Introduction

Producing second-generation ethanol from lignocellulosics such as agricultural and forestry residues, herbaceous and woody crops, weeds and waste paper, etc., has unique environmental, economic, and strategic benefits. The escalating demand for food, feed, and energy has raised several concerns about the potential use of food-based biofuels and their future sustainability, and global warming and energy security concerns have intensified the search for safe yet effective methods to commercially produce ethanol from other plants (Chandel et al. 2010a). Bioethanol is completely renewable in nature. Burning it releases carbon dioxide that is recycled back into plants, since plants use CO2 to synthesize cellulose during their photosynthesis cycle.

The wide variety of biomass is the backbone of biorefineries. The major types of biomass for ethanol production recognized to date are monoculture crops grown on fertile soils (such as sugarcane, corn, soya beans, oilseed rape, switch grass, willow, and hybrid poplar) (Farrell et al. 2006), waste biomass (such as straw, corn stover, and waste wood) (Kim and Dale 2004), and municipal solid waste (such as processed paper and newspaper; Kuhad et al. 2010). Another type of biomass is weedy cellulosics, viz. Eicchornia crassipes, Lantana camara, Prosopis juliflora, Saccharum spontaneum, Typha latifolia, Crofton, Chromolaena odorata, etc., which are promising and cheaper feedstocks for fuel ethanol production. These weedy cellulosic substrates do not require additional economic input as they grow on agriculturally degraded land or water bodies (Huber and Dale 2009).

No matter what plant it comes from, lignocellulosic biomass is composed of a complex mixture of cellulose, hemicellulose, and lignin (Fig. 1). After cellulose, hemicellulose is the fraction of the plant cell wall that has the most potential to serve as a source of bioethanol production (Chandel et al. 2007a; Kumar et al. 2008). The carbohydrate fractions of the plant cell wall can be converted into fermentable monomeric sugars through acidic and enzymatic (hemicellulase/cellulase) reactions, which have been exploited to produce ethanol, xylitol, and 2, 3-butanediol via microbial fermentation processes (Chandel et al. 2010b). The recalcitrance to saccharification is a one of the major limitations for conversion of lignocellulosic biomass to ethanol. The potential solution may lie in lignin modification, which could bypass the need for alkali or any microbial delignification step and thus facilitate the bioethanol process. In broader aspect, the future of biorefineries depends on low-input, high-diversity biomass feedstock that is rich in fermentable sugars and low in lignin (Tilman et al. 2007; Somerville et al. 2010). Basically, the bioconversion of lignocellulosics to ethanol includes three processes: (a) depolymerisation of structural polysaccharides into fermentable sugars via thermochemical and enzymatic routes, (b) fermentation of these sugars into ethanol, and (c) ethanol recovery (Fig. 2).

Fig. 1
figure 1

Molecular component of plant cell wall structure (Source: Rubin 2008, with permission and courtesy “Nature”)

Full size image
Fig. 2
figure 2

Future implications for bioethanol production from variety of lignocellulosic feedstocks

Full size image

For the long haul, it is necessary to understand the chemical compositions and structural hydrolysis of weedy substrates that are abundantly available on waste land for conversion to ethanol. This article aims to explore the chemical compositions of various weedy raw substrates for bioethanol formation. It will attempt to provide an in-depth understanding of the biotechnological aspects of lignocellulosic bioconversion from different biomass feedstocks in terms of the carbohydrate present in their cell walls, availability, feasible technologies for ethanol production, and new innovations involved in biorefineries.

Weedy lignocellulosic substrates: availability and chemical composition

Lignocellulosic biomass is an abundant renewable resource for the production of alternative biofuels, with 200 billion tons produced annually. It has a higher productivity rate per hectare than grains, oil seed, or sugars per unit of biomass produced (Kim and Dale 2004). Currently, the global yield of biomass crops, including woody and herbaceous crops grown in temperate and subtropical regions, varies from ~8 dry Mg/ha/year (for willow in Sweden) to 10–22 dry Mg/ha/year (for short rotation woody crops in the US). A conservative global biomass average would be ~10 dry Mg/ha/year, although some small-scale field trials have reported four times this level of biomass production (Perlack et al. 2005).

The production of bioethanol from agricultural residues and hays (wheat, barley, and triticale straws and barley, triticale, pearl millet, and sweet sorghum hays) is an attractive and feasible option (Kim and Dale 2004). Agro-residues are a very promising source of lignocellulosic feedstock for bioethanol production. Each source of biomass represents a technological challenge; the diversity of raw materials will allow the decentralization of fuel production with geopolitical, economic, and social benefits (Wyman 2007).

Plants using C4 photosynthesis tends to be productive in terms of fixing CO2 leading to increase photosynthesis, rapid growth even under extreme conditions such as drought and high temperatures. These plants can grow on marginal lands with high biomass density per unit area by using low nutrient and water needs (Rubin 2008). The photosynthesis reactions in C4 plants are the same as in C3. However, due to the dual carboxylase/oxygenase activity of RuBisCo in C3 plants, an amount of the substrate is oxidized rather than carboxylated. The oxidized substrate led to the loss of substrate and consumption of energy (i.e., photorespiration). In order to bypass the photorespiration, C4 plants developed a mechanism to efficiently deliver CO2 to the RuBisCO enzyme due to their specific leaf anatomy so called Kranz anatomy where chloroplasts exist, not only in the mesophyll cells in the outer part of their leaves but in the bundle sheath cells as well. C4 plants such as maize, sugarcane, sorghum, and millet efficiently fix CO2 during photosynthesis in turn storing high amount of carbohydrates.

The disadvantages of C4 plants are that they are rare in cold climates and unable to grow at temperatures less than 10°C. In these environments, trees (gymnosperms and angiosperms) that exclusively depend upon C3 photosynthesis are the only candidate species. The C3 group of potential energy crops includes trees such as poplar and eucalyptus that have relatively rapid growth potential in harsh conditions.

Perennial herbaceous energy crops make good feedstocks because they do not require annual reseeding once established, need fewer energy inputs (such as fertilizer and pesticides) than annual cropland, and can be grown on marginal croplands (Dien et al. 2005). They also have environmental benefits, including reduced soil erosion, enhanced carbon sequestration, and wildlife habitat (Lemus and Lai 2005). The major herbaceous energy crops that have been selected for bioethanol production in the US are switch grass (Panicum virgatum), miscanthus (Miscanthus spp. Anderss.), canary grass (Phalaris arundinacea), giant reed (Arundo donax L.), and alfalfa (Medicago sativa L.). They are considered to have energetic, economic, and environmental advantages over food crops for ethanol production (Hill et al. 2006). These dedicated energy crops have a fair amount of holocellulose (cellulose + hemicelluloses) in their cell walls, but their feedstock quality for livestock makes them unattractive options for fuel ethanol generation.

Major weedy substrates

Among the various forms of biomass (wood residues, agro residues, municipal solid wastes, and starchy substrates) available for ethanol production, weedy lignocelluloses seem to be the most promising as future biomass feedstock (Huber and Dale 2009). S. spontaneum (wild sugarcane) is a perennial weedy grass with deep roots and rhizomes that grows up to 4 m tall. It has worldwide distribution, extending across three geographic zones (the East Zone, Central Zone, and West Zone) and into other countries. In Asian countries like India, it has spread across millions of acres, often causing abandonment of fields. It can be an excellent biomass source for ethanol and cellulase production (Chandel et al. 2009b, 2010c; Scordia et al. 2010).

L. camara L. (Verbenaceae) is a noxious weed that can threaten land productivity, grazing for livestock, biodiversity, and consequently overall ecology. However, its luxuriant growth and vigorous survival give it potential economic value for utilization in value-added products such as ethanol (Pasha et al. 2007) and cellulose derivatives (Varshney et al. 2006). P. juliflora is a tree native to Mexico, South America, India, and the Caribbean that has become established as a weed in Asia, Australia, and elsewhere. It grows up to 12 m (39 ft) tall and has a trunk with a diameter of up to 1.2 m (3.9 ft), providing enough biomass for ethanol production (Gupta et al. 2009).

E. crassipes (water hyacinth) is a free-floating perennial aquatic plant native to tropical South America. The broad, thick, glossy, ovate leaves measure 10–20 cm across and float above the water surface. They have long, spongy, and bulbous stalks, and the plant may rise as much as 1 m above the surface of the water. The common water hyacinth is a vigorous grower that can double its population in 2 weeks. It is another potential biomass source for ethanol (Kumar et al. 2009a) and cellulase production (Sukumaran et al. 2009).

A perennial herbaceous plant, T. latifolia grows in temperate, subtropical, and tropical areas throughout the Northern Hemisphere. It grows in marshy areas and flowers in mid- to late summer. The plant is 1.5–3 m (5–10 ft) high and has 2–4 cm broad leaves. It may be a good carbon substrate for solid state fermentation to produce cellulase and ethanol (Chandel, unpublished data).

Eupatorium adenophorum (Crofton weed) is an erect, bushy, leafy, many-stemmed herbaceous perennial from Central America that grows to 2 m high. It is a highly invasive plant, forms dense stands, is tolerant of a wide range of conditions, and is common on roadsides and bush land edges and in wetlands. Zhao et al. (2007) studied pretreatment methods to enhance the enzymatic digestibility of this weed.

C. odorata (Siam weed or Christmas bush) is a species of flowering shrub native to North America, from Florida and Texas to Mexico and the Caribbean, and has been introduced to tropical Asia, West Africa, and parts of Australia. Recently, Zhao et al. (2010) explored its efficiency for ethanol production.

One of the most common noxious weeds, Parthenium (Asteraceae), is native to the tropical Americas and invades all disturbed land, including farms, pastures, and roadsides. The species Parthenium hysterophorus, also known as congress grass or gazar ghas, has become common in India, Australia, and parts of Africa and America (Everitt et al. 2007). As yet, there has been no report on ethanol production from this weed. We believe that harnessing it for biofuel use would be a legitimate application to promote a safe and clean environment.

Switch grass (P. virgatum) is a native tall prairie grass known for its rapid growth during the warm months to heights of 2–6 ft. Switch grass can be grown in most parts of the US, including swamplands, plains, and streams, and along the shores and interstate highways. It is self-seeding and resistant to many diseases and pests, and can produce high yields with low applications of fertilizer and other chemicals. It is also tolerant of poor soils, flooding, and drought; furthermore, it improves soil quality and prevents erosion due to its type of root system (Parrish and Fike 2009).

Miscanthus giganteus is another viable feedstock for cellulosic ethanol production. This species of grass is native to Asia and can grow up to 12 ft (3.7 m) tall with little water or fertilizer input. It is similar to switch grass with respect to cold and drought tolerance and water use efficiency (Ng et al. 2010). Miscanthus is commercially grown in the European Union as a combustible energy source.

Chemical composition

Lignocelluloses have three main components: cellulose, hemicelluloses, and lignin. Cellulose is the most abundant organic polymer on the earth, surpassing even starches and sugars; it is a homopolymer of sugars containing six carbon atoms linked together in a chain that constitutes the largest proportion of the plant cell wall. Hemicellulose is a heteropolymer consisting of xylose-linking compounds like arabinose, glucose, mannose, and other sugars through an acetyl chain (Chandel et al. 2010b). These compounds can be characterized as galactomannans, arabinoglucuronoxylans, or glucomannans based on their linkage with the main xylan backbone. Lignins are huge cross-linked jumbles of organic molecules that reinforce cellulose and hemicelluloses. They are complex, amorphous, three-dimensional polymers that have a phenyl propane structure (Rubin 2008; Fig. 1). Table 1 summarizes the basic cell wall composition of selected lignocellulosics. In general, hardwoods contain 18–25% lignin, 45–55% cellulose, and 24–40% hemicelluloses, while softwoods contain 25–35% lignin, 45–50% cellulose, and 5–35% hemicelluloses. Grasses normally contain 10–30% lignin, 25–40% cellulose, and 25–50% hemicelluloses (Betts et al. 1991). The structure and components of the cell walls of weeds are significantly different from those of most plant species, which may influence digestibility during the bioconversion process to bioethanol (Sarkar et al. 2009).

Table 1 Cell wall compositions of different lignocellulosic sources
Full size table

The critical parameters for selecting plants for fuel ethanol production include cell wall composition, growth rate, suitability for growth in different geographical regions, and resource–use efficiencies (Rubin 2008). Lignin and hemicelluloses differ in composition from species to species. Coniferous woods (gymnosperms) have a high proportion of mannose in their hemicelluloses, while deciduous wood species (angiosperms) have a high proportion of xylose (Sarkar et al. 2009). This complex composition can limit the potential of weedy substrates for use on an industrial scale. Hence, it is imperative to explore efficient and economical approaches to digesting complex chemicals.

Digestibility of weedy substrate

Pretreatment

Pretreatment is required to alter the macro- and microscopic size and structure of the biomass, as well as its submicroscopic chemical composition, so that the hydrolysis of the carbohydrate fraction to monomeric sugars can be achieved rapidly with greater yield (Kumar et al. 2009b). It solubilizes hemicellulose, reduces crystallinity, and increases the available surface area and pore volume of the substrate. In acid-catalyzed pretreatment, the hemicellulose layer is hydrolyzed, whereas in alkali-catalyzed treatment, a part of the lignin is removed and hemicelluloses are hydrolyzed using hemicellulases (Moiser et al. 2005; Kumar et al. 2009b). Various other types of pretreatment can be used, including mechanical, steam explosion, ammonia fiber explosion, and biological pretreatments (reviewed by Moiser et al. 2005).

A comparison of methods to assess the enzyme accessibility and hydrolysis of pretreated lignocellulosic substrates revealed one effective method for facilitating the enzymatic hydrolysis of a pretreated substrate (Chandra et al. 2009a). A lignocellulosic substrate of lodgepole pine chips was directly subjected to sulfite pretreatment to overcome the recalcitrance of lignocellulose pretreatment and then disk milled; the recovered cellulose substrate was quasi-simultaneously saccharified enzymatically and fermented into ethanol using commercial cellulases and Saccharomyces cerevisiae D5A (Zhu et al. 2010). Bak et al. (2010) proposed using rice straw that was fermented by the wood–rot fungus Dichomitus squalens as a biological pretreatment to increase the enzymatic digestibility of lignocellulose and promote cellulose hydrolysis. However, an efficient pretreatment process that can reduce the overall production cost of ethanol is still needed.

Removal of fermentation inhibitors from hemicellulosic hydrolysates

The acid hydrolysis of lignocellulosics releases xylose as the main sugar constituent in hydrolysates along with small fractions of arabinose, mannose, galactose, and glucose. Unfortunately, these hydrolysates also contain several fermentation inhibitors, such as furan derivatives from degradation of sugars, aliphatic acids released from hemicellulosic acetyl groups, phenolics from lignin, and metal traces if hydrolysis occurs in metal-based reactors. The compositional profile of hemicellulose hydrolysates depends upon the cell wall composition and the method employed for cell wall digestion (Chandel et al. 2007a, 2010b; Hahn-Hägerdal et al. 2007). These inhibitory compounds severely affect the fermentation performance of the biocatalyst used and reduce ethanol production. Several chemical, biological, and physical methods have been used to remove the inhibitors and increase the hydrolysate fermentability of lignocellulosic substrates (Chandel et al. 2007b). Parawira and Tekere (2010) reviewed the various physical, chemical, physico-chemical, and biotechnological strategies used for detoxification of lignocellulosic hydrolysates.

Enzymatic hydrolysis

After acid, alkaline, or fungal pretreatment, lignocellulosics can be saccharified enzymatically to obtain fermentable sugars. Microorganisms are potential sources of cellulases and hemicellulases, which can be used for the hydrolysis of pretreated lignocellulosics. Both bacteria and fungi are known to grow on these substrates in solid and submerged culture fermentation reactions. Table 2 summarizes the various lignocellulosics employed for cellulolytic enzyme production.

Table 2 Production of cellulolytic enzymes from various microorganisms using variety of lignocellulosic feedstock
Full size table

The enzyme cellulases act two orders of magnitude more slowly than other polysaccharidases. The action mechanism of cellulases needs to be deciphered at the molecular level. Studies must be done on mining of diversified cellulases and engineering proteins to make them penetrative. Enzymatic cocktails comprising cellulases, xylanases, mannanases, etc. are one option for efficient hydrolysis (Wilson 2009). The most important process improvement in the enzymatic hydrolysis of biomass was the introduction of simultaneous saccharification and fermentation, which has been improved to include the co-fermentation of multiple sugar substrates, and is now known as simultaneous saccharification and co-fermentation (Olofsson et al. 2010). Consolidated bioprocessing is another area of development, wherein the four steps—production of cellulases, enzymatic hydrolysis of lignocellulose, and conversion of hydrolysate (pentose and hexose) into ethanol—occur in a single step collectively (Lynd et al. 2005). The enzymatic hydrolysis in this process requires the use of cellulase, a multienzyme complex involving the synergistic action of endo-1,4-glucanase (EC 3.2.1.4), exo-alpha-1,4-glucanase (EC 3.2.1.91), and beta-glucosidase (EC 3.2.1.21). Cellobiose is a potent inhibitor of the cellulase enzyme. Beta-d-glucosidase thus provides a key catalytic activity for cellulase preparations and completes the saccharification of cellulose (Chandra et al. 2009b; Wilson 2009).

Bioethanol can be effectively economized by ensuring a maximum release of sugars from the pretreated substrate. Application of surfactants during enzymatic hydrolysis leads to an increase in the surface area of lignocellulosics and improves the yield of released sugars (Tabka et al. 2006). Nonionic surfactants like Tween-20 are more effective, and it is believed that surfactants change the nature of the substrate by increasing the available cellulose surface. The mechanism of surfactant activation may be due to their adsorption on hydrophobic surfaces composed of lignin fragments. Yang and Wyman (2006) reported that bovine serum albumin increased the surface area of pretreated corn stover and enhanced glucose yield (92%) at a loading of 7.5 FPU/g of cellulose. The enzymatic hydrolysis of lignocellulose can be limited by many factors, such as adsorption to surface areas, low fiber porosity, and low median pore size of fibers. Further limitations include the cellulase production, which is expensive and contributes significantly to the overall cost of saccharification.

Weedy substrate and microbial biosynthetic potential

Various sugars (pentose, hexose, and oligosaccharides) are derived from acid/enzymatic hydrolysis of the lignocellulosic sugar syrup derived from lignocellulosic biomass. The best-known alcohol-fermenting organisms, S. cerevisiae and Zymomonas mobilis, are capable of fermenting only hexose sugars and sucrose into ethanol. However, pentose-fermenting microorganisms such as Pichia stipitis, Candida shehatae, and Pachysolen tannophilus can produce ethanol from a variety of lignocellulosic substrates (Hahn-Hagerdaal et al. 2007). Anaerobic bacteria are able to ferment xylose, but are inhibited by high sugar and alcohol concentrations, producing excess byproducts that virtually lower the ethanol yield (Desai et al. 2004). Filamentous fungi are limited by their generation time, which would affect their overall ethanol yield on an industrial scale.

Microbial metabolic engineering

The production of alternative fuels can be enhanced by manipulating the metabolic intermediates in microbes that are mostly recognized in the cellular glycolysis pathway. Many native microorganisms have a distinct genetic system that is required for the synthesis of petroleum substitutes. However, these organisms lack a traditional usage that is economical and require genetic manipulation for industrial use. Metabolic engineering has played a pivotal role in the improvement of ethanol-producing microorganisms. Specific gene alteration was not possible through classical methods of genetic strain improvement, but industrial biotechnology can now provide pathways that extend the spectrum of usable industrial media (e.g., lignocellulosic hydrolysates) and enable the production of compounds not naturally formed by microorganisms.

Metabolic engineering of cellulase-producing microorganisms

Recombinant DNA technology offers significant potential for improving various aspects of lignocellulolytic enzymes to construct “synthetic” designer enzymes for specific applications. It may also be possible to fuse different lignocellulolytic genes or sections of genes from different organisms to produce novel chimeric proteins or enzymes with altered properties (Kumar et al. 2008).

With the advent of new biotechnologies and bioinformatics tools, searching for novel enzymes via metagenomic approaches may significantly contribute to their future economical production from renewable resources. Metagenomic analysis of the Nasutitermes hindgut reveals a rich diversity of bacterial genes encoding hitherto unknown glycosyl hydrolases. These enzymes constitute over 100 families of proteins that can break the glycosidic bonds between carbohydrates or between carbohydrate and non-carbohydrate entities (Warnecke et al. 2007). Later, Brulc et al. (2009) revealed forage-specific glycoside hydrolases that could be used in biofuel production through gene-centric metagenomics of the fiber-adherent bovine rumen microbiome. Alriksson et al. (2009) developed a recombinant Aspergillus niger strain expressing the Hypocrea jecorina endoglucanase Cel7B when grown on spent hydrolysates (stillage) from sugarcane bagasse and spruce wood. A. niger D15 [egI] displayed higher endoglucanase activity (2,100 nkat/ml) in the spent hydrolysates.

Martinez et al. (2008) performed a gene sequence analysis of the powerful cellulolytic fungus H. jecorina (Trichoderma reesei). Li et al. (2008a) compared function-based metagenome screening and sequence-based metagenome data mining as methods for discovering unusual enzymes related to the glycosyl hydrolase family from natural resources for degradation of recalcitrant lignocelluloses. Thermostable endocellulase (CelDR) was successfully cloned from a thermostable Bacillus subtillus and expressed into Escherichia coli BL21 (DE3), which showed almost three times the activity (0.78 U/ml).

Arrays of enzymes such as beta-glucosidases, endoglucanases, and cellobiohydrolases produced by T. reesei (Kumar et al. 2008; Martinez et al. 2008) and laccases and lignin peroxidases from white rot fungus (Larsson et al. 2001) were expressed in yeasts for enzymes and ethanol simultaneously. To identify new and useful enzymatic functions, researchers isolated a handful of microorganisms such as Z. mobilis, Clostridium phytofermentans, and Clostridium thermocellum and attempted to characterize their relative capacity for genetic manipulation and lignocellulosic conversion into ethanol (Warnicke et al. 2007).

Metabolic engineering of ethanol-producing microorganisms

To construct an efficient organism that can be used in large operations, important traits such as broad substrate utilization range simultaneously hydrolyzing the cellulose, high osmo-tolerance, high ethanol yields and productivity even at high temperatures, high ethanol tolerance, increased tolerance to inhibitors, and minimal nutrient supplementation are required (Zaldivar et al. 2001). An enormous amount of work has been done to search for suitable ethanologens from lignocelluloses, and efforts are underway to create a suitable microorganism that can be used on a larger scale in biorefineries. Hahn-Hagerdal et al. (2007) and Nevoigt (2008) elegantly reviewed the developments of recombinant yeast strains for simultaneous conversion of both pentose and hexose sugars from lignocellulose hydrolysates into ethanol.

The first xylose-fermenting S. cerevisiae strain was developed through the insertion and expression of xylose-metabolizing genes from P. stipitis (Kotter and Ciriacy 1993). Later, xylose-fermenting strains of S. cerevisiae were constructed by introducing the genes encoding xylose isomerase from the bacterium Thermus thermophilus (Walfridsson et al. 1996) and the anaerobic fungus Piromyces sp. (Kuyper et al. 2005), respectively. Ethanol production using lignocellulosic feedstock from recombinant and wild-type microorganisms is summarized in Table 3. Katahira et al. (2006) constructed a recombinant yeast strain that could ferment xylose and cellooligosaccharides by integrating genes for the intercellular expressions of xylose reductase and xylitol dehydrogenase from P. stipitis and xylulokinase from S. cerevisiae, as well as a gene for displaying β-glucosidase from Aspergillus acleatus on the cell surface. This strain produced 30 g/l ethanol from acid hydrolysate of wood chips (73 g/l total sugars).

Table 3 Fermentation of hydrolysates from different lignocellulosics into ethanol by recombinant and wild-type microorganisms
Full size table

Sanchez et al. (2010) developed a recombinant S. cerevisiae strain showing improved arabinose and xylose utilization by adopting evolutionary engineering. Jin et al. (2005) explored an inverse metabolic engineering approach to identify gene targets for improved xylose assimilation in recombinant S. cerevisiae expressing XYL1 and XYL2 from P. stipitis. The resulting recombinant strain exhibited a 100% increase in the growth rate and a 70% increase in ethanol production (0.033 versus 0.019 g ethanol/g cells·h) on xylose compared to the parental strain. Another industrially favorable microorganism, the recombinant S. cerevisiae D 452-2 strain, was developed for ethanol production from xylose expressing protein engineered NADH-preferring xylose reductase from P. stipitis NBRC 1687 (Watanabe et al. 2007).

Endo et al. (2008) identified the genes required for tolerance to vanillin in S. cerevisiae. Seventy-six deletion mutants were identified as vanillin-sensitive mutants and classified under the functional categories for chromatin re-modeling and vesicle transport, suggesting that these functions are important for vanillin tolerance. This study provided a biotechnological basis for molecular engineering as well as for screening of more robust yeast strains that may be useful in bioethanol fermentation.

The production of desirable compounds from microbes can often require a complete reprogramming of their innate metabolism. The evolution of such complex traits requires simultaneous modification of the expression levels of many genes, which may not be achievable by sequential multigene modifications. It could be helpful in the development of high ethanol-tolerant microbial strains. Alper et al. (2006) called this cellular engineering approach “global transcription machinery engineering”; it includes the alteration of key proteins regulating the global transcriptome and generates, through them, a new type of diversity at the transcriptional level. Following this, they observed 69%, 41%, and 15% improvement in volumetric and specific ethanol productivities and ethanol yield from an S. cerevisiae mutant compared with the wild species. Genome shuffling is a classical genetic engineering approach that uses iterative cycles of genome recombination and selection to combine the useful alleles of many parental strains into single cells showing the desired phenotype. P. stipitis was developed by genome shuffling several times for improved tolerance to hardwood spent sulfite liquor, resulting in improved ethanol production (Bajwa et al. 2010). The genome sequences of Z. mobilis ZM4 and P. stipitis revealed insights into the metabolic pathways responsible for pentose conversion into ethanol (Seo et al. 2005; Jeffries 2006).

Current status of genetic engineering in bioenergy crops

Genetic engineering of crops in order to increase structural carbohydrate content and reduce lignin levels is a promising path that may result in reduced pretreatment severity, facilitate the hydrolysis process, and help recover the maximum amount of sugars. In addition to this, cellulose and hemicellulose degradation enzymes are also being expressed in the cell wall, which decreases the overall cellulase enzyme load during saccharification of biomass (Sticklen 2008). Torney et al. (2007) reviewed the genetic engineering approaches to improve bioethanol production from maize. These approaches were intended to increase stress tolerance, photosynthesis rate, grain yield, and production of biomass conversion enzymes in planta (Table 4). These approaches could also be incorporated for the improvement of weedy crops in terms of increased biomass weight, cell wall composition, and biomass conversion assisted by enzyme expression in planta.

Table 4 Proposed routes to generate the high yielding and less calcitrant biomass for biofuel
Full size table

The lignin biosynthesis pathway has been a major area of research in plant biotechnology (Harris et al. 2009). It may be helpful to reduce the lignin content by increasing the amount of cellulose for improved digestion and pulping efficiency (Reddy et al. 2005). Chen and Dixon (2007) studied the downregulation of lignin biosynthetic genes in alfalfa, which revealed an increment in fermentable sugars for improved ethanol production; they advocated the downregulation of lignin-synthetic genes in other energy crops such as switch grass, Miscanthus, and poplar. Li et al. (2008b) constructed transgenic poplar plants using antisense technology, resulting in a 40% decrease in lignin and a 14% increase in cellulose content. Wei et al. (2001) reviewed the methods developed for altering lignin-biosynthetic genes in forest tree species. In another prospect for the genetic engineering of biofuel crops, Vega-Sanchez and Ronald (2010) suggested that the complete elucidation of lipid metabolism may facilitate the fatty acid biosynthetic pathways in cell wall synthesis. This could help in the development of the next generation of biofuel crops by increasing fatty acid contents and optimizing the hydrolysis of plant cell walls to release fermentable sugars for eventual conversion into bioethanol.

A genome sequence study on Populus trichocarpa (poplar), a potential bioenergy crop, reveals the potential of applying genomics to the challenge of optimizing energy crops. The shown traits will be used to maximize the biomass yield per unit land area (Tuskan et al. 2006). When using metagenomics, namely genetic material recovered directly from environmental samples, there is no need to cultivate cells. At the same time, the impetus to exploit “omics” approaches to capture new biotechnologies for plant cell wall deconstruction and the production of second-generation biofuels has reached new heights (Morrison et al. 2009).

Economic analysis of bioethanol production and commercialization

A steady state progress has been made in the bioconversion of lignocellulosic biomass for ethanol production. Despite the achievements made in the laboratory, the successful commercialization of ethanol remains a challenging task for commercialization (Wyman 2007; Himmel and Beyer 2009). The key issues relates to the cost and regular supply of feedstock and the balance between judicious usage of lignocellulosics, the economics of ethanol production and the environment (Banerjee et al. 2010). The economics of ethanol production using different raw materials are compared in Table 5. It is revealed that the cost of cellulosic ethanol is not competitive with grain-based ethanol as yet (Table 5).

Table 5 Comparison of the cost economics for ethanol production from various kinds of substrates
Full size table

Cellulosic ethanol commercialization is the process of building an industry out of methods of turning cellulose-containing organic matter into fuel. Companies such as Iogen Corporation, Mascoma Corporation, Lignol Energy Corporation, and Abengoa Bioenergy etc. are building refineries that can process biomass to turn into ethanol. Companies viz. Genencore Inc, Diversa Corporation, Novozymes Inc, and Dyadic Corporation are engaged producing enzymes that could enable a cellulosic ethanol in future. In recent years, the growth of commercial plants for bioethanol across the USA has mushroomed, including 26 new plants under construction in 2008 alone (Banerjee et al. 2010; Chandel et al. 2010a). The induction of cheap and surplus lignocellulosics having least economic and food/feed value (weedy materials, switch grass, Miscanthus, groundnut shell, sugarcane leaves, Brassica compestris stalks, cotton stalks, coffee spent, municipal solid waste, etc.) should be more explored.

The process integration, improved microbial traits for simultaneous production of cellulases and ethanol from mixed sugars, and improvements in the distillation process to get water-free ethanol will lead to a new manufacturing paradigm (Banerjee et al. 2010).

The implementation of bioethanol would generate more employment opportunities and income in rural areas and would reduce greenhouse gas emissions, which makes it worthwhile for the government to encourage biofuels by providing tax benefits (Himmel and Beyer 2009). It is recommended that appropriate policy objectives be imposed to foster bioethanol commercialization. These policy objectives could include the correction of certain tax anomalies, exemption from excise duty and sales tax, deregulation of feedstock and its pricing, and simplification of licensing for bioethanol production (Wyman 2007; Chandel et al. 2010a).

Future perspectives and challenges

Currently, the ability to produce biofuel is largely dependent upon lignocellulosic materials. Weedy materials may be the next-generation choice for biofuel, as they do not impose additional growth requirements for sustainability. It is advised to select C4 grasses such as sugarcane, switch grass, and Miscanthus, which have marginal requirements for growth. The increased demand for ethanol can be met by focused exploration of cheap lignocellulosic feedstock; pretreatment; elimination of detoxification steps (removal of fermentation inhibitors); a cost-effective, highly thermostable, synergistically acting enzyme mixture; development of robust fermentation microorganisms; and process integration to minimize process energy demand, including cost-efficient use of lignin (Fig. 2).

The development of efficient microorganisms can follow three paths: (1) making P. stipitis, C. shehatae, and recombinant E. coli more resistant to inhibitors; (2) genetic engineering of microorganisms (i.e., S. cerevisiae or Z. mobilis) for xylose fermentation and insertion of a laccase gene to eliminate the detoxification step for pentose hydrolysates; and (3) metagenomics of natural genes to produce an efficient fermentation process. In addition to optimize ethanol yields, a variety of microorganisms can be developed with the ability to utilize cellulosic and hemicellulosic sugars and tolerate high alcohol content and fermentation inhibitors. A more efficient distillation procedure for fermented broth must also be developed to economize the overall process. Developing a cheap process for ethanol recovery from lignocellulose hydrolysate fermented broth is one of the biofuel industry’s biggest challenges.

To create a sustainable generation of biofuels, using modern genetic engineering tools to produce tailor-made perennial plants and trees with increased amounts of biomass is an unavoidable necessity (Somerville et al. 2010; Harris et al. 2009). However, despite the promise of modern genetic engineering techniques, concerns about the environmental impact of genetically engineered plants cannot be ignored. In the USA, the FDA, USDA, and EPA are responsible for ensuring the safety of crops through regulations (Ragauskas et al. 2006). Several agencies in other countries monitor GE crops and frame guidelines for the safe application of recombinant genes in agro-industries (Singh 2010).

Conclusion

Lignocellulosic biomass is gaining popularity as a source of fermentable sugars for liquid fuel production. To use wood and/or weedy substrates as energy crops for commercial production, significant improvements will be required in the growth of feedstock. Recent advances in functional genomics and plant biotechnology have helped identify the genes and transcription factors that control wood formation and cellulase composition in fungi and bacteria. These advancements include potential approaches to develop ethnologic traits for fermenting pentose and hexose sugars and withstanding fermentative inhibitors, which may provide significant opportunities to genetically optimize tree crops as a cheap feedstock with high cellulase titers and high ethanol production.