Production of organic acids

A large number of organic acids with actual or potential uses are produced by microorganisms.e.g., Citric, itaconic, lactic, malic, tartaric, gluconic, mevalonic, salicyclic,gibberelic, diamino-pimelic, and propionic acids

Production of Citric Acid

Uses of Citric AcidCitric acid is used in the food industry, in medicine, pharmacy and in various other industries.

Uses in the food industry

(i) Citric acid is the major food acidulant used in the manufacture of jellies, jams, sweets, and soft drinks.(ii) It is used for artificial flavoring in various foods including soft drinks.(iii) Sodium citrate is employed in processed cheese manufacture.

Uses in medicine and pharmacy(iv) Sodium citrate is used in blood transfusion and bacteriology for the prevention of blood clotting.(v) The acid is used in efferverscent powers(vi) Since it is almost universally present in living things, it is rapidly and completely metabolized in the human body and can therefore serve as a source of energy.

Uses in the cosmetic industry(vii) It is used in astringent lotions such as aftershave lotions because of its low pH.(viii) Citric acid is used in hair rinses and hair and wig setting fluids.

Miscellaneous uses in industry

Citric acid has recently formed the basis of manufacture of detergents in place of phosphates, because the presence of the latter in effluents gives rise to eutrophication.

Biochemical Basis of the Production of Citric Acid

Citric acid is an intermediate in the citric acid cycle (TCA) . The acid can therefore be caused to accumulate by one of the following methods:(a) By mutation – giving rise to mutant organisms which may only use part of ametabolic pathway, or regulatory mutants; that is using a mutant lacking anenzyme of the cycle.

(b) By inhibiting the free-flow of the cycle through altering the environmentalconditions, e.g. temperature, pH, medium composition (especially the elimination of ions and cofactors considered essential for particular enzymes). e.g.,(i) By limiting the concentrations of ions or cofactors e.g., iron, manganese,

magnesium, zinc, and phosphate . To ensure their removal the medium is treated with ferro-cyanide or by ion exchange resins. These metal ions are required as prosthetic groups e.g., Mn++ or Mg++ by oxalosuccinic decarboxylase, Fe+++ is required for succinic dehydrogenase (ii) Limited aeration during fermentation: dehydrogenases, especially isocitrate dehydrogenase, are inhibited by anaerobiosis, (iii) Low pH and especially the presence of citric acid itself inhibits the TCA andhence encourages the production of more citric acid; the pH of the fermentationmust therefore be kept low throughout the fermentation (iii) Many of the enzymes of the TCA can be directly inhibited by variouscompounds and this phenomenon is exploited to increase citric acidproduction. E.g., isocitric dehydrogenase is inhibited by ferrocyanide as wellas citric acid; aconitase is inhibited by fluorocitrate and succinicdehydrogenase by malonate.

Fermentation for Citric Acid Production

Micro organism: Aspergillus niger , Asp. WentiPenicilliumCandida spp. (including Candida quillermondi)

Substrate:Malasses: Aspergillus niger , Asp. WentiPenicillium

Paraffin: Arthrobacter paraffineus and corynebacteria; the yeasts include Candida lipolytica and Candida oleiphila.

Fermentation with molasses and other sugar sources can be either surface orsubmerged. Fermentation with paraffins is submerged.

(a) Surface fermentation: Surface fermentation using Aspergillus niger Substrate: rice bran or in liquid solution in flat aluminium or stainless steel pans. The citric acid is extracted from the bran by leaching and is then

precipitated from the resulting solution as calcium citrate.

(b) Submerged fermentation: fermentor is made of acid-resistant materials such as stainless steel. Substrate: molasses MgSO4, 7H2 O and KH2PO4 at about 1% and 0.05-2% The pH is maintained at 3.5. Copper is used at up to 500 ppm as an antagonist of the enzyme aconitase 1-5% of methanol, isopranol or ethanol As high aeration is deleterious to citric acid production, mechanical agitation is not necessary and air may be bubbled through. Anti-form is added. Duration: five to fourteen days

Extraction

The broth is filtered until clear.

Calcium citrate is precipitated by the addition ofmagnesium-free (Ca(OH) 2. Since magnesium is more soluble than calcium, some acid may be lost in the solution as magnesium citrate if magnesium is added.

Calcium citrate is filtered and the filter cake is treated with sulfuric acid to precipitate the calcium.

The dilute solution containing citric acid is purified by treatment with activated carbon And passing through iron exchange beds.

The purified dilute acid is evaporated to yield crystals of citric acid.

Further purification may be required to meet pharmaceutical stipulations.

Lactic AcidLactic acid is produced by many organisms

Properties and chemical reactions of lactic acid

(i) Lactic acid is a three carbon organic acid: one terminal carbon atom is part of an acid or carboxyl group; the other terminal carbon atom is part of a methyl or hydrocarbon group; and a central carbon atom having an alcohol carbon group. Lactic acid exists in two optically active isomeric forms

(ii) Lactic acid is soluble in water and water miscible organic solvents but insoluble in other organic solvents.

(iii) It exhibits low volatility.

(iv) The acid reactions of lactic acid are those that form salts and undergo esterification with various alcohols.

Uses of lactic acid

(i) It is used in the baking industry(ii) In medicine it is sometimes used to introduce calcium in to the body in the form of calcium lactate, in diseases of calcium deficiency.(iii) Esters of lactic acid are also used in the food industry as emulsifiers.(iv) Lactic acid is used in the manufacture of rye bread.(v) Lactic acid is used as acidulant/ flavoring/ pH buffering agent or inhibitor ofbacterial spoilage in a wide variety of processed foods. It has the advantage, incontrast to other food acids in having a mild acidic taste.(vi) It is non-volatile odorless and is classified as GRAS (generally regarded as safe) by the FDA.(vii) It is a very good preservative. Addition of lactic acid aqueous solution to the packaging of poultry and fish increases their shelf life.(viii) The esters of lactic acid are used as emulsifying agents in baking foods (stearoyl-2- lactylate, glyceryl lactostearate, glyceryl lactopalmitate). The manufacture of these emulsifiers requires heat stable lactic acid, hence only the synthetic or the heat stable fermentation grades can be used for this application.

(x) Lactic acid has many pharmaceutical and cosmetic applications and formulations in topical ointments, lotions, anti acne solutions, humectants, parenteral solutions and dialysis applications, for anti carries agent.

(xi) Calcium lactate can be used for calcium deficiency therapy and as anti caries agent.

(xii) Its biodegradable polymer has medical applications as sutures, orthopaedic implants, controlled drug release, etc.

(xiii) Polymers of lactic acids are biodegradable thermoplastics.

(xiv) Lactic acid esters like ethyl/butyl lactate can be used as environment-friendly solvents. They are high boiling, non-toxic and degradable components.

Fermentation for lactic acid

Microorganisms:• No. of microbes produce lactic acid but in low conc.• Lactobacillus spp., especially L. delbruckii. • In recent times Rhizopus oryzae has been used. • Both organisms produce the L- form of the acid

Rhizopus fermentation has the advantage of being much shorter in duration; further, the isolation of the acid is much easier when the fungus is used.

Fermentation:• Lactic acid is very corrosive and the fermentor, which is usually between 25,000 and 110,000 liters in capacity is made of wood.• Alternatively special stainless steel may be used. • They are sterilized by steaming before the introduction of the broth as contamination with thermophilic clostridia yielding butanol and butyric acid is common.

Carbon source: The carbon source used in the broth has varied widely and have included whey, sugars in potato and corn hydrolysates, sulfite liquour, and molasses However, because of the problems of recovery for high quality lactic acid, purified sugar and a minimum of other nutrients are used.

pH is maintained around 5.5- 6.5 by adding calcium carbonate

Addition of vitamins and growth factors for growth: These requirements along with that of nitrogen are often met with ground vegetable materials such as ground malt sprouts or malt rootlets.

Fermentation with Lactobacillus delbruckii is usually for 5 to 10 days whereas withRhizopus oryzae, it is about two days.

Anaerobic process

The temperature of the fermentation is 45°C

ExtractionThe main problem in lactic acid production is not fermentation but the recovery of the acid. Lactic acid is crystallized with great difficulty and in low yield. The purest forms are usually colorless syrups which readily absorb water

At the end of the fermentation when the sugar content is about 0.1%, the beer ispumped into settling tanks. Calcium hydroxide at pH 10 is mixed in and the mixture is allowed to settle. The clear calcium lactate is decanted off and combined with the filtrate from the slurry. It is then treated with sodium sulfide, decolorized by adsorption with activated charcoal, acidified to pH 6.2 with lactic acid and filtered. The calcium lactate liquor may then be spray-dried.

For technical grade lactic acid the calcium is precipitated as CaSO4.2H2O which isfiltered off. It is 44-45% total acidity. Food grade acid has a total acidity of about 50%. It is made from the fermentation of higher grade sugar and bleached with activated carbon. Metals especially iron and copper are removed by treatment with ferrocyanide. It is then filtered. Plastic grade is obtained by esterification with methanol after concentration.

High-grade lactic acid is made by various methods: steam distillation under highvacuum, solvent extraction etc

Production of ergot alkaloids

Alkaloids are basic naturally occurring hetereocyclic organic nitrogen containing compounds which are biosynthesized from amino acids by plants, microorganisms and some animals.

The main precursors for alkaloid biosynthesis are ornithine, lysine, aspartic acid, phenylalanine, tyrosine and tryptophan. For example the alkaloid in tobacco, nicotine, is derived from ornithine while phenylalanine and tyrosine give rise to simple alkaloids such as ephedrine or more complex ones such as morphine

The name alkaloid (literally alkali like) derives from their basic nature, because of which they readily form salts with acids present in the natural sources from which they are derived.

The ergot alkaloids are so called because they were originally derived from ergot, a sclerotium (twisted mat of fungal hyphae) formed as a disease on the grain of rye (Secale cereale L.) a temperate cereal.

• ergot contains several (more than 40) highly potent alkaloids that result in ‘ergotism’,

• About 50 ergot alkaloids are known • While most of these alkaloids are derived from the Claviceps sclerotium formed on the rye grain, • hundreds of other cereals and grasses can serve as hosts for the fungus. • About 50 species of Claviceps itself are known.• alkaloids are also produced by other fungi including Aspergillus, Penicillium, and Rhizopus. •Furthermore, ergot alkaloids have recently been found in the seeds of some higher plants, Ipomea, Rivea, Agyreis which belong to Convulvulaceae

The life cycle of Claviceps • The sclerotium forms in the size and shape of the grain which it replaces. • These sclerotia fall to the ground at the end of the growing season and remain dormant till the beginning of the next growing season, when they germinate and form ascocarps (perithecia). • The ascospores are distributed by wind to the newly formed flowers of grains. • The germinated ascospores yield hyphae which produce masses of conidia supported in a sugary liquid which attracts insects. • These insects further help distribute the conidia to other plants

• The ergot alkaloids are classified as indole alkaloids, which are derived from tyrptophan. • the ergot alkaloids possess the basic tetracyclic (four-ringed) structure known as ergoline

The naturally occurring ergot alkaloids can be divided into two groups. (a) Lysergic acid derivaties and (b) clavine alkaloids.

Clavine alkaloids are much less known than the lysergic acid ones in terms of their fermentation and even in terms of pharmacological activity

The lysergic acid two types depending on the nature of the amide substituents • simple amide substituents and • peptide alkaloids in which a cyclic peptide is attached to lysergic acid. E.g., ergotamine

USES OF ERGOT ALKALOIDS AND THEIR DERIVATES

Ergot alkaloids and their derivates are powerful drugs and may be used as such or may be the basis of semi-synthetic preparations.

• Ergotamine blocks the sympathetic system and is used for treating strong headaches such as migraines.• The diethylamide derivate of lysergic acid (known as LSD) is a powerful hallucinogenic drug • Most of the clavine alkaloids do not possess strong pharmaceological properties.However a few of the didydro derivates have found use as strong stimulants of oxytoxic (milk secreting) activity.

Uses…………….

• ‘Nigericoline’, a derivate of lysergic acid which is a receptor-blocking agent and is used for treating peripheral and cerebral circulation disorder.

• ‘Lysenyl’ a diethyl derivate of isolysergic acid is used to treat hypertension and migraine, since it is a serotonin antagonist.

• In recent times it has been found that some ergot alkaloids affect functions controlled by the hypothalamic pituitary system particularly the release of prolactin – examined for certain breast cancers therapy.

• Some of newer ergot alkaloids have also been implicated as potential therapeutic agents in the treatment of diseases such as Parkinson’s disease, and cancer of the prostrate.

• The alkaloids have been used as models for the synthesis of several potent drugs.

Alkaloids used as drugs

PRODUCTION OF ERGOT ALKALOIDS

The methods for producing these drugs are:(a) Isolation from field cultivated ergot.(b) Fermentation of the ergot fungus(c) Partial or total chemical synthesis

(a) Isolation from field cultivated ergot (or parasitic production): • This method is widely used in Europe. • Inoculation of the rye plants with conidiospores of Claviceps and other

fungi takes place two to three weeks before flowering begins and may continue during flowering.

• Harvesting of ergotized ears of rye takes place about two months later.

disadvantages • Firstly, only one crop a year can be obtained.• Secondly, the yield of alkaloid in terms of quality (type) and quantity is highly unpredictable. • operation highly unstandardizable

(b) Fermentation production: History• Ergot alkaloids can be produced in submerged fermentation by Claviceps or Penicillium species. • Initial work in Japan showed that submerged cultures did not produce the typical alkaloids associated with the sclerotium but instead produced a series of new non-peptide bases (clavines) which did not possess any significant pharmacological action. • Attempts made latter by modification of the culture medium and the fungus strain. • The first pure ergot alkaloid, ergotamine, was obtained by Stoll in 1920. • Subsequently, others reported the discovery of the “water soluble uterotonic principle of ergot” which was subsequently determined to be ergonovine

Production of clavine alkaloids: Different species of Claviceps parasitizing a variety of grasses have been isolated and grown in liquid medium and the different alkaloids assayed.

Clavine alkaloids were obtained from Cl. litoralis, Cl. microcephals.

The medium : mannitol (5%) ammonium citrate (0.7%), KH2PO4 (0.1%)and MgSO4 (0.03%). The pH: 5.2. 10% sucrose Duration: 30 days to 40 days Yield: 1.0-1.5 gm/liter

Further improvement By addition of mixture of mannitol (6.5%) and glucose (1%) gave up to 1 gm/liter yield in about 14 days. A high carbon to nitrogen in the medium

Production of simple lysergic acid derivates: The production of simple lysergic acid derivates was achieved in 1961 using Claviceps paspali isolated from an infected Paspalum digitatum.

• Submerged fermentation• media: mannitol-succinic acid-mineral salts medium• Fumaric acid gave higher yields when it was used in place of succinic acid. • Vigorous aeration• Duration: nine days.• The addition of hydrophilic non-ionic surfactants improves yield

Peptide alkaloids: Microbes: Claviceps purpurea, C. litoralis, Elymus mollis.

• Improved ergotoxine yields were obtained by the mutation and selection of a strain of Claviceps purpurea. • This strain on a mannitol-ammonium-succinate medium produces up to 40 gm/liter.

PHYSIOLOGY OF ALKALOID PRODUCTION

(i) Induction: Tryptophan is the central precursor of all ergot alkaloids and it istherefore required in the medium. -In addition to being a direct precursor of ergot alkaloids, tryptophan is also a factor in the induction and derepression of enzymes necessary for alkaloid synthesis.

(ii) Feedback regulation: Not very successful

(iii) Phosphate repression: Like many secondary metabolites ergot alkaloid formation is inhibited by increasing the level of phosphate. However, addition of tryptophan nullify the effect of phosphate(iv) Catabolite regulation: High levels of glucose (5%) greatly inhibited enzyme production.

Alkaloid formation and morphological structures:

• interestingly, alkaloids seem to form in one structure and accumulated in another. e.g., in the plant genus Ipomea, alkaloids are formed in the leaves and accumulated in the seeds, which do not produce alkaloids at all. • In ergotoxine alkaloid fermentation, alkaloids are not elaborated until specific morphological structures, pellets, are formed in the medium. • The medium composition appears to influence the formation of these pellets. • Sucrose encourages their formation while they are poorly formed in malt. • The peptide alkaloids are found in these structures, while clavines and simpler derivates of lysergic acid are found in the medium.

Biosynthesis: Being secondary products, alkaloids are produced by pathways different from those of general metabolism

• synthesis initiates with carbon limitation. • The ergot skeleton is derived from tryptophan, mevalonic acid, and methionine. • The central precursor of the ergoline skeleton is tryptophan,• The five-carbon unit is obtained from mevalonic acid• Mevalonic acid may be formed from the malonyl COA pathway with biotin and decarboxylation.Lysergic acid itself is a key substrate in ergot alkaloid synthesis and the simpleramide as well as the peptide derivates come from it

enzymes involved in the synthesis of alkaloids

• Dimethyl tryptophan synthase (DMTPS) and • chanoclavine cyclase

•DMTPS catalyses the first specific reaction of the formation of 8-ergoline and introduces the isoprene residue to the C4 position of tryptophan. Analogues of tryptophan increase the activity of the enzyme. • The compound produced, dimethyl tryptophan (4-isoprenyltryptophan) is 5-10 times more effective as a precursor of the clavines than tryptophan.

Chanoclavine cyclase catalyses the cyclization of chanoclavine – 1 to the fourring 8-ergoline i.e. to elgmoclavine and agroclavine, both individually andsimultaneously.

• In general the sclerotia is dried, powdered, alkalinized, and extracted with tartaric acid. • For further purification, the tartaric acid solution is made alkaline and extracted with chloroform. •Chloroform layer contains all the alkaloids. • This has led to the terms water-soluble alkaloids which include the simple amide lysergic acid derivatives and the clavines, and water-insoluble alkaloids : the peptide-type• The chloroform extract is concentrated to a small volume, diluted with ether and extracted with concentrated H2SO4. • On neutralization with ammonia the water-soluble alkaloids can be extracted with water and the water insoluble ones with carbon-tetrachloride.

Extraction of the alkaloid Alkaloids are readily isolated at an alkaline pH by various organic solvents such as ether, chloroform, and ethyl acetate

Biosynthesis of alkaloids