Aerobic Biological Treatment of

Synthetic Municipal Wastewater in
Membrane-Coupled Bioreactors
Christian G. Klatt, Timothy M. LaPara
University of Minnesota, Department of Civil Engineering, 500 Pillsbury
Drive SE, Minneapolis, Minnesota 55455; telephone: 612-624-6028; fax:
612-626-7750; e-mail: lapar001@umn.edu
Received 27 July 2002; accepted 1 October 2002
DOI: 10.1002/bit.10572

Abstract: Membrane-coupled bioreactors (MBRs) offer
many benefits compared to conventional biological
wastewater treatment systems; however, their performance characteristics are poorly understood. Laboratory-scale MBRs were used to study bacterial adaptations in physiology and community structure. MBRs
were fed a mixture of starch, gelatin, and polyoxyethylene-sorbitan monooleate to simulate the polysaccharide,
protein, and lipid components of municipal wastewater.
Physiological adaptations were detected by measuring
ectoenzyme activity while structural dynamics were
studied by denaturing gradient gel electrophoresis of
PCR-amplified 16S rRNA gene fragments. As cell biomass accumulated in the MBRs, pollutant removal efficiency initially improved and then stabilized with respect
to effluent concentrations of chemical oxygen demand,
protein, and carbohydrate. Comparison of the MBR effluent to filtered reactor fluid indicated that a portion of the
observed pollutant removal was due to filtration by the
membrane rather than microbial activity. The rates of
ectoenzyme-mediated polysaccharide (␣-glucosidase)
and protein (leucine aminopeptidase) hydrolysis became
relatively constant once pollutant removal efficiency stabilized. However, the maximum rate of lipid hydrolysis
(heptanoate esterase) concomitantly increased more
than 10-fold. Similarly, ␣-glucosidase and leucine aminopeptidase ectoenzyme affinities were relatively constant,
while the heptanoate esterase affinity increased more
than 30-fold. Community analysis revealed that a substantial community shift occurred within the first 7 days
of operation. A Flavobacterium-like bacterial population
dominated the community (>50% of total band intensity)
and continued to do so for the remainder of the experiment. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 82: 313–
320, 2003.

Keywords: DGGE; ectoenzymes; enzyme affinity; PCR

INTRODUCTION
The biodegradation of organic compounds found in industrial and municipal wastewaters is necessary to protect surface water quality. Aerobic biological techniques, such as
the activated sludge process, have proven effective for the
Correspondence to: T. M. LaPara

© 2003 Wiley Periodicals, Inc.

removal of these pollutants. Activated sludge treatment consists of a well-mixed aeration basin coupled to a quiescent
chamber that separates the biomass from the treated effluent
so that the biomass can either be recycled back to the aeration chamber or be removed from the system altogether as
a waste byproduct. Treatment efficiency by the activated
sludge process depends on the development of microbial
aggregates that are susceptible to gravitational separation.
Problems with poor settling biomass, however, are common
(Jenkins et al., 1993), such that biomass clarification efficiency often limits activated sludge treatment efficiency
(Dick, 1970).
Membrane-coupled bioreactors (MBRs) are an alternative biotechnology for wastewater treatment. MBRs operate
in a similar fashion as the activated sludge process, except
that an ultra- or microfiltration membrane replaces the
gravitational clarifier to separate the biomass from the
treated effluent. MBRs have the potential to achieve complete biosolids separation and cell recycle, thereby eliminating the need for the formation of flocs or aggregates,
while simultaneously increasing the treatment capacity of
the system (Çiçek et al., 1998; Konopka et al., 1996; LaPara
et al., 2001a; Muller et al., 1995). MBRs can therefore treat
considerably greater amounts of wastewater per reactor volume compared to conventional batch, fed-batch, or continuous-flow bioreactors.
Despite these potentials advantages, the implementation
of MBRs for wastewater treatment has been infrequent due
to high membrane costs and large energy inputs for membrane operation (Visvanathan et al., 2000). A considerable
amount of research to date, therefore, has focused on membrane operation—specifically, on understanding membrane
fouling to reduce MBR operational costs (Bouhabila et al.,
2001; Defrance et al., 2000; Kang et al., 2002; Yoon et al.,
1999). In contrast, relatively little research on MBRs has
focused on the microbiological aspects of MBR operation,
with results thus far indicating that while pollutant removal
efficiency is very high, the biomass is less reactive to

Membrane permeate was pumped from the filter cartridge at a specific rate to maintain a liquid volume (700 ± 50 ml) in the bioreactor. Fluorescence output was measured on a TD-700 fluorometer (Turner Designs.0 L min−1 to keep the culture sufficiently mixed and aerated. 1971). pumped through the membrane cartridge. protein. Tappe et al. Dissolved oxygen concentrations were continuously maintained >80% of saturation (data not shown). 314 Schematic of the MBR system used in this study.4) and mixed with a fluorogenic substrate analog. A/G Technology. Data are presented as the arithmetic means.1 mL SL7 trace mineral solution (Biebl and Pfennig. VOL. followed by three consecutive freeze-thaw cycles. 1994).. All assays were performed in triplicate. Ectoenzyme activity (the combination of extracellular and surface attached enzyme activity) was quantified using fluorogenic substrate analogs. CA) per the manufacturer’s instructions. Genomic DNA was purified from these samples using the Fast DNA Spin Kit (Qbiogene. 2000. This synthetic feed media contained starch. The purpose of this study was to elucidate the relationships between microbial community structure and function in an aerobic MBR treating a synthetic municipal wastewater. Quantification of soluble protein was determined using the Hartree (1971) modification of the Lowry method using BSA as a protein standard. 150 mg ammonium chloride.0). 1998. IL).. Sunnyvale.2-␮m pore size polysulfone microfilter membrane cartridge (surface area ⳱ 0. PA) and potassium hydrogen phthalate as a standard. Konopka. Paul. 30 mg potassium phosphate. One unit of enzyme activity corresponded to the production of 1 ␮mol of product per minute. Aliquots of culture media were diluted with 10 mM Tris-HCl (pH 7. Vista. MN)... MBRs consisted of a 580-ml fermentor (CYTOLIFT glass airlift bioreactor. Reactor performance was investigated by measuring the removal of COD. Partial 16S rRNA genes were amplified from the ex- BIOTECHNOLOGY AND BIOENGINEERING.. MATERIALS AND METHODS Membrane-Coupled Bioreactors Laboratory-scale MBRs were inoculated with 1 ml of biomass from a 100-ml batch culture that had been fed synthetic wastewater medium and incubated overnight at 25°C. Soluble carbohydrate was determined using the anthrone method and glucose as a carbohydrate standard (Herbert et al. and polyoxyethylene-sorbitan monooleate to simulate the polysaccharide. and L-leucine-AMC (leucine aminopeptidase). NO. Aeration rates were delivered in the range of 0. 1993). Sterile feed medium was added using a peristaltic pump (Masterflex variable-speed console drive pump. the cell pellet was resuspended in 1 ml of lysis buffer (120 mM sodium phosphate buffer. Vernon Hills. protein. pH 8.5 h. Cells were lysed during a 90-min incubation at 70°C. 5% sodium dodecyl sulfate. Vineland. and returned to the bioreactor. 40 mg magnesium chloride. 60 mg calcium chloride.5–1. 100 mg sodium bicarbonate. the standard deviations for all samples were <5% of the mean (data not shown). S >> Km) or to produce a reaction velocity vs. substrate profile. Enzymatic activity for the hydrolysis of organic biopolymers was quantified using fluorogenic model substrates involving 4-methylumbelliferone (MUF) or 7-amino-4methylcoumarin (AMC) as substrate analogs (Hoppe. 2003 . This batch culture had been inoculated with 1 ml of cryopreserved activated sludge collected from the aeration tanks of the Metropolitan Wastewater Treatment Plant (St. 1996. (1951) method and bovine serum albumin (BSA) as a protein standard. 70 mg starch. and 0. 10 mg casamino acids.changes in reactor conditions compared conventional treatment bioreactors (Konopka et al. MAY 5. The amount of substrate analog was controlled to measure either enzyme activity under saturation conditions (i. This medium had a chemical oxygen demand (COD) of 400 mg l−1.011 m 2 . 1981). and lipid components of municipal wastewater (Raunkjær et al. Cole-Parmer. 25 mg sodium phosphate. Bethlehem. Community Analysis Biomass samples were collected from the MBR and centrifuged. MA) (Fig. COD analysis was determined colorimetrically using low-range accu-Test vials (Bioscience. gelatin. 3. The reactor hydraulic residence time was approximately 8. 1999). The model substrates used were MUF-␣glucopyranoside (␣-glucosidase). Culture fluid was rapidly (residence time <30 sec) withdrawn from the bioreactor. Needham. Analytical Methods Biomass was measured as particulate protein using the Lowry et al. Figure 1. NJ) coupled to 0. and carbohydrate as biomass accumulated in the MBR. 1). 120 mg polyoxyethylene-sorbitan monooleate 10 mg yeast extract. Enzymatic activity of each sample was determined from a linear regression of fluorescence units over time and correlated to a MUF or AMC standard curve. Bacterial community dynamics were investigated by denaturing gradient gel electrophoresis of PCR-amplified 16S rRNA gene fragments (PCRDGGE). MUF-heptanoate (heptanoate esterase). CA) using a long UV filter. 82.e. The feed medium was designed to represent low-strength municipal wastewater and contained the following (per liter of deionized water): 150 mg gelatin. Kontes.

Band intensities were quantified using LabWorks Image Acquisition software (UVP). Filtered carbohydrate concentrations were initially similar to that of the membrane permeate. 4 nmol deoxynucleoside triphosphates. Madison. the gel was stained with SYBR Green I (Molecular Probes. Soluble protein and carbohydrate levels in the membrane permeate were less than 10 mg l−1 and 2 mg l−1 throughout this experiment. The gross pollutant removals reported above are the sum of both biological (i.e. Specific PCR-DGGE bands were manually excised from the gel.5:1. S is the substrate analog concentration. IL) to the following equation: V = Vm S Km + S (1) where V is the enzymatic reaction velocity. Km is the half-saturation constant. Madison. WI). WI) prior to nucleotide sequence determination..000 in 0. conversion of nutrients to new biomass. was lost from each MBR due to sample collection and accidental cell loss caused by foaming. 2002) with the BLASTn program (Altschul et al. followed by 7 min at 72°C. and other microbial products) and physical (i.e. Nucleotide sequences were determined fully in both directions for each PCR-DGGE band using PRBA338F and PRUN518R as sequencing primers. Nucleotide sequences were checked for possible chimeric sequences with the CHECK_CHIMERA program at the Ribosomal Database Project website (Maidak et al. CO2.. DGGE was performed on a D-Code apparatus (BioRad. AF529281-AF529285.89) throughout the 35-day experiment. Filtered protein concentrations were 3–5-fold KLATT AND LAPARA: TREATMENT OF SYNTHETIC MUNICIPAL WASTEWATER 315 . The relationship between these two removal mechanisms was investigated in the second MBR experiment by comparing the protein and carbohydrate concentrations in the membrane permeate vs. PCR products were purified using the Wizard PCR preps DNA purification system (Promega. but then increased 4-fold and remained relatively constant throughout the duration of the experiment. coli positions 338–358) (Lane. Chicago. 2001). and ∼1 ng of template DNA. Gels were visualized on a UV transilluminator and photographed with a digital CCD camera (BioChemi System. biomass levels were approximately constant at 100–200 mg l−1 particulate protein for the first 20 days..5× TAE buffer (Sambrook et al. Photographs were enhanced for contrast and brightness using Adobe PhotoShop v 6.. Following electrophoresis. filtered (0. Electrophoresis was performed at 60°C. 1993) primers with a GC-clamp attached to the reverse primer (Muyzer et al.5× TAE). coli positions 534–518) (Muyzer et al. r2 ⳱ 0. CA). 1991) and PRUN518R (E. COD levels in the membrane permeate decreased initially and then remained reasonably constant at 130–150 mg l−1. 55°C for 30 sec. Samples containing approximately equal amounts of PCR amplicons were loaded onto 8% (wt/vol) polyacrylamide gels (37. and 72°C for 30 sec. The final 50-␮l reaction mixture contained: 1× PCR buffer with MgCl2 (Promega. PCR-DGGE was repeated on these samples until only a single band was detectable. 1989) using a denaturing gradient ranging from 30–55% denaturant (100% denaturant contains 7 M urea. 1993). suspended in 30 ␮l of sterile water.. but then declined so that over the next 30 days it was approximately 130 mg l−1. acrylamide:bisacrylamide) in 0.. All nucleotide sequences were determined at the Advanced Genetic Analysis Center at the University of Minnesota using an ABI 3100 Genetic Analyzer (Applied Biosystems. Hercules. OR. 2A). Soluble protein (10–30 mg l−1) and carbohydrate (<8 mg l−1) concentrations were again low throughout the duration of the experiment. Biomass density then increased linearly (27 mg l−1 d−1. Watertown. 1997).. The PCR protocol included a 5-min initial denaturation at 94°C. 3). and incubated overnight at room temperature. In the first experiment (Fig. Putative chimeric sequences were also manually split into subsections and resubmitted to the GenBank database to determine if the segments were from different phylogenetic groups. A final PCR step was performed without the GC-clamp attached to the reverse primer. The nucleotide sequences obtained in this study have been deposited in the GenBank database under accession nos. Reported nucleotide sequences do not include the original PCR primer sequence. Upland. MA) as described previously (LaPara et al. CA)..tracted genomic DNA by PCR using a PTC 100 thermal cycler (MJ Research.98) over the next 30 days. Reference nucleotide sequences were obtained from the GenBank database. biomass levels increased more than 10-fold in a quasilinear fashion (38 mg l−1 d−1. some biomass.0. Eugene. CA). Data Analysis Michaelis-Menten constants were calculated from the enzyme activity assays by nonlinear regression using SigmaPlot regression software (SPSS. 30 cycles of 92°C for 30 sec. r2 ⳱ 0. 40% (vol/vol) formamide in 0. and Vm is the reaction velocity when S >> Km. containment by the membrane) removal. COD levels in the membrane permeate reached a maximum of 235 mg l−1 on the fourth day. 25 pmol of forward and reverse primers. initially at 20 V (15 min) and then at 200 V (270 min). The variable V3 region of the 16S rRNA gene from members of the domain Bacteria was amplified using the PRBA338F (E. RESULTS Two different MBRs were operated with virtually 100% cell recycle.. however. In the second MBR experiment (Fig. Foster City. Enzyme affinity is used as a measure of the ability of an enzyme to catalyze a reaction at low substrate concentration and is calculated by the following equation: Affinity = Vm Km (2) Nucleotide sequences were compared with sequences in the GenBank database (Benson et al. diluted 1:5. 2B).2 ␮m pore size) reactor contents (Fig.5× TAE). 2000a). UVP.

Heptanoate esterase activity. 316 rating substrate concentrations (i. and carbohydrate during the first (A) and second (B) MBR experiments. 4). increased throughout the experiment in a complex pattern. in the second MBR experiment. followed by a gradual increase for the rest of the experiment. Metabolic activity of the MBR biomass was studied in both experiments by measuring ectoenzyme activity at satu- Figure 3. 䉭 ⳱ heptanoate esterase. ␣-Glucosidase and leucine aminopeptidase affinities were both reasonably constant throughout the experiment (Fig. ␣-Glucosidase activity initially decreased more than 90% in the first experiment. 䊊 ⳱ filtered reactor fluid. The accumulation of biomass with simultaneous removal of COD. Heptanoate esterase affinity then declined by almost 50% in the next 3 days.Figure 2. 6). 䊐 ⳱ effluent protein. In contrast. 82. 䊉 ⳱ membrane permeate. then declined by almost by 50% over the next 7 days. 䊊 ⳱ effluent COD. NO. 䉭 ⳱ effluent carbohydrate. Leucine aminopeptidase activity was 60–90 mU (g protein)−1 during the first 13 days and then decreased to 25–45 mU (g protein)−1 for the rest of the first MBR experiment. 䊐 ⳱ leucine aminopeptidase. MAY 5. Maximum ectoenzyme activity in replicate MBRs as a function of time during the first (A) and second (B) MBR experiments. Leucine aminopeptidase activity in the second MBR experiment was initially 17 mU (g protein)−1 and then slowly declined throughout the experiment to less than 2 mU (g protein)−1. ectoenzyme affinity was measured to help elucidate the physiological adaptations made by the biomass (Fig. The final Figure 4. In the second MBR experiment. VOL. followed by another increase in activity to more than 10fold higher than the heptanoate esterase activity at the beginning of the second experiment. 3. 2003 . V ⳱ Vm) related to the components of the synthetic wastewater media (Fig. heptanoate esterase affinity increased more than 15-fold in the first 4 days of the experiment. which was measured only in the second experiment. higher than membrane permeate levels after the tenth day of the experiment. Comparison between carbohydrate (A) and protein (B) concentrations in the membrane permeate and in filtered reactor fluid samples during the second MBR experiment.. ␣-glucosidase activity was maintained at 1–2 ␮U (g protein)−1 throughout the experiment. protein. 䊉 ⳱ cell protein. 䊊 ⳱ ␣-glucosidase. 5). Activity increased more than 6-fold in the first 4 days. after which it remained reasonably constant at 1–2 ␮U (g protein)−1. BIOTECHNOLOGY AND BIOENGINEERING.e.

Community profile of bacterial community structure as a function of time during the second MBR experiment as detected by denaturing gradient gel electrophoresis of PCR-amplified 16S rRNA gene fragments. DISCUSSION This research was performed to better understand the relationships between bacterial activity and bacterial community dynamics in MBRs used for biological wastewater treatment. the slope of the broken line shows enzyme affinity. MBRs impose increasingly stringent nutrient limitation by providing a constant nutrient supply while biomass accumulates. while the other three were from the Proteobacteria (␤. Band labels are shown in Figure 7. Two of the bands were associated with the CytophagaFlavobacteria-Bacteroides division of Bacteria (Bands A and C). 䊊 ⳱ Band B. a sub- Figure 8. Letters identify prominent bacterial populations. 7). Affinity of different ectoenzymes as a function of time during the second MBR experiment. Band B was identified as a chimeric sequence and excluded from further analysis (data not shown). KLATT AND LAPARA: TREATMENT OF SYNTHETIC MUNICIPAL WASTEWATER 317 . F. Bacterial community structure during the second MBR experiment was studied by denaturing gradient gel electrophoresis of PCR-amplified 16S rRNA gene fragments (PCR-DGGE) (Fig. and G). Numbers shown in each lane represent the time of sample collection (days). 䉭 ⳱ heptanoate esterase. 䊐 ⳱ leucine aminopeptidase. Band A became the dominant bacterial population (>55% of total band intensity) for the rest of the experiment (Fig. 8). Symbols represent measured enzyme activities. 䊐 ⳱ Band D. The fraction of total band intensity comprised by individual bacterial populations as detected by PCR-DGGE during the second MBR experiment. of which only one (Band C) remained detectable for the rest of the experiment.Figure 7. During the present research. Heptanoate esterase activity as a function of time and of substrate analog concentration during the second MBR experiment. heptanoate esterase affinity was more than 30-fold higher than the initial value. A substantial community shift occurred within the first 4 days of the experiment. 䊊 ⳱ ␣-glucosidase. An additional band (Band B) also became detectable during the last 2 days of the experiment. 䊏 ⳱ Band C. Figure 6. Band excision and sequence analysis from day 7 and day 35 confirmed that comigrating bands were the same bacterial population (data not shown). Several of the prominent PCR-DGGE bands were excised and their nucleotide sequences were determined (Table I). as all of the bacterial populations disappeared with one exception (Band A). Two additional bands were detected (Bands C and D) after 7 days.and ␥-subdivisions) (Bands E. solid lines represent the best fit to the Michaelis-Menten model. 䉭 ⳱ Band F. 䉱 ⳱ Band E. Figure 5. 䊉 ⳱ Band A.

2001a). The detection of this continuing physiological adaptation corresponded to a continuous structural shift. Phylogenetic relationship PCR-DGGE band Sequence length (bases) Most closely related sequence Bacterial division % Similarity A C D E F 155 155 163 160 160 Flavobacterium sp. the question remains why similar trends were not observed with the ␣-glucosidase and leucine aminopeptidase measurements. Here again. isolation of the relevant bacteria from complex ecosystems is often difficult (Amann et al. 2001a). Sequence length and closest phylogenetic affiliation of the PCR-DGGE bands analyzed in this study. 3. and a substantial shift in the dominant bacterial populations. VOL. 1995).. 82.. for a review) instead of the relatively biodegradable components of the feed media (starch. 1998). We are currently experimenting with MBRs fed a single substrate to elucidate the relationships between ectoenzyme activity. further increases in heptanoate esterase activity and affinity were detected. After this initial period. 1991).. In the present experiments.e. Our overall COD removal performance.. polysaccharide. CFB ⳱ Cytophaga-Flavobacteria-Bacteroides. 2001a). reinoculated with activated sludge. This suggests that virtually all of the COD measured in the membrane permeate was soluble microbial products (see Barker and Stuckey. and polyoxyethylene-sobitan BIOTECHNOLOGY AND BIOENGINEERING. Konopka et al. and incubated at 25°C for 30 days (data not shown). One of the goals of this research was to simulate the treatment of municipal wastewater that contains protein. 1998. The behavior of the MBR with respect to pollutant removal is consistent with previous results for MBR operation (Çiçek et al. the increases in heptanoate esterase activity and affinity in the second MBR experiment were substantially different from previous work where only moderate (10–25%) increases in normalized Vm rates were observed (LaPara et al. modulation of ectoenzyme activity and affinity.. 2000c). Effluent COD levels typically improve initially and then reach a pseudo-steady state. however. and stringency of nutrient limitation in an MBR (LaPara et al. increase Vm) (Matin. Unfortunately.. Isolation and physiological characterization of these bacterial populations would be enlightening. A thorough knowledge of these relationships between community structure and function that may be gained with further research would be helpful to understand the performance of MBRs and other full-scale wastewater treatment bioreactors in which mixed and dynamic bacterial communities are responsible for the biodegradation of mixtures of substrates. The proliferation of a single bacterial population (Band A) corresponded to the initial increase (<7 days) in heptanoate esterase activity and affinity. 1994).5 93. IsoA1 (AJ319015) Unidentified bacterium WP13 (AF303937) Delftia acidovorans (AF149849) Stenotrophomonas detusculanense (AF280434) Comamonas testosteroni Q10 (AF519533) CFB CFB ␤-Proteobacteria ␥-Proteobacteria ␤-Proteobacteria 100 93. 2001b). improved enzyme affinity) (Button.Table I. was quite low (∼70%) compared to full-scale municipal wastewater treatment bioreactors (>90%) (Tchobanoglous and Burton. suggesting that a combination of a structural shift to specialized bacteria and a physiological adaptation by the Band A-population were responsible for the continued increase in both heptanoate 318 esterase activity and affinity.. 1996. MAY 5. and bacterial community dynamics. Further reductions in COD concentrations were not detected when the membrane permeate was collected. While the modifications in heptanoate esterase activity and affinity reflect a physiological adaptation to the increasingly stringent nutrient limitation imposed by the MBR. and lipid as the principal organic constituents (Raunkjær et al.. bacterial community dynamics in wastewater treatment bioreactors are poorly understood (Amann et al. gelatin. 1999. 1998). nutrient uptake.4 100 GenBank accession numbers are shown in parentheses.. stantial shift in microbial activity and bacterial community structure was detected within the first several days of MBR operation. 2000b. but were not correlated to any specific operational variable. 1996. hydraulic residence time (LaPara et al. bacteria can gain an advantage by increasing the quantity of enzyme per cell (i.. In previous work. it is novel and may indicate a connection between bacterial community structure and biological wastewater treatment reactor function. the bacterial community continued to adapt its physiology to optimize bioreactor performance. LaPara et al.e. In the competition for a limited supply of nutrients.. These results are consistent with previous observations that both physiological adaptations and community structure shifts occur within the first few days of MBR operation (Konopka et al. 1994). indicating that although the performance of an MBR stabilized with respect to pollutant removal efficiency. Although this connection is circumstantial. two other bacterial populations (Bands C and D) were detected. LaPara et al. community adaptations were observed in laboratory bioreactors as functions of temperature (Konopka et al.8 99. 2001a). LaPara et al. NO.... however. suggesting that there is a connection between bacterial dynamics and bioreactor function. 1999. 1979) or by producing an enzyme that is more efficient at low substrate concentrations (i. This physiological and community adaptation was observed through improved pollutant removal efficiency. bacterial growth on individual compounds within a mixture of substrates is poorly understood (KovárovaKovar and Egli. 2003 . 1998. however.. In this study.

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