Source: UNIVERSITY OF ALABAMA IN HUNTSVILLE submitted to NRP
BIOLOGICAL CONVERSION OF BIODIESEL-DERIVED CRUDE GLYCEROL TO PRODUCE VALUE-ADDED INDUSTRIAL PRODUCTS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
Reporting Frequency
Annual
Accession No.
0210677
Grant No.
2007-35504-18253
Cumulative Award Amt.
(N/A)
Proposal No.
2007-02186
Multistate No.
(N/A)
Project Start Date
Sep 1, 2007
Project End Date
Nov 30, 2010
Grant Year
2007
Program Code
[71.2]- Biobased Products & Bioenergy Production Research
Recipient Organization
UNIVERSITY OF ALABAMA IN HUNTSVILLE
301 SPARKMAN DRIVE
HUNTSVILLE,AL 35899
Performing Department
(N/A)
Non Technical Summary
The rapidly expanding biodiesel market is dramatically altering the cost and availability of glycerol, positioning this compound to emerge as an important building block chemical. To further advance the production of biodiesel from renewable agricultural feedstocks, new and innovative processes for utilizing the glycerol co-product should be developed. The goal of this research is to evaluate the potential for using anaerobic fermentation to produce value-added products, including butanol, from crude glycerol generated during biodiesel production.
Animal Health Component
40%
Research Effort Categories
Basic
60%
Applied
40%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
51140102020100%
Knowledge Area
511 - New and Improved Non-Food Products and Processes;

Subject Of Investigation
4010 - Bacteria;

Field Of Science
2020 - Engineering;
Goals / Objectives
The goal of this research is to evaluate anaerobic fermentation for the production of bio-based value-added chemicals from crude glycerol generated during biodiesel production. Species of Clostridium utilize glycerol to produce a variety of compounds, including 1,3-propanediol, acetate, butyrate, lactate, butanol, ethanol, and acetone. This work will target butanol production, as it has applications as both a building block chemical and an alternative fuel. Research efforts will focus on identifying and quantifying the environmental conditions that elicit a shift from acid to solvent production to favor increased butanol production. Specific research objectives include: 1.) acclimating, enriching, and isolating a wild-type clostridial culture capable of utilizing high concentrations of crude glycerol; 2.) evaluating the effect of various nutrient and trace metal concentrations on product formation and developing a nutrient/trace metal formulation for improved butanol production; 3.) evaluating the effect of pH on product distribution and identifying the optimum pH for butanol production; and 4.) evaluating the effect of initial substrate concentration on production formation and determining the optimum concentration for butanol production.
Project Methods
Objective 1: Efforts will enrich and isolate a wild-type culture of clostridia capable of producing butanol from crude glycerol. Wild-type strains demonstrate improved glycerol utilization and tolerance to high substrate and product concentrations. Sludge samples will be obtained from the anaerobic portion of a wastewater treatment pond at the Alabama Biodiesel production facility and the anaerobic digester at a municipal wastewater treatment facility. Samples will be pasteurized to enrich for clostridial species and inactivate other competing microorganisms and used as inocula. Crude glycerol obtained from Alabama Biodiesel will be supplemented with a minimal nutrient solution. In successive batch experiments, the initial glycerol concentration will be increased from 5-50 g/L. Once the culture has acclimated, it will be recovered via centrifugation, resuspended in a small volume of liquid, and utilized in subsequent experiments to further enrich and isolate a butanol-producing culture. Objective 2: Iron limitation is known to inhibit butanol production. Experiments will evaluate the effect of iron by quantifying product formation over a range of non-limiting iron concentrations. Other metals such as nickel, molybdenum, and cobalt are important for cell growth and glycerol utilization but have not been evaluated with respect to product formation. In additional experiments, nutrient media will be supplemented with specific amounts of Co, Ni, and Mo. Product formation will be quantified, and the concentrations that favor butanol production will be utilized in subsequent experiments. Experiments will also evaluate the effect of providing excess macro-nutrients, specifically phosphorus and nitrogen. The optimum C:N ratio for glycerol utilization and butanol production will be determined. The effect of phosphorus will be evaluated by comparing results obtained under concentrations of excess phosphorus and phosphorus limitation. Objective 3: Species of clostridia are known to favor solvent production under acidic pH conditions. However, investigation on the effect of pH on pathway regulation during glycerol fermentation has so far been inconclusive. Experiments will evaluate the effect of pH on product formation by gradually acclimating the culture to acidic conditions . In successive experiments, the pH will be lowered from 7.5 to 4.5 in increments of 0.5, and product formation and distribution will be measured. The pH of the system will not be artificially contrilled, as pH decreases are known to trigger solvent formation during the anaerobic fermentation of glucose. Objective 4: Some species of clostridia can tolerate very high concentrations of pure and crude glycerol (>100 g/L). C. pasteurianum has exhibited similar tolerance to pure glycerol; however, utilization of crude glycerol as the sole substrate for butanol production has not been reported. Experiments will evaluate the ability of this culture to tolerate and utilize concentrations of crude glycerol up to 125 g/L. The optimum nutrient/trace metal composition and concentration and system pH determined during evaluation of Objectives 2-3 will be applied to these experiments.

Progress 09/01/07 to 11/30/10

Outputs
OUTPUTS: I was appointed as PI on this project on August 4, 2010, after PI Dr. Katherine Taconi had left the University of Alabama in Huntsville. The bulk of the research was conducted by Dr. Taconi and her results have been published, see below. Research results obtained between August 2010 and October 2010 were presented at: UAH Bioretreat, a symposium by the UAH Biological Sciences Department on October 1, 2010; Poster title: "The effect of Fatty Acids on the Metabolism of Biodiesel-derived Crude Glycerol on Clostridium pasteurianum ATCC 6013" and at the AIChE meeting that was held in Salt Lake City from November 8-12, 2010; Poster title: "The effect of Impurities in Biodiesel-derived Crude Glycerol on the Growth and Fermentation by Clostridium pasteurianum ATCC 6013". This award supported Ph.D. student Keerthi Venkataraman, Biotechnology Ph.D. program, and it supported in part undergraduate student Judy Boatman, Department of Chemical Engineering. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Audience interested in sustainable resources PROJECT MODIFICATIONS: PI Dr. Taconi left UAH, Dr. Scholz was responsible for finishing the project and mentoring the graduate student.

Impacts
My report will summarize the results achieved under my supervision. From August through November, 2010 we concentrated on the study of the impact of impurities on the butanol production by Clostridium pasteurianum. Crude glycerol contains a variety of impurities, mainly salts and residual fatty acids. We determined gravimetrically that the residual fatty acids content is approximately 10% by weight. We hypothesized that these residual fatty acids are the cause for the observed unsteady growth when the organism is grown on crude glycerol. Purifying crude glycerol by precipitating the fatty acids yielded a semi-crude glycerol on which the microbial growth rate and butanol production were identical to those of pure glycerol. The impact of other impurities (Methanol, KCl, K2SO4) was tested by adding these to pure glycerol and using the resulting mixtures in fermentation experiments. It was determined that none of these impurities impeded the growth rate or butanol production by this organism.

Publications

  • Taconi, K.A., Venkataraman, K.P., Johnson, D.T. Growth and Solvent Production by Clostridium pasteurianum ATCC 6013TM Utilizing Biodiesel-Derived Crude Glycerol as the Sole Carbon Source American Institute of Chemical Engineers, Environmental Progress and Sustainable Energy, 2009, 28 (1) 100-110.


Progress 09/01/08 to 08/31/09

Outputs
OUTPUTS: The PI continues to mentor one Ph.D. student who is completing the research as part of his dissertation requirements. This student has completed multiple experiments evaluating various parameters to optimize the conversion of biodiesel-derived crude glycerol into butanol. In addition, three undergraduate students have also been mentored on a special project to evaluate the liquid-liquid extraction of butanol from fermentation broth using biodiesel. One local community college student also completed an internship working on the project in the PI's lab. The Ph.D. student was awarded 1st place in a poster presentation competition at a regional biotechnology retreat. The PI has presented the research at one national conference and several invited conferences/seminars. The PI was also a featured speaker at the 2009 Genes to Products USDA CSREES annual project directors' meeting. PARTICIPANTS: Keerthi Venkataramanan, PhD student; Lalleh Adhami, Undergraduate student; Bennie Griggs, Undergraduate student; Patrick Himebrook, Undergraduate student; Patricia Curtis, Undergraduate Student TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
The PI's research group has demonstrated significant production of butanol by Clostridium pasteurianum using biodiesel-derived crude glycerol as the sole carbon source. In addition, butanol yields on crude glycerol are comparable to those obtained when purified glycerol is used, indicating that this organism has a good tolerance to the impurities found in crude glycerol. We believe we are the first research group to have demonstrated production of butanol using crude glycerol. We have also evaluated a number of parameters, including iron concentration, pH, and crude glycerol concentration, and are quantifying the effects of these parameters on product formation.

Publications

  • Taconi, K., Venkataramanan, K., and Johnson, D. "Growth and Solvent Production by Clostridium Pasteurianum ATCC 6013 Utilizing Biodiesel Derived Crude Glycerol as the Sole Carbon Source." Environmental Progress and Sustainable Energy. 2009. Vol. 28, No. 1, 100-110.
  • Adhami, L., Griggs, B., Himebrook, P., and Taconi, K. "Liquid-Liquid Extraction of Butanol from Dilute Aqueous Solutions using Soybean-Derived Biodiesel." Journal of the American Oil Chemists' Society. Published on-line August 7, 2009. DOI 10.1007/s11746-009-1447-7.


Progress 09/01/07 to 08/31/08

Outputs
OUTPUTS: Experiments evaluating the anaerobic fermentation of biodiesel-derived crude have been performed using both wild-type mixed cultures and pure cultures of Clostridium pasteurianum. Early results indicated that wilde-type cultures were able to produce 1,3-propanediol (PDO) and ethanol using crude glycerol; however, a disruption in graduate student personel resulted in the loss of these cultures. Efforts are now focusing on the utilization of pure cultures of C. pasteurianum for glycerol fermentation. Experiments have compared the growth of C. pasteurianum on purified glycerol and biodiesel-derived crude glycerol in batch culture at initial glycerol concentrations of 5, 10, and 25 g/L. Results indicated no significant differences in growth (as measured by optical density) between cultures grown on purified and crude glycerol. Our results have also produced a maximum butanol yield of 0.36 g/g glycerol, a maximum ethanol yield of 0.36 g/g glycerol, and a maximum PDO yield of 0.41 g/g glycerol. These yields are higher than those reported previously for the fermentation of purified glycerol by C. pasteurianum. The butanol yield is also higher than what is typically achieved by C. acetobutylicum or C. beijerinckii during glucose fermentation. Additionally, our results indicate that cultures can produce solvents, including butanol, using biodiesel-derived crude glycerol as the sole carbon source. This is believed to be the first demonstration of solvent production from crude glycerol. Although cultures can produce solvents from crude glycerol, the product yields have not been as high as those achieved using purified glycerol. Crude glycerol contains significant amounts of methanol and salts. In order to determine how methanol affects solvent production, future experiments will compare growth and solvent production using purified glycerol spiked with methanol. Experiments will continue to evaluate the effect of initial glycerol concentration on growth and solvent production, as experiments containing 50 g/L purified and crude glycerol are currently in progress. Efforts will also focus on determining the effect of pH and undissociated acid (specifically acetic and butyric) concentration on growth and solvent production. Finally, solvent producing cultures will be transferred to a semi-continuous fermentation system in an effort to maintain a stable, solvent-producing culture. PARTICIPANTS: Keerthi Venkataramanan - Ph.D. student, Biotechnology Science and Engineering Program, UA Huntsville Received an Alabama EPSCoR Graduate Research Fellowship to continue work on this project. Received 1st place in the Research Poster Competition at the 10th Annual Biotech Retreat, Huntsville, AL. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: The original research plan was to utilize mixed wild-type anaerobic cultures for glycerol fermentation. However, due to a problem with graduate student personnel, the wild-type cultures were lost. Subsequently, research efforts have focused on utilizing pure cultures of Clostridium pasteurianum for glycerol fermentation.

Impacts
In addition to the results described above, this research project is currently supporting one student, Keerthi Venkataramanan, who is pursuing a Ph.D. in Biotechnology Science and Engineering as well as one undergraduate research assistant. In previous semesters, one additional undergraduate research assistant was supported using grant funds. Ph.D. student Keerthi Venkataramanan as recently awarded an Alabama EPSCoR Graduate Research Fellowship from the Alabama EPSCoR office. This is a competitive fellowship program that provides a 1-year stipend to recipients. Ph.D. student Keerthi Venkataramanan also received a 1st place award in the research poster competition at the 10th Annual Bioretreat, hosted by the Partnership for Biotechnology Research, UA Huntsville, and the Hudson-Alpha Institute. Conference Presentations Taconi, Katherine; Venkataramanan, Keerthi. Production of Butanol from Biodiesel Derived Crude Glycerol Using Pure Cultures of C. Pasteurianum (ATCC 6013). American Institute of Chemical Engineers Annual National Meeting, Philadelphia, PA (November 2008). Taconi, Katherine; Johnson, Duane T. Fermentation of Biodiesel-Derived Crude Glycerol to Produce Value-Added Chemicals. American Institute of Chemical Engineers Annual National Meeting, Salt Lake City, UT (November 2007). Poster Presentations Venkataramanan, Keerthi; Taconi, Katherine. The Anaerobic Fermentation of Crude Glycerol from Biodiesel Plant into Butanol using Clostridium Pasteurianum ATCC 6013. Partnership for Biotechnology Research, UA Huntsville, Hudson-Alpha Institute 10th Annual Bioretreat. Huntsville, Al (September 2008). Taconi, Katherine; Venkataramanan, Keerthi. Production of Butanol from Biodiesel-Derived Crude Glycerol using Pure Cultures of C. Pasteurianum (ATCC 6013). 2008 Annual Alabama EPSCoR Conference. Montgomery, AL (July 2008). Taconi, Katherine; Venkataramanan, Keerthi. Fermentation of Biodiesel-Derived Crude Glycerol to Produce Value-Added Chemicals. Best of UAH Research Expo. Huntsville, AL (April 2008).

Publications

  • Taconi, K.A., Venkataramanan, K. P., and Johnson, D.T. "Growth and Solvent Production by Clostridium Pasteurianum ATCC 6013 Utilizing Biodiesel Derived Crude Glycerol as the Sole Carbon Source." Manuscript Submitted to Environmental Progress, September 2008.