M&E Papers in Press. Published online on March 18, 2011 doi:10.1264/jsme2.ME10205 1 2 3 Revised ME10205 4 5 6 Pyrosequencing Demonstrated Complex Microbial Communities in a Membrane 7 Filtration System for a Drinking Water Treatment Plant 8 1 1 1 1 9 SOONDONG KWON , EUNJEONG MOON , TAEK-SEUNG KIM , SEUNGKWAN HONG , and HEE- 1* 10 DEUNG PARK 11 12 1School of Civil, Environmental and Architectural Engineering,Proofs Korea University, Anam- 13 Dong, Seongbuk-Gu, Seoul 136-713, South Korea 14 15 (Received December 1, 2010 － Accepted ViewFebruary 17, 2011) 16 17 * Corresponding author. 18 E-mail: [email protected];Advance Tel: +82-2-3290-4861; Fax: +82-2-928-7656. 19 20 Running headline: Microbial Communities in a Filtration System 1 Copyright 2011 by the Japanese Society of Microbial Ecology / the Japanese Society of Soil Microbiology 1 Abstract 2 Microbial community composition in a pilot-scale microfiltration plant for drinking water 3 treatment was investigated using high-throughput pyrosequencing technology. Sequences of 4 16S rRNA gene fragments were recovered from raw water, membrane tank particulate matter, 5 and membrane biofilm, and used for taxonomic assignments, estimations of diversity, and the 6 identification of potential pathogens. Greater bacterial diversity was observed in each sample 7 (1,133 – 1,731 operational taxonomic units) than studies using conventional methods, 8 primarily due to the large number (8,164 – 22,275) of sequences available for analysis and 9 the identification of rare species. Betaproteobacteria predominated in the raw water (61.1%), 10 while Alphaproteobacteria were predominant in the membrane tank particulate matter 11 (42.4%) and membrane biofilm (32.8%). The bacterial communityProofs structure clearly differed 12 for each sample at both the genus and species levels, suggesting that different environmental 13 and growth conditions were generated duringView membrane filtration. Moreover, signatures of 14 potential pathogens including Legionella, Pseudomonas, Aeromonas, and Chromobacterium 15 were identified, and the proportions of Legionella and Chromobacterium were elevated in the 16 membrane tank particulate matter, suggesting a potential threat to drinking water treated by 17 membraneAdvance filtration. 18 19 Key words: membrane, drinking water, pathogen, pyrosequencing, microbial community 20 2 Copyright 2011 by the Japanese Society of Microbial Ecology / the Japanese Society of Soil Microbiology 1 One main objective of drinking water treatment is to remove pathogenic microorganisms (14). 2 Drinking water contaminated by pathogenic protozoa, bacteria, and viruses can cause 3 diseases (14, 15, 25, 26). Statistics indicate that 126 drinking water-related disease outbreaks, 4 429,000 cases of illness, 653 hospitalizations, and 58 deaths occurred in the United States 5 during the years 1991 - 1998 (4). 6 7 Sand filtration and disinfection are commonly used to purify drinking water. Sand filtration 8 removes particulate matter including microorganisms at the surface or in the middle of the 9 sand bed. Direct collisions, van der Waals force, surface charge attraction, and diffusion are 10 known to be involved in the capture of particulate matter by sand filters (12). Generally, the 11 filtration process is affected by several operational parameters (e.g.Proofs, linear velocity, backwash 12 rate, etc.) (12) and design conditions (e.g., grains size, depth of sand bed, etc.) (7). Current 13 increased water quality requirements make Viewit more difficult to design and utilize sand filters 14 for drinking water treatment (26). 15 16 Microfiltration (MF) or ultrafiltration (UF) with membranes is an attractive alternative to 17 sand filtrationAdvance for drinking water production mainly due to an excellent ability to remove 18 microorganisms as well as suspended solids and colloids, without the need for high 19 concentrations of disinfectants. Because the membranes used to purify drinking water have 20 pores that are smaller (typically 0.04 – 0.2 m) than microorganisms (typically 0.5 – 5.0 m), 21 microorganisms are effectively rejected through a sieving mechanism (30), although some 22 microorganisms (e.g., ultramicrobacteria (28)) can pass through the membranes. However, 23 defects on a membrane’s surface can decrease sieving efficiency, allow pathogens to pass 24 through the membrane, and affect public health, and it is important to test the integrity of 3 Copyright 2011 by the Japanese Society of Microbial Ecology / the Japanese Society of Soil Microbiology 1 membranes during the filtration process (1, 9, 10). A membrane integrity test is frequently 2 conducted by counting particles in filtered water and/or checking pressure-induced decay by 3 applying high pressure to the membranes. In addition, it is also important to know which 4 pathogens can persist in membrane systems to prepare for a possible entering of pathogens 5 into the public water supply. Pathogens are usually detected by culture and colony counting 6 methods (13), microscopic observation (35), and PCR (13). 7 8 In submersed membrane filtration operated in a dead-end mode for drinking water treatment, 9 membrane modules are installed in a tank (the membrane tank), and particulate matter is 10 concentrated in the membrane tank by filtration through the membrane. In addition, aeration 11 is frequently applied from the bottom of the membrane tank to minimizeProofs the accumulation of 12 foulants on the surface of the membrane during backwash and/or filtration periods (5, 29). 13 This operation can result in the concentrationView of particulate organic matter while oxygen is 14 dissolved in the tank, resulting in an environment suitable for the growth of aerobic 15 microorganisms, although biocidal treatment affects the growth of microorganisms in the 16 membrane system. The membrane tank therefore can behave like a bioreactor that facilitates 17 the growthAdvance of diverse microorganisms, including some pathogens. If the membrane tank 18 promotes the growth of pathogenic microorganisms and the membrane surface has some 19 defects, it would result in a potential threat to people who consume the water. 20 21 Although the potential exists for the growth of microorganisms in a membrane tank, such 22 growth has not been well reported or characterized. Thus, the objectives of this study were 1) 23 to investigate bacterial community composition and diversity and 2) to identify potential 24 pathogenic bacteria in membrane tanks. To this end, we collected biomass samples from a 4 Copyright 2011 by the Japanese Society of Microbial Ecology / the Japanese Society of Soil Microbiology 1 pilot-scale drinking water treatment plant that operates a low pressure submersible MF 2 system, and characterized bacterial 16S rRNA gene sequences using a high-throughput 3 pyrosequencing technique, and then analyzed the sequences using bioinformatics tools. 4 5 Materials and Methods 6 The pilot plant and its operating conditions 7 A pilot-scale drinking water treatment plant was set up at the Kuei municipal drinking water 3 -1 8 treatment plant (Seoul, South Korea) which produces 650,000 m d and provides treated 9 water to residents of northern Seoul. The Kuei treatment plant takes water from the Paldang 10 reservoir located in the upper parts of the Han River and treats the water by alum coagulation, 11 flocculation, sedimentation, sand filtration, and chlorination. AsProofs shown in Fig. 1, the pilot 3 12 plant primarily consisted of a membrane tank (working volume = 1.2 m ) and a produced 3 3 13 water tank (0.1 m ). Raw water was pumpedView to the membrane tank at a rate of 5.1 m /h. The 14 water was filtered through submersed hollow-fiber membranes by a suction pump at a rate of 3 -1 15 4.8 m h , and the filtered water was delivered to the produced water tank. At the bottom of 3 -1 16 the membrane tank, the concentrate was withdrawn at a rate of 0.3 m h . The pilot plant was -2 -1 17 operated Advanceat a flux of 60 LMH (L m h ). A daily total of 91 cycles of suction (15 min), 18 backwashing (0.5 min), and relaxation (0.17 min) were processed, followed by one cycle of 19 maintenance cleaning (2 min) and relaxation (15 min). During backwash, treated water from 20 the produced water tank was back-flowed across the membrane to the membrane tank at a 3 -1 21 rate of 7.2 m h , without addition of a chemical. Maintenance cleaning was practiced similar -1 22 to the backwash operation except for providing 15 mg L of NaOCl. A total of four ® 23 horizontal-type membrane modules were installed in the membrane tank (Cleanfil -S20H, 24 KOLON Industry, South Korea), and air was continuously supplied at the bottom of the 5 Copyright 2011 by the Japanese Society of Microbial Ecology / the Japanese Society of Soil Microbiology -1 1 membrane modules at a rate of 200 L min during filtration and backwash periods. The 2 module consisted of polyvinylidene fluoride hollow-fiber membranes with a nominal pore 2 3 size of 0.07 m. The overall membrane surface area of the four modules was 80 m . The pilot 4 plant had been operated since April, 2006 and had consistently produced water with low 5 levels of turbidity (0.04 0.01 Nephelometric Turbidity Units (NTU)) irrespective of highly 6 variable raw water quality (turbidity = 10.4 15.9 NTU). 7 8 Sampling, DNA extraction, and PCR amplification 9 After the pilot plant was operated for 30 months, during August 2009, particulate matter was 10 collected from the membrane tank using a bucket-type sampler. An attached biofilm sample 11 was also harvested by scraping the biofilm formed on the middleProofs of hollow-fiber membranes, 12 using a sterilized spatula after lifting the membrane modules. For a comparison of bacterial 13 communities, raw water was also sampled Viewfrom the source water equalization basin using a 14 bucket-type sampler.