GREENHOUSES AND POND LINERS

Role of pond lining in dynamics of sulfur recycling bacteria in Pacific White Shrimp, Penaeus vanammei grow out culture ponds

By: N. Manoharan, H.G. Solanki and A.K. Ray *

Plastic lining ponds provide better management and healthier environmental conditions, and sulfur cycling bacteria can be used as an indicator of pond environmental conditions. The present investigation, developed by researchers from Universities Bharathidasan and Navsar and the Aquatic Animal Health and Environment Division Central Institute of Brackishwater Aquaculture (CIBA) in India, compares observations on the trend of total heterotrophs, total vibrios, and the sulfur cycling bacteria in earthen and lined white legged shrimp Penaeus vannamei culture ponds and the influence of farm level interventions like application of exogenous probiotics.

Water quality in aquaculture systems to a large extent is controlled by the microbial biodegradation of organic residues (Avnimelech et al., 1995; Abraham et al., 2004) through the process of mineralization. Microorganisms are involved in the production and breakdown of organic matter and nutrient recycling in aquatic environments. Heterotrophic bacteria oxidize organic matter, whereas the autotrophic nitrifying and sulfur bacteria oxidize problematic compounds like ammonium, nitrite, or sulfide, respectively (Moriarty, 1997). Sulfur-reducing bacteria (SRB) and sulfuroxidizing bacteria (SOB) are predominately involved in the reduction and oxidation of sulfates and

Different water and soil probiotics, mineral mixtures, disinfectants used in culture systems.

hydrogen sulfide in pond bottom (Syed et al., 2006) and assist in maintaining a healthy pond environment in commercial shrimp culture ponds (Rao et al., 2000; Devaraja et al., 2002; Burford et al., 2003; Abraham et al., 2004, 2015; Fernandes et al., 2010; Patil et al., 2012). Studies on the role of sulfur cycling bacteria in P. vannamei culture systems are scanty.

Smith and Briggs (1998) suggested full pond liners (bitumen impregnated geotextile) against earthen pond for managing nutrient load in shrimp culture systems. Still, they did not discuss the role of pond lining on microbial dynamics, although pond lining is a highly intensive and high input system with zero water exchange. The present investigations compare observations on the trend of total heterotrophs, total vibrios, and especially the sulfur cycling bacteria (SOB and SRB), which play a significant role in understanding the ecosystem in commercial P. vannamei culture ponds.

Study Location

In group I, three earthen ponds (1.0 ha each) located 2.5 km from Gulf of Cambay at Onjal village of Navsari district, Gujarat (India) with P. Vannamei post larvae (PL 12) stocking density @ 30 nos. m-2 were selected for the study.

In Group II, three (1.0 ha each) fully lined ponds [Geosynthetic manufactured materials (GSE), GSE Lining Technology Co. Ltd., Thailand; 300 μm thickness] located 2 km from Gulf of Cambayat Chijgam village of Navsari district, Gujarat (India) with P. vannamei postlarvae (PL 12) stocking density @ 95 nos. m-2 were selected for the study.

Pond Preparation

Detailed procedure for the pond preparation is provided in Fig. 1. In both systems, an initial dose of water probiotics was given before stocking of seeds. The ponds were lined with 300 μm thickness of GSE (GSE Lining Technology Co. Ltd., Thailand). Aeration in earthen ponds was provided using paddlewheel aerators @ 8 HP ha-1 from 3 h early in the morning up to 30 DOC (days of culture), 8 h up to 50 DOC, 10 h up to 75, and 12 h till the end of harvest. Aeration in lined ponds was provided before stocking during the pond preparation period (rice ferment applications as well as probiotics applications). Then after, 24 h @ 6 HP ha-1 up to 30 DOC, 8 HP up to 50 DOC, 10 HP up to 75, and 12 HP till the end of harvest.

Shrimps were fed with commercial feed (30– 35% crude protein, 2.5–3.0% crude lipid, <3% crude fibre, <15% ash content and <12% moisture). Initially, blind feeding was done up to 28 DOC, which was then adjusted based on the feed consumptions in check trays (initially 60% of body weight and gradually reducing it to 1.8% of body weight at the end of culture period). Feeding was carried out in four equal rations per day at an interval of every 4 h.

Figure 1

Flow chart mentioning the pond preparation before stocking post larvae.

Total Bacterial Counts in soil and water samples of Lined and Earthen L. vannamei culture ponds.

Plastic lining ponds allow easy removal of the organic load, thereby permitting higher stocking densities and harvests than earthen ponds.

Figure 3

Total Vibrio Counts in soil and water samples of Lined and Earthen L. vannamei culture ponds.

Different water and soil probiotics, mineral mixtures, disinfectants used in culture systems are listed in Table 1 to maintain desirable conditions. Water probiotics included Bacillus sp., whereas soil [probiotics included Rhodobacter and Rhodococcus sp. (liquid formulations)] and Thiobacillus denitrificans (powder formulation). Apart from this, the accumulated sludge in the lining pond was removed regularly after 70 DOC through the centrally designed drainage system. Sample Collection

Water and sediment samples were collected from both the groups fortnightly from ponds in sterilized plastic bottles and plastic bags, respectively, and transported to the laboratory in insulated boxes containing pre-cooled gel ice packets. The samples were ponds compared with the water samples (Fig. 1).

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 they 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 systems. During the culture period, the values of TBC observed agreed 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 (pre stocking 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. 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, seen 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. As with TBC, the higher population of TVC in lined ponds may also be due to the longer duration of initial preparation time given before stocking, allowing the proliferation of the vibrio proliferation. The high vibrio load in lined ponds must be attributed to the higher stocking density in lined ponds throughout the culture period. This might be due to the steady increase in organic matter accumulation in the pond bottom (Moriarty, 1997; Sujatha, 2007), as witnessed by the total quantum of feed per pond in each group (Group I – 9.45 t and Group II – 10.35 t). A large amount of organic matter in shrimp culture ponds is possible due to high stocking density, overfeeding, uneaten feed, fecal matter, fertilizers, and over blooming (Kautsky et al., 2000).

Sulphur oxidising bacterial counts in soil and water samples of Lined and Earthen Penaeus vannamei culture ponds.

Sulphur reducing bacterial counts in soil and water samples of Lined and Earthen Penaeus vannamei culture ponds.

Figure 5

Sulfur-Oxidizing Bacteria (SOB) and Sulfate- Reducing Bacteria (SRB)

The SOB and SRB are essential in converting sulfur and sulfur-related compounds. The sulfur recycling bacteria that is SOB and the SRB were significantly lower (P < 0.01) in the 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 re-

Different bacterial population in shrimp culture ponds.

ports (Suplee and Cotner, 1996; Rao et al., 2000). However, Devaraja et al. (2002) and Abraham et al. (2004, 2015) reported even lower counts. The present study results reflect the intensification of culture practices and the effect of the stocking density. The counts of SOB in earthen ponds decreased up to 70 DOC then 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 the 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 the water column. The earlier stud-

ies 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 the water column might be attributed to creating anaerobic conditions at the center of micro-niche due to the 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 is supported by several researchers (Devaraja et al., 2002; Patil et al., 2012; Abraham et al., 2015). An 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 the regular exchange of bottom sludge using a central drainage system; otherwise, the SRB populations could have In the present study, the microbial population density differs significantly with the type of culture system despite the stocking density.

outnumbered the SOB populations (Smith, 1998).

Conclusions

In conclusion, the accumulation of organic matter (sludge) leads not only to increases in sediment oxygen demand but also to anaerobic conditions resulting in the production of undesirable gasses such as hydrogen sulfide. To avoid these unfavourable conditions in the pond environment, sludge has to be managed by removing at a certain period of time. With the intensification in aquaculture, the accumulation of a heavy organic load leads to the deterioration of the environment, leading to poor growth and survival of the cultured aquatic animal (Prawitwilaikul et al., 2006). Plastic lining ponds allow easy removal of the organic load, thereby permitting higher stocking densities and harvests than earthen ponds. It is important to study the dynamics of this recycling microorganism and its behavior in the present system of culture in commercial product applications. In the present study, the microbial population density differs significantly with the type of culture system despite the stocking density. It is mostly indicated by the corresponding load of sulfur cycling bacterial populations. Also, farm-level interventions like probiotics play a significant role in maintaining a 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.

N. Manoharan1, H.G. Solanki2 and A.K. Ray3* 1Marine Science Department, Bharathidasan University, Tiruchirappalli-620024, Tamil Nadu, India 2College of Fisheries, Navsari Agricultural University (NAU), Navsari-396450, Gujarat, India 3Aquatic Animal Health and Environment Division

Central Institute of Brackishwater Aquaculture (CIBA), Chennai-600028, Tamil Nadu, India. This is a summarized version developed by the editorial team of Aquaculture Magazine of the article titled Role of pond lining in dynamics of sulfur recycling bacteria in Pacific White Shrimp, Penaeus vanammei grow out culture ponds that was originally published on the Indian J. Comp. Microbiol. Immunol. Infect. Dis., Vol. 38 No. 2 (July-Dec), 2017: 85-91.

References cited by the authors are available under previous request to our editorial team.