Conversion of carbohydrates to lactic acid is one of the most employed fermentation processes in food industry. Applications of lactic acid fermentation are found in dairy industry, production of wine and cider, production of fermented vegetable products and meat industry.
The main markets for lactic acid have been in food, pharmaceutical and cosmetics industry, but presently the main growing application of lactic acid is in the production of biodegradable and renewable raw material based poly lactic acid (PLA) polymers. Production of lactate esters (
Production figure of 260,000 t as 100 % lactic acid for conventional (excluding PLA) markets in 2008 and forecast over 1 million ton annual production of lactic acid for conventional markets and PLA by 2020 has been presented in 2010 . DuPont patented PLA already in 1954 but it took almost 50 years before first large-scale production was started. The US-based NatureWorks is the largest producer of PLA having lactic acid production capacity of 180,000 t/a. The sustainability of the PLA product Ingeo® from NatureWorks has been evaluated . Greenhouse gas emissions and nonrenewable energy consumption for Ingeo from cradle to factory-gate are 1.3 kg CO2 eq./kg polymer and 42 MJ/kg polymer. These compare favorably with
The price of PLA is ca. 2.2 $/kg, the target being half of that . This means that the price of lactic acid in captive use should be less than 0.8 $/kg. A major cost factor is the raw material used in fermentation medium. This is especially the case with fastidious lactic acid bacteria. Processes based on cheap polymeric waste and side stream materials are indeed widely studied. So far research on alternative fermentation modes and reactor systems has been mainly academic. PLA production requires both optically and chemically pure lactic acid. Optical purity can be guaranteed with several microbial strains under optimized fermentation conditions. Chemical purity is mainly dependent on the constituents in the fermentation medium especially when cheap materials are being used. Contrary to many other fermentation products lactic acid yield on monosaccharides is usually very high (> 90 %) the main impurity being the cell mass itself, which is easily separated from the product. The key economic drivers in the fermentative production of lactic acid are optimization of the production medium, high product yields, productivity, and the concentration of products formed, which influences the down-stream processing costs .
Lactic acid bacteria (LAB) are a group of Gram-positive bacteria belonging to genera Aerococcus, Alloiococcus, Atopobium, Bifidobacterium, Carnobacterium, Enterococcus, Lactobacillus (Lb.), Lactococcus (L.), Leuconostoc (Leuc.), Oenococcus, Pediococcus, Streptococcus (S.), Tetragenococcus, Vagococcus and Weissella (W.). LAB are non-sporulating rods or cocci which produce lactic acid as the main fermentation product under suitable substrates. LAB are oxidase and benzidine negative, lack cytochromes, and do not reduce nitrates to nitrite . Most of the LAB are anaerobic, but some of them can shift to oxygen-dependent metabolism in aerobic conditions [10,11]. Lactic acid bacteria have complex nutrient requirements, including specific minerals, B vitamins, several amino acids, and purine and pyrimidine bases.
LAB ferment sugars via homo-, hetero-, or mixed acid fermentation. Homofermentative LAB produce lactic acid as main product from sugars, while hetero- or mixed acid fermentations produce also ethanol and/or acetic acid, formic acid and carbon dioxide. Although it is a common practice to divide LAB into homo- and heterofermentative strains, the division is not that straightforward as the actual metabolism is dependent on both the nature of the C/energy substrate (
Biotechnical production of lactic acid may be based on several alternative micro-organisms. In addition to lactic acid bacteria filamentous fungi (
In the wide literature on lactic acid production two examples based on other than lactic acid bacteria should be taken up. The first of them presents results with a thermotolerant
2. Future raw materials for production of lactic acid by LAB
The carboxylate platform is comprised of biological and chemical pathways that can be used in order to convert waste to bioproducts, such as lactic acid . Lactic acid is a relatively cheap product, and one of the major challenges in its large-scale fermentative production is the cost of the raw material. This is the situation even in case of so called low-cost substrates . Therefore, development of processes that utilize cheap raw materials at minimal costs have been under extensive studies. These substrates can be roughly classified as starch-based non-processed biomasses, lignocellulosic non-processed biomasses, and waste or side stream feedstocks. The former are nowadays generally considered as non-ideal feedstocks due to ethical reasons, and therefore they are not discussed in this review. Extensive reviews including starch-based feedstocks are available elsewhere . With respect to future applications, the most likely raw materials for the lactic acid production are industrial side-streams and lignocellulosic biomasses. Recent advances in case of both raw material groups are discussed in the following.
As in other bioconversion processes, also in lactic acid production the focus of research has turned towards the use of lignocellulosic feedstocks. The major driving forces are fossil fuel deprivation and general paradigm change to bioeconomy, and the abundancy of lignocellulose materials. Generally, the effective utilization of lignocellulosic biomass for biochemical processes is limited due to seasonal availability, scattered distributions and high logistics cost . The fermentation of lignocellulosic biomasses can also be hampered by inefficient pretreatment, high enzyme costs and end-product inhibition, formation of unwanted by-products under metabolism of pentoses, and carbon catabolite repression caused by the heterogeneous substrates. These challenges are further discussed in a recent review .
Paper industry residues and recycled paper products include various possible feedstocks for lactic acid production, which are together with agroindustrial residues discussed in a recent review . Due to economical and ecological reasons, an intensive research interest is currently devoted to complex industrial by-products. In this field the advances presented during the past five years include the utilization of cellulosic biosludges from a Kraft pulp mill [22,23], and recycled paper sludge . In both cases a nutrient supplement has increased the lactic acid productivity. LAB could be used for the bioconversion of hemicellulose fractions,
Food industry residues comprise a large variety of different biomasses and sludges that can be roughly categorized to agricultural wastes and food production wastes. Since the use of agricultural residues for lactic acid production is summarized in a recent review , it is not futher discussed here. Food production residues have been tested for bioconversion applications for ages, and the variety of used materials is large. Whey and other dairy industry residues are the prominent raw materials with respect to lactic acid production, reviewed in
Other quite often referred raw materials include brewery residues, especially spent grain , and winery wastes [31-35]. Additionally, there are various other proposed food industry residues that could fit to the lactic acid fermentation. The recently proposed include
The required supplements and their concentrations depend on the low-cost substrate. Drawbacks of complex supplements are their cost and extensive down-stream processing required for the purification of lactic acid from fermentation broth, especially in applications requiring high purity. Therefore, the optimization of supplement concentration is essential. Although yeast extract is often considered superior to other supplements in terms of efficiency, its major drawback is the relatively high cost, and therefore substitutive supplements have been suggested. Equal productivities may be achieved via use of cheaper alternatives, such as inorganic phosphates , and microbial lysates [46,47]. It is notable that the use of lysates in combination to
The modern biorefineries are looking into oceans in order to find new abundant and less land- or water-using biomasses for the production of commodities. Among the plenty marine biomasses, brown seaweed and especially species
3. Novel LAB strains
Metabolic engineering in general is applied when
Some of these studies are listed in a review on metabolic engineering for lactic acid production . The overexpression of L-LDH in
More straightforward work on lactic acid production has been performed to achieve high enantiomeric purity by expressing and deleting respective genes for LDH. There are several examples of these as discussed in the recent review , such as construction of two different strains of
Another straightforward target for the construction of genetically modified strains is widening of the raw materials for the production of lactic acid especially to lignocellulosic biomass-based materials. There are no reports on work to produce cellulolytic enzymes in LAB. Instead several groups have tried to produce xylanase in LAB . This is however focused on heterofermentative LAB as they are naturally able to utilize pentoses and especially
Typical LAB fermentations are run at minimum pH of 5 – 5.5, which is much higher than the pKa-value of lactic acid (
4. Novel process technologies
From fermentor design point of view lactic acid production by LAB is quite simple and conventional as the process requires no gassing, gas exchange or gas mass transfer. When the production strain and fermentation conditions are optimized for lactic acid production there is no or little formation of side products (metabolites, cellular mass, exopolysaccharides). Thus
Typical fermentation approaches other than simple batch include repeated batch and fed-batch fermentation and continuous fermentation with cell-recycle as solutions with free cells and the use of immobilized cells in different reactor types (fixed or fluidized bed). A novel fed-batch strategy was developed recently by combining pH-control and substrate feeding . The rationale behind the strategy was the linear relationship between the consumption amounts of alkali and that of substrate. Thus these two components were mixed together in the feeding liquid. This resulted in higher efficiency compared to batch fermentation, but the efficiency parameters were not especially high if compared with data from several other reports. By far the most studied method to increase the RP and/or separate cell growth from product formation is based on the immobilization of the cells. These have also been reviewed . Several immobilization methods have been applied including entrapment within gels such as alginate [77,78], modified alginate [79,80], or pectate , adsorption on granulated DEAE-cellulose  or porous glass , and biofilm formation on solid supports [84,85]. Solid incompressible supports and carrier materials such as granulated cellulose and porous glass may be applied in any scale and in various reactor designs while gels as compressible materials suit less well for larger scale especially in fixed-bed column reactors.
Immobilized cells may be utilized in various fermentation modes and reactor designs such as repeated batch or fed-batch, continuous fermentation with cell retention or recycle, in continuous stirred tank reactors (CSTR), fixed-bed or fluidized-bed reactors. High RP (19-22 g/lh) have been achieved in a two-stage process with immobilized cells . A special arrangement consisting of a CSTR for pH-control and substrate feeding and a fixed-bed reactor with immobilized cells was used in a concept with intermittent refreshing of the cells in a patent . Short residence time within the column was possible because of the incompressible nature of the carrier material. Chemically pure product was achieved by using a production medium with few nutrients. Once the productivity decreased below a threshold value based on the consumption of alkali the cells were refreshed with nutrients. Incompressible carriers for cell adsorption have obvious advantages. However, new solutions to secure cell adherence on the carrier are required. This would facilitate efficient use of fluidized-bed reactors with minimal pressure losses in the reactor. Biological means for cell adherence may be one solution which could offer a further advantage to selectively keep the productive cells in the reactor.
Another approach to increase RP is high cell-density fermentation with free cells recycled by membrane separation technique. This has been in use in industrial scale for lactic acid production already in 1980’s in Finland. Several academic reports on this approach have been since published demonstrating very high RP of 26 g/lh , 31.5 g/lh  and up to 57 g/lh .
It should be kept in mind that RP is affected by the concentration of lactic acid. Thus not all published figures are comparable. Product inhibition may be diminished by in-situ recovery of the product. Electrodialysis [91,92], nanofiltration  and ion-exchange [94,95] have thus been coupled with the fermentation system.
Conventional lactic acid recovery from fermentation broth consists of cell and other solids separation, lactic acid precipitation as calcium lactate and precipitate recovery, acidification of the precipitate by sulfuric acid and the separation of the gypsum precipitate formed. The amount of gypsum is usually higher than the amount of lactic acid produced. Lately NatureWorks has reported to have reduced the formation of gypsum significantly. Probably this has been achieved by performing the fermentation at lower pH
Lactic acid production in LAB has both cell mass and growth dependent portions. Typically LAB require several nutrient components for their growth increasing the fermentation and down-stream processing costs. Down-stream processing is especially important in the production of lactic acid for PLA. As RP is the a major investment factor affecting costs, the minimization of medium and product purification costs should be accompanied by methods increasing cell mass concentration without excess growth. For this several different strategies have been applied so far mainly in academia (cell immobilization, cell-recycling and cell-retention). As history shows some of these could be applicable in industrial production as well, however pilot and demonstration plant studies and some risk-taking are required.
The main C/energy source spectrum available for LAB has been widened significantly. Reports of new possible substrates are frequently published, and the utilization of industrial side streams is a growing trend. Into this direction major successes have also been achieved with metabolic engineering providing strains for efficient production of lactic acid from pentoses as well, which is to promote sustainable use of renewables.
In an ideal fermentation process product inhibition should be minimized so that high RP would be achieved even at high lactic acid concentrations resulting in feasible average productivities. For this purpose both acclimatization and mutagenesis has been applied successfully. However, it has to be considered how far can we go in respect to fermentation pH and lactic acid concentration. There are already remarkable alternatives to LAB with naturally better properties in this sense. Some success has been achieved with in-situ product recovery, but also these procedures lack experiences in any larger scale.
Conventional lactic acid production process with LAB is accompanied with the formation of large amounts of gypsum in the product recovery stage. Fermentation at lower pH diminishes this amount, but does not prevent its formation. Electrodialysis has been considered too expensive technique for the recovery of such cheap, bulk products as lactic acid. However, recent reports claim promising results with this technology. Forecasted figures for lactic acid market show up to one million tons per year. The growth would come mainly from the growth of PLA as a biodegradable polymer based on renewable raw materials. Economies of scale should decrease the production costs, but new technical approaches are also needed to reach these figures.
η % - Efficiency, i.e. the ratio of YP/S to the maximum theoretical value
D-LDH - D-lactate dehydrogenase
LAB – lactic acid bacteria
L-LDH - L-lactate dehydrogenase
NTG – Nitrosoguanidine
RP - Volumetric productivity g/l*h
SSF – Simultaneous saccharification and fermentation
PLA – poly lactic acid
PPP - Pentose phosphate pathway
PKP - Phosphoketolase pathway
YP/S – Yield of lactic acid per substrate consumed g/g
YP/X – Yield of lactic acid per cell mass g/g