Production of lipase and recovery of by - products in solid state fermentation by Aspergillus niger DSMZ 2466 using palm kernel cake as substrate

• Main Author: Nair , Anusha
• Format: Thesis
• Language: English
• Published: 2014
• Online Access: http://psasir.upm.edu.my/id/eprint/52513/1/FBSB%202014%2030RR.pdf; http://psasir.upm.edu.my/id/eprint/52513/

Bacteria, yeast, and fungi are known potential producers of extra cellular lipases. Fungal species are preferably cultivated in solid-state fermentation (SSF), while bacteria and yeast are usually cultivated in submerged fermentation (SLF). Lipase production in SLF systems has been well established. The use of SSF as a robust platform for upstream and downstream processing has yet to be reported. Thus, this research study focuses on the fungal growth morphology, fermentation and downstream processing of lipase as well as the analysis of other valuable products from the SSF system using palm kernel cake (PKC) as substrate. Lipase production by several fungal strains (Trichoderma viride SDTC EDF 002, Aspergillus niger SDTC SRW-4, Aspergillus terreus DSMZ 5770 and Aspergillus niger DSMZ 2466) using SSF and SLF systems in 500 mL flask were investigated. Lipase production in SSF was substantially higher compared to SLF except for Trichoderma viride SDTC EDF 002. The highest lipase activities and total protein content were obtained in SSF with A. niger SDTC SRW-4 (44.43 U g-1; 176.36 mg g-1) and A. niger DSMZ 2466 (42.05 U g-1; 176.28 mg g-1). The highest lipase activity (15.34 U mL-1) in SLF was also obtained with A. niger DSMZ 2466. Relationship studies showed positive correlation between spore diameter (quadratic, R2; 0.999) and branch diameter (linear, R2; 0.971) with lipase activity for both of these A. niger strains. Further enhancement of lipase production (305.09 U g-1; 7.3-fold) in SSF from one the best performing fungal strain, A. niger DSMZ 2466 was achieved by supplementing PKC with glucose (0.1%, w/w), NH4NO3 (0.05%, w/w) and palm oil (2%, v/w). Improvisation on extraction method showed that cellulase treatment and grinding of the solid fermented material as additional steps was able to increase enzyme recovery by 193% and 185%, respectively. The crude extracellular lipases extracted from SSF was purified using 10 kDa MWCO PLC membrane, QFF anion exchanger and gel filtration (Sephacryl S-200 HR) with a yield of 22% and purification fold of 44. Lipase from A. niger DSMZ 2466 has a molecular weight of 45.4 kDa, highly active and stable under moderate alkaline conditions (pH 6.0 to 8.0) and at moderate temperatures (35°C to 45°C). Citric acid (5.020 g kg-1), malic acid (2.370 g kg-1) and oxalic acid (1.435 g kg-1) were present in the buffer extract of the fermented substrate. The total amino acid and reducing sugars in the solid fermented material after lipase extraction process showed increment of 2-fold and 3-fold, respectively. The substantial increment in all essential amino acids (EAAs) content in the fermented SSF substrate suggests its suitability to be used as animal feed ingredient. The proposed SSF platform for lipase production in this study; in which lowcost agricultural by-product is used as substrate with the ability to utilize the solid fermented substrate as animal feed ingredient, has potential for industrial application. This platform is able to reduce the lipase production cost and also eliminate environmental problems related to solid agriculture wastes disposal. The potential added value of the fermented substrate for application as animal feed is another advantage of the SSF system for enzyme production as compared to SLF.