Lactic Acid Production

2.5. Lactic Acid Production

Preamble : Scheele (1780) first discovered the presence of lactic acid [CH 3 CH(OH)COOH)] in the sour milk. Pasteur (1857) i.e., almost a gap of 77 years after Scheele’s epoch making discovery proved and established that the souring of milk was due to microbiological fermentation by a causative organism. As on date, there are indeed several evidences to show that a plethora of microorganisms are capable of producing at least small amounts of lactic acid which is present in several fermented foods and beverages.

Subsequently, the first ever microbial production of an ‘organic acid’ was that of lactic acid carried out in the year 1880 i.e., almost after 23 years from Pasteur’s observation. Nowadays, both the chemical procedures and the biological processes are not only extremely competitive but also appreci- ably cost-effective.

Organisms for Lactic Acid Production : In fact, there are two different types of lactic acid organisms that have been duly recognized, namely : (a) Heterofermentative ; and (b) Homofermentative, which would be explained as under :

A. Heterofermentative Organisms : These organisms usually yield certain quantum of lactic acid, but simultaneously and most probably by virtue of the ensuing pentose-phosphate metabolic

pathway they may give rise to the production of several chemical substances, such as : ethanol, acetic acid (vinegar), CO 2 , and traces of a few other products.

Example : Leuconostoc mesenteroides — it is of no utility for the commercial lactic acid fermentative procedures due to the fact that a substantial quantum of the ‘substrate carbon’ is con- sumed in yielding products other than lactic acid.

B. Homofermentative Organisms : These strains of lactic acid producing bacteria invariably yield maximum quantum of lactic acid and only trace amount of other products. In fact, these organisms make use of the specific metabolic pathway to yield pyruvic acid which gets subsequently reduced by the corresponding enzyme, lactic dehydrogenase, to produce lactic acid. It has been observed that the overall percent conversion of hexose-sugar to lactic acid is almost equivalent to two moles of lactic acid

MICROBIAL TRANSFORMATIONS

279 Examples :

(1) Lactobacillus delbrueckii — it is used for the commercial production of lactic acid in fermentative procedures using corn-dextrose media.

(2) Lactobacillus bulgaricus — it makes use of ‘lactose’ as a source of carbon and finds usage in lactic acid production starting from whey media.

(3) Lactobacillus pentosus — it specifically utilizes the ‘pentoses’ obtained from the ‘sulphite- waste liquor’ for lactic acid production on a large-scale.

Other Potential Homofermentative Species : There are some other species which belong to the class of homofermentative organisms, such as : Lactobacillus casei ; Lactobacillus leichmannii ; and streptococcus lactis — all do possess potential industrial importance and recognition. These organ-

isms are essentially anaerobes ; however, they may withstand certain extent of O 2 . Interestingly, S. lactis is found to be relatively much less sensitive to O 2 ; and, hence, may be regarded as a facultative* aerobes rather than obligate anaerobes ; and, therefore, the bioreactors should be used in an absolutely O 2 -free atmosphere.

Theoretical Aspects : The biosynthetic pathway of lactic acid starting from glucose essentially takes the route via glyceraldehyde-3-P, 1, 3-di-P-glycerate, and pyruvate as given in Fig. 4.6. It may be further expatiated by the reducing power generated effectively during the oxidation of glyceraldehyde phosphate is eventually transferred with an NAD-dependent enzyme lactate dehydrogenase to the

corresponding pyruvate, and this ultimately gets reduced stereospecifically to give rise to the two optical isomers L(+) or D(–) lactic acid.

GLUCOSE (C H O ) 6 12 6

L A C TAT E P H O S P H AT E DEHYDROGENASE DEHYDROGENASE

Fig. 4.6. Lactic Acid Production from Glucose Using Lactobacillus delbrueckii.

Fermentation Medium : In USA, the C-source used commercially for the production of lactic acid from a variety of available media, such as : molasses, whey, and partially refined corn-sugar (containing dextrose) ; whereas, several other countries extensively employ either previously hydrolysed potato starch or other suitable C-substrates i.e., semirefined sugars, maltose, lactose, sucrose, and dextrose.**

* A microorganism may be facultative with respect to O 2 and thus be able to survive (live) either with or without O 2 .

** Inskeep GC et al. Ind. Eng. Chem., 44, 1955-1966.

PHARMACEUTICAL BIOTECHNOLOGY

Ideally, the fermentation medium must comprise of glucose (12-13%), diammonium acid phos- phate [(NH 4 ) 2 HPO 4 ] (0.25%), and small amount of B-vitamins. The fermentative operation is usually

initiated in huge fermentors (capacity : 25-120 m 3 ) at a temperature ranging between 45 to 50°C plus an excess of CaCO 3 (solid) supplemented to maintain the pH varying between 5.5 to 6.5 strictly. The entire

fermentation usually takes almost 3 days (72 hours) under the aforesaid experimental parameters. Becuase, lactic acid is found to be toxic to the organism, two specific procedures have been successfully tried and tested so as to remove the product (i.e., lactic acid) both simultaneously and continuously from the ‘fermented broth’, namely :

(a) Electrodialysis : A method of separating electrolytes from colloids by passing a current through a solution containing both.

(b) Continuous Culture : A continuous culture was duly carried out in a membrane reactor that ultimately led to the production of lactic acid to the extent of 80 g/l.h.

Extraction and Recovery : There are four distinct procedures that may be adopted for the extraction and recovery of lactic acid from the fermented broth as described under :

Method–1 : At harvest, a calculated amount of CaCO 3 is added to the fermented medium, pH adjusted to 10, and the contents are heated and filtered subsequently. By doing so a host of desirable goals are accomplished, such as :

(i) All of lactic acid gets converted to calcium lactate, (ii) Most organisms are killed and eliminated, (iii) Help in the complete coagulation of protein present in the medium,

(iv) Removes excess of CaCO 3 , if any, and

(v) Decomposes any residual sugar(s) present in the medium. Lactic acid is now recrystallized as calcium lactate and decolourized by adding activated carbon. The latter step may be accomplished alternatively by preparing the zinc salt of lactic acid which

happens to be comparatively less soluble. Method–2 : In this particular instance the ‘free lactic acid’ is subjected to extraction with isopropyl

ether (solvent) successively from the pre-heated and filtered fermentation broth. This phenomenon is based upon the principle of counter-current continuous extraction. In fact, the desired ‘lactic acid’ is finally recovered from the medium of isopropyl ether by further affecting counter-current extraction with water, in which the former is soluble because both are polar in nature.

Method–3 : The ‘lactic acid’ thus obtained is converted into its corresponding methyl ester

which is separated from the fermentation broth by distillation followed by hydrolysis of the corresponding ester by simply boiling in dilute aqueous medium.* Subsequently, the lactic acid is recovered from the aqueous medium by evaporation of the water, and the liberated methanol may be collected by distillation.

Method–4 : In this specific procedure, the lactic acid is obtained as its corresponding secondary

and tertiary alkylamine salts which are subsequently extracted from the aqueous medium with appro- priate organic solvents completely. The solvent is removed usually by distillation and the residual ‘salt’ is then decomposed carefully to obtain lactic acid.

* Methyl ester of lactic acid gets decomposed in water.

MICROBIAL TRANSFORMATIONS

281 Note : In general, it is quite important and necessary that the ‘recovery processing equip-

ment’ must be quite resistant to the corrossive action of the high concentration of lactic acid being accumulated. Hence, invariably one should make use of stainless-steel equip- ment for the recovery of lactic acid in its purest form and quality.

Grades of Lactic Acid : Depending upon the actual usage and application one may come across different ‘grades of lactic acid’ available commercially in trade, namely :

(1) ‘Crude’ or ‘Technical’ Grade : It is a coloured product solely meant for commercial appli- cation at various concentrations ranging between 20–80%. It may be prepared by using H 2 SO 4 to eliminate Ca 2+ from the calcium lactate salt obtained from the heated and filtered fermen- tation broth by adopting these steps sequentially viz., filtration, concentration, refiltration to

remove additional CaSO 4 . Hence, the crude or technical grade lactic acid comprises of several impurities and may be used where purity of the product is not so critical and essen- tial, such as : deliming of hides in leather industry.

(2) ‘‘Edible’’ Grade : It is usually having a straw-coloured appearance and mostly available at strengths ranging between 50–80%. It is mostly used in food and beverage industries to maintain optimum pH for better storage and shelf-life of products.

(3) ‘Plastic’ Grade : It is more or less colourless and available in strengths varying between 50– 80%. Invariably prepared from technical grade lactic acid via adequate refining processes.

(4) ‘USP’ Grade : It is a pharmaceutical grade lactic acid having a strength of 85% and mostly used in pharmaceutical formulations.