Abstract:
 
MBI has developed a corn-based, temperature-programmed, multi-stage, fermentation process for the manufacture of butanol. The process utilizes, a solvent-tolerant, improved bacterial strain of Clostridium acetobutylicum that produces butanol and total solvents in high concentrations. The patented strain has made the process stable, productive and high yielding. In a separate batch process, butanol was recovered by pervaporation process that resulted in phase separation. The permeating stream was enriched in butanol above the saturation limit (6%) and separated into a butanol-rich organic phase and lean aqueous phase. Pervaporation is less energy intensive and does not require large processing steps as in the distillation-based recovery process. Simultaneous removal of the product by pervaporation, during fermentation eliminates product inhibition, increases productivity and concentrates butanol selectively by phase separation. This project work will focus on i) integration of fermentation and pervaporation process units at pilot-scale for simultaneous fermentation and recovery of butanol, ii) design of process units for a commercial scale process, iii) completion of a first level economic analysis to obtain capital and manufacturing cost, and iv) projection of the impact of commercialization of butanol on utilization of corn and corn-processing byproducts, corn marketing price and additional income to corn producers. The project work involves an early on collaboration among technology developer MBI International, pervaporation unit developer Membrane Technology and Research, and the technology commercializer, Natural Chem Industries, Ltd. (Southwest) with the sole aim to validate the process at pilot-scale and economics, and to commercialize the process in a shortest possible time.
 

Key Words:

Butanol, acetone-butanol, ABE, solvent, pervaporation.

Impact Statement:

The acetone-butanol fermentation has a long history as a successful industrial fermentation process. The earliest was work performed by Pasteur in 1862. Butanol is a commodity chemical feedstock and solvent that was primarily produced by industrial fermentation. In the early 1900s, butanol was used to produce butadiene which was the most desirable raw material for synthetic rubber. The annual production of fermentation derived butanol was over 45 million pounds during 1945. But the fermentation-derived butanol process declined after World War II in the U.S. due both to changes in availability of renewable feedstocks (molasses, sugar cane) and the increase in availability of inexpensive petrochemical feedstocks. Butanol fermentation of beet molasses continued through the 1970s in the Soviet Union and fermentation of sugar cane molasses through the 1980s in South Africa.

Butanol is now synthesized chemically from petroleum derived ethylene, propylene and triethylaluminum or carbon monoxide and hydrogen. The major domestic producers of butanol and its derivatives are BASF, Chem Service Inc., Dow Chemical, Eastman Chemical, Hoechst Celanese, Shell, Union Carbide and Vista (Chem Marketing Report, 1991; Chemical Economic Handbook, 1990). The current U.S. production of butanol is more than 1.2 billion pounds per year and is experiencing a growth of 3-4% annually.

Domestic and international markets for butanol remains extremely tight, and the price is still firming because of low inventories and strong demand. Over the past two years, consumption of butanol has surged throughout the United States, Europe and Asia. The butanol producers see no let-up in demand in the foreseeable future due to the tight, hefty demand and supply. Butanol pricing has been rising steadily over the past few years due to propylene price increase and a dependance on housing and automotive markets (Chemical Economics Handbook, 1990). This type of situation represents an opportunity for the introduction of new fermentation-derived process technology based on an alternative feedstock whose supply is not limited, i.e., biomass.

Butanol prices vary according to contracts, but the bulk U.S. pricing is 40~504 per pound. Butanol prices are even higher in Europe and the Far East, way above 504 per pound. U.S. prices are moving up with worldwide increase in the supply and demand. The proposed butanol process consists of MBI's proprietary ABE fermentation process and microbial strain, and MTR's proprietary integrated recovery process. This process uses continuous, two-stage fermentation followed with a final batch stage fermentation, conventional cell recovery and pervaporation of acetone, butanol and ethanol. The production cost could be reduced (using corn starch at 64 per pound and pervaporation-based recovery process) to 25~304 per pound. This low production cost and availability of tremendous renewable resources will increase the production of fermentation-derived butanol.
 

In our opinion, market penetration of fermentation-based butanol will drive the utilization of corn from 7.2 million bushels in 1995 to 153 million bushels in 2010. This growth is based on a vary modest assumption of 4% increase per year in the butanol market. The estimated total butanol market, percent share for corn-based butanol and the quantity of corn required to meet the butanol market is provided in Table 1.

 

Table 1. Impact of Fermentation-based Butanol on Corn Utilization
	Year
	1995	1998	2000	2010
Total Butanol* Market (in million lbs)	1,300	1,460	1,580	2,300
Percentage of Corn-derived butanol in Market	5.0	20.0	40.0	60.0
Additional Usage of Corn (million bushels)	7.2	32.4	70.2	153.0
*assumed 4% growth annually

 

Communications Statement:

MBI has policy to communicate results of technology developments through press releases, brochures, institutional annual report, presentations at seminars, conferences and trade shows, and publications in reputed journals. The company has a program in place to oversee these activities. The goal is to effectively communicate to the public the progress made on different technologies at MBI. The communication statements routinely acknowledge the funding source such as ICMB, USDA, DOE, etc.

Budget Summary:

The totals from each funding source for this project are given as follows:

 

Illinois Corn Marketing Board	MBI1	MTR2	Natural Chem3
$149,000.00	$25,581.00	$25,000.00	$25,000.00

 

¹P.I.'s equal amount of time will be supported by MBI funds for this project. All the equipment except for pervaporation will be provided by MBI and is not charged in this proposal.

²Development of new class of pervaporation membranes for higher efficiencies.

³To develop and commercialize butanol process. The work includes market evaluation and market development, development of end-uses for the butanol and arranging for one or more ethanol plants or other facilities to serve as sites for commercial scale operations.

Objectives:

Production of butanol in China may still be through fermentative process but the last viable industrial acetone-butanol-ethanol (ABE) fermentation in the Western World was carried out by National Chemical Products in Germiston, South Africa, using the Clostridium acetobutylicum P262 strain. Batch fermentations, with molasses as the substrate, were run for 40-60 h. Although final solvent concentrations of 18-22 g/L were sometimes reached, the average concentration varied between 15 and 18 g/L. For batch ABE fermentation to be industrially viable, and to compete with the chemical process, concentrations of between 22 and 28 g/L must be obtained in 40-60 h (Woods, 1995). The economically competitive concentration will depend on the oil price, which can fluctuate.

In contrast, MBI has produced a total solvent concentration of more than 30 g/L using an improved mutant strain of C. acetobutylicum (U.S. Patent # 5,192,673) in a temperature programmed, multi-stage, continuous fermentation process (U.S. Patent # 5.063,156) in 48-72 h. Of this, concentration of butanol alone was about 20 g/L. This process was recently scaled-up by 30X. Butanol from the diluted fermentation broth was later recovered by pervaporation process. These processes, however, were run separately.

The goal of this project is to scale-up an integrated fermentation and pervaporation based recovery process using corn derived substrates. MBI International will conduct a feasibility study to gather design data to perform material and energy balance and also to calculate the energy savings created by this pervaporation based recovery process compared with other traditional processes. Membrane Technology and Research would invest their time and money in the development of new classes of pervaporation membranes for higher efficiencies and Natural Chem Industries, Ltd. would invest their time and money to analyze the project impact of commercializaiton of butanol on utilization of corn.

The fermentation microorganism could be a MBI International strain or any other strain recently developed by other experts in this field. Illinois Corn Marketing Board will decide and make available the strain for scale-up studies and MBI will destroy the strain upon completion of the project. The main objectives of this project are to:

A large number of publications on butanol-producing microbial cultures and the process demonstrates that enough research work has been done in past years (Linden et. al., 1985; Jones and Woods, 1986; Marlatt and Datta, 1986; Blaschek, 1986; Roffler et. al., 1987; Lovitt et. al., 1988; Laddisch, 1991; Maddox et. al., 1995; Qureshi and Maddox, 1991; Woods, 1995) on a continuous basis. It is time that the process be commercialized. Our goal is to fine-tune the process units and parameters and integrate both fermentation and recovery processes. We have the necessary pilot-scale facilities and fermentation and recovery experience. MBI will work with MTR and Natural Chem Industries and, together as a team, will reduce the time for commercialization of the butanol process.

Procedures:

The above-mentioned objectives will be achieved through combined efforts of scientists, engineers and business personnel from MBI, MTR and Natural Chem Industries.

Integration of fermentation and pervaporation process units

The goal is to allow simultaneous recovery of butanol from the broth during fermentation so that product inhibition can be eliminated. This will allow complete utilization of sugar and continual fermentation of the substrate at a relatively high rate. Removal of butanol will be achieved by integrating a pervaporation unit with a multi-stage fermentation system. A process schematic is provided in Figure 1. The scale of the process units for this integrated process demonstration at pilot-scale is shown in this figure. Approximate feed flow rate is targeted at 17-18 L/h. The recovery units will be sized according to this feed rate. The overall focus will be to operate continuously and to demonstrate long-term process stability. Pertinent issues to be addressed include: 1) membrane life (potential foulings) and replacement frequency in the cell recycle system; 2) maintenance of the strain activity in the stationary, solventogenic phase by control of environmental chemistry; and 3) control of fatty acid concentrations in stage one and solvent concentrations in stage two to minimize potential product inhibition.

The basic concept of pervaporation is illustrated in Figure 2. The dilute solvent broth is pumped through a membrane unit. There are many geometries but a suitable unit with high tangential liquid velocity will be used to prevent buildup of solids in the broth on the membrane surface. The membrane is selective in its transport for solvent components over water. The solvents which diffuse through the membrane evaporate on the other side into a low pressure chamber. The vapor is passed through a condenser at low temperature and recovered as a liquid and the vacuum pump is needed to maintain low pressure and to remove non-condensable gas. The membrane is an asymmetric structure with an ultra-thin hydrophobic layer on the liquid side. The best materials for high alcohol type selectivity are poly (dimethylsiloxane) PDMS, poly (1-trimethylsilypropyne) PTMSP, and poly (vinyl trimethylsilane) PVTMS. Since we have successfully separated butanol using a pervaporation unit (by MTR) in a separate batch process, we believe separation of solvents by pervaporation techniques in a multi-stage continuous process should be feasible without significant membrane fouling. In the process, the solvents will pass through the membranes preferentially and the organic acids and cells will remain on the liquid side and return to the fermentor. In this way, the organic acids will be returned to the fermentor for conversion to alcohols. The permeate stream, which is enriched in solvent relative to the feed, will separate into low butanol-aqueous phase and butanol-rich solvent phase. This aqueous phase could be recycled back to the feed tank. Pervaporation also provides for the retention of cells in the fermentor which improves volumetric productivity. A correlation between membrane flux and selectivity will be monitored.

The performance of the integrated system is expected to be a function of the cell concentrations in both reactors, the dilution rates, the pH values, and possibly, the butanol concentration in the stage two or subsequent fermentor. Concentrations of acids and alcohols in the liquid phase will be measured by gas chromatography.

Perform material and energy balance for the fermentation and pervaporation based recovery process and compare energy savings created by this pervaporation based recovery process.

During this process integration, data will be collected and analyzed to perform material and energy balances. Material and energy balances will be performed for each unit operation with experimental data. The energy savings will be calculated for this process and for another traditional recovery process. The energy savings will be compared and summarized in table form.

Project impact of commercialization of butanol

The project impact of commercialization of butanol will be analyzed with the commercial partner and with a corn wet miller. The amount of corn utilization, impact on corn production and social economic changes will be analyzed at the completion of this integrated process development. A proposal for a commercial demonstration will be prepared to identify a corn wet miller and client for butanol production.

Justification:

The demise of ABE fermentation was due to a number of intrinsic limitations in the process, which include low final solvent concentrations, low solvent yields, undesirable solvent ratios, low productivity, and relatively high substrate costs (Jones and Woods, 1986). In addition, with the advent of petrochemical processes and low cost petrochemical feedstocks, the fermentation-based processes became economically unattractive and most of the commercial installations were shut down in the 1950s.

The areas that must be addressed in developing a corn-based ABE fermentation are: (1) more efficient use of corn-derived substrate; (2) the production of strains that exhibit improved solvent yields and solvent ratios; (3) the development of strains that give superior performance and productivity in continuous and other fermentation systems; (4) the development of strains that have enhanced end-product tolerance, and can produce higher concentrations of solvents; and (5) the simple and economical butanol recovery process.

Butanol is primarily used as a feedstock chemical in the manufacture of lacquers, rayon, plasticizers, coatings, detergents, and brake fluids. It can also be used as a solvent for fats, waxes, resins, shellac and varnish. In addition, butanol may also be used as an extractant and solvent in the food industry.

The acetone-butanol fermentation currently has potential because butanol has many characteristics which make it a better liquid fuel extender than ethanol, now used in the formulation of gasohol. Motor fuels are of particular significance not only because transportation is important to our lifestyle, but also because motor fuels play a major role in our 12-digit balance of trade deficit that arises from our importation of foreign oil. Higher alcohols have several characteristics that are favorable for motor fuel use, either in gasoline blends or, in some cases, when used directly as fuel. They are miscible in gasoline; their heats of combustion per gallon are greater than those of methanol; and they have good octane-enhancing properties. As shown in Table 2, butanol has a heat of combustion 54% higher than methanol and 83% of that for gasoline. Although the octane numbers are less than that for methanol, the research octane number (RON) value of 100 is still above that of gasoline and therefore useful for fuel blends. Furthermore, butanol has a much lower vapor pressure than either methanol or ethanol, which would allow higher blend concentrations with much less evaporative loss due to component volatility. Butanol, which has low miscibility with water, exhibits high miscibility with both diesel and gasoline compared to methanol, an important factor when considering blend mixtures and choice of co-solvents. Owing to its high heat of combustion, butanol solutions containing as much as 20% (V/V) water, have the same combustion value as anhydrous ethanol. The water content of butanol solutions also results indirectly in a reduction of nitrogen oxide (NO) emissions by lowering the operating temperature of internal combustion engines. As additives, alcohols have the potential for reducing carbon monoxide emissions, improving the gasoline octane rating, acting as effective transportation fuels and/or extenders, and improving fuel utilization. Such compounds are prime targets for production from renewable resource such as corn.

 

Table 2. Characteristics of Alcohols for Use as Co-solvents and Octane Enhancers with Motor Fuels
Physical Property	Methanol	Ethanol	Butanol	Gasoline	Diesel
Heat of Combustion:  (k Btu/lb)	10.26	13.16	15.77	18.90	18.35
Vapor pressure:   (psi at 100°F)	4.64	2.35	0.32	variable	variable
Fuel Values:  (OCTANE)
RON	110	108	100	92	15
MON	91	90	87	83	--
Cetane #	3	8	25	8-14	45-55
Miscibility:
Gasoline	poor	fair	good	N/A	N/A
Diesel	poor	poor	good	N/A	N/A
Water	high	high	low	low	low
Adapted from S. Levy, U.S. Patent #4,260,936, April 1981; Perry's Handbook, 6th edition, McGraw Hill Book Company

 

In addition to its potential application as a liquid fuel, butanol has existing market as solvent and chemical intermediate (please see the Impact Statement).

Principal Investigators:

Mahendra K. Jain: - Director, Applied Biocatalysis. Dr. Jain, who is the Principal Investigator of this project, has about 23 years of research experience. He has focused on the use of microorganisms in both agricultural and industrial processes. He has worked on development of various corn-based anaerobic fermentations processes such as butyric acid, propionic acid, lactic acid, succinic acid and butanol. In addition, he has also developed processes for synthesis gas bioconversion to butanol, degradation of chlorinated toxic pollutants and coal comminution. Dr. Jain has obtained a solvent-tolerant improved butanol producing mutant strain and developed a multi-stage anaerobic continuous butanol process which have been patented. He has been awarded several grants and contracts from federal, state and industrial sources and, as a principal investigator and project manager, managed these projects successfully. Dr. Jain is an author on > 90 papers and 4 patents with additional 4 patents pending. In this project as a P.I., Dr. Jain will be responsible for overall project management and for continuous fermentation process and be the key contact person.

Ponnam Elankovan - Director, Bioprocess Engineering. Dr. Elankovan has about 12 years of experience in separation of microbial products from dilute fermentation broth. He is especially experienced in the development of cost-effective membrane-based separations for different products. Dr. Elankovan has developed electrodialysis method to separate and recover organic acids such as lactic acid and succinic acid. In addition, he has developed patented process for purification of the fermentation products. Dr. Elankovan has been awarded several federal and industrial contracts and managed these projects successfully at pilot-scale. In addition, he is also experienced in developing first level process economics for fermentation processes. Dr. Elankovan has published over 7 papers and has 3 patents. In this project as P.I., he will be responsible for integrated process scale-up, pervaporation based continuous separation of butanol, and development of first-level process economics.

Full Cvs for Drs. Jain and Elankovan are provided in Appendix A.

Consultants:

See Appendix A for supporting letters and background information on Natural Chem Industries, Ltd. and Membrane Research Technology & Research, Inc.

Patents and Publications:

Jain, M. K., D. Beacom and R. Datta. 1993. U.S. Patent #5,192,673. Mutant strain of Clostridium acetobutylicum and process for making butanol.

Glassner, D. A., M. K. Jain and R. Datta. 1991. U.S. Patent #5,063,156. Improved process for the fermentation production of acetone, butanol and ethanol.
 

Soni, B. K. and M. K. Jain. 1995. Comparison of Mutant and Parent Strains of Clostridium acetobutylicum Butyrate Uptake at Different Temperatures. J. Appl. Env. Microbiol. (being submitted).

Soni, B. K. and M. K. Jain. 1995. Influence of pH on Butyrate Uptake and Solvent Fermentation by a Mutant Strain of Clostridium acetobutylicum. J. Appl. Env. Microbiol. (being submitted).

Soni, B. K. and M. K. Jain. 1995. Butanol Production in a Temperature Programmed Multi-fermentor System: A Comparison of Mutant Strain and Parent Strain of Clostridium acetobutylicum (ATCC 4259). Biotech. and Bioengr. (being submitted).

Soni, B. K. and M. K. Jain. 1995. Influence of Dilution Rates on the Overall Performance of Multi-fermentor Chemostat Cultures by High Butanol Producing Mutant of Clostridium acetobutylicum. J. Appl. Microbiol. Biotechnol. (being submitted).

Soni, B. K. and M. K. Jain. 1995. High Substrate Utilization in Acetone-Butanol Fermentation by a Mutant Strain of Clostridium acetobutylicum. Appl. Microbiol. Biotechnol. (being submitted).

Grethlein, A. J., Soni, B. K., Worden, R. M. and Jain, M. K. 1992. Influence of hydrogen sulfide on the growth and metabolism of Butyribacterium methylotrophicum and Clostridium acetobutylicum. J. Appl. Biochem. 34/35:233-246.

Worden, R. M., Grethlein, A. J., Jain, M. K. and Datta, R. 1991. Production of butanol and ethanol from synthesis gas via fermentation. Fuel 70: 615-620.

Grethlein, A. J., Worden, R. M., Jain, M. K. and Datta, R. (1991). Evidence for production of n-butanol from carbon monoxide by Butyribacterium methylotrophicum. J. Ferment. Bioeng. 72(1): 58-60.

Worden, R. M. Grethlein, A. J., Jain, M. K., and Datta, R. (1990). Production of butanol and ethanol from synthesis gas via fermentation. In: Fuel Chemistry Division Preprints, American Chemical Society, Washington, D.C., August 26-31, 1990. 35(3):875-880.

Grethlein, A. J., Worden, R. M., Jain, M. K. and Datta, R. (1990). Continuous production of mixed alcohols and acids from carbon monoxide. J. Appl. Biochem. 24/25:875-884.

Jain, M. K., Datta, R. and Zeikus, J. G. (1989). High value organic acid fermentation - Emerging processes and products. In: T.K. Ghose (ed.), Bioprocess Engineering: First Generation, p. 366-389, Ellis Horwood Limited, Chichester, England.

Presentations:

Jain, M. K. Anaerobic Industrial and Environmental Processes. Invited Seminar, Department of Chemical Engineering, Wayne State University, Detroit, MI, October 13, 1995.

Chatterjee, S., Vick Roy, J., Grethlein, H. E., Worden, R. M., and Jain, M. K. Anaerobic fermentation of sulfur containing coal-derived synthesis gas to liquid fuels. Fourth International Symposium on biological processing of fossil fuels, September 21-23, 1993, Alghero, Italy.

Jain, M. K. Bioconversion of synthesis gas to liquid fuels. 33rd Annual Conference of Association of Microbiologists of India, November 5-7, 1992, Goa, India.

Jain, M. K. and Soni, B. K. Bioconversion of Externally Added Butyric Acid to Butanol by Clostridium acetobutylicum. Harnessing Biotechnology for the 21st Century - Ninth International Biotechnology Symposium and Exposition, August 16-21, 1992, Crystal City, Virginia.

Jain, M. K. Studies with a high butanol producing mutant of Clostridium acetobutylicum. Clostridia II, Second International Workshop on the Regulation of Metabolism, Genetics, and Development of the Solvent-Forming Clostridia, August 13-14, 1992, Blacksburg, Virginia.

Grethlein, A. J., Worden, R. M., Jain, M. K. and Datta, R. Regulation of butanol synthesis during carbon monoxide fermentation by an acidogenic anaerobe. Fourth Chemical Congress of North America and American Chemical Society National Meeting, August 25-30, 1991, New York, NY.

Grethlein, A. J., Soni, B. K., Worden, R. M., Jain, M. K. and Datta, R. Influence of hydrogen sulfide on the growth and metabolism of Butyribacterium methylotrophicum and Clostridium acetobutylicum. Thirteenth Symposium on Biotechnology for Fuels and Chemicals, May 6-10, 1991, Colorado Springs, Colorado.

Soni, B. K., Jain, M. K. and R. Datta. Influence of gas and cell recycle on the continuous acetone-butanol fermentation. Thirteenth Symposium on Biotechnology for Fuels and Chemicals, May 6-10, 1991, Colorado Springs, Colorado.

Jain, M. K., Worden, R. M., Grethlein, H. E. and Datta, R. Bioconversion of coal-derived synthesis gas to liquid fuels. Illinois Coal Development Board's 9th Annual Contractors' Technical Meeting, July 30 - August 1, 1991.

Grethlein, A. J., Worden, R. M., Jain, M. K., and Datta, R. Butanol production from carbon monoxide by Butyribacterium methylotrophicum. Bridging the Gap: Case Studies of University Technology Transfer to Industry, April 30 - May 1, 1990, East Lansing, MI.

Worden, R. M. Grethlein, A. J., Jain, M. K. and Datta, R. Production of butanol and ethanol from synthesis gas via fermentation. Fuel Chemistry Division, ACS, August 26-31, 1990, Washington, D.C.

Grethlein, A. J., Worden, R. M., Jain, M. K. and Datta, R. Butanol production from carbon monoxide by Butyribacterium methylotrophicum. Twelfth symposium on Biotechnology for Fuels and Chemicals, May 7-11, 1990, Gatlinburg, Tennessee.

Grethlein, A. J., Worden, R. M., Jain, M. K., Soni, B. K. and Datta, R. Two stage production of mixed solvents and organic acids by fermentation. Eleventh symposium Biotechnology for Fuels and Chemicals, Colorado Springs, Colorado, May 8-12, 1989.

Grethlein, A. J., Worden, R. M., Jain, M. K., and Datta, R. Continuous production of mixed alcohols and acids from carbon monoxide. Eleventh symposium Biotechnology for Fuels and Chemicals, Colorado Springs, Colorado, May 8-12, 1989.

Project Location:

The project work will be conducted at MBI International, Lansing, MI. MBI has a 20,000 ft² pilot-plant facility with fermentation capacity up to 1000 gallons fermentations.
 

Facilities and Equipment:

Technology development at MBI takes place in a 120,000 square-foot, state-of-the-art R&D center, including a 20,000 square-foot pilot plant. The fully equipped facility is capable of supporting multiple projects in microbiology, bioprocess engineering, materials science, and environmental engineering, and is well suited to conduct laboratory and pilot-scale research, development, and production. Besides the laboratories and pilot-plant, support services and administrative services are integrated into the facility, to efficiently coordinate project development. General features of the MBI facility include:

The microbiology laboratories are modern, fully-supplied laboratories. The majority of the culture isolation, media formulation, strain development, and culture optimization work is conducted in these laboratories. Each lab has several bench-tops with easy access to refrigerators, incubators, analytical balances, temperature-controlled shakers, bench-top and laboratory centrifuges, spectrophotometers, both gas and liquid chromatographs, and several modern microscopes. In addition, specific equipment for anaerobic work, including glove-boxes and media dispensing manifolds are located in these laboratories. Also located in the microbiology laboratories are equipment for culture lyophilization and freeze drying. Easy access to the fermentation laboratory allows for smooth transfer of samples and cultures to and from these respective areas.
 
Several environmental growth chambers are located at MBI, with temperatures ranging from -35 to 60°C. These chambers operate with independent refrigerated/heated air flow, and are large enough for limited bench-top experimentation. There are also three cryogenic storage freezers available, which operating at -80, -100, and -130°C, as well as several more -60°C freezers located in the microbiology laboratories. The facility also contains two large, general use autoclaves, located in the dish washing area and in the fermentation laboratory.

Chemistry laboratories contain chemical fume hoods, laboratory-size solvent storage cabinets, and refrigerator/freezer storage space. As with the microbiology laboratories, the chemistry labs contain numerous outlets for all house utilities except steam. These labs are also equipped with medium and high-temperature ovens, and bench-top analytical equipment such as pH meters, spectrophotometers, and centrifuges. Laboratory-scale gel-chromatography and purification, solvent extraction, distillation, crystallization, evaporation, and chemical isolation work is typically conducted in the chemistry laboratories.

Bench-scale fermentations of 1-15 L are conducted in a separate fermentation laboratory, with several bench tops for conducting experiments, a sink and wash area, a floor-length chemical fume hood, and also has all house utilities, including steam. Both 1-2 and 10-15 L fermentors are located in this area, all with separate ancillary equipment for temperature, pH, gas flow, and agitation control. The 10-15 L fermentation systems can be operated either on the bench-tops or on mobile carts, depending on the harvesting requirements. Sterilization of fermentors and equipment is conducted in an adjacent autoclave. Bench-scale fermentation experiments, from cleaning to sterilization to operation, can be conducted entirely in this laboratory. Samples from fermentation experiments are easily transported to either the microbiology and chemistry laboratories or the fermentation analysis laboratories. This area also allows easy access to the pilot-plant, for transfer of starter culture inocula to the larger volume fermentation systems.

The fermentation analysis laboratory houses the majority of analytical equipment for liquid phase products, including automated gas chromatography (flame-ionization, thermocouple, and hot-wire detectors available, as necessary), automated liquid chromatography (HPLC), visible and UV spectrophotometry, ambient and refrigerated centrifugation, and pH and viscosity measurement. A floor-length chemical fume hood for sample preparation is located in this laboratory, as well as refrigeration space for sample storage. Ample bench space is available for sample preparation and handling.

Other analytical capabilities available on a fee-for-services basis at Michigan State University (East Lansing, Michigan), include gas chromatography/mass spectrometry, atomic adsorption spectrometry, nuclear magnetic Resonance (NMR) spectrometry, and scanning, transmission, and x-ray diffraction microscopy.

MBI's 20,000 square-foot pilot facility serves the Institute's bioprocess scale-up needs for fermentation, separation, and purification. The three-level pilot facility, with a cargo elevator connecting the levels, is equipped with state-of-the-art equipment, instrumentation, and computer-control systems. A 5-ton capacity ceiling crane is available for movement of heavy processing equipment. The basement houses several mixing kettles and continuous sterilizer for media formulation and preparation. Sterilized media can then be pumped to either the first or second level, depending on which fermentation system is used. The first level contains much of the separation and purification equipment, including several evaporators, ultrafiltration systems, a centrifuge, and the 3700 L fermentor systems. The second floor houses the main fermentation bay, with several 80 L fermentors and the 500 L fermentation system. A 900 square-foot cold room in this area allows for large-scale handling of sensitive microorganisms, media, and products before, during, and after processing. The electrodialysis systems, gel-chromatography, and ion exchange columns are also located in a separate pilot-laboratory on the second floor.

Complete process utilities are available throughout the pilot-plant, including pressurized air, clean-steam (500 lbs/hr at 1575 psig and 603°F), water, purified water, gas, vacuum, and adequate drainage. Almost all of the pilot-plant equipment, except for the 500 and 3700 L fermentation systems, and the 500 L/hr vacuum evaporator, are either on wheels or attached to wheeled carts, and are thus completely mobile for relocation throughout the pilot facility. This allows great flexibility when designing and conducting bioprocess development, scale-up, and production in MBI's pilot-plant.

MBI also has its own machine and fabrication shop staffed by experienced crafts people, where specialized equipment and fittings can be manufactured from plastic, iron, aluminum, carbon steel, stainless steel, and various other materials.

Integration of Pilot Plant Operations

MBI has several core capabilities in bioprocess development which combine to form an integrated, fully functional pilot plant. These include:

These capabilities allow MBI to design, develop, and scale-up complete fermentation and recovery processes. Areas such as bacterial and fungal fermentation, biochemical product separations, chemical separations and recovery, and final product purification are emphasized. Together with laboratory microbial strain and culture development, a comprehensive approach to a wide-range of development issues in industrial biotechnology can be achieved.

Laboratory operations in culture handling, strain improvement, industrial media development and optimization, and optimization of bench scale fermentations are used prior to scale-up to take advantage of the lower costs and throughput of experiments associated with this scale of operations. Techniques such as mutagenesis, along with an appropriate screening protocol, are used to improve microbial strains for high product yield, to reduce or eliminate undesired by-products, or to alter other metabolic properties. Microbial nutritional requirements, particularly available nutrient levels, are carefully defined in the laboratory to develop precise media requirements for production media and to regulate product formation and yield. Cell growth and fermentation are optimized at the bench scale to maximize product concentration, obtain fermentation control points, and establish the best conditions for product formation. The work is conducted so that the fermentation, if desired, is ready for scale-up and process development in the pilot plant.

Successfully transferring experimental results and findings from the laboratory to the pilot plant involves integration of process optimization, process scale-up, and production, and requires significant scientific and engineering skill, as well as a suitable, versatile pilot facility. MBI's pilot plant is fully capable of conducting a variety of fermentation operations, including fermentation development, optimization, scale-up, process control, and process design. Fermentation capacities range from the shake flask to the 1000 gallon scale, and the equipment has been used for aerobic and anaerobic fermentation, recombinant organism fermentation, industrial and production media development, and many other processes. Engineering design work for fermentor configuration, media sterilization optimization, generation of process mass and energy balances, equipment fabrication, and development of P&ID's is routinely conducted by MBI's staff engineers. Equally important is the available experience in computer control of fermentation processes, on-line data acquisition, and process integration with downstream unit operations.

MBI's experience has shown that the recovery and purification processes are often where the bottlenecks and scale-up challenges occur, particularly in terms of cost. The pilot scale recovery capabilities have been developed to address this issue, and encompass both laboratory development expertise and pilot scale demonstration. Core capabilities to recover a wide range of intra- and extra-cellular products have been developed from both a facilities and a staff standpoint, and include centrifugation, membrane filtration, solvent extraction, cell disruption, distillation, ion exchange, column chromatography, electrodialysis, protein precipitation, evaporation, and crystallization. A large amount of process development experience in conducting bench feasibility, unit operation validation, and integrating unit operations with upstream fermentation development has been achieved. Comprehensive technical support and documentation for plant engineering and start-up is integrated with the recovery operations. As with fermentation, the recovery and purification expertise includes process scale-up, design of equipment, process optimization, process modeling and economics, and computer-controlled operation and data acquisition.

MBI's Available Pilot Plant Equipment and Capabilities

 

Quantity	Capacity	Manufacturer	Equipment Item
Fermentation:
1	500L	New Brunswick Scientific	Fermentor
1	350L	Vesselcraft	Fermentor
1	300L	Bioengineering	Sterile Media Tank/ Anaerobic Fermentor
1	80L	New Brunswick Scientific	Fermentor
2	50L	MBI	Fermentors
1	15L	New Brunswick Scientific	Fermentor
2	15L	MBI	Fermentors
1	15L & 5L	Braun	Fermentor
1	25 sq ft	Alfa-Laval/MBI	Continuous media sterilizer, spiral
1	10 sq ft	MBI	Continuous media sterilizer, tube-in-tube
1	3700L	Pfaudler	Sterile Media Tank/ Anaerobic Fermentor
Recovery and Downstream Processing:
1	25-100 sq ft	Amicon DC30P	Ultrafilter
1	0.5-5 sq ft	Amicon DC10	Ultrafilter
1		MBI	Ultrafilter, accepts a variety of manufacturers' cartridges
1	0.5 L	Westfalia SAOH-205	Disc-type, desludging centrifuge
1	1.7-13 sq ft	Sparkler 18-13	Pressure-leaf filter
1	0.3-0.9 sq ft	Sparkler 8-3	Pressure-leaf filter
1	2 sq ft	Emerson-Scheuring	Pressure/vacuum leaf filter
1	16 sq ft	Lydon Bros.	Tray drier
1	1.7sq ft	Pfaudler	Wiped-film evaporator
1		Whitlock Model E-66-7606	Recirculating evaporator
1		Precison Scientific Model F-10	Recirculating evaporator
6	0.8 cu. ft.	MBI	Ion exchange/absorption columns, PVC
14	2.5 cu. ft.	MBI	Ion exchange/absorption columns, PVC
1	30L	Buchi Model R152	Rotary evaporator, glass
1	2L	MBI	Batch glass distillation system
1	0.25 sq ft	EIMCO	Test-leaf filter, simulates rotary and belt vacuum filters
2	80 gal.	Lee Process Equipment	Hemispherical food kettles with lids and agitators
2	150 gal.	Lee Process Equipment	Hemispherical food kettles with lids and agitators
3		MBI	Electrodialysis skids. Several membrane stacks from commercial manufacturers are available. Includes control systems.
1		APV-Gaulin Model 90-30CD	Cell-disruption homogenizer
1		Dedert	Thin film evaporator, pilot scale
6	1.5 cu. ft.	MBI	Absorption/ion exchange columns. glass
1	15,000 psi	APV/Gaulin 30CD	Homogenizer
Polymer Processing:
1	40L	Pfaudler	Glass-lined reactor
1	3x10" Role Vertical Stack	Killion	Calendar
1	3/4" single screw 24L/D	Killion	Extruder
1	350 kN	Arburg	Injection molder
1	300 mm twin screw 10L/D	Baker-Perkins	Twin-screw extruder with pellitizer
1	1/3 HP (SA=150in2)	Masler	Drum dryer
1	1L	Parr	High pressure reactor
1	2L	Parr	High pressure reactor
! Available to MBI, through Michigan State University, are single- and twin-screw extruders, film casting equipment, reactive extrusion, and injection molding.
Waste Treatment:
1	40 ft	Envirex	Fluidized-bed bioreactor
1	12 ft	Envirex	Fluidized-bed bioreactor
2	6 cu. ft.	MBI	Composter
22	2-100L	MBI	Fluidized- and fixed-bed bioreactors, glass & PVC
Other Equipment and Capabilities:
3	55 gal	MBI	Tempered water baths (30-90°C)
1	24" dia.	Sweco	Continuous/batch screener
1		Fitzpatrick Co. JA-6	Homoloid mill
2	0-10 gpm	Tri-Clover	Lobe pumps
2	0-6 Lpm	Pulsafeeder 880	Diaphragm pump
1	0-10 gpm	Aro	Diaphragm pump
1	0-14 gpm	Warren-Rupp	Diaphragm pump
2	10 gpm	LaBour DZT-10	Centrifugal pumps
1	100 gal	International Tank	Wooden process tank w/ lid
1	50 gpm	Durco Type 2K2X1	Centrifugal pump
2	55-gal	Tranter	Drum heaters
1	8" dia.	C-E Tyler "Rotap"	Test sieves & shaker
1	1 cu. ft.	MBI	Batch steam exploder
1		MBI	Continuous-flow hydrolysis reactor, 25-300 C, 0-1200 psig, 1-60 sec. residence time
1		MBI	Automated ion exchange regeneration cart
3		Opto 22/ Paragon	Poratble systems for distributed control of process equipment
2	8" dia x 16" long	Hayward	Basket strainers
2	100 lb	OHaus	Bench scales
1	0-1 gpm	MicroMotion	Portable mass flow meter
1	1 sq ft	Buflovak	Rotary drum dryer
1	5000 lb	Fairbanks-Morse	Scale
1	1000 lb	Fairbanks-Morse	Portable drum scale
! A variety of polypropylene work tanks, with lids, having capacities from 7-250 gals. Agitators are available.
! A variety of magnetically-coupled centrifugal pumps with polypropylene housings, for flows between 0.1 and 50 gpm at 0-60 psig discharge head are available.
! A variety of peristaltic, diaphragm and piston metering pumps are available with flows of 0.1-600 L/hr at 0-200 psig discharge head.
Additional Equipment
2	25 cu. ft.	MBI	Absorption/ion exchange columns, PVC
1	3700L	MBI/Pfaudler	Fermentor

 

Itemized Budget:

MBI International

Itemized Budget for the Proposal Submitted to Illinois Corn Marketing Board

 

	Hours	Duration = 24 months
Salary and Wages  Principal Investigators  Ponnam Elankovan, Ph.D. Chemical Engineer  Mahendra Jain, Ph.D. Microbiologist Technical Personnel  Tonya Tiedje, Chemical Engineer  Deborah Burgdorf, Microbiologist  Leslie Neilson, Microbiologist Total Salaries and Wages    Parts and Raw Material  Analytical, cleaning and waste disposal  Rent for Pervaporation unit  Assistance for Pervaporation unit and  Consulting Fee  Travel Sub Total  Fee @6% Total    MBI Cost Sharing (50% of Principal Investigators Salary)  Fund from Illinois Corn Marketing Board	208 208   624 416 416 1872	133,874   12,825 2,000 10,000   4,000 2,000 164,699 9,882 174,581   25,581   149,000

 

References:

Blaschek, H. P. 1986. Genetic Manipulation of Clostridium acetobutylicum for Production of Butanol. Food Technology (October), 84-87.

Chemical Economic Handbook. 1990.

Chem Marketing Report. 1991.

Jones, D. T. and Woods, D. R. 1986. Microbiol. Rev., 50:484-524.

Ladisch, M. R. 1991. Fermentation-Derived Butanol and Scenarios for its Uses in Energy-Related Applications. Enzyme Microb. Technol., 13:280-283.

Linden, J. C., Moreira, A. R. and Lenz, T. Z. 1985. Acetone and Butanol. In: Comprehensive Biotechnology (H.W. Blanch, S. Drew, and D.I.C. Wang, vol. eds.), M. Moo-Young - ed.-in-chief, pp. 915-931, Pergamon Press, Oxford.

Lovitt, R. W., Kim, B. H., Shen, G.-J., and Zeikus, J. G. 1988. Solvent Production by Microorganisms. CRC Crit. Rev. Biotechnol., 7:107-186.

Maddox, I. S., Qureshi, N. and Roberts-Thomson, K. 1995. Production of Acetone-Butanol-Ethanol from Concentrated Substrates using Clostridium acetobutylicum in an Integrated Fermentation-Product Removal Process. Proc. Biochem., 30:209-215.

Marlatt, J. A. and Datta, R. 1986. Acetone-Butanol Fermentation Process Development and Economic Evaluation. Biotech. Progress., 2:23-28.

Qureshi, N. and Maddox, I. S. 1995. Continuous production of Acetone-Butanol-Ethanol using Immobilized Cells of Clostridium acetobutylicum and Integrated with Product Removal by Liquid-Liquid Extraction. J. Fermen. Bioeng., 2:185-189.

Roffler, S., Blanch, H. W. and Wilke, C. R. 1987. Extractive Fermentation of Acetone and Butanol: Process Design and Economic Evaluation. Biotechnol. Progress., 3:131-140.

Wood, D. R. 1995. The Genetic Engineering of Microbial Solvent Production. Trends in Biotechnol., 13:259-264.

Appendix A

Table of Contents

Page

Abstract: ii

Key Words: ii

Impact Statement: ii

Communications Statement: iv

Budget Summary: iv

Title: 1

Objectives: 1

Procedures: 2

Integration of fermentation and pervaporation process units 2

Project impact of commercialization of butanol 5

Justification: 5

Principal Investigators: 7

Patents and Publications: 7

Presentations: 9

Project Location: 10

Duration of Project: 10

Facilities and Equipment: 11

Itemized Budget: 19

References: 20

Appendix A