Enzyme and Microbial Technology 26 (2000) 87107

Review

Factors affecting the fermentative lactic acid production from

renewable resources1
Karin Hofvendahl, Barbel HahnHagerdal*
Department of Applied Microbiology, Lund Institute of Technology/Lund University, P.O. Box 124, SE-221 00 Lund, Sweden

Received 15 February 1999; received in revised form 11 August 1999; accepted 20 August 1999

Abstract

Parameters affecting the fermentative lactic acid (LA) production are summarized and discussed: microorganism, carbon- and nitrogen-
source, 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 Hemicellulose, in contrast to starch and cellulose, contains

Lactic acid (LA) is a versatile chemical, used 1) as an
acidulant, flavor and preservative in the food, pharmaceu-
tical, 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, D-
and 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 al-
ways 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 abun-
dant compounds in the world, and when hydrolyzed to mainly
glucose they are fermentable by a number of microorganisms.
1
This paper is based on the Ph.D thesis of Karin Hofvendahl (1998)
* Corresponding author. Tel.: 46-46-222-8428; fax: 46-46-222-4203.
E-mail address: Barbel.Hahn-Hagerdal@tmb.lth.se (B. HahnHagerdal)
pentoses, which give rise to by-products such as acetate and
ethanol, decreasing the LA yield. Fermentative LA production
from renewable resources comprises the following steps: pre-
treatment of substrate including hydrolysis to sugars, fermen-
tation of sugars to LA, separation of bacteria and solid particles
from the broth, and purification of LA.
The names of genera and strains used in this review were
updated to concur with our knowledge on LAB today, and
might therefore differ from those used by the authors cited.
The changes made are listed in Table 1. Today LAB consist
of the Gram-positive genera: Carnobacterium, Enterococ-
cus (Ent), Lactobacillus (Lb), Lactococcus (Lc), Leuconos-
toc (Leu), Oenococcus, Pediococcus (Ped), Streptococcus
(Str), Tetragenococcus, Vagococcus, and Weissella [4].
LAB are cocci, with the exception of lactobacilli and car-
nobacteria which are rods, unable to synthesize ATP by
respiration, and that have LA as the major end product from
energy-conserving fermentation of sugars. Most LAB are
facultatively anaerobic, catalase negative, nonmotile and
nonspore forming. They have high acid tolerance and sur-
vive pH 5 and lower. Their acid tolerance gives them a
competitive advantage over other bacteria. The optimal
temperature for growth varies between the genera from 20
to 45C [5,6]. Most of them are considered GRAS (gener-
ally regarded as safe), but some strains of e.g. streptococci
are pathogenic. All LAB genera belong to the Clostridium

0141-0229/00/$ see front matter 2000 Elsevier Science Inc. All rights reserved.
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88 K. Hofvendahl, B. HahnHagerdal / Enzyme and Microbial Technology 26 (2000) 87107

microorganism able to metabolize many different carbohy-

Nomenclature drates [8]. LAB have been used by humans for the fermen-
tation of food and feed products since ancient days, and
ATP adenosine triphosphate today their major applications are still in the food and feed
Ent Enterococcus industry, e.g. in the production of dairy products, pickles,
LA lactic acid meat and wine. The present technical applications of LAB
LA/tot La per total products (g/g) include the production of dextran from sucrose by Leu.
LAB lactic acid bacteria mesenteroides [9], the production of nisin by Lc. lactis spp.
Lb Lactobacillus lactis and the production of LA for different applications,
Lc Lactococcus see above. It has also been suggested that LAB could be
LDH lactate dehydrogenase used as oral vaccine vectors [10].
Leu Leuconostoc LAB ferment sugars via different pathways resulting in
NADH nicotinamide adenine dinucleotide, reduced homo-, hetero-, or mixed acid fermentation (Fig. 1). Homo-
form fermentation gives only LA as the end product of glucose
PDH pyruvate dehydrogenase metabolism, and the EmbdenMeyerhofParnas pathway is
Ped Pediococcus used (Fig. 1A) [11,12]. In heterofermentation equimolar
PFL pyruvate formate lyase amounts of LA, carbon dioxide and ethanol or acetate are
PLA poly-lactic acid formed from glucose via the phosphoketolase pathway (Fig.
QV maximum columetric productivity (g/lh) 1B) [13,14]. The ratio of ethanol and acetate formed is depen-
SSF simultaneous saccharification and fermentation dent on the redox potential of the system [15]. This pathway is
Str Streptococcus used by facultative heterofermenters, such as Lb. casei, for the
WWF whole wheat flour fermentation of pentoses, and for the fermentation of hexoses
YLA/tot yield of LA per substrate provided (g/g) 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].
subdivision of the Gram-positive bacteria; i.e. the GC Mixed acids are formed by homofermenters such as
content of the DNA is 55% [4]. lactococci during glucose limitation [16], and during growth
LAB have complex nutrient requirements, due to their on other sugars, e.g. Lc. lactis on maltose [1722], lactose
limited ability to synthesize B-vitamins and amino acids [7]. [23,24] and galactose [23,24], or at increased pH and de-
Therefore they are naturally found in nutrient-rich environ- creased temperature [25]. Ethanol, acetate and formate are
ments such as in plants, milk, and inside the human and formed in addition to LA. The homofermentative pathway
animal bodies. A complex natural environment renders a 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. plantarum DSM, ATCC

Lb. bulgaricus Lb. delbrueckii sp. bulgaricus 166
Lb. casei sp. rhamnosus Lb. rhamnosus 167, 168
Lb. cellobiosus Lb. fermentum 169
Lb. xylosus Lc. lactis sp. lactis 170
Str. diacetylactis Lc. lactis sp. lactis biovar diacetylactis 170
Str. cremoris Lc. lactis sp. cremoris 170
Str. faecalis Ent. faecalis 171
Str. faecium Ent. faecium 171
Str. lactis Lc. lactis sp. lactis 170
Str. salivarius sp. thermophilus Str. thermophilus 172
Lb. delbrueckii NRRL B-445 Lb. rhamnosus ATCC 10863 ATCC
Lb. casei sp. casei ATCC 393/DSM 20011 Lb. zeae ATCC 393 168
Lb. delbrueckii ATCC 9649 Lb. delbrueckii sp. delbrueckii ATCC 9649 ATCC
Lb. salivarius NRRL B-1950 Lb. salivarius sp. salivarius ATCC 11742 ATCC, NRRL
Lb. casei DSM 20244 Lb. paracasei sp. paracasei DSM 20244 167, DSM
Lb. casei ATCC 7469/IFO 3425 Lb. rhamnosus ATCC 7469 ATCC
Lb. casei ATCC 11443 Lb. rhamnosus ATCC 11443 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.
 K. Hofvendahl, B. HahnHagerdal / Enzyme and Microbial Technology 26 (2000) 87107 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.

which in addition to give LA is also metabolized into 2. Effectiveness of various processes

formate and acetyl-CoA by pyruvate formate lyase (PFL)
(Fig. 1C). In the presence of oxygen PFL is inactivated [26],
and an alternative pathway of pyruvate metabolism be-
comes active via pyruvate dehydrogenase (PDH), resulting
in the production of carbon dioxide, acetyl-CoA and NADH
[27]. LAB are also capable of forming other products, e.g.
flavors such as diacetyl and acetoin or bacteriocins, which is
not further discussed in this review.
Numerous processes of fermentative LA production have
been patented. They include the fermentation of industrial
starch waste, e.g. potato, with a mixture of five Lb. strains
in a continuous simultaneous saccharification and fermen-
tation process (SSF) with electrodialytic purification [28],
the use of whey permeate in a continuous process with cell
recirculation and electrodialysis [29], the use of lignocellu-
losic material and a recombinant strain of Lb. able to fer-
ment xylose [30], and the recovery of municipal solid waste
to solids and LA by fermentation [31]. Several patents claim
the production of optically pure D- [3234] or L-LA [32,35,
36]. In one patent, the development of a LA-tolerant strain
of Lb. delbrueckii is described [37]. Methods for the puri-
fication of LA from the fermentation broth with electrodi-
alysis [38,39], membrane separation [40], and esterification
[41] have also been patented.
The selection criteria for the material presented in this
review were fermentative LA production by LAB for the
purpose of industrial processes. Therefore, papers with a
metabolic approach have not been included. Many param-
eters influence the efficiency of a fermentation process,
several of which are discussed below. Due to the oxygen
tolerance of LAB most fermentations were performed with-
out aeration control. However, anaerobic conditions were
preferred to aerobic or microaerophilic conditions.
The effectiveness of a process can be measured as the
concentration of LA produced, as the yield of LA based on
substrate and as the productivity (LA production rate). The
yields presented in Tables 210 were calculated as g LA per
g substrate provided (YLA/tot), and the productivities are
given as the maximum volumetric productivity (QV) in g
LA per liter per hour. The overall most effective way to
produce LA in terms of concentration was to continuously
remove the acid by extraction, resulting in at most 771 g/l
[42]. The highest yields, 3.1 g/g, were achieved from whey
using electrodialysis [43] or a semicontinuous fermentation
mode [44]. The highest values of QV, 52-144 g/(lh), were
obtained by recirculating the cells, thus increasing the cell
density [45 48].
90 K. Hofvendahl, B. HahnHagerdal / Enzyme and Microbial Technology 26 (0) 87107

Table 2
Comparison of different strains for lactic acid production

Organism Substrate Temp LA YLA/tot Qv Ref

C g/l g/g g/(lh)

Lb. casei NRRL B-441 glucose 41 82 0.91 5.6 146

Lb. rhamnosus ATCC 10863 glucose 41 68 0.76 3.5 146
Lb. delbrueckii sp. lactis ATCC 8000 glucose 45 83 0.83 32
Lb. delbrueckii sp. lactis DSM 20073 glucose 45 82 0.82 32
Lb. delbrueckii sp. delbrueckii ATCC 9649 glucose 45 58 0.48 0.72 49
Lb. delbrueckii mutant DP3 glucose 45 77 0.64 1.7 49
Lb. delbrueckii mutant DP3, 19 glucose 45 68 0.57 1.2 49
Lb. rhamnosus ATCC 7469 glucose 45 28 0.93 8.0 51
Lb. salivarius sp. salivarius ATCC 11742 glucose 45 28 0.92 11 51
Lb. zeae ATCC 393 glucose 36 21 0.71 173
Lb. rhamnosus DSM 20024 glucose 36 22 0.74 173
Lb. rhamnosus ATCC 7469 glucose 36 24 0.80 173
Lb. zeae ATCC 393 glucose 36 37 0.98 4.0 50
Lb. coryniformis sp. torquens ATCC 25600 glucose 36 39 0.98 2.6 50
Lb. amylovorus ATCC 33622 hydr barley flour 37 93 0.52 2.0 92
Lb. casei NRRL B-441 hydr barley flour 37 120 0.67 1.5 92
Lb. delbrueckii sp. bulgaricus ATCC 11842 sorghum 42 4.5 100
Lb. plantarum ATCC 14917 sorghum 42 2.0 100
Lb. delbrueckii sp. lactis 447 lignocellulose hydr 37 55 0.85 113
Lb. rhamnosus CCM 1753 lignocellulose hydr 37 37 0.74 113
Lb. delbrueckii sp. bulgaricus AU molasses 47 20 0.45 89
Lb. delbrueckii sp. delbrueckii ATCC 9649 molasses 47 26 0.58 89
Lb. rhamnosus ATCC 7469 molasses 47 18 0.40 89
Lb. kefir paneer whey 43 9.8 0.20 174
Lb. acidophilus R paneer whey 43 8.6 0.17 174
Lb. casei SU No 22 whey 32 16 0.32 129
Le. lactis WS 1042 whey 32 11 0.22 129
Str. thermophilus whey permeate 42 18 0.50 5.9 137
Lb. delbrueckii sp. bulgaricus 5085 whey permeate 42 15 0.41 4.0 137
Lc. lactis sp. lactis 2432 whey permeate 30 8.3 0.21 2.1 137
Lb. delbrueckii sp. bulgaricus 5085 whey permeate 42 7.9 0.18 140
Str. thermophilus whey permeate 42 15 0.35 140
Lb. rhamnosus ATCC 7469 whey permeate 37 30 0.71 1.9 52
Lb. rhamnosus ATCC 10863 whey permeate 40 30 0.71 1.5 52
Lc. lactis sp. cremoris 2487 whey permeate 30 37 0.88 4.6 52
Lc. lactis sp. lactis 5085 whey permeate 30 37 0.88 2.4 52
Lb. casei SU No 22 deproteinised whey 32 20 0.39 2.0 175
Lc. lactis WS 1042 deproteinised whey 32 15 0.30 1.5 175
Str. thermophilus whey permeate 42 19 0.47 6.0 139
Lb. delbrueckii sp. bulgaricus 5085 whey permeate 42 16 0.38 4.4 139
Lc. lactis sp. lactis 2432 whey permeate 30 9.0 0.20 2.3 139
Lb. acidophilus CRL 640 skim milk 37 14 133
Lb. delbrueckii sp. bulgaricus CRL 870 skim milk 37 12 133
Str. thermophilus CRL 807 skim milk 37 8.5 133
Lb. rhamnosus ATCC 7469 lactose 37 21 0.38 5.6 53
Lc. lactis sp. cremoris SBT 1306 lactose 30 80 1.5 4.1 53
Lc. lactis sp. lactis ATCC 19435 hydr wheat flour 30 96 0.76 3.0 18
Lc. lactis sp. lactis AS211 hydr wheat flour 30 95 0.77 1.7 18
Lb. delbrueckii sp. delbrueckii ATCC 9649 hydr wheat flour 37 106 0.82 1.6 18
Lb. delbrueckii sp. bulgaricus ATCC 11842 hydr wheat flour 45 18 0.11 0.56 18
Lb. plantarum NRRL B-787 solid waste 32 17 0.42 111
Lb. plantarum NRRL B-788 solid waste 32 19 0.46 111
Lb. plantarum NRRL B-813 solid waste 32 18 0.43 111
Lb. plantarum NRRL B-531 solid waste 32 18 0.43 111
Lb. pentosus NRRL B-227 solid waste 32 21 0.51 111
Lb. pentosus NRRL B-473 solid waste 32 18 0.43 111
Lb. plantarum USDA 422 solid waste 32 18 0.43 111
Lc. lactis sp. lactis NRRL B-4449 solid waste 32 6.6 0.16 111
Ent faecium hydr cod corn syrup 37 11 0.45 0.10 176
Lb. plantarum hydr cod corn syrup 37 17 0.70 0.10 176
Ped. acidilacti hydr cod corn syrup 37 13 0.51 0.11 176

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.
 K. Hofvendahl, B. HahnHagerdal / Enzyme and Microbial Technology 26 (2000) 87107
3. Parameters affecting efficiency
3.1. Microorganism
Strains of lactobacilli were compared with regard to the
fermentation of various sugars (Table 2). Strains giving the
highest LA concentration and yield usually also showed the
highest productivity, but in some reports different strains
gave the highest yields and productivities [49 51]. Lb.
delbrueckii emerges as the most efficient strain.
On lactose, including whey and milk, Str. thermophilus
was in most studies superior to Lb. delbrueckii spp. bulgari-
cus and Lc. lactis, but not to Lb. acidophilus (Table 2). Lc.
lactis, on the other hand, gave higher LA concentrations and
yields than Lb. rhamnosus [52,53], and sometimes also a
higher productivity [53].
In wheat flour hydrolysate Lc. lactis showed the highest
productivity, whereas Lb. delbrueckii spp. delbrueckii re-
sulted in the highest LA concentration and yield (Table 2)
[18]. Generally the temperature used was the same for all
strains, but in some studies it was adjusted to the optimum
for each organism.
Metabolic engineering and traditional strategies for mu-
tation and selection can be used to alter the properties of
an organism. Lb. delbrueckii has been subjected to mu-
tagenesis to enhance its tolerance to LA (Table 2) [49].
The mutants resulted in better LA production than the
wild type.
3.2. Carbon source
A number of different substrates have been used for the
fermentative production of LA by LAB. The purest product
is obtained when a pure sugar is fermented, resulting in
lower purification costs. However, this is economically un-
favorable, because pure sugars are expensive and LA is a
cheap product. Instead waste products from agriculture and
forestry are utilized.
3.2.1. Whey
The most common substrate for fermentative LA pro-
duction is whey [54 59], a waste product from cheese
production normally used as animal feed [60]. It contains
proteins, salts, and lactose [60]. Whey can be hydrolyzed to
glucose and galactose [61,62], deproteinised by ultrafiltra-
tion [63 66], and demineralised [67]. Whey has been
supplemented with yeast extract [68 71], peptone [72,
73], milk powder [33,74], soy flour [36], or corn steep
liquor [75]. The strain most used for LA production from
whey was Lb. delbrueckii spp. bulgaricus [76 78], but in
many studies Lb. helveticus [79 81] or Lb. casei [82,83]
were utilized.
3.2.2. Molasses
Molasses, a by-product of the sugar manufacturing
process, is used as an animal feed, and for ethanol and
91
Fig. 2. A process of lactic acid production from wheat flour with simulta-
neous (SSF) (A) and separate (B) saccharification and fermentation. Pro-
cess parameters according to references [18,20,106].
yeast production [60]. In addition it could be used for LA
production [84 89]. The most abundant sugar is sucrose,
the high concentration of which makes the viscosity of
the liquid high [60]. Lb. delbrueckii has generally been
used.
3.2.3. Starch
Another common substrate for LA production is starch
from crops or wastes [18,90 92]. It has to be hydrolyzed to
glucose and maltose to be fermentable by LAB. Starch from
various origins has been used for LA production, including
wheat [20,93], corn [94,95], cassava [96], potato [97,98],
rice [93], rye [35], sorghum [99,100], and barley [95,101].
In some studies, starch either remained untreated or was
liquefied/gelatinized and then fermented by an amylase-
producing organism such as Lb. fermentum [98], Lb. amy-
lovorus [102,103], or Lb. amylophilus [104,105]. Amylases
can also be added to hydrolyze the starch and LAB fer-
mented the produced glucose to LA [99,106,107]. In one
study, amylase was produced by Aspergillus awamori and
Lc. lactis made LA from the glucose produced [108]. LAB
used for LA production from starch include: Lb. casei [92],
Lb. plantarum [109], Lb. delbrueckii [97,107], Lc. lactis
[18,108], and Lb. helveticus [28].
In the above mentioned reports hydrolysis was per-
formed simultaneously with fermentation (SSF) (Fig. 2A).
Another solution is to use pre-hydrolyzed starch as a sub-
strate [20,97]. This is an advantage if the saccharifying
enzymes and the bacterium have different temperature and
pH optima. This was the case in the fermentation by Lc.
lactis of wheat starch hydrolyzed by a commercial enzyme
preparation composed of - and gluco-amylase and a pro-
tease, with 30C, pH 6 and 55C, pH 5 as optima respec-
tively (Fig. 2B) [18]. Starch was often supplemented with
nutrients, mostly in the form of yeast extract [18] or peptone
[102]. In a few studies, the starchy material was considered
to be sufficient as nutritional source [18,20,92,95,97,98,104,
106,109]. In SSF of whole wheat flour (WWF) with Lc.
92 K. Hofvendahl, B. HahnHagerdal / Enzyme and Microbial Technology 26 (2000) 87107

Table 3
Influence of carbon source and substrate treatment on lactic acid production

Organism Substrate and treatment LA YLA/tot Qv Ref

g/l g/g g/(lh)

Lb. amylophilus ATCC 49845 glucose 21 0.95 1.6 104

hydr corn starch 33 0.73 0.88
Lb. amylovorus ATCC 33620 cassava starch 4.8 0.48 0.69 93
corn starch 10 1.0 1.2
potato starch 4.2 0.42 0.14
rice starch 7.9 0.79 0.86
wheat starch 7.8 0.78 1.2
Lb. amylovorus ATCC 33622 raw corn starch 45 0.82 8.6 102
liq corn starch 55 1.0 20
Lb. casei NRRL B-441 liq barley starch glucoam 112 0.68 101
liq barley starch glucoam alpha 162 0.87
Lb. casei NRRL B-441 barley flour 36 0.20 1.1 92
Lb. amylovorus NRRL B-4542 barley flour glucoam 114 0.63 2.0
Lb. delbrueckii IFO 3534 hydr newspaper 24 0.48 0.85 114
hydr pure cellulose 53 0.53 0.60
Lb. delbrueckii sp. bulgaricus CBS 743.84 glucose 35 0.85 33
lactose 37 0.82
Lb. delbrueckii sp. bulgaricus CNRZ 369 glucose 56 2.8 159
cellobiose 32 1.6
xylose 41 2.1
Lb. delbrueckii sp. delbrueckii glucose 87 0.87 5.5 118
fructose glucose 94 0.94 5.5
sucrose 85 0.85 6.2
Lb. delbrueckii sp. delbrueckii ATCC 9649 glucose 58 0.85 177
lactose 40 0.75
Lb. DSM 20605 MONT4, plasmid xylose 14 0.70 30
X1 XK reg
xylose glucose 11 0.55
Lb. helveticus sp. milano glucose 18 0.36 4.2 120
maltose 42 0.84 5.0
Lb. paracasei No 8 glucose 95 0.95 5.6 99
sweet sorghum 91 0.91 10
Lb. pentosus glucose 46 0.92 2.4 67
xylose 27 0.54 0.59
glucose xylose 90 1.8 4.0
hydr wood 40 0.70 1.3
Lb. pentosus NRRL B-227 glucose 6.0 0.60 111
galactose 7.4 0.74
mannose 6.8 0.68
hydr cellulose: glu, man, xyl, gal 0.51
Lb. pentosus NRRL B-473 glucose 6.9 0.69 111
galactose 5.9 0.59
mannose 7.4 0.74
xylose 1.4 0.14
hydr cellulose: glu, man, xyl, gal 0.43
Lb. plantarum hydr soluble starch 15 0.30 96
hydr tapioca starch 15 0.30
hydr tapioca flour 17 0.35
Lb. plantarum NRRL B-531 glucose 5.4 0.54 111
galactose 3.7 0.37
mannose 5.7 0.57
hydr cellulose: glu, man, xyl, gal 0.43
Lb. plantarum NRRL B-787 glucose 6.2 0.62 111
galactose 4.0 0.40
mannose 6.6 0.66
hydr cellulose: glu, man, xyl, gal 0.42
Lb. plantarum NRRL B-788 glucose 6.0 0.60 111
galactose 4.9 0.49
hydr cellulose: glu, man, xyl, gal 0.46
 K. Hofvendahl, B. HahnHagerdal / Enzyme and Microbial Technology 26 (2000) 87107 93

Table 3 (continued)

Organism Substrate and treatment LA YLA/tot Qv Ref

g/l g/g g/(lh)

Lb. plantarum NRRL B-813 glucose 7.3 0.73 111

galactose 4.7 0.47
mannose 8.3 0.83
hydr cellulose: glu, man, xyl, gal 0.43
Lb. plantarum USDA 422 glucose 5.2 0.52 111
galactose 3.1 0.31
mannose 6.2 0.62
xylose 1.3 0.13
hydr cellulose: glu, man, xyl, gal 0.43
Lb. rhamnosus ATCC 10863 glucose 17 0.86 88
fructose 14 0.71
glucose fructose 16 0.81
sucrose 15 0.73
Lb. rhamnosus ATCC 10863 alpha-cellulose 45 0.96 112
switch grass cellulose 28 0.50
LBM5 mix of 5 Lb strains glucose 99 0.90 28
starch 90 0.82
Lc. lactis IO-l JCM 7638 xylose 23 0.45 0.30 160
xylose glucose 28 0.70 2.2
Lc. lactis sp. lactis ATCC 13673 glucose 36 1.0 3.6 161
xylose 13 0.42 0.37
Lc. lactis sp. lactis ATCC 19435 glucose 4.9 0.86 2.5 25
maltose 3.2 0.70 1.0
Lc. lactis sp. lactis ATCC 19435 hydr wheat flour, 3 l enz/g starch 75 0.78 1.2 196
hydr wheat flour, 5 l enz/g starch 75 0.83 0.85
hydr wheat flour, 6 l enz/g starch 90 0.98 1.5
hydr wheat flour, 8 l enz/g starch 87 0.93 1.7
Lc. lactis sp. lactis NRRL B-4449 glucose 6.6 0.66 111
galactose 2.8 0.28
mannose 5.8 0.58
xylose 1.8 0.18
hydr cellulose: glu, man, xyl, gal 0.16

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 over-
come 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 Tricho-
derma reesei has also been reported [117].
3.2.5. Effectiveness
Comparing different carbon sources showed that glu-
cose resulted in higher LA concentrations and yields
compared with other sugars (Table 3). Xylose, galactose,
arabinose, lactose, fructose, and hydrolyzed cellulose
were less effective. However, mannose gave a higher LA
concentration and higher or the same yield as glucose
[111]. The simultaneous utilization of glucose and fruc-
tose was more effective than glucose alone with Lb.
delbrueckii [118], whereas the opposite was true for Lb.
rhamnosus [88]. When comparing glucose and maltose
fermentation by Lc. lactis, glucose resulted in higher LA
concentration, yield and productivity than maltose (Table
3) [21,25,119]. The main reason is that on maltose Lc.
lactis gives mixed acids, whereas on glucose the fermen-
tation is almost homofermentative [25]. Lb. helveticus,
on the other hand, showed higher values on maltose than
on glucose (Table 3) [120]. Amylase addition to starch
also resulted in enhanced LA production (Table 3) [20,
92,101], by increasing the concentration of sugar and
nutrients [25]. Also, increased amylase concentrations
increased the LA concentration, yield and productivity
(Table 3) [106].
The concentration of the substrate had no large effect on
yield and productivity [25].
94 K. Hofvendahl, B. HahnHagerdal / Enzyme and Microbial Technology 26 (2000) 87107

Table 4
Influence of nutrient type and concentration on lactic acid production

Organism Substrate and treatment Nutrients LA YLA/tot Qv Ref

g/l g/g g/(lh)

Lactobacterium delbrueckii sowjeskij sucrose 20% YE 21 1.1 1.0 122

sucrose 1% YE 0.5% pep 18 0.90 0.83
Lb. acidophilus R whey 13 0.22 174
whey YE 20 0.34
Lb. casei NRRL B-441 glucose malt sprouts 93 0.93 2.3 146
glucose YE 96 0.96 3.9
Lb. delbrueckii sp. bulgaricus ATCC 11842 hydr wheat flour, SSF 18 0.11 0.56 18
hydr wheat flour, SSF YE 26 0.18 0.9
Lb. delbrueckii sp. delbrueckii ATCC 9649 glucose MRS 1% YE 58 0.48 0.72 49
glucose MRS 3% YE 67 0.56 1.4
Lb. delbrueckii sp. delbrueckii ATCC 9649 hydr wheat flour, SSF 106 0.82 1.6 18
hydr wheat flour, SSF YE 109 0.91 3.6
Lb. delbrueckii sp. lactis ATCC 12315 hydr potato 100 1.0 28
hydr potato waste CSL 93 0.78
Lb. helveticus ATCC 15009 lactose MRS 17 0.38 178
whey 8.9 0.20
Lb. helveticus Milano whey permeate YE 36 0.75 5.8 123
whey permeate YE higher conc 36 0.75 9.4
whey permeate YE pep 40 0.83 12
Lb. helveticus sp. milano hydr whey YE 44 5.5 124
hydr whey, clarified CSL 41 4.4
whey, UF CSL 37 2.7
Lb. IMET 11466 glucose MRS 93 0.95 35
hydr rye 5% MRS 92 0.92
Lb. kefir whey 9.8 0.20 174
whey YE 14 0.28
Lb. paracasei No 8 sweet sorghum 106 0.79 10 99
sweet sorghum YE pep 91 0.91 10
Lb. pentosus glucose YE 45 0.90 2.3 67
glucose MRS 46 0.92 2.4
Lb. plantarum ATCC 14917 sorghum 5% vetch juice 2.2 100
sorghum 15% vetch juice 2.0
sorghum 25% vetch juice 2.8
Lb. rhamnosus ATCC 10863 hydr molasses, SSF 16 0.81 88
hydr molasses, SSF YE pep 14 0.70
Lb. rhamnosus ATCC 10863 glucose 0.25% YE 0.5% trp 57 0.81 156
glucose 0.5% YE 1% g/trp 58 0.95
Lb. rhamnosus ATCC 10863 hydr wood YE pep 27 0.96 2.3 115
hydr wood, SSF YE pep 29 1.0 1.5
Lb. rhamnosus ATCC 11443 glucose 0.4% YE 53 0.66 2.8 179
glucose 0.8% YE 53 0.66 3.7
Lb. rhamnosus ATCC 7469 glucose 25 0.83 0.2 180
glucose 0.2% YE 34 1.1 0.5
glucose 1% YE 26 0.81 2.6
Lb. salivarius sp. salivarius ATCC 11742 soy 4.2 0.76 87
soy molasses YE 5.5 0.85
Lc. lactis IFO 12007 Aspergillus awamori IFO 4033 potato starch 25 0.50 0.72 108
potato starch YE trp 10 0.20 0.43
Lc. lactis JO-l JCM 7638 glucose, cont substr feed 28 0.56 2.0 143
glucose, pH dep substr feed 34 0.68 2.1
Lc. lactis IO-l JCM 7638 glucose 0.3% YE 24 0.96 1.2 181
glucose 0.5% YE 37 0.74 2.1
glucose 1% YE 43 0.86 2.3
Lc. lactis sp. lactis AS211 hydr wheat flour, SSF 95 0.77 1.7 18
hydr wheat flour, SSF YE 107 0.91 2.4
Lc. lactis sp. lactis ATCC 19435 hydr wheat flour, SSF 96 0.76 3.0 18
hydr wheat flour, SSF YE 106 0.88 3.3
Lc. lactis sp. lactis ATCC 19435 hydr wheat flour, SSF 90 0.98 1.5 106
hydr wheat flour, SSF YE 87 0.96 3.3
Lc. lactis sp. lactis ATCC 19435 unhydr wheat flour glucose 75 1.0 2.1 20
unhydr wheat flour
hydr wheat flour 80 1.0 4.0
 K. Hofvendahl, B. HahnHagerdal / Enzyme and Microbial Technology 26 (2000) 87107 95

Table 4 (Continued)

Organism Substrate and treatment Nutrients LA YLA/tot Qv Ref

g/l g/g g/(lh)

Lc. lactis sp. lactis ATCC 19435 hydrwheatflourprotease 43 1.5 106

hydrwheatflourprotease vitamins 46 2.4
hydrwheatflourprotease amino acids 53 2.8
hydrwheatflourprotease peptides 44 2.2
hydrwheatflour 17 0.23

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 Substrate LA YLA/tot Qv Ref

mode g/l g/g g/(lh)

Ent. faecium batch alfalfa 27 0.91 130

semicont alfalfa 30 0.99
Lb. casei L100 batch, imm corn starch 50 0.83 94
cont, imm corn starch 40 0.67
Lb. casei SU No 22 batch whey 22 0.44 129
fed-b whey 45 0.45
Lb. delbrueckii IFO 3534 batch glucose 83 0.83 1.5 127
cont glucose 55 0.55 5.3
Lb. delbrueckii MIX several strains batch hydr maize barley 85 0.87 95
cont hydr maize barley 71 0.73
Lb. delbrueckii NCIM-2365 batch, imm glucose 90 0.9 131
cont, imm glucose 75 0.75
Lb. delbrueckii sp. bulgaricus batch whey 44 0.95 182
cont whey 13 0.28
Lb. delbrueckii sp. bulgaricus ATCC 11842 batch, imm whey 50 1.0 0.65 138
cont, imm whey 9.5 0.19 12
Lb. delbrueckii sp. bulgaricus Ch H 2217 batch whey 115 0.86 183
cont whey 117 0.87
Lb. delbrueckii sp. bulgaricus NRRL B-548 batch lactose 45 0.90 11 134
cont lactose 39 0.78 2.2
Lb. helveticus ATCC 15009 batch whey 49 1.1 1.3 116
cont whey 48 1.2 2.7
Lb. helveticus L89 batch whey 3.1 184
cont, imm whey 29
Lb. helveticus NCDO 1844 batch, dial whey 47 1.2 43
cont, dial whey 125 3.1
Lb. rhamnosus ATCC 10863 batch sucrose 77 0.73 1.7 185
cont, extract sucrose 80 0.74 8.0
Lb. rhamnosus ATCC 10863 batch glucose 80 0.89 5.1 141
cont, recirc, glucose 47 0.48 4.2
extract
Lb. rhamnosus ATCC 10863 batch glucose 38 0.76 186
cont glucose 10 0.20
Lc. lactis 65.1 batch glucose 39 0.75 187
cont glucose 28 0.56
Lc. lactis IFO 12007 batch, imm potato starch 25 0.50 0.72 108
cont, imm potato starch 10 0.20 0.43
Lc. lactis sp. lactis ATCC 19435 batch, extract glucose 0.29 0.3 126
rep b, extract glucose 0.50 0.4
Str. thermophilus batch lactose 40 7.1 125
fed-b lactose 39 1.4

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 K. Hofvendahl, B. HahnHagerdal / Enzyme and Microbial Technology 26 (2000) 87107

Table 6
Influence of cell recirculation on lactic acid production

Organism Fermentation Substrate LA YLA/tot Qv Ref

mode g/l g/g g/(lh)

Lb. casei cont, imm whey 22 0.44 7.3 72

cont, imm, recirc whey 28 0.57 9.4
Lb. rhamnosus ATCC 10863 cont glucose 26 0.65 142
cont, recirc glucose 32 0.80
Lb. rhamnosus ATCC 10863 cont glucose 17 0.68 5.1 141
cont, recirc glucose 47 0.48 4.2
Lc. lactis IO-l JCM 7638 cont glucose 27 0.54 3.4 143
cont, recirc glucose 45 0.90 10

Abbreviations as in Table 5 and: recirc recirculation of cells.

3.3. Nitrogen source
The medium composition has been investigated from
many aspects, including the addition of various concentra-
tions of nutrients in the form of e.g. yeast extract, peptone
or corn steep liquor [28,69]. The addition of nutrients and
higher nutrient concentrations generally had a positive ef-
fect on the LA production (Table 4). MRS medium, which
contains yeast extract, peptone and meat extract, was supe-
rior to yeast extract, which in turn was better than malt
extract. This reflects the complex nutrient demands of LAB,
being fastidious because of limited biosynthesis capacity
[121]. Yeast extract alone at high concentration gave higher
LA production than yeast extract and peptone in low
amounts [122], but the opposite resulted when the concen-
tration of yeast extract was kept constant and peptone was
added [123]. Whey treatment also affects the outcome of
fermentation (Table 4) [124]. WWF did not supply enough
nutrients for Lc. lactis [20]. This was overcome by adding
hydrolyzing enzymes, - and gluco-amylase and a protease,
releasing nutrients in forms of amino acids from the flour
[106].
3.4. Fermentation mode
LA is most commonly produced in the batch mode [111],
but numerous examples of continuous culture exist [90], as
well as some fed-batch [125] and semicontinuous/repeated
batch fermentations [126] (Table 5). When comparing batch
and continuous fermentation modes, the former gave higher
LA concentrations and yields in most of the studies (Table
5) [94]. This is mainly due to that all substrate is used in the
batch mode, whereas a residual concentration remains in the
continuous one. On the other hand, the continuous mode
generally resulted in higher productivities (Table 5) [127].
The major reason is probably that the continuous cultures
were run at a high dilution rate, where the advantage over
the batch mode is most pronounced [128]. Varying the dilution
rate (D) in continuous culture affects both the substrate and
nutrient concentrations. However, the effects on the yields and
productivities were inconclusive [25]. Fed-batch [129], semi-
continuous [130] and repeated batch mode [126] gave higher
yields than the batch mode (Table 5).
3.5. Immobilization and recirculation of cells
LAB cells can be recirculated or immobilized/supported
by solids in different ways to increase cell density (Table 6).
Immobilization of cells has not been very successful in
terms of increasing the LA yield and productivity [42,64,
67,73,131140]. In about half of the studies better results
were obtained using free cells. On the other hand, recircu-
lation of cells gave higher LA concentrations and higher or
equal yields (Table 6) [72,141143].
3.6. pH
The fermentation pH is either set at the beginning and
then left to decrease due to acid production, or it is con-
trolled by base titration, or by extraction, adsorption, or
electrodialysis of LA. The effect of pH has been studied by
fermenting at various pH values (Table 7). In all cases,
titration to a constant pH resulted in higher or equal LA
concentration, yield and productivity in comparison with
no pH control (Table 7) [130]. Removing LA by elect-
rodialysis and extraction, including aqueous two-phase
systems, were successful in some of the studies [42],
whereas in others titration gave the same or better results
[144]. The optimal pH for LA production varies between
5.0 and 7.0. A pH below 5.7 was only optimal for Lb.
strains, which are known to tolerate lower pH than lac-
tococci [145].
3.7. Temperature
The effect of temperature on the production of LA has
only been studied in a few reports (Table 8). The tempera-
ture giving the highest productivity was in some cases lower
than the temperature resulting in highest LA concentration
and yield [105,146], whereas in others the same temperature
gave the best results in all categories [20,146]. For Lb.
amylophilus, which is known to grow at 15C but not at
 K. Hofvendahl, B. HahnHagerdal / Enzyme and Microbial Technology 26 (2000) 87107 97

Table 7
Influence of initial pH and pH control on lactic acid production

Organism Substrate pH pH control LA YLA/tot Qv Ref

g/l g/g g/(lh)

Ent. faecium alfalfa 6.2 init 8.3 0.27 130

alfalfa 5.8 NH4OH 27 0.90
Lb. amylophilus ATCC 49845 glucose 5.4 NaOH titr 0.70 104
glucose 6.0 NaOH titr 1.6
glucose 6.5 NaOH titr 1.2
glucose 7.1 NaOH titr 1.0
glucose 7.8 NaOH titr 0.90
Lb. delbrueckii IAM 1928 glucose 4.2 NaOH 10 0.12 1.0 144
glucose 6.2 NaOH 36 0.42 6.5
glucose 6.2 init, extract 27 0.32 2.9
Lb. delbrueckii IFO 3534 glucose 5.6 0.06 2.3 127
glucose CaCO3 55 0.55 5.3
glucose 5.5 titr, dial 53 0.53 5.3
Lb. delbrueckii IFO 3534 glocose 5.0 NaOH titr 81 0.79 2.4 164
glucose 5.5 NaOH titr 92 0.89 1.9
glucose 5.5 titr, dial 88 0.81 4.3
glucose 6.0 NaOH titr 81 0.78 4.5
glucose 6.5 NaOH titr 54 0.52 1.5
glucose 7.0 NaOH titr 49 0.47 1.4
Lb. delbrueckii IFO 3534 cellulose 4.2 CaCO3 15 0.30 114
cellulose 5.0 CaCO3 26 0.52
cellulose 5.9 CaCO3 18 0.36
Lb. delbrueckii MIX several strains hydr 5.0 Na2CO3 59 0.61 95
maize barley
hydr 5.5 Na2CO3 87 0.90
maize barley
hydr 5.8 Na2CO3 85 0.87
maize barley
Lb. delbrueckii sp. bulgaricus ATCC 11842 sorghum 5.5 NH3 3.5 100
sorghum 6.0 NH3 4.5
sorghum 6.5 NH3 2.3
Lb. delbrueckii sp. bulgaricus ATCC 55163 whey 5.4 NH4OH 35 0.45 36
whey 6.0 NH4OH 50 0.64
Lb. delbrueckii sp. bulgaricus CNRZ 369 whey 3.5 init 25 0.48 159
whey 4.5 init 25 0.48
whey 5.5 init 31 0.60
whey 6.5 init 35 0.67
Lb. delbrueckii sp. bulgaricus NRRL B-548 lactose 4.5 NH4OH titr 25 0.50 0.68 134
lactose 5.0 NH4OH titr 45 0.90 3.1
lactose 5.6 NH4OH titr 45 0.90 11
Lb. delbrueckii sp. bulgaricus NRRL B-548 cellulose 4.2 NH4OH 27 0.27 0.23 117
cellulose 5.0 NH4OH 52 0.58 0.43
cellulose 5.8 NH4OH 33 0.33
Lb. helveticus NCDO 1844 whey 5.6 NaOH titr 31 0.78 1.3 43
whey 5.6 NaOH titr, dial 47 1.2 1.1
Lb. plantarum ATCC 14917 sorghum 5.5 NH3 2.1 100
sorghum 6.0 NH3 2.0
sorghum 6.5 NH3 2.8
sorghum 7.0 NH3 5.1
sorghum 7.5 NH3 1.9
Lb. rhamnosus ATCC 10863 sucrose 6.0 NaOH titr 77 0.73 1.7 185
sucrose 6.0 NaOH titr, extract 80 0.74 8.0
Lb. rhamnosus ATCC 10863 glucose 5.0 NaOH 65 0.65 2.3 153
glucose 5.5 NaOH 78 0.78 3.9
glucose 6.0 NaOH 79 0.79 4.9
glucose 6.5 NaOH 78 0.78 4.9
Lb. rhamnosus ATCC 10863 glucose 4.2 yes 25 0.91 2.5 188
glucose 4.2 yes, extract 19 0.79 0.91
Lb. rhamnosus ATCC 10863 gtlucose 6.0 NH4OH 71 1.3 42
glucose 6.0 extract 771 5.4
Lb. rhamnosus ATCC 10863 cellulose 4.3 NH4OH 45 0.31 112
cellulose 4.3 NH4OH, extract 28 0.96
98 K. Hofvendahl, B. HahnHagerdal / Enzyme and Microbial Technology 26 (2000) 87107

Table 7 (continued)

Organism Substrate pH pH control LA YLA/tot Qv Ref

g/l g/g g(lh)

Lb. rhamnosus ATCC 10863 glucose 5.5 NH4 16 132

glucose 6.3 NH4 23
glucose 7.5 NH4 17
Lb. rhamnosus ATCC 7469 glucose 6.2 init 7.5 0.21 0.30 180
glucose 6.2 CaCO3 26 0.81 2.6
Lb. salivarius sp. salivarius ATCC 11742 soy molasses 5.6 NaOH titr 5.5 0.85 87
soy molasses 6.0 NaOH titr 5.4 0.79
soy molasses 6.4 NaOH titr 4.9 0.82
Lc. lactis 65.1 glucose 6.5 init 5.1 1.0 189
glucose 6.5 init, extract 5.7 1.1
Lc. lactis 65.1 glucose 6.5 init 3.5 0.18 0.87 187
glucose 6.5 NaOH 19 0.81
glucose 6.5 init, extract 14 0.70 1.7
Lc. lactis IO-l JCM 7638 glucose 6.0 NaOH 45 0.90 10 143
glucose 6.0 NaOH, deal 35 0.70 15
Lc. lactis IO-l JCM 7638 glucose 6.0 yes 60 0.80 3.0 190
glucose 6.0 yes, dial 66 0.88 4.0
Lc. lactis IO-l JCM 7638 glucose 6.0 init, dial 60 0.75 2.4 191
glucose 6.0 NaOH, dial 60 0.75 5.1
Lc. lactis IO-l JCM 7638 glucose 6.0 NaOH 50 0.85 136
glucose 6.0 NaOH, dial 62 0.88
Lc. lactis sp. lactis ATCC 19435 glucose 5.0 NaOH titr 5.4 0.92 1.7 25
glucose 5.8 NaOH titr 5.3 0.90 3.4
glucose 6.5 NaOH titr 4.9 0.86 2.5
Lc. lactis sp. lactis ATCC 19435 maltose 5.0 NaOH titr 5.1 1.0 0.37 25
maltose 5.8 NaOH titr 4.2 0.82 1.2
maltose 6.5 NaOH titr 3.2 0.70 1.0
Lc. lactis sp. lactis ATCC 19435 hydr wheat 6.0 init 3.3 0.02 0.47 18
flour
hydr wheat 6.0 NaOH 96 0.76 3.0
flour
Lc. lactis sp. lactis ATCC 19435 hydr wheat 4.0 NaOH titr 7.0 0.041 0.23 20
flour
hydr wheat 5.0 NaOH titr 20 0.11 0.42
flour
hydr wheat 6.0 NaOH titr 105 0.58 2.9
flour
Lc. lactis sp. lactis biovar diacetylactis CNRZ 2125 lactose citrate 5.0 NaOH titr 7.0 0.13 0.83 152
lactose citrate 5.5 NaOH titr 37 0.71 1.9
lactose citrate 6.0 NaOH titr 37 0.71 4.5
lactose citrate 6.5 NaOH titr 38 0.73 7.7

Abbreviations as in Table 5 and: init initial pH set, then uncontrolled; titr titration.

45C [147], the optimal temperatures were 25 and 35C for
maximum productivity and yield, respectively [105]. For
Lb. casei and Lb. paracasei the optimal temperature was
reported to be between 37 and 44C [99,101,146], which is
contradictory to the information that the strains grow at 15
but not at 45C [147]. In agreement with previous observa-
tions [147,148], Lc. lactis and Lb. rhamnosus exhibited the
highest yields and productivities at 33 to 35C [20] and 41
to 45C [146], respectively.
4. Other effects on the processes
4.1. By-product formation
In addition to LA, LAB produce by-products, including
acetic acid, formic acid, carbon dioxide, and ethanol, de-
pending on the metabolic pathway used. For efficient indus-
trial 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 by-
products, 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
 K. Hofvendahl, B. HahnHagerdal / Enzyme and Microbial Technology 26 (2000) 87107 99

Table 8
Influence of temperature on lactic acid production

Organism Temp Substrate LA YLA/tot Qv Ref

C g/l g/g g/(lh)

Lb. amylophilus ATCC 49845 25 starch 26 0.52 0.54 105

28 starch 29 0.58 0.44
35 starch 30 0.60 0.33
Lb. casei NRRL B-441 30 glucose 80 0.89 3.2 146
37 glucose 80 0.89 5.6
41 glucose 82 0.91 5.6
45 glucose 42 0.47 1.2
Lb. casei NRRL B-441 37 hydr barley starch 140 0.98 101
41 hydr barley starch 117 0.82
Lb. paracasei No 8 30 sweet sorghum 1.5 99
36 sweet sorghum 1.9
44 sweet sorghum 2.2
Lb. rhamnosus ATCC 10863 30 glucose 67 0.74 3.3 146
37 glucose 70 0.78 3.3
41 glucose 68 0.76 3.5
45 glucose 75 0.83 3.3
Lc. lactis sp. lactis ATCC 19435 30 glucose 4.9 0.86 2.5 25
35 glucose 5.2 0.88 2.9
37 glucose 5.2 0.88 1.8
40 glucose 1.2 0.20
Lc. lactis sp. lactis ATCC 19435 30 maltose 3.2 0.70 1.0 25
35 maltose 3.7 0.73 1.2
37 maltose 4.0 0.80 1.1
Lc. lactis sp. lactis ATCC 19435 30 glucose 60 1.3 2.2 20
34 glucose 65 1.5 2.8
37 glucose 60 1.5 2.3
40 glucose 50 1.2 1.5

Abbreviations as in Table 2.

Lc. lactis [23,24]. Pentose fermentation results in the pro-
duction of acetate or ethanol and LA in equimolar amounts,
and values of LA/tot of 0.57 0.79 have been reported (Ta-
ble 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 (Ta-
ble 9).
4.2. LA isomery
amount of nutrients was changed [36] (Table 10). The
composition of the racemate formed by Lb. plantarum
changed with aeration and amount of NaCl [149]. Com-
paring batch with continuous culture, the amount of the
predominant isomer was higher in the former [51]. The
amount of the predominant isomer also increased with
increasing pH [18,20] and amount of substrate [20], but
decreased with increasing temperature [20] and when the
pH was uncontrolled [18].

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 mix-
ture, the composition of which varies. The lactate dehy-
drogenase (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 con-
stant 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
4.3. Cell density
The highest cell densities (48 103 g/l) were achieved by
recirculation [53,79,113,142,154], but also fermentations
without recirculation resulted in 60 and 77 g/l cells [27,155,
156]. Fermentation by LAB is normally accompanied by
increased cell mass that constitutes an undesired by-product
if the aim of the process is LA production. On the other
hand, the bacteria are the primary product in the production
of probiotics. At pH below 6.5 and temperatures above
30C, lower cell mass and cell yield were determined for Lc.
lactis fermenting glucose or maltose [25]. Similar results
were obtained for Lb. delbrueckii [157]. Maltose [25], lac-
tose [158], and mannose [158] gave more cells than glucose,
whereas fructose [158], cellulose [159], and xylose [159
100 K. Hofvendahl, B. HahnHagerdal / Enzyme and Microbial Technology 26 (2000) 87107

Table 9
Effect of process parameters on by-product formation

Parameter Organism Fermentation Substrate Substr g/l/ pH/ LA HAc EtOH HFo LA/tot Ref
studied mode Nutrients TempC g/l g/l g/l g/l g/g

carbon, Lb. IMET 11466 batch glucose hydr rye 92 trace 35

nutr 1/20 MRS
batch glucose MRS 93 0 1.0
carbon Lb. rhamnosus ATCC fed-b, recirc alpha-cellulose 45 1.0 0.98 112
10863
fed-b, recirc switch grass 28 0.50 0.98
cellulose
carbon Lc. lactis sp. lactis ATCC batch glucose 4.9 0.10 0.065 0.20 0.93 25
19435
batch maltose 3.2 0.49 0.36 0.72 0.67
conc Lb. amylophilus ATCC batch starch 50 29 0 0 0 1.0 105
49845
batch starch 70 45 0 0 0 1.0
batch starch 100 53 0 0 0 1.0
conc Lc. lactis IO-l JCM 7638 batch xylose 29 12 9.1 0.57 150
batch xylose 49 25 9.7 0.72
batch xylose 130 22 7.9 0.74
batch xylose 160 27 7.3 0.70
conc Lb. helveticus cont lactose 37 35 1.0 present 56
cont lactose 127 48 1.5 present
nutr Lb. delbrueckii sp. batch starch hydr wheat flour 18 10 0.64 18
bulgaricus
ATCC 11842
batch starch hydr wheat flour YE 26 15 0.63
nutr, O2 Lb. plantarum H4 batch, aer glucosecitrate 3.2 3.1 0.51 149
batch, aer glucosecitrate NaCl 6% 4.2 1.7 0.71
batch, aer glucosecitrate NaCl 8% 4.2 0.79 0.84
batch, ana glucosecitrate 10.0 1.3 0.88
batch, ana glucosecitrate NaCl 6% 4.5 0.58 0.89
batch, ana glucosecitrate NaCl 8% 5.5 0.56 0.91
nutr, pH Lb. plantarum MOP 3 batch glufrumalate HAc 49 mM 4.1 9.1 0 0 1.0 151
batch glufrumalate HAc 20 mM NaCl 3% 4.5 8.6 0 0 1.0
batch glufrumalate HAc 20 mM NaCl 6% 4.5 6.6 0 0 1.0
batch glufrumalate NaCl 6% 5.5 7.3 0 0 1.0
batch glufrumalate HAc 0.7 mM 6.0 13 0 0 1.0
mode Lb. rhamnosus ATCC batch alpha-cellulose 13 0 1.0 112
10863
fed-b, recirc alpha-cellulose 45 1.0 0.98
recirc Lb. rhamnosus ATCC cont glucose 40 32 0.44 0.29 0.66 0.96 142
10863
cont, recirc 59% glucose 40 32 0.66 0.45 1.1 0.94
cont, recirc 79% glucose 40 31 0.90 0.64 1.5 0.91
cont, recirc 78% glucose 40 32 0.49 0.30 0.74 0.95
pH Lb. rhamnosus ATCC batch glucose 5.0 65 0.35 0.10 0.99 153
10863
batch glucose 5.5 78 0.33 0.40 0.99
batch glucose 6.0 79 0.35 0.40 0.99
batch glucose 6.5 78 0.39 0.51 0.99
pH Lc. lactis sp. lactis batch glucose 5.0 5.4 0.028 0.20 0 0.96 25
ATCC 19435
batch glucose 5.8 5.3 0.067 0 0.16 0.96
batch glucose 6.5 4.9 0.10 0.065 0.20 0.93
pH Lc. lactis sp. lactis batch maltose 5.0 5.1 0.098 0.065 0.19 0.94 25
ATCC 19435
batch maltose 5.8 4.2 0.33 0.24 0.54 0.79
batch maltose 6.5 3.2 0.49 0.36 0.72 0.67
pH Lc. lactis sp. lactis batch glucose 4.0 18 0.90 15 1.0 0.46 106
19435
batch glucose 5.0 68 2.0 21 0.90 0.93
batch glucose 6.0 56 1.5 2.4 0.60 0.93
 K. Hofvendahl, B. HahnHagerdal / Enzyme and Microbial Technology 26 (2000) 87107 101

Table 9 (continued)

Parameter Organism Fermentation Substrate Substr g/l/ pH/ LA HAc EtOH HFo LA/tot Ref
studied mode Nutrients TempC g/l g/l g/l g/l g/g

T Lc. lactis sp. lactis ATCC batch glucose 30 4.9 0.10 0.065 0.20 0.93 25
19435
batch glucose 35 5.2 0.074 0.080 0.18 0.94
batch glucose 37 5.2 0.085 0.050 0.11 0.95
batch glucose 40 1.2 0.030 0 0 0.98
T Lc. lactis sp. lactis ATCC batch maltose 30 3.2 0.49 0.36 0.72 0.67 25
19435
batch maltose 35 3.7 0.48 0.38 0.78 0.69
batch maltose 37 4.0 0.36 0.26 0.60 0.77
T Lc. lactis sp. lactis ATCC batch glucose 30 60 1.0 1.0 0.97 20
19435
batch glucose 34 65 1.5 1.5 0.96
batch glucose 37 60 6.0 4.0 0.86
batch glucose 40 50 7.5 6.0 0.79
T Lb. amylophilus ATCC batch starch 25 26 0 0 0 1.0 105
49845
batch starch 28 29 0 0 0 1.0
batch starch 35 30 0 0 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.

161] fermentation resulted in lower cell masses than glu-
cose.
5. Process considerations
The best fermentation conditions are not always the most
favorable for the whole process from an economical point of
view, because the costs of substrate and downstream pro-
cessing are proportionally high. The substrate is usually
a question of geographic availability. Wastes from, for
example, agriculture and forestry are preferable to ex-
pensive, pure sugars for the low-price product LA. In
fermentative production of LA, renewable resources that
do not contribute to the greenhouse effect can be utilized.
A disadvantage is the infections, as observed when WWF
was fermented by Lc. lactis [20]. In Northern Europe
wheat is a major crop, whereas corn is used in the USA
and tapioca in Africa. Fractions lacking suitable polymer
qualities to be used for other purposes can be given added
value. The mode of operation has to be chosen to release
nutrients available in the substrate, e.g. enzymatic hydro-
lysis of starchy material.
For lignocellulosic substrates, a strain fermenting pen-
toses as well as hexoses such as Lb. pentosus is required
to maximize the yield [111]. Starch can either be both
hydrolyzed and fermented by certain amylase-producing
Lb. strains, such as Lb. amylovorus [102], or, after being
hydrolyzed to glucose, fermented by a number of organ-
isms [18]. A homofermentative strain maximizes the pro-
portion of LA produced. In contrast to the synthetic
racemate, an optically pure product can be produced by
fermentation by choosing the proper organism and
growth conditions.
Batch fermentation was superior to continuous fermen-
tation 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 oper-
ation [128], whereas the volumetric productivity is maxi-
mized 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 contin-
uous 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 hy-
droxide, but the regeneration of LA results in the production
of large amounts of solid calcium sulfate [3]. Better alter-
natives are ammonia or calcium carbonate, leading to pro-
duction 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 K. Hofvendahl, B. HahnHagerdal / Enzyme and Microbial Technology 26 (2000) 87107

Table 10
Effect of process parameters on the isomeric form of LA produced

Parameter Organism Fermentation Substrate Substr g/l/ pH/ % L-LA Ref

studied mode Nutrients TempC

carbon, conc Lb. delbrueckii sp. bulgaricus CBS 743.84 batch whey UF perm 43 1 33
batch centr whey 93 7
batch centr whey 78 10
carbon, nutr, T Lb. delbrueckii sp. bulgaricus CBS 743.84 batch lactose whey milk YE 37 1 33
batch lactose YE trp 44 3.0
carbon Lb. delbrueckii sp. bulgaricus DSM 2129 batch glucose 0 34
batch lactose 0
conc Lb. amylophilus ATCC 49845 batch starch 50 93 105
batch starch 70 93
batch starch 100 93
conc Lb. rhamnosus ATCC 10863 batch glucose 50 95 156
batch glucose 70 95
conc, recirc Lb. rhamnosus ATCC 10863 cont, recirc 78% glucose 40 96 142
cont, recirc 96% glucose 80 97
conc Lc. lactis sp. lactis ATCC 19435 batch glucose 85 96 20
batch glucose 174 99
nutr Lb. delbrueckii sp. delbrueckii ATCC 9649 batch glucose YE 1% 0 49
batch glucose YE 3% 0
nutr Lb. delbrueckii sp. delbrueckii ATCC 9649 batch starch hydr wheat flour 94 18
batch starch hydr wheat flour YE 95
nutr Lb. delbrueckii sp. bulgaricus ATCC 11842 batch starch hydr wheat flour 91 18
batch starch hydr wheat flour YE 95
nutr Lb. delbrueckii sp. bulgaricus ATCC 55163 batch lactose whey YE 100 36
batch lactose whey soy flour 100
nutr, O2 Lb. plantarum H4 batch, aer glucose citrate 48 149
batch, aer glucose citrate NaCl 6% 44
batch, aer glucose citrate NaCl 8% 43
batch, ana glucose citrate 45
batch, ana glucose citrate NaCl 6% 36
batch, ana glucose citrate NaCl 8% 33
nutr Lb. rhamnosus ATCC 10863 batch glucose YE 0.25% trp 0.5% 95 156
batch glucose YE 0.5% trp 1% 95
batch glucose YE 0.75% trp 1.5% 95
batch glucose YE 1% trp 2% 95
batch glucose YE 1.5% trp 3% 95
batch glucose YE 2% trp 4% 95
nutr Lb. rhamnosus ATCC 7469 batch glucose 98 180
batch glucose YE 0.2% 98
batch glucose YE 1% 97
nutr Lc. lactis sp. lactis AS211 batch starch hydr wheat flour 94 18
batch starch hydr wheat flour YE 100
nutr Lc. lactis sp. lactis ATCC 19435 batch glucose hydr wheat flour 99 20
batch glucose unhydr wheat flour 98
nutr Lc. lactis sp. lactis ATCC 19435 batch starch hydr wheat flour 100 18
batch starch hydr wheat flour YE 100
mode Lb. delbrueckii sp. bulgaricus DSM 2129 batch glucose 0 34
cont glucose lactose 0
mode Lb. salivarius sp. salivarius ATCC 11742 batch glucose 90 51
cont glucose 86
recirc Lb. rhamnosus ATCC 10863 cont, recirc 51% glucose 96 142
cont, recirc 79% glucose 95
recirc Lb. rhamnosus ATCC 10863 cont, recirc 78% glucose 96 142
cont, recirc 96% glucose 97
pH Lb. delbrueckii sp. bulgaricus ATCC 55163 batch lactose 5.4 100 36
batch lactose 6.0 100
pH Lb. rhamnosus ATCC 10863 batch glucose 5.0 98 153
batch glucose 5.5 98
batch glucose 6.0 98
batch glucose 6.5 97
pH Lc. lactis sp. lactis ATCC 19435 batch starch 6.0 init 97 18
batch starch 6.0 100
 K. Hofvendahl, B. HahnHagerdal / Enzyme and Microbial Technology 26 (2000) 87107 103

Table 10 (continued)

Parameter Organism Fermentation Substrate Substr g/l/ pH/ % L-LA Ref

studied mode Nutrients TempC

pH Lc. lactis sp. lactis ATCC 19435 batch glucose 6.0 99 20

batch glucose 5.0 99
batch glucose 3.0 97
T Lb. amylophilus ATCC 49845 batch starch 25 93 105
batch starch 28 93
batch starch 35 93
T Lc. lactis sp. lactis ATCC 19435 batch glucose 30 99 20
batch glucose 34 90
batch glucose 37 96
batch glucose 40 82

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 fluidized-
bed 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 com-
bination 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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