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Review
Abstract
Parameters affecting the fermentative lactic acid (LA) production are summarized and discussed: microorganism, carbon- and nitrogensource, fermentation mode, pH, and temperature. LA production is compared in terms of LA concentration, LA yield and LA productivity.
Also by-product formation and LA isomery are discussed. 2000 Elsevier Science Inc. All rights reserved.
Keywords: Lactic acid; Lactic acid bacteria; Lactococcus lactis; Lactobacillus; Starch hydrolysate; Lignocellulose; Whey; Nutrient; Fermentation mode;
Simultaneous saccharification and fermentation process; Cell recirculation; pH; Temperature; Productivity; Yield; By-product formation; Optical isomers;
Process optimization
1. Introduction
Lactic acid (LA) is a versatile chemical, used 1) as an
acidulant, flavor and preservative in the food, pharmaceutical, leather and textile industries, 2) for the production of
base chemicals, 3) and for polymerization to biodegradable
poly LA (PLA) [1,2]. LA exists as two optical isomers, Dand L-LA. Both isomeric forms of LA can be polymerized
and polymers with different properties can be produced
depending on the composition. Of the 80 000 tonnes of LA
produced worldwide every year about 90% are made by LA
bacterial fermentation and the rest is produced synthetically
by the hydrolysis of lactonitrile. Fermentative production
has the advantage that by choosing a strain of LA bacteria
(LAB) producing only one of the isomers, an optically pure
product can be obtained, whereas synthetic production always results in a racemic mixture of LA. It is also possible
to use renewable resources as substrates, such as starch and
cellulose in fermentative production. Renewable resources
do not give any net contribution of carbon dioxide to the
atmosphere, as do the limited oil- and fossil-fuel-based
sources. Cellulose, hemicellulose and starch are the most abundant compounds in the world, and when hydrolyzed to mainly
glucose they are fermentable by a number of microorganisms.
1
0141-0229/00/$ see front matter 2000 Elsevier Science Inc. All rights reserved.
PII: S 0 1 4 1 - 0 2 2 9 ( 0 0 ) 0 0 1 5 5 - 6
88
Nomenclature
ATP
Ent
LA
LA/tot
LAB
Lb
Lc
LDH
Leu
NADH
PDH
Ped
PFL
PLA
QV
SSF
Str
WWF
YLA/tot
adenosine triphosphate
Enterococcus
lactic acid
La per total products (g/g)
lactic acid bacteria
Lactobacillus
Lactococcus
lactate dehydrogenase
Leuconostoc
nicotinamide adenine dinucleotide, reduced
form
pyruvate dehydrogenase
Pediococcus
pyruvate formate lyase
poly-lactic acid
maximum columetric productivity (g/lh)
simultaneous saccharification and fermentation
Streptococcus
whole wheat flour
yield of LA per substrate provided (g/g)
microorganism able to metabolize many different carbohydrates [8]. LAB have been used by humans for the fermentation of food and feed products since ancient days, and
today their major applications are still in the food and feed
industry, e.g. in the production of dairy products, pickles,
meat and wine. The present technical applications of LAB
include the production of dextran from sucrose by Leu.
mesenteroides [9], the production of nisin by Lc. lactis spp.
lactis and the production of LA for different applications,
see above. It has also been suggested that LAB could be
used as oral vaccine vectors [10].
LAB ferment sugars via different pathways resulting in
homo-, hetero-, or mixed acid fermentation (Fig. 1). Homofermentation gives only LA as the end product of glucose
metabolism, and the EmbdenMeyerhofParnas pathway is
used (Fig. 1A) [11,12]. In heterofermentation equimolar
amounts of LA, carbon dioxide and ethanol or acetate are
formed from glucose via the phosphoketolase pathway (Fig.
1B) [13,14]. The ratio of ethanol and acetate formed is dependent on the redox potential of the system [15]. This pathway is
used by facultative heterofermenters, such as Lb. casei, for the
fermentation of pentoses, and for the fermentation of hexoses
and pentoses by obligate heterofermenters, organisms such as
Leu. [15]. According to Kandler, all LAB except lactobacilli of
type I (e.g. Lb. delbrueckii) are able to ferment pentoses, i.e.
they are facultative heterofermenters [15].
Mixed acids are formed by homofermenters such as
lactococci during glucose limitation [16], and during growth
on other sugars, e.g. Lc. lactis on maltose [1722], lactose
[23,24] and galactose [23,24], or at increased pH and decreased temperature [25]. Ethanol, acetate and formate are
formed in addition to LA. The homofermentative pathway
is used, but the difference is in the metabolism of pyruvate,
Table 1
Names of organisms changed in the material
Before change
After change
Reference
Lb. arabinosus
Lb. bulgaricus
Lb. casei sp. rhamnosus
Lb. cellobiosus
Lb. xylosus
Str. diacetylactis
Str. cremoris
Str. faecalis
Str. faecium
Str. lactis
Str. salivarius sp. thermophilus
Lb. delbrueckii NRRL B-445
Lb. casei sp. casei ATCC 393/DSM 20011
Lb. delbrueckii ATCC 9649
Lb. salivarius NRRL B-1950
Lb. casei DSM 20244
Lb. casei ATCC 7469/IFO 3425
Lb. casei ATCC 11443
Lb. plantarum
Lb. delbrueckii sp. bulgaricus
Lb. rhamnosus
Lb. fermentum
Lc. lactis sp. lactis
Lc. lactis sp. lactis biovar diacetylactis
Lc. lactis sp. cremoris
Ent. faecalis
Ent. faecium
Lc. lactis sp. lactis
Str. thermophilus
Lb. rhamnosus ATCC 10863
Lb. zeae ATCC 393
Lb. delbrueckii sp. delbrueckii ATCC 9649
Lb. salivarius sp. salivarius ATCC 11742
Lb. paracasei sp. paracasei DSM 20244
Lb. rhamnosus ATCC 7469
Lb. rhamnosus ATCC 11443
DSM, ATCC
166
167, 168
169
170
170
170
171
171
170
172
ATCC
168
ATCC
ATCC, NRRL
167, DSM
ATCC
ATCC
When different names of the same strain were used in different studies, they have been harmonized to one, where the ATCC No. is the highest in hierarchy,
followed by the DSM No.
89
Fig. 1. Catabolic pathways in lactic acid bacteria. Homofermentation (A), heterofermentation (B) and mixed acid fermentation (C). P phosphate, BP
bisphosphate, LDH lactate dehydrogenase, PFL pyruvate formate lyase, and PDH pyruvate dehydrogenase.
90
Table 2
Comparison of different strains for lactic acid production
Organism
Lb. casei NRRL B-441
Lb. rhamnosus ATCC 10863
Lb. delbrueckii sp. lactis ATCC 8000
Lb. delbrueckii sp. lactis DSM 20073
Lb. delbrueckii sp. delbrueckii ATCC 9649
Lb. delbrueckii mutant DP3
Lb. delbrueckii mutant DP3, 19
Lb. rhamnosus ATCC 7469
Lb. salivarius sp. salivarius ATCC 11742
Lb. zeae ATCC 393
Lb. rhamnosus DSM 20024
Lb. rhamnosus ATCC 7469
Lb. zeae ATCC 393
Lb. coryniformis sp. torquens ATCC 25600
Lb. amylovorus ATCC 33622
Lb. casei NRRL B-441
Lb. delbrueckii sp. bulgaricus ATCC 11842
Lb. plantarum ATCC 14917
Lb. delbrueckii sp. lactis 447
Lb. rhamnosus CCM 1753
Lb. delbrueckii sp. bulgaricus AU
Lb. delbrueckii sp. delbrueckii ATCC 9649
Lb. rhamnosus ATCC 7469
Lb. kefir
Lb. acidophilus R
Lb. casei SU No 22
Le. lactis WS 1042
Str. thermophilus
Lb. delbrueckii sp. bulgaricus 5085
Lc. lactis sp. lactis 2432
Lb. delbrueckii sp. bulgaricus 5085
Str. thermophilus
Lb. rhamnosus ATCC 7469
Lb. rhamnosus ATCC 10863
Lc. lactis sp. cremoris 2487
Lc. lactis sp. lactis 5085
Lb. casei SU No 22
Lc. lactis WS 1042
Str. thermophilus
Lb. delbrueckii sp. bulgaricus 5085
Lc. lactis sp. lactis 2432
Lb. acidophilus CRL 640
Lb. delbrueckii sp. bulgaricus CRL 870
Str. thermophilus CRL 807
Lb. rhamnosus ATCC 7469
Lc. lactis sp. cremoris SBT 1306
Lc. lactis sp. lactis ATCC 19435
Lc. lactis sp. lactis AS211
Lb. delbrueckii sp. delbrueckii ATCC 9649
Lb. delbrueckii sp. bulgaricus ATCC 11842
Lb. plantarum NRRL B-787
Lb. plantarum NRRL B-788
Lb. plantarum NRRL B-813
Lb. plantarum NRRL B-531
Lb. pentosus NRRL B-227
Lb. pentosus NRRL B-473
Lb. plantarum USDA 422
Lc. lactis sp. lactis NRRL B-4449
Ent faecium
Lb. plantarum
Ped. acidilacti
Substrate
glucose
glucose
glucose
glucose
glucose
glucose
glucose
glucose
glucose
glucose
glucose
glucose
glucose
glucose
hydr barley flour
hydr barley flour
sorghum
sorghum
lignocellulose hydr
lignocellulose hydr
molasses
molasses
molasses
paneer whey
paneer whey
whey
whey
whey permeate
whey permeate
whey permeate
whey permeate
whey permeate
whey permeate
whey permeate
whey permeate
whey permeate
deproteinised whey
deproteinised whey
whey permeate
whey permeate
whey permeate
skim milk
skim milk
skim milk
lactose
lactose
hydr wheat flour
hydr wheat flour
hydr wheat flour
hydr wheat flour
solid waste
solid waste
solid waste
solid waste
solid waste
solid waste
solid waste
solid waste
hydr cod corn syrup
hydr cod corn syrup
hydr cod corn syrup
Temp
C
LA
g/l
g/g
YLA/tot
41
41
45
45
45
45
45
45
45
36
36
36
36
36
37
37
42
42
37
37
47
47
47
43
43
32
32
42
42
30
42
42
37
40
30
30
32
32
42
42
30
37
37
37
37
30
30
30
37
45
32
32
32
32
32
32
32
32
37
37
37
82
68
83
82
58
77
68
28
28
21
22
24
37
39
93
120
0.91
0.76
0.83
0.82
0.48
0.64
0.57
0.93
0.92
0.71
0.74
0.80
0.98
0.98
0.52
0.67
55
37
20
26
18
9.8
8.6
16
11
18
15
8.3
7.9
15
30
30
37
37
20
15
19
16
9.0
14
12
8.5
21
80
96
95
106
18
17
19
18
18
21
18
18
6.6
11
17
13
0.85
0.74
0.45
0.58
0.40
0.20
0.17
0.32
0.22
0.50
0.41
0.21
0.18
0.35
0.71
0.71
0.88
0.88
0.39
0.30
0.47
0.38
0.20
0.38
1.5
0.76
0.77
0.82
0.11
0.42
0.46
0.43
0.43
0.51
0.43
0.43
0.16
0.45
0.70
0.51
Qv
g/(lh)
Ref
5.6
3.5
146
146
32
32
49
49
49
51
51
173
173
173
50
50
92
92
100
100
113
113
89
89
89
174
174
129
129
137
137
137
140
140
52
52
52
52
175
175
139
139
139
133
133
133
53
53
18
18
18
18
111
111
111
111
111
111
111
111
176
176
176
0.72
1.7
1.2
8.0
11
4.0
2.6
2.0
1.5
4.5
2.0
5.9
4.0
2.1
1.9
1.5
4.6
2.4
2.0
1.5
6.0
4.4
2.3
5.6
4.1
3.0
1.7
1.6
0.56
0.10
0.10
0.11
Abbreviations: LA lactic acid; YLA/tot yield of g LA per g substrate provided; Qv maximum volumetric LA productivity in g LA per l per h; Ref
reference; hydr hydrolysed.
91
Fig. 2. A process of lactic acid production from wheat flour with simultaneous (SSF) (A) and separate (B) saccharification and fermentation. Process parameters according to references [18,20,106].
92
Table 3
Influence of carbon source and substrate treatment on lactic acid production
Organism
LA
g/l
YLA/tot
g/g
Qv
g/(lh)
Ref
glucose
hydr corn starch
cassava starch
corn starch
potato starch
rice starch
wheat starch
raw corn starch
liq corn starch
liq barley starch glucoam
liq barley starch glucoam alpha
barley flour
barley flour glucoam
hydr newspaper
hydr pure cellulose
glucose
lactose
glucose
cellobiose
xylose
glucose
fructose glucose
sucrose
glucose
lactose
xylose
21
33
4.8
10
4.2
7.9
7.8
45
55
112
162
36
114
24
53
35
37
56
32
41
87
94
85
58
40
14
0.95
0.73
0.48
1.0
0.42
0.79
0.78
0.82
1.0
0.68
0.87
0.20
0.63
0.48
0.53
0.85
0.82
2.8
1.6
2.1
0.87
0.94
0.85
0.85
0.75
0.70
1.6
0.88
0.69
1.2
0.14
0.86
1.2
8.6
20
104
11
18
42
95
91
46
27
90
40
6.0
7.4
6.8
0.55
0.36
0.84
0.95
0.91
0.92
0.54
1.8
0.70
0.60
0.74
0.68
0.51
0.69
0.59
0.74
0.14
0.43
0.30
0.30
0.35
0.54
0.37
0.57
0.43
0.62
0.40
0.66
0.42
0.60
0.49
0.46
Lb. plantarum
xylose glucose
glucose
maltose
glucose
sweet sorghum
glucose
xylose
glucose xylose
hydr wood
glucose
galactose
mannose
hydr cellulose: glu,
glucose
galactose
mannose
xylose
hydr cellulose: glu,
hydr soluble starch
hydr tapioca starch
hydr tapioca flour
glucose
galactose
mannose
hydr cellulose: glu,
glucose
galactose
mannose
hydr cellulose: glu,
glucose
galactose
hydr cellulose: glu,
93
102
101
1.1
2.0
0.85
0.60
92
114
33
159
5.5
5.5
6.2
118
177
30
4.2
5.0
5.6
10
2.4
0.59
4.0
1.3
120
99
67
111
111
96
111
111
111
93
Table 3 (continued)
Organism
glucose
galactose
mannose
hydr cellulose: glu, man, xyl, gal
glucose
galactose
mannose
xylose
hydr cellulose: glu, man, xyl, gal
glucose
fructose
glucose fructose
sucrose
alpha-cellulose
switch grass cellulose
glucose
starch
xylose
xylose glucose
glucose
xylose
glucose
maltose
hydr wheat flour, 3 l enz/g starch
hydr wheat flour, 5 l enz/g starch
hydr wheat flour, 6 l enz/g starch
hydr wheat flour, 8 l enz/g starch
glucose
galactose
mannose
xylose
hydr cellulose: glu, man, xyl, gal
LA
g/l
7.3
4.7
8.3
5.2
3.1
6.2
1.3
17
14
16
15
45
28
99
90
23
28
36
13
4.9
3.2
75
75
90
87
6.6
2.8
5.8
1.8
YLA/tot
g/g
Qv
g/(lh)
0.73
0.47
0.83
0.43
0.52
0.31
0.62
0.13
0.43
0.86
0.71
0.81
0.73
111
111
88
0.96
0.50
0.90
0.82
0.45
0.70
1.0
0.42
0.86
0.70
0.78
0.83
0.98
0.93
0.66
0.28
0.58
0.18
0.16
Ref
112
28
0.30
2.2
3.6
0.37
2.5
1.0
1.2
0.85
1.5
1.7
160
161
25
196
111
Abbreviations as in Table 2 and: liq liquefied, glucoam glucoamylase; alpha alpha-amylase; Xl gene encoding xylose isomerase; XK gene
encoding xylulokinase; reg regulating gene xy/R; glu glucose; man mannose; xyl xylose; gal galactose; enz enzyme.
lactis, nutrient limitation occurred [106]. This was overcome by adding proteasereleasing nutrients present in the
flour or by increasing the flour concentration.
3.2.4. Lignocellulosic materials
Lignocellulosic materials have also been used for the
production of LA in similar ways as starch. It consists
mainly of the hexoses glucose, galactose, and mannose and
the pentoses xylose and arabinose, and has to by hydrolyzed
to monomers to be fermentable [110]. The lignocellulosic
materials included waste paper [111], plant material [112,
113], and wood [67]. SSF of cellulose has been studied with
Lb. delbrueckii [114] and Lb. rhamnosus [115,116]. A
mixed culture with the cellulase-producing fungus Trichoderma reesei has also been reported [117].
3.2.5. Effectiveness
Comparing different carbon sources showed that glucose resulted in higher LA concentrations and yields
compared with other sugars (Table 3). Xylose, galactose,
94
Table 4
Influence of nutrient type and concentration on lactic acid production
Organism
Nutrients
LA
g/l
YLA/tot
g/g
sucrose
sucrose
whey
whey
glucose
glucose
hydr wheat flour, SSF
hydr wheat flour, SSF
glucose
glucose
hydr wheat flour, SSF
hydr wheat flour, SSF
hydr potato
hydr potato waste
lactose
whey
whey permeate
whey permeate
whey permeate
hydr whey
hydr whey, clarified
whey, UF
glucose
hydr rye
whey
whey
sweet sorghum
sweet sorghum
glucose
glucose
sorghum
sorghum
sorghum
hydr molasses, SSF
hydr molasses, SSF
glucose
glucose
hydr wood
hydr wood, SSF
glucose
glucose
glucose
glucose
glucose
soy
soy molasses
potato starch
potato starch
glucose, cont substr feed
glucose, pH dep substr feed
glucose
glucose
glucose
hydr wheat flour, SSF
hydr wheat flour, SSF
hydr wheat flour, SSF
hydr wheat flour, SSF
hydr wheat flour, SSF
hydr wheat flour, SSF
unhydr wheat flour glucose
unhydr wheat flour
hydr wheat flour
20% YE
1% YE 0.5% pep
21
18
13
20
93
96
18
26
58
67
106
109
100
93
17
8.9
36
36
40
44
41
37
93
92
9.8
14
106
91
45
46
1.1
0.90
0.22
0.34
0.93
0.96
0.11
0.18
0.48
0.56
0.82
0.91
1.0
0.78
0.38
0.20
0.75
0.75
0.83
Lb. acidophilus R
Lb. casei NRRL B-441
Lb. delbrueckii sp. bulgaricus ATCC 11842
Lb. delbrueckii sp. delbrueckii ATCC 9649
Lb. delbrueckii sp. delbrueckii ATCC 9649
Lb. delbrueckii sp. lactis ATCC 12315
Lb. helveticus ATCC 15009
Lb. helveticus Milano
YE
malt sprouts
YE
YE
MRS 1% YE
MRS 3% YE
YE
CSL
MRS
YE
YE higher conc
YE pep
YE
CSL
CSL
MRS
5% MRS
YE
YE pep
YE
MRS
5% vetch juice
15% vetch juice
25% vetch juice
YE pep
0.25% YE 0.5% trp
0.5% YE 1% g/trp
YE pep
YE pep
0.4% YE
0.8% YE
0.2% YE
1% YE
YE
YE trp
0.3% YE
0.5% YE
1% YE
YE
YE
YE
80
16
14
57
58
27
29
53
53
25
34
26
4.2
5.5
25
10
28
34
24
37
43
95
107
96
106
90
87
75
1.0
0.95
0.92
0.20
0.28
0.79
0.91
0.90
0.92
0.81
0.70
0.81
0.95
0.96
1.0
0.66
0.66
0.83
1.1
0.81
0.76
0.85
0.50
0.20
0.56
0.68
0.96
0.74
0.86
0.77
0.91
0.76
0.88
0.98
0.96
1.0
4.0
Qv
g/(lh)
Ref
1.0
0.83
122
174
2.3
3.9
0.56
0.9
0.72
1.4
1.6
3.6
146
18
49
18
28
178
5.8
9.4
12
5.5
4.4
2.7
123
124
35
174
10
10
2.3
2.4
2.2
2.0
2.8
99
67
100
88
156
2.3
1.5
2.8
3.7
0.2
0.5
2.6
115
179
180
87
0.72
0.43
2.0
2.1
1.2
2.1
2.3
1.7
2.4
3.0
3.3
1.5
3.3
2.1
108
143
181
18
18
106
20
95
(Continued)
Organism
Nutrients
hydrwheatflourprotease
hydrwheatflourprotease
hydrwheatflourprotease
hydrwheatflourprotease
hydrwheatflour
vitamins
amino acids
peptides
LA
g/l
YLA/tot
g/g
43
46
53
44
17
Qv
g/(lh)
Ref
1.5
2.4
2.8
2.2
0.23
106
Abbreviations as in Table 2 and: dep dependent; YE yeast extract; pep peptone; CSL corn steep liquor; trp tryptone; SSF simultaneous
saccharification and fermentation; cont continuous; Substr substrate; UF ultrafiltrated.
Table 5
Influence of fermentation mode on lactic acid production
Organism
Fermentation
mode
Substrate
LA
g/l
YLA/tot
g/g
Ent. faecium
batch
semicont
batch, imm
cont, imm
batch
fed-b
batch
cont
batch
cont
batch, imm
cont, imm
batch
cont
batch, imm
cont, imm
batch
cont
batch
cont
batch
cont
batch
cont, imm
batch, dial
cont, dial
batch
cont, extract
batch
cont, recirc,
extract
batch
cont
batch
cont
batch, imm
cont, imm
batch, extract
rep b, extract
batch
fed-b
alfalfa
alfalfa
corn starch
corn starch
whey
whey
glucose
glucose
hydr maize barley
hydr maize barley
glucose
glucose
whey
whey
whey
whey
whey
whey
lactose
lactose
whey
whey
whey
whey
whey
whey
sucrose
sucrose
glucose
glucose
27
30
50
40
22
45
83
55
85
71
90
75
44
13
50
9.5
115
117
45
39
49
48
0.91
0.99
0.83
0.67
0.44
0.45
0.83
0.55
0.87
0.73
0.9
0.75
0.95
0.28
1.0
0.19
0.86
0.87
0.90
0.78
1.1
1.2
glucose
glucose
glucose
glucose
potato starch
potato starch
glucose
glucose
lactose
lactose
47
125
77
80
80
47
38
10
39
28
25
10
0.29
0.50
40
39
1.2
3.1
0.73
0.74
0.89
0.48
0.76
0.20
0.75
0.56
0.50
0.20
0.3
0.4
Qv
g/(lh)
Ref
130
94
129
1.5
5.3
127
95
131
182
0.65
12
138
183
11
2.2
1.3
2.7
3.1
29
134
116
184
43
1.7
8.0
5.1
4.2
185
141
186
187
0.72
0.43
108
126
7.1
1.4
125
Abbreviations as in Table 2 and: dial electrodialysis; extract extraction, adsorption; imm immobilised cells; fed-b fed-batch; rep b repeated
batch; cont continuous culture; semicont semicontinuous culture.
96
Table 6
Influence of cell recirculation on lactic acid production
Organism
Fermentation
mode
Substrate
LA
g/l
YLA/tot
g/g
Lb. casei
cont,
cont,
cont
cont,
cont
cont,
cont
cont,
whey
whey
glucose
glucose
glucose
glucose
glucose
glucose
22
28
26
32
17
47
27
45
0.44
0.57
0.65
0.80
0.68
0.48
0.54
0.90
imm
imm, recirc
recirc
recirc
recirc
Qv
g/(lh)
Ref
7.3
9.4
72
142
5.1
4.2
3.4
10
141
143
97
Table 7
Influence of initial pH and pH control on lactic acid production
Organism
Substrate
pH
pH control
Ent. faecium
alfalfa
alfalfa
glucose
glucose
glucose
glucose
glucose
glucose
glucose
glucose
glucose
glucose
glucose
glocose
glucose
glucose
glucose
glucose
glucose
cellulose
cellulose
cellulose
hydr
maize barley
hydr
maize barley
hydr
maize barley
sorghum
sorghum
sorghum
whey
whey
whey
whey
whey
whey
lactose
lactose
lactose
cellulose
cellulose
cellulose
whey
whey
sorghum
sorghum
sorghum
sorghum
sorghum
sucrose
sucrose
glucose
glucose
glucose
glucose
glucose
glucose
gtlucose
glucose
cellulose
cellulose
6.2
5.8
5.4
6.0
6.5
7.1
7.8
4.2
6.2
6.2
init
NH4OH
NaOH titr
NaOH titr
NaOH titr
NaOH titr
NaOH titr
NaOH
NaOH
init, extract
LA
g/l
8.3
27
YLA/tot
g/g
Qv
g/(lh)
0.27
0.90
5.5
5.0
5.5
5.5
6.0
6.5
7.0
4.2
5.0
5.9
5.0
CaCO3
titr, dial
NaOH titr
NaOH titr
titr, dial
NaOH titr
NaOH titr
NaOH titr
CaCO3
CaCO3
CaCO3
Na2CO3
10
36
27
5.6
55
53
81
92
88
81
54
49
15
26
18
59
0.12
0.42
0.32
0.06
0.55
0.53
0.79
0.89
0.81
0.78
0.52
0.47
0.30
0.52
0.36
0.61
5.5
Na2CO3
87
0.90
5.8
Na2CO3
85
0.87
5.5
6.0
6.5
5.4
6.0
3.5
4.5
5.5
6.5
4.5
5.0
5.6
4.2
5.0
5.8
5.6
5.6
5.5
6.0
6.5
7.0
7.5
6.0
6.0
5.0
5.5
6.0
6.5
4.2
4.2
6.0
6.0
4.3
4.3
NH3
NH3
NH3
NH4OH
NH4OH
init
init
init
init
NH4OH titr
NH4OH titr
NH4OH titr
NH4OH
NH4OH
NH4OH
NaOH titr
NaOH titr, dial
NH3
NH3
NH3
NH3
NH3
NaOH titr
NaOH titr, extract
NaOH
NaOH
NaOH
NaOH
yes
yes, extract
NH4OH
extract
NH4OH
NH4OH, extract
130
0.70
1.6
1.2
1.0
0.90
1.0
6.5
2.9
2.3
5.3
5.3
2.4
1.9
4.3
4.5
1.5
1.4
77
80
65
78
79
78
25
19
71
771
45
28
0.45
0.64
0.48
0.48
0.60
0.67
0.50
0.90
0.90
0.27
0.58
0.33
0.78
1.2
0.73
0.74
0.65
0.78
0.79
0.78
0.91
0.79
104
144
127
164
114
95
3.5
4.5
2.3
35
50
25
25
31
35
25
45
45
27
52
33
31
47
Ref
100
36
159
0.68
3.1
11
0.23
0.43
134
1.3
1.1
2.1
2.0
2.8
5.1
1.9
1.7
8.0
2.3
3.9
4.9
4.9
2.5
0.91
1.3
5.4
0.31
0.96
43
117
100
185
153
188
42
112
98
Table 7
(continued)
Organism
Substrate
pH
pH control
glucose
glucose
glucose
glucose
glucose
soy molasses
soy molasses
soy molasses
glucose
glucose
glucose
glucose
glucose
glucose
glucose
glucose
glucose
glucose
glucose
glucose
glucose
glucose
glucose
glucose
maltose
maltose
maltose
hydr wheat
flour
hydr wheat
flour
hydr wheat
flour
hydr wheat
flour
hydr wheat
flour
lactose citrate
lactose citrate
lactose citrate
lactose citrate
5.5
6.3
7.5
6.2
6.2
5.6
6.0
6.4
6.5
6.5
6.5
6.5
6.5
6.0
6.0
6.0
6.0
6.0
6.0
6.0
6.0
5.0
5.8
6.5
5.0
5.8
6.5
6.0
NH4
NH4
NH4
init
CaCO3
NaOH titr
NaOH titr
NaOH titr
init
init, extract
init
NaOH
init, extract
NaOH
NaOH, deal
yes
yes, dial
init, dial
NaOH, dial
NaOH
NaOH, dial
NaOH titr
NaOH titr
NaOH titr
NaOH titr
NaOH titr
NaOH titr
init
7.5
26
5.5
5.4
4.9
5.1
5.7
3.5
19
14
45
35
60
66
60
60
50
62
5.4
5.3
4.9
5.1
4.2
3.2
3.3
0.21
0.81
0.85
0.79
0.82
1.0
1.1
0.18
0.81
0.70
0.90
0.70
0.80
0.88
0.75
0.75
0.85
0.88
0.92
0.90
0.86
1.0
0.82
0.70
0.02
6.0
NaOH
96
0.76
3.0
4.0
NaOH titr
0.041
0.23
5.0
NaOH titr
20
0.11
0.42
6.0
NaOH titr
105
0.58
2.9
5.0
5.5
6.0
6.5
NaOH
NaOH
NaOH
NaOH
0.13
0.71
0.71
0.73
0.83
1.9
4.5
7.7
titr
titr
titr
titr
LA
g/l
7.0
7.0
37
37
38
YLA/tot
g/g
Qv
g(lh)
Ref
16
23
17
0.30
2.6
132
180
87
189
0.87
1.7
10
15
3.0
4.0
2.4
5.1
187
143
190
191
136
1.7
3.4
2.5
0.37
1.2
1.0
0.47
25
25
18
20
152
Abbreviations as in Table 5 and: init initial pH set, then uncontrolled; titr titration.
pending on the metabolic pathway used. For efficient industrial production of LA, by-product formation should be
avoided, or kept to a minimum. The ratio of g LA per g
total product (LA/tot) was higher in batch culture than in
fed-batch [112], and also under anaerobic conditions
compared with aerobic conditions [149] (Table 9). When
NaCl [149] and substrate concentrations [150] increased,
LA/tot also increased (Table 9). However, there was no
change in LA/tot when nutrients were varied (Table 9)
[18,151].
Different carbon sources give varying amounts of byproducts, so that maltose fermentation in Lc. lactis resulted
in values of LA/tot of 0.67, compared to 0.93 for glucose
fermentation (Table 9) [25]. Also, lactose and galactose
result in lower LA/tot-values than glucose in some strains of
99
Table 8
Influence of temperature on lactic acid production
Organism
Temp
C
Substrate
LA
g/l
YLA/tot
g/g
Qv
g/(lh)
Ref
25
28
35
30
37
41
45
37
41
30
36
44
30
37
41
45
30
35
37
40
30
35
37
30
34
37
40
starch
starch
starch
glucose
glucose
glucose
glucose
hydr barley starch
hydr barley starch
sweet sorghum
sweet sorghum
sweet sorghum
glucose
glucose
glucose
glucose
glucose
glucose
glucose
glucose
maltose
maltose
maltose
glucose
glucose
glucose
glucose
26
29
30
80
80
82
42
140
117
0.52
0.58
0.60
0.89
0.89
0.91
0.47
0.98
0.82
0.54
0.44
0.33
3.2
5.6
5.6
1.2
105
67
70
68
75
4.9
5.2
5.2
1.2
3.2
3.7
4.0
60
65
60
50
0.74
0.78
0.76
0.83
0.86
0.88
0.88
0.20
0.70
0.73
0.80
1.3
1.5
1.5
1.2
146
101
1.5
1.9
2.2
3.3
3.3
3.5
3.3
2.5
2.9
1.8
1.0
1.2
1.1
2.2
2.8
2.3
1.5
99
146
25
25
20
Abbreviations as in Table 2.
Lc. lactis [23,24]. Pentose fermentation results in the production of acetate or ethanol and LA in equimolar amounts,
and values of LA/tot of 0.57 0.79 have been reported (Table 9) [150]. Recirculation [53] of cells gave lower or
similar LA/tot-values as free cells, whereas the effects of pH
[151153] and temperature [20,105] were inconclusive (Table 9).
4.2. LA isomery
Most LAB produce only one isomeric form of LA, but
sometimes there is a slight production of the other isomer.
Lb. helveticus and Lb. plantarum produce a racemic mixture, the composition of which varies. The lactate dehydrogenase (LDH) is stereospecific, giving either D- or
L-LA [14]. Which isomeric form(s) of the enzyme present
in the LAB mainly determines the isomery of the LA
produced. For some applications, such as PLA synthesis,
an optically pure product or a racemic mixture of constant composition is desirable. For the L-LA-producing
Lb. amylophilus, Lb. delbrueckii and Lb. rhamnosus, no
D-isomer was produced when the pH was varied [36,153]
or when the amount of nutrients was changed [18,36]
(Table 10). On the other hand, only D-LA was formed by
Lb. delbrueckii spp. bulgaricus in batch and continuous
culture [34], from glucose and lactose [34], and when the
100
Table 9
Effect of process parameters on by-product formation
Parameter Organism
studied
Fermentation
mode
Substrate
Substr g/l/
Nutrients
carbon,
nutr
batch
glucose
carbon
batch
fed-b, recirc
glucose
alpha-cellulose
hydr rye
1/20 MRS
MRS
fed-b, recirc
switch grass
cellulose
glucose
carbon
conc
conc
conc
nutr
nutr, O2
nutr, pH
mode
recirc
pH
pH
pH
pH
maltose
starch
pH/
LA
TempC g/l
HAc
g/l
trace
35
93
45
0
1.0
1.0
0.98
28
0.50
0.98
0.93
25
3.2 0.49
29 0
0.36
0
0.72
0
0.67
1.0
105
0
0
0
0
1.0
1.0
0.57
0.72
0.74
0.70
70
100
29
49
130
160
37
127
hydr wheat flour
45
53
12
25
22
27
35
48
18
starch
glucosecitrate
glucosecitrate
glucosecitrate
glucosecitrate
glucosecitrate
glucosecitrate
glufrumalate
glufrumalate
glufrumalate
glufrumalate
glufrumalate
alpha-cellulose
26
3.2
4.2
4.2
10.0
4.5
5.5
9.1
8.6
6.6
7.3
13
13
3.1
1.7
0.79
1.3
0.58
0.56
0
0
0
0
0
0
45
32
1.0
0.44
5.0
32
31
32
65
0.66
0.90
0.49
0.35
glucose
glucose
glucose
glucose
5.5
6.0
6.5
5.0
78
79
78
5.4
0.33
0.35
0.39
0.028 0.20
glucose
glucose
maltose
5.8
6.5
5.0
maltose
maltose
glucose
5.8
6.5
4.0
4.2 0.33
3.2 0.49
18 0.90
0.24
0.36
15
glucose
glucose
5.0
6.0
68
56
21
2.4
alpha-cellulose
glucose
glucose
glucose
glucose
glucose
NaCl 6%
NaCl 8%
HAc 49 mM
4.1
HAc 20 mM NaCl 3% 4.5
HAc 20 mM NaCl 6% 4.5
NaCl 6%
5.5
HAc 0.7 mM
6.0
40
40
40
40
112
0.065 0.20
starch
starch
xylose
xylose
xylose
xylose
lactose
lactose
starch
NaCl 6%
NaCl 8%
LA/tot Ref
g/g
92
4.9 0.10
50
EtOH HFo
g/l
g/l
0
0
9.1
9.7
7.9
7.3
1.0
1.5
10
present
present
0.64
18
15
0
0
0
0
0
150
56
0.63
0.51
0.71
0.84
0.88
0.89
0.91
1.0
1.0
1.0
1.0
1.0
1.0
112
149
151
0.29
0.66
0.98
0.96
142
0.45
0.64
0.30
1.1
1.5
0.74
0.10
0.94
0.91
0.95
0.99
153
0.40
0.40
0.51
0
0.99
0.99
0.99
0.96
25
5.3 0.067 0
0.16
4.9 0.10 0.065 0.20
5.1 0.098 0.065 0.19
0.96
0.93
0.94
25
0.54
0.72
1.0
0.79
0.67
0.46
106
0.90
0.60
0.93
0.93
2.0
1.5
(continued)
Parameter
studied
Organism
Fermentation
mode
Substrate
batch
glucose
30
batch
batch
batch
batch
glucose
glucose
glucose
maltose
batch
batch
batch
101
Substr g/l/
Nutrients
pH/
TempC
LA
g/l
HAc
g/l
EtOH
g/l
HFo
g/l
LA/tot
g/g
Ref
4.9
0.10
0.065
0.20
0.93
25
35
37
40
30
5.2
5.2
1.2
3.2
0.074
0.085
0.030
0.49
0.080
0.050
0
0.36
0.18
0.11
0
0.72
0.94
0.95
0.98
0.67
25
maltose
maltose
glucose
35
37
30
3.7
4.0
60
0.48
0.36
1.0
0.38
0.26
1.0
0.78
0.60
0.69
0.77
0.97
20
batch
batch
batch
batch
glucose
glucose
glucose
starch
34
37
40
25
65
60
50
26
1.5
6.0
7.5
0
1.5
4.0
6.0
0
0.96
0.86
0.79
1.0
105
batch
batch
starch
starch
28
35
29
30
0
0
0
0
0
0
1.0
1.0
Abbreviations as in Table 6 and: carbon carbon source; nutr nutrients; O2 aeration; mode fermentation mode; T temperature; ana anaerobic;
aer aerobic; fru fructose; HAc acetic acid; EtOH ethanol; HFo formic acid; La/tot g LA per g total products.
Batch fermentation was superior to continuous fermentation in all respects but the volumetric productivity [127,
138]. Repeated or semicontinuous batch modes increase the
yield further [44]. If the substrate is expensive the yield
should be maximized, as in batch or semicontinuous operation [128], whereas the volumetric productivity is maximized by continuous operation if investment costs are high.
A high productivity is achieved by recycling the cells,
resulting in a high cell mass without reducing the yield
[47]. When using starch or lignocellulose, which must be
hydrolyzed before fermentation, the two steps can be
performed separately or simultaneously (SSF). In SSF the
hydrolyzing enzymes are not inhibited due to the continuous removal of the produced glucose, and less enzyme is
required. The time was only marginally reduced in SSF
of WWF by Lc. lactis [106]. Only one vessel is needed,
and only one temperature and pH has to be adjusted. In
SSF recirculation of cells is hampered by solid substrate
residues fouling the equipment. SSF of WWF requires
nutrient supplementation that is not demanded in separate
fermentation [106].
pH control is traditionally performed with calcium hydroxide, but the regeneration of LA results in the production
of large amounts of solid calcium sulfate [3]. Better alternatives are ammonia or calcium carbonate, leading to production of the fertilizer ammonium sulfate [162] or gaseous
carbon dioxide, respectively. Continuous removal of the
acid with extraction or electrodialysis results in even higher
LA concentrations and yields. The extracting material must
be bio-compatible so as not to harm the organism, and one
way of achieving this is aqueous two-phase systems, which
provide good separation of LA and cells when combined
with a tertiary amine [163]. Solid resins have also been
102
Table 10
Effect of process parameters on the isomeric form of LA produced
Parameter
studied
Organism
carbon, conc
Fermentation
mode
batch
batch
batch
carbon, nutr, T Lb. delbrueckii sp. bulgaricus CBS 743.84 batch
batch
carbon
Lb. delbrueckii sp. bulgaricus DSM 2129
batch
batch
conc
Lb. amylophilus ATCC 49845
batch
batch
batch
conc
Lb. rhamnosus ATCC 10863
batch
batch
conc, recirc
Lb. rhamnosus ATCC 10863
cont, recirc
cont, recirc
conc
Lc. lactis sp. lactis ATCC 19435
batch
batch
nutr
Lb. delbrueckii sp. delbrueckii ATCC 9649 batch
batch
nutr
Lb. delbrueckii sp. delbrueckii ATCC 9649 batch
batch
nutr
Lb. delbrueckii sp. bulgaricus ATCC 11842 batch
batch
nutr
Lb. delbrueckii sp. bulgaricus ATCC 55163 batch
batch
nutr, O2
Lb. plantarum H4
batch, aer
batch, aer
batch, aer
batch, ana
batch, ana
batch, ana
nutr
Lb. rhamnosus ATCC 10863
batch
batch
batch
batch
batch
batch
nutr
Lb. rhamnosus ATCC 7469
batch
batch
batch
nutr
Lc. lactis sp. lactis AS211
batch
batch
nutr
Lc. lactis sp. lactis ATCC 19435
batch
batch
nutr
Lc. lactis sp. lactis ATCC 19435
batch
batch
mode
Lb. delbrueckii sp. bulgaricus DSM 2129
batch
cont
mode
Lb. salivarius sp. salivarius ATCC 11742
batch
cont
recirc
Lb. rhamnosus ATCC 10863
cont, recirc
cont, recirc
recirc
Lb. rhamnosus ATCC 10863
cont, recirc
cont, recirc
pH
Lb. delbrueckii sp. bulgaricus ATCC 55163 batch
batch
pH
Lb. rhamnosus ATCC 10863
batch
batch
batch
batch
pH
Lc. lactis sp. lactis ATCC 19435
batch
batch
78%
96%
51%
79%
78%
96%
Substrate
Substr g/l/
Nutrients
whey UF perm
centr whey
centr whey
lactose
lactose
glucose
lactose
starch
starch
starch
glucose
glucose
glucose
glucose
glucose
glucose
glucose
glucose
starch
starch
starch
starch
lactose
lactose
glucose citrate
glucose citrate
glucose citrate
glucose citrate
glucose citrate
glucose citrate
glucose
glucose
glucose
glucose
glucose
glucose
glucose
glucose
glucose
starch
starch
glucose
glucose
starch
starch
glucose
glucose lactose
glucose
glucose
glucose
glucose
glucose
glucose
lactose
lactose
glucose
glucose
glucose
glucose
starch
starch
43
93
78
whey milk YE
YE trp
pH/
% L-LA Ref
TempC
37
44
50
70
100
50
70
40
80
85
174
YE 1%
YE 3%
hydr wheat flour
hydr wheat flour YE
hydr wheat flour
hydr wheat flour YE
whey YE
whey soy flour
NaCl 6%
NaCl 8%
NaCl 6%
NaCl 8%
YE 0.25% trp 0.5%
YE 0.5% trp 1%
YE 0.75% trp 1.5%
YE 1% trp 2%
YE 1.5% trp 3%
YE 2% trp 4%
YE 0.2%
YE 1%
hydr wheat flour
hydr wheat flour YE
hydr wheat flour
unhydr wheat flour
hydr wheat flour
hydr wheat flour YE
5.4
6.0
5.0
5.5
6.0
6.5
6.0 init
6.0
1
7
10
1
3.0
0
0
93
93
93
95
95
96
97
96
99
0
0
94
95
91
95
100
100
48
44
43
45
36
33
95
95
95
95
95
95
98
98
97
94
100
99
98
100
100
0
0
90
86
96
95
96
97
100
100
98
98
98
97
97
100
33
33
34
105
156
142
20
49
18
18
36
149
156
180
18
20
18
34
51
142
142
36
153
18
103
(continued)
Parameter
studied
Organism
Fermentation
mode
Substrate
pH
batch
batch
batch
batch
batch
batch
batch
batch
batch
batch
glucose
glucose
glucose
starch
starch
starch
glucose
glucose
glucose
glucose
Substr g/l/
Nutrients
pH/
TempC
% L-LA
6.0
5.0
3.0
25
28
35
30
34
37
40
99
99
97
93
93
93
99
90
96
82
Ref
20
105
20
Abbreviations as in Table 9 and: UF perm ultrafiltrated permeate; centr centrifuged; % L-LA % of LA in L-form.
used in combination with immobilized cells in fluidizedbed reactors, resulting in a LA concentration of up to 771
g/l [42]. Electrodialysis can be used in two ways: as a pH
controller producing the lactate anion [164], or in combination with a base, e.g. NaOH, to split the Na-lactate
into NaOH and LA [165]. The cells are removed by
filtration not to foul the membranes. The price of the
membranes is presently a considerable drawback. Both
aqueous two-phase systems and electrodialysis yield LA,
instead of lactate, which potentially could decrease the
purification costs.
Acknowledgments
This work was financially supported by the Swedish
National Board for Industrial and Technical Development
(NUTEK).
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