Paper mill sludge as a good substrate for enzyme production by (Pleurotus ostreatus) Dr. Mija Sežun 22nd Edition of International Conference on BIOTECHNOLOGY, Amsterdam 16 & 17 April, 2018

PULP AND PAPER INDUSTRY The pulp and paper industry plays an important role in the country’s economic growth a large contributor to environmental pollution large consumptions of energy and chemicals consumer standards are high, and manufacturing is competitive

Biotechnology for Pulp and Paper Industry the potential to increase the quality environmentally friendly processes reduce manufacturing costs create novel high-value products (Anon 2004, 2005 ; Mansfi eld and Esteghlalian 2003 ; Ojanpera 2004 ; Viikari et al. 2006, 2009 )

The use of enzymes in the process of BIOTECHNOLOGY effective in very small amounts – a few enzyme molecules will catalyze thousands of reactions per second unchanged and are not consumed in the reaction reduce the activation energy of a reaction increase the speed of reaction very specific to a reaction specific pH and temperature range that they are active in WHY TO USE ENZYMES?

Pulp and Paper Industry - DEINKING PROCESS A significant difficulty in dealing with secondary fiber is the removal of contaminants, particularly ink The difficulty of ink removal depends primarily on the ink type, printing process, and fiber type. large quantities of chemicals high wastewater treatment costs environmental regulations substantial amounts of solid and liquid waste

Enzymes Used in Deinking Process Enzyme-assisted deinking represent a potential environmentally friendly alternative (Anon 2010 ; Bajpai 1999 ; Bajpai and Bajpai 1998 ; Bajpai 2006 ; Ma and Jian 2002 ; Mohammed 2010 ; Puneet et al. 2010 ) . Enzymes Used in Deinking Process Lipases and esterases degrade vegetable-oil-based inks Pectinases, hemicellulases, cellulases, and ligninolytic enzymes alter the fiber surface or bonds in the vicinity of the ink particles release ink for removal by washing or flotation

Enzyme production by oyster mushroom (Pleurotus ostreatus) on pulp and paper sludge Paper-mill sludge is the most abundant by-product in the pulp and paper industry its disposal is a major solid-waste problem for the industry global production of paper-mill sludge will rise over the next 50 years by between 48% and 86% from current levels disposal of paper-mill sludge by landfill has become less possible in recent years

Fungi - oyster mushroom (Pleurotus ostreatus) can colonize and degrade a large variety of lignocellulose materials are easy to cultivate have great economical, ecological and medicinal value highly productive growth on cheap and abundant substrates ubsequent production of hydrolytic and oxidative enzyme degrade a wide variety of organic compounds structurally similar to lignin and other xenobiotic compounds

The aim of our study was: produce an appropriate mixture of the hydrolytic and oxidative enzymes by fungus Pleurotus ostreatus (oyster mushroom) through its cultivation on paper-mill sludge substrate selection and determination of optimal incubation time selection and determination of pH of the extraction buffer

Material and Methods Substrates, inoculum and experimental set-up The paper-mill DS (deinking) and PS (chemical and mechanical) were obtained from the Slovenian paper mill The PLAB P. ostreatus strain was obtained from the fungal culture collection of MycoMedica d.o.o. Substrate characterization dry matter total organic carbon (TOC) heavy metal contents : Cu, Zn, Ni, Hg, Cr, Pb cellulose, hemicellulose and lignin

Fungi cultivation P. ostreatus was grown on solid medium DS and PS, 200 g the moisture content was corrected from 50% to 65%. sterilization of the substrate(autoclave) and inoculated by P. ostreatus incubation at 23 °C for 61 days

Enzyme extraction enzymes were extracted by adding 2 mL of the appropriate buffer solution per 1 g fermented substrate, for 2 h at 4°C, while gentle shaking the effect of the pH of the extraction buffer on the enzyme activities was tested using nine different buffers with increasing pHs (pH 4.0-8.0) following the extractions, the samples were centrifuged at 13,200× g for 10 min at 4°C. the supernatants obtained were analyzed for the enzyme activities and for the total extracellular protein

Enzyme activities in P. ostreatus extracts enzyme extracts were measured using a spectrophotometric method all of the chemicals used for the enzyme analyses were from Sigma-Aldrich (USA) Cellulase and xylanase assays Lipase assay Peroxidase assay

Results and discussion Substrate characteristics The TOC contents for the DS and PS were 21.0% and 28.5%, respectively. Sludge substrate Heavy metal concentration (mg/kg dry matter)   Cu Zn Ni Cd Hg Cr Pb Deinking 165 58 2 0.15 <0.01 5 21 Primary 104 55 6 0.20 9 Sludge substrate Relative concentration (%)   Cellulose Lignin Hemicellulose Deinking 34 6 4 Primary 33 8

cellulase specific activities was higher for DS compared to PS Fungi cultivation cellulase specific activities was higher for DS compared to PS cellulase specific activity was highest after 53 and 61 days of incubation

xylanase specific activities was higher for DS compared to PS xylanase specific activity was highest at days 34 and 61 of incubation

the lipase specific activities decreased with incubation time for PS this trend was not seen for DS, where the lipase specific activities they were not stable

peroxidase specific activities were higher for PS compared to DS and were highest at acidic pH, peroxidase specific activities was highest at days 34 and 61 of incubation

Conclusions DS and PS, represent good substrates for growth of the edible oyster mushroom (P. ostreatus). DS and PS are suitable as supporting materials for deinking enzyme production by P. ostreatus. DS and PS substrates make this process economically interesting follows the guidelines for a recycling economy and the strategy behind a zero-waste society.