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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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><tbody><tr><td>culture flasks (120 mL, N20)</td><td>Ochs, Germany</td><td>102046</td><td></td></tr><tr><td>buty rubber septa (N20)</td><td>Ochs, Germany</td><td>102049</td><td></td></tr><tr><td>aluminium caps (N20)</td><td>Ochs, Germany</td><td>102050</td><td></td></tr><tr><td>N2 gas</td><td>Messer, Austria</td><td></td><td>purity 5.0</td></tr><tr><td>syringes + cannulae</td><td>various</td><td></td><td></td></tr><tr><td>crimper</td><td>Ochs, Germany</td><td>102051</td><td></td></tr><tr><td>de-crimper</td><td>Ochs, Germany</td><td>102052</td><td></td></tr><tr><td>GC2010</td><td>Shimadzu</td><td></td><td></td></tr><tr><td>Shin-carbon GC column</td><td>Restek</td><td></td><td>chromatographic separation of H2, O2, CH4, and CO2</td></tr><tr><td>HPLC Prominence</td><td>Shimadzu</td><td></td><td></td></tr><tr><td>Fast Fruit HPLC Column</td><td>Phenomenex</td><td></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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Research

JoVE Journal

Biochemistry

27.6K Views.  Universität Innsbruck. In tegenstelling tot aërobe organismen vereisen strikt anaerobe micro-organismen de afwezigheid van zuurstof. Aangezien zuurstof alomtegenwoordig is in de lucht, is het vasthouden van zuurstofvrije omstandigheden tijdens de teelt een vereiste voor anaerobe kweek. De toegepaste techniek is een eenvoudige en goedkope methode om anoxische voorwaarden te bieden om een gemengde microbiële populatie in vloeibare cultuur te cultiveren.Deze techniek kan ook worden toegepast voor de teelt van...

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 ...

Culturing and Maintaining Clostridium difficile in an Anaerobic Environment

Clostridium difficile is a Gram-positive, anaerobic, sporogenic bacterium that is primarily responsible for antibiotic associated diarrhea (AAD) and is a significant nosocomial pathogen. C. difficile is notoriously difficult to isolate and cultivate and is extremely sensitive to even low levels of oxygen in the environment. Here, methods for isolating C. difficile from fecal samples and subsequently culturing C. difficile for preparation of glycerol stocks for long-term storage are presented. Techniques for preparing and enumerating spore stocks in the laboratory for a variety of downstream applications including microscopy and animal studies are also described. These techniques necessitate an anaerobic chamber, which maintains a consistent anaerobic environment to ensure proper conditions for optimal C. difficile growth. We provide protocols for transferring materials in and out of the chamber without causing significant oxygen contamination along with suggestions for regular maintenance required to sustain the appropriate anaerobic environment for efficient and consistent C. difficile cultivation.

Culturing and Maintaining Clostridium difficile in an Anaerobic Environment

Clostridium difficile is a pathogenic bacterium that is a strict anaerobe and causes antibiotic associated diarrhea (AAD). Here, methods for isolating, culturing and maintaining C. difficile vegetative cells and spores are described. These techniques necessitate an anaerobic chamber, which requires regular maintenance to ensure proper conditions for optimal C. difficile cultivation.

Clostridium  difficile is a Gram-positive, anaerobic, sporogenic bacterium  that is primarily responsible for antibiotic associated diarrhea (AAD) and is ...

Immunology and Infection

In Situ Simulated Anaerobic Method for Culturing Environmental Microbes

A Set of In Situ Informed Simulated Medium Formats for Culturing Environmentally Acquired Anaerobic Microorganisms

Culture-dependent research of anaerobic microorganisms rests upon methodological competence. These methods must create and maintain suitable growth conditions (e.g., pH and carbon sources) for anaerobic microorganisms while also allowing samples to be extracted without compromising the artificial environment. To this end, methods that are informed by and simulate an in situ environment can be of great aid in culturing microorganisms from that environment. Here, we outline an in situ informed and simulated anaerobic method for culturing terrestrial surface and subsurface microorganisms, emphasizing anaerobic sample collection with minimal perturbation. This protocol details the production of a customizable anaerobic liquid medium, and the environmental&#160;acquisition and in vitro growth of anaerobic microorganisms. The protocol also covers critical components of an anaerobic bioreactor used for environmental simulations of sediment and anaerobic liquid media for environmentally acquired cultures. We have included preliminary Next Generation Sequencing data from a maintained microbiome over the lifespan of a bioreactor where the active culture dynamically adjusted in response to an experimental carbon source.

Author Spotlight: Unraveling the Mysteries of Terrestrial Anaerobic Microorganisms in Uncharted Environments by In Situ Culturing

The focus of this paper is to detail best practices for making media for fastidious anaerobic microorganisms acquired from an environment. These methods help manage anaerobic cultures and can be applied to support the growth of elusive uncultured microorganisms, the "microbial dark matter."

Culture-dependent research of anaerobic microorganisms rests upon methodological competence. These methods must create and maintain suitable growth ...

Environment

Modeling Methanotroph Niches – The Gradient Syringe Model Ecosystem

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

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

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

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

Aerobic methane-oxidizing bacteria, known as methanotrophs, serve important roles in biogeochemical cycling. Methanotrophs occupy a specific environmental ...

Automated Microbial Cultivation and Adaptive Evolution using Microbial Microdroplet Culture System (MMC)

Conventional microbial cultivation methods usually have cumbersome operations, low throughput, low efficiency, and large consumption of labor and reagents. Moreover, microplate-based high-throughput cultivation methods developed in recent years have poor microbial growth status and experiment parallelization because of their low dissolved oxygen, poor mixture, and severe evaporation and thermal effect. Due to many advantages of micro-droplets, such as small volume, high throughput, and strong controllability, the droplet-based microfluidic technology can overcome these problems, which has been used in many kinds of research of high-throughput microbial cultivation, screening, and evolution. However, most prior studies remain&#160;at the stage of laboratory construction and application. Some key issues, such as high operational requirements, high construction difficulty, and lack of automated integration technology, restrict the wide application of droplet microfluidic technology in microbial research. Here, an automated Microbial Microdroplet Culture system (MMC) was successfully developed based on droplet microfluidic technology, achieving the integration of functions such as inoculation, cultivation, online monitoring, sub-cultivation, sorting, and sampling required by the process of microbial droplet cultivation. In this protocol, wild-type Escherichia coli (E. coli) MG1655 and a methanol-essential E. coli strain (MeSV2.2) were taken as examples to introduce how to use the MMC to conduct automated and relatively high-throughput microbial cultivation and adaptive evolution in detail. This method is easy to operate, consumes less labor and reagents, and has high experimental throughput and good data parallelity, which has great advantages compared with conventional cultivation methods. It provides a low-cost, operation-friendly, and result-reliable experimental platform for scientific researchers to conduct related microbial research.

Automated Microbial Cultivation and Adaptive Evolution using Microbial Microdroplet Culture System MMC

This protocol describes how to use the Microbial Microdroplet Culture system (MMC) to conduct automated microbial cultivation and adaptive evolution. MMC can cultivate and sub-cultivate microorganisms automatically and continuously and monitor online their growth with relatively high throughput and good parallelization, reducing labor and reagent consumption.

Conventional microbial cultivation methods usually have cumbersome operations, low throughput, low efficiency, and large consumption of labor and ...

Bioengineering

Anaerobic Growth and Maintenance of Mammalian Cell Lines

Most mucosal surfaces along with the midpoints in tumors and stem cell niches are geographic areas of the body that are anoxic. Previous studies show that the incubation in normoxic (5% CO2 in air) or hypoxic (low oxygen levels) conditions followed by an anoxic incubation (an absence of free oxygen) results in limited viability (2&#8211;3 days). A novel methodology was developed that enables an anoxic cell cultivation (for at least 17 days; the maximum time tested). The most critical aspect of this methodology is to ensure that no oxygen is introduced into the system. This is obtained by the degassing of media, and by flushing tubes, dishes, flasks, and pipettes with an anaerobic gas mixture (H2, CO2, N2) followed by permitting the materials to equilibrate to the anoxic (non-oxygen) environment prior to usage.&#160;Additional care must be exercised when acquiring photomicrographs to ensure that the micrographs obtained do not contain artifacts. In the absence of oxygen, cell morphology is significantly altered. Two distinct morphotypes are noted for all anaerobically-grown cells. The ability to grow and maintain mammalian cells in the absence of oxygen can be applied to the analysis of cell physiology, polymicrobial interactions, and the characterization of biosynthetic pathways for novel cancer drug development.

Here, we describe a new method that enables the anaerobic long-term cultivation of established cell lines. The maximum survival time that was tested is 17 days. This method is suitable for the testing of cytotoxic agents and exploring the physiology of anoxically replicating cells.

Most mucosal surfaces along with the midpoints in tumors and stem cell niches are geographic areas of the body that are anoxic. Previous studies show that ...

Biology

Cultivation Strategies for Uncultured Microbial Diversity in the Chicken GI Tract

Strategies to Enhance Cultivation of Anaerobic Bacteria from Gastrointestinal Tract of Chicken

The gastrointestinal tract (GIT) of chicken is a complex ecosystem harboring trillions of microbes that play a pivotal role in the host&#39;s physiology, digestion, nutrient absorption, immune system maturation, and prevention of pathogen intrusion. For optimal animal health and productivity, it is imperative to characterize these microorganisms and comprehend their role. While the GIT of poultry holds a reservoir of microorganisms with potential probiotic applications, most of the diversity remains unexplored. To enhance our understanding of uncultured microbial diversity, concerted efforts are required to bring these microorganisms into culture. Isolation and cultivation of GIT-colonizing microorganisms yield reproducible material, including cells, DNA, and metabolites, offering new insights into metabolic processes in the environment. Without cultivation, the role of these organisms in their natural settings remains unclear and limited to a descriptive level. Our objective is to implement cultivation strategies aimed at improving the isolation of a diverse range of anaerobic microbes from the chicken&#39;s GIT, leveraging multidisciplinary knowledge from animal physiology, animal nutrition, metagenomics, feed biochemistry, and modern cultivation strategies. Additionally, we aim to implement the use of proper practices for sampling, transportation, and media preparation, which are known to influence isolation success. Appropriate methodologies should ensure a consistent oxygen-free environment, optimal atmospheric conditions, appropriate host incubation temperature, and provision for specific nutritional requirements in alignment with their distinctive needs. By following these methodologies, cultivation will not only yield reproducible results for isolation but will also facilitate isolation procedures, thus fostering a comprehensive understanding of the intricate microbial ecosystem within the chicken&#39;s GIT.

Author Spotlight: Advancing Intestinal Bacteria Cultivation for Poultry

The protocol presents two methodologies to improve the isolation of anaerobic intestinal bacteria. The first focuses on the isolation of a diverse range of bacteria using different culture media. The second focuses on the cultivation steps of a specific microbial group, possibly assimilating myo-inositol, to fully comprehend its ecological significance.

The gastrointestinal tract (GIT) of chicken is a complex ecosystem harboring trillions of microbes that play a pivotal role in the host&#39;s physiology, ...

Enrichment Cultures: Culturing Aerobic and Anaerobic Microbes on Selective and Differential Medias

Source: Christopher P. Corbo1, Jonathan F. Blaize1, Elizabeth Suter1 
1 Department of Biological Sciences, Wagner College, 1 Campus Road, Staten Island NY, 10301
Prokaryotic cells are able to inhabit nearly every environment on this planet. As a kingdom, they possess a great metabolic diversity, allowing them to use a wide variety of molecules for energy generation (1). Therefore, when cultivating these organisms in the lab, all necessary and specific molecules required to make energy must be provided in the growth media. While some organisms are metabolically diverse, others are able to survive in extreme environments such as high or low temperatures, alkaline and acidic pH, reduced or oxygen absent environments, or environments containing high salt (2,3,4). Termed &#34;extremophiles&#34;, these organisms often require these intense environments to proliferate. When scientists look to grow such organisms, the media components as well as any specific environmental conditions all need to be taken into account in order to successfully cultivate the organisms of interest.
Scientists are able to grow culturable organisms in the lab because they understand the specific requirements that those species need to grow. However, culturable organisms account for less than 1% of species estimated to be on the planet (5). Organisms that we have detected by gene sequencing but are not able to grow in the lab are considered unculturable (6). At this time, we do not know enough about the metabolism and growth conditions of these organisms to replicate their environment in the lab.
Fastidious organisms lie somewhere in between the former two. These organisms are culturable, but they require very specific growth conditions, such as specific growth media components and/or specific growth conditions. Two examples of such genera are Neisseria sp. and Haemophilus sp., both of which require partially broken-down red blood cells (also known as chocolate agar), as well as specific growth factors and an environment rich in carbon dioxide (7). Without all of the required specific components, these organisms will not grow at all. Often, even with all of their requirements, these organisms grow poorly.
Unlike eukaryotic cells, which are only able to grow in an aerobic, or oxygen containing, environment, prokaryotic cells are able to grow anaerobically using several fermentation pathways to generate ample energy (8). Other prokaryotes prefer a microaerophilic, or reduced oxygen environment, or even a capnophilic, or high carbon dioxide environment (9). These organisms are more challenging to enrich for, since the atmosphere must be altered. Scientists that frequently work with organisms sensitive to an oxygenated environment would normally work in an anaerobic chamber and incubator, where a heavy, inert gas such as argon is pumped in to displace the oxygen (10). Others make use of conventionally available sealed gas packet systems that use water to generate hydrogen and carbon dioxide, along with a catalyst like palladium to remove all atmospheric oxygen. These commercially available kits can create any of the above-mentioned atmospheric conditions (10).
Whether cultivating a pathogen to determine potential infection or looking to identify a specific species of bacteria present in a natural environment, one problem exists. No one bacterial species inhabits one habitat. Bacteria live as multicellular communities everywhere from the skin of humans to the oceans of our planet (11). When attempting to isolate one species of bacteria, scientists must work to exclude the numerous other organisms that are also inhabiting the isolated area. For this reason, enriched growth media for bacteria often carry out two functions. The first is to make the media selective. A selective agent will prevent some species from growing, while not inhibiting and often even promoting others to grow (12). The second function of media ingredients may be to work as differential agents. Such agents allow for the identification of a particular biochemical feature of an isolated organism. By pairing several different selective and differential medias along with appropriate growth conditions, scientists and diagnosticians are able to identify the presence of specific bacterial species from a particular isolate.
One example of a selective and differential media aiding in to identification is in the case of the clinically significant organism Staphylococcus aureus. This organism is typically cultured on mannitol salt agar. This media not only selects for only organisms which can live in a high salt environment, which include some gram positives like Staphylococcus, but it also inhibits any organisms sensitive to salt. The mannitol sugar is the differential component of this medium. Of all the clinically significant Staphylococcus species, only S. aureus is able to ferment mannitol. This fermentation reaction produces acid as a by-product which causes the red methyl red indicator in the media to turn yellow. Other Staphylococcus species (such as Staphylococcus epidermidis) although able to grow, will leave the media red in color.
This lab exercise demonstrates proper aseptic technique, as well as proper inoculation of growth media from broth. It also introduces the growth of common contaminant organisms on enrichment media, the use of a gas package anaerobic culture system for anaerobic bacteria, and the use of different selective and differential medias for the presumptive identification of gram positive and gram-negative bacteria.

Enrichment Cultures, Selective and Differential Media

Source: Christopher P. Corbo1, Jonathan F. Blaize1, Elizabeth Suter1
1 Department of Biological Sciences, Wagner College, 1 Campus Road, Staten Island NY, ...

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

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