1. 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

2. 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

3. 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 )

4. 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?

6. 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

7. 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 ) .
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

8. 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

9. 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

10. 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

11. 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

12. 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

13. 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 )
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

14. 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

15. 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

16. 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

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

18. 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

19. 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

20. 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.