L(+) Lactic acid and Its applications

L (+) lactic acid production is gaining interest due to its applications in food and pharmaceutical industries. At present world market is mainly captured by China and USA. Poly L(+) lactic acid is a biodegradable, biocompatible polymer that can be produced from the renewable resources via fermentation.

Different Lactobacillus species

Most homofermentative organisms produce both the stereoisomeric forms of lactic acid. Heterofermentative organisms may produce lactic acid along with other metabolites like ethanol, acetate, formate etc. anaerobically. The purification of one form of lactic acid from its racemic mixture is very difficult, which may involve expensive chromatography techniques. The most suitable way is to select the L(+) producer organism or use genetically modified strains. A few attempts have been made to improve and modify the production of L(+) lactic acid by metabolic engineering in Lactobacillus species. The ldhD gene in L. helveticus was inactivated. A two-fold increase in the amount of L(+) lactic acid was observed which restored the amount of total lactic acid to the level in the wild type strain. The ldhL gene was overexpressed in L. plantarum by increasing its copy number. This increased the activity of L-lactate dehydrogenase (L-LDH) but was found ineffective for L(+) lactic acid production. The total lactic acid production was not affected by the inactivation of ldhD gene or inactivating the ldhL gene. In another study, two ldhD^- strains of L.helveticus were constructed by deleting the promoter region of the ldhDgene in the first construct and by replacing the structural gene of ldhD gene by ldhL gene of the same species in the second construct. This has not only increased the enzyme activity of L-LDH by 53% and 93% respectively, but also enhanced L(+) lactic acid production. In Lactococcus lactis, ldhLgene is located as a part of the lac operon. An increase in copy number of the plasmid containing the whole operon resulted in a slight increase in lactic acid production. The ldhD gene of L. johnsonii was altered by an 8-bp deletion. The D-LDH enzyme activity was completely absent in the mutant strain and the remaining L-LDH enzyme activity rerouted the carbon flux to L-lactate with a marginal increase in the secondary end products.

Many of the genetically modified homofermentative lactic acid bacteria were unable to produce pure L(+) form of lactic acid. The present studies were planned to produce L(+) lactic acid as the only product using the heterofermentative organism Lactobacillus rhamnosus. This organism produces ethanol along with L(+) lactic acid under anaerobic conditions. Therefore its adh^- mutant may only give L(+) lactic acid. For this purpose adh^- mutant of L.rhamnosus was developed by chemical mutagenesis. Its behavior was examined in batch fermentation. The downstream processing for the separation of pure L(+) lactic acid from the fermentation broth can be made easier if a defined media is used. The concentration of amino acids, vitamins and trace metals solution were optimized by experimental design and response surface method and their batch kinetics were observed in the optimized media. These studies were focused on purified L(+) lactic acid production in chemically defined medium, so as by allowing minimum number of unit operations like only electrodialysis to get the purified lactic acid. The concentrated purified L(+) lactic acid can produce lactide that has great applications 

Mutagenesis and Selection of mutants

Actively growing cells harvested from the fermented medium, centrifuged, resuspended in citrate buffer, and then mutagenised by exposing these cells to N- methyl, N'-nitro, N-nitrosoguanidine. Thereafter, mutant cells were selected onto agar plates containing allyl alcohol. This compound inhibits wild type cells since cells with functional alcohol dehydrogenase activity convert allyl alcohol to the toxic compound acrolein.

Statistical experimental design

A Box Behnkehn experimental design with three variables, amino acids, vitamins, trace metals solution and the two parameters or responses, lactic acid and biomass were used which resulted in a total of 20 experiments. The real and coded values of the variables were obtained. Design Expert version 5.5 (Statease Inc, MN, USA) software was used for statistical experimental design, analysis of the results and drawing of the contour plots. The model was fitted by multiple linear regressions and selected by examining the 'Lack of Fit tests' in which low 'ROOT MSE' and 'PRESS' values describe the fitness of the model.

L(+) Lactic acid production

Batch lactic acid fermentations using mutant carried out in optimized medium under controlled conditions pH (6.2) and temperature 40ºC. This could able to exhibit the comparable yields of biomass and lactic acid in designed synthetic medium. The designed optimized medium is useful and does not cause hindrances in L(+) lactic acid polymerization.