Download Microbial Production of Organic Acids

Microbial Production of Organic Acids
Zulal ÖZDEMIR
Institute of Chemical Technology, Prague
Faculty of Food and Biochemical Technology,
Department of Biochemistry and Microbiology
166 28, Praha 6
CZECH REPUBLIC
May, 2014
Microbial products often classified as primary and secondary metabolites.
 Primary metabolites are considered essential to microorganisms for proper
growth. They include amino acids, nucleotides, and fermentation end
products such as ethanol and organic acids.
 Secondary metabolites do not play a role in growth, development, and
reproduction, and are formed during the end or near the stationary phase of
growth. Most antibiotics and the mycotoxins fall into this category.
What are the organic acids?
Organic acids are organic compounds with acidic properties. The most
common organic acids are the carboxylic acids whose acidity is associated
with their carboxyl group -COOH.
Organic acids have been used as
 Food additives
 Preservatives for preventing food deterioration and extending the shelf life
of perishable food ingredient.
The organic acids produced by various microbes via fermentation
Figure 1. Diagram of the Carboxylic acid group
What is fermentation?
Fermentation has always been an important part of our lives:
 Foods can be spoiled by microbial fermentations,
 Foods can be made by microbial fermentations, (alcoholic beverages or acidic
dairy products.)
 Muscle cells use fermentation to provide us with quick responses.(way of getting
energy without using oxygen)
But how fermentation actually works was not understood until the work of Louis
Pasteur in the latter part of the nineteenth century.
**The breaking down of complex organic substances into simpler ones.
Fermentation is a metabolic process that converts sugar to acids, gases
and/or alcohol. It occurs in yeast and bacteria, but also in oxygen-starved muscle
cells, as in the case of lactic acid fermentation.
What are advantages of fermentation over
chemical sythesis for production of organic
acids?
The chemical synthesis of organic acids :
 Requires very harsh conditions
 Involves many steps which make their large scale production impractical.
The microbial fermentation is:
 A very simple method to synthesize these organic acids in very pure form.
 Require less energy input and are cost effective due to simple media
formulations in many cases.
 All acids of the tricarboxylic acid (TCA) cycle can be produced microbially
in high yields(citric acid), other acids can be derived indirectly from the
Krebs cycle such as itaconic acid, or can be derived directly from glucose
(gluconic acid).
 Some acids are formed as the end products from pyruvate or ethanol (lactic
and acetic acid).
 Large-scale commercial production of a number of organic acids are citricgluconic- and itaconic acid. Other organic acids produced in lower scale are
lactic acid, malic acid, gibberellic acid, and kojic acid.
 Citric acid is by far the major organic acid in worldwide production.
Table 1. Worldwide organic acid production
Citric Acid
Citric acid (2-hydroxypropane-1,2,3-tricarboxylic acid) is one of the
world’s major fermentation products with the annual production of over
550,000 tonnes, and its demand is increasing at the rate of 2-3% every year.
 It was first isolated in 1784 from lemon juice and crystallized by Scheele.
Until the 1920s, citric acid was extracted from the lemon juice and referred
as ‘natural citric acid’.
Whehmer first time in 1923 described that citric acid is a metabolic
product of Penicillium and Mucor.
 In 1923, Pfizer became the first industry to produce citric acid through
fermentation based process in USA, by culturing Aspergillus niger in surface
culture in a medium containing sucrose and mineral salts.
As on today most of the citric acid is produced by fermentation, and the
major producers are located in Western Europe, USA and China.
Figure 2. Structure of Citric acid
 Produced by many microorganisms including
filamentus fungi, yeasts and bacteria. All of them
could be used to produce citric acid, however, the
mutants of A. niger are generally used for
commercial use..
 Carbohydrate sources such as beet molasses,
sucrose, commercial glucose, starch hydrolysis etc.
The raw material is diluted to 20-25 per cent sugar
concentration and mixed with nitrogen source and
other salts.
 The pH of the medium is maintained around 5.0,
when molasses are used and pH is adjusted at 3.0
when sucrose is used. The fermentation is carried
out either under the surface, submerged or under
solid state conditions.
 Citric acid fermentation by Aspergillus niger is
greatly enhanced in the presence of trace metals
such as iron, zinc, copper, manganese etc. in the
medium..
Table 2. Microorganisms which produce citric acid
The first stages of citric acid formation involve
the breakdown of hexoses to pyruvate in
glycolysis, followed by its decarboxylation to
produce acetyl CoA
CO2 released during this reaction is not lost, but
is recycled by pyruvate carboxylase which is
produced constitutively in Aspergillus.
Normally, oxaloacetate would largely be
supplied through the completion of the TCA
cycle, allowing recommencement of the cycle by
condensing with acetyl CoA to form citrate,
catalysed by citrate synthase. However, in order
to accumulate citrate, continuation of the cycle
must be blocked. This is achieved by inhibiting
aconitase, the enzyme catalysing the next step in
the TCA cycle. Inhibition is accomplished by
removal of iron, an activator of aconitase.
Consequently, during citrate accumulation, the
TCA cycle is largely inoperative beyond citrate
formation.
Colonies of Aspergillus niger
The black dots covering the colonies are Aspergillus spores.
The growth medium is Saboraud's Dextrose Agar (SDA)
Citric acid has GRAS (generally regarded as safe) status and its major
applications are summarized below
Lactic Acid
 Lactic acid (2-hydroxypropanoic acid) was discovered and isolated
in 1780 by the Swedish chemist Scheele from sour milk and the first
organic acid produced microbiologically in 1881 by Charles E.
Avery at Littleton, Massachusetts, USA.
 It is classified as GRAS (generally regarded as safe) by Food and
Drug Authority (FDA) in the USA and its annual consumption is
estimated to be 30 000 tonnes.
 Lactic acid is used in various industries for different applications
Figure 3. Structure of lactic acid
Major applications of lactic acid are summarized below
Lactic acid bacterias
There are two groups of lactic acid bacteria, one is heterofermentative and
other is homofermentative.
 The heterofermentative (e.g. Luconostoc mesenteroides) lactic acid bacteria
produce many byproducts other than lactic acid and are not suitable for
commercial processes.
 The homofermentative bacteria (Lactobacillus sp.), very little substrate is
used for producing cell mass and other metabolites and majority of the carbon
source is converted into lactic acid.
Some Lactobacillus species and their preferred carbon sources
Lactic acid fermentation
 Used carbohydrates: corn starch, potato starch, molasses and whey. When
starchy materials are used, they are initially hydrolysed to simple sugars.
 The medium is supplemented with a nitrogen source and calcium carbonate
and the fermentation is carried out by inoculation with homofermentative
Lactobacilli.
 During the fermentation the temperature is controlled at 40-50° C
depending on the organism. The medium is kept in constant agitation.
 After completion of the fermentation for 4-6 days, the fermented liquor is
heated to 82°C and then, filtration is done.
Itaconic Acid
In 1931, itaconic acid was first shown to be a metabolic product of Aspergillus
itaconicus and soon after, it was discovered that some strains of A. terreus also
excrete this type of organic acid. In today’s commercial production processes,
mutants of both strains are used.
Formed in a branch of the TCA cycle via decarboxylation of cis-aconitate
which is normally followed by its oxidation to itatartaric acid.
Onward metabolism of itaconic acid must be prevented in commercial
fermentations, otherwise yield is reduced. This is achieved by formulating the
medium with high levels of calcium ions, thereby inhibiting itaconic acid
oxidase, which catalyses the oxidation of itaconic acid to itatartaric acid.
Figure 4. Structure of lactic acid
Acetic Acid
Acetic acid production from alcoholic liquids has been carried out for
nearly 10,000 years.
 The Romans and the Greeks used diluted vinegar as a refreshing drink,
produced the vinegar by leaving wine open to the air.
 The first industrially manufactured vinegars were produced in flat open
vats. This process involved a film of bacteria forming over the surface of
the wine. This was a very slow process and it wasn’t until the nineteenth
century that surface fermentations were developed into more rapid
procedures.
 Many fermentative bacteria produce acetic acid, but only members of the
acetic acid bacteria group are used in commercial production. This group
can be divided into two genera, Gluconobacter and Acetobacter.
Figure 5. Structrure of acetic acid
Acetic Acid Fermentation
 Gluconobacter:
Oxidizes ethanol solely to acetic
acid. Members of the genus
Gluconobacter are not over
oxidizers. Mixed cultures appear
during production, even when the
inoculum is assumed pure.
 Acetobacter:
Oxidize ethanol first to acetic acid
and then further to CO2 and H2O.
Members of the genus Acetobacter
are gram negative and acid tolerant.
Gluconic Acid
 Gluconic acid (2,3,4,5,6-Pentahydroxycaproic acid) is used in the food and
beverage, pharmaceutical, detergent and construction industries.
 It is produced using the organism Aspergillus niger.
 The growth medium in this fermentation is based on glucose and corn
steep liquor with other nutritional requirements including ammonium salts
and urea. Too much nitrogen leads to excessive growth of organism
therefore producing lower acid yields.
 The production pH is maintained at 6.0-7.0 until optimum growth and a set
glucose oxidase level has been achieved. After this point, the pH may be
allowed to drop to 3.5. The temperature is maintained throughout the
process at 33°C.
 This fermentation requires a vast amount of aeration, as the process is
highly aerobic.
Figure 6. Structrure of gluconic acid
Gluconic Acid Fermentation
Gluconic acid is produced extracellularly!
 The spontaneous conversion of β-D-Glucose from α-D-Glucose is
accelerated by the enzyme mutarose in A. niger,
 β-D-Glucose is converted to D-glucono-δ-lactone by glucose oxidase. This
enzyme is induced by glucose presence at pH values >4 and is denatured
below pH 2.
 The conversion of D-glucono-δ-lactone to D-Gluconic acid is spontaneous at
neutral pH. At lower pH, this conversion is less effective and facilitated by
D-glucono-δ-lactonase by A. niger. This enzymatic process has the added
advantage that no product purification steps are required after the
fermentation.
Thank you for your attention! 