• ethanol production
• biofuels
• inulin
• inulinases
• metagenomic approach
• marine metagenomics

Ethanol has important uses as a solvent, topical disinfectant, principal psychoactive in alcoholic beverages, and as a biofuel. Currently, ethanol production consists of two/three-step processes using starchy foodstuffs or other feedstock, like sugarcane, cereal crops and maize, as starting materials. However, the production is costly and inefficient, and has a negative impact on agricultural price and food security due to the ongoing “food vs fuel” debate. An alternative way to obtain ethanol in a one step process involves the use of inulin, a polyfructan that usually has a terminal glucose residue and is found as a reserve carbohydrate in the roots and tubers of plants such as the Jerusalem artichoke, chicory, or dahlia. Inulinases can be produced by bacteria, yeasts and fungi and hydrolyze inulin, releasing fructose and glucose, which can be subsequently fermented to produce ethanol. Although several inulinases have been discovered and characterized, there is a need to discover new inulinases to improve the efficiency of conversion of inulin into ethanol. The aim of this study is detecting new inulinases from a previously constructed metagenomic library from the marine sponge Axinella dissimilis. The metagenomic library, consisting of 20,353 clones, was screened for inulinase production using a growth/no growth assay on a minimal medium, containing inulin as the sole carbon source. The 56 obtained positive clones were validated using a dye-linked plate screening technique to evaluate enzymatic activity. Based on this, three metagenomic library clones with the best activity were selected for further analysis. Fosmid DNA from each clone was purified and sub-cloning libraries were constructed and screened in pUC19 plasmid in an attempt to isolate the inulinase genes. Three plasmid subclones with apparent inulinase activity were identified and their inserts were fully sequenced. However, no putative inulinase genes could be identified despite adopting numerous bioinformatic approaches. Despite this, this is the first study in which inulinase activity was detected by metagenomic approach using marine sponge samples.