3rd International Conferences and Workshops on Basic and Applied Sciences 2011

ISBN: 978-979-19096-1-7

Exploration Of Indigenous Bacteria From Pond Brackish

Water At Gresik As Candidate Water Bioremediation
And Vibrio harveyi Biocontrol (As Agent Vibriosis At
Prawn)
Ainur Rofiq*, Nimatuzahroh, and Agoes Soegianto
*Department of Biology
Airlangga University, Surabaya, Indonesia
e-mail: ainurrofiq1978@yahoo.co.id

Keywords: Pond backish water, indigenous

bacteria, decomposer bacteria, Vibrio
harveyi, synergistic, antagonistic.

Abstract
The accumulation of organic material from
unconsumed food and metabolism in aquaculture
can cause decrease of water quality. The bad water
quality influence the growth of pathogenic bacteria
in water especially Vibrio harveyi (The most
important agent of Vibriosis in prawn).
Decomposer bacteria (as agent bioremediation) and
antagonism V. harveyi bacteria (as agent
biocontrol) are expected can repair water quality
and inhibit growth of V. harveyi. Indigenous
bacteria were expected to be more adaptable at their
origin environment and to be more friendly for
environment. This research is aimed to explore
indigenous bacteria from pond brackish water at
Gresik that have the ability as decomposer
(Amilolytic, Proteolytic, Lipolytic and Nitrifying)
and inhibit growth of V. harveyi. The bacteria are
isolated using salt basal medium (Bushnell-Hass +
2,8% NaCl) with adding 1% amilum which further
confirmed with iodine 1% as indicator for
amilolytic medium, 1% casein for proteolytic
medium, 1% olive oil for lipolytic medium.
Meanwhile, the medium for isolation nitrifying
bacteria is nitrat agar which further confirmed with
adding naphtylamine as indicator. Further, all
isolates bacteria are tested synergistic ability with
each other and antagonistic ability againts V.
harveyi. The exploration obtained 3 isolates
proteolytic bacteria (code P1, P3, P4), 3 isolates
amilolytic bacteria (code A1, A3, A5), 4 isolates
lipolytic bacteria (code L1, L4, L9, L12) and 2
isolates nitrifying bacteria (code N1, N6).
Surprisingly, the isolates bacteria with code N6 was
also able to inhibit (antagonism) to V. harveyi. All
isolates bacteria obtained can be sinergized with
each other.

Introduction

Prawn cultivation is one of economically inportant

aquaculture with high trading value at worldwide.
However. cultivation has faced many serious
problems such as prawn diseases and unsatisfactory
practices i.e inedequate control of water quality
etc. Viruses and bacteria are the main cause of
prawn diseases. Disease due to bacterial infections
particularly luminous vibriosis, is the major
problem for prawn cultivation. Among vibrios,
Vibrio harveyi is the main cause of prawn death.
The virulence of V. harveyi has at times caused a
100% loss in prawn production. V. harveyi is also
serious pathogen for a wide range of marine
animals and in other aquaculture systems
(Rattanachuay, 2010)
The accumulation of organic matter from feed
remains, fertilizer, prawn and other organism
metabolit excretion can setle in bottom of pond, if
not offset by good water management systems will
decrease carrying capacity to living of prawn
(Khasani, 2008). The bad environment will make
prawn become stress and susceptible to desease
caused by opportunistic bacteria that live in aquatic
environments where prawn live. This condition will
increase growth of pathogen (Kannapiran et al,
2009).
The use of disinfectans and antibiotics as a
treatment step of series of outbreaks of disease were
raised new problems in the prawn farming.
Disinfectans, whit its non spesific characteristic
sometimes not just turn off the target organism.
Application improper dosage of disinfectans can
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A. Rofiq, Exploration Of Indigenous Bacteria From Pond Brackish Water At Gresik As Candidate Water
Bioremediation And Vibrio harveyi Biocontrol (As Agent Vibriosis At Prawn)
also turn off other biota which is important for pond
(Khasani, 2008). The use of persistent synthetic
antibiotics can also cause resistance in bacteria,
leaving residue on prawn that could further
endanger to humans as consumers (Setyaningsih et
al, 2006).

This research is aimed to explore indigenous

bacteria from pond brackish water at Gresik that
have the ability as decomposer (Amilolytic,
Proteolytic, Lipolytic and Nitrifying) and inhibit
growth of V. harveyi.

The maintenance of good water quality is essential

for optimum growth and survival of prawn. Good
water quality is characterized by adequate of
oxygen and limited level of metabolites.
(Soundarapandian, 2010).

2 Methodology
Pond water from aquaculture areas in village
Tanjungwidoro, Gresik regency was taken
aseptically using steril sterile bottles at random at
three sampling points, then storing the water in the
ice box and the temperature is kept always in a state
about 4 0C and brought to the laboratory to do
further treatment . Vibrio harveyi isolates used tes
antagonist obtained from the Tropical Disease
Centre, Airlangga University. The research was
conducted at the laboratory of microbiology,
Departement of Biology Faculty of Science and
Technology, Airlangga University.

From many studies have been reported, it was the

addition of probiotics can effectively improve the
water quality that coukd ultimately enhance growth
and survival of prawn. This happens because
probiotics play a role in improving the health of
prawn, eliminating or reducing the pathogen
population in the prawn pond environment and
improve water quality through a more rapid
decomposition waste of organic material (Moriarty,
1999).

The bacteria are isolated using salt basal medium

(Bushnell-Hass + 2,8% NaCl) with adding 1%
amilum which further confirmed with iodine 1% as
indicator for amilolytic medium, 1% casein for
proteolytic medium, 1% olive oil for lipolytic
medium. Meanwhile, the medium for isolation
nitrifying bacteria is nitrat agar which further
confirmed with adding naphtylamine as
indicator. Further, all isolates bacteria are tested
synergistic ability with each other and antagonistic
ability to V. harveyi. Isolates bacteria are
considered to have potential proteolytic, amilolytic,
lipolytic, nitrification and antagonists V. harveyi
when bacteria are able to form a zone (halo) around
the colonies of bacteria.

The use of microbial consortia are considered more

advantageus in the process of improving the
environment (decomposition) when compared to
using pure culture due to the synergistic interaction
effect on the microbial association . The
decomposition of organic materials is usually done
by microbes that have the ability of proteolytic,
lipolytic, amilolytic and nitrification (Sohail et al,
2005 ; Aditiawati et al, 2001 ; Miyoshi et al, 1981
and Murtiati et al, 2007).
Commercial water probiotics have been widely
circulated in the market aimed at improving water
quality and disease fighting agents with various
trademark. But, the origin of microbes used can not
be known with certainty, so that optimal potency
and effectiveness can work in an environment
different from its origin still needs to be reviewed.
According Isnansetyo (2005) in Khasani (2008)
indegenous bacteria wich is extracted from
digestive tract fish and the water cultivation
medium that has been trough a series of screening
tests, more potential as a biocontrol, increased
digesbility of feed and bioremidiation agent if
compared with the strains of commercial probiotics
wich is obtained from habitats with different
physical-chemical charateristics.

Notes : Bushnell Hass medium are contains

MgSO4.7H2O 0,2 g/L, CaCl2 0,02 g/L, KH2PO4 1
g/L, K2HPO4 1 g/L, NH4NO3 1 g/L and Solution of
60% g/w FeCl3 2 drops.
3

Result

Bacteria potential proteolytic originally found 8

isolates. Furthermore, selected three isolates that
have the greatest ability to proteolytic first, second
and three base on hydrolysis index. The data are
presented in Table 1 and Figure 1.

Gresik regency is the area which in part territory

lies north Java coastal area and Madura strait.
Gresik including the largest producer of fishing
ponds in East Java besides Sidoarjo regency
(Anonymous, 2009). A third of Gresik regency is a
coastal and marine areas with huge potential for
business development in the maritime sector.
(Dirjen Perikanan Budidaya, 2010).

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3rd International Conferences and Workshops on Basic and Applied Sciences 2011

Table 1: Hydrolysis index of three isolates that

have ability to proteolytic

Table 3. Hydrolysis index of four isolates that

have ability to lypolytic.

Hydrolysis index
No

Isolates
code

Diameter
Halo
Colonies
(mm)
(mm)

P1

15

P3

P4

0,36

16

0,78

35

29

0,21

Hydrolysis index

Hydrolysis
index

11

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No

Isolates
code

1
2
3
4

L1
L4
L9
L12

Diameter
Halo Colonies
(mm) (mm)
13
11
10
13

Hydrolysis
index

9
8
6
7

0,44
0,38
0,67
0,86

Figure 1: Diagram of bacterial isolates

hydrolysis index protelytic code P1, P3 and P4.
Bacteria potential amilolytic found three isolates.
The data are presented in Table 2 and Figure 2.

Figure 3: Diagram of bacterial isolates

hydrolysis index liplolytic code L1, L4, L9 and
L12.

Table 2: Hydrolysis index of three isolates that

have ability to amilolytic.

Potential nitrification bacteria found two isolates.

The data are presented in Table 4.

Hydrolysis index
No

Isolates
code

Diameter
Halo
Colonies
(mm)
(mm)

Table 4: Hydrolysis index of four isolates that

have ability to nitrification.
Hydrolysis index

Hydrolysis
index

1
2

P1
A3

15
18

11
7

0,36
1,57

A5

10

1,00

No

Isolates
code

N1

15

15

N6

35

35

Diameter
Halo
Colonies
(mm)
(mm)

Hydrolysis
index
0,00 (Coloni is
red)
0,00 (Coloni is
red)

Antagonistic ability againts V. harveyi were

assessed by looking at inhibitory zone/ clear zona
around the colonies of bacteria isolates. The data
are presented in Table 5 and Figure 5.
Table 5: Inhibitory zone formed on antagonistic
test againts Vibrio harveyi on three
bacteria isolates
Diameter
Halo Colonies
(mm)
(mm)

Figure 2: Diagram of bacterial isolates

hydrolysis index amilolytic code A1, A3 and A5.

No

Isolates
code

Bacteria potential lypolytic found four isolates. The

data are presented in Table 3 and Figure 3.

A1

L9

17

14

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Inhibitory
zone(mm)

A. Rofiq, Exploration Of Indigenous Bacteria From Pond Brackish Water At Gresik As Candidate Water
Bioremediation And Vibrio harveyi Biocontrol (As Agent Vibriosis At Prawn)

N6

50

24

impairment in water quality is difficult. However,

addition of some commercial probiotics is reported
effectively deal with these subtances and that way
helpful in maintaining of water quality parameters
(bioremediation) thereby will improve of growth
rate and survival rate of prawn (Soundarapandian et
al, 2010).

26

Prawn feed contains a high amount of protein,

thereby in prawn ponds proteolytic bacteria are
likely to be one of bacterial groups that will govern
the water and sediment in prawn ponds, and have a
major influence on prawn health (Rattanachuay et
al, 2007). From this research was obtained three
isolates bacteria potential proteolytic. The largest
hydrolysis index was presented by isolate with code
P3 the value was 0.78. The characteristics of
isolates P3 are coccus, gram positive, catalase
negative, oxidase positive (weak) and arrangement
of cell is chain. According Bergeys manual of
Determinative Bacteriology, (1994) this isolate
near to Streptococcus sp.

Figure 4: Diagram halo diameter formed on

the antagonistic test againts V. harveyi on three
bacteria isolates code A1, L9 and N6.
The ability of synergistic among isolates assessed
by looking at the halo around the colonies of
bacteria isolates on the growth of other isolates, if
the halo is not formed around the colonies its means
the isolates can synergy each others. The data are
presented in table 6.

Amilolytic bacteria is bacteria capable to hydrolysis

starch into simpler molecules by removing amylase
enzyme. Amylolytic bacteria important in water
bioremediation to degrete prawn feed residue wich
content of starch. According (SITH, 2011)
percentage of starch content in prawn feed around
30% was the second largest after protein content.
From this research obtained three isolates potential
amilolytic bacteria. The largest hydrolysis index
was presented by isolate with code A1 the value
was 4.80. The characteristics of isolates A1 are rod,
gram positive, catalase negative, oxidase positive.
After compared among others biochemical test with
Bergeys manual of Determinative Bacteriology,
(1994) this isolate suspected near to Lactobacillus
sp.

Table 6: Data result of synergistic test, the sign

(-) indicates that a combination of
isolates do not cause the formation of
halo around the test isolates..
Isolates
code
A1
A3
P1
P3
L9
L12
N6

A1

A3

P1

P3

L9

L12

N6

Lypolytic bacteria are bacteria capable to hydrolyze

lypid into simpler molecules by removing enzyme
lypase. Bacteria proteolytic and lipolytic are
important parts of the solubilization of dead
organism, which contain a relative large part of
proteins and lipids (Miyoshi et al, 1981). From this
research obtained four isolates potential lipolytic
bacteria. The largest hydrolysis index was
presented by isolate with code L12 the value was
0.86.

Water quality plays an important role in

aquaculture production. A complete understanding
of relationship between water quality and aquatic
productvity is absolutely essential for optimum
growth and production. The water quality during
the culture period will deteriorate mainly due to the
accumulation of metabolic wastes of living
organisms, decomposition of unutilized feeds and
decay of of biotic materials. Generally, organism
are in a state of balance between potential disease
causing microorganism and their environment.
Change in this equilibrium through the way of
impairment in water quality parameters and can
influence survival of organism, so that they
become vulnerable to disease due to stress.
Eliminate of imbalances, which is caused by

Potential nitrifying bacteria code N6 was have

coloni diameter larger than N1, but both were have
not clear zone around colonies. Red color in the
both colonies after drops with naphtylamine
prove that bacteria can reduce nitrate to nitrite. The
use of nitrification and denitrification bacteria is to
maintain the balance of anorganic compaounds
(ammonia, nitrite and nitrate). This bioremidiation
approach is expected to balance the excess nitrogen
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3rd International Conferences and Workshops on Basic and Applied Sciences 2011

All candidates isolates bacteria has been obtained,

needs to do further testing wich involves testing of
pathogenitas on animals target, testing the
efectiveness in improving the water quality and
testing the ability of antagonistic againts V. harveyi
(jn vivo).

from the feed residue, metabolic excrete and further

release them as gaseous N2 / N2O to the atmosphere
(Badjoeri et al, 2008). The isolates bacteria with
code N6 was also able to inhibit (antagonistic) to V.
harveyi. The characteristics of isolates N6 are
coccus, gram positive, catalase negative, oxidase
positive and arrangement of cell was chain.
According Bergeys manual of Determinative
Bacteriology, (1994) this isolate near to
Streptococcus sp.

References
[1] Aditiawati,P.,Pikoli,M.R.,Indiani.D.,
2001,
Isolasi bertahap bakteri pendegradasi minyak
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The ability for mutual synergy among bacterial

isolates is to be one of the requirements for the
produce of probiotics candidate (Vine, 2004). The
combination of bacteria are expected to perform
functions of water bioremediation effectively.
Seven isolates were selected as candidates for water
bioremediation with proteolytic, amilolitik, lipolytic
and nitrification ability (Table 6), indicating that all
isolates can synergize with each other. This means
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Prawn will be healthy if the environment is not

polluted and is not attacked by the disease
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antagonistic againts V. harveyi with code A1, L9
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have the ability as an antagonistic againts V.
harveyi.
4

ISSN XXXX-XXXX

[5] Kannapiran.E, Ravindran .J, Chandrasekar. R,

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The exploration of indigenous bacteria from pond

brackish water at gresik obtained 3 isolates
proteolytic bacteria (code P1, P3, P4), 3 isolates
amilolytic bacteria (code A1, A3, A5), 4 isolates
lipolytic bacteria (code L1, L4, L9, L12) and 2
isolates nitrifying bacteria (code N1, N6).
Surprisingly, the isolates bacteria with code N6 was
also able to inhibit (antagonism) to V. harveyi. All
isolates bacteria obtained can be sinergized with
each other.

[9] Mukred, A.M., Hamid, A,A., Hamzah, A.,

Yusoff, W.M.W., 2008, Development three
bacteria consortium for the bioremediasion of
crude petrolium-oil in contaminated water,
Online Journal of Biological Sciences, 8 : 7379.

Bacteria with the ability amilolytic (code A1),

lipolytic (code L9) and nitrification (code N6) also
have the ability as an antagonistic againts Vibrio
harveyi.

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Bioremediation And Vibrio harveyi Biocontrol (As Agent Vibriosis At Prawn)
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