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J. Environ. Res. Develop.

Journal of Environmental Research And Development

Vol. 7 No. 1A, July-September 2012

ISOLATION, IDENTIFICATION AND CHARACTERIZATION


OF LACTIC ACID BACTERIA FROM
DAIRY SLUDGE SAMPLE

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Choksi Nikita and Desai Hemangi*

TIFAC-CORE In Environmental Engineering, Sarvajanik College of Engineering and Technology


Surat, Gujarat (INDIA)
*E-mail : hemangal4@yahoo.co.in
: nikkichoksi_01@yahoo.co.in

Received May 02, 2012

Accepted September 10, 2012

ABSTRACT

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Lactic Acid Bacteria (LAB) commonly used as starter cultures in food technology are known to
manufacture antimicrobial products having great potential. The aim of this study is isolation of lactic
acid bacterial strain which has been potential for lactic acid production, to identify, characterize it and
to optimize culture conditions for the process. For this study, we have isolated different strains of
Lactic Acid Bacteria (LAB) from dairy sludge samples which were collected from 3 different dairy
plant of Gujarat, (India). Therefore, characterization of isolated strains through morphological,
physiological, biochemical and carbohydrate fermentation test were done. Lactic Acid Bacteria (LAB)
are a group of Gram-positive, non- spore forming, cocci or rod shaped, catalase-negative and fastidious
organisms, considered as Generally Recognized As Safe (GRAS) organism. For isolation, samples
were serially diluted and plated on MRS agar (DE MAN, ROGOSA and SHARPE agar. HI Media)
Plate. Well-isolated colonies with typical characteristics were picked from each plate and were further
sub cultured until pure isolates were obtained and transferred to MRS broth (HI Media) for further
experiments. Identification of lactic acid bacteria belongs to family Lactobacillaceae was done first
at genus level in the Lactobacillus, Lactococcus, Streptococcus, Leuconostoc, Pediococcus.
Lactobacillus is rod shape and Streptococcus, Leuconostoc, Pediococcus are cocci shape. Most of
them are used in dairy starter culture, present in raw milk, milk-loving bacteria. Isolated colony was
identified by gram-staining, gram +ve and rod shape strain showed confirmation of lactobacillus .
L.bulgaricus and Streptococcus thermophilus were found as the most dominant species in common
sludge unit. While L.acidophillus was found as dominant species along with the L.bulgaricus and
Streptococcus thermophilus in the sludge of pro biotic acidophilus butter milk manufacturing unit
and L.casei, L.helveticus, L.brevis, L.lactis were present as the dominant species in the cheese
manufacturing unit along with the L.bulgaricus and Streptococcus thermophilus. Selected microbial
isolates obtained in this study will be used for production of lactic acid acts as monomer for the
synthesis of biodegradable polymer on my future study.
Key Words :LacticAcidBacteria(LAB),Isolation,Dairysludge,MRSagarplate,
MRS Broth, Bacterial strain

INTRODUCTION

to minimize the rate of pollution is by


manufacturing article from biodegradable material.
Biodegradable material are those which can be
degrade in the presence of environmental
condition like soil, moisture, microorganism, light,
heat etc. and end products of this is not harmful
to environment.
Dairy industry produces huge volumes of wastes,
both solid and liquids. This waste poses escalating

To overcome the problem of environmental


pollution through outcome of industries, waste and
pollutant from different industries, unused packing
material and one is the possible solution of these
problems is to minimize the use or to enhance the
reutilization of waste material. But simplest way
*Author for correspondence

J. Environ. Res. Develop.


Journal of Environmental Research And Development

dependent on the optically pure L(+) or D(-) lactic


acid which is suitable for commercial products.7,8
For the pilot scale production of lactic acid though
biotechnological route, there have been various
requirement for high productivity i.e. cheap raw
material , lactic acid producing microorganism,
fermentation approach, type of bioreactor and
finally purification of optically pure lactic acid for
production of high crystalline lactic acid.
So that production of PLA can be divided into
two step i.e. production of lactic acid and its
polymerization to PLA. Here, initially required
lactic acid producing bacteria which help to lactic
acid production with easily available raw material.
Lactic acid bacteria are a group of related bacteria
that produce lactic acid as a result of carbohydrate
fermentation. The concept of the group name
Lactic Acid Bacteria was created for bacteria
causing fermentation and coagulation of milk and
defines as those which produce lactic acid from
lactose. The family name Lactobacteriaceae
was applied by Orla-Jensen to a physiological
group of bacteria producing lactic acid alone or
acetic and lactic acids, alcohol and carbon dioxide.
Today, lactic acid bacteria are regarded as
synonymous by and large with the family
Lactobacteriaceae.
Lactic acid bacteria are widely distributed in the
nature. They could be isolated from soils, waters,
plants, silages, waste products and also from the
intestinal tract of animals and humans (Axelsson).
Lactic Acid Bacteria (LAB) are characterized
as Gram - positive, usually non-motile, non sporulating bacteria that produce lactic acid as a
major or sole product of fermentative metabolism.
Kandler and Weiss have classified Lactobacillus
isolates from temperate regions according to their
morphology, physiology and molecular characters9
Schleifer classified LAB based on the molecular
characteristics10 LAB from food and their current
taxonomical status have been described by many11,12
These microbes are broadly used by us in the
production of fermented food products, such as
yogurt (Streptococcus spp. and Lactobacillus
spp.), cheese (Lactococcus spp.), Sauerkraut
(Leuconostoc spp.) and sausage. T hese
organisms are heterotrophic and usually have
complex nutritional necessities because they lack

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disposal and pollution (High BOD) problems and


represents a loss of valuable biomass and
nutrients. However, despite their pollution and
hazard aspects, in many cases, dairy processing
wastes have a good potential of converting into
useful products of higher value as by-product, or
even as raw material for other industries. Organic
acids are examples of such valuable by-product
of the fermentation of high carbohydrate
containing industrial substrates. They therefore
could be utilized cheaply as substrate for
microorganisms producing intermediate volume
high value organic acids like lactic acid. Lactic
acid is under increasing demand in food,
pharmaceutical and chemical industries and for
production of polylactic acid polymers, which
possess outstanding biomedical applications.
Wastes generated from dairy plants may be
regarded as a viable option for meeting this
growing demand for lactic acid and lactic acid
has received attention for use if a wide range of
applications mostly as it acts as a monomer for
the production of biodegradable poly (lactic acid)
or polylactide (PLA). PLA can be produced
chemically and biotechnologically but
biotechnological routes are mostly favored
because of environmental concerns and limited
nature of petrochemical feedbacks. Worldwide
efforts have been made for the production of
lactic acid and PLA with good yield and low cost
management. 1,2
A long history record of lactic acid used in
fermentation, preservation of food stuffs is also
available. Lactic acid was firstly discovered by
Scheela in, 1780 in sour milk and named by
Lavoisier is acid lactique in 1789 which became
the origin of present terminology lactic acid.3 Lactic
acid has been approved by the US FDA as GRAS
(Generally Recognized As Safe) for the
consumption of it as a food additive, cosmetic,
pharmaceutical, medical implantation4 It can be
produced by chemically and biotechnologically
both.5,6 But due to several serious problems, its
biotechnology route is more favorable because
racemic DL-lactic acid is produced by chemical
synthesis from petro chemical sources whereas
an optically pure L(+) or D(-) lactic acid can be
obtained by microbial fermentation of renewable
sources and higher physical properties of
polymerized Poly Lactic Acid (PLA) is crucially

Vol. 7 No. 1A, July-September 2012

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Journal of Environmental Research And Development

Vol. 7 No. 1A, July-September 2012

incubated at 300C, 370C , 400C, 420C and 500C


for 48-72 hours. Well - isolated colonies with
typical characteristics with entire margins were
picked from each plate and transferred to MRS
broth. Lactobacilli isolated on MRS agar should
be further confirmed biochemically.

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many biosynthetic capabilities. Most species have


multiple requirements of amino acids and vitamins.
Because of this, lactic acid bacteria are generally
abundant only in communities where these
requirements can be provided. They are often
associated with animal oral cavities and intestines;
plant leaves as well as decaying plants or animal
matter, compost, etc.
The classification of lactic acid bacteria into
different genera is largely based on morphology,
mode of glucose fermentation, growth at different
temperatures and configuration of the lactic acid
produced ability to grow at high salt
concentrations and acid or alkaline tolerance.
Lactic acid bacteria are among the best studied
microorganisms for human health advantageous
effects and fermentation. Significant novel
developments have been made in the research of
lactic acid bacteria in the area of multidrug
resistance, bacteriocins, osmoregulation, autolysins
and bacteriophages. Advancement has also been
made in the production of food grade genetically
modified lactic acid bacteria.
In the previous studies, LAB could be isolated
from many kinds of sources such as milk products,
sugar cane plants 13 , fresh water fishes 14 , but
studies on isolation of LAB from dairy sludge
sample remain scare. Therefore, dairy isolates of
lactic acid bacteria capable of degrading dairy
waste as well as working with other waste raw
material and converting them into lactic acid are
considered to be the key to the development of a
workable microbial fermentation based value
addition process for dairy wastes containing lactic
acid bacteria.

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Identification of lactic acid bacteria at the


genus level
For identification of lactic acid bacteria, overnight
cultures of each isolate in MRS broth were used.
All isolates were initially tested for gram reaction,
catalase enzyme and production of acid from
glucose in Hugh and Leifson medium by oxidation
or fermentation rection (Harrigan and
MacCance). Only gram positive bacteria with
catalase negative reactions were observed
(Schillinger and Lucke, Garvie and Weiss.) and
the representative isolates were purified by
successive streaking on to the same agar
substrate. For the gram-positive, catalase negative
rods, growth at various temperature 100C, 150C
and 450C ,tolerance of different salt levels (2,4
and 6.5% w/v NaCl), hetero- and homofermentative activity (using MRS broth) with
inverted Durham tubes in MRS broth were
determined. Isolates from dairy sludge were then
selected based on the above tests for further
identification. The bacteria were characterized by
microscopic and by conventional biochemical and
physiological test. The culture were examined for
colony and cell morphology, motility, gram stain
and production of acid from glucose (Harrigan
and MacCance. In addition to the oxidation and
fermentation test according to Hugh and Leifson.
These preliminary test make it possible to classify
the isolates in genus on the basis of characteristic
and tests of identification mentioned by Harrigan
and MacCane, Hammes et al., Holzapfel and
Schillinger and Dicks et al.

MATERIAL AND METHODS

The present study used sludge samples collected


from 3 different well known dairy plants in Gujarat
at India. Sludge sample collected inside ice box
and transferred to the laboratory for
microbiological analysis MRS agar and broth were
used for enumeration and culture of LAB.15 The
sludge samples serially diluted and pour plated on
MRS plates. The MRS plates overlaid with MRS
agar and incubated at 37 C for 4872 h. This is
optimal temperature for bacterial growth but
different lactobacillus strains isolated at particular
temperature set up here for colony isolation and

Identification of lactic acid bacteria to the


species level
After their microscopic examination , gram +ve
and catalase ve lactobacillus were tested for
their sugar fermentation pattern, production of
ammonia from arginine in addition to their ability
of growth at 15 0 C and 45 0 C. according to
Harrigan and Maccance.16

J. Environ. Res. Develop.


Journal of Environmental Research And Development

RESULTS AND DISCUSSION

Vol. 7 No. 1A, July-September 2012

identification, most of them were determined as


representative of the family Lactobacillaceae
and genus Lactobacillus and rest were referred
to genus Lactococcus, Streptococcus,
Pediococcus, Leuconostoc and some of
Bifidobacteria, Staphylococcus etc. 17,18 A
summary of micro-organisms associated with
milk and milk products is given in Table 1.
This genus should be possible in sludge sample is
depend on many factors like dairy industries
region, raw milk sample, starter culture used for
production, production unit working such as crude
manufacturing, paneer manufacturing, different
types of cheese manufacturing, probiotic butter
milk, seasonal changes, temperature, weather
effects, drainage system etc. So, all time we could
not get same genus as well as same species

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Isolation and identification


Colonies of lactic acid bacteria were observed
on the surface of MRS plates. More than one
colony was observed in most of the cases.
Cultural and morphological characteristics were
examined with the help of microscope. Different
types of microorganisms were observed, majority
of them belonged to Gram+ve rods and cocci
shaped bacteria. The purification of isolates was
done by transferring Gram+ve rods and cocci
shaped bacteria to the plates of selective media
MRS respectively. These isolates were further
sub cultured until pure isolates were obtained.
Numbers of lactic acid bacterial cultures were
isolated from dairy sludge samples. After initial

Table 1 : Micro-organisms associated with milk and milk products


Family

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Section (According to 9th


edition of Bergeys Manual)
Bacteria

Spirillaceae
Pseudomonadaceae
Neisseriaceae

Enterobacteriaceae

Vibrionaceae

Rickettsiaceae
Microccaceae

Streptococcaceae
Bacillaceae

Lactobacillaceae

Propionibacteriaceae
Corynebacteriaceae

Actinomycetaceae
Mycobacteriaceae
Nocardiaceae

Genus
Campylobacter
Pseudomonas
Brucella
Acinetobacter
Moraxella-like organisms
Escherichia
Enterobacter
Salmonalla,Yersinia
Aeromonas
Chromobacterium
Flavobacterium
Vibrio
Coxiella
Micrococcus
Staphylococcus
Steptococcus
Leuconostoc
Bacillus
Clostridium
Lactobacillus
Listeria
Propionibacterium
Corynebacterium
Arthrobacter
Microbacterium
Actinomyces
Mycobacterium
Nocardia

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Journal of Environmental Research And Development

Vol. 7 No. 1A, July-September 2012

Although LAB are comprised of 11 genera,


only 6 of them are dairy associated
These are Lactococcus, Enterococcus, Streptococcus,

therefore, probability changes based on condition.


Here, we are interested to isolate lactic acid
bacteria which are potential for lactic acid
production in nutrient agar more of this were
isolated with it fungal and mould also grown and
contamination problem occurs. So MRS agar is
selected for study. Colonies were identified
according to their morphological, cultural,
physiological and biochemical characteristics 10,11.
This all genus grown on agar plate is used in dairy
starter culture and some of it was present in raw
milk also. Further, genus lactobacillus was
studied on subgenus and species level by
biochemical test as well as some particular
identification characteristics.

Leuconostoc, Pediococcus, and Lactobacillus.


(Axelsson, Garvie) Identification test for the

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same is shown in Fig. 1.


Pediococcus is a genus of gram-positive lactic
acid bacteria, placed within the family of
Lactobacillaceae. They usually occur in pairs
or tetrads and they are purely homofermentative.
Pediococci are used as probiotics, and are
commonly added as beneficial microbes in the
creation of cheeses and yogurts. Recently, lactose
positive pediococci have been used instead of
Streptococcus thermophilus. Lactococcus is a

Gram Positive, catalase -negative


Gas from gluco se

+ve

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ve
ve

Rods Lactoballius
ho mo fe r me nta tiv e

Coc c i

Coc c i
le uc o no sto r s

Tetrad

Growth @ 15C

+Ve
pediococcus

Roads
Lactobacillu Roads
he te r o te me nta tiv e

Ve

Ve
thermobacterium

+Ve
tr epto bacter ium

Gr owth@
45 C
Ve
+Ve
Growth@10C

+Ve
Lactococcus

Growth 6.5 NoCI

+Ve
Enterococcus

Ve
Lactococcus

Fig. 1 : Route for identification of lactic acid bacteria at genus level


cultures, or in mixed-strain cultures with other
lactic acid bacteria such as Lactobacillus and
Streptococcus. The bacteria also play a role in
the flavor of the final product.
Steptococcus is a genus of gram- positive lactic
acid bacteria. They are catalase-negative-cocci
usually found in chains or pairs. Some of species
are also great value as they used by dairy industry

genus of Gram-positive lactic acid bacteria that


were formerly included in the genus Streptococcus
Group N1. They are catalase-negative, non-motile
cocci that are found singly, in pairs, or in chains
and they are homofermentors. These organisms
are commonly used in the dairy industry in the
manufacture of fermented dairy products like
cheeses. They can be used in single-strain starter

5
s

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Vol. 7 No. 1A, July-September 2012

2. Growth at different temperatures (15C, 45C)


3. Growth at 6.5% NaCl concentration
4. Arginine hydrolysis
5. Carbohydrate fermentation

for the manufacturing of fermented dairy


products.
Leuconostoc is a genus of gram-positive bacteria,
placed within the family of Leuconostocaceae.
They are generally avoid cocci in pairs or often
forming chains. Leuconostoc spp. are catalasenegative (which distinguishes them from
Staphylococci). All species within this genus are
heterofermentative, i.e., glucose is fermented with
production of D(-) lactic acid, ethanol and CO2
and are able to produce dextran from sucrose.
They are generally slime-forming and used in
cheese manufacturing.
Lactobacillius are gram-positive rods, typically
non-motile, non-sporulating and non-acid fast,
lactobacilli are aerobic and facultatively
anaerobic, catalase negative and grow best at PH
6.0. the carbohydrates and polyalcohols are
changed by homofermentation to lactic acid or
by heterofermentation to lactic acid and acetic
acid, alcohols and carbon dioxide. The genus was
subdivided by Orla-jensen into three groups
(Thermo bacterium, Strepto bacterium and beta
bacterium).

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Gas production from Glucose


In order to further define ho mofermentative
isolates, CO2 production from glucose test was
performed. For this purpose, MRS broths and
inverted Durham tubes were used. 50l of
overnight cultures were transferred into the 8 ml
test media. After incubation for 5 days at 30 C,
gas accumulation in Durham tubes was taken as
the evidence for CO2 production from glucose.

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Voges-Proskauer (V-P) Test


Inoculate the medium Glucose Phosphate Broth
(GPB) with culture and incubate the medium at
370 C for 24-48 hours. After incubation, add 0.6
ml of a- naphthol and 0.2 ml of KOH solution per
ml of broth.(Reagents should be added in this
order only because a-naphthol exerts catalytic
effect only if added before the KOH). Shake well
after addition of each reagent and slope the tube
to increase the aeration. Development of pinkred color within 15 minutes or more, it means VP positive for particular species so, we could
concluded this lactobacillus strain either
thermobacterium or streptobacterium and V-P
negative identified betabacterium.

Identification test of Lactobacillus bacteria


All isolates were microscopically examined for
gram stain reaction, cell morphology and cellular
arrangement (Gerhardt et al., and Sneath et al.

Catalase test
A drop of 3% hydrogen peroxide was placed on
a clean microscopic slide. With a nicrome wire
loop pick up cells from the center of a well isolated
colony of the test culture and transfer them into
the drop of hydrogen peroxide. Both were mixed
and observed for gas bubble production. The
strains showing gram-positive and catalase
negative isolates were identified at species level.
Physiological and biochemical identification
Each isolate was activated in 5 ml MRS broth for
24 h at 30 C before use. Therefore, overnight
cultures were used during all the identification
procedures. Physiological and biochemical
identifications were performed according to the
methods and criteria of Sharpe and Fryer; Garvie,
Devriese et. al., Teuber.
For the identification of rod shaped isolates,
following tests were applied
1. Gas poduction from glucose

Growth at different temperatures


50l of overnight cultures were transferred into
the tubes which contain 5 ml temperature test
media. After inoculation, they were incubated for
7 days at 10 C, 40 C or 45 C. Cells growth at
any of these temperatures was detected by the
change in the color of the cultures, from purple to
yellow.
Growth at different NaCl concentrations
50l of overnight cultures were transferred into
the tubes which contain 5 ml NaCl test media.
Isolates were tested for growth at 2%, 4% or
6.5% NaCl concentrations. They were incubated
for 7 days at 30C. The change of the color from
purple to yellow taken as the evidence for cell
growth.
Arginine hydrolysis test
The arginine hydrolysis, arginine MRS broth was
used instead of Reddy broth. 50l overnight

J. Environ. Res. Develop.


Journal of Environmental Research And Development

cultures were inoculated into 5 ml arginine test


media, and were then incubated for 5 days at 30C.
After the incubation, ammonia production was
detected by using Nessler reagent. For this
purpose, 100 l of culture broth were pipetted into
each well of the microtitre plates and 100 l of
Nessler reagent were added. Immediate orange
color formation was taken as the indication for
ammonia production. No color change indicated
that the strain could not hydrolyse arginine.

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and sterilized. A single carbohydrate disc is added


to each tube aseptically and inoculated with test
organisms.
Incubation is carried out at 361.0 C for 18-48
hours and results are recorded at 18-24 hours and
again at 48 hours. The results should be frequently
observed since reversal of fermentation can take
place. In case of liquid medium gas produced
during fermentation is collected in the inverted
Durhams tube while acid production changes color
of the medium. In case of fermentation, the color
of sugar was changed from red to yellow,
reflecting the test as positive.
Percentage distribution of different genus of lactic
acid bacteria were collected from 3 different dairy
industries A, B, C. Dairy sludgeA1(common
sludge unit), Dairy sludgeA2 (acidophilus
buttermilk production unit). dairy sludgeB1
(common sludge unit), dairy sludgeB2(cheese
manufacturing unit),dairy sludgeC1(common
sludge unit), dairy sludgeC2(cheese manufacturing
unit). Differential characteristics of lactic acid
bacteria based on morphology and physiology
determined from various microbial and analytical
confirmation test is shown in rod shape
Lactobacillus is divided into three groups.
Summary of Differential character s of
lactobacillus is shown in Table 2. Rod shape
lactobacillus is divided into three groups.
Summary of Differential character s of
lactobacillus is shown in Table 3.
The best available microscopic results of the
experiments are shown in Fig. 2 to Fig.4.
Morphological and simple physiological
Characterization of LAB which were isolated from
the dairy sludge sample based on microscopic
characterization and analytical test is shown in
Table 5.
Results of sugar fermentation test and growth
stability at various temperature based on chemical
test is shown in Table 2.

Identification of Cocci
The characteristic used for the identification of
cocci shaped LAB in this study were presented.
Except for the arginine test, all the other tests
were the same as those for bacilli shaped LAB.

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Arginine hydrolysis and gas production from


citrate
In order to perform this test, 8 ml of Reddy broth
and inverted Durham tubes were used. Fifty l of
overnight cultures were inoculated into the Reddy
broth and were then incubated for 5 days at 30C.
Arginine hydrolysis
The cultures which utilize arginine, change the
color of the broth first to yellow due to the lactic
acid production and to violet because of the
ammonia production (Cardinal et al). On the other
hand, the cultures which do not utilize arginine
assume a deep-yellow color by producing lactic
acid only.
Gas production from citrate
The breakdown of the citrate results in production
of carbon dioxide. Gas accumulation in inverted
Durham tubes indicated citrate utilization.
Sugar fermentation tests

Carbohydrates differentiation discs (from Himedia) was used for sugar fermentation test.
Andr ade peptone water, liquid media are
dispensed in 5 ml amount in test tube with
inverted Durhams tube for testing fermentation

Fig. 2 : Lactic acid

Fig. 3 : Streptococcu

bacteria colony

thremophilus

Fig. 4 : Lactobacillus
bulgaricus

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Vol. 7 No. 1A, July-September 2012

Table 3 : The percentage distribution of different genus of LAB in dairy sludge sample
and differential characteristics of lactic acid bacteria based on morphology
Lactic acid bacteria (%)

Sample
Dairy SludgeA1

40

15

35

30

35

10

25

(common Sludge unit)


Dairy SludgeA2 (acidophilus
buttermilk production unit)
Dairy Sludge B1
(common sludge unit)
Dairy Sludge B2
(cheese manufacturing unit)
Dairy Sludge C1
(common sludge unit)
Dairy Sludge C2

Pediococcus

Lactobacillus

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Streptococcus Leuconostoc Lactococcus


10

45

50

50

10

55

45

10

55

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(Cheese manufacturing unit)


Characteristics

Morphology

Cocci

Cocci

Cocci

cocci in tetrads

Rods

10C

45C

6.5% NaCl

pH 4.4

pH 9.6

Lactic acid configuration

L, DL

D, L, DL

CO2fromglucose
Growth

+Positive; -negative; varies between pecies


*test for homo- or hetero fermentation of glucose : -homofermentation, + heterofermentation

Table 3 : Differential characters of the three group of Lactobacillus


Test

Thermo bacterium

Strepto bacterium

Beta bacterium

Motility

Growth at 5c

Growth at 15c

Growth at 45c

Gas from glucose

Voges- Proskaucer reaction

Nitrate reduction

Homofermentation

Homofermentation

Heterofermentation

Fermentation

+ = Positive reaction; = Negative reaction; V= Variable reaction

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9
Rods
Cocci

Cocci
Cocci

Lactobacillus spp.

Streptococcus spp.

Leuconostoc spp.

Pediococcus spp.

Rods

L. helviticus

Cocci

Rods

L. lactis

thermophillus

Rods

L. casei

Rods

Rods

L. acidophilus

L. brevis

Rods

Cell shape

L. bulgaricus

Species

G +ve

G +ve

G +ve

G +ve

G +ve

G +ve

G + ve

G+ ve

G +ve

G + ve

G + ve

reaction

stain

Gram

activity

Catalase

glucose

from

Acid

lactose

from

Acid

Cellular

Tetrads in pair or short chain


Cocci

Pair or short chain cocci

Cocci

Spherical and ovoid shape

Short or long chain rods

Pair or long chain Cocci

Single or in chain.

single, pairs and short chains

Long, single or in pairs

Short or long chain rods.

single, pairs and short chains

single, pairs and short chains

arrangement

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+

glucose

from

Co2

25-32C

15-35C

30-37C

30-50C

40-50C

30C

400-42C

40-43C

30C

35-38C

40C

temperature

Optimum

Table 4 : Morphological and simple physiological characterization of LAB isolated from the dairy sludge sample

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J. Environ. Res. Develop.

L.lactis

L.helveticus

L.brevis

S. thermophilus

L.acidophilus

L.casei

L.bulgaricus

Lactic acid

S/N

10

Morpho 5C
logy

15C

Growth at

45C Acid and


NH3 Arabi
gas from from nose
glucose arginine

Mann
itol

Sorbi
tol

Treha
lose

Mal
tose

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Meizi Melib Raffi


tose iose nose

Sugar fermentation

Table 5 : Results of sugar fermentation test

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+

Lact
ose

Gala
ctose

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6. Lunt J., Large scale production, properties


and commercial application of polyactic
acid polymers, Poly. Degrad Stabil,
59(2),145-152, (1998).
7. Soder G. and Stolt M., Properties of lactic
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CONCLUSION

SP
EC
IM
EN

CO
PY

Sludge samples were collected from 3 different


dairy plants. Identification of lactic acid bacteria
belongs to family Lactobacillaceae was done
first at genus level in the Lactobacillus,
Lactococcus, Streptococcus, Leuconostoc,
Pediococcus. Lactobacillus is rod shape and
Streptococcus, Leuconostoc, Pediococcus are
cocci shape. Most of them are used in dairy starter
culture, present in raw milk, milk-loving bacteria.
Isolated colony was identified by gram-staining,
gram +ve and rod shape strain showed
confirmation of Lactobacillus . L.bulgaricus and
Streptococcus thermophilus were found as the
most dominant species in common sludge unit.
While L.acidophillus was found as dominant
species along with the L.bulgaricus and
Streptococcus thermophilus in the sludge of pro
biotic acidophilus butter milk manufacturing unit
and L.casei, L.helveticus, L.brevis, L.lactis were
present as the dominant species in the cheese
manufacturing unit along with the L.bulgaricus
and Streptococcus thermophilus.

RECOMMENDATION

This species can be used in further study for


production of lactic acid from natural waste
resources.

REFERENCES

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