Program

Sea cucumber Apostichopus japonicus (Selenka) is one of the most important fishery resources in many Asian countries, especially in China. Recently, wild stock has drastically declined due to overfishing. Although the seed production is in urgent need of conservation, the huge variation in growth in their body sizes has delayed the development of intensive aquaculture. Unexpectedly, our recent study demonstrated that fecal bacteria, polyhydroxybutyrate (PHB) producers affiliated to Rhodobacterales, are a possible cause of the growth variation (Yamazaki et al., Sci. Rep. 6:21631, 2016). However, it is not clear whether Rhodobacterales is a member of natural fecal microbes of A. japonicus or is observed only in cultured individuals, and whether the bacteria are found in the feces of other sea cucumber species. To address these questions, we investigated community structures of the fecal and sediment microbiotas of sea cucumbers living at two different geographical sites (Menagawa and Ishigaki sites). A. japonicus was sampled from the Menagawa site and multiple species of sea cucumbers were sampled from the Ishigaki site. Although high-throughput 16S rRNA sequencing revealed the fecal and sediment microbial communities were significantly different, most of the fecal OTUs were also observed in sediments. Statistical comparisons showed specific taxa were significantly more abundant in feces than in sediments at each site. Interestingly, Rhodobacterales was more abundant in feces than in sediments at both sites. These results imply sea cucumbers shape their gut microbiota based on sediment microbiota and enrich beneficial bacteria (e.g. Rhodobacterales) in their guts. Since sea cucumbers repeat the process of ingesting sediments and excreting of feces many times in a day, this animal can modify sediment microbial communities to enrich the beneficial bacteria as a “microbial gardener”.

Mud crab Scylla serrata culture can produce accumulation of nitrogen wastes, that can affect the condition of D DON/ Dissolved Organic Nitrogen (protein) and DIN/ Dissolved Inorganic Nitrogen (NH4+ / NH3, NO2-, and NO3-). The availability of DIN in the recirculation aquaculture system is inseparable from microbiological process of nitrogen cycle and its microorganism activity, especially nitrification, denitrification and Dissimilatory Nitrate Reduction to Ammonia (DNRA) process. Utilization of the best-performing indigenous microorganisms, is expected to assist in the removal of harmful N-inorganic compounds in the recirculation aquaculture system. The method in this research consisted of isolation of nitrification bacteria, denitrification, DNRA with specific medium, activity test, potential rate, and 16S rRNA gene identification. The best of activity and activity rate of some bacteria were selected isolate groups, is three denitrification nitrification couple isolates with high value of activity rate HIB_ b rate of NH4+ 0.29 μM.h^-1, HIB_ d rate of NH4+ 0.39 μM.h^-1 and HIB_ f 0.45 μM.h^-1, one denitrification isolate HIB_7 with NO₃^-, 0.48 μM.h^-1, reduction rate and three DNRA isolates HIB_27 value of NO3-, 0.05 μM.h^-1 reduction rate; HIB_95 value of NO3- 0.07 μM.h^-1 reduction rate and HIB_96 with NO3- 0.07 μM.h^-1 reduction rate. Identification result is 99-100% of three selected isolates from coupled nitrification denitrification were included in the Pantoea calidagroup, Pseudomonas stutzerii and Halomonas sp. Potential denitrification bacteria found only one isolate were Stenotrophomonas maltophilia and three potential of DNRA bacteria ie Bacillus subtilis group, Staphylococcus nepalensis and Staphilococcus cohnii.
Keywords: nitrification, couple nitrification denitrification, denitrification, DNRA, aquaculture, recirculation, Scylla serrata

Ciprofloxacin (CIP) is a fluoroquinolone antibacterial drug which widely used in treatment of serious infections in humans. A significant quantity of CIP may be excreted unchanged and introduced into the environment through wastewater, predominantly from clinical settings. Therefore, studies on the biodegradability of drugs can be taken as a very first step of an environmental risk assessment. The present study reports the biodegradation of CIP by Lactobacillus gesseri, Enterobacter sp., Bacillus sp., Bacillus subtilius and Micrococcus luteus which were isolated as CIP resistance, nonpathogenic bacteria. The presence of antibiotic-resistant bacteria was identified using the 16s rRNA sequencing. A 0.5ml of overnight starved bacterial suspensions was introduced into medium containing CIP at 5 ppm. Triplicate samples were incubated at 280C with shaking at 100ppm. A 0.5 ml of subsamples was removed at 2 days interval for a period of 14 days. Samples were subjected to High Performance Liquid Chromatography (HPLC) analysis. Fourier Transform Infrared Spectroscopy (FTIR) analyses were carried out for each sample at the end of the 14 days to find structures of by-products. Complete degradation of CIP by L. gasserri was detected at the end of 14 days of incubation with average degradation rate of 0.182 ±0.15µg /day. Descending degradation rates were followed by Enterobacter sp. (0.75 ±0.03 d-1) and Bacillus sp. (0.41±0.02d-1) at 8 and 6 days respectively. However, clear cut degradation of CIP was not detected when B.subtilis and Micrococcus luteus were employed even after 14 days of incubation. Further, FTIR spectrum revealed that incubation of L. gesseri, Enterobacter sp. and Bacillus sp., changed the piperazine ring and quinolone part in the CIP structure while degradation occurred.

WHO estimated, globally around 1.5 billion people are suffer from waterborne diseases and 3.4 million people die directly or indirectly due to consumption of contaminated water. Salmonella spp., Shigella spp., and Campylobacter spp. are the bacteria which frequently cause waterborne diseases in worldwide. Kelani river basin caters as the main source of drinking water for over 2 million inhabitants in the capital city Colombo. Due to lack of information on groundwater quality data related to pathogenic bacteria, the present study was carried out to identify pathogenic bacterial contamination in the groundwater sources of the Kelani river basin. Shigella spp., Salmonella spp., Campylobacter spp., total coliform and feacal coliform were identified along with some physico-chemical parameters of water using standards methods. Seventy two groundwater sampling locations in the river basin were selected for the study during dry and wet seasons. The results showed that the entire Kelani river basin was contaminated with total and feacal coliform bacteria (colony count-200<) and the recorded numbers were greater than the threshold values given by SLS (Sri Lanka Standards) and WHO guideline for drinking water. It was detected that seventeen sampling locations were positive for Salmonella spp. and two were positive for Campylobacter spp. during the study period. It was found that bacterial contamination was high during the dry season. However, Shigella spp. was not recorded during the study. ANOVA test was employed to see the differentiation between two seasons and there was no significant difference between two seasons were detected (0.05<p). Thus, the result of the study revealed that people and stakeholders within the premises of the river basin should aware about ground water quality and responsibilities to safe guard aquifers of the river basin.

Keywords: Kelani river basin, Groundwater, Physico-chemical parameters, Shigella spp. Salmonella spp. and Campylobacter spp., ANOVA test.

There are high number of small-scaled gold mining in Ratatotok district and amalgamation-based mining processing still releases the mining wastes into the river flowing to Totok Bay. Metals are difficult to degrade and can be accumulated by algae so that a study needs to be done in order to know whether the algae can become phytoremediator and how far the metal affects the cell structure and the pigment content. This study was directed to analyze the metal content in Chlorodesmis sp., waters and sediment from Totok Bay and Blongko waters, the cell structure and pigment types of Chlorodesmis sp. from both waters. Metal analysis followed APHA method, pigment analysis applied Zapata method-based High Performance Liquid Chromatography, and the effect of metal on the cell structure of the green algae employed Transmission Electron Microscope following method. Results found that metal concentration in Chlorodesmis sp. from Totok Bay waters consisted of 4.80 ppm of Arsen, 0.40 ppm of cadmium, 1.00 ppm of chromium, 0.20 ppm of copper, 2.50 ppm of lead, 7.20 ppm of zinc, 0.63 ppm of mercury, respectively, while that from Blongko waters was < 0.50 ppm of As, 0.04 ppm of Cd, 1.00 ppm of Cr, < 0.50 ppm of Cu, 0.10 ppm of Pb, < 0.70 ppm of Zn, 0.006 ppm of Hg. The cell structure of Chlorodesmis sp. from Totok Bay waters had cell wall damage, the vacuola was reduced, and could occur heavy metal in the cell structure, while that from Blongko waters did not get any damage. In addition, pigment types in Chlorodesmis sp. were also detected as chlorophyll and carotenoid from both waters.

Screening of antibiotic resistance genes (ARG) are important factor for assessing risk of antibiotic resistance in aquaculture environment. In this study, tetracycline (tet A and tet M) and sulfanomide (sul 1 and sul 2) resistance genes were screened in 24 sampling locations including marine water samples (100m, 500m, 1000m), lagoons (Chilow, Puttalam), rivers (Kelani, Kalu, Walawe, Mahaweli) and Horton plains (2000m above from sea level). The Tetracycline (TET and OTC) and Sulfanomide (SUF) contamination levels were analyzed by using High Performance Liquid Chromatography (HPLC). DNA was extracted and Polymerase Chain Reaction (PCR) method was followed for screened the presence of ARGs in the samples. None of the environmental samples were shown contaminations of tetracycline and sulfonamides. However, the tet (M) gene was detected in seven sampling sites, while tet (A) was detected in three locations where Sul 1 and Sul 2 were found in two sites respectively. From the tested ARGs, both tet A and tet M were found at three marine samples (100m), two lagoon samples, whereas both sul 1 and sul 2 were recorded at only in one lagoon sample. No resistance genes were detected in water samples collected from greater than 100 m depth in marine water sample. In conclusion, the presence of tetracycline and sulphonamide resistance genes may limit the effectiveness of antibiotics use in aquaculture industry.

Key words:- Antibiotic resistance genes, Tetracycline, Sulphonamide