Italian Journal of Animal Science
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Effects of Biofloc Promotion on Water Quality, Growth, Biomass Yield and Heterotrophic Community in Litopenaeus Vannamei (Boone, 1931) Experimental Intensive Culture
Irasema E. Luis-Villaseñor, Domenico Voltolina, Juan M. Audelo-Naranjo, María R. Pacheco-Marges, Víctor E. Herrera-Espericueta & Emilio Romero- Beltrán
To cite this article: Irasema E. Luis-Villaseñor, Domenico Voltolina, Juan M. Audelo-Naranjo, María R. Pacheco-Marges, Víctor E. Herrera-Espericueta & Emilio Romero-Beltrán (2015) Effects of Biofloc Promotion on Water Quality, Growth, Biomass Yield and Heterotrophic Community in Litopenaeus Vannamei (Boone, 1931) Experimental Intensive Culture, Italian Journal of Animal Science, 14:3, 3726
To link to this article: http://dx.doi.org/10.4081/ijas.2015.3726
©Copyright I.E. Luis-Villaseñor et al. Published online: 17 Feb 2016.
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PAPER nated in both systems; in both some isolates Effects of biofloc promotion were potential pathogens, and diversity was Corresponding author: Dr. Juan M. Audelo- on water quality, growth, higher in the control than in the BFT treat- Naranjo, Universidad Autónoma de Sinaloa, ment. The advantages of BFT technology are Facultad de Ciencias del Mar, Mazatlán, 610 biomass yield and confirmed by the significantly lower TAN and Sinaloa, Mexico. Tel./Fax: +52.669.9828656. - E-mail: [email protected] heterotrophic community N-NO2 concentrations, as well as by the better in Litopenaeus vannamei shrimp performance in terms of growth, bio- mass yield, and food and protein conversion Key words: Biofloc; Shrimp growth; Nutrient recy- (Boone, 1931) experimental cling; Heterotrophic bacteria. intensive culture efficiency. Acknowledgments: supported by projects PRO- FAPI 2012/016, PROMEP/103.5/12/3368 and CIB- 1 Irasema E. Luis-Villaseñor, NOR AC0.38. Staff of the Shrimp and Fish Culture Domenico Voltolina,2 Introduction Academic Group (UAS-CA-134) helped with the Juan M. Audelo-Naranjo,1 field and analytical work. María R. Pacheco-Marges,1 Land-based aquaculture generates high vol- Víctor E. Herrera-Espericueta,1 umes of nutrient-rich water. These are dis- Received for publication: 5 November 2014. Emilio Romero-Beltrán3 charged to the natural environment and may Accepted for publication: 30 March 2015. 1 cause eutrophication of the receiving water Facultad de Ciencias del Mar, This work is licensed under a Creative Commons Universidad Autónoma de Sinaloa, bodies, which is a widespread source of con- Attribution NonCommercial 3.0 License (CC BY- Mazatlán, Sinaloa, Mexico cern. This requires a reassessment of the cur- NC 3.0). 2Cent ro de Investigaciones Biológicas del rent practices used in aquaculture, in order to Noroeste, Universidad Autónoma de maintain high levels of production and better ©Copyright I.E. Luis-Villaseñor et al., 2015 Sinaloa, Mazatlán, Sinaloa, Mexico water quality within the farms, as well as at the Licensee PAGEPress, Italy 3 respective points of discharge (Martínez- Italian Journal of Animal Science 2015; 14:3726 Centro Regional de Investigación doi:10.4081/ijas.2015.3726 Pesquera, Instituto Nacional de Pesca, Córdova et al., 2009; Martins et al., 2010). Mazatlán, Sinaloa, Mexico Apart from the cost involved, and of the poten- tial environmental impact, the high rates of activity may be achieved through the addition water exchange used to maintain an accept- of a carbon source, because in the pond envi- able level of water quality within the culture ronment organic carbon becomes limiting for system might be important points of entry for bacteria growth, while dissolved nitrogen is Abstract pathogenic organisms. Therefore, zero or lim- usually present in excess, and is in fact the ited water exchange systems shou ld be consid- 3 main source of deterioration of water quality Six 1.2-m tanks were stocked with an initial ered as viable options (Cohen et al., 2005). -3 (Emerenciano et al., 2013). Thus, this technol- biomass of 500 g m of Litopenaeus vannamei Among these, biofloc technology systems ogy contributes significantly to the health of juveniles (individual weight: 1.0±0.3 g), to (BFT) are considered highly efficient in inten- the industry, because by recycling nutrients it evaluate the effect of biofloc promotion on sive closed cultures, because they allow main- improves food use efficiency, reduces wastes water quality and on shrimp growth and pro- taining better water quality and limiting the and maintains good water quality and a duction, and to identify the dominant taxa in organic load of effluent waters at the time of the heterotrophic communities present in healthy environment within the farm and in Downloaded by [93.179.90.222] at 06:04 01 July 2016 harvest. Adoption of this technology increases experimental closed cultures. Feeding was ad the efficiency of feed utilization, because the surrounding water bodies (Stokstad, libitum twice daily with 35% protein shrimp organic and inorganic metabolit es, as well as 2010). Although it appears that these het- feed. Three tanks were managed as biofloc unused or partially used food, are recycled by erotrophic communities may exert a control- technology (BFT) systems, adding daily an microorganisms into microalgae and bacterial ling effect on pathogen growth (Defoirdt et al., amount of cornmeal equivalent to 50% of the biomass, which tend to coalesce into flocculat- 2007; Zhao et al., 2012; Aguilera-Rivera et al., shrimp feed supplied. The remaining three ed material (bioflocs). This is used by the 2014), there is insufficient information on the received only shrimp feed and served as con- organisms in culture as a protein and lipid- bacteria present in flocs. In this study we eval- trols. Experiment lasted 21 days. The mean rich food source (Avnimelech, 2007; Ballester uated the effect of biofloc promotion on water 3- concentrations of P-PO4 and inorganic dis- et al., 2010), contributing between 18 and 29% quality, on shrimp growth and related produc- - - solved N species (TAN, N-NO2 , N-NO3 ) were to the total daily food consumption even in tion parameters, and identified the main significantly lower (P<0.5) in BFT than in the intensive shrimp cultures (Burford et al., groups and the dominant taxa in the het- control. The individual final weight, increase 2004). erotrophic communities present in experimen- in biomass, food, and protein conversion rates This flocculated material consists of an tal Litopenaeus vannamei closed cultures with, were significantly better in BFT than in the organic matrix on which thrive heterotrophic or without promotion of biofloc. control (P<0.05). The mean N content of the and autotrophic microorganisms (De Schryver shrimp biomass gained in the BFT cultures et al., 2008; Ray et al., 2010). In particular, het- was equivalent to 45.7% of the protein-N added erotrophic bacteria degrade organic residues, as feed, and was significantly higher than the which are converted into new bacterial bio- Materials and methods 34.7% recycled into shrimp biomass in the con- mass. This is available as food for the next trol cultures. Bacterial concentrations were trophic levels, initiating the classical microbial On August 19, 2013, L. vannamei juveniles not significantly different. Vibrionaceae domi- loop (Azam et al., 1983). Regulation of their (1.0±0.3 g, wet weight), obtained from a local
[page 332] [Ital J Anim Sci vol.14:2015] Biofloc in L. vannamei culture
shrimp farm, were stocked in six 1.2-m3 high and dissolved organic N concentrations; partic- The number of strains of each species iden- density polyethylene tanks, previously (one ulate N was determined as in Holm-Hansen tified in each treatment served to calculate week) added a 10 cm-deep layer from the bot- (1968). Suspended solids were quantified Shannon-Weaver’s diversity index H= - pi lnpi, tom sediment of the shrimp farm, since in gravimetrically, floc volume was recorded with where pi= ni N-1 and ni and N are the number Mexico most shrimp cultures are kept in earth- Imhoff cones, and turbidity with the Secchi of strains of species i and N the total number en ponds, rather than in plastic-lined or con- disc. The N contents of shrimp feed and of of strains, respectively. crete tanks. In all cases the initial biomass was shrimp (initial and final) wet biomass, deter- At the end of the experiment all shrimps 500 g m-3. Bioflocs were started 18 days before mined with the Kjeldahl method (AOAC, 2005), were counted and weighed. Survival (S) and the experiment in bioreactor tanks fertilized were 5.6 and 3.58%, respectively. specific growth rates (SGR) were calculated as with 20 g of 35% protein shrimp feed and 20 g Bacteriological samples were obtained on the in Hernández et al. (2013), with the equations: of cornmeal (MASECA® Gruma, S.A.B. de C.V., same dates as the water samples, using pre- -1 Mexico, 7.7% proteins, 70% carbohydrates, 4.6 sterilized 20 mL test tubes that were capped S=100 (Nf Ni ) and SGR = 100 (ln Mf - ln -1 lipids, C:N ratio 31.5:1). Vigorous aeration was and brought to the laboratory for immediate Mi) t maintained with a 1 HP Sweetwater blower processing. After serial dilution (10-1-10-5), 0.1 (Aquatic Eco-Systems Inc., Apopka, FL, USA), mL subsamples were plated in duplicate on dif- where Nf and Ni are final and initial number of distributed through PVC tubing to each tank, ferent DIFCO media: Marine Agar for total organisms of each tank, Mf and Mi are the where fine bubbling was provided by Aero- marine bacteria, Tripticase Soy Agar with 2.5% individual final and initial wet weights, respec- TubeTM tubing (Water Management NaCl (TSA) for total bacteria, TCBS Agar for tively, and t = duration of the experiment Technologies, Inc., Baton Rouge, LA, USA). presumptive Vibrio detection and Cetrimide (days). Three days before stocking, the six experi- and MacConkey Agar for Pseudomonas and Food conversion and protein efficiency mental tanks received an initial 100 L inocu- total coliform bacteria, respectively (APHA, ratios FCR and PER were calculated as: lum of biofloc-containing water from the biore- 1992). -1 -1 actor and 900 L of 300- m filtered (Nytex) After 48 h at 30±2 °C, colonies were counted FCR = FS (Bf - Bi) and PER = (Bf - Bi) PS Mazatlán Bay seawater (35.3±0.1 salinity). In to obtain the respective concentrations (CFU all tanks shrimps were fed ad libitum twice mL-1). In the case of the final samples, bacteri- FS and PS = total food and total amount of daily (08:00 and 18:00 h) using feeding trays al colonies with different morphologies were protein supplied to each tank, (Bf - Bi) = and formulated 35% protein shrimp feed replated, cross-streak purified and six colonies increase in total biomass in each tank (all data (Camaronina 35%, Purina Mexico, Cd. of each bacterial strain were stored at room in grams). Obregon, Sonora, Mexico, 35% proteins, 8% temperature in a capped 1.5 mL Eppendorf The mean final values of survival, individual lipids, 30% carbohydrates, C:N ratio: 7.1:1). tube with 1-mL anhydrous (99.8%) ethanol weight and total biomass, FCR, PER, SGR cal- Three tanks, selected at random were man- (ETOH). culated for the BFT and control tanks were aged as BFT systems, adding daily to each tank Bacterial isolates were identified using mor- compared with t or Mann Whitney tests an amount of cornmeal, equivalent to 50% of phological criteria after Gram staining. For depending on the results of Kolmogorov- the shrimp feed supplied. The remaining three specific identification, 50 ng µL-1 of the DNA of Smirnov and Fisher’s F tests. Daily mean water tanks received only shrimp feed and served as ETOH-preserved strains were used for 16S characteristics and weekly mean bacteria and controls. Water lost through evaporation ARNr gene amplification with PCR, using the nutrient concentrations were compared using (approximately 6% weekly) was replaced with universal primers Forward 27f.1 (AGR GTT t tests for paired observations or non paramet- dechlorinated tap water to avoid salinity TGA TCM TGG CTC AG) and Reverse 1492R2 ric Wilcoxon tests, after ln and arcsine square root transformation for bacterial counts and Downloaded by [93.179.90.222] at 06:04 01 July 2016 increases and there were no additional water (GGT TAC CTT GTT ACG ACT T). The amplifi- changes or additions to the control and to the cation program was: 16S: 94 °C/2’ 35 cycles for data in percentage, respectively. All tests BFT cultures. (94 °C/1’ 56 °C/1’ 72 °C/1’) 72 °C/5’ 4 °C/ . were performed with a confidence level =0.05 Water temperature, dissolved oxygen, pH To identify the closest bacterial species, the (Zar, 1996). and salinity were determined twice daily sequences obtained were compared to the pub- immediately before feeding, using an YSI 57 lic databases GenBank® (BLAST) dissolved oxygen meter with temperature sen- (http://blast.ncbi.nlm.nih.gov/Blastcgi) and sor (YSI Inc., Yellow Spring, OH, USA), a EzTaxon (http://www.ezbiocloud.net/eztaxon). Results Hanna HI 98150 portable pH meter (Hanna The FASTA format of the sequences obtained, Instruments, Woonsocket, RI, USA) and an aligned with the multiple sequence program Temperature, salinity, pH and dissolved oxy- Atago S/Mill-E refractometer (Atago Co. Ltd., ClustalW (http://align.genome.jp), were used gen concentrations ranged from 32 to 34°C, 35.0 Tokyo, Japan), respectively. to obtain the respective phylogenetic trees. to 35.4 psu, 7.5 to 7.8 and 3.7 to 4.0 mg L-1, Water samples were obtained from each The molecular phylogeny was obtained using respectively, and there were no significant dif- tank on the initial day, and at weekly intervals version 5.1 of the program MEGA (Tamura et ferences between the mean values calculated until the end of the experiment that lasted 21 al., 2011), using the Neighbor-Joining test for BFT and control cultures. The mean con- 3- days (August 19-September 9, 2013). Samples (Saitou and Nei, 1987) with the p-distance centrations of P-PO4 and of all inorganic dis- - - for nutrient analysis (1 L) in polyethylene bot- method with 500 bootstrap repetitions, consid- solved N species (TAN, N-NO2 , N-NO3 ) were tles, were filtered through Whatman GF-C ering transitions and transversions. Programs significantly lower in the BFT t han in the con- glass fiber filters. Filters and particle-free BLAST (Altschul et al., 1997), and the procary- trol cultures, whereas dissolved and particu- water were stored frozen until analysis accord- otic strain database EzTaxon (Chun et al., late organic N (DON and PON, respectively), ing to Strickland and Parsons (1972) for P- 2007) were used for phylogenetic relationships suspended solids, biofloc volumes and turbidi- 3- + - - PO4 , N-NH4 + NH3 (TAN), N-NO2 , N-NO3 identification. ty were higher in the BFT tanks (Table 1).
[Ital J Anim Sci vol.14:2015] [page 333] Luis-Villaseñor et al.
Mean survival was similar in both treat- tains an appropriate C:N ratio for bacterial trol tanks. ments, but the individual final weight and all transformation of these toxic N compounds The second advantage is the direct or indi- remaining zootechnical indicators (total bio- into single cell protein (Ebeling et al., 2006; rect use of PON by the cultured organisms. In mass harvested, increase in biomass, SGR, Asaduzzaman et al., 2008). This was made evi- this case, this allowed a >22% higher mean amount of shrimp feed supplied, FCR and PER) dent by the significantly lower concentrations yield in the BFT cultures, even if the amount of were significantly better in the BFT than in the of dissolved inorganic N and P species, which food added was lower by close to 7%. In tradi- control cultures (Table 2). were used for bacterial and microalgae tional open systems, the percentage of protein- Bacterial concentrations varied widely and, growth, as shown by the concentrations of N added as feed that is converted into shrimp although at the time of ha rvest the mean final suspended particulate organic N (PON), biomass generally lies between 20 and 25% numbers of total and marine bacteria (TSA and which were higher in the BFT than in the con- (Piedrahita, 2003; Crab et al., 2007) whereas Marine Agar, respectively) were higher in the BFT (13.5±3.3x106 and 0.8±0.1x106 CFU mL-1) than in the control cultures (4.2±2.5x106, 0.4±0.1x106 CFU mL-1), there were no signifi- Table 1. Mean concentrations (±standard deviation) of inorganic dissolved nitrogen cant differences between the mean values cal- 3- species, dissolved and particulate organic nitrogen, dissolved P-PO4 , total suspended culated with the concentrations determined at solids, mean floc volume, turbidity determined in the biofloc technology, and control cul- weekly intervals throughout the experiment tures of L. vannamei juveniles. (Table 3). The samples obtained in the final day yield- BFT Control ed 58 Gram- and 2 unidentified Gram+ bacter- TAN, mg L-1 0.266±0.019a 0.424±0.015b -1 a b ial strains, 26 of which from the control and 34 N-NH3, mg L 0.011±0.007 0.020±0.007 - -1 a b from the BFT cultures. Vibrionaceae, followed N-NO2 , mg L 0.156±0.026 0.452±0.03 - -1 a b by Enterobacteriaceae were the dominant fam- N-NO3 , mg L 0.949±0.027 1.617±0.098 ilies in the BFT and in the control cultures, and DON, mg L-1 0.710±0.437b 0.150±0.089a the remaining strains pertained to PON, mg L-1 1.314±0.318b 0.777±0.206a 3- -1 a b Alteromonadaceae and Micrococcaceae in the P-PO4 , mg L 0.327±0.077 0.523±0.045 SST, mg L-1 400.8±30.4b 129.6±14.9a BFT, and Pseudoalteromonadaceae, -1 b a Rhodobacteriaceae and Alteromonadaceane i Floc volume, mL L 16.8±1.5 3.4±0.7 Turbidity, cm 18.2±1.5a 58.2±2.0 the control cultures, respectively. According to the numbers of strains isolated BFT, biofloc technology; DON, dissolved organic N; PON, particulate organic N. a,bDifferent letters indicate significant difference between data in the same row (t tests for paired observations, =0.05, a [page 334] [Ital J Anim Sci vol.14:2015] Biofloc in L. vannamei culture in a closed system at least part of the protein- teria, which Krummenauer et al. (2014) found growth rate (Coyer et al., 1996; Maida et al., N is recycled by microalgae and microorgan- effective in increasing survival and growth of 2013), and suggests that it might be able to isms, which increases the percentage of N Vibrio parahaemolyticus-infected L. vannamei outcompete pathogenic bacteria in the carbon converted in shrimp biomass. Since the mean in a BFT culture system. and nutrient-rich BFT environment. N content of shrimp biomass was 3.6%, the However, although in this work the patho- A second strain that deserves attention is close to 910 g of shrimp biomass gained in our genic Vibrio parahaemolyticus and Vibrio hepatarius, which was isolated from the BFT cultures represented 45.7% of the protein- Photobacterium damselae (Vandenberghe et control cultures, because it is known to allevi- N added as feed, which is 9% higher than the al., 1999) were isolated only from the control ate viral (white spot virus) infection and, 34.7% recycled into the 743 g of shrimp bio- cultures, the most common species from the when administered in mixture with other pro- mass gained in the control cultures. BFT tanks was V. rotiferianus, which has been biotics, confers resistance to Vibrio harveyi According to Teichert-Coddington et al. associated with mass mortalities of the and promotes L. vannamei growth (Balcázar et (1999), the impact on the environment repre- penaeid shrimp Fenneropenaeus chinensis al., 2006). sented by nutrient discharges may be consid- (Zhang et al., 2014). erably reduced by a short residence time in a Among the non-pathogenic species detected settling pond. This would be a further advan- in the BFT cultures, Vibrio natriegens deserves tage of BFT cultures in which, apart from the attention because it can utilize with high effi- Conclusions significantly lower concentration of dissolved ciency different carbon and nitrogen sources P, approximately 38.7% of the nitrogen present in the culture water was in the form of easily (Austin et al., 1978), and because of its ability Addition of organic carbon to closed inten- settleable particulate N, in comparison to the to increase the number of ribosomes in order sive shrimp culture increased heterotrophic 22.7% determined in the control cultures. to achieve high rates of protein synthesis bacterial activity, which resulted in low TAN - Therefore, a settling pond with a short resi- (Aiyar et al., 2002). This explains its high and N-NO2 concentrations, increased food uti- dence time would allow a lower environmental impact of BFT cultures at the time of harvest. The coefficients of variation of bacterial concentrations on marine and TS agar indicat- Table 4. Bacteria strains isolated in the biofloc technology cultures of L. vannamei juve- ed a slightly higher variability in the BFT than niles. in the control cultures. Concentrations were in Strain Species Class Family the same order of magnitude than the mean value (5.43x106 CFU mL-1) found by Kim et al. AM2-3-12 Vibrio rotiferianus Gammaproteobacteria Vibrionaceae (2014) in BFT-managed shrimp cultures, but AM4-2-14 Vibrio rotiferianus Gammaproteobacteria Vibrionaceae one order of magnitude lower than those deter- AM4-2-18 Vibrio rotiferianus Gammaproteobacteria Vibrionaceae mined in BFT-managed shrimp cultures by AM4-2-3 Vibrio rotiferianus Gammaproteobacteria Vibrionaceae Burford et al. (2004), although in that case AM4-2-8 Vibrio rotiferianus Gammaproteobacteria Vibrionaceae bacteria were counted with the epifluores- AM4-3-11 Vibrio rotiferianus Gammaproteobacteria Vibrionaceae cence technique which yields higher values AM4-3-16 Vibrio rotiferianus Gammaproteobacteria Vibrionaceae than the method used in this work (Maki and AM4-3-7 Vibrio rotiferianus Gammaproteobacteria Vibrionaceae Remsen, 1981). TC4-3-22 Vibrio rotiferianus Gammaproteobacteria Vibrionaceae Downloaded by [93.179.90.222] at 06:04 01 July 2016 The unsanitary conditions of the source of TC4-3-23 Vibrio rotiferianus Gammaproteobacteria Vibrionaceae the raw seawater used in this work (Alonso- TS2-3-35 Vibrio rotiferianus Gammaproteobacteria Vibrionaceae Rodríguez et al., 2000) are the most probable TS4-3-23 Vibrio rotiferianus Gammaproteobacteria Vibrionaceae explanation for the high numbers of coliforms, TS4-1-1 Vibrio mytili Gammaproteobacteria Vibrionaceae Pseudomonas and Vibrio colonies. The latter TS4-1-6 Vibrio mytili Gammaproteobacteria Vibrionaceae were two orders of magnitude higher than TS4-2-15B Vibrio mytili Gammaproteobacteria Vibrionaceae those determined by Moreira de Souza et al. TS4-3-10 Vibrio mytili Gammaproteobacteria Vibrionaceae (2014) in a similar experiment, in which TS4-2-24 Vibrio communis Gammaproteobacteria Vibrionaceae Vibrio concentrations were significantly lower TC4-3-12 Vibrio communis Gammaproteobacteria Vibrionaceae and less variable in the BFT cultures, which AM4-3-9 Vibrio natriegens Gammaproteobacteria Vibrionaceae does not correspond to the results of our exper- TS4-2-19 Photobacterium jeanii Gammaproteobacteria Vibrionaceae iment. TS4-1-8 Photobacterium jeanii Gammaproteobacteria Vibrionaceae One of the claimed advantages of BFT cul- TS4-1-2 Photobacterium gaetbulicola Gammaproteobacteria Vibrionaceae tures is the low occurrence of pathogenic TC4-2-18 Photobacterium leiognathi Gammaproteobacteria Vibrionaceae events or of high pathogen loads in compari- Mac4-1-10 Proteus mirabilis Gammaproteobacteria Enterobacteriaceae son with traditional cultures, possibly because Mac4-1-4 Proteus mirabilis Gammaproteobacteria Enterobacteriaceae of interspecific interactions, or of competition Mac4-1-8 Proteus mirabilis Gammaproteobacteria Enterobacteriaceae for substrate or essentials nutrients between Mac4-2-7 Proteus mirabilis Gammaproteobacteria Enterobacteriaceae pathogens and biofloc-forming microorgan- Mac4-3-1 Proteus mirabilis Gammaproteobacteria Enterobacteriaceae isms (Crab et al., 2010; Emerenciano et al., Mac4-3-5 Proteus mirabilis Gammaproteobacteria Enterobacteriaceae 2013). This effect may be particularly evident TS4-2-BIS Marinobacter goseongensis Gammaproteobacteria Alteromonadaceae when bioflocs are enriched with probiotic bac- AM2-3-18 Marinobacter goseongensis Gammaproteobacteria Alteromonadaceae [Ital J Anim Sci vol.14:2015] [page 335] Luis-Villaseñor et al. Table 5. Bacteria strains isolated in the control cultures of L. vannamei juveniles. namei in experimental closed systems with artificial substrates. Hidrobiológica Strain Species Class Family 22:1-7. AM1-2-13 Vibrio rotiferianus Gammaproteobacteria Vibrionaceae Austin, B., Zachary, A., Colwell, R.R., 1978. AM1-3-10Bis Vibrio rotiferianus Gammaproteobacteria Vibrionaceae Recognition of Beneckea natriegens AM1-3-5 Vibrio rotiferianus Gammaproteobacteria Vibrionaceae (Payne et al.) Baumann et al. as a member TC1-3-6 Vibrio rotiferianus Gammaproteobacteria Vibrionaceae of the genus Vibrio, as previously proposed TS1-3-23 Vibrio rotiferianus Gammaproteobacteria Vibrionaceae by Webb and Payne. Int. J. Syst. Bacteriol. TS1-3-5 Vibrio rotiferianus Gammaproteobacteria Vibrionaceae 28:315-317. TC1-3-4 Vibrio mytili Gammaproteobacteria Vibrionaceae Avnimelech, Y., 2007. Feeding with microbial TS1-2-31 Vibrio mytili Gammaproteobacteria Vibrionaceae flocs by tilapia in minimal discharge TS1-2-7 Vibrio mytili Gammaproteobacteria Vibrionaceae bioflocs technology ponds. Aquaculture TS1-3-20 Vibrio mytili Gammaproteobacteria Vibrionaceae 264:140-147. TC1-2-16 Vibrio hepatarius Gammaproteobacteria Vibrionaceae Azam, F., Fenchel, T., Field, J.G., Gray, J.S., TS1-2-4 Vibrio neptunius Gammaproteobacteria Vibrionaceae Meyer-Reil, L.A., Thingstad, F., 1983. The TS1-2-14 Vibrio communis Gammaproteobacteria Vibrionaceae ecological role of wat er-column microbes AM1-2-15 Vibrio parahaemolyticus Gammaproteobacteria Vibrionaceae in the sea. Mar. Ecol. Prog. Ser. 10:257-263. TC1-3-14 Photobacterium damselae Gammaproteobacteria Vibrionaceae Balcázar, J.L., Decamp, O., Vendrell, D., De TC1-3-7 Photobacterium gaetbulicola Gammaproteobacteria Vibrionaceae Blas, I., Ruiz-Zarzuela, I., 2006. Health and Mac1-2-2 Proteus mirabilis Gammaproteobacteria Enterobacteriaceae nutritional properties of probiotics in fish Mac1-2-6 Proteus mirabilis Gammaproteobacteria Enterobacteriaceae and shellfish. Microb. Ecol. Health D. Mac1-2-9 Proteus mirabilis Gammaproteobacteria Enterobacteriaceae 18:65-70. Mac1-3-3 Proteus mirabilis Gammaproteobacteria Enterobacteriaceae Ballester, E.L.C., Abreu, P.C., Cavalli, R.O., AM1-2-1 Pseudoalteromonas spongiae Gammaproteobacteria Pseudoalteromonadaceae Emerenciano, M., Abreu, L., Wasielesky, AM1-2-2 Pseudoalteromonas spongiae Gammaproteobacteria Pseudoalteromonadaceae W., 2010. 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