Methanogens - Science topic

I would say that there is not best in research in general. Everything is relative. And the results can also be dependent on what is your experiment about and what and how you design the experiment in terms of microbiology. As of your question, point 1 and 2 in not separate and can be done in parallel.

There are so many studies done on this topic which surely can help you in designing the study and to understand what is needed.

However, in the end everything would come to you what you are doing to do and how present the study.

Victor S Garcia-Rea. Thank you very much

got here years to late :-) We develloped that as a patent back in the nineties https://patents.google.com/patent/EP0337189A1/en

but the article

explained that a bit better

Philomina Mamley Adantey Arthur - specific methane activity value or range required to enhance performance of any aboaeribic digestion system based on any anaerobic technology was studied by me as well and it took a long time for compilation of my reports and works in that area however I found two more similar type of research activities on research gate and am sharing the links of both the papers for your reference and hope so these will be helpful to you as well

second pdf file is SMA under sludge salinity and interesting report as well

here is the final story i generated.

Hi Kate

If you have a DNA extract of pure culture and a qPCR method for the pmoA gene and another target that you know as a single copy, you can consider the following approach.

The distribution of positive PCR at very log copy numbers is different when you have two copies instead of one. It would help if you considered the Poison distribution of results in microbiology counts.

You have to do a dilution banc and a 10 PCR of the lowest levels, including a dilution where all the PCR are negatives for both targets. In the previous dilution to those with zero results in all replicates, you will find different positive ratios if the copy number is different. This experiment needs to be done several times to show significant results.

best regards

You have to disable methanogen for biohydrogen production. so you can use the acidic and thermal pretreatment.

The range reported for different types of microorganisms is as follows:

aerobes +300 to +500 mV; facultative anaerobes -100 to +300 mV; and anaerobes +100 to less than -250 mV

you can refer to the below papers for more information.

Of course, you should add microorganisms to your reactor due to the dynamics of anaerobic processes. You feed the waste to the digesters such as rice husks, rice straws, rice bran as SUBSTRATES and actually they're the food for your microorganisms. You have your microorganisms in your SEED SLUDGE (you can call it also 'inoculum'). You can determine the amount of seed sludge you put to your reactors from the literature.

Biological methanlization is taking place under anaerobic condition using microbes . So you can not mix with catalytic methanization as bacteria requires different environment for survival.

I know that faeces contains such microb, but addition of cow dung will facilitate the production of methane.

Please, can you explain what is the difference between biomass and methanogens? Methanogens are bacteria and it means that they are biomass, too.

Regards

Vit

Thank Biwen Annie An Stepec

It is helpful

High organic load and/or low HRT will be the efficiency to produce VFA.

Yann

Dear @ Chaitanya Choudhary

Find the links: http://mcb.illinois.edu/departments/microbiology/downloads/Wolfe.pdf https://aem.asm.org/content/aem/51/5/1056.full.pdf

Hi if you want to make your database just use this script! It will be fast and easy!

16S primers for Archaea or mcrA primers.

Hi,

Common methods use acetate (as a salt), cellulose, PEG 400, glucose etc. Roughly, 1 g of glucose generate 1 g of theoritical O2 demand (COD). Also 1 g of COD consummed generate approximatively 370-400 ml of CH4.

In our lab we used cellulose microcrystalline or sodium acetate.

Yann

Dear Cheng,

You can simply observe methanogens using fluorescence microscopy at wavelengths of 420-430 nm. The coenzymes F420 and F430 contained by most methanogenic species are autofluorescent, they absorb light at these wavelengths and become visible in bright green-blue color. I can not give you any concrete technical details because I am not a specialist in microscopy, but I easily and quickly observed coccus methanogens in wastewaters at these wavelengths (of course after a minimal preliminary study). I hope you get a more explicit answer from more experienced researchers. Regards.

Stoppered serum bottles are relatively convenient and inexpensive way to culture strictly anaerobic bacteria. Media can be prepared in large batches filling serum bottles which can then be stored at room temperature for several months ready for inoculation.Turn valve to the vacuum position and watch for the pressure to equilibrate to around -20 in Hg ( about 3 - 5min. For more information consult https://openwetware.com

If you want to analyze changes in the general pattern of proteins, you should go for 2-D gel analysis (isoelectric focusing +SDS-PAGE).You will no doubt find plenty of protocols on the Internet. SDS-PAGE by itself is not sufficient to resolve all protein components present in a cell.

Sadegh Hosseini If you control the residence time in the reactor to about 4 days you will get high VFAs without methane. This is used in the first of the two-stage fermentors.

Susie Lind I did not really understand what your real aim is now ?

Do you want to isolate and grow some new methanogens or want to cultivate some already known methanogens and analyse them for their methane producing potential?

Dear Dr. Towe, Thank you for your remarks. Let me please to address them. I did mean the use of methane as the fuel for cars and buses when I said "eco-friendly". As you could have seen many city buses are carrying the info like this : "we use methane instead of diesel". As you know the latter is very toxic.

Indeed, the finding of the ability of CB to produce methane can pose an interesting question to Astrobiological community: "Is the ability of cyanobacteria to generate CH4 a relic process in CB metabolism or they acquired methanogenesis from ancient methanogenic microbes by lateral gene transfer to protect them self of harsh UV?" I guess second option sounds more realistic.

Sincerely,

Igor Brown

The potential strategies for predicting or quantifying the amount of bioavailable ( Hg(II ) in water and sediment include measurements of the " dissolved" or filter-passing phase ( typically 0.2 or 0.45 microns nominal pore size ) or the solid - aqueous Hg partition coefficient where the aqueous fraction is defined by this filter - passing phase. Despite this widely employed approach, the filter - passing Hg concentraion rarely correlate with Hg methylation rates or MeHg concentration. Another approach is to infer Hg speciation using chemical equilibrium speciation models and assume the subset of dissolved species are available. For details consult Environmental Science and Tecchnology. Vol. 52 issue 15: pp. 8510 - 8520. http://dol.org/10121/acs.est.8b02515

In short you could refer the similar studies and try to do the same/similar with your data, follow tutorials and there are a lot of good ones available on the internet. And if you want any detailed and specific answer, that is not possible without looking on the data and careful analysis.

Dear Elisa,

It is always difficult to go from growth experiments to the identification of an active compound, especially if your model organism is hard and slow to grow, you lack a fast and simple assay for the activity, and if you are not based in a biochemical lab. However, there are some simple experiments that you can do to narrow down the nature of your active compound.

I would suggest that you try some simple growth addition experiments to demonstrate that the effect is real, and that cells are responding to this compound in a dosage-dependent manner. The easiest experiment is to add some filter-sterilised (FS) culture supernatant from cultures in which you see cell death or growth-inhibition to a fresh culture - you would expect to be able to inhibit the growth of this or see cell death occurring earlier than if you did not add FS culture supernatant.

If this works, then there are some simple tests you can do : test FS culture supernatants from cultures grown in different media, grown for different lengths of time, and grown to different cell densities. Try fractionating your FS culture supernatant using dialysis membrane or filters (with different pore sizes) to see if you can put a size limit on the active compound; try heat inactivating the compound, and precipitating or extracting it with alcohol, phenol, ammonium sulphate, etc.

The aim of all of this is to produce a semi-purified concentrated extract that you could then consider fractionating by HPLC and then identifying the compound by mass spectroscopy (if you had access tho this type of analytical equipment). Being able to demonstrate that you have a semi-purified extract plus a simple phenotype you can assay activity with would be a good end-point for a project where you could not do any further biochemical analyses.

Regards, Andrew.

Dear Shiva Shabanian, thank you very much for the reply. But the link you have send, I m unable o open. Please could you further send me the pdf copy of the same. my mail id is

Hi Martin,

I do agree with the previous opinions that your medium is extremely rich. Yeast extract, formate, acetate, sludge fluid and fatty acid mix all together is more than your organisms probably need for growth. Our M. hungatei e.g. grows on inorganic medium with only 0,1 % acetate supplement. The more organic you use, the riskier it is to get issues with cross-contamination. Especially yeast extract is a dangerous source concerning spores, therefore your idea on a contamination with spore-formers like Clostridia is justified. Also the acidification of your medium hints in that direction.

A solid medium suitable for the cultivation of methanogenic bacteria (from any environment, also marine) is described in the attached paper. It is a solid basal culture media, the formula is presented at page 520-521. Just be careful to seal the culture media against the oxygen from the air, the simplest way is to add pure paraffin oil on surface.

Dr. Martin Blaser,

Thank you very much for your valuable suggestion....

2-bromoethanesulfonic acid (BESA) with a concentration of 50 µM could be added into anaerobic sludge of the UASB. Herein, methanogens within sludge will be eliminated by BESA to prevent VFAs oxidation during the hydrolysis-acidification step. This method is one sure way to shut down the activities of methanogens in an AD system. However, i will advise that, you first conduct this experiment in batch mood to also appreciate the method scientifically before applying to your pilot scale system. To compartmentalize the UASB is quite a challenge although some reports had indicated this task as almost impossible. For instance, increasing the upflow velocity may reduce the contact time for methanogen to oxidize available VFAs but at the same time, this may cause washout of the hydrolyzable or acidogenic bacteria. On the other hand, increase or reduction in temperature may affect methanogens for a while but with time, they may acclimatize with the new environment and begin to produce methane upon contact with VFAs. Therefore the addition of BESA is highly suggested.

Hello dear 

I attached an article and a link for you. I hope be helpful

Im not sure what do you mean with "alternative sources" because it is not clear "alternative to what". I suggest you start whith what you have nearby: cattle manure, food waste, ...? Or if you can clarify your question maybe we can suggest acordingly.

Thank you Denny!!

are you using ultrapure (milli Q) water?

Also check if your Na2S 9H2O stock solutiuon is well prepared

The thermodynamic conditions in the AD process are now very complex. There is not one reaction; there are dozens of reactions that run parallel. This circumstance makes a theoretical consideration even more difficult. Also the data situation is not very good. The necessary thermodynamic data is only available for simple connections. Data from complex compounds such as proteins or fats are not present. Thus the reactions or thermodynamic aspects cannot be fully investigated. Only the reactions shortly before methane formation can be described. The calculation of the reactions under standard conditions also does not adequately reflect the situation. Calculated under biological conditions, the situations are partly fundamentally different. Reactions which are not possible under standard conditions can suddenly be possible under biological conditions.

A note gives only the measurement of the redox potential. But here, too, only the acquisition of a sum parameter is possible. A conclusion on the equilibrium is not possible because the valency of ions cannot be detected because of the large number of reactions taking place.

The calculation of the reaction enthalpy is therefore also only roughly possible. Depending on the nature of the substrates, the reaction enthalpy can also be quite significant.

Hello, we offer an anaerobic gassing station for Hungate tubes. You can download details from our website qcal.de.

Water.

Ref:

Long-Ping Zhang, Jian Zhang, Chuan-Hang Li, Jie Bao*. Rheological characterization and CFD modeling of corn stover-water mixing system at high solids loading for dilute acid pretreatment. Biochemical Engineering Journal, 2014, 90:324-332.

Weiliang Hou, Jian Zhang, Jie Bao*. Rheology evolution and CFD modeling of lignocellulose biomass during extremely high solids content pretreatment. Biochemical Engineering Journal. 2016, 105, 412-419.

Dear Ciara,

To my knowledge, without any experience in culturing pure hydrogenotrophic methanogen, formate can be cleaved into hydrogen, therefore, you can use formate if you worry about the low soluble of hydrogen. CO2 is easier to become bicarbonate or carbonate, that's not thing need to be worried. I have some experience in running reactor to stimulate the growth of hydorgenotrophic methanogens with applied voltage that produce hydrogen/electron. If you get interested in this, you can check my publication. I think use electrochemical method to feed bacteria can be a novel idea.

Good luck with you and your experiment. 

Dear Basma,

This mini-review summarizes the category, characteristics, and the application fields of the chemical methanogenic inhibitors. Usually, the chemical methanogenic inhibitors can be divided into "specific" and nonspecific inhibitors. The former group includes the structural analogs of coenzyme M and HMG-CoA inhibitors. The former group includes the structural analogs of coenzyme M and HMG-CoA inhibitors The nonspecific group includes many chemicals which can inhibit the activity of both methanogens and non-methanogens. The chemical inhibitors of methanogenesis have been widely used in the fields of understanding methane production and consumption in pure culture or in complex natural environment, production of value-added substances, such as volatile fatty acids and hydrogen, and reduction of energy loss and improvement of the efficiency of ruminal energetic transformations.The nonspecific group includes many chemicals which can inhibit the activity of both methanogens and non-methanogens. The chemical inhibitors of methanogenesis have been widely used in the fields of understanding methane production and consumption in pure culture or in complex natural environment, production of value-added substances, such as volatile fatty acids and hydrogen, and reduction of energy loss and improvement of the efficiency of ruminal energetic transformations. Finally, with an increasing understanding of the mechanistic effects of the chemical inhibitors of methanogenesis, it is possible that some could be used to develop into promising feed additives to reduce losses associated with enteric methane production or as useful tools to screen microbial consortia from various biotechnological applications to enhance hydrogen and acid production.

The question actually have a very simple answer, but one, many people ignore or do not realize: when it comes to metabolic activity, it is veru important to understand tne border of the main catabolic reactions, such as in methanogenic archae and marginal conversions, such as anabolic reactions or side (unspecific) processes. When it comes to biomethane (bio means that it is not thermogenic methane), the only real source are methanogens. No other organisms have the mcr catalyzing the final stage of the methyl group conversion into CH4. There are some evidences that tiny amounts of methane could be releazed as unspecific products from nitrogenase and in clostridia. In you particular case most probably methanogens were present in low numbers but you missed them, which often happen when 16S-rRNA gene is used as a marker. Therefore you must use the methogens-specific functional gene marker - mcrA

I suspect your alignment is too short (see example in Science 310, 1910-1911 [2005]) but it could also be that your data have too many sites that are incompatible in a phylogenetic sense (i.e., different sites supporting conflicting trees). Try to analyze the data using IQ-TREE (its maximum likelihood program); I've found it very useful.

Sir,

Methangenesis occur under anaerobic condition where microbes use oxygen from CO2. Whenever un-decomposed material is added to soil, under temporary submergence also, the methanogenesis will be occurring.  That is why farmers have to be advised to go for composting and then apply to field.  This process can be enhanced by adding some chemicals like alum.  The methano-bacteria is one microorganism used for the process.

CO2 + 4H2 → CH4 + 2H2O

Methane is 8-23 times danger than CO2 w.r.t. global warming.

Hence, the process needs to be stopped;

Pl. stop producing methane! SAVE EARTH!

Thanks on-be-half of all

Ferric Chloride will precipitate the sulphide in the digester that would otherwise form salts with essential trace elements required by the methanogens.  For instance there is a biological need for Selenium, Molybdenum, Cobalt, and Manganese to be present and available in the digester at levels typically below 1mg/l.  High levels of H2S/ sulphide in the digester precipitate these trace elements and cause a loss of enzyme based activity in the bacteria and a reduction in respiration.   Dosing with Ferric chloride produces a positive result for a known cost of chemical reagent and is predictable and therefore widely used in full scale digesters for this purpose.  Odour control in the digestate and corrosion control in a gas engine are useful consequences of this method of improving the respiration rate of the bacteria while removing hydrogen sulphide from the biogas.

Dr. Amin

may be your system is overloaded try to stop feeding couple of days and re-operate by lower concentration and higher retention time; it can help in many cases such yours.

best wishes

I see. In general, there are a few questions you want to answer before you proceed. The first of which is, "how anaerobic do you need your media?" Some methods will make your media anaerobic enough for most microbes, while some people are more stringent and go further by adding chemicals to their media that react with oxygen to remove it. In general, making your media anaerobic is a combination of boiling to drive off gasses dissolved in solution, bubbling with an inert gas to drive off oxygen, and adding a reducing agent to react with the remaining oxygen. 

You need the proper vessels to store your cultures. Bulch tubes are a standard, as are wheaton serum bottles. Both of these containers come with butyl rubber stoppers about 1.5-2cm thick. The benefit of these stoppers is that they are impenetrable to oxygen and can be pierced multiple times with a 23-21 gauge needle to insert or remove liquids. If you want to be very careful, people will also use glass or plastic syringes for this that have been kept in an anaerobic chamber for days so that any oxygen stuck to them degasses...I don't know how much of this is wishful thinking or fact.  

To make your media anaerobic

1) Some people will simply insert a probe into their media and bubble nitrogen through it for a few hours, or even overnight. They consider this to be anaerobic, I don't. But, it is good enough for many applications.

2) I would go a step further and add a reducing agent to remove the remaining oxygen from solution. Common reducing agents include sodium sulfide, titanium nitrilotriacetate, cysteine, or a combination of sodium sulfide and Cysteine. Cysteine is probably the most mild and preferred reducing agent, but it could easily be a carbon source. In that case, sulfide may be used. However, sulfide will produce sulfates upon reaction with the oxygen, so if you don't want sulfate, you may not want to use sulfide. Additionally, sulfate will form on the outside of the sulfide crystals, so many people wash the crystals in anaerobic water prior to use to remove the sulfate. If you're using a palladium catalyst in your anaerobic chamber, sulfide will destroy it. Titanium nitrilotriacetate is quite strong, but is toxic to some microbes....pick your poison.

3) use an indicator such as resazurin to tell you if the media is truly anaerobic. Resazurin will turn pink if oxygen is present. 

My preferred protocol is to bring the media, including resazurin, to a boil in a round bottom flask --> allow to boil for at least a few minutes. This will drive off most of the oxygen dissolved in the media. --> run a jet of nitrogen over the media while it cools or preferably bubble it through the solution. --> once the media is cool, quickly seal the container and put it in an anaerobic chamber. If an anaerobic chamber isn't available, the remaining steps will have to be done under a jet of nitrogen to keep the media anaerobic....anaerobic chambers, such as from COY, are a godsend. -->  add your reducing agent and wait until the resazurin has turned clear. It starts blue, turns pink once the reducing agent has been added, and then turns clear, indicating that your media is oxygen-free. However, sometimes it turns pink again, so wait a while the first few times you make media to see if this happens --> dispense media into serum bottles --> stopper the bottles and seal with aluminum crimp --> autoclave media --> once cool, add your microbes and relax. 

One more note. Some components of media, such as many common vitamins, are not autoclavable. If this is the case, filter sterilise your vitamins and add them after autoclaving. The reducing agent should take are of the minimal oxygen introduced since you typically don't add much volume of vitamin solution.

HRT should be designed considering the growth kinetics of the microbial consortia. Fermentative consortia have faster growth kinetics compared to methanogens in the second stage, thus the HRT in the methanogenic stage i.e. second stage is higher than the first stage. 

Gas is stored in coal as an adsorbed component on or within the coal matrix and as free gas within the micropore structure or cleats within a coal bed. The gas is held in place mainly by reservoir pressure; reducing the reservoir pressure allows gas to be released from the coal. (http://www.ags.gov.ab.ca/energy/cbm/coal_and_cbm_intro2.html) This coludo be by the size and porosity of the coal you used. Also, as Akuzuo said, since its biodegradability can be very slow and probably requires specific bacteria to degrade it (microflora present in water leached from coal mines were shown to generate methane), you have to evaluate the quality of your inoculum in terms of its affinity and capacity to degrade the coal. In the article of Strapòc et al (http://aem.asm.org/content/74/8/2424.full) you can read about the enrichment of the adecuate inoculum that you require for the improvement of your experiment, so you do not have this yields. Also If you want to keep your bacteria, you can try to aclimate them by using a media with the adecuate mineral and nitrogen concentration, so they can grow normaly and have the chance to produce the enzymes they need to degrade the coal and generate methane. You can find other factors in the article of Jones et al Stimulation of Methane Generation from Nonproductive Coal by Addition of Nutrients or a Microbial Consortium

Methane gas is not produced genetically but by simple reduction of carbon dioxide to methane gas by methanogens while utilizing various substrates like Hydrogen, Acetate or methyl compounds .

Methane production consist or 3 major stages liquification,acidification and methanification from which liquification and acidification  can be supported by protozoa but anaerobic bacteria are full-proof solution.

Those are all arch bacteria. does not need to go rumen for search but you can source it out from cattle dung.  

Hello Michal,

its not the ammonium nitrogen itself that has the impact on the microbial, especially the archaeal activity. Depending on the occurring process temperature and pH value, the cell-toxic free ammonia (NH3) is the parameter that lead to a decrease in the produced biogas yield because it has inhibitory effects. There are several references in the literature available dealing with this topic: e.g. Chen et al. 2008 Inhibition of anaerobic digestion process: a review. Bioresource Technology 99 , 4044–4064 / De Vrieze et al. 2015 Ammonia and temperature determine potential clustering in the anaerobic digestion microbiome. Water Research 75, 312-323 / Fortidis et al. 2013. The dominant acetate degradation pathways/methanogenic composition in full-scale anaerobic digesters operating under different ammonia levels. International Journal of Environmental Science and Technology. DOI: 10.1007/s13762-013-0407-9 / Niu et al. 2014 Effect of ammonia inhibition on microbial community dynamic and process functional resilience in mesophilic methane formation of chicken manure. Journal of Chemical Technology and Biotechnolgy. DOI: 10.1002/jctb.4527 / Lv et al. 2014 Stable isotope composition of biogas allows early warning of complete process failure as a result of ammonia inhibition in anaerobic digesters. Bioresource Technology 167, 251-259.

Am I allowed to ask what kind of feedstock / substrate do you used for your anaerobic digestions? Is it a protein- or urea-rich substrate?

I hope this helps you bit :-)

kind regards

Susanne

Dear Arun et al.,

It appears that there is some confusion. Free (dissolved) ammonia NH3 is indeed inhibitive/toxic for methanogens even at relatively low concentrations. However ammonium ions NH4+ are NOT and are in fact nutrients! As you probably know there is an equilibrium  between NH4+ and NH3 in water depending on the pH and temperature. Hence by controlling the pH, the operator can shift this equilibrium towards more NH4+ and less NH3.

In addition, there are complementing anaerobic biological ways to effectively remove NH4/NH3.

I/we have good experience in design and operation of high-rate anaerobic digestion (HRAD) on landfill leachate in China.

One good way to analyse diversity of any gene in a specie or group of them, or of isolates is:

- construct PCR primers that are directed to the flanking genes, don't go only for the gene, but also for the promotor sequence

- Amplify each fragment from each strain

- Sequence each fragment 

- analyse each sequencing result (blast or other algorithms). 

Good work 

I think these papers may be helpful

We wrote that methanogens is one of the options. Please read our article, which is fully available here on the RG :

Article:

N. S. Duxbury, S S Abyzov, V E Romanovsky, K Yoshikawa

A combination of radar and thermal approaches to search for methane clathrate in the Martian subsurface, 2004, Planetary and Space Science , 52,p. 109--115.

Thank you very much Pravin.

Are you looking for reactor scale or laboratory i.e. bench scale?

For both, You can inoculate some activated sludge into the liquid obtained afte fungal treatment. DO not areate  it. In few days you will find methonogenes growing in it. In fact they are slow growing. 

You can keep the activated sludge for a week without aeration. It will turn black color. You can use it to inoculate your liquid obtained after fungal treatment of bagasse. 

Thanks for your kind cooperation and the information @ Mustafa Vohra & Halina E Tegetmeyer.

Hope I can follow your referece

You CANNOT do this in a chamber! It's DANGEROUS! Very Explosive!

You can do it only in a tube, serum bottle or Oxoid anaerobic jar!!!

I agree with the idea of Sanjay Kumar, it is very difficult to isolate these organisms due to environmental, nutritional requirements and metabolic. However some methanogenic can be grown in tubes or bottles more easily: see if this text will help you in your search.

Sincerely

Dear Servios,

My thesis was with acid fermentation, pH 4.8 and 7.0 at the exit at the entrance. All colleagues offered effective contributions 2 bromoethanesulfonate, perhaps glycero (?), Heating 80 degrees inoculum, lower pH with substrate, oxygenate the medium. In my day was even told that the common detergent (appropriate dilution) eliminates the methanogenic.

However in my work, I made a "natural inoculation." Glucose solution (2g / l) at pH 7.0. Entered the reactor effluent and I returned for food (for input). So to get too impregnated support, the flow must be very low.

See details at:

  Milk, JAC, Fernandes BS, Pozzi And Barboza M, Zaiat M. 2008.Application of anaerobic packed-bed bioreactor for the production of hydrogen and organic acids. International Journal ...

International Journal of Hydrogen Energy 01/2008; 33: 579-586.

At your disposal.

Actually, the ultimate ratio of CH₄ to CO₂ produced from the degradation of organic material is independent of the pathway taken. If for example hydrogen is produced from the degradation of glucose, it does not matter whether this hydrogen is subsequently directly converted into methane, or first converted into acetate with the acetate subsequently converted into methane. In either case the mineralization of one mole of glucose yields 3 moles of CH₄ and 3 moles of CO₂ (see below).

Classical pathway:

C₆H₁₂O₆ + 2H2O --> 2CH₃COOH + 2CO₂ + 4H₂

CO₂ + 4H₂ --> CH₄ + 2H₂O

2CH₃COOH --> 2CH₄ + 2CO₂

SUM: C₆H₁₂O₆ --> 3CH₄ + 3CO₂

Pathway where hydrogen is removed by acetogens:

C₆H₁₂O₆ + 2H₂O --> 2CH₃COOH + 2CO₂ + 4H₂

2CO₂ + 4H₂ --> CH₃COOH + 2H₂O

3CH₃COOH --> 3CH₄ + 3CO₂

SUM: C₆H₁₂O₆ --> 3CH₄ + 3CO₂

The right feeding strategy is key. This open access mini-review by Professor Madalena Alves and co-workers in Microbial Biotechnology (doi: 10.1111/j.1751-7915.2009.00100.x) provides a concise introduction.

Professor Alves is world-leading in this area. You may want to also consult her more recent papers for the latest insights on his tricky topic.

Sorry I have even more ....

 Rocheleau, S, Greer, C. W., Lawrence, J. R., Cantin, C., Laramee, L., Guiot, S. R. 1999. Differentiation of Methanosaeta concilii and Methanosarcina barkeri in Anaerobic Mesophilic Granular Sludge by Fluorescent In Situ Hybridization and Confocal Scanning Laser Microscopy. Appl. Environ. Microbiol. 65: 2222-2229.

 Rogers, Shane W., Moorman, Thomas B., Ong, Say Kee. 2007. Fluorescent In Situ Hybridization and Micro-autoradiography Applied to Ecophysiology in Soil. Soil Sci Soc Am J 71: 620-631.

 Sekiguchi, Yuji, Kamagata, Yoichi, Nakamura, Kazunori, Ohashi, Akiyoshi, Harada, Hideki. 1999. Fluorescence In Situ Hybridization Using 16S rRNA-Targeted Oligonucleotides Reveals Localization of Methanogens and Selected Uncultured Bacteria in Mesophilic and Thermophilic Sludge Granules. Appl. Environ. Microbiol, 65: 1280-1288

 Song, H., W.P. Clarke, L.L. Blackall. 2005. Changes in relative populations of hydrolyzing bacteria and methanogens (Archaea) in biofilm formed during anaerobic digestion of crystalline cellulose. Biotech Bioeng 91:369-378.

 Sorensen, A. H., Torsvik, V. L., Torsvik, T., Poulsen, L. K., and Ahring, B. K. 1997. Whole-cell Hybridization of Methanosarcina Cells with Two New Oligonucleotide Probes. Applied and Environmental Microbiology, Vol. 63, No. 8, p. 3043-3050.

 Torsvik, V., and L. Øvreas. 2002. Microbial diversity and function in soil: from genes to ecosystems. Current Opinion in Microbiology 5: 240–245.

 Trejo, G., Hoffmann, R., Karim, K, Angenent, L. 2004. Fluorescent in situ hybridization (FISH) views of biomass from anaerobic digesters treating animal waste. [en línea], [citado 10/10/2007], Formato pdf. Disponible en: http://comp.uark.edu/~kkarim/FISH_paper.pdf

 Wagner, M., Amann, R., Lemmer, H., Schleifer, K. H. 1993. Probing activated sludge with proteobacteria-specific oligonucleotides: inadequacy of cultura-dependent methods for describing microbial community structure. Appl. Environ. Microbiol. 59, 1520-1525.

 Wagner, M., M. Horny and H. Daimsz. 2003. Fluorescence in situ hybridisation for the identification and characterisation of prokaryotes. Current Opinion in Microbiology 2003, 6:302–309.

 Wheeler Alm, E., D. Zheng, and L. Raskin. 2000. The Presence of Humic Substances and DNA in RNA Extracts Affects Hybridization Results. Appl Environ Microbiol., 66(10): 4547–4554.

 Woese, C. R., O. Kandler, and M. L. Wheels. 1990. Towards a natural system of organisms: proposal for the domains Archaea, Bacteria and Eucarya. Proc. Natl. Acad. Sci. USA 74: 5088-5090.

I agree with Juan, with the addition that besides the system's objective functions (methane, biohydrogen, valuable organic acids, oils, etc.) you should also take into consideration the nature of your fermentation substrate which could require a highly specific composition of your initial inocula.

All previous answers are right. The production of methane is a result of a mixture of bacteria. So there is no "one" bacteria which is the best but a consortium. If it is for a practical use the best is to take a sample of a similar bioreactor and to add it to yours. If it is for a study, it is almost impossible to isolate methanogenic bacteria.

Lipids will only provide a semiquantitative measure for archaeal abundance and the lipid pool will comprise compounds from alive and dead organisms (also looking at intact polar lipids will not necessarily give you a measure for alive biomass). I can give you a protocol for GC-detectable lipids (isoprenoidal di-ethers are in the "alcohol fraction"), which includes isoprenoidal glycerol di-ethers but I'd suggest to use a different approach such as fluorescence in situ hybridisation with which you can count eg archaeal cells by fluorescence microscopy. 

In UASB, mentaining shear force is difficult and need proper optimization,  One can use neutral gas bubbling to enhance granulation and then optimise required superficial velocity to stabilize granules.

It will give a quality library than a random library of aceto & acido community. it is good to study microbial communities by sequencing the gene of choice (V3, V4 regions). However  16S rDNA amplicon and whole genome sequencing approaches are more appropriate from population and community dynamics point of view.

Dear Aqil,

Excellent question!

ADM1 is a generic and recognized model for anaerobic digestion. Kinetic parameters are proposed for each trophic group involved, together with the respective amount of biomass. BUT assessing the actual amount of each trophic group and related kinetic parameters can only be made through experiments such as yours.

So, to my opinion, ADM1 is appropriate, but you need to tune the kinetic parameters differently in order to get the right balance between acetoclastic and hydrogenotrophic methanogens.

I think you can try some strains of Bacillus subtilis. 

As far as know, no. They have quite specific nutritional requirements. DSMZ has some media receipts for specific groups. For a more versatile medium take a look at

Khelaifia S, Raoult D, Drancourt M. 2013. A versatile medium for cultivating methanogenic archaea. PLoS One. 8(4):e61563

Vimac,

We have thought about this issue and reached the conclusion that understanding the process at the genome level is not very useful.  In anaerobic digestion you are dealing with an ecosystem that is dynamically altering itself to adapt to its environment.  The microbes are swapping DNA material between themselves to achieve survival.  Anaerobic digestion involves thousands of species of microbes including bacteria, yeast, anaerobic fungi, protozoa, etc.  All of theses species are needed to make the reaction work.  Most of these microbes can not be isolated as their dependence upon another species.  We have shown a more productive approach is to manage the system, not try to manage each microbe. 

That said we have been able to alter an anaerobic process by the introduction of select species of microbes that are compatible with the ecosystem.  This enables us to shift the production of acids from the low valued material of acetic acid to higher value buteric and hexanoic acids. Essentially we find compatible microbes and let nature take its course.  As one engineer put it, " in this process, you are either at the table or on the table".

I'm wondering why you don't have access to animal manure. It's basically everywhere and you can use slurry or diluted manure. If still struggle with animal by-products try to find sludge from wastewater treatment plant. They normaly provide it without problems. 

Gas chromatography with thermal conductivity detection (GC/TCD). Helium works best as a carrier gas for this analysis because of its large difference in thermal conductivity from methane. I use a portable GC/TCD for this analysis, which I switch out between He and Ar carrier gases for methane and hydrogen analysis, respectively.