Powering the Future: Biofuels

Activity: Cellulase enzymes Describe the breakdown of cellulose by cellulases and cellulose producing microbes Carry out quantitative assays of enzyme activity Assess the relative merits of immobilised cellulases and microbe produced cellulases

To help combat climate change the UK has a target to reduce carbon emissions by 80% by % of the UK renewable energy could come from biomass heat and electricity by To meet the European Renewable Energy Directive, the UK is aiming for 10% of transport energy to be from renewable sources by By 2020, 8% of our petrol and 5% of our diesel could come from crops grown in the UK. Facts and Figures

Bioenergy is the energy derived from harvesting biomass such as crops, trees or agricultural waste and using it to generate heat, electricity or transport fuels. © Rothamsted Research

Optimising the yield of fast growing energy crops that are not part of the food chain is one way scientists aim to make sustainable, green bioenergy replacements for fossil fuels a reality. © Rothamsted Research Ltd

Scanning Electron Microscope cross-section of straw: Non-edible waste from agriculture, such as straw, could be used in the future as a secure, green source of fuel without taking up land needed for growing food. Scientists will be looking at this as one possible way to provide sustainable, environmentally friendly bioenergy replacements for fossil fuels. © University of Cambridge

Improving barley straw for bioenergy production and transferring the new knowledge to other crops: Our challenge is to discover how the properties of lignin in barley straw can be changed, to make it easier to produce biofuel (or bioenergy) from this waste material without having any detrimental effects on the yield or quality of the crop.

Plant Cell Walls: Lignin is a strengthening and waterproofing material that encrusts the sugar based polymers in plant cell walls making them hard to access for biofuel production. © University of Dundee at SCRI

Plant Cell Walls: Woody plants, such as miscanthus and willow, convert much of the carbon that they capture into lignocellulosic polymers, which are not a readily fermentable form of carbohydrate. © University of Dundee at SCRI

Plant Cell Walls: Feedstocks rich in lignocellulose require treatment with acids, alkalis or steam explosion methods to hydrolyse hemicellulose and break down lignin, enabling access to the cellulose by enzymes. © University of Dundee at SCRI

The Gribble: This tiny seawater pest can destroy wooden boats and piers but remarkably the gut enzymes that allow it to eat wood are being harnessed by scientists to break down wood for conversion into green, sustainable bioenergy. © University of York

The Gribble: By examining genes that are expressed in the guts of gribble, researchers have demonstrated that its digestive system contains enzymes which could hold the key to converting wood and straw into liquid biofuels. © University of York

Bioenergy from sea pests: Gribbles are voracious consumers of lignocellulose and have all the enzymes needed for digestion of wood and straw. Scientists have already sequenced the genes that are expressed in the gribble gut and will study the enzymes and digestive process © University of York

Bioenergy from sea pests: Remarkably the little marine wood borer, or Gribble, that caused this damage could hold the secret to sustainable energy for us all. The gut enzymes that allow the bug to damage wooden sea structures such as piers will be harnessed by scientists to break down wood for sustainable bioenergy production © Richard Gribble University of York

Bioenergy from sea pests: The gribble digestive tract is dominated by enzymes that attack the polymers that make up wood. One of the most abundant enzymes is a cellulose degrading enzyme never before seen in animals. © University of York

Bioenergy from sea pests: Unlike termites and other wood-eating animals, gribble have no helpful microbes in their digestive system. This means that they must possess all of the enzymes needed to convert wood into sugars themselves. © G. Watson University of York

© Institute of Food Research Steam explosion unit: We need to optimise the release of sugars from agricultural and wood- industry wastes to produce a fermentable feedstock that microorganisms can use to produce fuels. Pretreatment of feedstocks with steam opens up the structures in plant cell walls to enable access by cellulase enzymes.

Food Waste: Scientists at the Institute of Food Research are investigating how our waste problem can be turned into an energy solution. © Institute of Food Research

Professor Katherine Smart BSBEC LACE Programme School of Biosciences University of Nottingham © University of Nottingham To harness the potential of lignocellulosic (plant cell wall) materials, we need to optimise the release of sugars from agricultural and wood-industry wastes to produce a fermentable feedstock that microorganisms can use to produce fuels. Developing robust microbial strains that can use these feedstocks will enable sustainable production of bioethanol.

Scanning Electron Microscope image of Yeast: Researchers are developing novel yeast strains and fermentation processes that optimise bioethanol production. © National Collection of Yeast Cultures

Researchers are using synthetic biology approaches to generate bacterial strains that can convert lignocellulose to fermentable sugars efficiently to maximise butanol productivity. © University of Nottingham

Method: Gel diffusion 1.Prepare an agar gel containing 1.7% agar and 0.5% CMC (carboxymethylcellulose). Pour this gel into petri dishes and allow it to set. 2.Prepare the fruit extracts by mashing a variety of ripe fruits in a mortar and pestle. 3.If testing enzymes or microbes using paper discs, prepare the paper discs by cutting them out of filter paper using a narrow cork borer or using a hole punch. Sterilise the discs by autoclaving wrapped in foil. 4.If testing enzymes or microbes immobilised in sodium alginate, make up the alginate the night before to allow it to fully dissolve. 5.After the agar gel has set, use a narrow cork borer to punch small cylinders in the gel. Then, using a mounted needle, remove each of these cylinders to create a series of similar sized wells in the agar. Four or more wells can be put in a single dish, provided they are spaced apart. 6.Place similar volumes of extracts of fruits in each of the wells. In one well, place some distilled water, as a control. 7.Mix the enzyme or microbes with the alginate shortly before adding it to the wells in the agar using a syringe. 8.Incubate the dishes for at least 24 hours at 30 °C. 9.After the incubation period is finished, use tap water to rinse out the contents of the wells, and then flood the dishes with Congo red solution for 15 minutes. Then rinse the dishes with 1 M sodium chloride solution for at least minutes.

Method: Viscosity reduction 1.Make up a 2% (w/v) wallpaper paste solution, sufficient to provide 25 ml for each sample to be tested. 2.Place 25 ml of the paste in a boiling tube and add 2 to 5 ml of fruit extract. Mix thoroughly. 3.Then pour the mixture into the barrel of a syringe, held in a retort stand, pointing downwards into a small beaker. Note the time taken for all the mixture to drain through the syringe nozzle into the beaker. 4.Incubate the fruit or enzyme-wallpaper paste mixture at different temperatures, such as in a water bath at 30°C, allow to return to room temperature and repeat the investigation, checking the change in viscosity. The more active the enzyme the greater the reduction in viscosity, and so the shorter the drainage times.

Activity: Cellulase enzymes Describe the breakdown of cellulose by cellulases and cellulose producing microbes Carry out quantitative assays of enzyme activity Assess the relative merits of immobilised cellulases and microbe produced cellulases

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