F.H. acknowledges support by the BMBF (Research Award &#8220;Next generation biotechnological processes - Biotechnology 2020+&#8221;) and the Helmholtz-Association (Young Investigators Group).

As the selection procedure in the bioelectrochemical fed-batch reactor is very specific due to the choice of substrate and anoxic conditions, working in sterile conditions is not necessary for the demonstrated example.
1. Preparation of Growth Medium
<ol>
	<li>Prepare a suitable amount of the growth medium by using a phosphate buffer according to Kim et al.13&#160;(see Table 1). Here, sodium acetate (10 mM) is serving as substrate. The growth medium used in this article is only one example, and depending on the experimental purpose can vary in its components.</li>
	<li>Add 1 ml&#160;of primary waste water per 20&#160;ml&#160;of growth medium as inoculum. The waste water sample is taken from a municipal waste water treatment plant after the mechanical filtration (see for example Figure 2) and can be stored up to three months until usage in a closed container at 4 &#176;C. 
	(Note: Depending on the objective of the experiment the exact initial cell count can be important and then should be determined. In the demonstrated example, the initial cell count is not important, as the biofilm reaches steady-state conditions.)</li>
	<li>Flush the medium including the inoculum with nitrogen for at least 30 min to deaerate it, by inserting a needle in the medium and turn on the nitrogen stream. &#160;The stream should be strong enough that you can observe a complete mixing of the medium. 
	(Note: Depending on the volume of the medium one should even consider extending the time of deaeration. During deaeration setup the fed-batch reactor (steps 2.1-2.2))</li>
</ol>
2. Setup of Fed-batch Reactor
<ol>
	<li>Take a suitable reactor vessel-&#160;here a modified 4-necked 250 ml round-bottom flask is used. 
	(Note: In general the vessel should have at least four inlets. These are necessary for the three electrodes and for sampling/ media exchange. Produced gas volumes can be neglected when the reactor is opened regularly due to sampling or medium change, alternatively a bubble counter should be installed. In order to avoid unwanted phototropic growth, the reactors should be shielded from direct light exposure.)</li>
	<li>For the three-electrode setup (see Figure 3 and section 3 for details) place the reference electrode (RE) (here Ag/AgCl, sat. KCl, 0.197 V vs. SHE), the working electrode (WE) and the counter electrode (CE) in the flask through individual inlets via silicone plugs &#8211; seal air-tight. 
	(Note: As WE and CE graphite rods are used. The wire of the electrodes is pulled through the silicon plug and fixed externally. The reference electrode is put through the silicon plug via a drilled hole. To avoid short-circuits, make sure that there is no physical contact of the electrodes, even while stirring or media recirculation. Place the electrodes that they are completely covered with medium solution. For sealing, wrap the silicon plug of every inlet with Parafilm.)</li>
	<li>Pour the growth medium including the inoculum (here 250 ml) through the sampling port into the reactor while flushing with nitrogen.</li>
	<li>Close the last inlet with a silicone plug and seal it air-tight. Note: Traces of oxygen will not harm the mixed species biofilms. They are able to withstand and even consume low levels of oxygen which in turn may decrease the CE.</li>
</ol>
3. Chronoamperometric Biofilm Growth
<ol>
	<li>Connect the electrodes to the respective cables of the potentiostat. Place the vessel in a temperature controlled chamber, here 35 &#176;C is used31, in order to ensure constant environmental conditions.</li>
	<li>Open the software to control the potentiostat and choose the technique: Chronoamperometry. Set the parameters for example as follows: A constant potential EWE of 0.2 V (vs. Ag/AgCl) for a duration t of 500 hr (&#8776; 20 days). The current is recorded in time steps dti every minute. 
	Note: The choice of the applied potential is dependent on the studied system/ microorganism. Generally anode processes are studied at positive potential and cathode processes at negative potentials. One can also deduce a suitable potential from the standard potentials of the electron donor/ acceptor and formal potential of the expected microbial electron transfer site, here as a starting point, please refer to recent literature.</li>
	<li>Start the chronoamperometry. A graph of the measured current over time is plotted by the software, the observed maximum current (imax) is an important online parameter, as discussed in sections 4 and 5.&#160;</li>
</ol>
4. Biofilm Maintenance
<ol>
	<li>Depending on the environmental conditions, e.g. the temperature, an oxidative current is starting to flow and reaches a plateau (see Figure 4). After reaching zero current again, pause the chronoamperometric measurement and replenish the medium solution. For a faster biofilm growth, replace the medium right after the plateau of the maximum current is reached. Open the sampling port and carefully, without destroying the thin biofilm, discharge the medium. While refilling the medium flush again with nitrogen. Close the sampling port and seal air-tight.</li>
	<li>Repeat the procedure of step 4.1. until the maximum oxidative current reaches a steady-state (see Figure 4). 
	(Note: A constant imax is believed to indicate a stable steady-state biofilm formation.)</li>
	<li>Take 1 ml&#160;samples of the fresh and the exchanged medium solutions for substrate analysis, e.g. by HPLC. The initial substrate (acetate) concentration (c0) as well as the concentration of the remaining substrate solution (c1) are subsequently needed for efficiency calculations (see section Data analysis). 
	Note: To achieve unaltered results, e.g.&#160;due to further substrate consumption, analyze the substrate samples immediately. If this is not possible, store the samples in a freezer at minimum -18 &#176;C for not longer than a month.)</li>
</ol>
5. Application of Cyclic Voltammetry to Study EET
<ol>
	<li>Perform cyclic voltammetry at the maxima and minima of the current flow as indicated (Figure 4). Depending on the question of interest the CV usually is performed when the system has reached steady-state to get comparable results. (Note: The generation of current is termed turnover conditions and related to the microbial oxidation of the substrate and subsequent electron transfer: microbial bioelectrocatalysis; whereas the nonturnover CVs are performed at (nearly) zero current flow in the CA.)</li>
	<li>Choose in the potentiostat controlling software the technique: Cyclic voltammetry, and set the appropriate parameters, here for instance: An initial potential of the EWE to Ei of -0.5 V vs. RE with a scan rate v=dE/dt of 1 mV/sec&#160;to a vertex potential E1 of 0.3 V vs. RE and back to the starting potential E2 = Ei of -0.5 V. 
	Note: When using cyclic voltammetry the applied voltage at the WE is changed linearly with time by the potentiostat (see Figure 5)- Box 2 provides principle rules for choosing the experiment parameters and troubleshooting).</li>
	<li>Start the cyclic voltammetry and record as many cycles as needed to perform a reproducible CV. Usually, at least 3 cycles are needed.&#160; The resulting plot is called a cyclic voltammogram (see also Figure 7). Note: Depending on the measurement conditions, different information can be gained as demonstrated in the next section.</li>
</ol>
6. Data Plotting
Here only the plotting of the data is discussed. A more detailed treatise follows in the next section. 
<ol>
	<li>Use appropriate data analysis software, here all data is exported as text and imported as ASCII file.</li>
	<li>Calculate and use preferentially current densities (j) and not absolute currents (i). 
	(Note: However, as different protocols for calculating j do exist, make clear which one was used. Here the current density per geometric, i.e. projected, surface area of the electrode is used.)</li>
	<li>Chronoamperometry: Plot the measured current density as function of the time.</li>
	<li>Cyclic voltammetry: Plot the measured current density as function of the potential of the WE Ewe.</li>
</ol>

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The authors declare that they have no competing financial interests.

The presented protocol shows an easy and straightforward way for growing electroactive microbial biofilms from waste water inoculate using a three-electrode setup in a fed-batch reactor. Chronoamperometry enables the growth and selection of electroactive microbial biofilms from diverse inocula as well as the fundamental characterization of the biofilms in term of maximum current density and coulombic efficiency. Cyclic voltammetry, being a noninvasive technique, allows performing fundamental studies on the EET thermodynamics of the microbial biofilm and thus to identify possible and actual EET transfer sites. Together, these parameters allow i) characterizing and comparing different microbial biofilms for identical conditions as well as ii) studying the impact of different operational conditions on identical electroactive microbial biofilms. However, these experiments can only be a starting point. On the one hand the multifaceted field of microbial bioelectrochemistry requires further and in-depth exploitation of electrochemical methods and protocols. This includes, for instance, models for extracting kinetic and mechanistic data as well as further methods like electrochemical impedance spectroscopy34,35. On the other hand electrochemical methods can provide no information on the molecular nature, the microbial composition, the spatial microbial organization, etc. Here further techniques being either noninvasive and coupled to electrochemistry or invasive are needed4.&#160; Box 2 provides a short discussion on the critical steps when performing the experiments as well as on initial troubleshooting.&#160;

From the engineering perspective it has to be taken into consideration that the electroactive biofilm anode is only one component of the current microbial bioelectrochemical systems and a multitude of other factors has to be analyzed for full systems36. Yet, we hope that this video-article will help to understand and tailor the core elements of BES, i.e. the microbial electrodes, in future.

The elucidation of the fundamentals of microbial extracellular electron transfer (EET) and its exploitation in engineered systems is a vital and rapidly increasing field of research and development1-3. The study of these electroactive (or bioelectrocatalytic or electrochemically active) microorganisms, including pure strains as well as defined co-cultures and complex consortia, requires a complex arsenal of techniques, methods and protocols4. These methods derive from diverse scientific disciplines, e.g. electrochemistry, materials science, microbiology, and provide insights on different hierarchical levels, i.e. from the entire microbial biofilm to single molecules. Thereby, electrochemistry and electrochemical methods represent the fundament of all activities.
Traditionally, fuel cell type setups were often used for the growth and maintenance of electroactive microbial cultures in the archetype of these engineered systems: microbial fuel cells (MFCs)5. Unfortunately, these types of MFCs often did not allow monitoring or even controlling the potential of a single electrode and thus, only limited insights in the electrode processes were possible-&#160;only the cell voltage was reported. Now it is more and more acknowledged that the individual monitoring and control of the potentials of single electrodes in MFCs represent a clear advantage, not only for fundamental research but also for engineering. Furthermore, with the diversification of the applications of microbial electrochemical technologies that now include, e.g. remediation, desalination, syntheses, and even biocomputing, in so-called microbial bioelectrochemical systems (BES)2,6,7 an external control of individual electrode potentials is often substantial. This control is usually achieved by using external power sources or potentiostats. Thereby the use of potentiostats allows-&#160;in contrast to other types of setups-&#160;an exact adjustment of the individual electrode potential. This is of high importance as the electrode represents the terminal microbial electron acceptor (for anodes) or electron donor (for cathodes) of the extracellular electron transfer. Figure 1 depicts the current state of knowledge on the microbial EET at anodes-&#160;see for details e.g.2,4,6,8,9&#160;The EET at the cathode is still mainly untapped10.
Thus the control of the electrode potential enables not only the use of a reproducible microbial culturing conditions but also its tailoring in terms of EET thermodynamics11. Within this article it is demonstrated how the characteristic parameters of electroactive microbial biofilms can be extracted from fed-batch reactor experiments using chronoamperometry (CA) and cyclic voltammetry (CV). This includes the performance parameters maximum current density (jmax) and coulombic efficiency (CE) as well as EET characteristics. Here the identification of the formal potentials (Ef) of possible and actual electron transfer sites will be in focus. The extraction of these parameters is shown on the example of waste water derived mixed culture biofilms that are dominated by Geobacter spec.12&#160;These waste water derived biofilms are gained by a simple electrochemical selection procedure&#160;as demonstrated by numerous research groups around the world13-16.
Dynamic electrochemical techniques to study microbial extracellular electron transfer
For a thermodynamic characterization of the EET of electroactive microbial cultures dynamic electrochemical techniques are needed. In general these techniques include potential controlled, i.e. voltammetric, and current controlled, i.e. galvanodynamic, techniques17. Thereby the potential controlled techniques are more prevalent and include for instance, square wave voltammetry and linear sweep voltammetry18. Most popular, however, is cyclic voltammetry (CV)19,20. CV is well-known and widespread in different fields of electrochemistry ranging from battery research via materials science to enzyme bioelectrochemistry21. Its application to living microbial cells can only be dated back more than one decade22 and increases significantly during the last years23,24. Especially the combination of CV and spectroscopic methods has provided unprecedented insights in the EET mechanisms and its molecular nature, e.g.25,26&#160;In parallel the theoretical framework for the extraction of thermodynamic and theoretical characteristics of the microbial EET was broadened and deepened27-30. In contrast, however, experimental mishandling or inadequate data-analysis often hamper the appropriate application of CV in the field of microbial bioelectrochemistry. Therefore, in addition to workshops (e.g. at the EU-ISMET 2012) and tutorials19, this video-article shall provide a basic guide for the conduction of cyclic voltammetric experiments on electroactive bacteria. Thereby it is focusing on the basic experimental steps and the fundamental data analysis and is not intended to provide the latest protocols of kinetic analysis. The most important termini for the following discussion are summarized in Box 1 (adapted from Harnisch and Freguia19 and modified).

<table><tbody><tr><td>Primary waste water</td><td>Waste water treatment plant</td><td></td><td>Taken after mechanical filtration</td></tr><tr><td>250 ml Round-bottom flasks with three additional inlets</td><td>Th.Geyer Berlin GmbH, Germany</td><td>9.011 845</td><td>Additional inlets were attached by our in-house glassblower</td></tr><tr><td>Ag/AgCl reference electrode (sat. KCl, 0.195 V vs. SHE) with ceramic diaphragm</td><td>Sensortechnik Meinsberg, Germany</td><td>SE 11</td><td></td></tr><tr><td>Graphite rods (&amp;Oslash;=1 cm, l=3 and 5 cm)</td><td>CP-Handels-GmbH, Wachtberg, Germany</td><td></td><td>Used as electrode material</td></tr><tr><td>Stainless steel, No. 1.4301, Wire Fe/Cr18/Ni10</td><td>Goodfellow GmbH, Bad Nauheim, Germany</td><td>155-923-05</td><td></td></tr><tr><td>Silicon plugs</td><td>Th.Geyer Berlin GmbH, Germany</td><td>9.230 512 and</td><td></td></tr><tr><td>Parafilm</td><td>neoLab, Heidelberg, Germany</td><td>3-1011</td><td>For sealing to maintain anaerobic conditions</td></tr><tr><td>Potentiostat/ Galvanostat Model MPG2</td><td>BioLogic Science Instruments, France</td><td></td><td></td></tr><tr><td>EC-Lab</td><td>BioLogic Science Instruments, France</td><td></td><td>software for performing electrochemistry measurements</td></tr><tr><td>Origin 8.6</td><td>OriginLab</td><td></td><td>data analysis software</td></tr></tbody></table>

waste water derived electroactive microbial biofilms: growth, maintenance, and basic characterization

The growth of anodic electroactive microbial biofilms from waste water inocula in a fed-batch reactor is demonstrated using a three-electrode setup controlled by a potentiostat. Thereby the use of potentiostats allows an exact adjustment of the electrode potential and ensures reproducible microbial culturing conditions. During growth the current production is monitored using chronoamperometry (CA). Based on these data the maximum current density (jmax) and the coulombic efficiency (CE) are discussed as measures for characterization of the bioelectrocatalytic performance. Cyclic voltammetry (CV), a nondestructive, i.e. noninvasive, method, is used to study the extracellular electron transfer (EET) of electroactive bacteria. CV measurements are performed on anodic biofilm electrodes in the presence of the microbial substrate, i.e. turnover conditions, and in the absence of the substrate, i.e. nonturnover conditions, using different scan rates. Subsequently, data analysis is exemplified and fundamental thermodynamic parameters of the microbial EET are derived and explained: peak potential (Ep), peak current density (jp), formal potential (Ef) and peak separation (&#916;Ep). Additionally the limits of the method and the state-of the art data analysis are addressed. Thereby this video-article shall provide a guide for the basic experimental steps and the fundamental data analysis.

1. Chronoamperometry
From Chronoamperometric measurements two operational characteristics can be calculated: the maximum current density (jmax), i.e. the maximum electron flow per second, and the coulombic efficiency (CE), i.e. the total current flow in relation to the consumed substrate. Figure 4 shows a typical chronoamperometric biofilm growth curve of several growth cycles using a fed-batch reactor. After the initial lag-phase a first current density maximum commences. Then the current decreases again due to substrate depletion. At (nearly) zero current flow the medium is exchanged as indicated. After this substrate replenishment the current density increases again and the following maximum current density is higher than the first one. After several growth cycles jmax is constant &#8211; which indicates a steady-state biofilm formation14. As the steady state jmax value is depended on several parameters, e.g. electrode material, reactor temperature and biofilm composition, it is often considered as a characteristic of a certain electroactive microbial biofilm/ electrode system. (In case of flow-through reactors not jmax, but the constant steady-state current density is used.) The second operational characteristic is the coulombic efficiency (CE) (Equation&#160;1). That is the number of electrons recorded as electric current flow, Qp , per fed-batch cycle related to the number of the theoretical maximum number of electrons Qth, which is calculated from substrate consumption during this cycle.
<img alt="Equation 1" fo:content-width="1in" src="/files/ftp_upload/50800/50800eq1.jpg" width="100" />
(Equation&#160;1)
The total transferred charge can be gained by integration of the CA curve (see also Figure 6):
<img alt="Equation 2" fo:content-width="1in" src="/files/ftp_upload/50800/50800eq2.jpg" width="100" />
(Equation&#160;2)
The theoretically possible electric charge is calculated by Faraday&#8217;s law:
<img alt="Equation 3" fo:content-width="1in" src="/files/ftp_upload/50800/50800eq3.jpg" width="100" />
(Equation&#160;3)
Where V is the volume of the growth medium, F the Faraday constant (F&#160;= 96,485.34 C/mol), z the released number of electrons of the oxidized substrate (in case of acetate 8 electrons are released during oxidation to CO2) and &#916;c = c0-c1, which represents the substrate consumption (is determined by chemical analysis methods, e.g. by HPLC).
2. Cyclic voltammetry:
Cyclic voltammograms are recorded for nonturnover conditions and for turnover conditions (as indicated in Figure 4). In both cases different types of information can be gained. In the following it is discussed how possible and actual EET sites can be identified.
2.1 Nonturnover conditions &#8211; identifying possible EET sites: 
Figure 7 shows typical CVs for nonturnover conditions, i.e. in the absence of substrate. Figure 7A shows the CV of a biofilm for high scan rates (here 50 mV/sec) where only one peak pair and thus one formal potential Ef can be identified. In general the formal potential of a redox couple can be calculated from the peak potential of the oxidation peak EpA and the reduction peak of the respective species EpC by forming the arithmetic mean (see also Figure&#160;7C):
<img alt="Equation 4" fo:content-width="1.5in" src="/files/ftp_upload/50800/50800eq4.jpg" width="120" />
(Equation&#160;4)
When applying a low enough scan rate to the identical biofilm (here 1 mV/sec) up to four redox pairs can be identified (the respective formal potentials are indicated in Figure 7B). The reason is that the capacitive background current is decreased when using lower scan rates, see also Box 1 and e.g.17,21 for details. However, the nonturnover CV only shows all redox-active compounds at the electrode and thus the Ef of the possible EET sites.&#160; Only the analysis of the turnover CV provides the Ef of the actual EET transfer sites (see step 2.2.).
When further analyzing the nonturnover CVs different other characteristic parameters like peak separation &#916;Ep (Equation&#160;5) or maximum peak current ipA and minimum peak current ipC can be analyzed (see Figure 7C).
<img alt="Equation 5" fo:content-width="1.5in" src="/files/ftp_upload/50800/50800eq5.jpg" width="120" />
(Equation&#160;5)
These parameters, especially when recorded for different scan rates, can be used for mechanistic and kinetic analysis of electron transfer processes at electrodes. However, this kinetic analysis that is well established for chemical systems is not straightforward for electroactive microbial biofilms, see e.g.19,30

<ol>
	<li value="2.2">Turnover conditions &#8211; identifying actual EET sites:</li>
</ol>
When performing CV measurement in the presence of substrate a turnover CV is obtained (see Figure 8A). After plotting the data you can see the typical s-shaped, so-called bioelectrocatalytic, curve17-19. When further analyzing the data, the maximum of the first derivative, i.e. the inflection point of the CV-curve, provides the formal potential Ef of the actual EET site(s) (see Figure 8B). In the case of a young, i.e. thin, Geobacteraceae-dominated biofilm, here 168 h after starting CA, two inflection points can be observed. Subsequently the derivative shows two maxima corresponding to the formal potential of the bioelectrocatalytic active EET sites: -0.376 V and -0.295 V vs. Ag/AgCl (sat. KCl, 0.197 V vs. SHE). In Figure 7B these sites are denominated as Ef,2 and Ef,3, respectively. This finding also shows that the redox processes associated with the formal potentials Ef,1 and Ef,4 (in the nonturnover CV) are not related to bioelectrocatalysis. As the biofilm grows and gets thicker the CV shows only one inflection point, i.e. meaning one maximum in the derivative curve. This formal potential Ef, here being is -0.32 V vs. Ag/AgCl (sat KCl), is (roughly) equal to the arithmetic mean of the two formal potentials assigned to EET of the thin biofilm (e.g. here Ef,2 and Ef,3) &#8211; see Figure 9. This shows that this very fundamental CV analysis does provide insights into the EET thermodynamics in a fast and noninvasive way, but noteworthy does not allow drawing further mechanistic or kinetic conclusions.
Notably biofilms gained by the specific conditions in this protocol are highly dominated by Geobacter species (not demonstrated here)12,15. Changing selected parameters e.g. substrate, temperature, etc. will also influence the microbial composition and thus the electrochemical properties.
Table 1.&#160;Growth medium according to Kim et al.32
<table border="1" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Component</td>
			<td>(mg/L)&#160;/ (ml/L)&#160;&#160;&#160;</td>
		</tr>
		<tr>
			<td>NaH2PO4&#8226;H2O</td>
			<td>2,690.00</td>
		</tr>
		<tr>
			<td>Na2HPO4</td>
			<td>4,330.00</td>
		</tr>
		<tr>
			<td>NH4Cl</td>
			<td>310.00</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>130.00</td>
		</tr>
		<tr>
			<td>sodium acetate</td>
			<td>820.00</td>
		</tr>
		<tr>
			<td>metal solution</td>
			<td>12.50</td>
		</tr>
		<tr>
			<td>vitamin solution</td>
			<td>12.50</td>
		</tr>
	</tbody>
</table>
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Table 2. Components of the metal and vitamin solution33.</td>
			<td></td>
		</tr>
		<tr>
			<td>Trace element solution:</td>
			<td>Vitamin solution:</td>
		</tr>
		<tr>
			<td>Component</td>
			<td>mg/L&#160;&#160;&#160;</td>
			<td>Component</td>
			<td>mg/L&#160;&#160;&#160;</td>
		</tr>
		<tr>
			<td>Nitriloacetic acid</td>
			<td>1,500.0</td>
			<td>Biotin</td>
			<td>2.0</td>
		</tr>
		<tr>
			<td>MgSO4&#8226;7H2O&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td>3,000.0</td>
			<td>Folic acid</td>
			<td>2.0</td>
		</tr>
		<tr>
			<td>MnSO4&#8226;2H2O&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td>500.0</td>
			<td>Pyridoxine hydrochloride</td>
			<td>10.0</td>
		</tr>
		<tr>
			<td>NaCl&#160;&#160;</td>
			<td>1,000.0</td>
			<td>Thiamine hydrochloride</td>
			<td>5.0</td>
		</tr>
		<tr>
			<td>FeSO4&#8226;7H2O&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td>100.0</td>
			<td>Riboflavin</td>
			<td>5.0</td>
		</tr>
		<tr>
			<td>CoSO4 or CoCl2&#160;&#160;&#160;&#160;&#160;</td>
			<td>100.0</td>
			<td>Nicotinic acid</td>
			<td>5.0</td>
		</tr>
		<tr>
			<td>CaCl2&#8226;2H2O&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td>100.0</td>
			<td>DL-Calcium pantothonate</td>
			<td>5.0</td>
		</tr>
		<tr>
			<td>ZnSO4&#160;&#160;&#160;</td>
			<td>130.0</td>
			<td>Vitamin B12</td>
			<td>0.1</td>
		</tr>
		<tr>
			<td>CuSO4&#8226;H2O</td>
			<td>10.0</td>
			<td>p-Aminobenzoic acid</td>
			<td>5.0</td>
		</tr>
		<tr>
			<td>AlK(SO4)2 &#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td>10.0</td>
			<td>Lipoic acid</td>
			<td>5.0</td>
		</tr>
		<tr>
			<td>H3BO3&#160;</td>
			<td>10.0</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Na2MoO4&#8226;2H2O&#160;&#160;&#160;&#160;&#160;</td>
			<td>10.0</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Note: Dissolve nitriloacetic acid with KOH to pH 6.5, and then proceed to add minerals.</td>
		</tr>
	</tbody>
</table>
 
<img alt="Box 1" fo:content-width="5in" src="/files/ftp_upload/50800/Box1.jpg" width="600" /> 
<img alt="Box 2" fo:content-width="5in" src="/files/ftp_upload/50800/Box2.jpg" width="600" />

<img alt="Figure 1" fo:content-width="5in" fo:src="/files/ftp_upload/50800/50800fig1highres.jpg" src="/files/ftp_upload/50800/50800fig1.jpg" /> 
Figure 1. Scheme of possible microbial extracellular electron transfer mechanisms. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50800/50800fig1highres.jpg" target="_blank">Click here to view larger image.</a>




<img alt="Figure 2" fo:content-width="5in" fo:src="/files/ftp_upload/50800/50800fig2highres.jpg" src="/files/ftp_upload/50800/50800fig2.jpg" /> 
Figure 2. Sampling at the waste water treatment plant after the grid collector (waste water treatment plant Germany).&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50800/50800fig2highres.jpg" target="_blank">Click here to view larger image.</a>
<img alt="Figure 3" fo:content-width="5in" fo:src="/files/ftp_upload/50800/50800fig3highres.jpg" src="/files/ftp_upload/50800/50800fig3.jpg" /> 
Figure 3. Three electrode setup.&#160;(A) Scheme, (B) real reactor; CE = counter electrode, RE = reference electrode, WE = working electrode with electroactive microbial biofilm, SP = sampling port. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50800/50800fig3highres.jpg" target="_blank">Click here to view larger image.</a>
<img alt="Figure 1" fo:content-width="5in" fo:src="/files/ftp_upload/50800/50800fig4highres.jpg" src="/files/ftp_upload/50800/50800fig4.jpg" /> 
Figure 4. Plot of a chronoamperometric fed-batch waste water inoculum based biofilm growth. The oxidative current reaches a steady-state after three cycles of medium change. Indicated are the lag-phase and the points of time of medium change and conduction of turnover cyclic voltammetry (CV) and nonturnover CV for steady-state biofilm conditions (raw data from&#160;Liu14). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50800/50800fig4highres.jpg" target="_blank">Click here to view larger image.</a>
<img alt="Figure 5" fo:content-width="5in" fo:src="/files/ftp_upload/50800/50800fig5highres.jpg" src="/files/ftp_upload/50800/50800fig5.jpg" /> 
Figure 5. Principle potential-time course during one cycle of cyclic voltammetry. CV is starting at the initial potential Ei. The applied voltage is then changed linearly with time with a scan rate of v. At a chosen vertex potential E1 the voltage returns to an end potential E2, here E2 = Ei. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50800/50800fig5highres.jpg" target="_blank">Click here to view larger image.</a>
<img alt="Figure 6" fo:content-width="5in" fo:src="/files/ftp_upload/50800/50800fig6highres.jpg" src="/files/ftp_upload/50800/50800fig6.jpg" /> 
Figure 6. Typical chronoamperogram of one growth cycle of a waste water derived biofilm; herein the maximum current density jmax, the flown (practical) electric charge Qp, the sampling of the initial substrate concentration c0 and the substrate concentration after one growth cycle c1 are indicated. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50800/50800fig6highres.jpg" target="_blank">Click here to view larger image.</a>
<img alt="Figure 7" fo:content-width="5in" fo:src="/files/ftp_upload/50800/50800fig7highres.jpg" src="/files/ftp_upload/50800/50800fig7.jpg" /> 
Figure 7. Nonturnover cyclic voltammograms of a G. sulfurreducens biofilm. Scan rate: (A) 50 mV/sec, (B) 1 mV/sec&#160;23; (C) cutout of B, demonstrating the peak current density (jpA) and the respective peak potential (EpA) of the anode at the maxima. Correspondingly at the minima the peak current density (jpC) and the respective peak potential (EpC) of the cathode are shown. The formal potential Ef and the peak separation &#916;Ep result from Equation&#160;4 and Equation&#160;5, respectively (for details see text below). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50800/50800fig7highres.jpg" target="_blank">Click here to view larger image.</a>
<img alt="Figure 8" fo:content-width="5in" fo:src="/files/ftp_upload/50800/50800fig8highres.jpg" src="/files/ftp_upload/50800/50800fig8.jpg" /> 
Figure 8. (A) Turnover cyclic voltammogram of a metabolizing G. sulfurreducens biofilm. The scan rate was 5 mV/sec. (B) First derivatives of the voltammetric curve23. Indicated are the formal potentials of the actual EET sites: Ef,2 and Ef,3. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50800/50800fig8highres.jpg" target="_blank">Click here to view larger image.</a>
<img alt="Figure 9" fo:content-width="5in" fo:src="/files/ftp_upload/50800/50800fig9highres.jpg" src="/files/ftp_upload/50800/50800fig9.jpg" /> 
Figure 9. (A) Turnover cyclic voltammogram and (B) first derivatives of a thicker metabolizing G. sulfurreducens biofilm.&#160;The scan rate was 5&#160;mV/sec&#160;(compare Figure 8). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50800/50800fig9highres.jpg" target="_blank">Click here to view larger image.</a>

Watch this Scientific Journal Video about Waste Water Derived Electroactive Microbial Biofilms: Growth, Maintenance, and Basic Characterization at JoVE.com

Waste Water Derived Electroactive Microbial Biofilms: Growth, Maintenance, and Basic Characterization

Chronoamperometric growth of anodic electroactive microbial biofilms in a fed-batch reactor using a three-electrode setup, controlled by a potentiostat, is demonstrated. The extracellular electron transfer is characterized using cyclic voltammetry in the presence of the electron donor and in the absence of the electron donor. Fundamental data analysis is demonstrated.

The growth of anodic electroactive microbial biofilms from waste water inocula in a fed-batch reactor is demonstrated using a three-electrode setup ...

waste-water-derived-electroactive-microbial-biofilms-growth

Nanyang Technological University

Research

JoVE Journal

Environment

13.3K 조회수.  UFZ - Helmholtz-Centre for Environmental Research. Chronoamperometric growth of anodic electroactive microbial biofilms in a fed-batch reactor using a three-electrode setup, controlled by a potentiostat, is demonstrated. The extracellular electron transfer is characterized using cyclic voltammetry in the presence of the electron donor and in the absence of the electron donor. Fundamental data analysis is demonstrated.

비디오: Waste Water Derived Electroactive Microbial Biofilms: Growth, Maintenance, and Basic Characterization

Electrochemically and Bioelectrochemically Induced Ammonium Recovery

Streams such as urine and manure can contain high levels of ammonium, which could be recovered for reuse in agriculture or chemistry. The extraction of ammonium from an ammonium-rich stream is demonstrated using an electrochemical and a bioelectrochemical system. Both systems are controlled by a potentiostat to either fix the current (for the electrochemical cell) or fix the potential of the working electrode (for the bioelectrochemical cell). In the bioelectrochemical cell, electroactive bacteria catalyze the anodic reaction, whereas in the electrochemical cell the potentiostat applies a higher voltage to produce a current. The current and consequent restoration of the charge balance across the cell allow the transport of cations, such as ammonium, across a cation exchange membrane from the anolyte to the catholyte. The high pH of the catholyte leads to formation of ammonia, which can be stripped from the medium and captured in an acid solution, thus enabling the recovery of a valuable nutrient. The flux of ammonium across the membrane is characterized at different anolyte ammonium concentrations and currents for both the abiotic and biotic reactor systems. Both systems are compared based on current and removal efficiencies for ammonium, as well as the energy input required to drive ammonium transfer across the cation exchange membrane. Finally, a comparative analysis considering key aspects such as reliability, electrode cost, and rate is made.
This video article and protocol provide the necessary information to conduct electrochemical and bioelectrochemical ammonia recovery experiments. The reactor setup for the two cases is explained, as well as the reactor operation. We elaborate on data analysis for both reactor types and on the advantages and disadvantages of bioelectrochemical and electrochemical systems.

We demonstrate the extraction of ammonium from an ammonium-rich stream using an electrochemical and a bioelectrochemical system. The reactor setup, operation and data analysis are discussed.

전기 및 Bioelectrochemically 유도 암모늄 복구

Streams such as urine and manure can contain high levels of ammonium, which could be recovered for reuse in agriculture or chemistry. The extraction of ...

연구

화학

Characterization of Aquatic Biofilms with Flow Cytometry

Biofilms are dynamic consortia of microorganism that play a key role in freshwater ecosystems. By changing their community structure, biofilms respond quickly to environmental changes and can be thus used as indicators of water quality. Currently, biofilm assessment is mostly based on integrative and functional endpoints, such as photosynthetic or respiratory activity, which do not provide information on the biofilm community structure. Flow cytometry and computational visualization offer an alternative, sensitive, and easy-to-use method for assessment of the community composition, particularly of the photoautotrophic part of freshwater biofilms. It requires only basic sample preparation, after which the entire sample is run through the flow cytometer. The single-cell optical and fluorescent information is used for computational visualization and biological interpretation. Its main advantages over other methods are the speed of analysis and the high-information-content nature. Flow cytometry provides information on several cellular and biofilm traits in a single measurement: particle size, density, pigment content, abiotic content in the biofilm, and coarse taxonomic information. However, it does not provide information on biofilm composition on the species level. We see high potential in the use of the method for environmental monitoring of aquatic ecosystems and as an initial biofilm evaluation step that informs downstream detailed investigations by complementary and more detailed methods.

Flow cytometry in combination with visual clustering offers an easy-to-use and fast method for studying aquatic biofilms. It can be used for biofilm characterization, detection of changes in biofilm community structure, and detection of abiotic particles embedded in the biofilm.

Cytometry와 수생 Biofilms의 특성

Biofilms are dynamic consortia of microorganism that play a key role in freshwater ecosystems. By changing their community structure, biofilms respond ...

환경과학

Characterizing Electron Transport through Living Biofilms

Here we demonstrate the method of electrochemical gating used to characterize electrical conductivity of electrode-grown microbial biofilms under physiologically relevant conditions.1 These measurements are performed on living biofilms in aqueous medium using source and drain electrodes patterned on a glass surface in a specialized configuration referred to as an interdigitated electrode (IDA) array. A biofilm is grown that extends across the gap connecting the source and drain. Potentials are applied to the electrodes (ES and ED) generating a source-drain current (ISD) through the biofilm between the electrodes. The dependency of electrical conductivity on gate potential (the average of the source and drain potentials, EG = [ED + ES]/2) is determined by systematically changing the gate potential and measuring the resulting source-drain current. The dependency of conductivity on gate potential provides mechanistic information about the extracellular electron transport process underlying the electrical conductivity of the specific biofilm under investigation. The electrochemical gating measurement method described here is based directly on that used by M. S. Wrighton2,3 and colleagues and R. W. Murray4,5,6 and colleagues in the 1980&#39;s to investigate thin film conductive polymers.

A protocol for measuring electrical conductivity of living microbial biofilms under physiologically relevant conditions is presented.

특성화 생활 Biofilms 통해 전자 전송

Here we demonstrate the method of electrochemical gating used to characterize electrical conductivity of electrode-grown microbial biofilms under ...

Methods for Characterizing the Co-development of Biofilm and Habitat Heterogeneity

Biofilms are surface-attached microbial communities that have complex structures and produce significant spatial heterogeneities. Biofilm development is strongly regulated by the surrounding flow and nutritional environment. Biofilm growth also increases the heterogeneity of the local microenvironment by generating complex flow fields and solute transport patterns. To investigate the development of heterogeneity in biofilms and interactions between biofilms and their local micro-habitat, we grew mono-species biofilms of Pseudomonas aeruginosa and dual-species biofilms of P. aeruginosa and Escherichia coli under nutritional gradients in a microfluidic flow cell. We provide detailed protocols for creating nutrient gradients within the flow cell and for growing and visualizing biofilm development under these conditions. We also present protocols for a series of optical methods to quantify spatial patterns in biofilm structure, flow distributions over biofilms, and mass transport around and within biofilm colonies. These methods support comprehensive investigations of the co-development of biofilm and habitat heterogeneity.

Biofilms have complex interactions with their surrounding environment. To comprehensively investigate biofilm-environment interactions, we present here a series of methods to create heterogeneous chemical environment for biofilm development, to quantify local flow velocity, and to analyze mass transport in and around biofilm colonies.

바이오 필름 및 서식지의 이질성의 공동 개발을 특성화하기위한 방법

Biofilms are surface-attached microbial communities that have complex structures and produce significant spatial heterogeneities. Biofilm development is ...

생체공학

A Microfluidic Platform to Study Bioclogging in Porous Media

Bacterial biofilms are found in several environmental and industrial porous media, including soils and filtration membranes. Biofilms grow under certain flow conditions and can clog pores, thereby redirecting the local fluid flow. The ability of biofilms to clog pores, the so-called bioclogging, can have a tremendous effect on the local permeability of the porous medium, creating a pressure buildup in the system, and impacting the mass flow through it. To understand the interplay between biofilm growth and fluid flow under different physical conditions (e.g., at different flow velocities and pore sizes), in the present study, a microfluidic platform is developed to visualize biofilm development using a microscope under externally-imposed, controlled physical conditions. The biofilm-induced pressure buildup in the porous medium can be measured simultaneously using pressure sensors and, later, correlated with the surface coverage of the biofilm. The presented platform provides a baseline for a systematic approach to investigate bioclogging caused by biofilms in porous media under flow conditions and can be adapted to studying environmental isolates or multispecies biofilms.

The present protocol describes a microfluidic platform to study biofilm development in quasi-2D porous media by combining high-resolution microscopy imaging with simultaneous pressure difference measurements. The platform quantifies the influence of pore size and fluid flow rates in porous media on bioclogging.

다공성 매체의 생물 막힘을 연구하기 위한 미세유체 플랫폼

Bacterial biofilms are found in several environmental and industrial porous media, including soils and filtration membranes. Biofilms grow under certain ...

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.

다양한 미생물 커뮤니티의 고밀도 배양위한 새로운 생물 반응기

A novel reactor design, coined a high density bioreactor (HDBR), is presented for the cultivation and study of high density microbial communities. Past ...

Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors

This work assesses different methodologies to study the impact of small molecule biofilm inhibitors, such as D-amino acids, on the development and resilience of Bacillus subtilis biofilms. First, methods are presented that select for small molecule inhibitors with biofilm-specific targets in order to separate the effect of the small molecule inhibitors on planktonic growth from their effect on biofilm formation. Next, we focus on how inoculation conditions affect the sensitivity of multicellular, floating B. subtilis cultures to small molecule inhibitors. The results suggest that discrepancies in the reported effects of such inhibitors such as D-amino acids are due to inconsistent pre-culture conditions. Furthermore, a recently developed protocol is described for evaluating the contribution of small molecule treatments towards biofilm resistance to antibacterial substances. Lastly, scanning electron microscopy (SEM) techniques are presented to analyze the three-dimensional spatial arrangement of cells and their surrounding extracellular matrix in a B. subtilis biofilm. SEM facilitates insight into the three-dimensional biofilm architecture and the matrix texture. A combination of the methods described here can greatly assist the study of biofilm development in the presence and absence of biofilm inhibitors, and shed light on the mechanism of action of these inhibitors.

Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors

This study presents the development of reproducible methodologies to study biofilm inhibitors and their effects on Bacillus subtilis multicellularity.

유학을위한 방법론<em&gt; B. 서브 틸리</em작은 분자 바이오 필름 억제제를 특성화 모델로&gt; 바이오 필름

This work assesses different methodologies to study the impact of small molecule biofilm inhibitors, such as D-amino acids, on the development and ...

면역학 및 감염병학

A New Method for Qualitative Multi-scale Analysis of Bacterial Biofilms on Filamentous Fungal Colonies Using Confocal and Electron Microscopy

Bacterial biofilms frequently form on fungal surfaces and can be involved in numerous bacterial-fungal interaction processes, such as metabolic cooperation, competition, or predation. The study of biofilms is important in many biological fields, including environmental science, food production, and medicine. However, few studies have focused on such bacterial biofilms, partially due to the difficulty of investigating them. Most of the methods for qualitative and quantitative biofilm analyses described in the literature are only suitable for biofilms forming on abiotic surfaces or on homogeneous and thin biotic surfaces, such as a monolayer of epithelial cells.
While laser scanning confocal microscopy (LSCM) is often used to analyze in situ and in vivo biofilms, this technology becomes very challenging when applied to bacterial biofilms on fungal hyphae, due to the thickness and the three dimensions of the hyphal networks. To overcome this shortcoming, we developed a protocol combining microscopy with a method to limit the accumulation of hyphal layers in fungal colonies. Using this method, we were able to investigate the development of bacterial biofilms on fungal hyphae at multiple scales using both LSCM and scanning electron microscopy (SEM). This report describes the protocol, including microorganism cultures, bacterial biofilm formation conditions, biofilm staining, and LSCM and SEM visualizations.

This protocol describes a new method to grow and qualitatively analyze bacterial biofilms on fungal hyphae by confocal and electron microscopy.

새로운 방법 사상 진균 식민지에 세균 바이오 필름의 질적 다중 스케일 분석을 위해 사용 공 촛점 및 전자 현미경

Bacterial biofilms frequently form on fungal surfaces and can be involved in numerous bacterial-fungal interaction processes, such as metabolic ...

Generation of Greater Bacterial Biofilm Biomass using PCR-Plate Deep Well Microplate Devices

Bacterial biofilms are difficult to eradicate from surfaces using conventional antimicrobial interventions. High-throughput 96-well microplate methods are frequently used to cultivate bacterial biofilms for rapid antimicrobial susceptibility testing to calculate minimal biofilm eradication concentration (MBEC) values. Standard biofilm devices consist of polystyrene pegged-lids fitted to 96-well microplates and are ideal for measuring biofilm biomass and MBEC values, but these devices are limited by available peg surface area for biomass accumulation and cost. Here, we outline a protocol to use self-assembled polypropylene 96-well deep well PCR-plate pegged-lid device to grow Escherichia coli BW25113 and Pseudomonas aeruginosa PAO1 biofilms. A comparison of 24-hour biofilms formed on standard and deep well devices by each species using crystal violet biomass staining and MBEC determination assays are described. The larger surface area of deep well devices expectedly increased overall biofilm formation by both species 2-4-fold. P. aeruginosa formed significantly greater biomass/mm2 on deep well pegs as compared to the standard device. E. coli had greater biomass/mm2 on standard polystyrene devices as compared the deep well device. Biofilm eradication assays with disinfectants such as sodium hypochlorite (bleach) or benzalkonium chloride (BZK) showed that both compounds could eliminate E. coli and P. aeruginosa biofilms from both devices but at different MBEC values. BZK biofilm eradication resulted in variable E. coli MBEC values between devices, however, bleach demonstrated reproducible MBEC values for both species and devices. This study provides a high throughput deep well method for growing larger quantities of biofilms on polypropylene devices for downstream studies requiring higher amounts of static biofilm.

This protocol presents methodology to perform biofilm growth and biomass measurements using self-assembled deep well PCR-plate devices for high-throughput 96-well pegged lid static biofilm screening.

PCR-플레이트 딥웰 마이크로플레이트 장치를 이용한 더 큰 박테리아 바이오필름 바이오매스의 생성

Bacterial biofilms are difficult to eradicate from surfaces using conventional antimicrobial interventions. High-throughput 96-well microplate methods are ...

Evaluation of the Efficacy of Organic Peroxyacids for Eradicating Dairy Biofilms Using an Approach Combining Static and Dynamic Methods

The presence of biofilms in the dairy industry is of major concern, as they may lead to the production of unsafe and altered dairy products due to their high resistance to most clean-in-place (CIP) procedures frequently used in processing plants. Therefore, it is imperative to develop new biofilm control strategies for the dairy industry. This protocol is aimed at evaluating the efficacy of organic peroxyacids (peracetic, perpropionic, and perlactic acids and a commercial peracetic acid-based disinfectant) for eradicating dairy biofilms using a combination of static and dynamic methods. All the disinfectants were tested on the strongest biofilm-producing bacteria in either a single or a mixed biofilm using the minimum biofilm eradication concentration (MBEC) assay, a static high-throughput screening method. A contact time of 5 min with the disinfectants at the recommended concentrations successfully eradicated both the single and mixed biofilms. Studies are currently ongoing to confirm these observations using the Center for Disease Control (CDC) biofilm reactor, a dynamic method to mimic in situ conditions. This type of bioreactor enables the use of a stainless-steel surface, which constitutes most industrial equipment and surfaces. The preliminary results from the reactor appear to confirm the efficacy of organic peroxyacids against biofilms. The combined approach described in this study may be used to develop and test new biological or chemical formulations for controlling biofilms and eradicating microorganisms.

This protocol describes an approach combining static and dynamic methods to evaluate the efficacy of organic peroxyacids for eradicating biofilms in the dairy industry. This approach may also be used to test the effectiveness of new biological or chemical formulations for controlling biofilms.

정적 방법과 동적 방법을 결합한 접근법을 사용하여 낙농 생물막을 박멸하기 위한 유기 과산화산의 효능 평가

The presence of biofilms in the dairy industry is of major concern, as they may lead to the production of unsafe and altered dairy products due to their ...