Joong Kyun Kim

Outbreaks and risks of infectious spleen and kidney necrosis virus disease in freshwater ornamental fishes

We examined the distribution of iridoviruses in 10 freshwater ornamental fish species hatched in Korea and imported from other Asian countries using both 1-step and 2-step polymerase chain reation (PCR). None of the 10 fish species analyzed were free of iridovirus as shown by 2-step PCR positive results, and 3 species yielded 1-step PCR positive results with associated mortality. Cloned PCR amplicons of the adenosine triphosphatase (ATPase) and major capsid protein (MCP) genes in genomic DNA of iridovirus showed the same nucleotide sequences as that of infectious spleen and kidney necrosis virus (ISKNV) isolated from the mandarinfish Siniperca chuatsi. These results indicate the presence of ISKNV disease in various ornamental fish as new host species and that the disease is widespread throughout different Asian countries including Korea, Singapore and China. Such infections were either clinical with associated mortality (and 1-step PCR positive) or asymptomatic in fish that were externally healthy (and only positive in 2-step PCR). Molecular analyses of the K2 region performed on iridovirus samples isolated from freshwater ornamental fishes revealed deletion/insertion of repetitive sequences of various lengths (42 to 339 bp), depending on the ISKNV isolates, without substitutions. Experimental infection of pearl gourami Trichogaster leeri and silver gourami T. microlepis with a tissue homogenate of pearl gourami infected by ISKNV induced 70 and 20% cumulative mortalities in the pearl and silver gourami, respectively.

Production of Candida utilis biomass on molasses in different culture types

Three different types of aerobic fermentations were performed for the mass production of C. utilis as aquafeeds. From the best fermentation result of each culture type, the biomass yield and productivity were calculated to be 0.67 and 0.24 for batch, 0.51 and 1.95 for fed-batch with sigmoidal feeding strategy, and 0.36 g g −1 and 2.15 g − 1 l −1 h −1 for continuous cultures, respectively. The cultivation of C. utilis using chemicals for industrial use resulted in considerable reduction of production cost. The fed-batch fermentation was found to be the best culture type for mass production of C. utilis. The total production cost of C. utilis cultivated in the fed-batch fermentation was estimated to be US

Mass production of Rhodopseudomonas palustris as diet for aquaculture

2.76 per kg of dry cells. The total production cost is favorably comparable with the sale price of the commercial yeast product.

Influence of temperature shifts on the onset and development of red sea bream iridoviral disease in rock bream Oplegnathus fasciatus.

Three different types of anaerobic fermentations were used for the mass production of the photosynthetic bacterium Rhodopseudomonas palustris as diet for aquaculture. The optimum agitation speed and malate concentration were 300 r.p.m. and 0.2% in the modified MYC medium, respectively. In batch fermentations of R. palustris, the maximum number of viable cells was 1.110 10 c.f.u. ml 1 with 2.65 g l 1 of DCW, and the maximum specific growth rate and biomass productivity were estimated to be 0.12 h 1 and 55 mg l 1 h 1 , respectively. Crude protein content of R. palustris was about 72‐74%. The composition of stearic acid and oleic acid of R. palustris was superior to those of Chlorella and yeasts, while that of other fatty acids tested was not. The amino acid profiles of the protein hydrolysate compared favorably with Food Agricultural Organization (FAO) guidelines. The biomass productivities from fed-batch experiments were found to be 50, 47 and 49 mg l 1 h 1 for linear, exponential, and sigmoidal feeding strategy, respectively. The maximum biomass productivity was found to be 112 mg l 1 h 1 in chemostat. Compared to growth in batch cultures, continuous fermentation yielded two times higher biomass productivity.

A continuous fermentation of Kluyveromyces fragilis for the production of a highly nutritious protein diet

The effects of various water temperature treatments on the development of red sea bream iridovirus disease (RSIVD) in rock bream Oplegnathus fasciatus challenged with iridovirus Sachun (IVS-1) were determined by measuring the mortality and the viral concentration in the spleen of infected fish. Experimental infections of rock bream with IVS-1 at water temperatures of 18, 21, and 25 degrees C resulted in a cumulative mortality of 100%, but infections at 13 degrees C resulted in 0% mortality, even after 45 d. The disease progressed more rapidly at higher water temperatures; at 25, 21, and 18 degrees C, the mean numbers of days until death were 17, 20, and 30 d, respectively. When the water temperature for fish infected with iridovirus by intramuscular injection was shifted from 13 to 25 degrees C, the cumulative mortality reached 100%, with rapid onset of the disease, independent of the time at which the temperature was shifted, i.e. 7, 14, or 30 d after injection at 13 degrees C. Real-time PCR data revealed that the viral genome copy number in the spleen of rock bream maintained at 13 degrees C increased with time, suggesting the occurrence of viral replication even at 13 degrees C. In the reverse experiment, when the water temperature for fish that were infected at a higher temperature was shifted to 13 degrees C, 3 or 7 d after injection at 25 degrees C, the fish showed 100% cumulative mortality, although the mean number of days until death was higher than that observed for fish maintained at a constant temperature of 25 degrees C. The viral DNA concentration in the spleen of rock bream that had been shifted down to 13 degrees C, 3 or 7 d after injection at 25 degrees C, was not suppressed, but increased and eventually reached levels sufficient to induce mortality at 13 degrees C. However, the level of viral genome copy numbers in the spleen of dead fish at 25 degrees C, regardless of whether those fish were held at a constant temperature of 25 degrees C or shifted up from 13 degrees C, appeared to be greater than the level found in the dead fish shifted down to 13 degrees C after inoculation at 25 degrees C.

Identification and characterization of microorganisms from earthworm viscera for the conversion of fish wastes into liquid fertilizer

To develop a novel yeast diet, production of Kluyveromyces fragilis has been carried out in aerobic continuous culture. With a 2.5% fructose medium, the maximum biomass productivity of the chemostat was 4.81 g l−1 per h. At low dilution rates (D<0.17 h−1) aerobic ethanol synthesis was almost not found and the maximum biomass yield was found to be 0.42 g dry biomass g−1 fructose with 5.5×109 viable yeasts per ml. The protein content of the yeast biomass was 50–55% at all dilution rates studied. Fermentation parameters of Ks, Dc, m, and Yx/s(G) were estimated by the Monod equation and found to be 0.1 g l−1, 0.79 h−1, 0.2 g fructose consumed per g dry biomass h−1, and 0.24 g dry biomass per g fructose, respectively. From the data, the specific productivity of biomass was estimated to be 0.74 h−1.

Characterization of denitrifying photosynthetic bacteria isolated from photosynthetic sludge

Five bacteria isolated from earthworm viscera and identified as Brevibacillus agri, Bacillus cereus, Bacillus licheniformis, and Brevibacillus parabrevis by 16S rRNA sequencing were employed in the conversion of fish wastes generated from a restaurant specializing in sliced raw fish into fertilizer. Within 120h after inoculation of autoclaved fish waste with 5.15 x 10(5) CFU ml(-1) mixed isolates, the amount of dry sludge decreased from 29.4 to 0.2g, the pH changed from 7.05 to 5.70, and the cell number reached 6.45 x 10(5) CFU ml(-1). Analyses of an 84-h culture of inoculated fish waste indicated low phytotoxicity in a seed germination test, an amino acid content of 5.71 g 100 g(-1), a low concentration of heavy metals (Pb, As, Cd, Hg, Cr, Cu, Ni and Zn), and a N/P/K level of 2.33%. Therefore the converted fish waste has the potential for use as liquid fertilizer, although the low NPK level is a concern. This is the first demonstration of the reutilization of fish wastes as a liquid fertilizer.

Scaled-up bioconversion of fish waste to liquid fertilizer using a 5 L ribbon-type reactor

Abstract Photosynthetic bacteria were isolated from photosynthetic sludge for use in a recirculating aquaculture system. A strong denitrifying photosynthetic bacterium was identified from four isolates. The useful strain was taxonomically identified to be Rhodopseudomonas palustris. The parameters of growth were characterized in anaerobic flask culture. The optimum pH, temperature, and illumination intensity were 5.5, 31°C, and 5000 lux, respectively. The maximum specific growth rate and the rate of gas production by denitrification were 0.095 h−1 and 0.2 ml N2 h−1, respectively. Dissimilatory nitrate reduction to nitrogen gas by the isolate started after the late-log phase, and a small amount of nitrite was accumulated at the end of the culture. The maximum number of viable cells was 14×108 cells ml−1 with 1.07 g l−1 of dry-cell weight, and the maximum concentration of bacteriochlorophyll a was 0.17 OD775 g−1 of dry-cell weight.

Cloning, expression analysis and enzymatic characterization of cathepsin S from olive flounder (Paralichthys olivaceus)

A scaled-up conversion process of fish waste to liquid fertilizer was performed in a 5 L ribbon-type reactor. Biodegradation was performed by inoculation of autoclaved fish waste with 5.84 × 10(5) CFU mL(-1) of mixed microorganisms for 96 h. As a result, the pH changed from 6.92 to 5.72, the cell number reached 7.28 × 10(5) CFU mL(-1), and approximately 430 g (28.3%) of fish waste was degraded. Analyses indicated that the 96 h culture of inoculated fish waste possessed comparable fertilizing ability to commercial fertilizers in hydroponic culture with amino acid contents of 6.91 g 100 g(-1). Therefore, the scaled-up production achieved a more satisfactory fish waste degradation rate (3.61 g h(-1)) than the flask-scale production (0.24 g h(-1)). The biodegraded broth of fish waste at room temperature did not undergo putrefaction for 6 months due to the addition of 1% lactate.

Application of the rpoS gene for the detection of Vibrio anguillarum in flounder and prawn by polymerase chain reaction.

Cathepsin S is a critical protease for the regulation of MHC class II immune responses, and thus is a potential target for developing immunosuppressive drugs in the pathogenesis of degenerative and autoimmune diseases. In this study, we cloned a cDNA encoding for cathepsin S (PoCtS) from the olive flounder, Paralichthys olivaceus. The 1170 bp PoCtS cDNA contained an open reading frame of 1014 bp, which consisted of a 25-residue putative signal peptide, a 96-residue propeptide and the 216-residue mature enzyme. The tissue-specific expression pattern of PoCtS, determined via RT-PCR and real-time PCR analysis, revealed ubiquitous expression throughout the entirety of healthy flounder tissues; however IL-1beta, IL-6, IL-8 and PoCtS expression increased significantly in muscle 6h post-injection of bacterial lipopolysaccharide (LPS). The cDNA encoding proenzyme of PoCtS was expressed in Escherichia coli as a fusion protein with glutathione S-transferase in a pGEX-4T-1 vector. Also, the recombinant proPoCtS protein was overexpressed in E. coli BL21(DE3) as a 60 kDa fusion protein. Cathepsin S activity was detected through the cleavage of synthetic fluorogenic peptide substrates, such as Z-Val-Val-Arg-AMC and Z-Phe-Arg-AMC. The optimum pH for the protease activity was determined to be 8. This is the first report that characterized the enzymatic properties and analyzed the expression of piscine cathepsin S.