Isolation of novel Quorum quenching bacteria for the control of membrane biofouling and the effect of Bacillus sp. T5 and Delftia sp. T6 on microbial community structure in membrane bioreactor

The outcomes of the increase in the World population and industrialization, waste discharging to non-receipt bodies or an insufficient treatment cause accumulation and rapid decrease in natural resources day by day. The shrinkage of these resources results in significant environmental issues. To overcome the water scarcity, there is a global effort to expand on water resources and reuse of wastewater. Membrane Bioreactor (MBR) technology is an advanced biological wastewater treatment technology. Nevertheless, biofouling on membrane surface remains one of the main obstacles to profitable MBR operation. Quorum Quenching (QQ) is an innovative approach attempting to solve the biofouling problem in MBRs. QQ applications prevent the group behavior of microorganisms therefore ceases/retards the formation of biofilm. Researchers have demonstrated that Quorum sensing and biofilm formation are strongly linked and act to regulate the biofilm formation on the membrane surface in MBR. C6-HSL and C8-HSL, 3-oxo-C8-HSL C4-HSL, C6-HSL, C8-HSL, 3-oxo-C8-HSL, C10- HSL, C12-HSL, 3-oxo-C12-HSL and C14-HSL have been detected from biofilm on the membrane surfaces and in the mix liquor of. Because biofilm formation on membranes can be controlled by QS, several studies have turned their attention to blocking AHL signals to either reduce membrane biofouling or improve reactor performance. In the last 10 years, research on QQ mediated strategies for mitigating membrane biofouling in MBRs has intensified and notable success has been achieved in this regard. Current thesis aims to isolate new and highly effective QQ bacteria from our national resources and to understand what are the Achyl-homoserine lactone (AHL) degradation pathways of the isolated new bacterial strains found in marine, pond and saltern environments that are capable of interfering with AHL-mediated Quorum Sensing. This study also evaluates applying the selected new QQ bacteria to the MBRs with QQ-fiber/beads for the purpose of controlling biofilm formation. Finally assess the interaction between the indigenous microbial community and two different QQ bacteria, Bacillus sp. T5 (Gram positive) and Delftia sp. T6 (Gram negative), in order to develop a comprehensive understanding of occurrence of QQ mechanisms in MBR step by step. A BLAST search of GenBank was performed to identify if there was any taxonomical relationship between the sequences available in the database and those of the 16S rRNA gene of the 25 new isolates. New isolates belonged predominantly to Gammaproteobacteria (G2, G4, T21, T22, T4, H1, H2, H3, H4, H7, H8, H9, H10), Betaproteobacteria (G6, T6, H5), Firmicutes (G5, T5), and Actinobacteria (G7). The assessment of the partial nucleotide sequence of the amplified 16S rRNA gene revealed a strong homology of 97-100 between this sequence and that of the bacteria from the following genera: Shewanella, Acinetobacter, Klebsiella, Bacillus, Deftia, Vibrio, Comamonas, Microbacterium, and Pseudomonas, Brevundimonas, Bosea, Stenotrofomonas and Tistrella. Minimal medium method was used which employs C8-HSL as the sole carbon source. AHL degradation activity was measured via Biotest. The activity test results indicated that Tistrella exhibited QQ activities for the first time, and strains T21, T22, T4, G2, G4 G5, G6, H1, H3, H7, H8, H10 presented lactonase activity which has not been recorded in the previous research. Strain T5 and T6 from saltern and strain S5 and S10 from leachate degraded more than 90% of the AHL, thus each of them exhibits significant AHL activity. Results point that QQ is the strategy that may typically be adopted in the different environmental sources to overcome stress conditions and achieve competitive advantages. After isolation of the QQ bacteria, a new immobilization medium named QQ-fiber was produced by hollow fiber membranes. MBRs was operated with QQ-fiber immobilized with Bacillus sp. T5 and biofilm formation was successfully mitigated with 25 % efficiency. To compare the immobilizations media and two different QQ bacteria, sodium alginate beads entrapped with chosen strains Bacillus sp. T5 and Delftia sp. T6. Total anti-biofouling effects of T5 and T6 beads were calculated as 85% and 75%, respectively. One of the major findings of the thesis was bacterial community changes during the QQ-MBR runs with QQ bacteria which investigated using metagenomic analysis. The effect of QQ on the bacterial community was examined in detail on the basis of phylum, class and genus. The gap in the understanding of bacterial community dynamics in MBRs was addressed by investigating the behavior of two types of QQ bacteria, Bacillus sp. T5 and Delftia sp. T6, within the control and QQ MBRs via Illumina's HiSeq method using 16S rRNA genes. The analysis of the results and comparison of relative abundances of taxa in MBR operations were revealed that different QQ bacteria have an effect on different taxa. Proteobacteria, Bacteroidetes, Chloroflexi, Firmicutes, Acidobacteria and Actinobacteria were most QQ affected phyla in the microcosms. Furthermore, when the QQ MBR was operated with gram-positive T5, a reduction in the abundance of gram-positive classes and an increase in the abundance of gram-negative classes was observed. On the contrary, when the QQ MBR was operated with gram-negative T6, a reduction in gram-positive classes and an increase in gram-negative classes was observed. As such, the outputs of the Illumina Hi-seq analysis were demonstrated that use of gram-negative QQ bacteria in the reactor induced a gram-positive microbial community and vice versa. This indicates a close interaction occurs between indigenous gram-negative and positive bacterial classes and Bacillus sp. T5/Delftia sp. T6 is fundamental to the performance of MBRs. To the best of the researchers' knowledge, this is the first study of its kind to demonstrate such a relationship. Molecular data of operation with mixed T5 and T6 was support this finding. To summarize, within the scope of this thesis, new and active species were isolated from different sources, used in MBR as an antibiofouling agent and successfully reduced biofilm and proven to be a highly effective biotechnological solution method for biofouling problem. Moreover, three different immobilization media (QQ-Fiber, SA Beads and PVA Beads) were applied with selected QQ bacteria. Finally, according to the results of the molecular analysis, different QQ bacteria were found to act by interacting with different microbial groups. The results of this thesis reveal valuable information about the QQ technologies that is the indispensable application for mitigating biofouling. It is anticipated that the findings of this thesis will lead to an enhanced knowledge of the relationships that exist between QQ bacteria and the bacterial community structures in MBRs. Additionally, it assistances to the researchers to select QQ bacteria/strategy in a different way from the past selection method (like only consideration of the QQ activity) for MBR operations and different biotechnological applications. Consequently, the results support the idea that molecular and metagenomic analysis in MBR could be essential for membrane biofouling control.