A reliable supply of sustainable energy is critical for the healthy, wealthy and peaceful future of our planet. Energy is the world’s largest market, with a political and strategic impact that is unmatched by any other sector. Most countries, including European nations, are currently highly dependent on the finite and non-renewable resources of fossil fuels for their energy needs. This allows countries rich in such resources to become major players in world politics, frequently at the expense of countries that lack them.
Biofuels constitute a major alternative to face this problem. For their obtention, among all the catalysts, nanocatalysts are very attractive ones as they greatly increase the surface-to-volume ratio compared to bulk materials. Thus, they hold promise for dramatically faster, cheaper, less toxic and environmentally friendly biofuel production. Recent advances in nanocatalysis have prompted a persistent shift in the economic and political balance of the fossil fuels market. As for all technological shifts, the control of the direction and magnitude of the change is in the hands of developers.
Furthermore, there are alternative energy sources (some of them renewable and sustainable) that remain to be exploited. One of them is fiber, the non-edible plant cell wall cellulosic biomass, which is the source for so-called second generation biofuels.
The synthesis of organic carbon is a major biological process and the primary source of energy for life. Using solar energy, photosynthetic organisms (like plants and algae) convert, through photosynthesis, inorganic carbon into organic carbon that can be processed by heterotrophic organisms. The major source of carbon and energy in the biosphere is fiber, formed by polysaccharides primarily composed of cellulose and hemicellulose. These two compounds are the first and second most abundant organic molecules on Earth, respectively, and offer a renewable and seemingly inexhaustible feedstock not only for the production of biofuels but also for a variety of fine chemicals. Most cellulose and hemicellulose is found in plant fiber, specifically in the primary cell wall of plants. The secondary cell wall (produced after the cell has stopped growing) also contains polysaccharides and is strengthened by the aromatic (non-polysaccharide) polymer lignin, covalently cross-linked to hemicellulose.
Cellulose is a linear homopolymer with (1→4) β-linked D-glucose, which is present in plant tissue primarily as an insoluble crystalline matrix of parallel glucan chains. On the other hand, “hemicellulose” is a polymer formed by a variety of compounds (e.g., xylans, xyloglucans, arabinoxylans and mannans) in complex branched structures with a spectrum of substituents (e.g., acetyl and feruloyl groups). Hemicelluloses are usually bound to cellulose and to other hemicelluloses via hydrogen bonding and hydrophobic interactions, which help stabilize the cell wall matrix.
However, lignocellulose is a recalcitrant carbohydrate, resistant to degradation. The transition to a more environmentally friendly economy has put the focus of research on enzymes capable of efficiently degrading this feedstock. Bacteria and fungi have evolved complex enzymatic systems enabling their growth on plant material rich in cellulose. Enzymatic conversion of crystalline polysaccharides (saccharification, see Figure 1) is also crucial for an environmentally sustainable bioeconomy, but microorganisms that produce these enzymes typically require weeks, months or even years to decompose fallen logs or tilled corn stalks. This process is consistent with Nature’s needs. However, for chemical or fuel production from these materials, industry requires affordable enzymatic systems that can do the job in a much shorter scale, of days or even hours.
CellulosomePlus project target. The focus of CellulosomePlus is the bottleneck in the production chain of value-added chemicals (such as biofuels) from lignocellulose feedstock: the inefficient saccharification. It proposes to use redesigned cellulosomes as nanocatalysts for this process. Thus, establishing an efficient enzymatic conversion process for crystalline polysaccharides is an active area of research. After saccharification, a variety of microorganisms can be chosen for fermenting the products of hydrolysis of polysaccharides to yield desirable end products such as alcohol. Agricultural residues, wood, herbaceous crops and municipal solid wastes have thus far been considered as feedstocks for ethanol production. These materials primarily consist of cellulose, hemicellulose and lignin. Once the cellulose is converted to glucose, this compound is easily fermented to ethanol by yeast, a process well developed by industry. Conversion of cellulosic feedstocks into ethanol has the advantages of the readily available feedstocks, the environmental friendliness of the alcohol product as a biofuel and the avoidance of incineration and transportation of waste products.
Currently, the search for biofuels (to be used in existing engines) derived from biomass, that could partially or fully substitute crude oil, mainly focuses on the processing of storage polysaccharides such as starch, thus competing with food resources. However, the majority of plant polysaccharides are stored in the plant cell wall matrix, a recalcitrant material composed of particularly complex structural polysaccharides (mainly cellulose, hemicellulose, and pectin, as well as the non-carbohydrate lignin). Due to its complex and crystalline structure, only selected classes of microorganisms have evolved the enzymatic means to digest this rich reserve of sugars. This vast and cheap source of energy is not currently used extensively by industry due to the current inefficient methodology for its degradation to simple fermentable sugars. Furthermore, this source of energy is carbon neutral (i.e. sustainable), can generate clean fuel derivatives (i.e. reduced pollution), has a wide geographic distribution, and it is a common residue (in agricultural, paper, and food processing industries) permitting also the recycling of common wastes. Lignocellulosic substrates of industrial interest include wheat straw, corn stover, sugarcane, poplar and other trees, switchgrass, in addition to municipal and industrial wastepaper. CellulosomePlus will focus on the OFMSW (currently used by ABNT) to demonstrate production of biofuels from it with an increased efficiency in comparison with current industrial methods.
The Broad Arsenal of Biological catalysts for Plant Cell Wall Degradation.Millions of years of microbial evolution have led to the design of multi-enzyme cellulosome complexes that efficiently degrade plant cell wall polysaccharides, enabling simpler sugars to be produced from crystalline cellulose. The modular nature of these polysaccharide-degrading enzymes in cellulosomes was discovered in the 1980s, and their ongoing atomic characterization is crucial to understand their structure-function relationships. Since the heterogeneity and flexibility of the full-length cellulases and cellulosomes are impediments to crystallization, small angle X-ray scattering (SAXS) measurements were performed, confirming the overall architecture hypothesized. Today, technical advances and automation used for X-ray diffraction (e.g., the systematic use of synchrotron radiation, the exploitation of anomalous diffraction methods in protein crystallography and the adaptation of beamlines to perform SAXS of proteins in solution) provide more sophisticated experimental approaches to study these challenging and intricate cellulolytic assemblies.
Because of the intricacies in composition of both cellulosic substrates and enzymes, the establishment of standard enzymatic assays on cellulosic substrates has been and continues to be a serious impediment to identify superior enzyme catalysts and for their subsequent analysis. Despite the long history of research on cellulases and related enzymes (e.g., hemicellulases, pectinases, carbohydrate esterases, etc.) and the vast range of assays established (which are critical to their study), there is no simple standard assay yet to monitor enzymatic activity during the degradation of crystalline cellulose or complex cellulosic substrates (e.g., natural cellulosic material such as wheat straw). Several factors complicate the achievement of this goal. First, the multiplicity of the different types and modes of action of cellulases and the wide variety of cellulose breakdown products (cellooligodextrins) interfere with facile analysis of cellulose activity. Second, there is confusion and misunderstanding (which are frequently misleading) among researchers because of the contrast between the difficulty in the degradation of crystalline cellulose and the relative ease of that of non-crystalline substrates. Third, the wide variety of hemicellulases and their tremendous array of oligosaccharide products interfere with analysis of the breakdown products and the interpretation of total hydrolysis of the relevant polysaccharides constituents of the plant cell wall. Thus, it is not surprising that currently there is no efficient high-throughput activity assay relating enzymes and substrates, which constitutes a serious impediment to advancing research on enzymes involved in the hydrolysis of plant-derived polysaccharides, particularly for those that could efficiently break down recalcitrant composite substrates.
Without reliable means for assaying cellulolytic activity in a high-throughput and precise fashion, the identification of superior enzyme catalysts, as well as improved DCs, remains a bottleneck in the advancement of the field. There are several factors that contribute to this problem that must be addressed during the course of research on energy production from lignocellulosic biomass: the complexity of the enzymatic substrate, the insoluble nature of cellulose and the cooperativity of cellulolytic enzymes.
There is a significant economic drive towards reducing the enzyme cost contribution in order to maximize the economic viability of lignocellulosic biomass ethanol. In the literature, values ranging from 0.10 to 1.50 US$ per gallon of ethanol or even higher are usually reported. It is generally accepted that the main challenge for the enzyme industry is to decrease the enzyme cost below 0.30–0.40 US$ per gallon of ethanol. In terms of enzymatic activity the goal will be to achieve better activities than the commercial enzymatic cocktails (e.g. Accellerase® TrioTM from Genencor®). As mentioned, it must be noted that standardization in the field of cellulases and related enzymes has not been well established. Therefore, it is really difficult to compare different commercial products when taking into account their specified activities. Indeed, companies in the area recommend nowadays the end-users to compare the hydrolysis of defined feedstock on the basis of gr enzyme/gr biomass. We will follow this recommendation to evaluate the improvement of DCs. Thus, we will concentrate in improving the cost of production per gr of DC and in getting higher yield and rates as measured by gr of DCs that hydrolyze gr of feedstock.
On the other hand, European Industrial Bioenergy Initiative (EIBI) strategy assumes to reduce ethanol selling price to 2.46 $/gallon by early 2017. Due to these figures, industry has to make a big effort to meet European challenge by increasing enzyme effectiveness while reducing the cost of the enzyme related to its consumption and/or production.