William S. Adney, Daniel van der Lelie, Alison M. Berry, and Michael E. Himmel

15.1 Introduction

Traditionally, microbiologists have taken a reductionist approach to understanding micro­bial biomass decay communities focusing on the analysis of individual genes, microorgan­isms, and biochemical reactions. Recent advances in molecular biology have identified many genetic components but have provided limited information on the mechanisms of biomass decay. In the area of biochemistry, advances in proteomics and high-throughput enzyme assays are providing new theories into the mechanisms of biomass decay, but limited in­formation on individual microorganisms and their interactions. Combined, however, these technologies are providing a new “systems biology” approach to understanding the biomass decay communities. This approach will allow microbiologists to envision and model micro­bial biomass decay as a set of interacting processes that when combined effectively degrade plant biomass.

The first major step towards clarifying the fundamental principles of biomass recalci­trance is to understand the scale and complexity of natural systems involved in biomass decomposition. Heterotrophic microorganisms are major players in biomass decomposi­tion and in the global cycling of terrestrial carbon. Our terrestrial biosphere depends on heterotrophs functioning in complex and dynamic communities to breakdown the natural accumulation of biomass. Although the subject of study for several decades, we still know little about the diversity and complex interrelationships of the individual organisms. How­ever, we now understand that these communities vary in spatial and temporal dimensions to control the biochemical rate of carbon cycling, as well as the cycling of other essential elements, like nitrogen, sulfur, and phosphorus. In fact, the production of carbon dioxide by chemoorganotrophs is the single most important contribution of CO2 to the atmosphere (1).

Microbial communities are complex networks of individual organisms that include every ecological relationship ever described, ranging from coexistence to commensalism, mutu­alism, and parasitism. There are direct symbioses between individual microorganisms and indirect symbioses in which metabolic processes of one species modify the habitat and/or physiology of another species. Studying microbial communities in most environments has

Biomass Recalcitrance: Deconstructing the Plant Cell Wall for Bioenergy. Edited by Michael. E. Himmel © 2008 Blackwell Publishing Ltd. ISBN: 978-1-405-16360-6

thus presented a challenge in that it is very difficult to simulate environmental conditions and ecological relationships adequately in the laboratory in order to satisfy the physiological requirements for the reproducible cultivation of a representative community. This maybe due to our inability to reproduce the physical, chemical, and temporal conditions needed for the multiplex interactions and unknown species.

Until recently, biodiversity estimates were based only on those species that could be culti­vated by using traditional in vitro microbiology techniques. Unfortunately, these techniques only allow 1-5% of the total community members to be examined (2, 3). Traditional mi­crobiology techniques were developed to study the growth and metabolic requirements of individual organisms in pure culture. As such, they provide limited information into the ac­tions of biomass-degrading communities determined by diverse and dynamic biochemical pathways. Recently, new molecular technologies have provided valuable information about the in situ biodiversity of plant decaying microbial communities. However, while genomic approaches provide important information about the diversity of individual species within populations they do not predict the biochemical outcome on functional terms. More than a biotic inventory of microorganisms is needed to develop a complete understanding of the organization and interactions within the community. Individual microorganisms display such strong interactions that new capabilities are needed to understand recalcitrance in comprehensive and integrated way. Therefore, a “systems biology” approach is needed to envision how biomass decomposition functions as a complete set of intersecting processes. This requires that we also understand the biochemical reactions and catalysts involved in the deconstruction of biomass. Clearly, no technique alone can provide the broad range of information needed to understand community structure and system function.

Plant biomass is a chemically diverse substrate varying in composition, but predominated by complex and interactive polymers of cellulose, hemicellulose, and lignin. Evolution has developed plants that are naturally recalcitrant to degradation by microbial communities. Major contributing biochemical features to the recalcitrance of terrestrial plants not shared by bryophytes and earlier plants are the lipid materials of the cuticle and its wax, and the diversity of phenolics in lignin and flavinoid compounds (4). The arrangement and density of the vascular bundles, the relative amount of sclerenchymatous (thick wall cell) tissue, the structural variation, and complexity of cell wall constituents also contribute to recalcitrance. The result is that the natural plant biomass decay involves diverse groups of heterotrophic bacterial and fungal communities that degrade and metabolize plant material. In short, Nature’s answer to the innate chemical and structural complexity found in plants is greater diversity and synergistic interactions. The full extent of this diversity is still up to debate.

Carbon turnover in the terrestrial biosphere occurs chiefly in the soil from complex and yet unclassified microbial communities. Turnover rates vary dramatically depending on environmental conditions such as temperature, water availability, inorganic nutrients, pH, organic carbon input such as plant exudates, biomass composition, and the presence of microorganisms producing hydrolytic enzymes such as cellulases and hemicellulases. The exact mechanisms of degradation carried out by complex interactions of members of biomass-degrading community remain elusive. The arrival of new biotechnology tools such as ribosomal rRNA gene sequencing, comparative metagenomics, transcriptome, and se — cretome analysis are allowing for habitat-specific fingerprinting of microbial communities. Genomic-based studies are providing vast amounts of sequence data from various environ­mental samples and have led to new insights into microbial populations. The improvement of sequencing technologies has made metagenome shotgun sequencing of an environmental sample feasible; however, most environmental communities are far too complex to be fully sequenced in this manner.

Metagenomic analysis of representative bacterial assemblages is beginning to provide a knowledge base and a source of genetic material for further studies on the ecology of ligno — cellulose degradation and biotechnology applications. One obvious limit of environmental metagenomic sequencing is the sheer diversity of the microbial communities that populate rich environments, which calls for large sequencing efforts, to assemble long DNA sequence data. This complexity can lead to bias through overrepresentation of only a few common genomes, and because of the difficulty of assembling long sequences for analysis. Also of sig­nificance is the increasing number of published genomes of biomass-degrading organisms. Genomics research is not only providing phylogenic information about microbial popula­tions but in conjunction with new biochemical tools is generating a broader understanding of the complete biophysical activity of the community. Together these new tools are rapidly advancing our understanding ofthe principles that underlie microbial biomass degradation and carbon cycling in nature. The knowledge gained from these studies is a stepping-stone to the development of optimized enzymes and microorganisms for the production of com­modities such as ethanol and hydrogen from biomass.