Periodic Reporting for period 2 - BIOrescue (Enhanced bioconversion of agricultural residues through cascading use) Reporting period: 2018-03-01 to 2019-08-31 Summary of the context and overall objectives of the project Total fresh mushroom production in Europe in 2016 amounted to approximately 1.2 million tonnes worth approximately €2 billion. For every tonne of mushrooms harvested approximately three times the amount of spent mushroom substrate (SMS) is produced. This means a disposal of more than three million tonnes of SMS per year and a significant economic and logistical problem for mushroom farms.On the other hand, current concerns over climate change and depletion of non-renewable resources are increasing drastically and have led to search for more sustainable products than fossil fuel-based ones which are known to disrupt the natural systems. BIOrescue aims to develop an innovative and integrated process concept based on the cascading use of biomass that will be applied to a conventional mushroom production farm, located in Tyholland (County Monaghan, Ireland), to turn it into an efficient, sustainable and innovative biorefinery. The main objectives are:1.Develop innovative efficient biorefinery technologies 2. Produce novel bio-based products from underutilised feedstocks3. Demonstrate an innovative biorefinery concept co-located on a mushroom farm and based on the cascading use of spent mushroom substrate supplemented by wheat straw (and other seasonal underutilised lignocellulosic feedstocks). Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far The main results achieved so far with the work performed from the beginning of the project (September 2016) to the end of the period covered by the report (August 2019) are:• A wide variety of underutilised lignocellulosic feedstocks from different European regions have been analysed to select the best combinations with mushroom compost. Based on these results, an optimal feedstock mixture of SMS compost, wheat, oat and barley straw has been proposed for a mushroom-farm-based biorefinery located in the Western region of Europe.• A rapid novel methodology based on Near Infrared (NIR) Spectroscopy that could be applied for online real-time characterisation of biomass feedstocks have been developed. • Extraction processes on mushroom compost have been carried out and shown the release of a low amount of soluble compounds. Moreover, enzymatic activities such as cellulase and xylanase have been measured in the extracted fraction although low values have been identified. On the other hand, the nitrogen levels in the liquid extract were also measured and turned to be quite low for its application as a fertiliser.• An optimisation of thermochemical pretreatment has been carried out on mushroom compost alone and in combination with wheat straw and other underutilised feedstocks (oat and barley straw) using different catalysts and process conditions with the aim of making the feedstocks more accessible to further enzymatic treatment. Cellulose hydrolysis yields above 96% have been achieved when using the optimised conditions. • The best enzyme cocktails for the hydrolysis of the carbohydrates available in combinations of mushroom compost and wheat straw have been selected. Results from the enzyme cocktail screening experiments were then used to assess techno-economically most suitable enzyme cocktail candidates for further pilot-scale hydrolysis assays. Moreover, an optimization of the enzymatic hydrolysis conditions has also been carried out. Furthermore, a library of 30,000 evolved cellulase mutants was created and screened for activity. Among these, 13 mutants showed 2-fold and 4-fold higher glucose yields than the wild type enzyme in the conversion of spent mushroom substrate-wheat staw (40:60). These enzymes were selected for their future use in an enzymatic cocktail in order to improve the bioconversion process and reducing its cost. In addition, a lytic polysaccharide monooxygenase (LPMO) was cloned and recombinantly expressed in Escherichia coli for future mutant library creation and screening• The liquid fraction obtained after the thermochemical pretreatment has been used as feedstock to produce two different types of biodegradable, enzyme-responsive lignin nanocarriers by miniemulsion polymerization for drug delivery. The nanocapsules were loaded with hydrophilic or hydrophobic drugs and the drug release was tested by enzymatic degradation. Moreover, the biopesticide produced during the project has been also encapsulated and the release rate was also tested.• A new strategy for solubilising the lignin from the pristine SMS and also from the recalcitrant solid obtained after enzymatic hydrolysis through an organosolv pretreatment has been implemented during the course of the project and included in the flow diagram of the biorefinery. Successful process for dissolving lignin contained in either pristine SMS (from 28% up to 38%) or recalcitrant (residue left after enzymatic hydrolysis) (from 30% up to 40%)• Validation of biopesticides production at pilot scale from BIOrescue sugars has been carried out. The biopesticides produced have shown high efficiency against two Lepidoptera species (Spodoptera littoralis, Spodoptera exigua.• Production of laccase using BIOrescue sugars to be used in lignin valorization tests. Lab-scale enzyme impact trials have been carried out using organosolv lignin from compost• Technological, technoeconomic and environmental assessments of two biorefinery configurations (i) nanocarriers production from SMS and ii) nanocarriers, enzymes and biopesticides production from SMS and wheat straw) have been developed and the final results have been used for an integrated sustainability assessment. • Finally, in terms of the exploitation of the results, a finalexploitation plan has been issued during the period, which includes the characterisation of the key results, risk analysis and overall intellectual property rights (IPR) strategy. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) BIOrescue project has developed and demonstrated a new innovative biorefinery concept that will avoid biomass disposal and allow for the production of some biodegradable bio-based products and bioactive compounds that will help to replace the existing ones based on fossil resources. This project will have direct impact for Mushroom farms, one of the major impacts of this project for mushroom farmers is reducing at least the €30,000,000/year cost for SMS disposal. Glucose and lignin obtained from the SMS treatment, can in turn be processed into higher value products such as biopesticides and nanocarriers which are proposed in the BIOrescue process. The potential yield of biopesticides per tonne of glucose is between 5-10% with a value of 4,500 and 9,000 ton and the potential yield of nanocarriers per tonne of lignin and xylose is between 70-100% with a value of 207,000 and 297,000 tons. A second major impact for the mushroom industry as a result of the BIOrescue project would be filling the technology gaps that will enable the co-location of biorefineries on commercial mushroom farms in order to be able to utilize SMS rather than disposing of it.