of liquid) is a suitable substrate for the fermenta
accomplished if a less expensive carbohydrate source can be tion on industrial scale to lactic acid and or lactate. Further, it
used than White sugar. To this end several research groups appears to be storage-stable and is not very sensitive to infec
tried to ferment cheaper byproducts and intermediates of a tions. Furthermore, fermentation to lactic acid is achievable
sugar production plant to lactic acid. HoWever, While the With the same yield, chemical purity, optical purity, clarity
fermentation of these crude sugar sources to ethanol can be and taste as lactic acid obtained from fermenting sucrose, i.e.
done readily, problems are encountered When trying to use White sugar.
these substrates on industrial scale for the fermentation to [0009] When processing sugar beet, the beet is usually
lactic acid. These problems lie in the ?eld of fermentability, Washed With Water, cut and the resulting cossettes are
storageistability, sensitivity to infections, the puri?cation of extracted With Water, from Which beet pulp is removed and the
the product of fermentation (i.e. the doWnstream processing) resulting raW juice is further processed to sugar by subsequent
juice puri?cation [i.e. traditionally by lime addition to a pH of
[0003] Examples of prior art Wherein the fermentation of approximately 11.2 in the pre-liming, folloWed by surplus
sugar beet juice to ethanol is described are Raw thick juice:
lime addition in the main liming (the alkaliZation), addition of
manufacture, storage and utilisation as feedstock in biotech
nological industry, G. Marke, P. V. Schmidt, R. Rieck, B. carbon dioxide in tWo subsequent stages (the carbonation),
Wherein after the ?rst carbonation the carbonation sludge/
Senge, B. Steiner, Zuckerindustrie (1992), 117 (12), 984-90,
The manufacture of alcohol from sugar beets, K. Antal, lime is removed] resulting in thin juice, said thin juice is
Zeitschrift fuer Spiritusindustrie (1911) 34, 239-40, 252-3 concentrated resulting in thick juice, said thick juice is sub
and Combined production of ethanol and white sugar, K. jected to one or more crystallization steps to form various
Austmeyer, H Roever, H. Zuckerindustrie (1988) 113 (9), sugar grades, and molasses as by-product.
765-72. [0010] The concentrated raW beet juice used in the process
[0004] The literature on fermentation of sugar beet juice to according to the invention is prepared by subjecting the raW
lactic acid on industrial scale is not so abundant. juice (approximately 16% sugar) to a heating step at a tem
[0005] For instance, in The industrial preparation oflactic perature betWeen 50 and 900 C. and concentrating the raW
acid from sugar beets, A. Bonelli, G. Gulinelli, lnd. Chim. beet juice to a Brix of at least 60 (that is, Without lime addition
Met. (1918), 5 121 -4, the fermentation of raW sugar beet juice to a pH of approximately 11.2 and surplus lime addition
to lactic acid is described. This raW sugar beet juice only has folloWed by carbon dioxide addition in tWo stages With
a concentration of about 16 Wt % sugar. First of all, as indi removal of carbonation sludge/lime). Optionally a liquid
cated in the publication this carbohydrate source is very sen solid separation step is performed betWeen the heating step
sitive to infections and often already infected to start With. and the concentration step and/or after the concentration step
This is con?rmed in for instance, F. Hollaus et Al: “Experi to remove residual soil, small beet particles and protein. The
mental studies on bacterial degradation in sugar ofsugar in concentration step is usually performed by evaporation at a
rawjuice andprelimingjuice.” Sucrerie beige, vol 99, No 5, temperature betWeen 50 and 1200 C. It is also possible to
(1980), p. 183. With sugar beet factories running in cam
combine the heating and concentration step. If the heating and
paigns and only active 3-4 months a year, it is clear that this
provides storage problems. Secondly, With a concentration of concentration steps are combined, the optional solid/liquid
only 16% the transport and storage costs Will render this separation is conducted either prior to the heating/concentra
process relatively expensive. These factors make raW sugar tion or after. The concentrated raW beet juice can be added in
beet juice un?t as a substrate for fermentation to lactic acid on diluted form to the fermentation. Usually the concentrated
an industrial scale. raW beet juice is diluted to a concentration of about 16-30
[0006] In Production of lactic acidfrom sugar beet and Brix, more preferably 20-30 Brix.
cases of inactivation of lactic acid fermentation, l. P. [0011] The processing of sugar beet to White sugar and the
Zakharov, M. F. Federova, Mikrobiologiya (1946), 15 (No. preparation of concentrated raW beet juice from the raW beet
US 2010/0062503 A1 Mar. 11, 2010

juice Which is an intermediate of the beet processing process [0017] After fermentation, the lactate and lactic acid-con
is schematically illustrated by FIG. 1. taining fermentation product must be separated from the bio
[0012] It Was found that molasses and thick juice prepared mass. Said lactic acid-containing fermentation product is in
during sugar beet processing is not very suitable for the fer the liquid form (i.e. liquid or in solution). Usually this biom
mentation to lactic acid on industrial scale. Without Wishing ass is separated by means of ?ltration, centrifuging, ?occu
to be bound to a theory We think that during the processing of lation, coagulation, ?otation or combinations thereof. After
the raW sugar beet juice to thick juice and further to molasses, biomass separation, the pH of the fermentation product is
several impurities are either concentrated in the molasses decreased by means of acid addition such as sulphuric acid so
and/or thick juice or are introduced by reactions occurring that lactic acid and a salt of the neutralizing base and the
during the alkalization, heat and carbon dioxide treatments added acid is formed. For example, if calcium hydroxide is
(e. g. the results of Maillard reactions). Said impurities inter used as neutralizing agent, the fermentation product Will
fere With fermentation reactions to lactic acid on industrial comprise both lactic acid and calcium lactate. Upon addition
scale. This is also shoWn in our experimental data. Sometimes of sulphuric acid, lactic acid and calcium sulphate (gypsum)
the impurities can be removed by various pre-treatments, but
Will be formed. The gypsum, or any other salt, is removed and
this requires additional, laborious and expensive puri?cation
steps. the lactic acid is isolated. The resulting lactic acid can be
[0013] Goeksungury et all: “Batch and continuous produc
subjected to further puri?cation steps.
tion of lactic acid from beet molasses lactobacillus del [0018] Conventional subsequent puri?cation steps are dis
brueckii [F0 3202’’ Journ. Chem. Techn. and Biotechn., vol tillation including short path distillation and vacuum distilla
69, No. 4, (1997) pp. 399-404, and Goeksungur y et all: tion, crystallization, salt SWAP, electrodialysis, extraction
“Production of lactic acidfrom beet molasses by calcium (both forWard and back-extraction), carbon treatment, ion
alginate immobilized lactobacillus delbrueckii [F0 3202” exchange and combinations thereof. These puri?cation steps
Journ. Chem. Techn. and Biotechn., vol 74, No. 2, (1999) pp. can be combined With intermediate concentration steps.
131-136, describe the production of lactic acid from pre
treated beet molasses With speci?c bacteria. The pretreatment [0019] The invention is further illustrated by means of
comprises acidi?cation With sulphuric acid, boiling, centri examples Which are not to be interpreted as limitative.
fusion, ?ltration and clari?cation, including pasteurization.
The experiments Were conducted in 250 cm3 and 500 cm3 EXAMPLE 1
?asks and it Was indicated that even With a loW initial sugar
concentration (28.2 g/l) the sugar Was not completely uti Fermentation to Lactic Acid Using Various Sub
lized. The authors attribute that to the complex nature of strates
molasses (i.e. impurities).
[0014] Monteagudo J. M. et Al: “Kinetics of lactic acid [0020] The fermentability of molasses, thick juice and con
fermentation by lactobacillus delbruekii grown on beet centrated raW juice substrates originating from a sugar factory
molasses Joum. Chem. Techn. And Biotechn., vol 68, No 3,
(5 samples With Brix values varying from 59.6 to 73.2, con
(1997), pp 271-276 describes the experiments of lactic acid
fermentation on beet molasses on lab scale (5 liter ?asks). centrated at temperatures of 55, 60, 75, and 850 C.), Was tested
Here also the sugar Was not completely utilised. in separate fermentations. For all substrates, the folloWing
procedure Was applied: the substrate Was diluted With Water to
[0015] El Sherbiny et al: “Utilisation ofbeet molasses in the
production of lactic acid ”, Egyptian Joum. Of Food SC. vol a sucrose concentration of approximately 300 g/l. To this
14, No 1 (1986), pp 91-100, also shoWs that in the fermenta solution, 6.0 g/l nutrients Were added and the pH Was adjusted
tion not all sugar is utilized and that several other organic to 6.4 using lime. Subsequently, the medium Was heated to
acids are formed besides lactic acid. 540 C. and the fermentation Was started With 10% (v/v) of a
[0016] The industrial fermentation to lactic acid requires sucrose-groWn inoculum of a lactic acid-producing microor
strict control of temperature and pH. Because of the forma ganism. During the fermentation diluted lime Was added to
tion of lactic acid the pH drops during fermentation. A drop in keep pH at 6.4 and the temperature Was controlled at 540 C.
pH beloW a critical value, depending on the microorganism [0021] A fermentation in Which White sugar (sucrose) Was
used in the process, could damage the microorganism’s meta
used as substrate, and otherWise applying the same condi
bolic process and bring the fermentation process to a stop.
Therefore, it is common practice to add a neutralizing agent, tions, Was carried out for comparison.
i.e. a base such as Ca(OH)2, Mg(OH)2, NaOH, KOH or [0022] Table 1 summarizes the results of these fermenta
ammonia to the fermentation reaction and thus produce a tions. Fermentations based on molasses hardly shoWed any
lactate salt such as calcium lactate, sodium lactate etcetera. activity; only a small fraction of the sugar Was consumed after
Normally both lactic acid and lactate salt are present in the 45 hrs and the productivity collapsed to virtually zero. The
fermentation product, depending on the pH of the fermenta fermentability of thick juice Was more active, but still only a
tion product. The fermentation can be done With conventional part of the sugar Was consumed. Concentrated raW juice could
lactic acid-producing microorganisms such as bacteria yeasts
be fermented completely. Compared to the reference sucrose
and fungi, such as lactobacilli, moderately thermophilic
bacilli, Rhizopus and Aspergillus. Preferred are the moder fermentation, the lactic acid yield and byproduct pro?le (or
ately thermophilic bacilli such as Bacillus coagulans, Bacil ganic acids) is similar; the chiral purity is loWer but still
lus thermoamylovorans, Geobacillus stearothermophylus acceptable. The residual sugar content is higher, because
and Bacillus smithii, because these types of micro-organisms concentrated raW juice contains some sugars that are not/
can ferment at relatively high temperature. sloWly fermented.
US 2010/0062503 A1 Mar. 11, 2010


Summary fermentation results for sucrose (reference), molasses, thick juice

and concentrated raw juice.

Composition ?nal broth

Lactate Residual sugars Polysaccharides

(% w/w of max. Chiral purity (% w/w of sugar (% w/w of lactate Total organic
Substrate obtainable) lactate (% S) input) formed) acids* (% w/w)

Sucrose*** 95% 99.7% 1% nd

Molasses nd nd 100% nd
Thickjuice nd nd >50% n.d.
Concentrated 95% 99.2% 3.5% <0.2%
raw juice**

*Sum of succinic, pyruvic, formic, 2-hydroxybutyric and acetic acid

**Average of 11 fermentations
***Average of 13 fermentations
n.d. = not determined

EXAMPLE 2 [0028] Both fermentations started with 275 g/l sucrose (and
a total amount of approximately 3 g/l glucose and fructose).
Concentrated Raw Sugar Beet Juice in Fermentation
on Industrial Scale
Biomass Removal
[0029] Flocculation, to remove bacterial cells, metals, pro
[0023] Two semi-industrial fermentations (scale 5000 teins etc., was performed with sugar free ?nal fermentation
liters) on concentrated raw beet juice were perfonned. Of the broth by alkaliZing the broth at a temperature of 75° C. with
?nal fermentation broth of these two fermentations biomass lime and adding ?occulant.
was removed and the lactate-containing product was acidu [0030] The clear top layer was siphoned off as soon as the
lated. settling of the biomass was complete. The clear alkaline top
layer was subsequently transferred to a second 5 m3 reactor
A. Materials and Methods for acidulation. The biomass containing bottom layer was
[0024] Sugar beets were cut, washed and extracted in water
at 70° C. The pulp was eliminated and the raw beet juice was [0031] The clear alkaline top layer was continuously stirred
subjected to heat treatment at 60° C., solid/liquid separation and sulphuric acid (96%) was added slowly using a pump.
and evaporation at 65° C. to make concentrated raw beet juice This was continued until a pH of 2.2 and a conductivity of
ofa Brix between 65-70. approximately 5 mS was reached. Precipitation of calcium
was checked using an ammonium oxalate solution (35.5 g/l).
[0025] The density of the batches of concentrated raw beet The acidulated broth was removed from the reactor and iso
juice was 1.334 kg/l and 1.335 kg/l at 66 Brix, respectively. lated from the formed gypsum.
[0032] The clear crude lactic acid solution was subjected to
Fermentation conventional puri?cation steps to make ?nal S-lactic acid
[0026] Fermentation of concentrated raw beet juice was (90%).
carried out in the 5000 l fermenter using a lactic acid-produc Results
ing microorganism. The medium composition of the fermen
tation is shown in table 2. [0033] Both fermentations performed as expected. The cul
ture followed the growth phase and the stationary or produc
tion phase until sugar free. The ?rst fermentation took 24
hours. The second fermentation ?nished after 21 hours.
The 5 m3 reactor received a medium of the following composition: [0034] Final fermentation broth of the ?rst fermentation
had a substantial higher amount of polysaccharides than ?nal
Medium composition added amount fermentation broth of the second fermentation. The differ
Water 2000 l ence in the amount of total nitrogen in ?nal fermentation
Concentrated raw beetjuice (appr. 70°Bx) 1000 1 broth from these two batches, is negligible. Optical purity of
Nutrient 18.16 kg ?nal fermentation broth was 99.3% for the ?rst fermentation
Start volume 3000 l
Inoculum 500 l and 99.4% for the second fermentation.
[0035] The results of the analyses of ?nal fermentation
broth samples are listed in table 3. The results show that
[0027] The temperature during the fermentation was kept at concentrated raw sugar beet juice can be fermented to lactic
54° C. The pH was controlled by adding a slurry of lime. acid on industrial scale.
US 2010/0062503 A1 Mar. 11, 2010

3. Process according to claim 1 wherein the concentrated

TABLE 3 raw beet juice is diluted to a Brix between 16 and 30 prior to
addition to the fermentation.
Lactate, total residual sugars, polysaccharides and optical purity of ?nal 4. Process according to claim 1 wherein the microorganism
fermentation broth is a moderately thermophilic bacillus.
5. Process according to claim 1 wherein the concentrated
Lactate residual beet juice is prepared by a procedure comprising the follow
5 m3 ?nal (% w/w of sugars Polysaccharides ing steps:
fermentation max. (% w/w (% w/w of Chiral purity a. washing and cutting sugar beet and extracting the result
broth obtainable) of input) lactate) (% S-lactate) ing cossettes with water,
l“ 95% 4.2% 4.0% 99.3% b. removing the beet pulp from the resulting raw beet juice,
2'” 95% 6.5% 2.6% 99.4% and
c. heat treating the raw beet juice at a temperature between
[0036] After puri?cation, the resulting lactic acid appeared 50 and 90 degrees Celsius, and
to ful?ll the speci?cations set for commercial lactic acid for d. concentrating the raw beet juice to at least 60 Brix.
food applications with respect to color, taste and optical 6. Process according to claim 5 wherein step c and step d
purity. are combined.
7. Process according to claim 5 wherein prior to and/or
1. Process for the preparation of lactic acid and/or lactate between, and/or after step c and d a liquid/solid separation
wherein a concentrated raw beet juice, beet juice which has step is conducted.
not been submitted to lime addition to a pH of approximately 8. Process according claim 1 wherein the lactic acid and/or
11.2 and surplus lime addition followed by carbon dioxide lactate resulting from the fermentation is subjected to one or
addition in two stages with removal of carbonation sludge/ more further puri?cation steps.
lime, having a concentration of at least 60 g sucrose per 100 9. Process according to claim 8 wherein the puri?cation
gram liquid (at least a Brix of 60) is used as a substrate for the steps comprise biomass separation, extraction, salt SWAP,
fermentation to lactic acid and/or lactate by means of a lactic distillation, ion exchange, carbon treatment, extraction, and/
acid-producing micro-organism. or concentration and combinations thereof.
2. Process according to claim 1 wherein the concentrated
* * * * *
raw beet juice has a Brix of at least 70.