The microbial landscape and architecture of aerobic granules, their life stages and practical ...

25 Pages·2014·2.19 MB·English

AEM Accepts, published online ahead of print on 21 March 2014
Appl. Environ. Microbiol. doi:10.1128/AEM.00250-14
Copyright © 2014, American Society for Microbiology. All Rights Reserved.
1 The microbial landscape and architecture of aerobic granules, their life
2 stages and practical implications
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4 Graciela Gonzalez-Gil# and Christof Holliger
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5 École Polytechnique Fédérale de Lausanne (EPFL), School of Architecture, Civil and Environmental o
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6 Engineering (ENAC), Laboratory for Environmental Biotechnology (LBE), Lausanne, Switzerland. lo
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8 # Corresponding author: [email protected] f
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9 [email protected]

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15 Running Title: Microbial architecture of aerobic granules N
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27 Abstract
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29 For the successful application of aerobic granules in wastewater treatment, granules
30 containing an appropriate microbial assembly able to remove contaminants should be retained
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31 and propagated within the reactor. To manipulate and/or optimize this process, a good
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32 understanding of the formation and dynamic architecture of the granules is desirable. Models n
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33 of granules often assume a spherical shape with an outer layer and an inner core but limited d
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34 information is available regarding the extent of deviations from such assumptions. We report f
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35 on new imaging approaches to gain detailed insights into the structural characteristics of

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36 aerobic granules. Our approach stained all components of the granule to obtain a high quality p
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37 contrast in the images; hence limitations due to thresholding in the image analysis were e
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38 overcome. A 3-D reconstruction of the granular structure was obtained that revealed the a
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39 mesoscopic impression of the cavern-like interior of the structure, showing channels and .o
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40 dead-end paths in detail. In “old” granules, large cavities allowed for the irrigation and growth /
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41 of dense microbial colonies along the path of the channels. Hence, in some areas, N
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42 paradoxically higher biomass content was observed in the inner part of the granule compared e
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43 to the outer part. Microbial clusters ‘rooting’ from the interior of the mature granule structure e
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44 indicate that granules mainly grow via biomass outgrowth and not by aggregation of small 8
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45 particles. We identify and discuss phenomena contributing to the life-cycle of aerobic 0
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46 granules. With our approach, volumetric tetrahedral grids are generated that may be used to b
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47 validate complex models of granule formation. u
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49 Keywords: microbial aggregates / aerobic granules/ granular sludge / phosphorus removal /
50 3D reconstruction model
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52 Introduction
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54 Granules are compact, quasi-spherical aggregates formed by self-immobilized, mixed-
55 microbial communities embedded in a matrix of extracellular bio-polymeric substances (1).
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56 The phenomenon of granule formation in wastewater treatment systems was first described in
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57 anaerobic bioreactors (2, 3). The technological success of anaerobic granules in the treatment n
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58 of highly concentrated industrial wastewaters (4) has been key to stimulating research in d
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59 developing their aerobic counterparts. Previous studies have shown that in sequencing batch f
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60 reactors and under certain operational conditions, which include high shear stress and short

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61 settling times (5), activated sludge can form aerobic granules (5, 6). Compared to flocs, p
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62 granules can accommodate five times the biomass concentration (7) and settle ten times faster e
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63 (8, 9), and these characteristics translate into small foot-print wastewater treatment plants. a
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64 Therefore, the use of aerobic granular sludge-based systems stands as a promising alternative .o
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65 technology to conventional activated sludge treatment systems (10). To guarantee the /
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66 successful application of the aerobic granular sludge technology, granules should be N
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67 physically stable and should contain an appropriate microbial assembly able to remove the e
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68 desired contaminants. Moreover, in the long term, this assembly should be retained and e
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69 should propagate within the reactor and should be resistant to environmental changes. Most 8
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70 research so far has focused on the engineering aspects of the reactors and on operational 0
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71 variables necessary to induce granulation (11-16). Recently at a molecular level, the makeup b
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72 of the microbial assemblies of the aggregates has been studied for aerobic granules under u
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73 different conditions (17-19). Aerobic granules exposed to temporal anaerobic-aerobic cycles t
74 display the ability to biologically remove phosphorus and nitrogen in a single reactor (7).
75 Mathematical simulations show that bulk oxygen concentrations of 2 mg l-1 and granules sizes
76 between 1.2 to 1.4 mm would be required for optimum nutrient removal because such
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77 conditions would result in an appropriate ratio of aerobic-anoxic volumes within the granule
78 (20). Conceptual and mathematical models of granules assume a spherical shape with an outer
79 layer and an inner core. Although this approach has increased our understanding of the
80 process, models are often only validated against macro-scale results with limited or no micro-
81 scale observations (21, 22). Despite the fact that the physical architecture of the granules is D
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82 intrinsically related to mass transport, conversion processes and niches partitioning within the n
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83 granules, few reports describe details on the micro-structure of aerobic granules (23, 24).
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84 Additionally, an effective approach to stain all the constituents of the aggregates is required
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85 because most previous research has focused on fluorescence methods to target specific m

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86 bacterial types. To better understand granule architecture and function we (i) explored a tp
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87 different approach to the staining and imaging of the granular structure and (ii) reconstructed
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88 the granular sludge in 3D. .a
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92 Materials and methods e
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94 Origin of granule samples 8
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95 Aerobic granules with highly enhanced biological phosphorus removal capacity were 0
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96 obtained from two 2.4 liters sequencing batch reactors fed with propionate or acetate as b
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97 described previously in detail (18). Briefly, the reactors were inoculated with activated sludge u
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98 from the Uetendorf sewage treatment plant, Thun, Switzerland; a plant that removes N and P t
99 biologically. The reactors were operated at 20 °C in a sequencing batch mode with 3 h-cycles
100 consisting of an anaerobic feeding phase at a flow rate of 1.2 L h-1(60 min), an aeration phase
101 with air flow rate at 4 L min-1(112 min), a settling phase (3 min) and a withdrawal phase (5
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102 min). The pH was controlled at 7 to 7.3. The composition of the synthetic wastewater used to
103 feed the reactors was as given previously (7). The organic loading rate for both reactors was
104 1.8 g COD liter-1 d-1. The oxygen concentration was maintained at saturation levels during the
105 aerobic phase.
106 D
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107 Preparation of thin sections of granules n
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108 Granules were fixed overnight at 4°C in 4% formaldehyde in phosphate-buffered saline
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109 (PBS), pH 7.4. The granules were then washed three times with PBS and stored overnight at
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110 20°C in a PBS-ethanol solution (1:1, vol/vol). The granules were then dehydrated using an m

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111 ethanol series of 50-80-100%, 20 min per step. Dehydrated samples were cleared with xylene tp
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112 to remove any remaining ethanol and then infiltrated with and embedded in paraffin wax.
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113 Granule sections 12 μm thick were obtained with a microtome (Microm, HM360) and placed .a
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114 on SuperfrostPlus slides (Menzel-Gläzer, Braunschweig). .
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116 Fluorescence in situ hybridization (FISH) N
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117 After removing the paraffin with xylene (2x20 min) followed by washing in 100% ethanol for e
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118 5 min, a section corresponding to the center of the granule was subjected to FISH procedures
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119 as reported earlier (25) and using a mixture of the following probes: Cy3-labelled PAO 8
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120 [PAO462, PAO651, PAO846] (26) and FAM-labeled GAO [GAOQ431, GAOQ989, 0
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121 GB_G2] (27, 28). The final concentration of each Cy3-labeled and FAM-labeled probe was 3 b
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122 ng ml-1 and 5 ng ml-1, respectively, and the hybridization conditions were as previously g
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123 described (26-28). After removing the unbound probes, the sections were counterstained with t
124 DAPI (20 μg/ml) for 20 min under dark conditions, washed with cold distilled water and air-
125 died before being mounted with citifluor AF1 antifading solution (Citifluour, England). The
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126 hybridized sections were observed and documented using a Leica DM5500 fluorescence
127 microscope and its associated software.
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129 Hematoxylin and eosin staining
130 For hematoxylin-eosin treatment, the sections were deparaffinized in xylene and hydrated. D
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131 Then, sections were stained with hematoxylin for 10 min, washed with water for 10 min and n
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132 counterstained with eosin for 2 min. Thereafter, the sections were dehydrated using an ethanol
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133 series (70-96-100 %, 3x2 min each), cleared with xylene (2x2 min) and mounted in Cytoseal
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134 60 (Micron International). The thin sections were observed using a Leica DM5500 m

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135 microscope in bright-field mode and documented using a DFC 320 R2 camera with the Leica tp
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136 Application Suite software. Dark-field observations were conducted using a Nikon Eclipse
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137 E800 microscope and NIS elements software. .a
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138 .
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139 Scanning electron microscopy (SEM) /

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140 Intact granules were prepared as described previously (29) and images were acquired using a
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141 Philips FEI XLF30 microscope at 5kV. v
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142 b
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143 Granule 3D reconstruction.
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144 Bright-field images of consecutive thin sections were used and registered (i.e., aligned) 0
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145 manually using Adobe Photoshop CS3. Thereafter, images were segmented to remove the
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146 background-related signal from the granule-related signal using the ImageJ software program g
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147 (30) with the Color Segmentation plug-in which is based on the k-mean algorithm t
148 (http://bigwww.epfl.ch/sage/soft/colorsegmentation/). From the results of the color
149 segmentation, a binary mask was obtained using the threshold function in ImageJ (intensity=1
150 and 0 for the granule and background-related signals, respectively). This binary mask was
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151 then multiplied by each channel of the RGB image (color segmented) and then the channels
152 were merged. The images were then stacked and 3D visualized using Avizo (Visualization
153 Sciences Group) image-processing software. The image processing and handling was
154 performed with a 64-bit, 32GB RAM work station.
155 D
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156 Estimation of granule characteristics from images n
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157 The fractal dimension of the granule was estimated by the box counting method as
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158 implemented in the ImageJ plugin BoneJ (31). This method counts the number of boxes
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159 required to cover the ribbon of the object at a given box scale. The scale is subsequently m

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160 changed and the box counting is repeated. The fractal dimension is computed as the ratio of tp
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161 the change in the log of the box count to the change in the log of the box scale (King et al
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162 2009). The fractal values obtained by this method vary between 1 and 2 with higher values .a
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163 indicating a more irregular shape. .
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164 Using ImageJ, the porosity was estimated for each binary image corresponding to a different /

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165 radial-location and calculated as the percentage of voids with respect to the total area of the
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166 granule. Further details can be found in the supporting material. v
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169 Results 0
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170 b
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171 Cluster structure of aerobic granules u
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172 The general appearance of mature aerobic granules is shown in Fig. 1A and 1B. The t
173 population of mature granules was comprised of (i) quasi-spherical entities showing large
174 cluster structures, (ii) quasi-spherical entities from which various clusters had been detached,
175 (iii) quasi-half broken granules, and (iv) small- and medium-sized newly growing granules
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176 from the detached clusters (Fig. 1C). These forms illustrate the life cycle of these mature
177 granules (see Fig. S1 in the supplemental material). Low- and high-magnification SEM
178 images show the cluster architecture of the granules in detail (Fig. 1D and 1E). The clusters
179 are comprised of dense bacterial populations with coccus- and rod-like morphologies. Few
180 bacterial filaments were found between cluster areas in which lower shear forces may exist D
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181 (Fig. 1F and 1G). n
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183 Fluorescence images of cross-sectioned propionate-cultivated granules showed that the large
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184 cluster structures were mainly composed of Accumulibacter-type bacteria, which are known m

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185 to accumulate poly-phosphate and are commonly found in enhanced biological phosphorus tp
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186 removal systems. These structures often protuberated from the “periphery of the granule”
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187 (Fig. 2A). A contiguous section of identical granules examined by hematoxylin-eosin (H-E) .a
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188 staining and observed under bright- (Fig. 2C) and dark-field (Fig. 2D) showed the spatial .
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189 location of a clear glue-like exopolymeric material on the edge of parts of the granule. /

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190 Similarly, fluorescence images of a large acetate-cultivated granule (3 to 4 mm in diameter)
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191 revealed that Accumulibacter also formed large cluster-structures which protuberated from the v
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192 “periphery of the granule” (Fig. 3A and B). The H-E images (Fig. 3C and D) revealed the b
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193 arrangement of cavities and the “root” of large clusters in the interior of the granules. In
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194 addition, these images showed that the overall structure of the granule was preserved after 0
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195 histological processing. In large granules, less substrate can penetrate, thus inherently less
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196 biomass can grow in the inner zones. The biomass present in those zones will eventually g
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197 decay leaving empty spaces, thus the granule may become porous. Although further t
198 confirmation tests using imaging under more natural conditions (i.e. hydrated state) than what
199 we used would be required, the granules in this study show cavities particularly in the inner
200 zones (Fig. 3 and Fig.4). The dense bacterial populations in the outer part of the granules are
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201 better contrasted in the dark-field mode in which they appear darker and closer in wavelength
202 to the strong pink and purple color of the original H-E staining (Fig. 3C and D).
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204 Images of a propionate-cultivated granule taken at the end of the experiment (day 380)
205 revealed various types of colonies and heterogeneous cell densities even on the surface of the D
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206 granule (Fig. 4A to 4C). Thin sections imaged from a complete granule sampled on day 380 n
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207 showed visible signs of erosion, e.g., the surface of an “old” granule (Fig. 4D) appeared more
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208 irregular than that of “mature” granules sampled on day 169 ( Fig. 2C and Fig. 3C). The “old”
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209 granules can most likely remain quasi intact for long periods of time before splitting. The m

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210 large cavities observed in the “old” granule allowed irrigation and growth of dense colonies tp
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211 along the paths of the channels (Fig. 4E). Hence, in some areas and along an x-axis,
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212 paradoxically higher biomass was observed in the inner part of the granule compared to the .a
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213 outer part (Fig. 4E). Observations at higher magnification revealed in more detail the “root” .
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214 of large clusters from the interior of the granules (Fig. 5). /

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216 The surface and porosity characteristics of granules e
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217 Because of the importance of surface characteristics for the hydrodynamics at the solid-liquid b
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218 interface (32) and the distinct surface characteristics observed in aerobic granules, we
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219 explored the possibility of quantifying the surface morphology at the granule structure-liquid 0
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220 interface reflected by the fractal dimension. Aerobic granules with an outer filamentous
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221 surface (i.e., hairy surface) are often observed in the initial phases during the granulation g
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222 process. The estimated fractal dimension for this type of filamentous granules was 1.3 ± 0.1, t
223 whereas the fractal dimension of mature aerobic granules was 1.08 ± 0.05 (Fig 6A). Stereo
224 microscopic images, however, do not display the internal structure-liquid interface of the
225 granules. Hence, the fractal dimension (1.4 ± 0.06) was estimated from images acquired using
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226 thin sections obtained from the center of granules (Fig. 6A aerobic internal structure) and
227 tended to be higher (i.e., a more complex structure) than at the surface (Fig. 6B). The porosity
228 as a function of the granule radial distance was estimated by using images from a series of
229 thin slices. The space occupied by voids in the central zone of the granule was approximately
230 35 to 40%, whereas estimated porosities in the outer zone were as low as 10% (Fig. 7). D
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231 n
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232 3D reconstruction
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233 A 3D model based on the measured information may allow for a better understanding of
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234 substrate supply and intermediates transport within the cavern-like architecture of the granules m

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235 (see Movie S1 in the supplemental material). Such models may offer the opportunity to tp
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236 explore the extent of dead-end pores and/or connectivity of the internal channeling structure.
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237 Because these features are key determinants in fluid flow within the granule, a 3D model of .a
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238 the aerobic granule was obtained using a series of 2D images (see Fig. S2 in the supplemental .
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239 material). The resulting 3D rendering allows a mesoscopic impression of the cavern-like /

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240 interior structure of the granule (Fig. 8).
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243 Discussion 8
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244 0
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245 Theories for anaerobic (3) and aerobic granule (33) structure formation suggest that b
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246 filamentous microorganisms serve as a backbone for granule structure. Also the floc-forming u
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247 bacteria Zoogloea are key players in activated sludge flocs formation, and aerobic granules t
248 dominated by this genus have been cultivated (34, 35). Indeed when the aerobic granules used
249 in the present study were formed, our previous molecular analysis identified the presence of
250 numerous filamentous bacteria (Thiothrix spp.) and floc-forming (e.g., Zoogloea spp.)
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