Proposal

10.27 Team 13 Athens Fitzcheung Date April 30th, 2012

A comparison between Diluted Acid and Ammonia Fiber Expansion pretreatment methods to quantify the effect of sugar to furfural selectivity on ethanol yields
Athens Fitzcheung
Second draft submitted March 16th 2012

Team members: Lauren Allen, Athens Fitzcheung, Shreesh Naik, Paul Uche Faculty Advisor: Jean Francois Hamel Teaching Assistant: Dongsook Chang

Proposal
10.27 Team 13 Athens Fitzcheung Date April 30th, 2012

Contents
1 2 3 4 5 List of Figures ........................................................................................................................................ 4 List of Tables ......................................................................................................................................... 5 Summary ............................................................................................................................................... 6 Introduction .......................................................................................................................................... 7 Literature Research and significance .................................................................................................. 10 5.1 Pretreatment method 1 Dilute Acid (DA) hydrolysis ............................................................ 11 What is DA........................................................................................................................... 11 Significance of DA................................................................................................................ 12

5.1.1 5.1.2 5.2

Pretreatment method 2 Ammonia fiber expansion (AFEX) .................................................. 13 What is AFEX ....................................................................................................................... 13 Significance of AFEX ............................................................................................................ 13

5.2.1 5.2.2 5.3

Studies on assessing the effectiveness of various pretreatment methods ................................ 14 Comparison of effects of leading pretreatment methods on sugar yields (Wyman et al 14 Pretreatment and enzymic saccharification of water hyacinth cellulose (Abdel-Fattah 16 Investigation of enzyme formulation on pretreated switchgrass (Falls et al 2011 ) .......... 17

5.3.1 2011) 5.3.2 2011) 5.3.3 5.4

Inhibitor formation ..................................................................................................................... 19 Furfural formation............................................................................................................... 19 How does furfural inhibit fermentation.............................................................................. 20 Acetate formation ............................................................................................................... 21

5.4.1 5.4.2 5.4.3 5.5

Studies of formation of inhibitant and its effect on fermentation ............................................. 21 Almarsdottir 2011 ............................................................................................................... 21 Bellido 2011 ........................................................................................................................ 23 Franden 2009 ...................................................................................................................... 25

5.5.1 5.5.2 5.5.3 5.6 5.7 5.8 6

Discussion of previous years (2010-2011) 10.27 project ........................................................... 27 Areas of improvements in previous years (2010-2011) 10.27 project ...................................... 29 Proposal rationale ....................................................................................................................... 30

Objectives............................................................................................................................................ 32

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10.27 Team 13 Athens Fitzcheung Date April 30th, 2012 7 Technical approach ............................................................................................................................. 32 7.1 7.2 7.3 7.4 7.5 7.6 7.7 Summary of Phases of experiment ............................................................................................. 32 Equipment design ....................................................................................................................... 33 Feedstock preparation ................................................................................................................ 35 Phase I ......................................................................................................................................... 36 Phase II ........................................................................................................................................ 36 Phase III ....................................................................................................................................... 37 Data Analysis, Kinetics calculation and Interpretation ............................................................... 37 Calculation and expectations of product concentration .................................................... 38

7.7.1 7.8 8

Safety .......................................................................................................................................... 39

Management requirement ................................................................................................................. 40 8.1 8.2 Work plan and schedule ............................................................................................................. 40 Budget ......................................................................................................................................... 41

Appendix ............................................................................................................................................. 43 9.1 Full table from Table 2, from Wyman et al 2011 ........................................................................ 43 Bibliography .................................................................................................................................... 43

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10.27 Team 13 Athens Fitzcheung Date April 30th, 2012

1 List of Figures
Figure 1 Schematic of the Ethanol production process .............................................................................. 10 Figure 2 Effect of pretreatment on accessibility of degrading enzymes [8] ............................................... 11 Figure 5 Glucan, xylan, and overall enzymatic yield (g component digested/100 g treated component) as a function of total enzyme loading (mg protein/g raw glucan). ................................................................. 18 Figure 6 Acid-catalyzed pathway for the hydrolysis of hemicellulose to xylose and the degradation of xylose to furfural. ........................................................................................................................................ 20 Figure 7 End product formation from glucose (20 mM) in the presence of different concentrations of furfural (A) and HMF (B) by strain AK17. Data represent average of two replicate experiments.............. 23 Figure 8 Fermentation profiles with furfural as inhibitor. ......................................................................... 24 Figure 9 Fermentation profiles with furfural as inhibitor. .......................................................................... 24 Figure 10 Inhibitory profile of HMF (A), furfural (B), acetate (C) and ethanol (D) on Z. mobilis. Final optical densities were taken after 46 h for HMF, 66 h for furfural, and 24 h for acetate and ethanol. Maximum growth rates ()and corrected final optical densities of wells as a percentage of cells grown without the inhibitor () ............................................................................................................................. 26

Figure 11 Plot of Sugar Concentration vs. Time for 190C Switchgrass Trial .............................................. 27 Figure 12 Plot of Furfural Concentration vs. Time for 190C Switchgrass Trial .......................................... 28 Figure 13 Ethanol Concentration after fermentation of hydrolysate ......................................................... 29 Figure 14 Schematic of experimental set up .............................................................................................. 33 Figure 15 Detail of Primary reactor design ................................................................................................. 34 Figure 16 Photo illustrating the grain size of feedstock after milling processes ........................................ 35 Figure 17 Gantt Chart for our project ........................................................................................................ 40

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10.27 Team 13 Athens Fitzcheung Date April 30th, 2012

List of Tables

Table 1 Yields of glucose and xylose with enzyme protein loading of 30mg/g glucan, in prewashed untreated Dacotah switchgrass [15] ........................................................................................................... 15 Table 2 Summary of optimal pretreatment conditions and corresponding solids compositions (excerpt of table presented in literature). For the full table, see section 9 Appendix. ................................................ 15 Table 3 Effect of water hyacinth pretreatment on its composition, cellulose conversion and saccharification ........................................................................................................................................... 16 Table 4 Composition and pretreatment yields. Note: AFEX and LHW were not washed after pretreatment. ............................................................................................................................................. 17 Table 5 Summary of Glucose and Furfural concentrations at t = 25 mins and t = 35 mins from Team 16 s work in last year 10.27 ................................................................................................................................ 30 Table 6 Estimate of sugar mass obtained from pretreatment of 25g of drymass. Dry mass to sugar yields adopted from Wyman et al 2009 [15], who used pre-washed Dacotah switchgrass, and processed the feedstock in two-stages: Stage 1 pretreatment and post-wash, while Stage 2 enzymatic hydrolysis. ...... 38 Table 7 What if scenarios ........................................................................................................................ 40 Table 8 Time Budget for our project .......................................................................................................... 41 Table 9 Financial Budget for our project.................................................................................................... 42

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10.27 Team 13 Athens Fitzcheung Date April 30th, 2012

3 Summary
As sustainability and climate change rise to the global agenda, the search for an alternative solution to fossil fuels becomes increasingly imminent. By 2025, the US Department of Energy targets a substitution of 30% of transport fossil fuel with biofuel. Within the ethanol production process, pretreatment chemistry is of central importance due to its impact on cellulosic biomass processing and biofuels conversion. Ammonia fiber expansion (AFEX) and dilute acid (DA) are the two frontrunning pretreatments, using alkaline and acidic pH that have clear differences in pretreatment reactions. Inhibitors are by-products of pretreatment and, unless separated, inhibits fermentation of hydrolysate and subsequently lowers ethanol production. Despite the significance of inhibitors to the quality of hydrolysate, current study of pretreatment process largely focuses on sugar conversion percentages, and little study has been done in the impact of inhibitors produced from these pretreatment processes on ethanol produced from microbial fermentation.

Our objective is to compare ethanol concentrations of two pretreatment processes: AFEX and DA, taking into account of furfural concentrations. In Phase I, the threshold furfural concentration that inhibits fermentation of xylose and glucose is investigated, through fermenting fixed glucose solutions mixed with varied concentrations of furfural. In Phase II and III both DA and AFEX pretreatment processes will be conducted with varying temperature and time, and xylose and glucose as well as furfural concentration will be measured. The furfural concentration data obtained from Phase I will give insights to the threshold furfural concentration, which is the level where inhibitation effects on fermentation remain low.

Proposal
10.27 Team 13 Athens Fitzcheung Date April 30th, 2012

By taking into account of inhibitor production, we believe that this study would introduce a new dimension of measuring effectiveness of pretreatment processes.

4 Introduction
Oil and petroleum supplies 37% of global energy consumption. With oil reserves being limited and the impact of fossil fuels on global warming, there needs to be an alternative fuel source for a sustainable future. The situation becomes more dire as the influence of China and India increases, as both countries experienced an average of 9% GDP growth rate in the past 10 years. The International Energy Agency World Energy Outlook, forecasts that the world will enter a peak oil dilemma by 2030, the point in time when the maximum rate of global petroleum extraction is reached, after which the rate of production enters terminal decline, regardless of price, which greatly destabilises the world economy. Furthermore, petroleum-based fuels contribute greatly to CO2 emissions, and climate change.

Thankfully, governments and private sectors have made in roads in mitigating dependence on oil, through legislation such as Energy Efficiency and Clean Energy Districts in New Hampshire, in 2011, Renewable Energy Rebates in Maine 2011 and Renewable Fuels Standard overseen by the Environmental Protection Agency in 2005. With the help of $57bn of government support, global biofuel production tripled between 2000 and 2007. According to the IEA, the growth of biofuels is expected to grow four-fold from 2008 to 2035, driven by continued rise in oil prices and government subsidy.

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10.27 Team 13 Athens Fitzcheung Date April 30th, 2012

While rapid expansion in biofuel production has increased expectations of potential substitutes for oil-based fuels, there have been growing concerns about the impact of rising commodity prices on the global food system. According to the International Monetary Fund, world food prices rose 10 % in 2006 because of increases in corn, wheat, and soybean prices, primarily from demand-side factors, including rising biofuel demand [1].

As such, there is great interest in developing non-food competing feedstock, also known as second-generation biofuels. The amount of biofuel that can be produced from an acre of land varies from 100 gallons per acre for EU rapeseed to 400 gallons per acre for U.S. corn and 660 gallons per acre for Brazilian sugarcane. However, cellulosic ethanol could raise per acre ethanol yields to more than 1,000 gallons, significantly reducing land requirements. The use of cellulosic materials such as wood, grasses, and natural wastes are necessary to lead to scaled production that is competitive with that of traditional fuels. As with other alternative fuels, the adoption of biofuels is a function of cost. Hence, the key to the sustainable future lies in the cost of production of ethanol [2] [3] [4]

Due to the high cost of enzymes and the sensitivity of ethanol yield to cost, the pretreatment process became the key driver to the overall costs of producing biofuels [4]. Innovation in pretreatment could have significant impact in reducing enzyme loadings, reducing hydrolysis duration, increasing sugar concentrations, and ultimately production of ethanol [3] [4] [5].

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10.27 Team 13 Athens Fitzcheung Date April 30th, 2012 Also, although pretreatment processes are set to reduce cellulose, hemicellulose and lignin matrix into monosaccharides, they often result in production of toxic compounds which inhibit subsequent microbial fermentation [6]. These toxic compounds include furfural and 5hydroxymethylfurfural (5-HMF or HMF), which reduces production of ethanol from fermentation and ultimately raises cost of production of ethanol [3] [6] [7] (see section 5.4)

Nonetheless many studies conducted today purely focus on sugar yields and sugar conversion ratio and not take into account of the impact of toxic inhibitors produced in the pretreatment process when assessing pretreatment methods. Such omission may lead to inaccurate assessments of effectiveness of pretreatment methods. For example, a longer DA pretreatment results more breakdown of polysaccharides, and higher glucose concentration, than if pretreated for a shorter period [6], which may be deemed desirable. But prolong treatment would also result further xylose degradation and production of inhibitors such as HMF and furfural, which would lower the amount of ethanol produced from fermentation. Hence, the highest sugar yields do not necessary translate to the highest quality of hydrolysate (see section 5.8).

Given the trade-off between higher sugar conversion and lower production of inhibitors, there is the question of what forms the optimal mix, or rather, the threshold furfural where fermentation is not severely compromised.

Proposal
10.27 Team 13 Athens Fitzcheung Date April 30th, 2012 The objective of our project is to introduce the measurement of furfural into the comparison between pretreatment methods DA and AFEX (Phase II) and to investigate the effects of furfural on fermentation (Phase I).

5 Literature Research and significance

The adoption of ethanol as a substitute for fossil fuels is driven primarily by cost of production of ethanol. One significant driver of cost is the amount of polysaccharides converted to ethanol achieved throughout the entire ethanol production process from pretreatment to fermentation process. Ethanol production from lignocellulosic biomass comprises of the following steps: hydrolysis of cellulose and hemicellulose, separation of lignin residue and, finally, sugar fermentation (see fig 1).

Lignocellulosic feedstock

Milling / Grinding

Pretreatment Chemical / Enzymatic Hydrolysis

Detoxification

Fermentation

Distillation and Dehydration

Figure 1 Schematic of the Ethanol production process

Cellulosic materials are a complex mixture of cellulose, hemicellulose, and lignin. The cellulose of these materials, in their original form, is not readily available for hydrolysis, so pretreatment is a crucial step to produce potentially fermentable sugars (mainly glucose and xylose) in the hydrolysis step.

The aim of the pretreatment is to break down and disrupt the crystalline structure of cellulose to enhance enzyme accessibility to the cellulose during hydrolysis step (see Figure 2) [7].

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10.27 Team 13 Athens Fitzcheung Date April 30th, 2012
Lignin Cellulose

Pre-treatment

Hemi- cellulose Figure 2 Effect of pretreatment on accessibility of degrading enzymes [8]

As per figure 2, the pretreatment process breaks down the shield formed by lignin and hemicellulose, disrupt the crystalline structure and reduce the degree of polymerization of cellulose. Pretreatment is essential in order to achieve high ethanol concentration from biological operations and this operation is projected to be the single, most expensive processing step, representing about 20% of the total cost [4]. Due to its significance, many studies of various pretreatment methods and its impact on fermentable sugar yield were made [8]. The major themes that are relevant in this field will be outlined, and explored in turn:

5.1 Pretreatment method 1 Dilute Acid (DA) hydrolysis

5.1.1 What is DA Normally DA pretreatment uses dilute sulfuric acid as the pretreatment chemical at concentration of 0.52.0% and reaction temperatures from 120 to 200 C, for 60 min [7]. This pretreatment penetrates the lignin structure and recovers hemicellulose as dissolved sugars. Recover of sugars, versus drymass, for DA reaches c. 90%. [3]

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10.27 Team 13 Athens Fitzcheung Date April 30th, 2012 The DA pretreatment process converts most of the hemicellulose to soluble sugars (xylose, mannose, arabinose, and glucose). These sugars are primarily recovered as monomers. Glucan in the hemicellulose and to some extent, cellulose are converted to glucose. [3] [9]. 5.1.2 Significance of DA

The National Renewable Energy Laboratory (NREL), who drives the global development of biomass ethanol, favours DA primarily because 80% to 90% of hemicellulose are recoverable, without the need of expensive, potentially dangers, explosive decompression [3] [4]. DA can be applied to a wide range of feedstocks, including softwood, hardwood, herbaceous crops, agricultural residues, wastepaper, and municipal solid waste. Sulfuric acid has been studied extensively due to its low cost and effectiveness. However, dilute sulfuric acid has its disadvantages, including: Corrosion, which then requires expensive materials for the construction of plant facility; Acidic hydrolysates must be neutralized before sugars proceed to be fermented; Current sulfuric acid price has increasingly become sensitive to global commodity markets [10] Moreover, DA further reduces glucose into degradation products like furfural and HMF and liberates acetic acid from the hemicellulose. In sufficiently high concentrations, these inhibitory compounds can have adverse effects on the fermenting organisms. More details of this will be discussed in section 5.4 below.

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5.2 Pretreatment method 2 Ammonia fiber expansion (AFEX)

5.2.1

What is AFEX In AFEX, the biomass is exposed to heated liquid ammonia under high pressure for a time duration and pressure is suddenly released. This quick pressure drop opens up the structure of lignocellulosic biomass leading to increased digestibility of biomass. AFEX pretreatment simultaneously decrystallizing cellulose, delignify and make soluble hemicellulose, but does not significantly remove hemicellulose. Thus, both micro- and macro-accessibilities of cellulose is affected [10]. Moreover the small amounts (12% of the dry weight of the cellulosic material) of ammonia left behind can serve as a nitrogen source in subsequent fermentations [4].

5.2.2

Significance of AFEX Ammonia is considered to be a promising catalyst for pretreatment of lignocellulosic biomass as its major advantages include its simple recovery process when compared to other catalysts used for pretreatment. About 9597% of ammonia used in the AFEX process can be recovered and reused, which translates to cost-saving. AFEX can achieve greater than 90% conversion of cellulose and hemicellulose to fermentable sugars for a variety of lignocellulosic materials including alfalfa, barley straw, corn residue, wheat straw, rice straw, sugarcane bagasse, switchgrass, coastal bermudagrass, and rye grass straw [4]. For most of these materials, it has been shown that AFEX permits essentially complete conversion of cellulose and hemicellulose to fermentable sugars at low enzyme concentration. AFEX produces lower toxicity degradation products, hence results less inhibition in ethanol fermentation. All these factors are advantageous for developing a cost effective cellulosic ethanol process. One disadvantage is

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10.27 Team 13 Athens Fitzcheung Date April 30th, 2012 that, AFEX has a slightly higher capital costs due to ammonia recovery step when compared to other pretreatments. [11]

5.3 Studies on assessing the effectiveness of various pretreatment methods

Research has been made on comparison of pretreatment processes, including DA [12], sulfur dioxide (SO2), ammonia fiber expansion (AFEX), and lime pretreatments [13] [14] [15]. These analysis were based on sugar yield, % recovered and / or capital cost. There has also been other studies that included the study of inhibitants such as furfural, HMF, acetate and ethanol and its impact on growth rate [15]. Although there is data which illustrates the apparent reduction in fermentation with increasing inhibitants concentration, there is less consideration in comparing the trade-off between increase sugar yields and increased inhibitor concentration and how that compares across pretreatment processes. To illustrate the point, here are several studies that have used purely sugar yield and % sugar recovered in assessing the effectiveness of pretreatment: 5.3.1 Comparison of effects of leading pretreatment methods on sugar yields (Wyman et al 2011)

Wyman et al [15]investigated various pretreatment methods including Dilute sulfuric acid (DA), sulfur dioxide (SO2), liquid hot water (LHW), soaking in aqueous ammonia (SAA), ammonia fiber expansion (AFEX), to measure glucose and xylose yields from pretreatment (stage I) and enzymatic hydrolysis (stage II). Yields and conditions for each pretreatment are found in the tables below:

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Table 1 Yields of glucose and xylose with enzyme protein loading of 30mg/g glucan, in prewashed untreated Dacotah switchgrass [15]

Table 2 Summary of optimal pretreatment conditions and corresponding solids compositions (excerpt of table presented in literature). For the full table, see section 9 Appendix.

Their optimal pretreatment condition for DA in [15] literature was 140Co for 40 min and expected yields were 79.2% (32.6% Xylose, 46.5% Glucose), which is a good representation of what is broadly seen in 120Co-180Co for 30 70 min [7]. For AFEX, these figures were 140Co for 30 min [15]. These conditions give a 79.2% combined sugar yield for DA, and 84.6% for AFEX. Their approach was mainly based on analytical methods and material balance approaches to compare sugar yields (in terms of percentage removal), without consideration on quality of hydrolysate and inhibitor concentration.

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10.27 Team 13 Athens Fitzcheung Date April 30th, 2012 5.3.2 Pretreatment and enzymic saccharification of water hyacinth cellulose (Abdel-Fattah 2011)

More recently, investigations were made on delignification of Water hyacinth, a water plant found across River Nile in Egypt. Abdel-Fattah et al [16]investigated the use of chemical pretreatments and enzymic saccharification on the feedstock. The results, and the definitions of conversion and saccharification is shown below:

Table 3 Effect of water hyacinth pretreatment on its composition, cellulose conversion and saccharification

Under NaOH, a material portion, 86.1%, of Cellulose is recovered, whereas much less proportion (14% and 12.5%) of Lignin and Hemicellulose is recovered. In his analysis, the author used recovered component, cellulose conversion % and saccharification % as measures to compare pretreatment with sodium hydroxide, peracetic acid, alkaline hydrogen peroxide and sodium chlorite. In section 3.1 of the paper the author concluded that the treatment of the chlorite sample with peracetic acid at 100 C was the most efficient as it gave the highest amounts of

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10.27 Team 13 Athens Fitzcheung Date April 30th, 2012 cellulose and hemicellulose all together with the highest cellulose conversion and high saccharification. Again, the author focuses purely on sugar recovery measures to measure efficiency of pretreatment method. 5.3.3 Investigation of enzyme formulation on pretreated switchgrass (Falls et al 2011 )

Falls et al studied the benefits of adding different enzyme cocktails (cellulase, xylanase, bglucosidase) to switchgrass pretreated under various treatments including AFEX, DA and liquid hot water (LHW) [17]. The composition and pretreatment yield of each pretreatment process is presented below:

Table 4 Composition and pretreatment yields. Note: AFEX and LHW were not washed after pretreatment.

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Figure 3 Glucan, xylan, and overall enzymatic yield (g component digested/100 g treated component) as a function of total enzyme loading (mg protein/g raw glucan).

Various amounts and proportions of cellulase and xylanase were then loaded to investigate the highest enzymatic yield, as defined below.

Lime pretreatment showed a maximum enzymatic yield of 64.2 g sugar digested/g protein loaded. The enzymatic yield increased to 91.3 g of sugar digested/g protein when ball-milling was added to the lime pretreatment. The author then proceeded with a cost analysis that concluded that Lime and ball milling was most cost effective at $0.8 / gal ethanol.

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10.27 Team 13 Athens Fitzcheung Date April 30th, 2012 In the study, pretreatment yield, sugar concentration and enzymatic yield and economics were used as measures to compare pretreatment processes and enzyme loading. Despite the use of DA, there was no consideration of inhibitants, in their enzymatic yield calculations nor subsequent economics analysis.

5.4 Inhibitor formation

Although pretreatment processes are designed to breakdown the cellulose, hemicellulose and lignin matrix, releasing monosaccharides and making the remaining polysaccharides available for enzymatic saccharification, they often result in production of toxic compounds which inhibit subsequent microbial fermentation.

The toxic inhibitors found in lignocellulosic hydrolysate results in increased ethanol production costs due to additional detoxification steps or constraints on solids loading in the fermentation step. The DA pretreatment process gives rise to organic acids, primarily acetic acid, sugar degradation products such as furfural and hydroxymethylfurfural (HMF), phenolics from lignin degradation as well as inorganic salts mainly arising from the pretreatment process. 5.4.1 Furfural formation

DA promotes rapid acid-catalyzed hydrolysis of polysaccharides to monosaccharides, which subsequently degrade to furfural, HMF, and other inhibitors. Acid hydrolysis of xylan is particularly problematic because it contains acetyl groups that upon hydrolysis form acetic acid

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10.27 Team 13 Athens Fitzcheung Date April 30th, 2012 (see fig 5). Xylan is also more readily hydrolyzed to monosaccharides (xylose) than cellulose, which leads to xylose degradation and the formation of furfural [18].

Figure 4 Acid-catalyzed pathway for the hydrolysis of hemicellulose to xylose and the degradation of xylose to furfural.

5.4.2

How does furfural inhibit fermentation

Furfural toxicity has been extensively studied in relation to ethanol production. For example Escherichia coli strain K011 has a maximum tolerance of 3 g/L. However, culture growth and ethanol production begin to be effected at concentrations greater than 1 g/L [18]. Other enteric bacteria, including those engineered for ethanol production, have been observed to

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10.27 Team 13 Athens Fitzcheung Date April 30th, 2012 have similar tolerances c. 3-4 g/L. Saccharomyces cerevisiae and xylose fermenting yeasts, Candida shehatae and Pichia stipitis, have been observed to be almost completely inhibited by furfural concentrations of 2-4 g/L. Possible mechanisms for furfural toxicity include chemical reactivity with cellular components, damage to the cellular membrane, and inhibition of metabolism. The toxicity of furfural appears to be a function of its hydrophobicity. Palmqvist et al. [19] demonstrated a correlation between toxicity and hydrophobicity for a number of inhibitors found in lignocellulose hydrolysates. Section 5.5 provide quantitative support on the effects of inhibitants Furfural, HMF and Acetate 5.4.3 Acetate formation Acetate, which is another inhibitor, and ethanol, hydrogen, and carbon dioxide, is formed from fermentation of carbohydrates [6] by strain Thermoanaerobacterium AK17:

Note that unlike furfural, which is formed in pretreatment, acetate is formed during fermentation. More quantitative studies of the effects of inhibitants on fermentation and ethanol concentration are highlighted below:

5.5 Studies of formation of inhibitant and its effect on fermentation

5.5.1 Almarsdottir 2011

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10.27 Team 13 Athens Fitzcheung Date April 30th, 2012 Almarsdottir (2011) [6] studied the ethanol production capacity of sugars and lignocellulosic biomass hydrolysates by Thermoanaerobacterium strain AK17, taking account of the inhibitory effects of furfural and HMF. The fermentation medium was supplemented with glucose (20mM) and either furfural or HMF were mixed in concentration varying from 0.0 to 6.0 g/L glucose. Ethanol and hydrogen concentrations were measured at the beginning and at the end of incubation time of 5 days.

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Figure 5 End product formation from glucose (20 mM) in the presence of different concentrations of furfural (A) and HMF (B) by strain AK17. Data represent average of two replicate experiments

The results indicate that at 0.5 g /L of furfural or HMF, the decrease in ethanol production was 17% and 15%, respectively (Fig. 6A,B). Furfural concentration at 2 g/L resulted in 50% reduction in ethanol production and at higher furfural concentrations, there was zero growth in the strain. For HMF, slightly less decrease was observed with increasing HMF concentrations and the minimum inhibitory concentration was 4 g/L. 5.5.2 Bellido 2011

The main objective of this work [20] is to analyze the influence of inhibitors formed during steam explosion of lignocellulosic biomass on ethanol fermentation by P. stipitis. This study focused on three inhibitors: acetic acid, furfural and HMF in a concentration range of (0.53.5 g/L), (0.52 g/L) and (0.10.5 g/L), respectively. Three different approaches were used: first, the inhibitory effect of acetic acid, furfural and HMF in a model substrate medium, was studied. Then, the fermentation of wheat straw hydrolysates was analyzed. Finally, the effect of steam explosion liquid addition and the influence of the presence of solid fraction during the fermentation process were also studied.

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Figure 6 Fermentation profiles with furfural as inhibitor.

Figure 7 Fermentation profiles with furfural as inhibitor.

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10.27 Team 13 Athens Fitzcheung Date April 30th, 2012 Results showed that an increase in acetic acid concentration led to a reduction in ethanol concentration and complete inhibition was observed at 3.5 g/L. Furfural produced a delay on sugar consumption rates with increasing concentration and HMF did not exert a significant effect. For whole slurry from steam exploded wheat straw, complete inhibition was seen at 1.5 g/L acetic acid, 0.15 g/L furfural and 0.05 g/L HMF. 5.5.3 Franden 2009

In this study, Franden et al [21] investigated the effects of furfural, HMF, acetate and ethanol on growth rate and final cell densities of straing Zymomonas mobilis. The author generated inhibitory profiles with various individual compounds and developed kinetic models that predicted toxic effects in the complex mixture of hydrolysates.

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10.27 Team 13 Athens Fitzcheung Date April 30th, 2012
Figure 8 Inhibitory profile of HMF (A), furfural (B), acetate (C) and ethanol (D) on Z. mobilis. Final optical densities were taken after 46 h for HMF, 66 h for furfural, and 24 h for acetate and ethanol. Maximum growth rates ()and corrected final optical densities of wells as a percentage of cells grown without the inhibitor ()

For HMF, the concentrations that resulted in 25%, 50% 75% and 100% inhibition of the microbial growth were 1.2, 2.8, 4.7 and 8.0 g/L respectively. At 1.7g/L, the concentration that was present in NH4-conditioned hydrolysate in the study, the projected inhibition is approximately 33% of the growth rate without inhibitors.

Furfural curve also followed a non-linear trend, similar to HMF inhibition, whereby growth rates decline with the increase of furfural concentration. Furfural is more toxic towards Z. mobilis cell growth than HMF on either molar or mass basis. Furfural was present at approximately 1.2 g/L in full-strength NH4-conditioned hydrolysate, and this concentration was sufficient to cause a 40% reduction of growth rate even in the absence of additional inhibitors. Furfural also caused reduction in final cell density measurements at 66 h of incubation up to 5 g/L at which point growth was completely inhibited.

For acetate, the maximum growth rates were plotted for each acetic acid concentration as shown in Fig. 9. Slight inhibition was observed up to 10 g/L, after which growth rates dramatically decrease.

The above three studies provide an empirical evidence on the inhibitory effect of furfural, HMF and acetate on product concentration and bacterial growth during fermentation.

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10.27 Team 13 Athens Fitzcheung Date April 30th, 2012 Whilst these studies draw the connection between pretreatment by-products and inhibition to fermentation and cell culture growth in a quantitative manner, there has been no direct connection between hydrolysate produced from pretreatment and ethanol concentration produced from fermentation of that hydrolysate.

5.6 Discussion of previous years (2010-2011) 10.27 project

Onyinyechi Okeke, Abhinav Jain, and Mary Zhang (Team 16) [22]were predecessors of our project. Last years proposal was to measure concentration of xylose and glucose concentrations in dilute sulfuric acid pretreatment, controlling various parameters: feedstock type (switchgrass and bagasse), temperature of the process, residence time, and concentration of dilute sulfuric acid. They also, albeit modestly, fermented their hydrolysate and measured ethanol concentrations.

Figure 9 Plot of Sugar Concentration vs. Time for 190C Switchgrass Trial

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10.27 Team 13 Athens Fitzcheung Date April 30th, 2012 At 190C, xylose and glucose concentration reached a maximum of 22.2 mg/mL and 30.0 mg/mL, at 35 min for both sugars. The team hypothesized that the two concentrations then decreased as the sugars degraded into furfural (Figure 10).

Figure 10 Plot of Furfural Concentration vs. Time for 190C Switchgrass Trial

Furfural production began at 15 min and steadily increased until it reached a peak of 7mg/ml at at 55 min. The team hypothesized that the furfural then started to degrade. It is noteworthy that the highest rate at which furfural is produced (steepest slope) was between 25-45 min, where the concentration of Glucose and Xylose was highest. The team interpreted this as the concentration of sugars peak, sugars was then converted to furfural. The decline of furfural concentration after 55 mins is an indication that furfural is being degraded.

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Figure 11 Ethanol Concentration after fermentation of hydrolysate

Hydrolysates from the various trials were fermented at 37C for 22 hours using Saccharomyces cerevisiae. There was limited variation of ethanol concentration across four samples, and the effects of furfural in the 190C trials were not apparent, nor discussed in the report.

5.7 Areas of improvements in previous years (2010-2011) 10.27 project

Team16 also discussed several challenges in their project: Non-continuous sampling The team commented that during several trials, due to build up of pressure and clogging of the needle valve, it was difficult to extract samples continuously. As such, they had to stop the trial everytime they extracted a sample, and restart the trial. This not only was time-consuming, but also increased susceptibility to experimental errors. This year, our team procured new sampling tube that has a built-in filter, increasing the resistance of the equipment against clogging and harsh conditions Insufficient Fermentation trials Due to insufficient time, the team was only able to conduct 4 trials of fermentation. Through better time budgeting and planning, we will strive to conduct more fermentation trials.

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10.27 Team 13 Athens Fitzcheung Date April 30th, 2012 Pretreatment starting prematurely The feedstock and DA was mixed in a container at room temperature and then heated to desired temperature (130C or 190C), which required approximately 20 minutes. During this ramp up time, the hydrolysis would have started prematurely. This year, we will endeavour to include a feature in our experimental design that allows us to control the injection solvent (H2SO4 or NH3) into the vessel (see section 7.2 for details) , which allows us to control the start of the hydrolysis reaction once the desired temperature is reached.

5.8 Proposal rationale

The final report of Team 16 (section 5.5) provides great food for thought [22]. As an illustration, taking results from DA at 190C, we find that at t=25 mins, glucose concentration is 22mg/ml, versus at t=35 mins, where glucose concentration is 31mg/ml. Based on higher glucose concentration, these measurements solely would lead to the conclusion that leaving the reaction to t=35 mins would result greater ethanol concentration from fermentation. Time (mins) 25 35 Glucose concentration (mg/ml) 22 31 Furfural concentration (mg/ml) 1.2 4.0 Ratio Glucose / Furfural ratio (x) 18.33 7.75

Table 5 Summary of Glucose and Furfural concentrations at t = 25 mins and t = 35 mins from Team 16 s work in last year 10.27

However, when we take account of furfural concentration, the results is less conclusive. At t = 35mins, although glucose level is higher, the more than proportionate increase in furfural concentration lead to lower glucose to furfural ratio 7.75x (vs. 18.33x at t=25 mins). At t=35 mins, despite higher glucose level, relatively higher furfural concentrations may mean more

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10.27 Team 13 Athens Fitzcheung Date April 30th, 2012 inhibited fermentation and lower ethanol yield. However, another possibility is that the furfural concentration, at 7.75x Glucose to furfural ratio, may not be high enough to significantly inhibit fermentation, resulting high ethanol concentration. The above illustrates a trade-off between higher sugar concentration leading to lower sugar / furfural ratio mix, and it is not apparent which leads to greater ethanol concentration. Kinetic analysis (discussed in section 8.8) would shed light to this question and fermentation (discussed in section 8.5) will provide empirical support.

Much of the research conducted to date focused only on the efficiency of enzymatic hydrolysis after pretreatment and failed to take into account of the inhibitory effects furfural, HMF and acetic acid. Without considering inhibitory effects, even a high conversion rate during enzymatic hydrolysis cannot prove that the pretreatment method is successful. Furthermore, studies that include the measure of inhibitants were conducted independently from pretreatment process, i.e. analysis is based on controlled amounts of inhibitants, rather than the hydrolysate (along with the inhibitants) from pretreatment.

In our present study, we will study ethanol concentrations from fermenting hydrolysates produced from various pretreatment conditions, taking into account of effects of inhibitants. In terms of the choice of inhibitor, we will use furfural as a starting point due to accessibility of material, ease of use (especially given the short time frame of our study) and relatively high toxicity as discussed in section 5.5. We will also build upon previous years work through more elaborate equipment design and time management.

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10.27 Team 13 Athens Fitzcheung Date April 30th, 2012

6 Objectives
The following are the primary objectives of our project Prevent clogging and allow for continuous data collection Improve mixing between feedstock and solvent through reactor design and 2-step grinding and reactor design Obtain Glucose concentration, Xylose concentration, Furfural concentration for 4 temperature standpoints for both DA and AFEX Quantify selectivity between sugar and furfural, and its influence on ethanol yield Assess accuracy of measuring furfural concentration using FTIR or HPLC Apply DA or ammonia at time t after target reaction temperature has been reached

7 Technical approach
7.1 Summary of Phases of experiment
The investigation will be divided into three phases. In Phase I, we will measure ethanol concentration across various controlled mixtures of glucose / furfural and xylose / furfural mix. In Phase II, concentrated (70%) H2SO4 will be used to pre-treat switchgrass. In Phase III, AFEX, Anhydrous ammonia will be heated in the reactor and pressure is then rapidly released at the end of the duration. Subsequently, hydrolysates from Phase II and III will be fermented to obtain an ethanol concentration. Special attention to feedstock preparation and reactor design was made to mitigate problems encountered in previous years team, listed in section 5.6. The

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10.27 Team 13 Athens Fitzcheung Date April 30th, 2012 following are more detail descriptions of the experimental set up, feedstock preparation and the three phases of experiments.

7.2 Equipment design

The schematic below illustrates the set up that consists of a primary reactor and secondary vessel.

sampling tube

Valve B

Primary reactor N2 Tank Secondary vessel Heater

Valve A

Schematic

Cooling source

Figure 12 Schematic of experimental set up

A more detailed schematic of the primary vessel is shown below:

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10.27 Team 13 Athens Fitzcheung Date April 30th, 2012

Thermocouple sampling tube

Solvent injection

Stirrer stabilizer Stirrer

Built in filter for sampling

Thermocouple

Primary reactor (enlarged diagram)

Figure 13 Detail of Primary reactor design

A reactor (Swagelok) made with stainless steel with a measured internal volume of 500 mL (outside diameter of 150 mm, length of 300 mm, and wall thickness of 12.4 mm) was used for this study. A working volume of 250 mL will be used to allow space for expanding liquid water at high temperature. The pressure vessel is equipped with a heater and an internal stirrer, and surrounded by an outer case, which acts as the heater for the vessel (see figure 12). In the primary reactor (see figure 13), there is one inflow and one outflow port to the reactor vessel: Inlet for injection of concentrated acid, contained in a secondary vessel, is pressurised by Nitrogen gas tank. Valves are placed in each end of the vessel that contains the concentrated acid to prevent backflow (Valve A and Valve B in figure 13).

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10.27 Team 13 Athens Fitzcheung Date April 30th, 2012 Sampling tube, (fig 14) with built-in filter to ensure that samples contain minimal solids. The external tubing will be coiled, dipped in cold water, to quench the extract. In terms of controlling solvent injection, once desired temperature is reached, the Nitrogen gas tank valve will be opened (Valve A in figure 13), to pressurise the concentrated acid end of the secondary vessel to a pressure greater than that in the primary pressure reactor. Once the pressure differential is achieved, the valve between the secondary vessel to the primary reactor (Valve B) will be opened, to allow the concentrated acid to flow into pressure reactor.

7.3 Feedstock preparation

Raw form

Double milled

Figure 14 Photo illustrating the grain size of feedstock after milling processes

Each phase would require feedstock preparation, which is performed through double-milling the switchgrass, procured from materials Team 16 from last year. The material was first grounded in an industrial Waring grinder for 2.5 minutes, manually mixed to ensure good contact with the impeller, and grounded for another 2.5 mins.

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10.27 Team 13 Athens Fitzcheung Date April 30th, 2012

The grinded material is further grinding in coffee grinder to grain size 1-2mm. This grain size was selected to reduce the probability of clumping during pretreatment, reducing the degree of random error. However, we note that it may also increase the risk of clogging of the sampling module. We will bear this in mind when we conduct our trials.

7.4 Phase I
In Phase I, concentration of sugars (60% glucose, 40% xylose) will be fixed at 5g/l and vary the concentration of furfural, from 0.5g/L to 5g/L at 0.5g/ and place in 250mL flasks. After being sterlised in an autoclave, the sample will be inoculated with Saccharomyces Cerevisiae and placed in New Brunswick Scientifics Innova 4430 incubator shaker for incubation in 45C for 72 hours. Data on ethanol concentration from fermentation of various sugar / furfural mix will be collected and will enable us to investigate the effect of furfural / sugar mix on ethanol concentration. Phase I can run in parallel, independent of other Phases.

7.5 Phase II
During pretreatment, the loading of the milled (1mm to 2mm particle size, see section 7.3) switchgrass will be 10% (w/w, 25g dry mass in 250 ml water). We will inject 3.6 ml of 70% concentrated acid to create 1% DA solution (250ml water in vessel), once desired temperature is achieved. Trials will be run at 125Co-200Co in 25C increments, for 90 minutes, with samples continuously taken at 10 minutes intervals (9 samples per run). The average of three trials will be taken. At the end of the pretreatment, the pretreatment spent liquor will be separated from

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10.27 Team 13 Athens Fitzcheung Date April 30th, 2012 the solid (pretreated substrate) by filtration and stored for HPLC, FTIR and fermentation study. The solid substrate will be collected on filter paper and washed thoroughly with water.

7.6 Phase III

In Phase III, AFEX will be studied. With 25g of feedstock, there will be 25g of NH3 (to achieve Ammonia loading: 1 g NH3 to 1 g dry feedstock) loaded into the reactor when the reaction starts. Samples will be taken out on 15,30,45,60 minutes and pressure and this will be repeated in temperatures 110Co and 135Co.

The 25g of Anhydrous ammonia in 5ml of water will first be deposited in the secondary vessel. As per procedure in DA, feedstock and water will be preheated to desired temperature. Once achieved, the secondary vessel will be pressurized, and NH3 solution will be injected into the main reactor to start the reaction. At the end of each time period, pressure is rapidly released through an exhaust value and under a fumehood. The sample will then be extracted and filtered, as per DA procedure. AFEX-pretreated feedstock will also be air dried in a fume hood overnight.

7.7 Data Analysis, Kinetics calculation and Interpretation

Kinetics analysis can be used to derive model that predicts ethanol yield from given sugar to furfural ratios. The kinetic study is based on the following relationship between cellulosic material, sugars and byproducts as follows:

Hemicellulose + Cellulose ------k1-----> Xylose + Glucose------k2----->

Furfural

(Eq 1)

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10.27 Team 13 Athens Fitzcheung Date April 30th, 2012

Where k1 and k2 are kinetic constants, which are determined by measuring xylose and glucose concentration through reaction time, collected from Phase II and III of the project. 7.7.1 Calculation and expectations of product concentration

In pretreatment, yields are defined based on the maximum potential sugars released from the feedstock. Wyman et al (2009) [15]used Dacotah switchgrass, with 65.6 g of 100 g as dry solids. Maximum potential xylose concentration was 39.4% and the maximum potential concentration of glucose was 60.6% on this basis. Dry mass (g) 25.0 25.0 25.0 Glucose mass (g) 15.0 11.6 12.0 Xylose mass (g) 10.0 8.3 9.2 Sugar mass (g) 25.0 19.9 21.2

Theoretical maximum DA AFEX

Table 6 Estimate of sugar mass obtained from pretreatment of 25g of drymass. Dry mass to sugar yields adopted from Wyman et al 2009 [15], who used pre-washed Dacotah switchgrass, and processed the feedstock in two-stages: Stage 1 pretreatment and post-wash, while Stage 2 enzymatic hydrolysis.

Using the 60:40 Glucose to Xylose ratio and 25g of dry mass (see section 7.5 7.6) and adopting yield data from Wyman et al (2009), we would expect total sugar (Glucose + Xylose) concentration form our DA experiments to be 19.9g, and for AFEX 21.2g.

For fermentation, last years team T16 maximum yield of ethanol from xylose to be 1.66mol ethanol/ mol xylose, through using stoichemistry. As reference, for simultaneous saccharification and fermentation (SSF), the maximal theoretical (stoichiometric) yield of ethanol from hexoses is

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10.27 Team 13 Athens Fitzcheung Date April 30th, 2012

This yield is less than 100% because part of the sugars is converted to cell mass and other byproducts by the organism. [23]

7.8 Safety
During our investigation, we will be predominantly be heating and pressurizing diluted sulphuric acid and liquid ammonia, both hazardous materials under MSDS data sheet, and skin contact should be avoided. Ammonia, in particular, is rated 3 for health, indicating poisonous, hence will be dealt with under the fume hood. A What if Safety analysis is found below:

Potential Issue

Result

Likelihood of Occurring
Low

Consequence s
Serious

Recommended Protocol
Monitor pressure Vent reactor if a dangerous level is reached Monitor pressure during sample extraction Quench reaction and disassemble equipment if clogging persists Frequently monitor equipment condition Ensure impeller will not inhibit any reactor components during the building of the equipment Purge prior sample out of collection tube

Pressure is too high in Hot liquid/vapor is the reactor during expelled dilute acid hydrolysis Feedstock jams No sample is sample collection collected tube while the Increased reaction is proceeding likelihood of explosion Iron contamination No sample is collected

Clogging: Medium Explosion: Low

Clogging: Minor Explosion: Serious

Pressure reactor becomes corroded Impeller inhibits sample collection tube

Low Low

Minor Medium

Sample collection tube is contaminated

Inaccurate data

High

Medium

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10.27 Team 13 Athens Fitzcheung Date April 30th, 2012 with previous samples Proceed to collect sample intended for analysis

Table 7 What if scenarios

8 Management requirement
We have highlighted our thoughts on time and budgetary resources necessary to implement our project:

8.1 Work plan and schedule

Phase I concerns the use of fermentation equipment and separate stock solution, which is independent of pretreatment processes in Phase II and Phase III, hence can be conducted whilst Phase II and Phase III occurs. The Gantt Chart below illustrates this:

Figure 15 Gantt Chart for our project

Note that as we are a group of 4, we are able to break into two groups of 2, and run a dual-track process. Potentially, the first pair can perform Phase II DA tests, whilst the second pair performs Phase III Fermentation trials. Weekly time budget is allocated as follows:

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10.27 Team 13 Athens Fitzcheung Date April 30th, 2012

Category Periodic

Item / task

hours r'qd / wk

Lab work Faculty meeting Internal Meeting Meeting preparation e.g. agenda / minutes Semi-periodic (on average per week) Reports (Interim Final) Memos Literature review Total weekly hours
Table 8 Time Budget for our project

8.0 1.0 2.0 1.0

1.5 1.5 0.5 15.5

8.2 Budget
The apparatus left behind from last years study will be altered, which requires new equipment, along with ancillary parts such as nuts plugs and bolts. Taking account of the solvents, substrates and fermentation equipment, the total costs comes to just under $1,517.

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10.27 Team 13 Athens Fitzcheung Date April 30th, 2012

Heading Item Equipment Parts

20 X nuts 20 X plugs

Unit Cost Total Cost $1 Quantity $

5.0 5.0 20.0 35.0 35.0 60.0 20.0

6 X 50cm stainless steel pipes 4 X arbor ferrule set (1/4 and 1/8) 4 X Unions (1/4 and 1/8) 1 X tank of nitrogen gas

4 X Unions Borethrough (1/4 and 1/8) 35.0 Innova 4430 incubator shaker holders 20.0

Erlenmeyer Flasks 250ml Stock ingredients

D-Glucose, G8270-1KG D-Xylose, X1500-500G Furfural, 185914-500ML, $28.56

20 20 6 4 4 4 1 10 10

100.0 100.0 120.0 140.0 140.0 140.0 60.0 200.0 200.0

36.8 101.5 28.5

1Kg 500g 500ml

36.8 101.5 28.5

Solvent2
Liquid anhydrous ammonia Sulphuric Acid 100.0 50.0

1 1

100.0 50.0

Total Cost
Table 9 Financial Budget for our project

1,517

Notes: 1. Estimated unit cost

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9 Appendix
9.1 Full table from Table 2, from Wyman et al 2011

10 Bibliography

[1] W. Coyle, "The Future of Biofuels: A Global Perspective," Amber Waves, [Online]. Available: http://www.ers.usda.gov/AmberWaves/November07/Features/Biofuels.htm. [Accessed 16 3 2012]. [2] International Energy Agency, "World Energy Outlook 2010," p. 18. [3] Cardona, Production of bioethanol from sugarcane bagasse: Status and perspectives, Bioresource Technology 101 (2010) 47544766, 2009.

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10.27 Team 13 Athens Fitzcheung Date April 30th, 2012 [4] B. Yang, "Pretreatment: the key to unlocking low-cost cellulosic ethanol," Biofuels Bioproducts and Biorefinery, vol. 10, no. 1002, pp. 26 - 40, 2007. [5] N. S. M. M. R. L. Youngmi Kim, "Comparative study on enzymatic digestibility of switchgrass varieties and harvests processed by leading pretreatment technologies," Bioresource Technology, vol. 102, p. 1108911096, 2011. [6] M. A. S. Arnheidur Ran Almarsdottir, "Effect of Various Factors on Ethanol Yields From Lignocellulosic Biomass byThermoanaerobacterium AK17," Biotechnology and Bioengineering, no. 10.1002, 2011. [7] Alvira, "Pretreatment technologies for an efficient bioethanol production process based on enzymatic hydrolysis: A review," Bioresource Technology 101, vol. 101, p. 48514861, 2010. [8] Taherzadeh, "Pretreatment of Lignocellulosic Wastes to Improve Ethanol and Biogas Production: A Review," Int. J. Mol. Sci, vol. 10.3390/ijms9091621, pp. 1621-1651, 2008. [9] Tao, "Process and technoeconomic analysis of leading pretreatment technologies for lignocellulosic ethanol production using switchgrass," Bioresource Technology, no. 102, p. 1110511114, 2011. [10] Y. Zheng, "Overview of biomass pretreatment for cellulosic ethanol production," Int J Agric & Biol Eng, vol. 2, no. 3, 2009. [11] Krishnan, "Alkali-Based AFEX Pretreatment for the Conversion of Sugarcane Bagasse and Cane Leaf residues to Ethanol," Biotechnology and Bioengineering, 2010. [12] Shuai, "Comparative study of SPORL and dilute-acid pretreatments of spruce for cellulosic ethanol production," Bioresource Technology, vol. 101, p. 31063114, 2010. [13] M. W. Lau, "The impacts of pretreatment on the fermentability of pretreated lignocellulosic biomass: a comparative evaluation between," BioMed Central, vol. 2, no. 30, pp. 2-30, 2009. [14] Y. Kim, "Comparative study on enzymatic digestibility of switchgrass varieties and harvests processed by leading pretreatment technologies," Bioresource Technology, vol. 102, p. 11089 11096, 2011. [15] C. E. Wyman, V. Balan and B. E. Dale, "Comparative data on effects of leading pretreatments and enzyme loadings and formulations on sugar yields from different switchgrass sources," Bioresource Technology, vol. 102, p. 1105211062, 2011.

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10.27 Team 13 Athens Fitzcheung Date April 30th, 2012 [16] Abdel-Fattah, "Pretreatment and enzymic saccharification of water hyacinth cellulose," Carbohydrate Polymers, vol. 87, p. 2109 2113, 2012. [17] Falls, "Investigation of enzyme formulation on pretreated switchgrass," Bioresource Technology, vol. 102, p. 1107211079, 2011. [18] Weil, "Removal of Fermentation Inhibitors Formed during Pretreatment of Biomass by Polymeric Adsorbents," Industrial Engineering , vol. 41, pp. 6132-6138, 2002. [19] Palmqvist, "p-Hydroxybenzoic Acid on Growth and Ethanol Productivity of Yeasts," Biotechnol. Bioeng, pp. 46-55, 1999. [20] C. Bellido, "Effect of inhibitors formed during wheat straw pretreatment on ethanol fermentation by Pichia stipitis," Bioresource Technology, vol. 102, p. 1086810874, 2011. [21] M. A. Franden, "Development of a high-throughput method to evaluate the impact of inhibitory compounds from lignocellulosic hydrolysates on the growth of Zymomonas mobilis," Journal of Biotechnology, vol. 144, p. 259267, 2009. [22] O. Okeke, A. Jain and M. Zhang, "Sulfuric Acid Hydrolysis pretreatment of sugarcane bagasse and switchgrass," MIT 10.27 Team 16, Cambridge, MA, 2011. [23] Hatzi, "Detailed Material Balance and Ethanol Yield Calculations for the Biomass-to-Ethanol Conversion Process," Humana Press, Vols. 0273-2289/96/5758--0443, 1996. [24] Z. Kadar, "Ethanol Fermentation of Various Pretreated and Hydrolyzed Substrates at Low Initial pH," Humana Press Inc, vol. 847858, p. 136140, 2007.

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