1

Final Report

For

Development of Sustainable Alternative Energy

Prepared For:

University of Wisconsin at Stevens Point

Prepared by:

American Science and Technology

Contract # AST#0408-101, UWSP PO#293364, TO2&TO3

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Table of Content

Task Page #

1.

Background 3

2.

Scope 3

3.

Task One, feasibility 4

3.1

TAT formation

3.2

Literature Search

3.3

Requirement Analysis

3.4

Economic Analysis

4. Task 2-3 5 4.1 Literature Search and Process Requirements Analysis 4.2 System Architecture 4.3 Pulp Optimization 4.4 Metabolic control model 4.5 Study of different enzymes in isoprenoid biosynthesis 4.6 Study of Growth Inhibitors 4.7 Bench Top Experimental Production 4.8 Production Process and Pilot Plant Design

5. 4.8 Computer Modeling and Simulation

Task 2 and 3 final report 7

6.

Attachment One (Task Order One Final report) 29

7. Attachment Two (Required analysis) 47

8. Attachment Three (Economic analysis) 53

9. Appendix A: Material Safety Data Base 54

10.

Appendix B: Health Questions 59

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1. Background The Energy Information Agency (EIA) believes that over the past several decades, the United States has become increasingly reliant on imported energy, primarily from petroleum and forecasts that U.S. dependence on petroleum imports will increase to 68 percent by 2025. DOD, the largest U.S. consumer of energy, also relies on foreign supplies of crude oil and the finished transportation fuels (such as military jet fuel) that are derived from it. Fuel represents more than half of the DOD logistics tonnage and more than 70 percent of the tonnage required to put the U.S. Army into position for battle. DOD’s heavy operational dependence on traditional fuel sources creates a number of decidedly negative effects such as exposing the department to price volatility, forcing it to consume unplanned resources that could be used to recapitalize an aging force structure and infrastructure. This is when United States bears many costs associated with the stability of the global oil market and infrastructure. The cost of securing Persian Gulf sources alone was estimated by the National Defense Council Foundation on 2003 to be $44.4 billion annually. In this environment of uncertainty about the availability of traditional fuel sources at a reasonable cost, DOD is facing increasing energy demand and support requirements that it must meet if it is to achieve its broader strategic goals such as establishment of a more mobile and agile force. Recent technological advances in energy efficiency and alternative energy technologies offer a unique opportunity for DOD to make progress toward reconciling its strategic goals with its energy requirements through reduced consumption of foreign fuel. To capitalize on this opportunity, DOD needs to implement an energy strategy that encompasses the development of innovative new concepts and capabilities to reduce energy dependence while maintaining or increasing overall war fighting effectiveness. Current commercial processes do not produce alternative fuels that meet the higher energy density and wide operating temperature range necessary for military aviation uses. Current biodiesel fuels are 25 percent lower in energy density than JP-8 and exhibit unacceptable cold-flow features at the lower extreme of the required JP-8 operating temperature range (minus 50 degrees Fahrenheit). Commercial bio-ethanol, the most prominent biofuel has 34% lower energy content than gasoline and can lead to corrosion in pipelines and fuel systems. Both biofuels are currently produced from agricultural products (soy and corn) and hence compete with food production. Because converting foodproducts to fuel is socially and ecologically unacceptable, substantial research is going into converting cellulosic biomass – plant material from sources other than food crops – into renewable liquid transportation fuels. The state of Wisconsin has a tremendous infrastructure to convert wood into energy and value-added products such as lumber, pulp and paper. The largest hurdles preventing the use of wood for biofuels is the recalcitrance of lignocellulose. Research is required to develop technologies for manufacturing chemical pulp that promotes the separation of wood components into lignin, hemicellulose and cellulose, and enzymatic saccharification of cellulose. Normally since glucose is the basis for ethanol production, the initial objective should be to provide the highest yield of glucose possible from cellulose. Lignin is an energy-dense material that can be developed into a diesel and JP-8 substitute. At the same time, because ethanol has lower energy content than gasoline, this effort must focus on converting glucose into products that are more valuable than ethanol such as methyl-butenol and isoprene.

2. Scope of work for January 2009 to June 2009

This has been a multiyear project planned to fully investigate, develop and integrate the advanced bio-crude and other alternative energy technologies that can drastically reduce the costs of Bio-JP-8 for DOD. Project tasks

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were included identification of all potential bio-sources, development of manufacturing technologies, design of pilot production plants, and launch production tasks that was expected to result in a significant sustainable and economically viable energy source for DOD. Previous research at UW-Stevens Point and other institutions suggests that the presence of lignin inhibits enzymatic saccharification of cellulose. The pulp and paper industry has developed several processes to separate wood into its three main components, cellulose, hemicellulose and lignin. This program used technologies developed to manufacture chemical pulp to promote the enzymatic saccharification of cellulose and provide the highest yield of glucose possible from cellulose. In a parallel path, team attempted to develop biological systems for the production of the hemiterpenoids isoprene (2-methyl 1,3 butadiene) and methyl butenol (MBO; 2-methyl-3-buten-2-ol). These 5-carbon natural products have outstanding market potential as precursors for organic bio fuels, polymers, and pharmaceuticals produced biologically from agricultural and forest biomass. Both chemicals are produced naturally from the biological precursor DMAPP (dimethylallyl diphosphate) that is made from glucose in bacteria and plants by the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway. At the end, the final process was supposed to be designed to convert wood into biofuel JP-8. To achieve the proposed objectives, the following task plan was designed such that each task can advance a certain part of the technology and act as a milestone that could lead the way for the following tasks. The five main tasks were:

Task One: Feasibility Study Task Two: Available Technology Enhancement and New Technologies Development Task Three: Bio-Crude and byproducts Process Development Task Four: Technology / Process Optimization Task Five: Integration, Pilot Production Plan and Technology Transfer

The following is a report that outlines the activities performed under task one, feasibility study. 3) Task One: Feasibility Study Report The following activities were performed during task one and previously reported as “TO-1-Final-Report”: 3.1) Technical Advisory Team (TAT) AST assisted UWSP to identify and introduce a team of experts from to form the Technical Advisory Team for his project. 3.2) Literature Search AST performed an initial literature search to find all available public domain information about state-of-the-art and emerging cellulose based bio fuel production technologies. 3.3) Requirement Analysis AST has worked closely with ARL and UWSP and together have developed the initial product requirements. 3.4) Economic and Technological Analysis As a part of feasibility study, AST has performed an economic study to determine if an integrated bio-refinery is a viable business. 3.5) Report and Recommendation At the end of task one, team of AST and UWSP has determined that this task is feasible and make economic sense. A copy of “TO-1-Final-Report” is attached to this report as Attachment One

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4) Task 2 and 3: Available Technology Enhancement and New Technologies Development

Although tasks 2 and 3 were designed to perform more basic and applied research and development, however, due to allocation of insufficient funds, project team decided to spend more time on development and enhancement of the manufacturing technologies. Based on that, the main goals of task two were modified as follow:

• Enhancement of using current pulp manufacturing and paper engineering technologies to maximize production of cellulose;

• Use metabolic models, enzyme expression techniques and bioreactor systems to develop an economically and environmentally sustainable process for the production of bio-crude from any sugar, starch or cellulose feedstock; and

• Develop a bench top system for test and evaluation.

And since the funding level for Task 3 was also well below the requested and expected amount, and because ARL was not able to allocate any more funds beyond the TO3, task three was basically became the continuation of task two but instead of using the laboratory scale equipment, it was decided to assemble a pilot plant, and use that as the test platform. So, as part of task three, AST attempted to assemble a pilot production plant for digestion system, a hydrolysis reactor, and a pilot fermenter to test the process back to back in a pilot setting. In specific, task orders two and three were included the following subtasks:

4.1) Literature Search and Process Requirements Analysis; AST assisted UWSP to continue the comprehensive literature search to first update the previously developed technology database and second to find any other available public domain information about state-of-the-art and emerging bio-crude and bio-JP-8 production technologies. In addition these tasks also provided information about the available technologies and processes to produce and handle various interested enzymes for bio-crude productions.

4.2) System Architecture; AST and its team designed and enhanced the system flow chart of the overall process from feed stock to pulp manufacturing, bio-crude production, and all other bi-products of biomass fractionation. (we don’t have anything to back it up)

4.3) Pulp Optimization; As part of task 2 and 3, by using the available equipment at UWSP, AST was able to drastically reduce the cycle time for fractionation process and reduce its costs. Meanwhile, UWSP continued to search for other technologies to manufacture pulp to better promote the enzymatic saccharification of cellulose with a maximum yield of glucose from cellulose.

4.4) Metabolic control model; AST assisted UWSP with manufacturing technologies and economic analysis so that UWSP team can continue to adapt the existing model of glycolysis in E. coli by adding the known biochemical steps in the MEP pathway. Enzyme kinetic data from published literature was used to assure the accuracy of the model. The model allowed for in silico experiments that vary the activity of individual enzymes and measure their effect on overall isoprene flux. The enzymes with the greatest control coefficients were used for genetic engineering.

4.5) Study of different enzymes in isoprenoid biosynthesis; The project team continued study effects of critical enzymes involved in the pathway and their effects on metabolic flux through the MEP pathway. Metabolic flux were measured using

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C metabolic flux analysis (13

C MFA); quantification of metabolic fluxes by using carbon-

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labeling experiments; and mathematical analysis of the labeling data. The E. coli bacteria were transformed with pre constructed plasmid containing eight genes in the MEP pathway and a second plasmid with isoprene synthase. Rates of isoprene synthesis among different transformants were compared. For all experiments, E. coli transformed with marker gene plasmids were used as a control.

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C MFA experiments were conducted in collaboration with an outside lab. 4.6) Study of Growth Inhibitors; Growing genetically modified bacteria were continued in flasks to measure isoprene production rate to determine the effect of isoprene presence on isoprene production rate (feedback). These were run in continuous-flow mode where new media and glucose will be continuously added and dead cells and old media will flow outward to maintain a continuous bacterial population size. Isoprene concentration in the airspace of the reactor was measured over time by gas chromatography, and cell counts were monitored. These data were used to relate isoprene concentration to productivity.

4.7) Bench Top Experimental Production; UWSP continued to attempt to grow genetically modified E. coli in bench top bioreactors using standard LB broth and glucose. These were run in continuous-flow mode where new media and glucose were continuously added and dead cells and old media was flow outward to maintain a continuous bacterial population size. Isoprene concentration in the airspace of the reactor was measured over time by gas chromatography and cell counts were monitored. These data were used to relate isoprene concentration to productivity. Upon completion of bench top experimentation, the process was successfully duplicated in AST’s pilot fermenter reactor.

4.8) Production Process and Pilot Plant Design; The process flow diagram and the conceptual design produced by the team, were used to modify some of the AST equipment and assemble a pilot production digestion system for technology evaluation. Because biomass is not as energy dense as petroleum, the cost of transporting biomass is an important issue. To make the final product affordable for DOD applications, team attempted to assemble a mobile field-portable digester that could potentially have civilian applications as well. This digester was planned to be a part of a small scale integrated bio-refinery that could be located throughout the operating regions and can take local biomass and refines it into fuels.

4.9) Computer Modeling and Simulation; A Chem CAD model developed by AST was reviewed for potential enhancement and used to simulate the process. The results were use to guide AST’s pilot plant design and assembly.

4.10) Technology Transfer As part of this effort, UWSP and AST have established collaborative relationships with other industries to further support the activities, promote manufacturing of bio-crude and bio-JP-8 fuel, and transfer the developed technologies. The following is a detailed report of AST activities during task orders 2 and 3.

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Task 2 and 3 Final Report A. Introduction:

With the worldwide availability of lignocellulosic biomass at 220 billion oven-dry ton per year, there has been a great deal of interest in utilizing lignocellulosic materials for the production of fuels or valuable chemicals. The bioconversion of lignocellulosic materials to fuels and chemicals is composed of three major steps; pretreatment (fractionation), enzymatic hydrolysis and fermentation. Pretreatment is done to recover lignin and hemicelluloses in useful form and increase accessibility of the cellulose to hydrolytic enzymes. The pretreatment and hydrolysis have been identified as one of the major economic barriers toward commercialization of lignocellulosic conversion. The fractionation is done by a process known as pulping. Chemical wood pulping involves the extraction of cellulose from wood by dissolving the lignin that binds the cellulose fibers together. The processes principally used in chemical pulping are kraft, sulfite, neutral sulfite semichemical (NSSC), and soda. The kraft process alone accounts for over 80 percent of the chemical pulp produced in the United States and is the dominant chemical pulping process, but it has some serious shortcomings, mainly air pollution and high capital cost. The shortcoming in kraft pulping has led to the development of several organosolv fractionations of the wood constituents. The organosolv pulping also has problems associated with chemical recovery of solvent. Economics of fractionation and sugar extraction, which are based only on cellulose, are not attractive and it is of vital importance to maximize the efficiency of the pulping process by recovering the by-products (lignin and hemicelluloses), which represent about 50wt% of the dry wood. In this context, we developed an organosolv processes, based on the use of organic solvents as delignification agents, in which it is possible to break up the lignocellulosic biomass to obtain cellulose fibers, high quality hemicelluloses and lignin. In general, a very simplified process flow diagram for fractionation of woody biomass is depicted below:

Separation BioreactorDepolymerization

CelluloseHemicelluloses

Lignin & solvent

Lignin +

solvent

Hemicelluloses

C5-C6 Sugars Fuels/

Chemicals

Lignocellulosic Biomass

Solvent

Catalyst

Tank

Solvent

Lignin Chemiclas

Decanter

As shown in the above process flow diagram, the overall process of biomass conversion consists of three major parts; fractionation, hydrolysis and fermentation. In the fractionation process, the major constituents of woody

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biomass are separated from wood and later the cellulose and hemicellulosse are depolymerized to make sugar monomers.

B. Pilot plant activities: Based on the above idea, a 20 kg/hr biomass deconstruction plant was designed and assembled. After commissioning the pilot plant, several digestion experiments were carried out to scale up the small scale experiments from UWSP. AST digestion system (Figure 1 & 2) is a fully controlled pressure cooker that can hold up to 350 PSI at 300 Fahrenheit. This system is being used mainly to remove lignin from the biomass and produce proper cellulose for sugar production.

Figure 1: Process flow diagram of the 20kg/hr biomass digestion plant.

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Figure 2, AST’s Digestion System After the digestion process is completed, the resultant output is a liquid phase (mixture of aqueous and organic layers) and a solid phase (cellulose and remaining lignin). The liquid phase is drained into a settling tank where the aqueous layer separates itself from organic layer (Figure 3).

Figure 3, Aqueous / Organic layers, and Cellulose

Cellulose

Lignin+ l

Hemicelluloses + water

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The optimized digestion process obtained from UWSP tests were scaled up using AST’s pilot plant.

Experiments were carried out using 9 - 20 kg wood chip loading. Temperature was kept around 178 C with an initial N2 pressure of 100 psi. The temperature was kept at 178 C for 30 min. Results were reproducible whether the pilot plant was run using half or full scale. All the experiments ran well without many issues. The results are listed in Table 1 & 2 which are very consistent and are in close agreement with the small scale experiments carried out at UWSP.

Table 1. results from AST pilot plant fractionation tests.

Table 2 summarizes the sugar analysis results of the aqueous layer obtained from our various pilot plant runs. The IC results look very consistent.

g/l AST-1 AST-2 AST-3 AST-4 AST-5

Arabinose 0.454 0.097 0.109 0 0.557

galactose 0.906 0.397 0.415 0.453 0.461

glucose 1.395 8.131 6.228 9.071 6.13

xylose 8.65 1.616 1.535 1.165 2.793

mannose 0.987 0 0.655 0.509 0.831

Total 11.938 10.144 8.833 11.198 10.215

Butanol 90.452 Acetic Acid 4.469

runs Dry (kg)

Mass balance, %

Lignin in the liquor, wt%

Sugar (butanol and others) in the Aq. layer (g/L, refractometer)

Kappa %saccharification (5% loading)

AST-2 9 105.74 9.32 70 - 84.3

AST-3 9 113.61 9.07 70 69 85.3

AST-4 20 104.33 10.35 80 57 81.2

AST-5 9.8 108.5 8 - 48 82

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Pulp yield: In these entire experiments pulp yield, liquor pH and aqueous layer pH were also very consistent (Figure 4).

Figure 4: AST pilot plant digestion results.

C. Sugar production: In this task, effect of amount and type of enzyme and also effect of different pulps were examined during a series of shake flask and reactor experiments. Effect of different enzymes on glucose release from pulp samples are shown in Figure 5. GC 220 was the least effective enzyme in releasing glucose from pulp samples. Effect of pH control on glucose release from pulp samples is shown in Figure 6, where control pH result in better glucose release from pulp samples. This effect becomes very pronounced after 20 hours of operation.

0

5

10

15

20

0 10 20 30 40 50Gluc

ose

Conc

entr

atio

n (g

/l)

Time (hr)

Glucose Libertion from pulp by Enzymes(shake flask exp.)

ACC1500 (85 μl/gr substrate)

CTec2 (40 μl/gr substrate)

GC220 (18 μl /gr substrate)

Figure 5. Glucose release from pulp sample by different enzymes

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0

10

20

30

40

50

0 10 20 30 40 50

Gluc

ose

Conc

entr

atio

n (g

/l)

Time (hr)

Glucose Libertion from Pulp by Enzymes(bioreactor exp.)

CTec2 (40 μl/gr substrate), pH controlledCTec2 (40 μl/gr substrate), pH NOT controlled

Figure 6. Effect of pH control on glucose release from pulp sample by CTec2© enzyme

Effect of different pulp samples on releasing glucose is depicted in Fig 7. These pulps were prepared under different operating conditions and the effect of process parameter is evident in glucose release by CTec 2 enzyme. The figure shows that BUOH 88 pulp had the best glucose release and 100% conversion (wt/wt) of pulp:sugar was achieved. Fig 8 shows comparison of two different enzymes; CTec2 and Accelerase1500. All processing condistions were idetical and the same pulp was used in both experiments. Based on the results obtained, CTec2 is better enzymes than Accelerase1500 in releasing glucose from pulp samples.

0

0.2

0.4

0.6

0.8

1

1.2

0 10 20 30 40 50

Gluc

ose

yiel

d (g

/g p

ulp)

Time (hr)

Glucose Libertion from pulp by Enzymes(shake flask exp.)

BUOH 88 (40 μl CTec2/gr substrate)

BUOH 84 (40 μl CTec2/gr substrate)

BUOH 47 (40 μl CTec2/gr substrate)

BUOH 83 (40 μl CTec2/gr substrate)

BUOH 81 (40 μl CTec2/gr substrate)

Figure 7. Glucose release from different pulp samples by CTec2© enzyme

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0

10

20

30

40

50

0 10 20 30 40 50

Gluc

ose

Conc

entr

atio

n (g

/l)

Time (hr)

Glucose Libertion from Pulp by Enzymes(bioreactor exp.)

ACC1500 (0.085 ml /gr substrate)

CTec2 (0.04 ml /gr substrate)

Figure 8. Glucose release from pulp samples by Accelerase 1500© and CTec2© enzymes

Fig 9 shows glucose release from different pulp samples. Two new enzymes; NS50012 and NS22002 were not effective at all and could not release any glucose from pulp samples. Based on these results, small variations in the amount of enzyme employed have a pronounced effect in releasing glucose from pulp samples. These results also indicate that autoclaving pulp samples does not offer any added advantage in releasing glucose.

Fig 9. Glucose release from pulp samples by combination of different enzymes

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Pre-treating pulp samples has a major impact in releasing glucose form the samples. Fig 10 shows the profiles of glucose release from pulp samples that had been pre-treated. Washing pulp samples with acetone to remove the lignin content of pulp was believed to be effective in releasing glucose from the samples. However, the results show contrary to our belief the pretreatment was not only effective, but hindered the glucose release. It is believed that drying the pulp samples has a major negative impact in releasing glucose from the pulp samples.

Figure 10. Glucose release from pulp samples prepared under different conditions

Concentrated sugar: ButOH 88E (47.7% dry pulp): 85.2 g (plan was to start with 190 g but But 88 pulp was not enough) of wet pulp 88 (22.79% solid) was dried for 2 h to make it 47.7% solid (40.7g). This was used to convert the pulp to sugar. The experiment was started with 15 g wet pulp, 0.94 mL Ctec 2 and 29 mL water (pH 4.8). After 4h 35 min, 5 g more pulp was added and after 5h (from the start) 0.94 ml Ctec 2 enzyme was added. After 6h 34min, 10g pulp; and the rest 10.7g more pulp was added after 22h 50 min. By this time 1.88 mL of Ctec 2 enzyme and 30.52 g of dry pulp was added (61 µL/dry g). The experiment was carried out in a 500 mL shake flask at 51 C for 48 h, and at 200-230 rpm shaking. Percentage of solid remained after the process was calculated by vacuum filtration of the remaining solid and followed by drying the solid in the oven. Experiment code is BuOH 88E. Based on solid to solid conversion (started with 19.41g dry, 7.3 g remained unreacted), 62.4% saccharification was obtained from this process. The lower % conversion is due to high solid and low enzyme dose used compared to 88D. Sugar solution yield was 47 mL. This should produce 186g/L sugar solution. IC data showed 236 g/L (23.6% concentration). Summary of concentrated sugar production: Table 3: comparison of experiments 88A to 88E. Expt %dry Feeding rate %saccharification Sugar 2d Sugar 1d

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pulp used (g/L) (g/L) 88A 22.79 slow 72 235 128 88B 22.79 slow 69.7 183 161 88C 22.79 fast 79.1 183 155 88D 31.3 slow 74 133 - 88E 47.7 slow 62.4 236 - Summarizing the results, Table 3, it is concluded that more than 20% sugar concentration can be obtained by this process if more dry pulp is used. A better pulp (like 84) and controlled atmosphere can further increase the sugar content and percentage of saccharification. Sugar production process was optimized in a small scale shake flask by rapid screening process. Organosolv fiber was used as the feedstock for this rapid screening process. Several process factors such as type of enzyme, mixed enzymes, reaction time, pH, time, mixing, solid loading are screened. Several commercial enzymes such as Ctec 2 (Novozymes); HTec2 (Novozymes) GC220 (Genencor); ACC1500 (Genencor); and Speczyme (Genencor) has been tested. Amongst these screened enzymes Ctec2 was found to be the most economical enzyme to produce lignocellulosic sugar syrup. The best process obtained from the rapid screening process was scaled up on a 150L hydrolysis unit using few Kg fiber under mild conditions (45-50C; pH 5-5.5). The sugar syrup produced using the organosolv cellulose was found (by GCMS analysis) to be free from inhibitors for fermentation process. The preliminary economic evaluation shows that using our optimized conditions sugar can be produced at 3-4 cents/lb. We also developed a multi-step feeding technology to produce more than 20% pure concentrated sugar syrup.

D. Inhibitor analysis: The pulp obtained from UWSP when hydrolyzed the sugar syrup contains inhibitor (Figures 11,12), however, the pulp produced from the AST pilot plant when hydrolyzed the sugar syrup does not have any inhibitors (Figure 13). This might be due to a better washing facility and centrifuge associated with the AST unit.

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Figure 11. GCMS of the sugar syrup obtained by the hydrolysis of But-84 pulp (UWSP run) using Ctec 2. The syrup contains hydroxymethyl furfural as the inhibitor for the fermentation process. A small amount of butanol and furfural were also detected.

Figure 12. GCMS of the sugar syrup obtained by the hydrolysis of mixed pulp (UWSP runs) using Ctec 2. The syrup contains hydroxymethyl furfural as the inhibitor for the fermentation process. A small amount of butanol and furfural were also detected.

Figure 13. GCMS of the sugar syrup obtained by the hydrolysis of AST-3 pulp using Ctec 2. The syrup does not contain any detectable amount of inhibitor

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E. Separation of products:

D1. Lignin Separation: Small Scale Optimization: Precipitaion of lignin from butanol liquor was optimized using three different precipitating agent, CaO, Ca(OH)2 and CaCO3. The results are summarized in the table 4. In all the expts, 100g lignin-butanol liquor (pH -2.65; lignin content 12-13 wt%) was used. Stirring was done using a mechanical stirrer at the rate of 200-300 rpm. All the expts were carried out at RT under ambient pressure. Table 4. Lignin precipitation from the butanol liquor (100g scale) using Ca based reagents. CaO wt%

Temp/time Wet lignin

Dry lignin

filtrate %lignin in the filtrate

pH Dry lignin-CaO

3 RT/45 min - - - 7.0 -

5 RT/45 min - - 62.0g 0.9 8.0

6 RT/45 min 29.15g 18.7g 68.5g 1.1 10.76 12.7g

5 40C/30 min

23.8g 15.9g 65.5g 1.0 - 10.9g

5 3C/45 min - 6.8g - 3.6 - 1.8g

4 60C/30 min

19.6g 13.1g 32.1g† 3.5 - 9.1g

3* 60C/15 min

13.8g 8.1g - 5.1 - 5.1g

†butanol evaporated due the expt is done in open system; *Ca(OH)2 CaO was found to be the best precipitation agent. Ca(OH)2 also works similar to CaO but as CaO is cheaper than Ca(OH)2, it is preferred to use CaO. CaCO3 did not work well. Large scale lignin separation: The best condition obtained from the small scale optimization studies were used to scale up the process in Kg scale.

1. Evaluate lignin concentration in the butanol liquor: Two separate expts were carried out to evaluate the lignin concentration in the butanol liquor. Butanol was evaporated by heating the liquor around 100 C and the weight of the left over solid mass was noted. Percentage of solid was calculated = [amount of solid left/amount of liquor used] x 100. From these two expts, it seems like the lignin concentration in the liquor is around 12-13% (Table 5).

Table 5. Lignin concentration in the liquor expt Amount of liquor used Lignin % 1 1g 12.3 2 2g 12.1

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2. Lignin precipitation using 1Kg of butanol liquor.:1000g lignin-butanol liquor (pH -2.65; lignin content 12-13 wt%) was used to separate the lignin from the solvent. 5 wt% (50 g) of CaO oxide was used as the precipitating reagent. Stirring was done using a mechanical stirrer at the rate of 1000 rpm. The experiment was carried out at RT (22 C). After 45 min stirring, the solid (precipitated lignin) and the liquid (butanol with left over lignin and chemicals) were separated by vacuum filtration using a 18 cm diameter Buchner funnel. The solid (brown colored) is wet lignin. The wet lignin was then dried in an oven at 80C for 24 h. The filtrate (butanol) is saved for recycling. A schematic of the process is shown in the Fig. 14. The lignin precipitation results are summarized in the table 6.

Fig. 14. Schematic of lignin precipitation process. Table 6. Lignin precipitation from the butanol liquor (1Kg scale) using CaO. Feed (g) Collected

after stirring

Loss during stirring Butanol collected after filtration

%lignin in the filtrate

Wet lignin

Loss in filtration

Dry lignin

lignin collected

butanol collected CaO Lignin-

Butanol Spatula+stirrer

bucket evaporated

50 1000 1041.6g 0.2g 4.4g 4.0g 672.5g 3.3 327g 40 g evaporated +1.1 g in filter paper s+ 1.5 g in the funnel

185g 18.5% 67.2

Wet lignin: lignin+left over adsorbed butanol+CaO; Dry lignin: Ca-lignin

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3. Lignin precipitation using 3Kg of butanol liquor: 3000g lignin-butanol liquor (pH -2.65; lignin content 12-13 wt%) was used to separate the lignin from the solvent. 6 wt% (180 g) of CaO oxide was used as the precipitating reagent. Stirring was done using a mechanical stirrer at the rate of 1100 rpm. The experiment was carried out at RT (22 C). After 60 min stirring, the solid (precipitated lignin) and the liquid (butanol with left over lignin and chemicals) were separated by vacuum filtration using a 18 cm diameter Buchner funnel. The solid (brown colored) is wet lignin. The wet lignin was then dried in an oven at 80C for 24 h. The filtrate (butanol) is saved for recycling. The lignin precipitation results are summarized in the table 7.

Table 7. Lignin precipitation from butanol liquor (3Kg scale) using CaO. Feed (g) Collecte

d after stirring

Loss during stirring Butanol collected after filtration

%lignin in the filtrate

Wet lignin

Loss in filtration

Dry lignin

% lignin collected

% butanol collected Ca

O Lignin-Butanol

Spatula+stirrer

bucket

evaporated

180 3000 3180 0.5g 2.0 g 0.0 1898.5 g 2.2 1071.5 g

206 g butanol evaporated +4.3 g in filter paper and funnel

548.5 (582.1 g butanol loss)

18.3 63

Wet lignin: lignin+left over adsorbed butanol+CaO; Dry lignin: Ca-lignin

4. Amount of CaO in the precipitated lignin: % CaO in the dried lignin (brown colored) prepared by CaO precipitation process (lignin from expt 2, 5% CaO) was calculated by combustion process. Two separate crucibles with 1g and 2g of Ca-lignin were kept in the box furnace at 830 c for 1h and then cooled down to RT. The weight of the remaining solid (CaO white) was around 25-30%.

D2. Butanol separation from the aqueous hemicelluloses layer: NaCl process: Hemicellulose present in the aqueous layer can be converted to C5 sugars using enzymes. However the presence of butanol (8-9 v%) in the hemicullose layer acts as an inhibitor and retards the enzymatic hydrolysis process to convert the hemicelluloses to C5 sugar monomers. So it is necessary to separate the butanol from the aqueous layer so that the whole process can be economical. In this experiment we used NaCl to push the dissolved butanol from the aqueous layer (hemicelluloses layer) to the butanol layer (lignin). The butanol liquor and the aqueous layer was taken in a beaker, to this NaCl was added under constant stirring. The more NaCl dissolves into the aqueous layer the thickness of the butanol layer (dark brown) increases; this indicates that butanol is being pushed from the aqueous layer to the butanol-lignin layer. This is also further confirmed by GC analysis. From GC analysis it is calculated that more than 90% of the butanol can be recovered by this process. Distillation: GCMS of furfural. A new method fine was developed to analyze furfural. Furfural GC analysis calibration (figure 15) is done: Five different furfural samples were prepared to calibrate the furfural analyses (1.5, 3, 6, 12 and 24 µL furfural in 1 mL water).

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Figure 16: Products in the aqueous liquor

Figure 15: Calibration curve for furfural analysis using GCMS. Furfural analysis is quantified, aqueous liquor contains 118 µL/L furfural and the recycled aqueous liquor (But 102) contains 173.5 µL/L of furfural. Comparing aqueous liquor and recycled aqueous liquor, there is a 1.17 time increase in butanol concentration in the recycled aqueous liquor and 1.47 times increase in furfural amount. This is minimal based on the amount of C5 sugars we are making the digestion process. This indicates most of our sugars are safe during the recycling run. Slight increase in the butanol concentration is not what we were expecting but it is not bad also as by consuming another 10% butanol we are able to make one more digestion (IC data did not show any increase in Butanol rather decrease). This saves 90% butanol if we are not recycling the aqueous layer. In addition to furfural, hydroxymethyl furfural (HMF) is also identified in the aqueous liquor. According to the literature (Virent work and U Madison work), HMF generally form from C6 sugars (we have some C6 sugars in the aq. liquor) can be converted to dimethyl furan (DME). Both furfural and DME can be substituted as petrochemical fuel with better calorific values than ethanol. A schematic of this secondary reaction is shown in the fig 16.

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F. Isoprene Capture BAC carbon obtained from Carbon Resources Corp. was tested for its ability to adsorb/desorb isoprene. Based on the results shown in 17, BAC carbon can adsorb and desorb isoprene under the condition of the experiments. Based on the data obtained, heating the BAC to 300oC in needed to release the isoprene from the solids. Varying the amount of BAC (from 0.2 to 0.1 gr) did not have a major effect in adsorption as shown in Fig 18, however and to our surprise, the desorption was not as effective. It has to be born in mind that due to the fluctuating nature of these experiments, some may need to be repeated to confirm the results. Fig 19 shown the profiles of isoprene adsorption/desorption by reusing BAC carbon. It was very interesting to see that upon prior desorption of isoprene, the BAC carbon can be reused to adsorb/desorb isoprene. The amount of desorption was approximated by measurement of the area under the curve and found to be 60%. More experiment must be done to confirm the results. Further lowering the amount pf BAC carbon was not effective as shown in Fig 20 . It can be concluded that 0.1-0.2 gr BAC carbon are required under the conditions tested.

0

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Isoprene Adsorption/desorption by BAC carbon, 0.2 gr

51 ppb

isoprene flow was discontinued, heating the reactor to 100 C

Heating the reactor to 300 C

Heating the reactor to 500 C

1305 ppb, isoprene was passed through the reactor

Figure 17. Isoprene adsorption by BAC carbon (0.2 gr)

0

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Isoprene Adsorption/desorption by BAC carbon, 0.1 gr

44.3 ppb

isoprene flow was discontinued, heating the resator to 300 C

heatingthe reactor to 500 C

976 ppb, isoprene was passed through the reactor

Figure 18. Isoprene adsorption by BAC carbon (0.1 g)

22

0

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0 200 400 600 800 1000 1200 1400 1600

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Isoprene Adsorption/desorption by BAC carbon, 0.1 gr

47.5 ppb

Heating the N2 line to 300 C

1290 ppb, isoprene was passed through the reactor

Figure 19. Isoprene adsorption/desorption by reusing BAC carbon

0

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Isoprene Adsorption/desorption by BAC carbon, 0.05 gr

490 ppb

isoprene flow was discontinued, heating the reactor to 200 C

Heating the reactor to 300 C

Heater malfunction

1605 ppb, isoprene was passed through the reactor

Figure 20. Isoprene adsorption/desorption using 0.05 gr BAC carbon

Several solid adsorbants were tested for their efficacy of adsorbing isoprene gas. For the first experiment, sand was used in the study. This was done in order to establish whether a drop in isoprene detection by sensor (an indication of adsorption) is because of material or an inherent function of any solids. Isoprene adsorption by activated carbon (AC) is shown in Figure 21A. For this experiment, 0.5 gram of AC was used inside the reactor. The experimental procedure was as explained earlier. The difference between AC and char and sand is vividly clear. The “L” shape graph (almost 1400 ppb drop in signal) indicates successful adsorption of isoprene by AC however, release of isoprene could not be achieved after heating up to 800 C. Figure 21B shows a similar isoprene capture experiment using a polymer-activated carbon hybrid material. This material adsorbs isoprene similar to that of activated carbon under ambient conditions. Unlike activated carbon, this material releases isoprene when heated to 300 C.

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(A) (B)

Figure 21: (A) Isoprene capture using activated carbon (surface area 1800 m2/g) as the adsorbent and (B) Isoprene capture using polymer activated carbon hybrid material (Carbon resources, surface area 1350 m2/gm) as the adsorbent.

Quantification of adsorption using pure Isoprene: 1.142g of BAC carbon (mesoporous polymer-activated carbon hybrid material) was activated overnight at 70C. The activated BAC material was then packed in glass reactor (tube) using ceramic wool. 5 mL of isoprene (Aldrich, 99%, d 0.681, B.P. 33-34 C) was taken in a 100 ml round bottomed flask. The glass reactor was placed at the top of this RB flask and the flask was heated constantly at 36C. The temperature was maintained using a water bath. In this process, the evaporated isoprene was allowed to pass through the reactor containing BAC carbon. The glass reactor was constantly heated (using a portable drier) to stop the condensation of the isoprene vapors (reduce the chances of liquid adsorption). After 2h, the solid was taken out from the reactor and the amount of isoprene adsorbed into the material was calculated by the weight difference, which was 0.31g. This is equivalent to 0.27 g (0.184 mL) of isoprene per 1g of solid adsorbant.

G. Commercialization Plan Summary The propose commercialization plan is going to use AST’s pilot plant equipments including 50 Lb digester, 150 Liter fermenter, 20 Lb pyrolysis, and various separation systems to refine the laboratory developed technologies for scale up. AST is planning to use its equipment first to refine the technologies for producing isoprene from sludge, and then using wood chips. The results will be used to design and manufacture a scaled up digestion, pyrolysis, and separation as well as purchasing fermenters that are required for high volume production. The raw materials for this process will be forestry, agricultural, and industrial biomass wastes. As renewable and carbon neutral, the biomass wastes can help in reduction of CO2 production. The proposed plan has examined the economic viability of these types of operations.

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Currently several permits have been issued to burn woodchips to create steam that can be used in an old fashion power plants to generate electricity. While this process is at least a carbon neutral power generation, but it may not be the best way to use our natural resources. Conversion of the biomass wastes to high value chemicals can reduce consumption of fossil fuel (that otherwise, must be used to make these chemicals).

Impact of Technology and Product on AST’s Business Growth AST is determined to establish its Bio-Isoprene production unit and a pilot Poly-Isoprene Rubber (PIR) manufacturing on its central Wisconsin manufacturing facilities due to its affiliation with Resilient Technologies LLC that is in business of producing airless tires. Resilient Technologies LLC is predicted to be one of the major users of PIR. Since isoprene is a commodity product, AST anticipates supplying other end users of isoprene such as various tire companies as well. Doing so, AST will add tens of high quality jobs around Wausau WI. Currently paper companies produce mill sludge that cost them hundreds of thousands of dollars to be landfielled. As an example, Thilmany pay about $140,000 per year to get rid of its sludge. The mill sludge is potentially a free biomass raw materials that can be use to produce isoprene. A supply of 20 tons per day of sludge could produce 3 tons of isoprene per day generating revenue in excess of $4 million per year. This production rate is based on a long term goal of 80% conversion efficiency of sugars to isoprene. If the conversion operation is located by the paper mill, the transportation costs also will be eliminated and therefore will increase the profitability of the process. Market Overview Global consumption of isoprene in 2004 was 717,000 tons. Prices have ranged from $1.3 to more than $4 per pound over the past ten years. High-purity isoprene is used almost entirely (90–95%) as a monomer for the production of polyisoprene rubber, styrenic thermoplastic elastomer block copolymers (styreneisoprene-styrene [SIS]) and butyl rubber. Polyisoprene rubber accounted for 41% of isoprene consumption in the United States, Western Europe and Japan in 2007. In Central and Eastern Europe (mainly Russia), polyisoprene rubber accounted for approximately 99% of isoprene consumption in 2007. The balance is used in the production of fine chemicals such as vitamins, pesticides, pharmaceuticals and flavors. Isoprene’s industrial use is constrained by its tight supply, and the longterm trends in petroleum use and environmental awareness will necessitate an increased supply. Overall, consumption of synthetic polyisoprene rubber has decreased significantly since the 1970s because of displacement by natural rubber, which has historically sold at a lower price. However, the purity of polyisoprene is higher, meaning its composition and properties are more consistent than those of natural rubber, giving polyisoprene advantages in certain tire, medical and other specialized applications. The use of latex is limited in medical applications due to natural impurities that can cause allergic reactions. The source of the natural impurities is the rubber plants that provide the natural rubber used to manufacture latex. Polyisoprene rubber is often substituted for natural rubber latex because it does not have natural impurities that can cause allergic reactions. Specific medical applications that benefit from the superior performance of Kraton Poly-Isoprene Rubber include catheters, surgical gloves, medical stoppers, medical tube connectors, dental dams, physiotherapy bands and personal care products. Industrial isoprene is produced as a byproduct of polyethylene (a petrochemical) production. For this reason, most synthetic rubbers are made from butadiene, a more readily available monomer, but one that is substantially more toxic than isoprene. Greater availability of isoprene in the marketplace will allow it to displace more toxic petroleum products. Furthermore, isoprene yields from petroleum have been decreasing due to the transition from light, sweet crude to less ideal petroleum sources. Global awareness of the importance of tropical forests will further impact the future market for isoprene. Rubber plantations deplete the tropical groundwater sources, reduce biodiversity, and eliminate natural forests.

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Market Potential The existing global isoprene market is approximately $2 billion. However, with an increase in supply and displacement of rubber sources the market will likely expand to over $30 billion. Most of the isoprene monomer is made from petroleum as a byproduct of ethylene synthesis. As supply of light sweet crude declines and is replaced with heavier grade oil, the yield of isoprene per barrel of oil is decreasing. Of course, the petroleum industry is currently under closer scrutiny, which will likely lead to less oil exploration and supply in the future. The other threatened source of rubber is natural latex from the rubber tree, Hevea brasiliensis. Displacing natural rubber for the production of a higher quality polyisoprene can potentially displace the natural rubber market as environmental concerns and disease risks put pressure on tropical rubber plantations. Thus, the challenges facing the petroleum and natural polyisoprene industries indicate that there is ample room for market expansion over the 21st century. The major market risks involve competition in the supply chain as well as development of bio-isoprene from competitors. As new bioprocessing and low-carbon energy sources are developed, the supply of biomass is likely to be constrained. The sludge to isoprene process should be more resilient in the face of these risks as the product is of relatively high value and the biomass supply comes from processing waste rather than newly-harvested biomass. Competition may increase as other players develop sugar to isoprene processes, which will drive down prices, but isoprene is anticipated to maintain a healthy margin over the price of fuel. From the completion of this project, pulp mills will be able to enter this market in 2-5 years. Saccharification, fermentation, and isoprene capture technologies currently exist. This project will use new biotechnology in existing technology to produce a relatively high value hydrocarbon from a waste stream. Barriers to Market Barriers to market include capital equipment and the large investment needed to bring this technology to market. We believe that capital equipment investments will not be a large hurdle as long as the isoprene yield is high enough to generate adequate return on investment. The largest barrier is the investment needed to bring the biotechnology to commercial scale. This project will bring the isoprene technology to 10% commercial scale. At that point, we anticipate the ability to attract significant private investment to develop the technology for commercial application. Cost analysis To be more realistic, for this analysis, we have assumed the long term planning of scale up integrated bio-refinery that can use wood chips as the input raw materials. The other assumptions are as follow:

1) Woody biomass will produce in average 45% Cellulose, 20% Hemi-cellulose, 30% lignin, and 5% wastes.

2) Isoprene production from cellulose yields 30% product at $1.30 per Lb;

3) Production of ethanol from hemi-cellulose yields 50% product with 11500 BTU/Lb;

4) The lignin will be fed to pyrolysis to produce 60% bio-oil (11500 BTU/Lb), 10% bio-char (12000 BTU/Lb), 25% Biogas (6000 BTU/Lb).

5) The price of energy is $0.025/K-BTU

6) All wastes are going to be dried and fed into pyrolysis

Based on the above assumptions, the following table presents net incomes from one, ten, and hundred dry tons of woody biomass per day.

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Required Capital $1,000,000 $4,000,000 $25,000,000

Raw Materials Dry

Ton/Day 1 10 100

PERFORMANCE

Plant Operating Days 350 350 350

Plant Availability Factor 85.0% 85.0% 85.0%

Production Lb/T Dry Wood Units(1)

#1 Butanol 720 Lb/Y 214,200 2,142,000 21,420,000

#2 Ethanol 200 Lb/Y 59,500 595,000 5,950,000

#3 Bio-Oil 648 Lb/Y 192,780 1,927,800 19,278,000

#4 Bio-Char 108 Lb/Y 32,130 321,300 3,213,000

Bio-Gas 216 Lb/Y 64,260 642,600 6,426,000

Feedstock Consumption Units(1)

A Short Rotation Hybrid

Poplar Ton/year 298 2,975 29,750

Utility Consumption

Net Electricity Consumed MWh 420 4,200 42,000 Water Consumed kGal 619 6,190 61,900 Enzymes 12.5 Tons 4 37 372 COMMODITY PRICES

Product Pricing Units(1)

#1 Butanol $0.32 $/Lb $0.32 $0.32 $0.32

#2 Ethanol $0.28 $/Lb $0.28 $0.28 $0.28

#3 Bio-Oil $0.29 $/Lb $0.29 $0.29 $0.29

#4 Bio-Char $0.30 $/Lb $0.30 $0.30 $0.30 Bio-Gas $0.15 $/Lb $0.15 $0.15 $0.15

Feedstock Pricing Units(1)

A Short Rotation Hybrid

Poplar $/Ton $75.00 $75.00 $75.00

Utility Consumption

Natural Gas Consumed $/MMBTU $10.49 $10.49 $10.49

Net Electricity Consumed $/MWh $54.53 $54.53 $54.53

Water Consumed $/kGal $1.25 $1.25 $1.25

Other Consumed $2,560.00 $/Ton $2,560.00 $2,560.00 $2,560.00

OPERATING REVENUES

Product Revenues

#1 Butanol $68,544 $685,440 $6,854,400

#2 Ethanol $16,660 $166,600 $1,666,000

#3 Bio-Oil $55,906 $559,062 $5,590,620

#4 Bio-Char $9,639 $96,390 $963,900

Bio-Gas $9,639 $96,390 $963,900

TOTAL OPERATING REVENUES $160,388 $1,603,882 $16,038,820

OPERATING EXPENSES

Variable Operating Costs

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Feedstock Costs

Short Rotation Hybrid

Poplar $22,313 $223,125 $2,231,250

Total Feedstock Costs $22,313 $223,125 $2,231,250

Utility Costs

Net Electricity Consumed $22,903 $229,026 $2,290,260

Water Consumed $774 $7,738 $77,375

Other Consumed $9,520 $95,200 $952,000

Total Utility Costs $33,196 $331,964 $3,319,635

Total Emissions Allowance

Costs $0 $0 $0

Total Feedstock

Transportation Cost $0 $0 $0

Total Miscellaneous

Supplies Cost $0 $0 $0

Total Variable Operating

Costs $55,509 $555,089 $5,550,885

$51,764 $517,639

Fixed Operating Costs

Payroll & Benefits % of

Revenue 54.40% 54.40% 54.40%

2

employees $110,000 $110,000 $330,000

Fixed Operating Expenses % of

Revenue 1.00% 1.00% 1.00%

$1,604 $16,039 $160,388

Annual Maintenance % of Capex 2.50% 2.50% 2.50%

$25,000 $100,000 $625,000

Total Fixed Operating

Costs $136,604 $226,039 $1,115,388

General & Administrative

Corporate Overhead % of

Revenue 8.00% 8.00% 8.00%

$12,831 $128,311 $1,283,106

Insurance % of Capex 0.50% 0.50% 0.50%

$5,000 $20,000 $125,000

Property Tax % of Capex 0.20% 0.20% 0.20%

$2,000 $8,000 $50,000

Management Fees % of

Revenue 4.00% 4.00% 4.00%

$6,416 $64,155 $641,553

IP Fees % of

Revenue 1.00% 1.00% 1.00%

$1,604 $16,039 $160,388

Total G&A Expenses $27,850 $236,505 $2,260,047

Contingency

Contingency % of O&M,

G&A 5.00% 5.00% 5.00%

$8,223 $23,127 $168,772

Total Contingency $8,223 $23,127 $168,772

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TOTAL OPERATING EXPENSES $228,186 $1,040,759 $9,095,092

NET OPERATING REVENUES ($67,798) $563,123 $6,943,728 Return of investment 10 years $100,000 $400,000 $2,500,000

Depreciation 10 Y/Linear $100,000 $400,000 $2,500,000

NET REVENUES ($267,798) ($236,877) $1,943,728

Based on the above numbers, to be able to scale up this technology, a large production plant is required (100 Dry Ton per Day or more). Small plants unless become more energy efficient, otherwise, will not make any profits.

Uncertainty Factors

A. Assumptions: Assumptions used in this report are based on our past experiences and the best currently available knowledge and business conditions as they currently exist in Northern Wisconsin. It is understood that assumptions may change based on differences relevant to proprietary technology and conditions unique to specific business groups and/or locations.

B. Critical Factors: Critical factors used in the economic analysis such as yield, capital cost, return on investment, overhead rates and etc. are consistent with those currently being used in other AST biomass conversion activities. These parameters may change as we build the scale up unit, with time, and/or local political developments.

C. Emerging Technology: The production of high value chemicals from biomass is in the development stage. The current calculations are based on a conservative prediction of the productivity. Any new development on emerging technologies may significantly shift this calculation toward a much higher profit activity D. Feedstock Availability: There is no assurance that existing markets for biomass wastes will not grow and/or that alternative uses of them will not be found. A competitive market for these wastes could result in a price increase, thus making the production of high value chemicals economically impractical. E. Energy Credits: Various State and Federal Agencies have enacted and/or proposed legislation to provide energy or carbon credits for specific applications. It is not certain that these credits will be enacted, but if they do, they will be applicable to a bio-chemical facility using currently available technology. For purposes of this report no benefits related to energy credits are assumed.

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Attachment One Final Report of Task Order One

Literature Review

1 - INTRODUCTION One of the emerging concepts in the process industry is the idea of a bio-refinery. One way of defining a bio-refinery is that it is a facility which converts biomass into liquid transportation fuels or valuable chemicals and other products. Because of the renewable nature of biomass, a bio-refinery is an important element in a circular economy which seeks to minimize negative ecological impact. The biomass used in biorefineries may be in different forms such as forest products, agricultural products, and bio-wastes. Over the past decade, scientists have raised concern about the sustainability of the traditional industries and how some practices may jeopardize our future. Depletion of natural resources and discharge of hazardous wastes are disconcerting factors. The greenhouse gas emission has also become a cause of concern (Mintzer and Schartz 2003). A bio-refinery can address some of these concerns because it involves the usage of renewable resources, it is part of a sustainable life cycle, and it normally results in less green house gas emissions compared to fossil-based processes. As such, a bio-refinery is regarded as a serious alternative to produce energy and chemicals in the near future. 2 - BACKGROUND In the 1970’s, there was an initial surge of interest in biomass-for-energy facilities following the energy crisis and the increase in crude oil prices. Many of the original ideas were not commercially pursued because of the subsequent decline in oil prices. However, recently the situation has changed with the increasing attention to global warming and the increasing prices of fossil fuels. The biomass-based fuels and products can make contributions to the U.S. environment, chemicals, power, and economy. As such, the concept of a biorefinery seems to be a serious option to improve the condition of our future while enhancing our economy. In this section, the background and literature survey will be presented in the following order: the current definition of biorefinery, characterization and availability of biomass feedstock, economic aspect, and chemical available through biorefinery. 2.1 Biorefinery Future refineries will encounter feedstock problems in terms of availability and cost. As the price of crude oil increases and the supplies diminish, there will be a need to find alternative sources of energy feedstocks. One solution of these issues is called biorefinery. The term biorefinery was recently coined to address refineries capable of converting biomass to valuable chemicals or energy. As more renewable sources are used, less waste is generated in the ecological cycle. Biomass products can range from biomaterials to fuels or important feedstocks for the productions of chemicals and other materials. Biorefineries can be based on a number of processing platforms using mechanical, thermal, chemical, and biochemical processes. Biorefinery conversion of biomass should be considered not only as a substitute for fossil resources but also as an integrated use of living organisms, microorganisms and enzymes in

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a cycle of producing foodstuff, fuels, feeds, value-added chemicals and industrial materials. In the literature (Sophonsiri and Morgenroth 2004), there are numerous examples showing that municipal wastes can be decomposed into valuable products such as energy, chemicals, and byproducts. 2.2 Characterization and availability of biomass feedstocks The biomass feedstock is divided in two categories, grains and lingo-cellulose. Grains feedstock is such as corn and other corps. Lignocellulosic feedstock is composed of cellulose and hemi-cellulose, which represent about 80% of biomass feedstock. Herbaceous and woody biomasses are composed of carbohydrate polymers such as cellulose and hemicellulose, lignin and small parts of acids, salts and minerals. The cellulose and hemicellulose compose about two-thirds of a dry biomass. The following is a typical composition of lignocelluloses (Scurlock 2003): Cellulose, between 40% and 60% of the dry biomass, is formed of glucose-glucose dimer. Hydrolysis is needed to break down the hydrogen bond (hard to break) and the product, glucose, is a six-carbon sugar. Hemicellulose, between 20% and 40% of dry biomass, consist of short highly branched chains of various sugars, mainly five-carbon and six-carbon as Xylose, Arbinose, galactose, glucose and mannose. Hemicelluloses are easy to break down during the hydrolysis. Lignin, between 10% and 25% of dry biomass, counted as a residue during the ethanol process. The key element for biorefinery is the availability of biomass. According to a recent study by the Department of Energy, the current quantity of biomass in the entire U.S. is about half of a billion dry tons (U.S. Department of Agriculture 2005). This study has shown the different categories of biomass and their quantities. The study by the Department of Energy also predicts that over one billion ton of biomass would be available after some modifications of the biomass. The forestland and agricultural land are the two largest potential biomass sources. Currently 1.3 billion dry ton per year is available, this can supply one-third of the actually demand of transport fuels. Biomass supplies about 3% of energy consumption in the United State from the pulp and paper industry and electric generation using forest industry residues and municipal solid waste.It is estimated that biomass will supply 5% of the nation’s power, 20% of its transportation fuels, and 25% of its chemicals by 2030 (U.S. Department of Agriculture and U.S. Department of Energy 2005). The surface area of agricultural land is 455 million acres, of which 349 million acres of land are in active use, 39 million acres are idle, and 67 million acres of cropland pasture over 48 states (U.S. Department of Agriculture and U.S. Department of Energy 2005). The biomass is not similar to the petroleum feedstock. Since the biomass is renewable resource. 2.3 Economics aspect of biomass feedstocks The other side of the biomass feasibility is the price at which biomass is available. The delivered price varies as a function of many factors including the type of biomass, harvesting and collection techniques, storage, the hauling distance, the region, and the available quantity. The other side of the economic aspects of biomass utilization is the potential credit and/or subsidy associated with the reduction in greenhouse gas (GHG) emissions. 2.3.1 Delivered cost of biomass Biomass feedstocks offer a distinct advantage being a renewable resource. For instance, every year corn has can be planted, grown, harvested and transported to the biorefinery to produce ethanol or other energy-related chemicals. The delivered cost of biomass depends on the expenses associated with growth, harvesting, and transportation. Walsh et al. (1999) published a report estimating the quantities and delivered cost of biomass for every state in the U.S.

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2.3.2 Greenhouse emission effect Renewable energy such as bio-derived ethanol and Butanol is produced from plants that use solar energy to grow. Combustion of a bio-fuel releases a portion of this energy; solar energy and the use of carbon dioxide are important steps in the life-cycle of an energy crop. This cycle entails the net release of a much smaller quantities of greenhouse gases (GHGs) compared to fossil fuels. Most of GHG emissions from process industries are related to combustion of fossil fuel. In U.S. several state are working on the legislations of GHG emissions. Therefore, bio-fuels represent one of the most promising options for reducing GHG emissions from the transportation sector. 2.4 Chemical potential of biomass feedstocks Today’s biomass uses include ethanol, Butanol, bio-diesel, biomass power and industrial process energy. In preparing biomass for bio-refineries, there are two primary pathways: hydrolysis of cellulosic biomass to sugars and lignin, and thermo-chemical conversion of biomass to synthesis gas. The sugar platform is composed of thermo-chemical pretreatment and enzymatic hydrolysis. The pretreatment is usually done with dilute acid and breaks down the hemicellulose into sugars such as Xylose Cellulose are enzymatically hydrolyzed to release glucose. Then, the produced sugar is available for fermentation to ethanol. Some forms of used biomass involve corn Stover, sorghum and seeds. Thermo-chemical technologies utilize catalysis and/or high pressure and temperature to convert biomass. Oils and bio-products from wood resources are used as such. The lignocellulosic biomass represents an energy potential. Gasification and pyrolysis are used to convert biomass into an energy fuel. It mostly used to produce electrical energy by cogeneration. Example of used biomass includes switch grass and wood. There are also other platforms such as biogas, carbon-rich chains, plant products and bio-oil which are beyond the scope of this work. Biogas platform is the decomposition of biomass into methane and carbon dioxide by anaerobic digesters (El-Halwagi 1986) Carbon-rich chains platform is the transesterification of vegetable oil or animal fat into fatty acid methyl ester, commonly known as bio-diesel. The plant products platform is the use of selective breeding and genetic engineering that can produce greater amounts of desirable feedstock and chemicals. Bio-oil may be produced through the pyrolysis of biomass to produce oil with similar characteristics to petroleum cuts. From these chemical, the potential derivatives of these chemicals is endless. For instance, it is possible to derive eight other chemicals from lactic acid and so and so. These chemicals represent an attractive target for new bio-based products. Waste is also considered as part of biomass, the municipal, industrial and agriculture wastes can be broken down into simple compounds such as proteins, carbohydrates, and lipids (Sophonsiri and Morgenroth 2004). A Bio-refinery may produce a wide variety of potential chemicals; one of the most promising bio-refinery products is ethanol. Ethanol is mainly produced by fermentation (95% fermentation, 5% synthetic from ethylene). Ethanol could be used as an alcoholic beverage, industrial alcohol, and/or fuel-related alcohol (Berg 2003). Ethanol represents an easy bio-fuels alternative for the near future. Ethanol is less expensive than the other oxygenates octane enhancers and often conventional gasoline. Ethanol is also seen as a way of reducing dependence on foreign sources of oil and gas. It can also lead to enhancing environmental quality. Ethanol reduces vehicle emissions. For instance, according to Dinneen (2005), if a10%-ethanol 90%-gasoline fuel blendes used, this will:

-Reduce tailpipe fine particulate matter (PM) emissions by 50% -Reduce secondary PM formation by diluting aromatic content in gasoline -Reduce carbon monoxide (CO) emissions by up to 30% -Reduce toxics content by 13% (mass)

The United States is not the only country that is closely considering expanding the use of ethanol as a bio-fuel. (Dinneen 2005) Many countries try to reduce petroleum imports, enhance the air conditions, and boost their

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agricultural economics. Ethanol addresses all of these objectives. The growth of ethanol production in these countries is estimated to accelerate as the countries account for the greenhouse gas emissions. The United States has witnesses an increasing level of interest in ethanol production. For 2005, the production of ethanol is estimated at 4,400 millions of gallons. It is expected that the U.S. will become the top producer of ethanol surpassing Brazil which has remained the world’s leading producer of ethanol for the past half a century The U.S. ethanol plants are mainly situated in 20 states throughout the country. The biggest plants are in the north of the U.S. in states such as Iowa, Illinois, Nebraska, and Minnesota. The fermentation process is the dominant process in the market (95% of ethanol production). Ethanol is fermented from grain and some from Lignocellulose. Grain-derived ethanol is produced via the wet-milling or dry-milling processes (BBI International 2003). In the dry-mill process, the corn is ground into flour and is processed without any separation of component parts. In the wet-mill process, the corn is soaked or steeped then separated into its component parts, which are recovered prior to fermentation. Both processes release carbon dioxide while producing ethanol. The key difference is in the set of byproducts. The demand for these byproducts is an important factor determining the type of the plant. Notwithstanding several advantages of bio-derived ethanol, these plants have limitations. These limitations may include the dependence on natural gas, the geographical position, the presence of sufficient market demand for the byproducts, competition and availability for feedstock, and the sensitivity of fermentation. When these limitations are relevant, they must be addressed and balanced against the benefits of bio-derived ethanol. 3. Butanol Liquid fuel use accounts for the single largest share of petroleum oil consumption in the United States. In 2006, the United States consumed more than 20 million barrels of crude oil per day; 66% of this total was used in the transportation sector. Motor vehicles alone consumed 140 billion gallons of gasoline and 50 billion gallons of diesel in 2006. Gasoline use has increased as a result of the growth in light-duty vehicle (LDV) travel in the past 20 years. The Energy Information Administration projected that transportation fuel use will continue to grow up to 30% by 2030 (Conti 2007). On the petroleum supply side, the United States relies heavily on foreign oil (13.7 million barrels per day, EIA 2007). The world’s most oil-rich region has become extremely unstable, which heightens energy security concerns. Furthermore, competition for petroleum oil has increased dramatically as a result of rapid economic growth in developing countries. Finally, exploration, production, and use of petroleum-based fuels generate greenhouse gas (GHG) emissions, which are the primary cause of global warming, as confirmed in a recent report prepared by the Intergovernmental Panel on Climate Change (IPCC 2007). Considering the challenges facing the United States in its continued reliance on fossil-based fuels in the transportation sector, many researchers are exploring other alternatives. Finding a liquid transportation fuel that (1) can be produced from domestic resources, (2) is carbon neutral, and (3) has minimal GHG impacts would allow the United States to reduce our dependence on foreign oil and decrease environmental burdens. The President stated his goal of displacing 20% of gasoline demand by renewable fuels and vehicle efficiency improvement — that translates to 35 billion gallons of biofuels and alternative fuels in 10 years. Following dramatic growth in the ethanol industry, corn ethanol (EtOH) production reached a record 4.9 billion gallons in 2006. Yet, this total accounts for only 2.3% of the total U.S. gasoline supply (in gallons of gasoline equivalent). Even considering a U.S. Department of Agriculture (USDA) projection that corn ethanol production could reach 12 billion gallons by 2017 (Collins 2007), a large gap remains to be filled by biofuels. Therefore, developments in feedstocks, processing technologies, and new biofuels are urgently needed if the United States is to meet the President’s target of 35 billion gallons per year by 2017. Among potential biofuels, Butanol (BuOH) produced from starch has gained visibility in recent years as a replacement for gasoline. Butanol has unique properties as a fuel. The energy content of Butanol — 99,840 Btu per gallon (low heating value [LHV]) — is 86% of the energy content of gasoline (on a volumetric basis) and 30% higher than the energy content of ethanol. The low

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water solubility of Butanol could minimize the co-solvency concern associated with ethanol, consequently decreasing the tendency of microbial-induced corrosion in fuel tanks and pipelines during transportation and storage. Butanol is much less evaporative than gasoline or ethanol, making it safer to use and generating fewer volatile organic compound (VOC) emissions. The majority of Butanol used as a chemical is produced from petroleum propylene through the Oxo process (in which synthetic gas [syngas] is reacted with propylene), and its ultimate end use is for surface coatings. The most dominant bio-Butanol production process has been Acetone-Butanol-Ethanol (ABE) fermentation. ABE fermentation by Clostridium Acetobutylicum was the route used to produce Butanol during World War II. It was phased out when more economical petrochemical routes emerged. Now, almost all Butanol in the world is produced from petrochemical feedstocks. Research interest in developing viable ABE fermentation processes has been rekindled recently as a result of the pursuit of non-fossil-based feedstocks. In the past 20 years, research and development (R&D) efforts have focused on various aspects of the ABE process. Molecular biology research has achieved major breakthroughs in strain/mutant development that dramatically improved microbial tolerance to Butanol toxicity, which resulted in a significant increase in ABE solvent production yield. Experimental and computational engineering efforts have included designing new schemes to minimize Butanol inhibition by using new fermentor configurations, improved downstream processing, and process integration. Huang et al. (2004) reported an experimental process that uses continuous immobilized cultures of Clostridium tyrobutyricum and Clostridium Acetobutylicum to maximize the production of hydrogen and butyric acid and convert butyric acid to Butanol separately in two steps. This process reportedly produced Butanol at a rate of 4.64 grams per liter of fermentation medium per hour (g/L/h) and used 42% glucose, compared with the up-to-25% glucose use rate in traditional ABE fermentation by Clostridium Acetobutylicum alone. In the early, 1990s Clostridium beijerinckii BA101 was developed by using chemical mutagenesis together with selective enrichment, which is able to produce twice as much Butanol as its parent strain (US Patent 6358717). Extensive studies have been performed to characterize this strain and develop an ABE process with various feedstocks and evaluate technologies for downstream product separation (Qureshi and Blaschek 1999; Parek et al. 1999; Qureshi and Blaschek 2001a and 2001b). Experimental and pilot-scale ABE fermentation processes by this organism resulted in up to 95.1% glucose utilization in fermentation. Using in-situ gas stripping for solvent removal from fermentor minimizes product inhibition and enables higher feed concentration — up to 500g/L of glucose (Ezeji et al. 2004). Solvent production in this process in a fed-batch mode reached 65:35:1 of Butanol: acetone: ethanol by weight, which is a significant increase in Butanol production from 6:3:1 in conventional ABE process. Liu (2003) presents an exhaustive survey of major research findings on ABE downstream processing. Recent studies have focused on integration of fermentation and product removal through in-situ gas stripping and fermentation gas recirculation (Qureshi and Blaschek 2000; Qureshi and Blaschek 2001a; Qureshi and Blaschek 2001b; Ezeji et al. 2004; and Ezeji et al. 2005). The latest development includes a DuPont patent (2007) describing a strain that produces Butanol from biological feedstocks while minimizing acetone production. Cellulosic feedstock for Butanol production has also been reported (Qureshi et al. 2007). Researchers have employed computer simulations in developing Butanol production processes, including ABE fermentation. The earliest efforts in downstream processing simulation of ABE fermentation were reported in Marlatt and Datta (1986) and Dadgar and Foutch (1988). In these studies, simulations were used to evaluate the economics of their processes. More recent studies were published in Liu (2003) and Liu et al. (2004 and 2006). In these studies, downstream processing systems were synthesized, simulated, and optimized in terms of cost. There are comparatively few publications on ABE fermentation process simulation. A corn-to- Butanol pathway has been modeled by NRC (Natural Resources Canada, Feb. 2007) recently, on the basis of earlier work of conventional ABE fermentation. The study examined corn-based Butanol used as 10% Butanol in a gasoline blend to fuel light-duty vehicles. Simulation results for fuel ethanol produced from corn via dry milling, such as those obtained by using USDA’s dry mill model (Kwiatkowski et al. 2006; McAloon 2006), are readily available. Since 1995, with support primarily from DOE’s Office of Energy Efficiency and Renewable Energy, Argonne has been developing the Greenhouse Gases, Regulated Emissions and Energy Use in Transportation (GREET) model.

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Argonne released the first version of the model — GREET 1.0 — in June 1996. GREET is a Microsoft® Excel™-based multidimensional spreadsheet model that addresses the well-to-wheels (WTW) analytical challenges associated with transportation fuels (including ethanol) and vehicle technologies. The latest versions— GREET 1.8 is capable of analyzing more than 100 transportation fuel pathways and 75 vehicle/fuel systems (Brinkman et al. 2005). The GREET model has been updated frequently to reflect new feedstocks, processing technologies, fuels, and vehicle systems. For a given vehicle and fuel system, GREET separately calculates:

• Consumption of total energy (energy in non-renewable and renewable sources), fossil fuels (petroleum, natural gas, and coal combined), petroleum, natural gas, and coal; • Emissions of carbon-dioxide (CO2) -equivalent GHGs — primarily CO2, methane (CH4), and nitrous oxide (N2O); and • Emissions of six criteria pollutants: VOCs, carbon monoxide (CO), nitrogen oxides (NOX), particulate matter measuring 10 micrometers or less (PM10), particulate matter measuring 2.5 micrometers or less (PM2.5), and sulfur oxides (SOX). These criteria pollutant emissions are further separated into total and urban emissions. This study was an attempt to evaluate the potential of the recent ABE process from a life- cycle perspective. This estimate provides a life-cycle analysis (LCA) of the production and use of corn-derived bio-Butanol as transportation fuel to displace petroleum gasoline. First, they developed an ABE process simulation model by using Aspen Plus®. They used the energy and mass balance resulting from the Aspen Plus® simulation as a basis for a life-cycle analysis of corn-based Butanol production and use. They estimated the life-cycle energy and GHG emissions impacts of corn-based Butanol (produced via the ABE process) when used to displace gasoline as a transportation fuel in LDVs. We also performed a “cradle-to-user” analysis for bio-acetone (which is co-produced with bio-Butanol) to address the impacts of displacing petroleum-based acetone with Research focuses on the production of higher (bio) alcohols and other compounds suitable as oxygenates (e.g. Butanol, pentanol, mixed alcohols; e.g. glycerin ethers, glycerol carbonate).

The objectives of the Butanol future production projects are:

1) To evaluate the old and novel procedures for microbiological production of Butanol, higher alcohols and oxygenates as fossil fuel substitutes in order to find the most efficient and feasible processes, 2) To develop and optimize catalytic materials and chemical reaction routes for the production of higher alcohols and other bio-derived compounds applicable as gasoline fuel and its additives, 3) To conduct a sustainability analysis of the processes to be developed. To analyze the atom economy of the new processes and to make a preliminary economical analysis and 4) To integrate the processes and know-how developed by the research groups.

Microbiological approach starts from biomass by means of fermentation. Several Clostridium species are able to metabolize carbon compounds to Butanol (butyric acid as an intermediate) in high yields. The main challenges are 1) digestion of the raw material (e.g., by-products of food or paper/pulp industry) to fermentable sugars, 2) complex multistage fermentation process, 3) inhibition caused by high solvent contents and 4) instability of solvent production. The recent progress in Butanol fermentation techniques (e.g., immobilization of microorganisms and separation of acidogenesis and solventogenesis to different bioreactors) has partly solved these problems. Also the separation of Butanol is a technological challenge. Butanol, although significantly less water soluble than ethanol, still exhibits water solubility and forms a separate phase only after the concentration exceeds 7%. A possibility for improving the efficiency is extraction by means of an organic or ionic liquid layer residing separated of the hydrophilic layer that has a high affinity for Butanol. Various immobilization techniques for microorganisms can be used to improve the catalytic activity and optimally

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facilitate also the phase-out of the product. They may also enable fixed bed technologies, separation and recycling of microorganisms. As a second approach, a chemical synthesis path starting from compounds such as glycerol (a by-product of the 1st generation biodiesel manufacturing) or methanol/ethanol by means of novel catalytic processes is studied. This approach is more challenging because a direct catalytic conversion route from glycerol involving e.g. chain elongation is currently not available in the literature, i.e. entirely new synthesis route and catalyst development is required. However, certain concepts adapted from recent literature are expected as key-steps for the desired goal. These technologies are expected to be applicable for the conversion of glycerol (a higher alcohol) and lower (bio) alcohols into high-value liquid fuels. The integration of both the fermentation and the chemical route for chemicals suitable as liquid fuel substitutes or additives is also considered. A .Summary of the results During the reporting period, research has been carried out both in fermentation as well as in the development of novel catalytic reaction pathways for biofuels and fuel components. An essential part of the project has been the development of analysis methods as well as characterization of catalysts. Biobutanol production is a two-stage fermentation process where acetic and butyric acid, CO2 and H2 are first produced in the acidogenic phase. Then the culture undergoes a metabolic shift to a solventogenic phase, and acids are converted to acetone and Butanol. The product inhibits the yield of Butanol and acids, making an integrated product separation process highly favorable. Literature studies have been performed on the properties of Butanol, and the production methods. The most common raw materials, pre-treatment methods, factors related to fermentation and methods for product recovery were covered with their applications, influences and limitations for the whole production process. The pre-treatment of biomass to fermentable sugars before the fermentation is a challenging task. Lignocellulosic materials (wood chips, bark sludge, bio sludge and fiber sludge from a pulp mill) were dissolved in the ionic liquid into sugars. The second aim was the successful fractionation of samples with liquid-liquid extraction for analysis. The real samples, if suitable for conversion to sugars, could then be utilized in the biofuel production. Sample pretreatment procedure was developed. Dried samples were characterized and dissolved in the ionic liquid [BMIM][Cl]. The fiber sludge dissolved totally. Instead, the bio sludge did not dissolve at all and the bark sludge dissolved only partly. The samples were fractionated by liquid-liquid extraction and the fractions were analyzed by GC-MS, ESI-MS and HPLC. Based on the results, glucose was found in some of the samples, while all samples showed the presence of cellulose. The fiber sludge is a potential fraction for further studies. Clostridial fermentation system was established including anaerobic reactor system (2 L) with temperature control and pH monitoring, cell immobilization unit, and fermentation medium circulation. Higher cell concentration and shorter lag phase prior to fermentation were achieved by cell immobilization. Without any further optimization, 2.1 g/L of Butanol was produced from whey permeate supplemented with a yeast extract. Simultaneously, butyric acid was produced 5.8 g/L and acetic acid 3.2 g/L, suggesting that the acidogenic phase was working more efficiently compared to the solventogenic phase. Also the two-step fermentation, where acid products from C. tyrobutyricum fermentation were fed to the C. Acetobutylicum, was tested. Fermentation system will be further developed and optimized, and experiments with whey and other substrates will be done. A product removal system is also included into the fermentation system. Development of analysis methods for liquid and gas phase reaction compounds have been performed The production of acetone and Butanol [AB fermentation] by solvent producing clostridia was one of the first large-scale industrial fermentation processes using a pure culture. It had been the major method to supply

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acetone and Butanol until the petroleum-based process became dominant in the early1960s [Jones and Woods, 1986]. Since the oil crisis of the 1970s, the use of renewable resources has received increased attention [Davison et al., 1997; Dürre, 1998; Finkelstein and Davison, 1998; Morris, 1993; Schilling, 1995]. The solvent-producing clostridia have been well studied with respect to their biochemistry and physiology. However, the genetic studies on clostridia and other obligate anaerobes have lagged behind those of aerobic species. Currently, attempts are underway to establish systems for genetic manipulation of Clostridium Acetobutylicum, Clostridium beijerinckii, and other solvent producing species [Minton et al., 1993; Papoutsakis and Bennett, 1993; Green et al., 1996; Wilkinson and Young, 1994; Johnson et al., 1997; Jones and Keis, 1995]. Recently, some significant advances in the study of the genetics of solvent-forming clostridia have been achieved. These include gene transfer [Lee et al., 1992; Li and Chen, 1995; Minton et al., 1993; Oultram and Young, 1985; Papoutsakis and Bennett, 1993]; homologous recombination [Green et al., 1996; Wilkinson and Young, 1994; Wong and Bennett, 1996]; physical maps of two G. LI Ó 1998 2species and the whole genomic sequence of C. Acetobutylicum ATCC 824 [Cornillot et al., 1997a; Wilkinson and Young, 1995; Smith et al., 1998]; a reporter system [Minton et al., 1993]; and genetically engineered Clostridial strains that produce high concentrations of solvents Mermelstein et al., 1992; Mermelstein et al., 1993; Green et al., 1996; Evans et al., 1998]. In this review, a brief history of solvent fermentation will be given. Attention will be focused on the progress of research on the metabolic switch from acid production to solvent production, gene regulation, genetic tools, and genetic engineering of solvent-producing bacteria. B. History of solvent production Chaim Weizmann isolated C. Acetobutylicum and developed the starch based Weizmann process at the University of Manchester, U. K., before the First World War [Jones and Woods, 1986]. The First World War stimulated the interest in acetone fermentation, which was used for the manufacture of cordite.Later on, it was realized that Butanol was an ideal solvent for quick-drying lacquer for the fast-growing automobile industry [Gabriel, 1928; Gabriel and Crawford, 1930]. Since the 1930s, strains belonging to other species, including C. beijerinckii, were isolated and used in the molasses-based fermentation process. After 1936, plants were also built in a number of other countries, including Australia, Canada, China, India, Japan, South Africa, and the USSR [Jones and Woods, 1986]. When the Second World War started, acetone fermentation was given top priority for the manufacture of munitions [McCutchan and Hichkey, 1954; Walton and Martin, 1979]. At that time, a plant in Illinois had G. LI Ó 1998 3 96 fermentors, each with a 50,000-gallon capacity. The fermentation process declined rapidly in the United States during the 1950s because of competition both from the petroleum-based synthetic industry and from the use of starch/molasses as animal feed. The most important economic factor for solvent fermentation is the cost of the substrate, which accounts for about 60% of the total cost. Two other factors are low solvent concentrations in the fermentation broth and strain degeneration, which refers to a strain losing its ability to produce solvents during serial transfers or prolonged cultivation [McCoy and Fred, 1941; Morris, 1993; Walton and Martin, 1979]. To revive solvent-fermentation by the clostridia, the metabolic pathways, gene regulation, and genetic manipulation have been investigated in the solvent-producing bacteria. C. Metabolic pathways and solvent production The metabolic pathways for solvent-producing clostridia have been reviewed [Chen, 1993; Dürre and Bahl, 1998; Girbal and Soucaille, 1998; Mitchell, 1998]. Acid- and solvent-producing pathways share the central reactions from acetyl-CoA to butyryl-CoA [Bennett and Rudolph, 1995]. Branch points arise from three key intermediates: acetyl-CoA, acetoacetyl-CoA, and butyryl-CoA. Metabolic switch from acid production to solvent production .In a batch culture, solvent-producing Clostridium species produce acetate, butyrate, carbon dioxide, and hydrogen during the acidogenic phase. The accumulation of acids results in a decrease in the pH of the culture medium at the early stage of growth. During the later part of exponential growth, the G. LI Ó 1998 4 metabolism of the cells undergoes a switch to solventogenesis to yield acetone, Butanol, and/or Isopropanol. A mechanism(s) for the switch from acid production to solvent production is to be defined. The activity of

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phosphotransacetylase, phosphotransbutyrylase, and acetatekinase rapidly decreases when the shift to solvent production occurs in C. Acetobutylicum [Hartmanis et al., 1984; Hartmanis and Gatenbeck, 1984], whereas the activity of solvent-forming enzymes increases 20 to 200 fold in C. Acetobutylicum and C. beijerinckii [Andersch et al., 1983; Ballongue et al., 1985; Grupe and Gottschalk, 1992; Yan et al., 1988]. During the solventogenic phase, conversion of preformed acids occurs concomitantly with the continued consumption of carbohydrate. Utilization of acetate and butyrate is directly coupled to the production of acetone by way of acetoacetyl-CoA: acetate/butyrate CoA-transferase [Girbla et al., 1995; Jones and Woods, 1986]. These reactions normally result in an increase in the pH of the culture medium. It would be impossible to obtain a good yield of Butanol without the production of acetone coupled to uptake of acids [Hartmanis et al., 1984]. Environmental signals and solvent production Organic acids, such as acetate and butyrate in the undissociated form are able to partition in the cell membrane and behave as uncouplers, which allow protons to enter the cell from the medium [Foster and McLaughlin, 1974; Freese, 1978; Huesemann and Papoutsakis, 1986; Kell et al., 1981]. When the concentration of the un dissociated acids reaches a level of 1.5 to 1.9 g/L, total inhibition of all metabolic functions in the vegetative cell was observed [Herrero et al., 1985; Monot et al. 1984]. The influence of pH on solvent production could be correlated with the central role of undissociated butyric acid [Fond et al., 1985; Monot et al., 1983; Monot et al., 1984]. The shift to solvent production seems to act as a detoxification mechanism. The undissociated acetic and butyric acids appear to be the essential factor in the regulation of solvent production. The concentrations and ratios of acetyl-CoA/CoA and NAD+/NADH have been postulated to play a key role in the regulation of the electron flow and to function as signals for both ATP regeneration and hydrogen production [Bennett and Rudolph, 1995; Boynton et al., 1994; Gottwald and Gottschalk, 1985; Jones and Woods, 1986]. A major consequence of the shift from acid to solvent production is a reduction in the net amount of ATP generated [Thauer et al., 1977]. The induction of solvent production is known to be associated with a reduction in growth [Spivey, 1978], thus there could be a relationship between the induction of solvent production and cell differentiation. Both events appear to be linked to the inhibition of vegetative growth and normal cell division [Brown et al., 1994; Jones and Woods, 1986]. It is still not clear whether or how the concentrations and the ratios of CoA and its derivatives, NAD[P]H/NAD[P] +, and ATP/ADP affect the metabolic shift at the molecular level. Solventogenesis can be induced by either high intracellular concentrations of acids [Foster and McLaughlin, 1974; Freese, 1978; Herrero et al., 1985; Huesemann and Papoutsakis, 1986; Kell et al., 1981; Monot et al. 1984], inhibition of hydrogen evolution [Kim et al., 1984; Doremus et al., 1985], or artificial electron carriers [Grupe and Gottschalk, 1992; Rao and Mutharasan, 1986]. Under conditions of high intracellular acid concentration, the formation of acetone and Butanol is switched on, whereas inhibition of hydrogen evolution only results in Butanol and ethanol production. A quick shift to butanol production is observed following the addition of methyl viologen to a growing culture of C. Beijerinckii or C. Acetobutylicum [Grupe and Gottschalk, 1992; Kim and Kim, 1988; Peguins et al., 1994; Rao and Mutharasan, 1986]. Artificial electron carriers, such as methyl viologen or neutral red, can increase ethanol yield and induce Butanol production [Grupe and Gottschalk, 1992; Girbal et al., 1995b]. The expression of the genes for solvent production is therefore believed to be regulated by environmental signals [Girbal et al., 1995a; Girbal and Soucaille, 1998; Rogers and Gottschalk, 1993; Yan et al., 1988]. However, the signal(s), which triggers the switch process from acid to solvent formation, remains to be defined.

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To investigate the shift process, it is important to distinguish between mechanisms that might be involved in the induction of enzyme biosynthesis from those that might be involved in the regulation of enzyme activity. During the switch process, the activity of solvent-forming enzymes increases 20 to 200 fold [Andersch et al., 1983; Ballongue et al., 1985; Grupe and Gottschalk, 1992; Yan et al., 1988]. The increase in activity of both butyraldehyde dehydrogenase and acetoacetate decarboxylase appears to require new protein synthesis because the addition of rifampin or chloramphenicol blocks the increase in activity of the enzymes in C. Acetobutylicum [Ballongue et al., 1985; Rogers, 1984; Welch et al., 1992]. D. Limiting factors for solvent fermentation The reason for low solvent concentration in fermentation broth may be due to the Butanol toxicity. In the solvent production phase, cell metabolism usually continues until the concentration of total solvents reaches inhibitory levels of around 20 g/liter. Butanol is the most toxic among the solvents produced. When Butanol concentration reaches 13 g/liter in the industrial fermentation process, cell growth is inhibited [Walton and Martin, 1979]. The addition of acetone and ethanol reduces growth of C. Acetobutylicum by approximately 50% at a concentration of around 40 g/liter, and total growth inhibition occurs at a concentration of about 70 g of acetone or 60 g of ethanol per liter [Costa and Oreira, 1983; Leung and Wang, 1981]. It might be possible to generate a Clostridial strain with high acetone production if the 3-hydroxybutyryl-CoA dehydrogenase gene is knocked out. Short chain aliphatic alcohols, such as ethanol, decrease membrane fluidity, whereas Butanol and other long chain aliphatic alcohols have the opposite effect and produce an increase in membrane fluidity [Bowles and Ellefson, 1985; Vollherbst-Schneck et al., 1984]. Both, the solubility of the alcohol in the membrane and its effect on membrane fluidity increase with increasing chain length. The increase in membrane fluidity in the presence of Butanol results in the destabilization of the membrane and disruption of membrane-linked functions. However, the sequence and relationship of these events are poorly understood. It will be ideal to get a strain that is tolerant to high Butanol concentrations. The tendency for solvent-producing clostridia to undergo degeneration was reported in the literature over 100 years ago [Grimbert, 1893; as cited in Kutzenok and Aschner, 1952]. One of the reasons for the lack of solvent production in C. Acetobutylicum ATCC 824 and ATCC 4259 is the loss of the 210-kb plasmid [Cornillot et al., 1997b; Cornillot and Soucaille, 1996;]. It was reported that truncation of peptide deformylase reduces growth rate and stabilizes solvent production in C. beijerinckii NCIMB 8052 [Evans et al. 1998]. Different degeneration mechanisms exist in C. beijerinckii and C. Acetobutylicum. Besides the loss of the megaplasmid, which can cause the lack of solvent production, another possibility for strain degeneration is either the loss or mutation of a structural gene[s], which encodes a solvent-forming enzyme[s] [Lebald et al., 1990; Stim-Herndon et al., 1996]. Because more and more genes related to this metabolic pathway are being cloned and sequenced, this possibility could easily be tested in C. beijerinckii and C. Acetobutylicum. Another possible reason is either the loss or mutation of a gene encoding a regulatory protein[s], which may regulate solvent-forming gene[s] or operon[s]. An additional possibility is either the loss or mutation in gene(s) encoding a development signal[s]. The last two possibilities may not be easily tested due to a lack of information about both regulatory proteins and signals that are responsible for the switch from acid to solvent formation. The study of strain degeneration in clostridia is an ongoing research effort in several laboratories. E. Operons, physical maps, and genome sequence. The metabolic pathways for solvent production are outlined. Most of the enzymes related to the pathways have been purified and characterized [Chen, 1993; Dürre and Bahl, 1998; Mitchell, 1998]. All of the genes for acid and solvent production from C. acetobutylicum, except those for additional aldehyde dehydrogenase and alcohol dehydrogenase, have been cloned and sequenced.

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The organization of the acid- and solvent-forming genes is therefore largely known in this species. Operons of acid- and solvent-forming genes In C. Acetobutylicum, the phosphotransacetylase and acetate kinase genes, which are for acetate production, are organized into one operon [Boynton et al., 1996]. The phosphotranbutyrylase and butyrate kinase genes, which are for butyrate formation, form another operon [Walter et al., 1993]. Solventproducing genes for the aldehyde/alcohol dehydrogenase (adhE), and the CoAtransferase subunits A and B (ctfA and ctfB) form a sol operon [Fisher et al., 1993; Petersen et al., 1993]. The adhE gene encodes a bi-functional Aldehydes /alcohol dehydrogenase. This enzyme is primarily for butanol formation [Nair et al., 1994; Sauer and Dürre, 1995]. The acetoacetate decarboxylase gene is adjacent to the sol operon, but it is transcribed from its own promoter and constitutes an operon [Gerischer and Dürre, 1990; Petersen et al., 1993]. The bdhA and bdhB genes are for the NADH-dependent butanol dehydrogenases [BDHI and BDHII], and each is transcribed from its own promoter [Walter et al, 1992]. The adhE and bdhB genes contribute to Butanol formation [Girbal and 10 Soucaille, 1998; Green and Bennett, 1996]. It is not clear how the operons are regulated. In C. beijerinckii NRRL B593, the ctfA, ctfB, and adc genes for acetone formation have been cloned and sequenced [Toth and Chen, 1998]. The ald and adh genes for Butanol and/or ethanol formation were also sequenced [Toth and Chen, 1998; Peretz et al., 1997]. The transcription of the adh, ald, ctfA, ctfB, and adc genes was investigated during this study [See Section III]. Physical maps and whole genome sequence The physical map of the C. Acetobutylicum ATCC 824 chromosome is available [Cornillot et al., 1997a]. Its genomic size is about 4.1 Mb and it contains a pSOL1 plasmid, which is 210kb in size. This plasmid carries the soloperon and the adc gene. Loss of the plasmid pSOL1 abolishes solvent production in C. Acetobutylicum ATCC 824 [Cornillot et al, 1997b]. C. Acetobutylicum ATCC 4259 has a 210-kb plasmid named pWEIZ. Loss of the pWEIZ plasmid in three mutants: WDS1, 2, and 3, correlates with the inability of the host strains to produce solvents [Cornillot and Soucaille, 1996]. The whole DNA sequence for C. Acetobutylicum ATCC 824 will be available soon [Smith et al., 1998]. It represents a great advance in the study of Clostridial genetics. The physical map of the C. beijerinckii NCIMB 8052 chromosome has also been constructed. Its genomic size is about 6.7 Mb, and no plasmid was detected in this strain. The solvent-producing genes that have been identified are located on the chromosome [Wilkinson and Young, 1995]. In C. beijerinckii NCIMB 8052, the loss of the ability to produce solvent during degeneration must not be due to the loss of a plasmid. The physical and genetic maps of clostridia, not to mention the whole genomic sequences, will facilitate a comparative study of the architecture of bacterial chromosomes and the regulation of genes for solventogenesis. F. Regulation of solvent-production genes To study the expression of genes encoding solvent-forming enzymes, both the level of mRNA from the corresponding genes and the enzyme activities have been monitored [Fisher et al., 1993; Gerischer and Dürre, 1992; Girbal et al., 1995a; Sauer and Dürre, 1995; Vasconcelos et al., 1994; Walter et al., 1992]. Analyses of mRNA show that the genes of the acetoacetate decarboxylase (adc) and the Butanol dehydrogenases A (bdhA) and B (bdhB) are induced about 4 hours before solvents can be detected in C. Acetobutylicum. The sol operon which is composed of genes for aldehyde/alcohol dehydrogenase (adhE), CoA transferase subunit A (ctfA) and subunit B (ctfB) is derepressed about 4 hours before solvents can be detected in C acetobutylicum [Fisher et al., 1993; Gerischer and Dürre, 1992; Sauer and Dürre, 1995; Ullmann et al., 1996; Walter et al., 1992]. These data indicate that the expression of the solventogenic genes is regulated at the transcriptional level. The question is how the expression of the solvent-forming genes is regulated. Changes in DNA topology are known to regulate the transcription of genes in many bacteria [Dorman, 1991; Higgins et al., 1990; Hulton, 1990]. The degree of DNA supercoiling might function as one of the transcription sensors that recognize and respond directly to environmental stimuli during the shift to solventogenesis [Ullmann and Dürre, 1996]. Changes in DNA conformation might reflect environmental signals such as changes in temperature, pH, osmolarity, and

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anaerobiosis, as well as intracellular conditions such as the ATP level [Hsieh et al., 1991; Matsushita et al., 1996]. How a Clostridial cell senses environmental signals and regulates solventogenic gene expression remains to be determined. Sigma factors and gene expression The s factor is a component of the DNA-dependent RNA polymerase. It determines the specificity of the holoenzyme of the RNA polymerase for a promoter. Constitutively expressed genes are transcribed by the s70 RNA polymerase in bacteria. Alternative s factors play a role in altering the program of transcription during developmental processes, such as sporulation and phage growth [Helmann, 1994; Losick and Pero, 1981; Sauer et al., 1995]. Most bacteria have a set of alternative s factors that control genes for specialized functions. These functions include endospore formation in gram-positive bacteria [Losick and Stragier, 1992; Santangelo et al., 1998], the stationary phase and stress response in the enteric bacteria and solvent-producing clostridia [Bahl, 1993; Bahl et al., 1995; Hengge-Aronis, 1993], control of nitrogen metabolism [Kustu et al., 1989], and expression of flagellar and chemotaxis genes [Helmann, 1991]. The question of whether or not the expression of solvent-producing genes is regulated by a sigma factor is to be answered. Two-component systems and signal transduction Bacteria live in precarious environments. Nutrients and toxin levels, acidity, temperature, osmolarity, and many other conditions can change rapidly and unexpectedly. Bacteria readily detect minute fluctuations in many chemical and physical conditions, which in turn trigger changes in gene expression or motility that enhance survival prospects of bacteria. Cells must sense and respond to their environment, a process that requires signal transduction across biological membranes. A major mechanism of signal transduction in bacteria involves the so-called two-component systems that have adopted phosphorylation as means of information transfer. A typical two-component system consists of two proteins functioning as a sensor and a response regulator [Albright et al., 1989]. Two-component systems are central to much of the cellular physiology that results from alterations in the environment. s54–dependent regulators are a distinct class of positive activators. All members of this family act in concert with s54 factor and are part of a two component sensor-response system [Parkinson and Kofoid, 1992]. An additional property of the s54–dependent regulators is that they usually bind to upstream activating sequences [UASs] located between 100- and 200-bp upstream from the promoters they regulate [Kustu et al., 1991; Morett and Segovia, 1993]. The binding sequences are often inverted repeats. The orientation of the UASs on the DNA double helix relative to that of the promoter is critical, although the 14 distance of the UASs from promoter is not [Morett and Segovia, 1993; Perez- artin et al., 1994]. The polypeptide of the s54–dependent transcription activators (e. g., NtrC) has an average size of 460 amino acid residues and is composed of three functional domains involving signal reception, transcription activation, and DNA binding [Morett and Segovia, 1993; North et al., 1993; Shingler, 1996]. The central domain of about 240 amino acid residues [AA] is highly conserved and involved in transcription activation, [Parkinson, 1993; Shingler, 1996]. The probable roles of this domain include binding and hydrolyzing ATP, oligomerization of the activator, and interacting with the s54 factor. The N terminal domain contains about 120 AA and is for signal reception. The C terminal domain has about 60 residues and a helix-turn-helix motif for DNA binding. Together with a subgroup of the s54–dependent transcription activators, a sensory histidine autokinase (e.g., NtrB) senses the signal and subsequently transduces it to activate the constitutively expressed transcription activator – the response protein. Activation of the response protein is achieved by transfer of a phosphate group to a conserved Asp residue of the regulatory Adomain [Alex and Simon, 1994; Stock et al., 1989; Stock et al., 1990]. This subgroup directly senses and responds by transcription activation in the presence of a small effector molecule [Shingler, 1996]. Modulation of the activity of transcription factors by derepression mechanisms appears to be a common strategy among prokaryotic transcription activators from different families [Shingler, 1996]. The solventogenic genes are regulated at the transcriptional level [Girbal et al., 1995a; Girbal and Soucaille, 1998; Welch et al., 1992]. However, the molecular mechanisms that regulate solventogenesis in

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solvent-producing clostridia are still to be elucidated. Multiple regulatory elements might be required to induce solvent production. Genetic tools will help us to answer this kind of question. G. Tools for genetic studies with the solvent-producing clostridia Vector construction Several shuttle vectors for solvent-producing clostridia and E. coli have been developed. The construction and features of the shuttle vectors have been reviewed [Minton et al., 1993; Papoutsakis and Bennett, 1993]. The basic components of these vectors are a selectable marker, two replicons, and cloning sites. Erythromycin- and tetracycline-resistance genes are most commonly employed as selectable markers in clostridia. The replicons from pIM13, pCS86, pAMb1, and pIP404 were used in the construction of vectors for use in the solvent-forming clostridia. There are two kinds of replication mechanisms for the vectors frequently used. One is the rolling circle model, which is used by pIM13, pCS86, and their derivatives [Minton et al., 1993]. The other is the unidirectional theta mechanism, which is utilized by pAMb1, pIP404, and their derivatives [Minton et al., 1993]. A wide variety of plasmid shuttle vectors for the clostridia and E. coli are now available [Lee et al., 1992; Minton et al., 1993; Papoutsakis and Bennett, 1993; Reysset and Sebald, 1993; Truffaut and Sebald, 1989]. Some of the shuttle vectors for C. beijerinckii and E. coli will be discussed further. A shuttle vector pMTL500E was constructed by Oultram et al. [1988]. Its size is 6.4 kb. It has an origin of a replication (ColE) and an ampicillin-resistance (Apr) gene for E. coli. It also contains the origin of replication and the erythromycin-resistance (Emr) gene for clostridia. There are 12 restrictionenzyme- cloning sites. pMTL 500E was introduced into C. beijerinckii NCIMB 8052 via electroporation [Minton et al., 1993]. Another shuttle vector pHR106 was developed [Robert et al., 1988]. It contains an origin of replication [ColE] and the Apr marker for E. coli and an origin of replication [pAMb1] and the chloramphenicol-resistance (catr) gene for the clostridia. It has 6 restriction-enzyme-cloning sites. The vector pHR106 was transformed into C. beijerinckii NRRL B592 via electroporation [Birrer et al., 1994]. The authors, however, could not successfully extract this plasmid from the cytosol of the transformant using various plasmid isolation techniques [Birrer et al., 1994]. A third Clostridium-E. coli shuttle vector pIMP1 was constructed by ligating the 2.1-kb HindIII fragment of pIM13 [Monod et al., 1986; Projan et al., 1987] to pUC9 [Mermelstein et al., 1992]. The 2.1-kb HindIII fragment contains the origin of replication (orfII) locus for the gram-positive bacteria and the Emr gene. There are 6 restriction-enzyme-cloning sites. The orientation of the fragments is such that the Emr and Apr genes are transcribed in the same direction. pIMP1 was transformed into C. acetobutylicum ATCC 824 via electroporation. Transformation Protoplast transformation is based on the introduction of DNA into a cell wall-less protoplast, which is capable of regenerating into walled vegetative cells. Protoplast transformation has been utilized in C. pasteurianum ATCC 6013 [Minton and Morris, 1983] and C. saccharoperbutylacetonicum N 1-4 [Yashinot et al., 1984; Reysset et al., 1987]. Polyethylene glycol-induced fusion of bacterial protoplasts has been performed in Clostridium P262 [Jones et al., 1985]. The frequency of protoplast regeneration is low. The protoplast transformation is also time-consuming compared with that of electroporation. Transformation by electroporation is now becoming the method of choice for transforming solvent producing clostridia. Transformation of C. beijerinckii NCIMB 8052 by electroporation was first reported by Oultram et al. [1988]. It turns out to be an efficient and relatively easy method for the transformation of solvent-producing clostridia [Birrer et al., 1994; Lee et al., 1992; Li and Chen, 1995; Mermelstein et al., 1992; Minton et al., 1993; Papoutsakis and Bennett, 1993a]. Before a shuttle plasmid is introduced into C. Acetobutylicum, in vivo methylation of the plasmid DNA is important to prevent restriction by an endonuclease found in C. acetobutylicum ATCC 824 [Mermelstein and Papoutsakis, 1993]. pAN1 contains a methyltransferase ( 3T1 ) gene and a p15A origin of replication [Noyer-Weidner et al., 1985], which can co-exist with pIM13-derivated vectors [Mermelstein and Papoutsakis, 1993]. Expression of the 3T1 gene from pAN1 in E. coli is sufficient to methylate co-resident Clostridium-E. coli shuttle vectors [Mermelstein and Papoutsakis, 1993]. Shuttle plasmids carrying clostridial genes have been transferred into C. acetobutylicum [Mermelstein et al., 1992; Mermelstein et al., 1993; Mermelstein and Papoutsakis, 1993b; Papoutsakis and Bennett, 1993; Petersen

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and Bennett, 1990] and C. beijerinckii [Li and Chen, 1995; Minton et al, 1993; Oultram et al., 1988] via electroporation. Homologous recombination Integration plasmids will be powerful and versatile tools for the genetic analysis of clostridia. They can be applied to [i] determining the map location and extent of cloned genes; [ii] achieving insertion mutagenesis, targeted cloning, and gene replacement; and [iii] stably expressing introduced foreign genes [Green et al., 1996; Perego, 1993; Wilkinson and Young, 1994]. It was reported that targeted integration of genes into C. beijerinckii NCIMB 8052 and C. acetobutylicum ATCC 824 chromosome occurred via homologous recombination [Green et al., 1996; Wilkinson and Young, 1994; Wong and Bennett, 1996]. Reporter systems to study the regulation of gene expression and signal transduction for the solventogenic switch, a good reporter system is desirable. Reporter systems have been developed for clostridia during the last five years. The cat gene encoding chloramphenicol transacetylase was used as a reporter gene for a clostridial reporter vector [Bullifent et al., 1995; Minton et al., 1993]. The cat gene product expressed in C. beijerinckii NCIMB 8052 is around 7% of the cell soluble protein [Minton et al., 1993]. The promoter region of the alpha-toxin gene of C. perfringens was inserted into the unique restriction site (ClaI) of the Clostridial reporter vector. The cat gene is expressed with the promoter of the alpha-toxin gene [Bullifent et al., 1995]. However, most protocols for the CAT assay require a elatively expensive radioactive substrate. The assays are time consuming to perform, and the sensitivity of CAT assays is relatively low. Furthermore, chloramphenicol will be reduced and inactivated by fast-growing solvent-producing Clostridium [O’ Brien and Morris, 1971]. An alternative reporter gene system for C. Acetobutylicum has been constructed to analyze the promoter region responsible for the regulation of the acetoacetate decarboxylase gene (adc) [Dürre et al., 1995]. For this purpose, the complete regulatory region has been fused to the structural gene of b–galactosidase from Thermoanaerobacterium thermosulfurigenes EM1. The plasmid carrying the gene fusion was transferred to C. acetobutylicum DSM 792. The recombinant strain expressed the b–galactosidase activity only when solvents started to appear in the medium. The wild type does not carry such a protein, but it is known to break down lactose by phospho-b–galactosidase. Thus, this reporter gene should allow determination of the promoter sequences responsible for the regulation of solvent formation [Dürre et al., 1995]. A clostridial promoter-probe vector pMTL710 was constructed based on the catechol-2,3–dioxygenase (xylE) gene [Minton et al., 1993]. Cells expressing this enzyme catalyze the conversion of catechol to 2-hydroxymuconic 20 semi-Aldehydes and turn yellow on the plate when sprayed with a 2% w/v catechol aqueous solution. Genomic DNA of B. subtilis and C. beijerinckii NCIMB 8052 ere, separately, digested with restriction enzymes. DNA fragments were ligated to the pMTL710 and the ligation mixtures were transformed into E. coli. About 2000 transformants were pooled and used to prepare a sample of heterogeneous bulk plasmid DNA. This DNA preparation was used to transform B. subtilis and C. beijerinckii NCIMB 8052. Colonies of the transformants were sprayed with catechol to see whether the cloned DNA fragments have the promoter function. Twelve putative promoter fragments have been characterized with regard to both the level of xylE expression in the two gram-positive hosts and the nucleotide sequences of these fragments [Minton et al., 1993]. Another reporter system that utilized the firefly luciferase gene was developed for C. botulinum [Schmidt et al., 1998]. The promoters of the neurotoxin gene and the nontoxic nonhemagglutinin gene were separately cloned into the reporter vector. Shuttle plasmids were transferred by conjugation from E. coli to C. botulinum. The luciferase gene in the shuttle plasmids was expressed in C. botulinum. H. Genetic engineering of solvent-producing clostridia The high cost of substrate and the low concentration of solvents in the fermentation broth are the main factors that led to the abandonment of AB fermentation. It is, therefore, attractive to use agricultural by-products or waste based biomass, such as whey and sulfite liquor, as substrates for the production of solvents and other chemicals [Beguin and Lemaire, 1996; Jones and Woods, G. LI 1998 21 1986; Morris, 1993; Schilling, 1995]. Recent progress in molecular biology and genetics has facilitated the study of solvent-forming clostridia. It is possible to genetically modify solvent-forming strains which could

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result in the improvement of the performance of the strains in the following areas: [i] efficient use of agricultural or industrial by-products such as whey, sulfite liquor, and Lignocellulose; [ii] high strain stability; and [iii] higher solvent concentrations and adjustable solvent ratios. Some progress has been made in genetic engineering of solvent producing clostridia [Dürre, 1998]. One way of genetically engineering solvent producing clostridia involves the use of plasmid-mediated amplification of acid and solvent-production genes. This approach might enable us to determine limiting enzyme activities and could aid in designing improved strains. For example, the acetoacetate decarboxylase (adc) gene and its promoter, which were cloned in a shuttle vector and expressed in C. Acetobutylicum ATCC 824, results in enzyme activity about 12-fold higher than that of C. Acetobutylicum ATCC 824 in the exponential phase of growth [Mermelstein et al., 1992]. However, the solvent-production pattern was not reported. Also, a shuttle plasmid, which contains an artificial ace operon in which the adc, ctfA and ctfB genes are transcribed from the adc promoter, has been introduced into C. Acetobutylicum ATCC 824 [Mermelstein and Papoutsakis, 1993]. Compared with the parent strain, the transformant of C. Acetobutylicum ATCC 824 produced 95%, 30%, and 90% higher final concentrations of acetone, Butanol and ethanol, respectively [Mermelstein et al., 1993 b; Walter et al., 1994]. Another way to improve solvent-producing clostridia is to inactivate some genes in the metabolic pathway, such as those genes involved in acid formation. It may then be possible to redirect carbon flow towards solvent production and so increase solvent yields. For example, inactivation of the butyrate kinase gene significantly decreases butyrate production and increases Butanol yield [Green et al., 1996]. It has been reported that transposon mutation of the gene encoding the peptide deformylase reduces the growth rate and frequency of degeneration in C. beijerinckii NCIMB 8052 [Evans et al., 1998]. The gene encoding the deformylase has also been cloned from C. Acetobutylicum ATCC 824 and characterized [Belouski et al., 1998]. REFERENCES

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Further readings

Acetone-butanol fermentation revisited.DT Jones, DR Woods - Microbiology and Molecular Biology Reviews, 1986 - Am Soc Microbiol 0146-0749/86/040484-41$02.00/0 Copyright C) 1986, American Society for Microbiology ... DAVID T. JONES AND DAVID R. WOODS* Department of Microbiology, University of Cape Town, Rondebosch 7700, South Africa

Genetics and biochemistry of Clostridium relevant to development of fermentation processesP Rogers - Advances in applied microbiology, 1986 - cat.inist.fr Genetics and biochemistry of Clostridium relevant to development of fermentation processes. P ROGERS Advances in applied microbiology 31, 1-60, Academic Press, 1986. Métabolisme; Metabolism; Metabolismo

Level of enzymes involved in acetate, butyrate, acetone and butanol formation by W Andersch, H Bahl, G Gottschalk - Applied Microbiology and Biotechnology, 1983 - SpringerSummary. Clostridium acetobutylicum cells were collected from chemostats which were run at pH 4.3 or 6.0 and which produced either acetone-butanol or acetate-butyrate; they were used to determine the level of enzymes involved

In vivo methylation in Escherichia coli by the Bacillus subtilis phage phi 3T I …nih.gov [PDF] LD Mermelstein, ET Papoutsakis - Applied and Environmental Microbiology, 1993 - Am Soc Microbiol Page 1. APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Apr. 1993, p. 1077-1081 0099-2240/93/041077-05$02.00/0 Copyright X 1993, American Society for Microbiology In Vivo Methylation in Escherichia coli by the Bacillus

Solvent production and morphological changes in Clostridium acetobutylicum nih.gov [PDF] DT Jones, A Van der Westhuizen, S Long, ER … - Applied and Environmental microbiology, 1982 - Am Soc Microbial Page 1. Vol. 43, No. 6

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APPLIED AND ENVIRONMENTAL MICROBIOLOGY, June 1982,p.1434-1439 099-2240/82/0614 34-06$02.00/0 Solvent Production and Morphological Changes in Clostridium acetobutylicum

Intermediary metabolism in Clostridium acetobutylicum: levels of enzymes involved in the … nih.gov [PDF] MGN Hartmanis, S Gatenbeck - Applied and Environmental Microbiology, 1984 - Am Soc Microbiol Page 1. APPLIED AND ENVIRONMENTAL MICROBIOLOGY, June 1984, p. 1277-1283 0099-2240/84/0601277-07$02.00/0 Copyright ©D 1984, American Society for Microbiology Vol. 47, No. 6 Intermediary Metabolism in Clostridium Acetobutylicum: Levels of

Expression of cloned homologous fermentative genes in Clostridium acetobutylicum ATCC LD Mermelstein, NE Welker, GN Bennett, ET … - Nature Biotechnology, 1992 - nature.com 1 Northwestern University, Dept. of Chemical Engineering, Evanston, IL 60208. ... 2 Dept. of Biochemistry, Molecular Biology and Cell Biology, Evanston, IL 60208. ... 3 Rice University, Dept. of Biochemistry and Cell Biology, PO.

Oxygen and the growth and metabolism of Clostridium acetobutylicum sgmjournals.org [PDF] RW O'brien, JG Morris - Microbiology, 1971 - Soc General Microbiol SUMMARY Clostridium acetobutylicum has been studied during batch cultivation at pH 7 and 35” in a glucose+casein hydrolysate+vitamins and salts medium kept (i) anaerobic (&, -400 to - 370 mV), (ii) aerated (&, - 50 to o mV; dissolved.

New insights and novel developments in clostridial acetone/butanol/isopropanol P Dürre - Applied Microbiology and Biotechnology, 1998 – Springer Abstract Clostridial acetone/Butanol fermentation used to rank second only to ethanol fermentation by yeast in its scale of production and thus is one of the largest biotechnological processes known. Its decline since about 1950.

The genes for butanol and acetone formation in Clostridium acetobutylicum ATCC 824 reside nih.gov [PDF] E Cornillot, RV Nair, ET Papoutsakis, P … - Journal of bacteriology, 1997 - Am Soc Microbiol Page 1. J OURNAL OF B ACTERIOLOGY , 0021-9193/97/$04.00 0 Sept. 1997, p. 5442–5447 Vol. 179, No. 17 Copyright © 1997, American Society for Microbiology The Genes for Butanol and Acetone Formation in Clostridium ...

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Attachment Two Requirement Analysis

Introduction

The Air Force is the largest single user of energy within the US federal government. Some 80% of that energy is aviation fuel for aircraft, representing over 50% of all fossil fuel use by the US government. The Air Force jet fuel bill alone runs some $5 billion annually for about 3 billion gallons of jet fuel. The US commercial aviation industry consumes another 20 billion gallons per year, representing 35% of global jet fuel consumption1. EIA estimates that building and transportation lead increases in primary energy use and transportation sector energy use will grow 0.5 percent annually from 2009 till 20302

Jet fuels for commercial aircrafts and military fleets are kerosene-based due to the fact that kerosene-based fuels; as opposed to wide-cut, have the best combination of properties

.

3, 4. The most common kerosene-based aviation fuels are Jet A and Jet A-1 which are produced to an internationally standardized set of specifications. Kerosene-type jet fuel (including Jet A and Jet A-1) has a carbon number distribution between about 8 and 16 carbon numbers; wide-cut or naphtha-type jet fuel (including Jet B), between about 5 and 15 carbon numbers5

JP-8 is the kerosene-type fuel introduced in 1979 to replace the wide-cut type JP4 for the US Air Force. JP-8, which is essentially Jet A with a larger additive package

.

6, 7, is currently the primary jet fuel used by the United States military. The United States Air Force is utilizing JP-8 as the fuel of choice for all its aircrafts and ground vehicles and the US Army plans a transition to a single fuel JP8 for all the ground vehicles and aircrafts as well8. It is estimated that 5 billion gallons of JP-8 jet fuel is used by the US DOD each year9 and the worldwide consumption is close to 60 billion gallons10

. The DOD and the North Atlantic Treaty Organizations (NATO) have declared that Jet Propulsion 8 (JP-8) will be the single battlefield fuel by 2010. The widespread use of JP8 has called for a renewed attention to physical and chemical properties of JP8.

Properties of Jet Fuels

1https://e-center2.doe.gov/iips/IIPSquestions.nsf, solicitation DE-PS36-09GO99038 2 Energy Information Administration, Annual Energy Outlook 2009, Report #: DOE/EIA-0383(2009), March 09 3 Handbook of Aviation Fuel Properties, 3rd ed.; Coordinating Research Council, Inc.: Alpharetta, GA, 2004 4 Edwards, T. “Advancements in gas turbine fuels from 1943 to 2005”, J. Eng. Gas Turbines Power 2007, 129, 13–20 5 J. Bacha, F. Barnes, M. Franklin, L. Gibbs, G. Hemighaus, N. Hogue, D. Lesnini, J. Lind, J. Maybury, J. Morris. “Technical Review Aviation Fuels, FTR3”, Chevron Products Company, 2000 6 Maurice, L. Q.; Lander, H.; Edwards, T.; Harrison, W. E., “Advanced aviation fuels: A look ahead via a historical perspective”, Fuel, 80, 747–756, 2001 7 Alessandro Agosta, “Development of a Chemical Surrogate for JP-8 Aviation Fuel Using a Pressurized Flow Reactor”, MS Thesis, Drexel University, 2002 8 Jason A. Widegren and Thomas J. Bruno, “Thermal Decomposition Kinetics of the Aviation Turbine Fuel Jet A”, Ind. Eng. Chem. Res., 2008, 47, 4342–4348 9 National Research Council of the National Academies, “Toxicological assessment of jet propulsion fuel 8”. Washington DC: 2003 10 http://www.gooptroop.com/gtroop/JetFuel/JP8%20pres%2017.8.01.pdf, accessed in June 2009

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The primary function of aviation fuels (jet fuels) is to power an aircraft and therefore, energy content and combustion quality are the most important fuel properties. Other significant properties are stability, lubricity, fluidity, volatility, non-corrosives and cleanliness.

Energy Content An aircraft jet engine generates power by converting chemical energy stored in the fuel into mechanical energy and heat. Since space is one of the most critical factors in most aircrafts, the amount of energy contained in a given quantity of fuel is important. Energy content of jet fuel can be measured by the amount of heat released from burning a known amount of fuel under specific conditions. Energy content can be measured either gravimetrically (energy per unit weight) or volumetrically (energy per unit volume). Generally, less dense jet fuels have higher gravimetric energy content and more dense fuels have higher volumetric energy content. The choice between higher gravimetric or volumetric energy content fuels depends on the type of application. For military applications where the aircraft takes off with full fuel tank, a fuel with high volumetric energy content maximizes the energy that can be stored in a fixed volume and thus provides a longer flight path.

Combustion Characteristics The main difference between piston and jet engines is that combustion is intermittent in a piston engine while it is continuous in a jet engine. Because of this, the engines have different fuel combustion quality requirements. In piston engines, combustion timing is critical to good performance. When combustion is continuous, combustion timing is no longer important. During combustion in a jet engine, small carbonaceous particles are formed. These particles continue to burn as they pass through the flame and can be completely consumed under suitable conditions. However, these particles become incandescent under the high temperature and pressure conditions of the combustion section and are absorbed by the combustor walls, augmenting the normal heat of the walls received from the combustion gases. High combustor wall temperatures or hot spots can lead to cracks and premature engine failures5. Fuels with high aromatics content, and especially fuels with high naphthalenes content, form more of these carbonaceous particles. Stability A fuel is stable when its properties remain unchanged. There are two kinds of stability; storage stability and thermal stability. Storage stability is the resistance to formation of gum and sediments under ambient condition while thermal stability is resistance to formation of deposits on hot surfaces within the fuel system11. In advanced military aircraft, jet fuel is used as a coolant. The hot fuel reacts with dissolved oxygen forming oxidized products. These oxidized products include gums and solid deposits which can coat fuel system surfaces resulting in filter plugging, fouling of close tolerance valves, valve hysteresis, and other problems12

. It is generally accepted that dissolved oxygen initiates the deposition process in freshly refined fuels. The presence of heteroatomic compounds such as sulfur, nitrogen and oxygen influence both thermal and storage stability of a fuel. Sulfur and oxygen compounds affect both storage and thermal stability of fuels.

Storage Stability

11 J. A. Anabtawi, M. Ashraf Ali, “Storage Stability of Fuels: A Comprehensive Literature Review”, Accessed through internet: http://www.kfupm.edu.sa/ri/RI_Research_Files/CRP/174_Storage_Stability_of_Fuels_ Comprehensive_Literature_Review.pdf 12 S. Zabarnick, M.S. Mick, R.C. Striebich, R.R. Grinstead, and S.P. Heneghan, “Studies of Silylation Agents as Thermal-oxidative Jet Fuel Additives” , http://www.anl.gov/PCS/acsfuel/preprint%20archive/Files/43_1_DALLAS_03-98_0064.pdf

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Instability of jet fuel during storage is generally not a problem because most fuel is used within weeks or months of its manufacture. Storage stability is an issue for the military, which often stores fuel for emergency use. Formation of gum and sediments is enhanced at higher storage temperature. It has been found13

that deposit formation occurs with almost all types of nitrogen compounds with the rise in temperature. Jet fuel that has been properly manufactured, stored, and handled should remain stable for at least one year. Jet fuel subjected to longer storage or to improper storage or handling should be tested to be sure it meets all applicable specification requirements before use. Because it is the more reactive fuel components that cause instability, storage stability is influenced by fuel composition. It is also influenced by storage conditions; instability reactions occur faster and to a greater extent at higher ambient temperatures. Antioxidants may be added to fuel to improve its storage stability.

Thermal Stability Thermal stability is one of the most important jet fuel properties because the fuel serves as a heat exchange medium in the engine and airframe. Jet fuel is used to remove heat from engine oil, hydraulic fluid, and air conditioning equipment. When jet fuel is subjected to thermal stress, it will undergo degradation either primarily by autoxidation if the fuel temperature is below 300ºC, or by pyrolytic degradation, if the fuel temperature exceeds 400ºC14

. This fuel degradation will form solid deposits that may develop as either filterable insolubles or as solid varnish-like deposits on fuel system surfaces. These insoluble compounds will be carried through the fuel system and collect in fuel filters and may agglomerate to form solid deposits. These gums and particles may deposit:

• On fuel filters, increasing the pressure drop across the filter and reducing fuel flow. • In fuel injector nozzles, disrupting the spray pattern, which may lead to hot spots in the combustion

chamber. • In the main engine control, interfering with fuel flow and engine system control. • On heat exchangers, reducing heat transfer efficiency and fuel flow.

These deposits may lead to operational problems and increased maintenance. Antioxidants that are used to improve fuel storage stability do not improve its thermal stability. Engine problems related to inadequate fuel thermal stability typically become evident only after hundreds or thousands of hours of operation. The long time and the large volume of fuel consumed make it impractical to test fuel thermal stability under conditions identical to those that exist in engines. Instead, the fuel is subjected to more severe conditions in a bench test in order to be able to see a measurable effect in a reasonable period of time. Use of hydroperoxide decomposing species to inhibit oxidation of jet fuel was studied by Zabarnick and Mick15. The study indicated that hydroperoxide decomposing species, such as alkyl sulfides, do not slow or delay oxidation in hydrocarbon solvents at 140 °C. However, when phenolic species are also present, such as those naturally occurring in fuel or by addition of hindered phenols, substantial delays in oxidation are observed. The study found that combination of hydroperoxide decomposer and hindered phenol can substantially inhibit oxidation of fuel under the studied conditions. A study by Heneghn and Zabernick16

13 Worstell, J H., S. R. Daniel, “Deposit Formation in Liquid Fuels. 2. Effect of Selected Compounds on the Storage Stability of Jet A Turbine Fuel” Fuel, 1981, 60, 481-484,

14 Balaster, L. M. and Balaster W. J., ”Thermal Stability of Jet-Fuel/Paraffin Blends”, Energy and Fuel, 1996, 10, 1176-1180, 15 S. Zabarnick and M. S. Mick, “Inhibition of Jet Fuel Oxidation by Addition of Hydroperoxide-Decomposing Species”, Ind. Eng. Chem. Res. 1999, 38, 3557-3563 16 S. P. Heneghan, S. Zabarnick, “Oxidation of jet fuels and the formation of deposit”, Fuel, 1994, 73(1), 35-43

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indicated that oxidative stability and thermal stability are inversely related; in other words fuels that oxidize easily are likely to be thermally stable whereas fuels that are not thermally stable are not easily oxidized. Lubricity Lubricity can be defined in many ways. “Lubricity is the ability of a liquid to provide hydrodynamic and /or boundary lubrication to prevent wear between moving parts” 17

17. It can also be defined as: “ The ability to reduce

friction between solid surfaces in relative motion” . Jet fuel must possess a certain degree of lubricity because jet engines rely on the fuel to lubricate some moving parts in fuel pumps and flow control units. “The lubrication mechanism is a combination of hydrodynamic lubrication and boundary lubrication”5. Hydrodynamic (Full Film) Lubrication is obtained when two mating surfaces are completely separated by a cohesive film of lubricant. The thickness of the film thus exceeds the combined roughness of the surfaces so, higher viscosity liquids provide more hydrodynamic lubrication than lower viscosity liquids. Boundary lubrication refers to the regime where a large portion of the mechanical loads are borne by the moving surface asperities. Under these conditions, lubricant molecules react with the surfaces to form lubricating films which control the wear. The coefficient of friction is lower in hydrodynamic lubrication than with boundary-layer lubrication. While jet fuel specifications do not include an explicit lower limit on viscosity, the distillation specification serves as a surrogate limit. Jet engines are designed to work with jet fuels within the normal viscosity range, and therefore, typical jet fuels provide adequate hydrodynamic lubrication. Fluidity Fluidity is not a defined physical property but in general term, it is the ability of a substance to flow. Viscosity and freezing point are the physical properties used to quantitatively characterize the fluidity of jet fuel. Jet fuel is exposed to very low temperatures both at altitude – especially on polar routes in wintertime – and on the ground at locations subject to cold weather extremes. The fuel must retain its fluidity at these low temperatures or fuel flow to the engines will be reduced or even stop. Viscosity Viscosity is a measure of the resistance of a fluid to deformation. It can be thought of as an internal resistance to flow processes. In simple terms, viscosity is “thickness” of a fluid. “Thin” liquids, like water or gasoline, have low viscosities; “thick” liquids, like maple syrup or motor oil, have higher viscosities. The viscosity of a liquid has inverse relation with temperature. Jet fuel at high pressure is injected into the combustion section of the turbine engine through nozzles. The nozzle system is designed to produce a fine spray of fuel droplets that evaporate quickly as they mix with air. The spray pattern and droplet size are influenced by fuel viscosity. High viscosity fluids can create problems for the aircraft engine. For this reason, jet fuel specifications place an upper limit on viscosity. Fuel viscosity influences the pressure drop in the fuel system lines. Higher viscosities result in higher line pressure drops, requiring the fuel pump to work harder to maintain a constant fuel flow rate.

Freezing Point Jet fuel and specifically JP8 is a mixture of more than a thousand individual hydrocarbons, each with its own freezing point. Jet fuel does not solidify at one temperature the way water does. As the temperature is lowered and fuel is cooled, the hydrocarbon components with the highest freezing points solidify first, forming wax crystals. Further cooling causes hydrocarbons with lower freezing points to solidify. Thus, the fuel changes from a homogenous liquid, to a liquid containing a few hydrocarbon (wax) crystals, to a slush of fuel and 17 Chevron U.S.A. Inc. 1998. Diesel Fuel Technical Review. (FTR-2). San Francisco, CA

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hydrocarbon crystals, and, finally, to a near-solid block of hydrocarbons. Thus the freezing point of fuel is well above the temperature at which it completely solidifies. The primary criterion for fuel system performance is pumpability – the ability to move fuel from the fuel tank to the engine. Pumpability is influenced both by fuel fluidity and fuel system design. Jet fuel typically remains pumpable approximately 4°C to 15°C (8°F to 27°F) below its freezing point. The U.S. Air Force is evaluating the use of additives that may prevent the formation of large wax crystals that are responsible for reduced fuel flow. Volatility Volatility is defined as a fuel’s tendency to vaporize. Two physical properties are used to characterize fuel volatility: vapor pressure and distillation profile. A more volatile fuel has a higher vapor pressure and lower initial distillation temperatures. Volatility is important because a fuel must vaporize before it can burn. However, too high a volatility can result in evaporative losses or fuel system vapor lock. Volatility is one of the major differences between kerosene-type and wide-cut jet fuel. Kerosene-type jet fuel is relatively non-volatile. It has a Reid vapor pressure 2 of about 1 kiloPascal (kPa) [0.14 pound per square inch (psi)]. Wide-cut jet fuel has a Reid vapor pressure as high as 21 kPa (3 psi). Wide-cut jet fuel is better suited for cold weather applications because it has a lower viscosity and freezing point than kerosene-type jet fuel. In such applications, evaporative losses are less of a concern. Non-corrosivity Jet fuel may come in contact with a variety of materials during its handling, storage and distribution. It is essential that the fuel not corrode any of these materials, especially those in aircraft fuel systems. Fuel tanks are typically made of aluminum but the fuel system contains steel, polymer materials and other metals. Corrosive compounds potentially present in jet fuel include organic acids and mercaptans. The specifications limit these classes of compounds. By-products of microbial growth also can be corrosive. Contamination from trace amounts of sodium, potassium, and other alkali metals in the fuel can cause corrosion in the turbine section of the engine.

Cleanliness (Microbial Growth) Because of high processing temperature, jet fuel is sterile when it is first produced but due to presence of microorganisms in the air jet fuel is susceptible to contamination. Microorganisms found in fuels include bacteria and fungi (yeasts and molds). These solid microorganisms can grow and plug fuel filters. Some microorganisms also generate acidic by-products that can accelerate metal corrosion. Since most microorganisms need free water to grow, their growth is usually concentrated at the fuel-water interface, when one exists. Some organisms need air to grow (aerobic organisms), while other grow only in the absence of air (anaerobic organisms). In addition to food (fuel) and water, microorganisms also need certain elemental nutrients. Jet fuel can supply most of these; phosphorus is the only one whose concentration might be low enough to limit growth of microorganisms. Higher ambient temperatures also enhance microbial growth. The best approach to microbial contamination is prevention and the most important preventive step is keeping the amount of free water in the fuel storage tank as low as possible. Biocides may be used under controlled conditions but biocides have their limits. A biocide may not work if a heavy biofilm has accumulated on the surface of the tank or other equipment, because then it doesn’t reach the organisms living deep within the biofilm. In such cases, the tank must be drained and mechanically cleaned. And even if the biocide effectively stops biogrowth, it still may be necessary to remove the accumulated biomass to avoid filter plugging. Since biocides are toxic, any water bottoms that contain biocides must be disposed of appropriately. Safety Properties

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Jet fuel can be hazardous if not handled properly. Jet fuel is easy to ignite and it burns rapidly. Exposure to jet fuel liquid or vapor should be limited. It should be kept in mind that liquid doesn’t burn; only vapor burns and vapor doesn’t always burn – the mixture of vapor and air must be within the flammable3 range. Mixtures with insufficient vapor (below the lower flammability limit) or too much vapor (above the upper flammability limit) will not burn. For kerosene-type jet fuel, the lower and upper flammability limits4 are 0.6 volume percent vapor in air and 4.7 volume percent vapor in air, respectively5 . For wide-cut jet fuel, the lower and upper flammability limits are 1.3 volume percent vapor in air and 8.0 volume percent vapor in air, respectively. (Appendices A and B) Flash Point The flash point is the lowest temperature at which the vapors above a flammable liquid will ignite on the application of an ignition source. At the flash point temperature, just enough liquid has vaporized to bring the vapor-air space over the liquid above the lower flammability limit. The flash point is a function of the specific test conditions under which it is measured. The flash point of wide-cut jet fuel is below 0°C (32°F) and is not typically measured or controlled. The minimum flash point of Jet A kerosene-type jet fuel is 38°C (100°F). Example of composition of additives used in JP8

Additive/

Ingredient

Chemical Function

Antioxidant Butylated

HydroxyToluene

Inhibit gum formation

Metal Deactivator N,N'-disalicylidene-1,2-

propanediamine

Complex with trace metals to reduce

catalysis of thermal oxidation reaction

Dispersant Trade secret Reduce size of solid particles, keep

solids in solution

Detergents Trade secret Remove solids from surfaces

Aromatic Solvent Alkylbenzenes,

hydroaromatics

Solvent

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Solvent Naphthalene Solvent

Solvent 1,2,4-trimethylbenzene Solvent

Paul Rawson, “AMRL Evaluation of the JP-8+100 Jet Fuel Thermal Stability Additive”, DSTO Aeronautical and Maritime Research Laboratory, Commonwealth of Australia 2001

Attachment Three Economic Analysis

For this economical analysis, the following assumptions have been made:

1) One ton dry wood will be used as raw materials, 2) Plant will work 350 days per year, 3) The refinery will use short rotation hybrid poplar crop for $50/dry ton delivered to refinery, 4) The price of final products (except Acetone) are based on energy price of $0.025/KBTU and for each

products will be: a. Butanol, $2.64/G b. Ethanol, $2.12/G c. Acetone, $16/G d. Bio-Oil, $1.76/G

5) Price of utilities are:

a. Electricity at $54.53 per MWh b. Water at $1.25 per KG c. Enzyme, Bacterial, and other Chemical at $2000 per Ton d. Waste Water costs at $1/G

6) Price of Acetone is $16/Gallon, and 7) The lignin can be either cracked to produce bio-oil and therefore Bio-JP8, or lignin can be used as low

energy fuel to provide required energy for the production plant. To perform the economic Analysis, a CHEM-CAD simulation has been performed and as a result it has been determined that a typical sustainable bio-refinery, using one ton dry feed stock, can produce the following products:

a. 19953 Gallons per year of Butanol b. 12317 Gallons per year of Ethanol c. 6792 Gallons per year of Acetone, and d. 16564 Gallons of Bio-Oil

And will use the following utilities:

a) Electricity, 420 MWh

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b) Water, 619 KG at c) Enzyme, Bacterial, and other Chemical ~45 Tons d) Creates 505 KG of Waste Water

Based on the above data and assumptions, a plant using one ton of raw materials per day will have a total operating revenue of $216,633 and a total variable cost of $461,681. In addition, it has been assumed that three technicians at $40,000 per year (plus 30% fringe) are required to work in this plant. The indirect costs of operation is 60% of the direct labor plus variable operating expenses. As a result the fixed operating cost will be $385,849. So, the total net income of the operation will be ($630,897) loss. To make the business profitable, it is necessary to increase production from one ton to 50 ton, reduce the cost and amount of the enzyme and chemicals by at least 30% each, and reduce the consumption of electricity by about 30%.

Appendix A

Material Safety Data Sheet SECTION 1 PRODUCT

JP-8 Product Use: Fuel SECTION 2 COMPOSITION/ INFORMATION ON INGREDIENTS

COMPONENTS CAS NUMBER AMOUNT Kerosene 8008-20-6 > 99 %weight Diethylene glycol monomethyl ether 111-77-3 < 1 %weight

SECTION 3 HAZARDS IDENTIFICATION

************************************************************************************************************************ EMERGENCY OVERVIEW Clear to light yellow liquid with petroleum odor. - COMBUSTIBLE LIQUID AND VAPOR - HARMFUL OR FATAL IF SWALLOWED - CAN ENTER LUNGS AND CAUSE DAMAGE

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- MAY CAUSE RESPIRATORY TRACT IRRITATION IF INHALED - CAUSES SKIN IRRITATION - TOXIC TO AQUATIC ORGANISMS ************************************************************************************************************************ IMMEDIATE HEALTH EFFECTS Eye: Not expected to cause prolonged or significant eye irritation. Skin: Contact with the skin causes irritation. Symptoms may include pain, itching, discoloration, swelling, and blistering. Contact with the skin is not expected to cause an allergic skin response. Not expected to be harmful to internal organs if absorbed through the skin. Ingestion: Because of its low viscosity, this material can directly enter the lungs, if swallowed, or if subsequently vomited. Once in the lungs it is very difficult to remove and can cause severe injury or death. May be irritating to mouth, throat, and stomach. Symptoms may include nausea, vomiting, and diarrhea. Inhalation: Breathing this material at concentrations above the recommended exposure limits may cause central nervous system effects. Central nervous system effects may include headache, dizziness, nausea, vomiting, weakness, loss of coordination, blurred vision, drowsiness, confusion, or disorientation. At extreme exposures, central nervous system effects may include respiratory depression, tremors or convulsions, loss of consciousness, coma or death. Mists of this material may cause respiratory irritation. Symptoms of respiratory irritation may include coughing and difficulty breathing. SECTION 4 FIRST AID MEASURES

Eye: No specific first aid measures are required because this material is not expected to cause eye irritation. As a precaution, remove contact lenses, if worn, and flush eyes with water. Skin: Wash skin with water immediately and remove contaminated clothing and shoes. Get medical attention if any symptoms develop. To remove the material from skin, use soap and water. Discard contaminated clothing and shoes or thoroughly clean before reuse. Ingestion: If swallowed, do not induce vomiting. Give the person a glass of water or milk to drink and get immediate medical attention. Never give anything by mouth to an unconscious person. Inhalation: Move the exposed person to fresh air. If not breathing, give artificial respiration. If breathing is difficult, give oxygen. Get medical attention if breathing difficulties continue. Note to Physicians: Ingestion of this product or subsequent vomiting may result in aspiration of light hydrocarbon liquid, which may cause pneumonitis. SECTION 5 FIRE FIGHTING MEASURES

See Section 7 for proper handling and storage. FIRE CLASSIFICATION: OSHA Classification (29 CFR 1910.1200): Combustible liquid. NFPA RATINGS: Health: 0 Flammability: 2 Reactivity: 0 FLAMMABLE PROPERTIES: Flashpoint: (Tagliabue Closed Cup) 100 ºF (38 C) (Min) Auto ignition: 410ºF (210ºC) Flammability (Explosive) Limits (% by volume in air): Lower: 0.7 Upper: 5 EXTINGUISHING MEDIA: Use water fog, foam, dry chemical or carbon dioxide (CO2) to extinguish flames. PROTECTION OF FIRE FIGHTERS: Fire Fighting Instructions: For fires involving this material, do not enter any enclosed or confined fire space without proper protective equipment, including self-contained breathing apparatus. Combustion Products: Highly dependent on combustion conditions. A complex mixture of airborne solids, liquids, and gases including carbon monoxide, carbon dioxide, and unidentified organic compounds will be evolved when this material undergoes combustion. SECTION 6 ACCIDENTAL RELEASE MEASURES

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Protective Measures: Eliminate all sources of ignition in the vicinity of the spill or released vapor. If this material is released into the work area, evacuate the area immediately. Monitor area with combustible gas indicator. Spill Management: Stop the source of the release if you can do it without risk. Contain release to prevent further contamination of soil, surface water or groundwater. Clean up spill as soon as possible, observing precautions in Exposure Controls/Personal Protection. Use appropriate techniques such as applying non-combustible absorbent materials or pumping. All equipment used when handling the product must be grounded. A vapor suppressing foam may be used to reduce vapors. Use clean non-sparking tools to collect absorbed material. Where feasible and appropriate, remove contaminated soil. Place contaminated materials in disposable containers and dispose of in a manner consistent with applicable regulations. Reporting: Report spills to local authorities and/or the U.S. Coast Guard's National Response Center at (800) 424-8802 as appropriate or required. SECTION 7 HANDLING AND STORAGE

Precautionary Measures: Liquid evaporates and forms vapor (fumes) which can catch fire and burn with explosive force. Invisible vapor spreads easily and can be set on fire by many sources such as pilot lights, welding equipment, and electrical motors and switches. Fire hazard is greater as liquid temperature rises above 85F. Do not get in eyes, on skin, or on clothing. Do not breathe vapor or fumes. Do not breathe mist. Do not taste or swallow. Wash thoroughly after handling. Do not use as a portable heater or appliance fuel. Toxic fumes may accumulate and cause death. General Handling Information: Avoid contaminating soil or releasing this material into sewage and drainage systems and bodies of water. Static Hazard: Electrostatic charge may accumulate and create a hazardous condition when handling this material. To minimize this hazard, bonding and grounding may be necessary but may not, by themselves, be sufficient. Review all operations which have the potential of generating an accumulation of electrostatic charge and/or a flammable atmosphere (including tank and container filling, splash filling, tank cleaning, sampling, gauging, switch loading, filtering, mixing, agitation, and vacuum truck operations) and use appropriate mitigating procedures. For more information, refer to OSHA Standard 29 CFR 1910.106, 'Flammable and Combustible Liquids', National Fire Protection Association (NFPA 77, 'Recommended Practice on Static Electricity', and/or the American Petroleum Institute (API) Recommended Practice 2003, 'Protection Against Ignitions Arising Out of Static, Lightning, and Stray Currents'. General Storage Information: DO NOT USE OR STORE near heat, sparks or open flames. USE AND STORE ONLY IN WELL VENTILATED AREA. Keep container closed when not in use. Container Warnings: Container is not designed to contain pressure. Do not use pressure to empty container or it may rupture with explosive force. Empty containers retain product residue (solid, liquid, and/or vapor) and can be dangerous. Do not pressurize, cut, weld, braze, solder, drill, grind, or expose such containers to heat, flame, sparks, static electricity, or other sources of ignition. They may explode and cause injury or death. Empty containers should be completely drained, properly closed, and promptly returned to a drum reconditioner or disposed of properly. SECTION 8 EXPOSURE CONTROLS/PERSONAL PROTECTION

GENERAL CONSIDERATIONS: Consider the potential hazards of this material (see Section 3), applicable exposure limits, job activities, and other substances in the work place when designing engineering controls and selecting personal protective equipment. If engineering controls or work practices are not adequate to prevent exposure to harmful levels of this material, the personal protective equipment listed below is recommended. The user should read and understand all instructions and limitations supplied with the equipment since protection is usually provided for a limited time or under certain circumstances. ENGINEERING CONTROLS: Use process enclosures, local exhaust ventilation, or other engineering controls to control airborne levels below the recommended exposure limits. PERSONAL PROTECTIVE EQUIPMENT Eye/Face Protection: No special eye protection is normally required. Where splashing is possible, wear safety glasses with side shields as a good safety practice.

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Skin Protection: Wear protective clothing to prevent skin contact. Selection of protective clothing may include gloves, apron, boots, and complete facial protection depending on operations conducted. Suggested materials for protective gloves include: 4H (PE/EVAL), Nitrile Rubber, Polyvinyl Alcohol (PVA) (Note: Avoid contact with water. PVA deteriorates in water.), Viton Respiratory Protection: Determine if airborne concentrations are below the recommended exposure limits. If not, wear a NIOSH approved respirator that provides adequate protection from measured concentrations of this material, such as: Air-Purifying Respirator for Organic Vapors Use a positive pressure, air-supplying respirator if there is potential for uncontrolled release, exposure levels are not known, or other circumstances where air-purifying respirators may not provide adequate protection. Occupational Exposure Limits: Component Limit TWA STEL Ceiling Notation Kerosene CHEVRON 350 mg/m3 1000 mg/m3 SECTION 9 PHYSICAL AND CHEMICAL PROPERTIES

Appearance and Odor: Clear to light yellow liquid with petroleum odor. pH: NA Vapor Pressure: 1 kPa (0.14 psi) @ 100 ºF Vapor Density (Air = 1): 5.7 Boiling Point: 160 - 300 ºC (320 - 572 F) Solubility: Low PPM range in water. Freezing Point: -47 ºC (-53 F) (Max) Density: 0.755 - 0.84 g/ml @ 15 ºC Viscosity: 8 cSt @ -20 ºC (Max) SECTION 10 STABILITY AND REACTIVITY

Chemical Stability: This material is considered stable under normal ambient and anticipated storage and handling conditions of temperature and pressure. Incompatibility With Other Materials: May react with strong oxidizing agents, such as chlorates, nitrates, peroxides, etc. Hazardous Decomposition Products: None known (None expected) Hazardous Polymerization: Hazardous polymerization will not occur. SECTION 11 TOXICOLOGICAL INFORMATION

IMMEDIATE HEALTH EFFECTS Eye Irritation: The eye irritation hazard is based on evaluation of data for similar materials or product components. Skin Irritation: The skin irritation hazard is based on evaluation of data for similar materials or product components. Skin Sensitization: The skin sensitization hazard is based on evaluation of data for similar materials or product components. Acute Dermal Toxicity: The acute dermal toxicity hazard is based on evaluation of data for similar materials or product components. Acute Oral Toxicity: The acute oral toxicity hazard is based on evaluation of data for similar materials or product components. Acute Inhalation Toxicity: The acute inhalation toxicity hazard is based on evaluation of data for similar materials or product components. SECTION 12 ECOLOGICAL INFORMATION

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ECOTOXICITY This material is expected to be toxic to aquatic organisms. ENVIRONMENTAL FATE Ready Biodegradability: This material is not expected to be readily biodegradable. SECTION 13 DISPOSAL CONSIDERATIONS

Use material for its intended purpose or recycle if possible. This material, if it must be discarded, may meet the criteria of a hazardous waste as defined by US EPA under RCRA (40 CFR 261) or other State and local regulations. Measurement of certain physical properties and analysis for regulated components may be necessary to make a correct determination. If this material is classified as a hazardous waste, federal law requires disposal at a licensed hazardous waste disposal facility. SECTION 14 TRANSPORT INFORMATION The description shown may not apply to all shipping situations. Consult 49CFR, or appropriate Dangerous Goods Regulations, for additional description requirements (e.g., technical name) and mode-specific or quantity-specific shipping requirements. DOT Shipping Name: FUEL, AVIATION, TURBINE ENGINE DOT Hazard Class: 3 (Flammable Liquid) DOT Identification Number: UN1863 DOT Packing Group: III SECTION 15 REGULATORY INFORMATION SARA 311/312 CATEGORIES: 1. Immediate (Acute) Health Effects: YES 2. Delayed (Chronic) Health Effects: NO 3. Fire Hazard: YES 4. Sudden Release of Pressure Hazard: NO 5. Reactivity Hazard: NO REGULATORY LISTS SEARCHED: 4A=IARC Group 1 12=TSCA Section 8(a) PAIR 21=TSCA Section 5(a) 4B=IARC Group 2A 13=TSCA Section 8(d) 25=CAA Section 112 HAPs 4C=IARC Group 2B 15=SARA Section 313 26=CWA Section 311 05=NTP Carcinogen 16=CA Proposition 65 28=CWA Section 307 06=OSHA Carcinogen 17=MA RTK 30=RCRA Waste P-List 09=TSCA 12(b) 18=NJ RTK 31=RCRA Waste U-List 10=TSCA Section 4 19=DOT Marine Pollutant 32=RCRA Appendix VIII 11=TSCA Section 8(a) CAIR 20=PA RTK The following components of this material are found on the regulatory lists indicated. Kerosene 17, 18, 20 Diethylene glycol monomethyl ether 17, 20, 25 CHEMICAL INVENTORIES: UNITED STATES: All of the components of this material are on the Toxic Substances Control Act (TSCA) Chemical Inventory. CANADA: All the components of this material are on the Canadian Domestic Substances List (DSL). WHMIS CLASSIFICATION:

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Class B, Division 3: Combustible Liquids Class D, Division 2, Subdivision B: Toxic Material - Skin or Eye Irritation SECTION 16 OTHER INFORMATION

NFPA RATINGS: Health: 0 Flammability: 2 Reactivity: 0 (0-Least, 1-Slight, 2-Moderate, 3-High, 4-Extreme, PPE:- Personal Protection Equipment Index recommendation, *- Chronic Effect Indicator). These values are obtained using the guidelines or published evaluations prepared by the National Fire Protection Association (NFPA) or the National Paint and Coating Association (for HMIS ratings). REVISION STATEMENT: REVISION STATEMENT: This document has been prepared using a new MSDS format and all 16 sections have been revised. Please read the entire document. Also, this is the first MSDS from Chevron Global Aviation, a division of Chevron USA, Inc. ABBREVIATIONS THAT MAY HAVE BEEN USED IN THIS DOCUMENT: TLV - Threshold Limit Value TWA - Time Weighted Average STEL - Short-term Exposure Limit PEL - Permissible Exposure Limit CAS - Chemical Abstract Service Number NDA - No Data Available NA - Not Applicable <= - Less Than or Equal To >= - Greater Than or Equal To

Prepared according to the OSHA Hazard Communication Standard (29 CFR 1910.1200) and the ANSI MSDS Standard (Z400.1).

The above information is based on the data of which we are aware and is believed to be correct as of the date hereof. Since this information may be applied under conditions beyond our control and with which we may be unfamiliar and since data made available subsequent to the date hereof may suggest modifications of the information, we do not assume any responsibility for the results of its use. This information is furnished upon condition that the person receiving it shall make his own determination of the suitability of the material for his particular purpose.

Appendix B

Health Questions This fact sheet answers some of the more common health questions about jet fuels, especially JP-8. Commercial (Jet A/A-1) and Military (JP-8) jet fuels are the most widely used fuels in the world. It is important to understand the health and safety information about jet fuel. Recognizing how jet fuel (JP-8) could harm you and what you can do to protect your health is a priority in the Department of Defense (DoD). Remember, the health effect of any exposure will vary depending on the dose or concentration of the substance, how long you are exposed, the way you are exposed (through skin contact, eating or drinking, or breathing it into your lungs) and your personal characteristics (including your age, gender, genetics and immune system status).

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What is JP-8?

JP-8 powers military aircraft and other high performance vehicles and equipment, including tanks, power generators and space heaters. JP-8 is very similar to Jet A/A-1 fuel, which is used in commercial aircraft around the world.

JP-8 is made by refining crude petroleum. Its primary ingredient is kerosene. The primary ingredient in JP-8 is kerosene, usually about 99.8% by weight. In addition to kerosene, JP-8 contains very small amounts of many other substances, such as benzene, and various additives to inhibit icing, prevent static charge buildup, avoid oxidation, and decrease corrosion. Why does the U.S. Air Force use JP-8?

Between 1976 and 1996, the Department of Defense began converting from JP-4 to JP-8 because JP-8 is safer to use. Planes using JP-8 are much less likely to explode if damaged in combat. How does JP-8 enter the environment? • Some of the components in JP-8 will evaporate into the air. • JP-8 in the air may form other compounds by reacting with other substances in the air or by being exposed to sunlight. • Bacteria and other organisms in soil or water can cause JP-8 to break down into other substances. • If JP-8 gets in the water, some of its components will attach to other particles in water and settle to the bottom. • If JP-8 gets in the soil, it may move slowly into the groundwater • JP-8 may stay in soil for years. How are people exposed to JP-8?

The most common way for people to be exposed to JP-8 is if they routinely work with it as part of their job. Some examples of how other people might be exposed to JP-8 are listed below. These potential exposures are, for the most part, much less than would be expected for people who work directly with the fuel. • Breathing air in an area where there is a JP-8 leak, such as a leaking storage tank or pipeline. • Drinking water containing JP-8. • Touching or eating soil containing JP-8. • Living near sites where JP-8 was improperly disposed. How can JP-8 affect my health?

Little is known about human health effects caused by JP-8. Various university and government scientists are presently doing research on JP-8.

The ability of a compound to affect your health depends on the way you were exposed, (skin, oral, or by breathing), how much you were exposed to, the length of the exposure and personal characteristics (age, gender, family traits, diet and other habits). We are able to make some predictions on possible health effects based on our knowledge of kerosene, the primary ingredient in JP-8. Possible health effects from JP-8 include: • Skin irritation and sensitization, resulting in itchy, red, peeling or tender skin. • Breathing in large amounts of jet fuel will make breathing painful and may cause you to feel like you are suffocating.

These high exposures can also cause headaches, difficulty concentrating, fatigue and trouble with balance or coordination.

• Breathing in small amounts of jet fuel vapors over a long period of time might result in sleep disturbances or dizziness.

• Accidentally swallowing a small amount of JP-8, such as could occur by not washing JP-8 off your • hands before eating a sandwich, has not been shown

to cause any significant health problems. • Drinking JP-8 is dangerous and can result in coma, convulsions and even death.

JP-8 Acute Exposure Study Page 2 General Facts and Information • • It isn't known whether JP-8 can affect fertility or cause birth defects. Does JP-8 cause cancer?

The International Agency for Research on Cancer (IARC) has concluded there isn't enough information available to determine if jet fuels cause cancer. Is there a medical test for JP-8?

There are laboratory tests that can identify various substances contained in the breath, blood or urine of people who have been exposed to JP-8. However, the tests are not widely available and are used primarily for research. Also, the substances that can be detected are found in other compounds, so positive tests do not necessarily mean that you were exposed to JP-8. Are there government standards to protect human health? There are no specific guidelines or regulations for jet fuel exposure and no definitive scientific study results available to help set appropriate limits. In the absence of such regulatory standards, the Air Force Medical Operations Agency (AFMOA) in the USAF Surgeon General's Office establishes standards. The current standard for JP-8 is: • An occupational exposure limit (OEL) of 350 mg/m3 for an 8-hour time weighted average exposure. • A 15-minute short-term exposure limit (STEL) of 1800 mg/m3. What should I do to protect my health? For people who work with JP-8, it is important to follow all existing regulations, guidance and technical orders, especially those concerning the wearing of personal protective equipment. As is the case for other potentially hazardous substances found in the workplace, personal hygiene remains one of the most effective means of protection—and it is completely within your control. If you get fuel on your uniform or coveralls, change into clean items as soon as you can. Be sure to launder any items of clothing that came in contact with fuel before reusing them. And finally, always wash your hands and face with soap and water before eating.

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Where can I get more information? Sources for more information include: Air Force Institute for Environment, Safety and Occupational Health Risk Analysis

(AFIERA) Phone: 1-888-232-ESOH (3764), Internet URL: http://afiera.afms.mil

U.S. Army Center for Health Promotion and Preventive Medicine (USACHPPM) Phone: 1-800-222-9698, Internet URL: http://chppm-www.apgea.army.mil

Navy Environmental Health Center (NEHC) Phone: 1-757-466-5500, Internet URL: http://www-nehc.med.navy.mil

Agency for Toxic Substances and Disease Registry (ATSDR) Division of Toxicology, Phone: 1-757-466-5500, Internet URL:

http://www.atsdr.cdc.gov