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Culture conditions improvement of Crassostrea gigas using a potential probiotic Bacillus sp strain
Accepted Manuscript Culture conditions improvement of Crassostrea gigas using a potential probiotic Bacillus sp strain Kais Fdhila, Najla Haddaji, Ibtissem Chakroun, Amel Dhiaf, Mohammed Ezz Edine Macherki, Bochra Khouildi, Faouzi Lamari, Kamel Chaieb, Nabil Abid, Hajer Marzougui, Sadok Khouadja, Hechmi Missaoui PII:
S0882-4010(16)30899-3
DOI:
10.1016/j.micpath.2017.07.017
Reference:
YMPAT 2355
To appear in:
Microbial Pathogenesis
Received Date: 18 December 2016 Revised Date:
10 July 2017
Accepted Date: 10 July 2017
Please cite this article as: Fdhila K, Haddaji N, Chakroun I, Dhiaf A, Edine Macherki ME, Khouildi B, Lamari F, Chaieb K, Abid N, Marzougui H, Khouadja S, Missaoui H, Culture conditions improvement of Crassostrea gigas using a potential probiotic Bacillus sp strain, Microbial Pathogenesis (2017), doi: 10.1016/j.micpath.2017.07.017. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Culture conditions improvement of Crassostrea gigas using a potential probiotic
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Bacillus sp strain
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Kais Fdhilaa, Najla Haddaji*a, Ibtissem Chakrouna, Amel Dhiafa, , Mohammed Ezz Edine
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Macherkia, Bochra Khouildib., Faouzi Lamaria, Kamel Chaieba, Nabil Abida, Hajer
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Marzouguib, Sadok Khouadjaa, Hechmi Missaouib.
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a
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Monastir.
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b
Fisheries Laboratory, Higher Institute of Agriculture Tunis.
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Laboratory of Analysis, Treatment and Valorization of pollutants Faculty of Pharmacy of
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⁎ Corresponding author:
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Najla Haddaji
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Laboratory of Analysis, Treatment and Valorization of Pollutants of the Environment and
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Products (LATVPEP).
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Faculty of Pharmacy, University of Monastir. Monastir 5000. Tunisia
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Tel.: (+216)73466244
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Fax: (+216) 73461830
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E-mail address: [email protected].
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ACCEPTED MANUSCRIPT Abstract
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It is well demonstrated that some probiotics improve rearing water quality and thereby have
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beneficial effects on reared organisms. We conducted this study to determine the effect of
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Bacillus consortium on Crassostrea gigas reared in contemned seawater with indigo dye
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priory treated with Bacillus or no treated. This effect was studied by assessing hemocytes
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death using flow cytometry analysis. We found that the percentage of decolorization of
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indigo dye in polluted seawater in presence of C. gigas increased from 41% to 90% when
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using Bacillus consortium. In these conditions, the hemocytes mortality of reared C. gigas
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decreased from 87% to 56%. We have demonstrated also that seawater contemned with
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priory treated indigo with Bacillus consortium is less toxic than seawater contemned with
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the no treated indigo. The percentage of hemocytes death is 81% for the contemned seawater
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with indigo and 56% for no contemned seawater. This consortium shows a protector effect
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of C. gigas against Vibrio harveyi contemning reared seawater.
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Key words: Crassostrea gigas, Bacillus consortium, indigo.
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1. Introduction Most of pollutants are thrown out on soil and get washed off during monsoon months and
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are thereby conducted to lagoons and coastal seawater. They lead to ecological and sanitary
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problems such as the decrease of aquatic biodiversity and the intoxication of aquatic
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organisms particularly filter feeders like oysters. The world annual production of dyes is
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estimated at more than 80000 tons used mainly in food industries, cosmetics, paper mills
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and especially in textile industries which absorb alone more than 70% of the produced total
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quantity [1]. The intense use of the synthetic dyes in these various industries generates
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sources of considerable pollution of the environment. Indeed, 10 to 15% of the quantity of
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dyes used is rejected into the natural environment [2]. These dyes, in addition to dangerous
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pollution that they can generate, constitute because of their toxicity a potential danger to
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both humans and environment [3]. Since the very early stages of the history of mankind,
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several natural dyestuffs have been used. In fact, the art of dyeing with indigo dates back
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several thousand years. The Indigo is classified as strongly toxic dyes [4]. The indigo can
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cause a major problem for aquaculture in general and particularly in Crassostrea gigas.
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The Pacific oyster Crassostrea gigas is one of the most important cultured species, with a
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high value and strong market demand [5]. Oyster farming practices usually include
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numerous transfers of oysters at all life stages, especially of spat and juveniles grown in
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hatchery and nursery systems before being transferred to oyster farming areas in open
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seawater [6]. Along with these farming practices, oysters must adapt to new abiotic and
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biotic environments. Biotic changes include interactions with new, potentially pathogenic or
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toxic microorganisms that infect oysters through the feeding and filtration process. The most
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important factor affecting aquaculture is the incidence of microbial pathologies, mainly
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bacterial in origin. Vibrio harveyi, and Vibrio anguillarum are most frequently isolated
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marine Vibrio species [7] [8], having been associated with large-scale losses of larval and
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juvenile penaeids [9] and also causing several opportunistic diseases to fishes [10] [11].
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Vibrios are gram-negative, ubiquitous in marine and estuarine ecosystems as well as
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aquaculture farms, and comprise one of the major microbiota of these ecosystems. Many
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vibrios are serious pathogens for animals reared in aquaculture [12] [13]. Vibriosis, caused
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by infection by Vibrio spp, is one of the most prevalent diseases in fishes and other
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aquaculture-reared organisms and is widely responsible for mortality in cultured aquaculture
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systems worldwide [14] [15].
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Disease outbreaks in shellfish aquaculture are managed using methods such as disease
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avoidance, frequent water changes, good husbandry, and the use of immune-stimulants and
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disease control. However, because of the emergence of antibiotic resistance and concerns
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about environmental pollution, alternatives to the use of antibiotics are needed [17], [18].
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One of these alternative methods is the use of nonpathogenic microorganisms called
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probiotics. Bacillus sp. is widely used as probiotic in aquaculture but it is not autochthonous
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in gastrointestinal tract. However, it may be active during intestinal transit [19]. In a recent
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study, two marine bacteria, Bacillus pumilus RI0695 and Phaeobacter gallaeciensis were
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previously reported to provide significant protection of the Eastern oyster larvae
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Crassostreae virginica when challenged with the shellfish pathogen Vibrio tubiashii [20].
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Additionally, Bacillus sp. had been shown to have health benefits for Dicentrachus labrax
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larvaes [21]. Probiotics have been tested successfully in shellfish culture. Chniti et al. (2003)
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[22] isolated two yeasts and one bacterial strain (designated SS1, AY1, and SY9, resp.) from
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the digestive tract of abalone (Haliotis midae). A diet was formulated with a mixture of the
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three putative probiotics. Each probiont was added to the feed to achieve a final
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concentration of approximately 107 cells g−1 of dry feed. The growth rate of small (20 mm)
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and large (67 mm) abalone was improved by 8% and 34%, respectively, in eight months
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cultures. Furthermore, abalones supplemented with probiotics had a survival rate of 62% to
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the pathogenic bacterium Vibrio anguillarum compared to 25% survival of untreated
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animals. Another study describes that the daily application of Phaeobacter inhibens and B.
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pumillus RI06-95 mixed with algal feed to culture tanks in the hatchery increased survival of
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oyster larvae to experimental challenge with V. coralliilyticus RE22 [23]. Probiotic
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microorganisms have the ability to release chemical substances with bactericidal or
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bacteriostatic effect on pathogenic bacteria that are in the intestine of the host, thus
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constituting a barrier against the proliferation of opportunistic pathogens. In general, the
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antibacterial effect is due to one or more of the following factors: production of antibiotics,
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bacteriocins, siderophores, enzymes (lysozymes, proteases) and/or hydrogen peroxide, as
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well as alteration of the intestinal pH due to the generation of organic acids [18]. According
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to these data, the genus Bacillus is one of dominant probiotics, which was commonly used in
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aquaculture. More and more studies confirmed that endogenous or exogenous Bacillus
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strains could be effective in improving growth, immunity and disease resistance of shellfish
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[24] [25] [20].
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This study thus investigated the possible protection effect of Bacillus sp for Crassostrea
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gigas reared in indigo polluted seawater and infected with Vibrio Harvey.
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2. Materials and methods 4
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2.1.Samples collection and bacterial characterization Three Bacillus strains, isolated from Tunisian hypersaline environments in a previous study
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in our laboratory [26], are used in this study as Bacillus consortium. Sequencing analyses
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showed that these bacteria corresponded to the genus Bacillus sp such as Bacillus subtilis, B.
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cereus, B. coagulans [26]. These strains were preserved in our laboratory and their purity
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was routinely checked. Stock cultures were stored at 80°C in powdered skimmed milk
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suspension with 25% glycerol. Bacillus strains were isolated, in this study, on nutrient plates
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and inoculated in nutrient broth prepared with seawater. It was incubated for 24 hours at
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20°C [27].
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Vibrio harveyi ATCC 14126 was routinely cultured in marine broth. It is used for control as
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positive pathogen.
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2.2.Screening and identification of micro-organisms with bioremediation activity
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Indigo was submitted to decimal dilution in a mineral liquid medium (MLM) prepared as
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described previously by [28] up to a concentration of 1.75 mg/ml.
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Isolated bacteria from sludge and wastewater of industrial textile plant were inoculated in
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flasks containing MlM medium with 1.75 mg/ml indigo dye concentration. Then flasks were
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incubated at 37°C under shaking conditions (120 rpm) in a rotary shaker. Samples were
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collected at different time to determine decolorization. Optical densities (OD) were
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determined using a spectrophotometer, Shimadzu UV-240I PC model Kyoto, Japan) at the
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max λ max 664 nm. The percentage of decolorization was calculated as following:
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decolorization (%) = [absorbance at t0 – absorbance at t1] * 100/ absorbance at t0.
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Crassortrea gigas were purchased from a rearing plant in a lagoon after an acclimatization
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period of one week at 25°C in aquariums containing 10 liters of seawater. Experiments were
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carried out in six aquariums, each containing six specimens.
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Aquarium 1 : containing C. gigas and seawater; Aquarium 2: containing C. gigas, seawater
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and indigo (1.74 mg/ml); Aquarium 3: containing C. gigas, seawater and filtrate from a
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culture of Bacillus consortium where indigo was degraded; Aquarium 4: Same content as
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the second aquarium but reared seawater is inoculated with Bacillus consortium (10 ml of
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Bacillus suspension 106 CFU/ml); Aquarium 5: seawater inoculated with Bacillus
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consortium (10 ml of Bacillus suspension 106 CFU/ml); Aquarium 6: the same content as
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the fifth aquarium but oysters are infected with V. harveyi ATCC 14126; Aquarium 7: the
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same content as the sixth aquarium but oysters are infected with V. harveyi ATCC 14126and
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added with Bacillus consortium (10 ml of Bacillus suspension 106 CFU/ml).
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2.4.Hemolymph collection C. gigas was collected from the lagoon of Bizerte (Tunisia). It is polluted with industrial and
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domestic wastewater.
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Hemolymph was collected from the adductor muscle following Chen method (1996). A
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notch was filed on the dorsal side of the shell valve, adjacent to the adductor muscle. 2ml of
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hemolymph were collected aseptically from each animal.
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A total of 10 000 hemocytes were analyzed using a flow cytometer BD FAC Caliber
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coupled with a Power Mac G3PC equipped with a Cell Quest Pro software
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2.5.Hemocytes viability assay
150 µl of hemolymph was mixed with 150 µl of an antiaggregant solution (AASH) and 5 ml
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of propidium iodide 1mg/l (PI) (Sigma) at a final concentration of 20µg/ml. The Propodium
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iodide binds to DNA double stranded and absorbs at wavelength above 630 nm. It enters and
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stains the cells but can’t cross the membrane of viable cells. Tubes were incubated with PI
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and AASH for 10 minutes at 20°C before analysis. Dead hemocytes are more fluorescent in
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flow cytometer’s orange light detector.
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We evaluate the percentage of dead heamocytes in proportion to the total number. 2.6. Statistical analysis
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Past 3.05 and Graphpad prism 6.05 softwares are used for statistical analysis and graphics.
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For all tests we used hypothetical risk α= 0.05 for alternative hypothesis acceptance. Bar
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chartwas plotted with standard deviation show.
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3. Results
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3.1.Screening and identification of bacterial strains decolorizing indigo dye
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More than 1500 biomasses from textile wastewater and sludge were tested for their ability to
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decolorize diluted indigo dye. Active strains associated with these biomasses were
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identified. Gram negative rods shape bacteria and some Gram positive bacteria were
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selected. Gram negative rods as Pseudomonas stutzeri ; Pseudomonas testosteroni;
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Pseudomonas
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Pseudomonas aeruginosa; Sphingomonas paucimobilis; Aeromonas hydrophila. And for
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Gram positive bacteria, we identified Staphylococcus epidermidi and Bacillus subtilis.We
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have been interested to Bacillus sp strains.
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vesicularis ;
Pseudomonasmaltophilia ;
Pseudomonas
acidovorans;
3.2.Biodegradation of indigo by Bacillus consortium
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The percentage of decolorization of indigo dye in polluted seawater in presence of C. gigas
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increased from 41% to 90% (Table 1, Aquarium 4) when using Bacillus consortium. These
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bacteria can survive in seawater because of their sporulation characteristics. In the absence 6
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indigo is 41% (Table 1, Aquarium 2).
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The Bacillus consortium has also improved the seawater quality of the aquarium
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contaminated with the previous product of indigo degradation: In aquarium 3, the percentage
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of decolorization was 74 %. Treated C. gigas with indigo in aquarium 2 accumulated this
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dye in its gills. This can have a harmful effect on consumers’ health. Table1 shows optical
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density variation of indigo in reared seawater of C.gigas under different conditions.
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3.3.Crassostrea gigas hemocytes protection by Bacillus consortium
Rearing seawater of the aquarium 1 and 5 is not contaminated with indigo dye so a low
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mortality of haemocytes was obtained in the aquarium1 and no mortality in the aquarium 5.
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We found that the indigo dye is very toxic for C. gigas and the mortality of haemocytes
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increased in the aquarium 2 c contaminated with diluted indigo from1.2 aqua. (1) up to 87%
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(aqua.2).
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Rearing seawater contaminated with treated indigo using Bacillus consortium is less toxic to
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C. gigas than that contaminated with untreated indigo. When added to contemned seawater
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with indigo, Bacillus consortium decreased the hemocytes mortality of reared C. gigas from
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87% to 56% (fig.1: Aquarium 2 and 4). It has also a protecting effect against pathogenic
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bacteria V. harveyi ATCC 14126. Less mortality is obtained in the aqu.6 containing Bacillus
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consortium than in the aqua.7. 4. Dicussion
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Effluent from the textile industry is loaded of non-biodegradable dyes which make them
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difficult to apply biological treatments. Several techniques have been employed to remove
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dyes from wastewater. These effluents have an impact on aquaculture. Aquaculture is a fast-
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growing food production sector and the use of probiotics for the improvement of aquatic
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environmental quality as major areas for further research in disease control in aquaculture
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[29], which would help to reduce the use of antimicrobial agents. However, the addition of
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probiotics is now also a common practice in commercial shrimp hatcheries in Mexico [30].
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The most studied probiotics are usually Bacillus and Lactobacillus species [31]. It has been
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reported that use of Bacillus sp. improved water quality, survival and growth rates, increased
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the health status of juvenile Penaeus monodon and reduced the pathogenic vibrios [32].
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Indeed, the probiotic effect of Bacillus sp was reported under in vitro and in vivo condition
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[33].
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Bacillus strains isolated from lagoons can survive in seawater because of their tolerance to
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high pH. It was found that they prefer pH>8.9. They also tolerate high salinity because of
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ACCEPTED MANUSCRIPT their origin: they were isolated from lagoons having a high salinity level [26]. These bacteria
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can survive in seawater because of their sporulation characteristics. This Bacillus
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consortium is as active as rot white fungi [34] but fungi need low pH and special nutrients to
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grow. Experiences conducted by von Verschuer (2000) [35] to improve seawater quality
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using Bacillus, had shown that they can easily multiply in seawater. So, Bacillus strains can
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be used in rearing media of aquatic organisms like filter feeds. When added to rearing water
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and is generally more effective in converting organic matter back to CO2 than Gram negative
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bacilli. Venkateswara et al.(2007) [36] had reported that microorganisms with probiotic
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effect can regulate microflora and improve the chemical quality of the water by
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decomposing pollutants, minimizing the toxic gases like NH3, N2O, H2O2. According to
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Kolndadacha et al. (2011) [37], Bacillus consortium improves water quality and increases
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heamocytes viability from 56% to 87% when added to rearing water of C.gigas. It can be
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considered as potential probiotic in rearing water of aquatic organisms. The percentage of
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decolorization of indigo dye in polluted seawater in presence of C. gigas increased from
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41% to 90% (Table 1, Aquarium 4) when using Bacillus consortium. Our results showed
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that indigo biodegradation products are harmless for C. gigas and decrease haemocytes
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mortality. Bacillus consortium is efficient in seawater control when added to the rearing
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medium. Mahdhi at al. (2007) [26] found similar results during Dicentrarchus labrax
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treatment by Bacillus. Bacillus strains isolated and used in this study may have an
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interesting antagonistic activity against pathogens. They have biomedical and antibacterial
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effects. In addition, they can be easily used in rearing seawater areas. These sporuling
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bacteria can resist to hostile conditions. In this investigation, we have shown that Bacillus
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sp. strains isolated from industrial textile waste water are able to degrade indigo. The
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optimization of their biodegradation activity in mixture showed that they need high pH and
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can be used in seawater. Experiments performed in rearing seawater of C. gigas with
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previously biodegraded indigo product water condition using Bacillus biomass show that
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indigo is more toxic for C. gigas and leads to higher haemocytes mortality rates than water
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contaminated with previously biodegraded indigo product.
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These strains have no toxic effect on C. gigas. No haemocytes mortality occurred in our
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assays. Similar results were found by Mahdhi et al. (2010) [26]. Bacillus consortium
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selected in our study, showed an antagonism effect against fish and shellfish pathogens [26].
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These results are in concordance with previous studies showed that B. cereus and B.
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thuringiensis mixture treatment resulted in survival rate of 100%, compared with 75% in the
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treatment without Bacillus. Authors concluded that B. cereus and B. thuringiensis have
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Furthermore, it was observed that the addition of B. subtilis to the rotifer culture water
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resulted in a significant increase in rotifer numbers and a reduction of Vibrio levels. B.
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subtilis appears to be a promising probiotic for rotifer cultures [39].
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Uncontrolled effect of antibiotics, their bad use and their high coast lead us to us to propose
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Bacillus consortium as an alternative substitute to antibiotics [40]. Several commercial
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consortia of probiotics (Bacillus or nitrifying bacteria) such as "Biostar"; BRF-1A; BRF 13
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A; Pond PrO-Vc are used to improve water quality and animal health. Experiments
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performed in C. gigas rearing seawater show that indigo is more toxic for C.gigas and lead
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to a higher haemocyte mortality rate than water contaminated with priory biodegradated
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indigo product. Bacillus consortium added to contaminated seawater with indigo improves
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seawater quality and results in a lower hemocytes mortality rate. It was reported that a
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commercially prepared bacterial mixture of Bacillus spp. mixed into the rearing water
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increased survival and production of channel catfish (Ictalurus punctatus) [33]. Currently
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there are no commercially available probiotics for shellfish aquaculture. Recent study
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describes efforts to create a stable formulation of B. pumilus RI0695 for delivery at shellfish
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hatcheries [20] [23]. Some effort to create a lyophilized probiotic formulation of P.
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gallaeciensis that provides reduced mortality of C. virginica larvae when exposed to the
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shellfish pathogen V. tubiashii RE22 [20].
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In this investigation, we have shown that three strains of Bacillus sp. isolates are able to
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degrade indigo.
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Experiments performed in rearing seawater showed that Bacillus consortium added to
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contaminated seawater with indigo dye or with Vibrio harvey improves seawater quality and
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results in lower haemocytes mortality rates.
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Rearing seawater contemned with indigo product is more toxic for C. gigas and leads to
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higher haemocyte mortality rates than water contaminated with previously biodegraded
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indigo product.
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Conflict of interest
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No conflicts of interests exist.
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Acknowledgement
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We thank all the members of both laboratories for their collaboration.
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References
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[22] Macey B, Coyne V. Improved growth rate and disease resistance in farmed Haliotis midae through probiotic treatment. Aquaculture. 2005;245:249-61. [23] Sohn S. Evaluation of the efficacy of candidate probiotics for disease prevention in shellfish hatcheries: University of Rhode Island; 2016. [24] Karim M, Zhao W, Rowley D, Nelson D, Gomez-Chiarri M. Probiotic Strains for Shellfish Aquaculture: Protection of Eastern Oyster, Crassostrea virginica, Larvae and Juveniles Againsl Bacterial Challenge. Journal of Shellfish Research. 2013;32:401-8. [25] Nemutanzhela ME, Roets Y, Gardiner N, Lalloo R. The use and benefits of Bacillus based biological agents in aquaculture. Sustainable Aquaculture Techniques: Intech; 2014. [26] Mahdhi A, Harbi B, Esteban MÁ, Chaieb K, Kamoun F, Bakhrouf A. Using mixture design to construct consortia of potential probiotic Bacillus strains to protect gnotobiotic Artemia against pathogenic Vibrio. Biocontrol science and technology. 2010;20:983-96. [27] Cabo M, Murado M, González MP, Pastoriza L. A method for bacteriocin quantification. Journal of applied microbiology. 1999;87:907-14. [28] Chniti N, Chriaa J, Dhief A, Bakhrouf A. Biodegradation of indigo and textile wastewater by acclimated biomasses. Journal Européen d'Hydrologie (France). 2003. [29] Subasinghe R. Fish health and quarantine. FAO Fisheries circular. 1997:45-9. [30] Rico-Mora R, Voltolina D, Villaescusa-Celaya JA. Biological control of Vibrio alginolyticus in Skeletonema costatum (Bacillariophyceae) cultures. Aquacultural Engineering. 1998;19:1-6. [31] Cordero H, Esteban MÁ, Cuesta A. Use of probiotic bacteria against bacterial and viral infections in shellfish and fish aquaculture. Sustain Aquac Tech. 2014;8:23. [32] Dalmin G, Kathiresan K, Purushothaman A. Effect of probiotics on bacterial population and health status of shrimp in culture pond ecosystem. Indian journal of experimental biology. 2001;39:939-42. [33] Balcázar JL, De Blas I, Ruiz-Zarzuela I, Cunningham D, Vendrell D, Múzquiz JL. The role of probiotics in aquaculture. Veterinary microbiology. 2006;114:173-86. [34] Gomaa EZ. Chitinase production by Bacillus thuringiensis and Bacillus licheniformis: their potential in antifungal biocontrol. The Journal of Microbiology. 2012;50:103-11. [35] von Verschuer C. Looking from within and without: Ancient and Medieval External Relations. Monumenta Nipponica. 2000;55:537-66. [36] Chandregowda V, Venkateswara Rao G, Chandrasekara Reddy G. Convergent approach for commercial synthesis of gefitinib and erlotinib. Organic Process Research & Development. 2007;11:813-6. [37] Kolndadacha O, Adikwu I, Okaeme A, Atiribom R, Mohammed A, Musa Y. The role of probiotics in aquaculture in Nigeria-a review. Continental Journal of Fisheries and Aquatic Science. 2011;5:8-15. [38] Masitoh MM, Hariati A, Fadjar M. Antimicrobial Activity of Bacillus cereus and Bacillus thuringiensis on Pathogenic Vibrio harveyi in Litopenaeus vannamei. 2016. [39] Murillo I, Villamil L. Bacillus cereus and Bacillus subtilis used as probiotics in rotifer(Brachionus plicatilis) cultures. Journal of Aquaculture Research & Development. 2011. [40] Rodgers C, Furones M. Antimicrobial agents in aquaculture: practice, needs and issues. Options Méditerranéennes. 2009;86:41-59.
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ACCEPTED MANUSCRIPT Table1 optical density variation of indigo in reared seawater of C.gigas under different conditions: Aquarium 2: C. gigas, seawater and indigo (1.74 mg/ml); Aquarium 3: C. gigas, seawater and previously degraded indigo using Bacillus consortium; Aquarium 4: Same content as the second aquarium but inoculated with Bacillus
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consortium (10 ml of Bacillus suspension 1012 CFU/ml).
Day1
Day2
Aquarium 2
0.1200±0,003
0.0700±0.003
Aquarium 3
0.0270±0,001
0.0070±0.001
74.0854±1,470
Aquarium 4
0.1260±0,003
0.0129±<0.001
89.7552±0,144
41.4377±4,550
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Decolorization
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Fig. 1 : Percentage of decolorization of reared seawater under different conditions: Aquarium 2: C. gigas, seawater and indigo (1.74 mg/ml); Aquarium 3: C. gigas, seawater and previously degraded indigo using Bacillus consortium; Aquarium 4: Same content as
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the second aquarium but inoculated with Bacillus consortium (10 ml of Bacillus
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suspension 1012 CFU/ml). Bars represent the SD for each point.
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Fig. 2: Bacillus Protection of heamocytes of Crassostrea gigas reared in seawater
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contaminated with indigo. 1; 2;....7: Aquarium number. Bars represent the SD for each point.
ACCEPTED MANUSCRIPT HIGHLIGHTS •
Bacillus sp used as safe probiotic
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Vibrio harveyi is widespread in aquaculture
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Indigo is a strongly toxic dyes
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Bacillus sp had an effect on immunity and resistance to Vibrio harveyi of Crassostrea gigas reared in
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indigo polluted seawater
S0882-4010(16)30899-3
DOI:
10.1016/j.micpath.2017.07.017
Reference:
YMPAT 2355
To appear in:
Microbial Pathogenesis
Received Date: 18 December 2016 Revised Date:
10 July 2017
Accepted Date: 10 July 2017
Please cite this article as: Fdhila K, Haddaji N, Chakroun I, Dhiaf A, Edine Macherki ME, Khouildi B, Lamari F, Chaieb K, Abid N, Marzougui H, Khouadja S, Missaoui H, Culture conditions improvement of Crassostrea gigas using a potential probiotic Bacillus sp strain, Microbial Pathogenesis (2017), doi: 10.1016/j.micpath.2017.07.017. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Culture conditions improvement of Crassostrea gigas using a potential probiotic
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Bacillus sp strain
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Kais Fdhilaa, Najla Haddaji*a, Ibtissem Chakrouna, Amel Dhiafa, , Mohammed Ezz Edine
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Macherkia, Bochra Khouildib., Faouzi Lamaria, Kamel Chaieba, Nabil Abida, Hajer
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Marzouguib, Sadok Khouadjaa, Hechmi Missaouib.
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a
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Monastir.
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b
Fisheries Laboratory, Higher Institute of Agriculture Tunis.
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Laboratory of Analysis, Treatment and Valorization of pollutants Faculty of Pharmacy of
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⁎ Corresponding author:
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Najla Haddaji
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Laboratory of Analysis, Treatment and Valorization of Pollutants of the Environment and
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Products (LATVPEP).
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Faculty of Pharmacy, University of Monastir. Monastir 5000. Tunisia
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Tel.: (+216)73466244
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Fax: (+216) 73461830
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E-mail address: [email protected].
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ACCEPTED MANUSCRIPT Abstract
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It is well demonstrated that some probiotics improve rearing water quality and thereby have
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beneficial effects on reared organisms. We conducted this study to determine the effect of
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Bacillus consortium on Crassostrea gigas reared in contemned seawater with indigo dye
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priory treated with Bacillus or no treated. This effect was studied by assessing hemocytes
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death using flow cytometry analysis. We found that the percentage of decolorization of
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indigo dye in polluted seawater in presence of C. gigas increased from 41% to 90% when
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using Bacillus consortium. In these conditions, the hemocytes mortality of reared C. gigas
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decreased from 87% to 56%. We have demonstrated also that seawater contemned with
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priory treated indigo with Bacillus consortium is less toxic than seawater contemned with
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the no treated indigo. The percentage of hemocytes death is 81% for the contemned seawater
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with indigo and 56% for no contemned seawater. This consortium shows a protector effect
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of C. gigas against Vibrio harveyi contemning reared seawater.
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Key words: Crassostrea gigas, Bacillus consortium, indigo.
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1. Introduction Most of pollutants are thrown out on soil and get washed off during monsoon months and
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are thereby conducted to lagoons and coastal seawater. They lead to ecological and sanitary
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problems such as the decrease of aquatic biodiversity and the intoxication of aquatic
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organisms particularly filter feeders like oysters. The world annual production of dyes is
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estimated at more than 80000 tons used mainly in food industries, cosmetics, paper mills
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and especially in textile industries which absorb alone more than 70% of the produced total
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quantity [1]. The intense use of the synthetic dyes in these various industries generates
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sources of considerable pollution of the environment. Indeed, 10 to 15% of the quantity of
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dyes used is rejected into the natural environment [2]. These dyes, in addition to dangerous
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pollution that they can generate, constitute because of their toxicity a potential danger to
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both humans and environment [3]. Since the very early stages of the history of mankind,
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several natural dyestuffs have been used. In fact, the art of dyeing with indigo dates back
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several thousand years. The Indigo is classified as strongly toxic dyes [4]. The indigo can
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cause a major problem for aquaculture in general and particularly in Crassostrea gigas.
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The Pacific oyster Crassostrea gigas is one of the most important cultured species, with a
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high value and strong market demand [5]. Oyster farming practices usually include
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numerous transfers of oysters at all life stages, especially of spat and juveniles grown in
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hatchery and nursery systems before being transferred to oyster farming areas in open
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seawater [6]. Along with these farming practices, oysters must adapt to new abiotic and
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biotic environments. Biotic changes include interactions with new, potentially pathogenic or
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toxic microorganisms that infect oysters through the feeding and filtration process. The most
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important factor affecting aquaculture is the incidence of microbial pathologies, mainly
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bacterial in origin. Vibrio harveyi, and Vibrio anguillarum are most frequently isolated
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marine Vibrio species [7] [8], having been associated with large-scale losses of larval and
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juvenile penaeids [9] and also causing several opportunistic diseases to fishes [10] [11].
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Vibrios are gram-negative, ubiquitous in marine and estuarine ecosystems as well as
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aquaculture farms, and comprise one of the major microbiota of these ecosystems. Many
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vibrios are serious pathogens for animals reared in aquaculture [12] [13]. Vibriosis, caused
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by infection by Vibrio spp, is one of the most prevalent diseases in fishes and other
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aquaculture-reared organisms and is widely responsible for mortality in cultured aquaculture
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systems worldwide [14] [15].
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Disease outbreaks in shellfish aquaculture are managed using methods such as disease
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avoidance, frequent water changes, good husbandry, and the use of immune-stimulants and
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disease control. However, because of the emergence of antibiotic resistance and concerns
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about environmental pollution, alternatives to the use of antibiotics are needed [17], [18].
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One of these alternative methods is the use of nonpathogenic microorganisms called
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probiotics. Bacillus sp. is widely used as probiotic in aquaculture but it is not autochthonous
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in gastrointestinal tract. However, it may be active during intestinal transit [19]. In a recent
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study, two marine bacteria, Bacillus pumilus RI0695 and Phaeobacter gallaeciensis were
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previously reported to provide significant protection of the Eastern oyster larvae
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Crassostreae virginica when challenged with the shellfish pathogen Vibrio tubiashii [20].
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Additionally, Bacillus sp. had been shown to have health benefits for Dicentrachus labrax
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larvaes [21]. Probiotics have been tested successfully in shellfish culture. Chniti et al. (2003)
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[22] isolated two yeasts and one bacterial strain (designated SS1, AY1, and SY9, resp.) from
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the digestive tract of abalone (Haliotis midae). A diet was formulated with a mixture of the
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three putative probiotics. Each probiont was added to the feed to achieve a final
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concentration of approximately 107 cells g−1 of dry feed. The growth rate of small (20 mm)
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and large (67 mm) abalone was improved by 8% and 34%, respectively, in eight months
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cultures. Furthermore, abalones supplemented with probiotics had a survival rate of 62% to
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the pathogenic bacterium Vibrio anguillarum compared to 25% survival of untreated
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animals. Another study describes that the daily application of Phaeobacter inhibens and B.
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pumillus RI06-95 mixed with algal feed to culture tanks in the hatchery increased survival of
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oyster larvae to experimental challenge with V. coralliilyticus RE22 [23]. Probiotic
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microorganisms have the ability to release chemical substances with bactericidal or
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bacteriostatic effect on pathogenic bacteria that are in the intestine of the host, thus
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constituting a barrier against the proliferation of opportunistic pathogens. In general, the
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antibacterial effect is due to one or more of the following factors: production of antibiotics,
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bacteriocins, siderophores, enzymes (lysozymes, proteases) and/or hydrogen peroxide, as
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well as alteration of the intestinal pH due to the generation of organic acids [18]. According
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to these data, the genus Bacillus is one of dominant probiotics, which was commonly used in
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aquaculture. More and more studies confirmed that endogenous or exogenous Bacillus
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strains could be effective in improving growth, immunity and disease resistance of shellfish
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[24] [25] [20].
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This study thus investigated the possible protection effect of Bacillus sp for Crassostrea
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gigas reared in indigo polluted seawater and infected with Vibrio Harvey.
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2. Materials and methods 4
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2.1.Samples collection and bacterial characterization Three Bacillus strains, isolated from Tunisian hypersaline environments in a previous study
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in our laboratory [26], are used in this study as Bacillus consortium. Sequencing analyses
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showed that these bacteria corresponded to the genus Bacillus sp such as Bacillus subtilis, B.
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cereus, B. coagulans [26]. These strains were preserved in our laboratory and their purity
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was routinely checked. Stock cultures were stored at 80°C in powdered skimmed milk
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suspension with 25% glycerol. Bacillus strains were isolated, in this study, on nutrient plates
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and inoculated in nutrient broth prepared with seawater. It was incubated for 24 hours at
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20°C [27].
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Vibrio harveyi ATCC 14126 was routinely cultured in marine broth. It is used for control as
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positive pathogen.
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2.2.Screening and identification of micro-organisms with bioremediation activity
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Indigo was submitted to decimal dilution in a mineral liquid medium (MLM) prepared as
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described previously by [28] up to a concentration of 1.75 mg/ml.
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Isolated bacteria from sludge and wastewater of industrial textile plant were inoculated in
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flasks containing MlM medium with 1.75 mg/ml indigo dye concentration. Then flasks were
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incubated at 37°C under shaking conditions (120 rpm) in a rotary shaker. Samples were
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collected at different time to determine decolorization. Optical densities (OD) were
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determined using a spectrophotometer, Shimadzu UV-240I PC model Kyoto, Japan) at the
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max λ max 664 nm. The percentage of decolorization was calculated as following:
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decolorization (%) = [absorbance at t0 – absorbance at t1] * 100/ absorbance at t0.
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Crassortrea gigas were purchased from a rearing plant in a lagoon after an acclimatization
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period of one week at 25°C in aquariums containing 10 liters of seawater. Experiments were
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carried out in six aquariums, each containing six specimens.
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Aquarium 1 : containing C. gigas and seawater; Aquarium 2: containing C. gigas, seawater
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and indigo (1.74 mg/ml); Aquarium 3: containing C. gigas, seawater and filtrate from a
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culture of Bacillus consortium where indigo was degraded; Aquarium 4: Same content as
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the second aquarium but reared seawater is inoculated with Bacillus consortium (10 ml of
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Bacillus suspension 106 CFU/ml); Aquarium 5: seawater inoculated with Bacillus
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consortium (10 ml of Bacillus suspension 106 CFU/ml); Aquarium 6: the same content as
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the fifth aquarium but oysters are infected with V. harveyi ATCC 14126; Aquarium 7: the
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same content as the sixth aquarium but oysters are infected with V. harveyi ATCC 14126and
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added with Bacillus consortium (10 ml of Bacillus suspension 106 CFU/ml).
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2.4.Hemolymph collection C. gigas was collected from the lagoon of Bizerte (Tunisia). It is polluted with industrial and
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domestic wastewater.
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Hemolymph was collected from the adductor muscle following Chen method (1996). A
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notch was filed on the dorsal side of the shell valve, adjacent to the adductor muscle. 2ml of
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hemolymph were collected aseptically from each animal.
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A total of 10 000 hemocytes were analyzed using a flow cytometer BD FAC Caliber
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coupled with a Power Mac G3PC equipped with a Cell Quest Pro software
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2.5.Hemocytes viability assay
150 µl of hemolymph was mixed with 150 µl of an antiaggregant solution (AASH) and 5 ml
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of propidium iodide 1mg/l (PI) (Sigma) at a final concentration of 20µg/ml. The Propodium
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iodide binds to DNA double stranded and absorbs at wavelength above 630 nm. It enters and
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stains the cells but can’t cross the membrane of viable cells. Tubes were incubated with PI
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and AASH for 10 minutes at 20°C before analysis. Dead hemocytes are more fluorescent in
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flow cytometer’s orange light detector.
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We evaluate the percentage of dead heamocytes in proportion to the total number. 2.6. Statistical analysis
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Past 3.05 and Graphpad prism 6.05 softwares are used for statistical analysis and graphics.
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For all tests we used hypothetical risk α= 0.05 for alternative hypothesis acceptance. Bar
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chartwas plotted with standard deviation show.
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3. Results
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3.1.Screening and identification of bacterial strains decolorizing indigo dye
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More than 1500 biomasses from textile wastewater and sludge were tested for their ability to
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decolorize diluted indigo dye. Active strains associated with these biomasses were
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identified. Gram negative rods shape bacteria and some Gram positive bacteria were
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selected. Gram negative rods as Pseudomonas stutzeri ; Pseudomonas testosteroni;
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Pseudomonas
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Pseudomonas aeruginosa; Sphingomonas paucimobilis; Aeromonas hydrophila. And for
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Gram positive bacteria, we identified Staphylococcus epidermidi and Bacillus subtilis.We
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have been interested to Bacillus sp strains.
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vesicularis ;
Pseudomonasmaltophilia ;
Pseudomonas
acidovorans;
3.2.Biodegradation of indigo by Bacillus consortium
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The percentage of decolorization of indigo dye in polluted seawater in presence of C. gigas
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increased from 41% to 90% (Table 1, Aquarium 4) when using Bacillus consortium. These
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bacteria can survive in seawater because of their sporulation characteristics. In the absence 6
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indigo is 41% (Table 1, Aquarium 2).
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The Bacillus consortium has also improved the seawater quality of the aquarium
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contaminated with the previous product of indigo degradation: In aquarium 3, the percentage
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of decolorization was 74 %. Treated C. gigas with indigo in aquarium 2 accumulated this
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dye in its gills. This can have a harmful effect on consumers’ health. Table1 shows optical
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density variation of indigo in reared seawater of C.gigas under different conditions.
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3.3.Crassostrea gigas hemocytes protection by Bacillus consortium
Rearing seawater of the aquarium 1 and 5 is not contaminated with indigo dye so a low
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mortality of haemocytes was obtained in the aquarium1 and no mortality in the aquarium 5.
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We found that the indigo dye is very toxic for C. gigas and the mortality of haemocytes
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increased in the aquarium 2 c contaminated with diluted indigo from1.2 aqua. (1) up to 87%
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(aqua.2).
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Rearing seawater contaminated with treated indigo using Bacillus consortium is less toxic to
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C. gigas than that contaminated with untreated indigo. When added to contemned seawater
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with indigo, Bacillus consortium decreased the hemocytes mortality of reared C. gigas from
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87% to 56% (fig.1: Aquarium 2 and 4). It has also a protecting effect against pathogenic
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bacteria V. harveyi ATCC 14126. Less mortality is obtained in the aqu.6 containing Bacillus
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consortium than in the aqua.7. 4. Dicussion
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Effluent from the textile industry is loaded of non-biodegradable dyes which make them
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difficult to apply biological treatments. Several techniques have been employed to remove
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dyes from wastewater. These effluents have an impact on aquaculture. Aquaculture is a fast-
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growing food production sector and the use of probiotics for the improvement of aquatic
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environmental quality as major areas for further research in disease control in aquaculture
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[29], which would help to reduce the use of antimicrobial agents. However, the addition of
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probiotics is now also a common practice in commercial shrimp hatcheries in Mexico [30].
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The most studied probiotics are usually Bacillus and Lactobacillus species [31]. It has been
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reported that use of Bacillus sp. improved water quality, survival and growth rates, increased
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the health status of juvenile Penaeus monodon and reduced the pathogenic vibrios [32].
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Indeed, the probiotic effect of Bacillus sp was reported under in vitro and in vivo condition
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[33].
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Bacillus strains isolated from lagoons can survive in seawater because of their tolerance to
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high pH. It was found that they prefer pH>8.9. They also tolerate high salinity because of
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can survive in seawater because of their sporulation characteristics. This Bacillus
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consortium is as active as rot white fungi [34] but fungi need low pH and special nutrients to
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grow. Experiences conducted by von Verschuer (2000) [35] to improve seawater quality
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using Bacillus, had shown that they can easily multiply in seawater. So, Bacillus strains can
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be used in rearing media of aquatic organisms like filter feeds. When added to rearing water
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and is generally more effective in converting organic matter back to CO2 than Gram negative
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bacilli. Venkateswara et al.(2007) [36] had reported that microorganisms with probiotic
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effect can regulate microflora and improve the chemical quality of the water by
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decomposing pollutants, minimizing the toxic gases like NH3, N2O, H2O2. According to
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Kolndadacha et al. (2011) [37], Bacillus consortium improves water quality and increases
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heamocytes viability from 56% to 87% when added to rearing water of C.gigas. It can be
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considered as potential probiotic in rearing water of aquatic organisms. The percentage of
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decolorization of indigo dye in polluted seawater in presence of C. gigas increased from
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41% to 90% (Table 1, Aquarium 4) when using Bacillus consortium. Our results showed
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that indigo biodegradation products are harmless for C. gigas and decrease haemocytes
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mortality. Bacillus consortium is efficient in seawater control when added to the rearing
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medium. Mahdhi at al. (2007) [26] found similar results during Dicentrarchus labrax
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treatment by Bacillus. Bacillus strains isolated and used in this study may have an
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interesting antagonistic activity against pathogens. They have biomedical and antibacterial
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effects. In addition, they can be easily used in rearing seawater areas. These sporuling
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bacteria can resist to hostile conditions. In this investigation, we have shown that Bacillus
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sp. strains isolated from industrial textile waste water are able to degrade indigo. The
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optimization of their biodegradation activity in mixture showed that they need high pH and
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can be used in seawater. Experiments performed in rearing seawater of C. gigas with
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previously biodegraded indigo product water condition using Bacillus biomass show that
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indigo is more toxic for C. gigas and leads to higher haemocytes mortality rates than water
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contaminated with previously biodegraded indigo product.
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These strains have no toxic effect on C. gigas. No haemocytes mortality occurred in our
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assays. Similar results were found by Mahdhi et al. (2010) [26]. Bacillus consortium
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selected in our study, showed an antagonism effect against fish and shellfish pathogens [26].
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These results are in concordance with previous studies showed that B. cereus and B.
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thuringiensis mixture treatment resulted in survival rate of 100%, compared with 75% in the
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treatment without Bacillus. Authors concluded that B. cereus and B. thuringiensis have
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Furthermore, it was observed that the addition of B. subtilis to the rotifer culture water
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resulted in a significant increase in rotifer numbers and a reduction of Vibrio levels. B.
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subtilis appears to be a promising probiotic for rotifer cultures [39].
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Uncontrolled effect of antibiotics, their bad use and their high coast lead us to us to propose
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Bacillus consortium as an alternative substitute to antibiotics [40]. Several commercial
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consortia of probiotics (Bacillus or nitrifying bacteria) such as "Biostar"; BRF-1A; BRF 13
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A; Pond PrO-Vc are used to improve water quality and animal health. Experiments
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performed in C. gigas rearing seawater show that indigo is more toxic for C.gigas and lead
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to a higher haemocyte mortality rate than water contaminated with priory biodegradated
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indigo product. Bacillus consortium added to contaminated seawater with indigo improves
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seawater quality and results in a lower hemocytes mortality rate. It was reported that a
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commercially prepared bacterial mixture of Bacillus spp. mixed into the rearing water
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increased survival and production of channel catfish (Ictalurus punctatus) [33]. Currently
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there are no commercially available probiotics for shellfish aquaculture. Recent study
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describes efforts to create a stable formulation of B. pumilus RI0695 for delivery at shellfish
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hatcheries [20] [23]. Some effort to create a lyophilized probiotic formulation of P.
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gallaeciensis that provides reduced mortality of C. virginica larvae when exposed to the
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shellfish pathogen V. tubiashii RE22 [20].
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In this investigation, we have shown that three strains of Bacillus sp. isolates are able to
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degrade indigo.
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Experiments performed in rearing seawater showed that Bacillus consortium added to
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contaminated seawater with indigo dye or with Vibrio harvey improves seawater quality and
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results in lower haemocytes mortality rates.
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Rearing seawater contemned with indigo product is more toxic for C. gigas and leads to
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higher haemocyte mortality rates than water contaminated with previously biodegraded
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indigo product.
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Conflict of interest
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No conflicts of interests exist.
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Acknowledgement
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We thank all the members of both laboratories for their collaboration.
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References
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ACCEPTED MANUSCRIPT Table1 optical density variation of indigo in reared seawater of C.gigas under different conditions: Aquarium 2: C. gigas, seawater and indigo (1.74 mg/ml); Aquarium 3: C. gigas, seawater and previously degraded indigo using Bacillus consortium; Aquarium 4: Same content as the second aquarium but inoculated with Bacillus
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consortium (10 ml of Bacillus suspension 1012 CFU/ml).
Day1
Day2
Aquarium 2
0.1200±0,003
0.0700±0.003
Aquarium 3
0.0270±0,001
0.0070±0.001
74.0854±1,470
Aquarium 4
0.1260±0,003
0.0129±<0.001
89.7552±0,144
41.4377±4,550
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Decolorization
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Fig. 1 : Percentage of decolorization of reared seawater under different conditions: Aquarium 2: C. gigas, seawater and indigo (1.74 mg/ml); Aquarium 3: C. gigas, seawater and previously degraded indigo using Bacillus consortium; Aquarium 4: Same content as
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the second aquarium but inoculated with Bacillus consortium (10 ml of Bacillus
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suspension 1012 CFU/ml). Bars represent the SD for each point.
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Fig. 2: Bacillus Protection of heamocytes of Crassostrea gigas reared in seawater
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contaminated with indigo. 1; 2;....7: Aquarium number. Bars represent the SD for each point.
ACCEPTED MANUSCRIPT HIGHLIGHTS •
Bacillus sp used as safe probiotic
•
Vibrio harveyi is widespread in aquaculture
•
Indigo is a strongly toxic dyes
•
Bacillus sp had an effect on immunity and resistance to Vibrio harveyi of Crassostrea gigas reared in
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indigo polluted seawater