Bacterial diversity and active biomass in full-scale granular activated carbon filters operated at low water temperatures.

Environmental Technology

ISSN: 0959-3330 (Print) 1479-487X (Online) Journal homepage: http://www.tandfonline.com/loi/tent20

Bacterial diversity and active biomass in full-scale granular activated carbon filters operated at low water temperatures Outi E. Kaarela, Heli A. Härkki, Marja R.T. Palmroth & Tuula A. Tuhkanen To cite this article: Outi E. Kaarela, Heli A. Härkki, Marja R.T. Palmroth & Tuula A. Tuhkanen (2015) Bacterial diversity and active biomass in full-scale granular activated carbon filters operated at low water temperatures, Environmental Technology, 36:6, 681-692, DOI: 10.1080/09593330.2014.958542 To link to this article: http://dx.doi.org/10.1080/09593330.2014.958542

Accepted online: 27 Aug 2014.Published online: 22 Sep 2014.

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Date: 11 September 2015, At: 20:24

Environmental Technology, 2015 Vol. 36, No. 6, 681–692, http://dx.doi.org/10.1080/09593330.2014.958542

Bacterial diversity and active biomass in full-scale granular activated carbon filters operated at low water temperatures Outi E. Kaarelaa∗ , Heli A. Härkkib , Marja R.T. Palmrotha and Tuula A. Tuhkanena† a Department

of Chemistry and Bioengineering, Tampere University of Technology, P.O. Box 541, FI-33101 Tampere, Finland; b HSY Helsinki Region Environmental Services Authority, P.O. Box 100, FI-00066 HSY, Finland

Downloaded by [Karolinska Institutet, University Library] at 20:24 11 September 2015

(Received 15 May 2014; final version received 21 August 2014 ) Granular activated carbon (GAC) filtration enhances the removal of natural organic matter and micropollutants in drinking water treatment. Microbial communities in GAC filters contribute to the removal of the biodegradable part of organic matter, and thus help to control microbial regrowth in the distribution system. Our objectives were to investigate bacterial community dynamics, identify the major bacterial groups, and determine the concentration of active bacterial biomass in full-scale GAC filters treating cold (3.7–9.5°C), physicochemically pretreated, and ozonated lake water. Three sampling rounds were conducted to study six GAC filters of different operation times and flow modes in winter, spring, and summer. Total organic carbon results indicated that both the first-step and second-step filters contributed to the removal of organic matter. Length heterogeneity analysis of amplified 16S rRNA genes illustrated that bacterial communities were diverse and considerably stable over time. α-Proteobacteria, β-Proteobacteria, and Nitrospira dominated in all of the GAC filters, although the relative proportion of dominant phylogenetic groups in individual filters differed. The active bacterial biomass accumulation, measured as adenosine triphosphate, was limited due to low temperature, low flux of nutrients, and frequent backwashing. The concentration of active bacterial biomass was not affected by the moderate seasonal temperature variation. In summary, the results provided an insight into the biological component of GAC filtration in cold water temperatures and the operational parameters affecting it.

Keywords: bacteria; biomass; drinking water; granular activated carbon (GAC); 16S rRNA gene

*Corresponding author. Email: outi.kaarela@tut.fi † Current address: Department of Biological and Environmental Science, University of Jyväskylä, P.O. Box 35, FI-40014 University of Jyväskylä, Finland. © 2014 Taylor & Francis


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Introduction Granular activated carbon (GAC) filtration is used in drinking water treatment to enhance the removal of natural organic matter (NOM) and micropollutants. The filtration process can effectively remove foul taste and odour compounds, precursors of harmful disinfection by-products, and biodegradable fractions of organic matter that promote microbial regrowth in the distribution system.[1,2] Adsorption is the major NOM removal mechanism in new GAC filters. When the filters are operated for longer periods of time, indigenous bacteria colonize the surface of GAC particles and form an active biofilm that can contribute to NOM removal via biodegradation.[3–6] GAC filtration is often assembled after a pre-ozonation step, which oxidizes the recalcitrant NOM molecules into biodegradable substances, and thus increases the biological activity in the GAC filters.[2] If two GAC filters are used in series, the biodegradation of organic matter in the first older filter is expected to delay the saturation of the sorption sites in the second recently regenerated filter and extend the service time of the filters.[7] Knowledge of the microbial ecology of the system is essential to improve and manage any biotechnological process, such as the biological component of GAC filtration.[8] Recent GAC filter studies have provided important new information on the major groups of cultivable bacteria in these filters,[9,10] the bacterial community dynamics during the start-up period of a filter,[4] and the bacterial diversity in different filtration systems.[11– 13] However, only a few studies have presented data on the composition and dynamics of bacterial communities in full-scale GAC filters,[14,15] which may differ from the often highly controlled pilot-scale systems due to different dimensions, flow parameters, backwashing regime, and fluctuation of temperature and influent water quality. Furthermore, research on the microbiology of full-scale GAC filters operated at cold temperatures typical for the high-latitude region is scarce although temperature is an important operational factor. Operating at cold temperatures may affect the rate of substrate metabolism,[16– 18] amount of biomass,[16,18] and microbial community structure [16,17] in biofiltration systems. Thus, there is a clear need for long-term studies focusing on the microbial communities of full-scale GAC filters operated at low water temperatures. In view of the above, the objectives of this study were to (a) investigate microbial community dynamics, (b) identify the major bacterial groups, and (c) determine the concentration of active bacterial biomass in GAC filters operated at cold temperatures in a full-scale drinking water treatment plant. In order to get a thorough picture of the bacterial communities in the GAC filtration stage of the plant, six GAC filters of different operation times and flow modes were followed in winter, spring, and summer. GAC samples were collected from two different filter depths and analysed by length heterogeneity polymerase

chain reaction (LH-PCR). The fingerprinting profiles were supported with sequence data, which allowed the assignment of phylogenetic groups to the predominant LH-PCR fragments. In addition, the concentration of active bacterial biomass in GAC samples was estimated by adenosine triphosphate (ATP) analysis and the total organic carbon (TOC) removal of the filters was quantified. To our knowledge, bacterial community composition and dynamics at different seasons have not been studied before in regularly backwashed first-step and second-step full-scale GAC filters, which are continuously operated for NOM removal at temperatures below 10°C.

Materials and methods Drinking water treatment plant and characteristics of GAC filters Samples were collected at the Vanhakaupunki water treatment plant (Helsinki Region Environmental Services Authority, Finland) that treats water from Lake Päijänne. The design capacity of this second largest drinking water treatment plant in Finland is 5000 m3 /h. The water purification process of the plant consists of coagulation, sedimentation, sand filtration, ozonation (0.3–0.4 mg O3 /mg TOC), GAC filtration, ultraviolet disinfection, and chloramination. The GAC filtration stage consists of a total of 20 filters, which are regenerated every 4 years. Each filter has a surface area of 42 m2 and a bed height of 2.8 m. The Vanhakaupunki treatment plant uses mainly twostep GAC filtration although some filters are operated in parallel to meet the increasing water demand. In the twostep filtration, the first-step filters filled with exhausted, 2–4 years old, GAC are operated in up-flow mode and the second-step filters containing fresher, 0–2 years old, GAC are operated in down-flow mode. Six GAC filters out of the 20 filters were studied. The flow scheme of the filters is presented in Figure 1. The filter medium was Chemviron Filtrasorb F400 (Chemviron Carbon, Feluy, Belgium), which is made of bituminous coal and has an effective granular size of 0.55–0.75 mm. The iodine number of regenerated GAC batches, which include approximately 15–50% of virgin carbon to achieve the desired volume and quality, is on average 900 mg/g upon receipt at the plant. The GAC in the studied filters had been regenerated three times in total before this study. The operation time of the filters after last regeneration was during the first sampling in February 2012 0.8 years for the filters 11, 13, and 15 and from 2.7 to 2.8 years for the filters 12, 14, and 16, respectively. The average empty bed contact time (EBCT) was 14 min for all of the filters. It should be noted that in the filters operated in series (filter pairs 12–11 and 16–15) the same water volume passed through 2 filters, and thus the average total EBCT was 28 min. The flow rate of water in the filters varied between 350 and 500 m3 /h. The first-step filters (12, 16), including the parallel filters (13, 14), were backwashed every 336 h and the second-step


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biomass quantification were transported to the laboratory in cooled containers and stored at 5°C ( < 24 h) until they were processed. Water analyses The organic matter in the influent and effluent water of the GAC filters was characterized in terms of TOC. TOC was analysed using a Shimadzu TOC-VCPH analyser (Shimadzu, Japan) at the FINAS (Finnish Accreditation Service) accredited laboratory of the waterworks according to a standard method.[19] Turbidity and pH in the influent water of the GAC filters were measured at the same laboratory according to standard methods.[20,21]

Figure 1. Flow scheme of the GAC filters. The first-step filters 12 and 16 were operated in up-flow mode and the second-step filters 11 and 15 in down-flow mode. The parallel filters 13 and 14 were down-flow filters.

filters (11, 15) every 504 h with chloraminated tap water (total chlorine 0.34–0.42 mg/L, NH4 –N 0.15–0.18 mg/L). Air scour was used in every fourth and in every seventh wash for the first-step and for the second-step filters, respectively.

Sampling Three types of samples were taken: water samples, GAC samples for the DNA analysis, and GAC samples for the active bacterial biomass analysis. All samples were collected on 14 February, 28 May, and 28 August 2012. For the water analysis, one grab sample was taken from the influent and effluent water of each filter on each sampling occasion. GAC samples were taken < 24 h before the backwashing of the filters. For the DNA analysis, two randomly selected GAC samples from the surface layer (upper 15 cm) and 2 m below the surface of the filter beds were taken from each filter, resulting in four replicates per each filter on each sampling time. In the filters 13 and 14, the August GAC samples were taken from 1 m and 2 m below the surface of the filter beds due to the low measured concentration of active bacterial biomass in the surface layers in February and May. The GAC samples for the DNA analysis were transported to the laboratory in carbon dioxide ice and stored at − 85°C. For the active bacterial biomass analysis, three random GAC samples were collected from the surface layer of each filter in February and May. In August, the samples for the active bacterial biomass analysis were taken from the same two depths as the samples for the DNA analysis: the surface layer and 2 m below the surface of the filter beds (filters 11, 12, 15, and 16) or 1 and 2 m below the surface of the filter beds (filters 13 and 14). The triplicate GAC samples for the active bacterial

Quantification of active bacterial biomass Concentration of active bacterial biomass in triplicate GAC samples was estimated by ATP analysis according to Velten et al.[22] In short, the GAC samples were rinsed in sterile phosphate buffer and 200 mg (wet weight) of GAC was transferred to an Eppendorf tube together with 100 μL of sterile phosphate buffer. The GAC tube and 300 μL of BacTiter-GloTM (Promega, Madison, WI, USA) reagent were placed in 30°C water bath for 3 min. Thereafter, BacTiter-GloTM reagent was added to the GAC tube and the mixture was incubated for further 1.5 min. Finally, 200 μL of the supernatant was transferred to a microplate and the resulting luminescence was measured with Chameleon Multilabel Detection Platform (Hidex Oy, Turku, Finland) after 30 s. Relative light units were converted to ATP concentrations using calibration curves. To prepare the calibration curves, the bacteria on GAC samples were inactivated by heat treatment (60°C, 21 h) and then the inactivated GAC was spiked with known amounts of ATP. A new calibration curve was made for each new batch of BacTiter-GloTM reagent and samples, as suggested by Velten et al.[22] Length heterogeneity polymerase chain reaction DNA was extracted from duplicate GAC samples according to the kit protocol (Powersoil DNA Isolation kit, MO BIO Laboratories, Inc, Carlsbad, CA, USA). LHPCR was performed separately for each DNA extract as described below and the data were then combined to calculate average relative quantities of LHPCR fragments in each filter at each time point. Universal bacterial primers IRD700 labelled 27F (5 AGAGTTTGATCMTGGCTCAG-3 ) [23] and 518R (5 ATTACCGCGGCTGCTGG-3 ) [24] were used to amplify an approximately 465–565 bp fragment of 16S rRNA gene, targeting the highly variable regions V1–V3.[25] Each 25 μL PCR reaction contained 2 μL of template DNA, 0.2 mM of each deoxynucleotide triphosphate, 0.3 μM of each primer, 1 × GoTaq reaction buffer, 0.2 mg/mL of bovine

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serum albumin, and 0.63 U of GoTaq DNA polymerase (Promega, Madison, WI, USA). The following thermocycler parameters were used: 95°C for 5 min, 25 × (94°C for 30 s, 55°C for 1 min, 72°C for 3 min), and 72°C for 1 min. Gel electrophoresis was performed with LI-COR 4300 DNA analyzer (LI-COR Biosciences, Lincoln, NE, USA) and the data were analysed using GelCompar II software (Applied Maths, Sint-Martens-Latem, Belgium). Size determination of the 16S rRNA gene fragments was based on using three similarly amplified and IRDye700 labelled PCR products of known length as standards.[25] Cloning and sequencing A clone library was created for the GAC samples of August 2012. The cloning was performed from the purified (QIAquick PCR purification kit, Qiagen, Hilden, Germany) LH-PCR products using pGEM-T Easy Vector System (Promega, Madison, WI, USA). The plasmids were isolated and purified by using the PureYield Plasmid Miniprep System (Promega, Madison, WI, USA). Inserts from clones were subjected to a LH-PCR analysis to choose clones of specific lengths to sequence. The sequencing was conducted by Macrogen (Macrogen, Seoul, South Korea). Sequences were compared with those submitted to the GenBank database using BLAST (http://ncbi.nlm.nih.gov/blast/). The existence of chimeras was analysed with the programme DECIPHER.[26] The sequences determined in this study were deposited in the GenBank database under accession numbers KC808713– KC808751.

Results and discussion Influent water quality and TOC removal Turbidity (0.07 ± 0.01 formazine turbidity units), pH (6.5 ± 0.1), and TOC (2.3 ± 0.1 mg/L) remained relatively constant in the influent water of the first-step GAC filters throughout the study. The influent water temperatures were 3.7°C, 4.5°C, and 9.5°C in February, May, and August, respectively. The average TOC removal in the studied GAC filters was 10 ± 4%. The TOC removal in individual GAC filters is presented in Figure 2. In February, the TOC removal was higher in the newer filters (11, 13, and 15), likely due to higher remaining adsorption capacity compared with the older exhausted filters (12, 14, and 16). The differences in the performances of the filters diminished by May, when the newer filters had been operated for over a year. The combined average TOC removal in filter pairs 12–11 and 16–15 was 17 ± 6% and 21 ± 6%, respectively, while the parallel filters (13 and 14) removed on average 9 ± 3% of the TOC. These results, although based on a small number of measurements, indicate that the overall TOC removal was higher in filter pairs with a total EBCT of 28 min,

Figure 2.

TOC removal in the GAC filters.

compared with the single parallel filters with an EBCT of 14 min. Even though several GAC filters were followed, the number of water samples in this study was limited. However, in a previous full-scale study at the same water treatment plant the TOC removal of four exhausted filters was quantified 11 times from March to August.[27] The average TOC removal rates in filters operated for 2 and 4 years were 8 ± 3% and 6 ± 2%, respectively, which confirms that the operation time has a very low impact on the TOC removal efficiency in exhausted GAC filters. The small differences in the performance of the filters of different operation times suggest that biological removal has a role in TOC reduction. The TOC removal results of this study and those of Ketola [27] are comparable to previous pilot-scale studies that reported 12–13% TOC removal in two-step GAC filtration in the same waterworks [28] and in another waterworks with similar influent water TOC concentration.[29] Velten et al. [5] reported higher 22% dissolved organic carbon (DOC) removal in a pilot-scale GAC filter operated at 7°C without backwashing but the results covered only the first six months after the start-up of the filter. In another long-term study conducted at 2–14°C, 23% TOC removal was achieved in a three-media filter that contained GAC, phonolith, and CaCO3 .[30] However, due to the different media, the results are not directly comparable to studies where solely GAC was used.

Concentration of active bacterial biomass The concentration of active bacterial biomass was measured in the surface layers of all the filters in February and May and in two different sampling depths in August (Figure 3). The concentrations of active biomass of these GAC filters were at the lower end of previously published studies, which reported 609 ng ATP/g GAC,[22] 25–5000 ng ATP/cm3 GAC,[31] and 0.2–0.7 nmol ATP/g GAC (approximately 100–400 ng ATP/g GAC).[10] Biomass



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Figure 3. Concentration of active bacterial biomass in the filter pairs 12–11 and 16–15 (a), and in the parallel filters 13 and 14 (b). In parallel filters 13 and 14, the August samples were taken 1 and 2 m below the surface of the filter beds due to the low measured concentration of active bacterial biomass in the surface layers in February and May. Error bars represent the standard deviation of three replicates.

quantity in GAC filters can be affected by many factors, including influent water quality, temperature, type of GAC, EBCT, and backwashing.[2,5,18,32,33] Thus, differences in biomass concentrations between different drinking water treatment systems are expected. Some studies have reported lower biomass concentrations in biofilters operated at 1–5°C than in the filters operated at 20– 25°C.[16,18] Conversely, Fonseca et al. [17] found no difference on biomass concentration in sand filters operated at 3°C and above 12°C. The moderate influent water temperature variation (3.7–9.5°C) did not have an apparent effect on active bacterial biomass levels in our filters. The concentration of active bacterial biomass did not differ between the first-step and second-step filters in the two filter pairs (Figure 3(a)), although in previous studies higher biomass densities in first-step filters have been reported.[10,31] The second-step filters had been operational for 9–10 months at the start of our study, which was long enough to allow an accumulation of biomass on the GAC. In addition, low temperature, low flux of nutrients, and more frequent backwashing of the first-step filters may have limited the biofilm growth, which was demonstrated by the overall low quantity of active bacterial biomass. Microbially available phosphorus was reported as being the limiting nutrient in Finnish chemically treated surface waters,[34] and phosphorus limitation in the studied filters is also possible. However, despite the low measured phosphorus concentration in the Lake Päijänne raw water, the addition of phosphorus did not increase TOC removal or active biomass concentration in a previous pilot-scale GAC filtration study at the same waterworks.[35] Backwashing of the filters with water that contained chloramine, a disinfectant, is another factor that could have affected the active bacterial biomass. Although only a minor impact of chlorinated backwash water was detected in a laboratoryscale GAC study at 5°C,[36] the effect may depend on the

concentration and type of disinfectant, that is, chloramine or chlorine, as well as the backwashing regime.[37] In the parallel filters 13 and 14, the surface layer was the first contact point of the ozonated influent water with the GAC, and the concentration of active bacterial biomass in these topmost layers was very low, 3–21 ng ATP/g GAC (Figure 3(b)). This was likely due to the effect of residual ozone (0.16–0.22 mg/L in the influent water) as suggested by Urfer and Huck.[38] The concentration of active bacterial biomass in the deeper layers of the parallel filters was within the range of active biomass concentration in the other filters. The first-step filters 12 and 16 also received ozone-containing water but the first contact point in these up-flow filters was the bottom of the 2.8 m deep filter beds. As samples were not taken deeper than 2 m below the surface of the filter beds, the effect of residual ozone was not observed in the up-flow filters. In the filter pair 12–11, the concentration of active bacterial biomass was clearly higher in the surface layer compared with the samples taken from 2 m depth (Figure 3(a)). Conversely, in the other filters there were no major differences between the two studied sampling depths. However, concentrations of active bacterial biomass in samples of both sampling depths were only measured once during the study. Thus, this difference in the homogeneity of biomass between the filters is difficult to explain, and it remains unclear whether it was a stable phenomenon. In a recent study, Gibert et al. [32] found that vertical stratification of biomass decreased with time in a regularly backwashed GAC filter operated at room temperature.

Bacterial community diversity LH-PCR analysis showed that bacterial communities in the GAC filters were diverse and considerably stable over

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Figure 4. LH-PCR analysis of 16S rRNA gene fragments from the GAC filters. Each bar represents the average relative quantity ( ± standard deviation) of an LH-PCR fragment in samples taken from two different sampling depths. In filters 13 and 14, only samples from 2 m depth were included in the analysis in February and May due to the low measured concentration of active bacterial biomass in the surface layers. Relative quantities of LH-PCR fragments comprising at least 2% of all fragments are shown.

time (Figure 4). Although there was some variation in the presence of minor bacterial groups, mostly the same LH-PCR fragment lengths dominated in each individual filter at all three sampling times. The relative quantities of LH-PCR fragments in samples taken from two different sampling depths were averaged in Figure 4. In

filters 13 and 14, the surface layer samples with very low measured concentration of active bacterial biomass were excluded from the analysis since they were presumably affected by residual ozone, and thus did not represent the overall microbial community structure in the filters.

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Environmental Technology Table 1. Affiliation of the cloned partial 16S rRNA genes from GAC filters and their determined LH-PCR length. Closest 16S rRNA gene sequence in GenBank (accession no.; similarity)b

Closest cultivated relative (accession no.; similarity)

α-Proteobacteria 13 468–469 14 469 15 469 16 469 17 471–472 18 471 19 471 20 471 21 472 22 472–473 23 472–473 24 472 25 472 26 474 27 475–476 28 486

Uncult. GAC filter clonec (AB638990; 100%) Uncult. lake sediment clone (DQ066976; 99%) Uncult. Hyphomicrobiaceaec (EF663224;100%) Uncult. biofilter clone (JN389761; 99%) Uncult. lake sediment clone (EU546762; 100%) Uncult. α-proteobacteriumc (EF664356; 99%) Uncult. Acetobacteraceaec (JN820013; 97%) Uncult. drinking water clone (GU756319; 99%) Sphingomonas sp. (AM989065; 100%) Uncult. α-proteobacteriac (CU926506; 100%) Uncult. soil clone (FJ409461; 99%) Uncult. soil clonec (JQ386396; 98%) Uncult. Nitratireductor sp.c (JQ791918; 99%) Afipia broomeae (JQ689188; 100%) Uncult. Reyranella sp.c (JQ177500; 100%) Uncult. α-proteobacteriumc (AF431132; 98%)

Hyphomicrobium sp. (GU479687; 95%) Woodsholea maritima (FM886859; 95%) Rhodoplanes sp. (GQ369128; 98%) Mesorhizobium sp. (JN119831; 93%) α-proteobacterium (AF236002; 89%) α-proteobacterium (FJ711221; 93%) Acidisphaera sp. (FJ915153; 95%) Bacterium Ellin5299 (AY234650; 94%) Sphingomonas sp. (AM989065; 100%) Hyphomicrobium sp. (FN667866; 94%) Pedomicrobium americanum (HM037996; 98%) Acidisphaera sp. (AF376024; 96%) Phenylobacterium composti (AB682432; 90%) Afipia broomeae (JQ689188; 100%) Reyranella sp. (JX458408; 99%) Bacterium Ellin363 (AF498745; 95%)

β-Proteobacteria 37 519 39 522 40 523 42 523 44 524–525 46 525 48 525

Uncult. river sediment clone (JN641372; 99%) Uncult. rice paddy clone (AB659867; 97%) Uncult. stream biofilm clonec (JF696119; 98%) Uncult. biofilter clonec (JN389733; 98%) Uncult. lake clonec (KC192426; 99%) Uncult. microbial mat clone (HQ827534; 98%) Uncult. biofilm clone (EU937925; 99%)

Comamonadaceae bacterium (FM886892;99%) Methylibium subsaxonicum (AM774413; 95%) β-proteobacterium (AY145571; 95%) Piscinibacter aquaticus (AB681749; 91%) Candidatus Nitrotoga arctica (DQ839562; 99%) Sterolibacterium sp. (FR666711; 95%) β-proteobacterium (AY145571; 95%)

Bacteroidetes 35 506 36 518 38 520–521 41 523 45 524

Uncult. soil clone (GQ397076; 95%) Uncult. Bacteroidetesc (JQ867299; 97%) Uncult. Flexibacteraceaec (AY509276; 99%) Uncult. soil clone (JQ372803; 97%) Uncult. soil clone (JQ372803; 98%)

Lewinella cohaerens (AB301614; 84%) Bacteroidetes bacterium (AB540001; 86%) Bacteroidetes bacterium (AB540001; 90%) Pedobacter sp. (KC768759; 88%) Pedobacter sp. (KC768759; 88%)

Nitrospira 43 47 49 50

Uncult. Nitrospirac (JQ791825; 100%) Uncult. biofilm clone (AB240472; 99%) Uncult. Nitrospirac (FN679237; 99%) Uncult. Nitrospirac (JF508304; 99%)

Nitrospira moscoviensis (X82558; 95%) N. moscoviensis (X82558; 95%) N. moscoviensis (X82558; 96%) N. moscoviensis (X82558; 96%)


No.a

Size (bp)

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Other phylogenetic groups 29 492 Uncult. rice paddy clonec (DQ830129; 97%) 30 492 Uncult. lake biofilm clone (FR667452; 99%) 31 492 Uncult. soil clonec (JX967656; 93%) 32 493 Uncult. groundwater clone (HM545462; 99%) 33 493 Uncult. rice paddy clone (AB659929; 99%) 34 503 Uncult. Acidobacteriac (GU257870; 100%) 51 544 Uncult. biofilter clone (JN389780; 96%) a Last two digits of accession numbers b Uncult. = uncultured. c Other matches of equal similarity.

Singulisphaera mucilaginosa (HM748856; 97%) Fimbriimonas ginsengisoli (CP002763; 88%) Nostocoida limicola III (AF244749; 91%) S. mucilaginosa (HM748856; 93%) Thiohalobacter thiocyanaticus (FJ482231; 94%) Acidobacteria bacterium (KF245634; 96%) Desulfatitalea tepidiphila (AB719404; 81%)

KC808713–KC808751.

A clone library was constructed to associate sequence data to the predominant LH-PCR fragment lengths. Based on LH-PCR analysis, 90 clones representing amplicon sizes of major LH-PCR fragment lengths ± 1 bp were chosen for sequencing, which resulted in 39 unique phylotypes using a 99.5% sequence similarity cut-off (Table 1). Out of these 39 phylotypes, 34 were unique at the 97%

level of sequence similarity. Most of the sequences had their closest relatives in uncultured bacterial clones. Short LH-PCR fragment lengths (469–476 bp) formed one of the most dominant groups in the LH-PCR profiles of GAC filters accounting for 20–40% of the total peak areas (Figure 4). Cloning and sequencing identified these amplicons as being associated with


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α-Proteobacteria, including genera Afipia, Hyphomicrobium, Pedomicrobium, and Sphingomonas frequently detected in the previous laboratory or pilot-scale studies of GAC filters using both traditional and non-culturebased methods.[9,10,12,13] Afipia broomeae isolated from prechlorinated GAC filters have been shown to degrade haloacetic acids, a common class of disinfection byproducts.[39] Sphingomonas spp. are able to degrade many types of recalcitrant environmental contaminants [40] and they are common in drinking water biofilms, including systems with a high chlorine residual.[41] Resistance to chlorine treatment may favour Sphingomonas in GAC filters backwashed with chlorinated water. LH-PCR fragment lengths 475–476 bp were closely related to Reyranella sp. (Table 1). This recently discovered genus has been associated with free-living amoebae in freshwaters, including drinking water.[42,43] Fragment lengths 518–525 bp formed another dominant group (Figure 4), which was associated with clones belonging to β-Proteobacteria, Nitrospira, and distantly related to members of Bacteroidetes. Previous studies have highlighted the dominance of β-Proteobacteria in GAC filters, whereas Bacteroidetes have been less abundant.[9,13,44] Fragment length 525 bp associated with β-Proteobacteria and Nitrospira comprised a considerable part of the PCR products in all of the filters and accounted for up to 32% of the total peak areas in filter 15 (Figure 4). Since β-Proteobacteria and Nitrospira were associated with the same LH-PCR fragment (Table 1), the relative quantity of Nitrospira in the filters cannot be determined based on our results. However, the clone libraries included several Nitrospira clones, suggesting that the proportion of these lithoautotrophic nitrite-oxidizing bacteria was substantial. In addition, one of the β-Proteobacteria sequences associated with fragment length 525 bp was closely related to Candidatus Nitrotoga arctica, a coldadapted bacterium that is able to oxidize nitrite to nitrate even at 4°C,[45] the lowest temperature of our study. Nitrification is common in chloraminated drinking water distribution networks.[46] The backwashing of the GAC filters with chloraminated water may have promoted nitrification in the filters. Nitrospira has also been previously detected in dual-media biofilters [44] and laboratory-scale GAC filters.[13,47] In addition to the above-mentioned two dominant groups, a 506 bp fragment was present in all of the filter samples and it comprised an especially high proportion of the PCR products in the older filters 12, 14, and 16 (Figure 4). This fragment length was associated with a clone that had no cultivated relatives of more than 85% similarity (Table 1). Furthermore, a 493 bp fragment was present at the proportion of 2–6% in most of the filter samples (Figure 4). The sequenced clones with a 492–493 bp length belonged to Planctomycetes, Gammaproteobacteria,

and Armatimonadetes (Table 1). A 480 bp fragment comprised 5–9% of the PCR products in filter 14 (Figure 4) but could not be assigned to any bacterial groups. The temporal stability of the microbial community structures in the GAC filters was not unexpected despite the moderate seasonal temperature variation, since other factors (e.g. TOC concentration in the influent water, backwashing regime, and EBCT) remained relatively constant over time. We found that the dominance of phylogenetic groups in the microbial community profiles of individual filters differed, which was most likely due to the differences in operation time, flow mode, and backwashing schedule of the filters. Overall, the filters shared a high number of phylogenetic groups, as was expected due to the common influent water and the same filter material. The results of this study were compared with previous molecular biology-based studies on bacterial community composition and dynamics in GAC filters that are operated to enhance NOM removal (Table 2). The focus was on filters supplied with physicochemically pretreated and ozonated surface water. The studies differed considerably in scale, filter media and operation time, organic carbon concentration in the influent water, EBCT, temperature, backwashing regime, if any, and other operational parameters. In addition, many investigations lacked data on some of these operational parameters, which makes the comparison of the studies difficult. Furthermore, some studies were based on a very limited number of samples. Despite these constraints, it can be concluded that all of the studies summarized in Table 2 underlined the dominance of Proteobacteria in GAC filters while the proportion of α-Proteobacteria and β-Proteobacteria in different filtration systems varied. Our study confirmed that the proportion of α-Proteobacteria can vary considerably even in GAC filters consisting of the same filter material and supplied with the same influent water. Moreover, we found that α-Proteobacteria and β-Proteobacteria also play an important role in filters operated below 5°C. In addition to this study, backwashing agent and schedule were only reported in two studies.[15,44] A higher proportion of Nitrospira was found in the study in which filters were backwashed with chloraminated water,[44] as in our study, compared with the study where the filter was backwashed with non-chlorinated water.[15] The effect of the backwashing agent on microbial community composition warrants further investigation. Only a few studies included data on temporal changes in microbial communities. Liao et al. [48] detected changes in the bacterial community composition at the beginning and end of the 160-day pilot-scale study although α-Proteobacteria remained the most abundant group. Conversely, the biomass concentration and DOC removal rate did not vary considerably with service time. Pinto et al. [44] reported high stability of the bacterial communities

Summary of published studies on bacterial community composition on GAC filters treating ozonated water.

Experimental set-up

GAC filter sampling

Influent water

T(°C)

TOC or DOC removal (%)

Reference

Dominating phyla Proteobacteria and Bacteroidetes. β-Proteobacteria the main class of Proteobacteria

[13]

Dominance of αProteobacteria, γ -Proteobacteria, and Acidobacteria

[12]

Clone library

Dominance of αProteobacteria in all of the samples. Spatial and temporal variation in other groups of bacteria

[48]

n. a.

Cultivation, sequencing

β-Proteobacteria predominant cultivable bacteria

[10]

n. a.

DGGE, selected bands sequenced

Dominance of Proteobacteria

[11]

Pyrosequencing

Dominating phyla Proteobacteria and Bacteroidetes. Dominating classes α-Proteobacteria, Sphingobacteriia, Cytophagia, and β-Proteobacteria. Temperature drove community dynamics

[15]

Cultivation, DNA fingerprinting, sequencing

β-Proteobacteria predominant cultivable bacteria

[9]

Laboratory-scale (one column)

Single sampling

Pretreated, ozonated SW, DOC 2.7–4.4 mg/L

n. a.

Pilot-scale (two columns)

Single sampling, two depths

Pretreated, ozonated SW, TOC/DOC n. a.

24–29

Pilot-scale (two columns)

Two samplings, two depths

Pretreated, ozonated SW, DOC 2.8–3.3 mg/L

n. a.

31–34

Pilot-scale (2 filters)

Six samplings, two replicates


n. a.

Pilot-scale (one column)

Single sampling, five depths


n. a.

Full-scale (one filter)

Eight samplings, four replicates

Pretreated, ozonated SW, TOC 1.0–1.2 mg/L

7.0–23.3

SW and GW, ozonated or without ozonation, DOC 1.5–5.5 mg/La

5–21a

Full-scale (21 filters 21 Filter beds in 9 DWTPs) sampled, details n.a.

Methods

Observations on bacterial community composition and dynamics

49–60

Pyrosequencing

n. a. (CODMn removal 37%) Clone library

37–46

n. a.



Table 2.

(Continued).

689

Continued

Experimental set-up

GAC filter sampling

Influent water

Full-scale (Dual-media GAC/sand filters)

Six samplings, one Pretreated, combined sample ozonated SW from three filters and GW, TOC 4.2–6.7 mg/L

Full-scale (six filters)

Three samplings, six filters sampled, four replicates

Pretreated, ozonated SW, TOC 2.3 ± 0.1 mg/L

T(°C)

TOC or DOC removal (%)

Methods

15–23

n. a.

Pyrosequencing

3.7–9.5

10 ± 4

Observations on bacterial community composition and dynamics Nine phylas detected. Dominance of α-Proteobacteria and βProteobacteria, other important groups γ -Proteobacteria, Bacteroidetes, and Nitrospira. Temporal stability of the bacterial community

LH-PCR, clone library Dominance of α-Proteobacteria, β-Proteobacteria, and Nitrospira. Temporal stability of bacterial communities. Different bacterial community structures in individual filters

Reference [44]

This study

Note: SW, surface water; GW, groundwater; n.a., not available; CODMn , chemical oxygen demand; DGGE, denaturing gradient gel electrophoresis; DWTP, drinking water treatment plant. a Range in individual DWTPs not reported.

O.E. Kaarela et al.


690

Table 2.


Environmental Technology in dual-media filters, which is in agreement with our findings although it should be noticed that the factors affecting microbial community dynamics in dual-media filters and in GAC filters are not necessarily the same. Water temperature variation between 7°C and 23°C was found to drive the bacterial community dynamics in a GAC filter located in the middle latitude region.[15] However, the results may not be comparable to the GAC filters of our study, where the water temperature stayed continuously below 10°C, and, thus, favoured micro-organisms adapted to low temperatures. Reported DOC and TOC removal efficiencies varied considerably in the studies cited in Table 2 as expected in such different systems in regard to operational parameters. While several systems had higher organic matter removal rates than in our study, removal rates closer to our study have been reported previously at low influent water temperatures,[5,28,30] as discussed before. In addition to temperature, not only the quantity but also the composition of organic matter in the influent water as well as availability of inorganic nutrients may significantly affect the removal efficiency.

Conclusions Bacterial community structure, concentration of active bacterial biomass, and TOC removal in full-scale GAC filters treating cold, physicochemically pretreated lake water were investigated in the winter, spring, and summer. The main conclusions of this study are: (1) TOC removal results from a limited number of samples indicated that also the older filters, which had been operational for over 2.7 years, contributed to TOC removal. This suggests that biodegradation had a role in TOC removal although the effect of adsorption processes cannot be ruled out. (2) The moderate water temperature variation (3.7– 9.5°C) did not affect the concentration of active bacterial biomass or bacterial community structures in the filters. (3) The active bacterial biomass accumulation in the filters was limited, up to 170 ng ATP/g GAC, due to low temperature, low flux of nutrients, and frequent backwashing with chloraminated water. Moreover, the concentration of active bacterial biomass did not differ between the first-step filters and the second-step filters despite their different operation times. (4) The bacterial communities in filters with different operation times and filtration modes differed but all the filters shared a high number of phylogenetic groups. α-Proteobacteria, β-Proteobacteria, and Nitrospira were the most abundant groups.

691

The results gave new insights into variation of microbial community structures and active bacterial biomass in GAC filtration under cold conditions. This information assists drinking water treatment plants in better understanding and harnessing the microbial resources in GAC filtration and provides a basis for further studies targeting the activity and function of specific bacterial groups.

Acknowledgements This study was funded by Finnish Doctoral Programme in Environmental Science and Technology and Finnish Cultural Foundation. We thank the staff at the Vanhakaupunki water treatment plant for assisting with sampling and for the water quality analyses. We are also grateful to M.Sc. Johanna Ojala for sharing her experience with LH-PCR and to Dr Marja Tiirola for providing the template DNA for the LH-PCR standards. Professors Jaakko Puhakka and Jukka Rintala are acknowledged for their valuable comments on the manuscript.

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The Bacterial Communities of Full-Scale Biologically Active, Granular Activated Carbon Filters Are Stable and Diverse and Potentially Contain Novel Ammonia-Oxidizing Microorganisms.

Seasonal changes in the invertebrate community of granular activated carbon filters and control technologies.

Microbial diversity and community structure of denitrifying biological filters operated with different carbon sources.

Bacterial community in the biofilm of granular activated carbon (GAC) PreBiofilter in bench-scale pilot plants for surface water pretreatment.

Removal of bromide and bromate from drinking water using granular activated carbon.

Adsorption removal of benzalkonium chloride by granular activated carbon for medical waste water treatment.

Assessment of the bacteriological activity associated with granular activated carbon treatment of drinking water.

Bactericidal Effects against S. aureus and Physicochemical Properties of Plasma Activated Water stored at different temperatures.

Propane biostimulation in biologically activated carbon (BAC) selects for bacterial clades adept at degrading persistent water pollutants.

Electrochemical sterilization of bacteria absorbed on granular activated carbon.

Enrichment of specific electro-active microorganisms and enhancement of methane production by adding granular activated carbon in anaerobic reactors.

Retention of titanium dioxide nanoparticles in biological activated carbon filters for drinking water and the impact on ammonia reduction.

Kinetics and thermodynamics of oxygen and carbon monoxide binding to simple ferrous porphyrins at low temperatures.

Root biomass and exudates link plant diversity with soil bacterial and fungal biomass.

Protein structure and function at low temperatures.

Effects of ultrasonic pretreatment on quantity and composition of bacterial DNA recovered from granular activated carbon used for drinking water treatment.

Factors influencing the capacity for photosynthetic carbon assimilation in barley leaves at low temperatures.

Flocking at a distance in active granular matter.

Collision dynamics of polyatomic molecules containing carbon rings at low temperatures.

Nitrosyl hemoglobin: EPR components at low temperatures.

C4 photosynthesis in Spartina townsendii at low and high temperatures.

Predicting trace organic compound breakthrough in granular activated carbon using fluorescence and UV absorbance as surrogates.

Trihalomethane hydrolysis in drinking water at elevated temperatures.

Is the exoproteome important for bacterial pathogenesis? Lessons learned from interstrain exoprotein diversity in Listeria monocytogenes grown at different temperatures.