Over the past decade, the research on biofuel production has dramatically increased due to the increases in oil prices and carbon dioxide levels[1]. Second generation biofuels have utilised biomass in the production of bioethanol. However, conversion of biomass to fermentable sugars faces many challenges due to the complexity of lignocellulose as a substrate.  Filamentous fungi are exploited commercially for their saprophytic activity and their ability to release cell wall degrading enzymes and have also been used in bioethanol production. Enzymes, such as cellulases from Trichoderma reesei, are part of the breakdown of biomass however, very little is still known about the physiological response of fungi to lignocellulose.

Aspergillus niger (CBS 513.88), a black-spored filamentous fungus generally involved in starch degradation, has been shown to respond to lignocellulosic substrate, wheat straw[2]. To further understand its response to the model lignocellulose substrate wheat straw, A.niger was placed under two different time growth conditions. Once A.niger spores had formed a mycelial mass, they were transferred to two separate liquid mediums, one containing 1% wheat straw and the other without.

Under these conditions, A.niger is be starved causing it to produce cell-wall degrading enzymes to break down the straw.Reverse transcription polymerase chain reaction (RT-PCR) was undertaken to analyse the transcription response to starvation. It was found that under starving conditions, A.

niger would produce a CBM containing enzymes, cbhA, cbhB, eglA and glaA at various time points to break down the straw to produce glucose. In the presence of a carbon source, cbhA and eglA are induced at 9 hours by XInR and cbhB at 6 hours.Without a carbon source, it was shown that cbhB and eglA are under partial control of XInR and induction of these genes is through starvation. Finally, glaA is independent of XInR control but is induced through the presence of starch in wheat straw.

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 Abbreviations: CAZy, Carbohydrate Active Enzyme; GH, Glycoside Hydrolase; CBM, Carbohydrate Binding Module; yefC, An07g02650; eglA, An01g11670; glaA, An03g06550. Key words: RNA-sequencing, Lignocellulose, Aspergillus niger, Starvation Response, Carbohydrate-Active-EnzymesSecond Generation BiofuelsCurrently, there are two main classes of biofuels, first generation biofuels and advanced (or second generation) biofuels. First generation biofuels has three commercial main types which include biodiesel, ethanol and biogas made from sugar, starch and vegetable oil. Advanced biofuels, in contrast, are a carbon-based fuel produced from biomass rather than food crops and has no impact on CO2 concentrations[3]. Despite second generation biofuels production being more sustainable than its prior generation, it is still not as cost effective[1] .

Biomass is organic (food biomass) and plant (non-food biomass) waste. Food biomass is made of mainly vegetable oil, starch cereal and sugars; the latter two can be made into biofuel or bioethanol through fermentation. On the other hand, production of fuel from non-food biomass is more complicated as it consists of lignocellulosic materials, non-edible oils and forest residue which are used to produce bio-oil, biofuel and biodiesel [3].

 Plant biomass is a one of the most abundant, unused biological resources and is composed of lignocellulose[4]. As a substrate, lignocellulose is a more complex substrate when compared to the simple substrates found in food crops however as a source of fuel, it is considered to be to one of the most efficient as the entire substrate is transformed into energy[5].In addition, plant biomass can be used in many ways for energy, such as simply burning biomass to produce heat and electricity [3] and the plant cell walls have an array of polysaccharides which can be broken down into simple sugars. Plant cell walls are composed of three large polysaccharides; pectin, cellulose and hemi-cellulose. These can be hydrolysed into glucose (and other sugars) and are converted into ethanol during biofuel production [6]. An example of a plant biomass is Triticum aestivum (wheat). Wheat is a traditional food crop with an abundance of sugars in both the stem and starch grains which can be used a resource for biofuels and wheat straw is a co-product of cereal grain production[2, 3].

The combination of these two results in a large feed stock for biofuel production.Finally, there are two main ways to produce liquid fuels from biomass; thermochemical processing or biochemical processing. Thermochemical processing converts all components of the biomass into its products through thermal decay and chemical reformation. The biomass is heated at a high temperature with varying levels of oxygen to give different products. In contrast, biochemical processing converts only the polysaccharide components of the biomass into sugars[4].

Bioethanol ProductionOne of the key alternatives to fossil fuels is bioethanol. Bioethanol is considered one of the cleanest liquid fuels and is made from ethanol fermentation which involves biomass, microorganisms and technology[7]. There are five main stages in bioethanol production: pre-treatment, enzyme breakdown, fermentation, distillations and recovery. Furthermore, fermentation of lignocellulosic materials produces bioethanol as a substitute for gasoline and gasification produces biodiesel; both bioethanol and biodiesel are carbon-based.Producing bioethanol from lignocellulosic biomass The production of bioethanol first requires the plant biomass to be converted into sugar, using enzymes, which are then converted into alcohols. A good example of this would be yeast-based fermentation where sugar and starch crops are fermented into ethanol.

One of the key stages in this type of fermentation is the enzymatic hydrolysis of lignocellulosic biomass due to its effect on the yield (and cost) of alcohol. Additionally, most of the commercially used enzymes for the conversion of lignocellulose to fermentable sugars are cellulases (CBH1 and CBH2) from Trichoderma reesei however; this fungus is limited in its hemicellulase content[8].Saccharification of lignocelluloses Saccharification is the hydrolysis of soluble polysaccharides into monosaccharides. However, lignocellulose is composed of lignin, cellulose and hemicellulose and only cellulose is soluble. For the saccharification of lignocellulose, lignin must be removed due to its insolubility, to allow cellulases to work[9] or any other kind of pre-treatment to remove the lignin to allow hydrolases to work[10]. In industry, the primary source cellulases come from Trichoderma reesei and A.niger is being used a model organism for hemicellulose breakdown[11].Aspergillus niger In industry, fungi have been exploited for their ability to secrete a vast number of proteins.

Filamentous fungi live a saprophytic lifestyle therefore they must secret of hydrolytic enzymes to break down polymeric organic material in order to survive[12]. The Aspergillus and Trichoderma species have commercially-used enzymes to breakdown polymers of the cell wall through extracellular digestion pectin, cellulose and hemicellulose xylan [13].A.niger is a filamentous, black-spored fungus which releases enzymes that are able to breakdown plant biomass. Conversely, in the fermentation industry, it is exploited for its production of enzymes and organic acid [14].

 For the breakdown of plant polysaccharides in Aspergilli, there are three main classes of enzymes; glycoside hydrolases (GH), carbohydrate esterases (CE) and polysaccharide lyases (PL). This classing of enzymes is from the Carbohydrate Active Enzyme database (CAZy – http://www.cazy.org) and subdivisions of these are based on the activity and enzyme sequence. GHs include endoglucanases, cellobiohydrolases and ?-Glucosidases and as its name suggest, hydrolyses glycosidic bonds. CEs are involved in catalysing deacylation of polysaccharides [15] and PLs use ?-elimination to cleave polysaccharides.