Research Areas

Trace metal biogeochemistry in surface seawater

The phytoplankton that grow in surface seawater form the basis of the marine food chain and are responsible for production of a large fraction of Earth’s atmospheric oxygen.  Therefore it is important to understand how individuals within the algal community and the community as a whole acquire the basic nutrients needed to survive and multiply.   My research group is particularly interested in micronutrients –  those elements that are present in low nanomolar concentrations in seawater but are essential cofactors in enzymes that form vital biochemical pathways.  Not only are trace metal concentrations very low in seawater but many metals are also complexed (chemically chelated or bound) by organic ligands.  This organic complexation is believed to control the geochemistry and biological availability of these trace metals.  In my group we are interested in determining whether algal cells are altering the chemistry of these metals with ligands (either purposefully or inadvertently) as a result of the cells’ management strategies for dealing with metals.  For example, we discovered in a recent study (Walsh and Ahner, 2014, Limnology and Oceanography), that the abundant coccolithophore, Emiliania huxleyi, appears to take up and exude Cu rapidly as part of its homeostatic Cu management strategy; we also know that this organism makes novel dipeptides that appear to be specific for Cu binding (Dupont et al. 2004; Walsh and Ahner 2013).  Interesting questions remain:  if this Cu is released from the cells as a ligand complex, how long does it stay in that form? Is it bioavailable to other algae?  Copper is a particularly fascinating trace metal because of its ability to rapidly cycle between two redox states—each with specific chemical affinities that influence biological uptake.

 

 

Figure 1. Mechanisms for copper reduction and uptake into an algal cell: (A) reduction of Cu(II) by cell surface reductases (Jones, Palenik et al. 1987); (B) photochemically-mediated production of Cu(I) (Moffett and Zika 1983); (C) thiol-facilitated reduction,(Walsh et al., in prep). (1) Low affinity uptake of the Cu2+ ion. (2) High affinity uptake of the Cu+ ion. (3) Cys-mediated uptake of Cu(I) (Walsh et al. in prep).

 

Algal biofuel systems: tools for improved productivity and co-product generation

Collaborator: Prof. Ruth Richardson, Civil & Environmental Engineering

As the world faces increasing crude oil prices and global warming, sustainable and carbon-neutral sources of energy and raw materials are critical for the global economy and our future environment.  Biomass-derived fuels –  biofuels –  are seen as a substantial portion of a sustainable energy portfolio.  Aquatic microalgae offer several unique features that make them an attractive prospect relative to land plants for biomass production.  They can be grown rapidly in reactors or ponds on marginal land.  A variety of species produce up to 77% of their total weight as energy-rich oils or carbohydrates.  Algae can be grown with less water than agricultural crops, and many species of algae have evolved to thrive in brackish or salty water.  Estimates of biodiesel production by algae on a land area basis typically exceed that of traditional crops by orders of magnitude.  Despite this potential, no large-scale facilities for commercial algal biofuel production currently exist.  Many private companies are currently attempting to commercialize algae production but appear to be facing significant challenges with respect to scale-up and profitability.

Optimization of algae biomass production in ponds or reactors requires diagnostic tools to interrogate various aspect of algae physiology; these tools range from rapid detection of intracellular lipid content of individual cells to RNA-based detection of specific biochemical stress responses.  In one project we are developing a new set of assays that could be used to improve production systems.  We are also working on the production of new transgenic strains of algae that will simultaneously produce high value proteins.  These functional proteins would provide added benefits to the use of algae protein in feed markets.  For example, we are currently expressing the enzyme phytase in algae to improve phosphorus digestion in animal diets.

Figure 2.  Lipid filled Chlorella sp. C596 cells.  Lipid droplet (stained with Nile Red) nearly fills the cell.  The chloroplast (white crescent) is pushed to the perimeter of the cell.  Mansfeldt et al. in prep

 

Optimizing the production of high value enzymes in transformed plant chloroplasts  

Collaborator: Prof. Maureen Hanson, Molecular Biology & Genetics

Accumulation of foreign protein in plant tissues is most often and most successfully achieved by inserting foreign genes in the chloroplast genome.  The comparative advantages of chloroplast transformation versus nuclear insertions include:  high chloroplast genome copy number, the ability to target insertion of genes to specific genome locations, and minimal gene silencing.  One of the disadvantages of chloroplast transformation for some protein products is that there is no post-translational modification in the chloroplast.  When using tobacco as a model plant species, foreign protein levels can accumulate as high as 70% total soluble protein (TSP), a level which compromises plant health (Oey et al. 2009), but there are many examples of plants accumulating 10-20% foreign protein without any negative change to phenotype (Gray et al. 2009; Gray et al. 2011; Maliga 2004).

We have shown in our work that careful selection of regulatory elements for use in the expression cassette can have a large influence on protein accumulation.  Some of these effects are well understood but others are still unexplained.  For example, we don’t fully understand why adding a short fusion to the 5’end of a protein can influence protein accumulation by orders of magnitude (See Gray et al. publications).  We are also interested in the effect of variable environmental conditions on the accumulation of protein, as these are easily manipulated in production facilities.  In our work we seek to understand basic biochemical processes as well as to find additional gene regulatory sequences that might help promote the use of transgenic plants in various industrial or pharmaceutical applications.  We have shown the potential for plant-based cellulase and beta-glucosidase production for use in the cellulosic ethanol industry.

 

Figure 3. Transgenic and wildtype plants growing with variable nitrogen concentrations. Richter et al. in prep.