Practical Manual

on
Food and Industrial Microbiology

• Anil Kumar Puniya

• Shilpa Vij

DAIRY MICROBIOLOGY DIVISION

NATIONAL DAIRY RESEARCH INSTITUTE
KARNAL ·132 001, HARYANA
 Preface
" There is nothing like searching, if you want to find knowledge and there is nothing like
telling, if you want to spread knowledge" .

This is the motto for writing thi s practical manual 011 "Food and Indu str ial Microbio logy".
This manual has been specially designed for un dergradu ate and postgraduate students. The
main purpose of thi s lab manual is to guide st uden ts through a process of development of
microbiological techniques, conducti ng experiment and interpretation of data. I have tried to
engage students in the learni ng process by applying knowledge. I have also given special
emphasis on individua l topics so that the st udents should not be required to re·read the
textbook s.

The manllal IS laid out III sections, e.g., microbiological examinations of different food
samples, fermenter In food industry, industrially important microbes and microbial
production, each contai ning a series of experiments. Every practical includes deta iled outline
in th e th eory, full list of materials and procedure. At the end of th e each sect ion, some review
questions are given wh ic h w ill enable the student to assess their potential of what has been
done. G lossary, which is given at th e end of the man ua l, covers detailed defi niti on of
important tenns used in th e manual.

The auth or sincerely thanks Dr. AK Srivastava, Director, NDR I, who en trusted LIS and
granted th e perm ission for w riting a practica l manual on 'Food and Industrial Microbiology'.
I also convey pe rsonal grati tude to Dr SL Goswami, (lOR), Dr GR Patd (lOA), Dr.
Rameshwar Singh (Head & Registrar), a nd all other co ll eagues of DM Division for their
support and guidance. Author a lso than ks ICAR for providing funds for manual preparation
under the sc heme for strengt heni ng and development of agr ic ultural educati on.

I also extend my thanks to research scho lars Dr Ravinder NagpaJ, Mr Sanjay Kumar, Sum it
Singh Dagar, Priti Devi a nd Malashree M, who imm ense ly contributed in retrieving literature
and co mpil ation of th e manual , wit hout their invo lvement, it wo uld have been very difficult
to complete the task effective ly in such a short period of tim e.

I wou ld also like to thank Monica a nd Nikhi l for their suppo rt in prepa rin g th e manual.

Shilpa Vij Anil Kumar Puniya

3.2 Production and assaying of microbia l proteases 40-4 2
3.3 Production and assay ing of microbial lipases 43 -45
3.4 Isolation of Anti biotic Producing Microbes from Soil 46-47
3.5 Isolation and screenin g of Streptomyces species as ant ibioti c producers 48-49
3.6 Productio n of Nisin from LactocooClIs lactis 50-52
3. 7 Productio n of anti microb ial substances from lacti c ac id bacteria 53-55
3.8 Isolati on of psychrophiles from milk samples 56-57
3.9 Isolation of salt lolera nt microorganisms from food samples 58-59
3. 10 Isolati on of salt tolerant microorganisms from food samp les 60-61
Rev iew question s 62-63
User's notes 64

Section D
Microbial Production

4. 1 Production of lactic acid from whey 66-68

4.2 App licati on of microbial consortia in food ferme ntati ons 69-70
4. 3 Prod ucti on of ethy l alcoho l from mo lasses and whey by yeasts 7 1-73
4.4 Citric ac id production from whey with sugars and add itives by 74-75
Asp ergillus niger
4.5 Productio n of sauerkraut by microorgani sms. 76-77
4.6 Production of sin gle cell protein s 78-79
Rev iew Questio ns 80
User's notes 81
AppendL,(-1 82-88
Media Composition
Appellllix -2 89-9 1
Reagents and Buffers
Most Probilble Number (MPN) Index 92-94
Glossary of Rel ated Terms 94- 103
 SECTION A:
MICROBIOLOGICAL EXAMINATIONS OF
DIFFERENT FOOD SAMPLES

1
Introduction:
Microorganisms are assoc iated with plants and animals in nature. They play important rol e
for survi val of plants and animals. But on th e oth er hand growt h of certain harmful
microorganisms in food ca n result in spoilage and sometimes ca use seve ral di seases on
cons umption of such food. Food spo ilage by mi croorganisms is due to in crease in their
numbers, utili zing nutri ents, ca using enzymati c changes resu ltin g in bad fla vo urs due to
brea kdown of some food material s or synth esis of new compounds. Due to such mi crobial
activit ies, food becomes unfit for human co nsumption. Also food acts as good medium for
tran smi ss ion of man y di seases. If th e food is co ntaminat ed by pathogenic microorganisms,
they ca n grow and increase their population and ca use di seases on co nsumpti on of such food .
Some tim e microorgani sms may not grow in food but th ey are transport ed throu gh food.
Several food born di seases are th e result of mi croorgani sm prese nt in food or the ir growth in
them. Th e presence of microorgani sms in food products, with their influence upon qualit y
and method s of preservation , is of grea t importance to food mi crobio logis ts. Con trol over
pathogeni c and infec tiou s organisms is esse nti al, but is not enoug h, sin ce the quality and
safety of man y food s depend upon the control of spoilage bacteria, yeas ts and moulds. So
th e microbial examina tion of food and food produ cts is of prime importance for assessi ng the
qual it y and safet y of food produclS.
1.1 Aim: To examine the microbial flora of vegetables and fruits.
Theory:
Fresh vege tab les & fruits are exposed to potential microbial contamination. It has been
estimated that 20% of all fruit s and vegetabl es harves ted for human consumption are lost
through microbial spoilage. The primal)' agen ts for mi crobial spoilage are the bacteria, yeasts
and moulds. The latter two are by Ihe fa r 1110St important et io logic agents of spoilage. Fruits
and vege tabl es carry microbial flora whi le pass in g from the farm to the table. The produce is
exposed to potential mi crobial co ntaminati on at eve I)' step in cludin g cu lti vation , harvesting,
tran sportin g, packaging, storage and se lling to the final consumers. Fruits and vegeta bles are
susceptible to mi crobial deco mposit ion beca use of their nutri ent ri ch characteristics and are
capabl e of supporting the growth of moulds, yeasts and bacteria. The average compos iti on of
vegetable and fruilS is: Water = 88.3 %, Carbohydrates = 8.6 %, Proteins = 2.0 %, Fat =
0. 8%, Ash = 0.3%. Microbial spoilage and co ntaminating pathogens pose a seriou s problem
in fo od safet y as well as eco nomy. The sa nital)' control of food quality is concerned with the
examination of food s for th e presence of pathogens. The ex amination of foods is performed
for th e presence of Iota I number of bacteria in the food by SPC, and the presence of colifonns
for indicating the presence of pathogens.
General microbiOlogical profile of harvested fruits:
• Bacteria: usually less than 10(,/ g

2
 SAMPLE DILUTION

B Iml

15ml ~
1ml

PI.1t11 eo..rt Aglr

"'~t. Ccu1t SlIm mn A r "

Figure 1.2 Enumeration steps for bacterial count in food sample

Source: 11"1\ "11 ' ••n 'har/au. ('0111

SAMPLE DILUTION CONRRMAnON

t;~
••••
Yeasts
r,
, .

B lml
i !
15ml l I -
i!
U
, ,
PenlCllium
1ml eGA or others

,-
Figure 1.3 Enumeration steps for fungal count in food sample
Source: U·U·II'.s('harluu.COIII

Observation table:
Type of Dilution Agar Incubation - Count per Total Average
count used temp .! time plate (cful g count count (cfu /g
or mil (cfu/g or or mil
mil

4
1.2 Aim: To study the microbial flora of meats and eggs.
Theory:
Meats and eggs are th e most peri shable of all important food s because of the presence of
ample amoun t of nutrients requi red for the growth of bacteria, yeasts and melds. Moreover
these nutrients are avai lable in more availab le form. It has al so been pointed out that during
slaughter, dressing and cutting. microorgani sms come chi efl y from th e exterior of an im als
and its intestinal tract but that more added from knives, cloth air, workers.
A great variety of kinds of organ isms are added and so it can assumed that under ordinary
condi ti ons most kind of potential spoilage organisms are present and it will be able to grow if
favourable conditi ons present thcm sc lves. Most commo n organ ism responsible for meat
spoilages are bacteria. meld s and yeasts respectively.
Bacteria most oftcn in vo lved in spoilage are AcinetoiJacter. H(~filia.
Campylohaeter.
EnterocoeclIs. Salmonella. Serratia. Staphylococcl/s. Vibrio. Pseudomonas. Vagococc//s.
Carnobacferiunl. clostridium. Aeromonas. Alcaligenes etc. with respect to fungal agcnt
invol ved Thal1ll1idiunI, Mucor. Penicillium. Rhizopus. Candida. Rhodotorula. CI1Iysosporilll11
and Cladospo rium.
The hen's egg is an exce ll ent examp le of a product that normally is well protec ted by its
intrinsic parameters. Extema ll y a fresh cgg ha s three structures outer waxy shell membrane,
thc shcll, and the inner shell membrane. Fres hl y laid eggs are genera lly sterile. However in a
relati ve ly short period afte r lay ing numerou s microorgani sms may be found on the outside
and under the proper condition Illay enter eggs, grow and cause spoilage.
Among bacteria found are th e members of following genera: Pseudomonas. Acinl1efObacter.
Proteus. Aeromollas. Alcaligenes. Escherichia. Micrococclfs. Salmonella. Serratia,
Emerobacter. Flavobaclerium ond SlaphylococclIs. Among the melds generally found are th e
members of the genera Mucor. Penicillium. !-Iormodendrum. Cladosporium alld Torula. The
entry ofmicrobcs in eggs is fa vo ured by high humidity. Under sllch co nditions growth on the
surface is also fa voured followed by the penet ration in shell and inner membrane.
The sanitary control of meat and eggs quality is primarily co nc erned with testing meat
products for the presence of specific microorgani sms. Meat products are the primary vehicle
respons ible for the tran smi ss ion of microbial di seases of the gastrointestinal system lik e
sa lmonellosis and botuli sm, for thi s reason, meal products are routinely examined for thc
presence of bacteria.
Materials Required:
Raw meat samples, 22.5 ml sa line, 9 Illl sa line, Plate Count Agar (PCA), Malt Extract Aga r
(MEA), Oxford Perfringens aga r, Bacillus cereus agar, VRBGA (plus 41111 overlay moltcn),
Steril c Petri di shes, Pipettes 0.1 and 11111 automatic plus tips, G lass rod spreader plus alcohol.

6
Proced ure:
Sampling:
• Take number of subsamples from different parts of the mcat and eggs including areas
known to be subject to contamination or particularly favourable for microbial grow th .
• Use ste rile swabs and temp lates for taking surface swab samples.
• Moisten th e first swab with steri le peptone water and rub firmly across the exposed area
seve ral times in all directions.
• Use the second swab dry and rub over the same area.
• Introduce both swabs into a bottle containing 3 or 4 glass beads and an appropriate known
vo lume of diluents (e.g .. 0.1 % peptone. 0.9% NaC I).
• Shake vigorously.
• Otherwise dircc tl y cut sma ll pieces of meat or egg yo lk and add it to the steril e diluents
Pla/ing:
Microbial flora of ra w meats
• Ana lyse two different raw meats.
• Check whether your aga r plates need pre-drying.
• Weigh 2.5 g of the raw meat provided into a Stomacher bag and add 22.5 ml of saline.
This is the 10,1 di lution. Homogenise using th e Stomacher for 30 seconds.
• Allow th e large particles to settl e (fatty mat erial will float to th e surface) and then dilute
your sa mp le in 9ml sterile sa line up to 10'11. dilution s.
Spre(lll pla/e inocII/a/iol1:
• Dispe nse O. lm l o f each di lution (10'11. to 10,1 to save on pipette usage) onto the surface of
dri ed Plate Co unt Agar (PCA), Malt Extract Agar (MEA). and Bacillus cereus agar.
POllr pltlle illocula/ioll :
• Pipette 1111 1 o f eac h dilution into 8 Petri dishes. Add approx. 20 ml mo lten Tryptose
Sulphite Cycloserine Agar (also known as Perfri nge ns agar).
• Ca refull y swi rl to mix the samples.
• Pipette I ml of each di luti on into 8 Petri di shes. Add approx. 20m lmo lten VRBGA.
• Carefull y swi rl to mix the samples.
NOTE: This will need a 4ml VRBGA overlay (stock vo lume 50ml).
• Incubate th e plates aerobically at 37°C; except for the TSCA aga r whi ch will be incubated
anaerobi ca ll y and the MEA which will be incubated at 25°C.
Observations:
• Arrange the plates in order of lowest to highest dilution.

7
• Count the number of co lonies on th e plates that ha ve co lonies 30 to 300. Des ignate plates
with fewer than 30 co loni es as too few to count and plates with more than 300 co lonies as
100 numerous to co unt.
• Calc ul ate the average num ber of bacte ri a per gram of sample as fo ll ows:
• Num ber of bacterial gram = Number of co lonies
Dilution fac tor x We igh t of sample
Observation Table:
Record in the table the number of colonies per plate.
Dilution Number of colonies/ plate
10--'

10-'

10-'

10 6

Results and interpretations:

8
1.3 Aim: To study the microflora of wheat and flour.
Theory:
The microbial flora of wheat, rye, corn and re lated produc ts may be expected to be that of
soi l, storage env iro nments, and those pi cked up dur ing the process ing of these commodities.
Whi le these products are high in proteins and carbohydrates, their low all is such as to restri ct
the growth of a ll microorganis ms if stored properly. Th e microbial flora of flour is relati ve ly
low, s ince so me of the b leachi ng agents reduce the load .
When conditi ons of aw favour growth of bac teria of genus bacillus and melds of severa l
genera are usually the ones that deve lop. Many aerobi c spore f0n11erS are capable of
producing amylase, which enables them to utili ze flour and related products as so urces of
energy, provided that sufficient moisture is present to allow growth to occ ur with less
moi sture, mould growth occurs and may be seen as typical mycelial growth and spore
formation. Members of th e gen ll s rhizopus are common and may be recogni zed by th eir
black spores . Most common micro flora of dough cons ist of mainl y lactic acid ba cteria more
than half belonging to th e gen us Lactobacillus, Leuconostoc and St reptococcus. Moulds are
genera Ily found in low numbers.
General microbiologi ca l profile of flour and baked goods (Breads) is:
A) Flour:

• Moulds '
~ W--IO 4/ g

,
• Yeast ~ 10-10-1 g

• Total Bacteria ~
'
10--10,1g

• Coli forms ~ 0-101 g

B) Baked Goods:
• Moulds ~ 10-10' 1 g

• Yea st ~ 1O-IO'/ g

• Total Bacteria ~ 10-10' 1 g

• Co lifonns ~ 10-1O' / g
Material Required:
Steril e sa mple bottle or po lyethy lene bags, sterile dilution blank (99 ml and 9 1ll1), Petri-
dishes, steril e pipette (I and 10 ml), nutri ent agar, potato dextrose agar, viole t red bile agar,
pestle and mortar, incubator, wax marking pencil
Procedure:
• Weigh II g of th e sa mple and put in steril e pestl e and mortar. Thoroughly mix the sa mple
into 99 ml ofsterile d iluents to make 10'1 di luti on.

9
• Transfer I ml of suspension from 10-1 dilution to a 9 ml sterile saline blank with a steri le
pipette to make 10- 2 dilution _Similarly, prepare se ri al dilu ti on up to IO-x_
• Transfer 1 ml aliquot from 10-(' and IO-x dilutions to sterile Petri dish for total bacterial
coun t, IO~3 and 10-5 for coli form count and yeast and melds co unt.
• Add approximately 15 ml of cooled medium (40°C) to each Petri-plate (Nutrie nt agar for
total bacterial co unt, VRBA for coli form count, acidified PDA for yeast and melds count )
and mix the content by gentle rotation of the Petri dish_
• In vert the plates after solidification and incubate at 37°C for 24-48 h for total bacteri al
count and coliform cou nt, and at 25 uC for 3-5 days for yeast and melds co un t.
Observation Table:
Type of Dilution Agar Incubation - Count per Total Average
count used temp.! time plate (cful count count
g or ml) (cfu/g or (cfu/g or
ml) mil
I. Bread

2. Flour

Results and Interpretations:

10
1.4 Aim: Detection of cndotoxins in food samples by Limulus Amoebocyte Lysate test.
Theory:
Microbial toxins are organic poisons produced by microorganisms. Wh en such poisons are
ingested , abso rbed, or otherwise introduced into the body of differen t li ving organisms, they
cause damage to ti ss ues and! or interfere with normal physiological function s. Microbial
toxins are complex in terms of stru cture and chemical composition, and they possess
antigenic properties. Numero us types of microbia l toxin s have been desc ribed; these are
bacterial toxins, fungal toxins (mycotoxins), a lgal toxin s (phycolOxins), etc. Va ri ous types of
toxins are produced by bacteria and potent toxins producers are COI),l1cbactcriulIl
dipl1t/Jeriae. Clostridiul1I teloni, Clostridillll1 botlilinllm, E. coli, Staphy/ococclls ourcus etc .
Exotoxi lls are so luble substances produced with in cells but secreted to the cell's exterio r
environment during periods of active growth. Endotoxins are bound to the bacterial cell wa ll
as a stru ctural co mponent - or, in certain types of microbes, contained within the cell's
cytoplasm.
E:r:l.doto:x:.irl.
Factor C --1-.. .A.cti vated factor C

FactorS Activated factor B

Proclotting enzyme Clot"ting enzyme

Soc-Leu-C7ly-.A..rg -pJ:--.,:f,A
--1-
--1-
oc-Leu.-G-ly-.A.rg -+- p'l"-JA

Diazo-coupling
--1-
a D 545 nrn.

Figure 1.4 Flow chart showing LAL test mechanism

SI!II n "I!: II I If} : 111I'u '\\ '. {'mil Ilf IX i lI.gII/X/If!ll/('. del/a /:li)g

Exotoxins are protei naceous substances, specially characterized by the ir lack of chemica l
association with other macromol ecul es. These are sensitive to denaturation by heat and other
chemical substances. Exotoxi lls can be neutrali zed by their homologous an tibodies.
Endotoxin is complex Ii po-polysaccharide-protein co mponent of the bacterial cells wall.
These are relati ve ly heat-stable and canllot be neutrali zed by homologous antibodies.
Limulus pol yphemus present in the blue blood of the horseshoe crab is a nucleated ccll.
called amoebocyte. The cytoplasm of amoebocyte is dense ly packed with granules. Limulus
lysate, extract of amoebocyte gran ul es, contains all th e necessary c lotting factors. Limu lus
lysate clots in th e prese nce of bacterial Ii po polysaccharides (e ndotoxins). Hence this test ca n
be used to rapidly detect th e endotoxins present in food sa mpl es. This test is ve ry spec ific
and sensitive and can detec t up to 10'\5 g cndotoxins per millilitre of sample.

11
Material required:
Food samp les, C lean and dry test tubes, Limulus lysate reagent , Wate r bath incubator (37°C),
wax marking pencil s, sterile 0. 1 and I Illi tips, auto-pi pettes
Procedure:
• Prepare th e serial decimal dilution of samples using normal sa line.
• Tak e eq ual volume of diluted sample and limulus lysate reagent in clea n and dry tes t
tubes and mi x the conten t ge ntly.
• In cubate the test tube in a water bath incubator at 37°C for 4 h,
• After incubation is over, in ve rt the test tube and see th e flow bcha viour of th e fluid in the
test tube.
• If the mixture remains unchanged and runs dowillh e test tube wall, indi cate the negative
LAL test and thu s endotoxins are abse nt in the sample dilution.
• If a firm and opaque ge l is fonned and sti cks to the bottom of the tube, ind icates th e
positi ve LAL test an d thus endolOxins are present in the sample d ilution.
Observation:
Sample(s) Dilutions Opaque Gel formed Results

Results and Interpretations:

12 I

l
1.5 Aim: To check the presence of antibiotics in milk samples through DSM DELVO
tcst.
Theory:
Antibi otic contamin ation in milk ca n se ri ously affect consumers ' hea lth by ca usin g al lergic
reac ti ons to res idues or by th e deve lopment o f res istant strains of mi croorga ni sms. Therefore,
subsequentl y antibi oti c cont am inati on in milk ca n al so cause signifi cant economi c losses for
produce rs and manufacturers o f milk and milk products,
For determin ati on of antibi oti c res idues in milk , use co mm erciall y ava il a bl e Antibi otic
Detec ti on Kit: Delvo-X -Press b L-II , CO PAN T EST, C HARM FARM T ES T or C HARM
AIM-96, BITA STA R KIT
The growth o f th e Bacillus sfearolhermophillls spores at 64°C initi ates an ac idi fic ati on
process whi ch causes th e turning of a pH indi cator from purpl e to ye ll ow, The presence of
antibacterial substances will ca use delay or inh ibi ti on of th e spores. dependin g on the
concelHrati on of the res idu es, In th e presence o f res idues the spores will not multipl y and th e
pH indicator wi ll re main pu rple,

Figure 1.5 Yellow colours: no antibiotic; Purple colour: antibiotic prescnt

S(JIII'('e: 1\'1\'1\'. ("\ '/11.1111111. edlll., .ldell·/ !ples Iph 's/1I1 nl/e.IIlIIII

Material Required:
DSM DELYO tes t kit , raw milk sa mpl es. wa ter bath , agar we ll strips
Procedure:
• Add I nutri ent ta bl et to eac h o f the agar we ll s in the strip ,
• In oc ul ate 100 IllI o f milk into the agar we ll plus nutrient ta bl et.
• Seal the we ll s for in cubatio n

13
• Incubate the strip o r wells in a wat er bath at 64°C ± O.5°C lo r 2 h 30 min * (at the time th e
negati ve control has been changed to ye ll ow)
• Ex amin e the strip for co lor change from purple to ye llow. A ye ll ow reading indica tes that
no inhibitory substan ces are present ; a purple readin g ind icates th at antibi otic res idu es are
present and a yellow/purple reading indica tes a doubtful res ult.
*For best se nsiti vity a co ntrol time readin g is advised usin g a negati ve co ntro l sa mpl e.
The sampl e incubation peri od is cru cial to th e acc uracy o f the Delvo® S P test method. Thi s
particul arl y appli es to th e detec ti on o f sulph onam ides as the sens iti vity to sulpho namides is
greatly reduced by an increased in cubati on tim e. An in cubati o n time o f 2 hr 30 min (at the
time the nega ti ve contro l has been changed to ye ll ow ) is reco mmended with a 15 minute
ex tension time of the test in the case o f a suspect sampl e. For readin g tim es o f 2 h 45 min or
3 hours the sens iti vity o f the test will dimini sh. Th e use o f antib ioti c free skim milk powder
as a control is al so ad vised . . It is essential that the co rrect temperature is mainta ined in th e
water bath (64°C ±O.5°C ). The use o f a proper wa ter· bath lid, a sloping lid is adv ised·
The temperature co ntrol in the water· bath should be di g ita l temperature read out . Good
circul atio n should be ma intained in the water-bath
Observations:
Samples Colour

Results and InterpretatIOns:

14
1.7 Aim: Presumptive, confirmatory and completed test for water quality.
• Determine the prese nce of coliform bacteria in a wa ter sampl e
• The poss ibl e number of coliform bacteria present in a wat er sa mpl e
• Desc ribe steps (i.e. pres uillpti ve, confirmed, and completed) for determining col ifonns in
a water sa mpl e.
Theory:
The number of total colifonns (Enrerohacler. Klebsiella. Cilrobacler. and Escherichia) in a
wate r sampl e can be determined by a stati sti cal es timation ca ll ed the most probable number.
This test involves a J11uhiple se ries of Durham fennentation tubes and is di vided into three
parts: th e pres umpti ve, co nfirm ed, and comp leted testS. In th e presumpti ve te st, dilution s
from the water sample are added to lactose or laury lllYptosc broth fermentation tubes. After
24 to 48 hours of incubation at 35°C, one looks for bacteria capable of fermenting lactose
with gas production, pres umably co lifonn s. (The lauryl try ptose broth is se lective for Gram-
nega tive bacteria due to th e presen ce of laury l sulfate.) In the confirmed tes t, one tran s fers
mat erial from the high est dilution o f those lactose broth tube s that showcd growth and gas
production int o brilliant green lactose bile broth. whic h is se lecti ve and differential for
colifoflllS. Th e tube is incubated for 48 ± 3 hours at 35°C. Gas formation in th e Durham tube
is a confi rm ed test for total co liforl11s. In th e complet ed te st. a sa mple from th e positi ve green
lactose bile broth is streak ed onto Levine's EMB or LES Endo aga r and incubat ed for 18 to
24 hours at 3 5°C. On EMB agar. colifonn s producc small co lonies wi th dark cen ters. On
LES Endo aga r. coli form s produce reddi sh co lonies. Samples are then inoculated int o -
brilliant green lactose bile broth and onto a nutri ent agar s lant. These tubes are incubated for
24 hours at 35°C. II' gas is produced in the lactose broth, and the iso lated bacterium is a
gram-negative (based on a Gram stain) non sporin g rod , the completed test is positi ve. An
estimate of the number of col iforms (mos t probable number) can a lso be done in the
presumptive test.
In thi s procedure, 15 lactose broth tubes are inocu lated with th e water sample. Five tubes
rece ive 10 ml of water. 5 tubes rece ive I 1111 of wat er, and 5 tubes rece ive 0.1 ml of water. A
cO llnt o f the number of tube s showing gas production is then made, and the fi gure is
compared to a table developed by th e American Public Health Assoc iatio n. The number is
the most probable number of co li forms pcr 1001111 of th e water sa mple. (Il should be no led
that th e MPN index usuall y comes from th e pres umpti ve test if raw sewage is being tested
and co mes from con finned or comp letcd te sts for other types of samples. )
Materials Required:
10-ml s in gle-strengt h lactose broth in Durham fermentation tubes (Iaury l tryptose broth)- J 0,
10-1111 double-strength lact ose broth in Durham fermcntation tubes-5 , 125-1111 water sampl e,

18
Gram-staining reagent s, Petri plate containing Le vin e's EMB agar, Tryptic agar slant, tubes
containing brilliant green lactose bile broth with Durham 's tubes, I sterile 10-ml pipettes, 2
sterile I-ml pipettes, wax pencil, te st-tube rack , 35°C incubator, inoculating loop and needle,
Bunsen burner,
Procedure:
I. Presllmptil1e Test
• Mix th e bottle of water to be tested 25 time s. Inoculate fiv e or the double-strength lactose
(or lauryl tryptose) broth tubes with 10 ml of the wate r sa mple; fi ve sin gle- st ren gt h tubes
with I ml or th e water sa mple: and five singl e-strength tubes with 0.1 1111 of the water
sampl e.
• Carefully mi x th e contents of each tube without spilling any of the broth by rolling th e
tubes between the palms of your hands.
• Using the wax pencil, label all tubes wit h your name, date. and the amount of water
added.
• Incubate th e three sets of tubes for 24 to 48 hours at 35°C.
• Observe after 24 ±2 and 48 ±3 hours. The presence of gas in any tube after 24 hours is a
positive presumptive te st.
• The formation of gas during the next 24 hours is a doubtful test. The absence of gas after
48 hours is a negative test.
• Determine the number of coliforms per! 00 Ill! Or\v<.lt~r sa lllp! ~.

• For instan ce, if gas was presen t in all five or the 10-1111 tubes, only in one o f the 1-1ll1
series, and non e in th e 0.1-1111 se ri es, yo ur test results would read 5- 1- 0.
• Standard tabl e (Page III) indi cates that the MPN for thi s readi ng would be 30 colironns
per 100 ml of water sampl e.
II. COlljirme(1 Test
• Using an inoculating loop, from the tube that ha s th e hi ghest dilution of water sampl e and
shows gas production tran sfer one loopful of culture to the brilliant-green lactose bile
broth tube. In cu bate for 48±3 ho urs at 35°C.
• The formation o f gas at any time within 48 hours constit ut es a pos itive confirmed test.
III. Complete(1 Test
• From th e positive brilliant green lactose bile broth tube, streak aLES En do or Lev ine's
EMB plale.
• Incubate the plate inverted for 24 hours at 35°C.

19
• If coli form s are present , select a well- iso lated co lony and inoculate a sin g le-strength ,
brilliant gree n lac tose bile broth tube and streak a nutrient aga r slant.
• Gram sta ins any bacteria found o n the s lant.
• The formation of gas in the lac tose broth and th e demonstratio n of gram-nega tive,
non sporing rods in the agar culture is a sati sfactorily co mpl eted test revea lin g the
presence ofco lifonns and indi cat ing tha t the water sa mpl e was polluted .
• This is a pos iti ve comp leted test.

~ w.~,
LJ~P.
Inoc ...... 1~ .ub.a: ~ w'i.1t 10ml of ....,ple. ~w ..,. 1 .0 ml 01 ..,.,p . . ...d ~ w,.n 0 . 1 ml." ~p • .

,. 10 10
,m,
10 1.0 1 .0 1.0 1 .0 1 .0 0. , 0 .1 0.1
(m' )
0 .1 0 .1

-.. . add._
The _-.ne.~.-.~.g
<:<>10,...,.,. _
bfO.... . . . -•
01 _
....toe... .
ab_t.
on -g :::,!.4~.":. . h.
1...,10_ broth _
of
...am",.d

-,- -.-
to, g • • p<OductlOflo
'n cub ... an
24 I'oou" 10 to • • u, ..

g
~:o'::.:o..:,,,,,-
-•... - ... g j

g
Co~fonn g'"" p _ _ t.

u_ po_ ..... contonned

-"~-- . A • •_ •••
"un ..... u_ 10
~."m. . ..
Inocu . . .

-'-
tut. •• , od........... MPN _ _ tube. 0 1 b ';!I;.,.. g ..... '-<: ... _

,,_.....
b;i.e bO'OCh. Incu o.t>on 10 ' 48 ~)
ho ...... 30 °C .

,J • to. 1&-2<l 1>0.......

~,-

~::: ~ A ..... 2<lhov .. 01"""'ub.1>Onm . . . .

1
IH'- _ 0.-.. __ ,""d."..",., tM ......
0,1....-y1 _ H,.,... b . a ........ g ...... -_o-o_.
trypto_
b<'ott> *"....
NU'lt>ent
IeM '-"toe.. _ com"'...." ......
-nep...... 1;J <Od. . . .d produ_ goe. hom
po ...... .
v..eol-...co!_
' 0 me..... nu1rien.
~.-.t
booth tub • .
...... .

Figuret.7 Flow Diagram for presumptive, confirmed and completed coliform test
SOllrce: PrescO/f el al. Microbio/ag\!. 1"1 Edilion, }OO}

20
User's Notes

23
 SECTION B:
FERMENTER IN FOOD INDUSTRY

24
2.1. Introduction:
The art of stud ying fermentation is called zymology or zymurgy. Louis Pasteur was one of
the first zymologists and referred to fermentation as "the result of life without air".
A general definition of fermentation is an energy-yielding anaerobi c metaboli c process in
which organisms conve rt nutrients (typically carbohydrates) to alcohols and acids (lactic acid
and acetic acid). Th e Illost commonly known definition for fermentation is the conversion of
sugar to alcohol, using yeast. under anaerobic conditions, as in the production of beer or
wine, vinegars and cider. Fermentation is among the oldest of hi storical biotechnological
processes thai people hav e been us ing for thousands of years. However. in biotechnology, the
teml is used more loose ly to refer to growth of microorganisms on food , tinder eith er aerobic
or anaerobic conditions.
The design of fermentation eq uipment has evo lved in a largely empiri cal manner. The
earliest fermentations required only rudimentary standards of hygiene, either beca use of the
nature of the substrate, or because the vigour of the des ired organism exceeded that of
potential competitors, or beca use the products of fermentation were inhibitory, or becau se the
expected shelf life of th e product was short. This applied to the fermentation processes
involved in the manufacture of wine. beer, cheese, yoghurt, vinegar. sauerkraut and so on.
Even with th ese products the tran sition from domestic art to commercial practice required
improved standards to increase shelf-life and to maintain acce ptabl y-consistent sta ndards of
product quality. but equipment and process control remain ed essentiall y simple. Some
sophisti cation occurred with the deve lopment of pure-culture technique s in beer making, but
the first reall y fastidious fermentation, the manufacture of acetone and butanol, was initiated
on a large scale less than s ixty years ago. In thi s system the maintenan ce of a stri ctly
anaerobic environment was essential. This provided protection against a wid e ran ge of
contaminants requiring atmospheric oxygen, and the principal hazard was contamination by
bacteriophage.
The nex t major deve lopment was th e adaptation of s ubl11er ge d~ c ulture techniques for the
product ion of penicillin, rapidly followed by processes for other antibioti cs. vitamins, and
amino acid s. and the conversion of steroid s. These processes have bee n developed over th e
past thirty years or so. and ha ve attracted the combined attention of microbiologists,
biochemists and enginee rs to reso lve a va riety of problems in order to improve both yie lds
and process e ffici ency. More recentl y, a good deal of attention has been devoted to problems
associated with the replacement of batch processes by continuous processes, and th e
utilization of gaseous and liquid hydrocarbons as substrates, instead of the traditional
carbohydrates. The latter processes, in particular, have stimulated in vesti gation s into the use
of fermenter configurations other than th e agitated cylindrical vessel which ha s bee n almost
universa ll y adopted for aerobic mi crobial processes and for some anaerobi c processes.

25
Among the new configurations, two in particular are the air-lift and the tower, have been
exploited commercially and hence attracted the attention of research workers.
2.2. Fermenter design and requirements of the microbial system
The function of the fennenter is to provide an environment suitable for the controlled growth
of a pure culture or of a defined mixture of organisms.
The materials of construction must be such that they will not adversely affect, nor be
adversely affected by. the des ired microbial activity, either by interaction with the
ferm entation medium or by harbouring unwanted organisms. They must be res istant to
corrosion by the nutrient medium and products, and to the effects of sterili zation
temperatures. The actual construction of the equipment from suitable materials must also
take account of these factors and of the stresses imposed by pressuri za tion and the weight of
the vessel contents.
There must be provision for the regulation of temperature and of the suppl y of air, for
charging and discharging the vesse l contents, for inoculation and also for samplin g and for
the control of pH and foaming. Frequently. even in batch wise systems. it will be necessary
to provide for controlled addition of nutrients or other materials during the course of the
fermentation. In continuous-culture systems additional faciliti es must be provided to control
culture volume and medium flow-rate. Depending on the method of control. some system
will be required to measure and control the cell concentration or the concentration of so me
rate-limiting substrate. These controls relate to the macroenvironment (the total cllilure) and
only measure or control the condition s of the microorgani sms in an indirect, empirical sense.
Some control systems, such as those based on the rate of oxygen uptake, evolution of carbon
dioxide or concentration of NA DH, re late more directly to cell activity, but none gives a
direct measure of the condition In the microenvironment, that is the envi ronment in the
immediate vicinity oft he cell.
It is an assumption implic it 111 all control systems that biochemically similar
macroen vironments produce biochemically-similar microenvironments. Anomalies and
difficuilies which arise in sca ling-up or in conducting a given fermentation in different types
of equipment indi cate that this assumption may be false. One poss ibl e source of anomalies is
differences in the extent of cell aggregation, which. in biochemica ll y-si milar environments.
may be strongl y affected by hydrodynamic factors.
Another likely source of error lies in systems for assessing conditions in the
macroen vironment. These will generally indicate a time-a verage va lue at a particular
sampling or sensing point and may conceal considerable variations from one element of
culture to another. Thus attempts to control conditions in the culture as a whole may have
somewhat attenuated effects on the microen vironment. At the same time, the activity of the
microorganism will itself ha ve effects on the microenvironment, both directly and through

26
effecting changes in the macroenvironment. The direct effects on the mi croenvironment will
result from:
(a) Consumpti on o f substrates
(b) Output o f products
(c) Producti on o fh ea t
(d) Aggregati on
Factors (a) and (b ) w ill result in defi ciency o f substrate and acc umulation of product, w ilillile
possibility of res ult ant inhibition, unless there is adequate interchange between th e region
immedi ately adjacent to the cell and the bulk o f the nui d.
A similar interchange is also necessary to prevent significant and possibl y damaging increase
in temperature. Aggregation of cell s will ad ve rsely affect both heat and mass transfer, partl y
by reducing th e area available for transfer, partl y by increasing th e length of th e transfer
paths and partl y by reducing the transfer coefficients.
The factors w ithin the control of th e designer and operator are:
(a) System geo metry
(b) Aerati on rate
(c) Intensity o f ag itati on
(d) Temperature
(e) Pressure
(I) Nutrient suppl y
(g) pH and oth er parameters in vo lvi ng specific io ns
(h) Dilution rate in continuous n ow systems
(i) Foamin g
When considering the design of vertical stirred vessels, the main variables in geometry are
the height-to-di ameter ratio, the number, type, dimensions and positions of impellers, the
number and breadth o f barn es, and th e design and location of coils for heatin g and cooling.
In relation to powe r input, th e geometri cal specificati on for th e impe ller and the degree o f
baming cannot be di vorced fro m the speed of rotati on. Some acc ount must also be taken o f
the rate of aerati on and degree o f gas hold-up in th e system, sincc thi s will affect th e density
of the culture which is the most significant property gove rnin g overall power input under
conditions of full y-developed turbulence.
Control of the other fac tors mentio ned above will have onl y minor effec ts on vessel design
arising from the introducti on of fac ilities fo r scnsing, sampling, addition and w ithdrawa l. A
major advantage o f the sti rred vessel ove r other designs is the degree of operation al
fl exibility which it prov ides even when install ed. This arises large ly because mixing and
mass transfer are innuenced both by the acti on o f th e impeller(s) and by th e rate o f ae rati on,
which can, within fairl y w ide limits, be vari ed independ entl y, albeit at th e ex pense of
changing the impeller speed or geometry. By contrast, in the air-lift mixing and mass transfer

27
are both dependent in a g ive n piece of equipme nt, on the rate of ae ration, a nd cannot readil y
be va ri ed independentl y. In the tower fennenter, unless provision is made for re-c yc ling,
mi xing and mass transfer are strongly a ffected by the diluti on rate, which must be determi ned
on bio logica l grounds, whereas the dilution ra te can be fi xed independentl y of considerations
of aerati on and mass transfer in sti rred vessels and air-lift fermcnters.
The o bj ecti ve of fennenter design and o perati on is to ensure that the des ired acti vit y of the
microorga ni sms concern ed sha ll not be restricted by the charac teri sti cs of the equipment. It
is, therefore, use ful to consider the problem in terms of the phys ical processes whi ch mi ght
limit microbial acti vity. Fennenters are constructed from materials that ca n withstand
repea ted steam sterili za ti on and cleaning cyc les. M aterials contac tin g th e ferm entati on
medium and brot h shou ld a lso be non-reac tive and non-a bsorpti ve. Glass is used to construct
fennenters up to abou t 30 liters capacity. The ad va ntages of g lass are that it is smooth, non-
tox ic, corrosion-proo f and transparent for easy inspec ti on o f th e vesse l contents. Because
entry ports arc required for medium, inoc ulum , ai r and instruments such as pH and
tem perature sensors, glass fe rm enters are usuall y equipped w ith sta inless steel head plates
containing many screw fitt ings. Most pilot- and large-sca le fe rmenters are made of
corrosion- r esistant 'stainless steel , a ltho ugh mild stee l with sta inless steel c laddin g has
a lso been used. C heaper grades of stai nless stee l may be used for the jacket and other
surfaces iso lated fro m th e broth . Interi or stee l surfaces are poli shed to a bright 'm irror' fini sh
to fac ilitate clea ning and steril izat ion of the reac tor; we lds on th e interi or of the vesse l are
gro und flus h before polishing. Electropolishing is preferred over mec hani cal polishing whi ch
leaves tin y ridges and grooves in th e metal to acc umul ate dirt and microorganisms.
The fe rm enter is equi pped w ith sparger, impeller and barnes that detelllline the
effecti veness of mi xing and oxygen mass transfer in stirred bioreactors. Aerobic cultures are
continuously sparged w ith air; however, most components of air are inert and leave direc tl y
through the exhaust gas line.
Barnes. whi ch are verti cal strips o f meta l mounted aga in st the wa ll of the tank , are insta lled
to reduce vo rt ex ing and sw irling of the li quid. Barn es are attached to the tank by means of
we lded brac kets; fo ur equa ll y-spaced barn es are usua ll y suffic ient to prevent vortex
form at ion. The opti mum bame w idth depends on the impe ll er des ign a nd fluid viscos ity but
is of the order 111 0-1 112 the tank dia meter. Effi c ient mi xing in the fennenter can be achieved
by the impellers. In standard des igns the im peller is located abo ut one impell er diameter, or
one-th ird the tank di ameter, above the bottom of th e tan k. Mi xing is fac ilitated when
c irc ulati on currents be low the impeller are sma ller than those a bove; flui d parti cles leav ing
the impe ll er at the same instant then take different peri ods of time to return and exc hange
materi a l. Rate of distri buti on thro ughout the vessel is increased when upper and lower
circulati on loops are asynchronous. A noth er device for improving mi xing is multiple
impe llers, a ltho ugh this requires an increase in power input.

28
During fermentation, the fermenter vessel containing medium is heated using steam and he ld
at the stcrili zation temperature for a period of time; cooling water is then used to bring the
temperature back to normal operating conditions. In addition, metabolic activity of cells
generates a substantial amount of heat in fermenters; this heat must be removed to avoid
temperature increases. Equipment used for heat exchange in bioreactors usuall y takes one of
the forms i.e., either ferment er may have an external jacket or coil through which steam or
cooling water is circulated . Alternatively, he lical or baffle coi ls may be located interna ll y.
External jackets on bioreactors provide sufficient heat-transfer are a for labo ratory and other
small-scale systems; however they are likely to be inadequate for large-sca le fermentations.
Intemal coils are frequently used in production vessels; the coil can be operated w ith high
liquid velocity and the entire tube surface is ex posed to the reactor contents prov iding a
relatively large heat-transfer area.

3te~ ____~___ ~========~~

___ pH conr:ro ller"

~~~~~~::-
;~ ~. c ido'l::::.:eo.:s:e
:&.nd pUfrlp re se t

Coo lir"'Q
'.V :So..be .....
out

Coolin..Q
w:9..tet'" In
--~ ===1 tl1+-+--- (sJ
Sp.BYge ....
.... b .... bble s )

Ste<9S..... - - - -_ c:===:: ___- - - S 00: til oS:! aJ t'"

Figure 2.1 A detailed diagram showing different parts of fermenter

As in fermenter cell s are provided with nutrients and very carefull y con trolled environment
to keep them in desired grow th stage. Nutrients and other materi als are fed in by va lve
operated pipelines. Conditions in the fermenter are carefully monitored to regulate cell
growth. Fermenter and all pipe work must be sterile before ferm entation begins. Thi s is
usually achieved by nushing the whole system with superheated steam before th e production
begins.

29
Interior is monitored by steril izable probes whi ch record temp. , pressure, stirrer speed, pH,
oxygen and carbon dioxide leve ls. These are a ll recorded and electronic control systems wi th
automatic valves will regulate them. E.g. if medium becomes too acidi c, bases can be added
from a reservo ir to correct the pH Incoming air is filtered and pumped into the base of the
fermenter - a valve releases the pressure from the top f the tank.
Antifoam Agents: Most ce ll cultures produce a variety of foam-producing and foam-
stabilizing age nts, such as proteins, polysaccharides and fatty acids. Foam bu ild-up In

fermenters is ve ry common, parti cul ar ly in aerobic systems. Foaming causes a range of

reactor operating problems; foam control is therefore an important consideration in
fermentation design. Excess ive foam overflowing from the top of the fermenter provides a
route for entry of contaminating organi sms and causes blockage of outlet gas lines. Liquid
and ce ll s trapped in the foam represent a loss of bi oreactor vo lume; conditions in the foa m
may not be favoura ble for metabolic activi ty. In addition , frag ile cell s can be damaged by
collapsing foam. Addition of special antifoam compounds to the med ium is the most
common method of reducing foam build-up in fennenters.
Mechanical foam breakers, such as high-speed discs rotating at the top of the vesse l and
centrifuga l foam destroyers, are suitable when foam development is moderate. Ho wever,
some of these devices need large quantities of power to operate in commercial-scale vessels;
their limited foam-destroying capac ity is a lso a problem with hi ghl y- foaming cultures. In
many cases, use of chemi ca l anti foam agents is unavo idab le.
Batch bioreactions
The majority of bioreactions are batch-wise. The first phase of batch bioreaction is
commonl y sterili zation, afte r which· the sterile culture medi um is inoculated wit h
microorganisms that have been culti vated to ac hi eve a speci fi c result.
During thi s dynamic reaction period, ce ll s, substrates (including the nutri ent sa lts and
vita mins) and concentrations of the products vary with time. Proper mi xing keeps the
differences in composition and temperature at acce ptable levels. To promote aerobi c
culti va tion, the medium is aerated to provide a continuous flo w of oxygen. Gaseo us
byprodu cts formed, such as C02 , are removed, and ae rat ion and gas-remo va l processes take
place semi continuously. Nex t, an acid or a lka li is added if the pH needs to be controlled. To
keep foaming to acceptable leve ls, anti foaming age nts may be added when indicated by a
foam sensor.
One of the first types o f batch systems is the tray fermenter, used in the earl y days of
commercial aerobic bioreactions for products slich as citric acid and penicillin. In thi s
system, the trays are loaded w ith the culture medium and the organisms, and the airflow
produces the bioreaction, during whi ch ex haust gas is discharged. When the bi oreaction is
compl ete, end product is removed from the trays. Because this method is inefficient for

30
produc ing la rge co mme rc ia l qua nti ti cs, it fe ll qui c kl y to the ways ide w ith the e me rgc nce of
subm erged ta nk sys te ms, w hi c h arc des igned to handl e signifi cantl y hi ghe r vo lumes.
Overall, batch bioreaction systems prov ide a number of advantages, including:
• Red uced risk of conta min ati on or cell mut ati on, due to a re la ti vely brief g rowth pe ri od .
• Lower ca p ital inves tm e nt w he n co m pa red to continu olls processes for th e sam e bi oreac to r
volume.
• Mo re fl ex ibilit y w ith vary ing produ ct/biological syste ms.
• Hi ghe r raw ma te ri a l convers ion levels, resultin g fro m a co nt ro ll ed growth pe ri od .
The disadvant a ges include:
• Lowe r prod uc ti vit y leve ls due to tim e fo r filling, hea tin g, ste rili zing, coolin g, e mpty ing and
cleanin g th e reac tor.
• Increased focus on instrumentation due to frequent sterili zati on.
• Greate r ex pe nse inc urred in pre parin g severa l subc ultu res for inoc ul ati on .
• Hi ghe r cos ts for labo r a nd/o r process co ntro l fo r thi s no n-stati o na ry proccss.
• Larger ind ustria l hyg ie ne ri sks du e to pote nti a l co ntac t w ith path oge ni c mi croorganism s or
toxin s.
Common a pplications for batch bioreactors include:
• Prod uc ts th a t mllst be prod uced w ith minima l ri sk o f cont a mination o r orga ni sm mutati on.
• Operati ons in w hi c h o nl y sma ll am o unts of produ c t a rc produ ced .
• Processes lIsing oll e reac to r to ma k e va ri o us produc ts.
• Processes in w hi c h batc h or se mi continuo us prod uct se para ti o n is adequ ate .
Continuous bioreactions
The de finin g c ha racte ri sti c of co ntinuo us bioreac ti o n is a pe rpe tu a l feeding process. A
culture medium th at is e ithe r s te ril e o r co mpri sed o f mi c roo rga ni sms is co ntinu o usly fed into
thc bioreac tor to m a inta in th e steady sta tc. Of co urse, th e produ ct is a lso d rawn co ntinuo us ly
from th e reactor. The reac ti on vari a bles and co ntrol pa ram ete rs re m ai n consiste nt,
establishin g a tim e-co nsta nt s ta te w it hin the reac to r. The res ult is co ntinuo us produc ti vity a nd
outpu1.
These systems provide a number of advanta ges, including:
• Increased pote nti a l fo r a uto ma ting th e process.
• Red uced labor ex pe nse, du e to a ut o ma ti o n.
• Less non-produc ti ve tim e ex pe nded in e mpty ing, fillin g and sterilizi ng th e reac tor.
• Consiste nt product q ua lit y due to in va ri able operat ing para me te rs.
• Dec reased tox icit y ri sks to staff, due to a uto mat ion .
• Reduced stress o n instrume nts du e to ste rili za ti on .
The disadvantages of continuous bioreactors include:
• Minima l fl ex ibility. since onl y s li ght va ri ati ons in th c proccss are possib le (th ro ughput.
medium co mpos itio n, oxyge n conce ntrat ion a nd te mp era ture).

31
• Mandatory uniformit y o f ra w material quality is necessary to ensure that the process
remains co ntinuous.
• Higher invest ment cos ts in co ntrol and automation equipment. and i ncreased expenses for
continuolls sterili zati on of th e medium .
• Grea te r process ing costs w ith continuolls replenishm e nt o f no n- sol ubl e, sol id substra tes
sti ch as straw.
Continuous biorcactioll is freq uentl y used for processes wi th hi gh-volume production: for
processes using gas. liquid or sol ub le sol id subs trates; a nd for processes invo lvi ng
microorga nisms with high I11lltation-stabili ty.
Review Question:
• Deline Biorcac lOf

• Give the advantage of lIsing glass for constructing fCrJn cnlcr body?

• What arc impell ers? Wh y they arc used in fcnncntcrs?

• What is ro lc of baffl cs in fcnncntcr'!

• Whal is the location of sparger in fefmclltcr?

• Givc the adva ntages of continllolls and batch fermcntcr.

• What arc pro bcs? How thcy hclp in fcrmcnter opcration?

• COllll11Cnt on computer controlled fcrmentation proccss.

• What arc anti foa m agcnts?

• Enli st the di ffo ront typos of fermentor.

• Diffcrcntiatc bctwee n continuolls and batch fermcnter.

• Discllss thc im portance of cooli ng coi ls in fefmcntcr.

• How heat is gcncratcd in fc rmcntcr'!

• Draw a wc ll labeled diagram of a fermcnter.

32
User's Notes

33
 SECTION C:
INDUSTRIALLY IMPORTANT MICROBES

34
Introduction:
Indust rial Mic robiology - Use of mi crobes to obta in a product or se rvice of economi c va lue
constitutes industrial mi crobiology . Any process mediated by or invol ving mi croorga nisms in
which a product of eco nomi c va lue is obtained is ca lled ferm entation. The terms industrial
microbiology and fermentation are virtually sy nonymous in th eir sco pe. object ives a nd
acti vities. The mi crobial product may be mi crobial cell s (li ving or dead), microbial biomass,
and components of microbial cel ls, intracellu lar or extrace llul ar enzy mes or chemicals
produced by the mi c robe s utili zing the medium constituents or the provided substrate .
The se rvices generated by mi croorgani sms ran ge from the degradation of orga ni c wastes,
detoxifi cation of industrial wastes and toxic co mpounds, to the degradati on of petroleum to
manage oil spill s, etc. Industri al mi crobiology also e ncompasses acti vities like producti on of
biocontrol agents. inoculan ts used as biofertili ze rs, etc.
Obviously, the sco pe and act iviti es of indu strial microbiology are too ex tens ive to be cove red
in any detail in a book like thi s scope; th ere fore. th e coverage in this c hapter remai ns
ge nerali zed and rather e le me ntary.
The activities in industrial mi crobiology begin with th e isolat iol1 of microorgani sms from
nature, th eir sc ree ning for product formation. imp roveme nt of product y ields. maintenan ce of
cultures, mass cul ture us ing bioreac tors, a nd usuall y end wi th th e recove ry of produc ts and
their purification .
Microbial Products of Potential Importan ce -
Product / Activity Examples
Products
I. Ami no acids L-g lutami c acid, L-Iys inc
2. Antibioti cs Stre ptom yc in , peni cillin. tetrac yc l ines. pol ymyx in
3. Beverages Wine, beer, di still ed beve rages
4. Biodegradable plasti c p-pol yhydrox ybuty rate
5. En zymes Amylase, proteases, pec tinases, in ve rtase, ce llul ase
6. Flavo uring age nts Monosodium g lutamate. nucleotides
7. Foods C heese, pickles, yoghurt, hread. vinegar
8. Gases CO, . H,.C H,
9. Organi c ac ids Lactic, citric. acetic. butyri c, fumari c
10. Orga ni c solve nts Acetone, ethanol , butanol , amyl alcohol
II. Othe rs G lyce rol, fat s, stero ids, gibbe rellins
II a. Vitami ns B 12, ribonavill, A
12. Recombinant proteins Insulin, interfe ron , subunit vacc ines
13 . Substrates A wide range of com pounds lIsed for c he mical sy nth eses of

35
 valuable products.
Ce ll s/Biomass
Food and feed yeast, other orga ni sms used as single celJ protein
14. Biomass
(SCP)
Biofertilizers, biocontrol agents, bacterial insec ticides,
15. Ce lls
mycorrhyzae
16. Vaccines A varie ty of viral and bacterial vaccines
Act iviti es
Biotransformat ion Steroids, antibiotics D-sorbitol
Di sposal of biological and indust rial wastes, detoxification of
Degradation
toxic compou nd s, petroleum
Impro ved recovery of oil and metals, di scovery of new oil
So Iubi Iiza t ion/accumu Ia t ion
re serves, removal of toxic metals

Thc mi croorgani sms of industrial importance are, generally, bacteria, actinomycctes, fungi
and algae. These organi sms occ ur virtua ll y everyw here, e.g., in air, water, soil , surfaces of
plants and animals, and plant and animals tis sucs. But most com mon so urces of industrial
mi croorga ni sms are soils, and lake and river mud.
Often the ecological habitat from which a desired microorganism is more likel y to be isolated
will depend on the characteri stic s of the product desired from it, and of process development.
For example, if the objectivc is to isolate a source of enzy mes, w hi c h can withstand high
temperatures, th e obvious place to look will be hot water springs.
A variety of complex isolation procedures ha ve been developed, but no single method can
reveal a ll the microorga ni sms prese nt in a sa mple. Many different microorgani sms ca n be
isolated by usin g speciali zed enri chm cnt techniqu es, e.g .. so il treatmcnt (UV irradiation , air
drying or heating at 70-120°C. filtration or continuous percolation, washi ngs from root
system s. treatment with dctergents or alcohol s, prcinoculation with toxic age nts), selecti ve
inhibitors (antimctabolites, antibiotics, etc.), nutritional (specific C and N sources). variations
in pH , temperature, aeration, etc.
The enrichment techniques are designcd for se lec tive multiplicat ion of onl y so mc of the
microorgani sms present in a sample. These approaches howe ver take a long time (20-40
day s), and require co nsiderablc labour and money.
The main isolation methods used routinely for isolation from soil samples arc: sponging (soil
direc tl y). dilution. gradie nt plate, aeroso l diluti on, notation , and differential centrifugation.
Ofte n th ese methods are used in conju nction with an enrichment technique.

36
3.1 Aim: Isolation of Am ylase producers from the environment.
Theory:
Amylases are enzymes th at break do wn starch o r g lycoge n. A my lases are produced by a
vari ety o f li vin g organi s ms, rang in g from bacteri a to plant s and human s. Bac te ria and fun g i
sec rete amy lases to th e outside o f th e ir ce ll s to ca rry o ut ex tra-ce llular di ges ti o n. Wh en th ey
have broke n down the insolubl e starc h. th e so luble e nd produc ts such as (g lu cose or ma ltose)
are absorbed int o th e ir ce ll s. A m y lases are c lass ifi ed based o n how th ey brea k down starch
mo lec ul es:
I. a -a my lase (alpha- a my lase) - Reduces th e viscosit y o f starch by brea kin g dow n th e bond s
at ra ndo m. th e re fo re produ c in g vari ed sized cha ins o f g lu cose.
11 . f3-a mylase (B eta-am ylase ) - Break s th e g lu cose-g lu cose bo nds d ow n by remov ing two
glu cose un its a t a tim e. th ereby pro duc in g ma lt ose.
III. Amy log lu cos idase (AMG ) - Brea ks s uccessive bo nd s from th e no n-reduc ing end o f th e
stra ig ht chain , producin g g lu cose M any mi crob ial am ylases usuall y co ntain a mi xture of
these a my lases.
Hum ans ex plo it mi cro bial a my lases fo r the fo ll ow in g purposes :
I. Hi gh Fru cto se Corn sy rup preparation
2. Additi ves to deterge nt s fo r re mo vin g sta ins
3. Saccharifi ca ti o n of s tarc h for alco ho l produc ti o n
4. Brew ing.
Th e soil co nt a ins a ri c h depos it of both bac teri a a nd fun gi, whi ch produce am y lases. Starch
hydro lyz in g fun gi o r bac teri a could be iso lated from th e so il. food s o r could be purc hased .
Buyin g saves tim e and e nsures a hi g h yie ldin g stra in . However, iso lating could be fun, and
constitutes a n additi o nal lab . A lthoug h man y mi croorga ni sms produ ce thi s e nzyme. the o nes
most comm o nl y used fo r the ir industr ial product ion are Bacillus SItIHilis. Bacillus
lichen{furmis. Bacilllts am,"/oliqu{jclciens and ASlle/gil/tls niger.

Figure 3. 1 Plate showing zone of starch hydrolysis

S(J/fr('e: jUJ/11('jwges. II ·m/c ·h. ('.},//- n !.\.I"h(/(·h/hif!.d I ] /LohPn/c ..

37
Materials Required :
Autoc lave or press ure coo ker, Hot Pla te or Microwave ove n, Nutri ent Aga r, Potato Dex trose
Agar. Soluble starch (1 % ). Gram 's Iodine. Ha nd trowel or disposable spoons. Sterile pipettes
(One each of 10 mL, 5 mL and I mL ), Pipe tt e pumps, S ix bott les of sterile wate r containing
90 mL eac h, Ste ril e G lass Pe tri dishes or Pre-ste rili zed d isposab le Pet ri-di shes, Bunsen
burn e r a nd matc hes, G lass spreade r
Procedure :
• Suspend abo ut 10 gra ms of e ither soi l or rolle n potato in 90 m L ste ril e d ist ill ed wtHer and ~
mi x prope rly ~
• Pipette 10 m L of the a bove and tra nsfer to a noth e r 90 m L o f wa ter
• Dilute fu rther in two more 90 !TI L sterile water blanks

For Fungi : Spread 0. 1 mL from th e d ilu tions o n Potato Dex trose Aga r pl ates (fort ified wi th
0. 1 mg/ mL st repto myci n s ul fate) w ith a g lass spreader. (The g lass spreader is qui ckly
steri li zed by d ippi ng in 95%) et hano l and putt ing in th e fl a me, so th at th e alco ho l burn s ofl)
Incubate at room temperat ure for abo ut 3 days
For Bacteria: Spread 0 .1 m L of the d iluted sam ples on Nutrie nt Agar p lates co ntai ning I %
w/v solu ble starc h and inc ubate at 30"C fo r 24 hours
• Starch-produ ci ng co lon ies wi ll have a n area o f c lea ri ng aroun d the m .
• Co nfirm by flood ing pl ates w ith G ra m's iodine.
• T ra nsfe r di stin g ui shab le, a my lase- prod uc ing fun gi to fres h p lates of Potato Dex trose aga r
• Contai n ing I % sta rc h, usi ng a ste ril ized dissec ting needl e. For bac teria, st rea k on a fresh
• Pla te o f Nutri e nt Agar co nta ining I % sta rc h.
• T ra nsfer yo ur isola ted amy lase-prod uci ng fungi to Potato Dex trose Ag ar slants, a nd the
bac te ria to Nutrien t Aga r. A llow bacte ri a to grow fo r 24 hours a nd fun gi to grow for 72
ho urs , the n store in th e re fr igerato r until needed .
Observation T a ble :
Sample Isolate A my lol ytic/ Non- Morpholog ical / Bioch emical characteristics
am y lol ytic of isolates

38
3.2 Aim: Production and assa ying of microbial proteases.
Theory:
Proteases are the key e nzy me in th e industri a l app lication. Mi c robi al proteases pl ay
impo rta nt role in biotechn ological process with worldw ide sale representin g about 60% of
th e tota l e nzy me ma rk ed. A numbe r of bac te ri a, fun gi and yea st have bee n repon ed for
protease producti on. Many o f the organi sms produce more th an one kind of protease. The
type of proteolyti c e nzyme formed may depe nd on th e composition o f th e medium . A
protease (a lso termed pepti dase or proteinase) breaks down proteins. A protease is an y
enzy me that condu cts proteolys is. that is. begi ns prote in cat a bolism by hydrol ysis o f th e
peptide bonds tha t link a min o ac ids togeth er in th e polypeptide cha in formin g th e prote in .
Pro teases wo rk bes t in ac idi c co nditi ons except a lkaline prot eases. It's o ptima l acti vit y shown
in a lka line (bas ic) pH.
The a lka line proteases are hi ghl y e xpl o ited enzy mes in food processi ng, lea th er, detergent,
pharmace ut ical , di agnosti cs, waste mana gement, sil ve r recovery medi ca l purposes as we ll as
feeds and chemical industries. The protease enzyme co nstitu tes two thi rds of to ta l enzymes
used in va ri ous industries. Of all proteases. alkaline proteases produced by Bac illus spec ies
are o f great import ance in deterge nt industry due to th e ir hi gh the rm ostability and pH
stabilit y.

...:.=-:'::':::. ~........
B a cillus sub tilis

Figure 3.2 Plate showing caseinolytic activities of Bacilllls whlilis

SOl/ree.- II IIp.- Ilacadem i c. pgcc, edl/l-kroherls/lI'ehl exnen=.l'me,,,lexoel1=.\ 'mes. 111111

For produ cti on of enzy me for industri al use, isolation and characteri za ti on of new promi sing
strains using cheap carbon and nitrogen source is a continuous process. The microbial
proteases o f Asperg illus species, in particul ar, ha ve been studi ed in detail since they are
know n for their capac it y to secrete hi gh levels of enzymes in their growth environment.
Several of these secreted enzy mes, produced in a large-scale submerged fe rmentation, have
been w ide ly used in the food a nd beverage industry for decades.

40
Materials Required:
Aspergillus niger or Bacillus slIbtilis, Potato Dex trose Agar Mediu m. Nu trient Aga r, Petri
plate sterili zed, Sal ine tu bes, Incubator, Inocul ating loop. Laminar air fl ow, Enzy me
production medium
Procedure:
Preparation of enzyme production medium:
A. Prod ucti on medium for Bacillifs sub/ilis:
Ingredients Compos ition (gl L)
Peptone I
NaCI 5
Skim Mil k 10
Agar 20
pH 7.0-7.2 at 25°C
.. ..
• Sten ltze peptone separately and add asepti ca ll y to the flask co nta1l1 J1lg the liqUId medium ,
att cr cool ing.
• Inoc ulate the med iulll (50ml in 250m I conical fl ask) with Iml of an overn ight cult ure of
Bacilllls Suhfi lis .
• Incubate at sone in a rotary shaker at 150 rpm fo r 12hr.
• At time intervals de termi ne th e turbidity of the cult ure meas uring the increase in optical
density at 450 11m wit h a spectro photometer.
B. Prod ucti on medi um fo r Aspergillll.\· niger
Ingredients Compos ition (gl L)
Ammon illll1 sul phate 1.0
Magnesium Sulp hate heptahydrate 5.0
Potassium di-Hyd rogen Phosphate 5.0
Ferrous Sulphate heptahydrate 0.005
Glucose 10.0
Jowar seeds
pH 5.0 at 25°C

• Inoculate the liq uid medium with overn igh t grown Aspe rgillus culture.
• Examine fer mentation duratio n for 24 to 120 hrs.
• Kep t the cult ure tl as k 0 11 ro tary shaker at 300 rp m at 28 1l C.
Product recovery a nd purification:
Protease is an extrace ll ular enzy me so its recovery is qui te easy. Aft er inc ubat ion cent rifuge
the production med ium at 12.200 rpm for 15 min to separate th e ce ll s. Collect the supern atant
as it wi ll contain the crude enzyme and store a14°C till furt he r use.

41
Enzyme assay:
Cascin so lution of 2% ( I ml) was incubated with 0.1 ml of enzyme so luti on and 0.9 ml of
sodium phosphate buffer (pH 7) for 10 minutes at 40°C. The reaction was stopped usi ng 10%
TCA solution. After 20 minutes th e mixture was centrifuged 10,000 rpm for 5 minutes. The
co lour intensit y of supernatant was read at 280 nm .
The enzyme activity was calcu lated frol11 standard curve of L-Tyrosine. The serial increase in
concentration of tyro sine (10- 1OOtglml) was read at 280 nm for th e standard graph. Enzyme
activity was depends on the temperature, particular pH , sub strate concentration and the active
site of enzyme.
Observation Table:
Sample Isolate Proteolyticl Non- Morphologicall Biochemical characteristics
proteolytic of isolates

Results and Interpretations:

42
3.3 Aim: Production and assaying of microbial Iipases.
Theory:
Lipases are ubiquito us enzymes of considerabl e ph ys io logical s ignifi ca nce and industri al
potential. Lipases find pro mi sing a pplica ti ons in o rga ni c chemi ca l processing, detergclH
formul ati ons, sy nth esis o f bios urfactants. in th e o leoc hemi cal industry, da iry industry,
agrochemi cal industry, bakery (fl avour imp rove ment ), beve rages (impro ved aroma), paper
manufacture, nutriti on, cos meti cs, and ph arm ace uti ca l process ing. Deve lopment of Ii pase-
based tec hno logies for th e syn th es is o f nove l compo unds is rapidly ex panding the uses of
these enzymes. O ne limiting fac to r is a shortage of lipases hav ing th e specific required
processing charac teri sti cs. A n increas ing number of lipases w ith suitable properti es are
becomin g ava ilable and effo rt s are und erway to co mmerciali ze biotrans form ati on and
syntheses based on lipases. The majo r commercial a ppli cati on for hydrol yti c lipases is th eir
use in laundty deterge nts. Deterge nt enzy mes make up nea rl y 32% o f the to tal lipase sales.
Lipase fo r use in deterge nts need s to be themlOstable and remain ac ti ve in th e a lk alin e
environment of a typ ical mac hine was h. An estimated 1000 to ns of lipases are add ed to
approximately 13 billion to ns of detergents produced each year. Mos t of th e industri al
microbiallipases are deri ved from fun g i and bacteria.
Some commerc iall y ava il abl e funga l lipases are Candida rugosa, Candida antarfica,
Thermomyces lanllgil1oslIs, RhizoJ11 l1cor fIl iehei and Bac terial lipases are BlIrkhulderia
cepacia, Pseudomonas alcaligenes. P5ielldomool1us mendocinCi and Ch rOlllObacter visc()slIm.

Figure 3.3 Plate showing lipidolysis by Staphylococcus epidermidi" in spirit blue agar
So 1/ ree: h//p:1/en 'adem ie. pgcc.edlll- k roher/si ll 'e We. roell=.I·mes!e.n)(!1/=.1 "/lies.It f 11/
Materials Required:
Medi a, Petripl ates, No rm al Sa line (9 mL ), Erl enmeye r fl as k (250 mL ), wax marking pe ncil,
steril e pipettes ( I and 10 m L). mec hani cal pippetor.

43
Medium A:
Ingredients Composition (gl L)
Oli ve oil 20.0
Ga ll powder 10.0
Ammonium Sulphate 5.0
Magnesium Sulphate 1.0
Aga r 20.0
* Heat and emul sify the mi xture, and adjust the pH to 8.7 wilh Na2C03.
Medi um B
Ingredients Composition (gl L)
Roasted soy bean meal 20.0
Com steep liquor (CS L) 10.0
Wheat starch 10.0
Di Potass ium hydrogen phos phate 5.0
Adjust the pH to 8.7 with NaOH

Procedure:
I. Iso/at ion Clnd cul/ivation:
• Suspend the soil sam ples in water and spread on plates of mediu m A
• Incubate the pl ates at 30°C fo r 72hr.
• Transfer the microorga nisms which form ed clear zones around the co lonies on the plates
to stock cultu re slants of medium A and nutrient aga r.
• In orde r to examine lipase prod uction, take 7ml of med ium B in a test tube ( 18 x 180
mm ) and inoc ulate wi th a loopful of an isolated microorganism and culture at 30°C for
40hr with shakin g.
• Use culture broth for the assay of lipase ac ti vity.
II. Assay methodjor lipase activity:
• Emulsify 10 ml of oli ve oil 90ml of a 10% gum aerobic solution.
• Make a reaction mi xture, consisti ng of 2.5 ml of oli ve oil emul sion, 2.0ml of glyc ine-
NaO H bu ffer ( I m, pH 8.7), 0.5ml of di stilled water and O. I ml of enzyme so luti on.
• Place reacti on mi xture in a 25 ml glass stoppered test tube and incubate for 10 min at
37°C.
• Add 15ml of a heptane- iso propanol mi xture ( 11:4) to the reaction mi xture.
• Shake vigo ro usly for 30 sec, and a llow to stand fo r more than 30 min .
• Pipette 5 ml of the upper laye r into a test tu be (20 x 130) and titrate under a nitrogen gas
stream aga inst 0.0 I N ethano lic potass ium hydrox ide so lution with th ymol blue as an
indica tor.

44
• Terminate the reaction by th e addition o f I ml of 2N sulphuri c ac id.
• A s a blank test, add 2N sulphuric acid solution to the reacti on mi xture before th e additi on
of enzy me soluti on.
• Subtract the titrati on va lue of the bl ank test fro m the prev ioll s titration va lue.
• Calculate th e amount of liberated fatty ac id from the standard line prepared us mg a
known amount of palmitic acid. One lipase unit is defin ed as the amount of enzyme
which liberates I 1111110 \ of fatty ac id in one minute under the assay conditions described
above.

Observation Table:
Sample Isolate Lipidolytic/ Non- Morphological/ Biochemical characteristics
lipidolytic of isolates

Results and Interpretations:

45
3.4 Aim: Isolation of Antibiotic Producing Microbes from Soil.
Theory:
Although pharmaceutical companies currently do much of their drug di scovery lI SlIlg

comp ute r modeling. traditionall y, antibiotics were di scove red by sc reening. In thi s approach.
a large number of isolates of poss ible antibioti c producing mi croorgan is ms are obtain ed from
nature in pure culture, and th ese iso lates are th en tes ted for antibiotic production.
Man y soil microorgani s ms produce anti-bacterial or anti-fungal chemica ls, compounds which
are termed antib iotics. It is re lative ly simpl e to id entify antib iotic producing microorganis ms
from isolated so il bacteria or fungi by testi ng them again st standard microbial strain s on Petri
di s hes containing a nutrient medium. In this act ivity antibiotic microorgani sms will be
identified by their abilit y to produce compounds wh ich inhibit the growth of a common
bacterium , non pathogenic staphy lococclIs.
Soil bacteria known as Streptomyces produce man y clinically use ful ant ibiotics, More than
500 Streptomyces spec ies are recogni zed, and nearly half of th em produce antibioti cs,
according to some studies. He re is a method for iso lating and sc reeni ng antibiotic producers.
This activ ity is intended to demon strate that common microorgani s ms iso lated from so ils are
sources of ant ibiotics. A simple growth method can demonstrate how th e effect of diffu sible
compou nd s from different mi c roorgani sms can be used to observe the inhibition of growth of
a common bacterium associated w ith human s, This sa me activity sca led up many times is
how the pharmace utical industry di sco ve rs new antibioti cs for human use.

" ,
Figure 3.4 Plate showing antibiotic producing RllOdococclI,"o-jascialls
SUII ree: II '\ \ 'II', fe( '/lIIologl 'reI 'ie\\', ("011 Il hh lIIu:di(' i nel 2 04 251

Material Required:
Steri le Petri di s hes, steril e so ft aga r and agar (Muller Hinton agar), inoculating loop, Bunsen
burne r, Laminar-air flow , Dis posable inoculating loops, Petri di s hes containing Mueller-
Hinton agar, microbial culture of non pathogenic Staphylococcus spp., so il sa mple

46
Procedure:
• Take a soil sa mpl e and seria ll y dilute it upto 10·' d ilutions.
• T ransfer one 1111 of diluted sample and pour pl ate w ith the agar
• Inc uba te th e pl ate at 35 ' C in incuba tor for 24-48 hours
• A ft er 24-48 hours incubati on in spec t the pl ate for th e growth of bac teri a.
• Ma ke replica copy o f the stand ard plate by to uching th e loo p to specific coloni es a nd
mark them on two repli ca plates
• O n one of th e plat e, indica tor organi sm seeded in so ft agar wi ll be poured (20 ml so ft
aga r w ith 5 0~1 o f c ulture)
• Incubate th e plate at 3YC in incubator for 18 hours and look fo r th e zone of clearance.
• O bserve th e co loni es w ith zone of clearance around them and cultivate the respec ti ve
co loni es from second repli ca pl ate for further check ing th e anti mi crobi al ac ti vity toward s
ot her indica tor microorgani sms.

Observation Table:
Sample Isolate Antibiotic Producers Morphological! Biochemical characteristics
(inhibition zone in dial of isolates

Results and Interpretations :

47
3.5 Aim: Isolation and screening of Streptomyces species as antibiotic producers.
Material required:
Streptomyces-se lecti ve medi a, soil sa mple, dilu tion bla nks (9 mL ), Steril e Petri plates,
In cubator (at 35-37°C), Wax markin g penc il, steril e pi pett es ( ImL, 10 mL) and mec hani cal
pipettor, Soft aga r seeded wi th fres h Slaphylococclfs epidermidis culture, ot her non
pathoge ni c cultures. Muller- Hinton agar, steril e inoc ul ating loop.
Procedure:
/. Isolalion o/StreplOmyces species./i-0111 soil
• Add a gra m of soil to 9 !TI L ste ril e water and m ix thoroughl y.
• Dilute the soi l mi xtu re further by transfe rri ng I mL dow n a series of dilut io n tubes.
• Selec t the most d ilu te mi xtures and add I mL of the liq uid to plates co nta ining
Streptomyces-selec ti ve med ia.
• Spread the sa mpl e evenl y, and th en incubate for 5-7 days at roo m tempe rature.
ii. Testing/or anfihiotic production
• To test fo r anti bioti c production, overl ay the plates wi th an indicator organi sm , such as
the non path oge ni c bac te rium Stap/~vlacocclls epidermhlis.
• Incubate th e plated at 3rC for 24 hours.
• Check th e plates for zo ne of inhibitio n surroun din g poten ti a l ant ib ioti c-produc ing
organi sms.
III. Test to check the antibiotic spectrum ala suspccled antibiotic producer
• Streak the suspec ted anti b ioti c prod ucer across a fresh Muller- Hint on agar pl ate.
• Incubate the plate to permit bacterial grow th and antib iotic producti on.
• C ross-strea k test orga nisms a long the p late.
• Incubate the pl ates at 37°C for overni ght. And check fo r grow th inhi bi tio n
• Obse rve the growth of th e test orga nisms.
Observation Table:
Sample Isolate Antibiotic producerl non- antibiotic producer Antibiotic spectrum

48
Results and Interpretations:

49
3.6 Aim: Production of Nisin from LaclococClis laclis.
Theory: 1
Nis in is ::l n::t fllr::t ll v o(,C' lIrri ll p :lllf;rnicrn hi til peptioe :lIld " ":-IS disco"crcu ill 1928. Nis in is a
polycyc lic pep tide antibacteria l w ith 34 amino aci d res idues used as a food preserva ti ve. It
contains the uncommon amino acids lanthionine, mcth yllanthionine, d idehydroa lanine and
di-dch yd roa min obut yri c ac id . These unusual amino ac ids arc int roduced by posttrans lational
mod ifi cati o n o f the prec urso r pe pt ide.
N isin is produced by ferme nta ti o n lIsing th e bacterium Lactococc us lact is. Commerciall y, it
is obtained from the culturin g o f Lac tocc us lac li s on natu ra l subs trates. slich as mil k or
dex trose, and is not chemi call y sy nth es ized. II is lIsed in processed cheese, meats, beverages,
etc. durin g produ cti on to ex tend she lf life by suppress ing G ram·positi ve spo il age and
pa th ogeni c bac teri a. Wh ile 1110s t bacteriocins genera lly inhi bit only closely re latcd spec ies,
N is in is a rare example of a "broad-s pectrum" bac te riocin effecti ve against ma ny G ram-
pos iti ve orga ni sms, inc ludin g lac ti c aci d bacteria (commo nl y assoc iated w ith spo ilage ).
Listeria !1l0110(\·togenes (a kn ow n pathogen). etc. However, when coupled wit h the che lator
EDTA. N isin has also bee n kn ow n to inhibit G ram· nega ti ve bac teria, as we ll. N is in is so luble
in wa ter and can be effec ti ve at levels nea ring th e parts per billio n ran ges. In foods. it is
co mmo n to lise

Figure 3.6 Plate showing antibacterial activity of Nisin

SO/ll"ce: 11'11 w.lC.'ag(lsc. it:!.. ./4541 !eopr-4 541 .£lsp
Nisin at levels rangi ng from - 1·25 ppm . de pending o n th e food type and regul atory a pprova l.
Due to its natura ll y selec ti ve spec trum o f ac ti vit y, it is a lso employed as a selecti ve age nt in
mi crobio logical media fo r th e isolation o f g ram-n egati ve bac teria. yeast. and mo ulds. S ubtilin
and Epidermin are related to N is in. All are membe rs of a class o f molec ul es kn ow n as
lanti b io ti cs. N isi n solubil ity and stab ilit y ,"creases substan ti a ll y wit h increase in ac idi ty.

50
Results and Interpretations:

52
3.7 Aim: Production of antimicrobial substances from lactic acid bacteria.
Theory:
Bac teria produ ce many inhi bitory co mpounds whi c h can inhibit the growth of potenti a l
spoil age or pathoge ni c mi croorgani sms. These substances include orga ni c ac ids (lac ti c ac id,
propi onic ac id, ace ti c ac id etc.), hyd roge n pe roxide and diace ty l, bac te rioc in s li ke Nisin,
Ac idophilin, a nd Di p lococc in e tc. if th ese anti mic robia l co mpounds arc prod uced in solid
medi a. Th e n clear zo nes are deve loped aro und the test organi sms grow in g in parti c ul ar so lid
medi a.
Material required:
L(lc1ic odd /)(1creria. indicat or Strain. MRS broth, MRS agar. Pctriplates. Erle nmeyer nask
(500 mL ), wax marking pe ncil, wate rbat h, inc uba tor. ce ntrifuge , filters (0.22-0.45)1 ), steril e
we ll borer, sterile tips (2 00 pL a nd 1.0 mL ), alltopipett e (2 00 pL and 1.0 mL), G ram stain ing
kit , g lass s lides
Procedure:
• C hec k the purit y of c ultures gram staining and c hec k fo r their purit y
• In oc ul ate the pure cultures obtain ed in a bove steps in M RS broth
u
• Incubate al 37 C for 4 8 hr.
• A fte r 4 8 hours of growth ce ntrifuge th e c ulture a t 7000 rpm for 10-\ 5 minut es
• Co ll ec t the supe rn ata nt and sterili ze us ing 0 .22 111m me mbra ne fi lte rs
• Use S.ul/reus. £. Coli. B. slIhfi/is as test organ isms.
Production of antimicrobial substances can be determined by 3 methods:
• We ll Assay Meth od
• Spotl Streak or Law n Method
• Disk Me thod

I. Well AsslIl' Method:

• Add 0. 1 1111 of test c ultures in so ft agar test tube
• Pour these test tubes on th e so lidified nutri ent agar and MRS med ium pl ate
• Inc ubate th e pl ates at 37 uC for 24 hrs.
• C UI we ll s on medi a us ing borer
• Add 0. 1 1111 o f ste rili zed supern atant i.e. antimi crob ial substance to each we ll and all ow
for diffusion under re fri ge rati on con diti ons
• Inc ubate th e pl ates at 37 uC fo r 24 hr an d observe for th e zone o f inhibiti on

II. Streak plate melllOd:

• Prepare MR S agar and nutri e nt agar pl ates
• Strea k th e cultu re to be tes led fo r a ntimi crobia l ac ti vity on the med ia

53
• All ow to grow at 37°C for 24 Itrs
• Mix 0 . 1 1'nl of test culture in soft agar test tube and \1om o\er the abo\'c streaked ?\a\cs
• Again incubate at '37u C for 24 hrs
• Observe for a zone of c learance

III. Disk Method:

• Prepare MRS agar and nutrient agar plates
• Mix 0.1 1111 of test cu lture in soft agar test tube and pour on the above plates
• Soak filter paper d isk (steriii zed) in the sllpematant obtained from different iactic acid
bacteria cultures
• Impregnate these di sk on th e above plates
• incllbate at 37"C for 24 hrs
• Observe for a zone of clearance and measure th e diameter

Figure 3.7 Agar well assays

SOli ree: 1\' \\'\\ '. ('VI1ICell .cum. lIlIl .. .lIJI(I.~' i I is 2(JO(j. 11/111

Figure 3.8 Disk diffusion methods

Source: f (Klllly.md.edll(jeariIMLlml-15. hlll/

54
Observation Table:

Lactic acid bacterial Test organism I Test organism 2 Test organism 3

Isolates (zone of inhibition in (zone of inhibition in (zone of inhibition
diameter) diameter) in diameter)

Results and Interpretations:

55
3.8 Aim : Isolation of psychrophiles from milk samples.
T heory:
Psychrophilic bacteria have bee n defin ed as bacte ria that grow wcll at O°C within 2 weeks
and based partl y on a standard method for de temlin in g psychrop hi li c bacteria. as bacteria that
grow at a rela ti vel y rapid rate at 7. 2°C, i.e., that for m visib le co lonies on plate s at this
temperature in 10 da ys. Fos ter defi nes th em as bacteria that grow relati ve ly rapid ly at 1.7 to
10°C, the temperature norma ll y used in commcrcial ho ldin g and d istribution channe ls. Some
in vestigato rs prefer to describe such bacte ria as psychrotrophic rat hcr Ihan psychroph ili c,
and there seems 10 be merit in using some tenn that indi cates that they mere ly are abl e to
grow at low temperatures rath er than Ihat they are co ld-l ov in g. Spoilage of paste uri zed mi lk
and mi lk product s o fte n results fro m the grow th of hea l-sensiti ve, gram-n egati ve.
nonsporcformin g bacte ria that enter prod ucts after pasteurization. Obviously, it wo uld be a
mi stak e 10 re lax e fforts to prevent nOilsporef0l111ing psyc hrophilic bacteria from getting into
mi lk and dairy products after pasteuri zati on. However, as attempts are made to ex tend th e
shelf- life o f n uid dairy produ cts, psyc hrophi lic spore formin g bacilli will become a greater
potentia l prob lem. Thi s is also important in regard 10 th e use of dairy prod ucts in other foods,
the deve lopment of aseptic fi ll in g, and the probabi lit y ora shift toward "sterili zation" of m ilk
and Ouid milk products.
Mate r ial required:
Refri ge rated Spoil ed milk sa mp le. Trypticase soya agar. Petrip lates. dil uti on blanks (9 mL ),
wax markin g penc il, Re fr igerator. sterile pipettes, mecha nical pipettor, Gram staining
reagents. Sc hae ffer-F ulton stai ning reagents.
Procedure:
• Tak e I mL of th e milk samp le and dilu te in 9 m L of di lu tio n buffer up lO 1000 to 10,000
times.
• Pour portio n ( I mL) of th e d iluted samp le from approp riate di lution to the steril e Pe tri
plate under asept ic condit ions and fina ll y pour TSA on it.
• Incubate the plate s al 4°C for 2 103 days.
• Transfer the co lonies to TSA s lant s, incubated at 4°C, and store in a refrigerator fo r
ident ifi cation .
• Perform Gram stainin g and Spore detection test.
O bse rva tion Ta ble:
Isolate Gram reaction Spore fo rmer Other obse r va tions

56
3.9 Isolation of salt tolerant microorganisms from food samples.
Theory:
Halotol erant or halophilic mi croorgan isms, able to li ve in sa line envi ronm ent s, offer a
multitude of actual or potential applications in various fields of biotechnology. The technical
a pplicatio ns of bacteriorhodopsin compri se holography. spatial light modulat ors. optical
computi ng, and optical memori es. Compati bl e solutes are usefu l as stab ili ze rs of
biolllolecuies and whole ce ll s, sa lt antagonists, or stress-protective agents. Biopolymers, suc h
as biosurfactants and exopo lysaccharides. are of interest for mi crobially enhanced oil
recovery. Other useful biosubstances are enzymes, suc h as new isomerases and hydrolases
that are active and stable at high salt con tent s. Hal otolerant mi croorga ni sms play an essential
role in food biotechnology for th e production of fermented rood and food supp leme nts. The
degradation or transfonllation of a ra nge of organic pollutants and th e production of
alternative energy are other fields o f applications of these groups of extrel11ophiles.
Material required: Food/ milk sa mple. Nutrient agar with 2, 5 and 10% Sod ium Chloride,
Potato Dextrose agar wit h 2, 5 and 10% Sodiu m Chloride. Petriplates, Ph ospha te bufTer
sa lin e (pl-l 7.0), wax marking pencil. steri le pipettes, mechanical pipeltor, Gram stai ning
reagents. Sc ha effer-F ult on sta inin g reagents, Lactophenol cotlon blue stai n, s lid es.
Procedure:
• Take 10 g of th e food sampl e and dilute in 90 mL of Ph ospha te burfer sa line (pH 7.0),
make further dilutions upto 1000 to 10,000 times.
• Pour portion (I mL) of the diluted sample from appropriate dilution to the sterile Petri
plate under aseptic co nditi ons and finall y pour Nu tri ent agar (salt co nce ntrat ion 2, 5 and
10%) and Potato Dextrose agar (salt co ncentrati on 2, 5 and 10%) on it.
• Incubate th e plates at 37°C for 2 to 3 days for bacteria and at 25-30 0 ( for 5-7 days for
fungi.
• Transfer the colonies to NA and PD A slants. incubated at respective temperatures, and
finally store in a refrige rat or for identifi catio n.
• Perform Gram sta inin g, Spore detection test and Biochemical test for the identification
according to Bergey' s Manual.
Observation Table:
Isolate Growth at salt concentration (in 0/0 ) Morphological and Biochemical analysis

58
Results and Interpretations:

59
3.10 Aim: Isolation of Sugar tolerant microorganisms from food samples.
Theory:
Osmotic concen trat ions of substrates where upon the micro-organisms grow help classifying
them as und er:
(i) Osmophobic: Those mi cro-orga ni sms that di e of dehydration if subj ec ted to substrates of
high osmotic co nce ntration s.
(i i) Osmophilic: Those m icroorga ni sm that best grown on substrate s of high osmo tic
concen tration s.
(iii) Halophilic: Halophilcs represent those mi crobes that preferably grow in high osmo ti c
concentrations produced by d isso lved sa lts.
(iv) Osmoduric: These are those mi crobes th at grow normally on substrates of moderate
osmoti c concentrations but, prove to be resistant to wide osmotic cha nges in their subst ratum.
It has been reported that high-sugar foods arc so metimes spoiled by sugar tol erant or
oSl11ophilic yeasts. It has been proposed that the yeasts which can grow in th e prese nce of 40-
70 % (w/w) sligar be called sugar-tole rant yeasts or osmophilic yeas ts. These yeas ts grow
very slowly near the minimal wa ter acti vit y (Aw) for growth, and very few spec ies can grow
in foods con tain ing 65-70 % (w/w) sugar such as neriyoka n (swee t bean curd), However,
sudden changes in atmosphe ric temperat ure may increa se the Aw of food by co ndensin g
moi sture o n the surface, thus accc\erat ing microb ial growth .
Most yeast grows bes t unde r ae robic condition s. but a large number of yeas ts ca n also grow
under low oxygen ten sion. T hu s. yeas ts may ca use fermentati ve spoilage of products und er
low oxygen conditions, such as gas-exchange packaged food s and vac uum-packaged foods.
This is an important problem in preserving high-sugar foods.
Material required :
Food sampl e, YM agar, Petri plates, wax marking penciL Inoc ul ating loop.
Procedure:
• Isolate yeasts by direct streaking on medium containing 25 or 40(10 (w/ w) glucose,
0.5% polypeptone, 0.3 % malt extract, 0.3 % yeast extract, and 2.5(Yo agar.
• Purify iso lated ye ast strains by conventional streaking technique using the sa me
media as used for isolation .
I. Test ofslIgar-lOlenll1ce qlthe isolates:
• Use the four kinds of agar media shown in Table 1 for testing sugar-tolerance. 2.
Inoculate actively growing cultures on YM agar on to agar plates.

Media
YM agar
25% (w/w) glueosc-po ly-peplO ne-yeas t ext.- malt ex t. agar

60
40% (w/w) gl ucose- poly-pe ptone-yeast ex t. -malt ex t. agar
50% (w/w) glu cose- poly-peptone-yeast ex t.-m alt ext. agar

• Tightly close the plates with rubber bands and incubate at 26°C for 2 weeks, and
me.Isure the diameter of each colony.
I/. Selection o,(veast slroins./iJr idelltf/koatioll:
• In vesti ga te all of the stra ins for fe rm ent ati on of gl ucose, ass imilation of maltose,
ga lac tose. sucrose. raffinose. lactose, and nitrate, grow th in vitamin free medium, and
grow th at 30, 37, and 42°C.

Observations:
Isolate 0/0 of Glucose Maltose Nitrate Growth Growth at
sugar fermentation fermentation reduction in different
tolerated vitamin temperatures
free
media

Results and Interpretation:

61
Review Questions:
• What are secondary metabo lites?

• Exp la in the ro le of bacteri oc in in food industry"

• A lthough antibioti cs are secondary metabo lites but industri all y th ey are produced in
continuous phase fennenler. Ex plain.

• In prin ciple, how do bac teriocin s such as ni sin functi on? Wh at bacterial genus produces
this impo rt ant polypepti de"

• Approx imate ly how many new antibio tics are being discovered per year" What port ion of
these is deri ved fro m Ac tin omycetes?

• Give some important spec ific compo unds th at are produ ced by the use of
microorganisms.

• What is the impo rt ance of crowded pl ate technique"

• Wh at is the fun cti o n o f li pascs?

• H ow ca n one determin e w heth er a bac teriulll is lipolyti c?

• What are two functions of lipids in bac teri al ce lls"

• G ive some exa mpl es of foods th at might be spoiled by lipolytic bac teri a.

• How is the ability of certa in bacteri a to attac k phospholipids re lated to path ogeni city?

• What is the difference between a tri g lyce rid e (triacy lglycerol ) and a phospho lipid?

• What are several path ways th at bac teri a use to metaboli ze lipids?

• Describe the fun cti on o f hyd ro lases.

62
• Describe the chemistry of starch hydro lys is.

• The chemica l used to detect microbia l starch hydro lys is on starch plates IS

• What does starch hydro lys is by a bacterium indicate"

• Amylase IS an enzyme th at att acks starch. The smallest prod uct of this hydrolys is IS

ca lled _ _ _ _ __

• How is it possibl e that bacteria may grow heavi ly on starch agar but not necessaril y
produce a-amy lase?

• What are the ingredients of starch agar?

• Define the foll ow ing term s:

• amino acid • protease
• casem • prote in
• hydrolysis • proteolysis
• pepti de bond
• How can plate count agar th at contains milk be used to demonstrate proteolysis?

• Wh y are some bacteria able to grow 0 11 pl ate count agar that contains milk even th ough
they do no t produce any proteases"

• Draw th e chemica l reaction for proteolyti c hydrolys is.

• Wh y was sterile skim milk llsed in th is ex periment?

• Wh y is milk white?

• What is the sig nificance of psychrop hiles in dairy industry"

• Explai n the ph ysiological adaptations of halop hiles"

• Give th e economi c importan ce of sugar tolerant bacteria in food industry?

63
Use r's No tes

64
 SECT ION D:
MICROBIAL PRODUCTION

'\~,\
\""

\
, ,
\ ' \'

... '
I

65
4.1 Aim: Production of lactic acid from whey.
Theory:
Whey is a by- product o f the c heese industry w hi c h is often di sposed as a was te in the past.
ca usin g high enviro nm ental contaminati o n. Co nsiderab le effort s ha ve been made over the
past yea rs to find new o utl ets for w hey utili zatio n and redu ce e nv ironmental pollutio n. Liquid
whey is co mposed of lactose (5%). wate r (93%). proteins (0.85%). minera ls (0.53 % ) and a
minimum amount o f fat (0.36%). The main w hey pro te ins are ~ -I ac t oglobulin (BLG) (58% )
and a- lacta lbumin (A LA) (130/0) whil e immunoglob ulin s, serum a lbumin s and proteose
pepto nes are prese nt in lesser exten t.
Lacti c acid bacteria (LAB) have been ex te nsive ly used as starter c ulture s in th e fermented
food industry due to th ei r metabo li c acti v ity o n prote ins. sugars and lipids, thu s co ntributing
to food dige sti bilit y and prese rva tion as we ll as the improvement of text ure and sensory
profile of the e nd product. These mi c roorga ni sms have complex nutrition a l requirement s.
The conce ntrati o n of free amin o acids in milk and w hey are very limit ed, thu s the s ustai ned
growth of LAB depends o n th e production ofpro te in ases. peptidases and specific peptide and
amin o aci d tran sport system s.
Whey fermentation by LAB could decrease th e hi gh lac tose content in whey, producing
mainl y lactic ac id and other metabolites such as aroma co mpo und s contributin g to the fla vor
and texture a nd in creasi ng ca rboh ydrat e so lu bility and sweetn ess of the end product.
The ho mo-ferme ntati ve lactic ac id bacteria cataboli ze 'glucose via th e Embde n-M eycrhof
pathway. T wo lactic acid mo lecules are produced fro m eac h molecule of g lucose, typically
w ith a y ie ld of better than 90 g per 100 g glucose. Pent ose sugars are a lso metabo li zed by
so me homo-fermentati ve s pecies sterospec ific a nd lact ic aci d are the products of thi s
metabolism. Organisms may produce D (-), L (+ ) or DL-Iact ic ac id.
C,H " OIo ~ 2C H,C HOH COO H
Method of Lactic Aci,/ Commercial Prot/lletioll:
Pasteuri zed whey is inoculated wit h a starter c ulture co ntai nin g lactobaci ll i, e.g.,
Lactobacillus hlllgariclIs and L. delbrueckii. To prepare a s uffici ent amOllnt of inoc ulum for
additi o n to main fermentation ta nk , the culture is success ively transferred in sterile skin milk,
paste uri zed skin milk , and finall y whe y. The inoculum from w hey is now added to th e main
fermentation tank con tai nin g large a mo unt of whey. The temperature o f the fe rme ntati on
u
take n is ma inta ined at 43-50 C to prevent th e growth of many ex traneou s mi croorga ni sms.
During th e fermentation , s lurry of lime [calc ium hydroxide; Ca(O Hh l is added intermittently
to pre vent the acc umulation of ac id othe rw ise the latte r would retard fermentation. Wh en the
fermentation is completed (in about 2-4 days), th e fermented liquid is boiled at about si'c to
a ll ow the coagulation o f prote in, the lactalbumin , w hi ch is the n filtered and processed for use
as a nimal-feed s uppl eme nt. The filtrate co ntainin g the calcium lactate is s pray dried a fter

66
• Compare the acid ity and pH with the in it ia l va lues

Observation Table :

Init ial ac idity = Vo lume orNaa H used x Normality orNaa B x 9

Vo lume of sample

Parameter Whey Whey with 5% Lactose broth M Rs broth

lactose

pH

Volume of NaOH
used (ml)
Acidity in % lactic
acid

Results and Inte rpretations :

68
4.2 Aim : Application of microbia l consort ia in food ferm enta tions.
T heory:
Microorgani sms necessary in food fermentations may be added as pure culture of mi xed
culture or in some instances a number of cultures may be added if the desired microorganism
is known to be present in suffi cient numbers in original raw material. Kn own mi xt ures of
pure cultu res sometimes are prepared; either they are grown together or grolVn separa tely and
mixed at the time of usc. A Ilumber of lac tic acid bac teri a are used in the form o f mixtures in
dai ry industry and are a good example of bacteria consortia. The most common mi crob ial
consort ia used as dai ry starters usually consist of a mix ture of strai ns of Luc/Ucoccils laClis
ssp. Lactis and Lellcol1oslOC mesellferiodes ssp cremoris for the production of lactic acid and
Lellcol1vslOC cremoris and LaclococclIS loclis ssp. diacetylaclis for the prod uction of fl avo ur
and aroma. The typ ica l yoghurt starter is a mi xture of Streptococcus thermophilus and
Lactobacillus bulgariclIs.
Materials req uired :
Skim milk, Erlenmeyer flask (500 mL), wax marking pencil, steril e pipette ( 10 mL )
Procedure:
• Take 100 1111 of skim milk in 250 1111conica l fl ask and keep fo r C:l utoc lav ing.
• Use both pure and mi xed Cll Itures.
• Inoculatc cultures separately into different conica l fl as ks containing steri lized skim milk
and incubated at 37"C for 24 hrs,
• Aftcr incubation analyze the sample fo r following parameters
• Titrablc ac idity
• Total bac teria l count
• Direct microscopic counts

I. Tilruh/e acidit l':

• Add Iml of phenolphthalein to 10 ml o f fermented milk and tit re aga inst N/9 aO H ti ll
pink colour appea rs.
• Record the volumc of NaO H lIsed and ca lculate % Lactic Ac id by using the foll owing
formu la:

% lactic acid = 9 x Normality of NaO H x volume of NaO H used

Vol ume of milk
II . Total Bacterial COIlI1l :

• Serially dilute I ml of the fermented milk sample upto 10·'

• Plate 10-4_ 10-5 samp les on nut rient agar medium
• Incubate at 37 u C for 24 hr in an invertcd posi tion.

69
Iff. Direct Microscopic Count:
• Clean DMC slide with alcohol
• Pipette out 0.0 I ml of fennented milk with breeds pipette and spread it in I cm l area
• Allow the slide to dir dry
• Add rew drops of methyl ene blue or Newmann's Stain on the slide
• Observe under IOOX object ive of compound microscope
• Calcu late number of microbes using microscopic factor:
MF ~ 100 X 100/ IT r'
• Calculate the average number of microorgani sm using thi s formula
Observation Table:
Inoculumn No. Titrable Acid ity Total count Direct microscopic count
10- 10-·

I
2
3
4
5

Results and Interpretations:

70
4.3 Aim: Production of ethyl alcohol from molasses and whey by yeasts.
Theory:
Ethy l alcohol is among 1110s1 co mmon so lve nt s and raw material used in a va riet y of chemical
industri es. It is also lIsed as a germicide, a beverage, antifreeze, a fuel , a depre ssant. and
especially because of its ve rsa tility as a chemical intermediat e for other organic chemica ls.
Ethanol is produced both as a petrochemica l, through the hyd ration of ethylene. and
biologically by microbial fermentation of c heap sugary substrates slich as molasses.
Howeve r, the microorgani sms lIsed I11l1st be tolerant to high sugars and high concentration of
alcohol and Illust grow vigorollsly to produce a large quantity of alcohol. Yeasts, particularl y
Saccharomyces cerevisiae, repre se nt the best known microorgani sms used in the production
of ethyl alcohol. In add it ion, Saccharomyces u\'C/rum, Kluyveromyces ji"(Jgilis and
Kluyveromyces lac/is ha ve also been used for alcoho l production . Several bacteria like
Escherichia coli. Klebsiella o:ryroca, and ZYlllomonas mobilis ha ve been gene tically
engineered 10 produce elhanaL The chemical reaclian Ihal resulis in Ihe microbial
fermentat ion of carbohydrate into alcohol is:
C"H "0,, ~ 2 CHJCH,OH + 2 CO,
COOH
I
c=o
I
CH,
Pyruvate
Pyruvat6 d6cBrboxyJaso

Acelal ehyde CO 2
NAOHj
alcohol dehydrogenase
NAO"
Ethanol
H2 C-OH
I
CH,
Figure 4.1 Steps in the Biosynthesis of Ethanol
Some of th e inexpensive sub strates used in alcohol industry are either crude cane molasses or
best mola sses which contain about 50 per cent fermentable suga rs. Wa ste sulphite liquor
from paper industries, whey from milk , starch y ield ing grams (corn), potatoes and grapes
may a lso be used as s ubstrat e . Some countries used sligar beet for the purpose. The
production process in vo lves the dilution of molasses to a suitabl e s ugar concentration (\5- [6
per cent sugars), addition of a small quantity of nitrogen source (urea, ammon ium s ulfate or
ammonium phos phate), adjustment of pH to about 5.0, and the , addition of an actively
growing yeas t cu lture. The ferme ntation is carri ed out in big deep tank s of steel or stainle ss

71
steel. The fermentation is allowed to cont inue for about 24-36 hours at 25°C-30°C after
whic h th e cells are a ll owed to sett le. The ferme nt ed mash is then di stilled and passed through
rectifyi ng columns to recove r ethy l a lco hol.
Molasses
+4---H,O
44----Nitrogen source
Yeast - - - - - - - . . +4---- (Urea, ammonium sulphale, etc.)
(SaccharmYC9S c9r9visiae)
44---- H,SO.
co, + 4 - - - - - - . 1
Fermenter

~
Distilling

~
Ethyl atcohol
Figure 4.2 Steps in the M2nufacturc of Ethyl Alcohol using Molasses as Fermentation
So u ret': ht tp://www.studentsguide.in/m icrob io logy Ii ndust ri a 1- microb io logy!
SuhSfrare:
Whey is another important waste material from w hich ethanol ca n be produced. The di sposa l
of whey is a wo rl dw ide problem. Large quantities of whey are produced as a by-product
durin g the manufa ctu re of cheese and casein. and thi s mu st be d isposed of or processed in an
enviro nm entally acceptab le way. Si nce most of the components are of small mo lec ular
weigh t and so luble. they ca n quickly deplete oxyge n le ve ls in natural wate r systems: the
COD (C hemical Oxygen Demand) of raw whey is about 60 kg m,J . The key to the utili za tion
of thi s reso urce ha s been cha ng ing th e perception of whey from a 'waste material' to an
'opportunity' for fu rther process ing. The yeast used is lac tose ferm entin g organism called
Kluveromyces ji·agilis. Thi s yeas t produces ~-galactosidase w hich breaks down lactose (a
di sacc haride) into its co mpon ent s ugars which are g lu cose and ga lactose.
CH,OH
I .

~
'H O
CH,OH , I ; CH ,OH CH,OH
Hi v ~ 'o
vtH0~ OH 0
:
i OH
' O OH
i. OH
I~ ' OH ~ H,O i ,. ) .
I OB OH
OH OH OH

ga ia":losc glllcose
Figure 4.3 Chemical reaction showing breakdown of lactose

72
Materials Required:
Molasses, Whey, C ulture ofSacc/wrDlI1yces cerevisiae (for molasses) and Kllfveroll1yces
ji-agilis (for whey) , Steril e water, Urea, Ammonium su lfate or Ammoni um ph os phate,
Erlenme yer nask (500 mL), wax marking pencil , steril e pipettes ( 10 mL), wate r bath(60°C),
Incubator
Procedure:
• Take 500 ml of pas teuri zed molasses and dilute to a su it ab le sugar concen tration ( 15- 16
per cent sugars).
• Add a smal1 quantity of nitrogen source (urea , ammon ium su1fate or ammonium
phosphate ).
• Take 500 ml of pasteurized whey.
• Adjust the p H to about 5.0
• In oculat e 1% act ively grow in g cu lture of Saccharomyces cerevisiae in pasteurized
molasses and Klllveromycesji'agifis in pa steuri zed w hey.
• In cubate at 30°C lor 3-7 days.
Observation Table:
1. Smell th e signs of fermentation (alcohol smell ).
2. Check for alcohol production.
Substrate Alcohol concentration (in percent)
Molasses
Whey
,
Results and InterpretatIOns:

73
4.4 Aim: Citric acid production from whey with sugars and additives by Aspergilllls
IlIger.
Theory: Citric ac id, a carboxy li c organi c acid, so lubl e in water w ith a pleasant taste, is the
most important acid used in th e food industri es. Until about 1920 , all commercial citric acid
was produced from lemon and lime juices. Later on it was reported that citric ac id can be
produced by fermentation process using spec ies of microorgani sms namely Aspergillus
niger, a fungus w hi ch was used commerc ially for the first time in 1923. They al so indicated
th at factors affectin g th e producti on o f citric ac id by fermentation include th e nutritional
composition o f th e medi a, environmental conditions, defi ciency of mangan ese and oth er
metals, pH , and di sso lved oxygen tension.
At present tim e citri c acid is produced commercially using mutant strains of Aspergillus
niger, and with a significant amount by Saccharomycopsis Upo(vlica, Pencilliul11
simp lic iss imlinJ and Asperg illus ./heilidlls. Other carbohydrates and wastes that have been
considered, ex perim entall y, to produce citric acid by Aspergillus niger includes inulin, date
fruit sy rup, suga r cane molasses, soya w hey, Carob pod and cheese whey.
Large amounts o f whey are produced world w ide as a by-product of cheese and oth er dai ry
products manufacturing. Whey in the Middle Eas tern region is generally considered a waste
and disposed in the sewage system leaving a small amount for drinking for domesti c animals.
The aim of thi s study was to produce citric ac id by Asperg illus niger from cheese whey
fortifi ed with different sucrose, tri ca lcium phosphates and ribolla vin in a liquid surface
Cll !ture process.
Materials required:
Pateuri zed cheese whey, Sucrose, tricalcium phos phate, ribolla vin, SOO ml Erlemyer Il ask,
Fres h Aspergillus niger c ulture (approx 10' spore suspens ion), steril e pipett e (10 mL), wax
marking pencil, incubator
Procedure:
• Take 100 mL cheese w hey in SOO mL Erl emyer Ilask and add sucro se ( ISg) and
tri ca lcium phosphate ( I g ).
• Pate uri zed cheese whey at 60°C for 30 minutes and add filt er sterili zed ribo lla vin (10
mg/L) to fortify the media . Adjust th e initial pH of the ferm entatio n medi a to 3.0 using I
N of HC I and/o r NaOH .
• Carry out surface liquid culture fermentation process by inoculating the media with the
fungal culture (approx 10J spore suspens ion) and incubate at 30°C for up to 20 days.
• Determine citric acid concentration titrating with 0. 1 N NaOH and phenolphthale in as
indicator and calculated as % accordin g to the foll owin g formula :
• Citric acid (in percent) = Normality X vo lume of NaOH X Equi v. wI. of CAl Weight of
sample (g) X 10

74
• For determining biomass, take the whole fungal culture growth and filter wit h Whatman
filter paper No.4, Wash with dist illed water (250 ml) and dry at 105°C to constant
weight.
• Measure cu lture pH by pH meter.
Observation Table:

Parameters Citric acid

Citric acid (in percentage)

Biomass (in gl L)

pH

Results and Interpretation :

75
4.5 Aim: Production of sauerkraut by microorganisms.
Theory:
Sauerkraut is defin ed as the c lean so ured product o f characteri sti c fl avo ur. o btained by full
ferm entati on of properl y pre pared and shreaded cabbage in presence o f not less than 2-3%
sa lt. Fin all y containin g not less than 1- 1. 5% ac id expresses as lactic ac id.
The bas ic process in vo lves fermentation of shredded cabbage by the mi xed activity of
Leuconustoc mesel/(eroides, Lactobacillus brevis, and Lactobacillus p lan/arum in the
presence o f 2.2-2.8% w!v NaC I. the latter play 3 important functions.
• It inhibits th e growth of initial spo ilage organi sms like pseudomonas
• It extrac ts moistu re fro m the shredd ed cabbage by osmosis to form the brine in which
ferm entation takes pl ace.
• It he lps to mainta in the cri sp texture o f the cabbage by condi tioning water and inhibit
endogeno us pectinolytic enzy mes which cause the prod uct to so ften.
The lac ti c ac id prod uced during fermentat ion performs two important functi ons
• It imprarts characteri stic fl avour to the product
• Ac t as a preservati ve by inhibitin g th e growth of food spoilage microorgani sms.
Material required:
Sterile Glass beaker (500 ml ). Cabbage. un iodi zed table salt. C lean Knife. weighing pan,
wooden boards. cheese cloth . rubb er bands! th read, pH strips or pH meter. plate count agar,
Nil 0 NaOH . phenolph tha lein ( I %)
Procedure :
• Trim the spotted and damaged o uter leaves fro m all the cabbage heads.
• Di vide each head into two ha lves with sterile kni fe and remove the core
• Wash th ese halves with clean runnin g water
• Weigh the shredded cabbage and di vide into two parts of 250 gm each
• We igh sodium chloride acco rding ly and di vide in to two parts of equal weig ht
• Pl ace the shre dded cabbage and slat in alternatin g layer in a w ide mouthed iar
• Place a wooden board over each mi xture and gently press to squeeze out the jui ce.
• Place a weight over the wooden boards
• Cover the i ar w ith cheese c loth by using rubber bands.
• Incubate the i ars at room temperatu re for 28 days for the fermentation of substrate
• Record the following res ults at an interva l of 7 and 14 days for the chemical and
microbi ological changes during sa uerkraut producti on
• Odo ur: Ac idi c. Earth y. Spicy Or Putrid
• Colo ur: Colourless, Brown. Pink, Straw. Yellow And Pale
• Tas te: Sour. Sa lty. Sweet And Bitter
• Tex tu re: Soft . Slim y And Rotten

76
• pH : Determin e by pH paper
• Total Ac id ity: Ex pressed as % lac ti c aci d by titrati on
• T ota l bacteria l co unt : By lIsing selecti ve medi a

Figurc 4.4: Sauerkraut preparation

5011 rce: 1\'\I'lI',lem'n i ng herhs, com/sa lIer/i rlllll_ I'eC ipe,'"III I
Observation Table:
Parameters 7 days 14 days
pH
Acidity (in percent)
Total bactcrial count
Odour
Colour
Taste
Texture

Results and Interpretation:

77
4.6 Aim: Production of single cell proteins.
Theory:
Increasing concern about pollution occurring from agricultural and industrial wastes has
stimulated interest in converting waste material s into commercially va luable products.
Additionally, th ere is a demand for the formulation of inno va ti ve and alternative
proteinaceous food so urces due to an insufficient suppl y from the traditional protein so urces
such as meat, fi sh or eggs.
Much interes t has been focu sed on the potential of converting soy milk wastes, potato
effluents, sugarcane bagasse, orange pee ls, shrimp-shell wastes, kimchi production wastes or
forestry wastes (e.g. wood hydrol ysates) to s ingle cell protein (SCP) T echnicall y, SCP is the
manufacture of cell mass using microorgani sms by culturing on abundantly avai lable wastes.
Algae, fungi and bacteria are the chief sources of microbial protein that can be utilized as
SCPo
The production of th e microbial biomass is done either by a subm erged or solid state
fermentation process. After fermentation, biomass is harvested and may be used as a protein
so urce or be subjected to processing steps like washing, cell disruption, protein extraction
and purification. In general, high production rates and protein yields as we ll as ease of
production control makes SCP more attractive as a protein so urce compared with
convent ional plant and anim al so urces.
Cheese whey, a by-product of the da iry industry, is the liquid effluent remaining following
the prec ipitation and remo va l of milk casein during cheese making. It re presents about 85-
95% of th e milk volume and retains a significant amount ( , .55%) of milk nutrients. Among
the most abundant of these nutri ents is lactose (4.5- 5% w/ v) wh ich is a suitable substrate fOl
the production of va lue-add ed products using biochemical conversion processes .
Whey also contains solubl e prote ins (0.6- 0.8% w/v ), lipids (OA- 0.5% w/ v) and mineral sa lt'
(8- 10% of dried ex tract), as well as appreciable quantities of other constituents, such as lacti,
and citric acids, non-protein nitrogen compounds, 8 group vitamins, etc.
Yeasts that ferment lactose are known for th e production of ethanol and SCP o Cheese whey i,
a cheap and largely avai lab le raw material for microbial biomass of SCP production b:
yeasts.
It is we ll known that the proteins are one of the main constituents of foods. In addition t.
their nutritional function , proteins contribute s ign ifi cantly to the ex pression of senso r:
attributes of foods. The functional properties of proteins are important in determining thei
usefuln ess in food systems.
There is limited information about s ingl e cell protein functionality and more knowledge i
needed in order to assess th eir potential uses in food s.
Considering that in exploring s ingle cell protein, iso lated from culti vation of yeasts on chees
whey, as a new so urce of food protein, its functional properties need to be determin ed.

78
Review Questions:
• How are bread, sauerkraut, and pickles produced?

• What microorganisms are most important in bread and pickles fermentations?

• Di sc uss the importance of th e specific sequential activity of th e microflora respons ible

for sauerkraut prod uction?

• What is th e function of the sa lt in sauerkraut production ?

• Wh y is uniodized salt used in this process?

• What arc SCPs?

• Give the advantage of Single cell protein.

• En li st sOl11e SCP available in the market.

• Defi ne liquid surface culture process.

• Define mother culture and seed culture.

• What is Ihe importance or BOD?

• What is th e sy mbiotic relationship between yoghurt cultures?

• G ive the th e rapeuti c value of fe rmented products.

• What would be the effect, if we di spose the whey in th e environment?

80
r
User's Notes

81
 APPENDIX-!
MEDIA COMPOSITION
BACILLUS CEREUS AGAR
Ingredients Grams / lit
Enzyma ti c Digest of Case in Ig
Mannitol 10 g
Sod ium Ch loride 2g
Magnes ium Su lfate 0.1 g
Disodium Phosphate 2.5 g
Monopotassium Phosphate 0.25 g
Bromthymol Blue 0.10 g
Sodi um Pyruvate 10 g
Agar 15 g
Sterile Egg Yolk Suspe ns ion 50 mL
Polymyxin B ( 100,000 units) (filtered 2 mL
sterili zed aqueous)
Final pH: 7.2 ± 0.2 at 25°C

BRILLIANT GREEN LACTOSE BILE BROTH

Ingredients Grams / lit
Peptic digest of animal tissue 5.0
Pancreatic digest of casein 5.0
Lactose 10.0
Sucrose 10.0
NaCI 5.0
Ox bile 20.0
Brilliant green 0.0025
Phenol Red 0.08
Final pH: 6.8 ±0.2 at 25°C

deMANN ROGOSA SHARPE (MRS) AGAR

Ingredients Grams / lit
Proteose Peptone No.3 10.0
Beef ex tract 10.0
Yeast extract 5.0
Dextrose 20.0
Polysorbate 80 1.0

82
Ammonium citrate 2. 0
Sodiulll ace tate 5.0
Magnes ium S ulfate 0.1
Mangan ese Sulfate 0.05
Dipotass ium Ph osphate 2.0
Aga r 15.0
Final pH: 6.5 ±0.2 at 25°C

deMANN ROGOSA SHARPE (MRS) BROTH

Ingredients Grams I lit
Proteose Peptone No.3 10.0
Beef ex trac t 10.0
Yeast extract 5.0
Dex trose 20.0
Polysorbate 80 1.0
AmmoniuJ11 citrate 2. 0
Sodium acetate 5.0
Magn es ium Sulfate 0. 1
Mangan ese Sulfate 0.05
Dipotass illlll Ph osphate 2.0
Final pH: 6.5±0.2 at 25°C

DOUBLE-STRENGTH LACTOSE BROTH (DSLB)

Ingredients Grams I lit
Pa ncrea tic Diges t of Ge latin /0.0
Bee f Ex tra ct 6.0
Lactose 10.0
Final pH: 6.5 ±0.2 at 25°C

GLUCOSE-POL Y -PEPTONE- YEAST EXT.-MAL T EXT. AGAR

Ingredients Grams I lit
Glucose 10.0
Polypeptone 5.0
Ma lt ex tract 3.0
Yeast ex trac t 3.0
Final pH : 5.5 ±0.2 at 25°C

83
LACTOSE BROTH
Ingredients Grams / lit
Pancreati c Digest of Gelatin 10.0
Beef Ex tract 6.0
Lactose 10.0
Final pH: 7.0±O.2 at 25°C

LAURYLTRYPTOSEBROTH
Ingredients Grams / lit
Tryptose 20.0
Lactose 5.0
Dipotassi um Phosphate 2.75
Monopotass ium Phosphate 2.75
Sodi um Chloride 5.0
Sod ium Lauryl Sulfate 0.1
Final pH: 7.0±O.2 at 25°C

LEVINE'S EOSIN METHYLENE BLUE AGAR

Ingredients Grams / lit
Pancreatic digest of case in 10.0
Lactose 5.0
Sucrose 5.0
K, HP04 2.0
Methylene Blue 0.065
Eosin Y 0.4
Agar 13.5
Final pH : 7.2±O.2 at 25°C

MI7BROTH
Ingredients Grams / lit
Case in enzyme hydro lysate 5.0
Papaic digest of soy bean meal 5.0
Yeast extract 2.5
Malt extract 5.0
Asco rb ic ac id 0.5
Magnes ium sulph ate 0.25
Di sodium ·p-glycerophosphate 19.0

84
I Agar 11.0

MALT EXTRACT AGAR (MEA)

Ingredients Grams I lit
Dextrin 2.75
Glycerol 2.35
Pepton e 0.78
Agar 15.0
Final pH: 4.7±0.2 at 25°C

MULLER HINTON AGAR

Ingredients Grams I lit
Beer Extract 2
Acid Casein hydrolysa te 17.5
Starch 1. 5
Agar 17.0
Final pH: 7.3±0.1 at 25°C

NUTRIENT AGAR (NA)

Ingredients Grams I lit
Peptic digest of animal ti ssue 5.0
Beef ex trac t 1.5
Yeast ex tract 1. 5
Sodium chloride 5.0
Agar 20.0
Final pH: 7.4±0.2 at 25°C

OXFORD PERFRINGENS AGAR

Ingredients Grams I lit
Enzymatic Diges t of Casein 15.0
Enzymatic Digest of Soybean Meal 5.0
Yeast Extract 5.0
Sodium Metabisulfite 1.0
Ferri c Ammonium C itrate 1.0
Agar 15 .0
Final pH: 7.6 ± 0.2 at 25°C

85
PLA TE COUNT AGAR (PC A)
Ingredients Grams / lit
Casein enzyme hydrol ysa te 5 .0
Yeas t ex trac t 2 .5
Dex trose 1.0
Aga r 15.0
Final pH : 7.0±0.2 at 25°C

POTATO DEXTROSE AGAR (PDA)

Ingredients Grams/ lit
Pee led Potato 200.0
Dex trose 20.0
Agar 15.0
Final pH 5.6 ± 0.2 at 25 °C

SINGLE-STRE NGTH LACTOSE BROTH (SSLB)

Ingredients Grams / lit
Pancreatic Digest of Gelatin 5.0
Beef Extract 3.0
Lactose 5.0
Final pH: 7.0 ±0.2 at 25°C

STREPTOMYC ES-SELECTIVE AGAR (SSA)

Ingredients Grams / lit
Beef Hea rt In fusion, Solids 10.0
T ryptose 10.0
Casei n enzy me hydro lysate 4.0
Yeast ex trac t 5.0
Dex trose 5.0
L-cysteine hydroc hl oride 1.0
Sta rch. soluble 1.0
Sodium Chl orid e 5. 0
Monopotass iulll phosphate 15.0
A mm o nium Sulph ate 1.0
Magnes ium Sulphate 0. 2
Calcium C hl oride 0 .Q2
Agar 20 .0

86
I Final pH: 6.9±0.2 at 25°C
TRYPTIC AG AR
Ingredients Grams I lit
Pancrea ti c Digest of Casei n 15.0 g
Enzy mati c Diges t of Soybean Mea l 5.0 g
Sodium Chloride 5.0 g
Agar 15.0 g
Final pH: 7.3±0.2 at 25°C

TRYPTICASE SOYA AGAR (TSA)

Ingredients Gramsl lit
Soy Peptone 5.0
Case in Tripti c Diges t 15.0
Sodium Chl oride 5.0
Bacteri ological Agar 15.0
Final pH 7.2 ± 0. 1 at 25°C

VIOL ET RED BILE AGAR (VRBA)

Ingredients Gramsl lit
Peptone fro m meat 7.0
Yeas t ex tract 3. 0
Sodium chl oride 5.0
Lac tose 10.0
Neut ra l red 0. 03
Bile salt m ix ture 1. 5
Crystal vio let 0.002
Agar 15.0
Final pH : 7.4 ± 0.2 at 25 °C

VIOLET R E D BLUE GLUCOSE AGAR (VRBGA)

Ingredients Gramsl lit
G lucose monohydra te 10.00
Pancreati c digest o f Ge latin 7.00
Sodium Chl oride 5.00
Yeas t Ex trac t 3.00
Bile Salts 1. 50

87
Neutral Red 0.03
Crystal Violet 0.002
Bacteriological Agar 15.00
Final pH: 7.4 ± 0.2 at 25°C

YEAST MALTOSE (YM) AGAR

Ingredients Grams/ lit
Yeast Ex tract 3.0
Malt extract 3.0
Bacteriological Aga r 15.0
Final pH: 5.5 ± 0.2 at 25°C

88
 APPEN DIX-]
REAG E NTS AND BUFFERS

CASEIN SOL UTION (2%)

Ingredients Grams
Case in 2. 0
Borate bu ffe r (0 . 1M) 80 m L
Adj ust th e pH 7.6 and comp le te ly di ssolved by hea tlll g o n a stea m bath fo r 15 mll1 . Cool tillS
so lution, adj ust th e pH to 7.6 a nd ma ke th e vo lume up to 100 m L by th e bo rate buffe r.

GRAM-STAINING REAGENTS
(A) C rystal Violet
Ingredients Grams
a Crysta l v io le t (85 % ) 2.0 g
e th y l a lcoho l (95%) 20.0 mL
b Am monium oxa late 0 .8 g
Distill ed wa ter 80.0 mL
Add so lut iO n a to so lut iOn b. Let sta nd for a da y, and th e n filte r. If th e c rysta l ViOlet IS too
concentra ted , so lutio n A Jllay be diluted a s much as 10 limes.
(B) Gram's Iodine Solution (mordant)
In gredients Grams
Iod ine crysta ls 1.0 g
Potass ium iodid e 2. 0 g
Di still ed wa ter 300.0 mL
Store In a n am ber bottl e; di sca rd whe n th e co lor beg l11s to fade.
(C) Safranin (counterstain) Solution
Ingredients Grams
Safranin 2.5 g
Ethy l a lco ho l (95%) 100 mL
.
For a worklll g so luti on, dilut e stoc k so lutIOn 1110 ( I Oml o f stoc k sa fra nll1 to 90 ml o f d istilled
wa ter).
(D) Decolorizer
95% Ace tone o f Eth y l a lcoho l
GUM ARABIC (10 % )
Ingredients Grams
G um Ara bic 10 .0 g
Ma ke th e Vo lume upto 100 mL w ith d istill ed wa te r

89
METHYLENE BLUE SOLUTION (1 :250,000)
The Methylene blue concentration used in I part of dye in 3,00,000 parts of milk . Presently
tablet forms are avai lable. One tablet dissolved in 200 ml of hot, di stilled wa ter produces the
stock dye solution for addition to the milk . Although this soluti on is stable when it is
refri gerated and protected from light, it is safer to prepare the solution weekl y.

NAOH (N/ IO)

Ingredients Grams
NaO H 0.4
Distilled wate r 100mL

NEWMANN'S STAIN
Ingredients Grams
Methylene blue chloride 0.6 g
Ethyl alcohol (95%) 52 ml
Tetrachlorethane 44ml
Glac ial acetic acid 4 ml

PHENOPTHALEIN (I %)
Ingredients Concentration
Phenolphthalein 0.1
Ethyl a lcohol 89-9 1
Methanol 4-6
Deioni zed wate r 4-6

SODIUM PHOSPHATE BUFFER (pH 7)

Solutions Ingredients Grams/ Lit
Stock solution A Monobasic sodium 276 .0
phosphate, monohydrate (2
M)
Stock solution B Dibasic sodium phosphate (2 284.0
M)
Final pH: 7.0±0.2 at 25°C
..
MlXlI1g an 39. 0 (mL) of A and 61.0 (mL) of B as dJlutmg to a total volume of 200 ml , a I M
phosphate buffer of the required pH at room temperature.

90
SOLUBLE STARCH (1%)
Ingredients Crams
Soluble starch 1.0
Heated Water 100.0

SULFURIC ACID (2N)

Ingredients mL
Sulfuric acid 55.0 mL
Di stilled Water 45.0 mL
Make final up to 1000 ml

TCA SOLUTION (10%)

Ingredients Grams
Trichloroacetic acid 10.0
Anhydrous sodium acetate 10.0
Glacial acetic acid 2mL
Make vo lume upto 100 mL

91
 Most Probable Number (MP N) Index for Various Combinations of Positive and
Negative Results When Five 10m I, Five I ml and five 0. 1 ml portions are used
No of tubes showing indi ca ti on of posili ve reac tion out of MPN index
5 of 10 In l sampl e each 5 of I ml sample eac h 5 of 0. 1 ml sample each per 100 ml

0 0 0 <2
0 0 I 2
0 I 0 2
0 2 0 4
I 0 0 2
I 0 I 4
I I 0 4
I I I 6
I 2 0 6
2 0 0 4
2 0 I 7
2 I 0 7
2 I I 9
2 2 0 9
2 3 0 12
3 0 0 8
3 0 I II
3 I 0 II
3 I I 14
3 2 0 14
3 2 I 17
3 3 0 17
4 0 0 13
4 0 I 17
4 I 0 17
4 I I 21
4 I 2 26

92
4 2 0 22
4 2 I 26
4 3 0 27
4 3 I 33
4 4 0 34
5 0 0 23
5 0 I 30
5 0 2 40
5 I 0 30
5 I I 50
5 I 2 60
5 2 0 50
5 2 I 70
5 2 2 90
5 3 0 80
5 3 I 11 0
5 3 2 140
5 3 3 170
5 4 0 130
5 4 I 170
5 4 2 220
5 4 3 280
5 4 4 350
5 5 0 240
5 5 I 300
5 5 2 500
5 5 3 900
5 5 4 1600
5 5 5 > 1600
SOllra: Swndard Melhod~' ./'01' Examination of 1h
. WaleI' alld Wastewater, \ 8 Edit ion. American Public Health
Association , New York, 1998

93
 Glossary of Related Terms

Acidophile:
A microorgani sm that has it s growth optimum between about pH 0 and 5.5.
Actinomycete:
A n ae robi c, G ram positi ve bac terium th at form s branching fil ament s and asex ual spores.
Aerobe:
A n organi sm th at grows in th e presence of atmos pheri c oxygen.
Aerobic respiration:
A metabolic process in w hi ch molec ul es. often organi c. is ox idi zed w ith oxygen as th e final
elec tron accep tor.
Agar:
A compl ex po lysacc hari de ex trac ted fro m red algae an d used as a solidifying agen t in culture
media preparati on.
Alcoholic fermentation:
A ferme ntati on process that produces ethanol and CO 2 from sugars.
Aliquot: Di spense an amount of liquid using a pipette.
Alkalophile:
A microorga nism th at grows best at pH s fro m abo ut 8.5 to 11 .5.
Anabolism:
The synthesis of compl ex mo lec ules from sim pler mo lec ul es w ith the input of energy.
Anaerobe:
A n organi sm that gro ws in th e absence of free oxygen.
Antibiotic:
A microbi al prod uct or its deri va ti ve th at kill s susceptibl e microo rgani sms or inhibits their
gro wth .
Antibod y (immunoglobulin):
A glyco protein produced in response to th e introducti on of an antigen; it has th e ability to
co mbine w ith the antigen th at stimul ated it s producti on. A lso kn own as an immunoglobulin
(I g).
Antimicrobial agent:
A n agent that kill s microorgani sms or inh ibit s th eir grow th .
Archaea:
The dom ain th at co nt ains prokaryotes with isopreno id g lyce ro l di eth er o r di g lyce ro l
Aseptic technique:
Proced ure to guarantee steri lit y and to reduce contamination
Autoclave:

94
An apparatus for ste rili zing objects by the use of steam under pressure. Its development
tremendously stimulated the growth of mi crob iol ogy.
Autotroph:
An organism that uses C02 as its sole or principal so urce of carbon.
Bacillus:
A rod-shaped bacterium.
Bacteria: The domain that co ntain s proca ryoti c cells with primaril y diacy l glycero l diesters
in their membranes and with bacterial rRNA.
Bacteriocin:
A protein produced by a bacterial strain that kill s other c lose ly related strai ns.
Bacteriophages: viruses that infect bacterial host ce lls; th ey usually co nsist of a nucleic acid
molecule enc losed by a protein coat.
Batch culture:
A culture of mi croorga ni sms produced by inoculating a closed cu lture vesse l co ntainin g a
single batch of medium.
Biochemical oxygen demand (BOD):
The amount of oxygen used by organisms in water under certain standard conditions; it
provides an index of the amount of microbially oxid izable organic matter present.
Biodegradation:
Metabolism of a substance by microorgani sms that yield min erali zed end products.
Biofilm:
Matrix-enclosed bacterial populations' adherent to each other and/ or to surface or interfaces.
Biological safety cabinet:
Cabinet use to protect perso nne l, product and the environment from exposure to biohazards
and cross co ntamination durin g routine procedures.
Bioremediation:
The use of biologically mediated processes to remove o r degrade pollutants from specific
environments. Bioremediation can be carried out by modification of the env ironment to
accelerate biological processes, eithe r with or without the addition of spec ifi c
microorganisms.
Biosensor:
The couplin g of a biological process with production of an electrical signal or li ght to
Biotransformation or microbial transformation:
The use of li ving organisms to modify s ubstances that are not nomlally used for growth.
BOD incubator:
Incubator meet different bio chemical oxygen demand te st in various fie lds including
medical , agricultural , industrial, researc h laboratori es, storage sensitive culture, vaCCll1es,

95
culture of bact eria, microorgani sm, serum incubation, seed gennination, vanous mauStnes
and more.
Broth:
Culture medium without agar.
Budding:
A vegetative outgrowth of yeast and some bacteria as a means of asexual reproduction.
CFU:
Colony-fomling units, i.e. colonies
Chemical oxygen demand (COO):
The amount of chemical oxidation required to convert organic matter in water and waste
water to C02.
Chemostat:
A continuous culture apparatus that feeds medium into th e culture vessel at the same rate as
medium containing microorgani sms is removed ; the medium in a chemostat contains one
essential nutrient in a limiting quantity.
Coccus:
A roughly spherical bacterial cell.
Coliform:
A gram-negative, nonspori ng, facultative rod that ferments lactose with gas formation within
48 hours at 35°C.
Colony:
A cluster or assemblage of microorgani sms growing on a so lid surface such as the surface of
an agar culture medium; the assemblage often is directly visible, but also may be seen on ly
microsco pically.
Colony forming units (CFU):
The numbers of microorgani sms that can form colonies when cultured using spread plates or
pour plates, an indication of the number of viable microorganisms
Complex medium:
Medium with some unknown ingredients or amounts, i.e. blood agar
Consortium:
A physical association of two different organisms, usually beneficial to both organisms.
Continuous culture system:
A culture sys tem with constant environmental conditions maintained
Culture medium:
A liquid or ge l, containing nutrients, that is used to cultivate microorganisms.
Defined medium: Culture medium made with components of known composition.
Deionised water:
Water that has had the ions remo ved.

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Diarrhoea:
An increase in the frequency of bowel movements and loss of body fluid .
Differential media:
Culture media that di stinguish between groups of microorgani sms based on differences In

their growth and metabolic products e.g. - MacConkey agar.

Diluents:
An inert substance used to dilute.
Dilution factor:
It is equal to the final volume di vided by the initial volume of solution.
Disease:
A dev iation or interruption of the normal stmcture or function of any part of th e body that is
manifested by a characteristic set of sy mptoms and signs.
Disinfectant:
An agent, usually chemi cal, that disinfec ts inanimate objects.
Distilled water:
Water from which both ionic and non-ionic components are removed .
Durham tube:
A small, in verted, liquid-filled test tube for collecting gas formed by mi crobial metabolism in
broth cultures. The Durham tube is used to test for the presence of organisms capable or
metaboli zing the broth nutrients under given conditions thorough observation for gas present
in th e tube.
Embden-Meyerhof pathway:
A pathway that degrades glucose to pyruvate; the six-carbon stage co nverts glucose to
fructose 1,6- bisphosphate, and the three-carbon sta ge produces ATP while changing
glyceraldehyde 3-phosphate to pyruvate.
Endospore:
An extremely heat- and chemical-resistant, dormant, thi ck-walled spore formed by bacteri a
for its survival in adverse conditions.
Endotoxin:
Component of th e cell wall of gram-negative bacteria that can cause adverse health effects.
Enriched media:
Enriched media contain the nutrients required to support the growth of a wide variety of
organisms, including some of the more fastidious ones.
Enteric bacteria:
Members of the family Enterobacteriaceae (Gram negati ve, peritrichous or nonmotile,
facultatively anaerobic, straight rods with simple nutritional requirements); also used for
bacteria that live in the intestinal tract.
Enterotoxin:
A tox in spec ificall y affec tin g th e cell s of th e intes tinal mucosa, causing vo miting and
di arrh ea.
Enzyme:
A pro tein cata lys t w ith spec ific it y fo r both the reacti on catalyzed and its substrates.
Exoenzymes:
Enzy mes that arc secreted by ce ll s.
Exotoxin:
A heat-l ab il e, tox ic protei n produ ced by a bacterium as a res ult of it s normal metabo lism or
beca use o f th e ac qui sition o f a pl as mid or prophage th at redirec ts it s metabo li sm.
Exponential phase: The phase of th e growth curve d urin g which th e microbi al gowth
Extremophiles:
M icroorga ni sms th at gro w under harsh or ex treme enviro nmental conditi ons such as very
hi g h temperatures o r low pH s.
Facultative anaerobes: Microorgani sms that do not requi re oxygen for growth , but do grow
be tter in its presence.
Faecal coliform: Coli fo rm bacte ri a w ith ability to ferm ent lac tose w ith th e producti on of
ac id and gas at 44.5°C w ithin 24 - 48 h.
Fastidious:
Hard-to-grow bac teria, requiring growth fac tors or parti cular nutri ents
Fecal coliform:
Co lifonlls whose norma l habitat is the intes tin a l trac t and that can grow at 44 .5°C.
Fecal enterococci :
Enterococci fo und in the intestin e o f humans and oth er wa rm- blooded anim als. Th ey are used
as ind icators of th e feca l po llutio n of wa ter
Fermentation:
A n energy yielding process in w hi ch an energy substrat e is ox idized w ithout an exogenous
e lec tro n acceptor. Us uall y o rga ni c mo lec ul es serve as both e lectron d onors and acceptors.
Food intoxication:
Food poisoning caused by microbial tox ins produced in a food pri or to consumpti on. Th e
presence of li ving bac teri a is not req uired.
Food poisoning:
A genera l term usually referrin g to a gastrointestin al disease caused by th e inges ti on of food
co nt aminated by path ogens or the ir tox ins.
Food-borne infection:
Gastrointestin a l illness ca used by inges ti on of microo rga ni sms, fo llowed by th e ir growth
w ithin th e hos t. Symptoms ari se from ti ssue in vas ion and/or tox in producti on.

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Fungus:
Achlorophyllous, heterotrophic, spore-bearing eukaryotes with absorpti ve nutrition; usuall y,
they have a wa lled th al lus.
Gastroenteritis:
An acute inflammation of tile lining of the stomach and intestines, characteri zed by ano rexia,
nausea, diarrhea, abdominal pain, and wea kness . It has various causes including food
poi sonin g due to such organisms as E. coli, S. a urellS, Campy/obaete,. (campyiobacte ri osis),
and Salmollella species; co nsumption of irritating food or drink ; or psyc hologica l fac tors
such as anger, stress, a nd fear. A Iso called cnterogastriti s.
Generation time:
The time required for a microb ial population to double in number.
Genus:
Category of orga ni sms with like features and closely related, di vided into species
Glycolysis:
The a nae robic conversion of glucose to lactic ac id by use of th e Embden- Meyerhof pathway.
Cram stain :
A differential staining procedure that divides bacteria into gram-positi ve and gram negati ve
gro ups based on their abi lity to retain crystal violet when deco lori zed with an organi c solve nt
such as ethanol.
Growth factors:
Organic compounds that must be suppli ed in the diet for growth because they are essential
cell components or precursors of such components and cannot be synthesized by the
organIsm.
Halobacteria or extreme halophiles:
A group of arc haea that ha ve an absol ute dependence on high NaC I concentrations for
growth and will not survive at a concentration below about 1.5 M NaC \.
Halophile:
A microorganism that requi res high levels of sodium chloride for growth.
Heterolactic fermenters:
Microorgan isms that femlent sugars to form lactate, and also other products such as ethanol
and CO, .
Homolactic ferl11cnters:
Organisms that ferm ent sugars almost compl etel y to lactic acid.
Identification:
The process of determining that a particular isolate or organism belongs to a recogni zed
taxo n.
Incubation period:
The peri od afte r pathoge n entry into a host and before signs and symptoms

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Indicator organism:
An organism whose presence indicates the condition ofa substance or
Infection:
The in vas ion of a host by a microorgani sm with subsequent establishment and multiplication
of the agent. An infection mayor may not lead to overt disease.
Intoxication:
A disease that results from the entrance of a specific toxin into the body of a host. The toxin
can induce the disease in the absence of the toxin-producing organism.
Intrinsic factors:
Food-related factors such as moi sture, pH, and available nutrients that influence microbial
growth.
Lactic acid fermentation:
A fermentation that produces lactic acid as the sole or primary product.
Microbiology:
The study of organisms that is usually too small to be seen with the naked eye.
Microorganism: An organism that is too small to be seen c learly with the naked eye.
Microscope:
Instrument used for viewing magnified image of the microorganisms, which are not visible
with aided eyes. It is of two main types: Light and Electron mi croscope.
Mold:
Any of a large group of fungi that cause mold or moldiness and that exists as multicellular
Most probable number (MPN):
The statistical estimation of the probable population in a liquid by d iluting and detennining
end points for microbial growth.
Mould:
Any of a large group of fungi that cause mould or mouldiness and that exists as multicellular
filamentous colonies; also the deposit or growth caused by such fungi. Moulds typically do
not produce macroscopic fruiting bodies.
Narrow-spectrum drugs:
Chemotherapeutic agents that is effective on ly against a limited
Nicoti namide adenine dinucleotide phosphate:
An electron-carrying coenzyme that most often participates as an electron carner m
biosynthetic metabolism.
Norma l saline:
Diluents used for enumeration, consisting of 0.85% NaCI concentration. Maintain osmotic
balance of ce ll.
Nutrient:
A substance that supports growth and reproduction.

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Osmophilic microorganisms:
Microorga nisms th at grows bes t in or on medi a o f hi gh solute concentrati on.
Os motolerant:
Orga nis ms th at grow over a fairl y wide ra nge of wa ter acti vit y or so lute co nce ntration.
Oxidation-reduction (redox) reactions:
Reacti ons in vo lvi ng elec tro n transfers; th e red uctant donates electro ns to an oxidant.
Pasteurization:
The process o f hea ting milk and other liquids to destroy mi croorga nisms th at ca n ca use
spoil age or di sease.
Pathoge n:
Any virus, bacterium, or oth er age nt that causes di sease.
PCA:
Plate co unt aga r medium, ge neral all -pu rpose enrichm ent
Penicillins:
A group o f anti bioti cs co nt aining a p- Iac tam ring, whi ch are ac ti ve agai nst gram-positi ve
bac teri a.
Pep tones:
Wat er- solu b le di ges ts or hydro lysat es of proteins that are used in the preparati on o f culture
medi a.
Petri dish:
A shallow dish co nsistin g of two ro und, overl apping halves that is used to grow
mi croo rganis ms on solid c ulture medium; the top is larger than th e bottom of the di sh to
preve nt contamin ati on o f the culture.
pH:
A meas ure o f the acidit y or alkalinit y o f a soluti on, numericall y eq ual to 7 for neut ra l
so lutions, increas ing with increasing alkalinit y.
Plate count agar:
Va ri a ti on of nutri ent agar, for optimi zing co unts o f bacte ri a in sa mpl es pop ul ation is growi ng
at a co nsta nt a nd max imum rate, di viding and doublin g at regul a r interva ls .
Pour plate:
Procedure where liquifi ed agar has bee n poured into a Pe tri dish after being mi xed with
bac teri a
Psychrophile:
A mi croorga ni sm that gro ws we ll at O°C and has an optimum growth te mpera tu re o f 15°C or
lowe r and a temperature max imum aro und 20°C.
Psychrotroph:
A microorga nism that grows at O°C. but has a gro wth optimum between 20 and 30°C, and a
maxi mum of aboul 35 °C.

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Pure culture:
A population of cells that is identical because they arise from a single cell.
Reducing agent or reductant:
The electron donor in an oxidation-reduct ion
Secondary metabolites:
Products of metabol ism that are synthesized after growt h has been
Selective media:
Luhure med13 th,n iavor the growth 0\ ~pec\l\c m\(;foorgan\:::.m::•. \n\~ rna) be accorn?\\shed
by inhibiting the growth of undesired mi croorgani sms.
Simple stain:
Single type of nucleic ac id, lack ing independent metaboli sm, and reproducing on ly within
Living host ce ll s.
Smear:
A uniform thin film of bacterial other suspension on glass slide.
Source:
The location or object from wh ich a pathogen is immed iately transmitted to the host, either
directly or through an intermediate agent.
Species:
Species of higher organisms are groups of interbreeding or potentially interbreeding natural
popul ati ons that are reproduct ively iso lated. Bacterial spec ies are co llections of strains that
have man y stable pro perties in common and differ signifi cantl y from other groups of strains.
Spore:
A differentiated, specialized form that can be used for dissemination, for survival of adve rse
conditions because of its heat and dessicat ion res istance, andlor for reprod uction. Spores are
usuall y uni cellular and may deve lop into vegetati ve organisms or gametes. They may be
produced asexually or sexually and are of many types.
Spread plate:
Procedure where pre-made agar plates have a sample of bacterium placed on top of the agar
and spread via a glass rod
Starter culture:
An inoculum, consisting of a mi xture of carefully selected mi croorganisms, used
Stationary phase:
The phase of mi crob ial growth in a batch culture when population growth ceases and the
growth curve level s off.
Sterilization:
The process by which all li ving cells, viabl e spores, vi ruses, and viroids are either destroyed
or removed from an object or habi tat .

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Strain:
A population of orga ni sms that desce nds from a single orga nism or pure c ulture isolate.
Streak plate:
Procedure whe re a bac teri a l spec imen is placed on a pre- made p la te and diluted out using
flame a nd multiple secti ons.
Streptomycin:
A bac teric idal 3minoglycos ide antibioti c produced by Streptomyces grisells.
Symbiosis:
The li ving togeth er Of c\ose assoc iati on of two di ssimilar organisms, each of these organisI11s
be ing kn own as a sym biont.
Thermophile:
A microorgani sm that can grow at temperatu res of 55 °C or higher: the min imum is usually
around 45°C.
Toxin:
A mi crobia l produ ct or comp onent th at ca n injure a noth er ce ll or orga nism at low
concentrati ons. Ofte n th e te rm refe rs to a poiso nous prote in , but tox ins ma y be lipids and
other substances.
Turbidostat:
A continuous culture system equipped with a photoce ll that adjusts the now of medium
through the culture vesse l so as to maintain a constant cell density or turbidity.
Virus:
An infectious age nt ha ving a simple acellular organization with a protein coat and a si ngle
type of nucleic ac id, lac king independent metabolism, and reprod ucing onl y within li ving
host cclls.
Vitamin:
An organic compound required by organi sms 111 minute quantiti es for gro wth and
reproduction because it cannot be synthes ized by the organism; vitamins often serve as
enzyme cofactors or parts of cofac tors.
Water activity (aw):
A quantitati ve measure of \vater availability in the h abitat ~ the water acti vity of a solution is
one-hundredth its relati ve humidity.
Yeast:
A unice llular fungus that has a single nucleus and rcproduces either asexuall y by budding or
sex ually.
Zone of inhibition:
Area of no bacterial growth around a chemica l on a disc, indicates sensit ivi ty

103