Chitosan production by psychrotolerant Rhizopus oryzae in non-sterile open fermentation conditions

• Use of low temperature and pH enables low-cost chitosan production.
• Non-sterile conditions provide a potential way for economic biopolymer production.
• An effective chitosan production by soil-derived psychrotolerant R. oryzae was achieved.
• Taguchi’s (L16 45) orthogonal array design proved to be efficient for optimization.

A new chitosan producing fungus was locally isolated from soil samples collected around Erzurum, Turkey and identified as Rhizopus oryzae PAS 17 (GenBank accession number KU318422.1). Cultivation in low cost non-sterile conditions was achieved by exploiting its ability to grow at low temperature and pH, thus, undesired microbial contamination could be eliminated when appropriate culture conditions (incubation temperature as 15 °C and initial pH of the medium as 4.5) were selected. Medium composition and culture conditions were optimized using Taguchi orthogonal array (OA) design of experiment (DOE). An OA layout of L16 (45) was constructed with five most influensive factors at four levels on chitosan production like, carbon source (molasses), metal ion (Mg2+), inoculum amount, agitation speed and incubation time. The optimal combinations of factors (molasses, 70 ml/l; MgSO4·7H2O, 0.5 g/l; inoculum, 6.7 × 106 spores/disc; agitation speed, 150 rpm and incubation time, 8 days) obtained from the proposed DOE methodology was further validated by analysis of variance (ANOVA) test and the results revealed the increment of chitosan and biomass yields of 14.45 and 8.58 folds from its unoptimized condition, respectively.