Turk J Biochem 2018; 43(3): 296–305 Research Article Melek Özkan*, Hilal Yılmaz, Merve Akay Çelik, Çişel Şengezer, Elif Erhan and Bülent Keskinler Application of Bdellovibrio bacteriovorus for reducing fouling of membranes used for wastewater treatment Atıksu arıtımında kullanılan membranların kirlenme probleminin azaltılması için Bdellovibrio bacteriovorus uygulaması https://doi.org/10.1515/tjb-2016-0302 showed that Alfa-proteobacteria are the most numerous Received April 28, 2016; accepted September 1, 2016; previously bacteria among the biofilm community on the membrane published online March 29, 2018 surface. Abstract Conclusion: Results suggested that cleaning of MBR mem- branes with B. bacteriovorus has a potential to be used as Background: Membrane bioreactor (MBR) systems used a biological cleaning method. for wastewater treatment (WWT) processes are regarded Keywords: Bdellovibrio bacteriovorus; Biofilm; Membrane as clean technologies. Degradation capacity of the preda- fouling; Wastewater treatment; Membrane bioreactor tor bacterium, Bdellovibrio bacteriovorus, was used as a (MBR) systems. cleaning strategy for reducing membrane fouling. Method: Wastewater with different sludge age and hydraulic retention time were filtered through Poly(ether) Özet sulphone (PES) membranes using dead end reactor. Change in filtration performance after cleaning of mem- Genel bilgi: Atıksu arıtımı için kullanılan membran biyore- brane surface by B. bacteriovorus was measured by com- aktör (MBR) sistemleri temiz teknolojiler olarak bilinmek- parison of flux values. Bacterial community of the sludge tedirler. Membran yüzeyinde biriken mikroorganizmalar was determined by 16SrRNA sequence analysis. Commu- nedeniyle membranın kirlenmesi MBR sistemlerin en önemli nity profile of membrane surface was analyzed by fluores- problemidir ve membranın kullanım ömrünün kısalmasına cent in situ hybridization technique. neden olmaktadır. Bdellovibrio bacteriovorus membranın Results: After cleaning of MP005 and UP150 membranes tıkanmasının temel nedeni olan biyofilm komunitedeki with predator bacteria, 4.8 L/m2 · h and 2.04 L/m2 · h hücre dışı polimerik maddelerin başlıca üreticilerinden olan increase in stable flux at steady state condition was Gram negatif bakterileri yiyerek beslenen avcı bir bakteridir. obtained as compared to the control, respectively. Aero- Amaç: Bu çalışmada avcı bakteri B. bacteriovorus ’un monas, Proteus, and Alcaligenes species were found to membranın tıkanma probleminin azaltılması amacıyla be dominant members of the sludge. Bdellovibrio bacte- kullanım potansiyeli araştırılmıştır. riovorus lysed pure cultures of the isolated sludge bacte- Yöntem: Farklı çamur yaşı ve hidrolik bekleme süre- ria successfully. FISH analysis of the membrane surface sine sahip atıksular Ölü Üç Reaktör hücresi kullanılarak Polietersülfon (PES) membranlardan filtre edilmişlerdir. *Corresponding author: Melek Özkan, Gebze Technical University, Bdellovibrio bacteriovorus ile temizleme işleminden sonra Environmental Engineering Department, Kocaeli, Turkey, filtrasyon performansındaki değişim membran akılarının e-mail: [email protected] kıyaslanması ile ölçülmüştür. Aktif çamurun bakteri Hilal Yılmaz, Merve Akay Çelik, Çişel Şengezer, Elif Erhan and Bülent Keskinler: Gebze Technical University, Environmental profili 16SrRNA dizi analizi ile belirlenmiştir. Membran Engineering Department, Kocaeli, Turkey, yüzeyindeki biyofilm komunitesi Floresan in Situ Hibridi- e-mail: [email protected] (H. Yılmaz) zasyonu tekniği ile analiz edilmiştir. Melek Özkan et al.: Application of Bdellovibrio bacteriovorus for reducing fouling of membranes 297 Bulgular: MP005 ve UP150 membranlarının avcı bakteri flushing (backwashing), a physical cleaning method, ile temizlenmesi sonucunda kontrol membranına kıyasla has been applied on fouled membranes in many cases. kararlı akıda sırasıyla 4,8 L/m2 · s ve 2,04 L/m2 · s ortalama This method successfully removes most of the revers- artış tespit edilmiştir. Aeromonas, Proteus, ve Alcalige- ible fouling due to pore blocking, and partially dislodges nes türlerinin çamurun baskın üyeleri olduğu görülmüş- loosely attached sludge cake from surface [5]. Respectively, tür. Bdellovibrio bacteriovorus’un izole edilen saf bakteri newer treatment method, air flushing (air sparging), is kültürlerini başarılı bir şekilde parçaladığı gözlenmiştir. another physical cleaning concept where air is used as a Membran yüzeyinin FISH analizi Alfa-proteobakterilerin back flushing medium. During the filtration process, depo- membran yüzeyinde çoğunlukta olduğunu göstermiştir. sition of particles and bacterial cells causes forming a slimy Tartışma ve Sonuç: Bdellovibrio bacteriovorus MBR sis- gel layer on the membrane surface that cannot be removed temlerin temizilenmesinde biyolojik temizleme ajanı ola- easily by physical cleaning methods. Different types of rak kullanım potansiyeline sahiptir. chemical cleaning methods may be recommended on a weekly/monthly/yearly basis to increase the membrane Anahtar kelimeler: Atıksu arıtımı; Bdellovibrio bacterio- life by removing the irreversible fouling which cannot be vorus; Biyofilm; Membran kirlenmesi; Membran Bioreak- removed by physical cleaning methods [6] Chemicals like tör (MBR) Sistemleri. HCl, HNO3, NaOCl, H2O2, citric acid have been employed on membranes to remove organic and inorganic fouling [7]. Biological control strategies are still at research and devel- Introduction opment stage. Inhibition of quorum sensing system, nitric oxide-induced biofilm dispersal, enzymatic disruption of Domestic and industrial wastewater discharges and the extracellular polysaccharides, proteins, and DNA, inhibi- contaminated rainwater run-off cause significant unfavora- tion of microbial attachment by energy uncoupling, use of ble impacts on the environment. There are many different cell wall hydrolases, and disruption of biofilm by bacte- technologies for wastewater treatment (WWT) including riophages can be listed under biological ways of microbial physico-chemical and/or microbial ones [1]. Membrane attachment control strategies [8]. bioreactors (MBRs) are now broadly favorable treatment WWT with MBRs can be regarded as clean technology technologies that combines membrane processes and sus- since it replaces secondary settlement tank. However, pended growth bioreactors [2]. MBR application in WWT harmful chemical agents are introduced to the environment is an activated sludge process serving as a replacement for during membrane cleaning process. Therefore, potential secondary settlement tanks. Because of more stringent dis- of biological ways of membrane cleaning methods should charge regulations, water reuse initiatives and decrease in be introduced for the aim of clean environment. Biofilms membrane cost, MBRs have gained increasing popularity. causing membrane fouling consist of multiple bacterial In MBR systems, membrane fouling is one of the most species including Gram-negative ones. Some previous important problems during operation which leads to a fre- researches have revealed that Bdellovibrio can consume quent cleaning and/or replacement of membranes, and Escherichia coli prey in simple biofilms, in some cases increases operation cost [3]. As soon as the membrane destroying the biofilms altogether [9]. Bdellovibrio bacte- surface contact with wastewater, deposition of particles riovorus is a Gram-negative, obligately aerobic bacterium and bacterial cells occurs onto membrane surface. The that preys upon a wide variety of other Gram-negative bac- microorganism and their products contribute to mem- teria [9]. Bdellovibrio life cycle consists of an attack phase brane fouling. As a dynamic process, biofouling occurs as cell that attaches to other Gram-negative bacteria, pene- a result of two main mechanisms: colonization of mem- trates their periplasm, multiplies in the periplasmic space brane surfaces with microorganisms and production of and finally bursts the cell envelope to start the new cycle. membrane foulants by microorganisms in the waste water Depending on the prey and environment, this life cycle [2]. Although this fouling and biofilm formation on mem- takes roughly 3–4 h. Dashiff et al. [10] showed lysis activ- brane surface enhances the removal of microbes by acting ity of B. bacteriovorus on biofilms formed by different bac- as a secondary barrier, it also reduces the flux and per- terial species, especially pathogenic ones. Although the meability of the membranes [4]. Thus, fouling needs to be main role of B. bacteriovorus in nature is not completely kept under control in order to increase the membrane life- known, there is evidence that these bacteria play a role in time and decrease the operational costs. the microbial ecology of natural environments, controlling In order to decrease the fouling problem, physical the population size of bacterial ecosystems. It is known and chemical cleaning methods are frequently used. Back that B. bacteriovorus efficiently degrades biofilms formed 298 Melek Özkan et al.: Application of Bdellovibrio bacteriovorus for reducing fouling of membranes by many Gram-negative bacteria including Aeromonas, plates. Wastewater from bench scale activated sludge Citrobacter, Enterobacter, Escherichia, Klebsiella, Proteus, reactor fed in laboratory was used for biofilm formation. Pseudomonas, Salmonella etc. [10]. Most of those bacteria Large solid particles of wastewater sludge was eliminated are members of wastewater sludge which contribute plug- by precipitation