A “Spike” in Biofuel Production: Mining the Porcupine Microbiome to Engineer a Softwood Feedstock Platform

Dwindling fuel resources and rising environmental concerns have catalyzed the development of biofuel production in microorganisms. In Nova Scotia, softwood waste from the lumber industry is an untapped source for low-cost biofuel feedstock; however, this waste cannot be utilized by traditional biofuel processes due to toxic compounds such as turpentines and unavailable carbon compounds such as cellulose. The porcupine microbiome provides a unique solution as it is capable of digesting bark and toxic products. Working with Schubenacadie Wildlife Park, we aim to not only identify cellulose and/or turpentine-degrading bacteria in the porcupine microbiome, but to also characterize microbial communities found within the Park’s mammal population. To achieve these goals, we are using fecal samples to construct a DNA library of the porcupine and to analyze each mammal’s microbial rRNA. Future experiments include introducing identified cellulose and/or turpentine-degrading pathways into E. coli to produce an economically viable and sustainable biofuel-generating organism.

Shubenacadie Wildlife Park:

The Shubenacadie Wildlife Park is home to 29 mammals and 35 birds on a 40 hectare piece of land. These animals were rescued as injured or sick from the wild, and were rehabilitated. Some animals will be unable to return to the wild and live out the rest of their lives at the park. These animals live in enclosures that simulate environments that are comfortable to them. For example, the rescued otters have a large pool which they can swim in. More about the wildlife park can be found here.

The wildlife park has been an invaluable resource. The animals are fed a supplemented diet, which consists mainly of natural diets with added food like fruits/veggies for the herbivores and protein supplements for the carnivores. Because they eat a supplemented diet, and diet does effect microbiome content, there is potential error associated with the use of the park. However, we considered this error to be minimized as the mammals are eating many of the same things they would in nature. The park was also the safest way for us to obtain fecal samples, so associated error was acknowledged as part of this process.

DNA Extraction

We obtained fecal samples from 21 mammals at the Shubenacadie Wildlife Park. These fecal samples contain a millions of each animal’s gut microbes, which make up the microbiome’s of their gut. The DNA extraction step allows us to identify these bacteria through the bacterial DNA found in each fecal sample. DNA extraction was done using MoBio’s PowerFECAL kit which uses both alkaline lysis and mechanical lysis. Alkaline lysis works through a detergent, which disrupts the membranes of the bacterial cells. Mechanical lysis works through small ceramic beads shaken in the tube at high speeds to mechanically break up bacterial membranes and release the bacterial cell contents. The broken up bacterial cells are run through a column, where DNA binds but all other cell material is left behind. The columns were washed with ethanol and eluted using a buffer provided by MoBio. We are left with clean, pure genomic DNA from all the cells in the fecal sample.

Polymerase Chain Reaction

In order to prepare our sample for illumina sequencing, we first had to create many copies of a specific region of the genomes found in the extracted DNA by polymerase chain reaction (PCR). The specific region we require for microbial identification is the 16S rRNA gene. This gene is used widely as a molecular phylogeny taxonomic identifier, giving us a good idea of what bacteria are present.

PCR works by using small oligonucleotides that are called primers, and a heat-stable DNA polymerase. These oligonucleotides are used to provide a free 5’-OH group, essentially priming the reaction, that the heat-stable polymerase uses to amplify DNA from a template strand. A machine called a thermocycler works to cycle the temperature of the reaction in order to make the reaction stop and start in a chain reaction. The cycle has three steps: denature, anneal and elongate. The denaturation step melts the double-stranded DAN molecule into single-stranded molecules. The annealing step allows the oligonucleotide primers to bind to the single-stranded molecules at their complementary sequence. The elongate step allows the heat-stable polymerase to extend the DNA sequence. After many cycles, we have exponential amplification of the segment of DNA that is flanked by the oligonucleotide primers. For illumina sequencing, the oligonucleotide primers also contain an adapter region that is added onto the amplified 16S rRNA gene DNA, allowing the DNA to be compatible with the illumina sequencer flowcell. The adapter addition is important because it allows us to be more specific in what is sequenced, as only DNA containing the adapter will be sequenced.

This will be a description of the second part of our project (isolation of cellulose and tree sap degrading bacteria from fecal samples)

This will be a description of the third part of out project (metagenomic library based on cells that degrade cellulose and sap)