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Project Overview

What would it take to...

Build a circuit using microbially catalyzed metal sulfide deposition?

Background: SRB, H₂S, and Copper Sulfides

The inspiration for this idea came from an ecological observation made of copper biomineralization by a species of Sulfate Reducing Bacteria. Sulfate Reducing Bacteria (SRB) comprise a family of chemolithotrophic bacteria that use sulfate as the terminal electron acceptor in anaerobic metabolism. As a result of sulfate reduction, these bacteria produce gaseous H₂S. Subsequently, microbially generated H₂S reduces copper in solution and precipitates soluble copper ions in the form of insoluble copper sulfide (CuS). It was discovered that by this method, some strains of SRB formed a CuS compound nearly identical to covellite - a natural superconductor (Weber, 2009). If this activity could be enhanced under spatial and temporal control, bacteria could be harnessed to deposit metal sulfide in specified geometries for manufacturing and engineering applications.

Building H₂S Production Activity into E. coli

Investigation of H₂S production in bacteria has been well documented in E. coli. Work by Dr. Jay Keasling at University of California, Berkeley, has shown that a gene encoding Thiosulfate Reductase from Salmonella enterica serovar Typhimurium has previously been expressed in E. coli to overproduce hydrogen sulfide from thiosulfate.

The gene phsABC functionally encodes Thiosulfate reductase. The gene itself contains an open reading frame for each of the three genes phsA, phsB, and phsC. The resulting complex of three transmembrane proteins constitutes Thiosulfate reductase and catalyzes the reduction of inorganic thiosulfate to hydrogen sulfide.

This activity can be monitored by growing bacteria on Triple Sugar Iron (TSI) Agar. Evolution of H₂S as a gas, results in the reduction of Iron Sulfate to an Iron Sulfide. Iron sulfide precipitates as a black solid and can be easily identified visually on the macroscopic scale. Bacteria that are actively producing H₂S will reduce the Iron in the medium, and turn the agar black.

Growing Bacteria In Copper Medium

Bacteria expressing Thiosulfate Reductase can then be grown in media containing both a source of sulfur and free copper in solution. Some strains of E. coli exhibit robust growth in copper containing media, despite toxicity of high levels of copper in solution. By measuring cell growth and viability at varying concentrations of copper, we can maximize the reaction of H₂S resulting from bacterial activity.

Correlating Bacterial Growth to Copper Deposition

In this experimental system, the formation of the copper sulfide should be directly related to the disappearance of Cu2+ with time as the insoluble metal sulfide formed is a complex of copper and sulfur. As H₂S production is turned on, there should be a subsequent decrease in soluble copper concentrations as copper is bound in the precipitated copper sulfide.

Localizing Copper Deposition

With bacteria able to affect CuS deposition, the next logical step is to control this activity spatially and temporally. Inspired by the work of Christine Jacobs-Wagner (Yale, MCDB), we built a mold for constructing agar channels in which to grow and constrain the active bacteria. A master mold was designed and constructed out of the polymer PDMS. This could then be used to iteratively create indented agar channels in our solid growth medium.

When bacteria are washed over these channels, they become trapped in the indentation. By imposing a physical restraint prior to the induction of metal deposition activity, it is possible to control where the metal sulfide is deposited. This is a necessary requirement in the construction of a circuit.

Modeling the Construction of Metal Sulfides

The metal sulfide can self-associate to form clusters of a mineral called covellite. This Copper Sulfide has a specific arrangements of unit cells. In an effort to model this, we used CAD to construct an idealized computer model for how this mineral deposition would effectively create a circuit.

This method can then be applied for the construction and manufacture of innumerable microscale structures, ranging from electrical components to microfluidic devices. Read on to find out more about our applications!