Isolation and Identification of Cronobacter spp. from Fish and Shellfish Sold in Retail Markets.

In this study, we investigated the incidence of Cronobacter spp. in seafood collected from retail fish markets of Mumbai, India. A total of 50 samples comprising fresh finfish (n = 32), shellfish (n = 6), dried fish (n = 9) and water (n = 3) were analyzed for Cronobacter spp. by selective enrichment, isolation and biochemical tests. Of 145 isolates presumptively identified as Cronobacter spp. by biochemical tests, 37 were confirmed as Cronobacter spp. by Polymerase Chain Reaction (PCR) specific to the internal transcribed spacer (ITS) regions. Based on the partial ITS gene sequence analysis, 35 isolates were identified as Cronobacter malonaticus and two as Cronobacter sakazakii. The highest incidence of Cronobacter spp. was in dried fish (55.6%), followed by shellfish (33.3%). The virulence gene ompA was detected in two Cronobacter sakazakii isolates. This is the first report of the incidence of Cronobacter spp. in fresh and dried seafood from India, which highlights the need to focus on this emerging pathogen in tropical seafood.

Evaluation of physicochemical and functional properties of spray-dried protein hydrolysate from non-penaeid shrimp (Acetes indicus).

BACKGROUND: Protein hydrolysate powder was prepared from non-penaeid shrimp (Acetes indicus) by enzymatic hydrolysis using Alcalase enzyme. Extraction conditions such as pH (6.5, 7.5 and 8.5), enzyme to substrate ratio (1.0, 1.5 and 2.0) and temperature (40, 50 and 60 °C) were optimized against the degree of hydrolysis using response surface methodology. RESULTS: Protein hydrolysate comprised of 740 g kg-1 protein, 150 g kg-1 ash and 90 g kg-1 fat contents. The amino acid score showed superior attributes with 56% essential amino acids. Furthermore, the functional properties of spray-dried protein hydrolysates were evaluated. Protein solubility was found to be the 90.20% at pH 2 and 96.92% at pH 12. Emulsifying properties were found to vary with the concentration of protein hydrolysates and the highest emulsifying capacity (26.67%) and emulsion stability (23.33%) were found at a concentration of 20 mg mL-1 . The highest and the lowest foaming capacity were observed at pH 6 and pH 10 with a concentration of 20 mg mL-1 . The water holding capacity of protein hydrolysate was found to increase with concentration, with a value of 5.4 mL g-1 at a concentration of 20 mg mL-1 . CONCLUSION: The results of the present study indicate that the use of A. indicus for the production of protein hydrolysate has good functional properties and nutritional value, rendering it suitable for broad industrial food applications. © 2019 Society of Chemical Industry.

Trophic state modeling for shallow freshwater reservoir: a new approach.

Lakes and reservoirs around the world are facing a substantial water quality degradation problem that poses significant environmental, social, and economic impacts. Reservoir productivity is influenced primarily by the climatic, morphometric, and hydro-edaphic features. High nutrient loadings in the reservoir from agriculture runoff often provide ideal conditions for algal blooms, leading to eutrophication. Reservoir and lake management to prevent or reduce eutrophication, therefore, has become the need of the hour. The traditional approach of trophic state monitoring by rigorous field surveys and eutrophication modeling has been revised in the present study by developing a new trophic state index (TSI)-based model for tropical shallow freshwater reservoirs. The new model has been constructed based on Carlson's Limnology and Oceanography, 22, 361-369, (1977) guidelines by establishing an empirical relationship between trophic parameters including total phosphorus (TP), Secchi disk depth (SDD), and chlorophyll (Chl-a). After comparing the new model with various earlier models for its applicability and validation with actual field conditions, it was found to be most precise over previous TSI models. Temporal and spatial fluctuations in the water quality of the Tiru reservoir were primarily attributed to the changing climatic conditions during the study period. Seasonal monsoon with less frequency, heavy nutrient loading from agriculture runoff, and increased turbidity due to a high level of sediment inflow during monsoon raised the TSI (SDD) values of the Tiru reservoir to place it in the hyper-eutrophic class. Average TSI values during winter for SDD, Chl-a, and TP were indicative of the meso-eutrophic to eutrophic state. Saturation of nutrients due to low water level during summer season caused the poly-eutrophic condition for TSI (SDD)- and TSI (TP)-based estimates and eutrophic condition as per TSI (Chl-a) estimates. However, seasonal deviations of the TSI values based on the relationship between TSI (Chl-a) and TSI (SD) indicated a predominance of smaller particles (non-algal turbidity) during all seasons. Even though TP present in the Tiru reservoir is controlling the algal production, it is also affected by low-light conditions due to non-algal turbidity. The recommendation from this study is that the TSI method for estimating the health of the water bodies is the efficient, cost-effective, and time-saving approach. The model developed during the study would help managers and policy makers to take necessary steps to reduce eutrophication levels in the reservoir and would be helpful for researchers in developing new concepts and protocols, mainly focusing on shallow freshwater reservoirs.

Effective remediation of fish processing waste using mixed culture biofilms capable of simultaneous nitrification and denitrification.

Fish processing waste water causes pollution and eutrophication of water bodies when released untreated. Use of bacteria capable of simultaneous nitrification and denitrification (SND) as biofilms on carriers in a moving bed bioreactor (MBBR) is a popular approach but seldom used for fish processing waste water remediation. Here, we studied the variations in biofilm formation and application activities by isolates Lysinibacillus sp. HT13, Alcaligenes sp. HT15 and Proteus sp. HT37 previously reported by us. While HT13 and HT15 formed significantly higher biofilms in polystyrene microtitre plates than on carriers, HT37 exhibited highest on carriers. A consortium of the three selected bacteria grown as biofilm on MBBR carriers exhibited better remediation of ammonia (200-600 ppm and 50 mM) than the individual isolates on carriers. The mixed biofilm set on the carriers was used for nitrogenous waste removal from fish processing waste water in 2 and 20 L setups. The total nitrogen estimated by elemental analysis showed complete remediation from 250 ppm in both 2 and 20 L waste water systems within 48 h. The usual toxic nitrogenous components-ammonia, nitrite and nitrate were also remediated efficiently.

Simultaneous nitrification and denitrification by novel heterotrophs in remediation of fish processing effluent.

Three isolates viz. Lysinibacillus sp. HT13, Alcaligenes sp. HT15 and Proteus sp. HT37 isolated from fish processing effluent and having a C/N ratio of 2, removed 218, 169, and 400 µg cell(-1) day(-1) NH4(+)-N, respectively without subsequent build up of nitrite or nitrate. Ability of the selected isolates in removing NH4(+)-N, NO2(-)-N, and NO3(-)-N was checked in the presence of four commonly reported and tested effluent carbon sources viz. pyruvate, glycerol, methanol, and acetate. Further, when supplemented to fish processing wastewater containing 234 ppm total Kjeldahl's nitrogen, Lysinibacillus sp. HT13, Alcaligenes sp. HT15, and Proteus sp. HT37 could remediate 95.74, 86.17, and 76.6% nitrogen, respectively in 48 h. This is the first report of a Lysinibacillus sp. carrying out aerobically the process of simultaneous nitrification and denitrification. The results demonstrate the potential of the isolates for use in treatment of fish processing effluents and demonstrating the efficient removal of ammonia.

A multivariate statistical approach for the evaluation of spatial and temporal dynamics of surface water quality from the small reservoir located in the drought-prone area of South-West India: a case study of Tiru reservoir (India).

With the use of different multivariate statistical analysis methods, spatio-temporal fluctuations in the water parameters of Tiru reservoir located at the Marathwada drought-prone area of Maharashtra, India, have been analysed and reported in this case study. Tiru reservoir, situated on the tributary of the Godavari River, was regularly monitored at five different sites from August 2017 to January 2019 for the estimation of 20 water quality parameters. Various multivariate methods such as pattern reorganisation using cluster analysis (CA), factor analysis/principal component analysis (FA/PCA), and discriminant analysis (DA) were used for handling complex datasets. CA extracted three different clusters from five sampling sites with similar water quality characteristics. FA/PCA extracted thirteen factors (65% of 20 measured) required to explain 74% of the data variability and identified the factors accountable for variation in water quality and also evaluated the prevalence of each cluster on the overall dissimilarity at five different sampling sites. Discriminant analysis extracted a total of 16 parameters with 97.7% right assignations. Varifactors (VFs) acquired by factor analysis recommended that the water quality parameters accounted for variation were linked to two groups. The first group included water quality parameters like T, DO, SDD, turbidity, TDS, PA, and MA, whereas the second group covered most of the nutrients Cl-, silicates, PP, TP, NO3-N, NO2-N, and NH3-N; hardness; and CHL-a and mainly entered the reservoir during surface runoff from agriculture fields and the surrounding area containing domestic as well as animal waste. Thus, the present work showed the efficiency of multivariate methods for the assessment of spatial as well as a temporal variation in the water quality of a small reservoir.

Effect of Sodium Hypochlorite on Biofilm-Forming Ability of Histamine-Producing Bacteria Isolated from Fish.

Histamine poisoning occurs when temperature-abused marine fish containing elevated levels of histamine are consumed. Histamine-producing bacteria found in fish can colonize processing surfaces and form biofilms. In this study, the biofilm-forming abilities of histamine-producing bacteria from Indian mackerel (Rastrelliger kanagurta) and the effect of hypochlorite treatment on biofilm formation were studied. The isolates of this study produced histamine in the range of 471 to 2,126 ppm. The histidine decarboxylase gene hdc was detected in all isolates producing histamine except in one strain each of Psychrobacter pulmonis and Proteus vulgaris. All isolates tested in this study produced moderate biofilms under control conditions, whereas exposure to 1 and 3 ppm of sodium hypochlorite significantly enhanced biofilm formation. However, exposure to 5 ppm of sodium hypochlorite showed an inhibitory effect on biofilm formation by all the isolates except Klebsiella variicola. The results of this study suggest that histamine-producing bacteria can form stable biofilms and that this activity may be enhanced by the application of low levels of sodium hypochlorite, a phenomenon that might influence the persistence of histamine-producing bacteria in fish processing areas.

Separation of marine bacteria according to buoyant density by use of the density-dependent cell sorting method.

The purpose of this study was to test whether some phylogenetic groups of natural marine bacteria have unique buoyant densities that allow them to be separated by the density-dependent cell sorting (DDCS) method. We first concentrated a natural bacterial assemblage to collect sufficient numbers of cells. They were separated into three fractions by DDCS, and the community structure in each was clarified by fluorescence in situ hybridization. The cells of Archaea tended to appear in the high-density fraction, whereas those of Cytophaga-Flavobacterium-Bacteroides were in the low-density fraction. We also calculated the sedimentation velocities of three typical marine bacteria (low density, middle density, and high density) using their buoyant density. The sedimentation velocities were approximately 10, 20, and 30 microm h(-1); these velocities have ecological implications when the heterogeneity of bacteria is considered at a microscale. To our knowledge, this is the first report on the buoyant density of natural marine bacteria.

Density-dependent sorting of physiologically different cells of Vibrio parahaemolyticus.

A pure bacterial culture is composed of clonal cells in different physiological states. Separation of those subpopulations is critical for further characterization and for understanding various processes in the cultured cells. We used density-dependent cell sorting with Percoll to separate subpopulations from cultures of a marine bacterium, Vibrio parahaemolyticus. Cells from cultures in the exponential and stationary phases were fractionated according to their buoyant density, and their culturability and ability to maintain culturability under low-temperature and low-nutrient stress (stress resistance) were determined. The buoyant density of the major portion of the cells decreased with culture age. The culturability of stationary-phase cells increased with increasing buoyant density, but that of exponential-phase cells did not. Stress resistance decreased with increasing buoyant density regardless of the growth phase. The results indicate that density-dependent cell sorting is useful for separating subpopulations of different culturabilities and stress resistances. We expect that this method will be a powerful tool for analyzing cells in various physiological states, such as the viable but nonculturable state.