GRANULE FORMATION MECHANISMS WITHIN AN AEROBIC WASTEWATER 1 SYSTEM ...

36 Pages·2010·3.76 MB·English

AEM Accepts, published online ahead of print on 17 September 2010
Appl. Environ. Microbiol. doi:10.1128/AEM.00864-10
Copyright © 2010, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.
1 GRANULE FORMATION MECHANISMS WITHIN AN AEROBIC WASTEWATER
2 SYSTEM OPERATING FOR PHOSPHORUS REMOVAL
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4 Jeremy J. Barr1,2*, Andrew E. Cook1, Phillip L. Bond1,2*
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6 1 The University of Queensland (UQ), Advanced Water Management Centre (AWMC), QLD 4072, Australia.
7 2Environmental Biotechnology Cooperative Research Centre, Sydney, Australia
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9 *Corresponding authors. Jeremy Barr: Ph.: [phone], E-mail: [email protected]. Phillip Bond:
10 Ph.: [phone], E-mail: [email protected]
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13 For submission to Applied and Environmental Microbiology lo
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14 Running Title
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15 Granule segregation in wastewater systems. o
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17 Subject Category a
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18 Environmental Microbiology .a
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19 Abstract
20 Granular sludge is a novel alternative for the treatment of wastewater and offers numerous
21 operational and economic advantages compared with conventional floccular sludge systems.
22 The majority of research on granular sludge has focused on optimisation of engineering
23 aspects relating to reactor operation with little emphasis on the fundamental microbiology. In
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24 this study we hypothesise two novel mechanisms for granule formation as observed in three o
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25 laboratory-scale sequencing batch reactors operating for biological phosphorus removal, lo
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26 treating two different types of wastewater. During the initial stages of granulation two distinct e
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27 granule types (white and yellow) were distinguished within the mixed microbial population. o
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28 White granules appeared as compact, smooth, dense aggregates dominated by 97.5% t
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29 Candidatus “Accumulibacter phosphatis” and yellow granules appeared as loose, rough, /a
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30 irregular aggregates with a mixed microbial population of 12.3% Candidatus .
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31 “Accumulibacter phosphatis” and 57.9% Candidatus “Competibacter phosphatis” among
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32 other bacteria. Microscopy showed white granules as homogenous microbial aggregates and /

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33 yellow granules as segregated, microcolony-like aggregates with phylogenetic analysis D
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34 suggesting granule types are likely not a result of strain-associated differences. Microbial e
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35 community composition and arrangement suggest different formation mechanisms occur for e
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36 each granule type. White granules are hypothesised to form by outgrowth from a single ,

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37 microcolony into a granule dominated by one bacterial type, while yellow granules are
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38 hypothesised to form via multiple microcolony aggregation into a microcolony-segregated
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39 granule with a mixed microbial population. Further understanding and application of these s
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40 mechanisms and the associated microbial ecology may provide conceptual information
41 benefiting start-up procedures for full-scale granular sludge reactors.
42
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43 Introduction
44 Activated sludge can be operated as an efficient biological process to treat and remove
45 unwanted nutrients such as carbon, nitrogen and phosphorus, from wastewater. Two common
46 processes applied in wastewater treatment are; enhanced biological phosphorus removal
47 (EBPR)(28), which is widely applied for carbon and phosphorus removal, and simultaneous
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48 nitrification denitrification and phosphorus removal (SNDPR)(40), which combines carbon, o
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49 phosphorus and nitrogen removal. Conventional plants operating for EBPR and SNDPR, lo
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50 typically operate as floccular sludge systems in which small aggregates (30-200 µm) of e
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51 microorganisms make up the suspended biofilms of activated sludge (10). Aerobic granular o
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52 activated sludge is a novel alternative for the treatment of wastewater and offers several t
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53 operational and economic advantages when compared with conventional floccular sludge a
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54 systems (9, 21). Granular sludges are typically larger suspended biofilm aggregates (200-2000 .a
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55 µm) and typically contain higher biomass concentrations within the same reactor vessels .
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56 compared to floccular sludge systems (4, 29). However, granular sludge has yet to be applied /
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57 to full-scale wastewater treatment plants (WWTP) partially due to a lack of the fundamental
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58 knowledge of the microbiology associated with the granulation process. m
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59 r

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60 For full-scale application of aerobic granular sludge systems the most likely strategy for the ,
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61 reactor start-up would be by seeding with a conventional floccular sludge. From the floccular 8

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62 seed there will be a transition period with the selection and eventual formation of granules. g
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63 During the start-up phase there is typically a high degree of process performance variability, s
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64 with biomass washout and large changes to the microbial community occurring due to certain
65 changes to operational parameters applied to the system (20, 21, 39). Thus the process
66 performance, start-up length and ultimately the optimisation of the start-up phase have
67 important implications for the application of full-scale granular sludge plants. Previous
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68 investigations of aerobic granular systems have visualised the microbial community and
69 aggregate structure during this start-up phase (39) and mathematical models have been
70 developed to predict the granule formation and growth (31, 36). While these studies provide
71 some models for granule formation, there has been little work to understand details of the
72 specific microbial aggregation mechanisms involved in the granulation process, which needs
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73 to be optimised for full-scale operation. o
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74 lo
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75 Aerobic granulation is often studied using laboratory-scale sequencing batch reactors (SBR) e
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76 fed with synthetic wastewater (18, 19). Within laboratory-scale SBR numerous operational o
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77 parameters can be manipulated to actively select for a granular sludge, these include, but are t
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78 not limited to; settling time (11, 15, 29), shear force (3, 34, 35) and high organic loading rates /a
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79 (4, 26). In contrast to studies of engineering process parameters, the fundamental .
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80 understanding of the microorganisms present within these systems has been under-
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81 investigated. Recently however, the use of modern molecular biology techniques is providing /

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82 novel insights into the formation and stability of these aerobic granular sludges within these D
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83 engineered systems and is helping to bridge the gap between successful reactor operation and e
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84 optimisation of the microbial ecology and stability (17, 27, 32, 38, 41). e
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85 ,

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86 This study uses an EBPR system enriched with Candidatus “Accumulibacter phosphatis” 1
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87 (Accumulibacter), which is one of the most important organisms responsible for phosphate
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88 removal (24), to investigate the microbial detail of granule formation from floccular sludge. s
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89 In the current study, two distinct granule types segregated from within a mixed-microbial
90 community on three occasions during granulation. This granulation process was intensely
91 investigated to deduce the mechanisms for this granule segregation. It is known that
92 microcolony structures present within activated sludge flocs have the potential for different
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93 microbial compositions and metabolic processes (30). Similarly, in anaerobic methanogenic
94 granules, microcolony cluster segregation occurs within the granular structure (12). However
95 the segregation of two different microbial granule types from within a mixed microbial
96 system has to our knowledge have not been previously reported. From our investigations of
97 the microbial ecology and structure of these two distinct granule types, two mechanisms for
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98 granulation were hypothesised. Further understanding and selection of these mechanisms and o
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99 understanding of the associated microbial ecology will provide insight towards facilitating the lo
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100 full-scale application of granular sludge. e
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101 Materials and methods
102 SBR Operation
103 Three laboratory-scale SBR were operated for granule formation. The first SBR, called
104 Synthetic-1 was operated for EBPR and fed with synthetic wastewater containing volatile
105 fatty acids (VFA) and orthophosphate. The reactor had a working volume of 8 L and 6 hr
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106 cycle time consisting of 6 min feed, 120 min anaerobic, 180 min aerobic, 4 min wasting, 40 o
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107 min settling and 10 min decanting. In each cycle, two litres of synthetic wastewater were fed lo
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108 into the reactor during the 6 min feed period, resulting in a hydraulic retention time (HRT) of e
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109 24 h. The solids retention time (SRT) was approximately 10 days. Dosing 0.5 M HCl and 2 M o
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110 NaOH controlled the pH during the anaerobic and aerobic phases between 7.8 and 8. The sole t
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111 carbon source in the synthetic feed alternated between acetate and propionate with a /a
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112 switching frequency of every two weeks, in order to provide selective advantages to .
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113 polyphosphate accumulating organisms (PAO) over glycogen accumulating organisms
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114 (GAO) (22). Further details of the synthetic feed, the feeding strategy and reactor operation /

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115 can be found in Lu, et al. (2006) (22). Chemical analyses were performed to detect the D
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116 biochemical transformations within cycles of the SBR operation (cycle study). The e
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117 orthophosphate (P-PO 3-) concentrations were analysed using a Lachat QuikChem8000 Flow e
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118 Injection Analyser (Lachat Instrument, Milwaukee, Wisconsin). VFAs were measured by ,

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119 Perkin-Elmer gas chromatography with column DB-FFAP 15 m x 0.53 mm x 1.0 µm (length 8

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120 x ID x film) at 140 °C.
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121 On day 0 of operation Synthetic-1 was inoculated with floccular sludge from a full-scale s
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122 wastewater treatment plant. Various SBR operational changes were implemented to influence
123 reactor performance and sludge selection during the 170-day operation. From day 63 onwards
124 the anaerobic phase was decreased from 120 min to 90 min and the aerobic phase was
125 increased from 180 min to 210 min to allow for further aerobic phosphorus uptake. From day
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126 98 onwards the reactor sludge wasting was adjusted to intentionally waste during the first four
127 minutes of the settle phase to selectively remove slower settling aggregates. From days 98 to
128 133 the volume of treated wastewater per cycle was increased from 2 L to 4 L. During this
129 period a diluted feed was used to maintain the same organic and nutrient loading as used
130 previously. After day-133 reactor operation was returned to day-63 operation.
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131 An additional two SBR were investigated at one time point only during this study and these o
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132 were termed Synthetic-2 (SBR treating synthetic wastewater for EBPR) and Domestic (SBR lo
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133 treating domestic wastewater for SNDPR). Synthetic-2 was operated with the same initial e
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134 parameters as Synthetic-1 (described above), except for the following; cycle time of 6 h o
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135 consisted of 6 min feed, 124 min anaerobic, 160 min aerobic, 65 min settling and decanting t
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136 and 5 min anaerobic idle period. For further details of Synthetic-2 operation see Zhou, et al. /a
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137 2008 (42). The Domestic reactor was operated with domestic wastewater feed, consisting of .
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138 by volume, 90% raw domestic sewage (see Supplementary Information (SI) Table S1 for
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139 details of composition of the domestic sewage) and 10% VFA feed, containing 1.5 g /

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140 NaAc·3H O/L. Domestic operation included two feed events such that the cycle time was 6 h D
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141 and consisted of 4 min feed, 100 min anaerobic, 130 min aerobic, 1 min feed, 50 min e
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142 anaerobic, 55 min aerobic, 7 min anoxic, 5 min settling and 5 min decanting. In each cycle 1 e
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143 L of wastewater was fed into the reactor resulting in a HRT of 12 hrs. The solids retention ,

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144 time (SRT) was approximately 18 days.
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145 Fluorescence in-situ hybridisation (FISH), light and stereo-microscopy analysis
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146 Granule samples were fixed and FISH was performed as previously described (1). For s
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147 quantitative FISH analysis of the different granule types, between five and ten granules of
148 each type were manually homogenised before analysis to provide an accurate representation
149 of bacterial populations present. For cryo-sectioned samples, fixed granules of the two
150 granule types were embedded in optimum cutting temperature compound prior to FISH
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151 (TissueTek, Sakura Finetek, Torrance, CA, USA), as previously described (27). Embedded
152 granules were frozen and sectioned into 10 µm thick slices using a cryotome operated at -20
153 °C (Kryo 1720, Leitz, Wetzler, Germany) and collected onto SuperFrost Plus microscope
154 slides (Menzel-Glaser, Braunschweig, Germany). All other samples were manually
155 homogenised before application onto Teflon-coated microscope slides. Samples were
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156 hybridised with FITC-labelled EUB Mix probes for detection of bacteria (2, 7), Cy3-labelled o
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157 GAO Mix probes (for Candidatus “Competibacter phosphatis” (Competibacter), a known lo
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158 GAO, (5)) and Cy5-labelled PAO Mix probes (for Accumulibacter, a known PAO, (6)) (See d

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159 SI Table S2 for detailed FISH probe information). Fluorescent DNA probes were visualized m

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160 and images captured with a Zeiss LSM 510 Meta confocal laser-scanning microscope tt
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161 (CLSM; Carl Zeiss, Jena, Germany) using a Zeiss Neofluor x 40/1.3 oil objective. Composite a
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162 images of cryo-sectioned granules were constructed using between 10 and 30 overlapping, .a
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163 consecutive images, which were reconstructed using Adobe Photoshop 7.0 (Adobe Systems, .
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164 San Jose, CA, USA). Images were analysed using daime version 1.2 (8). Image segmentation /
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165 was carried out using default parameters. Biovolumes were calculated from 20-30 images
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166 using the biovolume fraction function. The artefact rejection tool was set at a congruency m
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167 threshold of 75%. For stereo-microscopic examination, whole granules were photographed r

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168 using an Olympus SZH10 microscope and light-microscopic examinations on an Olympus ,
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169 BX51 microscope with a DP70 digital camera. 8

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170 Transmission electron microscopy (TEM) analysis g
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171 Granular sludge samples were first stabilised using 2.5% glutaraldehyde and 75 mM lysine in s
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172 0.1 M cacodylate buffer for 10 min (16), then high pressure frozen using a Leica EMPACT2
173 and then freeze substituted in 1.5% osmium tetroxide and 0.5% uranyl acetate in acetone at -
174 85 ˚C for 2 days. After warming to room temperature samples were washed in acetone,
175 submerged in Epon resin and polymerised at 60 ˚C for 2 days. Semi-thin sections of 500 nm
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176 were stained with 1% toluidine blue and 1% borax and viewed with an Olympus BX51 light-
177 microscope. Ultra-thin sections of 60 nm thicknesses were cut using a Leica Ultracut UC6
178 ultramicrotome and mounted on the Formvar-coated copper grids, stained with 5% uranyl
179 acetate in 5% methanol and Reynolds lead citrate and viewed using a JEOL (Tokyo, Japan)
180 1010 TEM operated at 80 kV.
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181 Physical analysis of activated sludge o
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182 The activated sludge aggregates particle size distribution was measured to monitor the lo
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183 distribution of granule size. 30 ml of well-mixed sludge was sampled from the SBR at the end e
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184 of the aeration period and applied to a Malvern laser light scattering instrument, Mastersizer o
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185 2000 series (Malvern Instruments, Worcestershire, UK). t
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186 DNA extraction /a
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187 White and yellow granules from Synthetic-1 and Domestic reactor were easily distinguishable .
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188 by colour and were manually separated prior to DNA extraction. Genomic DNA was
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189 extracted from white and yellow granule samples using the FastDNA SPIN for Soil Kit (MP /

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190 Biomedicals). D
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191 ppkI clone library construction and phylogenetic analysis e
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192 Amplification of Accumulibacter ppkI gene fragments from the Synthetic-1 white and yellow e
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193 granules was performed using the ACCppkI-254F and ACCppkI-1376R primers as previously ,

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194 described (25). The reaction mixtures contained 1X PCR buffer (New England Biolabs), 3
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195 mM MgCl2, 200 µM of each dNTP, 400 nM of each forward and reverse primer and 0.5 U/µl g
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196 of DNA Polymerase (New England Biolabs). The PCR was conducted using a MyCycler s
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197 (Bio-Rad) with an initial 10 min denaturation step at 96 °C, followed by 30 cycles of 96 °C
198 for 30 s, 64 °C for 1 min and 72 °C for 2 min and a final extension at 72 °C for 10 min. PCR
199 products (approximately 1100 bp) were visualised by agarose gel electrophoresis, purified
200 using the QIAquick PCR Purification Kit (Qiagen) and cloned into pGEM-T Easy Vector
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201 (Promega) using One Shot TOP10 (Invitrogen) competent cells. Twenty clones were selected
202 for further sequencing and phylogenetic analysis. An un-rooted phylogenetic tree was
203 constructed by the neighbor joining (21) method and evaluated by bootstrap re-sampling
204 (1000 replications) using MEGA (Molecular Evolutionary Genetics Analysis) version 4.0
205 (33) and CLUSTALX version 2.0.5 (37).
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206 16S rRNA gene terminal restriction fragment length polymorphism (tRFLP) o
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207 fingerprinting lo
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208 PCR was performed on white and yellow granules from the Domestic SBR using the bacterial e
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209 (63F and 1389R)(23) primer sets, except that the forward primer (63F) was labelled at the o
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210 5’end with a phosphoramidite dye 6-FAM. Reaction mixtures contained 1X PCR buffer (New t
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211 England Biolabs), 2 mM MgCl , 200 µM of each dNTP, 400 nM of each forward and reverse /a
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212 primer, 0.5 U/µl of DNA Polymerase (New England Biolabs), approximately 20 ng of .a
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213 template DNA and were performed in triplicate. PCR using the 16S rRNA gene primers was .
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214 conducted in a MyCycler (BIO-RAD) with an initial 10 min denaturation step at 96 °C,
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215 followed by 30 cycles at 96 °C for 30 s, 56 °C for 1 min and 72 °C for 2 min and a final
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216 extension at 72 °C for 10 min. PCR products were purified using the QIAquick PCR m
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217 Purification Kit (Qiagen) and used in separate endonuclease restriction digestions using the
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218 enzymes; AluI, HaeIII, MspI and Sau3AI. Digestions were performed as per the 2
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219 manufacturer’s instructions. 16S rRNA gene digests were analysed at the Australian Genome
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220 Research Facility (Glen Osmond, SA, Australia) on an AB3730 Genetic Analyzer (Applied g
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221 Biosystems, Foster City, CA, USA) fitted with a 36 cm array and using the GS500(-250)LIZ t
222 standard. Further details of the tRFLP analysis are in the SI.
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