Mikroalglerden enzimatik sulu ekstraksiyon yöntemi ile yağ eldesi
Enzymatic aqueous extraction of oil from microalgae
Yazar: YİĞİT AYHAN
Danışman: PROF. DR. MELEK TÜTER
İstanbul Teknik Üniversitesi / Fen Bilimleri Enstitüsü / Kimya Mühendisliği Ana Bilim Dalı
Derece: Yüksek Lisans, 2015
Konu: Kimya Mühendisliği = Chemical Engineering

Özet:
Teknolojinin gelişmesi ile birlikte insanların doğal kaynaklara yönelimi artmış ve doğada yaşayan birçok canlı türüne yönelme başlamıştır. Algler de insanların kendi yararları için kullanmaya başladığı doğal kaynaklar arasında yer almakta ve gün geçtikçe önemi artmaktadır.
Mikroalgler akuatik ekosistemlerdeki ekolojik ve de biyolojik rollerinin haricinde, gerek insan sağlığı gerekse de sucul hayvanlar için önemli besin maddeleri içermektedir. Günümüzde birçok mikroalg türü doymamış yağ asitlerinin zenginliği, yüksek oranda protein, vitamin vb. içeriklerinden dolayı birçok biyoteknolojik çalışma da önemli rol oynamaktadır. Bu çalışmaların önemi 1950'li yıllardan itibaren gün geçerek artmıştır.
Bu çalışmada, mikroalglerden yağ ekstraksiyonunda geleneksel olarak kullanılan çözücü ekstraksiyonu yöntemine alternatif olabilecek, daha sağlıklı, yüksek kalitede ve yüksek verimde yağ elde edilebilecek bir yöntem geliştirilmeye çalışılmıştır. Deneylerde içerisinde %18,6 oranında yağ içeren Schizochtrium sp. mikroalg türü kullanılmıştır. Alternatif ekstraksiyon yöntemi olarak enzimatik sulu ekstraksiyon yöntemi seçilmiş ve hücre duvarlarını parçalanması ve diğer etkileri arttırmak için proteaz enzimi kullanılmıştır. Proteaz enzimi için pH, enzim miktarı, sıcaklık ve süre parametreleri ele alınmış, uygun çalışma koşulları belirlenmiş ve ekstraksiyon verimine etkileri araştırılmıştır.
Deneysel çalışmalar, 30ml tampon çözelti, pH 5-8 aralığında, gram mikroalge karşılık 0,50-1,25 mL enzim miktarı, 30-50oC sıcaklık aralığında ve 4-24 saatlik deney sürelerinde gerçekleştirilmiştir. Gerçekleştirilen deneysel çalışmalar sonucunda elde edilen en uygun ekstraksiyon koşulları; pH: 8, enzim miktarı: 1 mLenzim/g mikroalg, sıcaklık: 50oC ve ekstraksiyon süresi: 12 saat olarak belirlenmiştir.  Elde edilen uygun enzimatik ekstraksiyon koşullarında ekstraksiyon verimi %61,47 olarak bulunmuştur.

Abstract:
In human nutrition, lipids are essential substances like carbohydrates and proteins that have vital roles in health. There are also fat-soluble vitamins in these lipids. Taking these vitamins is fundamental in terms of health and extremely necessary for the body. In addition, omega-3 fatty acids which can not be produced by the human body, is also plays an important role in human health and many other areas. On the other hand, these omega-3 fatty acids can be produced by algae and aquatic animals play the primal role of transporting these lipids into human body [1,2].
Algae are abundant and ancient organisms that can be found in virtually every ecosystem in the biosphere. They vary from tiny single-celled species one micrometer in diameter to giant seaweeds over 50 meters long. For billions of years algae have exerted profound effects on our planet, and they continue to do so today. Still, in many habitats algae often go unnoticed unless environmental conditions become favorable for the development of conspicuous and sometimes massive proliferations of a situation often brought about by human activity. People from many cultures, ancient and modern, have used algae for a variety of purposes. With the advent of biotechnology, algae are poised to play greater, although often subtle, roles in the day-to-day lives of human beings. 
For millennia people throughout the world have collected algae for food, fodder, or fertilizer. More recently algae have begun to play important roles in biotechnology. For example, they have been used to absorb excess nutrients from water streams, thereby reducing nutrient pollution in lakes and streams. Algae also generate industrially useful biomolecules, and serve as a human food source, either directly or indirectly, by supporting aquaculture of shrimp and other aquatic animals. Algae are increasingly being cropped in lab-based bioreactors, outdoor production ponds, and engineered offshore environments. Algae have provided science with uniquely advantageous model systems for the study of photosynthesis and other molecular, biochemical, and cellular-level phenomena of wider importance. Examples include Melvin Calvin's explanation of the light-independent reactions of photosynthesis in the green alga Chlorella. Studies of algae have been essential to our understanding of basic photosynthetic processes, and they continue to break new conceptual ground. The relative simplicity, antiquity, and vast diversity of algae, coupled with excellent fossil records in some cases, have also made algae invaluable systems for learning the organismal and organellar evolution and ecosystem function, and for understanding the effects of human disturbance upon the biosphere [3].
Algae are involved in global biogeochemical cycles and biotic associations that provide essential ecological services. Humans reap the benefits of these algal activities in the form of atmospheric oxygen, climate modulation, and fossil fuels, as well as finfish, shrimp, and shellfish harvests, which depend upon algal primary production. In addition, humans have learned to use algae in a wide variety of technological applications. Certain algal species have become priceless and irreplaceable model systems for research. Algae are used as environmental monitors, both to assess the health of modern aquatic systems and to understand environmental conditions of the past. Numerous food products are derived from algae, including food for cultivated shellfish, seaweeds eaten by humans, and protein and vitamin supplements produced from microalgae grown in pond or bioreactor cultivation systems. Some microalgae produce lipids that are potential sources of renewable fuels. Many algae manufacture compounds that have scientifically and industrially useful gelling properties. Ancient marine and lake diatom deposits (diatomite or diatomaceous earth) are mined for use in abrasives and industrial filtration. Algal phycobiliproteins can be used as fluorescent dyes in applications such as flow cytometry. Some algal compounds have potentially valuable antibiotic or antitumor activity. Finally, algae have been incorporated into engineering systems utilizing algal nutrient uptake and gas exchange properties to purify water or air [3].
Lipids are mostly extracted from seeds or the biomass itself by conventional methods such as mechanical pressing and/or solvent extraction. Generally n-hexane is used as solvent and high oil production yield is obtained. Although there is an advantage of good oil yield for the use of hexane, the oil product has low quality, high investment and management costs, and high energy requirement problems for its usage. Besides, organic solvent hexane is a toxic substance and has explosive property, it releases hazardous volatile materials to the atmosphere. Even though this traditional process for the extraction of oil is economically suitable, there are draw-backs like damage to the human health and environment and quality loss of finished products which cause to search for new techniques. Therefore, due to environmental safety regulations and public health risks associated with the use of hexane, alternative methods which are safe, environment-friendly, provide edible protein and qualified, highly efficient oil have been developed by researchers.
Many researchers have done a lot of work on aqueous enzymatic oil extraction from many different biomasses in order to obtain oil and different compounds and they have found that the yield value could be obtained the same or close the results of the solvent extraction processes. However, laboratory scale researches are needed to continue further researches to provide optimum conditions for extraction and separation stages of the process in order to take it to bring commercially more attractive.
Aqueous extraction method is carried out at lower temperatures with respect to solvent-based extraction method and more qualified oil can be obtained by it. However, due to low oil efficiency of aqueous extraction, enzymes are added to the extraction environment to increase the oil yield and to minimize byproducts. Enzymatic aqueous extraction makes use of enzymes to degrade the cell walls with water acting like a solvent. This enables much easier oil release and refining of the oil. Aim is not only to separate cellular or fluid lipids from other constituents, proteins, polysaccharides and macromolecules, but also to preserve these lipids for further analyses. The preservation of proteins also permits the pulp to be rich in proteins. Removing the non-lipid molecules without losing some lipids is a complete challenge. By means of enzymes, these difficulties are tried to be reduced. Although cost of enzymatic extraction process is estimated to be much more than hexane extraction, this situation may be overcome by recycling of the enzymes and using immobilized enzymes to decrease enzyme cost. If the oil to be extracted has high market value, again investment cost could be compensated. Enzymatic extraction can also be supported by ultrasonication for increasing the oil efficieny. Therefore, aqueous and enzyme-assisted aqueous extraction substitute the use of solvents and lead to obtain oil with high efficiency, so there is no more need for organic solvents in extraction process.
The aim of this study is the investigation of a method intended for elimination of toxic effects, obtaining high oil yield and quality and lowered economical issues which is an alternative to conventional solvent extraction. Microalgae (Schizochytrium sp.) is obtained from Vitatis Biyoteknoloji and contains %18,6 lipid of  the dried cell weight of microalgae. In this study, aqueous enzymatic extraction is selected as alternative extraction method and protease enzyme is used for degrading cell walls. Optimum conditions in respect to pH, enzyme amount, temperature and time are determined. 
Alcalase 2.5L Type-DX is used as enzyme in the experiments. Alcalase is a serine protease enzyme obtained from Bacillus licheniformis microorganisms. It has a high proteolitic activity of 2,5 AU/g (Anson Units/gram). The optimum conditions for enzyme activity are at the temperature between 55-70°C and pH 4-8 interval depending upon the substrate type.
Aqueous enzymatic extraction experiments are carried at 30 ml of pH 5-8 buffer, 0,50-1,25 mL/g microalgae enzyme amount, 30-50oC and between 4-24 hours. Optimum condition results for alcalase enzyme achieved at 50°C, 30 mL pH 8 buffer solution with 1,00 mL/g microalgae enzyme amount and duration of 12 hours. The extraction yield of microalgase oil at these conditions is determined as %61,47.