Chap 15 Organic Acids
Introduction
Various organic acids accumulate in eucaryotic and procaryotic cells.
For anaerobic bacteria: usually the organic acid accumulation is a means by which they regenerate NADH, and hence accompanies growth.
For aerobic bacteria and fungi: the organic acid accumulation resultsfrom incomplete substrate oxidation and is usually initiated by an imbalance in some essential nutrients.
Annual production of major organic acids treated in this chapterKilotons/year
Citric acid (酸味料) / 900
Gluconic acid (葡萄糖酸) / 60
Lactic acid (乳酸) / 50
L-Ascorbic acid (vitamin C) / 60
I.Citric Acid:
Originally found as a constituent of lemons, it is an intermediate of TCA cycle and can be found in almost all living organisms.
Fermentation of Aspergillus niger accounts for 95% of worldwide production.
Biochemical pathways
The molar yield of citric acid can be up to 90% of the consumed glucose, but the reason is not understood although the pathway is known.
Regulation of citric acid accumulation by nutrient parameters
Although citric acid can accumulate to high level, this can be achieved only under certain strict conditions.
Sugar type and concentration:
Sugars that can be quickly metabolized by fungus (e.g. sucrose, maltose or glucose) allow high yield and productivity.
Major carbon source raw materials: Beet (甜菜) and cane (果樹的主莖) molasses (糖漿). But the quality is variable from season to season and from refinery to refinery. Other alternatives have been reported such as starch, cotton waste(see Table 15.3).
If the carbon concentration is below 100g/l, the yield of citric acid from glucose decreases significantly. High sugar concentration can induce additional glucose transport system and improve the yield.
Trace metal ions
Usually trace metal ions (Fe, Zn, Cu, Mn, Co) are essential for A. niger growth, some of them (Mn2+, Fe3+, Zn2+) have to be present at growth-limiting concentrations (i.e. can’t be high) to give high yields.
The effect of Mn2+ is striking becauseits concentrationas low as 2 g/l can reduce the acid accumulation by 20%.
Since these metal ions are easily introduced together with the high concentration of carbon source, all carbon sources have to be purified to reduce metal concentrations by:
precipitation (for beet or cane molasses): can add ferrocyanide (氰化亞鐵) to complex the metal ions, then precipitate.
cation exchange chromatography (for glucose syrup)
The exact reason for this requirement for metal limitation is not clear. One of the explanations is that a low concentration of Mn2+ increases the flux of carbon through glycolysis, alters the composition of A. niger plasma membrane and impairs the electron transport chain (so citric acid produced can exit the TCA cycle).
pH
The pH has to be below 2.5 to get high yield. Due to the pK of citric acid, pH 1.8 is reached when it is accumulated in the medium that contains no other buffering agents. However, some carbon sources used (e.g. beet molasses) contain a lot of other a.a. (particularly glutamate) which strongly buffer the medium pH to 45. So pH needs to be controlled by addition of HCl.
DO tension
DO higher than the required amount for growth is essential, and even sparging with pure oxygen is in use.
Sudden interruption in the air supply can irreversibly impair citric acid production without harming the mycelial growth, thus close monitoring and control of DO are critical.
Nitrogen, phosphate (skip)
Production process for citric acid
Two major types: surface process and submerged process
- Surface process
carried out in aluminum trays, filled with medium to a depth of 50200 cm. Spores are distributed over the surface, sterile air is passed over them. A final yield of 0.7-0.9 g/g supplied sugar can be obtained within 7 to 15 days.
Older and labor-intensive, but is still in use due to the low requirement in power and high reproducibility.
- Submerged process
Higher efficiency due to higher degree of automation.
Disturbance in O2 supply makes it more difficult to manage.
Two types of reactors: stirred tank and tower fermenters.
Characteristic pattern:the germinating spores form stubby (粗短的), forked hyphae, which aggregate to small (0.2 to 0.5 mm) pellets (with a firm, smooth surface)and sediment quickly when harvested. This morphology is critical in obtaining high yields and is dependent on nutrient conditions. Microscopic observation is therefore a convenient indicator.
Applications of citric acid
Used in industry for food, sugar (21%) and beverages (45%) for its pleasant taste (酸味料) and low toxicity.
Also used in pharmaceutical (8%) and detergent/cleaning industry (19%).
II.Lactic Acid
First isolated from sour milk in 1798. Production by fermentation began in 1880.
Lactic acid bacteria associated with foods include Lactobacillus, Streptococcus(鏈球菌), Enterococcus (腸球菌), etc.
Production process
Raw materials often used include glucose syrup (from starch hydrolysis), maltose-containing materials, sucrose (e.g. from molasses) or lactose (whey乳清, 酸牛奶中去除凝乳部分).
Reactor volume: up to 100 m3
Carbon source: 120-180 g/l and appropriate N and P-containing salts and micronutrients.
Other nutrients: crude vegetable materials (e.g. malt sprouts) because lactate bacteria require vitamin B and some a.a.
Conditions: >45C with gentle stirring (lactic bacteria are anaerobic and O2 should be avoided).
pH: 5.5-6.0 by the addition of calcium carbonate.
Yields: 85-95% of the theoretical maximum is obtained after 4-6 days.
Applications: food additive (乳酸飲料), production of sour flour and dough (酸味料), intestine treatment, biodegradable polymers, etc.
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