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Plastic lining ponds provide better management and healthier environmental conditions. Sulphur cycling bacteria can serve as an indicator of pond environmental conditions.
Introduction
In aquaculture systems, water quality is largely controlled by microbial biodegradation of organic wastes (Avnimelech et al., 1995; Abraham et al., 2004) through mineralization. In aquatic environments, microorganisms play a role in nutrient recycling and organic matter formation and decomposition. Heterotrophic bacteria oxidize organic waste, while autotrophic nitrifying and sulphur bacteria handle troublesome chemicals such ammonium, nitrite, and sulphide (Moriarty, 1997).
Sulphate-reducing bacteria (SRB) and sulphur-oxidizing bacteria (SOB) primarily reduce and oxidize sulphates and hydrogen sulphide in pond bottoms, promoting a healthy environment in commercial shrimp culture ponds (Syed et al., 2006; Rao et al., 2000; Devaraja et al., 2002; Burford et al., 2003; Abraham et al., 2004, 2015; Fernandes et al., 2000). Limited research exists on the role of sulphur cycling bacteria in Penaeus vannamei culture systems.
In 1998, Smith and Briggs recommended full pond liners (bitumen impregnated geotextile) to manage nutrient load in shrimp culture systems.
However, they did not address the impact of pond lining on microbial dynamics, despite its high input and zero water exchange. These investigations compare observations on trends in total heterotrophs, vibrios, and sulphur cycling bacteria (SOB and SRB) are crucial for understanding the ecosystem in commercial P. vannamei cultivation ponds.
Materials and methods
Place of study
✓ Group I studied three earthen ponds (1.0 ha each) 2.5 km from Gulf of Cambay in Onjal village, Navsari district, Gujarat (India) with P. vannamei postlarval (PL 12) stocking density at 30 nos. m-2.
✓ Group II included three 1.0 ha fully lined ponds with P. vannamei post-larvae (PL 12) stocking density of 95 no. m-2, located 2 km from Gulf of Cambayat village in Navsari district, Gujarat (India) and lined with geosynthetic manufactured materials (GSE) (300 μm thickness).
Pond preparation
Both systems began with a water probiotic dose before seed stocking. The ponds were lined with 300 μm GSE. Aeration in earthen ponds was achieved using paddle wheel aerators at 8 HP/ha from 3 h in the morning to 30 days of culture (DOC), 8 h to 50 DOC, 10 h to 75 DOC, and 12 h until harvest.
Aeration was delivered in lined ponds before stocking during pond preparation for rice ferment and probiotic applications. Afterward, 24 hours @ 6 HP/ha up to 30 DOC, 8 HP to 50 DOC, 10 HP to 75 DOC, and 12 HP until harvest.
The shrimp were fed commercial feed (30-35% crude protein, 2.5- 3.0% crude lipid, < 3% crude fiber, < 15% ash, and < 12% moisture). Initially, blind feeding was done up to 28 DOC, later adjusted based on feed intake in check trays (from 60% to 1.8% at the conclusion of culture time).
Food was given in four equal meals daily at intervals of 4 hours. Water probiotics comprised Bacillus sp, while soil probiotics included Rhodobacter, Rhodococcus, and Thiobacillus denitrificans (liquid and powder formulations). Additionally, the central drainage system constantly cleaned accumulated sludge in the liner pond after 70 DOC (Table 1).
A sample collection
Every two weeks, water and sediment samples were collected from ponds in sterile plastic bottles and bags, respectively. They were transported to the lab in an insulated box with precooled gel ice packets. Samples were: pond drying, subsurface soil scraping and liming (Agril. lime).
To maintain a healthy pond, follow these steps: fill with sea water using a four-stage filtration system (20, 40, 60, 80 mesh), bleach with 30% chlorine at 400 kg/ ha, lime with agricultural lime at 100 kg/ha after 3 days, fertilize with fermented juice (100 kg rice bran + 10-kg jaggery + 100 g yeast per 100 L pond water), ferment juice weekly and add microbial products. Post-larvae shrimp stocking processed within 4 h of collection and stored for further analysis.
Physico-chemical analysis
Water samples were analyzed for pH, salinity, calcium, magnesium, total hardness, carbonate, bicarbonate, total alkalinity, nitrite nitrogen and total ammonia nitrogen and sediment samples were analyzed for pH, electrical conductivity, organic carbon, available nitrogen, and available phosphorus using standard procedures [American Public Health Association (APHA), 1998].
Bacteriological analysis
Water and sediment samples were analyzed for heterotrophic bacteria and presumptive vibrio counts on Zobell marine agar and thiosulfate-citratebile saltssucrose agar, respectively (Gilliland et al., 1976; Austin, 1988). Sulphur recycling bacteria, SOB and SRB by Most Probable Number MPN technique using specific medium (Rodina, 1972).
The media employed for the isolation of SOB include both composed of 3.0 g to 0.5 g (NH4 ) 2 SO4 , traces of FeSO4 in 1,000 ml distilled water with pH 8.0. Statistical analysis Statistical significance of difference between the treatments means and correlation analysis was computed using statistical package. Differences between means were determined and compared by Tukey’s test.
Results and discussion
Physico-chemical parameters of water
The levels of physico-chemical parameters like pH, salinity, CO3 -2, HCO3 -1, total alkalinity, NO2 –N and NH3 –N are well within the optimum values (MPEDA, 1992) in both the groups. The pH values in earthen ponds did not show wide variations throughout the culture period, whereas it showed a decreasing trend may be due to higher population of heterotrophic bacterial population in earthen ponds (Panjaitan, 2010).
Soil quality parameters in earthen ponds
The pH value ranged from 7.92 to 8.44 (8.204 ± 0.081), organic carbon percentage ranged from 0.43 to 0.94% (0.72 ± 0.007), available nitrogen content ranged from 101.91 to 230.5 kg/ha (158.5 ± 15.18), available phosphorous ranged from 16.99 to 88.73 kg/ha (56.86 ± 21.22) and available potassium ranged from 3,022.7 to 5,288 kg/ha (442.38 ± 590.47) were within the normal range.
Bacteriological population
The trend in the bacterial populations with the progress of culture is shown in Figures 1–4.
As shown in Table 2, the total bacterial counts (TBC), total vibrio count (TVC), SOB and SRB in both the systems were significantly different (p < 0.01). The average counts of bacterial populations in earthen pond sediments were found to be higher in all the ponds compared with the water samples.
The TBC outnumbered the TVC, SOB and SRB populations indicating the major role of abundant heterotrophic bacteria over the autotrophic beneficial bacterial populations. Similar results were also reported by other authors (Rao et al., 2000; Devaraja et al., 2002; Patil et al., 2012).
In both the groups, the applications of disinfectants, in the present study, though might have worked at the time of application have not helped in controlling the pathogenic bacteria that is vibrios or even the water quality deteriorating bacterial population in the long run and the system comes to its original state where it was before application.
Total bacterial population
The mean TBC of pond water and sediments were close to or above 6.0 log CFU/ml suggesting abundant availability of nutrients in both the systems. During the culture period, the values of TBC observed were in agreement with earlier studies (Abraham et al., 2004, 2015; Patil et al., 2012).
Rao et al. (2000) reported TBC of 3 log 1.40 to 4 log 3.40 CFU/ml in water samples and 3 log 2.60 to 5 log 6.10 CFU/ml in sediment samples. The earthen pond water sample showed the first peak of 6 log 8.60 ± 1.15 CFU/ml at 23 DOC and subsequently the second peak was observed at 104 DOC (6 log 8.37 ± 1.17 CFU/ml), whereas the highest peak in lined ponds was observed at 136 DOC (6 log 7.25 ± 2.05 CFU/ml).
After bleaching of pond water and 5 days prior to stocking of seeds (prestocking period), the TBC were 6 log 0.53 ± 0.06 CFU/ml and 6 log 3.73 ± 0.65 CFU/ml in earthen and lined ponds, respectively. The use of high aerations might be the reason for the high bacterial growth (Fernandes et al., 2010).
The sediment samples of earthen ponds showed slightly higher TBC than the water samples, so also in earlier reports (Abraham et al., 2004, 2015; Patil et al., 2012).
Total presumptive vibrio population
The total presumptive vibrio count was 2 log 0.73 ± 0.05 CFU/ml and 2 log 3.67 ± 0.98 CFU/ml in earthen and lined ponds, respectively. As with TBC, the higher population of TVC in lined ponds may also be due to longer duration of initial preparation time given before stocking, allowing the proliferation of the vibrio proliferation.
The high vibrio load in lined ponds throughout the culture period must be attributed to the higher stocking density in lined ponds. This might be due to the steady increase in the accumulation of organic matter in pond bottom (Moriarty, 1997; Sujatha, 2007) as is witnessed by total quantum of feed per pond in each group (Group I – 9.45 t and Group II – 10.35 t).
Large amount of organic matter in shrimp culture pond is possible due to high stocking density, overfeeding, uneaten feed, fecal matter, fertilizers and overblooming (Kautsky et al., 2000).
Sulphur-oxidizing bacteria (SOB) and sulphate-reducing bacteria (SRB)
The SOB and SRB are important in converting sulphur and sulphur-related compounds. The sulphur recycling bacteria that is SOB and the SRB were significantly lower (p < 0.01) in lined pond compared with earthen ponds throughout the culture period indicating the role of soil substratum requirement for the proliferation and favorable condition requirement for the growth of this bacteria (Abraham et al., 2004, 2015).
The SOB and SRB populations were 4 log 1.44 ± 6.87 CFU/ml, 3 log 8.50 ± 2.17 CFU/ml and 3 log 0.58 ± 0.25 CFU/ml, 3 log 0.85 ± 0.22 CFU/ml, respectively, for earthen and lined ponds. The levels of SOB and SRB counts in the present study were in accordance with Patil et al. (2012) but were much lower than the previous reports (Suplee and Cotner, 1996; Rao et al., 2000). However, Devaraja et al. (2002) and Abraham et al. (2004, 2015) reported even lower counts.
“The results of the present study reflect the intensification of culture practices and effect of the stocking density. The counts of SOB in earthen ponds decreased up to 70 DOC then after showed an increasing trend with a peak of 3 log 45.00 ± 16.46 CFU/ml at 172 DOC.”
A drastic increase after 70 DOC was observed which may be due to increased frequency of soil probiotics application and aeration.
The counts of SOB in lined ponds showed an increasing trend up to 120 DOC with a peak of 3 log 1.33 ± 0.21 CFU/ml and drop at the end of culture period (136 DOC). Though SRBs were considered anaerobic bacteria, they were present both in pond bottom sediments and water column. The earlier studies by Rao et al. (2000), Devaraja et al. (2002) and Patil et al. (2012) also supported the present observations.
The possible reason for higher SRB counts in water column might be attributed to creation of anaerobic conditions at the center of microniche due to higher activity of heterotrophic bacteria (Schramm et al., 1999).
The SRB counts in earthen pond water samples were almost stable up to 85 DOC, increased drastically with a peak at 172 DOC (4 log 2.53 ± 4.62 CFU/ml) indicating the pond deterioration in the second half of the culture. It registered a drastic drop at 133 DOC and then again increased till the end of culture.
Significant reduction in SRB counts coincides with the application of soil probiotics. The role of probiotic applications in improving the pond conditions are supported by several researchers (Devaraja et al., 2002; Patil et al., 2012; Abraham et al., 2015).
Almost similar trend was observed in pond sediments but with slightly higher SRB counts. An increasing trend of SRB in pond sediment samples up to 56 DOC also supports the assumption of deteriorating pond conditions and the drop in SRB count then after coinciding with the application of soil probiotics.
The SRB counts in lined ponds increased up to 59 DOC with a peak of 3 log 2.30 ± 0.70 CFU/ml and then decreased may be due to applications of soil probiotics and regular sludge removal. Also, the SRB populations in lined ponds were managed through regular exchange of bottom sludge using central drainage system otherwise the SRB populations could have outnumbered the SOB populations (Smith, 1998).
“In conclusion, accumulation of organic matter (sludge) leads not only to increases in sediment oxygen demand but also to anaerobic conditions resulting in production of undesirable gasses such as hydrogen sulphide. To avoid these unfavorable conditions in pond environment, sludge has to be managed by removing at certain period of time.”
With the intensification in aquaculture, the accumulation of heavy organic load leads to the deterioration of environment which in turn will lead to poor growth and survival of the cultured aquatic animal (Prawitwilaikul et al., 2006).
Plastic lining ponds provides an easy removal of the organic load thereby permitting higher stocking densities and harvests compared with earthen ponds. It is important to study the dynamics of this recycling microorganism and their behavior in the present system of culture in the context of commercial products application.
“In the present study, the microbial population density differs significantly with the type of culture system in spite of the stocking density and is mostly indicated by the corresponding load of sulphur cycling bacterial populations. Also, the farm level interventions like application of probiotics play a significant role in maintaining the healthier pond environment.”
Further research on managing these populations through suitable and appropriate bacterial consortiums (probiotics formulations), their dosage and schedule of application will aid in improving the water quality requirements of the aquatic organism in different systems of culture.
This informative version of the original article is sponsored by: REEF INDUSTRIES INC
References and sources consulted by the author on the elaboration of this article are available under previous request to our editorial staff.
This is a summarized version developed by the editorial team of Aquaculture Magazine based on the review article titled “ROLE OF POND LINING IN DYNAMICS OF SULPHUR RECYCLING BACTERIA IN PACIFIC WHITE SHRIMP, PENAEUS VANNAMEI GROW OUT CULTURE PONDS” developed by: MANOHARAN, N.- Bharathidasan University, India, SOLANKI, H.G.- Navsari Agricultural University, India and RAY, A.K.- Central Institute of Brackishwater Aquaculture, India.
The original article was published, including tables and figures, on JULY-DECEMBER, 2017, through INDIAN JOURNAL OF COMPARATIVE MICROBIOLOGY, IMMUNOLOGY AND INFECTIOUS DISEASES.
The full version can be accessed online through this link: 10.5958/0974-0147.2017.00014.9.
