Final Report: Microbial Solution: Application of Microorganisms for Biofuel Production and CO2 Mitigation

EPA Grant Number: SU833915
Title: Microbial Solution: Application of Microorganisms for Biofuel Production and CO2 Mitigation
Investigators: Markov, Sergei A. , Schiller, Joseph R.
Institution: Austin Peay State University
EPA Project Officer: Page, Angela
Phase: I
Project Period: August 15, 2008 through August 14, 2009
Project Amount: $10,000
RFA: P3 Awards: A National Student Design Competition for Sustainability Focusing on People, Prosperity and the Planet (2008) RFA Text |  Recipients Lists
Research Category: Pollution Prevention/Sustainable Development , P3 Challenge Area - Air Quality , P3 Awards , Sustainable and Healthy Communities


These students contributed to this project: Dawn Danielson, Luke Holliday, Nimit Patel, Ryan Willingham, Tracy Bisaquera (Department of Biology), Matthew Murphy (Department of Engineering Technology), and Barbara Waldron (Department of Chemistry), Austin Peay State University

The purpose of this project is to convert solar energy and waste CO2 (carbon dioxide that is released in power plants by burning fossil fuels) into an array of biofuels by sequential use of microorganisms in bioreactors.

An interdisciplinary student team first used microalgae in a photobioreactor to produce oil using CO2 and light as an energy source. Produced oil was converted into biodiesel by transesterification. Next, glycerol (also known as glycerin), a by-product of biodiesel production, was used as a substrate for making H2 (biohydrogen) and ethanol by immobilized bacteria in a bioreactor. Finally, students tested one of produced biofuels: biohydrogen in model fuel cell. While this proof of concept studies used purchased CO2, an abundant supply of waste CO2 from local power plants is available for commercial scale up.

Summary/Accomplishments (Outputs/Outcomes):

A 100 L photobioreactor for biodiesel generation from microalga Chlorella vulgaris was constructed from two parallel clear PVC 10 feet tubes (6’ diameter) with a small slope (10%). The gas mixture (5% CO2 and air) flowed up the top of the PVC tubes from the bottom as large gas bubbles. Photobioreactor was run in a batch mode for two weeks at room temperature. Continuous light was provided by cool white fluorescent lamps (136 - 186 μmol • m-2 • s-1 on the surface of the culture). Algal biomass was harvested at OD665 ~ 0.35 corresponding to a chlorophyll concentration of ~ 3 μg per ml, and concentrated by sedimentation with subsequent drying under 70◦C. Algal oil was extracted with hexane. The amount of oily material was expressed as a percentage of algal dry weight. Oil content in the algal cells in the bioreactor was found to be 10%. Algal oil was converted into biodiesel by transesterification with glycerol as a by-product. Next, glycerol was used as a substrate for making H2 and ethanol by bacterium Enterobacter aerogenes in batch culture (test tubes), and in hollow-fiber bioreactor. Highest H2 and ethanol production rates were observed under 2% glycerol, volume per volume (v/v), on a synthetic medium in test tubes. The yield of H2 from glycerol (0.9 mol/mol) was relatively high in batch culture (in test tubes). A lab-scale hollow-fiber bioreactor for conversion of glycerol into H2 and ethanol was constructed. The bioreactor was designed in a way that the glycerol diluted in growth medium was pumped from the outside of the fibers into the lumen (inside). Bacterial cells were readily adsorbed to the outer surface of the hollow fibers, and the cells consumed glycerol. Used growth medium was returned to the bioreactor medium reservoir, to create a close system in which it was possible to measure glycerol uptake and ethanol production by bacterial cells. The bioreactor was run under 30º C. Glycerol uptake efficiency by bacteria in the bioreactor was found to be 90% for 7 days. The assessment of H2 and ethanol-producing activity by bacteria in the hollow-fiber bioreactor in the presence of 2% glycerol (v/v) was made. Hydrogen production in the hollow-fiber bioreactor by E. aerogenes from glycerol was observed for 3 days at a maximum rate of 30 mL per hour (mL · h-1). Hydrogen from the hollow-fiber bioreactor was directly injected into a small fuel cell, and shown to be capable of generating enough electricity to power a small motor.


Our P3 Phase I project was successful in meeting its proposed purpose and objectives. We have demonstrated the possibility to produce biodiesel from microalgae in sustainable way with simultaneous consumption of CO2 and utilization of a by-product of biodiesel conversion - glycerol into H2 and ethanol. Most importantly, it was possible to produce biodiesel in a simple, inexpensive (under $300) photobioreactor, which was run on the light energy. We exceeded our expectations by obtaining higher H2 production rates from glycerol, compared to H2 rates from other microorganisms reported in published papers. That was possible by increasing mass transfer of glycerol into bacterial cells using hundreds of tiny hollow fibers in the bioreactor. The large surface-to-volume ratio of the hollow fibers allows to grow cells in high densities in bioreactors and to promote mass transport of glycerol to cells. Glycerol uptake efficiency was higher in our bioreactor (90%) compared to the batch culture experiment (60%). Our bioprocess for conversion of glycerol into H2 is ready for practical application. We were able to inject H2 from the hollow-fiber bioreactor directly into a small fuel cell, and generated enough electricity to power a small fan.
Proposed Phase II Objectives and Strategies:
For Phase II project we will concentrate on our bioreactor for H2 production from glycerol by bacteria. Hydrogen production by bacteria from glycerol will be optimized, and the hollow fiber bioreactor will be upgraded to a pilot-scale bioreactor. Our industrial partner will be the Applied Membrane Technology, Inc.
The purpose of this project is to optimize bacterial H2 production from glycerol wastes obtained after biodiesel manufacturing, and to scale up H2 production bioreactor to make it suitable for industrial uses.
An interdisciplinary student team will first optimize bacterial H2 production from glycerol wastes obtained after biodiesel manufacturing from algal oil and restaurant greases from a local cafeteria. Then, students will design and construct a pilot scale hollow-fiber bioreactor for continuous glycerol fermentation into H2 by bacteria. The assessment of H2 - producing activity by bacteria in this hollow-fiber bioreactor in the presence of glycerol wastes will be made. Finally, H2 will be compressed and filled onboard of the hydrogen-powered Toyota car. This will be done with the help of our partner from the Middle Tennessee State University (MTSU). In addition, the feasibility of producing other fuels (such as ethanol and butanol) and chemicals by bacteria from glycerol will be accessed.
Specific objectives of this project are: (1) to study bacterial fermentation of glycerol-containing wastes from biodiesel manufacturing processes into H2; (2) to scale up the hollow-fiber bioreactor for H2 production by bacteria; (3) to investigate the feasibility of production of other fuels (such as ethanol and butanol) and chemicals by bacteria from glycerol.

Journal Articles on this Report : 1 Displayed | Download in RIS Format

Other project views: All 4 publications 1 publications in selected types All 1 journal articles
Type Citation Project Document Sources
Journal Article Markov SA. Potential of using microalgae for biofuel production and CO2 removal from atmosphere. International Scientific Journal of Alternative Energy and Ecology 2009;2:83-91.
abstract available  
SU833915 (Final)
  • Abstract: ISJAEE
  • Supplemental Keywords:

    Alternative energy source, renewable energy, solar energy, renewable fuel, biodiesel, biohydrogen, bioethanol, waste to energy, RFA, Scientific Discipline, Air, Sustainable Industry/Business, POLLUTION PREVENTION, Sustainable Environment, Energy, Environmental Chemistry, climate change, Air Pollution Effects, Technology for Sustainable Environment, Atmosphere, environmental monitoring, sustainable development, environmental sustainability, alternative materials, biomass, alternative fuel, biodiesel fuel, energy efficiency, energy technology, carbon credits, alternative energy source

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