Microbial diversity in the sediment of a crab pond in Nanjing, China

Abstract

Chinese mitten crab (Eriocheir sinensis) is of great commercial significance and is bred extensively in Eastern and Northern China. Aquaculture is a complex ecosystem where microorganisms in the water, sediment and gastrointestinal tracts interact with each other to affect the health of aquatic animals (Cheng, Zhou, Xie, Ge, Zhu & Liu 2010). The microbial communities in crab pond water (Cheng et al. 2010) and in the gut of Chinese mitten crab (Li, Guan, Wei, Liu, Xu, Zhao & Zhang 2006) have been studied. However, there is no report on the microbial community in crab pond sediment. Generally a crab may require 12–16 moults to reach maturity (Zhang, Li & Cui 2001). Crab shell is rich in chitin, and the crab pond represents one of the most chitin-rich environments. Organisms produce chitinase for different physiological purposes. Bacterial chitinases play roles in nutrition and parasitism, and thus form one of the major sources of chitinases (Dahiya, Tewari & Hoondal 2006). Chitinase genes have been identified in a large pool of uncultured chitinolytic microorganisms in marine and soil environments and used as molecular markers (Cottrell, Wood, Yu & Kirchman 2000; Metcalfe, Krsek, Gooday, Prosser & Wellington 2002). Recently, chitinases of various aquatic habitats have been investigated (LeCleir, Buchan & Hollibaugh 2004; Hobel, Marteinsson, Hreggvidsson & Kristjansson 2005; Bhattacharya, Nagpure & Gupta 2007). Denaturing gradient gel electrophoresis (DGGE) analysis of bacterial 16S rRNA gene fragments generated by PCR is a reliable, rapid and an easy method to evaluate microbial diversity (Liu, Zhou, Yao, Shi, He, Benjamisen Holvold & Ringo 2008; Zhou, Liu, Shi, He, Yao & Ringo 2009) and is capable to detect the dominant bacterial species in the environments (Muyzer, Waal & Uitterlinden 1993). We used PCR-DGGE and PCR screening technologies to investigate the microbial community in crab pond sediments. The sediment sample was collected from a pond rearing Chinese mitten crab near the city of Nanjing, Jiangsu province, China, in July 2008. The depth of the pond was 1.5 m, with temperature of 30°C, pH 7.5 and the culture area was approximately 100 acres. The crabs were fed the commercial diet (Crude protein 36% and crude lipid 6% without dietary antibiotics) manufactured by Jiangsu Danyang Lekaihuai Feed Co., Ltd (Zhenjiang City, Jiangsu Province, China). The earth pond has been used for the crab culture for about 3 years. The pH of the water 50 cm below the surface was measured at 11:00 hours on the sampling day. Due that dissolved oxygen level in the crab pond water was above 5.0 mg L 1 and the surface of the pond sediment was sampled, the sediment sampled was not regarded to be anoxic. The total genomic DNA of the crab pond sediment was extracted as described by Brady (2007) with some modifications. 10 g of sediment was suspended in a 50 mL centrifuge tube containing 20 mL of pre-heated (70°C) lysis buffer [100 mM Tris-HCl, 100 mM EDTA, 1.5 M NaCl, 1% (w/v)