Category Archives: BIOETHANOL

Economic considerations

Although recent years have witnessed an increasing number of studies in the literature on BESR reaction, the commercialization of a BESR process for hydrogen production still faces many obstacles before it can become a reality. The major obstacle is the cost associated with the process. While the cost of the catalyst, which is usually precious-metal based, can be an inhibitive factor, a detailed analysis of the economics involved in the process and an understanding of the contribution of many cost factors are still lacking.

An economic analysis model based on the cost structures in the United States was thereafter developed by our laboratories based on a process for hydrogen production from bio-ethanol steam reforming. The process includes upstream feedstock considerations as well as downstream hydrogen purification strategies and is analyzed for two different capacity levels, namely a central production scheme (150,000 kg H2/ day) and a distributed (forecourt) production scheme (1,500 kg H2/ day). The analysis was based on several assumptions and input parameters provided by the US Department of Energy and involved sensitivity analyses of several input parameters and their effects on the hydrogen selling price.

The detailed methodologies for performing economic analysis and associated results and discussions can be found in our recent publication [158]. Here we just give a brief summary of what we have obtained from this study. The hydrogen selling price is determined to be $2.69/kg H2 at central hydrogen production scale. According to cost breakdown analysis, ethanol feedstock contributes almost 70% of the total cost. Nevertheless, this technique is still economically competitive with other commonly used hydrogen generation technologies at same production scale such as methane steam reforming ($1.5/kg H2), and biomass gasification ($1.77/kg H2). When the production scale is downsized to forecourt level, the hydrogen selling price is significantly increased up to $4.27/kg H2, which is mainly attributed to the significant increase of capital cost contribution. A series of sensitivity analyses have been performed in order to determine the most significant factor influencing the final hydrogen selling price. From the analyses, hydrogen yield has a major effect on the estimated selling price through variation on ethanol feedstock cost contribution, which is reasonable since higher yield would require less feedstock to produce the same amount of hydrogen. Feed dilution is another important impact on hydrogen selling price, particularly at higher dilution percentage. The exponential escalation of hydrogen selling price is clearly observed when the dilution percentage is higher than 50%. Higher dilution percentage means that larger amount of gas should be processed to get the same amount of hydrogen. The effect of molar ratio of ethanol to water variation on hydrogen selling price has also been evaluated. As expected, hydrogen selling price is increased along with increasing molar ratio of water to ethanol, because larger amount of water is required to be evaporated to get the same amount of hydrogen, resulting in the capital and operation cost increase. However, another factor that is not reflected in this analysis is the fact that excess water (i. e., larger water-to-ethanol ratios) would inhibit coking on the surface and extend the active catalyst life time. So, choosing a higher water input may have additional advantages not captured by this analysis. Finally, the effect of catalyst cost and associated performance on hydrogen selling price has also been intensively explored. The estimations indicate the significance of using transition metal based catalyst for hydrogen production from BESR. If noble metal based catalyst is used instead, the hydrogen selling price will jump up to $22.34/kg H2 from $4.27/kg H2 where transition metal (e. g., Co) based catalyst is employed assuming that their catalytic performance is comparable. In order to get the same hydrogen selling price, the noble metal based catalyst has to either be operated under gas hourly space velocity 100 times higher or has lifetime 100 times longer than those of transition metal based catalyst, which is almost impossible from a realistic viewpoint.

Solid waste

The wet cake has the total solid around 20-30% and contains a mixed component of cassava feedstock since no fractionation of cassava components is employed in dry milling process. The wet cake can be used to produce Dry Distillers Grains With Solubles or DDGS as developed in corn ethanol industry. However, cassava roots do not contain a high protein content as corn grains, cassava DDGS contains less protein contents (around 11-14% and 30% dry basis for cassava and corn DDGS; Sriroth et al., 2006). Though the solid waste from cassava chips is not as valuable as corn DDGS, it can be utilized in many ways:

— To produce biogas: this waste treatment has been practiced in China. The solid waste in the thick slop is sent to Biomethylation process. The results are successfully reported by Dai et al. (2006).

— To feed the burner: Another alternative design for Thai factories is that the solid waste from the thick slop is separated by a decanter so that the moisture content is around 50% of total solids (50% H2O). This semi-dry solid is then used as the feedstock for fuel production by direct burning.

— To supplement in animal feed: The solid waste contains some fibers, proteins and ash and can be used as animal feed fillers.

Steam explosion

The ground sorghum bagasse is rehydrated with steam at atmospheric pressure and impregnated with low amounts (up to 3% w/w) of sulfur dioxide (SO2) in plastic bags for 20-30 minutes in order to improve enzymatic saccharification (Sipos et al., 2009; Stenberg et al., 1998; Ohgren et al., 2005). The impregnated bagasse is introduced into a reactor and the temperature is maintained by injection of saturated steam, varying in a range of 170-210°C (Sipos et al., 2009; Stenberg et al., 1998; Ohgren et al., 2005). After 2 to 10 minutes, the blow­down valve is opened and the hydrolyzate is released into a cyclone (Stenberg et al., 1998). Sipos et al. (2009) achieved an extraction of 89% to 92% of cellulose with steam explosion, up to 18 g glucose, 23 g xylose and 5.5 g arabinose/L hydrolyzate. Ballesteros et al. (2003) used steam explosion pretreatment without sulfur dioxide and obtained around 50% of solids recovery and only 20% solubilization of the cellulose. Hemicellulose sugars were extensively solubilized because the raw material had originally 25% xylose and after the treatment only 2% remained on the fibrous residue.

Lignocellulosics: Variety and chemical composition

1.1 Lignocellulosic biomass as a renewable resource for energetic, chemicals and materials platform

Lignocellulosic biomass (LCB) is the most abundant renewable resource on Earth, comprising about 50% of world biomass. LCB is outside the human food chain and its energetic content exceeds many times world basic energy requirements. These features make it an important option as feedstock, as a relatively inexpensive raw-material, for bioethanol production, and for the development of other bioindustries, to face the international demand for biofuel market. In 2008 it was estimated that 200 x 109 tons of biomass were produced and only 3% were used in pulp and paper industries (Rutz et al. 2008; Sanchez et al. 2008; Zhang 2008).

The use of LCB as feedstock for bioethanol production results in significant reduction of gas emissions (Sanchez et al. 2008; Brehmer et al. 2009) and in economic profits increase due to low-cost raw-materials (Balat et al. 2008). LCB can be classified based on their origin: wood (softwoods and hardwoods) and shrubs, non-food agricultural crops (kenaf, reed, rapeseed, etc.) and residues (such as olive stones, wheat straw, corncobs, rise husk, sugarcane and winemaking residues, among others), and municipal solid wastes related to thinning, gardening, road maintenance, etc. (Demirbas 2005; Balat et al. 2008; Sanchez et al. 2008). Wastes from pulp and paper industries, as spent liquors, paper broke, fibres from primary sludge, waste newsprint and office paper or recycled paper sludge are another specific group of LCB to consider.

The conversion of LCB to fermentable monomeric sugars is much more difficult than the conversion of starch. Numerous studies on the development of large-scale production of


Fig. 1. Lignin potential utilization pathways, adapted from Zhang 2008

ethanol from LCB have been carried out around the world in the last years (Mussatto et al. 2004). The particular inherent structure of LCB is the main limiting factor of its conversion to ethanol. Besides cellulose, with a broad range of applications, lignin and hemicelluloses are also considered promising raw materials for the aforementioned purposes. The brief presentation of potential pathways of lignin and hemicelluloses is depicted in Fig. 1 and Fig. 2, respectively.

Reactor-level approaches

In this section, we discuss reactor-level strategies towards the enhanced ethanol productivity in two ways. First, we seek optimal ratios of glucose and xylose in batch and continuous cultures to maximize bioethanol productivity. Second, various configurations combining batch, fed-batch and continuous reactors are considered. Their maximum achievable productivities are assessed using the model for the original recombinant strain S. cerevisiae 1400 (pLNH33) (i. e., with no amplification of EM6 flux).

The ethanol productivity in a batch and continuous reactor is computed as follows:

Подпись: IXETH (tf) - XETH (0)] / (tf + ts) (batch) [XETH(t) - XETH,IN]D (continuous)
Подпись: PETH - Подпись: (9)

where xeth, in is the ethanol concentration in the feed (which is zero in our case), ts is the extra time taken for harvesting and preparation for the next batch. The normal range of ts is from 3 to 10 hours (Shuler & Kargi, 2002) and we set it to 6 hours.

Heterologous Expression and Extracellular Secretion of Cellulases in Recombinant Microbes

Parisutham Vinuselvi1 and Sung Kuk Lee12 1School of Nano-Bioscience and Chemical Engineering, Ulsan National Institute of Science and Technology (UNIST), Ulsan 2School of Urban and Environmental Engineering, Ulsan National Institute of Science and Technology (UNIST), Ulsan Republic of Korea

1. Introduction

Lignocellulose, starch, sucrose, and macroalgal biomass are different forms of plant biomass that have been exploited for bioethanol production. Among them, lignocellulose, found in both agricultural and forest waste, has attracted great attention because of its relative abundance in nature (Lynd et al. 2002). Lignocellulose is a complex polymer made up of cellulose, hemicellulose, and lignin. Efficient conversion of lignocellulose into bioethanol involves a series of steps, namely, the collection of biomass; pretreatment to dissolve lignin; size reduction to reduce the number of recalcitrant hydrogen bonds; enzymatic saccharification to yield simple sugars; and, finally, fermentation of the sugars to ethanol. The main hurdle in this process is the lack of low-cost technology to overcome the recalcitrance associated with lignocellulose (Lynd et al. 2002; Himmel et al. 2007; Xu et al. 2009). Pretreatment is needed to dissolve the lignin, and enzymes such as xylanases are needed to hydrolyze the hemicellulosic fraction that otherwise would prevent cellulases from accessing the cellulose (Wen et al. 2009) (Fig 1A). The half-life of crystalline cellulose at neutral pH is estimated to be one hundred million years (Wilson 2008). A cocktail of saccharification enzymes—with endoglucanases, exoglucanases and ^-glucosidases forming the major portion—is needed to disrupt the chemical stability of cellulose. The physical stability of lignocellulose, rendered by hydrogen bonds formed between adjacent cellulose polymers, is still a major obstacle to the efficient hydrolysis of cellulose. An additional challenge in cellulose hydrolysis is the relatively poor kinetics exhibited by cellulases (Himmel et al. 2007). Cellulases have lower specific activities than do other hydrolytic enzymes, because their substrate (cellulose) is insoluble, crystalline, and heterogeneous (Fig 1B) (Zhang and Lynd 2004; Wilson 2008). Activity of each of the cellulases in complex enzyme cocktails is inhibited by intermediates —such as cello-oligosaccharides and cellobiose, produced during cellulose hydrolysis —leading to discontinuity in the process. For example, exoglucanase action yields cellobiose, which inhibits endoglucanase (Fig 1C) (Lee et al. 2010).


Fig. 1. (A) Schematic representation of the barriers to access the cellulose present in plant cell wall. Adapted from Biotechnology and Bioengineering (Zhang and Lynd 2004). (B) Degree of solubility of various forms of cellulose. CD, cellodextrin; SS CDs, Semi soluble CD; CT, cotton linters; FP, filter paper; P, wood pulp; BC, bacterial cellulose; BMCC, bacterial microcrystalline cellulose; PASC, phosphoric acid swollen cellulose; NW, natural wood; NC, natural cotton. Reproduced with the permission from Biotechnology and Bioengineering (Zhang and Lynd 2004). (C) Schematic representation of enzymatic hydrolysis of cellulose.

Despite these hurdles, several species of Clostridium, Trichoderma, and Aspergillus can efficiently degrade cellulose. Exploitation of the innate potential of the microbial world might be an economical alternative to overcome the recalcitrance associated with lignocellulose (Alper and Stephanopoulos 2009). Two major strategies have been employed to hydrolyze lignocellulose by using microbial consortia. In the first strategy, native cellulolytic organisms like Clostridium spp. are engineered to produce bioethanol. In another approach, cellulolytic ability is imposed on efficient ethanol producers such as Escherichia coli, Saccharomyces cerevisiae, and Zymomonas mobilis (Xu et al. 2009). This chapter focuses mainly on the cellulolytic systems that have been engineered into recombinant microorganisms.

Carbon source

The cost of raw material is important and cannot be overlooked since it governs the total cost which represents more than 60% of total ethanol production cost (Ogbonna et al., 2001). Using cassava (Manihot esculenta) or tapioca starch as substrate in bioethanol production will reduce the production cost since cassava plants are abundant, cheap and can easily be planted. It is a good alternative at low production cost. It is a preferred substrate for bioethanol production especially in situation where water availability is limited. It tolerates drought and yields on relatively low fertility soil where the cultivation of other crops would be uneconomical especially on idle lands. Furthermore, the starch has a lower gelatinization temperature and offers a higher solubility for amylases in comparison to corn starch (Sanchez and Cardona, 2008).

Cassava is one of the richest fermentable substances and most popular choice of substrates for bioethanol production in the Asian region. The fresh roots of cassava contain 30% starch and 5% sugars while the dried roots contain about 80% fermentable substances. Its roots can yield up to an average 30-36 t/ha. Several other varieties of its non edible tubers maybe selected based on the cyanide content which can be categorized as sweet, bitter, non-bitter and very bitter cassava contains 40-130 ppm, 30-180 ppm, 80-412 ppm and 280-490 ppm, respectively (Food Safety Network, 2005). Since fresh cassava tubers cannot be kept long, it needs to be processed immediately or produced ethanol from dried root. Alternatively, its roots can be milled and dried to form pallet or flour. This will prolong its storage time and save storage spaces. Cassava tuber also can be kept in soil after maturating for several months unharvest without deteriorating. Besides the tuber, cassava waste also can be utilized for ethanol production due to its high content of cellulose, hemicelluloses and starch respectively at 24.99%, 6.67% and 30-50% (w/w) (Ferreira-Leitao et al., 2010).

One of the advantages of using starch such as cassava is that most of the plants can be intercropped with other plants such as cover crops (legume plant) or tree crops (such as cocoa plant and palm oil plant) which can simultaneously grow together (Aweto and Obe, 1993; Polthanee et al., 2007). Polthanee et al. (2007) discussed four possible ways of intercropping practice. They are i) mixed inter-cropping, simultaneous growing of two or more crop species; ii) row-intercropping where simultaneous growing of two or more crop species in a well-defined row arrangement in an irregular arrangement; iii) strip inter­cropping, simultaneous growing of two or more crop species in strips wide enough to allow independent cultivation but, at the same time, sufficiently narrow to induce crop interactions and iv) Relay inter-cropping, planting one or more crops within an established crop in a way that the final stage of the first crop coincides with the initial development of the other crops. This will improve the land productivity and better land usage without the need to explore new land which might lead to deforestation. Figures 2 and 3 show the row-


Fig. 2. Soyabean in four-year-old oil palm (Ismail et al., 2009)


Fig. 3. Cassava intercrop with oil palm (Ismail et al., 2009)

intercropping of immature oil palm plantation intercrop with soyabean and cassava, respectively by Malaysian Palm Oil Berhad (MPOB), Malaysia.

The challenge and perspectives to develop sugarcane ethanol in China

Sugarcane is mainly planted in southern China, such as Guangxi, Yunnan, Guangdong, Hainan et al, Its total planting areas were about 20 million acres in 2010 statistically, and Guangxi contribute about 60 percent of the total. Lands suitability for sugarcane is limited. It is very difficult to expand the land for sugarcane production because of the industrialization in China. An additional challenge is the harvesting. High investment requirements and difficulties with mechanization on, for example steep land, increase the risks of the implementation of mechanized harvest. About over 90 percent of the China sugarcane area was still manually harvested. Expansion of sugarcane areas will be affected by the cost/benefit of manual labor. Under the driving of the market opportunities, national policies giving incentives to the sugarcane agri-business, the further expansion of sugarcane areas forecasted for China is expected to about 2 million acres, which mustn’t reduce the availability of arable land for the cultivation of food and feed crops.

There are risks of environmental degradation in different stages of sugarcane ethanol production and processing. Negative impacts have been caused by the lack of implementation of best management practices and ineffective legislation and control. Nevertheless, further improvements are necessary.

A major concern of developing sugarcane ethanol in China is the threat to sugar security. Rapid expansion of bioethanol production could potentially reduce the availability of sugar production, causing a reduction in its supply and increase of sugar price. In recent years, the sugar productions are stably at about 12 million tons, the max exceeded 14.84 million tons in 2008. While the total demand for sugar is about 12 million tons in China. With the combination of the further expansion of about 2 million acres sugarcane areas, and applying the advanced technology, for example: genetically modified sugarcane and improved cultivation techniques, yields can be increased from 5 tons to about 6-7 tons. So the sugar productions in China are expected to over 16 million tons. Based on these estimates, without affecting the supply of sugar, the current potential of sugarcane ethanol production reached over 2 million tons.


Clearly, the hydrolysis step is affected by the type of pretreatment and the quality of this process — particularly by the accessibility of the lignocellulose.

Lignoculluloses can be solubilised by enzymatic or chemical hydrolysis (mainly with acids). Both the pretreatment and hydrolysis are performed in a single step during acid hydrolysis. Two types of acid hydrololysis are usually applied: concentrated and dilute acid hydrolysis (Wyman et al., 2004, Gray et al., 2006, Hendriks & Zeeman, 2009).

Cellulase enzymes from diverse fungi (e. g. like Trichoderma, Aspergillus) (Dashtban et al., Sanchez, 2009) and bacteria (e. g Clostridium, Bacillus) (Sun & Cheng, 2002) can release sugar from lignocellulose at moderate temperatures (45-50°C) with long reaction times (one to several days) (reviewed in Brethauer & Wyman, 2010; Balat, 2011).

Three different enzymes work synergistically — the endo-|3-1,4-glucanases (EC, exo-|3- 1,4-glucanases (EC and P-glucosidase (EC — to generate glucose molecules from cellulose (Lynd et al., 2002). In addition, enzymes like hemicellulases and ligninases improve the hydrolysis rate and raise the content of the fermentable sugar (Palonen & Viikari, 2004; Berlin et al., 2005).

Diverse factors inhibit the activity of the cellulase and thereby decrease the rate of hydrolysis and the effectiveness of the hydrolysis step: end-product inhibition, easily degradable ends of molecules are depleted, deactivation of the enzymes, binding of enzymes in small pores of the cellulose and to lignin (Brethauer & Wyman, 2010; Balat, 2011).

Hemicellulose is a highly complex molecule and multi-enzyme systems are needed like endoxylanase, exoxylanase, |3-xylanase, a-arabinofuranosidase, a-glucoronidase, acetyl xylan esterase and ferulic acid esterase (all produced by diverse fungi e. g. Aspergillus and bacteria e. g. Bacillus) for the enzymatic hydrolysis (reviewed in Balat, 2011).

Materials and methods

1.1 Furfuryl alcohol effect on glucose fermentation and microscopy

To assess the number of inactive yeast cells "blue staining" is usually done. Methylene blue is the most commonly used color agent. It is a redox indicator that turns colorless in the presence of the active enzymes produced by the yeast Saccharomyces cerevisiae. For verification of the results this compound is compared with erioglaucine (E133) (Brilliant blue No. 1).

In order to verify that erioglaucine is equal in quality to methylene blue, a dose response curve for the toxic compound fufuryl alcohol is produced, and the mortality is then examined with the two color agents. 6 fermentations of 100 ml are started with different levels of furfuryl alcohol (0.0, 1.5, 3.5, 6.0, 9.0 and 15.0) (mL/L) and 2 g/L Turbo yeast (from AlcoTec). All the fermentations are completed in blue cap flasks in Millipore water and with 100 g/L glucose monohydrate. The yeast is left to ferment at 25, 32, 40 and 45°C, using 100 rpm of stirring for 24 hours. Weight loss is measured during working hours to monitor the fermentation. After 24 hours samples are taken for HPLC analysis as described by Thomsen et al. (2009). A methylene blue and an erioglaucine solution are produced each of 0.29 g/L. The 6 samples are divided into 12 portions to perform double repetition. Six portions were mixed with methylene blue in a 9:1 ratio and six portions with erioglaucine in a 1:1 ratio. The samples are examined with microscopy at 1000x magnification just after the color agent was added.