This research was financially supported by the SIAM Gravitation Grant 024.002.002, the Netherlands Organization for Scientific Research and by the Dutch Technology Foundation (STW - Simon Stevin Meester 2013). The authors want to thank Mario Pronk for providing the granular sludge samples.

NOTE: AGS was collected from the Nereda pilot reactor at the wastewater treatment plant Utrecht, the Netherlands. The reactor was fed with municipal sewage. The granular sludge had a sludge volume index (SVI5min) of 59.5 ml/gVSS. The sludge was sampled in April at the end of an aerobic cycle. After sampling, the sludge was immediately transported to the laboratory, sieved and stored at -20 &#176;C until use.
1. EPS Extraction
NOTE: Centrifuge granular sludge at 4,000 &#215; g and 4 &#176;C for 20 min, and decant the supernatant. Collect granules in the pellet for the extractions. The total solids (TS) and volatile solids (VS) of the granules were determined by the standard methods23. The conversion factor between granule wet weight &#8212; the weight of the granules taken directly from the pellet &#8212; and the TS was determined prior to the extraction. All extractions were done in triplicates.
NOTE: 3 g wet granules were used for each extraction method. Their TS and VS values (0.39 g TS and 0.34 g VS), measured in triplicates, were used to calculate the extraction yield.
<ol>
	<li>Centrifugation extraction11
	<ol>
		<li>Transfer 3 g (wet weight) of granules into a centrifugation tube and fill up the centrifugation tube to 50 ml with demineralized water.</li>
		<li>Slightly shake the centrifugation tube by hand.</li>
		<li>Centrifuge the centrifugation tube containing the mixture at 4,000 &#215; g and 4 &#176;C for 20 min.</li>
		<li>Collect the supernatant in a glass beaker, discard the pellet and continue with the supernatant as described in section 1.7.</li>
	</ol>
	</li>
	<li>Sonication extraction10
	<ol>
		<li>Transfer 3 g (wet weight) of granules into a centrifugation tube and fill up the centrifugation tube to 50 ml with demineralized water.</li>
		<li>Apply pulsed sonication on ice for 2.5 min at 40 W to the mixture.</li>
		<li>Centrifuge the centrifugation tube containing the mixture at 4,000 &#215; g and 4 &#176;C for 20 min.</li>
		<li>Collect the supernatant in a glass beaker, discard the pellet and continue with the supernatant as described in section 1.7.</li>
	</ol>
	</li>
	<li>Ethylenediaminetetraacetic acid (EDTA) extraction11
	<ol>
		<li>Transfer 3 g (wet weight) of granules into a 100 ml glass bottle and fill up the bottle to 50 ml with 2% (w/v) EDTA solution.</li>
		<li>Slightly shake the bottle by hand and store it in the refrigerator at 4 &#176;C for 3 hr.</li>
		<li>Transfer the mixture into a 50 ml centrifugation tube.</li>
		<li>Centrifuge the centrifugation tube containing the mixture at 4,000 &#215; g and 4 &#176;C for 20 min.</li>
		<li>Collect the supernatant in a glass beaker, discard the pellet and continue with the supernatant as described in section 1.7.</li>
	</ol>
	</li>
	<li>Formamide &#8211; sodium hydroxide extraction (NaOH)13
	<ol>
		<li>Transfer 3 g (wet weight) of granules into a 100 ml glass bottle and fill up the bottle to 50 ml with demineralized water.</li>
		<li>Add 0.3 ml 99% formamide.</li>
		<li>Slightly shake the bottle by hand and store it in the refrigerator at 4 &#176;C for 1 hr.</li>
		<li>Add 20 ml 1 M NaOH to the granule suspension.</li>
		<li>Slightly shake the bottle by hand and store it in the refrigerator at 4 &#176;C for 3 hr.</li>
		<li>Transfer the mixture evenly into two 50 ml centrifugation tube.</li>
		<li>Centrifuge the centrifugation tubes containing the mixture at 4,000 &#215; g and 4 &#176;C for 20 min.</li>
		<li>Collect the supernatant in a glass beaker, discard the pellet and continue with the supernatant as described in section 1.7.</li>
	</ol>
	</li>
	<li>Formaldehyde &#8211; NaOH extraction11
	<ol>
		<li>Transfer 3 g (wet weight) of granules into a 100 ml glass bottle and fill up the bottle to 50 ml with demineralized water.</li>
		<li>Add 0.3 ml 37% formaldehyde.</li>
		<li>Slightly shake the bottle by hand and store it in the refrigerator at 4 &#176;C for 1 hr.</li>
		<li>Add 20 ml 1 M NaOH to the granule suspension.</li>
		<li>Slightly shake the bottle by hand and store it in the refrigerator at 4 &#176;C for 3 hr.</li>
		<li>Transfer the mixture evenly into two 50 ml centrifugation tube.</li>
		<li>Centrifuge the centrifugation tubes containing the mixture at 4,000 &#215; g and 4 &#176;C for 20 min.</li>
		<li>Collect the supernatant in a glass beaker, discard the pellet and continue with the supernatant as described in section 1.7.</li>
	</ol>
	</li>
	<li>High temperature &#8211; sodium carbonate extraction (Na2CO3)9,22,24
	<ol>
		<li>Pre-heat 150 ml tap water in a 1,000 ml glass beaker on a magnetic stirrer to 80 &#176;C.</li>
		<li>Transfer 3 g (wet weight) of granules in a 250 ml baffled flask and fill up the flask to 50 ml with demineralized water.</li>
		<li>Add 0.25 g Na2CO3 anhydrous or 0.67 g Na2CO3&#8226;10H2O into the flask to obtain a 0.5% (w/v) Na2CO3 concentration.</li>
		<li>Put the flask containing the mixture into the water bath. Cover the flask and the beaker glass separately with aluminum foil to prevent evaporation.</li>
		<li>Stir the mixture for 35 min at 400 rpm and 80 &#176;C.</li>
		<li>Transfer the mixture into a 50 ml centrifugation tube.</li>
		<li>Centrifuge the centrifugation tube containing the mixture at 4,000 &#215; g and 4 &#176;C for 20 min.</li>
		<li>Collect the supernatant and discard the pellet.</li>
	</ol>
	</li>
	<li>TS and VS measurement of all extracts according to the standard methods23.
	<ol>
		<li>Take the supernatant and dialyze it for 24 hr against 1,000 ml ultrapure water (dialysis bag with 3,500 Da molecular weight cut off (MWCO))11,12. Change the dialysis water after 12 hr to enhance the effect of the dialysis.</li>
		<li>Transfer a reasonable fraction (around 1/3) of the dialyzed supernatant to an aluminum dish for TS and VS measurement23. 
		NOTE: Dry the sample overnight at 105 &#176;C. The weight difference of the empty aluminum dish and the aluminum dish containing the dried sample is the TS content. Then burn the same aluminum dish containing the sample at 550 &#176;C for 2 hr. The weight difference between the empty aluminum dish and the aluminum dish containing the burned sample is the ash content. The difference between TS and ash content is the VS content.</li>
		<li>For each extract, transfer the residual fraction of the dialyzed supernatant to 10 ml glass beakers. Thicken the supernatant for 2 days at 60 &#176;C to a final volume of 1-2 ml to increase the polymer concentration in the supernatant.</li>
	</ol>
	</li>
</ol>
2. Alginate-like Extracellular Polymer (ALE) Extraction
<ol>
	<li>Dialyze the extract obtained in step 1.6.8 according to step 1.7.1.</li>
	<li>Transfer the dialyzed extract into a 250 ml glass beaker. Slowly stir the extraction at 100 rpm and room temperature. Constantly monitor pH changes with a pH electrode, while adding 1 M hydrochloric acid (HCl) to a final pH of 2.2 &#177; 0.05 to obtain ALE in the acidic form.</li>
	<li>After adjusting the pH to 2.2, transfer the extract into a 50 ml centrifugation tube and centrifuge at 4,000 &#215; g and 4 &#176;C for 20 min.</li>
	<li>Discard the supernatant and collect the gel-like pellet. The gel-like pellet is ALE in the acidic form.</li>
	<li>To obtain the sodium (or potassium) form of ALE, slowly add 0.5 M NaOH (or 0.5 M potassium hydroxide) to the gel obtained in step 2.4, while mixing the gel slowly with a glass stick by hand until pH 8.5 is reached.</li>
</ol>
3. Ionic Hydrogel Formation Test
NOTE: In order to check if the extracted EPS had ionic hydrogel formation properties, a bead formation test with Ca2+ ions was used25.
<ol>
	<li>After thickening of the extract in step 1.7.3 to a volume of 1-2 ml, slowly stir the mixture with a glass stick and adjust its pH to 8.5 with 0.5 M NaOH.</li>
	<li>Take the extract of step 3.1 or the sodium ALE of step 2.5 and slowly drip the extract with a Pasteur pipette into a 2.5% (w/v) calcium chloride (CaCl2) solution. 
	NOTE: If the extracted EPS has ionic hydrogel gel forming properties, drop-shaped (spherical) beads will be formed. If the extracted EPS has no ionic hydrogel gel forming properties, the extract will disperse in the CaCl2 solution.</li>
</ol>
4. Stability Test of the Ionic Hydrogel
NOTE: To further understand the role of the ionic EPS hydrogel in AGS structure formation, stability tests were performed on the ionic hydrogel beads of the Na2CO3 extraction, collected in step 3.2.
<ol>
	<li>Keep the hydrogel beads for 30 min in the CaCl2 solution.</li>
	<li>Use a spoon to take out the hydrogel beads from the CaCl2 solution and split the beads in four equal fractions.</li>
	<li>Store fraction 1 in 10 ml demineralized water for 4 hr at 4 &#176;C. 
	The following stability tests were performed in the same manner as described in the extraction methods 1.3 - 1.5.</li>
	<li>Store fraction 2 in 10 ml 2% (w/v) EDTA solution for 3 hr at 4 &#176;C.</li>
	<li>Store fraction 3 in 7.15 ml demineralized water with 60 &#181;l 99% formamide for 1 hr at 4 &#176;C. Then add 2.85 ml 1 M NaOH and store fraction 3 for 3 hr at 4 &#176;C.</li>
	<li>Store fraction 4 in 7.15 ml demineralized water with 60 &#181;l 37% formaldehyde for 1 hr at 4 &#176;C. Then add 2.85 ml 1 M NaOH and store fraction 4 for 3 hr at 4 &#176;C.</li>
	<li>Monitor if there is visible disintegration of the beads during the storage under the conditions described in 4.3 - 4.6 to evaluate if the beads withstand the extraction conditions.</li>
</ol>

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The authors have nothing to disclose.

Remarks for the protocol section 
The extraction of EPS/ALE is described for a volume of 50 ml and 3 g of granules. These values are intended as guidelines. Extractions with higher granule concentrations can decrease the yield of the extracted EPS. During the extraction of ALE the temperature should be kept constant at 80 &#176;C for 30 min. The time required for the mixture to heat up (around 5 min) is included in the protocol. Furthermore, the extraction efficacy is enhanced by using a magnetic stir bar of the same size as the diameter of the flask bottom. This will result in good mixing properties and milling effects, promoting the extraction of EPS.
Later in the protocol section, TS and VS yields of all extractions (supernatant collected in steps 1.1-1.6) are determined. Dialysis needs to be performed prior to TS and VS measurement to decrease possible errors owing to the presence of chemicals used for extractions. A MWCO of 3,500 Da is recommended to remove these chemicals while retaining the EPS macromolecules within the dialysis bag. The dialysis bag should have a larger volume than the volume of the extract. This is necessary, because the volume of the extract will increase during the dialysis (e.g., for EDTA extraction up to 40% volume increase). The extent of chemical removal by dialysis can be determined by measuring the pH in the sample prior and after dialysis. Alternatively, conductivity measurements of the dialysis water show the extent of ion removal.
To obtain ALE from the total extracted EPS (steps 1.6 and 2) the dialysis step is optional. Nevertheless, dialysis has three benefits: it reduces the amount of HCl needed for the precipitation, it improves the acid mass transfer in the extract and decreases the ash content of the obtained ALE. For the precipitation of ALE it is recommended to use a glass beaker with a much larger volume than the extract. Na2CO3 is normally overdosed in the extraction. The added HCl will first react with the Na2CO3 left in the extract, resulting in carbon dioxide formation and, if the sample was not dialyzed before, in foaming. During the addition of HCl, the extract should be stirred slowly with a magnetic stir bar of the same size as the bottom of the beaker. A stir bar of this size and slow stirring will result in even mixing without breaking the structure of the precipitate. If acidic gel clumps are formed in the extract, the beaker should be swirled slightly by hand. The precipitation is conducted with an acid concentration of 1 M to avoid a large volume increase of the extract while still obtaining a homogeneous distribution of the acid in the sample. Higher acid concentrations can result in a regional pH decrease and acidic gel clumps formation. A pH lower than 2.0 decreases the amount of ALE that can be recovered, probably due to structural changes of the polymers at lower pH. It is therefore important to keep the final pH at 2.20 &#177; 0.05.
Limitations 
The ALE extraction method aims to extract structural extracellular polymers of the EPS from AGS or biofilms in general and is not intended to extract all present EPS. To extract all EPS, a combination of more than one extraction method is necessary. Moreover, as shown with the increase of the VSEPS yield by applying a double and quadruple extraction, one single extraction will not extract all structural EPS. ALE extraction is a harsh EPS extraction method, combining constant mixing with heat and alkaline conditions. For this reason it is possible that some intracellular material is extracted together with the EPS. Although cell lysis can be caused by physical and chemical extraction techniques (sonication31,32, NaOH31,32, EDTA11,32, CER32, heat32 and high shear rates by mixing19), the presence of intracellular material in recovered EPS still needs to be verified. The ionic gel-forming property of the extracted EPS is the main focus of this research, whether the recovered EPS contains intracellular material was not analyzed. Future research will focus on identifying intracellular material in the extracted EPS.
Solubilizing the hydrogel matrix of AGS is crucial to extract structural EPS 
EPS forms a dense and compact hydrogel matrix in AGS. Although EPS contains various classes of organic macromolecules such as polysaccharides, proteins, nucleic acids, (phospho)lipids, humic substances and some intercellular polymers7,5,8, not all of them form a gel. Only those gel-forming polymers are here considered as structural polymers in EPS.
The aim of EPS extractions is to first solubilize EPS and then to collect the solubilized EPS. If the structural EPS (i.e., the EPS forming a hydrogel) is the target of the extraction, the gel matrix of AGS has to be solubilized first. Only methods that can solubilize the gel matrix are capable of extracting structural EPS. In this research, some frequently used EPS extraction methods such as centrifugation10-15, sonication10,14,15, EDTA10-12,14,15, formaldehyde + NaOH10-15 and formamide + NaOH13 could not efficiently isolate the structural EPS. This is due to the fact that the hydrogel matrix of the aerobic granules was not solubilized by these methods. For this reason, stability tests in section 4 were only performed with conditions present in EDTA, formamide + NaOH and formaldehyde + NaOH extraction. These three extractions were not capable of isolating structural EPS, but still obtained the highest VSEPS yield besides the Na2CO3 extraction. Conditions of the Na2CO3 extraction were not applied as this extraction method clearly solubilized the AGS matrix. Hence the applied conditions during the stability test were considered representative.
Extraction with cation exchange resin (CER), another frequently used EPS extraction method, was not considered for this comparison, as previous studies on EPS extraction with CER did not yield better results than the chemical extractions used here.
Gel-forming EPS in AGS 
Gel-forming EPS are considered as the structural EPS in the hydrogel matrix of AGS. It is worth pointing out that there are various kinds of hydrogels such as ionic gels, temperature-induced gels and pH induced gels. This study only focuses on EPS that form ionic gels. Regarding the large fraction of structural gel material extracted, this is likely to be the dominant structural EPS. There are certainly possibilities that other kinds of EPS that form different kinds of hydrogels (e.g., pH induced gel28) exist in the same or other type of aerobic granules. Nevertheless, no matter what kind of hydrogel is targeted, solubilizing the EPS gel matrix is the most important step to extract gel-forming EPS.
Currently, little research has been done on structural EPS of granular sludge. The ALE extraction described in this protocol is capable of extracting gel-forming EPS from AGS and will be used in future studies to characterize structural EPS. More research needs to be done on AGS, structural EPS and non-structural EPS to better understand the process and function of granulation and EPS. Especially the following three points need to be investigated: why microorganisms produce such a large amount of EPS, what is the exact composition of EPS and how is the composition of EPS modified depending on environmental changes. Detecting and analyzing all involved compounds and their interactions will help to understand biofilms and how to use them to our advantage.

In recent years the aerobic granular sludge (AGS) process has become a popular biological wastewater treatment process, successfully applied at several full-scale wastewater treatment plants1. In contrast to the conventional activated sludge process, in the AGS process the microorganisms form granules instead of flocs2. These granules have better settleability, are able to withstand higher organic loading rates, and have higher tolerance to toxicity than activated sludge flocs3.
Unlike biofilms, AGS is formed spontaneously without involvement of any carrier material4. In AGS, like in biofilms, microorganisms produce a significant amount of highly hydrated extracellular polymeric substances (EPS)5 to form a hydrogel matrix in which they are self-immobilized4-6. EPS are a complex mixture, consisting of polysaccharides, proteins, nucleic acids, (phospho)lipids, humic substances and some intercellular polymers5,7,8. These polymeric substances interact with each other through electrostatic forces, hydrogen bonds, attractive ionic forces and/or biochemical reactions, etc.5, forming a dense and compact tertiary network structure. The polymers in EPS which are able to form hydrogels4,9 and contribute to the formation of the tertiary network structure are in this respect considered as structural EPS, a subset of the total EPS.
EPS are responsible for the chemical structure and physical properties of granules5. It is therefore crucial to understand the function of each EPS compound. Various approaches are applied to extract EPS10-15. However, due to their extreme complexity, it is almost impossible to extract all the EPS components by one single method. To date, there is no &#34;one size fits all&#34; method for EPS extraction. The choice of the extraction method influences not only the total amount, but also the composition of the recovered polymers13,16-20. Depending on the type of sludge and the EPS of interest different methods are required.
Extracting gel-forming polymers, characterizing their properties and investigating their interactions with each other and with non-gel-forming EPS will help to reveal the role of EPS in aerobic granular sludge formation. Furthermore, the gel-forming polymers are also useful biopolymers in industrial applications. One possible application was already shown by using gel-forming polymers from AGS as a coating material to increase the water resistance of paper21.
Therefore, extraction methods, specific for gel-forming EPS are needed. The aim of this study is to develop a methodology to extract gel-forming EPS from AGS. Six extraction methods10-15,22, which are frequently used in literature, were selected to extract EPS from AGS. The total amount and the gel-forming property of the extracted EPS were compared for each methodology.

<table><tbody><tr><td>250 ml baffled flask</td><td>Kimble</td><td>25630-250</td><td></td></tr><tr><td>1,000 ml glass beaker</td><td>VWR</td><td>213-1128</td><td></td></tr><tr><td>RCT basic, magnetic stirrer with thermometer</td><td>IKA</td><td>3810000</td><td></td></tr><tr><td>sodium carbonate decahydrate</td><td>Merck KGaA</td><td>1063911000</td><td></td></tr><tr><td>50 ml centrifugation tubes</td><td>greiner bio-one</td><td>227261</td><td></td></tr><tr><td>Multifuge 1 S-R, centrifuge</td><td>Heraeus/Thermo Scientific</td><td>-</td><td></td></tr><tr><td>hydrochloric acid, 37%</td><td>Sigma-Aldrich</td><td>30721-1L-GL-D</td><td></td></tr><tr><td>250 ml glass beaker</td><td>VWR</td><td>213-1124</td><td></td></tr><tr><td>calcium chloride dihydrate</td><td>Merck KGaA</td><td>1023821000</td><td></td></tr><tr><td>1 ml Pasteur Pipette</td><td>Copan</td><td>201C</td><td></td></tr></tbody></table>

extraction of structural extracellular polymeric substances from aerobic granular sludge

To evaluate and develop methodologies for the extraction of gel-forming extracellular polymeric substances (EPS), EPS from aerobic granular sludge (AGS) was extracted using six different methods (centrifugation, sonication, ethylenediaminetetraacetic acid (EDTA), formamide with sodium hydroxide (NaOH), formaldehyde with NaOH and sodium carbonate (Na2CO3) with heat and constant mixing). AGS was collected from a pilot wastewater treatment reactor. The ionic gel-forming property of the extracted EPS of the six different extraction methods was tested with calcium ions (Ca2+). From the six extraction methods used, only the Na2CO3 extraction could solubilize the hydrogel matrix of AGS. The alginate-like extracellular polymers (ALE) recovered with this method formed ionic gel beads with Ca2+. The Ca2+-ALE beads were stable in EDTA, formamide with NaOH and formaldehyde with NaOH, indicating that ALE are one part of the structural polymers in EPS. It is recommended to use an extraction method that combines physical and chemical treatment to solubilize AGS and extract structural EPS.

EPS extraction 
The appearance of granules after applying different EPS extraction procedures is shown in Figure 1. The shape and gel structure of granules were intact after centrifugation (Figure 1a) and EDTA extraction (Figure 1c). Granules were broken into fragments of different sizes by sonication. The turbidity in the liquid phase could be due to suspension of small fragments (Figure 1b) as the turbidity highly decreased after centrifugation. Formamide and formaldehyde alone did not have any impact on changing the shape of the granule and its gel structure (data not shown). After the addition of NaOH, the liquid phase turned yellowish. Some fluffy material was detached from the surface of the granules and formed a layer on top of the settled granules (Figure 1d and 1e). Still, the shape of the granules was not changed. The addition of NaOH apparently improved EPS solubilization, but could not damage the gel matrix structure. In comparison, granules completely disappeared after Na2CO3 extraction (Figure 1f). Instead a mixture of sol-like liquid and tiny jelly-like particles were formed, showing the gel matrix of granules was indeed solubilized.
<img alt="Figure 1" src="/files/ftp_upload/54534/54534fig1.jpg" /> 
Figure 1. Aerobic granular sludge EPS extractions. For a better visualization of the impact of each extraction method on the granules, experiments were conducted in 25 ml glass bottles. After the extraction procedure, the extracts were kept for 1 hr at room temperature to allow suspended matter to settle. (a) Centrifugation extraction, (b) Sonication extraction, (c) EDTA extraction, (d) Formamide &#8211; NaOH extraction, (e) Formaldehyde &#8211; NaOH extraction, (f) High temperature &#8211; Na2CO3 extraction. <a href="https://www.jove.com/files/ftp_upload/54534/54534fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
EPS yield with respect to the VS fraction for each method is illustrated in Figure 2. The yield is presented in mg VSEPS per g initial VSgranule. The amount of EPS obtained by formaldehyde + NaOH, formamide + NaOH and Na2CO3 + heat + mixing was higher than that of centrifugation, sonication and EDTA extraction. Similar results for these extraction techniques were also shown by previous studies11-13,15 indicating that alkaline conditions enhance EPS solubility26,27. The amount of EPS recovered by Na2CO3 was the highest, more than 20 times of that obtained only by centrifugation. Additionally, the total EPS yield of the Na2CO3 extraction can be further enhanced by multiple extractions. A second extraction using the pellet discarded in step 1.6.8 (protocol section) of the first extraction increased the total yield by 28%, a quadruple extraction even increased the total yield by 46%.
<img alt="Figure 2" src="/files/ftp_upload/54534/54534fig2.jpg" /> 
Figure 2. Results of all extraction methods with respect to VS yield and ash content. For each extraction the first bar represents the VS yield in mg VSEPS per g initial VSgranule. The second bar represents the weight percentage of ash in the extracted TS. The error bars illustrate the standard deviation of the three extractions performed for each extraction technique. <a href="https://www.jove.com/files/ftp_upload/54534/54534fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Alginate-like extracellular polymer (ALE) extraction 
After the pH of the EPS extracted by the Na2CO3 extraction was adjusted to 2.2, 63% of the total VS was precipitated. The precipitate is acidic ALE25. The residual fraction was likely EPS which can be solubilized under the extraction conditions, but cannot form a precipitate at pH 2.2.
Ionic hydrogel formation test 
Aerobic granules have been described as being similar to a hydrogel. The granulation process has been regarded as a gel-forming phenomenon with glycosides as the gelling agent4,9,25,28. Normally, Ca2+ is one of the most common cations in wastewater. In addition, it easily binds with acidic polysaccharides (e.g., alginates and poly-galacturonic acid), presumably as a counter-ion to mediate gelation29. Thus resulting in an ionically cross-linked hydrogel. It was observed that the addition of Ca2+ ions can accelerate aerobic sludge granulation30. Therefore, Ca2+-EPS (ionic hydrogel) could play an important role in building up the gel matrix structure in aerobic granular sludge. In this respect, whether the extracted EPS forms an ionic hydrogel with Ca2+ ions could be used as a test to check if the extracted EPS is a structural polymer contributing to the formation of the gel matrix in aerobic granular sludge9.
In this research, for the EPS extracted from AGS (Figure 3a) by various methods, only the EPS extracted by Na2CO3 held the shape of a droplet in 2.5% (w/v) CaCl2 solution and formed stable ionic hydrogel beads. Moreover, the sodium ALE obtained from this EPS by additional steps (ALE polymer extraction, Figure 3b) displayed the same property as well. The color and morphology of the Ca2+-ALE gel beads (Figure 3c) are similar to aerobic granular sludge (Figure 3a). Apparently, the EPS extracted by the Na2CO3 method contributes to the formation of the gel matrix in aerobic granular sludge. ALE, which is a main component of this EPS are structural polymers, able to form an ionic hydrogel.
Stability test of the ionic hydrogel 
It was observed that during EPS extraction, aerobic granules kept their spherical shape in EDTA, formaldehyde + NaOH and formamide + NaOH (Figure 1). In order to understand if the extracted structural polymers play a role in the stability of the granules, Ca2+-ALE beads were treated exactly the same way as aerobic granules during the extraction. Interestingly, Ca2+-ALE beads displayed the similar stabilities as that of AGS (Figure 3d - 3f), i.e., Ca2+-ALE beads were extremely stable in EDTA. There was little amount of ALE detached from the surface of Ca2+-ALE beads (tiny brownish floc in Figure 3e and 3f), when the Ca2+-ALE beads had been soaked in formaldehyde + NaOH and formamide + NaOH for three hours, respectively. This similarity in terms of stability between Ca2+-ALE beads and aerobic granules indicates that ALE are one part of the important structural polymers forming the AGS gel matrix.
<img alt="Figure 3" src="/files/ftp_upload/54534/54534fig3.jpg" /> 
Figure 3. Aerobic granules and extracted ALE. (a) Granules in demineralized water prior extraction. (b) Acidic ALE (extracted according to the paragraphs 1.6 and 2) after centrifugation at 4,000 &#215; g and 4 &#176;C for 20 min. Results of the stability test of the ionic hydrogel. (c) Ca2+-ALE-beads stored in demineralized water for 4 hr at 4 &#176;C. (d) Ca2+-ALE-beads stored in 2% EDTA, for 3 hr at 4 &#176;C. (e) Ca2+-ALE-beads stored in formamide + NaOH for 4 hr at 4 &#176;C. (f) Ca2+-ALE-beads stored in formaldehyde + NaOH for 4 hr at 4 &#176;C. <a href="https://www.jove.com/files/ftp_upload/54534/54534fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Extraction of Structural Extracellular Polymeric Substances from Aerobic Granular Sludge at JoVE.com

Extraction of Structural Extracellular Polymeric Substances from Aerobic Granular Sludge

The protocol provides a methodology to solubilize aerobic granular sludge in order to extract alginate-like extracellular polymers (ALE).

To evaluate and develop methodologies for the extraction of gel-forming extracellular polymeric substances (EPS), EPS from aerobic granular sludge (AGS) ...

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Research

JoVE Journal

Environment

22.5K Views.  Delft University of Technology.  Het protocol voorziet in een methodologie om aëroob korrelslib oplosbaar te maken met het oog op alginaat-achtige extracellulaire polymeren (ALE) uit te pakken. 

Video: Extraction of Structural Extracellular Polymeric Substances from Aerobic Granular Sludge

Methods for Facilitating Microbial Growth on Pulp Mill Waste Streams and Characterization of the Biodegradation Potential of Cultured Microbes

The kraft process is applied to wood chips for separation of lignin from the polysaccharides within lignocellulose for pulp that will produce a high quality paper. Black liquor is a pulping waste generated by the kraft process that has potential for downstream bioconversion. However, the recalcitrant nature of the lignocellulose resources, its chemical derivatives that constitute the majority of available organic carbon within black liquor, and its basic pH present challenges to microbial biodegradation of this waste material. Methods for the collection and modification of black liquor for microbial growth are aimed at utilization of this pulp waste to convert the lignin, organic acids, and polysaccharide degradation byproducts into valuable chemicals. The lignocellulose extraction techniques presented provide a reproducible method for preparation of lignocellulose growth substrates for understanding metabolic capacities of cultured microorganisms. Use of gas chromatography-mass spectrometry enables the identification and quantification of the fermentation products resulting from the growth of microorganisms on pulping waste. These methods when used together can facilitate the determination of the metabolic activity of microorganisms with potential to produce fermentation products that would provide greater value to the pulping system and reduce effluent waste, thereby increasing potential paper milling profits and offering additional uses for black liquor.

Industrial wastes can be collected and modified to analyze microbial growth. Lignocellulose extraction techniques provide components to analyze specific biodegradation ability. Gas chromatography-mass spectrometry identifies fermentation products of microorganisms grown on pulping waste. These methods determine the metabolic capacity of microorganisms to degrade pulping waste.

The kraft process is applied to wood chips for separation of lignin from the polysaccharides within lignocellulose for pulp that will produce a high ...

Development of Sulfidogenic Sludge from Marine Sediments and Trichloroethylene Reduction in an Upflow Anaerobic Sludge Blanket Reactor

The importance of microbial sulfate reduction relies on the various applications that it offers in environmental biotechnology. Engineered sulfate reduction is used in industrial wastewater treatment to remove large concentrations of sulfate along with the chemical oxygen demand (COD) and heavy metals. The most common approach to the process is with anaerobic bioreactors in which sulfidogenic sludge is obtained through adaptation of predominantly methanogenic granular sludge to sulfidogenesis. This process may take a long time and does not always eliminate the competition for substrate due to the presence of methanogens in the sludge. In this work, we propose a novel approach to obtain sulfidogenic sludge in which hydrothermal vents sediments are the original source of microorganisms. The microbial community developed in the presence of sulfate and volatile fatty acids is wide enough to sustain sulfate reduction over a long period of time without exhibiting inhibition due to sulfide. 
This protocol describes the procedure to generate the sludge from the sediments in an upflow anaerobic sludge blanket (UASB) type of reactor. Furthermore, the protocol presents the procedure to demonstrate the capability of the sludge to remove by reductive dechlorination a model of a highly toxic organic pollutant such as trichloroethylene (TCE). The protocol is divided in three stages: (1) the formation of the sludge and the determination of its sulfate reducing activity in the UASB, (2) the experiment to remove the TCE by the sludge, and (3) the identification of microorganisms in the sludge after the TCE reduction. Although in this case the sediments were taken from a site located in Mexico, the generation of a sulfidogenic sludge by using this procedure may work if a different source of sediments is taken since marine sediments are a natural pool of microorganisms that may be enriched in sulfate reducing bacteria.

Microbial sulfate reduction is a process of great importance in environmental biotechnology. The success of the sulfidogenic reactors depends among other factors on the microbial composition of the sludge. Here, we present a protocol to develop sulfidogenic sludge from hydrothermal vents sediments in a UASB reactor for reductive dechlorination purposes.

The importance of microbial sulfate reduction relies on the various applications that it offers in environmental biotechnology. Engineered sulfate ...

Au-Interaction of Slp1 Polymers and Monolayer from Lysinibacillus sphaericus JG-B53 - QCM-D, ICP-MS and AFM as Tools for Biomolecule-metal Studies

In this publication the gold sorption behavior of surface layer (S-layer) proteins (Slp1) of Lysinibacillus sphaericus JG-B53 is described. These biomolecules arrange in paracrystalline two-dimensional arrays on surfaces, bind metals, and are thus interesting for several biotechnical applications, such as biosorptive materials for the removal or recovery of different elements from the environment and industrial processes. The deposition of Au(0) nanoparticles on S-layers, either by S-layer directed synthesis 1 or adsorption of nanoparticles, opens new possibilities for diverse sensory applications. Although numerous studies have described the biosorptive properties of S-layers 2-5, a deeper understanding of protein-protein and protein-metal interaction still remains challenging. In the following study, inductively coupled mass spectrometry (ICP-MS) was used for the detection of metal sorption by suspended S-layers. This was correlated to measurements of quartz crystal microbalance with dissipation monitoring (QCM-D), which allows the online detection of proteinaceous monolayer formation and metal deposition, and thus, a more detailed understanding on metal binding.
The ICP-MS results indicated that the binding of Au(III) to the suspended S-layer polymers is pH dependent. The maximum binding of Au(III) was obtained at pH 4.0. The QCM-D investigations enabled the detection of Au(III) sorption as well as the deposition of Au(0)-NPs in real-time during the in situ experiments. Further, this method allowed studying the influence of metal binding on the protein lattice stability of Slp1. Structural properties and protein layer stability could be visualized directly after QCM-D experiment using atomic force microscopy (AFM). In conclusion, the combination of these different methods provides a deeper understanding of metal binding by bacterial S-layer proteins in suspension or as monolayers on either bacterial cells or recrystallized surfaces.

Au-Interaction of Slp1 Polymers and Monolayer from Lysinibacillus sphaericus JG-B53 - QCM-D, ICP-MS and AFM as Tools for Biomolecule-metal Studies

To obtain basic information on the sorption and recycling of gold from aqueous systems the interaction of Au(III) and Au(0) nanoparticles on S-layer proteins were investigated. The sorption of protein polymers was investigated by ICP-MS and that of proteinaceous monolayers by QCM-D. Subsequent AFM enables the imaging of the nanostructures.

In this publication the gold sorption behavior of surface layer (S-layer) proteins (Slp1) of Lysinibacillus sphaericus JG-B53 is described. These ...

Chemistry

Looking Outwards: Isolation of Cyanobacterial Released Carbohydrate Polymers and Proteins

Cyanobacteria can actively secrete a wide range of biomolecules into the extracellular environment, such as heteropolysaccharides and proteins. The identification and characterization of these biomolecules can improve knowledge about their secretion pathways and help to manipulate them. Furthermore, some of these biomolecules are also interesting in terms of biotechnological applications. Described here are two protocols for easy and rapid isolation of cyanobacterial released carbohydrate polymers and proteins. The method for isolation of released carbohydrate polymers is based on conventional precipitation techniques of polysaccharides in aqueous solutions using organic solvents. This method preserves the characteristics of the polymer and simultaneously avoids the presence of contaminants from cell debris and culture medium. At the end of the process, the lyophilized polymer is ready to be used or characterized or can be subjected to further rounds of purification, depending on the final intended use. Regarding the isolation of the cyanobacterial exoproteome, the technique is based on the concentration of the cell-free medium after removal of the major contaminants by centrifugation and filtration. This strategy allows for reliable isolation of proteins that reach the extracellular milieu via membrane transporters or outer membrane vesicles. These proteins can be subsequently identified using standard mass spectrometry techniques. The protocols presented here can be applied not only to a wide range of cyanobacteria, but also to other bacterial strains. Furthermore, these procedures can be easily tailored according to the final use of the products, purity degree required, and bacterial strain.

Here, protocols for the isolation of cyanobacterial released carbohydrate polymers and isolation of their exoproteomes are described. Both procedures embody key steps to obtain polymers or proteins with high purity degrees that can be used for further analysis or applications. They can also be easily adapted according to specific user needs.

Cyanobacteria can actively secrete a wide range of biomolecules into the extracellular environment, such as heteropolysaccharides and proteins. The ...

Biochemistry

Optimizing Attachment of Human Mesenchymal Stem Cells on Poly(&#949;-caprolactone) Electrospun Yarns

Research into biomaterials and tissue engineering often includes cell-based in vitro investigations, which require initial knowledge of the starting cell number. While researchers commonly reference their seeding density this does not necessarily indicate the actual number of cells that have adhered to the material in question. This is particularly the case for materials, or scaffolds, that do not cover the base of standard cell culture well plates. This study investigates the initial attachment of human mesenchymal stem cells seeded at a known number onto electrospun poly(&#949;-caprolactone) yarn after 4 hr in culture. Electrospun yarns were held within several different set-ups, including bioreactor vessels rotating at 9 rpm, cell culture inserts positioned in low binding well plates and polytetrafluoroethylene (PTFE) troughs placed within petri dishes. The latter two were subjected to either static conditions or positioned on a shaker plate (30 rpm). After 4 hr incubation at 37&#160;oC, 5% CO2, the location of seeded cells was determined by cell DNA assay. Scaffolds were removed from their containers and placed in lysis buffer. The media fraction was similarly removed and centrifuged &#8211; the supernatant discarded and pellet broken up with lysis buffer. Lysis buffer was added to each receptacle, or well, and scraped to free any cells that may be present. The cell DNA assay determined the percentage of cells present within the scaffold, media and well fractions. Cell attachment was low for all experimental set-ups, with greatest attachment (30%) for yarns held within cell culture inserts and subjected to shaking motion. This study raises awareness to the actual number of cells attaching to scaffolds irrespective of the stated cell seeding density.

This article describes a range of set-ups for seeding human mesenchymal stem cells onto materials, in this case electrospun yarns, that do not cover the base of standard culture well plates in order to maximize and quantify the number of cells that initially attach compared to the known seeding density.

Research into biomaterials and tissue engineering often includes cell-based in vitro investigations, which require initial knowledge of the starting cell ...

Bioengineering

A Novel Bioreactor for High Density Cultivation of Diverse Microbial Communities

A novel reactor design, coined a high density bioreactor (HDBR), is presented for the cultivation and study of high density microbial communities. Past studies have evaluated the performance of the reactor for the removal of COD1 and nitrogen species2-4 by heterotrophic and chemoautotrophic bacteria, respectively. The HDBR design eliminates the requirement for external flocculation/sedimentation processes while still yielding effluent containing low suspended solids. In this study, the HDBR is applied as a photobioreactor (PBR) in order to characterize the nitrogen removal characteristics of an algae-based photosynthetic microbial community. As previously reported for this HDBR design, a stable biomass zone was established with a clear delineation between the biologically active portion of the reactor and the recycling reactor fluid, which resulted in a low suspended solid effluent. The algal community in the HDBR was observed to remove 18.4% of total nitrogen species in the influent. Varying NH4+ and NO3- concentrations in the feed did not have an effect on NH4+ removal (n=44, p=0.993 and n=44, p=0.610 respectively) while NH4+ feed concentration was found to be negatively related with NO3- removal (n=44, p=0.000) and NO3- feed concentration was found to be positively correlated with NO3- removal (n=44, p=0.000). Consistent removal of NH4+, combined with the accumulation of oxidized nitrogen species at high NH4+ fluxes indicates the presence of ammonia- and nitrite-oxidizing bacteria within the microbial community.

A novel reactor design, coined a high density bioreactor (HDBR), is presented for the cultivation and study of high density microbial communities. Here, the HDBR is successfully applied in a photobioreactor (PBR) configuration for the study of nitrogen metabolism by a mixed high density algal community.

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Modeling Methanotroph Niches – The Gradient Syringe Model Ecosystem

Agarose-Based Model Ecosystem for Cultivating Methanotrophs in a Methane-Oxygen Counter Gradient

Aerobic methane-oxidizing bacteria, known as methanotrophs, serve important roles in biogeochemical cycling. Methanotrophs occupy a specific environmental niche within methane-oxygen counter gradients found in soils and sediments, which influences their behavior on an individual and community level. However, conventional methods to study the physiology of these greenhouse gas-mitigating microorganisms often use homogeneous planktonic cultures, which do not accurately represent the spatial and chemical gradients found in the environment. This hinders scientists' understanding of how these bacteria behave in situ. Here, a simple, inexpensive model ecosystem called the gradient syringe is described, which uses semi-solid agarose to recreate the steep methane-oxygen counter gradients characteristic of methanotrophs' natural habitats. The gradient syringe allows for the cultivation of methanotrophic strains and the enrichment of mixed methane-oxidizing consortia from environmental samples, revealing phenotypes only visible in this spatially resolved context. This protocol also reports various biochemical assays that have been modified to be compatible with the semi-solid agarose matrix, which may be valuable to researchers culturing microorganisms within other agarose-based systems.

Author Spotlight: Designing Simple and Inexpensive Techniques to Grow Methane-Oxidizing Bacteria in the Laboratory

A protocol is described for preparing a simple model ecosystem that recreates the methane-oxygen counter gradient found in the natural habitat of aerobic methane-oxidizing bacteria, enabling the study of their physiology in a spatially resolved context. Modifications to common biochemical assays for use with the agarose-based model ecosystem are also described.

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A Facile and Eco-friendly Route to Fabricate Poly(Lactic Acid) Scaffolds with Graded Pore Size

Over the recent years, functionally graded scaffolds (FGS) gaineda crucial role for manufacturing of devices for tissue engineering. The importance of this new field of biomaterials research is due to the necessity to develop implants capable of mimicking the complex functionality of the various tissues, including a continuous change from one structure or composition to another. In this latter context, one topic of main interest concerns the design of appropriate scaffolds for bone-cartilage interface tissue. In this study, three-layered scaffolds with graded pore size were achieved by melt mixing poly(lactic acid) (PLA), sodium chloride (NaCl) and polyethylene glycol (PEG). Pore size distributions were controlled by NaCl granulometry and PEG solvation. Scaffolds were characterized from a morphological and mechanical point of view. A correlation between the preparation method, the pore architecture and compressive mechanical behavior was found. The interface adhesion strength was quantitatively evaluated by using a custom-designed interfacial strength test. Furthermore, in order to imitate the human physiology, mechanical tests were also performed in phosphate buffered saline (PBS) solution at 37 &#176;C. The method herein presented provides a high control of porosity, pore size distribution and mechanical performance, thus offering the possibility to fabricate three-layered scaffolds with tailored properties by following a simple and eco-friendly route.

A Facile and Eco-friendly Route to Fabricate PolyLactic Acid Scaffolds with Graded Pore Size

In this work, poly(lactic acid)/polyethylene glycol (PLA/PEG) scaffolds were prepared by using a combination of melt mixing and selective leaching. The method herein discussed permitted to develop three-layer scaffolds by highly controlling both porosity and pore size. The mechanical properties were also evaluated in a physiological environment.

Over the recent years, functionally graded scaffolds (FGS) gaineda crucial role for manufacturing of devices for tissue engineering. The importance of ...

Production of Extracellular Matrix Fibers via Sacrificial Hollow Fiber Membrane Cell Culture

Engineered scaffolds derived from extracellular matrix (ECM) have driven significant interest in medicine for their potential in expediting wound closure and healing. Extraction of extracellular matrix from fibrogenic cell cultures in vitro has potential for generation of ECM from human- and potentially patient-specific cell lines, minimizing the presence of xenogeneic epitopes which has hindered the clinical success of some existing ECM products. A significant challenge in in vitro production of ECM suitable for implantation is that ECM production by cell culture is typically of relatively low yield. In this work, protocols are described for the production of ECM by cells cultured within sacrificial hollow fiber membrane scaffolds. Hollow fiber membranes are cultured with fibroblast cell lines in a conventional cell medium and dissolved after cell culture to yield continuous threads of ECM. The resulting ECM fibers produced by this method can be decellularized and lyophilized, rendering it suitable for storage and implantation.

The goal of this protocol is production of whole extracellular matrix fibers targeted for wound repair which are suitable for preclinical evaluation as part of a regenerative scaffold implant. These fibers are produced by the culture of fibroblasts on hollow fiber membranes and extracted by dissolution of the membranes.

Engineered scaffolds derived from extracellular matrix (ECM) have driven significant interest in medicine for their potential in expediting wound closure ...

Assaying for Inorganic Polyphosphate in Bacteria

Inorganic polyphosphate (polyP) is a biological polymer found in cells from all domains of life, and is required for virulence and stress response in many bacteria. There are a variety of methods for quantifying polyP in biological materials, many of which are either labor-intensive or insensitive, limiting their usefulness. We present here a streamlined method for polyP quantification in bacteria, using a silica membrane column extraction optimized for rapid processing of multiple samples, digestion of polyP with the polyP-specific exopolyphosphatase ScPPX, and detection of the resulting free phosphate with a sensitive ascorbic acid-based colorimetric assay. This procedure is straightforward, inexpensive, and allows reliable polyP quantification in diverse bacterial species. We present representative polyP quantification from the Gram-negative bacterium (Escherichia coli), the Gram-positive lactic acid bacterium (Lactobacillus reuteri), and the mycobacterial species (Mycobacterium smegmatis). We also include a simple protocol for nickel affinity purification of mg quantities of ScPPX, which is not currently commercially available.

We describe a simple method for rapid quantification of inorganic polyphosphate in diverse bacteria, including Gram-negative, Gram-positive, and mycobacterial species.

Inorganic polyphosphate (polyP) is a biological polymer found in cells from all domains of life, and is required for virulence and stress response in many ...

Extraction of Structural Extracellular Polymeric Substances from Aerobic Granular Sludge

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