Activity of Proteolytic and Amylolytic Enzymes from Bacillus spp. Isolated from Shrimp Ponds

Abstract

Accumulation of feed excess in commercial shrimp ponds due to overfeeding could decrease water quality. Protein and starch are the primary components of shrimp feed. This study was conducted to characterize extracellular proteases and amylases of Bacillus spp. isolated from shrimp ponds. 72 proteolytic and amylolytic Bacillus spp. isolates were screened from shrimp ponds in Karawang, West Java. Ten isolates were selected for further characterization for their growth and ability to reduce total suspended solid generated from commercial shrimp feed. Bacillus sp. DA 5.2.3 and L5 showed excellent activity in reducing total suspended solid, by 37 and 30% respectively. Protease and α-amylase activities of Bacillus sp. DA 5.2.3 isolate were consistently higher than that of L5. Maximum total and specific protease activity of DA 5.2.3 isolate was 2.0 U mL-1 and 40.9 U mg-1 respectively, while the activities of the L5 isolate were 2.1 U mL-1 and 23.0 U mg-1 respectively. Based on its 16S rRNA gene sequences, Bacillus sp. DA 5.2.3 showed 99% similarity to Bacillus cereus XHJ-2-6. Bacillus sp. DA 5.2.3 could potentially be applied to maintain water quality by reducing total suspended solid in water columns of shrimp ponds. Overfeeding in aquaculture system can influence water quality that hampers animal growth. Generally, 10% of feed remainds as waste in the water. A high load of feed in pond water can increase biological oxygen demand (BOD), bacterial population, and decrease dissolved oxygen (DO) content. It can also increase total suspended solid (TSS) in the water. Reduction of water quality has a bad influence on survival and growth of shrimps. The used of bacterial probiotics in shrimp aquacultures has been studied and reported to have great impact on shrimp growth and survival. Some of these can maintain water quality by reducing the ammonia concentration in shrimp ponds, others can inhibit the growth of shrimp pathogens (Rengpipat et al. 1998; Vaseeharan and Ramasamy 2003; Decamp and Moriarty 2007), stimulate the immune system of the shrimp (Gullian et al. 2004) and increase digestive enzyme activity of the shrimps intestine (Ziaei-Nejad et al. 2006). Bacillus spp. have derived great attention as a probiotic application in aquaculture in the last decade (Rengpipat et al. 1998; Vaseeharan and Ramasamy 2003; Decamp and Moriarty 2007). Many researchers reported their roles as biological control agents in shrimp ponds (Verschuere et al. 2000; Decamp and Moriarty 2007). Recently, Ochoa-Solano and Olmos-Soto (2006) studied the contribution of Bacillus as part of the functional feed to enhance shrimp feed quality. Several studies of potential protease from Bacillus cereus isolated from fish gut for converting fish-wastes have been reported. However, information about the role of Bacillus spp. to reduce shrimp feed wastes, was very limited. The group of Bacillus bacteria is well known as a producer of a large variety of extracellular protease and amylase. Therefore, this study attempted to identify potential proteolytic and amylolytic enzymes of Bacillus spp. isolated from shrimp ponds for their potential application to maintain water quality in shrimp aquaculture. MATERIALS AND METHODS Sample Collection and Preparation. Nineteen samples of four different sample sources being soil, sediment and water and shrimp intestine were collected from shrimp ponds in Karawang, West Java, Indonesia. From samples of shrimp intestine were prepared by grinding and diluting in 0.85% (w/v) NaCl. Isolation and Screening of Bacillus. Serial dilution of each sample in 0.85% NaCl (w/v) was follow by heating at 80 °C for 15 min. Then, 100 mL aliquots solution was spread on half strength of solid seawater complete medium (SWC) (Atlas 1997) added with 1% (w/v) skimmed milk. Cultures were incubated at room temperature 24-48 h. Proteolytic activity was shown by formation of clearing zones around colonies. Amylolytic activities of Bacillus spp. were screened by a similar method except SWC medium was mixed with 1% (w/v) commercial cassava starch instead of glycerol was used as the carbon source. Clearing zones around the colonies were detected by iodine staining solution (15 g L-1 potassium iodide and 15 g L-1 iodine in distilled water). The proteolytic index (PI) or the amylolytic index (AI) value is the ratio of hydrolysis zone (clear zone) diameter (cm) formed by a bacterium colony and its colony diameter (cm). Proteolytic and amylolytic activity positive colonies were isolated on solid SWC medium to give a pure culture. Confirmation of amylolytic and proteolytic activity of the pure culture isolates were conducted using similar methods as to mention above. Growth Conditions of Selected Isolates. Selected high PI and AI value isolates were inoculated into 50 mL SWC medium in 250 mL flasks and then incubated on shaker (120 rpm) at room temperature (28 °C) to get 10 8 CFU mL-1 (OD 620nm = 0.4).