Cellulose breakdown in soil and in model soil environments

Cellulose is the major cell wall polymer of plants and is deposited in enormous quantities in terrestrial environments. Thus an understanding of the processes involved in cellulose decomposition is of importance with reference to carbon cycling in the biosphere.

In order to degrade a polymeric insoluble substrate such as cellulose which is discontinuously distributed in soil, a microorganism must rely on extracellular enzymes which are likely to be rapidly inactivated. Furthermore, constitutive production and secretion of extracellular enzymes by soil microorganisms in the prolonged absence of substrate is an inefficient and ultimately fatal use of cell energy. It has been suggested that, in addition to enzymes associated with viable microorganisms, an immobilized and hence stabilized enzyme fraction may have a significant role in substrate decomposition.

In this work, we have developed assays and measured cellulase activities (exo- and endoglucanase, 3_D-glucosidase) in air-dried soil using the substrates : Avicel, carboxymethyl cellulose, cellobiose and £-nitrophenyl-3-D-glucopyranoside. From an extensive characterization of these activities (in terms of pH-activity optima, thermostability and resistance to proteolysis), we have been able to speculate as to the origin and location of these enzymes in soil, and to evaluate the role of immobilized and other forms of enzymes in cellulolysis.

The persistence and efficacy (in terms of locating substrate) of 'free' (soluble), extracellular enzymes in soil was investigated using a novel technique developed for this purpose. This 'barrier ring plate' technique has allowed us to study extracellular enzyme diffusion and microbial growth through soil-like but carefully controlled environments. Using the barrier ring diffusion plates we demonstrated that whole soil and bentonite (a high unit surface area smectite clay) impeded the diffusion of both 0-D-glucosidase and endoglucanase and subsequent substrate hydrolysis. Whilst sand or kaolin (a low unit surface area clay) had no significant effect on enzyme diffusion or substrate hydrolysis.

We have further used -barrier ring plates to demonstrate how soil components differentially affect the radial growth of three fungal soil isolates : Trichoderma virdde, T. koningii.3 Botryotviohim piluliferum’, and a Streptcmyoes sp. However, the interpretation of the data from these growth experiments is limited by our poor understanding of the genetic and environmental factors which influence the morphology of the developing colony. The main advantages of the barrier ring plates lie in their versatility and in the fact that they provide a vectorial system in which the influence of soil components on the comparatively complex spatial interactions between microorganisms, enzymes and substrates may be studied.