Bioethanol: New opportunities for an ancient product

ARTICLE IN PRESS

Bioethanol: New opportunities for an ancient product Chen-Guang Liua,*, Kai Lia, Yuan Wena, Bo-Yu Genga, Qian Liua, Yen-Han Linb,* a

State Key Laboratory of Microbial Metabolism, Joint International Research Laboratory of Metabolic & Developmental Sciences of Ministry of Education, School of Life Sciences & Biotechnology, Shanghai Jiao Tong University, Shanghai, China b Department of Chemical and Biological Engineering, University of Saskatchewan, Saskatoon, SK, Canada *Corresponding authors: e-mail address: [email protected]; [email protected]

Contents 1. Overview of bioethanol 2. The first generation of bioethanol 2.1 Strains used in bioethanol production 2.2 Ethanol fermentation process 2.3 Debates on first-generation bioethanol 3. The second generation of bioethanol 3.1 Lignocellulose pretreatment 3.2 Cellulase and cellulase producing organisms 3.3 Fermentation and challenges 4. The third generation of bioethanol 4.1 Raw material 4.2 Treatment 4.3 Fermentation 4.4 Prospect of algae 5. Future perspective of bioethanol References

2 4 4 6 9 9 11 14 16 20 21 22 23 24 26 27

Abstract Ethanol as a key component of alcoholic beverage came along with human in an extremely long history. Nowadays, it has been the most successful sustainable alternative to fossil fuels, even beyond wind power and solar energy, to fuel vehicles and alleviate environmental pollution. Although almost all bioethanol produced by starch- or sugar-based biomass, also known as the first-generation bioethanol, is economically feasible nowadays, it still causes many controversial issues in food security and CO2 emission. In order to address these concerns, the second-generation bioethanol, taking lignocellulose such as forest waste and crop straw as the feedstocks, has emerged a promising future of ideal biofuel. Unfortunately, because of the challenges raised in technology and economy, the running of industrial plants for second-generation Advances in Bioenergy ISSN 2468-0125 https://doi.org/10.1016/bs.aibe.2018.12.002

#

2019 Elsevier Inc. All rights reserved.

1

ARTICLE IN PRESS 2

Chen-Guang Liu et al.

ethanol closely depends on the global price of crude oil. The third-generation bioethanol is derived from algae, which is considered as a valuable option for feedstock due to its potentially abounding output. In this chapter, we drew a sketch of threegeneration bioethanol in the regular production process and innovative research input globally. Bioethanol production, as a long story since the beginning of civilization, is destined to promise human a bright energy road.

1. Overview of bioethanol Since our ancestors happened to taste a mystical liquid with special aroma about 13,000 years ago (Liu et al., 2018), ethanol had been a well-known word in the world, which appeared in poems, novels, and songs in almost all civilizations. From beverage to sanitizer, form chemical to fuel, ethanol shifts its roles along with the forward steps of science and technology. The traces of alcohol found in stone mortars indicate the Natufian people, ancient seminomadic hunter–gatherers (13,000 years), had already produced the oldest bread and alcohol (Liu et al., 2018). It challenges the previous evidence that people consumed alcoholic beverages 9000 years ago in China. However, according to recorded history, people in China, India, the Middle East, and Europe all developed the distillation of wine and spread the technology to all over the world. Global industrialization and motorization have resulted in a steep rise for the demand for energy, which ramped up the environmental deterioration and threatened the sustainability of human society by using petroleum-based fuels. The immense consumption of petroleum-based fuels has led to massive emissions of CO2, SO2, and NOx which is the main cause of atmospheric pollution. Furthermore, petroleum reserves are very limited and will deplete in decades at such mining speed. Hence, the research for renewable energy sources is becoming a matter of widespread attention (Sadeghinezhad et al., 2014). Bioethanol has been recognized as a potential alternative to petroleum-derived transportation fuels. First, bioethanol is produced from biomass feedstocks which makes it renewable. Besides, ethanol is oxygenated leading to no particulate emissions when burning in engines. Then, it has a higher octane number, flame speed, vaporization heat, and broader flammability limit (Sathiyamoorthi and Sankaranarayanan, 2017). Although more bioethanol fuel than gasoline is required to generate a similar amount of heat due to the lower energy density, ethanol with higher octane number than gasoline leads to increased engine’s compression ratio and higher

ARTICLE IN PRESS Bioethanol: New opportunities for an ancient product

3

thermal efficiency. Higher octane number fuel can prevent the early ignition which causes cylinder knocks that may damage the internal combustion engines (ICEs). These merits benefit an ICE with a higher compression ratio, shorter burn time, whose theoretical efficiency advantages over gasoline (Khuong et al., 2017). The origin of ethanol usage happened in 1897. Nikolas Otto used it in an ICE invented by himself. However, it was in 1908, Henry Ford first expected using ethanol as an automobile fuel in Model T without practical application. Hence, ethanol was considered as fuel for vehicles in the United States in the 1930s again, and successfully used after 1970 (Shahir et al., 2014). Since the 1980s, ethanol has been blended with gasoline in any percentage to replace gasoline. In a variety of countries like the United States, Canada, and Brazil, bioethanol is of great importance to their energy system, which is used as an additive in fuels or complete transport fuel. According to EU quality standard EN 228, bioethanol can be used as a 5% blend with gasoline without engine modification. Once with engine modification, bioethanol can be blended at higher levels. For example, in several states of the United States, a little higher amount of bioethanol (10% by volume) was added to gasoline, called E10. In Brazil, bioethanol can be used pure or blended with gasoline in a mixture called gasohol (24% bioethanol, 76% gasoline). And the most popular blend for light-duty vehicles is known as E85, which contains 85% bioethanol and 15% gasoline. The U.S. Energy Information Administration has predicted that ethanol production will average 1.04 billion barrels per day in both 2018 and 2019, up from an average of 1.03 million barrels per day last year. Consumption of fuel ethanol is expected to average 950,000 barrels per day in 2018 and 2019, up from 940,000 barrels per day in 2017. Bioethanol can be produced from a large variety of potential feedstock all around the world. Up to now, the search of the suitable feedstock for bioethanol has led to the rise of the three generations. So far the first-generation ethanol is derived from edible crops (Ribeiro, 2013). Corn and sugarcane are used as feedstocks for bioethanol mainly by the United States and Brazil, while wheat and sugar beet are commonly used in Europe. However, there are some drawbacks of first-generation ethanol that utilizing food resources for fuels may threatened food supply and millions of people all over the world who are suffering from hunger. On the other hand, it can also increase food prices. Hereafter, here comes to the second-generation ethanol which is produced from lignocellulosic biomass (Aditiya et al., 2016), including forest residues, agricultural residues, and urban wastes. This sort of biomass

ARTICLE IN PRESS 4

Chen-Guang Liu et al.

extensively exists around the world with low cost and can be converted into high-value products by biorefinery. The lignocellulose is subjected to a pretreatment for easily enzymatic hydrolysis which converts lignocellulosic materials into reducing sugars and then into bioethanol by fermentation. However, the industrial scale up of second-generation ethanol experienced the main obstruction in some technological issues including the high cost of the pretreatment process and relatively low ethanol yield. Therefore, the requirement of advanced technologies and facilities is necessary to improve the conversion efficiency. There also exists a problem of feedstock collecting, in which logging is possibly needed and the act can destroy nature. Hence, there is a demanding challenge to develop bioethanol from marine plants as they have high potential to produce large amounts of biomass and it is called the third-generation ethanol ( Jambo et al., 2016). The relative mature technology of the first generation provokes a potential threat to the food supply for human beings (Bhatia et al., 2017). Therefore, the recent bioethanol researches has focused on the second- and third-generation feedstocks due to ease of availability and immense commercialization (Shastri, 2017).

2. The first generation of bioethanol The first-generation bioethanol is produced directly from starch and sugar crops, such as corn and sugarcane (Azapagic et al., 2004), which is considered as the most successfully commercialized biofuels. Besides, the feedstocks of bioethanol are also other nonfood plants like cassava, sorghum (Rendleman and Shapouri, 2007), from which ethanol generated can be called the 1.5-generation bioethanol. Moreover, sorghum is able to provide more fermentable sugars with less water required for producing ethanol, and its growth duration is shorter than sugarcane.

2.1 Strains used in bioethanol production Saccharomyces cerevisiae is commonly used as an ethanol producer thanks to its varieties of advantages such as fast sugar consumption, high ethanol yield from glucose, and robust resistance to ethanol and other inhibitors (Hasunuma et al., 2011). Zymomonas mobilis has also attracted increasing attention and been intensively studied over the past 30 years for some favored industrial characteristics over S. cerevisiae including unique Entner–Doudoroff (ED) pathway with less ATP and biomass generated (Ma et al., 2016), and thus many researchers have claimed that it can replace S. cerevisiae in bioethanol production.

ARTICLE IN PRESS Bioethanol: New opportunities for an ancient product

5

2.1.1 Saccharomyces cerevisiae For S. cerevisiae, the main metabolic pathway involved in the ethanol fermentation is glycolysis, and the generated pyruvate is further reduced to ethanol with the release of CO2 under anaerobic condition. Theoretically, the yield is 0.511 for ethanol and 0.489 for CO2 in term of a mass balance of glucose metabolized. Two ATPs produced in the glycolysis are used to drive the biosynthesis of yeast cells which involves a variety of energy-requiring bioreactions. Therefore, ethanol production is tightly coupled with yeast cell growth, so-called primary metabolite. Without the continuous consumption of ATPs by the growth of yeast cells, the glycolytic metabolism of glucose will be interrupted immediately because of the intracellular accumulation of ATP that inhibits phosphofructokinase, one of the most important regulation enzymes in the glycolysis. This basic principle contradicts the ethanol fermentation with the yeast cells immobilized by supporting materials, particularly by gel entrapments, which physically restrict the yeast cells and significantly retard their growth. In addition to ethanol and CO2, various by-products are also produced during ethanol fermentation such as glycerol and organic acids, and the production of these by-products, as well as the growth of yeast cells, inevitably redirects some glycolytic intermediates to the corresponding metabolic pathways, decreasing the ethanol yield. During the fermentation process, yeast cells are always exposed to many stresses like high temperature, high osmotic pressure, and product inhibition such as acetic acid and ethanol. In order to solve these problems, researches paid attention to improving the tolerance of S. cerevisiae through physical and chemical mutagenesis (Sridhar et al., 2002; Thammasittirong et al., 2013; Zhu et al., 2018), adaptation (Ailion and Thomas, 2003), protoplast fusion (Sakanaka et al., 1996), evolution engineering (C ¸ akar et al., 2005; Gonza´lezRamos et al., 2016), global transcription machinery engineering (Alper et al., 2006), and genome shuffling (Shi et al., 2009; Snoek et al., 2015). Besides, consolidated bioprocess enables the yeast directly convert cellulose to ethanol through simultaneously transforming the genes related to cellulases and a cellodextrin transporter (Chang et al., 2013), and other studies even improved thermal tolerance of yeast by consolidated bioprocess (Khatun et al., 2017). 2.1.2 Zymomonas mobilis Z. mobilis is an anaerobic, Gram-negative bacterium which produces ethanol from glucose via the ED pathway in conjunction with the enzymes pyruvate

ARTICLE IN PRESS 6

Chen-Guang Liu et al.

decarboxylase and alcohol dehydrogenase (Conway, 1992). This microorganism was originally discovered in fermenting sugar-rich plant saps, e.g., in the traditional pulque drink of Mexico, palm wines of tropical African, or ripening honey (Swings and Ley, 1977). Compared with the EMP pathway of S. cerevisiae, which involves the cleavage of fructose-1,6-bisphosphate by fructose bisphosphate aldolase to yield one molecule of glyceraldehydes3-phosphate and dihydroxyacetone phosphate respectively, the ED pathway forms glyceraldehyde-3-phosphate and pyruvate by the cleavage of 2-keto-3-deoxy-6-phosphogluconate by 2-keto-3-deoxy-gluconate aldolase, yielding only one molecule ATP per glucose molecule. As a consequence, Z. mobilis produces less biomass than S. cerevisiae, and more carbon is funneled to the ethanol fermentation. It was reported that the ethanol yield of Z. mobilis could be as high as 97% of the theoretical yield of ethanol to glucose, while only 90%–93% can be achieved for S. cerevisiae. Also, as a consequence of the low ATP yield, Z. mobilis maintains a higher glucose metabolic flux, and correspondingly, this guarantees its higher ethanol productivity, normally 3–5-folds higher than that of S. cerevisiae. However, Z. mobilis is still not suitable for the industrial ethanol production for the following reason: extremely narrow substrate spectrum only including glucose, fructose, and sucrose. In fact, the ethanol fermentation industry cannot use pure glucose as a raw material as many researchers did in their laboratory studies. Besides, Z. mobilis faces up with many stresses and presents weak tolerance. Taking into account these drawbacks, some investigations involving the ethanol fermentation with Z. mobilis seem misdirected, in spite of certainly scientific interest. Some researchers who concluded this species would replace S. cerevisiae are likely too optimistic in their assessments. To make the strain suitable for industrial application, the studies aiming to improve the tolerance of Z. mobilis includes identifying potential stress tolerant genes (Alvin et al., 2017; Charoensuk et al., 2017), digging out the tolerance mechanism (Ma et al., 2016), and constructing the robust strains by gene manipulation (Shui et al., 2015; Tan et al., 2016).

2.2 Ethanol fermentation process There are various traditional ethanol fermentation techniques like batch fermentation, semibatch fermentation, and continuous ethanol fermentation. Based on these traditional technologies, many innovative technologies in ethanol fermentation have been developed. Very high gravity (VHG)

ARTICLE IN PRESS Bioethanol: New opportunities for an ancient product

7

fermentation and cell immobilization by self-flocculation technologies were mentioned in this chapter, and their history, application, as well as drawbacks, were also discussed. 2.2.1 VHG fermentation High ethanol concentration has been continuously pursued in the industry, because significant energy savings can be achieved for the downstream distillation and waste distillage treatment. HG ethanol fermentation was proposed in the 1980s, and successfully commercialized thereafter, making the final ethanol concentration be increased dramatically from the previous level of 7%–8% (v/v) to the current value of 10%–12% (v/v). VHG fermentation, using the medium containing sugar in excess of 250 g/L to achieve over 15% (v/v) ethanol, makes it possible that high ethanol titer and significant energy savings can be achieved at the same time. High ethanol concentration is the major stress factor during VHG fermentation, but fortunately, many strains of S. cerevisiae showed great advantages to tolerate very high ethanol concentration even without genetic manipulation (Casey and Ingledew, 1986). Among many ethanol fermentation technologies, the VHG fermentation is very promising for its industrial application. On one hand, the energy cost is the second largest part in ethanol production only after the cost of the raw material consumption. On the other hand, the availability of the VHG mash in mass quantities is now economically feasible, because the low cost and highly efficient enzymes such as α-amylases, glucoamylases, and proteases are now available. Meanwhile, the process of the biorefinery requires the separation of most raw material solid residues in the pretreatment, especially for those ethanol fermentation plants with large processing capacities, which further guarantees the reliable supply of the VHG mash. The secret of VHG fermentation is to reduce osmotic pressure, keeping substrate in dextrin form, but in equilibrium with smaller amounts of glucose liberated by added glucoamylase enzyme. In this way, fermentation is prolonged and alcohol is produced at the fastest rate. In addition, the highest viable cell number of yeast (the catalyst) is allowed to be achieved, and bacterial and wild yeast are discouraged. The key to fast growth and high cell number is nutrition. Elaborated research has shown that usable nitrogenous compounds are the most important nutrients now at suboptimal levels in almost all mashes and, therefore, required as fermentation additives (yeast foods). Mashes made from all grains and especially from sugars and semipurified starches are devoid of usable nitrogen as well as other nutrients such

ARTICLE IN PRESS 8

Chen-Guang Liu et al.

as usable phosphorous and sulfur, and minerals and vitamins that yeast require for enzyme stability and activity. For this reason, yeast growth is poor and the amount of cellular biomass is insufficient to finish ferment even moderate amounts of carbohydrate in such prepared “mashes.” In order to further improve the economic and industrial feasibility of VHG fermentation, other issues have to be addressed. On one hand, high substrates with high osmotic pressure significantly inhibit the viability of yeast cells. Ingledew and Lin (2011) believed keeping starch-based substrates in dextrin form, but in equilibrium with smaller amounts of glucose liberated by addition of glucoamylase can reduce osmotic pressure (Ingledew and Lin, 2011). On the other hand, mashes made from starch are devoid of usable nitrogen and other nutrients that yeast requires for fast growth. Expensive supplements like amino acids, vitamins, sterols, and unsaturated fatty acids, commonly used in laboratory studies (Lebaka et al., 2014; Li et al., 2017), are not suitable for industrial application. Therefore, engineering design can help optimize the physiological environment for the yeast cells to grow under a variety of stresses during ethanol fermentation (Bai et al., 2004). 2.2.2 Cell immobilization by self-flocculation There exist many disadvantages of using free cells for ethanol fermentation such as substrate and product inhibition (Pa´tkova´ et al., 2000), extra time needed for inoculum preparation, and cleaning the reactor between batches. To overcome these limitations, immobilization was introduced in fermentation processes by using several low-cost support materials such as alginate, carrageen, and other new materials (Ariyajaroenwong et al., 2016; Chang et al., 2016; Mishra et al., 2016). Actually, the spontaneous flocculation of yeast cells is a kind of selfimmobilization technology, which is superior to other strategies where supporting materials are employed. First, no supporting material is consumed, which not only makes the process more simple and economically competitive compared with the yeast cell immobilization by supporting materials but also completely eliminates the potential contamination to the quality of the coproduct animal feed by the supporting materials. Second, the growth of yeast cells is not significantly affected, guaranteeing the ethanol fermentation to be carried out effectively. Third, the yeast flocs can be purged from the fermentors under controlled conditions, maintaining the biomass concentration inside the fermentors at designated levels. And finally, the yeast flocs purged from the fermentors can be recovered by sedimentation rather than centrifugation which is widely used in the recovery

ARTICLE IN PRESS Bioethanol: New opportunities for an ancient product

9

of free yeast cells, saving the capital investment for centrifuges as well as the energy consumption for centrifuge operation (Bai et al., 2008). The research on self-flocculating yeast in ethanol fermentation was relatively rare in the past decades since the 1990s. Bai et al. reported the fundamental progress like online monitoring and characterization of the self-flocculating yeast flocs (Bai et al., 2008; Hu et al., 2005; Xu et al., 2005a), the intrinsic and observed kinetics, and optimization of process design for ethanol fermentation (Xu et al., 2005b). Besides, they also reported tolerance improvement caused by self-flocculation (Xu et al., 2012), flocculation controlled by genetic manipulation (Xu et al., 2018) and redox potential (Liu et al., 2015a). By using self-flocculating yeast, the commercialization of ethanol fermentation was achieved in 2005 with an annual production capacity of 200,000 tons at BBCA, a famous fuel ethanol producer in China (Bai et al., 2008).

2.3 Debates on first-generation bioethanol In fact, we are supposed to take it into consideration that producing firstgeneration bioethanol using starch or sugarcane may not be feasible in many countries. For instance, low-cost substantial sugarcane-based ethanol production may not be replicated in some African countries and Latin America due to higher production cost and lower yield as compared to Brazil. Similarly, corn-based ethanol production may not be feasible on large scale outside the United States because of costs and issues related to sustainability. Apart from bioethanol, the other commonly used first-generation biofuels like biodiesel and biogas are derived mainly from corn, sugarcane, soybean, vegetable oil, and palm oil, which all leads to the issue of food vs fuel. The transition of land from crop cultivation to biofuel feedstock has also been attracting broad concern to the food crisis (Ritslaid et al., 2010). To solve these problems, investigations on developing the second-generation bioethanol have gained increasing attention and achievements in this field have been realized.

3. The second generation of bioethanol Since the large continuous increase of ethanol production with current grain crops based technology may not be practical as the competition with food, the second-generation ethanol, also known as cellulose ethanol, using agricultural and forestry wastes as the substrate is becoming more

ARTICLE IN PRESS 10

Chen-Guang Liu et al.

promising. Numerous researches have been reported about the conversion of lignocellulosic materials to bioethanol in the past 2 decades. Lignocellulose is the most abundant and inexpensive renewable resource in the world, which is mainly composed of cellulose, hemicellulose, and lignin, with a small quantity of extractive, ash, and so on. The most worthwhile cellulose and hemicellulose can be used by microorganisms to produce bioethanol and other high value-added products, and lignin is a beneficial source for production of the phenol-derived chemicals. As the main component of the plant cell wall, cellulose is the most abundant polysaccharide in nature. A long branched chain is formed by β-1,4linking glycosidic bonds from D-glucose monomer. The properties and functions of cellulose are mainly determined by the aggregation state of cellulose macromolecules, crystalline zone, and an amorphous region, which affect the enzymatic hydrolysis process. There are many ways to hydrolyze cellulose, including acid hydrolysis, enzymatic hydrolysis, microbial degradation, mechanical degradation, etc. Hemicellulose, also called noncellulosic carbohydrates, is a general term for all carbohydrate polymers except cellulose and pectin in plant cell walls. Unlike cellulose, hemicellulose is usually highly branched and consists of a series of subunits, including sugar and sugar acid. These complex structures distinguish the hemicellulose from other polysaccharides in the plant cell wall, and empower it water solubility, water absorbability, and crosslinking with other components such as cellulose and lignin. It has been reported that hemicelluloses, lignin, cellulose, and protein can be tightly integrated through the chemical connection. Hemicellulose and lignin are connected by ester bonds or ether bonds. The complex structure of hemicellulose determines that hemicellulose hydrolysis requires multiple hemicellulases to cooperate with each other. Xylose and other monosaccharides produced by enzymatic hydrolysis or acid hydrolysis from hemicellulose can be utilized by microorganisms to produce various chemical products. In addition to cellulose and hemicelluloses, lignin is another large class of macromolecules in the plant cell wall. Lignin exists between cell wall microfibrils and intercellular layers, thus enhances the strength and toughness of plant stem. Although lignin has been known for more than 100 years, its true chemical structure is still a mystery. At present, three basic units are recognized as lignin at home and abroad, namely: guaiacyl propane, syringing propane, and hydroxyphenyl propane. High chemical reactivity of lignin is attributed to the existence of various connections and complex groups,

ARTICLE IN PRESS Bioethanol: New opportunities for an ancient product

11

which cause lignin decomposed conveniently by strong oxidizers such as sodium hypochlorite and hydrogen peroxide, or by strong alkali at high temperature. Because of the structural features of lignocellulose, complex composition, and crystalline cellulose, the recalcitrance of lignocellulosic biomass prohibits the release of sugar for organism fermentation. The process of cellulosic ethanol with unique characteristics consists of biomass pretreatment, enzymatic hydrolysis, ethanol fermentation, and distillation.

3.1 Lignocellulose pretreatment Pretreatment is a crucial step for lignocellulosic biomass conversion to bioethanol. The main effect of pretreatment is to disintegrate the fiber structure into individual components consisting of cellulose, hemicellulose, and lignin. 3.1.1 Physical pretreatment The earliest lignocellulose pretreatment method was mechanical extrusion with heating together, which led to the outcome of gaseous products and char from the pretreated lignocellulosic biomass (Shafizadeh and Bradbury, 1979), Evon et al. studied mechanical, thermomechanical properties of oleaginous flax, and water resistance after mechanical extraction process (Evon et al., 2018). However, extrusion pretreatment required a significant amount of energy input, making it a cost-intensive method and difficult to scale up for the industrial purpose (Zhu and Pan, 2010). Mechanical grinding is another pretreatment method used for lignocellulose destruction. It mostly includes chipping, grinding, and/or milling techniques. Chipping cuts the biomass to 10–30 mm while grinding and milling decrease particle size to 0.2 mm. However, Chang et al. found that rate and yield of cellulose hydrolysis did not increase when biomass particle was below 0.4 mm (Chang et al., 1997). Lee et al. studied mechanical pretreatment of cellulose fluff pulp using a shear cutting mill with three cycles and energy input for the cutting process was about 894 kWh/Mg which was relatively low compared with other methods (Lee and Mani, 2017). Microwave irradiation, different from the conventional physical lignocellulose pretreatment methods, owns many advantages such as (1) easy operation, (2) low energy requirement, (3) high heating capacity in short duration of time, (4) minimum generation of inhibitors, and (5) degrading structural organization of cellulose fraction (Kumar and Sharma, 2017).

ARTICLE IN PRESS 12

Chen-Guang Liu et al.

Huang et al. studied effects of particle size, pretreatment conditions, and catalysis on microwave pyrolysis of corn stover (Huang et al., 2018). Zhao et al. optimized parameters of substrate concentration and pretreatment time using response surface methodology of microwave pretreatment of Hyacinth (Zhao et al., 2017). Ultrasound is a novel method for the pretreatment of lignocellulosic biomass and ultrasonic pretreatment combines physics and chemical effect for processing biomass. He et al. found ultrasound pretreatment is a good way to modify the physiochemical structure of wood and could increase the crystallinity of samples (He et al., 2017). The effect of ultrasound combined with other pretreatment methods will be better. Wu et al. enhanced the enzymatic hydrolysis of rice straw with ultrasound-assisted alkaline pretreatment (Wu et al., 2017). Wang et al. found ultrasound-assisted [TBA][OH] pretreatment is more efficient and environment-friendly compared with alkaline pretreatment for enhancing enzymatic saccharification (Wang et al., 2018). 3.1.2 Chemical pretreatment Dilute acid pretreatment is the most common and efficient method for lignocellulosic feedstocks to dissolve the hemicellulose of biomass (Sen et al., 2016). The usually used conditions are low acid concentration with high temperature or high acid concentration with low temperature (Sen et al., 2016). However, the generation of inhibitors such as carboxylic acid, phenolic acids, and aldehydes are harmful to the microorganism in fermentation. In addition, acids are highly corrosive on the equipment (Chen et al., 2017). Recently, organic acids showed the merits because of high-efficiency cellulose hydrolysis with less amount of inhibitors formation compared with sulfuric acid (Amnuaycheewa et al., 2017). Alkali pretreatment often performs at ambient temperature and pressure, and hydroxyl derivatives of sodium, potassium, calcium, and ammonium salts are regularly used. Owing to ideal solubility of lignin, alkali pretreated lignocellulosic biomass is more conveniently hydrolyzed by enzymes (Li et al., 2016a). Hosgun et al. removed 41.18% of lignin of hazelnut shells and enhanced the ethanol yield after low-temperature sodium hydroxide (NaOH) pretreatment (Hosgun et al., 2017). However, its drawback is a difficult downstream process such as wastewater treatment. Recently, many studies have shown that organosolv pretreatment could dissolute lignin and degrade hemicellulose. Unlike other pretreatment methods, organic solvent pretreatment with or without an acid/base catalyst is capable to dissolve lignin and degrade hemicellulose. Salapa et al. compared five solvents (ethanol, methanol, butanol, acetone, and diethylene

ARTICLE IN PRESS Bioethanol: New opportunities for an ancient product

13

glycol) in the pretreatment of wheat straw. Ethanol pretreatment at 180°C for 40 min resulted in a maximum cellulose conversion (Salapa et al., 2017). An ionic liquid (IL) is salt in liquid whose melting point is below 100°C. ILs became a fashionable lignocellulosic pretreatment method in the last decade due to good potential for lignin extraction and carbohydrate dissolution. As a kind of new solvent, IL plays an attractive role in lignocellulose fractionation through effectively dissolving and converting the biomass into various types of products (Yoo et al., 2017). Liu et al. increased the glucose yield to 70.35% with the addition of 1-ethyl-3-methylimidazolium acetate ([Emim]Ac) compared with the untreated rice straw (Liu et al., 2015b). Raj et al. characterized plant cell wall after IL pretreatment by using five ILs to deconstruct plant cell wall, and they found [C2mim][OAc] is the most effective in all ILs to release high sugars (Raj et al., 2017). 3.1.3 Physicochemical pretreatment Steam explosion is a popular physicochemical method for pretreatment of lignocellulosic biomass. In this process, biomass is contained in saturated steam with high pressure (0.7–4.8 MPa) and under elevated temperatures (160–260°C) conditions, and then the pressure is suddenly released in a few seconds to minutes. Owing to a combination of mechanical forces and chemical effects, steam explosion leads to partial hydrolysis and particle size reduction which increase the accessibility of enzymes to cellulose. This method is a combination of mechanical forces (particle size reduction) and chemical effects (release and hydrolysis of hemicellulose), however, the by-products produced during steam explosion pretreatment are massive. Liu et al. (2014) reported an innovative instant catapult steam explosion pretreatment method with very short depressurization time (<0.01 s), resulting in higher glucose recovery than normal steam explosion (Liu et al., 2014). Ammonia fiber expansion (AFEX) is one of the most promising methods for lignocellulosic biomass pretreatment. AFEX is similar to the steam explosion but ammonia is used instead of water (Eggeman and Elander, 2005). AFEX treatment rarely produces inhibitors, which is highly desirable for downstream processing. Meantime, detoxification, washing, and other steps are saved after pretreatment. Carlos et al. found that AFEX-pretreated agave residues can be effectively hydrolyzed at a high solid loading. Their results show that AFEX technology has considerable potential in the biorefinery process (Flores-G€ omez et al., 2018). Kamm et al. modified the AFEX process with aqueous ammonia (25%, w/v) instead of liquid ammonia which could improve enzymatic hydrolysis (Kamm et al., 2017).

ARTICLE IN PRESS 14

Chen-Guang Liu et al.

3.1.4 Biological pretreatment Different from chemical and physical pretreatment methods, biological pretreatment is carried out by microorganisms such as brown, white, and softrot fungi which synthesize enzymes to degrade lignin and hemicellulose, and thus this process is more environment-friendly and lowers energy input. Kumar and Wyman found that white-rot fungi degraded lignin by secreting peroxidases and laccases (Kumar and Wyman, 2009). However, hydrolysis rate of biomass is severely slow hampering industrial application. Meantime, the insufficient enzymes exposure and low accessibility on the limited surface area are the main hurdles in complete microbial degradation. Therefore, finding suitable microbial consortia and undertaking genetic modification to produce more enzymes should be feasible approaches to make the biological pretreatment more efficient.

3.2 Cellulase and cellulase producing organisms Hydrolysis is the next step after pretreatment and now is always carried out by enzymes. Back to one century ago, concentrated acid was used to degrade lignocellulose. Unfortunately, this process consequently generates inhibitory by-products that interfere downstream microbial fermentation. Enzyme hydrolysis of lignocellulose by cellulases owns many advantages compared with chemical treatment, such as low yield of by-products, mild reaction conditions, noncorrosion, and low energy demand. Lignocellulose has a very stable internal structure with strong resistance. Microorganism able to use the carbohydrates in lignocellulosic biomass has to secrete a series of enzymes to degrade the lignocellulose with compact structure into the available monosaccharide. General cellulase is actually a mixture of several enzymes called cellulase system made up with a variety of protein components. Cellulases hydrolyze cellulose β-1,4-glycosides and break long-chain cellulose into cellobiose and glucose for next process, fermentation by the microorganism. 3.2.1 Mechanism of cellulase action Cellulase, a mixture of several enzymes, can be categorized into three major groups: endoglucanases (EGs), cello-biohydrolases (CBHs), and βglucosidases (BGs). EG acts randomly on the cellulose chain to produce cello-oligosaccharides. CBH splits off cellobiose on exposed ends of cellulose chain. BG hydrolyzes cellobiose and soluble oligosaccharides to glucose. Besides, the commercial cellulase mixtures may also contain many other ingredients such as reducing agents and stabilizers.

ARTICLE IN PRESS Bioethanol: New opportunities for an ancient product

15

3.2.2 Cellulase producing organisms Trichoderma reesei is a widely used cellulase producing strain in the world. This strain was discovered during World War II due to the rapid degradation of tents in American military camp. In the next 80 years of study, T. reesei has become an industrial cellulase production strain by continuous mutagenesis breeding, and it has been reported that its extracellular protein production can reach 100 g/L (Cherry and Fidantsef, 2003). Genomic analysis showed that there were 200 glycosidase hydrolase genes in T. reesei, but only 36 cellulose-binding proteins, 11 exoglucanases and endoglucanases are the most important cellulase (Martinez et al., 2008). Neurospora crassa is another cellulase producing strain closely related to T. reesei, usually separated from branches, soil, straw, or corrupt fruit. As a model microorganism for cellulase production, N. crassa has been studied near 90 years. In 2003, its genome was published as the first sequenced fungus to produce cellulase with the unique advantage to be applied by gene modification (Cai et al., 2015). Cai et al. revealed the role of the cellooligosaccharide transporter CDT-2 mutant in the metabolism of solid cellulose and hemicellulose, and found that XLR-1 was the main regulation transcription factor of CDT-2 (Cai et al., 2015). In nature, bacteria produce less cellulase than fungi, but the cellulase from bacteria sometimes is better, because bacteria not only grow faster than fungi but also allow higher yield of recombinant enzymes. Besides, bacteria can survive harsh environment during bioconversion processes. There is a wide range of microorganisms with cellulose degradation ability in nature. The currently known cellulose-degrading microorganisms include bacteria, actinomycetes, fungi, and paleobacteria (Zverlov et al., 2015). Clostridium plays an important role in cellulose degradation, such as Clostridium thermocellum, which can directly decompose cellulose and convert it into ethanol. Recently, National Renewable Energy Laboratory (NREL) of the United States claimed that the cellulose degradation by CelA cellulase produced by CelA Caldicellulosiruptor bescii is twofold more efficient than that by Cel7A cellulase produced by T. reesei, which showed the highest efficiency of cellulose degradation until now (Brunecky et al., 2013). 3.2.3 Cellulase production At present, the commonly used ways to produce cellulase are solid-state fermentation and submerged fermentation. The condition of solid-state fermentation is close to the growth habit of the natural state, leading to higher cellulase yield and lower energy consumption. However, it is hard to be

ARTICLE IN PRESS 16

Chen-Guang Liu et al.

operated in large scale due to contamination and problem of mass transfer (Singhania et al., 2009). Chen et al. developed a new type of gas doubledynamic solid-state fermentation which can avoid those shortcomings and improve the yield of cellulase (Chen et al., 2014). The process of submerged fermentation is easy to control and difficult to be contaminated compared with solid-state fermentation. At the same time, the production efficiency is higher while the labor intensity is smaller. It is more suitable for large-scale industrial production. Li et al. (2017) performed cellulase fermentation by T. reesei Rut C30 in 7 L fermentor at low glucose condition through batch feeding of a mixture of glucose disaccharides, finally, 90.3 FPU/mL cellulase was achieved at the end of fermentation (Li et al., 2016b).

3.3 Fermentation and challenges At present, cellulose ethanol faces two problems. Firstly, inhibitors produced in the pretreatment significantly compromise cell growth and ethanol production (Kumar and Sharma, 2017). Secondly, xylose released from hemicellulose cannot be utilized properly (Chen et al., 2017). Thus S. cerevisiae should be qualified as high-stress tolerance and pentose utilization. 3.3.1 Inhibitor tolerance A lot of chemicals inhibiting the fermenting microorganism are formed during lignocellulosic pretreatment. These inhibitors are usually divided into three major groups: weak acids, furaldehydes, and phenolic compounds. Weak acids, related to uncoupling and accumulation of intracellular anions, cause cytoplasmic acidification. In order to pump H+ out of the cell, a large amount of ATPs are consumed, lowering cell activity. Current studies have focused on reducing the adsorption of acetic acid, enhancing the efflux of hydrogen ions and acetic acid ions, as well as activating the intracellular metabolism of acetic acid (Ullah et al., 2012). By overexpressing the gene AZR1, Tenreiro et al. enhanced the ability of cells to tolerate acetic acid by hindering the absorption of acetic acid and raising intracellular pH (Tenreiro et al., 2000). Overexpression of the gene ACS2 in S. cerevisiae improved the synthesis of acetyl coenzyme A with acetic acid as its precursor, thus enhanced the metabolism of acetic acid and growth rate of cells (Ding et al., 2015). Furfural induces the accumulation of reactive oxygen species, which damage cell protein, nucleic acid, fatty acid, and so on. By overexpression of gene ZWF1 (glucose-6-phosphate dehydrogenase) with NADPH generation, S. cerevisiae improved the growth in furfural because of removal

ARTICLE IN PRESS Bioethanol: New opportunities for an ancient product

17

of furfural by the increased enzyme activity of reductase which requires NADPH as a cofactor (Almeida et al., 2007). Phenolic substances may act on the structure of the membrane, damage the integrity of the cell membrane and disturb the electrochemical gradient of the mitochondrial membrane, thus breaking the selective permeability of the cell membrane (Heipieper et al., 1994). 3.3.2 Ethanol tolerance Yeast cells have to handle the increasing ethanol toxicity during ethanol fermentation. Ethanol destructs cell membrane structure and enhances membrane fluidity, thus hinders the adsorption of sugar, amino acids, and other nutrients. Besides, ethanol blocks metabolism of biological macromolecules and interferes enzymatic activity. Yoshikawa et al. (2009) identified the genetic phenotype of S. cerevisiae under ethanol pressure and found that 359 genes were associated with ethanol tolerance (Yoshikawa et al., 2009). Cao et al. expressed trehalose synthase gene TPS1 with the vector pMIRSC11 in S. cerevisiae and consequently obtained an excellent ethanol tolerance strain (Cao et al., 2014). Expression of ATP-binding efflux protein in S. cerevisiae also improves ethanol tolerance (Yang et al., 2013). However, the ethanol tolerance of S. cerevisiae is controlled by polygenes, and that is why only a single gene modification presents some limitations. Alper et al. found that the mutation of three amino acids in the SPT15 gene of S. cerevisiae RNA polymerase II transcription complex TFIID could greatly improve the tolerance of S. cerevisiae to ethanol, and for the first time proposed a global transcription engineering (Liu et al., 2011). 3.3.3 Heat tolerance The optimal fermentation temperature of traditional brewing yeast is 28–33°C, generally no more than 36°C, which restricts the ethanol industrial production due to dramatically raising of the cost for cooling, especially in summer. Besides, if yeast can efficiently work at more than 36°C high temperature, then the fermentation period would be shortened with the increase of fermentation rate (Hoshida and Akada, 2017). Yeast contains a very complex heat-resistant mechanism, which is closely related to changes in multiple physiological processes within the cell, maintaining the process of cell internal structural stability, including induction of heat shock protein (Estruch, 2000). Lee et al. found that heat resistance in a biological process tightly connect to heat shock protein synthesis and heat shock response (Lee and Key, 1994). Accumulation of heat shock

ARTICLE IN PRESS 18

Chen-Guang Liu et al.

protein benefits the improvement of cell heat tolerance. Lindquist et al. found that a large amount of HSP104 expressed in the cells showed a positive effect on the heat resistance of yeast (Lindquist and Kim, 1996). In addition to heat shock protein, trehalose can also improve the thermal stability of yeast. Trehalose can protect protein through a combination of hydrophilic groups that bind to the surface of a protein to inhibit the exposure of hydrophobic groups to achieve protein stability (Babazadeh et al., 2017). Simon et al. had clearly stated and proved that trehalose has a high-temperature resistance because of its ability to assist molecular chaperones Hspl04 to refold (Simola et al., 2000). A large number of genes related to the heat tolerance of S. cerevisiae were also found. Hiroyuki et al. got a recombinant yeast through the expression of multiple ubiquitin-binding enzymes with ubiquitin ligase Rsp5 which had a better tolerance than wild-type yeast (Hiraishi et al., 2006). Because yeast has a complex genetic background, it is difficult to control the function of a single or a few genes to obtain the desired effect. It has been reported that many genes can regulate yeast heat resistance. 3.3.4 Pentose utilization Lignocellulose can be degraded into hexose (glucose) and pentose such as xylose and arabinose. Microorganism traditionally used in alcoholic fermentation such as yeast can easily utilize glucose but not xylose. Making full use of xylose can increase 25% ethanol production on the basis of the original carbohydrate. Therefore, breeding cofermenting pentose and hexose strains to produce ethanol are meaningful for cellulosic ethanol industrialization (Katahira et al., 2008). Owing to lack of an enzyme to convert xylose into xylulose in S. cerevisiae, yeast cannot uptake xylose. There are two ways to convert xylose into xylulose: in fungi, xylose reductase and xylitol dehydrogenase catalyze this process. While in bacteria, xylose isomerase can realize the conversion in one step. The metabolic pathway of ethanol fermentation using xylose is shown in Fig. 1. A pentose is first reduced to xylitol by a NADH- or NADPH-dependent xylose reductase. Then xylitol is oxidized to xylulose by NAD-dependent xylitol dehydrogenase. Thereafter, xylulose-5-phosphate is further converted into glucose-6-phosphate and glycerol by transaldolase and transketolase thus enter the pentose phosphate pathway (PPP), and finally, pyruvate is reduced to ethanol (Chu and Hung, 2007).

ARTICLE IN PRESS Bioethanol: New opportunities for an ancient product

19

Fig. 1 Metabolic pathway of ethanol fermentation with xylose.

According to the idea of metabolic engineering, S. cerevisiae can be endowed with the ability to utilize xylose or L-arabinose by introducing the upstream pathway of pentose metabolism, but the efficiency is often low. Sugar transport is another important factor. The wild-type strain can use its own hexose transporter to absorb xylose nonspecifically (Wieczorke et al., 1999). There are 18 endogenous hexose transporters in S. cerevisiae, including 17 of the Hxt family (Hxt1p to Hxt17p) and 1 Gal2p protein. However, the affinity of these transporters to xylose is much lower than that to glucose, and they are strongly inhibited by glucose, which limits the utilization of xylose by S. cerevisiae mutants (Shin et al., 2015). So, more and more researches about the heterologous expression of high xylose affinity transporter in S. cerevisiae have been reported. The expression of Candida intermedia Gxf1p and Pichia stipitis Sut1p in wild S. cerevisiae significantly enhanced the growth and metabolic ability of the strain when xylose was used as a substrate (Leandro et al., 2006). After heterologous expression of the transporter genes, At5g59250 and At5g17010 from Arabidopsis thaliana, the xylose utilization rate of the strain in mixed sugar fermentation was 2.5 times higher than that of the wild type (Hector et al., 2008).

ARTICLE IN PRESS 20

Chen-Guang Liu et al.

4. The third generation of bioethanol Many countries such as America, Brazil, and China have their own industry of first-generation bioethanol using glucose contained in starch and sugar crops. However, the main disadvantage of first-generation bioethanol is the threat of food supply and safety for human around the world. Anyhow, hunger is still the primary problem in some areas. The secondgeneration bioethanol is still facing economic and technical bottlenecks. Therefore, marine plants are considered for bioethanol production as their potential on producing huge amounts of biomass. In the context of the previously mentioned challenges, marine algae (including macroalgae and microalgae) are an attractive renewable source for bioethanol production with many advantages over biomass from food or cellulosic materials. Algae include a wide variety of photosynthetic organisms living in diverse environments and present in all existing ecosystems on the Earth. Under normal conditions, autotrophic algae absorb sunlight and fix inorganic carbon from the atmosphere for assimilation in the form of carbohydrates that can be applied for bioethanol production. The marine algae have many advantages for renewable energy applications ( Jing et al., 2011; Ritslaid et al., 2010). First of all, marine algae with relatively higher photon conversion efficiency compared to other plants can rapidly synthesize biomass by absorbing numerous resources in nature such as sunlight, carbon dioxide, and inorganic nutrients. Production yields of algae per unit area thus are significantly higher than other biomass. Also, for a higher rate of carbon dioxide fixation of marine algae than terrestrial plants, they have shown great potential for carbon dioxide remediation (Teixeira et al., 2016). Second, marine algae are lack of hemicellulose and lignin, which are predominant components for physical support in most terrestrial plants, and thus the degradation of algae becomes relatively easier as compared to lignocellulosic biomass. Finally, the cultivation of marine algae needs rare land and it adapts a variety of aquatic environments including fresh water, salt water, or municipal wastewater. Growth in salt water or wastewater is the key feature for sustainable bioethanol production to avoid competition with food crops that require much more fresh water and cultivable land. Algae is a promising feedstock for bioethanol production thanks to the merits of high carbohydrate contents, easy cultivation in a wide variety of water environment, relatively low land usage and high carbon dioxide absorption ( Jambo et al., 2016). The maximum theoretical productivity of algal is calculated at

ARTICLE IN PRESS Bioethanol: New opportunities for an ancient product

21

365 ton dry biomass per hectare per year (Schenk et al., 2008). Therefore, marine plants or algae have been considered as the third-generation feedstock for bioethanol production (Teixeira et al., 2016).

4.1 Raw material Dinoflagellates, green algae (Chlorophyceae), golden algae (Chryosophyceae), and diatoms (Bacillariophyceae) are several main types of microalgae with high carbohydrate and lipid contents, which are influenced by light, temperature, nutrient, pH, O2 and CO2, salinity, and toxic chemicals. Through an acid or enzymatic hydrolysis, microalgae can be degraded to sugar and then fermented to bioethanol (Sirajunnisa and Surendhiran, 2016). It should be highlighted that the high photosynthetic efficiency of microalgae is able to relief global warming, even used in the area of food, fertilizer, and cosmetics (Lee and Lee, 2016). Macroalgae, usually known as seaweeds, include brown (Phaeophyceae) and red (Rhodophyceae). Among them, red algal with the high carbohydrates content is suitable for bioethanol production. Recently, plenty of studies have focused on seaweeds especially on the potential of its composition. A large ratio content of carbohydrates in seaweeds is represented and subsequently, the conversion of this composition into bioethanol is the key point during the production phase. On the other side, its ability to store sufficient carbon sources is also important in producing bioethanol. What is more attractive is that algae can be cultivated in wastewater, so wastewater treatment and ethanol production can be implemented simultaneously. Rapid urbanization results in considerable depletion of fresh water resources. Hence, for conserving the fresh water, wastewater treatment has been widely performed. With plenty of nutrients, wastewater is capable of supporting microbes. It is noticeable that municipal wastewater is more suitable for culturing microalgae (Sunwoo et al., 2018). As the daily generation of wastewater is enormous, it is highly economical to utilize the domestic wastewater for microalgae cultivation. Suspension and immobilized microalgae are the common forms for municipal wastewater treatment. Moreover, a system with microalgae and bacterial can increase the efficiency of wastewater treatment. Compared to domestic wastewater, industrial wastewater treatment by microalgae faces more challenges on the reduction of organic compounds and heavy metals. Cultivation in industrial wastewater is a part of new remediation technologies. With less quantity of phosphorous and ammonia and

ARTICLE IN PRESS 22

Chen-Guang Liu et al.

adequate amount of heavy metals, industrial wastewater is able to affect the microalgae growth rate and biomass accumulation badly (Tan and Lee, 2015). However, this negative effect can be relieved by growth simulators. Besides, subjecting the industrial wastewater to dilution with organic compounds can also overcome the limitations. Besides, algae are able to fix CO2 for the formation of biomass. An increase in greenhouse gases such as carbon dioxide (CO2), sulfur dioxide (SO2), and nitrogen oxide (NOx) occurred due to the huge demand for energy and the rapid industrial expansion. Among these greenhouse gases, carbon dioxide is the primary gas released by the industrial production. CO2 fixation technology is a powerful tool for mitigating the CO2 emission by capturing and converting it into a usable form. Nowadays, it is found that CO2 emitted from the industrial plant is possibly absorbed as a carbon source for the microalgal growth during photoautotrophic cultivation. Hence, for reliving greenhouse effect and CO2 fixation, microalgae play a crucial role as an environmental friendly CO2 mitigation strategy. Furthermore, the microalgal biomass is a promising source for various products such as biodiesel, bioethanol, pharmaceutical, and nutritional supplements (Teixeira et al., 2016).

4.2 Treatment Algae treatment is necessary to improve the usage efficiency and bioethanol yield before fermentation. In general, the first step drying is beneficial to preserve the crude extract and prevent the algae from gelling. The next step extraction is crucial to obtain a component of algae by depolymerizing cell walls. The final step hydrolysis of the polysaccharide is to get monomer molecules for fermentation commonly through the chemical and enzymatic approaches (Singh et al., 2018). Sulfuric acid (H2SO4) is a powerful chemical to release simple sugars from the polysaccharides component by breaking the long chains of polysaccharides (Sunwoo et al., 2018). It was found that polysaccharides of the three main kinds of macroalgae (brown, red, and green) can be easily hydrolyzed to monosaccharides by treatment of H2SO4 at optimal temperature. The role of acid in hydrolysis can be explained by its potential in breaking the bonds which help connect the long chains between polysaccharides ( Jambo et al., 2016). In the initial step, hydrogen bonds destruction happened for rupturing chains and turning feedstock into a completely amorphous state. The polysaccharide is extremely susceptible to hydrolysis

ARTICLE IN PRESS Bioethanol: New opportunities for an ancient product

23

at this point. Then, the acid will catalyze to cleave polysaccharide by hydrolyzing the glycosidic bonds. In the end, addition or dilution with water at a bit moderate temperature will provide complete and rapid hydrolysis. According to the mechanism of acid treatment, acid concentration, incubation time, and temperature should also be optimized to obtain a more efficient glucose release (Harchi et al., 2018). Enzymatic hydrolysis has less impact on the environment, resulting in a more than 80% sugar conversion (Tan and Lee, 2015). Cellulases are the most employed enzymes for degrading the polysaccharides into glucose, and it can be categorized into three main classes, which are endo-glucanases, exo-glucanases, and β-glucosidase. The mechanisms of endoglucanases are hydrolysis of the complex sugars of the feedstock by breaking the interior parts of the amorphous region of cellulose. As for exo-glucanases, they degrade cellulose by cleaving cellobiose units from the nonreducing end of a cellulose fiber to enable the enzyme hydrolysis. With the combination of β-glucosidase, the cellobiase residues are finally broken into two units of glucose. During the reaction, the structure of polysaccharides is destructed by binding the enzyme complex. Besides, algal-based feedstock has superior features in terms of its porosity which can enhance the contact of the enzyme during the hydrolysis. It has been found that the mechanism of accessibility of enzyme into the feedstock during hydrolysis is through the pores in the cell wall which influences the hydrolysis process efficiency. Consequently, the physical structure of the raw material and interaction with the enzymes are key factors that influence the treatment outcome (Harun and Danquah, 2011). Alternatively, pH and temperature are also essential in enzymatic hydrolysis (Shokrkar et al., 2018).

4.3 Fermentation Under the interaction with microbes, the simple sugars released for hydrolysis can be easily converted to bioethanol (Kiran et al., 2014). S. cerevisiae is the most widely used strain in bioethanol fermentation, due to its high selectivity, low accumulation of by-products, high ethanol yield, as well as high rate of fermentation ( Jambo et al., 2016). There are two separated examples of sugars produced after the hydrolysis and its pathway of ethanol production (Nguyen et al., 2016). The conversion of glucose and galactose into ethanol involves the Embden–Meyerhof pathway of glycolysis and Leloir pathway, respectively. In the Embden–Meyerhof pathway, there are two main stages. The first one is the conversion of the

ARTICLE IN PRESS 24

Chen-Guang Liu et al.

sugar to a common intermediate, which is glucose-6-phosphate followed by the second stage including the conversion of the intermediate into pyruvate. The class of the end product of this pathway depends upon the microorganism used. In the case when yeast is applied, it reduces the pyruvate to alcohol (ethanol) and CO2 by an enzyme-catalyzed two-step process, named as alcoholic fermentation. In the Leloir pathway process, galactose-1-phosphate is converted to glucose-1-phosphate followed by the conversion to glucose-6-phosphate. The resulted molecules then may be entered into both the Embden–Meyerhof pathway and the PPP which continued to produce ethanol. Leloir pathway in galactose metabolism is more complex than glycolysis, which results in the slow consumption of galactose when it is compared with the consumption of glucose during the fermentation. The distinction of these sugar’s metabolism influences the bioethanol yield especially during the fermentation of mixed sugars substrate using S. cerevisiae. In terms of the fermentation process, separated hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) are well known in bioethanol production. SHF is separated into two distinct processes: hydrolysis and fermentation. Hydrolysis is the first step for degrading the raw material into monomer molecules by enzymes, and microbes in fermentation convert the sugars to ethanol. High bioethanol titer can be achieved by this process (Nguyen et al., 2016). However, product inhibition caused by sugars is one of the major problems (Shen et al., 2011). SSF simultaneously undertakes hydrolysis and fermentation, where the feedstocks, enzymes, and microbes are mixed together so that the released sugars can be fermented into ethanol rapidly. SSF is able to relief the end-product inhibition by removing the residual sugar in order to obtain a higher bioethanol yield ( Jiang et al., 2017). Besides, with the support of novel combinations of bacteria and microalgae (Hamouda et al., 2018) and a mixture of hydrolytic enzymes (Sulfahri et al., 2016), SSF can approach the theoretical yield.

4.4 Prospect of algae The potential of third-generation bioethanol has caught the attention of the researchers around the world due to the sustainability of the algal feedstock and susceptibility to energy conversion. The innovative employment of micro- and macroalgae as the feedstocks in the bioethanol industry facilitates the process more commercially viable. Besides, suitability of microalgae for biofuels especially attributes to the high capacity of lipids production.

ARTICLE IN PRESS Bioethanol: New opportunities for an ancient product

25

Currently, macroalgae (seaweeds) are also reported to be one of the best feedstock that can be employed as the bioethanol substrate (Hamouda et al., 2018). Despite a significantly high level of carbohydrates, seaweeds also serve as a source of income, which help create job opportunities for the coastline countries. In addition, for the states that have a large number of aquaculture activities, seaweeds can be a very promising choice of renewable energy. Enzymatic hydrolysis has a lot of potentials to be applied in the production of bioethanol for its reasonable economical cost and less negative impact on the environment. Besides, the microorganisms used in fermentation also play an important role in enhancing the conversion of sugars into bioethanol. So the detailed study of optimization in enzymatic hydrolysis and fermentation is required further for the development of an efficient, advanced, and significant bioethanol production process from third-generation feedstock. But to some extent, the future of algae is difficult to interpret because more time and effort are required to explore the ability of third-generation feedstock for bioethanol world. However, what can be expected from algal bioethanol is its contribution to the decrease in the consumption of fossil fuels for a cleaner and more sustainable earth in the future. The employment of algae as the feedstock is innovative for the bioethanol industry. Currently, with a high level of carbohydrates, macroalgae show promising features as the bioethanol feedstock. Moreover, the third-generation bioethanol offsets the limitation of the first- and second-generation bioethanol, such as few impact on the environment, no threat of food supplying, and no need for occupying land (Velazquez-Lucio et al., 2018). However, there are still some challenges for algae bioethanol. Although seaweed has several favorable advantages as a renewable feedstock, challenges and potential impacts on ecosystems also need to be properly addressed. First, polymers in seaweed biomass are generally composed of mixed sugars, and some are not found in terrestrial biomass. Thus, although seaweed polysaccharides degrade relatively easily, efficient conversion of the unusual sugars to bioethanol is challenging, and continuing efforts are being made to develop strategies for solving this problem. Second, the application of seaweed bioethanol will result in increasing seaweed farming (expansion of farming area or increased intensity of current seaweed farming area), and thus additional attention should be paid to the potential impacts on marine and coastal environments. Potential impacts include alteration of natural habitats, change of hydrology (e.g., sedimentation and water movement), nutrient depletion, decrease of biodiversity (e.g., removal of mangrove or sea grass), degradation

ARTICLE IN PRESS 26

Chen-Guang Liu et al.

of water quality, and disturbance of coral reefs (Moheimani et al., 2012). Meanwhile, the environmental impacts of seaweed farming may be not much heavy in some cases and may even have beneficial effects on increasing populations of fish and invertebrates in the area. A balance must be made between seaweed biofuel production and its environmental cost. To compensate high cost in a highly controlled biomass growth environment, algal biomass productivity should be much higher. During circulate cultivation, for achieving such high biomass production level, an energy-efficient control system is required (Moheimani et al., 2012). Besides, the fundamental challenge is the limitation of biological photosynthetic efficiency of around 11% (Ugwu et al., 2008). For scale up production, the issue of mass transfer should be overcome during enzymatic hydrolysis for more carbohydrates.

5. Future perspective of bioethanol As one of the earliest biotechnologies, the fermentation of sugar into ethanol is employed by humans since prehistory for collecting the ingredient of alcoholic beverages. Although ethanol is the most successful sustainable biofuel nowadays, even beyond wind power and solar energy, the gap between ideal biofuel and the first-generation bioethanol is still enormously huge. Innovative technologies and gene-designed strains have been continuously demanded by industry to improve the economic feasibility of bioethanol. For example, thermal tolerant strain is expected for high-temperature ethanol fermentation, which could save the cooling energy, especially in summer. Immobilized microorganism with the property of flocculation is also welcomed to realize high-density cell fermentation to enhance the ethanol productivity. In order to solve the problem of wastewater treatment, stillage backset will be explored to reach the purpose of zero wastewater disposal. The human anchor the hope on the second- and the third-generation bioethanol to address the ethic and economic issues raised by first-generation bioethanol. Bioethanol produced from lignocellulosic biomass or algae not only highlights its environmental benefit in reducing greenhouse gas emissions but also carries moral significance by saving grains currently used for fuel ethanol production to feed people. Sometimes, to determine a suitable pretreatment method is a difficult choice due to various feedstock conditions, thus the energy efficiency needs to be improved based on a mechanistic understanding. Cellulase production by T. reesei is characterized as non-Newtonian fluid properties, which is very energy intensive for mixing

ARTICLE IN PRESS Bioethanol: New opportunities for an ancient product

27

and aeration. Furthermore, cellulase production is inducible, and cellulosebased inducers such as microcrystalline cellulose are expensive. Novel technologies for synthesizing more efficient inducers and optimizing process will be performed. Xylose is the major component of the lignocellulose hydrolysate that must be utilized for making full use of feedstock and reducing pollutant loads during the stillage treatment. The strains that can metabolize pentose will be engineered for the cofermentation of xylose, glucose, and other sugars. Another strategy for saving cellulase cost is to engineer ethanol-producing strains with cellulase production for developing consolidated bioprocessing (CBP) to produce ethanol directly from pretreated biomass without cellulase supplementation. The CBP strains integrate cellulase production, cellulose hydrolysis, and ethanol fermentation together, but strategies for heterologous expression of multiple genes encoding different components of cellulase and transcriptional factors need to be developed. Fortunately, it emerged a prospective future for innovative tech and harnessing the strains because of intensive research input globally. Bioethanol production, as a long story since the beginning of civilization, is destined to promise human a bright energy road.

References Aditiya, H.B., Mahlia, T.M.I., Chong, W.T., Nur, H., Sebayang, A.H., 2016. Second generation bioethanol production: a critical review. Renew. Sustain. Energy Rev. 66, 631–653. Ailion, M., Thomas, J.H., 2003. Isolation and characterization of high-temperature-induced Dauer formation mutants in Caenorhabditis elegans. Genetics 165, 127–144. Almeida, J.R., Modig, T., Petersson, A., Ha´hn-Ha´gerdal, B., Liden, G., Gorwa-Grauslund, M.F., 2007. Increased tolerance and conversion of inhibitors in lignocellulosic hydrolysates by Saccharomyces cerevisiae. J. Chem. Technol. Biotechnol. 82, 340–349. Alper, H., Moxley, J., Nevoigt, E., Fink, G.R., Stephanopoulos, G., 2006. Engineering yeast transcription machinery for improved ethanol tolerance and production. Science 314, 1565–1568. Alvin, A., Kim, J., Jeong, G.-T., Tsang, Y.F., Kwon, E.E., Neilan, B.A., Jeon, Y.J., 2017. Industrial robustness linked to the gluconolactonase from Zymomonas mobilis. Appl. Microbiol. Biotechnol. 101, 5089–5099. Amnuaycheewa, P., Rodiahwati, W., Sanvarinda, P., Cheenkachorn, K., Tawai, A., Sriariyanun, M., 2017. Effect of organic acid pretreatment on Napier grass (Pennisetum purpureum) straw biomass conversion. KMUTNB Int. J. Appl. Sci. Technol. 10, 107–117. Ariyajaroenwong, P., Laopaiboon, P., Salakkam, A., Srinophakun, P., Laopaiboon, L., 2016. Kinetic models for batch and continuous ethanol fermentation from sweet sorghum juice by yeast immobilized on sweet sorghum stalks. J. Taiwan Inst. Chem. Eng. 66, 210–216. Azapagic, A., Perdan, S., Clift, R., 2004. Sustainable Development in Practice: Case Studies for Engineers and Scientists. John Wiley & Sons. Babazadeh, R., Lahtvee, P.J., Adiels, C.B., Goks€ or, M., Nielsen, J.B., Hohmann, S., 2017. The yeast osmostress response is carbon source dependent. Sci. Rep. 7, 990.

ARTICLE IN PRESS 28

Chen-Guang Liu et al.

Bai, F., Chen, L., Anderson, W., Moo-Young, M., 2004. Parameter oscillations in a very high gravity medium continuous ethanol fermentation and their attenuation on a multistage packed column bioreactor system. Biotechnol. Bioeng. 88, 558–566. Bai, F., Anderson, W., Moo-Young, M., 2008. Ethanol fermentation technologies from sugar and starch feedstocks. Biotechnol. Adv. 26, 89–105. Bhatia, S.K., Kim, S.H., Yoon, J.J., Yang, Y.H., 2017. Current status and strategies for second generation biofuel production using microbial systems. Energy Convers. Manag. 148, 1142–1156. Brunecky, R., Alahuhta, M., Xu, Q., Donohoe, B.S., Crowley, M.F., Kataeva, I.A., Yang, S.J., Resch, M.G., Adams, M.W., Lunin, V.V., 2013. Revealing nature’s cellulase diversity: the digestion mechanism of Caldicellulosiruptor bescii CelA. Science 342, 1513–1516. Cai, P., Wang, B., Ji, J., Jiang, Y., Wan, L., Tian, C., Ma, Y., 2015. The putative cellodextrin transporter-like protein CLP1 is involved in cellulase induction in Neurospora crassa. J. Biol. Chem. 290, 788. C ¸ akar, Z.P., Seker, U.O., Tamerler, C., Sonderegger, M., Sauer, U., 2005. Evolutionary engineering of multiple-stress resistant Saccharomyces cerevisiae. FEMS Yeast Res. 5, 569–578. Cao, T.S., Chi, Z., Liu, G.L., Chi, Z.M., 2014. Expression of TPS1 gene from Saccharomycopsis fibuligera A11 in Saccharomyces sp. W0 enhances trehalose accumulation, ethanol tolerance, and ethanol production. Mol. Biotechnol. 56, 72–78. Casey, G.P., Ingledew, W.M., 1986. Ethanol tolerance in yeasts. CRC Crit. Rev. Microbiol. 13, 219–280. Chang, V.S., Burr, B., Holtzapple, M.T., 1997. Lime pretreatment of switchgrass. Appl. Biochem. Biotechnol. 63–65, 3–19. Chang, J.J., Ho, F.J., Ho, C.Y., Wu, Y.C., Hou, Y.H., Huang, C.C., Shih, M.C., Li, W.H., 2013. Assembling a cellulase cocktail and a cellodextrin transporter into a yeast host for CBP ethanol production. Biotechnol. Biofuels 6, 19. Chang, Z., Cai, D., Wang, Y., Chen, C., Fu, C., Wang, G., Qin, P., Wang, Z., Tan, T., 2016. Effective multiple stages continuous acetone–butanol–ethanol fermentation by immobilized bioreactors: making full use of fresh corn stalk. Bioresour. Technol. 205, 82–89. Charoensuk, K., Sakurada, T., Tokiyama, A., Murata, M., Kosaka, T., Thanonkeo, P., Yamada, M., 2017. Thermotolerant genes essential for survival at a critical high temperature in thermotolerant ethanologenic Zymomonas mobilis TISTR 548. Biotechnol. Biofuels 10, 204. Chen, H., Shao, M., Li, H., 2014. Effects of gas periodic stimulation on key enzyme activity in gas double-dynamic solid state fermentation (GDD-SSF). Enzym. Microb. Technol. 56, 35–39. Chen, H., Liu, J., Chang, X., Chen, D., Xue, Y., Liu, P., Lin, H., Han, S., 2017. A review on the pretreatment of lignocellulose for high-value chemicals. Fuel Process. Technol. 160, 196–206. Cherry, J.R., Fidantsef, A.L., 2003. Directed evolution of industrial enzymes: an update. Curr. Opin. Biotechnol. 14, 438–443. Chu, B.C.H., Hung, L., 2007. Genetic improvement of Saccharomyces cerevisiae for xylose fermentation. Biotechnol. Adv. 25, 425. Conway, T., 1992. The Entner–Doudoroff pathway: history, physiology and molecular biology. FEMS Microbiol. Lett. 103, 1–28. Ding, J., Holzwarth, G., Penner, M.H., Pattonvogt, J., Bakalinsky, A.T., 2015. Overexpression of acetyl-CoA synthetase in Saccharomyces cerevisiae increases acetic acid tolerance. FEMS Microbiol. Lett. 362, 1. Eggeman, T., Elander, R.T., 2005. Process and economic analysis of pretreatment technologies. Bioresour. Technol. 96, 2019–2025.

ARTICLE IN PRESS Bioethanol: New opportunities for an ancient product

29

Estruch, F., 2000. Stress-controlled transcription factors, stress-induced genes and stress tolerance in budding yeast. FEMS Microbiol. Rev. 24, 469–486. Evon, P., Barthodmalat, B., Gregoire, M., Vacamedina, G., Labonne, L., Ballas, S., Veronese, T., Ouagne, P., 2018. Production of fiberboards from shives collected after continuous fiber mechanical extraction from oleaginous flax. J. Nat. Fibers 15, 1–17. Flores-G€ omez, C.A., Silva, E.M.E., Zhong, C., Dale, B.E., Sousa, L.D.C., Balan, V., 2018. Conversion of lignocellulosic agave residues into liquid biofuels using an AFEX™-based biorefinery. Biotechnol. Biofuels 11, 7. Gonza´lez-Ramos, D., De Vries, A.R.G., Grijseels, S.S., Berkum, M.C., Swinnen, S., Broek, M., Nevoigt, E., Daran, J.-M.G., Pronk, J.T., Maris, A.J., 2016. A new laboratory evolution approach to select for constitutive acetic acid tolerance in Saccharomyces cerevisiae and identification of causal mutations. Biotechnol. Biofuels 9, 173. Hamouda, R.A., Sherif, S.A., Ghareeb, M.M., 2018. Bioethanol production by various hydrolysis and fermentation processes with micro and macro green algae. Waste Biomass Valoriz. 9, 1495–1501. Harchi, M.E., Kachkach, F.Z.F., Mtili, N.E., 2018. Optimization of thermal acid hydrolysis for bioethanol production from Ulva rigida with yeast Pachysolen tannophilus. S. Afr. J. Bot. 115, 161–169. Harun, R., Danquah, M.K., 2011. Enzymatic hydrolysis of microalgal biomass for bioethanol production. Chem. Eng. J. 168, 1079–1084. Hasunuma, T., Sanda, T., Yamada, R., Yoshimura, K., Ishii, J., Kondo, A., 2011. Metabolic pathway engineering based on metabolomics confers acetic and formic acid tolerance to a recombinant xylose-fermenting strain of Saccharomyces cerevisiae. Microb. Cell Factories 10, 2. He, Z., Wang, Z., Zhao, Z., Yi, S., Mu, J., Wang, X., 2017. Influence of ultrasound pretreatment on wood physiochemical structure. Ultrason. Sonochem. 34, 136–141. Hector, R.E., Qureshi, N., Hughes, S.R., Cotta, M.A., 2008. Expression of a heterologous xylose transporter in a Saccharomyces cerevisiae strain engineered to utilize xylose improves aerobic xylose consumption. Appl. Microbiol. Biotechnol. 80, 675–684. Heipieper, H.J., Weber, F.J., Sikkema, J., Keweloh, H., Bont, J.A.M.D., 1994. Mechanisms of resistance of whole cells to toxic organic solvents. Trends Biotechnol. 12, 409–415. Hiraishi, H., Mochizuki, M., Takagi, H., 2006. Enhancement of stress tolerance in Saccharomyces cerevisiae by overexpression of ubiquitin ligase Rsp5 and ubiquitin-conjugating enzymes. Biosci. Biotechnol. Biochem. 70, 2762–2765. Hosgun, E.Z., Bozan, B., Berikten, D., Kivanc, M., 2017. Ethanol production from hazelnut shells through enzymatic saccharification and fermentation by low-temperature alkali pretreatment. Fuel 196, 280–287. Hoshida, H., Akada, R., 2017. High-Temperature Bioethanol Fermentation by Conventional and Nonconventional Yeasts. Springer International Publishing. Hu, C., Bai, F., An, L., 2005. Effect of flocculence of a self-flocculating yeast on its tolerance to ethanol and the mechanism. Sheng Wu Gong Cheng Xue Bao—Chin. J. Biotechnol. 21, 123–128. Huang, Y.F., Kuan, W.H., Chang, C.Y., 2018. Effects of particle size, pretreatment, and catalysis on microwave pyrolysis of corn stover. Energy 143, 696–703. Ingledew, W., Lin, Y.-H., 2011. Ethanol from starch-based feedstocks. In: Comprehensive Biotechnology. vol. 3. Elsevier, pp. 37–49. Jambo, S.A., Abdulla, R., Azhar, S.H.M., Marbawi, H., Gansau, J.A., Ravindra, P., 2016. A review on third generation bioethanol feedstock. Renew. Sustain. Energy Rev. 65, 756–769. Jiang, H., Mei, C., Chen, Q., 2017. Rapid identification of fermentation stages of bioethanol solid-state fermentation (SSF) using FT-NIR spectroscopy: comparisons of linear and non-linear algorithms for multiple classification issues. Anal. Methods 9, 5769–5776.

ARTICLE IN PRESS 30

Chen-Guang Liu et al.

Jing, T., Yu, S.R., Wu, T.X., 2011. Review of China’s bioethanol development and a case study of fuel supply, demand and distribution of bioethanol expansion by national application of E10. Biomass Bioenergy 35, 3810–3829. Kamm, B., Lei, S., Sch€ onicke, P., Bierbaum, M., 2017. Biorefining of lignocellulosic feedstock by a modified ammonia fiber expansion pretreatment and enzymatic hydrolysis for production of fermentable sugars. ChemSusChem 10, 48. Katahira, S., Ito, M., Takema, H., Fujita, Y., Tanino, T., Tanaka, T., Fukuda, H., Kondo, A., 2008. Improvement of ethanol productivity during xylose and glucose co-fermentation by xylose-assimilating S. cerevisiae via expression of glucose transporter Sut1. Enzym. Microb. Technol. 43, 115–119. Khatun, M.M., Yu, X., Kondo, A., Bai, F., Zhao, X., 2017. Improved ethanol production at high temperature by consolidated bioprocessing using Saccharomyces cerevisiae strain engineered with artificial zinc finger protein. Bioresour. Technol. 245, 1447–1454. Khuong, L.S., Masjuki, H.H., Zulkifli, N.W.M., Mohamad, E.N., Kalam, M.A., Alabdulkarem, A., Arslan, A., Mosarof, M.H., Syahir, A.Z., Jamshaid, M., 2017. Effect of gasoline-bioethanol blends on the properties and lubrication characteristics of commercial engine oil. RSC Adv. 7, 15005–15019. Kiran, B., Kumar, R., Deshmukh, D., 2014. Perspectives of microalgal biofuels as a renewable source of energy. Energy Convers. Manag. 88, 1228–1244. Kumar, A.K., Sharma, S., 2017. Recent updates on different methods of pretreatment of lignocellulosic feedstocks: a review. Bioresour. Bioprocess. 4, 7. Kumar, R., Wyman, C.E., 2009. Effects of cellulase and xylanase enzymes on the deconstruction of solids from pretreatment of poplar by leading technologies. Biotechnol. Prog. 25, 302–314. Leandro, M., Goncalves, P., Spencer-Martins, I., 2006. Two glucose/xylose transporter genes from the yeast Candida intermedia: first molecular characterization of a yeast xylose-H + symporter. Biochem. J. 395, 543. Lebaka, V.R., Ryu, H.-W., Wee, Y.-J., 2014. Effect of fruit pulp supplementation on rapid and enhanced ethanol production in very high gravity (VHG) fermentation. Bioresour. Bioprocess. 1, 22. Lee, Y.R.J., Key, J.L., 1994. A soybean 101-kD heat shock protein complements a yeast HSP104 deletion mutant in acquiring thermotolerance. Plant Cell 6, 1889–1897. Lee, O.K., Lee, E.Y., 2016. Sustainable production of bioethanol from renewable brown algae biomass. Biomass Bioenergy 92, 70–75. Lee, H., Mani, S., 2017. Mechanical pretreatment of cellulose pulp to produce cellulose nanofibrils using a dry grinding method. Ind. Crop. Prod. 104, 179–187. Li, K., Qin, J.C., Liu, C.G., Bai, F.W., 2016a. Optimization of pretreatment, enzymatic hydrolysis and fermentation for more efficient ethanol production by Jerusalem artichoke stalk. Bioresour. Technol. 221, 188–194. Li, Y., Liu, C., Bai, F., Zhao, X., 2016b. Overproduction of cellulase by Trichoderma reesei RUT C30 through batch-feeding of synthesized low-cost sugar mixture. Bioresour. Technol. 216, 503–510. Li, Z., Wang, D., Shi, Y.-C., 2017. Effects of nitrogen source on ethanol production in very high gravity fermentation of corn starch. J. Taiwan Inst. Chem. Eng. 70, 229–235. Lindquist, S., Kim, G., 1996. Heat-shock protein 104 expression is sufficient for thermotolerance in yeast. Proc. Natl. Acad. Sci. U. S. A. 93, 5301. Liu, H., Liu, K., Yan, M., Xu, L., Ouyang, P., 2011. gTME for improved adaptation of Saccharomyces cerevisiae to corn cob acid hydrolysate. Appl. Biochem. Biotechnol. 164, 1150–1159. Liu, C.G., Liu, L.Y., Zi, L.H., Zhao, X.Q., Xu, Y.H., Bai, F.W., 2014. Assessment and regression analysis on instant catapult steam explosion pretreatment of corn stover. Bioresour. Technol. 166, 368–372.

ARTICLE IN PRESS Bioethanol: New opportunities for an ancient product

31

Liu, C.G., Liu, L.Y., Lin, Y.H., Bai, F.W., 2015a. Kinetic modeling for redox potentialcontrolled repeated batch ethanol fermentation using flocculating yeast. Process Biochem. 50, 1–7. Liu, C.G., Qin, J.C., Liu, L.Y., Jin, B.W., Bai, F.W., 2015b. Combination of ionic liquid and instant catapult steam explosion pretreatments for enhanced enzymatic digestibility of rice straw. ACS Sustain. Chem. Eng. 4, 577–582. Liu, L., Wang, J., Rosenberg, D., Zhao, H., Lengyel, G., Nadel, D., 2018. Fermented beverage and food storage in 13,000 y-old stone mortars at Raqefet cave, Israel: investigating Natufian ritual feasting. J. Archaeol. Sci. Rep. 21, 783–793. Ma, K., Ruan, Z., Shui, Z., Wang, Y., Hu, G., He, M., 2016. Open fermentative production of fuel ethanol from food waste by an acid-tolerant mutant strain of Zymomonas mobilis. Bioresour. Technol. 203, 295–302. Martinez, D., Berka, R.M., Henrissat, B., Saloheimo, M., Arvas, M., Baker, S.E., Chapman, J., Chertkov, O., Coutinho, P.M., Dan, C., 2008. Genome sequencing and analysis of the biomass-degrading fungus Trichoderma reesei (syn. Hypocrea jecorina). Nat. Biotechnol. 26, 553. Mishra, A., Sharma, A.K., Sharma, S., Bagai, R., Mathur, A.S., Gupta, R.P., Tuli, D.K., 2016. Lignocellulosic ethanol production employing immobilized Saccharomyces cerevisiae in packed bed reactor. Renew. Energy 98, 57–63. Moheimani, N.R., Mchenry, M.P., Mehrani, P., 2012. Microalgae Biodiesel and Macroalgae Bioethanol: The Solar Conversion Challenge for Industrial Renewable Fuels. John Wiley & Sons. Nguyen, T.H., Ra, C.H., Park, M.R., Jeong, G.T., Kim, S.K., 2016. Bioethanol production from seaweed Undaria pinnatifida using various yeasts by separate hydrolysis and fermentation (SHF). Korean J. Microbiol. Biotechnol. 44, 529–534. Pa´tkova´, J., Sˇmogrovicˇova´, D., D€ omeny, Z., Bafrncova´, P., 2000. Very high gravity wort fermentation by immobilised yeast. Biotechnol. Lett. 22, 1173–1177. Raj, T., Gaur, R., Lamba, B.Y., Singh, N., Gupta, R.P., Kumar, R., Puri, S.K., Ramakumar, S., 2017. Characterization of ionic liquid pretreated plant cell wall for improved enzymatic digestibility. Bioresour. Technol. 249, 139–145. Rendleman, C.M., Shapouri, H., 2007. New technologies in ethanol production. US Department of Agriculture, Office of the Chief Economist, Office of Energy Policy and New Uses. Ribeiro, B.E., 2013. Beyond commonplace biofuels: social aspects of ethanol. Energy Policy 57, 355–362. Ritslaid, K., K€ uu €T, A., Olt, J., 2010. State of the art in bioethanol production. Agron. Res. 8, 236–254. Sadeghinezhad, E., Kazi, S.N., Badarudin, A., Togun, H., Zubir, M.N.M., Oon, C.S., Gharehkhani, S., 2014. Sustainability and environmental impact of ethanol as a biofuel. Rev. Chem. Eng. 30, 51–72. Sakanaka, K., Yan, W., Kishida, M., Sakai, T., 1996. Breeding a fermentative yeast at high temperature using protoplast fusion. J. Ferment. Bioeng. 81, 104–108. Salapa, I., Katsimpouras, C., Topakas, E., Sidiras, D., 2017. Organosolv pretreatment of wheat straw for efficient ethanol production using various solvents. Biomass Bioenergy 100, 10–16. Sathiyamoorthi, R., Sankaranarayanan, G., 2017. The effects of using ethanol as additive on the combustion and emissions of a direct injection diesel engine fuelled with neat lemongrass oil-diesel fuel blend. Renew. Energy 101, 747–756. Schenk, P.M., Thomashall, S.R., Stephens, E., Marx, U.C., Mussgnug, J.H., Posten, C., Kruse, O., Hankamer, B., 2008. Second generation biofuels: high-efficiency microalgae for biodiesel production. Bioenergy Res. 1, 20–43.

ARTICLE IN PRESS 32

Chen-Guang Liu et al.

Sen, B., Chou, Y.P., Wu, S.Y., Liu, C.M., 2016. Pretreatment conditions of rice straw for simultaneous hydrogen and ethanol fermentation by mixed culture. Int. J. Hydrog. Energy 41, 4421–4428. Shafizadeh, F., Bradbury, A.G.W., 1979. Thermal degradation of cellulose in air and nitrogen at low temperatures. J. Appl. Polym. Sci. 23, 1431–1442. Shahir, S.A., Masjuki, H.H., Kalam, M.A., Imran, A., Fattah, I.M.R., Sanjid, A., 2014. Feasibility of diesel–biodiesel–ethanol/bioethanol blend as existing CI engine fuel: an assessment of properties, material compatibility, safety and combustion. Renew. Sustain. Energy Rev. 32, 379–395. Shastri, Y., 2017. Renewable energy, bioenergy. Curr. Opin. Chem. Eng. 17, 42–47. Shen, F., Kumar, L., Hu, J., Saddler, J.N., 2011. Evaluation of hemicellulose removal by xylanase and delignification on SHF and SSF for bioethanol production with steampretreated substrates. Bioresour. Technol. 102, 8945–8951. Shi, D.J., Wang, C.L., Wang, K.M., 2009. Genome shuffling to improve thermotolerance, ethanol tolerance and ethanol productivity of Saccharomyces cerevisiae. J. Ind. Microbiol. Biotechnol. 36, 139–147. Shin, H.Y., Nijland, J.G., Waal, P.P.D., Jong, R.M.D., Klaassen, P., Driessen, A.J.M., 2015. An engineered cryptic Hxt11 sugar transporter facilitates glucose–xylose co-consumption in Saccharomyces cerevisiae. Biotechnol. Biofuels 8, 1–13. Shokrkar, H., Ebrahimi, S., Zamani, M., 2018. Enzymatic hydrolysis of microalgal cellulose for bioethanol production, modeling and sensitivity analysis. Fuel 228, 30–38. Shui, Z.X., Qin, H., Wu, B., Ruan, Z.Y., Wang, L.S., Tan, F.R., Wang, J.L., Tang, X.Y., Dai, L.C., Hu, G.Q., He, M.X., 2015. Adaptive laboratory evolution of ethanologenic Zymomonas mobilis strain tolerant to furfural and acetic acid inhibitors. Appl. Microbiol. Biotechnol. 99, 5739–5748. Simola, M., H€anninen, A.L., Stranius, S.M., Makarow, M., 2000. Trehalose is required for conformational repair of heat-denatured proteins in the yeast endoplasmic reticulum but not for maintenance of membrane traffic functions after severe heat stress. Mol. Microbiol. 37, 42–53. Singh, S., Chakravarty, I., Pandey, K.D., Kundu, S., 2018. Development of a process model for simultaneous saccharification and fermentation (SSF) of algal starch to thirdgeneration bioethanol. Biofuels 1–9. https://doi.org/10.1080/17597269.2018.1426162. Singhania, R.R., Patel, A.K., Soccol, C.R., Pandey, A., 2009. Recent advances in solid-state fermentation. Biochem. Eng. J. 44, 13–18. Sirajunnisa, A.R., Surendhiran, D., 2016. Algae—a quintessential and positive resource of bioethanol production: a comprehensive review. Renew. Sustain. Energy Rev. 66, 248–267. Snoek, T., Picca Nicolino, M., Van Den Bremt, S., Mertens, S., Saels, V., Verplaetse, A., Steensels, J., Verstrepen, K.J., 2015. Large-scale robot-assisted genome shuffling yields industrial Saccharomyces cerevisiae yeasts with increased ethanol tolerance. Biotechnol. Biofuels 8, 32. Sridhar, M., Sree, N.K., Rao, L.V., 2002. Effect of UV radiation on thermotolerance, ethanol tolerance and osmotolerance of Saccharomyces cerevisiae VS1 and VS3 strains. Bioresour. Technol. 83, 199–202. Sulfahri, S., Amin, M., Sumitro, S.B., Saptasari, M., 2016. Bioethanol production from algae Spirogyra hyalina using Zymomonas mobilis. Biofuels 7, 621–626. Sunwoo, I.Y., Nguyen, T.H., Sukwong, P., Jeong, G.T., Kim, S.K., 2018. Enhancement of ethanol production via hyper thermal acid hydrolysis and co-fermentation using waste seaweed from Gwangalli beach, Busan, Korea. J. Microbiol. Biotechnol. 28, 401–408. Swings, J., Ley, J., 1977. The biology of Zymomonas. Bacteriol. Rev. 41, 1–46. Tan, I.S., Lee, K.T., 2015. Solid acid catalysts pretreatment and enzymatic hydrolysis of macroalgae cellulosic residue for the production of bioethanol. Carbohydr. Polym. 124, 311–321.

ARTICLE IN PRESS Bioethanol: New opportunities for an ancient product

33

Tan, F., Wu, B., Dai, L., Qin, H., Shui, Z., Wang, J., Zhu, Q., Hu, G., He, M., 2016. Using global transcription machinery engineering (gTME) to improve ethanol tolerance of Zymomonas mobilis. Microb. Cell Factories 15, 4. Teixeira, A.C.R., Sodr, J.R., Guarieiro, L.L.N., Vieira, E.D., Medeiros, F.F.D., Alves, C.T., 2016. A Review on Second and Third Generation Bioethanol Production. Congresso Sae Brasil. Tenreiro, S., Rosa, P.C., Viegas, C.A., Sa´-Correia, I., 2000. Expression of the AZR1 gene (ORF YGR224w), encoding a plasma membrane transporter of the major facilitator superfamily, is required for adaptation to acetic acid and resistance to azoles in Saccharomyces cerevisiae. Yeast 16, 1469–1481. Thammasittirong, S.N.-R., Thirasaktana, T., Thammasittirong, A., Srisodsuk, M., 2013. Improvement of ethanol production by ethanol-tolerant Saccharomyces cerevisiae UVNR56. Springerplus 2, 583. Ugwu, C.U., Aoyagi, H., Uchiyama, H., 2008. Photobioreactors for mass cultivation of algae. Bioresour. Technol. 99, 4021–4028. Ullah, A., Orij, R., Brul, S., Smits, G.J., 2012. Quantitative analysis of the modes of growth inhibition by weak organic acids in Saccharomyces cerevisiae. Appl. Environ. Microbiol. 78, 8377. Velazquez-Lucio, J., Rodr Guez-Jasso, R.M., Colla, L.M., Galindo, A.S., CervantesCisneros, D.E., Aguilar, C.N., Fernandes, B.D., Ruiz, H.A., 2018. Microalgal biomass pretreatment for bioethanol production: a review. Biofuel Res. J. 5, 780–791. Wang, Z., Hou, X., Sun, J., Li, M., Chen, Z., Gao, Z., 2018. Comparison of ultrasoundassisted ionic liquid and alkaline pretreatment of Eucalyptus for enhancing enzymatic saccharification. Bioresour. Technol. 254, 145–150. Wieczorke, R., Krampe, S., Weierstall, T., Freidel, K., Hollenberg, C.P., Boles, E., 1999. Concurrent knock-out of at least 20 transporter genes is required to block uptake of hexoses in Saccharomyces cerevisiae. FEBS Lett. 464, 123–128. Wu, H., Dai, X., Zhou, S.L., Gan, Y.Y., Xiong, Z.Y., Qin, Y.H., Ma, J., Yang, L., Wu, Z.K., Wang, T.L., 2017. Ultrasound-assisted alkaline pretreatment for enhancing the enzymatic hydrolysis of rice straw by using the heat energy dissipated from ultrasonication. Bioresour. Technol. 241, 70–74. Xu, T., Zhao, X., Bai, F., 2005a. Continuous ethanol production using self-flocculating yeast in a cascade of fermentors. Enzym. Microb. Technol. 37, 634–640. Xu, T., Zhao, X., Zhou, Y., Bai, F., 2005b. Continuous ethanol fermentation using selfflocculating yeast strain and bioreactor system composed of multi-stage tanks in series. Sheng Wu Gong Cheng Xue Bao—Chin. J. Biotechnol. 21, 113–117. Xu, G., Zhao, X., Li, N., Bai, F., 2012. Improvement of acetic acid tolerance of selfflocculating yeast by zinc supplementation. CIESC J. 63, 1823–1829. Xu, J., Zhao, X., Liu, C., Bai, F., 2018. Improving xylose utilization of Saccharomyces cerevisiae by expressing the MIG1 mutant from the self-flocculating yeast SPSC01. Protein Pept. Lett. 25, 202–207. Yang, K.M., Woo, J.M., Lee, S.M., Park, J.B., 2013. Improving ethanol tolerance of Saccharomyces cerevisiae by overexpressing an ATP-binding cassette efflux pump. Chem. Eng. Sci. 103, 74–78. Yoo, C.G., Pu, Y.Q., Ragauskas, A.J., 2017. Ionic liquids: promising green solvents for lignocellulosic biomass utilization. Curr. Opin. Green Sustain. Chem. 5, 5–11. Yoshikawa, K., Tanaka, T., Furusawa, C., Nagahisa, K., Hirasawa, T., Shimizu, H., 2009. Comprehensive phenotypic analysis for identification of genes affecting growth under ethanol stress in Saccharomyces cerevisiae. FEMS Yeast Res. 9, 32. Zhao, B.H., Chen, J., Yu, H.Q., Hu, Z.H., Yue, Z.B., Li, J., 2017. Optimization of microwave pretreatment of lignocellulosic waste for enhancing methane production: hyacinth as an example. Front. Environ. Sci. Eng. 11, 17.

ARTICLE IN PRESS 34

Chen-Guang Liu et al.

Zhu, J.Y., Pan, X.J., 2010. Woody biomass pretreatment for cellulosic ethanol production: technology and energy consumption evaluation. Bioresour. Technol. 101, 4992–5002. Zhu, L., Gao, S., Zhang, H., Huang, H., Jiang, L., 2018. Improvement of lead tolerance of Saccharomyces cerevisiae by random mutagenesis of transcription regulator SPT3. Appl. Biochem. Biotechnol. 184, 155–167. Zverlov, V.V., K€ ock, D.E., Schwarz, W.H., 2015. The role of cellulose-hydrolyzing bacteria in the production of biogas from plant biomass. In: Microorganisms in Biorefineries. Springer, pp. 335–361.