Sakarya Karaman atıksu arıtma tesisi çıkış suyunun mikrobiyolojik ve kimyasal olarak değerlendirilmesi
Microbiological and chemical evaluation of Sakarya Karaman wastewater treatment plant effluent
Yazar: DİĞDEM YELEK
Danışman: DR. ÖĞR. ÜYESİ KENAN TUNÇ
Sakarya Üniversitesi / Fen Bilimleri Enstitüsü / Biyoloji Ana Bilim Dalı
Derece: Yüksek Lisans, 2023
Konu: Biyoloji = Biology

Özet:
Bu çalışmada, 2021 Ağustos ile 2022 Nisan ayları arasında, MFS kullanılarak, Sakarya Karaman Atıksu Arıtma Tesisi (KAAT) çıkış suyundan alınan 12 adet numunede, mikrobiyolojik kirlilik göstergeleri olan koliform ve fekal koliform olarak Escherichia coli (E.coli), fekal streptekoklar, Pseudomonas aeroginosa (P. aeroginosa) ile Clostridium perfringens (C. perfringens) varlığı araştırılmıştır. Yine aynı numunelerde kimyasal kirlilik göstergeleri olarak Amonyum (NH₄), Nitrat (NO₃), Nitrit (NO₂), Fosfat (PO₄) düzeyleri ile pH, iletkenlik değerleri ölçülmüş, Kimyasal Oksijen İhtiyacı (KOİ) sonuçları ise atıksu arıtım tesisinden alınmıştır. 
Koliform ve fekal koliform analizi için Chromocult Coliform Agar (CCA) besiyerine konulan filtre kâğıdı 36˚C'de 24 saat inkübasyona bırakıldı. İnkübasyon sonunda pembemsi kırmızı renkli koloni oluşturanlar şüpheli koliform olarak; koyu mavi-menekşe renkli koloni oluşturanlar E. coli olarak değerlendirildi. Şüpheli koliform olarak değerlendirilen koloniler daha sonra çizgi ekim yöntemiyle 90 mm çaplı petrilere hazırlanan Yeast Extract Agar (YEA)'a alınarak, 24 saat daha 36 ˚C'de inkübasyona tabi tutuldu. Koliformlar oksidaz negatif özellik gösterdiğinden, inkübasyon sonunda öze ile üreme bölgesinden oksidaz test çubuklarına örnek alındı. Oksidaz test çubuğunu negatife döndürenler koliform olarak kabul edildi. 
Clostridium perfringens analizi için, membran filtrasyon sisteminden geçirilerek anaerobik jarda 42,5˚C'de 24 saat bekletilen Membrane Clostridium perfringens Agar (m-CP) besiyerinde üremiş şüpheli Clostridium perfringens kolonilerine, doğrulama işlemi için pastör pipeti yardımıyla petrinin kapağına amonyak solüsyonu damlatıldı. 20–30 saniye boyunca amonyum hidroksit buharına maruz bırakıldıktan sonra pembe veya kırmızı renge dönüşen opak sarı kolonilerin sayımı yapıldı.
Enterekokların analizini yapmak için Membran Filtrasyon Sistemi (MFS)'den geçirilerek Slanetz Bartley (SB) besiyerine yerleştirilen filtre kâğıdı, 36˚C'lik etüvde 48 saat inkübasyona bırakılmıştır. İnkübasyon sonucunda SB besiyerinde üreyen şüpheli Enterekok kolonileri içeren filtre kâğıdı, doğrulama işlemi için 60 mm'lik çaplı petrilere hazırlanmış Safra Eskülin Agar (SEA)'a steril pensle yerleştirilerek 42,5˚C de 2 saat boyunca inkübe edildi. SEA'da zeytin yeşiline dönüşen koloniler Enterokok kolonileri olarak kabul edildi.
P. aeruginosa analizi için 36˚C'de 48 saat inkübasyon sonunda üreme görülen CN besiyerindeki kolonilere önce UV ışık altında bakıldı. UV ışık altında floresan pigment gösteren koloniler, şüpheli P. aeruginosa olarak değerlendirilerek YEA'a çizgi yöntemiyle ekildi ve 24 saat 36˚C'de tekrar inkübasyona bırakıldı. İnkübasyon sonucunda YEA'daki üreme bölgesinden öze ile alınan örnekler, Acetamide Broth (AB) çözeltisine aktarılarak 36˚C'lik etüvde 24 saat boyunca acetamitten amonyak oluşumu için inkübasyona bırakıldı. İnkübasyon sonucunda nessler reaktifinden pastör pipetiyle birkaç damla alınarak AB'li tüplere damlatıldı. Derişime bağlı olarak sarıdan tuğla kırmızısına kadar değişen bir rengin meydana gelmesi amonyak üretiminin pozitif olduğunu gösterdiğinden, P. aeruginosa'nın bu test sonucunda petride ve örnekte bulunduğu sonucunu varıldı.
Sulardaki kimyasal kirlilik göstergeleri olan amonyak (NH₄), nitrit (NO₂), nitrat (NO₃) ve fosfat (PO₄) ölçümleri test kitleri yardımıyla spektrofotometrede yapılmıştır. pH ve iletkenlik değerleri pH metre cihazı kullanılarak ölçülmüş olup Kimyasal Oksiyen İhtiyacı (KOİ) değeri ise tesisteki cihazdan temin edilmiştir.
Alınan 12 adet numunenin mikrobiyolojik analizlerinin sonucunda koliform koloni sayısı 474000 (koloni oluşturan birim) kob/100 ml ile 17700 kob/100 ml aralığında; E. coli koloni sayısı 180000 kob/100 ml ile 4700 kob/100 ml arasında değiştiği görülmüştür. Doğada çok yaygın bulunan ve sporlarının canlılığını uzun süre sürdürebildiği fekal kontaminasyonun göstergesi olan C. perfringens'in değerlerinin 2000 kob/100 ml ile 300 kob/100 ml aralığında olduğu tespit edilmiştir. İntestinal enterekokların tespitinde ise bulunan koloni sayıları 60000 kob/100ml ile 4500 kob/100 ml aralığındadır. İnsan için fırsatçı patojen mikroorganizma olan P. aeruginosa' nın analiz sonuçlarında ise koloni sayısının 70000 kob/100 ml ile 5000 kob/100 ml aralığında değiştiği görülmüştür.
Nutrient maddelerden olan azotlu bileşiklerin ve fosfatın kimyasal ölçümlerde sınır değerlerde olduğu, iletkenlik düzeyinin 966 µS/cm ile 637 µS/cm arasında değiştiği görülmüştür. pH değerleri ise en yüksek 7,67; en düşük 6,95 olarak ölçülmüş olup evsel nitelikli atık suların parametre değerlerine uygun aralıkta bulunmuştur. Mikrobiyolojik kirliliğin daha azami seviyelere çekilerek çevre için daha korunaklı bir yapı oluşturulması için ikincil arıtıma ek olarak çeşitli dezenfeksiyon yöntemlerinin kullanılmasının yararlı olabileceği düşünülmektedir.

Abstract:
Water is one of the most essential substances for the life of living things. Cells, the smallest unit of our life, and most of the events that take place in the tissues formed by the cells and the organs formed by the tissues take place in a liquid environment, the origin of which is water.
About 0.3% of the water resources on earth are usable and drinkable. For this reason, it is of great importance that the water used is recycled and used in drinking, irrigation or industrial activities without harming the environment.
After the waste water is disposed of by sewerage, it is treated by institutions due to water efficiency, and recycling is ensured. These processes also provide a great deal of savings in the use of today's inadequate water resources. In addition, treated wastewater is seen as a resource in agricultural irrigation to feed the ever-increasing urban population.
Poor water purification is linked to the transmission of diseases such as hepatitis A, diarrhea, cholera, typhoid fever, dysentery and polio. Poor sanitation reduces human well-being, economic and social development by causing malnutrition and lost educational opportunities for children due to many neglected diseases, including intestinal worms, schistosomiasis and trachoma.
In our country, plans are being made by the Ministry of Environment, Urbanization and Climate Change in order to reuse domestic and urban treated wastewater as an alternative water source for purposes such as industrial water supply, urban irrigation, agricultural irrigation.
Physical properties such as odor, color, temperature in determining the qualities of domestic wastewater; Chemical properties such as pH, organic compounds, inorganic compounds; Biological features such as bacteria, viruses, protists, algae play a role. Eutrophication may occur in surface waters due to domestic wastewater discharged into the discharge environment without being adequately treated. Insufficient oxygen availability as a result of eutrophication negatively affects the life of aquatic organisms.
In this study, between August 2021 and April 2022, in 12 samples taken from Sakarya Karaman Wastewater Treatment Plant (KAAT) effluent using MFS, microbiological pollution indicators as coliform and fecal coliform, Escherichia coli (E.coli), fecal streptococci, Pseudomonas aeroginosa (P. aeroginosa) and Clostridium perfringens (C. perfringens) were investigated. Again, in the same samples, Ammonium (NH₄), Nitrate (NO₃), Nitrite (NO₂), Phosphate (PO₄) levels as well as pH, conductivity values were measured as chemical pollution indicators, and Chemical Oxygen Demand (COD) results were obtained from the wastewater treatment plant.
Bacteria used as indicators for faecal contamination of wastewater treated by conventional methods are total and fecal coliform bacteria. Coliform group bacteria are non-spore forming, gram negative, aerobic and facultative aerobes, producing gas and acid from lactose at 36˚C in 24-48 hours. In addition to these features, bacteria that are fecal coliform reproduce at 44˚C and produce gas and acid from lactose. Although these bacteria are found naturally in the human gut, some subtypes can threaten human health. These diseases are treated with antibiotics.
For coliform and fecal coliform analysis, filter paper placed in Chromocult Coliform Agar (CCA) medium was incubated for 24 hours at 36˚C. At the end of incubation, pinkish-red colonies forming suspicious coliforms; dark blue-violet colored colonies were evaluated as E. coli. Colonies that were considered as suspicious coliform were then transferred to Yeast Extract Agar (YEA) prepared for 90 mm diameter petri dishes by line sowing method and incubated at 36 ˚C for another 24 hours. Since the coliforms were oxidase negative, samples were taken from the growth area with loops to the oxidase test rods at the end of the incubation. Those that turned the oxidase test strip negative were considered coliform.
Since C. perfringens is a heat-resistant bacterium that can be found in spore form, its detection from water is used as an indicator of fecal pollution. They can survive in waters longer than E. coli and Enterococci. Unlike most Clostridium species, C. perfringens can reproduce anaerobically at 44˚C.
For Clostridium perfringens analysis, suspected Clostridium perfringens colonies grown on Membrane Clostridium perfringens Agar (m-CP) medium, which were kept in an anaerobic jar at 42.5˚C for 24 hours after being passed through a membrane filtration system, were dripped with ammonia solution on the petri dish with the help of a pasteur pipette for confirmation. Opaque yellow colonies that turned pink or red after exposure to ammonium hydroxide vapor for 20–30 seconds were counted.
All species known as Enterecoccus genus are heated to 60˚C for 30 minutes. They provide the growth criteria at pH 9.6 and 6.5% sodium chloride (NaCl), which can withstand the main growth at 10˚C to 45˚C. They live longer than E. coli and are more resistant to environmental conditions and chlorination. Enterococci are one of the indicators of fecal contamination in water, such as E. coli.
In order to analyze enterococci, filter paper was placed in Slanetz Bartley (SB) medium by passing through Membrane Filtration System (MFS) and incubated for 48 hours in a 36˚C oven. As a result of the incubation, filter paper containing suspicious Enterococcal colonies grown in SB medium was placed in Bile Esculin Agar (SEA) prepared in 60 mm diameter petri dishes with sterile forceps and incubated at 42.5˚C for 2 hours. Colonies that turned olive green in SEA were considered Enterococcal colonies.
P. aeruginosa is among the facultatively multiplying bacteria classified as gram negative. It has the ability to oxidize some carbohydrates, such as galactose, but cannot ferment it. Although the optimum growth temperature is 36˚C, it has been observed that this temperature can be up to 41˚C in the laboratory environment.

For the analysis of P. aeruginosa, colonies on CN medium that showed growth after 48 hours of incubation at 36˚C were first examined under UV light. Colonies that showed fluorescent pigment under UV light were evaluated as suspicious P. aeruginosa and seeded on YEA by streaking method and incubated again at 36˚C for 24 hours. As a result of the incubation, the samples taken from the growth area in YEA were transferred to Acetamide Broth (AB) solution and incubated for 24 hours in a 36˚C oven for the formation of ammonia from acetamide. At the end of the incubation, a few drops of Nesler's reagent were taken with a pasteur pipette and dropped into tubes with AB. It was concluded that P. aeruginosa was found in the petri dish and the sample as a result of this test, since a color ranging from yellow to brick red depending on the concentration indicates positive ammonia production.
Measurements of ammonia (NH₄), nitrite (NO₂), nitrate (NO₃) and phosphate (PO₄), which are indicators of chemical pollution in water, were made in a spectrophotometer with the help of test kits. The pH and conductivity values were measured using a pH meter device, and the Chemical Oxygen Demand (COD) value was obtained from the device in the facility.
As a result of microbiological analysis of 12 samples taken, the number of coliform colonies ranged from 474000 (colony forming unit) cfu/100 ml to 17700 cfu/100 ml; It was observed that the number of E. coli colonies varied between 180000 cfu/100 ml and 4700 cfu/100 ml. It has been determined that the values of C. perfringens, which is very common in nature and is an indicator of fecal contamination, whose spores can sustain their viability for a long time, are between 2000 cfu/100 ml and 300 cfu/100 ml. Colony numbers in the detection of intestinal enterococci range from 60000 cfu/100 ml to 4500 cfu/100 ml. In the analysis results of P. aeruginosa, which is an opportunistic pathogenic microorganism for humans, it was observed that the number of colonies varied between 70000 cfu/100 ml and 5000 cfu/100 ml.
It has been observed that nitrogenous compounds and phosphate, which are nutrient substances, are at limit values in chemical measurements, and the conductivity level varies between 966 µS/cm and 637 µS/cm. The highest pH values are 7.67; the lowest was measured as 6.95 and it was found in the appropriate range for the parameter values of domestic wastewater. It is thought that it may be useful to use various disinfection methods in addition to secondary treatment in order to know how to create a more sheltered structure for the environment by maximizing microbiological pollution.