br Acknowledgements We thank Joseph Granger and Bryan
Acknowledgements We thank Joseph Granger and Bryan Neumann for careful reading of this manuscript. This work is supported by NIH grants AG37609 and HL 60306 to TYC and CCYC.
Introduction Eleven million tonnes of waste are produced yearly by the European pulp and paper industry (Monte et al., 2009). Approximately 70% of these originate from manufacturing tissue paper from recovered fibre, leading to the generation of considerable amounts of deinking sludge (150 kg dry solids/t paper manufactured), which must be properly managed to avoid negative effects on the environment (Deviatkin et al., 2016). Deinking sludge (DIS) exists as a mixture of short cellulosic fibers and inorganic fillers, such as calcium carbonate and china clay, and residual chemicals dissolved in water (Likon and Saarela, 2012). DIS originating from printed recycle mills is high in ash content compared to sludge originating from corrugated recycle mills and virgin pulp mills (Boshoff et al., 2016). Traditional methods of DIS management include landfilling, landspreading, composting, incineration and pyrolysis, utilisation as construction material and landfill capping material (Likon and Trebše, 2012). However, due to high moisture content some of these recovery methods, such as incineration and pyrolysis, are expensive for large amounts of sludge while the environmental impact of others is questionable due to the possibility of hazardous substances leaking into the environment. As a result, numerous possibilities for biological valorisation of paper sludge waste, including its fermentation and trans-isomer digestion are currently being explored (Gottumukkala et al., 2016). These aim for efficient microbial transformation of cellulose waste into bioethanol (Boshoff et al., 2016), biomethane (Mohan et al., 2016), biohydrogen or other value added chemicals (Liguori and Faraco, 2016). The main advantage of exploiting paper sludges as sources of cellulose-derived energy and chemicals in comparison to other lignocellulose substrates is their amenability, which is associated with an extensive pulping process that removes the majority of the lignin and exposes cellulose fibers to enzymes. This results in substantial cost savings on energy for substrate pretreatment in comparison to other lignocellulose fuel production technologies (Gottumukkala et al., 2016). Studies on direct production of bioethanol from paper sludge have shown that tissue printed recycle sludge (a type of DIS) resulted in significantly lower ethanol yields when compared to corrugated recycle and virgin pulp mill sludges (Williams, 2017), so other possible ways for valorisation of this type of substrate need to be explored. One of the major limiting factors for bioconversion of cellulosic waste such as paper sludge to valuable products is the cost of the enzymes, as they are commercially produced using high cost feedstocks. Reducing the enzyme dosage per gram cellulose/feedstock has been a major research area for the past few years (Robus et al., 2016). Efficient transformation of cellulosic feedstock to value added products requires a mix of synergistically acting enzymes (CAZYmes) that are able to work at low dosages. White-rot fungi (WRF) are known to produce significant amounts of powerful extracellular oxidative and hydrolytic enzymes that degrade lignin and cellulose biopolymers (Manavalan et al., 2015). Major functional groups of glycoside hydrolases (cellulases and hemicellulases) produced by WRF involve endoglucanases (EG; EC 184.108.40.206), cellobiohydrolases (EC 220.127.116.11, EC 18.104.22.168), beta-glucosidases (22.214.171.124), endoxylanases (EX; EC 126.96.36.199), beta-xylosidases (EC 188.8.131.52) and alpha-glucuronidases (E.C. 184.108.40.206). On the other hand, lignin degradation enzyme systems are based on oxidative enzymes, such as lignin peroxidases (EC 220.127.116.11), manganese peroxidases (EC 18.104.22.168), versatile peroxidases (EC22.214.171.124), and laccases (EC 126.96.36.199) (Manavalan et al., 2015). Recently, oxidative enzymes, namely lytic polysaccharide monooxygenases, have also been shown to play an important role in the degradation of cellulose (Garajova et al., 2016). The composition of the enzyme mixtures produced on different substrates (ratios between different types of enzymes) reflects substrate composition (Elisashvili et al., 2008).