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

1
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.
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.
Figure 1. A Flow diagram showing the basic layout
of the project orange colouring indicates
current work, blue colouring indicates future
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
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.
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.

• References
• Centre for Lignocellulose Structure and Formation
  http//www.lignocellulose.org/CLSFsummary.pdf
• Wisconsin Biorefining Development Initiative
  http//www.biorefine.org/proc/fermlig.pdf
• Figure 2. http//pubs.acs.org/cen/_img/86/i49/8649
  cov_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