Microaerobic DO-induced microbial mechanisms responsible for enormous energy saving in upflow microaerobic sludge blanket reactor

Bioresource Technology 140 (2013) 192–198

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Microaerobic DO-induced microbial mechanisms responsible for enormous energy saving in upflow microaerobic sludge blanket reactor Shaokui Zheng ⇑, Cancan Cui, Ying Quan, Jian Sun School of Environment, MOE Key Laboratory of Water and Sediment Sciences, State Key Lab of Water Environment Simulation, Beijing Normal University, Beijing 100875, China

h i g h l i g h t s  About 90% energy saving was achieved in UMSB without sludge bulking.  Similar COD and NH3-N removals and sludge granulation at two DO levels.  UMSB induces the dominance of microaerophilies with higher oxygen affinity.  Upflow column-type configuration creates the novel wastewater treatment process.

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Article history: Received 1 March 2013 Received in revised form 21 April 2013 Accepted 23 April 2013 Available online 30 April 2013 Keywords: Upflow microaerobic blanket reactor Energy saving Domestic wastewater Clone library Oxygen affinity

a b s t r a c t This study experimentally examined the microaerobic dissolved oxygen (DO)-induced microbial mechanisms that are responsible for enormous energy savings in the upflow microaerobic sludge blanket reactor (UMSB) for domestic wastewater treatment. Phylogenetic and kinetic analyses (as determined by clone library analyses and sludge oxygen affinity analyses) showed that the microaerobic conditions in the UMSB led to the proliferation and dominance of microaerophilic bacteria that have higher oxygen affinities (i.e., lower sludge oxygen half-saturation constant values), which assured efficient COD and NH3-N removals and sludge granulation in the UMSB similar as those achieved in the aerobic control. However, the microaerobic DO level in the UMSB achieved significant short-cut nitrification, a 50–90% reduction in air supply, and an 18–28% reduction in alkali consumption. Furthermore, the disappearance of sludge bulking in the UMSB when it was dominated by ‘‘bulking-induced’’ filamentous bacteria should be attributed to its upflow column-type configuration. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction Since the development of the activated sludge process (ASP) in England in the early 1900s, it has become the most commonly used biological treatment process worldwide (Guo et al., 2010; Ma et al., 2009). However, conventional ASPs have high operational costs because of the need for aerobic aeration at a dissolved oxygen (DO) level P2 mg L1 to ensure complete chemical oxygen demand (COD) removal and nitrification. Furthermore, it has been well demonstrated that microaerobic DO levels in conventional ASPs, i.e., <1 mg L1, will inevitably lead to filamentous bacteria-related sludge bulking (and thus poor sludge settling and treatment performance) (Guo et al., 2010; Jenkins et al., 2004; Ma et al., 2009). In previous studies, we developed a novel energy-saving ASP process, the upflow microaerobic sludge blanket (UMSB) process, which is characterized by a microaerobic DO level <1 mg L1, sludge blanket ⇑ Corresponding author. Tel.: +86 10 58809266. E-mail address: [email protected] (S. Zheng). 0960-8524/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2013.04.088

development, sludge granulation, low excess sludge production, simultaneous nitrification–denitrification, and short-cut nitrification (Zheng and Cui, 2012; Zheng et al., 2011a). The UMSB process removed 97% of the 5-day biochemical oxygen demand from ammonia-rich high-organic-strength industrial wastewater at an influent organic loading of 6.2 kg COD m3 d1 (Zheng et al., 2011a), and achieved COD and NH3-N removals of 97% and 92%, respectively, from low-strength domestic wastewater (Zheng and Cui, 2012). When no aerobic controls were available, the UMSB was estimated to reduce the required air supply by 80% for industrial wastewater (Zheng et al., 2011a) and by 60% for domestic wastewater (Zheng and Cui, 2012). One of the most intriguing and complex questions regarding the UMSB is that of why it can operate without sludge bulking at a microaerobic DO level to achieve enormous energy savings, which is significantly different from conventional ASPs. Microaerophilic microorganisms have a competitive advantage over obligate aerobic bacteria in microaerobic environments (Mannisto and Puhakka, 2002), due to higher metabolic rates in the former group in response to physiological O2 limitation

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(Ludwig, 2004). These microbes were suspected to proliferate in, and dominate, the sludge microbial community in the UMSB (Zheng et al., 2011a). However, the microaerobic DO-induced microbial mechanisms that are responsible for the enormous energy savings in the UMSB need to be examined experimentally. In this study, the UMSB was individually operated at 0.3 kg COD m3 d1 (phase 1, used in a previous study (Zheng and Cui, 2012)) and 1.0 kg COD m3 d1 (phase 2, used in conventional ASPs (Guo et al., 2010; Ma et al., 2009)), and an identical reactor with an identical seed sludge was used as an aerobic control. In addition to assessing treatment performance, five clone libraries were constructed to document the evolution of the sludge bacterial community from the seed sludge to the final sludge samples in both systems. Because DO level (aerobic/microaerobic) is not widely used as a critical microbial taxonomic norm in current studies, it might be difficult to determine which bacterial species are microaerophic or obligately aerobic following microbial community analyses. Therefore, differences in oxygen affinity in these activated sludges, measured as the oxygen half-saturation constant (KO), were used to further explain the microbial mechanisms that are responsible for energy saving in the UMSB. In previous studies, differences in oxygen affinity among autotrophic nitrifying bacterial species have been successfully used to explain why partial and complete nitrification processes occur in response to microaerobic/aerobic DO levels (Ma et al., 2009). However, an overall survey of the literature produced no available information on the effect of DO level (aerobic/microaerobic) on the oxygen affinity of complex activated sludges that were dominated by heterotrophic bacteria rather than autrophic nitrifying bacteria. 2. Methods 2.1. Experimental setup and operating conditions A lucite aeration column with a conical-shaped bottom (Ø0.1  1 m, effective volume of 6.3 L) equipped with a metering pump and an air pump in conjunction with a gas flow meter constituted the lab-scale UMSB (or the aerobic control system). Air was introduced into diffusers at the bottom of the aeration column, to manually achieve the designated DO level in the UMSB (0.7– 0.9 mg DO L1) and the aerobic control system (2.5– 3.0 mg DO L1). No data on sludge age was available for these two systems without sludge return due to the presence of the sludge blanket. The synthetic domestic wastewater, with an approximate COD:N:P of 40:10:1, was prepared daily by dissolving 200 mg L1 food-grade glucose, 21 mg L1 KH2PO4, 236 mg L1 (NH4)2SO4, and 90 mg L1 NaHCO3 in groundwater containing 3–20 mg L1 NO3-N. The seed sludge was generated by mixing river sediment, field soil and activated sludge, and was incubated in batch mode in the aerated column containing 6.3 L of synthetic wastewater at 30 °C for 12 h. Subsequently, synthetic wastewater was continuously pumped into the aeration column without pH control, during which time water sampling for COD, TN, NH3-N, NO2-N, NO3-N, and TP measurements, and in situ DO and pH measurements were conducted daily. The sludge in the bottom of the two reactors was agitated at 60 rpm in order to avoid the anaerobic reactions. In this study, the highest DO levels were generally detected in the upper part of the reactors while they almost approximated to 0 mg L1 at the bottom of the reactors possibly due to the presence of plentiful biodegradable organic substances and microbial biomass. Therefore, the in situ DO levels were detected in the upper part of the two reactors. Both systems were subjected to a hydraulic retention time (HRT) of 24 h and a COD volumetric loading (CODLR) of 0.3 kg COD m3 d1 for 76 days during phase 1, which was subsequently changed to a HRT of 7 h and a CODLR of

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1.0 kg COD m3 d1 for the next 53 days during phase 2 (summarized in Table 1). A quasi-steady-state was achieved if the effluent pollutant levels varied by less than 10% over three consecutive samplings. The average values and standard deviations of the consecutive measurement sets collected under the quasi-steady-state conditions were considered to represent the corresponding treatment. Additionally, samples were collected along the height of the reactor to determine the sludge hold-up capacity, pollutant conversion, and the granulation process at the reactor bottom throughout the experimental period, as mentioned in previous studies (Zheng and Cui, 2012; Zheng et al., 2011a). The median diameter was used to represent the size of the sludge particles. 2.2. Construction of bacterial 16S rDNA clone libraries Two grams of wet sludge samples were bead-beaten twice at 250 rpm for 15 min in phosphate buffer (10 mM, pH 7.4). After centrifugation (5000 rpm, 5 min), the samples were homogenized at 250 rpm and 37 °C in 4-mL DNA extraction buffer (10 mM EDTA, 0.2 M Tris–HCl, 0.5 M NaCl, 1% SDS, pH 7.5) for 30 min. Following centrifugation, the solids were immersed in 4 mL of 20% (w/v) SDS solution in a 65 °C water bath for 2 h, and then frozen in liquid nitrogen (80 °C, 20 min) followed by three cycles of thawing in a water bath (65 °C, 20 min). Subsequently, equal volumes of lysis buffer and chloroform–isoamyl alcohol (CIA) (24:1, v/v) were added and samples were gently shaken for 5 min. After centrifugation, nucleic acids were extracted from the resulting supernatants with phenol–chloroform–isoamyl alcohol (25:24:1, v/v/v), and the residual phenol was removed with CIA. Nucleic acids were recovered by the addition of 1 mL of pre-cold 2-propanol (20 °C) and centrifugation for 30 min at 12,000 rpm at 4 °C. The pellets were rinsed with 70% (v/v) ethanol and resuspended in 50-lL Tris–EDTA buffer (10 mM Tris/HCl, pH 8.0, 1 mM EDTA). DNA integrity was confirmed by 0.8% agarose gel electrophoresis. For cloning, 16S rRNA genes were amplified between positions 27 and 1492 (Escherichia coli 16S rRNA gene sequence numbering), using the primers 27F (50 -AGA GTT TGA TCM TGG CTC AG-30 ) and 1492R (50 -GGT TAC CTT GTTACG ACT T-30 ) (Ferrera et al., 2004). All reactions were conducted in a 20-lL PCR mixture that contained 1 lL of extracted DNA, 2 lL of 10 PCR buffer, 2.5 mM of each dNTP, 5 lM of each primer, and 1 U of Taq polymerase. PCR was conducted using a 9600 thermal cycler (Perkin–Elmer) under the following conditions: denaturation at 95 °C for 5.0 min, followed by 25 cycles of 94 °C for 60 s, 54 °C for 45 s, and 72 °C for 90 s, with a final extension at 72 °C for 8 min. PCR amplification products were purified using a UNIQ-10 DNA purification kit (Shanghai Sangon Biological Engineering Technology & Services Co.), according to the manufacturer’s instructions. The purified PCR products were cloned into the pMD18-T vector (Promega) and transformed into E. coli DH5a High Efficiency Competent Cells (TaKaRa), according to the manufacturer’s instructions. Recombinant cells were plated onto Luria–Bertani medium containing ampicillin (final concentration, 150 lg mL1) to identify white-colored recombinant colonies. To confirm the presence of DNA inserts, approximately 100 white colonies per sample were selected at random, and subjected to colony PCR using the primers described previously. Depending on the PCR result, the amplification products from each positive clone were further digested individually with two DNA restriction endonuclease treatments (RsaI and HaeIII) (Zheng et al., 2011b). The 20-lL reactions contained 2 lL of 10 buffer, 1-lL RsaI, 1-lL HaeIII, 8 lL of DNA, and 8 lL of ddH2O, and were incubated at 37 °C for 4 h. The resulting restriction fragment length polymorphism (RFLP) products were separated by electrophoresis on an agarose gel. Based on these RFLP profiles, the sequences of representative 16S rDNA clones were

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S. Zheng et al. / Bioresource Technology 140 (2013) 192–198

Table 1 Operational conditions and treatment performance of the MR and AR during the two phases. Items

Microaerobic reactor

Aerobic reactor

Phase 1

Phase 2

Phase 1

Phase 2

Operational conditions

Periods DOa Airflowb HRTc CODLRd

D1–76 0.7–0.9 25–28 24 0.3

D76–129 0.7–0.9 50–90 7 1.0

D1–76 2.5–3.0 50–70 24 0.3

D76–129 2.5–3.0 600–800 7 1.0

COD

Periods Inffluenta Effluenta Removale

D52–76 219 ± 12 2±3 99 ± 1

D116–127 256 ± 9 6±0 98 ± 0

D57–76 215 ± 18 0±0 100 ± 0

D108–127 251 ± 12 0±0 100 ± 0

NH3-N

Periods Inffluenta Effluenta Removale

D52–76 54 ± 2 4±0 92 ± 1

D113–127 57 ± 1 4±0 93 ± 1

D60–76 55 ± 3 2±1 96 ± 1

D116–127 55 ± 2 2±0 97 ± 1

NO3-N

Periods Inffluenta Effluenta

D48–76 17 ± 1 21 ± 1

D119–127 18 ± 2 2±1

D60–76 13 ± 5 47 ± 5

D110–127 17 ± 1 52 ± 5

NO2-N

Periods Inffluenta Effluenta

D54–76 0.2 ± 0.1 7.8 ± 0.1

D110–127 0.2 ± 0.2 20.2 ± 4.5

D60–76 0.4 ± 0.1 0.1 ± 0.1

D110–127 0.2 ± 0.2 2.2 ± 2.8

Alkalinity

Periods Inffluenta Enffluenta Removale

D60–76 470 ± 16 259 ± 15 45 ± 4

D108–127 431 ± 14 211 ± 15 51 ± 4

D60–76 432 ± 79 209 ± 11 55 ± 2

D113–127 446 ± 31 128 ± 11 71 ± 2

MLSS

Periods Bottoma Topa

D62–76 2.3 ± 0.2 0.03 ± 0.00 106

D115–127 11.4 ± 1.4 0.12 ± 0.08 459

D62–76 2.3 ± 0.1 0.02 ± 0.02 278

D115–127 5.9 ± 0.4 0.11 ± 0.05 427

Final sludge particle size

f

Note: It took different time spans for every pollutant norms to achieve their individual quasi-steady-state periods, which is the reason why these data were collected during different experimental periods in this study. a mg L1. b mL min1. c h. d kg COD m3 d1. e %. f lm (particle size of seed sludge: 59 lm).

determined by a commercial laboratory (Beijing Sunbiotech Co.). All 16S rDNA sequences obtained were compared with 16S rRNA gene sequences available in the GenBank database using the NCBI Blast program to locate closely related bacterial sequences, and checked for chimeric artifacts using the CHIMERA CHECK program (version 2.7) from the Ribosomal Database Project. Sequences suspected of being chimeric were not included in further analyses. Sequences of the selected clones and closely related bacterial species obtained from the NCBI databases were aligned using the CLUSTALW package and corrected by manual inspection. For the phylogenetic analysis of cloned nucleotide sequences, a neighbor-joining tree with the Juke–Cantor correction was constructed using the MEGA package, version 4.1. Bootstrap resampling analysis for 1000 replicates was performed for confidence estimation of tree topologies. The clone library coverage (C) value for each clone library was calculated according to the following equation: C = 1  (n/N), where n is the number of unique clones and N is the total number of clones examined. All 16S rRNA gene sequences were deposited in the GenBank database under accession number JX271902–JX271918 (Library A), JX271961–JX272009 (Library B), JX271941–JX271960 (Library C), JX272010–JX272048 (Library D), and JX271919–JX271940 (Library E). 2.3. Determination of sludge KO value The KO value for each sludge sample was determined by fitting a Monod curve through experimental data determined using numerous batch experiments of about 10–30 min duration each, in

synthetic domestic wastewater containing various DO concentrations, with the substrate concentration kept in excess (well above the KS values in each case). For each DO concentration experiment, an average oxygen uptake rate (OUR) was determined. Non-linear least sum of squares regression was used to determine the optimum Monod model fit through the experimental data and the KO value and maximum OUR (OURMAX) values (Oridge V7.00). 2.4. Wastewater analysis DO and pH were measured in situ using a Hi9143 DO-meter and a Hi8424 pH-meter (Hanna), respectively. COD, NH3-N, NO2-N, NO3-N, mixed liquor suspended solids (MLSS), and total phosphorus (TP) were analyzed following standard methods (APHA, 2005). 3. Results 3.1. System performance during the two phases Table 1 summarizes the mean values and standard errors of COD, NH3-N, alkalinity, NO2-N, NO3-N, and TP removal in both systems, MLSS levels at the bottom and top of both systems, pH level in the effluent, and the mean sludge particle size at the bottom of both systems during the two quasi-steady-state periods (Figs. S1–S4 show their time courses during 127 days of continuous operation). Efficient removal of COD and NH3-N was consistently detected in both the UMSB (98–99% and 92–93%, respectively) and the aerobic control system (100% and 96–97%,

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respectively) during two phases with residual COD and NH3-N levels of <6 and 4 mg L1 for the UMSB and 0 and 2 mg L1 for the aerobic control system. By contrast, TP was removed only partly in both systems (9–27% and 16–23%, respectively). Parallel to the decrease in NH3-N concentration in the UMSB, there was a simultaneous increase in NO2-N and NO3-N concentrations, with NO2-N being the predominant form of newly-produced oxidized nitrogen (averaging 66%) during phase 1. During phase 2 there was an increase in NO2-N concentration (from 0.2 to 20.2 mg L1) and a decrease in NO3-N concentration (from 18 to 2 mg L1) with NO2-N as the sole form of newly-produced oxidized nitrogen. In comparison, in the aerobic control system, there was a consistently significant increase in the NO3-N (from 13–17 to 47– 52 mg L1), but not NO2-N, concentration; and NO3-N was always the predominant form of newly-produced oxidized nitrogen (>95%) during both phases. In this study, the increase in CODLR led to a significant increase in the biomass concentration at the reactor bottom in both systems, while the biomass concentration at the reactor top remained <0.5 g L1 (Figs. S1 and S2), indicating development of a sludge blanket in both systems. Corresponding to this sludge blanket development, the compartmentalization of pollutant transformation along the reactor height, including COD, NH3-N, NO2-N and NO3-N, was found in both systems (Fig. S3). Additionally, sludge granulation at the reactor bottom also occurred simultaneously in both systems (Fig. S4 and S5), with final sludge particle sizes of 459 and 427 lm, respectively. 3.2. Sludge microbial community structure analyses Five 16S rDNA clone libraries were constructed for the seed sludge (A) and sludge samples obtained at the end of continuous operation (B: sludge at the bottom of the UMSB; C: sludge at the bottom of the aerobic control system; D: sludge at the top of the UMSB; E: sludge at the top of the aerobic control system). A total of 118, 138, 86, 164, and 105 positive clones were individually obtained, and clustered into 25, 71, 30, 51, and 38 RFLP patterns in the five clone libraries. A representative clone of each pattern was sequenced. After grouping sequences that differed by 63% into the same operational taxonomic units (OTUs), these clones were finally divided into 17, 43, 20, 26, and 22 OTUs for the five clone libraries. Approximately 53–79% of sequences in these libraries exhibited <98% similarities to known sequences in the GenBank database, which is the species definition level (Chen et al., 2008). This low level of similarity indicates that these clones may be from newly detected organisms. The retrieved sequences, the number of clones in the library, closest relatives (including accession number), and similarity are summarized in Tables S1–S5. To precisely determine the phylogenetic positions and the relative distributions of these sequences, the phylogenetic analysis of the clonal sequences from these libraries, together with all known 16S rRNAs of bacterial species within the GenBank database, is illustrated in Figs. S6–S10. The C values for the five libraries remained as high as 92%, 81%, 88%, 92%, and 87%, respectively, suggesting that samples from these clone libraries provided full coverage of the bacterial diversity. Fig. 1 summarized the main differences between these five clone libraries, including the representative groups (% total clones), the dominated bacterial species (% total clones) and the detected denitrifiers (clone numbers). It indicates a significant difference in the bacterial communities between seed sludge and sludge samples, between sludge samples obtained from the two systems, and between sludge samples obtained at different heights in the reactor. 3.2.1. Clone library A The seed sludge (Table S1) comprised 17 OTUs from the b-Proteobacteria group (5 OTUs, 78 clones, 66% of total clones), the

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a-Proteobacteria group (5 OTUs, 20 clones, 17% of total clones), and another four groups (7 OTUs, 20 clones, 17% of total clones). A-OTU12 (73 clones, 62% of total clones) were affiliated with Sphaerotillus natans, one of the most widely reported filamentous bacteria that causes the poor settling problem (bulking and foaming difficulties) of activated sludge (Kiu et al., 2002). A-OTU10 (15 clones, 13% of total clones) were affiliated with Plemorphomonas oryzae, which produces acids from glucose by fermentative metabolism and exhibits nitrate-reduction activity (Xie and Yokota, 2005). 3.2.2. Clone library B The sludge sample from the bottom of the UMSB (Table S2) comprised 43 OTUs from the b-Proteobacteria group (12 OTUs, 75 clones, 54% of total clones), the a-Proteobacteria group (8 OTUs, 11 clones, 8% of total clones), the c-Proteobacteria group (5 OTUs, 12 clones, 9% of total clones), and another six groups (18 OTUs, 40 clones, 29% of total clones). B-OTU5 (48 clones, 35% of total clones) was affiliated with S. natans. B-OTU26 (9 clones, 7% of total clones) was closely related to Trichococcus pasteurii (99%), an aerotolerant lactate- and acetate-producing spherical bacterium originally isolated from anoxic digester sludge or soil (Jiang et al., 2010). B-OTU9 and B-OTU23 (two clones) were closely related to Planctomycetes, widely-reported anaerobic ammonium oxidation (anammox) bacteria that generally live in oxygen-limited environþ ments and can produce N2 gas from NO 2 and NH4 in the absence of oxygen (Kirkpatrick et al., 2006). B-OTU33 (one clone) was closely related to Nitrospira defluvii, a slow-growing nitrite-oxidizing bacterium (NOB) that converts nitrite to nitrate (Yu et al., 2010). 3.2.3. Clone library C The sludge sample from the bottom of the aerobic control system (Table S3) comprised 20 OTUs from the a-Proteobacteria group (3 OTUs, 26 clones, 30% of total clones), the b-Proteobacteria group (9 OTUs, 26 clones, 30% of total clones), the c-Proteobacteria group (3 OTUs, 13 clones, 15% of total clones), the Nitrospirales group (1 OTUs, 1 clones, 1% of total clones), and another two groups (4 OTUs, 20 clones, 23% of total clones). C-OTU16 (12 clones, 14% of total clones) was closely related to T. pasteurii (99%). C-OTU13 and C-OTU2 (11 clones, 13% of total clones) were closely related to Nitrosomonas sp., a fast-growing ammoniumoxidizing bacterium (AOB), while C-OTU6 (1 clone) was closely related to Nitrospira sp., a slow-growing NOB (Yu et al., 2010). C-OTU1 (22 clones, 26% of total clones) was related to Rhodobacter sp., a denitrifying purple non-sulfur photosynthetic bacterium (Do et al., 2003). 3.2.4. Clone library D The sludge sample from the top of the UMSB (Table S4) comprised 26 OTUs from the b-Proteobacteria group (5 OTUs, 104 clones, 63% of total clones), the c-Proteobacteria group (3 OTUs, 17 clones, 10% of total clones), and another nine groups (18 OTUs, 43 clones, 26% of total clones). D-OTU1 (100 clones, 61% of total clones) were affiliated with S. natans. Additionally, D-OTU5 (2 clones) was affiliated with Planctomycetes, while D-OTU3 (1 clone) was affiliated with N. defluvii. 3.2.5. Clone library E The sludge sample from the top of the aerobic control system (Table S5) comprised 22 OTUs from the a-Proteobacteria group (5 OTUs, 50 clones, 48% of total clones), the a-Proteobacteria group (6 OTUs, 16 clones, 15% of total clones), and another six groups (11 OTUs, 39 clones, 37% of total clones). E-OTU16 (46 clones, 44% of total clones) were affiliated with Rhodobacter sp., while EOTU3 (12 clones, 11% of total clones) was closely related to T. pasteurii (99%). Additionally, E-OTU4 (two clones) and E-OTU5

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S. Zheng et al. / Bioresource Technology 140 (2013) 192–198

The top (β- (63%) and γ- (10%) Proteobacteria

group,

other

9

groups (26%)): Sphaerotillus natans (61 %); Planctomycetes sp. (2 clones); Nitrospira defluvii (1 clone)

Sludge from the UMSB The bottom (β- (54%),γ- (9%), and α- (8%) Proteobacteria group, other six groups (29%)): Sphaerotillus natans (35 %); Trichococcus pasteurii (7%); Planctomycetes sp. (2 clones); Nitrospira defluvii (1 clone)

Seed

sludge

(β-

(66%),

α-Proteobacteria group (17%), other four groups (17%)):

The top (α- (48%) and β- (15%)

Sphaerotillus natans (62 %);

Proteobacteria

Plemorphomonas oryzae (13%)

group,

other

6

groups (37%)): Rhodobacter sp. (44%); Trichococcus pasteurii (12%); Nitrosomonas sp. (2 clones); Nitrospira defluvii (4 clones)

Sludge from the aerobic control system

The bottom (α- (30%), β(30%) and γ- (15%) Proteobacteria group, other 3 groups (25%)): Rhodobacter sp. (26%); Trichococcus pasteurii (14%); Nitrosomonas sp. (13%); Nitrospira sp. (1 clone)

Fig. 1. The main differences between these five clone libraries, including the representative groups (% total clones), the dominated bacterial species (% total clones) and the detected denitrifiers (clone numbers).

(four clones) were affiliated with Nitrosomonas sp. and N. defluvii, respectively.

(days 119, 123 and 127), were determined. The OUR was determined for each sludge sample at various DO concentrations (Fig. S11). The Monod kinetics equation, fitted by non-linear least sum of squares regression, fitted all sludge samples well, producing R2 values of 0.81–0.99. The KO, the OURMAX and R2 values of all sludge samples analyzed are shown in Table 2. It is likely that all these sludge samples experienced a similar overall activity, as demonstrated by the similar OURMAX values (42–65 mg g1 h1). At a seed sludge sample KO value of 1.48 42–65 mg L1, the KO

3.3. KO values of sludge samples To experimentally verify the oxygen affinity of sludge samples, the KO values of seed sludge and sludge samples obtained at the beginning of continuous operation (or at the end of the batch cultivation) (day 1), and during phases 1 (days 50, 61 and 76) and 2

Table 2 KO and OURMAX values of sludge samples obtained from the UMSB and aerobic control systems, using an identical seed sludge (KO: 1.48 mg L1; OURMAX: 65 mg g1 h1; R2: 0.88). UMSB

Day Day Day Day Day Day Day

1 50 61 76 119 123 127

Aerobic control

KO (mg L1)

OURMAX (mg g1 h1)

R2

KO (mg L1)

OURMAX (mg g1 h1)

R2

1.33 0.97 1.08 1.11 0.20 0.10 0.10

62 61 53 44 43 44 42

0.95 0.89 0.93 0.93 0.89 0.90 0.91

1.69 1.58 1.72 1.78 0.73 1.06 1.06

64 64 48 54 61 62 46

0.92 0.99 0.89 0.81 0.99 0.92 0.82

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value of sludge samples obtained at the beginning of continuous operation was 1.33 mg L1 for the UMSB and 1.69 mg L1 for the aerobic control system. Subsequently, the KO values of sludge samples from the UMSB were 0.97–1.11 and 0.10–0.20 mg L1, respectively, during phases 1 and 2, while those in the aerobic control system were 1.58–1.78 and 0.73–1.06 mg L1, respectively. The sludge samples obtained from the UMSB had a significantly higher oxygen affinity (i.e., lower KO values) than those obtained from the aerobic control system.

4. Discussion The traditional biological nitrogen removal processes involves the oxidation of ammonium (NH3-N) to nitrate (NO3-N) (nitrification), followed by nitrate reduction with an organic carbon source to nitrogen gas (N2) (denitrification). Both nitrification and denitrification involve nitrite (NO2-N) as an intermediate (Blackburne et al., 2008; Ma et al., 2009). AOBs and NOBs catalyze the first (ammonia oxidation to nitrite; nitritation) and second (nitrite oxidation to nitrate; nitritation) steps of nitrification, respectively (Blackburne et al., 2008). Nitritation in combination with denitrification from nitrite to nitrogen gas has been previously recognized as economically beneficial due to the 25% reduction in oxygen demand for nitrification (Blackburne et al., 2008). Besides, the short-cut nitrification can save 40% of the carbon source used in the subsequent denitrification stage, which would be particularly advantageous for the treatment of wastewater with a low COD to nitrogen ratio (Ma et al., 2009; Wang et al., 2007). The difficulty in the nitritation process lies in achieving specific inhibition or removal of the NOBs while retaining the AOBs (Blackburne et al., 2008; Ma et al., 2009). Rather than pH and temperature, control of microaerobic DO levels seems to be the most appropriate strategy for the continuous-flow nitritation process because it can be easily regulated via the manipulation of air supply (Ma et al., 2009). In recent years, microaerobic nitritation has been consistently achieved during the treatment of domestic wastewater, during which significant sludge bulking and thus poor sludge settling were frequently observed (Guo et al., 2010; Ma et al., 2009). Therefore, although the microaerobic nitration process implies a considerable energy saving, the poor sludge settleability and thus the requirement for a larger settler would offset the cost savings represented by reduced carbon and oxygen consumption (Ma et al., 2009). The results presented here also suggest that the DO level in both systems critically affected the conversion of NH3-N into NO2-N or NO3-N, i.e., the microaerobic DO levels triggered the complete transformation of NH3-N into NO2-N, particularly during phase 2. The microaerobic DO level in the UMSB resulted in the reduction in air supply of 50% and 90%, and in alkali consumption of 18% and 28%, during the two phases. Besides the nitritation process, the microaerobic DO level in the UMSB also induced the dominance of the typical bulking-induced filamentous bacterial species, S. natans. However, no filamentous bulking happened in the UMSB throughout the experimental period, and no significant difference in COD removals were observed in both systems. In fact, when S. natans induces filamentous sludge bulking in some cases, it can also cause filamentous bridging of the flocs, facilitating separation of the biomass from the mixed liquid (Kiu et al., 2002). The absence of filamentous sludge bulking in the UMSB is attributable to the upflow column-type configuration with hydraulic behavior and substrate regime different from the well-mixed tank in a conventional ASP. Inside this configuration operated at either aerobic or microaerobic DO levels, we simultaneously observed the compartmentalization of pollutant transformation, sludge blanket development, sludge granulation, and even the microbial community distribution along the height of the reactor, which were often

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observed in other upflow column-type configurations such as UASB (Kalyuzhnyi et al., 1996). The absence of mature granules was attributable to the low CODLR level (0.3–1.0 kg COD m3 d1). In addition to the well-known anaerobic granulation process, research on aerobic granulation has commenced in recent years, most of which has focused on sequencing batch reactors (Liu et al., 2004). It seems in this study that the granulation process in the continuous-flow upflow column-type configuration can happen at both DO levels (microaerobic/aerobic) with different shear forces. The AOB were assigned to the five recognized genera (Nitrosococcus, Nitrosomonas, Nitrosospira, Nitrosovibrio and Nitrosolobus), and the NOB to four recognized genera (Nitrobacter, Nitrospina, Nitrococcus and Nitrospira) (Juretschko et al., 1998). The fast-growing Nitrosomonas and Nitrobacter spp. were frequently the two dominant AOB and NOB, respectively, having adapted to short SRT and high concentrations of substrates (Yu et al., 2010). The higher oxygen affinity of AOB (indicated by lower KO values) compared to NOB was critical to achieving the microaerobic nitritation process (Blackburne et al., 2008). Oxygen limitation has been reported to influence the activity and growth rate of NOB to a greater extent than AOB, which resulted here in the washout of NOB from the system under the microaerobic conditions employed (Ma et al., 2009). Even in the case of AOB of the Nitrosomonas europaea lineage, the difference in DO levels (high, 8.5 mg L1 and low, 0.12– 0.24 mg L1) also resulted in the development of distinct communities (Park and Noguera, 2004). The results presented here also demonstrate the significant influence of DO concentration (aerobic and microaerobic) on the nitrifier communities in both systems. In this study, a Nitrosomonas sp. was the dominant AOB in the aerobic control system when a slow-growing Nitrospira sp. was the sole NOB detected in the identical sludge sample. However, no AOB were detected in the sludge samples from the UMSB possibly due to their lower sequence abundance. The presence of the anammox bacteria in the UMSB is of interest as it demonstrates their significance for reducing energy consumption. The phylogenetic analyses in this study revealed a diversified spectrum of heterotrophic bacterial divisions in the sludge samples; there were considerable differences between the predominant heterotrophic bacterial groups in both systems. By contrast, the diversity of the bacterial community in the UMSB was markedly greater than that in the aerobic control system. Furthermore, the microaerobic DO level in the UMSB induced the sharp proliferation and dominance of the microaerophilic filamentous bacterial species (S. natans) in activated sludge, while the aerobic DO level in the control system resulted in their disappearance from the reactor (Tables 2 and 3). It seems that the difference in DO levels between the systems was responsible for the occurrence, development and magnification of the differences in sludge microbial communities from seed sludge to sludge samples obtained in the two systems. When an identical seed sludge was used, the microaerophilic bacteria species (e.g., S. natans) with higher sludge oxygen affinity sharply proliferated to dominate in the UMSB while they gradually disppeared from the aerobic control system. This biological attribute of these microaerophilic bacterial species may ensure their survival in the microaerobic environments in the UMSB, and thus assure efficient COD and NH3-N removals compared to those achieved in the aerobic control system. In previous studies, the effect of DO levels (aerobic/microaerobic) on sludge fungal community has been also observed in a continuous-flow ASP (Zheng et al., 2011b) or during batch enrichment culture (Quan et al., 2012). The data presented here is the first report on the effect of these two DO levels on bacterial community structure and function of heterotrophic bacteria-dominated activated sludge. In this study, an increase in CODLR from 0.3 (phase 1) to 1.0 kg COD m3 d1 (phase 2) resulted in a 2- to 3-fold increase

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in the air supply in the UMSB and a 12-fold increase in the aerobic control system (Table 1). Compared with the aerobic control system, the UMSB air supply was reduced by 50% during phase 1 and 90% during phase 2. It seems that the UMSB can provide an enormous energy saving, which increases with an increasing CODLR level. In our previous research, the CODLR level in the UMSB amounted to 6.2 kg COD m3 d1 for high-organic-strength industrial wastewater (Zheng et al., 2011a), which is similar to that of the conventional anaerobic treatment process (typically >3 kg COD m3 d1 (Kalyuzhnyi et al., 1996)). Since the granulated sludge and sludge blanket in the UMSB guaranteed a good retention of active biomass inside the reactor, it is promising for the UMSB to treat domestic wastewater at higher CODLR levels than that in the conventional ASP, e.g., 2 kg COD m3 d1 or higher, and to provide even greater energy savings in the near future. On the other hand, conventional ASP frequently requires larger reaction volumes to fulfill biological reactions due to its low organic loading (typically 1.0 kg COD m3 d1 or less), which results in high capital costs. The potentially higher CODLR level in the UMSB will offer marked advantages compared to the conventional ASP in terms of both operational (lower aeration consumption) and capital (smaller reactor volume) costs. 5. Conclusion This study explained why the UMSB achieved enermous energy saving when no sludge expansion occurred under microaerobic conditions. The microaerobic conditions in the UMSB induced the proliferation and dominance of microaerophilic bacteria that have higher oxygen affinities, which assured efficient COD and NH3-N removals as well as short-cut nitrification, a significant reduction in air supply and alkali consumption. Furthermore, the disappearance of sludge bulking in the UMSB should be attributed to its upflow column-type configuration, which has a hydraulic behavior, and a substrate regime that differs from the well-mixed tanks that are used in conventional ASPs. Acknowledgements This study was supported by the New Century Excellent Talents in University (NECT-11-0044) and the Natural Science Foundation of China (21077011). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.biortech.2013.04. 088. References APHA, 2005. Standard Methods for the Examination of Water and Wastewater. American Public Health Association/American Water Works Association/Water Environment Federation, Washington, DC.

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