Utilisation of Local Penicillium SPP. In Consortium with Bacillus SPP. As Bioremediators for Shrimp Culture

Shrimp aquaculture industry is suffering from severe disease outbreaks, environmental degradation and poor management practices. This project was undertaken to investigate the use of Penicillium isolates as bioremediation consortium with potential Bacillus spp. for economical and environmental-friendly clean-up of shrimp culture tank water, maintenance of good water quality, biocontrol against pathogenic vibrios and enhancement of shrimp production in shrimp hatchery with zero water exchange. Two potential Penicillium spp. S6 and S48 were originally isolated from sediment samples. S6 was collected from Sungai Dina while S48 was collected from Teluk Adang, Johor. The Penicillium isolates were identified up to genus level based on colony morphology and were coded as Penicillium sp. S6 and Penicillium sp. S48. The Penicillium species S6 and S48 showed no inhibitory effect towards B. pumilus, B. subtilis and B. licheniformis and no mycotoxins were detected when the isolates were run on thin-layer chromatography against vomitoxin, aflatoxin B1, B2, G1 and G2 standard. The S6 colony produced amylase enzymes while S48 produced four types of major extracellular enzymes viz., amylase, protease, lipase and gelatinase. In a preliminary biocontrol experiment using disc diffusion methods, S6 showed a significant inhibitory effect on the growth of the pathogenic vibrios tested. Both potential isolates passed the non-pathogenicity test against shrimp postlarvae (PL15). Preliminary ammonia reduction experiment showed that S6 in its mycelial form and S48 in the spore forms reduced the total ammonia nitrogen (TAN) concentration better in the flasks. A cocktail of microorganisms containing S6 and S48 could reduce ammonia significantly than other cocktails when combination of Penicillium spp. (S6 andS48) and Bacillus spp. was tested. Results revealed that a microorganism cocktail containing S6 reduced ammonia significantly higher (p<0.05) than other combination of isolates. Hatchery tanks containing PL 15 to 36 grown for 3 weeks and treated with combination of Penicillium spp. (S6 and S48) showed the highest survival rate (41.17%) compared to other treatments. The TAN concentration of the hatchery tank treated with a combination of Penicillium spp. (S6 and S48) with final concentration of 0.721 mgl-1 and tanks treated with Penicillium sp. S6 (final concentration 0.829 mgl-1) also showed significant reduction of TAN compared to control tanks (final concentration 2.153 mgl-1 ), at 21 days of growth. The PL grown in Penicillium sp. S6 tanks and microorganism cocktail tanks (Penicillium spp. and Bacillus spp.) showed better stress tolerance (90%) compared to other treatments and control tanks (67%). Vibrio counts were significantly lower in tanks treated with Bacillus spp. (p<0.05) compared to other treatments. In addition, the vibrio counts for tanks treated with Penicillium sp. S6 also shown significant reduction (p<0.05) and good specific growth rate (15.32%) compared to the control (11.41%). Results showed that selected Penicillium spp. satisfied the criteria to qualify as bioremediation agent in marine shrimp culture

Developments of lactic acid bacteria as probiotic for bacterial diseases control in aquaculture

In the recent decades, lactic acid bacteria (LAB) in aquatic organism have been one of the major interesting research subjects due to their various existence strains in fish microbiota. Moreover, LABs are sometimes abundant in the intestine of several fish species. Many recent papers indicated that several LAB strains are harmless and have been reported for beneficial effects on fish health. There was also converging evidence that led us for more research and findings regarding LAB as a promising probiotics in aquaculture. This article provides an overview of the variability of LAB in gastrointestinal (GI) tract of fish and the development of this species as probiotics. LAB was known able to colonise the gut, and has antagonistic activity against some fish pathogens. This harmless bactreriocin-producing strain may confer benefits in increasing disease resistance, improving nutrient digestibility and growth of the host animals. In addition, these strains may reduce the need of antibiotics usage in future aquaculture industry

The application of garlic (Allium sativum) peel on African catfish (Clarias gariepinus) against Aeromonas hydrophila infection

This study aim to investigate the efficacy of dietary doses of garlic (Allium sativum) peel on disease resistance and the duration of protection of African catfish (Clarias gariepinus) juveniles against Aeromonas hydrophila infection. African catfish juveniles were fed twice daily for 4 weeks with 20 gkg-1 of garlic peels incorporated into fish formulated diet. Meanwhile 0 g of peels was served as control. After 4 weeks of post feeding, 15 fish were randomly selected for challenge with 108 cell/mL of A. hydrophila. During the challenge the fish were not fed with dietary of garlic. The duration of protection was observed at 7, 14 and 21 days following infection. The results demonstrated that dietary garlic peels provided protection toward African catfish against A. hydrophila after 14 days post feeding and slightly reduced protection after 21 days of post feeding. Significantly higher survival rate were recorded in dietary garlic peels (66%, 86% and 42%) when compared with the control. The result indicated that garlic peels able to enhance disease resistant of African catfish towards A. hydrophila infection

Antibacterial activity of garlic extracts on fish pathogenic bacteria

Aqueous and methanol extracts of Allium sativum (clove and peel) were investigated for its in vitro antibacterial properties against four marine pathogens, Aeromonas hydrophila, Vibrio anguillarum, Vibrio alginolyticus, and Vibrio harveyi. Antibacterial activity of different concentrations of aqueous and methanol garlic extracts were evaluated based on the inhibition zone using disc-diffusion method, minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) values. The aqueous extract of A. sativum (clove and peel) had no antibacterial effect against the pathogenic bacteria tested whereas the clove extract of methanol had inhibitory effects on the growth of all pathogenic bacteria tested. The maximum zone of inhibition was observed in A. hydrophila and the minimum was observed in V. anguillarum and V. harveyi. The MIC and MBC values revealed that A. hydrophila was able to be inhibited by supernatant extract of clove at lower concentration (0.6 mg/ml) while other pathogens are inhibited at slightly higher concentration. This study suggests that clove extract of A. sativum has the potential to be used as phytobiotics in controlling the growth of marine pathogens

Evaluation of potential bacteria isolated from marine shellfish as probiotic for Penaeus monodon larviculture against pathogenic Vibrio harveyi

Probiotics have been widely used as an alternative treatment in aquaculture. This study was carried out to determine the ability of two potential probionts; Bacillus strain I24 and Exiguobacterium strain S66 in producing biofilm and reducing hemolysin production in two Vibriosp;Vibrio alginolyticusand Vibrio harveyi.Biofilm assay was carried out to determine the ability of the potential probionts in producing biofilm; a slimy compound that facilitates bacterial attachment. Results showed that all potential probionts able to produce biofilm. Potential probionts started to produce high biofilm formation at 40 hr. For probiont S66, the highest production was recorded at 60 hr, and started to decrease at 70 hr. Meanwhile for potential probiont I24, increased of biofilm formation started at 30 hr until 70 hr. Furthermore, V. harveyi and V. alginolyticushad lower biofilm formation compared with the potential probionts. Thus, results indicated that potential probionts S66 and I24 possibly able to compete with pathogen for adhesion sites hence colonize the host. Hemolysin assay was carried out to determine the ability of potential probionts in reducing hemolysin production that caused hemolysis, which was one of the virulence factor in Vibriosp.. Incubation of potential probionts and pathogen showed no reduction in hemolysin production

Extracellular enzyme production of Probiotic Bacillus JAQ04 and Micrococcus JAQ07 isolated from tiger grouper (Epinephelus fuscoguttatus)

Tiger grouper has been identified as a good market price and most desired in the live fish trade market species especially in Southeast Asia region. However, intensive grouper aquaculture often triggering diseases by bacteria pathogen that leads to infectious diseases. Thus, to hinder this infectious disease, promising probiotic bacteria successfully isolated from intestine Tiger grouper juvenile. Previous studies showed that this bacterium has been shown to produce antibacterial activity against pathogenic marine bacteria Vibrio alginolyticus. Thus, this study aims to further identify the characteristic of Probiotic Bacillus JAQ04 and Micrococcus JAQ07 in-vitro. In this study the bacteria were screened for their extracellular enzyme production of protease, amylase and lipase. These bacteria may play a role in inhibiting the pathogen by production of extracellular enzyme and improve the feed digestion. The enzymes were shown positive for protease, amylase and lipase which exhibit the clear zone on skim milk agar, starch agar and spirit blue agar. However, in vivo study needs to be done to further confirm the enzymatic activity of the isolates in inhibiting of pathogen and improve feed digestion

Probiotic strains for shellfish aquaculture: protection of eastern oyster, Crassostrea virginica, larvae and juveniles againsl bacterial challenge

Bacterial pathogens, including several Vibrio spp. and Roseovarius crassostreae, cause severe mortality of larval and juvenile eastern oysters. The introduction of beneficial bacterial isolates in oyster hatcheries and nurseries for the biocontrol of bacterial diseases is a good alternative to the use of antibiotics. The goal of this study was to screen and characterize marine bacterial isolates as potential agents to prevent larval and juvenile mortality by the oyster pathogens Vibrio tubiashii and R. crassostreae. Screening of bacterial isolates from Rhode Island marine organisms and environment using agar-based assay methods for detection of antimicrobial activity against oyster pathogens led to the isolation of candidate probionts Phaeobacter sp. S4 and Bacillus pumilus RI06-95. Pretreatment of larval and juvenile oysters for 24 h with 102–106 cfu/mL Phaeobacter sp. S4 or B. pumilus RI06-95 protected larval oysters against mortality resulting from challenge with R. crassostreae and V. tubiashii (relative percent survival (RPS) range, 9%–56%). These probiotics also protected juvenile oysters against challenge with V. tubiashii (RPS, 37%–50%). Probiotic isolates had no negative impact on oyster survival. Protection conferred to larvae against bacterial challenge was short-lived, lasting for only 24 h after removal of the probiotics from the incubation water. These results suggest the potential of marine bacterial isolates Phaeobacter sp. S4 and B. pumilus RI06-95 to serve as biocontrol agents to reduce the impact of bacterial pathogens in the culture of Crassostrea virginica

Screening and evaluation of local bacteria isolated from shellfish as potential probiotics against pathogenic Vibrios

The present study was carried out to isolate, screen and evaluate potential candidates of local bacteria isolated from tiger shrimp Penaeus monodon and slipper cupped oysters Crassostrea iredalei as probiotics in shellfish aquaculture. A total of 144 of bacteria were successfully isolated from the intestine and stomach of 20 tails of healthy adult tiger shrimp P. monodon, while 136 were successfully isolated from the digestive tract, gills and inner shells of 10 healthy adult C. iredalei. The number of potential isolates was narrowed down to two from tiger shrimp, and one from slipper cupped oyster after in vitro screening assays. The three isolates, labeled as G11, I24 and S66, were identified as Virgibacillus sp., Bacillus sp. and Exiquobacterium sp., respectively, using 16S rDNA gene analysis. The antagonistic ability of the isolates towards Vibrio alginolyticus and Vibrio harveyi were conducted in stagnant and liquid modes via spot lawn and broth co-culture assay, respectively. In these assays, all the potential probionts were inhibitory to both pathogenic vibrios. In the in-vivo assay, Artemia was used as host and treated with different concentrations of potential probionts (10⁴, 10⁶ and 10⁸ CFU ml⁻¹), and challenged with V. alginolyticus ana V. harveyiat 10⁵ CFU ml⁻¹ Respectively, Artemia treated with probiont Gl 1 at all concentrations and challenged with V. alginolyticus had increased survival (70-80 %), which was significantly higher as compared with group with only the pathogen (20 %). Meanwhile, probiont I24 increased the survival of Artemia by 70 % at a concentration of 10⁸ CFU ml⁻¹ after being challenged with V. alginolyticus and Artemia treated with 10⁶ CFU ml⁻¹ of probiont S66 had increased survival of 90% after being challenged with V. harveyi. Thus, the three isolates might have potential applications as probiotics in shellfish aquaculture against vibriosis

Characterization of Enterococcus hirae isolated from the intestine of Seabass (Lates Calcarifer) as a new potential probiotic against pathogenic vibrios

In this study, we aimed to isolate, identify and characterize lactic acid bacteria (LAB) from the intestine of juvenile seabass (Lates calcarifer) as a new potential probiotic. Four strains of LABs were isolated from the intestines of ten healthy seabass juveniles. In the in vitro screening process using spot lawn assay, one isolate labeled as LAB3 showed inhibitory activity against Vibrio harveyi (ATCC 35,084). Strain LAB3 was determined to belong to the gram positive bacteria group with cocci shape and was identified as Enterococcus hirae using 16S rDNA analysis. This bacterium was able to grow at pH ranging from pH 2 to 10 with the best growth at pH 7. This strain was also able to grow at 0-4% NaCl after 24 h incubation and grew best at 1.5% NaCl. Enterococcus hirae strain LAB3 of the present study is worthy to be further characterized as a potential probiotic for use in seabass culture

Evaluation of Enterobacter sp. strain G87 as potential probiont against Vibrio harveyi infection in Artemia nauplii and Asian seabass (Lates calcarifer) larvae

Probiotic has gained many interests as an alternative method in preventing and treating diseases in aquaculture. The benefits include improving feed value, inhibition of pathogenic microorganisms, anti-mutagenic and anti-carcinogenic activity, growth promoting factors, and increase host immune response. This research was carried out in order to evaluate the potential of probiont Enterobacter sp. G87 in conferring protection to Artemia and seabass larvae against Vibrio harveyi infection. In preliminary in vivo test, Artemia nauplii was treated with Enterobacter sp. G87 at three different concentrations 104, 106 and 108 CFU mL-1 and challenged with V. harveyi at 105 CFU mL-1. After challenged, significant increased survival was found in Artemia (78±2%) treated with 106 CFU mL-1 of Enterobacter sp. G87 compared with challenged group with no probiont added (48±2%). From the results, two concentrations of probiont (106 and 108 CFU mL-1) were selected to be used in seabass larvae in vivo challenge assay. After challenged with V. harveyi at 105 CFU mL-1 highest survival was found in seabass larvae treated with 106 CFU mL-1 of Enterobacter sp. G87 (95±3%). Additionally, Enterobacter sp. G87 was also able to reduce Vibrio counts both in Artemia and seabass larvae culture. This study showed that probiont Enterobacter sp. G87 was able to protect Artemia nauplii and seabass larvae from Vibrio harveyi infection and has a potential to be further studied in a larger scale