Synthetic Polymers & Alkyl benzyl sulphonates

Synthetic Polymers

Eg., Plastics, polyethylene- PE, polyvinyl chloride -PVC and polystyrene - PS 

• Easily molded into complex shapes, have high chemical resistance and are more or less elastic
• Can be formed into fibres or thin, transparent films
• Popular in the manufacture of garments, durable and disposable goods, packaging materials
• More than 90% of plastic material in municipal garbage contains polyethylene, polyvinyl chloride (PVC) and polystyrene (PS)
• More than one third of total plastic production include disposable goods and packaging material; largest environmental impact
• These materials have molecular weights ranging from several thousand to lakhs and resist biodegradation indefinitely- Resistance to biodegradation is associated with excessive molecular size
• If molecular size of polyethylene is reduced to molecular weight under 500 daltons by pyrrolysis, the fragments are found susceptible to biodegradation 

• Other reports on plastic degradation focus on the degradation of plasticizers, the additives designed to render the plastic pliable

• Plasticizers- esters of long chain fatty acids and alcohols 
• Plasticizer biodegradation renders the material brittle but does not affect the polymer structure

• Plastics are biologically inert, so were regarded as an aesthetic nuisance but not hazard
• However, oceans dumped with plastic wastes and floating plastic spherules being ingested by small fish with consequent intestinal blockage- much concern
• Remains of plastic nets and ties also harm wildlife
• Agricultural practices involving plastics (film mulching technique)- covering the fields with plastic films in order to control weeds and conserve moisture
• Large accumulation of plastics in the field are undesirable-reuse or collection at the end of growing season not practical

• Research has now focused on bioplastics such as poly β-hydroxy alkoanates produced by Alcaligenes, Bacillus and various Pseudomonads
• These biodegradable intracellular storage products have thermoplastic properties and can be molded- properties can be influenced by the substrate fed to the bacteria
• β-hydroxy butyric and β-hydroxy valeric acid copolymer give the best properties
• High price (5-7 times higher than petrochemical based polyethylene) restrict to its use

• Modified starch and other natural polymers can be useful biodegradable plastics

• Synthetic caprolactone polyester contains hydrolyzable bonds-good candidate for biodegradable plastics

• Polyethylene, polybutene etc are susceptible to photochemical biodegradation- Materials that sustain sufficient photochemical damage during a growing season are later susceptible to microbial degradation
• Manufacturers sell these as degradable or environment friendly products – they contain photosensitizers alone or in combination with 3-15% modified starch

• Prolonged sun irradiation (weeks to months) causes breaks in the polymer making it brittle and susceptible to biodegradation

 Alkyl benzyl sulphonates/ Alkyl benzene sulphonates (ABS)

•  Main components of anionic detergents-surface active with polar (water soluble) sulphate and nonpolar alkyl end 

• Aid in emulsification of fatty substances and cleaning occurs while these molecules make a monolayer around lipophilic droplets or particles.

• Their molecules orient with their nonpolar end towards the lipophilic substance and the sulphonate end towards water.

• Nonlinear alkylbenzene sulphonates or Branched alkylbenzene sulfonates (BAS) are recalcitrants and resistant to biodegradation and cause foaming problems in sewage treatment and  in the rivers.

• ABS are easier to manufacture and bear superior detergent properties but the methyl branching of alkyl chain in them interferes with biodegradation of ABS,  because tertiary carbon atom blocks the normal β-oxidation.

• Nonlinear alkylbenzyl sulphonates or Branched alkylbenzene sulfonates (BAS) were replaced with linear alkylbenzene sulfonates (LAS) during the 1960s due to environmental concerns

• 

• 
  

LAS (linear alkylbenzyl sulphonates) are biodegradable

• It is biodegraded rapidly under aerobic conditions within weeks- oxidative degradation initiates at the alkyl chain.

• Under anaerobic conditions it degrades very slowly or not at all, accumulates in sewage sludge, but once oxygenated, it will rapidly degrade

• Thus, BAS to LAS conversion is a story where a synthetic molecule was redesigned to remove obstacles to biodegradation, while preserving the other useful characteristics of the compound.

Reference

Microbial Ecology: Fundamentals and Applications- Ronald M. Atlas, Richard Bartha

 

S. pneumoniae- Epidemiology, Pathogenicity, Laboratory Diagnosis, Prophylaxis, Treatment

Epidemiology

• ·         S. pneumoniae occurs in the throats of 50% of human population, any time 
•        Source of infection is the respiratory tract of carriers, and at times, of patients.
• ·         Transmitted by fingers or by inhalation of droplets
• ·         Dissemination is facilitated by crowding
• ·         Infection usually leads to pharyngeal carriage-- disease results when host resistance is lowered by viral infection, pulmonary congestion, stress, malnutrition, immunodeficiency or alcoholism

  Pathogenicity

• ·         Experimental infection in mice & rabbits by intraperitoneal inoculation - fatal infection – pneumococci demonstrable in peritoneal exudate and heart blood–death in 1-3 days

• ·         Colonise the human Nasopharynx – cause infection of middle ear, paranasal sinuses and respiratory tract – direct infection
• ·         Can spread through blood and lymphatic system– cause Meningitis – distant infections in the heart, peritoneum or joints
• ·         Infection is generally, endogenous in nature, can be Exogenous also

Common Pneumococcal infections 

• otitis media and sinusitis – prior respiratory infection or allergy can lead to these conditions

• common cause of lobar and bronchopneumonia –also cause acute tracheobronchitis and empyema (collection of pus in the pleural cavity)

•  Normal mucosal defense mechanisms (entrapment, cough, ciliary defense) prevent establishment of infection. But if these defense mechanisms are compromised by infections, anesthesia or chilling etc, Pneumococci can multiply and spread through lung and lymphatics- Bacteremia is common in lobar pneumonia

• Diffusion of capsular polysaccharide into the blood and tissues- cause toxemia.
•  SSS neutralized by anti-capsular antibodies produced by the patient- cause fall in temperature and general relief of symptoms
•  In adults, 50% fatalities are due to Pneumococcal bacteremia.

• Bronchopneumonia is always a secondary infection – damage to the respiratory epithelium and excessive bronchial secretions caused by the primary infection, lead to the invasion of Pneumococci to the bronchial tree- terminal event in aged and debilitated patients 

• Meningitis – most serious Pneumococcal infection
• Is a secondary infection after the primary infections like  pneumonia, otitis media, sinusitis or conjunctivitis - Occur at all ages 
• If untreated, highly fatal 
• 25% fatality even with antibiotic therapy

•  Pneumocci also cause suppurative lesions in different parts of the body- empyema, pericarditis, otitis media, sinusitis, conjunctivitis, suppurative arthritis, peritonitis , keratitis, dacryocystitis (lacrymal sacs infection)

Laboratory Diagnosis

• Clinical diagnosis of Pneumonia is easy -etiological diagnosis needs laboratory assistance- many organisms involved
• Specimen – Sputum, CSF, Blood and Urine
• Microscopy - In acute lobar pneumonia, rusty sputum contain Pneumococci in large numbers -hardly any other bacterium- Gram stain.

(Rusty sputum- sputum produced in pneumonia caused by S pneumoniae - contain bacteria, hemorrhage, mucus,  necrotic lung tissue.)

• In the pre-antibiotic era, direct serotyping with Quellung reaction followed by treatment with specific antiserum-routine process

• Culture – The sputum after homogenization, inoculated onto blood agar plates, incubated at 37^oC, under 5-10 % CO₂, overnight. In infants, laryngeal swabs may be used for culture. Gentamycin can be added to blood agar to facilitate isolation of Pneumococci.

• In acute pneumonia, the organism may be obtained from Blood culture in Glucose broth – isolation of pneumococci in blood indicates bad disease condition.

• Animal inoculation – Intraperitoneal inoculation in mice, if specimen contains scanty numbers of the organism and even if cultures are negative. Pneumococci may be demonstrated in the peritoneal exudates and heart blood. Inoculated mice die in 1-3 days.
• Test maybe negative if the strains are avirulent for mice (type 14 strain)

• In otitis media, Pneumococci demonstrated in fluid aspirated from the middle ear

• Meningitis- presumptive diagnosis from Gram stained films of CSF-Gram positive diplocoocci visible inside polymorphs and extracellularly. Diagnosis confirmed by culture. If negative in culture, SSS demonstrated in CSF by immunoprecipitation (with antisera)

• Antigen Detection  -SSS/Capsular polysaccharide -in blood, urine, CSF by Precipitation, counterimmunoelectrophoresis
• Antibody demonstrated by agglutination, precipitation, indirect haemagglutination, radioimmunoassay etc

Prophylaxis

• Immunity is type specific – antibodies against  capsular polysaccharide
• ` 90 serotypes- so complete polyvalent vaccine not practical
• A polyvalent polysaccharide vaccine with  capsular antigens of the 23 most prevalent serotypes gives 80 – 90 % protection 
• not meant for general use but for people at increased risk such as absent or dysfunctional spleen,  Sickle cell disease, chronic heart, renal, lung and celiac disease, Diabetes mellitus, HIV etc
• not recommended for children under two years of age and those with immunosuppressive therapies.

Treatment 

• Parenteral administration of Penicillin in serious cases and  Amoxycillin in mild cases (if the isolate is sensitive to penicillin)  
• Many Penicillin resistant (alteration in Penicillin Binding Protein)- strains are resistant to erythromycin and tetracycline
• For resistant isolates- Third generation cephalosporins and in life threatening illnesses with highly resistant strains, Vancomycin

 

Recalcitrant Nitroaromatics

• Nitroaromatics are common military explosives such as Trinitro toluene-TNT, solvents such as nitrobenzene and pesticides such as nitro phenols.
• Nitrosubstitution of aromatic rings decreases biodegradability.
• These compounds are highy toxic and mutagenic
• Compounds with several nitrosubstituents are recalcitrant

• Biodegradation of nitroaromatic compounds tend to be slow and leads to bound or polymerized residues in soils and sediments
• Extensively nitro-substituted aromatics are more easily transformed under anaerobic conditions than aerobic conditions.

Oxidative pathway/Aerobic

• Nitrobenzene is converted to diol by the action of dioxygenases which gets converted to catechol. Catechol ring is opened by meta-cleavage later.
• Nitrobenzene ^dioxygenaseàNitrobenzene diolà Catechol (ring opening by metacleavage) àoxalocrotonaldehyde--àOxalocrotonate                                                           

 

Reductive pathway/Anaerobic

• Release more energy
• Does not require strict anaerobic conditions
• Nitro group is stepwise reduced through nitrobenzene to phenylhydroxylamine to 2-aminophenol. The phenol ring subsequently opens up and oxalocrotonate is formed with the release of ammonia

Trinitro toluene -TNT

• no efficient biodegradation pathway
• Under anaerobic and microaerophilic conditions, nitro groups of TNT are reduced one by one to amino groups, each subsequent reduction slower and less complete 
• If conditions are aerobic, the partially reduced intermediates form very complex and mutagenic azo condensation products 
• No mineralization, but only polymerization or binding of residues

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Bioprospecting - Secondary metabolites, Antibiotics, antiviral agents, immunosuppressive agents and therapeutic agents

  Secondary metabolites

    Plants select competent endophytes from the environment for their own ecological benefit. Many diverse and highly specialized secondary metabolites and enzymes are produced by the inhabiting microorganisms. Microbial production of manifold molecules like phytohormones, antibiotics and quorum-sensing molecules has been reported in plants. Cytokinins and auxins are plant growth regulators expressed by a vast amount of microbes, especially bacteria. Microbial indole 3-acetic acid production can be of importance to plants, either beneficial by promoting root development or harmful as in crown gall formation induced by Agrobacterium tumefaciens.

 Antibiotics

    Penicillin, the classical example of  natural antibiotics is produced by Penicillium spp. Streptomycin is produced by Streptomyces spp. The production of such compounds is common among microorganisms. Examples of antimicrobial compounds synthesized by plant associated microbes include cyclosporine that is produced by the endophytic fungus Tolypocladium inflatum and shows antifungal activity.  Bioactive compounds produced by plant associated microbes display antiviral activity also. Xiamycin from Streptomyces sp. is reported to have anti-HIV properties.

Antifungal ecomycins derived from the plant endophyte Pseudomonas viridiflava. The ecomycins are active against such human-pathogenic fungi as Cryptococcus neoformans and C. albicans. Another group of antifungal compounds is the Pseudomycins, produced by a plant-associated pseudomonad. They are active against a variety of plant- and human-pathogenic fungi. Pseudomycins are also effective against a number of ascomycetous fungi, they are also being considered for agricultural use. Ambuic acid, a cyclohexenone produced by a number of isolates of Pestalotiopsis microspora found in rainforests around the world, possesses antifungal activity. 

Antifungal peptide cryptocandin, produced by Cryptosporiopsis quercina is active against a number of plant-pathogenic fungi. Cryptocandin and its related compounds are in use against a number of fungi causing diseases of skin and nails. Cryptocin, possesses potent activity against  plant-pathogenic fungi. Antibiotics Turbomycin A and B are isolated from a soil derived metagenomic library.

Antiviral Compounds

Endophytic microorganisms also produce antiviral agents which cause inhibition of viruses. Two novel human cytomegalovirus protease inhibitors, cytonic acids A and B, have been isolated from the endophytic fungus Cytonaema sp. The potential for the discovery of compounds, from endophytes, having antiviral activity is in its infancy. However, some compounds have been found is promising. The main limitation in compound discovery is related to the absence of appropriate antiviral screening systems in most compound discovery programs.

Immunosuppressive Compounds 

Immunosuppressive drugs are used today to prevent allograft rejection in transplant patients, and in the future they could be used to treat autoimmune diseases such as rheumatoid arthritis and insulin-dependent diabetes. The endophytic fungus Fusarium subglutinans, isolated from Tripterygium wilfordii, produces the immunosuppressive, non-cytotoxic diterpene pyrones subglutinol A and B. The fungus Tolypocladium inflatum from which cyclosporine, a beneficial immunosuppressant, was isolated is another example

Anticancer Agents from Endophytic Fungi

    Paclitaxel, the world's first billion-dollar anticancer drug, is produced by many endophytic fungi,  Taxomyces andreanae.  T. andreanae is found symbiotically associated living within yew tree, Taxus brevifolia. Paclitaxel is used to treat a number of other human tissue-proliferating diseases as well. Trichothecium sp, Pestalotiopsis microspora, Tubercularia and many novel endophytic fungal species produce paclitaxel to protect their respective host plant from degradation and disease caused by plant pathogens. Torreyanic acid, another anticancer agent was isolated from a Pestalotiopsis microspora strain. Fungal genera as Xylaria, Phoma, etc produce cytochalasins, compounds with antitumor and antibiotic activities

Antioxidants from Endophytes

Pestacin and Isopestacin, obtained from culture fluids of P. microspora, has antimicrobial as well as antioxidant activity.

Insecticidal compounds from Endophytes 

Nodulisporic acids, which are indole diterpenes exhibiting potent insecticidal properties are isolated from an endophyte, a Nodulisporium sp., from the plant Bontia daphnoides. Another endophytic fungus, Muscodor vitigenus, yields naphthalene as its major product. Naphthalene, the active ingredient in common mothballs, is a widely exploited insect repellant. 

Antidiabetic Agents from Rainforest Fungi

An endophytic fungus, Pseudomassaria sp. collected from an African rainforest produces an antidiabetic compound which acts like insulin but, unlike insulin, is not destroyed in the digestive tract and may be given orally. These results may lead to new therapies for diabetes

Haloaromatics- Halobenzenes, halophenols and halobenzoates

Recalcitrant Halocarbons

• Haloalkyls
• Haloaromatics

Chlorobenzenes are industrial solvents. Such halo aromatics have low biodegradability under aerobic conditions, with increasing number of halo substituents.

Under anaerobic conditions, such extensively halogenated aromatics are dehalogenated by dehalogenases. Sequential removal of halo substituents occurs, which in turn makes the product less susceptible to further dehalogenation. Thus, Hexachlorobenzene -HCB is most readily dehalogenated to 1,3,5-trichlorobenzene. Further dehalogenation to monochlorobenzene is possible but less readily catalyzed. 

Hexachlorobenzene à1,3,5 trichlorobenzene (dechlorination with ease)

1,3,5 trichlorobenzene à -à  1,4 dichloro benzene (further dechlorination slow and incomplete)

1,4 dichloro benzene -à monochlorobenzene (dechlorination slow and incomplete)

Monochlorobenzene resist dechlorination anaerobically and is stable under anaerobic conditions.

Aerobic degradation of tetra and trichlorobenzene is difficult. Mono and dichloro benzenes degraded with ease aerobically, by the dioxygenases of Pseudomonas and Alcaligenes species forming chlorocatechols.

Degradation of aromatic compounds requires the opening of the aromatic ring. Bacteria must possess enzymes that either

Ø  cleave the ring of the aromatic compound, in spite of the halogen substituents

Ø  catalyse the removal of the halogen substituents prior to the dearomatising reactions.

Chlorophenols

Highly chlorinated pentachlorophenol (PCP) is used as preservatives for wood and canvas. Other chlorophenols find uses in synthesis of pesticides, resins, dyes and pharmaceuticals. They resist degradation since they are highly toxic to microorganisms. In low concentrations, microorganisms degrade them under aerobic and anaerobic conditions.

Aerobically,

Highly halogenated aromatic compounds are particularly resistant to biodegradation by aerobic microorganisms because chlorine substituents interfere with the action of dioxygenase enzymes that oxidatively cleave aromatic rings. Therefore, biodegradation of these compounds requires initial removal of the halogen substituents from the aromatic rings.

The aerobic degradation of PCP proceeds by the action of monooxygenases, to produce tetrachloro-para- hydroquinone; further degradation continues by Pseudomonas, Sphingomonas sp. and Mycobacterium sp. removing chlorine substituents to yield trihydroxybenzene. Further ring opening yields simpler products such as CO₂ and water.

Anaerobically, Reductive dechlorination of PCP occurs in anaerobic sediments and in anaerobic sludges complete degradation of PCP has been observed. The degradation products 3,4,5-trichlorophenol, 2,5-dichlorophenol, and 3-chlorophenol are further subjected to ring opening producing CO₂ and water.

The sulfate-reducing bacterium Desulfomonile tiedjei reductively dehalogenates PCP and other chlorophenols.

Lactic Acid

•  First organic acid produced by microorganisms
• Commercial production by fermentation or synthetic processes

Uses

• Lactic acid is used as acidulant  (give sour, or acidic flavor to foods)/ flavouring/ pH buffering agent or inhibitor of bacterial spoilage in a wide variety of processed foods. It is non-volatile, odorless, has a mild acidic taste and is classified as GRAS (generally regarded as safe). 
• It is a very good preservative and pickling agent. 
• Addition of lactic acid aqueous solution to the packaging of poultry and fish increases their shelf life.
• The esters of lactic acid are used as emulsifying agents in baking foods (glyceryl lactostearate, glyceryl lactopalmitate). 
• Technical grade lactic acid is used in leather tanning industries. Various textile finishing operations and acid dying of food require low cost technical grade lactic acid for pH adjustment, hardening baths for cellophanes used in food packaging, terminating agent for phenol formaldehyde resins, lithographic and textile printing developers, adhesive formulations etc
• Lactic acid has many pharmaceutical and cosmetic applications and formulations in topical ointments, lotions, anti acne solutions, dialysis applications. 
• Calcium lactate can be used for calcium deficiency therapy and as anti caries agent. 
• Polymers of lactic acids are biodegradable thermoplastics. These polymers are transparent and their degradation can be controlled by adjusting the composition, and the molecular weight. Their properties approach those of petroleum derived plastics. 
• Polymers of lactic acids have medical applications as sutures (stitches), orthopaedic implants, controlled drug release etc.
• Poly L-lactic acid with low degree of polymerization can help in controlled release or degradable mulch films for large-scale agricultural applications.

Biosynthesis

Lactic acid fermentation is a metabolic process by which glucose is converted into pyruvate and then anaerobially, into lactate. Lactic acid fermentation is an anaerobic fermentation reaction that occurs in some bacteria and animal cells, such as muscle cells invoving lactate dehydrogenase enzyme

Theoretically, from I mole of glucose, 2 moles of lactate obtained - 90% yield obtained practically.

• Microorganisms

Commercial production using fermentaion with Lactic acid bacteria

Two kind:

Homofermentative-Maximum substrate conversion into lactic acid. Not much production of byproducts or cell biomass. Used in commercial lactic acid production. Eg. Lactobacillus delbrueckii, L. bulgaricus

Heterofermentative- Produce some lactic acid, and a great amount of byproducts like CO₂, ethanol, acetic acid etc. Not suitable for commercial lactic acid production. Eg. Leuconostoc mesenteroides

 Lactobacillus has complex nutritional requirements, as they have lost their ability to synthesize their own growth factors. They cannot grow solely on carbon source and inorganic nitrogen salts. Organisms such as Rhizopus oryzae have less limiting nutritional requirements and can utilize starch feed stocks. They are able to produce pure L (+) lactic acid. Saccharomyces cerevisiae and Kluyveromyces lactis also can produce of L (+) lactic acid.

The choice of an organism primarily depends on the carbohydrate to be fermented. Lactobacillus delbreuckii are able to ferment sucrose. Lactobacillus bulgaricus is able to use lactose/whey. Lactobacillus helveticus is able to use both lactose and galactose. Lactobacillus amylophylus and Lactobacillus amylovirus are able to ferment starch. Lactobacillus lactis can ferment glucose, sucrose and galactose. Lactobacillus pentosus have been used to ferment sulfite waste liquor.

 L. casei, Rhizopus oryzae and S. lactis are also employed. These are all facultative anaerobes and bioreactors need not be completely anaerobic

Stock culture maintained in skim milk media. Milk used for building up of inoculum since it is an ideal medium. Culture transferred and incubated in increasing amounts to sterile skim milk and finally whey.

Selection of microorganism depends on the carbon source being used

• Media and Conditions

1) Carbon Sources

1. Glucose, maltose, sucrose, lactose etc.
2. Crude substrates like Corn starch, potato starch, molasses, whey, sulfite lyes etc
3. Starch is pretreated with amylase/acid hydrolysis to break down into simple sugars (Glucose, maltose)
4. Sulphite lyes steam stripped to eliminate SO₂ and alkali treatment to remove lignin-pretreatment
5. Sugar conc. Usually adjusted to 5-20%, usually 12%.
6. Whey is a major source since it contains, lactose

Choice of carbon source depends on

Ø  Availability

  Ø Treatments required prior to fermentation

  Ø  Cost

        Whey

• Byproduct of dairy industry, cheese industry
• Value addition by converting whey into lactic acid
• It contains, carbohydrates, nitrogenous substances, vitamins and salts
• Large quantities produced annually as part of dairy industries

2) Nitrogen sources

Ammonium salts like ammonium hydrogen phosphate (0.25%)

3) Growth factors & mineral sources

• Complex nutrients, especially Vit B 12 complexes
• Supplied by enriching the medium with malt sprouts and other crude vegetable sources (care taken to avoid overheating of such materials, during drying)
• Minerals added as per the requirement of microorganisms

4)  pH

• 5.5-6.5 - maintained using CaCO₃
• The acid produced can accumulate and should be neutralized or removed for improved growth and yield
• At low pH values, no other bacterial contamination. High pH values can inhibit the gowth of Lactobacilli and inhibit fermentation

 5)  Temperature

• Depend on the microorganism used
• 45-50⁰C for Lactobacillus delbreuckii or Lactobacillus bulgaricus
• 30⁰C for Lactobacillus pentosus, L. casei, Streptococcus lactis

6) Aeration and Agitation

• anaerobic fermentation; No supply of sterile air
• Facultative anaerobes are used for fermentation so, complete air removal not needed
• Broth usually stirred to keep CaCO₃ in suspension

 7)  Time

• 5-10 days
• Usually completed using 12-13% glucose supply

8) Yield

• L⁺ lactic acid produced commercially.  93-95% of weight of glucose supplied is converted to lactic acid.
• Free lactic acid toxic to the organism- removed continuously by electro dialysis or continuous culture.
• Fermentation carried out in wooden lined tank/stainless steel tank to avoid corrosion.

 Recovery

Method of recovery depends on the type of grade required- food grade, technical grade or plastic grade. Broth heated to dissolve calcium lactate, then filtered. Calcium precipitated by adding H₂SO₄. Lactic acid concentrated and further purified.

 Plastic grade lactic acid by esterification with methanol after concentration

Technical grade lactic acid- Calcium present removed as calcium sulfate dihydrate by precipitation. Filtration to remove the precipitate of CaSO₄ dihydrate and further concentration to 35-40% lactic acid by evaporation.

Food grade lactic acid- Fermented broth filtered followed by acidification of filtrate. Calcium precipitated as CaSO_4, washed_, filtrate treated with activated carbon to remove organic impurities. Concentration of crude lactic acid to 25% solids by evaporation. Refining and evaporation repeated to obtain 65% total acidity. Sodium Ferro cyanide treatment to remove heavy metals (eg., copper) by precipitation to remove discolouration. Ion exchange chromatography finally to remove traces of contamination.

 References

• A Text Book of Industrial Microbiology: (2nd edition By Wulf Crueger & Anneliese Crueger)
• Industrial Microbiology: (By Casida L. E.New Age international (P) ltd publications)