RNAi suppression of lignin biosynthesis in sugarcane reduces recalcitrance for biofuel production from lignocellulosic biomass

Sugarcane is a prime bioethanol feedstock. Currently, sugarcane ethanol is produced through fermentation of the sucrose, which can easily be extracted from stem internodes. Processes for production of biofuels from the abundant lignocellulosic sugarcane residues will boost the ethanol output from sugarcane per land area. However, unlocking the vast amount of chemical energy stored in plant cell walls remains expensive primarily because of the intrinsic recalcitrance of lignocellulosic biomass. We report here the successful reduction in lignification in sugarcane by RNA interference, despite the complex and highly polyploid genome of this interspecific hybrid. Down‐regulation of the sugarcane caffeic acid O‐methyltransferase (COMT) gene by 67% to 97% reduced the lignin content by 3.9% to 13.7%, respectively. The syringyl/guaiacyl ratio in the lignin was reduced from 1.47 in the wild type to values ranging between 1.27 and 0.79. The yields of directly fermentable glucose from lignocellulosic biomass increased up to 29% without pretreatment. After dilute acid pretreatment, the fermentable glucose yield increased up to 34%. These observations demonstrate that a moderate reduction in lignin (3.9% to 8.4%) can reduce the recalcitrance of sugarcane biomass without compromising plant performance under controlled environmental conditions.     

Evaluation of pretreatment methods for lignocellulosic ethanol production from energy cane variety L 79-1002

Approximately half of the 80 billion tons of crop produced annually around the world remains as residue that could serve as a renewable resource to produce valuable products such as ethanol and butanol. Ethanol produced from lignocellulosic biomass is a promising renewable alternative to diminishing oil and gas liquid fuels. Sugarcane is an important industry in Louisiana. The recently released variety of “energy cane” has great potential to sustain a competitive sugarcane industry. It has been demonstrated that fuel-grade ethanol can be produced from post harvest sugarcane residue in the past, but optimized ethanol production was not achieved. Optimization of the fermentation process requires efficient pretreatment to release cellulose and hemicellulose from lignocellulosic complex of plant fiber. Determining optimal pretreatment techniques for fermentation is essential for the success of lignocellulosic ethanol production process. The purpose of this study was to evaluate three pretreatment methods for the energy cane variety L 79-1002 for maximum lignocellulosic ethanol production. The pretreatments include alkaline pretreatment, dilute acid hydrolysis, and solid-state fungal pretreatment process using brown rot and white rot fungi. Pretreated biomass was enzymatically saccharified and subjected to fermentation using a recombinant Escherichia coli FBR5. The results revealed that all pretreatment processes produced ethanol. However, the best result was observed in dilute acid hydrolysis followed by alkaline pretreatment and solid-state fungal pretreatment.     

Second generation ethanol in Brazil: can it compete with electricity production?

Much of the controversy surrounding second generation ethanol production arises from the assumed competition with first generation ethanol production; however, in Brazil, where bioethanol is produced from sugarcane, sugarcane bagasse and trash will be used as feedstock for second generation ethanol production. Thus, second generation ethanol production may be primarily in competition with electricity production from the lignocellulosic fraction of sugarcane. A preliminary technical and economic analysis of the integrated production of first and second generation ethanol from sugarcane in Brazil is presented and different technological scenarios are evaluated. The analysis showed the importance of the integrated use of sugarcane including the biomass represented by surplus bagasse and trash that can be taken from the field. Second generation ethanol may favorably compete with bioelectricity production when sugarcane trash is used and when low cost enzyme and improved technologies become commercially available.     

Perspective of the Sugarcane Industry in Brazil

The sugarcane industry in Brazil is experiencing a rapid shift towards creating the grounds for a green and sustainable biorefinary industry. After 30 years of ProAlcool, the federal government program that boosted Brazil’s sugarcane industry by creating a mandate to blend ethanol with gasoline, flex fuel engines now dominate Brazil’s automobile industry. Currently, bioethanol replaces around 30% of the gasoline consumed in the country and its demand is projected to more that double in the next 10 years. On another front, the sugarcane genomics program created by FAPESP in the late 1990s paved the way for the establishment of innovative biotechnology startup companies that attracted the attention of the largest agro-biotechnology sector companies of the world. Almost all of these companies now have their sugarcane research centers surrounding the city of Campinas, São Paulo. In addition, innovative synthetic biology companies are developing technologies to produce diesel, jet fuel and other high value molecules using sugarcane juice as a carbon source. The sugarcane industry also teamed with petrochemical companies and already established operating plants to produce bioplastics. Innovations have also occurred in the field of co-generation of electricity from sugarcane bagasse. Currently sugarcane supplies 4% of the electricity needs of the country. Collectively, these innovations suggest that Brazil’s sugarcane industry could supply over 30% of the country energy needs by 2021 and a significant fraction of new bioproducts produced by its nascent biorefinary plants.     

Preparation and properties of HDPE/sugarcane bagasse cellulose composites obtained for thermokinetic mixer

The use of natural fibers as reinforcement for thermoplastics has generated much interest due to their low cost, possibility of environmental protection and use of locally available renewable resources. In this work the mechanical and morphological properties of high density polyethylene/pre-treated and modified residues from sugarcane bagasse cellulose composites were analyzed. Composites were produced by a thermokinetic mixer. The microstructural analyses of fracture surface from composites can be easily evaluated by microscopic techniques. Results showed that the modification of sugarcane bagasse cellulose with zirconium oxychloride was successfully accomplished and that this reinforcement material with high density polyethylene showed tensile strength higher than non-modified sugarcane bagasse cellulose. Modification in the sugarcane bagasse cellulose influenced directly in mechanical properties of the composite material. This can be observed by the fracture surface, which showed that modified cellulose sugarcane bagasse improved interfacial adhesion between fiber and matrix.     

Ethanol production from lignocellulosic biomass of energy cane

Ethanol produced from lignocellulosic biomass is a renewable alternative to diminishing petroleum based liquid fuels. The release of many new sugarcane varieties by the United States Department of Agriculture to be used as energy crops is a promising feedstock alternative. Energy cane produces large amounts of biomass that can be easily transported, and production does not compete with food supply and prices because energy cane can be grown on marginal land instead of land for food crops. The purpose of this study was to evaluate energy cane for lignocellulosic ethanol production. Energy cane variety L 79-1002 was pretreated with weak sulfuric acid to remove lignin. In this study, 1.4 M sulfuric acid pretreated type II energy cane had a higher ethanol yield after fermentation by Klebsiella oxytoca without enzymatic saccharification than 0.8 M and 1.6 M sulfuric acid pretreated type II energy cane. Pretreated biomass was inoculated with K. oxytoca for cellulose fermentation and Pichia stipitis for hemicellulose fermentation under simultaneous saccahrification and fermentation (SSF) and separate hydrolysis and fermentation (SHF) conditions. For enzymatic saccharification of cellulose, the cellulase and ??-glucanase cocktail significantly increased ethanol production compared to the ethanol production of fermented acid pretreated energy cane without enzymatic saccharification. The results revealed that energy cane variety L 79-1002 produced maximum cellulosic ethanol under SHF (6995 mg/L) and produced 3624 mg/L ethanol from fermentation of hemicellulosic sugars.     

The Brazilian experience of sugarcane ethanol industry

Biomass has gained prominence in the last few years as one of the most important renewable energy sources. In Brazil, a sugarcane ethanol program called ProAlcohol was designed to supply the liquid gasoline substitution and has been running for the last 30 yr. The federal government’s establishment of ProAlcohol in 1975 created the grounds for the development of a sugarcane industry that currently is one of the most efficient systems for the conversion of photosynthate into different forms of energy. Improvement of industrial processes along with strong sugarcane breeding programs brought technologies that currently support a cropland of 7 million hectares of sugarcane with an average yield of 75 tons/ha. From the beginning of ProAlcohol to the present time, ethanol yield has grown from 2,500 to around 7,000 l/ha. New technologies for energy production from crushed sugarcane stalk are currently supplying 15% of the electricity needs of the country. Projections show that sugarcane could supply over 30% of Brazil’s energy needs by 2020. In this review, we briefly describe some historic facts of the ethanol industry, the role of sugarcane breeding, and the prospects of sugarcane biotechnology     

An-Overview on invertase in sugarcane

Saccharum officinarum is one of the most cultivated hybrid varieties among the sugarcane varieties. In sugarcane plant sucrose is the major carbohydrate which can be stored and transported. Different physiological and biochemical studies on this crop report that invertase activity and sucrose concentration some how are key limiting step in the process of sucrose accumulation. Significant efforts have been made in relation to the sucrose cycle by altering the sucrose phosphate synthetase, sucrose synthetase and invertase. In sugarcane two types of invertase enzymes have been reported on the basis of pH and cellular localization. Invertase breaks the sucrose into hexoses as a source of energy and carbon. It has also been reported that this enzyme is involved in the process of cell differentiation and plant development. Progress has been made for the understanding of invertase activity and its role in sugarcane plant. With the help of biotechnology it is possible to target the desired gene with genetic engineering approach to increase sucrose content by careful manipulation of invertase (enzyme) gene to increase the sucrose yield in sugarcane. Purpose of this mini review is to high-light the role of invertase in sugarcane and how to overcome sucrose recovery in sugarcane.     

Scientific challenges of bioethanol production in Brazil

Bioethanol (fuel alcohol) has been produced by industrial alcoholic fermentation processes in Brazil since the beginning of the twentieth century. Currently, 432 mills and distilleries crush about 625 million tons of sugarcane per crop, producing about 27 billion liters of ethanol and 38.7 million tons of sugar. The production of bioethanol from sugarcane represents a major large-scale technology capable of producing biofuel efficiently and economically, providing viable substitutes to gasoline. The combination of immobilization of CO₂ by sugarcane crops by photosynthesis into biomass together with alcoholic fermentation of this biomass has allowed production of a clean and high-quality liquid fuel that contains 93% of the original energy found in sugar. Over the last 30 years, several innovations have been introduced to Brazilian alcohol distilleries resulting in the improvement of plant efficiency and economic competitiveness. Currently, the main scientific challenges are to develop new technologies for bioethanol production from first and second generation feedstocks that exhibit positive energy balances and appropriately meet environmental sustainability criteria. This review focuses on these aspects and provides special emphasis on the selection of new yeast strains, genetic breeding, and recombinant DNA technology, as applied to bioethanol production processes.     

Potential for Bioenergy Production from Sugarcane in China

There has been rapid economic development in China in recent decades, and demand for energy has consequently been increasing rapidly. Development and utilisation of clean and renewable energy has been promoted by the Chinese government to help sustain long-term and stable development. Sugarcane is being increasingly used in several countries as feedstock for renewable energy products, and is a major and expanding crop in southern China. In this paper, we discuss the potential of sugarcane as a feedstock for bioenergy production in China. It includes a review of (1) the existing sugarcane industry in China and key bio-physical factors affecting the extent to which sugarcane-based industries could supply feedstock for renewable energy production in China, (2) the economic and policy factors which are likely to affect production of bioenergy from sugarcane in China, and (3) recommendations on actions and policies that may assist with appropriate development of bioenergy production from sugarcane in China. Existing and expected future economic conditions are unlikely to favour production of biofuel from the sugar component in cane. However, the fibre component of cane remains an under-utilised resource component. A conclusion is made that sugarcane fibre has potential to contribute towards renewable electricity production in China. However, at present, favourable incentives do not exist to encourage this production. It is suggested that policies to facilitate cost-effective production of renewable electricity by sugar mills, consistent with national objectives regarding production of renewable energy, be considered by governments. Priorities for future research are in improving biomass yields per unit area of land and technologies for low-cost conversion of lignocellulosic biomass into biofuel.     

Optimization of sugarcane farming as a multipurpose crop for energy and food production

Sugarcane is a multipurpose crop whose components may be used, in addition to sugar production, for various energy carriers or end‐products (electricity, liquid biofuels and heat) which enhance its economic potential. For many years, plant breeders and agronomists have focused on increasing sucrose yields per hectare and millers on increasing recoverable sucrose per ton of sugarcane in sugar mills. Attempting to exploit the energy potential of sugarcane more fully, calls for a more holistic approach focusing on both sucrose and lignocellulosic components of sugarcane biomass, and gaining some insight into the management practices required to optimize sugarcane cropping systems in these respects. Such options include genotype selection, harvest date with respect to the crop's growing cycle, crop type (plant crop vs. ratoon crops) and harvesting systems (mechanical vs. manual). The effects of these factors are strongly modulated by climate and soil properties, and these interactions are overall poorly known. Here, we set out to examine sugarcane infield management × environmental interactions with respect to (i) sugarcane yield and partitioning of the aboveground biomass; and (ii) sugarcane milling products (recoverable sucrose yield and amounts of coproducts) and their derived energy carriers. Three Saccharum cv. cultivars (R570, R579 and R585) were planted in three locations on La Reunion Island with contrasting management practices and climatological conditions. Quality characteristics of the samples were assessed by conventional and near infrared spectroscopy methods. Product, coproducts and potential energy production were measured and computed using transfer equations and a mill‐operating model. Yields and quality characteristics from cultivars and harvesting systems were affected differently by environmental factors – low temperature and radiation, and water stress. The current study also provides valuable information on how combinations between environments, genotypes and practices affect yield and partitioning of the aboveground biomass, and food and energy production.     

A rapid and highly efficient method for transformation of sugarcane callus

Modern sugarcane cultivars have complex genetic characteristics and low fertility that render their genetic improvement through traditional breeding difficult. Genetic engineering methodology to introduce foreign genes provides new opportunities for the genetic improvement of sugarcane cultivars. One of prerequisites for successful insertion of a gene cassette into the plant genome is the availability of an efficient transformation protocol. An improved protocol for Agrobacterium-mediated transformation of sugarcane is described. Between 85 and 100% of calli transformed using this procedure produced new calli, and 100% of them were positive for the inserted gene. The whole procedure permitted the production of transgenic calli in a short time (1.5 mo). The transformed calli can be cultured further for the production of the inserted gene-encoded enzyme by using cell culture, or they can be regenerated into transgenic plants. This protocol may be implemented also for the generation of transgenic plants from other species.     

Production of microbial levan from sucrose,sugarcane juice and beet molasses

Summary Bacillus polymyxa (NRRL-18475) produced a levan-type fructan (B, 26 fructofuranoside) when grown on sucrose, sugarcane juice, and sugarbeet molasses. The organism converted about 46% of the fructose moiety of sucrose to levan when grown on sucrose medium, however, the yields of levan from sugarcane juice and beet molasses were much less than sucrose solution. Such sugarcane juice and beet molasses can be made a good substrate for levan production by various modifications. Adding peptone to sugarcane juice or passing beet molasses through a column of gel filtration media improved levan yield to a level almost comparable to that obtained from sucrose.     

Sugarcane improvement: how far can we go?

In recent years, efforts to improve sugarcane have focused on the development of biotechnology for this crop. It has become clear that sugarcane lacks tools for the biotechnological route of improvement and that the initial efforts in sequencing ESTs had limited impact for breeding. Until recently, the models used by breeders in statistical genetics approaches have been developed for diploid organisms, which are not ideal for a polyploid genome such as that of sugarcane. Breeding programs are dealing with decreasing yield gains. The contribution of multiple alleles to complex traits such as yield is a basic question underlining the breeding efforts that could only be addressed by the development of specific tools for this grass. However, functional genomics has progressed and gene expression profiling is leading to the definition of gene networks. The sequencing of the sugarcane genome, which is underway, will greatly contribute to numerous aspects of research on grasses. We expect that both the transgenic and the marker-assisted route for sugarcane improvement will contribute to increased sugar, stress tolerance, and higher yield and that the industry for years to come will be able to rely on sugarcane as the most productive energy crop.     

Comparative Secretome Analysis of Trichoderma reesei and Aspergillus niger during Growth on Sugarcane Biomass

Background

Our dependence on fossil fuel sources and concern about the environment has generated a worldwide interest in establishing new sources of fuel and energy. Thus, the use of ethanol as a fuel is advantageous because it is an inexhaustible energy source and has minimal environmental impact. Currently, Brazil is the world''s second largest producer of ethanol, which is produced from sugarcane juice fermentation. However, several studies suggest that Brazil could double its production per hectare by using sugarcane bagasse and straw, known as second-generation (2G) bioethanol. Nevertheless, the use of this biomass presents a challenge because the plant cell wall structure, which is composed of complex sugars (cellulose and hemicelluloses), must be broken down into fermentable sugar, such as glucose and xylose. To achieve this goal, several types of hydrolytic enzymes are necessary, and these enzymes represent the majority of the cost associated with 2G bioethanol processing. Reducing the cost of the saccharification process can be achieved via a comprehensive understanding of the hydrolytic mechanisms and enzyme secretion of polysaccharide-hydrolyzing microorganisms. In many natural habitats, several microorganisms degrade lignocellulosic biomass through a set of enzymes that act synergistically. In this study, two fungal species, Aspergillus niger and Trichoderma reesei, were grown on sugarcane biomass with two levels of cell wall complexity, culm in natura and pretreated bagasse. The production of enzymes related to biomass degradation was monitored using secretome analyses after 6, 12 and 24 hours. Concurrently, we analyzed the sugars in the supernatant.

Results

Analyzing the concentration of monosaccharides in the supernatant, we observed that both species are able to disassemble the polysaccharides of sugarcane cell walls since 6 hours post-inoculation. The sugars from the polysaccharides such as arabinoxylan and β-glucan (that compose the most external part of the cell wall in sugarcane) are likely the first to be released and assimilated by both species of fungi. At all time points tested, A. niger produced more enzymes (quantitatively and qualitatively) than T. reesei. However, the most important enzymes related to biomass degradation, including cellobiohydrolases, endoglucanases, β-glucosidases, β-xylosidases, endoxylanases, xyloglucanases, and α-arabinofuranosidases, were identified in both secretomes. We also noticed that the both fungi produce more enzymes when grown in culm as a single carbon source.

Conclusion

Our work provides a detailed qualitative and semi-quantitative secretome analysis of A. niger and T. reesei grown on sugarcane biomass. Our data indicate that a combination of enzymes from both fungi is an interesting option to increase saccharification efficiency. In other words, these two fungal species might be combined for their usage in industrial processes.     

Chemical inhibition of acetyl coenzyme A carboxylase as a strategy to increase polyhydroxybutyrate yields in transgenic sugarcane

Polyhydroxybutyrate (PHB) is a naturally occurring bacterial polymer that can be used as a biodegradable replacement for some petrochemical‐derived plastics. Polyhydroxybutyrate is produced commercially by fermentation, but to reduce production costs, efforts are underway to produce it in engineered plants, including sugarcane. However, PHB levels in this high‐biomass crop are not yet commercially viable. Chemical ripening with herbicides is a strategy used to enhance sucrose production in sugarcane and was investigated here as a tool to increase PHB production. Class A herbicides inhibit ACCase activity and thus reduce fatty acid biosynthesis, with which PHB production competes directly for substrate. Treatment of PHB‐producing transgenic sugarcane plants with 100 μm of the class A herbicide fluazifop resulted in a fourfold increase in PHB content in the leaves, which peaked ten days post‐treatment. The minimum effective concentration of herbicide required to maximize PHB production was 30 μm for fluazifop and 70 μm for butroxydim when applied to saturation. Application of a range of class A herbicides from the DIM and FOP groups consistently resulted in increased PHB yields, particularly in immature leaf tissue. Butroxydim or fluazifop treatment of mature transgenic sugarcane grown under glasshouse conditions increased the total leaf biomass yield of PHB by 50%–60%. Application of an ACCase inhibitor in the form of a class A herbicide to mature sugarcane plants prior to harvest is a promising strategy for improving overall PHB yield. Further testing is required on field‐grown transgenic sugarcane to more precisely determine the effectiveness of this strategy.     

甘蔗基因克隆研究进展

甘蔗是重要的糖料和能源作物,在我国,其糖产量占全国食糖产量的94%。甘蔗是高光合效率C4作物,其蔗糖含量是所有植物中最高的,其基因组中蕴藏着大量重要基因。因此,甘蔗基因克隆对甘蔗品种定向改良具有重要意义。从蔗糖代谢、生长发育和抗逆性三个方面全面总结甘蔗基因克隆的研究进展,并提出今后研究的建议,以期为今后甘蔗基因研究提供参考。     

A Comparative Life Cycle Assessment Study of Polyethylene Based on Sugarcane and Crude Oil

A potential strategy for tackling the negative environmental impact of conventional plastics is to produce them from renewable resources. However, such a strategy needs to be assessed quantitatively, by life cycle assessment (LCA) for example. This screening LCA is intended to identify key aspects that influence the environmental impact of sugarcane low‐density polyethylene (LDPE) and compare these results against fossil‐based LDPE. The study showed that the major contributors to the environmental impact of sugarcane LDPE are ethanol production, polymerization, and long‐distance sea transport. The comparison between sugarcane‐ and oil‐based plastics showed that the sugarcane alternative consumes more total energy, although the major share is renewable. Moreover, for their potential impacts on acidification, eutrophication, and photochemical ozone creation, no significant difference between the two materials exists. However, with regard to global warming potential (GWP), the contribution of land use change (LUC) is decisive. Although the range of LUC emissions is uncertain, in the worst case they more than double the GWP of sugarcane LDPE and make it comparable to that of fossil‐based LDPE. LUC emissions can thus be significant for sugarcane LDPE, although there is need for a consistent LUC assessment method. In addition, to investigate the influence of methodological choices, this study performed attributional and consequential assessments in parallel. No major differences in key contributors were found for these two assessment perspectives.     

Surface polysaccharides and quorum sensing are involved in the attachment and survival of Xanthomonas albilineans on sugarcane leaves

Xanthomonas albilineans, the causal agent of sugarcane leaf scald, is a bacterial plant pathogen that is mainly spread by infected cuttings and contaminated harvesting tools. However, some strains of this pathogen are known to be spread by aerial means and are able to colonize the phyllosphere of sugarcane before entering the host plant and causing disease. The objective of this study was to identify the molecular factors involved in the survival or growth of X. albilineans on sugarcane leaves. We developed a bioassay to test for the attachment of X. albilineans on sugarcane leaves using tissue‐cultured plantlets grown in vitro. Six mutants of strain XaFL07‐1 affected in surface polysaccharide production completely lost their capacity to survive on the sugarcane leaf surface. These mutants produced more biofilm in vitro and accumulated more cellular poly‐β‐hydroxybutyrate than the wild‐type strain. A mutant affected in the production of small molecules (including potential biosurfactants) synthesized by non‐ribosomal peptide synthetases (NRPSs) attached to the sugarcane leaves as well as the wild‐type strain. Surprisingly, the attachment of bacteria on sugarcane leaves varied among mutants of the rpf gene cluster involved in bacterial quorum sensing. Therefore, quorum sensing may affect polysaccharide production, or both polysaccharides and quorum sensing may be involved in the survival or growth of X. albilineans on sugarcane leaves.     

C4 plants as biofuel feedstocks: optimising biomass production and feedstock quality from a lignocellulosic perspective

The main feedstocks for bioethanol are sugarcane (Saccharum officinarum) and maize (Zea mays), both of which are C(4) grasses, highly efficient at converting solar energy into chemical energy, and both are food crops. As the systems for lignocellulosic bioethanol production become more efficient and cost effective, plant biomass from any source may be used as a feedstock for bioethanol production. Thus, a move away from using food plants to make fuel is possible, and sources of biomass such as wood from forestry and plant waste from cropping may be used. However, the bioethanol industry will need a continuous and reliable supply of biomass that can be produced at a low cost and with minimal use of water, fertilizer and arable land. As many C(4) plants have high light, water and nitrogen use efficiency, as compared with C(3) species, they are ideal as feedstock crops. We consider the productivity and resource use of a number of candidate plant species, and discuss biomass 'quality', that is, the composition of the plant cell wall.