DEVELOPING A NOVEL CYANOBACTERIUM TO CONVERT CO2 INTO ETHANOL

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: TERMINATED
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 0217243
• Project No.: SD00H295-08IHG
• Project Start Date: Oct 1, 2008
• Project End Date: Sep 30, 2010

Project Director
Gibbons, W. R.

Recipient Organization
SOUTH DAKOTA STATE UNIVERSITY
PO BOX 2275A
BROOKINGS,SD 57007

Performing Department
BIOLOGY & MICROBIOLOGY

Non Technical Summary
Fermentation produces two molecules of ethanol and 2 molecules of carbon dioxide per molecule of glucose, and less than a quarter of ethanol plants capture carbon dioxide, with the rest venting it to the atmosphere. This greenhouse gas contributes to global warming. Because this carbon dioxide is available in a pure and concentrated form, it represents an underutilized resource. Several groups are promoting use of algae to produce oil from carbon dioxide, followed transesterification of oil to biodiesel. Because the oil accumulates in intracellular vacuoles, the algae must be killed to extract the oil. This reduces oil yield and increases costs, since carbon must be used to create the cell mass in the first place. One advantage of producing ethanol from carbon dioxide is that ethanol would be excreted from the cell to the liquid medium. Therefore, cells could continuously produce ethanol from carbon dioxide, so long as ethanol is continuously removed from the medium. This would provide the maximum ethanol yield. If an ethanol producing photosynthetic microbe were available, ethanol production from carbon dioxide could readily be integrated into ethanol plants. In addition, ethanol facilities have ample quantities of low-grade heat to maintain the temperature of greenhouse space. Ethanol could be efficiently recovered from even dilute streams using pervaporation membranes developed by our collaborator (Separation Kinetics). Their systems have concentrated ethanol from 10 to 70 percent in a single pass, and should work equally well at even lower ethanol concentrations (personal communication, John Thomas, Separation Kinetics). Various bioreactor designs have been developed for photosynthetic microbes. Among the more novel is a vertical plastic film design created by Valcent Products. The Vertigro system consists of horizontal tubules connected at alternating ends, in the form of plastic bi-layer sheets. Culture medium flows by gravity, back and forth through the tubules, which are hung in greenhouses to eliminate the need for costly artificial illumination. The equipment is available to support conversion of carbon dioxide to ethanol using photosynthetic microbes. The limiting factor has been the lack of a microbe to efficiently convert carbon dioxide to ethanol using sunlight.

Animal Health Component

(N/A)

Research Effort Categories

Basic

60%

Applied

40%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
511	4010	1040	60%
511	4010	1100	40%

Knowledge Area
511 - New and Improved Non-Food Products and Processes;

Subject Of Investigation
4010 - Bacteria;

Field Of Science
1100 - Bacteriology; 1040 - Molecular biology;

Keywords

anabaena

cyanobacteria

metabolic engineering

photobioreactor

ethanol

Goals / Objectives
This project will develop sufficient preliminary data to demonstrate the feasibility of converting carbon dioxide into ethanol using cyanobacteria. Specific objectives include developing a series of ethanol producing cyanobacteria, genetic modification of strains to improve ethanol production, screening strains to assess ethanol productivity, yield, and tolerance, and conducting benchtop photobioreactor trials with an ethanol separation membrane. We will estimate preliminary energy and mass balances, and cost projections. We anticipate favorable results, considering that no additional corn would be required and the process could use low-grade heat from the corn ethanol process. The significant boost in ethanol production should easily outweigh the costs for greenhouse space and equipment.

Project Methods
To transfer ethanologenic genes into cyanobacteria we will use pdc-zm and adhB-zm coding regions, with an engineered sequence (ribosomal binding site) upstream of their initiation codons. This DNA fragment will be fused to Anabaena nitrate inducible promoter (nir) in shuttle vector pNir (plasmid that replicates in Anabaena bears kanamycin resistance gene, Kan). This construct will be introduced into Anabaena by conjugation, and transformants will be selected in a nitrate-minus medium Kan plate. Ethanol production will be tested by first growing the transformants in a nitrate-minus medium (AA/8) to certain cell density and followed an addition of NaNO3 (500 mg/l) to induce ethanol synthesis. This shuttle vector approach will allow us to quickly screen for the best ethanologenic genes. Once the ideal ethanologenic genes and the best cyanobacter are found we will integrate these genes into the chromosome. Since carbon flux may limit ethanol production, further genetic manipulation in these competitive pathways is necessary. To block the pyruvate entering TCA cycle, we will knock out alr4745 using a double crossover knockout approach. DNA fragment encoding E3 subunit of PDH (Alr4745) will be PCR amplified from Anabaena genomic DNA and cloned into pRL278, a plasmid designed for conjugative transfer into Anabaena, to inactivate alr4745 through double crossover approach. Accumulation of pyruvate will be monitored and higher ethanol production will be expected. To block synthesis of glycogen, we will knock out the gene all4645, the only gene encoding ADP-glucose pyrophosphorylase in Anabaena. To draw more 3-PGA flow from Calvin cycle to convert to pyruvate, we will need to over-express three enzymes (phosphoglycerate mutase, enolase and pyruvate kinase) in glycolytic reactions that catalyze the conversion of 3-PGA to pyruvate. Three robust genes, from Z. mobilis) and from yeast strain S. cerevisiae will be constructed as an artificial operon fused to a PsbA1 promoter (a strongly constitutive expression promoter in Anabaena) and then cloned into an integrative vector to insert it within the coding region of alr4745). A screening protocol will select strains exhibiting traits needed for commercial production. Growth and ethanol production characteristics will be evaluated using a Sixfors multi-fermentor system that will be diurnally illuminated at an optimized light intensity. A cyanobacterial growth medium will be used, while carbon dioxide will be sparged at an optimized rate to provide the carbon source. Temperature will be maintained at 30 C, with agitation at 100 rpm. Strains exhibiting rapid growth and high ethanol yields and productivities will be subjected to secondary screening to individually examine process hardiness factors. The best strains will be cultured in an integrated photobioreactor membrane system to determine performance metrics under simulated industrial conditions.

Progress 10/01/08 to 09/30/10

Outputs
OUTPUTS: To improve ethanol production economics, we propose a dramatically different approach of converting the CO2 byproduct of ethanol production back into ethanol using a photosynthetic cyanobacterium. This project will develop sufficient preliminary data to demonstrate the feasibility of converting CO2 into ethanol using cyanobacteria. Specific objectives include: 1. Developing a series of ethanol producing cyanobacteria; 2. Genetic modification of strains to improve ethanol production; 3. Screening strains to assess ethanol productivity, yield, and tolerance. We have made substantial progress in objective 1 and 2. Objective 1:Both pdc and adhB genes from ethanologenic bacteriurm Z. mobilis have been transferred into Anabaena sp. 7120 by conjugation. Our first transgenic Anabaena has been capable of producing and excreting some ethanol into the medium using CO2 as the only carbon source. Objective 2: Reducing carbon flux to these competitive pathways, such as glycogen synthesis and pyruvate consumption, is necessary for boosting ethanol production. To do so we have been knocking out alr4745gene encoding a subunit of pyruvate dehydrogenase (PDH). Thus, more pyruvate will be redirected to PDC and ADH for ethanol production. Accumulation of pyruvate in this PDH-KO strain will be monitored by HPLC. Higher ethanol production will be expected in this strain. We are also working on blocking the synthesis of glycogen, the major storage carbohydrate in Anabaena. To block synthesis of glycogen, we are knocking out the gene all4645, the only one gene encoding ADP-glucose pyrophosphorylase in Anabaena genome, which controls the first step reaction of glycogen synthesis. Thus, more carbon flow in all4645 knockout (KO) strain can be redirected to ethanol production. Next, we will make a double knockout (alr4745 and all4645) in our ethanol-producing strain. The Co-PI Dr. Ruanbao Zhou mentored two undergraduate students Caryn Johansen, Jaimie Gibbons working on engineering cyanobacteria to produce ethanol using CO2 and sunlight. Dr. William Gibbons has been working on constructing the photobioreactor system to test the engineered cyanobacteria strains. PARTICIPANTS: Dr. Ruanbao mentored two undergraduate students Caryn Johansen, Jaimie Gibbons working on engineering cyanobacteria to produce ethanol using CO2 and sunlight. Dr. William Gibbons has been working on constructing the photobioreactor system to test the engineered cyanobacteria strains. TARGET AUDIENCES: Companies such as ICM, Inc, VeraSun Energy, Zymetis, Separation Kinetics, and KL Process Design are developing biomass to ethanol processes. These companies form the base of our private sector partnerships and will provide the most direct route to commercialization. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Outcomes: We have succeeded in creating an ethanol producing Anabaena strain. Our first transgenic Anabaena has been capable of producing and excreting some ethanol into the medium using CO2 as the only carbon source, but the yield was not high, only 0.1% ethanol was detected in the medium (the non-transgenic Anabaena did not produce any ethanol). Two undergraduate students Caryn Johansen, Jaimie Gibbons received extensive hands-on training on molecular biology and metabolic engneering from this project. Caryn Johansen et al presented a poster titled "Biosolar conversion of CO2 to ethanol by engineered cyanobacteria" and won the best poster award from North Central Center of Sun Grant. Impacts: A 100 million gallon per year corn ethanol plant releases over 45 tons/hr of CO2 and 686 million BTU/hr of heat. This huge resource is currently ignored. Our ethanol-producing cyanobacteria which we develop will reconvert these resources into ethanol in photobioreactors located in greenhouses adjacent to biorefineries, thus improving their economics, energy balance, and carbon balance. ICM, Inc has expressed interest in incorporating this technology in their ethanol process design. These benefits should increase investment in biofuel production, create additional jobs, and improve biofuel lifecycle issues.

Publications

• No publications reported this period

Progress 01/01/09 to 12/31/09

Outputs
OUTPUTS: To improve ethanol production economics, we propose a dramatically different approach of converting the CO2 byproduct of ethanol production into ethanol using a photosynthetic cyanobacterium, in a combined photobioreactor and membrane separation system. This project will develop sufficient preliminary data to demonstrate the feasibility of converting CO2 into ethanol using cyanobacteria. Specific objectives include: 1. Developing a series of ethanol producing cyanobacteria 2. Genetic modification of strains to improve ethanol production 3. Screening strains to assess ethanol productivity, yield, and tolerance 4. Conducting benchtop photobioreactor trials with an ethanol separation membrane In year 1, we have made substantial progress in objective 1 and 2. Objective 1: We have created an ethanol producing Anabaena strain. Both pdc and adhB genes from ethanologenic bacteriurm Z. mobilis were fused to Anabaena nitrate inducible promoter (Pnir) and have been transferred into Anabaena sp. 7120 by conjugation. These transgenic Anabaena clones are growing in a nitrate-minus (AA/8 medium) Kan plate. Their ethanol production was tested by addition of NaNO3 (500 mg/l) to medium for induction of ethanol synthesis. Ethanol production was measured by HPLC. Our first transgenic Anabaena has been capable of producing and excreting some ethanol into the medium using CO2 as a carbon source, but the yield was not high, only 0.1% ethanol was detected in the medium (the non-transgenic Anabaena did not produce any ethanol). Since Pnir promoter is well-conserved in the other three cyanobacterial strains listed above, so similarly, this ethanologenic gene construct will also be transferred into the other three cyanobacterial strains to screen for the best ethanol producing cyanobacter. Objective 2: Genetic modification of strains to improve ethanol production Competition among different pathways for the newly-fixed carbon flux, including glycogen synthesis and pyruvate consumption, may limit carbon flux to ethanol production. Therefore, reducing carbon flux to these competitive pathways is necessary for boosting ethanol production. To do so we have knocked out alr4745gene encoding a subunit of pyruvate dehydrogenase (PDH). Thus, more pyruvate will be redirected to PDC and ADH for ethanol production. As expected, this alr4745 knockout (KO) strain grows fairly normal. Accumulation of pyruvate in this PDH-KO strain will be monitored by mass spectrometry. Higher ethanol production will be expected in this strain. We have blocked the synthesis of glycogen, the major storage carbohydrate in Anabaena. To block synthesis of glycogen, we have knocked out the gene all4645, the only one gene encoding ADP-glucose pyrophosphorylase in Anabaena genome, which controls the first step reaction of glycogen synthesis. Thus, more carbon flow in all4645 knockout (KO) strain may be redirected to ethanol production. Next, we will make a double knockout (alr4745 and all4645) in our ethanol-producing strain. PARTICIPANTS: Dr. Ruanbao Zhou from Department of Bio-Microbiology is working on engineering cyanobacteria to produce ethanol using CO2 and sunlight. Dr. William Gibbons will construct and operate the photobioreactor system to test the engineered cyanobacteria strains. TARGET AUDIENCES: Companies such as ICM, Inc, VeraSun Energy, Zymetis, Separation Kinetics, and KL Process Design are developing biomass to ethanol processes. These companies form the base of our private sector partnerships and will provide the most direct route to commercialization. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
A 100 million gallon per year corn ethanol plant releases over 23 tons/hr of CO2 and 350 million BTU/hr of heat. The ethanol-producing cyanobacteria which we develop will reconvert these resources into ethanol in photobioreactors located in greenhouses adjacent to biorefineries, thus improving their economics, energy balance, and carbon balance. ICM, Inc has expressed interest in incorporating this technology in their ethanol process design. These benefits should increase investment in biofuel production, create additional jobs, and improve biofuel lifecycle issues.

Publications

• No publications reported this period