This research was funded by the Austrian Science Fund (FWF): project numbers P 29360 and P&#160;29143.&#160;Publication was supported by Publikationsfonds der Universit&#228;t Innsbruck. We greatly acknowledge EIG.

1. Preparation of medium
<ol>
	<li>Prepare redox indicator stock solution (0.1 g of resazurin/100 mL aqueous solution).</li>
	<li>Prepare vitamin solution (Table 1).</li>
	<li>Prepare trace element solution (Table 2). 
	NOTE: The order of addition is important; please refer to Table 2 and respective protocols.</li>
	<li>Prepare reducing agent stock solution (60 g Na2S/L aqueous solution).</li>
	<li>Weigh medium ingredients (mineral salt medium, Table 3) in an appropriate flask (e.g., 1 L screw cap lab flask). 
	NOTE: Depending on the experimental setup, the addition of a separate carbon source might be necessary.</li>
	<li>Add half-volume of distilled water (Table 3) and dissolve the ingredients.</li>
	<li>Add 1 mL of redox indicator solution according to Table 3.</li>
	<li>Add vitamin and trace element solution according to Table 3.</li>
	<li>Adjust the pH according to medium/organism requirements in Table 3. 
	NOTE: Color of redox indicator is pH dependent and might require some time to adjust.</li>
	<li>Bring to a final volume of 1 L with distilled water. 
	NOTE: Vitamin and trace element solutions can also be added after autoclaving by aseptically adding a filter-sterilized aliquot (diluted solutions, filter pore size &#60; 0.2 &#181;m) into previously closed and autoclaved serum flasks. However, this approach bears an elevated risk of contamination.</li>
</ol>
2. Filling of cultivation flasks
<ol>
	<li>Thoroughly clean and dry 120 mL serum flasks. 
	NOTE: Serum flasks are available in different volume capacities (e.g., 20, 60, 120, 250 mL).</li>
	<li>Thoroughly clean and dry butyl rubber septa.</li>
	<li>Weigh additional medium components (e.g., phenyl acid precursor substrates) in the cultivation flasks. 
	NOTE: Additional components depend on experimental setup and hypothesis.</li>
	<li>Fill the serum flasks with 50 mL of medium.</li>
</ol>
3. Reduction/removal of oxygen in the liquid phase
<ol>
	<li>Prepare a ~100&#176; C water bath.</li>
	<li>Set filled serum flasks in the water bath and incubate for approx. 20-30 min to reduce the solubility of O2 in the liquid phase.</li>
	<li>Flush the headspace immediately with N2 gas or alternatively with other gas or gas mixtures like N2/CO2. 
	CAUTION: Take care of appropriate room ventilation.</li>
	<li>Close the flasks with&#160;butyl rubber septa and fix with aluminum caps. 
	NOTE: Rubber septa might often fit better onto the neck of the flask by adding a drop of water/medium while drilling it in.</li>
	<li>Add 0.1 mL of reducing agent (stock solution) to each flask filled with 50 mL of medium to further reduce the redox potential (0.1 mL of reducing agent per 50 mL of medium).</li>
	<li>Autoclave for 20 min at 121 &#176;C. 
	CAUTION: An autoclave certified for the sterilization of closed vessels has to be used. Otherwise, overpressure derived from temperature increase might cause serum flasks to explode.</li>
</ol>
4. Inoculation of the medium
<ol>
	<li>Prepare inoculum from anaerobic digester.
	<ol>
		<li>Add 400 mL of distilled water into a flask and bring it to boil.</li>
		<li>Cool it down (&#60; 30 &#176;C) while permanently flushing the headspace with N2.</li>
		<li>Add approx. 100 g of sludge derived from an anaerobic digester. 
		NOTE: Avoid excessive contact of sludge with oxygen.</li>
		<li>Record the exact mass of added sludge for the exact determination of dilution.</li>
		<li>Exchange the flask&#8217;s headspace with N2 and close it with a butyl rubber septum.</li>
		<li>Shake the flask for 30 min at 120 rpm.</li>
	</ol>
	</li>
	<li>Remove 5 mL inoculum by using syringe + cannula and inject it into prepared serum flasks as described&#160;in step 1-3.</li>
</ol>
5. Incubation, sampling, and analysis
<ol>
	<li>Incubate inoculated serum flasks at a temperature that is appropriate for the respective experiment. 
	NOTE: Incubation temperature is dependent on experimental setup and used inoculum.
	<ol>
		<li>Drain overpressure resulting from the temperature increase using syringe + cannula, when the liquid in the serum flasks has equilibrated to incubation temperature (about 15 &#8211; 30 min, depending on incubation temperature). 
		CAUTION: Depending on the applied substrate, its concentration, temperature, incubation time, inoculum type and concentration, overpressure within flasks can rise by up to &#62; 2 bar pressure and might cause serum flaks to explode. Monitoring the overpressure by using a manometer and subsequently draining overpressure with a cannula is therefore mandatory.</li>
	</ol>
	</li>
	<li>Evaluate biogas production and composition during incubation time. 
	NOTE: Incubation period can span a few days to several weeks.
	<ol>
		<li>Record current atmospheric pressure.</li>
		<li>Prepare a manometer and evaluate the pressure within the flasks derived from microbial activity.</li>
		<li>Shake the flasks.</li>
		<li>Remove 1 mL of headspace gas using a syringe + cannula, and measure the H2, O2, CH4, and/or CO2 concentrations via gas chromatography. 
		NOTE: For the qualification and quantification of H2, O2, CH4, and CO2, a gas chromatograph was used applying operation temperatures of 160 &#176;C (column oven), 100 &#176;C (injector), and 180 &#176;C (thermal conductivity detector, TCD). N2 was used as a carrier gas. For details, refer to previous studies10.</li>
	</ol>
	</li>
	<li>Monitor concentrations of volatile fatty acids (VFA) and phenyl acids. For VFA and phenyl acid analysis, use a HPLC system equipped with an UV-detector (at 220 nm) running with 5 mM H2SO4 as a mobile phase. For method&#8217;s details and additional information on sample storage, please refer to previous studies11. 
	NOTE: The analysis of VFA is exemplary for many other physico-chemical analyses or microscopic evaluations. Moreover, molecular biological methods targeting the abundance of specific microorganisms and/or composition of the microbial community at a certain point of the experiment can be applied using the described procedure.
	<ol>
		<li>Remove 1 mL of liquid with syringe + cannula. 
		NOTE: Samples can be frozen (-20 &#176;C) immediately after withdrawal and analyzed at the end of the experiment11.</li>
		<li>Centrifuge at 15,000&#8211;20,000 x g and pass through 0.2 &#181;m RC (regenerated cellulose) filters.</li>
		<li>Inject 5-20 &#181;L onto a HPLC system and analyze for VFA composition and concentration of phenyl acids.</li>
	</ol>
	</li>
	<li>Drain flask&#8217;s overpressure using a cannula. 
	NOTE: After determining the pressure and gas composition as well as taking any necessary sample, place the cultivation flask back on respective temperature and do not drain overpressure before the liquid has achieved the incubation temperature.</li>
	<li>Calculate biogas and methane production VCH4N considering the ideal gas law using Equation 1-3. Please also refer to Table 4. 
	Equation 1: <img alt="Equation 1" src="/files/ftp_upload/60155/60155eq1v2.jpg" /> 
	whereby 
	Equation 2: <img alt="Equation 2" src="/files/ftp_upload/60155/60155eq2v2.jpg" /> 
	and 
	Equation 3: <img alt="Equation 3" src="/files/ftp_upload/60155/60155eq3v3.jpg" /> 
	NOTE: For the calculation of the total biogas production, the amount of CH4% and CH4%X in equation 2 and 3 has to be set to 100. NmL: normalized gas volume under standardized conditions (0 &#176;C, 1 atm), under which the molar gas volume is 22.414 NmL/mmol.</li>
</ol>

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	<li>Wiebe, W. J., Pomeroy, L. R., Wiegert, R. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anaerobic+Respiration+and+Fermentation.">Anaerobic Respiration and Fermentation.</a> The Ecology of a Salt Marsh. , 137-159 (1981).</li><li>Kim, B. H., Gadd, G. M., Kim, B. H., Gadd, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anaerobic+fermentation.">Anaerobic fermentation.</a> Bacterial physiology and metabolism. , 252-297 (2008).</li><li>Stolz, J. F., Oremland, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+respiration+of+arsenic+and+selenium).">Bacterial respiration of arsenic and selenium).</a> FEMS Microbiology Reviews. 23 (5), 615-627 (1999).</li><li>Lackner, N., Hintersonnleitner, A., Wagner, A. O., Illmer, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogenotrophic+Methanogenesis+and+Autotrophic+Growth+of+Methanosarcina+thermophila.">Hydrogenotrophic Methanogenesis and Autotrophic Growth of Methanosarcina thermophila.</a> Archaea. 2018 (5), 1-7 (2018).</li><li>Mutschlechner, M., Praeg, N., Illmer, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ecological+importance+of+grazing+to+methane+fluxes+and+engaged+microbial+communities+in+alpine+forest+soils.">The ecological importance of grazing to methane fluxes and engaged microbial communities in alpine forest soils.</a> FEMS Microbiology Ecology. , (2018).</li><li>Prem, E. M., Reitschuler, C., Illmer, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Livestock+grazing+on+alpine+soils+causes+changes+in+abiotic+and+biotic+soil+properties+and+thus+in+abundance+and+activity+of+microorganisms+engaged+in+the+methane+cycle.">Livestock grazing on alpine soils causes changes in abiotic and biotic soil properties and thus in abundance and activity of microorganisms engaged in the methane cycle.</a> European Journal of Soil Biology. 62, 22-29 (2014).</li><li>Praeg, N., Wagner, A. O., Illmer, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+fertilisation,+temperature+and+water+content+on+microbial+properties+and+methane+production+and+methane+oxidation+in+subalpine+soils.">Effects of fertilisation, temperature and water content on microbial properties and methane production and methane oxidation in subalpine soils.</a> European Journal of Soil Biology. 65, 96-106 (2014).</li><li>Wagner, A. O., Prem, E. M., Markt, R., Kaufmann, R., Illmer, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+phenylacetic+acid+and+phenylpropionic+acid+under+different+overload+conditions+during+mesophilic+and+thermophilic+anaerobic+digestion.">Formation of phenylacetic acid and phenylpropionic acid under different overload conditions during mesophilic and thermophilic anaerobic digestion.</a> Biotechnology for Biofuels. 12 (1), 359 (2019).</li><li>Lins, P., Malin, C., Wagner, A. O., Illmer, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduction+of+accumulated+volatile+fatty+acids+by+an+acetate-degrading+enrichment+culture.">Reduction of accumulated volatile fatty acids by an acetate-degrading enrichment culture.</a> FEMS Microbiology Ecology. 71 (3), 469-478 (2010).</li><li>Wagner, A. O., Hohlbrugger, P., Lins, P., Illmer, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+different+nitrogen+sources+on+the+biogas+production+-+a+lab-scale+investigation.">Effects of different nitrogen sources on the biogas production - a lab-scale investigation.</a> Microbiological research. 167 (10), 630-636 (2012).</li><li>Wagner, A. O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sample+preparation,+preservation,+and+storage+for+volatile+fatty+acid+quantification+in+biogas+plants.">Sample preparation, preservation, and storage for volatile fatty acid quantification in biogas plants.</a> Engineering in Life Sciences. 17 (2), 132-139 (2017).</li><li>Uzarski, J. S., DiVito, M. D., Wertheim, J. A., Miller, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Essential+design+considerations+for+the+resazurin+reduction+assay+to+noninvasively+quantify+cell+expansion+within+perfused+extracellular+matrix+scaffolds.">Essential design considerations for the resazurin reduction assay to noninvasively quantify cell expansion within perfused extracellular matrix scaffolds.</a> Biomaterials. 129, 163-175 (2017).</li><li>Costilow, R. N., Breznak, J. A., Marzluf, G. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physicochemical+Factors+in+Growth.">Physicochemical Factors in Growth.</a> Methods for General and Molecular Microbiology. , 309-329 (2007).</li><li>Le Mer, J., Roger, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production,+oxidation,+emission+and+consumption+of+methane+by+soils:+a+review.">Production, oxidation, emission and consumption of methane by soils: a review.</a> European Journal of Soil Biology. 37 (1), 25-50 (2001).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Verein+deutscher+Ingenieure+VDI+4630:+Fermentation+of+organic+material.">Verein deutscher Ingenieure VDI 4630: Fermentation of organic material.</a> VDI Richtlinien. , (2006).</li><li>Wagner, A. O., Reitschuler, C., Illmer, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+different+acetate:+Propionate+ratios+on+the+methanogenic+community+during+thermophilic+anaerobic+digestion+in+batch+experiments.">Effect of different acetate: Propionate ratios on the methanogenic community during thermophilic anaerobic digestion in batch experiments.</a> Biochemical Engineering Journal. 90, 154-161 (2014).</li></ol>

The authors have nothing to disclose.

The most important and critical step in culturing anaerobic microorganisms is to ensure oxygen-free conditions in cultivation media and flasks&#8217; headspace. An indicator like resazurin can be used to indirectly check the correct anaerobic filling of the flasks. Resazurin is a commonly used redox dye as it is inexpensive, non-toxic, and already effective in low doses and short incubation times 12. When incorporated to media, the blue colored resazurin first undergoes an irreversible reduction step to resorufin, which is pink at neutral pH values. This first reaction can occur when the media are heated&#160;13. Subsequently, resorufin is reduced to colorless dihydroresorufin in a reversible secondary reaction (Figure 7)12. The resorufin/dihydroresorufin redox system becomes completely colorless at a standard oxidation-reduction potential of about Eh = -110 mV and turns pink above a redox potential of -51 mV 13.
In order to further reduce the redox potential, for example, to facilitate the growth of methanogenic microorganisms known to require less than -200 mV14, a Na2S solution can be added. Alternatively, cysteine-HCl, sodium-thioglycolate, or sodium dithionite are commonly used. However, which reducing agent is appropriate to use depends on the respective experimental setup and might require special attention. For instance, sodium thioglycolate needs temperature activation (e.g., by autoclaving).
A well-balanced microbial consortium, comprised of various genera of Bacteria and Archaea, and an efficiently working anaerobic degradation cascade can further be evaluated by determining the headspace gas composition in the culture flasks via gas chromatography. When handling compounds like phenyl acids derived from different precursors, the assessment of the headspace is a fast way to check the methanogenesis process8. A headspace CH4 concentration of approx. 50-60% in the controls at the end of the incubation period indicates a successful utilization of the applied nutrients and thus a&#160;mineralization of organic material under anaerobic conditions. The theoretical methane production and expectable methane concentrations during the digestion process can be determined ex ante according to the Buswell-Boyle equation after elementary analysis of the substrate or by estimating the content of carbohydrates, proteins, and fats in the substrate. According to VDI 4630 15, carbohydrates can lead to a theoretical biogas production of 750 L kg-1 VSS (50% CH4 and 50% CO2), proteins to 800 L kg-1 VSS (72% CH4 and 28 % CO2), and fats to 1,390 L kg-1 VSS (60% CH4 and 40% CO2).
Moreover, formation and possible subsequent degradation of VFAs and phenyl acids were monitored. The degradation process can be evaluated by analyzing the VFA concentrations (e.g., acetate, propionate) at different time points. Accumulation of short-chain fatty acids like acetate and/or propionate can point to disturbances in the methanogenic community composition and to an overall reactor overload. However, a well-balanced microbial degradation cascade can even cope with very high VFA and acetate concentrations9. Besides, the acetate / propionate ratio might further provide information on the overall reactor condition16. However, there are many parameters suitable for process monitoring that have to be selected according to the proposed experimental hypotheses. In the present example, target variables were phenyl acid concentrations (Figure 6).

On earth molecular oxygen in notable concentrations is available in areas having direct contact with the atmosphere or in the presence of oxygenic phototrophs. Environments in which oxygen is absent are called anaerobic. However, energy conversion is still possible under anaerobic conditions via two different metabolic processes, fermentation and anaerobic respiration1.
While organisms undergoing aerobic respiration are using oxygen as a terminal electron acceptor, anaerobic respiration requires alternative electron acceptors like nitrate or sulphate2. In the so-called &#8220;electron tower&#8221;, redox couples are organized according to their redox potential, with the most negative ones located at the top (electron donors) and strongest oxidation agents with positive redox potential at the bottom (electron acceptors). The electron transfer between donors and acceptors leads to energy conservation via the so-called respiratory chain and electrons can be captured by electron acceptors - to stay in the picture - in different floors of the tower. Thereby, the higher the fall of electrons through the electron tower, the more energy can be conserved by the respective reaction. Therefore, respiration is also possible in anaerobic habitats, for example, with redox pairs including NO3-/NO2-, fumaric acid/succinic acid, SO32-/H2S, S&#176;/H2S, Mn(IV)/Mn(II), Fe(III)/Fe(II)2,3. First, the resulting energy is conserved as membrane potential, which is subsequently used by electron transport phosphorylation for adenosine-triphosphate (ATP) synthesis by membrane-bound ATP-synthases. In contrast to aerobic respiration, the amount of energy that can be conserved by anaerobic respiration can be dramatically reduced; however, the energy output of most anaerobic respirations is still higher compared to fermentation, an anaerobic energy conservation path in habitats lacking oxygen and other terminal electron acceptors2.
During fermentation, energy-rich, organic substrates are degraded to various fermentation products that often define the name of the overall process, for example, alcoholic fermentation. In contrast to respiration processes, ATP generation during fermentation is limited to substrate-level phosphorylation during which a phosphate group is transferred to adenosine-di-phosphate (ADP) from an energy-rich phosphorylated substrate2. Fermenting microorganisms play a central role in the anaerobic degradation of organic matter as they are key-players in substrate breakdown. The primary fermentation products, like organic acids, alcohols, CO2, and H2, can subsequently be used by secondary fermenting microorganisms to produce acetic acid, CO2, and H2. Examples for fermentation products include lactic acid, various volatile fatty acids (formic-, acetic-, propionic-, butyric-, valeric acid), n-butanol, 2,3-butandiol, acetone, and ethanol.
Cultivation of microorganisms under strictly anaerobic conditions requires completely different methods and equipment compared with the cultivation of aerobic organisms. While oxygen-tolerant organisms are often cultivated on agar dishes, so-called surface cultures, this is - with a few exceptions - hardly possible for strictly anaerobic microorganisms. Therefore, enrichment cultures of strictly anaerobic microorganisms are mainly established in liquid media applying culture vessels sealed with gas-tight septa that ensure an oxygen-free headspace atmosphere4,6,7.
The current protocol description will provide appropriate cultivation methods for target microorganisms of a mixed population derived from an anaerobic biogas plant. The isolation and cultivation of pure cultures is even more challenging but not part of this work.
Here, we show the procedure for cultivating an anaerobic microbial community based on a study regarding the formation of phenyl acids during anaerobic digestion of proteinaceous substrates8. The microbial community consisted of members from all four phases of anaerobic digestion: hydrolysis, acidogenesis, acetogenesis, and methanogenesis. A mineral salt medium supplemented with a carbon source, redox-indicator, vitamin and trace element solution, and reducing agent was applied9. The medium was amended with the respective proteinaceous phenyl acid precursor substrates8.

<table border="0" cellpadding="0" cellspacing="0" style="width:703px;" width="701">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">culture flasks (120 mL, N20)</td>
			<td style="width:187px;">Ochs, Germany</td>
			<td align="right" style="width:123px;">102046</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">buty rubber septa (N20)</td>
			<td style="width:187px;">Ochs, Germany</td>
			<td align="right" style="width:123px;">102049</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">aluminium caps (N20)</td>
			<td style="width:187px;">Ochs, Germany</td>
			<td align="right" style="width:123px;">102050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">N2 gas</td>
			<td style="width:187px;">Messer, Austria</td>
			<td style="width:123px;"></td>
			<td>purity 5.0</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">syringes + cannulae</td>
			<td style="width:187px;">various</td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">crimper</td>
			<td style="width:187px;">Ochs, Germany</td>
			<td align="right" style="width:123px;">102051</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">de-crimper</td>
			<td style="width:187px;">Ochs, Germany</td>
			<td align="right" style="width:123px;">102052</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">GC2010</td>
			<td style="width:187px;">Shimadzu</td>
			<td style="width:123px;"></td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Shin-carbon GC column</td>
			<td style="width:187px;">Restek</td>
			<td style="width:123px;"></td>
			<td>chromatographic separation of H2, O2, CH4, and CO2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">HPLC Prominence</td>
			<td style="width:187px;">Shimadzu</td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Fast Fruit HPLC Column</td>
			<td style="width:187px;">Phenomenex</td>
			<td style="width:123px;"></td>
			<td>chromatographic separation of VFAs, phenyl acids, etc.</td>
		</tr>
	</tbody>
</table>

medium preparation for the cultivation of microorganisms under strictly anaerobic/anoxic conditions

In contrast to aerobic organisms, strictly anaerobic microorganisms require the absence of oxygen and usually a low redox potential to initiate growth. As oxygen is ubiquitous in air, retaining O2-free conditions during all steps of cultivation is challenging but a prerequisite for anaerobic culturing. The protocol presented here demonstrates the successful cultivation of an anaerobic mixed culture derived from a biogas plant using a simple and inexpensive method. A precise description of the entire anoxic culturing process is given including media preparation, filling of cultivation flasks, supplementation with redox indicator and reducing agents to provide low redox potentials as well as exchanging the headspace to keep media free from oxygen. Furthermore, a detailed overview of aseptically inoculating gas tight serum flasks (by using sterile syringes and needles) and suitable incubation conditions is provided. The present protocol further deals with gas and liquid sampling for subsequent analyses regarding gas composition and volatile fatty acid concentrations using gas chromatography (GC) and high performance liquid chromatography (HPLC), respectively, and the calculation of biogas and methane yield considering the ideal gas law.

Cultivation flasks were filled with medium under anaerobic conditions according to the protocol described above, checked for the appropriate color (Figure 1), and used as miniature batch bioreactors conducting anaerobic digestion. These were amended with substrates potentially causing phenyl-acid formation and incubated using anaerobic digester sludge as inoculum (Figure 2). Tryptophan, tyrosine, and phenylalanine, as well as the complex proteinaceous precursor meat extract and casein were applied in two and three different concentrations, respectively. Controls were prepared without additional substrate supplementation. Different substrate concentrations aimed at the simulation of different stages of overload. Flasks were incubated at 37 &#176;C (mesophilic) for 4 weeks.
Biogas production and composition (H2, CH4, CO2) was monitored regularly via gas chromatography (GC TCD)10 and evaluation of headspace pressure. Figure 3 demonstrates differences in the cumulative methane production derived from digestion of the applied substrates in varied concentrations during 4 weeks of anaerobic incubation. Besides, methanogens were visualized by irradiating the coenzyme F420, an electron carrier in methanogenesis, exhibiting a blue-green fluorescence with an absorption maximum at 420 nm (Figure 4).
Concurrent to gas analysis, samples for VFA and phenyl acid concentration measurements via HPLC11 were withdrawn and stored frozen until further processing. Figure 5 shows the effect of different stages of overload as reflected by an accumulation in highly overloaded samples exemplarily depicted for acetate. Figure 6&#160;depicts&#160;the dynamics of phenyl acetate concentrations during the incubation period.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60155/60155fig1.jpg" /> 
Figure 1: Redox indicator. Correct redox potential in the cultivation flasks can be controlled by adding a redox indicator. <a href="https://www.jove.com/files/ftp_upload/60155/60155fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60155/60155fig2.jpg" /> 
Figure 2: Miniature batch bioreactors. Miniature batch bioreactors prepared in 120 mL cultivation flasks for anaerobic digestion experiments. Flasks were filled with medium and inoculated with diluted digester sludge. The reactors were gas-tightly sealed with butyl rubber stoppers and aluminum caps. <a href="https://www.jove.com/files/ftp_upload/60155/60155fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60155/60155fig3.jpg" /> 
Figure 3: Methane production. Cumulative methane production during 28 days of mesophilic incubation from reactors reflecting different overload conditions (low, medium, high). Cont: control; Tryp: tryptophan; Tyr: tyrosine; Phe: phenylalanine; ME: meat extract; Cas: casein. This is a modified figure originating from an earlier study8. <a href="https://www.jove.com/files/ftp_upload/60155/60155fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/60155/60155fig4.jpg" /> 
Figure 4: Fluorescing methanogens. Methanogens emit a blueish light when being excited with UV light. Here, methanogens are attached to plant particles (light green). Samples were taken from a batch reactor, diluted for microscopy, and immediately analyzed. <a href="https://www.jove.com/files/ftp_upload/60155/60155fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/60155/60155fig5.jpg" /> 
Figure 5: Acetate concentration. Acetate concentration during 28 days of mesophilic incubation in reactors reflecting different overload conditions (low, medium, high). Cont: control; Tryp: tryptophan; Tyr: tyrosine; Phe: phenylalanine; ME: meat extract; Cas: casein. This is a modified figure originating from an earlier study8. <a href="https://www.jove.com/files/ftp_upload/60155/60155fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/60155/60155fig6.jpg" /> 
Figure 6: Phenylacetate concentration. Phenylacetate concentration during 28 days of mesophilic incubation in reactors reflecting different overload conditions (low, medium, high). Cont: control; Tryp: tryptophan; Tyr: tyrosine; Phe: phenylalanine; ME: meat extract; Cas: casein. This is a modified figure originating from an earlier study8. <a href="https://www.jove.com/files/ftp_upload/60155/60155fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/60155/60155fig7.jpg" /> 
Figure 7: Resazurin reaction. Blue colored resazurin undergoes an irreversible reduction to resorufin (pink) and a further reversible reduction to the colourless dihydroresorufin according to Uzarski et al.12. <a href="https://www.jove.com/files/ftp_upload/60155/60155fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Cyanocobalamin</td>
			<td>0.050 g</td>
		</tr>
		<tr>
			<td>4-aminobenzoic acid</td>
			<td>0.050 g</td>
		</tr>
		<tr>
			<td>D-biotin</td>
			<td>0.010 g</td>
		</tr>
		<tr>
			<td>Nicotinic acid</td>
			<td>0.100 g</td>
		</tr>
		<tr>
			<td>Pyridoxine</td>
			<td>0.250 g</td>
		</tr>
		<tr>
			<td>D-pantothenic acid</td>
			<td>0.025 g</td>
		</tr>
		<tr>
			<td>Thiaminium chloride HCl</td>
			<td>0.18 g</td>
		</tr>
		<tr>
			<td>Distilled water</td>
			<td>1000 mL</td>
		</tr>
	</tbody>
</table>
Table 1: Vitamin solution.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>25% (w/v) HCl</td>
			<td>10.0 mL</td>
		</tr>
		<tr>
			<td>FeCl2 x 4 H2O</td>
			<td>1.50 g</td>
		</tr>
		<tr>
			<td>ZnCl2</td>
			<td>0.070 g</td>
		</tr>
		<tr>
			<td>MnCl2 x 4 H2O</td>
			<td>0.100 g</td>
		</tr>
		<tr>
			<td>H3BO3</td>
			<td>0.006 g</td>
		</tr>
		<tr>
			<td>CoCl2 x 6 H2O</td>
			<td>0.190 g</td>
		</tr>
		<tr>
			<td>CuCl2 x 2 H2O</td>
			<td>0.002 g</td>
		</tr>
		<tr>
			<td>NiCl2 x 6 H2O</td>
			<td>0.024 g</td>
		</tr>
		<tr>
			<td>Na2MoO4 x 2 H2O</td>
			<td>0.036 g</td>
		</tr>
		<tr>
			<td>Distilled water</td>
			<td>990.0 mL</td>
		</tr>
		<tr>
			<td>Preparation recommendation</td>
			<td>Add HCl and dissolve FeCl2, add 100 mL distilled water, dissolve the other ingredients, and make up to 1000 mL.</td>
		</tr>
	</tbody>
</table>
Table 2: Trace element solution.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>NaCl</td>
			<td>1.0 g</td>
		</tr>
		<tr>
			<td>MgCl2 x 6 H2O</td>
			<td>0.4 g</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>0.2 g</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>0.5 g</td>
		</tr>
		<tr>
			<td>CaCl2 x 2 H2O</td>
			<td>0.15 g</td>
		</tr>
		<tr>
			<td>L-cysteine</td>
			<td>0.5 g</td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>1.0 g</td>
		</tr>
		<tr>
			<td>Resazurin solution</td>
			<td>1 mL</td>
		</tr>
		<tr>
			<td>Vitamin solution</td>
			<td>1 mL</td>
		</tr>
		<tr>
			<td>Trace element solution</td>
			<td>1 mL</td>
		</tr>
		<tr>
			<td>Distilled water</td>
			<td>1000 mL</td>
		</tr>
		<tr>
			<td>pH</td>
			<td>7.2</td>
		</tr>
	</tbody>
</table>
Table 3: Minimal salt medium.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Variable</td>
			<td>Unit</td>
			<td>Description</td>
		</tr>
		<tr>
			<td>tY</td>
			<td>[d]</td>
			<td>Timepoint of measurement</td>
		</tr>
		<tr>
			<td>tX</td>
			<td>[d]</td>
			<td>Timepoint of preceding measurement</td>
		</tr>
		<tr>
			<td>pM</td>
			<td>[mbar]</td>
			<td>Measured overpressure at tY</td>
		</tr>
		<tr>
			<td>pA</td>
			<td>[mbar]</td>
			<td>Ambient pressure at tY</td>
		</tr>
		<tr>
			<td>pAX</td>
			<td>[mbar]</td>
			<td>Ambient pressure at tX</td>
		</tr>
		<tr>
			<td>pS</td>
			<td>[mbar]</td>
			<td>Standard pressure, 1013,25 mbar acc. DIN 1343</td>
		</tr>
		<tr>
			<td>TI</td>
			<td>[K]</td>
			<td>Incubation temperature</td>
		</tr>
		<tr>
			<td>TS</td>
			<td>[K]</td>
			<td>Standard temperature, 273,15 K (corresponds to 0&#176; C) acc. DIN 1343</td>
		</tr>
		<tr>
			<td>VH</td>
			<td>[ml]</td>
			<td>Headspace volume at tY</td>
		</tr>
		<tr>
			<td>VHX</td>
			<td>[ml]</td>
			<td>Headspace volume at tX</td>
		</tr>
		<tr>
			<td>CH4%</td>
			<td>[vol%]</td>
			<td>Methane concentration according to GC-measurement at tY</td>
		</tr>
		<tr>
			<td>CH4%X</td>
			<td>[vol%]</td>
			<td>Methane concentration according to GC-measurement at tX</td>
		</tr>
		<tr>
			<td>VCH4T</td>
			<td>[Nml]</td>
			<td>Total methane amount in the serum bottle at tY</td>
		</tr>
		<tr>
			<td>VCH4R</td>
			<td>[Nml]</td>
			<td>Residual methane amount in the headspace at tX</td>
		</tr>
		<tr>
			<td>VCH4N</td>
			<td>[Nml]</td>
			<td>Newly produced methane from tX to tY</td>
		</tr>
	</tbody>
</table>
Table 4: Description of variables in Equation 1 - 3.

Watch this Scientific Journal Video about Medium Preparation for the Cultivation of Microorganisms under Strictly Anaerobic/Anoxic Conditions at JoVE.com

Medium Preparation for the Cultivation of Microorganisms under Strictly Anaerobic/Anoxic Conditions

As obligate anaerobic organisms are unable to grow upon oxygen exposure, the use of anaerobic culturing techniques is indispensable. Here, we demonstrate a simple and effective method to cultivate a mixed culture derived from a biogas plant from media preparation to gas and volatile fatty acid quantification.

In contrast to aerobic organisms, strictly anaerobic microorganisms require the absence of oxygen and usually a low redox potential to initiate growth. As ...

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27.1K Views.  Universität Innsbruck. As obligate anaerobic organisms are unable to grow upon oxygen exposure, the use of anaerobic culturing techniques is indispensable. Here, we demonstrate a simple and effective method to cultivate a mixed culture derived from a biogas plant from media preparation to gas and volatile fatty acid quantification.

Video: Medium Preparation for the Cultivation of Microorganisms under Strictly Anaerobic/Anoxic Conditions

Preparation of Homogeneous MALDI Samples for Quantitative Applications

This protocol demonstrates a simple sample preparation to reduce spatial heterogeneity in ion signals during matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. The heterogeneity of ion signals is a severe problem in MALDI, which results in poor data reproducibility and makes MALDI unsuitable for quantitative analysis. By regulating sample plate temperature during sample preparation, thermal-induced hydrodynamic flows inside droplets of sample solution are able to reduce the heterogeneity problem. A room-temperature sample preparation chamber equipped with a temperature-regulated copper base block that holds MALDI sample plates facilitates precise control of the sample drying condition. After drying of sample droplets, the temperature of sample plates is returned to room temperature and removed from the chamber for subsequent mass spectrometric analysis. The areas of samples are examined with MALDI-imaging mass spectrometry to obtain the spatial distribution of all components in the sample. In comparison with the conventional dried-droplet method that prepares samples under ambient conditions without temperature control, the samples prepared with the method demonstrated herein show significantly better spatial distribution and signal intensity. According to observations using carbohydrate and peptide samples, decreasing substrate temperature while maintaining the surroundings at ambient temperature during the drying process can effectively reduce the heterogeneity of ion signals. This method is generally applicable to various combinations of samples and matrices.

A protocol for reducing spatial heterogeneities of ion signals in MALDI mass spectrometry by regulating substrate temperature during sample drying processes is demonstrated.

This protocol demonstrates a simple sample preparation to reduce spatial heterogeneity in ion signals during matrix-assisted laser desorption/ionization ...

Preparation and Delivery of Protein Microcrystals in Lipidic Cubic Phase for Serial Femtosecond Crystallography

Membrane proteins (MPs) are essential components of cellular membranes and primary drug targets. Rational drug design relies on precise structural information, typically obtained by crystallography; however MPs are difficult to crystallize. Recent progress in MP structural determination has benefited greatly from the development of lipidic cubic phase (LCP) crystallization methods, which typically yield well-diffracting, but often small crystals that suffer from radiation damage during traditional crystallographic data collection at synchrotron sources. The development of new-generation X-ray free-electron laser (XFEL) sources that produce extremely bright femtosecond pulses has enabled room temperature data collection from microcrystals with no or negligible radiation damage. Our recent efforts in combining LCP technology with serial femtosecond crystallography (LCP-SFX) have resulted in high-resolution structures of several human G protein-coupled receptors, which represent a notoriously difficult target for structure determination. In the LCP-SFX technique, LCP is recruited as a matrix for both growth and delivery of MP microcrystals to the intersection of the injector stream with an XFEL beam for crystallographic data collection. It has been demonstrated that LCP-SFX can substantially improve the diffraction resolution when only sub-10 &#181;m crystals are available, or when the use of smaller crystals at room temperature can overcome various problems associated with larger cryocooled crystals, such as accumulation of defects, high mosaicity and cryocooling artifacts. Future advancements in X-ray sources and detector technologies should make serial crystallography highly attractive and practicable for implementation not only at XFELs, but also at more accessible synchrotron beamlines. Here we present detailed visual protocols for the preparation, characterization and delivery of microcrystals in LCP for serial crystallography experiments. These protocols include methods for conducting crystallization experiments in syringes, detecting and characterizing the crystal samples, optimizing crystal density, loading microcrystal laden LCP into the injector device and delivering the sample to the beam for data collection.

We describe procedures for the preparation and delivery of membrane protein microcrystals in lipidic cubic phase for serial crystallography at X-ray free-electron lasers and synchrotron sources. These protocols can also be applied for incorporation and delivery of soluble protein microcrystals, leading to substantially reduced sample consumption compared to liquid injection.

Membrane proteins (MPs) are essential components of cellular membranes and primary drug targets. Rational drug design relies on precise structural ...

Preparation of Giant Vesicles Encapsulating Microspheres by Centrifugation of a Water-in-oil Emulsion

The constructive biology and the synthetic biology approach to creating artificial life involve the bottom-up assembly of biological or nonbiological materials. Such approaches have received considerable attention in research on the boundary between living and nonliving matter and have been used to construct artificial cells over the past two decades. In particular, Giant Vesicles (GVs) have often been used as artificial cell membranes. In this paper, we describe the preparation of GVs encapsulating highly packed microspheres as a model of cells containing highly condensed biomolecules. The GVs were prepared by means of a simple water-in-oil emulsion centrifugation method. Specifically, a homogenizer was used to emulsify an aqueous solution containing the materials to be encapsulated and an oil containing dissolved phospholipids, and the resulting emulsion was layered carefully on the surface of another aqueous solution. The layered system was then centrifuged to generate the GVs. This powerful method was used to encapsulate materials ranging from small molecules to microspheres.

Giant vesicles containing highly packed micrometer-sized components are useful cell models. The water-in-oil emulsion centrifugation method is a simple, powerful tool for the preparation of giant vesicles with encapsulated materials.

The constructive biology and the synthetic biology approach to creating artificial life involve the bottom-up assembly of biological or nonbiological ...

Hydrolysis of a Ni-Schiff-Base Complex Using Conditions Suitable for Retention of Acid-labile Protecting Groups

Unnatural amino acids, amino acids containing side-chain functionalities not commonly seen in nature, are increasingly found in synthetic peptide sequences. Synthesis of some unnatural amino acids often includes the use of a precursor consisting of a Schiff-base stabilized by a nickel cation. Unnatural side-chains can be installed on an amino acid backbone found in this Schiff-base complex. The resulting unnatural amino acid can then be isolated from this complex using hydrolysis of the Schiff-base, typically by employing reflux in strongly acidic solution. These highly acidic conditions may remove acid-labile side-chain protecting groups necessary for the unnatural amino acids to be used in microwave-assisted solid-phase peptide synthesis. In this work, we present an efficient hydrolysis and subsequent Fmoc protection of an amino acid isolated from a Ni-Schiff base complex. Hydrolysis conditions presented in this work are suitable for retention of acid-labile side-chain protecting groups and may be adaptable to a variety of unnatural amino acid substrates.

Here, we present an efficient hydrolysis and subsequent Fmoc protection of an amino acid isolated from a Ni-Schiff-base complex. Hydrolysis conditions presented here are suitable for use when retention of acid-labile side-chain protecting groups is required. This technique may be adaptable to a variety of unnatural amino acid substrates.

Unnatural amino acids, amino acids containing side-chain functionalities not commonly seen in nature, are increasingly found in synthetic peptide ...

Plate-based Large-scale Cultivation of Caenorhabditis elegans: Sample Preparation for the Study of Metabolic Alterations in Diabetes

Culturing Caenorhabditis elegans (C. elegans) in a large-scale manner on agar plates can be time-consuming and difficult. This protocol describes a simple and inexpensive method to obtain a large number of animals for the isolation of proteins to proceed with a western blot, mass spectrometry, or further proteomics analyses. Furthermore, an increase of nematode numbers for immunostainings and the integration of multiple analyses under the same culturing conditions can easily be achieved. Additionally, a transfer between plates with different experimental conditions is facilitated. Common techniques in plate culture involve the transfer of a single C. elegans using a platinum wire and the transfer of populated agar chunks using a scalpel. However, with increasing nematode numbers, these techniques become overly time-consuming. This protocol describes the large-scale culture of C. elegans including numerous steps to minimize the impact of the sample preparation on the physiology of the worm. Fluid and shear stress can alter the lifespan of and metabolic processes in C. elegans, thus requiring a detailed description of the critical steps in order to retrieve reliable and reproducible results. C. elegans is a model organism, consisting of neuronal cells for up to one-third, but lacking blood vessels, thus providing the possibility to investigate solely neuronal alterations independent of vascular control. Recently, early neurodegeneration in diabetic retinopathy was found prior to vascular alterations. Thus, C. elegans is of special interest for studying general mechanisms of diabetic complications. For example, an increased formation of advanced glycation end products (AGEs) and reactive oxygen species (ROS) is observed, which are reproducibly found in C. elegans. Protocols to handle samples of adequate size for a broader spectrum of investigations are presented here, exemplified by the study of diabetes-induced biochemical alterations. In general, this protocol can be useful for studies requiring large C. elegans numbers and in which liquid culture is not suitable.

Plate-based Large-scale Cultivation of Caenorhabditis elegans: Sample Preparation for the Study of Metabolic Alterations in Diabetes

This protocol describes a method for the large-scale cultivation of Caenorhabditis elegans on solid media. As an alternative to liquid culture, this protocol allows obtaining parameters of different scales under plate-based cultivation. This increases the comparability of results by omitting the morphological and metabolic differences between liquid and solid media culture.

Culturing Caenorhabditis elegans (C. elegans) in a large-scale manner on agar plates can be time-consuming and difficult. This protocol describes a simple ...

Preparation of Chloroplast Sub-compartments from Arabidopsis for the Analysis of Protein Localization by Immunoblotting or Proteomics

Chloroplasts are major components of plant cells. Such plastids fulfill many crucial functions, such as assimilation of carbon, sulfur and nitrogen as well as synthesis of essential metabolites. These organelles consist of the following three key sub-compartments. The envelope, characterized by two membranes, surrounds the organelle and controls the communication of the plastid with other cell compartments. The stroma is the soluble phase of the chloroplast and the main site where carbon dioxide is converted into carbohydrates. The thylakoid membrane is the internal membrane network consisting of grana (flat compressed sacs) and lamellae (less dense structures), where oxygenic photosynthesis takes place. The present protocol describes step by step procedures required for the purification, using differential centrifugations and Percoll gradients, of intact chloroplasts from Arabidopsis, and their fractionation, using sucrose gradients, in three sub-compartments (i.e., envelope, stroma, and thylakoids). This protocol also provides instructions on how to assess the purity of these fractions using markers associated to the various chloroplast sub-compartments. The method described here is valuable for subplastidial localization of proteins using immunoblotting, but also for subcellular and subplastidial proteomics and other studies.

Here, we describe a method to purify intact chloroplasts from Arabidopsis leaves and their three main sub-compartments (envelope, stroma, and thylakoids), using a combination of differential centrifugations, continuous Percoll gradients, and discontinuous sucrose gradients. The method is valuable for subplastidial and subcellular localization of proteins by immunoblotting and proteomics.

Chloroplasts are major components of plant cells. Such plastids fulfill many crucial functions, such as assimilation of carbon, sulfur and nitrogen as ...

Proteomic Analysis of Human Macrophage Polarization Under a Low Oxygen Environment

Macrophages are innate immune cells involved in a number of physiological functions ranging from responses to infectious pathogens to tissue homeostasis. The various functions of these cells are related to their activation states, which is also called polarization. The precise molecular description of these various polarizations is a priority in the field of macrophage biology. It is currently acknowledged that a multidimensional approach is necessary to describe how polarization is controlled by environmental signals. In this report, we describe a protocol designed to obtain the proteomic signature of various polarizations in human macrophages. This protocol is based on a label-free quantification of macrophage protein expression obtained from in-gel fractionated and Lys C/trypsin-digested cellular lysis content. We also provide a protocol based on in-solution digestion and isoelectric focusing fractionation to use as an alternative. Because oxygen concentration is a relevant environmental parameter in tissues, we use this protocol to explore how atmospheric composition or a low oxygen environment affects the classification of macrophage polarization.

We present a protocol to obtain proteomic signatures of human macrophages and apply this to determination of the impact of a low oxygen environment on macrophage polarization.

Macrophages are innate immune cells involved in a number of physiological functions ranging from responses to infectious pathogens to tissue homeostasis. ...

Preparation of High-Quality Fermented Fish Product

This protocol provides a method for preparation of industrialized fermented fish product with sturgeon (Aquilaria sinensis) meat product. The procedures were: (1) pretreatment of farmed sturgeon including decapitation, evisceration, skinning-off, cleaning and cutting; (2) marinating fish cubes in 6-12% (w/v) salt solution (1:1, fish cube mass to solution volume); (3) drying fish cubes to a water content of 50-60% by hot air (40-60 &#176;C) or by vacuum; (4) fermentation involving inoculating fish cubes with 0.4-1.6% (w/w) S. cerevisiae in flavor solution to fish cubes and fermenting at 25-35 &#176;C for 6-10 h; (5) sealing fish cubes in vacuum packages with marinating and fermenting solutions; (6) sterilizing at 115-121 &#176;C for 10-20 min. The sturgeon meat product prepared by this method has delicious taste which is mellow and thick, has various types and large amounts of volatile flavor compounds such as alcohols and esters which could mask musty and unpleasant odor from fish, has moderate salt content but good texture properties such as high springiness, gumminess and chewiness, and has bright russet color and attractive appearance. This new technique could also be applied in the processing of other fish to provide convenient fish snack foods which could be stored at room temperature. It is appropriate for both marine and freshwater fish.

The goal of the protocol is to provide an industrialized fish fermentation technique based on inoculation of Saccharomyces cerevisiae.

This protocol provides a method for preparation of industrialized fermented fish product with sturgeon (Aquilaria sinensis) meat product. The procedures ...

A Sample Preparation Pipeline for Microcrystals at the VMXm Beamline

The mounting of microcrystals (&#60;10 &#181;m) for single crystal cryo-crystallography presents a non-trivial challenge. Improvements in data quality have been seen for microcrystals with the development of beamline optics, beam stability and variable beam size focusing from submicron to microns, such as at the VMXm beamline at Diamond Light Source1. Further improvements in data quality will be gained through improvements in sample environment and sample preparation. Microcrystals inherently generate weaker diffraction, therefore improving the signal-to-noise is key to collecting quality X-ray diffraction data and will predominantly come from reductions in background noise. Major sources of X-ray background noise in a diffraction experiment are from their interaction with the air path before and after the sample, excess crystallization solution surrounding the sample, the presence of crystalline ice and scatter from any other beamline instrumentation or X-ray windows. The VMXm beamline comprises instrumentation and a sample preparation protocol to reduce all these sources of noise.
Firstly, an in-vacuum sample environment at VMXm removes the air path between X-ray source and sample. Next, sample preparation protocols for macromolecular crystallography at VMXm utilize a number of processes and tools adapted from cryoTEM. These include copper grids with holey carbon support films, automated blotting and plunge cooling robotics making use of liquid ethane. These tools enable the preparation of hundreds of microcrystals on a single cryoTEM grid with minimal surrounding liquid on a low-noise support. They also minimize the formation of crystalline ice from any remaining liquid surrounding the crystals. 
We present the process for preparing and assessing the quality of soluble protein microcrystals using visible light and scanning electron microscopy before mounting the samples on the VMXm beamline for X-ray diffraction experiments. We will also provide examples of good quality samples as well as those which require further optimization and strategies to do so.

The signal-to-noise ratio of data is one of the most important considerations in performing X-ray diffraction measurements from microcrystals. The VMXm beamline provides a low-noise environment and microbeam for such experiments. Here, we describe sample preparation methods for mounting and cooling microcrystals for VMXm and other microfocus macromolecular crystallography beamlines.

The mounting of microcrystals (&#60;10 &#181;m) for single crystal cryo-crystallography presents a non-trivial challenge. Improvements in data quality ...

Visualization and Quantification of TGF&#946;/BMP/SMAD Signaling under Different Fluid Shear Stress Conditions using Proximity-Ligation-Assay

Transforming Growth Factor &#946; (TGF&#946;)/Bone Morphogenetic Protein (BMP) signaling is tightly regulated and balanced during the development and homeostasis of the vasculature system Therefore, deregulation in this signaling pathway results in severe vascular pathologies, such as pulmonary artery hypertension, hereditary hemorrhagic telangiectasia, and atherosclerosis. Endothelial cells (ECs), as the innermost layer of blood vessels, are constantly exposed to fluid shear stress (SS). Abnormal patterns of fluid SS have been shown to enhance TGF&#946;/BMP signaling, which, together with other stimuli, induce atherogenesis. In relation to this, atheroprone, low laminar SS was found to enhance TGF&#946;/BMP signaling while atheroprotective, high laminar SS, diminishes this signaling. To efficiently analyze the activation of these pathways, we designed a workflow to investigate the formation of transcription factor complexes under low laminar SS and high laminar SS conditions using a commercially available pneumatic pump system and proximity ligation assay (PLA).
Active TGF&#946;/BMP-signaling requires the formation of trimeric SMAD complexes consisting of two regulatory SMADs (R-SMAD); SMAD2/3 and SMAD1/5/8 for TGF&#946; and BMP signaling, respectively) with a common mediator SMAD (co-SMAD; SMAD4). Using PLA targeting different subunits of the trimeric SMAD-complex, i.e., either R-SMAD/co-SMAD or R-SMAD/R-SMAD, the formation of active SMAD transcription factor complexes can be measured quantitatively and spatially using fluorescence microscopy.
The usage of flow slides with 6 small parallel channels, that can be connected in series, allows for the investigation of the transcription factor complex formation and inclusion of necessary controls. 
The workflow explained here can be easily adapted for studies targeting the proximity of SMADs to other transcription factors or to transcription factor complexes other than SMADs, in different fluid SS conditions. The workflow presented here shows a quick and effective way to study the fluid SS induced TGF&#946;/BMP signaling in ECs, both quantitatively and spatially.

Here, we establish a protocol to simultaneously visualize and analyze multiple SMAD complexes using proximity ligation assay (PLA) in endothelial cells exposed to pathological and physiological fluid shear stress conditions.

Transforming Growth Factor &#946; (TGF&#946;)/Bone Morphogenetic Protein (BMP) signaling is tightly regulated and balanced during the development and ...