Storage induced changes in coliform, heterotrophic groups of bacteria and nutrient levels of human urine for its safe use in biological production.

Human urine is a potential source of various nutrients, minerals and trace elements. Its use as a fertilizer is growing popular among farmers. Here, we examined the pattern of changes in the counts of coliform, heterotrophic bacteria as well as physico-chemical characteristics of human urine during different days of storage under closed conditions at ambient temperature. We observed that after 253 days of storage under closed condition, the coliform counts were reduced significantly and remained within the safe limit to be used as fertilizer. With increase in storage period, the concentration of phosphate showed decline coupled with rise in pH, alkalinity and electrical conductance. Our study revealed that human urine can be used as safe fertilizer after 8 months of storage under closed conditions at ambient temperature ranging 25-35ºC.

Use of human urine in phytoplankton production as a tool for ecological sanitation.

Measurements of primary productivity of phytoplankton and enumeration of the counts of coliform and heterotrophic bacteria (HB) were made in the water of 12 experimental tanks used for 3 treatments and control in triplicate as follows: (a) fresh human urine (0.02%), (b) stored human urine (0.02%), (c) mixed urine of fresh and stored human urine (0.02%) and (d) control without input of urine. The gross primary productivity of phytoplankton was highest in the stored urine treated tanks (508 mg C m(-2) h(-1)) followed by fresh urine (353 mg C m(-2) h(-1)), mixed urine (303 mg C m(-2) h(-1)) and control (215 mg C m(-2) h(-1)). Similar was the response of net primary production of phytoplankton. The mean count of HB observed in stored urine fed tanks was significantly higher (59-184%) than the remaining urine fed treatments. The mean count of Escherichia coli did not differ from urine treated tanks to control implying the good quality of water. The concentration of dissolved oxygen of water (7.6 to 12.8 mg L(-1)) in these tanks remained satisfactory for aquaculture. The mean concentration of ammonium-N observed in fresh urine treated tanks was more than 10 times higher than the remaining treatments employed. In contrast, the level of phosphate and electrical conductivity in the stored urine treated tanks were significantly higher than the remaining treatments. It is proposed that stored urine with a significantly reduced load of E. coli might be an effective low cost liquid fertilizer for algal biomass production.

Cadmium tolerance and antibiotic resistance in Escherichia coli isolated from waste stabilization ponds.

The incidence pattern of cadmium tolerance and antibiotics resistance by Escherichia coli was examined periodically from the samples of water, sludge and intestine of fish raised in waste stabilization ponds in a sewage treatment plant. Samples of water and sludge were collected from all the selected ponds and were monitored for total counts of fecal coliform (FC), total coliform (TC) and the population of Escherichia coli, which was also obtained from the intestine of fishes. Total counts of both FC and TC as well as counts of E. coli were markedly reduced from the facultative pond to the last maturation pond. Tolerance limit to cadmium by E. coli tended to decline as the distance of the sewage effluent from the source increased; the effective lethal concentration of cadmium ranged from 0.1 mM in split chamber to 0.05 mM in first maturation pond. E. coli isolated from water, sludge and fish gut were sensitive to seven out of ten antibiotics tested. It appears that holistic functions mediated through the mutualistic growth of micro algae and heterotrophic bacteria in the waste stabilization ponds were responsible for the promotion of water quality and significant reduction of coliform along the sewage effluent gradient.

Cadmium tolerance and antibiotic resistance of Pseudomonas sp. isolated from water, sludge and fish raised in wastewater-fed tropical ponds.

The numbers of Pseudomonas sp. isolated were counted in samples collected from water, sludge and intestine of fishes raised in different wastewater ponds along an effluent gradient in a sewage treatment plant. Total fish yield in the last maturation pond increased by 73% over the yield in first maturation pond or facultative pond. The number of Pseudomonas sp. isolated from the intestine of the tilapia (Oreochromis mssambicus) raised in facultative pond, was more than three times the counts (7.22 x 10(8)/g) observed in the last maturation pond (2.025 x 10(8)/g). The effective lethal concentration of cadmium for Pseudomonas sp. isolated from the intestine of the tilapia was 0.6 mM and 0.08-0.09 mM when the fish was procured from facultative pond and last maturation pond, respectively. The Pseudomonas sp. isolated from the intestine of the tilapia did not have resistance to any of the ten antibiotics tested. However, the bacterium isolated from raw sewage, water and sediment of the anaerobic pond was resistant to seven out of ten antibiotics tested.

Responses of succinate dehydrogenase and non-specific alkaline phosphatases and mortality of tilapia to ambient pH stress in a sewage-fed aquaculture pond.

The fish, tilapia (Oreochromis mossambicus) of 50-60 g body weight was experimentally exposed to effluent gradients of highly alkaline pH in a sewage-fed aquaculture farm for examining the pH stress-induced responses of mortality and the stress marker enzyme succinate dehydrogenase and the non-specific alkaline phosphatases of fish prior to death at different hours of intoxication. A second trial was performed after two months when water quality changed along the sewage effluent gradient. An in situ experiment was also performed for better understanding of the responses of enzymatic activities attributable to different levels of pH conditions. Time required for 100% mortality of fish tended to increase from 30 min in pH 11.6 to 22 hr in pH 10.2. There was no mortality of fish when water quality improved significantly (with pH ranging between 9.6 to 8.0) after two months. The activities of succinate dehydrogenase and intracellular alkaline phosphatases assayed in gills and liver prior to death of fish tended to reduce with increase in survival hour, following a pattern of decay curve. On the other hand, percent of enzymatic inhibition of the exposed fish over the control increased as the survival hour increased following a pattern of exponential curve. It appears that the highest water pH of 11.6. maximum ratio for ammonium to ammonium hydroxide (1: 21) and reduced level of dissolved oxygen (2.62 mg/l) were perhaps responsible for the 100% mortality of fish within 30 min of their exposure and the enzymatic activities in the gills and liver assayed prior to death of fish tended to reduce as the acclimatization period of fish increased and vice-versa.

Cadmium induced adaptive responses of certain biogeochemical cycling bacteria in an aquatic system.

The population growth of some biogeochemical cycling bacteria (heterotrophic bacteria (HB), ammonifying bacteria (AB), ammonia oxidizing bacteria (AOB), denitrifying bacteria (DNB) and cellulose decomposing bacteria (CDB)), as well as ammonification, and denitrification rates were determined in simulated pond systems treated with full doses (FDs) and split doses (SDs) of cadmium. Reductions in bacterial density and nitrogen activity rates were more severe for the FD and SD followed by a slight recovery in both. Among the test groups, reductions were maximal in the AOB and DNB and lowest for the CDB populations, suggesting that the latter had greater potential for cadmium resistance. Bicarbonate alkalinity of water was found to exert a profound influence in counteracting cadmium stress in the system, as strong negative correlations between this parameter and bacterial mass were evident in the SD treatment. Though the aquatic microbial populations were greatly altered by cadmium stress, the normal operating range of the system was restored at a later time with the CDB population showing higher degree of adaptive responses.

Distribution pattern of ambient cadmium in wetland ponds distributed along an industrial complex.

Water and sediment samples collected from 18 wetland ponds within and outside industrial areas were examined for cadmium concentration and water quality parameters during the period of January to July 1996. The Cd contents in gill, liver, mantle and shell of freshwater mussel (Lamellidens marginalis) as well as leaves and roots of water hyacinth Eichhornia those occurred in these ponds were also estimated. Cd concentration ranged from 0.006 to 0.7025 mg/l in water and from 7 to 77 microg/gdw in sediments of all the ponds investigated. The amount of Cd occurring in water and sediment was much higher in concentrations in the ponds located in Captain Bheri and Mudiali farm close to industrial areas, compared to remaining ponds located outside the industrial belt. Lamellidens marginalis procured from Mudiali and Captain Bheri ponds showed regardless of size, tissue and season of collection significantly higher Cd concentration than did those from other ponds. Likewise, tissue Cd in Eichhornia collected from Mudiali pond was as high as 125-152 microg/gdw in root and 21-63 microg/gdw in leaves compared to 40-108 microg/gdw in root and 9-43 microg/gdw in leaves in the remaining ponds. Seasonal variability of Cd was clear-cut; the concentration was relatively higher in water and sediment in all ponds during summer than during monsoon season or winter. Size-wise, smaller groups showed the highest concentrations of Cd in all tissues of Lamellidens compared with medium and large size groups. Concentration factor for all tissues of Lamellidens regardless of size and season, was inversely proportional with the ambient Cd concentrations. Concentration factor estimated for all tissues in all ponds and all seasons was in the order: liver>gill>shell>mantle. As all ponds located outside the industrial belt showed Cd concentrations ranging from 0.006 to 0.049 mg/l, it is suggested that these wetlands do not pose serious risk to the environment.

In situ cadmium reclamation by freshwater bivalve Lamellidens marginalis from an industrial pollutant-fed river canal.

The biofilter potential of the freshwater bivalve, Lamellidens marginalis was examined in cage experiments conducted in a river canal (Ichhapore, 24-Parganas, West Bengal, India) receiving industrial effluents from steel and metal factories as well as from an ordinance factory. Cadmium is one of the major contaminants in this river canal. Lamellidens collected from pollution free natural ponds, were sorted into three size groups (large: 59+/-3.2 g, 10+/-2.3 cm; medium: 30+/-2 g, 6+/-1.7 cm and small: 13+/-1.5 g, 4+/-1.2 cm) were held in cages at three different sites along a cadmium concentration gradient. Concentrations of cadmium were measured from water, sediment and different tissues of Lamellidens at weekly intervals using atomic absorption spectrophotometric methods. Cadmium uptake by Lamellidens in all media were highly concentration dependent in both summer and winter months. For all three size groups, cadmium uptake was maximum in the gills at the beginning of experiment, and liver at the later phase. Cadmium uptake was maximum in the small bivalves and minimum in the large bivalves groups. Cadmium uptake was 11-67% higher during summer than during the monsoon season for all tissues and size groups. Estimation of concentration factor revealed that tissues were saturated with cadmium during the 13-14th week after Lamellidens introduction during summer, but remained unsaturated during the monsoon season. It is concluded that Lamellidens might be considered as an efficient biofilter for reclamation of aquatic environment having sub-lethal concentrations of cadmium.

Hatching of common carp (Cyprinus carpio L.) embryos stored at 4 and -2 degrees C in different concentrations of methanol and sucrose.

The hatching performance of common carp (Cyprinus carpio L.) embryos was examined after 12-72-h storage at 4 and -2 degrees C using different concentrations of sucrose (0.1, 0.25, 0.5 and 1.0 M or 3.42, 8.55, 17.10 and 34.2%), methanol (MeOH) (0.5, 1.0, 1.5, 2.0, 2.5, 3.0 and 3.5 M or 1.6, 3.2, 4.8, 6.4, 8.0, 9.6 and 11.2%), or varying concentrations of methanol in 0.5 M (17.10%) sucrose. For sucrose, 0.5 M (17.10%) showed the maximum survival (41+/-1% (12 h) to 11+/-1.5% (72 h)) at 4 degrees C. No survival was observed at -2 degrees C with any concentration of sucrose. At both temperatures employed, hatching was higher with mixed combination of methanol (1.5 M or 4.8%) and 0.5 M (17.10%) sucrose (4 degrees C: 41+/-1.5% (12 h), 38+/-1.2% (72 h); -2 degrees C: 33+/-1.7% (12 h), 28+/-1.2% (72 h)) compared to methanol alone (4 degrees C: 38+/-1.5% (12 h), 35+/-2.5% (72 h); -2 degrees C: 31+/-2.5% (12 h), 25+/-2% (72 h)). The combination of 1.5 M (4.8%) methanol and 0.5 M (17.10%) sucrose produced the best results among all the concentrations tested at both temperatures.

Oxygen uptake and filtration rate as animal health biomarker in Lamellidens marginalis (Lamarck).

The freshwater bivalve, L. marginalis was experimentally exposed to 10 and 30 ppm concentrations of CdCl2 to examine filtration rate, oxygen uptake and glycogen level of liver and gills for health assessment for their reuse in the reclamation of cadmium intoxicated environments. In situ experiment was also performed for better appraisal of the filtration rate in the lake. Oxygen uptake in the treated group exceeded that of control by 15-22% during the early 24 hr after cadmium exposure, but followed an essential decline (23-30%) thereafter. The reduction of filtration rate ranged from 12-62% in laboratory to 83-85% in field trials. At the tissue level, glycogen content was reduced by 61-72% in liver and 52-63% in gill. In both tissues, glycogen content was inversely proportional to the cadmium contents of the animal. Critical appraisal of data suggests that the threshold values of cadmium in gill and liver were 50-80 microg/g dw for oxygen uptake and 50-60 microg/g for filtration rate because of marked reduction of these parameters beyond the values of cadmium. It is concluded that filtration rate, oxygen uptake of the freshwater bivalve, L. marginalis can be used as biomarker for animal health assessment and for possible reuse of the stock animals.

Distribution of nitrifying bacteria in fish culture tanks: influence of management factors.

Examination of microbial populations of ammonia and nitrite oxidizers in model tanks used for farming of air-breathing catfish (ABFFS), polyculture of herbivorous carps (PS) and control system (CS) revealed maximum abundance of ammonia and nitrite oxidizers in ABFFS and minimal in CS. In any of the two culture systems, the chemical concentration or density of bacteria developed due to feed application was many times higher than other management factors. At a given time, the bacteria of each group occurring in any management protocol of ABFFS was about 10% higher than PS. Culture of air-breathing fishes caused greater abundance of ammonia and nitrite oxidizers than the herbivorous carps. There was a strong positive correlation between each of the chemical concentration of produced nitrite (ammonia oxidizers) and density of nitrite oxidizers and ratio of total nitrogen to phosphate.

Stimulatory effect of herbicide 2,4-D on the heterotrophic microbial community in the water of three fish ponds.

Herbicide 2,4-D at a dosage of 500 mg l-1 stimulated the proliferation of the heterotrophic bacterial community present in the water of three fish ponds over a period of one year. 2,4-D due to its toxic action might kill the planktonic algal population occurring in the pond water, which on degradation generated more nutrients conductive to bacterial proliferation. The microbial density in different hours were closely correlated (p less than 0.01 less than 0.05) with the variations of each of NH4-N, NO2-N and specific conductivity of water, while the relationship between the former and PO4-P was reciprocal (P less than 0.001).

Stage-dependent hatching responses of rohu (Labeo rohita) embryos to different concentrations of cryoprotectants and temperatures.

Hatching performances of three embryonic stages of postfertilization rohu (Labeo rohita) (9-, 12-, and 15-h) were examined after treatment with various concentrations (0.5-4.5M) of two cryoprotectants (methanol and propylene glycol) supplemented with 0.1M trehalose. Different lengths of storage (1-48 h) and temperature (-4 degrees C to ambient) were studied. Of the three stages of embryonic development, the 12-h stage proved to be the most suitable stage for low temperature storage, showing the highest percentage of hatch out (72+/-2%) with 2.0M methanol and 0.1M trehalose. Methanol was more useful for storage at higher temperatures and propylene glycol at subzero temperatures. The maximum possible duration of effective storage of 12-h embryos was 31h in 2.0M methanol at 0 degrees C. No hatch out was found beyond 31h of storage with all concentrations of methanol at 0 degrees C. The results of interactions was that the optimal concentration of methanol was 3.0M at 4 degrees C, 2.0M at 0 degrees C, and 1.5M at 4 degrees C. Among three embryonic stages 12-h stage showed better results in trehalose treatment than sucrose. Among all concentrations of trehalose tested 0.1M gave the maximal survival rate of the rohu embryos.

The hatching of common carp (Cyprinus carpio L.) embryos in response to exposure to different concentrations of cryoprotectant at low temperatures.

The hatching performance of embryos of the common carp (Cyprinus carpio L.) was examined after 1, 7, 14, 21, or 28 days of storage at -8, -6, -4, -2, 0, 2, or 4 degrees C with different concentrations of methanol (0.5-7.0 M in 0.5 M steps) or varying concentrations of methanol in 0.1 M sucrose or trehalose. Preserved embryos failed to hatch after storage at -8 and -6 degrees C, regardless of the duration of storage or the concentrations tested. Likewise, there was no hatching out above 5.0 M concentration of methanol, even with the addition of sucrose or trehalose. After storage at 2 or 4 degrees C, the hatching rate was higher with mixtures of methanol (1.5 M) and trehalose (0.1 M) than with methanol plus sucrose or methanol alone. At 4 degrees C, the solution containing 1.5 M methanol supplemented with trehalose gave the highest hatching response of embryos stored for 14 days. Comparison of hatching after 24h of storage at the effective temperatures (-4, -2, 0, 2, and 4 degrees C) revealed that low concentrations of methanol were effective at high temperatures and high concentrations at sub-zero temperatures. The combination of 0.1 M trehalose with 1.5 M methanol gave the highest percentage hatching out both at 4 and 2 degrees C. At 0 degrees C, the highest percentage hatching occurred with 0.1 M trehalose plus 2.5 M methanol and at -2 and 4 degrees C, the best results were with 0.1 M trehalose plus 3.0 M methanol.

Biogeochemical cycling bacteria as indices of pond fertilization: importance of CNP ratios of input fertilizers.

AIMS: The influence of carbon-nitrogen and nitrogen-phosphorus ratios of input fertilizers, and that of pond water, on the growth of heterotrophic and phosphate-solubilizing bacteria of water and sediment, was examined in relation to fertilizer mineralization indices using different modes of fertilization through inorganic and organic sources. METHODS AND RESULTS: The first experiment used carbon-nitrogen-phosphorus ratios varying from 12 : 2 : 1 to 151 : 6 : 1, applied at the rate of 0.043 g l(-1) week(-1), whereas in the second ratios varied from 25.6 : 6.2 : 1 to 150 : 12 : 1 applied once at the rate of 3.33 g l(-1). Different fertilizers (cattle dung, poultry droppings, urea, single superphosphate and starch) were mixed in different proportions to achieve the desired carbon-nitrogen-phosphorus ratio. The heterotrophic and phosphate-solubilizing populations were more responsive to an early manuring phase than later, implying that pond fertilization was microbiologically more dynamic in the earlier phase. The carbon-nitrogen and nitrogen-phosphorus ratios of 11.8 (88.6 : 7.5) and 7.5 (7.5 : 1), respectively, of input fertilizers favoured growth of both heterotrophic and phosphate-solubilizing bacterial populations much better than the other ratios tested. Likewise, water carbon-nitrogen and nitrogen-phosphorus ratios of 11.9 and 3.34 induced bacterial growth. The carbon-nitrogen ratios of 12.63 (101 : 8) (input fertilizer)-4.54 (water), and nitrogen-phosphorus ratios of 8 (8 : 1) (input fertilizer)-2.93 (water), gave gross primary productivity values higher than the remaining ratios, exhibiting overall curvilinear relationships. The values of gross primary productivity were the direct function of values of fertilizer mineralization indices for carbon, nitrogen and phosphorus. CONCLUSION: It is concluded that the mixed fertilizer (carbon-nitrogen-phosphorus-88.6 : 7.5 : 1) comprising cattle dung (95%), poultry droppings (2.5%), urea (2%) and single superphosphate (0.5%), applied at the rate of 23,000 kg ha(-1) year(-1), was a suitable cost-effective fertilization option for aquaculture practices. SIGNIFICANCE AND IMPACT OF THE STUDY: As chemical fertilizers are expensive and cause some adverse effects on the soil structure, composition, microflora and other characteristics of the pond, mixed combinations of inorganic and organics with narrow range of carbon-nitrogen-phosphorus ratio can be suitable and cost-effective fertilization tools in aquaculture practices, which is to be linked with the microbial activities of the pond.