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Bio - ethanol — The fuel of tomorrow from the residues of today
Abstract
The increased concern for the security of the oil supply and the negative impact of fossil fuels on the environment, particularly greenhouse gas emissions, has put pressure on society to find renewable fuel alternatives. The most common renewable fuel today is ethanol produced from sugar or grain (starch); however, this raw material base will not be sufficient. Consequently, future large-scale use of ethanol will most certainly have to be based on production from lignocellulosic materials. This review gives an overview of the new technologies required and the advances achieved in recent years to bring lignocellulosic ethanol towards industrial production. One of the major challenges is to optimize the integration of process engineering, fermentation technology, enzyme engineering and metabolic engineering.
... However, the high chemical stability of glucose hampers its transformation to other chemicals. Therefore, many researches have proposed high-valueutilization strategies of cellulose regard not glucose but fructose as a substrate for the synthesis of fuels and chemicals [4][5][6][7][8][9]. Fructose, structurally similar to glucose, is chemically more active than glucose and is easily converted to different molecules such as 5-hydroxymethylfurfural (5-HMF) and levulinic acid (LA). ...
... The dried mixture was pyrolyzed in a tube furnace under N 2 atmosphere (the flow rate was 1.5 L/min) at a ramp rate of 10 °C/min from room temperature to 650 °C and then kept for 60 min at 650 °C. Finally, the Mg-Al-supported biochar catalysts with the Mg/ Al ratio of 0:1, 1:1, 2:1, 3:1, 4:1, 5:1 were obtained and are denoted as 0 Mg-1 Al-C, 1 Mg-1 Al-C, 2 Mg-1 Al-C, 3 Mg-1 Al-C, 4 Mg-1 Al-C, and 5 Mg-1 Al-C, respectively. ...
... The number of times of the catalyst used was denoted as X. For example, 4 Mg-1 Al-C-3 means that 4 Mg-1 Al-C catalyst was used three times. All experimental values represent the average of triplicate analyses, where the average standard deviation was 1.5%. ...
This work provides an innovative method for preparing different isomerization catalysts by impregnating different proportions of MgCl2 and AlCl3 and combining different K compounds on cellulose-derived biochar, followed by pyrolysis. Results show MgO and Al(OH)3 existing in 4Mg-1Al-C catalyst can obtain better catalytic effect on glucose isomerization than the singe of Al presenting in 0Mg-1Al-C catalyst. Moreover, the synergism effects of the multi-catalytic active sites such as β-, γ-Al(OH)3, KCl, MgO, and K4H2(CO3)3 in Mg-Al-KHCO3-C catalyst can further lead to an increase in glucose isomerization, compared to the 4Mg-1Al-C catalyst. The X-ray diffraction results present that the value of O/Al in Mg-Al-KHCO3-C catalyst is as high as 13.38, which provides many unsaturated acidic catalysis sites and benefits the glucose isomerization. Simultaneously, the TPD results reveal that the main active sites (MgO, Al(OH)3, and K4H2(CO3)3) in Mg-Al-KHCO3-C catalyst can provide weakly acidic and basic sites and avoid strongly acidic and basic sites to excessively attack the glucose. Based on the DFT analysis, the results indicate that the MgO has a great effect on the ring-opening reaction to form acyclic glucose, while Al(OH)³⁺ has a great effect on promoting acyclic glucose hydrogen transfer isomerized to form fructose. Compared to other carbon-based metal catalysts, the prepared Mg-Al-KHCO3-C has excellent catalytic performance, which gives a higher fructose yield (38.7%) and selectivity (87.72%) and glucose conversion (44.12%) at 100 °C in 30 min. In this study, we develop a highly efficient Mg-Al-K-biochar catalyst for glucose isomerization and provide an efficient method for cellulose valorization.
... Fermentation technologies are also mature when used to convert organic matter rich in directly fermentable sugars or starch into ethanol, a liquid biofuel [28]; however, the techno-economic challenges for the conversion of lignocellulosic biomass by fermentation are complex, and further research is required [28]. Major issues involved in the fermentation of lignocellulosic biomass are: (i) the need for extensive pre-treatment to yield fermentable sugars [29]; (ii) the low concentrations of ethanol, which require large amounts of energy for downstream separations and ethanol purification [30] (iii) the presence of both pentose and hexose sugars in the fermentation broth [31]; and (iv) the presence of toxic compounds that may act as inhibitors [31]. ...
... Fermentation technologies are also mature when used to convert organic matter rich in directly fermentable sugars or starch into ethanol, a liquid biofuel [28]; however, the techno-economic challenges for the conversion of lignocellulosic biomass by fermentation are complex, and further research is required [28]. Major issues involved in the fermentation of lignocellulosic biomass are: (i) the need for extensive pre-treatment to yield fermentable sugars [29]; (ii) the low concentrations of ethanol, which require large amounts of energy for downstream separations and ethanol purification [30] (iii) the presence of both pentose and hexose sugars in the fermentation broth [31]; and (iv) the presence of toxic compounds that may act as inhibitors [31]. ...
Biomass is a highly versatile and reliable source of firm, renewable energy, capable of generating heat, power and various biofuels. The technologies used to convert biomass into fuels or energy can be broadly divided into two categories: biochemical and thermochemical. Biochemical pathways for forest biomass conversion into fuels still face techno-economic challenges, requiring further research to make them economically attractive. In contrast, thermochemical conversion processes, including gasification, pyrolysis and combustion, are well suited for forest biomass conversion, with several technologies having reached a fully commercial stage. Combustion, the most common and mature thermochemical pathway, converts forest biomass into heat, power, or combined heat and power. While traditional, inefficient and polluting methods are still used for burning forest biomass, modern, cleaner, and more efficient combustion technologies are available and in use. Some pathways based on gasification and pyrolysis are also commercially viable, providing solid, liquid and gaseous biofuels. These options offer versatility across combustion systems, heat engines, fuel cells and synthesis applications. This chapter provides a comprehensive overview of forest biomass as an energy source, covering processing technologies, technology readiness levels, fuel characteristics and pre-treatment methods. It emphasizes the potential and challenges associated with using forest biomass for sustainable energy production.
... To this end, there has been a lot of interest in using other biomasses that do not compete with food utilization such as lignocellulosic and seaweed biomass. Lignocellulosic biomass is composed of cellulose, hemicellulose, and lignin [4] while seaweeds have a much more diverse composition of carbohydrates which often includes cellulose but can also include β-glucans such as laminarin, uronic-acid containing carbohydrates such as carrageenan, alginate, agarose, mannitol, and sulfated polysaccharides [5,6]. As with terrestrial biomass, the exact composition varies as a function of the type of seaweed in addition to other variables related to growth conditions. ...
... The ability of strain AK15 to degrade β-1,3-O glycosidic bonds was evaluated using lichenan and laminarin. The ability of strains to utilize the hemicellulose fraction was evaluated using xylan, mannan (β-O- (1)(2)(3)(4) linked mannose), galactan, and rhamnan. The strain did not utilize any of these substrates. ...
The present study is on the biotechnological potential of Thermoanaerobacter strain AK15 concerning its ability to produce ethanol and valuable alcohols from carbohydrates and amino acids as well as investigations on their enzyme capability. The strain AK15 was found to be positive for alkaline phosphate, C4 esterase, acid phosphatase, napthol-AS-BI-phosophohydrolase, and α-glucosidase. Strain AK15 is highly ethanologenic, producing a maximum of 1.57 mol ethanol per mol of glucose degraded at high liquid-gas phase ratios. The strain degrades most of the sugars tested as well as starch and pretreated hydrolysates of biopolymers (Whatman paper, newspaper, Timothy grass, Rhubarb leaves, and several macroalgae species) to ethanol mainly. The strains degraded serine and threonine when used as a single substrate, producing mainly acetate and ethanol as end products, and the branched-chain amino acids (leucine, isoleucine, valine) when cultivated in the presence of thiosulfate. The main end products from branched-chain amino acids were a mixture of their corresponding branched-chain fatty acids and alcohols. Finally, the strain was also shown to use butyrate as an electron sink during glucose degradation resulting in the reduced product butanol in addition to end products produced from glucose. Thus, strain AK15 is a promising candidate for ethanol and fine chemical production.
... (1) synthesis from ethylene and (2) fermentation of starchy and sugary materials. In recent years, the three-step process of producing ethanol from methanol has been considered (Hahn-Hagerdal et al. 2006;Ni et al. 2007;Balat et al. 2008;Taghizadeh-Damanabi and Bahadori 2017;Arshad 2018;Bahadori and Naalband-Oshnuie 2019;Kokkinos 2021;Ahmadi and Bahadori 2022;Kokkinos 2022). ...
... Considering that about 60% of acetic acid is produced by the carbonylation of methanol, the production rate of methyl acetate as a side product is significant. Methyl acetate is also produced by esterification of acetic acid with methanol in the presence of strong acids such as sulfuric acid (Cheung et al. 2002;Hahn-Hagerdal et al. 2006;Balat et al. 2008). Addition to its application as a solvent, methyl acetate is also considered an intermediary material for the production of other valuable products. ...
Ethanol production methods are expanding due to the importance of ethanol as a fuel or additive to fuels. One of these methods is converting methanol to ethanol in a three-step process. All of these steps need to deeply study and investigate to develop the process. In this research, the carbonylation of dimethyl ether to produce methyl acetate, which is the intermediate reaction of the three-step process of methanol to ethanol, has been simulated and optimized. The parameters of temperature, pressure, residence time, and feed ratio have been investigated as effective operational parameters of the process. It has been shown that the temperature and pressure of the process are more effective in the ranges of 220–280 °C and 30–50 bar, respectively. The simulation results showed a maximum point in dimethyl ether conversion in the feed ratio of 0.4–0.6, i.e., in temperature of 260 °C, residence time of 5 h, pressure of 45 bar, DME/CO/Ar = 30/67/3, and DME conversion about 22%. Also, it has been shown that increasing the residence time increases the effect of each of the above parameters. Optimization of the dimethyl ether carbonylation process has demonstrated that the combination of different ranges of the above parameters achieves the desired conversion, i.e., in pressure of 48.23 bar, temperature of 259.06 °C, residence time of 3.68 h, and dimethyl ether/feed of 0.461 vol%; conversion of dimethyl ether will be equal to 85.50%.
... Glucose can be converted into ethanol with the aid of the most important ethanol producers, mainly the yeast S. cerevisiae and the bacterium, Z. mobilis (Hahn-H€ agerdal, Galbe, Gorwa-Grauslund, Lid en, & Zacchi, 2006;Lin & Tanaka, 2006;Sarris & Papanikolaou, 2016). A study was performed on wild strains of Z. mobilis isolated from sugarcane molasses for bioethanol production and claimed a maximum ethanol concentration of 79.78 g/L (Pinilla et al., 2011). ...
Bioethanol is one of the most important renewable fuels contributing to the reduction of
negative environmental impacts generated by the worldwide utilization of fossil fuels.
Over the past 15years, studies have shown that the replacement of gasoline by bioethanol
or biofuel causes a net average reduction of greenhouse gases of 71% (Haq et al., 2016;
Koh & Ghazoul, 2008). Bioethanol is not a new energy source since it has been used
extensively in Europe and the United States in the early 1900s, but it was ignored
due to its high production cost compared to petrol. The bioethanol production was then
continued due to the oil crisis in the 1970s (Aditiya, Mahlia, Chong, Nur, & Sebayang,
2016). The production of bioethanol from microbial fermentation strategies is an alternative
method to meet the global demand. The production of bioethanol is a complicated
process consisting of several steps. First, its ability to exploit a variety of microorganisms
that are capable of efficient bioethanol production by fermentation; second, to utilize
various substrates such as sugars, starches, or celluloses derived from a variety of different
sources such as energy crops (corn, wheat, sugarcane, sugar beet, cassava, among others),
crop residues (e.g., rice straw, rice husk, corn stover, corn cobs), or waste biomass (for
instance, food waste, livestock waste, paper waste, construction-derived wood residues,
and others); and third, inexpensive sources of enzymes (ligninolytic, and cellulolytic
enzymes). Both fungi and bacteria are capable of efficiently converting sugars to ethanol
by fermentation processes (Cardona & Sa´nchez, 2007; Liu et al., 2018).
... The negative impact of fossil fuel on the environment is a global concern, particularly greenhouse gas emissions [1]. The challenges associated with the use of fossil fuel is enormous and costly in some developing oil-producing countries. ...
... such as industrial robustness, substrate utilization, productivity and yield. Bacteria have some advantages over yeasts (Hahn-Hägerdal et al. 2006;Khoja et al. 2014;Sadik and Halema 2014). Industrially, bacteria are preferred over fungal strains as bacterial strains show higher yield, higher tolerance, shorter generation time, lower biomass generation, more effective substrate utilization and simpler downstream processing steps than fungal strains (Yang et al. 2016). ...
... To this end, there has been a lot of interest in using other biomasses that do not compete with food utilization, such as lignocellulosic and seaweed biomass. Lignocellulosic biomass is composed of cellulose, hemicellulose, and lignin [4], while seaweeds have a much more diverse composition of carbohydrates, which often includes cellulose but can also include β-glucans, such as laminarin and ...
The conversion of lignocellulosic and algal biomass by thermophilic bacteria has been an area of active investigation. Thermoanaerobacter species have proven to be particularly capable in the production of bioethanol and biohydrogen from lignocellulosic biomass, although detailed studies of their abilities to utilize the full gamut of carbohydrate, amino acids, and proteins encountered in biomass hydrolysates are seldom comprehensively examined. Here, we re-evaluate the ability of Thermoanaerobacter strain AK15, a highly ethanologenic strain previously isolated from a hot spring in Iceland. Similar to other Thermoanaerobacter species, the strain degraded a wide range of mono- and di-saccharides and produced a maximum of 1.57 mol ethanol per mol of glucose degraded at high liquid–gas phase ratios. The ability of strain AK15 to utilize amino acids in the presence of thiosulfate is limited to the branched-chain amino acids as well as serine and threonine. Similar to other Thermoanaerobacter species, strain AK15 produces a mixture of branched-chain fatty acids and alcohols, making the strain of interest as a potential source of longer-chain alcohols. Finally, the strain was also shown to use butyrate as an electron sink during glucose degradation resulting in the reduced product butanol, in addition to end-products produced from glucose. Thus, strain AK15 is a promising candidate for ethanol and higher-order alcohols from a range of lignocellulosic and algal biomass.
... To this end, there has been a lot of interest in using other biomasses that do not compete with food utilization such as lignocellulosic and seaweed biomass. Lignocellulosic biomass is composed of cellulose, hemicellulose, and lignin [4] while seaweeds have a much more diverse composition of carbohydrates which often includes cellulose but can also include β-glucans such as laminarin, uronic-acid containing carbohydrates such as carrageenan, alginate, agarose, mannitol, and sulfated polysaccharides [5,6]. As with terrestrial biomass, the exact composition varies as a function of the type of seaweed in addition to other variables related to growth conditions. ...
Biotechnological potential of Thermoanaerobacter strain AK15 to produce ethanol and valuable alcohols from carbohydrates and amino acids were evaluated in present study. The strain is highly ethanologenic, producing a maximum of 1.57 mol ethanol per mol of glucose degraded at high liquid-gas phase ratios. The strain degrades most of the sugars tested as well as starch and pretreated hydrolysates of biopolymers (Whatman paper, newspaper, Timothy grass, Rhubarb leaves, and several macroalgae species) to ethanol mainly. The strains degraded serine and threonine when used as a single substrate, producing mainly acetate and ethanol as end products, and the branched-chain amino acids (leucine, isoleucine, valine) when cultivated in the presence of thiosulfate. The main end products from branched-chain amino acids were a mixture of their corresponding branched-chain fatty acids and alcohols. Finally, the strain was also shown to use butyrate as an electron sink during glucose degradation resulting in the reduced product butanol in addition to end products produced from glucose. Thus, strain AK15 is a promising candidate for ethanol and fine chemical production from a range of lignocellulosic and algal biomass.
... The microorganism should grow and produce ethanol in the presence of at least 4% (v/v) ethanol. 23 Ethanol tolerance of yeast cell is closely related to ethanol productivity. 24 The growth rates of many organisms decrease markedly with increasing ethanol concentrations. ...
In this research work, twenty two xylose-utilizing yeasts were isolated from various sources. Although all isolates could assimilate all tested sugars, they have variations in sugar fermentation pattern. In temperature tolerant activity, almost all yeast isolates could grow well at 40°C. Weak growth of seven yeast isolates (YP3, YP4, YP7, YP8, YP11, YP12 and YP15) was occurred at 45°C. Yeast isolates could grow at pH range (pH3 to pH6) and their optimum growth was occurred at pH3 and pH4. Moreover, isolated yeast strains were tolerant to ethanol concentration of 5%. Some yeast isolates could grow at 7% ethanol concentration. Among all isolates, YP5 and YP14 could produce 1.1% and 1.5% of ethanol concentration respectively at 14 days incubation period and YP17 could produce 0.6% at 3 days incubation period.
... Then, sources of 2 nd generation biofuels include agricultural, livestock, and forestry residues and their related industries [9]. Ethanol resulting from the fermentation of sugars obtained by hydrolysis of lignocellulosic biomass is indicated as a consolidated second-generation biofuel, whose treatment has one of the most promising development technologies [10,11]. ...
A significant amount of paper residue has continuously been discarded worldwide. This rsidue encompasses a high-cellulose content that can be converted into value-added chemicals. Hydrolysis can be applied as an effective method to produce chemicals from lignocellulosic materials. This work presents a study on biomass waste valorization that uses acid hydrolysis to convert the cellulose residue into raw materials. Commercial cardboard waste was used as a lignocellulosic material source to produce fermentable sugar. The original material was crushed to about 1 mm pieces before being submitted to the hydrolysis reaction. It was found 4.8 mg of calcium ion (Ca²⁺) per gram of cardboard by means of a complexometric method using direct titration. The calcium ion acted as an inhibitor for the action of yeasts during the fermentation process, and then Ca²⁺ ions were removed by calcium sulfate decantation. A low-cost ionic liquid (n-butylammonium acetate) was used to enhance the hydrolysis reactional media. The fermentable sugar was obtained by two-step cellulose hydrolysis. Sugar content was assessed by both the Fehling test and the 3,5-dinitro-salicylic acid (DNS) method. Without ionic liquid, the maximum sugar concentration was 6.09 and 0.85 g/L, respectively, to 10% and 35% sulfuric acid concentrations. In the presence of ionic liquid, the corresponding sugar concentrations were 6.56 and 5.68 g/L. The whole reaction conversion achieved 30%, which must be considered significant for cellulose encompassed in the cardboard waste. The high amount of sugar obtained by acid hydrolysis of cardboard waste using ionic liquid points to a proper recovery and discarding for cardboard waste. Results show paper residues as an alternative source to sugar production, which is feasible to be converted into bioenergy and biofuel.
Graphical Abstract
... Sugars obtained from the hydrolysis of lignocellulosic material are promising compounds for the production of important chemicals such as cellulosic ethanol, hydrocarbons, and starting materials for polymers associated with reducing CO 2 emissions and promoting sustainable green chemistry [1][2][3][4][5]. Substantial effort has been devoted to the development of appropriate hydrolysis schemes, including catalysis using mineral acids [6][7][8], enzyme-driven reactions [9,10], subcritical and supercritical water [11][12][13][14], carbon-based solid acid catalysts [3,15], magnetic mesoporous carbon-based solid acid catalysts [16], and cellulase-mimetic solid catalysts [17,18]. ...
The powder properties of a carbon-based solid acid catalyst, an amorphous carbon material bearing SO3H, COOH and OH groups, were investigated for the hydrolysis of cellulose. The Carr flowability and floodability indices, the angle of internal friction (adherence), and the particle size distribution and shape for the powder catalyst were determined. The need to develop a special reactor with a stirring apparatus for the hydrolysis of cellulose was determined based on the Carr flowability index. Insight into the interaction or adherence between the catalyst and crystalline cellulose during the hydrolysis process was gained by measuring the internal friction angle. The optimum moisture content in the catalyst to achieve the maximum adherence was investigated.
... It results in release of the recalcitrant hemicellulose portion of lignocellulose and solubilizes the pentose fraction. Pentose sugars can constitute 5-25% dry weight of lignocellulosic biomass [12][13][14][15][16] with xylose being the second most abundant sugar after glucose. Solubilization of sugars is directly proportional to the severity of thermo-acidic treatment which also leads to a proportional increase in concentration of inhibitors formed due to breakdown of the sugars [16][17][18][19][20][21] under harsh conditions. ...
Lignocellulosic biomass represents a carbon neutral cheap and versatile source of carbon which can be converted to biofuels. A pretreatment step is frequently used to make the lignocellulosic carbon bioavailable for microbial metabolism. Dilute acid pretreatment at high temperature and pressure is commonly utilized to efficiently solubilize the pentose fraction by hydrolyzing the hemicellulose fibers and the process results in formation of furans—furfural and 5-hydroxymethyl furfural—and other inhibitors which are detrimental to metabolism. The presence of inhibitors in the medium reduce productivity of microbial biocatalysts and result in increased production costs. Furfural is the key furan inhibitor which acts synergistically along with other inhibitors present in the hydrolysate. In this review, the mode of furfural toxicity on microbial metabolism and metabolic strategies to increase tolerance is discussed. Shared cellular targets between furfural and acetic acid are compared followed by discussing further strategies to engineer tolerance. Finally, the possibility to use furfural as a model inhibitor of dilute acid pretreated lignocellulosic hydrolysate is discussed. The furfural tolerant strains will harbor an efficient lignocellulosic carbon to pyruvate conversion mechanism in presence of stressors in the medium. The pyruvate can be channeled to any metabolite of interest by appropriate modulation of downstream pathway of interest. The aim of this review is to emphasize the use of hydrolysate as a carbon source for bioproduction of biofuels and other compounds of industrial importance.
... This integration approach seeks to digest all the sugars completely [130]. In SSCF, the glycolysis is accelerated by the continual discharge of hexose sugars from the enzymatic hydrolysis, resulting in a quicker and more efficient fermentation of the pentose sugars [13,54]. The primary issue with this method is that hexose-utilizing microbes reproduce more quickly than pentose-using microorganisms, which increases the conversion of hexoses to bioethanol. ...
The global demand for ethanol is rising tremendously because of fast industrialization and population expansion. The increased attention gained by ethanol is due to its application as a transport fuel because it is renewable, sustainable, eco-friendly, carbon neutral, and has a high-octane rating. The processes involved in its production include treatment and hydrolysis of substrates when starchy and lignocellulosic materials are used, microbial fermentation of substrates to bioethanol, and downstream processes such as distillation. The various steps are delineated in this chapter, but more consideration is given to the microbial fermentation of diverse substrates. Also presented in the chapter are the critical steps in adopting emerging technologies to improve the overall conversion system. Modeling and optimizing the pertinent operating variables involved in microbial fermentation to obtain bioethanol can make the process cost-effective and offer insight into understanding the behavior of the process. The benefits and downsides of some response surface methodology (RSM) screening approaches were discussed. The machine-learning techniques commonly used to model biochemical systems were also discussed extensively. It was noted that these data-mining techniques are better at handling the nonlinearity and multivariate nature of the microbial fermentation process for bioethanol production than the non-data-mining techniques, such as RSM.KeywordsBioethanolFermentationModelingOptimizationMachine learning technologyMicrobesEnzymesHydrolysisSaccharificationPretreatment
... After an experimental plant converted cellulosic feedstock to ethanol for the first time in 2014 (Menetrez 2014), POET-DSM and Abengoa Bioenergy (Hugoton, Kansas) founded a commercial-scale plant with cellulosic bioethanol producing capacity of up to 25 million gallons per year. However, the current cost of enzymes is a bottleneck for commercial-scale bioethanol production due to its significant contribution to the final product cost (Hahn-Hagerdal et al. 2006). Commercial cellulases are produced mainly from fungi Aspergillus niger, Trichoderma reesei, T. viride and T. longibrachium (Singhania et al. 2010, Reczey et al. 1996, although the Basidiomycota fungi are also able to synthesize enzymes which catalyze the breakdown of complex biopolymers, e.g., cellulose, hemicelluloses, lignin, pectin (Elisashvili et al. 2009, Floudas et al. 2012. ...
Enzymatic hydrolysis is an environmentally friendly technology to produce sugars from pretreated biomass. Here, we show that the new Il-11 Irpex lacteus strain can synthesize cellulases in a high quantity. The peptone and filter paper contained in the medium significantly enhanced activity of endo-1,4-β-D-glucanases (app. 50 IU/mL) and total cellulases (app. 9 IU/mL), whereas the medium with peptone and sodium carboxymethyl cellulose stimulated activity of exo-1,4-β-D-glucanases (33 IU/mL). The expression of cellulases reached its maximum within 96–144 hours, and the optimum pH is 3,7. Thermal treatment at 30 °C for 60 minutes activated endo-1,4-β-D-glucanases and total cellulases, while exo-1,4-β-D-glucanases activity was enhanced following 40 °C treatment. In total, the cellulases complex (300 IU/g) saccharified untreated cellulose by 38 % in 48 hours. Concentrate with filter paper activity 100 IU/g is the more balanced enzyme-substrate ratio (2 %), which allows prolonging the saccharification process that will have a positive effect on the cost of the final product.
... However, 1G feedstocks compete with food in terms of land occupancy and need to be improved to fulfill the growing demand for fuel. They negatively influence biodiversity and may cause deforestation to gain more farmland [15]. The retrograde impact of these circumstances has raised the necessity of designing second-generation bioethanol technology from lignocellulosic materials, which are plentiful renewable organic materials in the biosphere [16]. ...
Citation: Mahbubul, I.M.; Himan, M. Prospects of Bioethanol from Agricultural Residues in Bangladesh. Energies 2023, 16, 4657. https:// Abstract: Bangladesh is a middle-income country. With the development of the industrial and agricultural sectors, the demand for petroleum-based fuels in the transport sector has been steadily growing. Diesel, petrol, octane (C 8 H 18), liquid petroleum gas (LPG), and compressed natural gas are mainly used as fuels in the transportation sectors of Bangladesh. The government imports LPG as well as refined, crude, and furnace oil from abroad to meet the country's growing energy demand. Apart from that, Bangladesh has a shortage of natural gas reserves, which is a great concern. As a result, it is essential to find and use renewable fuel sources. Since Bangladesh is an agricultural country, bioethanol could be the best alternative fuel generated from agricultural residues and waste. Every year, a large amount of agricultural residue is generated in this country, from which a vast amount of bioethanol could be produced. Bioethanol derived from agricultural residue and waste can reduce dependency on fossil resources, reduce fossil fuel's environmental impact, and improve engine performance. This article comprehensively reviews the bioethanol production potential from agricultural residues and investigates the opportunities and possibilities in Bangladesh. The research outcomes reveal that in the fiscal year 2019-2020, approximately 46.5 million tons of agricultural residue were generated from the available major crops, from which about 19.325 GL (gigalitres) of bioethanol could be generated. This current study also investigates the practical methods of bioethanol production from different agricultural feedstocks and identifies the challenges related to bioethanol production in Bangladesh.
... Depending upon the technical implementation status, type of raw materials used, technological implementation status, type of main intermediates produced, and type of conversion processes applied to biorefineries, many researchers classify biorefineries into several different classes Kamm, 2004a,b, Benedé, 2014) hence led to an utmost confusing situation. To overcome this situation, IEA Bioenergy Task 42 classified biorefineries based on platforms, products, feedstocks, and processes (Hahn-Hagerdal et al., 2006;The Royal Society, 2008;Naik et al., 2010;Bell et al., 2014). ...
Nanotechnology for Biorefinery
2023, Pages 27-87
Chapter 2 - Nanomaterials used in biorefineries: types, properties, and synthesis methods
Author links open overlay panelBrandon Lowe, Amina Muhammad Ahmad, Jabbar Gardy, Ali Hassanpour
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Abstract
Countries are increasingly becoming aware of the urgent need to transition toward lower carbon dioxide emitting societies. While certain industries such as the transportation sector have been difficult to decarbonize thus far, continued development into biomass-derived fuels such as biodiesel will likely aid this transition. Traditionally, biodiesel has been made via a two-step homogeneous process, with acidic or basic catalysts required to improve reaction kinetics. More recent explorations into heterogeneous catalysis have facilitated simpler separation and recovery of catalyst from the produced biodiesel, without expensive cleanup prior to use.
The application of nanotechnology however has the potential to achieve even greater catalytic performances. By careful design of the nanomaterial, successful nanoscale catalysts have been synthesized with impressive yields, reaction kinetics, stability, impurity tolerance, and even magnetic separability. The field of nanocatalysis for biodiesel production continues to develop at pace and proves an exciting area of current research. The present chapter is focused on various types of nanomaterials that have been produced to date and their catalytic properties to the future potential for full-scale, industrial biorefinery operation.
... Commercially feasible biofuel production from a lignocellulosic biomass requires an optimized process whereby all the sugars present in the raw material are efficiently converted to ethanol. This implies that, independently of the chosen process configuration, the following key steps have to be optimized: pretreatment of the raw material, enzymatic hydrolysis of the pretreated material into fermentable sugars, ethanol fermentation, and product recovery by distillation [1]. ...
The commercial production of bioethanol from lignocellulosic biomass such as wheat straw requires utilizing a microorganism that can withstand all the stressors encountered in the process while fermenting all the sugars in the biomass. Therefore, it is essential to develop tools for monitoring and controlling the cellular fitness during both cell propagation and sugar fermentation to ethanol. In the present study, on-line flow cytometry was adopted to assess the response of the biosensor TRX2p-yEGFP for redox imbalance in an industrial xylose-fermenting strain of Saccharomyces cerevisiae during cell propagation and the following fermentation of wheat-straw hydrolysate. Rapid and transient induction of the sensor was recorded upon exposure to furfural and wheat straw hydrolysate containing up to 3.8 g/L furfural. During the fermentation step, the induction rate of the sensor was also found to correlate to the initial ethanol production rate, highlighting the relevance of redox monitoring and the potential of the presented tool to assess the ethanol production rate in hydrolysates. Three different propagation strategies were also compared, and it was confirmed that pre-exposure to hydrolysate during propagation remains the most efficient method for high ethanol productivity in the following wheat-straw hydrolysate fermentations.
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The use of bioethanol derived from lignocellulosic feedstocks has shown great potential as a renewable fuel option. This sustainable substitute to fossil fuels can be helpful to diminish greenhouse gas emissions. This chapter offers a comprehensive overview of ethanol production from lignocellulosic biomass, including recent technological advancements. It addresses the critical challenges faced in lignocellulosic ethanol production and highlights innovative technologies and strategies developed to overcome these challenges. Various pretreatment methods are also discussed along with enzymatic hydrolysis techniques and fermentation approaches. These methods aim to enhance the efficiency and economics of bioethanol production from lignocellulosic feedstock.
Ethanol produced through the fermentation of plant biomass is considered an environment friendly alternate to fossil fuels. Bioethanol and biodiesel, commonly known as second-generation biofuels, are produced through biological processes using agro-industrial waste and are considered sustainable, safe, and ecofriendly. These biofuels can minimize the emission of carbon dioxide and reduced the world’s dependence on fossil fuel. This review article focuses on three generations of biofuels, particularly the production of biofuel using fungal biocatalysts specifically Aspergillus niger and Saccharomyces cerevisiae and the mechanism by which they convert biomass into biofuel. A. niger is known for releasing cellulolytic and pectolytic enzymes to hydrolyze biomass and survive against toxins, while S. cerevisiae produces invertase and zymase enzymes to convert sucrose into fructose and glucose sugars, and then further convert fructose and glucose into ethanol. The main purpose of this review is to explore alternative techniques for generating biofuels, using as few harmful chemicals as possible and reducing time consumption.
One of the major limiting factors for the social and economic progress of a country is the energy. One of the biggest issues most emerging countries encounter is this crucial component. With the rapid increase in population, the energy demand is increasing linearly and is the main reason of conflicts between many nations. At present bioenergy crops constitute only a small fraction to the overall energy produced from biomass per year. Therefore, there is an urgent need to adopt the biotechnological approaches to mitigate the ever-increasing demand of energy by making living biomass energetically more feasible. Besides the utilization of biomass as a fuel can contribute to climate change mitigation by reducing fossil fuel CO2 emission in the atmosphere making it almost CO2 neutral energy. Energy crops also have a capability for fulfilling the upcoming energy demand globally. Bioenergy crops provide an inexpensive and eco-friendly means of production of energy. C4 plants such as silver grass, switch grass and sweet sorghum produce a huge biomass even in barren areas. So, these plants can be employed in a new type of farming called as energy farming. An energy farming can be carried out in complementation with the modern biotechnology to enhance biofuel production.
Diante da situação ambiental atual e todas as consequências que o planeta vem sofrendo devido, especialmente, ao uso dos combustíveis fósseis como principal fonte de energia, a busca por fontes alternativas na matriz energética e por combustíveis mais limpos vem ganhando espaço em nível mundial. Uma alternativa promissora é o aproveitamento do bagaço de cana de açúcar, por possuir uma composição química favorável não somente para o etanol de primeira geração, mas também para o etanol celulósico. Desta forma, o objetivo desse estudo foi avaliar as características físico-químicas entre variedades de cana-de-açúcar, visando o potencial para produção de etanol celulósico. A variedade de cana-de-açúcar CTC apresentou maior potencial para a produção de etanol celulósico, por possuir maior percentual de celulose (48,92%), contrapondo o valor encontrado na amostra de biomassa da cultivar RB (46,88%). No que tange a produção de bioeletricidade, considerando a produção de vapor pela caldeira como referência, a amostra de cana CTC9003 destacou-se pelo teor mais elevado de lignina (10,77%). No entanto, o poder calorífico superior apresentado pela amostra de biomassa CTC (180030 kJ/kg) também a torna uma importante candidata para a cogeração de bioenergia. Quando observadas as cultivares, evidencia-se que a mais viável para a obtenção de etanol celulósico é a CTC9003, uma vez que apresenta o maior percentual de celulose.
Diante da situação ambiental atual e todas as consequências que o planeta vem sofrendo devido, especialmente, ao uso dos combustíveis fósseis como principal fonte de energia, a busca por fontes alternativas na matriz energética e por combustíveis mais limpos vem ganhando espaço em nível mundial. Uma alternativa promissora é o aproveitamento do bagaço de cana de açúcar, por possuir uma composição química favorável não somente para o etanol de primeira geração, mas também para o etanol celulósico. Desta forma, o objetivo desse estudo foi avaliar as características físico-químicas entre variedades de cana-de-açúcar, visando o potencial para produção de etanol celulósico. A variedade de cana-de-açúcar CTC apresentou maior potencial para a produção de etanol celulósico, por possuir maior percentual de celulose (48,92%), contrapondo o valor encontrado na amostra de biomassa da cultivar RB (46,88%). No que tange a produção de bioeletricidade, considerando a produção de vapor pela caldeira como referência, a amostra de cana CTC9003 destacou-se pelo teor mais elevado de lignina (10,77%). No entanto, o poder calorífico superior apresentado pela amostra de biomassa CTC (180030 kJ/kg) também a torna uma importante candidata para a cogeração de bioenergia. Quando observadas as cultivares, evidencia-se que a mais viável para a obtenção de etanol celulósico é a CTC9003, uma vez que apresenta o maior percentual de celulose.
Microbial cell factories offer an eco-friendly alternative for transforming raw materials into commercially valuable products because of their reduced carbon impact compared to conventional industrial procedures. These systems often depend on lignocellulosic feedstocks, mainly pentose and hexose sugars. One major hurdle when utilizing these sugars, especially glucose, is balancing carbon allocation to satisfy energy, cofactor, and other essential component needs for cellular proliferation while maintaining a robust yield. Nearly half or more of this carbon is inevitably lost as CO2 during the biosynthesis of regular metabolic necessities. This loss lowers the production yield and compromises the benefit of reducing greenhouse gas emissions—a fundamental advantage of biomanufacturing. This review paper posits the perspectives of using CO2 from the atmosphere, industrial wastes, or the exhausted gases generated in microbial fermentation as a feedstock for biomanufacturing. Achieving the carbon-neutral or -negative goals is addressed under two main strategies. The one-step strategy uses novel metabolic pathway design and engineering approaches to directly fix the CO2 toward the synthesis of the desired products. Due to the limitation of the yield and efficiency in one-step fixation, the two-step strategy aims to integrate firstly the electrochemical conversion of the exhausted CO2 into C1/C2 products such as formate, methanol, acetate, and ethanol, and a second fermentation process to utilize the CO2-derived C1/C2 chemicals or co-utilize C5/C6 sugars and C1/C2 chemicals for product formation. The potential and challenges of using CO2 as a feedstock for future biomanufacturing of fuels and chemicals are also discussed.
A highly efficient reactor with a stirring device was specially designed with the intent of performing the hydrolysis of pure crystalline cellulose using a carbon-based solid acid catalyst. This catalyst comprised an amorphous carbon-based material bearing –SO3H, –COOH and –OH groups. The stirring apparatus had seven blades coated with polytetrafluoroethylene and arranged axially at regular intervals with a 60° offset. This design proved highly effective, providing double the glucose yield compared with conventional stirring systems. The basic properties of this novel reactor were investigated and analyzed and are discussed herein.
Due to its abundance and sustainability, lignocellulosic biomass is a possible replacement for petroleum oil in the production of energy and chemicals. Numerous thermochemical processes have been used in significant study to turn biomass into products with added value. One of the best methods for creating bio-fuels and bio-based compounds among them is hydrothermal liquefaction (HTL). However, a number of technological obstacles still need to be removed before HTL technology can be widely used in industry. Hydrothermal liquefaction is now thought to be amongst the most popular effective processes to converting moist biomass for bio crude, but it requires costly renovation procedures to be utilized as biofuel. It is crucial to employ catalysts that may straightforwardly improve the bio crude yield as well as the efficiency of the reaction process; the benefit of raising the operation’s overall production; the impacts of adding heterogeneous catalysts and how they affect the bio-crude yield. In lignocellulosic biomass hydrothermal liquefaction, a typical catalytic activity was discovered, dividing the various catalysts into four separate groups (transition metal, lanthanide oxide, alkaline metal oxide, and zeolite). The purpose study is to objectively evaluate the hydrothermal liquefaction of lignocellulosic biomass and know effecting of adding a zeolite catalyst on it, with a focus on increasing the production and efficiency of the biofuel. In addition, it has drawn attention to the natural stimulatory effects associated with zeolite catalysts.
Since the discovery of thermophilic and extremophilic bacteria, their potential in various biotechnological properties has been well studied. The present investigation dwells on the main areas of research where thermophilic bacteria are of great importance as well as discussion on possible future aspects of their utilization. The main use of thermophiles and extremophiles is within the production of biofuels (ethanol and hydrogen), their use in biorefineries (production of diols and branched-chain alcohols), production of thermostable enzymes, and industrial use in general. The main future prospect of the use of thermophiles is to upscale the production of ethanol (and hydrogen) to large-scale factories, using complex lignocellulosic biomass as substrate. Additionally, the production of most fine chemicals is not yet financially profitable as compared with the production from fossil fuels. The opportunities in this sector are most likely further genetic engineering work on present microorganism. Finally, although genetic engineering seems to be of more and more use in anaerobic thermophiles, the need for genetic tools is still lacking in their process.
div class="section abstract"> It is well known that ethanol can be used in spark-ignition (SI) engines as a pure fuel or blended with gasoline. High enthalpy of vaporization of alcohols can affect air-fuel mixture formation prior to ignition and may form thicker liquid films around the intake valves, on the cylinder wall and piston crown. These liquid films can result in mixture non-homogeneities inside the combustion chamber and hence strongly influence the cyclic variability of early combustion stages. Starting from these considerations, the paper reports an experimental study of the initial phases of the combustion process in a single cylinder SI engine fueled with commercial gasoline and anhydrous ethanol, as well as their blend (50%<sub>vol</sub> alcohol). The engine was optically accessible and equipped with the cylinder head of a commercial power unit for two-wheel applications, with the same geometrical specifications (bore, stroke, compression ratio). Ultra-violet (UV) natural emission spectroscopy measurements ranging from 250nm to 470nm wavelength and simultaneous thermodynamic analysis were used to better understand the effect of ethanol content on flame kernel inception and development. All experiments were conducted at wide open throttle (WOT), with stoichiometric air-fuel mixtures, fixing the engine speed at 2000rpm. Optical investigations allowed to follow the evolution of chemical species that marked the spark discharge (cyano CN and hydroxyl OH radicals) as well as flame front initial growth (OH and carbyne CH radicals). Vibrational and rotational temperatures were calculated during the arc and glow phase by the ratio between the emission intensity of CN and OH radicals. Results were compared with adiabatic flame temperature traces obtained by applying a two zone model.
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Biowaste is becoming a significant category in the global energy mix to mitigate the negative impacts of burning fossil fuels. The aim of this review paper was to investigate the potential, conversion mechanisms, benefits, and policy gaps related to the utilization of solid biowaste resources as renewable, clean, and affordable energy sources. Thus, a systematic review approach was employed to undertake a comprehensive analysis of the studies that dealt with solid biowaste resources for energy recovery. This review paper was conducted from November 2022 to June 2023. The relevant literature was searched using databases from scholarly journal publishers, online search engines, and websites. A total of 82 studies were determined to be eligible from 659 records. Ethiopia has a huge potential for biowaste resources, with an annual generation potential of 18,446.4 MJ per year. The multifaceted advantages associated with biowaste-to-energy conversion such as clean energy production, waste management, forest conservation, greenhouse gas emission reduction, and maintaining soil fertility using the digestate left after anaerobic digestion were mentioned. This review highlights various conversion technologies for converting solid biowastes into valuable forms of energy, such as thermochemical, biochemical, and physico-mechanical techniques. It also investigated the value-added products of the Solid Biowastes-to-Energy (SBWtoE) process, including bio-oil, syngas, bioethanol, biodiesel, biomethane, bio-briquettes, and pellets, with applications ranging from transportation to power generation. Furthermore, this review addresses the multifaceted challenges associated with implementing a circular economy, emphasizing the need to overcome policy, technological, financial, and institutional barriers. These efforts are crucial for harnessing the growing biowaste resources in Ethiopia, ultimately promoting sustainable and cost-effective energy production while advancing the nation's environmental objectives.
Bioethanol is the most widely used alternative transportation fuel to petrol. Bioethanol is considered a clean, renewable, and environmentally friendly fuel that can contribute to climate change mitigation, decreased environmental pollution, and enhanced energy security. Commercial bioethanol production is based on traditional agricultural crops such as corn, sugarcane, and sugarbeet, primarily used as food and feed. In order to meet the growing demand for this fuel and decrease competition in the food and biofuel sectors for the same feedstock, other raw materials and process technologies have been intensively studied. Lignocellulosic biomass is one of the most abundant renewable resources, with it being rich in compounds that could be processed into energy, transportation fuels, various chemical compounds, and diverse materials. Bioethanol production from lignocellulosic biomass has received substantial attention in recent decades. This review gives an overview of bioethanol production steps from lignocellulosic biomass and challenges in the production process. The following aspects of bioethanol production are covered here, including pretreatment methods, process strategies, strain development, ethanol isolation and purification, and technical hurdles.
Transportation currently takes up around a third of overall energy usage, of which the majority is petroleum-based gasoline. Petroleum is both a finite resource and a big contributor to the carbon emissions that are causing climate change. To continue to benefit from transportation whilst mitigating climate change it is essential to find alternatives to petroleum-based gasoline. Although a lot of recent developments have focused on electrifying transport the infrastructure for large scale uptake of electric vehicles is still lacking and it may be less practical in some parts of the world than others. Biofuels, therefore, still have a role to play in improving the sustainability of our transportation systems.
The term green gasoline refers to biofuels intended to be direct drop-in replacements for petroleum-based gasoline. Such products allow vehicles to run on biofuel without any engine modifications and, being made from biomass, they are both renewable and have a better carbon emission profile than petroleum-based gasoline.
Green Gasoline covers a range of new technologies being used to produce these biofuels and compares them to petroleum-based fuels in terms of sustainability. It will be an interesting read for those working in fuel chemistry as well as green chemists and anyone with an interest in transport sustainability.
A series of binder‐free mordenite (MOR) zeolites were successfully prepared by hydrothermal conversion of shaped precursors containing aluminosilicate and MOR seeds. In the synthesis of seed crystals, hexadecyltrimethylammonium bromide (CTAB) was used as a crystal growth inhibitor as well as a pore filler to adjust the morphology and introduce mesoporous pores. As a result, large aggregates, assembled nanosheets, and flowers‐like MOR were successfully synthesized with these MOR seeds. The effect of seed morphology on the performance of the binder‐free MOR was studied by scanning electron microscopy (SEM), N 2 adsorption–desorption, and NH 3 ‐TPD characterization techniques. It was found that the mesopore volume of formed MOR was positively correlated with those of the seeds. The synthesized binder‐free MOR showed high activity (0.28 g methyl acetate gcat ⁻¹ h ⁻¹ ), 100% selectivity, and high stability in dimethyl ether (DME) carbonylation to methyl acetate, which can be attributed to the larger surface area and more mesopores. The synthesis of MOR nano‐assemblies via this cost‐effective strategy has a wide range of potential applications.
Environmental issues such as overexploitation, pollution and degradation of natural resources have prompted us to look for ways to devise sustainable practices across industrial and public service sectors. Researchers and scientists all over the world are involved in developing strategies and techniques that help us achieve a sustainable environment. Technology for a Sustainable Environment presents an overview of various methods and techniques that can be adapted to sustain the environment. Chapters focus on techniques such as bioremediation, nanotechnology and biotechnology that can play a very important role in achieving environmental sustainability goals. The chapters also provide a detailed account about use of biotechnology, nanotechnology and other techniques in achieving environmental sustainability. Additionally, the book includes a discussion about emerging technologies that promote environmental sustainability like green technologies, biodegradable polymers and plastics. Readers will be able to understand how modern technologies can help in monitoring environmental pollutants, remediation of environmental pollution and prevention of environmental degradation.
The book is suitable for readers, professionals and scholars at all levels who require an understanding of the technology in environmental science, environmental engineering and environmental biotechnology.
Nowadays, there is a wide variety of pathways to transform biomass into biofuels. There are economically viable pathways that have been escalated to an industrial level and others still at the pilot and/or research and development scale. As well, considering a life cycle perspective for the environmental analysis, there are pros and cons that should be considered regarding the bioethanol environmental performance, since it has been considered that the use of bioethanol could help to reduce fossil fuels depletion and greenhouse gas emissions. In this chapter, some insights about techno-economic and environmental analyses of different bioethanol production methods developed in the last five years are determined. The techno-economic analysis details the potential economic viability of a selected technology at the research and development stage. The analysis considers the maturity level of the technologies available today: first, second, and third-generation bioethanol. Regarding the environmental performance of bioethanol from a life cycle perspective, it is clear that no biofuel is “climate neutral” because of the inputs of fossil fuels needed, in the agricultural and industrial processes, before the use stage. Given this background, a life cycle approach in a bioethanol environmental analysis facilitates an environmental profile containing all inputs and outputs that entailed in the bioethanol system, from the extraction of raw materials to the use of bioethanol in the engine of a vehicle. Life cycle assessment results allow informed decision-making and avoiding shifting environmental impacts.KeywordsBiofuelsEthanolCostsPoliciesSustainabilityEnvironment
A review of recent reports that focus on the selective hydrodeoxygenation of lignin biomass derived aromatic compounds is presented. Obtaining high value chemical feedstocks and fuels from lignin is recognized as an essential aspect of the economic feasibility of biorefineries. Lignin is both a non-edible part of biomass and a potential source of aromatic commodity chemicals. The selective catalytic conversion of lignin derived compounds to deoxygenated aromatic molecules represents the most direct route towards high value chemicals while also maximizing hydrogen use efficiency. This review aims to give an overview of reports within the last 3–4 years that have focused on the selective hydrodeoxygenation of lignin derived or lignin inspired oxygenated aromatic compounds. Many of these lignin derived dimers and monomers can be selectively obtained via reductive catalytic fractionation (RCF) of native lignin, thus a section of this review is dedicated to recent advancements of RCF. The observed trends with respect to catalyst composition and reaction conditions in these reports along with an outlook for selective catalytic hydrodeoxygenation is presented.
Electrochemical coupling hydrogen evolution with biomass reforming reaction (named electrochemical hydrogen and chemical cogeneration (EHCC)), which realizes green hydrogen production and chemical upgrading simultaneously, is a promising method to build a carbon-neutral society. Herein, we analyze the EHCC process by considering the market assessment. The ethanol to acetic acid and hydrogen approach is the most feasible for large-scale hydrogen production. We develop AuCu nanocatalysts, which can selectively oxidize ethanol to acetic acid (> 97%) with high long-term activity. The isotopic and in-situ infrared experiments reveal that the promoted water dissociation step by alloying contributes to the enhanced activity of the partial oxidation reaction path. A flow-cell electrolyzer equipped with the AuCu anodic catalyst achieves the steady production of hydrogen and acetic acid simultaneously in both high selectivity (> 90%), demonstrating the potential scalable application for green hydrogen production with low energy consumption and high profitability.
The main objective of this study relates to simple and economic process with stepwise enzymes treatment for the cost effective production of ethanol from Bagasse waste and Egg albumin waste. Sugarcane Bagasse waste was selected as inexpensive cellulosic feed stock, which has the general composition of 40-45% cellulose, 28-10% hemicellulose, and 19-21% lignin. Removal of lignin, tannin, to lose the bagasse fiber matrix by chemical treatment and stage wise addition of neutral and acid cellulose to facilitate release of reducing sugars by enzymatic treatment. Apparently the production of inexpensive sterile organic nitrogen from egg albumin waste with neutral protease and peptidase by enzymatic process. The obtained C: N was mixed in the ratio of 3:1used for the cost effective production of Ethanol from Saccharomyces cerevisiae. Further this study includes the unhydrolysed part of Bagasse processing for the production of insoluble cellulose fiber as dietary supplement. 50 % reducing sugars recovered from Sugarcane Bagasse by chemical-enzyme coupled treatment and 90 % free amino acids recovered from albumin protein by enzyme treatment. 3:1 ratio of Carbohydrate: Nitrogen mixed in definite proportion inoculated with yeast enhances the conversion of ethanol to 79g/L yield on batch mode.
Bioethanol is an appropriate alternate energy option due to its renewable, nontoxic, environmentally friendly, and carbon-neutral nature. Depending upon various feedstocks, bioethanol is classified in different various generations. First-generation ethanol created a food vs fuel problem, which was overcome by second-generation, third-generation and fourth-generation ethanol. The considerable availability of lignocellulosic biomass makes it a suitable feedstock, however, its recalcitrant nature is the main hurdle in converting it to bioethanol. The present study gives a comprehensive assessment of global biofuel policies and the current status of ethanol production. Feedstocks for first-generation (sugar and starch-based), second-generation (lignocellulosic biomass and energy crops), third-generation (algal-based) and fourth-generation (genetically modified algal biomass or crops) are discussed in detail. The study also assessed the process for ethanol production from various feedstocks, besides giving a holistic background knowledge on the bioconversion process, factors affecting bioethanol production, and various microorganisms involved in the fermentation process. Biotechnological tools also play a pivotal role in enhancing process efficiency and product yield. In addition, most significant development in the field of genetic engineering and adaptive evolution are also highlighted.
Lignocellulose is currently the most available raw material on the earth for the production of biofuels. The major enzyme component involved in the microbial production of fuels from lignocellulose is cellulase. Therefore efficient saccharification of lignocellulosic biomasses is critical for biofuel production. However, several chemical processes (acid/alkali pretreatment) may lead to incomplete saccharification, whereby lignin may be removed incompletely; thus the fuel produced is insufficient. Pretreatment is the most vital step in unlocking cellulose, thereby releasing lignin. Thus for these processes, the enzymes used are crucial, so a wide variety of cellulolytic and hemicellulolytic enzymes have been employed to get a proper and desired product yield, especially during saccharification. This chapter investigates the role of various cellulase forms in pretreatment and strategies to enhance enzymatic activity to get a better and more refined product.
As the Bioethanol Program at the Department of Energy (DOE) nears the end of two decades of research, it is time to take a hard look at where we have been and where we are going. This paper summarizes the status of bioethanol technology today and what we see as the future directions for research and development. All of this is placed in the perspective of strategic national issues that represent the drivers for our program - the environment, the economy, energy security and sustainability. The key technology pathways include the use of new tools for protein engineering and directed evolution of enzymes and organisms, as well as new approaches to physical/chemical pretreatment of biomass.
Cette étude fait le point des connaissances scientifiques et techniques dans le domaine de la production alcoolique à partir de susbstrats lignocellulosiques. Ce travail, réalisé dans le cadre d'Agrice (Agriculture pour la chimie et l'énergie), est une synthèse bibliographique qui a cherché à identifier les avancées capables de débloquer certains verrous technologiques et économiques liés à ce type de procédé. La biomasse lignocellulosique est un substrat complexe, constitué des trois principales fractions que sont la cellulose, les hémicelluloses et la lignine. Le procédé de production d'éthanol consiste à récupérer par hydrolyse le maximum de sucres issus à la fois des fractions cellulosiques et hémicellulosiques, puis de fermenter ces sucres en éthanol. Les premiers procédés d'hydrolyse utilisés étaient surtout chimiques, mais ils sont peu compétitifs à l'heure actuelle, en raison notamment du coût des réactifs et de la formation de nombreux sous-produits et de composés inhibiteurs rendant les hydrolysats peu fermentescibles. Ils sont désormais concurrencés par les procédés enzymatiques, plus spécifiques et qui permettent de meilleurs rendements d'hydrolyse dans des conditions moins sévères. Cependant, la biomasse lignocellulosique n'est pas directement accessible aux enzymes, et elle doit subir au préalable une phase de prétraitement dont l'objectif est d'améliorer la susceptibilité à l'hydrolyse enzymatique de la cellulose et éventuellement d'hydrolyser la fraction hémicellulosique en sucres monomères. Parmi les nombreuses méthodes de prétraitement qui ont été étudiées, nous en avons identifié trois répondant au mieux aux objectifs précédemment cités : le prétraitement à l'acide dilué, l'explosion à la vapeur avec utilisation d'un catalyseur, et la thermohydrolyse. Ces trois méthodes permettraient d'atteindre des rendements d'hydrolyse enzymatique de la cellulose proches de 100 %, tout en permettant un taux d'hydrolyse des hémicelluloses supérieur à 80 %, et en minimisant la formation de composés de dégradation. L'hydrolyse enzymatique doit encore être améliorée afin de réduire le coût lié à la consommation d'enzymes. Les principales voies de recherche devraient porter sur l'amélioration de l'activité des cellulases, afin de se rapprocher le plus possible de celles d'enzymes telles que les amylases. Le développement du procédé SFS (saccharification et fermentation simultanées) permet d'améliorer l'efficacité des enzymes en minimisant les réactions d'inhibition des enzymes par les produits formés. Son inconvénient est lié aux différences entre les températures optimales de l'hydrolyse enzymatique et de la fermentation. La recherche de micro-organismes conservant de bonnes performances fermentaires à température élevée doit donc se poursuivre. Un autre verrou technologique du procédé concerne la fermentation alcoolique des pentoses, qui peuvent représenter jusqu'à 25 à 40 % des sucres totaux contenus dans la biomasse lignocellulosique. C'est pourquoi il est indispensable de les valoriser en éthanol. Contrairement à la fermentation alcoolique du glucose, largement connue et maîtrisée, celle des pentoses n'est toujours pas résolue, en raison des performances fermentaires médiocres des micro-organismes utilisés. Le développement des outils génétiques et les nouvelles voies de recherche portant sur la transformation de Saccharomyces cerevisiae et de Zymomonas mobilis afin de leur faire acquérir la capacité à fermenter les pentoses, devraient permettre d'améliorer les performances, et éventuellement de se rapprocher de celles enregistrées sur glucose par Saccharomyces Cette étude fait le point des connaissances scientifiques et techniques dans le domaine de la production alcoolique à partir de susbstrats lignocellulosiques. Ce travail, réalisé dans le cadre d'Agrice (Agriculture pour la chimie et l'énergie), est une synthèse bibliographique qui a cherché à identifier les avancées capables de débloquer certains verrous technologiques et économiques liés à ce type de procédé. La biomasse lignocellulosique est un substrat complexe, constitué des trois principales fractions que sont la cellulose, les hémicelluloses et la lignine. Le procédé de production d'éthanol consiste à récupérer par hydrolyse le maximum de sucres issus à la fois des fractions cellulosiques et hémicellulosiques, puis de fermenter ces sucres en éthanol. Les premiers procédés d'hydrolyse utilisés étaient surtout chimiques, mais ils sont peu compétitifs à l'heure actuelle, en raison notamment du coût des réactifs et de la formation de nombreux sous-produits et de composés inhibiteurs rendant les hydrolysats peu fermentescibles. Ils sont désormais concurrencés par les procédés enzymatiques, plus spécifiques et qui permettent de meilleurs rendements d'hydrolyse dans des conditions moins sévères. Cependant, la biomasse lignocellulosique n'est pas directement accessible aux enzymes, et elle doit subir au préalable une phase de prétraitement dont l'objectif est d'améliorer la susceptibilité à l'hydrolyse enzymatique de la cellulose et éventuellement d'hydrolyser la fraction hémicellulosique en sucres monomères. Parmi les nombreuses méthodes de prétraitement qui ont été étudiées, nous en avons identifié trois répondant au mieux aux objectifs précédemment cités : le prétraitement à l'acide dilué, l'explosion à la vapeur avec utilisation d'un catalyseur, et la thermohydrolyse. Ces trois méthodes permettraient d'atteindre des rendements d'hydrolyse enzymatique de la cellulose proches de 100 %, tout en permettant un taux d'hydrolyse des hémicelluloses supérieur à 80 %, et en minimisant la formation de composés de dégradation. L'hydrolyse enzymatique doit encore être améliorée afin de réduire le coût lié à la consommation d'enzymes. Les principales voies de recherche devraient porter sur l'amélioration de l'activité des cellulases, afin de se rapprocher le plus possible de celles d'enzymes telles que les amylases. Le développement du procédé SFS (saccharification et fermentation simultanées) permet d'améliorer l'efficacité des enzymes en minimisant les réactions d'inhibition des enzymes par les produits formés. Son inconvénient est lié aux différences entre les températures optimales de l'hydrolyse enzymatique et de la fermentation. La recherche de micro-organismes conservant de bonnes performances fermentaires à température élevée doit donc se poursuivre. Un autre verrou technologique du procédé concerne la fermentation alcoolique des pentoses, qui peuvent représenter jusqu'à 25 à 40 % des sucres totaux contenus dans la biomasse lignocellulosique. C'est pourquoi il est indispensable de les valoriser en éthanol. Contrairement à la fermentation alcoolique du glucose, largement connue et maîtrisée, celle des pentoses n'est toujours pas résolue, en raison des performances fermentaires médiocres des micro-organismes utilisés. Le développement des outils génétiques et les nouvelles voies de recherche portant sur la transformation de Saccharomyces cerevisiae et de Zymomonas mobilis afin de leur faire acquérir la capacité à fermenter les pentoses, devraient permettre d'améliorer les performances, et éventuellement de se rapprocher de celles enregistrées sur glucose par Saccharomyces cerevisiae. The reported study intends to describe the state of the art in the domain of ethanol production from lignocellulosic biomass. It was sustained and managed by a specialized group of the French Agrice (Agriculture for Chemical and Energy Organization). Its first goal was to pinpoint the main technical and economical bottlenecks of the processes which are today under consideration, and to identify which research and development efforts could be implemented to overcome them (in the short or middle term). Lignocellulosic biomass is a complex substrate, and essentially made of cellulose, hemicellulose and lignin. The processes which have been considered, attempt to recover a maximum amount of sugars from the hydrolysis of cellulose and hemicellulose, and to ferment them into ethanol. The hydrolysis processes used in the past are essentially chemical processes, but the acid recovery costs and the formation of toxic products make them uncompetitive. They are now substituted by enzymatic processes, which are more specific and allow higher hydrolysis yields under less severe conditions. However, the cellulose that is the target of the enzymatic hydrolysis, is not directly accessible to the enzymes. It is the reason why a pretreatment step has to precede the enzymatic hydrolysis, in order to improve the enzymatic susceptibility of the cellulose, and to hydrolyse the hemicellulosic fraction. Different types of pretreatment have been studied, but three methods appear more efficient: dilute acid hydrolysis, steam explosion with catalyst addition and thermohydrolysis. These pretreatments could result in high hydrolysis yields of the cellulose fraction (close to 100%), and in a maximum recovery of the sugars from the hemicellulosic fraction. Enzymatic hydrolysis has yet to be improved in order to reduce the cost of consumption of the enzymes. Research works will have to focus upon the enzyme specific activity, in order to achieve higher efficiencies such as those obtained with amylases. The SSF (Saccharfication and Simultaneous Fermentation) process improves the enzyme efficiency by reducing the feed-back inhibition from the hydrolysis products. The screening of efficient fermentative microorganisms under high temperature conditions (45°C) has thus to be further implemented. The last technological barrier of the process concerns the ethanolic fermentation of the pentoses. Indeed, the pentoses, originating from the hemicellulosic fraction, can represent up to 40% of total sugars in some lignocellulosic substrates. Nobody has yet identified a microorganism which is able to ferment the pentoses into ethanol with performances similar to those of Saccharomyces cerevisiae on glucose. But recent genetic improvements focused on the transformation of Saccharomyces cerevisiae and Zymomonas mobilis could result in good fermentative performances on pentoses. The reported study intends to describe the state of the art in the domain of ethanol production from lignocellulosic biomass. It was sustained and managed by a specialized group of the French Agrice (Agriculture for Chemical and Energy Organization). Its first goal was to pinpoint the main technical and economical bottlenecks of the processes which are today under consideration, and to identify which research and development efforts could be implemented to overcome them (in the short or middle term). Lignocellulosic biomass is a complex substrate, and essentially made of cellulose, hemicellulose and lignin. The processes which have been considered, attempt to recover a maximum amount of sugars from the hydrolysis of cellulose and hemicellulose, and to ferment them into ethanol. The hydrolysis processes used in the past are essentially chemical processes, but the acid recovery costs and the formation of toxic products make them uncompetitive. They are now substituted by enzymatic processes, which are more specific and allow higher hydrolysis yields under less severe conditions. However, the cellulose that is the target of the enzymatic hydrolysis, is not directly accessible to the enzymes. It is the reason why a pretreatment step has to precede the enzymatic hydrolysis, in order to improve the enzymatic susceptibility of the cellulose, and to hydrolyse the hemicellulosic fraction. Different types of pretreatment have been studied, but three methods appear more efficient: dilute acid hydrolysis, steam explosion with catalyst addition and thermohydrolysis. These pretreatments could result in high hydrolysis yields of the cellulose fraction (close to 100%), and in a maximum recovery of the sugars from the hemicellulosic fraction. Enzymatic hydrolysis has yet to be improved in order to reduce the cost of consumption of the enzymes. Research works will have to focus upon the enzyme specific activity, in order to achieve higher efficiencies such as those obtained with amylases. The SSF (Saccharfication and Simultaneous Fermentation) process improves the enzyme efficiency by reducing the feed-back inhibition from the hydrolysis products. The screening of efficient fermentative microorganisms under high temperature conditions (45°C) has thus to be further implemented. The last technological barrier of the process concerns the ethanolic fermentation of the pentoses. Indeed, the pentoses, originating from the hemicellulosic fraction, can represent up to 40% of total sugars in some lignocellulosic substrates. Nobody has yet identified a microorganism which is able to ferment the pentoses into ethanol with performances similar to those of Saccharomyces cerevisiae on glucose. But recent genetic improvements focused on the transformation of Saccharomyces cerevisiae and Zymomonas mobilis could result in good fermentative performances on pentoses.
The U.S. Department of Energy (DOE) is promoting the development of ethanol from lignocellulosic feedstocks as an alternative to conventional petroleum-based transportation fuels. DOE funds both fundamental and applied research in this area and needs a method for predicting cost benefits of many research proposals. To that end, the National Renewable Energy Laboratory (NREL) has modeled many potential process designs and estimated the economics of each process during the last 20 years. This report is an update of the ongoing process design and economic analyses at NREL. We envision updating this process design report at regular intervals; the purpose being to ensure that the process design incorporates all new data from NREL research, DOE funded research and other sources, and that the equipment costs are reasonable and consistent with good engineering practice for plants of this type. For the non-research areas this means using equipment and process approaches as they are currently used in industrial applications. For the last report 1, published in 1999, NREL performed a complete review and update of the process design and economic model for the biomass-to-ethanol process utilizing co-current dilute acid prehydrolysis with simultaneous saccharification (enzymatic) and co-fermentation. The process design included the core technologies being researched by the DOE: prehydrolysis, simultaneous saccharification and co-fermentation, and cellulase enzyme production. In addition, all ancillary areas feed handling, product recovery and purification, wastewater treatment (WWT), lignin combustor and boiler-turbogenerator and utilities were included. NREL engaged Delta-T Corporation (Delta-T) to assist in the process design evaluation, the process equipment costing, and overall plant integration. The process design and costing for the lignin combustor and boiler turbogenerator was reviewed by Reaction Engineering Inc.
The National Renewable Energy Laboratory (NREL) has undertaken a complete review and update of the process design and economic model for the biomass-to-ethanol process based on co-current dilute acid prehydrolysis, along with simultaneous saccharification (enzymatic) and co-fermentation. The process design includes the core technologies being researched by the U.S. Department of Energy (DOE): prehydrolysis, simultaneous saccharification and co-fermentation, and cellulase enzyme production. In addition, all ancillary areas feed handling, product recovery and purification, wastewater treatment lignin burner and boiler-turbogenerator, and utilities are included. NREL engaged Delta-T Corporation to assist in the process design evaluation, the process equipment costing, and overall plant integration. The process design and costing for the lignin burner and boiler turbogenerator has been reviewed by Reaction Engineering Inc. and the wastewater treatment by Merrick & Company. An overview of both reviews is included here. The purpose of this update was to ensure that the process design and equipment costs were reasonable and consistent with good engineering practice for plants of this type using available technical data. For the non-research areas this means using equipment and process approaches as they are currently being used in industrial applications. For areas currently being researched by NREL, we used the best research estimates of near-term data to develop a process design and equipment specifications consistent with existing similar commercial operations.
Analysis is undertaken motivated by the question: “What are the likely features and cost of a facility producing ethanol from cellulosic biomass at a level of maturity comparable to a refinery?” This question is considered with respect to cost reductions arising from increased scale, lower-cost feedstock, and process improvements in pretreatment and biological conversion, but not other process steps. An “advanced technology” scenario is developed that represents our estimate of the most likely features of mature biomass ethanol technology. A “best-parameter” scenario, intended to be indicative of the potential for R&D-driven cost reductions, is also developed based on the best values for individual process parameters reported in the literature. Both scenarios involve large plants (2.7 million dry t feedstock/yr). Feedstock costs are taken to be $38.60/delivered dry t for the advanced scenario and $34.00/delivered dry t for the best-parameter scenario. Projected selling prices, including operating costs and capital recovery corresponding to a 14.2% return on investment, are 50¢/gal (pure ethanol basis) for the advanced technology case and 34¢/gal for the best-parameter case. These are markedly lower than the 118¢ /gal selling price projected for base-case technology, with the largest share of cost reductions due to improved conversion technology. Key conversion technology improvements include, in order of importance, consolidated bioprocessing, advanced pretreatment, elimination of seed reactors, and faster rates. First-law thermodynamic efficiencies based on the biomass high heating value and production of ethanol and electricity are 61.2% for the advanced case and 69.3% for the best-parameter case, as compared to 50.3% currently. Combining advanced ethanol production technology of the type presented here with advanced gas turbine-based power generation is a promising direction for future analysis and may offer still further cost reductions and efficiency increases.
Simultaneous saccharification and fermentation (SSF) processes for producing ethanol from lignocellulose are capable of improved
hydrolysis rates, yields, and product concentrations compared to separate hydrolysis and fermentation (SHF) systems, because
the continuous removal of the sugars by the yeasts reduces the end-product inhibition of the enzyme complex. Recent experiments
using Genencor 150L cellulase and mixed yeast cultures have produced yields and concentrations of ethanol from cellulose of
80% and 4.5%, respectively. The mixed culture was employed because B.clausenii has the ability to ferment cellobiose (further reducing end-product inhibition), while the brewing yeastS. cerevisiae provides a robust ability to ferment the monomeric sugars. These experimental results are combined with a process model to
evaluate the economics of the process and to investigate the effect of alternative processes, conditions, and organisms.
During hydrolysis of lignocellulosic materials a wide range of compounds which are inhibitory to microorganisms are formed or released. Based on their origin the inhibitors are usually divided in three major groups: weak acids, furan derivatives, and phenolic compounds. These compounds limit efficient utilisation of the hydrolysates for ethanol production by fermentation. If the inhibitors are identified and the mechanisms of inhibition elucidated, fermentation can be improved by developing specific detoxification methods, choosing an adapted microorganism, or optimising the fermentation strategy. The present review discusses the generation of inhibitors during degradation of lignocellulosic materials, and the effect of these on fermentation yield and productivity. Inhibiting mechanisms of individual compounds present in the hydrolysates and their interaction effects are reviewed.
A two-stage process was evaluated for the fermentation of polymeric feedstocks to ethanol by a single, genetically engineered microorganism. The truncated xylanase gene (xynZ) from the thermophilic bacterium Clostridium thermocellum was fused with the N terminus of lacZ to eliminate secretory signals. This hybrid gene was expressed at high levels in ethanologenic strains of Escherichia coli KO11 and Klebsiella oxytoca M5A1(pLOI555). Large amounts of xylanase (25 to 93 mU/mg of cell protein) accumulated as intracellular products during ethanol production. Cells containing xylanase were harvested at the end of fermentation and added to a xylan solution at 60 degrees C, thereby releasing xylanase for saccharification. After cooling, the hydrolysate was fermented to ethanol with the same organism (30 degrees C), thereby replenishing the supply of xylanase for a subsequent saccharification. Recombinant E. coli metabolized only xylose, while recombinant K. oxytoca M5A1 metabolized xylose, xylobiose, and xylotriose but not xylotetrose. Derivatives of this latter organism produced large amounts of intracellular xylosidase, and the organism is presumed to transport both xylobiose and xylotriose for intracellular hydrolysis. By using recombinant M5A1, approximately 34% of the maximal theoretical yield of ethanol was obtained from xylan by this two-stage process. The yield appeared to be limited by the digestibility of commercial xylan rather than by a lack of sufficient xylanase or by ethanol toxicity. In general form, this two-stage process, which uses a single, genetically engineered microorganism, should be applicable for the production of useful chemicals from a wide range of biomass polymers.
The genes encoding essential enzymes of the fermentative pathway for ethanol production in Zymomonas mobilis, an obligately ethanologenic bacterium, were inserted into Escherichia coli under the control of a common promoter. Alcohol dehydrogenase II and pyruvate decarboxylase from Z. mobilis were expressed at high levels in E. coli, resulting in increased cell growth and the production of ethanol as the principal fermentation product from glucose. These results demonstrate that it is possible to change the fermentation products of an organism, such as E. coli, by the addition of genes encoding appropriate enzymes which form an alternative system for the regeneration of NAD+.
Hemicellulose hydrolysates of agricultural residues often contain mixtures of hexose and pentose sugars. Ethanologenic Escherichia coli that have been previously investigated preferentially ferment hexose sugars. In some cases, xylose fermentation was slow or incomplete. The purpose of this study was to develop improved ethanologenic E. coli strains for the fermentation of pentoses in sugar mixtures. Using fosfomycin as a selective agent, glucose-negative mutants of E. coli KO11 (containing chromosomally integrated genes encoding the ethanol pathway from Zymomonas mobilis) were isolated that were unable to ferment sugars transported by the phosphoenolpyruvate-dependent phosphotransferase system. These strains (SL31 and SL142) retained the ability to ferment sugars with independent transport systems such as arabinose and xylose and were used to ferment pentose sugars to ethanol selectively in the presence of high concentrations of glucose. Additional fosfomycin-resistant mutants were isolated that were superior to strain KO11 for ethanol production from hexose and pentose sugars. These hyperproductive strains (SL28 and SL40) retained the ability to metabolize all sugars tested, completed fermentations more rapidly, and achieved higher ethanol yields than the parent. Both SL28 and SL40 produced 60 gl-1 ethanol from 120 gl-1 xylose in 60 h, 20% more ethanol than KO11 under identical conditions. Further studies illustrated the feasibility of sequential fermentation. A mixture of hexose and pentose sugars was fermented with near theoretical yield by SL40 in the first step followed by a second fermentation in which yeast and glucose were added. Such a two-step approach can combine the attributes of ethanologenic E. coli for pentoses with the high ethanol tolerance of conventional yeasts in a single vessel.
The Thermus thermophilus xylA gene encoding xylose (glucose) isomerase was cloned and expressed in Saccharomyces cerevisiae under the control of the yeast PGK1 promoter. The recombinant xylose isomerase showed the highest activity at 85 degrees C with a specific activity of 1.0 U mg-1. A new functional metabolic pathway in S. cerevisiae with ethanol formation during oxygen-limited xylose fermentation was demonstrated. Xylitol and acetic acid were also formed during the fermentation.
Cofermentation of glucose, xylose, and arabinose is critical for complete bioconversion of lignocellulosic biomass, such as agricultural residues and herbaceous energy crops, to ethanol. We have previously developed a plasmid-bearing strain of Zymomonas mobilis (206C[pZB301]) capable of cofermenting glucose, xylose, and arabinose to ethanol. To enhance its genetic stability, several genomic DNA-integrated strains of Z. mobilis have been developed through the insertion of all seven genes necessay for xylose and arabinose fermentation into the Zymomonas genome. From all the integrants developed, four were selected for further evaluation. The integrants were tested for stability by repeated transfer in a nonselective medium (containing only glucose). Based on the stability test, one of the integrants (AX101) was selected for further evaluation. A series of batch and continuous fermentations was designed to evaluate the cofermentation of glucose, xylose, and L-arabinose with the strain AX101. The pH range of study was 4.5, 5.0, and 5.5 at 30 degrees C. The cofermentation process yield was about 84%, which is about the same as that of plasmid-bearing strain 206C(pZB301). Although cofermentation of all three sugars was achieved, there was a preferential order of sugar utilization: glucose first, then xylose, and arabinose last.
The effect of process stream recirculation on ethanol production from steam- pretreated softwood based on simultaneous saccharification and fermentation (SSF) was investigated for two process configurations. In the first configuration, a part of the stillage stream after distillation was recycled and, in the second configuration, the liquid after SSF was recycled. The aim was to minimize the energy consumption in the distillation of the fermentation broth and in the evaporation of the stillage, as well as the use of fresh water. However, recirculation leads to an increased concentration of nonvolatiles in the first configuration, and of both volatiles and nonvolatiles in the second configuration. These substances might be inhibitory to the enzymes and the yeast in SSF. When 60% of the fresh water was replaced by stillage, the ethanol yield and the productivity were the same as for the configuration without recirculation. The ethanol production cost was reduced by 17%. In the second configuration, up to 40% of the fresh water could be replaced without affecting the final ethanol yield, although the initial ethanol productivity decreased. The ethanol production cost was reduced by 12%. At higher degrees of recirculation, fermentation was clearly inhibited, resulting in a decrease in ethanol yield while hydrolysis seemed unaffected.
The enzymatic digestibility of ammonia fiber explosion (AFEX)-treated rice straw was modeled by statistically correlating the variability of samples to differences in treatment using several different analytical techniques. Lignin content and crystallinity index of cellulose affect enzymatic hydrolysis the most. X-ray diffraction was used to measure the crystallinity index (CrI), while fluorescence and diffuse reflectance infrared (DRIFT) spectroscopy measured the lignin content of the samples. Multivariate analysis was applied to correlate the enzymatic hydrolysis results of the various samples with X-ray diffraction and spectroscopic data. Principal component analysis (PCA) and multilinear regression (MLR) techniques did not accurately predict the digestibility of the rice straw samples. The best correlation (R value of 0.775) was found between the treatment conditions of the AFEX process and the concentration of xylose at 24 h after enzymatic hydrolysis.
The fungal pathway for L-arabinose catabolism converts L-arabinose to D-xylulose 5-phosphate in five steps. The intermediates are, in this order: L-arabinitol, L-xylulose, xylitol and D-xylulose. Only some of the genes for the corresponding enzymes were known. We have recently identified the two missing genes for L-arabinitol 4-dehydrogenase and L-xylulose reductase and shown that overexpression of all the genes of the pathway in Saccharomyces cerevisiae enables growth on L-arabinose. Under anaerobic conditions ethanol is produced from L-arabinose, but at a very low rate. The reasons for the low rate of L-arabinose fermentation are discussed.
In addition to fermentable sugars, dilute-acid hydrolysates of lignocellulose contain compounds that inhibit fermenting microorganisms, such as Saccharomyces cerevisiae. Previous results show that phenolic compounds and furan aldehydes, and to some extent aliphatic acids, act as inhibitors during fermentation of dilute-acid hydrolysates of spruce. Treatment of lignocellulose hydrolysates with alkali, usually in the form of overliming to pH 10.0, has been frequently employed as a detoxification method to improve fermentability. A spruce dilute-acid hydrolysate was treated with NaOH in a factorial design experiment, in which the pH was varied between 9.0 and 12.0, the temperature between 5 and 80 degrees C, and the time between 1 and 7 h. Already at pH 9.0, >25% of the glucose was lost when the hydrolysate was treated at 80 degrees C for 1 h. Among the monosaccharides, xylose was degraded faster under alkaline conditions than the hexoses (glucose, mannose, and galactose), which, in turn, were degraded faster than arabinose. The results suggest that alkali treatment of hydrolysates can be performed at temperatures below 30 degrees C at any pH between 9.0 and 12.0 without problems with sugar degradation or formation of inhibiting aliphatic acids. Treatment with Ca(OH)2 instead of NaOH resulted in more substantial degradation of sugars. Under the harsher conditions of the factorial design experiment, the concentrations of furfural and 5-hydroxymethylfurfural decreased while the total phenolic content increased. The latter phenomenon was tentatively attributed to fragmentation of soluble aromatic oligomers in the hydrolysate. Separate phenolic compounds were affected in different ways by the alkaline conditions with some compounds showing an increase in concentration while others decreased. In conclusion, the conditions used for detoxification with alkali should be carefully controlled to optimize the positive effects and minimize the degradation of fermentable sugars.
The lack of industrially suitable microorganisms for converting biomass into fuel ethanol has traditionally been cited as a major technical roadblock to developing a bioethanol industry. In the last two decades, numerous microorganisms have been engineered to selectively produce ethanol. Lignocellulosic biomass contains complex carbohydrates that necessitate utilizing microorganisms capable of fermenting sugars not fermentable by brewers' yeast. The most significant of these is xylose. The greatest successes have been in the engineering of Gram-negative bacteria: Escherichia coli, Klebsiella oxytoca, and Zymomonas mobilis. E. coli and K. oxytoca are naturally able to use a wide spectrum of sugars, and work has concentrated on engineering these strains to selectively produce ethanol. Z. mobilis produces ethanol at high yields, but ferments only glucose and fructose. Work on this organism has concentrated on introducing pathways for the fermentation of arabinose and xylose. The history of constructing these strains and current progress in refining them are detailed in this review.
Consolidated bioprocessing (CBP) is a new approach to convert biomass into useful products. The approach involves consolidating cellulase production, cellulose hydrolysis, hexose formation, and pentose fermentation into a single process step. Thus, the approach relies on microbial rather than enzymatic processes. This technique can lower the cost of producing ethanol and other commodity fermentation products.
Copyright ©1999, Éditions Technip The reported study intends to describe the state of the art in the domain of ethanol production from lignocellulosic biomass. It was sustained and managed by a specialized group of the French Agrice (Agriculture for Chemical and Energy Organization). Its first goal was to pinpoint the main technical and economical bottlenecks of the processes which are today under consideration, and to identify which research and development efforts could be implemented to overcome them (in the short or middle tenu). Lignocellulosic biomass is a complex substrate, and essentially made of cellulose, hemicellulose and lignin. The processes which have been considered, attempt to recover a maximum amount of sugars from the hydrolysis of cellulose and hemicellulose, and to ferment them into ethanol. The hydrolysis processes used in the past are essentially chemical processes, but the acid recovery costs and the formation of toxic products make them uncompetitive. They are now substituted by enzymatic processes, which are more specific and allow higher hydrolysis yields under less severe conditions. However, the cellulose that is the targof the enzymatic hydrolysis, is not directly accessible to the enzymes. It is the reason why a pretreatment step has to precede the enzymatic hydrolysis, in order to improve the enzymatic susceptibility of the cellulose, and to hydrolyse the hemicelhtlosic fraction. Different types of pretreatment have been studied, but three methods appear more efficient: dilute acid hydrolysis, steam explosion with catalyst addition and thermohydrolysis. These pretreatments could result in high hydrolysis yields of the cellulose fraction (close to 100%), and in a maximum recovery of the sugars from the hemicellulosic fraction. Enzymatic hydrolysis has yto be improved in order to reduce the cost of consumption of the enzymes. Research works will have to focus upon the enzyme specific activity, in order to achieve higher efficiencies such as those obtained with amylases. The SSF (Saccharification and Simultaneous Fermentation) process improves the enzyme efficiency by reducing the feed-back inhibition from the hydrolysis products. The screening of efficient fermentative microorganisms under high temperature conditions (45°C) has thus to be further implemented. The last technological barrier of the process concents the ethanolic fennentation of the pentoses. Indeed, the pentoses, originating from the hemicellulosic fraction, can represent up to 40% of total sugars in some lignocellulosic substrates. Nobody has yidentified a microorganism which is able to ferment the pentoses into ethanol with performances similar to those of Saccharomyces cerevisiae on glucose. But recent genetic improvements focused on the transfonnation o/Saccharomyces cerevisiae and Zymomonas mobilis could result in good fermentative performances on pentoses.
Lignocellulosic materials pretreated using liquid hot water (LHW) (220 degrees C, 5 MPa, 120 s) were fermented to ethanol by batch simultaneous saccharification and fermentation (SSF) using Saccharomyces cerevisiae in the presence of Trichoderma reesei cellulase. SSF of sugarcane bagasse (as received), aspen chips (smallest dimension 3 mm), and mixed hardwood flour (-60 +70 mesh) resulted in 90% conversion to ethanol in 2-5 d at enzyme loadings of 15-30 FPU/g. In most cases, 90% of the final conversion was achieved within 75 h of inoculation. Comminution of the pretreated substrates did not affect the conversion to ethanol. The hydrolysate produced from the LHW pretreatment showed slight inhibition of batch growth of S. cerevisiae. Solids pretreated at a concentration of 100 g/L were as reactive as those pretreated at a lower concentration, provided that the temperature was maintained at 220 degrees C.
In two previous studies, optimal conditions were identified for two-step steam pretreatment of SO2- and H2SO 4-impregnated softwood. In the present study the yield of sugar and ethanol was determined in a process development unit where pretreatment was performed in a 10-L reactor and simultaneous saccharification and fermentation (SSF) or enzymatic hydrolysis (EH) were performed in 30-L reactors. The study showed that a steam pretreatment reactor should be larger than 2 L to yield acceptable results. Two pretreatment combinations were studied. In the H2SO4 case, the first pretreatment step was at 180°C for 10 min with 0.5% H2SO4 and the second step at 210°C for 2 min with 1% H2SO4. In the SO2 case, firt step was at 190°C for 2 min followed by a second step at 210°C for 5 min. The concentration of SO2 was 3% in both steps. EH and SSF were performed on the whole slurry after the second pretreatment step to determine the yield of sugars and ethanol. The liquid after the first pretreatment step was also analyzed and fermented. When SSF and EH were performed at the same dry matter content and enzymatic activity, the ethanol yield in SSF exceeded the yield obtained with EH in both pretreatment cases, even when 100% yield in the fermentation step was assumed. Thus SSF is a better process if yield is the main priority. Comparison of the yields with the two acid catalysts showed higher yields with SO2 in both SSF and EH. The overall ethanol yield following SSF of SO2-impregnated and pretreated wood reached 81% of the theoretical, that is, 357 liters per metric ton of dry raw material.
Conventional resources mainly fossil fuels are becoming limited because of the rapid increase in energy demand. This imbalance in energy demand and supply has placed immense pressure not only on consumer prices but also on the environment, prompting mankind to look for sustainable energy resources. Biomass is one such environmentally friendly renewable resource from which various useful chemicals and fuels can be produced. A system similar to a petroleum refinery is required to produce fuels and useful chemicals from biomass and is known as a biorefinery. Biorefineries have been categorized in three phases based on the flexibility of input, processing capabilities, and product generation. Phase I has less or no flexibility in any of the three aforementioned categories. Phase II, while having fixed input and processing capabilities, allows flexibility in product generation. Phase III allows flexibility in all the three processes and is based on the concept of high-value low-volume (HVLV) and low-value high-volume (LVHV) outputs. This paper reviews the concept of biorefinery, its types, future directions, and associated technical challenges. An approach of streamlining biorefineries with conventional refineries in producing conventional fuels is also presented. Furthermore, twelve platform chemicals that could be major outputs from an integrated biorefinery are also discussed.
Lignocellulosic materials pretreated using liquid hot water (LHW) (220°C, 5 MPa, 120 s) were fermented to ethanol by batch
simultaneous saccharification and fermentation (SSF) usingSaccharomyces cerevisiae in the presence ofTrichoderma reesei cellulase. SSF of sugarcane bagasse (as received), aspen chips (smallest dimension 3 mm), and mixed hardwood flour (−60 +70
mesh) resulted in 90% conversion to ethanol in 2–5 d at enzyme loadings of 15–30 FPU/g. In most cases, 90% of the final conversion
was achieved within 75 h of inoculation. Comminution of the pretreated substrates did not affect the conversion to ethanol.
The hydrolysate produced from the LHW pretreatment showed slight inhibition of batch growth ofS. cerevisiae. Solids pretreated at a concentration of 100 g/L were as reactive as those pretreated at a lower concentration, provided
that the temperature was maintained at 220°C.
We have performed a comparative study of xylose utilization in Saccharomyces cerevisiae transformants expressing two key enzymes in xylose metabolism, xylose reductase (XR) and xylitol dehydrogenase (XDH), and in a prototypic xylose-utilizing yeast, Pichia stipitis. In the absence of respiration (see text), baker's yeast cells convert half of the xylose to xylitol and ethanol, whereas P. stipilis cells display rather a homofermentative conversion of xylose to ethanol. Xylitol production by baker's yeast is interpreted as a result of the dual cofactor dependence of the XR and the generation of NADPH by the pentose phosphate pathway. Further limitations of xylose utilization in S. cerevisiae cells are very likely caused by an insufficient capacity of the non-oxidative pentose phosphate pathway, as indicated by accumulation of sedoheptulose-7-phosphate and the absence of fructose-1,6-bisphosphate and pyruvate accumulation. By contrast, uptake at high substrate concentrations probably does not limit xylose conversion in S. cerevisiae XYL1/XYL2 transformants.
A xylose-rich, dilute-acid-pretreated corncob hydrolase was fermented by Escherichia coli ATCC 11303, recombinant (rec) E. coli (pLOI 297 and KO11), Pichia stipitis (CBS 5773, 6054 adn R), Saccharomyces cerevisiae siolate 3 in combination with xylose isomerase, rec S. cerevisiae (TJ1, H550 and H477), and Fusraium oxysporum VIT-D-80134 in an interlaboratory comparison. The micro-organisms were studied according to three different options: (A) fermentation under consistent conditions, (B) fermentation under optimal conditions for the organisms, and (C) fermentation under optimal conditions for the organism with detoxification if the hydrolysate. The highest yields of tehanol, 0.24 g/g (A), 0.36 g/g (B) and 0.54 g/g (C), were obtained from rec E. coli B, KO11. P. stipitis and F. oxysporum were sensitive to the inhibitors present in the hydrolysate and produced a minimum yields of 0.34 g/g (C) and 0.04 g/g (B), respectively. The analysis of the corn-cob hydrolysate and aspects of process economy of the different fermentation options (pH, sterilization, nutrient supplementation, adaptation, detoxification) are discussed.
Phenolic compounds released and generated during hydrolysis inhibit fermentation of lignocellulose hydrolysates to ethanol by Saccharomyces cerevisiae. A wide variety of aromatic compounds form from lignin, which is partially degraded during acid hydrolysis of the lignocellulosic raw material. Aromatic compounds may also form as a result of sugar degradation and are present in wood as extractives. The influence of hydroxy-methoxy-benzaldehydes, diphenols/quinones, and phenylpropane derivatives on S. cerevisiae cell growth and ethanol formation was assayed using a defined medium and oxygen-limited conditions. The inhibition effected by the hydroxy-methoxybenzaldehydes was highly dependent on the positions of the substituents. A major difference in inhibition by the oxidized and reduced form of a diphenol/quinone was observed, the oxidized form being the more inhibitory. The phenylpropane derivatives were examined with respect to difference in toxicity depending on the oxidation-reduction state of the gamma-carbon, the presence and position of unsaturated bonds in the aliphatic side chain, and the number and identity of hydroxyl and methoxyl substituents. Transformations of aromatic compounds occurring during the fermentation included aldehyde reduction, quinone reduction, and double bond saturation. Aromatic alcohols were detected as products of reductions of the corresponding aldehydes, namely hydroxy-methoxy-benzaldehydcs and coniferyl aldehyde. High molecular mass compounds and the corresponding diphenol were detected as products of quinone reduction. Together with coniferyl alcohol, dihydroconiferyl alcohol was identified as a major transformation product of coniferyl aldehyde.
This study describes different detoxification methods to improve both cell growth and ethanol production by Baker's yeast,
Saccharomyces cerevisiae. A dilute-acid hydrolyzate of spruce was used for the all detoxification methods tested. The changes in the concentrations
of fermentable sugars and three groups of inhibitory compounds—aliphatic acids, furan derivatives, and phenolic compounds—were
determined and the fermentability of the detoxified hydrolyzate was assayed. The applied detoxification methods included:
treatment with alkali (sodium hydroxide or calcium hydroxide); treatment with sulfite (0.1% [w/v] or 1% [w/v] at pH 5.5 or
10); evaporation of 10% or 90% of the initial volume; anion exchange (at pH 5.5 or 10); enzymatic detoxification with the
phenoloxidase laccase; and detoxification with the filamentous fungus Trichoderma reesei. An ion exchange at pH 5.5 or 10, treatment with laccase, treatment with calcium hydroxide, and treatment with T. reesei were the most efficient detoxification methods. Evaporation of 10% of the initial volume and treatment with 0.1% sulfite
were the least efficient detoxification methods. Treatment with laccase was the only detoxification method that specifically
removed only one group of the inhibitors, namely phenolic compounds. Anion exchange at pH 10 was the most efficient method
for removing all three major groups of inhibitory compounds; however, it also resulted in loss of fermentable sugars.
Utilization of pentose sugars (d-xylose andl-arabinose) derived from hemi-cellulose is essential for the economic conversion of biomass to ethanol. Xylose-fermenting
yeasts were discovered in the 1980s, but to date, no yeasts have been found that fermentl-arabinose to ethanol in significant quantities. We have screened 116 different yeasts for the ability to fermentl-arabinose and have found the following species able to ferment the sugar:Candida auringiensis, Candida succiphila, Ambrosiozyma monospora, andCandida sp. (YB-2248). Though these yeasts produced ethanol concentrations of 4.1 g/L or less, they are potential candidates for
mutational enhancement ofl-arabinose fermentation. These yeasts were also found to fermentd-xylose.
A process for conversion of lignocellulosic biomass into acetone-butanol (ABE) involving steam explosion pretreatment of the raw material, cellulase production, enzymatic hydrolysis and acetone-butanol fermentation of hydrolysates has been developed. Procedures and scale-up of the steam-explosion pretreatment of corncobs are described. The influence of experimental parameters, time, temperature and addition of acids, on both the performance of enzymatic hydrolysis and the fermentability into ABE of the hydrolysates obtained were first studied in a batch pilot reactor. The same criteria of performance were applied in large scale development work which was completed using a continuous industrial plant (Stake II machine with a capacity of 2–4 t per h) located in the biomass conversion facilities at Soustons, France. Under optimal conditions, which were strongly dependent on the technology used, nearly quantitative hydrolysis yields could be obtained with moderate amounts of appropriate cellulase preparations.
Low-molecular weight aliphatic acids, furaldehydes and a broad range of different aromatic compounds are known to inhibit the fermentation of lignocellulose hydrolysates by yeasts. In this work, a cocktail of different lignocellulose-derived inhibitors was used to compare the inhibitor resistance of eleven different industrial and laboratory Saccharomycescerevisiae strains and two Zygosaccharomyces strains. The inhibitor cocktail was composed of two aliphatic acids, formic and acetic acid, two furaldehydes, furfural and 5-hydroxymethylfurfural (HMF), and two aromatic compounds, cinnamic acid and coniferyl aldehyde. Fermentations were performed under oxygen-limited conditions and with different levels (100, 75, 50, 25 and 0%) of the inhibitor cocktail present. The ethanol yield on initial glucose, the volumetric and specific ethanol productivity, the biomass yield and the glucose consumption rates were used as criteria for the performance of the strains. The results revealed major differences in inhibitor resistance between yeast strains within the same species. The ethanol yield of the S. cerevisiae strain that was least affected decreased only with 10% at an inhibitor cocktail concentration of 100%, while the decrease in ethanol yield for the most sensitive S. cerevisiae strain was more than 50% already at an inhibitor cocktail concentration of 25%. Ethanol formation was generally less affected than growth and ethanol yield less than ethanol productivity. The two most resistant strains were an S. cerevisiae strain isolated from a spent sulphite liquor plant and one of the laboratory S. cerevisiae strains. Additional fermentations with either HMF or coniferyl aldehyde revealed that the degree of resistance of different yeast strains was highly dependent on the inhibitor used. A mutant strain of S. cerevisiae displaying enhanced resistance against coniferyl aldehyde compared with the parental strains was identified.
The fermentation of xylose and xylulose with yeasts, bacteria and fungi as well as the combination of yeasts and bacterial xylose isomerase, has been reviewed. Methods to measure oxygenation in fermentation with yeasts are compared and the relationship between pentose metabolism in yeasts and oxygenation during fermentation is discussed. The influence of substrate concentration, substrate specificity, pH, temperature, inhibitors and other additives in yeast fermentation have been summarized. Batch fermentation has been compared to continuous fermentation, fermentation with cell recirculation and immobilization. The fermentation of lignocellulose and lignocellulose hydrolysates with bacteria, yeasts and fungi has also been reviewed. Throughout this review, product concentration, product yield and productivity have been compared with values generally achieved in batch fermentation of hexoses: 50 g l−1, 0.5, and 2 g l−1 h−1.
The present study is a review of published investigations regarding the economy of ethanol production from lignocellulosic material. The objective is to present relations between and tendencies observed in different cost estimates. The influence of plant capacity and overall product yield on the ethanol production cost is investigated, as well as variations in capital costs in the different processes. The underlying technical and economic assumptions show a large variation between the various studies published. The variation in the ethanol production cost is large, from 18 to 151 US¢/l. The most important factor for the economic outcome is the overall ethanol yield. Other important parameters are the feedstock cost, which was found to vary between 22 and 61 US$/dry metric ton, and the plant capacity, which influences the capital cost. It is shown that there is a tendency towards a decrease in ethanol production cost with an increase in plant capacity for the enzymatic processes. A high yield also results in a decrease in production cost for the enzymatic and dilute acid processes in the papers reviewed.
Softwood constitutes the main source of lignocellulosic material in Sweden which can be used for ethanol production from renewable resources. To make the biomass-to-ethanol process more economically feasible, it is preferable to include the sugar-rich prehydrolysate, i.e. the liquid obtained after the pretreatment step, in the enzymatic hydrolysis of the solid fraction. This study shows that the prehydrolysate inhibits cellulose conversion in the enzymatic hydrolysis step. When the prehydrolysate was included in the enzymatic hydrolysis, the cellulose conversion was reduced by up to 36%. However, this inhibition can be overcome by fermentation of the prehydrolysate prior to enzymatic hydrolysis.
Simultaneous saccharification and fermentation (SSF) was performed on the slurries resulting from steam pretreatment of non-, SO2- and H2SO4-impregnated Salix chips. The aim was to increase the ethanol concentration by running SSF at high dry-matter content, while maintaining a high ethanol yield. Using SO2-impregnated Salix chips in steam pretreatment resulted in the highest ethanol concentration in the subsequent SSF. At a water-insoluble solids concentration of 9%, 32 g/L ethanol was obtained after 78 h of SSF, using 3.3 g/L baker's yeast (Saccharomyces cerevisiae) cultivated on the pretreatment liquid. This corresponds to an overall ethanol yield of 76% of the theoretical, based on the glucan and mannan content in the raw material. The cultivation procedure made the yeast better adapted to the medium. However, increasing the substrate loading further resulted in decreased fermentability.
Cellulolytic enzymes have mostly been studied from a biotechnological point of view and not according to accepted biochemical criteria. The lack of standardized structurally defined substrates and the lack of standardization of activity determination methods have resulted in contradictory views about their properties. The cellulolytic enzymes are not very stable proteins and their interaction with each other and modification during cultivation and purification has added to the confusion about their individual roles in the hydrolysis of native cellulose. The nomenclature used is to some extent historical and does not describe the activity of the enzymes. It is suggested that the hydrolysis of insoluble native cellulose is accompanied by two enzymes acting in synergy. (Refs. 57).
Various wild-type yeasts and fungi were screened to evaluate their ability to ferment L-arabinose under oxygen-limited conditions when grown in defined minimal media containing mixtures of L-arabinose, D-xylose, and D-glucose. Although all of the yeasts and some of the fungi consumed arabinose, arabinose was not fermented to ethanol by any of the strains tested. Arabitol was the only major product other than cell mass formed from L-arabinose; yeasts converted arabinose to arabitol at high yield. The inability to ferment L-arabinose appears to be a consequence of inefficient or incomplete assimilation pathways for this pentose sugar.
Various techniques are available for the conversion of lignocellulosics to fuel ethanol. During the last decade processes based on enzymatic hydrolysis of cellulose have been investigated more extensively, showing good yield on both hardwood and softwood. The cellulase production of a filamentous fungi, Trichoderma reesei Rut C 30, was examined on carbon sources obtained after steam pretreatment of spruce. These materials were washed fibrous steam-pretreated spruce (SPS), and hemicellulose hydrolysate. The hemicellulose hydrolysate contained, besides water-soluble carbohydrates, lignin and sugar degradation products, which were formed during the pretreatment and proved to be inhibitory to microorganisms. Experiments were performed in a 4-L laboratory fermentor. The hydrolytic capacity of the produced enzyme solutions was compared with two commercially available enzyme preparations, Celluclast and Iogen Cellulase, on SPS, washed SPS, and Solka Floc cellulose powder. There was no significant difference among the different enzymes produced by T. reesei Rut C 30. However, the conversion of cellulose using these enzymes was higher than that obtained with Iogen or Celluclast cellulases using steam-pretreated spruce as substrate.
Whole tree chips obtained from softwood forest thinnings were pretreated via single- and two-stage dilute-sulfuric acid pretreatment. Whole-tree chips were impregnated with dilute sulfuric acid and steam treated in a 4-L steam explosion reactor. In single-stage pretreatment, wood chips were treated using a wide range of severity. In two-stage pretreatment, the first stage was carried out at low severity to maximize hemicellulose recovery. Solubilized sugars were recovered from the first-stage prehydrolysate by washing with water. In the second stage, water-insoluble solids from first-stage prehydrolysate were impregnated with dilute sulfuric acid, then steam treated at more severe conditions to hydrolyze a portion of the remaining cellulose to glucose and to improve the enzyme digestibility. The total sugar yields obtained after enzymatic hydrolysis of two-stage dilute acid-pretreated samples were compared with sugar yields from single-stage pretreatment. The overall sugar yield from two-stage dilute-acid pretreatment was approx 10% higher, and the net enzyme requirement was reduced by about 50%. Simultaneous saccharification and fermentation using an adapted Saccharomyces cerevisiae yeast strain further improved cellulose conversion yield and lowered the enzyme requirement.
Dilute-acid hydrolyzates from lignocellulose are, to a varying degree, inhibitory to yeast. In the present work, dilute-acid hydrolyzates from spruce, birch, and forest residue, as well as synthetic model media, were fermented by Saccharomyces cerevisiae in fed-batch cultures. A control strategy based on on-line measurement of carbon dioxide evolution (CER) was used to control the substrate feed rate in a lab scale bioreactor. The control strategy was based solely on the ratio between the relative increase in CER and the relative increase in feed rate. Severely inhibiting hydrolyzates could be fermented without detoxification and the time required for fermentation of moderately inhibiting hydrolyzates was also reduced. The feed rate approached a limiting value for inhibiting media, with a corresponding pseudo steady-state value for CER. However, a slow decrease of CER with time was found for media containing high amounts of 5-hydroxymethyl furfural (HMF). The success of the control strategy is explained by the conversion of furfural and HMF by the yeast during fed-batch operation. The hydrolyzates contained between 1.4 and 5 g/l of furfural and between 2.4 and 6.5 g/l of HMF. A high conversion of furfural was obtained (between 65-95%) at the end of the feeding phase, but the conversion of HMF was considerably lower (between 12-40%).
A plan has been put forth to strategically thin northern California forests to reduce fire danger and improve forest health. The resulting biomass residue, instead of being open burned, can be converted into ethanol that can be used as a fuel oxygenate or an octane enhancer. Economic potential for a biomass-to-ethanol facility using this softwood biomass was evaluated for two cases: stand-alone and co-located. The co-located case refers to a specific site with an existing biomass power facility near Martell, California. A two-stage dilute acid hydrolysis process is used for the production of ethanol from softwoods, and the residual lignin is used to generate steam and electricity. For a plant processing 800 dry tonnes per day of feedstock, the co-located case is an economically attractive concept. Total estimated capital investment is approximately $70 million for the co-located plant, and the resulting internal rate of return (IRR) is about 24% using 25% equity financing. A sensitivity analysis showed that ethanol selling price and fixed capital investment have a substantial effect on the IRR. It can be concluded that such a biomass-to-ethanol plant seems to be an appealing proposition for California, if ethanol replaces methyl tert-butyl ether, which is slated for a phaseout.
Softwood is an interesting raw material for the production of fuel ethanol as a result of its high content of hexoses, and it has attracted attention especially in the Northern hemisphere. However, the enzymatic hydrolysis of softwood is not sufficiently efficient for the complete conversion of cellulose to glucose. Since an improvement in the glucose yield is of great importance for the overall economy of the process, the influence of various parameters on the cellulose conversion of steam-pretreated spruce has been investigated. The addition of beta-glucosidase up to 50 IU g(-)(1) cellulose to the enzymatic hydrolysis process resulted in increased cellulose conversion at a cellulase loading up to 48 FPU g(-)(1) cellulose. Despite very high enzyme loading (120 FPU g(-)(1) cellulose) only about 50% of the cellulose in steam-pretreated spruce was converted to glucose when all of the material following pretreatment was used in the hydrolysis step. The influence of temperature, residence time, and pH were investigated for washed pretreated spruce at a dry matter (DM) content of 5% and a cellulase activity of 18.5 FPU g(-)(1) cellulose. The optimal temperature was found to be dependent on both residence time and pH, and the maximum degree of cellulose conversion, 69.2%, was obtained at 38 degrees C and pH 4.9 for a residence time of 144 h. However, when the substrate concentration was changed from 5% to 2% DM, the cellulose conversion increased to 79.7%. An increase from 5% to 10% DM resulted, however, in a similar degree of cellulose conversion, despite a significant increase in the glucose concentration from 23 g L(-)(1) to 45 g L(-)(1). The deactivation of beta-glucosidase increased with increasing residence time and was more pronounced with vigorous agitation.
Ethanol production was evaluated from wheat straw (WS) hemicellulose acid hydrolysate using an adapted and parent strain of Pichia stipitis. NRRL Y-7124. The treatment by boiling and overliming with Ca(OH)(2) significantly improved the fermentability of the hydrolysate. Ethanol yield (Yp/s) and productivity (Qp av) were increased 2.4+/-0.10 and 5.7+/-0.24 folds, respectively, compared to neutralized hydrolysate. Adaptation of the yeast to the hydrolysate resulted further improvement in yield and productivity. The maximum yield was 0.41+/-0.01 g(p) g(s)(-1), equivalent to 80.4+/-0.55% theoretical conversion efficiency. Acetic acid, furfurals and lignins present in the hydrolysate were inhibitory to microbial growth and ethanol production. The addition of these inhibitory components individually or in various combinations at a concentrations similar to that found in hydrolysate to simulated medium resulted a reduction in ethanol yield (Yp/s) and productivity (Qp av). The hydrolysate used had the following composition (expressed in g x l(-1)): xylose 12.8+/-0.25; glucose 1.7+/-0.3; arabinose 2.6+/-0.21 and acetic acid 2.7+/-0.33.
Corn stover, the most abundant agricultural residue in Hungary, is a potential raw material for the production of fuel ethanol as a result of its high content of carbohydrates, but a pretreatment is required for its efficient hydrolysis. In this article, we describe the results using various chemicals such as dilute H2SO4, HCl, and NaOH separately as well as consecutively under relative mild conditions (120 degrees C, 1 h). Pretreatment with 5% H2SO4 or 5% HCl solubilized 85% of the hemicellulose fraction, but the enzymatic conversion of pretreated materials increased only two times compared to the untreated corn stover. Applying acidic pretreatment following a 1-d soaking in base achieved enzymatic conversion that was nearly the theoretical maximum (95.7%). Pretreatment with 10% NaOH decreased the lignin fraction >95%, increased the enzymatic conversion more than four times, and gave a 79.4% enzymatic conversion. However, by increasing the reaction time, the enzymatic degradability could also be increased significantly, using a less concentrated base. When the time of pretreatment was increased three times (0.5% NaOH at 120 degrees C), the amount of total released sugars was 47.9 g from 100 g (dry matter) of untreated corn stover.
Evidence is presented that xylose metabolism in the anaerobic cellulolytic fungus Piromyces sp. E2 proceeds via a xylose isomerase rather than via the xylose reductase/xylitol-dehydrogenase pathway found in xylose-metabolising yeasts. The XylA gene encoding the Piromyces xylose isomerase was functionally expressed in Saccharomyces cerevisiae. Heterologous isomerase activities in cell extracts, assayed at 30 degrees C, were 0.3-1.1 micromol min(-1) (mg protein)(-1), with a Km for xylose of 20 mM. The engineered S. cerevisiae strain grew very slowly on xylose. It co-consumed xylose in aerobic and anaerobic glucose-limited chemostat cultures at rates of 0.33 and 0.73 mmol (g biomass)(-1) h(-1), respectively.
Corn fiber, a byproduct of corn wet milling, is an attractive feedstock for biomass ethanol production. Corn fiber was hydrolyzed by dilute sulfuric acid and neutralized by one of two methods: conventional lime treatment or neutralization by strongly basic anion exchange. The anion exchange neutralized (AEN) hydrolysate contained substantially lower levels of the inhibiting compounds furfural, 5-hydroxymethylfurfural, and acetic acid compared to the lime neutralized hydrolysate. In batch fermentations the ethanol yields and final ethanol concentration of the two hydrolysates were similar at 0.32-0.43 g/g and 29-44 g/l, respectively. Sugar consumption in the AEN fermentations was superior. Coupling of a membrane pervaporation unit to a fed-batch fermentation of AEN hydrolysate maintained the ethanol concentration below 25 g/l with complete sugar utilization for approximately 5 days. A concentrated ethanol stream of 17 wt.% ethanol was produced by the pervaporation unit.
The gene encoding L-lactate dehydrogenase from Thermoanaerobacterium saccharolyticum JW/SL-YS485 was cloned, sequenced, and used to obtain an L-ldh deletion mutant strain (TD1) following a site-specific double-crossover event as confirmed by PCR and Southern blot. Growth rates and final cell densities were similar for strain TD1 and the wild-type grown on glucose and xylose. Lactic acid was below the limit of detection (0.3 mM) for strain TD1 on both glucose and xylose at all times tested, but was readily detected for the wild-type strain, with average final concentrations of 8.1 and 1.8 mM on glucose and xylose, respectively. Elimination of lactic acid as a fermentation product was accompanied by a proportional increase in the yields of acetic acid and ethanol. The results reported here represent a step toward using metabolic engineering to develop strains of thermophilic anaerobic bacteria that do not produce organic acids, and support the methodological feasibility of this goal.