Genetic Engineering of Bacteria for an MFC

Marshall Porter - Biomolecular EngSai Edara - Biomolecular Eng

Aaron Maloney - Bioelectronics EngDavid Dillon - Biomolecular Eng

Christian Pettet - Biomolecular EngArjun Sandhu - Biomolecular Eng

Ansley Tanoto-MCD Bio/BioinformaticsAlex Ng- Biomolecular Eng

Addressing Energy DemandAccording to the 2013 International Energy Outlook, energy demands will increase 56% by 2040. This rapidly growing demand for energy has sparked a search for sustainable, renewable, and cheap energy sources. One intriguing new energy technology is the Microbial Fuel Cell (MFC), capable of turning wastewater treatment into an electricity generating process.

The bacteria Shewanella oneidensis

● Can live in both environments with or without oxygen

● Can reduce poisonous heavy metal

● Has “electrogenic” properties allowing it to generate electricity in a Microbial Fuel Cell (MFC).

http://www.newscientist.com/article/dn9526-bacteria-made-to-sprout-conducting-nanowires.html#.U9gp_LEzCM0

What is an MFC?● Microbes Break down

Carbohydrates● Transfer electrons to anode,

which then flow to the cathode● Protons pass through

permeable membrane● Protons and electrons react

with oxygen to make clean water

● Can be implemented into secondary treatment of waste-water to allow for power generation[5]

http://www.sciencebuddies.org/Files/3665/5/Energy_img033.jpg

Physical Design

● A lot of previous research has looked at structural design

● Two main points○ Large surface area on

electrode○ Close distance between

electrodes

http://2013.igem.org/Team:Bielefeld-Germany/Project/MFC

Our Project

● We believe the bacteria which drive the power generation of an MFC can be genetically engineered to increase power density

● Our goal is to design MFC with increased efficiency by○ Altering metabolism of our electrogenic bacteria○ Modifying growth pattern of biofilm formation

● Two pronged approach, each with potential to improve efficiency alone

Energy Balance and Coulombic Efficiency● The process of metabolism and electron transfer is

complex.● The cell itself uses up some of the energy in other

processes● One such process is metabolite generation, which

reduces coulombic efficiency.[1]● We plan to redirect metabolism toward a pathway

capable of harvesting the lost energy

● When Shewanella is grown without oxygen, it generates the metabolite acetate from acetyl-coa

Acetate generation

[3]

● “gate keeper” to the TCA cycle● Converts acetyl-CoA and

Oxaloacetate to Citrate● Diverts Acetyl-CoA from being

converted to Acetate (metabolite)

Citrate Synthase (GltA)

● Under anaerobic conditions Shewanella is capable of using the oxidative branch of TCA, which allows the bacteria to use the energy lost by metabolite generation

● Use of oxidative branch is reliant on Citrate Synthase activity

Oxidative branch

Oxidative branch of TCA

Citrate Synthase● Under the anaerobic conditions citrate synthase activity

reduced by over one half due to downregulation of the gltA gene coding for citrate synthase [3]

● In our project we will recover this activity using an expression plasmid

(gene deletion)[3]

● Magnitude of electron transfer reliant on surface area of the anode○ More surface area allows more bacteria

to transfer electrons○ Growth of bacteria in biofilm allows for a

dense community to grow in one area● Growth of Shewanella in anaerobic

conditions leads of down regulation of biofilm production, and biofilm density is lost

Biofilm

Biofilm● Biofilm formation in Shewanella is

controlled by the gene mxdA, which regulates levels of c-di-GMP

● Upon deletion of mxdA, biofilm biomass decreases (fig A, mxdA)

● Biomass also decreases when switching from oxic to anoxic growth (fig B, control) but is retained when a gene similar to mxdA is expressed (fig B, VCA0956)[7]

● We hope to express VCA0956 in Shewanella while it grows in the MFC anaerobically to increase biofilm density

A

B

[7]

Waste-water treatment ● Treatment of waste water can be divided into three main steps

1. Heavy and light materials are removed by separation in a holding tank2. Microorganisms are used to break down organic matter3. Water is disinfected to be reintroduced to environment

http://en.wikipedia.org/wiki/Sewage_treatment

Implementation ● A microbial fuel cell can be implemented into

secondary waste water treatment processes, and potentially in septic tanks as well

● There are other applications of electrogenic bacteria as well, including microbial electrolysis cells used to generate hydrogen fuel

Citations1. Korneel Rabaey, ed. Bioelectrochemical systems: from extracellular electron transfer to biotechnological

application. IWA publishing, 2010.2. Franks, Ashley E., and Kelly P. Nevin. "Microbial fuel cells, a current review." Energies 3.5 (2010): 899-919.3. Brutinel ED, Gralnick JA. Anomalies of the anaerobic tricarboxylic acid cycle in Shewanella oneidensis

revealed by Tn-seq. Mol Microbiol. 2012 Oct;86(2):273-83. doi: 10.1111/j.1365-2958.2012.08196.x. Epub 2012 Aug 27. PubMed PMID: 22925268.

4. Papagianni M. Recent advances in engineering the central carbon metabolism of industrially important bacteria. Microb Cell Fact. 2012 Apr 30;11:50. doi: 10.1186/1475-2859-11-50. Review. PubMed PMID: 22545791; PubMed Central PMCID: PMC3461431

5. Rabaey K, Verstraete W. Microbial fuel cells: novel biotechnology for energy generation. Trends Biotechnol. 2005 Jun;23(6):291-8. Review. PubMed PMID: 15922081.

6. Beliaev, Alex S., et al. "Gene and protein expression profiles of Shewanella oneidensis during anaerobic growth with different electron acceptors." Omics: a journal of integrative biology 6.1 (2002): 39-60.

7. Thormann, Kai M., et al. "Control of formation and cellular detachment from Shewanella oneidensis MR-1 biofilms by cyclic di-GMP." Journal of Bacteriology 188.7 (2006): 2681-2691.