Assignment Topic

Microbial Production of Organic acid

Submitted by:
Shakeela Rasheed
Bisma Arzoo hashmi

3493
3494

Submitted to:
Sir Afzaal
Smester:
7th Evening (E).
Department:
Home and Food science & Nutrition

G.C.University Faisalabad

Microbial production of organic acid

Organic acid
An organic acid is an organic compound with acidic properties. The most
common organic acids are the carboxylic acids, whose acidity is associated with
their carboxyl group COOH. Sulfonic acids, containing the group SO2OH, are
relatively stronger acids

List of Common Organic Acids

citric Acid
Tartaric acid
Glutamic Acid

Barbituric Acid
Gluconic Acid
Hexanoic Acid
Lactic Acid
Malic Acid
Odliec Acid
Folic Acid
Propionic Acid
Stearic Acid
Uric Acid
Acetic Acid
Gallic Acid

Microorganism
A microorganism (from the Greek word mikros, "small" and , organisms, "organism")
is a microscopic living organism which may be single called or multicellular. The study
of microorganisms is called microbioloy, a subject that began with the discovery of
microorganisms in 1674 by Antonie van Leeuwenhoek using a microscope of his
own design

Essential use of MicroorganismMicroorganisms play an important role in our

life: helps us to digest our food, decompose wastes and participate in various cycles.
microorganisms present in us than there are cells, and the various microorganisms are
bacteria, viruses, fungi and protozoa.
Applications of microorganisms in the food industry, mainly in the production of dairy
products are another example where microorganisms are beneficial to humans.
Lactobacillus bulgaricus and Streptococcus thermophilus converts lactose in milk into
lactic acid causing the milk to coagulate, during fermentation (reduction and oxidation
of organic molecules), and form yoghurt in the proces.

Production of Organic acids by Microorganism

Table
Sr.
#

List of Organic
acids

Microorganisms

Citric acid

Aspergillus niger

Gluconic acid

Acetic acid

Tartaric acid

Succinic acid

Glutamic acid

A.niger , Penicillum
spicies
Acetobacter ,
Acetomonas spicies
A.niger , Aspegillus
griseus
Anaerobiospirillum
succinicproducens
Arthrobacter globiformis

Lactic acid

Lactobacillus
planarum,lactic acid
bacteria

INTRODUCTION

Citric acid
Citric acid (C6H8O7, 2 - hydroxy - 1,2,3 - propane tricarboxylic acid), a natural
constituent and common metabolite of plants and animals, is the most versatile and
widely used organic acid in the field of food (60%) and pharmaceuticals (10%). It
has got several other applications in various other fields. Currently, the global
production of citric acid is estimated to be around 736000 tones/year (Qumica e
Derivados, 1997), and the entire production is carried out by fermentation. In Brazil,
almost the entire demand of citric acid is met through imports. There is constant
increase (3.5-4%) each year in its consumption, showing the need of finding new
alternatives for its manufacture.

Historical developments

Citric acid was first isolated by Karls Scheels in 1874, in England, from the lemon
juice imported from Italy.The industrial process was first open by Currie, in 1917,

who found that Aspergillus niger had the capacity to accumulate significant
amounts of citric acid in sugar based medium.

Applications of citric acid

Citric acid is mainly used in food industry because of its pleasant acid taste an its
high solubility in water. It is worldwide accepted as "GRAS" (generally recognized as
safe), approved by the Joint FAO/WHO Expert Committee on Food Additives.

MICRO-ORGANISMS USED FOR CITRIC ACIC PRODUCTION

A large number of micro-organisms including bacteria, fungi and yeasts have been
employed to produce citric acid. Most of them, however, are not able to produce
commercially acceptable yield.
only A. niger and certain yeasts such as Saccharomycopsis sp. are employed for
commercial production. However, the fungus A. niger has remained the organism of
choice for commercial production.
The main advantages of using this micro-organism are:
(a) its ease of handling,
(b) its ability to ferment a variety of cheap raw materials, and
(c) high yields.

PRODUCTION TECHNIQUES AND RAW MATERIALS

Although citric acid is mostly produced from starch or sucrose based media using
liquid fermentation, a variety of raw materials such as molasses, several starchy
materials and hydrocarbons have also been employed.
Rohr et al. (1983) classified raw materials used for citric acid production in to two
groups:
(i)

(ii)

with a low ash content from which the cations could be removed by
standard procedures (e.g. cane or beet sugar, dextrose syrups and
crystallized dextrose)
raw materials with a high ash content and high amounts of other non
sugar substances (e.g. cane and beet molasses, crude unfiltered starch
hydro-lysates).

Liquid fermentation
Submerged fermentation:

The submerged fermentation (SmF) process is the commonly employed technique

for citric acid production. It is estimated that about 80% of world production is
obtained by SmF. Several advantages such as higher yields and productivity and
lower labour costs are the main reasons for this.
Two types of fermenters,
1. conventional stirred fermenters and
2. tower fermenters are employeds.
In SmF, different kinds of media are employed such as sugar and starch based
media. Molasses and other raw materials demand pre-treatment, addition of
nutrients and sterilization. Inoculation is performed either by adding a suspension of
spores, or of pre-cultivated mycelia. When spores are used, a surfactant is added in
order to disperse them in the medium. For pre-cultivated mycelia, 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.

Surface fermentation:
The first individual process for citric acid production was the liquid surface culture
(LSC), which was introduced in 1919 by Socit des Produits Organiques in Belgium,
and in 1923 by Chas Pfizer & Co. in US. After that, other methods of fermentation,
such as submerged fermentation were developed. Although this technique is more
sophisticated, surface method required less effort in operation and installation and
energy cost (Grewal
In the classical process for citric acid manu-facture, the culture solution is held in
shallow trays (capacity of 50-100 L) and the fungus develops as a mycelial mat on
the surface of the medium. The trays are made of high purity aluminium or special
grade steel and are mounted one over another in stable racks. The fermentation
chambers are provided with an effective air circulation in order to control
temperature and humidity. Fermentation chambers are always in aseptic conditions,
which might be conserved principally during the first two days when spores
germinate. Frequent contamination are mainly caused by Penicilia, other Aspergilli,
yeast and lactic bacteria (Rohr et al, 1983; Morgant, 1988). Refined or crude
sucrose, cane syrup or beet molasses are generally used as sources of carbon.
When applied, molasses is diluted to 15-20% and is treated with hexacyanoferrate
(HFC). nd Kalra, 1995).

Solid-state fermentation
Solid-state fermentation (SSF) has been termed as an alternative method to
produce citric acid from agro-industrial residues (Pandey 1991, 1992, 1994, Soccol

1994, Pandey and Soccol 1998). 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 4.5-6.0 and the
temperature of incubation can vary from 28 to 30C. The most commonly organism
is A. niger.
However there also have been reports with yeasts (Maddox and Kingston, 1983;
Tisnadjaja et al., 1996). 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.
Different types of fermenters such as conical flasks, glass incubators and trays, etc.
have been used for citric acid fermentation in SSF. Vandenberghe et al. (1999a,b)
used Erlen-meyer flasks and glass columns for the production of citric acid from
gelatinized cassava bagasse. Higher yields were obtained in flasks without any
aeration, and very little sporulation was observed. The same yields were found in
column reactors only with variable aeration. This showed great perspective to use
SSF process for citric acid production in simple tray type fermenters.

Process parameters
pH
Generally, a pH below 2.0 is required for optimum production of citric acid. A low
initial pH has the advantage of checking contamination and inhibiting oxalic acid
formation. A pH of 2.2 was reported to be optimum for the growth of the mould as
well as for the production of citric acid (Srivastava and De, 1980) whereas, a higher
pH i.e. 5.4 and 6.0-6.5 has been found optimum for citric acid production in
molasses medium (Roukosu and Anenih, 1980)

Aeration
is performed during the whole fermentation with the same intensity through the
medium at a rate of 0.5 to 1.5 vvm. However, because of economic reasons, it's
usually preferred to start with a low aeration rate (0.1 to 0.4 vvm). High aeration
rates lead to high amounts of foam, especially during the growth phase. Therefore,
the addition of antifoaming agents and the construction of mechanical "defoamers"
are required to tackle this problem.

http://www.scielo.br/scielo.php?script=sci_arttext&pid=S151689131999000300001

Gallic Acid
Gallic acid (3, 4, 5-trihydroxybenzoic acid) is an organic substance occurring in
many plants either as a free molecule or as part of tannic acid molecule .

Gallic acid is extensively used as a precursor for the commercial production of an

anti-microbial drug trimethoprim, a food preservative propyl gallate and some
dyestuffs.
Besides this, gallic acid possesses wide range of biological activities, such as
antioxidant, antibacterial, antiviral, analgesic etc. As antioxidant gallic acid acts as
an antiapoptotic agent and helps to protect human cells against oxidative damage.
Gallic acid is also found to show cytotoxic activity against cancer cells, without
harming normal cells. Because of its several interesting properties and commercial
applications, gallic acid is a compound of great interest to both pharmaceutical and
chemical industries.

Production of Gallic Acid

Conventionally gallic acid is produced by acid hydrolysis of tannic acid but it has
cost, yield and low purity disadvantages. Alternatively, gallic acid can be produced
by the microbial hydrolysis of tannic acid by tannase (tannin-acyl-hydrolase EC
3.1.1.20), an inducible enzyme, secreted by microorganisms. Microbial production of
tannase, especially from fungi, is well documented , however, the reports on tannic
acid hydrolysis is limited. Mainly Aspergilli have been used for hydrolysis of tannic
acid
to
yield
gallic
acid
among
bacteria Klebsiella
pneumoniae and Corynebacterium sp. have been reported to produce gallic acid
from crude extract of tara gallotannin.

The present study proposes a protocol for gallic acid production in a 5 liter
fermenter at 40 gL1 of tannic acid concentration extracted from Quercus

infectoria gall nuts usingAspergillus fischeri MTCC 150, a new fungus which
has not been reported so far.
A strain of A.fischeri MTCC 150 showing high tannase activity at shake flask
level was selected and maintained on PDA slants supplemented with 0.01%
tannic acid. Dry powder of tannic acid was obtained by aqueous extraction of
Chinese gall nuts (Q. infectoria) in a soxhlet apparatus.
The experiment was performed in a 5 liter capacity, top driven BIOFLO III, NBS
fermenter. The bioconversion was divided into two phases: z
(1) Growth phase: Three liters of growth medium was inoculated with 300 mL of
pre-induced, 30 h old culture of A. fischeri MTCC 150. The values of physical
parameters during growth phase were temperature: 30C, initial pH: 5.6, aeration:
1 vvm and agitation: 300 rpm.
(2) Bioconversion phase: During this phase tannic acid was added in two
installments of 25 gL1 and 15 gL1 after 32 h and 50 h respectively. The values of
physical parameters during this phase were- temperature: 35C, pH: 4.0 to 3.5 (not
regulated), aeration: nil, agitation: 250 rpm. The fermentation was stopped after 77
h of fermentation, when there was no significant increase in gallic acid formation.
The samples were analyzed at regular intervals for biomass, residual tannic acid
and gallic acid formed. Biomass was estimated by dry cell weight method at 105C,
whereas, tannic acid and gallic acid were estimated by following the two
wavelengths simultaneous estimation method of Bajpai and Patil.
In a new approach to microbial gallic acid production by Aspergillus fischeri MTCC
150, 40gL1 of tannic acid was added in two installments during the bioconversion
phase of the process (25gL1 and 15gL1 at 32 and 44h respectively).

The optimum parameters

for the bioconversion phase were found to be temperature: 35C,
pH: slightly acidic (3.33.5),
aeration: nil and
agitation: 250 rpm.
A maximum of 71.4% conversion was obtained after 71h fermentation with 83.3%
product recovery.
The yield was 7.35 g of gallic acid per g of biomass accumulated and the fermenter
productivity was 0.56 g of gallic acid produced per liter of medium per hour.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3768451/

Acetic acid
Vinegar production dates back at least to 200 BC, and it is an
illustrative example of microbial biotransformation. However, vinegar
has always been seen as a leftover in the family of fermented
products. Vinegar has been part of the human diet as a condiment and
food preservative, as well as the basis for simple remedies for people
and animals, since remote antiquity.
As mentioned in the review on vinegar history in 1732, the Dutchman
Boerhaave noted that the mother of vinegar was a living organism,
although he did not specify the role of this organism in the process of
acidification. We shall refer to this process as acetification instead of
the more popular acetous fermentation due to its strict requirement
for oxygen.

Despite some small local differences, in general, food regulations

consider vinegar to be the result of a double fermentation (alcoholic
and acetous or acetification) of any sugar substrate. worldwide most of
the vinegar produced is white vinegar, that is, vinegar produced
directly from diluted alcohol.

Production technology of vinegar

Apart from their different substrates, vinegars can also be
differentiated by their production systems. In traditional vinegars, the
transformation of ethanol into acetic acid is performed by a static
culture of acetic acid bacteria at the interface between the liquid and
air. The barrels are filled to 2/3 capacity to leave an air chamber, which
is kept in contact with the outside air using one of various types of
openings. This production system is called surface culture, and this
process is considered the traditional method.
The vinegars produced by this traditional system are generally
considered of high quality because of their organoleptic complexity.
In fact, the product quality results from

(i)

the raw material (wine or other substrate),

(ii)

the metabolism of the acetic acid bacteria, which produce some

additional transformations (mostly oxidation reactions, but also
ester formations, e.g.) on top of the basic transformation
(ethanol to acetic acid),

(iii)

the interaction between the vinegar and the wood from the
barrels, and

(iv)

the aging process, which integrates all of the preSubmerged

culture systems provide a much faster alternative.

These systems rely on suitable turbines to generate a flow of air

bubbles into the wine or alcoholic solution. The oxidative process
occurs in the air-liquid interfaces of the air bubbles. Improvements to
this process generally involve engineering (maintenance and
persistence of the bubbles in the liquid, uniformity of the bubble size,
recovery of lost aromas, etc.). In this type of vinegar, the bacteria
become bioreactors for the transformation of alcohol into acetic acid,
with only very limited production of other metabolites. The airflow also
contributes to considerable loss of the volatile compounds present in
the original wine, resulting in more organoleptically limited product
that was produced at a significantly lower cost.
Although early containers for submerged culture processing were
made of wood, the most current containers are stainless steel, which is
more hygienic and resistant to wear. Although the wood containers
were meant to provide some organoleptic complexity, there was hardly
any transfer from the wood to the vinegar because of the imbalance
between contact surface and volume and the speed of the process.
This limitation can be compensated for by subsequent aging in barrels
or incubation with wood fragments or wood chips, which may
contribute to the recovery of some missing organoleptic characters.
Despite the loss in product quality, this methodology has two
important advantages: speed (the vinegar is produced in cycles of 24
hours) and acidity (the product can reach concentrations of acetic acid
of up to 2325%, compared to 613% achieved with other systems).
Higher acidity helps to reduce transportation costs by reducing water
transport.viously mentioned characteristics.

Acetic acid bacteria

Although acetic acid bacteria are feared among oenologists because of

their negative effects on grapes and on wine in general, they are the
main agents in the production of vinegar. Acetic acid bacteria are Gram
negative or Gram variable, ellipsoidal or cylindrical, and can be
observed under the microscope alone, in pairs or in aggregates and
chains. Acetic acid bacteria have aerobic respiratory metabolism, and
oxygen is generally used as the final electron acceptor; however, other
compounds may occasionally act as final electron acceptors, allowing
the bacteria to survive under nearly anaerobic conditions, such as the
ones present during wine fermentation.
Some of the transformations performed by acetic acid bacteria are of
great interest to the biotechnology industry. Despite this interest, the
role of these bacteria in vinegar production remains their most familiar
and extensively used industrial application.
http://www.hindawi.com/journals/tswj/2014/394671/

Lactic acid in Food

Lactic acid is naturally present in many foodstuffs. It is formed by natural fermentation in
products such as cheese, yogurt, soy sauce, sourdough, meat products and pickled
vegetables.
Lactic acid is also used in a wide range of food applications such as bakery products,
beverages, meat products, confectionery, dairy products, salads, dressings, ready meals,
etc. Lactic acid in food products usually serves as either as a pH regulator or as a
preservative. It is also used as a flavoring agent.

Beverages
Because of its mild taste, lactic acid is used as an acidity regulator in beverages such as
soft drinks and fruit juices.

Meat,
Poultry
&
Fish
Lactic acid can be used in meat, poultry and fish in the form of sodium or potassium
lactate to extend shelf life, control pathogenic bacteria (improve food safety), enhance
and protect meat flavor, improve water binding capacity and reduce sodium.