Bisbal’un Streptococcus Pyogenes Bakterisi Üzerine İnhibisyon Etkisi Ve Bisbal İçeren Membranlar İle Aktif Çamur Filtrasyonu
Bisbal’un Streptococcus Pyogenes Bakterisi Üzerine İnhibisyon Etkisi Ve Bisbal İçeren Membranlar İle Aktif Çamur Filtrasyonu
thumbnail.default.placeholder
Tarih
2014-02-26
Yazarlar
Durmaz, Gamze
Süreli Yayın başlığı
Süreli Yayın ISSN
Cilt Başlığı
Yayınevi
Fen Bilimleri Enstitüsü
Institute of Science and Technology
Institute of Science and Technology
Özet
Nüfusun her geçen gün hızla artmasına karşı su kaynaklarının giderek azalması sebebiyle su ihtiyacı giderek artmaktadır. Su kaynaklarını korumak, kalitesinin arttırmak ve artan su talebini karşılamak; su arıtma sistemlerin gelişiminin artmasına neden olmaktadır. Bu sebeple günümüzde geliştirilen arıtma teknolojileri ve metotları içerisinde yeni membranların geliştirilmesi, üretimi ve kullanımı çalışmaları artmaktadır. Membran biyoreaktörler konvansiyonel arıtma sistemleriyle kıyaslandığında yüksek kaliteli çıkış suyu, az alan ihtiyacı, ucuz işletme maliyeti, modül şeklinde kullanımı, yüksek konsantrasyonlara uygulanabilirliği ve taşınabilir olması gibi avantajlara sahip yeni arıtma sistemlerinden biridir. Fakat bu avantajlarına rağmen membran biyoreaktörlerinin sık kullanılmamasının en büyük sebebi membran kirlenmeleridir. Bu çalışmada bizmut elementinin lipofilik tiyol bileşikleri kombinasyonu sonucunda elde edilen etkili antibakteriyel özelliğinden faydalanılarak, membran biyokirliliğini engellemedeki etkinliğinin araştırılması amaçlanmaktadır. Membran kirlenmesi üzerine yapılan araştırmalara bakıldığında SMP (çözünmüş mikrobiyal ürünler) ve EPS’lerin (hücredışı polimerik maddeler) membranlar için temel kirletici kaynakları olduğu görülmektedir (Chang ve diğ., 2002). SMP çözünür hücresel madde metabolizması sırasında ortaya çıkan bileşenler ve benzerleri olarak tanımlanmıştır. EPS ise proteinler, karbonhidratlar, nükleik nasitler, lipidler ve humik maddelerden oluşur ve çözünebilir makro-moleküller ve kolloidler olarak bilinir (Laspidou & Rittmann, 2000). EPS bakterilerin membran yüzeyine tutunmasına neden olduğundan membranlarda biyofilm oluşumuna neden olurlar (Codony & Dominico, 2003). Membranların en önemli sorunu biyofilm oluşmasıdır. Biyofilmler düşük besin ve dezinfeksiyon koşullarına adapte olmuş çeşitli mikrobiyal topluluklardır. Genel olarak; biyofilmlerin özellikleri seyrek olarak kolonize olmuş yüzeylerden, hücre dışı polimerlerin iç içe geçmiş yapısından, mikrobiyal hücreler ve iyon kanallar tarafından oluşturulan çeşitli µm derinlikteki kalın kompleks katmanlara kadar büyük farklılıklar gösterir (LeChevallier, 1987; Stewart, 1993). Antibakteriyel metaller artık antibakteriyel amaçlı direkt yüzey olarak değil, çeşitli malzemelerin bünyesinde partiküler olarak yer almaktadırlar. Bu maddeleri seçmekteki neden bu maddelerin ucuz ve üretimde etkili olmasıdır. Bizmut tiyoller en önemli ve bilinen antibiyotiklerdir. EPS üretimini azaltıp, biyofilm oluşumunu engellerler (Dominico ve diğ., 2009). Yapılan çalışmalarda BisBAL (bizmut-2-3-dimerkapto-1-propanol) adı verilen bizmutun dimerkaptopropanol ile yapmış olduğu şelatın Staphylococcus, Klebsiella ve Pseudomonas gibi bakteriler tarafından salgılanan hücredışı polimerik maddelerin yani EPS’lerin hem polisakkarit hem de protein üretimlerini inhibe ettiği böylece membranlarda biyofilm oluşumunu azaltırken, membranların kullanım ömürlerini arttırdığı görülmüştür. BisBAL, laboratuvarda üretilen bir bizmut tiyol grubu olup, diğer üretilen inorganik bizmut bileşiklerinden 1000 kat daha fazla antibakteriyel aktivitesi olan bir bileşiktir. BisBAL saf bizmut nitrata ve benzer bileşiklere oranla 300 kat daha fazla antibakteriyel özelliğe sahiptir (Domenico P. T. J., 1999). Bu çalışmada BisBAL (bizmut-2-3-dimerkapto-1-propanol) adı verilen bizmutun dimerkaptopropanol ile yapmış olduğu şelatın Streptococcus pyogenes bakterisi tarafından salgılanan hücredışı polimerik maddelerin yani EPS’lerin hem polisakkarit hem de protein üretimleri üzerine İnhibisyonu ve BisBAL içeren membranların aktif çamur ile filtrasyonu gerçekleştirilerek performansları araştırılmıştır. İnhibisyon çalışmaları için Streptococcus pyogenes saf bakteri kültürü İstanbul Tıp Fakültesine bağlı Mikroorganizma Kültür Kolleksiyonları Araştırma ve Uygulama Merkezi (KÜKENS)’nden alınmıştır. BisBAL sentezi aşamasında 25 ᵒC ve 45 ᵒC sıcaklığı, 4-7-10 pH ve 1-2-3 Bis:BAL molar oran parametrelerine göre üretimleri yapılmış ve toplamda 18 farklı BisBAL çeşidi elde edilmiştir. İnhibisyon konsantrasyonu belirlenirken 5, 12, 15, 20, 30 ve 40 μM BisBAL konsantrasyonlarında çalışılmıştır. 5’er ml’lik sıvı besiyerlerine önce seyreltilmiş bakteri kültürü daha sonra 5, 12, 15, 20, 30 ve 40 µM’lık BisBAL konsantrasyonuna sahip olacak şekilde BisBAL eklenerek 37 °C de inkübatörde 1 gün inkübe edilerek numunelerin spektrofotometre cihazında 420 ve 570 nm dalga boylarında optik yoğunlukları (OD) ölçülmüştür. İnhibisyon sonuçlarına göre en etkili ve en iyi giderim verimi 25ᵒC’de pH 4’de, 1:1 Bis:BAL molar oranında 15 µM BisBAL içeren konsantrasyonda elde edilmiştir. İnhibisyon çalışmaları sonrası %16 ve %20 PES ve PSf içerikli 15 µM konsantrasyonuna sahip BisBAL’lu ve BisBAL’suz membran üretimi yapılmış ve aktif çamur filtrasyonu gerçekleştirilmiştir. Aktif çamur kullanılarak yapılan filtrasyon deneyleri sonucunda %16 PES (Polietersülfon) ve PSf (Polisülfon) içerikli membranlarda 15 µM konsantrasyonlarına sahip BisBAL’lu membranların biyokirlenmeyi azalttığı görülmüştür.
Water and wastewater treatment have been gaining importance day by day along with developments in industry, increasing in population and decreasing at natural sources. A lots of water and wastewater treatment systems have been developed and many researches have been continuing to develop new systems. Membrane bioreactors (MBRs) are one of the newest important treatment technologies to treat water and wastewater. MBRs are not widespread used comparing to conventional treatment systems despite of providing high quality effluent water, lower first investment and cheaper operational costs. The one of the main reason not using membranes is pollution of membranes. Pollution of membrane decreases flowrate and increases maintanence and operational coasts because of cleaning and need of chemical backwashing of membranes. The reason of limited usage of membranes todays is fouling of membrane. Sistematical approach on researching and identifiying parameters affecting membrane pollution are very important. High percantages of the MBR pollution studies showed that fundamental parameters fouling membranes are to soluble microbial products (SMP) and extracellular polymeric substances (EPS). Many parameters such as operational ones (hydrolic retention time, sludge age, organic loading rate etc.), membrane specifications (hydrophilicite, surface roughness, surface charge etc.), and handling of membranes with different chemical agents (polyelectrolyte, nanaoparticles, antibiotics etc.) are being investigated in order to decrease SMP and EPS effects on membrane fouling. The reserchers have evidence that bismuth dimercaptopropanole inhibit EPS synthesis both protein and carbohydrate fractions which release from gram positive and gram negative bacteria. The main goal of this thesis is to develop effective and new methods to prevent fouling of the membranes which are using for water and wastewater treatment. However, elemental bismuth chelated with lipophilic thiols exhibits antimicrobial properties even at very low concentrations and these unique properties will be used for preventing of membrane biofouling. The membranes which will be both synthesized and purchased commercially will be coated different methods and tested to inhibit SMP and EPS synthesis. Therefore the membrane’s life will increase. The most important problem of membrane is biofilms. Biofilms are microbial communities adapted to the conditions of low nutrient and disinfection. In general, biofilms features rarely colonized surfaces, the nested structure of the extracellular polymers, ion channels formed by the microbial cells and the complex layers of thick as large as the depth varies µm (LeChevallier, 1987; Stewart, 1993). Antibacterial metals for use directly as a surface, but as the particles are located within the various materials. The reason for choosing this material is cheaper and more efficient in production. Bismuth also used for medical treatments throughout history. Bismuth thiols are one of the most important and well known antibiotics. They are able to reduce production of EPS (extracellular polymeric substance) and biofilm formation. Concentrations of 0.6 to 1 ppm Bisbal capsule is known to inhibit the formation of biofilm 70-90%. It is safer and less toxic than other antibacterial products like silver (Domenico, 2009; Bierer, 1990 and Marshall, 1991). Biological treatment processes, with the increase of the biological activity of extracellular polymeric substances (EPS) and soluble microbial products (SMP) was increased (Chang, 2002, and Nagaoka, 1998). BisBAL, can greatly reduce rates of EPS substances secreted by bacteria such as; Staphylococcus, Klebsiella and Pseudomonas, thus reducing the formation of biofilm membranes increases the life span of membranes in the MBR (Badireddy et al., 2008). Within the scope of this study, optimization of BisBAL synthesis, inhibition tests on bacteria of BisBAL and manufacturing of antifouling polyethersulfone flat sheet membrane were done. For the optimization of the synthesis conditions of BisBAL; temperature, pH and Bis:BAL molar ratio were chosen as three different parameters. At room temperature (25 ᵒC) and high temperature conditions (45 ᵒC) BisBALs were synthesized. pH 4, 7 and 10 values were selected in order to represent the acidic, neutral and basic conditions respectively for the optimization of the pH. Another parameter which is effective in the inhibition of is ratio of bismuth and BAL. In order to optimize the molar ratio Bis:BAL, 3:1, 2:1 and 1:1 ratios were selected. BisBAL was synthesized by addition of bismuth nitrate pentahydrate (Bi(NO3).5H2O) solved in propylene glycol (C3H8O2) to 2,3-dimercapto-1-propanol. Stock solution was first prepared for the synthesis and then adjustments were made with 1 N NaOH addition and temperature adjustments were provided by using heating stirrer constantly. 18 combinations of BisBAL were synthesized. Streptococcus pyogenes was taken from “Microorganism Culture Collection Research and Application Center (KÜKENS) of Turkey”. Pure culture bacteria species were incubated at 37 °C in Standard Plate Medium. Activated sludge was taken from an advanced wastewater biological treatment plant in Istanbul. BisBAL inhibition tests on S. pyogenes and activated sludge had been run with different molar concentrations (5, 12, 15, 20, 30 and 40 µM). Inhibition experiments were carried out in 5 mL liquid media. Autoclaved medium was put in sterile tubes, inoculated with bacteria and various molar concentrations of BisBAL in order to see the effects on bacteria were added with sterile automatic pipettes. After 24 hour and 48 hour incubation of bacteria with BisBAL in broth medium by using Heraeus incubator, optical densities of samples were analyzed at 420 and 570 nm wavelengths. EPS and SMP extractions were done by using ultracentrifugation (15.000 and 9.000 ppm respectively). Protein analyses were run according to Lowry method and carbohydrate analyses were performed by using the phenol-sulfuric acid method (Dubois). 14 different membranes were casted. Polyethersulfone was used as polymer and PVP as solvent. Two different PES ratios were selected: 16% and 20%. PVP ratio was 8%. BisBAL additions were done according to the 15 and 30 µM. Activated Sludge with 4000 mg/L VSS was filtered by StarliTech Filtration system. After all inhibition experiments were performed, the best combination for BisBAL was chosen and synthesized. With these molar concentrations, EPS and SMP can be decreased up to 98% for both bacteria species. By using the membrane manufactured with BisBAL addition to polymer solution (PES as polymer and Polyvinylpyrrolidone (PVP) as solvent), flux value was increased from 200 L/m2/h to 400 L/m2/h and 500 L/m2/h for 15 µM and 30 µM BisBAL including membranes, respectively. Furthermore, change of inhibition effect of BisBAL on the bacteria with time has been examined. Effect had slightly been decreased during first 3 weeks and after 26 days from synthesis, minimum inhibition effect was seen and it was 30% less than 1th day effect. However, as long as 4 months inhibition effect had regularly been increased and maintain an effective inhibition. Experiments have been performed on S. pyogenes, and on activated sludge sample taken from an advanced domestic biological wastewater treatment plant. Minimum inhibitory concentration (MIC) values were found and membranes were synthesized according to these MIC values with small amounts of BisBAL. It was achieved to the target of decreasing of cake forming and biofouling and increasing flux values during activated sludge filtration by using membranes synthesized with the BisBAL. These are new and improving result for the prevention of cake formation in MBR systems.
Water and wastewater treatment have been gaining importance day by day along with developments in industry, increasing in population and decreasing at natural sources. A lots of water and wastewater treatment systems have been developed and many researches have been continuing to develop new systems. Membrane bioreactors (MBRs) are one of the newest important treatment technologies to treat water and wastewater. MBRs are not widespread used comparing to conventional treatment systems despite of providing high quality effluent water, lower first investment and cheaper operational costs. The one of the main reason not using membranes is pollution of membranes. Pollution of membrane decreases flowrate and increases maintanence and operational coasts because of cleaning and need of chemical backwashing of membranes. The reason of limited usage of membranes todays is fouling of membrane. Sistematical approach on researching and identifiying parameters affecting membrane pollution are very important. High percantages of the MBR pollution studies showed that fundamental parameters fouling membranes are to soluble microbial products (SMP) and extracellular polymeric substances (EPS). Many parameters such as operational ones (hydrolic retention time, sludge age, organic loading rate etc.), membrane specifications (hydrophilicite, surface roughness, surface charge etc.), and handling of membranes with different chemical agents (polyelectrolyte, nanaoparticles, antibiotics etc.) are being investigated in order to decrease SMP and EPS effects on membrane fouling. The reserchers have evidence that bismuth dimercaptopropanole inhibit EPS synthesis both protein and carbohydrate fractions which release from gram positive and gram negative bacteria. The main goal of this thesis is to develop effective and new methods to prevent fouling of the membranes which are using for water and wastewater treatment. However, elemental bismuth chelated with lipophilic thiols exhibits antimicrobial properties even at very low concentrations and these unique properties will be used for preventing of membrane biofouling. The membranes which will be both synthesized and purchased commercially will be coated different methods and tested to inhibit SMP and EPS synthesis. Therefore the membrane’s life will increase. The most important problem of membrane is biofilms. Biofilms are microbial communities adapted to the conditions of low nutrient and disinfection. In general, biofilms features rarely colonized surfaces, the nested structure of the extracellular polymers, ion channels formed by the microbial cells and the complex layers of thick as large as the depth varies µm (LeChevallier, 1987; Stewart, 1993). Antibacterial metals for use directly as a surface, but as the particles are located within the various materials. The reason for choosing this material is cheaper and more efficient in production. Bismuth also used for medical treatments throughout history. Bismuth thiols are one of the most important and well known antibiotics. They are able to reduce production of EPS (extracellular polymeric substance) and biofilm formation. Concentrations of 0.6 to 1 ppm Bisbal capsule is known to inhibit the formation of biofilm 70-90%. It is safer and less toxic than other antibacterial products like silver (Domenico, 2009; Bierer, 1990 and Marshall, 1991). Biological treatment processes, with the increase of the biological activity of extracellular polymeric substances (EPS) and soluble microbial products (SMP) was increased (Chang, 2002, and Nagaoka, 1998). BisBAL, can greatly reduce rates of EPS substances secreted by bacteria such as; Staphylococcus, Klebsiella and Pseudomonas, thus reducing the formation of biofilm membranes increases the life span of membranes in the MBR (Badireddy et al., 2008). Within the scope of this study, optimization of BisBAL synthesis, inhibition tests on bacteria of BisBAL and manufacturing of antifouling polyethersulfone flat sheet membrane were done. For the optimization of the synthesis conditions of BisBAL; temperature, pH and Bis:BAL molar ratio were chosen as three different parameters. At room temperature (25 ᵒC) and high temperature conditions (45 ᵒC) BisBALs were synthesized. pH 4, 7 and 10 values were selected in order to represent the acidic, neutral and basic conditions respectively for the optimization of the pH. Another parameter which is effective in the inhibition of is ratio of bismuth and BAL. In order to optimize the molar ratio Bis:BAL, 3:1, 2:1 and 1:1 ratios were selected. BisBAL was synthesized by addition of bismuth nitrate pentahydrate (Bi(NO3).5H2O) solved in propylene glycol (C3H8O2) to 2,3-dimercapto-1-propanol. Stock solution was first prepared for the synthesis and then adjustments were made with 1 N NaOH addition and temperature adjustments were provided by using heating stirrer constantly. 18 combinations of BisBAL were synthesized. Streptococcus pyogenes was taken from “Microorganism Culture Collection Research and Application Center (KÜKENS) of Turkey”. Pure culture bacteria species were incubated at 37 °C in Standard Plate Medium. Activated sludge was taken from an advanced wastewater biological treatment plant in Istanbul. BisBAL inhibition tests on S. pyogenes and activated sludge had been run with different molar concentrations (5, 12, 15, 20, 30 and 40 µM). Inhibition experiments were carried out in 5 mL liquid media. Autoclaved medium was put in sterile tubes, inoculated with bacteria and various molar concentrations of BisBAL in order to see the effects on bacteria were added with sterile automatic pipettes. After 24 hour and 48 hour incubation of bacteria with BisBAL in broth medium by using Heraeus incubator, optical densities of samples were analyzed at 420 and 570 nm wavelengths. EPS and SMP extractions were done by using ultracentrifugation (15.000 and 9.000 ppm respectively). Protein analyses were run according to Lowry method and carbohydrate analyses were performed by using the phenol-sulfuric acid method (Dubois). 14 different membranes were casted. Polyethersulfone was used as polymer and PVP as solvent. Two different PES ratios were selected: 16% and 20%. PVP ratio was 8%. BisBAL additions were done according to the 15 and 30 µM. Activated Sludge with 4000 mg/L VSS was filtered by StarliTech Filtration system. After all inhibition experiments were performed, the best combination for BisBAL was chosen and synthesized. With these molar concentrations, EPS and SMP can be decreased up to 98% for both bacteria species. By using the membrane manufactured with BisBAL addition to polymer solution (PES as polymer and Polyvinylpyrrolidone (PVP) as solvent), flux value was increased from 200 L/m2/h to 400 L/m2/h and 500 L/m2/h for 15 µM and 30 µM BisBAL including membranes, respectively. Furthermore, change of inhibition effect of BisBAL on the bacteria with time has been examined. Effect had slightly been decreased during first 3 weeks and after 26 days from synthesis, minimum inhibition effect was seen and it was 30% less than 1th day effect. However, as long as 4 months inhibition effect had regularly been increased and maintain an effective inhibition. Experiments have been performed on S. pyogenes, and on activated sludge sample taken from an advanced domestic biological wastewater treatment plant. Minimum inhibitory concentration (MIC) values were found and membranes were synthesized according to these MIC values with small amounts of BisBAL. It was achieved to the target of decreasing of cake forming and biofouling and increasing flux values during activated sludge filtration by using membranes synthesized with the BisBAL. These are new and improving result for the prevention of cake formation in MBR systems.
Açıklama
Tez (Yüksek Lisans) -- İstanbul Teknik Üniversitesi, Fen Bilimleri Enstitüsü, 2014
Thesis (M.Sc.) -- İstanbul Technical University, Institute of Science and Technology, 2014
Thesis (M.Sc.) -- İstanbul Technical University, Institute of Science and Technology, 2014
Anahtar kelimeler
BisBAL,
EPS,
SMP,
Streptococcus pyogenes,
Membran,
biyotıkanma