Publication: Evaluation of spent coffee grounds as feedstock for enzyme production
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2022
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Biyoproseslerin en önemli maliyetini, kullanılan karbon kaynakları oluşturmaktadır. Bu tez kapsamında, operasyonel maliyeti düşürmek ve sürdürülebilir bir biyoproses geliştirmek amacı ile kahve telvesi atıklarının mikrobiyal lipaz enzimi üretiminde karbon kaynağı olarak kullanımı hedeflenmiştir. Bu kapsamda altında, kahve telvesi atığının toplandıktan sonra yaklaşık olarak %60’ının sudan oluştuğu, kurutulmuş 1 gram kahve telvesi atığından heksan:izopropanol (1:1) çözeltisi kullanılarak 0,2 mL kahve yağı; %3 H2SO4 (v/v) çözeltisi kullanılarak ise %39,11±1,8 (w/w) indirgen şeker ve % 9,95 ± 0,3 gallik asit eşdeğeri elde edildiği tespit edilmiştir. Kahve telvesi atığının içeriği belirlendikten sonra, daha önce lipaz üreticisi bir maya olarak bilinen C. diffluens D44 ile kahve telvesi atıklarından lipaz üretimi koşulları Design Expert programında Box-Behnken tasarımı kullanılarak optimize edilmiştir. Yapılan çalışmalar sonucunda, en yüksek lipaz enzim aktivitesi, 29,4 °C'de, pH’ı 8,3 olan ve %8,7 (w/v) kullanılmış kahve telvesi içeren bir ortam ile 14,6 U/mL olarak bulunmuştur. Üretilen D44 lipaz enziminin optimum sıcaklığı ve pH'ı ise sırasıyla 37 °C ve 8,0 olarak belirlendi. Buna ek olarak, üretim sonucunda elde edilen enzim içeren üst faz sıvısına GC-FID analizi yapıldı ve ortamdaki yağ asitlerinin bileşimi %31,05 linoleik asit, %28,67 palmitik asit, %20,44 heptadekanoik asit ve %19,84 oleik asit olarak belirlendi. Ayrıca, kahve telvesi atıklarının bakteriler tarafından kullanımını incelemek için İstanbul’un Kadıköy bölgesindeki toprak örneğinden B. subtilis BT2 izole edilerek, %1 (w/v) kahve telvesi içeren besi ortamından lipaz üretimi gerçekleştirildi. Yapılan üretim sonucunda, üretilen bakteriyel lipaz aktivitesi 40.426 U/mL olarak bulundu. Üretim ortamına %0,1 Tween80 (v/v) eklenmesi ile üretilen lipaz aktivitesi yaklaşık %10 arttı ve lipaz aktivitesi 44.711 U/mL olarak belirlendi. Üretilen bakteriyel lipaz enziminin optimum sıcaklığı ve pH'ı ise sırasıyla 37 °C ve 8,0 olarak belirlendi. Buna ek olarak, kahve telvesi atığından ekstrakte edilen kahve yağı ve şekeri kullanılarak, BT2 lipazının katalizi ile şeker-yağ asidi esteri üretimi yapılarak, üretilen şeker-yağ asidi esterleri FTIR analizi ile belirlendi. Son olarak, maya ve bakteri tarafından lipaz enzimi üretiminde kullanılan kahve telvesi atığının tekrar kullanılabilirliğini araştırmak için kullanılan kahve telvesi atığı örneklerinin SEM görüntüsü çekildi ve yapısal değişimleri incelendi.
The most significant cost of bioprocesses is the carbon sources used. In this thesis, it was aimed to use SCG as a carbon source in the production of microbial lipase enzyme in order to reduce operational costs and develop a sustainable bioprocess. Under this scope, it has been determined that approximately 60% of the SCG consists of water after collection. In addition, 0.2 mL of coffee oil was extracted from 1 gram of dried SCG by using hexane: isopropanol (1:1) solution and 39.11 ± 1.8% (w/w) reducing sugar and 9.95% ± 0.3 GAE were obtained by using 3% H2SO4 (v/v) solution. After the content of the SCG was determined, the conditions for lipase production from SCG by C. diffluens D44, previously known as a lipase producing yeast, were optimized in the Design Expert program using the Box-Behnken design. As a result of the studies, the highest lipase enzyme activity was found to be 14.6 U/mL at 29.4 °C with a pH of 8.3 and a medium containing 8.7% (w/v) SCG. The optimum temperature and pH of the produced D44 lipase enzyme from SCG were determined as 37 °C and 8.0, respectively. Besides, GC-FID analysis was performed on the enzyme-containing upper phase liquid obtained as a result of production. The composition of fatty acids in the medium was determined as 31.05% linoleic acid, 28.67% palmitic acid, 20.44% heptadecanoic acid and 19.84% oleic acid. Furthermore, B. subtilis BT2 was isolated from the soil sample in Kadıköy, Istanbul to evaluate the use of SCG by bacteria and lipase was produced from a nutrient medium containing 1% (w/v) SCG. As a result of the production, the bacterial lipase activity produced was found to be 40.426 U/mL. Adding 0.1% Tween80 (v/v) to the production medium increased the bacterial lipase activity produced from SCG by approximately 10%, resulting in 44,711 U/mL. The optimum temperature and pH of the produced bacterial lipase enzyme were determined as 37 °C and 8.0, respectively. In addition, using coffee oil and sugar extracted from SCG, sugar-fatty acid ester production by BT2 lipase catalysis was determined by FTIR analysis. Finally, SEM images of the SCG samples used to investigate the reusability of SCG used in lipase enzyme production by yeast and bacteria were taken and their structural changes were examined.
The most significant cost of bioprocesses is the carbon sources used. In this thesis, it was aimed to use SCG as a carbon source in the production of microbial lipase enzyme in order to reduce operational costs and develop a sustainable bioprocess. Under this scope, it has been determined that approximately 60% of the SCG consists of water after collection. In addition, 0.2 mL of coffee oil was extracted from 1 gram of dried SCG by using hexane: isopropanol (1:1) solution and 39.11 ± 1.8% (w/w) reducing sugar and 9.95% ± 0.3 GAE were obtained by using 3% H2SO4 (v/v) solution. After the content of the SCG was determined, the conditions for lipase production from SCG by C. diffluens D44, previously known as a lipase producing yeast, were optimized in the Design Expert program using the Box-Behnken design. As a result of the studies, the highest lipase enzyme activity was found to be 14.6 U/mL at 29.4 °C with a pH of 8.3 and a medium containing 8.7% (w/v) SCG. The optimum temperature and pH of the produced D44 lipase enzyme from SCG were determined as 37 °C and 8.0, respectively. Besides, GC-FID analysis was performed on the enzyme-containing upper phase liquid obtained as a result of production. The composition of fatty acids in the medium was determined as 31.05% linoleic acid, 28.67% palmitic acid, 20.44% heptadecanoic acid and 19.84% oleic acid. Furthermore, B. subtilis BT2 was isolated from the soil sample in Kadıköy, Istanbul to evaluate the use of SCG by bacteria and lipase was produced from a nutrient medium containing 1% (w/v) SCG. As a result of the production, the bacterial lipase activity produced was found to be 40.426 U/mL. Adding 0.1% Tween80 (v/v) to the production medium increased the bacterial lipase activity produced from SCG by approximately 10%, resulting in 44,711 U/mL. The optimum temperature and pH of the produced bacterial lipase enzyme were determined as 37 °C and 8.0, respectively. In addition, using coffee oil and sugar extracted from SCG, sugar-fatty acid ester production by BT2 lipase catalysis was determined by FTIR analysis. Finally, SEM images of the SCG samples used to investigate the reusability of SCG used in lipase enzyme production by yeast and bacteria were taken and their structural changes were examined.
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kullanılmış kahve telvesi, kahve atığı, yemek atığı, lipaz, mikrobiyal enzim üretimi, yeşil teknoloji spent coffee grounds, coffee waste, food waste, lipase, microbial enzyme production, green technology