B003 a. Rofiq

3rd International Conferences and Workshops on Basic and Applied Sciences 2011 ISBN: 978-979-19096-1-7

B003

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*, Ni’matuzahroh, and Agoes Soegianto

*Department of Biology

Airlangga University, Surabaya, Indonesia e-mail: [email protected]

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 α – 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.

Keywords: Pond backish water, indigenous bacteria, decomposer bacteria, Vibrio harveyi, synergistic, antagonistic.

1 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

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)

B003

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).

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).

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 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.

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).

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.

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.

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 α – 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.

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.

3rd International Conferences and Workshops on Basic and Applied Sciences 2011 ISSN XXXX-XXXX

B003

Table 1: Hydrolysis index of three isolates that have ability to proteolytic

Hydrolysis index

Diameter No Isolates code Halo

(mm) Colonies

(mm) Hydrolysis

index

1 P1 15 11 0,36

2 P3 16 9 0,78

3 P4 35 29 0,21

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.

Table 2: Hydrolysis index of three isolates that have ability to amilolytic.

Hydrolysis index Diameter No Isolates

code Halo (mm)

Colonies (mm)

Hydrolysis index

1 P1 15 11 0,36 2 A3 18 7 1,57 3 A5 10 5 1,00


hydrolysis index amilolytic code A1, A3 and A5.

Bacteria potential lypolytic found four isolates. The data are presented in Table 3 and Figure 3.

Table 3. Hydrolysis index of four isolates that have ability to lypolytic.


code Halo (mm)

Colonies (mm)

Hydrolysis index

1 L1 13 9 0,44 2 L4 11 8 0,38 3 L9 10 6 0,67 4 L12 13 7 0,86


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

Potential nitrification bacteria found two isolates. The data are presented in Table 4.

Table 4: Hydrolysis index of four isolates that have ability to nitrification.


code Halo (mm)

Colonies (mm)

Hydrolysis index

1 N1 15 15 0,00 (Coloni is

red)

2 N6 35 35 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 No

Isolates code Halo

(mm) Colonies (mm)

Inhibitory zone(mm)

1 A1 8 6 2

2 L9 17 14 3


B003

3 N6 50 24 26

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.

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 0 - - - - - - A3 0 - - - - - P1 0 - - - - P3 0 - - - L9 0 - - L12 0 - N6 0

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

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).

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 Bergey’s manual of Determinative Bacteriology, (1994) this isolate near to Streptococcus sp.

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 Bergey’s manual of Determinative Bacteriology, (1994) this isolate suspected near to Lactobacillus sp.

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.

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 α – 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

3rd International Conferences and Workshops on Basic and Applied Sciences 2011 ISSN XXXX-XXXX

B003

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 Bergey’s manual of Determinative Bacteriology, (1994) this isolate near to Streptococcus sp.

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 that all isolates can be used as mixed consortium bacteria for water bioremediation candidates.

Prawn will be healthy if the environment is not polluted and is not attacked by the disease (Newman, 2000). Bacteria with antagonistic ability against V. harveyi is required to inhibit growth of V. harveyi (agent of vibriosis in prawn). Antagonistic mechanisms against other bacteria due to several factors, namely the production of antibiotics, bacteriocin, sidephores, lysozymes, proteases or produce organic acids (Verschuere et al, 2000). In this research was obtained three isolates with ability antagonistic againts V. harveyi with code A1, L9 and N6. Isolate with code N6 have the greatest antagonistic againts V. harveyi. From this results can be obtained information that the bacteria with the ability amilolytic, lipolytic and nitrification also have the ability as an antagonistic againts V. harveyi.

4 Conclusions

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.

Bacteria with the ability amilolytic (code A1), lipolytic (code L9) and nitrification (code N6) also have the ability as an antagonistic againts Vibrio harveyi.

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).

References

[1] Aditiawati,P.,Pikoli,M.R.,Indiani.D., 2001, Isolasi bertahap bakteri pendegradasi minyak bumi dari sumur bangko, Proceeding simposium nasional IATMI, Yogyakarta.

[2] Anonimous, 2009, Map of local economy potency, Jawa timur, www.cps-sss.org, 21 Desember 2010

[3] Bergey’s manual, 1994, Determinative bacteriology, 9th edition, Lippincott Williams & Wilkins, Philadelphia, USA.

[4] Dirjen perikanan budidaya, 2010, Periksa penyakit di laboratorium keliling, www.perikanan-budidaya.dkp.go.id/index.php?, 21 Desember 2010.

[5] Kannapiran.E, Ravindran .J, Chandrasekar. R, Kalaiarasi. A, 2009, Studies on luminous, Vibrio harveyi associated with shrimp culture system rearing Penaeus monodon, J. Environ. Biol., 30: 791-795

[6] Khasani,I.,2008, Aplikasi probiotik menuju sistem budidaya perikanan berkelanjutan, Probiotic aquaculture.blog.spot.com. 21 Maret 2010.

[7] Moriarty, D. J.W., 1999. Disease control in shrimp aquaculture with probiotic bacteria, Microbial interaction in aquaculture, Proceeding of the 8th International symposium on microbial ecology, Canada.

[8] Miyoshi, H., Handa, T., Date, S., 1981, Proteolytic and Lipolytic of marine bcteria from Uranouchi Bay, with special reference to the decomposition of dead organisms, Bulletin of Japanese Sosiety of Scientific Fisheries, 47 : 1361-1366.

[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 : 73-79.

[10] Murtiati, Simbolon. K., Wahyuni, T., Juyana, 2007, Penggunaan biokatalisator pada budidaya udang galah, Jurnal budidaya air tawar, 4 : 19-26.


B003

[11] Newman, S.G. 2000. Management and Prevention of Stress in Aquaculture with a focus on Farmed Shrimp. Fourth Latin American Aquaculture Congress and Seafood Trade Show, Oct 25-28, 2000.

[12] Rattanachuay,P.,Kantachote,D.,Tantirungkij, M., Nitoda, T., Kanzaki, H., 2010, Inhibion of shrimp pathogenic vibrios by extracellular compounds from a proteolytic bacterium Pseudomonas sp. W3, Electronic Journal of Biotechnology, 13 : no. 1.

[13] Setyaningsih, I., Panggabean, L.M., Riyanto, B., Nugraheny, N., 2006, Potensi antibakteri diatom laut Skeletonema costatum terhadap bakteri Vibrio sp. Bulletin Teknologi Hasil Perikanan, Vol IX Nomor 1

[14] Simpedal Kabupaten Gresik, 2010, Potensi kelautan dan perikanan Kabupaten Gresik, 203.77.237.21/einvest/homepage/3525/.../0/investasi.htm, 21 Desember 2010

[15] SITH, 2011, Teknologi produksi bahan baku pakan, Slide presentasi, ITB, Bandung,www.sith.itb.ac.id/.../bahan.../2_Teknologi%20Produksi%20Bahan%20Baku%20Pakan, 24 Juni 2011.

[16] Sohail, M., Ahmad, A., Shahzad, S., Khan, S.A., 2005, A survey of amylolytic bacteria and fungi from native environmental samples, Pak. J. Bot., 37 : 155-161.

[17] Soundarapandian, P., Babu, R.,2010, Effect of probiotic on the hatchery seed production of black tiger shrimp, Penaeus monodon (Fabricius), Int. J. Anim, Veter, Adv., 2 : 9-15.

[18] Verchuere. L., Rombaut, G., Sorgeloos, P., Verstraete, W., 2000, Probiotic bacteria as biological control agent in aquaculture, Microbiol. Mol. Biol. 64 : 655-671.

[19] Vine, G. N, 2004, Toward the development of protocol for the selection of probiotics in marine fish larviculture, Doctoral Thesis, Rhodes University, Shouth Africa.

Documents

B003 a. Rofiq