Optimization of Oil Content And Specific Fatty Acids Traits of Crambe And Camelina As Industrial Oil Crops. Erwin Fajar Hasrianda (Reg. no: 860728229010) Master Thesis Submitted to the Laboratory of Plant Breeding at Wageningen University In Partial Fulfilment Of The Requirements For The Degree Of Masters of Science in Plant Sciences Specialization Plant Breeding and Genetic Resources November 2016 Abstract Currently, our modern society is using high amount of petroleum oil every day. However, petroleum oil is a finite natural resources and causing a lot of problem such as air pollution and global warming. Green oil from plant production can be an alternative oil because green oil is more environmental friendly and can produce high quality oil composition. Green oil itself already has a potential market, especially for oleochemicals industrial sector. In European Union, industrial sector is demanding high quality oil with specific fatty acids composition. Erucic acid and gondoic acid are among the most valuable fatty acid in European Union market. These fatty acids has potential market as the feedstock material for plasticizers, paintings, cosmetics, coatings high quality nylon and lubricants industry. Crambe and camelina were chosen as the new crop species to produce erucic acid and gondoic acid. To answer this societal challenge, European Union consortium launched COSMOS (Camelina & crambe Oil crops as Sources for Medium-chain Oils for Specialty oleochemicals) project. This project is aiming to improve economical value of crambe and camelina, for producing specific fatty acids production and the derivative products of crambe and camelina. As a part of COSMOS project, this research is focused on gaining information of oil content and oil composition in current crambe and camelina cultivars from various cultivation site (Italy, Poland and Netherlands) by using GC test, NMR measurement and solvent extraction method. This research also collecting information about biomass and seed production (kg/Ha) of crambe and camelina field trial. Percentage oil content of crambe and camelina was significantly influenced by cultivation site and species factor while fatty acids composition in crambe and camelina oil were influenced country and species factor and interaction of those factor. For biomass (stem, leaves, pod, seed parameter) production in crambe and camelina, cultivar and harvest time were significantly influencing the biomass parameter. The information from this research is expcted to be used to design a better plant breeding project for crambe and camelina. Keyword : Crambe, camelina, gondoic acid, erucic acid, biomass production, GC test, NMR measurement, solvent extraction. 1. INTRODUCTION 1.1 Alternative oil source outside fossil oil for Europe Petroleum is playing major role in the modern society. Small amounts of it are used for chemical and petrochemical as their feedstock (raw material), while most of them (about 90%) is taken to energize transportation and electricity as heat source. Industry is using chemical feedstock from petroleum to make feedstocks, lubricants, synthetic rubbers, solvents, plastics, detergents, fibers, and others (Carlsson, 2009). Since petroleum can create environmental problems (increasing CO2 level in atmosphere), it is also a finite resource and an alternative yet more sustainable material which need to be explored so it can gradually replace petroleum-based material. Alternative oleochemicals came from plan oil sources can be a good option for feedstocks in petroleum-material based industry. With annual production of 129 metric tonnes, plant oil is important commodities in agriculture sector (FAO, 2008; Carlsson 2009). Current industrial feedstock is using approximately 15% of total plant oil production. It has diverse use in global market, such as soap, lubricants, detergents, solvents, chemical feedstocks, surfactants, paints, and cosmetics production (Carlsson, 2009). Although its very diverse function in industry sector, six main fatty acids are dominating the composition of most seed oil: oleic, linolenic, stearic, linoleic, and palmitic acid. They have 16, 12 and 18 carbon chains length (ibid.). Currently, high number of plant oil sources are coming from coconut and palm which grow in tropical area (tropical oil) and unable to grow in European Union (EU) environment. Attractive and promising approach come by developing wild plant oil species which produce great diversity of fatty acids. Those oil crops could be developed with modern plant breeding technique (Carlsson, 2009). In near future, these oil plat species are expected to produce a higher oil content with better fatty acids composition to fullfil the European needs of feedstock from oil crop. EU answered the question by launching COSMOS (Camelina & crambe Oil crops as Sources for Medium-chain Oils for Specialty oleochemicals), a project for developing potential oil crops which can be grown in diverse European soils and climate condition (Blaauw, 2014). Carlsson (2009) states there are several requirements for future oil plant producers: ability to produce high quality and quantity oil per hectare and demand only low amount of agricultural resources; and high compatibility with current agriculture infrastructure and less probability of oil crops admix with oil food species (for example, through cross-breeding or mixing in supply chain process). Long-time preservation character of this new oil crop final and broad range of the industrial application are two strong points for plant oil. Furthermore, availability of compatible modern plant breeding technique which may be applied to these new oil crops can be a big advantage to improve production in a long run. 1.2 Fatty acids market for industry in EU In general, oil from plants can be used for two main products: food purpose and non-food (industry) purpose. It is necessary to keep oil plants for industrial purpose are not mixed with its counterpart. In a review paper from Carlsson (2009), it is explained that the main reason for this is because industrial-purpose oil contains special properties carbon chains (specific fatty acids) which have unique molecular structure. The technical oil qualities are essential for industrial feedstock, but might have poisonous effect and indigestible for human organs. The example of this is various types of unusual, unsaturated (triple, mono, conjugated), and branched molecules structure. Furthermore, carbon chains with different length types (short-, medium-, or very long-chained) as well as functional groups or added side chains are also important characteristics in industrial-purpose oil. Plants with these oil characteristics should be specifically dedicated for industry sector usage. Two promising fatty acids in oil plant are erucic (C22:1) and gondoic acids (C20:0). Both are broadly used in industry sector as their derivative products can be essential feedstocks in lubricant industry, paintings, cosmetics, plasticizers, nylon industry, coatings, and lipochemical industrial preparations (Blaauw, 2014). By improving the production of erucic (C22:1) and gondoic acids (C20:0) in crambe and camelina, it can be alternative for filling the demand of EU industrial fatty acids market in more environmental friendly manner, compared to using fatty acids from fossil oil. In order to gain more stable oil plant supply for EU fatty acid market, it will be beneficial if the oil crop can be suitably planted at multiple locations in EU areas and have good result in terms of seed yield, compatibility level with the current cultivation practices, ease of harvesting, oil content, and low resource inputs (Blaauw, 2014). Potential oil crops for this purpose are crambe (Crambe abyssinica Hochst.) and camelina (Camelina sativa). 1.3 Crambe Crambe has already been well-adapted to grow in temperate climate and does not have probability to cross with any other present oil crop. Crambe oil can contain erucic acids around 60% while oil content is about 60% in its seeds (38% when including the pod). It produces a single seed inside a pot with oil yields up to 1 ton/ha (Carlson, 2007). Since its oil content is dominated by erucic acids, crambe has already been categorized as non-food oil plant producer. Other major fatty acids in crambe oil are palmitic (3%), stearic (5%), linoleic (9%), and oleic acid (16%) (Warwick and Gugel, 2003). The challenge in improving crambe as potential oil crop is its lack of genetic diversity (Mastebroek et al., 1994). Currently, breeding project for improving crambe genetic diversity is held (Blaauw, 2014). In taxonomy, crambe belongs to cruciferous family. Depending on plant’s density and plantation season, its height is around 1-2m. Commonly, its flowers are yellow or white. A thousand of crambe seeds weigh about 6-10g with its greenish brown colour inside little capsule. One capsule can only contain one seed. The diameter of crambe seed is o.8-2.6mm. Crambe species are natively from Ethiopia highland and able adapting very well with Europe’s cold weather. It can grow as winter crop in Mediterranean climate and spring crop in Northern Europe climate (Falasca, 2010). As explained by Castleman et. al., (1999), Crambe has great tolerance to frost and drought. It has short life cycle as it can be harvested in 90 days and bloom in 35 days. Mechanical harvest is possible in crambe since it has uniform maturity. Generally, the commercial cultivars of crambe take 83-105 days before the seed is ready to harvest. Meanwhile, Wang et al., (2000) state that Crambe in Chengdu area in China needs much longer time (212-224 days) before ready to be harvested. In addition, Falasca (2010) argues that crambe can be sowed in March or April (as winter crop after soybean) because its possibility in mechanized harvesting and its low cultivating costs,. Now, soybean farmers in Brazil are showing great in cultivating crambe in their field. Crambe usually grows in the site with rainfall range of 350 to1200 mm, with annual temperature average in the range of 5.7°C to 16.2°C, and with pH soils range of 5.0 to 7.8 [9: Falasca (2010)]. Crambe roots may reach more than 15 cm depth so it provides tolerance in dry periods.