Effect of Different Natural Substrates on Periphyton Production under Earthen Lined Seawater Pond for Periphyton based Aquaculture

Aquaculture mainly depends upon external inputs for production such as fertilizer and feed. Because of the high cost of foreign inputs, poorer sectors were unable to participate in production (O’Riordan, 1992). As a result, natural substrate-based aquaculture is receiving more attention in order to reduce production costs. Periphyton refers to any organism that is connected to a submerged substrate. On a clean surface, the formation of a periphyton layer is usually initiated by the deposition of a coating of dissolved organic compounds to which bacteria are drawn by hydrophobic interactions (Cowling et al., 2000). Before stocking fish, let at least 2 weeks for periphyton to grow on the substrates (Azim et al., 2003). Periphyton attachment is higher in bamboo than in PVC pipes or sugarcane bagasse bundles, according to Keshavanath et al., (2001). Natural substrates support periphyton growth more than artificial substrates (Keshavanath et al., 2012; Dutta et al., 2013). Using three plant substrates, sugarcane bagasse, paddy straw, and dried Eichhornia, periphyton had a good influence on rohu development (Wahab et al., 1999). Several authors have undertaken various research on the effects of substrates on shrimp growth and discovered a variety of outcomes, including improved water quality, more natural food supplementation, bacterial growth inhibition, shrimp rescue, and the addition of living space (Zarain-Herzberg et al., 2006; Kumlu et al., 2001).    

Several studies have shown that bacterial and microalgal biofilms growing on natural or manufactured submerged substrates (periphyton) can successfully be used as the primary or sole food source for a variety of freshwater and brackish water fish species (Azim et al., 2002, 2004; Keshavanath et al., 2004). Promoting the growth of natural planktonic or benthic microbial (bioflocs) and microalgal communities (periphyton) in the pond environment is another option. Because they consume nutrients in autotrophic and heterotrophic activities, which speed up the removal of organic and inorganic wastes, water quality improves, and their biomass can be used as a food source by cultured species (Azim et al., 2002). The increased fish production was ascribed to the formation of bacterial biofilm on the substrate, which provides food for zooplankton and fish. Umesh et al., (2000) investigated the effects of microbial biofilm development on the growth of common carp, tilapia, and rohu fingerlings in biodegradable substrate such as sugarcane bagasse, and found a considerable increase in fish growth in ponds with substrate. The good impact of substrate-based biofilm or periphyton on overall pond ecology, combined with higher production and lower input costs, makes periphyton-based ponds a reliable production method.

Periphyton growth on various types of natural substrate in seawater earthen line systems or ponds has received little attention. In a seawater earthen lined pond, the current study was conducted to evaluate periphyton biomass and chlorophyll ‘a’ pigment on various natural materials such as split bamboo poles, coconut coir, and coconut shell.

The experiments were carried out in two earthen lined ponds, each having water spread area of 30 m2 during the period 2018-19 at Marine Research Farm Facility, Tharuvaikulam, Thoothukudi, Tamil Nadu, India. The area is located at 8o89' N latitude and 78°17' E longitude. Seawater was filled upto 1.2 m depth and water is maintained in this depth over the study period. The experiment was conducted in duplicate with natural substrate for periphyton attachment on them.

In this experiment, three different natural substrates such as split bamboo pole, coconut coir and coconut shell were used for periphyton growth and they were collected locally. All the substrates were introduced in one pond and in duplicate ponds. The five bamboo poles were tied in nylon ropes (5 mm) and vertically immersed in the experimental ponds leaving 10 cm space between each bamboo poles. Coconut coir were tied in nylon rope (5 mm) and vertically immersed in the experimental ponds leaving 10 cm space between each coconut coir. 1.3 m length nylon twine was taken and five coconut shells were arranged in each line and hanged vertically. These five nylon twines were tied in nylon ropes and immersed vertically in the experimental ponds (5 coconut shell x 5 nylon twines). The space between each nylon twine is 10 cm. The mean diameter of the coconut shell, coconut coir and bamboo pole were 11.4 cm, 2.6 cm and 6.2 cm respectively.

After the substrates were installed, the ponds were filled with sea water upto 1.20 m depth. Liming is done by applying Quicklime (CaO) at the rate of 250 kg/ha (Azim et al., 2003). After three days of liming, fertilization is done by applying cow dung at the rate of 1,500 kg/ha. The same dosage rate of cow dung application was continued once in 10 days over the study period.

Periphyton samples were collected in morning 10.00 to 11.00 am on 15^th, 30^th and 45^th day of experiment. From each type of substrate periphyton samples were collected at each of three depths 40, 80 and 120 cm below the water surface from 2 x 2 cm2 area per substrate. While sample collection the areas were scrapped carefully with a scalpel blade to remove all periphyton without damaging the substrate. After sampling, the substrates were marked and placed in their original positions to exclude from subsequent sampling.

The composite samples of each depth were pooled. Pooled samples from three depth of each substrate were pre weighed and dried at 105°C until constant weight and kept in a desiccator. The dry matter of the samples was determined by weight difference. Apha (1995) standard methods were used for the determination.

The chlorophyll ‘a’ pigment in the periphyton was determined following standard method of Apha (1995). In addition to the samples taken from three depths, an extra 2 x 2 cm2 composite periphyton sample was collected from each depth and pooled. After removal, the pooled sample was immediately transferred to a tube containing 10 ml 90% acetone, sealed and transferred to the refrigerator for storing overnight. Following the samples collected in the morning were homogenized for 30 seconds with a tissue grinder and centrifuged for 10 min at 3000 rpm. The supernatant was transferred to the 3.5 ml glass cuvettes and then acidified by the addition of three drops of 0.1 N HCL and absorption was measured at 663, 645 and 630 nm using a UV-VIS spectrophotometer. Chlorophyll ‘a’ pigment was calculated using the trichromatic equation given in Apha (1995).
 

Chlorophyll ‘a’ = 11.64E₆₆₃ – 2.16E₆₄₅ + 0.10E₆₃₀

Water quality parameters were also checked for every two weeks by taking water samples between 10.00 and 11.00 am. Water quality parameters were checked by using mercury thermometer for temperature, hand refractometer for salinity, secchi disk for transparency and PH pen for measuring PH. Dissolved oxygen was measured following Winkler method Apha (1995) at lab. The water samples were filtered through GF/C Whatman glass fibre filter and the filtrate was obtained. The filtrate was analysed for Nitrate-N, Nitrite-N and total ammonia nitrogen (TAN) by using the standard procedures given by Apha (1995). Chlorophyll a content is analysed from non-filtered water column samples by following standard methods as given by Apha (1995). The water sample was estimated for Biological oxygen demand (BOD) by following standard methods as given by Apha (1995).

Statistical analysis such as One-way ANOVA, Duncan’s multiple range test using SPSS 20.0 and student’s ‘t’ test using Microsoft excel were used to analyse the data collected from this study.

The periphyton biomass from coconut coir was comparatively higher (3.4995±0.31201 mg/cm²) than the split bamboo poles (3.2011±0.30437 mg/cm²) and coconut shell (1.6297±0.22078 mg/cm²) during the experiment (Fig 1). The chlorophyll ‘a’ pigment recorded from coconut coir was comparatively higher (3.2949±0.27076 mg/cm²) than the split bamboo poles (3.0062±0. 26531 mg/cm²) and coconut shell (1.5569±0.19885 mg/cm²) during the experiment (Fig 2).



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One way ANOVA of the different data affirmed that significant difference (P<0.05) was observed in the dry matter content of the periphyton (Table 1) and chlorophyll ‘a’ pigment (Table 2) among the substrates. As per the Duncan’s multiple range test, dry matter content of periphyton (Table 1) and chlorophyll ‘a’ pigment (Table 2) recorded for coconut coir and split bamboo poles were significantly higher (P<0.05) than coconut shell. 



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As per the student’s ‘t’ test analysis of the data affirmed that highly significant was observed in periphyton biomass (Table 3) and chlorophyll ‘a’ pigment (Table 4) at various depth (40, 80 and 120 cm) among all the treatments over the study period.

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The mean values of periphyton biomass (mg/cm²) observed among all the treatments for 40, 80 and 120 cm depth over the study period is given in the following descending order:
 
40 cm depth
 
[Coconut Coir (4.145) > Spit bamboo pole (3.67166) > Coconut shell (2.01583)]
 
80 cm depth
 
[Coconut Coir (3.5325) > Spit bamboo pole (3.21083) > Coconut shell (1.66)]
 
120 cm depth
 
[Coconut Coir (2.821) > Spit bamboo pole (2.72083) > Coconut shell (1.21333)]

The mean values of chlorophyll ‘a’ pigment (mg/cm²) observed among all the treatments for 40, 80 and 120 cm depth over the study period was given in the following descending order:
 
40 cm depth
 
[Coconut Coir (4.10) > Spit bamboo pole (3.52) > Coconut shell (1.979)]
 
80 cm depth
 
[Coconut Coir (3.398) > Spit bamboo pole (2.88) > Coconut shell (1.58)]
 
120 cm depth
 
[Spit bamboo pole (2.60) > Coconut Coir (2.386) > Coconut shell (1.105)]

Student’s ‘t’ test analysis of the data relating to periphyton biomass and chlorophyll ‘a’ pigment for split bamboo pole, coconut coir and coconut shell recorded at different depths indicated that upper 0 to 40 cm depth had higher values than the other depths such as 40 to 80 and 80 to 120 cm.

In the present study, coconut coir had better surface structure for periphyton species for attachment. But, Keshavanath et al., (2001) indicated that bamboo pole is having a better surface structure for attachment of periphyton and provide nutrients beneficial to the growth of periphyton. Several authors found that, based on the substrate type, rate of fertilization, different environment and species composition periphyton quantity is varied significantly (Makarevich et al., 1993 and Keshavanath et al., 2001). Keshavanath et al., (2012) made the periphyton growth comparison study and reported that the periphyton attachment was better on coconut leaf than bamboo mat, sugarcane bagasse and palm leaf. Dutta et al., (2013) found that comparatively periphyton growth and attachment was better in palm leaf than nylon net. A positive effect of periphyton on growth of rohu was recorded using three plant substrates such as sugarcane bagasse, paddy straw and dried Eichhornia (Wahab et al., 1999). The higher production of fish was attributed to bacterial biofilm promoted on the substrate, which forms food for zooplankton and fish. Effects of microbial biofilm production on biodegradable substrate such as sugarcane bagasse on growth of common carp, tilapia and rohu fingerlings have been evaluated by Umesh et al., (2000) and recorded a significant increase in fish growth in pond with substrate. Shankar et al., (1998) indicated that, the increase in growth of fish in tanks with sugar bagasse, paddy straw were largely due to the increased zooplankton density in the water. This was influenced by a phenomenal increase in biofilm on the substrate on which both zooplankton and fish grazed.

Over all, higher values of periphyton biomass (Fig 3) and chlorophyll ‘a’ pigment (Fig 4) were recorded at upper 0 to 40 cm depth than the other depths (40 to 80 and 80 to 120 cm) in all the natural substrates. Similar results were found from the findings of Keshavanath et al., (2001), who highlighted the maximum periphytic biomass levels coinciding with photosynthetic compensation depths.





The physico-chemical water quality parameters were checked and the mean and standard deviation values are given in Table 5. In the periphyton experimental pond, there was no significant difference (P>0.05) observed in temperature, salinity, pH, BOD, nitrite and ammonia over the experimental period. But significant difference (P<0.05) was observed in secchi disk reading, dissolved oxygen, nitrate and chlorophyll ‘a’ over the experimental period (Table 5). The highest and lowest secchi disk reading was observed on 15th day (53.00±1.41cm) and final day (47.00±.00 cm). The lowest dissolved oxygen was recorded on 15^th day (5.76 ±0.22 mg/l), while it was highest in the final day (6.80± 0.34 mg/l). This shows the good sign of dissolved oxygen and provide good environment in the pond for shrimp culture. The highest and lowest level of Nitrate-N value was observed on 45th day (0.41±0.02 µg.at.NO3-N / l) and 15^th day (0.33±0.01 µg.at.NO3-N / l) of the experiment. The highest and lowest level of chlorophyll ‘a’ was observed on final day (75.25±5.89 mg/m³) and 15^th day (40.50±9.98 mg/m³) of the experiment. This result indicates the increment of periphyton production over the study period due to the natural substrate introduction. There is significant variations in biological oxygen demand (BOD) level among the sampling days and this manifest the differential rates of oxygen consumption over the experimental period by the organisms. Many authors proved that the BOD level was reduced in substrate installed pond compared to non substrates pond (control) (Keshavanath et al., 2001, 2002; Dharmaraj et al., 2002; Keshavanath et al., 2004). In the present study also, the BOD level was low in the substrate installed pond compared to non substrate pond and this coincides with the findings of above said reports. ANOVA results indicated that over the experimental period, significant variations were observed in the Nitrate - N and chlorophyll ‘a’ content. Phytoplankton productivity is positively correlated with nutrient concentrations in ponds and lakes (Boyd, 1990). In substrate treatments, higher chlorophyll ‘a’content indicates the phytoplankton production, which is the positive effect indication on plankton nutritional quality (Azim et al., 2002).

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In the study, the growth of periphyton is higher in coconut coir than split bamboo pole and coconut shell. Coconut coir is also enhancing as natural substrate for periphyton development in earthen lined seawater pond for aquaculture. Periphyton grown on this natural substrate serves as an important natural food source for the fishes and reduces the cost of production by reducing the artificial feed substantially in aquaculture ponds. By this way cost reduction can be done in feed in shrimp farming, hence increases the profit of culture. Further, research work is also required to determine the optimum density of natural substrates and economic viability of substrate based shrimp culture in earthen lined pond for adoption.

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