VOL. 11, NO. 4, FEBRUARY 2016 ISSN 1819-6608 ARPN Journal of Engineering and Applied Sciences ©2006-2016 Asian Research Publishing Network (ARPN). All rights reserved. www.arpnjournals.com 2698 THE EFFECT OF NITROGEN STRESS IN MEDIUMFOR INCREASING CARBOHYDRATE AS A BIOETHANOL SOURCE AND CAROTENOID AS AN ANTIOXIDANT FROM CHLORELLA ZOFINGIENSIS CULTURE Eko Agus Suyono 1,2 , Umi Muavatun 1, , Faridatul Husna 1 , Husnul Khotimah 1 , Ika Pratiwi 1 , Rahmah Husna 1 , Fitri Cahyani 1 , Yuni Purwanti 1 and Thoriq Teja Samudra 1 1 Faculty of Biology, Gadjah MadaUniversity Jl. Teknika Selatan, Sekip Utara, Yogyakarta, Indonesia 2 Centre for Energy Studies, GadjahMada University Sekip K1A, Kampus UGM, Yogyakarta, Indonesia E-Mail: eko_suyono@ugm.ac.id ABSTRACT Chlorella zofingiensis is a prospective microalgae because it is mainly ascarotenoid producer, such as astaxanthin. However, its carbohydrates could be also as promising source ofbioethanol. Furthermore, Nitrogen stress treatment is reported used for increasing both carbohydrate and carotenoid of some microalgae. Therefore, this study aimed to increase carbohydrate and carotenoid of microalgae C. zofingiensis by using low and high nitrogen excessin the growth medium. The mediums were consisted of local compound fertilizer (farmpion), urea and ZA with a ratio of 0.25:0.5:1(low nitrogen excess medium) and 0.5: 1: 2(high nitrogen excess medium). Its cells density, carotenoid, and carbohydrate were measured every day for 7 days. The cell density was calculated using haemocytometer under light microscope. The carotenoid was measured using spectrophotometer with absorbance at a wavelength of 470, 645 and 662 nm. The carbohydrate was measured using sulfuric acid method. The results showed that the nitrogen stress treatment was able to increase carbohydrates and carotenoids approximately twice in C. zofingiensis as culture as source of bioethanol and antioxidant. Keywords: chlorella zofingiensi, nitrogen, carotenoid, carbohydrate. INTRODUCTION Chlorella zofingiens is freshwater green algal that classified into class and family Chlorophyceae and ordo Chlorococcales [1] and indicates having high carbohydrate content as bioethanol source [2]. That product has been proposed as good alternative to non-renewable fossil fuels [3]. Microalgal is as a potential new generation of feedstock for biofuel production because of its high growthh rates and high photosynthetic efficiencies when grown on specific environments [4]. When the microalgae is cultivated and grown in extreme environmental conditions and under stress circumstances, it will synthesize various secondary metabolites to increase the possibility for surviving under those stress conditions [5, 6]. One of the secondary metabolites produced by the microalgae is carotenoid asantioxidant property [7]. Microalgae C. zofingiensis is reportedable accumulate the carotenod, especially astaxhanthin as a king of antioxidant [8], so that that the microalgae is potentially as astaxanthin producer [9]. Aprospective method for enhancing its valuable contents is by modifying Nitrogen concentration in the growth medium. Nitrogen is an essential nutrient for increasing C. Zofingiens is carbohydrate and carotenoid [10]. Nitrogen is used for producing chlorophyll which is an important compound to accelerate the photosynthesis rate. Therefore, the aim of this research is to increase carbohydrate and carotenoid of microalgae C. zofingiensis by nitrogen excess treatments in the medium. The research will be an alternative method for producing bioethanol from high carbohydrate microalgae as a future renewable energy as well as producing carotenoid as a valuable antioxidant. MATERIALS AND METHODS Materials This research used medium agricultural fertilizers: urea: ZA with a ratio of 0.5: 1: 2 (high excess nitrogen medium) and 0.25: 0, 5: 1(low excessnitrogen medium). Method The study was conducted in Wukir Sari, Cangkringan, Pakem, Sleman, Daerah Istimewa Yogyakarta (DIY). C. zofingiensis was cultured on a mass scale in the 3600 litre pool. Environmental parameters measured were temperature, pH, and density. The mediums were local agricultural fertilizer (farmpion), urea and ZA with ratio of 0.25: 0, 5: 1 (low excess nitrogen medium) and 0, 5: 1: 2 (high excess nitrogen medium). As a control, C. zofingiensis was cultivated in medium with local agricultural fertilizer (farmpion) without the addition of urea and ZA. Nitrogen content in fertilizers and ZA 21%, while the nitrogen contentinUrea46%. Samples were taken every day for 7 days. The parameters measured were cells density, carotenoid, and charbohydrate.