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- Herausgeber: Bentham Science Publishers
- Kategorie: Wissenschaft und neue Technologien
- Serie: Mycology: Current and Future Developments
- Sprache: Englisch
Mycology: Current and Future Developments is a book series that brings together latest contributions to research on the biology, genetics, and industrial use of fungi. Each book chapter is written by experts from around the world. The book series would be of interest to mycologists and allied researchers seeking to gain new knowledge perspectives about fungi.
This volume of the book series focuses chiefly on advances in biofuel production. Topics covered in this volume include an overview of biofuel production, the use of lignocelluloses in fungal biofuel production, fungal metabolic engineering, biomass pretreatment for biofuel refineries, and more. The volume also contains chapters about research on other fungi such as S. Cerevisiae. The reviews presented in this volume serve as a useful reference for researchers and readers interested in learning about new developments in biofuel production at a time when the need for alternative energy sources is ever increasing.
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Veröffentlichungsjahr: 2016
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Mycology: Current and Future Developments
(Volume 1)
Fungal Biotechnology for Biofuel Production
Edited by
Roberto Nascimento Silva
BENTHAM SCIENCE PUBLISHERS LTD.
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FOREWORD
Today’s chemical technology is still strongly dependent on fossil starting materials, fuel production being only one but a dominant example. Yet environmental concerns and political constraints have put the replacement of fossil carbon sources in industrial processes by compounds that are available in even larger amounts, but whose utilization can be performed in a carbon dioxide neutral way, at a top place in the list of technological advances of the 21st century. Quantitatively, this requirement can only be fulfilled by plant biomass since plants use carbon dioxide fixation via photosynthesis as the sole source of carbon. Furthermore, the dry plant biomass consists mainly of three polymers (cellulose, hemicelluloses and lignin), which – after depolymerization to the monomer constituents (hexoses, pentoses and phenylpropan substances) – can be used as a basis for the industrial production of ethanol or platform chemicals by fermentation or biocatalysis (biorefinery). Clearly, the production of these monomeric constituents by reliable processes and in high amounts that satisfy economic requirements and avoiding the production of hazardous byproducts is essential to the biorefinery concept. There is general consent that this can only be achieved by enzymatic hydrolysis.
Yet biological processes are slow on technological scales. Therefore, the composition of the enzymes used, their properties and the kinetics of the hydrolysis process are still major areas which need improvement and optimization. To do this on a science-driven basis, however, more basic knowledge is needed. Almost all of the enzymes used are derived from fungi. Fungi, the fifth kingdom, play a predominant role in the degradation of lignocellulose biomass in nature, and consequently some of them have therefore served as models or workhorses for the production of the respective enzymes for plant biomass hydrolysis in academic laboratories and on an industrial scale. This book therefore presents an overview of the progress and latest achievements in the understanding of these processes at genomic, transcriptomic and proteomic levels which are indispensable prerequisites for further improvement of the process, and highlights areas for further research development.
PREFACE
The world is going through major environmental changes and new energy demands are required. With the imminent shortage of energy sources based on fossil, new renewable energy source have been used for both purposes, as fuel as well as for industrial power generation. However, to access these renewable matrices, a full understanding of the process is necessary in order to reach biofuels at low cost. In this sense, biotechnology comes as a new science that combines biology of various knowledge areas such as biochemistry, microbiology, enzymology, molecular and systems biology. The use of microorganisms in the processing of biomass in order to produce biofuels such as ethanol or biodiesel, as well as derivatives with biotechnological potential has been an alternative to implement the so-called biorefinery.
In the following chapters of this work, the current panorama of biofuel and the use of fungi in biotechnology for the production of bioethanol and biodiesel will be discussed. In this sense, an overview of the process and the global biofuels market will be presented, followed by the basic biology of degradation of plant biomass, enzymology and regulation of gene expression of genes encoding enzymes that degrade plant biomass. In a second step, the processes already established will be presented in biorefinery such as large-scale production of hydrolytic enzymes as well as the general and specific aspects of biodiesel production. Subsequently, the presentation of enzymes used in the degradation of biomass in its immobilized form and the exploration of new enzymes will be discussed in addition to the benefits of using consortium of microorganisms for use in biorefinery. Finally, the current situation of biomass degradation processes and production of biofuel within the scope of ‘omics’ sciences and the integration of these processes from the point of view of synthetic and systems biology will be discussed.
We hope this book will contribute both in the academic field but also in the industrial area to the understanding and implementation of biofuel production worldwide.
The Panorama for Biofuels Biotechnology: Present and Future
Renato Graciano de Paula,Amanda Cristina Campos Antoniêto,Roberto N. Silva*Abstract
In 2013, the world production of ethanol was about 23.4 billion gallons. However, because of a global increase in fuel consumption, an increase in bioethanol production is necessary. The search for new energy sources increased the attention on biomass; now it is used directly for energy cogeneration by combustion and for the production of new fuels such as cellulosic ethanol or lignocellulosic ethanol, also called second-generation (2G) ethanol. Bioethanol production employing renewable sources is increasingly in demand worldwide because of the continuous depletion of fossil fuels, economic and political crises, and growing environmental safety concerns. Brazil and USA are the two largest producers and exporters of ethanol in the world. Nevertheless, other countries including China, India, Canada, Japan, Colombia, and Argentina have assumed featured positions in global fuel ethanol production. Therefore, this new world order may result in the development of an industrially suitable production strategy that will solve our energy crisis by producing more ethanol sustainably.
INTRODUCTION
Increases in population and industrialization have led to an increase in global demand for energy and raw materials. Fossil fuels are the main sources of global energy and chemicals, which affect the environment and cause economic and social problems. A decrease in the proportion of fossil fuels in the energy matrix is necessary to reduce the emission of greenhouse gases and consequently reduce
global warming. With the depletion of non-renewable petrochemical resources and an increase in concerns about environmental damage, renewable sources of energy have emerged as an important alternative to meet the energy needs of our present and future generations.
Biofuels are derived from renewable biomass, which can partially or fully substitute fossil fuels for use in combustion or energy generation. The two main liquid biofuels used are ethanol (bioethanol), which is extracted from sugar cane, and biodiesel, which is produced from vegetable oils or animal fats and added to diesel oil in varying proportions. In 2013, the global production of ethanol was about 23.4 billion gallons (www.ethanolrfa.org). However, because of a global increase in fuel consumption, a corresponding increase in bioethanol production is necessary. The search for new energy sources increased the popularity of biomass; now it is used directly for energy cogeneration by combustion and for production of new fuels such as cellulosic ethanol or lignocellulosic ethanol, also called second-generation (2G) ethanol.
The lignocellulosic ethanol production process has been widely studied in order to resolve bottlenecks in each step of the process, which are as follows: 1) characteristics and availability of biomass in raw materials, which determines the success of ethanol production; 2) selection of techniques to degrade the cell wall biomass, as this can be a challenging task; and 3) selection of efficient enzymes to obtain monosaccharide composition in the raw material, as cost and regularity on an industrial scale are key factors. In this chapter, we shall provide a global perspective of biofuel production, the pioneering experience of Brazil in this field, and the use of microorganisms in ethanol production. Furthermore, we will explore aspects of biorefineries and bottlenecks in implementation of this technology worldwide.
GLOBAL PERSPECTIVE OF BIOFUEL PRODUCTION
The global economy depends largely on energy derived from fossil carbon sources, mainly oil and coal. However, currently, the interest in natural gas has increased [1]. Bioethanol production employing renewable sources is increasingly in demand worldwide because of continuous depletion of fossil fuels, economic and political crises, and growing environmental safety concerns [2]. Biofuels have been used and produced in diverse regions worldwide. In this regard, the main determining factors of biofuel production are availability of biomass and presence of government incentives [3].
Presently, approximately 20 million barrels of gasoline are used globally every day. USA, Japan, China, India, and the European Union are the main consumers of gasoline. However, these countries are looking for alternative sources of energy to reduce consumption of fossil fuels in an attempt to reduce pollution. Diverse environmental, economic, political, and strategic factors suggest that bioethanol is the best alternative to gasoline. Therefore, many countries have begun to show interest in the production and consumption of bioethanol for use as vehicle fuel through programs and policies such as international agreements and incentives for domestic production and consumption. The global market for bioethanol fuel is still in its infancy and faces difficulties including supply security, lack of infrastructure and policies, and trade barriers in some regions. Nevertheless, a rapid increase in the demand for gasoline and oil price fluctuations are helping increase international trade of this renewable fuel [5].
The current biofuels industry produces around 57 billion toe (tonne of oil equivalent), which meets approximately 3% of the road transport sector’s energy requirements [5]. Currently, global production of biofuels has continued to increase, but the rate of production has slowed since 2008. Investments in biofuels are beginning to fall, mainly because of a constrained global economy and volatility of regulations governing the use of biofuels [3].
The most common biofuel, bioethanol, currently accounts for 75% of global biofuel production. Depending on the geographical region, different cereals, such as corn, or both sugar cane and beet are the main plants used to produce fuel ethanol employed in gasoline engines [5]. Brazil and USA are the two largest producers and exporters of ethanol in the world, with ethanol being produced from corn feedstocks in USA and from sugarcane in Brazil (Fig. 1). The USA is the main ethanol supplier in the world [6], while Europe, North America, and Latin America are the biggest consumers of fuel ethanol. In 2011, USA and Brazil consumed 24.6 million toe and 10.5 million toe of bioethanol, respectively [5].
Since 2010, USA has been a net exporter of bioethanol. Moreover, in 2011, because of poor-quality of the sugar cane harvest in Brazil, the ethanol exported by the USA reached record levels. Similarly, a significant volume of biofuels has been traded in Europe, which mainly imports biodiesel. Germany, France, and Spain are the most important importers of biofuels in Europe. Fifty percent of these imports come from Argentina, 39% from Indonesia, and less than 5% from USA [5]. Moreover, China, India, and Canada produce a significant amount of global fuel ethanol; in 2013, they collectively produced 1.764 million gallons [7].
Fig. (1))Global ethanol production by country/region and year. (Source:www.afdc.energy.gov/data/).
In December 2009, the Government of India (GOI) approved the National Policy on Biofuels. This policy encourages the use of renewable fuels to supplement transport fuels and proposes an indicative target to replace 20% of petroleum fuel consumption with biofuels (bioethanol and biodiesel) by the end of the 12th Five-Year Plan (2017) [8]. According Bryant [9], India is an emerging market for cellulosic bioethanol production. The new biofuel policy in India establishes an E20 (gasoline with 20% ethanol blended into it) target by 2017. Reaching this target will require the production of more than 4 billion gallons per year of cellulosic ethanol to meet the demand.
A nascent market for biofuels is Japan. The present government statistics indicate the use of 500 million liters of bioethanol in 2010 and a targeted use of 6000 million liters in 2030 [9]. In 2011, Japanese fuel ethanol production increased by an estimated 35%; additionally, biodiesel production expanded by 61% [10]. Peru, Colombia, and Central America are future suppliers with potential for ethanol production. For example, the Peruvian estimate for ethanol production in 2015 is approximately 250 million liters. This projection corresponds to a 2% increase compared to the 2014 estimate of 245 million liters. Furthermore, the forecasted domestic ethanol consumption in 2015 is 165 million liters. However, Peru does not produce biodiesel because of the availability of more affordable Argentine biodiesel, causing Peruvian biodiesel imports to remain flat at 283,000 metric tons [11].
Colombia’s sugarcane-based ethanol industry has a significant position in the western hemisphere, as it is the second most developed market. In 2010, expansion projects increased the daily production by almost 1 million gallons. Exports were responsible for most of the expansion in this market [12].
Argentina is currently the leading exporter of biodiesel and accounts for more than half of global biodiesel exports (1.36 Mt (megatonne), exporting mainly to Norway and the USA. United States Department of Agriculture (USDA)estimates suggest that Argentine biodiesel exports will increase by more than 70% of current levels to reach 8 Mt by 2020. Biodiesel production in Argentina reached 2.5 million tons in 2010 and is expected to produce 3 million tons by 2011. According Sapp (2014), Argentine biodiesel exports increased by 44% during the first nine months of 2014 compared to the same period last year. Moreover, ethanol production increased by approximately 43% [13].
The market for biofuels should continue to grow in the near future. As discussed above, new countries are poised as potential global biofuel suppliers. This may increase competition in the international market, which is important to protect against market oscillations. Moreover, for the successful growth of global biofuel production, investments are necessary in research, transfer of new technologies, and training of technical staff.
WORLD OIL CRISIS AND BIOFUELS INDUSTRY
Until mid-2014, the world production and oil prices had been relatively stable. However, recently the global oil prices have fallen sharply over the past seven months. In August 2014, the oil prices at around $110 a barrel. But since January 2015 prices have more than halved [14, 15]. Brent crude oil has now dipped below $50 a barrel for the first time since May 2009 and US crude is down to below $48 a barrel. This scenario affects of world economy of many countries which longer feel the effect. For example, Russia one of the world’s important oil producers, loses about $2 billion in revenues for every dollar in the oil price and its economy would shrink by at least 0.7% in 2015 if oil prices do not recover [14]. The reasons for the mundial scenario change are mainly the weak demand in many countries due to insipid economy growth, together with the US production, which reaches the highest levels since 1985. Furthermore, the fact that Opec’s members (Organization of the Petroleum Exporting Countries) do not cut production has also contributed to oil global depression. Concomitantly, the possibility of loss its niche market, does countries as Nigeria, the biggest oil producer in Africa and heavily oil-dependent, not decrease oil prices, contributing to increase the world crisis. The same way, Venezuela and Iran were important oil producers affected by this crisis. Moody’s and Fitch agencies bring down the Venezuelan credit to the default risk category due to the impact of falling oil prices on the balance of payments and the country's foreign exchange reserves [16].
In Brazil, the drop in the oil price also decrease the earnings with exploration projects in the pre-salt layer. On the other hand, the Brazilian oil company (Petrobras) has been able to reverse some of the accumulated loss last year the gap between fuel prices in the international market and those charged in the domestic market. Nowadays, the gasoline price is at least 70% over that international price. However, one question remains: What the impact of oil crisis on the world biofuel market? The first point is that gasoline and the oil price have reached their lowest levels in five years boosting world oil crisis. This scenario probably will permit the hit of cleaner alternative fuels. So, renewable energy remains as an important alternative to the future of the planet to meet the energy needs of our and future generations.
SUCCESS OF BRAZILIAN ETHANOL PRODUCTION
Brazil has been becoming the most competitive producer of bioethanol in the world. Recently, Ibeto and coworkers [3] highlighted the role of Brazil in international bioethanol production. This success is partially due to a well-developed domestic market. Moreover, the development of new sugarcane varieties, favorable weather, fertile soils, and agricultural technologies has supported the increase in Brazilian bioethanol production [17]. In 2013/14, Brazil produced 546 million tons of sugarcane, which yielded 31 million tons of sugar and 24 billion liters (6.336 billion gallons) of ethanol, which makes Brazil the world's largest sugar producer and second largest ethanol producer, behind USA [18].
The “ProAlcool” program launched by Brazilian government in 1975, stimulated the national production of bioethanol [19, 20]. Since then, the country has become a considerable bioethanol producer. Consequently, consumption of bioethanol as a fuel has surpassed consumption of gasoline in Brazil [21]. Currently, gasoline sold in Brazil contains 25% anhydrous bioethanol, and the expansion of ethanol consumption can be attributed to the growing fleet of light vehicles, especially flex fuel cars [21, 22].
The growing global demand for clean energy sources has placed the Brazil at the forefront of international bioethanol production [4]. According to reports of Brazilian ethanol exports in the 2014/2015 harvest season, of all the states, São Paulo was responsible for 94.96% (261.378 liters) of all exports, followed by Minas Gerais (5.48%) and Goiás (0.03%) [23]. Presently, Brazil is a pioneer in bioethanol production. Recent data showed that approximately 15% of Brazilian bioethanol is exported. As demonstrated in Table 1, the main export destinations of Brazilian bioethanol are USA, Korea, Nigeria, and Japan.
*Accumulated values from April 2014 to May 2015 of the 2014/2015 harvest season. Source: [27] UNICA (2014). Available from: http://www.unicadata.com.br/listagem.php?idMn=74.
In 2013, Brazilian sugarcane ethanol imported by the USA fell by 40% compared to 2012, to approximately 242 million gallons. Because Brazil is the largest supplier of ethanol imports to the USA, this drop caused USA to become a net exporter of bioethanol for the year. Export volumes of corn-based ethanol to Brazil declined, but were more than offset by higher export volumes to Canada and many other countries. Although the net level has varied monthly, since 2011, USA has both imported ethanol from and exported ethanol to Brazil [24].
Brazil is a successful story in terms of sugarcane ethanol production. Regional characteristics and a supply of adequate technologies ensures low-cost production of ethanol, which in the past few years has allowed for an impressive expansion of the Brazilian market and a change in focus of the market, which was previously predominantly based on the domestic market. To decrease dependence on fossil fuels, many countries have implemented the use of bioethanol in their energy matrix, have added bioethanol directly into gasoline, or have used bioethanol in the manufacture of oxygenated gasoline [4]. Remarkably, different countries have announced programs that have set biofuel use targets participation at its headquarters in less than 20 years. Therefore, Brazil needs to develop additional technical, economic, and political experience to respond to the soaring global demand of ethanol.
The rapid expansion of ethanol production from sugarcane in Brazil has raised a number of questions regarding its negative impacts and sustainability. Main positive effects include elimination of lead compounds from gasoline and reduction of CO2 emissions. However, negative impacts concern destruction or damage of high-biodiversity areas, degradation of soils, and deforestation [19].
Important contributors to the development of a successful national project in bioethanol production in Brazil include the follows: global increase in oil prices, growing global recognition of environmental consequences of global warming and its correlation with consumption of fossil fuels, and consistent reductions in costs of bioethanol production from sugarcane that are already competitive with gasoline prices of approximately $50 per barrel. Projections for oil import prices by the International Energy Agency [25] indicate values above $100 per barrel (for 2007) by 2020, which would make bioethanol more competitive in comparison with gasoline. Moreover, sugarcane and its main products are advantageous with regard to production value by hectare, revealing a preferred economic option [4].
Finally, others bioethanol producers, such as India and China, are less competitive as compared to Brazil because of weather conditions (rainfall and temperature). Sugarcane is well adapted to the soil and climate in some Brazilian regions [26]. Thus, Brazil has excelled in the world market for bioethanol production and has potential to grow further in the future.
FUNGAL BIOTECHNOLOGY FOR 2G-BIOETHANOL PRODUCTION: FROM THE FIELD TO THE TANK
The use of microorganisms for obtaining several primary and secondary metabolites by using different carbohydrates has been used throughout history. Presently, these metabolites have key roles in various domestic and industrial applications, including the production of 2G ethanol by microbial conversion [28].
An important step in bioethanol production is the hydrolysis of lignocellulolytic material. In this step, a variety of microorganisms, including bacteria and fungi, have the ability to obtain glucose monomers by degradation of plant biomass. Fungi are widely known as cellulase producers that can use cheap and surplus lignocellulosic raw materials as main carbon sources under several cultivation conditions [28]. The main cellulolytic and hemicellulolytic strains include Trichoderma (Trichoderma reesei, Trichoderma longibrachiatum, and Trichoderma harzianum), Penicillium (P. brasilianum, P. occitanis, P. funiculosum, and P. decumbans), Aspergillus (A. niger, A. nidulans, and A. oryzae), Fusarium (F. solani and F. oxysporum), Humicola (H. insolens and H. grisea), and Melanocarpus albomyces. Some yeast, such as the genus Trichosporium sp, are also producers of cellulases and xylanases [29-31]. Of these genera, Trichoderma have been most widely studied and are indisputable champions in cellulase production. The involvement of some of these species in bioethanol production will be described in greater detail below.
Trichoderma fungi are saprophytes and mesophilic and inhabit soil and decaying wood [32]. These microorganisms produce a number of enzymes, including cellulase and hemicellulase, that act synergistically to hydrolyze crystalline cellulose to smaller oligosaccharides and ultimately to glucose [33]. The Trichoderma reesei cellulolytic system is probably one of the most extensively studied systems, with regard to mechanisms of cellulase action. This system consists of cellobiohydrolases, endoglucanases, and β-glucosidases; thus, the synergistic action of these three groups of enzymes ensures efficient conversion of lignocellulosic biomass to glucose and other fermentable sugars [34].
Alternatively to the fungus Trichoderma reesei for the production of bioethanol, several species of Penicillium have been identified as potential cellulase producers. Many studies indicate that Penicillium spp. have a complete cellulolytic system with high β-glucosidase activity. P. funiculosum, for example, is known to produce glucose faster during hydrolysis of corn cobs as compared to marketed enzymes. Furthermore, P. citrinum has been employed in the production of alkali-tolerant and thermostable cellulases [31].
Species belonging to the genus Aspergillus, such as A. niger, are also commonly used in the industrial production of enzymes in recent times. These fungi were never considered efficient cellulase producers for the saccharification of plant biomass, although their genome has genes encoding hydrolytic enzymes such as endoglucanases and cellobiohydrolases. However, β-glucosidases produced by Aspergillus sp. are an interesting alternative for the lack of β-glucosidases often found in various strains of Trichoderma reesei [35].
Another group of fungi, which has attracted the attention of researchers as promising cellulase producers, consists of organisms capable of synthesizing thermostable enzymes, which cause increased catalytic efficiency and a consequent reduction in production costs of bioethanol. This group includes the species Talaromyces emersonii, Thermoascus aurantiacus, Sporotrichum thermophile, Chaetomium thermophilum, and Corynascus thermophilus. These microorganisms produce stable and active enzymes at elevated temperatures (above 60 °C), well above the optimal temperature for growth, which is approximately 30-55 °C [36].
Until now, the initial step of bioethanol production, hydrolysis of lignocellulosic material, was discussed. With simple sugars already available, the process of fermentation is initiated (see in more detail in section 1. V), which includes the participation of microorganisms.
Sugars from lignocellulosic hydrolysates comprise of a mixture of pentoses and hexoses, and economically efficient and sustainable biomass conversion to ethanol involves the use of microbial strains capable of fermenting not only glucose but also all sugars present in lignocellulosic hydrolysates, such as D-cellobiose, D-xylose, L-arabinose, mannose, and galactose, with high efficiency and productivity [29].
The most widely microorganism used in fermentation is the yeast Saccharomyces cerevisiae because of its ability to easily assimilate glucose from sugarcane or cellulosic biomass waste [37]. Saccharomyces cerevisiae has characteristics that make it a good candidate for fermentation processes, such as high fermentation rate, high ethanol tolerance, and a wide public acceptance. However, a disadvantage of this yeast is its inability to efficiently use xylose as a sole carbon source or ferment it to ethanol. Thus, several attempts have been made using genetic engineering to enhance the capacity of Saccharomyces cerevisiae to ferment xylose [36]. In contrast, Pichia stipitis has a natural ability to ferment the pentose sugar, xylose, and represents a relevant yeast species for biofuel research [38].
Among bacteria, the most promising is Zymomonas mobilis, which has high energy efficiency resulting in a high ethanol yield (greater than 90%) [39]. Escherichia coli is another bacterial species whose genome has been engineered to convert all hexoses and pentoses present in plant biomass. The resulting engineered strain showed increased ethanol tolerance and increased biofuel production effort at similar rates to those found in yeast [38].
Nowadays, the scientific community and the biofuels industry has made a major effort in searching for microorganisms with optimized functions, such as in the secretion of hydrolytic enzymes that act on lignocellulosic material or for the fermentation of simple sugars into ethanol [38]. In this context, genetic engineering plays a key role and is constantly improving the search for microbial strains that reconcile increased bioethanol production rate, reduction in environmental damage, and affordable cost.
BIOREFINERIES
The use of lignocellulosic biomass as an alternative to non-renewable energy sources has gained acceptance globally. Thus, the development and improvement of biorefineries is key to sustainable production of bioethanol that can economically compete with petrochemical fuels.
Biorefinery refers to an integral unit containing facilities, equipment, and processes using several biological nonfood feedstocks and converting them into many useful products including chemicals, fuels, and materials, with minimal waste generation and minimal emission of polluting gases [40]. It is closely analogous with the petroleum refinery, while employing renewable raw materials. Basic principles underlying a traditional petroleum refinery and a biorefinery are schematically represented in Fig. (2). Both are based on the same strategy: break down molecular complexes into their basic constituents and use them in the generation of new products. However, a petroleum refinery mainly supplies fuel for transport and energy, and only a small fraction of this fuel is used in the chemical industry. In contrast, the biorefinery produces a relatively greater amount of bioproducts (chemical and materials), besides producing bioenergy [41] (Fig. 2).
Fig. (2))Comparison of the basic-principles underlying a petroleum refinery and a biorefinery, showing raw materials used in each case and their respective products. (This figure is an adaptation of [41]).
A wide variety of natural resources, including hardwood, softwood, and residues from agricultural and forest activity, can be converted into functional materials through the process of biorefining [42, 43]. Lignocellulose, a major component of these raw materials, is predominantly composed of cellulose (40-50%), followed by hemicellulose (25-35%) and lignin (15-20%), which makes it extremely resistant to enzymatic digestion [44].
The varied composition of biomass sources make biorefineries capable of producing a larger class of products, which can be classified into two broad categories: material and energy products. The most important material products are chemicals, organic acids, polymers and resins, biomaterials, food, animal feed, and fertilizers. Energy products are used according to their energy content and enable transportation services, electricity production, and heat generation. This category comprises gaseous biofuels (biogas, syngas, hydrogen, and biomethane), solid biofuels (pellets, lignin, and charcoal), and liquid biofuels for transportation (bioethanol, biodiesel, Fischer–Tropsch fuels, and bio-oil) [45].
With regard to 2G ethanol production, lignocellulose is processed through four major steps, which are represented in Fig. (3): (1) the biomass is pretreated to remove lignin, which frees the other components, cellulose and hemicellulose, for the hydrolysis process; (2) cellulose and hemicellulose are hydrolyzed to produce fermentable sugars such as glucose, arabinose, galactose, xylose, and mannose; (3) 5- and 6-carbon sugars are fermented into ethanol by microorganisms; and (4) finally, ethanol is distilled, which purifies it and makes it suitable for use [46, 47].
Figure 3)The three major steps of lignocellulose processing for bioethanol production including pretreatment to remove lignin (1), hydrolyses of cellulose and hemicellulose (2), and fermentation of the resulting free sugars (3). (This figure is an adaptation of [47]).
The pretreatment step is performed under stringent conditions that make possible the removal of lignin and subsequent depolymerization of cellulose and hemicellulose, making the biomass more susceptible to attack by hydrolytic enzymes [48]. Four basic techniques can be used in this pretreatment step, namely physical, chemical, physicochemical, and biological; usually, a combination of these methods is used for the pretreatment step [46].
Enzymatic hydrolysis occurs through the action of microbial cellulases and hemicellulases that catalyze the hydrolysis of cellulose and hemicellulose, respectively, releasing 5- and 6-carbon sugars for subsequent fermentation [34]. Enzymatic hydrolysis can occur by two different ways: either hydrolysis takes place simultaneously with fermentation of monomeric sugars produced by the action of hydrolytic enzymes and this method is called simultaneous saccharification and fermentation (SSF); or, these processes occur at separate times and in separate compartments, and this method is called separate hydrolysis and fermentation (SHF). An advantage of using SSF is a lower production cost than SHF, since it has been demonstrated that the overall yield of ethanol produced by the lignocellulosic biomass is higher in the SSF process [48].
Fermentation, the last step of bioethanol production, involves conversion of several pentose and hexose sugars to ethanol [49]. The ideal ethanol-producing microorganism would ferment all biomass-derived sugars, possess resistance to inhibitory byproducts of the process, and, if possible, produce a synergistic combination of cellulases required for the complete hydrolysis of the cellulose polymer. As a result, fermentative microorganisms are subjected to continuous improvements, particularly by the use of genetic manipulation tools. Thus, several yeasts and bacteria, such as Saccharomyces cerevisiae and E. coli have been genetically engineered to enhance the fermentation of sugars such as glucose, xylose, and arabinose [50].
These technologies are still mostly expensive and offer low return on investment, which affects the production of chemicals and fuels at competitive costs. Accordingly, there is a constant requirement for development of new products and processes in biorefineries that allow for sustainable economic growth, are socially accessible, and reduce environmental impacts caused by the use of fossil fuels [42].
BOTTLENECKS IN 2G-BIOETHANOL PRODUCTION
Lignocellulosic materials, such as bagasse or straw, are mainly made up of three components: cellulose, hemicellulose, and lignin. The process of converting lignocellulosic biomass to ethanol consists of four stages, as discussed previously: pretreatment, enzymatic hydrolysis, fermentation, and distillation. Viability of 2G ethanol involves challenges on several fronts: agronomy, process engineering, logistics, equipment, and biotechnology. These challenges begin during the pretreatment step, and include genetic engineering to reduce lignin content in biomass, alteration of lignin composition, and identifying potential ligninases in natural sources (mainly fungi).
Furthermore, conversion of cellulosic components into fermentable sugars is a major technological and economic challenge in the production of biofuels. The cost of enzymatic hydrolysis is an important factor, as it depends on the efficiency, yield, pretreatment costs, synergistic actions of cellulase and accessory enzymes, and required addition of external enzymes [51]. Additionally, single-step biomass hydrolysis and sugar fermentation is an important goal for biorefinery technology.
Some pilot plants have already been implemented in the world, the most important of which are as follows: the National Renewable Energy Laboratory (NREL, USA), which produces approximately 120,000 liters of bioethanol/year; The Iogen Corporation (Canada), recently acquired by Novozymes, which produces approximately 320,000 liters/year using wheat straw as biomass. In Europe, in total, five plants will produce more than 1000 t/year ethanol, including plants in Germany (agriculture residues), Spain (straw and corn stover), Denmark (mainly wheat straw), Finland, and Italy (the biggest in the world, producing 75 million liter/year) [52].
Between 2014 and 2015, the first two Brazilian biorefineries producing 2G ethanol using cellulose as a raw material (bagasse and straw cane sugar) will be put into operation. The first plant is a GranBio unit with a capacity of 82 million liters/year using technology from ProesaTM Beta Renewables [53] and with investments of approximately $350 million [54]. The second plant, belonging to Raizen, is valued at $230 million and is being built in the State of São Paulo. The new unit will have the capacity to produce 40 million liters/year. Raizen claims to have plans to build seven cellulosic ethanol plants by 2024, all located near existing conventional power plants to facilitate logistics of straw and sugarcane bagasse [54]. With the applied technology, Brazil will become the largest biomass-derived bioenergy producer in the world. Currently, Brazil has about 450 million of hectares of underused arable land, which is far superior to any other country [55].
With a “global race” underway for 2G-ethanol production, China will bring new dimensions with the construction of a new refinery in partnership of Novozymes and Beta Renewables, which will be the largest in the world. With investments of approximately $325 million, the new plant will be ready by the end of 2016 in the city of Fuyang and will process between 970,000 and 1.3 million tons of biomass per year. This 2G-ethanol production plant will have the capacity to process approximately 235 million gallons of cellulosic ethanol per year [56].
A big challenge for the successful implementation of biorefineries is to reduce production costs for the conversion of biomass into sugars for fermentation. This requires the use of a cheap and efficient enzymatic cocktail. In this sense, cellulases produced by Trichoderma spp. play a key role in lignocellulosic biomass saccharification and are considered the best sources for industrial production. The US Department of Energy considers viable an enzyme cost of less than $0.12 per gallon ethanol to make lignocellulosic ethanol competitive [57]. However, enzyme producers work with optimistic values of approximately $0.35-$0.5 per gallon [58].
Presently, a large number of different cocktails are commercially available for biomass degradation. CellicTM CTec3 from Novozymes is one such easily available enzymatic cocktail, with better performance and competitive costs for the production of biofuels from biomass [59]. This cocktail is composed of enzymes such as cellulases (including GH61 and β-glucosidases) and a new range of hemicellulases that convert cellulose fibers and hemicellulose into fermentable sugars such as glucose and xylose [59, 60]. The identity of company producing microorganisms that generate these enzymes has not been disclosed. According Scharr (2013) [60], other companies that manufacture enzymes for cellulosic ethanol production include Codexis from USA (the company produces an enzyme package with cellulase activity called CodeXyme and is developing the product to meet technical requirements of cellulosic ethanol processes of Shell); Dyadic from USA (the company has developed C1 platform technology, which involves a strain of fungus, Chrysosporium lucknowense, originating from Russia, that grows in alkaline soil and can grow in extreme conditions); and Genencor from USA (the company has developed an enzyme called Accellerase 1000 with various enzymatic activities from a mixture of different enzymes, mainly exoglucanase, endoglucanase, hemicellulase, and β-glucosidase, and has also developed “Accellerase 1500”, an improved version enzyme from a genetically modified strain of Trichoderma reesei) [61].
Another limiting step in 2G-ethanol production, which should be overcome to reduce costs, is simplification or integration of processes. Currently, the production of enzymes, hydrolysis of biomass, and fermentation for ethanol production occur in separate steps. The SHF process includes the production of enzymes, hydrolysis of biomass, and subsequent fermentation of hexoses (C6) and pentoses (C5) in different tanks. However, accumulation of high glucose content can inhibit some enzymes, principally glucosidases. To solve this problem, the SSF process can be used as it leads to the removal of glucose by yeast during fermentation [62]. However, an ideal process for bioethanol production would be consolidated bioprocessing (CBP). Here, the same microorganism would be able to produce enzymes for biomass hydrolysis and conversion of sugars into ethanol [63]. However, this process is difficult to develop as it depends on genetic engineering to produce ethanologenic Saccharomyces cerevisiae that can also produce cellulases [64] or to convert the Trichoderma reesei into a good ethanol producer [63].
CONCLUDING REMARKS
2G-ethanol production is already a global reality. However, production costs still limit the commercial success of this product. The contribution of enzyme costs to the economics of 2G-ethanol production cannot be ignored. Commercial production of cellulase cocktails is often too great a bottleneck to be overcome. Increasing understanding of cellulase production is key for enzymatic hydrolysis of biomass. Biotechnological developments will allow for the production of a variety of enzyme cocktails at low costs. Furthermore, implementation of CBP will provide cost-effective biomass hydrolysis and sugar fermentation for biofuel and biorefinery technologies. Conversion of biomass to 2G ethanol depends on the robustness of efficient microbial enzyme systems; implementation of this process on a large scale should reduce production costs.
CONFLICT OF INTEREST
The author confirms that the author has no conflict of interest to declare for this publication.
ACKNOWLEDGEMENTS
This work was supported by The State of São Paulo Research Foundation (FAPESP) (proc. 2014/23653-2).
