ARACHIDONIC ACID AND OTHER FATTY ACID FROM

MICROORGANISM

Arachidonic acid

• Is a polyunsaturated fatty acid (PUFA) that is present in the phospholipids (especially phosphatidylethanolamine, phosphatidylcholine and phosphatidylinositides) of membranes of the body's cells, and is abundant in the brain, muscles, liver.

• in the human body usually comes from dietary animal sources—meat, eggs, dairy—or is synthesized from linoleic acid.

• essential fatty acids required by most mammals

Arachidonic acid (cont.)

• Some mammals lack the ability to or have a very limited capacity to convert linoleic acid into arachidonic acid

need supplement commercial source of arachidonic acid has been derived, from the fungus Mortierella alpina

Similarity of ARA (top) with prostaglandin hormone (bottom)

The importance of Arachidonic acid in body

• repair and growth of skeletal muscle tissue

• one of the most abundant fatty acids in the brain (similar quantities to DHA docosahexaenoic acid)

Microorganism for commercial production Mortierella alpina

Colony formation of filamentous fungi Mortierella. Genus Mortierella could be classified into two subgenera Mortierella (A) and Micromucor (B). Strains of subgenus Mortierella form rose-like colony on agar plate. While all the strains of subgenus Mortierella can produce C20 fatty acids, strains of subgenus Micromucor can only produce fatty acids up to C18. (A) Mortierella alpina 1S-4: (B) Mortierella isabellina CBS 194.28.

Growth of Mortierella fungi on agar plate containing tetrazolium salt. (A) When M. alpina 1S-4 grew on a agar medium containing triphenyltetrazolium chloride (TCC), oils formed could be stained red. (B) M. isabellina CBS 194.28 grown on the same medium.

Oil drops accumulated in the cells of Mortierella alpina 1S-4. A large number of oil drops could be observed, when M. alpina 1S-4 grown in a liquid medium

Mortierella alpina

Mortierella alpina

Oil globule

Other microorganisms

• Genetic engineered Yarrowia lipolytica capable of producing greater than 10% arachidonic acid (ARA, an omega.-6 polyunsaturated fatty acid) in the total oil fraction (Patent 7588931 )

• Mucor circinelloides (gamma linoleic acid)

• Rhizopus sp.(gamma linoleic acid)

Rhizopus spp Mucor circinelloides

Mucor circinelloides colonies incubated in various temperature

Biosynthesis of fat in microorganism

Production of ARA by microorganisms

• Submerged fermentation and Solid substrate

• Solid substrate using rice bran, wheat bran, peanut meal residue, and sweet potato residue

Production step

1. Culture Media and Conditions

2. Submerged Fermentation and Solid state fermentation

3. Downstream processing

Ad 1. Culture Media and Conditions

• Mortierella was grown at 20°C in a culture containing (mg /l): glucose, 10; yeast extract, 5 and agar, 20 at pH 6.5.

• Mycelia were harvested from culture and blended with a micro-Waring blender for mycelial suspension.

Ad 2. Submerged Fermentation

• Submerged basal medium contained (mg/l) soluble starch, 20; Bacto yeast extract, 5; KNO3, 10; KH2PO4, 1 and MgSO4•7H2O, 0.5 at pH 6.5.

• The broth was inoculated with 5% (v/v) mycelial suspension and shaken at 200 rev min-1 and at 20°C for 2 to 10 days. Each ml of mycelial suspension contained 1.0-1.5 × 106 mycelial fragments.

Ad 2. solid state fermentation

• Media contained (g) solid substrate (rice bran, wheat bran, peanut meal residue, sweet potato residue, or a mixture of sweet potato residue and rice bran) 100; Bacto yeast extract (Difco, Michigan) 2.5; KNO3 5; KH2PO4 0.5 and MgSO4•7H2O 0.25.

Factors affecting ARA production

1. Initial moisture content

2. Initial pH

3. Incubation temperature

4. Supplement of nitrogen

5. Supplement of oil

Analyzing of fatty acids

• The lipids were extracted with a 5 times volume of chloroform/methanol (2:1, v/v) by an ultrasonicator for 2 h and concentrated by rotary evaporator at 50°C.

• The residue was dissolved in 1 ml of 0.5 M KOH-methanol solution, and methylated with 1 ml of 20% (w/v) of BF3-methanol complex.

• The methylated fatty acids were separated from the water layer by adding saturated NaCl and anhydrous Na2SO4, and then dissolving in n-hexane.

Downstream Process

• Cells were homogenized to break the cells walls so that the products can be extracted

• Extraction by butane crude fatty acids are obtained

• Purification of fatty acids by hexane and citric acid followed by bleaching, deodorization and filtration

Procedure to analyze fatty acids from microorganisms to select potential microorganisms that produced highest fatty acid

Figure 1. Time course of biomass, pH, cell protein, and PUFA production in submerged fermentation with Mortierella alpina ATCC 3222. Culture medium was incubated at 20°C with orbital shaking at 200 rev min-1.

Figure 2. Time course of moisture content, pH, cell protein, and PUFA production in rice bran solid substrate fermentation with Mortierella alpina ATCC 3222. Solid substrate with an initial moisture content of 65% and an initial pH of 6.5 were statically incubated at 20°C.

Lipid formation and γ-linolenic acid production by Mucor circinelloides and Rhizopus sp., grown on

vegetable oil • The submerged cultivation were carried out in 250 mL Erlenmeyer

flasks with 50 mL of medium containing 1 - 4% oils (palm, canola, soybean oil that had been used for frying, sesame, or sunflower) or 1 - 4% carbohydrates (galactose, maltose, malt extract, sorbitol) and 1% yeast extract as nitrogen source.

• Each flask was inoculated with 1 ml of freshly prepared spore suspension. For M. circinelloides, malt extract was also used replacing maltose in the medium.

• The cultures were agitated continuously for 72 hours at 150 rpm, at 25ºC and allowed to stand for 48 hours without agitation at 12ºC.

• The biomass produced was separated using vacuum filtration in No. 1 Whatman filter paper. The wet biomass was placed in pre-weighed beakers at 105ºC for 48h to determine the dry weight.

• After part of the resulting biomass was set aside for later extraction of the fatty acids; it was dried by storage for 5 days in an oven at 55ºC.

Downstream process (cont.)

• Approximately 100 mg of mycelia were used to extraction of lipid using chloroform: methanol: water (2:1:0,8) and the solvent removed in a nitrogen atmosphere.

GLA = gamma linoleic acid, AA= arachidonic acid

Docosahexaenoic acid (DHA)

• is a long-chain polyunsaturated omega-3 fatty acid

• important for brain, eye and heart health throughout the lifecycle

• Can be produced by algae vegetarian DHA

DHA's structure

• a carboxylic acid(~oic acid) with a 22-carbon chain (docosa- is Greek for 22) and six (Greek "hexa") cisdouble bonds (-en~); the first double bond is located at the third carbon from the omega end.

• Its trivial name is cervonic acid, its systematic name is all-cis-docosa-4,7,10,13,16,19-hexa-enoic acid,

• shorthand name is 22:6(n-3) 

Applications and function

• infant formulas, products for pregnant and nursing women, food and beverage products and dietary supplements.

• DHA possesses a variety of immune modulating effects.

• DHA was found to inhibit growth of human colon carcinoma cells, more than other omega-3 PUFAs.

Source of DHA from microbial process

• microalgae; Crypthecodinium cohnii and another of the genus Schizochytrium (example : Schizochrytium limacinum)

• Pavlova lutheri seawater media atau synthetic medium

• Isochrysis galbana

• Phytium irregulare

• etc

Schematic representation of Crypthecodinium cohnii celldrawn from Perret et al. (1991). A Ventral view. B Dorsal view. Eepisome, H hyposome, L.F. longitudinal flagellum, T.F. transverseflagellum, C cingulum. Bar 5 μm. Reproduced with permission of theCompany of Biologists

Scanning electron micropgraph of a Crypthecodinium speciesshowing the ventral view (from Parrow et al. 2006). Reproduced withpermission of the Editor-in-Chief of the Afr. J. Mar. Sci.

Production of DHA from algae

Example of DHA media composition produced by algae

• Glucose or glycerol 10 g/l

• Yeast extract 1 g/l

• Pepton 1 g/l

Diluted in artificial seawater

Composition of artificial seawater/liter

• 18 g Na Cl• 2.44 g MgSO4.7 H2O• 0.16 g KCl• 1 g Tris buffer• 1 g NaNO3• 0.3 g CaCl2.2H2O• 0.005 g KH2PO4• 0.0027 g NH4Cl• 15 x 10-8 vit B12• 3 ml chelated iron solution• 10 ml trace element (Boron, Cobalt, Managanese, Zinc,

Molybdenum)• pH 7.5-8.0

Fermentation condition

• 20oC• 170 rpm• 10 % inoculum • pH 7.5 – 8.0

DHA is produced in early stage of stationary phase

Products of microbial fatty acids