Denitrifikasyon prosesinde yüksek sıcaklığın sistem bileşenleri üzerine olan etkilerinin araştırılması
The investigation of high temperature effects on system components of denitrification process
Yazar: TUĞBA DOĞAN
Danışman: PROF. DR. EMİNE ÇOKGÖR
İstanbul Teknik Üniversitesi / Fen Bilimleri Enstitüsü / Çevre Mühendisliği Ana Bilim Dalı / Çevre Bilimleri ve Mühendisliği Bilim Dalı
Derece: Yüksek Lisans, 2014
Konu: Çevre Mühendisliği = Environmental Engineering

Özet:
Son yıllarda artan su kullanımına karşılık su kaynaklarının tükenmeye başlaması doğal kaynakların korunmasının önemini arttırmaktadır. Kullanılabilir su kaynaklarının sürdürülebilirliğinin sağlanması için alıcı ortama yapılan deşarjlar kontrol altına alınmalıdır. Atıksu içerisindeki kirletici parametreler en az deşarj standartlarını sağlayacak değerlere düşürülmelidir. Bu parametrelerden birisi de azot parametresidir. Atıksulardan azot giderimini hedefleyen en yaygın yöntemlerden birisi biyolojik denitrifikasyon prosesidir.
Yapılan çalışma ile biyolojik azot gideren aktif çamur sistemlerinde yüksek sıcaklığın denitrifikasyon mekanizması üzerine olan etkilerinin araştırılması amaçlanmıştır. Pepton karışımı sentetik atıksu kullanılarak 25 oC, 30 oC ve 37 oC sıcaklıklarında çamur yaşı 4 gün ve 12 gün olan doldur boşalt reaktörler işletilmiştir. Aklimasyon süreci boyunca sistemler uçucu askıda katı madde, askıda katı madde madde, KOİ, nitrat ve pH parametreleri ile takip edilmiştir. Üç farklı sıcaklıkta işletilen sistemler kararlı hale ulaştığında, sistemlerde konvansiyonel parametreler, nitrat tüketim hızı, mikrobiyolojik analizler ve enzim aktiviteleri ölçülmüştür. Mikrobiyolojik analiz kapsamında yaş örnekleme ve gram boyama yapılmıştır. Enzim aktiviteleri yarı kantitatif metod olan API-ZYM ile gerçekleştirilmiştir.
Birinci bölümde çalışmanın anlam ve önemine değinilerek amaç ve kapsam hakkında bilgi verilmiştir.
İkinci bölümde azot bileşenleri ve azot çevrimi hakkında bilgilendirme yapıldıktan sonra denitrifikasyon mikrobiyolojisi, stokiyometrisi ve kinetiği açıklanmıştır. Literatürdeki çalışmalar araştırılarak sıcaklık düzeltme katsayıları ve denitrifikasyon hızları ile ilgili derleme yapılmıştır.
Üçüncü bölümde laboratuvarda uygulanan tüm analiz yöntemleri, reaktörlerin kurulumu ve işletimi ayrıntılı bir şekilde açıklanmıştır. 
Dördüncü bölümde yürütülen deneysel çalışmaların sonuçları ayrıntılı olarak verilmiş, mikrobiyolojik analizler ve enzim aktivitesinin ölçümüne ait görüntüler gösterilmiştir.Beşinci bölümde deneysel sonuçlar değerlendirilmiş ve verilerin literatür ile karşılaştırılması yapılmıştır.Altıncı bölümde genel değerlendirme yapılarak önerilerde bulunulmuştur.

Abstract:
Water use has been increasing due to the rapidly population growth and developing technology; thus, avaliable water resources have diminished day by day. For this reason, it is very important to use water efficiently and have optimum treatment techniques for protection of water sources and maintaining the long-term sustainability. In recent years, removal of nutrients such as nitrogen and phosphorus from wastewater has gained a great importance in terms of protection of water quality.
Excess nitrogen load to the receiving body cause some adverse effects to beneficial use. Some of these effects include dissolved oxygen depletion, high pH and eutrophication that is the most important adverse effects.
One of the most common method is biological denitrification process for removal of nitrogen from wastewater; since, it is efficient and economic. Nitrification and denitrification are sequential processes in activated sludge system for biological nitrogen removal. Denitrification is the second step of nitrogen removal. Temperature is one of the most important factor that affects the process kinetics. Studies in the literature are generally about the low temperature effects. Accordingly, the aim of this study is the investigation of high temperature effects on denitrification mechanism in activated sludge system. The aim of this study covers the following scopes: Literature review, reactor setup and operation, microbiological analysis, enzymatic activity analysis and Nitrogen Uptake Rate (NUR) analysis.
Nitrogen naturally exists with a valence ranging from -3 to +5. Organic nitrogen, ammonia (NH3) and nitrogen gas (N2) are unoxidized nitrogen forms, and nitrite (NO2) and nitrate (NO3-)  are oxidized forms of nitrogen. The biological oxidation of ammonia with oxygen into nitrite followed by the oxidation of these nitrites into nitrates is called nitrification. Nitrification process is carried out by autotrophic species that are Nitrosomonas and Nitrobacter, respectively.
Denitrification is the reduction of nitrate nitrogen to molecular nitrogen in anoxic conditions by heteretrophic microorganisms. Acetobacter, Denitrobacillus, Enterobacter, Moraxella and Pseudomonas are some of the denitirication bacteria species. Denitrification process includes four stages given in below.
NO3-  NO2-  NO  N2O  N2
Basically, the following four conditions are required for biological denitrification: Presence of nitrate of nitrite, absence of dissolved oxygen, a facultative biomass and presence of suitable electron donor. Organic carbon (electron donor) that is used as energy source can be obtained from three sources. These are external addition, organic matter present in the wastewater and lysis of biomass in the endogenous phase.
The process kinetics of denitrification are discussed under three circumstances which are growth of denitrifies, hydrolysis of prticulate organic matter and decay of denitrifiers (endogenous decay). Growth of denitrifiers is expressed with double Monod-type function that includes both organic matter and nitrate concentrations acting as carbon source and alectron acceptors in denitrification.
Denitrification rate is influenced by environmental factors such as dissolved oxygen, pH, alkalinity, carbon source, elektron acceptor, inhibitors and temperature. The presence of dissolved oxygen is detrimental to denitrification and it has inhibitory effects when the concentration is 0.2 mg/L. The optimum pH must be between 7.0 and 7.5. The relationship between temperature and kinetic coefficients are expressed with Arrhenius equation.
The amount of nitrate that can be biologically denitrified in the presence of  enough organic carbon is called denitrification potential. It is significant to use denitrification potential effectively. Denitrification potential is one of the basic principle of design. It is influenced by wastewater characterization and the ratio of anoxic and total volume.
The first stage of the study includes reactor setup and operation. Three reactors were operated during the study with synthetic wastewater. Laboratuary scale fill and draw reactors with the hydraulic retention time of 1.33 day was sustained at a sludge age 4 and 12 days respectively at steady state under aerobic and anoxic conditions. The temperatures of reactor was 25oC, 30oC  and 37oC. The reactors were operated 8 hours anoxic and 16 hours aerobic conditions. The peptone mixture used as organic carbon source was prepared with 16 g/L of peptone, 11 g/L of meat extract, 3 g/L of urea, 0.7 g/L of NaCl, 0.4/L of CaCl2. 2H2O, 0.2 g/L of MgSO4.7H2O and 2.8/L of K2HPO4. The solution A for macronutrients was prepared with 320 g/L K2HPO4, and 160 g/L KH2PO4. Also the nitrate solution was prepared with 72.22 mg KNO3/L for nitrate source. The feeding in the fill and draw reactor was adjusted to maintain around 600 mg COD/Land 50 mg N/L of total nitrogen.
During the operation, COD and NO3- samples were taken twice a week with Millipore membrane filter that have 0.45 µm and 0.22 µm pore sizes respectively. Also routine SS/VSS measurement were done. Everyday pH and temperature of the reactors were monitored. Standard methods were used during all of the measurements.
The second stage of study includes the most important experimental analysis which are NUR tests. NUR tests were conducted in order to determine denitrification rates. The NUR experiments were performed in a 2 L reactor for 25oC, 30oC  and 37oC and for both 4 and 12 days sludge age. Before starting nitrogen gas was supplied to the reactor in order to remove oxygen from reactor and keep anoxic conditions during the entire procedure and then reactor was closed. After peptone mixture and nitrate addition, NO3-, COD and PHAs samples were taken for every 15 and 45 minutes respectively. Then all of the samples were analysed and NUR profiles plotted. 
The third stage of the study includes microbiological analysis that were wet mount and gram staining. Wet mount was conducted in order to learn which types of microorganisms were in the reactor and observe the mobility of bacteria. Gram staining was conducted to have information about morphology of the microorganisms. 10x and 100x objectives were used for microscopic observations and microscopic images were taken.
The final stage of the study includes enzymatic activity analysis. Enzymatic activities were measured with API-ZYM method that semiquantitative micromethod designed for the research of enzymatic activities. The technique is applicable to all specimens and  it allows the systematic and rapid study of 19 enzymatic reactions using very small quantities. API ZYM system has strips with 20 cupules that the base of them contain the enzymatic substrate and buffer. The strips allow contact between the enzyme and substrate. Enzymatic activities were determined by color change in the cupules.
After the experiments were carried out, result were discussed. According to the routine analysis for monitoring, pH, SS/VSS and effluent COD values of  sludge age 4 days reactors were higher than sludge age 12 days reactor. It was found that, there is no nitrification in 25 oC and 37 oC reactor and partial nitrification in 30 oC reactor for sludge age 4 days. It was discussed that, increasing pH suddenly over 9 for a short time cause death of sensitive nitrification microorganisms. On the other hand, for 12 days, all of reactors had nitrification process. Denitrification process determined efficiently for all reactors by samples taken at the end of the anoxic time for the sludge age of 4 and 12 days.
Denitrification rates were calculated from NUR profiles and all of the calculations includes nitrite corrections. For sludge age 4 and 12 days, k2 is denitrification rate of together with SS1 and SH1, k3 is denitrification rate of SH2 and k4 is denitrification rate of endogenous decay. The processes were very fast; hence, SS1 and SH1 were undistinguishable. At the sludge age 4 days, k2 values were 0.034, 0.032, 0.037, k3 values were 0.021, 0.018, 0.015 and k4 values were unobserved, 0.0071, 0.0085 for 25 oC and 37 oC respectively. At the sludge age 12 days, k2 values were 0.091, 0.129, 0.103, k3 values were 0.022, 0.027, 0.011 and k4 values were 0,0058, 0.0066, 0.0053 for 25 oC and 37 oC respectively. SS1 and SH1 results for almost all experiments 393 mgCOD/L and SH2 values were 207 mgCOD/L. Unit of denitrification rates is mgN/mgVSS.hr. According to these results temperature correction factors were calculated by Arrhenius equation for sludge age 4 days. Temperature correction factors were 1-1.02, 0.97 and 1.026 for k1, k2 and k3 respectively. It is found that simplified Arrhenius equation was not valid for sludge age 12 days. Consequently, extended Arrhenius equation was applied  and according to the graph obtained, the denitrification rate decreased after 34 oC.
Microbiological analysis results showed that, Amoeba Arcella, Epistylis sp. and Opercularia were the most common species in the reactors. In addition, at the end of the gram staining, almost all of the images obtained with red color. This means bacterias were gram-negative.
Enzymatic avtivity analysis showed that, the activity of microorganisms of sludge age 4 days reactors were greater than 12 days. Lipase (c14), cystine arylamidase, α-galactosidase, ß-galactosidase, ß-glucuronidase, α-mannosidase ve α-fucosidase enzymes had no activity of very low activity for both 4 and 12 days sludge age.
Finally, there are very limited number of articles about high temperature effect on denitrification. Thus, this is one of the possible leading study explaining the impact of high temperature on denitrification process and could contribute to the literature with promising results.