EVALUATION OF ALGINATE MAGNETIC NANOPARTICLE BEADS FOR IMMOBILIZATION OF QUORUM QUENCHING BACTERIA ISOLATED FROM DAIRY INDUSTRY WASTEWATER TO ALLEVIATE BIOFOULING

Abstract

Membrane fouling is one of the prominent problem of membrane bioreactors (MBR) during wastewater treatment. Biofouling caused by the bacterial biofilm formation on the membrane surface is considered as one of the major contributor of the overall membrane fouling process. Reduction in hydraulic performance; increase in transmembrane pressure (TMP) and shortening of membrane’s life span are some of the widely encountered adverse effects of biofouling on membrane systems. Various approaches including membrane backwashing, cleaning of membranes employing numerous chemicals, variation in hydrodynamic conditions and membrane modification have been explored to mitigate fouling. But membrane biofouling being a complex multistage process is not effectively eradicated by these approaches. Additionally, the problems of chemical toxicity, foulant accumulation within membrane pores also facilitates the need for development of effective technologies for biofouling control. Bacterial acyl homoserine (AHL) based quorum sensing (QS) mechanism is considered as the regulatory phenomenon for biofilm formation. However, these signalling molecules are disrupted by a special group of bacteria termed the quorum quenching bacteria through the phenomena of quorum quenching (QQ). This phenomenon has been recognized as a promising method to control the problem of membrane biofouling. The QQ bacteria co-exist with the QS bacteria in a wide variety of habitats including rhizosphere, sewage, soil and many other sources but the occurrence of QQ bacteria in dairy waste activated sludge (WAS) remains unexplored. Keeping this in view, an attempt has been made to investigate the potential of QQ bacteria isolated from the dairy WAS collected from Verka milk industry effluent treatment plant situated in Bathinda, Punjab. The QQ bacteria showing higher AHL degrading potential was chosen to alleviate biofouling in MBR. Since, the bacteria are known to have low survival rate in the free-state, they were v immobilized onto the magnetic iron nanocomposite carriers. The nanoparticles in the magnetic nanocomposite beads possessed enough magnetic strength to enable their easy separation from the MBR during operation. The QQ bacteria present in dairy WAS was enriched in the KG medium supplemented with n-hexanoyl homoserine lactone (C6-HSL) as a sole source of carbon and nitrogen. Five bacterial isolates obtained after enrichment were identified as Klebsiella pneumoniae (JYQ1 and JYQ5), Acinetobacter baumannii JYQ2, Pseudomonas nitroreducens JYQ3, and Pseudomonas JYQ4 through 16S ribosomal deoxyribonucleic acid (16S rDNA) analysis. These isolates were submitted in Genbank under accession numbers KP189202 (JYQ1), KP340458 (JYQ2), KP340459 (JYQ3), KU555415 (JYQ4), and KP780263 (JYQ5). The C6-HSL degrading ability of the isolated QQ bacteria was determined quantitatively and qualitatively through biosensor (using Chromobacterium violaceum CV026) assay and GC-MS analysis, respectively. All the five isolates exhibited decolourization zone around the Chromobacterium violaceum CV026 spotted lawns indicating C6-HSL degradation. Maximum degradation percentage of 83.8% was shown by Pseudomonas JYQ4 within 6 h of incubation. Other isolates Klebsiella pneumoniae JYQ1 and JYQ5 showed around 81.5% and 81.4% of C6- HSL degradation, respectively within 24 h of incubation. Pseudomonas nitroreducens JYQ3 degraded 68.4% of C6-HSL in 12 h of exposure. The isolate Acinetobacter baumannii JYQ2 possessed both QS and QQ activity which is evident from its degradation percentage. The isolates Klebsiella pneumoniae JYQ1 and JYQ5 due to their pathogenic nature were exempted and the other three QQ bacterial isolates (Acinetobacter baumannii JYQ2, Pseudomonas nitroreducens JYQ3 and Pseudomonas JYQ4) were immobilized onto the IMN beads by encapsulating magnetic iron nanoparticles and QQ bacteria in sodium alginate mixture. The magnetic iron nanoparticles prepared by co-precipitation method were cubical in shape and ranged in size from 5-19 nm. The FTIR analysis indicated the presence of Fe and O functional groups in the nanoparticles. The nanoparticles exhibited polycrystalline structure with crystallite size of around 6.9 nm and saturation magnetization of 39 emu g-1 . The successful immobilization of QQ bacteria onto the magnetic nanocomposite beads was confirmed through the SEM analysis. The QQ bacteria IMN beads also showed C6-HSL degradation potential. Confirming the preliminary studies, the Pseudomonas JYQ4 IMN beads exhibited the maximum C6-HSL degradation of 90% within 6 h of incubation when compared to other isolates whose degradation percentage varied in the range between 73- 90%. The IMN beads of bacterial consortium (prepared by mixing JYQ2, JYQ3 and JYQ4) showed degradation percentage of 73.9%. The efficiency of QQ bacteria IMN beads in controlling the biofilm developed by Pseudomonas aeruginosa 3541 was then assessed. The SEM analysis demonstrated the growth of less number of bacterial cells on the surface of QQ IMN beads incubated membranes when compared to control. Among the different QQ IMN beads, Pseudomonas JYQ4 IMN beads indicated better biofilm reduction ability. Furthermore, the CLSM analysis confirmed the efficiency of QQ bacteria IMN beads in controlling the biofilm growth and development when compared to control which exhibited signs of Pseudomonas aeruginosa 3541 biofilm maturation in 10 vi days. Among the different QQ bacteria and consortium IMN beads incubated membranes, Pseudomonas JYQ4 IMN beads incubated membranes showed no signs of biofilm maturation till 30 days of incubation. The isolates Acinetobacter baumannii JYQ2 and consortium incubated membrane showed signs of biofilm maturation within 20 days, respectively unlike Pseudomonas nitroreducens JYQ3 that showed biofilm maturation in 30 days. The biofilm structural elucidation by COMSTAT software further supported the results of CLSM analysis by showing less biomass (0.015± 0.001 µm3 /µ2 ) and more surface to biovolume ratio (0.93± 0.003 µm2 /µ3 ) for biofilm developed by Pseudomonas aeruginosa 3541 in 30 days incubation period with Pseudomonas JYQ4 IMN beads when compared to control membrane that showed biomass volume of 0.06± 0.003 µm3 /µ2 and surface to biovolume ratio of 0.21± 0.005 µm2 /µ3 . Microcolony development and biofilm growth is indicated by more biomass volume and lesser surface to biovolume ratio. Further, the flux measurement of the incubated membranes confirmed the delayed biofilm formation in membranes incubated with Acinetobacter baumannii JYQ2, Pseudomonas nitroreducens JYQ3, Pseudomonas JYQ4 and consortium IMN beads that showed 10.4%, 17.7%, 20.3% and 8.1% higher flux, respectively compared to control. The potential of QQ bacteria IMN beads in controlling the biofilm formation by the sludge bacteria was also tested. The light microscopy analysis of glass slides incubated with QQ bacteria IMN beads revealed a significant reduction in the number of sludge bacterial cells when compared with the control slide (without IMN beads). Pseudomonas JYQ4 IMN beads were more efficient in controlling biofilm formation followed by Pseudomonas nitroreducens JYQ3, Acinetobacter baumannii JYQ2 and consortium. The CLSM analysis of the QQ bacteria IMN beads incubated membranes exhibited the less amount of biofilm on the membranes compared to control. Similar to Pseudomonas aeruginosa 3541 studies, Pseudomonas JYQ4 delayed the biofilm maturation up to 30 days showing better biofilm controlling ability compared to other isolates. The biomass volume and surface to biovolume ratio of sludge bacteria biofilm developed on the Pseudomonas JYQ4 IMN beads incubated membrane was calculated to be around 0.019± 0.015 µm3 /µ2 and 0.85± 0.65 µm2 /µ3 , respectively. The control 1 (without IMN beads) incubated membranes however, showed biomass volume of 0.065± 0.061 µm3 /µ2 and surface to biovolume ratio of 0.18± 0.14 µm2 /µ3 , respectively whereas control 2 (blank nanoparticle beads) incubated membranes showed biomass volume of 0.06± 0.02 µm3 /µ2 and surface to biovolume ratio of 0.16± 0.38 µm2 /µ3 , respectively. The flux studies showed that Pseudomonas JYQ4 IMN beads incubated membranes showed 22% higher flux followed by Pseudomonas nitroreducens JYQ3 (19% higher), Acinetobacter baumannii JYQ2 (16% higher) and consortium (12.6% higher) compared to control membranes within 30 days incubation. The efficiency of the QQ bacteria IMN beads in biofouling control was also investigated in MBR. A submerged aerobic MBR with polyethersulfone hollow fiber membrane of pore size 0.4 µm and working volume of 4.5 L was used for the study. The MBR was operated at hydraulic retention time (HRT) and flux of 8h and 12.5 L/ (m2h), respectively. Synthetic dairy industry wastewater with COD of 4800± 40 mg/L was used as substrate in MBR. The MBR performance was evaluated at three different MLSS (i.e.) 4000, 7000 and 10,000 mg/L. The MBR showed removal efficiencies in the range of 95.6- 99.2% for COD removal; 55.7- 88.4% for TSS removal; 93.6- 94.6% for NH3-N removal and 25.5- 33.2% for phosphate removal at vii the MLSS 4000, 7000 and 10,000 mg/L. The performance of MBR in terms of effluent quality remained the same in QQ-MBR and showed no significant change in removal efficiencies. The membrane fouling potential at three different MLSS (i.e.) 4000, 7000 and 10,000 mg/L were assessed through tightly bound (TB-EPS), loosely bound (LBEPS) and soluble EPS polysaccharides and proteins. At MLSS of 4000 mg/L, the polysaccharides and protein fraction in TB-EPS varied between 32- 42.6 mg/L and 34.2- 50.7 mg/L, respectively which increased to 59.9- 71.3 mg/L and 61.6- 80.3 mg/L at 7000 mg/L MLSS; and 76.6- 92.6 mg/L and 85.9- 115.9 mg/L at 10,000 mg/L MLSS. Likewise, the LB-EPS polysaccharides and proteins were in the range of 11.7- 17.7 mg/L and 38.5- 56.1 mg/L at 4000 mg/L MLSS; 25.5- 36.2 mg/L and 81.1- 115.9 mg/L at 7000 mg/L MLSS; and 40.4- 58.5 mg/L and 124.6- 186.5 mg/L at 10,000 mg/L MLSS. The polysaccharides and proteins in soluble EPS also increased with increase in MLSS and was observed to be 23.4- 31.8 mg/L and 30.3- 48.1 mg/L at 4000 mg/L MLSS; 40.2- 53.2 mg/L and 56.3- 75 mg/L at 7000 mg/L MLSS; and 56.4- 72.3 mg/L and 77.9- 101.1 mg/L at MLSS 10,000 mg/L. Among all the EPS, TB-EPS and LB-EPS showed higher levels of polysaccharides and proteins, respectively. TMP also showed continuous increase and reached 29.8 kPa during MLSS 10,000 mg/L. When the QQ-MBR (Pseudomonas nitroreducens JYQ3 and Pseudomonas JYQ4 IMN beads) was assessed, it showed decreased EPS production in terms of polysaccharide and protein fractions of TB-EPS, LB-EPS and soluble EPS. During the operation of QQ-MBR at 4000 mg/L, 75.3% and 63.2% decrease in polysaccharide and proteins fractions of TB-EPS was observed compared to that of control MBR. Likewise, the QQ-MBR showed 64% and 77% lower LB-EPS and soluble EPS polysaccharides, respectively. The protein of LB-EPS and soluble EPS decreased by 62.2% and 68.7%, respectively in QQ-MBR at MLSS of 4000 mg/L. At MLSS 7000 mg/L, the polysaccharide in TB-EPS, LB-EPS and soluble EPS reduced by 69.7%, 63.1% and 72.3% when compared to control MBR. Similarly, around 57.3%, 63.4% and 61.7% reduction in the protein fraction of TB-EPS, LBEPS and soluble EPS, respectively was attained in the QQ-MBR. At MLSS 10,000 mg/L, the QQ-MBR showed a decrease of around 57.3% and 42.7% in TB polysaccharides and proteins fractions, respectively. LB polysaccharides and proteins reduced by 52.2% and 59.5% and soluble polysaccharides and proteins showed a decrease of around 60.7% and 40.3% in the QQ-MBR at 10,000 mg/L MLSS compared to control. Also, the QQ-MBR showed comparatively slower TMP rise than control MBR. The results concluded that the QQ bacteria Pseudomonas nitroreducens JYQ3 and Pseudomonas JYQ4 can be used effectively for interrupting the QS mechanism in bacteria for controlling the membrane biofouling problem in MBR.