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Growth and Nitrogen Fixation Dynamics of Azotobacter Chroococcum in Nitrogen-Free and Omw Containing Medium

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Growth and Nitrogen Fixation Dynamics of Azotobacter Chroococcum in Nitrogen-Free and Omw Containing Medium GROWTH AND NITROGEN FIXATION DYNAMICS OF AZOTOBACTER CHROOCOCCUM IN NITROGEN-FREE AND OMW CONTAINING MEDIUM A THESIS SUBMITTED TO THE GRADUATE SCHOOL OF NATURAL AND APPLIED SCIENCES OF THE MIDDLE EAST TECHNICAL UNIVERSITY BY GÜL )ø'AN SARIBAY IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN THE DEPARTMENT OF FOOD ENGINEERING DECEMBER 2003 Approval of the Graduate School of Natural and Applied Sciences. Prof. Dr. Canan Özgen Director I certify that this thesis satisfies all the requirements as a thesis for the degree of Master of Science. ¢¡¤£¦¥¨§ © ¡ § !¡¤"# Head of Department We certify that we have read this thesis and in our opinion it is fully adequate, in scope and quality, as a thesis for the degree of Master of Science in Food Engineering. 3 ABSTRACT GROWTH AND NITROGEN FIXATION DYNAMICS OF AZOTOBACTER CHROOCOCCUM IN NITROGEN-FREE AND OMW CONTAINING MEDIUM $%&¤'()%+*,¦-/.0¦13254¦%+6 M.Sc., Department of Food Engineering 789):;=<)>#?A@B;DCFEG;¤@H¨IJK; ILNM OP8QNLNMRSM+RST U December 2003, 68 Pages Olive Mill Wastewater (OMW), by-product of oil industry, is a dark liquid with a characteristic fetid smell, bitter taste and bright appearance; having a high pollution potential, creating serious problems in countries producing olive oil. Azotobacter chroococcum as a Nitrogen-fixing bacteria can bioremediate OMW, by degrading its toxic constituents. With the help of this detoxification process OMW can be used as biofertilizer. In this study, the dynamics of growth and nitrogen fixation at different physiological conditions and nutrient requirements of A. chroococcum in chemically defined N-free medium was determined. These parameters were cultivation conditions such as pH, temperature and aeration and some additives such as inorganic salts, boric acid and nitrogen. Consequently, the iii maximum cell concentration were obtained when A. chroococcum was grown at neutral pH, 35°C, 150 rpm and in medium supplemented with manganese salt at 0.01% concentration. The maximum nitrogen fixation products were attained when A. chroococcum was grown under the same conditions except at pH 8. Further, bioremediation of OMW by A. chroococcum was examined. When A. chroococcum was cultivated in OMW containing basal medium at 10% OMW concentration, a cell density 12 times higher than in the OMW free medium was achieved. Also, it was found to have maximum increase in extracellular protein concentration (112 mg/l) at 10% OMW containing medium and maximum increase in ammonia concentration (9.05 mg/l) at 5% OMW containing medium. Key Words: Azotobacter chroococcum, Nitrogen Fixation, OMW, Bioremediation iv ÖZ AZOTOBACTER CHROOCOCCUM V5WKXZY\[]_^/`W\[ba cd[ecgfN^ehAX ikjKl\jNm)n\oKnZpqsrglKrgtvu_lxwejKy{zKj}|g~Ko\~Kyr\rjxukw|gjN gjKydj z\pAt\jxydp _p ¤)+¦/¦35¦+ K¦¨_ ¢¡\£P¤g¥¦§)¤¦#©¨5ª¦«¬¦¨¦­® +­S­±° £ Tez Yöneticisi: Prof. Dr. Haluk ¯ ²x³µ´ ¶P·5¸_¹º¦º»¼¦½)¾_¿)´ ÀÁ´ à ÄÅBÆÂÇÈŦÉÊË ÄÈÌ¢ÍΤÆϤÇÎ ÇÈ)ÇÈÉDÆ=ËÐΨÑÅBÑÒ¦Ó5ÉÈdÔFÄ+ÅBÆÂÇÈÕÐÉϵÉΤÑÅ¢ÑÖ×Ð)ÉϤÉ+ТÆØÄ+ϤÇεÆÂÇ!ÐbÙÇÏ Ú)Û#ÜÞÝߢÝàܤàáßâ5ã+äåæãFç èPåæÚ)ãé¤âPã+ÝëêìBé=í¦äíî âí)åæÝ)ßBï¢àðéµñäÝóòÛ!éôÜ èõ)èö¦èéø÷ùÝ)Û5é¤âPÛPâ#ÛÝ ÜAߢé=àäâ ÚßBúûã+äÜAÛ5ï¦ñ âPÛ!ä)ñKÜAã+ü)Û!ڮߦâãäò¦à±ÜAè5õ)èPå¢ýFñ+ïBúØÛäï¦ãFþèíé¤ñúûñäÿíâÝñâPñéµö¦ñ ç ÛPöö¦Û ar ortaya ¡£¢¥¤§¦©¨ ¦ ¤¦§¢¨ ¤¦©¨ ¦"!$# #%# ¨$()+*,-( 1( ¨$ &©' /.0 Azotobacter chroococcum 2§3 4 5£3£6)3£7%3©4$3©8:9,;7%<§6-<0=>;8?<%6-3 4 3©8?;A@06¥B7%B 9,;-6CDB©8FEB£G%;4IHKJMLNGB>EO<&8,P0;)Q ;83/PR7%<&S?;@06¥B©CT; 7%3 4UG%V¥C V7§6-3XWB£7&E;S 8§34 3PUL7FL 9,;7%<§6¥<0=©;8 Y"Z%94UB[<%6)3©4 3©8 8%L6A6¥3©SVA6C 3 8FE3G§V4H\JML 536-V)@$C 3£G%3 A. chroococcum ]^%_a`,bced%fgUfhDi§h¥f jUf©`lk§m£h-b¥j$h¥m©_%cTb)nKfoi"p g$^oqk§m£gbd§m j$b b¥r/m©j$b)gb_§s%m `b+sm/tbAnRb¥`uvbo£di%h-iwIb`x`§in$^h)h-f©j$y_§sf{z§m|k§m/gb_}b~FpObd%f£/y_§s%fk%%dF%cemzm{f©o&i"p k§f£th)f cefs%b_f cDb-tbk§m£h-b¥j$h-m _cTb)n pObj §^§f j$f c m>pAjUm£h¥m£j,k%dFcDm|`§i§n$^h)h-f©j$yi§h¥f©jUf `T% g0y-f `,h-y`~f z§f/h¥f _s%yjvcefzmk§f©o&y`§f©pA`yc f£ss§m£h eri olarak anorganik tuzlar, borik asit v ve azottur. Sonuç olarak, en yüksek hücre konsantrasyonu A. chroococcum nötr §,§ &¡1¢0£ ¡F¤) £"¢$¥% F¡§¦§£qD£©¡§£©¡¨¤)©%ª£©¬«)­-£©£¦§«-­D«)® pH, 35°C, ¯§°±0²³%°£´$²µ¶°·°£¸)¶%°¹°/¶%²-¸ºT²)»$¼²´½£¾¿µ|³FÀÁ,±°©Á ÃÄ"¼1¯Â£Å§¸¥Â º ÂÆÀ´ À%µ¸)°©´$²µ°·¶%°ÆÇ%ÈeÉ ÊµDËÌÄ%¸¥ºe±0Ê ¶&Í%´ ͺÎÍTÏ ´$²-Ð{³FµÊÑÁħ»$Í,¸-¸- ´ ¶ÂÒÍ,¸-Â/»Ê-¸ºTÊ)»$¼Ê´½§Ó©ϧ±Ä&µ%´  A chroococcum’un zeytin Á§Â ´ ± ͧ³FÍ%µÍÔ¯,²¥³%ħ¸¥Ä0Õ>²ÁÖħ¸¥Â©´ ÂÁײ)»R¸)°©ºe°"±0²Ø²µÙ°/¸-°©µ%ºT²)»$¼²´½ A chroococcum zeytin Á§Â ´ ± ͧ³FÍÚ²)У°©´$° µ¼° º °¸¿¯§°/±²³%°£´$²µ¶°DÛF°¸-²)»$¼²´$²A¸¥¶%²¥Å%²µ,¶°£Ü+ÝßÞ àxÃ"° ³&¼²µáÁ§Â ´$Â/±$ͧ³FÍâ²-а ´ ° µ ortamda zeytin karasuyu içermeyen ortamda elde edilen hücre Á§ÄFµ,±0©µF¼)´$±U³%Ä&µÍ%µÍ%µ Þ>ã Á©¼ÊµÂ Í,¸-Â/»Ê-¸ºTÊ)» ¼OÊ´½Öä̳%´$Ê¥Ù£ÂÜX°©µ ³FÀÁ1±R°©Á Ç´$Ä"¼°²µ Á§ÄFµ,±0©µF¼)´$±U³%Ä&µÍ ´O¼ÊA»0ʵ§Â å>æ%æIçéèDêëìí îïæ©ðéñ&ò$ó©ôõ¥ô§ö§ó}÷&ø©ù&úû¥ôýü§ó©ò ó"þ ÿ§ùFÿ û øò$ø ô£¢øþRû ¡ ¤¦¥¨§ © © !"©# $¤©#%¤¦&©!§'¥(©)*+¤¦,"© ¥-§- /.0 ©21¨346578 9;:=<?> ise %5 ¤¦¥*©!+ @©BA) )§¨CD+©#¥(©E,,$CGF#!¥* DHI C+¤¥-§ © +©B¥*©)(§=</©! '.(§- ¥04 Anahtar Kelimeler: Azotobacter chroococcum ;JDA;¤¦§KB©#L</©! ©¦MN#¦§-C ODP!Q(PRESTSIU¦VXW?TYZGY0[\W]_^-TWGZG`)a(b=WQdc` vi ACKNOWLEDGEMENT egfih(j+klnmGoprq/so#tuhwvxyq x{z}|(x~E|0o;pu2ro! o ln_t+x)q only for his guidance and supervision but also for his patience and support throughout this study. ¦* ) + }(E#0?D!-/0) *En¡0n¢£ (n¤nn/¦ (+E#0 ¥D¦#§¨©«ªD¦n¬­®¦!¯_¦ ¯+§¡°²±\±E³´µ#¶·©(³¸&µ#¹©0µ#¶º¯¦ ©¼»½¦n¬/º?­¾D¿³DÀ¦#Á+Âè?¯I± ¨GÀ)¿Ä·¦!Å+³¦ÆÄ®$Çȸ ÆÄ/Æ©( studies. I offer sincere thanks to my family members for encouraging me at all the stages of this study. Thanks and love to all laboratory my friends for creating such a peaceful and humorous atmosphere in the laboratory. I also want to thank to Aytekin Güler for his technical support for his helps. vii TABLE OF CONTENTS ABSTRACT..................................................................................................... iii ÖZ .................................................................................................................... v ACKNOWLEDGEMENT............................................................................... vii TABLE OF CONTENTS................................................................................. x LIST OF TABLES........................................................................................... xi LIST OF FIGURES ......................................................................................... xiii CHAPTER 1. INTRODUCTION ..................................................................................... 1 1.1. Organism; Azotobcater chroococcum ................................................ 1 1.1.1. Effect of External Environmental Factors on the Growth of the Genus Azotobacter ...................................................................... 2 1.1.1.1.pH....................................................................................... 2 1.1.1.2.Temperature ....................................................................... 3 1.1.1.3.Aeration ............................................................................. 3 1.1.1.4.Inorganic Salts ................................................................... 4 1.1.1.5.Nitrogen ............................................................................. 4 1.2. Biological Nitrogen Fixation.............................................................. 5 1.2.1. Significance of Biological Nitrogen Fixation.......................... 9 1.2.2. Factors Affecting Nitrogen Fixation........................................ 9 1.2.2.1.pH....................................................................................... 9 1.2.2.2.Temperature....................................................................... 10 1.2.2.3.Oxygen............................................................................... 10 1.2.2.4.Inorganic Salts ................................................................... 11 1.2.2.5.Nitrogen ............................................................................. 11 viii 1.2.3. Methods for Measuring Nitrogen Fixation .............................. 12 1.2.3.1.Measurement of Nitrogen Fixation with the Increase in Growth in N-free Medium .................................................. 12 1.2.3.2.Measurement of Nitrogen Fixation by the Nitrogen Difference Method ................................................................................ 12 1.2.3.3.Measurement of Nitrogen Fixation by Ammonia Determination...................................................................... 13 1.2.3.4.Measurement of Nitrogen Fixation by Stable Isotope (15N) Method ................................................................................ 13 1.2.3.5.Measurement of Nitrogen Fixation by the Acetylene Reduction Assay ................................................................. 13 1.3. Olive Mill Wastewater (OMW).......................................................... 14 1.3.1. Disposal and Treatment of OMW............................................ 14 1.3.2. Utilization of OMW by Azotobacter Chrooccocum................ 16 1.3.3. Bioremediation of OMW ......................................................... 16 1.4. Aim Of the Study................................................................................ 18 2. MATERIALS AND METHODS............................................................... 19 2.1. Materials ............................................................................................. 19 2.1.1. Organism.................................................................................
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