Investigation of Thermobifida fusca as a Novel Biofuel Agent
Joe Alvin
Dr. Stephen Fong
Dr. Ana Estevez
Bioinformatics and Bioengineering Summer Institute
Virginia Commonwealth University
St. Lawrence University
August 5th, 2008
I.
Introduction
The dependence on fossil fuels has brought first world and developing countries
to the cusp of energy economies. Cellulosic ethanol—one of many biofuels—is regarded
as a likely source for renewable energy in the future 1. Cellulose is the most abundant
carbohydrate polymer on the Earth and is found in all plant matter 2. It is normally
degraded by saprophytes via enzymes and then fermented into a number of by-products.
The second most abundant—chitin and chitosan—are found in terrestrial and marine
animal exoskeletons 3. These polymers constitute a vast pool of energy hitherto untapped
for lack of means or efficiency 3. Current common practice involves a treatment of the
polymer in acid and enzymes—breaking hydrolyzing the bonds—followed by
fermentation by yeast (ie. Saccharomyces cerevisiae) 1. In order to increase process
efficiency, one organism couples both enzymatic interactions and fermentation, such as
Thermobifida fusca.
Thermobifida fusca’s genome has been sequenced and the annotation provides a
bright outlook for T. fusca’s metabolism in the biofuel industry4. However, T. fusca’s
physiology and metabolism have not been studied in vivo in detail; many studies have
cloned cellulase genes into other organisms such as Escheria coli or Bacillus megaterium
5, 6
. Recently, the cellulases found in T. fusca have shown non-specific catabolism of
chitin/chitosan 3. Known for its highly active cellulases and potential to ferment digested
biomass into fuel, our laboratory is investigating T. fusca’s potential as a biofuel producer
7, 8
.
Metabolic engineering allows further enhancement through organisms designed
with increased enzyme activity and product yield. In the past, metabolic engineering has
been a time-consuming, hit-or-miss method of genetic modification. New methods
combining genome annotation and in silico metabolic flux analysis provide a working
model for the organism 9. In silico models enhance the process’ effectiveness by
eliminating experiments that are unfavorable to pursue in vitro 10, 11. Flux-balance
analysis (FBA) of an annotated genome involves a matrix of reactions, metabolites and
flux. The model is solved for a solution space of growth rate and the desired product
based on the model’s constraints (substrate, moles etc.). FBA of T. fusca will provide
ample information about the organism’s metabolic pathways and the applicability of
genomic engineering for biofuel production 12. In addition, the FBA will elucidate the
best genes and pathways for metabolic engineering.
II.
Progress and Future Work
a. Metabolic Flux-Balance Analysis of Thermobifida fusca
The annotated genome provided by the Integrated Microbial Genomes project
(IMG) database listed proteins connected with the Kyoto Encyclopedia of Genes and
Genomes. These proteins were cross-referenced for their individual reactions and
metabolites. All of these were compiled using MetModel software to generate a matrix
of reactions and metabolites to be solved with a biomass equation. The model currently
contains 320 unique metabolic, enzyme-catalyzed reactions that occur in T. fusca and
4,447 metabolites. This is an estimated 50% of the true reactions and metabolites within
the cellular model.
Future work will finalize the model: integrating fatty acid and lipid syntheses,
determining and adding the appropriate biomass reaction. This work will be continued
over the 08/09 academic year at St. Lawrence University with help from Dr. Seth
Roberts, Chris Gowen and Dr. Paul Brooks.
However, The working model will not be sufficient proof for the potential of
metabolic FBA in biofuel research. When a working model is built with the software,
single or double deletions of proteins will be made in silico and evaluated for efficiency.
The model will need to accurately predict the final phenotypes of individual pathway as
per the genomic modification. If the model fails to predict the phenotype, sequencing the
anomalous strain will help identify the shortcomings in the model or organic mutations
that prevented proper evolution. When several potential modifications to the metabolic
pathways have been identified, the knockout strains will be created. The model will then
be proven with experimental evidence based on identical deletions and resulting
phenotypes. This facet of my research is the most time-consuming, yet minimal in terms
of equipment and supplies, therefore, in this area that I will likely progress the most over
the 08/09 academic year.
b. Investigation of Chitin/Chitosan substrates
Deng Yu and I collaborated on research concerning the use of chitosan as a
fermentable carbon source for T. fusca. Pre-cultures containing 1% D-glucose and
essential nutrients were incubated for 36h. Two replicates of a 0.5% and 1% chitosan
where evaluated for their growth (determined by protein assays) and compared to those
on D-glucose and cellobiose media; both known to support normal growth of T. fusca.
The first experiment did not provide definitive results with error attributed to insufficient
culture density after pre-culture.
A second experiment—including identical replicates and an additional bioreactor
component—was designed and carried out. T. fusca’s growth rate and phylogenic
expression have been known to vary depending on the environmental conditions. The
results indicate a slow growth rate on chitosan as a carbon source.
Few resources are necessary to assay the substrates’ carbon source efficiencies
and thus minimal experimentation could be completed at St. Lawrence University.
Requirements are chitosan samples, proper incubation facilities, and total protein assay
kits.
c. Alternative Biofuel Production/Efficiency
A pathway to potentially generate additional biofuels (presently uncharacterized)
was uncovered, during the metabolism review for MFBA. This alternate product in
question was verified by HPLC and GC results. The samples were obtained from an
experiment that varied growth conditions to determine variety in growth rates and
metabolite secretion. The product has potential use in the biofuel industry and has not
been categorized in T. fusca and as such, a provisional patent is being written to secure he
intellectual property. Therefore, little information about this particular project can be
released.
Experiments were carried out—using the growth/product secretion rate data—in
an attempt to discover environmental parameters that generate the maximum amount of
product. Two parameter settings were chosen from previous experiments—yielding
maximum product secretion—and used in the bioreactor fermentation. The results
showed decreased yields with the combination of settings when compared to the maxima
of the independent settings.
Due to the lack of proper facilities at St. Lawrence University, most of the future
experimentation will be completed at Virginia Commonwealth University’s laboratories.
Limited experimentation may be possible depending on the environmental factors
(bioreactor vs. flask).
References
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