Rapid Testing of Resistance of Timber to Biodegradation by Marine Wood-Boring Crustaceans.

Wood-boring invertebrates rapidly destroy marine timbers and wooden coastal infrastructure, causing billions of dollars of damage around the globe every year. As treatments of wood with broad spectrum biocides, such as creosote and chromated copper arsenate (CCA), are now restricted in marine use by legislation, naturally durable timber species and novel preservation methods of wood are required. These methods undergo testing in order to meet regulatory standards, such as the European standard for testing wood preservatives against marine borers, EN 275. Initial investigation of durable timbers species or wood preservative treatments can be achieved quickly and inexpensively through laboratory testing, which offers many advantages over marine field trials that are typically costly, long-term endeavours. Many species of Limnoria (gribble) are marine wood-boring crustaceans. Limnoria are ideal for use in laboratory testing of biodegradation of wood by marine wood-borers, due to the practicality of rearing them in aquaria and the ease of measuring their feeding rates on wood. Herein, we outline a standardizable laboratory test for assessing wood biodegradation using gribble.

Hemocyanin facilitates lignocellulose digestion by wood-boring marine crustaceans.

Woody (lignocellulosic) plant biomass is an abundant renewable feedstock, rich in polysaccharides that are bound into an insoluble fiber composite with lignin. Marine crustacean woodborers of the genus Limnoria are among the few animals that can survive on a diet of this recalcitrant material without relying on gut resident microbiota. Analysis of fecal pellets revealed that Limnoria targets hexose-containing polysaccharides (mainly cellulose, and also glucomannans), corresponding with the abundance of cellulases in their digestive system, but xylans and lignin are largely unconsumed. We show that the limnoriid respiratory protein, hemocyanin, is abundant in the hindgut where wood is digested, that incubation of wood with hemocyanin markedly enhances its digestibility by cellulases, and that it modifies lignin. We propose that this activity of hemocyanins is instrumental to the ability of Limnoria to feed on wood in the absence of gut symbionts. These findings may hold potential for innovations in lignocellulose biorefining.

Structural characterization of a unique marine animal family 7 cellobiohydrolase suggests a mechanism of cellulase salt tolerance.

Nature uses a diversity of glycoside hydrolase (GH) enzymes to convert polysaccharides to sugars. As lignocellulosic biomass deconstruction for biofuel production remains costly, natural GH diversity offers a starting point for developing industrial enzymes, and fungal GH family 7 (GH7) cellobiohydrolases, in particular, provide significant hydrolytic potential in industrial mixtures. Recently, GH7 enzymes have been found in other kingdoms of life besides fungi, including in animals and protists. Here, we describe the in vivo spatial expression distribution, properties, and structure of a unique endogenous GH7 cellulase from an animal, the marine wood borer Limnoria quadripunctata (LqCel7B). RT-quantitative PCR and Western blot studies show that LqCel7B is expressed in the hepatopancreas and secreted into the gut for wood degradation. We produced recombinant LqCel7B, with which we demonstrate that LqCel7B is a cellobiohydrolase and obtained four high-resolution crystal structures. Based on a crystallographic and computational comparison of LqCel7B to the well-characterized Hypocrea jecorina GH7 cellobiohydrolase, LqCel7B exhibits an extended substrate-binding motif at the tunnel entrance, which may aid in substrate acquisition and processivity. Interestingly, LqCel7B exhibits striking surface charges relative to fungal GH7 enzymes, which likely results from evolution in marine environments. We demonstrate that LqCel7B stability and activity remain unchanged, or increase at high salt concentration, and that the L. quadripunctata GH mixture generally contains cellulolytic enzymes with highly acidic surface charge compared with enzymes derived from terrestrial microbes. Overall, this study suggests that marine cellulases offer significant potential for utilization in high-solids industrial biomass conversion processes.