INDUSTRIAL MICROBIOLOGY

1. Mentor: Ms. Renu NK Jaisinghani Prepared By : Ms.Shivani Shinde
Assistant Professor Ms. Priyanka Mahajan
Department of Microbiology Mr. Omkar Lobhi
Smt. CHM College

2.  Vinegar fermentation is the oldest fermentation occurring naturally – as unwanted
spoilage of Wine.
 The term Vinegar has originated from Dutch word Vinegar [Sour Wine].
Definition : Vinegar is fermentation derived food product containing not more
than 4gm acetic acid per 100 ml of vinegar with specific flavor and
aroma at 20ºC.

3. Types of vinegar
On the basis of substrate , used for getting alcohol it is named
accordingly
White vinegar - Grape wine
Spirit vinegar - Spirit
Alcohol vinegar - alcohol
Grain vinegar- grains- wheat, rice, and barley
Cider vinegar- apple cider
Malt vinegar- barley malt
Whey vinegar- whey
Fruit vinegar- fruits
Sugar vinegar- sugars syrup
Glucose vinegar- glucose
Rice vinegar- rice starch

4.  Vinegar production is 2 steps process-
1)Production of ethanol from sugar using
S. cereviciae var ellipsoid
2)Production of acetic acid (vinegar) from
alcohol using
Acetobacter aceti
Acetobacter curvum
Acetobacter orleanense
 Uses :
1) Used in home for preparation of salads and vegetables.
2) Used as preservatives and flavouring agents e.g. in pickles,
vegetables, fish , salads dressing etc.

5. 1) Natural processes or field processes or Let alone processes.
2) French method or slow method or Orleans method.
3) German method or quick method or packed generator (trickling
generator method).
4) Deep fermentation or submerged method or use of acetator.
First 3 methods are considered to be surface fermentation as
organisms grow as thick mat at the surface.

6. Large amount of oxygen is required as NADH2 released during reaction
enters in respiratory chain (ETC) and requires large amount of oxygen for
release of ATP, hence if oxygen is less than organisms cannot grow and
poor yield is obtained.
Biochemistry of Acetic Acid

7.  In natural processes, wine with alcohol content 10-13% is
taken in open pan and kept for few days at RT.
 Acetic acid bacteria grow as film (scum) at the surface
and carry out fermentation.
 Not good method - as quality and quantity varies with
each batch as no pure culture used or flora varies with
every batch.
 Also less yield is due to limited oxygen available.

8.  In this method , fermentation is carried out in big barrels.
 Initially barrels are filled with pure vinegar and 10-15 liters wine (alcohol
12%).
 Mixture is incubated at RT, pH =3.5 for 1 week is repeated for 5 weeks
and then after 5 weeks 10-15 liters wine is added and 10-15
liters fermented medium (vinegar) is withdrawal from barrel.
 Then processes becomes continuous there after.
 In this method , organisms grown as thick gelatinous mat ( called
mother of vinegar ) at the surface.
 To keep this mat growth at the surface for aerobic fermentation to
proceed, wooden raft or Frame is used to support mother of vinegar.
 Vinegar withdrawn is subjected to storage, aging and clarification.
Bottling and pasteurization.

9. Gives fine quality of vinegar.
- Slow hence costly
- Long incubation period hence non-acetobacter may
grow and produce lactic acid, propionic acid, esters etc.
- As use of open barrels there is loss of ethanol by
evaporation
- Poor yield
- Mother of vinegar may get disturbed while addition
of wine and this has effect on yield.
- It needs lots of attention and checking.

10. 1)In this, aerated medium ( wine with 12% alcohol and
pH=3.5) is inoculated with A. schutzanbachii culture and
trickle down through sprinkler over as generator packed
with beech wood shaving.
2)Organisms stick to solid support and start growing in
the form of zoogleal film.
This active zoogleal film of Acetobacter converts alcohol to
acetic acid, as it trickles down.
3)Trickling generator has cooling coils to control
temperature and collection chamber at the bottom to
collect product.
4)If vinegar collected is not of desirable strength
(concentration) then it can be pumped and taken to the
top of the generator and recycled till vinegar of desirable
strength is obtained.

11. 1) It consists of air tight tank, 14ft. In diameter and
15 ft. in height.
2) It has flat perforated bottom that divides tank
into upper fermenter and lower collection
chamber.
3) There is sprayer or disturbing arm fitted near the
top of the tank.
4) There is vent at the top and 10 air intakes with
air filter situated around the tank near the level of
wooden grate.
5) To control temperature, tank is equipped with
tubular cooling coils situated at the bottom of the
tank in the collection chamber. Thermometers are
inserted to control the temperature.

12. 6) The generator is packed with beech wood shavings upto 1.5ft. from the
top of the generator.
7) There are wooden frames inserted in between to support loose packing of
wood shavings.
8) Medium ( wine with 12% alcohol + glucose + NH4PO4 + MgSO4 + K-
citrate and pantothenic acid PH-3.5) is sprinkled over packing materials.
9) The vinegar stock or medias mixture is allowed to undergo circulation
through beech wood shavings for more and more alcohol to undergo
oxidation. This is continued till vinegar of desirable strength is obtained.
10)Conditions for fermentation
Temperature - RT
pH - 3.5
Time - 10 days

13. Trickling Generator

14. Trickling Generator

15. o Advantages :
1) higher yield
2) less time
3) being closed generator, no loss of ethanol
4) recycling of vinegar to get vinegar of desirable
strength
5) less space required
6) low cost
Trickling Generator

16. Trickling Generator
o Disadvantages :
1)Vinegar eel- Nematodes like Anguillula aceti
may enter through dust and insects.
They feed on zoogleal film.
When large in number , the putrefication of dead
nematodes can cause off flavor.
2)Vinegar mites- may also appear in large
numbers.
3) Acetobacter xylinum : Contamination by this
organism can cause clogging of generator and
thereby poor aeration as they produce excess of
extracellular polysaccharide or slime.

17. Deep/submerged fermentation-
Main advantages over surface are:
1) High level of aeration necessary for oxidative
conversion of alcohol to acetic acid can be provided
at low cost
2) Not required compressed air hence economic, low
energy input
3) Being closed tank , no loss of ethanol
4) High degree of automation and no manual operation
5) No packing materials hence no clogging
6) Very efficient with quick start up and shut down of
fermentation

18. Deep/submerged fermentation-
7) Very high yield 95%
8)Requires less space
9)Oxygen is consumed very fast hence no oxidative
browning of raw materials like apple cider or wine
mash
10)Low alcohol with less than 6% alcohol can also be
used as raw material
11)Vinegar withdrawn at the end of fermentation
contains suspended microorganisms which oxides
residual alcohol and carry out additional changes in
taste, flavor and aroma.

19. Inoculum Development:
Lyophilized stock culture of acetic acid
Sterile sweet cider with 4% alcohol
(R.T/24 hrs on shaker)
Step-by-step increases the volume
Maintenance of culture is as great problem because these organisms loose
efficiency if streaked on solid medium. Also on storage under refrigeration
organisms do not survive for long.
Hence , organisms are maintained on,
1) Sterile tomato juice agar slant in screw capped tubes with small amount
of sterile broth.
2) Lyophilized stock.
3) Maintained in liquor fermentation medium (small fermenter)

20. Fermentation Medium:
Alcohol- 13%
Vinegar- to adjust pH to 3-3.5
Small amount of glucose, NH4PO4,MgSO4, K-
citrate, pantothenic acid
Alcohol concentration should be between 10-
13%.At higher concentration acetic acid bacteria
may get inhibited and at lower concentration
poor yield because of complete oxidation of
alcohol to CO2 and water. Also ,loss of aroma
and flavor because of oxidation of ester.

21. Fermentation Conditions:
1) Temperature: R.T. is optimum.
o At low temperature: process is slow and time required is
more.
o At high temperature : loss of ethanol by evaporation and
decrease in efficiency of organisms.
 No development of flavor and Aromas
 Oxidative process of vinegar production is exothermic
hence lots of heat generated and temperature increases.
 Efficient cooling system is required.
2) pH: optimum 3-3.5
o It favors growth of acetic acid bacteria and prevents
growth of contaminants.

22. 3) Oxygen supply: From biochemistry of production of
vinegar it is clear large amount of oxygen is required for
generation of ATP and for oxidative process.
Acetator used has special aeration device that make use
of fresh air and bring about efficient aeration at low cost
and energy input.
Also, vigorous and uniform aeration is achieved.
4) Time:
Two days and operated in semi continuous fashion.
5) Yield: 15 gm %
After fermentation, vinegar withdrawn from fermenter is
subjected to storage, aging, clarification, bottling and
pasteurization.

23. Post harvest steps
1.Storage:
 Vinegar withdrawn is stored in barrel upto the rim
 Barrels are sealed to avoid contact with
atmospheric oxygen
 During storage further oxidation by organisms is
carried out of vinegar to ester, which gives flavor
and aroma but care is taken to avoid complete
oxidation of vinegar to CO2 and water.
 This results into decrease in strength of vinegar.

24. 2.Aging:
 Process that improves flavour and clarity of vinegar.
 During aging also esters are formed that add to flavour and
aroma and also reduce harsh flavour and odour.
3. Clarification:
 All suspended particles and microorganisms are removed if
not they may give turbidity or haziness to final product.
 This can be done by-
a. Filtration- RVF
b. By adding fining agents in proper proportion(Bentonite,
Spanish clay= 1to 5 lb/100 gals of vinegar)mixture is allowed
to stand till as clear vinegar appears at top.
Post harvest steps

25. 4. Bottling and pasteurization:
 Bottles are completely filled with vinegar and tightly
capped or corked to prevent entry of air.
 Bottles are then pasteurized at 60-66˚C for 30min.
5. Units used to express concentration of acetic acid in
vinegar
 Vinegar grain gallon: 1 gal of 100 grain (10%
acetic acid)
 Vinegar grain strength: 1 grain vinegar is 0.1 gm of
CH3COOH in 100 ml of vinegar at 20˚C i.e.
(10mg/ml) or ( 0.1 gm%)
Post harvest steps

26. It was 1st described by Hromatka
 Acetator means excellent performance by the
fermenter
It gives high yield 15gm% at low cost
It is cylindrical stainless steel fermenter i.e.
4m. in diameter and 9 to 13m in height.
It is operated in semi-continuous fashion.

27.  IMPORTANT PARTS OF ACETATOR
 Acetator used device for aeration consists of Hollow body
turbine surrounded by a stator.
 Hollow body is connected to fast rotating (1400-1700
rpm)ceramic disc in clockwise direction.
 It has 6 openings for escape of air. Arranged radially and open
against direction of rotation.
 Ceramic disc has upper and lower ring and upper and lower
stator for support. This stator rings are connected with vertical
baffles.
 Baffles direct air bubbles in upward directions.
 The other end of Hollow central pipe is connected to air
suction. Because of proper size and speed of rotor; sufficient
air get aspirated and dispersed outward radially in form of tiny
bubbles.
 It works by self-aspiration and doesn’t required compressed
air .

29.  Due to vigorous aeration lots of foam is formed.
 Chemical Antifoam agents are not used as final
product is food product, mechanical defoamer is
used.
 Defoamer mounted at the top of acetator.
 It consists of spiral housing in which rotor with wings
is fitted. This rotor rotates at 1000 rpm.
 As foam enters in spiral housing due to centrifugal
force of rotor, foam is thrown at periphery and break
gases released then leave the fermenter through gas
vent and liquid is drawn back to fermenter.

30.  It is automatic instrument for measuring the
percentage of alcohol in fermentation medium so as
to control complete process.
 It is made up of 2 analyzers vessels.
 Amount of fermentation medium flow continuously
through it .When it enters in the 1st vessel; it boils till
all ethanol evaporates.
 This boiling point is recorded (A) it is less as medium is
mixture of water and alcohol.

31.  Then liquid flows to 2nd vessel and boils again. Its boiling
point recorded again (B) this is always higher as it mainly
water in its.
 The difference between two boiling points (B-A) gives %
alcohol present in the medium and is recorded
automatically.
 As fermentation proceeds, % alcohol decreases. The other
parameters are also controlled automatically on the same
basis.
 i.e. When alcohol concentration drops down to 0.2- 0.3 %
alkograph gives signal and control the alcohol percentage.

33.  Discovered by Yeoman‘s company.
 It operates in continuous fashion.
 Aerator is made up of simple hollow body turbine with
arms and paddle, this driven from top using magnetic
coupling.
 Hollow shaft that helps in rotation of aerator.
 Cylindrical body around the shaft suck liquid, foam and
air from headspace and draws this emulsion to bottom of
the tube, thus cavity is formed.
 Air trapped in cavity is dispersed through medium.

34.  Casida L. E., "Industrial Microbiology” (2009) Reprint, New
Age International (P) Ltd, Publishers, New Delhi.
 Crueger W. and Crueger A. (2000) "Biotechnology -"A
Textbook of Industrial Microbiology", 2nd edition, Panima
Publishing Corporation, New Delhi.
 Prescott and Dunn's ‘’Industrial Microbiology’’(1982) 4th
edition, McMillan Publishers