Investigation of mircroorganisms colonising activated zeolites during anaerobic biogas production from grass silage

Abstract

The colonisation of activated zeolites (i.e. clinoptilolites) as carriers for microorganisms involved in the biogas process was investigated. Zeolite particle sizes of 1.0–2.5 mm were introduced to anaerobic laboratory batch-cultures and to continuously operated bioreactors during biogas production from grass silage. Incubation over 5–84 days led to the colonisation of zeolite surfaces in small batch-cultures (500 ml) and even in larger scaled and flow-through disturbed bioreactors (28 l). Morphological insights were obtained by using scanning electron microscopy (SEM). Single strand conformation polymorphism (SSCP) analysis based on amplification of bacterial and archaeal 16S rRNA fragments demonstrated structurally distinct populations preferring zeolite as operational environment. via sequence analysis conspicuous bands from SSCP patterns were identified. Populations immobilised on zeolite (e.g. Ruminofilibacter xylanolyticum) showed pronounced hydrolytic enzyme activity (xylanase) shortly after re-incubation in sterilised sludge on model substrate. In addition, the presence of methanogenic archaea on zeolite particles was demonstrated.

Introduction

In numerous studies grass silage has been recommended as an excellent substrate for biomethane production resulting from high energy yields and low energy input demand (Prochnow et al., 2009). The high potential of methane production from grass silage was confirmed both in batch and semi-continuous experiments and batch leach bed processes (Lehtomäki et al., 2008). In practice, grass silage is the most important substrate for agricultural biogas production following maize silage in Germany (Rösch et al., 2007). Though grass silage may be less energetically productive compared to maize silage, it still offers a good energy balance and environmental advantages (Gerin et al., 2008). The formation of inhibitory NH₃ and high chemical oxygen demand (COD) values in the course of grass biomethane production are drawbacks, which may be counteracted by the high ammonia binding capacity of zeolites as reported by Montalvo et al. (2005).

The addition of support materials like magnesium or aluminium silicates to anaerobic digestion processes has been reported to lead to higher methane yields or better gas quality (Pande and Fabiani, 1989). Furthermore, the immobilisation of microorganisms on various zeolite types was claimed to be beneficial, expanding the possibilities to support the process (Murray and van den Berg, 1981). Among natural and synthetic zeolites, clinoptilolite has a superior CO₂ adsorption capacity, regenerability and stability through several adsorption–desorption cycles, upgrading the quality of biogas by adjusting the CO₂/CH₄ ratio (Alonso-Vicario et al., 2010). Moreover, clinoptilolite has been shown to be suitable as operational environment for microorganisms in biogas production processes (Milán et al., 2003). Recently, we have demonstrated that the degradation of recalcitrant cell wall components can be enhanced by addition of hydrolytic bacterial populations immobilised on activated zeolite to the biogas process (Weiß et al., 2010). However, our understanding of the specificity of zeolites regarding the immobilisation of certain microbial populations is still poor. Therefore, we characterised the microbial populations colonising trace metal activated clinoptilolite during anaerobic digestion of grass silage and of a model substrate in this study.