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1 Departemen Teknologi Industri Pertanian FATETA - IPB
m.k TEKNOLOGI BIOINDUSTRI TIN 330 (2-3) ORGANIC ACID Production Technology and Its Prospect to be Developed in Indonesia Departemen Teknologi Industri Pertanian FATETA - IPB

2 ORGANIC ACID Mayor Organic Acid : Citric acid, lactic acid, L-ascorbic acid, gluconic acid  Table In anaerobic bacteria  their formation parallels growth (growth associated product) In aerobic bacteria and fungi  accumulation of organic acids is the result of incomplete substrate oxidation and is usually initiated by an imbalance in some essential nutrients, e.g mineral ions Different physiological prerequisites for formation of organic acids



5 CITRIC ACID Commercial production of citric acid is generally by submerged fermentation of sucrose or molasses using the filamentous fungus A. niger or synthetically from acetone or glycerol However synthetic methods proved to be unsuitable because of expensive or hazardous raw materials or an excessive number of reaction steps leading to low yields. C6H8O7 2-hydroxypropane-1,2,3-tricarboxylic acid

6 APPLICATION Citric acid is produced either in the anhydrous form (crystallization from hot aqueous solutions ) or as the monohydrate (crystallization at temperatures below 36.6 ° C). Food : a. The dominant use of citric acid is as a flavoring and preservative in food and beverages (soft drinks). b. Citrate salts used to deliver those minerals in a biologically available form in many dietary supplements. Pharmaceutical : Used with sodium bicarbonate in effervescent formulae,for example in antacid and soluble aspirin preparation.

7 Cleaning and Chelating Agent
excellent chelating agent, binding metals. It is used to remove scale from boilers and evaporators. soften water, which makes it useful in soaps and laundry detergents. By chelating the metals in hard water, it lets these cleaners produce foam and work better without need for water softening. active ingredient in some bathroom and kitchen cleaning solutions. Plastic Citric acid esters, particular triethyl, tributyl and acetyltributyl esters are employed as non-toxic plasticizers in plastic films used to protect food stuffs.

8 http://medicalmnemonics4u. blogspot. com/2009/11/citric-acid-cycle

9 Microorganisms Many microorganisms have been evaluated for the
production of citric acid including : Bacteria such as Bacillus licheniformis, B. subtilis, Corynebacterium spp. Fungi such as A. niger, A. awamori, A. foetidus, Penicillium restrictum Yeast such as Candida lipolytica, C. intermedia and Saccharomyces cerevisiae However, A. niger a filamentous fungus remained the organism of choice for citric acid production due to ease of handling, its ability to ferment a variety of cheap raw materials, and high yields of citric acid.

10 Medium : CHO source could be starch, starch hydrolysate, cane juice, glucose, sucrose or molasses  sucrose was the most favourable carbon source followed by glucose, fructose and galactose. Nitrogen source : ammonium salts are preferred, e.g. urea, ammonium sulfate, ammonium chlorite, peptone, malt extract, etc. Mineral : zinc, manganese, iron, copper and magnesium affect citric acid production. Potassium dihydrogen phosphate has been reported to be the most suitable phosphorous source.  Growth of A. niger on high concentrations of sugars and low concentrations of Fe3+ and Mn2+ gives high yield of citric acid

11 pH A low initial pH has the advantage of checking contamination and inhibiting oxalic acid formation. pH of 2.2 optimum for the growth of the mould as well as for the production of citric acid. Higher pH i.e. 5.4 and has been found optimum for citric acid production in molasses medium. Aeration : Increased aeration rates led to enhanced yields and reduced fermentation time. It is important to maintain the oxygen concentration above 25% saturation and interruptions in oxygen supply may be quite harmful. Aeration is performed during the whole fermentation with the same intensity through the medium at a rate of 0.5 to 1.5 vvm.


13 Submerged fermentation (SmF) :
Liquid fermentation Submerged fermentation (SmF) :  the commonly employed technique for citric acid production. It is estimated that about 80% of world production is obtained by SmF. Advantages : higher yields and productivity and lower labour costs are the main reasons for this. Two types of fermenters, stirred tank fermenters and tower fermenters are employed, although the latter is preferred due to the advantages it offers on price, size and operation

14 http://www. scielo. br/scielo. php

15 Inoculum (seed culture)
Inoculation by adding : a suspension of spores, or of pre-cultivated mycelia. Spores are produced in glass bottle on solid substrate at 250C with incubation time of days. When spores are used, a surfactant is added in order to disperse them in the medium. For pre-cultivated mycelia (germinate), an inoculum size of 10% of fresh medium is generally required. Normally, submerged fermentation is concluded in 5 to 10 days depending on the process conditions. It can be carried out in batch, continuous or fed batch systems, although the batch mode more frequently used.

16 Morphology of the mycelium :
is crucial not only in relation to the shape of the hyphae , but also in the aggregation of the growth into small spherical pellets. The mycelial pellets should be small (0.2 to 0.5 mm) with a hard surface. This state of affairs is brought about by a deficiency of manganese in the medium or the obviously related additions of ferrocyanide ion.

17 Where a separate inoculum stage is employed, a suspension of spores of A. niger, usuaIly grown on a solid medium, is introduced into sterilized medium in the inoculum fermenter. The medium is aerated and, in some processes, agitated and the mould allowed to grow at a temperature of about 30°C for a period of from 18 to 30 hours as judged by pH level reached . Downstream Process

18 Surface Fermentation with A. niger
 liquid surface culture (LSC) - Medium - beet molasses as raw material is still extensively employed by major manufacturers although somewhat labour intensive, the power requirements are less than in the submerged fermentation. - Additional nutrients and alkali ferrocyanide After sterilized and cooling, the prepared medium is run down into a series of trays supported on racks in a ventilated chamber. The trays are filled to a depth of between 0.05 and 0.20 m.

19 Surface Fermentation with A. niger
Inoculum Spores of A. niger are obtained by growing a selected strain on a sporulation medium. The spores are collected and distributed over the surface of the medium in the trays. Physical Conditions Sterile air is supplied to the fermentation chamber. The air performs the dual function of supplying oxygen and carrying away fermentation heat and the rate of flow of the air is regulated accordingly. A temperature in the region of 30 °C is often employed.

20 The mycelium forms a coherent film on the surface of the
liquid becoming progressively more convoluted. The removal of the heavy metals by the ferrocyanide severely restricts sporulation. · After a period of 7 to 15 days the trays are emptied and the mycelium separated from the fermented liquor. · The liquors are pumped forward to the recovery section. · Unwanted by-products of the process are gluconic and oxalic acids. In many processes oxalic, acid production is minimized by careful strain selection

21 Citric Acid Production
using Surface Fermentation

22 Surface fermentation of citric acid
Citric acid manufactures using microbial conversion of sugar beet molasses fermentation based on potassium hexacyanoferrate use for control of trace metal effect on fermentation ..

23 PRODUCT RECOVERY The recovery of citric acid from liquid fermentation is generally accomplished by three basic procedures : 1. Precipitation 2. Extraction Adsorption and Absorption (mainly using ion exchange resins). Separation of citric acid from the liquid : calcium hydroxide is added to obtain calcium citrate tetrahydrate → wash the precipitate→ dissolve it with dilute sulfuric acid, yield citric acid and calcium sulfate precipitate → bleach and crystallization → anhydrous or monohydrate citric acid.

24 Ca(OH)2 calcium citrate tetrahydrate Downstream Process of Citric Acid

25 Solid-state Fermentation (SSF)
Citric acid production by SSF (the Koji process) was first developed in Japan and is as the simplest method for its production. SSF can be carried out using several raw materials. Generally, the substrate is moistened to about 70% moisture depending on the substrate absorption capacity. The initial pH is normally adjusted to and the temperature of incubation can vary from 28 to 30°C. The most commonly organism is A. niger in simple tray type fermenters. One of the important advantages of SSF process is that the presence of trace elements may not affect citric acid production so harmfully as it does in SmF. Consequently, substrate pre-treatment is not required.

The use of alternative raw materials to produce citric acid by SmF, LSC, and SSF seems to be a suitable possibility. However, it is necessary to adapt the right type of raw material to the right technique e.g. cassava bagasse employed as substrate in SSF, or cellulose hydrolysate used in SmF. The need of some pre-treatment of raw materials may enhance the fermentation efficiency. Another area is the strain improvement with improved substrate utilization efficiency.


28 Lactic acid production (
Lactic acid is widely used as an acidulant and preservative in foods and as a precursor for production of emulsifiers, such as stearoyl-2-lactylates, for the baking industries. New applications such as its use as a monomer for production of biodegradable plastics (PLA) and as an environment-friendly chemical and solvent will increase future lactic acid demand The molecular formula for lactic acid is C3H6O3.

29 http://redalyc. uaemex. mx/redalyc/html/847/84711261007/84711261007_2

30 The group consist of a number of gram positive bacteria which include the genera; Aerococcus, Bifidobacterium, Carnobacterium, Enterococcus, Lactococcus, Lactobacillus, Lactosphaera, Leuconostoc, Microbacterium, Oenococcus, Pediococcus, Streptococcus, Tetragenococcus, Vagococcus and Weissella Two groups based on the amount of lactic acid produced as the end product : Homolactics.: those that produce lactic acid as its only or major end product using the glycolitic pathway (EMP ) Lactobacillus acidophilus, Lactobacillus bulgarius, Lactobacillus delbrueckii, Lactobacillus helveticus, Streptococcus thermophilus and Enterococcus faecium normally use this homolactic fermentation process

31 These homolactics are able to produce twice as much energy then heterolactics as homolactics produce two lactic acid molecules from one glucose molecule Heterolactics produce only one molecule of lactic acid along with carbon dioxide and ethanol or acetate as its major products. The heterolactic fermentation process (Fosfoketolase pathway) is normally used by Bifidobacterium breve, Bifidobacterium longum, Bifidobacterium infantis, Bifidobacterium thermophilum, Lactobacillus fermentum, Lactobacillus salivarius, Lactobacillus casei, Lactobacillus rhamnosus and Lactobacillus plantarum

32 BAL Homofermentatif Homolactic acid bacteria (Lactobacillus, Lactococcus and most streptococci)  used to ferment milk and milk products in the manufacture of yogurt, buttermilk, sour cream, cottage cheese, cheddar cheese, and most fermented dairy

33 2 CH3CHOHCOOH Asam laktat
Reaksi pada Fermentasi & Pemanenan Asam Laktat a) Fermentasi dan Netralisasi fermentasi C6H12O6  Karbohidrat  Ca (OH)2 Kalsium hidroksida      + (2CH3CHOHCOO- ) Ca2+ kalsium laktat + 2H2O (b) Hidrolisis oleh H2SO4 (2CH3CHOHCOO- ) Ca2+ Kalsium laktat + H2SO4 Asam sulfat  2 CH3CHOHCOOH Asam laktat + Ca SO4 Kalsium sulfat The broth containing calcium lactate is filtered to remove cells, carbon treated, evaporated and acidified with sulphuric acid to get lactic acid and calcium sulphate. The insoluble calcium sulphate is removed by filtration; lactic acid is obtained by hydrolysis, esterification, distillation and hydrolysis

34 2 CH3CHOHCOOH Asam laktat + CH3OH Metanol
(c) Esterifikasi 2 CH3CHOHCOOH Asam laktat + CH3OH Metanol  CH3CHOHCOOCH3 Metil laktat + 2H2O  distilasi (d) Hidrolisis oleh H2O CH3CHOHCOOCH3 Metil laktat + 2H2O 2 CH3CHOHCOOH Asam laktat + CH3OH Metanol 

35 (calcium lactate) Lactic Acid Downstream Process

36 Lactic Acid Production



39 Acetic Acid (Vinegar) Acetic acid is produced industrially both synthetically and by bacterial fermentation. About 75% of acetic acid made for use in the chemical industry is made by the carbonylation of methanol. In this process, methanol and carbon monoxide react to produce acetic acid according to the equation: CH3OH + CO → CH3COOH Previously, this acid was produced by oxidation of acetaldehyde. This remains the second-most-important manufacturing method, 2 CH3CHO + O2 → 2 CH3COOH The fermentation accounts for only about 10% of world production, but it remains important for the production of vinegar, as many food purity laws stipulate that vinegar used in foods must be of biological origin.

40 Vinegar is typically 4-18% acetic acid by mass.
Vinegar is used directly as a condiment, and in the pickling of vegetables and other foods. Table vinegar tends to be more diluted (4% to 8% acetic acid) Cellulose acetate, a synthetic textile also used for photographic film. The major use of acetic acid is for the production of vinyl acetate monomer (VAM). Vinyl acetate can be polymerized to polyvinyl acetate or to other polymers, which are components in paints and adhesives. The major esters of acetic acid are commonly used solvents for inks, paints and coatings. The esters include ethyl acetate, n-butyl acetate, isobutyl acetate, and propyl acetate.

41 Acetic Acid Production by Fermentation
Acetic acid bacteria of the genus Acetobacter have made acetic acid, in the form of vinegar. Given sufficient oxygen, these bacteria can produce vinegar from a variety of alcoholic foodstuffs. Commonly used feeds include apple cider, wine, and fermented grain, malt, rice, or potato mashes. C2H5OH + O2 → CH3COOH + H2O A dilute alcohol solution inoculated with Acetobacter and kept in a warm, airy place will become vinegar over the course of a few months. Industrial vinegar-making methods accelerate this process by improving the supply of oxygen to the bacteria.


43 The ‘slow’ Process Vinegar used to be produced by keeping wine in open, partially filled containers. Addition of some vinegar speeded up the reaction. This concept was already incorporated into the process known as the ‘Orleans process’, the ‘French method’ or the ‘slow process’. In this process, wooden barrels are partially filled with good quality vinegar which acts as a source of inoculum and wine is added at weekly intervals for four weeks. After five weeks, a portion of vinegar is withdrawn and the same amount of wine is introduced and the process is repeated resulting in a very slow continuous process.

44 The ‘slow’ Process

45 The ‘quick’ Process The idea that vinegar can be produced. rapidly by trickling wine through packed pumice was discovered. It was improved by Schiizen-bach to make it the ‘quick process’, also known as the ‘German process’, which is the basis for modern methods of manufacture of vinegar using a generator. The generator consists of a wooden or metal-coated tank packed with beech wood shavings on which cells are allowed to grow. The feed trickles from the top through the wood shavings. A large volume of air is sparged into the tank through perforations in the bottom. Employing 12% (v/v) alcohol, 98% conversion into acetic acid is attained in 5 days by this process.

46 Diagram of Trickling Generator for Vinegar

47 Frings Process Major improvements in the quick process with forced aeration and temperature control were introduced and the trickling generator widely used today emerged. The significant advantages of this process include the following: 1. the cost is low, it is relatively simple and easy to control 2. higher acetic acid concentrations are obtained 3. the tank occupies less space 4. evaporation losses are low


49 The Submerged Culture Process
The application of submerged fermentation to the oxidation of ethanol to acetic acid was the next technical advance in the commercial production of vinegar. Acetabacter species in submerged conditions were very sensitive to oxygen-deficiency and that the fermentation stopped when the level of oxygen in the gas phase was less than 5%. The success of this process depends largely on the efficiency of the aeration of the broth. Nowadays, most vinegar is made in submerged tank culture,

50 The advantages of submerged cultivation over the
trickling generator are: 1. the submerged cultivation permits 30 times faster oxidation of alcohol 2. a smaller reactor volume is needed (about 16% of the trickling generator) to produce an equivalent amount of vinegar 3. greater efficiency is achieved; yields are 5-8% higher and more than 90% of the theoretical yield is obtained 4. the process can be highly automated 5. increased economy owing to the elimination of clogging by shavings, interruptions, etc. The ratio of productivity to capital investment is much higher in the case of submerged cultivation.

51 Anaerobic Fermentation
The genus Clostridium or Acetobacterium can convert sugars to acetic acid directly, without using ethanol as an intermediate. C6H12O6 → 3 CH3COOH These acetogenic bacteria produce acetic acid from one-carbon compounds, including methanol, carbon monoxide, or a mixture of carbon dioxide and hydrogen: 2 CO2 + 4 H2 → CH3COOH + 2 H2O Clostridium bacteria are less acid-tolerant than Acetobacter. But at present, it remains more cost-effective to produce vinegar using Acetobacter than to produce it using Clostridium. As a result, although acetogenic bacteria have been known since 1940, their industrial use remains confined to a few niche applications.

52 The acetic acid bacteria grow on the surface of the liquid and a gelatinous zoogloeal mat known as the ‘mother of vinegar’ is formed; this contains a large number of bacteria. This process was modified by immobilization of the cells on a floating light wooden grating on the surface of the medium so that addition of alcoholic solution did not break the mat.

53 Recovery and Purification
The physical methods of separation of acetic acid from water include : i. Fractional distillation ii. Azeotropic dehydration distillation iii. Solvent extraction iv. Extractive distillation v. Caron adsorption In extractive distillation counter current washing of mixed vapors in a distillation column takes place via descending stream of high boiling point liquid, which is a preferential solvent for one of the components.

54 The most economic method for the recovery of acetic acid from a dilute aqueous stream is by extraction with a hydrocarbon followed by distillation. The extraction methods suggested are ion-exchange resins, solvent extraction and membrane separation. A membrane system has been developed having a hollow fiber membrane device consisting of silicone rubber tubing. This system is resistant to biological attack and does not suffer mechanical fatigue.


56 Kojic acid ( C6H6O4 ) Kojic acid is a by-product in the fermentation process of malting rice (koji process), for use in the manufacturing of sake, the Japanese rice wine As chelation agent produced by several species of fungi, especially Aspergillus oryzae Cosmetics : excellent effects in even toning the skin, fighting age spots, pregnancy marks, freckles as well as general skin pigmentation disorders

57 Microorganisms : flavus, A. oryzae , A. Tamarii and A. parasiticus were reported to have the ability to produce large amounts of kojic acid. Medium : A variety of carbon containing substrates may be used as carbon sources : starch, sucrose, maltose, glucose, fructose, mannose, galactose, xylose, arabinose, sorbitol, acetate, ethanol, glycerol and arabinose Organic nitrogen sources are generally better than inorganic nitrogen sources for kojic acid fermentation. Complex organic nitrogen sources such as peptone and yeast extract may contain vitamins, which act as a precursor for kojic acid production.

58 Furthermore, some organic nitrogen sources have a good buffering system, while inorganic nitrogen sources, such as ammonia, excessively reduce the culture pH during NH4+ absorption. Low pH may influence the synthesis of kojic acid during fermentation and inhibit its growth. Mineral : medium containing KH2PO4, KCl, NaNO3, MgSO4 and FeSO4.7H2O is favourable for the growth of kojic acid producing fungus (A. flavus and A. oryzae) and kojic acid production Batch submerged fermentation gives the highest efficiency in terms of yield, maximum kojic acid concentration and also overall productivities.


60 Vitamin C

61 Production of Ascorbic Acid (Vitamin C)
Vitamin C commercially produced by : extraction from plants, by chemical synthesis, by fermentation and by mixed fermentation/chemical synthesis methods. (Fermentation is a chemical reaction in which a microorganism or enzyme). The manufacture of vitamin C is now carried out in two ways : The Reichstein process & Two-stage Fermentation Process First Step : Glucose is reduced at high temperature to produce sorbitol . Sorbitol is oxidized into sorbose by fermentation.

62 The Reichstein process
Mixed fermentation/chemical synthesis method (employed by Roche, BASF and Takeda). Chemical Synthesis : Sorbose is reacted acetone  di-acetone sorbose - Di-acetone sorbose is then oxidized using chlorine and sodium hydroxide  di-acetoneketogulonic acid (DAKS). DAKS is dissolved in solvents and its structure is rearranged to form vitamin C, using an acid catalyst. In the last production step, the crude vitamin C is purified by recrystallization.

63 Two-stage Fermentation Process :
Developed in China and is used by all Chinese producers. The use of the process has also been licensed to a number of Western manufacturers, including Roche and a joint venture involving BASF and Merck. Second Fermentation step : Sorbose is fermented to produce keto gulonic acid (KGA). - KGA then undergo ring-closing lactonization via dehydration  Ascorbic acid (Vit C) The two-stage fermentation process makes less use of toxic solvents and reagents  reduction in the cost of processing waste products.

64 Methods of producing vitamin C
Reichstein process (mixed fermentation & chemical synthesis method.) Glucose *Sorbitol is made by hydrogenation glucose at high temperature. & pressure Sorbose + aceton  diaceton sorbose Diacetone sorbose, then oxidized using chlorine and sodium hydroxide to produce diacetoneketogulonic acid (DAKS). Purified by recrystallization Sorbitol* Fermentation by Acetobacter Sorbose Diaceton L-sorbose DAKS Raw vitamin C Pure vitamin C

65 Two-step Fermentation Process
Sorbitol fermentation Sorbose KGA Raw vitamin C Purified vitamin C KGA (keto gulonic acid) then undergo ring-closing lactonization via dehydration  Ascorbic acid (Vit C)

66 Many raw vitamins can be unstable if affected by temperature, light, acidity or alkalinity. To prevent degradation in use, these vitamins may be coated, combined with other products or, alternatively, various chemical derivatives of the vitamins may be produced. Producers market vitamin C in various forms : powder and granular forms, different purity levels, and chemical derivatives such as sodium ascorbate and calcium ascorbate. Sodium ascorbate was preferred as an antioxidant as it would not discolour meat. Calcium ascorbate is also commonly produced for use as a combined source of calcium and vitamin C in supplements.


68 REFERENSI ( ( industrialbiotechnology/lecture-notes/lecture-15.pdf) ( Rai University Luciana P. S. Vandenberghe I,II; Carlos R. Soccol I *; Ashok Pandey I; Jean-Michel Lebeault II Microbial Production of Citric Acid . Braz. arch. biol. Technol. vol.42 no.3  Curitiba  1999 Rosfarizan Mohamad, Mohd Shamzi Mohamed, Nurashikin Suhaili, Madihah Mohd Salleh and Arbakariya B. Ariff . Kojic acid: Applications and development of fermentation process for production. Biotechnology and Molecular Biology Reviews Vol. 5(2), pp , April 2010 The production of vitamin C (

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