www.beaconwales.org
Bioprospecting for Microorganisms and Enzyme Activities with
Biorefining Potential
Laura Lyons, Iain Donnison, Justin Pachebat, Kerrie Farrar
Biorefining is the generation of heat and power, transport fuels, chemicals and composites from plant biomass in order to mitigate the use
of fossil fuels. This study focuses on the secondary processing aspect after material has been processed, which encompasses microbe
identification and enzyme activities. This project aims to discover novel microbes and enzymes with potential to contribute to large-scale
biorefining. The project structure can be seen in Figure 1.
Miscanthus is a woody, rhizomatous perennial tall grass, originating from Asia. This fast growing plant exhibits high water efficiency and the
ability to grow on low quality lands, qualities which make it an ideal crop for biofuels. However, converting lignocellulosic material into
ethanol and other products can only be achieved when plant cell wall polysaccharides have been hydrolysed in to fermentable sugar
monomers. Specific enzymatic activity is needed for this hydrolysis as the plant cell wall lignocellulose is complex as shown in Figure 2.
Miscanthus
chip
Culturing
gDNA from
soil/compost
16S PCR
Bacterial single colony
collection
Isolate morphology
Enzyme discovery
The project will focus on identifying microbes and
efficient enzymes capable of releasing sugars from
lignocellulosic biomass (Figure 2). To achieve this,
environments such as Miscanthus chip, were
screened for novel culturable bacteria with
lignocellulosic degradation properties. Bacteria are
identified via 16S rRNA sequencing and further
characterised by morphology using Gram staining
and microscopy. Isolates are then selected based on
their performance in functional assays (Figure 3) and
growth curves (Figure 4), and interesting proteins
expressed (Figure 5) and characterised.
Protein characterisation
gDNA
Genome
extraction
sequencing
Figure 2. Harvested biomass comprises plant cell
wall elements consisting
primarily lignin,
cellulose and hemicellulose. Lignin is a phenolic
Fosmid libraries
compound which binds the cellulose microfibrils
providing rigidity to the cell wall but also making
it recalcitrant to degradation. Some fungi and
Figure 1. A Flow diagram showing the basic
bacteria produce ligninases that can biodegrade layout of the project; orange colouring indicates
the
polymer,
thereby
releasing
the
current work, blue colouring indicates future
carbohydrates.1+2
work, black colouring shows past work.
Figure 4. Growth curve of three isolates in MRS Broth over 12 hrs
and after 25 hrs using OD600 used to choose a strain for protein
extraction
75 kDa
50 kDa
Figure 3. Examples of functional screening of isolated bacteria from Miscanthus chip on
three carbon source media: xylan, cellulose and starch
Figure 5. a-amylase Ni-NTA purification after expression in E. coli, followed
by zymography showing that the enzyme is active
Repeating the carbon utilisation assays shown in Figure 3 is required, at
different pHs, to ascertain which bacteria would be best for isolation and
narrow down the microbes to a select few for protein characterisation.
Identification of all isolates using 16S sequencing will lead to the formation
of a library of bacteria providing information on isolates.
Future work will include genomic characterisation of isolates
by nextera XT Illumina sequencing and bio-informatic mining of
sequences for hydrolytic enzymes, or functional screening
through fosmid expression libraries. Sequences of proteins can
be obtained and maybe genetically engineered into
fermentation microbes such as Lactobacillus plantarum.
References:
1. Centre for Lignocellulose Structure and Formation http://www.lignocellulose.org/CLSFsummary.pdf
2. Wisconsin Biorefining Development Initiative http://www.biorefine.org/proc/fermlig.pdf
3. Figure 2. http://pubs.acs.org/cen/_img/86/i49/8649cov_6.gif
Acknowledgments: Naomi Cope-Selby, Sarah Hawkins, David Bryant, Sharon Huws, Jessica Adams, Ana
Winters, Colin Jackson, Laurence Jones, Alun Hughes, Michael Squance, Mike Hale, Pete Malvern