Amongst the different options for the conversion of xylose reported in the previous chap­ter, production of lactic acid via the microbial route is a vastly studied field [61—63] since currently, all of the production of lactic acid at an industrial scale in the world is biologi­cally based. Traditionally, the concept evolves around fermenting carbohydrate-based syr­up by homolactic organisms, mostly lactic acid bacteria (LAB). The most common carbohydrate-based substrates used to this purpose may be molasses, corn syrup, whey, sugarcane or even beet bagasse. Highly efficient LAB includes Lactobacillus delbrueckii, L.

amylophilus, L. bulgaricus and L. leichmanii. Mutant Aspergillus niger has also been reported to be effective at an industrial scale [64]. LAB have the particularity to possess an homo­fermentative metabolism producing only lactic acid as extracellular waste product, instead of the heterofermentative pathway yielding by-products such as aldehydes, organic acids and ketones. The catabolic pathway yielding lactic acid is essentially the same across all organisms; the pyruvate intermediate is converted to lactic acid by a lactate dehydrogen­ase (LDH). Thus for hexose sugars, the theoretical yield is 2 moles of lactate per mole of sugar (or 1g sugar for 1g lactate). This enzymatic catalysis has the advantage over its chemical counterpart to be stereospecific: both L-lactate-dehydrogenase (L-LDH) and D — lactate-dehydrogenase (D-LDH) exist, generating either L-lactate or D-lactate respectively [65]. Both are NAD-dependant (nicotinamide adenine dinucleotide) and may be found alone or together in wild lactate-producing microbial strains. Since optical purity of lac­tate is a major requirement for the lactate industry, research focuses on stereospecificity as much as yields and productivity [61,66—70].

An efficient lactate producer has to display specific attributes, mainly the adaptability to low-cost substrates, high selectivity of desired enantiomer (L, D or both), high optimal tem­perature for decreased contamination risks, low pH tolerance and high performances (yield and productivity). LAB display appreciable performances, but lack a low pH tolerance, which implies uses of a pH control apparatus during the fermentation process. LAB optimal pH is near neutral, but the pKa of lactic acid being 3.8, an alkali agent, usually Ca(OH)2 must be used thus generating calcium lactate. After typical batch fermentation, the medium is acidified with H2SO4 therefore regenerating and purifying the lactic acid [64]. Another drawback of LAB is their requirement for a complex growth medium, since they are auxo — troph for certain amino acids and vitamins [71]. In order to overcome this problem, many fungi were also investigated for lactate production. Strains of Rhizopus, Mucor and Monilla sp. have shown potential whilst other fungi even displayed amylolytic activity, which could lead to a direct starch-to-lactate conversion [72—74].

Most researches still focuses on hexose conversion, and research group have optimized strains and process strategies in order to obtain high lactate titers, yields and productivities. Ding and Tan [75] developed a glucose fed-batch strategy using L. casei and generating up to 210 g/L of lactic acid with a 97 % yield. Chang et al. [76] proposed a continuous high cell density reactor strategy yielding a titer of 212.9 g/L and productivity of 10.6 g/L/h with Lb. rhamnosus. Dumbrepatil et al. [77] created a Lb. delbrueckii mutant by ultraviolet (UV) muta­genesis producing 166 g/L with productivity of 4.15 g/L/h in batch fermentation. Genetically engineered non-LAB biocatalysts yet have to match the performances of highly efficient wild LAB. In fact, C. glutamicum, S. cerevisiae and E. coli recombinant have been developed, but with limited success [61].

The search for lignocellulose-to-lactate biocatalysts have led to the discovery of many strains of pentose-utilizing LAB. Lb. pentosus ATCC8041 [78, 79], Lb. bifermentans DSM20003 [80], Lb. brevis [81], Lb. Plantarum [82], Leuconostoc lactis [83, 84], and E. mundtii QU 25 [85, 86]. Lactic acid produced from xylose per say has been investigated by few [84,85, 87, 88], but with mitigated results, mainly due to the fact that the pentose-utilizing

LAB do not perform as well in pentoses as in hexoses-rich metabolism. This phenomenon is most likely due to the fact that pentoses are metabolized in the PK pathway (phospho — ketolase), thus for a given strain, even if hexoses are fermented through an homofermen­tative route, pentose will yield heterofermentative products (i. e. acetic and lactic acid) [78, 89]. Nevertheless, Tanaka et al. [84] have shown that in addition to the PK, L. lactis could metabolize xylulose-5-phosphate (X5P), an intermediate pentose catabolite, through the pentose phosphate pathway (PPP). The theoretical yield through the PPP is 5 moles of lac­tate for 2 moles of pentoses, but through the PK it decreases to 1:1 [61], thus, the conver­sion advantage of the PPP is obvious. Okano et al. [87,89] demonstrated this approach by creating a pentoses-utilizing Lb. plantarium recombinant in which the native L-lactate de­hydrogenase (L-LDH) gene was disrupted, leaving only the homologous D-lactate dehy­drogenase (D-LDH) active. However, this strain produced both acetic and D-lactic acid; hence the PK gene (xpk1) was substituted by a heterologous transketolase (tkt) from L. lac — tis, thereby shifting heterolactic fermentation to a homolactic one.

Modification of yeast strains in order to achieve xylose-to-lactate conversion has also been investigated, as an example Ilmen et al. [90] expressed the L-LDH gene from L. helveticus in P. stipitis and was able to reach a titer of 58 g/L of lactate with a yield of 58 %. These results were obtained despite the fact that no effort had been made to silence the native PDC/ADH (pyruvate decarboxylase/alcohol dehydrogenase) ethylic pathway, consequent­ly 4.5 g/L of ethanol was simultaneously produced as the endogenous PDC rivalled against the recombinant L-LDH for pyruvate. Tamakawa et al. [88] went further by trans­forming C. utilis, disrupting the native pdc1 gene, and expressing heterologous LDH, XR (xylose reductase), XDH (xylitol dehydrogenase) and XK (xylulokinase) enzymes. Further­more, to prevent the redox imbalance, they increased the XR’s NADH (reduced nicotina­mide adenine dinucleotide) affinity by site-directed mutagenesis. In batch culture this recombinant was able to yield titers up to 93.9 g/L of lactate at a yield of 91 %. Table 3 shows the most recent and most efficient strains developed for lactic acid production, both from hexoses and pentoses.

of bovine L-LDH.

Disruption of
PDCand PDH
genes.

K. lactis Glucose Semi-Batch 60 500 0.85 0.12 [99]

Expression of

bovine L-LDH

gene.

Disruption of endogenous PDC gene. Expression of heterologous LDH, XR, XDH

C. utilis and XK. XR gene site-specific mutation for preferential NADH cofactor utilization

Expression of

P. stipitis LDH from L. Xylose Batch 58 147 0.58 0.39 [90]

helveticus.

* No xylose consumption occurred

**SSF = simultaneous saccharification and fermentation

Table 3. Lactic acid concentration (LA), time of fermentation (Tf), yield and production rate for the most common microorganisms used for the biological conversion of xylose to lactic acid

Lactic acid seems to be, on the biological as well as on the chemical point of view the best possible compound that could be derived from a based-catalysed reaction of xylose. Race­mic mixtures of lactic acid (most probably derived from chemical synthesis) can be evaluat­ed to 1150 USD/tonne [100] whilst the pure isomer was reported to have a price market around 1750 USD/tonne [101]. As in many cases, the price will vary proportionally with pu­rity of the compound. Utilisation of lactic acid on the market is mostly related to polymers, food, pharmaceutical and detergents. The annual world demand for the compound should reach a little more than 367 Ktonnes/year by 2017 [102].