Method for measuring microbial degradation and mineralization of14C-labeled chitin obtained from the blue crab,Callinectes sapidus

Abstract

A method for measuring microbial degradation and mineralization of radiolabeled native chitin is described.¹⁴C-labeled chitin was synthesized in vivo by injecting shed blue crabs (Callinectes sapidus) with N-acetyl-D-[1-¹⁴C]-glucosamine and allowing for its incorporation into the exoskeleton. The cuticle had a total organic carbon content of 0.48 mg C mg⁻¹ with a specific radioactivity of 6,356 CPM mg⁻¹. Glucosamine, i.e., chitin content as determined colorimetrically, was 22% (w/w). Microbial degradation and mineralization rates were assessed in batch culture using¹⁴C-chitin as substrate and York River water as inoculum. Replicate flasks were sampled daily for enumeration of chitinoclastic bacteria and the radiolabel recovered as particulate¹⁴C-chitin or¹⁴CO₂. The amount of¹⁴CO₂ generated was directly proportional to the loss of particulate¹⁴C-chitin, with 96% of the added label recovered as the sum of both phases. The maximum rate of mineralization was 207 mg day⁻¹ g⁻¹ seeded¹⁴C-chitin at 20°C. Highest chitinoclastic bacterial counts corresponded to the period of maximum rate of chitinolysis. It is suggested that the rate of chitin mineralization is limited by exoenzymatic depolymerization and not by chitin concentration.