Organic Acids

Chapter 15, Basic Biotechnology

Most produced organic acids

1. Citric Acid: 1 600 000 tons/a - C6

2. Lactic Acid: 300 000 tons/a - C3

3. Acetic Acid: 190 000 tons/a - C2

They are most microbially produced. Adipic acid, in contrast, is mostly produced other way. 

Difference between fermentation and partial oxidation with the examples of the produces

Fermentation: microbial degradation of organic matter to produce energy without the involvement of external electron acceptors (O2) or nitrate (NO3-).

• Disproportionation, where compounds of higher and lower oxidation states occur - CO2.

• Anaerobic. 

• Lactate, Succinate.

Partial Oxidation: excretion of not fully oxidized produced, despite O2 being available as terminal hydrogen acceptor. 

• Aerobic.

• Citrate, Itaconic Acid, Gluconate. 

Energetic Result of glycolysis

Glucose → 2 Pyruvates

2 ATP, 2 NADH

What bacteria can produce lactic acid?

Lactococcus sp., Lactobacillus so., Streptococcus sp.

Gram+ and non sporous.

What is the purpose of LA starter culture in food?

To improve taste, smell, texture and/or preservation.

35% of daily consumed food is dairy.

Examples of products made with spontaneous inoculation

Cocoa, Coffee, Beer, and Wine

Examples of products made with directed fermentation and starter culture

Bread, Sauerkraut, Fermented milk products

Name cultures and their functions

Starter: fast pH reduction in milk

Maturing: flavour development

Protective: protection from other M/O

Probiotic: health

What are products from the sugar fermentation of heterfermentative LA bacteria? For what are they used?

CO2, acetic and lactic acids

They are used for flavour and texture.

Examplesof LA usage in food industry and chemistry

Food Industry: flavour, preservative, antioxidant

Chemistry: precursor for enantiomerically pure herbicides, biodegradable polymers (implants, food packaging, polymers)

How many lactates come from 2 pyruvate? What enzyme facilitates the reaction?

2, so 2 lactates per 1 mol of glucose
2 NAD+ are produced

Lactate dehydrogenase

Describe Lactic acid bacteria

Gram +

Obligate fermenters = anaerobic (Lactobacillus delbrueckii) OR facultative anaerobs (Lactobacillus plantarum)

GRAS

Homofermentative strains: Lactobacillus bulgaricus

In what form cna one buy LA bacteria?

Suspension (~10¹¹ /mL), cut proof masses (~10¹² /mL) or freeze-dried powder

Requirements to cultivate LA bacteria

1. LA → buffering or titration for the optimal range

2. Complex building blocks are needed (vitamins, purines) → complex media

3. Optimum pH 5.0 - 6.9

4. Mesophilic: 18-32 °C, 37-45 °C

Why are optimum start sugar conditions necessary?

If there is too little sugar (lactose), production might be insufficient, less dry matter.

If there is too much sugar, at the end, not all the sugar will be consumed, and growth will be inhibited (substrate = product inhibition).

22 g/L → 43 g/L → 82 g/L

How to determine the optimal time to harvest a culture of lactic acid bacteria (LAB) by looking at the titration curve?

Based on the peak NaOH addition, one observes the maximum of metabolic activity. After this sharp peak, the cells stop producing lactic acid, so the culture should be harvested immediately and cooled to preserve the viability of the culture (cold shock),

Most important anaplerotic reactions

Pyruvate → malate (Malic Enzyme)

Pyruvate → OAA (Pyruvate Carboxylase)

Phosphoenolpyruvate → OAA (OAA Carboxylase)

Malate → OAA (malate dehydrogenase)

What is glyoxylate cycle?

Isocitrate (Isocitrate Lyase) → Succinate

Isocitrate → Glyoxylate (Malate Syntase) → Malate

What is citrate-malate shuttle?

Citrate produced in mitochondria must be transported out into the cytosol, in exchange for malate.

Why is export out of сitrate from the cytoplasm to the medium important?

It helps to achieve pH homeostasis, pH < 2 → export

Reasons for product inhibition

• Osmotic Pressure

• pH change

• Exporters Overload

Name important steps in citric acid production

• separation of mycellium. Supernatant: citric acid, oxalic acid, residual sugar

• oxalic acid precipitation with Ca²⁺

• citric acid precipitation with Ca(OH)₂ → Calcium Citrate

• Ca₃-citrate + H₂SO₄ → citric acid + CaSO₄ (gypsum)

• Purification: activated carbon (decolourization) + ion exchange chromatography

What is an alternative to precipitation with gypsum?

Concentration with ion exchange chromatography or precipitation with other salts, e.g., ammonium that can be reused as fertilizer

Explain difference btwn “3+1” and “2+2” pathways for the production of succinic acid

3 + 1
Inputs:

• 3 carbons from PEP / pyruvate

• +1 carbon from CO₂ fixation

Core reactions:

PEP → OAA (PEP carboxylase)
OAA → malate (requires NADH)
Malate → fumarate
Fumarate → succinate (requires NADH)

2 + 2
Inputs:

• 2 carbons from acetyl-CoA

• 2 carbons via condensation with glyoxylate
  → total 4 carbons = succinate

2 + 2 is better because it generates NADH

Advantages of Saccharomyces over Bacteria for the succinic acid production

A process used by DSM/Roquette.

Low pH values production: so, during the downstream, one doesn’t have to adjust the pH.

Lower salts load due to pH correction

Lower by-products: ethanol, glycerol, malate

Easier preparation

Why is continuous process good for the production of organic acids with bacteria?

Many org.acids are inhibitory for bacteria. If the product is removed continuously, the production will be higher.

What acids are produced anaerobically, what aerobically?

Aerobic: citric, gluconic, itaconic

Anaerobic: lactic, acetic, succinic