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After the completion of in situ hybridization, some samples were stained with DAPI (4′, 6-diamidino-2-phenylindole) to enumerate total cell numbers ...
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Environ. Sci. Technol. 2008, 42, 7380–7386

Quantification of Cell Specific Uptake Activity of Microbial Products by Uncultured Chloroflexi by Microautoradiography Combined with Fluorescence In Situ Hybridization YUKI MIURA AND SATOSHI OKABE* Department of Urban and Environmental Engineering, Graduate School of Engineering, Hokkaido University, North-13, West-8, Kita-ku, 060-8628, Sapporo, JAPAN

Received February 25, 2008. Revised manuscript received June 5, 2008. Accepted July 10, 2008.

We, for the first time, quantitatively determined cell specific uptake activities of microbial products (bacterial cell detritus and extracellular polymeric substances, EPS) by the member of uncultured Chloroflexi by using a microautoradiography combined with fluorescence in situ hybridization (MAR-FISH) technique. For this MAR-FISH analysis, we prepared [14C]-labeled microbial products from biomass sludge obtained and bacterial strains (Pseudomonas sp. and Acinetobacter sp.) isolated from our pilot-scale membrane bioreactor (MBR) as tracer substrates, which probably represent the more realistic food source in the MBR. The quantitative MAR-FISH analyses clearly showed that most of the uncultured Chloroflexi could indeed uptake the bacterial detritus of the two isolated strains with rates of 1.7-3.5 × 10-17 g-C µm-2-surface area h-1 (corresponding to 1.2-1.7 mg-C-bacterial detritus L-1 h-1) in the cultures, which were, however, about 2 orders of magnitude lower than the uptake rates of simple monosaccharides (mannose, arabinose, fucose, and galactose). Based on these results and their high abundance (more than 20% of total bacteria detected with EUB338-mixed probes), it could be estimated that the uncultured Chloroflexi contributes 38-51% of the total degradation of microbial products occurred in the MAR-FISH cultures.

Introduction Membrane bioreactor (MBR) technologies offer many advantages over the conventional activated sludge process including excellent effluent quality, small footprint, and low sludge production. Nevertheless, one of the drawbacks in the MBR technologies is obviously membrane fouling, which causes declining permeate flux and increasing operation costs. It has been suggested that microbial products in either soluble or colloidal form are currently considered as main culprit substances of membrane fouling in MBRs (1, 2). Because of the low food to microorganism ratio (F/M ratio) and long solid retention time (SRT) in typical MBR operations, most of the microbial products occur as a result of biomass decay and cell lysis, which mainly consist of high molecular * Corresponding author phone: +81-11-706-6266; fax: +81-11706-6266; e-mail: [email protected]. 7380

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weight cellular macromolecules including polysaccarides and proteins (3). It is desirable to minimize the concentration of microbial products in MBRs. Although previous researches focused on impact of the microbial products on membrane fouling in MBRs (3, 4), only a few studies have attempted to identify specific microbial groups involved in biodegradation of the microbial products (5). We previously found that the uncultured filamentous Chloroflexi was one of the dominant phylogenetic groups in our pilot-scale MBR treating municipal wastewater (6, 7) and their population dynamics was closely related to accumulation of soluble carbohydrates (a major constituent of soluble microbial products) (5). Based on these results, we speculated that the uncultured Chloroflexi might play an important role in degradation of the microbial products. However, in our previous study, we could neither directly demonstrate uptake of the microbial products (bacterial cell detritus and extracellular polymeric substances (EPS)) derived from biomass decay and cell lysis by the uncultured Chloroflexi in the MBR nor quantify the uptake activities. There appears to have been no published work reporting such information in literature. If the uptake activity is quantitatively analyzed in real complex environments (i.e., MBR), such information can be very valuable for better understanding of in situ ecophysiology of the uncultured Chloroflexi in MBRs. Clones affiliated with the phylum Chloroflexi were often retrieved from various habitats such as freshwater (8), activated sludge (9, 10), anaerobic digesters (11, 12), and MBRs (5, 6). Most of the clones retrieved from wastewater treatment plants were belonged to the Chloroflexi subphylum 1 and 3 (5, 9, 10). Although the Chloroflexi phylum comprises genetically diverse members, only a few pure cultures were obtained so far, in particular the member of the Chloroflexi subphylum 1 (13). Despite an ecophysiological significance of Chloroflexi in wastewater treatments including MBRs, the basic ecophysiology of this group is still largely unknown. The aim of this study was, therefore, to quantitatively determine the cell specific uptake activities of microbial products (i.e., bacterial detritus and EPS) by the uncultured Chloroflexi present in MBRs based on quantitative microautoradiography combined with fluorescence in situ hybridization (MAR-FISH) (14, 15). To accomplish this goal, we, for the first time, prepared [14C]-labeled microbial products from the MBR sludge and two bacterial strains isolated from the MBR sludge for the MAR-FISH analysis. This technique exploits in situ simultaneous phylogenetic identification and substrate uptake activities of even uncultured microorganisms without the need of enrichment or isolation. Furthermore, to quantify the cell specific uptake rates of microbial products under in situ conditions, the number of silver grains accumulated around individual probe-detected Chloroflexi cells was correlated to concurrently obtained bulk 14C uptake activity measurements.

Materials and Methods Preparation of [14C]-Labeled Bacterial Detritus and EPS. In this study, [14C]-labeled bacterial detritus and EPS were prepared from the biomass sludge taken from our pilot-scale MBR treating real municipal wastewater (5) and two pure bacterial strains (i.e., Pseudomonas sp. and Acinetobacter sp.) isolated from the MBR sludge for MAR-FISH. This is because the 14C contents in the [14C]-labeled bacterial detritus and EPS derived from the MBR sludge was too low to accurately determine the cell specific uptake activity. The [14C]-labeled bacterial detritus was, therefore, prepared from the isolated pure bacterial strains, which contain higher radioactivity. 10.1021/es800566e CCC: $40.75

 2008 American Chemical Society

Published on Web 08/26/2008

FIGURE 1. Calibration curves of specific 14C-labeled substrate uptake activity of MAR positive cells in MBR sludge after four different lengths of exposure during the autoradiographic development (i.e., exposure time; ET ) 1, 2, 3, and 5 day). The MBR sludge samples were incubated with different amounts of [14C]-arabinose, [14C]-fucose, [14C]-galactose or [14C]-mannose for 6 h. The slopes of the calibration curves imply correlation factors between 14C taken in cells and the number of silver grains (CPM one-silver-grain-1) and were shown as “C”. Correlation coefficients of the calibration curves were shown as “R 2”.

FIGURE 2. Combined MAR and DAPI images showing 14C-labeled and heat-pasteurized bacterial detritus that incorporated [14C]-glucose (A). All bacterial cells were stained with only DAPI. Uptake of 14C-labeled microbial products (B: bacterial detritus; C: extracted EPS) derived from MBR sludge by uncultured Chloroflexi at day 6 (B) or at day 15 (C). In situ hybridizations were performed with a combination of FITC-labeled EUB338-mixed probes (green) and TRITC-labeled probes GNSB941 and CFX1223 (B) and TRITC-labeled probes GNSB941 and CFX1223 (C). The [14C]-labeled monosaccharides were also used to compare with the cell specific uptake activities of microbial products. First, bacterial detritus and EPS were prepared by growing the MBR sludge with [14C]-glucose (specific activity, 317 mCi mmol-1; Amersham Biosciences, Little Chalf-ont, UK) as the sole carbon source, because glucose was frequently detected as the major dissolved monocarbohydrate in the MBR mixed liquor during the operation (data not shown). Three mL of the MBR sludge samples (ca. 10 g-MLSS L-1) taken from the MBR were transferred to 10 mL glass serum vials. Each bottle was sealed with a gastight rubber stopper. The MBR sludge samples were then cultured on 0.85 mM glucose (hot/cold ratio, 10%) with shaking at 60 rpm for 6 h at 28 °C in the dark. Cells were harvested by centrifugation at 10 000g for 10 min and washed three times with 2 mL of phosphate-buffered saline (10 mM sodium phosphate buffer, 130 mM sodium chloride; pH 7.2). Then the biomass were incubated in the phosphate-buffered saline without glucose with shaking at 60 rpm for 1 day at 30 °C in the dark in order to deplete easily biodegradable storage material (e.g., glycogen) in the cells. Then the biomass was harvested by centrifugation at 10 000g for 10 min and washed three times with 2 mL of phosphatebuffered saline. The harvested biomass was heat-pasteurized at 100 °C for 30 min. On the other hand, [14C]-labeled EPS

was extracted from the nonheat-pasteurized biomass samples by the hot-phenol-water procedure (16). Second, bacterial detritus was prepared by growing two pure bacterial strains (Pseudomonas sp. and Acinetobacter sp.) isolated from the MBR sludge with [14C]-acetic acid (specific activity, 55 mCi mmol-1; Amersham Biosciences, Little Chalf-ont, United Kingdom) as the sole carbon source. Six mL of the M9 minimal salt medium (17) inoculated with isolated bacterial strains was transferred to 10 mL glass serum vials. Each bottle was sealed with a gastight rubber stopper. Isolated bacterial strains were grown on 6 mM acetic acid (hot/cold ratio, 10%) with shaking at 60 rpm for 2 days at 30 °C in the dark. After the incubation, cells were harvested by centrifugation at 10 000g for 10 min and washed three times with 6 mL of M9 minimal salt medium. Then, the cells were incubated in the M9 minimal salt medium without acetic acid, harvested, and washed three times as descried above. Then the cells were heat-pasteurized at 100 °C for 1 h. Incubation with Radioactive Substrates. For all MARFISH analyses, the MBR mixed liquor sludge samples were diluted to a final concentration range of 0.4-1.0 g-MLSS L-1 with filter-sterilized (0.2 µm pore-size polycarbonate) MBR mixed liquor. The diluted MBR sludge samples were adjusted to pH 7 with 1 N HCl. To investigate uptake of microbial products (bacterial detritus and EPS), the following radioactively labeled organic substrates were used: (i) [14C]-labeled bacterial detritus (14C contents, approximately 0.05%) and (ii) [14C]-labeled EPS (14C contents, approximately 0.01%), which were prepared from the MBR sludge. The 14C contents were determined based on the radioactivity and the total organic carbon (TOC) concentrations of these compounds. For each experiment, 5 mL of the diluted MBR sludge sample was incubated in 10 mL glass serum vials. The bottle samples were sealed with gastight rubber stoppers. Each bottle was supplemented with radioactively labeled substrates (0.3-1.1 µCi for the bacterial detritus and 0.2-0.3 µCi for the extracted EPS) on days 0, 2, 4, and 6. The samples were incubated for 15 days at room temperature (approximately 23 °C). To determine the cell specific uptake activity of bacterial detritus, the following radioactively labeled organic substrates were used: (i) [14C]-labeled bacterial detritus derived from VOL. 42, NO. 19, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 4. Frequency of cell-specific uptake rates of [14C]-arabinose, [14C]-fucose, [14C]-galactose, and [14C]-mannose for uncultured Chloroflexi (A) and other Eubacteria (B) at 28 °C and pH 7 within 6 h incubation. FIGURE 3. Changes in net 14C content in the culture medium during the incubation with 14C-labeled bacterial detritus (A) or 14C-labeled EPS (B) that were prepared culturing MBR sludge with [14C]-glucose. The net 14C contents were expressed as percentages of initial CPM (CPM0day). The 14C-labeled bacterial detritus or the 14C-labeled EPS were supplemented on day 0, 2, 4, and 6, as indicated with the arrows (A and B). The scale of the y-axis between panels A and B is different. Changes in the relative abundance of uncultured Chloroflexi and Proteobacteria (Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, and Deltaproteobacteria) that hybridized with group-specific probes and the total active cell numbers that hybridized with EUB338-mixed probes in culture medium during the incubation with the 14C-labeled bacterial detritus (C) or the 14C-labeled EPS (D). Uptake patterns of the 14C-labeled bacterial detritus (E) or the 14C-labeled EPS (F) by uncultured Chloroflexi and Proteobacteria that hybridized with groupspecific probes. Chloroflexi was hybridized with probes GNSB941 and CFX1223 (C, D, E, and F). Proteobacteria were hybridized with probes ALF968, BET42a, GAM42a, and DELTA495 (C, D, E, and F). Eubacteria were hybridized with EUB338-mixed probes (C and D). The fractions of probe-hybridized and MAR-positive cells that incorporated the 14C-labeled bacterial detritus or EPS were expressed as percentages of total bacteria hybridized with EUB338-mixed probes. The error bars indicate the standard deviations. Pseudomonas sp. (14C contents, approximately 5%) and (ii) Acinetobacter sp. (14C contents, approximately 5%). For each

experiment, 7 mL of the diluted MBR sludge samples were transferred to 10 mL glass serum vials and then supplemented with radioactively labeled substrates. The total 14C-labeled dead cell numbers in the samples were adjusted at ca. 1010 cells L-1. Therefore, the final radioactivities of bacterial detritus derived from Pseudomonas sp. and Acinetobacter sp. in each sample were 1.5 and 4.5 µCi, respectively. The samples were incubated for 15 days at 28 °C. To determine the cell specific uptake activity of monosaccharides, the following radioactively labeled organic substrates were used: (i) L-[14C]-arabinose (specific activity; 55 mCi mmol-1), (ii) L-[14C]-fucose (specific activity; 55 mCi mmol-1), (iii) D-[14C]-galactose (specific activity; 55 mCi mmol-1), and (iv) D-[14C]-mannose (specific activity; 55 mCi mmol-1). For each experiment, 3 mL of the diluted MBR sludge sample were transferred to 10 mL glass serum vials and then preincubated for 1 h with nonradioactive substrates. Subsequently, the samples were supplemented with radioactively labeled substrates (the final activity was 5 µCi) and unlabeled substrates (hot/cold ratio, 20%). The total substrate concentrations in the samples were fixed at 0.15 mM. Then the samples were incubated for 6 h at 28 °C. For all experiments, MBR mixed liquor sludge samples pasteurized at 100 °C for 30 min were incubated in the same way as negative controls to test for possible adsorption phenomena and chemography (18). All incubations were conducted in triplicate.

TABLE 1. Average Specific Radioactively Labeled Substrates ([14C]-arabinose, [14C]-fucose, [14C]-galactose and [14C]-mannose) Uptake Activities by Uncultured Chloroflexi and Other Eubacteria at 28°C and pH 7 specific 14C-uptake activity

incubation conditions for MAR-FISH substrate [14C]-arabinose [14C]-fucose [14C]-galactose [14C]-mannose

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Chloroflexi ( ×

10-15

g-C µm-2 h-1)

1.0 ( 0.5 0.6 ( 0.2 1.8 ( 0.5 4.1 ( 0.4

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Eubacteria ( × 10-15 g-C µm-2 h-1) 1.7 ( 1.6 0.4 ( 0.1 2.1 ( 0.9 4.2 ( 1.3

TABLE 2. Average Specific Radioactively Labeled Substrates ([14C] Labeled Bacterial Detritus Derived from Pseudomonas sp. or Acinetobacter sp.) Uptake Activities by Uncultured Chloroflexi and Other Eubacteria at 28 °C and pH 7 specific 14C-uptake activity

incubation conditions for MAR-FISH substrate [14C] labeled bacterial detritus

Chloroflexi ( × 10-17 g-C µm-2 h-1)

Eubacteria ( × 10-17 g-C µm-2 h-1)

Pseudomonas sp. Acinetobacter sp.

1.7 ( 0.3 3.5 ( 0.5

3.4 ( 1.1 4.7 ( 1.0

Liquid Scintillation Counting. The degradations of 14Clabeled microbial products were confirmed by measuring the 14C contents in the cultures by liquid scintillation counting during the incubation. The radioactivity was determined with Aloka model LSC-1000 liquid scintillation counter as recommended by the manufacturer (19). Sample Fixation and Washing. After incubation with radioactively labeled substrates, the samples were fixed for 3 h at 4 °C by adding 8% paraformaldehyde in equal proportions to the samples (20). In the same way, MBR mixed liquor samples were fixed for FISH analysis. After the fixation and washing steps, the samples were spotted on a gelatincoated glass coverslips (the samples for microautoradiography) or slide glasses (the MBR mixed liquor samples for FISH) (18, 19). Oligonucleotide Probes and In Situ Hybridization. The 16S and 23S rRNA targeted oligonucleotide probes used in this study and their hybridization conditions are listed in Supporting Information Table S1. The probes were labeled with fluorescein isothiocyanate (FITC), tetramethylrhodamine 5-isothiocyanate (TRITC), or the sulfoindocyanine dye Cy5 at the 5′ end. Dehydration and FISH were performed according to the procedure described by Okabe et al., (1999a) (20). For quantitative determination of microbial compositions, the ratio of the surface area of bacterial cells stained with the group- or subgroup-specific probes to the surface area of all bacterial cells stained with EUB338-mixed probes (21, 22) was determined after simultaneous hybridization by using image analysis software provided by Zeiss (23). After the completion of in situ hybridization, some samples were stained with DAPI (4′, 6-diamidino-2-phenylindole) to enumerate total cell numbers by the direct counting method (24). Total bacterial cells detected with EUB338-mixed probes were also enumerated as described by Okabe et al., (2007) (25). Autoradiographic Procedure. After FISH, the autoradiographic procedure was performed directly on the glass coverslips as previously described by Okabe et al. (2005) (19). The optimal exposure time was adjusted to between 1 and 5 days, depending on the percentage of radioactive substrates incorporated into the biomass. Microscopy and Enumeration by MAR-FISH. A model LSM510 confocal laser-scanning microscope (Carl Zeiss, Oberkochen, Germany) equipped with an Ar ion laser (458/ 488 nm) and two HeNe ion lasers (543 and 633 nm) was used to observe and record all MAR-FISH images (26). A MARpositive cell was defined as a cell covered with more than three silver grains in this study. The numbers of MAR-positive cells and total probe-hybridized cells were determined by directly counting in randomly chosen 20 microscopic fields of three slides prepared from each sample. The averages and standard deviations were then determined. Quantitative MAR-FISH. Quantitative MAR-FISH was performed according to the procedure described by Nielsen et al. (2003) (14) with the following modifications. The silver grain numbers around the FISH- and MAR-positive cells were directly counted. Only bacteria in the center of the slide were counted to avoid the effect of the uneven distribution of bacteria when the silver grain number was counted. The background silver grain number was checked in areas more

than 25 µm away from any MAR-positive bacteria. The back ground levels were always negligible in comparison with the numbers of the silver grains present around the FISH- and MAR-positive cells. In addition, all silver grain numbers were counted for only the samples with the same background level. The number of silver grains in close proximity to the active cells was developed as a function of exposure time (14). Therefore, the optimal length of exposure time had to be determined for each sample. In this study, fixed exposure times of 1, 2, 3, and 4 days were used. The accumulation of the silver grains was shown to be proportional to concurrently obtained bulk specific 14C uptake activity measurements (Figure 1). Because of the filamentous nature of Chloroflexi, the cell sizes were larger (>50 µm) than other bacterial cells and were quite variable among the cells (data not shown). Therefore, 14C uptake activity per unit cell surface area (mol µm-2-bacterial surface h-1) was determined to compare the activity levels of individual bacterial cells. The cell specific carbon uptake activity for each radiolabeled substrate was calculated from the following equation: Specific carbon uptake activity (mol µm-2-bacterial surface -1) h -1 -1 ) Agcell × C × Dtracer × R-1 × Acell × T-1

where, Agcell ) number of silver grains around a cell (silvergrain number cell-1), C ) a correlation factor between specific 14C uptake activity of cell and the number of silver grains around cells (count per minute (CPM) silver grain-1), which was determined from the calibration curves (Figure 1), Dtracer ) specific activity of tracer in incubation (CPM molracioactive-1 tracer-1), R ) hot to cold ratio (molradioactive-tracer moltotal-tracer ), Acell ) cell size (µm2 cell-1), T ) length of incubation (h).

Results Community Composition in the MBR. The microbial community composition in the MBR mixed liquor was first analyzed by FISH with various sets of oligonucleotide probes (Supporting Information Table S1). The total bacterial cell number detected with the EUB338-mixed probes (21, 22) was 3.1 ((0.9) × 109 ((SD) cell mL-1, accounting for 38 ( 14% of the total DAPI counts in the pilot-scale MBR mixed liquor. Members of Chloroflexi (detected with probes GNSB941 (8) and CFX1223 (10)), Alphaproteobacteria (detected with probe ALF968 (27)), Betaproteobacteria (detected with probe BET42a (27)), Gammaproteobacteria (detected with probe GAM42a (27)) and Deltaproteobacteria (detected with probes DELTA495mixed (28)) accounted for 25 ( 12%, 17 ( 6%, 27 ( 6%, 7 ( 3%, and 12 ( 8% of the total bacteria detected with the EUB338-mixed probes, respectively. Uptake of 14C-Labeled Bacterial Detritus and EPS Derived from MBR Sludge. In the preparation of [14C]-labeled bacterial detritus from the MBR sludge, various bacteria species in the sludge utilized [14C]-glucose and showed MARpositive. After heat pasteurization, all bacteria (i.e., the 14Clabeled bacterial detritus) were FISH-negative, but were stained with DAPI (Figure 2A). Liquid scintillation counting revealed that the bacterial detritus and the extracted EPS contained about 10 and 2% VOL. 42, NO. 19, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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of 14C in the [14C]-glucose originally added, respectively. Since the 14C contents (calculated from CPM and TOC of 14C-labeled substrates) in the microbial products derived from the MBR sludge were very low (the specific activities were approximately 1.9 mg-C-1 and 0.4 µCi mg-C1- in the bacterial detritus and in the extracted EPS, respectively), the 14C-labeled microbial products were supplemented four times during the incubation period to promote uptake of 14C-labeled microbial products by bacterial cells (Figure 3A and B). During the incubation, 68% (for the [14C]-labeled bacterial detritus) and 64% (for the [14C]-labeled EPS) of the radioactivity in the culture samples (biomass plus culture medium) were lost from the culture medium. This result indicates that microbial products (bacterial detritus and EPS) derived from MBR sludge were indeed degraded by bacteria present in the MBR. The total bacteria number detected with the EUB338mixed probes significantly decreased from 2.9 × 108 cells mL-1 at day 0 to 6.2 × 106 cells mL-1 and to 4.0 × 107 cells mL-1 at day 15 in the cultures supplemented with the [14C]labeled bacterial detritus and EPS, respectively (Figure 3C and D). Accordingly, the both cell numbers of Proteobacteria and Chloroflexi decreased 1-2 orders of magnitude during the incubation. The relative abundance of Proteobacteria (detected with ALF968, BET42a, GAM42a, and DELTA495mixed probes) decreased from 64 ( 16% at day 0 to 19 ( 16% and to 18 ( 6% at day 15 in the cultures supplemented with the [14C]-labeled bacterial detritus and EPS, respectively. In contrast, the relative abundance of Chloroflexi remained relatively constant (25 ( 12% at day 0 to 36 ( 24% and to 27 ( 18% at day 15 in the cultures supplemented with the [14C]-labeled bacterial detritus and EPS, respectively). The bacteria that could utilize 14C-labeled microbial products derived from MBR sludge were directly visualized by MARFISH (Figure 2B and 2C) and quantified (Figure 3E and 3F). At the beginning of incubation (day 0), no MAR- and FISHpositive bacteria were detected. The percentage of MARpositive Chloroflexi that hybridized with GNSB941 and CFX1223 significantly increased to more than 90% within 6 days, and thereafter this percentage was unchanged (Figure 3E). Similarly, the percentage of MAR-positive Proteobacteria cells also increased to more than 50% within 6 days, indicating that Proteobacteria are also important in degradation of microbial products. Similar results were obtained for the [14C]-labeled EPS (Figure 3F). Cell Specific Uptake Activities of 14C-Monosaccharides. [14C]-arabinose, [14C]-fucose, [14C]-galactose and [14C]-mannose were chosen as target substrates because these monosaccharides including glucose were frequently detected in the MBR mixed liquor during the operation (data not shown). Sixty-two to 91% percent of probe-detected Chloroflexi could utilize [14C]-arabinose, [14C]-fucose, [14C]galactose, and [14C]-mannose (Supporting Information Figure S1). To determine the cell specific uptake activity, the numbers of silver grains accumulated around the individual cells were enumerated (Figure 1), which were then converted to the cell specific uptake activities of arabinose, fucose, galactose and mannose (Table 1). The numbers of silver grains around the individual cells were shown to be proportional to the amount of 14C-labeled substrate uptake during the given incubation times (R2 ) 0.75 - 0.82, Figure 1). The cell specific mannose uptake rates of both Chloroflexi and other bacteria were higher than those for arabinose, fucose, and galactose (Table 1). More than 60% of all the [14C]-mannose utilizing Chloroflexi cells took up mannose at rates higher than 3.5 × 10-15 g-C µm-2 h-1 (Figure 4). The general distribution patterns of the cell specific mannose, arabinose, fucose or galactose uptake rates for Chloroflexi were similar to those for other bacteria (Figure 4). Thus, there were no significant differences in the specific monosaccharide uptake activities (g-C µm-2-cell surface area h-1) between the 7384

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FIGURE 5. Frequency of cell-specific uptake rates of [14C]-labeled bacterial detritus derived from Pseudomonas sp. and Acinetobacter sp. by uncultured Chloroflexi (A) and other Eubacteria (B) at 28 °C and pH 7 within 24 h incubation. uncultured Chloroflexi and other bacteria (t test, P < 0.05) (Table 1 and Figure 4). For precise quantification of cell specific substrate uptake activity by probe-detected bacteria via MAR-FISH, it is necessary to determine the appropriate incubation conditions (e.g., pH, temperature, and time), fixation protocol, and exposure time for each substrate used in advance (14, 15, 29). Cell Specific Activities of 14C-Bacterial Detritus Derived from Pure Bacterial Strains. After inoculation with [14C]labeled bacterial detritus derived from Pseudomonas sp. and Acinetobacter sp., the 14C contents in the culture samples (biomass plus culture medium) were monitored by liquid scintillation counting (Supporting Information Figure S2). After 15 day incubation with [14C]-labeled bacterial detritus, 84-87% of the radioactivity was lost from the culture medium. For 24 h incubation, more than 85% of the probe-detected Chloroflexi took up [14C]-labeled bacterial detritus, while less than 15% of other bacteria took up [14C]-labeled bacterial detritus (Supporting Information Figure S3). The cell specific uptake activity of the bacterial detritus by the individual cells was calculated based on quantitative MAR-FISH analysis (Table 2). Macromolecular fractions of bacterial detritus have been considered fairly inert substances, not easily biodegradable, as compared with simple carbohydrates. Indeed, the cell specific uptake rates of the bacterial detritus were about 2 orders of magnitude lower than those of monosaccharides (i.e., arabinose, fucose, galactose, and mannose) (Tables 1 and 2). Uptake distribution patterns showed that contribution at the lower range of the cell specific bacterial detritus uptake rates (less than 4.0 × 10-17 g-C µm-2 h-1) for Chloroflexi was higher than for the other bacteria (Figure 5). The variations of the cell specific uptake rates for other bacteria were larger than those for Chloroflexi (Figure 5). Thus, the specific uptake activity of bacterial detritus per unit cell surface area by Chloroflexi was slightly lower than that by other bacteria (P < 0.05) (Table 2). However, the absolute number of MAR-

positive Chloroflexi was higher than that of MAR-positive other bacteria in the cultures. The distributions of specific uptake activities of monosaccharides or bacterial detritus by the uncultured Chloroflexi and other bacteria showed large variations in the activity levels among cells (Figures 4 and 5). Similarly, the wide range distributions of specific leucine uptake activities by Cytophaga-Flavobacter group and SAR86 cluster were reported (29). The distribution of specific uptake activities may reflect the variation in growth rates of bacteria composing these phylogenetic groups. Moreover, a 3-fold variation in the specific uptake activities was reported even within a single bacterial population (14). Most of the probe-detected Chloroflexi cells immediately took up [14C]-labeled bacterial detritus derived from isolated bacterial strains and became MAR-positive within 24 h of incubation. However, the percentage of MAR- and FISHpositive other bacteria was still low at day 1 (Supporting Information Figure S3) and gradually increased to 18-43% at day 6. Thus, it is speculated that the member of Chloroflexi was most likely primary consumers of bacterial detritus and other bacteria may utilize the secondary metabolites of original bacterial detritus. These data are consistent with the previous studies, suggesting that the member of uncultured Chloroflexi is numerically important in utilization of Nacetylglucosamine (5), yeast-extract like substrates (13) and microbial products derived from nitrifying bacteria (19, 26). Alpha- and Gammaproteobacteria preferentially utilized low molecular weight (LMW) organic matter (i.e., acetic acid and amino acid) and most of the Betaproteobacteria could not utilize directly the microbial products derived from nitrifying bacteria (19, 26). It is, however, difficult to differentiate between primary bacterial detritus-degrading bacterial groups and the secondary bacterial groups that utilize the metabolites of the primary consumers due to substrate cross-feeding. Based on the cell specific uptake rate of bacterial detritus by the uncultured Chloroflexi and their abundance (more than 20% of total bacteria detected with the EUB338-mixed probe) in the culture, we estimated that the uncultured Chloroflexi could degrade the microbial products with rates of 1.2-1.7 mg-C-bacterial detritus L-1 h-1, accounting for 38-51% of the total degradation of bacterial detritus occurred in the cultures for MAR-FISH analysis. Thus, we strongly believe that the uncultured Chloroflexi could play a significant role in degradation of microbial products in our pilot-scale MBRs treating domestic wastewater (5). However, other bacteria (i.e., Proteobacteria) are also involved in degradation of microbial products. Therefore, further study is required to understand the synergistic degradation mechanisms of the microbial products by Chloroflexi and other different microbial groups in MBRs. To understand the fate of microbial products that cause membrane fouling in MBRs, it is important to identify the key bacteria that can degrade microbial products in the MBR mixed liquor. In this study, we could, for the first time, directly demonstrate that the uncultured Chloroflexi indeed utilize the microbial products, and quantitatively determine the cell specific uptake activities of microbial products by applying quantitative MAR-FISH with [14C]-labeled bacterial detritus derived from the pure bacterial strains isolated from the MBR, without the need of isolation. The radiolabeled substrates used for the MAR-FISH in this study represented the more realistic food source of the uncultured Chloroflexi found in the MBR treating real municipal wastewater.

Acknowledgments We thank the Central Institute of Isotope Science, Hokkaido University, for providing the facilities for all isotope experiments. We appreciate Y. Watanabe for providing the MBR sludge samples. We particularly thank T. Ito and K. Ono for

technical support for MAR-FISH analysis. Yuki Miura was financially supported by the 21st Century Center Of Excellence (COE) Program “Sustainable Metabolic System of Water and Waste for Area-Based Society” from the Ministry of Education, Science and Culture of Japan.

Supporting Information Available This material is available free of charge via the Internet at http://pubs.acs.org.

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